Protease-activatable receptors (PARs) engineered in T cells enable early cancer detection through dual-gated activation requiring both tumor-associated protease activity and antigen recognition, which reduces off-tumor activation by >75% in healthy tissues while driving secretion of synthetic urinary biomarkers that detect tumors as small as 10-30 mm³ with significantly improved sensitivity (AUC=0.96) compared to FDA-approved protein biomarkers (AUC=0.57) and circulating tumor DNA assays (AUC=0.67).

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 mm3 with significantly improved sensitivity than the protein biomarker CA 15-3 or a 20-plex circulating tumor DNA (ctDNA) assay.