-Background- At variance from Hemophilia, the development of inhibitory antibodies upon replacement therapy is very rare in coagulation factor VII (FVII) deficiency. So far, two cases have been reported and the inhibitory antibodies have not been characterized. Here, we investigate an anti-FVII inhibitory antibody that was developed at high titer (range 7-32 Bethesda Units) in a severe FVII deficient patient treated with rFVIIa or plasma-derived FVII. -Materials and Methods- Expression of recombinant FVII variants in BHK cells. Activated factor X (FXa) generation assays in plasma systems to assess FVII inhibition. ELISA-based assays to evaluate binding to FVII/FVIIa and competition. Bioplex 200 for IgG classification. -Results- The anti-FVII antibodies in patient’s plasma were polyclonal with a majority of IgG1. ELISA-based binding and competition assays showed that the antibodies recognized FVIIa (Kd 0.54±0.09 BU/ml) with higher affinity than FVII (Kd 0.77±0.07 BU/ml), thus pointing toward the hypothesis that rFVIIa was the major immunogen. Sequencing indicates that the patient was homozygous for the A294V-11125delC double mutation, which alters and extends the FVII carboxy-terminal sequence beyond position 404 and makes it different from that of the normal FVII. This information suggested the carboxyl terminal region of the normal molecule as a candidate epitope for the inhibitory antibodies. We therefore expressed the progressively truncated rFVII-406X, rFVII-405X and rFVII-404X variants that displayed a virtually normal specific activity. Upon incubation with the inhibitory antibodies the shortest variant showed the highest residual activity. As compared to that of rFVII-wt (12.4±0.8%), the FXa generation activity of the rFVII-406X, rFVII-405X and rFVII-404X variants were 28.1±8.9%, 25.1±9.9% and 56.5±6.7%, respectively. -Conclusions- This is the first characterization of an inhibitory antibody against FVII. The data obtained through the mutation-based approach support the carboxy-terminus region of FVII as a main functional epitope.
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|Titolo:||Immune response to treatment in a severe factor VII deficient patient: characterization of the inhibitory antibody and epitope-mapping|
|Autori interni:||BRANCHINI, Alessio|
|Data di pubblicazione:||2012|
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