In vivo characterization of recombinant factor VIII in a canine model of hemophilia A (factor VIII deficiency)

Infusion studies of recombinant factor VIII were performed in hemophilic (factor VIII-deficient) animals. Functional activity was determined using a standardized model of bleeding and kinetic characteristics charted by performing assays of factor VIII functional and antigenic activities over time. To obtain an adequate comparison with plasma-derived factor VIII, a crossover study in two animals was performed in which each animal received either recombinant factor VIII or a highly purified plasma-derived factor VIII on day 1 and the alternative three days later (day 4). Both factor VIII preparations were functionally effective with complete correction of the cuticle bleeding time occurring one hour after infusion. The observed recovery was full and close to predicted for both preparations. The survival curves obtained for both functional and antigenic activities for both preparations were virtually identical and within the anticipated range determined from previous experiments using infusions of conventional factor VIII preparations. The plasma half-disappearance time (T 1/2) for recombinant factor VIII and plasma-derived factor VIII was 9.2 and 7.9 hours, respectively. Plasma samples obtained following infusion were subjected to chromatography on Sepharose 4B. The elution profile of factor VIII antigen activity was compared with that obtained with the infusates. A clear shift in profile was observed with the plasma samples, suggesting complexing of the infused factor VIII material with circulating canine von Willebrand factor (vWF). The elution profile of vWF antigen was superimposable, thus providing further evidence in support of this assumption. The study provided evidence that recombinant factor VIII possesses full functional activity in vivo, binds to circulating vWF, and exhibits normal recovery and survival characteristics.     

Assaying the circulating factor VIII activity in hemophilia A patients treated with recombinant factor VIII products

Large discrepancies between factor VIII assay methods have been reported from pharmacokinetic studies of recombinant factor VIII concentrates. In the assay of postinfusion patient plasma samples, traditional activated partial thromboplastin time (aPTT)-based one-stage clotting methods usually give results that are 20 to 50% lower than those obtained by chromogenic substrate assays. Investigations into the cause of these discrepancies have shown that the choice of phospholipid in the one-stage assay is crucial. The use of platelets or liposomes resembling platelet factor 3 instead of traditional aPTT reagents results in an increase in the apparent one-stage activity and a fairly good correlation with the chromogenic results. These and other functional test results, antigen measurements as well as clinical data, support the view that the chromogenic assay most accurately reflects the therapeutic effect. In addition to the differences among assay methods, there is also a discrepancy between the World Health Organization (WHO) standards for concentrates and plasma. The use of product-specific standards, prepared by diluting the factor VIII concentrate into hemophilic plasma, when assaying postinfusion plasma samples seems to be a feasible approach to overcome the problems encountered in pharmacokinetic studies.     

Anti-inhibitor Coagulant Complex (Autoplex) for treatment of factor VIII inhibitors in hemophilia

Fourteen individuals with severe hemophilia complicated by factor VIII inhibitors (1 to 132 Bethesda Units) were treated for 33 bleeding episodes with a new activated prothrombin complex concentrate, Anti- Inhibitor Coagulant Complex (Autoplex, Hyland, Glendale, Calif.). Excellent or good results were observed in 21 of 25 minor bleeding episodes treated, which included joint, soft tissue, and mucous membrane hemorrhages. Eight major bleeding problems (an epidural bleed, a puncture wound, 2 serious soft tissue hemorrhages, 2 lacerations, and 2 major surgical procedures) were treated with excellent (6) or good (2) results. No serious complications were encountered, but two children developed transient hypofibrinogenemia following Autoplex infusion. Although some shortening of the prothrombin time and activated partial thromboplastin time was noted after infusion of Autoplex, there is no useful laboratory test for monitoring therapy. Despite the unknown mechanism of action for bypassing factor VIII, Autoplex appears to be a useful and needed interim product and is safe and effective. In view of the possible potentiation of thrombosis concurrent use of fibrinolytic inhibitors should be avoided.     

Prevention and treatment of factor VIII inhibitors in murine hemophilia A

Inhibitory antibody formation is a major complication of factor VIII replacement therapy in patients with hemophilia A. To better understand the pathogenesis of this immunologic reaction, we evaluated the role of T-cell costimulatory signals for antifactor VIII antibody formation in a murine model of hemophilia A. Repeated intravenous injections of factor VIII in these factor VIII-deficient mice induced an antifactor VIII inhibitor antibody response. This response was shown to be T-cell dependent by its absence in hemophilic mice also deficient for the T-cell costimulatory ligand B7-2. In separate experiments, injection of murine CTLA4-Ig completely blocked the primary response to factor VIII in hemophilic mice with intact B7 function. This reagent also prevented or diminished further increases in antifactor VIII when given to hemophilic mice with low antifactor VIII antibody titers. These studies suggest that strategies targeting the B7-CD28 pathway are potential therapies to prevent and treat inhibitory antifactor VIII antibodies. Moreover, because the development of antibodies to replaced proteins may limit the success of many human gene therapy approaches, our results may be broadly applicable. (Blood. 2000;95:1324-1329)     

Development of factor VIII:C antibodies in dogs with hemophilia A (factor VIII:C deficiency)

Classic hemophilia A (factor VIII:C deficiency) was diagnosed in a miniature Schnauzer dog and a breeding program established. Inbreeding and crossbreeding produced 16 hemophilic animals. All were initially treated with canine cryoprecipitate, as required, for sporadic hemorrhagic events. Five animals developed potent antibodies to canine factor VIII:C. All were the offspring of obligate carriers, resulting from the mating of a hemophilic purebred miniature Schnauzer male to a normal female Brittany spaniel. The mean age at first treatment and factor VIII exposure at the time of inhibitor development was 10.3 wk and 286.3 U, respectively. The remaining hemophilic animals have not developed antibodies, despite receiving a mean factor VIII dosage of 1.5 X 10(3) U. This group includes animals derived from a mating between the same purebred miniature Schnauzer hemophilic male and a purebred miniature Schnauzer carrier female. In each case, the antibodies recognize both canine and human but not porcine VIII:C. They are non-precipitating IgG immunoglobulins. Following inhibitor development, infusion of canine cryoprecipitate was hemostatically ineffective and factor VIII:C recovery at 30 min was negligible. Infusion of a concentrate of porcine factor VIII resulted in a correction of the hemostatic defect and optimal factor VIII:C recovery. All animals receiving porcine factor VIII:C subsequently developed antibodies to this protein. The chance occurrence of this complication should facilitate further studies directed at elucidating the pathogenesis and management of hemophilia complicated by the development of antibodies to factor VIII:C.     

Antibodies to factor VIII. V. Patterns of immune response to factor VIII in hemophilia A.

The natural history of factor VIII antibodies was studied in 20 severe, multitransfused hemophiliacs. Two patterns of humoral immune reactivity were observed. In one group of ten, who developed antibodies after an average of 22 cumulative exposure days to factor VIII, the antibody titers increased after each antigenic stimulation or persisted for years in the absence of transfusion. These patients were designated as high-responding hemophiliacs. In the second group of ten patients, the factor VIII neutralizing activity appeared after a longer exposure period (48 days). Antibody titers remained low, and there was no significant difference in individual titers before and 8--20 days following transfusion. Antibody affinity did not increase after renewed antigenic challenge. This pattern characterized low-responding hemophiliacs. The latter group of patients benefited from repeated placement therapy required by the clinical situation.     

Antibodies to factor VIII in hemophilia A patients

Inhibitor development represents the main complication in the treatment of haemophilia A. The risk of inhibitor formation is in part genetically determined by the type of the underlying factor VIII gene lesion but environmental factors may also play an important role. Due to the lack of efficiency of factor VIII in these patients other therapeutic agents must be used for the treatment of bleedings, however, none is as effective as factor VIII in a non-inhibitor patient. Therefore, the eridication of the inhibitor through immune tolerance therapy is the treatment of choice. Centralized national and international immune tolerance registries have collected the results and show cost effectiveness of this treatment.     

Safety and prolonged activity of recombinant factor VIII Fc fusion protein in hemophilia A patients

Current factor VIII (FVIII) products display a half-life (t(1/2)) of ～ 8-12 hours, requiring frequent intravenous injections for prophylaxis and treatment of patients with hemophilia A. rFVIIIFc is a recombinant fusion protein composed of a single molecule of FVIII covalently linked to the Fc domain of human IgG(1) to extend circulating rFVIII t(1/2). This first-in-human study in previously treated subjects with severe hemophilia A investigated safety and pharmacokinetics of rFVIIIFc. Sixteen subjects received a single dose of rFVIII at 25 or 65 IU/kg followed by an equal dose of rFVIIIFc. Most adverse events were unrelated to study drug. None of the study subjects developed anti-rFVIIIFc antibodies or inhibitors. Across dose levels, compared with rFVIII, rFVIIIFc showed 1.54- to 1.70-fold longer elimination t(1/2), 1.49- to 1.56-fold lower clearance, and 1.48- to 1.56-fold higher total systemic exposure. rFVIII and rFVIIIFc had comparable dose-dependent peak plasma concentrations and recoveries. Time to 1% FVIII activity above baseline was ～ 1.53- to 1.68-fold longer than rFVIII across dose levels. Each subject showed prolonged exposure to rFVIIIFc relative to rFVIII. Thus, rFVIIIFc may offer a viable therapeutic approach to achieve prolonged hemostatic protection and less frequent dosing in patients with hemophilia A. This trial was registered at www.clinicaltrials.gov as NCT01027377.     

Immune tolerance induction with recombinant factor VIII in hemophilia A patients with high responding inhibitors

Immune tolerance induction (ITI) eradicates inhibitors in patients with hemophilia A. This study was designed to investigate the success rate of ITI in high-responding inhibitor patients with severe hemophilia A using recombinant factor VIII (rFVIII). Twenty-six patients received different ITI regimens until a normal recovery (>66%) and half-life (>6 h) of infused FVIII was achieved. In order to maximize the chance of success, the initiation of ITI was deferred in the majority of patients until the inhibitor declined to <10 BU. Twenty-two patients (85%) had baseline inhibitor levels <10 BU (median 2.3 BU) when ITI began. Within a median of 6 months, immune tolerance was achieved in 19 of 26 patients (73%) including 12/17 (70%) with intron 22 inversion, 5/7 (71%) with other null mutations and two with small deletion/insertions in the F8 gene. In conclusion, recombinant FVIII induces a high rate of immune tolerance even in carriers of null F8 mutations.     

Mutations of factor VIII cleavage sites in hemophilia A

Hemophilia A is caused by a defect in coagulation factor VIII, a protein that undergoes extensive proteolysis during its activation and inactivation. To determine whether some cases of hemophilia are caused by mutations in important cleavage sites, we screened patient DNA samples for mutations in these sites by a two-step process. Regions of interest were amplified from genomic DNA by repeated rounds of primer- directed DNA synthesis. The amplified DNAs were then screened for mutations by discriminant hybridization using oligonucleotide probes. Two cleavage site mutations were found in a survey of 215 patients. A nonsense mutation in the activated protein C cleavage site at amino acid 336 was discovered in a patient with severe hemophilia. In another severely affected patient, a mis-sense mutation results in a substitution of cysteine for arginine in the thrombin activation site at amino acid 1689. This defect is associated with no detectable factor VIII activity, but with normal levels of factor VIII antigen. The severe hemophilia in this patient was sporadic; analysis of the mother suggested that the mutation originated in her gametes or during her embryogenesis. The results demonstrate that this approach can be used to identify factor VIII gene mutations in regions of the molecule known to be important for function.     

Clinical evaluation of moroctocog alfa (AF-CC), a new generation of B-domain deleted recombinant factor VIII (BDDrFVIII) for treatment of haemophilia A: demonstration of safety, efficacy, and pharmacokinetic equivalence to full-length recombinant factor VIII

Summary.  BDDrFVIII is a B-domain deleted recombinant factor VIII (rFVIII) product for haemophilia A. Manufacture uniquely includes purification chromatography by synthetic-affinity ligand rather than murine-based monoclonal antibody, as well as an albumin-free cell culture process. BDDrFVIII was studied in 204 patients, including 62 subjects <16 years old, in two studies. A double-blind, randomized, pharmacokinetic (PK) crossover study, utilizing a central laboratory assay (one-stage (OS)) for both drug potency assignment and plasma FVIII-activity measurements, demonstrated that BDDrFVIII was PK-equivalent to a full-length rFVIII. Favourable efficacy and safety were observed: during defined routine prophylaxis in a patient population significant for preexisting target joints, nearly half (45.7%) of patients had no bleeding, and a low-annualized bleed rate (ABR) was achieved (median 1.9); 92.5% of haemorrhages ( n  = 187) required ≤2 infusions. Three subjects (1.5%, across both studies) developed de novo inhibitors (low-titre, transient), and the primary safety endpoint, based on a prospective Bayesian analysis, demonstrated the absence of neoantigenicity for BDDrFVIII. The PK-equivalence, based on central testing to align test and reference articles, and the novel Bayesian analysis of inhibitor safety in these investigations reflect robust experimental designs with relevance to future studies. This extensive dataset demonstrates the safety and efficacy of BDDrFVIII for haemophilia A.     

High-dose factor VIII inhibits factor VIII-specific memory B cells in hemophilia A with factor VIII inhibitors

Hemophilia A in its severe form is a life-threatening hemorrhagic disease that is caused by mutations in the factor VIII (FVIII) gene (symbol F8). About 25% of patients who receive replacement therapy develop neutralizing antibodies that inhibit the function of substituted FVIII. Long-term application of high doses of FVIII has evolved as an effective therapy to eradicate the antibodies and to induce long-lasting immune tolerance. Little is known, however, about the immunologic mechanisms that cause the down-modulation of anti-FVIII antibodies by high doses of FVIII. We report that high doses of FVIII inhibit the restimulation of FVIII-specific memory B cells and their differentiation into antibody-secreting plasma cells in vitro and in vivo in a murine model of hemophilia A. The inhibition of memory B-cell responses is irreversible and not mediated by FVIII-specific T cells. Furthermore, it seems to involve the activation of caspases. We conclude that the inhibition of FVIII-specific memory B cells might be an early event in the down-modulation of anti-FVIII antibodies in patients with hemophilia A who receive high doses of FVIII.     

The role of plasma-derived factor VIII/von Willebrand factor concentrates in the treatment of hemophilia A patients

Besides preventing bleeding episodes, common goals of the treatment of hemophilia include integrating of patients into a normal social life and optimizing their quality of life. Sufficient amounts of factor VIII (FVIII) concentrates, whether recombinant or plasma-derived, are continuously needed. Guidelines for quality assurance of treatment will be a cornerstone to maintain optimal clinical management of patients especially considering financial aspects. Advances in manufacturing technologies have made possible general availability of modern concentrates for the management of hemophilia A patients. Safety, cost and continuous supply of concentrates must be considered when deciding on a product for replacement therapy. As todays' products have reached an excellent margin of safety with regard to virus transmission, the development and treatment of inhibitors is currently the main concern for physicians and patients. The incidence of inhibitors is influenced by various patient-related factors such as mutation type or severity of the disease. Plasma-derived FVIII concentrates containing von Willebrand factor (VWF) may have clinical advantages over pure FVIII concentrates with regard to inhibitor development and inhibitor eradication. Clinical trials comparing FVIII/VWF concentrates with pure FVIII concentrates are lacking, thus a lower inhibitor incidence has not yet been proven. Data from Germany on immune tolerance induction with FVIII/VWF concentrates indicate higher success rates with these than with pure FVIII concentrates. In addition FVIII/VWF concentrates are the therapy of choice when immune tolerance therapy with pure FVIII products is not successful.