Epitope Mapping during FVIII Inhibitor Elimination with Rituximab Revealed Conformational Epitopes on FVIII and Identifies Potentially Small Molecules for Novel Therapies.

Hemophilia A is an X-linked bleeding disorder. Mutations in the Factor VIII (FVIII) gene result in reduced or non-functional expression of FVIII. Patients are commonly treated by substitution of recombinant (rFVIII) or plasma derived (pdFVIII) FVIII preparations. However, a significant number of patients treated develop an antibody response (inhibitors) against substituted FVIII. Various Immune Tolerance Therapies (ITTs) are applied to overcome the FVIII specific immune response.

We recently reported on a novel approach to eliminate the FVIII specific immune response by several rounds of rituximab (Mabthera®) infusions after several failed ITT approaches.

In this study, phage displayed random peptide libraries were screened with sera from a patient before, under and after rituximab treatment. After several rounds of biopanning more than 100 phage clones specific for FVIII-inhibitors were identified and sequenced. One amino acid motif - EVPN - was most common in isolated clones. Computer based analysis identified a conformational epitope in the A2 domain of FVIII. The clones specifically inhibit binding of inhibitor to FVIII. Also sera from other inhibitor patients cross reacts with isolated phage clones. Inhibitor sera also bind to synthetic peptides, which sequences correspond to the peptide insert on isolated phage. Functional studies to target inhibitor specific B cells with isolated peptides are currently being performed.

Specific ligands for FVIII inhibitors were identified. The epitope was mapped to the A2 domain. When inhibitor relapsed after Rituximab treatment, the same epitope was recognized. Small peptides representing the epitope on the phage (mimotopes) bound to inhibitor positive sera and even cross-reactivity with heterologous sera was observed. The inhibitor specific peptide ligands may serve as useful tools to develop novel approaches for inhibitor elimination therapies.