Recombinant Porcine Factor VIII Research Articles

Acquired hemophilia A as a disease of the elderly: A comprehensive review of epidemiology, pathogenesis, and novel therapy

AbstractAcquired hemophilia A (AHA) is a rare autoimmune bleeding disorder characterized by the development of neutralizing autoantibodies (inhibitors) against coagulation factor VIII (FVIII). This review provides an in-depth exploration of AHA, covering its epidemiology, pathogenesis, clinical presentation, diagnosis, complications, and treatment strategies, focusing on recent advancements. AHA can manifest in both men and women with no prior bleeding history. The annual incidence is estimated to be approximately 1 case per million individuals in the general population. The incidence increases significantly with age: the incidence among individuals aged 60 years or older is approximately 3 to 4 cases per million individuals per year. Typically, patients present with an acquired bleeding disorder that is characterized by an isolated, prolonged activated partial thromboplastin time stemming from FVIII deficiency. Diagnosis relies on the detection of neutralizing antibodies using the Nijmegen-modified Bethesda assay. Hemostatic control strategies involve bypassing agents like recombinant activated factor VII, activated prothrombin complex concentrate, and recombinant porcine FVIII for bleeding patients. Emicizumab, a novel bypassing agent, exhibits several potential advantages. In the realm of immunosuppressive treatment for inhibitor eradication, the CyDRi regimen emerged as a remarkable advancement, significantly enhancing the outlook for the management of AHA even in the elderly frail population.

Acquired Hemophilia A: Bleeding Pattern and Hemostatic Therapeutic Strategies.

Acquired Hemophilia A (AHA) is a rare autoimmune disorder characterized by the onset of a sudden and unexpected bleeding episode in a patient with no personal or family history of bleeding diathesis, and with a typical laboratory feature, i.e., a prolonged activated partial thromboplastin time that is not otherwise explained. This bleeding disorder is caused by autoantibodies directed against the coagulation factor VIII (FVIII). AHA is idiopathic in 50% of cases and is secondary to well-defined diseases in the remaining 50%. AHA affects elderly patients although it has also been observed in the post-partum period. Bleeding manifestations are heterogeneous, ranging from mild to life-threatening bleeds involving limbs and organs. Severe bleeding with a significant decrease in hemoglobin levels must be promptly and adequately treated in order to avoid a worsening of the hemorrhages and their complications. According to international recommendations, the bypass agents (i.e., activated prothrombin complex concentrate and activated recombinant factor VII) and the replacement therapy with recombinant porcine FVIII are considered as the first-line therapy for bleeding control, due to their proven clinical efficacy. Plasma-derived or recombinant FVIII concentrates could be used as second-line treatments. Emicizumab may represent a valid and interesting therapeutic option for prophylaxis of bleeding recurrences.

Emicizumab in acquired hemophilia A: pros and cons of a new approach to the prevention and treatment of bleeding.

Emicizumab, a monoclonal bispecific antibody that mimics the function of activated factor VIII (FVIII), is currently licensed for prophylactic use in patients with congenital hemophilia A with and without inhibitors. Acquired hemophilia A (AHA) is a very rare bleeding disorder caused by the development of autoantibodies that inhibit FVIII activity in plasma; males and females are equally affected. Therapeutic options for patients with AHA currently include eradication of the inhibitor with immunosuppressive treatments and management of acute bleeding with bypassing agents or recombinant porcine FVIII. More recently, several reports described the off-label use of emicizumab in patients with AHA and a phase III study is ongoing in Japan. The aims of this review are to describe the 73 reported cases, and to highlight the advantages and disadvantages of this novel approach to the prevention and treatment of bleeding in AHA.

Utilization of emicizumab in acquired hemophilia A: A case report

BackgroundAcquired Hemophilia A (AHA) is a rare autoimmune disorder associated with the development of autoantibodies against factor VIII (FVIII). Although obtaining hemostatic control through the use of recombinant factor VIIa, activated prothrombin complex concentrate and recombinant porcine FVIII are cornerstones in the clinical management of AHA, these therapies have several disadvantages, including a higher risk for the development of thromboembolic events, unpredictable efficacy and short half-lives. While emicizumab has been FDA licensed for use in bleeding prophylaxis for patients with Congenital Hemophilia A (CHA) with and without inhibitors, it has not been approved for use in AHA, with only a few reports describing its use in this context. Case reportWe report our experience with the use of emicizumab in an 83-year old male with AHA, complicated by the onset of atrial fibrillation following admission, drug-induced thrombocytopenia, infectious complications, and the identification of a low-grade lymphoproliferative disorder, in which emicizumab prophylaxis was used for bleeding prophylaxis in the context of persistently elevated inhibitor titers without evidence of thrombotic events or thrombotic microangiopathy.

Rituximab Monotherapy Is Effective for Inhibitor Eradication with Concomitant Porcine Factor VIII Followed By Emicizumab for Bleed Control in Acquired Hemophilia a

▪IntroductionAcquired hemophilia A (AHA) is a rare bleeding disorder in which acquired auto-antibodies to endogenous Factor VIII (FVIII) resulting in decreased FVIII activity. AHA can lead to life-threatening bleeding, with effective treatment requiring both immunosuppressive therapy (IST) and bypassing agents such as recombinant activated Factor VII (rFVIIa) or activated prothrombin complex concentrates (APCC) (Tiede et al. Haematologica 2020). Some, including our group, have begun using emicizumab as well (Knoebl et al. Blood 2020). IST is required for inhibitor eradication, but regimens are heterogenous and have not been systematically compared in the literature. While there is no standard of care IST in these patients, most patients in the literature receive multiple agents, including corticosteroids, mycophenolate mofetil, cyclosporine, and/or rituximab in combination.We report in a prospective cohort that for IST, rituximab monotherapy is an effective strategy. An updated treatment algorithm is offered that has been effective for treatment of these patients at our institution, which adds emicizumab therapy after initial bleed control.MethodsWe analyzed clinical, pharmacy, and laboratory data from 24 patients treated with rpFVIII at the University of North Carolina for AHA from July 2015 to June 2021. All patients were initially treated according to our previously established dosing algorithm with recombinant porcine FVIII, and the last five patients have received emicizumab after initial factor dosing (see Figure 1). 17 of the patients who received rituximab and were followed at our center subsequently attained inhibitor eradication, six of those received only rituximab therapy. Investigational review board approval was obtained for our data collection and analysis.Patients who did not receive rituximab, failed to reach an inhibitor level <0.5 BU, or who were lost to follow up were excluded from the analysis. For patients that fit the inclusion criteria, the time between date of the first rituximab infusion and the date of inhibitor eradication was calculated.ResultsAll patients in our cohort who we followed until inhibitor eradication (17 of 24 patients) had eradication of inhibitors after a median of 143 days from initiation of immunosuppression. For patients treated with rituximab monotherapy for inhibitor eradication (6 of 17), this goal was reached in a median of 134.5 days (range 76-191 days). For those who received agents in addition to rituximab and have reached inhibitor eradication to date (9 of 17 patients), median days from initiation of immunosuppression to inhibitor eradication was 137.5 days (range 11-485) (P = 0.43 on Mann-Whitney test).Patients were treated as previously reported by our group per an algorithm that starts recombinant porcine FVIII without waiting for a porcine inhibitor and at lower than FDA recommended dosing. Subsequent doses for bleed control are titrated according to one-stage, clot based FVIII activity. This report also includes 5 new patients who, after initial bleed control per our algorithm, were initiated on emicizumab while awaiting inhibitor eradication.There was no correlation between time to rituximab initiation and time to inhibitor eradication in both those who received rituximab monotherapy and those who had multiple IST agents. There was also no significant difference in initial inhibitor titer between groups with median initial inhibitor titer of 104 BU in the rituximab monotherapy group, and 70 BU in the multiple IST agents group (see Figure 3).ConclusionsRituximab monotherapy appears to be an effective strategy for inhibitor eradication in acquired hemophilia A. In the context of bleed treatment with porcine factor, followed by emicizumab, a standardized, algorithmic approach can be effectively employed for these patients. Though any patients have inhibitor recurrence, as is described in the literature, with emicizumab available, bleeding can be avoided with regular monitoring. Emicizumab given while re-eradicating an inhibitor can prevent morbidity of this disease. [Display omitted] DisclosuresEllsworth: Takeda: Other: Salary supported as part of NHF-Takeda Clinical Fellowship Award. Key: Uniqure: Consultancy, Other: Participation as a clinical trial investigator; Grifols: Research Funding; Takeda: Research Funding; BioMarin: Honoraria, Other: Participation as a clinical trial investigator; Sanofi: Consultancy. Ma: Accordant: Consultancy; Takeda: Honoraria, Research Funding. OffLabel Disclosure:Emicizumab is not approved for use in Acquired Hemophilia A and this represents an OFF LABEL use of the drug.

Recombinant porcine FVIII for bleed treatment in acquired hemophilia A: findings from a single-center, 18-patient cohort.

Acquired hemophilia A (AHA) is a rare bleeding disorder in which acquired autoantibodies to endogenous factor VIII (FVIII) decrease FVIII activity and lead to a bleeding phenotype. A substantial majority of individuals who develop AHA present with severe bleeding. Effective treatment requires both immunosuppressive therapy and prompt hemostatic treatment. Bleeding is commonly treated with bypassing agents (BPAs) such as recombinant activated FVII (rFVIIa) or activated prothrombin complex concentrates Disadvantages to BPAs include the inability to monitor response with standard laboratory assays, inconsistent hemostatic efficacy, and thrombosis. Recombinant porcine FVIII (rpFVIII: Obizur, Baxter, Deerfield, IL) was approved by the US Food and Drug Administration (FDA) for bleed treatment in AHA in 2014, and has the advantage of laboratory monitoring of FVIII activity levels and known hemostatic efficacy in the presence of anti-human FVIII inhibitors and after failure of BPAs. Using an algorithm-based approach, rpFVIII has been used to successfully treat 18 patients with AHA at our center with substantially lower doses than the current FDA-recommended dosing. Additionally, data from our cohort show that the preexposure anti-porcine Bethesda titer does not reliably predict the clinical response to rpFVIII treatment and is not correlated with the anti-human Bethesda titer. We also present data showing lower total rpFVIII use for initial bleed resolution when rpVIII is used upfront, as compared with use as rescue therapy. We validated our dosing algorithm, which uses much lower than FDA-recommended doses with 14 more patients than in our previously reported patient series.

Experience with Continuous Infusion of Recombinant Porcine FVIII in Patients with Acquired Hemophilia a

Background: Acquired hemophilia A (AHA) is a rare disorder with high morbidity and mortality that results from the development of autoantibodies against factor VIII (FVIII). To manage acute bleeding, recombinant porcine FVIII (rpFVIII) can be administered intravenously as a bolus dose of 50IU/kg to 200IU/kg depending on bleeding severity and the level of baseline cross-reacting anti-porcine FVIII inhibitors(Kruse-Jarres et al., 2017). Here we describe the efficacy of rpFVIII used as a continuous infusion (CI) and compare total product utilization to bolus infusions (BI). Methods: Retrospective chart review was conducted on patients with AHA who met the International Society on Thrombosis and Hemostasis criteria for major bleeding and who received rpFVIII at our institution from 2015 to 2020(Kaatz, Ahmad, Spyropoulos, &amp; Schulman, 2015). Efficacy was defined as clinical bleeding control and achievement of adequate FVIII levels. Data were collected through electronic patient records and analyzed using simple descriptive (mean ± standard deviation (SD)) and inferential statistics (T-Test, alpha=0.05). Institutional Research Ethics Board approval was obtained. Results: During the study period, 13 patients received rpFVIII (CI n=3, BI n=10). The mean age of patients receiving CI and BI was 75.3 years (SD 2.5) and 72.0 years (SD 14.5) respectively. All patients who received rpFVIII as a CI received a bolus dose ranging between 102.6IU/kg-200.8IU/kg prior to initiation of the CI. CI rates between 3.4IU/kg/hr-11.5IU/kg/hr were administered. Two of the patients receiving rpFVIII as a CI had their infusion rates adjusted according to their clinical symptoms and FVIII levels. One patient had the rpFVIII CI stopped due to an increase in anti-rpFVIII antibodies which interfered with hemostatic efficacy. Thirty-three percent of CI patients and 60% of BI patients required a red blood cell (RBC) transfusion after starting rpFVIII. Sixty-six percent of CI patients and 20% of BI patients had worsening of bleeding after initiation of rpFVIII. Lastly, rpFVIII usage in the CI group (170.4 ± 25.9 IU/kg/day) was not significantly different compared to the BI group (120.9 ± 64.4 IU/kg/day) when accounting for the duration of admission and weight of the patients (P&amp;gt;0.05). No thromboembolic events occurred in either group while receiving rpFVIII. Conclusions: Our study shows that the total amount of rpFVIII administered to patients as a CI is not significantly different to those receiving BI. The CI group required less RBC transfusions but reported more exacerbation of bleeding. Thus, the efficacy of rpFVIII given as a CI requires further evaluation in future prospective studies. Disclosures Sholzberg: NovoNordisk: Honoraria, Other: Scientific Advisory Board; Novartis: Honoraria, Other: Scientific Advisory Board; Takeda: Honoraria, Other: Scientific Advisory Board, Research Funding; Octapharma: Honoraria, Other: Scientific Advisory Board, Research Funding; Amgen: Honoraria, Other: Scientific Advisory Board, Research Funding. OffLabel Disclosure: Antihemophilic Factor (Recombinant), Porcine Sequence (OBIZUR) is a purified protein produced by recombinant DNA that is a B-domain deleted recombinant factor VIII, porcine sequence, manufactured in tissue culture in baby hamster kidney (BHK) cells. OBIZUR is indicated for the treatment of bleeding episodes in patients with Acquired Hemophilia A (AHA). OBIZUR is administered as a bolus infusion.

Critical Bleeding in Acquired Hemophilia A: Bypassing Agents or Recombinant Porcine Factor VIII?

Acquired hemophilia A (AHA) is caused by autoantibodies neutralizing coagulation factor VIII (FVIII). In the presence of inhibitors against FVIII, acute bleeds can be managed with bypassing agents, including recombinant factor VIIa (eptacog alfa activated, NovoSeven) and activated prothrombin complex concentrate (FEIBA), as well as recombinant porcine FVIII (susoctocog alfa, Obizur). Studies comparing these agents directly are not available, and indirect evidence suggests an overall similar efficacy. Selecting an agent in clinical practice therefore depends on (1) availability of agent, (2) safety profile, (3) monitoring requirements, (4) cost, and (5) personal experience. This review examines available data and collects additional considerations to support decision making for bleeding emergencies in AHA.

International recommendations on the diagnosis and treatment of acquired hemophilia A.

Acquired hemophilia A (AHA), a rare bleeding disorder caused by neutralizing autoantibodies against coagulation factor VIII (FVIII), occurs in both men and women without a previous history of bleeding. Patients typically present with an isolated prolonged activated partial thromboplastin time due to FVIII deficiency. Neutralizing antibodies (inhibitors) are detected using the Nijmegen-modified Bethesda assay. Approximately 10% of patients do not present with bleeding and, therefore, a prolonged activated partial thromboplastin time should never be ignored prior to invasive procedures. Control of acute bleeding and prevention of injuries that may provoke bleeding are top priorities in patients with AHA. We recommend treatment with bypassing agents, including recombinant activated factor VII, activated prothrombin complex concentrate, or recombinant porcine FVIII in bleeding patients. Autoantibody eradication can be achieved with immunosuppressive therapy, including corticosteroids, cyclophosphamide and rituximab, or combinations thereof. The median time to remission is 5 weeks, with considerable interindividual variation. FVIII activity at presentation, inhibitor titer and autoantibody isotype are prognostic markers for remission and survival. Comparative clinical studies to support treatment recommendations for AHA do not exist; therefore, we provide practical consensus guidance based on recent registry findings and the authors’ clinical experience in treating patients with AHA.

Recombinant Porcine FVIII in Acquired Hemophilia: Decreased Product Usage When Used Upfront, Non-Utility of Baseline Porcine Bethesda Titer, and Confirmation of Dosing Algorithm

IntroductionAcquired hemophilia A (AHA) is a rare bleeding disorder in which acquired autoantibodies to endogenous Factor VIII (FVIII) decrease FVIII activity. AHA can lead to serious bleeding, with effective treatment requiring both immunosuppressive therapy (IST) and bypassing agents such as recombinant activated Factor VII (rFVIIa) or activated prothrombin complex concentrates (APCC). Disadvantages to bypassing agents include the inability to monitor response with standard lab assays, inconsistent hemostatic efficacy, and thrombosis (Astermark et al. 2007).Recombinant porcine FVIII (rpFVIII) ), by virtue of its incomplete homology in the A2 and C2 domains of the FVIII molecule, has decreased reactivity with anti-human FVIII (hFVIII) inhibitors (Mulliez et al. 2014), enabling its use in AHA. It also has the advantage of laboratory monitoring of FVIII activity levels and hemostatic efficacy after failure of bypassing agents (Trautmann-Grill et al. 2018).Between July 2015 and April 2018, 14 patients with AHA were treated with rpFVIII at the University of North Carolina. Data from our cohort show that the initial anti-porcine Bethesda titer (pBIA) does not reliably predict the clinical response to rpFVIII treatment and is not correlated with the anti-human Bethesda titer (hBIA). We also present data showing lower total rpFVIII use for initial bleed resolution when rpVIII is used up front, as compared to use as rescue therapy. Lastly, we validated our dosing algorithm which uses much lower than FDA-recommended doses with 10 more patients than in our previously reported patient series (Martin et al. 2016).MethodsWe analyzed clinical, pharmacy, and laboratory data from 14 patients treated with rpFVIII at the University of North Carolina for AHA from July 2015 to April 2018. All patients were initially treated according to our previously established dosing algorithm (Fig. 3). Investigational review board approval was obtained for our data collection and analysis.ResultsOf our 14 patients, 11 had a pBIA drawn prior to receiving rpFVIII, with 9/11 having a detectable porcine inhibitor, and only 2 of these showing no clinical response to rpFVIII infusion. There was no correlation between initial pBIA and FVIII response to rpFVIII infusion (Figs 1a-c). Only 2 patients lost their FVIII response and needed subsequent bypass agent use. One had a peak pBIA of 212 BU, and the other had a pBIA of 7, though 3 other patients who developed high titer pBIA did not lose their response. pBIA and hBIA showed poor correlation (r = 0.4, P = 0.2). 2/11 patients with available values had pBIA of <0.4, but had high titer hBIA.13/14 patients achieved excellent resolution of their presenting bleed at much lower than FDA-recommended doses (see Fig 1c and Table 1). In 12/14 patients, the inhibitor resolved after IST. rpFVIII was given as up front therapy in 6/14 patients (i.e., preceded by < 24 hours of bypassing agent), and they required less cumulative rpFVIII for resolution of the presenting bleed compared to the 8 patients given rpFVIII as rescue therapy (up front mean dose = 400 U/kg, rescue mean dose = 663 U/kg, P=0.06, see Fig 2).ConclusionOur data validate our previously established dosing algorithm, providing hemostatic efficacy at lower than FDA-approved doses, and allowing for rapid and standardized dosing adjustments based on laboratory and clinical response without delaying initial treatment. As the initial pBIA does not reliably predict response to rpFVIII, and is not correlated with the hBIA, our practice has been to initiate rpFVIII therapy without waiting for results of the pBIA. The pBIA can be useful in case of repeat bleeding events, though not in a predictable manner. Our data also suggest lower dose requirements when used up front, rather than as rescue therapy, which can also allow pharmacy cost savings. [Display omitted] DisclosuresKey:UniQure BV: Research Funding. Ma:CSL: Consultancy; Shire: Honoraria, Research Funding; Novo Nordisk: Consultancy; CVS: Honoraria.

Alternative therapies for the management of inhibitors.

The development of inhibitors to factor VIII (FVIII) or factor IX (FIX) remains a major treatment complication encountered in the treatment of haemophilia. Not all patients with even the same severity and genotype develop inhibitors suggesting an underlying mechanism of tolerance against FVIII- or FIX-related immunity. One mechanism may be central tolerance observed in patients in whom the FVIII mutation enables some production of the protein. The other is a peripheral tolerance mechanism which may be evident in patients with null mutation. Recently, recombinant porcine FVIII (rpFVIII, Obixur, OBI-1, BAX801) has been developed for the haemostatic treatment of both congenital haemophilia with inhibitor (CHAWI) and acquired haemophilia A (AHA). In 28 subjects with AHA with life-/limb-threatening bleeding, rpFVIII reduced or stopped bleeding in all patients within 24h. The cross-reactivity of anti-human FVIII antibodies to rpFVIII remains around 30-50%. Recently, new therapeutics based on the quite novel concepts have been developed and clinical studies are ongoing. These are humanized asymmetric antibody mimicking FVIIIa function by maintaining a suitable interaction between FIXa and FX (Emicizumab, ACE910), and small interfering RNAs (siRNA, ALN-AT3) suppress liver production of AT through post-transcriptional gene silencing and a humanized anti-TFPI monoclonal antibody (Concizumab). Their main advantages are longer half-life, subcutaneous applicability and efficacy irrespective of the presence of inhibitors which will make it easier to initiate more effective treatment especially early childhood.

Recombinant Porcine Factor VIII, Obi-1, Successfully Controlled Gastrointestinal Bleeding in a Patient with Acquired Hemophilia A

▪Introduction: Acquired hemophilia A (AHA) is a rare, life-threatening bleeding diathesis categorized by low levels of Factor VIII (FVIII) due to an acquired auto-antibody against FVIII. Treatment for AHA requires both control of the bleeding and eradication of the inhibitor. Recombinant activated factor VII (rFVIIa) maybe used to control the bleeding, but with only mixed results in controlling gastrointestinal bleeding (GIB). rFVIIa is associated with thrombosis risk and lack of ability to monitor. The recombinant porcine FVIII (rpFVIII), OBI-1, recently obtained regulatory approval based on the results of Phase 2/3 trial, but the patients in this cohort did not have GIBs. Here we present a patient with AHA admitted with a GIB and successfully treated with OBI-1.Case: A 69 year old African American female with T1N0M0 infiltrative ductal carcinoma of the left breast and Stage IVA Squamous cell carcinoma of the cervix, treated in 1997 and 2007 respectively, presented to the emergency department with two days of painless bright red blood per rectum. She was orthostatic (supine: HR 79, BP 112/77 and standing: HR 111, BP 100/72) with hemoglobin (Hb) of 5.7gms/dl. An EGD demonstrated a bleeding vessel and multiple arteriovenous malformations (AVM) in the gastric body that were secured with clipping. Over the next 5 days EGD was repeated twice and multiple endoclips were placed in an attempt to achieve hemostasis. She subsequently underwent angiography to identify a target vessel for embolization, but one could not be found. On hospital day 11, hematology was consulted for a prolonged aPTT (113.8 seconds) and inadequate correction with mixing studies (63.7 seconds). FVIII level was <1% with factor VIII inhibitor titer of 245 Bethesda Units/ml (BU). Prior to starting OBI-1, she received 9 units of packed red blood cells and 2 units of fresh frozen plasma. OBI-1 was started at 200u/kg q4h for first 2 days and then decreased to q8h, with cessation of bleeding within 24 hours. Upon completion of the bolus of OBI-1, her FVIII level was 14%, then 38% at 8 hours, 48% at 16 hours and 62% at 24 hours. Rituximab (375mg/m2 weekly) and corticosteroids (prednisone 1mg/kg daily x7 days) were given in an attempt to eliminate the inhibitor. She remained stable for 6 days but subsequently developed abdominal pain. A CT of the abdomen revealed free air prompting emergent surgical intervention. She had internal hernia in the pelvis, ischemic small bowel and perforated jejunum requiring extensive resection. During this time she remained on rpFVIII (200u/kg q8h) with FVIII pre-infusion levels ranging from 2% - 14% and post-infusion levels from 80% to 217% and had only 25ml estimated blood loss during surgery. Unfortunately, 1 day after surgery she developed multi-organ failure and expired on hospital day 19.Discussion: OBI-1 is a glycosylated, B-domain deleted recombinant porcine factor VIII that retains the ability to interact with Factor IXa and activate Factor X, but it is less inhibited by auto-antibodies to human FVIII. For FVIII inhibitor titers >5 BU, an inhibitor bypassing agent is usually necessary because human FVIII products are not able to overcome the inhibitor. Given the high FVIII inhibitor titers in this patient, administration of rFVIIa or OBI-1 was necessary. Our own institutional experience of treating GIBs in patients with AHA had poor outcomes. The efficacy of rFVIIa in treating GIB is inconclusive, with response rate ranging from 53% to 71%. However, rFVIIa is associated with a risk of thrombosis approaching 9.4%, especially in older patients and those with vasculopathy, a common demographic in AHA. Unlike rFVIIa, OBI-1 allows close monitoring of FVIII and in the aforementioned Phase 2/3 trial, there were no thrombotic complications or serious adverse events. We were successful in controlling the bleeding within 24 hours with OBI-1 and were able to achieve therapeutic levels of FVIII despite high Bethesda titers. Unfortunately, our patient died from an ischemic bowel and perforated jejunum that was likely related to hernia or repeated instrumentation in an attempt to stop the bleeding prior to the diagnosis of AHA.Conclusion: OBI-1 appears to effectively and rapidly overcome a high titer FVIII inhibitor and control bleeding in a patient with AHA and GIB. While this is a promising therapy, additional study is necessary to determine safety, efficacy, and cost-effectiveness. DisclosuresNo relevant conflicts of interest to declare.

Bioengineering Coagulation Factor VIII through Ancestral Protein Reconstruction

The development of transformative hemophilia A therapeutics has been hindered by the size, instability, immunogenicity and biosynthetic inefficiency of coagulation factor VIII (FVIII). Through the study of FVIII orthologs from existing vertebrate species, we discovered unique molecular, cellular and biochemical properties that can overcome the limitations of human FVIII. This approach facilitated the development of recombinant porcine FVIII for acquired hemophilia A and has enabled low resolution mapping and bioengineering of functional sequence determinants into human FVIII. To further extend this bioengineering approach, we employed a novel methodology termed ancestral protein reconstruction that provides certain advantages over 'rational design' approaches including a priori confidence that each ancestral FVIII is hemostatically functional. First, a mammalian FVIII phylogenetic tree with corresponding ancestral node (An) sequences was constructed through Bayesian inference using both DNA and amino acid-based models in PAML Version 4.1 (Figure 1). The limited availability of non-mammalian sequences precluded accurate ancestor prediction outside of this class. Initially, we selected 14 An-FVIII sequences for reconstruction and subsequent molecular, cellular, biochemical and immunological characterization. Each An-FVIII displayed activity in coagulation assays utilizing human hemophilia A plasma as a substrate thus demonstrating evolutionary mammalian compatibility. Infusion of highly purified preparations of several An-FVIIIs into hemophilia A mice also corrected the bleeding phenotype following a tail transection bleeding challenge confirming in vivo functionality. To study biosynthetic efficiency, secreted FVIII activity and mRNA transcript levels were analyzed following transfection of An-FVIII plasmids into HEK293 and BHK-M cell lines. An-53, common ancestor to rodents and primates, and An-68, ancestor to a subset of current rodents, displayed the highest FVIII biosynthetic efficiencies that were 12 and 15 fold greater than human FVIII, respectively (P = 0.002; Mann Whitney U test). These two An-FVIII sequences share 95 and 87% amino acid identity to human FVIII, respectively. In contrast, intermediate ancestors between An-53 and human FVIII, designated An-55, -56 and -57, do not display enhanced biosynthetic efficiency suggesting that the functional sequence determinant of high expression was lost during primate evolution. Predicting that high expression ancestral FVIIIs would be enabling to gene therapy approaches, An-53, An-68 and human FVIII cDNAs were placed in an AAV expression cassette under the control of a potent liver-specific promoter and the resulting plasmid DNA was infused hydrodynamically into hemophilia A mice. An-53 and An-68, but not human FVIII vector treated animals, achieved sustained, therapeutic plasma FVIII activity levels over 4 weeks (0.1 - 0.6 IU/ml versus <0.01 IU/ml, respectively). Recombinant An-FVIIIs were expressed, purified and biochemically characterized by SDS-PAGE, specific activity, decay following thrombin activation and inhibitor recognition. Early mammalian and all primate lineage thrombin-activated An-FVIII(a) displayed half-lives between 1.5 - 2.2 min that were not distinguishable from human FVIII. We have shown previously that modern murine, porcine, and ovine FVIIIa display significantly longer half-lives and thus this property may have evolved under positive selection. Supporting this conclusion, An-68 and An-78 display prolonged half-lives of 16 and 7 min, respectively. Lastly, the immune recognition of An-FVIIIs by a panel of A2 and C2 domain targeting inhibitory murine monoclonal antibodies as well as hemophilia A inhibitor patient plasmas was examined and many examples of reduced reactivity were revealed, which may enable the development of less immunogenic FVIII products. Herein, we report molecular discoveries that enhance our understanding of FVIII structure/function and provide a blueprint for bioengineering novel FVIII molecules with enhanced properties. These studies also show 'proof of concept' for ancestral protein reconstruction as a powerful approach to studying the biochemistry, molecular biology and evolution of the vertebrate coagulation system, which should enable identification of other new hematological drug targets and candidate biotherapeutics. [Display omitted] DisclosuresSpencer:Expression Therapeutics: Equity Ownership. Doering:Expression Therapeutics: Equity Ownership; Bayer Healthcare: Consultancy, Honoraria, Research Funding.

Porcine recombinant factor VIII (Obizur; OBI-1; BAX801): product characteristics and preclinical profile.

Acquired haemophilia A (AHA) is a rare, often severe, auto-immune bleeding disorder caused by the development of inhibitory antibodies (inhibitors) to factor VIII (FVIII). Bypassing agents, recombinant activated FVII or activated prothrombin complex concentrate, are currently recommended as first-line treatments to control bleeding events in patients with AHA. A plasma-derived porcine FVIII (Hyate:C, Ipsen, UK) was used as a first-line treatment for AHA but was discontinued in 2004 due to viral safety concerns. A recombinant pFVIII (rpFVIII), Obizur (OBI-1; BAX801), which is expected to have a similar efficacy profile to Hyate:C but with a superior safety profile was developed and recently approved by the US Food and Drug Administration for the treatment of AHA. Obizur manufacturing begins with the expression of B domain deleted rpFVIII by genetically modified baby hamster kidney-derived cells. The final purified and lyophilized drug product has a negligible risk of viral contamination and contains no animal-derived plasma proteins. Obizur was evaluated for immunogenicity, tolerability, pharmacokinetics and bleeding times in preclinical models including in haemophiliac dogs, cynomolgus monkeys and FVIII-knockout mice. Preclinical animal studies show that the efficacy and immunogenicity of Obizur are similar to that of Hyate:C and that Obizur has a more favourable safety profile. Obizur is a highly purified recombinant porcine FVIII drug product that has been demonstrated to have a favourable safety and efficacy profile when compared with Hyate:C and can be a valuable treatment option for control of bleeding in AHA patients.

Acquired hemophilia A: emerging treatment options.

Acquired hemophilia A is a rare autoimmune disorder caused by an autoantibody (inhibitor) to factor VIII (FVIII) that interferes with its coagulant function and predisposes to severe, potentially life-threatening hemorrhage. Disease management focuses on controlling bleeding, primarily with the use of bypassing therapy and recombinant porcine FVIII, and permanently eradicating the autoantibody using various immunosuppressants. Treatment challenges include delayed diagnosis, difficulty achieving hemostasis and durable remissions, and complications associated with the use of hemostatic and immunosuppressive therapy in a primarily older patient population.

Recombinant Porcine Factor VIII Corrects Thrombin Generation and Improves Clot Structure In Vitro in Plasma Containing Anti-Factor VIII Inhibitory Antibodies

Abstract 2282 Introduction:Treatment of bleeding episodes in patients with hemophilia A who have developed inhibitory antibodies can be challenging. Using human factor VIII (FVIII) and, historically, porcine FVIII in patients with a low inhibitor titer are therapeutic options, and provide ease of monitoring. A B-domain deleted recombinant porcine FVIII (rpFVIII; OBI-1), which may possess low cross-reactivity to anti-human FVIII antibodies, is being investigated for the treatment of bleeding episodes in individuals with congenital hemophilia A and inhibitors, and in those with acquired hemophilia. The in vitro capacity of this molecule to correct hemostasis has been further characterized. Methods:This is an international, multicenter in vitro study. Individuals with hemophilia A and inhibitor antibodies were recruited during routine out-patient visits between January 2011 and March 2011. Written and signed informed consent was obtained prior to venepuncture. Blood was obtained from volunteers with congenital hemophilia A and inhibitors attending routine visits at participating hemophilia treatment centers. A single blood sample was obtained from consenting individuals under protocols approved by Institutional Review Boards/Ethical Committees. In vitro spiking experiments with OBI-1 were conducted using FVIII-deficient plasma with and without anti-FVIII inhibitory activity. Three control inhibitor plasmas were provided, composed of FVIII deficient plasma to which the anti-C1 monoclonal antibody (MAb) to human FVIII (Sanquin, Amsterdam, the Netherlands) was added at two concentrations to reach anti-human FVIII inhibitory activity of 4.9 Bethesda Units (BU)/mL and 32.8 BU/mL with anti-porcine anti-FVIII inhibitory activity of 2.7 BU/mL and 19.1 BU/mL, respectively; and FVIII deficient plasma to which “polyclonal” mixture of the anti-C1 MAb, along with an anti-A2 and 2 anti-C2 MAbs was added. Plasma from eight patients with hemophilia A and inhibitors was tested. Hemostatic correction by OBI-1 was assessed by thrombin generation measurement (Calibrated Analytical Thrombography assay, Synapse BV, Maastricht, The Netherlands) and clot structure using electron microscopy. Epitope mapping of the inhibitor patient plasma was undertaken at a central laboratory (Atlanta, Georgia, USA) using an Enzyme-Linked Immunosorbent Assay (ELISA) with human/porcine FVIII hybrids as the antigen. Results:The results showed a dose-dependent and anti-porcine titer dependent correction of thrombin generation parameters (peak and ETP) with OBI-1 at concentrations equivalent to 100 IU/dL, 200 IU/dL, and 400 IU/dL, which paralleled a correction of the clot structure (number and diameter of fibrin fibres). These results were only dependent on the anti-porcine titer. In samples with high titers of anti-porcine inhibitor (>10 BU), little or no restoration of the diminished thrombin generation was observed when various OBI-1 concentrations were added to the plasma. In the plasmas with high anti-human titers (≥10 BU/mL) the dominant epitope was C2 in 3 plasmas, A2 in 1 plasma, and indeterminate in 3 plasmas. The plasmas with no restoration of the thrombin generation with even the highest dose of OBI-1 all had antibody detected to more than one domain of FVIII or were not able to be mapped due to high porcine cross-reactivity. Conclusion:In vitro data obtained with spiking experiments using OBI-1 indicate that it has the potential to correct surrogate markers of hemostasis depending on the anti-porcine FVIII titer which may translate into in vivo effectiveness. Further investigation into the epitope specificity of responsive and non-responsive inhibitor plasmas correlation with effectiveness is warranted. Disclosures:Negrier:Inspiration Biopharmaceuticals: Honoraria, Research Funding. Meeks:Inspiration Biopharmaceuticals: Research Funding. Oldenburg:SOBI: Membership on an entity's Board of Directors or advisory committees; Catalyst: Membership on an entity's Board of Directors or advisory committees; Inspiration: Consultancy, Honoraria, Research Funding; LFB: Consultancy; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novo Nordisk: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Grifols: Honoraria, Research Funding; CSL Behring: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Biotest: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Baxter: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Biogen Idec: Honoraria; Octapharma: Consultancy, Honoraria, Research Funding. Bordet:Inspiration Biopharmaceuticals: Research Funding. Poetzsch:Inspiration Biopharmaceuticals: Research Funding. Al Dieri:Synapse BV: Employment. Dargaud:Inspiration Biopharmaceuticals: Research Funding. Hemker:Synapse BV: Employment. Eckmann:Sanquin Diagnostic Services: Employment. Gomperts:Inspiration Biopharmaceuticals: Employment. Lee:Inspiration Biopharmaceuticals: Employment.