title: A highly stable polyethylene glycol-conjugated human single-chain antibody neutralizing granulocyte-macrophage colony stimulating factor at low nanomolar concentration.
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.
authors.affiliation: Micromet AG Staffelseestrasse 2, Munich, Germany.
authors.forename: Eva-Maria
authors.initials: EM
authors.lastname: Krinner
authors.forename: Julia
authors.initials: J
authors.lastname: Hepp
authors.forename: Patrick
authors.initials: P
authors.lastname: Hoffmann
authors.forename: Sandra
authors.initials: S
authors.lastname: Bruckmaier
authors.forename: Laetitia
authors.initials: L
authors.lastname: Petersen
authors.forename: Silke
authors.initials: S
authors.lastname: Petsch
authors.forename: Larissa
authors.initials: L
authors.lastname: Parr
authors.forename: Ioana
authors.initials: I
authors.lastname: Schuster
authors.forename: Susanne
authors.initials: S
authors.lastname: Mangold
authors.forename: Grit
authors.initials: G
authors.lastname: Lorenczewski
authors.forename: Petra
authors.initials: P
authors.lastname: Lutterbüse
authors.forename: Stefan
authors.initials: S
authors.lastname: Buziol
authors.forename: Inessa
authors.initials: I
authors.lastname: Hochheim
authors.forename: Jörg
authors.initials: J
authors.lastname: Volkland
authors.forename: Michael
authors.initials: M
authors.lastname: Mølhøj
authors.forename: Mirnalini
authors.initials: M
authors.lastname: Sriskandarajah
authors.forename: Markus
authors.initials: M
authors.lastname: Strasser
authors.forename: Christian
authors.initials: C
authors.lastname: Itin
authors.forename: Andreas
authors.initials: A
authors.lastname: Wolf
authors.forename: Amartya
authors.initials: A
authors.lastname: Basu
authors.forename: Karen
authors.initials: K
authors.lastname: Yang
authors.forename: David
authors.initials: D
authors.lastname: Filpula
authors.forename: Poul
authors.initials: P
authors.lastname: Sørensen
authors.forename: Peter
authors.initials: P
authors.lastname: Kufer
authors.forename: Patrick
authors.initials: P
authors.lastname: Baeuerle
authors.forename: Tobias
authors.initials: T
authors.lastname: Raum
mesh_terms: D000818:Animals; D004305:Dose-Response Relationship, Drug; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000737:chemistry*; D006358:Hot Temperature; D006801:Humans; D007128:Immunoglobulin Fragments / Q000737:chemistry*; D007135:Immunoglobulin Variable Region / Q000737:chemistry*; D007700:Kinetics; D051379:Mice; D019151:Peptide Library; D011092:Polyethylene Glycols / Q000737:chemistry*; D015202:Protein Engineering / Q000379:methods; D017434:Protein Structure, Tertiary; D051381:Rats; D017422:Sequence Analysis, DNA
chemical_list: D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D011092:Polyethylene Glycols; D016178:Granulocyte-Macrophage Colony-Stimulating Factor