Structural Modeling of Antibody Variant Epitope Specificity with Complementary Experimental and Computational Techniques

Abstract

Antibodies are key therapeutic biomolecules yet the principles underlying diverse paratopes binding to the same epitope remain unexplained. An insufficient understanding of the structural rules of antibody-antigen binding, due to a lack of experimentally resolved structures, leads to the current inability to characterize antibody variants in silico. Here we explore a rule-based antibody design strategy that relies on a thorough understanding of epitope-paratope interactions, in contrast to generative design based on millions of trials and errors. We identified the epitope of six affinity-verified complexes between HER2 and Trastuzumab paratope variants using cryo-EM and position-resolved HDX-MS. Computational analysis of modeled structural conformational ensembles replicates and expands experimental results and highlights the importance of flexibility in understanding high and low-affinity binders. Structural parameters calculated based on geometry, surface, and biochemical properties were able to stratify antibodies by affinity. Overall, our study describes the structural interfaces of the paratope variants, showing how antibodies with diverse sequences share similar binding rules.