1. Coagulation factor VII variants resistant to inhibitory antibodies.
- Author
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Branchini A, Baroni M, Pfeiffer C, Batorova A, Giansily-Blaizot M, Schved JF, Mariani G, Bernardi F, and Pinotti M
- Subjects
- Amino Acid Sequence, Antigen-Antibody Reactions, Blood Coagulation, Factor VII antagonists & inhibitors, Factor VII chemistry, Factor VII genetics, Factor VII Deficiency drug therapy, Factor VIIa chemistry, Factor VIIa therapeutic use, Factor Xa biosynthesis, Frameshift Mutation, Humans, Immunoglobulin G chemistry, Immunoglobulin G immunology, Immunoglobulin Isotypes chemistry, Immunoglobulin Isotypes immunology, Isoantibodies chemistry, Molecular Sequence Data, Protein Interaction Mapping, Protein Structure, Tertiary, Recombinant Proteins chemistry, Recombinant Proteins immunology, Recombinant Proteins therapeutic use, Sequence Deletion, Structure-Activity Relationship, Thrombin biosynthesis, Factor VII immunology, Factor VIIa immunology, Isoantibodies immunology
- Abstract
Replacement therapy is currently used to prevent and treat bleeding episodes in coagulation factor deficiencies. However, structural differences between the endogenous and therapeutic proteins might increase the risk for immune complications. This study was aimed at identifying factor (F)VII variants resistant to inhibitory antibodies developed after treatment with recombinant activated factor VII (rFVIIa) in a FVII-deficient patient homozygous for the p.A354V-p.P464Hfs mutation, which predicts trace levels of an elongated FVII variant in plasma. We performed fluorescent bead-based binding, ELISA-based competition as well as fluorogenic functional (activated FX and thrombin generation) assays in plasma and with recombinant proteins. We found that antibodies displayed higher affinity for the active than for the zymogen FVII (half-maximal binding at 0.54 ± 0.04 and 0.78 ± 0.07 BU/ml, respectively), and inhibited the coagulation initiation phase with a second-order kinetics. Isotypic analysis showed a polyclonal response with a large predominance of IgG1. We hypothesised that structural differences in the carboxyl-terminus between the inherited FVII and the therapeutic molecules contributed to the immune response. Intriguingly, a naturally-occurring, poorly secreted and 5-residue truncated FVII (FVII-462X) escaped inhibition. Among a series of truncated rFVII molecules, we identified a well-secreted and catalytically competent variant (rFVII-464X) with reduced binding to antibodies (half-maximal binding at 0.198 ± 0.003 BU/ml) as compared to the rFVII-wt (0.032 ± 0.002 BU/ml), which led to a 40-time reduced inhibition in activated FX generation assays. Taken together our results provide a paradigmatic example of mutation-related inhibitory antibodies, strongly support the FVII carboxyl-terminus as their main target and identify inhibitor-resistant FVII variants.
- Published
- 2014
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