Nanomechanical binding mechanism of ligands drives agonistic activity

Hannah Seferovic, Patricia Sticht, Lisa Hain, Rong Zhu, Sebastian Diethör, Christian Wechselberger, Florian Weber, David Bernhard, Birgit Plochberger, Yoo Jin Oh, Javier Chaparro-Riggers and Peter Hinterdorfer ()
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Hannah Seferovic: Johannes Kepler University
Patricia Sticht: Johannes Kepler University
Lisa Hain: Johannes Kepler University
Rong Zhu: Johannes Kepler University
Sebastian Diethör: Johannes Kepler University
Christian Wechselberger: Johannes Kepler University
Florian Weber: Upper Austria University of Applied Sciences
David Bernhard: Johannes Kepler University
Birgit Plochberger: Upper Austria University of Applied Sciences
Yoo Jin Oh: Johannes Kepler University
Javier Chaparro-Riggers: San Diego
Peter Hinterdorfer: Johannes Kepler University

Nature Communications, 2025, vol. 16, issue 1, 1-13

Abstract: Abstract Monoclonal antibodies and ligands targeting CD40 exhibit a wide range of agonistic activities and antitumor responses. Studies have shown that the flexibility and affinity of antibodies play a crucial role in their immunostimulatory activity. However, a systematic comparison with the natural ligand is yet missing and a detailed investigation with respect to molecular rigidity, binding kinetics, and bond lifetime has not been undertaken to date. Here, we study the dynamic binding features of clinically relevant anti-hCD40 antibody subclasses, ChiLob 7/4, and the trimeric human CD40L to hCD40 at the single-molecule level. We visualize resembling of hCD40 receptors into dimers and higher-order oligomers that are dynamically captured and released by both ChiLob 7/4 and hCD40L with their multiple binding sites. Thereby, ChiLob 7/4 acts as a nanomechanical calliper and rotates its Fab arms in a highly dynamic fashion to screen for hCD40 binding, while hCD40L undergoes significantly less conformational changes. Despite its minor molecular flexibility, hCD40L performs association, dissociation, and re-association of hCD40 ten times faster when compared to ChiLob 7/4. We uncover a distinct binding mechanism that may explain the enhanced cluster formation potential and agonistic activity of the natural ligand and will inspire the design of novel ligand formats.

Date: 2025
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DOI: 10.1038/s41467-025-61929-1

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