Long-Term Intravenous Treatment of Pompe Disease With Recombinant Human -Glucosidase From Milk

Objective. Recent reports warn that the worldwide cell culture capacity is insufficient to fulfill the increasing demand for human protein drugs. Production in milk of transgenic animals is an attractive alternative. Kilogram quantities of product per year can be obtained at relatively low costs, even in small animals such as rabbits. We tested the long-term safety and efficacy of recombinant human -glucosidase (rhAGLU) from rabbit milk for the treatment of the lysosomal storage disorder Pompe disease. The disease occurs with an estimated frequency of 1 in 40 000 and is designated as orphan disease. The classic infantile form leads to death at a median age of 6 to 8 months and is diagnosed by absence of -glucosidase activity and presence of fully deleterious mutations in the -glucosidase gene. Cardiac hypertrophy is characteristically present. Loss of muscle strength prevents infants from achieving developmental milestones such as sitting, standing, and walking. Milder forms of the disease are associated with less severe mutations and partial deficiency of -glucosidase. Methods. In the beginning of 1999, 4 critically ill patients with infantile Pompe disease (2.5–8 months of age) were enrolled in a single-center open-label study and treated intravenously with rhAGLU in a dose of 15 to 40 mg/kg/week. Results. Genotypes of patients were consistent with the most severe form of Pompe disease. Additional molecular analysis failed to detect processed forms of -glucosidase (95, 76, and 70 kDa) in 3 of the 4 patients and revealed only a trace amount of the 95-kDa biosynthetic intermediate form in the fourth (patient 1). With the more sensitive detection method, 35S-methionine incorporation, we could detect low-level synthesis of -glucosidase in 3 of the 4 patients (patients 1, 2, and 4) with some posttranslation modification from 110 kDa to 95 kDa in 1 of them (patient 1). One patient (patient 3) remained totally deficient with both detection methods (negative for cross-reactive immunologic material [CRIM negative]). The -glucosidase activity in skeletal muscle and fibroblasts of all 4 patients was below the lower limit of detection (<2% of normal). The rhAGLU was tolerated well by the patients during >3 years of treatment. AntirhAGLU immunoglobulin G titers initially increased during the first 20 to 48 weeks of therapy but declined thereafter. There was no consistent difference in antibody formation comparing CRIM-negative with CRIMpositive patients. Muscle -glucosidase activity increased from <2% to 10% to 20% of normal in all patients during the first 12 weeks of treatment with 15 to 20 mg/kg/week. For optimizing the effect, the dose was increased to 40 mg/kg/week. This resulted, 12 weeks later, in normal -glucosidase activity levels, which were maintained until the last measurement in week 72. Importantly, all 4 patients, including the patient without any endogenous -glucosidase (CRIM negative), revealed mature 76and 70-kDa forms of -glucosidase on Western blot. Conversion of the 110-kDa precursor from milk to mature 76/70-kDa -glucosidase provides evidence that the enzyme is targeted to lysosomes, where this proteolytic processing occurs. At baseline, patients had severe glycogen storage in the quadriceps muscle as revealed by strong periodic acid-Schiff–positive staining and lacework patterns in hematoxylin and eosin–stained tissue sections. The muscle pathology correlated at each time point with severity of signs. Periodic acid-Schiff intensity diminished and number of vacuoles increased during the first 12 weeks of treatment. Twelve weeks after dose elevation, we observed signs of muscle regeneration in 3 of the 4 patients. Obvious improvement of muscular architecture was seen only in the patient who From the *Department of Pediatrics, Division of Metabolic Diseases and Genetics, Erasmus MC-Sophia, Rotterdam, Rotterdam, The Netherlands; ‡Department of Clinical Genetics, Erasmus MC, Rotterdam, Rotterdam, The Netherlands; §Department of Pediatric Neurology, Erasmus MC-Sophia, Rotterdam, Rotterdam, The Netherlands; Hospital Pharmacy, Erasmus MC, Rotterdam, Rotterdam, The Netherlands; ¶Division of Pediatric Cardiology, Erasmus MC-Sophia, Rotterdam, Rotterdam, The Netherlands; #Division of Neonatology, Erasmus MC-Sophia, Rotterdam, Rotterdam, The Netherlands; **Department of Epidemiology and Biostatistics, Erasmus MC, Rotterdam, Rotterdam, The Netherlands; ‡‡Department of Neurology, Erasmus MC, Rotterdam, Rotterdam, The Netherlands; §§Division of Pediatric Intensive Care, Erasmus MC-Sophia, Rotterdam, Rotterdam, The Netherlands; Department of Pediatrics, Erasmus MC-Sophia, Rotterdam, Rotterdam, The Netherlands; ¶¶Pharming-Genzyme LLC, Leiden, The Netherlands; ##Department of Pediatrics, University Hospital Gasthuisberg, Leuven, Belgium; ***Department of Pediatrics, Division of Metabolic Diseases, University Medical Center Nijmegen, Nijmegen, The Netherlands; and ‡‡‡Department of Internal Medicine, Erasmus MC, Rotterdam, Rotterdam, The Netherlands. Received for publication Mar 21, 2003; accepted Dec 8, 2003. Reprint requests to (A.T.V.d.P.) Department of Pediatrics, Division of Metabolic Diseases and Genetics, Erasmus MC-Sophia, Dr Molewaterplein 60, 3015GJ Rotterdam, The Netherlands. E-mail: [email protected] PEDIATRICS (ISSN 0031 4005). Copyright © 2004 by the American Acad-