Quantitative Analysis of Therapeutic Antibody Interactions with Fcγ Receptors Using High-Speed Atomic Force Microscopy

This study employed high-speed atomic force microscopy to quantitatively analyze the interactions between therapeutic antibodies and Fc gamma receptors (Fc gamma Rs). Antibodies are essential components of the immune system and are integral to biopharmaceuticals. The focus of this study was on immunoglobulin G molecules, which are crucial for antigen binding via the Fab segments and cytotoxic functions through their Fc portions. We conducted real-time, label -free observations of the interactions of rituximab and mogamulizumab with the recombinant Fc gamma RIIIa and Fc gamma RIIa. The dwell times of Fc gamma R binding were measured at the singlemolecule level, which revealed an extended interaction duration of mogamulizumab with Fc gamma RIIIa compared with that of rituximab. This is linked to enhanced antibody -dependent cellular cytotoxicity that is attributed to the absence of the core fucosylation of Fc-linked N-glycan. This study also emphasizes the crucial role of the Fab segments in the interaction with Fc gamma RIIa as well as that with Fc gamma RIIIa. This approach provided quantitative insight into therapeutic antibody interactions and exemplified kinetic proofreading, where cellular discrimination relies on ligand residence times. Observing the dwell times of antibodies on the effector molecules has emerged as a robust indicator of therapeutic antibody efficacy. Ultimately, these findings pave the way for the development of refined therapeutic antibodies with tailored interactions with specific Fc gamma Rs. This research contributes to the advancement of biopharmaceutical antibody design and optimizing antibody -based treatments for enhanced efficacy and precision.