Affinity Enhancement in Discrete Multivalent MegaMolecules

In this study, we quantitatively investigated the binding properties of eighteen multivalent megamolecules functionalized with nanobodies targeting epidermal growth factor receptor (EGFR) or human EGFR 2 (HER2). We synthesized a series of structurally defined multivalent megamolecules containing up to six anti-HER2 or anti-EGFR Nbs with monovalent affinities varying by 2800-fold. These megamolecules exhibited improvements in apparent binding affinities from nano- or micromolar range to picomolar or sub-picomolar range (31- to ∼118,000-fold increase). Notably, a dendritic hexavalent megamolecule achieved sub-picomolar binding affinity, as determined by biolayer interferometry. To assess whether the enhanced affinity of this hexamer arose from its valency or extended conformation, variants with reduced valency were synthesized and exhibited lower affinity, confirming valency as the primary factor in affinity enhancement. Our research highlights the utility of megamolecules in synthesizing precisely defined multivalent structures with controlled valency and spatial presentation, providing a versatile platform for fundamental studies and development of advanced therapeutic agents.