Volume 11 - Issue 1, January 2016

Second Spring Forum Haemophilia 2015 Back

Meeting Highlights

The 2nd Spring Forum Haemophilia 2015 – ‘New Avenues in Human rFVIII Replacement: Perspectives and Challenges for the Years Ahead’– was held in Berlin, Germany, on 17 and 18 April 2015. The two-day educational meeting brought together an internationally renowned faculty and 270 participants from around the world to discuss current and future challenges in the treatment of haemophilia A, with a focus on new-generation recombinant FVIII (rFVIII) products, including Nuwiq® (Human-cl rhFVIII; simoctocog alfa). The Forum comprised three scientific sessions:

  • FVIII physiology, post-translational modifications (PTMs) and von Willebrand factor (VWF) interaction.
  • Inhibitor development in haemophilia A: Can the risks be minimised?
  • Opportunities and challenges in pharmacokinetic (PK)-guided personalised prophylaxis.

Dr Robert KlamrothThe meeting chairman, Dr Robert Klamroth, set the scene for the scientific programme by discussing the unmet needs in haemophilia A from a patient and clinical perspective. Patients demand confidence that their treatment will effectively prevent and treat bleeds, provide flexibility and convenience to fit into their lifestyle, and will not cause side effects or development of FVIII inhibitors. Important challenges from a clinical perspective include:

  • Development of inhibitors, particularly in previously untreated patients (PUPs).
  • Need for frequent venous access for FVIII injection.
  • Limitations of FVIII half-life due to its dependency on the half-life of its carrier protein, VWF.
  • Inefficiency of a ‘one-sizefits- all’ strategy for prophylaxis.
  • The need for product-specific assays for novel rFVIII products.

In Europe, rFVIII products produced in hamster cells have been available for the treatment of haemophilia A for over 20 years. With the approval of Nuwiq® by the European Medicines Agency (EMA) in July 2014, the first rFVIII produced in a human cell line became available for the treatment of haemophilia A in Europe. Nuwiq® is a new-generation rFVIII, without chemical modification or fusion with any other protein, produced in a human cell line cultured without additives of human or animal origin.1–3 The vision for Nuwiq® is to provide patients with a rFVIII product that has a reduced immunogenic potential in PUPs and that can enable personalised prophylaxis with the possibility of fewer injections (Figure 1).

FVIII physiology, PTMs and VWF interaction

Claude Négrier (Chair), Cécile Denis, Peter J. Lenting and Christoph Kannicht

Dr Claude NégrierDr Claude Négrier introduced the session with an overview of FVIII biology. FVIII is a crucial cofactor in the intrinsic coagulation pathway and circulates in the blood in a complex with VWF.4,5 FVIII is a ~300 kDa heterodimeric protein that undergoes extensive post-translational modifications (PTMs), including glycosylation and tyrosine sulphation.3–5 Sulphated residues play an important role in FVIII activation in the coagulation pathway and in the interaction between FVIII and VWF.4–6 Glycosylation alters the structural and functional properties of a protein by modulating physicochemical properties, immunological properties, receptor binding/ affinity and intracellular sorting.6 Approximately 95% of FVIII in the bloodstream is bound to VWF (Figure 2), which stabilises FVIII by preventing proteolytic degradation and considerably prolongs FVIII survival.5,7 The FVIIIVWF complex is thought to be mainly eliminated from the bloodstream by the specialised clearance receptor LRP (lowdensity lipoprotein receptor-related protein) located on hepatocytes and macrophages.5,8

Dr Cécile DenisDr Cécile Denis focused on the underlying mechanisms of VWF clearance. VWF has a dual role in haemostasis, as a carrier protein for coagulation FVIII in the circulation and in the recruitment of blood platelets to sites of vascular injury.8 VWF clearance is a complex process that is modulated by numerous cells and cell-surface receptors (Figure 3). Mouse models9–11 and genetic screening in population-based studies12 have identified LRP-1 as an important contributor to VWF, and consequently FVIII clearance.8 Other receptors that play a role in VWF clearance include CLEC4M, Siglec-5 and the asialoglycoprotein receptor (ASGPR).8 VWF clearance is also modified by factors such as reduced VWF glycosylation/ sialylation and blood group.8 Translating preclinical knowledge of VWF clearance into a therapeutic perspective will be a significant challenge as targeting a single receptor/mechanism involved in the complex VWF clearance process is unlikely to be successful.

We are only beginning to understand which receptors are involved in VWF clearance, and the interplay between these receptors remains to be determined.
Cécile Denis

Dr Peter J. LentingDr Peter J. Lenting further explored the complex relationship of VWF and FVIII. The VWF-FVIII complex combines two independent proteins with different haemostatic functions; FVIII acts as a cofactor for FIXa to promote formation of fibrin networks, whereas VWF plays a role in the formation of platelet-rich thrombi.4,7,13 Nevertheless, a number of factors support the hypothesis that VWF determines FVIII survival: (1) equilibrium dynamics force > 95% of FVIII in complex with VWF (Figure 2); (2) pre-infusion levels of VWF and VWFpropeptide can be used to predict FVIII half-life;14 (3) VWF and FVIII are targeted to the same macrophages following infusion.15 These factors may explain the limited ~1.5-fold half-life extension of modified rFVIII variants.16 As VWF and FVIII are cleared by a similar repertoire of scavengerreceptors (Figure 4) and the majority of FVIII is present in the FVIII-VWF complex, it may prove difficult to significantly extend FVIII half-life by modifying FVIII alone. In a haemophilia mouse model,17 pegylation of VWF was shown to prolong the half-life of PEG-VWF, but not FVIII, due to rapid redistribution of FVIII to endogenous wild-type VWF. It is therefore unlikely that this strategy will be successful in a clinical setting.

To really improve FVIII half-life, there is a need for a FVIII molecule that is cleared independently of endogenous VWF.
Peter Lenting

Haema_Octapharma_01_2016 KannichtDr Christoph Kannicht focused on the contribution of PTMs to FVIII quality. All plasma proteins undergo biochemical modifications after their translation from DNA to an amino acid sequence. These PTMs are species specific, and different expression systems (e.g., human or hamster cell lines) produce different PTMs for the same protein / amino acid sequence.3,6,18 Sulphation and glycosylation are of greatest importance for FVIII quality, with sulphation determining VWF binding and FVIII activity and glycosylation influencing its immunogenic properties.3,6 Tyrosine sulphation at six sites of the FVIII molecule is required for full FVIII activity.6,19 Sulphation at Tyr1680 is required for the interaction between FVIII and VWF whereas tyrosine sulphation at other sites within FVIII affects the rate of cleavage by thrombin at the respective thrombin cleavage sites.6,19 Nuwiq® has been shown to be fully sulphated at Tyr1680 and therefore to have a higher VWF binding affinity than comparator rFVIII products (Figure 5).3 As Nuwiq® is produced in a human cell line, it contains only human glycans and is devoid of immunogenic glycan epitopes, such as N-glycolylneuraminic acid or Gala1-3Galb1-GlcNAc-R (a-Gal), present in rFVIII products derived from hamster cell lines.3 A combination of higher VWF affinity and absence of antigenic glycans has the potential to optimise FVIII half-life towards VWF half-life and to reduce the overall immunogenic challenge.

The quality of rFVIII depends on accurate post-translational sulphation and glycosylation.
Christoph Kannicht

Inhibitor development in haemophilia A: Can the risks be minimised?

Antonio Coppola (Chair), Susanne Holzhauer and Sigurd Knaub

Dr Antonio CoppolaDr Antonio Coppola discussed the impact of inhibitor incidence in PUPs with haemophilia A. With the availability of effective and pathogen-free replacement FVIII products, the development of FVIII inhibitors is currently the most serious treatment-related clinical problem in the management of haemophilia A. Inhibitor development has major adverse implications for bleeding rates, morbidity, mortality, quality of life and treatment costs.20-24 FVIII inhibitors arise in patients with haemophilia A throughout life with a bimodal risk, being greatest in early childhood and, with a lower incidence peak, in old age.25 Approximately one-third of PUPs with severe haemophilia A develop inhibitors (~60% persistent and high-titre) and are at maximal risk during the first 20 exposure days (EDs).26,27 Inhibitor development is mediated by a complex interaction of currently unmodifiable hostrelated factors, such as haemophilia severity, family history, ethnicity, F8 genotype28-30 and potentially modifiable treatment- related factors, such as treatment intensity, FVIII dose, treatment regimen and product type. Additionally, the environment in which these interactions occur, including immunologic challenges, severe bleeding, infection and vaccination, needs to be considered.26,27,31,32 The influence of FVIII product type (plasma-derived FVIII [pdFVIII] vs rFVIII) on the incidence of inhibitors has been intensely debated over the past decade. Some studies33-35 and meta-analyses36,37 have reported an increased inhibitor risk with rFVIII compared with pdFVIII, whereas other studies26,27,38 and meta-analyses39,40 have reported no difference in inhibitor risk. Comparisons of inhibitor risk have been limited by the heterogeneity of patient populations (disease severity, gene mutation type, ethnicity and therapy regimen) and study design (retrospective vs prospective, frequency of inhibitor testing and length of the observation period). In the most recent attempt to overcome some of these methodological limitations, Marcucci and co-workers41 performed a meta-analysis based on individual patient data for 761 PUPs with severe or moderate haemophilia. Unadjusted inhibitor rates were higher in patients treated with rFVIII compared with patients treated with pdFVIII (40% vs 22%). However, this difference did not persist after adjustment for confounding factors. Following the publication of the ‘Research Of Determinants of INhibitor development’ (RODIN) study,27 the debate surrounding the influence of product type on inhibitor rates has extended to the influence of different rFVIII products. The RODIN study evaluated 574 consecutive patients with severe haemophilia A at 29 haemophilia centres in Europe, Israel and Canada. It was reported that a second-generation full-length rFVIII was associated with an increased risk of inhibitor development in PUPs compared with a thirdgeneration full-length rFVIII.27 The adjusted hazard ratio (HR) was 1.60 (95% confidence interval [CI]: 1.08, 2.37) for all inhibitors and 1.79 (95% CI: 1.09, 2.94) for high-titre inhibitors. Subsequent retrospective analyses from the United Kingdom42 and France43 supported the findings of the RODIN study.27 In contrast, the patientlevel meta-analysis by MarMarcucci and co-workers41 and the 4-year, prospective European Haemophilia Safety Surveillance (EUHASS) report38 did not support a difference in inhibitor risk with different rFVIII products. Whether pdFVIII or rFVIII, or individual rFVIII, products are associated with an increased inhibitor risk has not been conclusively established due to heterogeneity between studies and the lack data from randomised trials. The ongoing ‘Study on Inhibitors in Plasma-Product Exposed Toddlers’ (SIPPET)44, a randomised, investigatordriven, worldwide, prospective, open-label clinical trial in 300 PUPs or minimally treated patients with severe haemophilia A, will provide important data on inhibitor rates by product type. The results of this study are eagerly awaited.

Inhibitor development is the most important clinical problem in the management of haemophilia A.
Antonio Coppola

Dr Sigurd KnaubDr Sigurd Knaub gave an update on the NuProtect (GENA-05) study. Current rFVIII products produced in hamster cell lines are associated with a cumulative incidence of inhibitors of up to 38% for all inhibitors and up to 25% for high-titre inhibitors in PUPs.27 Thus, investigating the immunogenicity of Nuwiq® in PUPs is of considerable importance and it was a requirement of the EMA to initiate a PUP study before submitting a marketing authorisation application for Nuwiq® in Europe. NuProtect is a prospective, multicentre, multinational, open-label, non-controlled, phase III study in PUPs with severe haemophilia A (FVIII:C < 1%), i.e., those at highest risk of developing inhibitors. The study was initiated in 2013 and is being conducted in 18 countries and 47 centres worldwide. It is planned to enrol 100 PUPs of all ethnicities who will be under observation for their first 100 EDs or a maximum study participation of 5 years. NuProtect will investigate immunogenicity (primary objective), efficacy (during prophylaxis, treatment of breakthrough bleeding and during surgery), safety and tolerability. The trial will also look at health economic modelling analysis with resource use parameters. Intensive screening for inhibitors will take place every 3–4 EDs until 20 EDs, then every 10–12 EDs until 100 EDs, and then every 3 months until study completion. In addition, a number of optional substudies will be performed to identify predictive markers of inhibitor development. These sub-studies include epitope mapping, immunogenotyping, in vitro immunogenicity and RNA expression profiling.6,18 As of 13 April 2015, 42 of 47 centres in 16 of 18 countries have initiated the study. Of the planned 100 PUPs, 65 have been enrolled and 56 of these have begun treatment with Nuwiq®.

Opportunities and challenges in PK-guided personalised prophylaxis

Johannes Oldenburg (Chair), Gerry Dolan, Robert Klamroth, Michaela Praus and John Pasi

Dr Gerry DolanDr Gerry Dolan began the session with an overview of new-generation rFVIII concentrates. The new-generation rFVIII products include several modified FVIII products (not yet approved in Europe) and Nuwiq®, a rFVIII produced in a human cell line without chemical modification or fusion with any other protein,1-3,6,18 which was approved in Europe in July 2014 and subsequently approved in Canada and Australia. Clinical trials with modified rFVIII in previously treated patients (PTPs) have demonstrated effective control of bleeding together with an approximate 1.5-fold extension in half-life that appears to be generally consistent across products.16,46 Based on the one-stage FVIII assay, reported mean half-lives for the modified rFVIII products in adults range from ~14 hours to ~19 hours.45-49 Half-life data of modified rFVIII products in children are currently available only for rFVIIIFc with a mean half-life range from 12 hours in children aged 2 to 5 years to 14.6 hours in children aged 6 to 11 years.53 The relatively limited success in extending halflife through FVIII modification may be related to the fact that FVIII half-life is strongly influenced by the half-life of its carrier protein, VWF (half-life ~16–17 hours), and it is likely that most patients require at least twice weekly treatment with modified rFVIII products.16,47 There are a number of open issues that need to be addressed with new modified rFVIII products, such as longterm safety, immunogenicity in PUPs, the optimum methods for monitoring, including the need for product-specific standards, and cost effectiveness.16 Regulatory approval of Nuwiq® was based on clinical data in 135 adult and paediatric PTPs with severe haemophilia A enrolled primarily in the pivotal GENA-01, GENA-08 and GENA-03 clinical studies. In these clinical studies, Nuwiq® was effective in preventing and treating bleeds and no patients developed FVIII inhibitors or experienced treatment-related serious or severe adverse events (Figure 6).6,18 Additionally, the NuPreviq (GENA-21) study evaluating personalised prophylaxis with Nuwiq® in 66 PTPs has subsequently completed and the NuProtect study in PUPs is ongoing.6,18 With the completion of NuPreviq, there have been no cases of inhibitor development in more than 200 PTPs treated with Nuwiq®. In the context of new-generation rFVIII products, it is notable that Nuwiq® achieves a half-life of 15.1 to 17.1 hours in adults55,57 and a half-life of 11.9 to 13.1 hours in children,6,18 without chemical modifications or fusion with any other protein (Figure 7).1-3

Produced by human cells with a halflife of 15 to 17 hours, without any major modification, Nuwiq® may represent a significant clinical improvement.
Gerry Dolan

Dr Robert KlamrothDr Robert Klamroth presented final data of the NuPreviq (GENA-21) PK-guided prophylaxis study with Nuwiq®. NuPreviq was a prospective, open-label, multicentre phase IIIb study investigating the efficacy and safety of individualised, PK-guided prophylaxis with Nuwiq® in 66 adult (≥18 years) PTPs with severe haemophilia A. Patients were enrolled at 20 centres in 8 European countries. The study consisted of three phases: (1) initial PK evaluation phase; (2) a 1–3 month standard prophylaxis treatment phase; and (3) a 6-month PK-guided personalised prophylaxis phase. The prophylactic dose and dosing interval recommended for the personalised prophylaxis phase was based on the analysis of individual PK data obtained at the initial PK evaluation. Final data56 have been analysed for 66 evaluable adult PTPs (mean age 33.6 ± 9.9 years) with severe haemophilia A. Baseline mean haemophilia joint health score was 37.4 ± 25.3 and the prior mean annualised bleeding rate (ABR) was 38.9 ± 27.6, which indicates a very severe, poorly controlled patient population. During 6 months of personalised, PK-guided prophylaxis with Nuwiq®, median ABR was 0 (Mean ± SD: 1.45 ± 3.51) and 73% of patients did not experience a single bleed (Figure 8). Mean half-life was 15.1 ± 4.7 hours (one-stage assay). The median dosing interval was 3.5 days and the frequency of infusions was ≤ 2 per week for 58% of patients. In addition, dose requirement in the final two months of prophylaxis was 10% lower than in the standard prophylaxis phase. There were no cases of FVIII inhibitor development or treatment-related serious adverse events during the study. An ongoing, open-label, prospective, multicentre, multinational, non-interventional study (NIS) [NIS-NuPreviq] is evaluating personalised prophylaxis with Nuwiq® in routine clinical practice. A total of 50 patients are planned to be enrolled in Germany (already initiated) and 200 patients are planned worldwide.

We now have a solid base of clinical data and clinical experience that replacement FVIII can prevent and treat bleeds, so we can now take into account individual patient needs.
Robert Klamroth

Michaela PrausMichaela Praus provided further details of the PK analyses performed in the NuPreviq study. In the initial PK evaluation phase of NuPreviq, 60 ± 5 IU/kg (labelled potency) of Nuwiq® was administered to 66 patients after a 96 hour wash-out period. Blood samples were taken before infusion and at 0.5, 1, 3, 6, 9, 24, 30, 48 and 72 hours after end of infusion. All laboratory analyses were performed in the central laboratory and PK calculations were based on the one-stage assay. PK parameters calculated from data obtained in the PK evaluation phase were used to determine the individual PK-tailored dose and dosing interval for personalised prophylaxis. The goal was to determine the maximum prophylactic dosing interval that was capable of maintaining a FVIII trough level of ≥ 0.01 IU/mL, with a dose of not more than 60–80 IU/kg and a concentration at the end of infusion of < 2.0 IU/mL. The dose and dosing frequency opoptions that could achieve this goal were determined by applying a 1-compartment or 2-compartment PK model to data for each patient individually. A 1-compartment model assumes that the distribution of infused FVIII is restricted to a single central intravenous body compartment, whereas a 2-compartment model assumes that distribution within a central intravenous compartment is influenced by distribution to and from a peripheral compartment. PK data for 30 patients (45.5%) were best fitted by a 1-compartment model and PK data for 36 patients (54.5%) were best fitted by a 2-compartment model (Figure 9).

Johannes Oldenburg

We are entering an era where we can personalise haemophilia A treatment.
Johannes Oldenburg






Dr John PasiDr John Pasi presented his insight into possible future developments in personalised prophylaxis. Currently accepted targets are to maintain a minimum FVIII:C level of at least 1 IU/dL, convert severe to moderate disease, reduce the number of haemorrhages, prevent or delay arthropathy, protect from joint deterioration and improve quality of life. Historically, prophylaxis treatment has been based on the broad assumption that FVIII has a half-life of ~12 hours and should be given at a dose in the range 25–40 IU/kg on alternative days. However, many patient-, treatment- and product-related factors can influence therapy and therefore limit the effectiveness of a onesize- fits-all treatment strategy.57 For example, FVIII half-life varies between patients, ranging from 8 to 25 hours in one study,58 and between products.45 A more personalised approach to prophylaxis can embrace variation and adapt treatment to an individual’s lifestyle. The aim of personalised prophylaxis is to reduce the bleeding rate, reduce the number of infusions and reduce the FVIII consumption. An ideal treatment would be an individualised optimal balance of the three interacting aspects of bleeding rate, FVIII dose and dosing frequency, with the aim to keep the bleeding rate as low as possible. Until very recently, personalised prophylaxis has been limited to retrospective dosing adjustments in response to observed bleeding patterns and clinical response to treatment (clinical approach). In contrast, a PK-guided approach provides an opportunity to pro-actively set the dose and frequency to achieve a chosen trough FVIII level based on PK parameters. PK-guided approaches can be based on individual PK parameters (NuPreviq study)6,18 or population PK models.58 Given the large inter-patient variability in FVIII PK parameters,6,18,58 an approach based on a patient’s individual PK parameters would be expected to be more accurate than an approach based on average PK parameters of a wider population of patients. Dr Pasi described two patient cases to illustrate the key points of PK-tailored prophylaxis in the NuPreviq study (Figures 10 and 11). A one-size-fits-all approach is clearly not an ideal treatment approach for haemophilia A. In the coming years, a PK-guided personalised prophylaxis approach has the potential to improve patient care by optimising treatment to meet individual needs (e.g., different activities, lifestyles). Furthermore, such an approach could enable greater patient engagement and greater awareness of the importance of adherence to therapy.

We now have a solid base of clinical data and clinical experience that replacement FVIII can prevent and treat bleeds, so we can now take into account individual patient needs.
John Pasi


Writing support was provided by nspm ltd, Meggen, Switzerland, and funded by Octapharma AG, Lachen, Switzerland.


Antonio Coppola: research support (Bayer), consultancy (Bayer, Kedrion), speakers bureau (Novo Nordisk, Octapharma), scientific advisory boards (Bayer, Baxter, SOBI). Cécile Denis: travel support and honoraria (Octapharma). Gerry Dolan: research support (Baxter, Pfizer), travel support (Bayer, Pfizer), speakers bureau (Baxter, Bayer, Novo Nordisk, Octapharma, Pfizer, SOBI), scientific advisory boards (Baxter, Bayer, Biogen, Novo Nordisk, Octapharma, Pfizer). Christoph Kannicht is an employee of Octapharma Biopharmaceuticals GmbH, Berlin, Germany. Robert Klamroth: research support (Baxter, Bayer, CSL Behring, Novo Nordisk, Pfizer), travel support and speakers bureau (Baxter, Bayer, Biogen Idec, Biotest, CSL Behring, Grifols, Novo Nordisk, Octapharma, Pfizer, SOBI), scientific advisory boards (Baxter, Bayer, CSL Behring, Novo Nordisk, Pfizer, SOBI). Sigurd Knaub is an employee of Octapharma AG, Lachen, Switzerland. Peter Lenting: research support and consultancy (Novo Nordisk), travel support, honoraria (Octapharma). Claude Négrier: research support (Alnylam, CSL Behring, Novo Nordisk, Octapharma, Pfizer), travel support (Bayer, Novo Nordisk, Octapharma), scientific advisory boards (Baxter, Bayer, CSL Behring, LFB, Novo Nordisk, Octapharma, SOBI). Johannes Oldenburg: research support (Baxter, Bayer, Biotest, CSL Behring, Grifols, Novo Nordisk, Octapharma, Pfizer, SOBI) travel support, consultancy, honoraria and scientific advisory boards (Baxter, Bayer, Biogen Idec, Biotest, CSL Behring, Grifols, Novo Nordisk, Octapharma, Pfizer, SOBI), patents (Baxter). John Pasi: research support (Octapharma), travel support (Biogen, Octapharma, Pfizer, SOBI), honoraria (Novo Nordisk), scientific advisory boards (Bayer, Biogen, Octapharma, Pfizer, Sanofi, SOBI). Michaela Praus: supports Octapharma projects as employee of the CRO Accovion GmbH. Accovion is a service provider for Octapharma.

Speakers and chairs in alphabetic listing

Antonio Coppola
Federico II University
Department of Clinical Medicine & Surgery
Naples (Italy)

Cécile Denis
Institut National de la Santé et de la Recherche Médicale (INSERM)
Le Kremlin-Bicêtre (France)

Gerry Dolan
University Hospital
Nottingham Haemophilia Comprehensive Care Centre
Nottingham (United Kingdom)

Susanne Holzhauer
Charité Universitätsmedizin
Department of Pediatric Hematology and Oncology
Berlin (Germany)

Christoph Kannicht
Molecular Biochemistry
Berlin (Germany)

Robert Klamroth
Vivantes Klinikum Friedrichshain
Centre for Vascular Medicine, Internal Medicine – Angiology
and Coagulation Disorders
Haemophilia Treatment Centre
Berlin (Germany)

Sigurd Knaub
Clinical R&D Haematology
Lachen (Switzerland)

Peter J. Lenting
Institut National de la Santé et de la Recherche Médicale (INSERM)
Le Kremlin-Bicêtre (France)

Claude Négrier
Hospital Louis Pradel
Hemostasis Unit
Lyon (France)

Johannes Oldenburg
University Clinic Bonn
Institute of Experimental Haematology and Transfusion Medicine
Bonn (Germany)

John Pasi
The Royal London Hospital
Barts and The London School of Medicine and Dentistry
London (United Kingdom)

Michaela Praus
Accovion GmbH
Marburg (Germany)


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