Multiobjective V H H discovery through integrated high-throughput screening and AlphaFold3-guided structural prioritization
8 affiliations
- 1Department of Biotechnology and Biomedicine, Technical University of Denmark, Kongens Lyngby, Denmark.
- 2Center for Translational Protein Design, Technical University of Denmark, Kongens Lyngby, Denmark.
- 3Division of Toxicology, Department of Medicine, Chulalongkorn University and King Chulalongkorn Memorial Hospital, Thai Red Cross Society, 10330 Pathumwan, Bangkok, Thailand.
- 4Novonesis, Kongens Lyngby, Denmark.
- 5Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Brawijaya, Malang, Indonesia.
- 6Molecular Design Laboratory, ETH Zurich, Zurich, Switzerland.
- 7Department of Computer Science and Engineering, Chalmers University of Technology and University of Gothenburg, Gothenburg, Sweden.
- 8IMES, Massachusetts Institute of Technology, Cambridge, MA, USA.
Abstract
Finding therapeutic antibodies that bind multiple related targets with high affinity and favorable biophysical properties remains challenging and resource-intensive. For snakebite antivenoms, this challenge is critical as treatments must neutralize toxins across multiple snake species. We developed a pipeline combining high-throughput yeast screening, deep sequencing, and AlphaFold3 structure prediction to identify polyspecific variable domains of heavy chain–only antibodies (V H Hs) against long-chain α-neurotoxins. Multiplexed yeast display screening generated a dataset of diverse hits with varying binding specificities. AlphaFold3-generated V H H-toxin complex predictions enabled stringent high-precision structural triage of polyspecific V H Hs that bind conserved epitopes across multiple toxins, with a representative subset experimentally confirmed to block toxin binding to the acetylcholine receptor. These structural insights provided a starting point for computational optimization of affinity and soluble expression of the V H Hs, with experimental validation confirming that optimized V H H variants maintained broad binding specificity across toxins. This integrated approach supports structure-guided prioritization of polyspecific V H H hits, offering a high-precision filter that could reduce future reliance on extensive experimental specificity screening and providing a framework applicable to other therapeutic contexts, in which substantial antigen variation occurs and broad neutralization is essential.
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BibTeX
@article{overath2026multiobjective,
title = {{Multiobjective V H H discovery through integrated high-throughput screening and AlphaFold3-guided structural prioritization}},
author = {Max D. Overath and Suthimon Thumtecho and Esperanza Rivera-de-Torre and Melisa Benard-Valle and Darian S. Wolff and Rahmat Grahadi and Kasper H. Björnsson and Andreas S. H. Rygaard and Nils Hofmann and Jann Ledergerber and Anne Ljungars and Andreas H. Laustsen and Simon Olsson and Thomas J. Fryer and Timothy P. Jenkins},
journal = {Science Advances},
year = {2026},
month = oct,
doi = {10.1126/sciadv.aef5325},
url = {https://www.science.org/doi/10.1126/sciadv.aef5325},
note = {CC BY},
}
RIS
TY - JOUR
TI - Multiobjective V H H discovery through integrated high-throughput screening and AlphaFold3-guided structural prioritization
AU - Max D. Overath
AU - Suthimon Thumtecho
AU - Esperanza Rivera-de-Torre
AU - Melisa Benard-Valle
AU - Darian S. Wolff
AU - Rahmat Grahadi
AU - Kasper H. Björnsson
AU - Andreas S. H. Rygaard
AU - Nils Hofmann
AU - Jann Ledergerber
AU - Anne Ljungars
AU - Andreas H. Laustsen
AU - Simon Olsson
AU - Thomas J. Fryer
AU - Timothy P. Jenkins
PY - 2026
DA - 2026/10/02
JO - Science Advances
DO - 10.1126/sciadv.aef5325
UR - https://www.science.org/doi/10.1126/sciadv.aef5325
AB - Finding therapeutic antibodies that bind multiple related targets with high affinity and favorable biophysical properties remains challenging and resource-intensive. For snakebite antivenoms, this challenge is critical as treatments must neutralize toxins across multiple snake species. We developed a pipeline combining high-throughput yeast screening, deep sequencing, and AlphaFold3 structure prediction to identify polyspecific variable domains of heavy chain–only antibodies (V H Hs) against long-chain α-neurotoxins. Multiplexed yeast display screening generated a dataset of diverse hits with varying binding specificities. AlphaFold3-generated V H H-toxin complex predictions enabled stringent high-precision structural triage of polyspecific V H Hs that bind conserved epitopes across multiple toxins, with a representative subset experimentally confirmed to block toxin binding to the acetylcholine receptor. These structural insights provided a starting point for computational optimization of affinity and soluble expression of the V H Hs, with experimental validation confirming that optimized V H H variants maintained broad binding specificity across toxins. This integrated approach supports structure-guided prioritization of polyspecific V H H hits, offering a high-precision filter that could reduce future reliance on extensive experimental specificity screening and providing a framework applicable to other therapeutic contexts, in which substantial antigen variation occurs and broad neutralization is essential.
SN - 2375-2548
ER -