Production of a complement inhibitor possessing sialyl Lewis X moieties by in vitro glycosylation technology

Lawrence J. Thomas1,3, Krishnasamy Panneerselvam1,4, David T. Beattie3, Michele D. Picard3, Bi Xu3, Charles W. Rittershaus3, Henry C. Marsh, Jr.3, Russell A. Hammond3, Jun Qian4, Tom Stevenson4, David Zopf4 and Robert J. Bayer2,4

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


    Abstract
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Recombinant soluble human complement receptor type 1 (sCR1) is a highly glycosylated glycoprotein intended for use as a drug to treat ischemia-reperfusion injury and other complement-mediated diseases and injuries. sCR1-sLex produced in the FT-VI-expressing mutant CHO cell line LEC11 exists as a heterogeneous mixture of glycoforms, a fraction of which include structures with one or more antennae terminated by the sialyl Lewis X (sLex) [Neu5Ac{alpha}2-3Galß1-4(Fuc{alpha}1-3)GlcNAc]) epitope. Such multivalent presentation of sLex was shown previously to effectively target sCR1 to activated endothelial cells expressing E-selectin. Here, we describe the use of the soluble, recombinant {alpha}2-3 sialyltransferase ST3Gal-III and the {alpha}1-3 fucosyltransferase FT-VI in vitro to introduce sLex moieties onto the N-glycan chains of sCR1 overexpressed in standard CHO cell lines. The product (sCR1-S/F) of these in vitro enzymatic glycan remodeling reactions performed at the 10-g scale has approximately 14 N-glycan chains per sCR1 molecule, comprised of biantennary (90%), triantennary (8.5%), and tetraantennary (1.5%) structures, nearly all of whose antennae terminate with sLex moieties. sCR1-S/F retained complement inhibitory activity and, in comparison with sCR1-sLex produced in the LEC11 cell line, contained twice the number of sLex moieties per mole glycoprotein, exhibited a twofold increase in area under the intravenous clearance curve in a rat pharmacokinetic model, and exhibited a 10-fold increase in affinity for E-selectin in an in vitro binding assay. These results demonstrate that in vitro glycosylation of the sCR1 drug product reduces heterogeneity of the glycan profile, improves pharmacokinetics, and enhances carbohydrate-mediated binding to E-selectin.

Key words: glycoengineering / glycoprotein remodeling / glycosylation / glycosyltransferase


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Soluble complement receptor type 1 (sCR1) is a recombinant glycoprotein that has been shown to inhibit the progression of the complement cascade in both the classical and alternative pathways by inhibiting the stable formation of C3 and C5 convertases and by serving as a cofactor in the proteolytic degradation of C3b and C4b by Factor I (Weisman et al., 1990Go). The administration of sCR1 has been shown to be effective in a number of animal disease models of human complement–dependent ischemia-reperfusion injury for tissues, such as heart (Lazar et al., 1999Go), liver (Lehmann et al., 1998Go), hind limb (Kyriakides et al., 2001aGo), lung (Naka et al., 1997Go), and intestine (Williams et al., 1999Go). Complement inhibition by sCR1 has been shown to reduce hyperacute rejection (Pruitt et al., 1997Go) and enhance graft survival in many established transplant models (Kallio et al., 2000Go; Pratt et al., 1996Go; Stammberger et al., 2000Go).

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 (Neu5Ac{alpha}2-3Galß1-4[Fuc{alpha}1-3]GlcNAcß1-) (Lasky, 1995Go).

Previously we have described sCR1-sLex (Picard et al., 2000Go; Rittershaus et al., 1999Go), 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 {alpha}1-3 linkage to GlcNAc in oligosaccharide chains that terminate with either Galß1-4GlcNAcß1 ... or NeuAc{alpha}2-3Galß1-4GlcNAcß1 ...(Zhang et al., 1999Go). Of the 25 potential N-glycosylation sites within the sCR1 polypeptide sequence, 13–15 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., 2000Go; Rittershaus et al., 1999Go). 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., 1991Go; Jenkins et al., 1996Go).

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.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
In vitro remodeling of sCR1 glycans
sCR1 (250 mg) expressed in CHO cells was sialyated by treatment with ST3Gal-III plus CMP-sialic acid to give a product designated sCR1-S. After an aliquot was removed from the reaction mixture for analysis, the remaining sCR1-S was fucosylated in the same reaction vessel by the addition of FT-VI plus GDP-fucose to give a product designated sCR1-S/F. After purification by serial chromatography on ceramic hydroxyapatite and Q Sepharose, the reaction products had the same retention time and percent purity (98.5%) by reversed phase high-pressure liquid chromatography (RP-HPLC) as the starting material, sCR1 (data not shown). Chemical and functional properties of these molecules were compared with those of sCR1-sLex, a molecule previously produced in the FT-VI-expressing LEC11 CHO cell line and shown to contain some N-linked biantennary glycans terminated with the sLex tetrasaccharide (Picard et al., 2000Go).

From the mannose content of sCR1, sCR1-S, sCR1-S/F, and sCR1-sLex (Table I) it may be inferred that these molecules contain ~13–15 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., 2000Go). 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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Table I. Monosaccharide Content (mol/mol glycoprotein) by HPLC analysis

 


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Fig. 1. FACE profiling of oligosaccharides from sCR1-sLex and sCR1 before and after enzymatic remodeling: (1) Glyko oligosaccharide standard ladder, (2) sCR1, (3) sCR1-S, (4) sCR1-S/F, (5) sCR1-sLex. The oligosaccharide profile of sCR1 (lane 2) contains predominantly bands representing biantennary structures with two sialic acids (bottom band), one sialic acid (middle band), and no sialic acids (top band).

 
FACE analysis of glycans from sCR1-S/F, prepared by enzymatic fucosylation of sCR-S, suggests that N-glycans are predominantly biantennary and that fucosylation at both antennae is nearly complete (Figure 1). The dominant oligosaccharide band derived from sCR1-S/F was cut out and extracted from the gel. Sequential removal of monosaccharide residues from the extracted glycoprotein using specific glycosidases gave products with mobilities consistent with {alpha}1-6 core-fucosylated, biantennary N-glycans (Figure 2). Monosaccharide analysis of sCR1-S/F shows the presence of 39.3 moles fucose per mol sCR1-S/F, a figure in agreement with the prediction from theory that 39–45 fucose residues per mol protein would be present if all N-glycans were core fucosylated and enzymatic fucosylation of antennary GlcNAc residues were complete.



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Fig. 2. FACE analysis of oligosaccharides from sCR1-S/F after serial treatment with glycosidases. The dominant oligosaccharide band derived from sCR1-S/F (lane 2) was cut out and extracted from the gel. The resulting oligosaccharide preparation was digested sequentially to remove each monosaccharide residue starting at the terminal sialic acid residue and ending at the trimannosyl core: (1) Glyko oligosaccharide standard ladder; (2) total N-linked oligosaccharides of sCR1-S/F; (3) purified dominant band (band 1) from lane 2; (4) band 1 treated with NANaseIII (cleaves {alpha}2-3, 4, 6, 8, and 9 linked sialic acid); (5) band 1 treated with NANaseIII and FucaseIII (cleaves {alpha}1-3 and 4 fucose); (6) band 1 treated with NANaseIII, FucaseIII, and GalaseIII (cleaves terminal galactose); (7) band 1 treated with NANaseIII and FucaseIII, GalaseIII, and hexosaminidase; (8) standard trimannosyl core N-glycans with (upper band) and without {alpha}1-6 fucose.

 
The FACE oligosaccharide profile for sCR1-sLex, a glycoprotein produced in LEC11 CHO cells, shows at least seven bands (Figure 1) with some common to sCR1 and others shown previously (Picard et al., 2000Go) to represent core fucosylated structures with {alpha}1-3 fucosylation at one or more antennae. Heterogeneity in the degree of fucosylation of the N-glycan chains from sCR1-sLex also can be appreciated from the results of monosaccharide analysis (Table I). For example, it may be calculated (assuming 3 mannose residues per chain) that sCR1-sLex contains an average of 2.5 fucosyl residues per glycan chain. By contrast, the fucose content per glycan chain increases from 0.95 for sCR1-S to 3.3 for sCR1-S/F, a result that correlates well with the single band visualized by FACE analysis of sCR1-S/F (Figure 1).

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 {alpha}2-3 linked, as expected.



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Fig. 3. FACE analysis of oligosaccharides from sCR1-S/F treated with sialidases. The dominant oligosaccharide band (band 1) derived from sCR1-S/F (see Figure 2, lane 2) was cut out and extracted from the gel. The resulting oligosaccharide preparation was subjected to enzymatic digestion to remove terminal sialic acid: (1) Glyko oligosaccharide standard ladder; (2) band 1 from sCR1-S/F; (3) band 1 treated with NANaseI (cleaves {alpha}2-3 linked sialic acid); (4) band 1 treated with NANaseIII (cleaves {alpha}-3, 4, 6, 8, and 9 linked sialic acid).

 
Optimization of sialylation reaction for scale-up
To establish conditions for scaleup of sialylation, sCR1 (5 mg/ml) was incubated with varying amounts of ST3Gal-III (10, 25, 75, 100, 200, 300 and 400 U/ml) and 5 mM CMP–sialic acid plus a trace amount of radiolabeled CMP–sialic acid for 24 h at 32°C. At an ST3Gal-III concentration of 150 mU/ml, incorporation of radiolabeled sialic acid reached 91% of maximum after 24 h and 100% at 48 h. The lowest concentration of enzyme required to give nearly maximum incorporation (~40 mol sialic acid/mol protein) under these conditions was 25 mU/ml ST3Gal-III (Figure 4). It should be noted that the contribution of triantennary and tetraantennary species may be responsible for the observation that more than 30 moles of sialic acid was added per mole of sCR1. Increasing the CMP–sialic acid concentration from 5 mM to 10 mM did not affect the level of sialylation of sCR1 at any of the ST3Gal-III concentrations tested (data not shown). HPLC and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis of glycans released from sCR1-S revealed that at all concentrations of enzyme tested, the product contained predominantly disialylated, biantennary, core fucosylated N-glycans (data not shown). A concentration of 200 mU ST3Gal-III/ml was chosen for scale-up to ensure completeness of reaction.



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Fig. 4. Incorporation of sialic acid into sCR1 at increasing concentrations of ST3Gal-III in a 24-h reaction. The moles of sialic acid added are estimated from incorporation of radiolabeled CMP–sialic acid. Incorporated radiolabel is separated from free by gel filtration on a TSKG2000SWXL column.

 
Optimization of fucosylation
To establish conditions for scale-up of fucosylation, sCR1-S (5 mg/ml) was incubated with varying amounts of FT-VI (10, 20, 40, 60, 100, 220, 440 mU/ml) and 5 mM GDP-fucose plus a trace of radiolabeled GDP-fucose for 24 h at 32°C. The lowest concentration of enzyme required to give nearly maximum incorporation of fucose under these conditions was 100 mU/ml FT-VI (Figure 5). Increasing the GDP-fucose concentration from 5 mM to 10 mM did not increase fucose incorporation at several different FT-VI concentrations tested (data not shown).



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Fig. 5. Incorporation of fucose into sCR1-S at increasing concentrations of FT-VI in a 24-h reaction. The moles of fucose added are estimated from incorporation of radiolabeled GDP-fucose. Incorporated radiolabel is separated from free by gel filtration on a TSKG2000SWXL column.

 
For products of reactions run at all concentrations of FT-VI ≥ 100 mU/ml, the glycan structures identified by HPLC and MALDI-TOF MS were almost the same and essentially indistinguishable from the structures described next for sCR1-S/F produced at the 10-g scale.

Remodeling at 10-g scale
Purified sCR1 (10 g in a volume of 2 L) was incubated first with ST3Gal-III plus CMP–sialic 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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Fig. 6. RP-HPLC analysis of 2-AA-oligosaccharides before and after enzymatic remodeling at the 10-g scale: (A) sCR-1; (B) sCR1-S; (C) sCR1-S/F. MALDI-TOF MS analysis (data not shown) of 2-AA-oligosaccharides from sCR1-S (B) indicated that: peak a contains monosialylated biantennary glycans that lack terminal galactose on one antenna; peak b, constituting 12% of biantennary glycans, contains biantennary glycans with two galactose residues, but only one sialic acid; peaks c and d contain disialylated, biantennary glycans, with and without core fucose, respectively.

 

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Table II. HPLC data summary of large scale remodeling

 
After fucosylation of sCR1-S to create sCR1-S/F, HPLC and MALDI-TOF MS analyses (Table III and Figure 7) showed that more than 95% of the glycans were fucosylated by FT-VI. About 62% of the total N-glycans gained two fucose residues, and ~30% gained a single fucose residue. Failure to accept two fucosyl residues was in part due to missing galactosyl residues on one or more antennae. From these results it can be estimated that the sCR1-S/F molecules created by consecutive in vitro sialylation and fucosylation reactions contain, on average, 28 sLex epitopes per protein molecule, whereas sCR1-sLex, glycosylated and secreted by the FT-VI-expressing LEC11 CHO cell, contains ~14 sLex epitopes per protein molecule (Table I).


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Table III. sCR1-S/F glycans

 


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Fig. 7. MALDI-TOF analysis of total glycans from (A) sCR1, (B) sCR1-S, and (C) sCR1-S/F remodeled at the 10-g scale. The blue square is GlcNAc, the yellow filled circle is mannose, the green filled triangle is fucose, the red filled diamond is galactose, and the asterisk is sialic acid.

 
To check the stability of sCR1 under conditions of incubation with glycosyltransferases, a small amount of protein was analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) after each remodeling reaction. There was no evidence of degradation of the polypeptide after incubation with either ST3Gal-III or FT-VI (data not shown).

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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Fig. 8. The concentration of sCR1 and sCR1-S in plasma at various time points following bolus IV injection in rats.

 
In vitro antihemolytic activity
The IH50 values for sCR1, sCR1-S, sCR1-sLex, and sCR1-S/F as inhibitors of human complement–mediated lysis of sheep red blood cells were found to be similar (Figure 9 and Table IV), indicating that in vitro glycosylation of sCR1 to yield sCR1-S or sCR1-S/F does not significantly impact the complement inhibitory properties of the molecule in the classical pathway.



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Fig. 9. Inhibition of red cell lysis via the classical pathway as a function of the concentration of sCR1, sCR1-S, sCR1-sLex, and sCR1-S/F.

 

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Table IV. Antihemolytic activity of modified sCR1 and sCR1-sLex

 
In vitro binding to E-selectin
Figure 10 shows that sCR1-sLex and sCR1-S/F bind E-selectin in a concentration-dependent manner. The IC50 for sCR1-sLex from this plot is ~5 nM, and for sCR1-S/F ~0.4 nM. The observed 10-fold increase in inhibitory potency presumably is due to enhanced avidity, attributable to the increased density of sLex moieties on sCR1-S/F (28/mol) as compared with sCR1-sLex (14 per mol) (see Table I). The specificity of this binding was demonstrated by its calcium requirement and by the observation that sCR1 (which does not contain any sLex structures) does not inhibit E-selectin binding at concentrations as high as 10 µM (data not shown).



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Fig. 10. Inhibition of PAA-sLex binding to E-selectin coated microtiter plates in the presence of varying concentrations of sCR1-sLex or sCR1-S/F.

 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
sCR1, made by standard CHO production methods, possesses predominantly biantennary oligosaccharides that are incompletely sialylated. We previously described an alternately glycosylated form of sCR1 called TP20 or sCR1-sLex (Picard et al., 2000Go; Rittershaus et al., 1999Go), secreted by the FT-VI-expressing LEC11 CHO cell line and bearing sLex moieties on a fraction of its N-linked oligosaccharides. In this article we describe in vitro enzymatic remodeling of sCR1 by the stepwise application of two soluble recombinant glycosyltransferases in "one pot": The first step adds sialic acid to make sCR1-S, and the second adds fucose to make sCR1-S/F. The product of these glycan remodeling reactions contains an average of 28 sLex moieties per mol, as compared with 14 per mol found in CHO cell-produced sCR1-sLex.

That the sCR1 protein remains intact under conditions of glycan remodeling was demonstrated by RP-HPLC and SDS–PAGE 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 {alpha}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{alpha}2-3Galß1-4[Fuc{alpha}1-3]GlcNAcß1-) versus sLea (NeuAc{alpha}2-3Galß1-3[Fuc{alpha}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., 2001Go). Second, the acceptor specificity of FT-VI is known to be restricted to type 2 chains (Costache et al., 1997Go; Weston et al., 1992Go).

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, 1995Go).

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, 1998Go; Nemansky et al., 1995Go; Paulson et al., 1977Go; Raju et al., 2001Go; Thotakura et al., 1994Go).

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., 1999Go), a murine model of ischemic stroke (Huang et al., 1999Go), moderating skeletal muscle reperfusion injury (Kyriakides et al., 2001aGo), moderation of acid aspiration injury (Kyriakides et al., 2001bGo), reducing ischemia/reperfusion injury in rat lung grafts (Schmid et al., 2001Go), 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., 1999Go). It will be interesting to investigate whether sCR1-S/F is even more effective than sCR1-sLex in similar animal models.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Complement proteins, antibodies, enzymes, and other reagents
Purified sCR1 and sCR1-sLex were prepared as previously described (Rittershaus et al., 1999Go). Nucleotide sugars (CMP–sialic acid and GDP-fucose) were manufactured at Neose (Horsham, PA). CMP–sialic acid was prepared from CTP and sialic acid with recombinant CMP NeuAc synthetase (Shames et al., 1991Go). GDP-fucose was either made from GDP-mannose using GDP-mannose 4,6-dehydratase and GDP-4-keto-6-deoxymannose 3,5-epimerase/reductase, or purchased from Yamasa (Chiba, Japan). A gene encoding for a truncated, soluble form of ST3Gal-III (rat) was expressed in A. niger var. awamori dgr246 P2 using a variant of the expression vector pSL 1180 (Ward and Power, 2003Go). A 30–60% ammonium sulfate pellet was dissolved in 100 mM NaCl, 20 mM 2-(N-morpholino)ethanesulfonic acid (MES), pH 6, loaded on SP Sepharose (Amersham Biosciences, Piscataway, NJ), and eluted with 1 M NaCl, 20 mM MES, pH 6. rFT-VI (human) was expressed either in NSO cells or in A. niger as described as a soluble protein lacking the transmembrane domain. For the A. niger expressed protein, a 30–60% ammonium sulfate pellet was dissolved in 20 mM MES, pH 6, loaded on SP Sepharose (Amersham Biosciences, Piscataway, NJ), and eluted with a linear gradient from 0 to 1 M NaCl in 20 mM MES, pH 6. Soluble recombinant E-selectin was purchased from R&D Systems (Minneapolis, MN). Streptavidin–horseradish peroxidase conjugate (SA-HRP) was from Pierce (Rockford, IL), and biotinylated polyacrylamide polymer (PAA-sLex) was from GlycoTech (Rockville, MD). Anti-sCR1 monoclonal antibodies 6B1.H12 and 4D6.1 were prepared as previous described (Nickells et al., 1998Go). Standards and glycosidases used in FACE analyses were from Glyko (Novato, California).

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 CMP–sialic 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 Tris–HCl, 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 CMP–sialic 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 Tris–HCl, 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, 1994Go). Sialic acid content was determined after sodium bisulfate hydrolysis and reaction with o-phenylenediamine by C18 reverse-phase HPLC with fluorescence detection (Anumula, 1995Go).

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., 2000Go).

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)Go 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.

SDS–PAGE
sCR1 samples before and after in vitro enzymatic remodeling were separated on 8–16% 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., 1996Go).

E-selectin binding assay
E-selectin binding assays were performed according to previously reported methods (Weitz-Schmidt et al., 1996Go). 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., 1999Go). 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).


    Acknowledgements
 
The authors thank Michelle Richardson and Gang Yan for expert technical assistance.


    Footnotes
 
2 To whom correspondence should be addressed; e-mail: bbayer{at}neose.com

1 These authors contributed equally to this work. Back


    Abbreviations
 
2-AA, 2-anthranilic acid; AUC, area under the curve; CHO, Chinese hamster ovary; FACE, fluorophore-assisted carbohydrate electrophoresis; HBS, HEPES-buffered saline; MALDI-TOF MS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry; MES, 2-(N-morpholino)ethanesulfonic acid; RP-HPLC, reversed phase high-pressure liquid chromatography; SA-HRP, streptavidin–horseradish peroxidase; sCR1, soluble recombinant complement receptor type 1; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
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