Irreversible Blockade of Factor VIII Antibodies.

Abstract

High titer antibodies (Abs) to Factor VIII (FVIII) can render FVIII replacement therapy ineffective in severe hemophilia A patients. An antigen capable of specific and covalent binding is predicted to reverse the effects of pathogenic Abs potently compared to conventional noncovalently binding antigens, as dissociation of the former type of antigen from the immune complexes is precluded. Recently, we described the apparently universal presence of enzyme-like nucleophiles in the antigen binding sites of Abs (Planque et al, J Biol Chem, 2003). Here, we report a covalently reactive antigen analog (CRA) of FVIII that inactivates anti-FVIII Abs specifically. Recombinant FVIII was derivitized at Lys side chains with phosphonate diester groups, positioning these electrophiles within the antigenic epitopes expressed by FVIII. Comparable binding of FVIII-CRA and underivitized FVIII by a monoclonal Ab and polyclonal Abs from 5 hemophilia patients was evident, suggesting maintenance of the FVIII epitope structure necessary for noncovalent Ab recognition. FVIII-CRA formed immune complexes with polyclonal anti-FVIII Abs from hemophilia patients that were not dissociated by sodium dodecyl sulfate (2%), a detergent that disrupts noncovalent interactions. Removal of free FVIII-CRA from the reaction mixtures by affinity chromatography on Protein G did not restore the FVIII-binding activity of the Abs, confirming irreversible occupancy of the Ab binding sites. No binding of the Abs by an irrelevant peptidyl CRAs was observed, suggesting the role of noncovalent antigen binding in imparting specificity to the covalent reaction. Ab inhibition of the functional role of FVIII in coagulation was studied by the chromogenic assay. Treatment of the Abs with the FVIII-CRA followed by removal of free FVIII-CRA relieved the inhibitory effect of the Abs on FVIII mediated activation of FIXa/FX. An irrelevant peptidyl CRA did not relieve the Ab inhibitory effect. Compared to FVIII, the FVIII-CRA activated FIXa/FX poorly, suggesting that the integrity of the FIXa binding site had been disrupted. These observations encourage the development of electrophilic CRA analogs of FVIII for treatment of patients with inhibitory Abs to FVIII.