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Title:
SYNTHETIC DEGRADER SYSTEM FOR TARGETED PROTEIN DEGRADATION
Document Type and Number:
WIPO Patent Application WO/2022/169913
Kind Code:
A2
Abstract:
A fusion protein is provided having a binding element and a degradation initiator, where the binding element selectively binds a target molecule, and the degradation initiator has a sequence isolated or derived from an E3 ligase. A composition is provided comprising: (a) a first fusion protein comprising a first binding element; and (b) a second fusion protein comprising a second binding element; wherein: (1) the first fusion protein further comprises a degradation initiator or a functional variant thereof and the second fusion protein further comprises a target molecule; or (2) the first fusion protein further comprises a target molecule and the second fusion protein further comprises a degradation initiator or a functional variant thereof. The fusion proteins and compositions may be used for the targeted degradation of endogenous and exogenous proteins, optionally, in a cell or in vivo, for the treatment or prevention of a disease or disorder.

Inventors:
MOFFETT HOWELL (US)
NGUYEN DUY (US)
LANGAN ROBERT (US)
BOYKEN SCOTT (US)
LAJOIE MARC (US)
FOIGHT GLENNA (US)
Application Number:
PCT/US2022/014998
Publication Date:
August 11, 2022
Filing Date:
February 02, 2022
Export Citation:
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Assignee:
OUTPACE BIO INC (US)
International Classes:
C12N9/10; A61P35/00; C07K14/705; C07K14/725; C12N15/52; C12N15/62
Domestic Patent References:
WO2020117778A22020-06-11
Foreign References:
US20140242701A12014-08-28
Other References:
EUROPEAN BIOINFORMATICS INSTITUTE (EBI, Retrieved from the Internet
U.S. GOVERNMENT'S NATIONAL CENTER FOR BIOTECHNOLOGY INFORMATION BLAST, Retrieved from the Internet
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ZHU, L. ET AL., ELIFE, 2017
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GREEN ET AL.: "Cold Spring Harbor", 2014, COLD SPRING HARBOR LABORATORY PRESS, article "Molecular cloning: A laboratory manual"
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GAIDUKOV, L ET AL., NUCLEIC ACIDS RES, vol. 46, 2018, pages 4072 - 4086
KOZIK, P. ET AL., TRAFFIC, 2010
Attorney, Agent or Firm:
MAYER, Mika, R. (US)
Download PDF:
Claims:
Claims

1. A fusion protein comprising a binding element and a degradation initiator, wherein the binding element selectively binds a target molecule and wherein the degradation initiator comprises a sequence isolated or derived from an E3 ligase.

2. The fusion protein of claim 1 , wherein

(a) an E3 ligase comprises LNX1, RNF4, RNF43, RNF128, XNRF3, MARCH8, LRG1, NEDD4, SOCS2, CHIP, SPOP, FBXW7, FBXW1A, ELOC, TRAF6, VHL or any functional fragment thereof; or

(b) an E3 ligase comprises LNX1, RNF4, RNF43 or any functional fragment thereof; or

(c) an E3 ligase comprises LNX1 or any functional fragment thereof; or

(d) an E3 ligase comprises RNF4 or any functional fragment thereof; or

(e) an E3 ligase comprises RNF43 or any functional fragment thereof; or

(1) the sequence isolated or derived from an E3 ligase comprises a sequence of Table 10, a functional fragment thereof, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% identity to the sequence of Table 10 or the functional fragment thereof; or

(g) the sequence isolated or derived from an E3 ligase comprises the sequence of the E3 ligase of SEQ ID NOs: 74-114; or

(h) the sequence isolated or derived from an E3 ligase comprises any one of SEQ ID NOs: 159-163; or

(i) an E3 ligase comprising a motif of Table 14.

3. The fusion protein of claim 1 or 2, wherein, in a cell capable of expressing the fusion protein, the target molecule is an endogenous molecule.

4. The fusion protein of claim 3, wherein the target molecule is a naturally-occurring molecule.

5. The fusion protein of any one of claims 1-4, wherein the binding element comprises a DNA sequence, an RNA sequence, an amino acid sequence, or any combination thereof.

6. The fusion protein of claim 5, wherein a sequence of the binding element forms a nucleic acid duplex with a sequence of the target molecule.

7. The fusion protein of claim 6, wherein the binding element selectively binds to an epitope of the target molecule.

8. The fusion protein of claim 6 or 7, wherein the target molecule comprises a sequence in the file “016-TNP023PCT_SeqList_v2”, created on 2 February 2022 and having a size of 52,267 kilobytes, hereby incorporated by reference in its entirety, or a nucleic acid sequence encoding the target molecule.

9. The fusion protein of any one of claims 1-8, wherein the fusion protein or the binding element comprises a dimerization domain.

10. The fusion protein of claim 9, wherein the dimerization domain comprises a designed heterodimer (DHD) polypeptide.

11. The fusion protein of any one of claims 1-10, wherein the target molecule or a sequence encoding the target molecule is modified

(a) to comprise a binding element capable of forming a heterodimer with the dimerization domain of the fusion protein; or

(b) be operably-linked to a binding element capable of forming a heterodimer with the dimerization domain of the fusion protein.

12. The fusion protein of claim 11, wherein the binding element comprises a dimerization domain.

13. The fusion protein of claim 12, wherein the dimerization domain comprises a designed heterodimer (DHD) polypeptide.

14. The fusion protein of any one of claims 11-13, wherein the binding element of the fusion protein is a first binding element and the binding element of the target molecule is a second binding element, and (a) wherein the first binding element or the second binding element comprises a single helix; or

(b) wherein the first binding element or the second binding element comprises at least two-helices; or

(c) wherein the first binding element or the second binding element comprises 3,

4, 5, 6, 7, or 8 helices; or

(d) the first binding element comprises a single helix and the second binding element comprises three helices; or

(e) the first binding element comprises three helices and the second binding element comprises a single helix.

15. The fusion protein of any one of claims 11-13, wherein:

(a) the first binding element comprises a DHD-A and the second binding element comprises a DHD-B; or

(b) the first binding element comprises a DHD-B and the second binding element comprises a DHD-A.

16. The fusion protein of claim 15, wherein the DHD-B comprises a sequence of DHD37- short-B-KtoR (SEQ ID NO: 16).

17. The fusion protein of claim 11-16, wherein the first binding element or the second binding element comprises a non-helical element.

18. The fusion protein of claim 11-17, wherein the heterodimer comprises a non-helical element.

19. The fusion protein of claim 17 or 18, wherein the non-helical element comprises a small molecule.

20. A composition comprising:

(a) a first fusion protein comprising a first binding element; and

(b) a second fusion protein comprising a second binding element; wherein:

(1) the first fusion protein further comprises a degradation initiator or a functional variant thereof and the second fusion protein further comprises a target molecule; or (2) the first fusion protein further comprises a target molecule and the second fusion protein further comprises a degradation initiator or a functional variant thereof.

21. The composition of claim 20, wherein the first binding element and the second binding element are capable of forming a heterodimer.

22. The composition of claim 21 or 22, wherein the first binding element or the second binding element comprises a single helix.

23. The composition of claim 21 or 22, wherein the first binding element or the second binding element comprises at least two-helices.

24. The composition of claim 21 or 22, wherein the first binding element or the second binding element comprises 3, 4, 5, 6, 7, or 8 helices.

25. The composition of claim 21 or 22, wherein the first binding element comprises a single helix and the second binding element comprises three helices, or the first binding element comprises three helices and the second binding element comprises a single helix.

26. The composition of claim 21 or 22, wherein:

(c) the first binding element comprises a DHD-A and the second binding element comprises a DHD-B; or

(d) the first binding element comprises a DHD-B and the second binding element comprises a DHD-A.

27. The composition of any one of claims 21-26, wherein the first binding element or the second binding element comprises a non-helical element.

28. The composition of any one of claims 21-27, wherein the heterodimer comprises a non-helical element.

29. The composition of claim 27 or 28, wherein the non-helical element comprises a small molecule.

30. The composition of any one of claims 20-29, wherein the first binding element comprises a sequence of DHD37-short-B-KtoR (SEQ ID NO: 16).

31. The composition of claim 30, wherein the first binding element further comprises a sequence isolated or derived from an E3 ligase.

32. The composition of claim 31, wherein

(j) an E3 ligase comprises LNX1, RNF4, RNF43, RNF128, XNRF3, MARCH8, LRG1, NEDD4, S0CS2, CHIP, SPOP, FBXW7, FBXW1A, ELOC, TRAF6, VHL or any functional fragment thereof; or

(k) an E3 ligase comprises LNX1, RNF4, RNF43 or any functional fragment thereof; or

(l) an E3 ligase comprises LNX1 or any functional fragment thereof; or

(m)an E3 ligase comprises RNF4 or any functional fragment thereof; or

(n) an E3 ligase comprises RNF43 or any functional fragment thereof; or

(o) the sequence isolated or derived from an E3 ligase comprises a sequence of Table 10, a functional fragment thereof, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% identity to the sequence of Table 10 or the functional fragment thereof; or

(p) the sequence isolated or derived from an E3 ligase comprises the sequence of the E3 ligase of SEQ ID NOs: 74-114; or

(q) the sequence isolated or derived from an E3 ligase comprises any one of SEQ ID NOs: 159-163; or

(r) an E3 ligase comprising a motif of Table 14.

33. The composition of any one of claims 20-32, wherein the first binding element comprises a sequence of SEQ ID NO: 174.

34. The composition of any one of claims 20-29, wherein the first binding element comprises a sequence of any one of SEQ ID NOs: 1-73, or 121-125.

35. The composition of any one of claims 20-29, wherein the second binding element comprises a sequence of DHD37-short-B-KtoR (SEQ ID NO: 16).

36. The composition of claim 35, wherein the first binding element further comprises a sequence isolated or derived from an E3 ligase.

37. The composition of claim 36, wherein the sequence isolated or derived from an E3 ligase comprises any one of SEQ ID NOs: 159-163.

38. The composition of any one of claims 20-29 or 35-37, wherein the first binding element comprises a sequence of SEQ ID NO: 174.

39. The composition of any one of claims 20-29, wherein the second binding element comprises a sequence of any one of SEQ ID NOs: 1-73, or 121-125.

40. The composition of any one of claims 21-29, wherein a small molecule mediates formation of the heterodimer.

41. The composition of claim 40, wherein the first binding element or the second binding element binds the small molecule.

42. The composition of claim 40, wherein the first binding element and the second binding element bind the small molecule.

43. The composition of claim 41 or 42, wherein the first binding element and the second binding element do not directly bind each other.

44. The composition of any one of claims 40-43, wherein the small molecule increases formation of the heterodimer.

45. The composition of any one of claims 40-43, wherein the small molecule decreases formation of the heterodimer.

46. The composition of any one of claims 40-43, wherein a first small molecule and a second small molecule mediate formation of the heterodimer and wherein the second small molecule decreases formation of the heterodimer with the first small molecule by out- competing the first small molecule for binding either the first binding element or the second binding element.

47. The composition of any one of claims 21-29 or 40-46, wherein:

(a) the first binding element comprises an NS3a sequence and the second binding element comprises a DNCR2 sequence or a GNCR1 sequence; or

(b) the first binding element comprises a DNCR2 sequence or a GNCR1 sequence and the second binding element comprises an NS3a sequence.

48. The composition of any one of claims 29 or 40-47, wherein the small molecule comprises danoprevir or an analog thereof.

49. The composition of claim 48, wherein the first small molecule or the second small molecule comprises danoprevir or an analog thereof.

50. The composition of any one of claims 29 or 40-47, wherein the small molecule comprises grazoprevir or an analog thereof.

51. The composition of claim 50, wherein the first small molecule or the second small molecule comprises grazoprevir or an analog thereof.

52. The composition of any one of claims 20-51, wherein the target molecule comprises a synthetic molecule or an exogenous molecule.

53. The composition of claim 52, wherein the synthetic molecule or the exogenous molecule comprises a protein.

54. The composition of claim 52, wherein the synthetic molecule or the exogenous molecule comprises a chimeric protein, a chimeric receptor, or a chimeric antigen receptor.

55. The composition of claim 54, wherein the chimeric receptor comprises an extracellular domain comprising an antigen sensing domain, a transmembrane domain, and an intracellular domain.

56. The composition of claim 55, wherein the antigen sensing domain comprises one or more of

(a) a set of three complementarity determining regions (CDRs) of a heavy chain variable region;

(b) a set of three complementarity determining regions (CDRs) of a heavy chain variable region and a set of three complementarity determining regions (CDRs) of a light chain variable region;

(c) a fibronectin-protein based scaffold; wherein the antigen sensing region specifically binds a target antigen.

57. The composition of claim 55 or 56, wherein the antigen sensing region comprises one or more sequences isolated or derived from a mammalian sequence.

58. The composition of any one of claims 55-57, wherein the antigen sensing region comprises one or more sequences isolated or derived from a human sequence.

59. The composition of any one of claims 55-58, wherein the antigen sensing region comprises a humanized or fully human antibody.

60. The composition of any one of claims 55-59, wherein the antigen sensing region comprises a single chain variable fragment (scFv).

61. The composition of any one of claims 55-60, wherein the extracellular domain further comprises one or more of a hinge region, a spacer sequence, or a safety switch.

62. The composition of claim 61, wherein the hinge region comprises a sequence isolated or derived from a CD4 (cluster of differentiation 4) polypeptide, a CD8 (cluster of differentiation 8) polypeptide or a CD28 (cluster of differentiation 28) polypeptide.

63. The composition of claim 62, wherein the hinge region comprises a sequence isolated or derived from a human sequence.

64. The composition of claim 61, wherein the spacer sequence comprises a sequence isolated or derived from a CD4 polypeptide, a CD8 polypeptide, a CD28 polypeptide.

65. The composition of claim 64, wherein the spacer sequence comprises a sequence isolated or derived from a human sequence.

66. The composition of claim 61, wherein the safety switch comprises a sequence isolated or derived from an epidermal growth factor receptor (EGFR) polypeptide.

67. The composition of claim 66, wherein the safety switch comprises a truncated EGFR (EGFRt) polypeptide.

68. The composition of claim 66 or 67, wherein the safety switch comprises a sequence isolated or derived from a human sequence.

69. The composition of any one of claims 55-68, wherein the transmembrane domain comprises a sequence isolated or derived from a CD4 polypeptide, a CD8 polypeptide, a CD28 polypeptide.

70. The composition of claim 69, wherein the transmembrane domain comprises a sequence isolated or derived from a human sequence.

71. The composition of any one of claims 55-70, wherein the intracellular domain comprises one or more costimulatory domain(s).

72. The composition of claim 71, wherein the one or more costimulatory domain(s) comprises a sequence isolated or derived from a CD3^ (cluster of differentiation 3 zeta) polypeptide.

73. The composition of claim 71 or 72, wherein the one or more costimulatory domain(s) comprises a sequence isolated or derived from a CD28 polypeptide, a 4- IBB (cluster of differentiation 137) polypeptide, an ICOS (Inducible T Cell Costimulator) polypeptide, an 0X40 polypeptide, or a CD27 (cluster of differentiation 27) polypeptide.

74. The composition of claim 71 or 72, wherein the one or more costimulatory domain(s) comprises

(a) a first costimulatory domain comprising sequence isolated or derived from a CD28 polypeptide, a 4-1BB polypeptide, or an ICOS polypeptide; and

(b) a second costimulatory domain comprising sequence isolated or derived from a 4-lBB polypeptide, an 0X40 polypeptide, or a CD27 polypeptide.

75. The composition of any one of claims 71-74, wherein the one or more costimulatory domain(s) comprise(s) a sequence isolated or derived from a human sequence.

76. The composition of any one of claims 55-75, wherein the chimeric receptor comprises an intracellular domain further comprising an inducible cytokine domain.

77. The composition of claim 76, wherein the inducible cytokine domain comprises a nuclear factor of activated T-cells (NF AT) polypeptide capable of inducing expression of an IL- 12 cytokine.

78. The composition of any one of claims 55-75, wherein the chimeric receptor comprises an intracellular domain further comprising an intracellular domain of a cytokine receptor.

79. The composition of claim 78, wherein the intracellular domain of a cytokine receptor comprises an IL-2 receptor beta (IL-2RP) chain fragment.

80. The composition of claim 78 or 79, wherein the chimeric receptor comprises an intracellular domain further comprising a Signal Transducer and Activator of Transcription (STAT3/5) binding motif.

81. The composition of any one of claims 55-80, wherein the chimeric receptor comprises an intracellular domain further comprising at least one immunoreceptor tyrosine-based activation motif (ITAM) sequence.

82. A nucleic acid sequence encoding

(a) a fusion protein of any one of claims 1-19 or

(b) one or more elements of (a).

83. A nucleic acid sequence encoding

(a) a first fusion protein of any one of claims 20-81 ;or

(b) a second fusion protein of any one of claims 20-81; or

(c) one or more elements of (a); or

(d) one or more elements of (b).

84. A nucleic acid sequence encoding a first fusion protein and a second fusion protein of any one of claims 20-81.

85. The nucleic acid sequence of any one of claims 82-84, further comprising one or more of a non-coding sequence, an untranslated region, a regulatory element, a separation element, a polycistronic element or a post-translational element.

86. The nucleic acid sequence of any one of claims 82-84, further comprising at least one promoter capable of driving expression of the nucleic acid sequence in a mammalian cell.

87. The nucleic acid sequence of any one of claims 82-84, further comprising at least one promoter capable of driving expression of the nucleic acid sequence in a human cell.

88. The nucleic acid sequence of claim 86 or 87, wherein at least one promoter comprises a constitutive promoter.

89. The nucleic acid sequence of claim 88, wherein the constitutive promoter comprises a sequence isolated or derived from one or more of a MND promoter, a hPGK promoter, a CMV promoter, a CAG promoter, a SFFV promoter, an EFl alpha promoter, a UBC promoter, and a CD43 promoter.

90. The nucleic acid sequence of claim 86 or 87, wherein at least one promoter comprises an inducible promoter.

91. The nucleic acid sequence of claim 90, wherein the inducible promoter comprises a sequence isolated or derived from one or more of a YB TATA promoter, a human beta globin (huBG) promoter, a minIL2 promoter, a minimalCMV (minCMV) promoter, and a TRE3G promoter.

92. The nucleic acid sequence of claim 90 or 91, wherein the inducible promoter comprises a minimal sequence.

93. The nucleic acid sequence of any one of claims 90-92, wherein the inducible promoter further comprises a transcription factor-specific recognition sequence.

94. The nucleic acid sequence of claim 93, wherein the transcription factor-specific recognition sequence comprises a transcription factor-specific response element.

95. The nucleic acid sequence of claim 41, wherein the transcription factor response element comprises a sequence isolated or derived from an NF AT sequence.

96. The nucleic acid sequence of any one of claims 93-95, wherein the transcription factor-specific recognition sequence comprises at least one repeat of a transcription factorspecific response element.

97. The nucleic acid sequence of any one of claims 93-95, wherein the transcription factor-specific recognition sequence comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeats of a transcription factor-specific response element.

98. The nucleic acid sequence of any one of claims 86-97, wherein a first promoter drives expression of the first fusion protein; or a second promoter drives expression of the second fusion protein.

99. The nucleic acid sequence of claim 98, wherein the first promoter and the second promoter are identical.

100. The nucleic acid sequence of claim 98, wherein the first promoter and the second promoter are not identical.

101. The nucleic acid sequence of any one of claims 85-100, wherein the separation element comprises a ribosomal skipping sequence.

102. The nucleic acid sequence of claim 101, wherein the separation element comprises at least two ribosomal skipping sequences.

103. The nucleic acid sequence of claim 101 or 102, wherein the ribosomal skipping sequence comprises a P2a sequence or a T2a sequence.

104. The nucleic acid sequence of any one of claims 101-103, wherein the ribosomal skipping sequence comprises a T2a-RFP-P2a sequence, a P2a-T2a sequence, or a T2a-P2a sequence.

105. The nucleic acid sequence of any one of claims 85-100, wherein the polycistronic element comprises an internal ribosome entry site (IRES) sequence.

106. A vector comprising a nucleic acid of any one of claims 82-105.

107. A first vector comprising a nucleic acid encoding the first fusion protein of any one of claims 20-81 and a second vector comprising a nucleic acid encoding the second fusion protein of any one of claims 20-81.

108. The vector of claim 106 or 107, wherein the vector is an expression vector capable of expressing a nucleic acid in a mammalian cell.

109. The vector of claim 108, wherein the vector comprises a plasmid.

110. The vector of claim 106 or 107, wherein the vector comprises a delivery vector capable of introducing a nucleic acid to a mammalian cell.

111. The vector of claim 110, wherein the delivery vector comprises one or more of a viral vector, a non-viral vector, a liposome, a micelle, a polymersome, and a nanoparticle.

112. The vector of claim 111, wherein the viral vector comprises a sequence isolated or derived from a virus or a viral vector.

113. The vector of claim 112, wherein the viral vector comprises a sequence isolated or derived from one or more of an adenoviral vector, a lentiviral vector, a baculoviral vector, an Epstein Barr viral vector, a papovaviral (parvovirus) vector, a vaccinia viral vector, a herpes simplex viral vector, an adeno associated virus (AAV) vector, and a hepatitis B viral vector.

114. The vector of any one of claims 106-110, wherein the vector comprises a sequence isolated or derived from a transposition system.

115. The vector of claim 114, wherein the vector comprises a sequence isolated or derived from one or more of a piggy BAC transposition system, a Sleeping Beauty transposition system, a Tcl/mariner transposition system, a Tol2 transposition system, ahelraiser transposition system and a Tn7 transposition system.

116. The vector of any one of claims 106-115, wherein the vector comprises one or more sequences mediating homology directed repair.

117. A cell comprising the fusion protein of any one of claims 1-19.

118. A cell comprising the composition of any one of claims 20-81.

119. A cell comprising the nucleic acid sequence of any one of claims 82-105.

120. A cell comprising the vector of any one of claims 106-116.

121. The cell of any one of claims 117-120, wherein the cell stably expresses the fusion protein of any one of claims 1-19.

122. The cell of any one of claims 117-120, wherein the cell stably expresses the first fusion protein or the second fusion protein of any one of claims 20-81.

123. The cell of any one of claims 117-122, wherein the cell is a eukaryotic cell.

124. The cell of any one of claims 117-122, wherein the cell is a mammalian cell.

125. The cell of any one of claims 117-122, wherein the cell is a human cell.

126. The cell of any one of claims 117-125, wherein the cell is a stem cell.

127. The cell of any one of claims 117-126, wherein the stem cell is a hematopoietic cell.

128. The cell of any one of claims 117-125, wherein the stem cell is a mesenchymal cell.

129. The cell of claim 127 or 128, wherein the cell is an immune cell.

130. The cell of claim 129, wherein the immune cell is a T-cell, a Natural Killer (NK) cell, or an Innate Lymphoid Cell (ILC).

131. A composition comprising

(a) a fusion protein of any one of claims 1-19; or

(b) a composition of any one of claims 20-81; or

(c) a nucleic acid of any one of claims 82-105; or

(d) a vector of any one of claims 106-116; or

(e) a cell of any one of claims 117-130.

132. A pharmaceutical composition comprising a composition of claim 131 and a pharmaceutically-acceptable carrier.

132. The use of a fusion protein of any one of claims 1-19, a composition of any one of claims 20-81 or 131; a nucleic acid sequence of any one of claims 82-105, a vector of any one of claims 106-116, a cell of any one of claims 117-130 or a pharmaceutical composition of claim 132 in the manufacture of a medicament for the treatment of a disease or a disorder.

133. The use of a fusion protein of any one of claims 1-19, a composition of any one of claims 20-81 or 131; a nucleic acid sequence of any one of claims 82-105, a vector of any one of claims 106-116, a cell of any one of claims 117-130 or a pharmaceutical composition of claim 132 for the treatment of a disease or a disorder.

134. The use of claim 132 or 133, wherein the disease or disorder comprises one or more of an autoimmune disease or disorder; an inflammatory disease or disorder; an immunodeficiency disease or disorder; an ischemic disease or disorder; a blood disease or disorder; a bone disease or disorder; a neurological disease or disorder; a cardiac disease or disorder; a vascular disease or disorder; a metabolic disease or disorder; a dermatological disease or disorder; a digestive disease or disorder; a mitochondrial disease or disorder; a muscle disease or disorder; a liver disease or disorder; a kidney disease or disorder; a hearing disease or disorder; an ophthalmic disease or disorder; and a proliferative disease or disorder.

135. The use of claim 132 or 133, wherein the disease or disorder comprises a cancer.

136. The use of any one of claims 132-135, wherein the disease or disorder comprises an infection or a disease or disorder caused by the infectious disease.

137. The use of any one of claims 132-135, wherein the disease or disorder comprises a genetic disease or disorder.

138. A method of treating a disease or a disorder, comprising administering to a subject an effective amount of a fusion protein of any one of claims 1-19, a composition of any one of claims 20-81 or 131; a nucleic acid sequence of any one of claims 82-105, a vector of any one of claims 106-116, a cell of any one of claims 117-130 or a pharmaceutical composition of claim 132, wherein a severity of a sign or symptom of the disease or disorder is decreased, thereby treating the disease or disorder.

139. A method of preventing a disease or a disorder, comprising administering to a subject an effective amount of a fusion protein of any one of claims 1-19, a composition of any one of claims 20-81 or 131; a nucleic acid sequence of any one of claims 82-105, a vector of any one of claims 106-116, a cell of any one of claims 117-130 or a pharmaceutical composition of claim 132, wherein an onset or a relapse of a sign or symptom of the disease or disorder is delayed or inhibited, thereby preventing the disease or disorder.

140. The method of claim 138 or 139, wherein the disease or disorder comprises one or more of an autoimmune disease or disorder; an inflammatory disease or disorder; an immunodeficiency disease or disorder; an ischemic disease or disorder; a blood disease or disorder; a bone disease or disorder; a neurological disease or disorder; a cardiac disease or disorder; a vascular disease or disorder; a metabolic disease or disorder; a dermatological disease or disorder; a digestive disease or disorder; a mitochondrial disease or disorder; a muscle disease or disorder; a liver disease or disorder; a kidney disease or disorder; a hearing disease or disorder; an ophthalmic disease or disorder; and a proliferative disease or disorder.

141. The method of claim 138 or 139, wherein the disease or disorder comprises a cancer.

142. The method of any one of claims 138-141, wherein the disease or disorder comprises an infection or a disease or disorder caused by the infectious disease.

143. The method of any one of claims 138-141, wherein the disease or disorder comprises a genetic disease or disorder.

Description:
Synthetic Degrader System for Targeted Protein Degradation

Cross-reference to Related Applications

[0001] This application claims priority from U.S. Provisional Application Nos. US63/144895, filed February 2, 2021, and US63/248516, filed September 26, 2021. The entire contents of each of the prior applications are incorporated by reference herein.

Incorporation of the Sequence Listing

[0002] The Sequence Listing submitted 2 February 2022 as a text file named “016- TNP023PCT_SeqList_v2”, created on 2 February 2022 and having a size of 52,267 kilobytes, is hereby incorporated by reference in its entirety.

Field of the Disclosure

[0003] The disclosure relates to synthetic degrader systems for targeted degradation of a molecule of interest. The degrader systems include a degradation initiator fused to a binding element, wherein the binding element is used to recruit the degradation initiator to the molecule of interest to initiate degradation.

Background

[0004] Protein degradation is an essential cellular process that provides a mechanism of protein quality control and rapid response to a large number of cellular signals. Within cells, the proper coordination and synchronization of protein functions is controlled by their degradation in a spatial and temporal order. To develop next-generation cell and gene therapies that are efficacious and safe, there is an increasing need for technologies that enable precise control of protein stability and half-lives to regulate cellular processes.

Summary

[0005] The disclosure provides a fusion protein comprising a binding element and a degradation initiator, wherein the binding element selectively binds a target molecule and wherein the degradation initiator comprises a sequence isolated or derived from an E3 ligase. In some embodiments, (a) an E3 ligase comprises LNX1, RNF4, RNF43, RNF128, XNRF3, MARCH8, LRG1, NEDD4, SOCS2, CHIP, SPOP, FBXW7, FBXW1A, ELOC, TRAF6, VHL or any functional fragment thereof; or (b) an E3 ligase comprises LNX1, RNF4, RNF43 or any functional fragment thereof; or (c) an E3 ligase comprises LNX1 or any functional fragment thereof; or (d) an E3 ligase comprises RNF4 or any functional fragment thereof; or (e) an E3 ligase comprises RNF43 or any functional fragment thereof; or (f) the sequence isolated or derived from an E3 ligase comprises a sequence of Table 10, a functional fragment thereof, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% identity to the sequence of Table 10 or the functional fragment thereof; or (g) the sequence isolated or derived from an E3 ligase comprises the sequence of the E3 ligase of SEQ ID NOs: 74-114; or (h) the sequence isolated or derived from an E3 ligase comprises any one of SEQ ID NOs: 159-163; or (i) an E3 ligase comprising a motif of Table 14. In some embodiments, the fusion protein further comprises a DAP 10 sequence, optionally comprising a tag or the sequence of SEQ ID NO: 157. In some embodiments, the fusion protein further comprises a CD8 transmembrane domain, optionally, comprising the sequence of SEQ ID NO: 158. In some embodiments, in a cell capable of expressing the fusion protein, the target molecule is an endogenous molecule. In some embodiments, the target molecule is a naturally-occurring molecule. In some embodiments, the binding element comprises a DNA sequence, an RNA sequence, an amino acid sequence, or any combination thereof. In some embodiments, a sequence of the binding element forms a nucleic acid duplex with a sequence of the target molecule. In some embodiments, a sequence of the binding element forms a nucleic acid duplex with a sequence of the target molecule by hybridization of a DNA or RNA sequence of the binding element with a DNA or RNA sequence of the target molecule. In some embodiments, the binding element selectively binds to an epitope of the target molecule. In some embodiments, the binding element comprises an antibody or a functional fragment thereof, an antibody mimetic, a fibronectin domain, a protein scaffold, or an aptamer. In some embodiments, the target molecule comprises a sequence in the sequence listing file “016-TNP023PCT_SeqList_v2”, created on 2 February 2022 and having a size of 52,267 kilobytes, which is hereby incorporated by reference in its entirety, or a nucleic acid sequence encoding the target molecule.

[0006] In some embodiments of the fusion proteins of the disclosure, the fusion protein or the binding element comprises a dimerization domain. In some embodiments, the dimerization domain comprises a designed heterodimer (DHD) polypeptide. In some embodiments, the target molecule or a sequence encoding the target molecule is modified (a) to comprise a binding element capable of forming a heterodimer with the dimerization domain of the fusion protein; or (b) be operably -linked to a binding element capable of forming a heterodimer with the dimerization domain of the fusion protein. In some embodiments, the binding element comprises a dimerization domain. In some embodiments, the dimerization domain comprises a designed heterodimer (DHD) polypeptide. In some embodiments, the binding element of the fusion protein is a first binding element and the binding element of the target molecule is a second binding element, and (a) wherein the first binding element or the second binding element comprises a single helix; or (b) wherein the first binding element or the second binding element comprises at least two-helices; or (c) wherein the first binding element or the second binding element comprises 3, 4, 5, 6, 7, or 8 helices; or (d) the first binding element comprises a single helix and the second binding element comprises three helices; or (e) the first binding element comprises three helices and the second binding element comprises a single helix. In some embodiments, (a) the first binding element comprises a DHD-A and the second binding element comprises a DHD-B; or (b) the first binding element comprises a DHD-B and the second binding element comprises a DHD-A. In some embodiments, the DHD-B comprises a sequence of DHD37- short-B-KtoR (SEQ ID NO: 16). In some embodiments, the first binding element or the second binding element comprises a non-helical element. In some embodiments, the heterodimer comprises a non-helical element. In some embodiments, the non-helical element comprises a small molecule.

[0007] The disclosure provides a composition comprising: (a) a first fusion protein comprising a first binding element; and (b) a second fusion protein comprising a second binding element;

[0008] wherein: (1) the first fusion protein further comprises a degradation initiator or a functional variant thereof and the second fusion protein further comprises a target molecule; or (2) the first fusion protein further comprises a target molecule and the second fusion protein further comprises a degradation initiator or a functional variant thereof.

[0009] In some embodiments of the compositions of the disclosure, the first binding element and the second binding element are capable of forming a heterodimer. In some embodiments, the first binding element or the second binding element comprises a single helix. In some embodiments, the first binding element or the second binding element comprises at least two- helices. In some embodiments, the first binding element or the second binding element comprises 3, 4, 5, 6, 7, or 8 helices. In some embodiments, (a) the first binding element comprises a single helix and the second binding element comprises three helices, or (b) the first binding element comprises three helices and the second binding element comprises a single helix. In some embodiments, (a) the first binding element comprises a DHD-A and the second binding element comprises a DHD-B; or (b) the first binding element comprises a DHD-B and the second binding element comprises a DHD-A. In some embodiments, the first binding element or the second binding element comprises a non-helical element. In some embodiments, the heterodimer comprises a non-helical element. In some embodiments, the non-helical element comprises a small molecule.

[0010] In some embodiments of the compositions of the disclosure, the first binding element comprises a sequence of DHD37-short-B-KtoR (SEQ ID NO: 16). In some embodiments, the first binding element further comprises a sequence isolated or derived from an E3 ligase. In some embodiments, (a) an E3 ligase comprises LNX1, RNF4, RNF43, RNF128, XNRF3, MARCH8, LRG1, NEDD4, S0CS2, CHIP, SPOP, FBXW7, FBXW1A, ELOC, TRAF6, VHL or any functional fragment thereof; or (b) an E3 ligase comprises LNX1, RNF4, RNF43 or any functional fragment thereof; or (c) an E3 ligase comprises LNX1 or any functional fragment thereof; or (d) an E3 ligase comprises RNF4 or any functional fragment thereof; or (e) an E3 ligase comprises RNF43 or any functional fragment thereof; or (I) the sequence isolated or derived from an E3 ligase comprises a sequence of Table 10, a functional fragment thereof, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% identity to the sequence of Table 10 or the functional fragment thereof; or (g) the sequence isolated or derived from an E3 ligase comprises the sequence of the E3 ligase of SEQ ID NOs: 74-114; or (h) the sequence isolated or derived from an E3 ligase comprises any one of SEQ ID NOs: 159-163; or (i) an E3 ligase comprising a motif of Table 14. In some embodiments, the first fusion protein further comprises a DAP 10 sequence, optionally comprising a tag or the sequence of SEQ ID NO: 157. In some embodiments, the first fusion protein further comprises a CD8 transmembrane domain, optionally, comprising the sequence of SEQ ID NO: 158. In some embodiments, the first binding element comprises a sequence of SEQ ID NO: 174.

[0011] In some embodiments of the compositions of the disclosure, the first binding element comprises a sequence of any one of SEQ ID NOs: 1-73, or 121-125.

[0012] In some embodiments of the compositions of the disclosure, the second binding element comprises a sequence of DHD37-short-B-KtoR (SEQ ID NO: 16). In some embodiments, the first binding element further comprises a sequence isolated or derived from an E3 ligase. In some embodiments, (a) an E3 ligase comprises LNX1, RNF4, RNF43, RNF128, XNRF3, MARCH8, LRG1, NEDD4, S0CS2, CHIP, SPOP, FBXW7, FBXW1A, ELOC, TRAF6, VHL or any functional fragment thereof; or (b) an E3 ligase comprises LNX1, RNF4, RNF43 or any functional fragment thereof; or (c) an E3 ligase comprises LNX1 or any functional fragment thereof; or (d) an E3 ligase comprises RNF4 or any functional fragment thereof; or (e) an E3 ligase comprises RNF43 or any functional fragment thereof; or (I) the sequence isolated or derived from an E3 ligase comprises a sequence of Table 10, a functional fragment thereof, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% identity to the sequence of Table 10 or the functional fragment thereof; or (g) the sequence isolated or derived from an E3 ligase comprises the sequence of the E3 ligase of SEQ ID NOs: 74-114; or (h) the sequence isolated or derived from an E3 ligase comprises any one of SEQ ID NOs: 159-163; or (i) an E3 ligase comprising a motif of Table 14. In some embodiments, the second fusion protein further comprises a DAP 10 sequence, optionally comprising a tag or the sequence of SEQ ID NO: 157. In some embodiments, the second fusion protein further comprises a CD8 transmembrane domain, optionally, comprising the sequence of SEQ ID NO: 158. In some embodiments, the first binding element comprises a sequence of SEQ ID NO: 174.

[0013] In some embodiments of the compositions of the disclosure, the second binding element comprises a sequence of any one of SEQ ID NOs: 1-73, or 121-125.

[0014] In some embodiments of the compositions of the disclosure, a small molecule mediates formation of the heterodimer. In some embodiments, the first binding element or the second binding element binds the small molecule. In some embodiments, the first binding element and the second binding element bind the small molecule. In some embodiments, the first binding element and the second binding element do not directly bind each other.In some embodiments, the small molecule increases formation of the heterodimer. In some embodiments, the small molecule decreases formation of the heterodimer. In some embodiments, a first small molecule and a second small molecule mediate formation of the heterodimer and wherein the second small molecule decreases formation of the heterodimer with the first small molecule by out-competing the first small molecule for binding either the first binding element or the second binding element. In some embodiments, (a) the first binding element comprises an NS3a sequence and the second binding element comprises a DNCR2 sequence or a GNCR1 sequence; or (b) the first binding element comprises a DNCR2 sequence or a GNCR1 sequence and the second binding element comprises an NS3a sequence. In some embodiments, the small molecule comprises danoprevir or an analog thereof. In some embodiments, the first small molecule or the second small molecule comprises danoprevir or an analog thereof. In some embodiments, the small molecule comprises grazoprevir or an analog thereof. In some embodiments, the first small molecule or the second small molecule comprises grazoprevir or an analog thereof.

[0015] In some embodiments of the compositions of the disclosure, including those comprising a first fusion protein and a second fusion protein, the target molecule comprises a synthetic molecule or an exogenous molecule. In some embodiments, the synthetic molecule or the exogenous molecule comprises a protein. In some embodiments, the synthetic molecule or the exogenous molecule comprises a chimeric protein, a chimeric receptor, or a chimeric antigen receptor. In some embodiments, the chimeric receptor comprises an extracellular domain comprising an antigen sensing domain, a transmembrane domain, and an intracellular domain. In some embodiments, the antigen sensing domain comprises one or more of (a) a set of three complementarity determining regions (CDRs) of a heavy chain variable region; (b) a set of three complementarity determining regions (CDRs) of a heavy chain variable region and a set of three complementarity determining regions (CDRs) of a light chain variable region; (c) a fibronectin-protein based scaffold; wherein the antigen sensing region specifically binds a target antigen. In some embodiments, the antigen sensing region comprises one or more sequences isolated or derived from a mammalian sequence. In some embodiments, the antigen sensing region comprises one or more sequences isolated or derived from a human sequence. In some embodiments, the antigen sensing region comprises a humanized or fully human antibody. In some embodiments, the antigen sensing region comprises a single chain variable fragment (scFv).

[0016] In some embodiments of the compositions of the disclosure, the extracellular domain further comprises one or more of a hinge region, a spacer sequence, or a safety switch. In some embodiments, the hinge region comprises a sequence isolated or derived from a CD4 (cluster of differentiation 4) polypeptide, a CD8 (cluster of differentiation 8) polypeptide or a CD28 (cluster of differentiation 28) polypeptide. In some embodiments, the hinge region comprises a sequence isolated or derived from a human sequence. In some embodiments, the hinge region and the spacer region are the same region. In some embodiments, the hinge region and the spacer region are distinct regions.

[0017] In some embodiments of the compositions of the disclosure, the spacer sequence comprises a sequence isolated or derived from a CD4 polypeptide, a CD8 polypeptide, a CD28 polypeptide. In some embodiments, the spacer sequence comprises a sequence isolated or derived from a human sequence.

[0018] In some embodiments of the compositions of the disclosure, the safety switch comprises a sequence isolated or derived from an epidermal growth factor receptor (EGFR) polypeptide. In some embodiments, the safety switch comprises a truncated EGFR (EGFRt) polypeptide. In some embodiments, the safety switch comprises a sequence isolated or derived from a human sequence.

[0019] In some embodiments of the compositions of the disclosure, the transmembrane domain comprises a sequence isolated or derived from a CD4 polypeptide, a CD8 polypeptide, a CD28 polypeptide. In some embodiments, the transmembrane domain comprises a sequence isolated or derived from a human sequence.

[0020] In some embodiments of the compositions of the disclosure, the intracellular domain comprises one or more costimulatory domain(s). In some embodiments, the one or more costimulatory domain(s) comprises a sequence isolated or derived from a CD3^ (cluster of differentiation 3 zeta) polypeptide. In some embodiments, the one or more costimulatory domain(s) comprises a sequence isolated or derived from a CD28 polypeptide, a 4-1BB (cluster of differentiation 137) polypeptide, an ICOS (Inducible T Cell Costimulator) polypeptide, an 0X40 polypeptide, or a CD27 (cluster of differentiation 27) polypeptide. In some embodiments, the one or more costimulatory domain(s) comprises (a) a first costimulatory domain comprising sequence isolated or derived from a CD28 polypeptide, a 4-1BB polypeptide, or an ICOS polypeptide; and (b) a second costimulatory domain comprising sequence isolated or derived from a 4- IBB polypeptide, an 0X40 polypeptide, or a CD27 polypeptide. In some embodiments, the one or more costimulatory domain(s) comprise(s) a sequence isolated or derived from a human sequence.

[0021] In some embodiments of the compositions of the disclosure, the chimeric receptor comprises an intracellular domain further comprising an inducible cytokine domain. In some embodiments, the inducible cytokine domain comprises a nuclear factor of activated T-cells (NF AT) polypeptide capable of inducing expression of an IL-12 cytokine.

[0022] In some embodiments of the compositions of the disclosure, the chimeric receptor comprises an intracellular domain further comprising an intracellular domain of a cytokine receptor. In some embodiments, the intracellular domain of a cytokine receptor comprises an IL-2 receptor beta (IL-2RP) chain fragment. In some embodiments, the chimeric receptor comprises an intracellular domain further comprising a Signal Transducer and Activator of Transcription (STAT3/5) binding motif.

[0023] In some embodiments of the compositions of the disclosure, the chimeric receptor comprises an intracellular domain further comprising at least one immunoreceptor tyrosinebased activation motif (ITAM) sequence.

[0024] The disclosure provides a nucleic acid sequence encoding (a) a fusion protein of the disclosure or (b) one or more elements of (a). In some embodiments, the fusion protein comprises a binding element and a degradation initiator, wherein the binding element selectively binds a target molecule and wherein the degradation initiator comprises a sequence isolated or derived from an E3 ligase. In some embodiments, in a cell capable of expressing the fusion protein, the target molecule is an endogenous molecule. In some embodiments, the target molecule is a naturally-occurring molecule.

[0025] The disclosure provides a nucleic acid sequence encoding (a) a first fusion protein of the disclosure; or (b) a second fusion protein of the disclosure; or (c) one or more elements of (a); or (d) one or more elements of (b).

[0026] The disclosure provides a nucleic acid sequence encoding a first fusion protein of the disclosure and a second fusion protein of the disclosure.

[0027] In some embodiments of the nucleic acid sequences of the disclosure, the nucleic acid sequence further comprises one or more of a non-coding sequence, an untranslated region, a regulatory element, a separation element, a polycistronic element or a post- translational element.

[0028] In some embodiments of the nucleic acid sequences of the disclosure, the nucleic acid sequence further comprises at least one promoter capable of driving expression of the nucleic acid sequence in a mammalian cell. In some embodiments, the nucleic acid sequence further comprises at least one promoter capable of driving expression of the nucleic acid sequence in a human cell. In some embodiments, the at least one promoter comprises a constitutive promoter. In some embodiments, the constitutive promoter comprises a sequence isolated or derived from one or more of a MND promoter, a hPGK promoter, a CMV promoter, a CAG promoter, a SFFV promoter, an EFl alpha promoter, a UBC promoter, and a CD43 promoter. In some embodiments, the at least one promoter comprises an inducible promoter. In some embodiments, the inducible promoter comprises a sequence isolated or derived from one or more of a YB TATA promoter, a human beta globin (huBG) promoter, a minIL2 promoter, a minimalCMV (minCMV) promoter, and a TRE3G promoter. In some embodiments, the inducible promoter comprises a minimal sequence. In some embodiments, the inducible promoter further comprises a transcription factor-specific recognition sequence. In some embodiments, the transcription factor-specific recognition sequence comprises a transcription factor-specific response element. In some embodiments, the transcription factor response element comprises a sequence isolated or derived from an NF AT sequence. In some embodiments, the transcription factor-specific recognition sequence comprises at least one repeat of a transcription factor-specific response element. In some embodiments, the transcription factor-specific recognition sequence comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeats of a transcription factor-specific response element.

[0029] In some embodiments of the nucleic acid sequences of the disclosure, the (a) a first promoter drives expression of the first fusion protein; or (b) a second promoter drives expression of the second fusion protein. In some embodiments, the first promoter and the second promoter are identical. In some embodiments, the first promoter and the second promoter are not identical.

[0030] In some embodiments of the nucleic acid sequences of the disclosure, the separation element comprises a ribosomal skipping sequence. In some embodiments, the separation element comprises at least two ribosomal skipping sequences. In some embodiments, the ribosomal skipping sequence comprises a P2a sequence or a T2a sequence. In some embodiments, the ribosomal skipping sequence comprises a T2a-RFP-P2a sequence, a P2a- T2a sequence, or a T2a-P2a sequence.

[0031] In some embodiments of the nucleic acid sequences of the disclosure, the polycistronic element comprises an internal ribosome entry site (IRES) sequence. [0032] The disclosure provides a vector comprising a nucleic acid of the disclosure.

[0033] The disclosure provides a first vector comprising a nucleic acid encoding the first fusion protein of the disclosure and a second vector comprising a nucleic acid encoding the second fusion protein of the disclosure.

[0034] In some embodiments of the vectors of the disclosure, the vector is an expression vector capable of expressing a nucleic acid in a mammalian cell. In some embodiments, the mammalian cell is a human cell. In some embodiments, the vector comprises a plasmid

[0035] In some embodiments of the vectors of the disclosure, the vector comprises a delivery vector capable of introducing a nucleic acid to a mammalian cell. In some embodiments, the mammalian cell is a human cell. In some embodiments, the delivery vector comprises one or more of a viral vector, a non-viral vector, a liposome, a micelle, a polymersome, and a nanoparticle. In some embodiments, the viral vector comprises a sequence isolated or derived from a virus or a viral vector. In some embodiments, the viral vector comprises a sequence isolated or derived from one or more of an adenoviral vector, a lentiviral vector, a baculoviral vector, an Epstein Barr viral vector, a papovaviral (parvovirus) vector, a vaccinia viral vector, a herpes simplex viral vector, an adeno associated virus (AAV) vector, and a hepatitis B viral vector. In some embodiments, the vector comprises a sequence isolated or derived from a transposition system. In some embodiments, the vector comprises a sequence isolated or derived from one or more of a piggyBAC transposition system, a Sleeping Beauty transposition system, a Tcl/mariner transposition system, a Tol2 transposition system, a helraiser transposition system and a Tn7 transposition system.

[0036] In some embodiments of the vectors of the disclosure, the vector comprises one or more sequences mediating homology directed repair

[0037] The disclosure provides a cell comprising the fusion protein of the disclosure. In some embodiments, the fusion protein comprises a binding element and a degradation initiator, wherein the binding element selectively binds a target molecule and wherein the degradation initiator comprises a sequence isolated or derived from an E3 ligase. In some embodiments, in a cell capable of expressing the fusion protein, the target molecule is an endogenous molecule. In some embodiments, the target molecule is a naturally-occurring molecule. [0038] The disclosure provides a cell comprising the composition of the disclosure. In some embodiments, the composition comprises a first fusion protein and a second fusion protein.

[0039] The disclosure provides a cell comprising the nucleic acid sequence of the disclosure.

[0040] The disclosure provides a cell comprising the vector of the disclosure. In some embodiments, the cell stably expresses a fusion protein of the disclosure. In some embodiments, the fusion protein comprises a binding element and a degradation initiator, wherein the binding element selectively binds a target molecule and wherein the degradation initiator comprises a sequence isolated or derived from an E3 ligase. In some embodiments, in a cell capable of expressing the fusion protein, the target molecule is an endogenous molecule. In some embodiments, the target molecule is a naturally-occurring molecule.

[0041] The disclosure provides a cell comprising the vector of the disclosure. In some embodiments, the cell stably expresses a first fusion protein or a second fusion protein of the disclosure.

[0042] In some embodiments of the cells of the disclosure, the cell is a eukaryotic cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a stem cell. In some embodiments, the stem cell is a hematopoietic cell. In some embodiments, the stem cell is a mesenchymal cell. In some embodiments, the cell is an immune cell. In some embodiments, the immune cell is a T-cell, a Natural Killer (NK) cell, or an Innate Lymphoid Cell (ILC).

[0043] The disclosure provides a composition comprising (a) a fusion protein of the disclosure; or (b) a composition of any one of the disclosure; or (c) a nucleic acid of any one of the disclosure; or (d) a vector of any one of the disclosure; or (e) a cell of any one of the disclosure.

[0044] The disclosure provides a pharmaceutical composition comprising a composition of the disclosure and a pharmaceutically-acceptable carrier.

[0045] The disclosure provides a use of a fusion protein of any one of the disclosure, a composition of any one of the disclosure; a nucleic acid sequence of any one of the disclosure, a vector of any one of the disclosure, a cell of any one of the disclosure, or a pharmaceutical composition of the disclosure in the manufacture of a medicament for the treatment of a disease or a disorder.

[0046] The disclosure provides a use of a fusion protein of any one of the disclosure, a composition of any one of the disclosure; a nucleic acid sequence of any one of the disclosure, a vector of any one of the disclosure, a cell of any one of the disclosure, or a pharmaceutical composition of the disclosure for the treatment of a disease or a disorder.

[0047] In some embodiments of the uses of the disclosure, the disease or disorder comprises one or more of an autoimmune disease or disorder; an inflammatory disease or disorder; an immunodeficiency disease or disorder; an ischemic disease or disorder; a blood disease or disorder; a bone disease or disorder; a neurological disease or disorder; a cardiac disease or disorder; a vascular disease or disorder; a metabolic disease or disorder; a dermatological disease or disorder; a digestive disease or disorder; a mitochondrial disease or disorder; a muscle disease or disorder; a liver disease or disorder; a kidney disease or disorder; a hearing disease or disorder; an ophthalmic disease or disorder; and a proliferative disease or disorder.

[0048] In some embodiments of the uses of the disclosure, the disease or disorder comprises a cancer. In some embodiments, the cancer comprises one or more of Acute Lymphocytic Leukemia (ALL) in Adults, Acute Myeloid Leukemia (AML) in Adults, Adrenal Cancer, Anal Cancer, Basal and Squamous Cell Skin Cancer, Bile Duct Cancer, Bladder Cancer, Bone Cancer, Brain and Spinal Cord Tumors in Adults, Brain and Spinal Cord Tumors in Children, Breast Cancer, Breast Cancer in Men, Cancer in Adolescents. Cancer in Children, Cancer in Young Adults, Cancer of Unknown Primary, Cervical Cancer, Chronic Lymphocytic Leukemia (CLL), Chronic Myeloid Leukemia (CML), Chronic Myelomonocytic Leukemia (CMML), Colorectal Cancer, Endometrial Cancer, Esophagus Cancer, Ewing Family of Tumors, Eye Cancer (Ocular Melanoma), Gallbladder Cancer, Gastrointestinal Neuroendocrine (Carcinoid) Tumors, Gastrointestinal Stromal Tumor (GIST), Head and Neck Cancers, Hodgkin Lymphoma, Kaposi Sarcoma, Kidney Cancer, Laryngeal and Hypopharyngeal Cancer, Leukemia, Leukemia in Children, Liver Cancer, Lung Cancer, Lung Carcinoid Tumor, Lymphoma, Lymphoma of the Skin, Malignant Mesothelioma, Melanoma Skin Cancer, Merkel Cell Skin Cancer, Multiple Myeloma, Myelodysplastic Syndromes, Nasal Cavity and Paranasal Sinuses Cancer, Nasopharyngeal Cancer, Neuroblastoma, Non-Hodgkin Lymphoma, Non-Hodgkin Lymphoma in Children, Oral Cavity (Mouth) and Oropharyngeal (Throat) Cancer, Osteosarcoma, Ovarian Cancer, Pancreatic Cancer, Pancreatic Neuroendocrine Tumor (NET), Penile Cancer, Pituitary Tumors, Prostate Cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary Gland Cancer, Skin Cancer, Small Intestine Cancer, Soft Tissue Sarcoma, Stomach Cancer, Testicular Cancer, Thymus Cancer, Thyroid Cancer, Uterine Sarcoma, Vaginal Cancer, Vulvar Cancer, Waldenstrom Macroglobulinemia and Wilms Tumor.

[0049] In some embodiments of the uses of the disclosure, the disease or disorder comprises an infection or a disease or disorder caused by the infectious disease.

[0050] In some embodiments of the uses of the disclosure, the disease or disorder comprises a genetic disease or disorder.

[0051] The disclosure provides a method of treating a disease or a disorder, comprising administering to a subject an effective amount of a fusion protein of any one of the disclosure, a composition of any one of the disclosure; a nucleic acid sequence of any one of the disclosure, a vector of any one of the disclosure, a cell of any one of the disclosure, or a pharmaceutical composition of the disclosure, wherein a severity of a sign or symptom of the disease or disorder is decreased, thereby treating the disease or disorder.

[0052] The disclosure provides a disease or a disorder, comprising administering to a subject an effective amount of a fusion protein of any one of the disclosure, a composition of any one of the disclosure; a nucleic acid sequence of any one of the disclosure, a vector of any one of the disclosure, a cell of any one of the disclosure, or a pharmaceutical composition of the disclosure, wherein an onset or a relapse of a sign or symptom of the disease or disorder is delayed or inhibited, thereby preventing the disease or disorder.

[0053] In some embodiments of the methods of the disclosure, the disease or disorder comprises one or more of an autoimmune disease or disorder; an inflammatory disease or disorder; an immunodeficiency disease or disorder; an ischemic disease or disorder; a blood disease or disorder; a bone disease or disorder; a neurological disease or disorder; a cardiac disease or disorder; a vascular disease or disorder; a metabolic disease or disorder; a dermatological disease or disorder; a digestive disease or disorder; a mitochondrial disease or disorder; a muscle disease or disorder; a liver disease or disorder; a kidney disease or disorder; a hearing disease or disorder; an ophthalmic disease or disorder; and a proliferative disease or disorder. [0054] 141. In some embodiments of the methods of the disclosure, the disease or disorder comprises a cancer. In some embodiments, the cancer comprises one or more of Acute Lymphocytic Leukemia (ALL) in Adults, Acute Myeloid Leukemia (AML) in Adults, Adrenal Cancer, Anal Cancer, Basal and Squamous Cell Skin Cancer, Bile Duct Cancer, Bladder Cancer, Bone Cancer, Brain and Spinal Cord Tumors in Adults, Brain and Spinal Cord Tumors in Children, Breast Cancer, Breast Cancer in Men, Cancer in Adolescents. Cancer in Children, Cancer in Young Adults, Cancer of Unknown Primary, Cervical Cancer, Chronic Lymphocytic Leukemia (CLL), Chronic Myeloid Leukemia (CML), Chronic Myelomonocytic Leukemia (CMML), Colorectal Cancer, Endometrial Cancer, Esophagus Cancer, Ewing Family of Tumors, Eye Cancer (Ocular Melanoma), Gallbladder Cancer, Gastrointestinal Neuroendocrine (Carcinoid) Tumors, Gastrointestinal Stromal Tumor (GIST), Head and Neck Cancers, Hodgkin Lymphoma, Kaposi Sarcoma, Kidney Cancer, Laryngeal and Hypopharyngeal Cancer, Leukemia, Leukemia in Children, Liver Cancer, Lung Cancer, Lung Carcinoid Tumor, Lymphoma, Lymphoma of the Skin, Malignant Mesothelioma, Melanoma Skin Cancer, Merkel Cell Skin Cancer, Multiple Myeloma, Myelodysplastic Syndromes, Nasal Cavity and Paranasal Sinuses Cancer, Nasopharyngeal Cancer, Neuroblastoma, Non-Hodgkin Lymphoma, Non-Hodgkin Lymphoma in Children, Oral Cavity (Mouth) and Oropharyngeal (Throat) Cancer, Osteosarcoma, Ovarian Cancer, Pancreatic Cancer, Pancreatic Neuroendocrine Tumor (NET), Penile Cancer, Pituitary Tumors, Prostate Cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary Gland Cancer, Skin Cancer, Small Intestine Cancer, Soft Tissue Sarcoma, Stomach Cancer, Testicular Cancer, Thymus Cancer, Thyroid Cancer, Uterine Sarcoma, Vaginal Cancer, Vulvar Cancer, Waldenstrom Macroglobulinemia and Wilms Tumor.

[0055] In some embodiments of the methods of the disclosure, the disease or disorder comprises an infection or a disease or disorder caused by the infectious disease.

[0056] In some embodiments of the methods of the disclosure, the disease or disorder comprises a genetic disease or disorder.

[0057] The disclosure provides a polynucleotide set comprising: (a) a first polynucleotide encoding a first fusion protein comprising a first binding element; and (b) a second polynucleotide encoding a second fusion protein comprising a second binding element; and wherein: (i) the first or second fusion protein further comprises a degradation initiator or a functional variant thereof; and (ii) the other of the first or second fusion protein further comprises a target molecule of interest; an (iii) interaction of the first and second binding elements mediates recruitment of the degradation initiator to the molecule of interest to initiate degradation.

[0058] In some embodiments of the polynucleotide sets disclosure, the first and second binding elements comprise a pair of heterodimer proteins. In some embodiments, the pair of heterodimer proteins comprises dimers with one or both subunits comprised of one-helix subunits. In some embodiments, the pair of heterodimer proteins comprises dimers with one or both subunits comprised of two-helix subunits. In some embodiments, the pair of heterodimer proteins comprises dimers with one or both subunits comprised of multiple-helix subunits. In some embodiments, the pair of heterodimer proteins comprises a three-helix subunit and a single-helix subunit. In some embodiments, the pair of heterodimer proteins comprises non-helical subunits. In some embodiments, the first or second binding element is selected from the group consisting of: DHD37-short-A (SEQ ID NO: 11) and either DHD37- short-B (SEQ ID NO: 13), DHD37-short-B-Ntrunc (SEQ ID NO: 15), or DHD37-short-B- KtoR (SEQ ID NO: 16).

[0059] In some embodiments of the polynucleotide sets disclosure, the first and second binding elements are selected so that interaction of the first and second binding elements is mediated by the presence of a small molecule.

[0060] In some embodiments of the polynucleotide sets disclosure, the polynucleotide set of claim 1 wherein: (a) the first or second binding element comprises NS3a; (b) and the other of the first or second binding element is selected from the group consisting of DNCR2 and GNCR1; and (c) wherein the first and second binding elements are selected so that interaction of the first and second binding elements is mediated by the presence of a small molecule. In some embodiments, the small molecule mediates binding of the first and second binding elements. In some embodiments, the small molecule disrupts binding of the first and second binding elements. In some embodiments, a second small molecule disrupts binding of the first and second binding elements by out-competing the first small molecule. In some embodiments, interaction of the first and second binding element in a cell is titratable relative to administration of the small molecule to the cell. In some embodiments, the small molecule is selected from the group consisting of: danoprevir and grazoprevir and their analogs. [0061] In some embodiments of the polynucleotide sets disclosure, the molecule of interest comprises a synthetic or exogenous molecule.

[0062] In some embodiments of the polynucleotide sets disclosure, the synthetic or exogenous molecule comprises a protein.

[0063] In some embodiments of the polynucleotide sets disclosure, the first polynucleotide and the second polynucleotide on a single vector.

[0064] In some embodiments of the polynucleotide sets disclosure, the polynucleotide set comprises at least two vectors comprising: (a) a first vector comprising the first polynucleotide; and (b) a second vector comprising the second polynucleotide.

[0065] In some embodiments of the polynucleotide sets disclosure, the polynucleotide set is integrated into a vector backbone. In some embodiments, the vector backbone is selected from the group consisting of backbones of adenoviral vectors, lentiviral vectors, baculoviral vectors, Epstein Barr viral vectors, papovaviral vectors, vaccinia viral vectors, herpes simplex viral vectors, adeno associated virus (AAV) vectors, and transposon vectors. In some embodiments, the vector backbone comprises a homology directed repair vector.

[0066] In some embodiments of the polynucleotide sets disclosure, the polynucleotide set is integrated into a chromosome.

[0067] In some embodiments of the polynucleotide sets disclosure, the first and/or second polynucleotides encoding a first or second fusion protein is operatively linked to a polynucleotide component comprising one or more promoter sequences.

[0068] In some embodiments of the polynucleotide sets disclosure, (a) the first or second polynucleotide encoding a first or second fusion protein comprising the degradation initiator or functional variant thereof is operatively linked to a polynucleotide component encoding an inducible promoter sequence; and (b) the first or second polynucleotide encoding a first or second fusion protein comprising the molecule of interest is operatively linked to a polynucleotide component encoding a constitutive promoter sequence.

[0069] In some embodiments of the polynucleotide sets disclosure, (a) the first or second polynucleotide encoding a first or second fusion protein comprising the degradation initiator or functional variant thereof is operatively linked to a polynucleotide component encoding a first inducible promoter sequence; and (b) the first or second polynucleotide encoding a first or second fusion protein comprising the molecule of interest is operatively linked to a polynucleotide component encoding a second inducible promoter sequence.

[0070] In some embodiments of the polynucleotide sets disclosure, the first polynucleotide encoding the first fusion protein and the second polynucleotide encoding the second fusion protein comprise a set of two or more polynucleotide components.

[0071] The disclosure provides a polynucleotide set, wherein the first polynucleotide encoding the first fusion protein and the second polynucleotide encoding the second fusion protein comprise a single polynucleotide component. In some embodiments, the polynucleotide component encoding the first fusion protein and the polynucleotide encoding the second fusion protein are separated by a separation element comprising a polynucleotide sequence that prevents fusion of the first fusion protein and the second fusion protein. In some embodiments, the separation element comprises a polynucleotide sequence comprising a ribosomal skipping sequence. In some embodiments, the separation element comprises a polynucleotide sequence comprising at least two ribosomal skipping sequences. In some embodiments, the ribosomal skipping sequence comprises a polynucleotide sequence comprising P2a and/or T2a. In some embodiments, the separation element comprises a polynucleotide sequence selected from the group consisting of: P2a, T2a, T2a-RFP-P2a, P2a- T2a, T2a-P2a, and IRES. In some embodiments, the separation element comprises a polynucleotide sequence comprising a second constitutive promoter.

[0072] In some embodiments of the polynucleotide sets disclosure, the constitutive promoter sequence is selected from the group consisting of: MND, hPGK, CMV, CAG, SFFV, EFlalpha, UBC, and CD43.

[0073] In some embodiments of the polynucleotide sets disclosure, the inducible promoter sequence comprises a minimal promoter sequence selected from the group consisting of: YB TATA, human beta globin (huBG), minIL2, minimalCMV (minCMV), and TRE3G.

[0074] In some embodiments of the polynucleotide sets disclosure, the inducible promoter sequence comprises a transcription factor-specific recognition sequence comprising a transcription factor-specific response element. In some embodiments, the transcription factor response element comprises a polynucleotide selected from the group consisting of: NFAT- AP1. In some embodiments, the transcription factor response element is repeated. In some embodiments, the transcription factor response element is repeated 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times.

[0075] The disclosure provides a polynucleotide set comprising a single polynucleotide component encoding a fusion protein comprising: (a) a binding element specific for a native motif on a molecule of interest; and (b) a degradation initiator; and wherein binding of the fusion protein to the molecule of interest mediates recruitment of the degradation initiator to the molecule of interest to initiate degradation. In some embodiments, the molecule of interest comprises an endogenous molecule. In some embodiments, the endogenous molecule comprises a protein. In some embodiments, binding of the fusion protein to the molecule of interest comprises a conformation-specific interaction. In some embodiments, the binding of the fusion protein to the molecule of interest is in response to a specific modification on the molecule. In some embodiments, the specific modification on the molecule comprises a post-translation modification. In some embodiments, the posttranslation modification comprises phosphorylation. In some embodiments, the binding element comprises a domain that is specific for a protein-protein interaction domain.In some embodiments, the binding element comprises a single-chain variable fragment (scFVf). In some embodiments, the binding element comprises a monomeric variable antibody domain. In some embodiments, the binding element comprises a designed ankyrin repeat protein domain (DARPin). In some embodiments, the binding element comprises a variable lymphocyte receptor (VLR) domain.

[0076] In some embodiments of the polynucleotide sets disclosure, the degradation initiator comprises an E3 ubiquitin ligase domain or any functional variant thereof.

[0077] The disclosure provides a cell comprising a polynucleotide set of the disclosure. In some embodiments, the cell is a prokaryotic cell. In some embodiments, the cell is a yeast cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a human cell in vivo. In some embodiments, the cell is a human cell ex vivo. In some embodiments, the cell is a stem cell. In some embodiments, the cell is a pluripotent stem cell. In some embodiments, the cell is a multipotent stem cell. In some embodiments, the cell is a hematopoietic stem cell. In some embodiments, the cell is a mesenchymal stromal cell. In some embodiments, the cell is a mesenchymal cell. In some embodiments, the cell is an autologous cell selected for a cell therapy or is the progeny of an autologous cell selected for a cell therapy. In some embodiments, the cell is an allogeneic cell selected for a cell therapy or is the progeny of an allogeneic cell selected for a cell therapy

[0078] The disclosure provides a method of effecting stem cell differentiation comprising modifying a stem cell using a polypeptide set of the polynucleotide set of the disclosure. In some embodiments, the cell is a cancer cell. In some embodiments, the cell is a non-cancer cell from a human subject diagnosed with cancer. In some embodiments, the cell is an immune cell. In some embodiments, the cell is selected from the group consisting of leukocyte, lymphocyte, T cell, regulatory T cell, effector T cell, CD4+ effector T cell, CD8+ effector T cell, memory T cell, autoreactive T cell, exhausted T cell, natural killer T cell, B cell, dendritic cell, and macrophage. In some embodiments, the cell is selected from the group consisting of cardiac cell, lung cell, muscle cell, epithelial cell, pancreatic cell, skin cell, CNS cell, neuron, myocyte, skeletal muscle cell, smooth muscle cell, liver cell, kidney cell and glial cell.

[0079] The disclosure provides a cell genetically modified to express a CAR, comprising a polynucleotide set of the disclosure. In some embodiments, the cell is a T cell, a natural killer (NK) cell, a natural killer T (NKT) cell or an ILC cell.

[0080] The disclosure provides a viral capsid comprising the polynucleotide set of the disclosure. In some embodiments, the viral capsid is selected from capsids of an adenovirus, lentivirus, baculovirus, Epstein Barr virus, papovavirus, vaccinia virus, herpes virus, herpes simplex virus, and adeno-associated virus.

[0081] The disclosure provides a cell producing the viral capsid of the disclosure. In some embodiments, the viral capsid is selected from capsids of an adenovirus, lentivirus, baculovirus, Epstein Barr virus, papovavirus, vaccinia virus, herpes virus, herpes simplex virus, and adeno-associated virus.

[0082] The disclosure provides a composition comprising the polynucleotide set of the disclosure.

[0083] The disclosure provides a use of a composition of the disclosure, including a composition comprising the polynucleotide set of the disclosure, for treating a subject in need of a CAR therapy. [0084] The disclosure provides a kit comprising the polynucleotide set of the disclosure.

[0085] The disclosure provides a method of making an engineered cell comprising introducing the polynucleotide of any of the polynucleotide set of the disclosure into a cell. In some embodiments, the polypeptides are expressed in the cell. In some embodiments, the method further comprises administering the cell in a subject in need thereof. In some embodiments, the method further comprises administering the small molecule to the subject.

[0086] The disclosure provides a method of controlling a T cell-mediated immune response in a subject in need thereof comprising administering to the subject an effective amount of the cell of the disclosure.

[0087] The disclosure provides a method of providing an anti -tumor immunity in a subject in need thereof, the method comprising administering to the subject an effective amount of the cell of the disclosure. In some embodiments, the cell is a T cell. In some embodiments, the cell is an autologous T cell. In some embodiments, the cell is an allogeneic T cell. In some embodiments, the method further comprises administering to the subject the small molecule.

[0088] The disclosure provides a method of treating cancer in a subject in need thereof comprising administering to the subject an effective amount of the cell of the disclosure. In some embodiments, the cell is a T cell. In some embodiments, the cell is an autologous T cell. In some embodiments, the cell is an allogeneic T cell. In some embodiments, the method further comprises administering to the subject the small molecule.

[0089] The disclosure provides a gene therapy method wherein: (a) a polynucleotide set comprises a degradation initiator operatively linked to a small molecule regulated promoter; and (b) a therapeutic molecule; the method comprising administering to a subject in need thereof a therapeutically effective amount of the polynucleotide set of the disclosure. In some embodiments, the method further comprises administering to the subject the small molecule. In some embodiments, the method further comprises adjusting the dosage of the small molecule to adjust the level of the therapeutic molecule in the subject. In some embodiments, the method further comprises (a) monitoring production of the therapeutic molecule in the subject; and (b)adjusting dosage of the small molecule to adjust the level of the therapeutic molecule in the subject to the desired level. In some embodiments, the subject has a condition selected from the group consisting of: cancer, cystic fibrosis, heart disease, diabetes, hemophilia and AIDS.

[0090] The disclosure provides a use of the polynucleotide set of the disclosure for the manufacture of a medicament for treating cancer in a subject in need thereof.

Brief Description of Drawings

[0091] FIG. 1 is a diagram illustrating a 3+1 heterodimer binding element pair.

[0092] FIG. 2 is a diagram illustrating an example of a pair of small molecule-regulated polypeptide binding elements.

[0093] FIG. 3 is a diagram illustrating regulating an endogenous protein target using a binding element (“B”) that is specific to a native motif (“A”) on the target.

[0094] FIG. 4A is a diagram illustrating using a 3+1 DHD pair as binding elements for targeted degradation of a transmembrane protein.

[0095] FIG. 4B is a diagram illustrating using a 3+1 DHD pair as binding elements for targeted degradation of a cytoplasmic protein.

[0096] FIG. 5 is a diagram illustrating using a small molecule-regulated degrader system for degradation of a chimeric receptor.

[0097] FIG. 6A is a diagram illustrating using a transmembrane receptor domain as a binding element for targeted degradation of a chimeric transmembrane receptor.

[0098] FIG. 6B is a diagram illustrating using a phosphotyrosine-binding domain (PYBD) as a binder for targeted degradation of a post-translationally phosphorylated (“P”) transmembrane receptor.

[0099] FIG. 7A is a diagram illustrating a unidirectional forward configuration for encoding an inducible polynucleotide component and a constitutive polynucleotide component on a single vector.

[0100] FIG. 7B is a diagram illustrating a two-vector system for encoding an inducible polynucleotide component and a constitutive polynucleotide component. [0101] FIG. 8A is a plot showing the Kd determination from a competitive binding ELISA assay of DHD-A:DHD-B interaction for dimerization domain DHD-A (SEQ ID NO: 11) and DHD-B (SEQ ID NO: 13).

[0102] FIG. 8B is a plot showing the Kd determination from a competitive binding ELISA assay of DHD-A:DHD-B interaction for dimerization domain DHD-A (SEQ ID NO: 11) and DHD-B-Ntrunc (SEQ ID NO: 15).

[0103] FIG. 9 is a plot showing a comparison of DHD-B (SEQ ID NO: 13) and DHD37- short-B-KtoR (SEQ ID NO: 16) in a direct binding ELISA assay with DHD-A (SEQ ID NO: H).

[0104] FIG. 10A and FIG. 10B are plots showing the normalized gMFIs for membrane- associated DHD-E3 ligases and cytoplasmic DHD-E3 ligases, respectively.

[0105] FIG. 11 is a plot showing the normalized gMFI for cytoplasmic DHD-E3 ligases screened for degradation of a 3xFLAG-tagged DHD-B-BACH2.

[0106] FIG. 12 is a panel showing CAR surface staining and geometric mean fluorescence intensity (gMFIs) levels in transduced and control CD4+ T cells.

[0107] FIG. 13 is a plot showing inducible degradation of CAR-NS3a with LNX1-DNCR2, DNCR2-RNF4, and RNF43-DNCR2 in the presence of 500 nM danoprevir.

[0108] FIG. 14 is a diagram of single vector constructs encoding CAR-NS3a and RNF43- DNCR2 (i) and RNF43-DNCR2-KtoR-endo (ii) linked by P2A self-cleavage peptides.

[0109] FIG. 15 is a panel showing CAR surface staining and gMFI levels in SUP-T1 cells transduced with lentiviruses expressing the control RNF43-DNCR negative control (i) or the RNF43-DNCR-KtoR-endo (ii) constructs.

[0110] FIG. 16 is a pair of plots showing overlay histograms of TCR staining and gMFI levels, respectively, of cells transduced with TM CD3z -RNF43 constructs, a GFP control, and non-transduced cells using an anti-TCRa/p antibody (BV421).

[0111] FIG. 17A is an overlay histogram plot showing endogenous TCR staining levels on the cells expressing LNXl-nSH2-cSH2, nSH2-cSH2, and LNGFR control cells using an anti TCRot/p antibody. [0112] FIG. 17B is a bar plot showing gMFIs values in cells expressing LNXl-nSH2-cSH2, nSH2-cSH2, and LNGFR.

[0113] FIG. 18 is a pair of plots showing concentrations of IL-2 and IFNy, respectively, present in the supernatant after 24 hours of co-culture.

[0114] FIG. 19 is a plot showing the killing of Jekol target cells by T cells co-cultured at an effector to target ratio of 1 :4.

[0115] FIG. 20 is a panel of plots showing gMFI for surface expression of the CD39, PD-1, and Lag3 exhaustion markers on single or dual transduced CAR-T cells co-cultured with A549 target cells.

[0116] FIGS. 21A-C are a series of graphs demonstrating that the addition of a DHD to CAR does not change surface expression or function of CAR. As shown in this figure the label “CAR” is CAR without any DHD, the label “CAR-DHD S R2K” is CAR fused to SEQ ID NO: 170, the label “CAR-DHD L R2K” is CAR fused to SEQ ID NO: 172, and the label “CAR-DHD L” is CAR fused to SEQ ID NO: 173. (A) Comparison of CAR Surface expression in unmodified CAR and CAR-DHD constructs. A panel of designed Her2 CAR- DHD constructs were screened for surface expression of Her2 CAR. Primary T cells were transduced with lentiviruses expressing either Her2-CAR only or a panel of Her2 CAR-DHD constructs, or untransduced (Mock T), and cells were stained with a Her2 -Alexa Fluor 647 conjugate to detect the surface expression of CAR-DHD by flow cytometry. Surface expression measuring was made by comparing transduction marker positive Her2 CAR gMFI between the constructs. (B) Cytokine release following challenge by tumor cells in unmodified CAR. and CAR-DHD constructs. CAR T cells were co-cultured at an effector to target ratio of 1 : 1 with A549 cells. Concentration of IL2 (right Y axis) and interferongamma (left Y axis) present in the supernatant was measured after 24 hours of co-culture. (C) Measurement of T cell cytotoxicity in unmodified CAR. and CAR-DHD constructs. CAR. T cells were co-cultured at an effector to target ratio of 1:1 with A549 labeled with NucLight Red (NLR) cells positive for Her2 target in a 96 well plate. Plates were cultured in an Incucyte (Sartorius) for 72 hows. Tumor cell killing was determined via Incucyte measurement over time of total NLR+ cells/well compared to tumor cells alone.

[0117] FIGS. 22A-B are a series of graphs demonstrating that designed degraders facilitate efficient degradation of protein targets. (A) A panel of designed DHD-E3/LJb constructs were screened for efficient degradation of a Her2-specific CAR as a model target for degrading membrane proteins. Primary T cells were co-transduced with lentiviruses expressing each member of the panel and Her2 CAR-DHD, and cells were stained with a Her2- Alexa Fluor 647 conjugate to detect the surface expression of CAR-DHD by flow cytometry. The gMFI ratio of each construct was calculated by normalizing with the gMFI measured in the cells transduced with only the CAR constructs. As shown, from left to right, designed DHD-E3/Ub constructs (sequences provided in Table 10): F43 native RNF43 IX DHD-B; DAP10-V5 CD8TMD LNX1 2X DHD-B; DAP10-V5 CD8TMD LNX1 3X DHD-B; DAP10-V5 CD8TMD LNX1 4X DHD-B; DAP10-V5 CD8TMD LNX1 KioR 2X DHD-B; DAP10-V5 CD8TMD LNX1 KtoR 2X DHD-B; DAP10-V5 CD8TMD LNX1 KtoR 3X DHD-B;

DAP10-V5 CD8TMD LNX1 KtoR 4X DHD-B; LNX1 IX DHD-B; LNX1 2X DHD-B; LNX1 3X DHD-B; LNX1 KtoR IX DHD-B; LNX1 KioR 2X DHD-B; LNX1 KtoR 3X DHD-B; DAP10-V5 CD8TMD RNF4 KtoR IX DHD-B; DAP10-V5 CD8TMD RNF4 KtoR 2X DHD-B; DAP10-V5 CD8TMD RNF4 3X DHD-B; DAP10-V5 CD8TMD RNF44X DHD-B; DAP10-V5 CD8TMD RNF4 IX DHD-B; DAP10-V5 CD8TMD RNF42X DHD-B; DAP10-V5 CD8TMD RNF4 3X DHD-B; DAP10-V5 CD8TMD RNF44X DHD-B; RNF4 2X DHD-B; RNF4 3X DHD-B; RNF44X DHD-B; RNF4 2X DHD-B; RNF43X DHD-B; RNF44X DHD-B; DAP10-V5 RNF43 IX DHD-B; DAP10-V5 RNF43 2X DHD-B; DAP 10- V5 RNF43 3X DHD-B; DAP10-V5 RNF43 4X DHD-B; (B) Functional constructs were identified as having the normalized gMFIs below the 0.25 cutoff were rescreened in primary T cells from two normal donor. Bars show CAR gMFI in single transduced (CAR-DHD only) and dual transduced (+ DHD-E3) cells. As designated in this figure, the terms “IX”, “2X”, “3X” and “4X” refer to the number of repeats included of a G4S linker (of which a single or “IX” repeat has the sequence “GGGGS”) (see Table 10). As shown in this figure, the term “K2R” is interchangeable with the term “KtoR”, and does not denote an amino acid substitution at the second position of the sequence of any degrader shown. As shown in this figure the label “DHD37B” refers to “DHD37-short-B-KtoR” (SEQ ID NO: 16).

[0118] FIG. 23 is a graph demonstrating that the RtoK mutation increases down-regulation of CAR. Comparison of CAR Surface expression in CAR-DHD and CAR-DHD RtoK constructs. Primary T cells were co-transduced with lentiviruses expressing DHD-e3 constructs with a range of degradation activity and Her2 CAR-DHD, or a Her2 CAR-DHD with a RtoK mutation, and cells were stained with a Her2-Alexa Fluor 647 conjugate to detect the surface expression of CAR-DHD by flow cytometry. The gMFI ratio of each construct was calculated by normalizing with the gMFI measured in the cells transduced with only the CAR constructs, and the effect of RtoK mutation was assessed using Wilcoxon matched-pairs signed rank test. As shown in this figure, the term “R2K” is interchangeable with the term “RtoK”, and does not denote an amino acid substitution at the second position of the sequence of any degrader shown.

[0119] FIGS. 24A-C are a series of graphs demonstrating that C terminal fusion of coil-coil domains to CAR maintain CAR activity and enable targeted regulation by paired coil-coil domain-E3 fusions. A) Cytolytic activity of CART cells challenged with Jekol target cells showing control T cells, (Mock), parental CAR, and CAR constructs fused to the indicated coil-coil domains. B) Cytokine secretion of CAR constructs. C) Regulation of surface CAR expression by co-transduction with the indicated coil-coil-E3 fusion.

[0120] FIGS. 25A-C are a series of graphs demonstrating that regulation of CAR-DHD with a DHD-e3 construct enhances survival and blocks acquisition of activation/'exhaustion markers in a tumor cell co-culture model. A) Experimental design: T cells were transduced with a single lentiviral construct encoding CAR-DHD (CAR alone) encoding 2 different CAR-DHD designs (CAR DHD L and CAR_DHD_S), or co-transduced with CAR-DHD and DHD-E3 constructs. CAR T cells were challenged with repeated 4 day c-cultures with A549 adenocarcinoma cells, followed by flow cytometry analysis. B) Expression patterns of CD39, PD1, and Lag3 exhaustion/activation markers on CAR transduced cells. C) Percent of CD3+ cells which were CAR+ for the indicated transduction conditions after co-culture compared to input cells. As shown in this figure the label “DFID S” is SEQ ID NO: 171 and the label “DHD L. ” is SEQ ID NO: 173.

Detailed Description

Nucleic Acids and Related Terminology

[0121] In some embodiments, the terms “Nucleic acid,” “nucleic acid molecule,” “nucleotide,” “nucleotide sequence,” “polynucleotide,” and grammatical variants thereof may be used interchangeably and refer to the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine; “RNA molecules”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; “DNA molecules”), or any phosphoester analogs thereof, such as phosphorothioates and thioesters, in either single stranded form, or a double-stranded helix. Single stranded nucleic acid sequences refer to single-stranded DNA (ssDNA) or single-stranded RNA (ssRNA). Double stranded DNA- DNA, DNA-RNA and RNA-RNA helices are possible.

[0122] In some embodiments, “Nucleic acid,” and in particular a DNA or RNA molecule, may refer only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter aha, in linear or circular DNA molecules (e.g., restriction fragments), plasmids, supercoiled DNA and chromosomes. In discussing the structure of particular double-stranded DNA molecules, sequences are provided according to the normal convention of writing the sequence left to right in the 5’ to 3’ direction along the non-transcribed strand of DNA (i.e., the strand having a sequence homologous to the messenger RNA or mRNA). Unless otherwise indicated, all nucleic acid and nucleotide sequences are written left to right in 5’ to 3’ orientation.

[0123] Nucleotides are referred to by their commonly known one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Accordingly, ‘A’ represents adenine, ‘C’ represents cytosine, ‘G’ represents guanine, ‘T’ represents thymine, and ‘U’ represents uracil.

[0124] In some embodiments, the term “Polynucleotide” refers to polymers of nucleotides of any length or type, including ribonucleotides, deoxyribonucleotides, analogs thereof, or mixtures thereof. This term refers to the primary structure of the molecule. Thus, the term includes triple-, double- and single-stranded deoxyribonucleic acid (“DNA”) and ribonucleic acid (“RNA”). It also includes modified, for example by alkylation and/or by capping, and unmodified forms of the polynucleotide. More particularly, “polynucleotide” includes polydeoxyribonucleotides (containing 2-deoxy-D-ribose) and polyribonucleotides (containing D-ribose), including mRNA, whether spliced or unspliced, any other type of polynucleotide which is an N- or C-gly coside of a purine or pyrimidine base, and other polymers containing nucleotide backbones, for example, polyamide (e.g., peptide nucleic acids “PNAs”) and polymorpholino polymers, and other synthetic sequence-specific nucleic acid polymers providing that the polymers contain nucleobases in a configuration which allows for base pairing and base stacking, such as is found in DNA and RNA.

[0125] In some embodiments, a vector of the disclosure comprises a nucleic acid sequence of the disclosure and backbone sequence(s). [0126] In some embodiments, a polynucleotide includes a DNA, e.g., a DNA inserted in a vector. In other aspects, a polynucleotide includes an mRNA. In some aspects, the mRNA is a synthetic mRNA. In some embodiments, the synthetic mRNA includes at least one unnatural nucleobase. In some embodiments, all nucleobases of a certain class have been replaced with unnatural nucleobases (e.g., all uridines in a polynucleotide can be replaced with an unnatural nucleobase, e.g., 5-methoxy uridine).

[0127] In some embodiments, the term “Expression” refers to the transcription and/or translation of a particular nucleotide sequence driven by a promoter.

[0128] In some embodiments, the term “Expression vector” refers to a plasmid, virus, or other nucleic acid designed for polypeptide expression in a cell. The vector or construct is used to introduce a gene into a host cell whereby the vector will interact with polymerases in the cell to express the protein encoded in the vector/construct. The expression vector may exist in the cell extrachromosomally or may be integrated into the chromosome. Expression vectors may include additional sequences which render the vector suitable for replication and integration in prokaryotes, eukaryotes, or preferably both (e.g, shuttle vectors). The polynucleotides of the disclosure may be provided as components of expression vectors.

[0129] In some embodiments, the term “Cloning vector” refers to a plasmid, virus, or other nucleic acid designed for producing copies of a polynucleotide. Cloning vectors may contain transcription and translation initiation sequences, transcription and translation termination sequences and a poly adenylation signal. Such constructs will typically include a 5' LTR, a tRNA binding site, a packaging signal, an origin of second-strand DNA synthesis, and a 3' LTR or a portion thereof. The polynucleotides of the disclosure may be provided as components of cloning vectors, which may be used to produce the polynucleotides of the disclosure.

[0130] In some embodiments, “Promoter” refers to a nucleotide sequence which indicates where transcription of a gene is initiated and in which direction transcription will continue.

[0131] In some embodiments, “Encoding” refers to an ability of specific sequences of nucleotides in a polynucleotide (e.g. a gene, cDNA, or mRNA) to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (e.g., rRNA, tRNA and mRNA) or a defined sequence of amino acids. Thus, a gene, cDNA, or RNA, encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0132] Unless otherwise specified, a nucleotide sequence “encoding an amino acid sequence,” (including a polynucleotide “encoding” a chimeric polypeptide of the disclosure), includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence.

[0133 ] Polypeptides and Related Terminology

[0134] Amino acids are referred to by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. The amino acid residues are abbreviated as follows, where the abbreviations are shown in parentheses: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine (Cys; C), glutamic acid (Glu; E), glutamine (Gin; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Vai; V).

[0135] Amino acid sequences are written left to right in amino to carboxy orientation.

[0136] In some embodiments, the term “Polypeptide” refers to a sequence of amino acid subunits. In some embodiments, a “peptide” may comprise at most 50 amino acids. In some embodiments, a “peptide” may comprise about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids. The term “Polypeptide,” may refer to proteins, polypeptides, and peptides of any length, size, structure, or function. The terms “Polypeptide,” “peptide,” and “protein” may be used interchangeably.

[0137] In some embodiments, polypeptides of the disclosure comprise naturally or synthetically created or modified amino acids, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. In some embodiments, polypeptides of the disclosure comprise one or more amino acid residues are artificial chemical analogs of a corresponding naturally occurring amino acid (including, for example, synthetic amino acids such as homocysteine, ornithine, p-acetylphenylalanine, D-amino acids, and creatine), as well as other modifications known in the art. In some embodiments, polypeptides of the disclosure comprise gene products, homologs, orthologs, paralogs, fragments and other equivalents, variants, and analogs of the foregoing. In some embodiments, polypeptides of the disclosure comprise a single polypeptide or can be a multi- molecular complex such as a dimer, trimer or tetramer. In some embodiments, polypeptides of the disclosure comprise single-chain or multi-chain polypeptides. In some embodiments, polypeptides of the disclosure comprise disulfide linkages, which may be found in multichain polypeptides.

[0138] In some embodiments, the polypeptides of the disclosure include L-amino acids + glycine, D-amino acids + glycine (which are resistant to L-amino acid-specific proteases in vivo), or a combination of D- and L-amino acids + glycine. Polypeptides of the disclosure may be chemically synthesized or recombinantly expressed.

[0139] Polypeptides of the disclosure may include additional residues at the N-terminus, C- terminus, internal to the polypeptide, or a combination thereof; these additional residues are not included in determining the percent identity of the polypeptides of the disclosure relative to the reference polypeptide. Such residues may be any residues suitable for an intended use, including but not limited to tags.

[0140] In some embodiments, the term “Tags” refers to a detectable moieties, including but not limited to a fluorescent protein, an antibody epitope tag, a purification tag, a histidine tag, or a linker. In some embodiments, tagged therapeutic agents of the disclosure or tagged ligands of the disclosure comprise a detectable moiety suitable for purposes of purification, to drive localization of the polypeptide, and to add functionality to the polypeptides.

[0141] In some embodiments, “Chimeric polypeptide” refers to any polypeptide comprising a first amino acid sequence derived from a first source, which is operably -linked, covalently or non-covalently, to a second amino acid sequence derived from a second source, wherein the first and second source are not the same (two distinct sources). A first source and a second source that are not the same can include two different biological entities, or two different proteins from the same biological entity, or a biological entity and a non-biological entity. A chimeric protein of the disclosure may include a protein derived from at least 2 different biological sources. A biological source may include any non-synthetically produced nucleic acid or amino acid sequence (e.g. a genomic or cDNA sequence, a plasmid or viral vector, a native virion or a mutant or analog of any of the above). A synthetic source may include a protein or nucleic acid sequence produced chemically and not by a biological system (e.g. solid phase synthesis of amino acid sequences). A chimeric protein of the disclosure may include a protein derived from at least 2 different synthetic sources or a protein derived from at least one biological source and at least one synthetic source. A chimeric protein of the disclosure may include a first amino acid sequence derived from a first source, covalently or non-covalently linked to a nucleic acid, derived from any source or a small organic or inorganic molecule derived from any source. The chimeric protein can include a linker molecule between the first and second amino acid sequence or between the first amino acid sequence and the nucleic acid, or between the first amino acid sequence and the small organic or inorganic molecule.

[0142] In some embodiments, a “Fragment” of a polypeptide, or a “truncated polypeptide” may refers to an amino acid sequence of a polypeptide that is shorter than a reference sequence. In some embodiments, the reference sequence comprises or consists of a naturally- occurring sequence. In comparison to a reference sequence, the fragment may comprise an N- terminal or a C-terminal deletion (optionally referred to as a truncation). Alternatively, or in addition, in comparison to a reference sequence, the fragment may comprise an internal deletion at any one or more amino acid positions of the polypeptide. Polypeptides of the disclosure may be provided as a fragment or a truncated version of a reference polypeptide. Moreover, all possible fragments and truncated variants of the polypeptides of the disclosure are contemplated in the embodiments provided in this disclosure.

[0143] In some embodiments, the term “Functional fragment” refers to a polypeptide fragment that retains a function of the polypeptide. Accordingly, in some embodiments, a functional fragment of a bioactive peptide, such as an enzyme, retains the ability to catalyze a biological action, because the functional fragment comprises a catalytic domain of the enzyme. Polypeptides of the disclosure may be provided as a fragment or a truncated version of a reference polypeptide. Moreover, all possible fragments and truncated variants of the polypeptides of the disclosure are contemplated in the embodiments provided in this disclosure. In some embodiments, a functional fragment of the disclosure retains a function of the polypeptide even if the activity of the functional fragment or the efficacy of the functional fragment is modified when compared to the full-length polypeptide. In some embodiments, a functional fragment of the disclosure retains a function of the polypeptide even if the activity of the functional fragment or the efficacy of the functional fragment is decreased when compared to the full-length polypeptide.

[0144] In some embodiments, the term “Functional variant” refers to a modified form of a polypeptide, fragment, or a member of a class of polypeptides, which maintains the function of the polypeptide. In some embodiments, a functional variant of the disclosure retains a function of the polypeptide even if the activity of the functional variant or the efficacy of the functional variant is modified when compared to the unmodified polypeptide. In some embodiments, a functional variant of the disclosure retains a function of the polypeptide even if the activity of the functional variant or the efficacy of the functional variant is decreased when compared to the unmodified polypeptide.

[0145] In some embodiments, the term “Amino acid substitution” refers to replacing an amino acid residue present in a parent or reference sequence (e.g., a wild type sequence) with another amino acid residue. An amino acid may be substituted, for example, via chemical peptide synthesis or through recombinant methods known in the art. For example, substituting an amino acid residue with an alternative amino acid residue is conducted by substituting the codon encoding the first amino acid with a codon encoding the second amino acid. The various polypeptide components of the disclosure may be provided with amino acid substitutions.

[0146] In some embodiments, “Conservative amino acid substitution” is one in which one amino acid residue is replaced with an amino acid residue having a chemically similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including acidic side chains (e.g., aspartic acid, glutamic acid), basic side chains (e.g., lysine, arginine, histidine), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, if an amino acid in a polypeptide is replaced with another amino acid from the same side chain family, the substitution is considered to be conservative. In another aspect, a string of amino acids can be conservatively replaced with a chemically similar string that differs in order and/or composition of side chain family members. The various polypeptide components of the disclosure may be provided with conservative amino acid substitutions.

[0147] In some embodiments, the term “Non-conservative amino acid substitutions” may refer to those substitutions in which (i) a residue having an electropositive side chain (e.g., Arg, His or Lys) is substituted for, or by, an electronegative residue (e.g., Glu or Asp), (ii) a hydrophilic residue (e.g., Ser or Thr) is substituted for, or by, a hydrophobic residue (e.g., Ala, Leu, He, Phe or Vai), (iii) a cysteine or proline is substituted for, or by, any other residue, or (iv) a residue having a bulky hydrophobic or aromatic side chain (e.g., Vai, His, He or Trp) is substituted for, or by, one having a smaller side chain (e.g., Ala or Ser) or no side chain (e.g., Gly). The various polypeptide components of the disclosure may be provided with non-conservative amino acid substitutions. The likelihood that one of the foregoing non-conservative substitutions can alter functional properties of the protein is also correlated to the position of the substitution with respect to functionally important regions of the protein: some non-conservative substitutions can accordingly have little or no effect on biological properties. The various polypeptide components of the disclosure may be provided with non-conservative amino acid substitutions that do not significantly alter the functionality of the altered components.

[0148] In some embodiments, the terms “Transmembrane element” or “transmembrane domain” may refer to a polypeptide element between the extracellular element and the intracellular element. A portion of the transmembrane element exists within the cell membrane. Chimeric antigen receptors (CARs) of the disclosure include transmembrane elements.

[0149] In some embodiments, the terms “Intracellular element” or “intracellular domain” may refer to a polypeptide element that resides on the cytoplasmic side of a cell's cytoplasmic membrane, and transmits a signal into the eukaryotic cell. CARs of the disclosure include intracellular elements. In some embodiments, the cell is a eukaryotic cell.

[0150] In some embodiments, the terms “Intracellular signaling element” or “intracellular signaling domain” refers to a portion of the intracellular element that transduces the effector function signal and, which, subsequently directs a cell to perform a specialized function. In some embodiments, the cell is a eukaryotic cell. [0151] In some embodiments, the terms “Extracellular element” or “extracellular element” may refer to the polypeptide element that resides outside of a cell's cytoplasmic membrane. In a CAR-expressing cell, the extracellular element may comprise an antigen binding element of the CAR. In some embodiments, the cell is a eukaryotic cell.

[0152] Sequence Identity and Related Terminology

[0153] In some embodiments, two or more sequences are said to be “identical” if they are 100% identical to one another.

[0154] In some embodiments, “Identity” refers to the overall monomer conservation between polymeric molecules, e.g., between polypeptide molecules or polynucleotide molecules. “Identical” without any additional qualifiers, e.g., protein A is identical to protein B, implies the sequences are 100% identical (100% sequence identity). Describing two sequences as, e.g., “70% identical,” is equivalent to describing them as having, e.g., “70% sequence identity.”

[0155] In some embodiments, when a position in the first sequence is occupied by the same amino acid as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.

[0156] In certain aspects, the percentage identity (%ID) of a first amino acid (or nucleic acid) sequence to a second amino acid (or nucleic acid) sequence is calculated as %ID = 100 (Y/Z), where Y is the number of amino acid (or nucleobase) residues scored as identical matches in the alignment of the first and second sequences (as aligned by visual inspection or a particular sequence alignment program) and Z is the total number of residues in the second sequence. If the length of a first sequence is longer than the second sequence, the percent identity of the first sequence to the second sequence will be higher than the percent identity of the second sequence to the first sequence.

[0157] In some embodiments, calculation of the percent identity of two polypeptide sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes. For example, gaps can be introduced in one or both of a first and a second polypeptide sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes. In certain aspects, the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the length of the reference sequence. The amino acids at corresponding amino acid positions are then compared.

[0158] In some embodiments, the generation of a sequence alignment for the calculation of a percent sequence identity is not limited to binary sequence-sequence comparisons exclusively driven by primary sequence data. It will also be appreciated that sequence alignments can be generated by integrating sequence data with data from heterogeneous sources such as structural data (e.g., crystallographic protein structures), functional data (e.g., location of mutations), or phylogenetic data. A suitable program that integrates heterogeneous data to generate a multiple sequence alignment is T-Coffee, available at www.tcoffee.org, and alternatively available, e.g., from the European Bioinformatics Institute (EBI) at website ebi.ac.uk/Tools/psa. It will also be appreciated that the final alignment used to calculate percent sequence identity can be curated either automatically or manually.

[0159] Suitable software programs are available from various sources, and for alignment of both protein and nucleotide sequences. One suitable program to determine percent sequence identity is bl2seq, part of the BLAST suite of program available from the U.S. government’s National Center for Biotechnology Information BLAST website (blast.ncbi.nlm.nih.gov). B12seq performs a comparison between two sequences using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other suitable programs are, e.g., Needle, Stretcher, Water, or Matcher, part of the EMBOSS suite of bioinformatics programs and also available from the EBL Sequence alignments can be conducted using methods known in the art such as MAFFT, Clustal (ClustalW, Clustal X or Clustal Omega), MUSCLE, etc. Different regions within a single polynucleotide or polypeptide target sequence that aligns with a polynucleotide or polypeptide reference sequence can each have their own percent sequence identity. It is noted that the percent sequence identity value is rounded to the nearest tenth. For example, values from 80.11 to 80.14 are rounded down to 80.1, while values from 80.15 to 80. 19 are rounded up to 80.2. It also is noted that the length value will always be an integer. [0160] In some embodiments, “Similarity” refers to the overall relatedness between polymeric molecules, e.g. between polynucleotide molecules and/or between polypeptide molecules. Calculation of percent similarity of polymeric molecules to one another can be performed in the same manner as a calculation of percent identity, except that calculation of percent similarity takes into account conservative substitutions as is understood in the art. It is understood that percentage of similarity is contingent on the comparison scale used, for example whether the amino acids are compared, e.g., according to their evolutionary proximity, charge, volume, flexibility, polarity, hydrophobicity, aromaticity, isoelectric point, antigenicity, or combinations thereof.

[0161] In some embodiments, the term “linked” may refer to a fusion of a first moiety to a second moiety at the C-terminus or the N-terminus. In some embodiments, the term “linked” may refer to an insertion of the whole first moiety (or the second moiety) into any two points, e.g., amino acids, in the second moiety (or the first moiety, respectively). In some aspects, the first moiety is linked to a second moiety by a peptide bond or a linker. The first moiety can be linked to a second moiety by a phosphodiester bond or a linker. The linker can be a peptide, a polypeptide, a nucleotide, a nucleotide chain or any chemical moiety.

[0162] In some embodiments, the term “operably -linked” may refer to two or more elements that are functionally linked within a sequence. In some embodiments, two elements may be functionally linked in a folded protein that are not contiguous with respect to the linear sequence. In some embodiments, two elements may be functionally linked, following expression from a multicistronic sequence. In some embodiments, two elements may be functionally linked because one element can induce or inhibit the expression or function of another, either directly or indirectly.

[0163] In some embodiments, the term “non-naturally occurring” may refer to a polypeptide or a polynucleotide sequence that does not exist in nature. In some embodiments, the non- naturally occurring sequence does not exist in nature because the sequence is altered relative to a naturally occurring sequence. In some embodiments, the non-naturally occurring sequence does not exist in nature because it is a combination of two, naturally-occurring, sequences that do not occur together in nature (e.g., chimeric polypeptide). In some embodiments, a non-naturally occurring polypeptide is a chimeric polypeptide. In some embodiments, a polypeptide or a polynucleotide is not naturally occurring because the sequence contains a portion (e.g., a fragment) that cannot be found in nature, i.e., a novel sequence. Any of the polynucleotides described herein may be provided as non-naturally occurring sequences, e.g., having sequences which are altered relative to native sequences or provided as polynucleotides which are linked to other polynucleotides in a manner that does not exist in nature. Any of the polypeptides described herein may be provided as non- naturally occurring sequences, e.g., having sequences which are altered relative to native sequences or provided as polypeptides which are linked to other polypeptides in a manner that does not exist in nature.

Antibodies and Related Terminology

[0164] In some embodiments, the term “Antibody” may refer to various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, and antibody fragments so long as they exhibit the desired antigen-binding activity.

[0165] In some embodiments, the term “Antibody fragment’ ’may refer to a molecule other than an intact antibody that includes a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, scFv antibody fragments, linear antibodies, single domain antibodies such as sdAb (either VL or VH), camelid VHH domains, and multispecific antibodies formed from antibody fragments. Genes of interest of the disclosure, may for example, include antibody fragments.

[0166] In some embodiments, the term “Single chain antibody” (scFv) may refer to an antibody fragment that includes variable regions of heavy (VH) and light (VL) chains, which are linked by a flexible peptide linker.

[0167] In some embodiments, the term “Antigen binding molecule” may refer to a molecule that specifically binds an antigenic determinant. Genes of interest of the disclosure, may for example, include antigen binding molecules.

[0168] In some embodiments, the term “Antigen” may refer to a molecule that provokes an immune response.

[0169] In some embodiments, the term “Chimeric Antigen Receptor” or “CAR” may refer to a fusion protein including antigen recognition moieties and cell-activation elements. Polynucleotides of the disclosure may include genes of interest that produce CARs. [0170] In some embodiments, the term “CAR T cell” or “CAR T lymphocyte” may refer to a T cell containing the capability of producing a CAR polypeptide. For example, a cell that is capable of expressing a CAR is a T cell containing nucleic acid sequences for the expression of the CAR in the cell. Cells of the disclosure may be CAR T-cells.

[0171] In some embodiments, the terms “costimulatory element” or “costimulatory signaling domain” or “costimulatory polypeptide” may refer to an intracellular portion of a costimulatory polypeptide. In some embodiments, CARs comprising costimulatory domains demonstrate increased or enhanced T cell expansion, function, persistence and antitumor activity when expressed in a T-cell as compared to a CAR lacking a costimulatory domain. Costimulatory domains may be provided in CARs of the disclosure by incorporating intracellular signaling domains from one or more T cell costimulatory molecules, such as CD28 or 4-1BB.

[0172] In some embodiments, a costimulatory polypeptide may comprise a sequence isolated or derived from one or more of the following: a TNF receptor protein, an Immunoglobulin-like protein, a cytokine receptor, an integrin, a signaling lymphocytic activation molecule (SLAM proteins), and an activating natural killer cell receptor. In some embodiments, a costimulatory polypeptide may comprise a sequence isolated or derived from one or more of CD27, CD28, 4-1BB (CD137), 0X40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, and MyD88.

Therapeutically Effective and Related Technology

[0173] In some embodiments, the term “Therapeutically effective” may refer to the provision of a beneficial effect on the recipient, e.g., providing some alleviation, mitigation, or decrease in a clinical symptom in the subject or an improvement in a clinical state of a subject. Therapeutic effects need not be complete or curative, as long as some benefit is provided to the subject.

[0174] In some embodiments, the term “Therapeutically effective amount” may refer to a dose sufficient to impart a therapeutically effective benefit on the recipient. For example, fusion proteins, nucleic acids, vectors, cells, compositions, or pharmaceutical compositions of the disclosure may be administered in a therapeutically effective amount. A subject who has been administered fusion proteins, nucleic acids, vectors, cells, compositions, or pharmaceutical compositions of the disclosure may subsequently be administered a therapeutically effective amount of a small molecule of the disclosure to induce or disrupt dimer formation of the fusion proteins of the disclosure to effect the desired cellular outcome.

Cell Terminology

[0175] In some embodiments, the term “Stem cell” may refer to an undifferentiated or partially differentiated cell that can differentiate into various types of cells and proliferate indefinitely to produce more of the same stem cell.

[0176] In some embodiments, the term “Pluripotent stem cell” (PSC) may refer to a cell that can maintain an undifferentiated state indefinitely and can differentiate into most, if not all cells of the body.

[0177] In some embodiments, the term “Induced pluripotent stem cell” (iPS or iPSC) may refer to a pluripotent stem cell that can be generated directly from a somatic cell. This includes, but is not limited to, specialized cells such as skin or blood cells derived from an adult.

[0178] In some embodiments, the term “Multipotent” may refer to a cell that can develop into more than one cell type but is more limited than a pluripotent cell. For example, adult stem cells and cord blood stem cells may be considered as multipotent.

[0179] In some embodiments, the term “Hematopoietic cell” may refer to a cell that arises from a hematopoietic stem cell. This includes, but is not limited to, myeloid progenitor cells, lymphoid progenitor cells, megakaryocytes, erythrocytes, mast cells, myeloblasts, basophils, neutrophils, eosinophils, macrophages, thrombocytes, monocytes, natural killer cells, T lymphocytes, B lymphocytes and plasma cells.

[0180] In some embodiments, the term “T-lymphocyte” or T-cell” may refer to a hematopoietic cell that normally develops in the thymus. T-lymphocytes or T-cells include, but are not limited to, natural killer T cells, regulatory T cells, helper T cells, cytotoxic T cells, memory T cells, gamma delta T cells, and mucosal invariant T cells.

[0181] In some embodiments, the term “Mesenchyme” may refer to a type of animal tissue included of loose cells embedded in a mesh off proteins and fluid, i.e., the extracellular matrix. Mesenchyme directly gives rise to most of the body’s connective tissues including bones, cartilage, lymphatic system, and circulatory system.

[0182] In some embodiments, the term “Mesenchymal cell” may refer to a cell that is derived from a mesenchymal tissue. In some cases, cells of the disclosure may be mesenchymal cells.

[0183] In some embodiments, the term “Mesenchymal stromal cell” (MSC) may refer to a spindle shaped plastic-adherent cell isolated from bone marrow, adipose, and other tissue sources, with multipotent differentiation capacity in vitro. For example, a mesenchymal stromal cell can differentiate into osteoblasts (bone cells), chondrocytes (cartilage cells), myocytes (muscle cells), and adipocytes (fat cells which give rise to marrow adipose tissue). The term mesenchymal stromal cell is suggested in the scientific literature to replace the term “mesenchymal stem cell”. In some cases, cells of the disclosure may be mesenchymal stromal cells.

[0184] In some embodiments of the disclosure, an “autologous cell” is a cell obtained from the same individual to whom it may be administered as a therapy (the cell is autologous to the subject). Autologous cells of the disclosure include, but are not limited to, hematopoietic cells and stem cells, such as hematopoietic stem cells.

[0185] In some embodiments of the disclosure, an allogeneic cell is a cell obtained from an individual who is not the intended recipient of the cell as a therapy (the cell is allogeneic to the subject). Allogeneic cells of the disclosure may be selected from immunologically compatible donors with respect to the subject of the methods of the disclosure. Allogeneic cells of the disclosure may be modified to produce “universal” allogeneic cells, suitable for administration to any subject without unintended immunogenicity. Allogeneic cells of the disclosure include, but are not limited to, hematopoietic cells and stem cells, such as hematopoietic stem cells.

[0186] In some embodiments, the terms “Transfect” or “transform” or “transduce” may refer to a process by which exogenous nucleic acid is transferred or introduced into a cell or a host cell. A “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid or progeny of the cell. [0187] In some embodiments, the term “Cell therapy” may refer to the delivery of a cell or cells into a recipient for therapeutic purposes.

Small Molecule Terminology

[0188] In some embodiments, the term “Analog” may refer to a chemically modified form of a compound, or member of a class of compounds, described herein which maintains the binding properties of the compound or class. For example, an analog of danoprevir would include chemically modified forms of danoprevir that retains the ability to bind DNCR2and NS3a as described herein.

[0189] In some embodiments, the term “prodrug”, may include any covalently bonded carriers which release a small molecule of the disclosure in vivo when such prodrug is administered to a patient. Prodrugs of the disclosure may be prepared by modifying functional groups present in the compound in such a way that the modifications are cleaved, either in routine manipulation or in vivo, to the parent compound. The transformation in vivo may be, for example, as the result of some metabolic process, such as chemical or enzymatic hydrolysis of a carboxylic, phosphoric or sulphate ester, or reduction or oxidation of a susceptible functionality. Prodrugs within the scope of the disclosure include compounds wherein a hydroxy, amino, or sulfhydryl group is bonded to any group that, when the prodrug of the disclosure is administered to a mammalian subject, it cleaves to form a free hydroxyl, free amino, or free sulfhydryl group, respectively. Functional groups that may be rapidly transformed, by metabolic cleavage, in vivo form a class of groups reactive with the carboxyl group of the compounds of this disclosure. They include, but are not limited to, such groups as alkanoyl (such as acetyl, propionyl, butyryl, and the like), unsubstituted and substituted aroyl (such as benzoyl and substituted benzoyl), alkoxy carbonyl (such as ethoxy carbonyl), trialky silyl (such as trimethyl- and triethy silyl), monoesters formed with dicarboxylic acids (such as succinyl), and the like. The small molecules of the disclosure may be administered as prodrugs. The small molecules of the disclosure may be administered to a subject as a prodrugs. A therapeutically effective amount of such a prodrug of the disclosure may be administered. The prodrug may be administered contemporaneously with the administration of the polynucleotides, gene therapy vectors or cells of the disclosure or following the administration of the polynucleotides, gene therapy vectors or cells of the disclosure. Formulations and Related Technology

[0190] In some embodiments, the term “Pharmaceutically acceptable” may refer to compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit/risk ratio. For example, the small molecules, polynucleotides, polypeptides, gene therapy vectors or cells of the disclosure may be administered as part of a composition together with other pharmaceutically acceptable components, including pharmaceutically acceptable carriers.

[0191] In some embodiments, the term “Pharmaceutically acceptable salts” may refer to derivatives of the small molecules of the disclosure wherein the specified compound is converted to an acid or base salt thereof. Such pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. The pharmaceutically acceptable salts include the conventional non-toxic salts or the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluensulfonic, methanesulfonic, ethane dislfonic, oxalic, isethionic, and the like. For example, the small molecules of the disclosure may be provided as pharmaceutically acceptable salts.

[0192] In some embodiments, the term “Controlled release” may refer to part or all of a dosage form that can release one or more active pharmaceutical agents over a prolonged period of time (i. e. , over a period of more than 1 hour). The characteristic of controlled release (CR) may also be referred to as sustained release (SR), prolonged release (PR), or extended release (ER). When used in association with the dissolution profiles discussed herein, the term “controlled release” refers to that portion of a dosage form according to the disclosure that delivers active agent over a period of time greater than 1 hour. For example, the small molecules of the disclosure may be administered in a controlled release composition. [0193] In some embodiments, the term “Immediate release” may refer to part or all of a dosage form that releases active agent substantially immediately upon contact with gastric juices and that results in substantially complete dissolution within about 1 hour. The characteristic of immediate release (IR) may also be referred to as instant release (IR). When used in association with the dissolution profiles discussed herein, the term “immediate release” refers to that portion of a dosage form according to the disclosure that delivers active agent over a period of time less than 1 hour. The small molecules of the disclosure may be administered in an immediate release composition.

[0194] In some embodiments, the term “Excipients” may refer to pharmacologically inert ingredients that are not active in the body. See, for example, Hancock, B. C., Moss, G. P., & Goldfarb, D. J. (2020). Handbook of pharmaceutical excipients. London: Pharmaceutical Press, the entire disclosure of which is incorporated herein by reference. The small molecules of the disclosure may be mixed with pharmaceutically acceptable carriers, diluents, adjuvants, excipients, or vehicles, such as preserving agents, fillers, polymers, disintegrating agents, glidants, wetting agents, emulsifying agents, suspending agents, sweetening agents, flavoring agents, perfuming agents, lubricating agents, acidifying agents, and dispensing agents, depending on the nature of the mode of administration and dosage forms. Such ingredients, including pharmaceutically acceptable carriers and excipients that may be used to formulate oral dosage forms. Pharmaceutically acceptable carriers include water, ethanol, polyols, vegetable oils, fats, waxes polymers, including gel forming and nongel forming polymers, and suitable mixtures thereof. Examples of excipients include starch, pregelatinized starch, Avicel, lactose, milk sugar, sodium citrate, calcium carbonate, dicalcium phosphate, and lake blend. Examples of disintegrating agents include starch, alginic acids, and certain complex silicates. Examples of lubricants include magnesium stearate, sodium lauryl sulphate, talc, as well as high molecular weight polyethylene glycols. For example, the small molecules, polynucleotides, polypeptides, gene therapy vectors or cells of the disclosure may be provided and administered in compositions that include pharmaceutically acceptable excipients.

General Terminology

[0195] As used throughout the disclosure, the term “Subject” includes any mammal, including without limitation, humans. In some embodiments, the subject is a human. In some embodiments, the subject is a neonate, an infant, a toddler, a child, a teenager, an adult, a senior, a centenarian. In some embodiments, the subject has at least 1, 2, or 3 X chromosomes. In some embodiments, the subject has at least 1 or 2 Y chromosomes. In some embodiments, the subject is diagnosed with a disease or disorder of the disclosure, or otherwise, in need of treatment of the disclosure. In some embodiments, the subject is at risk of developing a disease or disorder of the disclosure, or otherwise, in need of preventing a disease or disorder of the disclosure.

[0196] In some embodiments, the subject is a non-human primate. In some embodiments, the subject is a mammal, including, but not limited to, livestock, a horse, a dog, a cat, a pig, a rabbit, a guinea pig, a rodent, a rat, a gerbil, and a mouse. In some embodiments, the subject is anon-primate mammal and the subject is genetically -modified.

[0197] "A", "an" and "the" include their plural forms unless the context clearly dictates otherwise.

[0198] “And” is used interchangeably with “or” unless expressly stated otherwise.

[0199] "And/or" is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, "and/or" as used in a phrase such as "A and/or B," includes "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, "and/or," as used in a phrase such as "A, B, and/or C," is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0200] In some embodiments, the term "About" means approximately, roughly, around, or in the regions of. When "about" is used with a numerical range, it may modify that range by extending the boundaries above and below the numerical values set forth.

[0201] In some embodiments, numeric ranges are inclusive of the numbers defining the range. Where a range of values is stated, each intervening integer value, and each fraction thereof, between the recited upper and lower limits of that range is also specifically disclosed, as is each subrange between such values. The upper and lower limits of any range can independently be included in or excluded from the range, and each range where either, neither or both limits are included is also encompassed within the disclosure. Thus, ranges are understood to be shorthand for all of the values within the range, inclusive of the recited endpoints. For example, a range of 1 to 10 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0202] Where a value is explicitly stated, it is to be understood that values which are about the same quantity or amount as the stated value are also within the scope of the disclosure. Where a combination is disclosed, each subcombination of the elements of that combination is also specifically disclosed and is within the scope of the disclosure. Conversely, where different elements or groups of elements are individually disclosed, combinations thereof are also disclosed. Where any element of a disclosure is disclosed as having a plurality of alternatives, examples of that disclosure in which each alternative is excluded singly or in any combination with the other alternatives are also hereby disclosed; more than one element of a disclosure can have such exclusions, and all combinations of elements having such exclusions are hereby disclosed.

[0203] Unless the context requires otherwise, throughout the description and the claims, the words ‘include’, ‘including’, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”.

[0204] Singular or plural words also include the plural and singular number, respectively. Thus, for example, where the specification describes a gene of interest, the disclosure includes polynucleotides with a single gene of interest or multiple genes of interest.

[0205] “Above,” and “below” and words of similar import refer to this application as a whole and not to any particular portions of the application.

[0206] “Set” includes sets of one or more elements or objects.

[0207] Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form.

[0208] Headings are included herein for reference and to aid in locating the various sections. These headings are not intended to limit the scope of the concepts described with respect to the headings. Such concepts may have applicability throughout the present specification. [0209] Although the disclosure is described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent to those skilled in the art that certain changes and modifications may be practiced. Reference to “the disclosure” or the like is intended as a reference to any of a wide variety of embodiments of, or aspects of, the disclosure, and not as limiting the disclosure to a single embodiment or aspect. As used throughout the disclosure, the terms “aspect” and “embodiment” are interchangeable. Features discussed in the context of “certain”, “some”, or “other” aspects or embodiments of the disclosure may be found in any embodiment of the disclosure, however, in these instances, the feature may be considered a preferred feature in these highlighted embodiments.

[0210] The description and examples should not be construed as limiting the scope of the disclosure to the embodiments and examples described herein, but rather as encompassing all modifications and alternatives falling within the true scope and spirit of the disclosure.

Synthetic Degrader System for Targeted Degradation

[0211] The disclosure provides a synthetic degrader system for targeted degradation of a molecule of interest. The system generally includes a polynucleotide encoding a degradation initiator fused to a binding element, wherein the binding element recruits the degradation initiator to the molecule of interest to initiate degradation.

[0212] In some embodiments, the system includes a polynucleotide set that includes a first polynucleotide and a second polynucleotide, wherein either the first or second polynucleotide encodes the degradation initiator fused to a first binding element and the other of the first or second polynucleotides encodes a synthetic or exogenous molecule of interest fused to a second binding element. For example, the first polynucleotide may encode the degradation initiator fused to a first binding element and the second polynucleotide may encode the synthetic or exogenous molecule of interest, wherein interaction of the first and second binding elements mediates recruitment of the degradation initiator to the molecule of interest to initiate degradation. The first and second polynucleotides may be provided as a single polynucleotide or as a set of two or more polynucleotides.

[0213] In some embodiments, the system includes a polynucleotide set that includes a polynucleotide encoding a degradation initiator fused to a binding element that is specific for a native motif on an endogenous molecule of interest. The binding domain may, for example, recognize and bind a specific region on the molecule of interest or a specific modification on the molecule of interest. Interaction of the binding element with the native motif on the molecule recruits the degradation initiator to the molecule to initiate degradation.

[0214] In some embodiments, a pair of designed heterodimer (DHD) proteins may be used as binding elements, wherein one DHD binding element is fused to a degradation initiator and the second DHD binding element is fused to a synthetic or exogenous molecule of interest. Dimerization of the DHD binding elements brings the degradation initiator into proximity with the targeted molecule, thereby facilitating degradation of the targeted molecule.

[0215] In some embodiments, a pair of small molecule-regulated polypeptides may be used as binding elements, wherein one binding element is fused to a degradation initiator and the second polypeptide is fused to a synthetic or exogenous protein molecule of interest. The first and second small molecule-regulated polypeptides may be selected so that interaction of the first and second polypeptides is mediated by the presence of a small molecule. For example, the first and second small molecule-regulated polypeptides may assemble, together with the small molecule, to form a dimerization complex. The formation of the dimerization complex brings the degradation initiator into proximity with the molecule of interest, thereby facilitating degradation of the targeted molecule.

[0216] In some embodiments, the system includes a polynucleotide set that includes a polynucleotide encoding a degradation initiator fused to a binding element that is specific for a native motif on an endogenous molecule of interest. The binding element may, for example, recognize and bind a specific region on the molecule of interest or a specific modification on the molecule of interest. Interaction of the binding element with the native motif on the molecule effectively recruits the degradation initiator to the molecule of interest to initiate degradation.

[0217] In some embodiments, the disclosure provides a synthetic degrader system for targeted degradation of a synthetic or exogenous protein of interest. The system generally includes a polynucleotide encoding an E3 ubiquitin ligase (“E3 ligase”) domain (or functional variant thereof) fused to a binding element, wherein the binding element is used to recruit the E3 ligase domain to a protein of interest for targeted degradation via a cellular ubiquitin pathway. [0218] In some embodiments, a pair of designed heterodimer (DHD) proteins is used as binding elements, wherein one DHD binding element is fused to an E3 ligase domain (or functional variant thereof) and the second DHD binding element is fused to a synthetic or exogenous protein target of interest. Dimerization of the DHD binding elements brings the modified E3 ligase into proximity with the targeted protein, thereby facilitating ubiquitination and subsequent degradation of the protein target.

[0219] In some embodiments, a pair of small molecule-regulated polypeptides is used as binding elements, wherein one binding element is fused to an E3 ligase domain (or functional variant thereof) and the second polypeptide is fused to a synthetic or exogenous protein target of interest. The first and second small molecule-regulated polypeptides may be selected so that interaction of the first and second polypeptides is mediated by the presence of a small molecule. For example, the first and second small molecule-regulated polypeptides may assemble, together with the small molecule, to form a dimerization complex. The formation of the dimerization complex brings the E3 ligase into proximity with the target protein, thereby facilitating degradation of the protein target.

[0220] In some embodiments, the system includes a polynucleotide set that includes a polynucleotide encoding an E3 ligase domain (or functional variant thereof) fused to a binding element that is specific for a native motif on an endogenous protein of interest. The binding element may, for example, recognize and bind a specific region on the protein of interest or a specific post-translational modification on the protein of interest. Interaction of the binding element with the native motif on the protein effectively recruits the E3 ligase domain to the protein for targeted degradation.

[0221] The synthetic degrader system of the disclosure is useful for regulating the activity of a range of target molecules (e.g., proteins) in various synthetic biology applications, such as therapeutic applications.

[0222] In one embodiment, the synthetic degrader system of the disclosure may be used in a cell therapy application to program a population of cells for performing and/or modulating a therapeutic function.

[0223] In one aspect, the synthetic degrader system of the disclosure may be used to regulate (degrade) T cell receptors (TCRs) and/or chimeric antigen receptors (CARs) cells used in cancer therapy. More specifically, the synthetic degrader system of the disclosure may be used to degrade CARs and/or TCRs and inhibit excessive CAR/TCR signaling that may result in undesirable exhaustion phenotypes observed in many existing T-cell therapies.

[0224] In one embodiment, the synthetic degrader system of the disclosure may be used in gene therapy applications.

Binding Elements

[0225] The disclosure provides binding elements that are designed to recruit a fused degradation initiator to a molecule of interest for targeted degradation. In some embodiments, a binding element is designed to specifically and/or selectively bind to a molecule of interest targeted for degradation. In some embodiments, a binding element(s) may be selected based on the molecule of interest targeted for degradation.

[0226] A binding element or a pair of binding elements for targeted degradation of a protein of interest may be designed using a de novo design strategy. In some embodiments, a binding element may be designed for targeted degradation of a specific protein target. In some embodiments, a binding element may be designed for targeted degradation of multiple proteins. For example, a binding element may be designed that is specific for a conserved motif present in multiple proteins (e.g., a protein family).

[0227] In some embodiments, a library-based binder panning approach may be used to identify a recombinant binding element that is specific for a protein of interest. A recombinant binding element may, for example, be an immunoglobulin derivative or a nonimmunoglobulin binder that is based on natural or designed protein scaffolds. Examples of immunoglobulin derived binding formats that may be used include, but are not limited to, single-chain variable fragments (scFvs) and nanobodies. Examples of non-immunoglobulin binder formats that may be used include, but are not limited to, monobodies, designed ankyrin repeat protein domains (DARPins), and variable lymphocyte receptor (VLR) domains.

Designed Heterodimers (DHDs)

[0228] A pair of designed heterodimer (DHD) proteins may be used as binding elements to recruit a fused degradation initiator to a synthetic or exogenous molecule of interest. For example, one DHD binding element may be fused to a degradation initiator and the second DHD binding element of the binding pair may be fused to the synthetic or exogenous molecule of interest. Dimerization of the pair of DHD binding elements brings the degradation initiator into proximity with the targeted molecule for targeted degradation.

[0229] A pair of designed DHD binding elements may, for example, be designed using the de novo Rosetta protein design software package and previously published methods (Chen, Z. et al., Nature (2019) doi:10.1038/s41586-018-0802-y, which is incorporated herein by reference in its entirety). Amino acid sequences of dimerization domains, including sequences of designed DHDs are provided in Table 1.

[0230] A DHD binding pair may, for example, include a three-helix protein bundle binding element and a single-helix protein binding element, i.e., a 3+1 heterodimer binding pair. An example of a 3+1 heterodimer binding pair is shown in FIG. 1.

[0231] FIG. 1 is a diagram 100 of an example of a 3+1 heterodimer binding element pair. In this example, the binding pair includes a three-helix protein bundle DHD-A and a singlehelix protein DHD-B. One binding element (e.g., “A”) may be fused to a molecule of interest (e.g., a protein; not shown) and the second binding element (e.g., “B”) of the binding pair may be fused to a degradation initiator (e.g., an E3 ligase domain; not shown) to provide a “protein only” dimerization system for targeted degradation the molecule of interest.

[0232] A pair of DHD binding elements with alternative topologies may also be used as binding elements. Alternative topologies include, but are not limited to, dimers with one or both subunits comprised of one-helix subunits, two-helix subunits, multiple-helix subunits, or subunits with non-helical topologies.

[0233] A pair of binding elements may, for example, include SYNZIPs. SYNZIPs are synthetic heterodimeric coiled coils that form a diverse set of interaction connectivities (Thompson, K.E., ACS Synth. Biol. (2012) 1: 118-129, which is incorporated herein by reference in its entirety).

[0234] A pair of binding elements may, for example, include two-helix coiled coils (Ghosh, I., et al., J. Am. Chem. Soc. (2000) 122: 5658-5659, which is incorporated herein by reference in its entirety).

[0235] A pair of DHD binding elements may, for example, include a two-helix subunit and a one-helix subunit (e.g., 2plusl_5400_dimerl.l_short). [0236] Surface lysine residues in the designed binding element sequences may in some embodiments be mutated to arginine to prevent auto-ubiquitination of the binding element without altering the structure and function of the binding element. Surface residues of the designed binding elements can be varied to alter charge and other properties that can influence expression levels (e.g. the ‘surface_redesign’ variants in Table 1). Mutations to the core of the designed binding elements can be used to alter the binding affinity of a given heterodimer (e.g. DHD37-short-B-Y8A; SEQ ID NO: 14).

Small Molecule-Regulated Polypeptides

[0237] In some embodiments, a pair of small molecule-regulated polypeptides may be used as binding elements to recruit a fused degradation initiator to a molecule of interest. The use of small molecule-regulated polypeptides extends the functionality of the degrader system and provides an extrinsic control mechanism (i.e., a chemical control mechanism) for regulating degradation of the targeted molecule.

[0238] For example, a first small molecule-regulated polypeptide may be fused to a degradation initiator and a second small molecule-regulated polypeptide may be fused to a synthetic or exogenous molecule of interest. The first and second small molecule-regulated polypeptides may be selected so that interaction of the first and second small molecule- regulated polypeptides to form a dimerization complex is mediated by the presence of the small molecule. In some cases, the small molecule may mediate assembly of the dimerization complex. In other cases, the small molecule may mediate disassembly of the dimerization complex. In still other cases, a first small molecule may mediate assembly of the dimerization complex while a second small molecule may displace the first small molecule and thereby mediate disassembly of the dimerization complex.

[0239] FIG. 2 is a diagram 200 illustrating an example of a pair of small molecule-regulated polypeptide binding elements. One binding element (e.g., “A”) may be fused to a molecule of interest (e.g., a protein; not shown) and the second binding partner (e.g., “B”) may be fused to a degradation initiator (e.g., an E3 ligase domain; not shown) to provide a small molecule-regulated dimerization system for targeted degradation of the molecule of interest.

[0240] As an example, a pair of small molecule-regulated binding elements may include the hepatitis C virus protease NS3a/4a protein (hereafter referred to as NS3a) or a functional variant thereof as a first binding element and a “reader” protein as a second binding element. The reader protein may, for example, be selected to recognize a specific drug-bound state of the NS3a protein. NS3a proteins and NS3a reader proteins have been described in Baker et al., International Patent Publication W02020117778, entitled “Reagents and Methods for Controlling Protein Function and Interaction,” published on June 11, 2020, which is incorporated herein by reference in its entirety.

[0241] NS3a can integrate multiple drug inputs and translate the drug inputs into diverse outputs using different engineered reader proteins as binding partners. NS3a proteins and pleiotropic response outputs from danoprevir/NS3a complex readers, grazoprevir/NS3a complex readers, and ANR/NS3a complex readers have been described in Foight, G.W., et al., Nature Biotechnology (2019) 37:1209-1216; Cunningham-Bryant, D. et al., Journal of the American Chemical Society (2019) 141: 3352-3355; and Kugler, J., et al., Journal of Biological Chemistry (2012) 287:39224-39232, which are incorporated herein by reference in their entireties.

[0242] Interaction between the NS3a and reader binding partners may be controlled by the presence of a small molecule drug. A reader may be selected to recognize and bind a specific NS3a/drug complex.

[0243] In some embodiments, the reader selected for the dimer is a danoprevir/NS3 complex reader (DNCR) polypeptide (or functional variants thereol) designed to recognize and bind NS3a in the presence of the small molecule drug danoprevir, thereby providing a druginducible transcription system. In one example the DNCR polypeptide is DNCR2. See Foight, G.W., et al., Nature Biotechnology (2019) 37:1209-1216.

[0244] In some embodiments, the reader selected for the binding element pair is a grazoprevir/NS3 complex reader (GNCR) polypeptide (or functional variants thereol) designed to recognize and bind NS3a in the presence of the small molecule drug grazoprevir, thereby providing a drug-inducible transcription system. In one example, the GNCR protein is GNCR1. See Foight, G.W., et al., Nature Biotechnology (2019) 37:1209-1216.

Native Motif Target-Specific Binding Element

[0245] An endogenous molecule of interest may be targeted for degradation using a degradation initiator fused to a binding element that interacts with a native motif on the molecule. [0246] In one embodiment, a binding element may be designed to bind the target molecule in a conformation-specific manner.

[0247] In one embodiment, a binding element may be designed to bind the molecule in response to a specific modification on the molecule. For example, a binding element may be designed to bind a target protein and/or protein complex in response to a post-translation modification of the protein. In one example, a binding element may be designed to bind a specific phosphorylation state of the target protein.

[0248] FIG. 3 is a diagram 300 of an example of regulating an endogenous molecule of interest using a binding element (“B”) that is specific to a native motif (“A”) on the target molecule. Endogenous targets may be regulated using binding elements B that are specific to the native motif A on the target, including conformational-specific binding or binding in response to a specific modification on the molecule.

Targeted Degradation of Proteins

[0249] The disclosure provides a synthetic degrader system for targeted degradation of a protein of interest. The system generally includes a polynucleotide encoding an E3 ligase domain (or functional variant thereof) fused to a binding element, wherein the binding element is used to recruit the E3 ligase domain to a protein of interest to initiate degradation.

E3 Ubiquitin Ligase

[0250] E3 ligases control substrate specificity and the topology of ubiquitination. For example, in a cellular ubiquitin proteasome system, an E3 ligase protein recruits an E2 ubiquitin-conjugating enzyme that has been loaded with ubiquitin, recognizes a protein substrate, and assists or directly catalyzes the transfer of ubiquitin from the E2 to the protein substrate, thereby targeting the protein for degradation. An E3 ligase domain and/or a variant thereof may be selected based on the intended protein targeted for degradation. For example, an E3 ligase and/or a set of E3 ligases may be selected for modulating the stability and/or half-life of a targeted transmembrane protein. In another example, an E3 ligase and/or a set of E3 ligases may be selected for modulating the stability and/or half-life of a cytoplasmic protein (e.g., a transcription factor).

[0251] Functional E3 ligases (as well as functional variants thereof) for efficient degradation of target proteins may be identified using a screening panel strategy. For example, a screening panel may include constructs that encode an E3 -ligase domain, or a ubiquitin variant fused to a binding element (e.g., DHD-B or DHD-A binding element). An example of a screening panel for identifying functional E3 ligases for degradation of target proteins is shown in Table 2. In this example, the panel of E3 ligase domains and ubiquitin variants includes several published ubiquitin and E3 ligase domains: 3xUb, LNX1, NEDD4, VHL, SPOP, SOCS2, Elongin C, CHIP, FBW1A, FBXW7, and RFN4 (Zhu, L., et al., Elife (2017) doi:10.7554/eLife.26403; Hatakeyama, E., et al., Cancer Res. (2005) doi: 10.1158/0008- 5472.CAN-05-1581; Lim, S. et al., Proc. Natl. Acad. Sci. U. S. A. (2020) doi: 10.1073/pnas.1920251117; Ibrahim, A.F.M., et al., Mol. Cell (2020) doi:10.1016/j.molcel.2020.04.032; and Deng, W. et al., Nat. Commun. (2020) doi:10.1038/s41467-019-14160-8; which are incorporated herein by reference in their entirety). The panel of E3 ligase domains and ubiquitin variants also includes several novel designs that incorporate the following domains: RNF43, RNF128, ZNRF3, MARCH8, TRAF6, and tandem ubiquitin mutants (K48R and K63R). This panel includes both cytoplasmic and membrane-associated DHD-E3 degrader designs.

[0252] A screening strategy may use, for example, co-transduction of cells with a DHD-E3 ligase construct or a DHD-ubiquitin variant construct in combination with a model target protein fused to the corresponding DHD binding element partner. For example, to screen the DHD-E3 ligase constructs or the DHD-ubiquitin variant constructs for efficient degradation of a transmembrane protein, a transmembrane receptor domain fused to a corresponding DHD binding element partner may be used. In one example, an ROR1 -specific chimeric antigen receptor fused to a DHD-A binding element may be used as a model protein target to screen for degradation of a transmembrane protein.

[0253] Similarly, to screen the DHD-E3 ligase constructs or the DHD-ubiquitin variant constructs for efficient degradation of a cytoplasmic protein, a model transcription factor domain fused to a corresponding DHD binding element may be used. In one example, a FLAG-tagged transcription factor BACH2 fused to a DHD-B binding element may be used as a model protein target to screen for degradation of a cytoplasmic protein.

[0254] Examples of model target protein fusion constructs that may be used to screen the binding element-E3 ligase constructs or the binding element-ubiquitin variant constructs for efficient degradation of a transmembrane protein or a cytoplasmic protein are shown in Table 3. Polynucleotides

[0255] A degrader system generally includes a polynucleotide encoding an E3 ubiquitin ligase (“E3 ligase”) domain (or functional variant thereof) fused to a binding element, wherein the binding element is used to recruit the E3 ligase domain to a protein of interest for targeted degradation.

Heterodimer Binding Elements

[0256] In various embodiments, a degrader system may include a polynucleotide set that includes a first polynucleotide and a second polynucleotide, wherein either the first or second polynucleotide encodes an E3 ligase domain fused to a first binding element and the other of the first or second polynucleotide encodes a synthetic or exogenous target protein of interest fused to a corresponding second binding element.

[0257] In some embodiments, the polynucleotide set includes a polynucleotide component that may include:

(i) a first polynucleotide encoding a first fusion protein that includes a first binding element and an E3 ligase domain,

(ii) a second polynucleotide encoding a second fusion protein that includes a second binding element and a synthetic or exogenous protein of interest,

(iii) one or more promoter sequences operatively linked to the first and/or second polynucleotides,

(iv) an optional separation element that includes a polynucleotide sequence that prevents fusion of the first fusion protein and the second fusion protein, and

(v) one or more optional regulatory sequences, wherein the first and second polynucleotides, promoter sequence(s), and optional separation element and regulatory elements are configured for expression and regulated degradation of the protein of interest.

[0258] The polynucleotides encoding the first and second fusion proteins, promoter sequence(s), and optional separation element and regulatory sequences may be configured in a vector backbone for expression and regulated degradation of the protein of interest. [0259] In various embodiments, the polynucleotide component encoding the fusion proteins may include a polynucleotide sequence encoding a separation element separating the fusion proteins.

[0260] In some embodiments, the separation element may include a ribosomal skipping sequence selected from the group consisting of P2a and T2a.

[0261] In some embodiments, the separation element may include a polynucleotide sequence that includes at least two ribosomal skipping sequences selected from the group consisting of T2a-RFP-P2a , P2a-T2a , and T2a-P2a .

[0262] In some embodiments, the separation element may include an internal ribosome entry site (IRES).

[0263] In some embodiments, the separation element may include a second promoter sequence.

[0264] In some embodiments, the polynucleotide component that includes the first and second polynucleotides encodes one or more constitutive promoter sequences operatively linked to the first and/or second polynucleotides.

[0265] In some embodiments, the polynucleotide component that includes the first and second polynucleotides encodes one or more inducible promoter sequences operatively linked to the first and/or second polynucleotides.

[0266] In some embodiments, the first polynucleotide encoding the first fusion protein including the E3 ligase domain also encodes an inducible promoter and the second polynucleotide encoding the second fusion protein including the protein of interest encodes a different inducible promoter.

[0267] In some embodiments, the polynucleotide encoding the first fusion protein including the E3 ligase domain encodes an inducible promoter sequence and the polynucleotide encoding the second fusion protein including the protein of interest encodes a constitutive promoter sequence.

[0268] In various embodiments, the constitutive promoter sequence may include a constitutive promoter sequence selected from the group consisting of MND (SEQ ID NO: 144), hPGK (SEQ ID NO: 145), CMV (SEQ ID NO: 146), CAG (SEQ ID NO: 147), SFFV (SEQ ID NO: 148), EFlalpha (SEQ ID NO: 149), UBC (SEQ ID NO: 150), and CD43 (SEQ ID NO: 151) (see Table 8).

[0269] In some embodiments, the inducible promoter sequence may include a minimal promoter sequence, such as for example, a minimal core promoter. In some embodiments, the minimal promoter sequence may be selected from the group consisting of YB TATA (SEQ ID NO: 152), human beta globin (huBG) (SEQ ID NO: 153), minIL2 (SEQ ID NO: 154), minimalCMV (minCMV) (SEQ ID NO: 155), and TRE3G (SEQ ID NO: 156) (see Table 9).

[0270] In some embodiments, the inducible promoter sequence includes a transcription factor-specific recognition sequence.

[0271] In some embodiments, the transcription factor-specific recognition sequence may include a response element that is repeated 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times.

[0272] In some embodiments, the transcription factor-specific recognition sequence may include a response element selected to respond to a cell-intrinsic stimulus.

[0273] In some embodiments, the transcription factor-specific recognition sequence may include a calcium-responsive NFAT-AP1 response element (Hooijberg, E., et al., Blood (2000) 96: 459-466, which is incorporated herein by reference in its entirety).

[0274] In certain embodiments, the inducible promoter sequence includes a minimal CMV (minCMV) promoter sequence and an NFAT-AP1 response element that is repeated 4 times (SEQ ID NO: 143) (see Table 7).

[0275] In some embodiments, the transcription factor-specific recognition sequence may include a response element selected to respond to an environmental stimulus.

[0276] In some embodiments, the first and/or second polynucleotides may encode one or more optional regulatory sequences selected from the group consisting of poly A, endo.

[0277] In some embodiments, the polynucleotide encoding the first and/or second fusion protein further includes a protein tag sequence (e.g., a FLAG-tag sequence). [0278] In some embodiments, the first or second polynucleotide encoding an E3 ligase domain and a first binding element further includes a flexible linker sequence separating the E3 ligase domain and the binding element. In one example, the flexible linker is a glycineserine linker.

[0279] In some embodiments, the first or second polynucleotide encoding a protein of interest and a second binding element further includes a flexible linker sequence separating the protein of interest and the binding element. In one example, the flexible linker is a glycine-serine linker.

[0280] In some embodiments, the first or second polynucleotide encodes a DHD-A binding element and the other of the first or second polynucleotide encodes a DHD-B binding element, wherein the DHD binding pair is selected from the group consisting of DHD37- short-A (SEQ ID NO: 11) and either DHD37-short-B (SEQ ID NO: 13), DHD37-short-B- Ntrunc (SEQ ID NO: 15), or DHD37-short-B-KtoR (SEQ ID NO: 16).

[0281] In some embodiments, the first or second polynucleotide encodes a binding element selected from the group consisting of Synzipl (SEQ ID NO: 66), Synzip2 (SEQ ID NO: 67), Synzip3 (SEQ ID NO: 68), Synzip4 (SEQ ID NO: 69), Synzip5 (SEQ ID NO: 70), Synzip6 (SEQ ID NO: 71), Coil-coil-CZ-A (SEQ ID NO: 72), or Coil-coil-NZ-B (SEQ ID NO: 73).

[0282] In various embodiments, either the first or second polynucleotide encodes a fusion protein including a binding element and an E3 ligase domain, which may include: RNF43 198- 783 -DHD37-short-B (SEQ ID NO: 74), RNF43 198- 364 -DHD37-short-B (SEQ ID NO: 75), RNF43 195-325 -DHD37-short-B (SEQ ID NO: 76), RNF43 198-317 -DHD37-short-B (SEQ ID NO: 77), RNF128 208-428 -DHD37-short-B (SEQ ID NO: 78), RNF128 208-333 -DHD37-short-B (SEQ ID NO: 79), RNF128 208 325 -DHD37-short-B (SEQ ID NO: 80), DHD37-short-B-3xUb (SEQ ID NO: 81), DHD37-short-B-3xUb K48R (SEQ ID NO: 82), DHD37-short-B-3xUb K63R (SEQ ID NO: 83), ZNRF3 219- 936 -DHD37-short-B (SEQ ID NO: 84), ZNRF3219-528-DHD37-short- B (SEQ ID NO: 85), ZNRF3 219- 345 -DHD37-short-B (SEQ ID NO: 86), DHD37-short-B- MARCH8 1- 154 -KLRG1 37-60 (SEQ ID NO: 87), DHD37-short-B-MARCH8 1-228 (SEQ ID NO: 88), MARCH8 1-228 -DHD37-short-B (SEQ ID NO: 89), LNXl 1- 171 -DHD37-short-B (SEQ ID NO: 90), DHD37-short-B-VHL 152-213 (SEQ ID NO: 91), DHD37-short-B-NEDD4 921- 1303 (SEQ ID NO: 92), DHD37-short-B-SPOP 167- 374 (SEQ ID NO: 93), DHD37-short-B- SOCS2 143- 198 (SEQ ID NO: 94), DHD37-short-B-CHIP 128-303 (SEQ ID NO: 95), FBXW7 1-293 - DHD37-short-B (SEQ ID NO: 96), ELOC 1- 87 -DHD37-short-B (SEQ ID NO: 97), FBW1A 1- 261 -DHD37-short-B (SEQ ID NO: 98), DHD37-short-B-RNF4 71- 190 (SEQ ID NO: 99), TRAF6 49-237 -DHD37-short-B (SEQ ID NO: 100), LNXl 1- 171 -DHD37-short-A (SEQ ID NO: 101), DHD37-short-A-VHL 152-213 (SEQ ID NO: 102), DHD37-short-A-NEDD4 921- 1303 (SEQ ID NO: 103), DHD37-short-A-SPOP 167-374 (SEQ ID NO: 104), DHD37-short-A-SOCS2 143- 198 (SEQ ID NO: 105), DHD37-short-A-CHIP 128-303 (SEQ ID NO: 106), FBXW7 1- 293 -DHD37- short-A (SEQ ID NO: 107), ELOC 1- 87 -DHD37-short-A (SEQ ID NO: 108), FBW1A 1-261 - DHD37-short-A (SEQ ID NO: 109), DHD37-short-A-RNF4 71- 190 (SEQ ID NO: 110), Synzip2-RNF4 (SEQ ID NO: 111), Synzip4-RNF4 (SEQ ID NO: 112), Synzip6-RNF4 (SEQ ID NO; 113), Coil-coil-NZ-RNF4 (SEQ ID NO: 114), RNF43 198-317 -DHD37-short-B-KtoR (SEQ ID NO: 121), RNF43 198-317 -DHD37-short-B-endo-varl (SEQ ID NO: 122), RNF43 198- 317 -DHD37-short-B-endo-var2 (SEQ ID NO: 123), RNF43 198-317 -DHD37-short-B-endo-var3 (SEQ ID NO: 124), or RNF43 198-317 -KtoR-DHD37-short-B-endo-varl (SEQ ID NO: 125). See Table 2 and Table 4.

[0283] In various embodiments, either the first or second polynucleotide encodes a fusion protein including a binding element and a target protein of interest.

[0284] In some embodiments, either the first or second polynucleotide encodes a fusion protein including a binding element and an ROR1-CAR protein, which may include: ROR1- CAR-DHD37-short-A (SEQ ID NO: 115), ROR1 CAR-Synzipl (SEQ ID NO: 116), ROR1 CAR-Synzip3 (SEQ ID NO: 117), ROR1 CAR-Synzip5 (SEQ ID NO: 118), or ROR1 CAR- Coil coil-CZ (SEQ ID NO: 119). See Table 2.

[0285] In certain embodiments, either the first or second polynucleotide encodes a fusion protein including a DHD polypeptide and a BACH2 polypeptide (SEQ ID NO: 120). See Table 2.

Small Molecule-Regulated Polynucleotides [0286] A pair of small molecule-regulated polypeptides may be used as binding elements to recruit a fused E3 ligase domain (or functional variant thereof) to a protein of interest.

[0287] For example, degrader system may include a polynucleotide set that includes a first polynucleotide and a second polynucleotide, wherein either the first or second polynucleotide encodes an E3 ligase domain fused to a first small molecule-regulated polypeptide and the other of the first or second polynucleotide encodes a synthetic or exogenous target protein of interest fused to a corresponding second small molecule-regulated polypeptide.

[0288] In various embodiments, the first or second polynucleotide may encode a binding element that includes NS3a (or a functional variant thereof) and the other of the first or second polynucleotide may encode a binding element selected from the group consisting of DNCR2 (or a modification thereof) and GNCRf (or modification thereof).

[0289] In some embodiments, the first or second polynucleotide encodes a binding element which may include an NS3a polypeptide that includes: NS3a.

[0290] In certain embodiments, the first or second polynucleotide encodes an ROR1-CAR- NS3a fusion protein that includes ROR1-CAR and an NS3a binding element (SEQ ID NO: 126). See Table 5.

[0291] In some embodiments, the first or second polynucleotide encodes a binding element which may include a DNCR2 polypeptide that includes: DNCR2.

[0292] In some embodiments, the first or second polynucleotide encodes a binding element which may include a GNCR1 polypeptide that includes: GNCR1.

[0293] In some embodiments, either the first or second polynucleotide encodes a fusion protein including a DNCR2 polypeptide and an E3 ligase domain, which may include: RNF431 98-3 i7- DNCR (SEQ ID NO: 127), DNCR-RNF4 71- 190 (SEQ ID NO: 128), LNX1 1- 171 - DNCR (SEQ ID NO: 129), RNF43 198- 317 -DNCR-endo (SEQ ID NO: 130), RNF43 198-317 - DNCR-KtoR (SEQ ID NO: 131), or RNF43 198-317 -DNCR-KtoR-endo (SEQ ID NO: 132). See Table 5.

Native Motif Polypeptide

[0294] In various embodiments, a polynucleotide encoding a fusion protein includes a binding element specific for a native motif on an endogenous protein of interest and an E3 ligase domain (or functional variant thereof).

[0295] In some embodiments, the polynucleotide may encode a binding element including a domain that is specific for a region of a transmembrane protein and/or protein complex. [0296] In one embodiment, the binding element includes a domain targeting an intracellular domain of a transmembrane protein and/or protein complex.

[0297] In one embodiment, the binding element includes a domain targeting a transmembrane domain of a transmembrane protein and/or protein complex.

[0298] In some embodiments, the transmembrane protein is a T cell receptor (TCR) complex.

[0299] In some embodiments, the polynucleotide may encode a binding element including a domain that is specific for the transmembrane domain of a TCR complex selected from the group consisting of TM CD3z .

[0300] In some embodiments, the polynucleotide may encode a binding element including a domain that is specific for a protein-protein interaction domain on an endogenous protein of interest. Examples of protein-protein interaction domains include, but are not limited to, a pleckstrin homology (PH) domain, a Src homology 2 domain (SH2), and a Src homology 3 domain (SH3).

[0301] In some embodiments, the polynucleotide may encode a binding element including a domain that is a single-chain variable fragment (scFV) that recognizes and binds a certain region of a protein of interest.

[0302] In some embodiments, the polynucleotide may encode a binding element including a single monomeric variable antibody domain or “nanobody” that recognizes and binds a certain region of a protein of interest.

[0303] In some embodiments, the polynucleotide may encode a binding element including a monobody that recognizes and binds a certain region of a protein of interest. Monobodies are synthetic binding proteins constructed using a fibronectin type III domain (FN3) as a molecular scaffold.

[0304] In some embodiments, the polynucleotide may encode a binding element including a designed ankyrin repeat protein domain (DARPin) that recognizes and binds a certain region of a protein of interest. [0305] In some embodiments, the polynucleotide may encode a binding element including a variable lymphocyte receptor (VLR) domain that recognizes and binds a certain region of a protein of interest.

[0306] In some embodiments, the polynucleotide encodes a fusion protein including a binding element and an E3 ligase domain, which may include: TM CD3z -RNF43 226-317 (vl) (SEQ ID NO: 133), TM CD3z-K54 R-RNF43 226-317 (v2) (SEQ ID NO: 134), TM CD3z -RNF43 236-317 (v3) (SEQ ID NO: 135), TM CD3Z-K54R -RNF43 236-317 (V4) (SEQ ID NO: 136), ZAP70 nSH2-cSH2 - RNF4 71-190 (SEQ ID NO; 137), ZAP7O cSH2 -RNF4 71-190 (SEQ ID NO: 138), LNX1 1-171 - ZA p70 nSH2-cSH2 (SEQ ID NO: 139), LNX1 1471 -ZAP70 CSH2 (SEQ ID NO: 140), NCK1 SH3 1 - RNF4 71-19 (SEQ ID NO: 141), or LNX1 1471 -NCK1 SH3. 1 (SEQ ID NO: 142). See Table 6.

Protein Targets

[0307] The synthetic degrader system of the disclosure is useful for regulating the activity of a range of target molecules. The combination of a binding element and a degradation initiator (e.g., an E3 ligase domain) used in a degrader system may be selected based on the protein targeted for degradation. A targeted protein may be a synthetic protein or an exogeneous protein. A targeted protein may be an endogenous protein and/or protein complex.

[0308] The degrader system of the disclosure may be used to provide constitutive regulation of a protein target of interest.

[0309] The degrader system of the disclosure may be used to provide dynamic regulation of a protein target of interest in a context-dependent manner. For example, the degrader system may be an inducible system that can be “turned off’ at one point in a cellular process wherein the function of the targeted protein is required and “turned on” at another point in the cellular process wherein the function of the targeted protein is not desired.

[0310] The degrader system of the disclosure may be used to regulate multiple proteins at the same time. For example, the targeted degrader system may use a degradation initiator and a single binding element that recognizes and binds a conserved domain that is present in multiple proteins (e.g., members of a protein family). The binding element may, for example, be a designed binding element that has promiscuous targeting properties. [0311] Proteins that may be targeted for degradation include, but are not limited to, metabolic enzymes such as enzymes involved in glycolysis, oxidative phosphorylation, and fatty acid metabolism; enzymes involved in cell signaling processes; transcription factors; scaffolding and adapter proteins; and mitochondrial proteins.

[0312] A certain domain and/or motif in a protein may be used to target a protein for degradation.

[0313] A certain post-transcriptional modification to a protein and/or protein domain may be used to target a protein for degradation.

Transmembrane receptors

[0314] The degrader system of the disclosure may be used for targeted degradation of an integral (intrinsic) membrane protein, such as a transmembrane receptor. Examples of transmembrane receptors that may be targeted for degradation include, but are not limited to, chimeric antigen receptors (CARs), T cell receptors (TCRs), receptor tyrosine kinases (RTKs), Notch, Notch-like receptors, growth factor family receptors, receptor serine/threonine kinases, G-protein coupled receptors (GPCRs), inhibitory receptors such as PD-1, TIGIT, LAG3, TGFβ, FAS, and cytokine receptors.

[0315] Examples of receptor tyrosine kinases (RTKs) that may be targeted for degradation include, but are not limited to, epidermal growth factor receptor (EGFR), ErbB, ERBB2 (HER2), fibroblast growth factor receptors (FGFRs), insulin-like growth factor 1 receptor (IGF1R), and vascular endothelial growth factor receptors (VEGFRs).

[0316] In some embodiments, a transmembrane protein targeted for degradation is a T cell receptor (TCR).

[0317] In some embodiments, a transmembrane protein targeted for degradation is a chimeric antigen receptor (CAR). In one example, a CAR protein targeted for degradation is an ROR1 -specific chimeric antigen receptor protein (ROR1-CAR).

[0318] In some embodiments, the cytokine receptor IL-7R may be targeted for degradation. Constitutive IL-7 receptor signaling has been shown to promote T-cell proliferation and persistence in killing tumor cells (Shum, et al., (20017) and Peng (2017), which are incorporated herein by reference in their entirety). Transcription Factors

[0319] The degrader system of the disclosure may be used for targeted degradation of a transcription factor. Examples of transcription factors (TFs) that may be targeted for degradation include, but are not limited to, TFs that regulate T cell receptor signaling, T cell exhaustion, T cell memory formation, T cell differentiation; TFs that regulate stem cell differentiation and/or sternness; TFs in the bzip family; TOX, TOX2, NR4A1, NR4A2, NR4A3, IRF4, BATF, XBP1, c-Jun, Fos, API, Bach2, Tcfl/Tcf7, FoxPl, and FoxP3.

[0320] In some embodiments, the NR4A family of transcription factors, NR4A1 (Nur77), NR4A2, and NR4A3, may be targeted for degradation using a binding element that recognizes and binds all three family members. The NR4A family of transcription factors has been shown to play roles in T cell exhaustion and knocking out (or decreasing) the expression of the three NR4A transcription factors shows promise for mitigating exhaustion and improving T cell efficacy (Chen et al., 2019, which is incorporated herein by reference in its entirety).

[0321] In some embodiments, the transcription factor TOX and/or TOX2 may be targeted for degradation in a context-dependent manner (i.e., dynamically regulated). TOX and TOX2 have been shown to play a role in T cell exhaustion but cannot be completely knocked out because of their essential role in T cell memory formation (Sekine et al., (2020), Seo et al., (2019), and Bordon (2019), which are incorporated herein by reference in their entirety).

[0322] In some embodiments, the transcription factor IRF4 may be targeted for degradation. IRF4 (Man et al., (2017) which is incorporated herein by reference in its entirety).

[0323] In some embodiments, the transcription factor BATF may be targeted for degradation. BATF (Kurachi et al. (2014) which is incorporated herein by reference in its entirety).

[0324] In some embodiments, the transcription factor XBP1 may be targeted for degradation. XBP1 (Song et al., (2018); American Association for Cancer Research, which is incorporated herein by reference in its entirety).

[0325] In some embodiments, the transcription factor c-Jun may be targeted for degradation. c-Jun (Lynn et al., (2019), which is incorporated herein by reference in its entirety). [0326] In some embodiments, the transcription factor Bach2 may be targeted for degradation. Bach2 (Utzschneider et al., (2020); Sidwell et al., (2020); Roychoudhuri et al., (2016); and Richer et al., (2016), which are incorporated herein by reference in their entirety).

Cytoplasmic Enzymes and Enzyme Regulators

[0327] A protein targeted for degradation may be a cytoplasmic enzyme or a regulator of an enzyme’s activity. Examples of cytoplasmic enzymes and/or regulators thereof that may be targeted for degradation include, but are not limited to, non-receptor tyrosine kinases (nRTKs), non-receptor serine/threonine kinases, phosphoinositide kinases, phosphatases, and E3 ubiquitin ligases.

[0328] Non-receptor tyrosine kinases that may be targeted for degradation include, but are not limited to, Src-family tyrosine kinases such as Lek, Src, Fyn, Yes, Fgr, Hck, Blk, and Lyn; Tec family tyrosine kinases, such as Tec, Itk, Btk, Rlk, and Bmx; C-terminal Src kinase (CSK), and Zap70.

[0329] In some embodiments, the non-receptor tyrosine kinase Lek may be targeted for degradation. Lek has been shown to be critical for initiating proximal T cell signaling (Salmond et al., (2009), which is incorporated herein by reference in its entirety).

[0330] In some embodiments, the non-receptor tyrosine kinase Fyn may be targeted for degradation. Fyn has been shown to be critical for initiating proximal T cell signaling and for CAR T cell signaling (Salmond et al., (2009), Salter et al., (2018), Bommhardt et al., (2019), Suryadevara et al., (2019), and Hartl et al., (2020), which are incorporated herein by reference in their entirety).

[0331] In some embodiments, the non-receptor tyrosine kinase Itk may be targeted for degradation. (Andreotti et al., (2010) and Andreotti et al., (2018), which are incorporated herein by reference in their entirety).

[0332] In some embodiments, the non-receptor tyrosine kinase CSK may be targeted for degradation. (Okada, (2012), which is incorporated herein by reference in its entirety).

[0333] In some embodiments, the non-receptor tyrosine kinase Zap70 may be targeted for degradation. (Yokosuka et al., (2005) and Wang et al., (2010)), which are incorporated herein by reference in their entirety). [0334] Non-receptor serine/threonine kinases that may be targeted for degradation include, but are not limited to, mitogen-activated protein kinase (MAPK), MAPK kinase (MAPKK), MAPKK kinase (MAPKKK), calcium-dependent kinases, cyclin-dependent kinase such as CDK1 and CDK2.

[0335] Phosphoinositide kinases that may be targeted for degradation include, but are not limited to, phosphoinositide 3-kinases (PI3K or PIK3), PI3Ka p85, PI3Ka pl 10, and phosphoinositide 5-kinase (PI5K or PIK5).

[0336] Phosphatases that may be targeted for degradation include, but are not limited to, PTEN, SHP1, SHP2, and PTPN2/TCPTP.

[0337] In some embodiments, the phosphatase PTEN may be targeted for degradation. (Newton and Turka, (2012); Lin et al., (2021); Amaria et al., (2016); Cheng et al., (2019); and Locke et al, (2013), which are incorporated herein by reference in their entirety).

[0338] In some embodiments, the phosphatase SHP1 may be targeted for degradation. (Johnson et al., (2013); and Brockdorff et al., (1999), which are incorporated herein by reference in their entirety).

[0339] In some embodiments, the phosphatase SHP2 may be targeted for degradation. (Yokosuka et al., (2012); and Strazza et al., (2021), which are incorporated herein by reference in their entirety).

[0340] In some embodiments, the phosphatase PTPN2/TCPTP may be targeted for degradation. PTPN2/TCPTP (LaFleur et al., (2019); and Wiede et al., (2020), which are incorporated herein by reference in their entirety).

[0341] E3 ubiquitin ligase proteins and/or components of an E3 ubiquitin ligase complex that may be targeted for degradation include, but are not limited to, von Hippel-Lindau protein (VHL), cytokine-induced SH2 protein (CISH), Cbl-b, c-Cbl, suppressor of cytokine signaling (SOCS) 1, SOCS2, SOCS3, SOCS4, SOCS5, SOCS6, SOCS7, Elongin B, Elongin C, and Elongin BC heterodimer.

[0342] In some embodiments, the E3 ubiquitin ligase complex protein VHL may be targeted for degradation. VHL has been shown to regulate hypoxia-inducible factors which regulate T cell differentiation and function (Cardote et al., (20177); Stebbins et al., (1999); Velixa et al., (2021); and McNamee et al., (2013) which are incorporated herein by reference in their entirety).

[0343] In some embodiments, the E3 ubiquitin ligase complex protein CISH may be targeted for degradation. CISH has been shown to downregulate T cell signaling by targeting PLCγ (and possibly other targets). Deletion of CISH in T cells increases T cell activity and cytotoxicity (Palmer et al., (2015); Palmer et al., (2014); and Guittard et al., (2018), which are incorporated herein by reference in their entirety).

[0344] In some embodiments, the E3 ubiquitin ligase complex proteins Cbl-b and/or c-Cbl may be targeted for degradation. Cbl-b and c-Cbl (Nguyen et al., (2021); Lutz-Nicoladoni et al., (2015); and Tang et al., (2019), which are incorporated herein by reference in their entirety).

[0345] In some embodiments, the E3 ubiquitin ligase complex protein SOCS1 may be targeted for degradation. SOCS1 (Takahashi et al., (2011); Takahashi et al., (2017); and Tamiya et al., (2011), which are incorporated herein by reference in their entirety).

[0346] In some embodiments, the E3 ubiquitin ligase complex protein SOCS2 may be targeted for degradation. SOCS2 (Tannahill et al., (2005); Greenhalgh et al., (2005); and Knosp et al., (2011), which are incorporated herein by reference in their entirety).

[0347] In some embodiments, the E3 ubiquitin ligase complex protein SOCS3 may be targeted for degradation. (Croker et al., (2003); and Chen et al., (2006), which are incorporated herein by reference in their entirety).

[0348] In some embodiments, the E3 ubiquitin ligase complex protein SOCS4 may be targeted for degradation. (Bullock et al. (2007), which is incorporated herein by reference in its entirety).

[0349] In some embodiments, the E3 ubiquitin ligase complex protein SOCS5 may be targeted for degradation. (Zhang et al. (2009), which is incorporated herein by reference in its entirety).

[0350] In some embodiments, the E3 ubiquitin ligase complex protein SOCS6 may be targeted for degradation. (Choi et al. (2010), which is incorporated herein by reference in its entirety). [0351] In some embodiments, the E3 ubiquitin ligase complex protein S0CS7 may be targeted for degradation. (Bondar et al. (2018), which is incorporated herein by reference in its entirety).

[0352] In some embodiments, the E3 ubiquitin ligase complex proteins Elongin B, elongin C, and/or Elongin BC heterodimer may be targeted for degradation. Elongin B, Elongin C, and Elongin BC heterodimer have been shown to play critical roles in regulating Cullin-Ring ubiquitin ligases (Okumura et al., (2012), which is incorporated herein by reference in its entirety).

Signaling Proteins

[0353] A protein targeted for degradation may be a signaling protein. Examples of signaling proteins that may be targeted for degradation include, but are not limited to, CIN85, PLCyl, PLCy2, Smad2, Smad3, and Smad4.

[0354] In some embodiments, the signaling protein PLCyl may be targeted for degradation. (Su et al., (2016); Bilal and Houtman (2015); and Braiman et al., (2006), which are incorporated herein by reference in their entirety).

[0355] In some embodiments, the signaling proteins Smad2 and/or Smad3 may be targeted for degradation. (Yang et al., (1999); Takimoto et al., (2010); Malhotra and Kang (2013); and Kashiwagi et al., (2015), which are incorporated herein by reference in their entirety).

Scaffolding and Adapter Proteins

[0356] A protein targeted for degradation may be a scaffolding or an adapter protein. Examples of scaffolding and adaptor proteins that may be targeted for degradation include, but are not limited to, SLP76, LAT, Grb2, GADS, Ste5p, KDS, HOP, PSD95, Cullin-Skpl, RACK1, and ADAP.

[0357] In some embodiments, the adapter protein LAT may be targeted for degradation. (Zhang et al., (1998); Williamson et al., (2011); and Liu et al., (1999), which are incorporated herein by reference in their entirety).

[0358] In some embodiments, the adapter protein SLP76 may be targeted for degradation. (Yokosuka et al., (2005), which is incorporated herein by reference in its entirety). [0359] In some embodiments, the adapter protein Grb2 may be targeted for degradation. (Zhang et al., (2000); Gong et al., (2001); and Bilal and Houtman (2015), which are incorporated herein by reference in their entirety).

[0360] In some embodiments, the adapter protein Gads may be targeted for degradation. (Liu et al., (1999); Zhang et al., (2000); and Yoder et al., (2001), which are incorporated herein by reference in their entirety).

Protein Domains as Targets

[0361] A functional and/or structural domain of a protein may be used to target the protein for degradation. Examples of protein domains that may be used to target a protein for degradation include, but are not limited to, signaling domains, regulatory domains, lipid membrane binding domains, phospholipid binding domains, and DNA binding domains. Specific examples of regulatory and signaling domains that may be targeted include, but are not limited to, Src homology 2 (SH2), src homology 3 (SH3), pleckstrin homology (PH), dbl homology (DH), DHPH, Bcl-2 homology (BH), PDZ domains, PDZ-binding, and C2 domains. Specific examples of lipid membrane binding domains that may be targeted include, but are limited to, DHPH, PH, and C2. Specific examples of phospholipid binding domains that may be targeted include, but are limited to, PH, DHPH, C2, PI(3,4,5)P3 binding, PI(3,4)P2 binding, and PI(4,5)P2. Specific examples of DNA binding domains that may be targeted include, but are limited to, zinc fingers (ZFs), TALEs, Cas9, and Casl2a.

SOCS-Box Motif

[0362] A SOCS-box motif may be used to target a SOCS-box containing protein for degradation. Examples of SOCS-box containing proteins include, but are not limited to, the suppressor of cytokines (SOC) family proteins, VHL, and CISH.

Chimeric Antigen Receptors

[0363] A target protein of interest may include chimeric antigen receptors (CARs). CARs can be fusion proteins including an extracellular antigen-binding/recognition element, a transmembrane element that anchors the receptor to the cell membrane and at least one intracellular element. These CAR elements are known in the art, for example as described in patent application US20140242701, entitled “Chimeric Antigen Receptors”, published on August 28, 2014, which is incorporated by reference in its entirety. The CAR can be a recombinant polypeptide expressed from a polynucleotide including at least an extracellular antigen binding element, a transmembrane element and an intracellular signaling element including a functional signaling element derived from a stimulatory molecule.

[0364] The stimulatory molecule may, for example, be the zeta chain associated with the T cell receptor complex.

[0365] The cytoplasmic signaling element may, for example, include one or more functional signaling elements derived from at least one costimulatory molecule.

[0366] The costimulatory molecule may, for example, be selected from 4-1BB (i.e., CD 137), CD27 and/or CD28.

[0367] The CAR may be a chimeric fusion protein including an extracellular antigen recognition element, a transmembrane element and an intracellular signaling element including a functional signaling element derived from a stimulatory molecule.

[0368] The CAR may include a chimeric fusion protein including an extracellular antigen recognition element, a transmembrane element and an intracellular signaling element including a functional signaling element derived from a co-stimulatory molecule, and a functional signaling element derived from a stimulatory molecule.

[0369] The CAR may be a chimeric fusion protein including an extracellular antigen recognition element, a transmembrane element, and an intracellular signaling element including two functional signaling elements derived from one or more co-stimulatory molecule(s) and a functional signaling element derived from a stimulatory molecule.

[0370] The CAR may include a chimeric fusion protein including an extracellular antigen recognition element, a transmembrane element, and an intracellular signaling element including at least two functional signaling elements derived from one or more co-stimulatory molecule(s) and a functional signaling element derived from a stimulatory molecule.

[0371] The CAR may include an optional leader sequence at the amino-terminus (N-term) of the CAR fusion protein. The CAR may further include a leader sequence at the N-terminus of the extracellular antigen recognition element, wherein the leader sequence is optionally cleaved from the antigen recognition element (e.g., a scFv) during cellular processing and localization of the CAR to the cellular membrane. Selection of a Degrader System

[0372] The combination of a binding element and a degradation initiator (e.g., an E3 ligase domain) used in a degrader system may be selected based on the protein targeted for degradation.

[0373] In some embodiments, a pair of DHD binding elements is used for targeted degradation of a synthetic or exogenous transmembrane protein. FIG. 4A is a diagram 400 of an example of using a 3+1 DHD pair as binding elements for targeted degradation of a transmembrane protein. A transmembrane protein (“TMP”) targeted for degradation is fused to one partner of the binding element pair (DHD-B), and an E3 ligase domain is fused to the second binding partner of the binding element pair (DHD-A). Interaction of the DHD-A and DHD-B binding partners brings the E3 ligase domain into proximity with the targeted protein, thereby facilitating the ubiquitination of the transmembrane protein. Ubiquitination of the transmembrane protein promotes subsequent receptor internalization, endosomal trafficking, and lysosomal degradation of the transmembrane protein.

[0374] In some embodiments, a pair of DHD binding elements is used for targeted degradation of a synthetic or exogenous cytoplasmic protein, such as a transcription factor. FIG. 4B is a diagram 410 of an example of using a 3+1 DHD pair as binding elements for targeted degradation of a cytoplasmic protein. The cytoplasmic protein (“Target”) targeted for degradation is fused to one partner of the of the binding element pair (DHD-B) and an E3 ligase domain is fused to the second binding partner of the binding element pair (DHD-A). Interaction of the DHD-A and DHD-B targeting domains of the fusion proteins brings the E3 ligase domain into proximity with the targeted protein, thereby facilitating the ubiquitination of the cytoplasmic protein target. Polyubiquitination (“Ub”) of the target protein is induced upon interaction with the DHD-E3 ligase and promotes downstream proteasomal degradation.

[0375] In some embodiments, a small molecule-regulated degrader system is used for targeted degradation of transmembrane receptor. In one example, the transmembrane receptor is a chimeric receptor, such as a CAR. FIG. 5 is a diagram 500 of an example of a small molecule-regulated degrader system for degradation of a chimeric receptor. In this example, the chimeric receptor targeted for degradation is fused to a NS3a polypeptide and an E3 ligase domain is fused to an DNCR2 polypeptide. In the presence of the small molecule danoprevir, the DNCR2 and NS3a polypeptide assemble, together with the small molecule danoprevir, to for a dimerization complex. The formation of the dimerization complex brings the E3 ligase into proximity with the targeted chimeric receptor, thereby facilitating ubiquitination (“Ub”) and subsequent degradation of the targeted receptor.

[0376] In some embodiments, a binding element may target a native motif on an endogenous protein of interest. In one example, a binding element may be a protein domain that interacts with a native motif on an intracellular region of a transmembrane protein of interest. In yet another example, a binding element may be a protein domain that interacts with a native motif on a transmembrane domain of a transmembrane protein of interest. The interaction of the binding element and the native motif on the protein of interest brings the E3 ligase into proximity with the targeted protein thereby facilitating ubiquitination and subsequent degradation of the protein target.

[0377] FIG. 6A is a diagram 600 illustrating a transmembrane receptor domain as a binding element for targeted degradation of a chimeric transmembrane receptor. In this example, an E3 ligase domain is fused to a receptor-targeting transmembrane domain that is designed to bind a native transmembrane motif on the chimeric receptor. The interaction of the receptortargeting transmembrane domain and the chimeric receptor brings the E3 ligase domain into proximity with the targeted receptor, thereby facilitating the ubiquitination of the chimeric receptor. Ubiquitination of the chimeric receptor promotes subsequent lysosomal trafficking and degradation of the receptor.

[0378] In some embodiments, a binding element may be a protein domain that interacts with a specific post-translational modification on a protein of interest. For example, a binding element may interact with a post-translationally phosphorylated region of the target protein.

[0379] In one embodiment, a synthetic degrader system of the disclosure uses a phosphotyrosine-binding domain (PYBD) as a binding element to recruit an E3 ligase domain to transmembrane receptor for degradation. FIG. 6B is a diagram 610 illustrating a phosphotyrosine-binding domain (PYBD) as a binder for targeted degradation of a post- translationally phosphorylated (“P”) transmembrane receptor. The interaction of the PYBD domain and the phosphorylated region of the transmembrane receptor brings the E3 ligase domain into proximity with the targeted receptor, thereby facilitating the ubiquitination of the chimeric receptor. Ubiquitination of the receptor promotes subsequent lysosomal trafficking and degradation of the receptor. [0380] In a specific example, a binding element may be used to target the endogenous T cell receptor (TCR) complex. For example, constructs encoding the CD3<" transmembrane domain (TM CD3z ) may be used to target an E3 ligase domain (e.g., RNF43) to the TCR complex, wherein the CD3<( transmembrane domain (TM CD3z ) is used to recognize and disrupt the TCR complex and the E3 ligase domain is used to facilitate degradation of the receptor, thereby by providing two mechanisms for regulating the activity of the TCR complex.

Vectors and Vector Configurations

[0381] The polynucleotides of the disclosure may be provided as part of a vector. Examples of suitable vectors include expression vectors, viral vectors, and plasmid vectors. Expression vectors can include plasmids, phagemids, viruses, and derivatives thereof. In some aspects, the polynucleotides of the disclosure may be provided as part of a homology directed repair vector.

[0382] In some aspects, the viral vectors may include polynucleotides encoding gene editing polypeptides, such as polypeptides useful for implementation of gene editing techniques. Examples of such gene editing techniques include RNA/DNA guided endonucleases (e.g., CRISPR (clustered regularly interspaced short palindromic repeats)), TALEN (transcription activator-like effector nucleases), ZFN (zinc finger nucleases), recombinase, meganucleases, or viral integration.

[0383] In some aspects, the polynucleotides of the disclosure may be provided as part of a homology directed repair (HDR) vector. A homology directed repair mechanism may be used to integrate a polynucleotide set into a chromosome. Examples of mechanisms that may be used to integrate a polynucleotide set into a chromosome include sequence-specific nucleases such as transposase, CRISPR/Cas9, ZF nucleases, TALE nucleases, recombinases, and other homologous recombination targeting vectors known in the art.

[0384] Vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. A vector for use in a eukaryotic host cell may also encode a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide of interest. The signal sequence selected is preferably one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. In mammalian cell expression, mammalian signal sequences as well as viral secretory leaders may be used. Expression vectors used in eukaryotic host cells will typically also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5' and, occasionally 3', untranslated regions of eukaryotic or viral DNAs or cDNAs. One useful transcription termination component is the bovine growth hormone polyadenylation region.

[0385] Expression and cloning vectors may contain a selection gene, also termed a selectable marker. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, where relevant, or (c) supply critical nutrients not available from complex media.

[0386] The polynucleotides of the disclosure may in some cases be provided as part of a single vector. The polynucleotides of the disclosure may be provided as part of a set of at least two vectors; a first vector including the first polynucleotide and a second vector including the second polynucleotide.

[0387] Examples of vectors suitable for use with the polynucleotides of the disclosure include adenoviral vectors, lentiviral vectors, baculoviral vectors, Epstein Barr viral vectors, papovaviral vectors, vaccinia viral vectors, herpes simplex viral vectors, adeno associated virus (AAV) vectors, and transposon vectors. The polynucleotides of the disclosure may be provided as part of a homology directed repair vector.

[0388] The disclosure provides a polynucleotide set that includes the following as part of one or more vectors:

(i) a first polynucleotide encoding a first fusion protein that includes a first binding element, and

(ii) a second polynucleotide encoding a second fusion protein that includes a second corresponding binding element, wherein:

(iii) the first or second fusion protein further includes a degradation initiator; and

(iv) the other of the first or second fusion protein further includes a molecule of interest; and (v) interaction of the first and second elements effectively recruits the degradation initiator to the molecule of interest for targeted degradation.

[0389] In some embodiments, the disclosure provides a polynucleotide set that includes the following as part of one or more vectors:

(i) a first polynucleotide encoding a first fusion protein that includes a first binding element, and

(ii) a second polynucleotide encoding a second fusion protein that includes a corresponding second binding element, wherein:

(iii) the first or second fusion protein further includes an E3 ligase domain (or functional variant thereof); and

(iv) the other of the first or second fusion protein further includes a protein of interest of interest; and

(v) interaction of the first and second binding elements effectively recruits the E3 ligase to the protein of interest for targeted degradation.

[0390] In some embodiments, the disclosure provides a polynucleotide set that includes the following as part of one or more vectors:

(i) a first polynucleotide encoding a first fusion protein that includes a first binding element and an E3 ligase domain (or functional variant thereof), and

(ii) a second polynucleotide encoding a second fusion protein that includes a corresponding second binding element and a synthetic or exogenous protein of interest, wherein interaction of the first and second binding elements effectively recruits the E3 ligase domain to the protein of interest for targeted degradation.

In some embodiments, the disclosure provides a polynucleotide set that includes the following as part of one or more vectors:

(i) a first polynucleotide encoding a first fusion protein that includes a DHD binding element and an E3 ligase domain (or functional variant thereof), and (ii) a second polynucleotide encoding a second fusion protein that includes a corresponding second DHD binding element and a synthetic or exogenous protein of interest, wherein interaction of the first and second binding elements effectively recruits the E3 ligase domain to the protein of interest for targeted degradation.

[0391] In some embodiments, the disclosure provides a polynucleotide set that includes the following as part of one or more vectors:

(i) a first polynucleotide encoding a first fusion protein that includes a binding element comprising a reader protein and an E3 ligase domain (or functional variant thereof), and

(ii) a second polynucleotide encoding a second fusion protein that includes an NS3a binding element and a synthetic or exogenous protein of interest, wherein interaction of the first and second binding elements is mediated by the presence of a small molecule, thereby recruiting the E3 ligase domain to the protein of interest for targeted degradation.

In some embodiments, the disclosure provides a polynucleotide set encoding a fusion protein that includes the following as part of a single vector:

(i) a degradation initiator; and

(ii) a binding domain specific for a native motif on an endogenous molecule of interest, wherein binding of the fusion protein to the molecule of interest mediates recruitment of the degradation initiator to the molecule of interest for targeted degradation.

[0392] In some embodiments, the disclosure provides a polynucleotide set encoding a fusion protein that includes the following as part of a single vector:

(i) an E3 ligase domain, and

(ii) a binding domain specific for a native motif on an endogenous protein of interest, wherein binding of the fusion protein to the protein of interest mediates recruitment of the E3 ligase to the protein of interest for targeted degradation.

Single Vector Configuration

[0393] FIG. 7A is a diagram illustrating a unidirectional forward configuration 700 for encoding an inducible polynucleotide component and a constitutive polynucleotide component on a single vector. In this example, the vector is configured to express an inducible polynucleotide component 710 encoding the first fusion protein including an E3 ligase domain fused to a first binding element and a constitutive polynucleotide component 715 encoding a second fusion protein including a target protein of interest fused to a second binding element. The first and second binding elements may, for example, be DHD polypeptides. The inducible promoter component consists of a minimal promoter with one or more 5’ response element repeats that are recognized and bound by a specific transcription factor in response to a stimulus. The inducible promoter component may also include optional regulatory sequences such as a polyA sequence. The constitutive polynucleotide component may also include optional regulatory sequences such as a polyA sequence.

[0394] In some embodiments, the vector further includes a transduction marker.

Two-Vector Configuration

[0395] In some embodiments, the polynucleotide set that includes the first polynucleotide and the second polynucleotide is integrated on two vectors, wherein:

(i) a first vector may include the first polynucleotide encoding the first fusion protein, and

(ii) a second vector may include the second polynucleotide encoding the second fusion protein.

[0396] In some embodiments, the first vector is configured to express an inducible polynucleotide component encoding the first fusion protein including a degradation initiator fused to a first binding element and the second vector is configured to express a constitutive polynucleotide component encoding a second fusion protein including a molecule of interest and a second binding element.

[0397] In some embodiments, the first vector is configured to express an inducible polynucleotide component encoding the first fusion protein including an E3 ligase domain (or functional variant thereof) fused to a first binding element and the second vector is configured to express a constitutive polynucleotide component encoding a second fusion protein including a target protein of interest and a second binding element.

[0398] FIG. 7B is a diagram illustrating a two-vector system 720 for encoding an inducible polynucleotide component and a constitutive polynucleotide component. In this example, a first vector 725 includes the inducible polynucleotide component 710 for expression of the first fusion protein including an E3 ligase domain fused to a first binding element and a second vector 730 includes the constitutive polynucleotide component 715 encoding the second fusion protein including a target protein of interest and a second binding element.

Compositions Including Vectors

[0399] A polynucleotide set of the disclosure may be provided as part of a vector. In some embodiments, the first and second polynucleotide components of the polynucleotide set may be provided as part of a single vector.

[0400] The disclosure provides a composition that includes a single vector including:

(i) a first polynucleotide component encoding a first binding element, and

(ii) a second polynucleotide component encoding a second binding element, wherein either the first or second polynucleotide component further encodes a degradation initiator and the other of the first or second polynucleotide component further encodes a molecule of interest and interaction of the first and second binding elements mediates recruitment of the degradation initiator to the molecule of interest for targeted degradation.

[0401] In some embodiments, the disclosure provides a composition that includes a single vector including:

(i) a first polynucleotide component encoding a first binding element, and

(ii) a second polynucleotide component encoding a second binding element, wherein either the first or second polynucleotide component further encodes an E3 ligase domain (or functional variant thereof) and the other of the first or second polynucleotide component further encodes a protein of interest and interaction of the first and second binding elements mediates recruitment of the E3 ligase to the protein of interest for targeted degradation.

[0402] In some aspects, the composition may be used for treating a subject in need of a therapy. The disclosure provides a pharmaceutical composition that includes:

(i) a single vector including a first polynucleotide component encoding a first binding element and a second polynucleotide component encoding a second binding element, wherein either the first or second polynucleotide component further encodes a degradation initiator and the other of the first or second polynucleotide component further encodes a molecule of interest, and

(ii) a pharmaceutically acceptable carrier, excipient, and/or stabilizer.

[0403] In some aspects, the disclosure provides a pharmaceutical composition that includes:

(i) a single vector including a first polynucleotide component encoding a first binding element and a second polynucleotide component encoding a second binding element, wherein either the first or second polynucleotide component further encodes an E3 ligase domain and the other of the first or second polynucleotide component further encodes a protein of interest, and

(ii) a pharmaceutically acceptable carrier, excipient, and/or stabilizer.

[0404] In some embodiments, the first and second polynucleotide components of the polynucleotide set may be provided as part of a set of at least two vectors, wherein, for example, a first vector includes the first polynucleotide component, and the second vector includes the second polynucleotide component.

[0405] The disclosure provides a composition that includes:

(i) a first vector including a first polynucleotide component encoding a first binding element, and

(ii) a second vector including a second polynucleotide component encoding a second binding element, wherein either the first or second polynucleotide component further encodes a degradation initiator and the other of the first or second polynucleotide component further encodes a molecule of interest, and interaction of the first and second binding elements mediates recruitment of the degradation initiator to the molecule of interest for targeted degradation.

[0406] In some aspects, the composition may be used for treating a subject in need of a therapy. The disclosure provides a pharmaceutical composition that includes: (i) a first vector including a first polynucleotide component encoding a first binding element, and

(ii) a second vector including a second polynucleotide component encoding a second binding element, wherein either the first or second polynucleotide component further encodes degradation initiator and the other of the first or second polynucleotide component further encodes a molecule of interest, and

(iii) a pharmaceutically acceptable carrier, excipient, and/or stabilizer.

[0407] In some aspects, the disclosure provides a pharmaceutical composition that includes:

(i) a first vector including a first polynucleotide component encoding a first binding element, and

(ii) a second vector including a second polynucleotide component encoding a second binding element, wherein either the first or second polynucleotide component further encodes an E3 ligase (or functional variant thereof) and the other of the first or second polynucleotide component further encodes a protein of interest, and

(iii) a pharmaceutically acceptable carrier, excipient, and/or stabilizer.

[0408] The disclosure provides a composition that includes a single vector including polynucleotide encoding a fusion protein that includes a degradation initiator and a binding element as part of a single vector, wherein the binding element is specific for a native motif on an endogenous molecule of interest.

[0409] In some aspects, the composition may be used for treating a subject in need of a therapy. The disclosure provides a pharmaceutical composition that includes:

(i) a vector including a polynucleotide encoding a fusion protein that includes a degradation initiator and binding element, wherein the binding element is specific for a native motif on an endogenous molecule of interest, and

(ii) a pharmaceutically acceptable carrier, excipient, and/or stabilizer.

[0410] In some embodiments, the disclosure provides a composition that includes polynucleotide encoding a fusion protein that includes an E3 ligase domain and binding element as part of a single vector, wherein the binding element is specific for a native motif on an endogenous protein of interest.

[0411] In some aspects, the composition may be used for treating a subject in need of a therapy. The disclosure provides a pharmaceutical composition that includes:

(i) a vector including a polynucleotide encoding a fusion protein that includes an E3 ligase domain and binding element, wherein the binding element is specific for a native motif on an endogenous protein, and

(ii) a pharmaceutically acceptable carrier, excipient, and/or stabilizer.

Cells

[0412] Expression vectors of the disclosure may be expressed in host cells. Host cells may, for example, be prokaryotic cells, such as bacteria cells; or eukaryotic cells, such as yeast cells, plant cells, or mammalian cells. Examples of mammalian cells suitable for use with the disclosure include human, mouse, rat, pig, rabbit, sheep, and goat cells. In some cases, the cells are synthetic cells.

[0413] A host cell may, for example, be selected from the group consisting of cardiac cell, lung cell, muscle cell, epithelial cell, pancreatic cell, skin cell, CNS cell, neuron, myocyte, skeletal muscle cell, smooth muscle cell, liver cell, kidney cell and glial cell.

[0414] In some embodiments, a host cell is a human cell ex vivo. In some embodiments, a host cell is a human cell in vivo.

[0415] In some embodiments, a host cell is a stem cell such as a pluripotent stem cell or a hematopoietic stem cell.

[0416] In some embodiments, a host cell is a multipotent cell or a mesenchymal cell or a mesenchymal stromal cell (MSC).

[0417] In some embodiments, a host cell is a stem cell and the polynucleotides of the disclosure are used to control differentiation for cell products being generated from pluripotent cells, such as pluripotent stem cells. A degrader system including an inducible E3 ligase fusion protein may, for example, be used to control the stability (e.g., half-life) of a protein driving the differentiation. [0418] In some embodiments, a host cell is not pluripotent and the polynucleotides of the disclosure are used to control reprogramming of the cell to modulate pluripotency. A degrader system including an inducible E3 ligase fusion protein may, for example, be used to control the stability (e.g., half-life) of a protein driving the reprogramming.

[0419] In some embodiments, a host cell is part of an organism. In addition to the therapeutic embodiments described elsewhere herein, the cells may be part of a model organism. A degrader system may, for example, be used to control the stability of a protein regulating a cellular process and producing a characteristic for scientific study, such as a disease characteristic or a biological enhancement. Examples of suitable model organisms include yeast, fruit flies, nematodes, frogs, mice and fish (such as zebrafish). The protein of interest may, for example, be a dysfunctional polypeptide, or a polypeptide that interacts with or modulates a gene of the organism, or that interferes with a metabolic process. In one example, the degrader system is inducible, and an inducing stimulus may be administered to modulate or titrate expression of an E3 ligase fusion protein and thus produce variation in the characteristic being studied.

[0420] In some embodiments, a host cell is a cancer cell and/or a non-cancer cell from a human subject diagnosed with cancer.

[0421] In some embodiments, a host cell is an immune cell selected from the group consisting of leukocyte, lymphocyte, T cell, regulatory T cell, effector T cell, CD4+ effector T cell, CD8+ effector T cell, memory T cell, autoreactive T cell, exhausted T cell, natural killer T cell, B cell, dendritic cell, and macrophage.

[0422] Host cells may be transformed with one or more polynucleotides or vectors of the disclosure and cultured in nutrient media. Nutrient media may be formulated for inducing promoters, selecting transformants, or amplifying the genes of interest.

[0423] In some embodiments, the cell is a mammalian cell or cell line. Non-limiting examples include African green monkey kidney cells (VERO-76, ATCC CRL-1587); baby hamster kidney cells (BHK, ATCC CCL 10); BALB/c mouse myeloma lines (NSO/I, ECACC No: 85110503); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); canine kidney cells (MDCK, ATCC CCL 34); Chinese hamster ovary (CHO) cell or cell line, CHO-K1 cell line (see, e.g., ATCC catalog no. CCL-61™ and Lewis, N.E. et al. (2013) Nat. Biotechnol. 31 :759-765); Chinese hamster ovary cells +/-DHFR (see. e.g., Urlaub, G. and Chasin, L.A. (1980) Proc. Natl. Acad. Sci. 77:4216-4220); FS4 cells; HEK 293 cells; HT-1080 cells (ATCC® CCL-121™); human cervical carcinoma cells (HeLa, ATCC CCL-2); human embryonic kidney cell lines (293 or 293 cells subcloned for growth in suspension culture, Graham et al, J. Gen Virol. 36:59 (1977)); human hepatoma line (Hep G2); human liver cells (Hep G2, HB 8065); human lung cells (W138, ATCC CCL 75); human retinoblasts (PER.C6, CruCell, Leiden, The Netherlands); monkey kidney cells (CV1 ATCC CCL 70); monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); mouse mammary tumor (MMT 060562, ATCC CCL51); MRC 5 cells; TRI cells (Mather et al., Annals N. Y. Acad. Sci. 383:44-68 (1982)); and engineered T cells and engineered natural killer cells.

Compositions Including Host Cells

[0424] A polynucleotide set of the disclosure may be provided in a host cell. The cells can be transiently or stably engineered to incorporate the polynucleotide set of the disclosure.

[0425] The disclosure provides a composition that includes a cell including a polynucleotide set that includes a first polynucleotide component encoding a first binding element and a second polynucleotide component encoding a second binding element, wherein either the first or second polynucleotide component further encodes a degradation initiator and the other of the first or second polynucleotide component further encodes a molecule of interest and interaction of the first and second binding elements mediates recruitment of the degradation initiator to the molecule of interest for targeted degradation.

[0426] In some embodiments, the disclosure provides a composition that includes a cell including a polynucleotide set that includes a first polynucleotide component encoding a first binding element and a second polynucleotide component encoding a second binding element, wherein either the first or second polynucleotide component further encodes an E3 ligase domain (or functional variant thereol) and the other of the first or second polynucleotide component further encodes a protein of interest and interaction of the first and second binding elements mediates recruitment of the E3 ligase to the protein of interest for targeted degradation.

[0427] The disclosure provides a composition that includes a cell including a polynucleotide encoding a fusion protein that includes degradation initiator and binding element, wherein the binding element is specific for a native motif on an endogenous molecule of interest. [0428] In some embodiments, the disclosure provides a composition that includes a cell including a polynucleotide encoding a fusion protein that includes an E3 ligase (or functional variant thereof) and binding element, wherein the binding element is specific for a native motif on an endogenous protein of interest.

[0429] In some aspects, a composition that includes a cell may be used for treating a subject in need of a therapy. The disclosure provides a pharmaceutical composition that includes:

(i) a cell which has been modified to express a polynucleotide set, and

(ii) a pharmaceutically acceptable carrier, excipient, or stabilizer.

[0430] The cells may include polynucleotides of the disclosure expressing a gene of interest that provides a therapeutic benefit. Expression of the gene of interest may confer the cells with the ability to attack tumor cells. The gene of interest may be a chimeric antigen receptor (CAR), e.g., a chimeric antigen receptor that targets tumor cells. The gene of interest may express a single-chain antibody fragment linked to a hinge linked to a transmembrane region. The transmembrane region may be linked to an intracellular signaling domain. The transmembrane region may be linked to a costimulatory domain.

[0431] The cells of the composition may, for example, be T cells. The cells of the composition may, for example, be CAR-T cells.

[0432] In some embodiments, the disclosure provides a cell composition including a means for reducing, ameliorating, or inhibiting exhaustion and/or dysfunction in a population of immune cells, e.g., immune cells expressing a CAR. In some aspects, the means include expressing the CAR as a gene of interest in a polynucleotide set and regulating the degradation of the CAR, thereby inhibiting excessive CAR signaling that may result in an undesirable exhaustion phenotype observed in many existing T-cell therapies.

Methods of Making Making Small Molecules

[0433] The small molecules of the disclosure may be synthesized using known techniques. Danoprevir ((2R,6S, 12Z, 13aS, 14aR, 16aS)- 14a-[(Cy clopropylsulfonyl)carbamoyl] -6-( { [(2- methyl-2-propanyl)oxy]carbonyl}amino)-5,16-dioxo- l,2,3,5,6,7,8,9,10,ll,13a,14,14a,15,16,16a-hexadecahydrocycl opropa[e]pyrrolo[l,2- a][l,4]diazacyclopentadecin-2-yl 4-fluoro-l,3-dihydro-2H-isoindole-2-carboxylate) may be synthesized using known techniques. See for example, Carreira, Erick Moran, Hisashi Yamamoto, and N.K. Yee. “Industrial Applications of Asymmetric Synthesis.” In Comprehensive Chirality 9, Amsterdam: Elsevier, 2012. Section 9.19.6, Danoprevir, the disclosure of which is incorporated herein by reference.

Making Polynucleotides

[0434] The disclosure provides methods of producing the polynucleotides of the disclosure, such as DNA vectors of the disclosure and their subcomponents, as well as packaging vectors and plasmids of the disclosure. Standard molecular biology techniques may be used to assemble the polynucleotides of the disclosure. Polynucleotides can be chemically synthesized.

Making Packaged Viral Capsids

[0435] The disclosure includes methods of making viral capsids containing polynucleotides of the disclosure. In general, viral capsids of the disclosure may be produced by supplying cells with packaging polynucleotides of the disclosure. The packaging polynucleotides may, for example, be supplied to packaging cells as plasmids. The packaging cells may be cultured to produce the viral capsids containing polynucleotides of the disclosure. Preferably the packaged viral capsids are replication incompetent.

[0436] A variety of commercially available kits are suitable for producing packaged viral capsids of the disclosure. Examples include: MISSION® Lentiviral Packaging Mix (available from Millipore Sigma); LV-Max Lentiviral Packaging Mix (available from ThermoFisher Scientific).

[0437] Viral capsids produced by packaging cells may be purified for use in downstream methods, such as delivery to cells for use in cell-based therapies, or delivery to subjects for gene therapy methods. Purification may include processing to eliminate contaminants from host cells or culture media. Purification steps may include steps based on physical and/or chemical characteristics of the plasmids. Chemical characteristics may include, for example, hydrophilicity-hydrophobicity. Physical characteristics may include, for example, size. Examples of purification strategies based on particle size include density-gradient ultracentrifugation, ultrafiltration, precipitation, two-phase extraction systems and size exclusion chromatography. In some cases, precipitation may be employed together with centrifugation, e.g., using polyethylene glycol, ammonium sulfate or calcium phosphate. In some cases, aqueous two-phase separation systems with PEG, dextran or polyvinyl alcohol may be used. In some cases, membrane-based tangential flow filtration techniques are used; examples include ultrafiltration, diafiltration and microfiltration. In other embodiments, chromatographic means may be used for purifying viral capsids. In still other embodiments, immunoaffinity methods may be used to capture capsids using monoclonal antibodies having specificity to the relevant capsids. See Morenweiser, R., “Downstream processing of viral vectors and vaccines,” Gene Therapy (2005) 12, S103-S110 (2005), the entire disclosure of which is incorporated herein by reference.

[0438] Examples of suitable viral capsids include, but are not limited to, adenovirus, retrovirus, Lentivirus, Sendai virus, baculovirus, Epstein Barr virus, papovavirus, vaccinia virus, herpes simplex virus, and adeno-associated virus (AAV).

Making Cells

[0439] The disclosure provides methods of making a modified cell to express a gene of interest.

[0440] In some embodiments, the disclosure provides a method of making a therapeutic cell that expresses a polynucleotide set for use in treating a subject in need of a cell therapy. In one aspect, the disclosure provides a method of generating or preparing a therapeutic cell that expresses a gene of interest from a polynucleotide set integrated into a single vector. In one aspect, the disclosure provides a method of generating or preparing a therapeutic cell that expresses a gene of interest from a polynucleotide set integrated into two (or more) vectors.

[0441] In some embodiments, the polynucleotides of the disclosure are maintained as extrachromosomal polynucleotides in the host cell. In some embodiments, the polynucleotides of the disclosure are present in a vector (e.g., expression vector) in the host cell. In some embodiments, the polynucleotides of the disclosure or a subset or subcomponents thereof, are integrated into a chromosome of the host cell.

[0442] Various methods can be used to introduce the expression vector of some embodiments of the disclosure into cells to produce cells of the disclosure. See for example, Green, et al., Molecular cloning: A laboratory manual. Cold Spring Harbor, NY : Cold Spring Harbor Laboratory Press (2014). [0443] Methods of introducing nucleic acid alterations to a gene of interest are well known in the art. Examples include targeted homologous recombination (e.g. “Hit and run”, “double-replacement”), site specific recombinases (e.g. the Cre recombinase and the Flp recombinase), PB transposases (e.g. Sleeping Beauty, piggyBac, Tol2 or Frog Prince), genome editing by engineered nucleases (e.g. meganucleases, Zinc finger nucleases (ZFNs), transcription-activator like effector nucleases (TALENs) and CRISPR/Cas system) and genome editing using recombinant adeno-associated virus (rAAV) platform. Agents for introducing nucleic acid alterations to a gene of interest can be designed using publicly available sources or obtained commercially from Transposagen, Addgene and Sangamo Biosciences. Vectors of the disclosure may make use of these methods for integrating polynucleotides of the disclosure into a host genome. Polynucleotides and vectors of the disclosure may include polynucleotides encoding polypeptides required for implementation of these methods for integrating polynucleotides of the disclosure into a host genome.

[0444] Various approaches suitable for integrating a polynucleotide(s) into a host cell genome are known in the art, including random integration or site-specific integration (e.g., a“landing pad” approach); see, e.g., Zhao, M. el al. (2018 ) Appl. Microbiol. Biotechnol. 102:6105-6117; Lee, J.S. et al. (2015) Sci. Rep. 5:8572; and Gaidukov, L. et al. (2018) Nucleic Acids Res. 46:4072-4086. Vectors of the disclosure may make use of these methods for integrating polynucleotides of the disclosure into a host genome. Vectors of the disclosure may include polynucleotides encoding polypeptides required for implementation of these methods for integrating polynucleotides of the disclosure into a host genome.

[0445] Examples of commercially available media suitable for culturing host cells of the disclosure include Ham's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RP MI- 1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma).

[0446] Culture media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN™ drug), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art. Culture conditions, such as temperature, pH, and the like, will be apparent to the ordinarily skilled artisan.

Cell Therapy Methods

[0447] The disclosure provides methods of treating a subject in need of a cell therapy. The method includes the steps of:

(i) administering to the subject an effective amount of a pharmaceutical composition including a therapeutic cell encoding a polynucleotide set that includes a degradation initiator expressed from an inducible promoter and a therapeutic molecule of interest; and

(i) administering a therapeutically effective amount of an inducing stimulus to the subject.

[0448] In some embodiments, the method includes the steps of

(i) administering to the subject an effective amount of a pharmaceutical composition including a therapeutic cell encoding a polynucleotide set that includes an E3 ligase (or functional variant thereof) expressed from an inducible promoter and a therapeutic molecule of interest; and

(i) administering a therapeutically effective amount of an inducing stimulus to the subject

[0449] In one aspect, the disclosure provides a method for treating a cancer, e.g., a tumor, in a subject in need thereof. Examples of cancers that can be treated using a pharmaceutical composition of the disclosure include, but are not limited to, melanomas, lymphomas, sarcomas, and cancers of the colon, kidney, stomach, bladder, brain (e.g., gliomas, glioblastomas, astrocytomas, medulloblastomas), prostate, bladder, rectum, esophagus, pancreas, liver, lung, breast, uterus, cervix, ovary, blood (e.g., acute myeloid leukemia, acute lymphoid leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, Burkitt's lymphoma, EBV-induced B-cell lymphoma).

[0450] In one aspect, the disclosure provides a method of controlling or modulating a T cell- mediated immune response in a subject in need thereof.

[0451] In one aspect, the disclosure provides a method of stimulating a T cell-mediated immune response to a target cell population or tissue in a subject. [0452] In one aspect, the disclosure provides a method of providing an anti-tumor immunity in a subject.

[0453] In one aspect, the disclosure provides a method preventing T cell exhaustion during CAR T cell cancer therapy in a subject.

Gene Therapy Methods

[0454] The disclosure provides methods of delivering a polynucleotide set of the disclosure to a subject. A polynucleotide set of the disclosure may be delivered into a cell of a subject. The method may include administering a pharmaceutically effective amount of the polynucleotide set to the subject. Administration may be via administration of viral particles including one or more polynucleotides of the disclosure. Administration may be via administration of a pharmaceutical composition including one or more polynucleotides of the disclosure.

[0455] In some embodiments, the method includes the steps of:

(i) administering to the subject an effective amount of a pharmaceutical composition including a polynucleotide set encoding a degradation initiator (e.g., an E3 ligase domain) expressed from small molecule regulated promoter and a therapeutic molecule of interest;

(ii) administering a therapeutically effective amount of the small molecule regulator to the subject;

(iii) monitoring the level of the therapeutic molecule in the subject; and

(iv) optionally, adjusting the dosage of the small molecule regulator to adjust level of the therapeutic molecule in the subject to a desired level.

[0456] The subject may be a mammalian subject. The subject may be a human subject.

[0457] Examples of conditions that may be selected for gene therapy include, but are not limited to, cancer, cystic fibrosis, heart disease, diabetes, hemophilia, and AIDS.

Kits

[0458] The disclosure provides kits or articles of manufacture including polynucleotides of the disclosure and a preparation for delivery of the polynucleotides to cells. The polynucleotides may be provided as part of a vector of the disclosure. In some embodiments, the kit or article of manufacture further includes instructions for using the set of the polynucleotides to transform cells to express a gene of interest to produce a polypeptide of interest and/or regulate the gene of interest.

[0459] The disclosure provides kits or articles of manufacture including a pharmaceutical composition that includes a therapeutic cell encoding a polynucleotide set of the disclosure. The kit or article of manufacture may further include instructions for administering the pharmaceutical composition to a subject.

[0460] In some cases, the kits or articles of manufacture may also include a small molecule regulator of the disclosure.

Tables

Table A Sequences

Table B

[0461] Table 1. Amino acid sequences of dimerization domains (binding elements), including sequences of designed heterodimers (DHDs).

[0463] Table 3. Amino acid sequences of target proteins used in the degradation screen. Optional tags and glycine-serine flexible linkers are underlined. Sequences of the DHDs and other binding elements are in bold and can be replaced with any of the binding element sequences in Table 1. Shorthand names used in figures and main text are in parentheses. Table 7. The DNA sequence of the NFAT-AP1 promoter. The minimal CMV promoter is underlined, and the sequence of 4 repeated NFAT-AP1 binding sites are in bold.

Table 9. The DNA sequence of constitutive promoters. Table 10. Amino Acid sequences of components for combinatorial screen(s).

Table 11. CAR-DHD Constructs, amino acid sequences of DHDs

Table 12. DHD-e3 Sequences

Table 13. Target Molecules

Table 14. E3 Ligase Motifs

[0467] For the E3 ligases of Table 14, the motif pattern uses the following nomenclature: ‘ specifies any amino acid type, ‘[X]’ specifies the allowed amino acid type(s) at that position, X at the beginning of the pattern specifies that the sequence starts with amino acid type X, [ ]’ means that the position can have any amino acid other than type X, numbers specified as the following ‘X{x,y} ’, where x and y specify the minimum and maximum number of ‘X’ amino acid type required at that position. ‘$’ sign implies the C-terminal of the protein chain. Conserved residue positions within the primary degron that are known to be posttranslationally modified (for example, phosphorylation and proline hydroxylation) are shown in boldface (PTM data from UniProt61).

Examples

DHD-A and DHD-B Binding Pairs

[0468] To evaluate the designed DHD-A and DHD-B binding element pairs described with reference to Table 1, enzyme-linked immunosorbent assays (ELISAs) were performed according to standard protocols. Briefly, 96-well Maxisorp plates (Nunc-Immuno, Sigma- Aldrich) were coated with NeutrAvidin (10 pg/ml) overnight at 4°C and subsequently blocked with BSA (2% w/v) for 1 hr at 20 °C. Two (2) nM biotinylated MBP-DHD-B was captured on the NeutrAvi din-coated wells for 30 min followed by the addition of FLAG- tagged DHD-A diluted in ELISA buffer (PBS, pH 7.4, 0.05% Tween-20, 0.2% BSA) for 30 min. The bound DHD-A proteins were then detected using a horseradish peroxidase (HRP)- conjugated anti-FLAG M2 monoclonal antibody (Sigma Aldrich) and a TMB substrate. For analysis, curves were fit using the Graphpad Prism software to a one-site specific binding model with Hill coefficient.

[0469] FIG. 8A is a plot showing the Kd determination from a competitive binding ELISA assay of DHD-A:DHD-B interaction for the DHD-A (SEQ ID NO: 11) and DHD-B (SEQ ID NO: 13) binding elements. FIG. 8B is a plot showing the Kd determination from a competitive binding ELISA assay of DHD-A:DHD-B interaction for binding elements DHD- A (SEQ ID NO: 11) and DHD-B-Ntrunc (SEQ ID NO: 15). The concentration of the bound DHD-B competitive inhibitor is plotted against the total concentration of the DHD-B competitive inhibitor. The binding curves were constructed and the Kd values were determined by non-linear curve fitting using the Graphpad Prism software. The data show that the designed binding elements exhibit high affinities with Kd in the nanomolar range.

[0470] Anticipating the auto-ubiquitination of a binding element upon fusing to a E3 ligase domains, the lysine (K) residues in the DHD-B protein (SEQ ID NO: 13) were changed to arginine (R) to generate DHD37-short-B-KtoR (SEQ ID NO: 16). FIG. 9 is a plot showing a comparison of DHD-B (SEQ ID NO: 13) and DHD37-short-B-KtoR (SEQ ID NO: 16) in a direct binding ELISA assay with DHD-A (SEQ ID NO: 11). The ELISA absorbance at 450 nm is ploted against the concentration of the added DHD-A. The binding curve was constructed by non-linear curve fitting using the Graphpad Prism software. The data show that the affinity of the binding element is not substantially altered upon the lysine-to-arginine mutations. Generally, surface lysine residues in the designed sequences can be mutated to arginine without altering structure and function (other than the intended effect of preventing auto-ubiquitination).

E3 Ligase Screening and Evaluation of Expression Constructs

[0471] Functional E3 ligase domains and ubiquitin variants (described hereinabove with reference to Table 2) that may be used to generate expression constructs for targeted protein degradation were evaluated using lentiviral transduction of T cells and flow cytometry analysis.

[0472] Jurkat, SupTl, A549 and Jekol T cell lines were obtained from American Type Culture Collection (Manassas VA). SupTl and Jekol cells were maintained in RPMI 1640 media with Glutamax (Gibco) containing 10% heat-inactivated fetal bovine serum (Gibco). For lentiviral transduction, SupTl and Jurkat cells were fed with fresh media 4-16 hours before transduction, then incubated with lentivirus in complete media + LentiBOOST at the manufacturers recommended concentration (Sirion Biotech). At 18 hours post transfection, lentivirus and LentiBOOST were diluted by addition of 1 volume fresh media.

[0473] Pre-selected, cryopreserved primary human CD4 and CD8 T cells, or mixed CD4/CD8+ T cells from normal donors were obtained from Bloodworks (Seatle WA) or Stemcell (Vancouver BC). Human T cells were cultured in OpTmizer medium (Thermo Fisher) supplemented with Immune Cell Serum Replacement (Thermo Fisher), 2mM L- glutamine (Gibco), 2mM Glutamax (Gibco), 200IU/ml IL-2 (R&D systems), 120 lU/ml IL-7 (R&D systems), and 20 lU/ml IL- 15 (R&D systems).

[0474] For lentiviral transduction, T cells were stimulated with a 1 : 100 dilution of T cell TransAct (Miltenyi) for 30 hours. Virus was then added to T cells for 18-24 hours. In some instances, virus was added to T cells after 24 hours of stimulation, and a second lentivirus was added after 30 hours of stimulation. Stimulation and viral infection were then terminated by addition of 7 volumes of fresh media without TransAct, and cells were cultured for 3-7 additional days before analysis. For danoprevir inducible dimerization of binding elements, cells were treated with 500 nM danoprevir (RG7227, product number A4024) or an equal amount of DMSO for 18-24 hours before analysis. For CD3/CD28 stimulation in cells expressing fusion constructs of E3 ligase domains and SH2 or SH3 domains, T cells were incubated with 1:100 dilution of T cell TransAct for 4 hours before analysis. For CD3 activation, a 24-well culture plate was pre-coated overnight at 4°C with a monoclonal CD3 agonist antibody (OKT3) in PBS. The plate was washed with PBS, and ~2xl0 6 transduced cells were incubated in each well of the plate for 1-2 hours before harvesting for flow cytometry and immunoblotting.

[0475] Flow cytometry was performed on a Ze5 cytometer (Biorad). To determine expression of cell surface markers, between IxlO 5 - 2xl0 5 total cells were transferred to a V bottom 96 well culture dish (Coming). Cells were washed twice with flow cytometry staining buffer (eBioscience), then stained with the relevant reagents in a total volume of 50 pL flow cytometry staining buffer for 30 minutes on ice. After staining, cells were washed twice with flow cytometry staining buffer, fixed in FluoroFix Buffer (Biolegend) and kept at 4°C in the dark until analysis. For experiments involving staining of intracellular BACH2, cells were surface stained as described above, then fixed, permeabilized and labeled according to the FoxP3 Transcription Factor Staining Buffer Set manufacturer’s protocol (eBioscience, # 00-5523-00). Purified recombinant ROR1 ectodomain linked to human Ig Fc was produced in-house and conjugated to Alexa 647 dye for detection. Purified recombinant Her2 ectodomain linked to human Ig Fc was produced in-house and conjugated to Alexa 647 dye for detection. eFluor 780 Fixable Viability dye (eBioscience) was included during primary antibody stain at a 1 : 8000 dilution. Flow cytometry data was analyzed using FlowJo 10 (Tree Star). A list of antibodies that were used is shown in Table 8.

[0476] A panel of designed DHD-E3/Ub constructs (see Table 2) was screened for efficient degradation of a ROR1 -specific CAR (ROR1 CAR-DHD-A, SEQ ID NO: 115; see Table 3, which was used as a model target for degrading membrane proteins. The panel of membrane- associated DHD-E3 ligases screened were as follows: RNF43 -vl (SEQ ID NO: 74), RNF43- v2 (SEQ ID NO: 77), RNF43-v3 (SEQ ID NO: 76), ZNRF3-vl (SEQ ID NO: 84), ZNRF3-v2 (SEQ ID NO: 85), ZNRF3-v3 (SEQ ID NO: 86), MARCH8-vl (SEQ ID NO: 87), MARCH8-v2 (SEQ ID NO: 88), MARCH8-v3 (SEQ ID NO: 89), RNF128-vl (SEQ ID NO: 78), RNF128-v2 (SEQ ID NO: 79), RNF128-v3 (SEQ ID NO: 80). The panel of cytoplasmic DHD-E3 ligase screened were as follows: ELOC (SEQ ID NO: 97), FBW1A (SEQ ID NO: 98), FBXW7 (SEQ ID NO: 96), LNX1 (SEQ ID NO: 90), CHIP (SEQ ID NO: 95), NEDD4 (SEQ ID NO: 92), S0CS2 (SEQ ID NO: 94 ), SPOP (SEQ ID NO: 93), VHL (SEQ ID NO: 91), RNF4 (SEQ ID NO: 99), TRAF6 (SEQ ID: 100), 3xUb (SEQ ID NO: 81), 3xUb K48R (SEQ ID NO: 82), and 3xUb K63R (SEQ ID NO: 83). Briefly, SupTl cells were co-transduced with lentiviruses expressing each member of the panel and either ROR1 CAR-DHD-A (black bars) or a control CAR lacking a dimerization domain (binding element; white bars), and cells were stained with a RORl-ECD-Fc Alexa Fluor 647 conjugate to detect the surface expression of CAR-DHD by flow cytometry. The gMFI ratio of each construct was calculated by normalizing with the gMFI measured in the cells transduced with only the CAR constructs. Functional constructs were identified as having the normalized gMFIs below the 0.25 cutoff.

[0477] FIG. 10A and FIG. 10B are a plot 1000 and a plot 1010 showing the normalized gMFIs for membrane-associated DHD-E3 ligases and cytoplasmic DHD-E3 ligases, respectively. Novel E3 ligase designs are denoted with asterisks (*). We identified both membrane-associated DHD-E3 designs (FIG. 10A) and cytoplasmic DHD-E3 designs (FIG. 10B) that are capable of degrading the CAR target. The designs that exhibited the most degradation activity are: RNF43, RNF4, LNX1, TRAF6 and a three-tandem linear chain of the ubiquitin K48R mutant (3xUb K48R ). The geometric mean fluorescence intensities (gMFIs) of the CAR-DHD expression were reduced to about 10-20% of the control CAR when CAR-DHD is co-expressed with these synthetic degraders.

[0478] The panel of the cytoplasmic DHD-E3 constructs (SEQ ID NOs are shown above with reference to FIG. 10) was screened for efficient degradation of a 3xFL AG-tagged DHD- B-BACH2 as a model target of cytoplasmic proteins. Briefly, SupTl cells were cotransduced with lentiviruses expressing DHD-B-BACH2 (SEQ ID NO: 120) (see Table 3) and each member of the panel, and then stained with an anti-FLAG Brilliant Violet 421 antibody conjugate to detect the ectopic expression of DHD-BACH2 by intracellular flow cytometry. The gMFI ratio of each construct was calculated by normalizing with the gMFI measured in the cells transduced with the wildtype BACH2 construct. Functional constructs were identified as having the normalized gMFIs around or below the 0.25 cutoff.

[0479] FIG. 11 is a plot 1100 showing the normalized gMFI for cytoplasmic DHD-E3 ligases screened for degradation of a 3xFLAG-tagged DHD-B-BACH2. The intracellular flow cytometry analysis of the FLAG-tagged BACH2 levels showed that the ectopically expressed BACH2 was efficiently degraded to about 10-20% of the negative control when the DHD-B fusion of LNX1, SOCS2, SPOP, VHL and RNF4 E3 domains were co-expressed with DHD-BACH2.

[0480] Referring now to FIG. 10A, FIG. 10B, and FIG. 11, the results of the two screening experiments enabled us to identify lead DHD-E3 candidate constructs for optimal degradation of both membrane receptors and cytoplasmic protein targets.

[0481] As the DHD subunits contain lysine residues that are potential sites for auto- ubiquitination, we hypothesized that the binding domain could be replaced with its lysine- depleted variant, DHD-KtoR (SEQ ID NO: 16), in order to stabilize the binding element-E3 fusion and improve degradation of the protein targets. In addition to the lysine-to-arginine mutagenesis (i. e. , lysine (K) to arginine R; KtoR), the use of a C-terminal endocytic motif to enhance the CAR degradation was explored. The endocytic motif (indicated by “endo”) contains a dileucine motif [D/E]xxxLL and a C-terminal valine residue that allows for efficient endoplasmic-reticulum export (Kozik, P., et al., Traffic (2010) doi:10.1111/j.l600- 0854.2010.01056.x, which is incorporated herein by reference in its entirety). Amino acid sequences of the optimized RNF43-DHD-B constructs that exhibit enhanced target degradation are shown in Table 4.

[0482] To test these hypotheses, primary CD4 + T cells were co-transduced with lentivirus expressing CAR-DHD and either RNF43-DHD, RNF43-DHD-endo (SEQ ID NO: 122), or the RNF43-DHD-KtoR variant (SEQ ID NO: 121) (see Table 4). Transduction of CD4+ T cells with CAR-DHD alone and co-transduction of CD4+ T cells with CAR control and RNF43-DHD-KtoR were used as controls. Non-transduced CD4+ T cells were used as a background CAR expression level control. The CAR expression levels in the transduced and non-transduced cells were evaluated using CAR surface staining and flow cytometry. [0483] FIG. 12 is a panel 1200 showing CAR surface staining and geometric mean fluorescence intensity (gMFIs) levels in transduced and control CD4+ T cells. Panel A is a table showing the “CAR” and “E3” expression constructs used for each CD4+ T cell transduction (i.e., row 1: CAR-DHD alone; row 2: CAR-DHD + RNF43-DHD; row 3: CAR- DHD + RNF43-DHD-endo; row 4: CAR-DHD + RNF43-DHD-KtoR; row 5: Car control + RNF43-DHD-KtoR, and row 6: non-transduced cells Panel B is an overlay histogram showing CAR surface staining levels for each CD4+ T cell transduction and non-transduced cells. Panel C is a histogram showing CAR gMFI for each CD4+ T cell transduction and non-transduced cells. The histograms shown in Panels B and C are aligned with the respective expression constructs (CAR and E3) shown in Panel A. The flow cytometry analysis shows that the CAR surface expression in CAR-DHD + RNF43-DHD-KtoR transduced cells is reduced to the same level as in the non-transduced control cells (-,-). The data also shows that the lysine-depleted variant RNF43-DHD-KtoR does not impact the surface expression of the CAR control (row 5: Car control + RNF43-DHD-KtoR). Similar to the RNF43-DHD-KtoR variant, the endocytic-motif containing construct RNF43-DHD-endo induced the degradation of CAR-DHD to the background level observed in the nontransduced control. We also observed a similar amount of degradation with two other endocytic motifs (data not shown). The gMFI measurements confirmed that the CAR expression was reduced close to the background level in the mock control when RNF43- DHD-KtoR or RNF43-DHD-endo was co-transduced with the CAR-DHD construct. The results from these two optimization approaches indicate that CAR degradation can be further promoted by increasing the cellular pool of DHD-E3 ligases by minimizing self- ubiquitination and by enhancing CAR endocytosis with the use of endocytic motifs.

Small Molecule-Recruited Polypeptides

[0484] The DHD degrader system does not require the addition of a small molecule for controlling dimerization of the binding element pair. However, the functional E3 ligase domains identified in our screens can be put under small molecule control by using “chemically induced dimers” (CID) in place of the DHDs. An example of a small molecule- controlled heterodimer system is described hereinabove with reference to FIG. 5. Amino acid sequences of DNCR-E3 and CAR-NS3a constructs used to evaluate the used of small molecule-recruited binding elements in the degrader system are shown in Table 5. [0485] To evaluate the use of small molecule-recruited binding elements in the degrader system, lentiviral constructs encoding CAR-NS3a (SEQ ID NO: 126) and DNCR2-E3 ligases including the E3 ligase domains RNF43, DNCR2-RNF4 (SEQ ID NO: 128) and LNX1- DNCR2 (SEQ ID NO: 129) (see Table 5) were generated and evaluated for their ability to facilitate degradation of the CAR in response to danoprevir. Briefly, SUP-T1 cells were cotransduced with lentiviruses expressing CAR-NS3a and each of the DNCR-E3 ligases. The gMFIs of the surface CAR expression were measured by flow cytometry 24 hours after danoprevir addition. SUP-T1 cells transduced with CAR-NS3a alone were used as a control. Functional constructs were identified as having gMFI values below 10% of the cells transduced with only CAR-NS3a.

[0486] FIG. 13 is a plot 1300 showing inducible degradation of CAR-NS3a with LNX1- DNCR2, DNCR2-RNF4, and RNF43-DNCR2 in the presence of 500 nM danoprevir. The flow cytometry analysis of CAR surface expression indicated that CAR-NS3a was degraded upon danoprevir addition to less than 10% of the original CAR level when the cells were transduced with RNF43-DNCR2 and DNCR2-RNF4.

[0487] We further optimized the design by introducing the lysine-to-arginine mutations (KtoR) and endocytic motif (endo) in single lentiviral constructs that a transduction marker, CAR-NS3a and RNF43-DNCR2-KtoR-endo (SEQ ID NO: 132) variant linked by P2A selfcleavage peptides. Single lentiviral constructs encoding RNF43-DNCR2 were used as a control. FIG. 14 is a diagram 1400 of single vector constructs encoding CAR-NS3a and RNF43-DNCR2 (i) and RNF43-DNCR2-KtoR-endo (ii) linked by P2A self-cleavage peptides.

[0488] SUP-T1 cells were transduced with lentiviruses expressing the control RNF43- DNCR2 control construct (i) or the DNCR2-KtoR-endo (ii) construct, incubated with 500 nM danoprevir. The gMFIs of the surface CAR expression were measured by flow cytometry 24 hours after danoprevir addition.

[0489] FIG. 15 is a panel 1500 showing CAR surface staining and gMFI levels in SUP-T1 cells transduced with lentiviruses expressing the control RNF43-DNCR negative control (i) or the RNF43-DNCR-KtoR-endo (ii) constructs. Panel A is a table showing the control (i), DNCR-KtoR endo (ii), and presence or absence of danoprevir (Dano) addition for each experimental condition. Panel B is an overlay histogram showing CAR surface staining levels for each SUP-T1 cell transduction and non-tranduced cells Panel C is a histogram showing CAR gMFI for each SUP-T1 cell transduction and non-transduced cells (NTD). The histogram shown in Panel B is aligned with the respective expression constructs (i and ii) shown in Panel A. Flow cytometry analysis of the transduced cells revealed that adding 500 nM danoprevir reduced the CAR gMFI of the cells transduce with construct (i) to less than about 10% of the negative. As expected with the single lentiviral format, the expression level of DNCR2-E3 was potentially limiting, and the CAR degradation upon drug treatment was thus observed to be only at about 32% of the original level. With the use of RNF43-DNCR2-KtoR variants, the level of CAR degradation was drastically reduced fourfold to about 8% of the original level. The data show that these optimized designs may be useful in applications in which extrinsic control of targeted degradation is required.

Native Motif Binding Elements for Recruiting E3 Ligase

[0490] In addition to using DHDs and small molecule-recruited polypeptide binding elements, it is possible to control degradation in our systems using protein domains that recruit E3 ligases to endogenous targets of interest, including domains that respond to post- translational modifications (as described hereinabove with reference to FIG. 6A and FIG. 6B). We generated sequences to test our identified E3 ligase domains with other proteinrecognition modules, including TCR-targeting transmembrane domains and protein binders that target phosphorylation sites. Amino acid sequences of the TM CD3z -, SH2 ZAP70 -, SH3 NCK1 -E3 fusion constructs are shown in Table 6.

[0491] To target the endogenous TCR complexes, constructs encoding the CD3<( transmembrane domain (TM CD3z ) and the E3 ligase domain of RNF43 were generated. Jurkat cells were transduced with the lentiviruses expressing these TM CD3z -RNF43 fusion proteins and the surface expression of the TCR complex was measured by flow cytometry.

[0492] FIG. 16 is a plot 1600 and a plot 1610 showing overlay histograms of TCR staining and gMFI levels, respectively, of cells transduced with TM CD3z -RNF43 constructs, a GFP control, and non-transduced cells using an anti-TCRa/p antibody (BV421). In this example, data is shown for four versions of the TM CD3z -RNF43 construct: vl (SEQ ID NO: 133), v2 (SEQ ID NO: 134), v3 (SEQ ID NO: 135), and v4 (SEQ ID NO: 136). The flow cytometiy analysis revealed that the gMFI of the endogenous TCR proteins was degraded to 10-20% of the non-transduced control cells. [0493] In addition, the use of SH2 domains from ZAP70 and SH3 domains from NCK1, which are known to interact with CD3<( and TCR-CD3e, respectively, in the activated TCR complex where also tested. Jurkat cells were transduced with the lentiviruses expressing the fusion constructs of phosphoresponsive domains and E3 ligase domains and stimulated for 4 hours with a polymeric nanomatrix conjugated to recombinant humanized CD3 and CD 28 agonists (TransAct). The surface expression of the TCR complex was measured by flow cytometry. The constructs tested were: LNXl-nSH2-cSH2 (SEQ ID NO: 139), nSH2-cSH2 and LNGFR (see Table 6).

[0494] FIG. 17A is an overlay histogram plot 1700 showing endogenous TCR staining levels on the cells expressing LNXl-nSH2-cSH2, nSH2-cSH2, and LNGFR control cells using an anti TCRot/p antibody. FIG. 17B is a bar plot 1710 showing gMFIs values in cells expressing LNXl-nSH2-cSH2, nSH2-cSH2, and LNGFR. The flow cytometry analysis revealed that the level of the endogenous TCR proteins was downregulated by about 70% in the unstimulated cells expressing the E3 fusion proteins with the tandem SH2 domain (nSH2- cSH2). Upon stimulation with a polymeric nanomatrix conjugated to humanized recombinant CD3 and CD28 agonist antibodies for 4 hours, the endogenous TCR level was further downregulated to about 5% and remained to be about 3 -fold lower than the levels observed in the non-transduced cells or the cells expressing the tandem SH2 domain. These results suggest that these fusion constructs could be used to regulate TCR signaling effectively in CAR-T applications (e.g., CAR-T cell therapy).

CAR Regulation in Primary T Cells

[0495] To confirm that our designed DHD-E3 ligase are able to regulate CAR activity in primary T cells, a 1:1 mixture of CD4 + /CD8 + primary cells were co-transduced with lentiviruses expressing ROR1 CAR-DHD (SEQ ID NO: 115) (designated “CAR-DHD”) and RNF43-DHD-KtoR (SEQ ID NO: 121) (designated “DHD-E3R”) or LNXl-DHD-KtoR (designated “DHD-E3L”) as follows: un-transduced (Mock-T), transduced with either CAR or CAR-DHD alone, or dual transduced with CAR-DHD and either DHD-E3R or DHD-ESL, or GFP control. Cells were assayed for cytokine secretion, immune cell killing, and marker expression.

[0496] To test the ability of DHD-E3 recruitment to regulate acute CAR activation, we challenged un-transduced (Mock-T), single, or dual-transduced T cells against co-culture Jekol target cells or ROR1 -knockout Jekol cells. Cells were co-cultured at an effector to target ratio of 1 :4 Jekol cells or control Jekol cells lacking the CAR target antigen (i.e., Jekol-KO). After 24 hours of co-culture, supernatant was collected from the cultured T cells, centrifuged for 5 minutes at 300xg to remove cells and debris, and then frozen at -80°C until analysis. Secretion of cytokines interleukin 2 (IL2) and interferon-gamma (IFNy) were measured using a custom V-plex human proinflammatory kit (human IFNy, IL2, TNFa;

Meso Scale Discovery (MSD), Rockville, MD). For cytokine detection, supernatant was diluted 1:5 with MDS calibrator diluent and analyzed according to manufacturer’s instructions.

[0497] FIG. 18 is a pair of plots 1800 and 1810 showing concentrations of IL-2 and IFNy, respectively, present in the supernatant after 24 hours of co-culture. The data show that coexpression of either DHD-E3 construct mediated a significant reduction in antigen-driven cytokine production from CAR-T cells, compared to the cells transduced with CAR-DHD only or CAR without DHD.

[0498] To test CAR-mediated cytolytic activity, target cell number in T-cell:Jeko-l cocultures was measured over time. Briefly, CAR T cells were seeded at a 1 : 1 or 1 :5 effector to target cell ratio with NucLight Red (NLR)-ROR1+ target cells or NLR-ROR1 JeKol-KO target cells, or A549 Nuclight Red (NLR) target cells in a Coming 96 well plate. Plates were cultured in an Incucyte system (Sartorius; Essen BioScience, Ann Arbor, MI) for 72 hours. Tumor cell (JeKol) killing was determined via Incucyte measurement over time for total NLR+ cells/well compared to tumor cells alone.

[0499] FIG. 19 is a plot 1900 showing the killing of Jekol target cells by T cells co-cultured at an effector to target ratio of 1 :4. The data show that co-expression of DHD-E3 constructs effectively blocked CAR-antigen driven target cell killing, with results close to those observed in control Mock T co-cultures. These results indicate that the expression of DHD- E3 fusions effectively blocks acute CAR activity.

[0500] We next sought to determine if the intrinsic control of CAR expression could prevent exhaustion induced by chronic CAR signaling. To test chronic signaling induced exhaustion in vitro, T cells were co-cultured with the A549 lung adenocarcinoma cell line (target cells) at an effector to target ratio of 1:3 for 7 days under conditions of constant antigen exposure, then surface markers of T cell exhaustion (CD39, PD-1, and Lag3) were examined by flow cytometry. [0501] FIG. 20 is a panel of plots 2000, 2010, and 2015 showing gMFI for surface expression of the CD39, PD-1, and Lag3 exhaustion markers on single or dual transduced CAR-T cells co-cultured with A549 target cells. The data show that co-expression of CAR- DHD and DHD-E3 constructs reduced expression of the exhaustion markers CD39, PD1, and Lag3, compared to CAR, CAR-DHD, or CAR-DHD + eGFP cultures. These results indicate that modular expression of DHD-E3 fusions regulates acute and chronic signaling from a paired CAR-DHD construct.

[0502] As regulated degradation of the CAR is triggered by expression of a paired E3 fusion that is recruited to the CAR, dynamic patterns of regulation could be generated by placing the engineered E3 module under control of an inducible promoter which responds to cell-intrinsic or environmental stimuli. In one example, expression of a DHD-E3 module is placed under a calcium responsive NF AT driven promoter (see Table 7). In this example, activation of the T cell by CAR signaling will induce calcium flux and expression of the regulatory DHD-E3 module, reducing CAR expression and terminating signaling. Since T cell exhaustion is considered a major factor limiting the efficacy of chimeric antigen receptor (CAR) T cells against cancer, we hypothesized that this intrinsic system of CAR degradation regulated by our designed E3 ligase fusions would enable transient cessation of CAR signaling and allow exhausted cells to regain their antitumor functionality and differentiation into memory states.