Subscribe to RSS
Factor VIIIa-mimetic cofactor activity of a bispecific antibody to factors IX/IXa and X/Xa, emicizumab, depends on its ability to bridge the antigensFinancial Support: This study was supported by Chugai Pharmaceutical Co., Ltd.
15 January 2017
Accepted after minor revision: 25 March 2017
28 November 2017 (online)
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.
Institution where the work was carried out: Research Division, Chugai Pharmaceutical Co., Ltd.
Supplementary Material to this article is available online at www.thrombosis-online.com.
- 1 Srivastava A, Brewer AK, Mauser-Bunschoten EP. et al. Guidelines for the management of hemophilia. Haemophilia 2013; 19: e1-e47.
- 2 Peyvandi F, Garagiola I, Young G. The past and future of haemophilia: diagnosis, treatments, and its complications. Lancet 2016; 388: 187-197.
- 3 Leissinger CA. Advances in the clinical management of inhibitors in hemophilia A and B. Semin Hematol 2016; 53: 20-27.
- 4 Kitazawa T, Igawa T, Sampei Z. et al. A bispecific antibody to factors IXa and X restores factor VIII hemostatic activity in a hemophilia A model. Nat Med 2012; 18: 1570-1574.
- 5 Lenting PJ, Donath MJ, van Mourik JA. et al. Identification of a binding site for blood coagulation factor IXa on the light chain of human factor VIII. J Biol Chem 1994; 269: 7150-7155.
- 6 Lapan KA, Fay PJ. Localization of a factor X interactive site in the A1 subunit of factor VIIIa. J Biol Chem 1997; 272: 2082-2088.
- 7 Fay PJ, Koshibu K. The A2 subunit of factor VIIIa modulates the active site of factor IXa. J Biol Chem 1998; 273: 19049-19054.
- 8 Soeda T, Nogami K, Nishiya K. et al. The factor VIIIa C2 domain (residues 2228–2240) interacts with the factor IXa Gla domain in the factor Xase complex. J Biol Chem 2009; 284: 3379-3388.
- 9 Takeyama M, Wakabayashi H, Fay PJ. Factor VIII light chain contains a binding site for factor X that contributes to the catalytic efficiency of factor Xase. Biochemistry 2012; 51: 820-828.
- 10 Griffiths AE, Rydkin I, Fay PJ. Factor VIIIa A2 subunit shows a high affinity interaction with factor IXa: contribution of A2 subunit residues 707–714 to the interaction with factor IXa. J Biol Chem 2013; 288: 15057-15064.
- 11 Sampei Z, Igawa T, Soeda T. et al. Identification and multidimensional optimization of an asymmetric bispecific IgG antibody mimicking the function of factor VIII cofactor activity. PLoS One 2013; 8: e57479.
- 12 Muto A, Yoshihashi K, Takeda M. et al. Anti-factor IXa/X bispecific antibody (ACE910): hemostatic potency against ongoing bleeds in a hemophilia A model and the possibility of routine supplementation. J Thromb Haemost 2014; 12: 206-213.
- 13 Muto A, Yoshihashi K, Takeda M. et al. Anti-factor IXa/X bispecific antibody ACE910 prevents joint bleeds in a long-term primate model of acquired hemophilia A. Blood 2014; 124: 3165-3171.
- 14 Shima M, Hanabusa H, Taki M. et al. Factor VIII-mimetic function of humanized bispecific antibody in hemophilia A. N Engl J Med 2016; 374: 2044-2053.
- 15 Uchida N, Sambe T, Yoneyama K. et al. A first-in-human phase 1 study of ACE910, a novel factor VIII-mimetic bispecific antibody, in healthy subjects. Blood 2016; 127: 1633-1641.
- 16 Igawa T, Ishii S, Tachibana T. et al. Antibody recycling by engineered pH-dependent antigen binding improves the duration of antigen neutralization. Nat Biotechnol 2010; 28: 1203-1207.
- 17 Presta LG, Chen H, O’Connor SJ. et al. Humanization of an anti-vascular endothelial growth factor monoclonal antibody for the therapy of solid tumors and other disorders. Cancer Res 1997; 57: 4593-4599.
- 18 Hamilton RG, Saini SS, MacGlashan D. Surface plasmon resonance analysis of free IgE in allergic patients receiving omalizumab (Xolair). J Immunol Methods 2012; 383: 54-59.
- 19 Kaymakcalan Z, Sakorafas P, Bose S. et al. Comparisons of affinities, avidities, and complement activation of adalimumab, infliximab, and etanercept in binding to soluble and membrane tumor necrosis factor. Clin Immunol 2009; 131: 308-316.
- 20 Wu H, Pfarr DS, Johnson S. et al. Development of motavizumab, an ultra-potent antibody for the prevention of respiratory syncytial virus infection in the upper and lower respiratory tract. J Mol Biol 2007; 368: 652-665.
- 21 Coagulation sequence and structure database (CoagBase). Available at http://www.isth.org/?page=CoagBase . Accessed January 15, 2017.
- 22 Lacy SE, DeVries PJ, Xie N. et al. The potency of erythropoietin-mimic antibodies correlates inversely with affinity. J Immunol 2008; 181: 1282-1287.
- 23 Davis CB, Tobia LP, Kwok DC. et al. Accumulation of antibody-target complexes and the pharmacodynamics of clotting after single intravenous administration of humanized anti-factor IX monoclonal antibody to rats. Drug Delivery 1999; 6: 171-179.
- 24 Sunnerhagen M, Drakenberg T, Forsen S. et al. Effect of Ca2+ on the structure of vitamin K–dependent coagulation factors. Haemostasis 1996; 26 (Suppl. 01) 45-53.
- 25 Lenting PJ, Christophe OD, Maat H. et al. Ca2+ binding to the first epidermal growth factor-like domain of human blood coagulation factor IX promotes enzyme activity and factor VIII light chain binding. J Biol Chem 1996; 271: 25332-25337.
- 26 Makino Y, Omichi K, Kuraya N. et al. Structural analysis of N-linked sugar chains of human blood clotting factor IX. J Biochem 2000; 128: 175-180.
- 27 Chevreux G, Tilly N, Faid V. et al. Mass spectrometry based analysis of human plasma-derived factor X revealed novel post-translational modifications. Protein Sci 2015; 24: 1640-1648.
- 28 Salvagno GL, Berntorp E. Thrombin generation testing for monitoring hemophilia treatment: a clinical perspective. Semin Thromb Hemost 2010; 36: 780-790.