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Recombinant factor VIII (rFVIII), rFVIIIFc and emicizumab are established treatment options in the management of hemophilia A. Each has its unique mode of action, which can influence thrombin generation kinetics and therefore also the kinetics of thrombin substrates. Such differences may potentially result in clots with different structural and physical properties. A starting observation of incomplete wound closure in a patient on emicizumab prophylaxis led us to employ a relevant mouse model in which we noticed that emicizumab-induced clots appeared less stable compared to FVIII-induced clots. We therefore analyzed fibrin formation in vitro and in vivo. In vitro fibrin formation was faster and more abundant in the presence of emicizumab than in the presence of rFVIII/rFVIIIFc. Furthermore, the time-interval between the initiation of fibrin formation and factor XIII activation was twice as long for emicizumab than as for rFVIII/rFVIIIFc. Scanning electron microscopy and immunofluorescent spinning-disk confocal microscopy of in vivo-generated clots confirmed increased fibrin formation in the presence of emicizumab. Unexpectedly, we also detected a different morphology between rFVIII/rFVIIIFcand emicizumab-induced clots. Contrary to the regular fibrin mesh obtained with rFVIII/rFVIIIFc, fibrin fibers appeared to be fused into large patches upon emicizumab treatment. Moreover, fewer red blood cells were detected in regions in which these fibrin patches were present. The presence of highly dense fibrin structures associated with a diffuse fiber structure in emicizumab-induced clots was also observed when using super-resolution imaging. We hypothesize that the modified kinetics of thrombin, fibrin and factor XIIIa generation contribute to differences in structural and physical properties between clots formed in the presence of FVIII or emicizumab.

Background: The effect of factor VIII (FVIII) or emicizumab on thrombin generation is usually assessed in assays using synthetic phospholipids. Here, we assessed thrombin generation at the surface of human arterial cells (aortic endothelial cells [hAECs] and aortic vascular smooth muscle cells [hVSMCs])., Objectives: To explore the capacity of hAECs (resting or stimulated) and hVSMCs to support thrombin generation by FVIII or emicizumab., Methods: Primary hVSMCs and hAECs were analyzed for tissue factor (TF)-activity and antigen, phosphatidylserine (PS)-exposure, tissue factor pathway inhibitor (TFPI)-content and thrombomodulin expression. Cells were incubated with FVIII-deficient plasma spiked with FVIII, emicizumab, activated prothrombin complex concentrate (APCC) or combinations thereof., Results: TF activity and PS-exposure were present on both hVSMCs and hAECs. In contrast, thrombomodulin and TFPI were expressed on hAECs, while virtually lacking on hVSMCs, confirming the procoagulant nature of hVSMCs. Tumor necrosis factor α-mediated stimulation of hAECs increased not only TF antigen, TF activity, and PS-exposure but also TFPI and thrombomodulin expression. As expected, FVIII and emicizumab promoted thrombin generation on nonstimulated hAECs and hVSMCs, with more thrombin being generated on hVSMCs. Unexpectedly, FVIII and emicizumab increased thrombin generation to a lesser extent on stimulated hAECs compared with nonstimulated hAECs. Finally, adding emicizumab to FVIII did not further increase thrombin generation, whereas the addition of emicizumab to APCC resulted in exaggerated thrombin generation., Conclusion: Tumor necrosis factor stimulation of hAECs increases both pro- and anticoagulant activity. Unexpectedly, the increased anticoagulant activity is sufficient to limit both FVIII- and emicizumab-induced thrombin generation. This protective effect disappears when emicizumab is combined with APCC., Competing Interests: Declaration of competing interests P.J.L. receives research funding to the institute from Hoffman-Laroche Ltd, Institut Roche, Pfizer, Sanofi, and Sobi. The other authors declare no conflict of interest., (Copyright © 2023 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc. All rights reserved.)

Background: Emicizumab is a bispecific, chimeric, humanized immunoglobulin G (IgG)4 that mimics the procoagulant activity of factor (F) VIII (FVIII). Its long half-life and subcutaneous route of administration have been life-changing in treating patients with hemophilia A (HA) with or without FVIII inhibitors. However, emicizumab only partially mimics FVIII activity; it prevents but does not treat acute bleeds. Emergency management is particularly complicated in patients with FVIII inhibitors receiving emicizumab prophylaxis in whom exogenous FVIII is inefficient. We have shown recently that Imlifidase (IdeS), a bacterial IgG-degrading enzyme, efficiently eliminates human anti-FVIII IgG in a mouse model of severe HA with inhibitors and opens a therapeutic window for the administration of exogenous FVIII., Objectives: To investigate the impact of IdeS treatment in inhibitor-positive HA mice injected with emicizumab., Methods: IdeS was injected to HA mice reconstituted with human neutralizing anti-FVIII IgG and treated with emicizumab., Results: IdeS hydrolyzed emicizumab in vitro and in vivo, albeit, at slower rates than another recombinant human monoclonal IgG4. While F(ab') 2 fragments were rapidly cleared from the circulation, thus leading to a rapid loss of emicizumab procoagulant activity, low amounts of single-cleaved intermediate IgG persisted for several days. Moreover, the IdeS-mediated elimination of the neutralizing anti-FVIII IgG and restoration of the hemostatic efficacy of exogenous FVIII were not impaired by the presence of emicizumab and polyclonal human IgG in inhibitor-positive HA mice., Conclusion: Our results suggest that IdeS could be administered to inhibitor-positive patients with HA receiving emicizumab prophylaxis to improve and ease the management of breakthrough bleeds or programmed major surgeries., Competing Interests: Declaration of competing interests S.L.D. and J.D.D. are inventors on patent EP18305971.6 related to the use of IdeS in the context of AAV-mediated gene therapy. Other authors declare no competing financial interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Transplacental delivery of maternal immunoglobulin G (IgG) provides humoral protection during the first months of life until the newborn's immune system reaches maturity. The maternofetal interface has been exploited therapeutically to replace missing enzymes in the fetus, as shown in experimental mucopolysaccharidoses, or to shape adaptive immune repertoires during fetal development and induce tolerance to self-antigens or immunogenic therapeutic molecules., Objectives: To investigate whether proteins that are administered to pregnant mice or endogenously present in their circulation may be delivered through the placenta., Methods: We engineered monovalent immunoglobulin G (FabFc) specific for different domains of human factor VIII (FVIII), a therapeutically relevant model antigen. FabFc was injected with exogenous FVIII into pregnant severe hemophilia A mice or pregnant mice expressing human FVIII following AAV8-mediated gene therapy. FabFc and FVIII were detected in the pregnant mice and/or fetuses by enzyme-linked immunosorbent assay and immunohistochemistry., Results: Administration of FabFc to pregnant mice allowed the maternofetal delivery of FVIII in a FcRn-dependent manner. FVIII antigen levels achieved in the fetuses represented 10% of normal plasma levels in the human. We identified antigen/FabFc complex stability, antigen size, and shielding of promiscuous protein patches as key parameters to foster optimal antigen delivery., Conclusion: Our results pave the way toward the development of novel strategies for the in utero delivery of endogenous maternal proteins to replace genetically deficient fetal proteins or to educate the immune system and favor active immune tolerance upon protein encounter later in life., Competing Interests: Declaration of competing interests J.M. and A.C.N. are inventors on a patent licensed to BioMarin. The remaining authors have no competing interests to disclose., (Copyright © 2023 International Society on Thrombosis and Haemostasis. All rights reserved.)

The bispecific antibody emicizumab is increasingly used for hemophilia A treatment. However, its specificity for human factors IX and X (FIX and FX) has limited its in vivo functional analysis to primate models of acquired hemophilia. Here, we describe a novel mouse model that allows emicizumab function to be examined. Briefly, FVIII-deficient mice received IV emicizumab 24 hours before tail-clip bleeding was performed. A second infusion with human FIX and FX, administered 5 minutes before bleeding, generated consistent levels of emicizumab (0.7-19 mg/dL for 0.5-10 mg/kg doses) and of both FIX and FX (85 and 101 U/dL, respectively, after dosing at 100 U/kg). Plasma from these mice display FVIII-like activity in assays (diluted activated partial thromboplastin time and thrombin generation), similar to human samples containing emicizumab. Emicizumab doses of 1.5 mg/kg and higher significantly reduced blood loss in a tail-clip-bleeding model using FVIII-deficient mice. However, reduction was incomplete compared with mice treated with human FVIII concentrate, and no difference in efficacy between doses was observed. From this model, we deducted FVIII-like activity from emicizumab that corresponded to a dose of 4.5 U of FVIII per kilogram (ie, 9.0 U/dL). Interestingly, combined with a low FVIII dose (5 U/kg), emicizumab provided enough additive activity to allow complete bleeding arrest. This model could be useful for further in vivo analysis of emicizumab., (© 2020 by The American Society of Hematology.)

Monitoring factor VIII (FVIII) activity has traditionally been complicated by discrepancies between assays for the various sorts of FVIII molecules. The advent of novel nonfactor therapies (emicizumab, fitusiran, and anti-tissue factor pathway inhibitor antibodies) in hemophilia A poses a new level of difficulty on the laboratory monitoring of these patients. To use the correct assays and for a proper interpretation of their results, it is pertinent to understand the mode of action of these nonfactor agents. Furthermore, the biochemical consequences for the different types of activity assays (whether it be specific FVIII activity assays or global coagulation assays) should be taken into account as well. In this review, these aspects will be discussed. In addition, the use of various animal models to estimate FVIII-equivalence of the nonfactor therapies will be presented., (© 2020 by The American Society of Hematology.)

Novel therapies for hemophilia, including non-factor replacement and in vivo gene therapy, are showing promising results in the clinic, including for patients having a history of inhibitor development. Here, we propose a novel therapeutic approach for hemophilia based on llama-derived single-domain antibody fragments (sdAbs) able to restore hemostasis by inhibiting the antithrombin (AT) anticoagulant pathway. We demonstrated that sdAbs engineered in multivalent conformations were able to block efficiently AT activity in vitro, restoring the thrombin generation potential in FVIII-deficient plasma. When delivered as a protein to hemophilia A mice, a selected bi-paratopic sdAb significantly reduced the blood loss in a model of acute bleeding injury. We then packaged this sdAb in a hepatotropic AAV8 vector and tested its safety and efficacy profile in hemophilic mouse models. We show that the long-term expression of the bi-paratopic sdAb in the liver is safe and poorly immunogenic, and results in sustained correction of the bleeding phenotype in hemophilia A and B mice, even in the presence of inhibitory antibodies to the therapeutic clotting factor., (© 2020 The Authors. Published under the terms of the CC BY 4.0 license.)

The treatment or prevention of bleeding in patients with hemophilia A relies on replacement therapy with different factor VIII (FVIII)-containing products or on the use of by-passing agents, i.e., activated prothrombin complex concentrates or recombinant activated factor VII. Emerging approaches include the use of bispecific anti-factor IXa/factor X antibodies, anti-tissue factor pathway inhibitor antibodies, interfering RNA to antithrombin, and activated protein C-specific serpins or gene therapy. The latter strategies are, however, hampered by the short clinical experience and potential adverse effects including the absence of tight temporal and spatial control of coagulation and the risk of uncontrolled insertional mutagenesis. Systemic delivery of mRNA allows endogenous production of the corresponding encoded protein. Thus, injection of erythropoietin-encoding mRNA in a lipid nanoparticle formulation resulted in increased erythropoiesis in mice and macaques. Here, we demonstrate that a single injection of in vitro transcribed B domain-deleted FVIII-encoding mRNA to FVIII-deficient mice enables endogenous production of pro-coagulant FVIII. Circulating FVIII:C levels above 5% of normal levels were maintained for up to 72 h, with an estimated half-life of FVIII production of 17.9 h, and corrected the bleeding phenotype in a tail clipping assay. The endogenously produced FVIII did however exhibit low specific activity and induced a potent neutralizing IgG response upon repeated administration of the mRNA. Our results suggest that the administration of mRNA is a plausible strategy for the endogenous production of proteins characterized by poor translational efficacy. The use of alternative mRNA delivery systems and improved FVIII-encoding mRNA should foster the production of functional molecules and reduce their immunogenicity., (Copyright© 2020 Ferrata Storti Foundation.)

Engineered recombinant factor X (FX) variants represent a promising strategy to bypass the tenase complex and restore hemostasis in hemophilia patients. Previously, a thrombin-activatable FX variant with fibrinopeptide-A replacing the activation peptide (FX-delAP/FpA) has been described in this regard. Here we show that FX-delAP/FpA is characterized by a sixfold shorter circulatory half-life compared with wild-type FX, limiting its therapeutical applicability. We therefore designed a variant in which the FpA sequence is inserted C-terminal to the FX activation peptide (FX/FpA). FX/FpA displayed a similar survival to wt-FX in clearance experiments and could be converted into FX by thrombin and other activating agents. In in vitro assays, FX/FpA efficiently restored thrombin generation in hemophilia A and hemophilia B plasmas, even in the presence of inhibitory antibodies. Expression following hydrodynamic gene transfer of FX/FpA restored thrombus formation in FVIII-deficient mice in a laser-induced injury model as well as hemostasis in a tail-clip bleeding model. Hemostasis after tail transection in FVIII-deficient mice was also corrected at 5 and 90 minutes after injection of purified FX/FpA. Our data indicate that FX/FpA represents a potential tenase-bypassing agent for the treatment of hemophilia patients with or without inhibitors., Competing Interests: O.D.C., G.C., P.G., and C.V.D. are inventors on a patent regarding FX/FpA. The other authors do declare to have no conflict of interest., (Georg Thieme Verlag KG Stuttgart · New York.)

Replacement therapy has been proven effective in the management of bleedings in haemophilia A. Nevertheless, this approach comes with several shortcomings, like the need for frequent intravenous infusions and the development of neutralizing antibodies in 20 to 30% of the patients with severe haemophilia A replacement. This has led to the development of novel strategies to expand the spectrum of treatment options, some of which are based on antibody technology. These include a bispecific antibody that bridges enzyme factor IXa and substrate factor X, monoclonal antibodies that block the function of tissue factor pathway inhibitor, and a factor VIII-nanobody fusion protein with strongly enhanced von Willebrand factor binding. In this review, functional and mechanistic considerations on the use of these antibody variants will be discussed., Competing Interests: Disclosure The authors report no conflicts of interest in this work., (Georg Thieme Verlag KG Stuttgart · New York.)

Competing Interests: Disclosure The authors report no conflicts of interest in this work.

Recombinant adeno-associated virus (rAAV) vectors encoding human factor VIII (hFVIII) were systematically evaluated for hemophilia A (HA) gene therapy. A 5.7-kb rAAV-expression cassette (rAAV-HLP-codop-hFVIII-N6) containing a codon-optimized hFVIII cDNA in which a 226 amino acid (aa) B-domain spacer replaced the entire B domain and a hybrid liver-specific promoter (HLP) mediated 10-fold higher hFVIII levels in mice compared with non-codon-optimized variants. A further twofold improvement in potency was achieved by replacing the 226-aa N6 spacer with a novel 17-aa peptide (V3) in which 6 glycosylation triplets from the B domain were juxtaposed. The resulting 5.2-kb rAAV-HLP-codop-hFVIII-V3 cassette was more efficiently packaged within AAV virions and mediated supraphysiologic hFVIII expression (732 ± 162% of normal) in HA knock-out mice following administration of 2 × 10(12) vector genomes/kg, a vector dose shown to be safe in subjects with hemophilia B. Stable hFVIII expression at 15 ± 4% of normal was observed at this dose in a nonhuman primate. hFVIII expression above 100% was observed in 3 macaques that received a higher dose of either this vector or the N6 variant. These animals developed neutralizing anti-FVIII antibodies that were abrogated with transient immunosuppression. Therefore, rAAV-HLP-codop-hFVIII-V3 substantially improves the prospects of effective HA gene therapy.

Background: Von Willebrand factor (VWF) is critical for the in vivo survival of factor VIII (FVIII). Since FVIII half-life correlates with VWF-antigen pre-infusion levels, we hypothesized that VWF levels are useful to predict FVIII half-life., Methodology: Standardized half-life studies and analysis of pre-infusion VWF and VWF-propeptide levels were performed in a cohort of 38 patients with severe haemophilia A (FVIII <1 IU/ml), aged 15-44 years. Nineteen patients had blood-group O. Using multivariate linear regression-analysis (MVLR-analysis), the association of VWF-antigen, VWF-propeptide, age and body-weight with FVIII half-life was evaluated., Principal Findings: FVIII half-life was shorter in blood-group O-patients compared to non-O-patients (11.5+/-2.6 h versus 14.3+/-3.0 h; p = 0.004). VWF-antigen levels correlated with FVIII half-life considerably better in patients with blood-group non-O than O (Pearson-rank = 0.70 and 0.47, respectively). Separate prediction models evolved from MVLR-analysis for blood-group O and non-O patients, based on VWF-antigen and VWF/propeptide ratio. Predicted half-lives deviated less than 3 h of observed half-life in 34/38 patients (89%) or less than 20% in 31/38 patients (82%)., Conclusion: Our approach may identify patients with shorter FVIII half-lives, and adapt treatment protocols when half-life studies are unavailable. In addition, our data indicate that survival of FVIII is determined by survival of endogenous VWF rather than VWF levels per se.

Background and Objectives: Patients with severe hemophilia A have considerably different factor VIII half-lives. Whether this is associated with their clinical characteristics has not been reported. The aim of this study was to describe the association of factor VIII half-lives with treatment and clinical characteristics of patients with severe hemophilia A, who have been treated with individually tailored prophylaxis., Design and Methods: Patients were selected from a single-center cohort of 214 patients with severe hemophilia, born between 1944 and 1995. To improve efficiency we measured factor VIII half-life in 42 patients selected from the extremes of the distribution of phenotypes of severe hemophilia. We assessed information on life-long joint bleeds and clotting factor consumption. Orthopedic outcome was assessed by the Pettersson score., Results: Among these patients with severe hemophilia, factor VIII half-life ranged from 7.4-20.4 hours (median 11.8 hours). A one-hour increase in factor VIII half-life was associated with 96 (95% confidence interval (CI) 2 -190) IU less clotting factor use per kg per year. Age was an important determinant of factor VIII half-life, and explained a large part of the association between factor VIII half-life and clotting factor consumption. The median number of joint bleeds per year and arthropathy were similar for patients with different half-lives., Interpretation and Conclusions: Among patients with severe hemophilia, treated prophylactically with clotting factor, those with a shorter factor VIII half-life required slightly more clotting factor to prevent joint bleeds and subsequent arthropathy than similar patients with a longer factor VIII half-life.

Résumé: Le facteur VIII (FVIII) est une protéine de la cascade de coagulation qui exerce une activité de cofacteur non enzymatique vis-à-vis du facteur IXa (FIXa), l'enzyme qui va promouvoir l'activation du facteur X (FX). Un déficit fonctionnel en FVIII conduit à l'hémophilie A, une pathologie hémorragique grave dont la prévalence se situe autour de 1-2 cas pour 10 000 naissances de garçons. La prise en charge des complications hémorragiques associées à l'hémophilie A s'effectue par une thérapie de substitution visant à remplacer le FVIII manquant, préférentiellement par prophylaxie. En raison des limites inhérentes à cette approche (avec la nécessité d'infusions intraveineuses fréquentes et l'apparition d'anticorps inhibiteurs contre le FVIII thérapeutique), de nouvelles thérapies ont récemment été développées. L'emicizumab, un anticorps bispécifique, en est un représentant. Cet anticorps a pour fonction de mimer l'activité cofacteur du FVIII en permettant un rapprochement dans l'espace du FIXa et du FX. Dans cette revue, nous discuterons des aspects fonctionnels et cliniques de l'emicizumab, nous comparerons son mode d'action à celui du FVIII et les implications pour le suivi biologique des patients. De plus, nous présenterons les données cliniques obtenues dans les essais pivots HAVEN qui ont montré un bénéfice significatif de l'emicizumab avec une réduction importante des saignements spontanés chez les patients traités. Finalement, nous discuterons des effets secondaires associés à l'utilisation de l'emicizumab et nous conclurons sur son utilisation potentielle au-delà de l'hémophilie A. Coagulation factor VIII (FVIII) functions as a non-enzymatic cofactor for the enzyme factor IXa (FIXa) in the activation of factor X (FX). The functional deficiency of FVIII is associated with a severe bleeding disorder known as hemophilia A, affecting 1-2 per 10,000 male births. Bleeding complications have long been managed via FVIII substitution therapy, preferably on a prophylactic basis. Due to limitations of this approach (i.e., the need for frequent intravenous infusions, development of neutralizing antibodies), novel treatment options for hemophilia A have been evaluated, including the use of the bispecific antibody emicizumab. Emicizumab is designed to mirror FVIII cofactor function by promoting the spatial approximation of FIXa and FX. In this review, we will discuss functional and clinical aspects of emicizumab. We will compare its mode of action to that of FVIII and consider the implications for laboratory monitoring of this antibody. In addition, we highlight the clinical data obtained in the pivotal HAVEN-trials, which have shown a clear clinical benefit by strongly reducing the occurrence of spontaneous bleeding episodes. Finally, we discuss some of the adverse effects that have been reported and also the potential use of emicizumab beyond patients with severe hemophilia A. [ABSTRACT FROM AUTHOR]