title: N-glycosylation as novel strategy to improve pharmacokinetic properties of bispecific single-chain diabodies.
abstract: The therapeutic efficacy of recombinant antibodies such as single-chain Fv fragments and small bispecific or bifunctional molecules is often limited by rapid elimination from the circulation because of their small size. Here, we have investigated the effects of N-glycosylation on the activity and pharmacokinetics of a small bispecific single-chain diabody (scDb CEACD3) developed for the retargeting of cytotoxic T cells to CEA-expressing tumor cells. We could show that the introduction of N-glycosylation sequons into the flanking linker and a C-terminal extension results in the production of N-glycosylated molecules after expression in transfected HEK293 cells. N-Glycosylated scDb variants possessing 3, 6, or 9 N-glycosylation sites, respectively, retained antigen binding activity and bispecificity for target and effector cells as shown in a target cell-dependent IL-2 release assay, although activity was reduced approximately 3-5-fold compared with the unmodified scDb. All N-glycosylated scDb variants exhibited a prolonged circulation time compared with scDb, leading to a 2-3-fold increase of the area under curve (AUC). In comparison, conjugation of a branched 40-kDa PEG chain increased AUC by a factor of 10.6, while a chimeric anti-CEA IgG1 molecule had the longest circulation time with a 17-fold increase in AUC. Thus, N-glycosylation complements the repertoire of strategies to modulate pharmacokinetics of small recombinant antibody molecules by an approach that moderately prolongs circulation time.
authors.affiliation: Institut für Zellbiologie und Immunologie, Universität Stuttgart, Allmandring 31, 70569 Stuttgart, Germany.
authors.forename: Roland
authors.initials: R
authors.lastname: Stork
authors.forename: Kirstin A
authors.initials: KA
authors.lastname: Zettlitz
authors.forename: Dafne
authors.initials: D
authors.lastname: Müller
authors.forename: Miriam
authors.initials: M
authors.lastname: Rether
authors.forename: Franz-Georg
authors.initials: FG
authors.lastname: Hanisch
authors.forename: Roland E
authors.initials: RE
authors.lastname: Kontermann
mesh_terms: D000818:Animals; D018033:Antibodies, Bispecific / Q000235:genetics / Q000276:immunology / Q000493:pharmacokinetics*; D002460:Cell Line; D005260:Female; D006031:Glycosylation; D007074:Immunoglobulin G / Q000235:genetics / Q000276:immunology; D007376:Interleukin-2 / Q000276:immunology; D051379:Mice; D011092:Polyethylene Glycols / Q000493:pharmacokinetics; D046188:Protein Modification, Translational; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology
chemical_list: D018033:Antibodies, Bispecific; D007074:Immunoglobulin G; D007376:Interleukin-2; D011092:Polyethylene Glycols