Development of a Pegylated Dimeric Recombinant Factor VIII-Fc Fusion Protein for the Treatment for Hemophilia a Patients

Hemophilia A is a hereditary bleeding disorder resulting from reduced factor FVIII activity. It occurred in 1/5000 male. Currently, the treatment option is with the factor FVIII replacement therapy. A long-acting recombinant monomeric FVIII-Fc fusion protein product (Eloctate®) has been approved in 2014 by the US FDA, it requires to infuse the drug for every 3 days or twice a week. There is a clinical need to develop longer half-life product to extend the treatment option to once a week infuse for hemophilia A patients. Recently, we have developed a dimeric recombinant factor VIII-Fc (drFVIII-Fc) fusion protein therapeutic candidate, which is entering the clinical development in China. To generate a longer half-life recombinant FVIII product, we have developed a PEGylation method to PEGylated this drFVIII-Fc fusion protein to PEGdrFVIII-Fc. We have analyzed and characterized the fusion protein by various analytic methods, as well as in vivo animal tests. It was shown that PEGdrFVIII-Fc fusion protein has been modified with about five Y type of 40kd PEG; the remaining activity is around 700 IU/mg, and the in vivo tests in cynomolgus monkey demonstrated that the fusion protein has a half-life of about 37 hours. The data also showed that there was no detectable affinity binding activity of vWF to a PEGdrFVIII-Fc fusion protein, as compared with the binding activity of 5.16X10-4M for the molecule of a drFVIII-Fc fusion protein. In conclusion, we are able to generate a PEGylated form of a drFVIII-Fc molecule with the relevant specific activity and has been shown the molecule with the prolonger half-life in the in vivo tests. The further biochemical analysis demonstrated PEGdrFVIII-Fc fusion protein with no detectable vWF binding activity, which might explain why its half-life is longer than vWF's ~15hours half-life in vivo. This molecule is likely to be used as a once-weekly treatment option for hemophilia A patients. Currently, we are in the development stage of an IND filing in China.