Fig. 1: Assessing the structure-function relationship of anti-CD20 therapeutic antibodies in the cellular context using RESI microscopy.

a The structural configuration of CD20 proteins and their interactions with therapeutic antibodies on the cell membrane influence the therapeutic efficacy of the antibodies. b CD20 proteins (magenta) in the membrane exist as a mixture of monomers and pre-formed dimers¹. Rituximab (RTX) (cyan) can bind with 2 Fabs per CD20 dimer, thereby inducing higher-order arrangements of CD20 dimers^2,3. However, the quantitative nature of the 3D cluster organization is still unknown. In contrast, Obinutuzumab (OBZ) (green) can bind with one Fab per single CD20 dimer, suggesting a terminal complex of up to CD20 tetramers by bridging two CD20 dimers. However, the resulting structural organization of CD20 – potentially forming monomers, dimers, trimers, or tetramers – remains to be fully elucidated. c We use two-target RESI super-resolution microscopy with four rounds per target to visualize and correlate the locations of mEGFP-tagged CD20 and ALFA-tagged therapeutic antibodies at single-protein resolution (sub-5 nm). CD20 is labeled in 1:1 stoichiometry and mAbs are labeled in a 2:1 stoichiometry. By performing eight consecutive DNA-PAINT imaging rounds and clustering localizations, we achieve precise RESI localizations (σ ≈ 0.6 nm) of the targets in their cellular context. Created with the help of BioRender (https://BioRender.com/y76v9f4 and https://BioRender.com/5575210).