Hypoxia-Responsive CAR-T Cells Exhibit Reduced Exhaustion and Enhanced Efficacy in Solid Tumors

Engineering CAR-T cells to upregulate CAR expression under hypoxic conditions induces metabolic reprogramming, reduces differentiation, and increases proliferation to enhance their antitumor activity, providing a strategy to improve efficacy and safety. Expanding the utility of chimeric antigen receptor (CAR)-T cells in solid tumors requires improving their efficacy and safety. Hypoxia is a feature of most solid tumors that could be used to help CAR-T cells discriminate tumors from normal tissues. In this study, we developed hypoxia-responsive CAR-T cells by engineering the CAR to be under regulation of hypoxia-responsive elements and selected the optimal structure (5H1P-CEA CAR), which can be activated in the tumor hypoxic microenvironment to induce CAR-T cells with high polyfunctionality. Hypoxia-responsive CAR T cells were in a "resting" state with low CAR expression under normoxic conditions. Compared with conventional CAR-T cells, hypoxia-responsive CAR-T cells maintained lower differentiation and displayed enhanced oxidative metabolism and proliferation during cultivation, and they sowed a capacity to alleviate the negative effects of hypoxia on T-cell proliferation and metabolism. Furthermore, 5H1P-CEA CAR-T cells exhibited decreased T-cell exhaustion and improved T-cell phenotype in vivo. In patient-derived xenograft models, hypoxia-responsive CAR-T cells induced more durable antitumor activity than their conventional counterparts. Overall, this study provides an approach to limit CAR expression to the hypoxic tumor microenvironment that could help to enhance CAR T-cell efficacy and safety in solid tumors.Significance: Engineering CAR-T cells to upregulate CAR expression under hypoxic conditions induces metabolic reprogramming, reduces differentiation, and increases proliferation to enhance their antitumor activity, providing a strategy to improve efficacy and safety.