3 AVANT Immunotherapeutics, 119 Fourth Ave., Needham, MA 02494, and 4 Neose Technologies, 102 Witmer Road, Horsham, PA 19044
Received on March 24, 2004; revised on June 4, 2004; accepted on June 4, 2004
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Abstract |
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Key words: glycoengineering / glycoprotein remodeling / glycosylation / glycosyltransferase
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Introduction |
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In some clinical situations, complement inhibition therapy could be more effective if it were targeted directly to sites of endothelial activation. At sites of inflammation, activated endothelial cells express E-selectin and P-selectin, surface adhesins with carbohydrate-binding domains that recognize the carbohydrate epitope, sLex (Neu5Ac2-3Galß1-4[Fuc
1-3]GlcNAcß1-) (Lasky, 1995
).
Previously we have described sCR1-sLex (Picard et al., 2000; Rittershaus et al., 1999
), a variant of the sCR1 glycoprotein conveniently produced in LEC11 cells transfected with the sCR1 gene. LEC11 is a mutant Chinese hamster ovary (CHO) cell line that expresses fucosyltransferase VI (FT-VI), a Golgi enzyme capable of adding fucose in
1-3 linkage to GlcNAc in oligosaccharide chains that terminate with either Galß1-4GlcNAcß1 ... or NeuAc
2-3Galß1-4GlcNAcß1 ...(Zhang et al., 1999
). Of the 25 potential N-glycosylation sites within the sCR1 polypeptide sequence, 1315 are occupied, the majority with biantennary chains, creating the possibility for as many as 30 sLex moieties per molecule of sCR1-sLex. However, a previously reported analysis of the N-glycans of sCR1-sLex showed heterologous oligosaccharides with a variety of partially sialylated and fucosylated structures yielding less than the maximal number of sLex moieties (Picard et al., 2000
; Rittershaus et al., 1999
). Similar heterogeneity of glycans in CHO-expressed glycoproteins has been described previously and attributed to incomplete Golgi processing, postsecretion degradation due to glycohydrolases released into cell culture media, or both (Goochee et al., 1991
; Jenkins et al., 1996
).
In this article we describe a process to introduce sLex moieties onto the N-glycan chains of sCR1 produced in standard CHO cell lines using in vitro enzymatic synthesis. This method employs serial treatment of sCR1 with soluble recombinant rat ST3Gal-III and human FT-VI to give an sCR1-sLex product, designated sCR1-S/F (for differentiation from the LEC11 product) in which the antennae of N-glycans are nearly uniformly terminated with sLex epitopes. The benefits of in vitro glycan remodeling include improved pharmacokinetics, enhanced binding to E-selectin, and a means to improve product homogeneity. Enzymatic remodeling is demonstrated at the 10-g scale.
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Results |
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From the mannose content of sCR1, sCR1-S, sCR1-S/F, and sCR1-sLex (Table I) it may be inferred that these molecules contain 1315 N-glycan chains per mol protein (assuming 3 mol mannose per N-glycan chain). The fluorophore-assisted carbohydrate electrophoresis (FACE) oligosaccharide profile for sCR1 (Figure 1) shows three major bands consistent with a biantennary structure containing zero, one, or two sialic acid residues, as described previously (Picard et al., 2000
). The monosaccharide composition of sCR1 (Table I) suggests that
57% of total galactosyl residues are substituted with sialic acid (19 mol sialic acid/33.2 mol galactose). By comparison, the FACE oligosaccharide profile for sCR1-S (Figure 1) shows one major band that migrates at a position consistent with a biantennary structure containing two sialic acid residues, and monosaccharide analysis reveals the galactose/sialic acid ratio to be 1:1 (Table I).
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Oligosaccharide sequencing using FACE
The linkage of terminal sialic acids on sCR1-S/F was assessed by digestion with specific neuraminidases (Figure 3). Complete removal of sialic acid by treatment of band 1 from sCR1-S/F with NANase I indicates that sialic acid residues are 2-3 linked, as expected.
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Remodeling at 10-g scale
Purified sCR1 (10 g in a volume of 2 L) was incubated first with ST3Gal-III plus CMPsialic acid at 32°C for 36 h and then, following addition of FT-VI plus GDP-fucose, incubated at 32°C for another 36-h period.
FACE analyses of glycans from sCR1, sCR1-S, and sCR1-S/F for reactions performed at the 10-g scale (data not shown) were essentially indistinguishable from FACE results obtained at the 250-mg scale (Figure 1), suggesting that occupancy of potential acceptor sites for ST3Gal-III and FT-VI on sCR1 at the 10-g scale was nearly complete.
HPLC profiles for 2-AA-derivatized glycans of sCR1, sCR1-S, and sCR1-S/F are shown in Figure 6 and the percentages of glycan species estimated from integrated peak areas are summarized in Table II. After in vitro sialylation with ST3Gal-III, neutral glycans, comprising 50% of carbohydrate chains in sCR1, are reduced to 2% of chains in sCR1-S, and monosialo-glycans likewise decrease to from 35% in sCR1 to 17.5% in sCR1-S (Figure 6 and Table II). Overall, about 90% of N-glycans are biantennary and these chains contain an average of 1.8 sialic acid moieties per glycan. Among the minority of biantennary glycans on sCR1-S that are monosialylated, some lack galactose on one antenna, whereas others contain two galactosyl residues, only one of which is sialylated. The remaining 10% of glycans are fully sialylated triantennary (8.5%) or tetraantennary (1.5%) structures.
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Pharmacokinetics
When sCR1-S prepared at the 250-mg scale was injected intravenously into rats, the observed area under the curve (AUClast) was twofold greater than the AUClast for sCR1 (p < 0.004), indicating a significantly greater exposure of the more completely sialylated form of the complement inhibitor to intravascular cells following dosing (Figure 8).
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Discussion |
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That the sCR1 protein remains intact under conditions of glycan remodeling was demonstrated by RP-HPLC and SDSPAGE analyses showing single polypeptides with expected molecular weights for sCR1-S and sCR1-S/F. Evidence for (1) conformational stability under conditions of the in vitro glycosylation reactions, and (2) preserved function despite variations in glycan structure, is provided by the observed near equivalence in bioactivity of sCR1, sCR1-S, sCR1-S/F, and CHO-produced sCR1-sLex in a standard complement inhibition assay.
The oligosaccharide structures associated with sCR1-S and sCR1-S/F were assessed by a number of methods. FACE profiling demonstrated a more fully sialylated set of glycoforms for sCR1-S as compared with sCR1 and nearly homogeneous, fully sialylated and fucosylated biantennary N-glycans for sCR1-S/F. Sequencing experiments using FACE provided supporting evidence that sialic acid was linked 2-3 to galactose and that the predominant, single oligosaccharide band derived from sCR1-S/F was BiNA2F2. The analyses we performed do not establish linkages between the terminal and penultimate sugars that define sLex (NeuAc
2-3Galß1-4[Fuc
1-3]GlcNAcß1-) versus sLea (NeuAc
2-3Galß1-3[Fuc
1-4]GlcNAcß1-). However, two factors make it likely that the glycans of sCR1-S/F do, in fact, terminate in sLex. First, it is known that in CHO cells, N-linked glycans are most commonly formed by ß4GalT-1, and hence have the type-2 structure, Galß1-4GlcNAcß1- (Lee et al., 2001
). Second, the acceptor specificity of FT-VI is known to be restricted to type 2 chains (Costache et al., 1997
; Weston et al., 1992
).
During optimization of the sialylation reaction, we noted that incubation of sCR1 with either a low concentration of ST3Gal-III (10 mU/ml) for 24 h or a higher concentration (75 mU/ml) for 1 h produced a nearly maximally sialylated product. Even after incubation at the highest concentration of sialyltransferase tested (600 mU/ml for 24 h), a small fraction of monosialylated biantennary species persisted, perhaps due to steric hindrance at particular sites. Improved pharmacokinetics observed for the fully sialylated sCR1-S molecule as compared with sCR1 is probably a consequence of the added sialic acid blocking the interaction of terminal galactosyl residues with hepatic asialoglycoprotein receptors (Stockert, 1995).
We observed that FT-VI at 25 mU/ml fucosylates most sialylated biantennary glycans within 24 h. No significant differences were observed in catalytic activities of FT-VI expressed in the NSO cell line versus Aspergillus niger expression systems. The sCR1 polypeptide was shown to be stable following prolonged incubation with enzyme from either source.
In vitro glycosylation of sCR1 at the 10-g scale was carried out at enzyme concentrations selected to ensure nearly complete reaction at each stage. Success with the single experiment reported is consistent with the ability to predict useful scaled-up reaction conditions over a range of at least 40-fold based on mass of starting substrate. Both the ST3Gal-III and FT-VI enzymes used to glycosylate 10 g sCR1 were produced in A. niger, an expression system widely used for the manufacture of industrial enzymes in ton quantities. Although further scale-up would require refinement of incubation conditions, it can be estimated from present results that glycosylation of 1 kg of sCR1 might require 40,000 U ST3Gal-III and 20,000 U FT-VI, amounts that seem plausible to produce at reasonable cost in an industrial setting. To our knowledge, this is the largest scale reported enzymatic glycosylation of a glycoprotein to date by several orders of magnitude (Fischer and Dorner, 1998; Nemansky et al., 1995
; Paulson et al., 1977
; Raju et al., 2001
; Thotakura et al., 1994
).
The optimized conditions chosen for scale-up were very similar to the conditions used to generate material used for in vivo and in vitro studies. Compared with sCR1-sLex, sCR1-S/F was shown to have twice the number of sLex moieties and about a 10-fold higher apparent affinity for binding to E-selectin. This higher affinity presumably results from increased cooperativity in a multivalent binding reaction wherein sLex moieties distributed widely over sCR1-S/F engage multiple immobilized E-selectin molecules. In certain clinical situations, the anticomplement inhibitory and anti-inflammatory activity of sCR1-S/F could be effectively targeted via a similar mechanism to sites of inflammation where endothelial cells have been activated and have up-regulated expression of adhesion molecules including P- and E-selectin. sCR1-sLex has been shown to be superior to sCR1 in a complement- and selectin-dependent lung injury model (Mulligan et al., 1999), a murine model of ischemic stroke (Huang et al., 1999
), moderating skeletal muscle reperfusion injury (Kyriakides et al., 2001a
), moderation of acid aspiration injury (Kyriakides et al., 2001b
), reducing ischemia/reperfusion injury in rat lung grafts (Schmid et al., 2001
), and a myocardial ischemia and reperfusion model in the rat. sCR1-sLex significantly reduced myocardial infarct size and was significantly more effective than sCR1 in reducing neutrophil infiltration into the infarction (Zacharowski et al., 1999
). It will be interesting to investigate whether sCR1-S/F is even more effective than sCR1-sLex in similar animal models.
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Materials and methods |
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Preparation of sCR1-S
Lyophilized sCR1 (250 mg) was reconstituted and buffer exchanged into 50 mM Tris, 0.15 M NaCl, 0.05% NaN3, pH 7.2, using gel filtration columns (PD-10, Amersham Biosciences), and the concentration of sCR1 was adjusted to 5 mg/ml with the same buffer. Following addition of ST3Gal-III (150 mU/ml) and CMPsialic acid (7 mM) the mixture was incubated at 32°C. A separate aliquot of the reaction mixture to which a trace amount of CMP[14C]sialic acid was added was incubated in parallel. From this, aliquot samples were withdrawn at various times and fractionated by isocratic HPLC/size-exclusion chromatography at 0.5 ml/min in 45% MeOH, 0.1% trifluoracetic acid (7.8 mm x 30 cm TSKG2000SWXL column, particle size 5 µm, TosoHaas). Incorporation of sialic acid into glycoprotein was calculated from the fraction of counts in the first eluted peak and the known concentration of sugar nucleotide.
Preparation of sCR1-S/F
After the sialylation reaction had proceeded for 48 h, GDP-fucose was added to a final concentration of 7 mM, MnCl2 to 5 mM, and rFT-VI to 0.1 U/ml. A trace amount of GDP-[14C]fucose was added to a separate aliquot, and both reaction mixtures were incubated at 32°C. Chromatography of the radiolabeled mixture as described showed the transfer of 44 moles/mole sCR1-S after 48 h and 47 moles after 48 h. The product was provisionally designated sCR1-S/F.
Removal of nucleotide sugars and residual glycosyltransferases using ceramic hydroxyapatite and Q Sepharose chromatography
Glycosyltransferases and nucleotide sugars were removed from remodeled sCR1-S and sCR1-S/F by chromatography on ceramic hydroxyapatite (type I; BioRad, Hercules, CA) followed by Q Sepharose (Amersham Biosciences). Purity was assessed by RP-HPLC on a Poros R1/10 column (4.6 mmD/100 mmL, Applied Biosystems, Framingham, MA).
Optimization of sialylation and fucosylation reactions prior to scale-up
sCR1 was thawed slowly at 4°C and buffer exchanged into 50 mM TrisHCl, pH 7.5, 150 mM NaCl, using a PD10 column. In vitro sialylation of sCR1 (5 mg/ml) was evaluated using varying amounts of ST3Gal-III, 5 mM CMPsialic acid, in the presence of 0.02% sodium azide at 32°C for 24 h. A trace amount of CMP-[14C]sialic acid was added to an aliquot to monitor incorporation of radioactive sialic acid as described.
To the product (sCR1-S) of the reaction performed at a sialyltransferase concentration of 100 mU/ml (still containing the sialylation reagents) was added MnCl2 and GDP-fucose, each to a final concentration of 5 mM, varing amounts of FT-VI, and a trace amount of GDP-[3H]fucose. The resulting reaction mixture was incubated at 32°C for 24 h. Incorporation of radioactive fucose into the product (sCR1-S/F) was monitored as described for sialic acid.
sCR1 remodeling at 10-g scale
Purified sCR1 (10 g) was dialyzed exhaustively at 4°C against 50 mM TrisHCl, 0.15 M NaCl, pH 7.5, adjusted to a concentration of 5 mg/ml with the same buffer, and incubated with ST3Gal-III (200 mU/ml) and CMP-sialic acid (5 mM) for 36 h at 32°C in a final volume of 2 L. After 36 h, an aliquot containing the sialylated product (sCR1-S) was withdrawn for analysis and the following reagents (final concentrations) were added: rFT-VI (100 mU/ml), GDP-fucose (5 mM), MnCl2, (5 mM). After further incubation at 32°C for 36 h, a precipitate (manganese phosphate) was removed by centrifugation at 3000 x g for 5 min, and the sialylated and fucosylated product (sCR1-S/F) was stored at 70°C.
Monosaccharide analysis by HPLC
The neutral and amino sugar composition of glycoproteins was determined after trifluoroacetic acid hydrolysis and reductive amination with anthranilic acid by C18 reverse-phase HPLC with fluorescence detection (Anumula, 1994). Sialic acid content was determined after sodium bisulfate hydrolysis and reaction with o-phenylenediamine by C18 reverse-phase HPLC with fluorescence detection (Anumula, 1995
).
Carbohydrate analysis by FACE
Carbohydrate sequencing and electrophoresis by FACE (Glyko and ProZyme, San Leandro, CA) was performed as previously described elsewhere (Picard et al., 2000).
Carbohydrate analysis by 2-AA HPLC and MALDI-TOF MS
Glycans were released by PNGaseF and labeled with 2-AA according to the method described by Anumula and Dhume (1998) except that the labeled glycans were purified on cellulose cartridges (Glyko) according to the manufacturer's instructions. 2-AA-labeled N-glycans were analyzed using a Shodex Asahipak NH2P-50 4D amino column (4.6 mm x 150 mm). The two solvents used for the separation were (A) 2% acetic acid and 1% tetrahydrofuran in acetonitrile and (B) 5% acetic acid, 3% triethylamine, and 1% tetrahydrofuran in water. The column was eluted isocratically with 70% A for 2.5 min, followed by a linear gradient from 70% to 5% A over a period of 97.5 min, and a final isocratic elution with 5% A for 15 min. Eluted peaks were detected using fluorescence detection with an excitation wavelength of 230 nm and an emission wavelength of 420 nm.
For MALDI-TOF analysis, a small aliquot of the 2-AA-labeled N-glycans was dialyzed for 45 min on an MF-Millipore membrane filter (0.025 µm pore, 47 mm diameter) floating on water. The dialyzed aliquot was dried in a vacuum centrifuge, redissolved in a small amount of water, and mixed with a solution of 2,5-dihydroxybenzoic acid (10 g/L) dissolved in water:acetonitrile (50:50). The mixture was dried onto the target and analyzed using an Applied Biosystems DE-Pro MALDI-TOF mass spectrometer operated in the linear/negative-ion mode. Glycan structures were assigned based on the observed mass-to-charge ratio and literature precedence. No attempt was made to fully characterize isobaric structures.
SDSPAGE
sCR1 samples before and after in vitro enzymatic remodeling were separated on 816% gradient Tris-glycine polyacrylamide gels and stained with colloidal blue Coomassie stain. Gels, staining solutions, and molecular weight standards were obtained from Invitrogen (Carlsbad, CA).
Assays of complement regulatory activity
The inhibition of complement-mediated lysis of antibody-sensitized sheep erythrocytes (classical pathway) was assessed as previously described (Scesney et al., 1996).
E-selectin binding assay
E-selectin binding assays were performed according to previously reported methods (Weitz-Schmidt et al., 1996). Flat-bottom 96-well microtiter plates were coated with 5 µg/ml recombinant human E-selectin (R&D Systems) in 150 mM NaCl, 1 mM CaCl2, 20 mM HEPES, pH 7.4 (HEPES-buffered saline, HBS). Coated wells were blocked with 2% bovine serum albumin/HBS. Varying concentrations of sCR1 or sCR1-sLex were added to the plate. A complex of a biotinylated polyacrylamide polymer containing sLex (PAA-sLex, GlycoTech) and SA-HRP was prepared. A dilution of this conjugate complex was added to the wells containing sCR1 or sCR1-sLex or buffer and incubated for 90 min at room temperature. The wells were washed with HBS/CaCl2 and 3,3',5,5'-tetramethylbenzidine substrate (KPL) was added to each well. Color was allowed to develop for 15 min, and the reaction was stopped with 2.0 N H2SO4. Bound PAA-sLex complex was measured by determining the absorbance at 450 nm with a microplate reader (Molecular Devices, Sunnyvale, CA).
Pharmacokinetic analysis in rats
Male Sprague-Dawley rats (250 g), with in-dwelling jugular vein cannulas were purchased from Taconic (Germantown, NY) or Harlan Sprague Dawley (Indianapolis, IN). The catheters were periodically flushed with 0.9% saline followed by either heparinized glycerol (1:4 glycerol/333 IU heparin/ml) or heparinized saline (333 IU/ml) to ensure patency.
Animals were injected with sCR1 or sCR1-S (10 mg/kg) via the lateral tail vein as a bolus at time 0. Blood samples were obtained at timed intervals from the jugular vein cannula. The levels of sCR1 and sCR1-S present in the plasma samples were measured by a previously described enzyme-linked immunosorbent assay (Rittershaus et al., 1999). Briefly, microtiter plates were coated with anti-sCR1 monoclonal antibody 6B1.H12 and captured sCR1 from a sample was detected with an HRP-conjugated anti-sCR1 monoclonal antibody 4D6.1. Pharmacokinetic data was analyzed using WinNonlin (Pharsight, Mountain View, CA).
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Acknowledgements |
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Footnotes |
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1 These authors contributed equally to this work.
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Abbreviations |
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References |
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