Crystal structure of the CD33/Fab-10C8 complex elucidates the mechanism of antibody antagonism in HBV-induced immunosuppression

BACKGROUND: Hepatitis B virus (HBV) infection persists through immune evasion strategies, including engagement of the inhibitory receptor CD33 (Siglec-3) by α2,6-linked sialoglycans on HBsAg. This interaction induces ITIM phosphorylation and SHP-1/2 recruitment, dampening myeloid cell activation. The monoclonal antibody 10C8 has been identified as a potent antagonist of CD33, but the structural basis for its inhibitory activity remains unclear. METHODS: We determined the 3.2 Å crystal structure of the human CD33 extracellular domain (CD33-ECD) in complex with the Fab fragment of 10C8 (Fab-10C8). The stoichiometry and assembly of the complex in solution were validated by size-exclusion chromatography and analytical ultracentrifugation (AUC). Structural comparisons with apo CD33 were performed to assess potential Fab-induced conformational changes, and buried surface area analyses were conducted to characterize binding interfaces. RESULTS: of surface area, while inter-Fab contacts stabilize a compact dimeric arrangement of CD33-ECD. This geometry differs markedly from the relaxed apo state, showing a ~21° reduction in the dimer angle. The antibody-binding epitope is adjacent to, but not overlapping with, the canonical sialic acid-binding cleft, leading to steric occlusion that prevents HBsAg engagement. Together with a potential Fab-induced ectodomain compaction, this conformation restricts CD33 clustering and likely precludes SHP-1/2 recruitment, providing a mechanistic explanation for the antagonistic activity of 10C8. CONCLUSIONS: This study provides the structural insight into antibody-mediated inhibition of CD33. By locking CD33 into a sterically occluded, signaling-refractory conformation, 10C8 effectively reverses HBV-induced immunosuppression, thereby restoring host antiviral activity. These findings establish a structural framework for rational design of Siglec-targeted immunotherapies against chronic viral infections and other immune regulatory disorders.