CARTyrin compositions and methods of use

文档序号：751362 发布日期：2021-04-02 浏览：26次中文

阅读说明：本技术 CARTyrin组合物和使用方法 (CARTyrin compositions and methods of use ) 是由 E·奥斯特塔 D·舍德洛克于 2019-03-07 设计创作，主要内容包括：公开了Centryin嵌合抗原受体(CARTyrins),编码本公开内容的CARTyrins的CARTyrin转座子,经修饰以表达本公开内容的CARTyrins的细胞,以及其制备方法和使用其用于过继细胞治疗的方法。在优选的实施方案中,本公开内容的CARTyrins特异性结合前列腺特异性膜抗原(PSMA)的序列。(Disclosed are centrysin chimeric antigen receptors (CARTyrins), CARTyrin transposons encoding the CARTyrins of the disclosure, cells modified to express the CARTyrins of the disclosure, as well as methods of making and using the same for adoptive cell therapy. In a preferred embodiment, the CARTyrins of the present disclosure specifically bind to a sequence of Prostate Specific Membrane Antigen (PSMA).)

1. A Chimeric Antigen Receptor (CAR) comprising:

(a) an extracellular domain comprising an antigen recognition region, wherein the antigen recognition region comprises at least one Prostate Specific Membrane Antigen (PSMA) -specific centrin;

(b) A transmembrane domain, and

2. The CAR of claim 1, wherein the extracellular domain of (a) further comprises a signal peptide.

3. The CAR of claim 1 or 2, wherein the extracellular domain of (a) further comprises a hinge between the antigen recognition region and the transmembrane domain.

4. The CAR of claim 2 or 3, wherein the signal peptide comprises a sequence encoding a human CD2, CD3 δ, CD3 ε, CD3 γ, CD3 ζ, CD4, CD8 α, CD19, CD28, 4-1BB, or GM-CSFR signal peptide.

5. The CAR of claim 2 or 3, wherein the signal peptide comprises a sequence encoding a human CD8 a signal peptide.

6. The CAR of claim 5, wherein the signal peptide comprises an amino acid sequence comprising MALPVTALLLPLALLLHAARP (SEQ ID NO: 18004).

7. The CAR of claim 5 or 6, wherein the signal peptide is encoded by a nucleic acid sequence comprising atggcactgccagtcaccgccctgctgctgcctctggctctgctgctgcacgcagctagacca (SEQ ID NO: 18005).

8. The CAR of any preceding claim, wherein the transmembrane domain comprises a sequence encoding a human CD2, CD3 δ, CD3 ε, CD3 γ, CD3 ζ, CD4, CD8 α, CD19, CD28, 4-1BB, or GM-CSFR transmembrane domain.

9. The CAR of any preceding claim, wherein the transmembrane domain comprises a sequence encoding a human CD8 a transmembrane domain.

10. The CAR of claim 9, wherein the transmembrane domain comprises an amino acid sequence comprising IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 18006).

11. The CAR of claim 9 or 10, wherein the transmembrane domain is encoded by a nucleic acid sequence comprising atctacatttgggcaccactggccgggacctgtggagtgctgctgctgagcctggtcatcacactgtactgc (SEQ ID NO: 18007).

12. The CAR of any preceding claim, wherein the endodomain comprises a human CD3 ζ endodomain.

13. The CAR of any preceding claim, wherein the at least one co-stimulatory domain comprises human 4-1BB, CD28, CD40, ICOS, MyD88, OX-40 intracellular segment, or any combination thereof.

14. The CAR of any preceding claim, wherein the at least one co-stimulatory domain comprises a human CD28 and/or a 4-1BB co-stimulatory domain.

15. The CAR of claim 13 or 14, wherein the CD3 ζ costimulatory domain comprises an amino acid sequence comprising

16. The CAR of claim 15, wherein the CD28 co-stimulatory domain is encoded by a nucleic acid sequence comprising

17. The CAR of claim 13 or 14, wherein the 4-1BB co-stimulatory domain comprises an amino acid sequence comprising KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 18011).

18. The CAR of claim 17, wherein the 4-1BB co-stimulatory domain is encoded by a nucleic acid sequence comprising

19. The CAR of any one of claims 14-18, wherein the 4-1BB co-stimulatory domain is located between the transmembrane domain and the CD28 co-stimulatory domain.

20. The CAR of any one of claims 2-19, wherein the hinge comprises a sequence derived from a human CD8 a, IgG4, and/or CD4 sequence.

21. The CAR of any one of claims 2-19, wherein the hinge comprises a sequence derived from a human CD8 a sequence.

22. The CAR of claim 20 or 21, wherein the hinge comprises an amino acid sequence comprising TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 18014).

23. The CAR of claim 22, wherein the hinge is encoded by a nucleic acid sequence comprising

24. The CAR of any preceding claim, wherein the at least one PSMA-specific Centyrin comprises at least one fibronectin type III (FN3) domain.

25. The CAR of any preceding claim, wherein the at least one PSMA-specific centrin is capable of specifically binding to a sequence of Prostate Specific Membrane Antigen (PSMA).

26. The CAR of claim 25, wherein the at least one PSMA-specific centrin comprises

Or consists of the amino acid sequence of (a).

27. The CAR of claim 26, wherein the at least one PSMA-specific centrin comprises a chemical bond with

Has an amino acid sequence of at least 70% identity.

28. The CAR of claim 25 or 26, wherein the at least one PSMA-specific centrin comprises

Or consists of a nucleic acid sequence of (a).

29. The CAR of claim 25, wherein the at least one PSMA-specific centrin comprises

Or consists of the amino acid sequence of (a).

30. The CAR of claim 29, wherein the at least one PSMA-specific centrin comprises a chemical bond with

Has an amino acid sequence of at least 70% identity.

31. The CAR of claim 25 or 29, wherein the at least one PSMA-specific centrin comprises

Or consists of a nucleic acid sequence of (a).

32. The CAR of claim 25, wherein the at least one fibronectin type III (FN3) domain is derived from a human protein.

33. The CAR of claim 32, wherein the human protein is tenascin-C.

34. The CAR of any one of claims 25 or 32-34, wherein the consensus sequence comprises

35. The CAR of any one of claims 25 or 32-34, wherein the consensus sequence comprises

36. The CAR of any one of claims 25 or 32-34, wherein the consensus sequence comprises a consensus sequence with

Has at least 70% identity to the amino acid sequence of (a).

37. The CAR of any one of claims 25 or 32-34, wherein the consensus sequence is modified at one or more positions within:

(a) an A-B loop comprising or consisting of amino acid residues TEDS (SEQ ID NO: 18020) at positions 13-16 of the consensus sequence;

(b) a B-C loop comprising or consisting of amino acid residues TAPDAAF at positions 22-28 of the consensus sequence (SEQ ID NO: 18021);

(c) a C-D loop comprising or consisting of amino acid residues SEKVGE (SEQ ID NO: 18022) at positions 38-43 of the consensus sequence;

(d) a D-E loop comprising or consisting of the amino acid residues GSER at positions 51-54 of the consensus sequence (SEQ ID NO: 18023);

(e) an E-F loop comprising or consisting of amino acid residues GLKPG at positions 60-64 of the consensus sequence (SEQ ID NO: 18024);

(f) a F-G loop comprising or consisting of amino acid residues KGGHRSN at positions 75-81 of the consensus sequence (SEQ ID NO: 18025); or

(g) Any combination of (a) - (f).

38. The CAR of any one of claims 25 or 32-37, which comprises a consensus sequence of at least 5 fibronectin type III (FN3) domains.

39. The CAR of any one of claims 25 or 332-38, which comprises a consensus sequence of at least 10 fibronectin type III (FN3) domains.

40. The CAR of any one of claims 25 or 32-39, comprising a consensus sequence of at least 15 fibronectin type III (FN3) domains.

41. The CAR of any one of claims 1-40, wherein the at least one Centryin is selected from less than or equal to 10^-9M, less than or equal to 10^-10M, less than or equal to 10^-11M, less than or equal to 10^-12M, less than or equal to 10^-13M, less than or equal to 10^-14M and less than or equal to 10^-15K of M_DBinds to the antigen with at least one affinity.

42. The CAR of claim 41, wherein the K is_DThe measurement was performed by surface plasmon resonance.

43. A composition comprising a CAR of any preceding claim and at least one pharmaceutically acceptable carrier.

44. A transposon comprising the CAR of any one of claims 1-42.

45. The transposon of claim 44, wherein the transposon further comprises a selection gene.

46. The transposon of claim 45 wherein the selection gene encodes a gene product essential for cell viability and survival.

47. The transposon of claim 45 wherein the selection gene encodes a gene product essential for cell viability and survival when challenged with selective cell culture conditions.

48. The transposon of claim 47 wherein the selective cell culture condition comprises a compound that is detrimental to cell viability or survival and wherein the gene product confers resistance to the compound.

49. The transposon of claim 44, wherein the selection gene comprises neo, TYMS (thymidylate synthase), MGMT (O (6) -methylguanine-DNA methyltransferase), a broad spectrum drug resistance gene (MDR1), DHFR, ALDH1 (aldehyde dehydrogenase family 1, member A1), FRANCF, RAD51C (RAD51 paralog C), GCS (glucosylceramide synthase), NKX2.2(NK2 homeobox 2), or any combination thereof.

50. The transposon of any one of claims 44-49, wherein the transposon comprises an inducible caspase polypeptide comprising

(a) A ligand-binding domain having a ligand-binding domain,

(b) a joint, and

51. The transposon of claim 50, wherein the non-human sequence is a restriction enzyme site.

52. The transposon of claim 50 or 51, wherein the ligand binding region inducible caspase polypeptide comprises an FK506 binding protein 12(FKBP12) polypeptide.

53. The transposon of claim 52, wherein the amino acid sequence of the FK506 binding protein 12(FKBP12) polypeptide comprises a modification at position 36 of the sequence.

54. The transposon of claim 53, wherein the modification is a substitution of valine (V) for phenylalanine (F) at position 36 (F36V).

55. The transposon of any one of claims 52-54, wherein the FKBP12 polypeptide is encoded by an amino acid sequence comprising

56. The transposon of claim 55, wherein the FKBP12 polypeptide is encoded by a nucleic acid sequence comprising

57. The transposon of any one of claims 50-56, wherein the linker region of the inducible pro-apoptotic polypeptide is encoded by an amino acid comprising GGGGS (SEQ ID NO: 18028).

58. The transposon of claim 57, wherein the linker region of the inducible pro-apoptotic polypeptide is encoded by a nucleic acid sequence comprising GGAGGAGGAGGATCC (SEQ ID NO: 18029).

59. The transposon of any one of claims 50-58, wherein the truncated caspase 9 polypeptide of the inducible pro-apoptotic polypeptide is encoded by an amino acid sequence that does not comprise arginine (R) at position 87 of the sequence.

60. The transposon of any one of claims 50-59, wherein the truncated caspase 9 polypeptide of the inducible pro-apoptotic polypeptide is encoded by an amino acid sequence that does not comprise alanine (A) at position 282 of the sequence.

61. The transposon of any one of claims 50-60, wherein the truncated caspase 9 polypeptide of the inducible pro-apoptotic polypeptide is comprised of

The amino acid code of (1).

62. The transposon of claim 61, wherein the truncated caspase 9 polypeptide of the inducible pro-apoptotic polypeptide is comprised of

The nucleic acid sequence of (a).

63. The transposon of any one of claims 50-62, wherein the inducible pro-apoptotic polypeptide is comprised of

The amino acid sequence of (1) encodes.

64. The transposon of claim 63, wherein the inducible pro-apoptotic polypeptide consists of

The nucleic acid sequence of (a).

65. The transposon of any one of claims 50-64, wherein the transposon comprises at least one self-cleaving peptide.

66. The transposon of any one of claims 50-65, wherein said transposon comprises at least one self-cleaving peptide, and wherein a self-cleaving peptide is located between said CAR and said selection gene.

67. The transposon of any one of claims 50-66, wherein said transposon comprises at least one self-cleaving peptide, and wherein self-cleaving peptide is located between said CAR and said inducible pro-apoptotic polypeptide.

68. The transposon of any one of claims 50-67, wherein the transposon comprises at least two self-cleaving peptides, and wherein a first self-cleaving peptide is located upstream of the inducible pro-apoptotic polypeptide and a second self-cleaving peptide is located downstream of the inducible pro-apoptotic polypeptide.

69. The transposon of any one of claims 50-68, wherein said transposon comprises at least one self-cleaving peptide, and wherein a first self-cleaving peptide is located upstream of said CAR and a second self-cleaving peptide is located downstream of said CAR.

70. The transposon of any one of claims 65-69, wherein the at least one self-cleaving peptide comprises a T2A peptide, a GSG-T2A peptide, an E2A peptide, a GSG-E2A peptide, an F2A peptide, a GSG-F2A peptide, a P2A peptide or a GSG-P2A peptide.

71. The transposon of claim 70, wherein the T2A peptide comprises an amino acid sequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO: 18034).

72. The transposon of claim 70, wherein the GSG-T2A peptide comprises an amino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 18035).

73. The transposon of claim 70, wherein the E2A peptide comprises an amino acid sequence comprising QCTNYALLKLAGDVESNPGP (SEQ ID NO: 18036).

74. The transposon of claim 70, wherein the GSG-E2A peptide comprises an amino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 18037).

75. The transposon of claim 70, wherein the F2A peptide comprises an amino acid sequence comprising VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 18038).

76. The transposon of claim 70, wherein said GSG-F2A peptide comprises an amino acid sequence comprising GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 18039).

77. The transposon of claim 70, wherein the P2A peptide comprises an amino acid sequence comprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 18040).

78. The transposon of claim 70, wherein the GSG-P2A peptide comprises an amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 18041).

79. The transposon of any one of claims 44 to 78, further comprising a sequence encoding a Chimeric Stimulatory Receptor (CSR).

80. The transposon of claim 79, wherein the CSR comprises:

(a) an extracellular domain comprising an activating component;

(b) a transmembrane domain; and

wherein the combination of (a), (b), and (c) is non-naturally occurring.

81. The transposon of claim 80 wherein the activating component of (a) is isolated or derived from a first protein.

82. The transposon of claim 81, wherein the at least one signaling domain of (c) is isolated or derived from a second protein.

83. The transposon of claim 81 or 82, wherein the first protein and the second protein are not identical.

84. The transposon of any one of claims 80-83, wherein the activating component comprises one or more of a component of a human transmembrane receptor, a human cell surface receptor, a T-cell receptor (TCR), a component of a TCR complex, a component of a TCR co-receptor, a component of a TCR co-stimulatory protein, a component of a TCR inhibitory protein, a cytokine receptor and a chemokine receptor.

85. The transposon of claim 84 wherein the activating component comprises a component of a T-cell receptor (TCR) to which an agonist of the activating component binds, a component of a TCR complex, a component of a TCR co-receptor, a component of a TCR co-stimulatory protein, a component of a TCR inhibitory protein, a cytokine receptor and a portion of one or more of a chemokine receptor.

86. The transposon of any one of claims 80-85, wherein the activation component comprises a portion thereof to which a CD2 protein or agonist binds.

87. The transposon of any one of claims 80-86, wherein the signal transduction domain comprises one or more of a component of a human signal transduction domain, a T-cell receptor (TCR), a component of a TCR complex, a component of a TCR co-receptor, a component of a TCR co-stimulatory protein, a component of a TCR inhibitory protein, a cytokine receptor and a chemokine receptor.

88. The transposon of claim 87, wherein the signaling domain comprises a CD3 protein.

89. The transposon of claim 88, wherein said CD3 protein comprises a CD3 ζ protein.

90. The transposon of any one of claims 80-89, wherein the endodomain further comprises a cytoplasmic domain.

91. The transposon of claim 90, wherein the cytoplasmic domain is isolated or derived from a third protein.

92. The transposon of claim 91, wherein the first protein and the third protein are the same.

93. The transposon of any one of claims 80-92, wherein said extracellular domain further comprises a signal peptide.

94. The transposon of claim 93, wherein the signal peptide is derived from a fourth protein.

95. The transposon of claim 94, wherein the first protein and the fourth protein are the same.

96. The transposon of any one of claims 80-95, wherein the transmembrane domain is isolated or derived from a fifth protein.

97. The transposon of claim 96, wherein the first protein and the fifth protein are the same.

98. The transposon of any one of claims 80-97, wherein the activator component does not bind to a naturally occurring molecule.

99. The transposon of any one of claims 80-97, wherein the CSR does not transduce a signal when the activating component binds to a naturally occurring molecule.

100. The transposon of claim 80, wherein the extracellular domain comprises a modification.

101. The transposon of claim 100, wherein the modification comprises a mutation or truncation of a sequence encoding the activator component when compared to the wild type sequence of the first protein.

102. The transposon of any one of claims 80-101, wherein the activating component binds to a non-naturally occurring molecule.

103. The transposon of any one of claims 80-102, wherein the CSR selectively transduces a signal when the activating component binds to a non-naturally occurring molecule.

104. The transposon of any one of claims 45-103, wherein the transposon is a piggyBac or piggyBac-like transposon.

105. The transposon of any one of claims 45-103, wherein the transposon is a TcBuster transposon.

106. The transposon of any one of claims 45-103, wherein the transposon is a Sleeping Beauty transposon.

107. The transposon of any one of claims 45-103, wherein the transposon is a helraisiser transposon.

108. The transposon of any one of claims 45-103, wherein said transposon is a Tol2 transposon.

109. A composition comprising the transposon of any one of claims 45-108.

110. The composition of claim 109, further comprising a plasmid comprising a sequence encoding a transposase.

111. The composition of claim 110, wherein the sequence encoding a transposase is an mRNA sequence.

112. The composition of any one of claims 109-111, wherein the transposon is a piggyBac or piggyBac-like transposon, and wherein the transposase is a piggyBac transposase or a piggyBac-like transposase.

113. The composition of claim 112, wherein the piggyBac transposase comprises a nucleic acid comprising SEQ ID NO: 14487 amino acid sequence.

114. The composition of claim 112 or 113, wherein the piggyBac transposase is a hyperactive variant, and wherein the hyperactive variant comprises SEQ ID NO: 14487 amino acid substitutions at one or more of positions 30, 165, 282, and 538.

115. The composition of claim 114, wherein said SEQ ID NO: the amino acid substitution at position 30 of 14487 is a substitution of valine (V) for isoleucine (I) (I30V).

116. The composition of claim 114, wherein said SEQ ID NO: 14487 the amino acid substitution at position 165 is a serine (S) to glycine (G) substitution (G165S).

117. The composition of claim 114, wherein said SEQ ID NO: the amino acid substitution at position 282 of 14487 is a substitution of valine (V) for methionine (M) (M282V).

118. The composition of claim 114, wherein said SEQ ID NO: the amino acid substitution at position 538 of 14487 is a substitution of asparagine (N) with lysine (K) (N538K).

119. The composition of any one of claims 110-118, wherein the transposase is a Super piggyBac (sPBo) transposase.

120. The composition of claim 119, wherein the Super piggyBac (sPBo) transposase comprises a sequence comprising SEQ ID NO: 14484, or a pharmaceutically acceptable salt thereof.

121. The composition of any one of claims 109-111 wherein the transposon is a TcBuster transposon and wherein the transposase is a TcBuster transposase.

122. The composition of claim 121, wherein said TcBuster transposase is a high activity TcBuster transposase.

123. The composition of claim 121 or 122, wherein the TcBuster transposase comprises a sequence having at least 75% identity to:

124. the composition of any one of claims 109-111, wherein the transposon is a Sleeping Beauty transposon, and wherein the transposase is a Sleeping Beauty transposase.

125. The composition of claim 124, wherein the Sleeping Beauty transposase comprises SEQ ID NO: 14485.

126. The composition of claim 124, wherein said Sleeping Beauty transposase is a high activity Sleeping Beauty transposase (SB 100X).

127. The composition of claim 126, wherein the high activity Sleeping Beauty transposase (SB100X) comprises SEQ ID NO: 14486.

128. The composition of any one of claims 109-111, wherein the transposon is a helraisier transposon and wherein the transposase is a helraisier transposase.

129. The composition of claim 128, wherein said helraisiser transposase comprises SEQ ID NO: 14501.

130. The composition of any one of claims 109-111 wherein the transposon is a Tol2 transposon and wherein the transposase is a Tol2 transposase.

131. The composition of claim 130, wherein the Tol2 transposase comprises SEQ ID NO: 14502.

132. A vector comprising the CAR of any one of claims 1-42.

133. The vector of claim 132, wherein the vector is a viral vector.

134. The vector of claim 133, wherein the viral vector comprises a sequence isolated or derived from a retrovirus, lentivirus, adenovirus, adeno-associated virus, or any combination thereof.

135. The vector of claim 133 or 134, wherein the viral vector comprises a sequence isolated or derived from an adeno-associated virus.

136. The vector of any one of claims 133-135 wherein the viral vector is a recombinant vector.

137. The carrier of claim 132, wherein the carrier is a nanoparticle carrier.

138. The vector of claim 137, wherein the nanoparticle vector comprises a nucleic acid, an amino acid, a polymer, a micelle, a lipid, an organic molecule, an inorganic molecule, or any combination thereof.

139. The vector of any one of claims 132-138 wherein the vector further comprises a selection gene.

140. The vector of claim 139 wherein the selection gene encodes a gene product essential for cell viability and survival.

141. The vector of claim 140 wherein the selection gene encodes a gene product essential for cell viability and survival when challenged with selective cell culture conditions.

142. The vector of claim 138 wherein the selective cell culture conditions comprise a compound detrimental to cell viability or survival and wherein the gene product confers resistance to the compound.

143. The vector of any one of claims 139-142 wherein the selection gene comprises neo, TYMS (thymidylate synthase), MGMT (O (6) -methylguanine-DNA methyltransferase), pan-drug resistance gene (MDR1), ALDH1 (aldehyde dehydrogenase family 1, member a1), FRANCF, RAD51C (RAD51 paralogous gene C), GCS (glucosylceramide synthase), NKX2.2(NK2 homeobox 2), or any combination thereof.

144. The vector of any one of claims 132-143, wherein the vector comprises an inducible caspase polypeptide comprising

(a) A ligand-binding domain having a ligand-binding domain,

(b) A joint, and

wherein the inducible caspase polypeptide does not comprise a non-human sequence.

145. The vector of claim 144, wherein the non-human sequence is a restriction enzyme site.

146. The vector of claim 144 or 145 wherein the ligand binding region inducible caspase polypeptide comprises an FK506 binding protein 12(FKBP12) polypeptide.

147. The vector of claim 146, wherein the amino acid sequence of the FK506 binding protein 12(FKBP12) polypeptide comprises a modification at position 36 of the sequence.

148. The vector of claim 147, wherein the modification is a substitution of valine (V) for phenylalanine (F) at position 36 (F36V).

149. The vector of any one of claims 147 or 148, wherein the FKBP12 polypeptide is encoded by an amino acid sequence comprising

150. The vector of claim 149, wherein the FKBP12 polypeptide is encoded by a nucleic acid sequence comprising

151. The vector of any one of claims 144-150 wherein the linker region of the inducible pro-apoptotic polypeptide is encoded by an amino acid comprising GGGGS (SEQ ID NO: 18028).

152. The vector of claim 151, wherein the linker region of the inducible pro-apoptotic polypeptide is encoded by a nucleic acid sequence comprising GGAGGAGGAGGATCC (SEQ ID NO: 18029).

153. The vector of any one of claims 144-152 wherein the truncated caspase 9 polypeptide of the inducible pro-apoptotic polypeptide is encoded by an amino acid sequence that does not comprise arginine (R) at position 87 of the sequence.

154. The vector of any one of claims 144-153 wherein the truncated caspase 9 polypeptide of the inducible pro-apoptotic polypeptide is encoded by an amino acid sequence that does not comprise alanine (a) at position 282 of the sequence.

155. The vector of any one of claims 144-154 wherein the truncated caspase 9 polypeptide of the inducible pro-apoptotic polypeptide is encoded by an amino acid comprising

156. The vector of claim 155, wherein the truncated caspase 9 polypeptide of the inducible pro-apoptotic polypeptide is encoded by a nucleic acid sequence comprising

157. The vector of any one of claims 144-156 wherein the inducible pro-apoptotic polypeptide is encoded by an amino acid sequence comprising

158. The vector of claim 157, wherein said inducible pro-apoptotic polypeptide is encoded by a nucleic acid sequence comprising

159. The vector of any one of claims 132-158 wherein the vector comprises at least one self-cleaving peptide.

160. The vector of any of claims 132-158 wherein the vector comprises at least one self-cleaving peptide and wherein the self-cleaving peptide is located between the CAR and the selection gene.

161. The vector of any one of claims 132-158 wherein the transposon comprises at least one self-cleaving peptide and wherein the self-cleaving peptide is located between the CAR and the inducible pro-apoptotic polypeptide.

162. The vector of any one of claims 132-158 wherein the transposon comprises at least two self-cleaving peptides and wherein a first self-cleaving peptide is located upstream of the inducible pro-apoptotic polypeptide and a second self-cleaving peptide is located downstream of the inducible pro-apoptotic polypeptide.

163. The vector of any one of claims 132-162, wherein the vector comprises at least one self-cleaving peptide, and wherein a first self-cleaving peptide is located upstream of the CAR and a second self-cleaving peptide is located downstream of the CAR.

164. The vector of any one of claims 159-163, wherein the at least one self-cleaving peptide comprises a T2A peptide, a GSG-T2A peptide, an E2A peptide, a GSG-E2A peptide, an F2A peptide, a GSG-F2A peptide, a P2A peptide, or a GSG-P2A peptide.

165. The vector of claim 164, wherein the T2A peptide comprises an amino acid sequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO: 18034).

166. The vector of claim 164, wherein the GSG-T2A peptide comprises an amino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 18035).

167. The vector of claim 164, wherein the E2A peptide comprises an amino acid sequence comprising QCTNYALLKLAGDVESNPGP (SEQ ID NO: 18036).

168. The vector of claim 164, wherein the GSG-E2A peptide comprises an amino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 18037).

169. The vector of claim 164, wherein the F2A peptide comprises an amino acid sequence comprising VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 18038).

170. The vector of claim 164, wherein the GSG-F2A peptide comprises an amino acid sequence comprising GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 18039).

171. The vector of claim 164, wherein the P2A peptide comprises an amino acid sequence comprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 18040).

172. The vector of claim 164, wherein the GSG-P2A peptide comprises an amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 18041).

173. A composition comprising the vector of any one of claims 132-172.

174. A cell comprising the CAR of any one of claims 1-42.

175. The cell of claim 174, further comprising a Chimeric Stimulus Receptor (CSR) comprising:

(a) an extracellular domain comprising an activating component;

(b) a transmembrane domain; and

wherein the combination of (a), (b), and (c) is non-naturally occurring.

176. The cell of claim 175, wherein said activating component of (a) is isolated or derived from a first protein.

177. The cell of claim 176, wherein said at least one signaling domain of (c) is isolated or derived from a second protein.

178. The cell of claim 176 or 177, wherein the first protein and the second protein are not the same.

179. The cell according to any one of claims 175-178, wherein the activating component comprises one or more of a component of a human transmembrane receptor, a human cell surface receptor, a T-cell receptor (TCR), a component of a TCR complex, a component of a TCR co-receptor, a component of a TCR co-stimulatory protein, a component of a TCR inhibitory protein, a cytokine receptor, and a chemokine receptor.

180. The cell of claim 179, wherein the activating component comprises a component of one or more of a T-cell receptor (TCR) to which an agonist of the activating component binds, a component of a TCR complex, a component of a TCR co-receptor, a component of a TCR co-stimulatory protein, a component of a TCR inhibitory protein, a cytokine receptor, and a portion of a chemokine receptor.

181. The cell of any one of claims 175-180 wherein the activating component comprises a portion thereof to which a CD2 protein or agonist binds.

182. The cell of any one of claims 175-181, wherein the signaling domain comprises one or more of a component of a human signaling domain, a T-cell receptor (TCR), a component of a TCR complex, a component of a TCR co-receptor, a component of a TCR co-stimulatory protein, a component of a TCR inhibitory protein, a cytokine receptor, and a chemokine receptor.

183. The cell of claim 182, wherein the signaling domain comprises a CD3 protein.

184. The cell of claim 183, wherein said CD3 protein comprises a CD3 ζ protein.

185. The cell of any one of claims 175-184, wherein the intracellular domain further comprises a cytoplasmic domain.

186. The cell of claim 185, wherein said cytoplasmic domain is isolated or derived from a third protein.

187. The cell of claim 186, wherein said first protein and said third protein are the same.

188. The cell of any one of claims 175-187, wherein the extracellular domain further comprises a signal peptide.

189. The cell of claim 188, wherein said signal peptide is derived from a fourth protein.

190. The cell of claim 189, wherein said first protein and said fourth protein are the same.

191. The cell of any one of claims 175-190, wherein the transmembrane domain is isolated or derived from a fifth protein.

192. The cell of claim 191, wherein said first protein and said fifth protein are the same.

193. The cell of any one of claims 175-192, wherein the activating component does not bind to a naturally occurring molecule.

194. The cell of any one of claims 175-192, wherein the CSR does not transduce a signal when the activating component binds to the naturally occurring molecule.

195. The cell of claim 194, wherein said extracellular domain comprises a modification.

196. The cell of claim 195, wherein the modification comprises a mutation or truncation of a sequence encoding an activating component when compared to the wild-type sequence of the first protein.

197. The cell of any one of claims 175-196, wherein the activating component binds to a non-naturally occurring molecule.

198. The cell of any one of claims 175-197, wherein the CSR selectively transduces the signal upon binding of the activating component to the non-naturally occurring molecule.

199. A cell comprising the transposon or transposase of any one of claims 44-95.

200. A cell comprising the vector of any one of claims 132-172.

201. The cell of any one of claims 198-200, wherein the cell expresses the CAR on the surface of the cell.

202. The cell of any one of claims 198-200, wherein the cell is an immune cell.

203. The cell of claim 202, wherein the immune cell is a T-cell, a Natural Killer (NK) -like cell, a cytokine-induced killer (CIK) cell), a hematopoietic progenitor cell, a Peripheral Blood (PB) -derived T cell, or a Umbilical Cord Blood (UCB) -derived T-cell.

204. The cell of claim 203, wherein the immune cell is a T-cell.

205. The cell of any one of claims 175-204, further comprising: a sequence encoding an inducible pro-apoptotic polypeptide.

206. The cell of claim 204 or 205, wherein the T-cell is an early memory cell, a stem-like T-cell, a T-cell_SCM-like cells, T_SCMOr T_CM。

207. The cell of claim 26, wherein said T-cell is T_SCM。

208. The cell of any one of claims 174-207, wherein the cell is an artificial antigen presenting cell.

209. The cell of any one of claims 174-207, wherein the cell is a tumor cell.

210. The cell of any one of claims 174-209, wherein the cell is autologous.

211. A composition comprising the cell of any one of claims 174-210.

212. A method of expressing a Chimeric Antigen Receptor (CAR) on the surface of a cell, comprising:

(a) obtaining a population of cells;

(b) contacting the population of cells with a composition comprising a CAR according to any one of claims 1-42, or a sequence encoding the CAR, under conditions sufficient for transfer of the CAR across the cell membrane of at least one cell in the population of cells, thereby producing a modified population of cells;

(d) expanding and/or selecting at least one cell from the modified population of cells that expresses a CAR on the cell surface.

213. The method of claim 212, wherein the cell population comprises leukocytes.

214. The method of claim 212 or 213, wherein the cell population comprises CD4+ and CD8+ leukocytes in an optimized ratio.

215. The method of claim 214, wherein said optimized ratio of CD4+ to CD8+ leukocytes is not naturally present in vivo.

216. The method of claim 212, wherein the transposon or vector comprises a CAR or a sequence encoding a CAR.

217. The method of claim 216, wherein the transposon of any one of claims 42-77 comprises a CAR or a sequence encoding a CAR.

218. The method of claim 216 or 217, wherein the transposon comprises a piggyBac transposon.

219. The method of claim 216 or 217, wherein the transposon comprises a TcBuster transposon.

220. The method of any one of claims 215-219, further comprising a composition comprising a plasmid comprising a sequence encoding a transposase.

221. The method of claim 220, wherein the sequence encoding the transposase is an mRNA sequence.

222. The method of any one of claims 220 or 221, wherein the transposase is a piggyBac transposase.

223. The method of claim 222, wherein the piggyBac transposase comprises a nucleic acid comprising SEQ ID NO: 14487 amino acid sequence.

224. The method of claim 222 or 223, wherein the piggyBac transposase is a hyperactive variant, and wherein the hyperactive variant comprises the amino acid sequence set forth in SEQ ID NO: 14487 at one or more of positions 30, 165, 282, and 538.

225. The method of claim 224, wherein in SEQ ID NO: the amino acid substitution at position 30 of 14487 is a substitution of valine (V) for isoleucine (I) (I30V).

226. The method of claim 224, wherein in SEQ ID NO: 14487 the amino acid substitution at position 165 is a serine (S) to glycine (G) substitution (G165S).

227. The method of claim 224, wherein in SEQ ID NO: the amino acid substitution at position 282 of 14487 is a substitution of valine (V) for methionine (M) (M282V).

228. The method of claim 224, wherein in SEQ ID NO: the amino acid substitution at position 538 of 14487 is a substitution of asparagine (N) with lysine (K) (N538K).

229. The method of any one of claims 220 or 221, wherein the transposase is a Super piggyBac (sPBo) transposase.

230. The method of claim 229, wherein the Super piggyBac (sPBo) transposase comprises a nucleic acid sequence comprising SEQ ID NO: 14484 in a pharmaceutically acceptable carrier.

231. The method of claim 200, wherein the vector comprises a CAR or a sequence encoding a CAR.

232. The method of any of claims 212-231, wherein the conditions sufficient to transfer the CAR-encoding sequence across the cell membrane of at least one cell in the population of cells comprise nuclear transfection.

233. The method of any of claims 212-231, wherein the conditions of (b) sufficient to transfer the CAR-encoding sequence across the cell membrane of at least one cell in the population of cells comprise the application of at least one of one or more electrical pulses at a specified voltage, a buffer, and one or more supplemental factors.

234. The method of claim 233, wherein the buffer comprises PBS, HBSS, OptiMEM, BTXpress, Amaxa nuclear transfection instrument, human T cell nuclear transfection buffer, or any combination thereof.

235. The method of claim 233 or 234, wherein the one or more supplemental factors comprise

(a) Recombinant human cytokines, chemokines, interleukins, or any combination thereof;

(b) a salt, a mineral, a metabolite, or any combination thereof;

(d) an inhibitor of cellular DNA signaling, metabolism, differentiation, signal transduction, one or more apoptotic pathways, or a combination thereof; and

(e) an agent that modifies or stabilizes one or more nucleic acids.

236. The method according to claim 235, wherein the recombinant human cytokine, chemokine, interleukin, or any combination thereof comprises IL2, IL7, IL12, IL15, IL21, IL1, IL3, IL4, IL5, IL6, IL8, CXCL8, IL9, IL10, IL11, IL13, GM-CSF, IFN- γ, IL-1 α/IL-1F 13, IL-1 β/IL-1F 72, IL-12p 13, IL-12/IL-35p, IL-13-17A/IL-17F 72, IL-17 p-17F 17, IL13, IL-17 p-17F 32, IL13, IL-17 p, IL13, IL, IL-32 γ, IL-33, LAP (TGF-. beta.1), lymphotoxin- α/TNF- β, TGF-. beta.TNF-. alpha., TRANCE/TNFSF11/RANK L, or any combination thereof.

237. The method of claim 235, wherein the salt, mineral, metabolite, or any combination thereof comprises HEPES, nicotinamide, heparin, sodium pyruvate, L-glutamine, MEM non-essential amino acid solution, ascorbic acid, nucleosides, FBS/FCS, human serum, serum replacement, antibiotics, pH adjuster, Earle's salt, 2-mercaptoethanol, human transferrin, recombinant human insulin, human serum albumin, nuclear stainer PLUS, KCL, MgCl2, Na2HPO4, NAH2PO4, sodium lactobionate, mannitol, sodium succinate, sodium chloride, CINa, glucose, Ca (NO3)2, Tris/HCl, K2HPO4, KH2PO4, polyethylenimine, polyethylene glycol, poloxamer 188, poloxamer 181, poloxamer 407, polyvinylpyrrolidone, Pop313, Crown-5, or any combination thereof.

238. The method of claim 235, wherein the cell culture medium comprises PBS, HBSS, OptiMEM, DMEM, RPMI 1640, AIM-V, X-VIVO 15, CellGro DC medium, CTS OpTimizer T cell expansion SFM, TexMACS medium, PRIME-XV T cell expansion medium, ImmunoCult-XF T cell expansion medium, or any combination thereof.

239. The method of claim 235, wherein the inhibitor of cellular DNA signaling, metabolism, differentiation, signal transduction, one or more apoptotic pathways, or a combination thereof comprises TLR9, MyD88, IRAK, TRAF6, TRAF3, IRF-7, NF-KB, type 1 interferon, Pro-inflammatory cytokines, cGAS, STING, Sec5, TBK1, IRF-3, RNA pol III, RIG-1, IPS-1, FADD, 1, TRAF3, AIM2, ASC, caspase 1, Pro-IL1B, PI3K, Akt, Wnt3A, an inhibitor of glycogen synthase kinase-3 β (GSK-3 β), TWS119, bavlomycin, chloroquine, quinacrine, AC-YVAD-CMK, Z-VAD-FMK, Z-IETD-FMK, or any combination thereof.

240. The method of claim 235, wherein the agent that modifies or stabilizes one or more nucleic acids comprises a pH modifier, a DNA binding protein, a lipid, a phospholipid, CaPO4, a net neutral charge DNA binding peptide with or without an NLS sequence, a TREX1 enzyme, or any combination thereof.

241. The method of any of claims 212-231, wherein the conditions suitable for integration of the CAR or CAR-encoding sequence comprise at least one of a buffer and one or more additional factors.

242. The method of claim 241, wherein the buffer comprises PBS, HBSS, OptiMEM, BTXpress, Amaxa nuclear transfection instrument, human T cell nuclear transfection buffer, or any combination thereof.

243. The method of claim 241 or 242, wherein the one or more supplemental factors comprise

(a) Recombinant human cytokines, chemokines, interleukins, or any combination thereof;

(b) a salt, a mineral, a metabolite, or any combination thereof;

(d) an inhibitor of cellular DNA signaling, metabolism, differentiation, signal transduction, one or more apoptotic pathways, or a combination thereof; and

(e) an agent that modifies or stabilizes one or more nucleic acids.

244. The method according to claim 243, wherein said recombinant human cytokine, chemokine, interleukin, or any combination thereof comprises IL2, IL7, IL12, IL15, IL21, IL1, IL3, IL4, IL5, IL6, IL8, CXCL8, IL9, IL10, IL11, IL13, GM-CSF, IFN- γ, IL-1 α/IL-1F 13, IL-1 β/IL-1F 72, IL-12p 13, IL-12/IL-35p, IL-13-17A/IL-17F 72, IL-17 p/IL-17F 72, IL-17F 17, IL13, IL-17 p-17F 72, IL13, IL-17 p, IL-32 γ, IL-33, LAP (TGF-. beta.1), lymphotoxin- α/TNF- β, TGF-. beta.TNF-. alpha., TRANCE/TNFSF11/RANK L, or any combination thereof.

245. The method of claim 243, wherein said salt, mineral, metabolite, or any combination thereof comprises HEPES, nicotinamide, heparin, sodium pyruvate, L-glutamine, MEM non-essential amino acid solution, ascorbic acid, nucleosides, FBS/FCS, human serum, serum replacement, antibiotics, pH adjuster, Earle's salt, 2-mercaptoethanol, human transferrin, recombinant human insulin, human serum albumin, nuclear stainer PLUS, KCL, MgCl2, Na2HPO4, NAH2PO4, sodium lactobionate, mannitol, sodium succinate, sodium chloride, CINa, glucose, Ca (NO3)2, Tris/HCl, K2HPO4, KH2PO4, polyethylenimine, polyethylene glycol, poloxamer 188, poloxamer 181, poloxamer 407, polyvinylpyrrolidone, Pop313, Crown-5, or any combination thereof.

246. The method of claim 243, wherein said cell culture medium comprises PBS, HBSS, OptiMEM, DMEM, RPMI 1640, AIM-V, X-VIVO 15, CellGro DC medium, CTS OpTimizer T cell expansion SFM, TexMACS medium, PRIME-XV T cell expansion medium, ImmunoCult-XF T cell expansion medium, or any combination thereof.

247. The method of claim 243, wherein said inhibitor of cellular DNA signaling, metabolism, differentiation, signal transduction, one or more apoptotic pathways, or a combination thereof comprises TLR9, MyD88, IRAK, TRAF6, TRAF3, IRF-7, NF-KB, type 1 interferon, Pro-inflammatory cytokines, cGAS, STING, Sec5, TBK1, IRF-3, RNA pol III, RIG-1, IPS-1, FADD, 1, TRAF3, AIM2, ASC, caspase 1, Pro-IL1B, PI3K, Akt, Wnt3A, an inhibitor of glycogen synthase kinase-3 β (GSK-3 β), TWS119, bavlomycin, chloroquine, quinacrine, AC-YVAD-CMK, Z-VAD-FMK, Z-IETD-FMK, or any combination thereof.

248. The method of claim 243, wherein the agent that modifies or stabilizes one or more nucleic acids comprises a pH modifier, a DNA binding protein, a lipid, a phospholipid, CaPO4, a net neutral charge DNA binding peptide with or without an NLS sequence, a TREX1 enzyme, or any combination thereof.

249. The method of any one of claims 212-248, wherein the expanding and selecting of (d) occurs sequentially.

250. The method of claim 249, wherein the expanding occurs prior to the selecting.

251. The method of claim 249, wherein the expanding occurs after the selecting.

252. The method of claim 249, wherein the further selecting occurs after the expanding.

253. The method of any one of claims 212-252, wherein the expanding and selecting of (d) occurs simultaneously.

254. The method of any of claims 212-253, wherein the expanding comprises contacting at least one cell of the modified population of cells with an antigen to stimulate the at least one cell by the CAR.

255. The method of claim 254, wherein the antigen is present on the surface of a substrate.

256. The method of claim 255, wherein the substrate is a bead or a plurality of beads.

257. The method of claim 256, wherein the bead or plurality of beads is isolated from the modified cell population after expansion.

258. The method of claim 257, wherein the antigen is presented on the surface of a cell.

259. The method of claim 258, wherein the antigen is presented on the surface of an artificial antigen presenting cell.

260. The method of any one of claims 212-259, wherein the transposon or vector comprises a selection gene and wherein the selecting step comprises contacting at least one cell of the modified population of cells with a compound to which the selection gene confers resistance, thereby identifying cells that express the selection gene as surviving the selection and identifying cells that fail to express the selection gene as failing to survive the selecting step.

261. The method of any one of claims 212-260, wherein the expanding and selecting steps are performed over a period of 10 to 14 days inclusive.

262. A composition comprising the expanded and selected cell population of any one of claims 212-261.

263. A method of treating cancer in a subject in need thereof, comprising administering to the subject the composition of any of claims 43, 109, 123, 173, 211, or 262, wherein the CAR specifically binds to an antigen on a tumor cell.

264. The method of claim 263, wherein the tumor cell is a malignant tumor cell.

265. The method of claim 263 or 264, comprising administering to the subject the composition of claim 211 or 262, wherein the cell or population of cells is autologous.

266. The method of claim 263 or 264, comprising administering to the subject the composition of claim 211 or 262, wherein the cell or population of cells is allogeneic.

267. A method of modifying cell therapy in a subject in need thereof, comprising administering to the subject a composition comprising a cell comprising the transposon of any one of claims 44 to 80, wherein apoptosis can be selectively induced in the cell by contacting the cell with an inducing agent.

268. A method of modifying cell therapy in a subject in need thereof, comprising administering to the subject a composition comprising a cell comprising the vector of any one of claims 96-136, wherein apoptosis can be selectively induced in the cell by contacting the cell with an inducing agent.

269. The method of claim 267 or 268, wherein the cells are autologous.

270. The method of any one of claims 267-269, wherein the cell therapy is adoptive cell therapy.

271. The method of any one of claims 267-269, wherein the modification is termination of cellular therapy.

272. The method of any one of claims 267-271, wherein the modification is the depletion of a portion of the cells provided in the cell therapy.

273. The method of any one of claims 267-272, further comprising the step of administering an inhibitor of an inducing agent to inhibit modification of the cell therapy, thereby restoring function and/or efficacy of the cell therapy.

Technical Field

The present disclosure relates to molecular biology, and more particularly, to chimeric antigen receptors, and to transposons comprising one or more CARTyrins, and methods of making and using the same.

Background

There has been a long but unmet need in the art for methods of directing immune cell specificity without the use of traditional antibody sequences or fragments thereof. The present disclosure provides superior chimeric antigen receptors.

Disclosure of Invention

The present disclosure provides a Chimeric Antigen Receptor (CAR) comprising: (a) an extracellular domain comprising an antigen recognition region, wherein the antigen recognition region comprises at least one centrin; (b) a transmembrane domain, and (c) an intracellular domain (endodomain) comprising at least one co-stimulatory domain. As used throughout this disclosure, a CAR comprising centrysin is referred to as CARTyrin. In certain embodiments, the antigen recognition region may comprise two centrins to produce a bispecific or tandem CARTyrin. In certain embodiments, the antigen recognition region may comprise three centrins to produce a trispecific CARTyrin. In certain embodiments, the extracellular domain may further comprise a signal peptide. Alternatively or additionally, in certain embodiments, the extracellular domain may further comprise a hinge between the antigen recognition region and the transmembrane domain. In certain embodiments, the extracellular domain may further comprise a signal peptide. Alternatively or additionally, in certain embodiments, the extracellular domain may further comprise a hinge between the antigen recognition region and the transmembrane domain.

The present disclosure provides a Chimeric Antigen Receptor (CAR) comprising: (a) an extracellular domain comprising an antigen recognition region, wherein the antigen recognition region comprises at least one centryrin, and wherein the at least one centryrin specifically binds to a sequence of Prostate Specific Membrane Antigen (PSMA); (b) a transmembrane domain, and (c) an endodomain comprising at least one costimulatory domain. As used throughout this disclosure, a CAR comprising centrysin is referred to as CARTyrin. In certain embodiments, the antigen recognition region may comprise two centrins to produce a bispecific or tandem CARTyrin. In certain embodiments, including those in which the antigen recognition region may comprise two centrins to produce a bispecific or tandem CARTyrin, one or both of the two centrins specifically bind to the sequence of PSMA. In some embodiments, the first centryrin may specifically bind to a sequence of the first PSMA and the second centryrin may specifically bind to a sequence of the second PSMA. In some embodiments, the sequence of the first PSMA and the sequence of the second PSMA are the same. In some embodiments, the sequence of the first PSMA and the sequence of the second PSMA are not identical. In certain embodiments, the antigen recognition region may comprise three centrins to produce a trispecific CARTyrin. In certain embodiments, including those in which the antigen recognition region may comprise three centrins, one, two, or three of which specifically bind PSMA, to produce a trispecific or tandem CARTyrin. In certain embodiments, the first centrin may specifically bind to a sequence of the first PSMA, the second centrin may specifically bind to a sequence of the second PSMA, and the third centrin may specifically bind to a sequence of the third PSMA. In certain embodiments, the extracellular domain may further comprise a signal peptide. In certain embodiments, two or more of the sequences of the first, second or third PSMA are the same. In certain embodiments, two or more of the sequences of the first, second or third PSMA are not identical. In certain embodiments, the sequence of the first PSMA, the sequence of the second PSMA, and the sequence of the third PSMA are not identical. Alternatively or additionally, in certain embodiments, the extracellular domain may further comprise a hinge between the antigen recognition region and the transmembrane domain. In certain embodiments, the extracellular domain may further comprise a signal peptide. Alternatively or additionally, in certain embodiments, the extracellular domain may further comprise a hinge between the antigen recognition region and the transmembrane domain. As used herein, the term "anti-PSMA CARTyrin" refers to a CARTyrin comprising at least one centrin that specifically binds to a sequence of PSMA.

In certain embodiments of the anti-PSMA CARTyrins of the present disclosure, the centrin comprises an amino acid sequence

Or a nucleic acid sequence

Or consist thereof.

In certain embodiments of the anti-PSMA CARTyrins of the present disclosure, the at least one PSMA-specific centrin comprises an amino acid sequence having at least 70% identity to the following amino acid sequence

In certain embodiments, the at least one PSMA-specific centrin comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 97%, 99%, or any percent identity therebetween, with the following amino acid sequence

In certain embodiments of the anti-PSMA CARTyrins of the present disclosure, the centrin comprises an amino acid sequence

Or a nucleic acid sequence

Or consist thereof.

In certain embodiments of the CARTyrins of the present disclosure, the signal peptide may comprise a sequence encoding a human CD2, CD3 δ, CD3 ∈, CD3 γ, CD3 ζ, CD4, CD8 α, CD19, CD28, 4-1BB, or GM-CSFR signal peptide. In certain embodiments of the CARTyrins of the present disclosure, the signal peptide may comprise a sequence encoding the human CD8 a signal peptide. The human CD8 a signal peptide may comprise an amino acid sequence comprising MALPVTALLLPLALLLHAARP (SEQ ID NO: 18004). The human CD8 a signal peptide may comprise an amino acid sequence comprising MALPVTALLLPLALLLHAARP (SEQ ID NO: 18004) or a sequence having at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising MALPVTALLLPLALLLHAARP (SEQ ID NO: 18004). The human CD8 a signal peptide may be encoded by a nucleic acid sequence comprising atggcactgccagtcaccgccctgctgctgcctctggctctgctgctgcacgcagctagacca (SEQ ID NO: 18005).

In certain embodiments of the CARTyrins of the present disclosure, the transmembrane domain may comprise a sequence encoding a human CD2, CD3 δ, CD3 ∈, CD3 γ, CD3 ζ, CD4, CD8 α, CD19, CD28, 4-1BB, or GM-CSFR transmembrane domain. In certain embodiments of the CARTyrins of the present disclosure, the transmembrane domain may comprise a sequence encoding a human CD8 a transmembrane domain. The CD8 a transmembrane domain may comprise an amino acid sequence comprising IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 18006) or a sequence having at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 18006). The CD8 a transmembrane domain may be encoded by a nucleic acid sequence comprising atctacatttgggcaccactggccgggacctgtggagtgctgctgctgagcctggtcatcacactgtactgc (SEQ ID NO: 18007).

In certain embodiments of the CARTyrins of the present disclosure, the endodomain may comprise a human CD3 ζ endodomain.

In certain embodiments of the CARTyrins of the present disclosure, the at least one co-stimulatory domain may comprise human 4-1BB, CD28, CD40, ICOS, MyD88, OX-40 intracellular segments, or any combination thereof. In certain embodiments of the CARTyrins of the present disclosure, the at least one co-stimulatory domain may comprise a CD28 and/or a 4-1BB co-stimulatory domain. The CD3 ζ costimulatory domain can comprise an amino acid sequence comprising

Or a sequence having at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising

The CD3 zeta costimulatory domain may be encoded by a nucleic acid sequence comprising

The 4-1BB co-stimulatory domain may comprise an amino acid sequence comprising KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 18011), or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to an amino acid sequence comprising KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 18012). The 4-1BB co-stimulatory domain may be encoded by a nucleic acid sequence comprising

The 4-1BB costimulatory domain may be located between the transmembrane domain and the CD28 costimulatory domain.

In certain embodiments of the CARTyrins of the present disclosure, the hinge can include sequences derived from human CD8 a, IgG4, and/or CD4 sequences. In certain embodiments of the CARTyrins of the present disclosure, the hinge can include a sequence derived from the human CD8 a sequence. The hinge may comprise a human CD8 a amino acid sequence comprising TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 18014), or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to an amino acid sequence comprising TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 18015). The human CD8 alpha hinge amino acid sequence may be encoded by a nucleic acid sequence comprising

The centrins of the present disclosure may comprise at least one fibronectin type III (FN3) domain. The centrins of the present disclosure may be capable of specifically binding to antigens. Preferred Centryrins of the present disclosure specifically bind to sequences of PSMA. The at least one fibronectin type III (FN3) domain may be derived from a human protein. The human protein may be tenascin-C. The consensus sequence may comprise

The consensus sequence may be modified at one or more positions within: (a) an A-B loop comprising or consisting of amino acid residues TEDS (SEQ ID NO: 18020) at positions 13-16 of the consensus sequence; (b) a B-C loop comprising or consisting of amino acid residues TAPDAAF at positions 22-28 of the consensus sequence (SEQ ID NO: 18021); (c) a C-D loop comprising or consisting of amino acid residues SEKVGE (SEQ ID NO: 18022) at positions 38-43 of the consensus sequence; (d) a D-E loop comprising or consisting of the amino acid residues GSER at positions 51-54 of the consensus sequence (SEQ ID NO: 18023); (e) comprisesAmino acid residues GLKPG at positions 60-64 of the consensus sequence (SEQ ID NO: 18024) or the E-F loop consisting thereof; (f) a F-G loop comprising or consisting of amino acid residues KGGHRSN at positions 75-81 of the consensus sequence (SEQ ID NO: 18025); or (g) any combination of (a) - (f). The centrins of the present disclosure may comprise a consensus sequence of at least 5 fibronectin type III (FN3) domains, at least 10 fibronectin type III (FN3) domains, or at least 15 fibronectin type III (FN3) domains. The centrins and/or CARTyrins of the present disclosure may be selected from less than or equal to 10 ^-9M, less than or equal to 10^-10M, less than or equal to 10^-11M, less than or equal to 10^-12M, less than or equal to 10^-13M, less than or equal to 10^-14M and less than or equal to 10^-15K of M_DBinds to the antigen with at least one affinity. K_DThe measurement can be performed by surface plasmon resonance.

The present disclosure provides compositions comprising the CARTyrin of the present disclosure and at least one pharmaceutically acceptable carrier.

The present disclosure provides transposons comprising the CARTyrin of the present disclosure.

The transposons of the present disclosure can comprise a selection gene for identifying, enriching and/or isolating cells that express the transposons. Exemplary selection genes encode any gene product (e.g., transcript, protein, enzyme) essential to cell viability and survival. Exemplary selection genes encode any gene product (e.g., transcript, protein, enzyme) essential to conferring resistance to drug challenge to which a cell is susceptible (or which may be lethal to the cell) in the absence of the gene product encoded by the selection gene. Exemplary selection genes encode any gene product (e.g., transcript, protein, enzyme) essential for viability and/or survival in a cell culture medium lacking one or more nutrients essential for cell viability and/or survival in the absence of the selection gene. Exemplary selection genes include, but are not limited to, neo (conferring resistance to neomycin), TYMS (encoding thymidylate synthase), MGMT (encoding O (6) -methylguanine-DNA methyltransferase), pan-drug resistance gene (MDR1), ALDH1 (encoding the aldehyde dehydrogenase family 1, member a1), FRANCF, RAD51C (encoding RAD51 Paralog C), GCS (encoding glucosylceramide synthase), and NKX2.2 (encoding NK2 homeobox 2).

The transposons of the present disclosure can comprise an inducible pro-apoptotic (proptotic) polypeptide comprising (a) a ligand-binding region, (b) a linker, and (c) a pro-apoptotic polypeptide, wherein the inducible pro-apoptotic polypeptide does not comprise a non-human sequence. In certain embodiments, the non-human sequence comprises a restriction enzyme site. In certain embodiments, the ligand binding region can be a ligand binding region of a multimer. The induced pro-apoptotic polypeptides of the present disclosure may also be referred to as "iC 9 safety switches (safety switches)". In certain embodiments, a transposon of the present disclosure can comprise an inducible caspase polypeptide comprising (a) a ligand binding region, (b) a linker, and (c) a caspase polypeptide, wherein the inducible pro-apoptotic polypeptide does not comprise a non-human sequence. In certain embodiments, a transposon of the present disclosure can comprise an inducible caspase polypeptide comprising (a) a ligand binding region, (b) a linker, and (c) a caspase polypeptide, wherein the inducible pro-apoptotic polypeptide does not comprise a non-human sequence. In certain embodiments, a transposon of the present disclosure can comprise an inducible caspase polypeptide comprising (a) a ligand binding region, (b) a linker, and (c) a truncated caspase 9 polypeptide, wherein the inducible pro-apoptotic polypeptide does not comprise a non-human sequence. In certain embodiments of the inducible pro-apoptotic polypeptides, inducible caspase polypeptides, or truncated caspase 9 polypeptides of the present disclosure, the ligand-binding region may comprise an FK506 binding protein 12(FKBP12) polypeptide. In certain embodiments, the amino acid sequence comprising the ligand binding region of the FK506 binding protein 12(FKBP12) polypeptide may comprise a modification at position 36 of the sequence. The modification may be a substitution of valine (V) for phenylalanine (F) at position 36 (F36V). In certain embodiments, the FKBP12 polypeptide is encoded by an amino acid sequence comprising

In certain embodiments, the FKBP12 polypeptide is encoded by a nucleic acid sequence comprising

In certain embodiments, the inducer specific for the ligand binding region of FK 506-binding protein 12(FKBP12) polypeptide that may comprise a substitution (F36V) of valine (V) to phenylalanine (F) at position 36 comprises AP20187 and/or AP1903, two synthetic drugs.

In certain embodiments of the inducible pro-apoptotic polypeptides, inducible caspase polypeptides, or truncated caspase 9 polypeptides of the present disclosure, the linker region is encoded by an amino acid comprising GGGGS (SEQ ID NO: 18028) or a nucleic acid sequence comprising GGAGGAGGAGGATCC (SEQ ID NO: 18029). In certain embodiments, the nucleic acid sequence encoding the linker does not comprise a restriction enzyme site.

In certain embodiments of the truncated caspase 9 polypeptides of the present disclosure, the truncated caspase 9 polypeptide is encoded by an amino acid sequence that does not include arginine (R) at position 87 of the sequence. In another aspect or in addition, in certain embodiments of the inducible pro-apoptotic polypeptides, inducible caspase polypeptides, or truncated caspase 9 polypeptides of the disclosure, the truncated caspase 9 polypeptide is encoded by an amino acid sequence that does not include alanine (a) at position 282 of the sequence. In certain embodiments of the inducible pro-apoptotic polypeptides, inducible caspase polypeptides, or truncated caspase 9 polypeptides of the disclosure, the truncated caspase 9 polypeptide is comprised of

Or comprise

The nucleic acid sequence of (a).

In certain embodiments of the induced pro-apoptotic polypeptides, wherein said polypeptide comprises a truncated caspase 9 polypeptide, the induced pro-apoptotic polypeptides are comprised of

Or comprises an amino acid sequence of

The nucleic acid sequence of (a).

Transposons of the present disclosure can comprise at least one self-cleaving peptide located, for example, between one or more centrin(s) or cartyrin(s) of the present disclosure and a selection gene of the present disclosure. The transposons of the present disclosure can comprise at least one self-cleaving peptide located, for example, between one or more centrin(s) or cartyrin(s) of the present disclosure and the inducible pro-apoptotic polypeptides of the present disclosure. The transposons of the present disclosure can comprise at least two self-cleaving peptides, a first self-cleaving peptide located, for example, upstream or immediately upstream of an inducible pro-apoptotic polypeptide of the present disclosure, and a second first self-cleaving peptide located, for example, downstream or immediately upstream of an inducible pro-apoptotic polypeptide of the present disclosure.

The at least one self-cleaving peptide may include, for example, a T2A peptide, a GSG-T2A peptide, an E2A peptide, a GSG-E2A peptide, an F2A peptide, a GSG-F2A peptide, a P2A peptide, or a GSG-P2A peptide. The T2A peptide may comprise an amino acid sequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO: 18034) or a sequence having at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO: 18035). The GSG-T2A peptide may comprise an amino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 18036) or a sequence having at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 18037). The GSG-T2A peptide may comprise a nucleic acid sequence comprising

The E2A peptide may comprise an amino acid sequence comprising QCTNYALLKLAGDVESNPGP (SEQ ID NO: 18039) or a sequence having at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising QCTNYALLKLAGDVESNPGP (SEQ ID NO: 18040). The GSG-E2A peptide may comprise an amino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 18041) or a sequence having at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 18042). The F2A peptide may comprise an amino acid sequence comprising VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 18043) or a sequence having at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 18044). The GSG-F2A peptide may comprise an amino acid sequence comprising GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 18045) or a sequence having at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 18046). The P2A peptide may comprise an amino acid sequence comprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 18047) or a sequence having at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 18048). The GSG-P2A peptide may comprise an amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 18049) or a sequence having at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 18050).

The transposons of the present disclosure may comprise first and second self-cleaving peptides, the first self-cleaving peptide being located, for example, upstream of one or more centryrin(s) or cartyrin(s) of the present disclosure, and the second self-cleaving peptide being located, for example, downstream of one or more centryrin(s) or cartyrin(s) of the present disclosure. The first and/or second self-cleaving peptide may include, for example, a T2A peptide, a GSG-T2A peptide, an E2A peptide, a GSG-E2A peptide, an F2A peptide, a GSG-F2A peptide, a P2A peptide, or a GSG-P2A peptide. The T2A peptide may comprise an amino acid sequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO: 18034) or a sequence having at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO: 18035). The GSG-T2A peptide may comprise an amino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 18036) or a sequence having at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 18037). The GSG-T2A peptide may comprise a nucleic acid sequence comprising

In some embodiments of the transposons of the present disclosure, including those comprising the CARs of the present disclosure, the transposons further comprise a sequence encoding a Chimeric Stimulatory Receptor (CSR). In some embodiments, the CSR comprises: (a) an extracellular domain comprising an activating component; (b) a transmembrane domain; and (c) an endodomain comprising at least one signaling domain; wherein the combination of (a), (b), and (c) is non-naturally occurring. In some embodiments, the activating component of (a) is isolated or derived from a first protein. In some embodiments, the at least one signaling domain of (c) is isolated or derived from a second protein. In some embodiments, the first protein and the second protein are not the same. In some embodiments, the activating component includes one or more of a component of a human transmembrane receptor, a human cell surface receptor, a T-cell receptor (TCR), a component of a TCR complex, a component of a TCR co-receptor, a component of a TCR co-stimulatory protein, a component of a TCR inhibitory protein, a cytokine receptor, and a chemokine receptor. In some embodiments, the activating component includes a component of one or more of a T-cell receptor (TCR) to which an agonist of the activating component binds, a component of a TCR complex, a component of a TCR co-receptor, a component of a TCR co-stimulatory protein, a component of a TCR inhibitory protein, a cytokine receptor, and a chemokine receptor. In some embodiments, the activation component comprises a portion thereof to which a CD2 protein or agonist binds. In some embodiments, the signal transduction domain includes one or more of a component of a human signal transduction domain, a T-cell receptor (TCR), a component of a TCR complex, a component of a TCR co-receptor, a component of a TCR co-stimulatory protein, a component of a TCR inhibitory protein, a cytokine receptor, and a chemokine receptor. In some embodiments, the signaling domain comprises a CD3 protein. In some embodiments, the CD3 protein comprises a CD3 ζ protein. In some embodiments, the endodomain further comprises a cytoplasmic domain. In some embodiments, the cytoplasmic domain is isolated or derived from a third protein. In some embodiments, the first protein and the third protein are the same. In some embodiments, the extracellular domain further comprises a signal peptide. In some embodiments, the signal peptide is derived from a fourth protein. In some embodiments, the first protein and the fourth protein are the same. In some embodiments, the transmembrane domain is isolated or derived from a fifth protein. In some embodiments, the first protein and the fifth protein are the same. In some embodiments, the activating component does not bind to a naturally occurring molecule. In some embodiments, the CSR does not transduce a signal when the activating component binds to a naturally occurring molecule. In some embodiments, the extracellular domain comprises a modification. In some embodiments, the modification comprises a mutation or truncation of the sequence encoding the activating component when compared to the wild-type sequence of the first protein. In some embodiments, the activating component is associated with a non-naturally occurring molecule. In some embodiments, the CSR selectively transduces the signal upon binding of the activating component to the non-naturally occurring molecule.

In some embodiments of the transposons of the present disclosure, the transposon is a piggyBac or piggyBac-like transposon.

In some embodiments of the transposon of the present disclosure, the transposon is a TcBuster transposon.

In some embodiments of the transposon of the present disclosure, the transposon is a Sleeping Beauty transposon.

In some embodiments of the transposons of the present disclosure, the transposon is a helraisier transposon.

In some embodiments of the transposon of the present disclosure, the transposon is a Tol2 transposon.

The present disclosure provides compositions comprising a transposon of the present disclosure. In certain embodiments, the composition may further comprise a plasmid comprising a sequence encoding a transposase. The sequence encoding the transposase can be an mRNA sequence.

The present disclosure provides compositions comprising a CAR of the present disclosure. In some embodiments, the composition further comprises a CSR of the disclosure or a sequence encoding the CSR. In some embodiments, the sequence encoding the CSR comprises DNA. In some embodiments, the sequence encoding the CSR comprises RNA. In some embodiments, the sequence encoding CSR comprises messenger rna (mrna). In some embodiments, the CSR or a sequence encoding the CSR is stably integrated by the cell upon introduction into the cell of the present disclosure. In some embodiments, the CSR or a sequence encoding the CSR is not stably integrated by the cell when introduced into the cell of the present disclosure. In some embodiments, the CSR or a sequence encoding the CSR is stably expressed by the cell when introduced into the cell of the present disclosure. In some embodiments, the CSR or a sequence encoding the CSR is transiently expressed by the cell upon introduction into the cell of the present disclosure. In some embodiments, the CSR or CSR-encoding sequence comprises RNA or mRNA and the CSR or CSR-encoding sequence is transiently expressed by the cell upon introduction into the cell of the present disclosure.

The disclosure provides cells comprising a CAR of the disclosure. In some embodiments, the cell further comprises a CSR of the disclosure or a sequence encoding the CSR. In some embodiments, the sequence encoding the CSR comprises DNA. In some embodiments, the sequence encoding the CSR comprises RNA. In some embodiments, the sequence encoding CSR comprises messenger rna (mrna). In some embodiments, the CSR or the sequence encoding the CSR is stably integrated into the genomic locus or loci of the cell. In some embodiments, the CSR or the sequence encoding the CSR is not stably integrated into the genomic locus or loci of the cell. In some embodiments, the CSR or a sequence encoding the CSR is stably expressed by the cell. In some embodiments, the CSR or a sequence encoding the CSR is transiently expressed by the cell. In some embodiments, the CSR or CSR-encoding sequence comprises RNA or mRNA, and the CSR or CSR-encoding sequence is transiently expressed by the cell.

The transposons of the present disclosure can include piggyBac transposons. In certain embodiments of the method, the transposon is a plasmid DNA transposon having a sequence encoding a chimeric antigen receptor flanked by two cis-regulatory insulator elements. In certain embodiments, the transposon is a piggyBac or piggyBac-like transposon.

Transposases of the present disclosure can include piggyBac transposases or compatible enzymes. Transposases of the present disclosure can include piggyBac-like transposases or compatible enzymes. In certain embodiments, and particularly those wherein the transposon is a piggyBac transposon, the transposase is piggyBac^TMOr Super piggyBac^TM(SPB) transposase. In certain embodiments, and in particular wherein the transposase is Super piggyBac^TM(SPB) those embodiments of the transposase, the sequence encoding the transposase is an mRNA sequence.

In certain embodiments of the methods of the present disclosure, the transposase is piggyBac^TM(PB) transposase. piggybac (pb) transposases can comprise or consist of an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99%, or any percentage therebetween identical to:

in certain embodiments of the methods of the present disclosure, the transposase is piggyBac^TM(PB) a transposase comprising or consisting of an amino acid sequence having an amino acid substitution at one or more of positions 30, 165, 282, or 538 of the sequence:

in certain embodiments, the transposase is piggyBac^TM(PB) a transposase comprising a peptide having the sequence set forth in SEQ ID NO: 14487 amino acid sequence consisting of or consisting of amino acid substitutions at two or more of positions 30, 165, 282, or 538 of the sequence. In certain embodiments, the transposase is piggyBac ^TM(PB) a transposase comprising a peptide having the sequence set forth in SEQ ID NO: 14487 amino acid sequence consisting of or consisting of amino acid substitutions at three or more of positions 30, 165, 282, or 538 of the sequence. In certain embodiments, the transposase is piggyBac^TM(PB) runA hydrolase comprising a hydrolase having the amino acid sequence set forth in SEQ ID NO: 14487 or by amino acid substitutions at each of the following positions 30, 165, 282, and 538 of the sequence of (a). In certain embodiments, in SEQ ID NO: 14487 is a valine (V) for isoleucine (I) amino acid substitution. In certain embodiments, in SEQ ID NO: 14487 is a serine (S) to glycine (G) substitution. In certain embodiments, in SEQ ID NO: 14487 is a valine (V) to methionine (M) substitution. In certain embodiments, in SEQ ID NO: 14487 is a substitution of lysine (K) for asparagine (N).

In certain embodiments of the methods of the present disclosure, the transposase is Super piggyBac^TM(sPbO) transposase. In certain embodiments, the Super piggyBac of the present disclosure ^TMThe (sPBo) transposase may comprise SEQ ID NO: 14487, wherein the amino acid substitution at position 30 is a substitution of valine (V) for isoleucine (I), the amino acid substitution at position 165 is a substitution of serine (S) for glycine (G), the amino acid substitution at position 282 is a substitution of valine (V) for methionine (M), and the amino acid substitution at position 538 is a substitution of lysine (K) for asparagine (N). In certain embodiments, Super piggyBac^TMThe (sPBo) transposase may comprise or consist of an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99%, or any percentage therebetween identical to:

in certain embodiments of the methods of the present disclosure, the transposase is a TcBuster transposon, and wherein the transposase is a TcBuster transposase. In some embodiments, the TcBuster transposase is a highly active TcBuster transposase. In some embodiments, the TcBuster transposase comprises a sequence having at least 75% identity to:

in certain embodiments of the methods of the present disclosure, the transposase is a Sleeping Beauty transposon, and the transposase is a Sleeping Beauty transposase. In some embodiments, the Sleeping Beauty transposase comprises SEQ ID NO: 14485. In some embodiments, the Sleeping Beauty transposase is a high activity Sleeping Beauty transposase (SB 100X). In some embodiments, the high activity Sleeping Beauty transposase (SB100X) comprises SEQ ID NO: 14486.

In certain embodiments of the methods of the present disclosure, the transposase is a helraisier transposon, and wherein the transposase is a helraisier transposase. In some embodiments, the helraisiser transposase comprises SEQ ID NO: 14501.

In certain embodiments of the methods of the present disclosure, the transposase is a Tol2 transposon, and wherein the transposase is a Tol2 transposase. In some embodiments, the Tol2 transposase comprises SEQ ID NO: 14502.

The present disclosure provides vectors comprising the CARTyrin of the present disclosure. In certain embodiments, the vector is a viral vector. The vector may be a recombinant vector.

The viral vectors of the present disclosure may comprise sequences isolated or derived from retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, or any combination thereof. The viral vector may comprise sequences isolated or derived from adeno-associated virus (AAV). The viral vector may comprise a recombinant aav (raav). Exemplary adeno-associated viruses and recombinant adeno-associated viruses of the present disclosure comprise two or more Inverted Terminal Repeat (ITR) sequences located in cis proximate to a sequence encoding a centryrin or a CARTyrin of the present disclosure. Exemplary adeno-associated viruses and recombinant adeno-associated viruses of the present disclosure include, but are not limited to, all serotypes (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV 9). Exemplary adeno-associated viruses and recombinant adeno-associated viruses of the present disclosure include, but are not limited to, self-complementary AAV (scaav) and AAV hybrids (e.g., AAV2/5, AAV-DJ and AAV-DJ8) containing a genome of one serotype and a capsid of another serotype. Exemplary adeno-associated viruses and recombinant adeno-associated viruses of the present disclosure include, but are not limited to, rAAV-LK 03.

The viral vectors of the present disclosure may comprise a selection gene. The selection gene may encode a gene product essential for cell viability and survival. The selection gene may encode a gene product essential for cell viability and survival when challenged with selective cell culture conditions. The selective cell culture conditions may comprise a compound that is detrimental to cell viability or survival, and wherein the gene product confers resistance to said compound. Exemplary selection genes of the present disclosure can include, but are not limited to, neo (conferring resistance to neomycin), TYMS (encoding thymidylate synthase), MGMT (encoding O (6) -methylguanine-DNA methyltransferase), a pan-drug resistance gene (MDR1), ALDH1 (encoding aldehyde dehydrogenase family 1, member a1), FRANCF, RAD51C (encoding RAD51 paralogous gene C), GCS (encoding glucosylceramide synthase), NKX2.2 (encoding NK2 homeobox 2), or any combination thereof.

The viral vector of the present disclosure may comprise an inducible pro-apoptotic polypeptide comprising (a) a ligand binding region, (b) a linker, and (c) a pro-apoptotic polypeptide, wherein the inducible pro-apoptotic polypeptide does not comprise a non-human sequence. In certain embodiments, the non-human sequence comprises a restriction enzyme site. In certain embodiments, the ligand binding region can be a ligand binding region of a multimer. The induced pro-apoptotic polypeptides of the present disclosure may also be referred to as "iC 9 safety switches". In certain embodiments, a viral vector of the present disclosure may comprise an inducible caspase polypeptide comprising (a) a ligand binding region, (b) a linker, and (c) a caspase polypeptide, wherein the inducible pro-apoptotic polypeptide does not comprise a non-human sequence. In certain embodiments, a viral vector of the present disclosure may comprise an inducible caspase polypeptide comprising (a) a ligand binding region, (b) a linker, and (c) a caspase polypeptide, wherein the inducible pro-apoptotic polypeptide does not comprise a non-human sequence. In certain embodiments, a viral vector of the present disclosure may comprise an inducible caspase polypeptide comprising (a) a ligand binding region, (b) a linker, and (c) a truncated caspase 9 polypeptide, wherein the inducible pro-apoptotic polypeptide does not comprise a non-human sequence. In certain embodiments of the inducible pro-apoptotic polypeptides, inducible caspase polypeptides, or truncated caspase 9 polypeptides of the present disclosure, the ligand-binding region may comprise an FK506 binding protein 12(FKBP12) polypeptide. In certain embodiments, the amino acid sequence comprising the ligand binding region of the FK506 binding protein 12(FKBP12) polypeptide may comprise a modification at position 36 of the sequence. The modification may be a substitution of valine (V) for phenylalanine (F) at position 36 (F36V). In certain embodiments, the FKBP12 polypeptide is encoded by an amino acid sequence comprising

In certain embodiments, the FKBP12 polypeptide is encoded by a nucleic acid sequence comprising

Or comprise

The nucleic acid sequence of (a).

In certain embodiments of the induced pro-apoptotic polypeptides, wherein said polypeptide comprises a truncated caspase 9 polypeptide, the induced pro-apoptotic polypeptides are comprised of

Or comprises an amino acid sequence of

The nucleic acid sequence of (a).

The viral vectors of the present disclosure may comprise at least one self-cleaving peptide. In some embodiments, the vector may comprise at least one self-cleaving peptide, and wherein the self-cleaving peptide is located between the carpyrin and the selection gene. In some embodiments, the vector may comprise at least one self-cleaving peptide, and wherein the first self-cleaving peptide is located upstream of carpyrin and the second self-cleaving peptide is located downstream of carpyrin. The viral vectors of the present disclosure can comprise at least one self-cleaving peptide located, for example, between one or more of the CARTyrin, CAR, or CAR of the present disclosure and the inducible pro-apoptotic polypeptides of the present disclosure. The viral vectors of the present disclosure may comprise at least two self-cleaving peptides, a first self-cleaving peptide located, for example, upstream or immediately upstream of an inducible pro-apoptotic polypeptide of the present disclosure, and a second first self-cleaving peptide located, for example, downstream or immediately upstream of an inducible pro-apoptotic polypeptide of the present disclosure. The self-cleaving peptide may include, for example, a T2A peptide, a GSG-T2A peptide, an E2A peptide, a GSG-E2A peptide, an F2A peptide, a GSG-F2A peptide, a P2A peptide, or a GSG-P2A peptide. The T2A peptide may comprise an amino acid sequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO: 18034) or a sequence having at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO: 18035). The GSG-T2A peptide may comprise an amino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 18036) or a sequence having at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 18037). The GSG-T2A peptide may comprise a nucleic acid sequence comprising

The present disclosure provides vectors comprising the CARTyrin of the present disclosure. In certain embodiments, the carrier is a nanoparticle. Exemplary nanoparticle carriers of the present disclosure include, but are not limited to, nucleic acids (e.g., RNA, DNA, synthetic nucleotides, modified nucleotides, or any combination thereof), amino acids (L-amino acids, D-amino acids, synthetic amino acids, modified amino acids, or any combination thereof), polymers (e.g., polymersomes), micelles, lipids (e.g., liposomes), organic molecules (e.g., carbon atoms, sheets, fibers, tubes), inorganic molecules (e.g., calcium phosphate or gold), or any combination thereof. The nanoparticle carrier can be transported across the cell membrane passively or actively.

The nanoparticle vectors of the present disclosure may comprise a selection gene. The selection gene may encode a gene product essential for cell viability and survival. The selection gene may encode a gene product essential for cell viability and survival when challenged with selective cell culture conditions. The selective cell culture conditions may comprise a compound that is detrimental to cell viability or survival, and wherein the gene product confers resistance to said compound. Exemplary selection genes of the present disclosure can include, but are not limited to, neo (conferring resistance to neomycin), TYMS (encoding thymidylate synthase), MGMT (encoding O (6) -methylguanine-DNA methyltransferase), a pan-drug resistance gene (MDR1), ALDH1 (encoding aldehyde dehydrogenase family 1, member a1), FRANCF, RAD51C (encoding RAD51 paralogous gene C), GCS (encoding glucosylceramide synthase), NKX2.2 (encoding NK2 homeobox 2), or any combination thereof.

The nanoparticle carrier of the present disclosure may comprise an inducible pro-apoptotic polypeptide comprising (a) a ligand binding region, (b) a linker, and (c) a pro-apoptotic polypeptide, wherein the inducible pro-apoptotic polypeptide does not comprise a non-human sequence. In certain embodiments, the non-human sequence comprises a restriction enzyme site. In certain embodiments, the ligand binding region can be a ligand binding region of a multimer. The induced pro-apoptotic polypeptides of the present disclosure may also be referred to as "iC 9 safety switches". In certain embodiments, the nanoparticle vectors of the present disclosure may comprise an inducible caspase polypeptide comprising (a) a ligand binding region, (b) a linker, and (c) a caspase polypeptide, wherein the inducible pro-apoptotic polypeptide does not comprise a non-human sequence. In certain embodiments, the nanoparticle vectors of the present disclosure may comprise an inducible caspase polypeptide comprising (a) a ligand binding region, (b) a linker, and (c) a caspase polypeptide, wherein the inducible pro-apoptotic polypeptide does not comprise a non-human sequence. In certain embodiments, the nanoparticle vectors of the present disclosure may comprise an inducible caspase polypeptide comprising (a) a ligand binding region, (b) a linker, and (c) a truncated caspase 9 polypeptide, wherein the inducible pro-apoptotic polypeptide does not comprise a non-human sequence. In certain embodiments of the inducible pro-apoptotic polypeptides, inducible caspase polypeptides, or truncated caspase 9 polypeptides of the present disclosure, the ligand-binding region may comprise an FK506 binding protein 12(FKBP12) polypeptide. In certain embodiments, the amino acid sequence comprising the ligand binding region of the FK506 binding protein 12(FKBP12) polypeptide may comprise a modification at position 36 of the sequence. The modification may be a substitution of valine (V) for phenylalanine (F) at position 36 (F36V). In certain embodiments, the FKBP12 polypeptide is encoded by an amino acid sequence comprising

In certain embodiments, the FKBP12 polypeptide is encoded by a nucleic acid sequence comprising

Or comprise

The nucleic acid sequence of (a).

In certain embodiments of the induced pro-apoptotic polypeptides, wherein said polypeptide comprises a truncated caspase 9 polypeptide, the induced pro-apoptotic polypeptides are comprised of

Or comprises an amino acid sequence of

The nucleic acid sequence of (a).

The nanoparticle carriers of the present disclosure may comprise at least one self-cleaving peptide. In some embodiments, the nanoparticle carrier can comprise at least one self-cleaving peptide, and wherein the self-cleaving peptide is located between the CARTyrin and the nanoparticle. In some embodiments, the nanoparticle carrier can comprise at least one self-cleaving peptide, and wherein the first self-cleaving peptide is located upstream of the CARTyrin and the second self-cleaving peptide is located downstream of the CARTyrin. In some embodiments, the nanoparticle carrier can comprise at least one self-cleaving peptide, and wherein the first self-cleaving peptide is located between the CARTyrin and the nanoparticle, and the second self-cleaving peptide is located downstream of the CARTyrin. In some embodiments, the nanoparticle vector can comprise at least one self-cleaving peptide, and wherein a first self-cleaving peptide is located between the CARTyrin and the nanoparticle, and a second self-cleaving peptide is located downstream of the CARTyrin, e.g., between the CARTyrin and the selection gene.

The nanoparticle carriers of the present disclosure may comprise at least one self-cleaving peptide positioned between, for example, one or more of centrins(s) and cartyrin(s) of the present disclosure and the inducible pro-apoptotic polypeptides of the present disclosure. The nanoparticle vectors of the present disclosure may comprise at least two self-cleaving peptides, a first self-cleaving peptide located, for example, upstream or immediately upstream of an inducible pro-apoptotic polypeptide of the present disclosure, and a second first self-cleaving peptide located, for example, downstream or immediately upstream of an inducible pro-apoptotic polypeptide of the present disclosure. The self-cleaving peptide may include, for example, a T2A peptide, a GSG-T2A peptide, an E2A peptide, a GSG-E2A peptide, an F2A peptide, a GSG-F2A peptide, a P2A peptide, or a GSG-P2A peptide. The T2A peptide may comprise an amino acid sequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO: 18034) or a sequence having at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO: 18034). The GSG-T2A peptide may comprise an amino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 18036) or a sequence having at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 18036). The GSG-T2A peptide may comprise a nucleic acid sequence comprising

The E2A peptide may comprise an amino acid sequence comprising QCTNYALLKLAGDVESNPGP (SEQ ID NO: 18039) or a sequence having at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising QCTNYALLKLAGDVESNPGP (SEQ ID NO: 18039). The GSG-E2A peptide may comprise an amino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 18041) or a sequence having at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 18041). The F2A peptide may comprise an amino acid sequence comprising VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 18043) or a sequence having at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 18043). The GSG-F2A peptide may comprise an amino acid sequence comprising GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 18045) or a sequence having at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 18045). The P2A peptide may comprise an amino acid sequence comprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 18047) or a sequence having at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 18047). The GSG-P2A peptide may comprise an amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 18050) or a sequence having at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 18050).

The present disclosure provides compositions comprising a carrier of the present disclosure. The present disclosure provides cells comprising the CARTyrin of the present disclosure. The present disclosure provides cells comprising a transposon of the present disclosure. In certain embodiments, a cell comprising a CARTyrin, transposon, or vector of the present disclosure can express a CARTyrim on the surface of the cell. The cells may be any type of cells. Preferably, the cell is an immune cell. The immune cell can be a T-cell, a Natural Killer (NK) -like cell, a cytokine-induced killer (CIK) cell), a hematopoietic progenitor cell, a Peripheral Blood (PB) -derived T-cell, or a Umbilical Cord Blood (UCB) -derived T-cell. Preferably, the immune cells are T-cells. The T-cell may be early memory cell, stem-like (stem-like) T-cell, T-cell_SCM-like cells, T_SCMOr T_CM. The T-cell may be T_SCM. The cells may be artificial antigen presenting cells, which optionally may be used to stimulate and expand the modified immune cells or T cells of the present disclosure. The cell may be a tumor cell, which optionally may be used as an artificial or modified antigen presenting cell.

The modified cells of the present disclosure that may be used for adoptive therapy may be autologous or allogeneic

The present disclosure provides a method of expressing CARTyrin on the surface of a cell, comprising: (a) obtaining a population of cells; (b) contacting the population of cells with a composition comprising a CARTyrin of the present disclosure or a sequence encoding the CARTyrin under conditions sufficient for the transfer of the CARTyrin across the cell membrane of at least one cell in the population of cells, thereby producing a modified population of cells; (c) culturing the modified population of cells under conditions suitable for transposon integration; and (d) expanding and/or selecting at least one cell from the modified cell population that expresses CARTyrin on the cell surface.

In certain embodiments of the methods of expressing CARTyrin, the cell population may comprise leukocytes and/or CD4+ and CD8+ leukocytes. The cell population may comprise CD4+ and CD8+ leukocytes in an optimized ratio. The optimized ratio of CD4+ to CD8+ leukocytes does not occur naturally in vivo. The cell population may comprise tumor cells.

In certain embodiments of the methods of expressing CARTyrin, the transposon or vector comprises CARTyrin or a sequence encoding CARTyrin. In certain embodiments, the transposon comprises an anti-PSMA CARTyrin or a sequence encoding an anti-PSMA CARTyrin. In certain embodiments, the transposon comprises a piggyBac transposon. In certain embodiments, the transposon further comprises a composition comprising a plasmid comprising a sequence encoding a transposase. In certain embodiments, including those wherein the transposase is a piggyBac transposase, the transposase is an mRNA sequence. In certain embodiments, the piggyBac transposase comprises a nucleic acid comprising SEQ ID NO: 18017 in a pharmaceutically acceptable carrier. In certain embodiments, the piggyBac transposase is a high activity variant, and wherein the high activity variant comprises the amino acid sequence set forth in SEQ ID NO: 18017 at one or more of positions 30, 165, 282 and 538. In certain embodiments, in SEQ ID NO: 18017 is a valine (V) to isoleucine (I) substitution (I30V). In certain embodiments, in SEQ ID NO: 18017 is a serine (S) to glycine (G) substitution (G165S). In certain embodiments, wherein in SEQ ID NO: 18017 is a valine (V) to methionine (M) substitution (M282V). In certain embodiments, in SEQ ID NO: 18017 is a lysine (K) to asparagine (N) substitution (N538K). In certain embodiments, the transposase is a Super piggyBac (sPBo) transposase. In certain embodiments, the Super piggyBac (sPBo) transposase comprises a nucleic acid sequence comprising SEQ ID NO: 14484 in a pharmaceutically acceptable carrier.

The present disclosure provides vectors comprising the CARTyrin or sequences encoding the CARTyrin of the present disclosure. In certain embodiments, the vector comprises an anti-PSMA CARTyrin or a sequence encoding an anti-PSMA CARTyrin of the present disclosure.

In certain embodiments of the methods of expressing CARTyrin, the conditions sufficient for transfer of a sequence encoding CARTyrin across the cell membrane of at least one cell in the population of cells comprise nuclear transfection (nucleofection).

In certain embodiments of the methods of expressing CARTyrin, wherein the conditions sufficient for transfer of a sequence encoding CARTyrin across the cell membrane of at least one cell in the population of cells comprise application of at least one of one or more electrical pulses at a specified voltage, a buffer, and one or more supplemental factors (supplemental factors). In certain embodiments, the buffer can include PBS, HBSS, OptiMEM, BTXpress, Amaxa nuclear transfection instrument (Nucleofector), human T cell nuclear transfection buffer, or any combination thereof. In certain embodiments, the one or more supplemental factors can include (a) a recombinant human cytokine, a chemokine, an interleukin, or any combination thereof; (b) a salt, a mineral, a metabolite, or any combination thereof; (c) a cell culture medium; (d) an inhibitor of cellular DNA signaling (sensing), metabolism, differentiation, signal transduction, one or more apoptotic pathways, or a combination thereof; and (e) an agent that modifies or stabilizes one or more nucleic acids. The recombinant human cytokine, chemokine, interleukin or any combination thereof may comprise IL2, IL7, IL12, IL15, IL21, IL1, IL3, IL4, IL5, IL6, IL8, CXCL8, IL9, IL10, IL11, IL13, GM-CSF, IFN- γ, IL-1 α/IL-1F 13, IL-1 β/IL-1F 13, IL-12 p 13, IL-12/IL-35 p 13, IL-13, IL-17/IL 17A/IL-17F 72, IL-17F-17, IL-17P-72, IL-17F-17, IL-17F-32, IL-13, IL-, LAP (TGF-. beta.1), lymphotoxin-. alpha./TNF-. beta., TGF-. beta., TNF-. alpha., TRANCE/TNFSF11/RANK L, or any combination thereof. The salt, mineral, metabolite, or any combination thereof may include HEPES, niacinamide, heparin, sodium pyruvate, L-glutamine, MEM non-essential amino acid solutions, ascorbic acid, nucleosides, FBS/FCS, human serum, serum substitutes, antibiotics, pH adjusters (adjust), Earle's Salts, 2-mercaptoethanol, human transferrin, recombinant human insulin, human serum albumin, nuclear stainer PLUS (Nucleofector PLUS Supplement), KCL, MgCl2, Na2HPO4, NAH2PO4, sodium lactobionate, mannitol (Manitol), sodium succinate, sodium chloride, CINa, glucose, Ca (NO3)2, Tris/HCl, K2HPO4, KH2PO4, Polyethylenimine (Polyethylenimine), polyethylene glycol, Poloxamer (Poloxamer)188, Poloxamer 181, Poloxamer 407, polyvinyl pyrrolidone, Pop313, Crown-5, or any combination thereof. The cell culture medium may include PBS, HBSS, OptiMEM, DMEM, RPMI 1640, AIM-V, X-VIVO 15, CellGro DC medium, CTS OpTimizer T cell expansion SFM, TexMACS medium, PRIME-XV T cell expansion medium, ImmunoCult-XF T cell expansion medium, or any combination thereof. Inhibitors of cellular DNA signaling, metabolism, differentiation, signal transduction, one or more apoptotic pathways, or combinations thereof include TLR9, MyD88, IRAK, TRAF6, TRAF3, IRF-7, NF-KB, type 1 interferon, Pro-inflammatory cytokines, cGAS, STING, Sec5, TBK1, IRF-3, RNA pol III, RIG-1, IPS-1, FADD, RIP1, TRAF3, AIM2, ASC, Caspase 1(Caspase1), Pro-IL1B, PI3K, Akt, inhibitors of Wnt3A, inhibitors of glycogen synthase kinase-3 β (GSK-3 β) (e.g., TWS119), baveromycin, chloroquine, quinacrine, AC-YVAD-CMK, Z-YVAD-FMK, Z-IETD-FMK, or any combination thereof. Agents that modify or stabilize one or more nucleic acids include pH modifiers, DNA binding proteins, lipids, phospholipids, CaPO4, net neutral charge DNA binding peptides with or without NLS sequences, TREX1 enzymes, or any combination thereof.

In certain embodiments of the methods of expressing CARTyrin, the conditions suitable for integration of the CARTyrin or a sequence encoding CARTyrin of the present disclosure comprise at least one of a buffer and one or more supplemental factors. In certain embodiments, the transposons or vectors of the disclosure comprise the CARTyrin or sequences encoding the CARTyrin of the disclosure. In certain embodiments, the buffer can include PBS, HBSS, OptiMEM, BTXpress, Amaxa nuclear transfection instrument (Nucleofector), human T cell nuclear transfection buffer, or any combination thereof. In certain embodiments, the one or more supplemental factors can include (a) a recombinant human cytokine, a chemokine, an interleukin, or any combination thereof; (b) a salt, a mineral, a metabolite, or any combination thereof; (c) a cell culture medium; (d) an inhibitor of cellular DNA signaling (sensing), metabolism, differentiation, signal transduction, one or more apoptotic pathways, or a combination thereof; and (e) an agent that modifies or stabilizes one or more nucleic acids. The recombinant human cytokine, chemokine, interleukin or any combination thereof may comprise IL2, IL7, IL12, IL15, IL21, IL1, IL3, IL4, IL5, IL6, IL8, CXCL8, IL9, IL1O, IL11, IL13, GM-CSF, IFN- γ, IL-1 α/IL-1F 13, IL-1 β/IL-1F 13, IL-12p 13, IL-12/IL-35p 13, IL-13, IL-17/17A/IL-17F 72, IL-17P/F-17, IL-17P-13, IL-17P-F-17, IL13, IL-32, IL-13, IL-17P-17F-32, IL-17P-17F-17, IL-17F-72, IL-17, LAP (TGF-. beta.1), lymphotoxin-. alpha./TNF-. beta., TGF-. beta., TNF-. alpha., TRANCE/TNFSF11/RANK L, or any combination thereof. The salt, mineral, metabolite, or any combination thereof may include HEPES, niacinamide, heparin, sodium pyruvate, L-glutamine, MEM non-essential amino acid solutions, ascorbic acid, nucleosides, FBS/FCS, human serum, serum substitutes, antibiotics, pH adjusters (adjust), Earle's Salts, 2-mercaptoethanol, human transferrin, recombinant human insulin, human serum albumin, nuclear stainer PLUS (Nucleofector PLUS Supplement), KCL, MgCl2, Na2HPO4, NAH2PO4, sodium lactobionate, mannitol (Manitol), sodium succinate, sodium chloride, CINa, glucose, Ca (NO3)2, Tris/HCl, K2HPO4, KH2PO4, Polyethylenimine (Polyethylenimine), polyethylene glycol, Poloxamer (Poloxamer)188, Poloxamer 181, Poloxamer 407, polyvinyl pyrrolidone, Pop313, Crown-5, or any combination thereof. The cell culture medium may include PBS, HBSS, OptiMEM, DMEM, RPMI 1640, AIM-V, X-VIVO 15, CellGro DC medium, CTS OpTimizer T cell expansion SFM, TexMACS medium, PRIME-XV T cell expansion medium, ImmunoCult-XF T cell expansion medium, or any combination thereof. Inhibitors of cellular DNA signaling, metabolism, differentiation, signal transduction, one or more apoptotic pathways, or combinations thereof include TLR9, MyD88, IRAK, TRAF6, TRAF3, IRF-7, NF-KB, type 1 interferon, Pro-inflammatory cytokines, cGAS, STING, Sec5, TBK1, IRF-3, RNA pol III, RIG-1, IPS-1, FADD, RIP1, TRAF3, AIM2, ASC, Caspase 1(Caspase1), Pro-IL1B, PI3K, Akt, inhibitors of Wnt3A, inhibitors of glycogen synthase kinase-3 β (GSK-3 β) (e.g., TWS 119), baveromycin, chloroquine, quinacrine, AC-YVAD-CMK, Z-YVAD-FMK, Z-IETD-FMK, or any combination thereof. Agents that modify or stabilize one or more nucleic acids include pH modifiers, DNA binding proteins, lipids, phospholipids, CaPO4, net neutral charge DNA binding peptides with or without NLS sequences, TREX1 enzymes, or any combination thereof.

In certain embodiments of the methods of expressing CARTyrin, the expanding and selecting steps occur sequentially. The augmentation may occur prior to the selection. The augmentation may occur after the selection, and optionally, further (i.e., second) selections may occur after the augmentation.

In certain embodiments of the methods of expressing CARTyrin, the expansion and selection steps can occur simultaneously.

In certain embodiments of the methods of expressing CARTyrin, expanding can comprise contacting at least one cell of the modified cell population with an antigen to stimulate the at least one cell by CARTyrin, thereby producing an expanded cell population. The antigen may be present on the surface of the substrate. The substrate may have any form including, but not limited to, a surface, a well, a bead or a plurality thereof, and a matrix. The substrate may further comprise a paramagnetic or magnetic component. In certain embodiments of the methods of expressing CARTyrin, the antigen may be present on the surface of a substrate, wherein the substrate is a magnetic bead, and wherein a magnet may be used to remove or separate the magnetic bead from the modified and expanded cell population. The antigen may be presented on the surface of a cell or an artificial antigen presenting cell. The artificial antigen presenting cells of the present disclosure may include, but are not limited to, tumor cells and stem cells.

In certain embodiments of the methods of expressing CARTyrin, wherein the transposon or vector comprises a selection gene, and wherein the selecting step comprises contacting at least one cell of the modified population of cells with a compound to which the selection gene confers resistance, thereby identifying the cell that expresses the selection gene as surviving the selection and identifying the cell that fails to express the selection gene as failing to survive the selecting step.

In certain embodiments of the methods of expressing CARTyrin, the expanding and/or selecting step can be performed for a period of 10 to 14 days, inclusive.

The present disclosure provides compositions comprising modified, expanded, and selected cell populations of the methods of the disclosure.

The present disclosure provides a method of treating cancer in a subject in need thereof, comprising administering to the subject a composition of the disclosure, wherein the CARTyrin specifically binds to an antigen on a tumor cell. In certain embodiments, the tumor cell can be a malignant tumor cell. In certain embodiments, comprising administering to a subject a composition comprising a modified cell or population of cells of the present disclosure, which may be autologous. In certain embodiments, comprising administering to a subject a composition comprising a modified cell or population of cells of the present disclosure, which may be allogeneic.

The present disclosure provides a method of treating cancer in a subject in need thereof, comprising administering to the subject a composition of the disclosure, wherein anti-PSMA CARTyrin specifically binds to a PSMA antigen on a tumor cell or a component of the vasculature of a tumor cell. In certain embodiments, the tumor cell is a prostate cell. In certain embodiments, the tumor cell can be a malignant tumor cell. In certain embodiments, comprising administering to a subject a composition comprising a modified cell or population of cells of the present disclosure, which may be autologous. In certain embodiments, comprising administering to a subject a composition comprising a modified cell or population of cells of the present disclosure, which may be allogeneic.

Methods of modifying cell therapy of the present disclosure can be used to terminate or inhibit therapy in response to, for example, signs of recovery or reduction in disease severity/progression, signs of disease remission/cessation and/or occurrence of adverse events. Cell therapy of the present disclosure can be restored by inhibiting the inducing agent if signs or symptoms of the disease reappear or increase in severity and/or adverse events are resolved.

Drawings

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the office upon request and payment of the necessary fee.

Figure 1 is a schematic depicting a 7738 base pair piggyBac CARTyrin P-PSMA-101 plasmid comprising the EF1 a promoter, safety switch (iC9), PSMA CARTyrin, and a selection cassette.

FIG. 2 is a schematic drawing depicting piggyBac CARTyrin comprising a P-PSMA-101 transposon comprising PSMA CARTyrin (comprising a CD8 a signal peptide, an anti-PSMA centryrin, a CD8 a spacer, a CD8 a transmembrane sequence, a 4-1BB co-stimulatory domain, and a CD3 zeta co-stimulatory domain).

FIG. 3A is a schematic representation of the amino acid sequence of the P-PSMA5-101 construct of the present disclosure.

Figure 3B is a schematic representation of the nucleic acid sequence of the P-PSMA5-101 construct of the present disclosure.

Figure 3C is a schematic representation of the nucleic acid sequence of the P-PSMA5-101 construct of the present disclosure.

FIG. 4A is a schematic representation of the amino acid sequence of the P-PSMA8-101 construct of the present disclosure.

FIG. 4B is a schematic representation of the amino acid sequence of the P-PSMA8-101 construct of the present disclosure.

FIG. 4C is a schematic representation of the amino acid sequence of the P-PSMA8-101 construct of the present disclosure.

Fig. 5 is a schematic diagram depicting the construction of CARTyrin of the present disclosure and a table comparing the characteristics of centrins and antibodies.

FIGS. 6A-6E show transient expression and function of PSMA CARTyrins. In vitro assays were performed to test the expression and function of lead PSMA CARTyrins for the production of P-PSMA5-101 and P-PSMA 8-101. PSMA CARTyrins were detected on the surface of primary human T cells transiently transfected with mRNA encoding PSMA CARTyrins the evening before (fig. 6A). Briefly, previously activated and then frozen whole T cells (Pan T cells) were thawed and rested in T cell culture media overnight, the next night cells were electroporated with 10 μ g of PSMA CARTyrin mRNA, and then the next morning surface expression analysis was performed by FACS using soluble recombinant human PSMA protein (rpma) for labeling. To test the function of these T cells in vitro, cells were co-cultured with a panel of PSMA-expressing cells for 4 hours, and target cell killing was then measured by expression of CD107a, CD107a being a marker of degranulation and a surrogate for T cell killing. The surface expression of PSMA was evaluated on K562 cells (K562.PSMA) and LNCaP (human prostate cancer cell line endogenously expressing PSMA) engineered to stably express PSMA (fig. 6B). PSMA CARTyrin-expressing T cells were able to degranulate against all PSMA-expressing cell lines (LNCaP, K562.PSMA) with little to no more than background degranulation against PSMA-cell lines (K562, PC-3) (fig. 6C). The PSMA-encoding mRNA was titrated into PSMA-cell line K562 to control the surface expression level of PSMA (fig. 6D). PSMA CARTyrin + cells showed strong cytotoxic function against K562 cells expressing various amounts of surface PSMA (fig. 6E). These data indicate that PSMA CARTyrins can be expressed on the surface of T cells and promote cytotoxic function against PSMA + cellular targets.

FIGS. 7A-7F show the phenotype and function of P-PSMA-101 produced by piggyBac. To support in vivo evaluation of guided PSMA CARTyrins, P-PSMA-101 was constructed using the piggyBac DNA modification system. PSMA CARTyrin was detected on the surface of primary human T cells from representative donors transposed with either the P-PSMA5-101 or P-PSMA8-101 plasmids, but not on cells transposed with the P-BCMA-101 plasmid control (fig. 7A). In both cases, most CD8+ CAR-T cells were double positive for expression of CD45RA and CD62L, markers typically associated with T stem cell memory phenotype (Tscm), after staining and FACS analysis (fig. 7B). Furthermore, these cells (gated according to CD8+ or CD4 +) expressed low to no levels of PD-1, Tim-3 and lang-3 by FACS analysis, which are molecules associated with activation and/or functional T cell depletion (fig. 7C). Next, effector function of these CAR-T cells was assessed in vitro after co-culture with PSMA-expressing cells. After 24 hours, IFN- γ secretion was measured by standard ELISA and detected in culture medium when CAR-T cells (from 3 independent donors) were incubated in the presence of their associated target antigens; P-BCMA-101 secreted IFN- γ only in the presence of K562 cells (K562.BCMA) that were artificially modified to express BCMA on the surface, whereas P-PSMA-101 secreted IFN- γ only in the presence of cell tumor lines (LNCaP and K562.PSMA) that expressed PSMA on the surface (fig. 7D). In addition, P-PSMA-101 exhibited strong cytotoxic function against LNCaP as measured by a standard killing assay, whereas P-BCMA-101 exhibited little killing ability; data from 2 independent donors (fig. 7E). Cell proliferation capacity when co-cultured with several tumor cell lines was assessed after 96 hours. P-PSMA-101 showed enhanced proliferative capacity against PSMA + LNCaP and 22Rv1, and P-BCMA-101 proliferated against BCMA + H929, while CAR-T cells did not proliferate against both PSMA-BCMA-cell lines K562 and DU145 (FIG. 7F). These data indicate that P-PSMA-101 cells express PSMA CARTyrin on the surface and display cytotoxic function and proliferative capacity in vitro against PSMA + cellular targets.

FIGS. 8A-8F show preclinical evaluation of P-PSMA8-101 using a murine xenograft model. Fig. 8A is a schematic of a treatment protocol. The in vivo anti-tumor efficacy of P-PSMA8-101 was evaluated using a murine xenograft model using a luciferase-expressing LNCaP cell line (LNCaP. luc) injected Subcutaneously (SC) into NSG mice. For these in vivo studies, all CAR-T cells were produced using the PB delivery and Poseida manufacturing process of the P-PSMA8-101 plasmid. Mice were injected in the axilla with LNCaP (n 25 to account for poor LNCaP "acceptance" rate) and treated as tumors were established (100 + 300mm measured by caliper)³17 days after implantation). By Intravenous (IV) injection, the injection is administered with a composition comprising ultra-low (1x10^6), 'stress' (5x10^6) and standard (10x10 ^6)6) Several doses of P-PSMA-101 at doses were administered to mice. Fig. 8B is a survival graph of antitumor activity. FIG. 8C is a bar graph showing expansion and detection of P-PSMA8-101 CD8+ T cells in blood. Fig. 8D is a series of line graphs showing the assessment of tumor volume by caliper measurement. Fig. 8E is a line graph showing bioluminescence of LNCaP tumors by BLI. Fig. 8F shows representative photographs of the bioluminescence of LNCaP tumors in mice quantified in fig. 8E.

FIGS. 9A-9G show preclinical evaluation of lead P-PSMA5-101 and P-PSMA8-101 candidates as "stress" doses using a murine xenograft model. Figure 9A is a schematic drawing depicting a study schedule for preclinical evaluation of P-PSMA-101 candidates for 'stress' doses using a murine xenograft model. The in vivo anti-tumor efficacy of P-PSMA5-101 and P-PSMA8-101 of 'stress' doses (4x10^6) total CAR-T cells was evaluated using a murine xenograft model using a luciferase-expressing LNCaP cell line (LNCaP. luc) injected Subcutaneously (SC) into NSG mice. For these in vivo studies, all CAR-T cells were produced using the Poseida manufacturing process using PB delivery of the P-PSMA5-101 or P-PSMA8-101 plasmids. Mice were injected in the axilla with LNCaP and treated as tumors were established (100-³). Mice were treated with 'stress' doses (4x10^6) of P-PSMA-101 by Intravenous (IV) injection to ascertain any possible differences in efficacy between PSMA5 and PSMA8 CARs. Antitumor activity was assessed by survival, expansion and detection of CD8+ T cells in blood, tumor volume assessment by caliper measurement, and bioluminescence of LNCaP tumors. The 'stressed' doses of P-PSMA5-101 and P-PSMA8-101 showed significantly enhanced antitumor efficacy and survival against SC lncap. luc solid tumors established in NSG mice compared to T cell (no CAR) control mice. Specifically, there was no survival in T cell (no CAR) control animals, 25% survival in P-BCMA-101 treated group, 75% survival in P-PSMA5-101 treated group, and 100% survival in animals treated with 'stressed' dose of P-PSMA 8-101. In peripheral blood, P-PSMA5-101 and P-PSMA8-101 expanded and generated differentiated effector CARTyrin + T cells, which were accompanied by a reduction in tumor burden to a detectable card Ruler and bioluminescence imaging limits below. These cells then shrink but remain present in the peripheral blood. Fig. 9B is a survival graph of antitumor activity. FIG. 9C is a bar graph showing expansion and detection of P-PSMA5-101 and P-PSMA8-101 CD8+ T cells in blood. Fig. 9D is a series of line graphs showing the assessment of tumor volume by caliper measurement. The left panel shows the mean of the tumor volume data shown in the series of panels on the right. Fig. 9E is a line graph showing bioluminescence of LNCaP tumors by BLI. Fig. 9F shows representative photographs of the bioluminescence of the LNCaP tumor mice quantified in fig. 9E. FIG. 9G is a series of flow cytometry plots showing P-PSMA-101 (T)_SCM/T_CM) Generates CARTyrin + T_CM、T_EMAnd T_effTo attack solid tumors. After solid tumor is eliminated, P-PSMA-101T continues to exist_SCMAnd (4) a group.

Fig. 10 is a series of flow cytometry plots depicting the abundance of cells moving from the region of viable cells (the lower right quadrant gated) to the region occupied by apoptotic cells (the upper left quadrant) as a function of increasing dose of an inducer (AP1903) in cells modified to express the therapeutic agent (CARTyrin), alone or in combination with the inducible caspase polypeptide of the present disclosure encoded by the iC9 construct (also referred to as a "safety switch") introduced into the cells by piggybac (pb) transposase, at day 12 post-nuclear transfection.

Fig. 11 is a series of flow cytometry plots depicting the abundance of cells moving from the region of viable cells (the lower right quadrant gated) to the region occupied by apoptotic cells (the upper left quadrant) as a function of increasing dose of the inducer (AP1903) in cells modified to express the therapeutic agent (CARTyrin), alone or in combination with the inducible caspase polypeptide of the present disclosure encoded by the iC9 construct (also referred to as a "safety switch") introduced into the cells by piggybac (pb) transposase, at day 19 post-nuclear transfection.

Fig. 12 is a pair of graphs depicting quantification of the integrated results shown in fig. 10 (left graph) or fig. 11 (right graph). Specifically, these figures show the effect of iC9 safety switch on percent cell viability as a function of the concentration of inducer of iC9 switch (AP1903) for each modified cell type at day 12 (fig. 10 and left) or day 19 (fig. 11 and right).

FIG. 13 is a bar graph depicting the knockdown efficiency of targeting various checkpoint signaling proteins that can be used to armor (armor) T-cells. Cas-CLOVER was used to knock out the checkpoint receptors PD-1, TGFBR2, LAG-3, TIM-3, and CTLA-4 in resting primary human whole (pan) T cells. Percent knockdown is shown on the y-axis. Gene editing results in 30-70% loss of protein expression at the cell surface as measured by flow cytometry.

FIG. 14 is a schematic representation of wild type, null and switch receptors (switch receptors) in primary T-cells and their effect on intracellular signaling (inhibitory or stimulatory). Binding of a wild-type inhibitory receptor endogenously expressed on T-cells to its endogenous ligand results in the transmission of inhibitory signals, which in part reduce T-cell effector function. However, mutation (mutational nullity) or deletion (truncated nullity) of the intracellular domain (ICD) of a checkpoint receptor protein, such as PD1 (upper panel) or TGFBRII (lower panel), reduces or eliminates its signaling ability when one or more cognate ligands bind. Thus, expression of the engineered mutated or truncated null receptor on the surface of the modified T cell results in competition with the endogenously expressed wild-type receptor for binding to one or more free endogenous ligands, thereby effectively reducing or eliminating the delivery of inhibitory signals by the endogenously expressed wild-type receptor. In particular, any binding by a mutated or null receptor will mask binding of one or more endogenous ligands to the wild-type receptor and result in a thinning of the total level of checkpoint signaling effectively delivered to the modified T-cell, thereby reducing or blocking checkpoint inhibition and functional depletion of the modified T-cell. The switch receptor was generated by replacing the wild-type ICD with ICDs from co-stimulatory molecules (e.g., CD3z, CD28, 4-1BB) or different inhibitory molecules (e.g., CTLA4, PD1, Lag 3). In the former case, binding of one or more endogenous ligands by the modified switch receptor results in delivery of a positive signal to the T-cell, thereby contributing to enhanced stimulation of the modified T-cell and possibly enhanced killing of the target tumor cell. In the latter case, binding of one or more endogenous ligands by the modified switch receptor results in delivery of a negative signal to the T-cell, thereby eliminating stimulation of the modified T-cell and possibly reducing target tumor cell killing. The signal peptide (purple arrow), extracellular domain (ECD) (bright green), transmembrane domain (yellow), intracellular signaling domain (ICD) (orange) and replacement ICD (green) are shown in the receptor map. "" indicates a mutated ICD. "+" indicates that a limit point signal is present. "-" indicates that there is no limit point signal.

Figure 15A is a schematic showing an example of a null receptor design with specific alterations that may help to increase receptor expression on the surface of modified T cells. Examples of PD1 and TGFBRII null receptors are shown, as are the signal peptide domain (SP), transmembrane domain (TM) and extracellular domain (ECD) of the truncated null receptors of PD1 (upper panel) and TGFBRII (lower panel). The first of the upper four molecules is the wild-type PD-1 receptor, which encodes wild-type PD-1 SP and TM. For the PD1 null receptor, a replacement of the PD1 wild-type SP or TM domain (green; light green) with the SP or TM domain of the human T-cell CD8a receptor (red) is depicted. The second molecule encodes CD8a SP along with native PD-1 TM, the third encodes wild-type PD-1 SP and the replacement CD8a TM, and the fourth encodes both the replacement CD8a SP and TM. Similarly, for the null receptor for TGF β RII, the SP domain of the human T cell CD8a receptor (red) was substituted for the wild-type TGFBRII SP (pink). The names of the constructs and the amino acid length (aa) of each construct protein are listed on the left side of the figure.

Fig. 15B is a series of histograms depicting expression of PD1 and TGFBRII null receptors on the surface of modified primary human T cells as determined by flow cytometry. Each of the six truncated null constructs from fig. 15A was expressed on the surface of primary human T cells. T cells were stained with anti-PD 1 (upper; blue histogram) or anti-TGF β RII (lower; blue histogram) or isotype control or secondary antibody only (grey histogram). Cells staining positive for PD-1 or TGF β RII expression were gated (frequency shown above the gate) and Mean Fluorescence Intensity (MFI) values are shown above each positive histogram. The name of the null receptor construct is depicted on the top of each figure. Two null receptor gene strategies, replacement of wild-type SP with the replacement CD8a resulted in successful expression. 02.8aSP-PD-1 and 02.8 aSP-TGF-. beta.RII resulted in the highest level of expression on the surface of T-cells. 02.8aSP-PD-1 null receptor showed an MFI of 43,680, which is 177-fold higher than endogenous T cell PD-1 expression and 2.8-fold higher than wild-type PD-1 null receptor. 02.8 aSP-TGF-. beta.RII null receptor showed MFI of 13, 809 that was 102-fold greater than endogenous T cell TGF-. beta.RII expression and 1.8-fold greater than wild-type TGF-. beta.RII null receptor. For both PD1 and TGRBRII, replacement of wild-type SP with the replacement CD8a SP resulted in null or increased surface expression of the switch receptor, which may help to maximize the reduction or blocking of checkpoint inhibition upon binding and masking of one or more endogenous ligands.

FIGS. 16A-B are a pair of schematic diagrams depicting NF-KB inducible vectors for expression in T-cells. Two T cell activation NF-KB inducible vectors are developed; one with the Gene Expression System (GES) in the forward orientation (a) and the other in the complementary orientation (B), both before the constitutive EF1a promoter. These vectors also direct the expression of the CAR molecule and DHFR selection gene separated by a T2A sequence. Both the conditional NF-KB inducible system and the EF1 a-directed gene are part of piggyBac transposons that can be permanently integrated into T cells using Electroporation (EP). Once integrated into the genome, T cells will constitutively express CAR on the membrane surface and DHFR within the cell, whereas expression of the NF-KB inducible gene GFP will only be expressed to the highest level upon T cell activation.

FIG. 17 is a pair of graphs depicting NF-KB inducible expression of GFP in activated T cells. T cells were nuclear transfected (nucleofected) with piggyBac vectors expressing anti-BCMA CAR and DHFR mutein genes under control of EF1a promoter and NF-KB inducible expression systems in the absence (no GES control) or in the presence of GFP expression driving either forward orientation (pfnfkb-GFP forward) or reverse orientation (pfkb-GFP reverse). Cells were cultured with methotrexate selection until the cells were almost completely quiescent (day 19) and GFP expression was assessed on days 5 and 19. On day 5, all T cells proliferated and were highly stimulated, while cells with NF-KB inducible expression cassettes produced high levels of GFP due to strong NF κ B activity. The GES-free control cells did not express detectable levels of GFP. By day 19, GES T cells were almost completely quiescent and GFP expression was significantly lower than day 5 (-1/8 MFI) due to lower NF κ B activity. GFP expression was still observed at day 19, possibly due to the long half-life (-30 hours) of the GFP protein, or basal levels of NF κ B activity signaled by, for example, TCR, CAR, cytokine receptor, or growth factor receptor.

Figure 18 is a series of graphs depicting NF-KB-inducible expression of GFP in the presence of BCMA + tumor cells anti-BCMA CAR-mediated activation. T cells were either unmodified (Mock) T cells) or nuclear transfected with piggyBac vector expressing anti-BCMA CAR and DHFR mutein genes under the control of EF 1a promoter and NF-KB inducible expression system in the absence (no GES control) or in the presence of GFP expression driving either forward orientation (pNFKB-GFP forward) or reverse orientation (pNFKB-GFP reverse). All cells were cultured for 22 days with or without methotrexate selection (mock T cells) until the cells were almost completely quiescent. Cells were then stimulated for 3 days in the absence (without stimulation) or presence of BCMA- (K562), BMCA + (RPMI 8226) or positive control anti-CD 3 anti-CD 28 activating reagent (CD3/28 stimulation). GFP expression was undetectable by either no GES control or mock T cells under all conditions. However, while pNFKB-GFP positively and reversely transposed cells showed a small amount of GFP expression when cultured with BCMA-K562 cells compared to the non-stimulated control, they all showed dramatic upregulation of gene expression in the presence of BCMA + tumor cells or under positive control conditions. Little difference in GFP expression was observed between pNFKB-GFP positive and reverse transposed cells co-cultured with BCMA + tumor cells.

FIG. 19 is a series of graphs demonstrating that the expression level of inducible genes can be regulated by the number of response elements preceding the promoter. T cells were nuclear transfected with piggyBac vectors encoding anti-BCMA CARTyrin plus a selection gene, both under the control of the human EF1a promoter. Further, the vector additionally encodes a conditional NF-KB inducible gene expression system driving expression of a truncated CD19 protein (dCD19), and includesA number of NFKB Responsive Elements (RE) varying from 0 to 5 were included, either no GES (no GES), or received electroporation pulses without piggyBac nucleic acids (mock). Only data on GES in the reverse (opposite) direction/orientation is shown. All cells were cultured for 18 days and included selection for piggyBac-modified T cells using methotrexate addition. Cells were then stimulated with anti-CD 3 anti-CD 28 bead activating reagent for 3 days and dCD19 surface expression was assessed by FACS at days 0, 3 and 18 and the data is shown as FACS histograms and MFI of target protein staining. The x-axis of each FACS histogram is in logarithmic scale (0, 10)³、10⁴、10⁵) And (4) depicting. The samples plotted per FACS histogram are NFKB-A08-DHFR _ Rev002.fcs, NFKB-A08_ DHFR _ Rev-RE4X _12.fcs, NFKB-A08_ DHFR _ Rev-RE3X _011.fcs, NFKB-A08_ DHFR _ Rev-RE2X _010.fcs, NFKB-A08_ DHFR _ Rev-RE1X _009.fcs, NFKB-A08_ DHFR _ Rev-RE0X _008.fcs, NFKB-A08_ DHFR _ v5_013.fcs, and NFKB-A08_ DHFR _ MOCK _014.fcs, from top to bottom. At day 0, low levels of surface dCD19 expression were detected in all T cells transposed with the GES-encoding vector. A dramatic upregulation of dCD19 expression was observed for all GES expressing T cells 3 days post stimulation, while the fold increase in surface expression was greater in those with higher numbers of REs. Thus, surface dCD19 expression is proportional to the number of REs encoded in GES. No dCD19 was detected on the surface of T cells without GES: no GES and mock control.

FIG. 20 is a schematic depiction of the Csy4-T2A-Clo051-G4S linker-dCas 9 construct map (embodiment 2).

FIG. 21 is a schematic depiction of a map of pRT1-Clo051-dCas9 dual NLS constructs (embodiment 1).

Figure 22 is a schematic depicting a time-course of a study conducted for preclinical evaluation of lead P-PSMA-101 candidates in a murine xenograft model of prostate cancer bone metastases. A murine xenograft model peritibial (peri-tibially) (IT) using a luciferase-expressing PC3 cell line (PC3.lucGFP. hPSMA) engineered to express human PSMA protein was injected into NSG mice and used to evaluate the in vivo anti-tumor efficacy of P-PSMA5-101 and P-PSMA8-101 at two different doses-a 'stress' dose of 4x10^6 and a standard of 12x10^6Total CAR-T cells. As negative controls, T cells alone (not expressing CAR; 12e6 dose) or P-BCMA-101T cells (expressing irrelevant anti-BCMA CAR; 12e6 dose) were also included. For these in vivo studies, all CAR-T cells were produced using the Poseida manufacturing process using PB delivery of the P-PSMA5-101 or P-PSMA8-101 plasmids. Mice were injected peritibial with Pc3.lucGFP. hPSAI and treated four days later with CAR-T cells (all tumor challenged mice had total luminous flux (p/sec/m) by the range 1-5x10^6 ²) A tumor detectable by bioluminescent imaging (BLI).

Figure 23 is a graph showing dose efficacy of P-PSMA5-101 and P-PSMA8-101 at 'stress' doses or standard doses showing anti-tumor efficacy against established IT pc3.lucgfp. hpsma solid tumors in NSG mice compared to T cell alone (no CAR) or P-BCMA-101 treated control mice.

FIG. 24 is a schematic drawing depicting piggyBac P-PSMA-101 nano-transposon (nanotransposon) of the present disclosure.

Fig. 25 is a graph showing that Electroporation (EP) delivery of P-PSMA-101 piggyBac plasmid (light grey bars) or P-PSMA-101 piggyBac nano-transposon (dark grey bars) in combination with super piggyBac transposase results in high transposition efficiency in human whole T cells (5 days post EP), as measured by surface expression of PSMA CARTyrin (percent transposition (%)).

Figure 26 is a series of graphs showing that human CAR-T cells generated using anti-PSMA CAR nano-transposons (NTs) are able to kill target tumor cells. anti-PSMA CAR T cells generated using either the full-size piggyBac plasmid (FP) or the piggyBac nano-transposon (NT) were produced using standard Poseida technology. Killing of K562(K562.PSMA) cells engineered to express PSMA by CAR-T cells at the indicated effector to target ratios. These data indicate that all CAR-T cells produced whether using FP or NT are able to kill target tumor cells in an antigen-dependent manner. This is true for CAR-T cells produced from human whole T cells from two different normal donors.

Figure 27 is a series of graphs showing that human CAR-T cells generated using anti-PSMA CAR nano-transposons (NTs) are comparable in phenotypic composition. anti-PSMA CAR T cells generated using either the full-size piggyBac plasmid (FP) or the piggyBac nano-transposon (NT) were made using standard Poseida technology. Phenotypic analysis of memory T cell markers and activation/depletion markers (data not shown) was performed. These data indicate that all CAR-T cells produced using FP or NT exhibit similar phenotypic compositions of CD45RA + CD62L + (Tsccm), CD45RA-CD62L + (Tcm), CD45RA-CD62L- (Tem), and CD45RA + CD62L- (Teff) cells. In addition, comparable expression levels of CCR7(CD197), CD127, CD27, LAG3, TIM3, CXCR3, PD-1, and CD25 were observed (data not shown). This is true for CAR-T cells produced from human whole T cells from two different normal donors.

Figure 28 is a series of graphs showing that human CAR-T cells generated using anti-PSMA CAR nano-transposons (NTs) have similar integrated copy numbers. anti-PSMA CAR T cells generated using either the full-size piggyBac plasmid (FP) or the piggyBac nano-transposon (NT) were made using standard Poseida technology. The average copy number of the integrated transposon was measured by quantitative PCR. These data indicate that all CAR-T cells, whether generated using FP or NT, exhibited similar integrated copy numbers of the transposon in the two different donors.

FIG. 29A is a schematic showing preclinical evaluation of P-PSMA-101 transposon using a murine xenograft model at 'stress' doses when delivered by Full Length Plasmid (FLP) versus Nano Transposon (NT). The in vivo antitumor efficacy of P-PSMA-101 transposons as delivered by Full Length Plasmid (FLP) or Nanotransposon (NT) at two different 'stress' doses (2.5x10^6 or 4x10^6) of total CAR-T cells from two different normal donors was evaluated using a murine xenograft model using luciferase-expressing LNCaP cell lines (LNCaP. luc) injected Subcutaneously (SC) into NSG mice. All CAR-T cells were produced using FLP or NT delivery using piggybac (pb) delivery of the P-PSMA-101 transposon. Mice were injected in the axilla with LNCaP and treated as tumors were established (100-³). Two different 'stress' doses (2.5x10^6 or 4x10^6) of P-PSMA-101 CAR by Intravenous (IV) injectionTs mice were treated for greater resolution in detecting possible functional differences in efficacy between transposon delivery by FLP and NT.

Figure 29B is a series of graphs showing tumor volume assessment of mice treated as described in figure 29A. Tumor volume estimates measured by calipers for control mice (black), donor #1FLP mice (red), donor #1NT mice (blue), donor #2 FLP mice (orange), and donor #2 NT mice (green) are shown as group means with error bars (top) and individual mice (bottom). The y-axis shows the tumor volume (mm) assessed by caliper measurement ³). The x-axis shows days after T cell treatment. Luc solid tumors compared to FLP and control mice, P-PSMA-101 transposons delivered by NT at 'stress' doses showed enhanced antitumor efficacy against established SC lncap.

Figure 30 is a schematic depicting T-cell receptor (TCR) and co-receptors CD28 and CD 2.

FIG. 31 is a schematic depicting the delivery of primary and secondary co-stimulation to T-cells via binding of agonist mAbs (anti-CD 3, anti-CD 28, and anti-CD 2). Complete T-cell activation is critically dependent on TCR engagement (TCR engagement) along with secondary signaling through co-stimulatory receptors that potentiate the immune response. Primary and secondary co-stimulation can be delivered to T-cells via treatment and conjugation of surface receptors with agents that display agonist mAbs (e.g., anti-CD 3, anti-CD 28, anti-CD 2, beads conjugated to these mAbs, multimeric complexes of these mAbs, etc.).

FIG. 32 is a schematic showing enhanced stimulation with expression of Chimeric Stimulatory Receptors (CSRs) in the absence of TCR. In the presence of one or more surface-expressed CSRs (transiently or stably expressed), enhanced primary and secondary co-stimulatory signals are delivered when T cells are treated with agents displaying agonist mAbs. T cell activation and expansion is enhanced due to more adequate T-cell activation via CSR-mediated stimulation signals.

Fig. 33A is a schematic diagram depicting an exemplary CSR CD28z of the present disclosure.

FIG. 33B is an amino sequence encoding the CSR CD28z shown in FIG. 33A.

FIG. 33C is a nucleotide sequence encoding the CSR CD28z set forth in FIG. 33A.

Fig. 34A is a schematic diagram depicting an exemplary CSR CD2z of the present disclosure.

FIG. 34B is an amino sequence encoding the CSR CD2z shown in FIG. 34A.

FIG. 34C is a nucleotide sequence encoding the CSR CD2z set forth in FIG. 34A.

Figure 35 is a series of graphs showing that CSRs are expressed on the surface of T cells and do not result in cell activation in the absence of exogenous stimulation. In standard T cell culture media, whole T cells from normal donors were stimulated with anti-CD 3/anti-CD 28 beads and then rested. These cells were then electroporated with 10 μ g of mRNA encoding CD28 CSR, CD2 CSR, or wild type CD19 control (BTX ECM 830 electroporator) @500V for 700 μ s). After two days, the surface expression of each molecule of the electroporated cells was examined by flow cytometry, and the data was displayed as a stacked histogram. In addition, cell size (FSC-a) and CD69 expression were evaluated as possible indicators of cell activation above control cells that mimic electroporation. Increased surface expression of CD28, CD2 and CD19 was detected in T cells electroporated with CD28z CSR, CD2z CSR or CD19, respectively. Expression of these molecules on the surface of T cells does not inherently activate the cells in the absence of exogenous stimuli.

Figure 36 is a graph showing that delivery of CSR enhances expansion of CAR-T cells. CSRs either transiently through mRNA or through piggyBac^TMStably delivered to CAR-T cells. Using piggyBac^TMDNA modification systems and standard Poseida procedures genetically modify whole T cells isolated from normal donor blood. The cells were co-electroporated (co-electroporated) in a single reaction with mRNA encoding Super piggyBac transposase (SPB), transposon encoding the BCMA CAR and selection gene, and additional mRNA encoding CSR (CD28z or CD2 z; resulting in transient expression) or CD19 mRNA control, or with transposon encoding the BCMA CAR, selection gene and CSR (CD28z or CD2 z; resulting in stable expression). The cells were then stimulated with agonist mAbs anti-CD 2, anti-CD 3, and anti-CD 28, and later cultured for 19 daysThe genetic modification is selected during the time period. At the end of the initial culture period, all T cells expressed CAR, indicating successful selection for genetically modified cells (data not shown). Bars represent total live CAR-T cells in wells, and numbers indicate fold enhancement of expansion above CAR-T cells produced without CSR or CD19 mRNA controls. CAR-T cells expanded to a greater extent in samples transiently or stably expressing CD2z or CD28z CSR.

Figure 37 is a series of bar graphs showing that expression of CSRs did not significantly affect the cytotoxicity of CAR-T cells. CSRs either transiently through mRNA or through piggyBac^TMStably delivered to CAR-T cells. Using piggyBac^TMDNA modification systems and standard Poseida procedures genetically modify whole T cells isolated from normal donor blood. The cells were co-electroporated in a single reaction with mRNA encoding Super piggyBac transposase (SPB), transposon encoding BCMA CAR and selection gene and additional mRNA encoding CSR (CD28z or CD2 z; resulting in transient expression), or with transposon encoding BCMA CAR, selection gene and CSR (CD28z or CD2 z; resulting in stable expression). The cells were then stimulated with agonist mAbs anti-CD 2, anti-CD 3, and anti-CD 28, and later selected for genetic modification during the 19 day culture period. At the end of the initial culture period, all T cells expressed CAR, indicating successful selection for genetically modified cells (data not shown). To assess the ability of CAR-T cells to kill, cells were co-cultured with artificially engineered K562-BCMA-luciferase (eK562-Luc. BCMA) or negative control K562-luciferase (eK562-Luc) at 10: 1, 3: 1, or 1: 1E: T ratios for 48 hours. Luciferase signal was measured to determine cytotoxicity. The bar graph on the left shows killing of eK562-Luc, while the bar graph on the right shows killing of eK562-Luc. All CAR ⁺T cells all expressed anti-BCMA specific CARs and showed similar cytotoxicity in vitro against BCMA + target cells. In summary, the activity was not significantly affected by transient or stable CSR co-expression.

Detailed Description

The present disclosure provides Chimeric Antigen Receptors (CARs) (CARTyrin) comprising at least one centrin. The chimeric antigen receptor of the present disclosure may comprise more than one centrin. For example, a bispecific CARTyrin may comprise two centrins that specifically bind to two different antigens. In a preferred embodiment, the CARTyrins of the present disclosure comprise at least one sequence that specifically binds PSMA, and, therefore, are referred to as PSMA-specific centrins. The CARTyrins of the present disclosure comprising at least one PSMA-specific Centryrin are referred to herein as anti-PSMA CARTyrins of a sequence for specifically binding PSMA.

The centrins of the present disclosure specifically bind to antigens. Chimeric antigen receptors of the present disclosure comprising one or more centrins that specifically bind to an antigen can be used to direct the specificity of a cell (e.g., a cytotoxic immune cell) to a particular antigen.

The centrins of the present disclosure may comprise a consensus sequence comprising

The chimeric antigen receptor of the present disclosure may comprise a signal peptide of human CD2, CD3 δ, CD3 ∈, CD3 γ, CD3 ζ, CD4, CD8 α, CD19, CD28, 4-1BB, or GM-CSFR. The hinge/spacer domain of the present disclosure may comprise the hinge/spacer/stem of human CD8 a, IgG4, and/or CD 4. The intracellular domain or endodomain of the present disclosure may comprise an intracellular signaling domain of human CD3 ζ, and may further comprise human 4-1BB, CD28, CD40, ICOS, MyD88, OX-40 intracellular segments, or any combination thereof. Exemplary transmembrane domains include, but are not limited to, human CD2, CD3 δ, CD3 ε, CD3 γ, CD3 ζ, CD4, CD8 α, CD19, CD28, 4-1BB, or GM-CSFR transmembrane domains.

The present disclosure provides genetically modified cells, such as T cells, NK cells, hematopoietic progenitor cells, Peripheral Blood (PB) -derived T cells (including T cells from G-CSF-mobilized (mobilized) peripheral blood), Umbilical Cord Blood (UCB) -derived T cells, that are made specific for one or more antigens by introducing the CARTyrin of the present disclosure into these cells. The cells of the disclosure can be modified by electrotransfering a transposon encoding a CARTyrin of the disclosure and a plasmid comprising a sequence encoding a transposase of the disclosure (preferably, the sequence encoding the transposase of the disclosure is an mRNA sequence).

Further modification of cellular compositions

The present disclosure provides a Chimeric Stimulatory Receptor (CSR) comprising: (a) an extracellular domain comprising an activating component and (b) a transmembrane domain or cell membrane attachment region, wherein the combination of (a) and (b) is non-naturally occurring. In some embodiments, the CSR binds to a component of the environment and alters the cellular consequences of the signaling by competing with the full-length or transmembrane form of the receptor to reduce the intracellular signal caused by the binding of the component of the environment to the activating component.

The present disclosure provides a Chimeric Stimulatory Receptor (CSR) comprising: (a) an extracellular domain comprising an activating component; (b) a transmembrane domain; and (c) an endodomain comprising at least one signaling domain; wherein the combination of (a), (b), and (c) is non-naturally occurring.

In some embodiments of the CSRs of the present disclosure, the activating component of (a) is isolated or derived from a first protein. In some embodiments, the signaling domain of (c) is isolated or derived from a second protein. In some embodiments, the first protein and the second protein are not the same.

In some embodiments of the CSRs of the present disclosure, the CSR is a switch receptor that switches extracellular binding activating components from inhibiting or transducing a signal that is qualitatively different from that which would be transduced by the wild-type, full-length, or transmembrane form of the first protein. Because CSR switch receptors are chimeric with respect to extracellular and intracellular domains, CSRs can switch the result of binding extracellular activating components from a naturally occurring situation to an artificially engineered, non-naturally occurring situation.

In some embodiments of the CSRs of the present disclosure, the activating component includes one or more of a component of a human transmembrane receptor, a human cell surface receptor, a T-cell receptor (TCR), a component of a TCR complex, a component of a TCR co-receptor, a component of a TCR co-stimulatory protein, a component of a TCR inhibitory protein, a cytokine receptor, and a chemokine receptor. In some embodiments, the activating component includes a component of one or more of a human transmembrane receptor, a human cell surface receptor, a T-cell receptor (TCR), a component of a TCR complex, a component of a TCR co-receptor, a component of a TCR co-stimulatory protein, a component of a TCR inhibitory protein, a cytokine receptor, and a chemokine receptor to which an agonist of the activating component binds.

In some embodiments of the CSRs of the present disclosure, the agonist comprises one or more of a small organic or inorganic molecule, nucleic acid, amino acid, antibody or fragment thereof, antibody mimetic (mimetic), aptamer, scaffold protein (scaffold protein), ligand, receptor, naturally occurring biomolecule, and non-naturally occurring molecule (organic or inorganic).

In some embodiments of the CSRs of the present disclosure, the activating component comprises a portion thereof to which a CD2 protein or agonist binds.

In some embodiments of the CSRs of the present disclosure, the signaling domain comprises one or more of a component of a human signaling domain, a T-cell receptor (TCR), a component of a TCR complex, a component of a TCR co-receptor, a component of a TCR co-stimulatory protein, a component of a TCR inhibitory protein, a cytokine receptor, and a chemokine receptor. In some embodiments, the signaling domain comprises a CD3 protein. In some embodiments, the CD3 protein comprises a CD3 ζ protein.

In some embodiments of the CSRs of the present disclosure, the intracellular domain further comprises a cytoplasmic domain. In some embodiments, the sequence encoding the cytoplasmic domain comprises a sequence encoding a costimulatory protein. In some embodiments, the cytoplasmic domain is isolated or derived from a third protein. In some embodiments, the first protein and the third protein are the same.

In some embodiments of the CSRs of the present disclosure, the extracellular domain further comprises a signal peptide. In some embodiments, the signal peptide is derived from a fourth protein. In some embodiments, the first protein and the fourth protein are the same.

In some embodiments of the CSRs of the present disclosure, the transmembrane domain is isolated or derived from a fifth protein. In some embodiments, the first protein and the fifth protein are the same.

In some embodiments of the CSRs of the present disclosure, the CSR comprises an extracellular domain comprising a signal peptide having a sequence isolated or derived from a CD2 protein and an activating component comprising a sequence isolated or derived from a portion thereof to which a CD2 protein or agonist binds, a transmembrane domain comprising a sequence isolated or derived from a CD2 protein, and an endodomain comprising a cytoplasmic domain comprising a sequence isolated or derived from a CD2 protein and a signal transduction domain comprising a sequence isolated or derived from a CD3 zeta protein.

In some embodiments of the CSRs of the present disclosure, the activating component does not bind to a naturally occurring molecule.

In some embodiments of the CSRs of the present disclosure, the CSR does not transduce a signal when the activating component binds to a naturally occurring molecule. In some embodiments, the extracellular domain comprises a modification. In some embodiments, the modification comprises a mutation or truncation of the sequence encoding the activating component when compared to the wild-type sequence of the first protein.

In some embodiments of the CSRs of the present disclosure, the activating component binds to a non-naturally occurring molecule.

In some embodiments of the CSRs of the present disclosure, the CSR selectively transduces a signal upon binding of an activating component to a non-naturally occurring molecule.

The present disclosure provides nucleic acid sequences encoding the CSRs of the disclosure.

The present disclosure provides vectors comprising a nucleic acid sequence encoding a CSR of the disclosure.

The present disclosure provides a transposon comprising a nucleic acid sequence encoding a CSR of the disclosure.

The present disclosure provides cells comprising a CSR of the present disclosure.

The present disclosure provides a cell comprising a nucleic acid encoding a CSR of the disclosure.

The present disclosure provides a cell comprising a vector comprising a nucleic acid sequence encoding a CSR of the disclosure.

The present disclosure provides a cell comprising a transposon comprising a nucleic acid sequence encoding a CSR of the disclosure.

The present disclosure provides compositions comprising CSRs of the present disclosure.

The present disclosure provides compositions comprising nucleic acids encoding CSRs of the disclosure.

The present disclosure provides compositions comprising a vector comprising a nucleic acid sequence encoding a CSR of the disclosure.

The present disclosure provides compositions comprising a transposon comprising a nucleic acid sequence encoding a CSR of the disclosure.

The present disclosure provides compositions comprising cells of the disclosure, including those comprising sequences encoding and/or expressing a CSR of the disclosure. The present disclosure provides compositions comprising a plurality of cells of the disclosure, including those comprising sequences encoding and/or expressing CSRs of the disclosure.

The present disclosure provides a modified cell comprising: (a) a sequence encoding a CSR of the disclosure, and (b) a sequence encoding an inducible pro-apoptotic polypeptide. In some embodiments of the modified cells of the present disclosure, the modified cells further comprise a sequence encoding a non-naturally occurring antigen receptor and/or a sequence encoding a therapeutic polypeptide.

In some embodiments of the modified cells of the present disclosure, including those in which the modified cells comprise a sequence encoding a non-naturally occurring antigen receptor, including a Chimeric Antigen Receptor (CAR). In some embodiments, the CAR comprises: (a) an extracellular domain comprising an antigen recognition region, (b) a transmembrane domain, and (c) an intracellular domain comprising at least one costimulatory domain. In some embodiments, the extracellular domain of (a) of the CAR further comprises a signal peptide. In some embodiments, the extracellular domain of (a) of the CAR further comprises a hinge between the antigen recognition region and the transmembrane domain. In some embodiments, the endodomain comprises a human CD3 ζ endodomain. In some embodiments, the at least one co-stimulatory domain comprises a human 4-1BB, CD28, CD40, ICOS, MyD88, OX-40 intracellular segment, or any combination thereof. In some embodiments, the at least one co-stimulatory domain comprises a human CD28 and/or 4-1BB co-stimulatory domain.

In some embodiments of the modified cells of the present disclosure, the transposon, vector, donor sequence, or donor plasmid comprises a sequence encoding a CSR and/or a sequence encoding an inducible pro-apoptotic polypeptide. In some embodiments, the transposon, vector, donor sequence, or donor plasmid further comprises a sequence encoding a non-naturally occurring antigen receptor or a sequence encoding a therapeutic protein. In some embodiments, the transposon, vector, donor sequence, or donor plasmid further comprises a sequence encoding a selectable marker. In some embodiments, the transposon is a piggyBac or piggy-Bac-like transposon.

In some embodiments of the modified cells of the present disclosure, the sequence encoding the CSR is transiently expressed in the cell, and wherein the sequence encoding the inducible pro-apoptotic polypeptide is stably expressed in the cell. In some embodiments, the sequence encoding the non-naturally occurring antigen receptor or the sequence encoding the therapeutic protein is stably expressed in the cell. In some embodiments, the first transposon, the first vector, the first donor sequence, or the first donor plasmid comprises a sequence encoding a CSR. In some embodiments, the second transposon, the second vector, the second donor sequence, or the second donor plasmid comprises one or more of a sequence encoding an inducible pro-apoptotic polypeptide, a sequence encoding a non-naturally occurring antigen receptor, a sequence encoding a therapeutic protein. In some embodiments, the first transformant, first vector, first donor sequence, or first donor plasmid further comprises a sequence encoding a first selectable marker. In some embodiments, the second transposon, the second vector, the second donor sequence, or the second donor plasmid further comprises a sequence encoding a second selectable marker. In some embodiments, the first selectable marker and the second selectable marker are not the same.

In some embodiments of the modified cells of the present disclosure, the selectable marker is a cell surface marker. In some embodiments, the cell surface marker distinguishes cells when sorted by marker or detectable label. In some embodiments, the detectable label is fluorescent or magnetic.

In some embodiments of the modified cells of the present disclosure, the selectable marker comprises a protein that is active in dividing cells and inactive in non-dividing cells. In some embodiments, the selectable marker comprises a metabolic marker. In some embodiments, the selectable marker comprises a dihydrofolate reductase (DHFR) mutant protease. In some embodiments, the DHFR mutant protease comprises or consists of the amino acid sequence:

in some embodiments, the amino acid sequence of the DHFR mutant protease further comprises a mutation at one or more of positions 80, 113 or 153. In some embodiments, the amino acid sequence of the DHFR mutant protease comprises one or more of a substitution of phenylalanine (F) or leucine (L) at position 80, a substitution of leucine (L) or valine (V) at position 113, and a substitution of valine (V) or aspartic acid (D) at position 153.

Chimeric Stimulating Receptors (CSRs)

The present disclosure provides a Chimeric Stimulatory Receptor (CSR) to deliver CD3z primary stimulation to T cells (and thus endogenous CD3 ζ) when stimulated with standard activating/stimulating reagents including agonist anti-CD 3 mAb.

The Chimeric Stimulatory Receptors (CSRs) of the present disclosure provide CD3 zeta stimulation to enhance activation and expansion of T cells. In some embodiments, CSRs of the present disclosure comprise an agonist mAb epitope outside the cell and a CD3 zeta stimulating domain inside the cell, and functionally convert an anti-CD 28 or anti-CD 2 binding event on the surface to a CD3z signaling event in allogeneic T cells modified to express CSRs. In some embodiments, the CSR comprises a wild-type CD28 or CD2 protein and a CD3z intracellular stimulation domain to produce CD28z CSR and CD2z CSR, respectively. In preferred embodiments, the CD28z CSR and/or CD2z CSR further express a non-naturally occurring antigen receptor and/or therapeutic protein. In a preferred embodiment, the non-naturally occurring antigen receptor comprises a chimeric antigen receptor.

In certain embodiments, the CD28z CSR is encoded by an amino acid sequence comprising:

In certain embodiments, the CD28z CSR is encoded by a nucleic acid sequence comprising:

in certain embodiments, the CD2z CSR is encoded by an amino acid sequence comprising:

in certain embodiments, the CD2z CSR is encoded by a nucleic acid sequence comprising:

the modified T cells of the disclosure comprising/expressing CSRs of the disclosure improve T cell expansion when compared to those cells not comprising/expressing CSRs of the disclosure.

Immune and immune precursor cells

In certain embodiments, the immune cells of the present disclosure include lymphoid progenitor cells, Natural Killer (NK) cells, T lymphocytes (T-cells), stem memory T cells (T cells)_SCMCells), central memory T cells (T cells) (central memory T cells)_CM) Stem cell-like T cells, B lymphocytes (B-cells), myeloid progenitor cells, neutrophils, basophils, eosinophils, monocytes, macrophages, platelets, erythrocytes (RBCs), megakaryocytes, or osteoclasts.

In certain embodiments, an immune precursor cell includes any cell that can differentiate into one or more types of immune cells. In certain embodiments, the immune precursor cells comprise pluripotent stem cells that can self-renew and develop into immune cells. In certain embodiments, the immune precursor cells comprise Hematopoietic Stem Cells (HSCs) or progeny thereof. In certain embodiments, the immune precursor cells include precursor cells that can develop into immune cells. In certain embodiments, the immune precursor cells comprise Hematopoietic Progenitor Cells (HPCs).

Hematopoietic Stem Cells (HSCs)

Hematopoietic Stem Cells (HSCs) are pluripotent, self-renewing cells. All differentiated blood cells from lymphoid and myeloid lineages are produced from HSCs. HSCs can be found in adult bone marrow, peripheral blood, mobilized peripheral blood, peritoneal dialysis effluent, and umbilical cord blood.

The HSCs of the present disclosure may be isolated or derived from primary or cultured stem cells. The HSCs of the present disclosure may be isolated or derived from embryonic stem cells, pluripotent stem cells, adult stem cells, or induced pluripotent stem cells (ipscs).

The immune precursor cells of the present disclosure may comprise HSCs or HSC progeny cells. Exemplary HSC progeny cells of the present disclosure include, but are not limited to, pluripotent stem cells, lymphoid progenitor cells, Natural Killer (NK) cells, T lymphocytes (T-cells), B lymphocytes (B-cells), myeloid progenitor cells, neutrophils, basophils, eosinophils, monocytes, and macrophages.

HSCs produced by the methods of the present disclosure may retain the characteristics of "naive" stem cells that, although isolated or derived from adult stem cells and despite committed to a single lineage, share the characteristics of embryonic stem cells. For example, "naive" HSCs produced by the methods of the present disclosure retain their "sternness" after division and do not differentiate. Thus, as adoptive cell therapy, "naive" HSCs produced by the methods of the present disclosure not only replenish their numbers, but also expand in vivo. The "naive" HSCs produced by the methods of the present disclosure may be therapeutically effective when administered as a single dose. In some embodiments, the original HSCs of the present disclosure are CD34 +. In some embodiments, the original HSCs of the present disclosure are CD34+ and CD 38-. In some embodiments, the original HSCs of the present disclosure are CD34+, CD38-, and CD90 +. In some embodiments, the original HSCs of the present disclosure are CD34+, CD38-, CD90+ and CD45 RA-. In some embodiments, the original HSCs of the present disclosure are CD34+, CD38-, CD90+, CD45RA-, and CD49f +. In some embodiments, the most primitive HSCs of the present disclosure are CD34+, CD38-, CD90+, CD45RA-, and CD49f +.

In some embodiments of the present disclosure, the original HSCs, and/or HSC progeny cells may be modified according to the methods of the present disclosure to express exogenous sequences (e.g., chimeric antigen receptors or therapeutic proteins). In some embodiments of the present disclosure, the modified naive HSCs, modified HSCs and/or modified HSC progeny cells may be forward differentiated to generate modified immune cells, including, but not limited to, modified T cells, modified natural killer cells and/or modified B-cells of the present disclosure.

T cells

Modified T cells of the present disclosure may be derived from modified Hematopoietic Stem and Promoter Cells (HSPCs) or modified HSCs.

Unlike traditional biologies and chemotherapeutic drugs, the modified T-cells of the present disclosure have the ability to rapidly multiply upon antigen recognition, thereby potentially eliminating the need for repeat therapy. To achieve this, in some embodiments, the modified T-cells of the present disclosure not only drive the initial response, but also persist in the patient as a stable population of viable memory T cells to prevent potential relapse. On the other hand, in some embodiments, the modified T-cells of the present disclosure do not persist in the patient when not desired.

Enhanced efforts have focused on developing antigen receptor molecules that do not lead to T cell depletion through antigen-independent (tone) signaling, and that contain early memory T cells, particularly stem cell memory (T)_SCM) Or stem cell-like T cells). The stem cell-like modified T cells of the present disclosure exhibit maximal self-renewal and multipotentiality to derive central memory (T)_CM) T cells or T_CMCell-like, effector memory (T)_EM) And effector T cells (T)_E) Resulting in better tumor eradication and long-term modified T cell engraftment. The linear differentiation pathway may be responsible for the generation of these cells: naive T cells (T)_N)＞T_SCM＞T_CM＞T_EM＞T_E＞T_TEWherein T is_NIs to directly generate T_SCMWhich then in turn directly produces T_CMAnd the like. Compositions of T cells of the present disclosure may comprise one or more of each parental T cell subpopulation, while T cells_SCMThe cells are most abundant (e.g., T)_SCM＞T_CM＞T_EM＞T_E＞T_TE)。

In some embodiments of the methods of the present disclosure, the immune cell precursor is differentiated into or capable of differentiating into early memory T cells, stem cell-like T-cells, naive T cells (T cells)_N)、T_SCM、T_CM、T_EM、T_EOr T_TE. In some embodiments, the immune cell precursor is an original HSC, or HSC progeny cell of the present disclosure.

In some embodiments of the methods of the present disclosure, the immune cell is an early memory T cell, a stem cell-like T-cell, a naive T cell (T cell)_N)、T_SCM、T_CM、T_EM、T_EOr T_TE。

In some embodiments of the methods of the present disclosure, the immune cell is an early memory T cell.

In some embodiments of the methods of the present disclosure, the immune cell is a stem cell-like T-cell.

In some embodiments of the methods of the present disclosure, the immune cell is T_SCM。

In some embodiments of the methods of the present disclosure, the immune cell is T_CM。

In some embodiments of the methods of the present disclosure, the methods modify and/or the methods produce a plurality of modified T cells, wherein at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or any percentage therebetween, of the plurality of modified T cells express one or more cell surface markers of early memory T cells. In certain embodiments, the plurality of modified early memory T cells comprises at least one modified stem cell-like T cell. In certain embodiments, the plurality of modified early memory T cells comprises at least one modified T _SCM. In certain embodiments, the plurality of modified early memory T cells comprises at least one modified T_CM。

In some implementations of the methods of the present disclosureIn a version, the method modifies and/or the method produces a plurality of modified T cells, wherein at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or any percentage therebetween, of the plurality of modified T cells express one or more cell surface markers of stem-like T cells. In certain embodiments, the plurality of modified stem cell-like T cells comprises at least one modified T_SCM. In certain embodiments, the plurality of modified stem cell-like T cells comprises at least one modified T_CM。

In some embodiments of the methods of the present disclosure, the methods modify and/or the methods produce a plurality of modified T cells, wherein at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or any percentage therebetween, of the plurality of modified T cells express stem memory T cells (T cells) _SCM) Of the cell surface marker(s). In certain embodiments, the cell surface markers include CD62L and CD45 RA. In certain embodiments, the cell surface markers include one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95, and IL-2R β. In certain embodiments, the cell surface markers include one or more of CD45RA, CD95, IL-2R β, CCR7, and CD 62L.

In some embodiments of the methods of the present disclosure, the methods modify and/orThe method produces a plurality of modified T cells, wherein at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or any percentage therebetween, of the plurality of modified T cells express naive T cells (T cells) _N) Of the cell surface marker(s). In certain embodiments, the cell surface markers include one or more of CD45RA, CCR7, and CD 62L.

In some embodiments of the methods of the present disclosure, the methods modify and/or the methods generate a plurality of modified T cells, wherein at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or any percentage therebetween, of the plurality of modified T cells express stem cell-like T cells, stem memory T cells (T cells) _SCM) Or central memory T cells (T)_CM) Of the cell surface marker(s).

In some embodiments of the methods of the present disclosure, the buffer comprises an immune cell or a precursor thereof. The buffer maintains or enhances the level of cell viability and/or stem-like (stem-like) phenotype of the immune cells or their precursors, including T-cells. In certain embodiments, the buffer maintains or enhances the level of cell viability and/or stem cell-like phenotype of the primary human T cells prior to nuclear transfection (nucleofection). In certain embodiments, the buffer maintains or enhances the level of cell viability and/or stem cells of the primary human T cells during nuclear transfectionCell-like phenotype. In certain embodiments, the buffer maintains or enhances the level of cell viability and/or stem cell-like phenotype of the primary human T cells following nuclear transfection. In certain embodiments, the buffer comprises KCl, MgCl in any absolute or relative abundance or concentration₂ClNa, glucose and Ca (NO)₃)₂And optionally, the buffer further comprises an additive selected from HEPES, Tris/HCl and phosphate buffer. In certain embodiments, the buffer comprises 5mM KCl, 15mM MgCl ₂90mM ClNa, 10mM glucose and 0.4mM Ca (NO)₃)₂. In certain embodiments, the buffer comprises 5mM KCl, 15mM MgCl₂90mM ClNa, 10mM glucose and 0.4mM Ca (NO)₃)₂And a supplement comprising 20mM HEPES and 75mM Tris/HCl. In certain embodiments, the buffer comprises 5mM KCl, 15mM MgCl₂90mM ClNa, 10mM glucose and 0.4mM Ca (NO)₃)₂And 40mM Na containing pH 7.2₂HPO₄/NaH₂PO₄The additive of (1). In certain embodiments, a composition comprising primary human T cells comprises 100 μ Ι buffer and 5 × 10⁶-25x10⁶The cell of (1). In certain embodiments, during the introducing step, the composition comprises a variable ratio of 250x10 per ml of buffer or other medium⁶Primary human T cells of (a).

In some embodiments of the methods of the present disclosure, the methods comprise contacting an immune cell of the present disclosure (including a T cell of the present disclosure) and a T-cell expansion composition. In some embodiments of the methods of the present disclosure, the step of introducing a transposon and/or transposase of the present disclosure into an immune cell of the present disclosure may further comprise contacting the immune cell with a T-cell expansion composition. In some embodiments, including those in which the introducing step of the method comprises an electroporation or nuclear transfection step, the electroporation or nuclear transfection step may be performed with immune cells of the present disclosure contacted with the T-cell expansion composition.

In some embodiments of the methods of the present disclosure, the T-cell expansion composition comprises phosphorus; one or more of caprylic acid, palmitic acid, linoleic acid and oleic acid; a sterol; and an alkane consisting essentially of, or consisting of.

In certain embodiments of the methods of producing a modified T cell of the present disclosure, the expansion supplement comprises one or more cytokines. The one or more cytokines may include any cytokine, including, but not limited to, lymphokines. Exemplary lymphokines include, but are not limited to, interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-15 (IL-15), interleukin-21 (IL-21), granulocyte macrophage colony stimulating factor (GM-CSF), and interferon- γ (INF γ). The one or more cytokines may include IL-2.

In some embodiments of the methods of the present disclosure, the T-cell expansion composition comprises human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, and an expansion supplement. In certain embodiments of this method, the T-cell expansion composition further comprises one or more of caprylic acid, niacinamide, 2, 4, 7, 9-tetramethyl-5-decyne-4, 7-diol (TMDD), diisopropyl adipate (DIPA), n-butylbenzenesulfonamide, 1, 2-benzenedicarboxylic acid, bis (2-methylpropyl) ester, palmitic acid, linoleic acid, oleic acid, stearic acid hydrazide, oleamide, sterol, and alkane. In certain embodiments of this method, the T-cell expansion composition further comprises one or more of caprylic acid, palmitic acid, linoleic acid, oleic acid, and sterol. In certain embodiments of this method, the T-cell expansion composition further comprises one or more of the following: caprylic acid at a concentration of 0.9mg/kg to 90mg/kg inclusive; palmitic acid at a concentration of 0.2mg/kg to 20mg/kg inclusive; linoleic acid at a concentration of 0.2mg/kg to 20mg/kg inclusive; oleic acid at a concentration of from 0.2mg/kg to 20mg/kg inclusive; and sterol at a concentration of from about 0.1mg/kg to about 10mg/kg inclusive. In certain embodiments of this method, the T-cell expansion composition further comprises one or more of caprylic acid at a concentration of about 9mg/kg, palmitic acid at a concentration of about 2mg/kg, linoleic acid at a concentration of about 2mg/kg, oleic acid at a concentration of about 2mg/kg, and sterol at a concentration of about 1 mg/kg. In certain embodiments of this method, the T-cell expansion composition further comprises one or more of the following: caprylic acid with concentration of 6.4-640 μmol/kg (inclusive); palmitic acid at a concentration of 0.7-70 μmol/kg inclusive; linoleic acid at a concentration of 0.75 μmol/kg to 75 μmol/kg inclusive; oleic acid at a concentration of from 0.75. mu. mol/kg to 75. mu. mol/kg inclusive; and sterol at a concentration of 0.25. mu. mol/kg to 25. mu. mol/kg inclusive. In certain embodiments of this method, the T-cell expansion composition further comprises one or more of caprylic acid at a concentration of about 64 μmol/kg, palmitic acid at a concentration of about 7 μmol/kg, linoleic acid at a concentration of about 7.5 μmol/kg, oleic acid at a concentration of about 7.5 μmol/kg, and sterol at a concentration of about 2.5 μmol/kg.

In certain embodiments, the T-cell expansion composition comprises one or more of human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, and expansion supplements to produce a plurality of expanded modified T-cells, wherein at least 2% of the plurality of modified T-cells express early memory T-cells, stem-like T-cells, stem memory T-cells (T-cells)_SCM) And/or central memory T cells (T)_CM) Of the cell surface marker(s). In certain embodiments, the T-cell expansion composition comprises or further comprises one or more of caprylic acid, niacinamide, 2, 4, 7, 9-tetramethyl-5-decyne-4, 7-diol (TMDD), diisopropyl adipate (DIPA), n-butylbenzene sulfonamide, 1, 2-benzenedicarboxylic acid, bis (2-methylpropyl) ester, palmitic acid, linoleic acid, oleic acid, stearic acid hydrazide, oleamide, sterol, and alkane. In certain embodiments, the T-cell expansion composition comprises one or more of caprylic acid, palmitic acid, linoleic acid, oleic acid, and a sterol (e.g., cholesterol). In certain embodiments, the T-cell expansion composition comprises one or more of the following: caprylic acid at a concentration of 0.9mg/kg to 90mg/kg inclusive; palmitic acid at a concentration of 0.2mg/kg to 20mg/kg inclusive; linoleic acid at a concentration of 0.2mg/kg to 20mg/kg inclusive; the concentration is 0.2mg/kg-20mg/k g (inclusive) oleic acid; and sterols at a concentration of about 0.1mg/kg to 10mg/kg inclusive (where mg/kg is parts per million). In certain embodiments, the T-cell expansion composition comprises one or more of caprylic acid at a concentration of about 9mg/kg, palmitic acid at a concentration of about 2mg/kg, linoleic acid at a concentration of about 2mg/kg, oleic acid at a concentration of about 2mg/kg, and sterol at a concentration of about 1mg/kg (where mg/kg is parts per million). In certain embodiments, the T-cell expansion composition comprises one or more of caprylic acid at a concentration of 9.19mg/kg, palmitic acid at a concentration of 1.86mg/kg, linoleic acid at a concentration of about 2.12mg/kg, oleic acid at a concentration of about 2.13mg/kg, and sterol at a concentration of about 1.01mg/kg (where mg/kg is parts per million). In certain embodiments, the T-cell expansion composition comprises caprylic acid at a concentration of 9.19mg/kg, palmitic acid at a concentration of 1.86mg/kg, linoleic acid at a concentration of 2.12mg/kg, oleic acid at a concentration of about 2.13mg/kg, and sterol at a concentration of 1.01mg/kg (where mg/kg is parts per million). In certain embodiments, the T-cell expansion composition comprises one or more of the following: caprylic acid with concentration of 6.4-640 μmol/kg (inclusive); palmitic acid at a concentration of 0.7-70 μmol/kg inclusive; linoleic acid at a concentration of 0.75 μmol/kg to 75 μmol/kg inclusive; oleic acid at a concentration of from 0.75. mu. mol/kg to 75. mu. mol/kg inclusive; and sterol at a concentration of 0.25. mu. mol/kg to 25. mu. mol/kg inclusive. In certain embodiments, the T-cell expansion composition comprises one or more of caprylic acid at a concentration of about 64 μmol/kg, palmitic acid at a concentration of about 7 μmol/kg, linoleic acid at a concentration of about 7.5 μmol/kg, oleic acid at a concentration of about 7.5 μmol/kg, and sterol at a concentration of about 2.5 μmol/kg. In certain embodiments, the T-cell expansion composition comprises one or more of caprylic acid at a concentration of about 63.75 μmol/kg, palmitic acid at a concentration of about 7.27 μmol/kg, linoleic acid at a concentration of about 7.57 μmol/kg, oleic acid at a concentration of about 7.56 μmol/kg, and sterol at a concentration of about 2.61 μmol/kg. In certain embodiments, the T-cell expansion composition comprises caprylic acid at a concentration of about 63.75. mu. mol/kg, palmitic acid at a concentration of about 7.27. mu. mol/kg, linoleic acid at a concentration of about 7.57. mu. mol/kg, oleic acid at a concentration of 7.56. mu. mol/kg, and oleic acid at a concentration of 2.61. mu. mol/kg Sterols.

As used herein, the term "fed T-cell expansion composition" or "T-cell expansion composition" may be used interchangeably with a culture medium comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, Iscove's MDM and expansion supplements at 37 ℃. Alternatively or additionally, the term "fed T-cell expansion composition" or "T-cell expansion composition" may be used interchangeably with a medium comprising one or more of the following elements: boron, sodium, magnesium, phosphorus, potassium, and calcium. In certain embodiments, the term "fed T-cell expansion composition" or "T-cell expansion composition" may be used interchangeably with a medium comprising one or more of the following elements present in respective average concentrations: 3.7mg/L boron, 3000mg/L sodium, 18mg/L magnesium, 29mg/L phosphorus, 15mg/L potassium and 4mg/L calcium.

As used herein, the term "fed T-cell expansion composition" or "T-cell expansion composition" is used interchangeably with a culture medium comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol and expansion supplements at 37 ℃. Alternatively or additionally, the term "fed T-cell expansion composition" or "T-cell expansion composition" may be used interchangeably with a medium comprising one or more of the following components: octanoic acid (CAS No. 124-07-2), nicotinamide (CAS No. 98-92-0), 2, 4, 7, 9-tetramethyl-5-decyne-4, 7-diol (TMDD) (CAS No. 126-86-3), diisopropyl adipate (DIPA) (CAS No. 6938-94-9), n-butylbenzenesulfonamide (CAS No. 3622-84-2), 1, 2-benzenedicarboxylic acid, bis (2-methylpropyl) ester (CAS No. 84-69-5), palmitic acid (CAS No. 57-10-3), linoleic acid (CAS No. 60-33-3), oleic acid (CAS No. 112-80-1), stearic acid hydrazide (CAS No. 4130-54-5), oleamide (CAS No. 3322-62-1), sterols (e.g., cholesterol) (CAS number 57-88-5) and alkanes (e.g., nonadecane) (CAS number 629-92-5). In certain embodiments, the term "fed T-cell expansion composition" or "T-cell expansion composition" may be used interchangeably with a medium comprising one or more of the following components: octanoic acid (CAS No. 124-07-2), nicotinamide (CAS No. 98-92-0), 2, 4, 7, 9-tetramethyl-5-decyne-4, 7-diol (TMDD) (CAS No. 126-86-3), diisopropyl adipate (DIPA) (CAS No. 6938-94-9), n-butylbenzenesulfonamide (CAS No. 3622-84-2), 1, 2-benzenedicarboxylic acid, bis (2-methylpropyl) ester (CAS No. 84-69-5), palmitic acid (CAS No. 57-10-3), linoleic acid (CAS No. 60-33-3), oleic acid (CAS No. 112-80-1), stearic acid hydrazide (CAS No. 4130-54-5), oleamide (CAS No. 3322-62-1), Sterols (e.g., cholesterol) (CAS number 57-88-5), alkanes (e.g., nonadecane) (CAS number 629-92-5), and phenol red (CAS number 143-74-8). In certain embodiments, the term "fed T-cell expansion composition" or "T-cell expansion composition" may be used interchangeably with a medium comprising one or more of the following components: octanoic acid (CAS No. 124-07-2), nicotinamide (CAS No. 98-92-0), 2, 4, 7, 9-tetramethyl-5-decyne-4, 7-diol (TMDD) (CAS No. 126-86-3), diisopropyl adipate (DIPA) (CAS No. 6938-94-9), n-butylbenzenesulfonamide (CAS No. 3622-84-2), 1, 2-benzenedicarboxylic acid, bis (2-methylpropyl) ester (CAS No. 84-69-5), palmitic acid (CAS No. 57-10-3), linoleic acid (CAS No. 60-33-3), oleic acid (CAS No. 112-80-1), stearic acid hydrazide (CAS No. 4130-54-5), oleamide (CAS No. 3322-62-1), Phenol red (CAS number 143-74-8) and lanolin alcohol.

In certain embodiments, the term "fed T-cell expansion composition" or "T-cell expansion composition" may be used interchangeably with a culture medium comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, and expansion supplements at 37 ℃. Alternatively or additionally, the term "fed T-cell expansion composition" or "T-cell expansion composition" may be used interchangeably with a medium comprising one or more of the following ions: sodium, ammonium, potassium, magnesium, calcium, chloride (chloride), sulfate, and phosphate.

As used herein, the term "fed T-cell expansion composition" or "T-cell expansion composition" is used interchangeably with a culture medium comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol and expansion supplements at 37 ℃. Alternatively or additionally, the term "fed T-cell expansion composition" or "T-cell expansion composition" may be used interchangeably with a medium comprising one or more of the following free amino acids: histidine, asparagine, serine, glutamic acid, arginine, glycine, aspartic acid, glutamic acid, threonine, alanine, proline, cysteine, lysine, tyrosine, methionine, valine, isoleucine, leucine, phenylalanine, and tryptophan. In certain embodiments, the term "fed T-cell expansion composition" or "T-cell expansion composition" may be used interchangeably with a medium comprising the respective average molar percentages of one or more of the following free amino acids: histidine (about 1%), asparagine (about 0.5%), serine (about 1.5%), glutamine (about 67%), arginine (about 1.5%), glycine (about 1.5%), aspartic acid (about 1%), glutamic acid (about 2%), threonine (about 2%), alanine (about 1%), proline (about 1.5%), cysteine (about 1.5%), lysine (about 3%), tyrosine (about 1.5%), methionine (about 1%), valine (about 3.5%), isoleucine (about 3%), leucine (about 3.5%), phenylalanine (about 1.5%) and tryptophan (about 0.5%). In certain embodiments, the term "fed T-cell expansion composition" or "T-cell expansion composition" may be used interchangeably with a medium comprising the respective average molar percentages of one or more of the following free amino acids: histidine (about.78%), asparagine (about 0.4%), serine (about 1.6%), glutamine (about 67.01%), arginine (about 1.67%), glycine (about 1.72%), aspartic acid (about 1.00%), glutamic acid (about 1.93%), threonine (about 2.38%), alanine (about 1.11%), proline (about 1.49%), cysteine (about 1.65%), lysine (about 2.84%), tyrosine (about 1.62%), methionine (about 0.85%), valine (about 3.45%), isoleucine (about 3.14%), leucine (about 3.3%), phenylalanine (about 1.64%), and tryptophan (about 0.37%).

As used herein, the term "fed T-cell expansion composition" or "T-cell expansion composition" may be used interchangeably with a culture medium comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, Iscove's MDM and expansion supplements at 37 ℃. Alternatively or additionally, the term "fed T-cell expansion composition" or "T-cell expansion composition" may be used interchangeably with a medium comprising one or more of phosphorus, caprylic fatty acid, palmitic fatty acid, linoleic fatty acid and oleic acid. In certain embodiments, the culture Medium comprises 10 times the amount of phosphorus as may be found, for example, in Iscove's Modified Dulbecco's Medium (IMDM); available as Cat No. 12440053 in ThermoFisher Scientific).

In certain embodiments, the term "fed T-cell expansion composition" or "T-cell expansion composition" may be used interchangeably with a medium comprising one or more of caprylic acid, palmitic acid, linoleic acid, oleic acid, and sterols (e.g., cholesterol). In certain embodiments, the term "fed T-cell expansion composition" or "T-cell expansion composition" may be used interchangeably with a medium comprising one or more of: caprylic acid at a concentration of 0.9mg/kg to 90mg/kg inclusive; palmitic acid at a concentration of 0.2mg/kg to 20mg/kg inclusive; linoleic acid at a concentration of 0.2mg/kg to 20mg/kg inclusive; oleic acid at a concentration of from 0.2mg/kg to 20mg/kg inclusive; and sterols at a concentration of about 0.1mg/kg to 10mg/kg inclusive (where mg/kg is parts per million). In certain embodiments, the term "fed T-cell expansion composition" or "T-cell expansion composition" may be used interchangeably with a medium comprising one or more of caprylic acid at a concentration of about 9mg/kg, palmitic acid at a concentration of about 2mg/kg, linoleic acid at a concentration of about 2mg/kg, oleic acid at a concentration of about 2mg/kg, and sterol at a concentration of about 1mg/kg (where mg/kg is parts per million). In certain embodiments, the term "fed T-cell expansion composition" or "T-cell expansion composition" may be used interchangeably with a medium comprising one or more of caprylic acid at a concentration of 9.19mg/kg, palmitic acid at a concentration of 1.86mg/kg, linoleic acid at a concentration of about 2.12mg/kg, oleic acid at a concentration of about 2.13mg/kg, and sterol at a concentration of about 1.01mg/kg (where mg/kg is one part per million). In certain embodiments, the term "fed T-cell expansion composition" or "T-cell expansion composition" may be used interchangeably with a medium comprising one or more of caprylic acid at a concentration of 9.19mg/kg, palmitic acid at a concentration of 1.86mg/kg, linoleic acid at a concentration of 2.12mg/kg, oleic acid at a concentration of about 2.13mg/kg, and sterol at a concentration of 1.01mg/kg (where mg/kg is one part per million). In certain embodiments, the term "fed T-cell expansion composition" or "T-cell expansion composition" may be used interchangeably with a medium comprising one or more of: caprylic acid with concentration of 6.4-640 μmol/kg (inclusive); palmitic acid at a concentration of 0.7-70 μmol/kg inclusive; linoleic acid at a concentration of 0.75 μmol/kg to 75 μmol/kg inclusive; oleic acid at a concentration of from 0.75. mu. mol/kg to 75. mu. mol/kg inclusive; and sterol at a concentration of 0.25. mu. mol/kg to 25. mu. mol/kg inclusive. In certain embodiments, the term "fed T-cell expansion composition" or "T-cell expansion composition" may be used interchangeably with a medium comprising one or more of caprylic acid at a concentration of about 64 μmol/kg, palmitic acid at a concentration of about 7 μmol/kg, linoleic acid at a concentration of about 7.5 μmol/kg, oleic acid at a concentration of about 7.5 μmol/kg, and sterol at a concentration of about 2.5 μmol/kg.

In certain embodiments, the term "fed T-cell expansion composition" or "T-cell expansion composition" may be used interchangeably with a medium comprising one or more of caprylic acid at a concentration of about 63.75 μmol/kg, palmitic acid at a concentration of about 7.27 μmol/kg, linoleic acid at a concentration of about 7.57 μmol/kg, oleic acid at a concentration of about 7.56 μmol/kg, and sterol at a concentration of about 2.61 μmol/kg. In certain embodiments, the term "fed T-cell expansion composition" or "T-cell expansion composition" may be used interchangeably with a medium comprising one or more of caprylic acid at a concentration of about 63.75 μmol/kg, palmitic acid at a concentration of about 7.27 μmol/kg, linoleic acid at a concentration of about 7.57 μmol/kg, oleic acid at a concentration of 7.56 μmol/kg, and sterol at a concentration of 2.61 μmol/kg.

Generation of modified T cells (e.g., Stem cell-like T cells, T) in the disclosure_SCMAnd/or T_CM) In certain embodiments of the methods of (1), the method comprises contacting the modified T cell with an inhibitor of the P13K-Akt-mTOR pathway. Modified T-cells of the disclosure, including modified stem cell-like T-cells, T-cells of the disclosure_SCMAnd/or T_CMIncubation, culturing, growth, storage or otherwise combining in any step of the method of the procedure may be performed with a growth medium comprising one or more inhibitors of a component of the PI3K pathway. Exemplary inhibitors of components of the PI3K pathway include, but are not limited to, inhibitors of GSK3 β, such as TWS119 (also known as GSK 3B inhibitor XII; having formula C) ₁₈H₁₄N₄O₂CAS number of 601514-19-6). Exemplary inhibitors of a component of the PI3K pathway include, but are not limited to, bb007 (BLUEBIRDBIO)^TM). Additional exemplary inhibitors of components of the PI3K pathway include, but are not limited to, allosteric Akt inhibitor VIII (also known as Akti-1/2 with Compound number 10196499), ATP competitive inhibitors (Orthosteric (Orthosteric) inhibitors that target the ATP-binding pocket of protein kinase B (Akt)), isoquinoline-5-sulfonamide (H-8, H-89, and NL-71-101), cycloheximide (Azepane) derivatives (a series of structures derived from (-) -balanol), aminofurazan (Aminofurazans) (GSK690693), heterocyclic rings (7-azaindole, 6-phenylpurine derivatives, pyrrolo [2, 3-d ] pyrrole]Pyrimidine derivatives, CCT128930, 3-aminopyrrolidine, anilinotriazole derivatives, spiroindoline (spiroindoline) derivatives, AZD5363, epratacin (iptastartib) (GDC-0068, RG7440), A-674563 and A-443654), phenylpyrazole derivatives(AT7867 and AT13148), thiophene carboxamide derivatives (Aflurorebertib (GSK2110183), 2-pyrimidinyl-5-amidothiophene derivatives (DC120), uprosertib (GSK2141795)), allosteric inhibitors (superior to orthosteric inhibitors, providing higher specificity, reduced side effects and less toxicity), 2, 3-diphenylquinoxaline analogs (2, 3-diphenylquinoxaline derivatives, triazolo [3, 4-f ] s ][1，6]Naphthyridin-3 (2H) -one derivatives (MK-2206)), alkylphospholipid (Edelfosine) (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine, ET-18-OCH₃) Ilofovir (ilmofosine) (BM 41.440), miltefosine (miltefosine) (hexadecylphosphocholine, HePC), piperafosine (perifosine) (D-21266), erucyl phosphocholine (erucyl phosphocholine) (ErPC), erufosine (ErPC 3), erucyl phosphocholine (erucyl phosphocholine)), indole-3-methanol analogs (indole-3-methanol, 3-chloroacetyl indole, diindolylmethane, 6-methoxy-5, 7-dihydroindolo [2, 3-b ] indolo [2, 3-b ] and their pharmaceutically acceptable salts]Diethyl carbazole-2, 10-dicarboxylate (SR13668), OSU-A9), sulfonamide derivatives (PH-316 and PHT-427), thiourea derivatives (PIT-1, PIT-2, DM-PIT-1, N- [ (1-methyl-1H-pyrazol-4-yl) carbonyl)]-N' - (3-bromophenyl)]) Thiourea), purine derivatives (Triciribine (TCN, NSC 154020), active analog of Triciribine monophosphate (TCN-P), 4-amino-pyrido [2, 3-d]Pyrimidine derivatives API-1, 3-phenyl-3H-imidazo [4, 5-b]Pyridine derivatives, ARQ 092), BAY 1125976, 3-methyl-xanthine, quinoline-4-carboxamide and 2- [4- (cyclohex-1, 3-dien-1-yl) -1H-pyrazol-3-yl ]Phenol, 3-oxo-tirucallonic acid, 3 α -and 3 β -acetoxy-tirucallonic acid, acetoxy-tirucallonic acid and irreversible inhibitors (antibiotics, lactoquinomycin, frenolicin B, kara-mycomycin, medemycin, Boc-Phe-vinyl ketone, 4-hydroxynonenal (4-HNE), 1, 6-naphthyridinone derivatives and imidazo-1, 2-pyridine derivatives).

Generation of modified T cells (e.g., Stem cell-like T cells, T) in the disclosure_SCMAnd/or T_CM) In certain embodiments of the methods of (1), the method comprises contacting the modified T cell with an inhibitor of T cell effector differentiation (T cell effector differentiation). Exemplary inhibition of T cell effector differentiationAgents include, but are not limited to, BET inhibitors (e.g., JQ1, thienotriazolodiazepine)(thienotriazolodiazepine)) and/or inhibitors of the BET protein family (e.g. BRD2, BRD3, BRD4 and BRDT).

Generation of modified T cells (e.g., Stem cell-like T cells, T) in the disclosure_SCMAnd/or T_CM) In certain embodiments of the methods of (1), the method comprises contacting the modified T cell with an agent that reduces nuclear cytoplasmic acetyl-coa. Exemplary agents that reduce nuclear cytoplasmic acetyl-CoA include, but are not limited to, 2-hydroxycitrate (2-HC) and agents that increase expression of Acss 1.

In certain embodiments of the methods of the present disclosure of producing a modified T cell (e.g., a stem cell-like T cell, TSCM, and/or TCM), the method comprises contacting the modified T cell with a composition comprising a Histone Deacetylase (HDAC) inhibitor. In some embodiments, the composition comprising the HDAC inhibitor comprises or consists of valproic acid, sodium phenylbutyrate (NaPB), or a combination thereof. In some embodiments, the composition comprising an HDAC inhibitor comprises or consists of valproic acid. In some embodiments, the composition comprising the HDAC inhibitor comprises or consists of sodium phenylbutyrate (NaPB).

Generation of modified T cells (e.g., Stem cell-like T cells, T) in the disclosure_SCMAnd/or T_CM) In certain embodiments of the methods of (a), the activation supplement may comprise one or more cytokines. The one or more cytokines may include any cytokine, including, but not limited to, lymphokines. Exemplary lymphokines include, but are not limited to, interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-15 (IL-15), interleukin-21 (IL-21), granulocyte macrophage colony stimulating factor (GM-CSF), and interferon- γ (INF γ). The one or more cytokines may include IL-2.

Within this disclosureGeneration of modified T cells (e.g.Stem-like T cells, T)_SCMAnd/or T_CM) In certain embodiments of the methods of (a), the activation supplement may comprise one or more activator protein complexes. Exemplary and non-limiting activin complexes may include monomeric, dimeric, trimeric or tetrameric antibody complexes that bind to one or more of CD3, CD28 and CD 2. In some embodiments, the activation supplement comprises or consists of an activator protein complex comprising a human, humanized, or recombinant antibody or a chimeric antibody. In some embodiments, the activation supplement comprises or consists of an activator protein complex that binds CD3 and CD 28. In some embodiments, the activating supplement comprises or consists of an activator protein complex that binds to CD3, CD28, and CD2,

natural Killer (NK) cells

In certain embodiments, the modified immune or immune precursor cell of the present disclosure is a Natural Killer (NK) cell. In certain embodiments, the NK cell is a cytotoxic lymphocyte differentiated from a lymphoid progenitor cell.

The modified NK cells of the present disclosure may be derived from modified Hematopoietic Stem and Progenitor Cells (HSPCs) or modified HSCs.

In certain embodiments, the non-activated NK cells are derived from leukapheresis (CD 14/CD19/CD56+ cells) that excludes CD 3.

In certain embodiments, NK cells are electroporated using a Lonza 4D nuclear transfection instrument (nucleofector) or BTX ECM 830(500V, 700 usec pulse duration, 0.2mm electrode gap, one pulse). All Lonza 4D nuclear transfectator procedures are contemplated to be within the scope of the methods of the present disclosure.

In certain embodiments, 5x10E6 cells were electroporated in a cup in 100 μ L P3 buffer per electroporation. However, for commercial manufacturing processes, this proportion of cells by volume is scalable.

In certain embodiments, the NK cells are stimulated by co-culturing with additional cell lines. In certain embodiments, the additional cell lines comprise artificial antigen presenting cells (aAPCs). In certain embodiments, the stimulating occurs on day 1, 2, 3, 4, 5, 6, or 7 after electroporation. In certain embodiments, the stimulation occurs on day 2 after electroporation.

In certain embodiments, the NK cells express CD 56.

B cell

In certain embodiments, the modified immune or immune precursor cell of the present disclosure is a B cell. B cells are a type of lymphocyte that expresses B cell receptors on the cell surface. B cell receptors bind to specific antigens.

The modified B cells of the present disclosure may be derived from modified Hematopoietic Stem and Progenitor Cells (HSPCs) or modified HSCs.

In certain embodiments, HSPCs are modified using the methods of the present disclosure and then primed for B cell differentiation in the presence of human IL-3, Flt3L, TPO, SCF, and G-CSF for at least 3 days, at least 4 days, at least 5 days, at least 6 days, or at least 7 days. In certain embodiments, HSPCs are modified using the methods of the present disclosure and then primed for B cell differentiation in the presence of human IL-3, Flt3L, TPO, SCF, and G-CSF for up to 5 days.

In certain embodiments, after priming, the modified HSPC cells are transferred to a feeder cell layer and are applied every two weeks, and transferred to a fresh feeder layer once a week. In certain embodiments, the feeder cells are MS-5 feeder cells.

In certain embodiments, the modified HSPC cells are cultured with MS-5 feeder cells for at least 7, 14, 21, 28, 30, 33, 35, 42, or 48 days. In certain embodiments, the modified HSPC cells are cultured with MS-5 feeder cells for 33 days.

Swivel mount system

Exemplary transposon/transposase systems of the present disclosure include, but are not limited to, piggyBac transposons and transposases, Sleeping Beauty transposons and transposases, helraisiser transposons and transposases, Tol2 transposons and transposases, and Tcbuster transposons and transposases.

piggyBac transposase recognizes transposon-specific Inverted Terminal Repeats (ITRs) at the ends of transposons and moves the content between ITRs into the TTAA chromosomal locus. For genes of interest that can be included between ITRs, the piggyBac transposon system has no payload restrictions. In certain embodiments, and, in particular, those embodiments in which the transposon is a piggyBac transposon, the transposase is piggyBac^TMOr Super piggyBac^TM(SPB) transposase. In certain embodiments, and, in particular, wherein the transposase is Super piggyBac^TM(SPB) transposase in those embodiments, the sequence encoding the transposase is an mRNA sequence.

In certain embodiments, the transposase is piggyBac^TM(PB) a transposase comprising the amino acid sequence set forth in SEQ ID NO: 14487 an amino acid sequence having or consisting of an amino acid substitution at two or more of positions 30, 165, 282, or 538 of the sequence. In certain embodiments, the transposase is piggyBac^TM(PB) a transposase comprising the amino acid sequence set forth in SEQ ID NO: 14487 with amino acid substitutions at three or more of positions 30, 165, 282, or 538 of the sequenceOr consists of the amino acid sequence of (a). In certain embodiments, the transposase is piggyBac^TM(PB) a transposase comprising the amino acid sequence set forth in SEQ ID NO: 14487 an amino acid sequence having or consisting of an amino acid substitution at each of the following positions 30, 165, 282, and 538. In certain embodiments, SEQ ID NO: 14487 is a valine (V) for isoleucine (I) amino acid substitution. In certain embodiments, SEQ ID NO: 14487 is a serine (S) to glycine (G) substitution. In certain embodiments, SEQ ID NO: 14487 is a valine (V) to methionine (M) substitution. In certain embodiments, SEQ ID NO: 14487 is a substitution of lysine (K) for asparagine (N).

In certain embodiments of the methods of the present disclosure, the transposase is Super piggyBac^TM(SPB) transposase. In certain embodiments, the Super piggyBac of the present disclosure^TM(SPB) the transposase can comprise SEQ ID NO: 14487, wherein the amino acid substitution at position 30 is a substitution of valine (V) for isoleucine (I), the amino acid substitution at position 165 is a substitution of serine (S) for glycine (G), the amino acid substitution at position 282 is a substitution of valine (V) for methionine (M), and the amino acid substitution at position 538 is a substitution of lysine (K) for asparagine (N). In certain embodiments, Super piggyBac^TMThe (SPB) transposase can comprise or consist of an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99%, or any percentage therebetween identical to:

in certain embodiments of the methods of the present disclosure, including those in which the transposase comprises the above-described mutations at positions 30, 165, 282, and/or 538, piggyBac^TMOr Super piggyBac^TMTransposaseMay further comprise the amino acid sequence set forth in SEQ ID NO: 14487 or SEQ ID NO: 14484 at one or more of positions 3, 46, 82, 103, 119, 125, 177, 180, 185, 187, 200, 207, 209, 226, 235, 240, 241, 243, 258, 296, 298, 311, 315, 319, 327, 328, 340, 421, 436, 456, 470, 486, 503, 552, 570, and 591. In certain embodiments, including those wherein the transposase comprises the above-described mutations at positions 30, 165, 282, and/or 538, piggyBac ^TMOr Super piggyBac^TMThe transposase can further comprise an amino acid substitution at one or more of positions 46, 119, 125, 177, 180, 185, 187, 200, 207, 209, 226, 235, 240, 241, 243, 296, 298, 311, 315, 319, 327, 328, 340, 421, 436, 456, 470, 485, 503, 552, and 570. In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of asparagine (N) for serine (S). In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a serine (S) to alanine (A) substitution. In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a threonine (T) to alanine (A) substitution. In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a tryptophan (W) to isoleucine (I) substitution. In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 the amino acid substitution at position 103 is a proline (P) to serine (S) substitution. In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a proline (P) to arginine (R) substitution. In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a cysteine (C) substituted with alanine (A). In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a leucine (L) to cysteine (C) substitution. In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a lysine (K) to tyrosine (Y) substitution. In some instances In embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 the amino acid substitution at position 177 is a histidine (H) to tyrosine (Y) substitution. In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a leucine (L) to phenylalanine (F) substitution. In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of isoleucine (I) for phenylalanine (F). In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a valine (V) to phenylalanine (F) substitution. In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a leucine (L) to methionine (M) substitution. In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a glycine (G) to alanine (A) substitution. In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a tryptophan (W) to phenylalanine (F) substitution. In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a valine (V) substitution by proline (P). In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a valine (V) substitution by phenylalanine (F). In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of phenylalanine (F) for methionine (M). In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 the amino acid substitution at position 235 is a substitution of arginine (R) for leucine (L). In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a lysine (K) to valine (V) substitution. In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a leucine (L) to phenylalanine (F) substitution. In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a lysine (K) to proline (P) substitution. In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 ammonia at position 258 The amino acid substitution is a substitution of asparagine (N) with serine (S). In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 the amino acid substitution at position 296 is a tryptophan (W) to leucine (L) substitution. In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a tyrosine (Y) to leucine (L) substitution. In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a phenylalanine (F) to leucine (L) substitution. In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a leucine (L) to methionine (M) substitution. In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of alanine (A) for methionine (M). In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a valine (V) to methionine (M) substitution. In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of isoleucine (I) for proline (P). In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a valine to proline (P) substitution. In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a lysine (K) to arginine (R) substitution. In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of threonine (T) with glycine (G). In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of arginine (R) for tyrosine (Y). In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a valine (V) to tyrosine (Y) substitution. In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 the amino acid substitution at position 340 is a substitution of glycine (G) for cysteine (C). In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a leucine (L) to cysteine (C) substitution. In some embodiments of the present invention, the substrate is, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a histidine (H) for aspartic acid (D). In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a valine (V) substitution with isoleucine (I). In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484, the amino acid substitution at position 456 is a tyrosine (Y) to methionine (M) substitution. In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of phenylalanine (F) for leucine (L). In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a lysine (K) to serine (S) substitution. In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a leucine (L) to methionine (M) substitution. In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of isoleucine (I) for methionine (M). In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a lysine (K) to valine (V) substitution. In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a threonine (T) to alanine (A) substitution. In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 the amino acid substitution at position 591 is a proline (P) for glutamine (Q) substitution. In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 the amino acid substitution at position 591 is a substitution of arginine (R) for glutamine (Q).

In certain embodiments of the methods of the present disclosure, including those in which the transposase comprises the above-described mutations at positions 30, 165, 282, and/or 538, piggyBac^TMThe transposase may comprise or Super piggyBac^TMThe transposase can further comprise a sequence set forth in SEQ ID NO: 14487 or SEQ ID NO: 14484 at one or more of positions 103, 194, 372, 375, 450, 509 and 570. In certain embodiments of the methods of the present disclosure, including wherein the transposase is contained inThose embodiments of the above mutations at positions 30, 165, 282 and/or 538, piggyBac^TMThe transposase may comprise or Super piggyBac^TMThe transposase can further comprise a sequence set forth in SEQ ID NO: 14487 or SEQ ID NO: 14484 at two, three, four, five, six or more of positions 103, 194, 372, 375, 450, 509 and 570 of the sequence. In certain embodiments, including those wherein the transposase comprises the above-described mutations at positions 30, 165, 282, and/or 538, piggyBac^TMThe transposase may comprise or Super piggyBac^TMThe transposase can further comprise a sequence set forth in SEQ ID NO: 14487 or SEQ ID NO: 14484 at positions 103, 194, 372, 375, 450, 509, and 570. In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 the amino acid substitution at position 103 is a proline (P) to serine (S) substitution. In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a valine (V) to methionine (M) substitution. In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of alanine (A) for arginine (R). In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 the amino acid substitution at position 375 is a substitution of alanine (A) for lysine (K). In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of asparagine (N) for aspartic acid (D). In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 the amino acid substitution at position 509 is a glycine (G) to serine (S) substitution. In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a serine (S) to asparagine (N) substitution. In certain embodiments, piggyBac ^TMThe transposase can be contained in SEQ ID NO: 14487 substitution of methionine (M) by valine (V) at position 194. In certain embodiments, included are wherein piggyBac^TMThe transposase can be contained in SEQ ID NO: 14487 those embodiments of valine (V) to methionine (M) substitution at position 194, piggyBac^TMThe transposase may beTo further comprise the amino acid sequence set forth in SEQ ID NO: 14487 or SEQ ID NO: 14484 at positions 372, 375 and 450. In certain embodiments, piggyBac^TMThe transposase can be contained in SEQ ID NO: 14487 substitution of valine (V) for methionine (M) at position 194, amino acid sequence set forth in SEQ ID NO: 14487 substitution of alanine (a) for arginine (R) at position 372 and the amino acid sequence set forth in SEQ ID NO: 14487 substitution of lysine (K) with alanine (A) at position 375. In certain embodiments, piggyBac^TMThe transposase can be contained in SEQ ID NO: 14487 substitution of valine (V) for methionine (M) at position 194, amino acid sequence set forth in SEQ ID NO: 14487 substitution of alanine (a) for arginine (R) at position 372, amino acid sequence set forth in SEQ ID NO: 14487 substitution of alanine (a) for lysine (K) at position 375 and SEQ ID NO: 14487 substitution of asparagine (N) for aspartic acid (D) at position 450.

The Sleeping Beauty transposon transposes into the target genome by a Sleeping Beauty transposase that recognizes ITRs and moves the contents between ITRs into the TA chromosomal locus. In various embodiments, SB transposon mediated gene transfer or gene transfer using any of a number of similar transposons can be used in the compositions and methods of the present disclosure.

In certain embodiments, and, in particular, those embodiments in which the transposon is a Sleeping Beauty transposon, the transposase is a Sleeping Beauty transposase or a high activity Sleeping Beauty transposase (SB 100X).

In certain embodiments of the methods of the present disclosure, the Sleeping Beauty transposase comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99%, or any percentage therebetween identical to:

in certain embodiments of the methods of the present disclosure, the high activity Sleeping Beauty (SB100X) transposase comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99%, or any percentage therebetween identical to the following sequence:

the Helraisier transposon was transposed by the Helitron transposase. Helitron transposase transferred the Helraisier transposon, an ancient element from the bat genome that was active about 3000 to 3600 ten thousand years ago. Exemplary helraisier transposons of the present disclosure include Helibat1 comprising a nucleic acid sequence comprising:

Unlike other transposases, the Helitron transposase does not contain an RNase-H like catalytic domain, but rather contains a ReHel motif consisting of a replication initiator domain (Rep) and a DNA helicase domain. The Rep domain is a nuclease domain of the HUH nuclease superfamily.

An exemplary Helitron transposase of the present disclosure comprises an amino acid sequence comprising:

in the Helitron transposition, the hairpin near the 3' end of the transposon acts as a terminator. However, this hairpin can be bypassed by the transposase, resulting in transduction of the flanking sequences. In addition, the helraisier transposition generates a covalently closed cyclic intermediate. In addition, the Helitron transposition may lack target site duplication. In the Helraiser sequence, the transposase is flanked by 5 'and 3' terminal sequences called LTS and RTS. These sequences terminate in a conserved 5 '-TC/CTAG-3' motif. A19 bp palindromic sequence with the potential to form a hairpin termination structure is located 11 nucleotides upstream of RTS and consists of sequence GTGCACGAATTTCGTGCACCGGGCCACTAG (SEQ ID NO: 14500).

The Tol2 transposon may be isolated or derived from the genome of medaka and may be similar to a transposon of the hAT family. An exemplary Tol2 transposon of the present disclosure is encoded by a sequence comprising about 4.7kb and contains a gene encoding Tol2 transposase, which contains four exons. An exemplary Tol2 transposase of the present disclosure comprises a sequence of amino acids comprising the following sequence:

An exemplary Tol2 transposon of the present disclosure, including inverted repeats, a terminator sequence and Tol2 transposase, is encoded by a nucleic acid sequence comprising:

exemplary transposon/transposase systems of the present disclosure include, but are not limited to piggyBac and piggyBac-like transposons and transposases.

piggyBac and piggyBac-like transposases recognize transposon-specific Inverted Terminal Repeats (ITRs) at the ends of transposons and move the content between ITRs into TTAA or TTAT chromosomal loci. For genes of interest that can be included between ITRs, the piggyBac or piggyBac-like transposon system has no payload restriction.

In certain embodiments, and, in particular, those embodiments in which the transposon is a piggyBac transposon, the transposase is piggyBac^TM、Super piggyBac^TM(SPB) transposase. In certain embodiments, and, in particular, wherein the transposase is piggyBac^TM、Super piggyBac^TM(SPB) in those embodiments, the transposase-encoding sequence is an mRNA sequence.

In certain embodiments of the methods of the present disclosure, the transposase is piggyBac or piggyBac-like transposase.

In certain embodiments of the methods of the present disclosure, the transposase is piggyBac or piggyBac-like transposase. piggyBac (pb) or piggyBac-like transposases can comprise or consist of an amino acid sequence that is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage therebetween identical to:

In certain embodiments of the methods of the present disclosure, the transposase is a piggyBac or piggyBac-like transposase comprising or consisting of an amino acid sequence having an amino acid substitution at one or more of positions 30, 165, 282, or 538 of:

in certain embodiments, the transposase is a piggyBac or piggyBac-like transposase comprising the amino acid sequence set forth in SEQ ID NO: 14487 an amino acid sequence having or consisting of an amino acid substitution at two or more of positions 30, 165, 282, or 538 of the sequence. In certain embodiments, the transposase is a piggyBac or piggyBac-like transposase comprising the amino acid sequence set forth in SEQ ID NO: 14487 an amino acid sequence having or consisting of an amino acid substitution at three or more of positions 30, 165, 282, or 538 of the sequence. In certain embodiments, the transposase is a piggyBac or piggyBac-like transposase comprising the amino acid sequence set forth in SEQ ID NO: 14487 an amino acid sequence having or consisting of an amino acid substitution at each of the following positions 30, 165, 282, and 538. In certain embodiments, SEQ ID NO: 14487 is a valine (V) for isoleucine (I) amino acid substitution. In certain embodiments, SEQ ID NO: 14487 is a serine (S) to glycine (G) substitution. In certain embodiments, SEQ ID NO: 14487 is a valine (V) to methionine (M) substitution. In certain embodiments, SEQ ID NO: 14487 is a substitution of lysine (K) for asparagine (N).

In certain embodiments of the methods of the present disclosure, the transposase is Super piggyBac^TM(SPB) or piggyBac-like transposase. In certain embodiments, the Super piggyBac of the present disclosure^TMThe (SPB) or piggyBac-like transposase can comprise SEQ ID NO: 14487, wherein the amino acid substitution at position 30 is a substitution of valine (V) for isoleucine (I), the amino acid substitution at position 165 is a substitution of serine (S) for glycine (G), the amino acid substitution at position 282 is a substitution of valine (V) for methionine (M), and the amino acid substitution at position 538 is a substitution of lysine (K) for asparagine (N). In certain embodiments, Super piggyBac^TMThe (SPB) or piggyBac-like transposase can comprise or consist of an amino acid sequence that is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage therebetween identical to:

in certain embodiments of the methods of the present disclosure, including those in which the transposase comprises the above-described mutations at positions 30, 165, 282, and/or 538, piggyBac ^TM、Super piggyBac^TMOr the piggyBac-like transposase can further comprise a mutation in SEQ ID NO: 14487 or SEQ ID NO: 14484 at one or more of positions 3, 46, 82, 103, 119, 125, 177, 180, 185, 187, 200, 207, 209, 226, 235, 240, 241, 243, 258, 296, 298, 311, 315, 319, 327, 328, 340, 421, 436, 456, 470, 486, 503, 552, 570, and 591. In certain embodiments, including those wherein the transposase comprises the above-described mutations at positions 30, 165, 282, and/or 538, piggyBac^TM、Super piggyBac^TMOr the piggyBac-like transposase can further comprise an amino acid substitution at one or more of positions 46, 119, 125, 177, 180, 185, 187, 200, 207, 209, 226, 235, 240, 241, 243, 296, 298, 311, 315, 319, 327, 328, 340, 421, 436, 456, 470, 485, 503, 552, and 570. In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of asparagine (N) for serine (S). In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a serine (S) to alanine (A) substitution. In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a threonine (T) to alanine (A) substitution. In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a tryptophan (W) to isoleucine (I) substitution. In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 the amino acid substitution at position 103 is a proline (P) to serine (S) substitution. In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a proline (P) to arginine (R) substitution. In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a cysteine (C) substituted with alanine (A). In some embodiments In the table, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a leucine (L) to cysteine (C) substitution. In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a lysine (K) to tyrosine (Y) substitution. In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 the amino acid substitution at position 177 is a histidine (H) to tyrosine (Y) substitution. In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a leucine (L) to phenylalanine (F) substitution. In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of isoleucine (I) for phenylalanine (F). In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a valine (V) to phenylalanine (F) substitution. In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a leucine (L) to methionine (M) substitution. In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a glycine (G) to alanine (A) substitution. In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a tryptophan (W) to phenylalanine (F) substitution. In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a valine (V) substitution by proline (P). In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a valine (V) substitution by phenylalanine (F). In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of phenylalanine (F) for methionine (M). In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 the amino acid substitution at position 235 is a substitution of arginine (R) for leucine (L). In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a lysine (K) to valine (V) substitution. In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 amino acid at position 241 The substitution is a substitution of leucine (L) for phenylalanine (F). In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a lysine (K) to proline (P) substitution. In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a serine (S) to asparagine (N) substitution. In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 the amino acid substitution at position 296 is a tryptophan (W) to leucine (L) substitution. In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a tyrosine (Y) to leucine (L) substitution. In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a phenylalanine (F) to leucine (L) substitution. In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a leucine (L) to methionine (M) substitution. In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of alanine (A) for methionine (M). In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a valine (V) to methionine (M) substitution. In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of isoleucine (I) for proline (P). In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a valine to proline (P) substitution. In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a lysine (K) to arginine (R) substitution. In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of threonine (T) with glycine (G). In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of arginine (R) for tyrosine (Y). In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a valine (V) to tyrosine (Y) substitution. In certain embodiments, SEQ ID NO : 14487 or SEQ ID NO: 14484 the amino acid substitution at position 340 is a substitution of glycine (G) for cysteine (C). In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a leucine (L) to cysteine (C) substitution. In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a histidine (H) for aspartic acid (D). In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a valine (V) substitution with isoleucine (I). In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484, the amino acid substitution at position 456 is a tyrosine (Y) to methionine (M) substitution. In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of phenylalanine (F) for leucine (L). In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a lysine (K) to serine (S) substitution. In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a leucine (L) to methionine (M) substitution. In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of isoleucine (I) for methionine (M). In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a lysine (K) to valine (V) substitution. In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a threonine (T) to alanine (A) substitution. In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 the amino acid substitution at position 591 is a proline (P) for glutamine (Q) substitution. In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 the amino acid substitution at position 591 is a substitution of arginine (R) for glutamine (Q).

In certain embodiments of the methods of the present disclosure, including those in which the transposase comprises the above-described mutations at positions 30, 165, 282, and/or 538, piggyBac^TMOr piggyBac-like transposase or may beInclusion or Super piggyBac^TMThe transposase can further comprise a sequence set forth in SEQ ID NO: 14487 or SEQ ID NO: 14484 at one or more of positions 103, 194, 372, 375, 450, 509 and 570. In certain embodiments of the methods of the present disclosure, including those in which the transposase comprises the above-described mutations at positions 30, 165, 282, and/or 538, piggyBac^TMOr the piggyBac-like transposase may comprise or be Super piggyBac^TMThe transposase can further comprise a sequence set forth in SEQ ID NO: 14487 or SEQ ID NO: 14484 at two, three, four, five, six or more of positions 103, 194, 372, 375, 450, 509 and 570 of the sequence. In certain embodiments, including those wherein the transposase comprises the above-described mutations at positions 30, 165, 282, and/or 538, piggyBac^TMOr the piggyBac-like transposase may comprise or be Super piggyBac^TMThe transposase can further comprise a sequence set forth in SEQ ID NO: 14487 or SEQ ID NO: 14484 at positions 103, 194, 372, 375, 450, 509, and 570. In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 the amino acid substitution at position 103 is a proline (P) to serine (S) substitution. In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a valine (V) to methionine (M) substitution. In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of alanine (A) for arginine (R). In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 the amino acid substitution at position 375 is a substitution of alanine (A) for lysine (K). In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of asparagine (N) for aspartic acid (D). In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 the amino acid substitution at position 509 is a glycine (G) to serine (S) substitution. In certain embodiments, SEQ ID NO: 14487 or SEQ ID NO: 14484 is a serine (S) to asparagine (N) substitution. In certain embodiments, piggyBa c^TMOr the piggyBac-like transposase can be contained in SEQ ID NO: 14487 substitution of methionine (M) by valine (V) at position 194. In certain embodiments, included are wherein piggyBac^TMOr the piggyBac-like transposase can be contained in SEQ ID NO: 14487 those embodiments of valine (V) to methionine (M) substitution at position 194, piggyBac^TMOr the piggyBac-like transposase can further comprise a mutation in SEQ ID NO: 14487 or SEQ ID NO: 14484 at positions 372, 375 and 450. In certain embodiments, piggyBac^TMOr the piggyBac-like transposase can be contained in SEQ ID NO: 14487 substitution of valine (V) for methionine (M) at position 194, amino acid sequence set forth in SEQ ID NO: 14487 substitution of alanine (a) for arginine (R) at position 372 and the amino acid sequence set forth in SEQ ID NO: 14487 substitution of lysine (K) with alanine (A) at position 375. In certain embodiments, piggyBac^TMOr the piggyBac-like transposase can be contained in SEQ ID NO: 14487 substitution of valine (V) for methionine (M) at position 194, amino acid sequence set forth in SEQ ID NO: 14487 substitution of alanine (a) for arginine (R) at position 372, amino acid sequence set forth in SEQ ID NO: 14487 substitution of alanine (a) for lysine (K) at position 375 and SEQ ID NO: 14487 substitution of asparagine (N) for aspartic acid (D) at position 450.

In certain embodiments, the piggyBac or piggyBac-like transposase is isolated or derived from an insect. In certain embodiments, the insect is Trichoplusia ni (Trichoplusia ni) (GenBank accession No. AAA87375; SEQ ID NO: 16796), Trichoplusia melanosporum nigricans (Argyromamma agnata) (GenBank accession No. GU477713; SEQ ID NO: 14534, SEQ ID NO: 16797), Anopheles gambiae (Anopheles gambiae) (GenBank accession No. XP-312615 (SEQ ID NO: 567); GenBank accession No. XP-320414 (SEQ ID NO: 16799); GenBank accession No. XP-310729 (SEQ ID NO: 16800)), Aphis gossypii (Aphis gossypii) (GenBank accession No. GU329918; SEQ ID NO: 16801, SEQ ID NO: 563202), Piper pisum sativum (Acyrospon suphon) (GenBank accession No. XP 2; GenBank accession No. 1687303; Bomori accession No. 16876; Bomori accession No. 16837; Boysio Bombyx bombycis persicae 16835; SEQ ID NO: 16837; SEQ ID NO: 16805; SEQ ID NO: 16835; SEQ ID NO: 16805; Boysiphe, SEQ ID NO: 16806) Drosophila melanogaster (Drosophila melanogaster) (GenBank accession No. aal3978; SEQ ID NO: 16807) Cotton bollworm (Helicoverpa armigera) (GenBank accession No. abs18391; SEQ ID NO: 14525) Heliothis virescens (GenBank accession No. abd 76335; SEQ ID NO: 16808) Silverworm moth (Macdunnoughia crassigna) (GenBank accession No. eu287451; SEQ ID NO: 16809, SEQ ID NO: 16810) Cotton bollworm (Pectinophora gossypiella) (GenBank accession No. gu270322; SEQ ID NO: 14530, SEQ ID NO: 16811) Tribolium castaneum (GenBank accession No. xp — 001814566; SEQ ID NO: 16812) The species trichoplusia agnata (ctenoplosia agnata) (also known as black spot trichoplusia agnata), messenger bauvieri, alfalfa leaf cutting bees (Megachile rotation), eastern Bombus americana (Bombus impatiens), cabbage looper (Mamestra brassicae), midge (mayetila destructor) or Apis mellifera (Apis mellia).

In certain embodiments, the piggyBac or piggyBac-like transposase is isolated or derived from an insect. In certain embodiments, the insect is Trichoplusia ni (AAA 87375).

In certain embodiments, the piggyBac or piggyBac-like transposase is isolated or derived from an insect. In certain embodiments, the insect is a silkworm (BAD 11135).

In certain embodiments, the piggyBac or piggyBac-like transposase is isolated or derived from a crustacean. In certain embodiments, the crustacean is Daphnia magna (Daphnia pulicaria) (AAM76342, SEQ ID NO: 16813).

In certain embodiments, the piggyBac or piggyBac-like transposase is isolated or derived from a vertebrate. In certain embodiments, the vertebrate is Xenopus tropicalis (Xenopus tropicalis) (GenBank accession No. BAF82026; SEQ ID NO: 14518), Homo sapiens (Homo sapiens) (GenBank accession No. NP-689808; SEQ ID NO: 16814), mouse (Mus musculus) (GenBank accession No. NP-741958; SEQ ID NO: 16815), cynomolgus monkey (Macaca fascicularis) (GenBank accession No. AB179012; SEQ ID NO: 16816, SEQ ID NO: l6817), rat (Rattus norvegicus) (GenBank accession No. XP-220453; SEQ ID NO: 16818), or small brown bats (Myotifusugus).

In certain embodiments, the piggyBac or piggyBac-like transposase is isolated or derived from a urodele. In certain embodiments, the urodele is Ciona intestinalis (GenBank accession No. XP-002123602; SEQ ID NO: 16819).

In certain embodiments, the piggyBac or piggyBac-like transposase inserts a transposon at the sequence 5 '-TTAT-3' (TTAT target sequence) within the chromosomal locus.

In certain embodiments, the piggyBac or piggyBac-like transposase inserts a transposon at the sequence 5 '-TTAA-3' (TTAA target sequence) within the chromosomal locus.

In certain embodiments, the target sequence of the piggyBac or piggyBac-like transposon comprises 5 ' -CTAA-3 ', 5 ' -TTAG-3 ', 5 ' -ATAA-3 ', 5 ' -TCAA-3 ', 5 ' -AGTT-3 ', 5 ' -ATTA-3 ', 5 ' -GTTA-3 ', 5 ' -TTGA-3 ', 5 ' -TTTA-3 ', 5 ' -TTAC-3 ', 5 ' -ACTA-3 ', 5 ' -agggg-3 ', 5 ' -CTAG-3 ', 5 ' -tga-3 ', 5 ' -AGGT-3 ', 5 ' -ATCA-3 ', 5 ' -CTCC-3 ', 5 ' -TAAA-3 ', 5 ' -TCTC-3 ', 5 ' -tga-3 5 '-AAAT-3', 5 '-AATC-3', 5 '-ACAA-3', 5 '-ACAT-3', 5 '-ACTC-3', 5 '-AGTG-3', 5 '-ATAG-3', 5 '-CAAA-3', 5 '-CACA-3', 5 '-CATA-3', 5 '-CCAG-3', 5 '-CCCA-3', 5 '-CGTA-3', 5 '-GTCC-3', 5 '-TAAG-3', 5 '-TCTA-3', 5 '-TGAG-3', 5 '-TGTT-3', 5 '-TTCA-3', 5 '-TTCT-3' and 5 '-TTTT-3'.

In certain embodiments of the methods of the present disclosure, the transposase is piggyBac or piggyBac-like transposase. In certain embodiments, the piggyBac or piggyBac-like transposase is isolated or derived from a silkworm. The piggyBac or piggyBac-like transposase can comprise or consist of an amino acid sequence that is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage therebetween identical to:

the piggyBac (pb) or piggyBac-like transposase can comprise or consist of an amino acid sequence that is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage therebetween identical to:

in certain embodiments, the piggyBac or piggyBac-like transposase is fused to a nuclear localization signal. In certain embodiments, the amino acid sequence of the piggyBac or piggyBac-like transposase fused to a nuclear localization signal is encoded by a polynucleotide sequence comprising:

in certain embodiments, the piggyBac or piggyBac-like transposase is highly active. A hyperactive piggyBac or piggyBac-like transposase is a transposase that is more active than the naturally occurring variant from which it is derived. In certain embodiments, the high activity piggyBac or piggyBac-like transposase is isolated or derived from a silkworm. In certain embodiments, the piggyBac or piggyBac-like transposase is SEQ ID NO: 14505. In certain embodiments, the high activity piggyBac or piggyBac-like transposase comprises a sequence that is at least 90% identical to the sequence:

In certain embodiments, the high activity piggyBac or piggyBac-like transposase comprises SEQ ID NO: 14576. in certain embodiments, the hyperactive piggyBac or piggyBac-like transposase comprises the following sequence:

in certain embodiments, the hyperactive piggyBac or piggyBac-like transposase comprises the following sequence:

in certain embodiments, the high activity piggyBac or piggyBac-like transposase is more stable than SEQ ID NO: 14505 is more active. In certain embodiments, the high activity piggyBac or piggyBac-like transposase is homologous to SEQ ID NO: 14505 are at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or any percentage therebetween the same.

In certain embodiments, a hyperactive piggyBac or piggyBac-like transposase comprises an amino acid substitution (relative to SEQ ID NO: 14505) at a position selected from 92, 93, 96, 97, 165, 178, 189, 196, 200, 201, 211, 215, 235, 238, 246, 253, 258, 261, 263, 271, 303, 321, 324, 330, 373, 389, 399, 402, 403, 404, 448, 473, 484, 507, 523, 527, 528, 543, 549, 550, 557, 601, 605, 607, 609, 610, or a combination thereof. In certain embodiments, a high activity piggyBac or piggyBac-like transposase comprises an amino acid substitution of Q92, V93, P96, F97, H165, E178, C189, a196, L200, a201, L211, W215, G219, Q235, Q238, K246, K253, M258, F261, S263, C271, N303, F321, V324, a330, L373, V389, S399, R402, T403, D404, N441, G448, E449, V469, C473, R484T 507, G523, I527, Y528Y 543, E549, K550, P557, E601, E605, D607, S609, L610, or any combination thereof. In certain embodiments, a hyperactive piggyBac or piggyBac-like transposase comprises an amino acid substitution of Q92, V93, P96, F97, H165, E178, C189, a196, L200, a201, L211, W215, G219, Q235, Q238, K246, K253, M258, F261, S263, C271, N303, F321, V324, a330, L373, V389, S399, R402, T403, D404, N441, G448, E449, V469, C473, R484T 507, G523, I527, Y528Y 543, E549, K550, P557, E601, E605, D607, S609, and L610.

In certain embodiments, a high activity piggyBac or piggyBac-like transposase comprises one or more substitutions of a non-wild-type amino acid, wherein the one or more substitutions to the wild-type amino acid comprises E4X, a12X, M13X, L14X, E15X, D20X, E24X, S25X, S26X, S27X, D32X, H33X, E36X, E44X, E45X, E46X, I48X, D49X, R58X, a62X, N63X, a64X, I65X, I66X, N68X, E X, D71X, S72, D X, D3676, P79X, R84, Q3685, a X, S88, N68X, E X, N69, N7, D X, N72, N185, N72, N185, N36, P85, N185, N72, N185, N72, N120, N72, N36, N72, N120, N72, N36, N120, N36, N72, N36, N72, N36, N72, N36, N207, N36, N72, N36, N72, N36, N240, P302, N303, P305, A306, K307, Y308, I310, K311, I312, L313, A314, L315, V316, D317, A318, K319, N320, F321, Y322, V323, V324, L326, E327, V328, A330, Q333, P334, S335, G336, P337, A339, V340, S341, N342, R343, P344, F345, E346, V347, E349, I352, Q355, A356, R357, N361, D365, W367, T369, G370, L373, M373, L375, H376, N379, E380, R382, V386, V389, N392, R394, Q395, S399, F400, I401, R402, D404, R405, Q513, P306, P415, P420, K415, K420, K78, K126, K78, K126, K150, K512, K150, K512, K150, K450, K512, K450, substitutions of L524, S525, I527, Y528, E529, H532, S533, N535, K536, K537, N539, I540, T542, Y543, Q546, E549, K550, Q551, G553, E554, P555, S556, P557, R558, H559, V560, N561, V562, P563, G564, R565, Y566, V567, Q570, D571, P573, Y574, K576, K581, S583, A586, A588, E594, F598, L599, E601, N602, C603, A604, E605, L606, D607, S608, S609 or L610 (relative to SEQ ID NO:). A list of highly reactive amino acid substitutions can be found in U.S. Pat. No.10,041,077, the contents of which are incorporated herein by reference in their entirety.

In certain embodiments, the excisable, integration deficient piggyBac or piggyBac-like transposase comprises one or more substitutions of non-wild-type amino acids, wherein the one or more substitutions to wild-type amino acids comprise R9X, a12X, M13X, D20X, Y21K, D23X, E24X, S25X, S26X, S27X, E28X, E30X, D32X, H33X, E36X, H37X, a39X, Y41X, D3642, T43X, E44X, E45X, E46X, R47X, D49X, S50X, S3655, a X, N63X, a64, 3666, a X, N67, N72, N55, X, N49, X D X, X D X, X, E178, T179, S180, Y182, Q184, E185, T187, L188, C189, L194, I195, a196, L198, L200, a201, L203, I204, K205, N207, Q209, L211, D213, L214, W215, R216, T217, G219, T220, V222, D223, 371, T228, F234, Q235, L237, Q238, N239, N240, N303, K304, I310, I312, L313, a314, L315, V316, D317, a318, K319, N320, F321, Y322, V323, V324, N325, L326, E327, V328, a330, G331, K332, Q333, S335, P337, P344, F345, E349, H392, N361, V362, D376, F365, Y372, E372, Y382, K382, N78, N444, N382, N78, N382, N78, N382, N78, N382, N78, N382, N401, N78, N185, N78, N382, N78, N401, N78, S461X, a464X, V466X, Q468X, V469X, C473X, a474X, N475X, N477X, K483X, R484X, P486X, T488X, L489X, G492X, V493X, M496X, I499X, I503X, Y505X, T507X, N510X, V511X, T512X, I513X, K514X, T516X, E517X, S521X, G36523 72, L524X, S525X, I527X, Y528X, L36531, H532X, S533, N535, N X, I X, T X, Y36553, Q546, Q X, N3656365636563672, N36563656365636563672, N365636563656365636563672, N3656365636563656365636563672, N36563656365636563656365636563672, N3656365636563656365636563656365636567, N3656365636563656365636563672, N36563656365636563656365636563672, N3656365636563656365636563656365636563656365636563656365636563672, N3656365636563656365636563656365636563672, N36563656365636563656365636563656365636563656365636563672, N X, N365636563656365636563656. A list of integration-deficient amino acid substitutions can be found in U.S. Pat. No.10,041,077, the contents of which are incorporated by reference in their entirety.

In certain embodiments, the integration-deficient piggyBac or piggyBac-like transposase comprises the following sequence:

in certain embodiments, the integration-deficient piggyBac or piggyBac-like transposase comprises the following sequence:

in certain embodiments, the integration-deficient transposase comprises a sequence that is complementary to SEQ ID NO: 14608 sequence at least 90% identical.

In certain embodiments, the piggyBac or piggyBac-like transposon is isolated or derived from a silkworm. In certain embodiments, the piggyBac or piggyBac-like transposon comprises the following sequences:

in certain embodiments, the piggyBac or piggyBac-like transposon comprises the following sequences:

in certain embodiments, piggyBac^TM(PB) or piggyBac-like transposons comprise the following sequences:

in certain embodiments, the piggyBac or piggyBac-like transposon comprises a nucleic acid sequence corresponding to SEQ ID NO: 14506 and a sequence corresponding to SEQ ID NO: 14507, or a sequence of the same. In certain embodiments, one piggyBac or piggyBac-like transposon ends in alignment with the nucleic acid sequence of SEQ ID NO: 14506 is at least 85%, at least 90%, at least 95%, at least 98%, at least 99% identical or any percentage therebetween, and the other piggyBac or piggyBac-like transposon end is identical to SEQ ID NO: 14507 are at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or any percentage therebetween the same. In certain embodiments, the piggyBac or piggyBac-like transposon comprises SEQ ID NO: 14506 and SEQ ID NO: 14507 or SEQ ID NO: 14509. in certain embodiments, the piggyBac or piggyBac-like transposon comprises SEQ ID NO: 14508 and SEQ ID NO: NO: 14507 or SEQ ID NO: 14509. in certain embodiments, the 5 ' and 3 ' transposon ends share at their ends a 16bp repeat of CCCGGCGAGCATGAGG (SEQ ID NO: 14510) immediately adjacent to the 5 ' -TTAT-3 target insertion site, which repeats are oppositely oriented at both ends. In certain embodiments, the 5 ' transposon ends begin with a sequence comprising 5 ' -TTATCCCGGCGAGCATGAGG-3(SEQ ID NO: 14511), and the 3 ' transposon ends with the reverse complement of the sequence comprising: 5'-CCTCATGCTCGCCGGGTTAT-3' (SEQ ID NO: 14512).

In certain embodiments, the piggyBac or piggyBac-like transposon comprises one end comprising the amino acid sequence of SEQ ID NO: 14506 or SEQ ID NO: 14508 at least 14, 16, 18, 20, 30 or 40 contiguous nucleotides. In certain embodiments, the piggyBac or piggyBac-like transposon comprises one end comprising the amino acid sequence of SEQ ID NO: 14507 or SEQ ID NO: 14509 of at least 14, 16, 18, 20, 30, or 40 contiguous nucleotides. In certain embodiments, the piggyBac or piggyBac-like transposon comprises a sequence that is identical to SEQ ID NO: 14506 or SEQ ID NO: 14508 one end with at least 90% identity. In certain embodiments, the piggyBac or piggyBac-like transposon comprises a sequence that is identical to SEQ ID NO: 14507 or SEQ ID NO: 14509 have one end at least 90% identical.

In certain embodiments, the piggyBac or piggyBac-like transposon comprises the following sequences:

in certain embodiments, the piggyBac or piggyBac-like transposon comprises the following sequences:

in certain embodiments, the piggyBac or piggyBac-like transposon comprises the sequence of CCCGGCGAGCATGAGG (SEQ ID NO: 14510). In certain embodiments, the piggyBac or piggyBac-like transposon comprises SEQ ID NO: 14510 ITR sequence. In certain embodiments, the piggyBac or piggyBac-like transposon comprises the sequence of TTATCCCGGCGAGCATGAGG (SEQ ID NO: 14511). In certain embodiments, the piggyBac or piggyBac-like transposon comprises a nucleic acid sequence from SEQ ID NO: 14511 at least 16 contiguous nucleotides. In certain embodiments, the piggyBac or piggyBac-like transposon comprises the sequence of CCTCATGCTCGCCGGGTTAT (SEQ ID NO: 14512). In certain embodiments, the piggyBac or piggyBac-like transposon comprises a nucleic acid sequence from SEQ ID NO: 14512 at least 16 contiguous nucleotides. In certain embodiments, the piggyBac or piggyBac-like transposon comprises one end comprising a nucleic acid sequence from SEQ ID NO: 14511, and one terminus comprising at least 16 contiguous nucleotides from SEQ ID NO: 14512 at least 16 contiguous nucleotides. In certain embodiments, the piggyBac or piggyBac-like transposon comprises SEQ ID NO: 14511 and SEQ ID NO: 14512. in certain embodiments, the piggyBac or piggyBac-like transposon comprises the sequence of TTAACCCGGCGAGCATGAGG (SEQ ID NO: 14513). In certain embodiments, the piggyBac or piggyBac-like transposon comprises the sequence of CCTCATGCTCGCCGGGTTAA (SEQ ID NO: 14514).

In certain embodiments, the piggyBac or piggyBac-like transposon can have a sequence comprising SEQ ID NO: 14506 and SEQ ID NO: 14507, or any one or both of these, or a variant of SEQ ID NO: 14506 or SEQ ID NO: 14507, and the piggyBac or piggyBac-like transposase has the amino acid sequence of SEQ ID NO: 14504 or SEQ ID NO: 14505, or a sequence corresponding to SEQ ID NO: 14504 or SEQ ID NO: 14505 have a sequence of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or any percentage identity therebetween. In certain embodiments, the piggyBac or piggyBac-like transposon comprises a heterologous polynucleotide inserted between a pair of inverted repeats, wherein the transposon is capable of hybridizing to a nucleic acid sequence of SEQ ID NO: 14504 or SEQ ID NO: 14505 have a piggyBac or piggyBac-like transposase transposition of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or any percentage identity therebetween. In certain embodiments, the transposon comprises two transposon ends, each comprising at the two transposon ends in opposite orientations the sequence of SEQ ID NO: 14510. in certain embodiments, each Inverted Terminal Repeat (ITR) is identical to SEQ ID NO: 14510 are at least 90% identical.

In certain embodiments, the piggyBac or piggyBac-like transposon is capable of being inserted at the sequence 5' -TTAT-3 within the target nucleic acid by a piggyBac or piggyBac-like transposase. In certain embodiments, one end of the piggyBac or piggyBac-like transposon comprises a nucleic acid sequence from SEQ ID NO: 14506 and the other transposon end comprises at least 16 contiguous nucleotides from SEQ ID NO: 14507 at least 16 contiguous nucleotides. In certain embodiments, one end of the piggyBac or piggyBac-like transposon comprises a nucleic acid sequence from SEQ ID NO: 14506 of at least 17, at least 18, at least 19, at least 20, at least 22, at least 25, at least 30 contiguous nucleotides, and the other transposon end comprises a contiguous sequence from SEQ ID NO: 14507 at least 17, at least 18, at least 19, at least 20, at least 22, at least 25, at least 30 contiguous nucleotides.

In certain embodiments, the piggyBac or piggyBac-like transposon comprises a nucleic acid sequence corresponding to SEQ ID NO: 14506 and SEQ ID NO: 14507 (each end comprising an ITR) and having a target sequence corresponding to 5 '-TTAT 3'. In certain embodiments, the piggyBac or piggyBac-like transposon also comprises a sequence encoding a transposase (e.g., SEQ ID NO: 14505). In certain embodiments, the piggyBac or piggyBac-like transposon comprises a nucleic acid sequence corresponding to SEQ ID NO: 14506 and a transposon end corresponding to SEQ ID NO: 14516. SEQ ID NO: 14516 and SEQ ID NO: 14507 are very similar but have a large insertion shortly before the ITR. Although the ITR sequences at the ends of both transposons are identical (they are both identical to SEQ ID NO: 14510), they have different target sequences: the second transposon has a target sequence corresponding to 5 '-TTAA-3', thereby providing evidence that altering the ITR sequence is not necessary to modify the target sequence specificity. piggyBac or piggyBac-like transposase associated with the 5 '-TTAA-3' target site (SEQ ID NO: 14504) differs from the 5 '-TTAT-3' -associated transposase (SEQ ID NO: 14505) only by 4 amino acid changes (D322Y, S473C, a507T, H582R). In certain embodiments, the piggyBac or piggyBac-like transposase associated with the 5 '-TTAA-3' target site (SEQ ID NO: 14504) has less activity on a transposon having a5 '-TTAT-3' terminus than the piggyBac or piggyBac-like transposase associated with the 5 '-TTAT-3' (SEQ ID NO: 14505). In certain embodiments, a piggyBac or piggyBac-like transposon having a5 ' -TTAA-3 ' target site can be converted to a piggyBac or piggyBac-like transposase having a5 ' -TTAT-3 target site by replacing the 5 ' -TTAA-3 ' target site with 5 ' -TTAT-3 '. Such transposons can be used in combination with piggyBac or piggyBac-like transposases that recognize 5 '-TTAT-3' target sequences as set forth in SEQ ID NO: 14504, or a variant of the transposase originally associated with the 5 '-TTAA-3' transposon. In certain embodiments, the high similarity between 5 '-TTAA-3' and 5 '-TTAT-3' piggyBac or piggyBac-like transposase demonstrates that minimal changes in the amino acid sequence of piggyBac or piggyBac-like transposase can alter target sequence specificity. In certain embodiments, a modification of any piggyBac or piggyBac-like transposon-transposase gene transfer system in which the 5 '-TTAA-3' target sequence is replaced with a5 '-TTAT-3' target sequence, the ITRs remain the same, and the transposase is the original piggyBac or piggyBac-like transposase or a variant thereof resulting from the use of low level mutagenesis to introduce mutations into the transposase. In certain embodiments, a piggyBac or piggyBac-like transposon transposase transfer system can be formed by modifying a5 '-TTAT-3' -active piggyBac or piggyBac-like transposon-transposase gene transfer system, wherein the 5 '-TTAT-3' target sequence is replaced by a5 '-TTAA-3' -target sequence, the ITRs remain the same, and the piggyBac or piggyBac-like transposase is the original transposase or a variant thereof.

in certain embodiments, the piggyBac or piggyBac-like transposon comprises the following sequences:

in certain embodiments, the transposon comprises a sequence from SEQ ID NO: 14577 and at least 16 contiguous bases from SEQ ID NO: 14578, and an inverted terminal repeat that is at least 87% identical to CCCGGCGAGCATGAGG (SEQ ID NO: 14510). In certain embodiments, the piggyBac or piggyBac-like transposon comprises the following sequences:

in certain embodiments, the piggyBac or piggyBac-like transposon comprises the following sequences:

in certain embodiments, the piggyBac or piggyBac-like transposon comprises SEQ ID NO: 14595 and SEQ ID NO: 14596 and consists of SEQ ID NO: 14505, transposase into a piggyBac or piggyBac-like transposase. In certain embodiments, SEQ ID NO: 14595 and SEQ ID: 14596 are not flanked by 5 '-TTAA-3' sequences. In certain embodiments, SEQ ID NO: 14595 and SEQ ID: 14596 are flanked by 5 '-TTAT-3' sequences.

In certain embodiments, the piggyBac or piggyBac-like transposon comprises the following sequences:

in certain embodiments, the piggyBac or piggyBac-like transposon comprises the following sequences:

in certain embodiments, the 5' end of the piggyBac or piggyBac-like transposon comprises SEQ ID NO: 14577. SEQ ID NO: 14595 or SEQ ID NOs: 14597, and 14599. In certain embodiments, the 5 'end of the piggyBac or piggyBac-like transposon is preceded by a 5' target sequence.

In certain embodiments, the piggyBac or piggyBac-like transposon comprises the following sequences:

in certain embodiments, the piggyBac or piggyBac-like transposon comprises the following sequences:

in certain embodiments, the 3' end of the piggyBac or piggyBac-like transposon comprises SEQ ID NO: 14578. SEQ ID NO: 14596 or SEQ ID NOs: 14600-14601. In certain embodiments, the 3 'end of the piggyBac or piggyBac-like transposon is followed by a 3' target sequence. In certain embodiments, the transposon consists of SEQ ID NO: 14505 is transposed. In certain embodiments, the 5 'and 3' ends of the piggyBac or piggyBac-like transposon share a common sequence of SEQ ID NO: 14510 of 16bp repeat sequence. In certain embodiments, the 5' transposon ends are from SEQ ID NO: 14510 and the 3' transposon ends in SEQ ID NO: the reverse complement 5'-CCTCATGCTCGCCGGG-3' of 14510 (SEQ ID NO: 14603) ends. In certain embodiments, the piggyBac or piggyBac-like transposon comprises a sequence that is identical to SEQ ID NO: 14510 or SEQ ID NO: 14603 has an ITR of at least 93%, at least 87%, or at least 81%, or any percentage identity therebetween. In certain embodiments, the piggyBac or piggyBac-like transposon comprises a sequence comprising a sequence selected from the group consisting of SEQ ID NOs: 14577. 14595 or 14597 and a sequence comprising SEQ ID NO: 14578 or 14596. In certain embodiments, the piggyBac or piggyBac-like transposon comprises a terminus comprising a sequence identical to SEQ ID NO: 14577, a sequence that is at least 90%, at least 95%, or at least 99% identical, or any percentage therebetween, and one terminus comprising a sequence that is at least 90%, at least 95%, or at least 99%, or any percentage therebetween, identical to SEQ ID NO: 14578 at least 90%, at least 95%, or at least 99%, or any percentage therebetween. In certain embodiments, one transposon end comprises a sequence from SEQ ID NO: 14577, and one transposon end comprises at least 14, at least 16, at least 18, or at least 20 contiguous bases from SEQ ID NO: 14578, at least 14, at least 16, at least 18, or at least 20 contiguous bases.

In certain embodiments, the piggyBac or piggyBac-like transposon comprises two transposon ends, wherein each transposon end comprises a sequence that is identical to the sequence of SEQ ID NO: 14510 sequences that are at least 81% identical, at least 87% identical, or at least 93% identical, or any percentage therebetween. One terminus may further comprise a sequence from SEQ ID NO: 14599 of at least 14, at least 16, at least 18, or at least 20 contiguous bases, and the other end may further comprise a nucleotide sequence from SEQ ID NO: 14601 of at least 14, at least 16, at least 18, or at least 20 contiguous bases. Can be determined by the method of SEQ ID NO: 14505 transposase transposes a piggyBac or piggyBac-like transposon, and the transposase can optionally be fused to a nuclear localization signal.

In certain embodiments, the piggyBac or piggyBac-like transposon comprises SEQ ID NO: 14595 and SEQ ID NO: 14596, and the piggyBac or piggyBac-like transposase comprises SEQ ID NO: 14504 or SEQ ID NO: 14505. in certain embodiments, the piggyBac or piggyBac-like transposon comprises SEQ ID NO: 14597 and SEQ ID NO: 14596, and the piggyBac or piggyBac-like transposase comprises SEQ ID NO: 14504 or SEQ ID NO: 14505. in certain embodiments, the piggyBac or piggyBac-like transposon comprises SEQ ID NO: 14595 and SEQ ID NO: 14578, and the piggyBac or piggyBac-like transposase comprises SEQ ID NO: 14504 or SEQ ID NO: 14505. in certain embodiments, the piggyBac or piggyBac-like transposon comprises SEQ ID NO: 14602 and SEQ ID NO: 14600 and the piggyBac or piggyBac-like transposase comprises SEQ ID NO: 14504 or SEQ ID NO: 14505.

In certain embodiments, the piggyBac or piggyBac-like transposon comprises a 5' terminus comprising 1, 2, 3, 4, 5, 6 or 7 sequences selected from ATGAGGCAGGGTAT (SEQ ID NO: 14614), ATACCCTGCCTCAT (SEQ ID NO: 14615), GGCAGGGTAT (SEQ ID NO: 14616), ATACCCTGCC (SEQ ID NO: 14617), TAAAATTTTA (SEQ ID NO: 14618), ATTTTATAAAAT (SEQ ID NO: 14619), TCATACCCTG (SEQ ID NO: 14620) and TAAATAATAATAA (SEQ ID NO: 14621). In certain embodiments, the piggyBac or piggyBac-like transposon comprises a 3 'terminus, the 3' terminus comprising 1, 2, or 3 nucleic acid sequences selected from SEQ ID NOs: 14617. SEQ ID NO: 14620 and SEQ ID NO: 14621.

In certain embodiments of the methods of the present disclosure, the transposase is piggyBac or piggyBac-like transposase. In certain embodiments, the piggyBac or piggyBac-like transposase is isolated or derived from xenopus tropicalis. The piggyBac or piggyBac-like transposase can comprise or consist of an amino acid sequence that is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage therebetween identical to:

In some embodiments, the piggyBac or piggyBac-like transposase is SEQ ID NO: 14517. In certain embodiments, the piggyBac or piggyBac-like transposase is SEQ ID NO: 14517 integration defect variant. The piggyBac or piggyBac-like transposase can comprise or consist of an amino acid sequence that is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage therebetween identical to:

in certain embodiments, the piggyBac or piggyBac-like transposase is isolated or derived from xenopus tropicalis. In certain embodiments, the piggyBac or piggyBac-like transposase is a high activity piggyBac or piggyBac-like transposase. In certain embodiments, the high activity piggyBac or piggyBac-like transposase comprises a sequence that is at least 90% identical to the sequence:

in certain embodiments, the piggyBac or piggyBac-like transposase is a high activity piggyBac or piggyBac-like transposase. A hyperactive piggyBac or piggyBac-like transposase is a transposase that is more active than the naturally occurring variant from which it is derived. In certain embodiments, the high activity piggyBac or piggyBac-like transposase is more stable than SEQ ID NO: 14517 transposase is more active. In certain embodiments, the hyperactive piggyBac or piggyBac-like transposase comprises the following sequence:

In certain embodiments, the hyperactive piggyBac or piggyBac-like transposase comprises the following sequence:

in certain embodiments, the hyperactive piggyBac or piggyBac-like transposase comprises the following sequence:

in certain embodiments, a high activity piggyBac or piggyBac-like transposase comprises an amino acid substitution (relative to SEQ ID NO: 14517) at a position selected from amino acid 6, 7, 16, 19, 20, 21, 22, 23, 24, 26, 28, 31, 34, 67, 73, 76, 77, 88, 91, 141, 145, 146, 148, 150, 157, 162, 179, 182, 189, 192, 193, 196, 198, 200, 210, 212, 218, 248, 263, 270, 294, 297, 308, 310, 333, 336, 354, 357, 358, 359, 377, 423, 426, 428, 438, 447, 450, 462, 469, 472, 498, 502, 517, 520, 523, 533, 534, 576, 577, 582, 583, or 587. In certain embodiments, a high activity piggyBac or piggyBac-like transposase comprises Y6C, S7G, M16G, S19G, S20G, E21G, E22G, F23G, S24G, S26G, S28G, V31G, a 34G, L67G, G73G, a 76G, D77G, P88G, N91G, Y141G, N145G, P146G, P148G, Y150G, H G, a 36179G, a G, L36182, L G, L193, L198, T G, S198, S72, N150G, N91G, N141G, Y150G, N36157G, N, a248N, L263M, Q270L, S294T, T297M, S308R, L310R, L333M, Q336M, a354H, C357V, L358F, D359N, L377I, V423H, P426K, K428R, S438A, T447G, T447A, L450V, a462H, I469H, I472H, Q498H, L502H, E5171, P520H, N523H, I533H, D534H, F576H, K577H, I582H, Y583H, L587H or L587H amino acid substitutions of at least 1, 2, 3, 576, 4, 576, 5, 1458, or all of these mutations or combinations thereof (relative to any one or all of the amino acid substitutions, 2, 3, 4, 6, 1458, or 10 ID: 16 of these mutations).

In certain embodiments, a high activity piggyBac or piggyBac-like transposase comprises one or more substitutions of an amino acid that is not wild-type, wherein the one or more substitutions to a wild-type amino acid comprises A2X, K3X, R4X, F5X, Y6X, S7X, a11X, a13X, C15X, M16X, a17X, S18X, S19X, S20X, E21X, E22X, F23X, S24X, G25X, 26X 27X, S28X, E29X, E42X, E43X, S44X, C46X, S47X, S48X, S3649, T50X, V X, S3652, a X, L X, N54, N72, N19, N72, N19X, N72, N19X, N72, P72, N19X, N72, N, L127, Q128, D129, M130, L132, Y133, V126, Y127, a138, E139, Q140, Y141, L142, Q144, N145, P146, L147, P148, Y150, a151, a155, H157, P158, I161, a162, V168, T171, L172, a173, M174, I177, a179, L182, D187, T188, T189, T190, L192, S193, I194, P195, V196, S198, a199, T200, S202, L208, L209, L210, R211, F212, F215, N217, N218, a219, T220, a221, V222, P224, D225, Q226, P227, H229, R231, H233, L235, P237, I239, D291, L242, S271, E254, R254, Y254, L250, Y150, L250, G293, S240, S293, S257, S240, S293, F260, S293, S240, S293, S257, S240, S293, F240, L150, F240, L150, R240, L150, R240, R150, R240, R150, R240, P313, G314, P316, P317, D318, L319, T320, V321, K324, E328, I330, S331, P332, L333, L334, G335, Q336, F338, L340, D343, N344, F345, Y346, S347, L351, F352, A354, L355, Y356, C357, L358, D359, T360, R422, Y423, G424, P426, K428, N429, K430, P431, L432, S434, K435, E436, S438, K439, Y440, G443, R446, T447, L450, Q451, N, T460, R461, A462, K465, V467, G468, I469, Y470, L471, I472, M474, A475, L476, R477, S479, Y480, V483, K520, K, Substitution of R558X, R559X, D560X, T561X, Y564X, P566X, K567X, P569X, R570X, N571X, L574X, C575X, F576X, K577X, P578X, F580X, E581X, I582X, Y583X, T585X, Q586X, L587X, H588X or Y589X (relative to SEQ ID NO: 14517). A list of highly reactive amino acid substitutions can be found in U.S. Pat. No.10,041,077, the contents of which are incorporated by reference in their entirety.

In certain embodiments, the piggyBac or piggyBac-like transposase is integration deficient. In certain embodiments, an integration-deficient piggyBac or piggyBac-like transposase is a transposase that can excise its corresponding transposon but integrates the excised transposon at a lower frequency than the corresponding naturally-occurring transposase. In certain embodiments, the piggyBac or piggyBac-like transposase is SEQ ID NO: 14517 integration defect variant. In certain embodiments, the integration-deficient piggyBac or piggyBac-like transposase is mutated relative to the sequence of SEQ ID NO: 14517 is defective.

In certain embodiments, the piggyBac or piggyBac-like transposase is active for excision, but is deficient in integration. In certain embodiments, the integration-deficient piggyBac or piggyBac-like transposase comprises a sequence that is at least 90% identical to the sequence:

in certain embodiments, the integration-deficient piggyBac or piggyBac-like transposase comprises a sequence that is at least 90% identical to the sequence:

in certain embodiments, the integration-deficient piggyBac or piggyBac-like transposase comprises SEQ ID NO: 14611. in certain embodiments, the integration-deficient piggyBac or piggyBac-like transposase comprises a sequence that is at least 90% identical to the sequence:

In certain embodiments, the integration-deficient piggyBac or piggyBac-like transposase comprises SEQ ID NO: 14612. in certain embodiments, the integration-deficient piggyBac or piggyBac-like transposase comprises a sequence that is at least 90% identical to the sequence:

in certain embodiments, the integration-deficient piggyBac or piggyBac-like transposase comprises SEQ ID NO: 14613. in certain embodiments, the integration-deficient piggyBac or piggyBac-like transposase comprises an amino acid substitution wherein the Asn at position 218 is replaced with Glu or Asp (N218D or N218E) (relative to SEQ ID NO: 14517).

In certain embodiments, the excisable, integration deficient piggyBac or piggyBac-like transposase comprises one or more substitutions of non-wild-type amino acids, wherein the one or more substitutions to wild-type amino acids comprise A2X, K3X, R4X, F5X, Y6X, S7X, A8X, E9X, E10X, a11X, a12X, a13X, H14X, C15X, M16X, a17X, S18X, S19X, S20X, E21X, E22X, F23X, S24X, G25X, 26X, S3628, E29X, V31, P32X, P33, X, a 3635, S3635, E36, S38, X, 3638, X, 3628, X, 3628, X, 3628, 36, V168, G169, L170, T171, L172, a173, M174, G175, L176, I177, K178, a179, N180, S181, L182, S184, Y185, D187, T188, T189, T190, V191, L192, S193, I194, P195, V196, F197, S198, a199, T200, M201, S202, R203, N204, R205, Y206, Q207, L208, L209, L210, R211, F212, L213, H241, F215, N216, N217, N218, a219, T220, a221, V222, P223, P224, D225, Q226, P227, G228, H229, D230, R231, H233, K234, L235, R236, L238, I239, D271, L242, S271, E255, R255, G228, H257, G255, H257, R240, R255, G255, F240, G293, S240, S293, S255, S240, S255, S293, S227, S150, S293, S150, S293, S150, e304, G305, K306, D307, S308, K309, L310, D311, P312, P313, G314, C315, P316, P317, D318, L319, T320, V321, S322, G323, K324, I325, V326, W327, E328, L329, I330, S331, P332, L333, L334, G335, Q336, F338, H339, L340, V342, N344, F345, Y346, S347, S348, I349, L351, T353, a354, Y356, C357, L358, D359, T360, P361, a362, C363, G364, I366, N367, R368, D369, K371, G372, L373, R375, a376, L377, L378, D379, K380, K447, L385, N382, N465, K78, R423, K391, K78, K26, N478, S479, Y480, V482Y 483, K484, A485, A486, V487, P488, G489, P490, K491, L492, S493, Y494, Y495, K496, Q498, L499, Q500, I501, L502, P503, A504, L505, L506, F507, G508, G509, V510, E511, E512, Q513, T514, V515, E517, M518, P519, P520, S521, D522, N523, V524, A525, L527, I528, G529, K530, F532, I533, D534, T535, L536, P537, P538, T539, P540, G541, F542, Q544, R562, P545, Q546, K547, G548, C549, K550, V551, C555, C558, R558, K483, R558, K9, K565, Y5, P2, P102, P518, P520, P521, P522, P523, P562, P578, K57586, K9, or K9 substitution relative to P9, P569, P9, K9, P569, K9, I569, K9, K567, K9, K565. A list of excision-competent, integration-deficient amino acid substitutions can be found in U.S. Pat. No.10,041,077, the contents of which are incorporated by reference in their entirety.

In certain embodiments, the piggyBac or piggyBac-like transposase is fused to a nuclear localization signal. In certain embodiments, SEQ ID NO: 14517 or SEQ ID NO: 14518 fusion with nuclear localization signals. In certain embodiments, the amino acid sequence of the piggyBac or piggyBac-like transposase fused to a nuclear localization signal is encoded by a polynucleotide sequence comprising:

in certain embodiments, the piggyBac or piggyBac-like transposon is isolated or derived from a xenopus tropicalis. In certain embodiments, the piggyBac or piggyBac-like transposon comprises the following sequences:

in certain embodiments, the piggyBac or piggyBac-like transposon comprises the following sequences:

in certain embodiments, the piggyBac or piggyBac-like transposon comprises SEQ ID NO: 14519 and SEQ ID NO: 14520. in certain embodiments, the piggyBac or piggyBac-like transposon comprises the following sequences:

in certain embodiments, the piggyBac or piggyBac-like transposon comprises the following sequences:

in certain embodiments, the piggyBac or piggyBac-like transposon comprises SEQ ID NO: 14520 and SEQ ID NO: 14519. SEQ ID NO: 14521 or SEQ ID NO: 14523. in certain embodiments, the piggyBac or piggyBac-like transposon comprises SEQ ID NO: 14522 and SEQ ID NO: 14519. SEQ ID NO: 14521 or SEQ ID NO: 14523. in certain embodiments, the piggyBac or piggyBac-like transposon comprises one end comprising a nucleic acid sequence from SEQ ID NO: 14519. SEQ ID NO: 14521 or SEQ ID NO: 14523 of at least 14, 16, 18, 20, 30, or 40 contiguous nucleotides. In certain embodiments, the piggyBac or piggyBac-like transposon comprises one end comprising a nucleic acid sequence from SEQ ID NO: 14520 or SEQ ID NO: 14522 of at least 14, 16, 18, 20, 30, or 40 contiguous nucleotides. In certain embodiments, the piggyBac or piggyBac-like transposon comprises a sequence that is identical to SEQ ID NO: 14519. SEQ ID NO: 14521 or SEQ ID NO: 14523 have one end at least 90% identical. In certain embodiments, the piggyBac or piggyBac-like transposon comprises a sequence that is identical to SEQ ID NO: 14520 or SEQ ID NO: 14522 have one end at least 90% identical. In one embodiment, one transposon end is identical to SEQ ID NO: 14519 are at least 90% identical and the other transposon end is identical to SEQ ID NO: 14520 are at least 90% identical.

In certain embodiments, the piggyBac or piggyBac-like transposon comprises the sequence of TTAACCTTTTTACTGCCA (SEQ ID NO: 14524). In certain embodiments, the piggyBac or piggyBac-like transposon comprises the sequence of TTAACCCTTTGCCTGCCA (SEQ ID NO: 14526). In certain embodiments, the piggyBac or piggyBac-like transposon comprises the sequence of TTAACCYTTTTACTGCCA (SEQ ID NO: 14527). In certain embodiments, the piggyBac or piggyBac-like transposon comprises the sequence of TGGCAGTAAAAGGGTTAA (SEQ ID NO: 14529). In certain embodiments, the piggyBac or piggyBac-like transposon comprises the sequence of TGGCAGTGAAAGGGTTAA (SEQ ID NO: 14531). In certain embodiments, the piggyBac or piggyBac-like transposon comprises the sequence of TTAACCYTTTKMCTGCCA (SEQ ID NO: 14533). In certain embodiments, one end of the piggyBac or piggyBac-like transposon comprises a nucleic acid sequence selected from SEQ ID NOs: 14524. SEQ ID NO: 14526 and SEQ ID NO: 14527, respectively. In certain embodiments, piggyBac^TM(PB) or piggyBac-like transposon comprises at one end a sequence selected from SEQ ID NO: 14529 and SEQ ID NO:14531, respectively. In certain embodiments, each inverted terminal repeat of the piggyBac or piggyBac-like transposon comprises the sequence of the ITR sequence of CCYTTTKMCTGCCA (SEQ ID NO: 14563). In certain embodiments, piggyBac ^TMEach end of the (PB) or piggyBac-like transposon comprises in opposite orientation the amino acid sequence of SEQ ID NO: 14563. in certain embodiments, one ITR of the piggyBac or piggyBac-like transposon comprises a sequence selected from SEQ ID NO: 14524. SEQ ID NO: 14526 and SEQ ID NO: 14527, respectively. In certain embodiments, one ITR of the piggyBac or piggyBac-like transposon comprises a sequence selected from SEQ ID NO: 14529 and SEQ ID NO: 14531, respectively. In certain embodiments, the piggyBac or piggyBac-like transposon comprises SEQ ID NOs: 14533.

in certain embodiments, the piggyBac or piggyBac-like transposon can have a sequence comprising SEQ ID NO: 14519 and SEQ ID NO: 14520, or a variant of either or both of these that is identical to SEQ ID NO: 14519 or SEQ ID NO: 14520 ends of a variant having at least 90% sequence identity, and a piggyBac or piggyBac-like transposase having the amino acid sequence of SEQ ID NO: 14517, or a sequence identical to SEQ ID NO: 14517 or SEQ ID NO: 14518 shows variants having at least%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or any percentage sequence identity therebetween. In certain embodiments, one piggyBac or piggyBac-like transposon end comprises a transposon from SEQ ID NO: 14519. SEQ ID NO: 14521 or SEQ ID NO: 14523, and the other transposon end comprises at least 14 contiguous nucleotides from SEQ ID NO: 14520 or SEQ ID NO: 14522 at least 14 contiguous nucleotides. In certain embodiments, one transposon end comprises a sequence from SEQ ID NO: 14519. SEQ ID NO: 14521 or SEQ ID NO: 14523, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 22, at least 25, at least 30 contiguous nucleotides, and another transposon end comprises a contiguous sequence from SEQ ID NO: 14520 or SEQ ID NO: 14522, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 22, at least 25, or at least 30 contiguous nucleotides.

In certain embodiments, the piggyBac or piggyBac-like transposase recognizes a polypeptide having an amino acid sequence corresponding to SEQ ID NO: 14519 and a sequence corresponding to SEQ ID NO: 14520 at the 3' end of the transposon. It will cleave DNA, including any heterologous DNA placed between them, from 5 '-TTAA-3' sequences at the 5 'end of one transposon end to 5' -TTAA-3 'at the 3' end of the second transposon end, excise the transposon from one DNA molecule, and insert the excised sequences into the second DNA molecule. In certain embodiments, truncated and modified forms of the 5 'and 3' transposon ends will also function as part of a transposon that can be transposed by a piggyBac or piggyBac-like transposase. For example, the 5' transposon ends can be substituted with a sequence corresponding to SEQ ID NO: 14521 or SEQ ID NO: 14523, the 3' transposon ends can be substituted with sequences corresponding to SEQ ID NO: 14522. In certain embodiments, the 5 ' and 3 ' transposon ends share at their ends an 18bp nearly perfectly repeated sequence (5 ' -TTAACCYTTTKMCTGCCA: SEQ ID NO: 14533) comprising a 5 ' -TTAA-3 ' insertion site, which sequences are oppositely oriented at both ends. That is, in SEQ ID NO: 14519 and SEQ ID NO: 14523, the 5 ' transposon ends at the start of sequence 5'-TTAACCTTTTTACTGCCA-3' (SEQ ID NO: 14524), or in SEQ ID NO: 14521, the 5 ' transposon ends starting at sequence 5'-TTAACCCTTTGCCTGCCA-3' (SEQ ID NO: 14526); the 3' transposon ends with the approximate reverse complement of this sequence: in SEQ ID NO: 14520, which terminates at 5 'TGGCAGTAAAAGGGTTAA-3' (SEQ ID NO: 14529), is set forth in SEQ ID NO: 14522, it terminates at 5'-TGGCAGTGAAAGGGTTAA-3' (SEQ ID NO: 14531). One embodiment of the invention is a transposon comprising a heterologous polynucleotide inserted between two transposon ends, each transposon end comprising in opposite orientations at both transposon ends the sequence of SEQ ID NO: 14533. in certain embodiments, one transposon end comprises a nucleic acid sequence selected from SEQ ID NOS: 14524. SEQ ID NO: 14526 and SEQ ID NO: 14527, respectively. In some embodiments, one transposon end comprises a sequence selected from SEQ ID NO: 14529 and SEQ ID NO: 14531, respectively.

In certain embodiments, piggyBac^TM(PB) or piggyBac-like transposons isolated or derived from Xenopus tropicalis. In certain embodiments, the piggyBac or piggyBac-like transposon comprises the following sequences:

in certain embodiments, the piggyBac or piggyBac-like transposon comprises the following sequences:

in certain embodiments, the piggyBac or piggyBac-like transposon comprises a nucleic acid sequence from SEQ ID NO: 14573 or SEQ ID NO: 14574 and an inverted terminal repeat of CCYTTTBMCTGCCA (SEQ ID NO: 14575).

In certain embodiments, the piggyBac or piggyBac-like transposon comprises the following sequences:

in certain embodiments, the piggyBac or piggyBac-like transposon comprises the following sequences:

In certain embodiments, the piggyBac or piggyBac-like transposon comprises the following sequences:

in certain embodiments, the piggyBac or piggyBac-like transposon comprises a nucleic acid sequence selected from SEQ ID NOs: 14573 and SEQ ID NOs: 14579-5' transposon end sequences of 14585. In certain embodiments, the 5 'transposon end sequence is preceded by a 5' target sequence. In certain embodiments, the piggyBac or piggyBac-like transposon comprises the following sequences:

in certain embodiments, the piggyBac or piggyBac-like transposon comprises the following sequences:

in certain embodiments, the piggyBac or piggyBac-like transposon comprises a nucleic acid sequence selected from SEQ ID NOs: 14574 and SEQ ID NOs: 14587 and 14590. In certain embodiments, the 3 'transposon end sequence is followed by a 3' target sequence. In certain embodiments, the 5 'and 3' transposon ends share a 14 repeat sequence (SEQ ID NO: 14575) adjacent to the target sequence that is oppositely oriented at both ends. In certain embodiments, the piggyBac or piggyBac-like transposon comprises a nucleic acid sequence comprising a target sequence and a sequence selected from SEQ ID NOs: 14582, -14584 and 14573, and a sequence selected from the group consisting of SEQ ID NOs: 14588 and 14590 and 14574.

In certain embodiments, the 5' transposon end of the piggyBac or piggyBac-like transposon comprises

(SEQ ID NO: 14591) and ITR. In certain embodiments, the 5' transposon end comprises

(SEQ ID NO: 14592) and ITR. In certain embodiments, the 3' transposon end of the piggyBac or piggyBac-like transposon comprises

(SEQ ID NO: 14593) and ITR. In certain embodiments, the 3' transposon end comprises

(SEQ ID NO: 14594) and ITR.

In certain embodiments, one transposon end comprises a sequence identical to SEQ ID NO: 14573, at least 90%, at least 95%, at least 99%, or any percentage therebetween, and the other transposon end comprises a sequence that is identical to SEQ ID NO: 14574 at least 90%, at least 95%, at least 99%, or any percentage therebetween. In certain embodiments, one transposon end comprises a sequence from SEQ ID NO: 14573, and one transposon end comprises at least 14, at least 16, at least 18, at least 20, or at least 25 contiguous nucleotides from SEQ ID NO: 14574 at least 14, at least 16, at least 18, at least 20, or at least 25 contiguous nucleotides. In certain embodiments, one transposon end comprises a sequence from SEQ ID NO: 14591 of at least 14, at least 16, at least 18, at least 20, and the other end comprises a sequence from SEQ ID NO: 14593 of at least 14, at least 16, at least 18, at least 20. In certain embodiments, each transposon end comprises in opposite orientation SEQ ID NO: 14575.

In certain embodiments, the piggyBac or piggyBac-like transposon comprises a nucleic acid sequence selected from SEQ ID NOs: 14573. SEQ ID NO: 14579. SEQ ID NO: 14581. SEQ ID NO: 14582. SEQ ID NO: 14583 and SEQ ID NO: 14588, and a sequence selected from SEQ ID NO: 14587. SEQ ID NO: 14588. SEQ ID NO: 14589 and SEQ ID NO: 14586, and the piggyBac or piggyBac-like transposase comprises SEQ ID NO: 14517 or SEQ ID NO: 14518.

in certain embodiments, the piggyBac or piggyBac-like transposon comprises ITRs of CCCTTTGCCTGCCA (SEQ ID NO: 14622) (5 'ITR) and TGGCAGTGAAAGGG (SEQ ID NO: 14623) (3' ITR) adjacent to the target sequence.

In certain embodiments of the methods of the present disclosure, the transposase is piggyBac or piggyBac-like transposase. In certain embodiments, the piggyBac or piggyBac-like transposase is isolated or derived from a cotton bollworm. The piggyBac or piggyBac-like transposase can comprise or consist of an amino acid sequence that is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage therebetween identical to:

In certain embodiments, the piggyBac or piggyBac-like transposon is isolated or derived from a cotton bollworm. In certain embodiments, the piggyBac or piggyBac-like transposon comprises the following sequences:

in certain embodiments, the piggyBac or piggyBac-like transposon comprises the following sequences:

in certain embodiments of the methods of the present disclosure, the transposase is piggyBac or piggyBac-like transposase. In certain embodiments, the piggyBac or piggyBac-like transposase is isolated or derived from pink bollworm. The piggyBac or piggyBac-like transposase can comprise or consist of an amino acid sequence that is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage therebetween identical to:

in certain embodiments, the piggyBac or piggyBac-like transposon is isolated or derived from pink bollworm. In certain embodiments, the piggyBac or piggyBac-like transposon comprises the following sequences:

in certain embodiments, the piggyBac or piggyBac-like transposon comprises the following sequences:

in certain embodiments of the methods of the present disclosure, the transposase is piggyBac or piggyBac-like transposase. In certain embodiments, the piggyBac or piggyBac-like transposase is isolated or derived from trichoplusia agnata. The piggyBac or piggyBac-like transposase can comprise or consist of an amino acid sequence that is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage therebetween identical to:

In certain embodiments, the piggyBac or piggyBac-like transposon is isolated or derived from trichoplusia agnata. In certain embodiments, the piggyBac or piggyBac-like transposon comprises the following sequences:

in certain embodiments, the piggyBac or piggyBac-like transposon comprises the following sequences:

in certain embodiments, the piggyBac or piggyBac-like transposon comprises the ITR sequence of CCCTAGAAGCCCAATC (SEQ ID NO: 14564).

In certain embodiments of the methods of the present disclosure, the transposase is piggyBac or piggyBac-like transposase. In certain embodiments, the piggyBac or piggyBac-like transposase is isolated or derived from black cutworm. The piggyBac (pb) or piggyBac-like transposase can comprise or consist of an amino acid sequence that is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage therebetween identical to:

in certain embodiments, the piggyBac or piggyBac-like transposon is isolated or derived from black cutworm. In certain embodiments, the piggyBac or piggyBac-like transposon comprises the following sequences:

In certain embodiments, the piggyBac or piggyBac-like transposon comprises the following sequences:

in certain embodiments of the methods of the present disclosure, the transposase is piggyBac or piggyBac-like transposase. In certain embodiments, the piggyBac or piggyBac-like transposase is isolated or derived from a lucerne aphanidermia. The piggyBac (pb) or piggyBac-like transposase can comprise or consist of an amino acid sequence that is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage therebetween identical to:

in certain embodiments, the piggyBac or piggyBac-like transposon is isolated or derived from a lucerne myrtle. In certain embodiments, the piggyBac or piggyBac-like transposon comprises the following sequences:

in certain embodiments, the piggyBac or piggyBac-like transposon comprises the following sequence

In certain embodiments of the methods of the present disclosure, the transposase is piggyBac or piggyBac-like transposase. In certain embodiments, the piggyBac or piggyBac-like transposase is isolated or derived from eastern american bombus. The piggyBac (pb) or piggyBac-like transposase can comprise or consist of an amino acid sequence that is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage therebetween identical to:

In certain embodiments, the piggyBac or piggyBac-like transposon is isolated or derived from bombus americana. In certain embodiments, the piggyBac or piggyBac-like transposon comprises the following sequences:

in certain embodiments, the piggyBac or piggyBac-like transposon comprises the following sequences:

in certain embodiments of the methods of the present disclosure, the transposase is piggyBac or piggyBac-like transposase. In certain embodiments, the piggyBac or piggyBac-like transposase is isolated or derived from spodoptera brassicae. The piggyBac (pb) or piggyBac-like transposase can comprise or consist of an amino acid sequence that is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage therebetween identical to:

in certain embodiments, the piggyBac or piggyBac-like transposon is isolated or derived from spodoptera brassicae. In certain embodiments, the piggyBac or piggyBac-like transposon comprises the following sequences:

in certain embodiments, the piggyBac or piggyBac-like transposon comprises the following sequences:

in certain embodiments of the methods of the present disclosure, the transposase is piggyBac or piggyBac-like transposase. In certain embodiments, the piggyBac or piggyBac-like transposase is isolated or derived from a wheat gall midge. The piggyBac (pb) or piggyBac-like transposase can comprise or consist of an amino acid sequence that is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage therebetween identical to:

In certain embodiments, the piggyBac or piggyBac-like transposon is isolated or derived from a wheat midge. In certain embodiments, the piggyBac or piggyBac-like transposon comprises the following sequences:

in certain embodiments, the piggyBac or piggyBac-like transposon comprises the following sequences:

in certain embodiments of the methods of the present disclosure, the transposase is piggyBac or piggyBac-like transposase. In certain embodiments, the piggyBac or piggyBac-like transposase is isolated or derived from an italian bee. The piggyBac (pb) or piggyBac-like transposase can comprise or consist of an amino acid sequence that is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage therebetween identical to:

in certain embodiments, the piggyBac or piggyBac-like transposon is isolated or derived from an italian bee. In certain embodiments, the piggyBac or piggyBac-like transposon comprises the following sequences:

in certain embodiments, the piggyBac or piggyBac-like transposon comprises the following sequences:

in certain embodiments of the methods of the present disclosure, the transposase is piggyBac or piggyBac-like transposase. In certain embodiments, the piggyBac or piggyBac-like transposase is isolated or derived from a Messor bouvieri. The piggyBac (pb) or piggyBac-like transposase can comprise or consist of an amino acid sequence that is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage therebetween identical to:

In certain embodiments, the piggyBac or piggyBac-like transposon is isolated or derived from a messenger bouvieri. In certain embodiments, the piggyBac or piggyBac-like transposon comprises the following sequences:

in certain embodiments, the piggyBac or piggyBac-like transposon comprises the following sequences:

in certain embodiments of the methods of the present disclosure, the transposase is piggyBac or piggyBac-like transposase. In certain embodiments, the piggyBac or piggyBac-like transposase is isolated or derived from trichoplusia ni. The piggyBac (pb) or piggyBac-like transposase can comprise or consist of an amino acid sequence that is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage therebetween identical to:

in certain embodiments, the piggyBac or piggyBac-like transposon is isolated or derived from trichoplusia ni. In certain embodiments, the piggyBac or piggyBac-like transposon comprises the following sequences:

in certain embodiments, the piggyBac or piggyBac-like transposon comprises the following sequences:

In certain embodiments, the piggyBac or piggyBac-like transposon comprises the following sequences:

in certain embodiments, the piggyBac or piggyBac-like transposon comprises the following sequences:

in certain embodiments, the piggyBac or piggyBac-like transposon comprises SEQ ID NO: 14561 and SEQ ID NO: 14562, and the piggyBac or piggyBac-like transposase comprises SEQ ID NO: 14558. in certain embodiments, the piggyBac or piggyBac-like transposon comprises SEQ ID NO: 14609 and SEQ ID NO: 14610 and the piggyBac or piggyBac-like transposase comprises SEQ ID NO: 14558.

in certain embodiments, the piggyBac or piggyBac-like transposon is isolated or derived from a cotton aphid. In certain embodiments, the piggyBac or piggyBac-like transposon comprises the ITR sequence of CCTTCCAGCGGGCGCGC (SEQ ID NO: 14565).

In certain embodiments, the piggyBac or piggyBac-like transposon is isolated or derived from chilo suppressalis. In certain embodiments, the piggyBac or piggyBac-like transposon comprises the ITR sequence of CCCAGATTAGCCT (SEQ ID NO: 14566).

In certain embodiments, the piggyBac or piggyBac-like transposon is isolated or derived from heliothis virescens. In certain embodiments, the piggyBac or piggyBac-like transposon comprises the ITR sequence of CCCTTAATTACTCGCG (SEQ ID NO: 14567).

In certain embodiments, the piggyBac or piggyBac-like transposon is isolated or derived from pink bollworm. In certain embodiments, the piggyBac or piggyBac-like transposon comprises the ITR sequence of CCCTAGATAACTAAAC (SEQ ID NO: 14568).

In certain embodiments, the piggyBac or piggyBac-like transposon is isolated or derived from Anopheles funestus (Anopheles stephensi). In certain embodiments, the piggyBac or piggyBac-like transposon comprises the ITR sequence of CCCTAGAAAGATA (SEQ ID NO: 14569).

DNA transposons in the hAT family are common in plants and animals. A number of active hAT transposon systems have been identified and found to be functional, including, but not limited to, the Hermes transposon, the Ac transposon, the hobo transposon, and the Tol2 transposon. The hAT family consists of two families that have been classified into the AC subfamily and the Buster subfamily based on their transposase primary sequences. Members of the hAT family belong to class II transposable elements. Class II mobile elements use a cut and paste (cut and paste) transposition mechanism. The hAT elements share a similar transposase, short terminal inverted repeat, and 8 base pair repeat of the genomic target.

The compositions and methods of the present disclosure may comprise a TcBuster transposon and/or a TcBuster transposase.

The compositions and methods of the present disclosure may comprise a TcBuster transposon and/or a highly active TcBuster transposase. A highly active Tcbuster transposase exhibits increased excision and/or increased insertion frequency when compared to the excision and/or insertion frequency of the wild-type Tcbuster transposase. A highly active Tcbuster transposase showed increased transposition frequency when compared with that of the wild type Tcbuster transposase.

In some embodiments of the compositions and methods of the present disclosure, the wild-type TcBuster transposase comprises or consists of the amino acid sequence:

(GenBank accession No. ABF20545 and SEQ ID NO: 17900).

In some embodiments of the compositions and methods of the present disclosure, a TcBuster transposase comprises or consists of a sequence having at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or any percent identity therebetween, which is a wild-type TcBuster transposase comprising or consisting of the amino acid sequence of:

(GenBank accession No. ABF20545 and SEQ ID NO: 17900).

In some embodiments of the compositions and methods of the present disclosure, the wild-type TcBuster transposase is encoded by a nucleic acid sequence comprising or consisting of:

(GenBank accession No. DQ481197 and SEQ ID NO: 17901).

In some embodiments of the compositions and methods of the present disclosure, a TcBuster transposase comprises or consists of a sequence having at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or any percent identity therebetween, with a wild-type TcBuster transposase encoded by a nucleic acid sequence comprising or consisting of:

(GenBank accession No. DQ481197 and SEQ ID NO: 17901).

In some embodiments of the compositions and methods of the present disclosure, the TcBuster transposase comprises or consists of a naturally occurring amino acid sequence.

In some embodiments of the compositions and methods of the present disclosure, the TcBuster transposase comprises or consists of a non-naturally occurring amino acid sequence.

In some embodiments of the compositions and methods of the present disclosure, the TcBuster transposase is encoded by a sequence comprising or consisting of a naturally occurring nucleic acid sequence.

In some embodiments of the compositions and methods of the present disclosure, the TcBuster transposase is encoded by a sequence comprising or consisting of a non-naturally occurring nucleic acid sequence.

In some embodiments of the compositions and methods of the present disclosure, the mutant TcBuster transposase comprises one or more sequence variations when compared to a wild-type TcBuster transposase. In some embodiments, the wild-type TcBuster transposase comprises SEQ ID NO: 17900 or consisting of the amino acid sequence thereof. In some embodiments, the wild-type TcBuster transposase consists of a nucleic acid comprising SEQ ID NO: 17901 or a sequence consisting thereof. In some embodiments, the one or more sequence variations comprise one or more of a substitution, inversion, insertion, deletion, transposition, and frameshift. In some embodiments, one or more sequence variations comprise modified, synthetic, artificial, or non-naturally occurring amino acids. In some embodiments, one or more sequence variations comprise a modified, synthetic, artificial, or non-naturally occurring nucleic acid.

In some embodiments of the compositions and methods of the present disclosure, the mutant TcBuster transposase comprises one or more sequence variations when compared to a wild-type TcBuster transposase. In some embodiments, the one or more sequence variations comprise amino acid substitutions that increase net charge neutral pH when compared to a wild-type TcBuster transposase. In some embodiments, the wild-type TcBuster transposase comprises SEQ ID NO: 17900 or consisting of the amino acid sequence thereof. In some embodiments, the wild-type TcBuster transposase consists of a nucleic acid comprising SEQ ID NO: 17901 or a sequence consisting thereof. In some embodiments, the one or more sequence variations comprise at least one of SEQ ID NOs: an amino acid substitution of aspartic acid (D) at position 223 of 17900 (D223), aspartic acid (D) at position 289 (D289), and aspartic acid (E) at position 589 (E289). In some embodiments, the one or more sequence variations comprise SEQ ID NO: 17900 amino acid substitutions within 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or any number therebetween of amino acids at positions 223, 289 and/or 289. In some embodiments, the one or more sequence variations comprise SEQ ID NO: 17900 amino acid substitutions within 70 amino acids at positions 223, 289 and/or 289. In some embodiments, the one or more sequence variations comprise SEQ ID NO: 17900 amino acid substitutions within 80 amino acids at positions 223, 289 and/or 289. In some embodiments, the one or more sequence variations comprise amino acid substitutions of aspartic acid (D) or aspartic acid (E) to a neutral amino acid, lysine (L), or arginine (R) (e.g., D223L, D223R, D289L, D289R, E289L, E289R of SEQ ID NO: 17900).

In some embodiments of the compositions and methods of the present disclosure, the mutant TcBuster transposase comprises one or more sequence variations when compared to a wild-type TcBuster transposase. In some embodiments, the one or more sequence variations comprise SEQ ID NO: 17900 of one or more of Q82, N85, D99, D132, Q151, E153, a154, Y155, E159, T171, K177, D183, D189, T191, S193, Y201, F202, C203, Q221, M222, I223, E224, S225, D227, R239, E243, E247, P257, Q258, E263, E274, S278, N281, L282, K292, V297, K299, a303, H322, a332, a358, D376, V377, L380, I398, F400, V431, S447, N450, I452, E469, K469, P517, P510, P517, P510, P590, P9, P559, P578, P559, P599, P578, P9, P599, P578, T599, P595, T599, P595, P599, P595, P59. In some embodiments, the one or more sequence variations comprise SEQ ID NO: 17900 of position 154, 155, 159, 171, 177, 183, 189, 191, 193, 201, 202, 203, 221, 223, 224, 225, 227, 239, 243, 247, 257, 258, 263, 274, 278, 281, 282, 292, 297, 299, 303, 322, 332, 358, 376, 377, 380, 398, 400, 431, 447, 450, 452, 469, 510, 517, 536, 553, 554, 559, 573, 578, 590, 595, 596, 598, 599, 615, 618, and 622 of amino acid substitution within 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or any number of amino acids therebetween.

In some embodiments of the compositions and methods of the present disclosure, the mutant TcBuster transposase comprises one or more sequence variations when compared to a wild-type TcBuster transposase. In some embodiments, when the sequence according to SEQ ID NO: 17900 when numbering, the one or more sequence variations comprise one or more of V377T, E469K and D189A.

In some embodiments of the compositions and methods of the present disclosure, the mutant TcBuster transposase comprises one or more sequence variations when compared to a wild-type TcBuster transposase. In some embodiments, when the sequence according to SEQ ID NO: 17900 the one or more sequence variations comprise one or more of K573E and E578L.

In some embodiments, when the sequence according to SEQ ID NO: 17900, the mutant TcBuster transposase contained the amino acid substitution I452K.

In some embodiments of the compositions and methods of the present disclosure, the mutant TcBuster transposase comprises one or more sequence variations when compared to a wild-type TcBuster transposase. In some embodiments, when the sequence according to SEQ ID NO: 17900 the one or more sequence variations comprise one or more of a 358K.

In some embodiments of the compositions and methods of the present disclosure, the mutant TcBuster transposase comprises one or more sequence variations when compared to a wild-type TcBuster transposase. In some embodiments, when the sequence according to SEQ ID NO: 17900 the one or more sequence variations comprise one or more V297K.

In some embodiments of the compositions and methods of the present disclosure, the mutant TcBuster transposase comprises one or more sequence variations when compared to a wild-type TcBuster transposase. In some embodiments, when the sequence according to SEQ ID NO: 17900 the one or more sequence variations comprise one or more of N85S.

In some embodiments of the compositions and methods of the present disclosure, the mutant TcBuster transposase comprises one or more sequence variations when compared to a wild-type TcBuster transposase. In some embodiments, when the sequence according to SEQ ID NO: 17900 when numbering, the one or more sequence variations comprise one or more of I452F, V377T, E469K and D189A.

In some embodiments of the compositions and methods of the present disclosure, the mutant TcBuster transposase comprises one or more sequence variations when compared to a wild-type TcBuster transposase. In some embodiments, when the sequence according to SEQ ID NO: 17900 when numbering, the one or more sequence variations comprise one or more of a358K, V377T, E469K and D189A.

In some embodiments of the compositions and methods of the present disclosure, the mutant TcBuster transposase comprises one or more sequence variations when compared to a wild-type TcBuster transposase. In some embodiments, when the sequence according to SEQ ID NO: 17900 when numbering, the one or more sequence variations comprise one or more of V377T, E469K, D189A, K573E and E578L.

In some embodiments of the compositions and methods of the present disclosure, the TcBuster transposase recognizes a 5' inverted repeat sequence comprising or consisting of:

in some embodiments of the compositions and methods of the present disclosure, the TcBuster transposase recognizes a 3' inverted repeat sequence comprising or consisting of:

in some embodiments of the compositions and methods of the present disclosure, the TcBuster transposase recognizes a polypeptide comprising SEQ ID NO: 17902 or a 5' inverted repeat sequence consisting thereof and a nucleic acid sequence comprising SEQ ID NO: 17903 or a 3' inverted repeat sequence consisting thereof.

In some embodiments of the compositions and methods of the present disclosure, the TcBuster transposase recognizes a 5' inverted repeat sequence comprising or consisting of:

In some embodiments of the compositions and methods of the present disclosure, the TcBuster transposase recognizes a 3' inverted repeat sequence comprising or consisting of:

in some embodiments of the compositions and methods of the present disclosure, the TcBuster transposase recognizes a polypeptide comprising SEQ ID NO: 17904 or a 5' inverted repeat consisting thereof and a nucleic acid sequence comprising SEQ ID NO: 17905 or a 3' inverted repeat sequence consisting thereof.

In some embodiments of the compositions and methods of the present disclosure, the TcBuster transposase recognizes a nucleic acid sequence comprising a sequence identical to SEQ ID NO: 17902. a sequence having or consisting of one or more of 17903, 17904, or 17905, a sequence of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% 97%, 99%, or any percent identity therebetween.

In some embodiments of the compositions and methods of the present disclosure, the TcBuster transposase recognizes an inverted repeat comprising or consisting of a sequence having at least one sequence. In some embodiments of the compositions and methods of the present disclosure, the TcBuster transposase recognizes a nucleic acid sequence comprising a sequence identical to SEQ ID NO: 17902. 17903, 17904, or 17905 or any portion thereof having 90-100% identity, a sequence of or consisting of at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 97, 99 or any number of contiguous nucleotides therebetween.

In some embodiments of the compositions and methods of the present disclosure, the TcBuster transposase recognizes a nucleic acid sequence comprising a sequence identical to SEQ ID NO: 17902. 17903, 17904, or 17905 or any portion thereof having 90-100% identity, a sequence of or consisting of at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 97, 99 or any number of discontinuous nucleotides therebetween.

In some embodiments of the compositions and methods of the present disclosure, the TcBuster transposon comprises a 3 'inverted repeat and a 5' inverted repeat. In some embodiments of the compositions and methods of the present disclosure, the TcBuster transposase recognizes a TcBuster transposon comprising a 3 'inverted repeat sequence and a 5' inverted repeat sequence, the 3 'inverted repeat sequence and the 5' inverted repeat sequence comprising a sequence identical to SEQ ID NO: 17902. 17903, 17904, or 17905 or any portion thereof having 90-100% identity, a sequence of or consisting of at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 8085, 90, 95, 97, 99 or any number of discontinuous nucleotides therebetween.

Genetically modified non-transposition-based methods

In some embodiments of the methods of the present disclosure, the modified HSCs or modified HSC progeny cells of the present disclosure may be produced by introducing a transgene into the HSCs or HSC progeny cells of the present disclosure. The introducing step can comprise delivering the nucleic acid sequence and/or the genome editing construct via a non-transposable delivery system.

In some embodiments of the methods of the present disclosure, introducing the nucleic acid sequence and/or genome editing construct into the HSC or HSC progeny cells ex vivo, in vitro, or in situ comprises one or more of local delivery, adsorption, uptake, electroporation, spin-infection (spin-infection), co-culture, transfection, mechanical delivery, sonic delivery, vibrational delivery, magnetic transfection (Magnetofoction), or delivery mediated by nanoparticles. In some embodiments of the methods of the present disclosure, introducing the nucleic acid sequence and/or genome editing construct into the HSC or HSC progeny cells ex vivo, in vitro, or in situ includes lipofection, calcium phosphate transfection, fugene transfection, and dendrimer mediated transfection. In some embodiments of the methods of the present disclosure, introducing the nucleic acid sequence and/or genome editing construct into the HSC or HSC progeny cells ex vivo, in vitro, or in situ by mechanical transfection comprises cell extrusion, cell bombardment, or gene gun techniques. In some embodiments of the methods of the present disclosure, introducing the nucleic acid sequence and/or genome editing construct into the HSC or HSC progeny cells ex vivo, in vitro, or in situ via nanoparticle-mediated transfection comprises liposome delivery, delivery via micelles, and delivery via polymersomes.

In some embodiments of the methods of the present disclosure, the nucleic acid sequence and/or genome editing construct is introduced into the HSC or HSC progeny cells, including non-viral vectors, ex vivo, in vitro, or in situ. In some embodiments, the non-viral vector comprises a nucleic acid. In some embodiments, the non-viral vector comprises plasmid DNA, linear double-stranded DNA (dsdna), linear single-stranded DNA (ssdna), DoggyBone^TMDNA, nanoplasmids, mini-circle DNA, single stranded oligodeoxynucleotides (ssODN), DDNA oligonucleotides, single stranded mrna (ssrna), and double stranded mrna (dsrna). In some embodiments, the non-viral vector comprises a transposon of the present disclosure.

In some embodiments of the methods of the present disclosure, the nucleic acid sequence and/or genome editing construct is introduced into the HSC or HSC progeny cells, including viral vectors, ex vivo, in vitro, or in situ. In some embodiments, the viral vector is a non-integrating, non-chromosomal vector. Exemplary non-integrative, non-chromosomal vectors include, but are not limited to, adeno-associated virus (AAV), adenovirus, and herpes virus. In some embodiments, the viral vector is an integrative chromosomal vector. Integrative chromosomal vectors include, but are not limited to, adeno-associated vectors (AAV), lentiviruses, and gamma retroviruses.

In some embodiments of the methods of the present disclosure, the nucleic acid sequence and/or genome editing construct is introduced into the HSC or HSC progeny cells, including combinations of vectors, ex vivo, in vitro, or in situ. Exemplary non-limiting combinations of vectors include: viral and non-viral vectors, many non-viral vectors or many viral vectors. Exemplary, but non-limiting, combinations of vectors include: combinations of DNA-derived and RNA-derived vectors, combinations of RNA and reverse transcriptase, combinations of transposons and transposases, combinations of non-viral vectors and endonucleases and combinations of viral vectors and endonucleases.

In some embodiments of the methods of the present disclosure, comprising introducing the nucleic acid sequence and/or the genome editing construct into the HSC or HSC progeny cells in vitro, in vivo, in vitro, or in situ stably integrates the nucleic acid sequence, transiently integrates the nucleic acid sequence, produces site-specific integration of the nucleic acid sequence, or produces biased integration of the nucleic acid sequence. In some embodiments, the nucleic acid sequence is a transgene.

In some embodiments of the methods of the present disclosure, comprising introducing the nucleic acid sequence and/or the genome editing construct into the HSC or HSC progeny cell genome modification stably integrates the nucleic acid sequence ex vivo, in vitro, or in situ. In some embodiments, stable chromosomal integration may be random integration, site-specific integration, or biased integration. In some embodiments, site-specific integration may be non-assisted or assisted. In some embodiments, the assisted site-specific integration is co-delivered with a site-directed nuclease. In some embodiments, the site-directed nuclease comprises a transgene having 5 'and 3' nucleotide sequence extensions that contain percent homology to regions upstream and downstream of the genomic integration site. In some embodiments, transgenes with homologous nucleotide extensions enable genomic integration by homologous recombination, micro-homology (microhomology) -mediated end joining, or non-homologous end joining. In some embodiments, site-specific integration occurs at a safe harbor site (safe harbor site). The genomic safe harbor site is capable of accommodating the integration of new genetic material in a manner that ensures that the newly inserted genetic element functions reliably (e.g., is expressed at therapeutically effective expression levels) and does not introduce deleterious alterations to the host genome that pose a risk to the host organism. Potential genomic safety harbors include, but are not limited to, the intron sequence of the human albumin gene, adeno-associated virus site 1(AAVS1), the naturally occurring integration site of AAV viruses on chromosome 19, the site of the chemokine (C-C motif) receptor 5(CCR5) gene, and the site of the human ortholog of the mouse Rosa26 locus.

In some embodiments, site-specific transgene integration occurs at a site that disrupts expression of a target gene. In some embodiments, disruption of target gene expression occurs by site-specific integration at an intron, an exon, a promoter, a genetic element, an enhancer, a repressor, an initiation codon, a stop codon, and a response element. In some embodiments, exemplary target genes targeted by site-specific integration include, but are not limited to, TRAC, TRAB, PDI, any immunosuppressive gene, and genes associated with allograft rejection (allo-rejection).

In some embodiments, site-specific transgene integration occurs at a site that results in enhanced expression of a target gene. In some embodiments, the enhancement of target gene expression occurs by site-specific integration at an intron, an exon, a promoter, a genetic element, an enhancer, a repressor, an initiation codon, a stop codon, and a response element.

In some embodiments of the methods of the present disclosure, enzymes may be used to generate strand breaks in the host genome to facilitate delivery or integration of the transgene. In some embodiments, the enzyme produces a single-strand break. In some embodiments, the enzyme generates a double strand break. In some embodiments, examples of cleavage-inducing enzymes include, but are not limited to: transposase, integrase, endonuclease, CRISPR-Cas9, transcription activator-like effector nuclease (TALEN), Zinc Finger Nuclease (ZFN), Cas-CLOVER ^TMAnd CPF 1. In some embodiments, the break-inducing enzyme may be encoded in DNA, encoded in mRNA, delivered to the cell as a protein, as a nucleoprotein complex with a guide rna (grna).

In some embodiments of the methods of the present disclosure, site-specific transgene integration is controlled by vector-mediated integration site bias. In some embodiments, the vector-mediated integration site bias is controlled by the selected lentiviral vector. In some embodiments, the vector-mediated integration site bias is controlled by the selected gamma-retroviral vector.

In some embodiments of the methods of the present disclosure, the site-specific transgene integration is an unstable chromosomal insertion. In some embodiments, the integrated transgene may become silenced, removed, excised, or further modified.

In some embodiments of the methods of the present disclosure, the genomic modification is unstable transgene integration. In some embodiments, the unstable integration may be transient non-chromosomal integration, semi-stable non-chromosomal integration, semi-persistent non-chromosomal insertion, or unstable chromosomal insertion. In some embodiments, the transient non-chromosomal insertion may be extrachromosomal (epi-chromosomal) or cytoplasmic.

In some embodiments, the transient non-chromosomal insertion of the transgene is not integrated into the chromosome, and the modified genetic material is not replicated during cell division.

In some embodiments of the methods of the present disclosure, the genomic modification is a semi-stable or persistent non-chromosomal integration of the transgene. In some embodiments, the DNA vector encodes a scaffold/matrix attachment region (S-MAR) assembly that binds to nuclear matrix proteins for episomal maintenance of the non-viral vector, thereby allowing autonomous replication in the nucleus of the dividing cell.

In some embodiments of the methods of the present disclosure, the genomic modification is chromosomal integration of a labile transgene. In some embodiments, the integrated transgene may become silenced, removed, excised, or further modified.

In some embodiments of the methods of the present disclosure, modification of the genome by transgene insertion may occur via host cell directed double-strand break repair (homology-directed repair) by Homologous Recombination (HR), microhomology-mediated end joining (MMEJ), nonhomologous end joining (NHEJ), transposase-mediated modification, integrase-mediated modification, endonuclease-mediated modification, or recombinase-mediated modification. In some embodiments, modification of the genome by transgene insertion can occur via CRISPR-Cas9, TALENs, ZFNs, Cas-close, and cpf 1.

In gene editing systems that include insertion of new or existing nucleotides/nucleic acids, in addition to a nicking enzyme (e.g., a nuclease, recombinase, integrase, or transposase), an insertion tool (e.g., a DNA template vector, transposable element (transposon or retrotransposon) must be delivered into a cell. But are not limited to, piggyBac transposons, Sleeping Beauty transposons, and L1 retrotransposons.

In certain embodiments of the methods of the present disclosure, the transgene is delivered in vivo. In certain embodiments of the methods of the present disclosure, in vivo transgene delivery may occur by: local delivery, adsorption, absorption, electroporation, rotational infection, co-culture, transfection, mechanical delivery, sonic delivery, vibrational delivery, magnetic transfection, or delivery mediated by nanoparticles. In certain embodiments of the methods of the present disclosure, in vivo transgene delivery by transfection may occur by lipofection, calcium phosphate transfection, fugene transfection, and dendrimer mediated transfection. In certain embodiments of the methods of the present disclosure, in vivo mechanical transgene delivery may occur through cell extrusion, bombardment, and gene gun. In certain embodiments of the methods of the present disclosure, in vivo nanoparticle-mediated transgene delivery may occur via liposome delivery, via micelle delivery, and via polymersome delivery. In various embodiments, nucleases that can be used as cleavage enzymes include, but are not limited to, Cas9, transcription activator-like effector nucleases (TALENs), and zinc finger nucleases.

In certain embodiments of the methods of the present disclosure, the non-viral vector is used for transgene delivery. In certain embodiments, the non-viral vector is a nucleic acid. In certain embodiments, the nucleic acid non-viral vector is plasmid DNA, linear double-stranded DNA (dsdna), linear single-stranded DNA (ssdna), DoggyBone^TMDNA, nanoplasmids, mini-circle DNA, single stranded oligodeoxynucleotides (ssODN), DDNA oligonucleotides, single stranded mrna (ssrna), and double stranded mrna (dsrna) in certain embodiments, the non-viral vector is a transposon. In certain embodiments, the transposon is piggyBac^TM。

In certain embodiments of the methods of the present disclosure, transgene delivery may occur via a viral vector. In certain embodiments, the viral vector is a non-integrating, non-chromosomal vector. Non-integrative non-chromosomal vectors may include adeno-associated virus (AAV), adenovirus and herpes virus. In certain embodiments, the viral vector is an integrative chromosomal vector. Integrative chromosomal vectors may include adeno-associated vectors (AAV), lentiviruses, and gamma retroviruses.

In certain embodiments of the methods of the present disclosure, transgene delivery may occur through a combination of vectors. Exemplary, but non-limiting, combinations of vectors can include: a virus plus a non-viral vector, more than one non-viral vector, or more than one viral vector. Exemplary, but non-limiting, combinations of vectors can include: DNA-derived plus RNA-derived vectors, RNA plus reverse transcriptase, transposons and transposases, non-viral vector plus endonucleases and viral vector plus endonucleases.

In certain embodiments of the methods of the present disclosure, the genomic modification may be stable integration of the transgene, transient integration of the transgene, site-specific integration of the transgene, or biased integration of the transgene.

In certain embodiments of the methods of the present disclosure, the genomic modification may be stable chromosomal integration of the transgene. In certain embodiments, stable chromosomal integration may be random integration, site-specific integration, or biased integration. In certain embodiments, site-specific integration may be non-assisted or assisted. In certain embodiments, the assisted site-specific integration is co-delivered with a site-directed nuclease. In certain embodiments, the site-directed nuclease comprises a transgene having 5 'and 3' nucleotide sequence extensions that contain homology to regions upstream and downstream of the genomic integration site. In certain embodiments, transgenes with homologous nucleotide extensions enable genomic integration by homologous recombination, microhomology (microhomology) -mediated end joining, or non-homologous end joining. In certain embodiments, site-specific integration occurs at a safe harbor site (safe harbor site). The genomic safe harbor site is capable of accommodating the integration of new genetic material in a manner that ensures that the newly inserted genetic element functions reliably (e.g., is expressed at therapeutically effective expression levels) and does not introduce deleterious alterations to the host genome that pose a risk to the host organism. Potential genomic safety harbors include, but are not limited to, the intron sequence of the human albumin gene, adeno-associated virus site 1(AAVS1), the naturally occurring integration site of AAV viruses on chromosome 19, the site of the chemokine (C-C motif) receptor 5(CCR5) gene, and the site of the human ortholog of the mouse Rosa26 locus.

In certain embodiments, site-specific transgene integration occurs at a site that disrupts expression of a target gene. In certain embodiments, disruption of target gene expression occurs by site-specific integration at an intron, an exon, a promoter, a genetic element, an enhancer, a repressor, an initiation codon, a stop codon, and a response element. In certain embodiments, exemplary target genes targeted by site-specific integration include, but are not limited to, TRAC, TRAB, PDI, any immunosuppressive gene, and genes associated with allograft rejection (allo-rejection).

In certain embodiments, site-specific transgene integration occurs at a site that results in enhanced expression of a target gene. In certain embodiments, the enhancement of target gene expression occurs by site-specific integration at an intron, an exon, a promoter, a genetic element, an enhancer, a repressor, an initiation codon, a stop codon, and a response element.

In certain embodiments of the methods of the present disclosure, enzymes may be used to generate strand breaks in the host genome to facilitate delivery or integration of the transgene. In certain embodiments, the enzyme produces a single-strand break. In certain embodiments, the enzyme generates a double strand break. In certain embodiments, examples of cleavage-inducing enzymes include, but are not limited to: transposase, integrase, endonuclease, CRISPR-Cas9, transcription activator-like effector nuclease (TALEN), Zinc Finger Nuclease (ZFN), Cas-CLOVER ^TMAnd cpf 1. In certain embodiments, the break-inducing enzyme may be encoded in DNA, encoded in mRNA, delivered to the cell as a protein, as a nucleoprotein complex with a guide rna (grna).

In certain embodiments of the methods of the present disclosure, site-specific transgene integration is controlled by vector-mediated integration site bias. In certain embodiments, the vector-mediated integration site bias is controlled by the selected lentiviral vector. In certain embodiments, the vector-mediated integration site bias is controlled by the selected gamma-retroviral vector.

In certain embodiments of the methods of the present disclosure, the site-specific transgene integration is an unstable chromosomal insertion. In certain embodiments, the integrated transgene may become silenced, removed, excised, or further modified. In certain embodiments of the methods of the present disclosure, the genomic modification is unstable transgene integration. In certain embodiments, the unstable integration may be transient non-chromosomal integration, semi-stable non-chromosomal integration, semi-persistent non-chromosomal insertion, or unstable chromosomal insertion. In certain embodiments, the transient non-chromosomal insertion may be extrachromosomal (epi-chromosomal) or cytoplasmic. In certain embodiments, the transient nonchromosomal insertion of the transgene is not integrated into the chromosome and the modified genetic material does not replicate during cell division.

In certain embodiments of the methods of the present disclosure, the genomic modification is a semi-stable or persistent non-chromosomal integration of the transgene. In certain embodiments, the DNA vector encodes a scaffold/matrix attachment region (S-MAR) module that binds to nuclear matrix proteins for episomal maintenance of the non-viral vector, thereby allowing autonomous replication in the nucleus of dividing cells.

In certain embodiments of the methods of the present disclosure, the genomic modification is chromosomal integration of a labile transgene. In certain embodiments, the integrated transgene may become silenced, removed, excised, or further modified.

In certain embodiments of the methods of the present disclosure, modification of the genome by transgene insertion may occur via host cell directed double-strand break repair (homology-directed repair) by Homologous Recombination (HR), microhomology-mediated end joining (MMEJ), nonhomologous end joining (NHEJ), transposase-mediated modification, integrase-mediated modification, endonuclease-mediated modification, or recombinase-mediated modification. In certain embodiments, modification of the genome by transgene insertion can occur via CRISPR-Cas9, TALENs, ZFNs, Cas-close, and cpf 1.

In certain embodiments of the methods of the present disclosure, the cell having in vivo or ex vivo genomic modification may be a germline cell or a somatic cell. In certain embodiments, the modified cell may be a human, non-human, mammalian, rat, mouse, or dog cell. In certain embodiments, the modified cell may be differentiated, undifferentiated, or immortalized. In certain embodiments, the modified undifferentiated cell may be a stem cell. In certain embodiments, the modified cell may be differentiated, undifferentiated, or immortalized. In certain embodiments, the modified undifferentiated cell may be an induced pluripotent stem cell. In certain embodiments, the modified cell may be a T cell, hematopoietic stem cell, natural killer cell, macrophage, dendritic cell, monocyte, megakaryocyte, or osteoclast. In certain embodiments, the modified cell may be modified while the cell is quiescent, in an activated state, quiescent, in an interphase, in a pre-division, in a metaphase, in a anaphase, or in a telophase. In certain embodiments, the modified cells may be fresh, cryopreserved, bulk, sorted into subpopulations, from whole blood, from leukapheresis (leukapheresis), or from immortalized cell lines.

Centyrins

The centrins of the present disclosure may be derived from fibronectin type III (FN3) repeat proteins, encoding or complementary nucleic acids, vectors, host cells, compositions, combinations, formulations, devices, and methods of making and using the same. In a preferred embodiment, the centryrin comprises a consensus sequence of multiple FN3 domains from human tenascin-C (hereinafter "tenascin"). In a further preferred embodiment, the protein scaffold of the invention is a consensus sequence of 15 FN3 domains. The centrins of the present disclosure can be designed to bind to various molecules, e.g., cellular target proteins. In preferred embodiments, the centrins of the present disclosure may be designed to bind to epitopes of wild-type and/or variant forms of the antigen.

The centrins of the present disclosure may include additional molecules or moieties, for example, the Fc region of an antibody, albumin binding domain, or other moieties that affect half-life. In further embodiments, the centrins of the present disclosure may be associated with nucleic acid molecules that may encode the centrins.

The present disclosure provides at least one method for expressing at least one centrin based on a consensus sequence of multiple FN3 domains in a host cell, comprising culturing a host cell as described herein under conditions wherein at least one protein scaffold is expressed in detectable and/or recoverable amounts.

The present disclosure provides at least one composition comprising (a) a centrin and/or encoding nucleic acid based on a consensus sequence of multiple FN3 domains, as described herein; and (b) a suitable and/or pharmaceutically acceptable carrier or diluent.

The present disclosure provides methods of generating a centryrin library based on a fibronectin type III (FN3) repeat protein, preferably a consensus sequence of multiple FN3 domains, and more preferably a consensus sequence of multiple FN3 domains from human tenascin. The library is formed by the sequential production of centryrins by altering (by mutation) the amino acids or the number of amino acids in a molecule at a specific position in the part of the centrin, e.g. in the loop region. Libraries can be generated by altering the amino acid composition of a single loop or simultaneously altering additional positions of multiple loops or centrin molecules. The altered loop may be lengthened or shortened accordingly. Such libraries can be generated to include all possible amino acids at each position, or a subset of designed amino acids. Library members can be screened by display, such as in vitro or CIS display (DNA, RNA, ribosome display, etc.), yeast, bacterial and phage display.

The centrins of the present disclosure provide enhanced biophysical properties, such as stability under reducing conditions and solubility at high concentrations; they can be expressed and folded in prokaryotic systems such as e.coli, eukaryotic systems such as yeast, and in vitro transcription/translation systems such as rabbit reticulocyte lysate systems.

The present disclosure provides methods for generating centryrin molecules that bind to a particular target by panning a centryrin library of the invention with the target and detecting the binders (binders). In other related aspects, the disclosure includes screening methods that can be used to generate or affinity mature proteins having a desired activity, e.g., centrins that are capable of binding a target protein with an affinity. Affinity maturation can be accomplished by repeated rounds of mutagenesis and selection using systems such as phage display or in vitro display. Mutagenesis in this process may be the result of site-directed mutagenesis of specific centrin residues, random mutagenesis due to error-prone PCR, DNA shuffling, and/or a combination of these techniques.

The present disclosure provides isolated, recombinant, and/or synthetic centrins based on the consensus sequence of fibronectin type III (FN3) repeat proteins, including, but not limited to, mammalian-derived centrins, as well as compositions and encoding nucleic acid molecules comprising at least one polynucleotide encoding a centrin based on the consensus FN3 sequence. The present disclosure further includes, but is not limited to, methods of making and using such nucleic acids and centrins, including diagnostic and therapeutic compositions, methods and devices.

The centrins of the present disclosure provide advantages over conventional treatments such as the ability to be administered topically, orally, or across the blood brain-barrier, the ability to be expressed in e.coli, thereby allowing for increased protein expression as a function of resource as compared to mammalian cell expression, the ability to be engineered into bispecific or tandem molecules that can bind to multiple targets or multiple epitopes of the same target, the ability to be conjugated to drugs, polymers, and probes, the ability to be formulated at high concentrations, and the ability of such molecules to effectively penetrate diseased tissues and tumors.

In addition, centrins possess many antibody properties related to their ability to mimic the folding of antibody variable regions. This orientation enables the FN3 loop to be exposed similar to antibody Complementarity Determining Regions (CDRs). They should be able to bind to cellular targets and the loops can be altered, e.g., affinity matured, to improve certain binding or related properties.

Three of the six loops of the centryrin of the present disclosure correspond topologically to the complementarity determining regions (CDRs 1-3) of the antibody, i.e., the antigen binding regions, while the remaining three loops are surface exposed in a manner similar to the CDRs of the antibody. These loops span SEQ ID NO: 18018, residues 13-16, 22-28, 38-43, 51-54, 60-64, and 75-81. Preferably, the amino acid sequence of SEQ ID NO: 18018, residues 22-28, 51-54 and 75-81 or the surrounding loop region. One or more of these loop regions are randomized with other loop regions and/or other chains that maintain their sequence as part of the backbone to provide libraries, and effective binders can be selected from libraries that have high affinity for a particular protein target. One or more of the loop regions may interact with a target protein, similar to the interaction of antibody CDRs with the protein.

In certain embodiments of the present disclosure, PSMA-specific centrins are designed, evolved, and/or selected for their ability to specifically bind to the sequence of PSMA. In certain embodiments, PSMA-specific centrins are capable of binding to a sequence of PSMA with an affinity similar to that with which an anti-PSMA antibody binds an epitope of PSMA. In certain embodiments, PSMA-specific centrins are capable of binding to a sequence of PSMA with greater affinity than that with which an anti-PSMA antibody binds an epitope of PSMA. In certain embodiments, PSMA-specific centrins are capable of binding to sequences of PSMA to which an antibody is not capable of binding. For example, the sequence of PSMA may be discontinuous or may have a secondary structure.

Production and Generation of CARTyrins

As is well known in the art, at least one CARTyrin of the present disclosure can optionally be produced from a cell line, a mixed cell line, an immortalized cell, or a clonal population of immortalized cells. See, e.g., Ausubel et al, eds, Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, N.Y. (1987-2001); sambrook et al, Molecular Cloning: a Laboratory Manual, 2 nd edition, Cold Spring Harbor, N.Y. (1989); harlow and Lane, Antibodies, a Laboratory Manual, Cold Spring Harbor, N.Y. (1989); colligan et al, eds., Current Protocols in Immunology, John Wiley & Sons, Inc., NY (1994-2001); colligan et al, Current Protocols in Protein Science, John Wiley & Sons, NY, N.Y. (1997-2001).

Amino acids from CARTyrin can be altered, added, and/or deleted to reduce immunogenicity or to reduce, enhance or modify binding, affinity, association rate, dissociation rate, avidity, specificity, half-life, stability, solubility, or any other suitable characteristic, as known in the art.

Optionally, CARTyrins can be engineered while retaining high affinity for antigens and other favorable biological properties. To achieve this goal, CARTyrins can optionally be prepared by an analytical process of the parental sequences and various conceptually engineered products using three-dimensional models of the parental and engineered sequences. Three-dimensional models are commonly available and familiar to those skilled in the art. Possible three-dimensional conformational structures of selected candidate sequences are exemplified and shown, and computer programs are available that can measure possible immunogenicity (e.g., the immunopilter program by Xencor, inc., of monprovia, calif.). Examination of these displays allows analysis of the likely role of the residues in the functional performance of the candidate sequence, i.e., analysis of residues that affect the ability of the candidate centrin or CARTyrin to bind its antigen. In this way, residues can be selected from the parent and reference sequences and combined to obtain a desired characteristic, such as affinity for one or more target antigens. Alternatively, or in addition to the above procedures, other suitable modification methods may be used.

Screening of CARTyrin protein

Screening for specific binding of centrins or CARTyrins to similar proteins or fragments can be conveniently accomplished using nucleotide (DNA or RNA display) or peptide display libraries, e.g., in vitro display. This method involves screening a large panel of peptides against individual members having a desired function or structure. The displayed nucleotide or peptide sequence may be 3 to 5000 or more nucleotides or amino acids in length, often 5-100 amino acids in length, and often about 8-25 amino acids in length. In addition to direct chemical synthesis methods for generating peptide libraries, have been describedSeveral recombinant DNA methods are described. One type involves the display of peptide sequences on the surface of a phage or cell. Each bacteriophage or cell contains a nucleotide sequence encoding a particular displayed peptide sequence. The centrorins CARTyrin of the present disclosure can bind human or other mammalian proteins with a wide variety of affinities (KD). In preferred embodiments, at least one centrin of the invention may optionally bind a target protein with high affinity, e.g., with a KD equal to or less than about 10^-7M, such as, but not limited to, 0.1-9.9 (or any range or value therein) X10^-8、10^-9、10^-10、10^-11、10^-12、10^-13、10^-14、10^-15Or any range or value therein, as determined by surface plasmon resonance or the Kinexa method, as practiced by those skilled in the art.

The affinity or avidity of centryrin or CARTyrin for an antigen can be determined experimentally using any suitable method. (see, e.g., Berzofsky et al, "Antibody-Antibody Interactions," In Fundamental Immunology, Paul, W.E., eds., Raven Press: New York, N.Y. (1984); Kuby, Janis Immunology, W.H.Freeman and Company: New York, N.Y. (1992); and methods described herein). The measured affinity of a particular centryrin-antigen or centryrin-antigen interaction can vary if measured under different conditions (e.g., salt concentration, pH). Thus, measurements of affinity and other antigen binding parameters (e.g., KD, Kon, Koff) are preferably performed with standardized solutions of centrins or CARTyrins and antigen, as well as standardized buffers such as those described herein.

Competitive assays can be performed with the centryrin or CARTyrin of the present disclosure to determine what proteins, antibodies and other antagonists compete with the centryrin or CARTyrin of the present invention for binding to a target protein and/or sharing epitope regions. These assays, as readily known to those of ordinary skill in the art, evaluate competition between antagonists or ligands for a limited number of binding sites on the protein. Before or after the competition, the proteins and/or antibodies are immobilized or insolubilized and the sample bound to the target protein is separated from the unbound sample, for example, by decantation (where the proteins/antibodies are not solubilized beforehand) or by centrifugation (where the proteins/antibodies are precipitated after the competitive reaction). Likewise, competitive binding can be determined by whether binding or non-binding of the centrin or CARTyrin to the target protein alters function, e.g., whether the centrin or CARTyrin molecule inhibits or enhances enzymatic activity of, e.g., a label. ELISA and other functional assays can be used as is well known in the art.

Nucleic acid molecules

Nucleic acid molecules of the present disclosure encoding centrins or CARTyrins can be in the form of RNA, e.g., mRNA, hnRNA, tRNA, or any other form, or can be in the form of DNA, including, but not limited to, cDNA and genomic DNA obtained by cloning or produced synthetically, or any combination thereof. The DNA may be triplex, double stranded or single stranded or any combination thereof. Any portion of at least one strand of the DNA or RNA may be the coding strand, also referred to as the sense strand, or it may be the non-coding strand, also referred to as the antisense strand.

An isolated nucleic acid molecule of the present disclosure can include a nucleic acid molecule comprising an Open Reading Frame (ORF), optionally with one or more introns, such as, but not limited to, at least one designated portion of at least one CARTyrin; a nucleic acid molecule comprising a coding sequence for CARTyrin; and nucleic acid molecules comprising a nucleotide sequence that is significantly different from the nucleotide sequences described above, but which, due to the degeneracy of the genetic code, still encodes a CARTyrin as described herein and/or as known in the art. Of course, the genetic code is well known in the art. Thus, it will be routine for one skilled in the art to generate such degenerate nucleic acid variants that encode a particular CARTyrins of the invention. See, e.g., Ausubel et al, supra, and such nucleic acid variants are included in the present invention.

As shown herein, nucleic acid molecules of the present disclosure comprising a CARTyrin-encoding nucleic acid can include, but are not limited to, those that independently encode the amino acid sequence of a centryrin fragment; the coding sequence of the entire CARTyrin or a portion thereof; a coding sequence for the centryrin, fragment or portion and additional sequences, such as at least one signal leader sequence or fusion peptide, with or without the aforementioned additional coding sequences, such as at least one intron, together with additional non-coding sequences, including but not limited to non-coding 5 'and 3' sequences, such as transcribed non-translated sequences that function in transcription, mRNA processing, including splicing and polyadenylation signals (e.g., ribosome binding and stability of mRNA); additional coding sequences that encode additional amino acids, such as those that provide additional functionality. Thus, a sequence encoding CARTyrin can be fused to a marker sequence, for example, a sequence encoding a peptide that facilitates purification of a CARTyrin comprising a centryrin fragment or portion of the fusion.

Polynucleotides that selectively hybridize to polynucleotides as described herein

The present disclosure provides isolated nucleic acids that hybridize under selective hybridization conditions to the polynucleotides disclosed herein. Thus, the polynucleotides of this embodiment can be used to isolate, detect and/or quantify nucleic acids comprising such polynucleotides. For example, the polynucleotides of the invention can be used to identify, isolate or amplify partial or full length clones in a deposited library. In some embodiments, the polynucleotide is a genomic or cDNA sequence isolated from or otherwise complementary to cDNA from a human or mammalian nucleic acid library.

Preferably, the cDNA library comprises at least 80% of the full-length sequence, preferably at least 85% or 90% of the full-length sequence, and more preferably, at least 95% of the full-length sequence. The cDNA library can be normalized to increase the representation of rare sequences. Low or medium stringency hybridization conditions are generally, but not exclusively, used with sequences having reduced sequence identity relative to the complementary sequence. For sequences of higher identity, medium and high stringency conditions can optionally be used. Low stringency conditions allow selective hybridization of sequences with about 70% sequence identity and can be used to identify orthologous or paralogous sequences.

Optionally, a polynucleotide of the invention will encode at least a portion of the CARTyrin encoded by the polynucleotides described herein. Polynucleotides of the invention include nucleic acid sequences that can be used to selectively hybridize to a polynucleotide encoding a CARTyrin of the invention. See, e.g., Ausubel, supra; colligan, supra, each of which is incorporated by reference herein in its entirety.

Construction of nucleic acids

Isolated nucleic acids of the present disclosure can be prepared using (a) recombinant methods, (b) synthetic techniques, (c) purification techniques, and/or (d) combinations thereof, as are well known in the art.

The nucleic acid may conveniently comprise a sequence other than a polynucleotide of the invention. For example, a multiple cloning site comprising one or more endonuclease restriction sites can be inserted into a nucleic acid to aid in the isolation of the polynucleotide. Likewise, translatable sequences may be inserted to aid in the isolation of translated polynucleotides of the present disclosure. For example, a hexahistidine tag sequence provides a convenient means of purifying the proteins of the present disclosure. In addition to the coding sequence, the nucleic acids of the disclosure are optionally vectors, adaptors, or linkers for cloning and/or expressing the polynucleotides of the disclosure.

Additional sequences may be added to such cloning and/or expression sequences to optimize their function in cloning and/or expression, to aid in the isolation of the polynucleotide, or to improve the introduction of the polynucleotide into the cell. The use of cloning vectors, expression vectors, adaptors, and linkers is well known in the art. (see, e.g., Ausubel, supra; or Sambrook, supra).

Recombinant method for constructing nucleic acids

The isolated nucleic acid compositions of the present disclosure can be obtained from biological sources, e.g., RNA, cDNA, genomic DNA, or any combination thereof, using a number of cloning methods known to those of skill in the art. In some embodiments, oligonucleotide probes that selectively hybridize under stringent conditions to polynucleotides of the invention are used to identify a desired sequence in a cDNA or genomic DNA library. The isolation of RNA and the construction of cDNA and genomic libraries is well known to those of ordinary skill in the art. (see, e.g., Ausubel, supra; or Sambrook, supra).

Nucleic acid screening and isolation method

Probes can be used to screen cDNA or genomic libraries based on the sequence of the polynucleotides of the disclosure. Probes can be used to hybridize to genomic DNA or cDNA sequences to isolate homologous genes in the same or different organisms. Those skilled in the art will appreciate that various degrees of hybridization stringency can be employed in the assay; and the hybridization or wash medium may be stringent. As hybridization conditions become more stringent, a greater degree of complementarity must exist between the probe and target in order for duplex formation to occur. The degree of stringency can be controlled by one or more of temperature, ionic strength, pH and the presence of partially denaturing solvents such as formamide. For example, the stringency of hybridization can be conveniently varied by changing the polarity of the reactant solution, for example by manipulating the concentration of formamide in the range of 0% to 50%. The degree of complementarity (sequence identity) required for detectable binding will vary depending on the stringency of the hybridization medium and/or wash medium. The degree of complementarity will optimally be 100% or 70-100%, or any range or value therein. However, it will be appreciated that minor sequence variations in the probes and primers may be compensated for by reducing the stringency of the hybridization and/or wash medium.

Methods of amplification of RNA or DNA are well known in the art and can be used in accordance with the present disclosure without undue experimentation based on the teachings and guidance presented herein.

Known methods of DNA or RNA amplification include, but are not limited to, Polymerase Chain Reaction (PCR) and related amplification methods (see, e.g., U.S. Pat. Nos.4,683,195, 4,683,202, 4,800,159, 4,965,188, to Mullis et al; 4,795,699 and 4,921,794, to Tabor et al; 5,142,033, to Innis; 5,122,464, to Wilson et al; 5,091,310, to Innis; 5,066,584, to Gyllensten et al; 4,889,818, to Gelfand et al; 4,994,370, to Silver et al; 4,766,067, to Biswas; 4,656,134, to Ringold) and RNA-mediated amplification using antisense RNA against a target sequence as a template for double-stranded DNA synthesis (U.S. Pat. No.5,130,238, to Malek et al, under the trade name of BA), the entire disclosures of which are incorporated herein by reference. (see, e.g., Ausubel, supra; or Sambrook, supra.)

For example, Polymerase Chain Reaction (PCR) techniques can be used to amplify the sequences of polynucleotides and related genes of the present disclosure directly from genomic DNA or cDNA libraries. PCR and other in vitro amplification methods may also be useful, for example, for cloning nucleic acid sequences encoding proteins to be expressed, such that the nucleic acids serve as probes for detecting the presence of a desired mRNA in a sample, for nucleic acid sequencing, or for other purposes. Examples of techniques sufficient to guide the skilled artisan in vitro amplification methods are described in Berger, supra, Sambrook, supra, and Ausubel, supra, and Mullis et al, U.S. Pat. No.4,683,202 (1987); and Innis et al, PCR Protocols A guides to Methods and Applications, eds., Academic Press Inc., San Diego, Calif. (1990). Commercially available kits for genomic PCR amplification are known in the art. See, for example, Advantage-GC genomic PCR kit (Clontech). In addition, for example, the T4 gene 32 protein (Boehringer Mannheim) can be used to increase the yield of long PCR products.

Synthetic methods for constructing nucleic acids

Isolated nucleic acids of the disclosure can also be prepared by direct chemical synthesis by known methods (see, e.g., Ausubel et al, supra). Chemical synthesis typically produces single-stranded oligonucleotides that can be converted to double-stranded DNA by hybridization to a complementary sequence or by polymerization with a DNA polymerase using the single strand as a template. One skilled in the art will recognize that although chemical synthesis of DNA may be limited to sequences of about 100 bases or more, longer sequences may be obtained by ligating shorter sequences.

Recombinant expression cassette

The present disclosure further provides recombinant expression cassettes comprising a nucleic acid of the present disclosure. Nucleic acid sequences of the disclosure, such as cDNA or genomic sequences encoding the CARTyrin of the disclosure, can be used to construct recombinant expression cassettes, which can be introduced into at least one desired host cell. A recombinant expression cassette will generally comprise a polynucleotide of the present disclosure operably linked to a transcription initiation regulatory sequence that will direct transcription of the polynucleotide in the intended host cell. Both heterologous and non-heterologous (i.e., endogenous) promoters can be used to direct expression of the nucleic acids of the disclosure.

In some embodiments, an isolated nucleic acid used as a promoter, enhancer, or other element may be introduced into an appropriate location (upstream, downstream, or in an intron) of a non-heterologous form of a polynucleotide of the present disclosure in order to up-regulate or down-regulate expression of the polynucleotide of the present disclosure. For example, endogenous promoters may be altered in vivo or in vitro by mutation, deletion, and/or substitution.

Nano transposon

The present disclosure provides a nano-transposon, comprising: (a) a sequence encoding a transposon insert comprising a sequence encoding a first Inverted Terminal Repeat (ITR), a sequence encoding a second Inverted Terminal Repeat (ITR), and an intra-ITR sequence; (b) a sequence encoding a backbone, wherein the sequence encoding a backbone comprises a sequence encoding an origin of replication having from 1 to 450 nucleotides (inclusive) and a sequence encoding a selectable marker having from 1 to 200 nucleotides (inclusive), and (c) an inter-ITR sequence. In some embodiments, the inter-ITR sequence of (c) comprises the sequence of (b). In some embodiments, the intra-ITR sequence of (a) comprises the sequence of (b).

In some embodiments of the nano-transposons of the present disclosure, the sequence encoding the backbone comprises 1 to 600 nucleotides (inclusive). In some embodiments, the sequence encoding the backbone consists of 1 to 50 nucleotides, 50 to 100 nucleotides, 100 to 150 nucleotides, 150 to 200 nucleotides, 200 to 250 nucleotides, 250 to 300 nucleotides, 300 to 350 nucleotides, 350 to 400 nucleotides, 400 to 450 nucleotides, 450 to 500 nucleotides, 500 to 550 nucleotides, 550 to 600 nucleotides, each inclusive.

In some embodiments of the femto transposons of the present disclosure, the inter-ITR sequence comprises 1 to 1000 nucleotides (inclusive). In some embodiments, the inter-ITR sequence consists of 1 to 50 nucleotides, 50 to 100 nucleotides, 100 to 150 nucleotides, 150 to 200 nucleotides, 200 to 250 nucleotides, 250 to 300 nucleotides, 300 to 350 nucleotides, 350 to 400 nucleotides, 400 to 450 nucleotides, 450 to 500 nucleotides, 500 to 550 nucleotides, 550 to 600 nucleotides, 600 to 650 nucleotides, 650 to 700 nucleotides, 700 to 750 nucleotides, 750 to 800 nucleotides, 800 to 850 nucleotides, 850 to 900 nucleotides, 900 to 950 nucleotides, or 950 to 1000 nucleotides, each range being inclusive.

In some embodiments of the nanotransposons of the present disclosure, including Short Nanotransposons (SNTs) of the present disclosure, the inter-ITR sequence comprises 1 to 200 nucleotides (inclusive). In some embodiments, the inter-ITR sequence consists of 1 to 10 nucleotides, 10 to 20 nucleotides, 20 to 30 nucleotides, 30 to 40 nucleotides, 40 to 50 nucleotides, 50 to 60 nucleotides, 60 to 70 nucleotides, 70 to 80 nucleotides, 80 to 90 nucleotides, or 90 to 100 nucleotides, each inclusive.

In some embodiments of the nano-transposons of the present disclosure, a selectable marker having 1 to 200 nucleotides (inclusive) comprises a sequence encoding a sucrose selectable marker. In some embodiments, the sequence encoding the sucrose selection marker comprises a sequence encoding an RNA-OUT sequence. In some embodiments, the sequence encoding the RNA-OUT sequence comprises or consists of 137 base pairs (bp). In some embodiments, a selectable marker having 1 to 200 nucleotides (inclusive) comprises a sequence encoding a fluorescent marker. In some embodiments, a selectable marker having 1 to 200 nucleotides (inclusive) includes a sequence encoding a cell surface marker.

In some embodiments of the nanotransposons of the present disclosure, the sequence encoding an origin of replication having from 1 to 450 nucleotides (inclusive) comprises a sequence encoding a small origin of replication (minor of replication). In some embodiments, the sequence encoding an origin of replication having from 1 to 450 nucleotides (inclusive) comprises a sequence encoding an R6K origin of replication. In some embodiments, the R6K origin of replication comprises the R6K γ origin of replication. In some embodiments, the R6K origin of replication comprises a R6K small origin of replication. In some embodiments, the R6K origin of replication comprises the R6K γ small origin of replication. In some embodiments, the R6K γ small origin of replication comprises or consists of 281 base pairs (bp).

In some embodiments of the nano-transposons of the present disclosure, the sequences encoding the backbone do not comprise recombination sites, excision sites, ligation sites, or a combination thereof. In some embodiments, neither the sequences of the nano-transposon and the coding backbone comprise products of recombination sites, excision sites, ligation sites, or a combination thereof. In some embodiments, neither the sequences of the nano-transposon nor the coding backbone are derived from recombination sites, excision sites, ligation sites, or a combination thereof.

In some embodiments of the femto transposons of the present disclosure, the recombination sites comprise sequences resulting from a recombination event. In some embodiments, the recombination site comprises a sequence that is the product of a recombination event. In some embodiments, the recombination event comprises an activity of a recombinase (e.g., a recombinase site).

In some embodiments of the nano-transposons of the present disclosure, the sequences encoding the backbone do not further comprise sequences encoding exogenous DNA.

In some embodiments of the femto transposons of the present disclosure, the inter-ITR sequences do not comprise recombination sites, excision sites, ligation sites, or a combination thereof. In some embodiments, the inter-ITR sequences do not comprise the product of a recombination event, a cleavage event, a ligation event, or a combination thereof. In some embodiments, the inter-ITR sequences are not derived from a recombination event, a cleavage event, a ligation event, or a combination thereof.

In some embodiments of the femto transposons of the present disclosure, the inter-ITR sequences comprise sequences encoding exogenous DNA.

In some embodiments of the femto transposons of the present disclosure, the sequences within the ITRs comprise at least one sequence encoding an insulator and a sequence encoding a promoter capable of expressing the exogenous sequence in a mammalian cell. In some embodiments, the mammalian cell is a human cell.

In some embodiments of the femto transposons of the present disclosure, the sequences within the ITRs comprise a first sequence encoding an insulator, a sequence encoding a promoter capable of expressing the exogenous sequence in a mammalian cell, and a second sequence encoding an insulator.

In some embodiments of the nano-transposons of the present disclosure, the sequences within the ITRs comprise a first sequence encoding an insulator, a sequence encoding a promoter capable of expressing the exogenous sequence in a mammalian cell, a poly adenosine (polyA) sequence, and a second sequence encoding an insulator.

In some embodiments of the nano-transposons of the present disclosure, the sequences within the ITRs comprise a first sequence encoding an insulator, a sequence encoding a promoter capable of expressing the exogenous sequence in a mammalian cell, at least one exogenous sequence, a poly adenosine (polyA) sequence, and a second sequence encoding an insulator.

In some embodiments of the nano-transposons of the present disclosure, the sequence encoding a promoter capable of expressing the exogenous sequence in a mammalian cell is capable of expressing the exogenous sequence in a human cell. In some embodiments, the sequence encoding a promoter capable of expressing the exogenous sequence in a mammalian cell comprises a sequence encoding a constitutive promoter. In some embodiments, the sequence encoding a promoter capable of expressing the exogenous sequence in a mammalian cell comprises a sequence encoding an inducible promoter. In some embodiments, the internal ITR sequence comprises a first sequence encoding a first promoter capable of expressing the exogenous sequence in the mammalian cell and a second sequence encoding a second promoter capable of expressing the exogenous sequence in the mammalian cell, wherein the first promoter is a constitutive promoter, wherein the second promoter is an inducible promoter, and wherein the first sequence encoding the first promoter and the second sequence encoding the second promoter are oriented in opposite directions. In some embodiments, the sequence encoding a promoter capable of expressing the exogenous sequence in a mammalian cell comprises a sequence encoding a cell-type or tissue-type specific promoter. In some embodiments, the sequence encoding a promoter capable of expressing the exogenous sequence in a mammalian cell includes a sequence encoding an EF1a promoter, a sequence encoding a CMV promoter, a sequence encoding an MND promoter, a sequence encoding an SV40 promoter, a sequence encoding a PGK1 promoter, a sequence encoding an Ubc promoter, a sequence encoding a CAG promoter, a sequence encoding an H1 promoter, or a sequence encoding a U6 promoter.

In some embodiments of the nano-transposons of the present disclosure, the poly adenosine (polyA) sequences are isolated or derived from viral polyA sequences. In some embodiments, a poly adenosine (polyA) sequence is isolated or derived from a (SV40) polyA sequence.

In some embodiments of the femto transposons of the present disclosure, the at least one exogenous sequence comprises an inducible pro-apoptotic polypeptide. In some embodiments, the inducible caspase polypeptide comprises (a) a ligand binding region, (b) a linker, and (c) a caspase polypeptide, wherein the inducible pro-apoptotic polypeptide does not comprise a non-human sequence. In some embodiments, the inducible caspase polypeptide comprises (a) a ligand binding region, (b) a linker, and (c) a truncated caspase 9 polypeptide, wherein the inducible pro-apoptotic polypeptide does not comprise a non-human sequence.

In some embodiments of the nanotransposons of the present disclosure, including those in which the at least one exogenous sequence comprises an inducible pro-apoptotic polypeptide, the ligand binding region comprises an FK506 binding protein 12(FKBP12) polypeptide. In some embodiments, the amino acid sequence of the ligand binding region comprises an FK506 binding protein 12(FKBP12) polypeptide. In some embodiments, the FK506 binding protein 12(FKBP12) polypeptide comprises a modification at position 36 of the sequence. In some embodiments, the modification comprises a substitution of valine (V) for phenylalanine (F) at position 36 (F36V). In some embodiments, the FKBP12 polypeptide is encoded by an amino acid sequence comprising:

In some embodiments, the FKBP12 polypeptide is encoded by a nucleic acid sequence comprising:

in some embodiments of the nanotransposons of the present disclosure, including those in which at least one of the exogenous sequences comprises an inducible pro-apoptotic polypeptide, the linker region is encoded by an amino acid comprising GGGGS (SEQ ID NO: 14637) or a nucleic acid sequence comprising GGAGGAGGAGGATCC (SEQ ID NO: 14638). In some embodiments, the nucleic acid sequence encoding the linker does not comprise a restriction enzyme site.

In some embodiments of the nano-transposons of the present disclosure, including those in which at least one exogenous sequence comprises an inducible pro-apoptotic polypeptide, the truncated caspase 9 polypeptide is encoded by an amino acid sequence that does not comprise arginine (R) at position 87 of the sequence. In some embodiments, the truncated caspase 9 polypeptide is encoded by an amino acid sequence that does not include alanine (a) at position 282 of the sequence. In some embodiments, the truncated caspase 9 polypeptide is encoded by an amino acid comprising:

in some embodiments, the truncated caspase 9 polypeptide is encoded by a nucleic acid sequence comprising:

In some embodiments, the inducible pro-apoptotic polypeptide is encoded by a nucleic acid sequence comprising:

in some embodiments of the nanotransposons of the present disclosure, including those in which at least one exogenous sequence comprises an inducible pro-apoptotic polypeptide, the exogenous sequence further comprises a sequence encoding a selectable marker. In some embodiments, the sequence encoding the selectable marker comprises a sequence encoding a detectable marker. In some embodiments, the detectable label comprises a fluorescent label or a cell surface label. In some embodiments, the sequence encoding the selectable marker comprises a sequence encoding a protein that is active in dividing cells and inactive in non-dividing cells. In some embodiments, the sequence encoding the selectable marker comprises a sequence encoding a metabolic marker. In some embodiments, the sequence encoding the selectable marker comprises a sequence encoding a dihydrofolate reductase (DHFR) mutant protease. In some embodiments, the DHFR mutant protease comprises or consists of the amino acid sequence:

in some embodiments, the DHFR mutant protease is encoded by a nucleic acid sequence comprising or consisting of:

In some embodiments, the amino acid sequence of the DHFR mutant protease further comprises a mutation at one or more of positions 80, 113 or 153. In some embodiments, the amino acid sequence of the DHFR mutant protease comprises one or more of a substitution of phenylalanine (F) or leucine (L) at position 80, a substitution of leucine (L) or valine (V) at position 113, and a substitution of valine (V) or aspartic acid (D) at position 153.

In some embodiments of the nanotransposons of the present disclosure, including those in which at least one exogenous sequence comprises an inducible pro-apoptotic polypeptide and/or the exogenous sequence comprises a sequence encoding a selectable marker, the exogenous sequence further comprises a sequence encoding a non-naturally occurring antigen receptor and/or a sequence encoding a therapeutic polypeptide. In some embodiments, the non-naturally occurring antigen receptor comprises a T Cell Receptor (TCR). In some embodiments, the sequence encoding the TCR comprises one or more of an insertion, deletion, substitution, inversion, transposition, or frameshift as compared to the corresponding wild-type sequence. In some embodiments, the sequence encoding the TCR comprises a chimeric or recombinant sequence. In some embodiments, the non-naturally occurring antigen receptor comprises a Chimeric Antigen Receptor (CAR). In some embodiments, the CAR comprises: (a) an extracellular domain comprising an antigen recognition region, (b) a transmembrane domain, and (c) an intracellular domain comprising at least one costimulatory domain. In some embodiments, the extracellular domain of (a) of the CAR further comprises a signal peptide. In some embodiments, the extracellular domain of (a) of the CAR further comprises a hinge between the antigen recognition region and the transmembrane domain. In some embodiments, the endodomain comprises a human CD3 ζ endodomain. In some embodiments, at least one co-stimulatory domain comprises a human 4-1BB, CD28, CD40, ICOS, MyD88, OX-40 intracellular segment, or any combination thereof. In some embodiments, the at least one co-stimulatory domain comprises a human CD28 and/or a 4-1BB co-stimulatory domain. In some embodiments, the antigen recognition region comprises one or more of scFv, VHH, VH, and centryrin.

In some embodiments of the nanotransposons of the present disclosure, including those in which at least one of the exogenous sequences comprises an inducible pro-apoptotic polypeptide and/or the exogenous sequence comprises a sequence encoding a selectable marker, the exogenous sequence further comprises a sequence encoding a transposase.

In some embodiments of the femto transposons of the present disclosure, the sequences within the ITRs comprise a sequence encoding a selectable marker, an exogenous sequence, a sequence encoding an inducible caspase polypeptide, and at least one sequence encoding a self-cleaving peptide. In some embodiments, the at least one sequence encoding a self-cleaving peptide is located between one or more of: (a) a sequence encoding a selectable marker and an exogenous sequence, (b) a sequence encoding a selectable marker and an inducible caspase polypeptide, and (c) an exogenous sequence and an inducible caspase polypeptide. In some embodiments, the first sequence encoding a self-cleaving peptide is located between the sequence encoding the selectable marker and the exogenous sequence, and the second sequence encoding a self-cleaving peptide is located between the exogenous sequence and the inducible caspase polypeptide.

In some embodiments of the nanotransposons of the present disclosure, including those in which the at least one exogenous sequence comprises one or more of an inducible pro-apoptotic polypeptide, a sequence encoding a selectable marker, and an exogenous sequence, the sequence encoding a first Inverted Terminal Repeat (ITR) or the sequence encoding a second Inverted Terminal Repeat (ITR) is recognized by a piggyBac transposase or a piggyBac-like transposase. In some embodiments, the sequence encoding the first Inverted Terminal Repeat (ITR) or the sequence encoding the second Inverted Terminal Repeat (ITR) is recognized by piggyBac transposase. In some embodiments, the sequence encoding the first Inverted Terminal Repeat (ITR) or the sequence encoding the second Inverted Terminal Repeat (ITR) is recognized by a piggyBac-like transposase. In some embodiments, the sequence encoding the first Inverted Terminal Repeat (ITR) or the sequence encoding the second Inverted Terminal Repeat (ITR) comprises a TTAA, TTAT, or TTAX recognition sequence. In some embodiments, the sequence encoding the first Inverted Terminal Repeat (ITR) or the sequence encoding the second Inverted Terminal Repeat (ITR) comprises a TTAA, TTAT, or TTAX recognition sequence and a sequence at least 50% identical to a sequence isolated or derived from a piggyBac transposase or a piggyBac-like transposase. In some embodiments, the sequence encoding the first Inverted Terminal Repeat (ITR) or the sequence encoding the second Inverted Terminal Repeat (ITR) comprises at least 2 nucleotides (nts), 3nts, 4nts, 5nts, 6nts, 7nts, 8nts, 9nts, 10nts, 11nts, 12nts, 13nts, 14nts, 15nts, 16nts, 17nts, 18nts, 19nts or 20 nts.

In some embodiments of the nanotransposons of the present disclosure, including those in which the at least one exogenous sequence comprises one or more of an inducible pro-apoptotic polypeptide, a sequence encoding a selectable marker, and an exogenous sequence, the sequence encoding a first Inverted Terminal Repeat (ITR) or the sequence encoding a second Inverted Terminal Repeat (ITR) is recognized by a piggyBac transposase or a piggyBac-like transposase. In some embodiments, the sequence encoding the first Inverted Terminal Repeat (ITR) or the sequence encoding the second Inverted Terminal Repeat (ITR) comprises the sequence of CCCTAGAAAGATAGTCTGCGTAAAATTGACGCATG (SEQ ID NO: 17096) or a sequence having at least 70% identity to the sequence of CCCTAGAAAGATAGTCTGCGTAAAATTGACGCATG (SEQ ID NO: 17096). In some embodiments, the sequence encoding the first Inverted Terminal Repeat (ITR) or the sequence encoding the second Inverted Terminal Repeat (ITR) comprises the sequence of CCCTAGAAAGATAATCATATTGTGACGTACGTTAAAGATAATCATGCGTAAAATTGACGCATG (SEQ ID NO: 17097). In some embodiments, the sequence encoding the first Inverted Terminal Repeat (ITR) or the sequence encoding the second Inverted Terminal Repeat (ITR) comprises the sequence of CCCTAGAAAGATAGTCTGCGTAAAATTGACGCATG (SEQ ID NO: 17096) and comprises the sequence of CCCTAGAAAGATAATCATATTGTGACGTACGTTAAAGATAATCATGCGTAAAATTGACGCATG (SEQ ID NO: 17097). In some embodiments, the sequence encoding the first Inverted Terminal Repeat (ITR) or the sequence encoding the second Inverted Terminal Repeat (ITR) comprises the sequence of CCCTAGAAAGATAGTCTGCGTAAAATTGACGCATG (SEQ ID NO: 17096) and comprises the sequence of CCCTAGAAAGATAATCATATTGTGACGTACGTTAAAGATAATCATGTGTAAAATTGACGCATG (SEQ ID NO: 17098). In some embodiments, the sequence encoding the first Inverted Terminal Repeat (ITR) or the sequence encoding the second Inverted Terminal Repeat (ITR) comprises the sequence of CCCTAGAAAGATAGTCTGCGTAAAATTGACGCATG (SEQ ID NO: 17096) and comprises the sequence:

In some embodiments, the sequence encoding the first Inverted Terminal Repeat (ITR) or the sequence encoding the second Inverted Terminal Repeat (ITR) is recognized by a piggyBac transposase having the following amino acid sequence

In some embodiments of the nanotransposons of the present disclosure, including those in which at least one of the exogenous sequences comprises one or more of an inducible pro-apoptotic polypeptide, a sequence encoding a selectable marker, and an exogenous sequence, the sequence encoding a first Inverted Terminal Repeat (ITR) or the sequence encoding a second Inverted Terminal Repeat (ITR) is recognized by a Helitron transposase.

The present disclosure provides cells comprising the nano-transposons of the present disclosure. In some embodiments, the cell further comprises a transposase composition. In some embodiments, the transposase composition comprises a transposase or a sequence encoding a transposase capable of recognizing the first ITR or the second ITR of a nano-transposon. In some embodiments, the transposase composition comprises a nano-transposon, which comprises a sequence encoding a transposase. In some embodiments, the cell comprises a first nano-transposon comprising the exogenous sequence and a second nano-transposon comprising a sequence encoding a transposase. In some embodiments, the cell is an allogeneic cell.

The present disclosure provides compositions comprising the nano-transposons of the present disclosure.

The present disclosure provides compositions comprising cells of the present disclosure. In some embodiments, the cell comprises a femto transposon of the present disclosure. In some embodiments, the cell is not further modified. In some embodiments, the cells are allogeneic.

The present disclosure provides compositions comprising a plurality of cells of the present disclosure. In some embodiments, at least one cell of the plurality of cells comprises a femto transposon of the present disclosure. In some embodiments, a portion of the plurality of cells comprises a femto transposon of the present disclosure. In some embodiments, the portion comprises at least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or any percentage therebetween of the plurality of cells. In some embodiments, each cell of the plurality of cells comprises a femto transposon of the present disclosure. In some embodiments, the plurality of cells does not comprise a modified cell of the present disclosure. In some embodiments, at least one cell of the plurality of cells is not further modified. In some embodiments, none of the plurality of cells is further modified. In some embodiments, the plurality of cells are allogeneic. In some embodiments, a plurality of cells that are allogeneic are generated according to the methods of the present disclosure. In some embodiments, the plurality of cells are autologous. In some embodiments, a plurality of cells are generated autologous according to the methods of the present disclosure.

The present disclosure provides a modified cell comprising: (a) a nano transposon of the present disclosure; (b) a sequence encoding an inducible pro-apoptotic polypeptide; and wherein the cell is a T cell, (c) a modification of an endogenous sequence encoding a T Cell Receptor (TCR), wherein the modification reduces or eliminates the level of expression or activity of the TCR. In some embodiments, the cell further comprises: (d) a modification comprising an HLA class I histocompatibility antigen, a non-naturally occurring sequence of alpha chain E (HLA-E), and (E) an endogenous sequence encoding β -2-microglobulin (B2M), wherein the modification reduces or eliminates the level of expression or activity of Major Histocompatibility Complex (MHC) class I (MHC-I).

The present disclosure provides a modified cell comprising: (a) a nano transposon of the present disclosure; (b) a sequence encoding an inducible pro-apoptotic polypeptide; (c) a modification comprising an HLA class I histocompatibility antigen, a non-naturally occurring sequence of alpha chain E (HLA-E), and (E) an endogenous sequence encoding β -2-microglobulin (B2M), wherein the modification reduces or eliminates the level of expression or activity of Major Histocompatibility Complex (MHC) class I (MHC-I).

In some embodiments of the modified cells of the present disclosure, the non-naturally occurring sequence comprising HLA-E further comprises a sequence encoding a B2M signal peptide. In some embodiments, the non-naturally occurring sequence comprising HLA-E further comprises a linker, wherein the linker is located between the sequence encoding the sequence of the B2M polypeptide and the sequence encoding HLA-E. In some embodiments, the non-naturally occurring sequence comprising HLA-E further comprises a sequence encoding a peptide and a sequence encoding a B2M polypeptide. In some embodiments, the non-naturally occurring sequence comprising HLA-E further comprises a first linker between the sequence encoding the B2M signal peptide and the sequence encoding the peptide, and a second linker between the sequence encoding the B2M polypeptide and the sequence encoding HLA-E.

In some embodiments of the cells, unmodified cells, and modified cells of the present disclosure, the cells are mammalian cells.

In some embodiments of the cells, unmodified cells, and modified cells of the present disclosure, the cells are human cells.

In some embodiments of the cells, unmodified cells, and modified cells of the present disclosure, the cells are stem cells.

In some embodiments of the cells, unmodified cells, and modified cells of the present disclosure, the cells are differentiated cells.

In some embodiments of the cells, unmodified cells, and modified cells of the present disclosure, the cells are somatic cells.

In some embodiments of the cells, unmodified cells, and modified cells of the present disclosure, the cell is an immune cell or an immune cell precursor. In some embodiments, the immune cell is a lymphoid progenitor cell, a Natural Killer (NK) cell, a cytokine-induced killer (CIK) cell, a T lymphocyte (T cell), a B lymphocyte (B cell), or an Antigen Presenting Cell (APC). In some embodiments, the immune cell is a T cell, an early memory T cell, a stem cell-like T cell, a stem memory T cell (Tscm), or a central memory T cell (Tcm). In some embodiments, the immune cell precursor is a Hematopoietic Stem Cell (HSC). In some embodiments, the cell is an Antigen Presenting Cell (APC).

In some embodiments of the cells, unmodified cells, and modified cells of the present disclosure, the cells further comprise a gene-editing composition. In some embodiments, the gene editing composition comprises a sequence encoding a DNA binding domain and a sequence encoding a nuclease protein or a nuclease domain thereof. In some embodiments, the gene editing composition comprises a sequence encoding a nuclease protein or a sequence thereof encoding a nuclease domain. In some embodiments, the e sequence encoding the nuclease protein or the sequence encoding the nuclease domain thereof comprises a DNA sequence, an RNA sequence, or a combination thereof. In some embodiments, the nuclease or nuclease domain thereof comprises one or more of a CRISPR/Cas protein, a transcription activator protein-like effector nuclease (TALEN), a Zinc Finger Nuclease (ZFN), and an endonuclease. In some embodiments, the CRISPR/Cas protein comprises a nuclease-inactivated Cas (dcas) protein.

In some embodiments of the cells, unmodified cells, and modified cells of the present disclosure, the cells further comprise a gene-editing composition. In some embodiments, the gene editing composition comprises a sequence encoding a DNA binding domain and a sequence encoding a nuclease protein or a nuclease domain thereof. In some embodiments, the nuclease or nuclease domain thereof comprises a nuclease-inactivated cas (dcas) protein and an endonuclease. In some embodiments, the endonuclease comprises Clo051 nuclease or a nuclease domain thereof. In some embodiments, the gene-editing composition comprises a fusion protein. In some embodiments, the fusion protein comprises a nuclease-inactivated Cas9(dCas9) protein and Clo051 nuclease or a Clo051 nuclease domain. In some embodiments, the gene editing composition further comprises a guide sequence. In some embodiments, the guide sequence comprises an RNA sequence. In some embodiments, the fusion protein comprises the amino acid sequence:

Or a nucleic acid comprising or consisting of:

or consist thereof. In some embodiments, the fusion protein comprises the amino acid sequence:

or a nucleic acid comprising or consisting of:

or consist thereof.

In some embodiments of the cells, unmodified cells, and modified cells of the present disclosure, the nano-transposon comprises a gene editing composition comprising a guide sequence and a sequence encoding a fusion protein comprising a sequence encoding an inactivated Cas9(dCas9) and a sequence encoding a Clo051 nuclease or a nuclease domain thereof.

In some embodiments of the cells, unmodified cells, and modified cells of the present disclosure, the cells transiently express the gene-editing composition.

In some embodiments of the cells, unmodified cells, and modified cells of the present disclosure, the cells are T cells and the guide RNA comprises a sequence complementary to a target sequence encoding an endogenous TCR. In some embodiments, the guide RNA comprises a sequence complementary to a target sequence encoding a B2M polypeptide.

In some embodiments of the cells, unmodified cells, and modified cells of the present disclosure, the guide RNA comprises a sequence complementary to a target sequence within a safe harbor site of a genomic DNA sequence.

In some embodiments of the cells, unmodified cells, and modified cells of the present disclosure, Clo051 nuclease or a nuclease domain thereof induces single-or double-stranded breaks in the target sequence. In some embodiments, the sequence within the donor sequence, donor plasmid or donor nano-transposon ITRs is integrated at the location of a single or double strand break within the target sequence and/or at the location of cell repair.

The present disclosure provides compositions comprising modified cells according to the present disclosure. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier.

The present disclosure provides compositions comprising a plurality of modified cells according to the present disclosure. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier.

The present disclosure provides compositions of the present disclosure for use in treating a disease or disorder.

The present disclosure provides for the use of the compositions of the present disclosure for treating a disease or condition.

The present disclosure provides a method of treating a disease or disorder comprising administering to a subject in need thereof a therapeutically effective amount of a composition of the present disclosure. In some embodiments, the subject does not develop graft versus host (GvH) and/or host versus graft (HvG) after administration of the composition. In some embodiments, the administration is systemic. In some embodiments, the composition is administered by an intravenous route. In some embodiments, the composition is administered by intravenous injection or intravenous infusion.

The present disclosure provides a method of treating a disease or disorder comprising administering to a subject in need thereof a therapeutically effective amount of a composition of the present disclosure. In some embodiments, the subject does not develop graft versus host (GvH) and/or host versus graft (HvG) after administration of the composition. In some embodiments, the administration is topical. In some embodiments, the composition is administered by an intratumoral route, an intraspinal route, an intraventricular route, an intraocular route, or an intraosseous route. In some embodiments, the composition is administered by intratumoral injection or infusion, intraspinal injection or infusion, intraventricular injection or infusion, intraocular injection or infusion, or intraosseous injection or infusion.

In some embodiments of the methods of treating a disease or disorder of the present disclosure, the therapeutically effective dose is a single dose, and wherein the allogeneic cells of the composition are engrafted and/or sustained for a sufficient time to treat the disease or disorder. In some embodiments, a single dose is one of at least 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or any number therebetween, made simultaneously.

In some embodiments of the methods of treating a disease or disorder of the present disclosure, the therapeutically effective dose is a single dose, and wherein autologous cells of the composition are engrafted and/or continued for a sufficient time to treat the disease or disorder. In some embodiments, a single dose is one of at least 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or any number therebetween, made simultaneously.

Vectors and host cells

The disclosure also relates to vectors comprising the isolated nucleic acid molecules of the disclosure, host cells genetically engineered with recombinant vectors, and the production of at least one centrin or CARTyrin by recombinant techniques, as is well known in the art. See, e.g., Sambrook et al, supra; ausubel et al, supra, each incorporated herein by reference in its entirety.

For example, the PB-EF1a vector may be used. The vector comprises the following nucleotide sequence:

the polynucleotide may optionally be ligated into a vector containing a selectable marker for propagation in a host. Typically, the plasmid vector is introduced into a precipitate, such as a calcium phosphate precipitate, or a complex with a charged lipid. If the vector is a virus, it may be extracorporeally packaged using an appropriate packaging cell line and then transduced into a host cell.

The DNA insert should be operably linked to a suitable promoter. The expression construct will further contain sites for transcription initiation, termination, and, in the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcript expressed by the construct will preferably include a translation initiation at the beginning and a stop codon (e.g., UAA, UGA, or UAG) appropriately positioned at the end of the mRNA to be translated, with UAA and UAG preferably being used for mammalian or eukaryotic cell expression.

The expression vector will preferably, but optionally, include at least one selectable marker. Such markers include, for example, but are not limited to, ampicillin, zeocin (Sh bla gene), puromycin (pac gene), hygromycin B (hygB gene), G418/geneticin (neo gene), mycophenolic acid or glutamine synthetase (GS, U.S. Pat. No. 5,122,464; 5,770,359; 5,827,739), blasticidin (bsd gene), resistance genes and ampicillin, zeocin (Sh bla gene), puromycin (pac gene), hygromycin B (hygB gene), G418/geneticin (neo gene), kanamycin, spectinomycin, streptomycin, carbenicillin, bleomycin, erythromycin, polymyxin B or tetracycline resistance genes for cultivation in E.coli and other bacteria or prokaryotes (all of which are hereby incorporated by reference). Suitable media and conditions for the above-described host cells are known in the art. Suitable vectors will be apparent to the skilled person. Introduction of the vector construct into the host cell can be achieved by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other known methods. Such methods are described in the art, for example, in Sambrook, supra, chapters 1-4 and chapters 16-18; ausubel, supra, chapters 1, 9, 13, 15, 16.

The expression vector will preferably, but optionally, include at least one selectable cell surface marker for isolating cells modified by the compositions and methods of the present disclosure. The selective cell surface markers of the present disclosure include surface proteins, glycoproteins, or proteomes that distinguish a cell or cell subpopulation from another defined cell subpopulation. Preferably, the selective cell surface marker distinguishes those cells modified by the compositions or methods of the present disclosure from those cells not modified by the compositions or methods of the present disclosure. Such cell surface markers include, for example, but are not limited to, "antigenic differentiation" or "classification determinant" proteins (often abbreviated as "CD"), such as truncated or full-length forms of CD19, CD271, CD34, CD22, CD20, CD33, CD52, or any combination thereof. The cell surface marker further included the "suicide" gene marker RQR8(Philip B et al, blood.2014, 8/21; 124 (8): 1277-87).

The expression vector will preferably, but optionally, include at least one selectable drug resistance marker for use in isolating cells modified by the compositions and methods of the present disclosure. The selectable drug resistance marker of the present disclosure may include wild-type or mutated Neo, TYMS, FRANCF, RAD51C, GCS, MDR1, ALDH1, NKX2.2, or any combination thereof.

The at least one centryrin or CARTyrin of the present disclosure may be expressed in a modified form, such as a fusion protein, and may include not only a secretion signal, but also additional heterologous functional regions. For example, additional amino acids, particularly regions of charged amino acids, can be added to the N-terminus of CARTyrin to improve stability and persistence in the host cell during purification or during subsequent handling and storage. Likewise, a peptide moiety can be added to the CARTyrin of the present disclosure to facilitate purification. Such regions may be removed prior to final formulation of the CARTyrin or at least one fragment thereof. This method is described in many standard laboratory manuals, such as Sambrook, supra, chapters 17.29-17.42 and 18.1-18.74; ausubel, supra, chapters 16, 17 and 18.

One of ordinary skill in the art is knowledgeable in the numerous expression systems that can be used to express nucleic acids encoding proteins of the disclosure. On the other hand, a nucleic acid of the present disclosure can be expressed in a host cell by actuating (by manipulation) in a host cell containing endogenous DNA encoding a centrin or a CARTyrin of the present disclosure. Such methods are well known in the art, for example, as described in U.S. Pat. Nos.5,580,734, 5,641,670, 5,733,746, and 5,733,761, which are incorporated herein by reference in their entirety.

Illustrative of cell cultures for producing CARTyrin, designated portions or variants thereof are bacterial, yeast and mammalian cells as known in the art. Mammalian cell systems will often exist as monolayers of cells, although mammalian cell suspensions or bioreactors may also be used. Many suitable host cell lines capable of expressing the entire glycosylated protein have been developed in the art and include COS-1 (e.g., ATCC CRL 1650), COS-7 (e.g., ATCC CRL-1651), HEK293, BHK21 (e.g., ATCC CRL-10), CHO (e.g., ATCC CRL 1610), and BSC-1 (e.g., ATCC CRL-26) cell lines, Cos-7 cells, CHO cells, hep G2 cells, P3X63Ag8.653, SP2/0-Ag14, 293 cells, HeLa cells, and the like, which are readily available from, for example, the American Type Culture Collection, Manassas, Va. (www.atcc.org). Preferred host cells include cells of lymphoid origin, such as myeloma and lymphoma cells. Particularly preferred host cells are P3X63Ag8.653 cells (ATCC accession number CRL-1580) and SP2/0-Ag14 cells (ATCC accession number CRL-1851). In a particularly preferred embodiment, the recombinant cell is a P3X63Ab8.653 or SP2/0-Ag14 cell.

Expression vectors for use in these cells may include one or more expression control sequences, such as, but not limited to, an origin of replication; promoters (e.g., late or early SV40 promoter, CMV promoter (U.S. Pat. No.5,168,062; 5,385,839), HSV tk promoter, pgk (phosphoglycerate kinase) promoter, EF-1 a promoter (U.S. Pat. No.5,266,491), at least one human promoter, enhancers, and/or processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites (e.g., SV40 large T Ag plus poly A site), and transcription termination sequences see, e.g., Ausubel et al, supra; Sambrook et al, supra. other cells useful for producing the nucleic acids or proteins of the invention are known and/or available, e.g., from American Type Culture Collection catalog (American Type Collection of cells and hybrids) (www.atcc.org) or other known or commercial sources.

When eukaryotic host cells are used, polyadenylation or transcription termination sequences are typically incorporated into the vector. An example of a termination sequence is the polyadenylation sequence from the bovine auxin gene. Sequences for accurate splicing of transcripts may also be included. An example of a spliced sequence is the VP1 intron from SV40 (Sprague et al, J.Virol.45: 773-781 (1983)). In addition, gene sequences that control replication in a host cell can be incorporated into the vector, as is known in the art.

Purification of CARTyrin

Centryrin or CARTyrin can be recovered and purified from recombinant cell cultures by well-known methods, including, but not limited to, protein a purification, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography. High performance liquid chromatography ("HPLC") can also be used for purification. See, e.g., Colligan, Current Protocols in Immunology, or Current Protocols in Protein Science, John Wiley & Sons, NY, N.Y. (1997-2001), e.g., chapters 1, 4, 6, 8, 9, 10, each of which is incorporated herein by reference in its entirety.

The centrins or CARTyrins of the present disclosure include purified products, products of chemical synthesis procedures, and products produced by recombinant techniques from prokaryotic or eukaryotic hosts including, for example, escherichia coli, yeast, higher plant, insect, and mammalian cells. Depending on the host used in the recombinant production procedure, the CARTyrin of the present disclosure may or may not be glycosylated. Such methods are described in a number of standard laboratory manuals, e.g., Sambrook, supra, sections 17.37-17.42; ausubel, supra, chapters 10, 12, 13, 16, 18 and 20, Colligan, Protein Science, supra, chapters 12-14, all of which are incorporated herein by reference.

Amino acid code

The amino acids that form the carpyrins of this disclosure are often abbreviated. Amino acid names can be shown by representing The amino acid by its single letter code, its three letter code, name, or one or more trinucleotide codons, as is well known in The art (see Alberts, b. et al, Molecular Biology of The Cell, third edition, Garland Publishing, inc., New York, 1994). As specified herein, the CARTyrin of the present disclosure can comprise one or more amino acid substitutions, deletions, or additions from spontaneous or mutated and/or human manipulation. Amino acids essential for function in the CARTyrin of the present disclosure can be identified by methods known in the art, such as site-directed mutagenesis or alanine-partition mutagenesis (e.g., Ausubel, supra, chapters 8, 15; Cunningham and Wells, Science 244: 1081-1085 (1989)). The latter approach introduces a single alanine mutation at each residue in the molecule. The resulting mutant molecules are then tested for biological activity, such as, but not limited to, at least one neutralizing activity. Sites critical for CARTyrin binding can also be identified by structural analysis, such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith et al, J.mol.biol.224: 899-904(1992) and de Vos et al, Science 255: 306-312 (1992)).

As will be understood by those of skill in the art, the present invention includes at least one biologically active CARTyrin of the present disclosure. The biologically active CARTyrin has a specific activity that is at least 20%, 30% or 40%, and preferably at least 50%, 60% or 70%, and most preferably at least 80%, 90% or 95% -99% or more of the specific activity of native (non-synthetic), endogenous or related and known CARTyrin. Methods for determining and quantifying measures of enzymatic activity and substrate specificity are well known to those skilled in the art.

In another aspect, the disclosure relates to centrins and fragments as described herein, which are modified by covalent attachment of an organic moiety. Such modifications can result in a CARTyrin fragment with improved pharmacokinetic properties (e.g., increased serum half-life in vivo). The organic moiety may be a linear or branched hydrophilic polymer group, a fatty acid group, or a fatty acid ester group. In particular embodiments, the hydrophilic polymer group can have a molecular weight of about 800 to about 120,000 daltons, and can be a polyalkanediol (e.g., polyethylene glycol (PEG), polypropylene glycol (PPG)), a carbohydrate polymer, an amino acid polymer, or polyvinylpyrrolidone, and the fatty acid or fatty acid ester group can include about 8 to about 40 carbon atoms.

The modified CARTyrin and fragments of the present disclosure may comprise one or more organic moieties covalently bonded, directly or indirectly, to an antibody. Each organic moiety bonded to the CARTyrin or fragment thereof of the present disclosure may independently be a hydrophilic polymer group, a fatty acid group, or a fatty acid ester group. As used herein, the term "fatty acid" includes monocarboxylic acids and dicarboxylic acids. As used herein, the term "hydrophilic polymer group" refers to an organic polymer that is more soluble in water than octane. For example, polylysine is more soluble in water than octane. Accordingly, the present disclosure includes centryrin or CARTyrin modified by covalent attachment of polylysine. Hydrophilic polymers suitable for modifying centrins or CARTyrins of the present disclosure may be straight or branched chain and include, for example, polyalkanediols (e.g., PEG, monomethoxy-polyethylene glycol (mPEG), PPG, etc.), carbohydrates (e.g., dextran, cellulose, oligosaccharides, polysaccharides, etc.), polymers of hydrophilic amino acids (e.g., polylysine, polyarginine, polyaspartic acid, etc.), polyalkylene oxides (e.g., polyethylene oxide, polypropylene oxide, etc.), and polyvinylpyrrolidone. Preferably, the hydrophilic polymer modifying the CARTyrin of the present disclosure has a molecular weight of about 800 to about 150,000 daltons as a separate molecular entity. For example, PEG5000 and PEG 20,000 can be used, where the subscripts are the average molecular weight of the polymer in daltons. The hydrophilic polymer groups may be substituted with one to about six alkyl, fatty acid, or fatty acid ester groups. Hydrophilic polymers substituted with fatty acid or fatty acid ester groups can be prepared by employing suitable methods. For example, a polymer comprising an amine group can be coupled to a carboxylic acid ester of a fatty acid or fatty acid ester, and an activated carboxylic acid ester on the fatty acid or fatty acid ester (e.g., activated with N, N-carbonyl diimidazole) can be coupled to a hydroxyl group on the polymer.

Fatty acids and fatty acid esters suitable for modifying the cardurins of the present disclosure may be saturated or may contain one or more units of unsaturation. Fatty acids suitable for modifying the carpyrins of the present disclosure include, for example, n-dodecanoic acid (C12, lauric acid), n-tetradecanoic acid (C14, myristic acid), n-octadecanoic acid (C18, stearic acid), n-eicosanoic acid (C20, arachidic acid), n-docosanoic acid (C22, behenic acid), n-triacontanoic acid (C30), n-tetracosanoic acid (C40), cis- Δ 9-octadecanoic acid (C18, oleic acid), all cis- Δ 5, 8, 11, 14-eicosatetraenoic acid (C20, arachidonic acid), suberic acid, tetradecanedioic acid (tetracanedioic acid), octadecanedioic acid (octadecedioic acid), docosanedioic acid, and the like. Suitable fatty acid esters include monoesters of dicarboxylic acids containing a linear or branched lower alkyl group. The lower alkyl group may contain 1 to about 12, preferably 1 to about 6 carbon atoms.

Modified CARTyrins and fragments can be prepared using a suitable method, for example, by reacting with one or more modifying agents. As used herein, the term "modifier" refers to a suitable organic group (e.g., hydrophilic polymer, fatty acid ester) that contains an activating group. An "activating group" is a chemical moiety or functional group that can react with a second chemical group under appropriate conditions to form a covalent bond between the modifying agent and the second chemical group. For example, amine-reactive activating groups include electrophilic groups such as tosylate, mesylate, halo (chloro, bromo, fluoro, iodo), N-hydroxysuccinimide esters (NHS), and the like. Activating groups that can react with thiols include, for example, maleimide, iodoacetyl, acryloyl, pyridyl disulfide, 5-thiol-2-nitrobenzoic acid thiol (TNB-thiol), and the like. The aldehyde functional group can be coupled to an amine-or hydrazide-containing molecule, and the azide group can react with a trivalent phosphorous group to form a phosphoramidate or a phosphoramidite (phosphorimide) linkage. Suitable methods for introducing activating groups into molecules are known in the art (see, e.g., Hermanson, G.T., Bioconjugate Techniques, Academic Press: San Diego, Calif. (1996)). The activating group can be directly bonded to an organic group (e.g., a hydrophilic polymer, a fatty acid ester), or through a linker moiety, e.g., a divalent C1-C12 group, in which one or more carbon atoms can be replaced by a heteroatom such as oxygen, nitrogen, or sulfur. Suitable linker moieties include, for example, tetraethylene glycol, - (CH2)3-, -NH- (CH2)6-NH-, - (CH2)2-NH-, and-CH 2-O-CH2-CH2-O-CH2-CH 2-O-CH-NH-. Modifiers comprising a linker moiety can be generated, for example, by reacting a mono-Boc-alkyldiamine (e.g., mono-Boc-ethylenediamine, mono-Boc-diaminohexane) with a fatty acid in the presence of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) to form an amide bond between the free amine and the fatty acid carboxylate. The Boc protecting group can be removed from the product by treatment with trifluoroacetic acid (TFA) to expose a primary amine, which can be coupled with another carboxylic acid ester, as described, or can be reacted with maleic anhydride, and the resulting product cyclized to produce an activated maleimide derivative of the fatty acid. (see, e.g., Thompson et al, WO 92/16221, the entire teachings of which are incorporated herein by reference.)

Modified CARTyrins and fragments of the present disclosure can be produced by reacting a CARTyrin protein or fragment with a modifying agent. For example, the organic moiety can be bonded to the CARTyrin protein in a non-site specific manner by using an amine-reactive modifier, such as an NHS ester of PEG. Modified CARTyrin proteins and fragments comprising an organic moiety bonded to a specific site of CARTyrin of the present disclosure can be prepared using suitable methods, such as reverse proteolysis (reverse proteolysis) (Fisch et al, Bioconjugate chem., 3: 147-153 (1992); Werlen et al, Bioconjugate chem., 5: 411-417 (1994); Kumaran et al, Protein Sci.6 (10): 2233-2241 (1997); Itoh et al, bioorg. chem., 24 (1): 59-68 (1996); Capella et al, Biotechnol. Bioeng., 56 (4): 456-463), and in Hermanson, G.T., Bioconjugate Techniques, academy Press: san Diego, Calif. (1996).

CARTyrin compositions comprising a further therapeutically active ingredient

The centryrin or CARTyrin compounds, compositions, or combinations of the present disclosure may further comprise at least one of any suitable adjuvant, such as, but not limited to, diluents, binders, stabilizers, buffers, salts, lipophilic solvents, preservatives, adjuvants, and the like. Pharmaceutically acceptable adjuvants are preferred. Non-limiting examples and methods of preparation of such sterile solutions are well known in the art, such as, but not limited to, Gennaro, eds, Remington's Pharmaceutical Sciences, 18 th edition, Mack Publishing Co. (Easton, Pa.) 1990. Pharmaceutically acceptable carriers suitable for the mode of administration, solubility and/or stability of the centrin or CARTyrin, fragment or variant composition may be routinely selected, as is well known in the art or as described herein.

Pharmaceutical excipients and additives that may be used in the present compositions include, but are not limited to, proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars, including mono-, di-, tri-, tetra-, and oligosaccharides; derivatized sugars, such as sugar alcohols, aldonic acids, esterified sugars, and the like; and polysaccharides or sugar polymers), which may be present alone or in combination, constituting 1-99.99% by weight or volume, alone or in combination. Exemplary protein excipients include serum albumin, such as Human Serum Albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and the like. Representative amino acid/protein components that may also play a role in buffering capacity include alanine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and the like. One preferred amino acid is glycine.

Carbohydrate excipients suitable for use in the present invention include, for example, monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose and the like; disaccharides such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides such as raffinose, melezitose, maltodextrin, dextran, starch, and the like; and sugar alcohols such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol), inositol, and the like. Preferred carbohydrate excipients for use in the present invention are mannitol, trehalose and raffinose.

The CARTyrin composition may further comprise a buffer or a pH-adjusting agent; typically, the buffer is a salt prepared from an organic acid or base. Representative buffers include organic acid salts, such as salts of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid; tris, butanetrialkanolamine hydrochloride or phosphate buffer. Preferred buffers for use in the present compositions are organic acid salts, such as citrate.

In addition, the CARTyrin compositions of the invention may comprise polymeric excipients/additives such as polyvinylpyrrolidone, ficoll (polymeric sugar), dextrates (dextrates) (e.g. cyclodextrins, such as 2-hydroxypropyl- β -cyclodextrin), polyethylene glycol, flavouring agents, antimicrobial agents, sweeteners, antioxidants, antistatic agents, surfactants (e.g. polysorbates, such as "tween 20" and "tween 80"), lipids (e.g. phospholipids, fatty acids), steroids (e.g. cholesterol) and chelating agents (e.g. EDTA).

These and additional pharmaceutical excipients and/or additives known to be suitable for use in the centrin or CARTyrin, partial or variant compositions according to the invention are known in the art, for example, as described in "Remington: the Science & Practice of Pharmacy, 19 th edition, Williams & Williams, (1995) and The Physician's Desk Reference, 52 th edition, Medical Economics, Montvale, N.J. (1998), The disclosures of which are incorporated herein by Reference in their entirety. Preferred carrier or excipient materials are carbohydrates (e.g., sugars and sugar alcohols) and buffers (e.g., citrate) or polymeric agents. An exemplary carrier molecule is mucopolysaccharide, hyaluronic acid, which may be used for intra-articular delivery.

Isolation of T cells from Leukapheresis (Leukapheresis) products

Leukapheresis products or blood can be collected from subjects at a clinical site using closed systems and standard methods (e.g., the COBE Spectra Apheresis System). Preferably, the product is collected in a standard leukapheresis collection bag according to standard hospital or regulatory leukapheresis procedures. For example, in preferred embodiments of the methods of the present disclosure, no additional anticoagulant or blood additive (heparin, etc.) is included other than those typically used during leukapheresis.

On the other hand, White Blood Cells (WBCs)/Peripheral Blood Mononuclear Cells (PBMCs) (using biosafee Sepax 2 (Closed/Automated)) or T cells (using Biosafe Sepax 2) can be directly isolated from whole bloodProdigy (Closed/Automated))). However, in certain subjects (e.g., those diagnosed and/or treated for cancer), WBC/PBMC yields may be significantly lower when isolated from whole blood than when isolated by leukapheresis.

Leukapheresis procedures and/or direct cell isolation procedures can be used for any subject of the present disclosure.

The leukapheresis product, blood, WBC/PBMC composition and/or T-cell composition should be packaged in an insulated container and maintained at a controlled room temperature (+19 ℃ to +25 ℃) according to standard hospitals approved for the prescribed blood collection procedures used for clinical protocols. The leukapheresis product, blood, WBC/PBMC compositions and/or T-cell compositions should not be cryopreserved.

The leukocyte separation product, blood, WBC/PBMC composition and/or T-cell composition should not have a cell concentration greater than 0.2x10 during transport⁹Individual cells/mL. Intensive mixing of the leukapheresis product, blood, WBC/PBMC composition and/or T-cell composition should be avoided.

If the leukapheresis product, blood, WBC/PBMC composition and/or T-cell composition must be stored, for example overnight, it should be kept at a controlled room temperature (the same as above). The concentration of leukapheresis product, blood, WBC/PBMC composition and/or T-cell composition should never exceed 0.2X10 during storage⁹Individual cells/mL.

Preferably, the cells of the leukapheresis product, blood, WBC/PBMC composition and/or T-cell composition should be stored in autologous plasma. In certain embodiments, the leukocyte isolation product, blood, WBC/PBMC composition, and/or T-cell composition are present at a cell concentration greater than 0.2x10⁹Individual cells/mL, the product is diluted with autologous plasma.

Preferably, the leukapheresis product, blood, WBC/PBMC composition and/or T-cell composition should not age more than 24 hours when the labeling and separation procedure is initiated. Closed and/or automated systems (e.g., CliniMACS Prodigy) can be used to process and/or prepare leukapheresis products, blood, WBC/PBMC compositions, and/or T-cell compositions for cell labeling.

The automated system may perform additional buffy coat separations, possibly via ficollization (ficollation) and/or washing of cell products (e.g., leukapheresis products, blood, WBC/PBMC compositions, and/or T cell compositions).

Closed and/or automated systems may be used to prepare and label cells for T-cell separation (from, for example, leukapheresis products, blood, WBC/PBMC compositions, and/or T-cell compositions).

Although WBC/PBMCs can be directly transfected into nuclei (which is easier and saves additional steps), the methods of the present disclosure can include first isolating T cells prior to nuclear transfection. The easier strategy of direct nuclear transfection of PBMCs requires selective expansion of the CARTyrin + cells mediated by CARTyrin signaling, which independently proved to be a poor expansion method that directly reduces the in vivo efficiency of the product by functionally depleting T cells. The product may be a heterogeneous composition of CARTyrin + cells, including T cells, NK cells, NKT cells, monocytes, or any combination thereof, which increases the variability of the product from one patient to another and makes dosing and CRS management more difficult. Since T cells are considered to be the major effectors of tumor inhibition and killing, T cell isolation for production of autologous products may result in significant benefits over other, more heterogeneous compositions.

T cells can be isolated directly by enrichment for labeled cells or depletion of labeled cells in a one-way labeling procedure, or indirectly in a two-step labeling procedure. According to certain enrichment strategies of the present disclosure, T cells may be collected in a Cell Collection Bag (Cell Collection Bag) and non-labeled cells (non-target cells) collected in a Negative Fraction Bag (Negative Fraction Bag). In contrast to the enrichment strategy of the present disclosure, non-labeled cells (target cells) are collected in a cell collection bag and labeled cells (non-target cells) are collected in a negative fraction bag or a non-target cell bag, respectively. The selection reagent may include, but is not limited to, antibody coated beads. The antibody-coated beads may be removed prior to the modification and/or expansion step or may remain on the cells prior to the modification and/or expansion step. One or more of the following non-limiting examples of cell markers can be used to isolate T-cells: CD3, CD4, CD8, CD25, avidin, CD1c, CD3/CD19, CD3/CD56, CD14, CD19, CD34, CD45RA, CD56, CD62L, CD133, CD137, CD271, CD304, IFN- γ, TCR α/β and/or any combination thereof. Methods for isolating T-cells may include one or more reagents that specifically bind to and/or detectably label one or more of the following non-limiting examples of cellular markers useful for isolating T-cells: CD3, CD4, CD8, CD25, avidin, CD1c, CD3/CD19, CD3/CD56, CD14, CD19, CD34, CD45RA, CD56, CD62L, CD133, CD137, CD271, CD304, IFN- γ, TCR α/β and/or any combination thereof. These agents may or may not be of "good manufacturing practice" ("GMP") grade. Reagents may include, but are not limited to, Thermo DynaBeads and Miltenyi CliniMACS products. The methods of isolating T-cells of the present disclosure may include multiple repetitions of the labeling and/or isolation steps. At any point in the methods of isolating T-cells of the present disclosure, unwanted cells and/or unwanted cell types can be excluded from the T cell product compositions of the present disclosure by positive or negative selection for unwanted cells and/or unwanted cell types. The T cell product compositions of the present disclosure may contain additional cell types that may express CD4, CD8, and/or one or more additional T cell markers.

The methods of the present disclosure for T cell nuclear transfection may eliminate the step of T cell isolation by, for example, a process of T cell nuclear transfection in a population or composition of WBC/PBMCs that includes an isolation step or selective expansion step by TCR signaling after nuclear transfection.

Certain cell populations may be excluded by positive or negative selection before or after T cell enrichment and/or sorting. Examples of cell compositions that can be excluded from the cell product composition can include myeloid cells, CD25+ regulatory T cells (T Regs), dendritic cells, macrophages, erythrocytes, mast cells, γ - δ T cells, Natural Killer (NK) -like cells (e.g., cytokine-induced killer (CIK) cells), Induced Natural Killer (iNK) T cells, NK T cells, B cells, or any combination thereof.

The T cell product compositions of the present disclosure may include CD4+ and CD8+ T-cells. During the isolation or selection procedure, CD4+ and CD8+ T-cells may be isolated into separate collection bags. CD4+ T cells and CD8+ T cells, respectively, may be further processed or processed after reconstitution (combined into the same composition) in specific ratios.

The particular ratio at which CD4+ T cells and CD8+ T cells can be reconstituted depends on the type and efficacy of the expansion technique used, the cell culture medium, and/or the growth conditions used to expand the T-cell product composition. Examples of possible ratios of CD4+ to CD8+ include, but are not limited to, 50% to 50%, 60% to 40%, 40% to 60% to 75% to 25%, and 25% to 75%.

CD8+ T cells exhibit potent tumor cell killing, while CD4+ T cells provide many cytokines required to support the proliferative capacity and function of CD8+ T cells. Because T cells isolated from normal donors are predominantly CD4+, the T-cell product composition is artificially adjusted in vitro with respect to the ratio of CD4+ to CD8+ to modify the ratio of CD4+ T cells to CD8+ T cells that would otherwise be present in vivo. The optimized ratio can also be used for ex vivo expansion of autologous T-cell product compositions. Given the artificially adjusted CD4 +: CD8+ ratio of T-cell product compositions, it is important to note that the product compositions of the present disclosure can be significantly different and provide significantly greater benefit than any endogenously present T-cell population.

Preferred methods for T cell isolation may include negative selection strategies for generating intact whole T cells, meaning that the resulting T-cell composition includes T-cells that have not been manipulated and contain endogenously present T-cell varieties/proportions.

Reagents that can be used for positive or negative selection include, but are not limited to, magnetic cell separation beads. The magnetic cell separation beads may or may not be removed or depleted from the selected CD4+ T cells, CD8+ T cell population, or mixed population of both CD4+ and CD8+ T cells prior to performing the next step of the T-cell separation method of the present disclosure.

T cell compositions and T cell product compositions can be prepared for cryopreservation, storage in standard T cell culture media, and/or genetic modification.

The T cell composition, T cell product composition, unstimulated T cell composition, resting T cell composition, or any portion thereof can be cryopreserved using standard cryopreservation methods optimized for storing and recovering human cells with high recovery, viability, phenotype, and/or functional capacity. Commercially available cryopreservation media and/or protocols can be used. Cryopreservation methods of the present disclosure can include a DMSO-free cryopreservation agent (e.g., cryoSOFree)^TMCryopreservation media without DMSO) reduces the toxicity associated with freezing.

The T cell composition, T cell product composition, unstimulated T cell composition, resting T cell composition, or any portion thereof can be stored in a culture medium. The T cell culture media of the present disclosure can be optimized for cell storage, cell genetic modification, cell phenotype, and/or cell expansion. The T cell culture medium of the present disclosure may comprise one or more antibiotics. Because the inclusion of antibiotics in the cell culture medium may reduce transfection efficiency and/or cell yield after genetic modification by nuclear transfection, the particular antibiotic (or combination thereof) and one or more of its respective concentrations may be varied for optimal transfection efficiency and/or cell yield after genetic modification by nuclear transfection.

The T cell culture media of the present disclosure can comprise serum, and in addition, serum composition and concentration can be varied for optimal cell outcome. Human AB serum is preferred over FBS/FCS for T cell culture because FBS/FCS can introduce xenogeneic proteins (xeno-proteins) although intended for use in the T cell culture media of the present disclosure. Serum can be isolated from the blood of a subject to whom the T-cell composition in culture is intended to be administered, and thus, the T-cell culture medium of the present disclosure can comprise autologous serum. Serum-free media or serum substitutes can also be used in the T-cell media of the present disclosure. In certain embodiments of the T-cell culture media and methods of the present disclosure, serum-free media or serum substitutes can provide benefits over supplementing the media with xenogenic serum (xeno-serum), including, but not limited to, healthier cells with greater viability, nuclear transfection with greater efficiency, exhibit greater viability following nuclear transfection, display a more desirable cell phenotype, and/or expand more/faster when expansion techniques are added.

T cell culture media can include commercially available cell growth media. Exemplary commercially available cell growth media include, but are not limited to, PBS, HBSS, OptiMEM, DMEM, RPMI 1640, AIM-V, X-VIVO 15, CellGro DC medium, CTS OpTimizer T-cell expansion SFM, TexMACS medium, PRIME-XV T-cell expansion medium, ImmunoCult-XF T-cell expansion medium, or any combination thereof.

T cell compositions, T cell product compositions, unstimulated T cell compositions, resting T cell compositions, or any portion thereof can be prepared for genetic modification. Preparation of T cell compositions, T cell product compositions, unstimulated T cell compositions, resting T cell compositions, or any portion thereof for genetic modification can include cell washing and/or resuspension in a desired nuclear transfection buffer. The cryopreserved T-cell compositions can be thawed and prepared for genetic modification by nuclear transfection. Cryopreserved cells can be thawed according to standard or known procedures. Thawing and preparation of cryopreserved cells can be optimized to produce cells with greater viability, with more efficient nuclear transfection, exhibit greater viability following nuclear transfection, exhibit a more desirable cell phenotype, and/or expand more/faster with the addition of expansion techniques. For example, Grifols albumin (25% human albumin) can be used in the thawing and/or preparation process.

Genetic modification of autologous T cell product compositions

The T cell composition, T cell product composition, unstimulated T cell composition, resting T cell composition, or any portion thereof can be genetically modified using, for example, a nuclear transfection strategy such as electroporation. The total number of cells to be nuclear transfected, the total volume of the nuclear transfection reaction, and the precise timing of sample preparation can be optimized to produce cells with greater viability, nuclear transfection with greater efficiency, exhibit greater viability following nuclear transfection, display a more desirable cell phenotype, and/or expand more/more quickly when expansion techniques are added.

The core transfection and/or electroporation may be accomplished using, for example, Lonza Amaxa, MaxCyte pulseAgile, Harvard Apparatus BTX, and/or Invitrogen Neon. Non-metallic electrode systems including, but not limited to, plastic polymer electrodes may be preferred for nuclear transfection.

Prior to genetic modification by nuclear transfection, the T cell composition, T cell product composition, unstimulated T cell composition, resting T cell composition, or any portion thereof can be resuspended in a nuclear transfection buffer. The nuclear transfection buffers of the present disclosure include commercially available nuclear transfection buffers. The nuclear transfection buffers of the present disclosure can be optimized to produce cells that have greater viability, nuclear transfection with greater efficiency, exhibit greater viability following nuclear transfection, exhibit a more desirable cell phenotype, and/or expand more/faster upon the addition of expansion techniques. The nuclear transfection buffer of the present disclosure may include, but is not limited to, PBS, HBSS, OptiMEM, BTXpress, Amaxa nuclear transfectants (Nucleofector), human T cell nuclear transfection buffers, and any combination thereof. The nuclear transfection buffer of the present disclosure may include one or more supplemental factors (supplemental factors) to produce cells with greater viability, with more efficient nuclear transfection, exhibit greater viability following nuclear transfection, exhibit a more desirable cell phenotype, and/or expand more/faster upon the addition of expansion techniques. Exemplary complementing factors include, but are not limited to, recombinant human cytokines, chemokines, interleukins, and any combination thereof. Exemplary cytokines, chemokines and interleukins include, but are not limited to, IL2, IL7, IL12, IL15, IL21, IL1, IL3, IL4, IL5, IL6, IL8, CXCL8, IL9, IL10, IL11, IL13, GM-CSF, IFN- γ, IL-1 α/IL-1F 13, IL-1 β/IL-1F 13, IL-12p 13, IL-12/IL-35p 13, IL-13, IL-17/IL-17A/17F 72, IL- β/F-17F-32, IL-17F-72, IL-17F-17, IL-32, IL-13, IL-, LAP (TGF-. beta.1), lymphotoxin-alpha/TNF-. beta.TGF-. beta.TNF-. alpha.TRANCE/TNFSF 11/RANK L, and any combination thereof. Exemplary supplemental factors include, but are not limited to, salts, minerals, metabolites, or any combination thereof. Exemplary Salts, minerals, and metabolites include, but are not limited to, HEPES, nicotinamide, heparin, sodium pyruvate, L-glutamine, MEM non-essential amino acid solutions, ascorbic acid, nucleosides, FBS/FCS, human serum, serum substitutes, antibiotics, pH adjusters (adjust), Earle's Salts, 2-mercaptoethanol, human transferrin, recombinant human insulin, human serum albumin, nuclear stainer PLUS additives (Nuclear of PLUS Supplement), KCL, MgCl2, Na2HPO4, NAH2PO4, sodium lactobionate, mannitol (Manitol), sodium succinate, sodium chloride, CINa, glucose, Ca (NO3)2, Tris/HCl, K2HPO4, KH2PO4, Polyethylenimine (Polyethylenimine), polyethylene glycol, Poloxamer (Poloxamer)188, Poloxamer 181, Poloxamer 407, polyvinylpyrrolidone, Pop313, Crown-5, and any combination thereof. Exemplary supplemental factors include, but are not limited to, media such as PBS, HBSS, OptiMEM, DMEM, RPMI 1640, AIM-V, X-VIVO 15, CellGro DC medium, CTS OpTimizer T cell expansion SFM, TexMACS medium, PRIME-XV T cell expansion medium, ImmunoCult-XF T cell expansion medium, and any combination thereof. Exemplary supplemental factors include, but are not limited to, inhibitors of cellular DNA signaling (sensing), metabolism, differentiation, signal transduction, apoptotic pathways, and combinations thereof. Exemplary inhibitors include, but are not limited to, TLR9, MyD88, IRAK, TRAF6, TRAF3, IRF-7, NF-KB, type 1 interferon, Pro-inflammatory cytokines, cGAS, STING, Sec5, TBK1, IRF-3, RNA pol III, RIG-1, IPS-1, FADD, RIP1, TRAF3, AIM2, ASC, Caspase 1(Caspase1), Pro-IL1B, PI3K, Akt, inhibitors of Wnt3A, inhibitors of glycogen synthase kinase-3 β (GSK-3 β) (e.g., TWS119), baviromycin, chloroquine, quinacrine, AC-YVAD-CMK, Z-VAD-FMK, Z-IETD-FMK, and any combination thereof. Exemplary supplemental factors include, but are not limited to, agents that modify or stabilize one or more nucleic acids in a manner that enhances cellular delivery, enhances nuclear delivery or transport, enhances facilitated transport of the nucleic acid into the nucleus, enhances degradation of extrachromosomal nucleic acids, and/or reduces DNA-mediated toxicity. Exemplary agents that modify or stabilize one or more nucleic acids include, but are not limited to, pH modifiers, DNA binding proteins, lipids, phospholipids, CaPO4, net neutral charge DNA binding peptides with or without NLS sequences, TREX1 enzymes, and any combination thereof.

A transposing reagent comprising a transposon and a transposase can be added to the nuclear transfection reaction of the present disclosure before, simultaneously with, or after the addition of cells to the nuclear transfection buffer (optionally contained in the nuclear transfection reaction vial or cup). Transposons of the present disclosure can comprise plasmid DNA, linearized plasmid DNA, PCR products, DOGGYBONE^TMDNA, mRNA template, single-or double-stranded DNA, protein-nucleic acid combination, or any combination thereof. Transposons of the present disclosure may comprise one or more genes encoding one or more TTAA sites, one or more Inverted Terminal Repeats (ITRs), one or more Long Terminal Repeats (LTRs), one or more insulators, one or more promoters, one or more full-length or truncated genesOne or more polyA signals, one or more self-cleaving 2A peptide cleavage sites, one or more Internal Ribosome Entry Sites (IRES), one or more enhancers, one or more regulators, one or more origins of replication, and any combination thereof.

The transposons of the present disclosure can comprise one or more sequences encoding one or more full-length or truncated genes. The one or more full-length and/or truncated genes introduced by the transposons of the present disclosure can encode one or more of a signal peptide, CARTyrin, anti-PSMA CARTyrin, centryrin, PSMA-specific Centryin, hinge, transmembrane domain, co-stimulatory domain, Chimeric Antigen Receptor (CAR), chimeric T-cell receptor (CAR-T, CARTyrin or anti-PSMA CARTyrin), receptor, ligand, cytokine, drug-resistant gene, tumor antigen, allo-or autoantigen, enzyme, protein, peptide, polypeptide, fluorescent protein, mutein, or any combination thereof.

Transposons of the disclosure can be made in water, TAE, TBE, PBS, HBSS, culture medium, a complementing factor of the disclosure, or any combination thereof.

The transposons of the present disclosure can be designed to optimize clinical safety and/or improve manufacturability. As non-limiting examples, the transposons of the present disclosure can be designed to optimize clinical safety and/or improve manufacturability by eliminating unnecessary sequences or regions and/or including non-antibiotic selection markers. The transposons of the present disclosure may or may not be GMP grade.

The transposase of the present disclosure can be encoded by one or more sequences of plasmid DNA, mRNA, protein-nucleic acid combination, or any combination thereof.

Transposases of the disclosure can be prepared in water, TAE, TBE, PBS, HBSS, culture media, supplemental elements of the disclosure, or any combination thereof. The transposases or sequences/constructs encoding or delivering them of the present disclosure may or may not be GMP grade.

The transposons and transposases of the present disclosure can be delivered to a cell by any means.

Although the compositions and methods of the present disclosure include delivering the transposons and/or transposases of the present disclosure to cells via plasmid DNA (pdna), the use of plasmids for delivery may result in integration of the transposons and/or transposases into the chromosomal DNA of the cells, which may result in continuous transposase expression. Thus, the transposons and/or transposases of the present disclosure can be delivered to cells as mRNA or protein to eliminate any possibility of chromosomal integration.

The transposons and transposases of the present disclosure may be pre-incubated alone or in combination with each other prior to introducing the transposons and/or transposases into the nuclear transfection reaction. The absolute amounts, as well as the relative amounts, e.g., the ratio of transposon to transposase, of each of the transposons and transposases can be optimized.

After preparation of the nuclear transfection reaction, optionally in a vial or cup, the reaction can be loaded into a nuclear transfection instrument device and activated for delivery of electrical pulses according to the manufacturer's protocol. The electrical pulse conditions used to deliver the transposons and/or transposases of the present disclosure (or sequences encoding the transposons and/or transposases of the present disclosure) to cells can be optimized to produce cells with enhanced viability, higher nuclear transfection efficiency, greater viability following nuclear transfection, desired cell phenotype, and/or greater/faster expansion upon addition of expansion technology. When using Amaxa nucleofector technology, each of the various nucleofection procedures of Amaxa 2B or 4D nucleofectors are contemplated.

Following the nuclear transfection reaction of the present disclosure, the cells may be gently added to the cell culture medium. For example, when T cells undergo a nuclear transfection reaction, the T cells may be added to a T cell culture medium. The post-nuclear transfection cell culture medium of the present disclosure can include any one or more commercially available media. The post-nuclear transfection cell culture media of the present disclosure (including the post-nuclear transfection T cell culture media of the present disclosure) can be optimized to produce cells that have greater viability, higher nuclear transfection efficiency, exhibit greater viability following nuclear transfection, exhibit more desirable cell phenotypes, and/or expand more/faster when expansion techniques are added. Post-nuclear transfection cell culture media of the present disclosure (including post-nuclear transfection T cell culture media of the present disclosure) may include PBS, HBSS, OptiMEM, DMEM, RPMI 1640, AIM-V, X-VIVO 15, CellGro DC medium, CTS OpTimizer T cell expansion SFM, TexMACS medium, PRIME-XV T cell expansion medium, ImmunoCult-XF T cell expansion medium, and any combination thereof. Post-nuclear transfection cell culture media of the present disclosure (including post-nuclear transfection T cell culture media of the present disclosure) can include one or more supplemental factors of the present disclosure to enhance viability, nuclear transfection efficiency, post-nuclear transfection viability, cell phenotype, and/or greater/faster expansion upon addition of expansion techniques. Exemplary complementing factors include, but are not limited to, recombinant human cytokines, chemokines, interleukins, and any combination thereof. Exemplary cytokines, chemokines and interleukins include, but are not limited to, IL2, IL7, IL12, IL15, IL21, IL1, IL3, IL4, IL5, IL6, IL8, CXCL8, IL9, IL10, IL11, IL13, GM-CSF, IFN- γ, IL-1 α/IL-1F 13, IL-1 β/IL-1F 13, IL-12p 13, IL-12/IL-35p 13, IL-13, IL-17/IL-17A/17F 72, IL- β/F-17F-32, IL-17F-72, IL-17F-17, IL-32, IL-13, IL-, LAP (TGF-. beta.1), lymphotoxin-alpha/TNF-. beta.TGF-. beta.TNF-. alpha.TRANCE/TNFSF 11/RANK L, and any combination thereof. Exemplary supplemental factors include, but are not limited to, salts, minerals, metabolites, or any combination thereof. Exemplary salts, minerals, and metabolites include, but are not limited to, HEPES, niacinamide, heparin, sodium pyruvate, L-glutamine, MEM nonessential amino acid solutions, ascorbic acid, nucleosides, FBS/FCS, human serum, serum substitutes, antibiotics, pH adjusters, Earle's salts, 2-mercaptoethanol, human transferrin, recombinant human insulin, human serum albumin, nuclear stainer PLUS additives, KCL, MgCl2, Na2HPO4, NAH2PO4, sodium lactobionate, mannitol, sodium succinate, sodium chloride, CINa, glucose, Ca (NO3)2, Tris/HCl, K2HPO4, KH2PO4, polyethylenimine, polyethylene glycol, poloxamer 188, poloxamer 181, poloxamer 407, polyvinylpyrrolidone, Pop313, Crown-5, and any combination thereof. Exemplary supplemental factors include, but are not limited to, media such as PBS, HBSS, OptiMEM, DMEM, RPMI 1640, AIM-V, X-VIVO 15, CellGro DC medium, CTS OpTimizer T cell expansion SFM, TexMACS medium, PRIME-XV T cell expansion medium, ImmunoCult-XF T cell expansion medium, and any combination thereof. Exemplary supplemental factors include, but are not limited to, inhibitors of cellular DNA signaling, metabolism, differentiation, signal transduction, apoptotic pathways, and combinations thereof. Exemplary inhibitors include, but are not limited to, TLR9, MyD88, IRAK, TRAF6, TRAF3, IRF-7, NF-KB, type 1 interferon, Pro-inflammatory cytokines, cGAS, STING, Sec5, TBK1, IRF-3, RNA pol III, RIG-1, IPS-1, FADD, RIP1, TRAF3, AIM2, ASC, caspase 1, Pro-IL1B, P13K, Akt, inhibitors of Wnt3A, inhibitors of glycogen synthase kinase-3 beta (GSK-3 beta) (e.g., TWS119), bafilomycin, chloroquine, quinacrine, AC-YVAD-CMK, Z-VAD-FMK, Z-IETD-FMK, and any combination thereof. Exemplary supplemental factors include, but are not limited to, agents that modify or stabilize one or more nucleic acids in a manner that enhances cellular delivery, enhances nuclear delivery or transport, enhances facilitated transport of the nucleic acid into the nucleus, enhances degradation of extrachromosomal nucleic acids, and/or reduces DNA-mediated toxicity. Exemplary agents that modify or stabilize one or more nucleic acids include, but are not limited to, pH modifiers, DNA binding proteins, lipids, phospholipids, CaPO4, net neutral charge DNA binding peptides with or without NLS sequences, TREX1 enzymes, and any combination thereof.

The post-nuclear transfection cell culture media of the present disclosure (including post-nuclear transfection T cell culture media of the present disclosure) can be used at room temperature or pre-warmed to, for example, 32 ℃ -37 ℃ (inclusive). The post-nuclear transfection cell culture media of the present disclosure (including post-nuclear transfection T cell culture media of the present disclosure) can be preheated to any temperature that maintains or enhances cell viability and/or expression of the transposons or portions thereof of the present disclosure.

The post-nuclear transfection cell culture media of the present disclosure (including the post-nuclear transfection T cell culture media of the present disclosure) may be contained in tissue culture bottles or dishes, G-Rex bottles, bioreactors or cell culture bags, or any other standard receptacle. Post-nuclear transfection cell cultures of the present disclosure (including post-nuclear transfection T cell cultures of the present disclosure) may remain stationary, or alternatively they may be perturbed (e.g., shaken, vortexed, or vibrated).

The cell culture may comprise genetically modified cells after nuclear transfection. The post-nuclear transfection T cell culture may comprise genetically modified T cells. The genetically modified cells of the present disclosure can be rested for a defined period of time or stimulated for expansion by, for example, the addition of T cell Expander (Expander) technology. In certain embodiments, the genetically modified cells of the present disclosure can be rested for a defined period of time or immediately stimulated for expansion by, for example, the addition of T cell expander technology. Genetically modified cells of the disclosure can be allowed to rest to provide them with sufficient time to comply, time for transposition to occur, and/or time for positive or negative selection to occur, resulting in cells with enhanced viability, higher nuclear transfection efficiency, greater viability following nuclear transfection, a desired cell phenotype, and/or greater/faster expansion upon addition of expansion technology. The genetically modified cells of the disclosure can be quiescent, e.g., for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or more hours. In certain embodiments, the genetically modified cells of the present disclosure can be quiescent, e.g., overnight. In certain aspects, the overnight is about 12 hours. The genetically modified cells of the disclosure can be quiescent, e.g., for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more days.

The genetically modified cells of the present disclosure can be selected after the nuclear transfection reaction and prior to the addition of the extender technique. For optimal selection of genetically modified cells, the cells can be allowed to rest in cell culture media for at least 2-14 days following nuclear transfection to facilitate identification of modified cells (e.g., to distinguish modified cells from non-modified cells).

When the transposon of the present disclosure is successfully transfected into a nucleus, the expression of the centryrin or CARTyrin and the selectable marker of the present disclosure can be detected in the modified T cell as early as 24 hours after the nuclear transfection. Due to the extrachromosomal expression of the transposon, expression of the selectable marker alone may not distinguish modified T cells (those cells that have successfully integrated the transposon) from unmodified T cells (those cells that have not successfully integrated the transposon). When extrachromosomal expression of the transposon obscures detection of the modified cell by the selectable marker, the nuclear transfected cells (both modified and unmodified) can be allowed to sit for a period of time (e.g., 2-14 days) to allow the cells to stop expressing or lose all extrachromosomal transposon expression. After this extended period of rest, only the modified T cells should remain positive for expression of the selectable marker. The length of this extended resting period can be optimized for each nuclear transfection reaction and selection process. When extrachromosomal expression of the transposon obscures detection of the modified cells by the selectable marker, selection can be performed without this extended period of rest, however, additional selection steps can be included at later time points (e.g., during or after the expansion phase).

Selection of the genetically modified cells of the present disclosure can be by any means. In certain embodiments of the methods of the present disclosure, selection of genetically modified cells of the present disclosure can be performed by isolating cells that express a particular selectable marker. The selectable marker of the present disclosure may be encoded by one or more sequences in a transposon. As a result of successful transposition, the selectable marker of the present disclosure can be expressed by the modified cell (i.e., not encoded by one or more sequence sequences in the transposon). In certain embodiments, the genetically modified cells of the present disclosure comprise a selectable marker that confers resistance to a deleterious compound of the cell culture medium following nuclear transfection. Harmful compounds may include, for example, antibiotics or drugs that would cause cell death in the absence of resistance conferred by the selectable marker to the modified cell. Exemplary selectable markers include, but are not limited to, wild-type (WT) or mutant forms of one or more of the following genes: neo, DHFR, TYMS, ALDH, MDR1, MGMT, FANCF, RAD51C, GCS, and NKX 2.2. Exemplary selectable markers include, but are not limited to, surface-expressed selectable markers or surface-expressed tags, which can be targeted by Ab-coated magnetic bead technology or column selection, respectively. Cleavable tags such as those used in protein purification can be added to the selection markers of the present disclosure for efficient column selection, washing, and elution. In certain embodiments, the selectable markers of the present disclosure are not endogenously expressed by the modified cells (including modified T cells) and thus may be useful in physically isolating the modified cells (by, for example, cell sorting techniques). Exemplary selectable markers of the present disclosure that are not endogenously expressed by modified cells (including modified T cells) include, but are not limited to, full-length, mutated or truncated forms of CD271, CD19 CD52, CD34, RQR8, CD22, CD20, CD33, and any combination thereof.

The genetically modified cells of the present disclosure can be selectively expanded following a nuclear transfection reaction. In certain embodiments, a modified T cell comprising CARTyrin can be selectively expanded by CARTyrin stimulation. Modified T cells comprising CARTyrin can be stimulated by contact with a target-overlaying agent (e.g., a tumor line or normal cell line expressing the target or an extender bead that is overlaid with the target). On the other hand, modified T cells comprising CARTyrin can be stimulated by contact with irradiated tumor cells, irradiated allogeneic normal cells, irradiated autologous PBMCs. To minimize contamination of the cell product composition of the present disclosure by target-expressing cells for stimulation, for example, expansion agent beads coated with a CARTyrin target protein can be used for stimulation when the cell product composition can be administered directly to a subject. Selective expansion of modified T cells comprising CARTyrin stimulated by CARTyrin can be optimized to avoid functionally depleting modified T-cells.

Selected genetically modified cells of the present disclosure may be cryopreserved, rested for a defined period of time, or stimulated for expansion by the addition of cell expander technology. Selected genetically modified cells of the present disclosure can be cryopreserved, rested for a defined period of time, or immediately stimulated for expansion by the addition of cell expander technology. When the selected genetically modified cell is a T cell, the T cell can be stimulated for expansion by the addition of T-cell expander technology. Selected genetically modified cells of the disclosure can be quiescent, e.g., for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or more hours. In certain embodiments, selected genetically modified cells of the present disclosure can be quiescent, e.g., overnight. In certain aspects, the overnight is about 12 hours. Selected genetically modified cells of the disclosure can be quiescent, e.g., for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more days. Selected genetically modified cells of the present disclosure can be made to quiesce for any period of time resulting in cells with enhanced viability, higher nuclear transfection efficiency, greater viability following nuclear transfection, desired cell phenotype, and/or greater/faster expansion when expansion techniques are added.

Selected genetically modified cells (including selected genetically modified T cells of the disclosure) can be cryopreserved using any standard cryopreservation method that can be optimized for storage and/or recovery of human cells with high recovery, viability, phenotype, and/or functional capacity. The cryopreservation methods of the present disclosure can include commercially available cryopreservation media and/or protocols.

Transposition efficiency of selected genetically modified cells, including selected genetically modified T cells of the present disclosure, can be assessed by any means. For example, expression of a transposon by selected genetically modified cells, including selected genetically modified T cells of the present disclosure, can be measured by Fluorescence Activated Cell Sorting (FACS) prior to application of the expander technology. Determining the transposition efficiency of selected genetically modified cells, including selected genetically modified T cells of the present disclosure, can include determining the percentage of selected cells that express a transposon (e.g., CARTyrin). Alternatively or additionally, the purity of the T cell, the Mean Fluorescence Intensity (MFI) of transposon expression (e.g., CARTyrin expression), the ability of CARTyrin (delivered in the transposon) to mediate degranulation and/or killing of target cells expressing a CARTyrin ligand, and/or the phenotype of selected genetically modified cells (including selected genetically modified T cells of the present disclosure) can be assessed by any means.

The cell product compositions of the present disclosure can be delivered for administration to a subject when certain delivery (release) criteria are met. Exemplary payment criteria can include, but are not limited to, a specific percentage of modified, selected, and/or expanded T cells that express detectable levels of CARTyrin on the cell surface.

Genetic modification of autologous T cell product compositions

Genetically modified cells (including genetically modified T cells) of the present disclosure can be expanded using expander technology. The extender technology of the present disclosure may include commercially available extender technology. Exemplary expander technologies of the present disclosure include stimulating genetically modified T cells of the present disclosure via a TCR. While all means for stimulating the genetically modified T cells of the present disclosure are contemplated, stimulation of the genetically modified T cells of the present disclosure by a TCR is a preferred method, resulting in a product with superior levels of killing ability.

To stimulate genetically modified T cells of the present disclosure via a TCR, the expression of a TCR can be expressed as a ratio of 3: bead to T cell ratio of 1 Thermo Expander DynaBeads was used. If the extender beads are not biodegradable, the beads can be removed from the extender composition. For example, the beads may be removed from the extender composition after about 5 days. To stimulate genetically modified T cells of the present disclosure via TCR, Miltenyi T Cell Activation/Expansion reagents (Miltenyi T Cell Activation/Expansion Reagent) can be used. To stimulate genetically modified T cells of the present disclosure via TCR, ImmunoCult human CD3/CD28 or CD3/CD28/CD 2T cell activator agents from StemCell Technologies may be used. This technique may be preferred because soluble tetrameric antibody complexes will degrade over time and will not need to be removed from the process.

Artificial Antigen Presenting Cells (APCs) can be engineered to co-express a target antigen and can be used to stimulate cells or T-cells of the disclosure by the TCRs and/or the CARTyrin of the disclosure. The artificial APCs may comprise or be derived from a tumor cell line (including, for example, the immortalized myeloid leukemia line K562), and may be engineered to co-express a variety of co-stimulatory molecules or techniques (e.g., CD28, 4-1BBL, CD64, mbiL-21, mbiL-15, CAR target molecules, etc.). When the artificial APCs of the present disclosure are combined with co-stimulatory molecules, conditions can be optimized to prevent the development or emergence of undesirable phenotypic and functional capacity, i.e., terminally differentiated effector T cells.

Irradiated PBMCs (autologous or allogeneic) may express some target antigen, such as CD19, and may be used to stimulate cells or T-cells of the disclosure by the TCRs and/or the CARTyrin of the disclosure. Alternatively or additionally, irradiated tumor cells may express some target antigens and may be used to stimulate cells or T-cells of the disclosure by the TCR and/or the CARTyrin of the disclosure.

Plate-bound and/or soluble anti-CD 3, anti-CD 2, and/or anti-CD 28 stimulation may be used to stimulate cells or T-cells of the disclosure by TCRs and/or CARTyrin of the disclosure.

The antigen-coated beads can display a target protein and can be used to stimulate cells or T-cells of the disclosure by the TCRs and/or the CARTyrin of the disclosure. Alternatively or additionally, expander beads coated with a CARTyrin target protein can be used to stimulate cells or T-cells of the disclosure by a TCR and/or CARTyrin of the disclosure.

Expansion methods are directed through TCR or CARTyrin and stimulate cells of the disclosure or T-cells by genetically modifying CD2, CD3, CD28, 4-1BB and/or other markers expressed on the upper surface of the T-cells.

Expansion techniques can be applied to cells of the present disclosure immediately after nuclear transfection until about 24 hours after nuclear transfection. Although a variety of cell culture media can be used during the expansion procedure, the desired T cell expansion media of the present disclosure can produce cells with, for example, higher viability, cell phenotype, total expansion, or higher capacity for in vivo persistence, engraftment, and/or CAR-mediated killing. The cell culture medium of the present disclosure can be optimized to improve/enhance the expansion, phenotype, and function of the genetically modified cells of the present disclosure. Preferred phenotypes of expanded T cells may include a mixture of T stem cell memory, T central and T effector memory cells. The expander Dynabeads may primarily generate central memory T cells that may lead to superior performance in the clinic.

Exemplary T cell expansion media of the present disclosure may include, in part or in whole, PBS, HBSS, OptiMEM, DMEM, RPMI 1640, AIM-V, X-VIVO 15, CellGro DC medium, CTS OpTimizer T cell expansion SFM, TexMACS medium, PRIME-XV T cell expansion medium, ImmunoCult-XF T cell expansion medium, or any combination thereof. The T cell expansion medium of the present disclosure may further comprise one or more supplemental factors. Supplemental factors that can be included in the T cell expansion media of the present disclosure enhance viability, cell phenotype, total expansion, or increase the ability to be used for persistence, engraftment, and/or CARTyrin-mediated killing in vivo. Additional factors that can be included in the T cell expansion media of the present disclosure include, but are not limited to, recombinant human cytokines, chemokines, and/or interleukins, e.g., IL2, IL7, IL12, IL15, IL21, IL1, IL3, IL4, IL5, IL6, IL8, CXCL8, IL9, IL10, IL11, IL13, GM-CSF, IFN- γ, IL-1 α/IL-1F 72, IL-1 β/IL-1F 13, IL-12 p-72, IL-12 p-72, IL 13/p-17F 17, IL 13/p-1F 17, IL13, IL-24, IL-32 β, IL-32 γ, IL-33, LAP (TGF-. beta.1), lymphotoxin-. alpha./TNF-. beta., TGF-. beta., TNF-. alpha., TRANCE/TNFSF11/RANK L, or any combination thereof. Supplemental factors that can be included in the T cell expansion medium of the present disclosure include, but are not limited to, salts, minerals, and/or metabolites, for example, HEPES, niacinamide, heparin, sodium pyruvate, L-glutamine, MEM non-essential amino acid solutions, ascorbic acid, nucleosides, FBS/FCS, human serum, serum replacement, antibiotics, pH modifiers, Earle's salts, 2-mercaptoethanol, human transferrin, recombinant human insulin, human serum albumin, nuclear stainer PLUS additives, KCL, MgCl2, Na2HPO4, NAH2PO4, sodium lactobionate, mannitol, sodium succinate, sodium chloride, CINa, glucose, Ca (NO3)2, Tris/HCl, K2HPO4, KH2PO4, polyethylenimine, polyethylene glycol, 188, poloxamer 181, poloxamer 407, polyvinylpyrrolidone, Pop313, Crown-5, or any combination thereof. Additional factors that may be included in the T cell expansion medium of the present disclosure include, but are not limited to, inhibitors of cellular DNA signaling, metabolism, differentiation, signal transduction, and/or apoptotic pathways, e.g., TLR9, MyD88, IRAK, TRAF6, TRAF3, IRF-7, NF-KB, type 1 interferon, Pro-inflammatory cytokines, cGAS, STING, Sec5, TBK1, IRF-3, RNA pol III, RIG-1, IPS-1, FADD, RIP1, TRAF3, AIM2, ASC, caspase 1, Pro-IL1B, PI3K, Akt, inhibitors of 3A, inhibitors of glycogen synthase kinase-3 β (GSK-3 β) (e.g., TWS), 119 bavlomycin, chloroquine, AC-YVAD-FMK, Z-TD-IEK, or any combination thereof.

Supplemental factors that may be included in the T cell expansion medium of the present disclosure include, but are not limited to, agents that modify or stabilize nucleic acids in a manner that enhances cellular delivery, enhances nuclear delivery or transport, enhances facilitated transport of nucleic acids into the nucleus, enhances degradation of extrachromosomal nucleic acids, and/or reduces DNA-mediated toxicity, e.g., pH regulators, DNA binding proteins, lipids, phospholipids, CaPO4, net neutral charge DNA binding peptides with or without NLS sequences, TREX1 enzymes, or any combination thereof.

The genetically modified cells of the present disclosure can be selected during the expansion process by using selective drugs or compounds. For example, in certain embodiments, when a transposon of the present disclosure can encode a selection marker that confers resistance to a drug added to the culture medium to genetically modified cells, selection can occur during the expansion process and may require about 1-14 days of culture for selection to occur. Examples of drug-resistant genes that can be used as selectable markers encoded by the transposons of the present disclosure include, but are not limited to, wild-type (WT) or mutant forms of the genes neo, DHFR, TYMS, ALDH, MDR1, MGMT, FANCF, RAD51C, GCS, NKX2.2, or any combination thereof. Examples of corresponding drugs or compounds that may be added to the medium to which the selection marker may confer resistance include, but are not limited to, G418, puromycin, ampicillin, kanamycin, methotrexate, melphalan (Mephalan), temozolomide, vincristine, etoposide, doxorubicin, bendamustine, fludarabine, adata (sodium pamidronate), carmustine (carmustine), BiCNU (carmustine), Bortezomib (Bortezomib), Carfilzomib (Carfilzomib), nitrosourea mustard (carmustine), carmustine, cyclophosphamide (cyclophosphamide), cyclophosphamide, adriamycin (cyclophosphamide), Daratumumab (darazazalex), darunavir (darunavir), Doxil (doxorubin hydrochloride liposome), doxorubin hydrochloride liposome, Dox-SL (doxorubin hydrochloride), rituzumab (eltamiloride), emtuzumab (eletuzumab), geruzumab (riturit (rituximab) Evacet (doxorubicin liposome HCl), Farydak (Panobinostat), Ixazomib Citrate (Ixazomib Citrate), Kyprolis (carfilzomib), Lenalidomide (Lenalidomide), LipoDox (doxorubicin liposome HCl), Mozobil (Plerixafor), Neosar (cyclophosphamide), Nilaro (ixazofamide Citrate), sodium pamidronate, Panobinostat, plerixafu, Pomalidomide (Pomalidomide), Pomallyst (maduramide), Revlimid (lenalimide), Synovir (thalidomide), thalidomide (thalidomide), Vetiade (Bortezomib), Zoledronic Acid (Zodronic Acid), and Zoomelatic Acid (Zosteradian Acid), or any combination thereof.

The T cell expansion process of the present disclosure may take place in a WAVE bioreactor, a G-Rex flask, or in any other suitable container and/or reactor in a cell culture bag.

The cells or T-cell cultures of the present disclosure may remain stable, agitated, gyrated, or vibrated.

The cell or T-cell expansion process of the present disclosure may optimize certain conditions including, but not limited to, culture duration, cell concentration, schedule of T-cell culture medium addition/removal, cell size, total cell number, cell phenotype, purity of the cell population, percentage of genetically modified cells in the growing cell population, use and composition of additives, addition/removal of expander technologies, or any combination thereof.

The cell or T-cell expansion process of the present disclosure may be continued until a predetermined endpoint before formulating the resulting expanded cell population. For example, the cell or T-cell expansion process of the present disclosure may continue for a predetermined amount of time: at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 hours; at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 days; at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks; at least 1, 2, 3, 4, 5, 6 months or at least 1 year. The cell or T-cell expansion process of the present disclosure may continue until the resulting culture reaches a predetermined total cell density: 1, 10, 100, 1000, 104, 105, 106, 107, 108, 109, 1010 cells per volume (μ Ι, ml, L) or any density in between. The cell or T-cell expansion process of the present disclosure may continue until the resulting genetically modified cells of the culture exhibit a predetermined level of expression of the transposon of the present disclosure: a threshold expression level of 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% or any percentage therebetween (indicating the lowest, highest or average expression level at which the resulting genetically modified cell is clinically effective). The cell or T-cell expansion process of the present disclosure may continue until the ratio of genetically modified cells to unmodified cell portions of the resulting culture reaches a predetermined threshold: at least 1: 10, 1: 9, 1: 8, 1: 7, 1: 6, 1: 5, 1: 4, 1: 3, 1: 2, 1: 1, 2: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 110: 1, or any ratio therebetween.

Analysis of genetically modified autologous T cells for delivery

The percentage of genetically modified cells can be assessed during or after the expansion process of the present disclosure. Cellular expression of the genetically modified cells of the disclosure to the transposon can be measured by Fluorescence Activated Cell Sorting (FACS). For example, FACS can be used to determine the percentage of cells or T cells that express the CARTyrin of the present disclosure. Alternatively or additionally, the purity of the genetically modified cell or T cell, the Mean Fluorescence Intensity (MFI) of the CARTyrin expressed by the genetically modified cell or T cell of the present disclosure, the ability of the CARTyrin to mediate degranulation and/or killing of a target cell expressing a CARTyrin ligand, and/or the phenotype of the CARTyrin + T cell can be assessed.

A composition of the present disclosure intended for administration to a subject may need to meet one or more "delivery criteria" that indicate that the composition is safe and effective for formulation into a pharmaceutical product and/or administration to a subject. Delivery criteria can include requiring that a composition of the disclosure (e.g., a T-cell product of the disclosure) comprise a particular percentage of T cells that express a detectable level of the CARTyrin of the disclosure on their cell surface.

The expansion process should continue until certain criteria have been met (e.g., reaching a certain total cell number, reaching a certain memory cell population, reaching a certain size population).

Certain criteria signal the point at which the augmentation process should terminate. For example, once a cell reaches a cell size of 300fL, it should be formulated, reactivated or cryopreserved (otherwise, cells reaching a size above this threshold may begin to die). Cryopreservation, which is performed immediately once the cell population reaches an average cell size of less than 300fL, can result in better cell recovery when thawed and cultured, since the cells have not reached a fully quiescent state prior to cryopreservation (a fully quiescent size of about 180 fL). Prior to expansion, the T cells of the present disclosure may have a cell size of about 180fL, but may have their cell size more than four-fold to about 900fL at 3 days post expansion. During the next 6-12 days, the T-cell population will slowly reduce cell size to complete quiescence at 180 fL.

Methods for preparing a cell population for formulation may include, but are not limited to, the following steps: concentrating the cells of the cell population, washing the cells, and/or further selecting the cells by magnetic bead sorting for drug resistance or for specific surface expressed markers. The method for preparing a cell population for formulation may further comprise a sorting step to ensure safety and purity of the final product. For example, if tumor cells from a patient have been used to stimulate genetically modified T cells of the present disclosure or have been genetically modified to stimulate genetically modified T cells of the present disclosure that are being prepared for formulation, it is critical that the tumor cells from the patient are not included in the final product.

Infusion and/or cryopreservation of cell products for infusion

The pharmaceutical formulations of the present disclosure may be dispensed into bags for infusion, cryopreservation, and/or storage.

The pharmaceutical formulations of the present disclosure may be cryopreserved using standard procedures and optionally infusible cryopreservation media. For example, cryopreservatives without DMSO (e.g., CryoSofree)^TMCryopreservation media without DMSO) can be used to reduce the toxicity associated with freezing. The cryopreserved pharmaceutical formulations of the present disclosure may be stored for infusion to a patient at a later date. Effective treatment may require multiple administrations of the pharmaceutical formulation of the present disclosure, and thus, the pharmaceutical formulation may be packaged in pre-aliquoted "doses" that can be stored frozen but separated for thawing individual doses.

The pharmaceutical formulations of the present disclosure may be stored at room temperature. Effective treatment may require multiple administrations of the pharmaceutical formulations of the present disclosure, and thus, the pharmaceutical formulations may be packaged in pre-aliquoted "doses" that may be stored together but separated for administration of the individual doses.

The pharmaceutical formulations of the present disclosure may be archived for subsequent re-expansion and/or selection for use in generating additional doses to the same patient in the case of allotherapy, who may require administration on a future date, e.g., after remission and relapse.

Preparation

As noted above, the present disclosure provides stable formulations, preferably comprising a phosphate buffer with saline or selected salts, and a preserved solution, and a preservative-containing formulation and a multi-use preserved formulation suitable for pharmaceutical or veterinary use, comprising at least one CARTyrin in a pharmaceutically acceptable formulation. The preserved formulation contains at least one known preservative or is optionally selected from at least one phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, phenylmercuric nitrite, phenoxyethanol, formaldehyde, chlorobutanol, magnesium chloride (e.g., hexahydrate), alkyl parabens (methyl, ethyl, propyl, butyl, etc.), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate, and thimerosal, a polymer, or a mixture thereof in an aqueous diluent. Any suitable concentration or mixture may be used, for example about 0.0015%, or any range, value, or fraction thereof, as is known in the art. Non-limiting examples include, without preservatives, about 0.1-2% m-cresol (e.g., 0.2, 0.3, 0.4, 0.5, 0.9, 1.0%), about 0.1-3% benzyl alcohol (e.g., 0.5, 0.9, 1.1, 1.5, 1.9, 2.0, 2.5%), about 0.001-0.5% thimerosal (e.g., 0.005, 0.01), about 0.001-2.0% phenol (e.g., 0.05, 0.25, 0.28, 0.5, 0.9, 1.0%), one or more alkyl parabens (e.g., 0.00075, 0.0009, 0.001, 0.002, 0.005, 0.0075, 0.009, 0.01, 0.02, 0.05, 0.09, 0.1, 0.75%, etc.).

As noted above, the present invention provides an article of manufacture comprising a packaging material and at least one vial containing a solution of at least one CARTyrin having a defined buffer and/or preservative, optionally in an aqueous diluent, wherein the packaging material comprises a label indicating that such solution can be preserved during a period of 1, 2, 3, 4, 5, 6, 9, 12, 18, 20, 24, 30, 36, 40, 48, 54, 60, 66, 72 hours or longer. The present invention further encompasses an article of manufacture comprising a packaging material, a first vial comprising lyophilized at least one CARTyrin and a second vial comprising an aqueous diluent of a specified buffer or preservative, wherein the packaging material comprises a label instructing a patient to reconstitute the at least one CARTyrin in the aqueous diluent to form a solution that can be stored over a period of twenty-four hours or more.

As described herein or as known in the art, at least one CARTyrin for use according to the invention may be produced by recombinant means, including from mammalian cells or transgenic preparations, or may be purified from other biological sources.

The range of at least one CARTyrin in the product of the invention includes amounts that upon reconstitution (if in a wet/dry system) result in a concentration of about 1.0 μ g/ml to about 1000mg/ml, although lower and higher concentrations are feasible and depending on the intended delivery vehicle, for example, solution formulations will be different from transdermal patch (patch), pulmonary, transmucosal or osmotic or micropump methods.

Preferably, the aqueous diluent optionally further comprises a pharmaceutically acceptable preservative. Preferred preservatives include those selected from the group consisting of phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, alkyl parabens (methyl, ethyl, propyl, butyl, etc.), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate, and thimerosal, or mixtures thereof. The concentration of preservative used in the formulation is a concentration sufficient to produce an antimicrobial effect. Such concentrations depend on the preservative selected and are readily determined by one skilled in the art.

Other excipients, for example, isotonicity agents, buffers, antioxidants and preservative enhancers (enhancers) may optionally and preferably be added to the diluent. Isotonic agents, such as glycerol, are generally used at known concentrations. A physiologically tolerable buffer is preferably added to provide improved pH control. The formulation may comprise a wide range of pHs, for example, from about pH 4 to about pH 10, and a preferred range from about pH 5 to about pH 9, and a most preferred range from about 6.0 to about 8.0. Preferably, the formulations of the present invention have a pH of about 6.8 to about 7.8. Preferred buffers include phosphate buffered saline, most preferably sodium phosphate, especially Phosphate Buffered Saline (PBS).

Other additives, e.g. pharmaceutically acceptable solubilizers, e.g. Tween 20 (polyoxyethylene (20) sorbitan monolaurate), Tween 40 (polyoxyethylene (20) sorboseAlcohol anhydride monopalmitate), tween 80 (polyoxyethylene (20) sorbitan monooleate), Pluronic F68 (polyoxyethylene polyoxypropylene block copolymer) and PEG (polyethylene glycol) or a non-ionic surfactant, such as polysorbate 20 or 80 or poloxamer 184 or 188,polyls, other block copolymers, and chelating agents (e.g., EDTA and EGTA) may optionally be added to the formulation or composition to reduce aggregation. These additives are particularly useful if the formulation is to be administered using a pump or a plastic container. The presence of a pharmaceutically acceptable surfactant reduces the tendency of the protein to aggregate.

The formulations of the present invention may be prepared by a process comprising mixing at least one CARTyrin and a preservative selected from the group consisting of phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, alkyl parabens (methyl, ethyl, propyl, butyl, etc.), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate, and thimerosal, or mixtures thereof in an aqueous diluent. The mixing of the at least one CARTyrin and the preservative in the aqueous diluent is carried out using conventional dissolution and mixing procedures. To prepare a suitable formulation, for example, a measured amount of at least one CARTyrin in a buffered solution is combined with a desired preservative in a buffered solution in an amount sufficient to provide the desired concentration of protein and preservative. One of ordinary skill in the art will recognize variations of this approach. For example, the order of addition of the components, whether additional additives are used, the temperature and pH at which the formulation is prepared are all factors that can be optimized for the concentration and mode of administration used.

The claimed formulations may be provided to a patient as a clear solution or as a dual vial comprising a vial of lyophilized at least one CARTyrin reconstituted with a second vial containing water, preservatives and/or excipients, preferably phosphate buffer and/or saline and selected salts, in an aqueous diluent. Either the single solution vial or the dual vial requiring reconstitution can be reused multiple times and can meet a single or multiple cycles of patient treatment and therefore can provide a more convenient treatment regimen than is currently available.

The presently claimed articles are useful for application over a period of time from immediately up to 24 hours or more. Thus, the presently claimed article provides significant advantages to patients. The formulations of the invention can optionally be safely stored at temperatures of about 2 ℃ to about 40 ℃ and maintain the biological activity of the protein for extended periods of time, thereby allowing for the indication of packaging labels that the solution can be stored and/or used over a period of 6, 12, 18, 24, 36, 48, 72, or 96 hours or more. Such labels may include up to 1-12 months, half a year, and/or two years of use if a preserved diluent is used.

The solution of at least one CARTyrin of the present invention can be prepared by a method comprising mixing at least one CARTyrin in an aqueous diluent. Mixing was performed using conventional dissolution and mixing procedures. To prepare a suitable diluent, for example, a measured amount of at least one CARTyrin in water or buffer is combined in an amount sufficient to provide the desired concentration of protein and optionally a preservative or buffer. One of ordinary skill in the art will recognize variations of this approach. For example, the order of addition of the components, whether additional additives are used, the temperature and pH at which the formulation is prepared are all factors that can be optimized for the concentration and mode of administration used.

The claimed product may be provided to a patient as a clear solution or as a dual vial comprising a vial of lyophilized at least one CARTyrin reconstituted with a second vial containing an aqueous diluent. Either the single solution vial or the dual vial requiring reconstitution can be reused multiple times and can satisfy a single or multiple cycles of patient treatment and thus provide a more convenient treatment regimen than is currently available.

The claimed product may be provided to the patient indirectly by providing a clear solution or a dual vial comprising a vial of lyophilized at least one CARTyrin reconstituted with a second vial containing an aqueous diluent to a pharmacy, clinic or other such public facility and institution. In this case, the clear solution may be up to one liter or even larger in size, thereby providing a large reservoir from which a smaller portion of the at least one CARTyrin solution may be extracted one or more times for transfer into a smaller vial and provided by a pharmacy or clinic to its patron and/or patient.

Recognized devices including single vial systems include pen-injector devices for delivering solutions, such as BD Pens, BD Pen，AndGenotronormHumatroRoferon J-tip Needle-Freefor example, as described by Becton Dickinson (Franklin Lakes, N.J., www.bectondickenson.com), Disetronic (Burgdorf, Switzerland, www.disetronic.com; Bio Ject, Portland, Oreg. (www.bioject.com); National Medical Products, Weston Medical (Peterborough, UK, www.weston-medical.com), Medi-Ject Corp (Minneapolis, Minn., Minn, Min, Minn, Min,www.mediject.com), manufactured or developed, and the like. Recognized devices including dual vial systems include those pen injector systems for reconstituting lyophilized drugs in cartridges for delivering the reconstituted solution, e.g.Examples of other suitable devices include pre-filled syringes, auto-injectors, needle-free injectors, and needle-free IV infusion sets.

The claimed product comprises a packaging material. In addition to the information required by regulatory agencies, packaging materials also provide conditions under which products can be used. The packaging material of the present invention provides instructions to a patient for reconstituting at least one CARTyrin in an aqueous diluent to form a solution for two vials of a wet/dry product and using the solution over a period of 2-24 hours or more. For single vial solution products, the label indicates that such solutions can be used over a period of 2-24 hours or more. The product as claimed in the invention can be used for human pharmaceutical product applications.

The formulations of the present invention can be prepared by a method comprising mixing at least one CARTyrin and a selected buffer, preferably a phosphate buffer containing saline or a selected salt. The mixing of the at least one CARTyrin and the buffer in the aqueous diluent is carried out using conventional dissolution and mixing procedures. To prepare a suitable formulation, for example, a measured amount of at least one CARTyrin in water or buffer is combined with a desired buffer in water in an amount sufficient to provide the desired concentration of protein and buffer. One of ordinary skill in the art will recognize variations of this approach. For example, the order of addition of the components, whether additional additives are used, the temperature and pH at which the formulation is prepared are all factors that can be optimized for the concentration and mode of administration used.

The claimed stable or preserved formulations may be provided to patients as clear solutions or as dual vials including a vial of lyophilized CARTyrin reconstituted with a second vial containing a preservative or buffer and excipients in an aqueous diluent. Either the single solution vial or the dual vial requiring reconstitution can be reused multiple times and can satisfy a single or multiple cycles of patient treatment and thus provide a more convenient treatment regimen than is currently available.

Other formulations or methods of stabilizing CARTyrin can result in the production of clear solutions other than lyophilized powders comprising CARTyrin. In a non-transparent solution there is a formulation comprising a suspension of particles, which are CARTyrin-containing compositions with structures of variable size and variously known as spheroids, microparticles, nanoparticles, nanospheres (nanospheres) or liposomes. Such relatively homogenous, substantially spherical particle formulations containing an active agent may be formed by contacting an aqueous phase containing the active agent and polymer with a non-aqueous phase followed by evaporation of the non-aqueous phase to cause the particles to coalesce from the aqueous phase, as taught in U.S. patent No.4,589,330. Porous microparticles may be prepared using a first phase containing the active agent and polymer dispersed in a continuous solvent and removing the solvent from the suspension by freeze-drying or dilution-extraction-precipitation, as taught in U.S. patent No.4,818,542. Preferred polymers for use in such formulations are natural or synthetic copolymers or polymers selected from the group consisting of: gelatin agar, starch, arabinogalactan, albumin, collagen, polyglycolic acid, polylactic acid, glycolide-L (-) lactide-polyepsilon-caprolactone, poly (epsilon-caprolactone-CO-lactic acid), poly (epsilon-caprolactone-CO-glycolic acid), poly (beta-hydroxybutyric acid, polyethylene oxide, polyethylene, polyalkyl-2-cyanoacrylate, polyhydroxyethylmethacrylate, polyamide, polyamino acid, poly 2-hydroxyethyl DL-asparagine, polyesterurea, poly (L-phenylalanine/ethylene glycol/1, 6-diisocyanatohexane) (poly (L-phenylalanine/ethylene glycol/1, 6-diisocyanatohexane)), and polymethylmethacrylate. Particularly preferred polymers are polyesters such as polyglycolic acid, polylactic acid, glycolide-L (-) lactide polyepsilon-caprolactone, poly (epsilon-caprolactone-CO-lactic acid) and poly (epsilon-caprolactone-CO-glycolic acid solvents useful for dissolving polymers and/or active (active) include water, hexafluoroisopropanol, methylene chloride, tetrahydrofuran, hexane, benzene or hexafluoroacetone sesquihydrate.

Dry powder formulations may be produced by methods other than lyophilization, for example by spray drying or by evaporation or by precipitation of a crystalline composition followed by one or more steps to remove solvent extraction of the aqueous or non-aqueous solvent. The preparation of spray dried CARTyrin formulations is taught in U.S. patent No.6,019,968. Dry powder compositions based on CARTyrin can be produced by spray drying a solution or slurry of CARTyrin and optionally excipients in a solvent under conditions to provide a respirable dry powder. The solvent may include polar compounds such as water and ethanol, which may be easily dried. The CARTyrin stability can be enhanced by performing a spray drying procedure in the absence of oxygen, for example under a nitrogen blanket or by using nitrogen as the drying gas. Another relatively dry formulation is a dispersion of a plurality of porous microstructures dispersed in a suspension medium, typically comprising a hydrofluoroalkane (hydrofluoroalkane) propellant, as taught in WO 9916419. The stable dispersion can be administered to the lungs of a patient using a metered dose inhaler. Equipment that can be used for commercial production of spray-dried drugs is manufactured by Buchi ltd.

At least one CARTyrin in a stable or preserved formulation or solution described herein can be administered to a patient according to the present invention by a variety of delivery methods, including SC or IM injections; transdermal, pulmonary, transmucosal, implantation, osmotic pump, cartridge, micropump, or other means known to those skilled in the art, as is well known in the art.

Therapeutic applications

The invention also provides methods of using at least one CARTyrin of the invention to modulate or treat a disease in a cell, tissue, organ, animal or patient as known in the art or as described herein, for example, administering or contacting a therapeutically effective amount of a CARTyrin to the cell, tissue, organ, animal or patient with a therapeutically effective amount of a CARTyrin. The invention also provides methods for modulating or treating diseases in cells, tissues, organs, animals or patients, including but not limited to malignant diseases.

The present invention also provides a method for modulating or treating at least one malignancy in a cell, tissue, organ, animal or patient, including, but not limited to, at least one of: leukemia, Acute Lymphoblastic Leukemia (ALL), acute lymphocytic leukemia, B-cell, T-cell or FAB ALL, Acute Myelogenous Leukemia (AML), acute myelogenous leukemia, Chronic Myelogenous Leukemia (CML), Chronic Lymphocytic Leukemia (CLL), hairy cell leukemia, myelodysplastic syndrome (MDS), lymphoma, Hodgkin's disease, malignant lymphoma, non-Hodgkin's lymphoma, Burkitt's lymphoma, multiple myeloma, Kaposi's sarcoma, colorectal cancer, pancreatic cancer, nasopharyngeal cancer, malignant histiocytosis, tumor-related disorders/malignant hypercalcemia, solid tumors, bladder cancer, breast cancer, colorectal cancer, endometrial cancer, head cancer, neck cancer, hereditary non-polyposis cancer, Hodgkin's lymphoma, liver cancer, lung cancer, non-small cell lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, testicular cancer, adenocarcinoma, sarcoma, malignant melanoma, hemangioma, metastatic disease, cancer-related bone resorption, cancer-related bone pain, and the like.

Any method of the present invention can comprise administering an effective amount of a composition or pharmaceutical composition comprising at least one CARTyrin to a cell, tissue, organ, animal, or patient in need of such modulation, treatment, or therapy. Such methods may optionally further comprise simultaneous administration (co-administration) or combination therapy for treating such disease or disorder, wherein the administration of the at least one CARTyrin, designated portion or variant thereof further comprises prior, simultaneous and/or subsequent administration of at least one selected from at least one alkylating agent, mitotic inhibitor and radiopharmaceutical. Suitable dosages are well known in the art. See, e.g., Wells et al, eds, Pharmacotherapy Handbook, 2 nd edition, Appleton and Lange, Stamford, Conn. (2000); PDR Pharmacopoeia, Tarascon Pocket Pharmacopoeia 2000, Deluxe Edition, Tarascon Publishing, Loma Linda, Calif. (2000); nursing 2001 Handbook of Drugs, 21 st edition, Springhouse Corp., Springhouse, Pa., 2001; health Professional's Drug Guide 2001, ed., Shannon, Wilson, Stang, prentic-Hall, Inc, Upper saddleriver, n.j., each of which is incorporated herein by reference in its entirety.

Preferred doses may optionally include about 0.1-99 and/or 100-500 mg/kg/administration, or any range, value or fraction thereof, or serum concentrations up to about 0.1-5000 μ g/ml serum concentration per single or multiple administration, or any range, value or fraction thereof. Preferred dosages of the CARTyrin of the present invention range from about 1mg/kg of patient body weight up to about 3, about 6, or about 12mg/kg of patient body weight.

On the other hand, the dose administered may vary depending on known factors, such as the pharmacokinetic profile of the particular agent and the mode and route of administration thereof; age, health, and weight of the recipient; the nature and extent of the symptoms, the type of concurrent treatment, the frequency of treatment, and the desired effect. Generally, the dosage of the active ingredient may be about 0.1 to 100mg per kg body weight. Usually 0.1 to 50 and preferably 0.1 to 10 milligrams per kilogram per administration or sustained release form is effective to obtain the desired result.

As a non-limiting example, treatment of a human or animal can be provided as a single, infused, or repeated dose, as one or periodic doses of about 0.1 to 100mg/kg or any range, value, or fraction thereof of at least one CARTyrin of the present invention per day on at least one of days 1-40, or, on the other hand or in addition, at least one week 1-52 weeks, or, on the other hand or in addition, at least one year 1-20 years, or any combination thereof.

Dosage forms (compositions) suitable for internal administration typically contain from about 0.001 mg to about 500 mg of the active ingredient per unit or container. In such pharmaceutical compositions, the active ingredient will generally be present in an amount of from about 0.5 to 99.999 percent by weight, based on the total weight of the composition.

For parenteral administration, the CARTyrin can be formulated in combination with a pharmaceutically acceptable parenteral carrier as a solution, suspension, emulsion, granule, powder or lyophilized powder or provided separately. Examples of such carriers are water, saline, ringer's solution, dextrose solution, and about 1-10% human serum albumin. Liposomes and non-aqueous vehicles, such as fixed oils, can also be used. The carrier or lyophilized powder may contain additives that maintain isotonicity (e.g., sodium chloride, mannitol) and chemical stability (e.g., buffers and preservatives). The formulation is sterilized by known or suitable techniques.

Suitable Pharmaceutical carriers are described in the latest edition of Remington's Pharmaceutical Sciences, a.

Alternative administration

Many known and improved means can be used according to the invention for administering a pharmaceutically effective amount of at least one CARTyrin according to the invention. Although pulmonary administration is used in the following description, other modes of administration may be used in accordance with the present invention with appropriate results. The CARTyrin of the present invention can be delivered in a carrier, as a solution, emulsion, colloid, or suspension, or as a dry powder, using any of a variety of devices and methods suitable for administration by inhalation or other means described herein or known in the art.

Parenteral formulations and administration

Formulations for parenteral administration may contain sterile water or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, hydrogenated naphthalenes and the like as common excipients. Aqueous or oily suspensions for injection may be prepared according to known methods by using appropriate emulsifying or wetting agents and suspending agents. The agents for injection may be non-toxic, non-oral diluents, such as aqueous solutions in solvents, sterile injectable solutions or suspensions. As a usable carrier or solvent, water, ringer's solution, isotonic saline, etc. are permissible; as a general solvent or suspending solvent, sterile, fixed oils may be used. For these purposes, any kind of non-volatile oils and fatty acids may be used, including natural or synthetic or semi-synthetic fatty oils or fatty acids; natural or synthetic or semisynthetic mono-or di-or triacylglycerols. Parenteral administration is known in the art and includes, but is not limited to, conventional injection means such as gas pressurized needle-free injection devices as described in U.S. patent No.5,851,198 and laser perforator devices as described in U.S. patent No.5,839,446, which is incorporated herein by reference in its entirety.

Alternative delivery

The invention further relates to administering at least one CARTyrin parenterally, subcutaneously, intramuscularly, intravenously, intraarticularly (intrabronchial), intrabronchially, intraperitoneally, intracapsularly, intracartilaginous, intracavitary, intracelial, intracelebellar, intracerebroventricular, intracolic, intracervical, intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic, intrapericardiac, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, intralesional, bolus, vaginal, rectal, buccal, sublingual, intranasal, or transdermal. At least one CARTyrin composition may be prepared for parenteral (subcutaneous, intramuscular or intravenous) or any other administration, in particular in the form of a liquid solution or suspension; for vaginal or rectal administration, particularly in semi-solid forms, such as, but not limited to, creams and suppositories; for buccal or sublingual administration, for example, but not limited to, in tablet or capsule form; or intranasally, for example, but not limited to, in the form of a powder, nasal drops or aerosol or certain agents; or transdermally, such as, but not limited to, a gel, ointment, lotion, suspension, or patch delivery system with a chemical enhancer (e.g., dimethyl sulfoxide) to modify the structure of the skin or increase the concentration of the Drug in a transdermal patch (juninger et al, "Drug performance Enhancement;" Hsieh, d.s., editions, pages 59-90 (Marcel Dekker, Inc. New York 1994, incorporated herein by reference in its entirety), or with an oxidizing agent (WO 98/53847) that enables the application of a formulation containing proteins and peptides to the skin, or the application of an electric field to create an instantaneous transport pathway, such as electroporation, or to increase the rate of migration of charged drugs through the skin, such as iontophoresis, or the application of ultrasound, such as sonophoresis (U.S. Pat. Nos.4,309,989 and 4,767,402) (the above publications and patents are incorporated herein by reference in their entirety).

Infusion of modified cells as adoptive cell therapy

The present disclosure provides modified cells expressing one or more CARs and/or cartyrinns of the present disclosure that have been selected and/or expanded for administration to a subject in need thereof. The modified cells of the present disclosure can be formulated for storage at any temperature, including room temperature and body temperature. The modified cells of the present disclosure may be formulated for cryopreservation and subsequent thawing. The modified cells of the present disclosure can be formulated in a pharmaceutically acceptable carrier for direct administration to a subject from sterile packaging. The modified cells of the present disclosure can be formulated in a pharmaceutically acceptable carrier with an indicator of cell viability and/or CAR/CARTyrin expression level to ensure a minimum level of cellular function and CAR/CARTyrin expression. The modified cells of the disclosure can be formulated in a pharmaceutically acceptable carrier at a defined density with one or more agents to inhibit further expansion and/or prevent cell death.

Inducible pro-apoptotic polypeptides

The inducible pro-apoptotic polypeptides of the present disclosure are superior to existing inducible polypeptides because the inducible pro-apoptotic polypeptides of the present disclosure are much less immunogenic. Although the inducible pro-apoptotic polypeptides of the present disclosure are recombinant polypeptides, and thus non-naturally occurring, the sequences that are recombined to produce the inducible pro-apoptotic polypeptides of the present disclosure do not comprise non-human sequences that the host human immune system can recognize as "non-self and thus induce an immune response in a subject receiving the inducible pro-apoptotic polypeptides of the present disclosure, cells comprising the inducible pro-apoptotic polypeptides, or compositions comprising the inducible pro-apoptotic polypeptides or cells comprising the inducible pro-apoptotic polypeptides.

The present disclosure provides an inducible pro-apoptotic polypeptide comprising a ligand binding region, a linker, and a pro-apoptotic peptide, wherein the inducible pro-apoptotic polypeptide does not comprise a non-human sequence. In certain embodiments, the non-human sequence comprises a restriction enzyme site. In certain embodiments, the pro-apoptotic peptide is a caspase polypeptide. In certain embodiments, the caspase polypeptide is a caspase 9 polypeptide. In certain embodiments, the caspase 9 polypeptide is a truncated caspase 9 polypeptide. The inducible pro-apoptotic polypeptides of the present disclosure may be non-naturally occurring.

Caspase polypeptides of the disclosure include, but are not limited to, caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, caspase 11, caspase 12, and caspase 14. Caspase polypeptides of the disclosure include, but are not limited to, those caspase polypeptides associated with apoptosis, including caspase 2, caspase 3, caspase 6, caspase 7, caspase 8, caspase 9, and caspase 10. Caspase polypeptides of the disclosure include, but are not limited to, those that initiate apoptosis, including caspase 2, caspase 8, caspase 9, and caspase 10. Caspase polypeptides of the disclosure include, but are not limited to, those that perform apoptosis, including caspase 3, caspase 6, and caspase 7.

The caspase polypeptides of the disclosure may be encoded by amino acid or nucleic acid sequences having one or more modifications as compared to the wild-type amino acid or nucleic acid sequence. Nucleic acid sequences encoding the caspase polypeptides of the disclosure may be codon optimized. One or more modifications to the amino acid and/or nucleic acid sequence of a caspase polypeptide of the disclosure may increase the interaction, cross-linking, cross-activation, or activation of the caspase polypeptide of the disclosure compared to a wild-type amino acid or nucleic acid sequence. Alternatively or additionally, one or more modifications to the amino acid and/or nucleic acid sequence of a caspase polypeptide of the disclosure may reduce the immunogenicity of the caspase polypeptide of the disclosure compared to a wild-type amino acid or nucleic acid sequence.

The caspase polypeptides of the disclosure may be truncated compared to a wild-type caspase polypeptide. For example, caspase polypeptides may be truncated to eliminate sequences encoding Caspase Activation and Recruitment Domains (CARDs) to eliminate or minimize the likelihood of activating local inflammatory responses in addition to initiating apoptosis in cells comprising the inducible caspase polypeptides in the present disclosure. In contrast to wild-type caspase polypeptides, nucleic acid sequences encoding the caspase polypeptides of the disclosure may be spliced to form variant amino acid sequences of the caspase polypeptides of the disclosure. The caspase polypeptides of the disclosure may be encoded by recombinant and/or chimeric sequences. Recombinant and/or chimeric caspase polypeptides of the disclosure may comprise sequences from one or more different caspase polypeptides. Alternatively or additionally, recombinant and/or chimeric caspase polypeptides of the disclosure may comprise sequences from one or more species (e.g., human and non-human sequences). The caspase polypeptides of the disclosure may be non-naturally occurring.

The ligand binding region of an inducible pro-apoptotic polypeptide of the present disclosure may include any polypeptide sequence that facilitates or promotes dimerization of a first inducible pro-apoptotic polypeptide of the present disclosure with a second inducible pro-apoptotic polypeptide of the present disclosure, which dimerization activates or induces cross-linking of the pro-apoptotic polypeptides and initiation of apoptosis in a cell.

The ligand binding ("dimerization") region may include any polypeptide or functional domain thereof that will permit induction using an endogenous or non-naturally occurring ligand (i.e., and inducer), such as a non-naturally occurring synthetic ligand. Depending on the nature of the inducible pro-apoptotic polypeptide and the choice of ligand (i.e., inducer), the ligand binding region may be internal or external to the cell membrane. A wide variety of ligand binding polypeptides and their functional domains, including receptors, are known. The ligand binding regions of the present disclosure may include one or more sequences from a receptor. Of particular interest are ligand binding regions whose ligands (e.g., small organic ligands) are known or can be readily generated. These ligand binding regions or receptors may include, but are not limited to, FKBPs and cyclophilin receptors, steroid receptors, tetracycline receptors, and the like, as well as "non-naturally occurring" receptors that may be obtained from antibodies, particularly heavy or light chain subunits, mutated sequences thereof, random amino acid sequences obtained by random procedures, combinatorial synthesis, and the like. In certain embodiments, the ligand binding region is selected from the group consisting of an FKBP ligand binding region, a cyclophilin receptor ligand binding region, a steroid receptor ligand binding region, a cyclophilin receptor ligand binding region, and a tetracycline receptor ligand binding region.

As an endogenous domain or truncated active portion thereof, a ligand binding region comprising one or more receptor domains can be at least about 50 amino acids, and less than about 350 amino acids, typically less than 200 amino acids. The binding region may be, for example, small (< 25kDa to allow efficient transfection in a viral vector), monomeric, non-immunogenic, with synthetically accessible, cell permeable, non-toxic ligands that may be configured for dimerization.

Depending on the design of the inducible pro-apoptotic polypeptide and the availability of suitable ligands (i.e., inducers), the ligand binding region comprising one or more receptor domains may be intracellular or extracellular. For hydrophobic ligands, the binding region may be on either side of the membrane, but for hydrophilic ligands, particularly protein ligands, the binding region will generally be outside the cell membrane, unless there is a transport system that internalizes the ligand in a form that it can be used to bind. For intracellular receptors, the inducible pro-apoptotic polypeptides or transposons or vectors comprising the inducible pro-apoptotic polypeptides may encode a signal peptide and transmembrane domain 5 ' or 3 ' of the receptor domain sequence, or may have a lipid attachment signal sequence 5 ' of the receptor domain sequence. Where the receptor domain is between the signal peptide and the transmembrane domain, the receptor domain will be extracellular.

Antibodies and antibody subunits, e.g., heavy or light chains, particularly fragments, more particularly all or portions of the variable regions, or fusions of heavy and light chains that produce high affinity binding, can be used as ligand binding regions of the present disclosure. Contemplated antibodies include those that are ectopically expressed human products, such as extracellular domains that will not trigger an immune response and are not normally expressed peripherally (i.e., outside the CNS/brain regions). Such examples include, but are not limited to, low affinity nerve growth factor receptor (LNGFR) and embryonic surface protein (i.e., carcinoembryonic antigen). Still further, antibodies can be prepared against physiologically acceptable hapten molecules and individual antibody subunits screened for binding affinity. The cDNA encoding the subunits may be isolated and modified by deletion of the constant region, portion of the variable region, mutagenesis of the variable region, etc., to obtain a binding protein domain with appropriate affinity for the ligand. Thus, almost any physiologically acceptable hapten compound can be used as a ligand or to provide an epitope of a ligand. Instead of an antibody unit, an endogenous receptor may be used, wherein the binding region or domain is known and there is a useful or known ligand for binding.

To multimerize the receptor, the ligand of the ligand-binding region/receptor domain of the inducible pro-apoptotic polypeptides may be multimeric in the sense that the ligand may have at least two binding sites, each of which is capable of binding to a ligand-receptor region (i.e., a ligand having a first binding site capable of binding to the ligand-binding region of a first inducible pro-apoptotic polypeptide and a second binding site capable of binding to the ligand-binding region of a second inducible pro-apoptotic polypeptide, wherein the ligand-binding regions of the first and second inducible pro-apoptotic polypeptides are the same or different). Thus, as used herein, the term "ligand binding region of a multimer" refers to the ligand binding region of an inducible pro-apoptotic polypeptide of the present disclosure that binds to a multimeric ligand. Multimeric ligands of the present disclosure include dimeric ligands. Dimeric ligands of the present disclosure may have two binding sites capable of binding to the ligand receptor domain. In certain embodiments, the multimeric ligands of the present disclosure are dimers or higher oligomers of small synthetic organic molecules, typically no more than about tetramers, each molecule generally being at least about 150Da and less than about 5kDa, typically less than about 3 kDa. A variety of synthetic ligand and receptor pairs can be used. For example, in embodiments involving endogenous receptors, dimeric FK506 may be used with FKBP12 receptor, dimeric cyclosporin a may be used with cyclophilin receptor, dimeric estrogen with estrogen receptor, dimeric glucocorticoid with glucocorticoid receptor, dimeric tetracycline with tetracycline receptor, dimeric vitamin D with vitamin D receptor, and the like. Alternatively, higher ligands, such as trimers, can be used. For embodiments involving non-naturally occurring receptors, e.g., antibody subunits, modified antibody subunits, single chain antibodies comprising tandem heavy and light chain variable regions separated by a flexible linker, or modified receptors, mutated sequences thereof, and the like, any of a wide variety of compounds may be used. A significant feature of the units comprising the multimeric ligands of the present disclosure is that each binding site is capable of binding to the receptor with high affinity, and preferably, they are capable of being chemically dimerized. As such, such methods are available to balance the hydrophobicity/hydrophilicity of the ligands, thereby enabling them to dissolve at functional levels in serum, yet diffuse through the plasma membrane for most applications.

Activation of an inducible pro-apoptotic polypeptide of the present disclosure can be accomplished, for example, by Chemically Induced Dimerization (CID) mediated by an inducer to produce a conditionally controlled protein or polypeptide. The pro-apoptotic polypeptides of the present disclosure are not only inducible, but the induction of these polypeptides is also reversible, due to degradation of labile dimerizing agents or administration of monomeric competitive inhibitors.

In certain embodiments, the ligand binding region comprises an FK506 binding protein 12(FKBP12) polypeptide. In certain embodiments, the ligand binding region comprises an FKBP12 polypeptide having a substitution of valine (V) to phenylalanine (F) at position 36 (F36V). In certain embodiments, wherein the ligand binding domain comprises an FKBP12 polypeptide having a substitution of valine (V) to phenylalanine (F) at position 36 (F36V), the inducing agent may comprise AP1903, a synthetic drug (CAS index name: 2-Piperidinecarboxylic acid (2-Piperidinecarboxylic acid), 1- [ (2S) -1-oxo-2- (3, 4, 5-trimethoxyphenyl) butyl ] -, 1, 2-ethanediylbis [ imino (2-oxo-2, 1-ethanediyl) oxy-3, 1-phenylene [ (1R) -3- (3, 4-dimethoxyphenyl) propylene ] ] ester, [2S- [1 (R), 2R [ S [1 (R), 2R ] ] ] - (9Cl) CAS number 195514-63-7; formula: C4O 3578H 20; molecular weight: 1411.65): molecular weight: (9 Cl): 195514-63-7) . In certain embodiments, wherein the ligand binding region comprises an FKBP12 polypeptide having a substitution (F36V) of valine (V) for phenylalanine (F) at position 36, the inducing agent can comprise AP20187(CAS registry number: 195514-80-8 and molecular formula: C82H107N5O 20). In certain embodiments, the inducing agent is an AP20187 analog, such as, for example, AP 1510. As used herein, the inducers AP20187, AP1903 and AP1510 may be used interchangeably.

The AP1903 API was produced by autophora Research inc, and the AP1903 drug product for injection was prepared by Formatech inc. It was formulated as a 5mg/mL solution of AP1903 in a 25% non-ionic solubilizer Solutol HS 15 solution (250mg/mL, BASF). At room temperature, this formulation was a clear slightly yellow solution. Upon freezing, this formulation undergoes a reversible phase change, resulting in a milky solution. Upon re-heating to room temperature, this phase change reversed. The loading was 2.33mL in 3mL glass vials (approximately 10mg total AP1903 per vial for injection). In determining the need to administer AP1903, a single fixed dose of AP1903 for injection (0.4mg/kg) can be administered to the patient by Intravenous (IV) infusion over a 2 hour period, for example, using a non-DEHP, non-ethylene oxide sterilized infusion set. The dosage of AP1903 was calculated individually for all patients and was not recalculated unless the body weight fluctuation was ≧ 10%. Prior to infusion, the calculated dose was diluted in 100mL in 0.9% saline. In a previous phase I study of AP1903, 24 healthy volunteers were treated with a single dose of AP1903 for injection at dosage levels of 0.01, 0.05, 0.1, 0.5, and 1.0mg/kg by intravenous infusion over a 2 hour period. The AP1903 plasma levels were dose-proportional over the dose range of 0.01-1.0mg/kg, with mean Cmax values ranging from about 10-1275 ng/mL. After the initial infusion phase, the blood concentration showed a rapid distribution phase, with plasma levels decreasing to about 18%, 7% and 1% of the maximum concentration at 0.5, 2 and 10 hours post-dose (post-dose), respectively. AP1903 for injection showed a safe and well tolerated at all dose levels and showed a favorable pharmacokinetic profile. Iuliucci J D et al, J Clin Pharmacol.41: 870-9, 2001.

The fixed dose of AP1903 used for injection may be, for example, 0.4mg/kg infused intravenously over a 2 hour period. The amount of AP1903 required for efficient cell signaling in vitro was 10-100nM (1600 Da MW). This is equivalent to 16-160. mu.g/L or 0.016-1.6. mu.g/kg (1.6-160. mu.g/kg). In the phase I study of AP1903 above, doses up to 1mg kg were well tolerated. Therefore, 0.4mg/kg may be a safe and effective dose of AP1903 for this phase I study in combination with therapeutic cells.

The amino acid and/or nucleic acid sequences of the present disclosure encoding ligand binding may contain one or more modifications of the sequence compared to the wild-type amino acid or nucleic acid sequence. For example, the amino acid and/or nucleic acid sequence encoding the ligand binding region of the present disclosure can be a codon optimized sequence. The one or more modifications can increase the binding affinity of the ligand (e.g., an inducing agent) to the ligand binding region of the disclosure as compared to the wild-type polypeptide. Alternatively or additionally, the one or more modifications may reduce the immunogenicity of the ligand binding region of the disclosure compared to the wild-type polypeptide. The ligand binding region of the present disclosure and/or the inducer of the present disclosure may be non-naturally occurring.

An inducible pro-apoptotic polypeptide of the present disclosure comprises a ligand binding region, a linker, and a pro-apoptotic peptide, wherein the inducible pro-apoptotic polypeptide does not comprise a non-human sequence. In certain embodiments, the non-human sequence comprises a restriction enzyme site. The linker may comprise any organic or inorganic material that, when the ligand binding region dimerizes, allows for interaction, cross-linking, cross-activation or activation of the pro-apoptotic polypeptides such that the interaction or activation of the pro-apoptotic polypeptides initiates apoptosis in the cell. In certain embodiments, the linker is a polypeptide. In certain embodiments, the linker is a polypeptide comprising a G/S-rich amino acid sequence ("GS" linker). In certain embodiments, the linker is a polypeptide comprising the amino acid sequence GGGGS (SEQ ID NO: 18028). In a preferred embodiment, the linker is a polypeptide and the nucleic acid encoding the polypeptide does not contain a restriction site for a restriction endonuclease. The linker of the present disclosure may be non-naturally occurring.

The inducible pro-apoptotic polypeptides of the present disclosure may be expressed in a cell under the transcriptional regulation of any promoter capable of initiating and/or regulating the expression of the inducible pro-apoptotic polypeptides of the present disclosure in the cell. The term "promoter" as used herein refers to a promoter that serves as an initiation binding site for RNA polymerase to transcribe a gene. For example, an inducible pro-apoptotic polypeptide of the present disclosure may be expressed in a mammalian cell under the transcriptional regulation of any promoter capable of initiating and/or regulating the expression of an inducible pro-apoptotic polypeptide of the present disclosure in a mammalian cell, including, but not limited to, native, endogenous, exogenous, and heterologous promoters. Preferred mammalian cells include human cells. Thus, an inducible pro-apoptotic polypeptide of the present disclosure may be expressed in a human cell under the transcriptional regulation of any promoter capable of initiating and/or regulating the expression of an inducible pro-apoptotic polypeptide of the present disclosure in a human cell, including, but not limited to, a human promoter or a viral promoter. Exemplary promoters for expression in human cells include, but are not limited to, the human Cytomegalovirus (CMV) immediate early gene promoter, the SV40 early promoter, the rous sarcoma virus long terminal repeat, the β -actin promoter, the rat insulin promoter, and the glyceraldehyde-3-phosphate dehydrogenase promoter, each of which can be used to obtain high levels of expression of the inducible pro-apoptotic polypeptides of the disclosure. The use of other viral or mammalian cell or bacteriophage promoters, well known in the art, is also contemplated to achieve expression of the inducible pro-apoptotic polypeptides of the disclosure, provided that the expression level is sufficient to initiate apoptosis in the cell. By using promoters with well-known properties, the level and pattern of expression of the protein of interest following transfection or transformation can be optimized.

Regulated actuation in response to specific physiological or synthetic signalsThe selection of subunits may allow for inducible expression of the inducible pro-apoptotic polypeptides of the present disclosure. The ecdysone system (Invitrogen, Carlsbad, Calif) is one such system. Such systems are designed to allow for the regulated expression of genes of interest in mammalian cells. It consists of a tightly regulated expression mechanism that does not actually allow basal level expression of the transgene, but allows over 200-fold inducibility. The system is based on the heterodimeric ecdysone receptor of Drosophila and when an ecdysone or analog such as Milasterone A (muristerone A) binds to the receptor, the receptor activates the promoter to turn on expression of the downstream transgene, resulting in high levels of mRNA transcripts. In this system, both monomers of the heterodimeric receptor are constitutively expressed from one vector, while the ecdysone-responsive promoter driving expression of the gene of interest is on the other plasmid. Thus, it may be useful to adapt this type of system to the vector of interest. Another inducible system that may be useful is Tet-Off, originally developed by Gossen and Bujard ^TMOr Tet-On^TMSystems (Clontech, Palo Alto, Calif.) (Gossen and Bujard, Proc. Natl. Acad. Sci. USA, 89: 5547-. This system also allows for high levels of gene expression to be modulated in response to tetracycline or tetracycline derivatives (e.g., doxycycline). At Tet-On^TMIn the system, gene expression is turned on in the presence of doxycycline and at Tet-Off^TMIn the system, gene expression is turned on in the absence of doxycycline. These systems are based on two regulatory elements derived from the tetracycline resistance operon of E.coli: a tetracycline operator sequence to which the tetracycline repressor binds and a tetracycline repressor protein. The gene of interest is cloned into a plasmid after the promoter in which the tetracycline responsive element is present. The second plasmid contains a regulatory element called the tetracycline-controlled transactivator, which is at Tet-Off^TMThe system consists of the VP16 domain from herpes simplex virus and the wild-type tetracycline repressor. Thus, in the absence of doxycycline, transcription is constitutively on. At Tet-On^TMIn the system, the system is provided with a plurality of sensors,the tetracycline repressor is not wild-type and activates transcription in the presence of doxycycline. For gene therapy vector production, Tet-Off can be used ^TMA system whereby the producer cell can grow in the presence of tetracycline or doxycycline and prevent expression of potentially toxic transgenes, but when the vector is introduced into a patient, gene expression will be constitutively on.

In some cases, it is desirable to regulate the expression of a transgene in a gene therapy vector. For example, different viral promoters with different strengths of activity are utilized depending on the desired expression level. In mammalian cells, the CMV immediate early promoter is often used to provide strong transcriptional activation. CMV promoter in Donnelly, j.j. et al, 1997 annu.rev.immunol.15: 617-48. When reduced transgene expression levels are desired, modified forms of the less potent CMV promoter have also been used. Retroviral promoters, such as the LTRs from MLV or MMTV, are often used when it is desired to express a transgene in hematopoietic cells. Other viral promoters used depending on the desired effect include SV40, RSV LTR, HIV-1 and HIV-2 LTR, adenovirus promoters (e.g., from E1A, E2A or MLP regions), AAV LTR, HSV-TK, and avian sarcoma virus.

In other examples, promoters may be selected that are developmentally regulated and active in particular differentiated cells. Thus, for example, a promoter may not be active in a pluripotent stem cell, but, for example, where the pluripotent stem cell differentiates into a more mature cell, the promoter may then be activated.

Similarly, tissue-specific promoters are used to achieve transcription in specific tissues or cells, thereby reducing potential toxic or undesirable effects on non-targeted tissues. These promoters may lead to reduced expression, but also to more limited expression and immunogenicity, compared to stronger promoters such as the CMV promoter (Bojak, A. et al, 2002. vaccine.20: 1975-79; Cazeaux, N. et al, 2002.Vaccine 20: 3322-31). For example, tissue-specific promoters such as PSA-related promoters or prostate-specific gonadal kallikrein, or muscle creatine kinase genes may be used where appropriate.

Examples of tissue-specific or differentiation-specific promoters include, but are not limited to, the following: b29(B cells); CD14 (monocytes); CD43 (leukocytes and platelets); CD45 (hematopoietic cells); CD68 (macrophages); desmin (muscle); elastase-1 (pancreatic acinar cells); endoglin (endothelial cells); fibronectin (cells in differentiation, tissues in healing); and Flt-1 (endothelial cells); GFAP (astrocyte).

In certain indications, it is desirable to activate transcription at a specific time after administration of the gene therapy vector. This is accomplished using promoters such as those regulated by hormones or cytokines. Cytokine and inflammatory protein responsive promoters which may be used include K and T kininogen (Kageyama et al, (1987) J.biol.Chem., 262, 2345-2351), C-fos, TNF- α, C-reactive protein (Arcone et al, (1988) Nucl.acids Res., 16(8), 3195-3207), haptoglobin (Oliviero et al, (1987) EMBO J., 6, 1905-1912), serum amyloid A2, C/EBP α, IL-1, IL-6(Poli and Cortese, (1989) Proc.Nat' L acid. Sci.USA, 86, 8202-8206), complement C3(Wilson et al, (1990) mol.cell.biol., 6181-6191), 1L-8, alpha-1 acid glycoprotein (Prows and Baumn, 1988), Cell-2408, Lipoprotein, Cell-12, Cell Lipoprotein, Cell-2408, Cell-12, Cell-Lipoprotein, Cell-12, Cell-2, (1991) cell.biol., 2887-2895), fibrinogen, c-jun (inducible by phorbol ester, TNF- α, uv, retinoic acid and hydrogen peroxide), collagenase (inducible by phorbol ester and retinoic acid), metallothionein (inducible by heavy metals and glucocorticoids), stromelysin (inducible by phorbol ester, interleukin-1 and EGF), α -2 macroglobulin and α -1 antichymotrypsin. Other promoters include, for example, SV40, MMTV, human immunodeficiency virus (MV), moloney virus, ALV, EB virus, rous sarcoma virus, human actin, myosin, hemoglobin, and creatine.

It is contemplated that any of the above promoters, alone or in combination with one another, may be useful depending on the desired effect. Promoters and other regulatory elements are selected so that they are functional in the desired cell or tissue. In addition, this list of promoters should not be construed as exhaustive or limiting; other promoters for use in conjunction with the promoters and methods disclosed herein.

Armored T cell "knockdown" strategy

The T-cells of the present disclosure may be genetically modified to enhance their therapeutic potential. Alternatively or additionally, the T-cells of the present disclosure may be modified to make them less susceptible to immune and/or metabolic limitations. This type of modification "armor" the T cells of the present disclosure, which may be referred to herein as "armored" T cells after modification. Armored T cells of the present disclosure may be produced by, for example, blocking and/or thinning certain endogenous checkpoint signals (i.e., checkpoint suppressions) delivered to T-cells, for example, within a tumor immunosuppressive microenvironment.

In some embodiments, the armored T-cells of the present disclosure are derived from T-cells, NK cells, hematopoietic progenitor cells, Peripheral Blood (PB) -derived T-cells (including T-cells isolated or derived from G-CSF-mobilized peripheral blood), or Umbilical Cord Blood (UCB) -derived T-cells. In some embodiments, the armored T-cells of the present disclosure comprise one or more of the chimeric ligand receptors (CLRs comprising a protein scaffold, antibody, ScFv, or antibody mimetic) of the present disclosure/chimeric antigen receptors (CARs comprising a protein scaffold, antibody, ScFv, or antibody mimetic), CARTyrin (CARs comprising centryrin), and/or VCAR (CARs comprising a camelid VHH or single domain VH). In some embodiments, the armored T-cell of the present disclosure comprises an inducible pro-apoptotic polypeptide comprising (a) a ligand binding region, (b) a linker, and (c) a truncated caspase 9 polypeptide, wherein the inducible pro-apoptotic polypeptide does not comprise a non-human sequence. In some embodiments, the non-human sequence is a restriction enzyme site. In some embodiments, the ligand binding region inducible caspase polypeptide comprises an FK506 binding protein 12(FKBP12) polypeptide. In some embodiments, the amino acid sequence of the FK506 binding protein 12(FKBP12) polypeptide comprises a modification at position 36 of the sequence. In some embodiments, the modification is a substitution of valine (V) for phenylalanine (F) at position 36 (F36V). In some embodiments, the armored T-cells of the present disclosure comprise exogenous sequences. In some embodiments, the exogenous sequence comprises a sequence encoding a therapeutic protein. Exemplary therapeutic proteins may be nuclear proteins, cytoplasmic proteins, intracellular proteins, transmembrane proteins, cell surface bound proteins or secreted proteins. Exemplary therapeutic proteins expressed by armored T cells may modify the activity of the armored T cell or may modify the activity of a second cell. In some embodiments, the armored T-cells of the present disclosure comprise a selectable gene or selectable marker. In some embodiments, the armored T-cells of the present disclosure comprise a synthetic gene expression cassette (also referred to herein as an inducible transgene construct).

In some embodiments, the T-cells of the present disclosure are modified to silence or reduce the expression of one or more genes encoding one or more receptors for inhibitory checkpoint signals to produce armored T-cells of the present disclosure. Examples of inhibitory checkpoint signals include, but are not limited to, PD-L1 ligand that binds to a PD-1 receptor on CAR-T cells of the present disclosure or TGF β cytokine that binds to a TGF β RII receptor on CAR-T cells. Receptors for inhibitory checkpoint signals are expressed on the cell surface or within the cytoplasm of T-cells. Silencing or reducing expression of a gene encoding a receptor for inhibitory checkpoint signaling results in loss of protein expression of the inhibitory checkpoint receptor on the surface or within the cytoplasm of the armored T-cells of the present disclosure. Thus, armored T cells of the present disclosure having silenced or reduced expression of one or more genes encoding inhibitory checkpoint receptors are resistant, non-receptive, or insensitive to checkpoint signaling. In the presence of these inhibitory checkpoint signals, the resistance or reduced sensitivity of armored T cells to inhibitory checkpoint signals enhances the therapeutic potential of armored T cells. The inhibitory limit point signals include, but are not limited to, the examples listed in table 1. Exemplary inhibitory checkpoint signals that may be silenced in armored T cells of the present disclosure include, but are not limited to, PD-1 and TGF β RII.

Table 1. exemplary inhibitory checkpoint signals (as well as proteins that induce immunosuppression). The CSR of the present disclosure may comprise the endodomain of any one of the proteins of the table.

In some embodiments, a T-cell of the present disclosure is modified to silence or reduce the expression of one or more genes encoding intracellular proteins associated with checkpoint signaling to produce an armored T-cell of the present disclosure. The activity of the T-cells of the present disclosure may be enhanced by targeting any intracellular signaling protein associated with a checkpoint signaling pathway, thereby effecting checkpoint inhibition or interference of one or more checkpoint pathways. Intracellular signaling proteins involved in checkpoint signaling include, but are not limited to, the exemplary intracellular signaling proteins listed in table 2.

Table 2. exemplary intracellular signaling proteins.

In some embodiments, T-cells of the present disclosure are modified to silence or reduce the expression of one or more genes encoding transcription factors that interfere with therapeutic efficacy to produce armored T-cells of the present disclosure. The activity of armored T-cells can be enhanced or modulated by silencing or reducing the expression (or repression function) of transcription factors that interfere with therapeutic efficacy. Exemplary transcription factors that can be modified to silence or reduce expression or suppress their function include, but are not limited to, the exemplary transcription factors listed in table 3. For example, expression of the FOXP3 gene can be silenced or reduced in armored T cells of the present disclosure to prevent or reduce the formation of T regulatory CAR-T cells (CAR-Treg cells), the expression or activity of which may reduce therapeutic efficacy.

TABLE 3 exemplary transcription factors.

In some embodiments, T-cells of the present disclosure are modified to silence or reduce expression of one or more genes encoding a cell death or apoptosis receptor to produce armored T-cells of the present disclosure. The interaction of the death receptor with its endogenous ligand leads to the initiation of apoptosis. The destruction of cell death and/or expression, activity or interaction of apoptosis receptors and/or ligands makes the armored T-cells of the present disclosure less receptive to death signals, thus making the armored T-cells of the present disclosure more effective in a tumor environment. An exemplary cell death receptor that can be modified in the armored T cells of the present disclosure is Fas (CD 95). Exemplary cell death and/or apoptosis receptors and ligands of the present disclosure include, but are not limited to, the exemplary receptors and ligands provided in table 4.

Table 4. exemplary cell death and/or apoptosis receptors and ligands.

In some embodiments, T-cells of the present disclosure are modified to silence or reduce the expression of one or more genes encoding metabolic signaling proteins (metabolic sensing proteins) to produce armored T-cells of the present disclosure. Disruption of the metabolic signaling sense of the immunosuppressive tumor microenvironment (characterized by low levels of oxygen, pH, glucose, and other molecules) by the armored T-cells of the present disclosure results in prolonged maintenance of T-cell function, and thus, more killed tumor cells per armored T-cell. For example, HIF1a and VHL play a role in T-cell function when in a hypoxic environment. Armored T-cells of the present disclosure may have silenced or reduced expression of one or more genes encoding HIF1a or VHL. Genes and proteins involved in the perception of metabolic signaling include, but are not limited to, the exemplary genes and proteins provided in table 5.

TABLE 5 exemplary Metabolic Signal sensory genes (and encoded proteins).

In some embodiments, T-cells of the present disclosure are modified to silence or reduce expression of one or more genes encoding proteins that confer sensitivity to cancer therapy, including monoclonal antibodies, to produce armored T-cells of the present disclosure. Thus, armored T-cells of the present disclosure may function and may exhibit superior function or efficacy in the presence of cancer therapy (e.g., chemotherapy, monoclonal antibody therapy, or another anti-tumor therapy). Proteins involved in conferring sensitivity to cancer therapy include, but are not limited to, the exemplary proteins provided in table 6.

Table 6 exemplary proteins conferring sensitivity to cancer treatment.

In some embodiments, a T-cell of the present disclosure is modified to silence or reduce expression of one or more genes encoding a growth advantage factor (growth advantage factor) to produce an armored T-cell. Silencing or reducing expression of oncogenes may confer a growth advantage on the armored T-cells of the present disclosure. For example, silencing or reducing the expression (e.g., disrupting expression) of the TET2 gene during CAR-T manufacturing results in the production of armored CAR-T that has significant ability to expand and subsequently eradicate tumors when compared to unarmored CAR-T that lacks such ability for expansion. This strategy may be coupled with a security switch (e.g., iC9 security switch of the present disclosure) that allows directional destruction of armored CAR-T-cells in the event of adverse reactions from the subject or uncontrolled growth of armored CAR-T. Exemplary growth advantage factors include, but are not limited to, the factors provided in table 7.

TABLE 7 exemplary growth advantage factors.

Full name	Abbreviations	SEQ ID NO：
			10-11 metathesis 2	TET2	16603
DNA (cytosine-5) -methyltransferase 3A	DNMT3A	16604
			Transformed protein RhoA	RHOA	16605
Protooncogene vav	VAV1	16606
			Rhombotin-2	LMO2	16607
T-cell acute lymphocytic leukemia protein 1	TAL1	16608
			Suppressor of cytokine signaling 1	SOCS1	16609
Herpes virus entry mediators	HVEM	16610
			T cell death-related gene 8	TDAG8	16611
BCL6 co-inhibitors	BCOR	16612
			B and T cell attenuators	BTLA	16613
SPARC-like protein 1	SPARCL1	16614
			Msh homeobox 1-like proteins	MSX1	16615

Armored T-cell 'null or switch receptor' strategy

In some embodiments, T-cells of the present disclosure are modified to express modified/chimeric checkpoint receptors to produce armored T-cells of the present disclosure.

In some embodiments, the modified/chimeric checkpoint receptor comprises a null receptor, a decoy receptor, or a dominant negative receptor. The null receptor, decoy receptor, or dominant negative receptor of the present disclosure may be a modified/chimeric receptor/protein. The null receptors, decoy receptors, or dominant negative receptors of the present disclosure may be truncated for expression of an intracellular signaling domain. Alternatively or in addition, the null, decoy or dominant negative receptors of the disclosure may be mutated within the intracellular signaling domain at one or more amino acid positions that are determinative or desirable for effective signaling. Truncation or mutation of the null, decoy or dominant negative receptors of the present disclosure may result in a loss of the ability of the receptor to transmit or transduce a checkpoint signal to or within a cell.

For example, thinning or blocking of immunosuppressive checkpoint signals from PD-L1 receptors expressed on the surface of tumor cells may be achieved by expressing on the surface of armored T-cells of the present disclosure a modified/chimeric PD-1 null receptor that effectively competes with endogenous (non-modified) PD-1 receptors also expressed on the surface of armored T-cells to reduce or inhibit transduction of the immunosuppressive checkpoint signals of endogenous PD-1 receptors by armored T-cells. In this exemplary embodiment, competition between two different receptors for binding PD-L1 expressed on tumor cells reduces or diminishes the level of effective checkpoint signaling, thereby enhancing the therapeutic potential of armored T-cells expressing PD-1 null receptors.

In some embodiments, the modified/chimeric checkpoint receptor comprises a null receptor, decoy receptor, or dominant negative receptor as a transmembrane receptor.

In some embodiments, the modified/chimeric checkpoint receptor comprises a null receptor, decoy receptor, or dominant negative receptor that is a membrane-bound or membrane-linked receptor/protein.

In some embodiments, the modified/chimeric checkpoint receptor comprises a null receptor, decoy receptor, or dominant negative receptor that is an intracellular receptor/protein.

In some embodiments, the modified/chimeric checkpoint receptor comprises a null receptor, decoy receptor, or dominant negative receptor that is an intracellular receptor/protein. Exemplary null, decoy, or dominant negative intracellular receptors/proteins of the present disclosure include, but are not limited to, inhibitory checkpoint signals (as, e.g., provided in tables 1 and 2), transcription factors (as, e.g., provided in table 3), cytokines or cytokine receptors, chemokine or chemokine receptors, cell death or apoptosis receptors/ligands (as, e.g., provided in table 4), metabolic signaling sensory molecules (as, e.g., provided in table 5), proteins conferring sensitivity to cancer therapy (as, e.g., provided in table 6), and signaling components downstream of oncogenes or oncogenes (as, e.g., provided in table 7). Exemplary cytokines, cytokine receptors, chemokines, and chemokine receptors of the present disclosure include, but are not limited to, the cytokine and cytokine receptors and chemokine receptors provided in table 8.

Table 8 exemplary cytokines, cytokine receptors, chemokines, and chemokine receptors.

In some embodiments, the modified/chimeric checkpoint receptor comprises a switch receptor. Exemplary switch receptors may include modified/chimeric receptors/proteins of the disclosure in which a native or wild-type intracellular signaling domain is switched or replaced with a different intracellular signaling domain that is not native to the protein and/or is not a wild-type domain. For example, replacement of the inhibitory signaling domain with a stimulatory signaling domain will convert the immunosuppressive signal to an immunostimulatory signal. On the other hand, replacement of an inhibitory signaling domain with a different inhibitory domain may reduce or enhance the level of inhibitory signaling. The expression or overexpression of the switch receptor may result in the thinning and/or blocking of the cognate checkpoint signal by competing with the endogenous wild-type checkpoint receptor (not the switch receptor) for binding to the cognate checkpoint receptor expressed in the immunosuppressive tumor microenvironment. The armored T cells of the present disclosure may comprise sequences encoding the switch receptors of the present disclosure, resulting in the expression of one or more switch receptors of the present disclosure and thus altering the activity of the armored T cells of the present disclosure. Armored T-cells of the present disclosure may express switch receptors of the present disclosure that target checkpoint receptors, transcription factors, cytokine receptors, death receptors, metabolic signaling sensory molecules, cancer therapies, oncogenes and/or oncosuppressors or intracellularly expressed proteins downstream of the gene of the present disclosure.

Exemplary switch receptors of the present disclosure may include or may be derived from proteins including, but not limited to, the following: inhibitory checkpoint signals (as, e.g., provided in tables 1 and 2), transcription factors (as, e.g., provided in table 3), cytokine or cytokine receptors, chemokine or chemokine receptors, cell death or apoptosis receptors/ligands (as, e.g., provided in table 4), metabolic signaling sensory molecules (as, e.g., provided in table 5), proteins that confer sensitivity to cancer therapy (as, e.g., provided in table 6), and signaling components downstream of oncogenes or oncogenes (as, e.g., provided in table 7). Exemplary cytokines, cytokine receptors, chemokines, and chemokine receptors of the present disclosure include, but are not limited to, the cytokine and cytokine receptors and chemokine receptors provided in table 8.

Armored T-cell "synthetic gene expression" strategy

In some embodiments, the T-cells of the present disclosure are modified to express a Chimeric Ligand Receptor (CLR) or a Chimeric Antigen Receptor (CAR) that mediates conditional gene expression to produce armored T-cells of the present disclosure. The combination of CLR/CAR and conditional gene expression system in the armored T cell nucleus constitutes a synthetic gene expression system that is conditionally activated upon binding of one or more cognate ligands to CLR or one or more cognate antigens to CAR. This system can help to armor' or enhance the therapeutic potential of modified T cells by reducing or limiting the synthetic gene expression of ligands or antigen binding sites, for example, at or within the tumor environment.

Exogenous receptor

In some embodiments, the armored T-cell comprises a composition comprising (a) an inducible transgene construct comprising a sequence encoding an inducible promoter and a sequence encoding a transgene, and (b) a receptor construct comprising a sequence encoding a constitutive promoter and a sequence encoding an exogenous receptor, such as CLR or CAR, wherein the exogenous receptor is expressed upon integration of the construct of (a) and the construct of (b) into the genomic sequence of the cell, and wherein the exogenous receptor transduces an intracellular signal upon binding a ligand or antigen that directly or indirectly targets the inducible promoter regulating expression of the inducible transgene (a) to modify gene expression.

In some embodiments of the synthetic gene expression systems of the present disclosure, the composition modifies gene expression by reducing gene expression. In some embodiments, the composition modifies gene expression by transiently modifying gene expression (e.g., for a duration of binding of a ligand to an exogenous receptor). In some embodiments, the composition modifies gene expression in an acute phase (e.g., a ligand reversibly binds to an exogenous receptor). In some embodiments, the composition modifies gene expression long-lasting (e.g., ligand binds to exogenous receptor irreversibly).

In some embodiments of the compositions of the present disclosure, the exogenous receptor of (b) comprises an endogenous receptor with respect to a genomic sequence of the cell. Exemplary receptors include, but are not limited to, intracellular receptors, cell surface receptors, transmembrane receptors, ligand-gated ion channels, and G protein-coupled receptors.

In some embodiments of the compositions of the present disclosure, the exogenous receptor of (b) comprises a non-naturally occurring receptor. In some embodiments, the non-naturally occurring receptor is a synthetic, modified, recombinant, mutated, or chimeric receptor. In some embodiments, the non-naturally occurring receptor comprises one or more sequences isolated or derived from a T-cell receptor (TCR). In some embodiments, the non-naturally occurring receptor comprises one or more sequences isolated or derived from a scaffold protein. In some embodiments, including those in which the non-naturally occurring receptor does not comprise a transmembrane domain, the non-naturally occurring receptor interacts with a second transmembrane, membrane-bound, and/or intracellular receptor that transduces an intracellular signal upon contact with the non-naturally occurring receptor.

In some embodiments of the compositions of the present disclosure, the exogenous receptor of (b) comprises a non-naturally occurring receptor. In some embodiments, the non-naturally occurring receptor is a synthetic, modified, recombinant, mutated, or chimeric receptor. In some embodiments, the non-naturally occurring receptor comprises one or more sequences isolated or derived from a T-cell receptor (TCR). In some embodiments, the non-naturally occurring receptor comprises one or more sequences isolated or derived from a scaffold protein. In some embodiments, the non-naturally occurring receptor comprises a transmembrane domain. In some embodiments, the non-naturally occurring receptor interacts with an intracellular receptor that transduces an intracellular signal. In some embodiments, the non-naturally occurring receptor comprises an intracellular signaling domain. In some embodiments, the non-naturally occurring receptor is a Chimeric Ligand Receptor (CLR). In some embodiments, the CLR is a Chimeric Antigen Receptor (CAR).

In some embodiments of CLR/CARs of the present disclosure, the signal peptide comprises a sequence encoding a human CD2, CD3 δ, CD3 ε, CD3 γ, CD3 ζ, CD4, CD8 α, CD19, CD28, 4-1BB, or GM-CSFR signal peptide. In some embodiments, the signal peptide comprises a sequence encoding a human CD8 a signal peptide. In some embodiments, the signal peptide comprises an amino acid sequence comprising MALPVTALLLPLALLLHAARP (SEQ ID NO: 18004). In some embodiments, the signal peptide is encoded by a nucleic acid sequence comprising atggcactgccagtcaccgccctgctgctgcctctggctctgctgctgcacgcagctagacca.

In some embodiments of CLR/CARs of the present disclosure, the transmembrane domain comprises a sequence encoding a human CD2, CD3 δ, CD3 ε, CD3 γ, CD3 ζ, CD4, CD8 α, CD19, CD28, 4-1BB, or GM-CSFR transmembrane domain. In some embodiments, the transmembrane domain comprises a sequence encoding a human CD8 a transmembrane domain. In some embodiments, the transmembrane domain comprises an amino acid sequence comprising IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 18006). In some embodiments, the transmembrane domain is encoded by a nucleic acid sequence comprising atctacatttgggcaccactggccgggacctgtggagtgctgctgctgagcctggtcatcacactgtactgc (SEQ ID NO: 18007).

In some embodiments of CLR/CARs of the present disclosure, the endodomain comprises the human CD3 ζ endodomain. In some embodiments, the at least one co-stimulatory domain comprises human 4-1BB, CD28, CD40, ICOS, MyD88, OX-40 intracellular segment, or any combination thereof. In some embodiments, the at least one co-stimulatory domain comprises a human CD28 and/or a 4-1BB co-stimulatory domain. In some embodiments, the CD3 ζ costimulatory domain comprises an amino acid sequence comprising

In some embodiments, the CD3 ζ costimulatory domain is encoded by a nucleic acid sequence comprising a sequence

In some embodiments, the 4-1BB co-stimulatory domain comprises an amino acid sequence comprising KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 18011). In some embodiments, the 4-1BB co-stimulatory domain is encoded by a nucleic acid sequence comprising

In some embodiments, the 4-1BB co-stimulatory domain is located between the transmembrane domain and the CD28 co-stimulatory domain.

In some embodiments of the CLR/CARs of the present disclosure, the hinge comprises sequences derived from human CD8 a, IgG4, and/or CD4 sequences. In some embodiments, the hinge comprises a sequence derived from the human CD8 a sequence. In some embodiments, the hinge comprises an amino acid sequence comprising

In some embodiments, the hinge is encoded by a nucleic acid sequence comprising

In some embodiments, the at least one protein scaffold specifically binds the ligand.

In some embodiments of the compositions of the present disclosure, the exogenous receptor of (b) comprises a non-naturally occurring receptor. In some embodiments, the CLR is a Chimeric Antigen Receptor (CAR). In some embodiments, the chimeric ligand receptor comprises: (a) an extracellular domain comprising a ligand recognition region, wherein the ligand recognition region comprises at least a scaffold protein; (b) a transmembrane domain, and (c) an endodomain comprising at least one costimulatory domain. In some embodiments, the at least one protein scaffold comprises an antibody, an antibody fragment, a single domain antibody, a single chain antibody, an antibody mimetic, or a centrin (referred to herein as CARTyrin). In some embodiments, the ligand recognition region comprises one or more of an antibody, an antibody fragment, a single domain antibody, a single chain antibody, an antibody mimetic, and a centryrin. In some embodiments, the single domain antibody comprises or consists of a VHH or VH (referred to herein as VCAR). In some embodiments, the single domain antibody comprises or consists of a VHH or VH comprising human Complementarity Determining Regions (CDRs). In some embodiments, the VH is a recombinant or chimeric protein. In some embodiments, the VH is a recombinant or chimeric human protein. In some embodiments, the antibody mimetic comprises or consists of an affibody, an affilin, an affimer, an affitin, an alphabody, an anticalin, an avimer, a DARPin, a Fynomer, a Kunitz domain peptide, or a monobody. In some embodiments, the centryrin comprises or consists of at least one consensus sequence of fibronectin type III (FN3) domain.

In some embodiments, the consensus sequence comprises

In some embodiments, the consensus sequence is modified at one or more positions within: (a) an A-B loop comprising or consisting of amino acid residues TEDS (SEQ ID NO: 18020) at positions 13-16 of the consensus sequence; (b) a B-C loop comprising or consisting of amino acid residues TAPDAAF at positions 22-28 of the consensus sequence (SEQ ID NO: 18021); (c) a C-D loop comprising or consisting of amino acid residues SEKVGE (SEQ ID NO: 18022) at positions 38-43 of the consensus sequence; (d) a D-E loop comprising or consisting of the amino acid residues GSER at positions 51-54 of the consensus sequence (SEQ ID NO: 18023); (e) an E-F loop comprising or consisting of amino acid residues GLKPG at positions 60-64 of the consensus sequence (SEQ ID NO: 18024); (f) is contained in Amino acid residues KGGHRSN at positions 75-81 of the consensus sequence (SEQ ID NO: 18025) or the F-G loop consisting thereof; or (g) any combination of (a) - (f). In some embodiments, the centryrin comprises a consensus sequence of at least 5 fibronectin type III (FN3) domains. In some embodiments, the centryrin comprises a consensus sequence of at least 10 fibronectin type III (FN3) domains. In some embodiments, the centryrin comprises a consensus sequence of at least 15 fibronectin type III (FN3) domains. In some embodiments, the scaffold is selected from less than or equal to 10^-9M, less than or equal to 10^-10M, less than or equal to 10^-11M, less than or equal to 10^-12M, less than or equal to 10^-13M, less than or equal to 10^-14M and less than or equal to 10^-15K of M_DBinds to the antigen with at least one affinity. In some embodiments, the K is_DMeasured by surface plasmon resonance.

Inducible promoters

In some embodiments of the compositions of the present disclosure, the sequence encoding an inducible promoter of (a) comprises a sequence encoding NF_KB promoter sequence. In some embodiments of the compositions of the present disclosure, the sequence encoding an inducible promoter of (a) comprises a sequence encoding an Interferon (IFN) promoter or a sequence encoding an interleukin-2 promoter. In some embodiments, the Interferon (IFN) promoter is an IFN γ promoter. In some embodiments of the compositions of the present disclosure, the inducible promoter is isolated or derived from a cytokine or chemokine promoter. In some embodiments, the cytokine or chemokine comprises IL2, IL3, IL4, IL5, IL6, IL10, IL12, IL13, IL17A/F, IL21, IL22, IL23, transforming growth factor beta (TGF β), colony stimulating factor 2(GM-CSF), interferon gamma (IFN γ), tumor necrosis factor (TNF α), LT α, perforin, granzyme C (gzmc), granzyme b (gzmb), C-C motif chemokine ligand 5(CCL5), C-C motif chemokine ligand 4(CCL4), C-C motif chemokine ligand 3(CCL3), X-C motif chemokine ligand 1(Xcl1), and LIF interleukin 6 family cytokines (LIF).

In some embodiments of the compositions of the present disclosure, the inducible promoter is isolated or derived from a promoter of a gene comprising a surface protein associated with cell differentiation, activation, depletion, and function. In some embodiments, the genes include CD69, CD71, CTLA4, PD-1, TIGIT, LAG3, TIM-3, GITR, MHCII, COX-2, FASL, and 4-1 BB.

In some embodiments of the compositions of the present disclosure, the inducible promoter is isolated or derived from a promoter of a gene involved in CD metabolism and differentiation. In some embodiments of the compositions of the present disclosure, the inducible promoter is isolated or derived from the promoters of Nr4a1, Nr4a3, Tnfrsf9(4-1BB), Sema7a, Zfp36l2, Gadd45b, durp 5, durp 6, and Neto 2.

Inducible transgenes

In some embodiments, the inducible transgene construct comprises or drives expression of a signal transduction component downstream of an inhibitory checkpoint signal (as, e.g., provided in tables 1 and 2), a transcription factor (as, e.g., provided in table 3), a cytokine or cytokine receptor, a chemokine or chemokine receptor, a cell death or apoptosis receptor/ligand (as, e.g., provided in table 4), a metabolic signaling sensory molecule (as, e.g., provided in table 5), a protein that confers sensitivity to cancer therapy (as, e.g., provided in table 6 and/or table 9), and an oncogene or an oncosuppressor gene (as, e.g., provided in table 7). Exemplary cytokines, cytokine receptors, chemokines, and chemokine receptors of the present disclosure include, but are not limited to, the cytokine and cytokine receptors and chemokine receptors provided in table 8.

Table 9 exemplary therapeutic proteins (and proteins that enhance the efficacy of CAR-T).

Cas-Clover

The present disclosure provides compositions comprising a guide RNA and a fusion protein or a sequence encoding a fusion protein, wherein the fusion protein comprises dCas9 and a Clo051 endonuclease or nuclease domains thereof.

Small Cas9(SaCas9)

The present disclosure provides compositions comprising a small, Cas9(Cas9) operably linked to an effector. In certain embodiments, the present disclosure provides a fusion protein comprising, consisting essentially of, or consisting of a DNA localization component and an effector molecule, wherein the effector comprises a small, Cas9(Cas 9). In certain embodiments, the small Cas9 constructs of the present disclosure may comprise an effector comprising a type IIS endonuclease.

An amino acid sequence of Staphylococcus aureus (Staphylococcus aureus) Cas9 with an active catalytic site.

Deactivated, small, Cas9(dSaCas9)

The present disclosure provides compositions comprising an inactivated, small, Cas9(dSaCas9) operably linked to an effector. In certain embodiments, the present disclosure provides a fusion protein comprising, consisting essentially of, or consisting of a DNA localization component and an effector molecule, wherein the effector comprises a small, inactivated Cas9(dSaCas 9). In certain embodiments, the small, inactivated Cas9(dSaCas9) constructs of the present disclosure may comprise an effector comprising a type IIS endonuclease.

dSaCas9 sequence: the D10A and N580A mutations (bold, capital letters and underlined) inactivate the catalytic site.

Inactivated Cas9(dCas9)

The present disclosure provides compositions comprising an inactivated Cas9(dCas9) operably linked to an effector. In certain embodiments, the present disclosure provides a fusion protein comprising, consisting essentially of, or consisting of a DNA localization component and an effector molecule, wherein the effector comprises an inactivated Cas9(dCas 9). In certain embodiments, an inactivated Cas9(dCas9) construct of the present disclosure may comprise an effector comprising a type IIS endonuclease.

In certain embodiments, the dCas9 of the present disclosure comprises dCas9 isolated or derived from staphylococcus pyogenes. In certain embodiments, dCas9 includes dCas9 with substitutions at positions 10 and 840 of the amino acid sequence of dCas9 that inactivate the catalytic site. In certain embodiments, these substitutions are D10A and H840A. In certain embodiments, the amino acid sequence of dCas9 comprises the following sequence:

in certain embodiments, the amino acid sequence of dCas9 comprises the following sequence:

clo051 endonuclease

An exemplary Clo051 nuclease domain may comprise, consist essentially of, or consist of the amino acid sequence of seq id no:

Cas-Clover fusion proteins

In certain embodiments, an exemplary dCas9-Clo051 fusion protein (embodiment 1) may comprise, consist essentially of, or consist of the following amino acid sequence (Clo051 sequence underlined, linker in bold italics, dCas9 sequence (streptococcus pyogenes)) italics):

in certain embodiments, an exemplary dCas9-Clo051 fusion protein (embodiment 1) may comprise, consist essentially of, or consist of the following nucleic acid sequences (dCas 9 sequence derived from streptococcus pyogenes):

in certain embodiments, the nucleic acid sequence encoding the dCas9-Clo051 fusion protein (embodiment 1) of the present disclosure may comprise DNA. In certain embodiments, the nucleic acid sequence encoding the dCas9-Clo051 fusion protein (embodiment 1) of the present disclosure may comprise RNA.

In certain embodiments, an exemplary dCas9-Clo051 fusion protein (embodiment 2) may comprise, consist essentially of, or consist of the following amino acid sequence (Clo051 sequence underlined, linker in bold italics, dCas9 sequence (streptococcus pyogenes) italics):

In certain embodiments, an exemplary dCas9-Clo051 fusion protein (embodiment 2) may comprise, consist essentially of, or consist of the following nucleic acid sequences (dCas 9 sequence derived from streptococcus pyogenes):

in certain embodiments, the nucleic acid sequence encoding the dCas9-Clo051 fusion protein (embodiment 2) of the present disclosure may comprise DNA. In certain embodiments, the nucleic acid sequence encoding the dCas9-Clo051 fusion protein (embodiment 2) of the present disclosure may comprise RNA.

Examples

Example 1: construction and in vitro characterization of PSMA5 and PSMA8 CARTyrins

PSMA5 and PSMA8 CARTYRins were constructed as shown in FIGS. 1, 2, 3A-3C and 4A-4C. Figure 5 depicts the structure of anti-PSMA CARTyrins.

Surface expression of the cardurins of the present disclosure was assessed by flow cytometry 24 hours after electroporation of sequence mRNA encoding PSMA5 or PSMA8 cardurin into human whole T cells (fig. 6A). Surface PSMA protein expression was detected by flow cytometry on LNCaP tumor cell lines and K562 cell lines transfected with PSMA (fig. 6B). The functionality of CARTyrin-expressing T cells was measured by degranulation against tumor cells. Degranulation of RNA-electroporated PSMA CARTyrin T cells was observed against the PSMA + tumor cell line (fig. 6C). PSMA surface protein expression was detected by flow cytometry after transfection of K562 cell lines with increasing amounts of PSMA mRNA (fig. 6D). Degranulation of RNA-electroporated PSMA CARTyrin T cells was observed against K562 expressing various amounts of PSMA protein (fig. 6E). Together, these data indicate that PSMA5 and PSMA8 CARTyrins can be expressed on the surface of T cells and promote cytotoxic function against PSMA + cellular targets.

To support in vivo evaluation of PSMA CARTyrins, P-PSMA5-101 and P-PSMA8-101 were constructed using the piggyBac DNA modification system. PSMA CARTyrin was detected on the surface of primary human T cells from representative donors transposed with either the P-PSMA5-101 or P-PSMA8-101 plasmids (fig. 7A). Flow cytometry analysis using surface-expressed markers indicated that PSMA CARTyrin-expressing T cells had T stem cell memory phenotypes rather than markers of activated and/or functional T cell depletion phenotypes (fig. 7B-7C). ELISA analysis showed that PSMA CARTyrins expressing T cells resulted in IFN γ secretion in PSMA expressing cells (LNCaP and k562.PSMA cells), showing effector function of T cells (fig. 7D). T cells expressing PSMA CARTyrins showed strong cytotoxic function by standard killing assays and cell proliferation assays (fig. 7E-7F). Together, these data indicate that surface expression of P-PSMA5-101 and P-PSMA8-101 PSMA CARTyrins on T cells shows cytotoxic function and proliferative capacity in vitro against PSMA + cellular targets. In accordance with this strong in vitro performance, the ability of PSMA CARTyrins to function in vivo was evaluated.

Example 2: in vivo characterization of PSMA5 and PSMA8 CARTyrins using a murine xenograft model

Figure 8A depicts the treatment regimen of the in vivo study in mice using P-PSMA8-101 CARTyrin. Antitumor activity was assessed by survival (fig. 8B), expansion and detection of P-PSMA8-101 CD8+ T cells in blood (fig. 8C), tumor volume assessment by caliper measurement (fig. 8D), and bioluminescence of LNCaP tumors (fig. 8E-F). P-PSMA8-101 at 'stress' and standard doses showed significantly enhanced antitumor efficacy and survival against established SC lncap. Specifically, there was no survival in control animals, 50% survival in animals treated with 'ultra low' doses of P-PSMA8-101, and 100% survival in animals treated with 'stressed' or standard doses of P-PSMA 8-101. The standard dose of P-PSMA8-101 eliminated established LNCaP tumors in 100% of the animals for the duration of the study (42 days post-treatment), while 2/3 animals receiving the 'stress' dose remained tumor-free. In peripheral blood, P-PSMA8-101 expanded and generated differentiated effect PSMA8 CARTyrin + T-cells, which were accompanied by a reduction in tumor burden below detectable calipers and bioluminescence imaging limits. P-PSMA8-101 then contracts, but still persists in the peripheral blood. Together, these data demonstrate that PSMA8 CARTyrin-expressing animals show reduced tumor burden compared to controls.

FIG. 9A depicts the therapeutic regimen of in vivo studies of P-PSMA5-101 and P-PSMA8-101 CARTyrins in mice using a 'stress' dose of (4x10^6) using a murine xenograft model. Antitumor activity was assessed by survival (fig. 9B), expansion and detection of CD8+ T cells in blood (fig. 9C), tumor volume assessment by caliper measurement (fig. 9D), and bioluminescence of LNCaP tumors (fig. 9E-F). The 'stressed' doses of P-PSMA5-101 and P-PSMA8-101 showed significantly enhanced antitumor efficacy and survival against SC lncap. luc solid tumors established in NSG mice compared to T cell (no CAR) control mice. Specifically, there was no survival in T cell (no CAR) control animals, 25% survival in P-BCMA-101 treated group, 75% survival in P-PSMA5-101 treated group, and 100% survival in animals treated with 'stressed' dose of P-PSMA 8-101. In peripheral blood, P-PSMA5-101 and P-PSMA8-101 expanded and generated differentiated effector CARTyrin + T cells, which were accompanied by a reduction in tumor burden below detectable calipers and bioluminescence imaging limits. These cells then shrink but remain present in the peripheral blood.

Example 3: expression and function of piggyBac-integrated iC9 safety switch into human whole T cells

Human whole T cells were nuclear transfected with one of the four piggyBac transposons using an Amaxa 4D nuclear transfectator. Modified T cells that received "mock" conditions were nuclear transfected with empty piggyBac transposons. The modified T cells received a piggyBac transposon containing only the therapeutic agent (the sequence encoding CARTyrin) or a piggyBac transposon containing the integrated iC9 sequence and the therapeutic agent (the sequence encoding CARTyrin).

Figure 8 provides a schematic of an iC9 safety switch containing a ligand binding region, a linker and a truncated caspase 9 polypeptide. Specifically, iC9 polypeptide contains a ligand binding region that comprises an FK506 binding protein 12(FKBP12) polypeptide, including a substitution of valine (V) for phenylalanine (F) at position 36 (F36V). In certain embodiments, the FKBP12 polypeptide is encoded by an amino acid sequence comprising:

in certain embodiments, the FKBP12 polypeptide is encoded by a nucleic acid sequence comprising:

in certain embodiments, the inducer specific for the ligand binding region of FK 506-binding protein 12(FKBP12) polypeptide that may comprise a substitution (F36V) of valine (V) to phenylalanine (F) at position 36 comprises AP20187 and/or AP1903, two synthetic drugs.

Or comprise

The nucleic acid sequence of (a).

In certain embodiments of the induced pro-apoptotic polypeptides, wherein said polypeptide comprises a truncated caspase 9 polypeptide, the induced pro-apoptotic polypeptides are comprised of

Or comprises an amino acid sequence of

The nucleic acid sequence of (a).

To test the iC9 safety switch, each of the four modified T cells was incubated with 0, 0.1nM, 1nM, 10nM, 100nM or 1000nM AP1903 (inducer of AP 1903) for 24 hours. Viability was assessed by flow cytometry using the fluorescent intercalator 7-amino-actinomycin D (7-AAD) as a marker for cells undergoing apoptosis.

Cell viability was assessed at day 12 (see figure 9). The data show the movement of the cell population from the lower right to the upper left quadrant with increasing concentration of the inducer in the cells containing the iC9 construct; however, this effect was not observed in cells lacking iC9 construct (those that received CARTyrin only), where cells were evenly distributed in both regions regardless of the concentration of the inducer. In addition, cell viability was assessed on day 19 (see figure 10). The data revealed the same trend as shown in fig. 9 (day 12 post-nuclear transfection); however, at this later time point (day 19 post-nuclear transfection), the population moved more significantly to the upper left quadrant.

Quantification of the combined results was performed and is provided in fig. 11, which shows the significant effect of iC9 safety switch on percent cell viability as a function of the concentration of the inducer of iC9 switch (AP1903) for each modified cell type at day 12 (fig. 9 and left panels) or day 19 (fig. 10 and right panels). By day 12, the presence of the iC9 safety switch induced apoptosis in most cells, and by day 19, this effect was even more dramatic.

The results of the study indicate that iC9 safety switch is extremely effective in cells that abrogate activity when contacted with an inducing agent (e.g., AP1903) because AP1903 induces apoptosis even at the lowest concentration studied (0.1 nM). In addition, the iC9 safety switch may be functionally expressed as part of a tricistronic vector.

Example 4: knock down efficiency of checkpoint signaling proteins on armored T-cells

To generate armored T cells with enhanced therapeutic potential, genetic modifications may be made to render the T-cells less susceptible to immune and/or metabolic restriction points. One mechanism for creating armored T-cells is to inhibit checkpoint signaling by knocking out various checkpoint receptors. Cas-CLOVER ^TMThe platform is used to target and knock-out the checkpoint receptors PD-1, TGF β R2, LAG-3, Tim-3, and CTLA-4 in resting (or quiescent) primary whole T cells. Gene editing resulted in 30-70% loss of protein expression at the cell surface as measured by flow cytometry (fig. 13). These results indicate that Cas-CLOVER^TMKnockouts of these genes can be targeted effectively, resulting in loss of target protein expression on the surface of the T-cell. Knockout efficiency can be significantly improved by further optimizing the guide RNA pairs, or by using additional guide RNA pairs that target the regulators or promoters of the same gene and/or target gene.

Example 5: strategies for expressing null or switching intracellular signaling proteins on armored T-cells

Another strategy to generate armored T-cells is to reduce or inhibit endogenous checkpoint signaling by expressing various modified/chimeric checkpoint receptors with altered or absent intracellular signaling domains. The checkpoint signals that can be targeted using this strategy include PD-1 or TGF β RII of T-cells, which bind PD-L1 ligand and TGF β cytokine, respectively. Figure 14 shows a schematic of various strategies for generating decoy/null/dominant-negative receptors (null receptors) for two different inhibitory receptors (PD-1 (top panel) and tgfbetarii (bottom panel)). To design a null receptor, the intracellular domain (ICD) of PD1 or TGF β RII may be mutated (mutated null) or deleted (truncated null). As a result, binding of one or more cognate ligands of the null receptor does not result in delivery of a checkpoint signal to the T-cell. In addition, because the null receptor competes with the wild-type receptor for binding to the one or more endogenous ligands, any binding by the null receptor will mask binding of the one or more endogenous ligands to the wild-type receptor. This results in a thinning of the overall level of checkpoint signaling that is effectively delivered to the T-cells, thereby reducing or blocking checkpoint inhibition. FIG. 15 also shows the switch receptor design strategy for the inhibitory receptor PD-1 (top panel) and TGF β RII (bottom panel). In the switch receptor, wild-type ICDs are replaced by ICDs from either an immunostimulatory molecule (costimulatory switch) or a different inhibitory molecule (inhibitory switch). Immunostimulatory molecules include, but are not limited to, CD3z, CD28, 4-1BB, and the examples listed in Table 1. Inhibitory molecules include, but are not limited to, CTLA4, PD1, lad 3, and the examples listed in tables 1 and 9. In the former case, binding of endogenous ligands by the modified switch receptors results in delivery of positive signals to T-cells, thereby helping to enhance stimulation of T-cells, thereby promoting continuation of tumor targeting and killing. In the latter case, binding of the endogenous ligand by the modified switch receptor results in delivery of a negative signal to the T-cell, thereby contributing to reduced stimulation and activity of the T-cell.

Example 6: enhancing PD1 and TGF β RII null or switching intracellular signaling proteins on armored T-cells Surface expression of

To generate armored T-cells, a number of truncated null receptors expressing alternative Signal Peptides (SP) and transmembrane domains (TM) were designed and tested for their maximal expression on the surface of modified T-cells. FIG. 15A shows a schematic representation of several null receptor constructs for PD-1 (top panel) and TGF β RII (bottom panel). The extracellular domain (ECD) of these proteins was modified such that the wild-type Signal Peptide (SP) and/or transmembrane domain (TM) were replaced with a domain from the human T cell CD8a receptor (red arrow). Each of the six truncated null constructs shown in fig. 15A was DNA synthesized and then subcloned into mRNA IVT DNA vector (pRT). High quality mRNA was produced by IVT for each. Transfection of mRNA encoding each of the six molecules was performed using Electroporation (EP) delivery into primary human T cells, and FACS analysis was performed 24 hours after EP to assess the expression level of each construct on the cell surface (fig. 15B). Replacement of WT SP (02.8aSP-PD-1 and 02.8aSP-TGF β RII) with the replacement CD8a resulted in the highest level of expression on the T cell surface by flow cytometry. 02.8aSP-PD-1 null receptor showed an MFI of 43,680, which is 177-fold higher than endogenous T cell PD-1 expression and 2.8-fold higher than WT PD-1 null receptor. 02.8 aSP-TGF-. beta.RII null receptor showed an MFI of 13,809 that was 102-fold higher than endogenous T cell TGF-. beta.RII expression and 1.8-fold higher than WT TGF-. beta.RII null receptor. These results indicate that replacement of wild type SP with the alternative CD8a SP resulted in either null or enhanced surface expression of the switch receptor for both PD1 and the tgfbetarii inhibitory protein. This in turn will maximize either checkpoint inhibition or co-stimulation upon binding of one or more endogenous ligands, respectively.

Example 7: design of NF-KB inducible vectors for expression in modified T-cells

Two T cell activating NF-KB inducible vectors were developed (fig. 16A and 16B); one with the Gene Expression System (GES) in the forward orientation (a) and the other in the complementary orientation (B), both before the constitutive EF1a promoter. These vectors also direct the expression of the CAR molecule and DHFR selection gene separated by a T2A sequence. Both the conditional NF-KB inducible system and the EF1 a-directed genes are part of piggyBac transposons that can be permanently integrated into T cells using EP. Once integrated into the genome, T cells constitutively express CAR on the membrane surface and DHFR inside the cell, whereas expression of the NF-KB inducible gene GFP will only be expressed to the highest level upon T cell activation.

Example 8: NF-KB inducible vectors for GFP expression in modified T-cells

T cells were nuclear transfected with piggyBac vectors expressing anti-BCMA CAR and DHFR mutein genes under control of EF1a promoter and NF-KB inducible expression systems in the absence (no Gene Expression System (GES) control) or in the presence of GFP expression driving either forward orientation (pNFKB-GFP forward) or reverse orientation (pNFKB-GFP reverse). Cells were cultured with methotrexate selection until the cells were almost completely quiescent (day 19) and GFP expression was assessed on days 5 and 19. On day 5, all T cells proliferated and were highly stimulated, while cells with NF-KB inducible expression cassettes produced high levels of GFP due to strong NF κ B activity (see figure 17). The GES-free control cells did not express detectable levels of GFP. By day 19, GES T cells were almost completely quiescent and GFP expression was significantly lower than day 5 (-1/8 MFI) due to lower NF κ B activity. GFP expression was still observed at day 19, possibly due to the long half-life (-30 hours) of the GFP protein, or basal levels of NF κ B activity signaled by, for example, TCR, CAR, cytokine receptor, or growth factor receptor.

Example 9: NF-KB inducibility for anti-BCMA CAR-mediated GFP expression in modified T-cells Carrier

T cells were either unmodified (mock T cells) or nuclear transfected with piggyBac vectors expressing anti-BCMA CAR and DHFR mutein genes under control of EF1a promoter and NF-KB inducible expression systems in the absence (no GES control) or in the presence of GFP expression driving either forward orientation (pNFKB-GFP forward) or reverse orientation (pNFKB-GFP reverse). All cells were cultured for 22 days with or without methotrexate selection (mock T cells) until the cells were almost completely quiescent. Cells were then stimulated for 3 days in the absence (without stimulation) or presence of BCMA- (K562), BMCA + (RPMI 8226) or positive control anti-CD 3 anti-CD 28 activating reagent (CD3/28 stimulation). GFP expression was undetectable by either no GES control or mock T cells under all conditions. However, while the pNFKB-GFP positively and reversely transposed cells showed a small amount of GFP expression when cultured with BCMA-K562 cells compared to the non-stimulated control, they all showed dramatic upregulation of gene expression in the presence of BCMA + tumor cells or under positive control conditions (FIG. 18). Little difference in GFP expression was observed between pNFKB-GFP positive and reverse transposed cells co-cultured with BCMA + tumor cells.

Example 10: control of anti-BCMA CAR-mediated expression in modified T-cells

The level of expression of an inducible gene can be regulated by the number of responsive elements upstream or preceding the inducible promoter. T cells were nuclear transfected with piggyBac vectors encoding anti-BCMA CARTyrin plus a selection gene, both under the control of the human EF1a promoter (fig. 19). Further, the vector additionally encodes a conditional NF-KB inducible gene expression system driving expression of a truncated CD19 protein (dCD19) and includes a number of NFKB Response Elements (RE) varying from 0 to 5, no GES (GES), or receives electroporation pulses without piggyBac nucleic acids (mock). Only data on GES in the reverse (opposite) direction/orientation is shown. All cells were cultured for 18 days and included selection for piggyBac-modified T cells using methotrexate addition. Cells were then stimulated with anti-CD 3 anti-CD 28 bead activating reagent for 3 days and dCD19 surface expression was assessed by FACS at days 0, 3 and 18 and the data is shown as FACS histograms and MFI of target protein staining. At day 0, low levels of surface dCD19 expression were detected in all T cells transposed with the GES-encoding vector. A dramatic upregulation of dCD19 expression was observed for all GES expressing T cells 3 days post stimulation, while the fold increase in surface expression was greater in those with higher numbers of REs. Thus, surface dCD19 expression is proportional to the number of REs encoded in GES. No dCD19 was detected on the surface of T cells without GES: no GES and mock control.

Incorporated by reference

Each document cited herein, including any cross-referenced or related patent or application, is hereby incorporated by reference in its entirety unless expressly excluded or otherwise limited. The citation of any document is not an admission that it is prior art with respect to any invention disclosed or claimed herein or that it teaches, suggests or discloses any such invention alone or in combination with any other reference or references. Further, to the extent that any meaning or definition of a term in this document conflicts with any meaning or definition of the same term in a document incorporated by reference, the meaning or definition assigned to that term in this document shall govern.

Other embodiments

While particular embodiments of the present disclosure have been illustrated and described, various other changes and modifications can be made without departing from the spirit and scope of the disclosure. The scope of the appended claims includes all such changes and modifications as fall within the true scope of the disclosure.

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