Laboratory of Immunobiochemistry

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Laboratory of Immunobiochemistry

• Allergenic Products Advisory Committee, April 8,
  2003

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Allergenic Products Advisory Committee, April 8,
2003

• 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

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Lab overview

• Staffing
• Lot release
• Reference replacement

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• Principal Investigators
• Jay E. Slater, MD, Lab Chief Supervisory
  Medical Officer (4)
• Ronald Rabin, MD - Senior Staff Fellow (2)
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• Post Doctoral Fellows
• Jonny Finlay, PhD - IRTA (2)
• Bo Chi, MD - Visiting Associate (lt1)
•  

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• Research Technicians
• Albert Gam Biologist (2)
• Mona Febus Microbiologist (3)
• Marc Alston Biologist (2)
• Cherry Valerio Biologist (2)
• Katia Dobrovolskaia Visiting associate (2)
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LIB staffing 1998-2003
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Routine regulatory activities

• Lot release
• Reference distribution
• Reference maintenance
• semiannual checks
• replacement

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• 1 withdrawn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

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Lot release protocols submitted
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Reference distribution
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Laboratory of Immunobiochemistry

• Operational issues

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Operational issues

• Replacement of cat and ragweed antisera
• Transition to ISO compliance

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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

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Immunization protocol
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Immunization protocol
Immunization doses
plasmapheresis
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Transmissible spongiform encephalopathies

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

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Transmissible spongiform encephalopathies

• Human
• kuru
• Creutzfeldt-Jakob disease
• classic sporadic
• familial
• iatrogenic
• new variant (a/w bovine TSE)
• Gerstmann-Sträussler-Scheincker
• fatal familial insomnia
• sporadic fatal insomnia

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Scrapie

• In Europe for gt250 years
• In US since 1947
• gt1000 flocks
• Vertical transmission
• Horizontal transmission, presumably by
  contamination with placenta and blood during
  lambing season
• Long incubation period
• No human transmission

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

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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

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• 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 gt30 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

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Current approach

• Begin immunizing two new sheep
• Process plasma from ewes 6747 and 7777 now
• Conserve current stocks

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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

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Current approach

• Advantages
• Highest degree of safety
• Large supplies for future
• Disadvantages
• Possibility of two serum switches in short period
• Time
• Expense

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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

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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 3rd party audit

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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

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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

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Elements of ISO compliance

• Management of
• People
• Equipment
• Documents
• Processes

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Elements of ISO compliance

• Management of People
• Defined roles
• Appropriate training
• Demonstrated proficiency
• Initial and ongoing
• Authorization process

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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

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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

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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

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CBERs 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

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What will this mean to LIB?

• No substantive protocol revisions
• Documentation
• Formatting
• Substance
• Formal separation of research and regulatory
  equipment
• Internal audits
• External audits

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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)

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Laboratory of Immunobiochemistry

• Research/regulatory update

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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

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Publications

• Patterson ML, Slater JE. Characterization and
  comparison of commercially available German and
  American cockroach allergen vaccines. Clin Exp
  Allergy 200232721-727
• 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 200232583-589
•  Trivedi B, Valerio C, Slater JE. Endotoxin
  content of standardized allergen vaccines. J
  Allergy Clin Immunol 2003 (in press).

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Publications (reviews)

• Lockey RF, Slater JE, Esch R. Preparation and
  standardization of allergen vaccines. In
  Middletons Allergy Principles and Practice, 6th
  ed. St. Louis Mosby (in press).

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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 111S153.
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• Valerio C, Slater JE. The effects of
  lipopolysaccharide (LPS) on immune responses in
  C57Bl/6 mice. J Allergy Clin Immunol 2003
  111S166.
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• Slater JE, Valerio C, Trivedi B. Endotoxin in
  standardized allergen vaccines. J Allergy Clin
  Immunol 2003 111S243.
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• Finlay WJJ, Rabin RL, Slater JE. Analysis of IgE
  heavy chain V-gene usage in human tonsil. J
  Allergy Clin Immunol 2003 111S313.

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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 64526-533)

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Products exempted in 21 CFR 610.13(b)

• Blood products
• Horse serum
• Bacterial, rickettsial and viral vaccines
• Toxoids
• Toxins
• Allergenic extracts

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LAL gel-clot method
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LAL gel-clot method
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ENDOTOXIN
(1,3) bD- glucan
Factor C
Act. factor C
Factor B
Factor G
Act. factor B
Act. factor G
Proclotting enzyme
Clotting enzyme
Coagulogen
Coagulin
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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

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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)

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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 95C for 15 min,
  followed by LAL assay

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P
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Heat inactivation
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Conclusions

• The observed LAL gel-clot activity probably
  represents real endotoxin content, not ?-glucan
  or protease activity

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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 128Suppl-64. Michie
HR, et al. N Engl J Med 1988 Jun 9 3181481-6.
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Specific endotoxin limits

• Product-specific
• Generally based on
• Drug dose
• 5.0 EU/kg limit

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Specific endotoxin limits
Based on USP limits and estimated maximum
therapeutic doses
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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

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Conclusions

• Pollens lt cat and mite
• Cat hair lt cat pelt
• D. pteronyssinus ltlt D. farinae
• Bioburden?
• Endogenous heat-stable ENP-binding LAL activator
  in D. farinae?
• Endogenous ENP in D. pteronyssinus?

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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

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Cockroach allergen standardization

• Clinical studies
• Developing the appropriate surrogate
• Correlation
• Depletion studies
• IgE combinatorial library to cockroach

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Cockroach allergen standardization

• Clinical studies
• Developing the appropriate surrogate
• Correlation
• Depletion studies
• IgE combinatorial library to cockroach

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Cockroach allergen standardization

• Clinical studies
• Developing the appropriate surrogate
• Correlation
• Depletion studies
• IgE combinatorial library to cockroach

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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

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Allergen standardization

• Establish a US standard, and
• Establish a testing procedure
• Manufacturers may use the established procedure,
  or may develop equivalent procedures

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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.

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Allergens and asthma

• Indoor allergens
• dust mites
• cat
• cockroach
• molds
• dog
• Outdoor allergens
• molds
• already standardized

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Cockroaches
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Why is cockroach allergy important?

• Ubiquitous
• Difficult to control
• Associated with asthma

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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 cant measure it, you
  cant study it)
• Pathophysiology
• Epidemiology
• Environmental control
• To the FDA
• Safer, more effective product

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Phase I - Laboratory

• Develop/adapt methods for allergen determination
• Compare allergen content of different lots

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Goals

• Determine consistency of available US products
• protein content
• specific allergen content
• overall allergenicity
• Determine best lot release measures

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Extracts used as reference

• E2-Cg and E2-Ca
• Previously characterized
• Limited skin test data
• Lyophilized available in large quantities

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Cockroach extracts studied

• From all nine allergen extract manufacturers

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Relative Potency of All Cockroach Extracts
(determined by competition ELISA)
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Relative Potency of All Cockroach Extracts
(determined by competition ELISA)
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Relative Potency of All Cockroach Extracts
(determined by competition ELISA)
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Relative Potency of All Cockroach Extracts
(determined by competition ELISA)
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Bla g 1 and Bla g 2 Levels in glycerinated German
cockroach
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Correlation of protein concentrations and ELISA
results
Protein concentration
Relative potency
R2 0.83
R2 0.92
R2 0.96
R2 0.92
R2 0.93
R2 0.92
Bla g 1 levels
Bla g 2 levels
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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

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Conclusions

• Established that cockroach allergen vaccines need
  to be standardized

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What we need now

• New cockroach references
• Characterize the references
• ID50EAL testing (Intradermal Dilution for 50 mm
  Sum of Erythema Determines Bioequivalent Allergy
  Units)
• Proceed to next phase

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Phase II - Clinical

• skin testing, histamine release, IT data
• establish biological unitage and ideal dosing
  ranges

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ID50EAL testing

• Proficiency
• Recruitment
• Testing
• Analysis

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ID50EAL testing
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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 (SE)
  to the allergen concentrate of 30 mm.

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Recruitment

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

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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
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d for D50 should be 10
BAU/mL 3-(14 - mean D50) 100,000
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? for D50 is about 10-20
Smith et al. Annals Allergy Asthma Immunol 1995
75317-323
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? for D50 is about 10-20
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How many study subjects?

• Estimate n 40 to establish D50 for each extract
  (based on ?/d 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)

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Conclusions

• Established that cockroach allergen vaccines need
  to be standardized
• Need to establish the potency of candidate US
  reference materials by bioassay (ID50EAL)

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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
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NIAID Inner City Asthma Consortium steering
committee

• Busse, William W., MD Chair
• Adams, Kenneth, PhD
• Eggleston, Peyton A., MD
• 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
• Pongracic, Jacqueline A., MD
• Sampson, Hugh A., MD
• Smartt, Ernestine, RN
• Strunk, Robert C., MD
• Szefler, Stanley J., MD

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Timetable

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

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Endotoxin content of cockroach vaccines
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Conclusions

• Established that cockroach allergen vaccines need
  to be standardized
• Need to establish the potency of candidate US
  reference materials by bioassay (ID50EAL)
• Endotoxin issue to be studied in depth

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Will overall allergenicity measurements be
sensitive to changes in specific allergen levels?

• In mite stability study (1998-1999), RP was
  stable at -20C and 4C for up to 12 months
• Degradation of specific allergens (group 1 and 2
  specific bands) was observed at 4C

Soldatova LN, Paupore EJ, Burk SH, Pastor RW,
Slater JE. J Allergy Clin Immunol 2000
105482-8.
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RID with monospecific antiserum

• Examples cat, ragweed
• Advantages
• quantitative
• monospecific
• Disadvantages
• need to identify relevant allergen(s)

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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

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Specific loss of a single allergen
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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

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SDS-Page of Bla g 1 Absorbed Cockroach Antigen
Cr Sham Abs
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Selective adsorption of Bla g 1
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Selective adsorption of Bla g 1 does not reduce
the RP as measured by competition ELISA
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Conclusions

• Established that cockroach allergen vaccines need
  to be standardized
• Need to establish the potency of candidate US
  reference materials by bioassay (ID50EAL)
• Endotoxin issue to be studied in depth
• Surrogate test may not be the competition ELISA

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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