Methods for non-viral manufacturing of engineered immune cells
By targeting and integrating transgenes into specific sites within immune cells using column-based stimulation and non-viral methods, the challenges of inefficient gene delivery in immune cell engineering are overcome, facilitating efficient production from whole blood samples.
Patent Information
- Authority / Receiving Office
- HK · HK
- Patent Type
- Applications
- Current Assignee / Owner
- JUNO THERAPEUTICS GMBH
- Filing Date
- 2026-05-20
- Publication Date
- 2026-07-17
AI Technical Summary
Existing methods for genetically engineering immune cells are limited by inefficient gene targeting and integration, particularly when using non-viral gene delivery methods, and there is a need for improved production methods from whole blood samples.
Genetically engineering immune cells by targeting and integrating transgenes into specific sites within the cells, utilizing column-based stimulation and non-viral gene delivery methods.
Enhances the efficiency and effectiveness of gene targeting and integration in immune cells, enabling production from whole blood samples.
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Abstract
Description
This document provides methods for producing genetically engineered immune cells (e.g., T cells). In some aspects, immune cells are genetically engineered by targeting and integrating transgenes into the target sites of genes within the immune cells. In some embodiments, the genetically engineered immune cells are produced from whole blood samples. In some aspects, the immune cells are genetically engineered after column-based stimulation. In some aspects, immune cells are genetically engineered using non-viral gene delivery methods. Related cells, compositions, and uses are also provided. Abstract
Claims
Claims1. A method for producing genetically engineered T cells, comprising:(a) adding a whole blood sample comprising a plurality of T cells to a stationary phase in an internal cavity of a chromatography column, the stationary phase comprising a selection agent that specifically binds to a selection marker expressed on the surface of the plurality of T cells, wherein specific binding of the selection agent to the selection marker effects the immobilization of the plurality of T cells on the stationary phase;(b) adding a T cell stimulatory reagent to the plurality of T cells immobilized on the stationary phase, wherein the T cell stimulatory reagent comprises (i) a primary agent that specifically binds to a member of a TCR / CD3 complex and (ii) a secondary agent that specifically binds to a T cell costimulatory molecule;(c) incubating the plurality of T cells immobilized on the stationary phase in the presence of the T cell stimulatory reagent under conditions to stimulate T cells of the plurality of T cells;(d) collecting T cells of the plurality of T cells from the chromatography column that are no longer immobilized after the incubating; and(e) introducing by non-viral gene delivery a nucleic acid molecule comprising a transgene encoding a recombinant protein under conditions for targeted integration of the transgene into a target site of a gene in one or more of the collected T cells; wherein the method produces genetically engineered T cells expressing the recombinant protein.
2. The method of claim 1, wherein the method comprises further incubating the collected T cells prior to the introducing of the nucleic acid molecule.
3. The method of claim 1 or claim 2, wherein the nucleic acid molecule is a DNA molecule, optionally a single-stranded DNA molecule or a double-stranded DNA molecule.
4. The method of any one of claims 1-3, wherein the targeted integration is by Programmable Addition via Site-specific Targeting Elements (PASTE).
5. The method of claim 4, wherein the PASTE comprises introducing one or more gene-editing agents for editing the gene in the one or more of the collected T cells.
6. The method of any one of claims 1-3, wherein the targeted integration is by homology directed repair (HDR).
7. The method of claim 6, wherein the HDR comprises introducing one or more gene-editing agents for inducing a genetic disruption in the gene in the one or more of the collected T cells.
8. The method of any one of claims 1-7, wherein the introducing of the nucleic acid molecule and / or the one or more gene-editing agents is by electroporation.
9. The method of any one of claims 1-8, wherein the conditions for targeted integration comprise cultivating the collected T cells under conditions to integrate the transgene into the target site.
10. A method for producing genetically engineered T cells, comprising:(a) adding a whole blood sample comprising a plurality of T cells to a stationary phase in an internal cavity of a chromatography column, the stationary phase comprising a selection agent that specifically binds to a selection marker expressed on the surface of the plurality of T cells, wherein specific binding of the selection agent to the selection marker effects the immobilization of the plurality of T cells on the stationary phase;(b) adding a T cell stimulatory reagent to the plurality of T cells immobilized on the stationary phase, wherein the T cell stimulatory reagent comprises (i) a primary agent that specifically binds to a member of a TCR / CD3 complex and (ii) a secondary agent that specifically binds to a T cell costimulatory molecule;(c) incubating the plurality of T cells immobilized on the stationary phase in the presence of the T cell stimulatory reagent under conditions to stimulate T cells of the plurality of T cells;(d) collecting T cells of the plurality of T cells from the chromatography column that are no longer immobilized after the incubating;(e) further incubating the collected T cells;(f) after the further incubating, introducing into T cells of the collected T cells (i) a nucleic acid molecule comprising a transgene encoding a recombinant protein, wherein the nucleic acid molecule is a DNA molecule, optionally a single-stranded DNA molecule or a double-stranded DNA molecule, and the introducing of the nucleic acid molecule is by non- viral gene delivery, and (ii) one or more gene-editing agents for inducing a genetic disruption in a gene in the T cells of the collected T cells, wherein the introducing of the nucleic acid molecule and / or the one or more gene-editing agents is by electroporation; and(g) cultivating the collected T cells under conditions to integrate by homology directed repair (HDR) the transgene into a target site of the gene in one or more of the collected T cells; wherein the method produces genetically engineered T cells expressing the recombinant protein.
11. The method of any one of claims 1-10, wherein the nucleic acid molecule is a double-stranded DNA molecule, a naked DNA molecule, and / or a closed-ended DNA molecule.
12. The method of any one of claims 1-11, wherein the nucleic acid molecule is a closed-ended linear duplex (CELiD) DNA molecule, a minicircle DNA molecule, a minimalistic immunological-defined gene expression (MIDGE) DNA molecule, a ministring DNA molecule, a dumbbell-shaped linear duplex closed-ended DNA molecule, or a doggybone™ DNA molecule.
13. The method of any one of claims 1-12, wherein the T cell stimulatory reagent is added in a cell medium.
14. The method of any one of claims 1-13, wherein the T cell stimulatory reagent is added in an amount between or between about 0.1 pg and 20 pg, 0.4 pg and 8 pg, 0.8 pgand 4 jug, or 1 pg and 2 pg, each inclusive and each per 106T cells of the plurality of T cells immobilized or expected to be immobilized on the stationary phase.
15. The method of any one of claims 1-14, wherein the binding capacity of the stationary phase is between or between about 0.5 billion and 5 billion T cells expressing the selection marker, 0.5 billion and 3 billion T cells expressing the selection marker, or 1 billion and 2 billion T cells expressing the selection marker, each inclusive.
16. The method of any one of claims 1-15, wherein the T cell stimulatory reagent is added in an amount between or between about 0.1 mg and 20 mg, 0.4 mg and 8 mg, 0.8 mg and 4 mg, or 1 mg and 3 mg, each inclusive.
17. The method of any one of claims 1-16, wherein the adding of the T cell stimulatory reagent is carried out within or within about 60 minutes, 30 minutes, or 15 minutes after the adding of the whole blood sample.
18. The method of any one of claims 1-17, wherein the incubating is carried out in a cell medium.
19. The method of any one of claims 1-18, wherein the incubating is carried out at a temperature between or between about 35°C and about 39°C.
20. The method of any one of claims 1-19, wherein the incubating is carried out for between or between about 0.5 hour and 8 hours, 2 hours and 6 hours, or 3 hours and 5 hours, each inclusive.
21. The method of any one of claims 1-20, wherein the collecting comprises adding a wash buffer to the stationary phase to collect the T cells of the plurality of T cells.
22. The method of claim 21, wherein the wash buffer is a cell medium.
23. The method of claim 21 or claim 22, wherein the wash buffer does not comprise a competition agent.
24. The method of any one of claims 1-23, wherein the collecting is carried out between or between about 0.5 hours and 8 hours, 2 hours and 6 hours, or 3 hours and 5 hours, each inclusive, after the adding of the T cell stimulatory reagent.
25. The method of any one of claims 2-24, wherein the further incubating is carried out in the presence of the T cell stimulatory reagent.
26. The method of any one of claims 2-25, wherein the further incubating is carried out in a cell medium.
27. The method of any one of claims 2-26, wherein the further incubating is carried out at a temperature between or between about 35°C and about 39°C.
28. The method of any one of claims 2-27, wherein the further incubating is carried out for between or between about 10 hours and 30 hours, 16 hours and 24 hours, or 18 hours and 22 hours, each inclusive.
29. The method of any one of claims 5 and 7-28, wherein the method comprises removing the T cell stimulatory reagent from the collected T cells prior to the introducing of the one or more gene-editing agents.
30. The method of any one of claims 1-29, wherein the method comprises removing the T cell stimulatory reagent from the collected T cells prior to the introducing of the nucleic acid molecule.
31. The method of claim 29 or claim 30, wherein the removing is carried out after the further incubating.
32. The method of any one of claims 29-31, wherein the removing comprises washing the collected T cells.
33. The method of any one of claims 5 and 7-32, wherein: the T cell stimulatory reagent comprises an oligomer of streptavidin or a streptavidin mutein molecule; the primary agent comprises a first streptavidin-binding partner that is bound to a streptavidin or streptavidin mutein molecule of the oligomer; the secondary agent comprises a second streptavidin-binding partner that is bound to a streptavidin or streptavidin mutein molecule of the oligomer; and the method comprises disrupting the binding between the first and second streptavidin-binding partners and the streptavidin or streptavidin mutein molecules prior to the introducing of the one or more gene-editing agents.
34. The method of any one of claims 1-33, wherein: the T cell stimulatory reagent comprises an oligomer of streptavidin or a streptavidin mutein molecule; the primary agent comprises a first streptavidin-binding partner that is bound to a streptavidin or streptavidin mutein molecule of the oligomer; the secondary agent comprises a second streptavidin-binding partner that is bound to a streptavidin or streptavidin mutein molecule of the oligomer; and the method comprises disrupting the binding between the first and second streptavidin-binding partners and the streptavidin or streptavidin mutein molecules prior to the introducing of the nucleic acid molecule.
35. The method of claim 33 or claim 34, wherein the disrupting is carried out after the further incubating.
36. The method of any one of claims 33-35, wherein the disrupting is by adding a competition agent to the collected T cells that reverses the binding between the first and second streptavidin-binding partners and the streptavidin or streptavidin mutein molecules.
37. The method of any one of claims 23-36, wherein the competition agent is biotin.
38. The method of any one of claims 5 and 7-37, wherein the introducing of the one or more gene-editing agents is carried out prior to or concurrently with the introducing of the nucleic acid molecule.
39. The method of any one of claims 5 and 7-38, wherein the introducing of the one or more gene-editing agents is carried out between or between about 12 hours and 36 hours, 18 hours and 30 hours, or 22 hours and 26 hours, each inclusive, after the adding of the T cell stimulatory reagent.
40. The method of any one of claims 1-39, wherein the nucleic acid molecule is introduced in a cell medium comprising the nucleic acid molecule.
41. The method of any one of claims 1-40, wherein the introducing of the nucleic acid molecule is carried out between or between about 12 hours and 36 hours, 18 hours and 30 hours, or 22 hours and 26 hours, each inclusive, after the adding of the T cell stimulatory reagent.
42. The method of any one of claims 9-41, wherein the cultivating is carried out in the presence of the nucleic acid molecule.
43. The method of any one of claims 9-42, wherein the cultivating is carried out in a cell medium.
44. The method of any one of claims 9-43, wherein the cultivating is carried out at a temperature between or between about 35°C and about 39°C.
45. The method of any one of claims 9-44, wherein the cultivating is carried out for between or between about 12 hours and 36 hours, 18 hours and 30 hours, or 22 hours and26 hours, each inclusive.
46. The method of any one of claims 13-45, wherein the cell medium is a basal medium.
47. The method of any one of claims 13-46, wherein the cell medium is a serum free medium.
48. The method of any one of claims 13-47, wherein the cell medium comprises no cytokines or comprises recombinant IL-2, IL-7, and IL-15.
49. The method of any one of claims 1-48, wherein the method comprises harvesting the genetically engineered T cells expressing the recombinant protein.
50. The method of claim 49, wherein the harvesting is carried out between or between about 36 hours and 60 hours, 42 hours and 54 hours, or 46 hours and 50 hours, each inclusive, after the adding of the whole blood sample.
51. The method of claim 49 or claim 50, wherein the harvesting is carried out between or between about 36 hours and 60 hours, 42 hours and 54 hours, or 46 hours and 50 hours, each inclusive, after the adding of the T cell stimulatory reagent.
52. The method of any one of claims 49-51, wherein the harvesting is carried out between or between about 12 hours and 36 hours, 18 hours and 30 hours, or 22 hours and 26 hours, each inclusive, after the introducing of the one or more gene-editing agents.
53. The method of any one of claims 49-52, wherein the harvesting is carried out between or between about 12 hours and 36 hours, 18 hours and 30 hours, or 22 hours and 26 hours, each inclusive, after the introducing of the nucleic acid molecule.
54. The method of any one of claims 49-53, wherein the method comprises formulating the harvested genetically engineered T cells for cryopreservation or administration to a subject.
55. The method of claim 54, wherein the harvested genetically engineered T cells are formulated in the presence of a cryoprotectant or a pharmaceutically acceptable excipient.
56. The method of any one of claims 1-55, wherein the plurality of T cells are primary T cells from a human subject.
57. The method of any one of claims 1-56, wherein the selection marker is selected from the group consisting of CD3, CD4, CD8, CD45RA, CD27, CD28, and CCR7.
58. The method of any one of claims 1-57, wherein the selection marker is CD3, CD4, or CD8.
59. The method of any one of claims 1-58, wherein the selection marker is CD3.
60. The method of any one of claims 1-59, wherein the selection agent comprises an antibody or antibody fragment that specifically binds to the selection marker.
61. The method of claim 60, wherein the antibody or antibody fragment of the selection agent is a monovalent antibody fragment.
62. The method of claim 60 or claim 61, wherein the antibody or antibody fragment of the selection agent is a Fab fragment.
63. The method of any one of claims 1-32 and 37-62, wherein: the T cell stimulatory reagent comprises an oligomer of streptavidin or a streptavidin mutein molecule;the primary agent comprises a first streptavidin-binding partner that is bound to a streptavidin or streptavidin mutein molecule of the oligomer; and the secondary agent comprises a second streptavidin-binding partner that is bound to a streptavidin or streptavidin mutein molecule of the oligomer.
64. The method of any one of claims 33-63, wherein the T cell stimulatory reagent consists or consists essentially of the oligomer, primary agent, and secondary agent.
65. The method of any one of claims 33-64, wherein the oligomer comprises between or between about 500 and 5000 tetramers, 1000 and 4000 tetramers, or 2000 and 3000 tetramers, each inclusive, of the streptavidin or streptavidin mutein molecule.
66. The method of any one of claims 33-65, wherein the oligomer is of the streptavidin mutein molecule.
67. The method of any one of claims 33-66, wherein the streptavidin mutein molecule comprises the amino acid sequence IGAR (SEQ ID NO: 133) or VTAR (SEQ ID NO: 134) at sequence positions corresponding to positions 44 to 47 of the sequence of amino acids set forth in SEQ ID NO: 1.
68. The method of any one of claims 33-67, wherein the streptavidin mutein molecule begins N-terminally in the region of amino acid positions 10 to 16 of SEQ ID NO: 1 and terminates C-terminally in the region of amino acid positions 133 to 142 of SEQ ID NO: 1.
69. The method of any one of claims 33-68, wherein the streptavidin mutein molecule comprises the amino acid sequence set forth in any one of SEQ ID NO: 3-6, 27, 28, 104, 105, and 136.
70. The method of any one of claims 33-69, wherein the streptavidin mutein molecule comprises the amino acid sequence set forth in SEQ ID NO: 6.
71. The method of any one of claims 33-70, wherein: the first streptavidin-binding partner is at the C-terminus of the primary agent; and / or the second streptavidin-binding partner is at the C-terminus of the secondary agent.
72. The method of any one of claims 33-71, wherein the first and / or second streptavidin-binding partner is a streptavidin-binding peptide.
73. The method of claim 72, wherein the streptavidin-binding peptide of the first and / or second streptavidin-binding partner comprises the amino acid sequence set forth in any one of SEQ ID NO: 7, 8, and 15-19.
74. The method of claim 72 or claim 73, wherein the streptavidin-binding peptide of the first and / or second streptavidin-binding partner comprises the amino acid sequence set forth in SEQ ID NO: 16.
75. The method of any one of claims 1-74, wherein the member of the TCR / CD3 complex is CD3.
76. The method of any one of claims 1-75, wherein the T cell costimulatory molecule is CD28, CD90 (Thy-1), CD95 (Apo- / Fas), CD137 (4-1BB), CD154 (CD40L), ICOS, LAT, CD27, 0X40, or HVEM.
77. The method of any one of claims 1-76, wherein the T cell costimulatory molecule is CD28.
78. The method of any one of claims 1-77, wherein: the primary agent comprises an antibody or antibody fragment that specifically binds to the member of the TCR / CD3 complex; and / or the secondary agent comprises an antibody or antibody fragment that specifically binds to the T cell costimulatory agent.
79. The method of claim 78, wherein: the antibody or antibody fragment of the primary agent comprises a heavy chain, and the first streptavidin-binding partner is fused to the C-terminus of the heavy chain of the primary agent; and / or the antibody or antibody fragment of the secondary agent comprises a heavy chain, and the second streptavidin-binding partner is fused to the C-terminus of the heavy chain of the secondary agent.
80. The method of claim 78 or claim 79, wherein the antibody or antibody fragment of the primary and / or secondary agent is a monovalent antibody fragment.
81. The method of any one of claims 78-80, wherein the antibody or antibody fragment of the primary and / or secondary agent is a Fab fragment.
82. The method of any one of claims 1-81, wherein the primary agent comprises an anti-CD3 antibody or antibody fragment, and the secondary agent comprises an anti-CD28 antibody or antibody fragment.
83. The method of any one of claims 1-82, wherein the primary agent comprises an anti-CD3 Fab fragment, and the secondary agent comprises an anti-CD28 Fab fragment.
84. The method of any one of claims 1-83, wherein the gene is the T cell receptor alpha constant (TRAC) gene.
85. The method of any one of claims 1-84, wherein the target site is within the sequence set forth in SEQ ID NO: 250.
86. The method of any one of claims 1-3 and 6-85, wherein the nucleic acid molecule comprises a 5’ homology arm and a 3’ homology arm comprising sequenceshomologous to nucleic acid sequences surrounding the target site, the nucleic acid molecule comprising the structure [5’ homology arm]-[transgene]-[3’ homology arm].
87. The method of claim 86, wherein the 5’ homology arm and the 3’ homology arm comprise sequences homologous to sequences of the TRAC gene surrounding the target site.
88. The method of claim 86 or claim 87, wherein the 5’ homology arm comprises a sequence comprising at least or at least about 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 contiguous nucleotides of a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence set forth in SEQ ID NO: 248.
89. The method of any one of claims 86-88, wherein the 5’ homology arm comprises at least or at least about 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 contiguous nucleotides of the sequence set forth in SEQ ID NO: 248.
90. The method of any one of claims 86-89, wherein the 5’ homology arm comprises the sequence set forth in SEQ ID NO: 248.
91. The method of any one of claims 86-90, wherein the 3’ homology arm comprises a sequence comprising at least or at least about 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 contiguous nucleotides of a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence set forth in SEQ ID NO: 249.
92. The method of any one of claims 86-91, wherein the 3’ homology arm comprises at least or at least about 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 contiguous nucleotides of the sequence set forth in SEQ ID NO: 249.
93. The method of any one of claims 86-92, wherein the 3’ homology arm comprises the sequence set forth in SEQ ID NO: 249.
94. The method of any one of claims 1-93, wherein transcription of the integrated transgene is under the control of a promoter comprised by the nucleic acid molecule.
95. The method of claim 94, wherein the promoter is a human elongation factor 1 alpha (EFla) promoter.
96. The method of claim 94 or claim 95, wherein the promoter comprises the sequence set forth in SEQ ID NO: 247.
97. The method of any one of claims 1-96, wherein the recombinant protein is a recombinant receptor.
98. The method of claim 97, wherein the recombinant receptor is a T cell receptor or a chimeric antigen receptor.
99. The method of any one of claims 5 and 7-98, wherein the one or more geneediting agents comprise (i) a gene-editing nuclease or nuclease combination or (ii) a nucleic acid molecule comprising one or more sequences encoding the gene-editing nuclease or nuclease combination.
100. The method of any one of claims 5 and 7-99, wherein the one or more geneediting agents comprise a gene-editing nuclease or nuclease combination.
101. The method of claim 99 or claim 100, wherein the gene-editing nuclease or nuclease combination specifically recognizes a nucleic acid sequence near or comprising the target site.
102. The method of any one of claims 99-101, wherein the gene-editing nuclease or nuclease combination specifically recognizes a nucleic acid sequence comprising the target site.
103. The method of claim 101 or claim 102, wherein the nucleic acid sequence comprising the target site comprises the sequence set forth in SEQ ID NO: 250.
104. The method of any one of claims 99-103, wherein the gene-editing nuclease or nuclease combination is a zinc finger nuclease, a transcription activator-like effector nuclease, or a CRISPR-Cas combination.
105. The method of any one of claims 99-104, wherein the gene-editing nuclease or nuclease combination is a CRISPR-Cas combination.
106. The method of claim 104 or claim 105, wherein the CRISPR-Cas combination comprises a CRISPR-Cas nickase, reverse transcriptase, and serine integrase.
107. The method of claim 105 or claim 106, wherein the CRISPR-Cas combination comprises a guide RNA comprising a targeting sequence that is complementary to the nucleic acid sequence comprising the target site.
108. The method of claim 105 or claim 107, wherein the CRISPR-Cas combination is a ribonucleoprotein complex comprising the guide RNA and a Cas protein.
109. The method of claim 108, wherein the Cas protein is a S. pyogenes Cas protein.
110. The method of any one of claims 104-109, wherein the CRISPR-Cas combination is a CRISPR-Cas9 combination or a CRISPR-Casl2 combination.
111. The method of any one of claims 107-110, wherein the targeting sequence comprises the sequence set forth in any one of SEQ ID NO: 144-175.
112. The method of any one of claims 107-111, wherein the targeting sequence comprises the sequence set forth in SEQ ID NO: 148.
113. A genetically engineered T cell produced by the method of any one of claims 1-112, wherein the genetically engineered T cell expresses the recombinant protein.
114. The genetically engineered T cell of claim 113, wherein the transgene is integrated into the target site of the gene in the genetically engineered T cell.
115. The genetically engineered T cell of claim 114, wherein the gene is the T cell receptor alpha constant (TRAC) gene.
116. The genetically engineered T cell of claim 114 or claim 115, wherein the target site is within the sequence set forth in SEQ ID NO: 250.
117. The genetically engineered T cell of any one of claims 113-116, wherein the recombinant protein is a recombinant receptor.
118. The genetically engineered T cell of claim 117, wherein the recombinant receptor is a T cell receptor or a chimeric antigen receptor.
119. A population of T cells comprising a plurality of the genetically engineered T cell of any one of claims 113-118.
120. The population of claim 119, wherein the plurality of genetically engineered T cells are at least 10%, 15%, or 20% of the population of T cells.
121. The population of claim 119 or claim 120, wherein the gene is disrupted in at least 85%, 90%, or 95% of the T cells of the population of T cells.
122. The population of claim 121, wherein the gene is the T cell receptor alpha constant (TRAC) gene.
123. A pharmaceutical composition comprising the population of T cells of any one of claims 119-122 and a pharmaceutically acceptable excipient.
124. A method of treatment, comprising administering to a subject having a disease or condition the pharmaceutical composition of claim 123.
125. The method of claim 124, wherein the recombinant protein is a recombinant receptor that targets an antigen expressed on a target cell associated with the disease or condition.
126. A method of cytolytic killing of a target cell, comprising contacting a target cell with the population of any one of claims 119-122.
127. A method of cytolytic killing of a target cell, comprising contacting a target cell with the pharmaceutical composition of claim 123.
128. The method of claim 126 or claim 127, wherein the contacting is performed ex vivo.
129. The method of claim 126 or claim 127, wherein the contacting is performed in vivo.
130. The method of claim 129, wherein the contacting is by administering the pharmaceutical composition to a subject having a disease or condition.
131. The method of claim 130, wherein the target cell is associated with the disease or condition, and the recombinant protein is a recombinant receptor that targets an antigen expressed on the target cell.
132. The method of claim 125 or claim 131, wherein the recombinant receptor is a T cell receptor or a chimeric antigen receptor.
133. The pharmaceutical composition of claim 123 for use in treating a disease or disorder in a subject.
134. The pharmaceutical composition of claim 133, wherein the recombinant protein is a recombinant receptor that targets an antigen expressed on a cell associated with the disease or condition.
135. The pharmaceutical composition of claim 134, wherein the recombinant receptor is a T cell receptor or a chimeric antigen receptor.
136. Use of the pharmaceutical composition of claim 123 for treating a disease or disorder in a subject.
137. Use of the pharmaceutical composition of claim 123 for the manufacture of a medicament for treating a disease or disorder in a subject.
138. The use of claim 136 or claim 137, wherein the recombinant protein is a recombinant receptor that targets an antigen expressed on a cell associated with the disease or condition.
139. The use of claim 138, wherein the recombinant receptor is a T cell receptor or a chimeric antigen receptor.