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

Interaction of multiple entities and receptors, or multivalency is widely applied to achieve high affinity ligands for diagnostic and therapeutic purpose. However, lack of knowledge on receptor distribution in living subjects remains a challenge for rational structure design. Here, we developed a force measurement platform to probe the distribution and distance of the cell surface vascular endothelial growth factor receptors (VEGFR) in live cells, and used 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 about 2000‐fold improvement in the binding affinity to VEGFR with IC50 value of 25 pM. 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. It showed that radiolabelled ZD‐4 resulted in 12 times increase in the tumor uptake than radiolabelled parent drug ZD6474. Our study suggests that combining statistical and chelate effect determined by receptor‐receptor distance on live cells for the magnification of multivalent binding is effective. The force measurement platform we describe would be also amenable to other cell‐surface multivalent ligand design.

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