Atomically accurate de novo design of antibodies with RFdiffusion
Nathaniel R. Bennett, Joseph L. Watson, Robert J. Ragotte, Andrew J. Borst, DéJenaé L. See, Connor Weidle, Riti Biswas, Yutong Yu, Ellen Shrock, Russell Ault, Philip J. Y. Leung, Buwei Huang, Inna Goreshnik, John Kit Chung Tam, Kenneth D. Carr, Benedikt Singer, Cameron Criswell, Basile I. M. Wicky, Dionne Vafeados, M. SANCHEZ, Ho Min Kim, Susana Vázquez Torres, Sidney Chan, Shirley M. Sun, Timothy T. Spear, Yi Sun, K. O'Reilly, John M. Maris, Nikolaos G. Sgourakis, Roman A. Melnyk, Chang C. Liu, David Baker
Abstract
Despite the central role of antibodies in modern medicine, no method currently exists to design novel, epitope-specific antibodies entirely in silico. Instead, antibody discovery currently relies on immunization, random library screening or the isolation of antibodies directly from patients1. Here we demonstrate that combining computational protein design using a fine-tuned RFdiffusion2 network with yeast display screening enables the de novo generation of antibody variable heavy chains (VHHs), single-chain variable fragments (scFvs) and full antibodies that bind to user-specified epitopes with atomic-level precision. We experimentally characterize VHH binders to four disease-relevant epitopes. Cryo-electron microscopy confirms the binding pose of designed VHHs targeting influenza haemagglutinin and Clostridium difficile toxin B (TcdB). A high-resolution structure of the influenza-targeting VHH confirms atomic accuracy of the designed complementarity-determining regions (CDRs). Although initial computational designs exhibit modest affinity (tens to hundreds of nanomolar Kd), affinity maturation using OrthoRep3 enables production of single-digit nanomolar binders that maintain the intended epitope selectivity. We further demonstrate the de novo design of scFvs to TcdB and a PHOX2B peptide–MHC complex by combining designed heavy-chain and light-chain CDRs. Cryo-electron microscopy confirms the binding pose for two distinct TcdB scFvs, with high-resolution data for one design verifying the atomically accurate design of the conformations of all six CDR loops. Our approach establishes a framework for the computational design, screening and characterization of fully de novo antibodies with atomic-level precision in both structure and epitope targeting. The combination of computational design, laboratory-based screening and biophysical validation enables the de novo generation of variable heavy-chain antibody fragments and antibodies that precisely target chosen disease-related molecules.