A Protease Resistant Factor VIII Analog for Blockade of Inhibitory Antibodies

Abstract 3361

High titer inhibitory antibodies (Abs) to Factor VIII (FVIII) generated in response to FVIII replacement therapy can render the therapy ineffective in hemophilia A (HA) patients. Abs to FVIII contain serine protease-like nucleophilic sites that react covalently with electrophilic phosphonate diester groups placed on the Lys side chains of FVIII (E-FVIII), resulting in restoration of the ability of exogenous FVIII to act as a cofactor for blood coagulation (Planque et al, J Biol Chem 2008; 283, p11876-11886). Irreversible inactivation of Abs by E-FVIII could potentially permit administration of FVIII for treatment of acute bleeding episodes in HA patients with high titer inhibitory antibodies. Another favorable effect of E-FVIII is its ability to block the production of anti-E-FVIII Abs by cultured memory B cells (Smith et al., J Thromb Haemost 2007; 5 Supplement 2: O-M-063). Here we report the resistance of E-FVIII to proteolytic clearance, a property predicted to enable prolonged blockade of pathogenic anti-FVIII immunity. E-FVIII was degraded by the serine proteases thrombin, trypsin and chymotrypsin at rates 100, 29 and 23-fold less rapidly, respectively, compared to FVIII, determined by electrophoretic generation of protein fragments. The effect of E-FVIII on thrombin catalyzed VAL-PRO-ARG-aminomethylcoumarinamide hydrolysis followed classical irreversible inhibitory kinetics, consistent with covalent bonding of the phosphonate group to the thrombin active site. Thrombin affinity (Ki) for E-FVIII and the rate of covalent bond formation (k2), respectively, were 1331 nM and 1.8 min^-1. E-FVIII resistance to thrombin should reduce its consumption in the coagulation pathway, increasing its availability for the reaction with anti-FVIII Abs. FXa, an enzyme that contributes in the metabolic clearance of FVIII from blood, also degraded E-FVIII more slowly compared to FVIII. The half-life of E-FVIII in human serum and human plasma in vitro was substantially superior to that of FVIII. Only weak binding of E-FVIII to human von Willebrand Factor (VWF) was detected, suggesting that stabilizing interactions with VWF are not the reason for increased E-FVIII resistance to the proteases. E-FVIII did not prolong the time to clot formation determined by the APTT test and did not inhibit FXa generation in the chromogenic Coamatic test. Therefore, E-FVIII does not cause a global inhibition of the serine protease pathways required for blood coagulation. The reaction model entails covalent bonding of the E-FVIII phosphonate if productive noncovalent contact with the enzyme is established. As the E-FVIII is present at limiting concentrations (<Ki), generalized protease inhibition does not occur. These data suggest that clearance of E-FVIII in vivo by blood-borne proteases should be limited, potentially affording a longer half-life and prolonged blockade of pathogenic antibody effects. Reduced E-FVIII digestion by intracellular proteases may also attenuate the presentation of pathogenic FVIII antigenic epitopes to T cells, thereby, reducing the production of inhibitory anti-FVIII antibodies.