The recent clinical success of inhibitors against immune checkpoint proteins (e.g. CTLA-4, PD-L1), which are thought to stimulate T cell responses against tumors, has revolutionized cancer therapy. Yet even among patients with high tumor mutational burden, only approximately 20-30% of patients achieve deep response, and discerning responders from non-responders is challenging with conventional imaging. On this basis, there is an urgent unmet need to develop biomarkers that distinguish responsive and treatment resistant patients, as well as identify patients at risk for undesired immune related adverse events. We hypothesized that an imaging biomarker capable of selectively measuring the biology that T cells use to impart cytotoxicity might address these unmet needs. Since antitumor T cell cytotoxicity is conferred primarily by the pro-apoptotic serine protease granzyme B, we have developed a peptide-based chemosensor we term “restricted interaction peptide” that enables spatiotemporal measurements of granzyme B proteolytic activity as the enzyme traverses the immunological synapse between T cell and target cell. Upon proteolytic cleavage of the full length, pro-form of the restricted interaction peptide (termed GB1) by granzyme B, a radiolabeled antimicrobial peptide is liberated and undergoes a spontaneous conformational shift that results in stable (and non-toxic) membrane association. We have shown that radiolabeled GB1 detects T cell activation in tumors and normal tissues elicited by systemic immune checkpoint inhibitors. Following on these encouraging preclinical data, we have now assembled a multidisciplinary team to conduct translational studies to evaluate the utility of granzyme B biochemistry as a biomarker. Over three specific aims, we will (1) perform IND enabling studies for 64Cu-GB1, (2) conduct a phase 0 first in human study to determine tracer safety, pharmacokinetics, and dosimetry, and (3) execute a phase I study to determine the accuracy for detection of urothelial and renal cancers undergoing a productive immune response due to treatment with standard of care immune checkpoint inhibitors. If successful, this project will establish a new paradigm for the measurement of T cell cytotoxicity in vivo that could have implications for the clinical management of other problematic human disorders like bacterial or viral (HIV, SARS-CoV) infections. Moreover, the imaging approach is entirely new, and favorable data emerging from this project could motivate further studies to develop restricted interaction peptides to measure the enzymology of other disease associated proteases in vivo with PET.