Integrative computational analysis of glycan and ROS-linker effects on Pertuzumab-HER2 stability for multimodal ADC architectures

Abstract

ADCs rely on linker chemistry to balance systemic stability with conditional activation. Glycoengineering provides additional design opportunities by incorporating terminal sialylated glycans that support multimeric interactions and PEG conjugation. Here, we applied an integrated computational workflow combining structural prediction with AlphaFold3, glycan construction with GLYCAM, docking with DiffDock, and all-atom free-energy perturbation (FEP) simulations in GROMACS using a double-decoupling framework to evaluate the effects of three Fc-linked glycan architectures: a short sialic acid glycan, a branched sialylated glycan with a PEG-accessible branch, and a branched sialylated glycan incorporating both ROS-cleavable branch and a PEG-accessible branch, on Pertuzumab in complex with HER2. Short glycans conferred modest stabilization but limited conformational engagement, while branched variants produced the strongest stabilization, linking structural effects with PEG accessibility. In branched variants, the ROS-responsive branch exhibited reduced stabilization consistent with a conditionally labile architecture, while the PEG-accessible branch remained structurally intact. These findings demonstrate how glycan branching and linker architecture influence relative Fc stabilization and structural behavior within modeled systems and support the use of an integrated computational workflow combining structural modeling, docking, and free-energy calculations to prioritize complex linker chemistries for further experimental validation.