Towards the Care of Hemophilia a Patients Using Induced Pluripotent Stem Cell (iPSC)-Derived Megakaryocytes (iMks) Expressing Coagulation Factor (F) VIII

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2266-2266
Author(s):  
Randolph B Lyde ◽  
Li Zhai ◽  
Karen Vo ◽  
Danuta Jadwiga Jarocha ◽  
Spencer Sullivan ◽  
...  
Keyword(s):  
Whole Blood ◽  
Neutralizing Antibodies ◽  
Research Funding ◽  
Hemophilia A ◽  
Specific Activity ◽  
Coagulation Factor ◽  
Clot Formation ◽  
Patient Specific ◽  
Furin Cleavage ◽  
Furin Cleavage Site

Abstract We and others have shown that FVIII expressed ectopically in platelets (pFVIII) is stored in α-granules, released at sites of vascular injury and restores hemostasis in FVIIInull mice, even in the presence of neutralizing antibodies to FVIII. These studies support the concept that unlike therapeutic interventions that correct plasma FVIII, pFVIII may be a useful therapy in hemophilia A with intractable inhibitors and significant bleeds. We have also demonstrated this approach has several limitations that may make pFVIII gene therapy bone marrow transplantation (BMT) strategies problematic: 1) pFVIII is not equivalent to plasma FVIII and its efficacy in joint and intracranial bleeds has yet to be shown, especially in the presence of inhibitors, and 2) pFVIII expressed during megakaryopoiesis can cause injury to the Mks, potentially exacerbating post-BMT thrombocytopenia. We propose an alternative strategy: interval prophylactic infusions of FVIII-containing platelets generated from patient-specific iMks expressing either human B-domain-deleted (BDD) FVIII or variants of this FVIII that have greater stability and longer half-lives; making them especially efficacious as pFVIII as we previously demonstrated. iPSCs are a renewable source of cells that can be pre-screened prior to clinical usage for lines that express optimal levels of pFVIII and also release optimal numbers of platelets after differentiation into iMks. Such iPSCs were transfected with a self-inactivating lentivirus containing cDNA for one of three FVIII variants: wildtype BDD FVIII (WT FVIII), R1645H PACE/furin cleavage site FVIII (FVIIIR1645H), and amino acid 1645 to 1648 deletion FVIII (FVIIIΔ). FVIIIR1645H and FVIIIΔ show greater stability and consequently greater specific activity with no increase in injuring Mks. All FVIII variants were expressed using the MK-specific Cxcl4 promoter and were shown to be effective in several bleeding models in FVIIInull mice. Differentiated and transduced iMKs were analyzed for RNA and protein expression. All of the FVIII variant iMKs expressed at least forty-fold higher levels of mRNA compared to the non-transduced control (n=6) and protein was expressed at >550 pg/106 CD42b+ iMKs (n=6). Transduced MKs released FVIII into the supernatant when activated by thrombin showing the pFVIII was likely stored in α-granules. Annexin staining was the same between FVIII-expressing iMKs and control iMks suggesting that the level of pFVIII did not cause the iMks to become apoptotic. To test the ability of FVIII-expressing iMKs to correct the coagulopathy in hemophilia A, 5x105 iMKs were added to FVIIInull murine whole blood and evaluated for clot formation using rotational thromboelastometry (ROTEM). Each FVIII variant showed a decrease in clotting time, clot formation time, and an increase in maximum clot firmness when compared to the non-transduced control (n=4). These data show that iMKs expressing FVIII variants can improve hemostasis in a whole blood clotting assay. Our next goal is to generate sufficient platelets from these iMKs to test for correction of the bleeding diathesis in immunodeficient FVIIInull mice and to determine their efficacy in improving hemostasis in a number of clinically relevant hemostatic models. Disclosures Arruda: Pfizer: Consultancy, Patents & Royalties, Research Funding; Spark Therapeutics: Patents & Royalties. Camire:Pfizer: Consultancy, Patents & Royalties, Research Funding; NovoNordisk: Research Funding; Spark Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Hemophilia a Clots Generated with Recombinant Factor VIIa Differed in Structure and Composition from Those Formed with Factor VIII

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3798-3798
Author(s):  
Lilley Leong ◽  
Irina N. Chernysh ◽  
Yifan Xu ◽  
Cornell Mallari ◽  
Billy Wong ◽  
...  
Keyword(s):  
Factor Viii ◽  
Whole Blood ◽  
Neutralizing Antibodies ◽  
Research Funding ◽  
Thrombin Generation ◽  
Hemophilia A ◽  
Clot Formation ◽  
Factor Vii ◽  
Wild Type ◽  
Independent Mechanism

Abstract Patients with severe factor VIII (FVIII) deficiency (hemophilia A [HemA]) develop neutralizing antibodies (inhibitors) against FVIII in up to ~30% of cases. For HemA patients with inhibitors, activated recombinant factor VII (rFVIIa) is a treatment option. High levels of rFVIIa are required for treating HemA patients with inhibitors to induce direct activation of factor X on the surface of activated platelets via a tissue factor (TF)-independent mechanism (Hoffman M, Monroe DM. Thromb Res. 2010;125(suppl 1):S16-S18). To assess how rFVIIa-mediated clot formation in HemA patients with inhibitors may differ from unaffected individuals, we compared the effect of rFVIIa on HemA versus control (or HemA supplemented with 100% FVIII) clot formation in human and/or mouse systems. By TF-induced thrombin generation assay, increasing rFVIIa from 5 nM to 100 nM did not appreciably alter the kinetics or extent of thrombin generation compared with the same human HemA plasma containing 100% FVIII. Confocal microscopy of human HemA plasma clots generated with 75 nM rFVIIa and TF showed few branching fibrin fibers and an open fibrin meshwork. In contrast, TF-induced coagulation of the same HemA plasma containing 100% FVIII formed fibrin clots with numerous branches, interconnecting to form a dense meshwork. To confirm that these findings reflect rFVIIa-mediated clot formation in vivo, we assessed the intrinsic coagulation of mouse HemA whole blood collected without anticoagulant and spiked with rFVIIa. Intrinsic coagulation with rFVIIa was assessed by T2 magnetic resonance (T2MR), a technique capable of monitoring the separation of whole blood into serum, loose-clot, and tight-clot compartments during coagulation (Skewis et al. Clin Chem. 2014;60:1174-1182; Cines et al. Blood. 2014;123:1596-1603). By T2MR, rFVIIa induced the separation of HemA whole blood into the serum and clot compartments, indicating that the reduced fibrin generation with rFVIIa did not interfere with whole blood coagulation. Furthermore, saphenous vein puncture of HemA mice treated with rFVIIa showed a dose-dependent decrease in clot times. Scanning electron microscopy of the clots extracted from these HemA mice indicated markedly different composition than clots extracted from wild-type mice. In wild-type clots, fibrin and polyhedral erythrocytes formed a large proportion of the total structures. In contrast, clots from rFVIIa-treated HemA mice consisted primarily of platelets and erythrocytes with forms intermediate between discoid and polyhedral but, surprisingly, low fibrin content. Taken together, these data suggest that rFVIIa-mediated clot formation may require greater activated platelet involvement, which would be consistent with the TF-independent mechanism of action proposed for rFVIIa in HemA. Finally, the compositional difference between clots from wild-type versus HemA mice dosed with rFVIIa suggest that evaluating HemA therapies for their ability to form more physiologic clots could be an approach to improve treatment options for patients with HemA. Disclosures Leong: Bayer: Employment. Xu:Bayer: Employment. Mallari:Bayer: Employment. Wong:Bayer: Employment. Sim:Bayer: Employment. Cuker:Stago: Consultancy; Genzyme: Consultancy; Amgen: Consultancy; Biogen-Idec: Consultancy, Research Funding; T2 Biosystems: Research Funding. Marturano:T2 Biosystems: Employment. Lowery:T2 Biosystems: Employment. Kauser:Bayer: Employment. Weisel:Bayer: Research Funding.

Immunogenicity of BAX 855 in Previously Treated Patients with Congenital Severe Hemophilia Α

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2594-2594
Author(s):  
Frank Michael Horling ◽  
Peter Allacher ◽  
Herwig Koppensteiner ◽  
Werner Engl ◽  
Fritz Scheiflinger ◽  
...  
Keyword(s):  
Adverse Events ◽  
Neutralizing Antibodies ◽  
Research Funding ◽  
Hemophilia A ◽  
De Novo ◽  
Coagulation Factor ◽  
Severe Hemophilia ◽  
Increased Risk ◽  
Previously Treated Patients ◽  
Previously Treated

Abstract Background and objectives BAX 855 (Antihemophilic Factor [Recombinant] pegylated, rurioctocog alfa pegol) is an extended half-life (EHL) recombinant human coagulation factor VIII (rFVIII) modified with polyethylene glycol (PEG) (Turecek et al., 2012). It was recently approved in the US and Japan for on-demand treatment of bleeding events and for prophylactic treatment for patients with congenital severe hemophilia A. The efficacy and safety of BAX 855 were extensively studied during clinical development of this compound (Konkle et al., 2015). The assessment of BAX855 immunogenicity was of particular interest because the development of neutralizing antibodies (FVIII inhibitors) is the most serious complication following replacement therapies with FVIII products. FVIII inhibitors develop in about 20-32% of previously untreated patients (Gouw SC et al., 2013) and with a rate of 1.55- 3.8 per 1000 patients per year in previously treated patients (Kempton CL, 2010) with severe hemophilia A. To fully understand the potential of BAX855 to induce antibody responses, both FVIII inhibitors and total FVIII-binding antibodies were assessed. Furthermore, potential antibody development against PEG-FVIII, PEG and CHO proteins was investigated. Methods The clinical protocols (ClinicalTrials.gov identifier: NCT02585960, NCT02210091, NCT01736475, NCT01913405, NCT01945593, NCT01599819, NCT02615691) and the methods used for antibody analytics (Whelan et al 2013; Lubich et al 2016) were previously described. ELISA technologies were used for the analysis of total binding antibodies, the Nijmegen modification of the Bethesda assay was used for the detection of FVIII inhibitors. Correlation analyses were done to assess any potential correlation between the development of antibodies and potential adverse events. Results None of the 243 subjects (6 PUPs and 237 PTPs) included in the analysis developed FVIII inhibitors (≥ 0.6 BU/mL) A total of 44 subjects tested positive for binding antibodies against FVIII, PEG-FVIII or PEG at single time points. 28 of these 44 subjects showed pre-existing antibodies against FVIII, PEG-FVIII, or PEG prior to first exposure to BAX 855, which disappeared during the study. 13 subjects who tested negative at screening developed transient antibodies against FVIII, PEG-FVIII, or PEG at one or two consecutive study visits after exposure to BAX 855. Antibodies were transient and not detectable at subsequent visits or at completion of the study. Five subjects showed positive results for binding antibodies at study completion or at the time of the data cutoff. No conclusion can be drawn whether these antibodies are of transient or persistent nature. There was no confirmed causal relationship between the appearance of binding antibodies against FVIII, PEG or PEG-FVIII and adverse events, nor was there an impact on hemostatic efficacy in any of the 44subjects. No subject had pre-existing antibodies or developed de novo antibodies to CHO proteins during the study at any time point. Conclusion Our data indicate that BAX855 did not show an increased risk for PTPs to develop FVIII inhibitors. We did not see any FVIII inhibitor development in PUPs, but the small number of overall exposures does not allow general conclusions for PUPs. Importantly, the data suggest that BAX855 did not induce immune responses associated with impaired treatment efficacy or with altered PK parameters. Disclosures Horling: Shire: Employment. Allacher:IMC Krems: Research Funding. Koppensteiner:Shire: Employment. Engl:Shire, formerly Baxalta and Baxter: Employment, Equity Ownership. Scheiflinger:Shire: Employment, Research Funding. Abbuehl:Baxalta (now part of Shire): Employment. Reipert:Shire: Employment.

scholarly journals Infusion of Coagulation Factor VIII-Containing Induced Pluripotent Stem Cell (iPSC)-Derived Megakaryocytes (iMks) Shows Potential As Hemophilia a Treatment

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2559-2559
Author(s):  
Randolph B Lyde ◽  
Hyunsook Ahn ◽  
Karen K Vo ◽  
Danuta Jadwiga Jarocha ◽  
Li Zhai ◽  
...  
Keyword(s):  
Carotid Artery ◽  
Factor Viii ◽  
Flow Rate ◽  
Cleavage Site ◽  
Research Funding ◽  
Hemophilia A ◽  
Furin Cleavage ◽  
Furin Cleavage Site

Abstract Ectopically expressed factor VIII (FVIII) in megakaryocytes (MKs) and platelets (pFVIII) is stored in a-granules and released at sites of vascular injury by activated platelets (Plts), restoring hemostasis in FVIIInull mice, even in the presence of neutralizing inhibitors. These studies support the idea that unlike therapies that correct plasma levels of FVIII, pFVIII may be a useful therapy in patients with hemophilia A who have intractable inhibitors and significant bleeds. Expressing FVIII in Plts, however, has limitations that make pFVIII gene therapy through bone marrow transplantation (BMT) problematic: 1) pFVIII expressed during megakaryopoiesis can injure the Mks, potentially exacerbating post-BMT thrombocytopenia, and 2) pFVIII's efficacy in joint and intracranial bleeds has yet to be shown, especially in the presence of inhibitors. Due to these limitations we propose an alternative strategy: infusing patient-specific iMks derived from personalized iPSCs and expressing either human B-domain-deleted (BDD) FVIII or variants of FVIII that have greater stability and longer half-lives. Our group has shown that infusing in vitro-grown Mks into mice releases functional Plts in the recipient animals. iPSCs are a renewable source of stem cells that can be pre-screened to select clones that both express high levels of pFVIII and also release high numbers of Plts after differentiation into iMks. As proof-of-principle, iMks were transfected with a self-inactivating lentivirus containing cDNA for 1 of 3 FVIII variants: wildtype BDD FVIII (WT FVIII), a PACE/furin cleavage site FVIII (FVIIIR1645H) variant, and an amino acid 1645 to 1648 deletion FVIII (FVIIIΔ) variant that removes the entire PACE/furin cleavage site. FVIIIR1645H and FVIIIΔ showed greater stability and consequently greater specific activity with no increase in injuring Mks. We previously published that hemophilia A mice expressing pFVIIIR1645H were more hemostatically corrected than comparable mice expressing WT pFVIII. All of the FVIII variant iMks expressed at least a 40-fold higher level of mRNA compared to the non-transduced control (N=6) and integration levels show the same number of viral copies between the groups (N=6). All variants expressed >550 pg FVIII/106 CD42b+ iMKs (N=6). Upon activation with thrombin, transduced Mks released the FVIII into the supernatant. To examine whether this pFVIII injured the developing Mks, baseline PAC-1 binding for Mk activation in culture (N=3), TUNEL staining and Annexin-5 binding for apoptosis (N=4) were analyzed with no differences observed with WT Mks not expressing pFVIII. To test the ability of FVIII-expressing iMks to correct the coagulopathy in hemophilia A, 5x105 iMks were added to FVIIInull murine whole blood (0.11 ml) and evaluated for clot formation using rotational thromboelastometry (ROTEM). Each pFVIII iMk variant showed a decrease in clotting time, clot formation time, and an increase in maximum clot firmness when compared to the non-transduced control (p<0.007 for each, N=4). These FVIII expressing iMks were also tested in vivo in a FeCl3 carotid artery injury murine model. 24 hours prior to infusion, recipient hemophilia A mice were treated with clodronate liposomes to eliminate circulating monocytes and to improve the survival of infused human iMks and their released Plts. Immediately post iMks (5x106)infusion, a 20% FeCl3 solution was applied to the carotid artery for 3 mins and flow rate through the injured vessel was measured for 30 mins. Both WT FVIII and FVIIIR1645H showed a significant decrease in blood flow through the injured vessel from 1.2 ml/min seen in FVIIInull mice receiving control iMks to 0.4 ml/min (p<0.05, N=10). Wild-type mice had a flow rate of 0.13 ml/min. These data indicate that pFVIII within iMKs or their derived Plts expressing FVIII can improve hemostasis in vitro and in vivo. These studies provide the groundwork to examine whether infused iMks pFVIII can improve hemostasis in the setting of inhibitors. Disclosures Arruda: Pfizer: Patents & Royalties, Research Funding. Sabatino:Spark Therapeutics: Research Funding. Camire:Bayer: Consultancy; Spark Therapeutics: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Pfizer: Consultancy, Patents & Royalties, Research Funding; Novo Nordisk: Research Funding.

Development of neutralizing antibodies in application of various concentrates of blood coagulation factor VIII in the treatment of hemophilia A

2021 ◽  
Author(s):  
Н.И. Зозуля
Keyword(s):  
Factor Viii ◽  
Cell Line ◽  
Neutralizing Antibodies ◽  
Hemophilia A ◽  
Chinese Hamster ◽  
Coagulation Factor ◽  
Human Cell Line ◽  
Chinese Hamster Cell ◽  
Coagulation Factor Viii ◽  
Recombinant Fviii

Серьезным осложнением, связанным с лечением гемофилии А, является развитие ингибиторов. В последние годы был проведён ряд исследований, посвящённых данной проблеме: RODIN, INSIGHT, FranceCoag, SIPPET и NuProtect. В данном обзоре суммируются основные результаты этих исследований. Согласно результатам рандомизированного исследования SIPPET, препараты плазматического фактора свертывания крови VIII (FVIII) обладают меньшей иммуногенностью, чем препараты рекомбинантного FVIII, синтезированного из клеточной линии китайских хомячков, что следует учитывать при выборе стратегии лечения. Согласно результатам исследования NuProtect, опубликованным в 2019 г., концентрат рекомбинантного FVIII, полученный из клеточной линии человека, демонстрирует профиль иммуногенности, сходный с таковым у препаратов плазматического FVIII. У ранее нелеченых пациентов с ненулевыми мутациями при применении симоктоког альфа не наблюдалось образования ингибиторов, также как и в случае применения препаратов плазматического FVIII в исследовании SIPPET. Inhibitor development is a serious complication associated with hemophilia A therapy. A number of studies have been carried out of this issue — RODIN, INSIGHT, FranceCoag, SIPPET, and NuProtect. This review summarizes the main results of these studies. According to the results of the SIPPET randomized trial, plasma-derived coagulation factor VIII (FVIII) products are less immunogenic than recombinant FVIII products synthesized from a Chinese hamster cell line; this fact should be taken into account in choosing a treatment strategy. According to the results of NuProtect study published in 2019, the concentrate of human cell line-derived recombinant FVIII demonstrates immunogenicity profi le similar to the one in plasma-derived FVIII products. Previously untreated patients with non-zero mutations receiving simoctocog alfa did not show development of inhibitors as well as in case of administration of plasma-derived FVIII products in SIPPET study.

scholarly journals Structure, function and antigenicity of the SARS-CoV-2 spike glycoprotein

2020 ◽  
Author(s):  
Alexandra C. Walls ◽  
Young-Jun Park ◽  
M. Alexandra Tortorici ◽  
Abigail Wall ◽  
Andrew T. McGuire ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Viral Entry ◽  
Neutralizing Antibodies ◽  
Target Cells ◽  
Furin Cleavage ◽  
Humoral Immune ◽  
Binding Domains ◽  
Furin Cleavage Site ◽  
Recent Emergence ◽  
Novel Coronavirus

SUMMARYThe recent emergence of a novel coronavirus associated with an ongoing outbreak of pneumonia (Covid-2019) resulted in infections of more than 72,000 people and claimed over 1,800 lives. Coronavirus spike (S) glycoprotein trimers promote entry into cells and are the main target of the humoral immune response. We show here that SARS-CoV-2 S mediates entry in VeroE6 cells and in BHK cells transiently transfected with human ACE2, establishing ACE2 as a functional receptor for this novel coronavirus. We further demonstrate that the receptor-binding domains of SARS-CoV-2 S and SARS-CoV S bind with similar affinities to human ACE2, which correlates with the efficient spread of SARS-CoV-2 among humans. We found that the SARS-CoV-2 S glycoprotein harbors a furin cleavage site at the boundary between the S1/S2 subunits, which is processed during biogenesis and sets this virus apart from SARS-CoV and other SARS-related CoVs. We determined a cryo-electron microscopy structure of the SARS-CoV-2 S ectodomain trimer, demonstrating spontaneous opening of the receptor-binding domain, and providing a blueprint for the design of vaccines and inhibitors of viral entry. Finally, we demonstrate that SARS-CoV S murine polyclonal sera potently inhibited SARS-CoV-2 S-mediated entry into target cells, thereby indicating that cross-neutralizing antibodies targeting conserved S epitopes can be elicited upon vaccination.

Thromboelastography in Children with Coagulation Factor Deficiencies.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2139-2139 ◽  
Author(s):  
Meera B. Chitlur ◽  
Indira Warrier ◽  
Madhvi Rajpurkar ◽  
Wendy Hollon ◽  
Lolita Llanto ◽  
...  
Keyword(s):  
Whole Blood ◽  
Thrombin Generation ◽  
Hemophilia A ◽  
Coagulation Factor ◽  
Response To Treatment ◽  
Factor Vii ◽  
P Value ◽  
Factor V ◽  
Mean Values ◽  
Significant Difference

Abstract The thromboelastograph produces a continuous profile of the rheological changes that occur during the process of coagulation using whole blood. This information can be transformed into a dynamic velocity profile of the changes in blood elasticity occurring during clotting. We used the TEG® hemostasis analyzer in patients with hemophilia A or B with and without inhibitors and other coagulation factor deficiencies (OFD), to study the thromboelastographic profiles in these patients. Materials and Methods: 62 children (6 months-19 years old) were enrolled according to IRB regulations. 29 children had severe hemophilia A (SHA), 4 moderate hemophilia A or B (Mod.H), 2 severe factor VII deficiency, 1 combined factor V and VIII deficiency, 1 VWD (type II B), 1 severe factor V deficiency, 1 Severe PAI deficiency, 19 normal controls (NC), and 4 SHA with inhibitors (SHA+I). All patients were studied 72 hours after the last dose of factor. Citrated whole blood was activated using recombinant human tissue factor (Innovin, Dade Behring Inc®) and recalcified using 0.2M CaCl2. In patients with central lines with heparin, a heparinase cup was used. The TEG® was run for ≥ 90 min. CBC with differential was obtained on all subjects. Results: There was no significant difference in the CBC parameters among patients. Analysis of the TEG data revealed the following: Table 1 TEG Parameters (mean values) SHA (n=29) Mod.H (n=4) SHA+I (n=4) OFD (n=6) Control(n=19) MTG:Max rate of thrombin generation; TMG: Time to MTG; R: Reaction Time; K: Time to reach an amplitude of 20mm; MA: Max. Amplitude MTG(mm*100/sec) 8.7 9.6 1.3 9 17 TMG(min) 27.5 16.6 62.7 17.5 8.9 R(min) 22 14 56 15 7 K(min) 7 4 41 4 2 Max.Amplitude, MA (mm) 59 56 12 58 62 The rate of thrombin generation as visualized by plotting the 1st derivative of the TEG course, in patients with SHA without inhibitors, showed that they could be divided into 2 groups based on MTG (</>9). When analysed the 2 groups showed the following characteristics (5 representative curves from each group are shown): Figure Figure Table 2 TEG Parameters (Mean values) MTG < 9 (n=16) MTG > 9 (n=13) p value TMA: Time to MA; MTG(mm*100/sec) 5.5 12.6 <0.001 TMG (min) 33 20 0.009 R(min) 26 16 0.004 K(min) 9 3.4 0.03 MA(mm) 56.1 62.3 0.01 TMA(min) 60 38 0.006 13/29 children with SHA had target joints and 69%of patients with target joints had a MTG<9. Conclusions: SHA patients have variable bleeding tendencies as seen by the variation in MTG. A lower MTG is associated with a higher incidence of target joints. This may provide a clue as to which patients may have the greatest benefit from primary prophylaxis. Patients with OFD have a TEG® profile similar to Mod.H patients. SHA+I have poor thrombin generation as seen by a significantly longer TMG and R time (p <0.05), compared to all subjects. The TEG may provide valuable clues to the severity of bleeding tendencies in patients with factor deficiencies. In additional observations (not shown), it appears that the TEG may be used to monitor the response to treatment with factor concentrates and tailor treatment with rFVIIa.

Site-Specific PEGylation of Factor VIII (PEG-FVIII) Preserves Full Clotting Activity and Extends Therapeutic Efficacy in HemophiliaA Dogs

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 511-511 ◽  
Author(s):  
Tongyao Liu ◽  
David Lillicrap ◽  
Xin Zhang ◽  
Andrea Labelle ◽  
Sandra Powell ◽  
...  
Keyword(s):  
Factor Viii ◽  
Whole Blood ◽  
Half Life ◽  
Therapeutic Efficacy ◽  
Neutralizing Antibodies ◽  
Hemophilia A ◽  
Volume Of Distribution ◽  
Pharmacokinetic Analysis ◽  
Site Specific ◽  
Intravenous Injections

Abstract To improve the effectiveness of Factor VIII replacement therapy for Hemophilia A, we sought to develop a PEGylated Factor VIII that would effectively treat bleeding episodes, while reducing the frequency of intravenous injections required for prophylaxis. Previously, we found that the site-specific PEGylation of Factor VIII (PEG-FVIII) preserves full clotting activity, prolongs circulating half-life and extends therapeutic efficacy in a number of bleeding models in hemophilic mice. To further characterize its activity, four naïve Hemophilia A dogs were treated with either PEG-FVIII or unmodified BDD-FVIII in a cross-over study design. All treated dogs showed normalized Whole Blood Clotting Time (WBCT), whole blood Thromboelastograph (TEG) profile, and Cuticle Bleeding Time within 30 min from dosing. Pharmacokinetic analysis of the decay of plasma FVIII activity and antigen levels showed that PEG-FVIII achieved 2-fold longer half-life and reduced clearance and volume of distribution relative to BDD-FVIII. Consistently, PEG-FVIII also demonstrated significantly prolonged efficacy relative to BDD-FVIII by measurement of WBCT and TEG. Both BDD-FVIII and PEG-FVIII were well tolerated in naïve HemA dogs, normal hematology and serum chemistry values were observed following administration. However, two naive dogs that received BDD-FVIII and one naive dog that received PEG-FVIII developed detectable neutralizing antibodies to human FVIII as early as on day 9 post-treatment. In summary, consistent with our previously reported findings in hemophilic mice, in comparison to BDD-FVIII, PEG-FVIII demonstrated superior half-life, full activity in stopping acute bleeding and prolonged efficacy in hemophilia A dogs. Taken together, the results support the use of site-specific PEGylation to create a homogeneous therapeutic for both prophylactic and on-demand treatment of patients with Hemophilia A.

The EPIC Study: A Clinical Trial To Assess Whether Early Low Dose Prophylaxis In The Absence Of Immunological Danger Signals Reduces Inhibitor Incidence In Previously Untreated Patients (PUPs) With Hemophilia A

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 576-576 ◽  
Author(s):  
Guenter Auerswald ◽  
Karin Kurnik ◽  
Jan Blatny ◽  
Armin J Reininger
Keyword(s):  
Research Funding ◽  
Low Dose ◽  
Hemophilia A ◽  
Venous Access ◽  
Patient Specific ◽  
Missense Mutations ◽  
Severe Hemophilia ◽  
Prophylactic Regimen ◽  
Inhibitor Development ◽  
Danger Signals

Abstract Background Inhibitor development is a complex, multifactorial immune response involving both patient-specific and treatment-related factors. Of the known risk factors, intensive treatment at an early age has been shown to be significant, and clinical observations have suggested that early prophylaxis (i.e. first exposure to FVIII in the absence of a bleed in the first year of age) may protect patients from inhibitor development by inducing FVIII tolerance. Aim This study aimed to assess prospectively if a once-weekly, low-dose prophylactic regimen started before 1 year of age and before the onset of a severe bleeding phenotype (i.e. joint bleed), together with the minimization of immunological danger signals, could reduce the incidence of inhibitor formation in PUPs with severe and moderately severe hemophilia A to 15% or less. Methods The EPIC study was a Phase 3b, prospective, single arm, historically-controlled, international multicenter study to assess the incidence rate of inhibitor formation in PUPs with severe and moderately severe hemophilia A during the first 50 exposure days (EDs) of treatment with ADVATE starting with a once-weekly, low-dose (ADVATE 25 IU/kg once weekly), prophylactic regimen. If clinically indicated, it was permissible to increase the frequency of dosing to 2 or 3 times per week. In addition, infusions during the first 20 EDs had to be given 3 to 4 days before or after any vaccinations, which had to be given subcutaneously, not intramuscularly; infusions had to be avoided if the subject had high fever (above 38°C [100°F]). Main enrolment criteria were: severe and moderately severe hemophilia A (FVIII ≤2%), age <1 year, ≤3 EDs to any FVIII-containing product used for treatment of minor bleeds or for precautionary infusions following injury, adequate venous access (without need for central venous access device), no life-threatening conditions (intracranial hemorrhage, severe trauma) or requirement for surgery at the time of enrollment, no evidence of inhibitor ≥0.6 BU in Nijmegen-modified Bethesda assay at study start, no hemostatic defect other than hemophilia A, no clinically significant chronic disease other than hemophilia A,. Information about type of FVIII gene defect was obtained in 17 subjects. FVIII inhibitor tests were performed at screening, at study infusion #3, 6, 10, 15, 20, 30, 40, and 50, and at any other time point if an inhibitor was suspected. Positive inhibitor testing had to be confirmed by 2 positive inhibitor tests on samples drawn at least 1 week apart. Results A total of 22 subjects were enrolled in the study. Of 20 subjects who met all entry criteria, 19 received treatment; of these, all had severe hemophilia A (FVIII<1%). At study entry 11 of these 19 patients were never exposed to FVIII before (PUPs), while the remaining 8 patients had been treated with FVIII concentrates before. FVIII gene mutation analysis revealed intron 22 inversions in 8 out of 17 subjects, hemizygous missense mutations resulting in a stop-codon in 2 subjects, frame-shift mutations in 2 subjects, and hemizygous missense mutations in 5 subjects. A total of 8 subjects developed a confirmed inhibitor: 2 of these 8 subjects had only borderline positivity at inhibitor testing (never above 0.6 BU) with absence of any anti-FVIII antibodies (IgG, IgA, IgM and IgG subclasses) as tested by ELISA. Thus incidence of inhibitors >0.6 BU in PUPs were 27%. A total of 67 major protocol deviations (PD) were reported in 15 patients: 44 PDs of these were reported in 10 subjects and were related to the treatment regimen and therefore have contrasted with the protocol intention, which was to minimize immunological danger signals and low dose prophylactic regimen. As a result of the observed inhibitor incidence the study was terminated based on futility analysis, i.e. the probability to achieve the primary end-point of inhibitor rate reduction to ≤15%. Details on inhibitor patients will be presented. Discussion The EPIC study showed no safety issue as confirmed by the Data Safety Monitoring Board. To align treatment decisions in the presence of danger signals (which are not completely avoidable in children around 1 year of age) with a demanding study protocol was found to be challenging. Thus the hypothesis that an early low dose prophylaxis in the absence of immunological danger signals might reduce inhibitor incidence in PUPs with hemophilia A could neither be verified nor disproved within this study. Disclosures: Auerswald: Novo Nordisk: Consultancy, Research Funding; Biotest: Consultancy, Research Funding; CSL-Behring: Consultancy, Research Funding; Bayer: Consultancy, Research Funding; Baxter: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding. Kurnik:Baxter: Consultancy, Research Funding; Bayer: Consultancy, Research Funding; Biotest: Consultancy, Research Funding; CSL-Behring: Consultancy, Research Funding; Novo Nordisk: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding. Blatny:Baxter: speaker fee Other. Reininger:Baxter Innovations GmbH: Employment.

scholarly journals Surgical Experience from the Phase III STASEY Trial of Emicizumab Prophylaxis in Persons with Hemophilia A with FVIII Inhibitors: Final Analysis

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 344-344
Author(s):  
Giancarlo Castaman ◽  
Jerzy Windyga ◽  
Hazza Alzahrani ◽  
Susan Robson ◽  
Fabian Sanabria ◽  
...  
Keyword(s):  
Research Funding ◽  
Hemophilia A ◽  
Knee Prosthesis ◽  
Coagulation Factor ◽  
Left Knee ◽  
Phase Iii ◽  
Postoperative Treatment ◽  
Research Centre ◽  
Case Report Form ◽  
Knee Arthrodesis

Abstract Background: Emicizumab, a bispecific monoclonal antibody, bridges activated factor (F) IX and FX, replacing the function of missing activated FVIII in persons with hemophilia A (PwHA). The Phase IIIb, multicenter, single-arm STASEY study (NCT03191799) assessed the safety and efficacy of emicizumab prophylaxis in PwHA with FVIII inhibitors. Surgical experiences in STASEY are reported here. Methods: Following informed consent and ethics committee approval, PwHA aged ≥12 years with FVIII inhibitors received 3 mg/kg/week emicizumab for 4 weeks (loading dose), then 1.5 mg/kg/week for the remaining 2-year treatment period. Minor and major surgeries were managed per the investigators' discretion. The type and number of procedures performed, additional prophylaxis, and frequency and management of postoperative bleeds were analyzed. Surgeries occurring up to 28 days after the last dose of emicizumab were included, due to emicizumab's ~28-day half-life (Emicizumab Prescribing Information, United States Food and Drug Administration, 2017). Surgeries were documented using an electronic case report form by the treating physicians and classified as minor or major based on manual medical review (Santagostino, et al. Haemophilia, 2015). Bleed and prophylactic hemophilia medication data were recorded in the electronic Bleed Medication Questionnaire by participants. Case narratives were provided by trial investigators. Results: Overall, 46 patients reported ≥1 on-study surgery. Thirty-seven patients had 56 minor surgeries (central venous access device [CVAD], n=9; dental, n=20; joint, n=4; other, n=23) (Figure), one of which (skin laceration and suture insertion on Day 9) was performed during the loading phase. Twenty-four surgeries (42.9%) were managed with additional prophylactic medications (Table). Of these, 11/24 (45.8%) resulted in postoperative bleeds, of which 6/11 were treated (54.5%). Of surgeries managed without additional prophylactic medications, 15/32 (46.9%) resulted in postoperative bleeds, of which 5/15 (33.3%) were treated. A total of 13 patients had 22 major on-study surgeries (arthroplasty, n=13; other, n=9). 'Other' included hemorrhoid operations, coronarography, sigmoidectomy, colostomy, laparotomy and polypectomy. Eighteen (81.8%) major surgeries, including all arthroplasties, were managed with additional prophylactic medications (Table). Of these, 12/18 (66.7%) resulted in postoperative bleeds (including 10/13 arthroplasties), of which six (50.0%) were treated (all arthroplasties). Four (18.2%) major surgeries were managed without additional prophylactic medication, including three hemorrhoid operations in one patient, and a coronarography in a patient with acute myocardial infarction. One hemorrhoid operation resulted in a postoperative treated bleed. Major surgeries included a 55-year-old male with Grade 4 device dislocation of left knee prosthesis on Day 7, who was diagnosed with recurrent infection and prosthesis misalignment on Day 62. Amputation of the left leg above the knee was performed, with treatment including tranexamic acid and rFVIIa. A 61-year-old male with left knee prosthesis infection underwent left knee arthrodesis on Day 457, vacuum-assisted closure therapy on Day 495, skin grafting on Day 512, and left knee arthrodesis with skin flap placement on Day 527. Throughout these surgeries, the individual experienced recurrent joint bleeding and received rFVIIa. Neither of these individuals had a change in their study treatment due to these events. No thrombotic events (TEs) or thrombotic microangiopathies (TMAs) related to surgeries were observed. Conclusions: In the STASEY study of PwHA with FVIII inhibitors receiving emicizumab prophylaxis, most minor surgical procedures were performed without additional prophylactic coagulation factor and did not result in postoperative treated bleeds. Therefore, emicizumab alone provided adequate hemostatic coverage for some PwHA undergoing certain types of minor surgery, such as tooth extraction and CVAD removal. Major surgeries were safely performed with additional coagulation prophylaxis. Management of surgeries with rFVIIa did not result in TE or TMA. In case of bleeds, a bleed management plan should be in place. Effects of emicizumab on coagulation and assays may persist for up to 6 months after the last dose, which may be relevant when planning postoperative treatment. Figure 1 Figure 1. Disclosures Castaman: Uniqure: Honoraria; Bayer: Honoraria; Sobi: Honoraria; CSL Behring: Honoraria; Novo Nordisk: Honoraria; Kedrion: Honoraria; LFB: Honoraria; Grifols: Honoraria; Werfen: Honoraria; Biomarin: Honoraria; Sanofi: Honoraria; F Hoffmann-La Roche Ltd: Honoraria. Windyga: Swixx BioPharma: Honoraria; Octapharma: Honoraria, Research Funding; Sobi: Honoraria, Research Funding; Sanofi-Aventis: Honoraria, Research Funding; Werfen: Honoraria; Bayer AG: Honoraria; Aspen: Honoraria; Alfasigma: Honoraria; Takeda: Honoraria, Research Funding; Shire: Honoraria, Research Funding; Alnylam Pharmaceuticals: Research Funding; Sanofi/Genzyme: Honoraria, Research Funding; F. Hoffmann-La Roche Ltd: Honoraria, Research Funding; Alexion: Honoraria; CSL Behring: Honoraria; Rigel Pharmaceuticals: Research Funding; Novo Nordisk: Honoraria, Research Funding; Baxalta: Honoraria, Research Funding. Alzahrani: Sobi: Consultancy, Honoraria, Speakers Bureau; Pfizer: Consultancy, Honoraria, Speakers Bureau; Bayer: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria; King Faisal Specialist Hospital and Research Centre: Current Employment. Robson: F. Hoffmann-La Roche Ltd: Current Employment, Ended employment in the past 24 months. Sanabria: F. Hoffmann-La Roche Ltd: Current Employment, Current holder of individual stocks in a privately-held company. Howard: F. Hoffmann-La Roche Ltd: Current Employment, Current equity holder in publicly-traded company. Jiménez-Yuste: Octapharma: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding; Sobi: Consultancy, Honoraria, Research Funding; BioMarin: Consultancy; CSL Behring: Consultancy, Honoraria, Research Funding; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; NovoNordisk: Consultancy, Honoraria, Research Funding; Grifols: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria, Research Funding.

scholarly journals Emizicumab Promotes Factor Xa Generation on Activated Endothelium in a Blood Cell-Independent Manner

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3182-3182
Author(s):  
Patrick Ellsworth ◽  
Dougald Monroe ◽  
Maureane Hoffman ◽  
Nigel S Key
Keyword(s):  
Clinical Trial ◽  
Endothelial Cells ◽  
Growth Medium ◽  
Research Funding ◽  
Hemophilia A ◽  
Coagulation Factor ◽  
Standard Of Care ◽  
Factor Ix ◽  
Adhesive Film ◽  
Trial Investigator

Abstract Introduction Hemophilia A (HA) is an inherited bleeding disorder caused by the deficiency of coagulation factor VIII (FVIII) resulting in severe hemorrhage if untreated. Recombinant and plasma derived FVIII products have long been the standard of care in hemophilia. However, approximately 25-30% of patients with severe HA develop inhibitors, neutralizing alloantibodies to FVIII, a significant complication in the treatment of patients with HA that leads to bleeding despite factor therapy. First approved for bleed prophylaxis in HA with inhibitors in the US by the FDA in 2018, emicizumab (Genentech, USA) has initiated a new era of HA treatment. This drug is a bispecific, monoclonal antibody that binds to activated Factor IX (FIXa) and Factor X (FX), mimicking activated FVIII (FVIIIa) by bringing FIXa and FX into proximity to enable FX activation, even in the presence of inhibitors. Emicizumab prophylaxis drastically reduces bleed episodes. However, thromboses and thrombotic microangiopathy (TMA) were observed in trials, all associated with concomitant use of activated prothrombin complex concentrates (aPCC) (Callaghan et al., 2021). The mechanism of this devastating condition is uncertain, as emicizumab is not known to bind to phospholipid or vascular surfaces. We report that FX is more readily activated by FIXa and emicizumab on endothelium that has been activated by tumor necrosis factor alpha (TNF). This finding may partially explain the development of TMA in these patients. Methods We utilized novel, microfluidic devices that are inexpensive to manufacture and were modified from a technique previously described (Alapan et al. 2016). These devices are fabricated using a laser cut double-sided adhesive film sandwiched between a clear, gas-permeable polymer (Ibidi, Germany) and an acrylic top that is laser cut (Universal Laser Systems Inc., USA) (Figure 1). Human umbilical endothelial cells (HUVEC, Lonza, Switzerland) were harvested at passage 3 to 4 and seeded into the devices. These were then cultured under flow conditions using a non-peristaltic, air-driven pump (Ibidi GmbH, Germany) to achieve a confluent and quiescent endothelial surface. HUVEC are then activated by incubating with 5 nM TNF in serum-free growth medium for 4 hours. This treatment induced markers of endothelial activation without gross apoptosis. Non-activated HUVEC were incubated with endothelial cell growth medium (2% serum) until time of experiments. Factors IXa, X (Haemtech, USA), and/or emicizumab (discarded clinical material) were mixed in HEPES-buffered saline with 5 mM calcium chloride for all experimental conditions. Concentrations used of FIXa (30 nM), FX (170 nM), and emicizumab (55 ug/mL) were constant for all conditions. Combinations of factors and emicizumab were then incubated in the endothelialized device for 30 minutes at 37° C. The entire volume of the mixture was then aspirated (20 uL) and stored at -80° C. FXa activity was assayed on the effluent for 60 minutes using a chromogenic FXa substrate (Pefachrome, Pentapharm, Switzerland). Results No significant generation of Xa was noted in the presence of healthy or activated endothelium with emicuzumab alone, emicizumab and FIXa, emicizumab and FX, or factors IXa and X. Median Xa generation observed with the combination of emicizumab, FIXa, and FX on healthy endothelium was 2 nM. Median Xa generation with the same combination on activated endothelium was 8.1 nM, a four-fold increase (P = 0.028, Mann-Whitney test) (Figure 2). Discussion Emicizumab represents an evolving standard of care for hemophilia A. Considering data showing diminishing FVIII expression in the months to years after AAV gene therapy, (Pasi et al., 2020) it may well be the dominant treatment paradigm in HA for some time. However, much remains to be answered in the use of emicizumab, and the mechanism of thrombosis and TMA with concomitant aPCC use has resulted in the avoidance of aPCC use for breakthrough bleeding in patients on emicizumab therapy, even up to 6 months after cessation. Our data demonstrate that activated endothelial cells promote FX activation more readily than quiescent endothelial cells in the presence of FIXa and emicizumab. These findings demonstrate the potential of thrombotic angiopathy in an in vitro system. Further investigation of the interaction of endothelium with FIXa, FX, and FVIIIa-mimetic antibodies is warranted. Figure 1 Figure 1. Disclosures Ellsworth: Takeda: Other: Salary supported as part of NHF-Takeda Clinical Fellowship Award. Monroe: Medexus Pharmaceuticals: Consultancy; Takeda: Consultancy; Otello Medical: Current equity holder in publicly-traded company. Hoffman: Takeda: Research Funding; CSL Behring: Consultancy; Sanofi: Consultancy; BPL (Bio Products Laboratory): Consultancy. Key: BioMarin: Honoraria, Other: Participation as a clinical trial investigator; Takeda: Research Funding; Grifols: Research Funding; Uniqure: Consultancy, Other: Participation as a clinical trial investigator; Sanofi: Consultancy.

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