Effect of Cryopreservation on Autologous Chimeric Antigen Receptor T Cell Characteristics

As clinical applications for chimeric antigen receptor T cell (CART) therapy extend beyond early phase trials, commercial manufacture incorporating preservation steps becomes a logistical necessity. The effect of cryopreservation on CART characteristics is unclear. We retrospectively evaluated the effect of cryopreservation on product release criteria and in vivo characteristics in 158 autologous CART products from 6 single-center clinical trials. Further, from 3 healthy donor manufacturing runs, we prospectively identified differentially expressed cell surface markers and gene signatures among fresh versus cryopreserved CARTs. Within 2 days of culture initiation, cell viability of the starting fraction (peripheral blood mononuclear cells [PBMNCs]) decreased significantly in the cryo-thawed arm compared to the fresh arm. Despite this, PBMNC cryopreservation did not affect final CART fold expansion, transduction efficiency, CD3%, or CD4:CD8 ratios. In vivo CART persistence and clinical responses did not differ among fresh and cryopreserved final products. In healthy donors, compared to fresh CARTs, early apoptotic cell-surface markers were significantly elevated in cryo-thawed CARTs. Cryo-thawed CARTs also demonstrated significantly elevated expression of mitochondrial dysfunction, apoptosis signaling, and cell cycle damage pathways. Cryopreservation during CART manufacture is a viable strategy, based on standard product release parameters. The clinical impact of cryopreservationrelated subtle micro-cellular damage needs further study To study T cell phenotypic changes and gene expression prospectively, 3 healthy donor PBMNCs were used to manufacture CARTs using 4 different manufacturing schemes: (1) starting manufacturing with fresh PBMNCs and analysis of fresh CARTs, (2) starting with fresh PBMNCs and analysis of cryopreserved CARTs, (3) starting with cryopreserved PBMNCs and analysis of fresh CARTs, and (4) starting with cryopreserved PBMNCs and analysis of cryopreserved CARTs.