A Customizable Platform to Integrate CAR and Conditional Expression of Immunotherapeutics in T Cells

The potential of chimeric antigen receptor (CAR)-based immunotherapy as a promising therapeutic approach is often hindered by the presence of highly immunosuppressive tumor microenvironments (TME). Combination therapies with either co-administration or built-in expression of additional TME-modulating therapeutic molecules to potentiate the functions of CAR-T cells can cause systemic toxicities due to the lack of control over the delivery of biologics. Here, we present a proof-of-concept engineered platform in human Jurkat T cells that combines CAR with a therapeutic gene circuit capable of sensing <i>β</i>-galactosidase (a reported cancer-associated signal) and subsequently activate the production of customized therapeutic gene products. We have demonstrated the integration of the chemically induced proximity (CIP) and associated signal sensing technologies with CAR in this study. A <i>β</i>-galactosidase-activatable prodrug was designed by conjugating a galactose moiety with a CIP inducer abscisic acid (ABA). We showed that Jurkat T cells engineered with CAR and the ABA-inducible genetic circuits can respond to recombinant <i>β</i>-galactosidase to drive the production and secretion of various immunotherapeutics including an anti-cancer agent, an immunomodulatory cytokine, and immune checkpoint inhibitors. Our design is highly modular and could be adapted to sense different cancer-related signals to locally produce antitumor therapeutics that can potentially boost CAR-T efficacy and persistence.