Development and characterization of synthetic human antibodies for sensing cellular metabolic states

Metabolic reprogramming of cancer is reflected in aberrant activity of the hexosamine biosynthetic pathway that produces UDP‐GlcNAc (a donor substrate used to form the O‐GlcNAc modification on proteins). Rather than acting as a switch that turns signaling pathways on or off, O‐GlcNAc should be considered as a “rheostat” that controls the intensity of intracellular signals according to the nutrient and stress conditions. There is evidence that some cell surface proteins contain O‐GlcNAc modification in their extracellular regions. However, there is a lack of understanding for the role of extracellular O‐GlcNAcylation in biological processes. Tuning the level of O‐GlcNAcylation is one way that tumor cells can adapt to varying nutrient and environmental conditions. We hypothesize that cancer cells can “sense” stress through the O‐GlcNAc pathway and, consequently, reprogram the cell surface and adapt to tougher conditions. Neo‐epitopes on the cell surface that emerge from tuning O‐GlcNAc pathway activity may be prime candidates as readouts that reliably report the status of metabolic reprogramming. These nutrient‐dependent cell surface epitopes may have functional roles in defining cancer cell identity. In order to advance understanding of metabolic reprogramming from the perspective of cell surface, we combined genetics and advanced protein engineering using phage‐displayed synthetic human antibody libraries to generate binders that respond to changes in the O‐GlcNAc rheostat. Our method for discovery of s urface and n utrient‐ a ssociated p rotein s ensors (SNAPS) has allowed us to identify multiple antibodies that can detect different cellular metabolic states.