BPAC 2010 Laboratory of Hemostasis

1. BPAC 2010 Laboratory of Hemostasis Basil Golding M.D. Director, Division of Hematology OBRR/CBER/FDA

4. Laboratory of Hemostasis Research/Regulator • Chava Kimchi-Sarfaty, Ph.D., Senior Staff Fellow • Nobuko Katagiri, Ph.D., Staff Fellow • Mikhail Ovanesov, Ph.D., Visiting Scientist • Andrey Sarafanov, Ph.D., Chemist • Zuben Sauna, Ph.D., Visiting Scientist

5. Laboratory of Hemostasis Full-time Regulatory Reviewers • Natalya Ananyeva, Ph.D., Visiting Scientist • Roman Drews, Ph.D., Chemist • Don Lebel, B.S., Medical Technologist ( Retired in June 2010) • Tim Lee, Ph.D., Chemist/Acting Chief • Ze Peng, Ph.D., Visiting Associate • Laura Wood, M.S. Biologist

6. Licensed products regulated by LH • Coagulation Factors • Factors VIII and IX (Human plasma-derived & Recombinant) • Factor VIII/von Willebrand Factor Complex (Human plasma-derived) • Fibrinogen Concentrate • “Bypassing” Products • AICC (e.g., FEIBA) • Recombinant activated Factor VII • Hemostatic Agents • Thrombin (Bovine, Human & Recombinant) • Fibrin Sealant • CryoSeal FS System • Fibrin Sealant Patch • Anti-coagulants • Protein C • Antithrombin III (Human plasma-derived & Transgenic)

7. Laboratory of HemostasisRegulatory Responsibilities • Review applications for: • Investigational products • Marketing of new products • Changes in manufacturing of, or indications for licensed products • Lot release testing of licensed products • Inspection of manufacturing facilities • Compliance and enforcement actions

8. Laboratory of HemostasisRegulatory Responsibilities (Cont.) • Review of Biological Product Deviation Reports • Assessment of risk and response • Develop policy and guidance • Product safety and efficacy • Patient and physician information • Current Good Manufacturing Practices • Pre-submittal Support • Review of briefing material • Meeting with sponsors • Preparation of summaries

9. Regulatory Activities 2005 - 2010 BLA Approvals • CEPROTIN (p.d. human Protein C) • RECOTHROM (rec. human thrombin) • EVITHROM (p.d. human thrombin) • XYNTHA (rec. human Factor VIII) • ARTISS (p.d. human fibrin sealant) • RiaSTAP (p.d. human fibrinogen) • ATRYN (transgenic human ATIII) • WILATE (p.d. human vWF/FVIII complex) • TachoSil (Fibrin Sealant Patch) • Fibrinogen BDS (p.d. human) • Thrombin BDS (p.d. human)

10. Regulatory Activities 2005 – 2010 cont’d Reviewed over • 400 BLA Supplements • 100 Annual Reports • 100 other miscellaneous submissions • 1600 lots tested for release • 50 original IND’s & their associated amendments Participated in • Facility inspections on-site and by phone • International calibration studies for reference standards

11. Coagulation Factor Standards

12. Emerging Products • Recombinant porcine Factor VIII • PEGylated coagulation factors • Fc coagulation factor fusions • Improve potency, expression, clearance • Proteins for rare bleeding disorders: • Factor XIII • Factor XI • Factor X • rADAMTS13 • von Willebrand Factor (recombinant)

13. Laboratory of HemostasisResearch Activities

14. Toward More Effective Treatment of Blood Clotting Disorders: Pharmacogenomic Studies of ADAMTS13 and Related Proteins Chava Kimchi-Sarfaty, Ph.D. Laboratory of Hemostasis DH / CBER / FDA

15. FDA Mission Relevance:Pharmacogenomics of Products Regulated by FDA New concept paper/guidance on clinical pharmacogenomics discusses the following issues: • FDA’s current view on whether or not clinical pharmacogenomic studies should be performed • General strategies for using pharmacogenomic information in drug development • Design of clinical pharmacogenomic studies • Incorporation of pharmacogenomics information on the drug label However, currently there are no regulations or guidelines vis à vis the choice of sequence in the development of a recombinant therapeutic protein

16. What is a SNP? • SNP: Single-nucleotide polymorphism • Synonymous SNP: No change in amino-acid sequence • Non-synonymous SNP: Altered amino-acid sequence • Polymorphism: DNA variation in which sequence is present in at least 1% of the population • Most common type of genetic variation • May occur every 100-300 bases

17. Potential effect of SNPs on protein structure and function Effect on protein EFFICACY Implications For Protein Therapeutics Protein Expression Protein sequence Protein conformation Truncated protein SAFETY STABILITY IMMUNOGENICITY

18. Aims • Understand the consequence of synonymous and non-synonymous mutations and SNPs on the development of recombinant therapeutic proteins • Develop tools to predict the consequences of changes made to wild-type sequence during manufacture of recombinant therapeutic proteins

19. Non-synonymous Synonymous Frame shift 2280 SNPs in the ADAMTS13 protein that were investigated in our laboratory Metaloprotease CUB1 and 2 TSPs Disintegrin Spacer SNP studied by our lab • Bolded SNPs indicate syn. SNPs • Red letter indicates the nucleotide that was changed within the variant codon

20. In Silico Studies I. ADAMTS13, the von Willebrand Factor (VWF) cleaving protease: We have completed the in silico studies of twelve Single Nucleotide Polymorphisms (SNPs) in ADAMTS13, located in various domains of the protein: The results of these in silico studies were compared to in vitro studies

21. In vitro studies of twelve ADAMTS13 SNPs Trypsin Digestion Western Blotting Real Time- PCR Flow Cytometry FRETS- VWF73 mRNA levels Intracellular protein levels & conformation changes ADAMTS13 activity Protein conformational changes Intracellular and extracellular protein levels • High correlation between in silico & in vitro studies provides tools to examine the effect of mutations on therapeutic proteins • We demonstrate that both synonymous and non-synonymous mutations affect protein expression, conformation and/or function

22. II. Factor IX: In silico and in vitro studies of synonymous mutation Val107Val in F9 gene, found in five unrelated Swedish Hemophilia B patients: In silico and in vitro assays performed as described previously for ADAMTS13 Conclusions: • Computational analyses suggest that the synonymous V107V mutation in F9, located at a region critical for protein folding, could change translation rate/rhythm, therefore affecting the protein’s conformation. • Experimental analyses demonstrated a significant change in conformation between Val107Val mutation and WT.

23. Future Studies • Develop new assays that can differentiate between different molecular forms of ADAMTS13, VWF, FVIII, and FIX based on increased sensitivity • Characterize ADAMTS13 SNPs in healthy, non-TTP individuals and correlate the in vitro analyses with in vivo characterization • Characterize additional FIX SNPs and mutations using in silico and in vitro analyses • Study WT vs. V107V FIX in hemophilia B knock-down mice

24. The Immunogenicity of Protein Therapeutics Zuben E. Sauna Ph.D.

25. Importance of project to the FDA • The FDA has been regulating biotechnology-derived protein products since the 1980s • More than 200 biopharmaceutical proteins have now been approved • Widespread use of biopharmaceuticals has demonstrated that nearly all biologicals can elicit antibody responses • Inhibitory antibodies to therapeutic proteins compromise efficacy and also cross-react with endogenous factors to cause serious toxicity • Even non-inhibitory antibodies can complicate interpretation of the toxicity, pharmacokinetic and pharmacodynamic data

26. Specific aims • Develop computational and in vitro methods for the pre-clinical identification of T-cell epitopes on the FVIII protein • Determine the role of sequence mismatch between the endogenous (albeit non-functional) and infused FVIII in eliciting immunogenicity • Pharmacogenetics and the development of inhibitory antibodies to FVIII: Evaluate genetic variability in the endogenous F8 gene vis- à-vis polymorphisms as a risk-factor for immunogenicity Evaluate HLA restriction of patients (in the context of sequence mismatch) as a risk-factor for immunogenicity

27. Accomplishments (1) • Computational predictions of peptide binding to specific MHC proteins, using 56 FVIII peptides, correlate well with in vitro binding measurements using purified recombinant MHC Class II alleles (p value = 0.000065). • Historical data shows that some missense mutations in the FVIII gene are associated with inhibitory antibodies while others are not. A missense mutation results in a localized mismatch between the sequence of the endogenous and infused FVIII (i.e. peptides from this region are foreign). An ‘immunogenicity score’ devised by us discriminates between mutations that do and do not elicit inhibitory antibodies (p value = 0.008). • A recent report suggests that African Americans develop inhibitory antibodies at higher frequencies due to the greater diversity in F8 gene polymorphisms in these patients. Peptides that span SNPs associated with inhibitory antibodies bind with higher affinity to MHC alleles commonly found in African Americans.

28. Accomplishments (2) • Demonstration that cells and tissue samples from hemophilia A (HA) patients with the intron 22 inversion (I22I) synthesize the entire FVIII polypeptide chain; however the primary sequence is contained in two polypeptide chains • The I22I represents ~50% of severe HA patients; the immunological implication of our finding is that these patients (despite a large gene alteration) may be tolerized to the FVIII protein Conclusions A pharmacogenetic approach, based on individual patients, is necessary for the accurate prediction of immunogenicity The extent and position of sequence mismatch between the endogenous and infused FVIII (due to disease causing mutations and SNPs) and the HLA restriction of the patient are important risk-factors for immunogenicity

29. Future Plans • Develop a hierarchical decision making tool for individualized immunogenicity risk assessment for HA patients using clinical samples to test our hypotheses • Develop strategies to reduce the disproportionate frequency of adverse alloimmune events in vulnerable populations • Determine whether principles developed in FVIII for a personalized assessment of immunogenicity risk can be applied to therapeutic proteins in general

30. Mikhail V Ovanesov, PhD Study of the regulation of blood coagulation kinetics

31. Public Health Issue: • Existing clinical assays of blood coagulation have poor predictive value • Coagulation testing can be improved to reflect physiological conditions Research Approach • Development and optimization of novel assays that measure ‘global’ hemostasis function • Investigation of potency and thrombogenicity of FVIIa, FIXa and FXIa Regulatory Contribution: • Product evaluation • Enhancing the assurance of the efficacy, safety and quality of coagulation products

32. Program is technique-based: a 7-year plan Assay development Assay application 2009 2010 2011 2012 2013 2014 years Thrombin generation test Mechanism of Action, Potency, Thrombogenicity 1. Clot growth assay (videomicroscopic) 2. Coagulation in mouse (deep tissue imaging) 3.

33. Development of the Thrombin Generation Test to facilitate the characterization and regulation of plasma-derived products • Funded by the FDA CBER Modernization Science Research goals: • Optimization of the assay for hemophilia treatments and thrombogenicity • Comparative analysis of commercially available and in house TGT variants • Application to outstanding regulatory issues • e.g.compare products with known controversies in potency assessment (Factor VIII and Factor VIIa variants)

34. Applying Thrombin Generation Test to study thrombogenicity of IGIV samples(Case study) • Octagam® (Octapharma): immunoglobulin intravenous (IGIV) • 2010: High incidence of Thrombotic Adverse Events (TAE): strokes, myocardial infarctions • Our testing indicated a possible root cause for TAEs: Factor XIa is the likely contaminant • As a result of TAEs and TGT-based testing, Octapharma voluntarily withdrew the product from the U.S. market (September 2010)

35. Research in progress: application of videomicroscopy to IGIV thrombogenicity testing Thrombosis-implicated lots Control non-implicated lots Negative control (buffer) • 40 minutes of video shooting • Factor XI-deficient plasma • supplemented with IGIV product • (4 different lots) or buffer

36. Ongoing efforts of our research team • Distribution of the CBER TGT protocol among all major IGIV manufacturers • Screening of marketed plasma derived products for thrombogenic contaminants • Collaboration with other regulatory agencies and industry • Development of thrombogenicity standards (with NIBSC, PEI, EMA)

37. Andrey Sarafanov Ph.D. Identification of Binding Sites on Coagulation Factor VIII (FVIII) for its Catabolic Receptors: Low-Density Lipoprotein Receptor (LDLR) Low-Density Lipoprotein Receptor-Related Protein (LRP)

38. Relevance of the project to FDA mission: • Deficiency in Factor VIII (FVIII) results in coagulation disorder (Hemophilia A). The disease is treated by infusions of FVIII products • Currently, new FVIII products with prolonged time in the circulation are under development by pharmaceutical companies • Regulation of these products requires an understanding ofthe mechanisms of FVIII clearance from the circulation

39. The Highlights of the Project LRP and LDLR: Expressed in the liver, and catabolize various ligands (plasma lipoproteins etc). Cooperate in clearance of FVIII: deficiency in both receptors results in prolongation of the half-life of FVIII by 5 times in mice The ongoing project involves: establishing expression of FVIII, LRP and LDL fragments, performing scanning mutagenesis of FVIII, testing interaction of the FVIII mutants with LRP and LDL in various in vitro and in vivo assays to determine the binding epitope(s) on FVIII. Future Plans: Testing possibility of generation of "improved" (long-lasting) FVIII for better treatment of Hemophilia A. The “improved” FVIII would have a decrease in the affinities for LDLR and LRP, while retaining activity, but not more immunogenic

40. LRP and LDLR • Evolutionarily related and similar in structure • Expressed in the liver, and serve a function to catabolize various ligands (plasma lipoproteins etc.) • Cooperate in clearance of FVIII: deficiency in both receptors results in prolongation of the half-life of FVIII by 5 times in mice • Molecular interactions of FVIII with LRP and LDLR are similar

41. Mapping the LRP- and LDLR-binding Site(s) on FVIII: Selection of FVIII amino acid residues for scanning mutagenesis Expression of fragments of FVIII, LDLR, LRP and FVIII mutants Performing binding assays for FVIII variants and receptors’ fragments Testing receptor-mediated internalization of FVIII variants in cell culture Testing clearance of FVIII variants in mice (normal, FVIII- and vWF-deficient) Determination of critical residues of FVIII for binding to LRP

42. A1 A2 A1 A2 I. Ongoing Experiments: Expression of FVIII and its variants Heavy Chain Light Chain Ca2+ FVIII (~300kDa) C2 A3 B C1 A1 A2 C 1 372 740 2332 1648 1649 2019 2173 LRP (LDLR) -binding region Constructs to be Generated FVIII variants (B-domain deleted) C2 A3 C1 Introduction of point mutations Heavy Chain C2 A3 C1 Light Chain variants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

43. LDLR N C (~160 kDa) Ligand-binding part Hepatic cell membrane β-propeller domain EGF-like repeats Complement-type repeats Transmembrane domain Cytoplasmic domain O-linked sugar domain II. Ongoing Experiments: Expression of LDLR Fragments Generated constructs: (a minimal ligand-binding motif is composed by two adjacent CRs) 1) CR 1-7 . . . . 2) CR 1-2 . . . . 3) CR 2-3. . . . . . . 4) CR 3-4 . . . . . . . . . 5) CR 4-5 . . . . . . . . . . . 6) CR 5-6 . . . . . . . . . . . . . . 7) CR 6-7 . . . . . . . . . . . . . . . .

44. Future Plans Generation of FVIII having a decrease in affinities for LDLR and LRP yet retaining the functional activity, and not increased immunogenicity. The “improved” FVIII would have a longer half-life in the circulation, thus requiring less frequent infusions into Hemophilia A patients, and a reduced cost.

45. Thank you