Glycobiology Advance Access originally published online on June 9, 2004
Glycobiology 2004 14(10):883-893; doi:10.1093/glycob/cwh112
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Glycobiology vol. 14 no. 10 © Oxford University Press 2004; all rights reserved.
Production of a complement inhibitor possessing sialyl Lewis X moieties by in vitro glycosylation technology
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
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
2-3Galß1-4(Fuc1-3)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 2-3 sialyltransferase ST3Gal-III and the 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.
1 These authors contributed equally to this work.
2 To whom correspondence should be addressed; e-mail: bbayer{at}neose.com
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  Y. Yamakoshi, Y. Lu, J. C.-C. Hu, J.-W. Kim, T. Iwata, K. Kobayashi, T. Nagano, F. Yamakoshi, Y. Hu, M. Fukae, et al. Porcine Dentin Sialophosphoprotein: LENGTH POLYMORPHISMS, GLYCOSYLATION, PHOSPHORYLATION, AND STABILITY J. Biol. Chem., May 23, 2008; 283(21): 14835 - 14844. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Ma, J. L. Simala-Grant, and D. E. Taylor Fucosylation in prokaryotes and eukaryotes Glycobiology, December 1, 2006; 16(12): 158R - 184R. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. DeFrees, Z.-G. Wang, R. Xing, A. E. Scott, J. Wang, D. Zopf, D. L. Gouty, E. R. Sjoberg, K. Panneerselvam, E. C. M. Brinkman-Van der Linden, et al. GlycoPEGylation of recombinant therapeutic proteins produced in Escherichia coli Glycobiology, September 1, 2006; 16(9): 833 - 843. [Abstract] [Full Text] [PDF]
|
|