Effect of glycoPEGylation on Factor VIIa Binding and Internalization

Frequent spontaneous joint bleedings in severe hemophilia leads to chronic arthopathy, which severely reduces the quality of life of severe hemophiliacs. Prophylactic administration of FVIII/FIX concentrates was introduced in the early 1960’s to convert severe hemophilia into a moderate form that cause less frequent joint bleedings. Recent data in hemophilia patients with inhibitors have provided evidence for that daily dose of rFVIIa can act in secondary prophylaxis in patients with frequent bleeds. The plasma half-life of rFVIIa is approx 2–3 hours which thus may limit its efficacy and convenience in prevention of bleeds. Therefore, development of rFVIIa molecules that could remain in the circulation for a prolonged period may potentially improve prophylactic options of rFVIIa. PEGylation is an established and clinically proven strategy for prolonging the circulatory life-time of bio-therapeutic proteins. Recently, GlycoPEGylation technology was used to generate PEGylated rFVIIa derivatives (Stennicke et al., 2008 Thromb. Haemost, in press) and they appeared to activate factor X at a similar rate as of rFVIIa (Stennicke et al., 2008 Thromb. Haemost, in press; and Ghosh et al., 2008, J. Thromb. Haemost, in press). In the present study, we further characterized the glycoPEGylated rFVIIa, rFVIIa-10K PEG and rFVIIa-40K PEG, particularly in reference to their interaction with tissue factor (TF) and endothelial cell protein C receptor (EPCR) on cell surfaces and their catabolism. rFVIIa and glycoPEGylated rFVIIa were labeled with ¹²⁵I and the radio-iodinated proteins were used to monitor rFVIIa binding and uptake in endothelial cells, CHO cells stably transfected with EPCR, and fibroblasts. PEG modification of rFVIIa resulted in a marked decrease in the rate of rFVIIa uptake in endothelial cells. The reduction in the uptake of rFVIIa following attachment of PEG was primarily due to the reduced association of rFVIIa to the cell surface. The level of glycoPEGylated rFVIIa binding to endothelial cells was about 20 to 30% of that was obtained with the rFVIIa. No significant differences were found between rFVIIa-10K PEG and rFVIIa-40K PEG in their association with endothelial cells. rFVIIa-40K PEG uptake was slightly lower compared to rFVIIa-10K PEG. The reduction in the uptake of glycoPEGylated rFVIIa by endothelial cells appeared to be the result of a decreased rate of glycoPEGylated rFVIIa binding to EPCR. Consistent with this glycoPEGylated rFVIIa binding, relative to rFVIIa, was markedly lower to CHO cells expressing EPCR whereas no significant differences were found in the basal binding of rFVIIa and glycoPEGylated rFVIIa to wild-type CHO cells. The radioligand binding studies also revealed marked differences between rFVIIa and glycoPEGylated rFVIIa binding to TF on fibroblasts. Although the total amounts of radioligands associated with cell surface TF at saturating concentrations were similar, the affinity of rFVIIa-10K PEG and rFVIIa-40K PEG to TF, relative to rFVIIa affinity to TF, was lowered by about 6 to 20-fold, respectively. Surprisingly, if the binding affinities were evaluated in factor X activation studies, the differences between rFVIIa and glycoPEGylated rFVIIa were minimal. The differences between rFVIIa and glycoPEGylated rFVIIa binding to TF in these two different assay systems suggest that rFVIIa and glycoPEGylated rFVIIa may interact with active TF with equal affinity whereas they interact differently with cryptic TF. Alternatively, glycoPEGylated rFVIIa binds initially to cell surface TF with a similar affinity as of rFVIIa, but may dissociate readily from TF upon the washing that is required to measure the binding of radioligands to cells. Additional studies yielded data that favor the later possibility. Overall the present data suggest that the reduced affinity of rFVIIa associated with glycoPEGylation on binding to EPCR and TF is likely attributable to either steric hindrance or changes in electrostatic binding properties rather than modification of binding sites itself. Further, the reduced uptake of glycoPEGylated rFVIIa by cells stems primarily from the decreased association of the modified rFVIIa to cell surfaces rather than specific impairment in their internalization.