An in silico method to assess antibody fragment polyreactivity

Antibodies are essential biological research tools and important therapeutic agents, but some exhibit non-specific binding to off-target proteins and other biomolecules. Such polyreactive antibodies compromise screening pipelines, lead to incorrect and irreproducible experimental results, and are generally intractable for clinical development. We designed a set of experiments using a diverse na{\"ıve synthetic camelid antibody fragment (‘nanobody’) library to enable machine learning models to accurately assess polyreactivity from protein sequence (AUC > 0.8). Moreover, our models provide quantitative scoring metrics that predict the effect of amino acid substitutions on polyreactivity. We experimentally tested our model’s performance on three independent nanobody scaffolds, where over 90% of predicted substitutions successfully reduced polyreactivity. Importantly, the model allowed us to diminish the polyreactivity of an angiotensin II type I receptor antagonist nanobody, without compromising its pharmacological properties. We provide a companion web-server that offers a straightforward means of predicting polyreactivity and polyreactivity-reducing mutations for any given nanobody sequence.Competing Interest StatementC.C.L is a co-founder of K2 Biotechnologies Inc., which applies continuous evolution technologies to antibody engineering. D.S.M. is an advisor for Dyno Therapeutics, Octant, Jura Bio, Tectonic Therapeutic and Genentech, and is a co-founder of Seismic Therapeutic. A.C.K. is a co-founder and consultant for Tectonic Therapeutic and Seismic Therapeutic and for the Institute for Protein Innovation, a non-profit research institute.