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1. WO2020118076 - REGULATABLE CELL SURFACE RECEPTORS AND RELATED COMPOSITIONS AND METHODS

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[ EN ]

WHAT is CLAIMED is:

1 . An engineered cell surface receptor comprising:

an extracellular binding domain;

a transmembrane domain;

an intracellular signaling domain; and

a protease cleavage site disposed between the extracellular binding domain and the intracellular signaling domain.

2. The cell surface receptor of claim 1 , wherein the cell surface receptor is a chimeric antigen receptor (CAR).

3. The cell surface receptor of claim 2, wherein the protease cleavage site is disposed between the extracellular binding domain and the transmembrane domain.

4. The cell surface receptor of claim 2, wherein the protease cleavage site is disposed between the transmembrane domain and the intracellular signaling domain.

5. The cell surface receptor of any one of claims 2 to 4, wherein:

the intracellular signaling domain is a first intracellular signaling domain;

the CAR further comprises a second intracellular signaling domain disposed between the transmembrane domain and the first intracellular signaling domain; and the protease cleavage site is disposed between the second intracellular signaling domain and the first intracellular signaling domain.

6. The cell surface receptor of claim 5, wherein the first and second intracellular signaling domains are independently selected from the group consisting of: a Oϋ3z intracellular signaling domain, a CD28 intracellular signaling domain, a 4-1 BB intracellular signaling domain, an OX-40 intracellular signaling domain, an inducible co-stimulator (ICOS) intracellular signaling domain, an CD27 intracellular signaling domain, and a MyD88/CD40 intracellular signaling domain.

7. The cell surface receptor of claim 6, wherein the first intracellular signaling domain is a Oϋ3z intracellular signaling domain and the second intracellular signaling domain is a CD28 intracellular signaling domain.

8. The cell surface receptor of claim 6, wherein the first intracellular signaling domain is a Oϋ3z intracellular signaling domain and the second intracellular signaling domain is a 4-1 BB intracellular signaling domain.

9. The cell surface receptor of any one of claims 2 to 8, wherein the extracellular binding domain of the CAR comprises a single chain antibody.

10. The cell surface receptor of claim 9, wherein the single chain antibody is a single chain variable fragment (scFv).

1 1 . The cell surface receptor of any one of claims 2 to 10, wherein the extracellular binding domain of the CAR specifically binds an antigen on the surface of a cancer cell.

12. The cell surface receptor of claim 1 1 , wherein the antigen on the surface of the cancer cell is a tumor-associated antigen or a tumor-specific antigen.

13. The cell surface receptor of claim 1 1 , wherein the antigen on the surface of the cancer cell is selected from the group consisting of: B7-H3 (CD276), CD19, GD2, CD22, and HER2.

14. The cell surface receptor of claim 1 , wherein the cell surface receptor is an engineered T cell receptor (TCR).

15. The cell surface receptor of claim 14, wherein the protease cleavage site is disposed between the extracellular binding domain and the transmembrane domain.

16. The cell surface receptor of claim 14, wherein the protease cleavage site is disposed between the transmembrane domain and the intracellular signaling domain.

17. The cell surface receptor of any one claims 14 to 16, wherein the engineered TCR specifically binds a tumor-associated antigen or a tumor-specific antigen.

18. A cell surface receptor comprising:

an extracellular binding domain;

a transmembrane domain;

an intracellular signaling domain; and

a protease cleavage site disposed between the extracellular binding domain and the intracellular signaling domain.

19. The cell surface receptor of claim 18, wherein the protease cleavage site is disposed between the extracellular binding domain and the transmembrane domain.

20. The cell surface receptor of claim 18, wherein the protease cleavage site is disposed between the transmembrane domain and the intracellular signaling domain.

21 . The cell surface receptor of any one of claims 18 to 20, wherein the cell surface receptor is a stem cell receptor.

22. The cell surface receptor of any one of claims 18 to 21 , wherein the cell surface receptor is an immune cell receptor.

23. The cell surface receptor of claim 22, wherein the immune cell receptor is selected from the group consisting of: a T cell receptor, a B cell receptor, a natural killer (NK) cell receptor, a macrophage receptor, a monocyte receptor, a neutrophil receptor, a dendritic cell receptor, a mast cell receptor, a basophil receptor, and an eosinophil receptor.

24. The cell surface receptor of claim 23, wherein the immune cell receptor is a T cell receptor (TCR).

25. The cell surface receptor of claim 24, wherein the protease cleavage site is disposed: between the variable region of the alpha chain (av) and the constant region of the alpha chain (ac); between the constant region of the alpha chain (ac) and the transmembrane region of the alpha chain (at); between the variable region of the beta chain (bn) and the constant region of the beta chain (bo); between the constant region of the beta chain (bo) and the transmembrane region of the beta chain (pt); if a Oϋ3z polypeptide is present, between the transmembrane domain of the Oϋ3z polypeptide and the cytoplasmic domain of the Oϋ3z polypeptide; and any combination thereof.

26. The cell surface receptor of any one of claims 1 to 25, wherein the protease cleavage site is a viral protease cleavage site.

27. The cell surface receptor of claim 26, wherein the viral protease cleavage site is for a viral protease derived from hepatitis C virus (HCV) nonstructural protein 3 (NS3).

28. The cell surface receptor of claim 27, wherein the viral protease further comprises a cofactor polypeptide derived from HCV nonstructural protein 4A (NS4A).

29. The cell surface receptor of any one of claims 26 to 28, wherein the viral protease cleavage site is selected from the group consisting of: an NS4A/4B junction cleavage site, an NS3/NS4A junction cleavage site, an NS4A/NS4B junction cleavage site, an NS4B/NS5A junction cleavage site, an NS5A/NS5B junction cleavage site, and variants thereof cleavable by the viral protease.

30. The cell surface receptor of any one of claims 1 to 29, wherein the cell surface receptor further comprises a protease dimerization domain that dimerizes with a domain of a protease, wherein the protease cleavage site is a cleavage site for the protease.

31 . The cell surface receptor of claim 30, wherein the protease dimerization domain comprises a leucine zipper domain, a helix-loop-helix domain, or both.

32. The cell surface receptor of any one of claims 1 to 29, wherein the cell surface receptor further comprises a protease, and wherein the protease cleavage site is a cleavage site for the protease.

33. A cell comprising the cell surface receptor of any one of claims 1 to 32.

34. The cell of claim 33, wherein the cell is a mammalian cell.

35. The cell of claim 34, wherein the cell is a human cell.

36. The cell of any one of claims 33 to 35, wherein the cell is a stem cell.

37. The cell of claim 36, wherein the stem cell is selected from the group consisting of: a hematopoietic stem cell (HSC), an induced pluripotent stem cell (iPSC), a mesenchymal stem cell (MSC), and a neural stem cell (NSC).

38. The cell of any one of claims 33 to 35, wherein the cell is an immune cell.

39. The cell of claim 38, wherein the immune cell is selected from the group consisting of: a T cell, a B cell, a natural killer (NK) cell, a macrophage, a monocyte, a neutrophil, a dendritic cell, a mast cell, a basophil, and an eosinophil.

40. The cell of claim 38, wherein the immune cell is a T cell.

41 . The cell of claim 40, wherein the cell surface receptor is a CAR.

42. The cell of claim 41 , wherein the cell surface receptor is the CAR of any one of claims

2 to 13.

43. The cell of claim 40, wherein the cell surface receptor is a TCR.

44. The cell of any one of claims 33 to 43, wherein the cell surface receptor further comprises a protease, and wherein the protease cleavage site is a cleavage site for the protease.

45. The cell of any one of claims 33 to 44, wherein the protease cleavage site is disposed between the transmembrane domain and the intracellular signaling domain.

46. The cell of claim 45, wherein the cell further comprises a protease intracellularly tethered to the cell membrane, and wherein the protease cleavage site is a cleavage site for the protease intracellularly tethered to the cell membrane.

47. The cell of claim 46, wherein the protease is intracellularly tethered to the cell membrane via a transmembrane domain.

48. The cell of claim 47, wherein the protease intracellularly tethered to the cell membrane comprises a transmembrane domain of the same type as the transmembrane domain of the cell surface receptor.

49. The cell of claim 48, wherein the protease and cell surface receptor each comprise a CD8a transmembrane domain.

50. The cell of claim 48, wherein the protease and cell surface receptor each comprise a CD28 transmembrane domain.

51 . The cell of any one of claims 48 to 50, wherein the protease and cell surface receptor each further comprise a hinge domain of the same type.

52. The cell of claim 46, wherein the protease comprises a domain that is post-translationally modified with a moiety that tethers the protease to the cell membrane.

53. The cell of claim 52, wherein the domain that is post-translationally modified is a domain that is palmitoylated or myristoylated.

54. The cell of any one of claims 45 to 53, wherein the cell comprises a soluble cytosolic protease, and wherein the protease cleavage site is a cleavage site for the soluble cytosolic protease.

55. The cell of any one of claims 33 to 44, wherein the protease cleavage site is disposed between the extracellular binding domain and the transmembrane domain.

56. The cell of claim 55, wherein the cell further comprises a protease extracellularly tethered to the cell membrane, and wherein the protease cleavage site is a cleavage site for the protease extracellularly tethered to the cell membrane.

57. The cell of claim 56, wherein the protease is extracellularly tethered to the cell membrane via a transmembrane domain.

58. The cell of claim 57, wherein the protease extracellularly tethered to the cell membrane comprises a transmembrane domain of the same type as the transmembrane domain of the cell surface receptor.

59. The cell of claim 56, wherein the protease is extracellularly tethered to the cell membrane via a glycolipid anchor.

60. The cell of claim 59, wherein the glycolipid anchor is a glycosylphosphatidylinositol (GPI) anchor.

61 . The cell of any one of claims 46 to 58, wherein the protease comprises a dimerization domain that dimerizes with a domain of the cell surface receptor to promote association of the protease and cell surface receptor.

62. The cell of claim 61 , wherein the dimerization domain comprises a leucine zipper domain, a helix-loop-helix domain, or both.

63. A pharmaceutical composition, comprising:

the cell of any one of claims 33 to 62; and

a pharmaceutically-acceptable carrier.

64. A nucleic acid encoding the cell surface receptor of any one of claims 1 to 32.

65. An expression vector comprising the nucleic acid of claim 64.

66. A cell comprising the nucleic acid of claim 64 or the expression vector of claim 65.

67. The cell of claim 66, wherein the cell is a mammalian cell.

68. The cell of claim 67, wherein the cell is a human cell.

69. The cell of any one of claims 66 to 68, wherein the cell is an immune cell.

70. The cell of claim 69, wherein the immune cell is selected from the group consisting of: a T cell, a B cell, a natural killer (NK) cell, a macrophage, a monocyte, a neutrophil, a dendritic cell, a mast cell, a basophil, and an eosinophil.

71 . The cell of claim 69, wherein the immune cell is a T cell.

72. The cell of claim 71 , wherein the cell surface receptor is a CAR.

73. The cell of claim 72, wherein the cell surface receptor is the CAR of any one of claims

2 to 13.

74. The cell of claim 71 , wherein the cell surface receptor is a TCR.

75. The cell of any one of claims 66 to 74, wherein the cell surface receptor further comprises a protease, and wherein the protease cleavage site is a cleavage site for the protease of the cell surface receptor.

76. The cell of any one of claims 66 to 75, wherein the protease cleavage site is disposed between the transmembrane domain and the intracellular signaling domain.

77. The cell of claim 76, wherein the cell further comprises a protease tethered intracellularly to the cell membrane of the cell, and wherein the protease cleavage site is a cleavage site for the protease tethered intracellularly to the cell membrane of the cell.

78. The cell of claim 77, wherein the protease is intracellularly tethered to the cell membrane via a transmembrane domain.

79. The cell of claim 78, wherein the protease intracellularly tethered to the cell membrane comprises a transmembrane domain of the same type as the transmembrane domain of the cell surface receptor.

80. The cell of claim 79, wherein the protease and cell surface receptor each comprise a CD8a transmembrane domain.

81 . The cell of claim 79, wherein the protease and cell surface receptor each comprise a CD28 transmembrane domain.

82. The cell of any one of claims 79 to 81 , wherein the protease and cell surface receptor each further comprise a same type of hinge domain.

83. The cell of claim 77, wherein the protease comprises a domain that is post-translationally modified with a moiety that tethers the protease to the cell membrane.

84. The cell of claim 83, wherein the domain that is post-translationally modified is a domain that is palmitoylated or myristoylated.

85. The cell of any one of claims 76 to 84, wherein the cell comprises a soluble cytosolic protease, and wherein the protease cleavage site is a cleavage site for the soluble cytosolic protease.

86. The cell of any one of claims 66 to 75, wherein the protease cleavage site is disposed between the extracellular binding domain and the transmembrane domain.

87. The cell of claim 86, wherein the cell comprises a protease tethered extracellularly to the cell membrane of the cell, and wherein the protease cleavage site is a cleavage site for the protease tethered extracellularly to the cell membrane of the cell.

88. The cell of claim 87, wherein the protease is extracellularly tethered to the cell membrane via a transmembrane domain.

89. The cell of claim 88, wherein the protease extracellularly tethered to the cell membrane comprises a transmembrane domain of the same type as the transmembrane domain of the cell surface receptor.

90. The cell of claim 87, wherein the protease is extracellularly tethered to the cell membrane via a glycolipid anchor.

91 . The cell of claim 90, wherein the glycolipid anchor is a glycosylphosphatidylinositol (GPI) anchor.

92. The cell of any one of claims 77 to 91 , wherein the protease comprises a dimerization domain that dimerizes with a domain of the cell surface receptor to promote association of the protease and cell surface receptor.

93. The cell of claim 92, wherein the dimerization domain comprises a leucine zipper domain, a helix-loop-helix domain, or both.

94. A method of making the cell of any one of claims 66 to 93, comprising introducing the nucleic acid of claim 64 or the expression vector of claim 65 into the cell.

95. A method of making the cell of any one of claims 75 to 93, comprising:

introducing the nucleic acid of claim 64 or the expression vector of claim 65 into the cell, wherein the nucleic acid further encodes the protease.

96. A method of making the cell of any one of claims 75 to 93, comprising:

introducing the nucleic acid of claim 64 or the expression vector of claim 65 into the cell; and

introducing a nucleic acid or expression vector that encodes the protease into the cell.

97. A method for regulating signaling of a cell surface receptor, comprising:

contacting the cell of any one of claims 75 to 93 with an inhibitor of the protease when signaling through the cell surface receptor is desired.

98. The method according to claim 97, wherein the protease is derived from HCV NS3, and wherein the inhibitor of the protease is selected from the group consisting of: asunaprevir (ASV), danoprevir (DPV), simeprevir (SPV), grazoprevir (GPV), and any combination thereof.

99. The method according to claim 97 or claim 98, further comprising ceasing the contacting when signaling through the cell surface receptor is no longer desired.

100. The method according to any one of claims 97 to 99, wherein the method is performed in vitro.

101 . The method according to any one of claims 97 to 99, wherein the method is performed ex vivo.

102. The method according to any one of claims 97 to 99, wherein the method is performed in vivo.

103. A pharmaceutical composition, comprising:

the cell of any one of claims 75 to 93; and

a pharmaceutically-acceptable carrier.

104. A method of making the pharmaceutical composition of claim 103, comprising introducing the expression vector of claim 65 into cells obtained from an individual.

105. A method of administering a regulatable cell-based therapy to an individual in need thereof, comprising administering to the individual the pharmaceutical composition of claim 103.

106. The method according to claim 105, further comprising administering to the individual an inhibitor of the protease when signaling through the cell surface receptor is desired.

107. The method according to claim 106, wherein the inhibitor of the protease is administered concurrently with the pharmaceutical composition.

108. The method according to claim 106 or claim 107, wherein the inhibitor of the protease is administered subsequently to administration of the pharmaceutical composition.

109. The method according to any one of claims 106 to 108, further comprising ceasing administration of the protease inhibitor when signaling through the cell surface receptor is no longer desired.

1 10. The method according to any one of claims 106 to 109, wherein the protease portion of the membrane-tethered protease is derived from HCV NS3, and wherein the inhibitor of the membrane-tethered protease is selected from the group consisting of: asunaprevir (ASV), danoprevir (DPV), simeprevir (SPV), grazoprevir (GPV), and any combination thereof.

1 1 1 . The method according to any one of claims 105 to 1 10, wherein the pharmaceutical composition comprises immune cells comprising the expression vector of claim 65.

1 12. The method according to claim 1 1 1 , wherein the expression vector encodes a protease, wherein the protease cleavage site is a cleavage site for the protease.

1 13. The method according to claim 1 12, wherein the cell surface receptor and the protease are expressed from the same promoter.

1 14. The method according to any one of claims 1 1 1 to 1 13, wherein the immune cells are selected from the group consisting of: T cells, B cells, natural killer (NK) cells, macrophages, monocytes, neutrophils, dendritic cells, mast cells, basophils, and eosinophils.

1 15. The method according to any one of claims 1 1 1 to 1 13, wherein the immune cells are T cells.

1 16. The method according to claim 1 15, wherein the cell surface receptor is a CAR.

1 17. The method according to claim 1 15, wherein the cell surface receptor is a TCR.

1 18. The method according to any one of claims 105 to 1 17, further comprising producing the pharmaceutical composition.

1 19. The method according to claim 1 18, wherein producing the pharmaceutical composition comprises introducing the expression vector of claim 65 into cells or progeny thereof obtained from the individual.

120. The method according to claim 1 19, wherein the expression vector encodes a protease, wherein the protease cleavage site is a cleavage site for the protease.

121 . The method according to claim 1 18, wherein producing the pharmaceutical composition comprises co-introducing the expression vector of claim 65 and an expression vector that encodes the protease into cells or progeny thereof obtained from the individual.

122. The method according to any one of claims 1 18 to 121 , comprising contacting the cells obtained from the individual, or progeny thereof, with an inhibitor of the protease prior to administering the pharmaceutical composition to the individual.

123. A kit, comprising:

the nucleic acid of claim 64 or the expression vector of claim 65; and

instructions for introducing the nucleic acid or expression vector into a cell.

124. The kit of claim 123, wherein the expression vector encodes a protease, and wherein the protease cleavage site is a cleavage site for the protease.

125. The kit of claim 124 wherein the expression vector is configured to express the cell surface receptor and the protease from the same promoter.

126. The kit of claim 123, further comprising a nucleic acid or expression vector that encodes a protease, wherein the protease cleavage site is a cleavage site for the protease.

127. The kit of any one of claims 123 to 126, wherein the instructions further comprise instructions for regulating signaling through the cell surface receptor.

128. The kit of claim 127, wherein the instructions comprise instructions for contacting the cell or progeny thereof with an inhibitor of the protease when signaling through the cell surface receptor is desired.

129. The kit of any one of claims 123 to 128, further comprising an inhibitor of the protease.