Antibody-mediated enzyme replacement therapy targeting both lysosomal and cytoplasmic glycogen in Pompe disease

Abstract

Pompe disease is characterized by accumulation of both lysosomal and cytoplasmic glycogen primarily in skeletal and cardiac muscles. Mannose-6-phosphate receptor-mediated enzyme replacement therapy (ERT) with recombinant human acid α-glucosidase (rhGAA) targets the enzyme to lysosomes and thus is unable to digest cytoplasmic glycogen. Studies have shown that anti-DNA antibody 3E10 penetrates living cells and delivers Bcargo^proteins to the cytosol or nucleus via equilibrative nucleoside transporter ENT2. We speculate that 3E10-mediated ERT with GAA will target both lysosomal and cytoplasmic glycogen in Pompe disease. A fusion protein (FabGAA) containing a humanized Fab fragment derived from the murine 3E10 antibody and the 110 kDa human GAA precursor was constructed and produced in CHO cells. Immunostaining with an anti-Fab antibody revealed that the Fab signals did not co-localize with the lysosomal marker LAMP2 in cultured L6 myoblasts or Pompe patient fibroblasts after incubation with FabGAA. Western blot with an anti-GAA antibody showed presence of the 150 kDa full-length FabGAA in the cell lysates, in addition to the 95-and 76 kDa processed forms of GAA that were also seen in the rhGAA-treated cells. Blocking of mannose-6-phosphate receptor with mannose-6-phosphate markedly reduced the 95-and the 76 kDa forms but not the 150 kDa form.

Targeting Pathogenic Lafora Bodies in Lafora Disease Using an Antibody-Enzyme Fusion

“…This antibody-enzyme fusion (AEF) strategy has proven successful for the replacement of a cytosolic enzyme and rescue of X-linked myotubular myopathy in mice (Lawlor et al, 2013). A Fab-GAA fusion has similar efficacy to reduce glycogen in Pompe mice with the additional advantage of targeting both lysosomal and cytosolic glycogen and is now in Phase 1/2 clinical trials (Valerion Therapeutics, 2016;Yi et al, 2017). Rather than utilizing the AEF platform to deliver a replacement enzyme, we generated an AEF designed to penetrate cells and degrade pathogenic LBs.…”

Targeting pathogenic Lafora bodies in Lafora disease using an antibody-enzyme fusion

“…While the glycogen in some GSDs is of normal structure like in Pompe disease, other GSDs such as Cori disease and Andersen disease are characterized by polyglucosans, like LD (7). Two AEF drugs (VAL-0417 and Fab-GAA) have now been shown to target, respectively, cytosolic polyglucosan (the present study) and normal glycogen in both the cytosol and lysosome (47). These drugs could be repurposed and/or modified to target glycogen in other GSDs.…”

“…In the first study, the deficient lipid phosphatase myotubularin fused to an Fv fragment was delivered by intramuscular injection and improved muscle function in a mouse model of myotubular myopathy (46). In the second, a Fab fragment fused to GAA delivered intravenously reduced glycogen load and alleviated pathology in GAA-deficient mice (47). These two studies illustrate that enzyme replacement therapy, which can be problematic for cytosolic enzymes, can be enhanced by fusion of the replacement enzyme with a cell-penetrating antibody fragment.…”

Pompe Disease: New Developments in an Old Lysosomal Storage Disorder

“…This monoclonal anti-DNA antibody were shown to penetrate living cells and move to the nucleus through the equilibrative nucleoside transporter 2 (ENT2) [113][114][115]. Experiments in cultured L6 myoblasts and fibroblasts derived from Pompe disease patients as well as in vivo studies in KO mice suggested a potential benefit of this fusion protein [116]. However, in our experience, VAL-1221 in KO mice did not show any improvement in muscle glycogen content (unpublished data).…”