Method for enhancing production of genetically engineered autologous T cells
The present invention relates to a method for enhancing production of autologous genetically engineered T cells for use in cell therapy applications.
1. A method for producing genetically engineered autologous T cells that have reduced background activation and target irrelevant toxicity, the method comprising
a) adding culture media comprising about 300 IU per ml IL-2 to a closed single use bioreactor bag;
b) inoculating the bioreactor bag with apheresed donor cells and one or more soluble T cell activators, wherein at least one soluble T-cell activator is bound to at least one donor cell at the time of inoculation and the bioreactor bag is part of a rocking bioreactor platform;
c) culturing the apheresed donor cells in the closed single use bioreactor bag containing culture media comprising about 300 IU per ml IL-2 and continuously rocking the bag at a rate of 2 rpm at a 2° angle;
d) transducing the cells in the bioreactor bag with at least one soluble viral vector comprising a polynucleotide which encodes a protein of interest, the bioreactor bag containing culture media comprising about 300 IU per ml IL-2 and continuously rocking the bioreactor bag at a rate of 2 rpm at a 2° angle; and
e) expanding the transduced cells in the bioreactor bag by feeding the transduced cells in the bioreactor bag with culture media comprising about 300 IU per ml IL-2 until Day 5 or Day 6 of the culture, after which the culture is fed with culture media comprising no IL-2, increasing the culture volume and the rocking rate as needed to maintain the culture until harvest,
wherein harvested genetically engineered autologous T cells have reduced background activation and target irrelevant toxicity compared to engineered autologous T cells expressing the same protein of interest from the same apheresed donor cells produced by the same method in which IL-2 is maintained in the culture media at a concentration of about 300 IU per ml until harvest.
2. The method of claim 1 , wherein the apheresed donor cells comprise cells from peripheral blood.
3. The method of claim 2 , wherein the apheresed donor cells comprise nucleated and non-nucleated cells.
4. The method of claim 1 , wherein the apheresed donor cells comprise leukocytes and erythrocytes.
5. The method of claim 4 , wherein the apheresed donor cells also comprise granulocytes and/or platelets.
6. The method of claim 1 , wherein the apheresis is leukapheresis.
7. The method of claim 1 , wherein the apheresed donor cells are washed and resuspended in a culture media.
8. The method of claim 1 , wherein at least one T cell activator is an anti CD3 antibody or binding fragments thereof.
9. The method of claim 1 , wherein the T cell activator comprises an anti CD3 antibody and an anti CD28 antibody, or binding fragments thereof.
10. The method of claim 1 , wherein the T cell activator comprises at least an anti CD3 antibody, an anti CD28 antibody, and an anti CD2 antibody, or binding fragments thereof.
11. The method of claim 1 , wherein the T cell activator comprises at least an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex, and an anti-human CD2 monospecific tetrameric antibody complex.
12. The method of claim 1 , wherein the concentration of at least one soluble T cell activator is at least 0.001 μg/ml to at least 10 μg/ml.
13. The method of claim 12 , wherein the concentration of at least one soluble T cell activator is at least 0.1 μg/ml to at least 5 μg/ml.
14. The method of claim 1 , wherein the number of nucleated cells within the apheresed donor cells is about 1.0E9 to about 1.4E9.
15. The method of claim 14 , wherein the number of nucleated cells within the apheresed donor cells is about 1.2E9 to about 1.4E9.
16. The method according to claim 1 , wherein the bioreactor bag contains at least 300 ml of culture media at inoculation.
17. The method according to claim 1 , wherein the apheresed donor cells are cultured in the bioreactor bag for about 12-24 hours in part c).
18. The method of claim 1 , wherein the viral vector is a retroviral vector.
19. The method of claim 1 , wherein the viral vector is a lentiviral vector.
20. The method of claim 19 , wherein the lentiviral vector is added at a MOI of 0.25-10.
21. The method of claim 19 , wherein the lentiviral vector is added at a MOI of 1.
22. The method of claim 1 , wherein the cells are transduced for about 20-24 hours.
23. The method of claim 1 , following transduction half of the culture media is removed from the bioreactor bag and replaced with an equal volume of fresh culture media.
24. The method of claim 1 , wherein during expansion the culture is fed by fed batch and/or perfusion.
25. The method of claim 24 , wherein during expansion the culture is perfused at a rate is one half to double the bioreactor bag volume per day.
26. The method of claim 1 , wherein during expansion the volume of the culture media in bioreactor is incrementally increased to 500 ml to maintain a cell density of at least 4E6 nucleated cells/ml.
27. The method of claim 1 , wherein at harvest the volume of culture media in the single use closed bioreactor bag is 500 ml rocking at a rate of 4 rpm at a 4° angle to 6 rpm at a 6° angle.
28. The method according to claim 1 , wherein the expansion begins at a culture volume of about 300 ml of culture media at a rocking rate of about 2 rpm at a 2° angle until the cells reach a predetermined cell density, the cell expansion is continued at a second culture volume of about 400 ml of culture media at a rocking rate of about 5 rpm at a 5° angle until the cells reach a predetermined cell density, and the cell expansion is continued at a third culture volume of about 500 ml at a rocking rate of about 6 rpm at a 6° angle until the cells reach a predetermined cell density after which the culture is maintained until harvest.
29. The method according to claim 28 , wherein the predetermined cell density is 4E6 cells/ml.
30. The method of claim 1 , wherein the cells are expanded for until they reach a target harvest cell density.
31. The method of claim 30 , wherein the target harvest cell density is about 10 billion cells.
32. The method of claim 1 , wherein the culture, transduction, and/or expansion steps are performed at 34-37° C.
33. The method of claim 1 , wherein the protein of interest is a cell surface receptor.
34. The method of claim 33 , wherein the cell surface receptor a T cell receptor, or chimeric antigen receptor.
35. The method of claim 33 , wherein the cell surface receptor recognizes an antigenic target associated with a target cell.
36. The method of claim 35 , wherein the target cell is a cancer cell.
37. The method of claim 1 , wherein the genetically engineered autologous T cells are used to treat an indication in a patient in need.