Fig. 3: Quantitative analysis and structural modeling of therapeutic antibody-CD20 complexes.

a DBSCAN analysis (marked in gray) of RTX (cyan)-CD20 (magenta) clusters shows distinct higher-order structures in 2D. b Quantitative analysis in 2D reveals a linear relationship between the number of RTX molecules and CD20 dimers. Two ALFA-Nbs correspond to one RTX per cluster. A linear fit yields 0.38, suggesting that approximately one RTX binds per CD20 dimer, when correcting for the labeling efficiency (for details, see Methods). c Nearest-Neighbor Distance (NND) analysis indicates a higher-order organization, which can be modeled by a flexible-chain arrangement. d The model includes anchor points at the RTX hinge regions, linear segments connecting these points, CD20 dimers located centrally along these segments and ALFA-Nbs located at the hinge regions. e The flexible-chain model can explain how RTX-CD20 interactions lead to the formation of U-shaped clusters to facilitate C1q binding. The number of hexameric RTX platforms was determined by counting each possible binding configuration of C1q (see Methods). f DBSCAN cluster analysis for OBZ (green) with CD20 in 2D shows smaller clusters compared to RTX-CD20. g Quantitative analysis reveals specific OBZ to CD20 stoichiometries, without a linear relationship between the number of OBZ and CD20 molecules. Two ALFA-Nbs correspond to one OBZ per cluster. h NND analysis for CD20 complexes suggests that CD20 does not form higher-order structures beyond tetramers at the cell surface. NND analysis of the ALFA-Nbs labeling OBZ reveals only a first NND peak, representing two ALFA-Nbs bound per single OBZ. The absence of a second NND peak excludes a higher-order arrangement of OBZ-CD20 clusters. i Simulations with CD20 monomers, dimers, trimers and tetramers, taking into account the labeling efficiency of the GFP-Nb, for this representative cell result in 35 % monomers, 48 % dimers, 10 % trimers and 7 % tetramers after OBZ treatment. j, We observe a 25° angle between OBZ bound to CD20 and the xy-plane of the cell membrane. This suggests the necessity of membrane bending to allow for OBZ binding to CD20 with two Fab arms simultaneously. Created with the help of BioRender (https://BioRender.com/y76v9f4 and https://BioRender.com/5575210).