Auto‐inducible expression of chimeric antigen receptor T cells using the <scp>NR4A1</scp> promoter

Chimeric antigen receptor (CAR) T cell therapies have shown remarkable efficacy in hematological malignancies, yet translation to solid tumors has been hindered by immunosuppressive tumor microenvironments, reduced T cell persistence and on-target/off-tumor toxicities. Constitutive CAR expression, typically driven by strong promoters such as EF1α, promotes tonic signaling, receptor clustering and antigen-independent activation, contributing to T cell exhaustion and adverse events. Inducible promoter systems have been proposed to improve control over CAR expression. NR4A1, a transcription factor (TF) activated during early T cell receptor (TCR) signaling, governs pathways central to T cell activation and dysfunction, making its promoter an attractive candidate for conditional CAR regulation. We compared constitutive (EF1α), synthetic inducible (6NFAT-NFκB and 2NFAT-2NurRE) and NR4A1 promoters to drive expression of a second-generation FRP5-CAR. NR4A1-driven CARs demonstrated low basal expression that was rapidly induced upon antigen encounter, reaching levels equivalent to EF1α-driven CARs while showing minimal antigen-independent signaling. Functionally, NR4A1-driven CARs mediated potent tumor lysis, preserved a less exhausted (PD-1^low and TIM-3^low) and more memory-like phenotype (CD62L^high and CD45RA^high), and sustained robust antitumor responses in vitro and in vivo. These findings establish the NR4A1 promoter as a native, activation-inducible system to fine-tune CAR expression, while maintaining therapeutic efficacy comparable to constitutively expressed CAR T cells. This strategy provides a promising framework for advancing CAR T cell therapies against solid tumors.