Project Summary Chimeric Antigen Receptor (CAR)-T cell therapy has had unprecedented success in patients with B- cell malignancies, demonstrating upwards of 100% complete remission rate in some cases. Nearly all these patients, however, subsequently relapse with therapy-resistant tumors caused by antigen escape. In addition, the widespread utility of CAR-T cell therapy has been met with additional safety and efficacy challenges, including on-target/off-tumor toxicity, cytokine release syndrome caused from hyperactive CAR- T cells, and T cell exhaustion. A precise, well-balanced, and controlled CAR-T cell response is required to navigate these obstacles, avoid non-cancerous bystander tissues, limit dangerous side effects, and still identify and destroy tumor cells. New therapeutic approaches that permit the user-controlled, fine-tuned regulation of cell-autonomous CAR-T cell activity are needed to tune the CAR-T cell response to maintain this balance. To address these needs, and through the experiments outlined in this proposal, I will create a system for programmable and drug-controllable antigen-dependent cellular response coined PAGER (Programmable Adhesion GPCR-based, Exogenously Regulated). To further meet these needs, I will also add drug controllability to existing CARs. To create PAGER, I will engineer an adhesion G Protein-Coupled Receptor (GPCR) so that its structural rearrangements upon activation are coupled to the activity of a fused protease, allowing it to release a transcription factor upon receptor activation. Furthermore, to add drug- controllability to both PAGER and existing CARs, I will add a druggable protease-dependent degradation signal to synthetic receptors to create “drug-on” tunable PAGERs and CARs. I will utilize high-throughput screening, protein engineering, and directed evolution to accomplish these goals. Application of drug-controlled PAGERs and CARs in CAR-T cells will permit fine-tuned regulation of CAR-T cell activity to limit or prevent unwanted CAR-T cell responses. I will demonstrate proof-of-concept for using these synthetic receptors to precisely control customized CAR-T cell behaviors to an unprecedented resolution. PAGER will be used to control CAR expression and activity, cytokine secretion profile, and local delivery of therapeutic antibodies in a user-controlled cell-autonomous manner. The wide spectrum of cellular responses that can be programmed using PAGER highlights its great potential to assist in overcoming many of the safety and efficacy challenges that currently face CAR-T cell therapy.