Accelerating multi-objective VHH discovery via integrated high-throughput selection and AlphaFold3-guided structure prediction

Overath, M. D.; Thumtecho, S.; Rivera-de-Torre, E.; Benard Valle, M.; Wolff, D. S.; Grahadi, R.; Björnsson, K. H.; Rygaard, A. S. H.; Hofmann, N.; Ledergerber, J.; Ljungars, A.; Laustsen, A. H.; Olsson, S.; Fryer, T. J.; Jenkins, T. P.

doi:10.64898/2026.01.19.700436

bioRxiv · 10.64898/2026.01.19.700436

Accelerating multi-objective VHH discovery via integrated high-throughput selection and AlphaFold3-guided structure prediction

Overath, M. D.·Thumtecho, S.·Rivera-de-Torre, E.·Benard Valle, M.·Wolff, D. S.·Grahadi, R.·Björnsson, K. H.·Rygaard, A. S. H.·Hofmann, N.·Ledergerber, J.·Ljungars, A.·Laustsen, A. H.·Olsson, S.·Fryer, T. J.·Jenkins, T. P.

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Abstract

Discovering therapeutic antibodies that bind multiple related targets with high affinity and favourable biophysical properties remains challenging and resource intensive. For snakebite antivenoms, this challenge is critical as treatments must neutralise toxins across multiple snake species. We developed a pipeline combining high-throughput yeast screening, deep sequencing, and AlphaFold3 structure prediction to rapidly identify poly-specific variable domains of heavy-chain-only antibodies (VHHs) against long-chain -neurotoxins. Multiplexed yeast display screening generated a dataset of diverse candidates with varying binding specificities. AlphaFold3-generated VHH-toxin complex predictions enabled structure-based prioritisation that accurately predicted poly-specific binders targeting conserved epitopes across multiple toxins. These structural insights enabled computational optimisation of both affinity and solubility without disrupting target recognition. Experimental validation confirmed improved variants maintained broad specificity across toxins. This integrated approach accelerates multi-objective antibody discovery by predicting which candidates will bind multiple targets before extensive laboratory testing, providing a generalisable strategy applicable beyond antivenoms to any therapeutic requiring broad target coverage.

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Overath, M. D., Thumtecho, S., Rivera-de-Torre, E., Benard Valle, M., Wolff, D. S., Grahadi, R., Björnsson, K. H., Rygaard, A. S. H., Hofmann, N., Ledergerber, J., Ljungars, A., Laustsen, A. H., Olsson, S., Fryer, T. J., Jenkins, T. P.. 2026-01-20. Accelerating multi-objective VHH discovery via integrated high-throughput selection and AlphaFold3-guided structure prediction. https://doi.org/10.64898/2026.01.19.700436

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