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

Summary 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.

Modified clot waveform analysis to measure plasma coagulation potential in the presence of the anti‐factor IXa/factor X bispecific antibody emicizumab

“…This modification increased the sensitivity of the data and reduced the differences between sample batches. Emicizumab does not moderate FVIII:C, but the antibody has a weaker affinity than FVIII for FIXa and FX [6,7], and it was difficult to evaluate coagulation potential in the presence of both emicizumab and FVIII. The Ad| min1| parameter in modified CWA, however, appeared to quantify the complex inter-reactions of these factors in the clotting mechanism.…”

Effects and interferences of emicizumab, a humanized bispecific antibody mimicking activated factor VIII cofactor function, on lupus anticoagulant assays

“…7 The mechanism of DRVVT prolongation is likely due to a weak steric interference with coagulation reactions in which FXa is generated, due to the binding of FX by emicizumab. 8 The lack of effect of emicizumab on TVT was expected, as emicizumab acts further upstream in the coagulation cascade.…”

Emicizumab: Review of the literature and critical appraisal

“…Neither clinical findings nor laboratory abnormalities indicating hypercoagulability were observed. The PK and PD profiles of emicizumab were similar in healthy Japanese and white subjects suggesting that emicizumab will be an effective and convenient prophylactic treatment of haemophilia A The clinical development programme for emicizumab included the five phase 3 trials (HAVEN-1, HAVEN-2, HAVEN-3, HAVEN-4, HAVEN-5 and STASEY) summarized in Tables 1 26,29,[35][36][37][38][40][41][42][43][44][45][46][47][48]54 and 2. 26,[49][50][51][52] In these tables, the level of evidence of the studies reported so far can be seen.…”