In Vivo Cleavage of Recombinant VWF Upon Intravenous Administration in Preclinical and Clinical Settings

Abstract 1222

Von Willebrand factor (VWF) is composed of a series of multimers with a molecular size ranging from 600 to 20,000 kDa. The ultra-high molecular weight (UHMW) portion of VWF multimers is more efficient in platelet interactions than the lower molecular weight portion, and as such it might have a thrombogenic potential under certain pathologic conditions. The concentration of UHMW multimers in healthy individuals' blood is low because the circulating specific metalloprotease ADAMTS13 rapidly cleaves UHMW VWF upon its release into the circulation.

A recombinant VWF (rVWF) expressed in CHO cells has been developed as a new drug candidate for treating patients with VWF deficiency. Since this protein is not cleaved by ADAMTS13 during the expression phase, it has a high portion of UHMW multimers, similar to the human VWF stored in Weibel-Palade bodies of endothelial cells.

During the development phase of rVWF in vitro experiments showed that when treated under mild denaturing conditions or under shear, the molecule was susceptible to ADAMTS13 of human or animal origin.

Here we present data from preclinical studies where analyzing the ex vivo samples showed the ADAMTS13-mediated cleavage of intravenously applied UHMW-containing rVWF. In good correlation with the in vitro results, studies in rabbits and cynomolgus monkeys showed that the ADAMTS13-specific cleavage fragments appeared as soon as 15 minutes after application, as visualized by immunoblot analysis. The appearance of cleavage bands coincided with the decrease in multimer numbers, indicating that rVWF had been specifically cleaved by endogenous ADAMTS13 in the circulation. The effect was species-dependent, only a low or minimal cleavage was observed in different mice strains.

A recently completed Phase I clinical study clearly demonstrated that rVWF administered intravenously to severe VWD patients in a dose-range of 7.5 to 50 VWF:RCo U/kg body weight was rapidly processed in the circulation, resulting in the disappearance of the UHMW multimers with visible cleavage fragments, as shown by immunoblot and high resolution multimer analysis. No thrombotic adverse events were seen during the study, underlying the suitability of the UHMW-containing rVWF for treating VWD patients.