Ligand-Induced Degradation of a CAR Permits Reversible Remote Control of CAR T Cell Activity In Vitro and In Vivo

Abstract

Chimeric antigen receptor (CAR)-modified Tcells are endowed with novel antigen specificity and are most often administered to patients without an engineered mechanism to control the CAR Tcells once infused. "Suicide switches" such as the small molecule-controlled, inducible caspase-9 (iCas9) system afford the ability to selectively eliminate engineered Tcells; however, these approaches are designed for all-or-none, irreversible termination of an ongoing immune response. In order to permit reversible and adjustable modulation, we have created a CAR that is capable of on-demand downregulation by fusing the CAR to a previously developed ligand-induced degradation (LID) domain. Addition of a small molecule ligand triggers exposure of a cryptic degron within the LID domain, resulting in proteasomal degradation of the CAR-LID fusion protein andloss of CAR on the surface of Tcells. This fusion construct allowed for reversible and "tunable" inhibition of CAR Tcell activity invitro. Delivery of the triggering molecule in CAR-LID-treated tumor-bearing mice temporarily reduced CAR activity through modulation of CAR surface expression. The ability to more flexibly modulate CAR Tcell expression through a small molecule provides a platform for controlling possible adverse side effects, as well as preclinical investigations of CAR Tcell biology.