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

Alexandre Fontayne; Karen Vanhoorelbeke; Inge Pareyn; Isabel Van Rompaey; Muriel Meiring; Seb Lamprecht; Jan Roodt; Johan Desmet; Hans Deckmyn

doi:10.1160/TH06-06-0297

Thrombosis and Haemostasis, Table of Contents

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

¹Laboratory for Thrombosis Research, IRC, KU Leuven Campus Kortrijk, Belgium

,

Karen Vanhoorelbeke

¹Laboratory for Thrombosis Research, IRC, KU Leuven Campus Kortrijk, Belgium

,

Inge Pareyn

¹Laboratory for Thrombosis Research, IRC, KU Leuven Campus Kortrijk, Belgium

,

Isabel Van Rompaey

²Thromb-X NV, Leuven, Belgium

,

Muriel Meiring

³Department of Hematology and Cell Biology, Faculty of Health Sciences, University of the Free State, South Africa

,

Seb Lamprecht

³Department of Hematology and Cell Biology, Faculty of Health Sciences, University of the Free State, South Africa

,

Jan Roodt

³Department of Hematology and Cell Biology, Faculty of Health Sciences, University of the Free State, South Africa

,

Johan Desmet

⁴Algonomics NV, Gent, Belgium

,

Hans Deckmyn

¹Laboratory for Thrombosis Research, IRC, KU Leuven Campus Kortrijk, Belgium

› Author Affiliations

Abstract

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.

Keywords

Antithrombotic - computer modeling - humanization - glycoprotein Ib - platelet

Full Text

References

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