Development Of Inhibitory Antibodies Research Articles

99.3% of Inhibitors in Severe Hemophilia a Develop before Exposure Day 75. Time to Change Definition of Previously Treated Patients; Data from 1038 Patients with Severe Hemophilia a of the Pednet Registry

99.3% of Inhibitors in Severe Hemophilia a Develop before Exposure Day 75. Time to Change Definition of Previously Treated Patients; Data from 1038 Patients with Severe Hemophilia a of the Pednet Registry

Efficacy and Safety Results from a Phase 3b, Open-Label, Multicenter, Continuation Study of Rurioctocog Alfa Pegol for Prophylaxis in Previously Treated Patients with Severe Hemophilia A

Background: Patients with severe hemophilia A experience substantial morbidity and mortality due to frequent spontaneous and traumatic bleeding episodes, especially in joints. Prophylaxis with standard half-life factor VIII (FVIII) is the standard of care to prevent bleeds. Extended half-life products benefit patients by reducing the number of infusions without impacting the treatment efficacy.Methods: This phase 3b, prospective, open-label, multicenter, continuation study (NCT# 01945593) investigated the safety and efficacy of a PEGylated recombinant FVIII with an extended half-life, rurioctocog alfa pegol (SHP660, BAX 855, ADYNOVATE®, Shire, Lexington, MA, USA), for prophylaxis and treatment of bleeding in patients with severe hemophilia A (FVIII <1%). Eligible children and adults were ≤75 years of age and had either completed a previous rurioctocog alfa pegol study (NCT# 01599819, 01736475, 02210091, 02615691, 01913405, or 02585960) and were willing to immediately transition to the continuation study, or were naïve to rurioctocog alfa pegol but had received treatment with plasma-derived or recombinant FVIII for ≥150 (in patients ≥6 years of age) or ≥50 (in patients <6 years of age) exposure days. Patients received prophylactic rurioctocog alfa pegol at least twice weekly, either at a fixed dose (FD; 45 ± 5 IU/kg in patients ≥12 years of age; 50 ± 10 IU/kg in those <12 years of age; dose adjustment ≤80 ± 5 IU/kg allowed) or with pharmacokinetically (PK)-tailored dosing (≤80 ± 5 IU/kg) to maintain FVIII trough levels ≥3%. The co-primary endpoints assessed were the incidence of FVIII inhibitory antibody development (as measured by ≥0.6 BU in the Nijmegen modification of the Bethesda assay) and the spontaneous annualized bleed rate (ABR). Secondary endpoints included overall hemostatic efficacy ratings and occurrence of adverse events (AEs).Results: The study began in October 2013 and completed in March 2018. Overall, 216 patients receiving prophylaxis were eligible and included in the safety/full analysis dataset (≥1 dose received). Of these, 215 were male, the mean (SD) age at enrollment was 22.8 (15.7) years, the mean (SD) number of documented previous rurioctocog alfa pegol exposure days was 57.0 (39.6), the total ABR over 3-6 months prior to enrollment in the continuation study (including patients naïve to rurioctocog alfa pegol or receiving on-demand or prophylactic treatment with rurioctocog alfa pegol) was mean (SD) 4.7 (12.6), median (range) 0.0 (0-69). Most patients (206; 95.4%) had participated in a previous rurioctocog alfa pegol study. Overall, 215 patients received ≥1 dose in the FD regimen and 25 received ≥1 dose in the PK regimen. These patients received a mean (SD) of 1.72 (0.29) and 2.11 (0.61) prophylactic infusions per week, respectively, with a mean (SD) dose per infusion of 51.15 (8.11) IU/kg and 52.14 (17.03) IU/kg, respectively.None of the patients developed a confirmed FVIII inhibitor in this study and only 1 treatment-related allergic or hypersensitivity reaction (a nonserious mild AE) was reported. Nonserious AEs assessed by the investigators to be related to treatment occurred in 11/216 (5.1%) patients. Serious AEs (SAEs) occurred in 33 (15.3%) patients; there were no SAEs assessed by the investigators to be related to treatment. Descriptive statistics on spontaneous, joint, and total ABR by prophylactic regimen are shown in the Table. The overall total ABR in all patients was mean (SD) 2.5 (3.1), median (range) 1.6 (0.0-19.5). Overall hemostatic efficacy was rated as good or excellent in 88.5% of all bleeds and 89.4% of all bleeds were treated with 1 or 2 infusions.Conclusions: These results show that in previously treated patients with severe hemophilia A, rurioctocog alfa pegol prophylaxis in FD- and PK-tailored regimens was well tolerated and effective. None of the patients developed FVIII inhibitory antibodies, and a decrease in mean total ABR was reported in these patients compared with baseline. [Display omitted] DisclosuresMullins:Shire: Honoraria, Membership on an entity's Board of Directors or advisory committees. Konkle:Genentech: Consultancy; CSL Behring: Consultancy; Gilead: Consultancy; Pfizer: Research Funding; Spark: Consultancy, Research Funding; BioMarin: Consultancy; Bioverativ: Research Funding; Shire: Research Funding; Sangamo: Research Funding. McGuinn:CSL Behring: Consultancy; BioMarin: Consultancy; Bioverativ: Membership on an entity's Board of Directors or advisory committees, Research Funding; Spark: Consultancy, Research Funding; Genentech: Consultancy; Shire: Research Funding; Pfizer: Research Funding. Engl:Shire: Employment, Equity Ownership. Tangada:Shire: Employment, Equity Ownership.

Inhibitor Formation in Congenital Hemophilia A: an Immunological Perspective.

The immunogenicity of therapeutic factor VIII (FVIII) in patients with hemophilia A has been puzzling scientific and clinical communities for more than 3 decades. Indeed, the development of inhibitory antibodies to FVIII remains a major clinical challenge and is associated with enormous societal costs. Thus, the reasons for which a presumably innocuous, short-lived, intravenously administered glycoprotein triggers such a deleterious, long-lasting neutralizing immune response is an enigma. This review does not pretend to bring an answer to this challenging question. It will however summarize the latest findings regarding the molecular interactions at play in the recognition of FVIII by the immune cells, the validity of the proposed risk factors for FVIII alloimmunization, and the different solutions that allow induction of FVIII-specific tolerance in preclinical models of hemophilia A.

Dexamethasone promotes durable factor VIII-specific tolerance in hemophilia A mice via thymic mechanisms.

The development of inhibitory antibodies to factor VIII is the most serious complication of replacement therapy in hemophilia A. Activation of the innate immune system during exposure to this protein contributes to inhibitor development. However, avoidance of factor VIII exposure during innate immune system activation by external stimuli (e.g., vaccines) has not been consistently shown to prevent inhibitors. We hypothesized that dexamethasone, a drug with potent anti-inflammatory effects, could prevent inhibitors by promoting immunologic tolerance to factor VIII in hemophilia A mice. Transient dexamethasone treatment during ainitial factor VIII exposure reduced the incidence of anti-factor VIII immunoglobulin G in both a conventional hemophilia A mouse model (E16KO, 77% vs. 100%, P=0.048) and a hemophilia A mouse model with a humanized major histocompatibility complex type II transgene (E17KO/hMHC, 6% vs. 33%, P=0.0048). More importantly, among E17KO/hMHC mice that did not develop anti-factor VIII immunoglobulin G after initial exposure, dexamethasone-treated mice were less likely to develop a response after re-exposure six (7% vs. 52%, P=0.005) and 16 weeks later (7% vs. 50%, P=0.097). Similar results were obtained even when factor VIII re-exposure occurred in the context of lipopolysaccharide (30% vs. 100%, P=0.069). The ability of these mice to develop immunoglobulin G to human von Willebrand factor, a structurally unrelated antigen, remained unaffected by treatment. Transient dexamethasone administration therefore promotes antigen-specific immunologic tolerance to factor VIII. This effect is associated with an increase in the percentage of thymic regulatory T cells (12.06% vs. 4.73%, P<0.001) and changes in the thymic messenger ribonucleic acid transcription profile.

Immune tolerance induction: What have we learned over time?

Development of inhibitory antibodies to infused factor VIII (FVIII) concentrates continues to be the most serious complication of haemophilia A management. Induction of immune tolerance by administering high doses of FVIII concentrate (antigen) and prothrombin complex concentrates to control bleeding was originated in the 1970s in Bonn, Germany, by Dr Hans-Hermann Brackmann, and became known as the Bonn protocol. ITI transformed the life of the index patient, who was 19years of age when he began treatment, and dramatically improved the medical landscape for all patients with haemophilia and inhibitors. Over the past 40years, variations to the Bonn protocol have been proposed. All protocols are effective although some are better suited than others for use in certain situations. The specific molecular defect in FVIII and the human leucocyte antigen (HLA) type of an individual with haemophilia are major codependent determinants to inhibitor development. Given the range of potential molecular defects and the staggering number of potential HLA types, it is likely that treatment arms of randomized studies in haemophilia represent highly diverse populations, which reduces the power of a study to demonstrate differences between treatments. Although available clinical guidelines and consensus recommendations for ITI therapy are not always in complete agreement, collectively the guidelines provide a reasonable level of guidance for administering ITI therapy under different clinical scenarios. Several studies of ITI therapy are ongoing with the aim of clarifying unresolved issues in haemophilia management including the role of von Willebrand factor in inhibitor eradication.

Comparative profiling of HLA-DR and HLA-DQ associated factor VIII peptides presented by monocyte-derived dendritic cells.

The development of anti-factor VIII antibodies is a major complication of the treatment of patients with hemophilia A. Generation of high affinity anti-factor VIII antibodies is dependent on help provided by CD4+ T cells that recognize factor VIII-derived peptides presented on class II major histocompatibility complex on the surface of antigen-presenting cells. In order to identify the immune-dominant epitopes that can be presented to CD4+ T cells, we previously developed a mass spectrometry-based method to identify factor VIII-derived peptides that are presented on human leukocyte antigen (HLA)-DR. In the present work, we compared the repertoire of FVIII-derived peptide presented on HLA-DR and HLA-DQ. Monocyte-derived dendritic cells from nine HLA-typed healthy donors were pulsed with recombinant factor VIII. HLA-DR and HLA-DQ molecules were purified using monoclonal antibodies. Our data show that HLA-DQ and HLA-DR present a similar repertoire of factor VIII-derived peptides. However, the number of peptides associated with HLA-DQ was lower than that with HLA-DR. We also identified a peptide, within the acidic a3 domains of factor VIII, which is presented with higher frequency on HLA-DQ. Interestingly, this peptide was found to have a higher predicted affinity for HLA-DQ than for HLA-DR. Taken together, our data suggest that HLA-DQ participates in the presentation of factor VIII peptides, thereby contributing to the development of inhibitory antibodies in a proportion of patients with severe hemophilia A.

Enhanced uptake of blood coagulation factor VIII containing immune complexes by antigen presenting cells

Background A major complication in the treatment of hemophilia A is the development of inhibitory antibodies targeting coagulation factor VIII (FVIII). Eradication of these inhibitors can be established by immune tolerance induction (ITI), which consists of daily administration of high dosages of FVIII. FVIII immune complexes (FVIII-IC) could be formed following FVIII infusion in patients with pre-existing anti-FVIII antibodies. Objectives Here we studied endocytosis of FVIII-IC by bone marrow-derived dendritic cells (BMDCs). Methods BMDCs were pulsed with FVIII/FVIII-IC and uptake was assessed by flow cytometry and confocal imaging. Results BMDCs were able to efficiently internalize FVIII-IC in a dose-dependent manner, 3-4-fold more efficiently when compared with equimolar concentrations of non-complexed FVIII. Uptake of FVIII-IC, but not FVIII alone, could be inhibited with anti-Fcγ receptor (FcγR) antibody 2.4G2, indicating functional involvement of FcγR. No internalization of FVIII-IC was observed in BMDCs lacking FcγRI, FcγRIIb, FcγRIII and FcγRIV. Genetic ablation of FcγRIIb, FcγRIII or FcγRIV individually did not affect the ability of anti-FVIII IgG to promote the uptake of FVIII. BMDCs lacking FcγRI showed lower FVIII-IC uptake levels when compared with other single FcγR null BMDCs. Expression of the inhibitory FcγRIIb alone was sufficient to internalize FVIII-IC more efficiently than FVIII. Conclusions FcγR are critical in the internalization of FVIII-IC by BMDCs and multiple FcγR can contribute independently to this process. Our findings provide a basis for future studies to address whether the outcome of ITI is dependent on the interplay between FVIII-IC and inhibitory and activating FcγR.

Oral Tolerance: Another Reason to Eat Your Veggies!

Development of inhibitory antibodies directed against infused clotting factors (termed inhibitors) is a major complication in the current treatment of hemophilia.1 Inhibitor formation occurs in about 30% of hemophilia A and 2%–5% of hemophilia B patients, making protein replacement therapy ineffective and requiring expensive and inconvenient bypass treatments to manage bleeds. Development of inhibitors in hemophilia B is also associated with several side effects, such as nephropathic syndrome and anaphylactic reactions, which complicate immune tolerance induction (ITI).

Design and characterization of an APC-specific serpin for the treatment of hemophilia

AbstractHemophilia is a bleeding disorder caused by deficiency in factors VIII or IX, the two components of the intrinsic Xase complex. Treatment with replacement factor can lead to the development of inhibitory antibodies, requiring the use of bypassing agents such as factor VIIa and factor concentrates. An alternative approach to bypass the Xase complex is to inhibit endogenous anticoagulant activities. Activated protein C (APC) breaks down the complex that produces thrombin by proteolytically inactivating factor Va. Defects in this mechanism (eg, factor V Leiden) are associated with thrombosis but result in less severe bleeding when co-inherited with hemophilia. Selective inhibition of APC might therefore be effective for the treatment of hemophilia. The endogenous inhibitors of APC are members of the serpin family: protein C inhibitor (PCI) and α1-antitrypsin (α1AT); however, both exhibit poor reactivity and selectivity for APC. We mutated residues in and around the scissile P1-P1′ bond in PCI and α1AT, resulting in serpins with the desired specificity profile. The lead candidate was shown to promote thrombin generation in vitro and to restore fibrin and platelet deposition in an intravital laser injury model in hemophilia B mice. The power of targeting APC was further demonstrated by the complete normalization of bleeding after a severe tail clip injury in these mice. These results demonstrate that the protein C anticoagulant system can be successfully targeted by engineered serpins and that administration of such agents is effective at restoring hemostasis in vivo.

Targeting Factor Xa Expression in Platelets As a Potential Gene Therapy Strategy for Hemophilia with Inhibitors

The development of inhibitory antibodies (inhibitors) is a critical issue in the replacement therapy of hemophilia. The effective therapy of hemophilia with inhibitors is still an urgent demand. In the coagulation cascade, factor X (FX) is the common point of both intrinsic and extrinsic pathway. FX is activated to factor Xa (FXa) by either factor IXa (FIXa)/factor VIIIa (FVIIIa) or factor VIIa (FVIIa)/tissue factor (TF) complexes. FXa forms a prothrombinase complex with factor Va (FVa), and activates prothrombin to thrombin. Since FXa activates prothrombin very efficiently and this process bypasses FIX and FVIII, it has been tried as a bypassing agent in the treatment of hemophilia with inhibitors for a long time. It was found that infusion of FXa in the plasma could alleviate the bleeding phenotype of hemophilia animals. However, studies disclosed that the half-life of FXa is too short to be an effective bypassing agent. Furthermore, improved plasma FXa concentration also increases the risk of disseminated intravascular coagulation (DIC). A protein ectopically expressed in platelets can be stored in platelets, and it can be released during the activation of platelets. Here, we hypothesize that if FXa can be targeted expressed and stored in platelets, the half-life of FXa could be increased, while at the same time the risk of DIC could be largely limited. When platelets are activated at the injury site, the stored FXa can be released and activate prothrombin on the surface of platelets, so as to initiate the blood coagulation. Thus, this platelet-stored FXa could be used in the treatment of hemophilia A (HA) and hemophilia B (HB) with inhibitors. To prove our hypothesis, we constructed a platelet-targeted FXa expression cassette (2bFXa) by putting FXa under the control of GPαIIb promoter. Then we made the corresponding lentiviral vector with this 2bFXa cassette. The 2bFXa lentiviral vector was used to transduce hematopoietic stem cells (HSCs) collected from HA and HB mice, the modified HSCs were then transplanted back into the lethally irradiated corresponding HA and HB recipient mice. We collected plasma and platelets from the recipients. FXa was successfully detected in the platelets but not in the plasma of both HA and HB recipients by either an FXa activity assay or FX ELISA. The level of platelet-stored FXa remains constant throughout the experiment. Platelet release assay indicates almost 85.7% stored FXa can be released after the stimulation of platelets. The storage of FXa in platelets was also demonstrated by confocal microscopy and flow cytometry analysis. We then carried out a tail clip survival test to evaluate the phenotypic correction of recipients, all HA and HB recipient mice survived the tail clip challenge, demonstrating the platelet-stored FXa is biologically functional. We also tested the same strategy on HA mice with inhibitors and acquired the same results, indicating that the strategy is effective for HA with inhibitors. During the whole period of experiment, the recipient mice were all in good health. We also kept on monitoring the complete blood cell counts for 48 weeks and the cell numbers remained in normal range, indicating that the blood cells were successfully recovered in the recipients. Taken together, our proof of principle studies suggest that targeting FXa expression in platelets could facilitate the storage of FXa in platelets and would not cause obvious FXa leakage. The platelet-stored FXa is active and can be released with the activation of platelets and corrects the bleeding phenotype of HA and HB mice. This new gene therapy method could eliminate the shortage of FXa and be functional in the treatment of hemophilia with inhibitors. DisclosuresNo relevant conflicts of interest to declare.

The Impact of VWF on FVIII Immune Responses in Hemophilia a Mice with Pre-Existing Anti-FVIII Immunity

Development of inhibitory antibodies (inhibitors) against FVIII is the significant complication in protein replacement therapy for hemophilia A (HA). Currently, immune tolerance induction (ITI) with aggressive infusion of high-dose FVIII represents the only effective therapeutic approach for eradication of FVIII inhibitors and results in restoration of normal FVIII pharmacokinetics in inhibitor patients. Whether the use of FVIII products containing VWF will affect the efficacy of the ITI is still a debated issue in the treatment of inhibitor patients. In this study, we explored the impact of VWF on FVIII immune responses in HA with pre-existing anti-F8 immunity using both in vitro and in vivomodels.Since the FVIII immune response is CD4+ T cell dependent, we first investigated how VWF affects FVIII-primed CD4+ T cells in response to FVIII restimulation. To address this question, we used a T cell proliferation assay. FVIIInull (F8null) mice were immunized with recombinant human FVIII (rhF8) to induce inhibitor development. Splenocytes from primed mice were labeled with CellTrace™ Violet and cultured with rhF8 with or without rhVWF. Four days later, cells were analyzed by FACS to assess the daughter (proliferated) cell population. The percentage of daughter CD4+ T cells (14.0±7.5%) in the condition cultured with 1 U/ml of rhF8 was significantly higher than without rhF8 (3.7±1.7%, n=6). The daughter cells further increased to 21.5±10.3% when cells were incubated with 10 U/ml of rhF8. However, when rhVWF was added to the culture media in addition to rhF8, percentages of daughter CD4+ T cells were significantly decreased in both the 1 U/ml and 10 U/ml rhF8 treatment groups (10.4±7.1% and 15. 8±8.4%, respectively). To further explore how VWF affects the FVIII immune response, we analyzed cytokine profiles in T cell culture supernants using a multiplex ELISA assay. The levels of IFNg and IL10 in the groups cultured with rhF8 in the presence of rhVWF were significantly lower than in the groups cultured with rhF8 only. The levels of TNFa, IL4, IL5, and IL12 in the groups cultured with rhF8 together with rhVWF were not significantly different than those in rhF8 groups without VWF. These results demonstrated that VWF significantly suppresses rhF8-primed CD4+ T cell proliferation in response to rhF8 restimulation and the inhibition is via the Th1 pathway.In a setting of pre-existing anti-F8 immunity, how FVIII-specific memory B cells respond to FVIII-restimulation and mature to antibody secreting cells (ASCs) is the critical pathway in terms of the clinical efficacy of FVIII infusion. To investigate how VWF affects memory B cell maturation upon FVIII restimulation, we used ELISPOT-based assay. Splenocytes from rhF8-primed HA mice were used as the source to prepare F8-specific memory B cell pools. CD138+ cells were depleted and the remaining cells were used as a pool of memory B cells. To stimulate the maturation of F8-specific memory B cells into ASCs, memory B cell pools from primed F8null mice were cultured with rhF8 with or without rhVWF for 6 days. After culture, newly formed ASCs were assessed by the ELISPOT assay. There were 54.4±19.5 ASCs/106 cells when cells from memory B cell pool were cultured with 0.05 U/ml rhF8. In contrast, there was only 15.6±1.6 ASCs/106cells after the cells were cultured with rhF8 together with rhVWF, indicating that memory B cell maturation is suppressed in the presence of rhVWF.We then used an in vivo model to further evaluate the impact of VWF on the immunogenicity of FVIII in HA with pre-existing immunity. Since we are unable to mimic the human ITI in F8null mice, we transferred memory B cells from rhF8-primed F8null splenocytes into immunocompromised F8null mice followed by rhF8 immunization in the presence or absence of rhVWF. Blood samples were collected one week after immunization for analysis. The inhibitor titer in animals that received rhF8-primed memory B cell pool followed by rhF8 immunization was 45.9±63.0 BU/ml (n=11), which was significantly higher than the titer in animals immunized with rhF8 together with rhVWF (23.9±38.4, P<.01). These results demonstrate that VWF suppressed the anti-F8 memory response in vivo.In summary, our ex vivo and in vivo data demonstrated that VWF attenuates F8-primed CD4+T cells and memory B cells in response to rhF8 restimulation, suggesting that infusion of FVIII together with VWF might reduce anti-F8 memory responses in HA with inhibitors. DisclosuresNo relevant conflicts of interest to declare.

Elimination of FVIII-Specific B Cells By Immunotoxins Comprised of a Single FVIII Domain Fused to Pseudomonas Exotoxin a

The development of inhibitory antibodies (inhibitors) against coagulation factor VIII (FVIII) is the most serious complication for patients with hemophilia A that undergo FVIII replacement therapy. In addition, healthy individuals can spontaneously develop inhibitory anti-FVIII auto-antibodies, which results in acquired hemophilia A. The current standard therapy for patients with hemophilia A and inhibitors, named immune tolerance induction (ITI), is based on frequent and mostly high dose administrations of FVIII. Unfortunately, the eradication of inhibitors can only be achieved in about 70% of patients. Alternative treatment of inhibitor patients with the monoclonal anti-CD20 antibody rituximab results in complete eradication of inhibitors; however, depletion of the entire CD20-positive B cell population is potentially accompanied by severe side effects. Recent studies in hemophilic FVIII knockout mice showed that the application of a FVIII-toxin conjugate resulted in (i) prevention of inhibitor development in naïve mice and (ii) long-term eradication of inhibitors in FVIII-immunized mice.As the use of FVIII for cell targeting of immunotoxins is presumably limited by its high molecular weight (250 kDa) and adhesiveness (off-target reactivity) we explored the potential use of alternative immunotoxins in the current study. The introduced immunotoxins are comprised of a single FVIII domain fused to the Exotoxin A (ETA) from Pseudomonas aeruginosa.The rationale for the use of a single domain instead of full length FVIII as cell-binding component is that immunodominant domains like A2 and C2 might still allow targeting of sufficient amounts of FVIII-specific B-cells by immunotoxins. For proof of concept studies, we generated a histidine-tagged C2 domain-ETA fusion protein (C2-ETA) that was bacterially expressed and purified by affinity chromatography. Purified C2-ETA was recognized by a panel of commercially available monoclonal anti-C2 antibodies in ELISA suggesting proper folding of the C2 domain in the bacterially expressed protein.To test the capacity of C2-ETA to eliminate FVIII-specific B-cells, splenocytes of FVIII-immunized FVIII knockout mice were re-stimulated with FVIII ex vivo in presence and absence of different concentrations of C2-ETA and ETA alone (as control). Re-stimulation of FVIII-specific memory B cells to FVIII- and C2-specific antibody secreting cells (ASC) was analyzed in anEnzyme linked immunospot (ELISPOT) assay using FVIII and C2 as antigens. While differentiation to FVIII-specific ASC was only partially inhibited by C2-ETA, differentiation to C2-specific ASC was completely blocked in a dose-dependent manner. In contrast, the use of ETA alone had no effect. Further analysis of the FVIII domain specificity of antibodies in plasma of FVIII-immunized FVIII knockout mice used for depletion studies revealed a strong contribution of C2-specific antibodies to the overall FVIII-specific immune response.In summary, our results show that the developed C2-ETA immunotoxin is able to specifically eliminate FVIII C2 domain-specific B cells ex vivo. Currently, C2-ETA is tested for its capacity to eliminate FVIII-specific B cells in FVIII knockout mice and additional FVIII domain-ETA immunotoxins are developed. DisclosuresNo relevant conflicts of interest to declare.

Fitusiran, an Investigational RNAi Therapeutic Targeting Antithrombin for the Treatment of Hemophilia: Updated Results from a Phase 1 and Phase 1/2 Extension Study in Patients with Inhibitors

Background: Development of inhibitory antibodies, also known as "inhibitors," to replacement factor is considered the most serious unmet need in hemophilia and occurs in up to 30% of persons with severe hemophilia A, and 3-5% of persons with severe hemophilia B. Once inhibitors are present in high titer, treatment or prevention of bleeding can become more difficult due to the decreased responsiveness to factor concentrates, requiring bypassing agents (BPA) for bleed management. Current BPAs have a short half-life and are sub-optimally effective. Fitusiran is a subcutaneously (SC) administered investigational RNA interference (RNAi) therapeutic targeting the endogenous anticoagulant antithrombin (AT) as a means to improve thrombin generation and promote hemostasis in patients with hemophilia. Preliminary data from an ongoing Phase 1, multi-center, study showed that fitusiran was generally well tolerated in patients with hemophilia A or B with and without inhibitors and that administration of once-monthly SC doses of fitusiran led to dose-dependent AT lowering, thrombin generation increase, and decrease in bleeding frequency (Pasi KJ, et al. Haemophilia 2016, 22[Suppl 4]). Here we report the updated safety, pharmacodynamic (PD) effect, and clinical activity of fitusiran in patients with hemophilia with inhibitors as well as long term data from the Phase 1/2 extension study.Methods: We are conducting a multi-center Phase 1, four part (Part A: healthy volunteers; Parts B and C: patients with moderate to severe hemophilia A or B; Part D: patients with hemophilia A or B with inhibitors) study (NCT02035605) followed by a multi-center Phase 1/2 extension study (NCT02554773). Primary endpoints include safety and tolerability; secondary endpoints include AT activity, thrombin generation and exploratory evaluation of bleed pattern. In Part D, patients with inhibitors received once-monthly SC fixed doses of 50 or 80mg fitusiran. After receiving 3 monthly doses in the Phase 1 study, all patients were eligible to continue monthly dosing in the Phase 1/2 extension study. Utilization of BPA for breakthrough bleed management was permissible in these patients.Results: Part D of the Phase 1 study included 12 hemophilia A or B patients with inhibitors in 2 dosing cohorts (50mg SC, qM dosing cohort, n=6; 80mg SC, qM dosing cohort, n=6). Within the 50mg dosing cohort there were five patients with severe hemophilia A with inhibitors and one patient with severe hemophilia B with inhibitors; mean age: 33 ± 7 years; mean weight: 73 ± 17kg. Previously reported safety data from the 50mg dosing cohort demonstrated fitusiran was generally well tolerated in hemophilia A or B patients with inhibitors and that there were no serious adverse events related to study drug and no thromboembolic events. Monthly administration of fitusiran at the 50mg dose led to a mean maximal AT lowering of 81 ± 2% and mean maximal thrombin generation increase of 368 ± 113%. A preliminary, post-hoc analysis suggested a 49-100% reduction in bleeding frequency at the lower dose of 50mg during initial follow-up in the Phase 1 study. As of July 2016, the 80mg dose cohort has been fully enrolled and includes 6 patients with hemophilia A with inhibitors; mean age: 39 ± 15 years; mean weight: 75 ± 19kg, and 5 of the 6 patients in the initial 50mg cohort have transitioned to the Phase 1/2 extension study. Follow-up in the Phase 1, 80mg cohort and Phase 1/2 extension study is ongoing. Updated safety, tolerability and clinical activity from the Phase 1 and Phase 1/2 extension studies among all 12 patients with inhibitors will be presented.Conclusions: Emerging clinical data suggest that targeting AT is generally safe and could be a promising approach for promoting hemostasis in patients with hemophilia with inhibitors. Furthermore, the potential for low volume SC administration, monthly dosing, and applicability to patients with hemophilia A and B with and without inhibitors make fitusiran a potentially encouraging investigational therapy. DisclosuresPasi:Biogen: Consultancy, Honoraria; Octapharma: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria; Genzyme: Consultancy, Honoraria; SOBI: Honoraria, Membership on an entity's Board of Directors or advisory committees. Georgiev:Alnylam Pharmaceuticals: Consultancy. Chowdary:Bayer: Honoraria; Baxalta: 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; Sobi: Honoraria; Biogen: Honoraria, Membership on an entity's Board of Directors or advisory committees; CSL Behring: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novo Nordisk: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Ragni:Novo Nordisk: Research Funding; Biomarin: Consultancy; Biogen: Consultancy, Research Funding; Alnylam Pharmaceuticals: Consultancy, Research Funding; Genentech: Research Funding; SPARK: Research Funding; Baxalta: Research Funding; CSL Behring: Research Funding; Shire: Consultancy; Vascular Medicine Institute: Research Funding; Tacere Benitec: Consultancy; OPKO: Research Funding. Soh:Alnylam Pharmaceuticals: Employment, Equity Ownership. Akinc:Alnylam Pharmaceuticals: Employment, Equity Ownership. Partisano:Alnylam: Employment, Equity Ownership. Sorenson:Alnylam Pharmaceuticals: Employment, Equity Ownership.

The C1 and C2 domains of blood coagulation factor VIII mediate its endocytosis by dendritic cells.

The development of inhibitory antibodies to therapeutic factor VIII is the major complication of replacement therapy in patients with hemophilia A. The first step in the initiation of the anti-factor VIII immune response is factor VIII interaction with receptor(s) on antigen-presenting cells, followed by endocytosis and presentation to naïve CD4+ T cells. Recent studies indicate a role for the C1 domain in factor VIII uptake. We investigated whether charged residues in the C2 domain participate in immunogenic factor VIII uptake. Co-incubation of factor VIII with BO2C11, a monoclonal C2-specific immunoglobulin G, reduced factor VIII endocytosis by dendritic cells and presentation to CD4+ T cells, and diminished factor VIII immunogenicity in factor VIII-deficient mice. The mutation of basic residues within the BO2C11 epitope of C2 replicated reduced in vitro immunogenic uptake, but failed to prevent factor VIII immunogenicity in mice. BO2C11 prevents factor VIII binding to von Willebrand factor, thus potentially biasing factor VIII immunogenicity by perturbing its half-life. Interestingly, a factor VIIIY1680C mutant, that does not bind von Willebrand factor, demonstrated unaltered endocytosis by dendritic cells as well as immunogenicity in factor VIII-deficient mice. Co-incubation of factor VIIIY1680C with BO2C11, however, resulted in decreased factor VIII immunogenicity in vivo In addition, a previously described triple C1 mutant showed decreased uptake in vitro, and reduced immunogenicity in vivo, but only in the absence of endogenous von Willebrand factor. Taken together, the results indicate that residues in the C1 and/or C2 domains of factor VIII are implicated in immunogenic factor VIII uptake, at least in vitro Conversely, in vivo, the binding to endogenous von Willebrand factor masks the reducing effect of mutations in the C domains on factor VIII immunogenicity.

Factor VIII associated with lipidic nanoparticles retains efficacy in the presence of anti-factor VIII antibodies in hemophilia A mice.

The development of inhibitory antibodies against factor VIII (FVIII) is a major challenge in hemophilia A (HA) therapy. Such antibodies develop in nearly 30% of patients receiving replacement FVIII, abrogating therapeutic efficacy. This work evaluated whether B-domain deleted FVIII encapsulated in phosphatidylinositol containing lipid nanoparticles (PI-BDD FVIII) could serve as an efficacious FVIII replacement therapy in the presence of inhibitors. The HA mice were given clinically relevant doses of FVIII to develop inhibitors. The efficacy of free and PI-BDD FVIII was studied in inhibitor-positive HA mice using a tail clip assay. Mathematical modeling of these data was conducted to evaluate the hypothesis that lipid association sterically shields the protein from inhibitor binding. The immunization protocol resulted in a mean inhibitory titer level of 198±52 BU/ml. Free BDD FVIII was ineffective at controlling blood loss in inhibitor-positive HA mice as early as 2h post dose. In contrast, PI-BDD FVIII treated animals retained partial hemostatic efficacy as long as 18h post dose. Mathematical modeling supports the hypotheses that a greater fraction of lipid-associated FVIII remains unbound to inhibitors and that PI-BDD FVIII has lower binding affinity to inhibitors than does the free protein. In addition, the modeling approaches extend current efforts to model the impact of immunogenicity on PK and the therapeutically meaningful endpoint of efficacy, thereby addressing an important knowledge gap, particularly in the FVIII scientific literature. Clinical translation of these findings could result in a significant improvement in the quality of care of inhibitor-positive HA patients. Copyright © 2016 John Wiley & Sons, Ltd.

688. Supplemental Immune Suppression Is Required for AAV-F8 ITI in Hemophilia A Mice with Pre-Existing Inhibitors

Hemophilia is an inherited coagulation disorder resulting in the loss of functional clotting factors VIII (hemophilia A) or IX (hemophilia B). Patients with the severe hemophilia (less than 1% residual clotting factor activity) develop spontaneous bleeds into the joints and closed spaces and have a high incidence of morbidity if not treated. Current treatment involves frequent infusion of short-lived exogenous plasma derived or recombinant clotting factors. A major complication with protein therapy is the development of inhibitory antibodies in 25-30% hemophilia A and 3-5% hemophilia B patients that prevent normalization of hemostasis with continued protein therapy. Inhibitor patients are treated with either bypassing agents or immune tolerance induction (ITI) therapy requiring daily infusions (months to years) of supraphysiological levels of clotting factor protein. ITI therapy is effective in up to 70% of hemophilia A patients, while it often fails in hemophilia B patients due to the development of pathogenic antibodies that induce anaphylaxis and nephrotic syndrome. Both treatment options for inhibitor patients have high associated financial and health related costs for the patient. We have previously shown that hemophilia B mice with pathogenic inhibitory antibodies are effectively treated with a novel immune tolerance induction protocol using a liver directed adeno-associated virus expressing the gene encoding for human factor IX protein (AAV8-F9). AAV8-F9 ITI rapidly eliminates pre-existing inhibitors and provided sustained therapeutic levels of FIX protein even following repeated exposure to recombinant FIX protein. Based on our success with hemophilia B, we sought to develop a complementary protocol for hemophilia A mice with pre-existing inhibitors. Our initial studies were conducted using a codon-optimized F8 (coF8) gene, which has been shown to enhance the levels of transcribed FVIII protein. In the first set of experiments we tested two different vector doses (1×1011 and 1×1012 vg) of an AAV8-cohF8 vector for factor VIII expression levels and tolerance induction in hemophilia A mice on two different genetic backgrounds (BALB/c-F8−/Y and 129/BL6-F8−/Y). Since BALB/c-F8−/Y mice develop lower titer FVIII inhibitors following intravenous hFVIII protein challenge compared to 129/BL6-F8−/Y mice we expected better outcomes with AAV8-cohF8 vector treated BALB/c-F8−/Y mice. Surprisingly BALB/c-F8−/Y mice spontaneously developed FVIII inhibitors approximately 4 weeks following vector delivery. In contrast,129/BL6-F8−/Y mice expressed vector dose dependent therapeutic levels FVIII protein without inhibitors and 75% of the mice remained inhibitor free following four weekly IV FVIII protein challenges. Subsequent studies for AAV8-cohF8 ITI were conducted in 129/BL6-F8−/Y mice. We generated a group of inhibitor mice and treated a subset of these mice with 1×1012 vg AAV8-coF8. AAV8-cohF8 ITI alone was not sufficient to eliminate inhibitors. Therefore we designed two additional studies to determine if 1×1012 vg AAV8-coF8 ITI would be more effective if combined with transient immune suppression using oral rapamycin (4mg/kg) or anti-CD20 (10mg/kg) mediated B cell depletion. While rapamycin adjunct therapy failed to lower inhibitors, preliminary data show that anti-CD20 combined with AAV8-coF8 ITI leads to a sharp reduction in high titer inhibitors. Given that FVIII protein invokes a stronger immune response in mice and patients, our data suggests that successful gene therapy for hemophilia A may require supraphysiological levels of FVIII protein to prevent inhibitor formation and transient immune suppression to be effective as an ITI therapy.

Susoctocog Alfa: A Review in Acquired Haemophilia A.

Intravenous susoctocog alfa (Obizur(®)) is a recombinant, B-domain deleted, porcine sequence antihaemophilic factor VIII (FVIII) product that has recently been approved for the treatment of bleeding episodes in adults with acquired haemophilia A (AHA). Intravenous susoctocog alfa was an effective and generally well tolerated treatment for serious bleeding episodes in adult patients with AHA in a multinational, phase II/III trial (n=28 evaluable). Patients received an initial dose of susoctocog alfa 200 U/kg, with subsequent dosages based on target FVIII trough levels and clinical assessments. All patients had a positive haemostatic response (based on predefined criteria) of the primary bleed 24 h after the first infusion of susoctocog alfa, with the bleed successfully controlled at the time of final dosing in 86% of patients. The most frequently reported adverse reaction (incidence >5%) was the development of inhibitory antibodies against susoctocog alfa (porcine FVIII). Overall, 25% of antibody naive patients developed anti-susoctocog alfa antibodies during the study. No serious treatment-related adverse events, thrombotic events or allergic reactions were reported during the trial. In conclusion, intravenous susoctocog alfa is a useful addition to the limited treatment options available for the management of acute bleeding episodes in adults with AHA.

The Impact of GPIba on the Efficacy of Platelet-Targeted FVIII Gene Therapy in Hemophilia a with Pre-Existing Anti-FVIII Immunity

Platelets play fundamental roles in hemostasis through surface proteins, e.g. GPIb, and storage proteins, such as von Willebrand factor (VWF). Our previous studies have demonstrated that FVIII ectopically targeted to platelets under control of the platelet-specific aIIb promoter (2bF8) is therapeutic in hemophilia A mice even in the presence of anti-FVIII inhibitory antibodies (inhibitors). Our recent studies have shown that VWF is essential in platelet-targeted FVIII gene therapy of hemophilia A with inhibitors. Without VWF, the clinical efficacy of platelet-FVIII was aborted. In primary hemostasis, platelets adhere to the vessel wall through the binding of GPIb to VWF. Thus, we wanted to explore whether GPIb is critical for maintaining the clinical efficacy of platelet-FVIII gene therapy of hemophilia A in the presence of inhibitors. To address this question, recipient FVIIInull (F8null) mice were immunized with recombinant FVIII (rhF8) to induce anti-FVIII inhibitory antibody development (the inhibitor model). Hematopoietic stem cells (HSCs) from GPIba knockout (Ibnull) mice were transduced with 2bF8 lentivirus and transplanted into rhF8-primed F8null mice under 1100 cGy total body irradiation (2bF8-Ibnull/F8null). As an inhibitor model control (2bF8-F8null/F8null), HSCs from F8null mice were transduced with 2bF8 and transplanted into rhF8-primed recipients under the same preconditioning. After bone marrow transplantation and reconstitution, animals were assessed for transgene expression and phenotypic correction. PCR analysis showed that the 2bF8 transgene was detected in all of the transduced recipients, demonstrating the viability of 2bF8-transduced engraftment. The level of functional platelet-FVIII expression as determined by a Chromogenic assay was 6.37 ± 5.62 mU/108 platelets (n = 6), which appeared to be higher than the level obtained in the inhibitor control group (1.97 ± 0.96 mU/108 platelets, n = 5), but there was no significant difference between these two groups. Since the size of Ibnull platelets is larger and the platelet number is lower than those in F8null mice, we converted FVIII levels into mU/ml whole blood and the level was 10.36 ± 5.33 mU/ml in 2bF8-Ibnull/F8null mice, which is not significantly different from the 2bF8-F8null/F8null mice (9.04 ± 3.38 mU/ml). When the tail bleeding test was used to grade phenotypic correction of the F8null coagulation defect, all transduced recipients in both groups survived beyond a 6-hour tail clipping test and the remaining hemoglobin level in the 2bF8-Ibnull/ F8null group was 46.4 ± 8.5%, which is not significantly different from the 2bF8-F8null/F8null group (48.3 ± 9.7%). The remaining hemoglobin levels in both groups were significantly higher than in the untransduced F8null control, in which only 5 of 12 animals survived beyond the 6-hour tail clipping test with a remaining hemoglobin level of 34.8 ± 7%. Thus, our data suggest that a lack of platelet GPIba does not negate the clinical efficacy of platelet FVIII gene therapy in hemophilia A in the presence of inhibitors. DisclosuresMontgomery:Baxter: Consultancy; Bayer: Consultancy; CSL: Consultancy; Biogen: Consultancy; Octapharma: Consultancy; Grifols: Consultancy. Shi:BloodCenter of Wisconsin: Patents & Royalties: METHOD OF INDUCING IMMUNE TOLERANCE THROUGH TARGETTED GENE EXPRESSION..

IDO1 suppresses inhibitor development in hemophilia A treated with factor VIII.

The development of inhibitory antibodies to factor VIII (FVIII) is a major obstacle in using this clotting factor to treat individuals with hemophilia A. Patients with a congenital absence of FVIII do not develop central tolerance to FVIII, and therefore, any control of their FVIII-reactive lymphocytes relies upon peripheral tolerance mechanisms. Indoleamine 2,3-dioxygenase 1 (IDO1) is a key regulatory enzyme that supports Treg function and peripheral tolerance in adult life. Here, we investigated the association between IDO1 competence and inhibitor status by evaluating hemophilia A patients harboring F8-null mutations that were either inhibitor negative (n = 50) or positive (n = 50). We analyzed IDO1 induction, expression, and function for any relationship with inhibitor occurrence by multivariable logistic regression and determined that defective TLR9-mediated activation of IDO1 induction is associated with an inhibitor-positive status. Evaluation of experimental hemophilic mouse models with or without functional IDO1 revealed that tryptophan metabolites, which result from IDO1 activity, prevent generation of anti-FVIII antibodies. Moreover, treatment of hemophilic animals with a TLR9 agonist suppressed FVIII-specific B cells by a mechanism that involves IDO1-dependent induction of Tregs. Together, these findings indicate that strategies aimed at improving IDO1 function should be further explored for preventing or eradicating inhibitors to therapeutically administered FVIII protein.

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

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