Pachmayr Isabelle, Masullo Luciano A, Reinhardt Susanne C M, Kwon Jisoo, Llop Maite, Skořepa Ondřej, Herter Sylvia, Bacac Marina, Klein Christian, Jungmann Ralf
Max Planck Institute of Biochemistry, Planegg, Germany.
Department of Biochemistry, Ludwig Maximilian University, Munich, Germany.
Nat Commun. 2025 Jul 23;16(1):6768. doi: 10.1038/s41467-025-61893-w.
Monoclonal antibodies (mAb) are key therapeutic agents in cancer immunotherapy and exert their effects through Fc receptor-dependent and -independent mechanisms. However, the nanoscale receptor reorganization resulting from mAb binding and its implications for the therapeutic mode of action remain poorly understood. Here, we present a multi-target 3D RESI super-resolution microscopy technique that directly visualizes the structural organization of CD20 receptors and the Type I (e.g., Rituximab) and Type II (e.g., Obinutuzumab) anti-CD20 therapeutic antibodies and quantitatively analyze these interactions at single-protein resolution in situ. We discover that, while Type I mAbs promote higher-order CD20 oligomerization, Type II mAbs induce limited clustering, leading to differences in therapeutic function. Correlating RESI with functional studies for Type II antibodies with different hinge region flexibilities, we show that the oligomeric CD20 arrangement determines the Type I or Type II function. Thus, the nanoscale characterization of CD20-mAb complexes enhances our understanding of the structure-function relationships of therapeutic antibodies and offers insights into the design of next-generation mAb therapies.
单克隆抗体（mAb）是癌症免疫治疗中的关键治疗药物，通过依赖Fc受体和不依赖Fc受体的机制发挥作用。然而，由mAb结合导致的纳米级受体重组及其对治疗作用方式的影响仍知之甚少。在此，我们提出了一种多靶点3D RESI超分辨率显微镜技术，该技术可直接可视化CD20受体以及I型（如利妥昔单抗）和II型（如奥妥珠单抗）抗CD20治疗性抗体的结构组织，并在原位以单蛋白分辨率定量分析这些相互作用。我们发现，虽然I型mAb促进高阶CD20寡聚化，但II型mAb诱导有限的聚集，导致治疗功能存在差异。将RESI与具有不同铰链区灵活性的II型抗体的功能研究相关联，我们表明寡聚化的CD20排列决定了I型或II型功能。因此，CD20-mAb复合物的纳米级表征增强了我们对治疗性抗体结构-功能关系的理解，并为下一代mAb疗法的设计提供了见解。
