OC 33.3 - Evaluating the impact of anti-PEG antibodies on activation of PEGylated therapeutic FVIII products

Background: Hemophilia A is an X-linked bleeding disorders caused by deficiency of coagulation factor VIII (FVIII) protein. Hemophilia replacement therapy with either recombinant (rFVIII) or plasma-derived (pdFVIII) therapeutics is the standard treatment for patients. Currently, two main classes of FVIII products are available: Standard Pd- or –rFVIII and extended half-life rFVIII therapeutics. Attachment of polyethylene glycol (PEG) polymers has is a successful strategy to increase protein half-life and stability. However, the development of antidrug antibodies with regard to the PEG moiety or pre-existing anti-PEG antibodies in “treatment-naïve” patients is a rising concern.

Aims: To evaluate the inhibition pattern of PEGylated rFVIII therapeutics by anti-PEG antibodies.

Methods: In the presence and absence of 3 IgM and 5 IgG anti-PEG antibodies, we evaluated 1) their inhibitory potential using a modified Nijmegen Bethesda (NBA) assay and 2) discrepancy of the FVIII activity (FVIII:C) levels using the one-stage clotting (OSC) and chromogenic (CSA) assays 3) the activation of PEGylated rFVIII drugs with thrombin by western blotting.

Results: The analysis revealed different inhibition patterns of anti-PEG antibody isotypes. Overall the IgM isotype showed higher level of inhibition (50% for 20PEG-FVIII and 90-100% for 40PEG- and 60-PEGFVIII). IgG antibodies did not inhibit 20PEGFVIII. Different degrees of inhibition was observed when comparing the OSC and CSA for 40PEGFVIII and 60PEGFVIII. Using OSC a reduction of FVIII:C between 70-80% was observed, whereas the inhibition was masked when tested with CSA. The thrombin activation on SDS-Blot showed differences in activation of the PEGylated products with respect to thrombin concentration and anti-PEG antibody isotype.

Conclusion(s): Commercially available anti-PEG antibodies inhibit the activation of all PEGylated FVIII therapeutics but to different degrees. Our result suggest, that the close vicinity of the PEG moiety to thrombin cleavage site in the B-Domain-Deleted PEGylated products might explain differences in inhibition pattern and differences between OSC and CSA.