ZAP327 signaling domain–driven chimeric antigen receptor generates robust and long-term antitumor immunity in mouse models

Chimeric antigen receptor (CAR) T cell therapy has shown impressive clinical responses in the treatment of blood cancers, but high percentages of disease relapse 1 year after T cell infusion and severe toxicities associated with CAR T cell therapy remain major issues. Here, we report the construction of CARs with a ZAP70-derived signaling domain (ZAP327) that enhances therapeutic antitumor activity with increasing in vivo T cell persistence. ZAP327-driven CAR T cells reduced cytokine release and expression of T cell exhaustion markers but maintained similar or better cytolytic activity against tumor cells compared with conventional CAR T cells. The costimulatory domains, such as CD28 and 4-1BB, in the ZAP327 CAR constructs are required for providing a spacer between the transmembrane domain and ZAP327 kinase domain for optimal kinase structure folding and function, as well as costimulatory signaling. Furthermore, ZAP327-driven CAR T cells outperform conventional and several recently improved CAR T cells in therapeutic antitumor immunity, particularly in an antigen-low expression tumor model, which is clinically relevant and important for immune escape and disease relapse. Mechanistically, we show that the ZAP327 domain tuned down TCR signaling, increased the pools of stem-like memory T cells, and exhibited metabolic features associated with memory T cells by using the oxidative phosphorylation pathway. These results highlight the therapeutic potential of ZAP327-driven CAR T cells to overcome the limitations of the current CAR T cell therapies and enhance the potency and persistence of antitumor T cell responses in solid tumors as well.