Impact of Clustered Antigenic Architectures on the Adhesion of Monoclonal Antibodies: Insights from Biomimetic Model Surfaces.

Monoclonal antibodies, thanks to their high affinity and specificity for their antigen targets, are increasingly used as highly selective therapeutics. While antigen expression and clustering at the cell surface critically influence therapeutic activity, quantifying these effects remains challenging due to the lack of well-defined in vitro platforms that enable precise control over antigen spatial organization and density. Surface-based affinity techniques, therefore, provide a powerful approach to address this challenge. For example, the monoclonal antibody rituximab (RTX) recognizes the human cluster of differentiation 20 (CD20) antigen through the formation of tight CD20 dimers and secondary antibody-antibody interactions. We previously reported an optimal inter-CD20 spacing of nearly 2 nm on gold surfaces for efficient interaction with RTX, consistent with concomitant crystallographic studies. However, these studies did not account for the heterogeneous distribution of antigens at the cell surface. Herein, we present well-defined and tunable biomolecular surface assemblies as analytical models of CD20 clusters grafted on self-assembled monolayers via click chemistry, followed by characterization using quartz-crystal microbalance coupled to spectroscopic ellipsometry and surface plasmon resonance. Inspired by biological systems, the present platform represents a simplified and static model that captures selected structural features (e.g., clustering and spacing).This study revealed that CD20 dimerization promotes strong and highly selective binding with RTX, providing a controlled and tunable model for investigating the impact of antigen clustering on antibody binding.