Project Abstract: The human granzymes are a somewhat mystical class of five serine proteases (A, B, H, K, M) that are expressed and conditionally secreted by select lymphocytes like natural killer (NK) and cytotoxic T cells (CTL). Based largely on data for A and B, granzymes have been historically regarded as pro-apoptotic effectors whereby they are presented transiently by NK and CTLs at the immunological synapse with a problematic target cell (e.g. cancer cell, pathogen infected cell), and immediately shuttled into the cytosol via perforin to initiate cell death. However, emerging data has begun to challenge this canon and present a more complex model in which secreted granzymes perform essential signaling functions in extracellular space, including activation of macrophages as part of host defense. Interestingly, dysregulation of granzyme presentation and/or proteolytic activity may also underlie the pathobiology of debilitating diseases like neurodegenerative or systemic autoimmune diseases. Thus, more fully elaborating the biology of granzymes is an important unmet need, and requires technologies to study granzymes in the most clinically relevant animal models and humans themselves. To this end, we developed a novel imaging approach we term “restricted interaction peptides” (RIP) to detect granzyme proteolytic activity in vivo with PET. Mechanistically, RIPs are administered as an inactive pro-form, whereupon internal cleavage of the RIP by the target endoprotease releases a radiolabeled peptide that immediately associates with nearby phospholipid membranes (i.e. the plasma membrane of the target cell). Thus, accumulation of the radiolabeled cleavage product adjacent to the endoprotease provides a readout of the relative units of enzyme activity within a region of interest. As preliminary data, we developed 64Cu-GB1, a RIP that measures granzyme B activity on PET. Our proof of concept data show that 64Cu-GB1 detects the cytotoxic pool of granzyme B activity trafficking to pathogenic target cells, but also an unexpected non-cytotoxic pool elicited as part of an inflammatory response to an endotoxin. During this project, we will expand upon these data in several important directions. First, we will test in Aim 1 if 64Cu-GB1 can be applied to study granzyme B proteolytic activity in the spontaneous immune responses arising due to pathogen stimulation. These data will be crucial to understanding the utility of this methodology beyond simply detecting pharmacologically augmented anticancer immune responses. During Aim 2, we will expand the RIP toolkit to develop and study probes targeting the tryptase proteolytic activities of granzymes A and K. During the final Aim, we will carry out the late stage preclinical experiments required to judge the feasibility of human imaging with RIP probes. As a model system, we will study our lead candidate 64Cu-GB1. If successful, this project will confer new translational technologies at a crucial inflection po...