Recombinant FVIII Products Research Articles

Treatment characteristics and the risk of inhibitor development: a multicenter cohort study among previously untreated patients with severe hemophilia A.

The development of inhibitory antibodies against infused factor (F) VIII is a major complication of treatment of patients with severe hemophilia A. This study was set up to examine the effects of treatment-related factors on inhibitor development among previously untreated patients with severe hemophilia A. In this multicenter cohort study, we combined individual patient data obtained from four recombinant FVIII product registration studies (Kogenate, Kogenate Bayer, Recombinate, ReFacto) that were performed between 1989 and 2001. From the databases we selected all 236 previously untreated patients with severe hemophilia A who were subsequently treated with FVIII on at least 50 days. Clinically relevant inhibitor development, defined as the occurrence of at least two positive inhibitor titers and a decreased recovery. 67 patients (28%) developed clinically relevant inhibitors (44 high-titer) at a median of ten exposure days. Age at first exposure was not associated with inhibitor development. Peak treatment moments and surgical procedures were related to an increased inhibitor risk [adjusted relative risk 1.6 (95% confidence interval 1.0-2.6) and 2.7 (95% confidence interval 1.3-5.7), respectively]. A shorter duration between exposure days was associated with an increased risk of inhibitor development. There was a possible association between dosing of FVIII and inhibitor development, which largely disappeared after adjustment for confounding factors. These findings show that intensive treatment periods are associated with an increased risk of inhibitor development in previously untreated patients with severe hemophilia A. Our results do not support the notion that age at first exposure is associated with the risk of developing inhibitors.

ReFacto®1 and Advate®2: a single‐dose, randomized, two‐period crossover pharmacokinetics study in subjects with haemophilia A

ReFacto is a recombinant B-domain-deleted, monoclonal antibody-purified, solvent-detergent-treated factor VIII (BDDrFVIII) with no albumin added to the final formulation. Although ReFacto has been shown to be bioequivalent to a plasma-derived FVIII product (Hemophil-M) in a randomized, crossover pharmacokinetic (PK) study, the comparability of ReFacto with the full-length (complete sequence) recombinant FVIII (FLrFVIII, Advate) product has not been previously examined in this manner. The primary objective of this study was to compare the PKs of ReFacto with those of Advate in patients with severe haemophilia A. This was a third-party unblinded, randomized, multicentre, two-period crossover PKs study of ReFacto and Advate in subjects with severe haemophilia A (FVIII:C < or =1%). Blood samples were collected over a 48-h period after i.v. administration of each of the FVIII products. FVIII:C was determined using the chromogenic substrate assay (CSA) in a central laboratory. The plasma FVIII:C PK parameters of ReFacto and Advate were determined using non-compartmental analysis. Bioequivalence was assessed on maximum plasma concentration (C(max)) and the area under the plasma concentration vs. time curves (AUCs) using an anova. The two products were judged to be equivalent if the 90% confidence limits of the ratio of the geometric mean values of C(max) and AUCs fell within the interval of 80-125%. Results from this PKs comparison of two different rFVIII products, using chromogenic substrate assay to measure FVIII:C, showed that ReFacto and Advate are bioequivalent to each other.

1 Bioengineering of Coagulation Factor VIII for Improved Secretion

PURPOSE: Mechanisms that limit recombinant factor VIII (rFVIII) within heterologous systems (eg. commercial rFVIII production or gene therapy applications) include: 1) inefficient expression of FVIII mRNA, 2) inefficient folding of the primary translation product within the endoplasmic reticulum (ER) with retention by chaperone proteins and 3) a requirement for facilitated transport from the ER to the Golgi apparatus. The B domain of FVIII shares no homology with any identified protein, is dispensable for functional activity and is extensively glycosylated (18 asparagine (N)-linked glycosylation attachment sites). B domain-deleted (BDD)-FVIII generates higher concentrations of mRNA compared to full-length FVIII and therefore increased primary translation product but exhibits a reduced rate of secretion and increased intracellular retention. Bioengineering of the FVIII B domain effectively improves the trafficking of rFVIII through facilitated ER-Golgi transport. METHODS: We have used a series of B domain variants to map key regions of the B domain that regulate mRNA levels and secretion efficiency. Each construct has increased B domain size (ranging from 29 to 774 amino acids (aa) and increased number of N-linked oligosaccharides (from 1 through 18). RESULTS: BDD-FVIII, FVIII wild-type (WT) and FVIII B domain variants were transfected into COS and CHO cell lines and the media harvested for analysis of FVIII activity and antigen. The addition of even a few N-linked oligosaccharides within a short B domain spacer (optimal at 226 aa and 6 N-linked oligosaccharides) improves secretion of BDD-FVIII approximately 5 to 10-fold. With additional increase in B domain size and oligosaccharide content, secretion efficiency declined in a stepwise fashion to similar levels observed for FVIII WT. An alternative construct was prepared in which the human FVIII B domain was replaced with the FVIII B domain from puffer fish (fugu rubripes). The fugu FVIII B domain is 224 aa and shares only 6% sequence identity to the human FVIII B domain, yet has a high density of N-linked oligosaccharides (11). This construct was secreted as efficiently as the 226aa/N6 human construct. CONCLUSIONS: The secretion efficiency of FVIII can be regulated by the size and oligosaccharide content of the B domain. The results suggest that it is primarily the presence of N-linked oligosaccharides that improves secretion efficiency. FVIII bioengineered for improved secretion will be an alternative for gene therapy strategies as well as recombinant FVIII production in manufacturing or transgenic strategies.

Inhibitor Incidence in Previously Untreated Patients (PUP) with Hemophilia A and B - a 10-Year-Follow up of the Prospective Multicenter Study.

The development of neutralizing FVIII-antibodies is one of the most serious complication in the replacement therapy with FVIII and IX concentrates in hemophilia A and B patients. In order to observe different parameters with possible impact on inhibitor development in PUPs with hemophilia A and B a prospective study was started in 1993 by the GTH (German, Swiss and Austrian Society on Thrombosis and Haemostasis Research).The study protocol provides frequent inhibitor testing particularly during the initial 200 exposure days (ED) according to the modified Bethesda method (central lab Prof. Budde, Hamburg) and correlates inhibitor development with age at first exposure, therapy regimen, dosage, mutation type, type of concentrate and further variables which may have an impact on inhibitor development. Until September 2003, 293 patients with hemophilia A and B have been enrolled in this ongoing study. 206 have been treated (176 hemophilia A, 30 hemophilia B) at least once with plasma derived (pd) or recombinant (r) factor (F) VIII or IX concentrates.Out of 176 hemophilia A patients 35 developed inhibitors (16 high titer >5 Bethesda Units/BU, 17 low titer >0.6–5 BU, 2 transient low titer inhibitors) after 12 exposure days (ED) in median (range 1–56) at the age of 0.9 years (median, range 0.25–10.8). Inhibitor development was predominantly observed in severe hemophilia A patients (31%) and to a lesser extent in moderate patients (7.7%). Inhibitor incidence was higher in patients receiving recombinant FVIII products than in those treated with plasma derived concentrates (p=0,14). No correlation between age at first exposure and inhibitor development was found. Early prophylaxis resulted in a lower inhibitor incidence than on-demand treatment (p=0.003).Out of 16 severely affected hemophilia B patients 2 developed inhibitors.

Characterization of the Pharmacokinetics of rFVIII in Young Pre-School Children with Hemophilia, Including an Analysis of the Influence of Age and Body Weight.

Commencement of prophylaxis at an early age for management of severe hemophilia A is widely recommended in order to prevent or minimize disabling arthropathy. Knowledge of the pharmacokinetics of replacement clotting factors is key to achieving optimal dosing regimens. However, the majority of pharmacokinetic studies on replacement FVIII have been conducted in older children and adults, and evidence on the pharmacokinetics of FVIII in pre-school children is primarily anecdotal. This study is the first to evaluate the pharmacokinetics of rFVIII in a large cohort of children < 6 y of age. The evaluation was part of an open-label, multicenter study of 53 subjects with FVIII ≤ 2% and ≥ 50 prior FVIII exposure days. The mean age of the cohort was 3.1 ± 1.5 y (range, 1.1–6.0 y). Standard pharmacokinetic parameters were assessed in accordance with the guidelines for pediatric patients of the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis. A single dose was infused of 50 IU/kg of a recently-developed recombinant FVIII product, ADVATE rAHF-PFM, a full-length FVIII concentrate prepared without the addition of human or animal plasma proteins. In an intent-to-treat analysis of results from 52 subjects, mean plasma half-life (t1/2) was 9.7 ± 1.9 h, the adjusted in vivo recovery (IVR) was 1.9 ± 0.4 IU/dL/IU/kg, and clearance was 0.04 ± 0.01 dL/kg•h. These data contrast with those of a recent study of rAHF-PFM in individuals with a median age of 18 (range, 10–65) in which the t1/2 was 12.0 ± 4.3 y and IVR was 2.4 ± 0.5 (Tarantino et al, Haemophilia, 2004, in press). No previous studies have examined whether pharmacokinetic changes in t1/2 and IVR related to age or weight occur in young pediatric patients. There was no significant correlation between IVR and age (r = −0.09, p = 0.54); however, there was a moderate correlation between IVR and body mass index (BMI) (r =.049; p < 0.001). There was also a weak correlation between t1/2 and age (r =0.34; p = 0.02). A significant inverse correlation was found between IVR and volume of distribution at steady state (Vss) (r = −0.82, p < 0.001). Subsequent assessment of the safety, efficacy and immunogenicity of rAHF-PFM is ongoing in these patients. In an interim analysis, after a total of 3,698 infusions, no inhibitors or other adverse events related to rAHF-PFM have been reported (median exposure days = 74). Of 210 bleeding episodes, 92.4% required ≤ 2 infusions. Median numbers of annual total bleeding episodes (joint + non-joint) for patients on either standard (n = 17) or modified (n = 25) prophylaxis or on-demand treatment (n = 4) were 2.85, 2.31, and 20.07, respectively. This study demonstrates for the first time in pre-school children and toddlers detectable correlations in the pharmacokinetics of rFVIII between age and t1/2, and between IVR and BMI, and that ADVATE rAHF-PFM is efficacious, well-tolerated and non-immunogenic in young children. Further research is required to define factors affecting inter-individual variation in t1/2 of rFVIII and the accelerated pharmacokinetics in children < 6 y old.

Bioengineering of coagulation factor VIII for improved secretion

AbstractFactor VIII (FVIII) functions as a cofactor within the intrinsic pathway of blood coagulation. Quantitative or qualitative deficiencies of FVIII result in the inherited bleeding disorder hemophilia A. Expression of FVIII (domain structure A1-A2-B-A3-C1-C2) in heterologous mammalian systems is 2 to 3 orders of magnitude less efficient compared with other proteins of similar size compromising recombinant FVIII production and gene therapy strategies. FVIII expression is limited by unstable mRNA, interaction with endoplasmic reticulum (ER) chaperones, and a requirement for facilitated ER to Golgi transport through interaction with the mannose-binding lectin LMAN1. Bioengineering strategies can overcome each of these limitations. B-domain-deleted (BDD)-FVIII yields higher mRNA levels, and targeted point mutations within the A1 domain reduce interaction with the ER chaperone immunoglobulin-binding protein. In order to increase ER to Golgi transport we engineered several asparagine-linked oligosaccharides within a short B-domain spacer within BDD-FVIII. A bioengineered FVIII incorporating all of these elements was secreted 15- to 25-fold more efficiently than full-length FVIII both in vitro and in vivo. FVIII bioengineered for improved secretion will significantly increase potential for success in gene therapy strategies for hemophilia A as well as improve recombinant FVIII production in cell culture manufacturing or transgenic animals. (Blood. 2004;103: 3412-3419)

Validation of a quantitative SPR assay for recombinant FVIII.

Surface plasmon resonance was employed to establish a quantitative assay for recombinant FVIII (rFVIII) products using rFVIII as standard. The anti-FVIII monoclonal antibody ESH4 was immobilized onto a carboxymethyldextran surface. A range of rFVIII concentrations were injected over the surface and the binding response enhanced by the addition of a further monoclonal antibody ESH8. Validation using National Institute of Biological Standards and Controls (NIBSC) sixth International rFVIII concentrate standard gave inter- and intra-assay coefficient of variations (CVs) of 7.5 and 3.68% respectively for ESH4-rFVIII binding alone. Enhancement of the binding signal by secondary addition of ESH8 produced inter- and intra-assay CVs of 2.75 and 1.5%. The ESH4 immobilized chip was found to retain binding capacity following regeneration for at least 75 cycles. The assay was found to be unsuitable for quantitation of plasma derived FVIII product but may prove useful for monitoring of rFVIII production.

Development of improved factor VIII molecules and new gene transfer approaches for hemophilia A.

Hemophilia A, the most common inherited bleeding disorder, is caused by deficiency or functional defects in coagulation factor VIII (fVIII). Conventional treatment for this disease involves intravenous infusions of plasma-derived or recombinant fVIII products. Although replacement therapy effectively stops the bleeding episodes, it has a risk of transmission of viral blood-borne diseases and development of neutralizing antibodies that inactivate the administered fVIII protein. Hemophilia A is an attractive candidate for application of gene therapy approaches because the therapeutic window is wide and even modest elevation of fVIII levels will correct the hemophilic phenotype. Ongoing preclinical investigations utilize animal models of hemophilia A, including genetically fVIII-deficient mice and naturally fVIII-deficient dogs, to optimize vectors, transgenes and target cell populations for Phase I clinical trials. In this review, we outline the progress in understanding the mechanisms of fVIII turnover, which provides a basis for development of improved fVIII molecules with prolonged half-life in the circulation. We discuss the possibility of incorporating these improved fVIII molecules as transgenes into self-inactivating lentiviral vectors carrying chromatin insulator sequences, representing a new generation of gene delivery vehicle, to target hematopoietic stem cells and endothelial cells. The use of hematopoietic stem cells as the target cell population may prevent inhibitor formation to transduced fVIII by induction of immune tolerance. Alternatively, endothelial cells may support optimal synthesis of fVIII and myeloablative conditioning of patients with radiation or chemotherapy may not be required for efficient engraftment of the engineered cells. Collectively, these proposed advances represent promising prophylactic strategies toward long-term correction of the coagulation defect in this progressively debilitating, life-threatening disease.

Inhibitor development and FVIII gene mutation analysis in a pediatric cohort treated with sucrose formulated, full‐length recombinant Factor VIII

Treatment of previously untreated patients (PUPs) and minimally treated patients (MTPs) with severe hemophilia A using FVIII concentrates is complicated by FVIII inhibitor formation in ∼30% of patients. The incidence of FVIII inhibitors was determined in a prospective clinical trial of sucrose‐formulated full‐length recombinant FVIII (rFVIII‐FS, KOGENATE® Bayer; Kogenate® FS) in pediatric patients.Methods: PUPs and MTPs (=4 exposure days‐EDs) with hemophilia A (&lt;2% FVIII) were enrolled from 19 EU and 13 US centers. Sixty patients were evaluable for inhibitor formation (EU, 31; US, 29). Patients were tested regularly with the Nijmegen‐modified Bethesda assay (negative, ≤0.6 BU; Low Titer, &gt;0.6‐5 BU; High Titer, &gt;5 BU).Results: In the EU cohort (31 Caucasian), 4 patients developed inhibitors (3 Low; 1 High). Five high titer inhibitors developed in the US cohort (17 Caucasian; 5 Black; 7 Other). Median EDs at inhibitor detection was 8 [range, 3‐16], and at study end 1 EU and 4 US patients had &lt;20 EDs. The incidence of inhibitors in patients achieving 20 ED was 16.4% (9/55).Conclusions: The rate of inhibitor formation in pediatric patients with severe hemophilia A treated with rFVIII‐FS is consistent with that observed with plasma‐derived and other recombinant FVIII products. Major gene disruptions were observed in all inhibitor patients.

Anaphylaxis following the use of a plasma‐derived immunopurified Monoclate‐P®, and the recombinant Recombinate® and Kogenate® factor VIII: a therapeutic challenge

We report the first case of an anaphylactic shock to three different FVIII concentrates, one immuopurified plasma‐derived (Monoclate‐P®) and two recombinant FVIII products (Recombinate® and Kogenate®). These shocks appeared despite the use of antihistaminic drugs. The use of a highly purified plasma‐derived FVIII allowed successful on‐demand therapy without any antiallergic drug or desensitization.

Choosing a Host Cell for Active Recombinant Factor VIII Production Using Vaccinia Virus

The recent molecular cloning of factor VIII (FVIII or antihaemophilic factor)1–3 has brought new hope to haemophilia A patients of a safer therapeutic preparation. The choice of a host cell for the expression of such a large and unstable protein4 in a biologically active form is limited however because of the time consuming process of screening cell lines. Having cloned the FVIII cDNA, we have used the vaccinia virus (VV) expression system5,6 in order to screen rapidly several different cell lines for active recombinant FVIII production. We show here that the ability (or inability) to express active FVIII is dependent on the host cell used. Baby hamster kidney 21 (BHK21) cells have given the most encouraging results so far. The stabilizing effect of von Willebrand factor (vWf) on the recombinant FVIII when added to the culture medium is also demonstrated.