In Silico Design and in Vitro Validation of a Mimotope for Blocking a FVIII-inhibitor Antibody ESH8

Abstract number: OR208

Zabe-KühnM¹ , Hoffmann^1,2 D, Hohm¹ T, Albert³ T, Schwaab³ R

^{1,2 11}Stiftung caesar, Research Group Functional Peptides, Bonn, Germany ¹¹Stiftung caesar, Research Group Functional Peptides, Bonn, Germany ³³University of Bonn, Institute of Experimental Hematology and Transfusion Medicine, Bonn, Germany

We hypothesize that it is possible to rationally design peptides that can neutralize inhibitory antibodies of blood coagulation factor VIII (FVIII), currently the major problem in FVIII replacement therapy of hemophilia A. We have developed bioinformatics methods to support the search for FVIII peptide mimotopes and we have applied these methods to the computational design and optimization of peptidic mimotopes based on peptide ECIVYGKTALCT (Villard et al., JBC, 2002) that binds to inhibitor antibody ESH8. The sequence of this peptide has similarity to sequence motif 2234–2238 of FVIII. We have transplanted this motif (FQKTM) in silico into a WW domain, a small b-sheet, of a putative protein from S. cerevisiae (Macias et al., Nat Struct Biol, 2000), and then computationally introduced several mutations to stabilize the motif in its native FVIII conformation. In this way we could propose four peptides that promise to present the FVIII motif in a nearly native conformation. Further optimization may be necessary since the peptides expose several hydrophobic residues which may lead to solubility problems. Additionally, we have designed a peptide of five amino acids deduced from the above motif where the outer hydrophobic residues of the motif have been exchanged for two Cys that form a stabilizing disulfide bridge. The proposed peptides are being tested for their ability to bind to ESH8 with ELISA. Kinetics are being tested with surface plasmon resonance techniques. Additionally we are investigating the anti-inhibitory potential of the peptides towards ESH8 in a functional Bethesda assay. This work is partly funded by the Bayer Hemophilia Special Project Award to RS.