Nijmegen-modified Bethesda Assay Research Articles

Pharmacokinetics, Safety and Preliminary Efficacy of Multiple Doses of Pegylated Recombinant Human Coagulation Factor VIII-Fc Fusion Protein (FRSW117) in Previously Treated Adult and Adolescent Subjects with Severe Hemophilia a: Results from an Open-Label, Multicenter, Phase 2 Study

Introduction ：Prophylaxis of severe hemophilia A (HA) with standard half-life Factor VIII (FVIII) products necessitates frequent administration, and low trough FVIII activity levels lead to higher risk of bleeds. FRSW117 is a PEGylated recombinant human coagulation factor VIII Fc fusion protein (PEGylated rhFVIII-Fc), in a phase 1 study, single-dose FRSW117 was well tolerated and provided a prominently longer t1/2 with FVIII activity maintaining at ≥ 1% for more than 7 days. This open-label, multicenter phase 2 study (NCT05265286) aimed to evaluate the PK, safety, and preliminary efficacy of multiple 4 doses of FRSW117 in previously treated subjects with severe HA. Methods ：Severe HA (FVIII:C <1%) patients aged 12 (inclusive) to 65 years, previously treated with FVIII products ≥ 150 exposure days (EDs) were enrolled in two cohorts (M1, 40IU/kg; M2, 50IU/kg), to receive 4 once-weekly doses of FRSW117 on day 1, 8, 15, and 22 (ED1 to ED4), respectively. The PK characteristics, safety, and immunogenicity of FRSW117 were evaluated. PK samples were collected pre-dose on day 1, 8, 15, 22, and at multiple times after infusion on day 1 and day 22. The primary endpoints were the PK parameters, the occurs of AEs, inhibitor development, and other immunogenicity profiles; the secondary endpoints were the bleeding episodes during multiple dosing. FVIII activity was determined using one-stage clotting assay. FVIII inhibitor was tested using the Nijmegen-modified Bethesda assay. PK analyses were performed using noncompartmental analysis (Phoenix WinNonlin 8.3). Results ：Between April 2022 and August 2022, a total of 15 male subjects were enrolled in cohort M1 (N=8) and M2 (N=7). 13/15 (86.7%) of the subjects were adults, 2 of them were adolescent, both in M1. The mean age was 28.1 (range: 13, 44) in M1 and 30.6 (range: 23, 36) in M2. 14 subjects completed the study, with 1 subject in M2 withdrew early after the PK sampling of ED1 due to personal reasons. Baseline-corrected PK parameters were summarized in Table 1. Geometric mean elimination half-life (t 1/2) of M1 and M2 were 29.536h and 31.545 h, respectively, consisting with the first-in-human single dose study. Area under the activity-time curve (AUC 0-t), and steady-state maximum concentration (C max) after 4 weekly doses for M1 and M2 were 4557.1 and 4937.9 h*IU/dL, 101.26 and 122.76 IU/dL, respectively. The mean FVIII activity at 168h after dose for M1 and M2 were summarized in Table 2, and it were 3.25 and 2.88 IU/dL on 168 h (7 day) after the dose on D22. Baseline-corrected FVIII activity over time of 2 cohorts are shown in Figure 1. The accumulation index based on AUC tau were 1.008 and 1.098, respectively, indicating there was minimal accumulation after 4 once-weekly doses. A total of 10 (66.7%,10/15) subjects reported 20 treatment emergent adverse events (TEAE) during the study. No ≥ grade 3 adverse events, serious adverse events (SAE), adverse event leading to treatment discontinuation, or AESI were reported. No positive FVIII inhibitor was detected during the study. Pre-existing Anti-PEG-rhFVIII-Fc antibodies (ADA) were observed in 4/15 (26.7%) subjects.Pre-existing anti-PEG antibodies were observed in 3/15 (20.0%) subjects. No treatment emergent ADA and anti-PEG antibodies were observed, no treatment-boost ADA and anti-PEG antibodies were observed in subjects with pre-existing ADA and anti-PEG antibodies. No significant impact of pre-existing ADA and/or anti-PEG antibodies on PK variable, safety profile and preliminary efficacy was observed. 12/15 (80.0%) patients experienced zero bleeding episodes during the 4-week FRSW117 treatment period (from day 1 through day 28, 7 days after D22 dosing). Three of 15 (20.0%) subjects experienced 6 bleeding events among which only 1 spontaneous bleeding episode in cohort M1. 2 bleeds episodes were treated with FRSW117 of mean dose of 30.01 IU/kg and both were controlled after one infusion, with a hemostatic response rate of 100%. Conclusion: The therapy of 4 once-weekly doses of FRSW117 displayed favorable safety profile and provided about 3% mean FVIII activity on day 7 postdose with minimal accumulation. These results suggest that FRSW117 may offer extended bleeding protection with weekly dose interval and support further assessment of FRSW117 in phase 3 study.

PB1228 Inhibitor Detection in Acquired Hemophilia A: A Comparison between the Nijmegen Modified Bethesda Assay and an Anti-FVIII ELISA

PB1228 Inhibitor Detection in Acquired Hemophilia A: A Comparison between the Nijmegen Modified Bethesda Assay and an Anti-FVIII ELISA

Antidrug antibodies against the polyethylene glycol moiety inhibit the procoagulant activity of therapeutic polyethylene glycolated factor VIII

BackgroundThe standard therapy for patients with hemophilia A (HA) is the replacement with factor VIII (FVIII) therapeutics. To overcome the limitation of short half-life of wild-type FVIII protein, polyethylene glycol (PEG) can be coupled to therapeutic FVIII to improve pharmacokinetics. ObjectivesWe aimed to characterize antibodies developed against a FVIII therapeutic PEGylated with a 40-kDa PEG (40PEG-BDDFVIII) in 2 patients with mild HA. MethodsAn inhouse bead-based immunoassay was developed to characterize and confirm the specificity of the detected antibodies. The neutralizing nature of the antibodies toward PEGylated therapeutics was determined by a modified Nijmegen-Bethesda assay. ResultsTwo out of 46 patients treated with 40PEG-BDDFVIII developed inhibitory antibodies toward the drug. Switching to a non-PEGylated FVIII successfully increased the FVIII activity in both patients. In patient 1, antibodies were raised against FVIII and PEG. Anti-FVIII antibodies were of the immunoglobulin (Ig)G isotype, whereas anti-PEG antibodies were of IgG, IgM, and IgA isotypes. In patient 2, antibodies of IgG and IgA isotypes were directed only against the PEG moiety. Competitive assays confirmed the specificity of the antibodies against PEG. The applied Nijmegen-Bethesda assay revealed that patients’ anti-PEG antibodies and AGP3, an antibody against the backbone of PEG, can inhibit all currently available PEGylated therapeutics but to different degrees. No inhibitory FVIII antibodies were detected. ConclusionAntibodies against the PEG moiety of 40PEG-BDDFVIII abolished the efficacy of the drug. This is the first report on real-world experiences with the development of neutralizing anti-PEG antibodies after treatment with PEGylated FVIII therapeutics in mild HA.

Impact of different factor VIII inhibitor kinetic profiles on the inhibitor titer quantification using the modified Nijmegen–Bethesda assay

BackgroundCoagulation factor VIII (FVIII) inhibitor titer quantification is vital for optimizing care in people with hemophilia A. ObjectivesThis study analyzed the impact of the different kinetic profiles of four FVIII monoclonal antibodies on inhibitor titer quantification using the modified Nijmegen–Bethesda assay. MethodsConcentration‐related and time‐related profiles of FVIII antibodies (4A4, BO2C11, 2‐54, ESH‐8) were evaluated in vitro. FVIII residual activity was measured using a one‐stage clotting assay and chromogenic substrate assay. Profiles of the FVIII antibodies were compared with the theoretical kinetic model: the ideal log (residual activity)‐linear (inhibitor concentration) relationship. Different theoretical kinetic model–dependent and –independent criteria to calculate FVIII inhibitor titer were compared. ResultsFactor VIII monoclonal antibodies had different concentration‐related and time‐related profiles, ideal for comparative analysis using the modified Nijmegen–Bethesda assay. The kinetic profile of 4A4 was similar to the theoretical kinetic model, while BO2C11 showed a steeper curve, and 2‐54 and ESH‐8 a flatter curve, than the model. In the modified Nijmegen–Bethesda assay, conversion of measured FVIII residual activities for different inhibitor dilutions into FVIII inhibitor titer is based on the theoretical kinetic model. Therefore, titer calculations for FVIII inhibitors that deviate from the model are prone to underestimation or overestimation. Calculating a theoretical dilution at 50% FVIII residual activity by sigmoidal regression reflecting different kinetic inhibition profiles can provide a more accurate titer result. ConclusionKinetic profiles of FVIII antibodies can deviate from the theoretical kinetic model in the modified Nijmegen–Bethesda assay, leading to differences in FVIII inhibitor titer quantification.

Interim Analysis of Immunogenicity to the Vector Capsid and Transgene-Expressed Human FVIII in a Phase-1/2 Clinical Study of Bmn 270, an AAV5-Mediated Gene Therapy for Hemophilia a

▪When assessing the immunogenicity of gene therapy drug products, two components need to be considered: immunogenicity toward the vector delivery system and immunogenicity specific for the transgene product. BMN 270 is an AAV5-mediated gene therapy indicated for the treatment of Hemophilia A, and encodes for a codon-optimized B-domain deleted human FVIII protein (hFVIII-SQ) under control of a liver specific promoter. In study BMN 270-201, the first-in-human clinical trial, prospective patients with severe Hemophilia A were screened for both pre-existing antibodies directed against the AAV5 vector and inhibitors of transduction using a cell-based in vitro transduction inhibition (TI) assay. Patients testing positive in either assay were excluded from the trial, as were patients who had a history of FVIII inhibitors. Following infusion of BMN 270, patient plasma was analyzed for total antibody (TAb) responses specific for the AAV5 capsid and TAb responses directed against FVIII, using bridging ECLA immunoassays. Development of FVIII inhibitors was monitored using the Nijmegen-modified Bethesda assay. Additionally, peripheral blood mononuclear cells were collected for analysis by IFN-γ and TNF-α ELISpot assays for detection of capsid-specific and hFVIII-SQ specific cellular immune responses. The available data indicate that, as expected, all patients develop anti-AAV5 TAb by Week 8 post BMN 270 infusion, the first immunogenicity time-point assessed. One patient screened and confirmed positive at a single time point in the anti-FVIII TAb assay. This response was below the minimum required dilution to determine a titer, and was negative at all subsequent time points. No patients have tested positive in the Bethesda assay for FVIII inhibitors.We investigated the possibility that cell mediated immunity may explain the observed elevation in hepatic enzymes. To date, several patients dosed with BMN 270 experienced asymptomatic elevation of alanine aminotransferase (ALT) laboratory values ranging from 44 to 141 U/L (normal, 6-43 U/L) that resolved without sequelae. Analysis of cellular immune response in these patients by IFN-γ ELISpot assay against peptides spanning AAV5 capsid or the hFVIII-SQ protein was negative, as it was for all other subjects in the study across all time points tested to date. Unlike the IFN-γ assay, intermittent responses were detected across several patients in response to hFVIII-SQ peptide pools in the TNF-α ELISpot assay. In general, these responses were self-limiting and were not temporally associated with increases in ALT or a decline in FVIII activity measures, and did not correlate with results from the IFN-γ assay. Similar TNF-α responses to hFVIII-SQ peptides were also observed in a subset of healthy donors. Overall, immune responses to BMN 270 were characterized by the production of anti-AAV5 antibodies, which is an expected finding following viral vector administration. In conclusion, no consistent association could be made with increases in ALT and cellular immune responses. Our data to date indicate that although antibodies to the AAV5 capsid develop in all patients, concomitant cellular immunity is not readily detectable, and has not been associated with liver function abnormalities nor changes in transgene expression levels. DisclosuresLong:BioMarin: Employment. Kim:BioMarin: Employment. Wong:BioMarin: Employment. Yang:BioMarin: Employment. Vettermann:BioMarin: Employment. Pryer:BioMarin: Employment. Hardet:Genethon: Employment. Kuranda:Genethon: Employment. Mingozzi:Genethon: Employment. Pierce:Roche: Consultancy; BioMarin: Consultancy. Schweighardt:BioMarin: Employment.

Centralized Laboratory Testing for Inhibitors in the U.S. Hemophilia Treatment Centers Network: Testing Protocol and Results on 10,178 Specimens

Background: The incidence and prevalence of inhibitors among hemophilia patients in the U.S. has not been determined. The Centers for Disease Control and Prevention (CDC) has established a national inhibitor surveillance program called the Community Counts Registry for Bleeding Disorders Surveillance, through a collaboration with the American Thrombosis and Hemostasis Network and the U.S. Hemophilia Treatment Center (HTC) Network, to collect data and specimens to monitor inhibitors.Methods: A centralized laboratory was established at the CDC to perform inhibitor tests using a protocol based on accepted methods and published findings from a pilot project, the Hemophilia Inhibitor Research Study. Specimens are collected annually and when an inhibitor is detected or suspected locally from patients with hemophilia A (HA), hemophilia B (HB), and Type 3 von Willebrand disease (VWD3) who have received treatment and are enrolled at one of 134 HTCs. In the testing protocol, a modified Nijmegen-Bethesda assay with preanalytical heat inactivation (CDC-NBA) for factor VIII (FVIII) or factor IX (FIX) is first performed. On specimens with low positive or “gray zone” results of 0.5-1.9 Nijmegen-Bethesda units (NBU) for FVIII and 0.3-0.9 NBU for FIX, chromogenic Bethesda assay (CBA) for FVIII, fluorescence immunoassay (FLI) for anti-FVIII or anti-FIX antibodies, and dilute Russell's viper venom test (DRVVT) for lupus anticoagulants are performed. Repeat specimens are requested for positive or ambiguous results in patients with negative history.Results: Initial results on 10,178 specimens from 5,931 HA patients, 1,541 HB patients, and 134 VWD3 patients are shown in the Table. Additional testing on 141 low positive or “gray zone” FVIII specimens showed 74% to be CBA positive and 26% to be CBA negative. Of those CBA positive, 99% were also positive for IgG1 or IgG4 by FLI and were classified as true positives (104/141, 74%). Of CBA-negative specimens, 10 were also IgG1 and IgG4 negative and were classified as false positives (10/141, 7%). FLI results were positive on 26 CBA-negative specimens, which could not be classified (26/141, 18%). Final results on classified specimens are shown in the Table. Fourteen specimens were DRVVT positive, 13 of which were also CBA and FLI positive.Follow up was conducted on two groups of specimens: Arm 1 included specimens with positive or ambiguous results at CDC from patients who were reported to have no history of inhibitor, and Arm 2 included specimens from patients with new inhibitors locally detected at the HTC within the study period. In Arm 1, of 366 positive or ambiguous FVIII specimens, 20 were from patients with negative history and 17 of 18 repeated were confirmed; and of 44 positive FIX specimens, 3 were from patients with negative history and 2 of 3 repeated were confirmed. In Arm 2, 38 locally detected positive FVIII specimens and 6 locally detected positive FIX specimens were confirmed.Conclusions: From December 2013 through December 2016, newly positive test results on 55 FVIII and 8 FIX patients were identified and confirmed in two specimens, at least one of which was tested at the central laboratory. A cohort of 6,456 patients with negative history who were confirmed negative by central testing will be followed prospectively for inhibitor development. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Characteristics of Males with Hemophilia in the United States Hemophilia Treatment Center Network (USHTCN) with New-Onset Elevated Inhibitor Titers, December 15, 2013 - December 31, 2016

Background: Hemophilia inhibitors are associated with increased morbidity, mortality and health care costs. Although inhibitors are adverse reactions to pharmaceutical products, it is likely that they are under-reported. Data are needed on the burden of inhibitors in the U.S. to aid in prevention efforts.Methods: The Centers for Disease Control and Prevention (CDC) integrated centralized inhibitor testing along with extensive inhibitor data collection as part of a public health surveillance system called Community Counts established in the USHTCN beginning in December 2013. HTC staff collect data on demographic (e.g., age, sex, race and ethnicity) and clinical (e.g., hemophilia type, severity, prescribed treatment) characteristics at annual comprehensive visits using standardized data forms. Data collected on inhibitors include the dates of first detection for any participants with a previous inhibitor history, results of the most recent local inhibitor test and changes in treatment such as use of bypassing agents or immune tolerance therapy. For the surveillance or for central lab confirmation of a locally identified recent new-onset elevated inhibitor titer, a blood sample is prepared and shipped to CDC where it undergoes testing using a validated modified Nijmegen Bethesda assay as previously described (Miller CH, et. al., JTH, 2012). Newly detected inhibitor titer elevations either from local testing (above lab threshold) or from CDC testing (≥0.5 NBU or ≥0.3 NBU for hemophilia A (HA) or B (HB), respectively) were identified prospectively and tests were repeated with a new blood specimen for confirmation. Specimens tested at CDC with titers above the cutoff but below 2 NBU received further testing to rule out false positive tests. Additional participants with a recent history of first elevated inhibitor titer within the 3 year surveillance period were also identified retrospectively; historical and treatment information and central lab testing were used to confirm the elevated titer. Participants with a new-onset elevated titer confirmed by at least one elevated titer by CDC testing and no reported history of an inhibitor prior to December 2013 were considered cases for surveillance purposes. The characteristics of the cases according to subgroups based on age and hemophilia type and severity are shown as the proportion of those with elevated titers among all participants tested in the subgroup with no previous inhibitor history.Results: During the period December 15, 2013 through December 31, 2016, a total of 7,718 males with HA or HB were enrolled in the surveillance, approximately 27% of hemophilia patients seen in the USHTCN. At enrollment 1124/6135 HA and 75/1583 HB patients had a history of inhibitor for a baseline inhibitor prevalence of 18.3% and 4.7% for HA and HB, respectively (including all severities). During the study period there were 55 cases of confirmed new-onset elevated inhibitor titers among 5,011 HA and 8 cases among 1,508 HB participants for overall proportions of 1% and 0.5%, respectively. Table 1 shows the proportions of cases among all participants with no inhibitor history by age, type and severity subgroup. As expected, the highest proportions of new cases were observed among young children with severe HA, totaling 8.6% in children < 6 years, compared to 4.4% in severe HB. Notable findings include: 1) 3.2% cases in children < 6 years with moderate HA; 2) 8.7% cases in severe HB < 2 years; 3) in participants > 5 years, cases comprised 0.25% and 0.63% of participants with severe HA and HB, respectively; and 4) cases beyond age 5 were observed in all age groups and severities of HA while new cases were identified in 0/364 mild and only 1/579 moderate HB participants.Conclusions: These preliminary data provide the first measure of the burden of inhibitors among the U.S. population receiving care in the USHTCN. Sub-analyses for race, ethnicity and other risk factors will be possible when the data set matures. A prospective study of a large cohort was able to identify inhibitor risk in rare populations including participants who are > 5 years with HB or with moderate and mild HA or HB. These data will be useful in targeting populations for research and evaluating the effectiveness of public health prevention programs.The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention. [Display omitted] DisclosuresSharathkumar:CSL Behring: Consultancy; Shire: Consultancy. Kempton:Genentech: Membership on an entity's Board of Directors or advisory committees.

Simoctocog Alfa (Nuwiq) in Previously Untreated Patients with Severe Haemophilia A: Final Results of the NuProtect Study

Introduction FVIII inhibitor development is the most serious contemporary treatment complication in haemophilia A, particularly in previously untreated patients (PUPs). No inhibitors developed in clinical trials in previously treated patients treated with simoctocog alfa (Nuwiq), a fourth-generation recombinant FVIII produced in a human cell line. Methods The NuProtect study investigated the immunogenicity of simoctocog alfa in PUPs. NuProtect was a prospective, multinational, open-label, non-controlled, phase III study. PUPs with severe haemophilia A (FVIII:C <1%) of any age and ethnicity were treated with simoctocog alfa for 100 exposure days or a maximum of 5 years. Patients were true PUPs without prior exposure to FVIII concentrates or blood components. Inhibitor titres were measured with the Nijmegen-modified Bethesda assay; cut-off for positivity was 0.6 BU mL −1 (≥0.6 to <5 low-titre, ≥5 high titre). Results A total of 108 PUPs with a median age at first treatment of 12.0 months (interquartile range: 8.0–23.5) were treated with simoctocog alfa. F8 mutation type was known for 102 patients (94.4%) of whom 90 (88.2%) had null F8 mutations and 12 (11.8%) had non-null mutations. Of 105 PUPs evaluable for inhibitor development, 28 (26.7%) developed inhibitors; 17 high titre (16.2%) and 11 low titre (10.5%). No PUPs with non-null F8 mutations developed inhibitors. Conclusion In the NuProtect study, the rate of inhibitor development in PUPs with severe haemophilia A treated with simoctocog alfa was lower than the rate reported for hamster-cell-derived recombinant factor VIII products in other recent clinical trials. No inhibitors were reported in PUPs with non-null F8 mutations.

Clinical utility of sample preheat treatment in a modified Nijmegen-Bethesda assay (mNBA) for inhibitor monitoring in congenital and acquired haemophilia A: A single-centre four-year experience.

Laboratory monitoring for factor VIII inhibitors ideally requires samples with the lowest possible factor VIII (FVIII) level, potentially challenging in patients with congenital haemophilia A (CHA) receiving regular prophylaxis and acquired haemophilia A (AHA) patients with endogenous FVIII. Inactivation of FVIII by preheating (preheat treatment, PHT) of patient plasma has been suggested to facilitate monitoring. To evaluate the clinical utility of PHT prior to inhibitor analysis by modified Nijmegen-Bethesda assay (mNBA) in patients with CHA and AHA. Inhibitor screening by mNBA under standard conditions and with PHT at 56°C for 30, 60 and 90 minutes was evaluated. FVIII inhibitor results between 2007 and 2010 without PHT (720 results from 222 CHA and AHA patients), and between 2011 and 2014 post-PHT (1102 results from 302 patients) were available for analysis. Of total 1822 results available, 61% were from severe HA patients, 22% from mild and moderate HA and 16% from AHA. Pre-PHT, 74% of samples were analysed by the mNBA, and the remaining 26% were not tested as FVIII levels were>20 IU/dL as per local protocol. Postintroduction of PHT (90 and 60 minutes), 96% of samples received were analysed for an inhibitor. Post-PHT in patients with AHA (n = 26), 69% of samples tested with factor VIII levels >20 IU/dL were found to have detectable inhibitor. FVIII inhibitor testing using PHT at 56°C for 60 minutes facilitates inhibitor surveillance of CHA on prophylaxis. Potentially, 30 minutes at 56°C might be equally efficacious. In AHA receiving immunosuppression, monitoring of inhibitor titre after initial factor VIII response might enable personalized immunosuppression.

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.

The Frequency and Effect of Baseline Cross-Reacting and De Novo Inhibitors to Recombinant Porcine FVIII in Patients with Congenital and Acquired Hemophilia a

Background Patients with severe congenital hemophilia A (CHA) have a 25-40% lifetime risk of alloantibody (inhibitor) development to FVIII. Patients with acquired hemophilia A (AHA) spontaneously develop neutralizing autoantibodies to factor VIII. In both cases, patients require pro-hemostatic therapy with bypassing agents: recombinant factor VIIa (rFVIIa), activated prothrombin complex concentrate (aPCC) and more recently recombinant porcine factor VIII (rpFVIII). Anti-human FVIII (hFVIII) inhibitors typically bind to the A2 and C2 domains of the FVIII molecule. RpFVIII is an effective pro-hemostatic treatment for AHA and CHA given the immunologic difference in the A2 and C2 domains of the rpFVIII while maintaining sufficient hFVIII homology to act as an effective cofactor to human FIX in the intrinsic tenase. However, some anti-hFVIII antibodies cross-react with rpFVIII and may interfere with its hemostatic function. Cross-reacting antibodies were reported in 35% of subjects in a phase II/III trial prior to initiation of rpFVIII. Moreover, de novo rpFVIII inhibitors may develop during or after the treatment with rpFVIII and may affect its hemostatic function. Here we describe the largest case series to date on baseline cross-reactivity of rpFVIII inhibitors and post-treatment de novo inhibitor development in patients with CHA and AHA to address the paucity of published literature in this area. Aim First, we describe the frequency of baseline cross-reacting rpFVIII inhibitors in patients with AHA and CHA (with inhibitors) at our institution. Second, we describe the effect of baseline rpFVIII antibodies on FVIII recovery after treatment with rpFVIII. We also describe the frequency and timing of de novo rpFVIII inhibitor development after exposure to rpFVIII. Methods Institutional research ethics board approval was obtained. Electronic charts of patients admitted to our institution with AHA or CHA who underwent testing for rpFVIII inhibitors were reviewed retrospectively. RpFVIII inhibitor assay is performed in the special coagulation laboratory using the Nijmegen modified Bethesda assay. The patient sample is initially heat-treated at 57 Results Twenty-seven patients (7 CHA, 20 AHA) underwent testing for porcine inhibitors since assay availability in 2016. 61% (5/7 CHA, 11/20 AHA) of patients had a detectable rpFVIII inhibitor prior to exposure to rpFVIII; median titer 1.6 BU/ml (range 0.6-192). Eight patients with AHA with baseline cross-reacting inhibitors received rpFVIII. Of those, three achieved an initial FVIII recovery beyond 100% (132%, 148% and 177%) after approximately 100U/kg of rpFVIII and all three had very low anti-rpFVIII Bethesda titers (0.70, 0.85 and 0.9 BU/ml). Five patients did not achieve a FVIII recovery above 50% (46%, 46%, 40%, 36% and 0%) despite approximately 100U/kg of rpFVIII. Most patients who received rpFVIII were tested weekly for the duration of their treatment or hospital stay. Upon discharge, patients who were seen in clinic for follow up were tested for anti-hFVIII and anti-rpFVIII. Two AHA patients without a baseline inhibitor who received rpFVIII treatment developed a de novo inhibitor after 20 days (1 BU/ml) and 133 days (12 BU/ml), respectively. One AHA patient had a rise in baseline anti-rpFVIII titer after exposure to rpFVIII. Conclusion In conclusion, we found that 61% of patients with AHA and CHA tested for rpFVIII inhibitors had a detectable baseline cross-reacting inhibitor which is higher than previously described. Of those patients with a baseline inhibitor treated with rpFVIII, only 37.5% of patients had an appropriate rise in FVIII. Finally, 13% of patients without baseline inhibitors developed a de novo inhibitor after exposure to rpFVIII, an incidence comparable to previously published findings. Disclosures Pavenski: Bioverativ: Research Funding; Alexion: Honoraria, Research Funding; Octapharma: Research Funding; Shire: Honoraria; Ablynx: Honoraria, Research Funding. Teitel:BioMarin: Consultancy; CSL Behring: Consultancy; Octapharma: Consultancy; Novo Nordisk: Consultancy; Shire: Consultancy; Pfizer: Consultancy, Research Funding; Bayer: Consultancy, Research Funding. Sholzberg:Takeda: Honoraria, Research Funding; Baxter: Honoraria, Research Funding; Baxalta: Honoraria, Research Funding. OffLabel Disclosure: Recombinant porcine factor VIII is used to treated patients with congenital hemophilia A with allo inhibitors

Immune Tolerance Induction (ITI) in Paediatric and Adult Patients with Haemophilia a (HA) and High-Titre (HT) Factor VIII (FVIII) Inhibitor - Real Life Data

Introduction: In Poland, the principles for the management of rare bleeding disorders as well as financing of medication are regulated by the National Program for the Management of Haemophilia and Related Bleeding Disorders. One objective of the Program for 2012-2018 was to include the financing of ITI for all haemophilia patients with inhibitor. The qualification for ITI was performed by a physician from a Haemophilia Treatment Centre (HTC). Goal: the aim of the study is/was prospective assessment of the efficacy of ITI in real life setting. Patients and Methods: The study comprised 30 patients with severe and one with moderate HA and HT FVIII inhibitors. Factor VIII activity (FVIII:C) was measured by one-stage clotting assay and Factor VIII inhibitors by a modified Nijmegen-Bethesda assay and expressed in Bethesda Units/ml (BU/ml). Cut-off level for positive inhibitor was ≥0.5BU/ml. The following data was collected: historical peak inhibitor titre (HPT), pre-ITI titre (measured 1 month prior to ITI), peak titre recorded during the course of ITI, time between inhibitor detection and ITI start, and ITI duration. During ITI course blood samples were collected every 4 weeks. Treatment regimen with dosing and type of FVIII concentrate was at the discretion of the physician in each HTC. ITI success was defined as inhibitor titre below 0.5 BU/ml with FVIII half-life ≥6h and FVIII recovery ≥66% of normal. Partial response to ITI was defined as clinical response to FVIII therapy with no anamnestic response despite the presence of FVIII inhibitor or FVIII half-life &lt;6h and FVIII recovery &lt;66% of normal. Results: 31 patients with HA and inhibitor were divided into two groups: Group 1 comprised 12 adults with severe HA, median age 36 years (mean 40.1±15.1 and Group 2 included 19 boys, (&lt;18 years) (18 with severe, one with moderate HA), median age 8 years (mean 8.1±4.3) The median (mean) duration time between inhibitor detection and ITI start was 108 months (102±91) in Group 1 and 10 months (9±10) in Group 2. The median (mean) HPT before ITI was 68 BU/ml (156±193) and 50 BU/ml (126±242) in Group 1 and 2, respectively. ITI is still ongoing in one patient (Group 1) and in 3 boys (Group 2); these 4 patients were excluded from outcome analysis. Two adult patients (16,6%) of Group 1 and 2 boys (10,5%) of group 2 discontinued ITI therapy for a variety of reasons such as death (one patient), poor compliance or loss to follow-up; those patients were however included in the outcome analysis. The median (mean) duration of ITI was 24 months (30.8±31.7) (1-120) in Group 1 and 28 months (33.4±21.4) (6-84) in Group 2. The median (mean) peak titre during ITI was 40 BU/ml (225.7±185.7) in Group 1 and 85 (160±171.7) in Group 2. Immune tolerance to FVIII (success) was achieved in 3/11 adult (27.3%) and in 6/16 paediatric patients (37.5%). Median HPT, median time between inhibitor detection and ITI start, median peak inhibitor titre during ITI and median duration of ITI in patients who achieved immune tolerance were 380 BU/ml, 120 months, 8 BU/ml, 33 months in Group 1 and 22.5 BU/ml, 9.5 months, 205 BU/ml, 25 months in Group 2, respectively. No partial response to ITI was observed in our patients. The most common initial ITI regimen in both groups was 100 IU/kg/d of FVIII (50 - 200 IU/kg/d). In 28/31 (90.3%) patients only plasma derived FVIII concentrates were used which contained various amounts of von Willebrand factor. In two patients recombinant FVIII was administered. Recombinant, plasma derived and EHL FVIII concentrates were used in the case of one boy. In two patients immunosuppressive therapy (prednisone or rituximab) was concomitantly applied. Conclusions: it is our belief that the presented real-life data reflects i) the challenge of ITI therapy in clinical practice and ii) wide variety of approaches to ITI strategy in individual haemophilia treatment centres. Disclosures Windyga: Octapharma: Honoraria, Research Funding; Rigel Pharmaceuticals: Honoraria, Research Funding; Ferring Pharmaceuticals: Honoraria, Research Funding; Roche: Honoraria, Research Funding; Alnylam: Honoraria, Research Funding; Alexion: Honoraria, Research Funding; Werfen: Honoraria, Research Funding; Novo Nordisk: Honoraria, Research Funding; Bayer: Honoraria, Research Funding; CSL Behring: Honoraria, Research Funding; Baxalta/Shire (a Takeda company): Honoraria, Research Funding; Sanofi: Honoraria, Research Funding; Siemens: Honoraria, Research Funding; Sobi: Honoraria, Research Funding.

On the Role of F8 Sequence Mismatch and Class-II Human Leukocyte Antigen Binding in the Development of Neutralizing Antibodies (“Inhibitors”) Directed Against Therapeutic Factor VIII Proteins (tFVIIIs): Evidence from the PATH Study

The development of inhibitors against tFVIIIs represents a serious impediment to efficacious management of bleeding episodes in patients with hemophilia A (HA). It is therefore critical to understand the etiology of inhibitors to improve HA outcomes. Our group has recently presented evidence (Diego et al. Res Pract Thromb Haemost. 2019;3(Suppl. 1):50-51) on the validity of the Sequence Mismatch hypothesis, which posits that a sequence mismatch between the patient's endogenous FVIII sequence (i.e., due to the presence of a non-HA-causing non-synonymous single-nucleotide variation) and that underlying their tFVIII increases the risk that inhibitors will be induced during FVIII replacement therapy. For our study, 442 North American HA patients (237 Whites &amp; 205 Blacks; 88% severely affected) were: 1) Immuno-Chip genotyped at ~167,000 SNPs in genes implicated in autoimmune disease risk; 2) Evaluated by Sanger DNA sequencing and assays for the recurrent intron (I)1- and I22-inversions to identify their F8-causal-mutations; and 3) Tested with the Nijmegen-modified Bethesda assay to determine their inhibitor status. The Immuno-Chip genotypes were used to construct a genetic-relationship matrix (GRM), and the F8 sequence data along with results from the I1- and I22-inversion (I22I) assays were used to construct a shared F8-mutation matrix (FMM). These matrices were used to estimate the heritable genetic and shared F8-mutation effects. Importantly, modeling a F8-mutation effect has the added advantage of accounting for the mutational heterogeneity in F8-mutations. We found that heritability and F8-mutation effects respectively accounted for 50% and 23% of the phenotypic variance in inhibitor (both p &lt; 0.0001). Under the Sequence Mismatch hypothesis, it is assumed that tFVIII-derived peptides spanning the sequence mismatch must first be bound and presented on HLAcII molecules. In Diego et al. (2019), we reported a significant sequence mismatch effect but did not account for the effect of HLAcII binding. In a previous study of PATH data, it was shown using an area under the curve (AUC) estimate from receiver operator characteristic (ROC) curve analysis that HLAcII binding affinities estimated from the then-current version of NetMHCIIpan algorithm with respect to 15-mer peptides spanning sequence mismatches significantly predicted inhibitor status in a sample of 25 patients with the I22I mutation (Pandey et al. Nat Med. 2013;19(10): 1318-24). Here we adopt two strategies to extend this latter analysis to account for genetic relatedness and mutational heterogeneity. In the first strategy, we initially performed a Cholesky decomposition of the phenotypic covariance matrix expressed as a function of the GRM and FMM and their associated heritability and F8-mutation effect estimates, respectively. Following this, the derived Cholesky factor was used to decorrelate the HLAcII binding data, and then a bootstrap with replacement followed by ROC curve analysis each time (for 1,000 resampling's of the data) was used to generate an empirical distribution of the estimated AUC estimates. In the second strategy, we stratified the sample into the subset with the I22I mutation. We next performed a Cholesky decomposition of the GRM multiplied by the heritability and then used the resulting Cholesky factor to decorrelate the data for this subset. Finally, bootstrap with replacement followed by ROC curve analysis each time was used to generate an empirical distribution of the estimated AUC estimates. The p-values for both strategies are determined as the number of AUC estimates greater than that for the original transformed sample plus 1 divided by the total number of bootstraps plus 1. For the first time, we report results on the validity of the sequence mismatch hypothesis while modeling the effects of genetic relatedness, mutational heterogeneity, and HLAcII binding affinity. Disclosures Luu: Haplogenics Corporation: Employment. Chitlur:CSL-Behring: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda/Shire: Consultancy, Membership on an entity's Board of Directors or advisory committees; Agios: Research Funding; Bayer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bioveritiv/Sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novo Nordisk Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees; Octapharma: Consultancy, Membership on an entity's Board of Directors or advisory committees. Dinh:Haplogenics Corporation: Employment. Mead:CSL Behring: Employment. Powell:Haplogenics: Membership on an entity's Board of Directors or advisory committees. Escobar:Novo Nordisk: Consultancy, Membership on an entity's Board of Directors or advisory committees; Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; National Hemophilia Foundation: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees. Howard:Haplogenics Corporation: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

HLA-DRB1*01:01, but not HLA-DRB1:1503 or HLA-DRB1*11, is associated with decreased inhibitor risk in Iranian hemophilia A patients

Background/objectiveHemophilia A is a genetic disorder through which patients suffer from recurrent bleeding. This can be caused by a defect in human plasma coagulation factor VIII. High incidence of FVIII inhibitors in some severe hemophilia A patients after FVIII therapy is a considerable complication. Determination of good predictive factors can improve the safety of this treatment. HLA-II have been shown as a predictive element for inhibitor development. The goal of this study is to determine the association between HLA-DRB1*15:03, HLA-DRB1*11 and HLA-DRB1*01:01 alleles and FVIII inhibitors in severe hemophilia A patients in Iran. Materials/methodsHLA-DRB1 genotyping was performed using Multiplex sequences Specific Primers (PCR-SSP) in two groups of severe hemophilia A patients comprising 51 and 50 individuals with and without FVIII inhibitors respectively. The levels of inhibitor were determined through Nijmegen-modified Bethesda assay. HLA-DRB1 allele frequencies were compared between groups by using multiple logistic regression models. ResultsHLA-DRB1*01:01 allele frequency was significantly higher in patients without inhibitor ORadj: 2.7 (95%CI: 1.08, 6.97; P = 0.034). There wasn’t any statistically significant difference in HLA-DRB1*11 allele frequency between groups ORadj: 0.7 (95%CI: 0.27, 1.82; P = 0.47). There was no connection between HLA-DRB1*15:03 and inhibitor development ORadj: 0.94 (95%CI: 0.38, 2.35; P = 0.94). ConclusionAn association between HLA-DRB1*01:01 and paucity of FVIII inhibitor showed that this allele has probably a protective effect in severe hemophilia A patients in Iran. Determination of the predictive and protective alleles are beneficial in pre-treatment activities and decrease the risk of unsuccessful therapy with FVIII in each population.

A Standardized Kit for a Chromogenic Modified Nijmegen-Bethesda Assay: Repeatability, Reproducibility, and Analytical Sensitivity

Antibody-based inhibitor response to Factor VIII replacement therapy is one of the most common complications in the treatment of persons with hemophilia A (PwHA). Accordingly, accurate quantification of FVIII inhibitor antibody titer is essential in guiding patient treatment. Testing for FVIII inhibitors is not standardized; typically, FVIII inhibitor titers are measured using non-standardized LDTs (Laboratory Developed Tests). These LDTs vary in the processing of patient plasma, the reagents used to perform the assay, and the method of FVIII activity measurement.Here we report the development of a standardized set of reagents to perform a chromogenic modified Nijmegen-Bethesda assay (MNBA). The kit consists of imidazole-buffered pooled normal plasma (IB-PNP) with a consistent normal level of FVIII activity, and an imidazole-buffered bovine serum albumin solution (IB-BSA) as a diluent. Also included in the kit are a positive FVIII inhibitor plasma control (1.2 - 1.8 BU/mL) and an inhibitor-free negative plasma control.To perform the assay, test plasma was heat inactivated at 56 °C for 30 minutes to minimize residual FVIII activity. The inactivated plasma was then centrifuged (2700 x g,5 min.) and the supernatant used to make a series of two-fold serial dilutions with IB-BSA. The dilutions were each mixed with an equal volume of IB-PNP to form test mixes and incubated in a 37 °C water bath to allow time for the inhibitor antibodies from the patient plasma to inactivate FVIII in the IB-PNP. After two hours, the reaction was halted by cooling the test mixes in an ice bath. The FVIII activity of the test mixes was then measured using a Siemens FVIII Chromogenic Assay on a Siemens BCS XP analyzer. The residual FVIII activity of each test mix was calculated by comparing its FVIII activity to the activity of a control mix containing no test plasma (1:1 mixture of IB-BSA and IB-PNP).Here we report the use of this kit to perform a comprehensive study examining the repeatability, reproducibility, and analytical sensitivity of a chromogenic MNBA performed according to CLSI guidelines (CLSI EP05 and EP17). Inhibitor-negative and inhibitor-positive plasma from PwHA were combined to yield a panel of test plasmas at four different levels of inhibitor: below cutoff level (~0.3 BU/mL), low positive (~1.2 BU/mL), mid positive (~5 BU/mL) and a high positive (~8 BU/mL). To assess the repeatability of the assay, we measured each of these test plasmas using three lots of MNBA kit and one lot of FVIII chromogenic kit for a total of 240 titer determinations (3 lots × 20 days × 2 runs × 2 replicates). Inhibitor measurements of the panel of test plasmas showed within-lot precision of less than 10% (CV), and the kit controls were identified correctly each time. The reproducibility of the assay was determined in a study conducted across three different laboratories using different BCS XP analyzers and operators but the same panel of test plasmas. The limit of blank (LoB) and the limit of detection (LoD) of the assay were also determined using multiple low titer plasmas from unique donors. The assay system can reliably distinguish inhibitor titers as low as 0.2 BU/mL (LoD) in plasma from PwHA with no history of inhibitors.The chromogenic MNBA kit shows potential for labs seeking repeatable and reproducible FVIII inhibitor measurement that can otherwise vary significantly within or between labs. Standardization of reagents and protocol yields consistent results and is suitable for multi-center inhibitor studies in PwHA. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Role of lupus anticoagulants in immediate acting inhibitor positivity in congenital haemophilia A patients

ObjectivesPresence of lupus anticoagulants (LA) in haemophilia and their interference in coagulation assays is well-known. Factor VIII (FVIII) inhibitors are generally time and temperature dependent whereas LAs are immediate acting inhibitors (IAIs). The present study reports the challenges in laboratory detection of both progressive and non-progressive, specific FVIII inhibitors in the presence of LA. MethodsFrom 2012 through 2015, 4900 HA patients were screened for inhibitors. APTT based inhibitor screening tests and Nijmegen-modified Bethesda assay (NBA) were done in all samples. LA test and FVIII inhibitors by ELISA were done in patients with IAIs. ResultsOut of 451 patients positive for inhibitors in the initial screening tests, classical and progressive FVIII inhibitors were observed in 398 patients while 53 had IAIs showing no/partial correction in 1:1 mixtures of NPP and patient plasma. In 27 patients, both FVIII and FIX activity levels were <1%, resulting in difficulty in diagnosis. In 48 HA patients with IAIs, 42 were LA positive. 4 patients were found to have only LA with false positive results in NBA while 38 had a combination of LA and FVIII inhibitors. Six patients were LA negative and had only FVIII IAIs. Five (62.5%) of 8 HA patients initiated on immune tolerance induction (ITI) also were positive for IAIs. ConclusionThe findings emphasizes the presence of specific FVIII inhibitors in congenital HA with absence of time dependent inactivation kinetics in a small proportion of cases. ELISA or chromogenic assays along with LA testing can offer accurate laboratory diagnosis in patients with coexisting LA.

Clinicopathological parameters influencing inhibitor development in patients with hemophilia A receiving on-demand therapy.

Development of inhibitors to transfused factor VIII in patients with hemophilia A continues to be a challenge for professionals involved in hemophilia care. The majority of patients in India receive 'on-demand' rather than prophylactic therapy. The present study was done to assess the prevalence of factor VIII inhibitors in patients with hemophilia A (PWHA) receiving 'on-demand' therapy in a North Indian population and to study the clinicopathological parameters influencing the development of inhibitors. The study group comprised of 300 PWHA. Detailed clinical parameters, treatment history, bleeding profile including family history were recorded. Diagnosis of hemophilia A was confirmed by relevant coagulation tests. Inhibitors were screened using mixing based studies followed by quantification by Bethesda assay and Nijmegen modified Bethesda assay. Samples were collected from five cities in North India where a free supply of factor VIII was available and effectively used in three of these cities. In the 300 PWHA, disease phenotype was severe in 219 (73%), moderate in 62 (20.67%) and mild in 19 (6.34%), based on the factor VIII bioassay. Inhibitor prevalence was 9.6% (n = 29) and seen only in the severe phenotype. Inhibitor titers ranged from 0.8 to 108.8 BU/ml. A total of 12 PWHA had low and 17 had high titers. Correlation of various clinicopathological parameters in inhibitor-positive versus negative PWHA showed significant correlation with age at onset of disease, severity of disease, age at first exposure to treatment, annual factor intake (IU/kg/year), intense treatment episodes and bleeding manifestations like central nervous system bleed and hematuria. The total study sample had blood group B in 33.34% PWHA, followed by O (27.34%), A (24.34%) and AB (15%), however, in inhibitor-positive samples, significant inhibitor formation was associated with the ABO subtype A (19/29, 65.51%). Factor VIII inhibitor prevalence in PWHA receiving 'on-demand' therapy was 9.6%. Clinicopathological correlates of inhibitor development in such PWHA have been analyzed in this novel study.

Inhibitor Development in Previously Untreated Patients with Severe Hemophilia a Treated with Nuwiq®, a New Generation Recombinant FVIII of Human Origin

Inhibitor Development in Previously Untreated Patients with Severe Hemophilia a Treated with Nuwiq®, a New Generation Recombinant FVIII of Human Origin

Long Term Safety and Efficacy of Recombinant Factor IX Fc Fusion Protein (rFIXFc) Prophylaxis in Children with Hemophilia B: Interim Results of the B-Yond Extension Study

Introduction: The phase 3 Kids B-LONG study (NCT01440946) demonstrated the safety, efficacy, and pharmacokinetics of prophylactic recombinant factor IX Fc fusion protein (rFIXFc) for the prevention and treatment of bleeding episodes in children with severe hemophilia B. The ongoing rFIXFc extension study B-YOND (NCT01425723) is evaluating long-term safety and efficacy of rFIXFc in children and adults with hemophilia B. The safety and efficacy data for pediatric subjects from the second B-YOND interim data cut (11 Sep 2015) are reported here. Methods: The Kids B-LONG study enrolled males aged Results: At the time of the second B-YOND interim data cut, 27 subjects had completed Kids B-LONG and enrolled in B-YOND ( Conclusion: These data confirm the long-term safety of rFIXFc with maintenance of low ABRs and extended-interval prophylaxis in children with hemophilia B. This research was funded by Biogen and Sobi. Biogen and Sobi reviewed and provided feedback on the abstract. The authors had full editorial control of the abstract and provided their final approval of all content. Disclosures Ducore:Octapharama: Membership on an entity9s Board of Directors or advisory committees; Baxalta (Shire): Membership on an entity9s Board of Directors or advisory committees; LFB: Membership on an entity9s Board of Directors or advisory committees; Bayer: Membership on an entity9s Board of Directors or advisory committees; Pfizer: Membership on an entity9s Board of Directors or advisory committees; CSL Behring: Membership on an entity9s Board of Directors or advisory committees; Biogen: Membership on an entity9s Board of Directors or advisory committees. Fischer:Baxter: Consultancy, Research Funding, Speakers Bureau; NovoNordisk: Consultancy, Research Funding, Speakers Bureau; Octapharma: Speakers Bureau; Biogen: Consultancy; Biotest: Consultancy, Speakers Bureau; Baxalta/Baxter: Consultancy, Research Funding, Speakers Bureau; Wyeth: Research Funding; Pfizer: Consultancy, Research Funding, Speakers Bureau; CSL Behring: Consultancy, Speakers Bureau; Bayer: Consultancy, Research Funding, Speakers Bureau; Freeline: Consultancy. Kulkarni:Kedrion: Membership on an entity9s Board of Directors or advisory committees; BPL: Membership on an entity9s Board of Directors or advisory committees; Biogen: Research Funding, Speakers Bureau; Pfizer: Membership on an entity9s Board of Directors or advisory committees; Novo Nordisk: Membership on an entity9s Board of Directors or advisory committees, Research Funding; Bayer: Membership on an entity9s Board of Directors or advisory committees, Research Funding; Baxter: Membership on an entity9s Board of Directors or advisory committees, Research Funding. Nolan:Sobi: Research Funding; Biogen: Research Funding. Perry:Biogen: Consultancy, Honoraria; Novo Nordisk: Consultancy, Membership on an entity9s Board of Directors or advisory committees. Yuan:Biogen: Employment, Equity Ownership. Ramirez-Santiago:Biogen: Employment, Equity Ownership. Ferrante:Sobi: Employment. Lethagen:Sobi: Employment.

Longitudinal Analysis of Long-Term Safety and Efficacy of Recombinant Factor VIII Fc Fusion Protein (rFVIIFc) in Adults/Adolescents with Severe Hemophilia a

Longitudinal Analysis of Long-Term Safety and Efficacy of Recombinant Factor VIII Fc Fusion Protein (rFVIIFc) in Adults/Adolescents with Severe Hemophilia a