(747b) Covert Cancer Therapeutics: Engineering T Cells to Interrogate Intracellular Tumor Antigens

Adoptive T-cell therapy has shown tantalizing promise as a cancer treatment strategy, with several clinical trials reporting that T cells expressing chimeric antigen receptors (CARs) can eradicate tumors in patients with relapsed disease. However, CAR-T cells rely on receptor-mediated recognition of surface-bound antigens that are seldom tumor-exclusive, resulting in severe on-target, off-tumor toxicities that have led to patient deaths in clinical trials. There is a growing consensus that the lack of suitable antigens poses a major obstacle to the broad application of engineered tumor-targeting T cells. The ability to overcome T cells’ reliance on surface antigen presentation and interrogate intracellular disease signatures would significantly expand the pool of detectable tumor markers and improve tumor-targeting specificity. Here, we present a novel strategy to reprogram T-cell–mediated cytotoxicity to interrogate intracellular disease signatures. We have engineered a switchable form of the cytotoxic protein Granzyme B (GrB) that is produced and delivered by T cells into target cells, but becomes active if and only if a tumor-associated protease is present inside the target cell. As a proof of concept, we have developed a GrB switch that responds to Sentrin-specific protease 1 (SENP1), an oncoprotein known to be overexpressed in prostate, pancreatic, and thyroid oncocytic tumor cells. We demonstrate that this GrB switch, termed cytoplasmic oncoprotein verification evaluator and response trigger (COVERT), is efficiently expressed and packaged by human T cells and properly trafficked to the immunological synapse between T cells and target cells. Furthermore, we show that COVERT is produced as an enzymatically inert protein that is activated by SENP1 in a dose-dependent manner. Finally, we describe designs to adapt COVERT into a modular platform technology that will expand the repertoire of candidate target antigens. We envision that COVERT can be utilized in combination with existing CAR technology to improve the tumor-targeting precision of cell-based immunotherapy.