Hemophilia A is an X-chromosome linked bleeding disorder resulting from the absence or nonfunctional expression of coagulation factor VIII (FVIII). About 30% of severe hemophiliacs develop neutralizing antibodies (inhibitors) to FVIII upon treatment with exogenous factor preparations. Specificity of these antibodies is often restricted to functional determinants in the A2 and C2 domain. Phage displayed random peptide libraries were screened with various plasma samples of high titer inhibitor patients and inhibitor specific peptide ligands were selected. In silco mapping of consensus amino acid motifs among peptides revealed conformational epitopes in A2 and C2. Selected ligands partially restored FVIII activity. Equimolar combination of these ligands enhanced blocking of inhibitors in autologous and heterologous patients’ plasma. Peptide ligands were fused to the C-terminal multimerization domain of the C4bp alpha-chain and expressed as multimers in 293T cells. Peptide multimers revealed improved binding of anti-FVIII IgG and prolonged in vitro half-life in comparison to single synthetic peptides. Selected peptide ligands were combined in heteromultimers by co-transfection of respective vectors, resulting in molecules binding both A2- and C2- specific IgG and blocking up to 100% of antibody binding to FVIII. Those molecules could provide a basis for the generation of novel peptide-based therapeutic approaches.

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