Division of Hematology

1. Division of Hematology Basil Golding M.D., Division Director Office Site Visit 2005

2. Division of Hematology Research/Review Units LBVB LCH LH LPD CRB Biochemistry & Vascular Biology Cellular Hematology Hemostasis Plasma Derivatives Clinical Review Branch PIs: 3 2 2 4 Total = 11

3. Division of Hematology Scope of Regulation Products (Biologics, Drugs, Devices) • Cellular components of blood e.g. platelets • Plasma-derived (Cohn-Oncley Fractionation) • Analogous recombinant e.g Factor VIII Clinical indications • Bleeding disorders • Shock/hypovolemia • Infectious diseases/immunological deficits • Replacement therapy in congenital or acquired deficiencies

4. Research Priorities:Critical Path • Safety • Product toxicity (HBOCs) • Contaminants (Microbial) • Viral transmission (HCV) • Efficacy • Standards, assays (HBOCs) • Animal models (platelets) • Counter-Terrorism (anthrax, smallpox) • In vitro assays of potency • Animal models

5. Hemoglobin-Based O2 Carriers Public Health Impact: Oxygen delivery in situations when blood is not immediately available or acceptable • Trauma (battlefield, rural areas) • Religious reasons • Blood shortages

6. Hemoglobin-Based O2 Carriers (LBVB) • Regulatory and Scientific Challenges: • Characterization of HBOC structure-function • Effects of chemical modifications • Development of pre-clinical models to evaluate HBOC safety

7. O-R-PolyHbA0Hallmarks of Functional Abnormality • Non-sigmoidal oxygen equilibrium curve • Non-saturating • Non-cooperative (Hill coefficient = 1.0 vs. 2.5) • pH insensitivity Biochemistry (2002)

8. O-R-PolyHbA0Identification of the Origin of Altered Function O2 O2 Locked (T) State O2 O2 O2 O2 Tense (T) Oxy O2 O2 O2 Normal Conformational Change O2 O2 Tense (T) Deoxy Relaxed (R) Oxy Tetragonal Heme Fe Rhombic Heme Fe (1) Heme Destabilization O-R-PolyHbA0 (EPR) HbA0 (2) Protein Destabilization (locked T state) MALDI-MS Biochemistry (2002), Biochemical J. (2004)

9. O-R-PolyHbA0:Actual Chemical Modification • Non-specific cross-link • Non-uniform O-raffinose • Modified cysteines Boykins,Buehler, Alayash. Proteins (2005)

10. HBOC-Induced Endothelial Cytotoxicity Redox Active aaHb Medium D’Agnillo, Am J Physiol. (2004), Blood (2001)

11. HBOCs: Outcomes (LBVB) • Establishing methodologies that distinguish between functional and non-functional HBOCs • Development of an endothelium-based assay that correlates with HBOC toxicity in vivo

12. Alpha-1-Proteinase Inhibitor (a1-PI)(LBVB) Public Health Impact: • Most patients with hereditary a1-PI deficiency develop fatal emphysema and about 15% develop severe liver disease. • Such deficient patients (approx. 4,000) can benefit from augmentation therapy.

13. a1-PI: Safety Regulatory and Scientific Challenges: Aggregated a1-PI can cause adverse events and decreases potency of a1-PI. • How do a1-PI polymers form? • How can polymer formation be avoided during manufacture?

14. Crystal Structure of a1-PI A b-sheet flexible reactive loop reactive center loop a1-PI polymerization is initiated by partial unfolding of monomer. free sulfhydryl Original Loop-Sheet Model, based on indirect evidence

15. Dimer Formed By Partial Unfolding of Monomer Continues to Polymerize After Refolding to Form Aggregate. High Aggregate SE-HPLC New model explains spontaneous polymerization of dimer to form aggregate. Marszal, Danino, and Shrake: J. Biol. Chem. (2003)

16. a1-PI: Outcomes • New understanding ofa1-PI polymer formation. • This knowledge can be used to minimize unfolding of monomers and dimer formation during manufacturing, e.g. adding appropriate stabilizers during heat treatment . Future plans Investigate the conformational change in dimers that results in spontaneous polymerization.

17. Immune Globulin Intravenous(LPD) Public Health Impact: Product Contamination • Infusion of IGIV causes adverse events – 25% • Sterile filtration of final product does not remove microbial components, e.g. LPS, DNA. • Microbial contaminants  proinflammatory cytokines  side effects

18. Immune Globulin IntravenousProduct Contamination Regulatory and Scientific Challenges: • Need for rapid high throughput assays to detect microbial components • Adapt cell lines expressing Toll-like receptors as a detector system

19. Toll-Like Receptors (TLRs) and Their Ligands Pathogen Recognition NF-B-luciferase NF-B Nucleus Nucleus Takeda and Akira Inf. & Imm. 2005

20. HEK-293 Cells Transfected with TLRs and a Reporter Gene Respond to Microbial Ligands Huang et al, J. Immunol. 171(3):1441-6. 2003

21. IGIV: Outcomes • Cell lines expressing multiple TLRs and a reporter gene can detect microbial components. • These cell lines will be used to develop a rapid high throughput system for testing IGIVs and other CBER products for microbial contaminants.

22. Cellular Components: Platelets (LCH) • Public Health Impact: • 10 million PLT units are transfused annually • in the US. • Transfusion of suboptimal products leads to decreased circulation time  more transfusions  increased risk of infection and alloimmunization.

23. Cellular Components: Platelets(LCH) Regulatory and Scientific Challenges: • Testing for Efficacy • In vitro tests - informative but not predictive • In vivo survival of radio-labelled platelets in humans is burdensome • Adverse Effects • Possibly due to microparticles

24. 1 day 7 day Animal Model for Evaluating Damage to Human Platelets During Collection, Processing and Storage In vivo performance of 1 and 7 day old human platelets in SCID mice Survival of human platelets in Control and SCID mice Control SCID

25. Platelets: Outcomes(LCH) • New in vivo assay in SCID mice was developed for measuring platelet survival. • Flow cytometric assays were developed to detect microparticles in platelet products: this will enable us to determine whether MPs in products are associated with adverse events (thrombosis, inflammation).

26. Viral Detection and Antiviral Antibodies: Immune globulins(LPD) Public Health Impact: An estimated 2 million Americans suffer from hepatitis C infection. • ~ 70% chronic hepatitis • Sequelae liver fibrosis  cirrhosis  HCC

27. Viral Detection and Antiviral Antibodies: Immune globulins(LPD) Regulatory and Scientific Challenges: • Screening plasma for HCV • Measuring HCV neutralizing Abs in HCIGIV • no in vitro system or small animal model is available for HCV infectivity • chimpanzee is the only model

28. HCV E1/E2 CMV gag-pol y GFP HCV Pseudoparticle System Step 1: Transfection and particle production CMV 293 T cell(s) Step 2:Infection of target cells GFP expression HCVpp entry Huh-7 cells Step 3: Quantification of infection by FACS

29. Pseudoparticle Assay Correlates With In Vivo Chimp Data Sample Anti-HCV V.I. Pseudoparticle Chimpanzee <1 : 20 neg S/D Not protected Control IGIV pos HCIGIV S/D 1 : 320 Protected HCIGIV (HCV RNA+) pos None Not infectious 1 : 320 Yu et al, PNAS 2004

30. Anti-HCV Screening Removed Nt Abs to HCV and Compromised Safety of IGIV Hepatitis Cases IGIV (Not V.I.) Pseudoparticle Non implicated lots made from anti-HCV unscreened plasma 1988-1990 1:320 0 0 1:320 0 1:320 0 1:320 4 <1: 20 Implicated lots made from anti-HCV screened plasma in 1993 2 <1: 20 <1: 20 60 18 <1: 20 Yu et al, PNAS 2004

31. HCIGIV: Outcomes (LPD) • HCV neutralization in pseudoparticle assay correlates with protection or lack of infection in chimps. • Pseudoparticle neutralization assay will facilitate development of new HCIGIV products. Future Plans • Identification and characterization of neutralizing epitopes

32. Inhibitory Antibodies to Factors VIII/IX (LH) Public Health Impact: Neutralizing antibodies to coagulation factors complicate the use of Factor VIII in ~20% of patients with severe hemophilia.

33. Inhibitory Antibodies to Factors VIII/IX (LH) Research and Regulatory Challenges: • Understanding the genetic factors that control whether patients make antibodies to Factor VIII/IX • Developing pre-clinical models to predict product efficacy

34. Mouse Pre-Clinical Models Using genetically well-characterized inbred mouse strains we showed that: • MHC genes, T-cell receptors, and zinc-a-2-glycoprotein 1 genes influence the antibody response to human factor VIII. • MHC genes and to a lesser extent, cytokine genes (IL10, Interferon-g) control the antibody response to human factor IX. Lozier, et al., Blood 2005;105:1029-1035.

35. Hemophilia A Dog Pre-Clinical Model The Chapel Hill hemophilia A dogs have a genetic defect identical to that in ~40% of humans with severe hemophilia A. The bleeding phenotype is identical to human hemophilia A. The dogs make inhibitors when treated with dog factor VIII. Lozier et al, PNAS 2002;99:12991-12996

36. Factors VIII/IX: Outcomes (LH) • Mouse genetic studies provide clues for probing the genetic predisposition to induction of inhibitory antibodies in humans. • The Chapel Hill hemophilia A dogs are an ideal model for preclinical evaluation of product potential to induce inhibitory antibodies.

37. Immune Globulins:Counter-Terrorism (LPD) Public Health Impact: • Anthrax is a major threat to public health and security. • Smallpox poses a potential bioterrorism threat. Widespread vaccination is expected to cause fatalities in susceptible individuals • Immune-compromised • Eczema

38. Immune Globulins:Counter-Terrorism (LPD) • Regulatory and Scientific Challenges: • In vitro and in vivo models are required to • assess efficacy of: • Anthrax Immune Globulins • Vaccinia Immune Globulins

39. Anthrax Immune Globulins • Inhalational anthrax • In 2001, 5/11 patients died despite antibiotic treatment. • Combined antibiotics and anti-anthrax antibodies may improve survival • Antibiotic targets the bacillus. • Antibodies target the toxins.

40. Toxins of Bacillus anthracis And Or Edema Factor Lethal Factor Protective Antigen (PA) Protective Antigen (PA) heptamers Cell lysis Cytokines LT Edema ET

41. Pilot studies in sheep Various anthrax immunogens DBA/2 Purified sheep antibodies Sheep were immunized with various proteins (PA or LF in adjuvant) or the Sterne strain agricultural vaccine. (FEMS Immunology and Med Micro, 2003)

42. Sheep-Derived Anthrax IG Protect DBA-2 mice From a Lethal Sterne Spore (1x106) Challenge (IP) 10 9 Control Ab 8 Anti PA 7 6 Cipro Number of mice surviving 5 Anti PA + Cipro 4 3 2 1 0 1 2 3 4 5 6 7 8 9 10 11 Days after challenge Antibodies (25 mg/kg) given day of challenge and Cipro (10mg/kg) given day after challenge-both given daily

43. Protective Effects of Anthrax Immune Globulin (LBVB) 120 100 MEK1 Cleavage Assay 80 Resistance (% control) 60 Medium Medium LT LT + AIG 40 LT 20 LT + AIG 0 0 20 40 60 80 Time (h)

44. Anthrax Immune Globulins: Outcomes (LPD/LBVB) • “Proof of Concept” that polyclonal antibodies made in animals can protect against anthrax toxins • Established in-house in vitro and in vivo assays for testing efficacy of Anthrax Immune Globulin products

45. Vaccinia Immune Globulins Public Health Impact: • Complications of smallpox vaccination: • Progressive vaccinia – • VIG reduces fatality: 100%  50% • Eczema Vaccinatum • VIG reduces fatality: 30%  3%

46. VIG Products: Efficacy (LPD) • Regulatory and Scientific Challenges: • How can efficacy and potency for VIG products be assessed? • Need for an animal model of severe vaccinia in an immunodeficient host

47. SCID Mouse Model of Progressive Vaccinia: Similarities to Human Disease Day 7 Day 14 • Mimics human route of exposure • Non-healing primary lesion • Systemic spread of virus • Lethality Day 21 Day 28

48. VIGIV Testing in Scarified SCID Mice: Post-exposure Prophylaxis Efficacy 4 long-term disease-free survivors Vaccinia day 0 - VIGIV day 2, 5, 10, 15

49. VIG: Outcomes(LPD) • SCID model used to demonstrate that VIGIV can reduce vaccinia lethality in pre- and post-exposure treatment. • SCID model adopted by industry in support of licensure.

50. Reference Standards Established Through International Collaborations (I) • 1st International Standard for von Willebrand Factor Concentrate • Mega 2/EP BRP Batch 3 International Working Standard for FVIII Concentrates • 7th International Standard for Factor VIII Concentrate • 2nd International Standard/FDA Standard Lot K for Thrombin