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Concept: Factor VIII

426

Background Patients with hemophilia A rely on exogenous factor VIII to prevent bleeding in joints, soft tissue, and the central nervous system. Although successful gene transfer has been reported in patients with hemophilia B, the large size of the factor VIII coding region has precluded improved outcomes with gene therapy in patients with hemophilia A. Methods We infused a single intravenous dose of a codon-optimized adeno-associated virus serotype 5 (AAV5) vector encoding a B-domain-deleted human factor VIII (AAV5-hFVIII-SQ) in nine men with severe hemophilia A. Participants were enrolled sequentially into one of three dose cohorts (low dose [one participant], intermediate dose [one participant], and high dose [seven participants]) and were followed through 52 weeks. Results Factor VIII activity levels remained at 3 IU or less per deciliter in the recipients of the low or intermediate dose. In the high-dose cohort, the factor VIII activity level was more than 5 IU per deciliter between weeks 2 and 9 after gene transfer in all seven participants, and the level in six participants increased to a normal value (>50 IU per deciliter) that was maintained at 1 year after receipt of the dose. In the high-dose cohort, the median annualized bleeding rate among participants who had previously received prophylactic therapy decreased from 16 events before the study to 1 event after gene transfer, and factor VIII use for participant-reported bleeding ceased in all the participants in this cohort by week 22. The primary adverse event was an elevation in the serum alanine aminotransferase level to 1.5 times the upper limit of the normal range or less. Progression of preexisting chronic arthropathy in one participant was the only serious adverse event. No neutralizing antibodies to factor VIII were detected. Conclusions The infusion of AAV5-hFVIII-SQ was associated with the sustained normalization of factor VIII activity level over a period of 1 year in six of seven participants who received a high dose, with stabilization of hemostasis and a profound reduction in factor VIII use in all seven participants. In this small study, no safety events were noted, but no safety conclusions can be drawn. (Funded by BioMarin Pharmaceutical; ClinicalTrials.gov number, NCT02576795 ; EudraCT number, 2014-003880-38 .).

Concepts: Factor VIII, Virus, Gene, Central nervous system, Nervous system

169

Development of inhibitory antibodies to coagulation factor VIII (fVIII) is the primary obstacle to the treatment of hemophilia A in the developed world. This adverse reaction occurs in 20-30% of persons with severe hemophilia A treated with fVIII-replacement products and is characterized by the development of a humoral and neutralizing immune response to fVIII. Patients with inhibitory anti-fVIII antibodies are treated with bypassing agents including recombinant factor VIIa (rfVIIa). However, some patients display poor hemostatic response to bypass therapy and improved treatment options are needed. Recently, we demonstrated that fVIII inhibitors display widely variable kinetics of inhibition that correlate with their respective target epitopes. Thus, it was hypothesized that for antibodies that display slow rates of inhibition, supplementation of rfVIIa with fVIII would result in improved thrombin generation and be predictive of clinical responses to this novel treatment regimen. In order to test this hypothesis, 10 murine monoclonal antibodies (MAbs) with non-overlapping epitopes spanning fVIII, differential inhibition titers, and inhibition kinetics were studied using a thrombin generation assay. Of the 3 MAbs with high inhibitory titers, only the one with fast and complete (classically defined as “type I”) kinetics displayed significant inhibition of thrombin generation with no improvement upon supplementation of rfVIIa with fVIII. The other two MAbs that displayed incomplete (classically defined as “type II”) inhibition did not suppress the potentiation of thrombin generation by fVIII. All antibodies that did not completely inhibit fVIII activity demonstrated potentiation of thrombin generation by the addition of fVIII as compared to rfVIIa alone. In conclusion, fVIII alone or in combination with rfVIIa corrects the thrombin generation defect produced by the majority of anti-fVIII MAbs better than single agent rfVIIa. Therefore, combined fVIII/rfVIIa therapy may provide better hemostatic control than current therapy in some patients with anti-fVIII inhibitors.

Concepts: Factor VIII, Enzyme inhibitor, Coagulation system, Thrombin, Protein C, Coagulation, Factor X, Immune system

168

Hemophilia A is a common X chromosome-linked genetic bleeding disorder caused by abnormalities in the coagulation factor VIII gene (F8). Hemophilia A patients suffer from a bleeding diathesis, such as life-threatening bleeding in the brain and harmful bleeding in joints and muscles. Because it could potentially be cured by gene therapy, subhuman animal models have been sought. Current mouse hemophilia A models generated by gene targeting of the F8 have difficulties to extrapolate human disease due to differences in the coagulation and immune systems between mice and humans. Here, we generated a porcine model of hemophilia A by nuclear transfer cloning from F8-targeted fibroblasts. The hemophilia A pigs showed a severe bleeding tendency upon birth, similar to human severe hemophiliacs, but in contrast to hemophilia A mice which rarely bleed under standard breed conditions. Infusion of human factor VIII was effective in stopping bleeding and reducing the bleeding frequency of a hemophilia A piglet but was blocked by the inhibitor against human factor VIII. These data suggest that the hemophilia A pig is a severe hemophilia A animal model for studying not only hemophilia A gene therapy but also the next generation recombinant coagulation factors, such as recombinant factor VIII variants with a slower clearance rate.

Concepts: Hemostasis, Haemophilia B, Thrombin, Haemophilia A, Factor VIII, Factor X, Protein C, Blood

168

Complex formation between coagulation factor VIII (FVIII) and von Willebrand factor (VWF) is of critical importance to protect FVIII from rapid in vivo clearance and degradation. We have now employed a chemical footprinting approach to identify regions on VWF involved in FVIII binding. To this end, lysine amino acid residues of VWF were chemically modified in the presence of FVIII or activated FVIII(a), which does not bind VWF. Nano-LC-mass spectrometry analysis showed that the lysine residues of almost all identified VWF peptides were not differentially modified upon incubation of VWF with FVIII or FVIIIa. However, Lys773 of peptide Ser766-Leu774 was protected from chemical modification in the presence of FVIII. In addition, peptide Ser764-Arg782, which comprises the first 18 amino acid residues of mature VWF, showed a differential modification of both Lys773 and the alpha amino group of Ser764. To verify the role of Lys773 and the N-terminal Ser764 for FVIII binding, we employed VWF variants in which either Lys773 or Ser764 was replaced by an alanine. Surface plasmon resonance analysis and competition studies revealed that VWF-K773A exhibits reduced binding to FVIII and FVIII light chain, which harbors the VWF binding site. In contrast, VWF-S764A revealed more effective binding to FVIII and FVIII light chain as compared to WT-VWF. The results of our study show that the N-terminus of VWF is critical for the interaction with FVIII, and that the residues Ser764 and Lys773 have an opposite role in the binding mechanism.

Concepts: Platelet, Lysine, Surface plasmon resonance, Factor VIII, Von Willebrand disease, Protein, Von Willebrand factor, Amino acid

165

This phase 3 pivotal study evaluated the safety, efficacy, and pharmacokinetics of a recombinant FVIII Fc fusion protein (rFVIIIFc) for prophylaxis, treatment of acute bleeding, and perioperative hemostatic control in 165 previously treated males aged ≥12 years with severe hemophilia A. The study had three treatment arms: arm 1, individualized prophylaxis (25-65 IU/kg every 3-5 days, n = 118); arm 2, weekly prophylaxis (65 IU/kg, n = 24); and arm 3, episodic treatment (10-50 IU/kg, n = 23). A subgroup compared rFVIII and rFVIIIFc pharmacokinetics. Endpoints included annualized bleeding rate (ABR), inhibitor development, and adverse events. The terminal half-life of rFVIIIFc (19.0 h) was extended 1.5-fold versus rFVIII (12.4 h; P<.001). Median ABRs observed in arms 1, 2, and 3 were 1.6, 3.6, and 33.6, respectively. In arm 1, the median weekly dose was 77.9 IU/kg; approximately 30% of subjects achieved a 5-day dosing interval (last 3 months on study). Across arms, 87.3% of bleeding episodes resolved with one injection. Adverse events were consistent with those expected in this population; no subjects developed inhibitors. rFVIIIFc was well-tolerated, had a prolonged half-life compared with rFVIII, and resulted in low ABRs when dosed prophylactically 1-2 times per week. This study is registered at www.clinicaltrials.gov, ID: NCT01181128.

Concepts: Pharmacology, The Terminal, Inhibitor, Dose, Factor VIII

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ACE910 is a recombinant humanized bispecific antibody that binds to activated factor IX and factor X and mimics the cofactor function of factor VIII (FVIII). This first-in-human study examined the safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of ACE910 in healthy male adults. A total of 40 Japanese and 24 Caucasian subjects were randomized to receive a single subcutaneous injection of ACE910 (Japanese: 0.001, 0.01, 0.1, 0.3, or 1 mg/kg; Caucasian: 0.1, 0.3, or 1 mg/kg; n = 6 per dose group) or placebo (n = 2 per dose group). ACE910 exhibited a linear PK profile and had a half-life of approximately 4 to 5 weeks. In FVIII-neutralized plasma, ACE910 shortened activated partial thromboplastin time and increased peak height of thrombin generation in a dose-dependent manner. All adverse events were non-serious and did not lead to any subject’s withdrawal. Neither clinical findings nor laboratory abnormalities indicating hypercoagulability were observed. Two of 48 subjects receiving ACE910 (1 Japanese and 1 Caucasian) were positive for anti-ACE910 antibodies (anti-drug antibodies; ADA). One subject tested positive for ADA both before and after ACE910 administration, whereas the other became ADA-positive after receiving ACE910. The PK and PD profiles of ACE910 were similar in healthy Japanese and Caucasian subjects, and suggest that ACE910 will be an effective and convenient prophylactic treatment for hemophilia A. This trial was registered at http://www.clinicaltrials.jp (JapicCTI-121934).

Concepts: Coagulation system, Clinical trial, Thrombin, Haemophilia B, Pharmacology, Factor VIII, Factor X, Coagulation

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Background The development of neutralizing anti-factor VIII alloantibodies (inhibitors) in patients with severe hemophilia A may depend on the concentrate used for replacement therapy. Methods We conducted a randomized trial to assess the incidence of factor VIII inhibitors among patients treated with plasma-derived factor VIII containing von Willebrand factor or recombinant factor VIII. Patients who met the eligibility criteria (male sex, age <6 years, severe hemophilia A, and no previous treatment with any factor VIII concentrate or only minimal treatment with blood components) were included from 42 sites. Results Of 303 patients screened, 264 underwent randomization and 251 were analyzed. Inhibitors developed in 76 patients, 50 of whom had high-titer inhibitors (≥5 Bethesda units). Inhibitors developed in 29 of the 125 patients treated with plasma-derived factor VIII (20 patients had high-titer inhibitors) and in 47 of the 126 patients treated with recombinant factor VIII (30 patients had high-titer inhibitors). The cumulative incidence of all inhibitors was 26.8% (95% confidence interval [CI], 18.4 to 35.2) with plasma-derived factor VIII and 44.5% (95% CI, 34.7 to 54.3) with recombinant factor VIII; the cumulative incidence of high-titer inhibitors was 18.6% (95% CI, 11.2 to 26.0) and 28.4% (95% CI, 19.6 to 37.2), respectively. In Cox regression models for the primary end point of all inhibitors, recombinant factor VIII was associated with an 87% higher incidence than plasma-derived factor VIII (hazard ratio, 1.87; 95% CI, 1.17 to 2.96). This association did not change in multivariable analysis. For high-titer inhibitors, the hazard ratio was 1.69 (95% CI, 0.96 to 2.98). When the analysis was restricted to recombinant factor VIII products other than second-generation full-length recombinant factor VIII, effect estimates remained similar for all inhibitors (hazard ratio, 1.98; 95% CI, 0.99 to 3.97) and high-titer inhibitors (hazard ratio, 2.59; 95% CI, 1.11 to 6.00). Conclusions Patients treated with plasma-derived factor VIII containing von Willebrand factor had a lower incidence of inhibitors than those treated with recombinant factor VIII. (Funded by the Angelo Bianchi Bonomi Foundation and others; ClinicalTrials.gov number, NCT01064284 ; EudraCT number, 2009-011186-88 .).

Concepts: Regression analysis, Multivariate statistics, Epidemiology, Von Willebrand disease, Von Willebrand factor, Factor VIII

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The determination of factor VIII (FVIII) potency in FVIII concentrates can be performed using both manual and automated methods. This work aimed to validate the use of the chromogenic kit Coamatic(®) FVIII (Chromogenix) on the automated ACL(®) Elite PRO analyzer for evaluating the potency of FVIII in commercial preparations in pharmaceutical analytical laboratories. After setting the activation and reading times to 2 min and 3 min, respectively, the validation parameters, according to the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use guideline Q2 (R1), were as follows: linearity, expressed by the adjusted model: log (Absorbance) = 1.848 + 0.777∙log (Concentration), with r(2)  = 0.998; accuracy was verified (P-value = 0.6959); and the coefficient of variation for repeatability and intermediate precision was ≤6.5%. The Coamatic(®) FVIII kit method has been adapted to the ACL Elite PRO analyzer with improved performance compared with a manual microplate method.

Concepts: Performance, Factor VIII, Pharmaceutical drug, Psychometrics, Verification, Pharmacology, Verification and validation, Validation

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Host cell lines used for recombinant protein expression differ in their ability to perform post-translational modifications (PTMs). The currently available recombinant human FVIII (rhFVIII) products are produced in mammalian, non-human cell lines. For rhFVIII, glycosylation and sulfation are vital for functionality and von Willebrand factor (VWF)-binding affinity. Here we present the characterisation of the PTMs of a novel, human cell line-derived recombinant human FVIII (human-cl rhFVIII). rhFVIII expression in a human cell line avoids expression of undesirable mammalian glycoforms like Galα1-3Galβ1-GlcNAc-R (α-Gal) and N-glycolylneuraminic acid (Neu5Gc), which constitute epitopes antigenic to humans.

Concepts: Biotechnology, Posttranslational modification, Factor VIII, Human, Cell biology, DNA, Von Willebrand factor, Cell

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Antihemophilic factor (recombinant), plasma/albumin-free method (rAHF-PFM) was evaluated during routine practice in Germany, among consenting subjects of any age with hemophilia A (HA) and no prior exposure to rAHF-PFM. The treating physician chose the dosing and inhibitor testing frequency. Data were captured for 12 months/subject from diaries and clinic records. Of 152 subjects, 69 % had severe HA, and 89 % had >150 exposure 6 days (ED) at baseline. The majority of subjects (63 %) were treated by continuous prophylaxis (CP). Assignment to CP was more likely for subjects ≥2 years of age and for those with FVIII ≤ 2 %. Median FVIII consumption was 3,548 IU/kg/year for CP and 999 IU/kg/year for continuous on-demand (OD) therapy. Median annual bleed rate was 0.82 for CP and 4.06 for OD. Of 1,218 bleeds, 97 % were home-treated and 68 % of evaluable bleeds involved joints. Based on evaluable subjects' worst ratings, 83/91 (91 %) on CP had a rating of excellent/good for all prophylactic assessments, 55/59 (93 %) on CP and 41/42 (98 %) on OD had a rating of excellent/good for all bleeding assessments. The de novo high-titer FVIII inhibitor rate in subjects with >50 ED at baseline was 1/144 (0.69 %; 95 % CI, 0.02 % to 3.81 %). No high-titer inhibitor occurred in patients with severe HA and >50 ED at baseline. Reduced HRQOL physical scores were predicted by older age (p < 0.0001), HIV positivity (p = 0.02), and presence of ≥1 target joint (p = 0.003). ADVATE rAHF-PFM is safe and efficacious for routine CP or OD management of patients with HA.

Concepts: Factor VIII, HIV, Bleeding