Enriching leukapheresis improves T cell activation and transduction efficiency during CAR T processing

Abstract The majority of CD19 directed CAR T-cell products are manufactured using an autologous process. Although using patient’s apheresis reduces risks of rejection, it introduces variability in starting material composition and the presence of cell populations that might negatively affect the production CAR T-cells, such as myeloid cells. In this work the impact of monocytes (CD14) on the level of activation, growth and transduction efficiency was monitored across well-plate and culture bag platforms using healthy donor apheresis. Removal of monocytes from leukapheresis improved the level of activation 2-fold, achieving the same level of activation as when initiating the process with a purified T-cell starting material. Two activation reagents were tested in well-plate cultures, revealing differing sensitivities to starting material composition. Monocyte depletion in culture bag systems had a significant impact on transduction efficiency, improving consistency and increasing the level of CAR expression by up to 64% compared to leukapheresis. Cytotoxicity assays revealed that CAR T-cell products produced from donor material depleted of monocytes and isolated T-cells consistently outperformed those made from unsorted leukapheresis. Analysis of memory phenotypes and gene expression indicated that CAR T-cells produced using depleted starting material displayed a more rested and naive state.