cMET CAR T-cell Manufacturing and Structure
WAVE
STERILITY
Autologous peripheral blood lymphocytes were obtained via leukapheresis collection at the Apheresis Unit at the HUP, then transferred to the CVPF at the University of Pennsylvania (Philadelphia, PA) for manufacturing. The apheresis product was processed with the CaridianBCT Elutra, a closed system which utilizes counter-flow centrifugal force to separate cell populations based on size and density, to enrich the lymphocyte population. The lymphocytes collected were then washed using the Haemonetics Cell Saver 5 system and resuspended in modified XVIVO-15 media (Lonza). Lymphocytes were stimulated with magnetic Dynabeads conjugated with mouse anti-human CD3 and CD28 (Gibco) and cultured in static tissue culture flasks. On day 5 of culture, cells were transferred to the WAVE Bioreactor. On the final day of the culture (day 9), the magnetic beads were removed (Baxter MaxSep) and the cells were concentrated using the Cell Saver 5. The next day, cells were washed and resuspended in Electroporation Buffer and loaded into the Maxcyte GT Flow Transfection System. Cells were electroporated with the cMet RNA, and allowed to recover for 4 hours and then formulated in infusible cryopreservation media. The cells were not released from the CVPF until FDA-specified release criteria (e.g., cell purity, sterility, potency, pyrogenicity, etc.) were verified.The final cMET CAR T-cell product thus consists of autologous T cells that have been expanded 