title: Production of a complement inhibitor possessing sialyl Lewis X moieties by in vitro glycosylation technology.
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-sLe(x) 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 (sLe(x)) [Neu5Acalpha2-3Galbeta1-4(Fucalpha1-3)GlcNAc]) epitope. Such multivalent presentation of sLe(x) was shown previously to effectively target sCR1 to activated endothelial cells expressing E-selectin. Here, we describe the use of the soluble, recombinant alpha2-3 sialyltransferase ST3Gal-III and the alpha1-3 fucosyltransferase FT-VI in vitro to introduce sLe(x) 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 sLe(x) moieties. sCR1-S/F retained complement inhibitory activity and, in comparison with sCR1-sLe(x) produced in the LEC11 cell line, contained twice the number of sLe(x) 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.
authors.affiliation: AVANT Immunotherapeutics, 119 Fourth Ave., Needham, MA 02494, USA.
authors.forename: Lawrence J
authors.initials: LJ
authors.lastname: Thomas
authors.forename: Krishnasamy
authors.initials: K
authors.lastname: Panneerselvam
authors.forename: David T
authors.initials: DT
authors.lastname: Beattie
authors.forename: Michele D
authors.initials: MD
authors.lastname: Picard
authors.forename: Bi
authors.initials: B
authors.lastname: Xu
authors.forename: Charles W
authors.initials: CW
authors.lastname: Rittershaus
authors.forename: Henry C
authors.initials: HC
authors.lastname: Marsh
authors.forename: Russell A
authors.initials: RA
authors.lastname: Hammond
authors.forename: Jun
authors.initials: J
authors.lastname: Qian
authors.forename: Tom
authors.initials: T
authors.lastname: Stevenson
authors.forename: David
authors.initials: D
authors.lastname: Zopf
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Bayer
mesh_terms: D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D019040:E-Selectin / Q000378:metabolism; D005647:Fucosyltransferases / Q000378:metabolism / Q000494:pharmacology; D006031:Glycosylation; D006801:Humans; D009844:Oligosaccharides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011134:Polysaccharides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011951:Receptors, Complement / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000080506:Sialyl Lewis X Antigen; D012799:Sialyltransferases / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D000094663:beta-Galactoside alpha-2,3-Sialyltransferase
chemical_list: D019040:E-Selectin; D009844:Oligosaccharides; D011134:Polysaccharides; D011951:Receptors, Complement; D011994:Recombinant Proteins; D000080506:Sialyl Lewis X Antigen; C082885:soluble complement inhibitor 1; D005647:Fucosyltransferases; D012799:Sialyltransferases; D000094663:beta-Galactoside alpha-2,3-Sialyltransferase