German cockroach allergen standardization

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German cockroach allergen standardization
progress report
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Cockroaches
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Biological potency by ID50EAL testing
(Intradermal Dilution for 50 mm Sum of Erythema
Determines Bioequivalent Allergy Units)

• Highly allergic individuals
• Serial 3-fold dilutions
• Establish dilution at which SE 50 mm (D50)

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Biological potency by ID50EAL testing
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Testing

• Screen puncture test (bifurcated needle) with
  concentrates
• Intradermal testing with serial 3-fold dilutions
• Record wheal and erythema size
• Calculate ?E

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Calculation of D50
D50
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ID50EAL method

• D50 14 100,000 BAU/mL
• BAU/mL 100,000 x 3 (D50 - 14)

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Testing

• ID testing with serial 3-fold dilutions
• Calculate ?E
• The skin response (SE) should fall within the
  limits of gt0 to lt 125 mm
• Each more concentrated dilution should produce a
  graded erythema response
• The four dilutions selected should span a wide
  range of SE (for example from 0-20 mm to 80-125
  mm) and bracket SE of 50 mm

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Manufacturing data
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Preclinical data

• Bla g 1
• Bla g 2
• Bla g 5
• Relative potency
• Human
• Rabbit
• Anti-CR
• Pooled anti-specific allergens

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Specific allergen data (two-site ELISA)
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Overall allergenicitycompetition ELISA
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IND 11319 German Cockroach Allergen
Standardization Evaluation (CASE)

• Sponsor DAIT/NIAID
• Four sites
• Baltimore
• Washington DC
• Chicago
• Denver

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Thanks to

• Inner City Asthma Consortium
• Robert L. James, M.S., M.Stat., Rho
• Herman Mitchell, PhD, Rho
• Peyton A. Eggleston, MD, Baltimore
• Andrew H. Liu, MD, Denver
• Jacqueline A. Pongracic, MD, Chicago
• Sampson Sarpong, MD, Washington, DC

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IND 11319 German Cockroach Allergen
Standardization Evaluation (CASE)

• Purpose
• Determination of the biological potency of three
  (3) commercially available German cockroach
  allergen extracts and test of their
  bioequivalence.
• Patient Population
• Adults with a history of allergenic disease or
  asthma and a demonstrated sensitivity to German
  cockroach allergen tested.

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IND 11319 German Cockroach Allergen
Standardization Evaluation (CASE)

• Design
• Multi-center, open-label
• Number Planned 61
• Number Entered 62
• Number Completed 62

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IND 11319 German Cockroach Allergen
Standardization Evaluation (CASE)

• Age Range 18-59
• Gender Male15, Female47
• Race/ethnicity
• Black 35
• Hispanic 14
• White 5
• Asian 1
• Native American 0
• Other 1
• More than one race 6

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Approaches to analysis

• Linear regression
• Standard method included in the protocol
• In previous CBER studies using this protocol,
  poor correlation coefficients were a major source
  of data invalidation
• In the current study, several subjects data were
  non-linear

D50
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Another approach Linear interpolation

• Rather than assume a mathematical relationship, a
  simpler method would take the serial dilution
  number at which the plots cross the 50 mm line
• Linear interpolation between two data points the
  data point immediately before and the data point
  after the 50 mm line is crossed

D50
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Linear interpolation rules

• Use the first crossing followed by at least two
  consecutive non-missing dilutions above the 50mm
  SE line.
• If the above criterion does not apply and the
  datas most concentrated dilution is above the 50
  mm SE, then assume that any more concentrated
  dilutions (had they been collected) would also
  have remained above 50mm. Use this last crossing
  for calculating D50.
• If the extract for a subject does not cross the
  50 mm SE line at any serial dilution tested, or
  if whenever the line is crossed, first two
  criteria do not apply, then the D50 is not
  calculated for that subjects extract.

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Regression vs interpolation
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A third approach Four-parameter logistic model
min minimum erythema
diameter possible at conc 0
max maximum erythema diameter possible at
conc infinity EC50
extract concentration at
which half of the
change (max-min) in
S(erythema) is expected. D
Hill
coefficient or slope factor.
(When D1, the equation reduces
to the 3-parameter logistic equation.)
D50
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D50 data
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Potency data (BAU/mL)
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Conclusions

• The D50s calculated using the interpolation and
  4-parameter methods had slightly tighter
  standard deviations than the D50s calculated
  using the regression method.
• Using the interpolation or regression methods
  maximized the number of study subjects whose data
  could be analyzed.
• Bioequivalence (d lt 20) using all methods, the
  three extracts were bioequivalent.
• The A vs B and the B vs C had significantly
  different D50s even though they were
  bioequivalent. The A and C extracts were not
  statistically different from each other.

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Which in vitro data best reflect in vivo potency?
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All data (normalized to extract B)
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All data (normalized to extract B)

• In aggregate, Bla g 2 data appear to best reflect
  overall potency
• In order to determine the best test, we will need
  to examine IgE-specificities of individual sera
  and correlate to individual D50 data

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Where does this fit?

• Grass pollens 100,000 BAU/mL
• Ragweed 30,000-100,000 BAU/mL
• Based on estimate of 350 Amb a 1 U/mL100,000
  BAU/mL
• Bermuda grass 10,000 BAU/mL
• Cat 5,000-10,000 BAU/mL
• German cockroach 3,300 BAU/mL

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Where does this fit?

• Skin test doses
• 100-1000 BAU/mL
• Ann Allergy Asthma Immunol. 1995 75553
• IT doses
• 100-2500 BAU
• Clin Allergy Immunol 1999 12321
• 2000-4000 BAU
• Ann Allergy Asthma Immunol. 200390(1 Suppl 1)1

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Where does this fit?
Calculated from Nelson, chapter 24, in Lockey and
Bukantz, Allergens and Allergen Immunotherapy,
3rd ed, 2004
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Next steps

• Obtain sera from the study subjects
• Determine IgE-specificity (using Westerns, ELISA,
  ELISA inhibition)
• Determine appropriate surrogate tests for
  standardization
• Determine appropriate reference standards