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Keywords:

  • factor IX;
  • factor VIII;
  • fusion proteins;
  • half-life;
  • hemophilia;
  • polyethylene glycols;
  • recombinant FVIIa

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Bioengineering strategies
  5. Fusion proteins technologies
  6. Factor VIII
  7. Factor IX
  8. Recombinant factor VIIa
  9. Conclusion
  10. Acknowledgement
  11. Disclosure of Conflict of Interest
  12. References

Over a million patients worldwide currently suffer from hemophilia and other congenital clotting factor deficiencies. Patients affected with hemophilia A and B are treated by intravenous replacement therapy of factor VIII and factor IX, respectively. Current hemophilia treatments have favorably supported their efficacy, tolerability, and safety profiles. The onset of alloantibodies inactivating the infused coagulation factor is the main problem in hemophilia patients rendering replacement therapies ineffective; another disadvantage is the short half-life of the infused clotting factors with the need for multiple and frequent infusions to manage a bleeding episode. Now, the challenge in the management of hemophilia treatment is the prolongation of the half-life and reduction in the immunogenicity of recombinant clotting factors. The bioengineering strategies, previously applied successfully to other therapeutic proteins, encourage the current efforts to produce novel coagulation factors with more prolonged bioavailability, with increased potency and resistance to inactivation and potentially reduced immunogenicity.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Bioengineering strategies
  5. Fusion proteins technologies
  6. Factor VIII
  7. Factor IX
  8. Recombinant factor VIIa
  9. Conclusion
  10. Acknowledgement
  11. Disclosure of Conflict of Interest
  12. References

The intrinsic coagulation cascade is a tightly regulated protease and cofactor-dependent amplification system that ensures the formation of stable clots after injury. Deficiencies of the coagulation cofactor factor VIII (FVIII) or the corresponding coagulation protease factor IX (FIX) lead to the X-chromosomal inherited bleeding disorders hemophilia A and hemophilia B, respectively. These coagulation disorders are associated with bleeding episodes affecting soft tissue, joints, and muscles. Repeated hemorrhages result in chronic arthropathy, with loss of joint movement, fixed flexion contracture, and severe muscle wasting [1]. Current therapy for hemophilia is based on the infusion of clotting factor concentrates either at the time of bleeding (on demand therapy) or in a prophylactic schedule to prevent bleeding episodes. This replacement therapy with infusion of solutions enriched for the missing clotting factor has been possible since the 1960s. The 1970s saw radical improvement in hemophilia care with the development and availability of plasma-derived clotting factor concentrates. These freeze-dried concentrates of FVIII and FIX made prophylaxis and home therapy possible and considerably improved patients' quality of life [2]. For the first time, people with hemophilia were able to travel, work, and attend school with regularity. In the 1980s, the cloning and sequencing of FVIII and FIX led to the development of recombinant FVIII (rFVIII) [3, 4] and FIX (rFIX) products [5]. Recombinant products have dramatically reduced the risk of blood-borne pathogen transmission that had become a major concern after the widespread contamination of plasma products with HIV and hepatitis viruses and secured an adequate supply of FVIII and FIX products [6]. The advent of recombinant concentrates has further improved the safety and availability of therapy, and the focus of care has shifted gradually from management of acute bleeding episodes toward prevention of bleeding and management of disease sequelae. However, hemostatic protection with current products is limited in several ways: first, temporally because of the short half-life (t1/2) of 8–12 h for FVIII and 18–24 h for FIX; second, the frequent occurrence of neutralizing antibodies rendering replacement therapy ineffective; third, by the necessity for intravenous administration and hence good venous access. Prophylactic injections 2–3 times per week or every other day are required for most patients to maintain circulating FVIII and FIX above 1% of normal level (1 IU dL−1), which is protective against most spontaneous bleeding episodes [7]. Neutralizing antibodies occur in approximately 30% of severe hemophilia A patients [8] and up to 3% of hemophilia B patients [9].

In young children, frequent intravenous administration may necessitate the insertion of a central venous access device to facilitate home infusions by parents but infection, sepsis, and thrombosis are the concomitants risks [10, 11].

The bioengineering technologies that have been previously applied successfully to other therapeutic proteins, such as the addition of polyethylene glycol (PEG) polymers [12] and polysialic acids (PSAs) [13], alternative formulations with PEG-modified liposomes (PEG-Lip) [14], and fusion proteins technologies [15], now are applied to FVIII, FIX, and FVIIa. Additionally, alternative therapeutic strategies are being considered, such as targeting the inhibitors of coagulation, tissue factor pathway inhibitor (TFPI) [16, 17], activated protein (APC) [18], and antithrombin (AT) [19]. Finally, the use of RNA interference (RNAi) is also being investigated as an approach for hemophilia treatment. Insights into the structure and function of clotting factors have allowed targeted modifications of the protein to increase the duration of its cofactor activity and to reduce its clearance in vivo.

This article summarizes the new technologies and products that are currently employed to improve the quality of treatment of patients affected with coagulation disorders. Tables 1–8 list ongoing preclinical and clinical trials at www.clinicaltrials.gov in hemophilia A and B and English language publications (manuscripts and abstracts) using modified proteins, update until 10 April 2013.

Table 1. Preclinical studies with modified long-acting rFVIII
Coagulation factorModificationProductManufacturerHalf-life (t1/2)References
  1. FVIII, factor VIII; PEG, polyethylene glycol; PSA, polysialic acid.

FVIIIPEG-LipBAY79–4980 (FL-rFVIII)Bayer1.5-fold longer than FL-rFVIII [30]
Random PEGBDD-rFVIIIShorter than BDD-rFVIII [34]
Site-specific PEGBAY94–9027 (BDD-rFVIII)Bayer2-fold longer than BDD-rFVIII [35]
N8-GP (BDD-rFVIII)Novo Nordisk1.3- to 2.7-fold longer than BDD-rFVIII [37, 38]
BAX855 (FL-rFVIII)Baxter1.8- to 3-fold longer than FL-rFVIII [40]
PolysialylationPSA-rFVIII4-fold longer than rFVIII [42, 43]
Fc fusionrFVIII-Fc (BDD-rFVIII)Biogen Idec1.8- to 2.2-fold longer than BDD-rFVIII [44, 45]
Table 2. Clinical studies with modified long-acting rFVIII
Coagulation factorModificationProductManufacturerPhaseStatusResultsReferences
Half-life (t1/2)Adverse events (AEs)
  1. N/A, data not available; FVIII, factor VIII; PEG, polyethylene glycol.

FVIIIPEG-LipBAY79–4980 (FL-rFVIII)BayerI (NCT00629837)CompletedSimilar to FL-rFVIIIIncreased blood triglycerides (2/13) [32]
II (NCT00623727)TerminatedFailure regarding the primary endpoint (< 9 bleeds per year) [33]
Site-specific PEGBAY94–9027 (BDD-rFVIII)BayerI (NCT01184820)Completed1.5-fold longer than BDD-rFVIII [36]
II/III (NCT01580293)Is currently recruitingN/AN/A
III (pediatric study; NCT01775618)Not yet recruitingN/AN/A
N8-GP (BDD-rFVIII)Novo NordiskI (NCT01205724)Completed1.6-fold longer than BDD-rFVIII [39]
III (treatment, prophylaxis; NCT01480180)Is currently recruitingN/AN/A
III (surgical study; NCT01489111)N/AN/A
III (pediatric study; NCT01731600)Is currently recruitingN/AN/A
BAX855 (FL-rFVIII)BaxterI (NCT01599819)CompletedN/AN/A
II/III (NCT01736475)Is currently recruitingN/AN/A
Fc fusionrFVIII-Fc (BDD-rFVIII)Biogen IdecI (NCT01027377)Completed1.5- to 1.7-fold longer than BDD-rFVIII [46]
II/III (NCT01181128)1.5-fold longer than BDD-rFVIII [47]
III (pediatric study; NCT01458106)Is currently recruitingN/AN/A
Table 3. Preclinical studies with modified long-acting rFIX
Coagulation factorModificationProductManufacturerHalf-life (t1/2) compared with rFIXReferences
  1. FIX, factor IX; PEG, polyethylene glycol.

FIXSite-specific PEGN9-GPNovo Nordisk2.4- to 6.2-fold longer [48]
Fc fusionrFIX-FcBiogen Idec2.7- to 6-fold longer [51]
Albumin fusionCSL654CSL Behring1.2- to 4.7-fold longer [54, 55]
Table 4. Clinical studies with modified long-acting rFIX
Coagulation factorModificationProductManufacturerPhaseStatusResultsReferences
Half-life (t1/2) compared with rFIXAdverse events (AEs)
  1. N/A, data not available; FIX, factor IX; PEG, polyethylene glycol.

FIXSite-specific PEGN9-GPNovo NordiskI (NCT00956345)Completed5-fold longerHypersensitivity reaction (1/16) [49]
III (treatment, prophylaxis; NCT01333111)Is ongoing but not recruiting participantsN/AN/A
III (long-term exposure; NCT01395810)Is currently recruitingN/AN/A
III (surgical study; NCT01386528)
III (pediatric study; NCT01467427)
Fc fusionrFIX-FcBiogen IdecI (NCT00716716)Completed3-fold longer [52]
II/III (NCT01027364)2- to 3-fold longer [53]
III (treatment, prophylaxis; NCT01425723)Is currently recruitingN/AN/A
III (pediatric study; NCT01440946)Is currently recruitingN/AN/A
Albumin fusionCSL654CSL BehringI (NCT01233440)Completed5-fold longer [56]
I/II (NCT01361126) [57]
II/III (NCT01496274)Is currently recruitingN/AN/A
III (pediatric study; NCT01662531)N/AN/A
Table 5. Preclinical studies with modified long-acting rFVIIa
Coagulation factorsModificationProductManufacturerHalf-life (t1/2) compared with rFVIIaReferences
  1. PEG, polyethylene glycol.

FVIIaPEG-LipPEG-Lip-FVIIaBayer1.4-fold longer [58]
Site-specific PEGN7-GPNovo Nordisk4.2- to 10-fold longer [60-62]
N-linked glycansBAY86-6150Bayer5-fold longer [65]
Fc fusionrFVIIa-FcBiogen IdecLonger [67]
Albumin fusionCSL689CSL Behring5.8- to 6.7-fold longer [68, 69]
Table 6. Clinical studies with modified long-acting rFVIIa
Coagulation factorsModificationProductManufacturerPhaseStatusResultsReferences
Half-life (t1/2) compared with rFVIIaAdverse events (AEs)
  1. N/A, data not available; PEG, polyethylene glycol. *Clinical trial not reported in www.clinicaltrials.gov.

FVIIaPEG-LipPEG-Lip-FVIIaBayerI/II*CompletedSimilar [59]
Site-specific PEGN7-GPNovo NordiskI (healthy subjects; NCT01272206)Completed5-fold longerAntibodies against N7-GP and cross-reacting to FVIIa (1/30) [63]
I (NCT00922792)Completed5-fold longer [64]
II (NCT00951405)Completed5-fold longer
N-linked glycansBAY86-6150BayerI*Completed2- to 3-fold longerProteinuria (1/33) [66]
II/III (NCT01625390)Is currently recruitingN/AN/A
Albumin fusionCSL689CSL BehringI (NCT01542619)CompletedN/AN/A
Table 7. Preclinical studies: alternative approaches to target inhibitors of coagulation
Inhibitors of coagulationModificationProductLaboratory assays to test efficacyResultsReferences
  1. AT, antithrombin; PEG, polyethylene glycol; TFPI, tissue factor pathway inhibitor. *Non-human primate saphenous vein bleeding. Rabbit cuticle bleeding.

TFPIPEG-aptamerBAX499

Prothrombin time

Thrombin generation

Thromboelastography

Bleeding time test*

Restore normal clotting parameters and bleeding time [17, 70]
mAbmAb 2021

Prothrombin time

Thrombin generation

Thromboelastography

Bleeding time test

Shortened clotting time [16]
APCLNP-siRNALNP-siRNAInhibition of PC mRNA Reduction in circulatory levels of PC [18]
ATsiRNAALN-AT3Thrombin generation4-fold increased thrombin generation [19]
Table 8. Clinical studies: alternative approaches to target inhibitors of coagulation
Inhibitors of coagulationModificationProductPhaseStatusResultsReferences
  1. N/A, data not available; PEG, polyethylene glycol; TFPI, tissue factor pathway inhibitor.

TFPIPEG-aptamerBAX499I/II (NCT01191372)Prematurely stopped due to an increased number of bleeding events

Increased TFPI plasma levels

Reduced thrombin generation

[71]
mAbmAb 2021I (Healthy subjects; NCT01555749)CompletedN/A
I (Explorer 2; NCT01631942)This study has suspended participants recruitment (awaiting protocol amendement)N/A

Bioengineering strategies

  1. Top of page
  2. Summary
  3. Introduction
  4. Bioengineering strategies
  5. Fusion proteins technologies
  6. Factor VIII
  7. Factor IX
  8. Recombinant factor VIIa
  9. Conclusion
  10. Acknowledgement
  11. Disclosure of Conflict of Interest
  12. References

Many approaches to improve antihemophilic drugs have been explored for FVIII, FIX, and FVIIa. These include the modification of amino acid sequences to reduce degradation by blood plasma and liver enzymes and to reduce immunogenic side effects. Peptides bound to immunoglobulin or albumin may increase half-life, and in addition, they can be covalently coupled with PEG chains to the molecule (PEGylation) [12]. In the late 1970s, Davis and Abuchowski generated the first PEGylated molecule, introducing PEG molecules to bovine serum albumin to reduce its immunogenicity [20].

PEGylation usually employs functionalized mono-methoxy PEG (mPEG) molecules to covalently attach to available surface groups on the protein, most commonly through multisite binding to the ε-amino group of lysine residues (random PEGylation) or through site-specific binding to free cysteine residues or via protein engineering (site-specific PEGylation; Fig. 1A). PEGylation increases drug efficacy, which has been attributed to a combination of increased molecular size reducing glomerular filtration and to masking of the protein surface decreasing immunorecognition response and proteolytic degradation. PEGylation of proteins is considered to be safe and well tolerated [21], although in animals the occurrence of renal tubular vacuolization has been observed due to accumulation of the non-degradable PEG chains in the kidney [22, 23].

image

Figure 1. A schematic representation of the protein of interest formulated with Polyethylene glycol (PEG; A), PEGylated Liposomes (B) and polysialic acid (PSA; C).

Download figure to PowerPoint

PEGylated liposome (PEG-Lip) technology is an approach, alternative to direct PEGylation of proteins, to improve the pharmacodynamic properties of therapeutic proteins. PEG-Lip are used as carriers with the protein bound non-covalently but with high specificity to the outer surface (Fig. 1B). Modifying the liposome surface with molecule such as PEG prevents adsorption of plasma proteins to the liposome surface and interferes with recognition and uptake by the reticuloendothelial system. This results in the generation of liposomes with a dramatically extended circulation time [14].

Polysialic acids (PSAs) are an attractive alternative method to PEGylation. PSAs are linear, hydrophilic polymers of N-acetylneuraminic acid that produces a ‘watery cloud’ around the therapeutic molecule protecting it from immune-mediating cells, proteolytic enzymes, and clearance receptors [24, 25] (Fig. 1C). PSA is found in abundance on the surface of mammalian cells and therefore is a biocompatible and biodegradable natural polymer [13]. PEGylation and sialylation prolong half-life, improve enzymatic stability, and decrease renal excretion by increasing molecular mass [26].

Fusion proteins technologies

  1. Top of page
  2. Summary
  3. Introduction
  4. Bioengineering strategies
  5. Fusion proteins technologies
  6. Factor VIII
  7. Factor IX
  8. Recombinant factor VIIa
  9. Conclusion
  10. Acknowledgement
  11. Disclosure of Conflict of Interest
  12. References

An alternative strategy to extend the half-life of proteins is fusing them to another protein with a much longer half-life (Fig. 2A), such as the fragment crystallizable (Fc) region of an immunoglobulin (IgG) [27]. Fc-containing proteins that are internalized by endothelial cells bind to the neonatal Fc receptor (FcRn) present in the acidified endosome in a pH-dependent manner and are then recycled back to the cell surface, avoiding catabolism in the lysosome, and they are subsequently released back into plasma at physiologic pH [13, 25]. These approaches markedly increase molecular weight, which reduces renal clearance. An increasing number of Fc fusion proteins are developed for therapeutic applications [15].

image

Figure 2. A schematic representation of the protein of interest directly linked to the Fc monomer (A) and albumin fused to the C-terminus of protein of interest through a cleavable linker (B).

Download figure to PowerPoint

Albumin fusion technology yields an altered version of a protein by fusing the gene for human albumin to the gene that encodes the active protein drug (Fig. 2B). This technology has initially been applied to cytokines (interferons, interleukins) and increases its molecular weight prolonging the half-life in vivo. The albumin molecule also masks the protein rendering it resistant to proteases and less immunogenic [28].

Factor VIII

  1. Top of page
  2. Summary
  3. Introduction
  4. Bioengineering strategies
  5. Fusion proteins technologies
  6. Factor VIII
  7. Factor IX
  8. Recombinant factor VIIa
  9. Conclusion
  10. Acknowledgement
  11. Disclosure of Conflict of Interest
  12. References

FVIII formulated with PEGylated liposomes

Encapsulation of therapeutic agents inside microparticle carriers such as liposomes is a way to improve their half-life [29]. Because liposomes are cleared quickly from the circulation, they are chemically modified with PEG to extend their half-life. The rFVIII molecules remain unmodified, so there is no loss of normal protein–protein interactions (including rFVIII–VWF interaction) and functional activities.

Preclinical study

Preclinical studies in hemophilia A mouse models using rFVIII formulated with PEGylated liposomes (rFVIII-PEG-Lip) showed prolongation of the half-life (approximately 1.5-fold) and increased protection from bleeding [30].

Clinical studies of BAY79–4980

The efficacy and safety of PEG-Lip-rFVIII-FS using various quantities of PEGylated liposomes with a fixed FVIII dose were evaluated in hemophilia patients confirming the data obtained in animal models [31]. Despite these promising results, a subsequent phase I trial demonstrated that rFVIII-PEG-Lip and standard rFVIII have similar pharmacokinetic parameters (NCT00629837) [32]. The potential efficacy of this formulation was investigated later in a randomized controlled, double-blind prophylaxis trial in patients with severe hemophilia A comparing once-a-week dosing of rFVIII-PEG-Lip with three-time per-week dosing with standard rFVIII (NCT00623727) [33]. Endpoints were occurrence of hemorrhages. The new compound proved inferior to rFVIII with 72.1% (49/68) patients experiencing less than 9 hemorrhages per year in the rFVIII control group compared with 38.1% (24/63) of BAY79–4980-treated subjects. A similar difference was seen in annualized joint bleeds. The investigational treatment arm was prematurely discontinued due to failure to achieve the primary endpoint [33].

FVIII PEGylation

In vitro study

The first PEGylation research of rFVIII was conducted by Rostin et al. using attachment of mPEG molecules to the lysine residues on a B-domain-deleted rFVIII (rFVIII-SQ). A substantial reduction in coagulant activity was observed as a consequence of random PEG coupling, probably due to steric effects in combination with changes of charge. Moreover, random attachment of PEG on FVIII disrupts the VWF-binding site causing impairment of FVIII binding to VWF [34].

Preclinical studies of BAY94–9027

A more precise selection of the sites for PEG conjugation avoids interference of functional protein–protein interactions. A B-domain-deleted rFVIII has been engineered introducing surface-exposed cysteines to which a PEG polymer was specifically conjugated. Site-directed PEGylation of rFVIII preserved coagulation efficacy and did not much reduce VWF binding. These bioengineered rFVIII molecules (BAY94–9027) exhibited improved pharmacokinetics in hemophilic mice and rabbits and prolonged efficacy in bleeding models in mice consistent with an enhanced half-life in vivo [35].

Clinical study of BAY94–9027

In a subsequent open-label phase I study (NCT01184820), BAY94–9027 demonstrated improved pharmacokinetics, with a half-life of 19 h (approximately 1.5-fold longer than B-domain deleted rFVIII) [36].

Preclinical studies of N8-GP

A novel site-specific PEGylation attaches a 40-kDa PEG to a unique O-glycan in the truncated B-domain of rFVIII (N8-GP). When activated by thrombin, the B-domain containing the PEGylation is cleaved off, generating active FVIIIa that is similar in structure to native FVIIIa [37]. The N8-GP pharmacokinetic parameters were estimated in different animal models. In hemophilia A dogs [37], mice, rabbits, and monkeys [38], a nearly 2-fold prolongation of half-life was demonstrated, but the effects were less pronounced in rats (approximately 1.3-fold) [37].

Clinical study of N8-GP

In a phase I clinical study, N8-GP showed a half-life of 19 h, representing approximately 1.6-fold prolongation compared with patients previously treated with conventional FVIII products (mean half-life of 11.7 h; NCT01205724) [39].

Preclinical study of BAX855

BAX855 is produced by the reaction of an activated novel PEG reagent with accessible amino groups on the FVIII protein. Preclinical animal studies (knockout mice and macaques) showed improved pharmacokinetic behavior and clinically prolonged efficacy (approximately 1.8-fold in knockout mice and approximately 3-fold in macaques) without signs of toxicity or increased immunogenicity [40].

FVIII polysialylation

PSA is a biocompatible and biodegradable natural polymer. The chemical addition of PSA to proteins may represent another means to improve the circulating half-life without compromising functional activity [41]. The effect of polysialylation of rFVIII on binding to low-density lipoprotein-receptor-related protein 1 (LRP1) has been investigated in murine models [42]. LRP1 is involved in the in vivo clearance and regulation of FVIII in plasma. Surface plasmon resonance was used to investigate the binding between polysialylated rFVIII to LRP1. Data suggested that a drastically reduction of the binding to LRP1 contributes to the increased in half-life of FVIII modified. Studies in VWF-FVIII double knockout mice demonstrated a half-life of polysialylated rFVIII that was approximately 4-fold higher than that of rFVIII [43]. No clinical trials of polysialylated FVIII protein have been performed in humans.

FVIII–Fc fusion

Preclinical studies of rFVIII-Fc

An alternative long-acting FVIII molecule has been constructed based on Fc fusion monomer technology. rFVIII-Fc is a recombinant fusion of a single B-domain-deleted FVIII molecule to the dimeric constant region (Fc) of IgG1. rFVIII-Fc exhibits approximately 2-fold extension in half-life in hemophilia A mice and dogs [44]. Furthermore, rFVIII-Fc has been characterized biochemically and functionally by Peters et al. [45]. Several methods were used to confirm the primary sequence and post-translational modifications to FVIII, confirming that rFVIII-Fc contains the expected sequence and N-linked glycosylation and tyrosine sulfation. VWF binding and specific activity were similar to other recombinant FVIII molecules. Ex vivo rotational thromboelastometry (ROTEM®) studies demonstrated that rFVIII-Fc activity was prolonged in hemophilia A mice in comparison with rFVIII [45].

Clinical studies of rFVIII-Fc

A phase 1/2 clinical study has been performed in hemophilia A patients demonstrating that rFVIII-Fc improves half-life approximately 1.5- to 1.7-fold (NCT01027377) [46]. Recently, a phase III study (NCT01181128) confirmed that the half-life of rFVIII-Fc was approximately 1.5-fold longer than rFVIII [47].

Factor IX

  1. Top of page
  2. Summary
  3. Introduction
  4. Bioengineering strategies
  5. Fusion proteins technologies
  6. Factor VIII
  7. Factor IX
  8. Recombinant factor VIIa
  9. Conclusion
  10. Acknowledgement
  11. Disclosure of Conflict of Interest
  12. References

In an attempt to make a longer-acting FIX, the same strategies that have been investigated to extend the half-life of FVIII have been applied to FIX.

FIX glycoPEGylation

Preclinical study of N9-GP

A rFIX product has been modified by site-directed glycoPEGylation in which a 40-kDa PEG molecule has been attached to the FIX activation peptide, which fusion protein is called N9-GP. Upon N9-GP activation by FXIa, the activation peptide is cleaved off leaving the wild-type activated FIX (FIXa). In a preclinical study, N9-GP exhibited an over 2-fold increased in vivo recovery and a markedly prolonged half-life in minipigs and hemophilia B dogs compared with rFIX. Moreover, N9-GP showed an efficacy similar to rFIX in stopping acute hemorrhage in hemophilia B mice suggesting that it may be efficacious for acute treatment of bleeding episodes in humans [48].

Clinical studies of N9-GP

In a phase I clinical study, N9-GP showed a half-life of 93 h, that is, five times longer than in hemophilia B patients previously treated with conventional FIX products (plasma derived FIX 18 h and rFIX 19 h; NCT00956345). The novel molecule was generally well tolerated, although one patient (of 16) developed transient hypersensitivity symptoms during administration of N9-GP leading to the withdrawal of the drug [49]. Pharmacokinetic modeling predicted that N9-GP dosed at 10 or 40 U kg−1 would maintain trough FIX plasma concentrations at a level previously associated with bleeding prevention with less frequent injections and, because it has conventional FIX concentrate as its source material, with lower factor concentrate consumption [50].

FIX–Fc fusion

Preclinical studies of rFIX-Fc

Traditionally, Fc fusion proteins have been made as dimers, while Peters et al. [51] generated a rFIX-Fc constructed as a single FIX molecule attached to the Fc domain of human IgG1. This long-acting molecule exhibited a half-life approximately 3- to 4-fold longer than rFIX across a range of species including normal mice, rats, and monkeys as well as FIX-deficient mice and dogs. In addition, the pharmacokinetics were examined in two FIX-deficient species, that is, mice and dogs. In whole-blood samples, 15 min after administration, normal clotting was observed in all FIX-deficient mice given rFIX-Fc or rFIX. Clotting activity in blood from rFIX-Fc-treated mice returned to baseline by 144 h in all animals, twice as long as the time in the rFIX-treated mice; similar results were observed in FIX-deficient dogs [51].

Clinical studies of rFIX-Fc

The safety and pharmacokinetics of rFIX-Fc were subsequently evaluated in previously treated human adults with severe hemophilia B showing a half-life that was 3-fold longer than that reported for rFIX (NCT00716716) [52]. No serious adverse events related to the study drug were observed, specifically no inhibitory antibody development occurred in 14 treated individuals [52]. In a recently completed phase II/III clinical study (NCT01027364), rFIX-Fc was well tolerated and effective in the treatment of bleeding, routine prophylaxis, and perioperative management. The half-life of rFIX-Fc was 2- to 3-fold longer than conventional rFIX [53].

FIX–albumin fusion

Preclinical studies of rFIX-FP

Genetic fusion of recombinant human albumin to rFIX was obtained through a linker sequence that contains a cleavable sequence identical to the activation site for FIX (rIX-FP, CSL654) [54]. Upon activation, albumin and the cleavable linker would be liberated allowing the resultant FIXa to function normally. Preclinical studies with rIX-FP showed that the half-life was not or not much affected in FIX-deficient mice (1.2-fold increase), while studies in rats and rabbits showed markedly prolonged half-lives (3- to 4-fold increase) compared with rFIX [54], as in cynomolgus monkeys and hemophilic dogs (2- to 3-fold increase) [55].

Clinical studies of rFIX-FP

The clinical safety and pharmacokinetic properties of rFIX-FP were subsequently tested in 25 previously treated patients with hemophilia B (NCT01233440). The terminal half-life of rFIX-FP was five times higher than for the previously used FIX product, without allergic reactions or inhibitors [56]. A phase I/II open-label, multicenter, clinical study of rFIX-FP has been completed in 10 previously treated patients with severe hemophilia B (FIX ≤ 2%; NCT01361126). These results demonstrated clinical efficacy of rFIX-FP for once-weekly routine prophylaxis to prevent spontaneous bleeding episodes. No allergic reaction, inhibitors to FIX, or antibodies to rFIX-FP were reported [57].

Recombinant factor VIIa

  1. Top of page
  2. Summary
  3. Introduction
  4. Bioengineering strategies
  5. Fusion proteins technologies
  6. Factor VIII
  7. Factor IX
  8. Recombinant factor VIIa
  9. Conclusion
  10. Acknowledgement
  11. Disclosure of Conflict of Interest
  12. References

The management of bleeding in hemophilia patients with inhibitors requires therapy with bypassing agents such as recombinant factor VIIa (rFVIIa). Its short plasma half-life, approximately 2–4 h, has led to efforts to produce a product with FVIIa properties with a prolonged half-life. Several bioengineering strategies have been implemented in an attempt to extend the half-life of rFVIIa through substitution of one or more amino acids within the protein formulation with PEGylated liposomes, PEGylation, and binding to fusion proteins.

FVIIa formulated with PEGylated liposomes and glycoPEGylation

Preclinical study of PEG-Lip-formulated FVIIa

In preclinical experiments conducted in rats, the half-life of a PEG-Lip-formulated FVIIa (PEG-Lip-FVIIa) was slightly longer (1.4 times) than that of rFVIIa [58].

Clinical study of PEG-Lip-formulated FVIIa

An open-label, crossover phase I/II study revealed that the pharmacokinetics of FVIIa and PEG-Lip-FVIIa were almost identical. Thrombin generation assays show that PEG-Lip-FVIIa induced faster thrombin generation and higher peak levels of thrombin than standards FVIIa. This effect lasted up to 5 h postinfusion. Therefore, an enhanced efficacy cannot be explained by changes in circulatory half-life or clearance [59].

Preclinical studies of glycoPEGylated rFVIIa (N7-GP)

GlycoPEGylation technology has been adopted to prolong the circulatory half-life of rFVIIa. Ghosh et al. [60] demonstrated that glycoPEGylated rFVIIa variants retain their catalytic activity and interact efficiently with tissue factor (TF), factor X (FX), and plasma inhibitors. The plasma half-life of rFVIIa-40k PEG was substantially prolonged in animal models (5–6 times longer than rFVIIa in rabbits) [61]. Moreover, in mice, dogs, and minipigs, the pharmacokinetics of the glycoPEGylated variant exhibited a decrease in clearance coinciding with an increased half-life (4.2- to 10-fold) [62].

Clinical studies of glycoPEGylated rFVIIa (N7-GP)

A phase I trial of rFVIIa-40k PEG (N7-GP) in healthy men (NCT01272206) showed that the pharmacokinetics of N7-GP are dose proportional in the range investigated [63]. N7-GP had a plasma half-life in humans approximately 5-fold longer than rFVIIa. No serious adverse events including thrombotic events or neutralizing antibodies against N7-GP were reported in 30 men. However, in one plasma sample, a positive antibody response for antibodies binding to N7-GP and cross-reacting with FVIIa was detected [63]. In a phase I (NCT00922792) trial, single and multiple doses of N7-GP were tested in eight high-titer inhibitors patients with hemophilia A and B [64]. The mean half-life of N7-GP was 15.6 h, in accordance with the data from the clinical trial in the healthy subjects. No adverse events and no binding or neutralizing antibodies were detected in any of the patients [64]. A phase II (NCT00951405) trial in hemophilia A and B patients with inhibitors has been completed, and the data demonstrated that N7-GP was well tolerated and half-life was approximately 15.0 h.

Modification of amino acid sequence of FVIIa protein

Preclinical study of BAY86–6150

A bioengineered rFVIIa (BAY86–6150) with two additional mutations (T106N and V253N) that introduce two more N-linked glycans has been generated. In preclinical studies, BAY86–6150 was well tolerated and showed a 5-fold increased half-life compared with currently available rFVIIa products [65].

Clinical study of BAY86–6150

A phase I clinical study showed a half-life of 2- to 3-fold longer than rFVIIa confirming a good tolerance with no serious adverse events. One of the six patients in the study showed trace of protein in urine [66]. A phase II/III (NCT01625390) is currently recruiting and results are not available yet.

FVIIa–Fc fusion

Preclinical study of rFVIIaFc

A recombinant FVIIa–Fc fusion protein (rFVIIaFc) has been developed similar to rFIX-Fc in which a single FVIIa molecule is fused to one Fc dimer. The rFVIIaFc activity showed an extended terminal half-life compared with rFVIIa in hemophilia A mice and showed similar efficacy in the tail clip bleeding model in hemophilia A mice [67].

FVIIa–albumin fusion

Preclinical study of rFVIIaFP

A fusion protein of FVIIa and albumin has been generated through a flexible 31 amino acid glycine–serine linker to the C-terminus of FVII (rFVIIa-FP, CSL689) [68]. In rats, the fusion of rFVIIa to albumin led to a approximately 6-fold prolonged half-life and 2-fold increase in recovery compared with rFVIIa [69].

Clinical study of rFVIIaFP

The results of an ongoing phase I study are not available yet (NCT01542619).

Alternative therapeutic strategies

An alternative approach to increase hemostatic efficacy is to target inhibitors of coagulation. The inhibition of TFPI will promote thrombin generation and may be effective in the treatment of bleeding in hemophilia. TFPI has been effectively targeted by antibodies (anti-TFPI) [16] and synthetic inhibitors (aptamers and peptides) of TFPI [17]. Inhibition of APC and AT could also enhance thrombin generation, and both aptamers and RNAi silencing have been used [18, 19].

Preclinical study

ARC19499 (BAX499) is a PEG-conjugated, modified oligonucleotide aptamer that potently and specifically inhibits TFPI [17].

Preclinical results have shown that ARC19499 specifically inhibits TFPI in vitro and in vivo and restores normal clotting parameters in hemophilic monkeys. Recently, a study on the mechanism of action of the aptamer ARC19499 showed that after binding of the aptamer to TFPI, the TFPI/BAX499 complex retained FXa inhibitory activity, albeit with reduced affinity [70]. When tested in an extrinsic Xase activity assay, BAX499 delayed TFPI-mediated inhibition of the extrinsic Xase activity. In addition, BAX499 reversed TFPI inhibition of the prothrombinase complex. BAX499 shortened the diluted prothrombin time in FVIII-deficient plasma, and when added to freshly drawn hemophilia A blood either with or without a FVIII inhibitor, the whole-blood clotting time was also shortened.

Clinical study

BAX499 was recently tested in a phase I clinical trial (NCT01191372). The study was prematurely stopped due to an increased number of bleeding events. Subsequently, several analysis was performed to investigate why clinical safety was inconsistent with preclinical observations. BAX499 induced a considerable increase in full-length TFPI plasma levels (25-fold higher with the highest dose of BAX499) reducing thrombin generation. An explanation of elevation of TFPI in plasma levels can be that BAX499 releases intracellularly stored TFPI, impacts its metabolism, and prolongs the circulatory half-life of full-length TFPI, most likely due to binding of BAX499 to the Kunitz 3-C-terminus domain of TFPI, a region required for full-length TFPI clearance. The net results of these effect are elevated plasma full-length TFPI, which even at a molar excess of BAX499 retains anticoagulant activity [71].

A second molecule mAb 2021 (NN7415, NNC172-2021) is being evaluated and a single dose phase 1 clinical trial has been concluded (results to be reported). A multiple dose phase 1b trial has been paused in order to allow further assessment of non-clinical data prior to proceeding into higher dose cohorts.

RNAi targeting AT

Hemophilia patients who co-inherited a prothrombotic mutation, such as factor V Leiden, protein C deficiency, or prothrombin G20210A, have a later onset of disease, lower risk of bleeding, and reduced requirements for FVIII or FIX replacement. AT acts as an important endogenous anticoagulant by inactivating FXa and thrombin.

RNAi (RNA interference) is a natural process of gene silencing that occurs in organisms ranging from plants to mammals. A short interfering RNA (siRNA), ALN-AT3, employing a hepatocyte targeting ligand has been developed against AT, which demonstrates potent activity in both wild-type and hemophilia mice after single subcutaneous administration. Treatment of hemophilic mice with ALN-AT3 normalized thrombin generation.

Preclinical study

Preclinical data presented recently [19] showed that ALN-AT3 yields potent, dose-dependent, and durable knockdown of AT in non-human primates with an up to 4-fold increase in thrombin generation.

Bispecific antibody (termed ACE910) against activated FIXa and FX

An alternative approach is a humanized bispecific antibody to FIXa and FX, termed hBS23 (ACE910) that is able to restore FVIII hemostatic activity in a hemophilia A model mimicking the cofactor function of FVIII in vitro and its hemostatic activity in vivo [72]. The hBS23 binds to the enzyme FIXa with one arm and to FX with the other, placing both in spatially appropriate positions and promoting FIXa-catalyzed FX activation. In vitro studies with hBS23/ACE910 led to shortened clotting times in human FVIII-deficient plasma both with and without inhibitory antibodies. The FVIII-mimetic cofactor activity of hBS23/ACE910 was evaluated by thrombin generation studies in FVIII-deficient plasma, which showed that treatment with the bispecific antibody yielded similar results to increasing plasma levels of FVIII to 1–10 IU dL−1 [72].

Preclinical studies

A pharmacokinetic study of hBS23/ACE910 with a single intravenous or subcutaneous administration of 3 mg kg−1 in cynomolgus monkeys reduced bleeding symptoms and revealed a long half-life of 14 days, which is consistent with the general pharmacokinetics of IgG antibodies [73]. Moreover, the subcutaneous bioavailability of ACE910 was nearly 100% [72], with no development of anti-FVIII inhibitors.

Clinical study

A phase I clinical study with hBS23/ACE910 has started and is currently recruiting in Japan (JapicCTI-121934; http://www.clinicaltrials.jp).

Rare bleeding disorders

Rare bleeding disorders (RBDs) such as afibrinogenemia, FII, FV, FV + VIII, FVII, FX, FXI, and FXIII deficiencies are autosomal recessive diseases and represent 3%–5% of all inherited coagulation deficiencies [74]. The absence of longitudinal clinical data and the limitations of available laboratory assays make it difficult to develop evidenced-based guidelines for the diagnosis and treatment of RBDs. The information gathered on 489 patients in European Network of Rare Bleeding Disorders (EN-RBD) has shown a strong association between coagulation factor activity level and clinical bleeding severity for fibrinogen, FX, FXIII, and combined FV and FVIII deficiencies. For FV and FVII deficiencies, this association was only weak, whereas it was absent for FXI. The minimum coagulation factor activity levels for patients to remain asymptomatic differed substantially between the various deficiencies [75].

Whereas advances in the manufacture of safe and effective products have vastly improved the quality of life of patients affected with hemophilia A and B [76], patients with RBDs have seen less progress. Fresh–frozen plasma (FFP) and cryoprecipitate therefore have remained the backbone of RBD treatment worldwide. Prothrombin and FX deficiencies are often treated with prothrombin complex concentrates (PCCs), which also contain, often, uncontrolled amounts of FII, FVII, and FX. Specific plasma-derived concentrates are currently available only for fibrinogen, FVII, FXI, and FXIII, and they are licensed only in some European countries. A new freeze-dried human FX concentrate has been recently developed and has facilitated prophylaxis in patients with FX deficiency [77]. No specific FV concentrate is available and because FV is not present in cryoprecipitate or PCCs, treatment options in patients with FV and combined FV and FVIII deficiencies are limited to the infusion of FFP.

Only two recombinant products are currently available for treatment of RBDs: rFVIIa, licensed for the treatment of FVII deficiency [78], which has been also used as an off-label therapy in a few cases of FV and FXI deficiency [79-83] and a more recently developed rFXIII. A phase III clinical trial (NCT00713648) has recently been completed proving that rFXIII is safe and effective in preventing bleeding episodes in patients with congenital FXIII-A subunit deficiency [84].

Information on the safety and efficacy of the few available products is scarce, and only few clinical trials are ongoing evaluating the use of specific concentrates of fibrinogen (NCT0091656), FX (NCT01086852), and FXI (NCT01701947).

The development of novel therapeutic approaches as RNAi, as described above, could also be useful for the treatment of patients with RBDs and no specific concentrate available, such as FV, FX, and FII.

Conclusion

  1. Top of page
  2. Summary
  3. Introduction
  4. Bioengineering strategies
  5. Fusion proteins technologies
  6. Factor VIII
  7. Factor IX
  8. Recombinant factor VIIa
  9. Conclusion
  10. Acknowledgement
  11. Disclosure of Conflict of Interest
  12. References

There are several new products for congenital bleeding disorders in various stages of development and testing that may offer significant improvement in care. Whereas patients with hemophilia in the industrialized world currently enjoy effective and safe treatment, this is not the case for the RBDs [85]. The first group of patients have a near normal life expectancy, and novel treatments will mainly focus on improving quality of life by reducing the number of infusions per time period and improving the ease of administration (from venous to subcutaneous). The major medical benefit in hemophilia will be adequate treatment options for patients with inhibitors and treatments without the risk of inhibitor development, as treatment for patients in areas of the world that cannot afford the currently available options.

Despite a good performance of long-acting FIX and FVIIa products, the prolongation of FVIII half-life remains unsatisfactory yet. Although the bioengineering strategies appear to be having an impact on the catabolism of FVIII, there has been continued reliance on a VWF association similar to the native FVIII molecule. The absence of VWF alters the limit on the half-life extension of PEGylated FVIII and rFVIII-Fc, as shown in VWF knockout mice [35, 44]. Whether PEG or Fc fusion on FVIII can effectively attenuate the clearance of the FVIII–VWF complex is an area that merits further investigation.

The different strategies to prolong half-life need to be based on solid knowledge of the influence of proteolytic enzymes encountered with systemic circulation as well as the renal clearance of the drug [86]. Most natural peptides and proteins are rapidly cleared from the circulation, requiring impracticably large or frequent doses for therapeutic activity. The correlation between PEG molecular weight and glomerular filtration has been investigated by a few researches groups, and glomerular filtration of macromolecules is related not only to their size and charge but also to shape and rigidity [22, 23]. A key goal of pharmaceutical design is to maintain a drug's concentration within the therapeutic window. Drug concentrations above this range cause toxicity, while those below it result in insufficient efficacy.

Finally, all drugs have side effects, and new drugs may have unexpected side effects, which should not damped enthusiasm but lead to a cautious approach. The potential for new protein immunogenicity with molecular manipulation requires harmonization of postmarketing data collection. Because the pre-approval studies are performed in selected populations, most serious complications of safety and efficacy do not appear until after the product has been approved and used on a large scale. Rigorous and prolonged independent surveillance studies may replace some of the pre-approval studies and speed up the approval process while at the same time improve the likelihood of identifying complications and side effects [87].

In conclusion, the greatest unresolved issue in hemophilia treatment is the lack of availability of factor concentrates for two-thirds of the world's patient population. Production of biosimilar products and prioritization of their registration could help to reduce the cost of available products and make recombinant proteins more accessible also for those patients coming from countries with low economic resources.

Acknowledgement

  1. Top of page
  2. Summary
  3. Introduction
  4. Bioengineering strategies
  5. Fusion proteins technologies
  6. Factor VIII
  7. Factor IX
  8. Recombinant factor VIIa
  9. Conclusion
  10. Acknowledgement
  11. Disclosure of Conflict of Interest
  12. References

The authors are grateful to Stella Forti, Roberta Palla, and Marzia Menegatti for their help and information on European Network on Rare Bleeding disorders and to Professor Frits Rosendaal for his critical and constructive comments on the text of this article.

Disclosure of Conflict of Interest

  1. Top of page
  2. Summary
  3. Introduction
  4. Bioengineering strategies
  5. Fusion proteins technologies
  6. Factor VIII
  7. Factor IX
  8. Recombinant factor VIIa
  9. Conclusion
  10. Acknowledgement
  11. Disclosure of Conflict of Interest
  12. References

F.P. has received honoraria from Baxter BioScience, CSL Behring, and Novo Nordisk, and research funding from Novo Nordisk.

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  2. Summary
  3. Introduction
  4. Bioengineering strategies
  5. Fusion proteins technologies
  6. Factor VIII
  7. Factor IX
  8. Recombinant factor VIIa
  9. Conclusion
  10. Acknowledgement
  11. Disclosure of Conflict of Interest
  12. References
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