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Glycobiology Advance Access published online on June 9, 2004
Glycobiology, doi:10.1093/glycob/cwh112
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Received March 24, 2004
Revised June 4, 2004
Accepted June 4, 2004

ORIGINAL ARTICLES

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

Lawrence J. Thomas 1, David T. Beattie 1, Michele D. Picard 1, Bi Xu 1, Charles W. Rittershaus 1, Henry C. Marsh Jr.1, Russell A. Hammond 1, Krishnasamy Panneerselvam 2, Jun Qian 2, Tom Stevenson 2, David Zopf 2, Robert J. Bayer 2*

1 AVANT Immunotherapeutics, Inc., 119 Fourth Ave., Needham, MA 02494, USA
2 Neose Technologies, Inc., 102 Witmer Road, Horsham, PA 19044, USA

* To whom correspondence should be addressed. E-mail: bbayer{at}neose.com.


  Abstract

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) [NeuNAc{alpha}2-3Gal{beta}1-4(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 gram scale has approximately 14 N-glycan chains per sCR1 molecule, comprised of biantennary (90%), triantennary (8.5%), and tetra-antennary (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 two-fold 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


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