Rihtar, E.; Fink, T.; Belak, M.; Udvanc, R.; Jerala, R.

Regulation of therapeutic cell response is important for safe and effective therapy, particularly for immunotherapy. Ideally, the regulators should be based on human proteins using compounds that have already been approved for human use. Regulation of protein degradation by small molecules enables fast cellular response and a small genetic footprint of genetic constructs. Here, we present a clinically compatible strategy for reversible pharmacological control of chimeric antigen receptor (CAR) T cell function using human estrogen receptor (ER)-based degron domains and FDA-approved small molecules. By fusing tamoxifen-responsive ER ligand-binding domains to CARs, we generate ligand-inducible ON-switch CARs whose stability, signaling, and effector functions are precisely controlled by 4-hydroxytamoxifen. Furthermore, we demonstrate that ER-tagged CARs can be selectively degraded using the FDA-approved ER-targeting PROTAC ARV-471, establishing a complementary OFF-switch mechanism that suppresses CAR expression and effector function. Notably, these results expand the bioorthogonal ON-OFF switch platform for post-translational control of therapeutic proteins, offering new opportunities to improve the safety and precision of cellular immunotherapies.