What’s Up with Bleeding 2012

1. What’s Up with Bleeding2012 Michael Recht, MD, PhD Director, The Hemophilia Center Oregon Health & Science University

2. Disclosures • Research funding directly to OHSU from • NovoNordisk • Baxter • Biogen Idec • Pfizer

3. Agenda • Bio-engineering of coagulation proteins • Chemical modification • Fusion to protein conjugates • Alternative hemostatic approaches • Gene transfer

4. History of hemophilia • The existence of hemophilia has been known for centuries—first mentioned in the Talmud ~2000 years ago • “If she circumcised her first child and he died, and a second one also died, she must not circumcise her third child”

5. History of hemophilia therapy • Prior to the 1940’s-supportive care and whole blood transfusion • Low concentration of factors VIII and IX • Individuals suffered significant pain and morbidity • Average lifespan 27 years • 1964-Judith Graham Pool described method to produce cryoprecipitate • Rich in FVIII and fibrinogen • Beginning of home infusions • Average lifespan 40 years

6. History of hemophilia therapy • 1970’s-plasma-derived factor concentrates • Pools of 20,000+ donors • Made school, work, and travel possible • Average lifespan 60 years

7. However…. • Hepatitis B and C were known to be in plasma supply • Thought to be an “acceptable” risk in light of drastic improvement in quality of life • First individual with hemophilia that died from HIV infection reported in 1982 • Only retrospectively was it discovered that plasma-derived factor was vector • HIV was isolated in 1984 • Heat treatment of plasma-derived factor became standard practice in 1985

8. Recombinant factor VIII • FVIII gene sequenced and cloned in 1984 • First clinical trial of recombinant FVIII concentrate reported in 1990 • First recombinant FVIII concentrate marketed for clinical use in 1992

9. Recombinant factor IX • Human FIX gene was cloned in 1982 • Minor differences in post-translational sulfation between recombinant FIX and plasma derived FIX • 30% lower in vivo recovery of recombinant FIX • Became available for clinical use in 1994

10. Engineering of coagulation proteins • Impetus derives from clinical limitations of current therapies • Require IV access • Must be given repeatedly due to short half-lives • Associated with development of inhibitory antibodies • 30% of patients with hemophilia A • <5% of patients with hemophilia B (though associated with anaphylaxis) • Available therapy for those with inhibitors is suboptimal

11. Rational design of new products • Increase half-life • Improve ease of delivery • Reduce immunogenicity • Increase potency

12. Wish list Fogarty, Hematology 2011

13. Chemical modification

14. PEGylation • Covalent conjugation of polyethylene glycol (PEG) to a therapeutic protein • Creates “cloud” around protein • Shields from exposure to: • Proteolytic enzymes • Clearance receptors • Immune effector cells • Effective at prolonging half-life of many other biopharmaceuticals • Interferon • Asparaginase • G-CSF

15. PEGylation concerns • PEG polymers may interfere with the peptide interaction with substrates, reducing activity • Randomly PEGylated B-domain-deleted recombinant factor VIII Rostin, BioconjugateChem, 2000

16. Site-directed PEGylation Mei, Blood, 2010

17. Glyco-PEGylation • Recombinant factor VIIa(Ghosh, J ThrombHaemost, 2009) • Increased factor X cleavage at lower concentrations • Reduced interaction with phospholipids • NN7128-three phase 1/2 clinical trials complete • Recombinant factor IX • Equivalent potency to recombinant factor IX • Better duration of hemostasis after bleeding challenge

18. Glyco-PEGylation • Recombinant factor IX • Equivalent potency to recombinant factor IX • Better duration of hemostasis after bleeding challenge in mice • Half-life prolonged to 93 hours Negrier, Blood, 2011

19. Liposomal PEGylation (PEGLip) • Liposomes are small vesicles consisting of a phospholipid bilayer surrounding aqueous interior • PEGylation of the liposome decreases clearance • No difference in clotting or other laboratory parameters between study drug and standard recombinant factor VIII concentrate. • There was a trend towards fewer bleeding episodes 14 days after infusion of PEGLip-recFVIII concentrate • Pulled from further trials Powell, J ThrombHaemost, 2008

20. Long term use of PEGylated proteins • Accumulation of PEG polymers has been documented in some preclinical studies • Whether accumulation will be evident in those receiving these medications over a life-time is unknown • In other compounds, anti-PEG antibodies have been demonstrated

21. Fusion to protein conjugates

22. rFVIII:Fc-IgG fusion (rFVIIIFc) • IgG molecule has a prolonged t1/2 secondary to continual recycling • Fc domain of IgG is a “natural” molecule with no known toxicity • rFVIIIFc is a single molecule of rFVIII covalently linked to human IgG1 Fc domain • Compared to full length rFVIII, rFVIIIFc had a 1.7-fold longer t1/2 Powell, Blood, 2012 preprint

23. rFIX:Fc-IgG fusion (rFIXFc) • Same technology as used in rFVIIIFc • Compared to rFIX, t1/2 was extended from 18 hours to 60 hours Shapiro, Blood, 2012 preprint

24. Prophylaxis schedule with rFIXFc Shapiro, Blood, 2012 preprint

25. Alternative hemostatic approaches • Non-peptide molecules may provide opportunities to enhance hemostasis in bleeding disorders patients • These molecules have the potential for oral or subcutaneous administration

26. Aptamers • Ribosomal or deoxyribosomaloligonucleic acids that can be produced to any length or 3D conformation • Allow binding to an unlimited array of physiologic targets

27. ARC19499 • Aptamer antagonist of tissue factor pathway inhibitor • Improved thrombin generation in plasma from subjects with FVIII deficiency • Improved clot times as measured by thromboelastography in monkeys depleted of FVIII Waters, Blood, 2009

28. Fucoidans • Also known as non-anticoagulant-sulfated polysaccharides (NASPs) • Heterogeneously sized anionic compounds that derive from marine plants (brown seaweed) • Have demonstrated pharmacologic activity: • Anti-cancer • Anti-inflammatory • Anti-angiogenesis • Anti-coagulant

29. AV513 • Fucoidan with anti-TFPI activity • Fed to hemophilia A dogs: • Improved clotting time as measured by TEG • Improved cuticle bleeding time • Phase 1 trial of BAX513 in healthy subjects is underway Prasad, Blood, 2008

30. Read-through of nonsense mutations • Nonsense mutations (premature termination codons [PCTs]) account for 11% of known mutations in severe hemophilia • First described in 1964, new compounds are being developed to promote ribosomal read-through of PCTs • Has been shown to be safe and effective in restoring the cystic fibrosis and Duchenne muscular dystrophy proteins

31. Ataluren (PCT124) • No major adverse events reported in CF or Duchenne MD • Currently phase 2 trial of 28 day treatment cycle with Ataluren in hemophilia A and B is recruiting patients

32. AAV-vector mediated gene transfer in hemophilia B • AAV8 vector • Packaged as complementary dimers within single virionmediate transgene expression at higher levels than single-stranded AAV vectors • AAV8 has lower seroprevalence that previous AAV vectorsdecreasedhumoral immunity • Since AAV8 has tropism for liver, able to administer via peripheral vein Nathwani, NEJM, 2011

33. Study design Inclusion Criteria Dose level cohorts Cohort 1: 2 x 1011 vg/kg Cohort 2: 6 x 1011 vg/kg Cohort 3: 2 x 1012 vg/kg • 18-60 years of age • Factor IX activity < 1% • No history of inhibitor or anaphylaxis to FIX • No immunity to AAV8

34. Results • AAV-mediated expression of FIX at 2-11% of baseline levels was observed in all participants • Four of the six discontinued FIX prophylaxis • In the other two, time between prophylactic infusions has increased • Both participants receiving highest dose had asymptomatic elevations of AST treated with short course of prednisone

35. Results Nathwani, NEJM, 2011

36. Conclusions • Peripheral vein infusion of AAV8 vector resulted in FIX expression at levels sufficient to improve bleeding phenotype with few side effects • Immune-mediated clearance of AAV-transduced hepatocytes remains a concern

37. Benefits of correcting hemostatic defect in hemophilia Fogarty, Hematology 2011

38. Benefits of novel therapeutics • Prophylaxis • Half of US treatment centers do not follow the advice of MASAC of NHF to start three times weekly prophylaxis in those with severe hemophilia • Requirement of frequent infusions and need for central venous access in children most common reason given • Fewer infusions and non-intravenous alternatives may improve adherence and long term outcome

39. Benefits of novel therapeutics • Joint health • Early three times weekly prophylaxis has been demonstrated to protect the joints of children with severe hemophilia A • Follow-up to the initial study is ongoing, but preliminary data indicate that this protective effect continues into adolesence • Decreasing infusion frequency may increase adherence

40. Benefits of novel therapeutics • Quality of life • QoL increases in children with severe hemophilia A on prophylaxis compared to those receiving factor on-demand • The potential impact of improved hemostasis with new products is substantial • Decreased missed work or school • Decreased pain

41. Questions?

42. Conclusions • Great strides have been made in the treatment of hemophilia • Understanding of the inherent limitations of currently available therapies • Unmet clinical needs of the affected population • Novel agents hold promise for: • Improved safety and efficacy • Improved joint health • Improved quality of life • Increased tolerability

43. Contact information • The Hemophilia Center at OHSU • 503-494-8716 (office) • 503-494-0714 (fax) • rechtm@ohsu.edu