Hemophilia A is a bleeding disorder resulting from coagulation factor VIII (FVIII) deficiency. Exogenously provided FVIII effectively reduces bleeding complications in patients with severe hemophilia A. In approximately 30% of such patients, however, the 'foreignness' of the FVIII molecule causes them to develop inhibitory antibodies against FVIII (inhibitors), precluding FVIII treatment in this set of patients. Moreover, the poor pharmacokinetics of FVIII, attributed to low subcutaneous bioavailability and a short half-life of 0.5 d, necessitates frequent intravenous injections. To overcome these drawbacks, we generated a humanized bispecific antibody to factor IXa (FIXa) and factor X (FX), termed hBS23, that places these two factors into spatially appropriate positions and mimics the cofactor function of FVIII. hBS23 exerted coagulation activity in FVIII-deficient plasma, even in the presence of inhibitors, and showed in vivo hemostatic activity in a nonhuman primate model of acquired hemophilia A. Notably, hBS23 had high subcutaneous bioavailability and a 2-week half-life and would not be expected to elicit the development of FVIII-specific inhibitory antibodies, as its molecular structure, and hence antigenicity, differs from that of FVIII. A long-acting, subcutaneously injectable agent that is unaffected by the presence of inhibitors could markedly reduce the burden of care for the treatment of hemophilia A.

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.

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.

Summary. Background: We previously reported that a humanized anti-factor IXa/X bispecific antibody, hBS23, mimics the function of FVIII even in the presence of FVIII inhibitors, and has preventive hemostatic activity against bleeding in an animal model of acquired hemophilia A. After further molecular engineering of hBS23, we recently identified an improved humanized bispecific antibody, ACE910, for clinical investigation. Objectives: To elucidate the in vivo hemostatic potency of ACE910 by examining its effect against ongoing bleeds, and to determine its pharmacokinetic parameters for discussion of its potency for prophylactic use. Methods: A nonhuman primate model of acquired hemophilia A was established by injecting anti-primate FVIII neutralizing antibody. When bleeds emerged following an artificial bleed-inducing procedure, either ACE910 or recombinant porcine FVIII (rpoFVIII) was intravenously administered. rpoFVIII was additionally administered twice daily on the following 2 days. Bleeding symptoms were monitored for 3 days. A pharmacokinetic study and multiple-dosing simulations of ACE910 were also performed. Results: A single bolus of 1 or 3 mg kg À1 ACE910 showed hemostatic activity comparable to that of 10 U kg À1 (twice daily) rpoFVIII against ongoing bleeds. The determined ACE910 pharmacokinetic parameters included a long half-life (3 weeks) and high subcutaneous bioavailability (nearly 100%). The simulation results based on pharmacokinetic parameters indicated that the above hemostatic level could be maintained with once-weekly subcutaneous administration of ACE910, suggesting the possibility of more effective prophylaxis. Conclusions: ACE910 may offer an alternative on-demand treatment option for patients with hemophilia A, as well as user-friendly and aggressive routine supplementation.

Background Emicizumab is an anti-activated factor IX/FX bispecific antibody that mimics activated FVIII cofactor function. Emicizumab does not require activation by thrombin, and its effect on shortening the activated partial thromboplastin time (APTT) is much greater than that of FVIII. Therefore, the APTT has limited utility in hemophilia A (HA) patients treated with emicizumab. Aim To evaluate the global coagulation potential of emicizumab. Methods Clot waveform analysis (CWA) with prothrombin time (PT)/APTT mixed reagents was used to define hemostatic monitoring protocols in HA patients. A modified parameter, adjusted-|min1| (Ad|min1|), was developed. Maximum and minimum percentage transmittance were defined as 100% and 0% in the precoagulation and postcoagulation phases, respectively. Ad|min1| was calculated as an index of the maximum velocity of the coagulation process. Results Ad|min1| obtained with mixed-trigger reagent (PT/APTT/buffer, 1 : 15 : 135) in the presence of emicizumab optimally corresponded to the conversion rate estimated in animals; 0.2-0.4 IU dL equivalent FVIII per 1 μg mL emicizumab). Ex vivo addition of emicizumab to HA plasma with or without inhibitors resulted in concentration-dependent increases in Ad|min1|, with some individual variations. The addition of various concentrations of FVIII to HA plasma mixed with emicizumab resulted in dose-dependent increases in Ad|min1|. Similarly, mixtures of activated prothrombin complex concentrate and emicizumab added to HA plasma resulted in dose-dependent increases in Ad|min1|. In contrast, enhanced coagulation potential appeared to be better defined by the clot time than by Ad|min1| in experiments using recombinant activated FVII. Conclusion The PT/APTT reagent-triggered adjusted CWA could provide a useful means of assessing global coagulation potential in emicizumab-treated HA patients, with enhanced activity neither masking nor being masked by FVIII or bypassing agents.

Summary.  Background: Thrombus formation through the activation of tissue factor (TF) and factor (F) XI is a critical event in the onset of cardiovascular disease. TF expressed in atherosclerotic plaques and circulating blood is an important determinant of thrombogenicity that contributes to fibrin‐rich thrombus formation after plaque disruption. However, the contribution of FXI to thrombus formation on disrupted plaques remains unclear. Methods: A mouse monoclonal antibody against FXI and activated FXI (FXIa) (XI‐5108) was generated by immunization with activated human FXI. Prothrombin time (PT), activated partial thromboplastin time (APTT), bleeding time, and ex vivo platelet aggregation in rabbits were measured before and after an intravenous bolus injection of XI‐5108. We investigated the role of FXI upon arterial thrombus growth in the rabbit iliac artery in the presence of repeated balloon injury. Results: The XI‐5108 antibody reacted to the light chain of human and rabbit FXI/FXIa, and inhibited FXIa‐initiated FXa and FXIa generation. Fibrin‐rich thrombi developed on the injured neointima that was obviously immunopositive for glycoprotein IIb‐IIIa, fibrin, TF, and FXI. Intravenous administration of XI‐5108 (3.0 mg kg−1) remarkably reduced thrombus growth, and the APTT was significantly prolonged. However, PT, bleeding time and platelet aggregation were not affected. Conclusions: These results indicate that plasma FXI plays a potent role in thrombus growth on the injured neointima. Inhibition of plasma FXI activity might help to reduce thrombus growth on ruptured plaques without prolonging bleeding time.

Background Emicizumab is an anti-factor (F)IXa/X bispecific monoclonal antibody (mAb), mimicking the factor (F)VIIIa cofactor activity. Emicizumab does not require activation by thrombin and its shortening effect on the activated partial prothrombin time (APTT) is more pronounced than that of factor (F)VIII. APTT-based FVIII activity (FVIII:C) and FVIII inhibiter titer measurements are influenced by the presence of emicizumab. Aim To establish a reliable APTT-based assay to measure FVIII in the presence of emicizumab. Methods Plasmas from hemophilia A (HA) patients without or with inhibitors were studied using one-stage FVIII:C and Bethesda inhibitor assays. Two recombinant anti-idiotype mAbs to emicizumab (anti-emicizumab mAbs) were prepared, rcAQ8 to anti-FIXa-Fab and rcAJ540 to anti-FX-Fab. Results The combined anti-idiotype mAbs (2000 nm each) eliminated the effects of emicizumab on APTTs of HA plasmas without or with inhibitor by competitive inhibition of antibody binding to FIX(a)/FX(a). Measurements of FVIII coagulation activity in HA plasmas without inhibitor were overestimated in the presence of emicizumab (1 μm = ~150 μg mL ) at all reference levels of FVIII. The addition of anti-emicizumab mAbs to the assay mixtures completely neutralized the emicizumab and facilitated accurate determination of FVIII:C. Anti-FVIII inhibitor titers were undetectable in the presence of emicizumab in HA plasmas with inhibitor or normal plasmas mixed with anti-FVIII neutralizing antibodies. These effects of emicizumab were completely counteracted by the addition of the anti-idiotype mAbs, allowing accurate assessment of inhibitor titers. Conclusion The in vitro inclusion of anti-emicizumab mAbs in the standard one-stage coagulation assays prevented interference by emicizumab and enabled accurate measurements of FVIII:C and inhibitor titers.

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) dosedependently (K d ‫؍‬ 108 nM). This binding ability was optimal under physiological conditions. A monoclonal antibody against the Gla domain of factor IXa inhibited binding by ϳ95%, 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 proteasecleaved 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.