Immune effector cells stably and transiently expressing nucleic acids
Immune effector cells engineered with specific nucleic acid molecules address the challenge of treating diseases with elevated antigen expression by modulating immune responses, offering targeted treatment solutions.
Patent Information
- Application Number
- HK62026124859
- Authority / Receiving Office
- HK · HK
- Patent Type
- Applications
- Current Assignee / Owner
- Priority Date
- 2023-02-07
- Filing Date
- 2026-06-16
- Publication Date
- 2026-07-31
AI Technical Summary
Existing technologies lack effective methods for treating diseases associated with elevated antigen expression, particularly through the use of immune effector cells engineered to target specific antigens.
The development of immune effector cells comprising specific nucleic acid molecules encoding cell surface antigen receptors and immune effector cell activators, enabling targeted treatment of diseases by modulating immune responses.
The engineered immune effector cells provide targeted and effective treatment of diseases by modulating immune responses against elevated antigen expression.
Smart Images
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Abstract
Description
This invention relates to immune effector cells comprising a first nucleic acid molecule and a second nucleic acid molecule, cell compositions and pharmaceutical compositions comprising such immune effector cells, and methods for generating such immune effector cells, wherein the first nucleic acid molecule comprises a first nucleotide sequence encoding a first cell surface antigen receptor, and the second nucleic acid molecule comprises a second nucleotide sequence encoding an immune effector cell activator molecule. Furthermore, the invention relates to particles comprising a first nucleic acid molecule and a second nucleic acid molecule, and complexes comprising such particles, wherein the first nucleic acid molecule comprises a first nucleotide sequence encoding a first cell surface antigen receptor, and the second nucleic acid molecule comprises a second nucleotide sequence encoding an immune effector cell activator molecule. Further, the invention relates to methods for treating subjects suffering from diseases, conditions, or illnesses associated with the expression or elevated expression of antigens using said immune effector cells, particles, or complexes.
Claims
We claim:
1. An immune effector cell comprising a first nucleic acid molecule comprising a first nucleotide sequence encoding a first cell-surface expressed antigen receptor and a second nucleic acid molecule comprising a second nucleotide sequence encoding an immune effector cell-activator molecule, wherein (i) the second nucleotide sequence is not integrated into a genomic nucleic acid molecule of the immune effector cell and / or (ii) the activator molecule is transiently expressed.
2. The immune effector cell according to claim 1, wherein the immune effector cell is isolated.
3. The immune effector cell according to claim 1 or claim 2, wherein the first nucleic acid molecule is DNA or RNA.
4. The immune effector cell according to any one of claims 1 to 3, wherein the first nucleotide sequence is integrated into a genomic nucleic acid molecule of the immune effector cell.
5. The immune effector cell according to any one of claims 1 to 4, wherein the genomic nucleic acid molecule is a chromosome, an episome, or a non-viral episome.
6. The immune effector cell according to claim 4 or claim 5, wherein the first nucleotide sequence is integrated into the genomic nucleic acid molecule via a DNA-based transposon system, a viral-based retrotransposon system, or a poly-A-based retrotransposon system.
7. The immune effector cell according to any one of claims 1 to 6, wherein the first nucleotide sequence is comprised within a transposable element.
8. The immune effector cell according to any one of claims 1 to 7, wherein the immune effector cell further comprises a third nucleic acid molecule comprising a third nucleotide sequence encoding a molecule having transposase activity.
9. The immune effector cell according to claim 8, wherein the third nucleic acid molecule is DNA or RNA.
10. The immune effector cell according to claim 8 or claim 9, wherein the third nucleic acid molecule is mRNA.
11. The immune effector cell according to any one of claims 8 to 10, wherein the molecule having transposase activity is Sleeping Beauty, PiggyBac, Frog, Prince, Himarl, Passport, Minos, hAT, Toll, Tol2, AciDs, PIF, Harbinger, Harbinger3-DR, Hsmarl, or a functionally equivalent variant thereof having transposase activity.
12. The immune effector cell according to any one of claims 8 to 12, wherein the molecule having transposase activity is Sleeping Beauty transposase SB100X.
13. The immune effector cell according to any one of claims 8 to 12, wherein (i) the third nucleic acid molecule is not integrated into a genomic nucleic acid molecule of the immune effector cell and / or (ii) the encoded molecule having transposase activity is transiently expressed.
14. The immune effector cell according to any one of claims 1 to 13, wherein first cell- surface expressed antigen receptor is stably expressed.
15. The immune effector cell according to any one of claims 1 to 14, wherein the activator molecule allows for the activation, expansion, differentiation and / or proliferation of the immune effector cell.
16. The immune effector cell according to any one of claims 1 to 15, wherein the activator molecule is a non-coding RNA or protein.
17. The immune effector cell according to any one of claims 1 to 16, wherein the activator molecule binds to the extracellular portion of the first cell-surfaced expressed antigen receptor.
18. The immune effector cell according to any one of claims 1 to 16, wherein the activator molecule is a cytokine.
19. The immune effector cell according to any one of claims 1 to 16, wherein the activator molecule is a second cell-surface expressed antigen receptor, wherein the extracellular portions of the first and second cell-surfaced expressed antigen receptors do not bind to the same binding target.
20. The immune effector cell according to claim 19, wherein the immune effector cell further comprises a fourth nucleic acid molecule comprising a fourth nucleotide sequence encoding the binding target of the first cell-surface expressed antigen receptor.
21. The immune effector cell according to claim 19 or claim 20, wherein the immune effector cell further comprises a fifth nucleic acid molecule comprising a fifth nucleotide sequence encoding the binding target of the second cell-surface expressed antigen receptor.
22. The immune effector cell according to claim 20 or claim 21 , wherein the fourth nucleic acid molecule or the fifth nucleic acid molecule comprise both the fourth nucleotide sequence encoding the binding target of the first cell-surface expressed antigen receptor and the fifth nucleotide sequence encoding the binding target of the second cell-surface expressed antigen receptor.
23. The immune effector cell according to any one of claims 20 to 22, wherein (i) the fourth and / or fifth nucleic acid molecule is not integrated into a genomic nucleic acid molecule of the immune effector cell and / or (ii) the encoded binding target of the fourth and / or fifth nucleic acid molecule is transiently expressed.
24. The immune effector cell according to any one of claims 20 to 23, wherein the fourth and / or fifth nucleic acid molecule is DNA or RNA.
25. The immune effector cell according to any one of claims 20 to 24, wherein the fourth and / or fifth nucleic acid molecule is mRNA.
26. The immune effector cell according to any one of claims 1 to 25, wherein the first cell- surface expressed antigen receptor is a chimeric antigen receptor (CAR) or a T cell receptor (TCR).
27. The immune effector cell according to any one of claims 19 to 26, wherein the second cell-surface expressed antigen receptor is a chimeric antigen receptor (CAR) or a T cell receptor (TCR).
28. The immune effector cell according to any one of claims 19 to 27, wherein the binding target of the second cell-surface expressed antigen receptor is expressed on or from cells different from cells expressing the binding target of the first cell-surface expressed antigen receptor.
29. The immune effector cell according to any one of claims 1 to 28, wherein the binding target of the first cell-surface expressed antigen receptor is a tumor-associated antigen or an antigen of an infectious agent, or an epitope thereof.
30. The immune effector cell according to any one of claims 19 to 30, wherein the binding target of the second cell-surface expressed antigen receptor is a cell-surface-expressed protein or a soluble protein, or an epitope thereof.
31. The immune effector cell according to claim 30, wherein the cell-surface expressed protein is a glycoprotein or a cell-surface expressed cytokine.
32. The immune effector cell according to claim 30 or claim 31, wherein the cell-surface expressed protein is a cluster of differentiation (CD) protein.
33. The immune effector cell according to claim 30, wherein the soluble protein is a soluble cytokine.
34. The immune effector cell according to any one of claims 19 to 34, wherein the binding target of the second cell-surface expressed antigen receptor is a cell-surface protein expressed on a blood cell, which blood cell is preferably another immune effector cell.
35. The immune effector cell according to claim 34, wherein the blood cell is a T cell, a NK cell, a dendritic cell, a macrophage, or a B cell.
36. The immune effector cell according to claim 34 or claim 35, wherein the cell-surface protein is CD 19.
37. The immune effector cell according to claim 30, wherein the cell-surface protein is CLDN18.2.
38. The immune effector cell according to any one of claims 1 to 37, wherein the second nucleic acid molecule is DNA or RNA.
39. The immune effector cell according to any one of claims 1 to 38, wherein the second nucleic acid molecule is mRNA40. The immune effector cell according to claim 3, 9, 10, 24, 25, 38 or 39, wherein the RNA or mRNA comprises a ribonucleobase other than A, C, G and U.
41. The immune effector cell according to claim 40, wherein the ribonucleobase is pseudouridine, preferably 1-methyl-pseudouridine.
42. The immune effector cell according to claim 3, 9, 10, 24, 25, 38 or 39, wherein the RNA comprises a 5’ cap structure.
43. The immune effector cell according to claim 42, wherein the 5’ cap structure is a natural occurring cap.
44. The immune effector cell according to claim 42, wherein the 5’ cap structure is a cap analog.
45. The immune effector cell according to claim 43 or claim 44, wherein the 5’ cap structure is one of the following: capO, capl, cap2, cap3, cap4, ARCA (Anti-Reverse Cap Analogs), modified ARCA, inosine, N1-methyl-guanosine, 2’-fluoro-guanosine, 7- deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine and 2-azido- guanosine.
46. The immune effector cell according to claim 45, wherein the 5’ cap structure is capO, which ism7G(5')ppp(5').
47. The immune effector cell according to claim 45, wherein the 5’ cap structure is capl, which ism7G(5')ppp(5')(N12'-OMe).
48. The immune effector cell according to claim 47, wherein the N1is chosen from A, C, G or U.
49. The immune effector cell according to claim 47 or 48, wherein the capl further comprises a second nucleotide N2, which is a cap proximal A, G, C or U at position +2 and is represented asm7G(5')ppp(5')(N12'-OMe)pN2.
50. The immune effector cell according to any one of claims 1 to 49, wherein the immune effector cell is a T cell, a B cell, a dendritic cell, or a NK cell.51 . The immune effector cell according to any one of claims 1 to 50, wherein the immune effector cell is a CD8+ and / or CD4+ T cell.
52. The immune effector cell according to any one of claims 1 to 51, wherein the immune effector cell is a cytotoxic T cell.
53. The immune effector cell according to any one of claims 1 to 52, wherein the second nucleic acid is not inherited by the progeny cells of the immune effector cell in the same manner as chromosomes are inherited, and / or wherein the second nucleic acid is diluted out compared to the total number of cells in each generation of progeny cells of the immune effector cell, and / or wherein after one round of cell division, the amount of the second nucleic acid molecule is less in each daughter cell compared to the amount in the parental cell.
54. An immune effector cell comprising (i) a DNA molecule comprising a transposable element, which element comprises a nucleotide sequence encoding a first T cell receptor or chimeric antigen receptor which binds to a tumor-associated antigen; (ii) an mRNA molecule encoding a second T cell receptor or chimeric antigen receptor which binds to a target different from the tumor-associated antigen; and (iii) an mRNA molecule encoding for a transposase.
55. An immune effector cell comprising (i) a nucleotide sequence encoding a first T cell receptor or a chimeric antigen receptor which binds to a tumor-associated antigen, which nucleotide sequence is integrated into the genome of the immune effector cell or is comprised within an episome present in the immune effector cell; and (ii) a nucleic acid molecule encoding a second T cell receptor or a chimeric antigen receptor which binds to a target different from the tumor-associated antigen, which nucleic acid molecule is not integrated into a genomic nucleic acid molecule of the immune effector cell.
56. An immune effector cell comprising (i) a DNA sequence encoding a first chimeric antigen receptor which binds to a tumor-associated antigen, which DNA sequence is integrated into the genome of the immune effector cell; and (ii) an mRNA molecule encoding a second chimeric antigen receptor which binds to a target different from the tumor-associated antigen.
57. The immune effector cell according to any one of claims 54 to 56, wherein the immune effector cell is a CD8+ cytotoxic T cell.
58. The immune effector cell according to any one of claims 54 to 57, wherein the immune effector cell is activated by the binding of the second T cell receptor or chimeric antigen receptor binding to its target.
59. The immune effector cell according to any one of claims 1 to 58, wherein the immune effector cell does not comprise a DNA nucleotide sequence encoding for the activator molecule.
60. The immune effector cell according to any one of claims 1 to 59, wherein the immune effector cell has reduced cell-surface expression of the endogenous T cell receptor to a level that prevents graft-versus-host activity of the immune effector cell when administered to a subject different from the subject from whom the immune effector cell was derived.
61. The immune effector cell according to any one of claims 1 to 60, wherein the immune effector cell does not express its endogenous T cell receptor on its cell-surface.
62. The immune effector cell according to any one of claims 1 to 61, wherein the immune effector cell has reduced cell-surface expression of the endogenous HLA complex to a level that prevents host-versus-graft activity in a subject to whom the immune effector cell has been administered, wherein the subject administered the immune effector cell is different from the subject from whom the immune effector cell was derived.
63. The immune effector cell according to any one of claims 1 to 62, wherein the immune effector cell does not express its endogenous HLA complex on its cell-surface.
64. A cellular composition comprising the immune effector cell according to any one of claims 1 to 63.
65. The cellular composition according to claim 64, further comprising a cryopreservation agent.
66. A pharmaceutical composition comprising the immune effector cell according to any one of claims 1 to 63 or the cellular composition according to claim 64 or claim 65, and a pharmaceutically acceptable carrier.
67. The immune effector cell according to any one of claims 1 to 63, the cellular composition according to claim 64 or claim 65, or the pharmaceutical composition according to claim 66 for use in a method of treating a subject having a disease, disorder or condition associated with expression or elevated expression of the binding target of the first cell-surface expressed antigen receptor, wherein the method comprises administering the immune effector cell, the cellular composition or the pharmaceutical composition to the subject.
68. The immune effector cell, the cellular composition or the pharmaceutical composition for use according to claim 67, wherein the disease, disorder or condition is cancer.
69. The immune effector cell, the cellular composition or the pharmaceutical composition for use according to claim 68, wherein the cancer is a solid cancer.
70. The immune effector cell, the cellular composition or the pharmaceutical composition for use according to claim 67, wherein the disease, disorder or condition is an infection.
71. The immune effector cell, the cellular composition or the pharmaceutical composition for use according to claim 70, wherein the infection is a viral infection.
72. The immune effector cell, the cellular composition or the pharmaceutical composition for use according to any one of claims 67 to 71 , wherein the immune effector cell is autologous or heterologous to the subject being administered the immune effector cell, the cellular composition or the pharmaceutical composition.
73. A particle comprising (i) a first nucleic acid molecule comprising a first nucleotide sequence encoding a first cell-surface expressed antigen receptor, which first nucleotide sequence is comprised within a transposable element; and (ii) a second nucleic acid molecule comprising a second nucleotide sequence encoding an immune effector cell- activator molecule, wherein the second nucleotide sequence not comprised within a transposable element.
74. The particle according to claim 73, wherein the particle further comprises a third nucleic acid molecule comprising a third nucleotide sequence encoding a molecule having transposase activity, wherein the third nucleotide sequence is not comprised within a transposable element.
75. A particle comprising (i) a DNA episome comprising a first nucleotide sequence encoding a first cell-surface expressed antigen receptor, preferably a non-viral episome; and (ii) a second nucleic acid molecule comprising a second nucleotide sequence encoding an immune effector cell -activator molecule, wherein the second nucleic acid molecule, when present in a cell provides for transient expression of the activator molecule.
76. The particle according to claim 73 or claim 74, wherein the first nucleic acid molecule is DNA or RNA.
77. The particle according to any one of claims 73 to 76, wherein the first nucleic acid molecule or episome is a DNA minicircle or a linear DNA molecule.
78. The particle according to any one of claims 73 to 77, wherein the transposable element in the first nucleic acid is derived from a DNA-based transposon system, a viral-based transposon system, or a poly-A-based retrotransposon system.
79. The particle according to claim 74, wherein the third nucleic acid molecule is DNA or RNA.
80. The particle according to claim 78, wherein the third nucleic acid molecule is mRNA.
81. The particle according to any one of claims 74 or 76 to 80, wherein the molecule having transposase activity is Sleeping Beauty, PiggyBac, Frog, Prince, Himarl, Passport, Minos, hAT, Toll, Tol2, AciDs, PIF, Harbinger, Harbinger3-DR, Hsmarl, or a functionally equivalent variant thereof having transposase / transposition activity.
82. The particle according to any one of claims 74 or 76 to 81 , wherein the molecule having transposase activity is Sleeping Beauty transposase SB100X.
83. The particle according to any one of claims 73 to 82, wherein the second nucleic acid molecule is RNA.
84. The particle according to any one of claims 73 to 83, wherein the second nucleic acid molecule is mRNA.
85. The particle according to any one of claims 73 to 84, wherein the first cell-surface expressed antigen receptor binds to a tumor-associated antigen or an antigen of an infectious agent, or epitope thereof.
86. The particle according to any one of claims 73 to 85, wherein the first cell-surface expressed antigen receptor is a chimeric antigen receptor (CAR) or T cell receptor (TCR).
87. The particle according to any one of claims 73 to 86, wherein the activator molecule allows for the activation, expansion, differentiation and / or proliferation of the immune effector cell.
88. The particle according to any one of claims 73 to 87, wherein the activator molecule is a non-coding RNA or protein.
89. The particle according to any one of claims 73 to 88, wherein the activator molecule binds to the extracellular portion of the first cell-surfaced expressed antigen receptor.
90. The particle according to any one of claims 73 to 88, wherein the activator molecule is a cytokine.
91. The particle according to any one of claims 73 to 88, wherein the activator molecule is a second cell-surface expressed antigen receptor, wherein the extracellular portions ofthe first and second cell-surfaced expressed antigen receptors do not bind to the same binding target.
92. The particle according to claim 91, wherein the particle further comprises a fourth nucleic acid molecule comprising a fourth nucleotide sequence encoding the binding target of the first cell-surface expressed antigen receptor, wherein the fourth nucleotide sequence is not comprised within a transposable element.
93. The particle according to claim 91 or claim 92, wherein the particle further comprises a fifth nucleic acid molecule comprising a fifth nucleotide sequence encoding the binding target of the second cell-surface expressed antigen receptor, wherein the fifth nucleotide sequence is not comprised within a transposable element.
94. The particle according to claim 92 or claim 93, wherein the fourth nucleic acid molecule or the fifth nucleic acid molecule comprise both the fourth nucleotide sequence encoding the binding target of the first cell-surface expressed antigen receptor and the fifth nucleotide sequence encoding the binding target of the second cell-surface expressed antigen receptor.
95. The particle according to any one of claims 92 to 94, wherein the fourth and / or fifth nucleic acid molecule is DNA or RNA.
96. The particle according to any one of claims 92 to 95, wherein the fourth and / or fifth nucleic acid molecule is mRNA.
97. The particle according to any one of claims 91 to 96, wherein the second cell-surface expressed antigen receptor is a chimeric antigen receptor (CAR) or a T cell receptor (TCR).
98. A particle comprising the first, second and third nucleic acid molecule specified in any one of claims 1 to 72.
99. The particle according to claim 98, which particle further comprises the fourth and / or fifth nucleic acid molecule specified in any one of claims 20 to 72.
100. A particle comprising the first, second, third and fourth nucleic acid molecule specified in any one of claims 19 to 72.
101. The particle according to claim 100, which particle further comprises the fifth nucleic acid molecule specified in any one of claims 19 to 72.
102. The particle according to any one of claims 73 to 101, wherein the particle comprises a polyalkyleneimine or a lipid.
103. The particle according to any one of claims 73 to 102, wherein the particle comprises a lipid, preferably comprising a lipid with a cationic headgroup.
104. The particle according to any one of claims 73 to 103, wherein the particle comprises a pH responsive lipid.
105. The particle according to any one of claims 73 to 104, wherein the particle comprises a PEGylated-lipid.
106. The particle according to any one of claims 73 to 105, wherein the particle is a lipid particle, polymer particle, or a mixture thereof.
107. The particle according to any one of claims 73 to 106, wherein the particle is a nanoparticle.
108. The particle according to any one of claims 73 to 107, wherein the particle is a lipid nanoparticle (LNP), a lipoplex, (LPX), a polyplex (PLX), or a lipopolyplex (LPLX) particle.
109. The particle according to any one of claims 73 to 108, wherein the particle further comprises at least one phosphatidylserine.
110. The particle according to any one of claims 73 to 110, wherein the particles are nanoparticles, in which:(i) the number of positive charges in the nanoparticles does not exceed the number of negative charges in the nanoparticles and / or(ii) the nanoparticles have a neutral or net negative charge and / or(iii) the zeta potential of the nanoparticles is 0 or less.
111. The particle according to any one of claims 73 to 102, wherein the particle comprises polyalkyleneimine.
112. The particle according to claim 111, wherein (a) the molar ratio of the number of nitrogen atoms (N) in the polyalkyleneimine to the number of phosphor atoms (P) in the first, second, and optionally third nucleic acid molecules (N:P ratio) is 2.0 to 15.0, preferably 6.0 to 12.0; or (b) the molar ratio of the number of the number of nitrogen atoms (N) in the polyalkyleneimine to the number of phosphor atoms (P) in the first, second, and optionally third nucleic acid molecules (N:P ratio) is at least about 48, optionally about 48 to 300, about 60 to 200, or about 80 to 150.
113. The particle according to claim 111, wherein the ionic strength of the composition is 50 mM or less, preferably wherein the concentration of monovalent cationic ions is 25 mM or less and the concentration of divalent cationic ions is 20 pM or less.
114. The particle according to any one of claims 111 to 113, wherein the particle is a polyplex particle.
115. The particle according to any one of claims 73 to 114, wherein the particle comprises a hydrophobic moiety having a binding moiety covalently attached thereto.
116. The particle according to claim 115, wherein the hydrophobic moiety having a binding moiety covalently attached thereto and the particle are non-covalently associated with each other.
117. The particle according to claim 115 or claim 116, wherein the hydrophobic moiety having a binding moiety covalently attached thereto is an integral part of the particle.
118. The particle according to any one of claims 115 to 117, wherein the hydrophobic moiety having a binding moiety covalently attached thereto comprises a polymer.
119. The particle according to any one of claims 115 to 118, wherein the hydrophobic moiety having a binding moiety covalently attached thereto comprises a compound of Formula IL-X1-P-X2-B (I) whereinP comprises a polymer;L comprises a hydrophobic moiety attached to a first end of the polymer;B comprises a binding moiety attached to a second end of the polymer; X1 is absent or a first linking moiety; andX2 is absent or a second linking moiety.
120. The particle according to claim 119, wherein X1 comprises a carbonyl group.
121. The particle according to claim 119 or claim 120, wherein X2 comprises the reaction product of a maleimide group with a thiol or cysteine group of a compound comprising the binding moiety.
122. The particle according to any one of claims 115 to 121 , wherein the hydrophobic moiety is or is comprised in a lipid.
123. The particle according to any one of claims 118 to 122, wherein the polymer provides stealth property, extends circulation half-life and / or reduces non-specific protein binding or cell adhesion.
124. The particle according to any one of claims 118 to 123, wherein the polymer comprises polyethyleneglycol (PEG).
125. The particle according to any one of claims 115 to 124, wherein the hydrophobic moiety having a binding moiety covalently attached thereto comprises a compound of Formula IIwherein B comprises the binding moiety.
126. The particle according to claim 125, wherein B comprises a moiety comprising the structure -N-peptide-C(O)-NH2.
127. The particle according to any one of claims 115 to 126, wherein the binding moiety covalently attached to the hydrophobic moiety comprises an antibody or an antibody derivative.
128. The particle according to any one of claims 73 to 127, wherein the particle is complexed with the nucleic acid molecules and / or encapsulates the nucleic acid molecules.
129. A pharmaceutical composition comprising the particle according to any one of claims 73 to 128, and a pharmaceutically acceptable carrier.
130. The particle according to claim 73 to 128 or the pharmaceutical composition according to claim 129 for use in a method of treating a subject having a disease, disorder or condition associated with expression or elevated expression of the binding target of the first cell-surface expressed antigen receptor, wherein the method comprises administering the particle or the pharmaceutical composition to the subject.
131. The particle or the pharmaceutical composition for use according to claim 130, wherein the disease, disorder or condition is cancer.
132. The particle for use according to claim 131, wherein the cancer is a solid cancer.
133. The particle or the pharmaceutical composition for use according to claim 130, wherein the disease, disorder or condition is an infection.
134. The particle for use according to claim 133, wherein the infection is a viral infection.
135. A complex comprising(a) the particle according to any one of claims 73 to 128, wherein the particle comprises a hydrophobic moiety having a binding moiety covalently attached thereto, and (b) acompound comprising (i) a moiety binding to the binding moiety covalently attached to the hydrophobic moiety and (ii) a moiety targeting a cell-surface antigen.
136. The complex according to claim 135, wherein the moiety binding to the binding moiety covalently attached to the hydrophobic moiety comprises an antibody or an antibody derivative.
137. The complex according to claim 136, wherein the binding moiety covalently attached to the hydrophobic moiety comprises a peptide comprising an ALFA-tag; and the moiety binding to the binding moiety covalently attached to the hydrophobic moiety comprises an antibody or an antibody derivative comprising a VHH domain comprising the CDR1 sequence VTISALNAMAMG, the CDR2 sequence AVSERGNAM, and the CDR3 sequence LEDRVDSFHDY.
138. The complex according to any one of claims 135 to 137, wherein (i) the moiety binding to the binding moiety covalently attached to the hydrophobic moiety and (ii) the moiety targeting a cell-surface antigen are linked to each other.
139. The complex according to any one of claims 135 to 138 , wherein the compound under (b) comprises a peptide or polypeptide.
140. The complex according to any one of claims 135 to 139, wherein the moiety targeting a cell-surface antigen comprises an antibody or an antibody derivative.
141. The complex according to any one of claims 135 to 140, wherein the cell-surface antigen is characteristic for an immune effector cell.
142. The complex according to any one of claims 135 to 141 , wherein the cell-surface antigen comprises CD4 and / or CD8.
143. The complex according to any one of claims 135 to 142, wherein the cell-surface antigen comprises CD3.
144. The complex according to any one of claims 135 to 143 for use in the treatment of a subject having a disease, disorder or condition associated with expression or elevated expression of the binding target of the first cell-surface expressed antigen receptor.
145. A method of producing an immune effector cell expressing a first antigen receptor on the cell-surface, the method comprising contacting an immune effector cell with (i) a first nucleic acid molecule comprising a first nucleotide sequence encoding a first cell-surface expressed antigen receptor, and (ii) a second nucleic acid molecule comprising a second nucleotide sequence encoding an immune effector cell activator molecule, wherein the second nucleotide sequence is not comprised within atransposable element, and wherein the first cell-surface expressed antigen receptor is stably expressed in the cell and the activator molecule is transiently expressed in the cell.
146. The method according to claim 145, wherein the first nucleic acid molecule is DNA or RNA.
147. The method according to claim 145 or claim 146, wherein the method further comprises integrating the first nucleotide sequence into a genomic nucleic acid molecule of the immune effector cell.
148. The method according to any one of claims 145 to 147, wherein the first nucleotide sequence is comprised within a transposable element.
149. The method according to any one of claims 145 to 148, wherein the method further comprises contacting the immune effector cell with a third nucleic acid molecule comprising a third nucleotide sequence encoding a molecule having transposase activity, wherein the third nucleotide sequence is not comprised within a transposable element.
150. The method according to claim 149, wherein the third nucleic acid molecule is DNA or RNA.
151. The method according to claim 149 or claim 150, wherein the third nucleic acid molecule is mRNA.
152. The method according to any one of claims 149 to 151, wherein the molecule having transposase activity is Sleeping Beauty, PiggyBac, Frog, Prince, Himarl, Passport, Minos, hAT, Toll, Tol2, AciDs, PIF, Harbinger, Harbinger3-DR, Hsmarl, or a functionally equivalent variant thereof having transposase / transposition activity.
153. The method according to any one of claims 149 to 151, wherein the molecule having transposase activity is Sleeping Beauty transposase SB100X.
154. The method according to claim 145 or claim 146, wherein the first nucleic acid is an epi some.
155. The method according to claim 154, wherein the episome is a non- viral episome.
156. The method according to any one of claims 145 to 155, wherein the activator molecule allows for the activation, expansion, differentiation and / or proliferation of the immune effector cell.
157. The method according to any one of claims 145 to 156, wherein the activator molecule is a non-coding RNA or protein.
158. The method according to any one of claims 145 to 157, wherein the activator molecule binds to the extracellular portion of the first cell-surfaced expressed antigen receptor.
159. The method according to any one of claims 145 to 158, wherein the activator molecule is a cytokine.
160. The method according to any one of claims 145 to 159, wherein the activator molecule is a second cell-surface expressed antigen receptor, wherein the extracellular portions of the first and second cell-surfaced expressed antigen receptors do not bind to the same binding target.
161. The method according to claim 160, wherein the method further comprises contacting the immune effector cell with a fourth nucleic acid molecule comprising a fourth nucleotide sequence encoding the binding target of the first cell-surfaced expressed antigen receptor.
162. The method according to claim 160 or claim 161, wherein the method further comprises contacting the immune effector cell with a fifth nucleic acid molecule comprising a fifth nucleotide sequence encoding the binding target of the second cell- surfaced expressed antigen receptor.
163. The method according to claim 161 or claim 162, wherein the fourth nucleic acid molecule or the fifth nucleic acid molecule comprise both the fourth nucleotide sequence encoding the binding target of the first cell-surfaced expressed antigen receptor and the fifth nucleotide sequence encoding the binding target of the second cell-surfaced expressed antigen receptor.
164. The method according to any one of claims 161 to 163, wherein the fourth and / or fifth nucleic acid molecule is DNA or RNA.
165. The method according to any one of claims 161 to 164, wherein the fourth and / or fifth nucleic acid molecule is mRNA.
166. The method according to any one of claims 145 to 165, wherein the first cell- surfaced expressed antigen receptor is a chimeric antigen receptor (CAR) or a T cell receptor (TCR).
167. The method according to any one of claims 160 to 166, wherein the first cell- surfaced expressed antigen receptor and / or the second cell-surfaced expressed antigen receptor is a chimeric antigen receptor (CAR) or a T cell receptor (TCR).
168. The method according to any one of claims 145 to 167, wherein the binding target of the second cell-surfaced expressed antigen receptor is expressed on or fromcells different from cells expressing the binding target of the first cell-surfaced expressed antigen receptor.
169. The method according to any one of claims 145 to 168, wherein the binding target of the first cell-surfaced expressed antigen receptor is a tumor-associated antigen or an antigen of an infectious agent, or an epitope thereof.
170. The method according to any one of claims 145 to 169, wherein the second nucleic acid molecule is DNA or RNA.
171. The method according to any one of claims 145 to 170, wherein the second nucleic acid molecule is mRNA.
172. The method according to claim 146, 150, 151 , 164, 165, 170 or 171 , wherein the RNA or mRNA comprises a ribonucleobase other than A, C, G and U.
173. The method according to claim 172, wherein the ribonucleobase is pseudouridine, preferably 1 -methyl-pseudouridine.
174. The method according to claim 146, 150, 151, 164, 165, 170 or 171, wherein the RNA comprises a 5’ cap structure.
175. The method according to claim 174, wherein the 5’ cap structure is a natural occurring cap.
176. The method according to claim 174, wherein the 5’ cap structure is a cap analog.
177. The method according to claim 175 or claim 176, wherein the 5’ cap structure is one of the following: capO, capl , cap2, cap3, cap4, ARCA (Anti-Reverse Cap Analogs), modified ARCA, inosine, N1-methyl-guanosine, 2 ’-fluoro-guanosine, 7-deaza- guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine and 2-azido- guanosine.
178. The method according to any one of claims 145 to 177, wherein the immune effector cell is a T cell, a B cell, a dendritic cell, or a NK cell.
179. The method according to any one of claims 145 to 178, wherein the immune effector cell is a CD8+ and / or CD4+ T cell.
180. The method according to any one of claims 145 to 179, wherein the immune effector cell is a cytotoxic T cell.
181. The method according to any one of claims 145 to 180, wherein the second nucleic acid is not inherited by the progeny cells of the immune effector cell in the same manner as chromosomes are inherited, and / or wherein the second nucleic acid is diluted out in each generation of progeny cells of the immune effector cell, and / or wherein afterone round of cell division, the amount of the second nucleic acid molecule is less in each daughter cell compared to the amount in the parental cell.
182. A method of producing an immune effector cell expressing a first antigen receptor on the cell-surface, the method comprising contacting an immune effector cell with a particle according to any one of claims 73 to 134 or a complex according to any one of claims 135 to 144.
183. The method according to any one of claims 145 or 182, wherein the contacting occurs in vitro.
184. The method according to any one of claims 160 to 183, wherein the method further comprises, after contacting the nucleic acid molecules to the immune effector cell, a step of contacting the immune effector cell with the binding target of the second cell-surface expressed antigen receptor or a cell expressing the binding target.
185. A method of producing an immune effector cell expressing two antigen receptors on the cell-surface, the method comprising contacting, in vitro or ex vivo, an immune effector cell with (i) a DNA molecule comprising a first nucleotide sequence encoding a first cell-surface expressed antigen receptor, which first nucleotide sequence is comprised with a transposable element; (ii) an RNA molecule comprising a second nucleotide sequence encoding a second cell-surface expressed antigen receptor, which second nucleotide sequence is not comprised within a transposable element; and (iii) an RNA molecule comprising a third nucleotide sequence encoding a transposase, which third nucleotide sequence is not comprises within a transposable element, wherein the extracellular domains of the first and second cell-surface expressed antigen receptors bind to different targets, preferably wherein the binding target of the first cell-surface expressed antigen receptor is a tumor or tumor-associated antigen and the binding target of the second cell-surface expressed antigen receptor is expressed on the surface of a blood cell.
186. The method according to claim 185, further comprising contacting the immune effector cell with the binding target of the second cell-surface expressed antigen receptor or with a cell expressing the binding target of the second cell-surface expressed antigen receptor.
187. A method of producing an immune effector cell expressing two antigen receptors on the cell-surface, the method comprising contacting an immune effector cell with a particle, which particle comprises (i) a DNA molecule comprising a first nucleotide sequence encoding a first cell-surface expressed antigen receptor, which first nucleotidesequence is comprised with a transposable element; (ii) an mRNA molecule comprising a second nucleotide sequence encoding a second cell-surface expressed antigen receptor; and (iii) an mRNA molecule comprising a third nucleotide sequence encoding a transposase; wherein the extracellular domains of the first and second cell-surface expressed antigen receptors bind to different targets, preferably wherein the binding target of the first cell-surface expressed antigen receptor is a tumor or tumor-associated antigen and the binding target of the second cell-surface expressed antigen receptor is expressed on the surface of a blood cell.
188. The method according to claim 187, wherein the contacting is in vivo.
189. A method of treating a subject having a disease, disorder or condition associated with expression or elevated expression of an antigen, the method comprising administering to the subject a first nucleic acid molecule comprising a first nucleotide sequence encoding a first cell-surface expressed antigen receptor and a second nucleic acid molecule comprising a second nucleotide sequence encoding an immune effector cell activator molecule, wherein the binding target of the first cell-surface expressed antigen receptor is the antigen that is associated with the disease, disorder or condition, wherein (i) the second nucleotide sequence does not integrate into a genomic nucleic acid molecule of the cells of the subject or is comprised within an episome present in the cells of the subject and / or (ii) the activator molecule is transiently expressed in the subject.
190. The method according to claim 189, wherein the nucleic acid molecules are in particle comprising a lipid.
191. A method of treating a subj ect having a disease, disorder or condition associated with expression or elevated expression of an antigen, the method comprising administering to the subject a particle, which particle comprises (i) a DNA molecule comprising a first nucleotide sequence encoding a first cell-surface expressed antigen receptor, which first nucleotide sequence is comprised with a transposable element and wherein the binding target of the first cell-surface expressed antigen receptor is the antigen that is associated with the disease, disorder or condition; (ii) an mRNA molecule comprising a second nucleotide sequence encoding a second cell-surface expressed antigen receptor; and (iii) an mRNA molecule comprising a third nucleotide sequence encoding a transposase; wherein the extracellular domains of the first and second cell- surface expressed antigen receptors bind to different targets.
192. A method of treating a subject having a disease, disorder or condition associated with expression or elevated expression of an antigen, the method comprising administering to the subject the immune effector cell according to any one of claims 1 to 63, the cellular composition according to claim 64 or claim 65, or the pharmaceutical composition according to claim 66, wherein the binding target of the first cell-surface expressed antigen receptor is the antigen that is associated with the disease, disorder or condition.
193. A method of treating a subject having a disease, disorder or condition associated with expression or elevated expression of an antigen, the method comprising administering to the subject a particle according to any one of claims 73 to 128 or the pharmaceutical composition according to claim 129, wherein the binding target of the first cell-surface expressed antigen receptor is the antigen that is associated with the disease, disorder or condition.
194. A method of treating a subject having a disease, disorder or condition associated with expression or elevated expression of an antigen, the method comprising administering to the subject a complex according to any one of claims 135 to 144, wherein the binding target of the first cell-surface expressed antigen receptor is the antigen that is associated with the disease, disorder or condition.
195. The method according to any one of claims 189 to 194, wherein the antigen associated with a disease, disorder or condition is a tumor-associated antigen.
196. The method according to any one of claims 189 to 195, which is a method for treating or preventing cancer in a subject.
197. The method according to any one of claims 189 to 194, wherein the antigen associated with a disease, disorder or condition comprises an antigen of an infectious agent.
198. The method according to claim 197, wherein the infectious agent is a virus.
199. The method according to any one of claims 189 to 194, 197 or 198, which is a method for treating or preventing an infection in a subject.