Degron-Based bioPROTACs for Controlling Signaling in CAR T Cells

Chimeric antigen receptor (CAR) T cells have made a tremendous
impact in the clinic, but potent signaling through the CAR can be
detrimental to treatment safety and efficacy. The use of protein degradation
to control CAR signaling can address these issues in preclinical models.
Existing strategies for regulating CAR stability rely on small molecules
to induce systemic degradation. In contrast to small molecule regulation,
genetic circuits offer a more precise method to control CAR signaling
in an autonomous cell-by-cell fashion. Here, we describe a programmable
protein degradation tool that adopts the framework of bioPROTACs,
heterobifunctional proteins that are composed of a target recognition
domain fused to a domain that recruits the endogenous ubiquitin proteasome
system. We develop novel bioPROTACs that utilize a compact four-residue
degron and demonstrate degradation of cytosolic and membrane protein
targets using either a nanobody or synthetic leucine zipper as a protein
binder. Our bioPROTACs exhibit potent degradation of CARs and can
inhibit CAR signaling in primary human T cells. We demonstrate the
utility of our bioPROTACs by constructing a genetic circuit to degrade
the tyrosine kinase ZAP70 in response to recognition of a specific
membrane-bound antigen. This circuit can disrupt CAR T cell signaling
only in the presence of a specific cell population. These results
suggest that bioPROTACs are powerful tools for expanding the CAR
T cell engineering toolbox.