1. U.S. Food and Drug Administration Notice: Archived Document The content in this document is provided on the FDA’s website for reference purposes only. It was current when produced, but is no longer maintained and may be outdated.

2. 2 Legal Status and Regulation of Allergen Products in the United States Ronald L. Rabin, MD Chief, Laboratory of Immunobiochemistry CBER/OVRR/DBPAP

3. 3 Outline 1.Change of Potency Assay for Standardized Short Ragweed Pollen and Cat Allergen Extracts 2. Environmental Exposure Units for Phase 3 Studies

4. 4 Outline 1.Change of Potency Assay for Standardized Short Ragweed Pollen and Cat Allergen Extracts 2. Environmental Exposure Units for Phase 3 Studies

5. 5 Standardized products are controlled for potency and stability House Dust Mites D. farinae D. pteronyssinus Cat Cat hair Cat pelt Hymenoptera Honey bee Wasp Yellow jacket Yellow hornet White-faced hornet Mixed vespid Short ragweed pollen Grass pollens Bermuda grass Red top June (Kentucky blue) Perennial rye Orchard Timothy Meadow fescue Sweet vernal

6. 6 Unitage for standardized allergens Standardized allergenUnitage Venomsµg protein Short Ragweed PollenUnits Amb a 1 / mL MiteAU / mL CatBAU / mL Units Fel d 1 / mL GrassesBAU / mL

7. 7 Allergen standardization (21 CFR 680.3(e)) Establishes that: potency refers to the allergenic activity of an extract once defined, the potency of each lot of that extract must be determined To satisfy 21 CFR 680.3(e), CBER has established a U.S. Standard and a testing procedure Manufacturers may use the CBER-established procedure, or may develop equivalent procedures

8. 8 Surrogate Assays for Potency Allergen(s)Current D. farinae and D. pteronyssinusCompetition ELISA Protein Cat pelt and cat hairRID - Fel d 1 IEF Protein GrassesCompetition ELISA IEF Protein Short ragweedRID - Amb a 1 HymenopteraHyaluronidase Phospholipase

9. 9 Procedure antibodies specific to the major allergen added to agar (1% agar + 1% azide) solidify agar glass slide punch wells in agar add equal amounts of antigen to wells incubate 72 hrs in humidified chamber (antigen complexes with antibody) immerse in 10% acetic acid for 2 min measure diameter Radial Immunodiffusion (RID)

10. 10 Radial Immunodiffusion (RID)

11. 11 Potential Advantages of ELISA vs. RID Much less time consuming RID readers are obsolete and no longer in production ELISA plates much easier to set up than gel slides Automated reader Specifications and reproducibility (e.g. correlation coefficient) much higher for ELISA Less reagent used per test Broader dynamic range

12. 12 Development of Sandwich ELISA for measurement of Fel d 1 and Amb a 1 Capture with one of three specific single chain variable fragments (scFv, 20 mg/mL)* Detect with sheep polyclonal antiserum (1:1000) Reveal with HRP conjugated anti-sheep IgG (1:1000) *deVore NC et al, Ann Allergy Asthma Immunol. 105:351; 2010

13. 13 Sandwich ELISA for measurement of Fel d 1 1 10 100 1000 native Fel d 1 (ng/mL) Concentration

14. 14 Sandwich ELISA for measurement of Fel d 1 from Cat Hair Extract Dilution of Cat Hair Extract Concentration

15. 15 Sandwich ELISA for measurement of Amb a 1 OD 450 Dilution of Short Ragweed Extract Concentration

16. 16 Next Steps for FDA to Adopt the Sandwich ELISA for testing of standardized Cat (Fel d 1) and ragweed (Amb a 1) allergenic extracts Select one of three scFv Western blot (cat scFv) Basophil stimulation assay (both) Prepare a validation protocol LIB perform the validation Invite manufacturers participate in the assay validation Summarize validation in a report Prepare at least 20g of each scFv

17. 17 Outline 1.Change of Potency Assay for Standardized Short Ragweed Pollen and Cat Allergen Extracts 2. Environmental Exposure Units for Phase 3 Studies

18. 18 Demonstration of efficacy of allergen immunotherapy for seasonal allergens Well designed Double Blind Placebo Controlled studies Comparison to placebo and to a “baseline” year FDA accepts combined symptom and medication scores as the primary endpoint Studies must be adequately powered taking into consideration: -expected differences between treatment and placebo (small) -expected variability of each group (high)

19. 19 Impediments to demonstrating efficacy of allergen immunotherapy for seasonal allergens Subjective nature of symptom scores Accepted differences between placebo and treatment groups ↓ Pivotal trials to prove efficacy of immunotherapy must be large; this requires multiple study sites. However: To induce symptoms, pollen levels at each site must be high, possibly for two consecutive years Studies of effective agents may fail due to poor pollen seasons

20. 20 Grass and ragweed pollen profiles in the Washington D.C. area, 1998-2007 Kosisky SE, Marks, MS, and Nelson MR, Ann Allergy, Asthma and Immunol 104:223; 2010

21. 21 Pollen Counts are Variable Pollen counts are highly variable within a single region The challenge of pollen variability increases with the number of study sites Variability in pollen seasons increases the variability of clinical symptoms enhancing the possibility of a failure to detect efficacy (type II error)

22. 22 Environmental Exposure Units Contained rooms in which exposure to airborne substances is controlled Advantages studies are not limited to the period of natural pollination controlled and uniform allergen exposure no impact of weather conditions no impact of personal context (participation in outdoor activities, etc) ensured compliance timed symptom assessments

23. 23 Consideration of EEU for clinical trials in support of licensure in the United States In March, 2009, a group of allergists met to explore the use of EEU for clinical trials to support licensure: Consensus EEU can provide uniform distribution of pollen Larger units require more monitoring Peak Nasal Inspiratory Flow may be an efficacy variable in addition to combined medication/symptom scores A priming phase must be included for seasonal allergens Mono-allergic subjects are unnecessary Natural exposure studies may also be necessary

24. 24 Clinical trials in support of licensure in the US In February, 2010, the NIAID met with stakeholders to further discuss the use of EEU in Phase 3 studies Consensus Standards for EEU need to be set and harmonized Requires sharing of data (but not protected technology)

25. 25 Clinical trials in support of licensure in the US An NIAID-hosted workshop with stakeholders in June, 2010 Attendees agreed to prepare a publishable document to discuss the need for EEU validation Consider preparing collaborative grant applications to fund EEU validation studies Study 1: Comparison of the Effectiveness of Common Seasonal Allergic Rhinitis Medications in the EEU and Under Natural Seasonal Allergen Exposure Study #2: Determining Inter-EEU Variability Under Standardized Allergen Exposure Conditions

26. 26 Approach towards allergenics: Controlled vs. Natural Exposure Allergen TypeExposure - Controlled Exposure - Natural Food+- Hymenoptera+- Pets / Molds+/-+ Pollen?+

27. 27 Summary EEU are potentially an attractive tool for proving efficacy of novel products for allergen immunotherapy EEU studies alone may not be sufficient for demonstrating efficacy of immunotherapeutics; natural exposure studies may continue to be required Outstanding issues: Validation of even distribution of pollen throughout an EEU Harmonization of standards among the different facilities in North America and Europe Contribution of behavioral aspects of a group to bias of data

28. 28 Acknowledgements Environmental Exposure Units FDA Jay Slater Paul Richman DAIT/NIAID Alkis Togias Marshall Plaut Matthew Fenton Cat/Ragweed ELISA Jay Slater Nicolette deVore Taruna Khurana Sandra Menzies Susan Huynh