Development and application of antibody framework region-targeted cross-linking mass spectrometry for epitope...

Identification of antibody-binding epitopes on antigens is essential for understanding the pathogenic roles of immune complexes (ICs) in autoimmune diseases and for the development of diagnostics and therapeutics. However, methods for direct epitope characterization of IC-antigens formed in vivo remain limited. Here, we investigated antibody framework region (FR)-targeted cross-linking mass spectrometry (XL-MS) as an approach to identify epitopes in patient-derived ICs. Four IC models with known epitopes (tumor necrosis factor alpha-adalimumab, tumor necrosis factor alpha-infliximab, human epidermal growth factor receptor 2-pertuzumab, and human epidermal growth factor receptor 2-trastuzumab) were analyzed using three cross-linkers of different lengths: bis(sulfosuccinimidyl) suberate (BS3), bis(sulfosuccinimidyl) glutarate (BS2G), and 1,1'-carbonyldiimidazole (CDI). BS3 preferentially generated antigen-FR cross-links, whereas CDI predominantly produced antigen-complementarity-determining region cross-links. Several cross-linked peptides contained antigen sequences overlapping previously reported epitopes, supporting the validity of this approach for epitope mapping. As a proof-of-concept clinical application, serum samples from patients with primary biliary cholangitis were analyzed using a targeted database containing four disease-associated autoantigens (PDC-E2, BCOADC-E2, OGDC-E2, and E3BP). Two candidate epitope peptides were identified. One corresponded to a known immunodominant epitope, whereas the other represented a novel candidate site supported by computational epitope prediction analysis. These findings demonstrate that FR-targeted XL-MS provides a strategy for identifying epitopes within patient-derived IC-antigens through the use of conserved antibody FRs. This approach may contribute to understanding antigen recognition mechanisms in autoimmune diseases and support the discovery of disease-relevant therapeutic targets.