The development and regulatory approval of Myozyme (alglucosidase alfa) for the treatment of Pompe disease represents the first major scientific and clinical breakthrough in the treatment of a life-threatening human myopathy. Several different forms of rhGAA were evaluated prior to the selection of CHO-GAA (as described within) for final clinical development and commercialization. In addition to CHO-GAA, these included rhGAA produced in milk of transgenic rabbits (tgGAA) and a carbohydrate engineered form of rhGAA containing high levels of M6P (HP-GAA) also produced in CHO cells. The HP-GAA, prepared by enzymatic addition of M6P to mannose residues, was of particular interest because effective targeting of GAA to the lysosomal compartment of cardiac and skeletal muscle depends upon M6P receptor mediated uptake [29
During trafficking to the lysosome, GAA undergoes complex processing during which glycans are remodeled and peptide segments are excised from the parent polypeptide to yield a multi-subunit complex [18
]. Previous studies have shown that the processed forms of GAA lack the M6P targeting signal and are not effectively taken up by human fibroblasts [26
]. Accordingly, the rhGAAs used in this study were restricted to the precursor form of the enzyme containing varying levels of M6P to ensure more effective targeting of exogenous enzyme to the lysosomal compartment of skeletal muscle.
The N-terminus of the CHO and HP-GAAs were located at residue 57, which was confirmed to be a pyroglutamic acid residue. The N-terminus of tgGAA was located at aspartic acid 67. Heterogeneity at the N-terminus of other GAA preparations either expressed in CHO cells or purified from human urine and placenta has been previously described and does not appear to impact enzyme function [24
]. Biochemical analyses of the various rhGAA preparations revealed that the oligosaccharides of HP-GAA, unlike those of CHO-GAA or tgGAA, were predominantly oligomannose and phosphorylated oligomannose structures. M6P analysis of these different preparations indicated levels of ~1.2 mol/mol for CHO-GAA, ~1.3 mol/mol for tgGAA and ~3.5 mol/mol for HP-GAA. Although the M6P levels of CHO and tgGAAs were comparable, the M6P residues of tgGAA were partially covered by N-acetylglucosamine (GlcNAc). Previous studies have shown that oligosaccharides containing GlcNAc-covered M6P structures have low affinity for the M6P receptor [34
]. The relative binding of the various rhGAAs to the immobilized sCIMPR, as judged by surface plasmon resonance analyses, was consistent with the level of exposed M6P (HP-GAA > CHO-GAA > tg-GAA). Likewise, the relative binding and uptake of the various forms of rhGAA by cells in vitro
demonstrated a strong correlation with the level of exposed M6P, with HP-GAA exhibiting an enhanced cellular uptake compared to either CHO-GAA or tgGAA. However, binding of HP-GAA to the mannose receptor was significantly higher than the binding of either tgGAA or CHO-GAA. The enhanced affinity of HP-GAA for the mannose receptor can be attributed to the predominance of oligomannose structures or glycan isoforms found at each of the seven N-linked sites of HP-GAA.
The relative efficacy of these different rhGAAs to reduce accumulated muscle glycogen in vivo
was assessed in a GAA knockout mouse model of Pompe disease [17
]. Systemic administration of all three forms of rhGAA resulted in a dose-dependent reduction of glycogen content in the cardiac and skeletal muscles of these animals as judged by histological analyses [22
]. Glycogen clearance from the cardiac muscle was significantly greater than that observed in skeletal muscle following administration of all three enzymes. Both CHO-GAA and HP-GAA were significantly more effective in clearing glycogen from the heart and skeletal muscle than was tgGAA. Furthermore, CHO-GAA appeared to be more effective, on a dose-dependent basis, than HP-GAA in clearing accumulated glycogen from the myocardium. Infantile clinical studies have shown that both CHO-GAA and tgGAA were effective in treating the myocardium [6
], and overall, our results suggest that all three recombinant enzymes are relatively effective in this tissue. The response in skeletal muscle is more difficult to interpret, partly because histopathologic findings from infantile muscle indicate significantly greater pathology than in GAA knockout mice [13
]. Comparison of these findings with human data is complicated by the relatively low glycogen load in Pompe mouse quadriceps (≈2–5%). Glycogen content in the quadriceps muscle of Pompe infants pre-treatment, measured by the same histomorphometric method, varied from ≈10–65% [13
]. Furthermore, infant muscle displays serious destructive intracellular pathology in correlation with glycogen load to the point of almost total obliteration of recognizable intracellular morphology in the worst cases, and this adds an additional factor with regards to therapeutic response. Our skeletal muscle data from the mouse clearly separated the in vivo
pharmacodynamic behavior of the three recombinant enzymes tested; however, translation of these results to human skeletal muscle is complicated by the considerable difference in the pathology of GAA deficiency in the two species.
Interestingly, heart and skeletal muscle tissue levels of the various rhGAA preparations did not correlate with efficacy in terms of glycogen clearance since the tissue levels of both tgGAA and HP-GAA were significantly higher than was observed for CHO-GAA. Western blot analysis indicated that all three enzymes were properly processed in the lysosomes of homogenized tissue. These data suggest that the higher levels of tgGAA and HP-GAA, compared to CHO-GAA, in target tissues may be a consequence of unproductive uptake of enzyme by resident endothelial cells and/or fibroblasts rather than glycogen-containing myocytes. This is particularly likely in the case of HP-GAA in which higher levels of exposed mannose may result in unproductive targeting of the enzyme to endothelial cells via the mannose receptor. Of note, recent studies demonstrated that chemically attaching synthetic bis-M6P containing glycans to oxidized sialic acid residues of CHO-GAA (neo-rhGAA) resulted in improved glycogen reduction from the skeletal muscles of GAA knockout mice [30
]. Since neo-rhGAA contains significantly less exposed mannose residues as compared to HP-GAA, these data demonstrate that if non-productive targets can be avoided, the concept of further exploiting uptake via the cation independent M6P receptor may offer a path to an improved therapeutic.
Finally, all of the enzyme preparations tested induced anti-GAA specific antibodies in the serum of the treated mice. The observation was not unexpected given that the mice were repeatedly administered high doses of a human recombinant protein to mice. Development of anti-GAA specific antibodies has also been observed in Pompe patients treated with ERT [10
]. However, it has been possible to clinically manage the immune response to ERT in both Pompe patients and GAA knockout mice in a manner that decreases infusion associated adverse reactions and in a large number of patients appears to provide for glycogen clearance from the muscle even in the presence of circulating antibodies.
Taken together, our in vitro and in vivo studies provided clear evidence that neither cell binding/uptake experiments nor the level of enzyme activity in whole tissues were predictive of efficacy in terms of clearance of accumulated muscle glycogen in the myocytes of GAA knockout mice. These results serve to emphasize the risks and uncertainties associated with using in vitro or tissue enzyme activity endpoints to predict efficacy in animal models of ERT for treatment of lysosomal storage disorders. Finally, the data presented in this report were instrumental in selecting the CHO-GAA candidate for further development and registration, leading to the first approved therapy for Pompe disease (Myozyme, alglucosidase alfa).