Peptides derived from a secretory yeast library restore factor VIII activity in the presence of an inhibitory antibody.
The development of autoantibodies against factor VIII represents one of the major complications in the treatment of hemophilia A patients. We have employed a novel library system to obtain peptides that specifically neutralize the interaction between factor VIII and these inhibitors. The random peptides are presented as carboxy-terminal extensions of the eukaryotic initiation factor 5a, an intracellular protein with a molecular mass of 18 kDa. These random peptides formed an unique binding site, as demonstrated by molecular simulations using the computer programs InsightII and GROMACS. The library was screened to identify peptides binding to the murine monoclonal anti-factor VIII antibody ESH8 and to inhibitors derived from patients with factor VIII antibodies. Ten peptides binding to ESH8 were identified. Their specificity was confirmed by displacement assays. Two peptides with the sequences STKTLGRPLHGPAGPVEGGALAGVAEDADLVTAVSGR and YHCKREDLTDRDATCALRQPPQAVRGLGPRVTAVSGR showed the ability to restore the factor VIII activity from 33% up to approximately 90% in functional tests performed in vitro. Three candidates for binding to factor VIII antibodies derived from four different patient's sera were achieved. Three fusion proteins with the peptide sequences PQLGSRRSTTPSLTFQNASWFPAGGPCARSNRG, SGSRQVCKLARSLQPF and WERGRRVGAQVRHARHLVARVLDGAGHQARLTAVNGP bound to inhibitors derived from different patients. Furthermore, two of the obtained fusion proteins with the peptide sequences RHWTALGPAPTHTCADLNYPLLS and WERGRRVGAQVRHARHLVARVLDGAGHQARLTAVNGP did also bind to the monoclonal antibody ESH8. This study demonstrates the potential of this system to identify peptides that inhibit the activity of potent inhibitory antibodies and also shows potential as a method for screening of bioactive peptides.
Mersich
Christa
C
Department of Biotechnology, University of Natural Resources and Applied Life Sciences Vienna, Vienna, Austria.
Billes
Werner
W
Pabinger
Ingrid
I
Jungbauer
Alois
A
D000906:Antibodies / Q000276:immunology*; D005169:Factor VIII / Q000276:immunology*; D006467:Hemophilia A / Q000276:immunology*; D006801:Humans; D019151:Peptide Library; D010455:Peptides / Q000235:genetics / Q000276:immunology*; D015202:Protein Engineering / Q000379:methods*; D015003:Yeasts / Q000235:genetics / Q000378:metabolism*
D000906:Antibodies; D019151:Peptide Library; D010455:Peptides; D005169:Factor VIII