Development of a Factor VIII-Specific Immunotherapy for Hemophilia A

Abstract 3361

The development of neutralizing allo-antibodies against coagulation factor VIII (FVIII) is currently the most serious complication for hemophilia A patients that undergo FVIII replacement therapy. Non-hemophiliacs can spontaneously develop inhibitory auto-antibodies to FVIII, which results in a condition called acquired hemophilia A. The control of the allo- or autoimmune response to FVIII apparently includes the elicitation of an anti-idiotypic immune response. Immune tolerance induction (ITI) by frequent administration of high doses of FVIII is successful in most patients. The remaining up to 20% failing ITI face increased morbidity and mortality.

To elucidate the capacity of single-chain variable antibody fragments (scFvs) for neutralization of inhibitory anti-FVIII antibodies (FVIII inhibitors), scFvs that specifically interact with strong inhibitory murine monoclonal anti-human FVIII antibodies (anti-hFVIII mAbs) were selected by screening synthetic scFv phage displayed libraries. Since the majority of inhibitory anti-hFVIII mAbs are directed against the FVIII A2 or C2 domain, selection of scFvs was performed for two anti-A2 and two anti-C2 mAbs. Affinity selection identified several specific, potential anti-idiotypic scFvs for each anti-hFVIII mAb, which were further analyzed. ScFvs expressed as IgG fusion protein bind to mAbs with affinities up to 0,1nM. Binding of mAbs to immobilized FVIII was inhibited via specific scFvs by more than 90%. In the functional Bethesda Assay FVIII activity was fully restored when corresponding anti-idiotypic scFvs were added to plasma spiked with FVIII-specific mAbs. In addition, the cross-reactivity of scFvs with heterologous mAbs specific for A2 and C2 was tested and confirmed the exclusive interaction of the selected scFvs with their respective mAb.

Overall, anti-idiotypic scFvs specific for anti-hFVIII mAbs can be successfully selected from phage displayed libraries and efficiently neutralize FVIII inhibitors. ScFv anti-idiotypes may therefore facilitate the development of specific immunotherapies for hemophilia patients with inhibitors.