Bowchro (@bowchro.bsky.social)

In silico discovery of nanobody binders to a G-protein coupled receptor using AlphaFold-Multimer Antibodies are central mediators of the adaptive immune response, and they are powerful research tools and therapeutics. Antibody discovery requires substantial experimental effort, such as immunization campaigns or in vitro library screening. Predicting antibody-antigen binding a priori remains challenging. However, recent machine learning methods raise the possibility of in silico antibody discovery, bypassing or reducing initial experimental bottlenecks. Here, we report a virtual screen using AlphaFold-Multimer (AF-M) that prospectively identified nanobody binders to MRGPRX2, a G protein-coupled receptor (GPCR) and therapeutic target for the treatment of pseudoallergic inflammation and itch. Using previously reported nanobody-GPCR structures, we identified a set of AF-M outputs that effectively discriminate between interacting and non-interacting nanobody-GPCR pairs. We used these outputs to perform a prospective in silico screen, identified nanobodies that bind MRGPRX2 with high affinity, and confirmed activity in signaling and functional cellular assays. Our results provide a proof of concept for fully computational antibody discovery pipelines that can circumvent laboratory experiments. ### Competing Interest Statement E.P.H., J.S.S., J.D.H., K.J.S., and A.C.K. are co-inventors on a patent application for MRGPRX2 antagonist nanobodies. D.S.M. is an advisor for Dyno Therapeutics, Octant, Jura Bio, Tectonic Therapeutic, and Genentech, and is a co-founder of Seismic Therapeutic. J.C.W. is a co-founder of MOMA therapeutics, a company in which he has a financial interest. A.C.K. is a co-founder and consultant for biotechnology companies Tectonic Therapeutic and Seismic Therapeutic, and for the Institute for Protein Innovation, a non-profit research institute. J.S.S. is an advisor / investigator for Biogen. The remaining authors declare no competing interests. https://www.biorxiv.org/content/10.1101/2025.03.05.640882v1