A highly stable polyethylene glycol-conjugated human single-chain antibody neutralizing granulocyte-macrophage colony stimulating factor at low nanomolar concentration.
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.
Krinner
Eva-Maria
EM
Micromet AG Staffelseestrasse 2, Munich, Germany.
Hepp
Julia
J
Hoffmann
Patrick
P
Bruckmaier
Sandra
S
Petersen
Laetitia
L
Petsch
Silke
S
Parr
Larissa
L
Schuster
Ioana
I
Mangold
Susanne
S
Lorenczewski
Grit
G
Lutterbüse
Petra
P
Buziol
Stefan
S
Hochheim
Inessa
I
Volkland
Jörg
J
Mølhøj
Michael
M
Sriskandarajah
Mirnalini
M
Strasser
Markus
M
Itin
Christian
C
Wolf
Andreas
A
Basu
Amartya
A
Yang
Karen
K
Filpula
David
D
Sørensen
Poul
P
Kufer
Peter
P
Baeuerle
Patrick
P
Raum
Tobias
T
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
D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D011092:Polyethylene Glycols; D016178:Granulocyte-Macrophage Colony-Stimulating Factor