Highlights d A dual oxygen-sensing switch provides stringent hypoxiadependent regulation of a CAR d HypoxiCAR T cells deliver tumor-selective CAR expression and anti-tumor efficacy d HypoxiCAR T cells prevent on-target, off-tumor activation and cytokine release syndrome d HypoxiCAR provides a strategy to expand the CAR repertoire for solid malignancies

There has been significant interest in the prospects of chimeric antigen receptor (CAR) T-cell therapy in the treatment of solid malignancies, and multiple clinical trials are in progress1. However, the scope of these trials has been restricted by the lack of availability of tumorspecific targets to direct CAR binding. Tumor specificity is crucial as on-target off-tumor activation of CAR T-cells in healthy tissues can result in potentially lethal toxicities due to uncontrolled cytokine release syndrome2. Here we engineer a stringent hypoxia-sensing CAR T-cell system which achieves selective expression of a pan-ErbB-targeted CAR within a solid tumor, a microenvironment characterized by an inadequate oxygen supply. Using murine xenograft models, we demonstrate that despite widespread expression of ErbB receptors in healthy organs, the approach provides anti-tumor efficacy without off-tumor toxicity. This dynamic on/off oxygen-sensing safety switch has the potential to facilitate the unlimited expansion of the CAR T-cell target repertoire for treating solid malignancies.

Summary Exploiting hypoxia in solid malignancies to restrict expression of chimeric antigen receptors (CARs) on engineered T cells to the tumor microenvironment overcomes the risk of on-target off-tumor toxicity and minimizes tonic signaling, which promotes CAR T cell exhaustion. This protocol summarizes the synthetic biology underlying the development of a stringent oxygen-sensitive CAR for in vitro and in vivo preclinical characterization. For complete details on the use and execution of this protocol, please refer to Kosti et al. (2021) .