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PEDS Advance Access published online on July 25, 2006
Protein Engineering Design and Selection, doi:10.1093/protein/gzl031
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org
Received February 16, 2006
Revised June 1, 2006
Accepted June 12, 2006

Article

A highly stable polyethylene glycol-conjugated human single-chain antibody neutralizing granulocyte-macrophage colony stimulating factor at low nanomolar concentration

Eva-Maria Krinner 1, Julia Hepp 1, Patrick Hoffmann 1, Sandra Bruckmaier 1, Laetitia Petersen 1, Silke Petsch 1, Larissa Parr 1, Ioana Schuster 1, Susanne Mangold 1, Grit Lorenczewski 1, Petra Lutterbüse 1, Stefan Buziol 1, Inessa Hochheim 1, Jörg Volkland 1, Michael Mølhøj 1, Mirnalini Sriskandarajah 1, Markus Strasser 1, Christian Itin 1, Andreas Wolf 1, Amartya Basu 2, Karen Yang 2, David Filpula 2, Poul Sørensen 3, Peter Kufer 1, Patrick Baeuerle 1, and Tobias Raum 1 *

1 Micromet AG, Staffelseestr. 2, 81477 Munich, Germany
2 Enzon Pharmaceuticals, 20 Kingsbridge Road, Piscataway, NJ 08854-3969, USA
3 LEO Pharma A/S, Industriparken 55, DK-2750 Ballerup, Denmark

* To whom correspondence should be addressed.
Tobias Raum, E-mail: tobias.raum{at}micromet.de


  Abstract

GM-CSF (granulocyte-macrophage colony stimulating factor) plays a central role in inflammatory processes. Treatment with antibodies neutralizing murine GM-CSF showed significant therapeutic effects in mouse models of inflammatory diseases. We constructed by phage display technology a human scFv, which could potently neutralize human GM-CSF. At first, a human VL repertoire was combined with the VH domain of a parental GM-CSF-neutralizing rat antibody. One dominant rat/human scFv clone was selected, neutralizing human GM-CSF with an IC50 of 7.3 nM. The human VL of this clone was then combined with a human VH repertoire. The latter preserved the CDR 3 of the parental rat VH domain to retain binding specificity. Several human scFvs were selected, which neutralized human GM-CSF at low nanomolar concentrations (IC50 ≥ 2.6 nM). To increase serum half-life, a branched 40 kDa PEG-polymer was coupled to the most potent GM-CSF-neutralizing scFv (3077) via an additional C-terminal cysteine. PEG conjugation had a negligible effect on the in vitro neutralizing potential of the scFv, although it caused a significant drop in binding affinity owing to a reduced on-rate. It also significantly increased the stability of the scFv at elevated temperatures. In mouse experiments, the PEGylated scFv 3077 showed a significantly prolonged elimination half-life of 59 h as compared with 2 h for the unconjugated scFv version. PEGylated scFv 3077 is a potential candidate for development of a novel antibody therapy to treat pro-inflammatory human diseases.

Keywords: GM-CSF; neutralization; PEGylation; phage display; single chain antibody.
E.-M.Krinner and J.Hepp contributed equally to this work
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