Low Molecular Weight Peptides Restore the Procoagulant Activity of Factor VIII in the Presence of the Potent Inhibitor Antibody ESH8

摘要

Following repeated administration of factor VIII (FVIII), a significant number of hemophilia A patients develop antibodies (Abs), inhibiting the procoagulant activity of infused FVIII. We have designed an approach based on the blocking of the deleterious activity of these Abs by peptide decoys mimicking the anti-FVIII Ab epitopes. Here, the well characterized inhibitory monoclonal Ab ESH8 served as a model. Several phage peptide libraries were screened for specific binding to ESH8. Seven constrained dodecapeptide sequences were obtained. Six sequences carried the consensus motif, hydrophobic-(Y/F)GKTXL. This motif showed a certain similarity with the2231QVDFQKTMKV2240 sequence of the C2 domain. In the seventh sequence, YCNPSIGDKNCR, the residues GDKN are similar to the sequence2267DGHQ2270. Upon inspection of the C2 domain crystallographic structure, the two stretches QVDFQKTMKV and DGHQ appeared close together in space and might constitute a discontinuous epitope. Corresponding synthetic peptides were able to inhibit the binding of ESH8 to FVIII in a specific and dose-dependent manner. Moreover, the ability of the selected peptides to neutralize the inhibitory activity of ESH8 was demonstrated in functional tests as well as in vivoin a murine model of hemophilia A. This study demonstrates the potential of this approach to neutralize the activity of potent inhibitory Abs. Following repeated administration of factor VIII (FVIII), a significant number of hemophilia A patients develop antibodies (Abs), inhibiting the procoagulant activity of infused FVIII. We have designed an approach based on the blocking of the deleterious activity of these Abs by peptide decoys mimicking the anti-FVIII Ab epitopes. Here, the well characterized inhibitory monoclonal Ab ESH8 served as a model. Several phage peptide libraries were screened for specific binding to ESH8. Seven constrained dodecapeptide sequences were obtained. Six sequences carried the consensus motif, hydrophobic-(Y/F)GKTXL. This motif showed a certain similarity with the2231QVDFQKTMKV2240 sequence of the C2 domain. In the seventh sequence, YCNPSIGDKNCR, the residues GDKN are similar to the sequence2267DGHQ2270. Upon inspection of the C2 domain crystallographic structure, the two stretches QVDFQKTMKV and DGHQ appeared close together in space and might constitute a discontinuous epitope. Corresponding synthetic peptides were able to inhibit the binding of ESH8 to FVIII in a specific and dose-dependent manner. Moreover, the ability of the selected peptides to neutralize the inhibitory activity of ESH8 was demonstrated in functional tests as well as in vivoin a murine model of hemophilia A. This study demonstrates the potential of this approach to neutralize the activity of potent inhibitory Abs. factor VIII antibodies monoclonal antibody enzyme-linked immunosorbent assay phosphate-buffered saline N-(9-fluorenyl)methoxycarbonyl thrombin generation Coagulation defects attributed to the absence or dysfunction of blood coagulation factor VIII (FVIII)1 are observed in 0.01–0.02% of the male population (1Hoyer L.W. Hum. Pathol. 1987; 18: 153-161Crossref PubMed Scopus (31) Google Scholar). This recessiveX-linked bleeding disorder is known as hemophilia A. The adequate treatment of this disease relies on infusions of human FVIII concentrates. However, a serious treatment complication is the development of a humoral immune response to FVIII in ∼25% of patients with hemophilia A. These antibodies (Abs), also called inhibitors, block the FVIII procoagulant activity and complicate seriously the medical care of the patients by precluding further FVIII injections (2Lusher J.M. Blatt P.M. Penner J.A. Aledort L.M. Levine P.H. White G.C. Warrier A.I. Whitehurst D.A. Blood. 1983; 62: 1135-1138Crossref PubMed Google Scholar). This immune response is not only polyclonal but also heterogeneous in its specificity (3Gilles J.G. Arnout J. Vermylen J. Saint-Remy J.M. Blood. 1993; 82: 2452-2461Crossref PubMe