Abstract B083: Novel mRNA manufacturing techniques using modification of nucleosides and purification improves the efficacy of mRNA chimeric antigen receptor T cells

Abstract

Abstract Chimeric Antigen Receptor (CAR) T-cells made with mRNA offer a transient and safe alternative to viral CARs, mitigating the concern for persistent unwanted side effects from constitutively active T-cells. Previous studies have shown that mRNA CARs are transiently effective, but lack CAR persistence and have struggled to show success across tumor types. We hypothesized the efficacy of mRNA CARs could be improved by utilizing recent advancements in RNA technology including the use of modified nucleosides and novel purification methods of the RNA to prevent double stranding that induces toxicity. Using the established CD19 CAR model in B-cell leukemia, we created mRNA CARs using previously described methods and compared them to mRNA CARs created using the modifications of pseudo-uridine or N-methyl pseudo-uridine instead of uridine, and purification to remove any aberrant double stranded mRNA. Comparing the modified and purified mRNA CAR T cells to those created using prior methods of making mRNA, the novel mRNA CAR T cells showed a two-fold increase in expression of the CAR on their surface initially, as well as a two-fold improvement in cytotoxic killing of leukemia cells that persisted for up to five days. The modified and purified mRNA CAR T cells also showed reduced expression of negative checkpoint regulators compared to original RNA CAR T cells. In vivo studies using a leukemia mouse model with a single dose of CAR T cells also showed marked improvement using modified and purified mRNA CAR T cells, with purified RNA showing the most robust 2-log enhanced suppression of leukemic burden. Our results provide a novel technique that can be easily incorporated into RNA production for use in clinical trials, and poise RNA CARs for increased efficacy as new CAR targets emerge and are being tested. Citation Format: Jessica Foster, Namrata Choudhari, David Barrett, Adam Resnick. Novel mRNA manufacturing techniques using modification of nucleosides and purification improves the efficacy of mRNA chimeric antigen receptor T cells [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B083.