Swedish Orphan Biovitrum Research Articles

Patients with Familial Thrombophilia of Unknown Origin Show Low APC Response to In Vivo Thrombin Formation in Stimulated Hemostasis Activity Pattern Evaluation (SHAPE)

Background: In a large proportion of patients with unprovoked venous thromboembolism (VTE), no risk factors can be identified. Recently, we have shown that an increased activated protein C (APC) response to in vivo thrombin formation reduces the thrombotic risk of factor V Leiden (FVL) carriers but not of prothrombin G20210A mutation (PTM) carriers. In vivo thrombin formation was triggered by low-dose administration of recombinant activated factor VII (rFVIIa) followed by hemostasis biomarker monitoring. Aim of the present study was to extend this SHAPE approach to patients with thrombophilia of unknown origin. Methods: The study population consisted of 21 subjects with a positive self-history of unprovoked VTE (VTE+) as well as a positive family history, in whom no established thrombophilic risk factor was detectable (thrombophilia of unknown origin, TUO). A second cohort included 27 VTE+ patients tested positive for FVL (n=14) or PTM (n=13). None of the subjects was under anticoagulant treatment at the time of analysis. The control group consisted of 18 healthy volunteers. Blood samples were collected immediately before and during a period of 8 hours following injection of 15 µg/kg rFVIIa. Plasma levels of free thrombin and APC were quantified using oligonucleotide-based enzyme capture assays (OECAs). Prothrombin activation fragment 1+2 (F1+2), thrombin-antithrombin complex (TAT), and D-dimer were measured additionally. Results: Injections of rFVIIa were well-tolerated by all subjects and median D-dimer levels remained within the reference range in all three cohorts. Plasma levels of F1+2 increased after rFVIIa injection, showing no significant differences between the groups. A comparable increase of TAT was observed in both TUO patients and VTE+ mutation carriers, from a median of 21.3 to 51.1 pmol/L (P=.022), and from 21.3 to 43.9 pmol/L, respectively (Figure 1A). Median levels of TAT in the controls and median levels of free thrombin in all cohorts remained below their respective quantifiable limits after rFVIIa injection. APC increased from 0.80 to 3.84 pmol/L (P=.001) in TUO patients, from 1.14 to 5.82 pmol/L (P<10-4) in VTE+ mutation carriers, and from 0.87 to 3.39 pmol/L (P=.001) in the control group. The APC increase was significantly smaller in the TUO cohort than in the VTE+ mutation carriers (P=.019) (Figure 1B). Conclusion: A low APC response to an increased in vivo thrombin formation is a frequent finding among patients with thrombophilia of unknown origin. This finding is in line with previous data suggesting that higher APC levels protect FVL carriers from thrombosis development. The results of the present study further suggest that a low APC response might be an independent risk factor of VTE. Further studies are warranted to identify the factors that modulate the APC response. Disclosures Rühl: Sanofi Genzyme: Honoraria; SOBI: Honoraria; Shire: Honoraria; Bayer: Honoraria; CSL Behring: Honoraria; Grifols: Honoraria. Reda:Shire: Honoraria; Grifols: Honoraria. Winterhagen:SOBI: Honoraria. Berens:Pfizer: Honoraria; Shire: Honoraria; Biotest: Honoraria; CSL Behring: Honoraria; NovoNordisk: Honoraria; Baxter: Honoraria. Müller:Roche: Membership on an entity's Board of Directors or advisory committees; Siemens: Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees; NovoNordisk: Membership on an entity's Board of Directors or advisory committees. Oldenburg:Grifols: Consultancy, Speakers Bureau; Roche: Consultancy, Speakers Bureau; Bayer: Consultancy, Research Funding, Speakers Bureau; Swedish Orphan Biovitrum: Consultancy, Speakers Bureau; NovoNordisk: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Speakers Bureau; Octapharma: Consultancy, Research Funding, Speakers Bureau; Biotest: Consultancy, Research Funding, Speakers Bureau; Chugai: Consultancy, Speakers Bureau; CSL Behring: Consultancy, Research Funding, Speakers Bureau; Takeda (Shire): Consultancy, Research Funding, Speakers Bureau.

TMIC-44. IL-1 ANTAGONIST ANAKINRA INHIBITS GLIOBLASTOMA PROLIFERATION BY ANTAGONIZING THE PROINFLAMMATORY MICROENVIRONMENT

Abstract INTRODUCTION The recombinant IL-1 receptor antagonist anakinra prevents binding of IL-1 to its receptor and blocks IL-1β-mediated proinflammatory signaling. It is currently used in the treatment of autoinflammatory diseases. As inflammation is a major driver of malignancy, we hypothesized that anakinra – by ameliorating the inflammatory tumor environment – might be capable to mitigate glioblastoma (GBM) progression. METHODS T98G GBM cells were cultivated with PBMC, separated by cell culture inserts with or without anakinra and incubated 48h in 5% O2 mimicking the GBM microenvironment. T-cells were obtained by magnetic bead isolation. mRNA expression levels were measured by quantitative Real-Time-PCR (qRT-PCR). Cytokine production was assessed by ELISA. Cell proliferation was analyzed by flow cytometric quantification of Ki-67 protein expression. Anakinra has been provided by Swedish Orphan Biovitrum AB (Sweden). Results are presented as mean±SEM, p-Values were calculated using Student’s t-test. RESULTS After administration of anakinra, mRNA expression levels of the inflammatory and proangiogenic genes IL-1β, COX-2, CCL2 and IL-8 were reduced in T98G (-64%±19.1%, -56.2%±21.3%, -87.1%±28.8%, -91.7%±27.2%, n=7, p< 0.05). Surprisingly, no changes of these targets were detectable in PBMC. However, in T-cells, anakinra induced mRNA expression of Th2 transcription factor GATA3 and the antiinflammatory cytokines IL-4 and IL-10 (+6,7%±4,6%, +36,1%±22,9%, +69,1%32,8%, n=10, p< 0.05). The expression of the protumorigenic genes IL-22, IL-17 as well as IFNγ was dramatically repressed (-63.2%±21.9%, -88.1%±21.7%, -85.6%±45.2%, n=10, p< 0.05), protein secretion of IL-22 and IFNγ was strongly blocked (-36.3%±9.1%, -13.9%±0.5%, n=6, p< 0.05). Importantly, treatment with anakinra markedly attenuated GBM cell proliferation (-20.1%±5%, n=4, p< 0.05). CONCLUSION Anakinra indeed puts the brake on proinflammatory signaling pathways in GBM cells and simultaneously promotes a shift towards an anti-inflammatory T-cell phenotype. As a result, GBM proliferation is diminished. Hence, administration of anakinra might be an interesting and novel therapeutic approach to reduce Glioblastoma aggressiveness.

5233Interleukin-1 blockade with Anakinra in ST-segment elevation acute myocardial infarction: Results from the VCUART3 study

Abstract Background ST-segment elevation myocardial infarction (STEMI) is associated with an intense inflammatory response that predicts an increased risk of death and heart failure (HF). In the current study we tested whether anakinra, a recombinant Interleukin-1 (IL-1) receptor antagonist, given once daily (standard dose) or twice daily reduced systemic inflammation in patients with STEMI. Methods We enrolled patients with STEMI within 12 hours of presentation at 3 sites. After revascularization, patients were randomly assigned to receive anakinra 100 mg twice daily, anakinra 100 mg once daily alternating with placebo once daily every 12 hours, or placebo twice daily, for 14 days in a 1:1:1 ratio. The primary efficacy outcome was the area under the curve for C-reactive protein levels (CRP-AUC) using a high-sensitivity assay at 14 days comparing anakinra (both arms) versus placebo followed by a comparison between each of the anakinra arms with placebo. Two pre-specified exploratory clinical efficacy endpoints, adjudicated by a blinded event committee, were assessed: a composite endpoint of all-cause death for any reason or incidence of HF (defined as new-onset HF requiring hospitalization or a new prescription of a loop diuretic, D+HF) and a composite endpoint of death and HF hospitalization (D+HHF) at 1 year. Data are expressed as median and interquartile range or number and percentage. Kaplan-Meyer survival curves were compared using Log-rank test (Mantel-Cox). (ClinicalTrials.gov number, NCT01950299) Results Of 311 patients screened, 99 subjects (80 [81%] males, 57 [58%] Caucasians, 55 [49–62] years of age) were randomly assigned to anakinra twice daily (N=31), anakinra once daily (N=33) or placebo (N=35). There were no significant imbalances in the demographic characteristics between groups (all P>0.05). The CRP-AUC was significantly lower in the anakinra group than in the placebo group (67 [39–120] versus 214 [131–394] mg/dl, P<0.001; and P<0.001 for each anakinra arm versus placebo separately, without significant differences between the two anakinra arms, P=0.41). Treatment with anakinra was associated with a significant reduction versus placebo in the incidence of D+HF (6/64 [9.4%] versus 9/35 [25.7%], P=0.046), and of D+HHF (0/64 [0] versus 4/35 [11.4%], P=0.011), without any significant difference between the two anakinra arms. Anakinra was not associated with any treatment-related serious adverse events, nor with excess infections compared with placebo (14.1% vs 14.3%, P=0.87). Conclusions Among patients with STEMI, IL-1 blockade significantly reduced the systemic inflammatory response compared with placebo, without any significant difference between standard or high dose regimens. Prespecified exploratory analyses on clinical endpoints demonstrate reduced incidence of HF and reduced HF hospitalizations, supporting the concept of beneficial effects with IL-1 blockade in patients with acute myocardial infarction. Acknowledgement/Funding Funded by NHLBI 1R34HL121402; Drug supply from Swedish Orphan Biovitrum

253Effects of Interleukin-1 blockade with Anakinra on cardiac function in ST-segment elevation acute myocardial infarction: results from the VCUART3 echocardiography study

Abstract Background ST-segment elevation myocardial infarction (STEMI) is associated with an intense acute inflammatory response further promoting cardiac dysfunction and heart failure (HF). Pilot proof-of-concept studies with anakinra, recombinant Interleukin-1 (IL-1) receptor antagonist, have shown feasibility and safety of IL-1 blockade in patients with STEMI. In the current study we analyzed the effects of anakinra on left ventricular (LV) dimensions and function in patients with STEMI. Methods We enrolled patients with STEMI within 12 hours of presentation at 3 sites in the United States of America. After revascularization, patients were randomly assigned to receive anakinra 100 mg twice daily, anakinra 100 mg once daily alternated with placebo once daily every 12 hours, or placebo twice daily, for 14 days in a 1:1:1 ratio. A transthoracic echocardiogram was completed within 24 hours of admission and at 1 year follow up to measure LV end-diastolic and end-systolic volumes (LVEDV and LVESV, respectively), stroke volume (SV) and ejection fraction (LVEF). (ClinicalTrials NCT01950299) Results Paired echocardiography studies (follow up study obtained 362 days [336–375] after the baseline study) were available in 63 of the 99 patients (63%): 23 of 35 patients in the placebo group (66%) and 40 of the 64 patients in the anakinra group (62%, P>0.05 for missing studies between the 2 groups; P>0.05 for duration of follow up). Baseline LVEDV, LVESV, SV and LVEF was not significantly different comparing placebo and anakinra (all P>0.05). Patients treated with anakinra had a significant improvement in LVEF from 49.8% (41.8–60.0%) to 54.0% (46.0–58.4%, P=0.028) and SV from 43.6 ml (37.6–52.1 ml) to 48.7 ml (40.9–62.5 ml, P=0.008), whereas no significant changes occurred within the placebo group (LVEF: from 51.7% [40.1–56.0%] to 53.5% [43.4–59.4%], P=0.25; SV: from 47.7 ml [40.1–56.8 ml], to 53.0 ml [44.9–57.4 ml], P=0.81). The between-groups differences, however, were not statistically significant. No significant changes were noted in LVEDV and LVESV in either group. The interval changes in CRP between admission and 72 hours, expression of the acute inflammatory response, inversely correlated with the LVEF at follow up (R=-0.30, P=0.026), with higher levels of CRP corresponding to lower LVEF values Conclusions A significant improvement in cardiac systolic function was seen in patients treated with IL-1 receptor antagonist, anakinra, following STEMI, and not in patients with placebo. Further studies are however required to determine whether the benefits of IL-1 blockade in the prevention and treatment of HF are mediated by the effects on cardiac function. Acknowledgement/Funding Funded by NHLBI 1R34HL121402; Drug supply from Swedish Orphan Biovitrum

5947Predictive role of C-reactive protein levels in patients with ST-segment elevation acute myocardial infarction for heart failure related events

Abstract Background ST-segment elevation myocardial infarction (STEMI) is associated with an intense acute inflammatory response and an increased risk of death and heart failure (HF). C-reactive protein (CRP) is the inflammatory biomarker most commonly used for risk stratification in patients with cardiovascular diseases. CRP levels are known to rise and fall during STEMI in response to myocardial injury. In this study, we analyzed whether admission CRP or delayed CRP (measured at 72 hours after admission) held a greater predictive value for adverse HF events in patients with STEMI. Methods We analyzed data from the VCUART3 clinical trial enrolling 99 patients with STEMI within 12 hours of presentation at 3 sites in the United States of America treated with anakinra or placebo. CRP levels were measured with a high-sensitivity assay at time of admission and again at 72 hours later. A dedicated committee composed of individuals not involved in the conduct of the trial adjudicated HF events including a composite endpoint of death from any reason or incidence of HF defined as new-onset HF requiring hospital admission or a new prescription for a loop diuretic (D+HF) and a composite endpoint of death and HF hospitalization (D+HHF) at 1 year. We used a time-dependent Cox-regression analysis to determine the association of CRP at admission or at 72 hours with the outcomes of interest in univariate and multivariate analysis. Data are presented as median and interquartile range. (ClinicalTrials NCT01950299) Results CRP levels from admission and 72 hours were available in 90 and 87 subjects respectively and they increased from 4.6 [2.8–8.5] mg/L to 11.6 [4.6–24.5] mg/L (P<0.001). Both admission CRP (CRP0) and CRP at 72 hours (CRP72) were associated with the risk of D+HF (P=0.011 and <0.001, respectively) and of D+HHF (P=0.010 and P<0.001, respectively); however at multivariate analysis, only CRP72 remained significantly associated with the risk of D+HF (P=0.001) and D+HHF (P=0.004) while CRP0 was not. CRP72 significantly correlated with NTproBNP levels at 72 hours (NTproBNP72, Spearman rho R=+0.37, P=0.001). NTproBNP72 predicted D+HF (P=0.030) but not independently of CRP72 (P=0.096 for NTproBNP72 and P=0.007 for CRP72 at multivariate analysis including the 2 variables). NTproBNP72 did not predict D-HHF. Conclusions Among contemporary patients with STEMI, the levels of CRP at 72 hours after admission was superior to admission CRP levels for predicting the incidence of HF events, and independent of NTproBNP levels. Our results indicate the importance of the inflammatory response during STEMI, supporting the concept of inhibiting the inflammatory response as a therapeutic strategy. Acknowledgement/Funding Funded by NHLBI 1R34HL121402; Drug supply from Swedish Orphan Biovitrum

P6388Effects of Interleukin-1 blockade with anakinra in patients with ST-segment elevation acute myocardial infarction on recurrent ischemic events: results from the VCUART3 study

Abstract Background ST-segment elevation myocardial infarction (STEMI) is associated with an intense acute inflammatory response and an increased risk of recurrent ischemic events. Prior studies of IL-1 blockade have shown conflicting results regarding the risk of future events. Methods We enrolled patients with STEMI within 12 hours of presentation at 3 sites in the United States of America. After revascularization, patients were randomly assigned to receive anakinra 100 mg twice daily, anakinra 100 mg once daily (standard dose) alternated with placebo once daily every 12 hours, or placebo every 12 hours for 14 days in 1:1:1 ratio. Prespecified exploratory endpoints for recurrent ischemic events, adjudicated by an independent committee, evaluated the composite risk of subsequent acute myocardial infarction (AMI, World Health Organization classification Type 1), unstable angina, or urgent revascularization. Data are expressed as median and interquartile range or number and percentage. Cox regression analysis was used to generate unadjusted hazard ratios and confidence intervals. (ClinicalTrials.gov number, NCT01950299) Results Of 311 patients screened, 99 subjects (81% males, 58% Caucasians, 55 [49–62] years of age) were randomly assigned to anakinra twice daily (N=31), anakinra once daily (N=33) or placebo (N=35). The cohort included patients with hypertension (57%), tobacco use (55%), diabetes mellitus (30%), and prior diagnosis of coronary artery disease (21%) without statistically significant imbalances in the demographic characteristics between groups (all P>0.05). Discharge medications for the index STEMI admission, in addition to the study medication, included aspirin (100%), statins (100%), P2Y12 inhibitors (100%), beta-blockers (90%), and angiotensin converting enzyme inhibitor/angiotensin receptor blocker (84%), without statistically significant imbalances between the 3 groups. Over the 1-year follow-up, recurrent ischemic events occurred in 5/35 (14.3%) patients treated with placebo and 6/64 (9.1%) patients treated with anakinra (hazard ratio = 0.68 [0.20–2.24], P=0.53). No differences were observed between high- and low-dose anakinra treatment groups. Conclusions A two week treatment with IL-1 receptor antagonist, anakinra, did not significantly decrease or increase recurrent ischemic events over the course of a 1-year follow-up in patients with STEMI. Acknowledgement/Funding Funded by NHLBI 1R34HL121402; Drug supply by Swedish Orphan Biovitrum

Prospective observation of Clostridium histolyticum collagenase for the treatment of Dupuytren\u2019s disease in 788 patients: the Austrian register

IntroductionSince March 2011, the microbial collagenase of Clostridium histolyticum (Xiapex®, Swedish Orphan Biovitrum AB, Stockholm, Sweden) has become available in the European Union for treatment of Dupuytren’s disease. The purpose of this study was to evaluate potential safety risks of Xiapex® and to contribute to a better understanding for its use.MethodsA prospective, non-interventional, observational study using Xiapex® for Dupuytren’s disease named XIANIS was conducted between 1.10.2011 and 01.10.2017. Treatment was conducted in accordance to the manufacturer information. Patients were invited for follow-up after 1 week, 1 month, 3 months and 1 year. Demographic data, treatment data, pain levels, anaesthetic application during passive manipulation, subjective function improvement, subjective satisfaction and adverse events were recorded.Results788 patients with 814 treatments were included who suffered from Dupuytren’s contracture for a mean of 64 months. The metacarpophalangeal joint was affected in 57% of cases and the PIP joint in 40.8% with a mean contracture of 39° and 56°, respectively. A change in the contracture down to 0°–5° was reported in 66.5% of cases, while 25.5% achieved a partial improvement. The pain during the injection was rated 4.5 and 3.3 during passive manipulation. Adverse events were reported in the majority of treated patients with skin tears being one main common event (26%). Further adverse outcomes were bleeding/hematoma, joint swelling, injection-site swelling, pressure sensitivity, erythema, injection-site pain, peripheral edema, blood blisters, blisters, painless lymphadenopathy, painful lymphadenopathy, axillary pain, arthralgia and sensory abnormality. There were no reported tendon ruptures, anaphylactic reactions or ligament injuries. On 1-year follow-up, 29% showed an increased contracture of a mean of 24° with the need for surgical treatment in 2% of patients. 74% of patients were very satisfied and 72% showed a high functional improvement.ConclusionThe injectable collagenase Clostridium histolyticum (Xiapex®) proved to be effective and safe in patients with Dupuytren’s disease. Minor adverse events disappeared within 30 days and the need for surgical treatment within 1 year was very low (2%). No major complications or rare side effects were seen in this prospective observational study.

Emapalumab: First Global Approval.

Emapalumab-Izsg (hereafter referred to as emapalumab) [Gamifant®] is a monoclonal antibody directed against interferon gamma that is available as an intravenous infusion. Emapalumab is being developed by Novimmune and Swedish Orphan Biovitrum for the treatment of haemophagocytic lymphohistiocytosis (HLH). In November 2018, emapalumab received its first global approval in the USA, for the treatment of paediatric (newborn and older) and adult patients with primary HLH, who have refractory, recurrent or progressive disease or intolerance to conventional HLH therapy. Emapalumab is under regulatory review in the EU for the treatment of primary HLH. This article summarizes the milestones in the development of emapalumab leading to this first global approval for HLH in the USA.

Accurate measurement of extended half-life and unmodified factor VIII low levels with one-stage FVIII assays is dependent on the matrix of calibration curves.

The monitoring of factor VIII (FVIII) replacement therapy relies on the accurate measurement of FVIII activity over a large concentration range. However, unexplained overestimation of low FVIII levels has recently been reported with extended half-life recombinant FVIIIs. The objective of this study was to confirm previous publications indicating that the reagents used to generate the calibration curves determine the accuracy of the measurement of low FVIII levels. We generated FVIII calibration curves with FVIII-deficient plasmas or a commercial diluent buffer. We then measured FVIII levels in FVIII-deficient plasma spiked with plasma FVIII, a full-length recombinant FVIII (Advate® , Shire, Brussels, Belgium) and two extended half-life recombinant FVIIIs (Elocta® , Swedish Orphan Biovitrum, Woluwe Saint-Lambert, Belgium and Afstyla® , CSL Behring, Mechelen, Belgium). FVIII levels were also analyzed in spiked samples prediluted two- and fourfold, either in diluent buffer or in FVIII-deficient plasma to evaluate parallelism. Coagulation times of calibration curves generated with diluent buffer were longer than those with FVIII-deficient plasmas. This resulted in an overestimation of FVIII levels lower than 25IU/dL in spiked samples and in detection of FVIII activity (≥1IU/dL) in FVIII-deficient plasma. Predilution of samples with diluent buffer rather than with FVIII-deficient plasma also led to discordant results. Our data confirm that the generation of calibration curves by dilution in FVIII-deficient plasma is crucial for the accurate measurement of low FVIII levels. When serial dilutions of samples are analyzed, predilution in FVIII-deficient plasma is required to respect the parallelism criteria. These methods should be more generally implemented for coagulation instruments.

The Variable Clinical Expressivity of Factor V Leiden, but Not of Prothrombin G20210A, Correlates with the Extent of the APC Response to In Vivo Thrombin Formation

Background: The factor V Leiden mutation (FVL) and the prothrombin G20210A mutation (PRO) are the most frequent thrombophilic mutations among Caucasians. Both mutations show a highly variable expressivity, ranging from a lifelong asymptomatic course to severe venous thromboembolism (VTE). Recently, we have shown increased generation rates of thrombin and activated protein C (APC) in response to in vivo thrombin formation in asymptomatic FVL carriers (VTE-). In vivo thrombin formation was triggered by low-dose administration of recombinant activated factor VII (rFVIIa). Aim of the present study was to extend this stress test approach to carriers of PRO and to FVL carriers with a history of VTE (VTE+).Methods: The study population consisted of 29 FVL carriers (thereof 25 heterozygotes: 13 VTE- and 12 VTE+) and 21 PRO carriers (thereof 19 heterozygotes: 12 VTE- and 7 VTE+). None of the subjects was under anticoagulant treatment at time of analysis. The control group consisted of 13 healthy volunteers tested negative for FVL and PRO. Blood samples were collected immediately before and during a period of 8 hours following injection of 15 µg/kg rFVIIa. Plasma levels of free thrombin and APC were quantified using oligonucleotide-based enzyme capture assays (OECAs). Prothrombin activation fragment 1+2 (F1+2), thrombin-antithrombin complex (TAT), and D-dimer were measured additionally.Results: Injections of rFVIIa were well-tolerated by all subjects and median D-dimer levels remained within the reference range in all groups. Compared with the controls, peak plasma levels of F1+2 and TAT were significantly higher after rFVIIa injection in FVL carriers (p=.009 and p=1.0∙10-4, respectively), and in PRO carriers (p=.045 and p=6.0·10-4, respectively), but they did not differ between the FVL and PRO cohorts, and between the VTE+ and VTE- subgroups. Median plasma concentrations of thrombin were below the limit of detection at baseline, and rFVIIa did not induce statistically significant changes of thrombin levels in any group. APC increased to peak levels 30 minutes after rFVIIa injection, from 0.63 (0.54-1.16) to 3.14 (2.55-3.55) pmol/L (p=.017) in the controls, and from 1.39 (1.14-1.86) to 7.86 (5.55-11.55) pmol/L (p<1.0∙10-4) in the FVL group. In PRO carriers, plasma levels of APC increased from 1.07 (0.86-1.23) pmol/L at baseline to a peak of 5.86 (4.44-6.41) pmol/L 1 hour after rFVIIa injection p=4.0·10-4). Peak plasma levels of APC were higher in both FVL carriers and PRO carriers in comparison to the controls (p<1.0∙10-4 and p=4.0∙10-4, respectively), and lower in PRO carriers compared with those in FVL carriers (p=.003). With peak levels of 8.11 (7.59-11.77) vs. 5.62 (4.94-6.49) pmol/L, APC was significantly higher in the VTE- subgroup than in the VTE+ subgroup (p=.004) in heterozygous FVL carriers, while in heterozygous PRO carriers, APC peak levels did not differ between the VTE- subgroup and the VTE+ subgroup (Figure 1).Conclusion: The APC response to in vivo thrombin formation is significantly lower in symptomatic than in asymptomatic FVL carriers, implying that higher APC levels protect FVL carriers from thrombosis development. This hypothesized effect appears to be specific for FVL as prothrombin G20210A carriers do not show differences in the APC response related to VTE history. Further studies are warranted to identify the factors that modulate the APC response in FVL carriers. [Display omitted] DisclosuresRühl:Swedish Orphan Biovitrum: Consultancy, Research Funding; Sanofi Genzyme: Research Funding; Shire: Research Funding; Grifols: Research Funding; CSL-Behring: Research Funding. Berens:Pfizer: Research Funding; Sanofi Genzyme: Research Funding; Shire: Research Funding; CSL-Behring: Research Funding; Biotest: Research Funding. Winterhagen:Swedish Orphan Biovitrum: Consultancy, Research Funding. Müller:Swedish Orphan Biovitrum: Consultancy, Research Funding. Oldenburg:Roche: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Octapharma: Consultancy, Honoraria, Research Funding; Novo Nordisk: Consultancy, Honoraria; Grifols: Consultancy, Honoraria, Research Funding; CSL-Behring: Consultancy, Honoraria, Research Funding; Chugai: Consultancy, Honoraria; Biotest: Consultancy, Honoraria; Biogen Idec: Consultancy, Honoraria; Bayer: Consultancy, Honoraria, Research Funding; Shire: Consultancy, Honoraria, Research Funding; Swedish Orphan Biovitrum: Consultancy, Honoraria.

Platelet-Dependent Thrombin Generation Induced By ADP or Activated Factor X Does Not Contribute to Increased In Vivo Thrombin Formation in Myeloproliferative Neoplasms

Introduction: Myeloproliferative Neoplasms (MPN), including the clinical entities Polycythemia Vera (PV), Essential Thrombocythemia (ET), and Primary Myelofibrosis (PMF), are characterized by an increased thrombotic risk, the pathomechanisms of which are not well-understood. It has been suggested that an increased sensitivity of platelets to adenosin diphosphate (ADP) contributes to the hypercoagulable state in PV and ET through increased thrombin generation. In the present study we analyzed plasma levels of thrombin and platelet-dependent thrombin generation in MPN patients with an additional focus on prothrombin activation by the prothrombinase complex.Methods: A total of 33 blood samples were obtained from patients with MPN (PV, n=18; ET, n=5; PMF, n=10) and from 33 healthy blood donors that served as controls. In vitro thrombin generation in platelet-rich plasma (PRP) and platelet-poor plasma (PPP) was assessed using the Calibrated Automated Thrombogram (CAT) assay. To induce thrombin generation either ADP (1 µmol/L final concentration) or activated factor X (FXa, 10 ng/mL final concentration) were applied. To further characterize the MPN-associated hypercoagulable state in vivo, plasma levels of free thrombin were measured using an oligonucleotide-based enzyme capture assay (OECA). Prothrombin activation fragment 1+2 (F1+2), thrombin-antithrombin complex (TAT), and D-dimer were measured additionally.Results: In PRP of MPN patients a slightly higher ADP-induced peak thrombin concentration (Cpeak) was observed than in the healthy controls, with 106 (79-130) vs. 84 (65-110) nmol/L (median and interquartile range, p=.026). There was no statistically significant difference in the ADP-induced endogenous thrombin potential (ETP) in MPN patients (1445, 1194-1643 nmol/L·min) compared with the controls (1417, 1258-1814 nmol/L·min). There was no statistically significant difference in the FXa-induced Cpeak and ETP between MPN patients and controls, with 106 (79-127) vs. 97 (82-128) nmol/L, and 1424 (1165-1560) vs. 1641 (1193-1841) nmol/L·min, respectively. With 0.68 (<0.46-1.20) pmol/L, plasma levels of free thrombin were significantly higher (p=.025) in MPN patients than in the control group, in which median thrombin levels were below the limit of detection. Plasma levels of F1+2 and TAT were also higher in the MPN group than in healthy controls, with 0.31 (0.17-0.50) vs. 0.18 (0.13-0.25) nmol/L (p=.002) and 4.36 (2.53-6.76) vs. 2.36 (<2.00-2.68) ng/mL (p=.003), respectively.Conclusion: Increased plasma levels of thrombin, F1+2, and TAT indicate enhanced in vivo thrombin formation in MPN patients. Despite a slightly increased ADP sensitivity of MPN-platelets, the total amount of thrombin generated in PRP from MPN patients is not increased. This makes it unlikely that a ‘hyperreactivity’ of MPN platelets, resulting in increased activities of the prothrombinase complex on the platelet surface, contributes to the increased thrombin formation in MPN patients. DisclosuresBerens:Shire: Research Funding; Biotest: Research Funding; Pfizer: Research Funding; Sanofi Genzyme: Research Funding; CSL-Behring: Research Funding. Rühl:Shire: Research Funding; Swedish Orphan Biovitrum: Consultancy, Research Funding; Grifols: Research Funding; Sanofi Genzyme: Research Funding; CSL-Behring: Research Funding. Müller:Swedish Orphan Biovitrum: Consultancy, Research Funding. Oldenburg:Roche: Honoraria, Research Funding; Grifols: Honoraria, Research Funding; Chugai: Honoraria, Research Funding; Novo Nordisk: Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding; Shire: Honoraria, Research Funding; Octapharma: Honoraria, Research Funding; CSL Behring: Honoraria, Research Funding; Biogen: Honoraria, Research Funding; Biotest: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Swedish Orphan Biovitrum: Honoraria, Research Funding.

Practical Utilisation of Octapharma FVIII Concentrates in Previously Untreated and Minimally Treated Haemophilia a Patients Entering Routine Clinical Treatment with Nuwiq®, Octanate® or Wilate® — the Protect-NOW Study

Introduction/Objective: The development of FVIII inhibitors remains the greatest challenge to the treatment of previously untreated patients (PUPs) with haemophilia A. Uncontrolled studies in PUPs have suggested that immunogenicity of FVIII concentrates varies between different products. In the SIPPET study, the first and only large randomised controlled study to examine the impact of FVIII product type on immunogenicity, the cumulative incidence of high-titre inhibitors in PUPs and minimally treated patients (MTPs) treated with hamster cell-derived recombinant FVIII (rFVIII) products was 28.4%, compared with 18.6% for plasma-derived FVIII/von Willebrand factor (pdFVIII/VWF) products [1]. However, SIPPET did not include all currently available FVIII products, limiting the applicability of its conclusions to the current haemophilia treatment landscape.In previous clinical studies of true PUPs treated with the pdFVIII/VWF concentrates octanate® [2] and wilate®, the cumulative incidences of high-titre inhibitors were 8.0% and 11.3%, respectively. For the human-cell derived rFVIII Nuwiq®, the cumulative incidence of high-titre inhibitors was 12.8% (data from a preplanned interim analysis) [3]. These incidences suggest a favourable immunogenicity profile compared to products in the SIPPET study. However, there is a need for more real-life data on treatment effectiveness and safety in PUPs and MTPs.The ongoing, non-interventional, multi-centre Protect-NOW study is a prospective and retrospective study evaluating real-life treatment patterns, effectiveness and safety, including inhibitor development, in PUPs and MTPs with severe haemophilia A who are treated with Octapharma's pdFVIII or rFVIII products.Methods: One hundred and forty PUPs (no previous treatment) and MTPs (<5 previous EDs with other FVIII products) with severe haemophilia A of all ages and ethnicities will be studied for 100 EDs or up to 3 years. Treatment effectiveness will be evaluated for regular prophylaxis, treatment of bleeding episodes, and surgical prophylaxis. Optional sub-studies, including epitope mapping, detection of non-neutralising inhibitors, and gene mutation analysis, will assess factors potentially associated with inhibitor development and eradication in patients with severe haemophilia A. Optional sub-studies will be carried out at the central laboratory at the Institute of Experimental Haematology in Bonn. Protect-NOW is planned to include around 17 countries and 50 centres worldwide. In the US, the Protect-NOW will be performed as part of the ATHN-8 study.Results: Recruitment is ongoing, with seven patients recruited at two German centres to date. The study has been approved by central and/or local ethics committees in Germany, US, UK, Spain and Russia, and is under ethical review in Canada. Final data collection is expected in 2022.Conclusions: Protect-NOW will collect real-life clinical experience with Octapharma's FVIII products in PUPs and MTPs. This study will contribute real-world data to the current debate on the relevance of FVIII concentrate type in inhibitor induction.ReferencesPeyvandi F et al. N Engl J Med 2016; 374:2054-64.Klukowska A et al. Haemophilia 2018; 24:221-28.Liesner R et al. Haemophilia 2018; 24: 211-20. DisclosuresOldenburg:Novo Nordisk: Honoraria, Research Funding; Roche: Honoraria, Research Funding; Swedish Orphan Biovitrum: Honoraria, Research Funding; Chugai: Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding; Biogen: Honoraria, Research Funding; Biotest: Honoraria, Research Funding; CSL Behring: Honoraria, Research Funding; Octapharma: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Shire: Honoraria, Research Funding; Grifols: Honoraria, Research Funding. Pavlova:Octapharma: Honoraria; Novo Nordisk: Honoraria. Halimeh:Bayer healthcare, Baxalta Innovations, Biotest, CSL Behring, Novartis, Novo Nordisk, Octapharma, LFB, Pfizer: Honoraria; Bayer Healthcare, Baxalta Innovations, Biotest, CSL Behring, Novo Nordisk, Octapharma, Pfizer: Research Funding. Klamroth:Baxalta (Shire), Bayer, CSL Behring, Novo Nordisk, Octapharma, Pfizer, Shire, and SOBI: Research Funding; Baxalta (Shire), Bayer, CSL Behring, Novo Nordisk, Octapharma, Pfizer, Shire, and SOBI: Consultancy. Versteden:Octapharma AG: Employment. Jansen:Octapharma: Employment. Belyanskaya:Octapharma AG: Employment.

Immunogenicity of Two Plasma-Derived FVIII Products and Simoctocog Alfa in Previously Untreated Patients According to F8 Mutation Type

Background: The SIPPET study investigated inhibitor development in 251 previously untreated patients (PUPs) treated with either plasma-derived FVIII products containing von Willebrand factor (pdFVIII/VWF; n = 125), or recombinant FVIII (rFVIII; n = 126) from hamster cell lines. Amongst PUPs with non-null F8 mutations, none developed inhibitors when treated with pdFVIII/VWF while the cumulative inhibitor incidence was 43% in those treated with hamster-cell-derived rFVIII. In patients with null F8 mutations, the cumulative inhibitor incidences were 31% and 47% in patients treated with pdFVIII/VWF and rFVIII, respectively. In patients with null mutations the cumulative incidences of high-titre inhibitors were 22% and 30% with pdFVIII/VWF and rFVIII, respectively.Aim: To investigate the relationship between inhibitor development and F8 mutation type in PUPs with severe hemophilia A treated with either a rFVIII from a human cell line (Nuwiq®; simoctocog alfa) or either of two pdFVIII/VWF products, one with a VWF/FVIII ratio of 0.4 (octanate®) the other with a VWF/FVIII ratio of 1.0 (wilate®).Materials and Methods: Data from completed multicenter, prospective trials with octanate® and wilate® and interim data from the NuProtect study with Nuwiq® were analyzed. Data on F8 mutation type were available for 50/51, 27/28 and 58/66 patients in each of the studies. All patients in the three studies had no previous treatment with FVIII concentrates or other blood products containing FVIII.Results: In the three studies, 18% (9/50), 7.4% (2/27) and 19%.0% (11/58) of patients had non-null mutations. None of the patients with non-null mutations developed inhibitors with octanate®, wilate® or Nuwiq®. In patients with null mutations, 9.8% (4/41), 12.0% (3/25), and 17.0% (8/47) developed high-titre inhibitors.Conclusions: PUPs with non-null F8 mutations did not develop inhibitors when treated with octanate®, wilate® or Nuwiq®. Whilst the different studies are not directly comparable, the findings with these products, two pdFVIII/VWF and a rFVIII from a human cell line, show similar behavior to the SIPPET trial where no patients with non-null mutations treated with pdFVIII/VWF products developed inhibitors. DisclosuresLiesner:Bayer: Consultancy, Research Funding; Baxalta: Consultancy, Research Funding; Novo Nordisk: Research Funding, Speakers Bureau; Roche: Research Funding; Sobi: Speakers Bureau; Octapharma: Consultancy, Other: Clinical study investigator for NuProtect Study (Octapharma sponsored), Research Funding, Speakers Bureau. Versteden:Octapharma AG: Employment. Lowndes:Octapharma AG: Employment. Belyanskaya:Octapharma AG: Employment. Oldenburg:Grifols: Honoraria, Research Funding; Novo Nordisk: Honoraria, Research Funding; Biotest: Honoraria, Research Funding; Biogen: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Roche: Honoraria, Research Funding; Octapharma: Honoraria, Research Funding; Shire: Honoraria, Research Funding; CSL Behring: Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding; Chugai: Honoraria, Research Funding; Swedish Orphan Biovitrum: Honoraria, Research Funding. Pavlova:Novo Nordisk: Honoraria; Octapharma: Honoraria.

ASPIRE Final Results Confirm Established Safety and Sustained Efficacy for Up to 4 Years of Treatment With rFVIIIFc in Previously Treated Subjects With Severe Hemophilia A

Introduction: Prophylactic replacement of coagulation factor VIII (FVIII) is the standard of care in hemophilia A, given its natural mechanism of action, strict homeostatic regulation, and consistent safety profile. Recombinant FVIII Fc fusion protein (rFVIIIFc) is an extended half-life therapy that demonstrated safety and efficacy in previously treated pediatric, adolescent, and adult subjects with severe hemophilia A in the Phase 3 A-LONG and Kids A-LONG trials (NCT01181128 and NCT01458106, respectively) (Young et al, J Thromb Haemostas, 2015; Mahlangu et al, Blood, 2014). Herein, the final results are reported from ASPIRE (NCT01454739), the long-term extension trial of those 2 studies.Methods: This was an open-label, multicenter, long-term trial of previously treated subjects of all ages with severe hemophilia A. Subjects received 1 of the 4 following regimens: individualized prophylaxis (IP; rFVIIIFc 25‒60 IU/kg every 3-5 days, or twice weekly), weekly prophylaxis (WP; rFVIIIFc 65 IU/kg every 7 days), modified prophylaxis (MP; personalized dosing), or episodic treatment (ET; on-demand dosing based on bleeding episodes). Subjects <12 years of age were eligible for only IP or MP. Investigators could switch a subject's treatment group at any time; therefore, each subject may appear in >1 treatment group. The primary endpoint was development of inhibitors. Secondary endpoints included annualized bleeding rates (ABRs), joint ABRs, spontaneous joint ABRs, exposure days (ED), and factor consumption. Descriptive statistics were used for analysis. Analyses were performed separately based on parent study.Results: A total of 211 subjects (150 were from A-LONG and 61 were from Kids A-LONG) enrolled in ASPIRE, and 186 subjects (132 from A-LONG and 54 from Kids A-LONG) completed the study. Subjects from Kids A-LONG had a median (range) age of 6.0 (2-12) years. Of the subjects from the A-LONG study enrolled to ASPIRE, the median (range) age was 31.0 (13-66) years. Most subjects received IP regardless of parent study (Kids A-LONG: IP [n=59], MP [n=3]; A-LONG: IP [n=110], WP [n=27], MP [n=21], ET [n=13]).No inhibitors were observed throughout the study, and the overall safety profile of rFVIIIFc was consistent with the parent studies and prior interim analyses. ABRs remained low throughout the entirety of ASPIRE in subjects prescribed IP (Table 1). For subjects from the Kids A-LONG study, the median (range) number of ED during ASPIRE was 332.0 (18.0-467.0) days. Total median (range) treatment duration was 166.6 (14.4-203.0) weeks. The median (range) number of ED for A-LONG subjects was 267.5 (8.0-660.0) days. Total median treatment duration was 201.4 (5.1-274.6) weeks. Overall, the median (range) duration of treatment with rFVIIIFc was 4.1 (0.4, 5.9) years.From the end of the parent study to the end of ASPIRE, the rFVIIIFc dosing interval increased for 6.6% and 21.1% of subjects from Kids A-LONG and A-LONG, respectively (Table 2). There was no change in median weekly factor consumption from the end of either parent study to the end of ASPIRE. For subjects from Kids A-LONG, the median (IQR) was 0.0 (0.0‒3.2), while the median (IQR) for those from A-LONG was 0.0 (0.0‒0.0).Conclusions: Throughout up to 4 years of treatment with rFVIIIFc in the ASPIRE extension study, no inhibitors were reported, and low ABRs and extended dosing intervals were sustained. These data are consistent with the well-characterized safety profile and durable efficacy of rFVIIIFc prophylaxis in previously treated subjects of all ages with hemophilia A. [Display omitted] DisclosuresNolan:Bayer: Research Funding; CSL Behring: Research Funding; Sobi: Research Funding. Mahlangu:Sanofi: Research Funding, Speakers Bureau; Roche: Consultancy, Research Funding, Speakers Bureau; Alnylam: Consultancy, Research Funding, Speakers Bureau; Bayer: Research Funding; Biogen: Research Funding, Speakers Bureau; Chugai: Consultancy; Catalyst Biosciences: Consultancy, Research Funding; Amgen: Consultancy; Biomarin: Research Funding, Speakers Bureau; CSL Behring: Consultancy, Research Funding, Speakers Bureau; NovoNordisk: Consultancy, Research Funding, Speakers Bureau; LFB: Consultancy; Shire: Consultancy, Research Funding, Speakers Bureau; Sobi: Research Funding, Speakers Bureau; Spark: Consultancy, Research Funding. Young:Bayer: Consultancy; Bioverativ: Consultancy, Honoraria; Kedrion: Consultancy; Novo Nordisk: Consultancy, Honoraria; CSL Behring: Consultancy, Honoraria; Genentech/Roche: Consultancy, Honoraria; Shire: Consultancy, Honoraria. Konkle:Sangamo: Research Funding; Gilead: Consultancy; Spark: Consultancy, Research Funding; BioMarin: Consultancy; CSL Behring: Consultancy; Genentech: Consultancy; Bioverativ: Research Funding; Pfizer: Research Funding; Shire: Research Funding. Pasi:Shire: Speakers Bureau; Alnylam: Honoraria, Research Funding; Bayer: Speakers Bureau; Octapharma: Honoraria; Pfizer: Speakers Bureau; Biomarin: Honoraria, Research Funding; Sobi: Honoraria; Bioverativ: Honoraria, Research Funding; NovoNordisk: Speakers Bureau; Catalyst Bio: Honoraria; Apcintex: Honoraria. Oldenburg:Novo Nordisk: Honoraria, Research Funding; Chugai: Honoraria, Research Funding; Roche: Honoraria, Research Funding; Biotest: Honoraria, Research Funding; Biogen: Honoraria, Research Funding; Shire: Honoraria, Research Funding; Grifols: Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding; Octapharma: Honoraria, Research Funding; CSL Behring: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Swedish Orphan Biovitrum: Honoraria, Research Funding. Nogami:Chugai Pharmaceutical Co., Ltd: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: Anti-FIXa/X bispecific antibodies , Research Funding, Speakers Bureau. Tripkovic:Sobi: Employment. Rudin:Bioverativ: Employment, Equity Ownership.

Immune Tolerance Induction for FIX Inhibitors Using Combined B and T Cell Immune Modulation Therapy in Severe Hemophilia B

IntroductionDevelopment of an inhibitor to Factor IX is a potentially life-threatening complication that occurs in 2-4% of severe hemophilia B patients. Attempt to eradicate these neutralizing alloantibodies with immune tolerance induction (ITI) regimens using prolonged repeated exposure to FIX fails to induce tolerance in ~70% of cases, and is limited by severe complications including FIX hypersensitivity and nephrotic syndrome. Cases of FIX ITI with immunosuppressive drugs have been reported infrequently. In light of increased understanding of the role of active T lymphocyte regulation of clotting factor-directed B lymphocyte-mediated humoral immunity, an approach combining immune modulating agents and frequent FIX infusions was reported by Beutel, et al (Haemostaseologie, 2009; 2014). We report a multi-institutional retrospective experience with combination immune modulation therapy (CIT), including B cell suppression with anti-CD20 monoclonal antibody Rituximab and T cell modulation using mycophenylate mofetil (MMF) with FIX ITI in 11 males with severe congenital hemophilia B.MethodsThe originally published regimen of Beutel, et al involves a 50 day course including FIX 100 IU/kg twice daily, rituximab 375 mg/m2 x 4 doses, mycophenolate (MMF) 300 mg/m2/dose daily, dexamethasone 6 mg/m2/dose pulses and IVIG 1 g/kg x 6 doses. Following individual Institutional Review Board approval, data on 11 patients was contributed by the INPH investigators and via outreach internationally and collected retrospectively using a uniform data collection form. Cases were included in the CIT series if the combination of FIX, rituximab and MMF was used. Approaches varied in the concomitant use of IVIG and pulse corticosteroids (dexamethasone or prednisone).ResultsCIT was the first immune tolerance therapy for 9/11 patients. Patients were 14-222 months of age at time of CIT, with a median time from inhibitor diagnosis to CIT 20 (range 0-207) months. Median historical peak inhibitor titer prior to initiation of CIT was 3.2 (range 1-42) BU/ml. Inhibitor titers at the initiation of CIT ranged from undetectable to 7 BU/ml. Eighty-onepercent of patients had a history of FIX hypersensitivity reaction. Prior to initiation of therapy, 27% of the patients underwent FIX desensitization during the initial course.The patients received FIX BID (9/11 patients) or QD (2/11 patients), 4 doses of rituximab 375 mg/m2, MMF for a minimum of 49 days (varying duration from 49-1247 days). IVIG was infused in 10/11 patients, with most receiving 3-7 doses during each course of CIT. 6/11 patients received between 2-10 courses of pulse corticosteroids during a CIT course.Each course of CIT achieved disappearance of the titer of FIX inhibitor at a median time of 1 month (range 1-41 months) Hypersensitivity reactions did not limit the courses of CIT, but did recur with inhibitor recurrence in 1 patient. Recurrence occurred in 6/11 patients at a median time of 11 months from time of CIT, in some cases soon after documented B cell recovery with a median inhibitor titer of 1.8 (range 0.7-7) BU/ml. In cases of recurrence, a negative inhibitor titer was achieved again in 1 patient by increasing FIX dose and in 4 patients who received additional courses of CIT. Repeat recurrence was seen in these patients following repeat CIT, although at low titer with a median of 1 BU/ml (range 1-4), allowing ongoing management with FIX in 3 of the 4. Overall, patients have had a median follow-up of 61 months following CIT, 9/11 patients are currently managed with factor IX for prophylaxis and bleeding, while 2/11 use bypassing agents.The most common complication was hospitalization for central venous catheter-related bacterial infection that occurred from < 1 to > 12 months after the use of rituximab in 3/11 patients. Nephrotic syndrome occurred in 3/11 patients. In each case, nephrotic syndrome responded to corticosteroids or re-initiation of CIT and FIX could be resumed.ConclusionsFIX ITI with CIT targeting both B and T cells in hemophilia B is an effective means of achieving a negative inhibitor titer with tolerable safety in this series, allowing return to use of FIX for hemostasis and prophylaxis in most patients. Although recurrence is common and longer follow up is needed, when compared to the experience with using FIX alone, outcomes appear to be improved using CIT with FIX ITI as an initial approach. DisclosuresAbajas:Bayer: Honoraria; CSL Limited: Honoraria. Allen:Shire: Employment, Equity Ownership. Oldenburg:Pfizer: Honoraria, Research Funding; Biogen: Honoraria, Research Funding; CSL Behring: Honoraria, Research Funding; Grifols: Honoraria, Research Funding; Octapharma: Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding; Shire: Honoraria, Research Funding; Chugai: Honoraria, Research Funding; Biotest: Honoraria, Research Funding; Roche: Honoraria, Research Funding; Swedish Orphan Biovitrum: Honoraria, Research Funding; Novo Nordisk: Honoraria, Research Funding.

The Role of Antithrombin Lowering in Patients with Hemophilia: Hemostatic Control Pre- and Post-Fitusiran Dosing Interruption

IntroductionHemophilia A and hemophilia B are rare bleeding disorders characterized by insufficient thrombin generation due to deficiencies in factors VIII or IX, respectively. Standard treatment for hemophilia is currently based on replacement of the deficient factor with factor concentrate. This, unfortunately, subjects the patient to the risk of developing neutralizing antibodies, or inhibitors, against replacement factor VIII or IX. Individuals with inhibitors become refractory to standard replacement therapy, a serious complication for as many as one-third of patients with severe hemophilia A and a lower proportion of patients with hemophilia B. Fitusiran is a once-monthly subcutaneously administered investigational RNA interference therapeutic that targets antithrombin (AT) to improve thrombin generation (TG) and promote hemostasis in patients with hemophilia A or hemophilia B with or without inhibitors. In September 2017, fitusiran dosing in the Phase 2 open-label extension (OLE) study was temporarily suspended to investigate a case of fatal cerebral venous sinus thrombosis. Following this investigation, fitusiran dosing resumed in December 2017 with protocol amendments for bleed management dosing and safety monitoring. During the temporary dosing suspension, AT levels, TG, bleeding events, and frequency of factor replacement and bypassing agents were assessed.MethodsBefore the dosing suspension, 33 patients (hemophilia A=27, hemophilia B=6) were enrolled, of whom 28 patients continued treatment over 20 months in the Phase 2 OLE study, with a median of 11 months on study. As of June 2018, data collected monthly during the clinical hold included AT levels, TG, and description and management of treated bleeding events before and during the clinical hold, which were used to estimate annualized bleeding rates (ABRs) and bleeding rates per month.ResultsAT activity in patients previously receiving fitusiran demonstrated a progressive increase during the interruption of fitusiran dosing. Median %AT increased to >60% after a 5-month period compared with the last data point before dosing interruption. These data confirm our previous findings that discontinuation of fitusiran results in gradual recovery of AT activity over time. Subjects also demonstrated a concomitant steady decrease in TG during the interruption of fitusiran dosing, which occurred over a similar time course to that of AT recovery. Consistent with both the increase in AT activity and decrease in TG, preliminary analysis shows the median overall ABR increased from 1.43 events/year before the dosing interruption to 6.07 events/year during the dosing interruption.ConclusionsDuring a period of fitusiran dosing suspension, recovery of AT activity was accompanied by a decrease in TG and an increase in bleeding events. These observations provide support for the therapeutic hypothesis that AT activity lowering by fitusiran leads to an increase in TG and improved hemostasis. Phase 3 studies of fitusiran are ongoing. DisclosuresRagni:Bioverativ: Consultancy, Research Funding; MOGAM: Membership on an entity's Board of Directors or advisory committees; CSL Behring: Research Funding; SPARK: Consultancy, Research Funding; Shire: Research Funding; Alnylam: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novo Nordisk: Research Funding; Sangamo: Research Funding; Biomarin: Membership on an entity's Board of Directors or advisory committees, Research Funding. Georgiev:Alnylam: Consultancy. Lissitchkov:Novo Nordisk: Other: Investigator fees as a participant of the clinical trial. Austin:Pfizer: Research Funding. Chowdary:Novo Nordisk: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; CSL Behring: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Freeline: Consultancy; Bayer: Honoraria; Baxalta (Shire): Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Swedish Orphan Biovitrum AB (Sobi): Honoraria; Biogen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Foster:Sanofi Genzyme: Employment. Yu:Sanofi Genzyme: Employment. Benson:Sanofi Genzyme: Employment. Madigan:Alnylam Pharmaceuticals Inc: Employment. Nguyen:Alnylam Pharmaceuticals Inc: Employment. Ali:Sanofi Genzyme: Employment. Kadam:Sanofi Genzyme: Employment. Jain:Sanofi Genzyme: Employment. Pasi:Octapharma: Honoraria; Catalyst Bio: Honoraria; Bayer: Speakers Bureau; Shire: Speakers Bureau; NovoNordisk: Speakers Bureau; Sobi: Honoraria; Apcintex: Honoraria; Biomarin: Honoraria, Research Funding; Pfizer: Speakers Bureau; Alnylam: Honoraria, Research Funding; Bioverativ: Honoraria, Research Funding.

Adeno-Associated Mediated Gene Transfer for Hemophilia B:8 Year Follow up and Impact of Removing “Empty Viral Particles” on Safety and Efficacy of Gene Transfer

▪Background: We have previously shown that a single intravenous administration of a self-complementary adeno-associated virus (scAAV) vector containing a codon-optimised factor IX gene, under control of a synthetic liver specific promoter and pseudotyped with serotype 8 capsid, (scAAV2/8-LP1-hFIXco) resulted in a dose-dependent increase in plasma FIX levels in all 10 enrolled severe hemophilia B (HB) patients (ClinicalTrials.gov:NCT00979238; Nathwani et al 2011). FIX activity was stably maintained for at least 3 years (Nathwani et al 2014) but concerns over FIX expression declining over time remain. This is because AAV-mediated transgene expression is mediated mainly by episomally retained viral genomes, which may be lost with natural hepatocyte turn-over. The only vector-associated adverse event was an asymptomatic rise in liver enzymes associated with a decline in FIX levels, occurring within 3 months of gene transfer in two-thirds of the patients treated at a dose of 2x1012 vector genomes(vg)/kg. Liver enzymes normalized with corticosteroids without complete loss of transgene expression. There was no long-lasting toxicity over a period of 3 years but further follow-up is required. The vector preparation used contained an excess of empty capsids, which lacked a full-length viral genome, and are therefore, non-functional but capable of provoking an immune response against transduced hepatocytes. Therefore, a new clinical preparation of scAAV2/8-LP1-hFIXco was manufactured from which most of the empty particles were removed by caesium chloride density centrifugation in the hope that this would reduce the risk of hepatotoxicity. We report on the evaluation of this new vector preparation in severe HB patients and provide an update on up to 8 years follow-up of our original cohort of patients.Methods: Ten subjects were recruited in 2010-2012 to the initial dose-escalation/extension study arm, which entailed a single intravenous infusion of scAAV2/8-LP1-hFIXco (full: empty capsid ratio ~1:10) at a dose of either 2x1011vg/kg, 6x1011vg/kg or 2x1012vg/kg. Two severe HB patients (FIX <1%) were enrolled into the first and mid-dose cohorts, with six patients treated at the high dose. In a follow-on study arm, two severe HB subjects received a dose of 2x1012vg/kg of the new scAAV2/8-LP1-hFIXco preparation (full: empty capsid ratio 1:3) whilst the next 2 patients were treated at a dose of 5x1012vg/kg. In both arms, vector was administered without prophylactic immunosuppression but corticosteroids (starting at 60mg/day) were commenced if liver enzymes increased to ≥2 fold over baseline levels after gene transfer.Results: Transgenic FIX activity levels have remained stable in all 10 subjects treated in the initial dose escalation/extension arm over a median follow-up of 6.7±1.0 years with mean levels in the three dose cohorts at the time of reporting of 1.9±0.6, 2.3±0.3 and 5.1±1.4 IU/l respectively. Over this period, annual FIX concentrate usage has dropped by 66% and annual bleed rate has declined by 82% when compared to pre-gene therapy levels. No late toxicity was observed. Neutralising antibodies to FIX were not detected in any patient but anti-AAV8 capsid-specific antibody levels persisted at high titres in 9 of 10 patients. In patients treated with the new preparation of scAAV2/8-LP1-hFIXco (median follow up = 2.1±1.4 years), mean FIX activity in the 2x1012vg/kg dose cohort was 2.6±0.7 IU/l. This is lower than observed previously at this dose level, but the difference is not statistically significant. Mean steady state FIX levels in the 5x1012vg/kg cohort were 17±5 IU/l. FIX antigen to activity ratio was 1:1. Elevation of serum alanine aminotransferase was observed in 3 of 4 patients treated with the new vector preparation, requiring treatment with corticosteroids.Conclusion: This is the first report to demonstrate stable therapeutic expression of FIX in patients with severe HB over a period of 8 years following systemic administration of scAAV2/8-LP1-hFIXco without late toxicities. We show for the first time that reducing the capsid load by removing empty AAV capsids does not appear to reduce the incidence of hepatotoxicity in patients with severe HB suggesting that other factors are involved in the aetiology of this process. DisclosuresNathwani:Freeline: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; BioMarin: Consultancy, Patents & Royalties; UniQure: Patents & Royalties. Tuddenham:BioMarin: Consultancy, Patents & Royalties; Freeline: Consultancy. Chowdary:Freeline: Consultancy; Baxalta (Shire): Honoraria, Membership on an entity's Board of Directors or advisory committees; Bayer: Honoraria; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Biogen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novo Nordisk: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; CSL Behring: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Swedish Orphan Biovitrum AB (Sobi): Honoraria. McIntosh:Freeline: Consultancy. Recht:Biogen: Research Funding; Shire: Research Funding; Novo Nordisk: Membership on an entity's Board of Directors or advisory committees, Research Funding; Kedrion: Membership on an entity's Board of Directors or advisory committees.

Pharmacodynamics, Pharmacokinetics and Safety of Bay 1093884, an Antibody Directed Against Human TFPI, in Patients with Factor VIII or IX Deficiency (With and Without Inhibitors): A Phase 1 Study

Background: BAY 1093884 is a fully human monoclonal antibody directed against the K1 and K2 domains of human tissue factor pathway inhibitor (TFPI). TFPI regulates the initiation of coagulation and is important for regulation of normal hemostatic response. In factor (F)VIII and FIX deficiency (hemophilia A [HA] and B [HB], respectively), lack of adequate amplification (FXa generation) in tandem with intact regulation contributes to reduced clotting. Inhibition of TFPI has the potential to normalize coagulation in hemophilia even in the absence of FVIII or FIX and it is expected to result in normalization of thrombin generation and consequently improvement in clot formation. TFPI is an attractive therapeutic target as it is unaffected by FVIII or FIX inhibitors and can be targeted in both HA and HB. A potential advantage of targeting TFPI is that downstream mechanisms that protect from excessive coagulation (including antithrombin), remain intact.Methods: BAY 1093884 was evaluated in a multicenter, open-label study in patients with severe HA or HB, with or without inhibitors. Safety, pharmacokinetics (PK) and pharmacodynamics (PD) were assessed after single intravenous (IV) (0.3 and 1 mg/kg) and subcutaneous (SC) (1, 3, and 6 mg/kg) doses. A multiple-dose (MD) cohort received BAY 1093884 150 mg SC once weekly for 6 weeks. Total and free TFPI protein levels and TFPI activity were measured. Thrombin generation assay (TGA) parameters were measured using a calibrated automated thrombogram. Clot formation was assessed using rotational thromboelastometry (ROTEM). A mechanistic physiology-based PK/PD (PBPK/PD) model was developed based on single-dose PK data observed in the study, and used to determine dosing in the MD cohort. Data for HA, HB, Japanese and inhibitor patient subgroups were analyzed separately.Results: Thirty-two patients were enrolled; 30 with HA and 2 with HB, of which 4 (3 HA, 1 HB) were included in the MD cohort. At least 4 patients were treated in each administration method/dose group. Clot formation using ROTEM was assessed in 6 patients. Total and free TFPI-related parameters showed dose-dependent effects, with the concentrations being below the lower limit of quantification for longer duration at higher doses. Improvements in TGA parameters were observed following both IV and SC administration; duration of improvement was longer at higher doses. BAY 1093884 led to shortening of the clotting time in all 6 patients tested. Dose-dependent increases in plasma concentration exceeding dose proportionality were seen following IV and SC administration, consistent with expectations of target-mediated drug disposition for BAY 1093884. Moderate accumulation was observed after MD. Limited information on subgroups (patients with inhibitors [n=4], patients with HB [n=2] and Japanese origin [n=1]) suggests that the PK and PD data for these subgroups were consistent with those from non-inhibitor HA patients. The PBPK/PD model adequately described the concentration time course and total TFPI following single-dose IV and SC administration of BAY 1093884; the PK and PD data from the MD cohort were as predicted by the model. The model also supported that the PK/PD of BAY 1093884 are independent of inhibitor status. No serious adverse events, deaths, or adverse events leading to discontinuation were observed. Fibrinogen and platelet counts remained within normal range at most visits and no meaningful changes in levels of antithrombin, protein C, or FV were seen. The mechanisms for anticoagulation and fibrinolysis were intact.Conclusions: Overall, BAY 1093884 gave no safety concerns in the dose range evaluated. Results suggest that BAY 1093884 shifts hemostatic balance towards greater thrombin generation and clot formation as intended. PK/PD characteristics were similar for inhibitor and HB patients as compared with HA patients, potentially supporting use in all of these patient types. The PBPK/PD model developed can be used for dose selection in future studies. Treatment efficacy will be evaluated in a future study. DisclosuresChowdary:Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Swedish Orphan Biovitrum AB (Sobi): Honoraria; Baxalta (Shire): Honoraria, Membership on an entity's Board of Directors or advisory committees; Novo Nordisk: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Freeline: Consultancy; CSL Behring: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Biogen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bayer: Honoraria. Lissitchkov:Novo Nordisk: Other: Investigator fees as a participant of the clinical trial. Willmann:Bayer: Employment. Schwers:Bayer: Employment. Michaels:Bayer: Employment. Shah:Bayer: Employment.

GO-8: Preliminary Results of a Phase I/II Dose Escalation Trial of Gene Therapy for Haemophilia a Using a Novel Human Factor VIII Variant

▪Background: Haemophilia A (HA), the most common inherited bleeding disorder, is well suited for gene therapy because a modest increase in the plasma factor VIII (FVIII) levels to ≥1% of normal levels will substantially ameliorate the bleeding diathesis and improve quality of life. Earlier gene transfer strategies for FVIII replacement approaches using plasmid electroporation, retroviral vector, or adenoviral vector failed to achieve persistent phenotypic correction of bleeding. We have recently shown that a single peripheral vein administration of adeno-associated viral (AAV) vectors expressing the FIX transgene results in stable long-term expression of transgenic FIX at therapeutic levels without long term toxicity in patients with severe haemophilia B (ClinicalTrials.gov:NCT00979238). However, the use of AAV vectors for HA gene therapy has been limited by inefficient expression of transgenic FVIII and the large size of the FVIII cDNA. To overcome these obstacles, we developed two AAV-FVIII expression cassettes containing a small synthetic liver specific promoter (HLP) driving the expression of codon optimized FVIII variants. These vectors mediated therapeutic expression of FVIII in murine and non-human primate models (McIntosh et al 2013). The first of these constructs, AAV-HLP-hFVIII-SQ, encoding a B-domain deleted FVIII variant, was recently shown (Rangarajan et al, 2017) to mediate sustained (>1 year) normalisation of factor VIII activity in six of seven participants following a single intravenous infusion of AAV serotype 5 pseudotyped vector. However, high vector doses (6x1013 vector genomes/kg [vg/kg]) were required for efficacy, possibly because this product was manufactured using the insect cell/baculovirus system. In this report we describe the preliminary results of our on-going Phase I/II clinical trial (GO-8) evaluating the second FVIII cassette (AAV-HLP-hFVIII-V3), which contains a 17 amino-acid peptide comprising six N-linked glycosylation motifs from the human FVIII B-domain that are highly conserved through evolution. In murine studies, AAV-HLP-hFVIII-V3 mediated expression of FVIII at 3-fold higher levels when compared to AAV-HLP-hFVIII-SQ.Methods: The safety and efficacy of a single intravenous infusion of AAV8-HLP-hFVIII-V3, pseudotyped with AAV serotype 8 capsid was assessed in three adult men with severe hemophilia A (FVIII activity levels ≤1% of normal) in the context of an Investigator led, Phase I/II, open-label, non-randomized, dose-escalation trial (ClinicalTrials.gov: NCT03001830GO-8). The first subject received a dose of 6x1011vg/kg and the subsequent two patients each received a dose of 2x1012vg/kg. AAV8-HLP-hFVIII-V3 was manufactured in mammalian HEK 293T cells. The subjects have been followed up for 13-47 weeks after vector administration.Results: Peripheral vein administration of AAV8-HLP-hFVIII-V3 was well tolerated in all patients with no infusion-related reactions. Transgenic FVIII was detectable within two weeks and was more than 5 IU/dl by 6 weeks of gene transfer in all three subjects. Factor VIII activity (one stage clotting assay) levels have remained stable at 7±1IU/dl in patient 1 over a period of 47 weeks. The second participant is 20 weeks following administration of 2x1012 vg/kg of AAV8-HLP-hFVIII-V3 and has steady-state FVIII activity of 6±2IU/dl. In the third subject, who was also treated at a dose of 2x1012 vg/kg, the steady state FVIII activity is almost 10 times higher at 69±7 IU/dl. Elevation of serum alanine aminotransferase was observed in patients 1 and 3 at between weeks 4-6 after gene transfer, reaching peak levels that were 1.5 X upper limit of the normal range. Both patients were treated with corticosteroids within 48 hours of the onset of transaminitis with no loss of transgene expression. No participant has developed a FVIII inhibitor.Conclusion: Our preliminary results from the ongoing Phase I/II study demonstrate FVIII activity levels >5% in all three subjects with normalization of FVIII:C levels in one patient. These levels are sufficient to reduce/prevent spontaneous hemorrhage and have been achieved using relatively lower doses of AAV8-HLP-FVIII-V3 than reported previously with a related FVIII expression cassette. No Grade III (CTCAE v4.03) or greater adverse events have been observed over a period of 47 weeks after administration of AAV8-HLP-hFVIII-V3. DisclosuresNathwani:Freeline: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Tuddenham:Freeline: Consultancy; BioMarin: Consultancy, Patents & Royalties. Chowdary:Biogen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Baxalta (Shire): Honoraria, Membership on an entity's Board of Directors or advisory committees; Swedish Orphan Biovitrum AB (Sobi): Honoraria; Novo Nordisk: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Honoraria; CSL Behring: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Freeline: Consultancy. McIntosh:Freeline: Consultancy.

Interleukin-1 Blockade in Acute Decompensated Heart Failure: A Randomized, Double-Blinded, Placebo-Controlled Pilot Study.

Heart failure is an inflammatory disease. Patients with acute decompensated heart failure (ADHF) exhibit significant inflammatory activity on admission. We hypothesized that Interleukin-1 blockade, with anakinra (Kineret, Swedish Orphan Biovitrum), would quench the acute inflammatory response in patients with ADHF. We randomized 30 patients with ADHF, reduced left ventricular ejection fraction (<40%), and elevated C reactive protein (CRP) levels (≥5 mg/L) to either anakinra 100 mg twice daily for 3 days followed by once daily for 11 days or matching placebo, in a 1:1 double blinded fashion. We measured daily CRP plasma levels using a high-sensitivity assay during hospitalization and then again at 14 days and evaluated the area-under-the-curve and interval changes (delta). Treatment with anakinra was well tolerated. At 72 hours, anakinra reduced CRP by 61% versus baseline, compared with a 6% reduction among patients receiving placebo (P = 0.004 anakinra vs. placebo). Interleukin-1 blockade with anakinra reduces the systemic inflammatory response in patients with ADHF. Further studies are warranted to determine whether this anti-inflammatory effect translates into improved clinical outcomes.