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J. Biol. Chem., Vol. 281, Issue 46, 35186-35201, November 17, 2006

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PEGylation and Multimerization of the Anti-p185^HER-2 Single Chain Fv Fragment 4D5

EFFECTS ON TUMOR TARGETING^*

Susanne Kubetzko¹, Ela Balic², Robert Waibel^¶, Uwe Zangemeister-Wittke³, and Andreas Plückthun⁴

From the Department of Biochemistry, University of Zürich, CH-8057 Zürich, the Department of Oncology, University Hospital, University of Zürich, CH-8044 Zürich, and the ^¶Paul Scherrer Institute, CH-5232 Villigen (PSI), Switzerland

A major goal in antibody design for cancer therapy is to tailor the pharmacokinetic properties of the molecule according to specific treatment requirements. Key parameters determining the pharmacokinetics of therapeutic antibodies are target specificity, affinity, stability, and size. Using the p185^HER-2 (HER-2)-specific scFv 4D5 as model system, we analyzed how changes in molecular weight and valency independently affect antigen binding and tumor localization. By employing multimerization and PEGylation, four different antibody formats were generated and compared with the scFv 4D5. First, dimeric and tetrameric miniantibodies were constructed by fusion of self-associating, disulfide-linked peptides to the scFv 4D5. Second, we attached a 20-kDa PEG moiety to the monovalent scFv and to the divalent miniantibody at the respective C terminus. In all formats, serum stability and full binding reactivity of the scFv 4D5 were retained. Functional affinity, however, did change. An avidity increase was achieved by multimerization, whereas PEGylation resulted in a 5-fold decreased affinity. Nevertheless, the PEGylated monomer showed an 8.5-fold, and the PEGylated dimer even a 14.5-fold higher tumor accumulation than the corresponding scFv, 48 h post-injection, because of a significantly longer serum half-life. In comparison, the non-PEGylated bivalent and tetravalent miniantibodies showed only a moderate increase in tumor localization compared with the scFv, which correlated with the degree of multimerization. However, these non-PEGylated formats resulted in higher tumor-to-blood ratios. Both multimerization and PEGylation represent thus useful strategies to tailor the pharmacokinetic properties of therapeutic antibodies and their combined use can additively improve tumor targeting.

Received for publication, May 1, 2006 , and in revised form, August 24, 2006.

^* This work was supported in part by Schweizerischer Nationalfonds Grant 31-65344.01 and the Cancer League of the Kanton Zürich. 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.

The on-line version of this article (available at http://www.jbc.org) contains supplementary methods.

¹ Present address: Kinderspital Zürich, Abteilung für Infektions- & Krebskrankheiten, August-Forel-Str. 1, CH-8008 Zürich, Switzerland.

² Present address: Inst. of Pharmacology and Toxicology, University of Zürich, CH-8057 Zürich, Switzerland.

³ Present address: Dept. of Biochemistry, University of Zürich, CH-8057 Zürich, and Dept. of Pharmacology, University of Bern, Friedbühlstr. 49, CH-3010 Bern, Switzerland.

⁴ To whom correspondence should be addressed: Dept. of Biochemistry, University of Zürich, CH-8057 Zürich, Switzerland. Tel.: 41-44-635-5570; Fax: 41-44-635-5712; E-mail: plueckthun{at}bioc.unizh.ch.

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