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J. Biol. Chem., Vol. 283, Issue 12, 7804-7812, March 21, 2008

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N-Glycosylation as Novel Strategy to Improve Pharmacokinetic Properties of Bispecific Single-chain Diabodies^*

Roland Stork, Kirstin A. Zettlitz¹, Dafne Müller, Miriam Rether, Franz-Georg Hanisch, and Roland E. Kontermann²

From the Institut für Zellbiologie und Immunologie, Universität Stuttgart, Allmandring 31, 70569 Stuttgart, Germany and the Institut für Biochemie II, Medizinische Fakultät, Zentrum für Molekulare Medizin Köln, Universität Köln, Joseph-Stelzmann-Strasse 52, 50931 Köln, Germany

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 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.

Received for publication, November 8, 2007 , and in revised form, January 9, 2008.

^* This work was supported by a grant from the Deutsche Forschungsgemeinschaft (Ko1461/2). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Present address: Multimmune GmbH, c/o Klinikum Rechts der Isar der TU München, Ismaninger Strasse 22, 81675 München, Germany.

² To whom correspondence should be addressed: Institut für Zellbiologie und Immunologie, Universität Stuttgart, Allmandring 31, 70569 Stuttgart, Germany. Fax: 49-711-685-67484; E-mail: roland.kontermann{at}izi.uni-stuttgart.de.

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