© 2014 Direct One Communications, Inc. All rights reserved. 1 Advances in Clotting Factors: From Bench to Bedside Anthony Sung, MD Duke University Medical Center, Durham, North Carolina A REPORT FROM THE 65 TH ANNUAL MEETING OF THE NATIONAL HEMOPHILIA FOUNDATION (NHF 2013) AND THE 55 TH ANNUAL MEETING OF THE AMERICAN SOCIETY OF HEMATOLOGY (ASH 2013)

© 2014 Direct One Communications, Inc. All rights reserved. 2 History of Clotting Factors Understanding the coagulation cascade is essential to appreciating the role of individual clotting factors in the intrinsic and extrinsic pathways.

© 2014 Direct One Communications, Inc. All rights reserved. 3 History of Clotting Factors The history of clotting factors is tied to the history of hemophilia: » 1840s: transfusion of whole blood can treat hemophilia- associated bleeding » 1911: factor VIII (FVIII) detected in plasma » 1937: role of FVIII described in hemostasis and the coagulation cascade » 1940s: development of plasma transfusions » 1950s: development of plasma concentrates » 1960s: development of cryopreciptiate as well as freeze- dried FVIII for home use » 1992 and 1997: US Food and Drug Administration (FDA) approval of recombinant FVIII and FIX (rFVIII, rFIX)

© 2014 Direct One Communications, Inc. All rights reserved. 4 Limitations of Recombinant Factors Short half-life requiring infusion 3–4 times/wk Venous access often requires the placement of a port or other device, with risk of infections and other complications Significant time commitment and problems with adherence to infusion schedule Development of alloimmune inhibitory antibodies (inhibitors)

© 2014 Direct One Communications, Inc. All rights reserved. 5 Strategies for Increasing Coagulation Factor Half-life Based on a presentation by Randal J. Kaufman, PhD, at ASH 2013

© 2014 Direct One Communications, Inc. All rights reserved. 6 PEGylation PEGylation is achieved by covalent attachment of polyethylene glycol (PEG) to residues on target proteins such as lysine or N-terminal amines. Site-directed PEGylation via attachment of PEG- maleimide to cysteine residues has shown improved results with many proteins. PEGylated products may vary both at the site of PEGylation as well as type of PEG. » PEG molecules may vary in size, with smaller molecules being more rapidly cleared than larger ones. » Tissue penetration may also vary, with smaller molecules having increased permeability. Alconcel SNS et al. Polym Chem. 2011;2:1442; Horton S et al. Expert Opin Biol Ther. 2012;12:235

© 2014 Direct One Communications, Inc. All rights reserved. 7 Fusion Proteins By covalently fusing clotting factors with proteins that have a much longer half-life (eg, Ig, albumin), the half-life of the resulting molecule may be extended. The Fc domain can improve the solubility and stability of the partner molecule and allows for easy and cost-effective purification by protein-G/A affinity chromatography. Kaufman RJ, Powell JS. Blood. 2013;122:3568

© 2014 Direct One Communications, Inc. All rights reserved. 8 Novel Coagulation Factor Products A recombinant single-chain FVIII protein prevents dissociation of the two chains of FVIII, resulting in higher affinity for von Willebrand factor (vWF). Reducing the immunogenicity of recombinant FVIII molecules by changing or substituting glycan and sulfyl groups. Factor Xase mimetics can bypass FVIII. » A humanized bispecific monoclonal antibody to FIXa and FX has demonstrated both efficacy and prolonged half-life (2 weeks) in a simian model of acquired hemophilia. Schulte S. Thromb Res. 2013;131:S2; Kannicht C et al. Thromb Res. 2013;131:78; Kitazawa T et al. Nat Med. 2012;18:1570

© 2014 Direct One Communications, Inc. All rights reserved. 9 Inhibition of Inhibitory Pathways Tissue factor pathway inhibitor (TFPI) inhibits FVIIa and FXa; by inhibiting TFPI, hemostasis may be achieved. » This concept has been demonstrated by a monoclonal antibody to one of the Kunitz domains of TFPI in a rabbit hemophilic model, which demonstrated decreased bleeding. » Other TFPI inhibitors include nucleic acid aptmers, non- anticoagulant sulfated polysaccharide (NASP), and small antagonist peptides. Anti-thrombin 3 (AT3) inhibits thrombin. » A synthetic GalNAc-conjugated RNAi (ALN-AT3) has been shown to suppress hepatic production of AT3 mRNA. Hilden I et al. Blood. 2012;119:5871; Gorczyca ME et al. J Thromb Haemost. 2012;10:1581; Prasad S et al. Blood. 2008;111:672; Peraramelli S et al. J Thromb Haemost. 2013;11:704; Kaufman RJ, Powell JS. Blood. 2013;122:3568

© 2014 Direct One Communications, Inc. All rights reserved. 10 Long-lasting Recombinant Factor VIII Proteins for Patients with Hemophilia A Based on a presentation by Amy D. Shapiro, MD, at ASH 2013

© 2014 Direct One Communications, Inc. All rights reserved. 11 PEG-FVIII Conjugate: BAY BAY is a B-domain deleted recombinant FVIII (PEG-BDD-rFVIII ( identifier NCT ) In a phase 1 study of two cohorts (25 IU/kg twice weekly or 60 IU/kg once weekly) of seven patients per cohort with severe hemophilia A, BAY was well tolerated, efficacious, and caused no serious adverse events. The half-life was 19 hours, representing a 50% increase over standard products, and it was well tolerated without immunogenicity. Phase 2/3 studies are underway. Ivens IA et al. Haemophilia. 2013;19:11

© 2014 Direct One Communications, Inc. All rights reserved. 12 PEG-FVIII Conjugate: BAX 855 BAX 855 is a PEGylated full-length rFVIII ( identifier NCT ). The product is reported to retain all of the physiologic properties of FVIII except binding to the low-density lipoprotein receptor-related protein clearance receptor. Preclinical testing revealed normal coagulation factor activity and a prolonged half-life compared with that of unmodified recombinant FVIII. Turecek PL et al. Hamostaseologie. 2012;32:S29

© 2014 Direct One Communications, Inc. All rights reserved. 13 PEG-FVIII Conjugate: N8-GP N8-GP is a glycol-PEGylated rFVIII ( identifier NCT ). A dose-escalation study in 26 previously treated patients with severe FVIII showed it to be well- tolerated at all dose levels (25, 50, and 75 IU/kg), and none of the patients developed an inhibitor or binding antibodies to FVIII or N8-GP. N8-GP exhibited a dose-linear pharmacokinetic profile with a mean half-life of 19 hours (range, 11.6–27.3 hours), representing a 1.6-fold increase over the half-life of standard rFVIII. Tiede A et al. J Thromb Haemost. 2013;11:670; Møss J et al. J Thromb Haemost. 2011;9:1368

© 2014 Direct One Communications, Inc. All rights reserved. 14 Fc Fusion Protein: rFVIIIFc rFVIIIFc was developed by fusing a single B-domain deleted rFVIII to the dimeric Fc region of IgG1 without intervening linker sequences. Preclinical studies showed prolonged in vivo activity compared with existing rFVIII products and comparable binding to vWF. Animal studies demonstrated approximately a twofold increase in half-life over that of rFVIII in murine and canine models of hemophilia. Peters RT et al. Blood. 2010;115:2057; Peters RT et al. J Thromb Haemost. 2013;11:132; Dumont JA et al. Blood. 2012;119:3024

© 2014 Direct One Communications, Inc. All rights reserved. 15 Fc Fusion Protein: rFVIIIFc A phase 1/2 study of rFVIIIFc in 16 patients with severe hemophilia A with either 25 or 65 IU/kg rFVIII followed by an equal dose of rFVIIIFc showed a 1.5- to 1.7-fold increase in mean half-life (18.8 hours for both doses) for rFVIIIFc compared with rFVIII (12.2 hours for the lower dose and 11.0 hours for the higher dose). No drug-related adverse events, inhibitors, or severe bleeding were observed. A phase 3 multicenter study of rFVIIIc (A-LONG) was recently completed, and results are pending. A similar study is ongoing in children (Kids A-LONG). Powell JS et al. Blood. 2012;119:3031

© 2014 Direct One Communications, Inc. All rights reserved. 16 The Old and the New: PCCs, rFVIIa, and Long-lasting Coagulation Proteins Based on a presentation by Margaret V. Ragni, MD, MPH, at ASH 2013

© 2014 Direct One Communications, Inc. All rights reserved. 17 Bypassing Agents Prothrombin complex concentrates (PCCs) contain combinations of clotting factors as well as proteins C and S. » Four-factor PCCs, such as Beriplex, Octaplex, and Kcentra, contain factors II, VII, IX, and X. » Three-factor PCCs, such as Bebulin and Profiline, contain factors II, IX, and X but little VII. FEIBA is a formulation of PCCs with activated clotting factors to enhance hemostasis. rFVIIa is thought to produce a “thrombin burst” through activation of FIX, FX, and FII on the surface of activated platelets.

© 2014 Direct One Communications, Inc. All rights reserved. 18 Surgical and Trauma-related Bleeding PCCs appear to be superior to rFVIIa and warfarin in reversing warfarin-related bleeding or stemming surgical and trauma-related bleeding. Although the combination of PCCs and fresh frozen plasma or PCCs and rFVIIa may correct laboratory abnormalities such as the INR even more rapidly than either agent alone, extreme caution must be exercised in using these agents, given the significant thrombotic risk of each agent in and of itself. These agents should be avoided in patients with recent thromboembolism (< 3 months). Ragni MV. Hematology Am Soc Hematol Educ Program. 2013:44; Holland L et al. Transfusion. 2009;49(6):1171

© 2014 Direct One Communications, Inc. All rights reserved. 19 Surgical and Trauma-related Bleeding Even in the absence of a thromboembolic history, dosing should be judicious. For example, for four-factor PCCs: » 25 U/kg for INR 2.0–3.9 » 35 U/kg for INR 4.0–6.0 » 50 U/kg for INR > 6.0 The lowest effective dose of rFVIIa has not been established. Pabinger I et al. J Thromb Haemost. 2008;6:622; Levy JH et al. Anesthesiology. 2008;109:918

© 2014 Direct One Communications, Inc. All rights reserved. 20 Bleeding Due to New Oral Anticoagulants It is unclear if bleeding due to FXa and FIIa inhibitors such as dabigitran and rivaroxaban can be reversed by PCCs or rFVIIa. Although they may be considered, caution should be exercised, as there appears to be little correlation between correction of laboratory abnormalities and reversal of bleeding, and dosing and monitoring are not established. New antidotes are under development but are experimental at this time; transfusion support and surgical hemostasis should be provided if indicated. Pragst I et al. J Thromb Haemost. 2012;10:1841; Eerenberg ES et al. Circulation. 2011;124:1573; Weitz JI et al. Circulation. 2012;126:2428; Baglin T et al. J Thromb Haemost. 2013;11:756

© 2014 Direct One Communications, Inc. All rights reserved. 21 Conclusion Advances in synthetic clotting factors promise to increase the half-life, decrease the frequency of infusions, and potentially improve compliance and access while decreasing complications. PCCs, FEIBA, and rFVIIa may help decrease the risk of life-threatening bleeding for hemophilia patients as well as those on warfarin or new oral anticoagulants with surgical or traumatic bleeding. As with all these agents, caution must be exercised, given the risk of upsetting the balance between hemostasis and thrombosis; nonetheless, the coming years promise major advances in clotting factor treatments.