Pre-Existing Anti-FVIII Immunity Alters Therapeutic Platelet-Targeted FVIII Engraftment in the System Preconditioned with Busulfan Alone through Cytotoxic CD8 T Cells

The development of anti-FVIII inhibitory antibodies (inhibitors) is a significant problem in FVIII protein replacement therapy in hemophilia A (HA). We have developed a platelet-targeted FVIII gene therapy approach, in which human FVIII expression is driven by the platelet-specific αIIb promoter (2bF8) and demonstrated that 2bF8 gene therapy can restore hemostasis and induce FVIII-specific immune tolerance in FVIII null mice even with pre-existing anti-FVIII immunity when an effective preconditioning regimen is employed. Since busulfan, an alkylating agent with potent effects on primitive hematopoietic cells, is an important component of many hematopoietic stem cell (HSC) transplantation preparative regimens in humans, we evaluated the efficacy of busulfan conditioning regimens in 2bF8 gene therapy. We found that busulfan conditioning alone resulted in sustained therapeutic levels of platelet-FVIII expression in FVIII null mice that received 2bF8-transduced HSCs in the non-inhibitor model but not in the inhibitor model. In the current study, we explored the mechanism of platelet FVIII loss upon busulfan conditioning in the FVIII inhibitor model. FVIII null mice were immunized with recombinant human FVIII (rhF8) to induce anti-FVIII inhibitor development to establish the inhibitor model. Once the inhibitor titers were confirmed, animals received busulfan preconditioning at the dose of 50 mg/kg followed by transplantation of either whole bone marrow or Sca-1 + cells from 2bF8 transgenic (2bF8 Tg) mice. After 4 weeks of bone marrow reconstitution, platelet-FVIII expression levels in recipients transplanted with 2bF8 Tg whole bone marrow cells were 7.19±8.59 mU/10 8 platelets (n=5), which were significantly higher than those obtained from animals transplanted with 2bF8 Tg Sca-1 cells (0.55±1.02 mU/10 8 platelets [n=15]). The differences in platelet-FVIII expression between the whole bone marrow and Sca-1 groups were maintained during the study period for 6 months. When CD8 T cells were depleted in addition to busulfan preconditioning, platelet-FVIII expression was significantly enhanced in rhF8-primed recipients that received 2bF8 Tg Sca-1 cells (2.14±2.25 mU/10 8 platelets [n=8]) and sustained during the study period. We then explored which subset of cells from 2bF8 Tg mice could activate rhF8-primed CD8 T cells using the mouse IFNγ ELISpot assay. rhF8-primed CD8 T cells were stimulated with platelets, Sca-1 + cells, or megakaryocytes sorted from either 2bF8 Tg or FVIII null mice. We found that CD8 T cells from rhF8-primed FVIII null mice were efficiently activated by Sca-1 + cells from 2bF8 Tg mice and secreted IFNγ but not by platelets or megakaryocytes. These results suggest that 2bF8 Tg-Sca-1 + cells could be a potential target for rhF8-primed CD8 T cells. As a control, Sca-1 + cells from FVIII null mice did not activate rhF8-primed CD8 T cells, suggesting that IFNγ production from rhF8-primed CD8 T cells stimulated with 2bF8 Tg-Sca-1 + cells was a FVIII-specific response. To explore whether the elimination of platelet-FVIII expression in the inhibitor model relies on antibody-dependent cellular cytotoxicity (ADCC), we transplanted 2bF8 Tg-Sca-1 + cells into rhF8-primed B-cell deficient μMT mice preconditioned with busulfan. We found that no platelet-FVIII was detected in μMT recipients even though they did not produce anti-FVIII antibodies, suggesting that the loss of platelet-FVIII expression in the inhibitor model is not mediated by the ADCC pathway. In summary, our studies demonstrate that pre-existing anti-FVIII immunity can alter the engraftment of 2bF8-genetically-manipulated Sca-1 + hematopoietic stem/progenitor cells via the cytotoxic CD8 T-cell killing pathway. Sufficient eradication of FVIII-primed CD8 T cells is critical for the success of platelet-targeted gene therapy in hemophilia A with pre-existing immunity. Disclosures No relevant conflicts of interest to declare.

Rurioctocog Alfa Pegol Use in Immune Tolerance Induction: Interim Results from an Open-Label Multicenter Clinical Trial in Previously Untreated Patients with Severe Hemophilia a

Background The development of inhibitors to exogenous factor VIII (FVIII) is a serious treatment complication in patients with hemophilia A. Immune tolerance induction (ITI) is the only proven method for the eradication of FVIII inhibitors. This prospective, multicenter, open-label, phase 3 study (NCT02615691) is being conducted to determine the safety, immunogenicity, and efficacy of the extended half-life (EHL) recombinant FVIII rurioctocog alfa pegol (Adynovate ®; Baxalta US Inc., a Takeda company, Lexington, MA, USA) in previously untreated patients (PUPs) with severe hemophilia A. The data presented here aims to evaluate the efficacy and safety of ITI therapy with rurioctocog alfa pegol in patients who developed FVIII inhibitors. Methods Eligible patients were ˂6 years of age with severe hemophilia A (FVIII <1%) and <3 exposure days (ED) to rurioctocog alfa pegol, octocog alfa, or plasma transfusion at any time prior to screening. Patients with detectable FVIII inhibitory antibodies at screening or a history of FVIII inhibitors prior to screening (≥0.6 Bethesda units [BU]) were excluded from the study. Patients received intravenous rurioctocog alfa pegol as prophylaxis (25-50 IU/kg, up to 80 IU/kg ≥1 × weekly) and/or on-demand therapy (10-50 IU/kg, up to 80 IU/kg depending on bleed severity). Patients who developed a high-titer FVIII inhibitor (>5.0 BU) or low-titer FVIII inhibitor (≥0.6 BU to ≤ 5.0 BU) plus poorly controlled bleeding despite increased FVIII doses and/or bypassing agents, were eligible for ITI therapy. Dosing for ITI therapy ranged between 50 IU/kg 3 × weekly (low dose) and 100-200 IU/kg daily (high dose) at investigator discretion. This protocol-specified interim analysis was conducted after 50 patients had completed ≥50 EDs without developing confirmed inhibitors to rurioctocog alfa pegol or had developed a confirmed FVIII inhibitor at any time. The data cut-off was 30 August 2019. The primary endpoint of this study was the success rate of ITI with rurioctocog alfa pegol. Success was defined as an inhibitor titer persistently <0.6 BU, FVIII incremental recovery (IR) ≥66% of baseline following 84- to 96-hour wash-out, and FVIII half-life ≥6 hours (dependent on protocol version). Secondary endpoints included the rates of partial success and failure of ITI, and annualized bleeding rate (ABR) during ITI. The number and percentage of patients reporting adverse events (AEs) and serious AEs (SAEs) were recorded for patients treated with ITI. Informed consent and ethics approval were obtained. Results As of the data cut-off, 59 (73.8%) of 80 enrolled patients had received ≥1 dose of rurioctocog alfa pegol; 18 patients did not meet the eligibility criteria (screen failures) and 4 discontinued prior to treatment. 10 patients developed an inhibitor to rurioctocog alfa pegol (high titer: n=5; low titer: n=5), of these, 6 patients were enrolled to receive ITI and only 5 of these (83.3%) actually received ≥1 dose of rurioctocog alfa pegol for the treatment of FVIII inhibitors (low dose: n=3; high dose: n=2). Of these 5 patients, 1 completed high-dose ITI therapy and this was successful (based on negative inhibitor titer and IR ≥66% of baseline). The remaining 4 patients were continuing in the study at the time of the data cut-off. Of the 5 patients who received ≥1 dose of ITI, 4 (80.0%) had a total of 17 AEs, 3 (60.0%) experienced 8 SAEs, and 1 experienced a treatment-related SAE of FVIII inhibition. It is important to note that the onset date of FVIII inhibitor development in this patient occurred prior to initiation of ITI. One patient experienced 2 catheter-related AEs, both of which resolved, and no patients experienced thrombotic AEs, study procedure-related AEs, or AEs leading to discontinuation of treatment. Discussion This is the first prospective study of the EHL recombinant FVIII rurioctocog alfa pegol for the treatment of PUPs with severe hemophilia A. These preliminary results demonstrate that rurioctocog alfa pegol has a safety profile consistent with previous studies. In addition, these interim data suggest that using a high-dose regimen for ITI therapy is potentially efficacious in PUPs who have developed FVIII inhibitors, although only 1 patient had completed ITI at the time of this interim analysis. Disclosures Sidonio: Pfizer: Consultancy; Octapharma: Consultancy, Research Funding; Catalyst: Consultancy; Novo Nordisk: Consultancy; Bayer: Consultancy; Guardian Therapeutics: Consultancy; Genentech: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; Biomarin: Consultancy. Thompson: Global Blood Therapeutics: Current equity holder in publicly-traded company; CRISPR Therapeutics: Research Funding; Vertex: Research Funding; Editas: Research Funding; Graphite Bio: Research Funding; Novartis: Research Funding; Agios: Consultancy; Beam: Consultancy; Celgene/BMS: Consultancy, Research Funding; Biomarin: Research Funding; Baxalta: Research Funding; bluebird bio, Inc.: Consultancy, Research Funding. Peyvandi: Bioverativ: Honoraria; Sanofi: Consultancy, Honoraria; Sobi: Consultancy, Honoraria; Spark: Honoraria; Takeda: Honoraria; Roche: Honoraria; Grifols: Honoraria. Yeoh: Grifols: Honoraria; Roche: Honoraria; Pfizer: Honoraria; Takeda: Honoraria. Lam: Takeda: Consultancy, Honoraria; Roche: Honoraria; Bayer: Honoraria; Pfizer: Consultancy, Honoraria. Maggiore: IQVIA: Current Employment. Engl: Takeda: Current equity holder in publicly-traded company; Baxalta Innovations GmbH, a Takeda company: Current Employment. Allen: Takeda: Current equity holder in publicly-traded company; Takeda Development Center Americas, Inc.: Current Employment. Tangada: Takeda Development Center Americas, Inc: Current Employment; Takeda: Current equity holder in publicly-traded company.

Serum TNF-α Level as a Possible Predictor of Inhibitor Levels in Severe Hemophilia A

Background. The development of factor VIII (FVIII) inhibitor in patients with hemophilia A (PWHA) is a great challenge for hemophilia care. Both genetic and environmental factors led to complications in PWHA. The development of inhibitory antibodies is usually induced by the immune response. Tumor necrosis factor α (TNF-α), one of the cytokines, might contribute to its polymorphism. In this study, we investigated the clinical factors, level of serum TNF-α, and polymorphism of c . − 308 G > A TNF − α gene in inhibitor development in severe PWHA. Methods. A cross-sectional study was conducted among all PWHA in West Java province. The clinical parameters, FVIII, FVIII inhibitor, and serum TNF-α level were assessed. The genotyping of − 380 G > A TNF-α gene polymorphism was performed using polymerase chain reaction and Sanger sequencing. Results. Among the 258 PWHA, 216 (83.7%) were identified as severe PWHA. The FVIII inhibitor was identified in 90/216 (41.6%) of severe PWHA, consisting of 45 high-titer inhibitors (HTI) and 45 low-titer inhibitors (LTI). There was a significant correlation between serum TNF-α level and the development of HTI ( p = 0.043 ). The cutoff point of serum TNF-α level, which can be used to differentiate between HTI and LTI, was 11.45 pg/mL. The frequency of FVIII replacement therapy was significant only in HTI of severe PWHA regarding serum TNF-α level ( p = 0.028 ). There is no correlation between polymorphisms of − 380 G > A TNF-α gene and inhibitor development ( p = 0.645 ). Conclusions. The prevalence of FVIII inhibitor in severe PWHA in West Java, Indonesia, was 41.6%. The frequency of replacement therapy is a risk factor for inhibitor development. Serum TNF-α level might be used to differentiate between high and low inhibitor levels in severe hemophilia A, and this might support decision making regarding treatment options for inhibitor in severe hemophilia A.

Small-Volume Noncontact Assessment of Blood Coagulation Via Acoustic Tweezing Coagulometry

Introduction: Blood coagulation analysis is routinely performed to assess bleeding and thrombotic risks in surgical and critical care patients as well as in patients with diseases that cause coagulation abnormalities (e.g., hemophilia, thrombophilia and sickle cell disease). Majority of coagulation assays are based on photo-optical measurement of coagulation onset in blood plasma such as prothrombin time (PT), international normalized ratio (INR), and activated partial thromboplastin time (aPTT) and viscoelastic measurement of coagulating whole blood, often referred to as "global coagulation analysis", mostly done by thromboelastography (TEG, ROTEM) but they require large sample volume (> 0.5ml) requiring venipuncture, have poor standardization, and are unreliable tools to predict bleeding/thrombotic risk. Acoustic tweezing coagulometry (ATC) is an innovative noncontact drop-of-blood coagulation analysis technique that can perform both photo-optical and viscoelastic coagulation analysis with a sample volume as low as 4 μl to provide a comprehensive set of clinically relevant coagulation parameters such as blood viscosity, elasticity, reaction time, clotting rate, maximum clot stiffness, fibrin formation rate and cross-linking kinetics helpful for diagnosis and prediction of bleeding and thrombotic risks. ATC is particularly valuable for the pediatric patients as it enables safe and reliable point of care coagulation assessment with minimal sample volume. Materials and Methods: In this project, we demonstrate the feasibility of ATC for coagulation analysis by validation and standardization of the technique using whole blood collected from healthy adult volunteers and commercially purchased blood plasma. Further, we present the ability of ATC to assess bleeding risk in commercial blood plasma with coagulation FVIII deficiency with and without inhibitors, as well as whole blood collected from pediatric Hemophilia A patients without inhibitors. The time dependent changes in elasticity (elastic tweezograph, Figure 1A) and viscosity (viscous tweezograph, Figure 1B) of coagulating blood plasma or whole blood sample are used to extract the following coagulation parameters: clot initiation time (CIT), clotting rate (CR), clotting time (CT), time to firm clot formation (TFCF), and maximum clot stiffness (MCS) from elastic tweezograph; reaction time (RT), fibrin formation rate (FFR), and maximum fibrin level (MFL) from viscous tweezograph. Results and Discussion: Figure 1C shows the elastic tweezograph and figure 1D shows the viscous tweezograph of the healthy plasma, plasma with coagulation FVIII deficieny and plasma with inhibitors for coagulation FVIII activated via the intrinsic pathway of coagulation. The tweezographs suggest that the clot initiation is faster in healthy plasma compared to the FVIII deficient plasma and FVIII inhibitor plasma. The clotting rate is highest for healthy plasma followed by the FVIII deficient plasma and is the lowest for the plasma with FVIII inhibitors suggesting a delayed clot formation in the deficient and inhibitor groups. They all reach a similar final clot stiffness, but the time to firm clot formation is least in healthy plasma as expected and increases in the FVIII deficient group and further increases in the FVIII inhibitor group. Conclusions: Acoustic tweezing coagulometry can successfully measure the viscosity, elasticity and coagulation of whole blood and blood plasma with only a drop of the sample. This technique can successfully assess the bleeding risks in pediatric and adult patients with Hemophilia. Acknowledgements: This study has been supported by American Heart Association pre doctoral fellowship 20PRE35210991, U.S. National Science Foundation grant 1438537, American Heart Association Grant-in-Aid 13GRNT17200013, and Tulane University intramural grants. The acoustic tweezing technology is protected by pending patents PCT/US14/55559, PCT/US2018/014879 and PCT/US21/15336. Figure 1 Figure 1. Disclosures Kasireddy: Levisonics Inc.: Current Employment. Rafique: Pfizer Inc.: Consultancy; CSL Behring: Consultancy; HEMA Biologics: Consultancy. Khismatullin: Levisonics Inc.: Current equity holder in publicly-traded company; Levisonics Inc.: Patents & Royalties: PCT/US14/55559 (pending); Levisonics Inc.: Patents & Royalties: PCT/US2018/014879 (issued) ; Levisonics Inc.: Patents & Royalties: PCT/US21/15336 (pending)..

Extra-Splenic Role of B Cell Activating Factor Blockade in Prevention of Factor VIII Inhibitors

The development of neutralizing antibodies (inhibitors) to factor VIII (FVIII) remains the most challenging complication of protein-based replacement therapy in hemophilia A (HA). Elevated levels of the B cell survival cytokine B cell activating factor (BAFF) are associated with inhibitors in HA patients and BAFF levels decrease with successful immune tolerance induction (Doshi BS et al, J Clin Invest 2021). In HA mice, localization of labeled FVIII to the spleen and subsequent depletion of splenic leukocytes has been shown to reduce and/or temporarily prevent FVIII inhibitors. In contrast, transient depletion of BAFF prevented inhibitors upfront and promoted long-term tolerance to FVIII in HA mice. Given BAFF's ability to modulate B cell pools in secondary lymphoid organs, here we compared the splenic versus extra-splenic role of BAFF in regulating the FVIII immune response in order to localize its mechanism of action. As BAFF could regulate survival of innate-like splenic-resident marginal zone B cells, initial experiments compared depletion of BAFF or marginal zone B cells in HA mice and showed more complete inhibitor prevention with BAFF depletion (median 0 vs 16.9 Bethesda Units [BU]/ml, respectively, p < 0.01). These data supported our hypothesis that BAFF's regulatory effect is not solely reliant on its modulation of the splenic-resident marginal zone B cell compartment in HA mice. To further understand the extra-splenic role of BAFF in FVIII immunogenicity, wild-type (WT) or severe HA mice (with F8 exon 16 knockout) on the C57Bl/6 background, were treated according to three groups (n=9-11/group): 1) sham controls, 2) splenectomy alone, and 3) anti-BAFF monoclonal antibody (2mg/kg IP once) 4 days prior to splenectomy ("combined therapy"). Following splenectomy, mice were injected with recombinant full length human FVIII at 80 IU/kg weekly for six weeks. Bethesda titers were measured one week following the final immunization. In order to provide hemostasis in HA mice without FVIII exposure prior to splenectomy, mice were given liver-directed gene therapy with AAV8-mFVIIa at 5e11 vg/mouse two weeks prior to splenectomy. Circulating mFVIIa levels of 5.8 ± 2.4 ug/ml at day 10 were observed. These levels normalized hemostasis allowing successful splenectomy in 85% of animals, which is similar to our experience in WT mice. In the combined therapy group, only a single HA mouse developed a high titer inhibitor (7.4 BU/ml) and no WT mice developed high titer inhibitors. As the presence of endogenous FVIII antigen in WT mice does not seem to significantly alter BAFF's role in preventing the alloantibody response to xenoantigen (human FVIII protein), data for WT and HA mice were combined for analysis. Although splenectomy decreased inhibitors titers compared to sham controls (23.1 vs 78.7 BU/ml), the addition of anti-BAFF antibody therapy to splenectomy significantly reduced the risk of high titer inhibitors (RR 0.15, 95% CI 0.02-0.66, p < 0.01). The median Bethesda titer in HA and WT splenectomized mice was 23.1 (0-139.4) versus 0 (0-7.4) with addition of BAFF antibody therapy. Of note, inhibitor prevention with combination splenectomy and anti-BAFF antibody therapy seen here is similar to our prior data using just anti-BAFF antibody monotherapy for FVIII inhibitor prevention in HA mice (RR 0.23, 95% CI 0.08-0.57) suggesting that splenectomy does not significantly improve FVIII inhibitor prevention in the setting of BAFF inhibition. Our data, using a challenging HA mouse model, suggest that anti-BAFF therapy may prevent FVIII inhibitors by inhibiting BAFF's function in non-splenic lymphoid compartments. As certain B cell subtypes, including marginal zone B cells, reside in non-splenic compartments in humans, our model mimics the anticipated human immune response to FVIII. Ongoing investigations into the B and T cell compartments in lymph nodes and bone marrow will provide further insights into the location of BAFF's regulatory role in the FVIII immune response and may provide insights into potential alternative pathways in the initial FVIII immune response. Disclosures Doshi: Janssen: Consultancy; Spark Therapeutics: Speakers Bureau.

Immunogenicity, Efficacy and Safety of Rurioctocog Alfa Pegol in Previously Untreated Patients with Severe Hemophilia a: Interim Results from an Open-Label Multicenter Clinical Trial

Background Management of severe hemophilia A includes on-demand treatment or prophylaxis with replacement factor VIII (FVIII) concentrate. FVIII inhibitors can develop following exposure to exogenous FVIII in approximately 30% of previously untreated patients (PUPs), typically in the first 50 exposure days (EDs), with serious complications. This is the first study evaluating the safety, immunogenicity, and hemostatic efficacy of rurioctocog alfa pegol (Adynovate ®; Baxalta US Inc., a Takeda company, Lexington, MA, USA), an extended half-life (EHL) recombinant FVIII, in PUPs with severe hemophilia A. Methods This prospective, open-label, multi-center, phase 3 study (NCT02615691) was conducted in patients ˂6 years of age with severe hemophilia A (FVIII <1%). Patients were previously untreated, or had <3 EDs to rurioctocog alfa pegol, octocog alfa, or plasma transfusion at any time prior to screening. Patients with detectable FVIII inhibitory antibodies at screening or a history of FVIII inhibitory antibodies prior to screening (≥0.6 Bethesda units) were not eligible. Patients received intravenous rurioctocog alfa pegol as prophylaxis (25-50 IU/kg, up to 80 IU/kg ≥1 × weekly) and/or on-demand therapy (10-50 IU/kg, up to 80 IU/kg depending on bleed severity). Prophylaxis was started before 3 years of age or after a maximum of 2 joint bleeds, whichever occurred first. The primary endpoint was the incidence of FVIII inhibitor development. Secondary endpoints included safety and efficacy (annualized bleeding rate [ABR] and hemostatic efficacy). This protocol-specified interim analysis was conducted after 50 patients had completed ≥50 EDs without developing an inhibitor to FVIII or had developed a confirmed FVIII inhibitor at any time. The data cut-off was 30 August 2019. Demographic and baseline characteristics were summarized using continuous and categorical data. The incidence of FVIII inhibitor development was calculated using the Clopper Pearson exact 95% CI computed for the proportion of patients who developed FVIII inhibitors during the study. ABR was analyzed by point and interval estimates derived from a negative binomial model with treatment regimen as a covariate. The number and percentage of patients reporting adverse events (AEs) and serious AEs (SAEs) was recorded for all patients receiving rurioctocog alfa pegol. Informed consent and ethics approval were obtained. Results As of the data cut-off, 59 (73.8%) of 80 enrolled patients had received ≥1 dose of rurioctocog alfa pegol; 18 patients (screen failures) did not meet the eligibility criteria and 4 discontinued prior to treatment. 54 patients received prophylaxis and 35 received on-demand treatment at any time during the study period. The mean (SD) patient age at baseline was 11.8 (8.2) months. The number of patients with 0 EDs prior to screening was 36 (61.0%), with 9 (15.3%) patients having 1 ED and 14 (23.7%) having 2 EDs. Overall, 32 patients had a family history of hemophilia A. A large deletion, intron 1 or intron 22 inversion, or substitution nonsense hemophilia gene mutation was present in 29 (49.2%) patients and 21 (35.6%) had either a small deletion, small duplication, or substitution missense gene mutation. Of the 52 patients who qualified for this interim analysis, 10 developed an inhibitory antibody to rurioctocog alfa pegol during the study; the incidence of inhibitor development was 0.192 (95% CI, 0.096-0.325) (10/52). Rurioctocog alfa pegol exposure data and ABRs for patients receiving prophylaxis or on-demand treatment are presented in Table 1. At bleed resolution, hemostatic efficacy was rated by patients as "excellent" for 88/269 bleeds (32.7%) and "good" for 73/269 bleeds (27.1%). Overall, 52 (88.1%) patients receiving rurioctocog alfa pegol experienced a total of 283 AEs, and 13 patients experienced 14 rurioctocog alfa pegol-related AEs (including 10 SAEs). SAEs occurred in 24 patients, 10 of whom experienced 10 treatment-related SAEs of FVIII inhibitor development. Discussion This is the first prospective study of the EHL recombinant FVIII rurioctocog alfa pegol for the treatment of PUPs with severe hemophilia A. These preliminary results demonstrate a relatively low inhibitor rate compared with other EHL recombinant FVIII products and a safety and efficacy profile consistent with that previously observed for rurioctocog alfa pegol in the treatment of bleeding episodes in patients with hemophilia A. Figure 1 Figure 1. Disclosures Sidonio: Guardian Therapeutics: Consultancy; Pfizer: Consultancy; Bayer: Consultancy; Octapharma: Consultancy, Research Funding; Novo Nordisk: Consultancy; Biomarin: Consultancy; Genentech: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; Catalyst: Consultancy. Peyvandi: Takeda: Honoraria; Spark: Honoraria; Sobi: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Roche: Honoraria; Bioverativ: Honoraria; Grifols: Honoraria. Stasyshyn: Octapharma: Consultancy, Research Funding, Speakers Bureau; Pfizer: Consultancy, Research Funding, Speakers Bureau; CSL Behring: Consultancy, Research Funding; Shire: Consultancy; Grifols: Consultancy, Speakers Bureau; Takeda: Consultancy, Research Funding, Speakers Bureau; Novo Nordisk: Consultancy, Research Funding, Speakers Bureau. Antmen: Takeda: Consultancy; Pfizer: Consultancy; Roche: Consultancy; Novo Nordisk: Consultancy. Yeoh: Takeda: Honoraria; Pfizer: Honoraria; Roche: Honoraria; Grifols: Honoraria. Maggiore: IQVIA: Current Employment. Engl: Baxalta Innovations GmbH, a Takeda company: Current Employment; Takeda: Current equity holder in publicly-traded company. Allen: Takeda Development Center Americas, Inc.: Current Employment; Takeda: Current equity holder in publicly-traded company. Tangada: Takeda: Current equity holder in publicly-traded company; Takeda Development Center Americas, Inc: Current Employment.

Antigen-specific immunotherapy with apitopes suppresses generation of FVIII inhibitor antibodies in HLA-transgenic mice

Haemophilia A (HA) is a blood clotting disorder caused by various genetic deficiencies in the factor VIII (FVIII) encoding F8 gene. Patients receiving FVIII replacement therapy are at risk of developing neutralizing antibodies (FVIII inhibitors) rendering the FVIII replacement therapy ineffective. Immunological tolerance towards FVIII can be achieved through immune tolerance induction (ITI) protocols in some patients but this is a lengthy and costly desensitization programme. Long-term eradication of inhibitors in HA patients could be achieved by antigen-specific immunotherapy targeting CD4+ T cells since formation of FVIII inhibitors is T cell dependent. Here, we report a peptide-based, antigen-specific immunotherapy designed to specifically re-establish immune tolerance to FVIII through the development of antigen-processing-independent epitopes (apitopes). We identified two FVIII immunodominant peptides in immunised human leukocyte antigen (HLA) DRA*0101/DRB1*1501 transgenic (HLA-DR2tg) mice that were optimised for tolerogenicity. These modified peptide analogues were initially screened for recognition using FVIII-specific T cell hybridoma clones from FVIII-immunised HLA-DR2tg mice. The FVIII apitopes were promiscuous and bound common human HLA-DRB1*haplotypes. The combination of these two FVIII apitopes (ATX-F8-117), administered according to a dose escalation protocol, promoted T cell tolerance towards FVIII in HLA-DR2tg mice. Furthermore, treatment with ATX-F8-117 significantly reduced FVIII inhibitor formation. ATX-F8-117 regulates both anti-FVIII T cell and B cell responses, specifically the generation of FVIII inhibitors, revealing peptide-based antigen-specific immunotherapy as a promising approach to both suppress and treat inhibitor formation in susceptible HA patients.

512 A CASE OF AN IDIOPATHIC ACQUIRED HEMOPHILIA A IN AN ELDERLY WOMAN

Introduction Acquired Hemophilia is a bleeding diathesis caused by autoantibodies that interfere with factor VIII (FVIII). Reasons for autoantibodies production are not clear but may be related to gene polymorphisms and/or CD4+ T lymphocytes. 1.3 to 1.5 cases per million population per year are reported in the UK. Half of the cases are secondary to malignancy, pregnancy related conditions, connective tissue disorders or drug reactions while the rest are idiopathic. Case Report We report a case of an acquired hemophilia A in an 86-year-old lady with underlying type 2 diabetes, hypertension, and cognitive impairment, being treated as the left lower limb cellulitis with antibiotics. She was found to have a sudden hemoglobin drop and her CT (Abdomen) confirmed a spontaneous intra-abdominal hematoma. Clotting profile showed prolonged APTT to 168.5 seconds, being not corrected at mixing study, with normal PT and INR. The FVIII assay was reduced to 18.4 iU/dL with FVIII inhibitor concentration of 0.7 Bu. Viral and autoimmune screenings were negative. The idiopathic acquired hemophilia A was diagnosed. Red blood cell transfusions, bypassing agents (FEIBA) and oral tranexamic acid were given for acute bleeding episode. Concomitantly, oral prednisolone was used to reduce the inhibitor levels. Repeated FVIII assay showed 121 iU/dL and 199iU/dL on day 6 and 12, respectively. Steroid was continued for the next 4 weeks and then gradually tapered. No further bleeding episode was noted. Conclusion The diagnosis of acquired hemophilia should be considered in any elderly patient with prolonged APTT. Mixing study is to measure the presence of inhibitors of coagulation or to detect coagulation factor deficiency. Quantitative coagulation factor assays and Bethesda Assays are performed for definitive diagnosis. Immunosuppressive regimens are the mainstay treatment. However, premorbid conditions and co-morbidities should be taken into consideration before initiating the aggressive immunosuppressive therapy in the elderly patients.

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

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

Inhibitor Index in the Clot Waveform Analysis-Based Mixing Test Differentiates among Hemophilia A without and with Inhibitors, and Lupus Anticoagulant

Background The mixing test is used to identify the pathway to follow-up testing and is also useful for the investigation of lupus anticoagulant (LA) positivity. “To completely correct” indicates clotting factor deficiency, while “to not correct” indicates the presence of a clotting factor inhibitor including LA. “Index of circulation anticoagulant” and/or “percent correction” is used to interpret the results of mixing studies, but it does not accurately differentiate factor inhibitors from LA. Aim To precisely differentiate hemophilia A (HA), HA with inhibitor (HA-inh), and LA using the clot waveform analysis (CWA)-based mixing test. Methods Plasma samples from HA, LA, and HA-inh including acquired HA were incubated with normal plasma in 9:1, 1:1, and 1:9 mix ratios. From activated partial thromboplastin time CWA at 0-minute (immediately) and 12-minute incubation, the ratios of CWA parameters at 12 minutes/0 minute (inhibitor index) were assessed. Results The inhibitor index values of CWA parameters obtained using the mixing test in a 1:1 ratio demonstrated a significant difference between HA-inh and LA but could not differentiate LA from HA-inh completely. Plasmas used for the mixing tests in 9:1 and 1:9 ratios were able to fully distinguish between HA-inh (>0.5 BU/mL) and LA. These indices significantly correlated with inhibitor titer below 40 BU/mL (r > 0.90), possibly estimating FVIII inhibitor titer from the inhibitor index. Plasmas in HA and LA could be distinguished by mixing in a 1:1 ratio at 0 minute (immediately). Conclusion The inhibitor index from CWA-based mixing tests with a 12-minute incubation could differentiate among HA, HA-inh, and LA quickly.