Redirecting T-Cell Specificity to EGFR Using mRNA to Self-limit Expression of Chimeric Antigen Receptor.

Abstract Potential for on-target, but off-tissue toxicity limits therapeutic application of genetically modified T cellsconstitutively expressing chimeric antigen receptors(CARs) from tumor-associated antigens expressed in normal tissue, such as epidermal growth factor receptor (EGFR). Curtailing expression of CAR through modification of T cellsby in vitro-transcribed mRNA species is one strategy to mitigate such toxicity. We evaluated expression of an EGFR-specific CAR coded from introduced mRNA in human T cellsnumerically expanded ex vivo to clinically significant numbers through coculture with activating and propagating cells (AaPC) derived from K562 preloaded with anti-CD3 antibody. The density of AaPC could be adjusted to affect phenotype of T cellssuch that reduced ratio of AaPC resulted in higher proportion of CD8 and central memory T cellsthat were more conducive to electrotransfer of mRNA than T cellsexpanded with high ratios of AaPC. RNA-modified CAR T cellsproduced less cytokine, but demonstrated similar cytolytic capacity as DNA-modified CAR T cellsin response to EGFR-expressing glioblastoma cells. Expression of CAR by mRNA transfer was transient and accelerated by stimulation with cytokine and antigen. Loss of CAR abrogated T-cellfunction in response to tumor and normal cells expressing EGFR. We describe a clinically applicable method to propagate and modify T cellsto transiently express EGFR-specific CAR to target EGFR-expressing tumor cells that may be used to limit on-target, off-tissue toxicity to normal tissue.