A highly stable polyethylene glycol-conjugated human single-chain antibody neutralizing granulocyte-macrophage colony stimulating factor at low nanomolar concentration.
Protein Eng Des Sel
; 19(10): 461-70, 2006 Oct.
Article
em En
| MEDLINE
| ID: mdl-16868004
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 V(L) repertoire was combined with the V(H) 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 V(L) of this clone was then combined with a human V(H) repertoire. The latter preserved the CDR 3 of the parental rat V(H) domain to retain binding specificity. Several human scFvs were selected, which neutralized human GM-CSF at low nanomolar concentrations (IC50 > or = 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.
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Bases de dados:
MEDLINE
Assunto principal:
Polietilenoglicóis
/
Região Variável de Imunoglobulina
/
Fragmentos de Imunoglobulinas
/
Fator Estimulador de Colônias de Granulócitos e Macrófagos
Tipo de estudo:
Prognostic_studies
Limite:
Animals
/
Humans
Idioma:
En
Revista:
Protein Eng Des Sel
Assunto da revista:
BIOQUIMICA
/
BIOTECNOLOGIA
Ano de publicação:
2006
Tipo de documento:
Article
País de afiliação:
Alemanha