Nanomechanical binding mechanism of ligands drives agonistic activity

TL;DR

The study reveals how the natural CD40 ligand interacts with receptors more efficiently than antibodies, offering insights for better drug design.

Contribution

The paper introduces a novel nanomechanical binding mechanism of CD40 ligands that explains their superior agonistic activity.

Findings

ChiLob 7/4 dynamically rotates its Fab arms to screen for hCD40 binding.

hCD40L forms and releases hCD40 clusters ten times faster than ChiLob 7/4.

The natural ligand's binding mechanism could inspire new drug formats with enhanced activity.

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.

Monoclonal antibodies and ligands targeting CD40 exhibit diverse agonistic and antitumor activities that are influenced by their design. Here, the authors identify mechanistic differences between clinically relevant anti-CD40 subclasses and CD40L, focusing on the dynamics and strengths of multi-bond formation at the single-molecule level.

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