Abstract
Genetically modified, induced pluripotent stem cells (iPSCs) offer a promising allogeneic source for the generation of functionally enhanced, chimeric antigen receptor (CAR) T cells. However, the signaling of CARs during early T cell development and the removal of the endogenous T cell receptor required to prevent alloreactivity pose significant challenges to the production of mature conventional CAR T cells from iPSCs. Here, we show that TCR-null, CD8αβ CAR T cells can be efficiently generated from iPSCs by engineering stage-specific onset of CAR expression and signaling to both permit conventional T cell development and to induce efficient positive selection. CAR T cells produced using this approach displayed a uniform, naïve T cell phenotype and demonstrated superior antigen-specific cytotoxicity compared to iPSC-derived effector memory CAR T cells. Multimodal sequencing revealed CAR-mediated positive selection induced the persistent upregulation of key transcription factors involved in naïve T cell development. Achieving precise control of CAR expression and signaling in developmentally sensitive T precursors will be critical to realizing the full potential for "off-the-shelf", iPSC-derived cellular therapies.