Chimeric antigen receptor (CAR) T cells typically use a strong constitutive promoter to ensure maximal long-term CAR expression. However, recent evidence suggests that restricting the timing and magnitude of CAR expression is functionally beneficial, whereas constitutive CAR activation may lead to exhaustion and loss of function. We created a self-driving CD19-targeting CAR, which regulates its own function based on the presence of CD19 antigen engaged by the CAR itself, by placing self-driving CAR19 constructs under transcriptional control of synthetic AP1-NFκB or STAT5 promoters. CD19 antigen-regulated expression was observed for self-driving AP1-NFκB-CAR19, with CAR19 upregulation within 18 hours after exposure to target CD19, and corresponded to the level of tumor burden. Self-driving CAR-T cells showed enhanced tumor-dependent activation, expansion, and low exhaustion in vitro as compared to constitutively-expressed EF1α and MSCV CARs, and mediated tumor regression and survival in Raji-bearing NSG mice. Long-term CAR function correlated with upregulated CAR expression within 24 hours of exposure to tumor antigen. The self-driving AP1-NFκB-CAR19 circuit was also used to inducibly express dominant-negative TGFβRII, which effectively countered the negative effects of TGFβ on CAR-T activation. Thus, a self-driving CAR approach may offer a new modality to express CAR and auxiliary proteins, by enhancing CAR-T functional activity and limiting exhaustion.
Copyright © 2021. Published by Elsevier Inc.

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