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

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 (K_D = 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 K_D-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 K_D-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.

Figure 1 Schematic illustrations of the interactions of FVIIIa or emicizumab with FIX/FIXa and FX/FXa

. A) Interactions of FVIIIa with FIXa and FX reported previously (5–10). B) Interactions of emicizumab with FIX/FIXa and FX/FXa. The illustrations do not necessarily indicate precise molecular structures.

Figure 2: Antigen domain recognised by emicizumab and emicizumab’s specificity to the antigens

. A) FIX/FIXa-related proteins used. B) The electrophoretic pattern of FIX/FIXa-related proteins stained by CBB in a non-reduced condition and the results of immunoblotting with the anti-FIX/FIXa arm of emicizumab. Bands above 100 kDa are considered to be the aggregated multimers. C) FX/FXa-related proteins used. D) The electrophoretic pattern of FX/FXa-related proteins stained by CBB in a non-reduced condition and the results of immunoblotting with the anti-FX/FXa arm of emicizumab. Bands above 100 kDa are considered to be the aggregated multimers. E) The results of ELISA to detect binding of emicizumab to immobilised FVII, FIX, FX, FXII, and Protein C. Data are shown as mean ± SD (n = 3).

Figure 3: K_D -based simulation of an equilibrium state in the presence of the standard plasma concentrations of FIX and FX

. Concentrations of FIX–emicizumab–FX ternary complex (A), and the percent ratio of monomer, binary complex, and ternary complex of FIX to total FIX (B) or those of FX to total FX (C), which were simulated on the basis of the K _D values at varied concentrations of emicizumab.

Figure 4: Emicizumab concentration-dependency of a parameter of an intrinsic pathway-triggered TG assay in FVIII-deficient plasma

. Peak Height analysed from the intrinsic pathway-triggered TG assay at varied concentrations of emicizumab in FVIII-deficient plasma. Data are shown as mean ± SD (n = 3).