Author:

Keywords:

antithrombotic, computer modeling, humanization, glycoprotein ib, platelet, platelet glycoprotein ib, von-willebrand-factor, murine monoclonal-antibodies, polymerase chain-reaction, variable domains, framework residues, nonhuman-primates, factor-viii, receptor, binding, Science & Technology, Life Sciences & Biomedicine, Hematology, Peripheral Vascular Disease, Cardiovascular System & Cardiology, glycoprotein Ib, PLATELET GLYCOPROTEIN IB, VON-WILLEBRAND-FACTOR, MURINE MONOCLONAL-ANTIBODIES, POLYMERASE CHAIN-REACTION, VARIABLE DOMAINS, FRAMEWORK RESIDUES, NONHUMAN-PRIMATES, FACTOR-VIII, RECEPTOR, BINDING, Animals, Antibodies, Monoclonal, Cloning, Molecular, Computer Simulation, Drug Design, Fibrinolytic Agents, Humans, Immunoglobulin Fab Fragments, Mice, Models, Molecular, Papio, Platelet Glycoprotein GPIIb-IIIa Complex, Protein Conformation, Protein Engineering, 1102 Cardiorespiratory Medicine and Haematology, 1103 Clinical Sciences, Cardiovascular System & Hematology, 3201 Cardiovascular medicine and haematology, 3202 Clinical sciences

Abstract:

Fab-fragments of the monoclonal antibody 6134, raised against human glycoprotein lb alpha (GPIb alpha), have a powerful antithrombotic effect in baboons by blocking the GPIb alpha 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 GPIb alpha by variable-domain resurfacing guided by computer modeling. First, the genes coding for the variable regions of the heavy and light chains of 6134 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 hereto humanize 6134 by resurfacing resulted in a fully active derivative, which is now ready for further development.