Methods for transducing immune cells
Provided herein are improved methods for transducing immune cells, such as T cells, with retroviral vectors to express exogenous gene products, such as chimeric antigen receptors (CARs). Provided herein are methods that increase transduction efficiency thereby increasing the percentage of immune cells in a population expressing the exogenous gene product. Associated cells, cell populations, compositions and methods of use are also provided.
1. A method for transducing a population of human cells with a retroviral vector, the vector comprising a nucleic acid exogenous to the cells, the method comprising:
culturing the population of human cells with the retroviral vector, in a cell culture media at a starting pH in a starting pH range of 7.0 to 7.4 and maintaining the starting pH in the starting pH range for at least a first hour of the transduction culturing step to result in a transduced cell population comprising cells expressing a gene product encoded by the exogenous nucleic acid, wherein the population of human cells are T lymphocytes, helper T cells, memory T cells, cytotoxic T cells, natural killer T cells, peripheral blood lymphocytes, peripheral blood mononuclear cells, dendritic cells, T cells derived from induced pluripotent stem cells, natural killer cells or mixtures thereof.
2. The method according to claim 1 , wherein the starting pH is maintained in the starting pH range for at least about 1, about 2, about 4, about 6, about 8, about 10, about 12, about 14, about 16, about 18, about 20, about 22, about 24, about 36, about 48, about 72 or about 96 hours to no more than about 168 hours.
3. The method according to claim 1 , wherein the starting pH is maintained above about 7.0 or is maintained in the starting pH range through the end of the transduction culturing step.
4. The method according to claim 3 , wherein the transduction culturing step is conducted for at least about 1, about 2, about 4, about 6, about 8, about 12, about 14, about 16, about 18, about 20, about 22, about 24, about 36, about 48, about 72 or about 96 hours.
5. The method according to claim 1 , wherein the cell culture media does not comprise a co-localization agent.
6. The method according to claim 1 , wherein the cell culture media does not comprise fibronectin or a fibronectin derivative.
7. The method according to claim 1 , wherein the cell population is cultured at a MOI of about 0.25, about 0.5, about 1, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about, or about 50 to about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 125, about 150, about 175, about 200, about 225, or about 250.
8. The method according to claim 1 , wherein the exogenous nucleic acid encodes a chimeric antigen receptor.
9. The method according to claim 1 , wherein the cell population is cultured in a vessel, wherein the vessel is a cell culture plate, cell culture deep well plate, a cell stack, a controlled bioreactor, a shake flask or a gas permeable bag.
10. The method according to claim 1 , wherein the retroviral vector is a lentiviral vector.
11. The method according to claim 1 , wherein the transduction culturing step comprises culturing the cell population and the retroviral vector in a volume of about 0.5 liters to a volume of about 10 liters of cell culture media.
12. The method according to claim 1 , wherein, at least about 35% to about 95%, about 40% to about 95%, about 50% to about 95%, about 60% to about 95%, or about 70% to about 95%, of the transduced cell population expresses the exogenous nucleic acid gene product about 3 to about 18 days after initiation of the transduction culturing step.
13. The method according to claim 1 , wherein, at least about 35% to about 95%, about 40% to about 95%, about 50% to about 95%, about 60% to about 95%, or about 70% to about 95%, of the transduced cell population expresses the exogenous nucleic acid gene product about 7 to about 18 days after initiation of the transduction culturing step.
14. The method according to claim 1 , wherein the starting pH range is maintained passively.
15. The method according to claim 1 , wherein the starting pH range is maintained actively.
16. The method according to claim 1 , wherein the cell population is an allogeneic cell population.
17. The method according to claim 1 , wherein the cell population is an autologous cell population.
18. A method for transducing a first and a second cell population, wherein the first and second cell population are transduced by the method of claim 1 , whereby the percent of the transduced cell population expressing the exogenous nucleic acid gene product in the first and second transduced cell populations does not vary more than about 2% to about 5%, about 5% to about 10%, about 10% to about 20%, or about 20% to about 30%.
19. The method according to claim 1 , wherein a polycation is not added to the cell culture media.
20. The method according to claim 1 , wherein polybrene, protamine sulfate or DEAE-dextran is not added to the culture media.
21. The method according to claim 1 , wherein fibronectin or a fibronectin derivative is not added to the cell culture media.
22. The method of claim 1 , wherein the starting pH is 7.1, 7.2, 7.3 or 7.4.
23. A method for transducing a population of human cytotoxic T cells or human peripheral blood mononuclear cells with a retroviral vector, the vector comprising a nucleic acid exogenous to the cytotoxic T cells, the method comprising culturing the human cytotoxic T cell population or the human peripheral blood mononuclear cell population with the retroviral vector, in a cell culture media at a starting pH in a starting pH range of 7.0 to 7.4 and maintaining the starting pH in the starting pH range for at least a first hour of the transduction culturing step to result in a transduced cytotoxic T cell population or a transduced peripheral blood mononuclear cell population comprising cells expressing the gene product encoded by the exogenous nucleic acid, wherein the cell culture media does not comprise a co-localization agent.
24. The method according to claim 23 , wherein the cell culture media does not comprise fibronectin or a fibronectin derivative.
25. The method of claim 23 , wherein the starting pH is maintained actively.
26. The method of claim 23 , wherein the starting pH is 7.1, 7.2, 7.3 or 7.4.
27. A method for transducing a population of human T cells derived from induced pluripotent stem cells with a retroviral vector, the vector comprising a nucleic acid exogenous to the derived T cells, the method comprising culturing the derived T cell population with the retroviral vector, in a cell culture media at a starting pH in a starting pH range of 7.0 to 7.4 and maintaining the starting pH in the starting pH range for at least a first hour of the transduction culturing step to result in a transduced derived T cell population comprising cells expressing the gene product encoded by the exogenous nucleic acid, wherein the cell culture media does not comprise a co-localization agent.
28. The method of claim 27 , wherein the starting pH is maintained actively.
29. The method of claim 27 , wherein the starting pH is 7.1, 7.2, 7.3 or 7.4.