Discovery and engineering of a nanobody targeting the M3 muscarinic acetylcholine receptor

Abstract

G protein-coupled receptors (GPCRs) are a superfamily of integral membrane proteins that regulate cellular signaling across a wide range of distinct, key physiological functions – ranging from vision to neurotransmission to cardiac output. As such, they are valuable drug targets, with current estimates suggesting that 30-40% of all FDA-approved drugs target GPCRs and the signaling pathways they regulate. Historically, these well-studied receptors have been targeted using small-molecule ligands that bind to a canonical ligand-binding pocket known as the orthosteric site. However, recent efforts have demonstrated the potential of antibody-based binders that recognize the extracellular face of GPCRs. Single-domain antibodies (nanobodies) have been extensively used in GPCR biochemical studies as tools for structure determination, biosensors, and ligand development. This dissertation discusses the discovery efforts undertaken to identify an extracellular nanobody for the M3 muscarinic acetylcholine receptor (M3R) – a prototypical Class A GPCR belonging to the muscarinic receptor subfamily. A longstanding challenge in the muscarinic receptor subfamily field has been the lack of subtype-selective tools for studying and modulating these receptors in a subtype-selective fashion. However, identifying novel nanobodies that target the therapeutically relevant extracellular region of GPCRs has remained difficult. In Chapter 2, I validate a previously identified intracellular G protein-mimetic nanobody for the M3R and describe early efforts to identify an extracellular nanobody targeting the M3R using an epitope-directed strategy designed to bias novel nanobody discovery toward extracellular receptor epitopes. Although these early efforts were unsuccessful, they ultimately informed optimization strategies for antigen preparation that enabled subsequent successful novel nanobody discovery campaigns for the M3R. In Chapter 3, I showcase an epitope-directed selection strategy in a fully in vitro yeast display platform that successfully identified an extracellular nanobody binder (M3102) targeting the M3R. In this chapter, I highlight M3102’s highly selective binding profile for the M3R over the other muscarinic receptors – addressing the longstanding need for a subtype-selective tool within the muscarinic receptor field. In Chapter 4, I explore the potential applications of M3102 as a subtype-selective agonist for M3R activation in vitro and its uses in studying M3R via flow cytometry-based detection methods for the receptor. Collectively, this work provides a blueprint for future epitope-directed strategies for identifying novel antibody-based binders to specific GPCR epitopes while also addressing a longstanding challenge in the muscarinic receptor field through the development of a subtype-selective tool for studying M3R.