PROJECT SUMMARY/ABSTRACT Pancreatic ductal adenocarcinoma (PDAC), soon to be the second leading cause of cancer deaths in the U.S, has an overall 9% survival. PDAC is characterized by a tumor microenvironment that is densely fibrotic and acidic. Effects of low pH can occur through a variety of mechanisms, including activation of proton-sensing G protein-coupled receptors, such as GPR65 and GPR68. GPR65, GPR68 and GPR132 (another proton-sensing GPCR) are more highly expressed in PDAC tumors (data from The Cancer Genome Atlas, TCGA) than in normal pancreas (GTEx database). GPCRs as a class are generally not targeted for cancers and are an unexplored area of high interest for therapeutic development, especially for cancers, such as PDAC. Proton-sensing GPCRs may be novel targets for treating PDAC and involvement of GPR65/GPR68 in PDAC provides an excellent model system for a focused pilot study. Their prominent up-regulation in PDAC tumors involves different cell types in the tumor microenvironment (TME): GPR65 in immune cells and GPR68 in cancer- associated fibroblasts (CAFs). GPR65 is predicted to be immunosuppressive via low-pH promoted increase in cyclic AMP (cAMP). Low pH activation of GPR68, acting via cAMP in patient-derived CAFs, increases production of collagens, fibrotic markers, and IL-6, which promotes PDAC proliferation. GPR68 is thus a hub of a previously unrecognized positive feedback loop between PDAC cells and CAFs. Inhibition of CAF-expressed GPR68 activity is predicted to blunt PDAC cell proliferation, fibrosis and reduce tumor size. We propose to use a novel approach to generate nanobodies (Nbs) directed at GPR65 and GPR68 on the extracellular surface. We seek to identify Nbs that can inhibit low pH-promoted signaling and functional changes, including in cells from patient-derived PDAC tumors. Such Nbs may also aid in the detection of PDAC tumors in their acidic TME. If successful, our approach could be generalized to find potential Nb antagonists to other GPCRs and receptors, minimizing (or eliminating) the need for purified receptor proteins as antigens. The project has two goals: (1) obtain a panel of Nbs specific to GPR65 and GPR68 on the cell surface and (2) define the specificity and biological activity of the GPR65 and GPR68-targeted Nbs, characterizing and validating them using cell-based assays that will interrogate their impact on their signaling and action in human cells. The goal is to generate Nbs that will inhibit the actions of GPR65 and GPR68 as a first step toward ultimately generating Nbs directed at all 4 of the proton-sensing GPCRs. In addition to having biological activities, such Nbs could aid in structural studies of the proton-sensing class of GPCRs and serve as IDG-generated reagents.