1. © 2014 Direct One Communications, Inc. All rights reserved. 1 Treatment of Hemophilia: What’s in the Pipeline? Kerry Hege, MD Indiana University School of Medicine, Indianapolis, Indiana 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)

2. © 2014 Direct One Communications, Inc. All rights reserved. 2 Treatment of Hemophilia: Past, Present, and Future The past four decades have witnessed great strides in hemophilia therapy. Franchini M, Mannucci C. Orphanet J Rare Dis. 2012;7:2

3. © 2014 Direct One Communications, Inc. All rights reserved. 3 Goals of the Hemophilia Pipeline Improving patient outcomes with more effective bleeding control and preservation of joint function Reducing the burden of factor administration through reduction in dosing frequency and more cost-effective therapy Individualizing therapy by adapting to individual pharmacokinetics and personalizing treatment regimens based on variability of bleeding phenotype, and lifestyle Identifying, monitoring, and preventing age-related comorbidities Developing a cure for hemophilia through gene therapy

4. © 2014 Direct One Communications, Inc. All rights reserved. 4 Recombinant Factors in Clinical Trials Note: Since this table was published, the US Food and Drug Administration approved the use of recombinant factor IX Fc fusion protein (rFIXFc; Alprolix) in adults and children with hemophilia B. Peyvandi F et al. J Thromb Haemost. 2013;11(suppl 1):84; Pipe SW. What's in the pipeline? NHF 2013; ClinicalTrials.gov Web site: http://clinicaltrials.gov

5. © 2014 Direct One Communications, Inc. All rights reserved. 5 Bioengineering Approaches Bioengineering approaches to extending the half-life of recombinant coagulation factors have employed several strategies, including: Reduction of exposure to clearance receptors through PEGylation with polyethylene glycol (PEG) Rescue of endocytosed proteins from intracellular degredation by Fc fusion and albumin fusion proteins Enhanced interactions with von Willebrand factor (vWF)

6. © 2014 Direct One Communications, Inc. All rights reserved. 6 PEGylation PEGylation improves drug efficacy via the covalent attachment of polyethylene glycol molecules (PEG) to the protein of interest—in this case, recombinant factor proteins. Peyvandi F et al. J Thromb Haemost. 2013;11(suppl 1):84

7. © 2014 Direct One Communications, Inc. All rights reserved. 7 PEGylated Liposomes Another approach to prolonging the half-life of recombinant factor proteins is by attaching them to the outer surface of PEGylated liposomes via noncovalent binding. Peyvandi F et al. J Thromb Haemost. 2013;11(suppl 1):84

8. © 2014 Direct One Communications, Inc. All rights reserved. 8 Polysialylation When polysialic acid polymers are attached to recombinant factor proteins, they attract water and produce a watery “cloud” that surrounds the protein to protect it from clearance receptors, proteolytic enzymes, and immune- mediating cells. Peyvandi F et al. J Thromb Haemost. 2013;11(suppl 1):84

9. © 2014 Direct One Communications, Inc. All rights reserved. 9 Fc Fusion Peyvandi F et al. J Thromb Haemost. 2013;11(suppl 1):84 Fusion of recombinant factor to the crystallizable fragment (Fc) portion of immunoglobulin G protects the factor protein from lysosomal degradation via interactions with neonatal Fc receptors when internalized by endothelial cells.

10. © 2014 Direct One Communications, Inc. All rights reserved. 10 Albumin Fusion Peyvandi F et al. J Thromb Haemost. 2013;11(suppl 1):84 Albumin fusion binds human albumin to factor protein, lengthening its half-life, protecting it from proteolytic degradation, and shielding the factor protein from exposure to immune-mediating cells, further prolonging its half-life and decreasing its immunogenicity.

11. © 2014 Direct One Communications, Inc. All rights reserved. 11 Enhanced Interactions with vWF Pabinger-Fasching I, Pipe S. Thromb Res. 2013;131:S1; Zollner SB et al. Thromb Res. 2013;132:280 The primary determinant of the half-life of FVIII is interaction with vWF, which naturally protects it from degradation. A unique recombinant, single-chain rFVIII (CSL627) has shown improved stability and higher affinity for vWF when compared with other rFVIII proteins. In preclinical studies, CSL627 has demonstrated safety and efficacy with hemostatic activity equivalent to that of full-length rFVIII formulations. The novel single-chain design provides for higher intrinsic stability and affinity for vWF.

12. © 2014 Direct One Communications, Inc. All rights reserved. 12 Bispecific Antibodies Lillicrap D. Nat Med. 2012;18:1460; Kitazawa T et al. Nat Med. 2012;18:1570 In addition to improving factor proteins themselves, a novel approach has been taken to replace FVIII cofactor function by a small molecule, creating a bispecific antibody capable of mimicking activated factor VIII (FVIIIa) activity. In preclinical studies, this bispecific antibody to FIXa and FX (hBS23) had a terminal half-life of 14 days and a subcutaneous bioavailability of nearly 100%.

13. © 2014 Direct One Communications, Inc. All rights reserved. 13 Gene Therapy Hemophilia makes a good candidate for gene therapy because it represents a monogenetic disease that requires the production of only a small fraction of normal factor activity to ameliorate or cure the bleeding phenotype in hemophilia. The most recent human trials of gene therapy in patients with hemophilia have achieved long-term expression of therapeutic factor levels. The field is exploring ways to improve gene delivery, minimize vector immunogenicity, prolong gene expression, and raise factor activity levels. High KA. J Thromb Haemost. 2011;9(suppl 1):2; High KA. Blood. 2012;120:4482; Chuah MK et al. J Thromb Haemost. 2013;11:99

14. © 2014 Direct One Communications, Inc. All rights reserved. 14 Where Are We Today? At least three different FIX products created using three different bioengineering approaches reveal terminal half-lives as much as fivefold greater than those of standard rFIX products. The available data on novel recombinant FVIII products, also using different engineering methods, show terminal half-lives up to 1.8 times longer than those of standard rFVIII products. Randomized clinical trials are currently underway to determine whether the extended half-lives of these new recombinant products will translate into fewer factor infusions, as much as two weeks apart.

15. © 2014 Direct One Communications, Inc. All rights reserved. 15 Conclusion Early pharmacokinetic data for new factor products are promising, but ultimately the ongoing phase 1–3 trials will establish what effect these new products will really have on: Patient’s individual dosing schedules Overall use of factor replacement products Patient adherence to factor replacement therapy Inhibitor development Need for venous access And the overall cost of factor replacement therapy