Automated Rapid CAR-T Cell Manufacturing Process, Starting from Whole Blood, on a Novel Closed Platform

Background: Autologous CAR-T (Chimeric Antigen Receptor T) cell therapy has been a great success in treating hematological cancers and making progress in treating autoimmune diseases as well as solid tumors. However, the high manufacturing cost and complicated logistics have prevented the therapy from being accessible by general patients. Recently, there are discoveries and developments in the manufacturing process to reduce cost, including reduction of manufacturing time from 7-14 days to one day by elimination of in vitro cell expansion. Researchers have learned that the “rapid” manufacturing process maintains stemness of the CAR-T cells that achieved a higher potency after infusion. Additionally, changing the cell source from leukapheresis products to peripheral whole blood also helps reduce processing time and overall cost. Finally, having CAR-T cells manufactured at “point-of-care” facilities that are closer to patients will greatly reduce logistical overhead. An automated closed platform that achieves: (1) “point-of-care” manufacturing, (2) starting from whole blood, and (3) completes CAR-T cell manufacturing within 1~2 days would be ideal to reduce the CAR-T therapy cost to 10% of its current cost, and potentially further to 1% when becoming a generally practiced method with volume.

Method: We have developed a novel cell manufacturing platform (MARS Atlas), which integrates T cell isolation, viral transduction and cell washing/formulation processes on one platform. We demonstrated the CAR-T manufacturing process starting from whole blood can be completed within 24 hours. Using healthy donor’s peripheral blood, we isolated T cells with CD4 and CD8 directly conjugated magnetic beads. The T cells were then activated and transduced with lentiviral vector containing CAR-19 gene. After overnight incubation, product cells were washed and harvested in either the medium that is suitable for injection or medium for cryopreservation. As a comparison, we also performed a standard 7-day expansion of transduced T cells in culture.

Results: CAR-T cell count, CAR-T cell phenotype, and CAR-T cell cytotoxic activity were monitored and compared of the 24-hour process and 7-day process. In the rapid process we harvested an average of 20million T cells after 24 hours from 30mL of blood with purity >94%. The product T cells have twice more naïve population (CD45RA+CCR7+) than the T cells harvested after 7- day process. Transduction efficiency was 60%-70% (CAR expression was stable after day 2). The function of CAR-T cells manufactured in 24 hours were verified by in vitro killing assay. By repeated stimulation with target cells CAR-T cells was observed to kill target cells in day 7.

Conclusion: In this study, we demonstrated an automated 24-hour LV-based CAR-T cell manufacturing workflow produced functioning CAR-T cells and this process has great potential for high potency and extreme low-cost CAR-T therapy.