PEGylation of Factor VIII Reduces the Inhibitory Effects of Human Antibody Inhibitors on the Factor VIII Molecule.

Replacement therapy with plasma-derived or recombinant Factor VIII (FVIII) has successfully reduced mortality and morbidity and improved the quality of life for patients with hemophilia A. However, up to 30% of patients develop antibody inhibitors to the FVIII molecule. Inhibitors reduce the efficacy of FVIII, increase the cost of treatment, and greatly increase the risk for life-threatening bleeding events in these patients. In the hopes of developing a more efficacious FVIII therapeutic for hemophilia A patients with inhibitors, we initiated studies on the PEGylation of B-region deleted FVIII (FVIII-BDD) with different sizes of polyethylene glycol (PEG) and investigated the inhibitory effects of anti-FVIII inhibitors on the activity of PEGylated FVIII-BDDs. Applying a site-specific mutagenesis technique to the FVIII-BDD molecule, an amino-acid residue at the inhibitor binding site was changed to cysteine. Mutated FVIII-BDD was purified and PEG molecules of different sizes were added at the mutation sites through a chemical reaction with the maleimide group on the activated PEG. PEGylated FVIII-BDD molecules were further purified and tested for FVIII activity using the chromogenic assay. To investigate the effects of antibody inhibitors on the PEGylated FVIII-BDD, studies were carried out utilizing inhibitory plasmas collected from 8 hemophilia A patients with confirmed inhibitors and different monoclonal antibodies as controls. Of the 8 patient plasmas tested, 43 kD PEGylated FVIII-BDD was more resistant to antibody inhibitors in 4 patient plasma samples than unmodified FVIII-BDD. In one sample, pre-incubation of FVIII-BDD with inhibitor patient plasma (1:15 dilution) reduced FVIII activity to 5% of the originally activity added, according to the chromogenic assay. By contrast, approximately 20% of original activity was retained for monoPEGylated FVIII-BDD and >40% activity was retained for diPEGylated FVIII-BDD. Overall, the results suggest that PEGylated FVIII-BDDs may retain more FVIII activity in the presence of some FVIII antibody inhibitors compared to FVIII-BDD. It is important to note that there was a positive correlation between the size of PEG added to FVIII-BDD and the amount of FVIII activity retained. This study indicates that the addition of PEG to FVIII molecules through site-specific PEGylation has the potential to increase the resistance of FVIII to the effects of some antibody inhibitors in patients with hemophilia A.