Single‐Molecule Force Measurement Guides the Design of Multivalent Ligands with Picomolar Affinity

Abstract Interaction of multiple entities and receptors, or multivalency is widely applied to achieve high affinity ligands for diagnostic and therapeutic purposes. However, lack of knowledge on receptor distribution in living subjects remains a challenge for rational structure design. Herein, we develop a force measurement platform to probe the distribution and separation of the cell surface vascular endothelial growth factor receptors (VEGFR) in live cells, and use this to assess the geometry of appropriate linkers for distinct multivalent binding modes. A tetravalent lead ZD‐4, which was developed from an antitumor drug ZD6474 (Vandetanib) with combined hybrid binding effects, yielded a 2000‐fold improvement in the binding affinity to VEGFR with IC 50 value of 25 p m . We confirmed the improved affinity by the associated increase of tumor uptake in the VEGFR‐targeting positron emission tomography (PET) imaging using U87 tumor xenograft mouse model.