Big Benefits from Linking Small Antibodies (Nanobodies) to Small Molecule GPCR Ligands

Abstract ID 92209 Poster Board 146 G protein-coupled receptors (GPCRs) are an important class of targets for therapeutic development and fundamental physiological studies. Investigations of GPCR function require the identification of ligands that can activate or block receptor function. Such ligands are often drawn from biological sources, such as genetically encoded polypeptides or naturally occurring small molecules. However, such naturally derived ligands often show promiscuity, activating multiple GPCRs and GPCR-coupled pathways, thereby complicating their application in mechanistic studies of GPCR function and therapeutic development. We hypothesized that by linking a promiscuous GPCR ligand to an antibody that is high specific for the receptor of interest we could improve specificity. Towards this end, we have undertaken the development of chemical and enzymatic methodology to facilitate the linkage of GPCR ligands to antibodies (or antibody fragments). Past work from my research group has shown that the linkage of peptide agonist ligands for GPCRs to single domain antibodies (nanobodies) provides substantial enhancements in receptor specificity, activation potency, and pathway selective signaling. This approach has not been demonstrated using small molecule agonists of GPCR signaling. In this work, we demonstrate for the first time that the linkage of a small molecule agonist (CGS21680, CGS) for the A2A adenosine receptor (A2AR) to a nanobody that binds to an engineered version of this receptor provides semi-synthetic nanobody-ligand conjugates with interesting and useful properties. Nanobody-CGS conjugates exhibit high potency, long lasting agonism of engineered A2A adenosine receptor. Additionally, these conjugates show specificity for the engineered receptor over wild type counterpart, suggesting a path towards the generation of engineered receptors preferentially activated by engineered ligands. Finally, we demonstrate an approach with nanobody-ligand conjugates that facilitates preferential targeting of receptor assemblies. We create conjugates comprised of CGS21680 and nanobodies that target other GPCRs co-expressed in certain tissues alongside A2AR. Such nanobody-CGS conjugates exhibit activity that is contingent upon expression of both A2AR and the target of nanobody binding. This approach offers a path for the preferential activation of GPCRs only in cells or tissues that co-express the protein targeted by the nanobody found within nanobody-ligand conjugates. This research was supported by the Intramural Research Program of the NIH, The National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)