Laboratory of Plasma Derivatives Research Overview

1. Laboratory of Plasma Derivatives Research Overview Dorothy Scott, M.D. Chief, Laboratory of Plasma Derivatives DH/OBRR/CBER Blood Products Advisory Committee December 15, 2010

2. LPD Mission Statement To meet the public health needs for safe and effective products by performing high quality research that directly impacts the safety, effectiveness and availability of plasma derivatives.

3. Research Projects Cross Sections

4. Regulated Products Immune Globulin (10 Products) – Primary Immune Deficiency, ITP, CIDP, Kawasaki Disease, secondary immune deficiencies Specific Immune Globulins – for HBV, CMV, HAV, tetanus, rabies, measles, vaccinia, infant botulism, prevention of newborn hemolytic disease

5. Regulated Products Antivenoms and antitoxins – coral snake, rattlesnake, and black widow spider envenomation, botulism, digitalis intoxication Anti-thymocyte globulins – treatment of transplant rejection Alpha-1 Proteinase Inhibitor – treatment of emphysema in A1PI deficiency

6. Standards and Reagents for release testing • Development of international standards and test methods to increase safety of plasma derivatives • Ongoing: • Testing of new CBER potency standard for Immune Globulins • Development and testing of new international B19 virus NAT standards for plasma testing • Characterization of the first A1PI international standard • Research on potency testing and safety correlates • HCV immune globulin • Vaccinia immune globulin • Influenza immune plasma • Characterization methods for aggregates in plasma derivatives

7. Developing Predictive Models for Preclinical Product Evaluation Of Safety and Efficacy • HCVIGIV and SARSIGIV– predicting product efficacy based on neutralizing activity • Influenza – more rapid test methods for neutralizing antibodies • Developing and evaluating test methods to characterize product aggregates and to assess aggregate immunogenicity

8. Evaluating Efficacy and Safety of Immune Globulins for Pandemic and Counterterror Response • Animal model evaluation of VIG products and therapies • Evaluation of animal models for counterterrorism-related immune globulins • Influenza antibody testing of plasma collected from donors receiving influenza vaccine or post-influenza infection • Predicting donor characteristics likely to be associated with highest neutralizing antibody titers

9. Research Framework Enables LPD to Scientifically Address Regulatory Issues • Evaluation of IG products reported to cause clinically relevant hemolysis • Biochemical changes in A1PI products were detected and analyzed, leading to changes in product manufacture • Evaluation of thrombosis-associated IGIV product lots (LPD/LH) • Identification of B19 virus transmission by a coagulation factor • Identification of bacterial constituent contaminant in a product using TLR-transfected cells

10. Immunogenicity – addressing the lack of predictive animal models Improving IGIV safety, efficacy, potency Enhancing neutralizing potency (HCVIGIV) Characterizing aggregates and their impact on potency, safety, and immunogenicity Facilitating development of potency assays most relevant to IGIV used for primary immune deficiency patients Rapid collection and testing of plasma for manufacturing specific immune globulins in pandemic or BT scenarios Developing preclinical models for inhaled A1PI efficacy and immunogenicity Preparing for the Future

11. Public Health Problem – Improving Treatment for Progressive Vaccinia(D. Scott, M. Kennedy, R. Fisher) • If there is a smallpox outbreak, mass vaccination is likely • In this setting, immune compromised people are likely to be exposed to vaccinia • Inadvertent vaccination • Contact exposure • Treatment and post-exposure prophylaxis for progressive vaccinia are not well-defined • High mortality rate/need for intensive support

12. Development and characterization of an animal model to study pathogenesis and treatment of Progressive Vaccinia Animal model can be used to assess new therapies and current products Need to understand mechanism of VIG potency - Improve product - Improve potency assays for product release

13. SCID mouse model results • Lesions similar to human lesions • Slow progression, lethality • Responsive to VIGIV • Early treatment eliminates infection • VIGIV + topical cidofovir • long-term survival with delayed treatment • Treatment effect does not require complement

14. Conclusions SCID PV model simulates many features of human PV* SCID PV model should be useful for testing combined treatments and new antibody treatments Studies support the likelihood that early PEP may prevent PV in susceptible hosts Ongoing studies – Identify VIGIV mechanism(s) of action Animal model studies of maternal/fetal vaccinia complications (funded, FDA Office of Women’s Health) * In press, Clinical and Vaccine Immunology, January 2011

15. Neutralization of Viruses Relevant to Immune Globulin Products (P. Zhang, Ph.D.) HCV Infection: About 170 million people worldwide including 4 million in the U.S. are infected with HCV. HCV-associated cirrhosis is the leading indication for liver transplantation in the U.S. Public Health Problem Lack of vaccine No immunoglobulin product for prophylaxis or treatment Treatment (IFN + ribavirin) is only 50% effective Addressing the Problem Identifying critical epitopes for HCV neutralization Unexpected finding of interfering antibodies

16. Presence of Neutralizing and Non-Neutralizing Antibodies in Experimental HCIGIV Preparations Binding Assay (ELISA) A450 nm Control IGIV AE BE CE DE HCIGIV A B C HCV E2 Peptides D Epitope I Epitope II 412 426 434 446 HCV E2 ---QLINTNGSWHINSTALNCNESLNTGWLAGLFYQHKF--- Interference? AE , BE DE HCIGIV Eluates

17. Epitope-Specific Neutralization and Interference of HCV by HCIGIV Eluates Infectivity (% of Negative IGIV Control) Infectivity (% of Negative IGIV Control) 150 150 100 100 50 50 0 0 DE AE+DE Control IGIV AE DE Control IGIV DE DE AE Epitope I Epitope I Epitope II Epitope II HCV E2 HCV E2 Neutralization Interference

18. Additional Findings • Depletion of interfering antibodies increases HCVIGIV neutralizing activity • Depletion of interfering antibodies in infected chimpanzee sera reveals cross-genotype neutralizing activity • Evidence that interfering antibodies found more in chronic than recovered HCV patients • Evidence from serial samples from one chronic HCV patient that interfering antibodies arise prior to neutralizing antibodies

19. Public Health Impact • HCVIGIV preparations may be improved by removing interfering antibodies and enriching neutralizing antibodies • Epitope-based binding assays can be designed to measure HCV neutralizing antibodies in sera (for clinical trials) and HCVIGIV products • Vaccine design may be improved by developing antigens that will provide neutralizing but not interfering antibody responses

20. Use of Toll-like Receptor (TLR) Assays to Detect and Identify Microbial Contaminants in Biological Products Basil Golding M.D. Site Visit June 15, 2010

21. Objective To develop a panel of TLR-expressing cell lines that can detect the presence of microbial components in biological products including cellular and acellular vaccines, cells used in gene therapy, plasma-derived, recombinant and transgenic proteins.

22. A Case Study A recombinant product made from E. coli linked to adverse events in early clinical trials. Testing Plan using TLR-expressing Cell Lines • A panel of blinded product samples labeled as A, B, C, and D • Two protein product samples made from two different manufacturing methods – both samples had passed lot release testing: LAL test, Total residual DNA assay and Host cell protein ELISA. • One LPS positive sample • One negative control • Various TLR ligands

23. Results • Sample B was found to activate cell lines expressing TLR5 • The ligand for TLR5 is flagellin • Sample B was shown to contain flagellin by Western Blot and confirmed by Mass Spectrometry

24. Conclusions • We have established a sensitive human cell-based assay which enabled the detection of trace amounts of a microbial contaminant in a biological product that was not detectable by standard lot release tests (LAL, DNA, HCP). • The TLR assay results suggested that the putative contaminant was flagellin. • This was confirmed by Western Blot and Mass Spectrometry.

25. Public heath problem - in vitro and in vivo aggregation of A1PI (E. Marszal, Ph.D.) • Protein aggregation • Causes human disease • Affects quality of biological products • Protein aggregates in biologics are generally not well characterized • Structural and biochemical understanding of A1PI polymers is critical • To enable development of therapies for A1PI ZZ-associated liver disease • To improve product stability (understanding polymerization of “normal” A1PI) • To address scientific questions: • Investigation of the mechanism of a1-PI polymerization in vitro and in vivo • Assay evaluation and development; characterization of a1-PI products Collaborative project FDA/academia/manufacturers

26. Applied Research – Heterogeneity of a1-PI products IEF, desialylated plasma products HPLC DLS Polymer presence Adverse events: rash, chills, fever, flu-like symptoms immunogenicity? Characterization of protein aggregates in a1-PI products Evaluation of methods and methods development First product class characterization Absorbance 215 nm Elutiontime Western blot Marszal BPAC 2005 Silver stain Marszal &Shrake 2006 Marszal 2010

27. Investigation of the structure of a1-PI polymers 55ºC 65ºC pH 4.1 Gu 1.4M 55ºC 65ºC 65ºC 55ºC Tet Tri D M Methods used: Gel electrophoresis CD Fluorescence Mass spectrometry AFM Load Eluate Tet Tri D M Tet Tri D M Tet Tri D M native PAGEnative PAGE Gaczynska, Osmolski, AFM Marszal, 2009

28. Outcomes and plans • A new model of a1-PI polymerization was proposed. • Proposed polymerization model of a1-PI had impact on the selection of formulation buffer for a product. • Continued investigation a1-PI polymer structure using AFM will help to establish: • If there are one or more polymer types in A1PI- deficient patients • If polymers formed in different tissues have the same structure • Physiological relevance of model polymers and of PiZZ transgenic mouse model of liver disease • AFM method for polymer characterization may potentially be used for personalized treatment (screening drugs that could be used to dissociate patient’s polymers). • Methods used to characterize polymers will include H/D exchange mass spectrometry, which may have a role in assessing future products. • Characterization of protein aggregates in a1-PI products (collaborative study) will increase understanding of the methods and the products; potential impact on measuring protein aggregates in biologics.

29. Alpha-1 Proteinase Inhibitor and Innate Immune Responses (J. Reed, D. Scott) Public Health Issue: • Intravenous A1PI is approved to treat emphysema in patients with A1PI deficiency • Original licensure based on achievement of serum A1PI levels • Long-term clinical studies challenging • Treated A1PI-deficient patients may continue to have declining pulmonary function/exacerbations • Need to better understand • Effects of A1PI in lung tissue beyond elastase inhibition • Dose and route of A1PI therapy – relationship to efficacy

30. Pulmonary Macrophages Express Abundant A1PI normal adult A1PI CD68 RSV LRI of infancy A1PI CD68 cystic fibrosis A1PI CD68

31. A1PI Phenotypes of Lung Macrophages homeostatic phagocytic model cell line: U937 28SC in situ differentiation recently recruited monocyte chemokines ROS proinflammatory susceptible to apoptosis model cell line: THP-1 MM6

32. A1PI expression is inversely related to markers of inflammation in human macrophage cell lines

33. Research Hypothesis/Ongoing Work 1. A1PI regulates macrophage function • Nitric oxide scavenging, inhibiting activating signal amplification (ongoing) • Inhibition of inflammasome activation and/or apoptosis • Hepcidin scavenging and inhibition of iron utilization (ongoing) • Confirmation in primary monocytes/macrophages (pending) 2. Recruitment and maturation of lung macrophages are dysregulated in A1PI deficient patients 3. Increasing A1PI expression in patients with normal A1PI expression could modify macrophage responses to injury and infectious insult Implications: • Delivery of A1PI to lungs could be optimized • Potency assays could be developed to reflect additional A1PI activities • Improved understanding of pathogenesis A1PI deficiency • Identifying A1PI pathway as target in ameliorating lung disease

34. Thank You!

35. Extra slides

36. Post-exposure Treatment with VIG Causes Primary Lesion Regression and Improved Survival VIG 10 mg/mouse on days 2, 5, 10, and 15 post-infection

37. Long-term survival of mice receiving VIG + topical Cidofovir SCID mouse treatments: Scarify with 106 PFU vaccinia VIG 10 mg/mouse on days 7, 10, 15, 20 Topical cidofovir 1% in dermovan 2 x daily for 2 weeks starting on day 7

38. Immunology Section Activities • Regulation of Immune Globulins, antivenoms, antitoxins, A1PI • Investigating product safety/efficacy issues • Allergic reactions • Thrombotic events • Product contaminations • Lack of efficacy reports for immune globulins • Product potency testing and standards (A1PI, VIG) • Shortage management (Rabies Immune Globulin, Immune Globulin, Digibind, DigiFab, Thymoglobulin, Coral Snake antivenom) • Policy Issues – guidances, workshops, cross-office/center policy • Immunogenicity • Animal Efficacy Rule implementation • Measles antibody activity decline in immune globulins • Licensing orphan products: Varicella Zoster Immune Globulin, Coral Snake antivenom • Animal models and licensure – cross-Center policy for anthrax immune globulins • Aggregates and particulates in products • Responses to Congressional and international regulatory agencies • CDC consultations (vaccinia, anthrax cases); ACIP working groups (rabies, RSV mAb’s • Emergency responses (Haiti, hurricane Katrina, influenza pandemic)