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.