Coagulation factor VII variants resistant to inhibitory antibodies

Alessio Branchini; Marcello Baroni; Caroline Pfeiffer; Angelika Batorova; Muriel Giansily-Blaizot; Jean F. Schved; Guglielmo Mariani; Francesco Bernardi; Mirko Pinotti

doi:10.1160/th14-03-0198

Thrombosis and Haemostasis, Inhaltsverzeichnis

Thromb Haemost 2014; 112(05): 972-980
DOI: 10.1160/th14-03-0198

Blood Coagulation, Fibrinolysis and Cellular Haemostasis

Schattauer GmbH

Coagulation factor VII variants resistant to inhibitory antibodies

Alessio Branchini^*

¹Department of Life Sciences and Biotechnology and LTTA Centre, University of Ferrara, Italy

,

Marcello Baroni^*

¹Department of Life Sciences and Biotechnology and LTTA Centre, University of Ferrara, Italy

,

Caroline Pfeiffer

²Department of Biological Haematology, CHU of Montpellier, Montpellier, France

³UMR 3145 SysDiag CNRS/Bio-Rad, Parc Euromédecine, Montpellier, France

,

Angelika Batorova

⁴Department of Haematology and Transfusion Medicine, National Haemophilia Centre, University Hospital, Bratislava, Slovakia

,

Muriel Giansily-Blaizot

²Department of Biological Haematology, CHU of Montpellier, Montpellier, France

,

Jean F. Schved

²Department of Biological Haematology, CHU of Montpellier, Montpellier, France

,

Guglielmo Mariani

¹Department of Life Sciences and Biotechnology and LTTA Centre, University of Ferrara, Italy

,

Francesco Bernardi

¹Department of Life Sciences and Biotechnology and LTTA Centre, University of Ferrara, Italy

,

Mirko Pinotti

¹Department of Life Sciences and Biotechnology and LTTA Centre, University of Ferrara, Italy

› Institutsangaben

Abstract

Summary

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.

Keywords

Inhibitory antibodies - replacement therapy - factor VII deficiency - carboxyl-terminus - recombinant proteins

Volltext

Referenzen

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