Antibody Epitope of Human α‐Galactosidase A Revealed by Affinity Mass Spectrometry: A Basis for Reversing Immunoreactivity in Enzyme Replacement Therapy of Fabry Disease |
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Authors: | Dr. Zdenek Kukacka Dr. Marius Iurascu Loredana Lupu Hendrik Rusche Dr. Mary Murphy Lorenzo Altamore Fabio Borri Dr. Stefan Maeser Prof. Dr. Anna Maria Papini Prof. Dr. Julia Hennermann Prof. Dr. Michael Przybylski |
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Affiliation: | 1. Steinbeis Centre for Biopolymer Analysis and Biomedical Mass Spectrometry, Rüsselsheim am Main, Germany;2. Department of Chemistry, University of Konstanz, Konstanz, Germany;3. Ametek Reichert Technologies, Depew, NY, USA;4. French–Italian Interdepartmental Laboratory of Peptide and Protein Chemistry and Biology, Università degli Studi di Firenze, Sesto Fiorentino, Italy;5. Dipartimento di Chimica “Ugo Schiff”, Università degli Studi di Firenze, Sesto Fiorentino, Italy;6. PeptLab@UCP and Laboratory of Chemical Biology EA4505, Université Paris-Seine, Cergy-Pontoise, France;7. Department of Pediatrics, Villa Metabolica, Universit?tsmedizin Mainz, Mainz, Germany |
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Abstract: | α‐Galactosidase (αGal) is a lysosomal enzyme that hydrolyses the terminal α‐galactosyl moiety from glycosphingolipids. Mutations in the encoding genes for αGal lead to defective or misfolded enzyme, which results in substrate accumulation and subsequent organ dysfunction. The metabolic disease caused by a deficiency of human α‐galactosidase A is known as Fabry disease or Fabry–Anderson disease, and it belongs to a larger group known as lysosomal storage diseases. An effective treatment for Fabry disease has been developed by enzyme replacement therapy (ERT), which involves infusions of purified recombinant enzyme in order to increase enzyme levels and decrease the amounts of accumulated substrate. However, immunoreactivity and IgG antibody formation are major, therapy‐limiting, and eventually life‐threatening complications of ERT. The present study focused on the epitope determination of human α‐galactosidase A against its antibody formed. Here we report the identification of the epitope of human αGal(309–332) recognized by a human monoclonal anti‐αGal antibody, using a combination of proteolytic excision of the immobilized immune complex and surface plasmon resonance biosensing mass spectrometry. The epitope peptide, αGal(309–332), was synthesized by solid‐phase peptide synthesis. Determination of its affinity by surface plasmon resonance analysis revealed a high binding affinity for the antibody (KD=39×10?9 m ), which is nearly identical to that of the full‐length enzyme (KD=16×10?9 m ). The proteolytic excision affinity mass spectrometry method is shown here to be an efficient tool for epitope identification of an immunogenic lysosomal enzyme. Because the full‐length αGal and the antibody epitope showed similar binding affinities, this provides a basis for reversing immunogenicity upon ERT by: 1) treatment of patients with the epitope peptide to neutralize antibodies, or 2) removal of antibodies by apheresis, and thus significantly improving the response to ERT. |
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Keywords: | affinity mass spectrometry α -galactosidase A enzyme replacement therapy epitope identification Fabry disease |
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