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

Fab-fragments of the monoclonal antibody 6B4, raised against human glycoprotein Ibalpha (GPIbalpha), have a powerful antithrombotic effect in baboons by blocking the GPIbalpha 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 GPIbalpha 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.