第IX(a)因子および第X因子に対する二重特異性抗体であるエミシズマブはin vitroで第XI因子欠乏血漿における凝固機能を増強する

Essentials Emicizumab mimics factor (F)VIIIa cofactor function, augments the intrinsic tenase activity. We assessed the emicizumab-driven hemostatic function in FXI-deficient plasmas. Emicizumab improved the coagulation potentials in severe FXI-deficient plasma. Emicizumab may provide a possibility for clinical application in patients with FXI deficiency. SUMMARY: Background Patients with factor (F)XI deficiency commonly present with markedly prolonged activated partial thromboplastin times (APTT), although bleeding phenotypes are heterogeneous. Emicizumab, a bispecific monoclonal antibody to FIX/FIXa and FX/FXa, mimics FVIIIa cofactor function on phospholipid (PL) surfaces. Antibody reactions were designed, therefore, to augment mechanisms during the propagation phase of blood coagulation. Aim To assess emicizumab-driven hemostatic function in FXI-deficient plasmas. Methods and Results Standard ellagic acid (Elg)/PL-based APTTs of different FXI-deficient plasmas (n = 13; FXI activity, < 1 IU dl-1 ) were markedly shortened dose dependently by the presence of emicizumab. To further analyze the effects of emicizumab, clot waveform analysis (CWA) in FXI-deficient plasmas with emicizumab, triggered by tissue factor (TF)/Elg demonstrated improvements in both clot times, reflecting the initiation phase, and coagulation velocity, which represents the propagation phase. Emicizumab also enhanced the TF/Elg-triggered thrombin generation in FXI-deficient plasmas dose-dependently although the degree of enhancement varied in individual cases. Thrombin generation with either FVII-deficient plasma or FIX-deficient plasma treated with anti-FXI antibody showed little or no increase by the co-presence of emicizumab, suggesting that the accelerated thrombin generation in FXI-deficient plasmas by emicizumab should depend on the FIXa-involved coagulation propagation initially triggered by FVIIa/TF. The ex vivo addition of emicizumab to whole blood from three patients with severe FXI deficiency demonstrated modest, dose-dependent improvements in Ca2+ -triggered thromboelastograms (NATEM mode). Conclusion Emicizumab appeared to improve coagulation function in severe FXI-deficient plasma, and might provide possibilities for clinical application in patients with FXI deficiency.博士（医学）・乙第1427号・平成31年3月15日© 2018 International Society on Thrombosis and HaemostasisThis is the pre-peer reviewed version of the following article: [https://onlinelibrary.wiley.com/doi/full/10.1111/jth.14334], which has been published in final form at [http://dx.doi.org/10.1111/jth.14334]. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions

エミシズマブは凝固第VIII因子と同様にフィブリン構造およびその安定性を向上させる

Introduction: Emicizumab is an antifactor (F)IXa/FX bispecific antibody, mimicking FVIIIa cofactor function. Emi prophylaxis effectively reduces bleeding events in patients with haemophilia A. The physical properties of emicizumab-induced fibrin clots remain to be investigated, however. Aim: We have investigated the stability and structure of emicizumab-induced fibrin clots. Methods: Coagulation was initiated by activated partial thromboplastin time (aPTT) trigger and prothrombin time (PT)/aPTT-mixed trigger in FVIII-deficient plasma with various concentrations of emicizumab or recombinant FVIII. The turbidity and stability of fibrin clots were assessed by clot waveform and clot-fibrinolysis waveform analyses, respectively. The resulting fibrin was analysed by scanning electron microscopy (SEM). Results: Using an aPTT trigger, the turbidity was decreased and the fibrinolysis times were prolonged in the presence of emicizumab dose-dependently. Scanning electron microscopy imaging demonstrated that emicizumab improved the structure of fibrin network with thinner fibres than in its absence. Although emicizumab shortened the aPTT dramatically, the nature of emicizumab-induced fibrin clots did not reflect the hypercoagulable state. Similarly, using a PT/aPTT-mixed trigger that could evaluate potential emicizumab activity, emicizumab improved the stability and structure of fibrin clot in a series of experiments. In this circumstance, fibrin clot properties with emicizumab at 50 and 100 µg/mL appeared to be comparable to those with FVIII at ~12 and ~24-32 IU/dL, respectively. Conclusion: Emicizumab effectively improved fibrin clot stability and structure in FVIII-deficient plasma, and the physical properties of emicizumab-induced fibrin clots were similar to those with FVIII.博士（医学）・甲第787号・令和3年3月15日© 2020 John Wiley & Sons Ltd.This is the peer reviewed version of the following article: https://onlinelibrary.wiley.com/doi/10.1111/hae.13961, which has been published in final form at https://doi.org/10.1111/hae.13961. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions

活性化第VIII 因子のC2 ドメイン(残基2228-2240) はFactor Xase 複合体における活性化第IX 因子のGla ドメインと相互作用する

Factor VIIIa functions as a cofactor for factor IXa in the phospholipid surface-dependent activation of factor X. Both the C2 domain of factor VIIIa and the Gla domain of factor IXa are involved in phospholipid binding and are required for the activation of factor X. In this study, we have examined the close relationship between these domains in the factor Xase complex. Enzyme-linked immunosorbent assay-based and surface plasmon resonance-based assays in the absence of phospholipid showed that Glu-Gly-Arg active site-modified factor IXa bound to immobilized recombinant C2 domain (rC2) dose-dependently (Kd = 108 nm). This binding ability was optimal under physiological conditions. A monoclonal antibody against the Gla domain of factor IXa inhibited binding by approximately 95%, and Gla domainless factor IXa failed to bind to rC2. The addition of monoclonal antibody or rC2 with factor VIIIa inhibited factor IXa-catalyzed factor X activation in the absence of phospholipid. Inhibition was not evident, however, in similar experiments in the absence of factor VIIIa, indicating that the C2 domain interacted with the Gla domain of factor IXa. A fragment designated C2-(2182-2259), derived from V8 protease-cleaved rC2, bound to Glu-Gly-Arg active site-modified factor IXa. Competitive assays, using overlapping synthetic peptides encompassing residues 2182-2259, demonstrated that peptide 2228-2240 significantly inhibited both this binding and factor Xa generation, independently of phospholipid. Our results indicated that residues 2228-2240 in the factor VIIIa C2 domain constitutes an interactive site for the Gla domain of factor IXa. The findings provide the first evidence for an essential role for this interaction in factor Xase assembly.博士（医学）・乙第1297号・平成24年5月28日Copyright © 2009 American Society for Biochemistry and Molecular Biolog

Identification and Multidimensional Optimization of an Asymmetric Bispecific IgG Antibody Mimicking the Function of Factor VIII Cofactor Activity

<div><p>In hemophilia A, routine prophylaxis with exogenous factor VIII (FVIII) requires frequent intravenous injections and can lead to the development of anti-FVIII alloantibodies (FVIII inhibitors). To overcome these drawbacks, we screened asymmetric bispecific IgG antibodies to factor IXa (FIXa) and factor X (FX), mimicking the FVIII cofactor function. Since the therapeutic potential of the lead bispecific antibody was marginal, FVIII-mimetic activity was improved by modifying its binding properties to FIXa and FX, and the pharmacokinetics was improved by engineering the charge properties of the variable region. Difficulties in manufacturing the bispecific antibody were overcome by identifying a common light chain for the anti-FIXa and anti-FX heavy chains through framework/complementarity determining region shuffling, and by pI engineering of the two heavy chains to facilitate ion exchange chromatographic purification of the bispecific antibody from the mixture of byproducts. Engineering to overcome low solubility and deamidation was also performed. The multidimensionally optimized bispecific antibody hBS910 exhibited potent FVIII-mimetic activity in human FVIII-deficient plasma, and had a half-life of 3 weeks and high subcutaneous bioavailability in cynomolgus monkeys. Importantly, the activity of hBS910 was not affected by FVIII inhibitors, while anti-hBS910 antibodies did not inhibit FVIII activity, allowing the use of hBS910 without considering the development or presence of FVIII inhibitors. Furthermore, hBS910 could be purified on a large manufacturing scale and formulated into a subcutaneously injectable liquid formulation for clinical use. These features of hBS910 enable routine prophylaxis by subcutaneous delivery at a long dosing interval without considering the development or presence of FVIII inhibitors. We expect that hBS910 (investigational drug name: ACE910) will provide significant benefit for severe hemophilia A patients.</p> </div

Improvement of pharmaceutical properties of bispecific antibodies.

<p>(A) Antibody solution profiles of hBS376 and hBS560 at different antibody concentrations, pH, and NaCl concentrations. The antibody solution under each condition was photographed and the state determined (P, precipitation; L, liquid–liquid phase separation; –, clear liquid). (B) Cation exchange chromatography of hBS560 and hBS660 before (black) and after incubation at 40°C for 2 weeks (red). Acidic peak indicating deamidation at HCDR3 increased after incubation at 40°C for 2 weeks for hBS560. No marked increase of acidic peak was observed for hBS660.</p

Improvement of therapeutic potential of the bispecific antibody.

<p>(A) Improving FVIII-mimetic activity of the bispecific antibody. Effect of hBS1 (circles), hBS106 (squares), and hBS910 (diamonds) on FX activation in the presence of FIXa, FX, and synthetic phospholipid is shown. The Y-axis indicates the absorbance at 405 nm of the chromogenic substrate assay (in many cases, the bars depicting s.d. are shorter than the height of the symbols). (B) Improving pharmacokinetics of the bispecific antibody. Time profiles of plasma concentration of hBS106 (circles), hBS128 (squares), and hBS228 (diamonds) in mice after subcutaneous injection at a dose of 1 mg/kg are shown. All the data were collected in triplicate and are expressed as mean ± s.d.</p

Therapeutic potential of multidimensionally optimized bispecific antibody, hBS910.

<p>(A) FVIII-mimetic activity of hBS910 in thrombin generation assay (TGA). Effect of hBS910 (circles) or recombinant human FVIII (squares) on thrombin generation in FVIII-deficient plasma is shown. The reaction was triggered by FXIa, synthetic phospholipid, and Ca²⁺. The Y-axis indicates the peak height, a thrombin generation parameter (in many cases, the bars depicting s.d. are shorter than the height of the symbols). Data were collected in triplicate for each plasma lot and are expressed as mean ± s.d. (B) Pharmacokinetics of hBS910 in cynomolgus monkeys. Time profiles of plasma concentration of hBS910 after intravenous (circles) or subcutaneous (squares) injection are shown.</p