Inhibitor Epitopes Identified from Inhibitor Positive Plasma of a Hemophilia B Patient Change during ITT.

Hemophilia B is an X-linked bleeding disorder. Substitution of recombinant (rFIX) or plasma derived (pdFIX) FIX preparations are required to restore coagulation. A rare but severe complication is the development of FIX inhibitors that are often accompanied by allergic reactions. Immune tolerance therapies (ITTs) for inhibitor elimination are difficult and can induce further allergic reactions. ITTs for FIX inhibitors are less established and less successful than for FVIII inhibitors.

In this study, three phage displayed random peptide libraries were screened by biopanning to isolate small peptides that mimic the epitope of FIX inhibitors. In the patient, the inhibitor was eliminated by ITT but reappeared again. Plasma samples before ITT and at reappearance of inhibitor were used to isolate inhibitor specific phage clones. Phage clones showing specific reactivity with inhibitor positive plasma compared to control plasma by ELISA were amplified and DNA was sequenced to determine the peptide sequence displayed on the phage. Before ITT, 31 isolated phage clones specific for FIX inhibitors have been sequenced so far. 10/31 contain a common motif. After ITT, 24 clones specific for patient plasma have been sequenced. 10/24 contain a different common motif. Synthetic peptides have been generated according to the phage displayed sequence to demonstrate binding of inhibitors and test for cross reactivity.

The surface of the model of FIX was screened for these motifs with a novel software tool. Different conformational epitopes could be identified on the surface of the molecule before and after ITT.

Small peptides (mimotopes) have been identified that mimic epitopes for FIX inhibitors in a hemophilia B patient. The sequences can be aligned to different conformational epitopes on FIX. It appears that the epitope has changed in the course of the ITT. The mimotopes can also be used to target inhibitor specific B cells for a novel approach in inhibitor elimination in hemophilia B.