Doses Of FVIII Research Articles

The Severe Spontaneous Bleeding Phenotype in a Novel Hemophilia a Rat Model with an Inversion Mutation Is Rescued By Platelet-Targeted FVIII Expression

Previous studies by our group and others have demonstrated that platelet targeted FVIII expression can rescue the bleeding phenotype in hemophilia A (HA) mice in both the inhibitor and non-inhibitor models. The clinical efficacy has been further proven in a dog HA non-inhibitor model. While these are informative, neither the canine nor mouse model entirely mimic human disease because 1) neither HA mice nor dogs exhibit the frequent spontaneous bleeding in severe HA patients, and 2) canine platelets do not contain FVIII carrier protein VWF and therefore store and release FVIII differently, thus precluding examining the efficacy of platelet gene therapy in the presence of inhibitors.The efficacy of platelet-FVIII gene therapy, therefore, needs to be examined in an animal model more representative of human disease. Since the inversion of FVIII gene is the most common mutation that causes severe HA in patients, using a CRISPR/Case9 strategy we generated a novel HA rat model in a Dahl/Salt Sensitive (Dahl) inbred congenic background (Dahl-F8KO) with the nearly entire murine FVIII gene inverted causing a translational stop 6 amino acids after the signal sequence of FVIII. There is no detectable FVIII in plasma in Dahl-F8KO rats (LOD is 1 U/dL). Spontaneous bleeding in the soft tissue, muscles, or joints occurred in 56.5% of homozygous affected animals (n=46) by the age of 8-weeks and up to 82.6% by the age of 32-weeks. This was significantly higher (P<0.0001) than the occurrence of spontaneous bleeding in another colony in Sprague Dawley (SD) background developed by Nielsen et al. using zinc finger nuclear strategy (J Thromb Haemost 2014) with a 13-bp deletion in exon 16 of FVIII. In SD-F8KO rat model, spontaneous bleeds occur in 23.3% and 42.1% of homozygous affected animals (n=85) that were housed in our facility by the age of 8- and 32-weeks, respectively. Likewise, the percentages of homozygous affected animals with 2, 3, or 4 bleeding episodes in the Dahl-F8KO colony were significantly higher than those in the SD-F8KO colony. In contrast, none of the heterozygous (F8+/-) animals ever have a spontaneous bleed. Similar to SD-F8KO rats, Dahl-F8KO rats can develop anti-FVIII inhibitory antibodies after 2 doses of recombinant human FVIII (rhF8) treatment. Thus, our novel Dahl-F8KO rat model is a unique animal model with the severest spontaneous bleeding phenotype yet reported.To investigate if platelet-derived FVIII can rescue the spontaneous bleeding phenotype in HA rats, we generated transgenic rats (2bF8Tg) in both SD- and Dahl-background in which FVIII expression is under control of the platelet-specific αIIb promoter (2bF8) using lentivirus-mediated oocyte transduction transgenesis. Bone marrow (BM) mononuclear cells were isolated from femurs of 2bF8Tg rats and transplanted into F8-/- rats preconditioned with 950 R TBI. Animals were closely monitored after BM transplantation (BMT), and blood samples were collected for FVIII assays and whole blood thrombin generation assay (wbTGA). After BMT from SD-2bF8Tg rats, no spontaneous bleeding was observed in SD-F8KO recipients within the entire study course (1 year at the time of this report) with a sustained platelet-FVIII expression of 20.9±8.1 mU/108 platelets (n=3). Similarly, we performed BMT from Dahl-2bF8Tg rats into Dahs-F8KO and found that 1 of 3 recipients had a bruise at the early stage of BM reconstitution. However, no any other spontaneous bleeding has yet been observed (5 months). To confirm that the bleeding diathesis in F8-/- rats was ameliorated after 2bF8 gene expression, wbTGA was performed. All parameters, including Lag Time, Peak Time, Peak Thrombin, Endogenous Thrombin Potential in 2bF8Tg-BMT recipients were significantly different compared to F8-/- control rats, but were similar to those obtained in WT control animals. Of note, neither detectable levels of plasma FVIII nor anti-F8 antibodies were detected in F8-/- recipients after receiving BMT from 2bF8Tg rats.In summary, we have developed a novel HA rat model with both the pathophysiology and clinical phenotype found in severe HA patients, which could be an ideal model for evaluating the clinical efficacy of new therapeutics. Our studies demonstrated that transplantation of BM cells that are genetically modified to express FVIII only in platelets can efficiently prevent the severe spontaneous bleeding in HA rats with no anti-F8 antibody development. DisclosuresMontgomery:BCW: Patents & Royalties: GPIbM assay patent to the BloodCenter of Wisconsin.

A Modified Thrombin Generation Analysis to Measure the Plasma Coagulation Potency in the Presence of Anti-FIXa/FX Bispecific Antibody, Emicizumab

【Introduction】 Emicizumab is a humanized anti-factor (F)IXa/FX bispecific antibody with FVIIIa cofactor function. Since emicizumab, unlike FVIII, does not require activation by thrombin, its aPTT-shortening effect is much greater than that of FVIII. Thus, aPTT, a conventional assay to assess intrinsic coagulation potency, would have limited utility in emicizumab-administered hemophilia A-patients (HA-pts), because emicizumab would mask the effect of residual FVIII or a FVIII agent on aPTT. Also, aPTT cannot differentiate between different levels of emicizumab.Recently we reported that clot waveform analysis (CWA) using a trigger reagent comprising a balanced mixture of ellagic acid (Elg) and tissue factor (TF) to reflect both intrinsic and extrinsic coagulation activities could provide a useful means of assessing plasma coagulation potential in HA-pts treated with emicizumab with enhanced activity neither masking nor being masked by FVIII or bypassing agents (BPAs)1). Thrombin generation assay (TGA) can provide multidimensional plasma coagulation potencies as well as CWA. We considered the possibility, therefore, that the TGA would also overcome the above issue on aPTT. In this study, we aimed to assess TGA using Elg/TF trigger and two reference triggers (FXIa, TF) to evaluate the coagulation potency of patients with HA receiving emicizumab.【Method】 TGA was assayed using Elg/TF trigger consisted of TF (0.5 pM) and Elg (0.3 μM) with phospholipids vesicles (PL, 4 μM), FXIa trigger consisted of FXIa (0.47 nM) with PL (20 μM), and TF trigger (PPP-Reagent LOW®). Various concentrations of emicizumab, FVIII agent, or BPAs (rFVIIa, aPCC) in clinical dosages were spiked into commercially available FVIII-deficient plasmas (George King) for testing. Then, FVIII or BPA was added to FVIII-deficient plasmas containing emicizumab.【Result】 Emicizumab or FVIII showed dose-dependent increase in thrombin peak height in Elg/TF trigger as well as FXIa and TF triggers, but TF trigger had weak sensitivity to emicizumab and low range FVIII (1-10 IU/dl). Spiking both rFVIIa or aPCC showed dose dependent increase in thrombin peak height under Elg/TF trigger and TF trigger conditions, but the values were lower than the level of normal peak height. On the other hand, rFVIIa and aPCC resulted in little increase in peak height under FXIa trigger condition, suggesting that the FXIa trigger is not suitable for assessment of BPAs. These results indicated that Elg/TF trigger system was the most useful to evaluate the single-spiked effect of these agents.Then, we examined the additional effects of FVIII or BPAs in the presence of emicizumab by Elg/TF trigger system. The combination of FVIII and emicizumab showed additive increase in peak thrombin height, but this effect was saturated at high dose of FVIII more than 100 IU/dl. rFVIIa and emicizumab showed additive effect on increased peak thrombin height, achieving normal level even at very low dose rFVIIa (0.67 μg/ml), but remains within normal ranges at high dose (6.0 μg/ml). aPCC and emicizumab showed increased peak thrombin height, but in this case, exceeding the normal level even at clinical dosage of aPCC (0.5-1.0 U/ml).【Conclusion】 TGA with Elg/TF trigger could provide a useful monitoring tool of assessing global coagulation potential during emicizumab prophyaxis including concomitant therapy with FVIII or BPAs.1) Nogami K, et al. Modified clot waveform analysis to measure plasma coagulation potential in the presence of the anti-factor IXa/factor X bispecific antibody emicizumab. J Thromb Haemost. 2018 Jun;16(6):1078-1088. DisclosuresOgiwara:CSL Behring: Research Funding. Nogami:Chugai Pharmaceutical Co., Ltd: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: Anti-FIXa/X bispecific antibodies , Research Funding, Speakers Bureau. Matsumoto:Shire Japan Co. Ltd: Research Funding. Noguchi-Sasaki:Chugai: Employment. Soeda:Chugai: Employment, Patents & Royalties: Patents related to emicizumab. Matsumoto:Chugai Pharmaceutical Co., Ltd: Employment, Equity Ownership. Hirata:Chugai Pharmaceutical Co., Ltd: Employment, Equity Ownership. Shima:Chugai Pharmaceutical Co., Ltd: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: Anti-FIXa/X bispecific antibodies , Research Funding, Speakers Bureau; F. Hoffmann-La Roche Ltd: Membership on an entity's Board of Directors or advisory committees.

Anti‐C1 domain antibodies that accelerate factor VIII clearance contribute to antibody pathogenicity in a murine hemophilia A model

Background The development of neutralizing anti-factorVIII (FVIII) antibodies remains a challenging complication of modern hemophiliaA care. Invitro assays are the primary method used for quantifying inhibitor titers, predicting bleeding risk, and determining bleeding management. However, other mechanisms of inhibition are not accounted for in these assays, which may result in discrepancies between the inhibitor titer and clinical bleeding symptoms. Objectives To evaluate FVIII clearance invivo as a potential mechanism for antibody pathogenicity and to determine whether increased FVIII dosing regimens correct the associated bleeding phenotype. Methods FVIII-/- or FVIII-/- /von Willebrand factor (VWF)-/- mice were infused with anti-FVIII mAbs directed against the FVIII C1, C2 or A2 domains, followed by infusion of FVIII. Blood loss via the tail snip bleeding model, FVIII activity and FVIII antigen levels were subsequently measured. Results Pathogenic anti-C1 mAbs that compete with VWF for FVIII binding increased the clearance of FVIII-mAb complexes in FVIII-/- mice but not in FVIII-/- /VWF-/- mice. Additionally, pathogenic anti-C2 mAbs that inhibit FVIII binding to VWF increased FVIII clearance in FVIII-/- mice. Anti-C1, anti-C2 and anti-A2 mAbs that do not inhibit VWF binding did not accelerate FVIII clearance. Infusion of increased doses of FVIII in the presence of anti-C1 mAbs partially corrected blood loss in FVIII-/- mice. Conclusions A subset of antibodies that inhibit VWF binding to FVIII increase the clearance of FVIII-mAb complexes, which contributes to antibody pathogenicity. This may explain differences in the bleeding phenotype observed despite factor replacement in some patients with hemophiliaA and low-titer inhibitors.

Intensity of factor VIII treatment and the development of inhibitors in non‐severe hemophilia A patients: results of the INSIGHT case–control study

Background Inhibitor development is a major complication of treatment with factor VIII concentrates in hemophilia. Findings from studies among severe hemophilia A patients suggest that intensive treatment episodes increase the risk of developing inhibitors. Objectives We set out to assess whether intensive treatment is also associated with an increased risk of inhibitor development among non-severe hemophilia A patients. Patients/Methods We performed a nested case-control study. A total of 75 inhibitor patients (cases) and 223 control patients were selected from 2709 non-severe hemophilia A patients (FVIII:C, 2-40%) of the INSIGHT cohort study. Cases and controls were matched for date of birth and cumulative number of exposure days (EDs) to FVIII concentrates. Conditional logistic regression was used to calculate both unadjusted and adjusted odds ratios (aOR); the latter were adjusted for a priori specified confounders. Results Peak treatment of 5 or 10 consecutive EDs did not increase inhibitor risk (aOR, 1.0; 95% confidence interval (CI), 0.4-2.5; and aOR, 1.8; CI, 0.6-5.5, respectively). Both surgical intervention (aOR, 4.2; CI, 1.7-10.3) and a high mean dose (> 45 IU kg-1 /ED) of FVIII concentrate (aOR, 7.5; CI, 1.6-35.6) were associated with an increased inhibitor risk. Conclusions Our findings suggest that high-dose FVIII treatment and surgery increase the risk of inhibitor development in non-severe hemophilia A. Together with the notion that non-severe hemophilia A patients are at a lifelong risk of inhibitor development, we suggest that in the future physicians will review the arguments for the FVIII dose and elective surgery extra critically.

Very High Dose Factor VIII Needed to Prevent Bleeding in the Presence of a Low Titer Anti-C1 Domain Antibody

Introduction: Up to 30% of patients with severe hemophilia A will develop inhibitory antibodies to factor VIII (fVIII inhibitors). Patients with congenital hemophilia who develop inhibitors usually have a polyclonal antibody response directed against the A2 and C2 domains of fVIII, but antibodies to all domains have been reported. We recently reported that anti-C1 domain antibodies were present in approximately 50% of plasmas from patients with hemophilia A and inhibitors. M6143 is a low titer (180 BU/mg IgG), tight-binding, anti-C1 monoclonal antibody that inhibits fVIII binding to von Willebrand Factor but not phospholipid. Surprisingly in a murine tail-snip bleeding model M6143 was pathogenic despite having a low inhibitory titer of 2.3 BU/ml (0.5 mg M6143/kg mouse body weight) and treatment with 180 U/kg of B-domain deleted (BDD) fVIII. The presence of M6143 resulted in increased clearance of fVIII/M6143 complexes. Given this data we hypothesized M6143 would remain pathogenic until fVIII was added in molar excess of M6143.Methods: 8-12 week old E16 hemophilia A mice received 100 µl injections of M6143 at a concentration of 0.5 mg/kg (65 nM estimated peak plasma concentration), 0.25 mg/kg (~32.5 nM), 0.125 mg/kg (~16 nM), 0.0625 mg/kg (8 nM), or normal saline, followed by BDD fVIII 15 minutes later at a dose of 180 U/kg (~2.5 nM estimated peak plasma concentration), 360 U/kg (~5 nM), 720 U/kg (~10 nM), or 1440 U/kg (~20 nM). Tails were warmed then transected at 4 mm distally two hours post fVIII injection. Blood loss per mouse body weight (mg/g) following tail snips was measured using a pre-weighed 15 ml conical tube containing normal saline.Results: Hemophilia A mice treated with 180 U/kg in the absence of antibody had minimal blood loss. M6143 at 0.5 mg/kg (2.3 BU/ml) produced significant bleeding at fVIII doses ranging from 180-1440 U/kg. Subsequently the concentration of M6143 was decreased while continuing extremely high fVIII dosing of 1440 U/kg (~20 nM). At all decreased concentrations of M6143, with high dose FVIII, bleeding was corrected (Figure 1).Conclusion: M6143 is a minimally inhibitory antibody whose pathogenicity is only overcome in the presence of very high dose fVIII. The amount of fVIII needed relative to the inhibitor titer is paradoxical to the typical clinical strategy that low titer inhibitors can be overwhelmed with modest increases in the fVIII dosing. Inhibitors with similar characteristics to M6143 may be responsible for the subset of patients with low titer inhibitors who do not respond to treatment with fVIII. [Display omitted] DisclosuresMeeks:CSL Behring: Membership on an entity's Board of Directors or advisory committees; Biogen: Membership on an entity's Board of Directors or advisory committees; Genentech: Membership on an entity's Board of Directors or advisory committees; Grifols: Membership on an entity's Board of Directors or advisory committees; Bayer Healthcare: Membership on an entity's Board of Directors or advisory committees; Shire: Membership on an entity's Board of Directors or advisory committees.

Effects of Joint Blood Volume and Hemostasis Correction on Vascular Changes in the Joint after Induced Hemarthrosis in Hypocoagulable Mice

Introduction:Hemophilia causes recurrent bleeds into 'target joints' despite clotting factor replacement. Vascular remodeling associated with hemarthrosis is thought to contribute to vessel "leakiness" and perpetuated bleeding, but the underlying mechanisms remain unknown. Here, we studied the effects of joint blood volume and timing of hemostasis correction on vascular changes after induced hemarthrosis in FVIII-deficient mice, and in BALB/c mice treated temporarily with warfarin and anti-FVIII to create reversible hemostasis suppression.Methods: Mice were injured by sub-patellar puncture of the right knee to induce hemarthrosis. FVIII-deficient mice were treated with two doses of recombinant human FVIII (rhFVIII) or saline with an 8-12 hour interval. The first dose of rhFVIII was given i) 2 hours before injury (prophylaxis), ii) 2 hours after injury (early), iii) 8 hours after injury (intermediate), or iv) 16 hours after injury (late). BALB/c mice were administered 10 µg/ml warfarin in drinking water for 1 week plus 0.25 mg/kg anti-FVIII 2 hours prior to injury. This dose of warfarin yielded a ~4-fold increase in prothrombin time that was reversed with 100 IU/kg 4-Factor prothrombin complex concentrate on day 2 post-injury in one group of mice, and maintained in another. Clearance of anti-FVIII within 4 days was indicated by normalization of blood loss in tail clip experiments. On day 2 after injury, the hematocrit was determined as a measure of joint bleeding and incapacitance was monitored by left:right weight bearing. Vascularity was assessed at baseline and 1-2 weeks post-injury by high resolution musculoskeletal ultrasound with Power Doppler (MSKUS/PD) and histology with Safranin-O-Fast Green staining. Statistical analyses were conducted by Mann-Whitney test.Results: In FVIII-deficient mice, knee injury significantly reduced the mean hematocrit from 47 % to ~25 % for mice receiving late or no rhFVIII treatment (p<0.001). Hematocrit remained at 47 % with prophylaxis, and decreased to 40 % (p=0.01) and 37 % (p<0.0001) with early and intermediate rhFVIII treatment, respectively. In the saline-only group, hemarthrosis caused a 3.1-fold increase in left:right weight bearing of the hind legs (p=0.008). Earlier treatments with rhFVIII alleviated this incapacitance and mice receiving rhFVIII prophylaxis had no deficit. In contrast, vascular changes were similar across treatment groups, regardless of hematoma size. Mean vessel number increased ~3-fold compared to baseline for all groups (p<0.05). This was accompanied by moderate increases in mean vessel diameter and percentage of vessels with a diameter ≥ 20 µm. Vascular perfusion measured by MSKUS/PD increased significantly (~2.5-fold) with early (p=0.01), intermediate, late or no rhFVIII treatment (p<0.001), but not with prophylaxis.Bleeding tendency and weight bearing deficits of the injured leg in BALB/c mice treated with warfarin and anti-FVIII were comparable to those observed in FVIII-deficient mice. Mean hematocrit decreased to 28 % (p<0.0001) and left:right weight bearing ratio increased to 2.6 (p=0.003). Mean vessel number in injured knees increased to a similar extent in the warfarin continuation and warfarin reversal groups (~1.5-fold; p=0.02). In contrast, mean vessel diameter and percentage of vessels with a diameter ≥ 20 µm were elevated only in mice continued on warfarin (p=0.008 and p=0.02, respectively).Conclusions: FVIII prophylaxis or early treatment reduced blood loss into joints of FVIII-deficient mice and preserved joint function. However, neovascularization and vascular changes consistent with remodeling were not proportional to joint blood volume and were mostly unresponsive to short-term FVIII replacement, regardless of timing. Similarly, neovascularization in joints of hemostatically compromised BALB/c mice after induced hemarthrosis persisted despite normalization of hemostasis. Changes in vessel architecture, however, could be abrogated. This suggests that long-term hemostasis correction may protect against irreversible vascular remodeling in the joint, thought to underlie re-bleeding and development of hemophilic arthropathy. Further investigations are required to determine the effects of prolonged hemostasis correction on re-bleeding tendencies and joint health in hemophilia. DisclosuresMosnier:The Scripps Research Institute: Patents & Royalties; Hematherix LLC: Membership on an entity’s Board of Directors or advisory committees; Bayer: Honoraria, Speakers Bureau; Baxalta: Honoraria, Speakers Bureau. von Drygalski:Hematherix LLC: Membership on an entity’s Board of Directors or advisory committees; Pfizer: Consultancy, Honoraria, Speakers Bureau; Novo Nordisk: Consultancy, Honoraria, Speakers Bureau; CSL-Behring: Consultancy, Honoraria, Speakers Bureau; Biogen: Consultancy, Honoraria, Speakers Bureau; Bayer: Consultancy, Honoraria, Speakers Bureau; Baxalta/Shire: Consultancy, Honoraria, Speakers Bureau.

A population pharmacokinetic model for perioperative dosing of factor VIII in hemophilia A patients.

The role of pharmacokinetic-guided dosing of factor concentrates in hemophilia is currently a subject of debate and focuses on long-term prophylactic treatment. Few data are available on its impact in the perioperative period. In this study, a population pharmacokinetic model for currently registered factor VIII concentrates was developed for severe and moderate adult and pediatric hemophilia A patients (FVIII levels <0.05 IUmL-1) undergoing elective, minor or major surgery. Retrospective data were collected on FVIII treatment, including timing and dosing, time point of FVIII sampling and all FVIII plasma concentrations achieved (trough, peak and steady state), brand of concentrate, as well as patients' and surgical characteristics. Population pharmacokinetic modeling was performed using non-linear mixed-effects modeling. Population pharmacokinetic parameters were estimated in 75 adults undergoing 140 surgeries (median age: 48 years; median weight: 80 kg) and 44 children undergoing 58 surgeries (median age: 4.3 years; median weight: 18.5 kg). Pharmacokinetic profiles were best described by a two-compartment model. Typical values for clearance, intercompartment clearance, central and peripheral volume were 0.15 L/h/68 kg, 0.16 L/h/68 kg, 2.81 L/68 kg and 1.90 L/68 kg. Interpatient variability in clearance and central volume was 37% and 27%. Clearance decreased with increasing age (P<0.01) and increased in cases with blood group O (26%; P<0.01). In addition, a minor decrease in clearance was observed when a major surgical procedure was performed (7%; P<0.01). The developed population model describes the perioperative pharmacokinetics of various FVIII concentrates, allowing individualization of perioperative FVIII therapy for severe and moderate hemophilia A patients by Bayesian adaptive dosing.

A Cross-Sectional Analysis of Cardiovascular Disease in the Hemophilia Population

A Cross-Sectional Analysis of Cardiovascular Disease in the Hemophilia Population

Adeno-Associated Viral Vector Delivery of Optimized Human Factor VIII Achieves Therapeutic Factor VIII Levels in Non-Human Primates

Clinical studies of adeno-associated viral (AAV)-mediated gene transfer of factor IX for hemophilia B have demonstrated long term expression of therapeutic levels of factor IX but revealed that the AAV vector dose may be limiting due to anti-AAV immune responses (Nathwani, 2011). While there is significant interest in moving this approach forward for hemophilia A, it is challenging to express high levels of human factor VIII (hFVIII) due to its intrinsic properties that result in lower expression levels compared to similarly sized proteins (Lynch, 1993). Approaches using codon optimization and variants of hFVIII with enhanced function (increased activity, stability and/or secretion) may provide strategies to increase hFVIII expression to support AAV clinical studies for hemophilia A. For example, we previously developed a codon-optimized hFVIII (CO3) that expressed 5-8-fold higher protein levels than wild type hFVIII after AAV delivery in the context of an optimized expression cassette utilizing a modified transthyretin (TTRm) promoter. Introduction of a PACE-furin (P/F) variant (Siner, 2013) that deletes residues 1645-47 (Δ3) or 1645-48 (Δ4) of the PACE-furin recognition site in CO3 resulted in hFVIII expression after AAV delivery that was 18 (Δ3) or 12-fold (Δ4) better than wild type hFVIII. To date, only one published study has reported clinically relevant levels of human FVIII following AAV treatment in a large animal model. This study used a hFVIII variant that contained a 17 amino acid synthetic sequence flanked by 14-amino acid SQ residues from the N- and C-terminal ends of the B domain (McIntosh, 2013). While the presence of the synthetic spacer allowed for an increase in circulating hFVIII levels, the use of a non-wild-type FVIII sequence in hemophilia A patients may increase the risk of development of neutralizing antibodies to FVIII due to its potential neo-antigenicity. Our goal in this study was to generate an AAV-hFVIII vector capable of expressing therapeutic doses of FVIII at a clinically relevant vector dose without adding any neoantigens to the protein. To this end, we generated 26 codon-optimized hFVIII-SQ constructs under the control of the TTRm promoter. Hydrodynamic delivery of the pAAV-TTRm-hFVIII plasmids identified 11 candidates that expressed FVIII 2-7 fold higher than CO3. Nine of these FVIII expression constructs were made into AAV vectors and delivered to hemophilia A/CD4 KO mice (1x1011 vg/mouse) using a novel capsid, AAV-Spark100. At 4 weeks post vector administration, 2/9 constructs were similar to CO3, 5/9 were 3-4 fold higher than CO3 and 2/9 (SPK-8003 and SPK-8005) were 4-6 fold higher than CO3. To determine if the deletion of the PACE-furin site would result in higher FVIII expression, the Δ4 P/F deletion was introduced into SPK-8003. The levels of FVIII expression after AAV-TTRm-SPK-8003-Δ4 P/F delivery were 2-fold higher than AAV-TTRm-SPK-8003. In order to evaluate the potency of these novel cassettes in a large animal model, SPK-8005 was administered as a single dose via intravenous infusion to male cynomolgus macaques and followed for 8 weeks of observation. At two weeks after gene transfer, NHPs transduced with 2x1012 vg/kg of SPK-8005 expressed hFVIII antigen levels of 12.7 ± 2.1% (average ± standard error of the mean, n=3). Average FVIII expression after treatment with 5x1012 vg/kg was 22.6 ± 0.8% (n=2). Finally, at the highest tested dose of 1x1013 vg/kg, hFVIII antigen levels of 54.1 ± 15.6% were observed two weeks after AAV infusion (n=3). As anticipated, hFVIII expression declined in approximately one third of the animals around week 4, concomitant with the appearance of inhibitory antibodies to human factor VIII in these macaques. In summary, these data using highly active, novel codon-optimized FVIII constructs devoid of potential neoantigens demonstrate the feasibility of lowering the AAV capsid load for a gene-based therapeutic approach for hemophilia A to a dosage level that appears to be efficacious and safe in the treatment of hemophilia B. DisclosuresAnguela:Spark Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties. Elkouby:Spark Therapeutics, Inc.: Employment, Equity Ownership. Toso:Spark Therapeutics, Inc.: Employment, Equity Ownership. DiPietro:Spark Therapeutics, Inc.: Employment, Equity Ownership. Davidson:Spark Therapeutics: Consultancy. High:Spark Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties: AAV gene transfer technology. Sabatino:Spark Therapeutics, Inc.: Research Funding.

Anti-C1 Domain Antibodies Are Pathogenic in a Murine Tail Snip Model Despite Low Inhibitor Titers

Introduction: Hemophilia A is an X-linked bleeding disorder characterized by a deficiency or absence of blood coagulation protein factor VIII (fVIII). Inhibitor development is the most common complication of fVIII treatment occurring in up to 30% of individuals with severe hemophilia A. Although the C2 and A2 domains are considered the predominant immunogenic domains of fVIII, prior work in our lab using a hemophilia A mice model immunized with fVIII showed that ~10% of the 506 hybridomas isolated produced antibodies to the C1 domain. Further studies have shown that the C1 domain participates in binding to activated platelets, phospholipid membranes, and von Willebrand factor (VWF) as well as fVIII uptake by dendritic cells. Two recent studies postulated that VWF primarily interacts with fVIII at the C1 domain using negative-stain electron microscopy. However the pathogenicity of anti-C1 antibodies have yet to be fully explored.Methods: E16 knockout hemophilia A mice were immunized with B-domain deleted fVIII. Spleens from the mice were harvested and fused with NS-1 myeloma cells to produce hybridomas using standard Köhler-Milstein technology. Hybridomas producing antibodies to the C1 domain were isolated and purified for monoclonal antibodies (MAbs). The effect of these anti-C1 MAbs on fVIII function and bleeding phenotype in mice were analyzed.Results: We have produced 8 anti-C1 MAbs from 5 different spleens. Two non-overlapping B-cell epitopes were identified, group A and B, using a competition sandwich ELISA. The 7 group A MAbs recognize an overlapping B-cell epitope despite 5 different spleens of origin, providing evidence for an immunodominant B-cell epitope. The group A MAbs weakly inhibit fVIII with inhibitor titers of < 180 BU/mg IgG by Bethesda assay. In contrast anti-A2 MAb 4A4 has an inhibitor titer of 40,000 BU/mg IgG. Five of these 7 MAbs inhibit fVIII binding to VWF and 1 MAb inhibits fVIII binding to phospholipids by competition ELISA. MAb B136, a group B MAb, binds to an epitope distinct from the group A MAbs and has an inhibitor titer of 700 BU/mg IgG. An additional human-derived anti-C1 MAb KM33 competes with group A and B MAbs and has an inhibitor titer of 97 BU/mg IgG. MAbs B136 and KM33 potently inhibit fVIII binding to VWF and phospholipids. All of the anti-C1 MAbs have a strong binding affinity to fVIII with dissociation constants (KD) of 0.1 - 10 nM determined by surface plasmon resonance (SPR) spectroscopy despite relatively low titers of inhibition (Table 1). MAbs 2A9, F156, I84, M6143, and B136 also inhibit peak thrombin generated and endogenous thrombin potential in the thrombin generation assay and fVIIIa incorporation into the intrinsic Xase assay in a similar fashion as the one-stage clotting assay.Table 1MAbGroupSpleen OriginInhibitor titer (BU/mg IgG)VWF Binding IC50 (µg/ml)Phospholipid Binding IC50 (µg/ml)KD (nM)Median Blood Loss/Body Weight (mg/g)2A9A1231.10.90.929.8B153A23>10>1010-F156A37>10>102.91.7I41A4152.7>101.9-I84A4Indeterminate3.3>101.9-I88A431.7>101.0-M6143A51800.6>100.241B136B27000.40.040.143.9KM33ABN/A970.03-0.145.9The pathogenicity of anti-C1 MAbs were assessed in a tail snip bleeding model. Hemophilia A mice received injections of anti-C1 MAb at saturating levels of 0.5 mg/kg (~65 nM) followed by 180 U/kg (~2.5 nM) fVIII and tail transection 2 hours later. Mice that received MAbs 2A9 (group A), M6143 (group A), B136 (group B), and KM33 (group AB) induced bleeding with median blood loss per mouse body weight of 29.8, 41, 43.9, and 45.9 mg/g respectively compared to 1.2 mg/g in mice who received fVIII alone (p = 0.049, 0.011, 0.008, and 0.004 respectively, Mann-Whitney). MAb F156 did not induce bleeding. MAbs F156, 2A9, M6143, B136, and KM33 produced differing inhibitor titers of 0.01, 0.3, 2.3, 8.8, and 1.2 BU/ml respectively in this system. Despite low inhibitor titers of 0.3, 2.3, and 1.2 BU/ml and a high fVIII dose of 180 U/kg, MAbs 2A9, M6143, and KM33 induced bleeding in mice. Previously we have shown that group A MAbs with epitopes that overlap 2A9 were found in 42% of patients with hemophilia A and inhibitors.Conclusion: Although anti-C1 MAbs show weak inhibition in in-vitro assays and low inhibitor titers, they bind tightly to fVIII and induce bleeding in a tail snip model. These pathogenic low titer inhibitors are found in patients with hemophilia A and inhibitors. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Long-Term Follow-up of Hemophilia a Patients Who Previously Showed Complete or Partial Success in Immune Tolerance Induction Therapy with a Single Plasma-Derived FVIII/VWF Product: Long-Term ITI Study

IntroductionInhibitor development against infused FVIII is a serious complication in hemophilia A that greatly increases the risk of developing major bleeding episodes. Immune tolerance induction (ITI) is the standard therapy for the management and eradication of inhibitors and return to effective FVIII therapy. Little evidence exists on the long-term status of patients who eradicated the inhibitor totally or partially by ITI. Therefore, the aim of this study is to investigate the clinical status and sustainability of tolerance in patients who succeeded on ITI therapy.MethodsThe Long-term ITI study was an international, multicenter, observational, retrospective study performed on hemophilia A patients who received primary or rescue ITI therapy using a single pdFVIII/VWF concentrate (Fanhdi®, Grifols, Spain) and who showed partial or total success in the Grifols-ITI study (Oldenburg J et al. Haemophilia 2014;20:83, Rangarajan S et al. J Thromb Haemost 2013;11 suppl 2;1081). In the G-ITI study, 60 patients from Germany, Italy and Spain showed a success rate of 85% and nine other G-ITI patients from UK obtained a 78% of ITI success. Demographics and clinical data relevant for the Long-term ITI study were extracted from the medical records of eligible patients. Primary endpoints were to describe the long-term status and post-ITI outcomes (including comorbidities, joint status, bleeding episodes, inhibitor relapses, venous access complications, hospitalizations and surgeries). Secondary endpoints included regular hemophilia therapy as well adjuvant and concomitant therapy used. Statistical analysis performed was descriptive.ResultsForty-four out of the 57 eligible patients signed the informed consent and were included in the study. The mean follow up period after ITI success was 9.3 ±3.3 years (range: 2.1-17.7). At least one co-morbidity was reported in 17 (38.6%) patients, which included liver and lung cancer, hypertension, liver disease, hepatitis C and HIV infection. Twenty-one (47.7%) patients had a total of 37 affected -joints of which 5 patients had 2 affected joints, 1 patient had 3 affected joints and 3 patients had 4 affected joints. In 38 (86.4%) patients at least 1 bleeding episode was reported (mean annual bleeding rate: 1.0±1.2; range 0.1-3.9). A total of 330 bleeding episodes were described, of which 271 were treated with Fanhdi. Four (9.0%) patients had inhibitor relapse. Of those, 2 patients did not attempt rescue ITI because in one case the inhibitor (0.5 BU) disappeared after the next infusion with Fanhdi, and the other was considered of low titer (3.5 BU). The 2 patients who attempted rescue ITI (one under prophylaxis with Kogenate and the other treated on demand with Emoclot at the time of inhibitor relapse) were both successfully tolerized, one completely after 3.9 months of ITI with Fanhdi and the other partially after 9.1 months of ITI with Haemate P. Twenty venous access complications were reported in 10 (22.7%) patients. Thirty-nine surgical/invasive procedures were reported in 22 (50.0%) patients, 37 procedures requiring extra treatment with FVIII (Fanhdi), 25 of them for more than 3 days and 14 for more than 5 days. Hospitalizations reported were 79, 17 of them due to bleeding, 18 due to venous access complications, 33 due to surgical/invasive procedures, and 11 due to other reasons. Treatment product for hemophilia after ITI success was always Fanhdi in 29 (65.9%) patients, while 13 (29.5%) other patients were first treated with Fanhdi and then switched to other FVIII products, and 2 patients were immediately switched post-ITI to other FVIII products. The most common regimen used was prophylaxis (42 patients, 95.4%), and the most common treatment frequency was every 48 hours (16 patients, 36.4%). The FVIII dose ranged from 20 to 85 IU/Kg. Use of NSAIDs was reported for 6 patients and immunesuppressors for 5 patients.ConclusionsAfter an average of more than 9 years of post-ITI success follow up of patients participating in the G-ITI Study, all of them continue with regular FVIII treatment for hemophilia. The few inhibitor relapses were successfully overcome. DisclosuresJiménez-Yuste:Baxter, Bayer, CSL-Behring, Grifols, Novo Nordisk, Octapharma and Pfizer: Consultancy, Honoraria, Research Funding, Speakers Bureau. Oldenburg:Baxter, Bayer, Biogen Idec, Biotest, CSL-Behring, Grifols, Novo Nordisk, Octapharma, Swedish Orphan Biovitrum and Pfizer: Consultancy, Honoraria, Research Funding, Speakers Bureau. Rangarajan:Baxter, Grifols, Biotest and Pfizer: Consultancy, Honoraria, Research Funding, Speakers Bureau. Peiró:Grifols: Employment. Santagostino:Bayer, Baxter, Pfizer, CSL Behring, Novo-Nordisk, Sobi/Biogen, Octapharma, Kedrion, Roche and Grifols: Consultancy, Honoraria, Research Funding, Speakers Bureau.

Decreasing the Humoral Response to Factor VIII By Targeted Deletion of Factor VIII - Specific B Cells

The development of neutralizing anti-factor VIII (fVIII) antibodies (inhibitors) remains the most significant complication in the treatment of hemophilia A patients. Treatment of inhibitor patients consists of management of bleeding episodes using bypassing agents or porcine fVIII. Inhibitors can be eradicated by immune tolerance induction (ITI) using thrice-weekly administration of large doses of fVIII. However, ITI fails in approximately 30% of patients. Additionally, the median time to tolerance in successful cases is ~18 months, making ITI expensive and inconvenient. In the current study, we used a murine E16 hemophilia A model to test a novel approach to both prevent and eradicate fVIII inhibitors. We hypothesized that conjugation of fVIII to the toxin saporin, a Type I ribosome-inactivating protein, would target fVIII-specific cell surface immunoglobulin and selectively delete fVIII-specific naïve and memory B cells. Recombinant full-length fVIII was covalently linked to saporin using the heterobifunctional crosslinker N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP). To test for eradication of existing fVIII inhibitors by fVIII-saporin, an adoptive transfer protocol was developed to measure fVIII-specific memory B cells. Hemophilia A donor mice were immunized with 2 μg of full-length fVIII by intravenous injection every other week for 8 weeks, followed by a final dose of 4 μg at ten weeks. Four weeks later, the mice were randomized into three treatment groups to receive equimolar doses of saporin, fVIII, or fVIII-saporin. Seven days after treatment, the mice were sacrificed and 4 x 106 plasma cell CD138+-depleted splenocytes were adoptively transferred as a source of fVIII-specific memory B cells into naïve recipient hemophilia A mice. At 24 hours, recipient mice were given a single injection of 0.5, 1.0 or 2.0 μg of recombinant full-length fVIII by tail vein injection. Anti-fVIII IgG antibodies in recipient mice were measured by ELISA 2 and 5 weeks following the fVIII injection. In the absence of fVIII-specific memory B cells from donor mice, naïve hemophilia A mice did not produce detectable anti-fVIII antibodies. Recipient hemophilia A mice receiving splenocytes from fVIII donor and saporin donor mice displayed a dose-dependent increase in anti-fVIII antibodies. In contrast, the slope of the anti-fVIII titer versus dose of fVIII was significantly decreased in recipient mice receiving splenocytes from fVIII-saporin donor mice. To test for prevention of fVIII inhibitor formation by fVIII-saporin, naïve hemophilia A mice were divided into three treatment groups to receive a single dose of saporin, fVIII, or fVIII-saporin by tail vein injection. Seven days after treatment, the mice were immunized by tail vein injection with 2 μg of full-length fVIII every other week for 10 weeks. Anti-fVIII IgG antibodies were measured 1 week after the fourth and sixth injections of fVIII. Anti-fVIII antibody titers were significantly lower in the fVIII-saporin group compared to the fVIII group (1,900 vs. 21,400 (p=0.027, n=4, Mann-Whitney test, see figure) after the fourth injection. After 6 injections, the average anti-fVIII titer of the fVIII group was 23,000 compared to 4,000 in the fVIII-saporin group (p=0.057, n=4, Mann-Whitney test, see figure). In conclusion, our results suggest that infusion of fVIII-saporin results in the depletion of both fVIII-specific naïve B cells and memory B cells. FVIII-saporin potentially could be used in the treatment of congenital hemophilia A patients with inhibitors and patients with acquired hemophilia A. In addition, fVIII-saporin potentially could be used in previously untreated patients with hemophilia A to prevent inhibitor development. Similar therapeutic strategies could be extended to other antigen-specific immune disorders. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A Cross-Sectional Analysis of Cardiovascular Disease in the Hemophilia Population

A Cross-Sectional Analysis of Cardiovascular Disease in the Hemophilia Population

A Comparison of Efficacy Between Recombinant Activated Factor VII (ARYOSEVEN TM) and NOVOSEVEN® in Patients with Hereditary FVIII Deficiency with Inhibitor

Introduction: Concentrated factor VIII replacement therapy in severe hemophilia prevents disabling arthropathy, life-threatening bleeding, improves health related quality of life and increase life expectancy. Patients with neutralizing antibodies (inhibitors) did not respond to usual doses of FVIII and they need higher doses of FVIII or bypassing agents (eg. rFVIIa, FEIBA) to control bleeding. The aim of this study was to compare the efficacy of a new biosimilar recombinant factor VIIa (Aryoseven ™) with Novoseven® in controlling bleeding episodes in hemophilia A patients with inhibitors.Methods: A randomized double blind clinical trial study was conducted in eight comprehensive hemophilia care centers in Iran. We randomized 66 male patients in two groups, with 4 consecutive block randomization when bleeding occurred. All patients entered the study with only one bleed. Informed consent signed by all patients or their parents. Group A (31 patients, 47%) received Aryoseven ™ and group B (35 patients, 53%) received Novoseven®. rFVIIa dosage was 90 -120 μg/kg intravenously every 2 hours.Results: During the study 66 patients (All male) were enrolled in two arms. The mean age was 20.5±10.9 years. Comparison of baseline data between the two showed no significant difference. The distributions of these variables were similar between the two groups. Other factors, such as vital signs, coagulation tests, liver, renal function tests, and blood count were not significantly different between the two groups. Median plasma level of FVII clotting activity (FVII: C) in group A and B was 103.8±38.4 IU/ dl and 98.6±26.7 IU/dl before injection respectively. Median plasma level of FVIII inhibitor in group A and B was 15.0 BU (IQR: 8.8-58.0) and 19.0 BU (IQR: 11.0-31.0). The average time from onset of bleeding to start treatment were 1246± 1104 and 2301 ± 1693 minutes (p = 0.311) in group A and B respectively.This study results showed that increased levels of factor VII were comparable between two groups after rFVIIa injection. A comparison of the Kavakli global response scores after injection and the treatment success rate in terms of achieving to score 6 or higher showed that both groups were comparable in treatment success rates. The global treatment response rate was 96.8% in group A and 91.4% in group B. Administration of either Aryoseven™ or Novoseven® had comparable effect on controlling pain and joint mobility. Reported side effects were minor (headache, nausea, and rash) and occurred in similar frequency.Discussion: The present study showed increased FVII levels and clinical efficacy in the control of the bleeding episodes in Hemophilia A patients with inhibitor for both drugs. Some similarities exist between our findings and the previous clinical trials designed for Novoseven®. In other studies, the treatment success rate by using the global response scoring system has been 70% -86% by different rFVIIa dosage regimen, in our study was above 90% in both treatment groups. In most studies, it was reported more than 90% achieving to cessation of bleeding at 9 hours after first injection of rFVIIa as response time which was also similar to our study.Conclusion: This study showed non-inferiority of Aryoseven ™ compared to Novoseven® in cessation of bleeding episodes in hemophilia A patients with inhibitor. There was no evidence of significant difference between the efficacy and safety of two drugs. There were no clinically meaningful differences between the biosimilar product and the original product in terms of the safety, purity, and efficacy. Biosimilar rFVIIa is effective in treatment of acute joint bleeding in patients with inhibitor in comparison to Novoseven®. DisclosuresMehrvar:Aryogen Zist Darou: Employment. Khoein:Aryogen Zist Darou: Employment. Kaymar:Aryogen Zist Darou: Employment. Mahbodi:Aryogen Zist Darou: Employment. Vaziri:Aryogen Zist Darou: Employment.

A Native Whole Blood Thrombin Generation Assay Allows Discrimination of Whole Blood Samples with FVIII Levels below 1%

Monitoring the correction of abnormal bleeding tendencies during the treatment of patients with hemostatic disorders is essential to evaluate success of therapy. While single clotting factor assays provide valuable information, global coagulation assays are desirable to better understand the overall hemostatic condition of patients. In Hemophilia A, severity of the clotting defect is traditionally evaluated by determining FVIII activity using chromogenic or clotting assays. Evaluation of thrombin generation in plasma samples for the assessment of bleeding tendencies in hemophilic patients has been suggested. Discriminating between samples with FVIII levels below 1%, however, has been challenging using FVIII activity and thrombin generation assays. We previously reported a native whole blood thrombin generation assay (nWB-TGA) that uses recalcification of whole blood samples without the addition of tissue factor to initiate clotting. We have shown that this assay is sensitive to varying levels of FVIII in vitroand to platelet targeted FVIII gene therapy in a murine model of Hemophilia A. The objective of the present study was to determine if the nWB-TGA can be used to monitor Hemophilia A patients during FVIII therapy and if this assay allows discrimination of whole blood samples with FVIII levels below 1%.Using the nWB-TGA we evaluated thrombin generation in a severe hemophilia A patient carrying an intron 22 inversion. Numerous data points were obtained from 15 different FVIII infusions, each targeting a FVIII level of 50%. Samples collected at least 72 hours (hrs) post infusion (>6 half-lives, calculated FVIII levels <1%) significantly differed from healthy control samples in all thrombin generation parameters. Compared with healthy controls (6.9 ± 0.6 min; mean ± SEM) the hemophilic patient had a lag time (LT) of 24.8 ± 3.4 min. Peak time in healthy controls and the patient was 10.1 ± 1 min and 35 ± 5 min, peak thrombin was 528 ± 78 nM and 124 ± 20 nM, endogenous thrombin potential (ETP) was 1949 ± 117 nM and 1201 ± 50 nM, and thrombin generation rate was 196 ± 58 nM/min and 21 ± 6 nM/min, respectively. While previous studies on thrombin generation in plasma samples mainly reported on differences in peak thrombin and ETP, spiking of hemophilic blood with increasing concentration of recombinant FVIII in vitro revealed that the LT was FVIII dose dependent in our assay. When hemophilic blood was reconstituted with FVIII to a 2%, 5% and 50% level, the LT was 22.5 ± 1.6 min, 16.1 ± 1.7 min and 8.8 ± 0.6 min, respectively. All other thrombin generation parameters were FVIII dose dependent as well. A FVIII dependent LT was also apparent in vivo, when we monitored the patient after FVIII infusions. LT was 6.4 ± 0.2 min at 15 min, 8.5 ± 0.4 min at 24 hrs, and 13.8 ± 0.5 min at 48 hrs post FVIII treatment. We identified the lack of tissue factor as being key to a FVIII dose dependent LT. At all post infusion time points the LT was approximately 5 min when tissue factor was added to the assay. To our surprise, looking at specific time points equal to or greater than 72 hrs post infusion enabled us to discriminate these samples based on the LT (72 hrs: LT= 13.0 ± 0.6 min, 84 hrs: LT= 19.5 ± 0.8 min, 96 hrs: 36.0 ± 4.4 min). The ETP, commonly used as a variable parameter in previous thrombin generation reports, however, was not different among 72, 84 and 96 hrs post FVIII infusion samples. Strikingly, FVIII activity determined by chromogenic and one stage clotting assay was below detection limit (1% FVIII:C) in samples obtained 72 hrs post infusion or later. Thus, the patient in our study displayed considerable thrombin generation determined by the nWB-TGA at post FVIII infusion time points when FVIII levels were considered undetectable with currently available technology. Our data suggest that the different LT observed in 72, 84 and 94 hrs post infusion samples is possibly related to differences in residual FVIII levels below 1%.In conclusion, the nWB-TGA provides a useful tool to monitor efficacy of FVIII replacement therapy and might assist in tailoring individual FVIII treatment regimens. This close to physiological whole blood assay allows distinguishing blood samples with FVIII levels below 1% in vivo, and might help to explain the heterogeneity in bleeding phenotypes observed in severe hemophilia A patients. This assay may also be useful in assessing therapeutic benefit of “long acting” FVIII or FIX products. DisclosuresNo relevant conflicts of interest to declare.

The haemophilia certification system in Canada.

Inherited bleeding disorders are caused by the deficiency or dysfunction of plasma proteins required for the development of a physiological hemostatic process1,2. Von Willebrand disease (vWD), haemophilia A and haemophilia B are the most common inherited disorders of hemostasis. In their severe forms, these disorders are life-threatening. However advanced treatment modalities have enabled even severely affected patients to reach the same life expectancy of the general male population at least in high-income countries3,4. Indeed, after the dramatic events of widespread blood-borne virus transmission in the 1970s–1980s in haemophilic patients, leading to a 35-to-40-year reduction in life expectancy in Canadian patients5, there has been a strong drive towards continuous improvement, primarily in the safety of replacement therapy6. There is now a very high degree of safety from risk of blood borne transmission of pathogens as evidenced by the absence of reported transmission of blood-borne viruses in persons with haemophilia since the late 1980s to date7. The safety of current replacement therapies was recently confirmed by a prospective surveillance program ongoing since 2008 and based on regular monitoring of 22,242 European patients (European Haemophilia Safety Surveillance System; www.euhass.org). Regular factor prophylaxis as a therapeutic regimen has been proven effective in preventing haemophilic arthropathy8. Even in the presence of neutralizing antibodies (inhibitors) directed against the deficient clotting factor, particularly in haemophilia A, bleeding can usually be treated successfully using activated variants of coagulation factors9. Moreover, immune tolerance induction (ITI), based on the long-term intravenous infusion of large doses of FVIII, eradicates inhibitors in as many as two-thirds of patients10. Therefore, improving quality of life has become the primary objective of care in developed countries; thus multiple modalities (psychosocial support, physiotherapy, integration into community life, etc), not just the infusion of the deficient factor, are made available to the patient and his family, to allow them to fully experience good health.

Anti-Factor VIII A2 Epitopes In a Murine Bleeding Model

Up to 30% of patients with severe hemophilia A will develop inhibitory antibodies to factor VIII (fVIII inhibitors). In addition, autoimmune antibodies to fVIII can develop in non-hemophiliacs, producing acquired hemophilia A, which frequently produces life- or limb-threatening bleeding. Patients with congenital hemophilia who develop inhibitors usually have a polyclonal antibody response directed against the A2 and C2 domains of fVIII. Patients with acquired hemophilia typically have a more limited B-cell epitope response with antibodies directed against the A2 or C2 domain but not both. We have shown that within the C2 domain of fVIII antibody epitope is more important than inhibitory titer in predicting pathogenicity in a murine in vivo bleeding model. In this project we investigated the pathogenicity of a diverse panel of anti-A2 monoclonal antibodies (Mabs) in the murine in vivo bleeding model. We have previously characterized anti-A2 antibodies into groups A, AB, B, BCD, C, D, DE, and E based on the pattern of overlap on the B-cell epitopes in a competition ELISA. Table 1 shows the characteristics of the anti-A2 Mabs. Mabs were injected retro-orbitally into Exon 16 hemophilic (E16) mice at a dose of 0.5 umg per g body weight (∼ 65nM plasma concentration). Fifteen minutes later, mice were injected with B-domain deleted human fVIII at a dose of 180U/kg (∼ 2.5nM plasma concentration). In addition, a subset of Mabs has also been tested at a high dose of 360U/kg (∼5 nM). Two hours after fVIII injection, the mice were anesthetized and a 4mm tail snip was performed. Blood was collected in a tube of normal saline over 40 minutes and measured. 4A4, 2-76, and 1D4 are all high inhibitory titer, type I Mabs that produced significant bleeding with 180 U/kg fVIII when compared to control. In addition, 2-76 and 4A4 were tested at the higher dose of fVIII and significant bleeding was again seen. In comparison, the high titer type II Mab 2-54 had a similar inhibitory titer but no significant bleeding at either dose of fVIII. B94 is a type II inhibitor with a similar inhibitory profile to 2-54, but maximum inhibition is 45% as compared to 82% for 2-54. B94 also was not pathogenic at either fVIII dose tested. Both 4C7—a non-inhibitory Mab—and B25—a very low titer Mab that would be predicted to have residual fVIII activity at the Mab concentration tested—did not produce significant bleeding. The inhibitory titer alone did not predict bleeding phenotype within a diverse panel of anti-A2 Mabs. This discrepancy combined with similar findings in the C2 domain stress the importance of inhibitor properties not detected in the standard Bethesda assay in predicting response to fVIII therapy. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Epitope Specific Discrepancies Between Antibody Mediated Inhibition Of Recombinant Factor VIII and Plasma Derived Factor VIII Containing Von Willebrand Factor Affecting All Domains Of Factor VIII

BackgroundPrevious results from our laboratory demonstrate an epitope specific response to high doses of FVIII within anti-C2 antibodies that was independent of antibody titer. In addition, for a panel of monoclonal antibodies (MAbs) directed against all FVIII domains the kinetics of inhibition influenced response to combinations of FVIII and recombinant factor VII (rFVIIa). The influence of inhibitor kinetics on response to treatment has also been demonstrated in inhibitor patient plasmas. The Bethesda assay detects the inhibitory capacity of anti-factor VIII (FVIII) antibodies to neutralize FVIII after 2 hours of incubation with pooled normal plasma (PNP). In this assay patient antibody is added to PNP as compared to the clinical scenario where recombinant FVIII (rFVIII) or plasma derived FVIII containing von Willebrand Factor (pdFVIII/VWF) is infused into the patient where antibody is already present. In this case the infused product is immediately available to interact with both VWF and anti-FVIII antibodies as compared to the Bethesda assay where VWF is already bound to FVIII when the antibody is added. MethodsIn this study we investigated the inhibitory kinetics of a panel of 20 anti-FVIII MAbs (Table) with known epitope specificity and inhibitory activity in a standard Bethesda assay. Inhibitor plasma consisted of a single MAb added to FVIII deficient plasma at 5 µg/ml. rFVIII and pd-FVIII/VWF were added to each inhibitor plasma and samples were sequentially removed at intervals between 5 and 90 minutes. FVIII activity was measured by a one-stage aPTT based assay. ResultsOf the MAb panel, 2 anti-A2 MAbs, 1D4 and 4A4, and the anti-A3 MAb F147 had full neutralization of both rFVIII and pd-FVIII/VWF. All 3 of these MAbs exhibit high inhibitory titers in the Bethesda assay. The majority of the other MAbs had improved neutralization kinetics and thus higher residual FVIII activity with pd-FVIII/VWF when compared to rFVIII. The figure below shows the residual FVIII activity at 15 minutes following the addition of rFVIII or pdFVIII/VWF into the inhibitor plasmas. Similar patterns were seen at the other time points. [Display omitted] Three MAbs from the panel, two anti-A2 (4C7 and B157) and one anti-C2 (2-117), had significant inhibition of FVIII activity when rFVIII was added to the inhibitor plasma. This was not demonstrated in the standard Bethesda assay or when pd-FVIII/VWF was added to the inhibitor plasma. This demonstrates that the order of binding of VWF and anti-FVIII antibodies may be clinically relevant for a subset of FVIII epitopes. ConclusionThe Bethesda assay in isolation neither predicts inhibitory kinetics nor defines response to various FVIII sources. FVIII source dependent neutralization kinetics and epitope mapping may be applied as additional tools for tailoring therapy in patients with inhibitors. Disclosures:No relevant conflicts of interest to declare.

High rate of spontaneous inhibitor clearance during the long term observation study of a single cohort of 524 haemophilia A patients not undergoing immunotolerance.

BackgroundThe natural history of inhibitors in patients with haemophilia A not undergoing immune tolerance induction (ITI) is largely unknown. A recent randomized controlled trial suggests that the higher the FVIII dose used for ITI, the faster the clearance and the lower the rate of bleeding, without any difference in the rate of tolerance. We aimed at assessing the rate of spontaneous inhibitor clearance in a large cohort of patients not undergoing ITI.MethodsA retrospective analysis of anti-FVIII inhibitors of long-term registry data in a single centre cohort of 524 haemophilia A patients considered for synovectomy was performed. Patients were tested for inhibitors before and 15 days after any and each surgical episode and thereafter did not undergo immune tolerance at any time.ResultsThe cumulative incidence of inhibitors overall was 34% (180 out of 524) with the highest percentage of 39% (168 out of 434) in severe patients which represented 83% of the cohort. Among the 180 inhibitor patients: 63 had permanent inhibitors; 70 fulfilled current criteria for transient inhibitors but a third category of 47 additional patients cleared the alloantibody spontaneously in >6 months. At logistic regression, both the inhibitor titre and the gene mutation were shown to predict time to clearance.ConclusionsSpontaneous clearance of inhibitors over variable time in the absence of ITI treatment was found in up to 2/3 of the cases.

Risk factors for high-titer inhibitor development in children with hemophilia A: results of a cohort study.

Among the discussed risk factors for high-titre inhibitor (HRI) development in patients with hemophilia A (HA) are high dose FVIII replacement therapy and use of recombinant FVIII concentrates (rFVIII). The aim of this study was to evaluate the aforementioned risk factors for HRI development in children with hemophilia A ≤2%. About 288 ascertained PUPs (Israel and Germany) were followed after initial HA diagnosis over 200 exposure days. Inhibitor-free survival, hazard ratios (HR), and 95% confidence intervals (CIs) were calculated. Adjustment was performed for factor VIII concentrates, median single dose over the first three months of treatment, first FVIII administration before the age of three months, presence of risk HA gene mutations, “intensive treatment moments” and “year of birth” (proxy for different treatment periods). HRI occurred in 71/288 children (24.7%). In multivariate analysis adjusted for “year of birth”, underlying risk gene mutations (HR/CI: 2.37/1.40–3.99), FVIII dose, measured per one IU increase per kgbw (HR/CI: 1.05/1.04–1.07), and first FVIII administration before the age of three months showed a significant impact on HR development. The risk of HRI development was similar for recombinant or plasmatic FVIII products. Children at risk should be treated with carefully calculated lower dose regimens, adapted to individual bleeding situations.