2024/03/18 by Nathaniel R. Bennett, Joseph L. Watson, Robert J. Ragotte +29 · 2 voices · 16 citations
Biochemistry, Genetics and Molecular Biology · Medicine · Physics and Astronomy · #Force Microscopy Techniques and Applications #Monoclonal and Polyclonal Antibodies Research #Protein purification and stability
paper · pdf · doi:10.1101/2024.03.14.585103
openalex publication_date 2024/03/18 · openalex created_date 2024/03/20 · openalex updated_date 2026/07/25
Abstract Despite the central role that antibodies play in modern medicine, there is currently no method to design novel antibodies that bind a specific epitope entirely in silico . Instead, antibody discovery currently relies on animal immunization or random library screening approaches. Here, we demonstrate that combining computational protein design using a fine-tuned RFdiffusion network alongside yeast display screening enables the generation of antibody variable heavy chains (VHHs) and single chain variable fragments (scFvs) that bind user-specified epitopes with atomic-level precision. To verify this, we experimentally characterized VHH binders to four disease-relevant epitopes using multiple orthogonal biophysical methods, including cryo-EM, which confirmed the proper Ig fold and binding pose of designed VHHs targeting influenza hemagglutinin and Clostridium difficile toxin B (TcdB). For the influenza-targeting VHH, high-resolution structural data further confirmed the accuracy of CDR loop conformations. While initial computational designs exhibit modest affinity, affinity maturation using OrthoRep enables production of single-digit nanomolar binders that maintain the intended epitope selectivity. We further demonstrate the de novo design of single-chain variable fragments (scFvs), creating binders to TcdB and a Phox2b peptide-MHC complex by combining designed heavy and light chain CDRs. Cryo-EM structural data confirmed the proper Ig fold and binding pose for two distinct TcdB scFvs, with high-resolution data for one design additionally verifying the atomically accurate conformations of all six CDR loops. Our approach establishes a framework for the rational computational design, screening, isolation, and characterization of fully de novo antibodies with atomic-level precision in both structure and epitope targeting.