Sex-based antibody subclass maturation drives direct enzymatic inhibition in fabry disease patients receiving enzyme replacement therapy

Abstract

BACKGROUND: Enzyme replacement therapy (ERT) for Fabry disease can elicit anti-drug antibodies (ADAs) that may diminish efficacy and limit clinical benefit. OBJECTIVES: To develop and apply a multiplexed proteomic assay for quantitative, subclass-specific ADA and complement profiling in Fabry disease to inform personalized ERT selection. METHODS: We created a targeted LC-MS/MS platform to quantify ADA binding across IgG1-4, IgM, and IgA1, and complement proteins C1Qc-C9, in serum from Fabry patients (n = 39) and healthy controls. Neutralizing capacity was measured via enzymatic inhibition assay. Subclass-specific cross-reactivity was assessed for agalsidase alfa, agalsidase beta, and pegunigalsidase alfa. RESULTS: IgG4 binding was significantly higher in Fabry males (p = 0.007), with no sex-based differences for other Ig classes. Complement binding (C1Qc, C3) was elevated in ~25% of patients, with IgG1, IgG2, IgM, and IgA1 correlating with C1Qc (r > 0.6). Seven patients (three female) exhibited >50% ERT inhibition; IgG4 binding correlated with enzymatic inhibition (p 50% of immune complexes for agalsidase alfa/beta but only 25% for pegunigalsidase alfa, indicating a potentially distinct immunogenic profile. CONCLUSION: Quantitative subclass-specific ADA and complement profiling reveals sex-specific IgG4 patterns, neutralizing capacity, and ERT-specific immunogenic differences, supporting its utility for personalized therapy in Fabry disease. CAPSULE SUMMARY: A novel multiplex LC-MS/MS assay quantifies ADA subclasses and complement in Fabry disease, uncovering distinct IgG4 patterns and ERT-specific profiles that enhance understanding of treatment immunogenicity.