Factor VIIIa-mimetic cofactor activity of a bispecific antibody to factors IX/IXa and X/Xa, emicizumab, depends on its ability to bridge the antigens

Thromb Haemost. 2017 Jun 28;117(7):1348-1357. doi: 10.1160/TH17-01-0030. Epub 2017 Apr 28.

Abstract

Emicizumab, a humanised bispecific antibody recognising factors (F) IX/IXa and X/Xa, can accelerate FIXa-catalysed FX activation by bridging FIXa and FX in a manner similar to FVIIIa. However, details of the emicizumab-antigen interactions have not been reported so far. In this study, we first showed by surface plasmon resonance analysis that emicizumab bound FIX, FIXa, FX, and FXa with moderate affinities (KD = 1.58, 1.52, 1.85, and 0.978 µM, respectively). We next showed by immunoblotting analysis that emicizumab recognised the antigens' epidermal growth factor (EGF)-like domains. We then performed KD-based simulation of equilibrium states in plasma for quantitatively predicting the ways that emicizumab would interact with the antigens. The simulation predicted that only a small part of plasma FIX, FX, and emicizumab would form antigen-bridging FIX-emicizumab-FX ternary complex, of which concentration would form a bell-shaped relationship with emicizumab concentration. The bell-shaped concentration dependency was reproduced by plasma thrombin generation assays, suggesting that the plasma concentration of the ternary complex would correlate with emicizumab's cofactor activity. The simulation also predicted that at 10.0-100 µg/ml of emicizumab-levels shown in a previous study to be clinically effective-the majority of plasma FIX, FX, and emicizumab would exist as monomers. In conclusion, emicizumab binds FIX/FIXa and FX/FXa with micromolar affinities at their EGF-like domains. The KD-based simulation predicted that the antigen-bridging ternary complex formed in circulating plasma would correlate with emicizumab's cofactor activity, and the majority of FIX and FX would be free and available for other coagulation reactions.

Keywords: Coagulation factors; drug design; factor VIII; haemophilia therapy.

MeSH terms

  • Antibodies, Bispecific / blood
  • Antibodies, Bispecific / immunology*
  • Antibodies, Bispecific / pharmacology*
  • Antibodies, Monoclonal, Humanized / blood
  • Antibodies, Monoclonal, Humanized / immunology*
  • Antibodies, Monoclonal, Humanized / pharmacology*
  • Antibody Specificity
  • Antigen-Antibody Reactions
  • Binding Sites
  • Biomimetic Materials / pharmacology
  • Computer Simulation
  • Factor IX / antagonists & inhibitors
  • Factor IX / immunology
  • Factor IXa / antagonists & inhibitors
  • Factor IXa / immunology
  • Factor VIIIa / immunology*
  • Factor X / antagonists & inhibitors
  • Factor X / immunology
  • Factor Xa / immunology
  • Factor Xa Inhibitors / blood
  • Factor Xa Inhibitors / immunology
  • Factor Xa Inhibitors / pharmacology
  • Humans
  • Models, Immunological

Substances

  • Antibodies, Bispecific
  • Antibodies, Monoclonal, Humanized
  • Factor Xa Inhibitors
  • Factor VIIIa
  • emicizumab
  • Factor IX
  • Factor X
  • Factor IXa
  • Factor Xa

Grant support

Financial Support: This study was supported by Chugai Pharmaceutical Co., Ltd.