Determination of the Epitopes of Alpha-Glucosidase Anti-Drug Antibodies in Pompe Disease Patient Plasma Samples

Abstract

Pompe disease is a rare autosomal-recessive neuromuscular disorder caused by a deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA), leading to the pathological accumulation of glycogen and impaired autophagy. Enzyme replacement therapy (ERT) with recombinant human alpha-glucosidase (rhGAA) has been available since 2006, but may lead to the formation of anti-drug antibodies (ADAs) against the recombinant human enzyme, which, in turn, may adversely affect the response to ERT. Knowledge of the antigenic determinants of rhGAA involved in interaction with ADAs may facilitate the development of strategies to attenuate the anti-drug immune response in patients. Here, we determined the rhGAA ADA epitopes in the plasma of Pompe disease patients using a series of affinity purifications combined with epitope extraction and label free quantitation LC-MS.

Keywords: anti-drug antibodies; enzyme replacement therapy; epitope mapping; label-free quantitation; mass spectrometry.

Figure 1

Analytical scheme of the anti-rhGAA anti-drug antibodies epitopes determination. (A) rhGAA immobilization. (B) anti-rhGAA ADA pull-down from patient plasma samples. (C) Wash. (D) Competitive elution of anti-rhGAA ADAs with rhGAA trypsin digest. (E) anti-rhGAA ADA pull-down by Protein A/G. (F). Wash. (G). Low pH elution of ADA-bound rhGAA epitope-containing tryptic peptides for subsequent identification by LC-MS. Anti-rhGAA ADAs are shown in red, non-specific immunoglobulins are shown in blue; epitope-containing and non-epitope-containing rhGAA peptides are shown as thick and thin lines, respectively.

Figure 2

The epitopes of rhGAA for anti-drug antibodies determined by epitope extraction analysis. Epitope-containing peptide sequences are highlighted in red.