Epitope Mapping of FIX Inhibitors Identify Contact Residues in the Protease Domain.

Abstract 3172

Poster Board III-112

Hemophilia B is an X-linked bleeding disorder that requires regular substitution of FIX preparations to restore coagulation. A rare but severe complication is the development of FIX inhibitors affecting about 3% of hemophilia B patients. Possible allergic reactions and challenging but often unsuccessful immune tolerance protocols complicate the treatment of affected patients. In the present study, five samples from patients with inhibitors against FIX (including one acquired inhibitor) were screened against random peptide libraries displayed on phage. A biopanning protocol was established to isolate phagotopes specific for FIX inhibitors and thus small peptides that mimic the epitopes of the inhibitors. Binding was confirmed by ELISA and the peptide sequences were determined by translating the DNA sequence of the phage. In total 65 peptide sequences were determined. For all patients except for the patient suffering from acquired FIX inhibitors consensus motifs were identified. These motifs include the amino acids FTK(T), TSL(T) and LRQ. In order to compare the motifs to the surface of FIX for epitope identification a novel structural model of human FIX was generated. Using this model epitopes were identified on the surface of the protease domain of FIX. The residues comprising the conformational epitopes are found in the regions AA290 - 330 and 380 - 390. Small peptides have been identified that mimic the epitopes for FIX inhibitors in hemophilia B patients. These mimotopes mimic conformational epitopes on the surface of the protease domain on FIX. Identifying inhibitor epitopes and isolating molecules that can possibly interfere with inhibitor binding might help to understand and to develop novel approaches to treat this rare but life-threatening complication.