Alpha-galactosidase (αGal) is a lysosomal enzyme that hydrolyses the alphagalactosyl moiety from glycosphingo-lipids. Mutations in the αGal genes lead to defect enzyme resulting in substrate accumulation and pathophysiology. The deficiency of αGal, called Fabry's Disease (FD), belongs to the lysosomal storage diseases. Effective treatment of FD has been developed by enzyme replacement therapy (ERT) by infusion of recombinant enzyme to increase enzyme levels and reduce accumulated substrate. Immuno-reactivity and IgG antibody formation are major, therapy-limiting complications of ERT. Here we report the antibody epitope identification of human αGal, αGal(309-332), using a combination of proteolytic excision of the immobilized immune complex and surface plasmon resonance (SPR)- biosensor mass spectrometry. The epitope peptide, αGal(309-332) was synthesized by solid-phase peptide synthesis; its affinity was determined by SPR (KD, 39 x 10-9 M) nearly identical to that of the full length enzyme (KD, 16 x 10-9 M). Proteolytic excision- mass spectrometry is shown to be an efficient tool for epitope identification of immunogenic lysosomal enzymes. Since the full length enzyme and the antibody epitope showed comparable binding affinities, this provides a basis for reversing immunogenicity by (i), treatment of patients with epitope peptide to neutralizing antibodies, or (ii), removal of antibodies by apheresis, thus significantly improving the response to ERT.

Antibody Epitope of human α-Galactosidase A revealed by affinity-mass spectrometry: a basis for reversing immunoreactivity in enzyme replacement therapy of Fabry Disease / Kukacka, Zdenek, Iurascu, Marius , Lupu, Loredana, Rusche, Hendrik, Murphy, Mary, Altamore, Lorenzo, Borri, Fabio, Maeser, Stefan, Papini, Anna-Maria, Hennermann, Julia, Przybylski, Michael. - In: CHEMMEDCHEM. - ISSN 1860-7179. - STAMPA. - 13:(2018), pp. 909-915. [10.1002/cmdc.201800094]

Antibody Epitope of human α-Galactosidase A revealed by affinity-mass spectrometry: a basis for reversing immunoreactivity in enzyme replacement therapy of Fabry Disease

ALTAMORE, LORENZO;BORRI, FABIO;Papini Anna-Maria;
2018

Abstract

Alpha-galactosidase (αGal) is a lysosomal enzyme that hydrolyses the alphagalactosyl moiety from glycosphingo-lipids. Mutations in the αGal genes lead to defect enzyme resulting in substrate accumulation and pathophysiology. The deficiency of αGal, called Fabry's Disease (FD), belongs to the lysosomal storage diseases. Effective treatment of FD has been developed by enzyme replacement therapy (ERT) by infusion of recombinant enzyme to increase enzyme levels and reduce accumulated substrate. Immuno-reactivity and IgG antibody formation are major, therapy-limiting complications of ERT. Here we report the antibody epitope identification of human αGal, αGal(309-332), using a combination of proteolytic excision of the immobilized immune complex and surface plasmon resonance (SPR)- biosensor mass spectrometry. The epitope peptide, αGal(309-332) was synthesized by solid-phase peptide synthesis; its affinity was determined by SPR (KD, 39 x 10-9 M) nearly identical to that of the full length enzyme (KD, 16 x 10-9 M). Proteolytic excision- mass spectrometry is shown to be an efficient tool for epitope identification of immunogenic lysosomal enzymes. Since the full length enzyme and the antibody epitope showed comparable binding affinities, this provides a basis for reversing immunogenicity by (i), treatment of patients with epitope peptide to neutralizing antibodies, or (ii), removal of antibodies by apheresis, thus significantly improving the response to ERT.
2018
13
909
915
Kukacka, Zdenek, Iurascu, Marius , Lupu, Loredana, Rusche, Hendrik, Murphy, Mary, Altamore, Lorenzo, Borri, Fabio, Maeser, Stefan, Papini, Anna-Maria, Hennermann, Julia, Przybylski, Michael
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Utilizza questo identificatore per citare o creare un link a questa risorsa: https://hdl.handle.net/2158/1113462
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