Caffeine-regulated molecular switches for functional control of CAR T cells in vivo

Abstract The limited controllability of CAR T cells in patients represents a key challenge of this highly potent immunotherapy. A molecular ON-switch, which can be regulated with a non-toxic and readily available small molecule drug, would represent a major advance towards controllable CAR T therapeutics. For that purpose, we engineered caffeine-responsive heterodimeric ON-switches (CaffSwitches) and demonstrate their high caffeine-dependency and virtually absent leakiness. When incorporating these CaffSwitches into CARs, the resulting CaffCARs were efficiently activated by caffeine concentrations achieved in human plasma after drinking one cup of coffee. Moreover, CaffCAR T cells also showed efficient tumor clearance in an in vivo mouse model, which was completely abolished in the absence of caffeine. This tight control was even observed with c-Jun overexpressing CaffCAR T cells, despite their massive expansion. Together, we anticipate that these novel CaffSwitches will be valuable tools for the development of safe and efficient next generation CAR T cells. Competing Interest Statement M.L. and M.W.T. receive funding from Miltenyi Biotec. E.S, B.S., C.U.Z., M.L. and M.W.T. have filed a patent application related to this work. J.Z. is a founder, shareholder, and scientific adviser of Quantro Therapeutics. The Zuber Lab receives research support and funding from Boehringer Ingelheim. A.M.-L., F.E. and B.E. are full-time employees of Miltenyi Biotec. J.M. was employee of Miltenyi Biotec at the time of this study. The remaining authors declare no competing interests.