阅读说明：本技术 可诱导的嵌合细胞激素受体 (Inducible chimeric cytokine receptors ) 是由 A·R·纳格尔 S·帕克 里格斯 J·F·查帕罗 R·J·林 T·J·范布拉孔 于 2019-03-01 设计创作，主要内容包括：本发明提供对例如小分子或蛋白质的配体有反应的可诱导的嵌合细胞激素受体,所述受体用于改进包含所述可诱导的嵌合细胞激素受体的如T细胞的经遗传修饰的免疫细胞的功能活性的用途,及包含所述细胞的组合物。(The present invention provides inducible chimeric cytokine receptors responsive to ligands such as small molecules or proteins, the use of the receptors to improve the functional activity of genetically modified immune cells such as T cells comprising the inducible chimeric cytokine receptors, and compositions comprising the cells.)

1. An inducible chimeric cytokine receptor comprising:

a dimerization domain;

a tyrosine kinase activation domain; and

a tyrosine effector domain.

2. The inducible chimeric cytokine receptor of claim 1, wherein the tyrosine kinase activation domain comprises a janus kinase (JAK) binding domain of a protein (or derived therefrom).

3. The inducible chimeric cytokine receptor of claim 1, wherein the tyrosine kinase activation domain comprises a tyrosine kinase domain of (or derived from) a Receptor Tyrosine Kinase (RTK).

4. The inducible chimeric cytokine receptor of claim 2 or 3, wherein the tyrosine kinase activation domain comprises a transmembrane domain.

5. The inducible chimeric cytokine receptor of any one of claims 1-4, wherein the tyrosine effector domain comprises at least one STAT activation domain of the receptor (or derived therefrom).

6. The inducible chimeric cytokine receptor of any one of claims 1-5, wherein the tyrosine effector domain comprises at least two STAT activation domains of (or derived from) both receptors.

7. The inducible chimeric cytokine receptor of any one of claims 1-6, wherein the tyrosine effector domain comprises a portion of the cytoplasmic tail (or derived therefrom) of at least one Receptor Tyrosine Kinase (RTK).

8. The inducible chimeric cytokine receptor of any one of claims 1-7, wherein the dimerization domain binds to ligand AP1903, AP20187, dimeric FK506, or a dimeric FK 506-like analog.

9. The inducible chimeric cytokine receptor of any one of claims 1-8, wherein the dimerization domain comprises an FKBP polypeptide.

10. The inducible chimeric cytokine receptor of claim 9, wherein the FKBP polypeptide is an FKBP12 polypeptide.

11. The inducible chimeric cytokine receptor of claim 10, wherein the FKBP12 polypeptide comprises the amino acid substitution F36V (SEQ ID NO: 218).

12. The inducible chimeric cytokine receptor of any one of claims 1-8, wherein the dimerization domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: (i) an FKBP polypeptide comprising one or more amino acid substitutions, (ii) two or three tandem repeats of an unmodified FKBP polypeptide, and (iii) two or three tandem repeats of an FKBP polypeptide comprising one or more amino acid substitutions.

13. The inducible chimeric cytokine receptor of any one of claims 1-8, wherein the dimerization domain comprises a dimerization domain sequence selected from the group consisting of SEQ ID NOs 69-87.

14. The inducible chimeric cytokine receptor of any one of claims 1-8, wherein the dimerization domain comprises an FKBP dimerization domain sequence selected from SEQ ID nos. 69-73.

15. The inducible chimeric cytokine receptor of any one of claims 1-8, wherein the dimerization domain comprises the amino acid sequence of (or derived from) a polypeptide selected from the group consisting of: FKBP12, FKBP12(F36V), the extracellular domain of OX-40 and the extracellular domain of TNFR2 superfamily receptors.

16. The inducible chimeric cytokine receptor of claim 15, wherein the TNFR2 superfamily receptor is BCMA, TACI, or BAFFR.

17. The inducible chimeric cytokine receptor of any one of claims 1-7, wherein the dimerization domain binds a small molecule.

18. The inducible chimeric cytokine receptor of any one of claims 1-7, wherein the dimerization domain binds a protein.

19. The inducible chimeric cytokine receptor of any one of claims 1-7, wherein the dimerization domain comprises the amino acid sequence of (or derived from) a protein selected from the group consisting of: FKBP, cyclophilins, steroid binding proteins, estrogen binding proteins, glucocorticoid binding proteins, vitamin D binding proteins, tetracycline binding proteins, extracellular domains of cytokine receptors, receptor tyrosine kinases, TNFR family receptors, and immune co-receptors.

20. The inducible chimeric cytokine receptor of claim 19, wherein the immune co-receptor is selected from the group consisting of: erythropoietin receptor, prolactin receptor, growth hormone receptor, thrombopoietin receptor, granulocyte colony stimulating factor receptor, GP130, common gamma chain receptor, common beta chain receptor, IFN alpha receptor, IFN gamma receptor, IFN lambda receptor, IL2/IL15 receptor, IL3 receptor, IL4 receptor, IL5 receptor, IL7 receptor, IL9 receptor, TSLP receptor, G-CSF receptor, GM-CSF receptor, CNTF receptor, OSM receptor, LIF receptor, CT-1 receptor, TGFBR 9/ALKL 9, TGFBR 9, EGFR/HER 9, ERBB 9/9, FGFR/9, VEGFR 1/VEGFR 72, FGFR/9, FG, FGFR-3, FGFR-4, CCK4, TRKA, TRKB, TRKC, MET, RON, EPHA1, EPHA2, EPHA3, EPHA4, EPHA5, EPHA6, EPHA7, EPHA8, EPHB1, EPHB2, EPHB3, EPHB4, EPHB5, EPHB6, AXL, MER, TYRO3, TIE, TEK, RYK, DDR1, DDR2, RET, LTK, ALK, RODcR 1, ROR2, MUSK, TYAAK, AATYK2, RTK106, TNFR2, Fas, TRAILR2, NGFR, DR2, TNFR2, TNFR, TROB 2, TROCK 2, TROCL 2, TROCR 36X 2, TROCL 3614, TROCL 2, TROCL 36X 2, TROCL 36X 2, TROCL 36X 2.

21. The inducible chimeric cytokine receptor of claim 2, wherein the protein is a receptor.

22. The inducible chimeric cytokine receptor of claim 21, wherein the receptor is a hormone receptor.

23. The inducible chimeric cytokine receptor of claim 2, 21, or 22, wherein the protein or the receptor is selected from the group consisting of EPOR, GP130, PRLR, GHR, GCSFR, and TPOR/MPLR.

24. The inducible chimeric cytokine receptor of claim 3, wherein the RTK is selected from the group consisting of: EGFR/HER, ERBB/HER, ERRB/HER, INSR, IGF-1R, IRR, PDGFRA, PDGFRB, CSF-1/SCFR, FLK/FLT, VEGFR, FGFR-1, FGFR-2, FGFR-3, FGFR-4, CCK, TRKA, TRKB, TRKC, MET, RON, EPHA, EPHB, AXL, MER, TYRO, TIE, TEK, RYK, DDR, RET, ROS, LTK, ALK, ROR, MUSK, AATYK, TYK, and RTK 106.

25. The inducible chimeric cytokine receptor of claim 3 or 24, wherein the RTK is EGFR.

26. The inducible chimeric cytokine receptor of any one of claims 1-25, wherein the tyrosine kinase activation domain comprises a tyrosine kinase activation domain sequence selected from SEQ ID No. 88-133.

27. The inducible chimeric cytokine receptor of any one of claims 4 to 26, wherein the transmembrane domain comprises a transmembrane domain of (or derived from) a protein selected from the group consisting of: EPOR, GP130, PRLR, GHR, GCSFR, PD-1, and TPOR/MPLR.

28. The inducible chimeric cytokine receptor of any one of claims 4-27, wherein the transmembrane domain comprises a transmembrane domain derived from TPOR/MPLR.

29. The inducible chimeric cytokine receptor of any one of claims 4-27, wherein the transmembrane domain is derived from amino acids 478-582 of the native TPOR/MPLR sequence of SEQ ID No. 64.

30. The inducible chimeric cytokine receptor of any one of claims 4-27, wherein the transmembrane domain comprises a deletion variant of amino acid region 478-582 of the native TPOR/MPLR sequence of SEQ ID No. 64.

31. The inducible chimeric cytokine receptor of claim 30, wherein the deletion variant comprises a deletion of 1 to 18 amino acids in region 478-582 of the native TPOR/MPLR sequence of SEQ ID No. 64.

32. The inducible chimeric cytokine receptor of claim 30 or 31, wherein the deletion variant comprises a deletion of 1 to 18 amino acids from region 489-510 of the native TPOR/MPLR sequence of SEQ ID No. 64.

33. The inducible chimeric cytokine receptor of any one of claims 4-27, wherein the transmembrane domain comprises an insertion variant of amino acid region 478-582 of the native TPOR/MPLR sequence of SEQ ID No. 64.

34. The inducible chimeric cytokine receptor of claim 33, wherein the insertion variant comprises an insertion of 1 to 8 amino acids in region 478-582 of the native TPOR/MPLR sequence of SEQ ID No. 64.

35. The inducible chimeric cytokine receptor of claim 33 or 34, wherein the insertion variant comprises an insertion of 1 to 8 amino acids in region 489-510 of the native TPOR/MPLR sequence of SEQ ID No. 64.

36. The inducible chimeric cytokine receptor of any one of claims 33 to 35, wherein the amino acid inserted in the insertion variant is selected from the group consisting of: leucine, valine and isoleucine.

37. The inducible chimeric cytokine receptor of any one of claims 1-25, wherein the tyrosine kinase activation domain comprises a sequence selected from the group consisting of SEQ ID No. 104-133.

38. The inducible chimeric cytokine receptor of claim 5 or 6, wherein the receptor is a hormone receptor.

39. The inducible chimeric cytokine receptor of claim 5 or 6, wherein the receptor is a cytokine receptor.

40. The inducible chimeric cytokine receptor of any one of claims 5-7 and 38-39, wherein the receptor is selected from the group consisting of: BLNK, IL2RG, EGFR, EpoR, GHR, IFNAR1, IFNAR2, IFNAR1/2, IFNLR1, IL10R1, IL12Rb1, IL12Rb2, IL21R, IL2Rb, IL2small, IL7R, IL7Ra, IL9R, IL15R and IL 21R.

41. The inducible chimeric cytokine receptor of claim 1, wherein the tyrosine kinase activation domain comprises a transmembrane domain and a janus kinase (JAK) binding domain and the tyrosine effector domain comprises at least one STAT activation domain of (or derived from) a receptor.

42. The inducible chimeric cytokine receptor of claim 41,

the dimerization domain comprises an FKBP polypeptide;

the transmembrane domain comprises a transmembrane domain of (or derived from) a protein selected from the group consisting of: EPOR, GP130, PRLR, GHR, GCSFR, PD-1, and TPOR;

The JAK binding domain comprises a JAK binding domain of (or derived from) a protein selected from the group consisting of: EPOR, GP130, PRLR, GHR, GCSFR, and TPOR; and

the STAT activation domain comprises at least one STAT activation domain of (or derived from) a receptor selected from the group consisting of: BLNK, IL2RG, EGFR, EpoR, GHR, IFNAR1, IFNAR2, IFNAR1/2, IFNLR1, IL10R1, IL12Rb1, IL12Rb2, IL21R, IL2Rb, IL2small, IL7R, IL7Ra, IL9R, IL15R and IL 21R.

43. The inducible chimeric cytokine receptor of claim 42, wherein the tyrosine effector domain comprises at least two STAT activation domains of (or derived from) at least two receptors.

44. The inducible chimeric cytokine receptor of any one of claims 1-43, wherein the tyrosine effector domain comprises a tyrosine effector domain sequence selected from the group consisting of SEQ ID NO 134-176.

45. The inducible chimeric cytokine receptor of any one of claims 1-44, wherein the dimerization domain is located at the N-terminus of the inducible chimeric cytokine receptor.

46. The inducible chimeric cytokine receptor of any one of claims 1-44, wherein the dimerization domain is located at the C-terminus of the inducible chimeric cytokine receptor.

47. The inducible chimeric cytokine receptor of any one of claims 1-46, wherein the inducible chimeric cytokine receptor comprises a membrane-targeting motif.

48. The inducible chimeric cytokine receptor of claim 47, wherein the membrane-targeting motif comprises a myristoylation motif.

49. The inducible chimeric cytokine receptor of any one of claims 1-48, wherein the receptor is myristoylated.

50. The inducible chimeric cytokine receptor of claim 1, comprising the sequences disclosed in table 2A or table 2B.

51. A polynucleotide comprising a nucleic acid sequence encoding the inducible chimeric cytokine receptor of any one of claims 1-50.

52. An expression vector comprising the polynucleotide of claim 51.

53. An engineered immune cell comprising at least one inducible chimeric cytokine receptor according to any one of claims 1 to 50 or at least one polynucleotide according to claim 51.

54. The engineered immune cell of claim 53, wherein the cell comprises at least two inducible chimeric cytokine receptors of any one of claims 1-50 or at least two polynucleotides of claim 51.

55. The engineered immune cell of claim 53 or 54, wherein the cell comprises at least three or four inducible chimeric cytokine receptors of any one of claims 1-50 or at least three or four polynucleotides of claim 51.

56. The engineered immune cell of any one of claims 53 to 55, wherein, when more than one inducible chimeric cytokine receptor is present, the dimerization domain, the tyrosine kinase activation domain and the tyrosine effector domain of each receptor can be the same or different.

57. The engineered immune cell of any one of claims 53-56, wherein the cell further comprises a Chimeric Antigen Receptor (CAR) or a polynucleotide encoding a CAR.

58. The engineered immune cell of any one of claims 53-57, wherein the immune cell is selected from the group consisting of: t cells, dendritic cells, killer dendritic cells, mast cells, NK cells, macrophages, monocytes, B-cells and immune cells derived from stem cells.

59. The engineered immune cell of any one of claims 53-58, wherein the immune cell is a T cell.

60. A method of modulating an engineered immune cell in an individual, the method comprising administering a ligand to an individual to whom an engineered immune cell according to any one of claims 53 to 59 has been previously administered, wherein the dimeric ligand binds to the dimerization domain of the inducible chimeric cytokine receptor.

61. The method of claim 60, wherein the ligand is AP 1903.

62. A method of making an engineered immune cell, the method comprising introducing the polynucleotide of claim 51 or the expression vector of claim 52 into an immune cell.

63. The method of claim 62, wherein the immune cell is selected from the group consisting of: t cells, dendritic cells, killer dendritic cells, mast cells, NK cells, macrophages, monocytes, B-cells and immune cells derived from stem cells.

64. The method of claim 62 or 63, wherein the immune cell is a T cell.

65. An isolated immune cell comprising:

(i) at least one inducible chimeric cytokine receptor comprising a dimerization domain, a tyrosine kinase activation domain, and a tyrosine effector domain; and

(ii) A Chimeric Antigen Receptor (CAR) comprising an extracellular ligand-binding domain, a transmembrane domain, and an intracellular signaling domain.

66. The isolated immune cell of claim 65, wherein the inducible chimeric cytokine receptor is the inducible chimeric cytokine receptor of any one of claims 1-50.

67. The isolated immune cell of claim 66, wherein the cell comprises at least two, three, or four inducible chimeric cytokine receptors.

68. The isolated immune cell of claim 65, wherein the extracellular ligand-binding domain of the CAR specifically binds BCMA, EGFRvIII, Flt-3, WT-1, CD20, CD23, CD30, CD38, CD70, CD33, CD133, LeY, NKG2D, CS1, CD44v6, ROR1, CD19, claudin-18.2 (claudin-18A 2 or claudin 18 isoform 2), DLL3 (3-like protein, Drosophila homolog 3, 3), Muc17(Mucin 2, Muc3, Muc3), FAP α (fibroblast activation protein α), Ly6G6D (lymphocyte antigen 6 complex gene seatin G6d, c6orf23, G6D, MEGT1, 1), RNF 72 (RNF 1-RNF 1, or RNRIF 43).

69. The isolated immune cell of any one of claims 65-68, wherein the immune cell is selected from the group consisting of: t cells, dendritic cells, killer dendritic cells, mast cells, NK cells, macrophages, monocytes, B-cells and immune cells derived from stem cells.

70. The isolated immune cell of any one of claims 65-68, wherein the immune cell is a T cell.

71. The isolated immune cell of any one of claims 65-70, wherein the isolated immune cell exhibits improved persistence relative to a persistence of an isolated immune cell that does not express the inducible chimeric cytokine receptor after contact with a ligand that binds to a dimerization domain.

72. The isolated immune cell of any one of claims 65-71, wherein the isolated immune cell exhibits increased activation of STAT upon contact with a ligand that binds to a dimerization domain relative to activation of STAT exhibited by an isolated immune cell that does not express the inducible chimeric cytokine receptor.

73. The isolated immune cell of claim 72, wherein the STAT is STAT1, STAT2, STAT3, STAT4, STAT5, STAT6, or a combination thereof.

74. The isolated immune cell of claim 72 or 73, wherein activation of STAT by the isolated immune cell increases with dose of ligand, after contact with ligand that binds to a dimerization domain, as compared to activation of STAT exhibited by an isolated immune cell that does not express the inducible chimeric cytokine receptor.

75. The isolated immune cell of any one of claims 65-74, wherein the isolated immune cell exhibits increased cytotoxicity after contact with a ligand that binds to a dimerization domain as compared to the cytotoxicity exhibited by an isolated immune cell that does not express the inducible chimeric cytokine receptor.

76. The isolated immune cell of any one of claims 65-75, wherein the isolated immune cell expands upon contact with a ligand that binds to a dimerization domain as compared to an isolated immune cell that does not express the inducible chimeric cytokine receptor.

77. The isolated immune cell of any one of claims 65-76, wherein the level of cellular markers of stem cell memory (Tsccm) and/or central memory (Tcm) on the isolated immune cell is increased or unchanged upon contact with a ligand that binds to a dimerization domain as compared to the level of these markers on an isolated immune cell that does not express the inducible chimeric cytokine receptor.

78. The isolated immune cell of any one of claims 71-77, wherein the isolated immune cell is a T cell.

79. A method of producing an isolated immune cell comprising the inducible chimeric cytokine receptor of any one of claims 1-50, wherein the method comprises the steps of:

(a) Providing an immune cell;

(b) introducing into the immune cell a polynucleotide encoding a Chimeric Antigen Receptor (CAR) comprising an extracellular ligand binding domain, a transmembrane domain, and an intracellular signaling domain; and

(c) introducing into said immune cell a polynucleotide encoding said inducible chimeric cytokine receptor.

80. The method according to claim 79, wherein step c) comprises stably expressing the inducible chimeric cytokine receptor in a cell.

81. The method of claim 79 or 80, wherein in step c) the polynucleotide encoding the inducible chimeric cytokine receptor is introduced into the cell by a transposon/transposase system, a virus-based gene transfer system, or electroporation.

82. The method of any one of claims 79 to 81, wherein in step b) the polynucleotide encoding the chimeric antigen receptor is introduced into the cell by a transposon/transposase system or a virus-based gene transfer system.

83. The method of claim 81 or 82, wherein the virus-based gene transfer system comprises a recombinant retrovirus or lentivirus.

84. The method of any one of claims 79 to 83, wherein step (b) is performed before step (c).

85. The method of any one of claims 79 to 83, wherein step (c) is performed prior to step (b).

86. The method of any one of claims 79 to 85, wherein the immune cell is selected from the group consisting of: t cells, dendritic cells, killer dendritic cells, mast cells, NK cells, macrophages, monocytes, B-cells and immune cells derived from stem cells.

87. The method of any one of claims 79-86, wherein the immune cell is a T cell.

88. A pharmaceutical composition comprising the isolated immune cell of any one of claims 65-78.

89. A method for treating a disorder in an individual, wherein the method comprises administering to the individual the isolated immune cell of any one of claims 65-78 or administering to the individual the pharmaceutical composition of claim 88.

90. The method of claim 89, wherein the cell or pharmaceutical composition is provided to the subject more than once.

91. The method of claim 89 or 90, wherein the cells or the pharmaceutical composition are provided to the individual at least about 1, 2, 3, 4, 5, 6, 7, or more days apart.

92. The method of any one of claims 89-91, wherein the individual has been previously treated with a therapeutic agent prior to administration of the isolated immune cell or the pharmaceutical composition thereto.

93. The method of claim 92, wherein the therapeutic agent is an antibody or a chemotherapeutic agent.

94. The method of any one of claims 89 to 93, wherein the disorder is a viral disease, a bacterial disease, a cancer, an inflammatory disease, an immune disease, or an aging-related disease.

95. The method of claim 94, wherein the cancer is a hematological malignancy or a solid cancer.

96. The method of claim 95, wherein the hematological malignancy is selected from Acute Lymphoblastic Leukemia (ALL), Acute Myeloid Leukemia (AML), Chronic Myeloid Leukemia (CML), Chronic Eosinophilic Leukemia (CEL), myelodysplastic syndrome (MDS), non-Hodgkin's lymphoma (NHL), or Multiple Myeloma (MM).

97. The method of claim 95, wherein the solid cancer is selected from cholangiocarcinoma, bladder cancer, bone and soft tissue cancer, brain tumor, breast cancer, cervical cancer, colon cancer, colorectal adenocarcinoma, colorectal cancer, desmoid tumor, embryonic cancer, endometrial cancer, esophageal cancer, gastric adenocarcinoma, glioblastoma multiforme, gynecological tumor, head and neck squamous cell carcinoma, liver cancer, lung cancer, malignant melanoma, osteosarcoma, ovarian cancer, pancreatic ductal adenocarcinoma, primary astrocytoma, primary thyroid cancer, prostate cancer, renal cell carcinoma, rhabdomyosarcoma, skin cancer, soft tissue sarcoma, testicular germ cell tumor, urothelial cell carcinoma, uterine sarcoma, or uterine cancer.

98. Use of the isolated immune cell of any one of claims 65-78 or the pharmaceutical composition of claim 88 for treating a disorder.

99. The use of claim 98, wherein the cell or the pharmaceutical composition is provided to the subject more than once.

100. The use of claim 98 or 99, wherein the cells or the pharmaceutical composition are provided to the subject at least about 1, 2, 3, 4, 5, 6, 7 or more days apart.

101. The use of any one of claims 98-100, wherein the individual has been treated with a therapeutic agent prior to administration of the isolated immune cell or the pharmaceutical composition thereto.

102. The use of claim 101, wherein the therapeutic agent is an antibody or a chemotherapeutic agent.

103. The use of any one of claims 98-102, wherein the disorder is a viral disease, a bacterial disease, a cancer, an inflammatory disease, an immune disease, or an aging-related disease.

104. The use of claim 103, wherein the cancer is a hematological malignancy or a solid cancer.

105. The use of claim 104, wherein the hematological malignancy is selected from Acute Lymphoblastic Leukemia (ALL), Acute Myeloid Leukemia (AML), Chronic Myeloid Leukemia (CML), Chronic Eosinophilic Leukemia (CEL), myelodysplastic syndrome (MDS), non-hodgkin's lymphoma (NHL), or Multiple Myeloma (MM).

106. The use of claim 104, wherein the solid cancer is selected from cholangiocarcinoma, bladder cancer, bone and soft tissue cancer, brain tumor, breast cancer, cervical cancer, colon cancer, colorectal adenocarcinoma, colorectal cancer, desmoid tumor, embryonic cancer, endometrial cancer, esophageal cancer, gastric adenocarcinoma, glioblastoma multiforme, gynecological tumor, head and neck squamous cell carcinoma, liver cancer, lung cancer, malignant melanoma, osteosarcoma, ovarian cancer, pancreatic ductal adenocarcinoma, primary astrocytoma, primary thyroid cancer, prostate cancer, renal cell carcinoma, rhabdomyosarcoma, skin cancer, soft tissue sarcoma, testicular germ cell tumor, urothelial cell carcinoma, uterine sarcoma, or uterine cancer.

107. The engineered immune cell of any one of claims 53-59, wherein the cell is an autologous T cell.

108. The engineered immune cell of any one of claims 53-59, wherein the cell is an allogeneic T cell.

109. The isolated immune cell of any one of claims 65-78, wherein the cell is an autologous T cell.

110. The isolated immune cell of any one of claims 65-78, wherein the cell is an allogeneic T cell.

Technical Field

The present invention relates generally to inducible chimeric cytokine receptors for use with immune cells (e.g., T cells) to treat diseases.

Background

Chimeric antigen receptor T (CAR-T) cells have entered the clinic and have demonstrated very promising results (Maus, m. et al, 2014, Blood (Blood) 123, 2625-35). Although most individuals have been treated with autologous CAR-T cells derived from the individual's own T cells, allogeneic CAR-T cells derived from healthy donors offer a more commercially viable, off-the-shelf option with the potential to treat a wider range of individuals.

Allogeneic CAR-T cells are generated by conferring T cells from healthy donors with CARs specifically activated by tumor-associated antigens. Does not express a functional TCR (example)E.g., knocked out or knocked down) allogeneic CAR-T cells lack basal TCR signaling. Basal TCR signaling increases persistence. TCR mobilization of Ca²⁺Eventually leading to NFAT and NFkB activation. Although cytokines can increase persistence via STAT5, this does not reproduce native TCR signaling. Thus, there is a need for compositions and methods that improve the persistence of allogeneic CAR-T cells.

Disclosure of Invention

The present invention provides inducible chimeric cytokine receptors responsive to ligands, e.g., small molecules or proteins, for use in improving the functional activity of genetically modified T cells (e.g., genetically modified antigen-specific T cells, such as chimeric antigen receptor T (CAR-T) cells), cells comprising inducible chimeric cytokine receptors, and compositions comprising the cells. In particular, the invention provides methods and compositions for enhancing the therapeutic efficacy of CAR-T cells.

In one aspect, the present invention provides an inducible chimeric cytokine receptor comprising: a dimerization domain; a tyrosine kinase activation domain; and a tyrosine effector domain.

In some embodiments, the tyrosine kinase activation domain comprises a janus kinase (JAK) binding domain of the protein (or derived therefrom). In certain of these embodiments, the tyrosine kinase activation domain further comprises a transmembrane domain.

In some embodiments, the tyrosine kinase activation domain comprises a tyrosine kinase domain of (or derived from) a Receptor Tyrosine Kinase (RTK). In certain of these embodiments, the tyrosine kinase activation domain further comprises a transmembrane domain.

In some embodiments, the tyrosine effector domain comprises a STAT activation domain of (or derived from) at least one receptor. In some embodiments, the tyrosine effector domain comprises at least two STAT activation domains for (or derived from) both receptors. In some embodiments, the tyrosine effector domain comprises at least three, four, or more STAT activation domains of (or derived from) the receptor.

In some embodiments, the tyrosine effector domain comprises a portion of the cytoplasmic tail of (or derived from) at least one Receptor Tyrosine Kinase (RTK).

In some embodiments, the dimerization domain binds to a ligand, such as AP1903, AP20187, dimeric FK506, or dimeric FK 506-like analogs.

In some embodiments, the dimerization domain comprises an FKBP polypeptide. In some embodiments, the FKBP polypeptide is an FKBP12 polypeptide. In some embodiments, the FKBP12 polypeptide comprises the amino acid substitution F36V (SEQ ID No.: 218).

In some embodiments, the dimerization domain comprises an amino acid sequence selected from the group consisting of seq id no: (i) an FKBP polypeptide comprising one or more amino acid substitutions, (ii) two or three tandem repeats of an unmodified FKBP polypeptide, and (iii) two or three tandem repeats of an FKBP polypeptide comprising one or more amino acid substitutions.

In some embodiments, the dimerization domain comprises a dimerization domain sequence selected from the group consisting of SEQ ID nos 69-87.

In some embodiments, the dimerization domain comprises an FKBP dimerization domain sequence selected from SEQ ID NO. 69-73.

In some embodiments, the dimerization domain comprises the amino acid sequence of (or derived from) a polypeptide selected from the group consisting of: FKBP12, FKBP12(F36V), the extracellular domain of OX-40 and the extracellular domain of TNFR2 superfamily receptors. In exemplary embodiments, the TNFR2 superfamily receptor is BCMA, TACI, or BAFFR.

In some embodiments, the dimerization domain binds to a small molecule. In exemplary embodiments, the small molecule is AP1903, AP20187, dimeric FK506, or dimeric FK 506-like analog. In some embodiments, the dimerization domain binds to a protein.

In some embodiments, the dimerization domain comprises the amino acid sequence of (or derived from) a protein selected from the group consisting of: FKBP, cyclophilins, steroid binding proteins, estrogen binding proteins, glucocorticoid binding proteins, vitamin D binding proteins, tetracycline binding proteins, extracellular domains of cytokine receptors, receptor tyrosine kinases, TNFR family receptors, and immune co-receptors.

In some embodiments, the immune co-receptor of the derivatized dimerization domain is selected from the group consisting of: erythropoietin receptor, prolactin receptor, growth hormone receptor, thrombopoietin receptor, granulocyte colony stimulating factor receptor, GP130, common gamma chain receptor, common beta chain receptor, IFN alpha receptor, IFN gamma receptor, IFN lambda receptor, IL2/IL15 receptor, IL3 receptor, IL4 receptor, IL5 receptor, IL7 receptor, IL9 receptor, TSLP receptor, G-CSF receptor, GM-CSF receptor, CNTF receptor, OSM receptor, LIF receptor, CT-1 receptor, TGFBR 9/ALKL 9, TGFBR 9, EGFR/HER 9, ERBB 9/9, FGFR/9, VEGFR 1/VEGFR 72, FGFR/9, FG, FGFR-3, FGFR-4, CCK4, TRKA, TRKB, TRKC, MET, RON, EPHA1, EPHA2, EPHA3, EPHA4, EPHA5, EPHA6, EPHA7, EPHA8, EPHB1, EPHB2, EPHB3, EPHB4, EPHB5, EPHB6, AXL, MER, TYRO3, TIE, TEK, RYK, DDR1, DDR2, RET, LTK, ALK, RODcR 1, ROR2, MUSK, TYAAK, AATYK2, RTK106, TNFR2, Fas, TRAILR2, NGFR, DR2, TNFR2, TNFR, TROB 2, TROCK 2, TROCL 2, TROCR 36X 2, TROCL 3614, TROCL 2, TROCL 36X 2, TROCL 36X 2, TROCL 36X 2.

In some embodiments, the tyrosine kinase activation domain comprises a JAK binding domain of (or derived from) a receptor. In one exemplary embodiment, the receptor is a hormone receptor.

In some embodiments, the tyrosine kinase activation domain comprises a JAK binding domain of (or derived from) a protein or receptor selected from the group consisting of EPOR, GP130, PRLR, GHR, GCSFR, and TPOR/MPLR.

In some embodiments, the tyrosine kinase activation domain comprises a tyrosine kinase domain of (or derived from) an RTK, wherein the RTK is selected from the group consisting of: EGFR/HER, ERBB/HER, ERRB/HER, INSR, IGF-1R, IRR, PDGFRA, PDGFRB, CSF-1/SCFR, FLK/FLT, VEGFR, FGFR-1, FGFR-2, FGFR-3, FGFR-4, CCK, TRKA, TRKB, TRKC, MET, RON, EPHA, EPHB, AXL, MER, TYRO, TIE, TEK, RYK, DDR, RET, ROS, LTK, ALK, ROR, MUSK, AATYK, TYK, and RTK 106. In an exemplary embodiment, the RTK is EGFR.

In some embodiments, the tyrosine kinase activation domain comprises a tyrosine kinase activation domain sequence selected from SEQ ID nos. 88-133.

In some embodiments, the transmembrane domain present in the tyrosine kinase activation domain comprises a transmembrane domain of (or derived from) a protein selected from the group consisting of: EPOR, GP130, PRLR, GHR, GCSFR, PD-1, and TPOR/MPLR.

In some embodiments, the transmembrane domain comprises a transmembrane domain derived from TPOR/MPLR. In some embodiments, the transmembrane domain is derived from amino acids 478-582 of the native TPOR/MPLR sequence of SEQ ID NO. 64.

In some embodiments, the transmembrane domain comprises a deletion variant of amino acid region 478-582 of the native TPOR/MPLR sequence of SEQ ID NO. 64. In some embodiments, the deletion variant comprises amino acid region 478-582 of the native TPOR/MPLR sequence of SEQ ID NO. 64. In some embodiments, the deletion variant comprises the deletion of 1 to 18 amino acids from region 489-510 of the native TPOR/MPLR sequence of SEQ ID NO. 64.

In some embodiments, the transmembrane domain comprises an insertion variant of amino acid region 478-582 of native TPOR/MPLR of SEQ ID NO. 64. In some embodiments, the insertion variant comprises amino acids 478-582 of the native TPOR/MPLR of SEQ ID NO. 64. In some embodiments, the insertion variant comprises amino acids 478-582 of the native TPOR/MPLR of SEQ ID NO. 64. In exemplary embodiments, the amino acids inserted in the insertion variant are selected from the group consisting of: leucine, valine and isoleucine.

In some embodiments, the tyrosine effector domain comprises at least one STAT activation domain of (or derived from) the receptor. In some embodiments, the tyrosine effector domain comprises at least two STAT activation domains of (or derived from) both receptors. In some embodiments, the tyrosine effector domain comprises a STAT activation domain of (or derived from) at least three, four, or more effectors. In some embodiments, the receptor is a hormone receptor and/or a cytokine receptor.

In some embodiments, the tyrosine effector domain comprises at least one, two, three, four or more STAT activation domains of (or derived from) a receptor, wherein the receptor is selected from the group consisting of: BLNK, IL2RG, EGFR, EpoR, GHR, IFNAR1, IFNAR2, IFNAR1/2, IFNLR1, IL10R1, IL12Rb1, IL12Rb2, IL21R, IL2Rb, IL2small, IL7R, IL7Ra, IL9R, IL15R and IL 21R.

In some embodiments, the tyrosine effector domain comprises a cellular tail (a portion of the cytoplasmic tail of a receptor comprising one or more tyrosine residues that may be phosphorylated) of (or derived from) at least one, two, three, four, or more receptors, wherein the receptors are cytokine receptors, hormone receptors, and/or RTKs.

In some embodiments, the inducible chimeric cytokine receptor comprises a dimerization domain; a tyrosine kinase activation domain comprising a transmembrane domain and a JAK binding domain; and a tyrosine effector domain comprising at least one STAT activation domain of (or derived from) the receptor. In certain of these embodiments, the tyrosine effector domain may comprise at least two, three, four, or more STAT activation domains of (or derived from) the receptor.

In some embodiments, the inducible chimeric cytokine receptor comprises a dimerization domain; a tyrosine kinase activation domain comprising a transmembrane domain and a JAK binding domain; and a tyrosine effector domain comprising at least one cell tail of (or derived from) the receptor. In certain of these embodiments, the tyrosine effector domain may comprise a cell tail of (or derived from) at least two, three, four, or more receptors.

In some embodiments, the inducible chimeric cytokine receptor comprises a dimerization domain comprising an FKBP polypeptide; a tyrosine kinase activation domain comprising a transmembrane domain and a JAK binding domain, wherein the transmembrane domain comprises a transmembrane domain of (or derived from) a protein selected from the group consisting of: EPOR, GP130, PRLR, GHR, GCSFR, PD-1, and TPOR, and the JAK binding domain comprises a JAK binding domain of (or derived from) a protein selected from the group consisting of: EPOR, GP130, PRLR, GHR, GCSFR, and TPOR; and a tyrosine effector domain comprising at least one STAT activation domain of (or derived from) a receptor selected from the group consisting of: BLNK, IL2RG, EGFR, EpoR, GHR, IFNAR1, IFNAR2, IFNAR1/2, IFNLR1, IL10R1, IL12Rb1, IL12Rb2, IL21R, IL2Rb, IL2small, IL7R, IL7Ra, IL9R, IL15R and IL 21R. In certain of these embodiments, the tyrosine effector domain comprises STAT activation domains of (or derived from) at least two, three, four, or more receptors.

In some embodiments, the tyrosine effector domain comprises a tyrosine effector domain sequence selected from the group consisting of SEQ ID NO. 134-176.

In some embodiments, the dimerization domain is located N-terminal to the inducible chimeric cytokine receptor.

In some embodiments, the dimerization domain is located C-terminal to the inducible chimeric cytokine receptor.

In some embodiments, an inducible chimeric cytokine receptor provided herein comprises a membrane-targeting motif. In an exemplary embodiment, the membrane-targeting motif comprises a myristoylation motif.

In some embodiments, the inducible chimeric cytokine receptor provided herein is myristoylated.

In some embodiments, the inducible chimeric cytokine receptor comprises the sequences disclosed in table 2A or 2B. In some embodiments, the inducible chimeric cytokine receptor comprises a sequence selected from the group consisting of SEQ ID NO. 1-58, 187-215, and 225-311.

In another aspect, the invention provides a polynucleotide comprising a nucleic acid sequence encoding an inducible chimeric cytokine receptor as described herein. In another aspect, the invention provides an expression vector comprising the polynucleotide.

In another aspect, the invention provides an engineered immune cell comprising at least one inducible chimeric cytokine receptor as disclosed herein. In some embodiments, the engineered immune cell comprises at least two inducible chimeric cytokine receptors. In some embodiments, the engineered immune cell comprises at least three or four inducible chimeric cytokine receptors disclosed herein. When more than one inducible chimeric cytokine receptor is present in an immune cell, the dimerization domain, tyrosine kinase activation domain, and tyrosine effector domain of each receptor may be the same or different.

In another aspect, the invention provides an engineered immune cell comprising at least one polynucleotide encoding an inducible chimeric cytokine receptor as disclosed herein.

In some embodiments, the engineered immune cell further comprises a Chimeric Antigen Receptor (CAR) or a polynucleotide encoding a CAR.

In some embodiments, the immune cell is selected from the group consisting of: t cells, dendritic cells, killer dendritic cells, mast cells, NK cells, macrophages, monocytes and B cells.

In some embodiments, the immune cell is derived from a stem cell. In exemplary embodiments, the immune cell is derived from an adult stem cell, a non-human stem cell, a cord blood stem cell, a progenitor cell, a bone marrow stem cell, an induced pluripotent stem cell, a totipotent stem cell, or a hematopoietic stem cell.

In some embodiments, the immune cell is a T cell. In some embodiments, the immune cell is an autologous T cell. In some embodiments, the immune cell is an allogeneic T cell.

In another aspect, the invention provides a method of modulating an engineered immune cell in an individual, the method comprising administering a ligand to an individual to whom an engineered immune cell as described herein has been previously administered, wherein the dimeric ligand binds to the dimerization domain of an inducible chimeric cytokine receptor. In an exemplary embodiment, the ligand is AP 1903.

In another aspect, provided herein is a method of making an engineered immune cell, the method comprising introducing into an immune cell a polynucleotide or expression vector comprising a polynucleotide encoding an inducible chimeric cytokine receptor. In one exemplary embodiment, the immune cell is selected from the group consisting of: t cells, dendritic cells, killer dendritic cells, mast cells, NK cells, macrophages, monocytes, B cells and immune cells derived from stem cells. In one exemplary embodiment, the immune cell is a T cell.

In another aspect, the invention provides an isolated immune cell comprising: (i) at least one inducible chimeric cytokine receptor comprising a dimerization domain, a tyrosine kinase activation domain, and a tyrosine effector domain as disclosed herein; and (ii) a Chimeric Antigen Receptor (CAR) comprising an extracellular ligand binding domain, a transmembrane domain, and an intracellular signaling domain.

In some embodiments, the isolated immune cell comprises at least two inducible chimeric cytokine receptors. In some other embodiments, the isolated immune cell comprises three or four inducible chimeric cytokine receptors.

In some embodiments, the isolated immune cells of the invention exhibit improved persistence relative to an isolated immune cell that does not express an inducible chimeric cytokine receptor upon contact with a ligand that binds to a dimerization domain.

In some embodiments, the isolated immune cells of the invention exhibit increased activation of STAT relative to the activation of STAT exhibited by an isolated immune cell that does not express an inducible chimeric cytokine receptor after contact with a ligand that binds to a dimerization domain. The STAT activated in the cell may be STAT1, STAT2, STAT3, STAT4, STAT5, STAT6, or a combination thereof.

In some embodiments, activation of STAT by an isolated immune cell of the invention increases with the dose of ligand after contact with ligand that binds to the dimerization domain, as compared to activation of STAT exhibited by an isolated immune cell that does not express an inducible chimeric cytokine receptor.

In some embodiments, the isolated immune cells of the invention exhibit increased cytotoxicity upon contact with a ligand that binds to the dimerization domain as compared to the cytotoxicity exhibited by isolated immune cells that do not express an inducible chimeric cytokine receptor.

In some embodiments, the isolated immune cells of the invention expand upon contact with a ligand that binds to the dimerization domain as compared to isolated immune cells that do not express an inducible chimeric cytokine receptor.

In some embodiments, the level of cellular markers of stem cell memory (Tscm) and/or central memory (Tcm) on the isolated immune cells of the invention is increased or unchanged upon contact with a ligand that binds to the dimerization domain compared to the level of these markers on isolated immune cells that do not express an inducible chimeric cytokine receptor.

In one aspect, provided herein is a method of producing an isolated immune cell comprising an inducible chimeric cytokine receptor as disclosed herein, comprising the steps of: (a) providing an immune cell; (b) introducing into an immune cell a polynucleotide encoding a Chimeric Antigen Receptor (CAR) comprising an extracellular ligand binding domain, a transmembrane domain, and an intracellular signaling domain; (c) introducing into an immune cell a polynucleotide encoding an inducible chimeric cytokine receptor.

In some embodiments, step c) of the above methods comprises stably expressing the inducible chimeric cytokine receptor in the cell.

In some embodiments, in step c) of the above method, the polynucleotide encoding the inducible chimeric cytokine receptor is introduced into the cell by a transposon/transposase system, a virus-based gene transfer system, or electroporation.

In some embodiments, in step b) of the above method, the polynucleotide encoding the chimeric antigen receptor is introduced into the cell by a transposon/transposase system or a virus-based gene transfer system

In some embodiments, the virus-based gene transfer system comprises a recombinant retrovirus or lentivirus.

In some embodiments of the above method, step (b) occurs before step (c) or step (c) occurs before step (b).

In one aspect, the invention provides a pharmaceutical composition comprising an isolated immune cell described herein.

In one aspect, the invention provides a method for treating a disorder in an individual, wherein the method comprises administering an isolated immune cell comprising an inducible cytokine receptor as disclosed herein or administering a pharmaceutical composition comprising such an immune cell.

In another aspect, the invention provides the use of an isolated immune cell or a pharmaceutical composition comprising an isolated immune cell disclosed herein for treating a disorder.

In some embodiments, the cell or pharmaceutical composition is provided to the subject more than once.

In some embodiments, the cells or pharmaceutical compositions are provided to the individual at intervals of at least about 1, 2, 3, 4, 5, 6, 7, or more days.

In some embodiments, the individual has been previously treated with a therapeutic agent prior to administration of the isolated immune cells or pharmaceutical composition. In one exemplary embodiment, the therapeutic agent is an antibody or a chemotherapeutic agent.

In some embodiments, the disorder treated using the methods of the invention is a viral disease, a bacterial disease, a cancer, an inflammatory disease, an immunological disease, or an aging-related disease. The cancer may be a hematological malignancy or a solid cancer.

In some embodiments, the hematological malignancy treated using the methods of the invention is selected from Acute Lymphoblastic Leukemia (ALL), Acute Myelogenous Leukemia (AML), Chronic Myelogenous Leukemia (CML), Chronic Eosinophilic Leukemia (CEL), myelodysplastic syndrome (MDS), non-hodgkin's lymphoma (NHL), or Multiple Myeloma (MM).

In some embodiments, the solid cancer treated using the methods of the invention is selected from cholangiocarcinoma, bladder cancer, bone and soft tissue cancer, brain tumor, breast cancer, cervical cancer, colon cancer, colorectal adenocarcinoma, colorectal cancer, desmoid tumor, embryonic cancer, endometrial cancer, esophageal cancer, gastric adenocarcinoma, glioblastoma multiforme, gynecological tumor, head and neck squamous cell carcinoma, liver cancer, lung cancer, malignant melanoma, osteosarcoma, ovarian cancer, pancreatic ductal adenocarcinoma, primary astrocytoma, primary thyroid cancer, prostate cancer, renal cell carcinoma, rhabdomyosarcoma, skin cancer, soft tissue sarcoma, testicular germ cell tumor, urothelial cell carcinoma, uterine sarcoma, or uterine cancer.

Drawings

Figure 1 depicts a schematic of an exemplary inducible chimeric cytokine receptor.

FIG. 2A depicts a schematic of FKBP binding to rapamycin (rapamycin) to inhibit mTOR.

FIG. 2B depicts FKBP^F36VSchematic of binding to rapamycin like compounds.

FIG. 2C depicts AP1903 reacting FKBP^F36VSchematic representation of dimerization.

Figure 3 depicts a schematic of an exemplary inducible chimeric cytokine receptor.

Figure 4 depicts a bar graph summarizing the results of a STAT5 assay testing the function of the inducible chimeric cytokine receptor shown.

Figure 5A depicts a graph summarizing the results of a cell-based reporter assay testing the function of the inducible chimeric cytokine receptor shown.

Figure 5B depicts a graph summarizing the results of a cell-based reporter assay testing the function of the inducible chimeric cytokine receptor shown.

Figure 6 depicts a graph summarizing the results of a cell-based reporter assay testing the function of the inducible chimeric cytokine receptor shown.

Figure 7 depicts a graph summarizing the results of a cell-based reporter assay testing the function of the inducible chimeric cytokine receptor shown.

Figure 8 depicts a graph summarizing the results of a cell-based reporter assay testing the function of the inducible chimeric cytokine receptor shown.

Figure 9 depicts a graph summarizing the results of a cell-based reporter assay testing the function of the inducible chimeric cytokine receptor shown.

Figure 10A depicts a graph summarizing the results of a cell-based reporter assay testing the function of the inducible chimeric cytokine receptor shown.

Figure 10B depicts a graph summarizing the results of a cell-based reporter assay testing the function of the inducible chimeric cytokine receptor shown.

Fig. 10C depicts a schematic showing a conventional method of engineering chimeric cytokine receptors.

Figure 10D depicts a schematic showing the method used in the present invention for engineering chimeric cytokine receptors.

Fig. 10E depicts a schematic of the chimeric receptor tested in the cell-based reporter assay of example 5C.

Figure 10F depicts a graph summarizing the results of a cell-based reporter assay testing the function of the inducible chimeric cytokine receptor shown.

Figure 11 depicts a graph summarizing the results of a cell-based reporter assay testing the function of the inducible chimeric cytokine receptor shown.

FIG. 12 depicts a schematic of AP-1 binding to NFAT of the promoter sequence.

FIG. 13 depicts a schematic of the MEK/ERK pathway leading to upregulation of Fos via Myc/Max.

Figure 14 depicts a graph summarizing the results of a cell-based reporter assay testing the function of the inducible chimeric cytokine receptor shown.

Figure 15 depicts a schematic of the BTK pathway.

Figure 16 depicts a graph summarizing the effect of the inducible chimeric cytokine receptor on cell growth shown.

Figure 17 depicts a graph summarizing the effect of the inducible chimeric cytokine receptor on cell survival shown.

Figure 18 depicts a graph summarizing the effect of the inducible chimeric cytokine receptor shown on cell phenotype.

Figure 19 depicts a graph summarizing the effect of the inducible chimeric cytokine receptor shown on cell phenotype.

Figure 20 depicts a graph summarizing the effect of the inducible chimeric cytokine receptor on cytokine release shown.

Figure 21 depicts a graph summarizing the effect of the inducible chimeric cytokine receptor on cytokine release shown.

Figure 22 depicts a graph summarizing the effect of inducible chimeric cytokine receptors on proliferation shown in the absence of signaling.

Figure 23 depicts a graph summarizing the effect of inducible chimeric cytokine receptors shown on cytokine release in the absence of signaling.

Figure 24 depicts a graph summarizing the effect of inducible chimeric cytokine receptors shown on cytokine release in the absence of signaling.

Figure 25 depicts a graph summarizing the effect of the inducible chimeric cytokine receptor on cell growth shown.

Figure 26 depicts a schematic of an exemplary inducible cytokine receptor with dual tyrosine effector domains.

Figure 27A depicts a graph summarizing the results of a cell-based reporter assay testing the function of the inducible chimeric cytokine receptor shown.

Figure 27B depicts a graph summarizing the results of a cell-based reporter assay testing the function of the inducible chimeric cytokine receptor shown.

Figure 28A depicts a schematic of an exemplary construct comprising a CAR and an inducible cytokine receptor.

Fig. 28B shows the transduction efficiency of T cells transduced with vectors comprising the constructs shown in fig. 28A.

Fig. 28C depicts a graph summarizing the results of FACS analysis testing function for the inducible chimeric cytokine receptors shown.

Figure 29 depicts a graph summarizing the results of FACS analysis testing the function of the inducible chimeric cytokine receptors shown.

Figure 30A depicts a schematic of an exemplary construct comprising a CAR and an inducible cytokine receptor.

Figure 30B depicts a graph summarizing the results of in vitro assays testing the cytotoxicity of indicated CAR-T cells.

Figure 30C depicts a graph summarizing the results of in vitro assays testing the cytotoxicity of the indicated CAR-T cells.

Figure 30D depicts a graph summarizing the results of in vitro assays testing the cytotoxicity of indicated CAR-T cells.

Figure 30E depicts a graph summarizing the results of in vitro assays testing the cytotoxicity of the indicated CAR-T cells.

Fig. 31A depicts a graph summarizing the results of tumor volume analysis for the treatment groups shown.

Fig. 31B depicts a graph summarizing the results of tumor volume analysis for the treatment groups shown.

Fig. 31C depicts a graph summarizing the results of tumor volume analysis for the treatment groups shown.

Fig. 31D depicts a graph summarizing the results of tumor volume analysis for the treatment groups shown.

Fig. 31E depicts a graph summarizing the results of tumor volume analysis for the treatment groups shown.

Fig. 31F depicts a graph summarizing the results of tumor volume analysis for the treatment groups shown.

Fig. 31G depicts a graph summarizing the results of tumor volume analysis for the treatment groups shown.

Fig. 31H depicts a graph summarizing overall survival for the treatment groups shown.

Figure 32A depicts a graph summarizing the expansion of CAR-T cells shown.

Figure 32B depicts a graph summarizing the effect of the inducible chimeric cytokine receptor on cell phenotype shown.

Figure 33A depicts a graph summarizing the results of a cell-based reporter assay testing the function of the inducible chimeric cytokine receptor shown.

Figure 33B depicts a graph summarizing the results of a cell-based reporter assay testing the function of the inducible chimeric cytokine receptor shown.

Figure 33C depicts a graph summarizing the results of a cell-based reporter assay testing the function of the inducible chimeric cytokine receptor shown.

Figure 33D depicts a graph summarizing the results of a cell-based reporter assay testing the function of the inducible chimeric cytokine receptor shown.

Figure 34A depicts a schematic of an exemplary construct comprising a CAR and an inducible cytokine receptor.

Figure 34B depicts a graph summarizing the results of in vitro assays testing the cytotoxicity of indicated CAR-T cells.

Figure 34C depicts a graph summarizing the results of in vitro assays testing the cytotoxicity of indicated CAR-T cells.

Figure 34D depicts a graph summarizing the results of in vitro assays testing the cytotoxicity of indicated CAR-T cells.

Figure 34E depicts a graph summarizing the results of the analysis testing the indicated expansion of CAR-T cells.

Figure 34F depicts a graph summarizing the results of the analysis testing the expansion of CAR-T cells shown.

Figure 35A depicts a schematic of an exemplary construct comprising a CAR and an inducible chimeric cytokine receptor.

Figure 35B depicts a graph showing expansion of control CAR-T cells comprising the indicated inducible chimeric cytokine receptors.

Figure 35C depicts a graph showing expansion of CAR-T cells comprising the indicated inducible chimeric cytokine receptors.

Figure 35D depicts a graph showing expansion of CAR-T cells comprising the indicated inducible chimeric cytokine receptors.

Figure 35E depicts a graph showing expansion of CAR-T cells comprising the indicated inducible chimeric cytokine receptors.

Figure 36A depicts a schematic of an exemplary inducible chimeric cytokine receptor.

Figure 36B depicts a graph summarizing the results of a cell-based reporter assay testing the function of the inducible chimeric cytokine receptor shown.

Figure 36C depicts a graph summarizing the results of a cell-based reporter assay testing the function of the inducible chimeric cytokine receptor shown.

Figure 37A depicts a schematic of an exemplary inducible chimeric cytokine receptor.

Figure 37B is a schematic representation of the interaction between receptors BCMA, TACI and BAFFR and their ligands BAFF and APRIL.

Figure 37C depicts a graph summarizing the results of a cell-based reporter assay testing the function of the inducible chimeric cytokine receptor shown.

Figure 37D depicts a graph summarizing the results of a cell-based reporter assay testing the function of the inducible chimeric cytokine receptor shown.

Figure 38A shows the amino acid sequences of wild-type TpoR and various transmembrane deletion variants.

Figure 38B shows the amino acid sequences of wild-type TpoR and various transmembrane insertion variants.

Figure 38C depicts a graph summarizing the results of a cell-based reporter assay testing the function of the inducible chimeric cytokine receptor shown.

Figure 39A depicts a schematic of an exemplary inducible chimeric cytokine receptor.

Figure 39B depicts a graph summarizing the results of a cell-based reporter assay testing the function of the inducible chimeric cytokine receptor shown.

Figure 40A depicts a graph summarizing the results of an in vitro cytotoxicity assay of CAR-T cells comprising the indicated inducible chimeric cytokine receptors.

Figure 40B depicts a graph summarizing the results of an in vitro cytotoxicity assay of CAR-T cells comprising the indicated inducible chimeric cytokine receptors.

Figure 40C depicts a graph summarizing the results of an in vitro cytotoxicity assay of CAR-T cells comprising the indicated inducible chimeric cytokine receptors.

Figure 40D depicts a graph summarizing the results of an in vitro cytotoxicity assay of CAR-T cells comprising the indicated inducible chimeric cytokine receptors.

Figure 40E depicts a graph summarizing the results of an in vitro cytotoxicity assay of CAR-T cells comprising the indicated inducible chimeric cytokine receptors.

Figure 40F depicts a graph summarizing the results of an in vitro cytotoxicity assay of CAR-T cells comprising the indicated chimeric receptors.

Figure 41A depicts a graph summarizing the effect of the inducible chimeric cytokine receptor on cytokine release shown.

Figure 41B depicts a graph summarizing the effect of the inducible chimeric cytokine receptor on cytokine release shown.

Figure 42A depicts a graph summarizing the effect of the inducible chimeric cytokine receptor on cytokine release shown.

Figure 42B depicts a graph summarizing the effect of the inducible chimeric cytokine receptor on cytokine release shown.

Figure 43 depicts a graph summarizing CAR-T cell enrichment comprising the chimeric cytokine receptors shown.

Figure 44A depicts a graph summarizing the memory subset distribution of CAR-T cells comprising the chimeric cytokine receptors shown.

Figure 44B depicts a graph summarizing the memory subset distribution of CAR-T cells comprising the chimeric cytokine receptors shown.

Figure 44C depicts a graph summarizing the memory subset distribution of CAR-T cells comprising the chimeric cytokine receptors shown.

Figure 44D depicts a graph summarizing the memory subset distribution of CAR-T cells comprising the chimeric cytokine receptors shown.

Figure 44E depicts a graph summarizing the memory subset distribution of CAR-T cells comprising the chimeric cytokine receptors shown.

Figure 44F depicts a graph summarizing the memory subset distribution of CAR-T cells comprising the chimeric cytokine receptors shown.

Figure 44G depicts a graph summarizing the memory subset distribution of CAR-T cells comprising the chimeric cytokine receptors shown.

Figure 45A depicts a graph summarizing the effect of the inducible chimeric cytokine receptor shown on cell phenotype.

Figure 45B depicts a graph summarizing the effect of the inducible chimeric cytokine receptor on cell phenotype shown.

Figure 45C depicts a graph summarizing the effect of the inducible chimeric cytokine receptor on cell phenotype shown.

Figure 45D depicts a graph summarizing the effect of the inducible chimeric cytokine receptor shown on cell phenotype.

Figure 45E depicts a graph summarizing the effect of the inducible chimeric cytokine receptor on cell phenotype shown.

Figure 45F depicts a graph summarizing the effect of the inducible chimeric cytokine receptor on cell phenotype shown.

Figure 45G depicts a graph summarizing the effect of the inducible chimeric cytokine receptor shown on cell phenotype.

Detailed Description

The present invention provides chimeric receptors and their use for improving the in vivo persistence and therapeutic efficacy of immune cells. Provided herein are receptors sensitive to ligands, such as small molecules (e.g., AP1903) or proteins (e.g., Epo, Tpo, or PD-L1). Also provided are cells comprising such inducible chimeric cytokine receptors, compositions comprising such cells, and methods for improving the functional activity of an isolated T cell (e.g., a CAR-T cell). Also provided herein are CAR-T cells with improved persistence, and methods of treating disorders using such CAR-T cells.

General techniques

Practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are well described in the literature, e.g., Molecular Cloning A Laboratory Manual, second edition (Sambrook et al, 1989) Cold Spring Harbor Press; oligonucleotide Synthesis (oligo Synthesis) (eds. m.j. goal, 1984); methods in molecular Biology (Methods in molecular Biology), Humana Press; academic Press, Inc. (Cell Biology: analytical Notebook), Academic Press; animal Cell Culture (Animal Cell Culture), ed.r.i. freshney, 1987; introduction to Cell and Tissue cultures (Introduction to Cell and Tissue Culture) (J.P.Mather and P.E.Roberts, 1998) Plenum Press; cell and Tissue Culture handbook (Cell and Tissue Culture: laboratory procedures) A.Doyle, J.B.Griffiths and D.G.Newell eds, 1993 and 1998 John Wiley father publishing company (J.Wiley and Sons); methods in Enzymology (Methods in Enzymology), academic Press; handbook of Experimental Immunology (compiled by d.m.weir and c.c.blackwell); gene Transfer Vectors for Mammalian Cells (Gene Transfer Vectors for Mammalian Cells) (eds. J.M.Miller and M.P.Calos, 1987); current Protocols in molecular Biology (Current Protocols in molecular Biology) (eds. F.M. Ausubel et al, 1987); PCR: polymerase Chain Reaction (PCR: the polymerase Chain Reaction), ed. (Mullis et al eds., 1994); current specifications in Immunology (J.E.Coligan et al, 1991); short Specifications for Molecular Biology (short protocols in Molecular Biology) (Willd-published, 1999); immunobiology (Immunobiology) (c.a. janeway and p.travers, 1997); antibodies (Antibodies) (p.finch, 1997); antibodies: practical methods (Antibodies: a practical approach) (D.Catty. eds., IRL Press, 1988-; monoclonal antibodies: practical methods (Monoclonal antibodies: a practical approach), compiled by P.shepherd and C.dean, Oxford University Press, 2000; using antibodies: a laboratory Manual (Using Antibodies: a laboratory Manual) (E.Harlow and D.Lane (Cold spring harbor laboratory Press, 1999); Antibodies (The Antibodies) (M.Zantetti and J.D.Capra, eds., Harwood and nucleic acids Publishers, 1995).

Definition of

As used herein, "autologous" means a cell, cell line, or cell population derived from the individual or derived from a Human Leukocyte Antigen (HLA) compatible donor for use in treating an individual.

As used herein, "allogenic" means that the cell or population of cells used to treat an individual is not derived from the individual but is derived from a donor.

As used herein, the term "endogenous" refers to any material that is derived from or produced within an organism, cell, tissue, or system.

As used herein, the term "exogenous" refers to any material introduced from or produced outside of an organism, cell, tissue, or system.

As used herein, "immune cell" refers to a cell of hematopoietic origin that is functionally involved in the initiation and/or execution of an innate and/or adaptive immune response. Examples of immune cells include T cells, e.g., α/β T cells and γ/T cells, B cells, Natural Killer (NK) cells, Natural Killer T (NKT) cells, invariant NKT cells, mast cells, bone marrow-derived phagocytes, dendritic cells, killer dendritic cells, macrophages, and monocytes. As used herein, the term "immune cell" also refers to a cell derived from (e.g., without limitation) a stem cell. The stem cells may be adult stem cells, non-human embryonic stem cells, more particularly non-human stem cells, cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells, totipotent stem cells or hematopoietic stem cells.

The terms "polypeptide", "oligopeptide", "peptide" and "protein" are used interchangeably herein to refer to a chain of amino acids of any length, preferably a relatively short chain (e.g., 10-100 amino acids) and longer chains comprising about 10-250, 10-500, 10-1000, 50-200, 50-500 or 50-1000 amino acids. The chain may be a straight or branched chain, which may comprise modified amino acids, and/or may be interrupted by non-amino acids. The term also includes amino acid chains that have been modified either naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation or any other manipulation or modification, such as binding to a labeling component. Also included in the definition are, for example, polypeptides comprising one or more analogs of an amino acid, including, for example, an unnatural amino acid, and the like, as well as other modifications known in the art. It is understood that the polypeptide may exist as a single chain or related chain.

As used herein, the term "expression" refers to the transcription and/or translation of a particular nucleotide sequence driven by a promoter.

As used herein, "expression vector" refers to a vector comprising a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. Expression vectors include all expression vectors known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate recombinant polynucleotides.

As used herein, "operably linked" refers to the association of nucleic acid sequences on a single nucleic acid fragment such that the function of one nucleic acid sequence is affected by another nucleic acid sequence. For example, a promoter may be operably linked to a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter).

As used herein, "expression control sequence" means a nucleic acid sequence that directs transcription of a nucleic acid. The expression control sequence may be a promoter (e.g., a constitutive or inducible promoter) or an enhancer. The expression control sequence may be operably linked to the nucleic acid sequence to be transcribed.

"promoter" and "promoter sequence" are used interchangeably and refer to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. Typically, the coding sequence is located 3' to the promoter sequence. It will be appreciated by those skilled in the art that different promoters may direct the expression of genes in different tissues or cell types, or at different stages of development, or in response to different environmental or physiological conditions.

In any of the vectors of the present invention, the vector optionally comprises a promoter disclosed herein.

"host cell" includes a single cell or cell culture that may be or has been used for incorporation into a polynucleotide insert vector recipients. Host cells include progeny of a single host cell, and due to natural, accidental, or deliberate mutation, the progeny may not necessarily be identical (in morphology or in genomic DNA complement) to the original parent cell. Host cells include cells transfected in vivo with a polynucleotide of the invention.

As used herein, the term "extracellular ligand binding domain" refers to an oligopeptide or polypeptide capable of binding a ligand. Preferably, the domain is capable of interacting with a cell surface molecule. For example, extracellular ligand binding domains can be selected to recognize ligands that serve as cell surface markers on target cells associated with a particular disease state.

The terms "stalk domain" or "hinge domain" are used interchangeably herein and refer to any oligopeptide or polypeptide used to link a transmembrane domain to an extracellular ligand-binding domain. In particular, the stem domain serves to provide greater flexibility and accessibility to the extracellular ligand-binding domain.

The term "intracellular signaling domain" refers to the portion of a protein that transduces effector signaling function signals and directs a cell to perform a specific function.

As used herein, "co-stimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a co-stimulatory ligand, thereby mediating a co-stimulatory response of the cell, such as (but not limited to) proliferation. Costimulatory molecules include, but are not limited to, mhc class i molecules, BTLA, and Toll ligand receptors. Examples of co-stimulatory molecules include CD27, CD28, CD8, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, antigen-1 associated with lymphocyte function (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83 and the like.

"costimulatory ligand" refers to a molecule on an antigen presenting cell that specifically binds to a cognate costimulatory signaling molecule on a T cell, thereby providing a signal in addition to the primary signal provided by, for example, the TCR/CD3 complex binding to a peptide-loaded MHC molecule, mediating T cell responses including, but not limited to, proliferation activation, differentiation, and the like. Costimulatory ligands can include, but are not limited to, CD7, B7-1(CD80), B7-2(CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible costimulatory ligand (ICOS-L), intracellular adhesion molecules (ICAM, CD30L, CD40, CD70, CD83, HLA-G, MICA, M1CB, HVEM, lymphotoxin beta receptor, 3/TR6, ILT3, ILT4, agonists or antibodies that bind to Toll ligand receptors, and ligands that specifically bind to B7-H3. costimulatory ligands also include, inter alia, antibodies that specifically bind to costimulatory molecules present on T cells, such as, but not limited to, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LTGHT, NKG2C, B7-H3, a ligand that specifically binds to CD 83.

An "antibody" is an immunoglobulin molecule that is capable of specifically binding to a target (e.g., a carbohydrate, polynucleotide, lipid, polypeptide, etc.) through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, the term includes not only intact polyclonal or monoclonal antibodies, but also antigen-binding fragments thereof (e.g., Fab ', F (ab')₂And Fv), and any other modified configuration of an immunoglobulin molecule comprising an antigen recognition site, including, for example, but not limited to, single chain (scFv) and domain antibodies, including, for example, shark and camelid antibodies, and fusion proteins comprising an antibody, antibody mimetic, or any protein that provides a particular protein-protein interaction.

As used herein, the term "antigen-binding fragment" or "antigen-binding portion" of an antibody refers to one or more fragments of an intact antibody that retain the ability to specifically bind to a given antigen. The antigen binding function of an antibody can be performed by fragments of an intact antibody. Examples of binding fragments encompassed within the term "antigen-binding fragment" of an antibody include Fab; fab'; f (ab')₂(ii) a An Fd fragment consisting of the VH and CH1 domains; (ii) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; single domain antibody (dAb) fragments (Ward et al, Nature 341:544-546, 1989) and isolated Complementarity Determining Regions (CDRs).

Antibodies, antibody conjugates, or polypeptides that "preferentially bind" or "specifically bind" (used interchangeably herein) to a target (e.g., a BCMA protein) are well known terms in the art, and methods of determining such specific or preferential binding are also well known in the art. A molecule is said to exhibit "specific binding" or "preferential binding" if it reacts or associates more frequently, more rapidly, for a longer duration of time, and/or with greater affinity to a particular cell or substance than it reacts or associates with a replacement cell or substance. An antibody "specifically binds" or "preferentially binds" to a target if it binds with greater affinity, binding, more easily, and/or for a longer duration than it binds to other substances. It will also be appreciated that by reading this definition, for example, an antibody (or portion or epitope) that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. Thus, "specific binding" or "preferential binding" does not necessarily require (although it may include) exclusive binding. Typically, but not necessarily, reference to binding means preferential binding.

As known in the art, "polynucleotide" or "nucleic acid" as used interchangeably herein refers to a strand of nucleotides of any length, and includes DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or analogs thereof, or any substrate that can be incorporated into a strand by a DNA or RNA polymerase. Polynucleotides may comprise modified nucleotides, such as methylated nucleotides and analogs thereof. Modifications to the nucleotide structure, if present, may be imparted before or after assembly of the strands. The sequence of nucleotides may be interspersed with non-nucleotide components. The polynucleotide may be further modified after polymerization, e.g., by conjugation with a labeling component. Other types of modifications include, for example, "capping", substitution of one or more of the natural nucleotides with an analog, internucleotide modifications, such as modifications with uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.) and modifications with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), modifications comprising pendant moieties (such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), modifications with intercalators (e.g., acridine, psoralen, etc.), modifications comprising chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.), modifications comprising alkylating agents, modifications with modified (e.g., alpha bond isomerization nucleic acids, etc.), and unmodified forms of the polynucleotide. Any hydroxyl groups typically present in the sugar may be replaced, for example, with phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to make additional linkages to additional nucleotides, or may be bound to a solid support. The 5 'and 3' terminal OH groups may be phosphorylated or may be amine or 1 to 20 carbons The organic end capping moiety of the atom. Other hydroxyl groups may also be derivatized as standard protecting groups. Polynucleotides may also comprise similar forms of ribose or deoxyribose commonly known in the art, including, for example, 2 '-O-methyl-, 2' -O-allyl, 2 '-fluoro-or 2' -azido-ribose, carbocyclic sugar analogs, alpha-or beta-allosteric sugars, epimeric sugars, such as arabinose, xylose, or lyxose, pyranose, furanose, sedoheptulose (sedoheptulose), acyclic analogs, and abasic nucleoside analogs (e.g., methyl nucleosides). One or more phosphodiester linkages may be replaced with an alternative linker. Such alternative linkers include, but are not limited to, those wherein the phosphate ester is substituted with P (O) S ("thioester"), P (S) S ("dithioate"), (O) NR₂("amidates"), P (O) R, P (O) OR', CO OR CH₂Examples of ("methylal") substitutions, wherein each R or R' is independently H or a substituted or unsubstituted alkyl (1-20C), aryl, alkenyl, cycloalkyl, cycloalkenyl, or aralkyl optionally containing an ether (-O-) linkage. Not all linkages in a polynucleotide need be identical. The foregoing description applies to all polynucleotides mentioned herein, including RNA and DNA.

As used herein, "transfection" refers to the uptake of exogenous or heterologous RNA or DNA by a cell. When the RNA or DNA has been introduced into the cell, the cell has been "transfected" with exogenous or heterologous RNA or DNA. When transfected RNA or DNA affects a phenotypic change, the cell has been "transformed" by exogenous or heterologous RNA or DNA. The transformed RNA or DNA may be integrated (covalently linked) into the chromosomal DNA that constitutes the genome of the cell.

As used herein, "transformation" refers to the transfer of a nucleic acid fragment into the genome of a host organism, resulting in genetically stable inheritance. Host organisms containing transformed nucleic acid fragments are referred to as "transgenic" or "recombinant" or "transformed" organisms.

As used herein, "substantially pure" refers to a material that is at least 50% pure (i.e., free of contaminants), more preferably, at least 90% pure, more preferably, at least 95% pure, yet more preferably, at least 98% pure, and most preferably, at least 99% pure.

As used herein, "treatment" is a method for obtaining a beneficial or desired clinical result. For purposes of the present invention, beneficial or desired clinical results include (but are not limited to) one or more of the following: reducing the proliferation of (or destroying) a tumor or cancer cell, inhibiting metastasis of a tumor cell, shrinking or reducing tumor size, resolving a disease (e.g., cancer), reducing symptoms caused by a disease (e.g., cancer), improving the quality of life of a person suffering from a disease (e.g., cancer), reducing the dose of other drugs required to treat a disease (e.g., cancer), delaying the progression of a disease (e.g., cancer), curing a disease (e.g., cancer), and/or extending the survival of an individual having a disease (e.g., cancer).

By "ameliorating" is meant reducing or ameliorating one or more symptoms as compared to no treatment. "improving" also includes shortening or reducing the duration of symptoms.

As used herein, an "effective dose" or "effective amount" of a drug, compound, or pharmaceutical composition is an amount sufficient to achieve any one or more beneficial or desired results. For prophylactic use, beneficial or desired results include elimination or reduction of risk, lessening the severity, or delaying the onset of the disease (including biochemical, histological, and/or behavioral symptoms of the disease, complications thereof, and intermediate pathological phenotypes that occur in the development of the disease). For therapeutic use, beneficial or desired results include clinical results, such as reducing the incidence of or ameliorating one or more symptoms of various diseases or disorders (e.g., cancer), reducing the dosage of other drugs required to treat the disease, enhancing the effect of another drug, and/or delaying the progression of the disease. An effective dose may be administered in one or more administrations. For the purposes of the present invention, an effective dose of a drug, compound or pharmaceutical composition is an amount sufficient to accomplish prophylactic or therapeutic treatment, either directly or indirectly. As is understood in clinical situations, an effective dose of a drug, compound, or pharmaceutical composition may or may not be achieved in combination with another drug, compound, or pharmaceutical composition. Thus, an "effective dose" may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be administered in an effective amount, if combined with one or more other agents, to achieve a desired result or to achieve a desired result.

As used herein, the term "individual" refers to any vertebrate animal, including, but not limited to, humans and other primates (e.g., chimpanzees, cynomolgus monkeys, and other apes), farm animals (e.g., cows, sheep, pigs, goats, and horses), sport animals, pets (including domestic mammals, e.g., dogs and cats), laboratory animals (e.g., rabbits, rodents (e.g., mice, rats, and guinea pigs)), and birds (e.g., poultry, pheasants, and game birds, such as chickens, turkeys, and other gallinaceous chickens, ducks, geese, and the like). In some embodiments, the subject is a mammal. In an exemplary embodiment, the subject is a human.

As used herein, "vector" means a construct capable of delivery, and preferably expression, of one or more genes or sequences of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells (e.g., producer cells).

As used herein, "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" includes any material that, when combined with an active ingredient, allows the ingredient to retain biological activity and not react with the immune system of an individual. Examples include, but are not limited to, any standard pharmaceutical carrier such as phosphate buffered saline solution, water, emulsions (such as oil/water emulsions) and various types of wetting agents. Preferred diluents for aerosol or parenteral administration are Phosphate Buffered Saline (PBS) or physiological (0.9%) saline. Compositions comprising such carriers are formulated by well-known conventional methods (see, e.g., Remington's Pharmaceutical Sciences, 18 th edition, eds., a. gennaro, Mack Publishing co., Easton, PA, 1990, and Remington, The Science and Practice of Pharmacy, 21 st edition, mark Publishing press, 2005).

Reference herein to "about" a value or parameter includes (and describes) embodiments that are directed to the value or parameter itself. For example, a description referring to "about X" includes a description of "X". Numerical ranges include the numbers defining the range.

It should be understood that the phrase "including" is used herein to describe any of the embodiments and other similar embodiments described as "consisting of and/or" consisting essentially of are also provided.

Where aspects or embodiments of the present invention are described in terms of Markush groups (Markush groups) or other alternative groups, the present invention includes not only the entire group listed as a whole, but not every member of the respective group and all possible subsets of the primary group, but also the primary group absent one or more of the group members. The present invention also contemplates the explicit exclusion of one or more of any group member of the claimed invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control. In the present specification and claims, the word "comprise" or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. Unless the context requires otherwise, singular terms shall include the plural and plural terms shall include the singular.

Exemplary methods and materials are described herein, however, methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. The materials, methods, and examples are illustrative only and not intended to be limiting.

Inducible chimeric cytokine receptor and improved isolated immune cells

Provided herein are inducible chimeric cytokine receptors, cells comprising such receptors, and methods comprising such cells. Also provided is the use of such inducible chimeric cytokine receptors for improving the functional activity of an isolated immune cell (e.g., an isolated T cell, such as an isolated T cell comprising a Chimeric Antigen Receptor (CAR)). The methods and compositions provided herein are useful for improving the in vivo and in vitro persistence, cytotoxicity, memory phenotype, and/or therapeutic efficacy of immune cells comprising a CAR (e.g., CAR-T cells).

In some embodiments, the inducible chimeric cytokine receptors provided herein comprise, in any order: dimerization domain, tyrosine kinase activation domain and tyrosine effector domain. Optionally, the inducible chimeric cytokine receptor provided herein can include a membrane-targeting motif. Ligand-mediated dimerization of inducible chimeric cytokine receptors provided herein induces receptor-mediated signaling events in host cells comprising the inducible chimeric cytokine receptors. In some embodiments, this signaling may result in improved persistence. For example, in one exemplary embodiment, the inducible chimeric cytokine receptors provided herein will activate the JAK-STAT pathway through ligand dimerization and mimic signaling induced by the native cytokine receptor. By "mimicking" is meant that the signaling cascade activated by the inducible chimeric cytokine receptor of the present invention is similar to that activated by the native cytokine receptor, whereas the degree of activation induced by the chimeric cytokine receptor of the present invention may differ from that induced by the native cytokine receptor. For example, if both the inducible chimeric cytokine receptor and the native cytokine receptor of the present invention activate STAT transcription factors; it is possible that the levels of these two receptor activated STATs may be similar or different.

The "dimerization domain" of an inducible chimeric cytokine receptor may be any amino acid sequence that can induce dimerization or even trimerization or multimerization by a ligand that can bind to the dimerization domain. Thus, the dimerization domain is a ligand binding domain. In some embodiments, the dimerization domain of an inducible chimeric cytokine receptor provided herein can be present outside the cell membrane. In some embodiments, the dimerization domain of the inducible chimeric cytokine receptor may be present inside the cell membrane.

In some embodiments, the ligand that binds to the dimerization domain is a dimeric ligand. For example, the dimerization domain may comprise the amino acid sequence of an FK506 binding protein ("FKBP"). The FKBP protein binds specifically to the drug FK 506. A ligand that is a multimeric analog of FK506 (i.e., a ligand that comprises at least two copies of FK506 or a derivative thereof) can bind to a first protein and a second protein through the ligand and thereby cause them together to induce dimerization of the first protein and the second protein, each comprising an amino acid sequence of FKBP. The first protein and the second protein may be the same or different. Thus, the inducible chimeric cytokine receptors provided herein can induce dimerization by exposing the inducible chimeric cytokine receptor to a suitable dimeric ligand that binds to the dimerization domain of the inducible chimeric cytokine receptor.

In some embodiments, the dimerization domain of an inducible chimeric cytokine receptor provided herein can comprise (or be derived from) an amino acid sequence of, for example, FKBP, cyclophilin, steroid binding protein, estrogen binding protein, glucocorticoid binding protein, vitamin D binding protein, or tetracycline binding protein. As used herein, "FKBP polypeptide", "cyclophilin polypeptide", or the like, refers to a polypeptide having the amino acid sequence of the respective protein, or a portion or variant thereof, wherein the portion or variant thereof retains the ability to bind to the corresponding ligand (e.g., ligand FK506 and related molecules, in the case of FKBP polypeptides) with high affinity.

In some embodiments, the dimerization domain of an inducible chimeric cytokine receptor provided herein may comprise the amino acid sequence of (or derived from) an extracellular domain of a cytokine receptor such as (for example, but not limited to): erythropoietin receptor, prolactin receptor, growth hormone receptor, thrombopoietin receptor, granulocyte colony stimulating factor receptor, GP130, common gamma chain receptor, common beta chain receptor, IFN alpha receptor, IFN gamma receptor, IFN lambda receptor, IL2/IL15 receptor, IL3 receptor, IL4 receptor, IL5 receptor, IL7 receptor, IL9 receptor, IL10 receptor, IL12 receptor, IL13 receptor, IL20 receptor, IL21 receptor, IL22 receptor, IL23 receptor, IL27 receptor, TSLP receptor, G-CSF receptor, GM-CSF receptor, CNTF receptor, OSM receptor, LIF receptor, CT-1 receptor, TGFBR1/ALKL5, and TGFBR 2. In some embodiments, the dimerization domain of an inducible chimeric cytokine receptor provided herein may comprise the amino acid sequence of (or derived from) the extracellular domain of a Receptor Tyrosine Kinase (RTK), such as: EGFR/HER, ERBB/HER, ERRB/HER, INSR, IGF-1R, IRR, PDGFRA, PDGFRB, CSF-1/SCFR, FLK/FLT, VEGFR, FGFR-1, FGFR-2, FGFR-3, FGFR-4, CCK, TRKA, TRKB, TRKC, MET, RON, EPHA, EPHB, AXL, MER, TYRO, TIE, TEK, RYK, DDR, RET, ROS, LTK, ALK, ROR, MUSK, AATYK, TYK, and RTK 106. The extracellular domains of the cytokine receptors and RTKs may be dimerized by corresponding ligands or agonists (e.g., in the case of thrombopoietin receptor polypeptides, corresponding ligands include, for example, TPO, eltrombopag, and related molecules).

In some embodiments, the dimerization domain of an inducible chimeric cytokine receptor provided herein can comprise, for example, the amino acid sequence of (or derived from) the extracellular domain of a TNFR family receptor such as: TNFR1, Fas, TRAILR1, TRAILR2, NGFR, DR3, DR6, EDAR, TNFR2, LTbR, OX40, CD40, CD27, CD30, 4-1BB, RANK, Fn14, TACI, BAFFR, HVEM, BCMA, GITR, TROY, RELT, XEDAR, TRAILR3, TRAILR4, OPG, Dcr 3. The extracellular domain of TNFR family receptors multimerizes upon binding to trimeric TNFR ligands. An exemplary dimerization domain amino acid sequence derived from a TNFR family receptor (comprising a BCMA ectodomain) is: MLQMAGQCSQNEYFDSLLHACIPCQLRCSSNTPPLTCQRYCNASVTNSVKGTNA (SEQ ID NO: 216). In some embodiments, the dimerization domain of an inducible chimeric cytokine receptor provided herein can comprise or be derived from (or derived from) an amino acid sequence of an extracellular domain of an immune co-receptor or ligand, e.g., as follows: PD-1, CD80, CD86, ICOS-L, ICOS, CTLA-4, BTLA, CD160, LAG3 or TIM 3. The extracellular domain of the immune co-receptor aggregates upon binding to cells displaying the corresponding ligand. An exemplary dimerization domain amino acid sequence derived from an immune co-receptor (comprising extracellular PD-1) is: MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLV (SEQ ID NO: 217).

In some embodiments, the dimerization domain may comprise an FKBP polypeptide amino acid sequence. FKBP is a group of proteins that have prolyl isomerase activity and bind to the drug FK506 and other related drugs.

Optionally, the FKBP may be human FKBP12 (also known as FKBP 1A; GenBank: CAG 46965.1). Optionally, the FKBP12 polypeptide may comprise an F36V mutation. FKBP12, which contains the F36V mutation, binds with high affinity to dimeric ligand AP1903(Jemal, A. et al, CA: J. Clinic.A.) -58, 71-96(2008), Scher, H.I. and Kelly, W.K., J.Clinic.Oncology 11, 1566-72 (1993)). In addition, FKBP12, which contains the F36V mutation, binds to AP1903 with much higher affinity than wild-type FKBP12 binds to AP 1903.

An exemplary dimerization domain amino acid sequence (comprising an FKBP12 polypeptide having an F36V mutation) is: GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLES (SEQ ID NO: 218).

In some embodiments, the dimerization domain of the inducible chimeric cytokine receptor may comprise the amino acid sequence of an FKBP comprising a modification selected from the group consisting of: (i) an FKBP polypeptide comprising one or more amino acid substitutions, (ii) two or three tandem repeats of an unmodified (native) FKBP amino acid sequence, and (iii) two or three tandem repeats of an FKBP polypeptide comprising one or more amino acid substitutions. In some embodiments, the FKBP protein is a human FKBP protein (GenBank: CAG46965.1) and the modification of FKBP described herein is performed on the human FKBP protein. In some embodiments, the one or more amino acid substitutions in the FKBP comprise one or more of the following: F36V, L106P, E31G, R71G and K105E, residues of the reference human FKBP protein (GenBank: CAG 46965.1). In such embodiments, where the dimerization domain comprises two or three tandem repeats of the dimerization domain sequences disclosed herein, each repeat may comprise a different mutation of the sequence. For example, in one exemplary embodiment, the dimerization domain comprises three tandem repeats of an FKBP sequence, wherein one of the repeats comprises a native FKBP sequence, the second repeat comprises an FKBP comprising a F36V substitution, and the third repeat comprises an FKBP comprising a F36V and a L106P substitution, in any order.

In some embodiments, the dimerization domain of the inducible chimeric cytokine receptor may comprise the amino acid sequence of an FKBP comprising a modification selected from the group consisting of: (i) an FKBP polypeptide comprising a F36V substitution, (ii) an FKBP polypeptide comprising a F36V and a L106P substitution, (iii) an FKBP polypeptide comprising a E31G, F36V, R71G, and K105E substitution, and (iv) two or three tandem repeats of any of these FKBP polypeptides.

In some embodiments, the dimerization domain may be a cyclophilin polypeptide amino acid sequence. Cyclophilins are proteins that bind to cyclosporine (cyclosporin a). Cyclophilins include, for example, cyclophilin a and cyclophilin D.

In some embodiments, the dimerization domain may have any of the features of the ligand binding regions disclosed in U.S. patent No. 9434935, which is incorporated herein by reference for all purposes.

In some embodiments, the dimerization domain of the inducible chimeric cytokine receptor of the present invention may comprise the amino acid sequence of (or derived from) a polypeptide selected from the group consisting of: FKBP12(F36V), the extracellular domain of OX-40, and the extracellular domain of TNFR2 superfamily receptors (e.g., BCMA, TACI, BAFFR).

In some embodiments, the dimerization domain of an inducible chimeric cytokine receptor provided herein comprises the dimerization domain amino acid sequence disclosed in table 1B.

As used herein, a "dimeric" ligand may optionally comprise more than two copies of a suitable binding molecule (i.e., the ligand may be multimeric); however, such ligands may still be considered to be "dimeric" as used herein, based on the ability of such ligands to dimerize the corresponding binding molecule. Similarly, in some embodiments, a dimerization domain as provided herein may be capable of supporting multimerization (e.g., where multiple copies of the dimerization domain are provided in the same molecule); however, based on the ability of such domains to dimerize, as used herein, such domains may still be considered "dimerization domains". In general, apoptotic protease-9 signaling can be efficiently induced upon dimerization of the apoptotic protease-9 molecule (i.e., without the need for trimerization or other multimerization). Furthermore, reference herein to "ligand" refers to dimeric ligand (e.g., when referring to "ligand" that induces dimerization of the chimeric apoptotic protease-9 proteins provided herein), unless the context clearly indicates otherwise.

As used herein, the tyrosine kinase activation domain of the inducible chimeric cytokine receptors provided herein may comprise the transmembrane domain of an RTK, followed by a janus kinase (JAK) binding domain or tyrosine kinase. JAK and RTK kinases are activated by multimerization. JAKs include JAK1, JAK2, JAK3 and TYK2, and bind to the membrane proximal motif consisting of Box 1 and Box 2 motifs. An exemplary tyrosine kinase activation domain amino acid sequence that activates JAK2 kinase (including the erythropoietin receptor transmembrane, Box 1 and Box 2 motifs) is: SEPVSGPTPSDLDPLILTLSLILVVILVLLTVLALLSHRRALKQKIWPGIPSPESEFEGLFTTHKGNFQLWLYQNDGCLWWSPCTPFTEDPPASLEVLSERC (SEQ ID NO: 219). In some embodiments, the transmembrane domain may contain a mutation that reduces ligand-independent dimerization.

In some embodiments, the tyrosine kinase activation domain of an inducible chimeric cytokine receptor provided herein comprises a JAK binding domain of a protein (or derived therefrom). In some embodiments, the protein is a receptor. In some embodiments, the protein is a hormone receptor or a cytokine receptor.

In some embodiments, the tyrosine kinase activation domain comprises a JAK binding domain of (or derived from) a protein selected from the group consisting of: prolactin receptor (PRLR), Growth Hormone Receptor (GHR), thrombopoietin receptor/myeloproliferative leukemia protein receptor (TPOR/MPLR), erythropoietin receptor (EPOR), Granulocyte Colony Stimulating Factor Receptor (GCSFR), or GP 130. The term "derived from" means that one or more modifications are made to the native sequence. For example, only a portion of the native sequence may be used, or the native sequence may be modified to include substitution, insertion, and/or deletion mutations, or a combination of such modifications. In some embodiments, the JAK binding domain comprises a JAK binding domain of (or derived from) TPOR or EPOR.

In some embodiments, the tyrosine kinase activation domain of an inducible chimeric cytokine receptor provided herein comprises a transmembrane domain of (or derived from) a protein selected from the group consisting of: PRLR, GHR, TPOR/MPLR, EPOR, GCSFR, and GP 130.

In some embodiments, the tyrosine kinase activation domain of an inducible chimeric cytokine receptor provided herein comprises the transmembrane domain of (or derived from) PD-1. In these embodiments, the tyrosine kinase activation domain further comprises a JAK binding domain or a tyrosine kinase domain of (or derived from) a receptor as described herein.

In some embodiments, the tyrosine kinase activation domain of an inducible chimeric cytokine receptor provided herein comprises a transmembrane domain and a JAK binding domain or a transmembrane domain and a tyrosine kinase domain, wherein the transmembrane domain is derived from a cytokine/hormone receptor (e.g., TpoR), a monomeric cytokine/hormone receptor (e.g., EpoR L241G L242P), or a monomeric receptor (e.g., PD 1). As used herein, "monomeric cytokine/hormone receptor" refers to a cytokine receptor or hormone receptor that is a homodimer or heterodimer in its native form, but is mutated to exist as a monomeric receptor.

In some embodiments, the tyrosine kinase activation domain of an inducible chimeric cytokine receptor provided herein comprises the transmembrane domain of (or derived from) TPOR/MPLR. An exemplary full-length sequence of native TPOR/MPLR is shown in Table 1A (SEQ ID No.: 64). In some embodiments, the tyrosine kinase activation domain comprises a transmembrane domain of (or derived from) the TPOR/MPLR sequence shown in table 1A. For example, in some embodiments, the tyrosine kinase activation domain comprises amino acids 478-582 of TPOR/MPLR shown in Table 1A (this sequence is also shown in Table 1C as "TPOR/MPLR (478-582) (wild-type sequence)").

In some other embodiments, the tyrosine kinase activation domain comprises the sequence shown in Table 1A for amino acids 478-582 derived from TPOR/MPLR. In these embodiments, the tyrosine kinase activation domain of the inducible chimeric cytokine receptor comprises the sequence derived from amino acids 478-582 of TPOR/MPLR shown in Table 1A, wherein the sequence comprises one or more mutations selected from the group consisting of: substitutions, deletions, insertions, and combinations thereof. In an exemplary embodiment, the tyrosine kinase activation domain comprises a deletion variant of amino acid sequence 478-582 of TPOR/MPLR shown in Table 1A. In some embodiments, deletion variants include the deletion of 1, 1 to 20, 1 to 19, 1 to 18, 1 to 17, 1 to 16, 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2 amino acids from region 478-582 of TPOR/MPLR shown in table 1A. In some embodiments, deletion variants include the deletion of 1, 1 to 20, 1 to 19, 1 to 18, 1 to 17, 1 to 16, 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2 amino acids from region 489-510 of TPOR/MPLR shown in table 1A. In an exemplary embodiment, the tyrosine kinase activation domain comprises a deletion variant of amino acid sequence 478-582 of TPOR/MPLR shown in Table 1C.

In some embodiments, the tyrosine kinase activation domain comprises an insertion variant of amino acid sequence 478-582 of TPOR/MPLR shown in Table 1A. In some embodiments, the insertion variant comprises the insertion of 1, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2 amino acids in region 478-582 of TPOR/MPLR shown in Table 1A. In an exemplary embodiment, the tyrosine kinase activation domain comprises an insertion variant of amino acid sequence 478-582 of TPOR/MPLR shown in Table 1C. In some embodiments, the amino acid inserted in the insertion variant is selected from the group consisting of: leucine, valine and isoleucine.

In some embodiments, the tyrosine kinase activation domain of an inducible chimeric cytokine receptor comprises the sequence derived from amino acids 478-582 of TPOR/MPLR shown in Table 1A, wherein the sequence comprises a combination of deletions and insertions of amino acids in this region. In one exemplary embodiment, the tyrosine kinase activation domain of the inducible chimeric cytokine receptor comprises the sequence derived from amino acids 478-582 of TPOR/MPLR shown in Table 1A, wherein the sequence comprises the deletion of 1 to 18 amino acids from region 478-582 and the insertion of 1 to 8 amino acids in region 478-582. In another exemplary embodiment, the tyrosine kinase activation domain of the inducible chimeric cytokine receptor comprises the sequence derived from amino acids 489-510 of TPOR/MPLR shown in Table 1A, wherein the sequence comprises the deletion of 1 to 18 amino acids from region 489-510 and the insertion of 1 to 8 amino acids in region 489-510. In some embodiments, the amino acid inserted in the insertion variant is selected from the group consisting of: leucine, valine and isoleucine.

In some embodiments, the tyrosine kinase activation domain of the inducible chimeric cytokine receptor comprises the sequences disclosed in table 1C.

In some embodiments, the tyrosine kinase activation domain of the inducible chimeric cytokine receptor comprises a variant with deletion and/or insertion of the transmembrane domain of PRLR, GHR, EPOR, GCSFR, or GP 130. Exemplary full-length sequences of native PRLR, GHR, EPOR, GCSFR and GP130 and their accession numbers are disclosed in table 1A. According to the invention, transmembrane domains of PRLR, GHR, EPOR, GCSFR and GP130 can be located and insertion and/or deletion variants of these transmembrane domains can be prepared.

In some embodiments, the transmembrane and/or JAK-binding domain of the tyrosine kinase activation domain may be derived from, for example, a common gamma chain receptor, a common beta chain receptor, an IFN α receptor (IFNAR), an IFN γ receptor (IFNGR), an IFN λ receptor (IFNLR), an IL2/IL15 receptor (IL2R/IL15R), an IL3 receptor (IL3R), an IL4 receptor (IL4 4), an IL4 receptor (IL5 4), an IL4 receptor (IL7 4), an IL4 receptor (IL9 4), an IL4 receptor (IL10 4), an IL4 receptor (IL12 4), an IL4 receptor (IL13 4), an IL4 receptor (IL20 4), an IL4 receptor (IL21 4), an IL4 receptor (IL22 4), an IL4 receptor (IL 3623 4), an IL4 receptor (IL27 4), a TSLP-CSF receptor (gcg-CSF receptor), a tsgm-CSF receptor (gmf-receptor), a CNTFR), a cstfr (cstfr), a cstfr, a receptor (cstfr), a cstfr), or a receptor.

In some embodiments, the tyrosine kinase activation domain of an inducible chimeric cytokine receptor provided herein comprises a tyrosine kinase domain of (or derived from) a Receptor Tyrosine Kinase (RTK). Exemplary tyrosine kinase activation domains (comprising the epidermal growth factor receptor transmembrane and kinase domain) from RTKs that activate RTK kinases are:GLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFMRRRHIVRKRTLRRLLQERELVEPLTPSGEAPNQALLRILKETEFKKIKVLGSGAFGTVYKGLWIPEGEKVKIPVAIKELREATSPKANKEILDEAYVMASVDNPHVCRLLGICLTSTVQLITQLMPFGCLLDYVREHKDNIGSQYLLNWCVQIAKGMNYLEDRRLVHRDLAARNVLVKTPQHVKITDFGLAKLLGAEEKEYHAEGGKVPIKWMALESILHRIYTHQSDVWSYGVTVWELMTFGSKPYDGIPASEISSILEKGERLPQPPICTIDVYMIMVKCWMIDADSRPKFRELIIEFSKMARDPQRYL (SEQ ID NO: 220). In some embodiments, the tyrosine kinase activation domain comprises a transmembrane domain and a tyrosine kinase domain derived from other RTKs, such as the following: EGFR/HER, ERBB/HER, ERRB/HER, INSR, IGF-1R, IRR, PDGFRA, PDGFRB, CSF-1/SCFR, FLK/FLT, VEGFR, FGFR-1, FGFR-2, FGFR-3, FGFR-4, CCK, TRKA, TRKB, TRKC, MET, RON, EPHA, EPHB, AXL, MER, TYRO, TIE, TEK, RYK, DDR, RET, ROS, LTK, ALK, ROR, MUSK, AATYK, TYK, AATYK, or RTK 106.

In some embodiments, the tyrosine kinase activation domain comprises a tyrosine kinase domain of (or derived from) EGFR.

In some embodiments, the tyrosine kinase activation domain comprises the tyrosine kinase activation domain sequences disclosed in table 1B.

In some embodiments, the tyrosine effector domain may contain a portion of the cytoplasmic tail (cell tail) of at least one receptor, such as: cytokine receptorsHormone receptors, or RTKs, or tyrosine kinase adaptor proteins. As used herein, a "cell tail" is a moiety that comprises one or more tyrosine residues that are capable of being phosphorylated by a kinase upon activation. Tyrosine within the cell tail or adaptor protein is phosphorylated by activated tyrosine kinases. Phosphorylated tyrosine motifs recruit signal transduction factors. Exemplary tyrosine effector amino acid sequences from cytokine receptors (including the distal cell tail of IL2 Rb) are: VTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLV(SEQ ID NO:221)。

The cell tails of certain receptors, such as cytokine receptors and hormone receptors, contain a STAT activation domain (also referred to herein as a STAT binding domain). In some embodiments, the tyrosine effector domain of an inducible chimeric cytokine receptor provided herein comprises at least one STAT activation domain of (or derived from) the receptor. In some embodiments, the tyrosine effector domain of an inducible chimeric cytokine receptor provided herein comprises at least two, three, four, or more STAT activation domains of (or derived from) two, three, four, or more receptors. The receptor may be a cytokine receptor and/or a hormone receptor.

In some embodiments, the tyrosine effector domain of the inducible chimeric cytokine receptor comprises at least one, two, three, four, or more receptors (e.g., a cytokine receptor, a hormone receptor, or an RTK) or the cellular tail of (or derived from) a tyrosine kinase adaptor (a portion of the cytoplasmic tail comprising one or more tyrosine residues capable of being phosphorylated by a kinase). In one exemplary embodiment, the tyrosine effector domain of the inducible chimeric cytokine receptor comprises a cell tail of (or derived from) a cytokine receptor and a cell tail of (or derived from) a hormone receptor. In another exemplary embodiment, the tyrosine effector domain of the inducible chimeric cytokine receptor comprises a cellular tail of (or derived from) the cytokine receptor and a tail of (or derived from) the RTK. In yet another exemplary embodiment, the tyrosine effector domain of the inducible chimeric cytokine receptor comprises a cell tail of the hormone receptor (or derived therefrom) and a cell tail of the RTK (or derived therefrom). In yet another exemplary embodiment, the tyrosine effector domain of the inducible chimeric cytokine receptor comprises the cell tail of (or derived from) the hormone receptor; a cellular tail of (or derived from) an RTK; and the cell tail of (or derived from) a cytokine receptor. Similar combinations of cell tails are contemplated in which at least one of the cell tails comprises a phosphorylatable tyrosine-containing moiety of a tyrosine kinase adaptor protein. When the tyrosine effector domain of the inducible chimeric cytokine receptor comprises more than one cell tail, the cell tails may be present in any order.

In some embodiments, the tyrosine effector domain of the inducible chimeric cytokine receptor comprises the STAT activation domains of (or derived from) two cytokine receptors. In some embodiments, the tyrosine effector domain of the inducible chimeric cytokine receptor comprises a STAT activation domain of (or derived from) at least one receptor selected from the group consisting of: BLNK (B-cell connexin), IL2RG, EGFR, EpoR, GHR, IFNAR1, IFNAR2, IFNAR1/2, IFNLR1, IL10R1, IL12Rb1, IL12Rb2, IL21R, IL2Rb, IL2small, IL7R, IL7Ra, IL9R, IL15R and IL 21R. When the tyrosine effector domain comprises more than one STAT activation domain, the STAT activation domains are in tandem, with one domain being membrane proximal and the other domain being membrane distal.

In some embodiments, the tyrosine effector domain of the inducible chimeric cytokine receptor comprises a cell tail of (or derived from) a first receptor selected from the group consisting of: BLNK, IL2RG, EGFR, EpoR, GHR, IFNAR1, IFNAR2, IFNAR1/2, IFNLR1, IL10R1, IL12Rb1, IL12Rb2, IL21R, IL2Rb, IL2small, IL7R, IL7Ra, IL9R, IL15R and IL21R, and the cellular tail of (or derived from) a second receptor selected from the group consisting of: BLNK, IL2RG, EGFR, EpoR, GHR, IFNAR1, IFNAR2, IFNAR1/2, IFNLR1, IL10R1, IL12Rb1, IL12Rb2, IL21R, IL2Rb, IL2small, IL7R, IL7Ra, IL9R, IL15R and IL 21R. In these embodiments, the cell tail from the first receptor may be membrane proximal and the cell tail from the second receptor may be membrane distal, or vice versa. The present invention encompasses similar embodiments in which the tyrosine effector domain of the inducible chimeric cytokine receptor comprises more than two (e.g., three, four, or more) cytoplasms from the receptors described in this paragraph.

In some embodiments, the tyrosine effector domain of the inducible chimeric cytokine receptor comprises at least one tyrosine effector domain sequence disclosed in table 1B. In some embodiments, the tyrosine effector domain comprises at least two tyrosine effector domain sequences disclosed in table 1B. When there is more than one tyrosine effector domain sequence disclosed in table 1B, any sequence may be membrane proximal and other sequences may be membrane distal.

In some embodiments, the tyrosine effector domain comprises a sequence from the cell tail of a cytokine receptor such as: common gamma chain receptors, common beta chain receptors, IFN alpha receptors, IFN gamma receptors, IFN lambda receptors, IL2/IL15 receptors, IL3 receptors, IL4 receptors, IL5 receptors, IL7 receptors, IL9 receptors, IL10 receptors, IL12 receptors, IL13 receptors, IL20 receptors, IL21 receptors, IL22 receptors, IL23 receptors, IL27 receptors, TSLP receptors, G-CSF receptors, GM-CSF receptors, CNTF receptors, OSM receptors, LIF receptors, CT-1 receptors, erythropoietin receptors, growth hormone receptors, prolactin receptors, thrombopoietin receptors or GP 130. Exemplary tyrosine effector amino acid sequences from RTKs (containing EGFR distal cell tail) are: VIQGDERMHLPSPTDSNFYRALMDEEDMDDVVDADEYLIPQQGFFSSPSTSRTPLLSSLSATSNNSTVACIDRNGLQSCPIKEDSFLQRYSSDPTGALTEDSIDDTFLPVPEYINQSVPKRPAGSVQNPVYHNQPLNPAPSRDPHYQDPHSTAVGNPEYLNTVQPTCVNSTFDSPAHWAQKGSHQISLDNPDYQQDFFPKEAKPNGIFKGSTAENAEYLRVAPQSSEFIGA (SEQ ID NO: 222). In some embodiments, the tyrosine effector domain comprises a sequence from the cellular tail of an RTK such as: GFR/HER, ERBB/HER, ERRB/HER, INSR, IGF-1R, IRR, PDGFRA, PDGFRB, CSF-1/SCFR, FLK/FLT, VEGFR, FGFR-1, FGFR-2, FGFR-3, FGFR-4, CCK, TRKA, TRKB, TRKC, MET, RON, EPHA, EPHB, AXL, MER, TYRO, TIE, TEK, RYK, DDR, RET, ROS, LTK, ALK, ROR, MUSK, AATYK, or RTK 106. Exemplary tyrosine effector domain amino acid sequences from tyrosine kinase adaptor proteins (comprising the BLNK tyrosine domain) are: ASESPADEEEQWSDDFDSDYENPDEHSDSEMYVMPAEENADDSYEPPPVEQETRPVHPALPFARGEYIDNRSSQRHSPPFSKTLPSKPSWPSEKARLTSTLPALTALQKPQVPPKPKGLLEDEADYVVPVEDNDENYIHPTESSSPPPEKAPMVNR (SEQ ID NO: 223). In some embodiments, the tyrosine effector domain comprises a sequence from or bound to an adaptor protein such as: ALX, BLNK, Grb7, Nsp, SLP-76, SOCS, TSAD, APS, Bam32, Crk, Gads, Grb2, Nck, SLAP, Shc, FRS2, Dab, Dok, IRS, eps8, AFAP110, Gab, ADAP, Carma1, Cas, CIN85, Cortactin, E3B1, Vinexin, SKAP-55, BANK, BCAP, Dof, Paxillin, LAT, LAX, LIME, NTAL, PAG, SIT, or TRIM.

In some embodiments, the tyrosine effector domain of an inducible chimeric cytokine receptor provided herein can comprise sequences from one or more cytokine receptors, RTKs, and/or adaptor proteins. In some embodiments, the sequences may be in tandem. In some embodiments, the tyrosine effector domain may comprise shorter tyrosine-containing peptides, e.g., from a cytokine receptor, an RTK, or a tyrosine kinase adaptor protein. In some embodiments, the tyrosine effector domain may be a synthetic sequence capable of binding to one or more proteins including, for example: a phosphor-tyrosine binding Protein (PTB) domain, a Src homology 2(SH2) domain, a C2 domain and/or a Src homology 3(SH3) domain.

In some embodiments, an inducible chimeric cytokine receptor provided herein comprises a dimerization domain; a tyrosine kinase activation domain comprising a transmembrane domain and a JAK binding domain; and a tyrosine effector domain comprising at least one STAT activation domain of (or derived from) a receptor. In some of these embodiments, the dimerization domain comprises an FKBP polypeptide; the transmembrane domain comprises a transmembrane domain of (or derived from) a protein selected from the group consisting of: EPOR, GP130, PRLR, GHR, GCSFR, PD-1, and TPOR; the JAK binding domain comprises a JAK binding domain of (or derived from) a protein selected from the group consisting of: EPOR, GP130, PRLR, GHR, GCSFR, and TPOR; and the STAT activation domain comprises a STAT activation domain of (or derived from) at least one receptor selected from the group consisting of: BLNK, IL2RG, EGFR, EpoR, GHR, IFNAR1, IFNAR2, IFNAR1/2, IFNLR1, IL10R1, IL12Rb1, IL12Rb2, IL21R, IL2Rb, IL2small, IL7R, IL7Ra, IL9R, IL15R and IL 21R.

In some embodiments, the inducible chimeric cytokine receptor provided herein comprises a dimerization domain selected from table 1B, a tyrosine kinase activation domain selected from table 1B or table 1C, and a tyrosine effector domain selected from table 1B.

In some embodiments, the inducible chimeric cytokine receptor provided herein comprises a sequence selected from table 2A or table 2B. In some embodiments, the inducible chimeric cytokine receptor comprises a sequence selected from the group consisting of SEQ ID NO. 1-58, 187-215, and 225-311.

Table 1A provides exemplary full-length sequences of the native receptors provided in the present invention from which transmembrane proteins are derived. The sequences provided in table 1A are reference sequences, and the later mutations associated with the reference sequences are shown, for example, in tables 1B and 1C.

Table 1A: exemplary Natural receptors

Exemplary amino acid sequences useful in the inducible chimeric cytokine receptors provided herein are shown in table 1B.

TABLE 1B

Exemplary transmembrane and JAK binding sequences useful in (or derived from) the inducible chimeric cytokine receptors provided herein are shown in table 1C.

TABLE 1C

In another aspect, provided herein are isolated immune cells comprising one or more inducible chimeric cytokine receptors disclosed herein. In other embodiments, an isolated immune cell provided herein comprises (i) one or more inducible chimeric cytokine receptors disclosed herein and (ii) a Chimeric Antigen Receptor (CAR). Advantageously, the isolated immune cells provided herein exhibit improved persistence upon contact with a ligand that binds to the dimerization domain relative to cells that do not express an inducible chimeric cytokine receptor. In some embodiments, the isolated immune cells provided herein exhibit improved cytotoxicity, increased expansion, and/or increased levels of memory phenotypic markers upon contact with a ligand that binds to the dimerization domain relative to cells that do not express an inducible chimeric cytokine receptor. The improvement in persistence, cytotoxicity, expansion, and/or memory phenotypic markers displayed by isolated immune cells comprising an inducible chimeric cytokine receptor described herein can be in vitro or in vivo. In some embodiments, the isolated immune cell is selected from the group consisting of: t cells, dendritic cells, killer dendritic cells, mast cells, NK cells, macrophages, monocytes and B cells.

In some embodiments, the isolated immune cell is an isolated T cell. In some embodiments, an isolated T cell provided herein comprises one or more inducible chimeric cytokine receptors disclosed herein. In other embodiments, an isolated T cell provided herein comprises (i) one or more inducible chimeric cytokine receptors disclosed herein and (ii) a Chimeric Antigen Receptor (CAR). Advantageously, the isolated T cells provided herein exhibit improved in vivo persistence upon contact with a ligand that binds to the dimerization domain relative to cells that do not express an inducible chimeric cytokine receptor. In some embodiments, the isolated T cells provided herein exhibit improved cytotoxicity, increased expansion, and/or increased levels of a memory phenotypic marker upon contact with a ligand that binds to the dimerization domain relative to cells that do not express an inducible chimeric cytokine receptor. The modification of one or more of these features may be in vitro or in vivo.

In some embodiments, an isolated immune cell comprising one or more inducible chimeric cytokine receptors disclosed herein exhibits (i) increased in vivo persistence, (ii) increased STAT activation, (iii) increased cytotoxicity, (iv) increased levels of a memory phenotypic marker, (v) increased expansion (proliferation), or a combination of these functional characteristics, upon contact with a ligand that binds to the dimerization domain, relative to an isolated immune cell that does not express an inducible chimeric cytokine receptor. In some embodiments, the improvement in one or more of the functional characteristics described herein varies with dose, i.e., the functional activity of immune cells comprising an inducible chimeric cytokine receptor increases upon contact with an increasing dose of ligand that binds to the dimerization domain. In some embodiments, the STAT activated by the inducible chimeric cytokine receptor comprises STAT1, STAT2, STAT3, STAT4, STAT5, STAT6, or a combination thereof. Activation of STAT includes recruitment of STAT, phosphorylation of STAT, and/or dimerization of STAT or translocation of STAT. In some embodiments, the memory phenotype markers that are increased or maintained by immune cells comprising an inducible chimeric cytokine receptor include a stem cell memory (Tscm) marker and a central memory (Tcm) marker.

In some embodiments, one or more functional characteristics exhibited by an immune cell comprising an inducible chimeric cytokine receptor provided herein is improved by at least about 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 125-fold, 150-fold, 200-fold, 250-fold, 300-fold, 350-fold, 400-fold, 450-fold, or even about 500-fold, including values and ranges therebetween, as compared to an immune cell that does not express an inducible chimeric cytokine receptor.

In some embodiments, one or more functional characteristics exhibited by an immune cell comprising an inducible chimeric cytokine receptor provided herein is improved by at least about 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%, 90%, 100%, 125%, 150%, 200%, 250%, 300%, 350%, 400%, or even about 500%, including values and ranges therebetween, as compared to an immune cell that does not express an inducible chimeric cytokine receptor.

In some embodiments, an isolated immune cell (e.g., an isolated T cell) of the invention comprises an inducible chimeric cytokine receptor shown in table 2A.

Table 2A: exemplary inducible chimeric cytokine receptor sequences

In some embodiments, an isolated immune cell (e.g., an isolated T cell) of the invention comprises an inducible chimeric cytokine receptor as set forth in table 2B.

Table 2B: exemplary inducible chimeric cytokine receptor sequences

The present invention encompasses modifications to the inducible chimeric cytokine receptors of the embodiments of the invention shown in tables 2A and 2B, including functionally equivalent proteins with modifications that do not significantly affect their properties and variants with enhanced or reduced activity and/or affinity. Modification of polypeptides is routine practice in the art and need not be described in detail herein. Examples of modified polypeptides include polypeptides having conservative substitutions of amino acid residues, one or more deletions or additions of amino acids that do not significantly adversely alter functional activity, or their use to mature (enhance) the affinity of the polypeptide for its ligand, or chemical analogs.

Amino acid sequence insertions include amino-and/or carboxy-terminal fusions ranging in length from one residue to polypeptides containing one hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with N-terminal methionyl residues or antibodies fused to epitope tags.

Substitution variants have at least one amino acid residue deleted from the inducible chimeric cytokine receptor and a different residue inserted at its position. Conservative substitutions are shown in table 3 under the heading "conservative substitutions". If such substitutions result in an alteration in biological activity, more substantial alterations, referred to in Table 3 as "exemplary substitutions" or as further described below with reference to amino acid classes, can be introduced and the products screened.

Table 3: amino acid substitutions

In some embodiments, the inducible chimeric cytokine receptor can be synthesized in situ in an isolated immune cell (e.g., a CAR-T cell) upon introduction of a polynucleotide encoding the inducible chimeric cytokine receptor into the cell. Alternatively, the inducible chimeric cytokine receptor can be produced outside the cell and then introduced into the cell. Methods for introducing polynucleotide constructs into cells are known in the art. In some embodiments, stable transformation methods can be used to integrate the polynucleotide construct into the genome of the cell. In other embodiments, transient transformation methods can be used to transiently express the polynucleotide construct, and the polynucleotide construct is not integrated into the genome of the cell. In other embodiments, virus-mediated methods may be used. The polynucleotide can be introduced into the cell by any suitable method, such as recombinant viral vectors (e.g., retrovirus, adenovirus), liposomes, and the like. Transient transformation methods include, for example, but are not limited to, microinjection, electroporation, or particle bombardment. The polynucleotide may be included in a vector, such as a plasmid vector or a viral vector.

In some embodiments, an isolated immune cell (e.g., an isolated T cell) of the invention can comprise at least one inducible chimeric cytokine receptor and at least one CAR. In some embodiments, an isolated immune cell (e.g., an isolated T cell) can comprise at least one distinct population of inducible chimeric cytokine receptors and at least one CAR. For example, a population of different inducible chimeric cytokine receptors present in isolated immune cells may include receptors with the same dimerization domain but different tyrosine kinase activation domains and different tyrosine effector domains, or receptors with the same dimerization domain, the same tyrosine kinase activation domain but different tyrosine effector domains, or receptors with all three domains that are different from each other, and the like. In some embodiments, an isolated immune cell (e.g., an isolated T cell) can comprise at least one inducible chimeric cytokine receptor and a population of CARs, each CAR comprising a different extracellular ligand-binding domain.

Introduction of different inducible populations of chimeric cytokine receptors into immune cells may allow manipulation of cell functional outcomes and/or phenotypes. For example, different inducible chimeric cytokine receptors present in isolated immune cells can activate different intracellular signaling events, each of which results in a particular functional outcome and/or directs the cell to a particular phenotype. By manipulating the population of inducible chimeric cytokine receptors introduced into the cells, the functional outcome and/or phenotype of the cells can be manipulated. For example, an inducible population of chimeric cytokine receptors introduced into immune cells can be manipulated to contain a greater number of receptors that activate one STAT transcription factor over other STATs, thereby biasing the functional outcome and/or phenotype of the cell towards the receptor controlled by the STAT.

In some embodiments of an isolated immune cell (e.g., an isolated T cell provided herein), a CAR can comprise an extracellular ligand-binding domain (e.g., a single-chain variable fragment (scFv)), a transmembrane domain, and an intracellular signaling domain. In some embodiments, the extracellular ligand-binding domain, transmembrane domain, and intracellular signaling domain are in one polypeptide, i.e., in a single chain. Also provided herein are multi-chain CARs and polypeptides. In some embodiments, the multi-chain CAR comprises: a first polypeptide comprising a transmembrane domain and at least one extracellular ligand-binding domain, and a second polypeptide comprising a transmembrane domain and at least one intracellular signaling domain, wherein the polypeptides are assembled together to form a multi-chain CAR.

The extracellular ligand-binding domain of the CAR specifically binds to a target of interest. The target of interest can be any molecule of interest including, for example, but not limited to, BCMA, EGFRvIII, Flt-3, WT-1, CD20, CD23, CD30, CD38, CD70, CD33, CD133, LeY, NKG2D, CS1, CD44v6, ROR1, CD19, tight junction protein-18.2 (tight junction protein-18 a2 or tight junction protein 18 isoform 2), DLL3 (like protein 3, drosophila gt homolog 3, 3), Muc17(Mucin17, Muc3, Muc3), FAP α (fibroblast activation protein α), Ly6G6D (lymphocyte antigen 6 complex locus protein G6d, c6orf23, G6D, meng 72, ubiquitin NG 1), RNF 1-protein ligase 1, RNF 1.

In some embodiments, the extracellular ligand-binding domain of the CAR comprises an scFv comprising a light chain Variable (VL) region and a heavy chain Variable (VH) region of a target antigen-specific monoclonal antibody connected by a flexible linker. Single chain variable region fragments were prepared by linking the light and/or heavy chain variable regions using short linking peptides (Bird et al, Science 242:423-426, 1988). An example of a linker peptide is a GS linker having the amino acid sequence (GGGGS)3(SEQ ID NO:224, which bridges about 3.5nm between the carboxy terminus of one variable domain and the amino terminus of another variable domain. linkers that have been designed and used with other sequences (Bird et al, 1988, supra.) typically, the linker may be a short, flexible polypeptide, and preferably comprises about 20 or less amino acid residues Followed by). The resulting scFv can be isolated using standard protein purification techniques known in the art.

The intracellular signaling domain of the CAR according to the present invention is responsible for intracellular signaling upon binding of the extracellular ligand-binding domain to the target, resulting in activation of the immune cell and an immune response. The intracellular signaling domain has the ability to activate at least one normal effector function of an immune cell in which the CAR is expressed. For example, the effector function of a T cell may be cytolytic activity or helper activity, including secretion of cytokines.

In some embodiments, the intracellular signaling domain used in the CAR can be, for example (but not limited to), cytoplasmic sequences of T cell receptors and co-receptors that act synergistically to trigger signal transduction following antigen receptor participation, as well as any derivative or variant of these sequences and any synthetic sequence with the same functional capacity. The intracellular signaling domain contains two distinct classes of cytoplasmic signaling sequences: sequences that elicit antigen-dependent primary activation, and sequences that function in an antigen-independent manner to provide secondary or costimulatory signals. The primary cytoplasmic signaling sequence may contain a signaling motif known as the immunoreceptor tyrosine-based activation motif of ITAM. ITAMs are well-defined signaling motifs found in the intracytoplasmic tail of multiple receptors that serve as binding sites for tyrosine kinases of the syk/zap70 class. Examples of ITAMs for use in the present invention may include, as non-limiting examples, ITAMs derived from TCR ζ, FcR γ, FcR β, FcR, CD3 γ, CD3, CD3, CD5, CD22, CD79a, CD79b, and CD66 d. In some embodiments, the intracellular signaling domain of the CAR can comprise a CD3 zeta signaling domain. In some embodiments, the intracellular signaling domain of a CAR of the invention comprises a domain of a co-stimulatory molecule.

In some embodiments, the intracellular signaling domain of a CAR of the invention comprises a portion of a co-stimulatory molecule selected from the group consisting of fragments of 41BB (GenBank: AAA53133.) and CD28(NP _ 006130.1).

The CAR is expressed on the surface membrane of the cell. Thus, the CAR may comprise a transmembrane domain. Suitable transmembrane domains for the CARs disclosed herein have the following capabilities: (a) expressed at the surface of a cell, preferably an immune cell, such as, for example, but not limited to, a lymphocyte or a Natural Killer (NK) cell, and (b) interacts with the ligand binding domain and the intracellular signaling domain to direct the cellular response of the immune cell to a predetermined target cell. The transmembrane domain may be derived from a natural source or from a synthetic source. The transmembrane domain may be derived from any membrane-bound or transmembrane protein. By way of non-limiting example, the transmembrane polypeptide may be a subunit of a T cell receptor, such as α, β, γ or, a polypeptide constituting the CD3 complex, the IL-2 receptor p55(a chain), p75(β chain) or γ chain, a subunit chain of an Fc receptor, in particular Fc γ receptor III or a CD protein. Alternatively, the transmembrane domain may be synthetic and may comprise predominantly hydrophobic residues (e.g., leucine and valine). In some embodiments, the transmembrane domain is derived from a human CD8 a chain (e.g., NP _ 001139345.1). The transmembrane domain may further comprise a stalk domain between the extracellular ligand-binding domain and the transmembrane domain. The stalk domain may comprise up to 300 amino acids, preferably 10 to 100 amino acids and most preferably 25 to 50 amino acids. The stem region may be derived from all or part of a natural molecule, such as all or part of an extracellular region derived from CD8, CD4, or CD28, or from all or part of an antibody constant region. Alternatively, the stem domain may be a synthetic sequence corresponding to a native stem sequence, or may be a fully synthetic stem sequence. In some embodiments, the stalk domain is part of a human CD8 a chain (e.g., NP _ 001139345.1). In another particular embodiment, the transmembrane and hinge domain comprises a portion of the human CD8 a chain. In some embodiments, a CAR disclosed herein can comprise an extracellular ligand binding domain that specifically binds BCMA, CD8 a human hinge and transmembrane domains, a CD3 zeta signaling domain, and a 4-1BB signaling domain. In some embodiments, the CAR can be introduced into an immune cell as a transgene through a plasmid vector. In some embodiments, the plasmid vector may also comprise, for example, a selectable marker that provides for identification and/or selection of the cells that receive the vector.

Table 4 provides exemplary sequences of CAR components that can be used in the CARs disclosed herein.

Table 4: exemplary sequences of CAR Components

Upon introduction of the polynucleotide encoding the CAR polypeptide into the cell, the CAR polypeptide can be synthesized in situ in the cell. Alternatively, the CAR polypeptide can be produced outside the cell and then introduced into the cell. Methods for introducing polynucleotide constructs into cells are known in the art. In some embodiments, stable transformation methods can be used to integrate the polynucleotide construct into the genome of the cell. In other embodiments, transient transformation methods can be used to transiently express the polynucleotide construct, and the polynucleotide construct is not integrated into the genome of the cell. In other embodiments, virus-mediated methods may be used. The polynucleotide can be introduced into the cell by any suitable method, such as recombinant viral vectors (e.g., retrovirus, adenovirus), liposomes, and the like. Transient transformation methods include, for example, but are not limited to, microinjection, electroporation, or particle bombardment. The polynucleotide may be included in a vector, such as a plasmid vector or a viral vector.

Also provided herein are isolated immune cells comprising at least one inducible chimeric cytokine receptor described herein. The isolated immune cell may further comprise a Chimeric Antigen Receptor (CAR). Isolated immune cells modified to express an inducible chimeric cytokine receptor and/or CAR as referred to in the specification are also interchangeably referred to as engineered immune cells. These isolated immune cells can be prepared according to any of the methods described herein. Any immune cell capable of expressing heterologous DNA can be used for the purpose of expressing an inducible chimeric cytokine receptor and the CAR of interest. In some embodiments, the isolated immune cell is a T cell. In some embodiments, the immune cells can be derived from (e.g., without limitation) stem cells. The stem cell may be an adult stem cell, a non-human embryonic stem cell (more particularly a non-human stem cell), a cord blood stem cell, a progenitor cell, a bone marrow stem cell, an induced pluripotent stem cell, a totipotent stem cell or a hematopoietic stem cell. Representative human cells are CD34+ cells. In some embodiments, the isolated immune cell can be a dendritic cell, a killer dendritic cell, a mast cell, an NK cell, a macrophage, a monocyte, a B cell, or a T cell. In some embodiments, the isolated immune cell may be a T cell selected from the group consisting of an inflammatory T lymphocyte, a cytotoxic T lymphocyte, a regulatory T lymphocyte, or a helper T lymphocyte. In some embodiments, the cells may be derived from the group consisting of CD4+ T-lymphocytes and CD8+ T-lymphocytes. In some embodiments, the isolated immune cells are autologous T cells. In some embodiments, the isolated immune cells are allogeneic T cells.

In some embodiments, a CAR immune cell (e.g., a CAR-T cell) of the invention comprises a polynucleotide encoding a suicide polypeptide (e.g., RQR 8). See, for example, WO2013153391A, which is incorporated herein by reference in its entirety. In some embodiments, the suicide polypeptide is expressed on the surface of a cell. In some embodiments, the suicide polypeptide is comprised in a CAR construct. In some embodiments, the suicide polypeptide is not part of the CAR construct.

In some embodiments, the extracellular domain of any one of the CARs disclosed herein can comprise one or more epitopes specific for (specifically recognized by) a monoclonal antibody. These epitopes are also referred to herein as mAb-specific epitopes. Exemplary mAb-specific epitopes are disclosed in international patent publication No. WO 2016/120216, which is incorporated herein by reference in its entirety. In these embodiments, the extracellular domain of the CAR comprises an antigen binding domain that specifically binds to a target of interest and one or more epitopes that bind to one or more monoclonal antibodies (mabs). The CAR comprising the mAb-specific epitope can be single-chain or multi-chain.

The inclusion of an epitope specific for a monoclonal antibody in the ectodomain of the CAR described herein allows for sorting and elimination of engineered immune cells expressing the CAR. In some embodiments, elimination is allowed to provide a safe conversion in the event of deleterious effects, for example, upon administration to an individual.

Prior to propagation and genetic modification, the source of the cells can be obtained from the individual by various non-limiting methods. Cells can be obtained from a number of non-limiting sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue, and tumors. In some embodiments, any number of immune cell lines, such as T cell lines, available and known to those of skill in the art can be used. In some embodiments, the cells may be derived from a healthy donor, from an individual diagnosed with cancer, or from an individual diagnosed with an infection. In some embodiments, the cells may be part of a mixed population of cells exhibiting different phenotypic characteristics.

Also provided herein are cell lines obtained from transformed immune cells (e.g., transformed T cells) according to any of the methods described herein. In some embodiments, an isolated immune cell (e.g., an isolated T cell according to the present invention) comprises a polynucleotide encoding an inducible chimeric cytokine receptor. In some embodiments, an isolated immune cell according to the invention comprises a polynucleotide encoding an inducible chimeric cytokine receptor and a polynucleotide encoding a CAR. In some embodiments, an isolated immune cell according to the invention comprises a polynucleotide encoding an inducible chimeric cytokine receptor, a polynucleotide encoding a CAR, and a polynucleotide encoding an NK cell antagonist.

The isolated immune cells of the invention (e.g., isolated T cells) can be used prior to or after genetic modification of T cells using methods as generally described, for example (but not limited to) U.S. patent 6,352,694; 6,534,055, respectively; 6,905,680, respectively; 6,692,964, respectively; 5,858,358, respectively; 6,887,466, respectively; 6,905,681, respectively; 7,144,575, respectively; 7,067,318, respectively; 7,172,869, respectively; 7,232,566, respectively; 7,175,843, respectively; 5,883,223, respectively; 6,905,874, respectively; 6,797,514, respectively; 6,867,041, respectively; and U.S. patent application publication No. 20060121005 for activation and proliferation. Immune cells may be propagated in vitro or in vivo. Generally, the T cells of the invention can be expanded, for example, by contacting with an agent that stimulates the CD3 TCR complex and costimulatory molecules on the surface of the T cell, to generate an activation signal for the T cell. For example, chemicals such as calcium ionophore A23187, phorbol 12-myristate 13-acetate (PMA) or mitotic lectins (e.g. Phytohemagglutinin (PHA)) can be used to generate activation signals for T cells.

In some embodiments, the immune cell population can be stimulated in vitro by contact with a suitable antibody or antigen-binding fragment thereof. For example, a population of T cells can be stimulated in vitro by contact with, for example, an anti-CD 3 antibody or antigen-binding fragment thereof, or an anti-CD 28 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryostatin) bound to a calcium ionophore. To co-stimulate accessory molecules on the surface of T cells, ligands that bind the accessory molecules are used. For example, a population of T cells can be contacted with an anti-CD 3 antibody and an anti-CD 28 antibody under conditions suitable to stimulate proliferation of T cells. Suitable conditions for T cell culture include suitable media (e.g., minimal requirement medium or RPMI medium 1640, or X-vivo 5 (Longza))), which may contain factors necessary for proliferation and activity, including serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN- γ, IL-4, IL-7, GM-CSF, IL-10, IL-2, IL-15, TGF β and TNF, or any other additive known to the skilled artisan for cell growth. Other additives for cell growth include, but are not limited to, surfactants, plasma agents (plasmanates), and reducing agents (e.g., N-acetyl-cysteine and 2-mercaptoethanol). The culture medium may include RPMI 1640, A1M-V, DMEM, MEM, a-MEM, F-12, X-Vivo 1, and X-Vivo 20(Optimizer) with added amino acids, sodium pyruvate and vitamins, serum (or plasma) or a defined set of hormones in serum-free or supplemented amounts, and/or cytokines in amounts sufficient to grow and proliferate T cells. Antibiotics (e.g., penicillin and streptomycin) are included only in the experimental cultures and not in the cell cultures to be infused into the individual. The target cells are maintained under conditions necessary to support growth, e.g., suitable temperature (e.g., 37 ℃) and atmosphere (e.g., air plus 5% CO ₂). T cells that have been exposed to different stimulation times may exhibit different characteristics.

In some embodiments, the cells of the invention may be expanded by co-culture with tissue or cells. The cells can also be expanded in vivo in the blood of an individual, for example, after administration of the cells to the individual.

In another aspect, the invention provides a composition (e.g., a pharmaceutical composition) comprising any of the cells of the invention. In some embodiments, the composition comprises an isolated T cell comprising a polynucleotide encoding any of the inducible chimeric cytokine receptors described herein and a polynucleotide encoding a CAR.

Further described herein are the administration of expression vectors, and polynucleotide compositions.

In another aspect, the invention provides a method of making any of the polynucleotides described herein.

Polynucleotides complementary to any such sequence are also encompassed by the invention. Polynucleotides may be single-stranded (coding or antisense) or double-stranded, and may be (genomic, cDNA, or synthetic) DNA, or RNA molecules. RNA molecules include HnRNA molecules (which contain introns and correspond in a one-to-one manner to DNA molecules) and mRNA molecules (which do not contain introns). Additional coding or non-coding sequences may (but need not) be present within the polynucleotides of the invention, and the polynucleotides may (but need not) be linked to other molecules and/or support materials.

The polynucleotide may comprise a native sequence (i.e., an endogenous sequence encoding an antibody or portion thereof) or may comprise a variant of such a sequence. Polynucleotide variants comprise one or more substitutions, additions, deletions and/or insertions such that the immunoreactivity of the encoded polypeptide is not diminished relative to the naturally-occurring immunoreactive molecule. The effect on the immunoreactivity of the encoded polypeptide can generally be assessed as described herein. The variant preferably exhibits at least about 70% identity, more preferably, at least about 80% identity, yet more preferably, at least about 90% identity, and most preferably, at least about 95% identity to the polynucleotide sequence encoding the native antibody or a portion thereof.

Two polynucleotide or polypeptide sequences are considered "identical" if the sequences of nucleotides or amino acids in the two sequences are identical when aligned for maximum correspondence as described below. Comparison between two sequences is typically performed by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity. As used herein, a "comparison window" refers to a fragment of at least about 20 contiguous positions, typically 30 to about 75, or 40 to about 50, where a sequence can be compared to a reference sequence of the same number of contiguous positions after optimally aligning the two sequences.

The best alignment of the sequences for comparison can be performed using the Megalign program in the Lasergene bioinformatics software suite (DNASTAR, Madison, Wis.) using default parameters. This program embodies several alignment schemes described in the following references: dayhoff, M.O., 1978, "protein evolution Change model-matrix for detecting distant relationships" (A model of evolution change in proteins-matrix for detecting differences), Dayhoff, M.O. (eds.) protein sequences and Structure maps (Atlas of protein sequences and Structure), National biomedical research Foundation (National biomedical research Foundation), Washington DC Vol.5, supplement 3, p.345 and 358; hein J., 1990, Unifie applied to Alignment and phylogenetic (Unifie) Unifie Association, 626. 645 Methods in Enzymology, 183, academic Press, Inc., San Diego, Calif.; higgins, D.G. and Sharp, P.M., 1989, CABIOS 5: 151-; myers, E.W. and Muller W., 1988, CABIOS 4: 11-17; robinson, E.D., 1971, "Combined theory (comb. Theor.) 11: 105; santou, N., Nes, M., 1987, molecular biology and evolution (mol. biol. Evol.) 4: 406-425; sneath, p.h.a. and Sokal, r.r., 1973, numerical classification: principles and practices of Numerical classification (Numerical Taxonomy the Principles and Practice of Numerical Taxonomy), Fremann Press, San Francisco, Calif.; wilbur, W.J., and Lipman, D.J., 1983, Proc. Natl. Acad. Sci., USA 80: 726-730.

Preferably, the "percent sequence identity" is determined by comparing two optimally aligned sequences over a comparison window of at least 20 positions, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20% or less, typically 5% to 15%, or 10% to 12%, as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the same nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence (i.e., the window size) and multiplying the result by 100 to yield the percentage of sequence identity.

The variant may also or alternatively be substantially homologous to the native gene or a portion thereof or to the complement. Such polynucleotide variants are capable of hybridizing to a native DNA sequence encoding a native antibody (or a complementary sequence) under moderately stringent conditions.

Suitable "moderately stringent conditions" include a pre-wash in a solution of 5 XSSC, 0.5% SDS, 1.0mM EDTA (pH 8.0); hybridization overnight at 5 XSSC at 50 ℃ to 65 ℃; followed by two washes each with 2X, 0.5X and 0.2 XSSC containing 0.1% SDS at 65 ℃ over 20 minutes.

As used herein, "high stringency conditions" are conditions as follows: (1) washing with low ionic strength and high temperature, e.g. 0.015M sodium chloride/0.0015M sodium citrate/0.1% sodium lauryl sulfate, at 50 ℃; (2) denaturing agents such as formamide, e.g., 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer (pH6.5) with 750mM sodium chloride, 75mM sodium citrate at 42 ℃; or (3) washing with 50% formamide, 5 XSSC (0.75M NaCl, 0.075M sodium citrate), 50mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 XDandard's solution, sonicated salmon sperm DNA (50. mu.g/ml), 0.1% SDS and 10% polydextrose sulfate at 42 ℃ in 0.2 XSSC (sodium chloride/sodium citrate) and at 55 ℃ in 50% formamide, followed by a high stringency wash consisting of 0.1 XSSC with EDTA at 55 ℃. The skilled artisan will recognize how to adjust the temperature, ionic strength, etc. as needed to accommodate factors such as probe length, etc.

One of ordinary skill in the art will appreciate that due to the degeneracy of the genetic code, there are many nucleotide sequences that encode a polypeptide as described herein. Some of these polynucleotides have minimal homology to the nucleotide sequence of any native gene. Nevertheless, the present invention specifically encompasses polynucleotides that vary due to differences in codon usage. Furthermore, alleles of genes comprising the polynucleotide sequences provided herein are within the scope of the invention. Alleles are endogenous genes that are altered by one or more mutations (e.g., deletions, additions and/or substitutions of nucleotides). The resulting mRNA and protein may (but need not) have altered structure or function. Alleles can be identified using standard techniques, such as hybridization, amplification, and/or database sequence comparison.

The polynucleotides of the present invention can be obtained using chemical synthesis, recombinant methods, or PCR. Methods of chemical polynucleotide synthesis are well known in the art but need not be described in detail herein. One of skill in the art can use the sequences provided herein and a commercial DNA synthesizer to generate the desired DNA sequence.

For the use of recombinant methods to prepare polynucleotides, polynucleotides comprising the desired sequences can be inserted into suitable vectors, and the vectors can in turn be introduced into suitable host cells for replication and propagation, as discussed further herein. The polynucleotide may be inserted into the host cell by any method known in the art. Cells are transformed by direct uptake, endocytosis, transfection, F-mating, or electroporation by introduction of exogenous polynucleotide. Once introduced, the exogenous polynucleotide may be maintained intracellularly as a non-integrating vector (e.g., a plasmid) or integrated into the host cell genome. The polynucleotide so amplified can be isolated from the host cell by methods well known in the art. See, e.g., Sambrook et al, 1989.

Alternatively, PCR allows for the reproduction of DNA sequences. PCR techniques are well known in the art and are described in U.S. patent nos. 4,683,195, 4,800,159, 4,754,065, and 4,683,202, and PCR: polymerase chain reaction, compiled by Mullis et al, Birkauswer Press, Boston, 1994.

RNA can be obtained by using the isolated DNA in a suitable vector and inserting it into a suitable host cell. When the cell replicates and the DNA is transcribed into RNA, then the RNA can be isolated using methods well known to those skilled in the art (e.g., Sambrook et al, 1989, supra).

Suitable cloning vectors may be constructed according to standard techniques, or may be selected from a large number of cloning vectors available in the art. Although the cloning vector selected may vary depending on the host cell intended for use, useful cloning vectors typically have the ability to self-replicate, may have a single target for a particular restriction endonuclease, and/or may carry a gene that can be used to select for markers that comprise the clones of the vector. Suitable examples include plasmids and bacterial viruses such as pUC18, pUC19, Bluescript (e.g., pBS SK +) and derivatives thereof, mp18, mp19, pBR322, pMB9, ColE1, pCR1, RP4, phage DNA, and shuttle vectors such as pSA3 and pAT 28. These and many other cloning vectors are available from commercial suppliers such as burle (BioRad), Strategene and Invitrogen.

Expression vectors are generally replicable polynucleotide constructs comprising a polynucleotide according to the invention. This means that the expression vector must be replicable in the host cell either as episomes or as an integral part of the chromosomal DNA. Suitable expression vectors include, but are not limited to, plasmids, viral vectors, including adenoviruses, adeno-associated viruses, retroviruses, cosmids, and the expression vectors disclosed in PCT publication No. WO 87/04462. The carrier component may typically include (but is not limited to) one or more of the following: a signal sequence; an origin of replication; one or more marker genes; suitable transcriptional control elements (e.g., promoters, enhancers, and terminators). To achieve expression (i.e., translation), one or more translation control components, such as a ribosome binding site, a translation initiation site, and a stop codon, are also typically required.

Vectors comprising a polynucleotide of interest can be introduced into a host cell by any of a number of suitable methods, including electroporation, transfection using calcium chloride, rubidium chloride, calcium phosphate, DEAE-polydextrose, or other substances; bombardment by micro-bullet; carrying out liposome transfection; and infection (e.g., where the vector is an infectious agent, such as a vaccine virus). The choice of vector or polynucleotide to introduce will generally depend on the characteristics of the host cell.

Polynucleotides encoding the inducible chimeric cytokine receptors or CARs disclosed herein can be present in an expression cassette or expression vector, e.g., a plasmid for introduction into a bacterial host cell, or a viral vector, such as a baculovirus vector for transfection of insect host cells, or a plasmid or viral vector, such as a lentivirus for transfection of mammalian host cells. In some embodiments, the polynucleotide encoding the inducible chimeric cytokine receptor and/or CAR is introduced into the isolated immune cell using a non-viral vector. Exemplary non-viral vectors that can be used in the methods of the invention include, but are not limited to, transposon-based vectors, such as piggybac^TMFrog Prince, Sleeping Beauty (e.g., SB100X vector), and the like. In some embodiments, the polynucleotide encoding the inducible chimeric cytokine receptor and/or CAR is integrated into the genome of the cell. In some embodiments, the integration is site-specific. Exemplary methods of providing site-directed integration include methods using genome editing nucleases, such as meganucleases, Zinc Finger Nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered, regularly interspaced short palindromic repeat-associated nucleases (CRISPRs), such as Cas9 endonuclease.

In some embodiments, the polynucleotide or vector can include a nucleic acid sequence encoding a ribosome skipping sequence, such as, but not limited to, a sequence encoding a 2A peptide. The 2A peptides identified in the foot-and-mouth disease virus (aphthvirus) subset of picornaviruses cause ribosomes to "hop" from one codon to the next without forming a peptide bond between the two amino acids encoded by the codon (see (Donnelly and Elliott 2001; Atkins, Wills et al 2007; Doronina, Wu et al 2008)). "codon" means three nucleotides on an mRNA (or on a sense strand of a DNA molecule) that are translated by ribosomes into one amino acid residue. Thus, when the polypeptides are separated by in-frame 2A oligopeptide sequences, two, three, four or more polypeptides can be synthesized from a single contiguous open reading frame within the imRNA. This ribosome skipping mechanism is well known in the art and is known to be used by several vectors for expression of several proteins encoded by a single messenger RNA.

To direct the transmembrane polypeptide into the secretory pathway of a host cell, in some embodiments, a secretory signal sequence (also referred to as a leader sequence, prepro sequence, or pre sequence) is provided in the polynucleotide sequence or vector sequence. The secretion signal sequence may be operably linked to the transmembrane nucleic acid sequence, i.e., the two sequences are linked and positioned in the correct reading frame to direct the newly synthesized polypeptide into the secretory pathway of the host cell. Secretion signal sequences are typically located 5' to the nucleic acid sequence encoding the polypeptide of interest, although certain secretion signal sequences may be located elsewhere in the nucleic acid sequence of interest (see, e.g., Welch et al, U.S. Pat. No. 5,037,743; Holland et al, U.S. Pat. No. 5,143,830). In some embodiments, the signal peptide comprises the amino acid sequence shown as SEQ ID NO:318 or 329. One skilled in the art will recognize that, given the degeneracy of the genetic code, there may be considerable sequence variation between these polynucleotide molecules. In some embodiments, the nucleic acid sequences of the invention are codon optimized for expression in mammalian cells, preferably human cells. Codon optimization refers to the exchange of sequences of interest for codons that are typically rare in highly expressed genes of a given species by codons that are typically common in highly expressed genes of these species (these codons encode amino acids as the exchanged codons).

Provided herein are methods of preparing immune cells for use in immunotherapy. In some embodiments, the method comprises introducing an inducible chimeric cytokine receptor and a CAR into an immune cell, and expanding the cell. In some embodiments, the invention relates to a method of engineering an immune cell, the method comprising: providing a cell and expressing an inducible chimeric cytokine receptor, and expressing at least one CAR at the surface of the cell. In some embodiments, the method comprises: transfecting a cell with at least one polynucleotide encoding an inducible chimeric cytokine receptor and at least one polynucleotide encoding a CAR, and expressing the polynucleotides in the cell. In some embodiments, the method comprises: transfecting a cell with at least one polynucleotide encoding an inducible chimeric cytokine receptor, at least one polynucleotide encoding a CAR, and expressing the polynucleotide in the cell.

In some embodiments, the polynucleotides encoding the inducible chimeric cytokine receptor and CAR are present in one or more expression vectors to achieve stable expression in the cell. In some embodiments, the polynucleotide is present in a viral vector to achieve stable expression in a cell. In some embodiments, the viral vector can be, for example, a lentiviral vector or an adenoviral vector.

In some embodiments, the polynucleotide encoding a polypeptide according to the invention may be an mRNA, which is introduced directly into the cell, e.g., by electroporation. In some embodiments, cytoPulse technology (e.g., PulseAgile) can be used to transiently permeabilize a living cell to deliver material into the cell (e.g., http:// cytoPulse. com; US 6,078,490; PCT/US 2011/000827; and PCT/US 2004/005237). Parameters can be modified to determine conditions for high transfection efficiency but minimal mortality.

Also provided herein are methods of transfecting immune cells (e.g., T cells). In some embodiments, the method comprises: contacting an immune cell with RNA and applying to the immune cell an agile pulse sequence consisting of: (a) an electrical pulse having a voltage in the range of about 2250 to 3000V per cm; (b) the pulse width is 0.1 ms; (c) the pulse time interval between the electrical pulses of steps (a) and (b) is about 0.2 to 10 ms; (d) an electrical pulse having a voltage in the range of about 2250 to 3000V, a pulse width of about 100ms and a pulse time interval between the electrical pulse of step (b) and the first electrical pulse of step (c) of about 100 ms; and (e) four electrical pulses, a voltage of about 325V and a pulse width of about 0.2ms and a pulse interval of 2ms between each of the 4 electrical pulses. In some embodiments, a method of transfecting an immune cell comprises contacting the immune cell with RNA and applying an agile pulse sequence to the immune cell, the agile pulse sequence comprising: (a) an electrical pulse at a voltage of about 2250, 2300, 2350, 2400, 2450, 2500, 2550, 2400, 2450, 2500, 2600, 2700, 2800, 2900 or 3000V per cm; (b) the pulse width is 0.1 ms; (c) the pulse time interval between the electrical pulses of steps (a) and (b) is about 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 ms; (d) an electrical pulse having a voltage range of about 2250, 2300, 2350, 2400, 2450, 2500, 2550, 2400, 2450, 2500, 2600, 2700, 2800, 2900 or 3000V, a pulse width of 100ms and a pulse time interval between the electrical pulse of step (b) and the first electrical pulse of step (c) of 100 ms; and (e)4 electrical pulses, a voltage of about 325V and a pulse width of about 0.2ms and a pulse time interval between each of the 4 electrical pulses of about 2 ms. Any value contained within the above range of values is disclosed herein. The electroporation medium may be any suitable medium known in the art. In some embodiments, the conductivity of the electroporation medium is in a range spanning from about 0.01 to about 1.0 milliSiemens (milliSiemens).

In some embodiments, the method may further comprise the step of genetically modifying the cell by inactivating at least one gene expression, such as, but not limited to, a component of a TCR, a target of an immunosuppressive agent, an HLA gene, and/or an immune checkpoint protein (such as PDCD1 or CTLA-4). By inactivating a gene, it is intended that the gene of interest is not expressed in the form of a functional protein. In some embodiments, the gene to be inactivated is selected from the group consisting of, for example, but not limited to, TCR α, TCR β, CD52, GR, deoxycytidine kinase (DCK), PD-1, and CTLA-4. In some embodiments, the method comprises inactivating one or more genes by introducing into the cell a rare-cutting endonuclease that can selectively inactivate the genes by selective DNA cleavage. In some embodiments, the rare-cutting endonuclease can be, for example, a transcriptional activator-like effector nuclease (TALE-nuclease) or a Cas9 endonuclease.

In another aspect, the step of genetically modifying the cell can comprise: modifying an immune cell by inactivating at least one gene expressing a target of an immunosuppressive agent; and optionally proliferating said cells in the presence of an immunosuppressive agent. Immunosuppressive agents are agents that inhibit immune function through one of several mechanisms of action. Immunosuppressive agents can reduce the extent and/or vorticity (voracity) of the immune response. Non-limiting examples of immunosuppressive agents include calcineurin inhibitors, targets of rapamycin, interleukin-2 alpha-chain blockers, inhibitors of inosine monophosphate dehydrogenase, inhibitors of dihydrofolate reductase, corticosteroids, and immunosuppressive antimetabolites. Some cytotoxic immunosuppressants act by inhibiting DNA synthesis. Others may act by activation of T cells or by inhibiting activation of helper cells. The method according to the invention allows to confer immunosuppressive resistance on T cells for immunotherapy by inactivating the target of immunosuppressive agents in the T cells. As a non-limiting example, the target of the immunosuppressant can be a receptor for an immunosuppressant, such as, for example, but not limited to, CD52, Glucocorticoid Receptor (GR), FKBP family gene members, and cyclophilin family gene members.

Method of treatment

The isolated immune cells obtained by the above-described method or cell lines derived from such isolated immune cells may be administered to an individual in need thereof and used as a medicament. In some embodiments, the isolated immune cell is a T cell. In some embodiments, such drugs may be used to treat a disorder, such as a viral disease, a bacterial disease, a cancer, an inflammatory disease, an immune disease, or an aging-related disease. In some embodiments, the cancer may be selected from the group consisting of: gastric cancer, sarcoma, lymphoma, leukemia, head and neck cancer, thymus cancer, epithelial cancer, salivary gland cancer, liver cancer, gastric cancer, thyroid cancer, lung cancer, ovarian cancer, breast cancer, prostate cancer, esophageal cancer, pancreatic cancer, glioma, leukemia, multiple myeloma, renal cell carcinoma, bladder cancer, cervical cancer, choriocarcinoma, colon cancer, oral cancer, skin cancer, and melanoma. In some embodiments, the individual is a previously treated adult individual with locally advanced or metastatic melanoma, Squamous Cell Head and Neck Cancer (SCHNC), ovarian cancer, sarcoma, or recurrent or refractory classic hodgkin's lymphoma (cHL).

In some embodiments, an isolated immune cell (e.g., an isolated T cell according to the invention or a cell line derived from an isolated immune cell) can be used in the manufacture of a medicament for treating a disorder in an individual in need thereof. In some embodiments, the disorder can be, for example, a cancer, an autoimmune disorder, or an infection.

Also provided herein are methods for treating an individual. In some embodiments, the method comprises providing an isolated immune cell comprising an inducible chimeric cytokine receptor of the invention to an individual in need thereof. In some embodiments, the method comprises the step of administering the isolated immune cells of the invention to an individual in need thereof. In one exemplary embodiment, the method comprises providing an individual in need thereof with an isolated T cell comprising an inducible chimeric cytokine receptor of the invention. In some embodiments, the method comprises the step of administering the isolated T cell of the invention to an individual in need thereof.

In some embodiments, the isolated immune cells of the invention can undergo robust in vivo cell proliferation and can last for an extended period of time.

The methods can further comprise administering one or more therapeutic agents to the individual prior to administering the engineered immune cells bearing the CAR and the inducible chimeric cytokine receptor provided herein. In certain embodiments, the agent is a lymphodepletion (preconditioning) regimen. For example, a method of lymphoablation in an individual in need of such therapy comprises administering to the individual a prescribed beneficial dose of cyclophosphamide (200 mg/m) ²Daily to 2000mg/m²A day, about 100mg/m²Daily to 2000mg/m²A day; for example, about 100mg/m²A day of about 200mg/m²A day, about 300mg/m²A day, about 400mg/m²A day, about 500mg/m²A day, about 600mg/m²A day of about 700mg/m²A day, about 800mg/m²A day of about 900mg/m²A day, about 1000mg/m²A day of about 1500mg/m²Daily or about 2000mg/m²Day) and the indicated dose of fludarabine (20 mg/m)²Daily to 900mg/m²A day, about 10mg/m²Daily to about 900mg/m²A day; for example, about 10mg/m²About 20 mg/m/day²About 30 mg/m/day²About 40 mg/m/day²About 40 days per daymg/m²About 50 mg/m/day²About 60 mg/m/day²About 70 mg/m/day²About 80 mg/m/day²About 90 mg/m/day²A day, about 100mg/m²A day, about 500mg/m²Daily or about 900mg/m²Day). An exemplary dosing regimen involves treating an individual, including prior to administering a therapeutically effective amount of engineered immune cells to the patient, in combination or at about 30mg/m²Administering to the patient about 300mg/m daily before or after the daily dose of fludarabine²Cyclophosphamide was given a day for three days.

In some embodiments, especially where the engineered cells provided herein have been genetically edited to eliminate or minimize surface expression of CD52, lymphodepletion further comprises administration of an anti-CD 52 antibody, such as alemtuzumab (alemtuzumab). In some embodiments, the CD52 antibody is administered at a dose of about 1-20 mg/day IV (e.g., about 13 mg/day IV) for 1, 2, 3, or more days. The antibody may be administered in combination with, before or after administration of other components of a lymphocyte depletion protocol (e.g., cyclophosphamide and/or fludarabine).

In certain embodiments, a composition comprising a CAR-expressing immune effector cell disclosed herein can be administered in combination with any number of chemotherapeutic agents.

The methods of treatment of the present invention may be ameliorating, treating or preventing. The methods of the invention may be part of an autoimmune therapy or part of an allogeneic immunotherapy treatment. The invention is particularly applicable to allogeneic immunotherapy. In one exemplary embodiment, T cells from a donor can be transformed into non-alloreactive cells using standard protocols and regenerated as needed, thereby generating CAR-T cells that can be administered to one or more individuals. Such CAR-T cell therapies are available as "off-the-shelf" therapeutic products.

In another aspect, the invention provides a method of inhibiting tumor growth or progression in an individual having a tumor, the method comprising administering to the individual an effective amount of an isolated immune cell as described herein. In another aspect, the invention provides a method of inhibiting or preventing metastasis of cancer cells in an individual, the method comprising administering to an individual in need thereof an effective amount of an isolated immune cell as described herein. In another aspect, the invention provides a method of inducing tumor regression in an individual having a tumor, the method comprising administering to the individual an effective amount of an isolated immune cell as described herein. In one exemplary embodiment, the isolated immune cell is a T cell.

In some embodiments, the isolated immune cells can be administered parenterally in an individual. In some embodiments, the subject is a human.

Also provided is the use of any of the isolated immune cells provided herein in the manufacture of a medicament for treating cancer or for inhibiting tumor growth or progression in an individual in need thereof. In one exemplary embodiment, the isolated immune cell is a T cell.

In some embodiments, the treatment may be administered to an individual undergoing immunosuppressive therapy. Indeed, the present invention preferably relies on cells or cell populations that have become resistant to at least one immunosuppressant due to inactivation of the gene encoding the receptor for such immunosuppressant. In this regard, immunosuppressive therapy should facilitate the selection and proliferation of immune cells according to the invention in an individual. Administration of the cells or cell populations according to the invention may be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. The compositions described herein can be administered to an individual subcutaneously, intradermally, intratumorally, intranodal, intramedullary, intramuscularly, by intravenous or intralymphatic injection, or intraperitoneally. In some embodiments, the cell compositions of the present invention are preferably administered by intravenous injection.

In some embodiments, administering the cell or population of cells can comprise administering, for example, about 10 per kg body weight⁴To about 10⁹And (b) a cell, including all integer cell numbers within the range. In some embodiments, administering the cell or population of cells can comprise administering about 10 per kg body weight⁵To 10⁶And (b) a cell, including all integer cell numbers within the range. Said thinThe cells or cell populations may be administered in one or more doses. In some embodiments, the effective amount of the cells can be administered in a single dose. In some embodiments, the effective amount of cells may be administered in more than one dose over a period of time. The time of administration is within the discretion of the administering physician and depends on the clinical condition of the individual. The cell or population of cells may be obtained from any source, such as a blood bank or donor. Although individual requirements vary, the optimal range for an effective amount of a given cell type is determined for a particular disease or condition within the skill of the art. An effective amount means an amount that provides a therapeutic or prophylactic benefit. The dose administered will depend on the age, health and weight of the recipient, the type of concurrent treatment (if any), the frequency of treatment and the nature of the desired effect. In some embodiments, an effective amount of the cells or compositions comprising the cells are administered parenterally. In some embodiments, the administration can be administered intravenously. In some embodiments, administration can be directly by intratumoral injection.

Reagent kit

The invention also provides kits for use in the methods of the invention. The kits of the invention comprise one or more containers comprising an isolated immune cell comprising one or more polynucleotides encoding an inducible chimeric cytokine receptor and a CAR as described herein, and instructions for use according to any of the methods of the invention described herein. Typically, these instructions include a description of administering the isolated immune cells for the therapeutic treatment described above. In one exemplary embodiment, the kit can comprise isolated T cells.

Instructions related to the use of isolated immune cells as described herein generally include information regarding the dosage, dosing regimen, and route of administration desired to be treated. The containers may be in unit doses, bulk packages (e.g., multi-dose packages), or sub-unit doses. The instructions provided in the kits of the invention are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disc) are also acceptable.

The kit of the invention is in a suitable package. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. Packaging is also contemplated for use in combination with a particular device, such as an inhaler, a nasal administration device (e.g., a nebulizer) or an infusion device (e.g., a micropump). The kit may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The container may also have a sterile access port (e.g., the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an isolated immune cell comprising an inducible chimeric cytokine receptor and a CAR. The container may further comprise a second pharmaceutically active agent.

The kit may optionally provide additional components, such as buffers and explanatory information. Typically, the kit comprises a container and a label or package insert on or associated with the container.

The following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims.

Numbering example

The invention disclosed herein may be defined with reference to the following numbered exemplary embodiments.