Experimental Validation of Variant Antibodies Guided by a Large Language Model and Optimized through Combinatorial Algorithms 2307181

Abstract Introduction Machine-learning—guided antibody design offers a promising approach for rapidly generating high-affinity therapeutics against emerging pathogens. Our group has developed Ab-Affinity, a large language model that predicts antibody—antigen binding and, together with genetic algorithms and simulated annealing, designs variants with markedly improved predicted stability and affinity for a SARS-CoV-2 spike epitope. Computational analyses indicate over a 160-fold affinity enhancement compared to experimentally derived sequences. This work focuses on experimentally validating these predictions through expression, purification, and functional characterization of the model-designed antibodies Methods Three antibodies–Ab-14-seed and the optimized variants Ab-14-SA-PSSM1 and Ab-14-SA-PSSM6–designed through Ab-Affinity were expressed in Pichia pastoris and purified using FPLC. Protein expression and purity were confirmed by SDS-PAGE and Western blot. Binding affinities are being assessed using ELISA and surface plasmon resonance to evaluate interactions with the target SARS-CoV-2 spike peptide. Results All three designed antibodies have been successfully expressed in Pichia pastoris and purified. Upcoming binding affinity assays will determine whether these variants demonstrate the enhanced binding affinities predicted by Ab-Affinity. Conclusion This study integrates large language model-guided antibody design with experimental validation to assess the real-world performance of computationally optimized antibodies. The results will clarify how effectively AI-generated sequences translate into functional high-affinity binders, informing the development of future therapeutics. Funding Source DOE Topic Categories Vaccines and Immunotherapy (VAC)