High-titer Inhibitors Research Articles

Factor VIII-Fc Activates Natural Killer Cells via Fc-Mediated Interactions With CD16.

The most challenging complication associated with Factor VIII (FVIII) replacement therapy is the development of neutralizing anti-drug antibodies, or inhibitors, which occur in 23-35% of severe (FVIII level <1%) hemophilia A (HA) patients and are a serious hindrance to effective management of HA. Consequently, strategies that can either prevent anti-FVIII inhibitors from developing or “tolerize” individuals who develop such antibodies represent a clinically important unmet need. One intervention for patients with high-titer inhibitors is immune tolerance induction (ITI) therapy. Although ITI therapy is the only clinically proven strategy to eradicate anti-FVIII inhibitors, mechanisms of inhibitor reduction remain unknown. Factor VIII Fc-fusion (rFVIIIFc) is an enhanced half-life antihemophilic factor used in replacement therapy for HA. Fc-fusion is a successful protein bio-engineering platform technology. In addition to enhancement of plasma half-life via neonatal Fc receptor (FcRn) binding, other Fc-mediated interactions, including engagement with Fc gamma receptors (FcγR), may have immunological consequences. Several case reports and retrospective analyses suggest that rFVIIIFc offers superior outcomes with respect to ITI compared to other FVIII products. Previously we and others demonstrated rFVIIIFc interactions with activating FcγRIIIA/CD16. Here, we investigated if rFVIIIFc activates natural killer (NK) cells via CD16. We demonstrated rFVIIIFc signaling via CD16 independent of Von Willebrand Factor (VWF):FVIII complex formation. We established that rFVIIIFc potently activated NK cells in a CD16-dependent fashion resulting in IFNγ secretion and cytolytic perforin and granzyme B release. We also demonstrated an association between rFVIIIFc-mediated NK cell IFNγ secretion levels and the high-affinity (158V) CD16 genotype. Furthermore, we show that rFVIIIFc-activated CD16+ NK cells were able to lyse a B-cell clone (BO2C11) bearing an anti-FVIII B-cell receptor in an antibody-dependent cellular cytotoxicity (ADCC) assay. These in vitro findings provide an underlying molecular mechanism that may help explain clinical case reports and retrospective studies suggesting rFVIIIFc may be more effective in tolerizing HA patients with anti-FVIII inhibitors compared to FVIII not linked to Fc. Our in vitro findings suggest a potential use of Fc-fusion proteins acting via NK cells to target antigen-specific B-cells, in the management of unwanted immune responses directed against immunogenic self-antigens or therapeutic protein products.

Immune Tolerance Induction in Hemophilia A Patients Using Wilate® —a Canadian 10 Year Study (PREVAIL)

Introduction:Hemophilia A patients with inhibitors experience a higher morbidity, a worse orthopedic condition and a decreased quality of life. In children with severe hemophilia A, attempting to eliminate inhibitors with immune tolerance induction (ITI) is standard of care; however, in adults with longstanding inhibitors ITI has been less extensively studied. Although practical guidelines in adults are scarce there has been an increase in case reports highlighting the success of ITI in patients with long-standing inhibitors.The possibility that the type of factor VIII (FVIII) product used for ITI could influence the likelihood of successful tolerization is the subject of intense debate. It has been postulated that FVIII products containing von Willebrand factor (VWF) may improve the ITI success rate in some patients with inhibitors, especially those with poor prognostic characteristics, i.e., a historical peak inhibitor level >200 BU/mL, an inhibitor level >10 BU/mL at the start of ITI, age >5 years, or >24 months between inhibitor detection and start of ITI.Study RationaleWilate® is the most extensively used plasma-derived FVIII/VWF concentrate for hemophilia A in Canada. Wilate® has been successful in eliminating inhibitors and reducing bleeding in Canadian children and adults who had developed inhibitors during treatment with other FVIII concentrates, including poor-prognosis patients. Wilate® was well tolerated at the high doses required for ITI. Furthermore, anecdotal clinical experience with Wilate® ITI therapy suggest that patients may have less bleeding, which may limit the need for bypassing agents.This research study was designed to evaluate the efficacy of Wilate® in achieving complete or partial ITI success in severe and moderate hemophilia A patients with inhibitors. Secondary objectives are to determine: the time to achieve immune tolerance; bleeding frequency while on Wilate® ITI treatment; quality of life while on Wilate® ITI treatment; joint health while on Wilate® ITI treatment; relapse rate; and time to relapse following successful ITI using Wilate® .Additional optional sub-studies have been incorporated into this study to gain insight into the biology of ITI, including biomarkers that might predict ITI success or failure: genotyping, anti-FVIII antibody characterization including epitope mapping, and thrombin generation.Study ProceduresThis 10-year Canadian study is an open-label, uncontrolled, multi-centre observational trial with both prospective and retrospective cohorts. A total of at least 80 hemophilia A patients with inhibitors will be enrolled into this study.The dose and frequency of ITI treatment will be at the discretion of the treating physician. However, use of risk-determined treatment protocols is recommended. Patients with high-titre inhibitors (defined as one with historical peak titre ≥5 BU/m) who have inhibitor titre ≤10 BU/mL at the start of ITI are “good-risk” for ITI success, and those who have inhibitor titre >10 BU/mL at the start of ITI are “poor-risk”. Patients with high-titre inhibitors who are good-risk should receive 50 IU FVIII/kg BW every other day or 3 times per week; daily treatment may also be considered. Patients with high-titre inhibitors who are poor-risk should receive 100-200 IU FVIII/kg BW daily. Patients with low-titre inhibitors (defined as one with historical peak titre < 5 BU/mL) should receive 50 IU FVIII/kg BW every other day to 3 times per week.For complete success, an individual must achieve the following three criteria: inhibitor titre <0.6 BU (at least 2 separate blood samplings; incremental recovery of FVIII in the normal range (≥66% of normal); FVIII half-life ≥6 hours. For partial success an individual must meet two of the three criteria. In a partial response, one of the three criteria above is met. In a partial failure : none of the three criteria are met, but inhibitor titre has decreased to <5 BU. In a complete failure none of the three criteria are met, and inhibitor titre is still ≥5 BU.ConclusionThis research study aims to evaluate and document data on the success of Wilate ® ITI in hemophilia patients with FVIII-inhibitors. It is the first 10-year study of its kind in a Canadian setting. The results of this study will provide further insights into addressing one of the most serious complications of hemophilia A treatment. DisclosuresMoorehead:Bioverativ: Honoraria; Pfizer: Speakers Bureau; Octapharma: Honoraria; Bayer: Honoraria; Baxalta: Honoraria, Speakers Bureau. Klaassen:Baxalta: Honoraria; Agios Pharmaceuticals: Consultancy; Hoffman-La Roche LTD: Consultancy; Biogen Canada LTD: Consultancy; Amgen: Consultancy; Octapharma: Honoraria. Carcao:Octapharma: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Novo Nordisk: Honoraria, Research Funding; Biogen: Honoraria, Research Funding; Baxter: Honoraria, Research Funding; Bayer: Honoraria, Research Funding; CSL Behring: Honoraria, Research Funding; Biotest: Honoraria.

Evaluation of Plasma-Derived FVII Efficacy for the Treatment of Bleeding in a Murine Model of Hemophilia a

BackgroundRecombinant human activated factor VII (rhFVIIa) has been used successfully to treat hemophilic patients with neutralizing antibodies to FVIII or FIX. High doses of FVIIa are known to be effective in opposing bleeding in hemophilia B mice and in a mouse model of antibody-induced hemophilia A. The therapeutic indications for human plasma-derived coagulation factor VII (pdFVII) include the prophylaxis and treatment of bleeding disorders in isolated congenital or acquired factor VII deficiency. These preparations are considered to be ineffective in hemophilia A in the presence of inhibitors as they do not contain activated factor VII and therefore cannot be used in haemophiliac patients with inhibitors.AimHere, we investigated the potential use of pdFVII to induce hemostasis in a mouse model of congenital hemophilia A with high-titer inhibitors and antibody-induced hemophilia A in wild-type mice.MethodsF8 KO mice were given 2 IU recombinant human FVIII intravenously weekly for 4 weeks. All mice that developed high-titer inhibitors (>5 BU/ml) were selected to be used in tail transection experiments. Additionally, a model of antibody-induced hemophilia was used by injecting C57BL/6 wild-type mice with 2 doses of murine plasma containing high-titer inhibitors. Mice were anesthetized and tails transected at a cross-sectional diameter of 1.5 mm; the tail was immediately placed in thermostated saline, and the duration of active bleeding was recorded. Bleeding was monitored for 10 minutes. Blood loss was determined by measuring the amount of released haemoglobin in the saline. Mice were pre-treated 15 minutes before haemostatic challenge with a single i.v. injection of saline, rhFVIIa (4 mg/kg) or pdFVII (500 IU/kg).ResultsAdministration of both rhFVIIa and pdFVII corrected the bleeding tendency in both animal models. In particular, pdFVII and rFVIIa treatments caused a marked reduction in the mean bleeding index score of hemophilia A mice with inhibitors in comparison to mice treated with vehicle only (vehicle, mean=5750, s.e.m.= 438; pdFVII, mean=1301, s.e.m.= 819; rFVIIa, mean=1101, s.e.m.=565; P < 0.001 for pdFVII vs vechicle and rFVIIa vs vehicle comparisons, NS for rFVIIa vs pdFVII; ANOVA followed by Bonferroni's multiple comparison test). Comparable results were obtained in the antibody-induced hemophilia model. No statistically significant differences were observed between groups treated with rFVIIa or pdFVII in both models.ConclusionsOur results provide evidence that pharmacological doses of both rFVIIa and pdFVII are effective in promoting hemostasis in animal models of congenital hemophilia A with inhibitors and antibody-induced hemophilia A in wild-type mice. DisclosuresNo relevant conflicts of interest to declare.

Novel Correlation between B Cell Survival Cytokines and Inhibitors in Hemophilia A

The most significant complication of factor VIII (FVIII) replacement in hemophilia A (HA) is the development of neutralizing antibodies (inhibitors) in 20-30% of severe HA patients. While several studies have investigated links between polymorphisms in cytokine regulatory elements and inhibitors, very few have measured patients' serum cytokine levels to understand their contribution. As an antibody-mediated process, inhibitor development must rely on the maturation and proliferation of FVIII-specific B cells, but details of this response remain poorly understood. One of the principal mechanisms of B cell tolerance is regulated at the transitional (TR) B cell stage when newly emerging B cells from the bone marrow remain susceptible to clonal deletion. TR B cell survival is highly dependent upon the abundance of the cytokine BAFF whereas BCMA, another member of the same family, is key to plasma cell survival (Parsons et al, Transplant Rev 2010). Inherited elevated levels of BAFF increase the risk of multiple sclerosis and lupus, where levels of 2 ng/mL (~2x normal) are associated with poor outcome (Steri et al, NEJM 2017, Cheema et al, Arthritis Rheum 2001). BAFF is expressed by dendritic cells, monocytes and macrophages and stimulates B cell proliferation, differentiation and survival (Litinskiy et al, Nature Immunology 2002). High levels of BAFF have been observed during immune reconstitution after rituximab therapy in allograft transplant recipients and may potentiate alloreactive B-cell immunity to promote rejection (Rickert et al, Transplant Rev 2010). We hypothesized that BAFF and/or BCMA may contribute to 1) the development of inhibitors in HA and 2) persistence of inhibitors in refractory patients. Here we analyze correlation of BAFF, BCMA, and 10 other cytokines with inhibitors in human HA samples and BAFF levels in murine inhibitor models.Samples from HA patients were collected longitudinally on an IRB approved protocol. Plasma BAFF and BCMA levels were measured by multiplex ELISA (R&D Systems). Nijmegen-modified Bethesda assays for inhibitor titer and anti-FVIII IgG subclasses were measured as described previously (Hofbauer et al, Blood 2015). A cohort of samples was analyzed for additional cytokines (IL2, IL4, IL5, IL6, IL10, IL12p70, IL17A, IL21, IFNγ, and TNFα) via a customized Luminex based multiplex microbead array (Millipore). C57Bl/6 HA mice were immunized with human FVIII 1 mcg weekly for 4 weeks and Bethesda assays tested at 8 weeks. Plasma from mice with high titer inhibitors (BU > 5, n=8) and non-inhibitor controls (n=2) was analyzed for murine BAFF via ELISA (R&D Systems). Data was analyzed with Prism using ANOVA, χ2 for categorical or t-test for continuous variables and are reported as mean ± SEM.Fifty-seven HA patients, 24 with and 33 without inhibitors, were enrolled with no difference in age, disease severity, or race (p = 0.13, 0.08 and 0.39, respectively by χ2); 2 were treated with rituximab. Patients with inhibitors had a higher frequency of large deletions or inversions. Excluding rituximab patients, BAFF levels were higher in patients undergoing immune tolerance induction (ITI) (1.53 ± 0.16) versus non-inhibitor patients (1.11 ± 0.09), and normalized in those who tolerized (0.98 ± 0.14 ng/mL, p = 0.024). BCMA levels were higher in patients with inhibitors (22.56 ± 1.95 vs 17.59 ± 0.59 ng/mL, p = 0.005) and correlated with Bethesda titer (r = 0.202, p = 0.024). Notably, none of the additional cytokines tested were associated with inhibitors. Consistent with allograft rejection models, BAFF levels rose 4-5 fold from baseline post-rituximab and did not normalize until anti-FVIII IgG was undetectable in rituximab-exposed patients. Consistent with human data, HA mice with inhibitors had higher BAFF levels than non-inhibitor mice (6.96 ± 0.37 vs 5.76 ± 0.08 ng/mL, p = 0.015, Welch's t-test).Here we uncover a novel underlying mechanism for modulation of inhibitor formation. BAFF may allow for the persistence of anti-FVIII antibodies during ITI and after rituximab therapy in HA inhibitor patients. Soluble BCMA levels correlate with inhibitor titer, suggesting it plays a role in anti-FVIII plasma cell survival. BAFF levels also increase in HA mice with inhibitor formation. Preclinical testing of antibody or cellular therapies against these cytokines may facilitate translational studies with FDA approved anti-BAFF therapies and enhance tolerance induction in HA. DisclosuresLacey:Novartis: Research Funding; Genentech: Honoraria. Chen:Novartis: Research Funding. Raffini:CSL Behring: Consultancy; Genetech: Consultancy; Green Cross Inc: Consultancy; Bayer: Consultancy.

US Economic Impact of Recombinant FVIII Vs Plasma-Derived FVIII/VWF in Previously Untreated Hemophilia a Patients

INTRODUCTIONInhibitor development to FVIII hemophilia therapy results in increased complications and substantial economic costs. The SIPPET study found higher inhibitor rates in previously untreated patients (PUPs) treated with certain recombinant FVIII (rFVIII) than in PUPs treated with plasma-derived FVIII with Von Willebrand factor (pdFVIII/VWF). The purpose to this study was to quantify differences in FVIII and bypassing agent utilization and to quantify the economic impact of treating PUPs with pdFVIII/VWF vs rFVIII. METHODSAn Excel-based clinical and economic model using SIPPET study data was developed from a United States healthcare payer perspective over a 5-year treatment period. For the model, it was assumed that 80% of PUPs were treated with FVIII prophylaxis and 20% received on-demand FVIII. FVIII-treated prophylaxis patients received 3 infusions of 40 IU/kg/week and were assumed to have no bleeds. Patients treated with on-demand FVIII had an annualized bleed rate (ABR) of 18.0 bleeds, 10% of which were serious bleeds (ie, requiring hospitalization). Serious bleeds were treated with an average of 1.5 infusions of 50 IU/kg, while minor/moderate bleeds were treated with 1.0 infusion of 25 IU/kg. Inhibitor development rates from the SIPPET study resulted in 18.6% of pdFVIII/VWF patients and 28.4% of rFVIII patients developing high-titer inhibitors. The model also assumed that patients developed their inhibitors over a period of 6 months after treatment initiation (ie, 50 exposure-days) and that those who developed high-titer inhibitors initiated immune tolerance induction (ITI) 3 months after the inhibitor diagnosis. Patients with high-titer inhibitors received bypassing agents (rFVIIa or aPCC) either prophylactically to prevent bleeds or on-demand to treat bleeds. It was assumed that prior to initiating ITI, 50% of patients received prophylaxis; during ITI, all patients received prophylaxis; and after unsuccessful treatment with ITI, all patients received bypassing agents prophylactically. Inhibitor patients successfully treated with ITI returned to FVIII treatment and were assumed to receive FVIII prophylaxis consisting of 3 infusions of 40 IU/kg per week. All cost inputs were identical between the 2 arms, except for the costs of the 2 antihemophilic agents. Treatment costs were calculated monthly using wholesale acquisition cost for medications and were based on patient weight, with total costs being summed over 5 years. RESULTSInitiating treatment with pdFVIII/VWF instead of rFVIII in severe hemophilia A PUPs significantly reduced the treatment costs of FVIII replacement, with total FVIII costs of $596,700 and $868,420 respectively. The use of bypassing agents was 35% lower in the pdFVIII than the rFVIII cohort, with costs of $212,947 vs $325,146, respectively. Hospital costs, exclusive of antihemophilic agents, were $19,173 in the pdFVIII/VWF cohort and $17,364 in the rFVIII cohort. Total cumulative costs per patient over the 5-year period were $828,821 for pdFVIII/VWF patients and $1,210,929 for rFVIII patients, representing a total savings of $382,108 per patient over the 5-year period for an average annual savings of $76,422 per patient. This amounted to greater than 30% savings. CONCLUSIONTreatment of hemophilia A PUPs with pdFVIII/VWF over a 5-year period may lead to lower FVIII and bypassing agent utilization than patients treated with rFVIII, ultimately resulting in significant cost-savings for pdFVIII/VWF-treated patients when compared to rFVIII-treated patients. Further research is warranted to corroborate these findings in real-world analyses. DisclosuresSidonio:Shire: Consultancy, Research Funding; Bioverativ: Research Funding; Grifols: Research Funding; CSL Behring: Consultancy; Bioverativ: Consultancy; Novo Nordisk: Consultancy; Bayer: Consultancy. Runken:Grifols: Employment. O'Day:Xcenda: Consultancy, Other: Employed by Xcenda a consulting company that received financial support to conduct the analysis on behalf of Grifols.. Meyer:Xcenda: Consultancy, Other: Employed by Xcenda a consulting company that received financial support to conduct the analysis on behalf of Grifols.. Spears:Grifols: Employment.

Estimating the Cost Savings and Cost Effectiveness of Low Dose Immune Tolerance Induction (ITI) Therapy with Bypassing Agent Prophylaxis Compared to High Dose ITI and Low Dose ITI

Introduction: The International Immune Tolerance Study (I-ITI) demonstrated comparable success rates between low (FVIII 50 IU/kg/TIW) and high dose (FVIII 200 IU/kg/day) ITI regimens for hemophilia A patients with high-titer inhibitors. While costlier, the high dose ITI regimen was associated with shorter time-to-treatment success and with fewer bleeding episodes compared to the low dose ITI regimen. Kenet (2017) estimated that adding aPCC prophylaxis to a low dose ITI regimen may be cost saving to achieve negative titer compared to high dose ITI, while achieving comparable bleeding outcomes. Low dose ITI is certainly the least costly treatment option; however, due to increased bleeds in this arm, many treaters are now using low dose ITI with bypassing-agent prophylaxis (BAP). The choice of which ITI regimen to use is enhanced by a cost effectiveness analysis (CEA) comparing the cost and clinical outcomes of low dose ITI with and without BAP.Methods: An economic model was developed to compare all three regimens: high dose ITI, low dose ITI and low dose ITI with BAP. All model inputs were derived from clinical trials. In the I-ITI study, patients on the high and low dose regimens were treated for a median of 4.6/6.9/10.6 versus 9.2/13.6/15.5 months, respectively, until achieving negative inhibitor titer/first normal recovery/tolerance, and experienced an average of 0.28/0.09/0.03 versus 0.62/0.13/0.15 bleeds/month, respectively, in each phase. Based on the BAP trials, the addition of aPCC (85 U/kg/TIW) and rFVIIa (90 μg/kg/day) achieved a 62% and 45% reduction in bleeding frequency, respectively. The CEA is based on a Markov model structure and simulates costs and outcomes for each treatment regimen over three years to allow for the average patient to complete ITI and achieve tolerance on either regimen. The analysis uses US third party payer perspective, applies a 3% discount rate, and regimen costs are limited to drug costs. One-way and probabilistic sensitivity analyses were performed.Results: The estimated treatment costs over three years were $216,024, $57,366, and $30,506 per kilogram body weight for high dose ITI, low dose ITI with aPCC prophylaxis, and low dose ITI alone, respectively. The estimated number of bleeds that occur with each regimen over three years were 3.9, 6.1, and 10.9, respectively.Adding aPCC prophylaxis to a low dose ITI regimen was estimated to prevent 4.8 bleeds and cost $5,642 per kg per bleed avoided relative to low dose ITI alone in the base case. The addition of rFVIIa prophylaxis to low dose ITI also improved bleeding with a higher cost; however, compared to aPCC prophylaxis, low dose ITI with rFVIIa prophylaxis was associated with higher cost, and less improvement in outcomes. Model results were robust in the majority of sensitivity analyses performed. Given the addition of aPCC prophylaxis is expected to reduce the risk of bleeding during ITI, this may improve the time to negative titer, and thus further lower the cost per bleed, i.e. a 50% reduction in time to negative titer results in a 52.8% reduction in cost per bleed avoided due to aPCC prophylaxis.Conclusion: A low dose ITI regimen with aPCC prophylaxis may be cost saving compared to a high dose ITI regimen, with a comparable risk for breakthrough bleeds. While more costly than low dose ITI alone, adding aPCC prophylaxis to a low dose ITI regimen may be a cost-effective option to control bleeding during ITI, especially if the addition of aPCC prophylaxis leads to a subsequent reduction in the length of time until a negative titer is achieved. DisclosuresKenet:Alnylam: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Shire: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Opko biologics: Consultancy, Research Funding; Bayer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; CSL Behring: Membership on an entity's Board of Directors or advisory committees. Oladapo:Shire: Employment. Epstein:Shire: Consultancy. Christin:Shire: Consultancy. Novack:Shire: Employment, Equity Ownership. Nugent:Genentech: Consultancy; Novo Nordisk: Consultancy; Shire: Consultancy; Bayer: Consultancy.

Emicizumab Prophylaxis in Adolescent/Adult Patients with Hemophilia A Previously Receiving Episodic or Prophylactic Bypassing Agent Treatment: Updated Analyses from the HAVEN 1 Study

Emicizumab Prophylaxis in Adolescent/Adult Patients with Hemophilia A Previously Receiving Episodic or Prophylactic Bypassing Agent Treatment: Updated Analyses from the HAVEN 1 Study

Bleeding Events and Safety Outcomes in Pediatric Persons with Hemophilia A with Inhibitors: The First Non-Interventional Study (NIS) from a Real-World Setting

Bleeding Events and Safety Outcomes in Pediatric Persons with Hemophilia A with Inhibitors: The First Non-Interventional Study (NIS) from a Real-World Setting

Epitope Mapping of Inhibitor Patient Plasmas during Immune Tolerance Induction

Background The most significant complication in the management of patients with severe hemophilia A continues to be the development of anti-factor VIII antibodies. The standard of care for inhibitor eradication is immune tolerance induction (ITI). ITI refers to frequent and prolonged exposure to fVIII, often at high doses, as a method to induce tolerance. Patients with severe hemophilia A typically develop a polyclonal B-cell response to fVIII, with antibodies to the A2 and C2 domains being the most prevalent. We have previously identified 5 non-overlapping B cell epitopes with unique characteristics in the A2 domain, and 3 in the C2 domain. Specifically within the A2 and C2 domain we have shown that epitope is a better predictor of response to treatment than the Bethesda titer. Using monoclonal antibodies (MAbs) with known epitopes, we tracked the change in A2 and C2 epitope response over time in 9 high-titer inhibitor patients during ITI.Methods B-cell epitopes were mapped using a previously published competition ELISA. For each epitope tested, a single well characterized biotinylated anti-fVIII MAb was competed against antibodies present in patient plasma. FVIII deficient plasma was used as a no antibody control. In the A2 domain, 4 of the 5 non-overlapping epitopes were tested (groups A, B, D, and E). All three non-overlapping epitopes in the C2 domain were tested (A, B, and C). Absorbance was measured at 405 nm and ELISA titers were calculated using an O.D. value of 0.3. Samples were considered positive if there was at least a 20% decrease in the ELISA titer ratio between patient and control plasma.Results We tested 85 plasma samples from 9 patients, with each individual patient contributing between 4-13 samples over the course of ITI. At the point of highest titer, patients had a mean of 4.4 of 7 tested epitopes. The most frequently detected epitopes were 2-76 (group A) and 4F4 (group B) in the A2 domain and 2-117 (group C) in the C2 domain. All patients (9 of 9) had antibodies to these epitopes at one or more time points during ITI. The 1D4 (group E) and 2-54 (group D) epitopes within the A2 domain and the I54 (group A) and 1B5 (group B) epitopes within the C2 domain were less frequent but still prominent, with 8 of 9 patients having the I54 epitope, 7 of 9 having the 1D4 epitope, and 6 of 9 having the 2-54 and 1B5 epitopes.Eighty of the 85 patient samples tested were found to have a positive Bethesda titer, indicating inhibitory antibodies were present. Higher inhibitory titers correlated with more epitopes detected. Of the 80 inhibitory samples, 64% of them showed antibodies to the 2-76 epitope, 59% to the 4F4 epitope, and 53% of them showed antibodies to the 2-117 epitope. The less frequent 1D4, 2-54, I54, and 1B5 epitopes were seen in 28%, 18%, 23%, and 16% respectively. The number of epitopes and the titers for those epitopes detected increased or decreased with the Bethesda titer over time. However, the most prevalent epitopes remained dominant even at times of relapse.Conclusion A broad antibody spectrum was seen in patients with hemophilia A and inhbitors. Multiple different epitopes within the A2 and C2 were present. Given our previous studies that showed epitope was more important than titer in the pathogenicity of these antibodies, tracking epitopes over time may provide valuable knowledge regarding response to therapy and may direct the clinician to adjust ITI strategy. Ultimately, knowledge of the dominant epitope that persists or recurs after ITI may be a target for individualized treatment. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Molecular Characterization of Two Homozygous Factor VII Variants Associated with Intracranial Bleeding.

Clinical parameters have been extensively studied in factor (F) VII deficiency, but the knowledge of molecular mechanisms of this disease is scarce. We report on three probands with intracranial bleeds at an early age, one of which had concomitant high titer of FVII inhibitor. The aim of the present study was to identify the causative mutations and to elucidate the underlying molecular mechanisms. All nine F7 exons were sequenced in the probands and the closest family members. A homozygous deletion in exon 1, leading to a frame shift and generation of a premature stop codon (p.C10Pfs*16), was found in proband 1. Probands 2 and 3 (siblings) were homozygous for a missense mutation in exon 8, resulting in a glycine (G) to arginine (R) substitution at amino acid 240 (p.G240R). All probands had severely reduced FVII activity (FVII:C < 1 IU/dL). Treatment consisted of recombinant FVIIa and/or plasma concentrate, and proband 1 developed a FVII inhibitor shortly after initiation of treatment. The FVII variants were overexpressed in mammalian cell lines. No FVII protein was produced in cells expressing the p.C10Pfs*16 variant, and the inhibitor development in proband 1 was likely linked to the complete absence of circulating FVII. Structural analysis suggested that the G to R substitution in FVII found in probands 2 and 3 would destabilize the protein structure, and cell studies demonstrated a defective intracellular transport and increased endoplasmic reticulum stress. The molecular mechanism underlying the p.G240R variant could be reduced secretion caused by protein destabilization and misfolding.

Initial experience of Emicizumab prophylaxis in children with inhibitor positive severe hemophilia A from a single center in India

BackgroundAlthough improvement in the access to clotting factor concentrates and provision of comprehensive care has dramatically improved the quality of life of patients with severe hemophilia, inhibitor-positive patients still develop significant morbidity in terms of joint involvement, progression to disability and life threatening bleeds due to poor access to bypassing agents and immune tolerance induction. ObjectiveTo analyze the response of patients with severe hemophilia A and high-titer inhibitors started on Emicizumab prophylaxis. MethodsPatients with inhibitor-positive severe Hemophilia A were followed up for bleed frequency, joint involvement and health-related quality-of-life assessment (HR-QOL) using PedHAL questionnaire. The data from 1 year prior to start of Emicizumab or from diagnosis of inhibitor status (if shorter period) was compared to that 4 weeks after start of the drug. Emicizumab was administered as per standard guidelines. Results4 patients were initiated on Emicizumab prophylaxis and followed for a median period of 34 weeks (19–57 weeks). The average bleed frequency reduced from 2 to 3.5 per month to 0 after initiation of prophylaxis. No significant adverse events were reported. Improvement in raw and normalized data for all 7 domains of PedHAL which measures activities of daily living was observed. Functional improvement was noted as early as 4 weeks into treatment. ConclusionEmicizumab is a promising agent for prophylaxis of patients with severe hemophilia A and high titre inhibitors.

Safety evaluation of emicizumab prophylaxis in individuals with haemophilia A

ABSTRACT Introduction Emicizumab is a bispecific-humanized monoclonal antibody that improves hemostasis by bridging activated factor IX and factor X to substitute for the function of missing activated FVIII. It is an alternative to prophylaxis with factor VIII replacement and is associated with improved outcomes in individuals with hemophilia A with and without inhibitors. Areas covered Emicizumab is efficacious in reducing bleeding events when compared to on-demand treatment and factor-based prophylaxis. Except for the few thrombotic microangiopathy and thrombotic event cases mainly seen in the HAVEN 1 trial, emicizumab has an overall excellent safety profile with minimal side effects. Expert opinion Knowledge gaps include the efficacy and safety of emicizumab in younger age groups and those with mild or moderate hemophilia A. Future directions for research include exploring the risk of inhibitor recurrence in patients with a history of high titer inhibitor who have been successfully tolerized, who switch from factor prophylaxis to emicizumab, as well as conducting ‘real world studies’ to evaluate the patient’s perception of emicizumab in regard to ease and tolerability in order to optimize individualized treatment plans.

Patients with haemophilia A with inhibitors in China: a national real‐world analysis and follow‐up

The development of alloantibodies (inhibitors) against coagulation factor VIII (FVIII) is the most serious complication of FVIII replacement therapy in patients with haemophilia A (HA). We carried out a nationwide study focussing on patients with HA with inhibitors in China to evaluate the condition and management of this population. The study retrospectively analysed patient characteristics, clinical history, manifestation, treatment strategy as well as individual haemophilia care of 493 patients with inhibitors (466 with severe HA and 27 with non-severe HA) registered all over China. The median (interquartile range) age at diagnosis of FVIII inhibitors was 13(5-28)years in patients with severe HA and 24(10·5-39·5)years in patients with non-severe HA. Most patients (85%) had high-titre inhibitors. Prothrombin complex concentrate and recombinant activated coagulation factor VII were used respectively in 76·2% and 29·2% of patients for acute bleeding. Only 22·3% of patients underwent immune tolerance induction (ITI) treatment, of whom 64·9% achieved negative inhibitor titre. In patients who did not undergo ITI, the inhibitors turned negative in 17·7%, and patients with low peak inhibitor titre were more likely to acquire negative titre spontaneously (odds ratio 11·524, 95% confidence interval 5·222-25·432; P=0·000). We recorded that 3·2% of the patients died from haemophilia-related life-threatening bleeding.

Immunoadsorption for the Treatment of Acquired Hemophilia: New Observational Data, Systematic Review, and Meta-Analysis.

The treatment of patients with acquired hemophilia is challenging due to life-threatening hemorrhages, delayed response, and adverse effects to immunosuppressive agents. Even though immunoadsorption (IA) rapidly removes autoantibodies against factor VIII, this intervention's effectiveness is still a matter of debate. We aimed to study important outcomes of IA as adjunctive treatment in patients with acquired hemophilia. We performed comprehensive literature searches in MEDLINE and EMBASE databases. Clinical and laboratory data of all patients treated in our institution were additionally included. Literature searching yielded 498 records, of which 10 studies describing 106 patients were finally included. The number of patients varied from 1 to 65, and patients' ages ranged between 14 and 89. Treatment criteria in most patients were (1) failed response to immunosuppressive treatment alone, and/or (2) uncontrollable bleeding episodes, and/or (3) high inhibitor titer. Methodological quality was moderate. The number of IA sessions varied from 1 to 24. Within our institution, 12 patients have been treated since 2002; median age was 76 years (range 34-86); median titer of factor VIII inhibitor was 20 Bethesda units (range 3-214). Pooled estimates, modeling a random-effect binominal distribution incorporating the Freeman-Tukey double arcsine transformation, were 86% in case of factor VIII recovery (95% confidence interval 76%-94%), 95% for reduction of factor VIII inhibitor (83%, 100%), and 7% in case of death (0%, 18%). Our data suggest that IA might be a beneficial adjunctive treatment in patients with high-risk acquired hemophilia, but future studies shall confirm this observation.

Inhibitor in Congenital Factor VII Deficiency; a Rare but Serious Therapeutic Challenge-A Systematic Literature Review.

Background: Congenital factor (F) VII deficiency is a rare coagulation factor deficiency with an estimated incidence of 1 per 500,000 individuals. Patients with severe FVII deficiency present a broad range of clinical presentations. Alloimmunization against exogenous FVII, as the main challenge of replacement therapy, is an extremely rare phenomenon that is accompanied by a high rate of life-threatening bleeding, that renders replacement therapy less effective. Due to the importance of the issue, we performed a systematic literature review in order to assess incidence, molecular basis, clinical presentations, and therapeutic challenge and management of inhibitor in congenital FVII deficiency. Strategy of search: This systematic review was performed in accordance with PRISMA guidelines. We performed an English-language literature review in the PubMed, EMBASE, Scopus, and Google Scholar databases, using the following keywords: “factor VII inhibitor”, “factor VII inhibitors”, “FVII inhibitors”, “congenital FVII deficiency”, “recombinant factor VII”, “anti rFVIIa”, “replacement therapy”, and “alloantibody”. Results: Out of 380 patients in the 13 studies, 27 had inhibitor against FVII; 18 were male, 7 were female, while the sex of 2 was not stated. The majority (92%) developed a high-titer inhibitor (Bethesda Unit > 5). All patients had severe FVII deficiency (FVII:C < 10%), and the majority received recombinant FVII prior to inhibitor development (N: 24, 89%). Among ten patients with a detected mutation, three subjects had a common non-sense (30%), and two had a deletion (20%). Conclusions: Inhibitor development is a relatively rare phenomenon seen only in severe FVII deficiency, where it is associated with severe and life-threatening presentations, treatment challenge, and economic burden on the patients and their families.

Case report of nasal pseudotumor – a rare presentation in severe haemophilia A with high titre inhibitors

Abstract Haemophilia patients with inhibitors suffer from increased morbidity and mortality due to the ineffectiveness of factor VIII replacement. Pseudotumors are rare but dangerous complications in these patients, and nasal pseudotumors are even rarer. Here, we present the case of a young child with severe haemophilia A with high titre inhibitors who developed a nasal pseudotumor. When immune tolerance therapy was not possible due to financial constraints, he was treated with FEIBA prophylaxis and rituximab. The pseudotumor was managed with surgical excision. We conclude that epistaxis in haemophiliacs can be due to an underlying nasal pseudotumor, and highlight the use of rituximab for the eradication of inhibitors.

Emicizumab in severe hemophilia A

Emicizumab is a recombinant, humanized, bispecific, asymmetric monoclonal antibody that bridges activated factor IX and factor X (FX) and leads to activation of FX, thus mimicking the hemostatic function of activated factor VIII (FVIIIa). The clinical trial program showed that emicizumab prophylaxis maintains low bleed rates and is well tolerated by patients with hemophilia A of all ages with and without factor VIII (FVIII) inhibitors. Emicizumab prophylaxis in severe hemophilia A patients with high titer inhibitor against FVIII was launched in Poland in 2020. As of April 2021, 42 patients were receiving emicizumab in Poland, not including clinical trials. The aim of this paper was to review the most recent data on the role of emicizumab in the management of patients with severe hemophilia A.

Real-World Clinical Outcomes in Previously Untreated and Minimally Treated Patients with Congenital Factor VIII Deficiency: The San Diego Experience

Introduction: Emicizumab is a bi-specific, humanized, monoclonal antibody that mimics the function of activated Factor VIII (FVIII). It is approved for prophylaxis in persons of all ages with hemophilia A (HA) both with and without a FVIII inhibitor. The data on the use of emicizumab in previously untreated patients (PUPs) and minimally treated patients (MTPs) are scarce, with only one PUP reported in the pediatric clinical trials for the drug. The objective of this retrospective study was to describe our real-world institutional experience in prescribing emicizumab prophylaxis for PUPs and MTPs. Methods: This analysis includes six patients between 1-23 months of age with severe HA prescribed emicizumab prophylaxis at Rady Children's Hospital San Diego (RCHSD) Hemophilia and Thrombosis Treatment Center between November 2017 to April 2020. Data collected through April 2020 included demographics, inhibitor history, bleeding and treatment history prior to initiation of emicizumab prophylaxis, reason for initiation of emicizumab, age, weight, and dosing regimen at the beginning of emicizumab prophylaxis, bleeding and surgical history after initiation of emicizumab, and adverse reactions to emicizumab. Results: Our cohort of six patients included 1 PUP and 5 MTPs with less than 20 FVIII exposure days (ED) prior to the initiation of emicizumab prophylaxis. All MTPs patients were previously treated with on-demand treatment prior to the initiation of emicizumab. Demographic and clinical characteristics are shown in Table 1. The median age and weight at initiation of emicizumab were 8 months (range: 1 to 23 months) and 9.5 kg (range: 4.4 to 11.8 kg) respectively. One patient who was treated on-demand was diagnosed with a high-titer FVIII inhibitor in the setting of a mouth bleed and started emicizumab prophylaxis after bleed management. The most common reason for initiation of emicizumab prophylaxis in non-inhibitor patients was the ability to administer medication without reliable venous access. Following four once weekly loading doses with emicizumab, five patients continued weekly maintenance dosing and one patient continued every 28 day maintenance dosing. Patients were followed for a median of 9 months (range 3-24 months) after emicizumab initiation. Four patients, including the patient with a known FVIII inhibitor prior to starting emicizumab, had zero treated bleeds following initiation of emicizumab prophylaxis. Two patients were diagnosed with high-titer FVIII inhibitors on routine inhibitor surveillance after initiation of emicizumab prophylaxis and were the only patients who experienced treated bleeds during the study period. The majority (6 of 8) of treated bleeds were traumatic. The patient with the highest FVIII inhibitor titer experienced a disproportionately high number of the total treated bleeds though three of the seven bleeds were prior to FVIII inhibitor diagnosis. No patients required central venous catheter placement. One patient underwent circumcision with peri-operative FVIII replacement and did not have post-op bleeding. In terms of medication safety, one patient reported injection site bruising. No patient experienced severe adverse reactions including thrombotic microangiopathy, thrombosis, or clinical evidence of anti-drug neutralizing antibodies. All patients continued emicizumab prophylaxis during the study period. Conclusions: We found that prophylaxis with emicizumab is efficacious and well-tolerated in our cohort of young children with HA under the age of 24 months both with and without high-titer FVIII inhibitors. Two patients were diagnosed with high-titer FVIII inhibitors on routine surveillance after initiation of emicizumab prophylaxis, which highlights the importance of continuing FVIII inhibitor surveillance after initiation of emicizumab. More data are needed on the use of emicizumab, particularly in PUPs and MTPs, with regards to safety, efficacy, and prevention of inhibitors in this patient population. Disclosures Thornburg: American Thrombosis and Hemostasis Network: Research Funding; National Hemophilia Foundation: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer Pharmaceuticals: Research Funding; Biomarin: Consultancy, Speakers Bureau; Genentech: Speakers Bureau; NovoNordisk: Research Funding; Sanofi Genzyme: Consultancy, Other: Data Safety Monitoring Board, Research Funding; Spark Therapeutics: Consultancy; Bluebird Bio: Consultancy; Ironwood Pharmaceuticals: Consultancy, Other: Data Safety Monitoring Board.

Assay Interference By Emicizumab in Aptt and Chromogenic Based FVIII:C and Inhibitor Assays

Background:Emicizumab, a humanized bispecific antibody, has been approved for the prophylactic treatment of hemophilia A in patients with and without inhibitors. Both activated partial thromboplastin time (APTT) based FVIII:C and inhibitor assays, as well as select chromogenic FVIII:C assays have been reported to be affected in the presence of emicizumab. Aims:To assess the effect of emicizumab spiked at 0, 25, 50, and 100 ug/mL into congenital FVIII deficient plasma, containing FVIII inhibitor at 0, 1 and 5 BU, in seven commonly used APTT and chromogenic based FVIII:C assays. Samples were also tested in an APTT and chromogenic based FVIII Nijmegen inhibitor assay. Methods:Four IVD approved, APTT based FVIII:C assays and three chromogenic FVIII:C assays were evaluated (Table 1). In addition, FVIII inhibitor levels were measured by Nijmegen Bethesda assay using either APTT (Actin®FSL, Siemens Healthcare) or chromogenic based (FVIII Chromogenic Assay, Siemens Healthcare) FVIII:C assays. Results:Reagent and dose-dependent increases in FVIII:C were observed for all APTT reagents tested as well as for the BIOPHEN FVIII:C chromogenic assay. The presence of low or high titer inhibitors did not impact APTT based FVIII:C measured. Neither of the two chromogenic FVIII:C assays, using bovine based FX and FIXa (i.e. FVIII Chromogenic Assay and Coatest®SP4) demonstrated measurable FVIII levels at any of the emicizumab concentrations tested. FVIII Nijmegen Bethesda inhibitor results using APTT based FVIII:C, when tested in the presence of emicizumab, consistently produced false negative results (Table 1) whereas results determined using the FVIII Chromogenic Assay measured inhibitor titers at expected concentrations. Conclusion:Confirming previously published results, emicizumab interferes in the APTT based FVIII:C and Nijmegen Bethesda inhibitor assays. Even at low emicizumab concentrations, false negative inhibitor titers are measured in APTT based inhibitor assays. Using a bovine based chromogenic FVIII:C assay is imperative when measuring both FVIII:C and FVIII inhibitors in samples containing emicizumab. Disclosures Tiefenbacher: Laboratory Corporation of America Holdings:Current Employment, Current equity holder in private company;Novo Nordisk:Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees;Shire:Honoraria;Octapharma:Honoraria;Siemens Healthcare:Membership on an entity's Board of Directors or advisory committees;BioMarin:Membership on an entity's Board of Directors or advisory committees.

Molecular Characterization of Two Homozygous Factor VII Variants Associated with Intracranial Bleeding

Introduction Congenital factor (F) VII deficiency is an inherited bleeding disorder with an autosomal recessive inheritance pattern. We report on two probands who had intracranial bleedings as toddlers. One of them had concomitant high titer FVII inhibitor that developed soon after initiation of treatment with recombinant FVII (eptacog alfa, NovoSeven). The underlying molecular mechanisms of these variants by recombinant overexpression in human cell lines were characterized. Methods All nine F7 exons, including exon/intron boundaries, were sequenced, and the sequences were aligned with the NCBI reference sequence for F7. Clotting factor activity, FVII antigen (FVII:Ag) and thrombin generation (TG) were measured in patient plasma. Structural analysis was based on the previously determined crystal structure of the complex of FVII with soluble tissue factor (TF) (PDB ID: 1DAN) and the molecular visualization system PyMOL. The mutations were created by site-directed mutagenesis and human embryonic kidney 293 or Chinese hamster ovary K1 cells were transiently transfected with lipofectamine. FVII:Ag was measured in cells and in supernatants. Endoplasmic reticulum (ER) stress was evaluated by a luciferase reporter assay, and by quantitative RT-PCR. Intracellular localization of FVII was assessed using confocal immunofluorescence microscopy. Results Proband 1 (P1) was homozygous for a deletion of two nucleotides in exon 1, c.27_28delCT, leading to a frame shift and a premature stop codon at amino acid 16 (p.C10Pfs*16), causing nonsense-mediated mRNA decay (NMD). Proband 2 (P2) was homozygous for a missense mutation in exon 8, c.718G&amp;gt;C, resulting in a glycine to arginine substitution at amino acid 240 (p.G240R). The latter variant has previously been described in the literature, but caused by a c.718G&amp;gt;A transition. Both probands had FVII activity &amp;lt; 1 IU/dL and FVII:Ag was concomitantly reduced, i.e. a type I deficiency. P1 developed a high-titer FVII inhibitor (&amp;gt;50 Bethesda Units/mL) shortly after initiation of treatment with NovoSeven. In such plasma with a high-titer of FVII inhibitor, thrombin was not generated within 90 min after the addition of TF and phospholipids to the re-calcified plasma. In P2, TG, especially the parameter lag time, was severely reduced. The amino acid G240 is located in the hydrophobic core within the catalytic domain. Our structure analysis suggests that a glycine to arginine substitution at this position likely will cause steric interactions and destabilization of the catalytic domain. The intracellular levels of FVII-c.27-28delCT were non-detectable and the levels of FVII-c.718 was reduced by 35%, relative to FVIIwt. FVII:Ag secreted to the conditioned medium of both variants was non-detectable. The ER stress luciferase assay and analysis of spliced (s) X-box binding protein (XBP) 1 mRNA demonstrated significantly increased ER stress levels in cells expressing FVII-c.718 compared to FVIIwt. Confocal immunofluorescence microscopy showed that the FVII-c.718 was localized in the ER and not in the Golgi, whereas FVIIwt could be found in both compartments. Conclusions The deficient secretion of the FVII-c.27_28delCT variant was caused by a lack of synthesis of the FVII protein, as a consequence of NMD. The inhibitor development in P1 was likely linked to the complete absence of circulating FVII. The molecular mechanism underlying the FVII-c.718 mutation could be reduced secretion caused by protein destabilization and misfolding. Disclosures No relevant conflicts of interest to declare.