Developing Superselective Homobivalent Ligands Differentiating Membrane Antigen Density by Exploring the Spatial Limit of the Bivalency on Cells

Developing highly selective molecular recognition tools to distinguish cancer cells from healthy cells remains a considerable challenge. Here, we engineered a series of homobivalent ligands by positioning monomeric aptamers at the 3'-termini of DNA duplex scaffolds and systematically investigated the key factors influencing their interactions with cells. These factors included the monomer binding affinity, intersection angle, scaffold length and flexibility, and membrane antigen density. Our findings revealed a spatial constraint on bivalency, primarily governed by monomer affinity and membrane antigen density. Guided by both experimental data and statistical mechanics modeling, we designed superselective homobivalent ligands that effectively discriminated PTK7^high CCRF-CEM cells ((2.5 ± 0.26) × 10⁵ PTK7/cell) from PTK7^low HEL cells ((1.0 ± 0.05) × 10⁵ PTK7/cell) in both in vitro and in vivo models. This study provides new insights into the fundamental principles of homobivalent ligand-cell interactions and establishes a framework for designing superselective ligands for cancer diagnosis and therapy based on membrane antigen density.