High‐throughput CRISPR for Generating Therapeutic Nanobodies

Abstract

Engineered antibodies have had a widespread impact throughout biomedical sciences, from their use as fundamental research tools to their employment as therapeutics. Closely related nanobodies, derived from heavy‐chain only antibodies, have begun gaining much attention as alternatives with improved pharmacological properties. Containing only the antigen recognition components, nanobodies maintain the exceptional specificity of conventional antibodies but their simplistic design leads to increased stability and decreased toxicity. In recent years, nanobodies have proven especially useful for studying G protein‐coupled receptors (GPCRs), a class of membrane receptors responsible for activating intracellular proteins (e.g., Gα proteins) and initiating subsequent signaling pathways. Most GPCR‐targeting nanobodies to date have been designed as crystallographic chaperones for stabilizing the GPCR‐Gα interaction for in‐depth structural insights. However, harnessing the intrinsic specificity provided by nanobodies for therapeutic purposes has been limited to few receptors. In this study, we present a unique platform for discovering functionally selective nanobodies for a broad range of GPCRs. Using a yeast model proven invaluable for discovering GPCR ligands and structure‐stabilizing nanobodies, we engineered a system for rapidly identifying nanobody‐based therapeutic candidates. In this system, each cell uniquely contains 1) a tethered nanobody displayed outside the cell membrane, 2) a human GPCR, 3) one of ten Gα proteins for characterizing GPCR‐Gα interactions, and 4) a fluorescent reporter induced upon GPCR/Gα activation. By combining all components into a single cell, we can directly access the therapeutic potential of each nanobody‐GPCR combination by monitoring the (in)activity of relevant signaling pathways inspired from this interaction. To date, we have completed the initial engineering stages of this platform and showcased its utility through pharmacological activation of various GPCRs (e.g., SSTR5, AGTR1, CXCR4, and C5aR) via tethered peptide/protein agonists. Next, we will use high‐throughput CRISPR to generate an extensive nanobody library for rapidly identifying and functionally quantifying GPCR‐targeting nanobodies. Using this approach for a large variety of GPCRs, this platform will provide a vast collection of nanobody‐based therapeutic candidates, and with minimal modifications may be expanded for identifying therapeutics for other classes of membrane receptors.