German cockroach allergen standardization – progress report

2/51 Cockroaches

3/51 Biological potency by ID 50 EAL testing (Intradermal Dilution for 50 mm Sum of Erythema Determines Bioequivalent Allergy Units) Highly allergic individuals Serial 3-fold dilutions Establish dilution at which  E = 50 mm (D 50 )

4/51 Biological potency by ID 50 EAL testing

5/51 Testing Screen: puncture test (bifurcated needle) with concentrates Intradermal testing with serial 3-fold dilutions Record wheal and erythema size Calculate  E

6/51 Calculation of D50 D50

7/51 ID 50 EAL method D50 = ,000 BAU/mL BAU/mL = 100,000 x 3 (D )

8/51 Testing ID testing with serial 3-fold dilutions Calculate  E The skin response (  E) should fall within the limits of >0 to < 125 mm Each more concentrated dilution should produce a graded erythema response The four dilutions selected should span a wide range of  E (for example from 0-20 mm to mm) and bracket  E of 50 mm

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23/51 Manufacturing data

24/51 Preclinical data Bla g 1 Bla g 2 Bla g 5 Relative potency Human Rabbit Anti-CR Pooled anti-specific allergens

25/51 Specific allergen data (two-site ELISA)

26/51 Overall allergenicity competition ELISA

27/51 IND 11319: German Cockroach Allergen Standardization Evaluation (CASE) Sponsor: DAIT/NIAID Four sites Baltimore Washington DC Chicago Denver

28/51 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

29/51 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. IND 11319: German Cockroach Allergen Standardization Evaluation (CASE)

30/51 IND 11319: German Cockroach Allergen Standardization Evaluation (CASE) Design Multi-center, open-label Number Planned: 61 Number Entered: 62 Number Completed: 62

31/51 IND 11319: German Cockroach Allergen Standardization Evaluation (CASE) Age Range: Gender: Male=15, Female=47 Race/ethnicity Black = 35 Hispanic = 14 White = 5 Asian = 1 Native American = 0 Other = 1 More than one race = 6

32/51 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 50 D50

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34/51 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 50 D50

35/51 Linear interpolation rules Use the first crossing followed by at least two consecutive non-missing dilutions above the 50mm  E  line. If the above criterion does not apply and the data ’ s most concentrated dilution is above the 50 mm  E  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  E 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 subject ’ s extract.

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37/51 Regression vs interpolation

38/51 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  (erythema) is expected. D = Hill coefficient or slope factor. (When D=1, the equation reduces to the 3-parameter logistic equation.) 50 D50

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41/51 D50 data

42/51 Potency data (BAU/mL)

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44/51 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 (  < 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.

45/51 Which in vitro data best reflect in vivo potency?

46/51 All data (normalized to extract B)

47/51 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

48/51 Where does this fit? Grass pollens: 100,000 BAU/mL Ragweed: 30, ,000 BAU/mL [Based on estimate of 350 Amb a 1 U/mL=100,000 BAU/mL] Bermuda grass: 10,000 BAU/mL Cat: 5,000-10,000 BAU/mL German cockroach: 3,300 BAU/mL

49/51 Where does this fit? Skin test doses BAU/mL Ann Allergy Asthma Immunol. 1995; 75:553 IT doses: BAU Clin Allergy Immunol 1999; 12: BAU Ann Allergy Asthma Immunol. 2003;90(1 Suppl 1):1

50/51 Where does this fit? Calculated from Nelson, chapter 24, in Lockey and Bukantz, Allergens and Allergen Immunotherapy, 3 rd ed, 2004

51/51 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