PROJECT SUMMARY Some types of cancers, as exemplified by estrogen receptor positive breast cancer, can recur as metastatic disease many years or even decades following a dormancy period where the patient displays no clinical symptoms. Late recurrence is thought to arise from disseminated tumor cells (DTCs) that were not killed by initial treatment and that lie dormant at metastatic sites such as the bone marrow until they reawaken. Strikingly, meta- analysis of over 60,000 early-stage ER-positive breast cancer patients treated with endocrine therapy revealed that the relative risk of recurrence progressively increases over a period of at least 20 years, indicating that patients in complete remission with no evidence of disease could harbor dormant cancer and remain at risk of metastatic relapse for the remainder of their life. Currently, there is no widely used method to monitor the dormant state nor its reawakening. The arrival of cancer immunotherapy has revealed exciting possibilities using engineered T cells as living medicines. T cells designed with tumor-targeting receptors and sophisticated genetic circuits have led to striking treatment responses in patients with certain types of cancers that were previously untreatable. This moment is an opportunity to not only build a future where T cells are engineered as therapies, but also as living sensors that can detect cancer with sensitivities and specificities beyond what is currently possible. The activation and growth of dormant tumor cells into micro-metastases require the hallmark expression of proteases during key steps such as angiogenesis. To exploit protease dysregulation, this project will develop engineered receptors that sense proteolysis to detect reawakening. These contain an extracellular single chain antibody that is blocked by a peptide mimotope such that they can bind to their cognate antigens only after the blocking peptide is removed by proteolysis, restricting T cell activation to the specific condition where the cognate protease and tumor antigen are both present. Following activation, T cell sensors amplify the release of synthetic biomarkers (blood, urine and imaging) for detection. Genetically encoded libraries (>10^4) of protease-activatable receptors will allow in vivo screening of 1000s of candidate T cell sensors to positively and negatively select for constructs that can report on awakening in different microenvironments such as the bone marrow and lungs. The adoptive transfer of T cell sensors with memory phenotype could lead to life-long T cell sensors that continuously monitor for future disease. These technological breakthroughs will have huge implications in understanding how and when dormant cells reawaken and guide therapeutic interventions at the earliest stages of reactivation.