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Background As the first non-factor replacement therapy for persons with congenital hemophilia A (PwcHA), emicizumab's safety profile is of particular interest to the community. Objectives We applied an algorithm for categorization of fatal events contemporaneous to emicizumab using reporter-assessed causality documented in the Roche Emicizumab Global Safety Database. Patients/methods All fatalities in PwcHA reported to the database (from clinical trials, pre-market access, and spontaneous post-marketing reports) were categorized into: associated with hemophilia A-hemorrhagic, thrombotic, human immunodeficiency virus (HIV)/hepatitis C virus (HCV), hepatic (non-HCV); associated with general population-trauma/suicide, non-HA-associated conditions; or, unspecified. Reported cause of death was not reassessed. Results As of cut-off May 15, 2020, 31 fatalities in PwcHA taking emicizumab were reported. Median age at death was 58 years; 51% had factor VIII inhibitors. Fifteen fatalities were considered associated with HA; overall, the most frequent category was hemorrhage (11/31). Of these, six had a history of life-threatening bleeds, and four had a history of intracranial hemorrhage. The remaining HA-associated fatalities were related to HIV/HCV (3/31) and other hepatic causes (1/31). No cases were categorized as thrombotic. Of 10 cases considered not associated with HA, two were categorized as cardiovascular (non-thrombotic), five as infection/sepsis, and one each of trauma/suicide, pulmonary, and malignancy. Six cases were unspecified. Conclusions No unique risk of death was associated with emicizumab prophylaxis in PwcHA. The data reveal that mortality in PwcHA receiving emicizumab was primarily associated with hemorrhage or non-HA-associated conditions, and was not reported by treaters to be related to emicizumab treatment.

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.

Essentials Emicizumab (Emi) affects the APTT-based assays of factor (F)VIII activity and inhibitor titer. A mixture of two anti-Emi monoclonal antibodies (mAb) effectively neutralized the Emi activity. Anti-Emi mAbs completely eliminated the influence of Emi on FVIII activity and inhibitor titer. The inclusion of anti-Emi mAbs in routine FVIII assays would be useful for Emi-treated patients. Summary 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-1 ) 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.

SummaryBackground Vatreptacog alfa, a recombinant human factor VIIa (rFVIIa) analog developed to improve the treatment of bleeds in hemophilia patients with inhibitors, differs from native FVIIa by three amino acid substitutions. In a randomized, double-blind, crossover, confirmatory phase III trial (adept™2), 8/72 (11%) hemophilia A or B patients with inhibitors treated for acute bleeds developed anti-drug antibodies (ADAs) to vatreptacog alfa. Objectives To characterize the formation of anti-vatreptacog alfa ADAs in hemophilia patients with inhibitors. Methods/patients This was a post hoc analysis of adept™2. Immunoglobulin isotype determination, specificity analysis of rFVIIa cross-reactive antibodies, epitope mapping of rFVIIa single mutant analogs and pharmacokinetic (PK) profiling were performed to characterize the ADAs. Results Immunoglobulin isotyping indicated that the ADAs were of the immunoglobulin G subtype. In epitope mapping, none of the rFVIIa single mutant analogs (V158D, E296V or M298Q) contained the complete antibody epitope, confirming that the antibodies were specific for vatreptacog alfa. In two patients, for whom PK profiling was performed both before and after the development of ADAs, vatreptacog alfa showed a prolonged elimination phase following ADA development. During the follow-up evaluation, the rFVIIa cross-reactivity disappeared after the last vatreptacog alfa exposure, despite continued exposure to rFVIIa as part of standard care. Conclusions Results from the vatreptacog alfa phase III trial demonstrate that the specific changes made, albeit relatively small, to the FVIIa molecule alter its clinical immunogenicity.

Summary Background The clinical phenotype of individuals with acquired factor V (A-FV) inhibitors varies from asymptomatic (non-B group) to life-threatening bleeding (B group), but the mechanism(s) underlying this variation in hemorrhagic phenotype are poorly understood. Objective To investigate coagulation mechanistically in a range of patients with A-FV antibodies. Methods and Results Ten cases of A-FV inhibitors in the non-B (n = 5) and B groups (n = 5) were studied. Thrombin generation assays in these plasmas revealed little thrombin generation, despite similar FV activity levels in both groups. However, prothrombin time-based clot waveform analysis revealed that the clot times were significantly prolonged and the maximum velocity and acceleration of coagulation were lower in the B group than in the non-B group, suggesting that this technique might be useful for predicting and monitoring hemorrhagic symptoms. A-FV inhibitors from the non-B group recognized predominantly the FV heavy chain, whereas those from the B group recognized the light chain. Purified anti-FV autoantibodies (autoAbs) from the B group inhibited FV binding to phospholipid by 60–90%, whereas there was little effect on this reaction in the non-B group. In addition, anti-FV autoAbs from the non-B group impaired the activated protein C (APC) cofactor activity of FV in FVIIIa inactivation mechanisms, and delayed APC-catalyzed cleavage of FVa at Arg306, but not at Arg506, indicating the presence of APC resistance in the non-B group. Conclusions The results suggest that the different hemorrhagic phenotypes in A-FV inhibitors depend on the specific epitope of anti-FV autoAbs, and appear to be associated with an imbalance of procoagulant and anticoagulant function.

Background Vatreptacog alfa, a recombinant factor VIIa (rFVIIa) analog with three amino acid substitutions and 99% identity to native FVIIa, was developed to improve the treatment of hemophilic patients with inhibitors. Objectives To confirm the safety and assess the efficacy of vatreptacog alfa in treating bleeding episodes in hemophilic patients with inhibitors. Patients and methods In this international, multicenter, randomized, double-blind, active-controlled, crossover, confirmatory phase III trial (adept™2) in patients with hemophilia A or B and inhibitors, bleeds were randomized 3 : 2 to treatment with vatreptacog alfa (one to three doses at 80 μg kg−1) or rFVIIa (one to three doses at 90 μg kg−1). Treatment failures after three doses of trial product (TP) were managed according to the local standard of care. Results In the 72 patients enrolled, 567 bleeds were treated with TP. Both vatreptacog alfa and rFVIIa gave 93% effective bleeding control at 12 h. Vatreptacog alfa was superior to rFVIIa in secondary efficacy outcomes, including the number of doses used to treat a bleed and sustained bleeding control 24–48 h after the first dose. Eight patients (11%) developed antibodies against vatreptacog alfa, including four with cross-reactivity against rFVIIa and one with an in vitro neutralizing effect to vatreptacog alfa. Conclusions This large randomized controlled trial confirmed the well-established efficacy and safety profile of rFVIIa, and showed that vatreptacog alfa had similar or better efficacy than rFVIIa. However, because of the development of anti-drug antibodies, a positive benefit–risk profile is unlikely to be achieved with vatreptacog alfa.

Summary Background Assays to determine the optimal hemostatic effects of bypass therapy in hemophilia A (HA) patients with inhibitors are difficult to compare. Clot waveform analysis (CWA), based on the continuous monitoring of routine coagulation parameters (prothrombin time/activated partial thromboplastin time), offers a useful method for assessing global clotting function. Objectives To investigate the technique of CWA for the hemostatic monitoring of bypass therapy in HA patients with inhibitors. Methods and Results Ellagic acid (Elg), tissue factor (TF) or both (Elg/TF) were used as trigger reagents in CWA. The standard parameters – clot time (CT), maximum coagulation velocity (|min1|), and acceleration (|min2|) – were recorded. Optimal monitoring was defined as: (i) a significant difference in these parameters between plasma from HA patients with inhibitors and normal plasmas; and (ii) a significant improvement in these indices in HA patients with inhibitors after bypass therapy. Experiments in vitro demonstrated that there were significant differences between plasma from HA patients with inhibitors and normal plasma with various triggers, in the order Elg > Elg/TF >> TF. Addition of therapeutically achievable concentrations of bypassing agents, however, showed significant improvements in the different parameters only with Elg/TF, suggesting that this reagent provided the most appropriate assay. A total of 20 plasmas from HA patients with inhibitors in which bypassing agents were infused were evaluated ex vivo by Elg/TF CWA. The postinfusion parameters CT and |min2| reflected clinical effects, and were close to normal levels. Furthermore, Elg/TF CWA facilitated quantitative evaluation of perioperative hemostatic management of bypass therapy in HA patients with inhibitors. Conclusions CWA is a promising method for the quantitative monitoring of bypass therapy during routine automated clotting assays with a modified trigger reagent comprising a well-balanced mixture of Elg and TF.

SummaryBackground 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 non-human 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−1 ACE910 showed hemostatic activity comparable to that of 10 U kg−1 (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.

Summary. Background: Hemophilia A is an X-chromosomelinked recessive bleeding disorder resulting from an F8 gene abnormality. Although various gene therapies have been attempted with the aim of eliminating the need for factor VIII replacement therapy, obstacles to their clinical application remain. Objectives: We evaluated whether embryonic stem (ES) cells with a tetracycline-inducible system could secrete human FVIII. Methods and results: We found that embryoid bodies (EBs) developed under conditions promoting liver differentiation efficiently secreted human FVIII after doxycycline induction. Moreover, use of a B-domain variant F8 cDNA (226aa/ N6) dramatically enhanced FVIII secretion. Sorting based on green fluorescent protein (GFP)–brachyury (Bry) and c-kit revealed that GFP–Bry + /c-kit + cells during EB differentiation with serum contain an endoderm progenitor population. When GFP–Bry + /c-kit + cells were cultured under the liver cellpromoting conditions, these cells secreted FVIII more efficiently than other populations tested. Conclusion: Our findings suggest the potential for future development of an effective ES cellbased approach to treating hemophilia A.

Factor (F)V is converted into its active form, FVa, by limited proteolysis. Thrombin-catalyzed activation of FV is essential for its full cofactor activation. Previously, we reported that thrombin was bound to the C2 domain in the light chain of FVIII. As FV has a similar domain structure to FVIII, we focused on the FV C2 domain as a possible binding region for thrombin. Kinetic parameters, measured by surface plasmon resonance, revealed that the K(d) values of anhydro-thrombin for FV, FVa, and the FV C2 domain were 66, 240, and 670 nmol L(-1), respectively. FV activation was increased by approximately 9-fold by the addition of thrombin. In the presence of the FV C2 domain, this increase of the FV activation was inhibited. However, FV activation was not inhibited by the addition of the FVIII C2 domain. FV was cleaved into a 105-kDa heavy chain and a 71/74-kDa light chain by thrombin-catalyzed proteolysis at Arg709, Arg1018 and Arg1545. In the presence of the FV C2 domain, the cleavage was inhibited at all sites. Proteolysis was not affected by the addition of the FVIII C2 domain. These results indicated that the FV C2 domain contains a major binding site for thrombin and that this domain is necessary for the proteolysis at all cleavage sites. Furthermore, the present results also suggested that thrombin has an independent binding site for FV different from that for FVIII.

Hemophilias A and B are X chromosome-linked bleeding disorders, which are mainly treated by repeated infusions of factor (F)VIII or FIX, respectively. In the present review, we specify the limitations in expression of recombinant (r)FVIII and summarize the bioengineering strategies that are currently being explored for constructing novel rFVIII molecules characterized by high efficiency expression and improved functional properties. We present the strategy to prolong FVIII lifetime by disrupting FVIII interaction with its clearance receptors and demonstrate how construction of human-porcine FVIII hybrid molecules can reduce their reactivity towards inhibitory antibodies. While the progress in improving rFIX is impeded by low recovery rates, the authors are optimistic that the efforts of basic science may ultimately lead to higher efficiency of replacement therapy of both hemophilias A and B.