1. Laboratory of Immunobiochemistry Allergenic Products Advisory Committee, April 8, 2003

2. Lab overview Staffing Lot release Reference replacement Operational issues Sheep serum replacement issues ISO certification report Research/regulatory update Endotoxin studies Cockroach antigens and antibodies

3. Lab overview Staffing Lot release Reference replacement

4. Principal Investigators Jay E. Slater, MD, Lab Chief – Supervisory Medical Officer (4) Ronald Rabin, MD - Senior Staff Fellow (2) Post Doctoral Fellows Jonny Finlay, PhD - IRTA (2) Bo Chi, MD - Visiting Associate (<1)

5. Research Technicians Albert Gam – Biologist (2) Mona Febus – Microbiologist (3) Marc Alston – Biologist (2) Cherry Valerio – Biologist (2) Katia Dobrovolskaia – Visiting associate (2)

6. LIB staffing 1998-2003

7. “ Routine ” regulatory activities Lot release Reference distribution Reference maintenance semiannual checks replacement

8. Lot release activities 357 protocols submitted and reviewed 1 withdrawn Reference distribution 2002: 1978 vials in 107 shipments sent to manufacturers

9. Lot release protocols submitted

10. Reference distribution

11. Laboratory of Immunobiochemistry Operational issues

12. Replacement of cat and ragweed antisera Transition to ISO compliance

13. Ragweed and cat antisera need to be replaced S2a cat Released in 1998 S6 ragweed Released in 2000 Replacement programs for both initiated spring 2002

14. Immunization protocol

15. plasmapheresis Immunization doses

18. Transmissible spongiform encephalopathies Animal scrapie (sheep and goats) chronic wasting disease (mule deer, elk) transmissible mink encephalopathy bovine spongiform encephalopathy feline spongiform encephalopathy

19. Transmissible spongiform encephalopathies Human kuru Creutzfeldt-Jakob disease classic sporadic familial iatrogenic new variant (a/w bovine TSE) Gerstmann-Str ä ussler-Scheincker fatal familial insomnia sporadic fatal insomnia

20. Scrapie In Europe for >250 years In US since 1947 >1000 flocks Vertical transmission Horizontal transmission, presumably by contamination with placenta and blood during lambing season Long incubation period No human transmission

21. Prion polymorphisms associate with different susceptibility, especially at codon 171 QQ (glutamine/glutamine) susceptible RR (arginine/arginine) resistant QR (glutamine/arginine) intermediate

22. Scrapie eradication program Preclinical testing and surveillance Live animal test (third eyelid biopsy) May take months to years to turn positive Tracking of infected and exposed animals Cleanup strategies: identify and genotype exposed animals Destroy QQ exposed QR or RR exposed are tracked but may be slaughtered for human consumption

23. Not believed to pose any risk to humans No recognized human transmission in three centuries of exposure in Europe This serum is safe to use because No documented transmission to humans Contact with affected sheep was limited Not in adjoining pens; >30 feet distant Normal BSL 2 precautions in place for all work with animal sera However, in order to maintain a serum reagent that is as safe as possible …

24. Current approach … Begin immunizing two new sheep Process plasma from ewes 6747 and 7777 now Conserve current stocks

25. Current approach … If new sera are available before we run out, sera from 6747 and 7777 will be saved frozen for possible future use If we have a shortfall, sera from 6747 and/or 7777 will be used until the new sera are available

26. Current approach … Advantages Highest degree of safety Large supplies for future Disadvantages Possibility of two serum switches in short period Time Expense

28. CBER Laboratory Quality Management Initiative  Seek ISO-17025 compliance for official product testing  ISO – International Organization for Standardization, Geneva, Switzerland 17025: “ General requirements for the competence of testing and calibration laboratories ” (1999) Set of guidelines for labs that do testing and calibration to show operation of a quality system to assure technical competence and production of valid results

29. CBER Laboratory Quality Management Initiative Establish policy with Center level Quality Manual  Audit for compliance with ISO-17025  Obtain Test/Lab Accreditation where appropriate “ Accreditation – successful 3 rd party audit

30. Why is CBER becoming ISO compliant? Establish recognized competence, assure the trust and value of our data and processes We require the manufacturers ’ labs to be in GMP compliance International efforts at harmonization are attempting to equate GMP and ISO requirements ISO is an internationally recognized standard

31. Why is CBER becoming ISO compliant? (continued) Implement Laboratory Quality Management policies and practices for official testing activities To document a high level of training, competence, and proficiency To establish a consistent product testing process

32. Elements of ISO compliance Management of People Equipment Documents Processes

33. Elements of ISO compliance Management of People Defined roles Appropriate training Demonstrated proficiency Initial and ongoing Authorization process

34. Elements of ISO compliance Management of Equipment Program for equipment calibration Maintenance Process to assure that only calibrated and maintained equipment is used Assurance of measurement traceability to accepted standards

35. Elements of ISO compliance Management of Documents Includes policies, procedures, specifications, equipment manuals, certifications Must be reviewed, issued and controlled Approved and issued prior to use

36. Elements of ISO compliance Management of Processes Approval of testing materials Environmental specifications and monitoring Handling of samples Validation and suitability Handling of data (including non-conforming data) Corrective action, Preventive action systems Internal Audits Management review Recording and handling complaints

37. CBER ’ s commitment to implementation of a Lab Quality System Establishment of CBER Quality Board Development of Quality Assurance structure within CBER Appointment of Center Level Quality Manager Hiring Office quality Managers Appointment and hiring of Division Quality Coordinators Preparation of CBER Quality Manual Purchase of Integrated Quality Management computer software

38. What will this mean to LIB? No substantive protocol revisions Documentation Formatting Substance Formal separation of research and regulatory equipment Internal audits External audits

39. Timetable (tentative) Implementation in stages over the next 3 years … Seek accreditation in 2005 Software operational during 2003 Policies and Quality Manual issued 2003 Initiation of compliance audits (ongoing) Training and process development (ongoing)

40. Laboratory of Immunobiochemistry Research/regulatory update

41. Active research projects PI Slater Cockroach allergen standardization Determination of optimal surrogate test Depletion analysis of CR extracts Cockroach IgE combinatorial library Endotoxin in allergen vaccines PI Rabin MDR proteins in T cell activation RSV responses in human tonsil

42. Publications Patterson ML, Slater JE. Characterization and comparison of commercially available German and American cockroach allergen vaccines. Clin Exp Allergy 2002;32:721-727 Sutherland MF, Drew A, Rolland JM, Slater JE, Suphioglu C, O'Hehir RE. Specific monoclonal antibodies and human IgE show Hev b 5 is an abundant allergen in high protein powdered latex gloves. Clin Exp Allergy 2002;32:583-589 Trivedi B, Valerio C, Slater JE. Endotoxin content of standardized allergen vaccines. J Allergy Clin Immunol 2003 (in press).

43. Publications (reviews) Lockey RF, Slater JE, Esch R. Preparation and standardization of allergen vaccines. In Middleton ’ s Allergy: Principles and Practice, 6 th ed. St. Louis: Mosby (in press).

44. Abstracts Rabin RL, Alston MA, Huang H, Slater JE. Cytokine secretion by activated T cells is dependent on multidrug resistance protein-1 (MRP-1). J Allergy Clin Immunol 2003; 111:S153. Valerio C, Slater JE. The effects of lipopolysaccharide (LPS) on immune responses in C57Bl/6 mice. J Allergy Clin Immunol 2003; 111:S166. Slater JE, Valerio C, Trivedi B. Endotoxin in standardized allergen vaccines. J Allergy Clin Immunol 2003; 111:S243. Finlay WJJ, Rabin RL, Slater JE. Analysis of IgE heavy chain V-gene usage in human tonsil. J Allergy Clin Immunol 2003; 111:S313.

45. Endotoxin content of allergen vaccines Allergenic extracts are not required to undergo evaluation for the presence of pyrogens (21CFR 610.13(b)) Prior studies confirmed variable endotoxin content (Siraganian, et al. J Allergy Clin Immunol 1979; 64:526- 533)

46. Products exempted in 21 CFR 610.13(b) Blood products Horse serum Bacterial, rickettsial and viral vaccines Toxoids Toxins Allergenic extracts

47. LAL gel-clot method

49. Factor CAct. factor C Factor B Proclotting enzyme Clotting enzyme Factor GAct. factor BAct. factor G CoagulogenCoagulin ENDOTOXIN  –D- glucan

50. Endotoxin content of allergen vaccines – possible interference Non-endotoxin (1,3)-  -D-glucans may induce clotting by an alternative pathway in the standard LAL assays Proteases (especially in cat and mite extracts) may also induce clotting

51. Endotoxin content of allergen vaccines - approach Determine endotoxin content using gel- clot method Assess the contribution of non- endotoxin components ((1,3)-  -D- glucans, enzymes)

52. Study design Standardized allergen vaccines LAL gel-clot assay Adsorption of selected allergens with ENP- silica resin, followed by LAL assay Endotoxin neutralizing protein (ENP) is a ~12 kDa, cationic, amphipathic protein that binds to and neutralizes the biological activity of lipopolysaccharide. Pre-treat selected allergens at 95°C for 15 min, followed by LAL assay

53. GP G G G G G G G G G G G G G G P

54. G G G G G P P P

55. G G G G G G G

56. G G G G G G G G G G G G

58. Heat inactivation

61. Conclusions The observed LAL gel-clot activity probably represents real endotoxin content, not  -glucan or protease activity

62. Is this amount of endotoxin physiologically significant? Mean endotoxin content: 1,900 EU/mL (allergen immunotherapy dose 0.5 – 2.0 mL per month) 40 – 50 EU/kg (2,800 to 3,500 EU) (administered IV) can elicit a rise in temperature, heart rate, and white blood cell count Wolff SM. J Infect Dis 1973; 128:Suppl-64. Michie HR, et al. N Engl J Med 1988 Jun 9 318;1481-6.

63. Specific endotoxin limits Product-specific Generally based on Drug dose 5.0 EU/kg limit

64. Specific endotoxin limits * Based on USP limits and estimated maximum therapeutic doses

65. The clinical consequences of endotoxin in allergen vaccines have not been studied No data that adverse events from IT are associated with endotoxin levels No data to support a beneficial effect of endotoxins Future studies of allergen IT should be controlled for endotoxin dose Role of endotoxin in safety and efficacy of IT should be assessed

66. Conclusions Pollens < cat and mite Cat hair < cat pelt D. pteronyssinus << D. farinae Bioburden? Endogenous heat-stable ENP-binding LAL activator in D. farinae? Endogenous ENP in D. pteronyssinus?

67. Plan Expand study of endotoxin content Additional standardized and non- standardized extracts Different methods (GC mass spec) Investigate differences between D. farinae and D. pteronyssinus

68. Cockroach allergen standardization Clinical studies Developing the appropriate surrogate Correlation Depletion studies IgE combinatorial library to cockroach

69. Cockroach allergen standardization Clinical studies Developing the appropriate surrogate Correlation Depletion studies IgE combinatorial library to cockroach

70. Cockroach allergen standardization Clinical studies Developing the appropriate surrogate Correlation Depletion studies IgE combinatorial library to cockroach

71. Current standardized allergens D. farinae D. pteronyssinus Cat hair Cat pelt Short ragweed pollen Hymenoptera Honey bee Wasp Yellow jacket Yellow hornet White-faced hornet Mixed vespid Grass pollens Bermuda grass Red top June (Kentucky blue) Perennial rye Orchard Timothy Meadow fescue Sweet vernal

72. Allergen standardization Establish a US standard, and Establish a testing procedure Manufacturers may use the established procedure, or may develop equivalent procedures

73. Which allergens should be standardized? Impact criteria … Availability of stable, preferably lyophilized material for use as long-term reference extracts. Consistency of currently marketed product. Widespread use as a diagnostic and/or therapeutic reagent in the U.S. Number of manufacturers producing the product. Potential use in immunotherapy or diagnostics. Public health impact of correct diagnosis and/or adequate treatment.

74. Allergens and asthma Indoor allergens dust mites* cat* cockroach molds dog Outdoor allergens molds * = already standardized

75. Cockroaches

76. Why is cockroach allergy important? Ubiquitous Difficult to control Associated with asthma

77. Why is cockroach allergen standardization important? To the patient More accurate diagnosis Safer and more effective immunotherapy To the physician/scientist Better science (if you can ’ t measure it, you can ’ t study it … ) Pathophysiology Epidemiology Environmental control To the FDA Safer, more effective product

78. Phase I - Laboratory Develop/adapt methods for allergen determination Compare allergen content of different lots

80. Goals Determine consistency of available US products: protein content specific allergen content overall allergenicity Determine best lot release measures

81. Extracts used as reference E2-Cg and E2-Ca Previously characterized Limited skin test data Lyophilized; available in large quantities

82. Cockroach extracts studied From all nine allergen extract manufacturers

83. Relative Potency of All Cockroach Extracts (determined by competition ELISA)

87. Bla g 1 and Bla g 2 Levels in glycerinated German cockroach

88. Correlation of protein concentrations and ELISA results Protein concentration Bla g 1 levelsBla g 2 levels Relative potency R 2 = 0.83 R 2 = 0.92 R 2 = 0.93 R 2 = 0.96 R 2 = 0.92

89. Conclusions Commercially available cockroach allergen extracts: vary widely in protein content, Bla g 2 content, SDS-PAGE banding pattern, and overall allergenicity. appear to be less potent and contain less Bla g 1 than the candidate reference extracts

90. Conclusions Established that cockroach allergen vaccines need to be standardized

91. What we need now … New cockroach references Characterize the references ID 50 EAL testing (Intradermal Dilution for 50 mm Sum of Erythema Determines Bioequivalent Allergy Units) Proceed to next phase

92. Phase II - Clinical skin testing, histamine release, IT data establish biological unitage and ideal dosing ranges

93. ID 50 EAL testing Proficiency Recruitment Testing Analysis

94. ID 50 EAL testing

95. Recruitment  Inclusion criteria  18 to 65 years of age  history of allergic disease, such as allergic rhinitis, related to exposure to the allergen of interest  puncture sum of erythema diameter responses (ΣE) to the allergen concentrate of ≥30 mm.

96. Recruitment Exclusion criteria Asthma with use of systemic steroids in the past 12 months Peak flow < 75% predicted at the time of testing Skin coloring or condition that would preclude the measurement of erythema responses Dermographism (> 4 mm ΣE following saline skin test) Immunotherapy – past or present - with the test allergen Current use of antihistamines, tricyclic antidepressants, MAO inhibitors, and beta-blockers

97. How many study subjects? Rabin et al., Sample Size Considerations for Establishing Clinical Bioequivalence of Allergen Formulations. Arb.Paul Ehrlich Inst.Bundesamt Sera Impfstoffe Frankf.A.M, in press

98.   for D50 should be 10% BAU/mL= 3 -(14 - mean D50) * 100,000

99.  for D50 is about 10-20% Smith et al. Annals Allergy Asthma Immunol 1995; 75:317-323

100.  for D50 is about 10-20%

101. How many study subjects? Estimate n = 40 to establish D50 for each extract (based on  /  = 1.5 ): 80 Geographic diversity: 80 x 3 = 240 Overlap between American CR and German CR allergic subjects may permit reduction in n (150-200)

102. Conclusions Established that cockroach allergen vaccines need to be standardized Need to establish the potency of candidate US reference materials by bioassay (ID 50 EAL)

103. NIAID Inner City Asthma Consortium “…established in FY 2002 to explore and evaluate promising new strategies for the treatment of asthma among minority children residing in the inner city. This consortium of basic scientists and clinical investigators will conduct clinical studies to elucidate the immunopathogenesis and natural history of asthma in this population.” From the FY 2003 Budget Justification Narrative, NIAID, http://www.niaid.nih.gov/director/congress/2002/cj/narrative.htm

104. NIAID Inner City Asthma Consortium steering committee Busse, William W., MD Chair Busse, William W., MD Adams, Kenneth, PhD Eggleston, Peyton A., MD Gruchalla, Rebecca S., MD, PhD Gruchalla, Rebecca S., MD, PhD Kattan, Meyer, MD Kercsmar, Carolyn M., MD Liu, Andrew H., MD Malveaux, Floyd J., MD, PhD Mitchell, Herman, PhD Morgan, Wayne, MD, CM O'Connor, George T., MD, MS O'Connor, George T., MD, MS Pongracic, Jacqueline A., MD Pongracic, Jacqueline A., MD Sampson, Hugh A., MD Smartt, Ernestine, RN Strunk, Robert C., MD Szefler, Stanley J., MD

105. Timetable Steering committee approval - done Study centers identified Order extracts IRB approvals IND approval Distribute materials Proficiency testing Proceed with study

106. Endotoxin content of cockroach vaccines

107. Conclusions Established that cockroach allergen vaccines need to be standardized Need to establish the potency of candidate US reference materials by bioassay (ID 50 EAL) Endotoxin issue to be studied in depth

108. Will overall allergenicity measurements be sensitive to changes in specific allergen levels? In mite stability study (1998-1999), RP was stable at -20°C and 4°C for up to 12 months Degradation of specific allergens (group 1 and 2; specific bands) was observed at 4°C Soldatova LN, Paupore EJ, Burk SH, Pastor RW, Slater JE. J Allergy Clin Immunol 2000; 105:482-8.

109. RID with monospecific antiserum Examples: cat, ragweed Advantages quantitative monospecific Disadvantages need to identify relevant allergen(s)

110. Competition ELISA with pooled allergic human sera Examples: mites, grasses Advantages quantitative reflects spectrum of allergen recognition does not require identification of relevant allergens Disadvantages use of pooled sera effects of fluctuations in individual allergens difficult to measure

111. Specific loss of a single allergen

112. Will overall allergenicity measurements be sensitive to changes in specific allergen levels? Depletion analysis Raise specific antibodies to Bla g 1, 2, 4 and 5 Selectively adsorb Test for specific allergen levels Test for overall allergenicity

113. SDS-Page of Bla g 1 Absorbed Cockroach Antigen Cr Sham Abs

114. Selective adsorption of Bla g 1 Bla g 1 ELISA Sham adsorbed Adsorbed with anti-Bla g 1

115. Selective adsorption of Bla g 1 does not reduce the RP as measured by competition ELISA

116. Conclusions Established that cockroach allergen vaccines need to be standardized Need to establish the potency of candidate US reference materials by bioassay (ID 50 EAL) Endotoxin issue to be studied in depth Surrogate test may not be the competition ELISA

117. Conclusions Standardized German and American cockroach allergen vaccines will facilitate definitive studies on the role of cockroach allergens in inner city asthma, and on the best methods for eradication and treatment make for safer and more effective products