Allosteric Antibody Modulation of EGFR Activity: Bridging Experiment and In Silico Modeling.

Abstract

Allosteric regulation provides a powerful framework for modulating receptor signaling in both physiological and therapeutic contexts. The epidermal growth factor receptor (EGFR), a receptor tyrosine kinase frequently dysregulated in cancer, undergoes activation through conformational transitions that couple extracellular ligand binding to intracellular kinase signaling. Here, we explore how camelid derived VHH (variable domain of the heavy chain of a heavy chain-only)-antibodies can exploit this allosteric architecture to inhibit EGFR function. Using a panel of single domain monospecific and biparatopic antibodies, targeting non-overlapping EGFR epitopes, we combined experimental assays with structure-based modeling to dissect their effects on EGFR signaling and internalization. AlphaFold3-predicted EGFR-antibody complexes were analyzed using the Structure-Based Statistical Mechanical Model of Allostery (SBSMMA) to compute residue-level allosteric modulations induced upon binding. The resulting profiles revealed that only a subset of epitope combinations produced long-range allosteric responses reaching the juxtamembrane segment and the kinase domain. These patterns correlated with effective inhibition of downstream ERK and AKT signaling in cellular assays. In contrast, some constructs with high internalization capacity induced minimal allosteric propagation and weak signaling suppression, indicating a mechanistic decoupling of receptor uptake from conformational regulation. Together, these results define distinct allosteric modes of EGFR modulation by VHH-antibodies and show how computational modeling based on energetic propagation can complement experimental screening to guide the design of next-generation allosteric biologics.