NR AXTF
AU Müller-Schiffmann,A.; Leliveld,S.R.; Petsch,B.; Stitz,L.; Korth,C.
TI Functional Recombinant Single Chain Antibody Against PrP
QU International Conference - Prion 2007 (26.-28.9.2007) Edinburgh International Conference Centre, Edinburgh, Scotland, UK - Book of Abstracts: Protein Misfolding P01.71
IA http://www.prion2007.com/pdf/Prion Book of Abstracts.pdf
PT Konferenz-Poster
AB
Prion diseases are characterized by the transformation of normal cellular PrPc into the transmissible isoform PrPsc. While it has been shown that passive immunization with monoclonal antibodies (mAbs) to PrPc are able to prevent prion disease after peripheral prion infection they have not shown to do so after intracerebral prion infection. A complicating circumstance was reported in that bivalent mAbs but not monovalent mAb fragments were neurotoxic.
Our objective was therefore to generate a monovalent high-affinity anti-PrP antibody fragment with anti-prion activity.
mAb W226 was generated by immunization of PrP ko mice with purified PrPsc and standard hybridoma technology. Amplified VL and VH fragments of this IgG1 were
cloned into pET22b including c-terminal c-myc and His6-tags for expression in the periplasm of E.coli. Soluble fractions were purified by IMAC- and mouse-PrP-affinity chromatography to a high level of purity (>98%), obtaining up to 15mg of purified scFv from 1L culture.
Binding of scFv-W226 to recombinant mouse PrP was verfied by ELISA and thermostability assays revealed that the scFv was stable at 37° in serum for at least 28 days. Moreover, 50% ELISA reactivity was still present at 60° indicating a particularly high thermostability; thermostability has been reported to correlate with in vivo half life time. Inhibition of prion propagation was analysed in cultured mouse neuroblastoma cells infected with PrPsc (ScN2a). The scFv was anti-prion active with an EC50 in the low nM range.
Thus a high affinity anti-prion active recombinant scFv has been constructed, which is soluble, thermostable to a high grade and may pass the blood brain barrier. In vivo studies in mice infected with PrPsc are currently underway and results will be reported.
AD A. Müller-Schiffmann, S.R. Leliveld, C. Korth, Heinrich Heine University of Düsseldorf, Institute for Neuropathology, Germany; B. Petsch, L. Stitz, Bundesforschungsinstitut für Tiergesundheit, Friedrich-Löffler-Institut, Germany
SP englisch
PO Schottland
EA pdf-Datei und Poster (zusätzliche Autoren: A. Salwierz und D. Riesner)