Programming T cells for Early Cancer Detection with Customized Protease-Activatable Receptors

Abstract Early cancer detection has the potential to reduce cancer mortality, yet endogenous tumor-shed biomarkers lack sensitivity for early-stage disease. We report OncoSCOUT, a cancer detection strategy using T cells engineered with protease-activatable receptors (PARs) that conditionally recognize tumor cells and release a synthetic biomarker for detection in urine. These PARs comprise masked synthetic Notch receptors in which antigen binding is blocked by a peptide mimotope tethered via a protease-cleavable linker. We demonstrate that requiring both extracellular protease activity and tumor antigen recognition improves spatial specificity and minimizes off-tumor activation of PAR T cells in vivo . To identify tumor-selective PARs, we adoptively transferred a HER2-targeted PAR library displaying ∼160,000 unique 4-mer amino acid linkers and discovered multiple variants significantly enriched in a HER2-positive cancer xenograft model. Using a single customized PAR, we show that OncoSCOUT can detect total tumor burdens as small as 10–30 mm 3 with significantly improved sensitivity than the protein biomarker CA 15-3 or a 20-plex circulating tumor DNA (ctDNA) assay.