Seferovic Hannah, Sticht Patricia, Hain Lisa, Zhu Rong, Diethör Sebastian, Wechselberger Christian, Weber Florian, Bernhard David, Plochberger Birgit, Oh Yoo Jin, Chaparro-Riggers Javier, Hinterdorfer Peter
Institute of Biophysics, Johannes Kepler University, Linz, Austria.
Division of Pathophysiology, Institute of Physiology and Pathophysiology, Medical Faculty, Johannes Kepler University, Linz, Austria.
Nat Commun. 2025 Jul 19;16(1):6674. doi: 10.1038/s41467-025-61929-1.
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.
靶向CD40的单克隆抗体和配体表现出广泛的激动活性和抗肿瘤反应。研究表明，抗体的灵活性和亲和力在其免疫刺激活性中起着关键作用。然而，目前尚缺乏与天然配体的系统比较，并且迄今为止尚未对分子刚性、结合动力学和键寿命进行详细研究。在此，我们在单分子水平上研究了临床相关的抗人CD40抗体亚类ChiLob 7/4和三聚体人CD40L与人CD40的动态结合特征。我们观察到hCD40受体形成二聚体和高阶寡聚体，ChiLob 7/4和hCD40L通过其多个结合位点动态捕获和释放这些寡聚体。因此，ChiLob 7/4充当纳米机械卡尺，并以高度动态的方式旋转其Fab臂以筛选hCD40结合，而hCD40L的构象变化明显较少。尽管hCD40L的分子灵活性较小，但与ChiLob 7/4相比，hCD40L与人CD40的缔合、解离和重新缔合速度快十倍。我们发现了一种独特的结合机制，这可能解释了天然配体增强的簇形成潜力和激动活性，并将激发新型配体形式的设计。
