Thromb Haemost 2006; 96(05): 671-684
DOI: 10.1160/TH06-06-0297
New Technologies, Diagnostic Tools and Drugs
Schattauer GmbH

Rational humanization of the powerful antithrombotic anti-GPIbα antibody: 6B4

Alexandre Fontayne
1   Laboratory for Thrombosis Research, IRC, KU Leuven Campus Kortrijk, Belgium
,
Karen Vanhoorelbeke
1   Laboratory for Thrombosis Research, IRC, KU Leuven Campus Kortrijk, Belgium
,
Inge Pareyn
1   Laboratory for Thrombosis Research, IRC, KU Leuven Campus Kortrijk, Belgium
,
Isabel Van Rompaey
2   Thromb-X NV, Leuven, Belgium
,
Muriel Meiring
3   Department of Hematology and Cell Biology, Faculty of Health Sciences, University of the Free State, South Africa
,
Seb Lamprecht
3   Department of Hematology and Cell Biology, Faculty of Health Sciences, University of the Free State, South Africa
,
Jan Roodt
3   Department of Hematology and Cell Biology, Faculty of Health Sciences, University of the Free State, South Africa
,
Johan Desmet
4   Algonomics NV, Gent, Belgium
,
Hans Deckmyn
1   Laboratory for Thrombosis Research, IRC, KU Leuven Campus Kortrijk, Belgium
› Author Affiliations

Financial support: This work was supported by a grant from the Instituut voor de Aanmoediging van Innovatie door Wetenschap en Technologie in Vlaanderen (IWT 020473) and by a Bilateral Collaboration grant between Flanders and South Africa (BIL/04/56). A.F. is a EU-RTN postdoctoral fellow and K.V.a postdoctoral fellow of the ‘Fonds voor Wetenschappelijk Onderzoek (FWO) Vlaanderen’ Belgium.
Further Information

Publication History

Received 01 June 2006

Accepted after revision 11 September 2006

Publication Date:
01 December 2017 (online)

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Summary

Fab-fragments of the monoclonal antibody 6B4, raised against human glycoprotein Ibα (GPIbα), have a powerful antithrombotic effect in baboons by blocking the GPIbα binding site for von Willebrand factor (VWF), without significant prolongation of the skin bleeding time. In order to bring this antibody to the clinic,we here humanized for the first time an anti-human GPIbα by variable-domain resurfacing guided by computer modeling. First, the genes coding for the variable regions of the heavy and light chains of 6B4 were cloned and sequenced. Based on this,a three-dimensional structure of the Fv-fragment was constructed by using homology-based modeling, and with this and comparison with antibodies with known structure,”murine” putative immunogenic residues which are exposed, were changed for “human-like” residues. The humanized Fab-fragment, h6B4-Fab, was constructed in the pKaneo vector system, expressed and purified and showed in vitro an unaltered, even slightly higher binding affinity for its antigen than the murine form as determined by different ELISA set-ups and surface plasmon resonance. Finally, injection of doses of 0.1 to 1.5 mg/kg of h6B4-Fab in baboons showed that both pharmacokinetics and ex-vivo bio-activity of the molecule were to a large extent preserved.In conclusion, the method used here to humanize 6B4 by resurfacing resulted in a fully active derivative, which is now ready for further development.