Preferential Binding of Anti-Neutrophil Cytoplasmic Antibodies to an Unexpected Epitope of a Chimeric Proteinase 3 Mutant

Abstract

AbstractProteinase 3 (PR3) is the major antigen for anti-neutrophil cytoplasmic antibodies (ANCAs) in the systemic autoimmune vasculitis, granulomatosis with polyangiitis (GPA). PR3-targeting ANCAs (PR3-ANCAs) recognize different epitopes on PR3 and are thought to be pathogenic for the development of the necrotizing vasculitis. To identify epitopes recognized by PR3-ANCAs, we pursued a strategy based on human-murine chimeric PR3 mutants. Interestingly, rather than observing reduced binding of PR3-ANCAs to Epitope 5 on a PR3 mutant (iHm5-Val103) with chimeric mutations in Epitope 5, we found substantially increased binding of the majority of PR3-ANCAs to iHm5-Val103 compared with the PR3 mutant (iPR3-Val103) clinically used to detect PR3-ANCAs. More interestingly, using iHm5-Val103 we identified a monoclonal antibody (moANCA518) from a patient with GPA that bound selectively to iHm5-Val103. Inhibition experiments using epitope-specific monoclonal antibodies and their antigen-binding fragments mapped the binding sites of moANCA518 and PR3-ANCAs (from patients displaying preferential binding to iHm5-Val103 over iPR3-Val103) to Epitope 3 on iHm5-Val103, a mutation-free epitope located far from the mutation sites in Epitope 5. These results demonstrate that the selective binding of moANCA518 (and likely the preferential binding of PR3-ANCAs from patients) to iHm5-Val103 is conferred by increased antigenicity of Epitope 3 on iHm5-Val103 caused by distal mutations, indicating that PR3-ANCAs bind to epitopes of a folded antigen conducive to allosteric effects of mutations—a previously unrecognized characteristic with implications for studying antibody-mediated autoimmune diseases and novel treatment approaches.