Cell and Protein Therapeutics, Genzyme Corporation, P.O. Box 9322, Framingham, MA 01701-9322, USA
Received on October 8, 2002; revised on November 19, 2002; accepted on November 22, 2002
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Abstract |
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Key words:
Fabry disease
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-galactosidase A
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glycosphingolipid
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lysosomal storage
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mannose-6-phosphate
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Introduction |
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Following initial studies which identified -galactosidase A as the enzyme deficient in Fabry disease, early attempts were made to replace the enzyme using human
-galactosidase A from a variety of sources (Brady et al., 1973
; Mapes et al., 1970
). These early studies demonstrated the potential utility of enzyme replacement therapy based on the clearance of GL-3 from the plasma of treated Fabry patients. Further progress in the treatment of this disease was limited by the lack of sufficient quantities of purified human protein.
The isolation and sequencing of the cDNA encoding human -galactosidase A in 1986 (Bishop et al., 1986
), the production of large amounts of recombinant human enzyme (Ioannou et al., 1992
) in Chinese hamster ovary (CHO) cells, and the development of a Fabry mouse model (Ohshima et al., 1997
; Wang et al., 1996
) provided the means to address enzyme supply issues and further explore the efficacy of enzyme replacement therapy (Ioannou et al., 2001
). These initial preclinical studies with the recombinant enzyme demonstrated that enzyme replacement therapy could clear accumulated GL-3 from both plasma and the affected tissues (kidney, spleen, and heart) of diseased animals in a dose-dependent manner.
These results, in combination with the success in treatment of Gaucher diseaseanother lysosomal storage diseasewith recombinant human ß-glucocerebrosidase (Cerezyme), led to the commercial development of two highly purified recombinant human -galactosidase A preparations. Both have been produced by genetic engineering techniques. One form of
-galactosidase A (Fabrazyme) is produced by established recombinant engineering techniques in a CHO cell line (Ioannou et al., 1992
), as had been successfully used to produce Cerezyme. The second form (Replagal) is produced utilizing a human cell line expression system. Although some biochemical properties have been reported for a CHO-derived human
-galactosidase A (Ioannou et al., 2001
), no biochemical information is available on the commercial preparations of either the CHO or human cell line-derived human
-galactosidase A. Clinically, both products are administered by IV infusion every 2 weeks. Fabrazyme is a lyophylized powder in 35-mg vials that are reconstituted and diluted prior to infusion at a dose of 1 mg/kg every 2 weeks. Replagal is provided as liquid formulation at a concentration of 1 mg/ml in 3.5-ml vials that are diluted prior to infusion at 0.2 mg/kg every 2 weeks. The two forms of human
-galactosidase A have both been approved for treatment of Fabry disease in Europe, albeit at the different doses, and are undergoing further clinical testing and review in the United States.
Although both enzyme preparations have demonstrated efficacy in human clinical trials, the different endpoints and dosing regimens used in the trials prevent direct comparison of the relative efficacies of the two products (Pastores and Thadhani, 2002).
Given the disparate clinical data, the lack of comparative preclinical and biochemical data, and the fact that head-to-head clinical trials have not yet been reported, we conducted biochemical, pharmacological, and immunological studies of the two proteins to determine if there are significant structural and/or functional differences that could account for the different recommended doses for the two products. Because both proteins are derived from the human -galactosidase A gene, any structural differences are likely to arise from differences in posttranslational modifications, particularly glycosylation, which can influence biodistribution, cellular uptake, stability, and potentially immunogenicity. We therefore focused this study on the comparison of posttranslational modifications and their potential impact on receptor binding, cell uptake, and biodistribution.
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Results |
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Reversed Phase HPLC analysis gave a complex profile with both preparations having three distinct protein peaks (Figure 2). To determine the identities of the three peaks, liquid chromatography mass spectrometry (LC/MS) analysis of tryptic digests was performed. This showed that both proteins had the predicted amino acid sequence, based on the published cDNA sequence, with the exception of the C-terminal residues. Both proteins displayed some degree of C-terminal heterogeneity with truncated species lacking either one or two C-terminal residues present in each preparation. Peak one in the chromatogram is -galactosidase A lacking both C-terminal leucine residues, peak two is lacking a single C-terminal leucine, and peak three is the full length protein (Figure 2). The relative amounts of these forms differed between the two preparations; in the Replagal sample the predominant molecular species (73%) lacked the C-terminal leucine, whereas the predominant species (70%) of Fabrazyme was the full-length protein.
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Changes in glycosylation are known to have a significant impact on the biological properties of recombinant glycoproteins. These differences can arise as a result of cell culture conditions as well as the expression system used to produce the protein (Goochee, 1992; James et al., 1995
; Jenkins and Curling, 1994
; Jenkins et al., 1996
). Because protein glycosylation can have a significant impact on the efficacy of protein therapeutics, mammalian cell lines are the preferred expression system for recombinant glycoprotein production. Although both enzyme preparations are produced from the same cDNA sequence by genetic modification of mammalian cells, the cell types used are different. Fabrazyme is produced in a CHO cell line, and Replagal is produced in a human cell line of undisclosed origin. As expected, in each case the resultant protein preparations are a heterogeneous mixture of glycoforms. To determine if these two expression systems lead to different glycosylation patterns that could affect biological properties, we analyzed both the type and site-specific location of the oligosaccharide chains for each enzyme preparation.
Monosaccharide analysis (Table III) indicated that there were differences in the ratio of complex to oligomannose oligosaccharides between the two preparations. The data also showed differences in the extent of phosphorylation and sialylation of these oligosaccharides. The higher level of fucose, N-acetylglucosamine, and galactose in Replagal indicated a higher percentage of complex oligosaccharides compared with Fabrazyme. Both proteins had the same amount of sialic acid on a mole/mole of protein basis; however, the higher amount of complex chains in Replagal resulted a lower sialic acid:galactose ratio than was observed for Fabrazyme. Consequently, Replagal would be considered less completely sialylated than the Fabrazyme.
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The differences in glycosylation implied by the monosaccharide compositions were confirmed by LC/MS analysis of the individual N-linked glycosylation sites. The sites of glycosylation on the two enzymes, that is, asparagine residues 108, 161, and 184, were the same. The predominant oligosaccharide species present at each of the glycosylation sites was identical for the two enzymes, although the relative ratios of these species did vary between Fabrazyme and Replagal (Table IV). Complex oligosaccharides were observed at Asn 108 in both proteins. Though the types of complex chains present at this residue were the same for each protein, significant differences were observed in the extent of sialylation. At asparagine 161 the predominant oligosaccharide species was monophosphorylated oligomannose 7 in both proteins. However, at this site there were considerably more complex type oligosaccharides present in Replagal (52%) than Fabrazyme (25%). Likewise at asparagine 184 the major oligosaccharide species was a phosphorylated oligomannose structure, but, as observed at asparagine 161, there were more complex oligosaccharides present in Replagal (34%) than in Fabrazyme (4%). The relative distribution of sialylated and phosphorylated oligosaccharides at each glycosylation site in Replagal and Fabrazyme is shown in Table V. At all three glycosylation sites there is a higher degree of sialylation and/or phosphorylation in Fabrazyme.
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Receptor binding and cell uptake
Based on current knowledge, one would predict that of the differences observed between the two proteins, the variation in glycosylation, in particular mannose-6-phosphate levels, had the greatest potential to influence bioactivity. This is because mannose-6-phosphate is required for both the binding of -galactosidase A to target cells via the mannose-6-phosphate receptor and the subsequent delivery to the lysosome. To determine if the observed differences in the level of phosphorylated oligomannose chains influenced the functional activity of the proteins, we performed receptor binding and Fabry fibroblast uptake studies. Receptor binding was evaluated using surface plasmon resonance to measure the interaction of
-galactosidase A with immobilized bovine soluble cation-independent mannose-6-phosphate receptor (sCIMPR). As predicted, from the higher level of mannose-6-phosphate, substantially more Fabrazyme bound to the receptor than did Replagal at all concentrations studied (Figure 3A). Cell uptake studies demonstrated that improved binding to the mannose-6-phosphate receptor correlated with enhanced uptake of Fabrazyme into Fabry fibroblasts. Although the uptake was similar at saturating concentrations of both proteins (Figure 3B), Fabrazyme clearly exhibited the more potent dose-response curve of the two enzyme preparations.
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Discussion |
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With the approval in Europe of Fabrazyme and Replagal, there is now a second lysosomal storage disease that can be treated by enzyme replacement therapy. Despite the fact that the active ingredient in both products is human -galactosidase A, they have been approved for use at different doses. Fabrazyme is recommended for use at 1 mg/kg and Replagal at 0.2 mg/kg. Because the protein sequence is the same for both products, the difference in recommended dosing most likely arises from different posttranslational modifications or is the outcome of different clinical strategies used to support product approval.
To determine if the fivefold difference in dose arises from biochemical differences between the two enzyme preparations, we performed the detailed structural and functional comparisons reported herein. Considering the different expression systems used for production, the two proteins were remarkably similar, even to the level of site-specific glycosylation. Differences were observed in the ratio of oligomannose to complex oligosaccharides at two of the three N-linked glycosylation sites and also in the levels of terminal sugar residues, with Fabrazyme having a higher percentage of phosphorylated oligomannose chains and a higher percentage of fully sialylated complex oligosaccharides. As would be expected, these differences in glycosylation resulted in improved binding of Fabrazyme to the cation-independent mannose-6-phosphate receptor, uptake into Fabry fibroblasts, and higher enzyme levels in the kidney, heart, and spleen when tested at the same dose in Fabry mice. These differences in glycosylation did not lead to different antigenic profiles that could affect efficacy because Fabry patient antibodies do not discriminate between the two proteins.
The studies reported herein demonstrate that biochemically and structurally the two enzyme preparations tested are very similar and, based on present knowledge of both Fabry disease and the role of glycosylation in protein clearance/distribution, provide no biochemical evidence to predict a fivefold difference in potency.
One of the major challenges in developing new therapies in general is the design of clinical trials that establish a new agent as both a safe and effective treatment for a particular disease. This is especially true in such diseases as the lysosomal storage disorders, for which no effective therapy exists. The limited number of patients, their often heterogeneous nature, and in some cases the slow progression of the diseases have encouraged the use of streamlined dosing strategies and surrogate endpoints to monitor clinical effects of enzyme replacement therapy. The magnitude of the challenges associated with establishing appropriate dosing are evident in the treatment of Gaucher's disease, where, despite several studies over the past 10 years, the optimal dosing regimen is still debated.
The results of the clinical trials on which Fabrazyme and Replagal were approved illustrate the difficulty of determining appropriate clinical endpoints and doses for diseases of this type (Eng et al., 2001b; Pastores and Thadhani, 2002
; Schiffmann et al., 2001
). Two entirely different clinical strategies were used in gaining European approval. The Fabrazyme pivotal (phase III) clinical trial evaluated the ability of enzyme replacement therapy to clear accumulated substrate (GL-3) from the affected tissues. The dose of 1 mg/kg chosen for this trial was based on a classical dose ranging study with three dose levels and two dosing regimens (Eng et al., 2001a
). A dose-response effect was observed for all doses (0.3, 1.0, and 3 mg/kg) with respect to both GL-3 clearance and infusion reactions. The 1 mg/kg dose every 2 weeks was chosen as the optimum from a clearance standpoint while limiting the infusion reactions.
In contrast, the Replagal trial evaluated the ability of enzyme replacement to therapy to reduce the pain associated with Fabry disease (Schiffmann et al., 2001). In this trial a dose of 0.2 mg/kg every 2 weeks was chosen. Because the phase I clinical trial for Replagal was based on a single infusion of 0.34.7 U/kg (0.070.11 mg/kg) (Schiffmann et al., 2000
), the rationale for choosing the 0.2 mg/kg dose for later studies is not clear, especially given the fact that the material used in the later studies was produced using a different expression system.
Both enzyme preparations were reportedly successful in reaching the primary endpoint chosen for their respective clinical trial, but neither was as successful in meeting the primary endpoint of the other trial. Fabrazyme was successful in clearing the renal micovascular endothelium of GL-3. Although an improvement in pain score was observed, it was not significant compared to the placebo group, though it was noted that the patients were not required to discontinue their pain medications during the trial (Eng et al., 2001b). Likewise, Replagal was reported successful in improving pain scores but was less effective in clearing GL-3. Given these disparate clinical results, there has been significant debate on the relative efficacy of the two products.
Indeed, when the clinical data for plasma GL-3 clearance are compared, both products appear to have similarly biological activity. Twelve 0.2 mg/kg infusions of Replagal reduced plasma GL-3 by 50% (Schiffmann et al., 2001) compared to a 7090% reduction following 5 0.3 mg/kg infusions of Fabrazyme (Eng et al., 2001a
) and a 100% reduction following 11 infusions at 1 mg/kg (Eng et al., 2001b
). These data suggest that plasma GL-3 clearance is dose-dependent and that equivalent reduction in GL-3 levels are seen with both enzyme preparations.
The GL-3 clearance data in conjunction with the biochemical analysis reported here support structural and functional equivalence of the two proteins. This suggests that the different dosing regimens currently used for the two preparations in Europe are a result of the different clinical trial designs rather than a functional difference between the two proteins.
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Materials and methods |
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Reversed-phase HPLC
Reversed-phase HPLC analysis was performed using a YMC 2.1x100 mm octyl column maintained at a temperature of 40°C. Protein (7.5 µg load) was eluted using a linear trifluoracetic acid/acetonitrile gradient at a flow rate of 0.25 ml/min with UV detection at 280 nm.
IEF
Ten micrograms of each protein were loaded on a pH 37 IEF gel run with premade buffer (Invitrogen, Carlsbad, CA). Focusing was for 1 h at 100 V, 1 h at 200 V, and 30 min at 500 V. Gels were stained for 1 h with PhastGel Coomassie Blue R-350 (Pharmacia, Uppsala, Sweden) and destained for 2 h in methanol/acetic acid.
Specific activity and kinetic measurements
Specific activities of the two enzymes were measured by the hydrolysis of the artificial substrates 4MU-Gal (Bishop and Desnick, 1981) and p-nitrophenyl-
-D-galactopyranoside (pNP-Gal) at 37°C at pH 4.6 in 20 mM citrate, 30 mM sodium phosphate, 0.1% bovine serum albumin, 0.67% ethanol, 0.02% sodium azide. For 4MU-Gal, the reaction was carried out at a substrate concentration of 10 mM and quenched after 15 min with NaOH and fluorescence read (excitation 365 nm, emission 460 nm) against a 4MU standard curve. For pNP-Gal, the reaction was carried out at a substrate concentration of 33 mM and quenched after 10 min with NaOH and the absorbance at 405 nm determined. Kinetic parameters for each substrate were based on measurements at five concentrations and Eadie-Hofstee plots were used to calculate Km and Vmax.
Monosaccharide analysis
Samples were dialyzed into 50 mM sodium phosphate pH 7.0, then 80 µg were removed and hydrolyzed in 2 M trifluoroacetic acid at 100°C for 2 h. Samples were cooled, evaporated, reconstituted in HPLC-grade water, and filtered through a 0.45-µm centrifugal filter (Durapore filters produced by Millipore, Bedford, MA). Analysis was performed using high-pH anion exchange chromatography with pulsed amperometric detection (Dionex). Mannose-6-phosphate was analyzed using a 10-min linear gradient of 170400 mM sodium acetate in 100 mM NaOH on a CarboPacPA10 column (Dionex Corp, Sunnyvale, CA). For sialic acid, hydrolysis was performed in 0.5 M formic acid at 80°C for 1 h, and elution was with a 20-min linear gradient of 50180 mM sodium acetate in 100 mM NaOH. Galactose, N-acetylglucosamine, mannose, and fucose were measured using a CarboPacPA1 column and Amino Trap precolumn (Dionex), and elution was with 200 mM sodium hydroxide over 45 min. Quantitation was performed relative to the appropriate monosaccharide standard curve. Mannose-6-phosphate and N-acetylneuraminic acid were from Sigma (St. Louis, MO); D-galactose, D-mannose, N-acetylglucosamine, and L-fucose were from Pfanstiehl Laboratories (Waukegan, IL).
LC/MS analysis
Samples were reduced and alkylated with 4-vinylpyridine (Sigma) prior to overnight digestion with Endoproteinase Lys-C (Roche, Indianapolis, IN) at a 1:25 enzyme:protein ratio. Digests (0.6 µg) were loaded onto a 320 µmx150 mm Vydac C18 column and peptides eluted with a linear gradient of acetonitrile in 1% formic acid at a flow rate of 4 µl/min. The eluant was fed directly into the electrospray source of a Bruker Esquire ion-trap mass spectrometer. For calculating glycoform distribution, spectra were averaged across the glycopeptide peak, and the amount of each glycoform was calculated as percentage of total response.
Receptor binding
The relative affinities for the sCIMPR were determined by surface plasmon resonance (BIAcor AB, Uppsala, Sweden). sCIMPR purified from fetal bovine serum was immobilized on a CM5 chip via the primary amine groups of lysine residues. Samples were serially diluted to 20, 10, 5, 2.5, 1.25, 0.63, and 0.31 µg/ml in 10 mM HEPES, pH 7.4, 3 mM ethylenediamine tetra-acetic acid, 150 mM NaCl, and 0.005% Tween 20. Binding was determined following a 3-min injection at a flow rate of 20 µl/min. The sCIMPR surface was regenerated prior to the next injection with two 30-s injections of 300 mM NaCl, 10 mM sodium citrate, pH 5.0, at a flow rate of 75 µl/min.
Fibroblast uptake
Fabry fibroblasts (NIGMS Coriell Institute) were seeded in 48-well plates at 1x104 cells/well in Earle's modified Eagle's medium (MEM), 10% fetal bovine serum, 1% MEM vitamin mix, 1% MEM nonessential amino acids, and 2% amino acids (Gibco, Gaithersburg, MD) and incubated overnight at 37°C and 5% CO2. Serial dilutions of samples were added to the cells and incubated for 24 h at 37°C. Following incubation, medium was aspirated and the cells lysed by freeze thawing. The activity of the cell lysates was determined at pH 4.4 using 4MU-Gal. Total protein content of the cell lysates was determined using a BCA total protein assay (Pierce, Rockford, IL).
Biodistribution
Three-month-old mice lacking the -galactosidase A gene (Wang et al., 1996
) were dosed intravenously with 3 mg/kg of each enzyme via the tail vein. Tissue samples were placed on ice, 10 volumes of lysate buffer (14.5 mM citric acid, 30 mM sodium phosphate, 0.15% Triton-X100, pH 4.4) added, and tissues homogenized for approximately 1 min using an IKA Ultra-Turrax T8 homogenizer set at approximately 25,000 rpm. Homogenates were centrifuged at 3500 rpm for 10 min at 4°C, and the supernatants were transferred to clean test tubes and centrifuged again at 3500 rpm for 5 min. Serial dilutions of tissue homogenate supernatant were prepared and assayed for
GAL activity using the pNP-Gal substrate.
Immunoreactivity with patient serum
Microtiter plates (96 well) plates were coated with a 5 µg/ml solution of either Fabrazyme or Replagal. After coating, plates were blocked with a 0.1% solution of human serum albumin in phosphate buffered saline and dilutions of Fabry patient serum added to the plates and incubated for 1 h. Plates were washed and binding of -galactosidase A-specific antibodies detected using goat anti-human IgG-horseradish peroxidase. Orthophenylenediamine substrate was added to each plate and titer determined by measuring the absorbance at 490 nm.
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Acknowledgements |
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1 To whom correspondence should be addressed; e-mail: tim.edmunds{at}genzyme.com
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Abbreviations |
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References |
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