Project Summary Extracellular proteolysis is a hallmark of cancer. Proteolysis has major functional consequences for remodeling cell surface proteins and matrix including receptor activation and shedding of receptor extracellular domains. Of the more than 600 human proteases, over half are on the cell surface or secreted reflecting their significance for processing the extracellular space. Yet we understand very little about the targets for cell surface proteolysis (CSP), the proteases responsible, and resulting neo- cleaved products that could serve as new biomarkers or drug targets. What has been missing are robust technologies for unbiased identification of the CSP targets, their specific sites of cleavage, the proteases responsible for promoting disease, and the generation of neo-epitope specific antibodies. My group has engineered a novel peptide ligase, called subtiligase, and shown it robustly labels sites of proteolysis of soluble proteins. Our preliminary data show that this powerful tool can be used to determine the targets and precise sites of cleavage for CSP. We propose this novel CSP technology can be used to determine CSP signatures in cancer. We will initially focus on KRAS, the most dominant oncogene, especially in pancreatic cancer. We will determine KRAS- induced proteolysis in isogenic pancreatic cell lines, with and without mutant KRAS, grown in hypoxic and normoxic conditions in the context of two- and three-dimensional culture. We will identify the surface proteases up-regulated using cell surface capture (CSC) proteomics and confirm their activities using CRISPRi or small molecule inhibitors. We will characterize their specificities using new substrate phage technology coupled with NGS and match these to neo-epitopes we discover. Lastly, we will further exploit differential antibody phage display selection technology, and generate antibodies specific to the cleaved forms of various identified targets, including CDCP1. We will develop important technologies for new neo-epitope biomarkers and potential immune-oncology reagents for the characterization of these important cleavage events. Impact: Our studies will provide robust protein engineering-based technologies for unbiased insights into how CSP remodels cells, the proteases responsible, and actionable oncogene specific neo-epitopes to generate antibodies as potential biomarkers and therapeutics. We believe these approaches will be generally applicable to characterizing proteolysis in other solid tumor cancers.