Mapping of inhibitory antibodies directed to the carboxy-terminus of FVIIa in severe FVII deficiency with elongated C-terminal variant (p.A354V-p.P464Hfs†)

Background The relationship between the development of inhibitory antibodies and gene defects, extensively investigated in Hemophilia patients, is still an open issue. We studied inhibitory antibodies in severe factor VII (FVII) deficiency, a rare bleeding disorder characterized by a low incidence of this complication. Aim To correlate the F7 gene mutation to major epitopes of anti-FVII inhibitory antibodies developed after replacement therapy. Patient and Methods The proposita (female, 59 years old) has been treated with plasma derived FVII (pdFVII) and recombinant activated FVII (rFVIIa) and developed high titers of anti-FVII inhibitory antibodies. The patient displayed very low circulating FVII protein levels (FVII:Ag 1.2%) and undetectable FVII activity (FVII:C <1%), in accordance with the homozygous condition for the F7 p.A354V-p.P464Hfs† mutation. Functional assays were performed in plasma samples and in FVII-deficient plasma upon addition of rFVIIa or engineered FVII recombinant variants. The affinity of antibodies in patient’s plasma for rFVIIa, pdFVII and recombinant variants was evaluated by binding and competition studies, using ELISA-based assays and Bio-Plex technology. Results Isotypic analysis showed a large prevalence of IgG subtype 1 in patient’s plasma and functional assays indicated a type II kinetics. Activated factor X (FXa) and thrombin generation activity assays showed a major impact of inhibitory antibodies on the coagulation initiation phase, leading to prolonged lag times. Binding assays revealed a 1.5 fold higher affinity of the antibody for rFVIIa than for pdFVII. Noticeably, once activated pdFVII was recognized similarly to rFVIIa in competition assays. The p.A354V-p.P464Hfs† mutation causes frameshift from residue 464 and predicts an elongated carboxy-terminal region (491 vs 466 residues in FVII-wt). The inferred structural differences between the elongated FVII molecules circulating in patient and the infused factors led us to hypothesize the carboxy-terminal region as bearing a candidate epitope for the antibodies. Binding assays demonstrated that denatured and reduced FVII was not recognized by antibodies, thus supporting the presence of a tridimensional epitope. To assess whether the carboxy-terminal region contributed to shape the major antibody epitopes we performed functional assays i) in plasma from a patient homozygous for the p.R462X nonsense mutation, which produces low circulating levels of a truncated FVII with increased specific activity (Branchini et al, 2012) and ii) by deletion scanning leading to the truncated recombinant FVII-466X, FVII-465X and FVII-464X variants, displaying normal specific activity. Strikingly, functional assays showed that the natural FVII-462X was less inhibited than FVII in pooled normal plasma. Moreover, studies with the truncated recombinant variants demonstrated that the carboxy-terminus length was related to the inhibition rate, being the shortest rFVII-464X the least inhibited variant, as indicated by a significant difference (p<0.0001) in lag time prolongation in FXa generation assays. Accordingly, binding assays on the Bio-plex platform indicated that the rFVII-464X was the least recognized variant by the inhibitory antibodies. Conclusions Taken together our findings, which represent the first characterization of an anti-FVII inhibitor, indicated that antibodies were directed toward the carboxy-terminal region of infused FVII molecules. Data may suggest a relation between the F7 mutation and major inhibitor epitopes.