Method for enhancing production of genetically engineered autologous T cells

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.