Expansion Microscopy Reveals the Spatial Association of Therapeutic Antibody–Receptor Complexes With Cholesterol‐Rich Membrane Domains

ABSTRACT Understanding the complex interplay between therapeutic monoclonal antibodies (mAbs) and target receptors within the plasma membrane is essential for improving immunotherapy efficacy. However, direct visualization of lipid nanodomains remains challenging due to their nanoscale dimensions and dynamic behavior. Here, we combine expansion microscopy (ExM) with a fluorescent perfringolysin O domain‐4 probe to map cholesterol‐rich nanodomains in the membrane of whole intact cells with a spatial resolution approaching ∼40 nm on a confocal setup. We demonstrate that cholesterol‐rich domains predominantly localize to actin‐supported membrane protrusions in COS‐7 cells. We directly visualize how the binding of therapeutic mAbs rituximab (RTX) and daratumumab (DARA) redistributes CD20 and CD38 on lymphoma and multiple myeloma cells, respectively, into cholesterol‐rich nanodomains. Accumulation of receptor/mAb complexes within lipid nanodomains creates Fc fragment‐dense regions that enhance signal transduction, apoptotic signaling, and complement‐dependent cytotoxicity. Furthermore, RTX binding induces pronounced B‐cell polarization and accumulation of CD20/RTX complexes in cholesterol‐rich membrane nanodomains, whereas CD38/DARA complexes exhibit a more homogeneous membrane distribution indicating that mAb binding‐induced receptor reorganization occurs in a cell‐type‐ and receptor‐dependent manner at the nanoscale. Our results show how ExM can be used advantageously to improve our understanding of the complex interplay of mAbs and lipid nanodomains.