Revisiting the Ofatumumab Epitope on CD20 through Integrative Molecular Dynamics and Flow Cytometry Analyses

Ofatumumab is a fully human anti-CD20 antibody used in B-cell malignancies and autoimmune disorders, yet its precise epitope recognition remains incompletely defined. Here, molecular dynamics simulations, binding free-energy analyses, and flow cytometry were combined to elucidate the structural basis of CD20 binding. The single-chain variable fragment recognizes a membrane-proximal epitope primarily centered on extracellular loop 2b/extracellular helix 2, stabilized by hydrophobic and electrostatic interactions involving the Y105-Y107-Y169 cluster and Arg228, while mutations at Asp99 and Tyr107 disrupted paratope stability and markedly reduced cellular binding. These results refine the molecular model of ofatumumab single-chain variable fragment-CD20 recognition and provide atomic-level insights that may assist the design of next-generation anti-CD20 and chimeric antigen receptor T-cell therapeutics.