Full-length FVIII Research Articles

Development of a rapid and fully automated factor VIII inhibitor assay, insensitive to emicizumab, and a lowest level of quantification of 0.2 BU/mL

BackgroundFactor (F)VIII inhibitors are measured using labor- and resource-expensive Nijmegen or Bethesda assays, which lack sensitivity for low-titer inhibitors and show high variations in quality surveys, mainly because of manual assay procedures. ObjectivesThe goal of this study was the development of a fast and fully automated FVIII inhibitor assay by using recombinant (r)FVIII as substrate and dedicated equipment for execution of the test. MethodsA new rapid, fully automated, FVIII inhibitor assay is presented, the core of which is use of full-length recombinant FVIII (rFVIII; Kovaltry, Bayer) as inhibitor substrate instead of plasma FVIII, resulting in rapid binding of inhibitors to rFVIII due to absence of von Willebrand factor. Dramatic shortening of incubation time facilitated full automation on an analyzer capable of 3 subsequent sample dilution steps and 3 reagent additions. Equal volume mixtures of sample and rFVIII (1.0 U/mL) were incubated for 10 minutes at 37 °C, whereafter remaining FVIII activity was analyzed with a kinetic chromogenic assay, allowing inhibitor activity calculation without preceding FVIII activity calibration, using a Ceveron s100 analyzer (Technoclone). ResultsMean titer in 60 nonhemophiliacs was 0.0 BU/mL (SD, 0.1), yielding a limit of blank of 0.1 BU/mL and lower limit of quantification of 0.2 BU/mL. Analyses were performed with the new method and a Nijmegen assay in 28 inhibitor-positive clinical samples, 14 containing emicizumab and 14 without. Correlation coefficient in emicizumab-free type I inhibitor samples was 1.0. Emicizumab dependency of the method was excluded in spiking experiments with inhibitor-positive samples. Reproducibility was tested by analyzing 7 samples in 3 laboratories for 5 days, twice daily; coefficients of variation of all samples were <15%. ConclusionWe present development data of a sensitive and specific rapid, automated FVIII inhibitor assay generating results within 20 minutes that is less resource-intensive than standard assays with potential to improve assay variability.

The self-reactive FVIII T cell repertoire in healthy individuals relies on a short set of epitopes and public clonotypes.

Non-mutated FVIII-specific CD4 T cell epitopes have been recently found to contribute to the development of inhibitors in patients with hemophilia A (HA), while auto-reactive CD4 T cells specific to FVIII circulate in the blood of healthy individuals at a frequency close to the foreign protein ovalbumin. Thus, although FVIII is a self-protein, the central tolerance raised against FVIII appears to be low. In this study, we conducted a comprehensive analysis of the FVIII CD4 T cell repertoire in 29 healthy donors. Sequencing of the CDR3β TCR region from isolated FVIII-specific CD4 T cells revealed a limited usage and pairing of TRBV and TRBJ genes as well as a mostly hydrophobic composition of the CDR3β region according to their auto-reactivity. The FVIII repertoire is dominated by a few clonotypes, with only 13 clonotypes accounting for half of the FVIII response. Through a large-scale epitope mapping of the full-length FVIII sequence, we identified 18 immunodominant epitopes located in the A1, A3, C1, and C2 domains and covering half of the T cell response. These epitopes exhibited a broad specificity for HLA-DR or DP molecules or both. T cell priming with this reduced set of peptides revealed that highly expanded clonotypes specific to these epitopes were responsible individually for up to 32% of the total FVIII repertoire. These FVIII T cell epitopes and clonotypes were shared among HLA-unrelated donors tested and previously reported HA patients. Our study highlights the role of the auto-reactive T cell response against FVIII in HA and its similarity to the response observed in healthy individuals. Thus, it provides valuable insights for the development of new tolerance induction and deimmunization strategies.

The role of proteolytic cleavage at Arg336 and Arg372 of the A1 domain in factor VIIa/tissue factor-catalyzed reactions of B domain-deleted factor VIII

BackgroundWe previously demonstrated that factor (F)VIII was rapidly activated through proteolytic cleavage at Arg372 and Arg740 by activated FVII (FVIIa)/tissue factor (TF) in very early coagulation phase, followed by inactivation by cleavage at Arg336. The influence of the absence of FVIII B domain in this series of FVIIa/TF-catalyzed reaction remains unclear, however. AimTo examine the FVIIa/TF-catalyzed reaction of B domain-deleted (BDD-)FVIII. Methods and resultsThe FVIII activity (FVIII:C) of commercial full-length (FL-)FVIII and BDD-FVIII increased by ∼1.7-fold within 0.5 min after addition of FVIIa/TF (1 nM/0.1 nM). FVIII:C decreased to initial levels with inactivation rate constant (k; ∼0.035) within 15 min of FL-FVIII activation, but decreased gradually to initial levels (k; ∼0.017) within 30 min of BDD-FVIII activation. SDS-PAGE analyses demonstrated that the FVIIa/TF-catalyzed cleavage of BDD-FVIII occurred at Arg336 within 0.5 min in parallel with elevation of FVIII:C, but cleavage at Arg372 was not evident. FVIIa/TF-catalyzed activation of both recombinant BDD-FVIII R336A and R372A mutants that were prepared, were similar to that of wild-type (WT) BDD-FVIII. However, FVIII:C returned to initial levels (k; ∼0.046) within 30 min of R336A mutant activation, but little reduction of FVIII:C was observed with R372A mutant (k; ∼0.0046). SDS-PAGE analysis indicated that FVIIa/TF-catalyzed cleavage of WT and R372A mutant was predominant at Arg336, whereas that of R336A mutant was observed at Arg372. ConclusionsFVIIa/TF-catalyzed activation of BDD-FVIII was initiated by cleavage at Arg336, and the FVIII B domain appeared to control FVIIa/TF-catalyzed reactions by altering pattern of cleavage at Arg336 and Arg372.

The Tfh/Tfr Pathway Is Pivotal in FVIII Inhibitor Development in Mice

The development of neutralizing anti-FVIII antibodies (inhibitors) is one major complication of FVIII protein replacement therapy in hemophilia A patients. Our previous study demonstrated that the induction of activated follicular helper T (Tfh) cells in FVIII null mice is critical for FVIII inhibitor development. Follicular regulatory T (Tfr) cells are a specialized subset of Foxp3 + T cells identified in the germinal center (GC) that can modulate Tfh cell activation of B cells and antibody development. However, the role of Tfr cells in controlling anti-FVIII inhibitor development in vivo is still unclear. Here, we explored the activation and induction of Tfr cells and their functions in anti-FVIII inhibitor development in FVIII-immunized mice. We first characterized the features of activated Tfr cells in the spleen of immunized FVIII null mice by flow cytometry analysis. Activated Tfr cells were defined as CD4 +Foxp3 +CD44 +CD62L -CXCR5 +PD-1 +. Tfr cells express a high level of transcription factor BCL6 and costimulatory receptor ICOS. Activated Tfr cells also express a high level of costimulatory receptor CTLA4, which is similar to non-Tfr effector Treg cells, but CD25 expression is low on Tfr cells. FVIII immunization increases the frequency and the absolute number of activated Tfr cells in the spleen of mice that produce inhibitors. The percentage of activated CXCR5 hiPD-1 hi Tfr cells within the CD4 population significantly increased in inhibitor-producing mice compared to saline-treated controls and non-inhibitor-producing mice (0.88 ± 0.2 %, 0.33 ± 0.16 %, and 0.36 ± 0.2%, respectively). The Tfh/Tfr ratio also significantly increased in FVIII inhibitor-producing mice, suggesting a preferential expansion of Tfh cells relative to Tfr cells in inhibitor-producing mice. Interestingly, similar to Tfh cells as we reported before, the emergence of Tfr cells correlated with titers of anti-FVIII inhibitors in FVIII immunized mice. This indicates that a functional relationship exists between Tfr cell activation and FVIII inhibitor production after FVIII immunization. Using Tfr deficient Foxp3-Cre Bcl6 fl/fl (BCL6FC) mice, we determined the role of Tfr cells in inhibitor generation in vivo. Loss of Tfr cells led to significantly decreased FVIII inhibitor levels, with FVIII inhibitor titers in BCL6FC mice markedly lower than in wild-type (WT) mice upon recombinant human full-length FVIII (rhFVIII, Kogenate 200 U/kg/week IV x4) immunization (24 ± 16 and 131 ± 114 BU/ml, respectively). However, the total anti-FVIII IgG titers were similar between the BCL6FC and WT groups, suggesting that Tfr cells help generate neutralizing anti-FVIII antibodies. FVIII immunization could still efficiently induce Tfh cell activation in BCL6FC mice. However, both GC Tfh cells and GC B cells were reduced in FVIII immunized BCL6FC mice, suggesting that Tfr cells promote FVIII-specific GC responses. To further investigate the regulatory function of Tfr cells on FVIII immune responses, mice with phosphatase Pten deficiency in Treg cells (Foxp3-Cre Pten fl/fl [PtenFC]), a model with augmented Treg and Tfr cells, were immunized with rhFVIII and anti-FVIII antibody titers were measured at 1 week after the 4 th weekly immunization. Of note, FVIII inhibitor titers (8.06 ± 8.6 BU/ml) were significantly lower than those in WT controls under the same immunization protocol. There were significant positive correlations between the inhibitor titers and total anti-FVIII IgG in the WT and PtenFC groups but not in the BCL6FC group after rhFVIII immunization. To confirm the important role of the Tfh/Tfr pathway in FVIII inhibitor development, we used CD4-Cre Bcl6 fl/fl (CD4-Bcl6-cKO) mice, a model with Bcl6 deficiency in all CD4 + T cells. As expected, none of the CD4-Bcl6-cKO mice developed detectable levels of FVIII inhibitors, demonstrating that the transcription repressor BCL6 is pivotal for FVIII inhibitor development. In conclusion, we found that FVIII immunization induces Tfr cell activation and expansion. Tfr cells have a dual function in regulating FVIII immune responses in mice, and the Tfh/Tfr pathway is essential for FVIII inhibitor development in hemophilia A mice.

Prospective Hemophilia Inhibitor PUP Study reveals distinct antibody signatures during FVIII inhibitor eradication.

Factor VIII (FVIII) inhibitor formation is a major clinical concern during replacement therapy in patients with hemophilia A. Immune tolerance induction (ITI) is the only therapeutic approach to attempt inhibitor eradication and establishment of long-term immune tolerance to FVIII. Hemophilia Inhibitor Previously Untreated Patient (PUP) Study (HIPS) was a prospective clinical trial to investigate changes in the immune system of PUPs with severe hemophilia A. Five patients who developed persistent FVIII inhibitors during HIPS entered an ITI extension arm (HIPS-ITI). During HIPS-ITI, inhibitor patients received ITI with the same FVIII product (a single source of recombinant, human full-length FVIII) used in HIPS until successful tolerance, declared failure, or a maximum of 2 years after HIPS-ITI enrollment, whichever came first. Blood samples and clinical data were collected monthly. Longitudinal FVIII-binding antibody signatures, associated binding specificities, and apparent affinities were determined for each patient at each sampling time point. ITI was successful or partially successful in 2 patients and failed in 3. Both groups presented with distinct FVIII-specific antibody signatures. ITI success required the disappearance of FVIII inhibitors, which was associated with the eradication or sustained titer minimization of high-affinity FVIII-specific antibodies, particularly of the immunoglobulin G1 (IgG1) and IgG4 subclasses. In contrast, ITI failure, as reflected by FVIII inhibitor persistence, was associated with persistent high-affinity FVIII-specific antibodies. Interestingly, 1 patient with partial ITI success and 1 patient with ITI failure developed apparent oligoreactive FVIII-binding antibodies during ITI. The explanation of the true nature of these antibodies requires more comprehensive follow-ups in future studies. This trial was registered at www.clinicaltrials.gov as #NCT01652027.

In-Silico Characterization of von Willebrand Factor Bound to FVIII

Factor VIII belongs to the coagulation cascade and is expressed as a long pre-protein (mature form, 2351 amino acids long). FVIII is deficient or defective in hemophilic A patients, who need to be treated with hemoderivatives or recombinant FVIII substitutes, i.e., biologic drugs. The interaction between FVIII and von Willebrand factor (VWF) influences the pharmacokinetics of FVIII medications. In vivo, full-length FVIII (FL-FVIII) is secreted in a plasma-inactive form, which includes the B domain, which is then proteolyzed by thrombin protease activity, leading to an inactive plasma intermediate. In this work, we analyzed through a computational approach the binding of VWF with two structure models of FVIII (secreted full-length with B domain, and B domain-deleted FVIII). We included in our analysis the atomic model of efanesoctocog alfa, a novel and investigational recombinant FVIII medication, in which the VWF is covalently linked to FVIII. We carried out a structural analysis of VWF/FVIII interfaces by means of protein–protein docking, PISA (Proteins, Interfaces, Structures and Assemblies), and protein contact networks (PCN) analyses. Accordingly, our computational approaches to previously published experimental data demonstrated that the domains A3-C1 of B domain-deleted FVIII (BDD-FVIII) is the preferential binding site for VWF. Overall, our computational approach applied to topological analysis of protein–protein interface can be aimed at the rational design of biologic drugs other than FVIII medications.

Acquired Hemophilia a Relapse Is Related to Th2 Response, and Increased Expression of B-Cell Activating Factor (BAFF)

Acquired hemophilia A (AHA) is a rare autoimmune disorder caused by the development of autoantibodies against the factor (F)VIII of coagulation with higher incidence in elderly individuals. The treatment includes immunosuppressive alternatives, and the majority of patients reach complete remission of symptoms. Although the role of immune T and B cells in the physiopathology of several autoimmune diseases is widely established, few studies have explored the T and B cells immune response pathway in AHA. This study aimed to determine the role of T cells through their cytokines expression and B cells through their expression of proteins that activate the B cell receptor (BCR), such as B-cell activating factor (BAFF) and A-proliferation inducing ligand (APRIL), in a longitudinal evaluation of patients with AHA.We included in this study 11 patients, 4 (36%) male, with the diagnosis of AHA, based on the occurrence of low FVIII levels (median FVIII level at the diagnosis was 0.10 IU/dL; range 0-18.8) and anti-FVIII inhibitor titer positive (≥0.6 Bethesda units (BU)/mL) (median inhibitor titer at the diagnosis was 60BU/mL; range 7.6-1000.2). The median age at the diagnosis was 59 years (range 9-75). Idiopathic AHA was evident in 72.7% of cases, while autoimmune diseases (18.2%), and pregnancy (9.1%) were the other underlying etiologies. All patients received immunosuppressive therapy (IST). Eight (72.7%) patients used corticosteroid (CS) plus cyclophosphamide (CP), one (9.1%) received only CP, one (9.1%) used CS plus rituximab and one (9.1%) received CS, CP and three cycles of rituximab. The response to IST was defined as complete remission (CR) with anti-FVIII < 0.6BU/mL and FVIII >50IU/dL, without further IST. Relapse was defined when anti-FVIII titer became >0.6BU/mL after previous remission. In this cohort, we observed 64% of sustained CR and 4(36%) AHA patients had at least one relapse, including two AHA patients considered idiopathic and two with other autoimmune diseases associated.Peripheral blood mononuclear cells (PBMC) were collected from all AHA patients at the diagnosis previously IST (baseline), when patient achieved CR, and at the occurrence of relapse. PBMCs were maintaining frozen in liquid nitrogen. Cells were taw and cultivated with RMPI-1640 medium in 7.5x10 5 cells/well in a 48-well plate. After 24h, cells were stimulated with a 1IU/well of a full-length recombinant FVIII concentrate (rFVIII) or phorbol myristate acetate (PMA) (1µM) with ionomycin (1µM). After 24h incubation, cells were analyzed by flow cytometry at BD FACSCalibur™.To determine T CD4 + cells' cytokine production profile, we stained T cells with anti-CD4, anti-interleukins (IL)-17a, IL-4, and IL-21, anti-TGF-b, and anti-TNF-a. We observed a significant increase of IL-17a production in the rFVIII-stimulated cells isolated at baseline from all patients compared to cells isolated from healthy individuals (P=0.01). However, IL-17a expression at baseline was not different between cells from patients who sustained CR to those who relapsed. Interestingly, when we compared cells isolated at baseline from patients that relapsed after different immunosuppressive attempts, we observed a significant difference for IL-4 (P=0.009), IL-21 (P=0.02), and TGF-b (P=0.01) expression in comparison to cells from healthy individuals. However, this difference was not observed in cells from sustained CR vs. cells from healthy individuals (figure 1). Thus, suggesting that all patients present Th17 response, while only patients that relapsed also showed Th2 response at the diagnosis.We also evaluated B cells' expression of BAFF and APRIL. The cells were stained with anti-CD19, BAFF, and APRIL. Only cells isolated at baseline from AHA relapsed patients after rFVIII stimulation or without any stimuli presented increased expression of BAFF (P=0.01 with rFVIII-stimulated and P=0.007 for unstimulated cells) when compared to PBMCs isolated from healthy individuals (figure 1). BAFF is responsible for controlling B-cell maturation and is associated with autoantibody production of different autoimmune conditions.Our finds reveal an increase in the Th17 response in patients with AHA at the diagnosis. However, the presence of Th2 response and increase of BAFF expression was observed only at the diagnosis of patients with recurrence of autoimmune response to FVIII, which suggests a potential biomarker for AHA evaluation. [Display omitted] DisclosuresOzelo: BioMarin: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Shire: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Sanofi: Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bayer: Speakers Bureau; Novo Nordisk: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Grifols: Other: Grant review.

CD4+ regulatory and naïve T cells specific for factor VIII stand vis-à-vis to balance the immune response in healthy individuals.

Additionally, inhibitory antibodies to FVIII rarely occur in healthy individuals resulting in acquired HA. [...]very few healthy individuals (0.2–1.0 per one million) [ 2], as well as one-third of HA patients, mount an aggressive auto- or alloimmune response to FVIII. Following stimulation of peripheral blood mononuclear cells (PBMCs) isolated from buffy coats of healthy individuals with full-length FVIII (rFVIII), the fusion protein (rFVIIIFc) and MOG for 16 h, the proportion of samples responding to the stimuli based on surface expression of CD154 (non-Treg) or CD137 (Treg) was compared. Both, rFVIII and rFVIIIFc stimulation (median of 68.4%) showed a higher proportion of activated naive T cells compared to the unstimulated control (median of 57.4%) (p < 0.001 and p = 0.002). [...]in this setting naive CD4+ T cells showed higher responsiveness towards either rFVIII product. Concerning reactivity towards FVIII, our data suggest that a significant proportion of healthy individuals possesses FVIII-specific T cells and that specific Tregs are produced during early tolerance development to bridle this cell population. [...]situations in which Treg functions are impaired might result in the formation of robust FVIII-specific T-cell responses eventually leading to the formation of FVIII-specific antibodies.

The Impact of Specific Factor VIII Domains on Uptake By Dendritic Cells

Introduction: The role of dendritic cells in the presentation of foreign antigens to induce an adaptive immune response is critical for protection from potentially harmful pathogens. However in the treatment of hemophilia A, the recognition, processing, and presentation of factor VIII (fVIII) by dendritic cells that results in anti-fVIII inhibitors is a devastating complication of modern hemophilia A care. FVIII structure consists of A1, A2, B, A3, C1, and C2 domains. While prior studies have described a role for the C1 and C2 domains in mediating fVIII uptake by dendritic cells, the contribution of the remaining fVIII domains has not been fully explored. In this study we analyzed the full spectrum of fVIII domains on uptake by dendritic cells as a means to better understand the pathogenic innate immune response to fVIII. Methods: Bone marrow cells were harvested from the femurs and tibias of 8-10 week old E16 hemophilia A knockout mice, stimulated with GM-CSF, and cultured at 37°C for 6-7 days to induce bone marrow derived dendritic cells (BMDCs). BMDCs were incubated with dylight650-conjugated recombinant full length fVIII (DyL650-rfVIII) in combination with a panel of 17 previously characterized anti-fVIII monoclonal antibodies (MAbs) from our repertoire of anti-human murine derived MAbs representing B cell epitopes from all fVIII domains. Internalization of 10 nM fVIII by CD11c+ BMDCs in the presence of molar excess of individual anti-fVIII MAbs (80 nM) following a 30 minute incubation at 37°C was compared to uptake of fVIII alone. FVIII uptake by BMDCs at 4°C was used as a negative control. FVIII internalization was analyzed by flow cytometry and imaged using an Imagestream X Mark II flow cytometer. A supraphysiologic dose of fVIII was utilized in these experiments for optimal visualization and measurement of fVIII internalization by flow cytometry, though similar trends in uptake pattern at more physiologic concentrations of fVIII have also been observed in previous studies performed. Results: The panel of 17 anti-fVIII MAbs tested included 1 anti-A1, 2 anti-A2, 2 anti-B, 2 anti-A3, 3 anti-C1, and 7 anti-C2 domain MAbs. In this model, the presence of anti-C1 domain and anti-C2 domain MAbs reduced fVIII uptake by BMDCs as shown in prior studies. Anti-C1 domain MAb B136, a group B MAb that binds a critical structural epitope within the 2077-2084 region of the C1 domain, reduced fVIII uptake by 56%. Additionally classical group A anti-C2 domain MAb 3E6 reduced fVIII uptake by 35%. Both MAbs B136 and 3E6 bind to epitopes that are important for fVIII binding to von Willebrand factor and phospholipid surfaces suggesting that these epitopes may play a role in fVIII recognition by dendritic cells, particularly in instances when fVIII is unbound by either ligand in plasma. Interestingly anti-A1 domain MAb 2-116 and anti-A3 domain MAb I92 that binds residues 1818-1916 located at the C terminal end of the A3 domain significantly increased fVIII uptake by BMDCs two-fold (113% and 95% respectively) compared to fVIII alone. These findings indicate that epitopes within the A1 and A3 domains may play a role in limiting fVIII recognition by dendritic cells. All of the anti-A2 and anti-B domain MAbs, 2 of the 3 anti-C1 MAbs, and 5 of the 7 anti-C2 MAbs tested did not alter fVIII uptake. The figure below depicts a bar graph summarizing fVIII internalization by BMDCs with or without the anti-fVIII MAbs tested (figure 1A) and a representative Imagestream image of a dendritic cell (Ch01), CD11c+ surface staining of the dendritic cell (Ch07), DyL650-rfVIII alone without an anti-fVIII MAb (Ch11), and a merged image of CD11c+ dendritic cell and DyL650-rfVIII (Ch07/Ch11) (figure 1B). Conclusions: The C1 and C2 domains of fVIII primarily mediate fVIII uptake by dendritic cells. However the A1 and A3 domains may play a protective role in antigen uptake and ultimately antigen presentation by dendritic cells. Further investigation into the innate immune response to fVIII in patients with hemophilia A and differences between patients who do and do not develop inhibitors would provide insight into key aspects of the immune response that regulates fVIII tolerance. Download : Download high-res image (136KB) Download : Download full-size image Figure 1 . Disclosures Batsuli: Genentech, Octapharma: Membership on an entity's Board of Directors or advisory committees.

Utilization of a novel Sendai virus vector in ex vivo gene therapy for hemophilia A.

Sendai virus (SeV) vectors are being recognized as a superior tool for gene transfer. Here, we report the transfection efficacy of a novel, high-performance, replication-defective, and persistent Sendai virus (SeVdp) vector in cultured cells and in mice using a near-infrared fluorescent protein (iRFP)-mediated in vivo imaging system. The novel SeVdp vector established persistent infection, and strong expression of inserted genes was sustained indefinitely in vitro. Analysis of iRFP-expressing cells transplanted subcutaneously into NOG, nude, and ICR mice suggests that innate immunity was involved in the exclusion of the transplanted cells. We also evaluated the feasibility of this novel SeVdp vector for hemophilia A gene therapy. This system enabled insertion of full-length FVIII genes, and transduced cells secreted FVIII into the culture medium. Transient FVIII activity was detected in the plasma of mice after intraperitoneal transplantation of these FVIII-secreting cells. Further improvement in methods to evade immunity, such as simultaneous expression of immunomodulatory genes, would make this novel vector a very useful tool in regenerative medicine.

BAY 81-8973 prophylaxis and pharmacokinetics in haemophilia A: Interim results from the TAURUS study.

ObjectivesTo report interim data from TAURUS, a study assessing real‐world prophylactic treatment with unmodified, full‐length recombinant FVIII BAY 81‐8973 (Kovaltry®; Bayer) indicated for haemophilia A.MethodsTAURUS (NCT02830477) is an international, open‐label, prospective, non‐interventional, single‐arm study with a one‐year observation period (target N = 350). Patients have moderate or severe haemophilia A (FVIII ≤5% or ≤1%) and ≥50 exposure days to any FVIII product. Clinician‐ and patient‐reported outcomes are captured on previous product use, changes in prophylaxis dose and dosing frequency, FVIII consumption, reported bleeding rates, treatment satisfaction and adherence, pharmacokinetic (PK) data (if available) and safety data.ResultsAt cut‐off, baseline data were available from 160 patients (89 had ≥6 months of follow‐up data). Most patients had severe haemophilia A (85%), infused BAY 81‐8973 ≥ 3×/wk (59%) and experienced a median number of total bleeds of 2.0 (non‐annualised; 246 days median documentation period). Good levels of treatment satisfaction (Hemo‐SATA,P) and adherence (VERITAS‐Pro) were maintained. TAURUS demonstrated a favourable PK profile of BAY 81‐8973 in comparison with other standard half‐life rFVIIIs and supported the WAPPS PopPK model. No patients developed inhibitors.ConclusionsTAURUS data demonstrate effective prophylaxis with BAY 81‐8973 in the real world without compromising patient satisfaction or adherence.

A Novel Approach for Generating Platelet-Delivered FVIII: Role of Transient LRP1 Expression during Megakaryopoiesis

Human platelets endocytose coagulation factor (F) V into their α-granules, in part, via low-density lipoprotein (LDL) receptor-related protein 1 (LRP1). In contrast to humans, mouse FV is endogenously expressed during megakaryopoiesis and stored in α-granules. The FV-related coagulation factor FVIII is not endogenously expressed in megakaryocytes (Mks) and thought not to be endocytosed by human or murine Mks/platelets. The lack of FVIII endocytosis is surprising as LRP1 on multiple cell types is involved in FVIII uptake and clearance. We and others have shown that FVIII can be ectopically expressed during human or murine megakaryopoiesis and stored in α-granules. These platelets are effective in the delivery of FVIII to sites of vascular injury and improve outcome in hemophilia A (HA) mice even in the presence of circulating inhibitors. Thus, it has been proposed that lentiviral bone marrow gene therapy to ectopically express FVIII during megakaryopoiesis may be a curative strategy especially for patients with HA and intractable inhibitors. However, we have shown that pFVIII has a toxic effect on Mks during intracellular processing in the endoplasmic reticulum/Golgi, limiting platelet yield and pFVIII levels. We have recently shown that exogenous FV can be taken up by in vitro differentiated CD34+- and by induced pluripotent stem cell (iPSC)-derived Mks and asked if the same can be done with FVIII. We found that Mks can take up full-length FVIII (Advate) (Fig. 1A), B-domainless (BD) FVIII (Xyntha, not shown) and BD FVIIIR1645H (not shown), a FVIII mutant we have shown is particularly effective when released by platelets. Uptake is half maximum at 0.25 IU/ml following overnight incubation. Endocytosed FVIII colocalizes with labeled fibrinogen uptake (Fig. 1A), supporting its localization to α-granules, and this FVIII uptake can be blocked by receptor-associated protein (RAP), a blocker of LDL receptor family members, including LRP1 (Fig. 1A) or by including FVIII's carrier protein von Willebrand factor (VWF) (Fig. 1B). To test the biological efficacy of endocytosed pFVIII, we took advantage of our previous studies showing that infused Mks into mice release highly functional platelets after becoming entrapped in the lungs. To focus our studies on the released human platelets and the hemostatic efficacy of the endocytosed pFVIII, we used immunodeficient NOD-scid IL2rgnull (NSG) mice that were also FVIII-deficient and that only expressed mutant VWF that binds human, but not mouse, platelet glycoprotein Ib/IX (VWFR1326H) (N/8/V mice). We infused 1x106 human FVIII-endocytosed Mks into these N/8/V mice resulting in ~1-10% being human platelets in recipient mice. We studied hemostatic efficacy by rotational thromboelastography (ROTEM) and demonstrated that the in vivo-released FVIII from released human platelets within the N/8/V blood corrected hemostasis in this system (Fig. 1C). These FVIII-containing platelets fully corrected clotting as well in a FeCl3 carotid artery injury model (Fig. 1D). In summary, we found that in vitro-grown human Mks can endocytose FVIII from the media into their α-granules in sufficient amounts to have potential clinical application in the care of HA patients. This endocytosis is likely via LRP1. FVIII is not found normally in platelets likely because of a combination of the following: 1) in plasma, FVIII it is bound to VWF, 2) circulating platelets also lack LRP1, and 3) in the marrow, there is little or no free FVIII for endocytosis by LRP1-positive Mks. We propose that endocytosed FVIII by Mks can be an important clinical application of commercial, in vitro-grown Mks for patients with HA and inhibitors who need supplemental long-lasting, hemostatic support to their emicizumab without the thrombotic risks of FVIII-bypassing agents. Disclosures Sabatino: Spark Therapeutics: Patents &amp; Royalties. Camire:Pfizer: Research Funding.

Clotting and chromogenic factor VIII assay variability in post‐infusion and spiked samples containing full‐length recombinant FVIII or recombinant factor VIII Fc fusion protein (rFVIIIFc)

Variability in FVIII measurement is a recognized problem. There are limited data for samples containing recombinant Factor VIII Fc fusion protein (rFVIIIFc). Many studies use samples for which factor concentrate has been spiked into FVIII deficient plasma in vitro. This approach requires validation. Four samples were distributed in a UK National External Quality Assessment Scheme for Blood Coagulation (NEQAS BC) survey. One contained Advate (full-length recombinant FVIII) (rFVIII) added to FVIII deficient plasma, one was from a severe haemophilia A patient after infusion of Advate, one was prepared by addition of rFVIIIFc (marketed as Elocta/Eloctate) to FVIII deficient plasma and the fourth was collected from a severe haemophilia A patient following rFVIIIFc (Eloctate) infusion. Fifty-three haemophilia centres (UK and Scandinavia) performed one-stage FVIII assays and 27 performed chromogenic FVIII assays. One-stage assays gave significantly lower results than chromogenic assays by 7% (P<0.01) and 13%(P<0.001) for post-Advate and Advate spiked samples, and by 22% (P<0.001) and 23% (P<0.001) for post-rFVIIIFc and rFVIIIFc spiked samples. The interlaboratory variation was similar for all samples, with CVs of 12%-16% (chromogenic) and 10%-13% (one stage). The data indicate that either product can be safely monitored by one-stage or chromogenic assay. Spiked samples behaved in a similar way to post-infusion samples for both products and could be substituted for post-infusion samples for use in proficiency testing exercises (ie, samples were commutable).

Bay 81-8973 in the Real World: Clinical Effectiveness and Safety in Patients with Hemophilia Α across the US and Europe

Background: BAY 81-8973 (Kovaltry®, Bayer) is an unmodified full-length recombinant FVIII launched in 2016 in Europe and the US for the prophylaxis and treatment of bleeds in patients with hemophilia A. BAY 81-8973 has been extensively studied in clinical trials, which have demonstrated consistent efficacy and safety in both children and adults in the LEOPOLD trials, and since launch, 6750 patient-years of experience have been accumulated. Pharmacokinetic analyses have demonstrated an increased half-life for BAY 81-8973 compared with standard factor VIII products (rFVIII-FS and rFVIII [rAHF-PFM]). Real world evidence demonstrating the effectiveness and safety of BAY 81-8973 across age groups is being collected, though aggregated international data have yet to be published. The primary objective of this analysis was to describe the effectiveness of BAY 81-8973 in the real world setting in children and adults from Europe and the US, as captured in the Cost of Haemophilia: a Socioeconomic Survey (CHESS) study.Methods: The CHESS 2018 program investigated the economic and psychological burden of moderate and severe hemophilia (FVIII < 5%) in Europe and the USA. The pediatric cohort (CHESS Pediatric) included 1050 males aged 1-17 years, with moderate or severe hemophilia, while CHESS US included 568 adults with severe hemophilia aged > 18 years. Clinical and patient reported data were obtained via medical chart abstraction and cross-sectional surveys sent to both physicians and patients. Data were collected between December 2017 and April 2018, and captured 12 months of clinical retrospective data. The current study is a descriptive analysis of patients treated with BAY 81-8973 from the CHESS pediatric and adult cohorts.Results: At the data cut off (May 2018), 49 patients were being treated with BAY 81-8973. The majority of pediatric patients were aged 6-11 years (51.7%, n=15), while 17.2% (n=5) were aged 0-5 years and 31% (n=9) were aged 12-17 years. In the adult US cohort, most (60%, n=12) were aged 18-35 years, 35% (n=7) were aged 36-59 years, and 5% (n=1) were aged > 60 years. The vast majority of patients across cohorts had severe disease (93.1% of children and 100% of adults); however, 89.7% (26/29) of children and 80% (16/20) of adults had no target joints. In this population, 75.9% (22/29) of children and 55.0% (11/20) of adults were receiving regular prophylaxis with BAY 81-8973. Children and adults on prophylaxis both had a mean (± SD) of 2 (± 1) infusions/week, with median (Q1; Q3) weekly doses of 84 (67; 110) and 62 (29; 144) IU/kg, respectively. Overall, 20.7% (6/29) of children and 35% (7/20) of adults treated with BAY 81-8973 had zero bleeds and mean annualized bleed rate (ABR) was 2.66 (± 2.06) in children and 1.45 (± 1) in adults; 68.9% (20/29) of children and 95.0% (19/20) of adults had experienced ≤ 3 bleeds in the previous year. There were no reports of inhibitor development in either the pediatric or adult cohorts while on BAY 81-8973.Conclusions: These observations suggest that BAY 81-8973 is an effective and well-tolerated treatment for adults and children with moderate and severe hemophilia A, with ABR < 3 in children and < 2 in adults and with many patients free from bleeds despite only 67% of patients studied receiving regular prophylaxis. These data confirm the observations from the LEOPOLD trials, suggest BAY 81-8973 provides good protection from bleeding across a range of patient types in routine clinical practice and illustrate how the established pharmacokinetic profile of BAY 81-8973 translates to clinical benefits for patients with hemophilia A.Study supported by Bayer DisclosuresHirst:Bayer: Employment. Rauchensteiner:Bayer: Employment.

Determinants of Prophylaxis Regimen Choice for Patients with Hemophilia Α Switching to Bay 81-8973: Real World Findings from the Taurus Non-Interventional Study

BACKGROUNDProphylaxis dosing regimens with standard half-life FVIII products range from 2-3 times per week to every other day regimens, and are adjusted according to patient needs. BAY 81-8973 (Kovaltry®, Bayer) is an unmodified full-length recombinant FVIII indicated for prophylaxis and treatment of bleeds in patients with hemophilia A; BAY 81-8973 was launched in 2016 and has since accumulated 6765 patient-years of exposure. The TAURUS study (NCT02830477) was established to investigate BAY 81-8973 prophylaxis use in clinical practice. OBJECTIVETo report reasons for choosing dose regimens at the time of switching to BAY 81-8973 treatment, and patient and disease characteristics associated with choice of less frequent (≤ 2 times per week [≤ 2 xW]) versus more frequent (≥ 3xW) treatment. METHODSTAURUS is an international, open label, prospective, non-interventional, single arm study with a target recruitment of 350 previously treated patients with haemophilia A of all ages with moderate or severe hemophilia A (≤ 5% FVIII:C) with ≥ 50 exposure days to any FVIII who have been recently switched to prophylaxis with BAY 81-8973. At baseline, physicians document clinical information including their reason for choosing a specific prophylaxis regimen. The full study will run until 2020. A scheduled interim analysis (30% of patients recruited) was conducted using a cut-off date of 2 July 2018 to investigate the determinants of choosing a specific regimen when initiating BAY 81-8973. RESULTSAt the cut-off date, 160 patients were enrolled: patient characteristics are shown in the table. For patients in the ≥ 3xW dosing category at baseline (n = 94, 59%), BAY 81-8973 regimens were: every day, n =2; 4xW, n= 2; every other day, n = 24; 3xW, n = 66. for patients dosed ≤ 2xW (n = 52, 33%) were: 2xW, n = 43; every 4th day, n = 1; 1.5xW, n = 1; 1xW, n = 7. Dosing regimen details were missing for 14 patients. Median patient age was 22 years. Pre-study, the majority of patients had been on prophylaxis (97%; 83% on rFVIII-FS). The most common reason for switching to BAY 81-8973 was ‘physician decision’ (in 79 patients, 49%). Most patients (79%) had been receiving BAY 81-8973 for less than 3 months prior to study entry. At baseline, ≥3xW patients were younger than ≤2xW patients (21.5 vs 27.0 years), had been on prophylaxis for a longer period pre-study (13 vs. 9 years), had severe haemophilia (88% vs. 79%), and more had a history of inhibitors (7% vs. 4%). The proportion of patients with ≥1 target joint and bleeding in the year prior to study was similar for both groups at baseline. The reasons for prescribing the specific dosing frequencies are shown in the table. ‘Patient/caregiver preference’ and ‘bleeding history’ were cited less frequently for ≤2xW patients, while ‘current treatment regimen’, ‘adherence/compliance history’, and ‘activity level’ were cited more frequently for ≤2xW patients vs ≥3xW patients. No recruited patients developed inhibitors with BAY 81-8973. CONCLUSIONSThese real-world data from 160 patients show patients treated more frequently (≥3xW) with BAY 81-8973 were younger and had a longer prophylaxis treatment history than those treated less frequently ≤2xW. Patient preference, bleeding history, and current regimen, were the most frequently cited reasons for choosing a specific BAY 81-8973 regimen. Taken together, the data indicate that BAY 81-8973 treatment may be successfully individualized according to patient need and disease characteristics. DisclosuresMaes:Bayer: Honoraria. Rauchensteiner:Bayer: Employment. Wang:CSL Behring: Consultancy; Terumo BCT: Other: CPC Clinical Research; Bayer, Novo Nordisk, Octapharma, Genentech, HEMA Biologics, Shire, CSL Behring: Honoraria; Bayer, Bioverative, Novo Nordisk, Octapharma, Shire, Genentech, Biomarain, Pfizer, CSL Behring, HEMA Biologics, Daiichi Sankyo: Research Funding; Novo Nordisk: Consultancy; Bayer: Consultancy.

Straightforward Transition to Bay 81-8973 Prophylaxis in Patients with Hemophilia A: Prospective Real-World Data from the Taurus Non-Interventional Study Show That Number of Infusions per Week Is Maintained or Reduced

BACKGROUNDBAY 81-8973 (Kovaltry®, Bayer) is an unmodified full-length recombinant FVIII indicated for prophylaxis and treatment of bleeds in patients with hemophilia A; BAY 81-8973 was launched in 2016 and has since accumulated 6765 patient-years of exposure. The TAURUS study (NCT02830477) was established to investigate BAY 81-8973 prophylaxis dosing regimens chosen in clinical practice and confirm the established safety and efficacy results from the LEOPOLD clinical trials in a real world setting. OBJECTIVESTo analyse the proportion of patients on specific BAY 81-8973 prophylaxis regimens, bleeds, and patient-reported outcomes at baseline and most recent follow-up. METHODSTAURUS is an international, open label, prospective, non-interventional, single arm study with a target recruitment of 350 previously treated patients with hemophilia A of all ages with moderate or severe hemophilia A (≤ 5% FVIII:C) with ≥ 50 exposure days to any FVIII product who have been switched to prophylaxis with BAY 81-8973. At baseline, physicians document clinical information including age, BMI, severity of hemophilia, number of target joints, prior treatment regimen, bleed history, inhibitor history, and reason for choosing a specific prophylaxis regimen. Patients/caregivers reported bleeds in ongoing patient diaries, and completed questionnaires on treatment satisfaction (HEMOSAT) and adherence (VERITAS-PRO) at baseline and follow-up. A scheduled interim analysis (30% of patients recruited) was conducted with data collected up to 2 July 2018. RESULTSAt the cut-off, 160 enrolled patients were included in the baseline analysis set, of whom 89 had ≥ 6 months of follow-up data available (median observation period 201 days), 33% of whom had completed one year of the study. Median (range) patient age was 22 (2‒69) years, time since diagnosis was 15 (0.5‒64) years, and most patients (76/89, 85%) had baseline FVIII level of <1%. Treatment assignments are shown in the table. All patients had received pre-study prophylaxis, for a median of 15 years, with 66% of patients using rFVIII-FS as their most recent FVIII treatment prior to BAY 81-8973. Most (91%) had been treated with BAY 81-8973 for <3 months prior to study entry. Pre-study, 72% of patients were treated ≥3 times per week (xW). At baseline, most patients (59%) were assigned treatment ≥3xW (every day, 1%; every other day, 16%; 3xW, 41%). The majority remained on their previous regimen (78% on ≤2xW and 97% on ≥3xW); any changes were mainly a reduction in frequency on BAY 81-8973 vs previous treatment (22%), with only 2% increasing frequency on BAY 81-8973. At last follow up, most patients remained on the same regimen: 60% on ≥3xW (≥ every other day, 17%; 3xW, 42%). Most patients (92%) did not alter their dosing frequency. Of the 8% who changed dosing frequency, the majority (6 patients) changed from ≥3xW to ≤2xW; 1 patient changed from ≤2xW to ≥3xW. The median prescribed weekly dose was 52 IU/kg (64 IU/kg for ≥3xW and 43 IU/kg for ≤2xW) on study, slightly lower than those with previous product: 56 IU/Kg overall, 64 IU/kg for ≥3xW and 50 IU/kg for ≤2xW. Median (Q1; Q3) patient diary-reported annualized joint bleed rates were 1.5 (0.0; 5.3), 1.2 (0.0; 5.3) and 1.4 (0.0; 6.1); for ≤2xW, ≥3xW, and all patients, respectively. HEMO-SAT and VERITAS-PRO data will be presented in the poster. No recruited patients developed inhibitors with BAY 81-8973. CONCLUSIONSThese real-world data from 89 patients show that the range of dosing options available for BAY 81-8973 allowed the majority of patients to become established quickly on this treatment upon switching. In the few instances where patients changed dosing frequency either upon switching to BAY 81-8973 or once established on treatment, most moved to less frequent treatment. Joint bleeding rates confirm and extend findings from the clinical trials and speak to effective bleeding prophylaxis with BAY 81-8973 in a real-world setting. Therefore, BAY 81-8973 treatment may be successfully individualized according to patient need and disease characteristics. DisclosuresWang:Terumo BCT: Other: CPC Clinical Research; CSL Behring: Consultancy; Bayer, Bioverative, Novo Nordisk, Octapharma, Shire, Genentech, Biomarain, Pfizer, CSL Behring, HEMA Biologics, Daiichi Sankyo: Research Funding; Bayer: Consultancy; Bayer, Novo Nordisk, Octapharma, Genentech, HEMA Biologics, Shire, CSL Behring: Honoraria; Novo Nordisk: Consultancy. Maes:Bayer: Honoraria. Rauchensteiner:Bayer: Employment.

Elevated Von Willebrand Factor May Abrogate Bleeding Phenotype in a Male with a Non-Null F8 Mutation

Introduction: Although FVIII activity (FVIII:C) correlates with phenotype in most Hemophilia A males, some have bleeding phenotypes milder than predicted by FVIII:C. Analysis of these individuals may provide additional insight into the relationship between F8 genotype and bleeding phenotype, identify additional factors that modulate FVIII, refine the classification of Hemophilia A and improve clinical management.We present a male subject who carries a c.5999G>C mutation in F8. This mutation has been reported in an individual with a severe bleeding phenotype in the EAHAD F8 Variant Database (www.factorviii-db.org). Our subject was serendipitously diagnosed at age 80, and has never had abnormal bleeding despite numerous challenges, including multiple dental extractions, adult circumcision, internal hemorrhoid ligation and cardiac catheterization. In an effort to resolve this genotype-phenotype discrepancy, we performed FVIII and von Willebrand Factor (VWF) assays, quantitated the extent of alternative splicing in F8, and screened for mutations in additional genes known to impact hemostasis. We validated a partial splicing defect that is consistent with mild Hemophilia A, but surprisingly cannot explain the lack of a bleeding phenotype.Methods: Peripheral blood was tested by one-stage FVIII:C and chromogenic FVIII assays, FVIII antigen ELISA, VWF antigen and activity assays, and von Willebrand Disease (VWD) Type 2 Normandy binding assay.To evaluate F8 splice variants, quantitative RT-PCR (qRT-PCR) was performed on RNA isolated from peripheral blood using primers that distinguish FVIII isoforms. Samples were tested in quadruplicate, with sample input normalized relative to GAPDH. Screening for exonic mutations in F8, F5, F2 and VWF genes was undertaken by Next Generation Sequencing (NGS).Results and Conclusions: In contrast to the previously reported severe c.5999G>C patient, this subject has reduced FVIII activity consistent with mild Hemophilia A measured by both FVIII activity assays. FVIII antigen assessment by ELISA gave similar results (0.35 IU/mL).To better understand FVIII:C levels in this individual, we sought to characterize F8 transcript isoforms. The c.5999G>C resides within the splice acceptor site of exon 19 and may result in two FVIII isoforms. One isoform produces a full-length FVIII containing a missense mutation (p.Gly2000Ala). The second isoform skips the 117 bp exon 19, but is predicted to retain an open reading frame; however, its location in the A3 domain likely disrupts secretion or function (Donadon et al., Haematologica 2018). The relative abundance of F8 variants evaluated by qRT-PCR demonstrated that despite a modest overall increase in F8 transcript relative to a normal control (1.6 fold higher expression of F8 at exon 18), the predominant isoform in this subject lacks exon 19, resulting in substantial depletion of the full-length isoform (0.3 relative to a normal control). These data indicate the full-length protein likely retains function, with transcript levels that explain residual FVIII activity.In an effort to explain absent bleeding phenotype, NGS was performed. The c.5999G>C F8 mutation was confirmed, while the thrombophilic Factor V Leiden and Prothrombin G20210A mutations were excluded. Sequencing also excluded mutations implicated in Type 2 Normandy VWD. Additional clinical testing may provide an explanation. VWF was elevated as indicated by VWF antigen and Ristocetin cofactor activity (>400%, >100%, respectively). Furthermore, VWF:FVIII binding activity by ELISA showed a higher than normal binding ratio (>1.42). VWF multimer analysis indicated normal distribution. Therefore, we propose elevated VWF abrogates this subject's bleeding phenotype.Altogether, our study expands our understanding of the functional consequences of this mutation by demonstrating that this mutation can result in FVIII levels predicting mild Hemophilia A. This case also underscores the complexities of predicting phenotype for mutations that result in partial splicing defects. Perhaps more intriguing is the lack of bleeding phenotype in this elderly individual with numerous hemostatic challenges, that might be related to increased VWF. These data support additional studies investigating the role of VWF in ameliorating bleeding risk in Hemophilia A. DisclosuresNo relevant conflicts of interest to declare.

Missense mutations near the N-glycosylation site of the A2 domain lead to various intracellular trafficking defects in coagulation factor VIII

Missense mutation is the most common mutation type in hemophilia. However, the majority of missense mutations remain uncharacterized. Here we characterize how hemophilia mutations near the unused N-glycosylation site of the A2 domain (N582) of FVIII affect protein conformation and intracellular trafficking. N582 is located in the middle of a short 310-helical turn (D580-S584), in which most amino acids have multiple hemophilia mutations. All 14 missense mutations found in this 310-helix reduced secretion levels of the A2 domain and full-length FVIII. Secreted mutants have decreased activities relative to WT FVIII. Selected mutations also lead to partial glycosylation of N582, suggesting that rapid folding of local conformation prevents glycosylation of this site in wild-type FVIII. Protease sensitivity, stability and degradation of the A2 domain vary among mutants, and between non-glycosylated and glycosylated species of the same mutant. Most of the mutants interact with the ER chaperone BiP, while only mutants with aberrant glycosylation interact with calreticulin. Our results show that the short 310-helix from D580 to S584 is critical for proper biogenesis of the A2 domain and FVIII, and reveal a range of molecular mechanisms by which FVIII missense mutations lead to moderate to severe hemophilia A.

Potential role of a new PEGylated recombinant factor VIII for hemophilia A.

Hemophilia A, a deficiency in the activity of coagulation factor (F) VIII, is an X-linked bleeding disorder with an approximate incidence of one in 5,000 male infants. Bleeding-related complications often result in greater severity of disease, poor quality of life, surgical interventions for severe joint destruction, and shortened life span. With the availability of plasma-derived and recombinant FVIII products, the benefits of primary prophylaxis were demonstrated and is now the standard of care for patients with severe factor deficiencies. Current hemophilia research is focusing on the creation of new factor replacement therapies with longer half-lives; accessing alternative mechanisms to achieve desired hemostasis and enhance bypassing activity; and limiting the immunogenicity of the protein. PEGylation involves the covalent attachment of polyethylene glycol (PEG) to a protein, peptide, or a small molecule drug. PEG effectively increases the molecular weight and size of the protein by creating a hydrophilic cloud around the molecule. This molecular change may reduce susceptibility of the molecule to proteolytic activity and degradation. It is also believed that PEGylation changes the surface charge of the protein that ultimately interferes with some receptor-mediated clearance processes. The half-life of PEGylated factor is more prolonged when compared to non-PEGylated full-length recombinant FVIII. The dawn of a new era in the care of hemophilia patients is upon us with the release of recombinant FVIII products with extended half-lives, and products with even more extended half-life will become available in a very short time. With all the promise of these new agents, many questions still remain.

Prospective evaluation of treatment regimens, efficacy and safety of a recombinant factor VIII concentrate in haemophilia A: the German EffeKt study.

Collecting clinical data under routine conditions remains important to monitor continuously efficacy and tolerability of an established product. This prospective observational study aimed at evaluating efficacy and tolerability of full-length sucrose-formulated recombinant FVIII (rFVIII-FS; KOGENATE(®) Bayer/KOGENATE(®) FS) in routine use and analysing concomitant diseases and medication. Haemophilia A patients treated with rFVIII-FS were followed up for 24 or 36 months. Efficacy, tolerability, concomitant medication and diseases were assessed at yearly intervals. Two hundred and twenty-one documented patients were mainly pretreated, predominantly with prophylaxis (74%). On study, 54 (31%) patients in the efficacy set (n = 174) documented regular prophylaxis, 91 patients (52.3%) failed to document their prescribed prophylactic infusions, 25 (14.4%) received on-demand treatment and four patients (2%) inhibitor adapted therapy. Patients on regular prophylaxis reported 8.4% of infusions for bleeding treatment and on-demand patients reported 55.3%. Young patients experienced mainly trauma-related bleedings and older patients reported spontaneous bleedings. Joint status was good overall. Median annual spontaneous joint bleeding rate in all groups was zero. A total of 79.0% of all bleedings were successfully treated with one or two infusions. Degenerative arthropathy and arterial hypertension were the most prominent concomitant diseases. Eighty-seven patients (40.5%) took at least one concomitant drug on study, mostly drugs for pain treatment related to haemophilic arthropathy. Two patients presented with positive inhibitor titres on study, one of them was a previously untreated patient and the other had a positive inhibitor history. This study corroborates the tolerability and efficacy profile of rFVIII-FS.