Kitazawa Takehisa, Esaki Keiko, Tachibana Tatsuhiko, Ishii Shinya, Soeda Tetsuhiro, Muto Atsushi, Kawabe Yoshiki, Igawa Tomoyuki, Tsunoda Hiroyuki, Nogami Keiji, Shima Midori, Hattori Kunihiro
Takehisa Kitazawa, Research Division, Chugai Pharmaceutical Co., Ltd., 200 Kajiwara, Kamakura, Kanagawa 247-8530, Japan, Tel.: +81 467 47 2260, Fax: +81 467 46 7795, E-mail:
Thromb Haemost. 2017 Jun 28;117(7):1348-1357. doi: 10.1160/TH17-01-0030. Epub 2017 Apr 28.
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 = 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-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-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.
依美珠单抗是一种识别因子（F）IX/IXa和X/Xa的人源化双特异性抗体，它可以通过以类似于FVIIIa的方式桥接FIXa和FX来加速FIXa催化的FX激活。然而，迄今为止尚未报道依美珠单抗与抗原相互作用的细节。在本研究中，我们首先通过表面等离子体共振分析表明，依美珠单抗以中等亲和力（K分别为1.58、1.52、1.85和0.978 μM）结合FIX、FIXa、FX和FXa。接下来，我们通过免疫印迹分析表明，依美珠单抗识别抗原的表皮生长因子（EGF）样结构域。然后，我们对血浆中的平衡状态进行了基于K的模拟，以定量预测依美珠单抗与抗原相互作用的方式。模拟预测，血浆中只有一小部分FIX、FX和依美珠单抗会形成抗原桥接的FIX-依美珠单抗-FX三元复合物，其浓度与依美珠单抗浓度呈钟形关系。血浆凝血酶生成试验重现了这种钟形浓度依赖性，表明三元复合物的血浆浓度与依美珠单抗的辅因子活性相关。模拟还预测，在先前研究中显示具有临床疗效的10.0 - 100 μg/ml依美珠单抗水平下，血浆中大多数FIX、FX和依美珠单抗将以单体形式存在。总之，依美珠单抗在其EGF样结构域以微摩尔亲和力结合FIX/FIXa和FX/FXa。基于K的模拟预测，循环血浆中形成的抗原桥接三元复合物将与依美珠单抗的辅因子活性相关，并且大多数FIX和FX将是游离的，可用于其他凝血反应。
