Discovery and validation of nanobody antagonists to the itch receptor MRGPRX2 using AlphaFold-Multimer

Summary: Abstract Body: The purpose of this study was to investigate if in silico antibody screening methods could identify high-affinity antibodies to a cellular surface receptor. VHH antibodies, also known as nanobodies, are single-domain antibodies naturally derived from camelids but which can be humanized and have recently received FDA approval. The smaller size and biochemical stability of nanobodies offer certain advantages, such as increased tissue penetration, relative to conventional antibody biologics. We computationally screened 10,000 nanobodies against the G protein-coupled receptor (GPCR) MRGPRX2, a promising target for the treatment of itch and chronic spontaneous urticaria. Based on our in silico confidence metrics, we selected ten nanobody candidates to purify and biochemically evaluate. Our results show that 3/10 nanobodies exhibited nanomolar affinity for MRGPRX2. Binding affinity was tested on both ROSA mast cells at endogenous receptor levels and on HEK293T cells overexpressing MRGRPX2 relative to cells lacking receptor. We validated that these nanobodies are apparent physiological antagonists through various approaches. Pretreating mast cells with the three nanobodies resulted in a significant reduction in agonist-induced mast cell degranulation (p<0.05, one-way ANOVA) as well as a rightward shift in EC50 and reduced Emax in G protein activation relative to control non-binding nanobodies (p<0.05, two-way ANOVA). Structural predictions suggest that the three nanobodies block the MRGPRX2 orthosteric binding pocket, which was supported by mutagenesis studies. Our results demonstrate a computational antibody discovery pipeline for a GPCR that can effectively bypass certain laboratory experiments. Jeffrey S. Smith^{1, 2}, Edward Harvey¹, Joseph Hurley¹, Alyana Granados³, Ernst Schmid¹, Jason Liang-Lin¹, Steffanie Paul¹, Emily M. Meara^{1, 2}, Matthew Ferguson¹, Victor Calvillo-Miranda¹, Debora Marks¹, Johannes Walter^{1, 4}, Katherine Susa³, Andrew C. Kruse¹ 1. Harvard Medical School, Boston, MA, United States. 2. Brigham and Women's Hospital, Boston, MA, United States. 3. University of California San Francisco, San Francisco, CA, United States. 4. Howard Hughes Medical Institute, Boston, MA, United States. Translational Studies: Cell and Molecular Biology