PEDS Advance Access originally published online on July 25, 2006
Protein Engineering Design and Selection 2006 19(10):461-470; doi:10.1093/protein/gzl031
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A highly stable polyethylene glycol-conjugated human single-chain antibody neutralizing granulocyte-macrophage colony stimulating factor at low nanomolar concentration
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
4To whom correspondence should be addressed. E-mail: tobias.raum{at}micromet.de
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
Received February 16, 2006; revised June 1, 2006; accepted June 12, 2006.