Blood test ID’s where antibodies bind to Pompe disease ERT
Pinpointing location may help reduce antibody-generating immune response
Locations where antibodies bind to enzyme replacement therapy (ERT) and potentially reduce its effectiveness in treating Pompe disease can now be identified using a drop of blood, a study reports.
Knowing where anti-ERT antibodies bind may help find interventions to diminish the immune responses that generate them and also guide the development of next-generation ERTs, said the researchers, who noted the approach may also be used to characterize antibodies against other protein-based therapies. The study, “Determination of the Epitopes of Alpha-Glucosidase Anti-Drug Antibodies in Pompe Disease Patient Plasma Samples,” was published in Antibodies.
Pompe is caused by mutations in the GAA gene, which provides instructions for making the acid alpha-glucosidase (GAA) enzyme, which is responsible for breaking down the complex sugar molecule glycogen into glucose for use in generating energy. When GAA is missing or deficient, glycogen builds up to toxic levels inside primarily muscle cells, resulting in muscle weakness and other disease symptoms.
ERT for Pompe disease is a class of medications that provide a lab-made version of GAA. Regular treatment can clear excess glycogen from muscle tissues, ease symptoms, and improve motor outcomes and life expectancy. Because it’s lab-made, however, it can trigger an immune response that generates anti-drug antibodies, or ADAs that can dampen its effectiveness and increase the likelihood of side effects.
Knowing where on the surface of the GAA enzyme those ADAs bind — called epitopes — may help researchers find ways to lower this immune response and/or guide the development of next-generation ERTs. Few studies, however, have located epitopes on enzymes using ADAs directly from patient blood samples because these antibodies’ levels in blood can be very low and multiple ADAs may bind to different locations on GAA.
Locating antibody binding sites
Researchers at McGill University in Canada have developed a method to find ADA epitopes on ERT proteins using a minimal amount of patient blood.
They used a drop of blood from a Pompe patient who was treated in Germany with Myozyme (alglucosidase alfa), an approved Pompe ERT sold in the U.S. as Lumizyme. The sample was mixed with tiny beads covered in the treatment protein itself (alglucosidase alfa, or rhGAA), which allowed antibodies in the blood that recognized rhGAA — the problematic anti-drug antibodies — to stick to the beads.
Using this method, the researchers could isolate the patient’s ADAs bound to rhGAA. The enriched ADA-rhGAA mixture was then treated with chopped-up pieces of the same treatment protein, which let the ADAs bind to the fragments containing their own epitopes.
Once the antibodies were bound to the fragments, the team pulled these complexes out using another type of bead, then isolated the fragments and examined them. Blood from a healthy person was also processed to confirm the findings.
The researchers identified nine epitopes that bound ADAs, each of which was mapped onto the surface of the three-dimensional structure of rhGAA to find where they were located in the native protein.
Most epitopes were located in protrusions on the surface of the rhGAA enzyme and were clustered at two locations. The first location was close to the site that interacted with glycogen, indicating some ADAs may reduce ERT effectiveness by blocking enzyme activity. The second cluster was close to the site that interacts with the mannose-6-phosphate receptor, which is needed to move rhGAA into cells. Thus, ADAs may prevent the protein from binding that receptor and uptake of rhGAA into cells, the researchers suggested.
“We have developed an analytical workflow for the determination of the epitopes of protein drug ADAs from a minimal amount of patient plasma,” the researchers wrote. “The developed approach is generally applicable for the characterization of ADAs in other protein-based therapies.”
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