Scientific Contribution 2018

Transcription

1 Allergy in a changing world Scientific Contribution th Congress of the European Academy of Allergy and Clinical Immunology EAACI, May 218, Munich, Germany

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3 Dear Congress delegate, On behalf of HAL Allergy, it is our pleasure to welcome you to the EAACI 218 Congress in Munich. HAL Allergy will present the latest results of its research & dlopment program both in the format of oral and poster presentations in the scientific program of the conference as well as by organizing a company sponsored symposium. The HAL Allergy symposium Allergy in a changing world will be held on Monday May 28, 218 from 13.45h to 15.15h. Harry Flore, PhD Chief Executive Officer Dirk-Jan Opstelten, PhD Chief Scientific Officer The theme for this year s EAACI Congress is Innovative solutions for allergy. Innovation lies at the heart of progress in the field of allergy and will help and propel health care professionals, researchers and industry to offer allergic patients a better quality of life. As one of Europe s leading players in the field of allergen immunotherapy HAL Allergy is pleased to present its contribution to allergy research and clinical practice to the participants of the Congress. At this year s conference we will provide an update on HAL Allergy s clinical dlopment program to demonstrate the clinical efficacy and safety of our immunotherapies and support their registration. The phase II and phase III study results obtained in the clinical program of liquid birch pollen extract for Sublingual Immunotherapy (SLIT) support its use in patients with allergic rhinitis/rhinoconjunctivitis with or without asthma. In a phase II allergen exposure chamber (AEC) trial the efficacy of SLIT with different doses of Phleum pratense liquid pollen extract in grass pollen allergic patients was demonstrated. Next to this in a prospective non-interventional study an overall good tolerability of a subcutaneous rush up-dosing scheme with modified allergens in pollen allergic subjects in day-to-day practice was shown. Apart from the clinical research we also present interesting data on an IgE based assay used to determine individual reactions to purified and modified single peanut allergens as well as an IgG based assay to be used in stability studies of formulated allergoids. Posters on the production of recombinant Bet v1 in a CHO system and on the use of handheld pollen counters are in line with the theme of our satellite symposium Allergy in a changing world. Together these data underpin HAL Allergy s continuous quest to move allergy immunotherapy and, consequently, the field of allergy as a whole into the future. In summary, our presented work provides an update on HAL Allergy s product dlopment program, which is designed to enhance the wellbeing of allergic patients, by providing them high quality products with demonstrated efficacy and safety. We wish you a fruitful congress; if you require further information about our products or our R&D program, please be invited to meet us at our symposium or visit our Scientific Corner in the HAL Allergy booth in the exhibition area. Kind regards, Harry Flore, PhD Chief Executive Officer Dirk-Jan Opstelten, PhD Chief Scientific Officer

4 Scientific Contribution 218 HAL Allergy Symposium Monday 28 May, 218, 13:45 15:15 Room 5 Allergy in a changing world Chairs: Ronald van Ree, The Netherlands Dirk-Jan Opstelten, The Netherlands Climate Change; its effect on the allergy epidemic Jeroen Buters, Germany Biologics versus AIT; Who will win? Nikos Papadopoulos, United Kingdom AIT: fixing current and future needs Moisés Calderón, United Kingdom

5 Scientific Contribution 218 Abstracts and poster presentations Contents Clinical Clinical program of a liquid birch pollen extract for SLIT: concordance with current regulatory requirements of EMA... 6 Sublingual Immunotherapy with a liquid Phleum pratense extract is effective in grass pollen allergic patients Results of a phase II allergen exposure chamber (AEC) trial... 8 Dose response and onset of action of sublingual Immunotherapy with a liquid Phleum pratense extract in grass pollen allergic patients... 1 Efficacy of SLIT with different doses of a liquid phleum pratense extract was shown during the grass pollen season and in an Allergen Exposure Chamber (AEC) Tolerability of a two week Rush up-dosing with modified allergens in pollen allergic subjects in the day-to-day practice Tolerability of a two week Rush up-dosing with modified Trees, modified Grasses or modified Grasses / Trees mixture in pollen allergic subjects in the day-to-day practice Dlopment ielisa as a tool to measure IgE binding towards single modified peanut allergens An IgG inhibition ELISA to measure the stability of alum-adsorbed grass pollen allergoids... 2 Glycosylation of rbet v 1.11 by Chinese Hamster Ovary cells affects proteolytic stability towards endo/lysosomal degradation Towards personalized pollen exposure measurements using hand held pollen samplers... 24

6 Clinical program of a liquid birch pollen extract for SLIT: concordance with current regulatory requirements of EMA O. Pfaar 1,2, L. Klimek², D. Boot³, P.J. de Kam³, A. Narkus⁴, D. Yu⁵, A. Larionov⁵ and D.J. Opstelten⁵ 1 Department of Otorhinolaryngology, Head and Neck Surgery, Universitätsmedizin Mannheim, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany. ²Center for Rhinology and Allergology, Wiesbaden, Germany. ³At time of clinical trial HAL Allergy BV, Leiden, The Netherlands, ⁴MC Narkus, Bleckede, Germany, ⁵HAL Allergy BV, Leiden, The Netherlands Background According to the current EMA guideline (CHMP/EWP/1854/26) a dose-response relationship for clinical efficacy with different doses in sral study-arms should be established e.g. by nasal challenge tests. Subsequently, confirmatory trials should be performed using a randomized placebo-controlled design with a combined symptom and medication score as primary endpoint to show a predefined and justified clinically relevant difference in the primary endpoint between test and control population. Here we report on the clinical dlopment program of a sublingual birch immunotherapy product. Methods In a first randomized, double-blind, placebo-controlled (DBPC), multi-center, dose tolerability, dose-range-finding trial, patients with birch pollen-induced rhinitis/rhinoconjunctivitis (ARC) with or without concomitant asthma (n=269, 18-6 years of age) were treated for 2 weeks with 4 doses of the liquid birch extract (3,333 AUN/ml; 1, AUN/ml; 2, AUN/ml; 4, AUN/ml) or placebo (ClinicalTrials.gov: NCT ). In a subsequent randomized, DBPC, parallel-group study, patients with birch pollen induced ARC with or without asthma (n=46, years of age) were treated with the liquid birch pollen extract (4, AUN/mL) or placebo pre- and co-seasonally (ClinicalTrials.gov NCT223137). Results In phase II the primary efficacy results demonstrated a clear dose response curve in the titrated nasal challenge (primary endpoint), with all active doses responding better than placebo and the 4, AUN/ml dose proved to be the most effective dose. These results were supported by the secondary efficacy parameters (Pfaar et al. Allergy 216;71(1):99-17). In phase III a clinically relevant (32%) and statistically significant (p<.1) reduction in the CSMS (primary endpoint) in patients treated with the liquid birch extract (4, AUN/mL) compared to placebo were observed (Pfaar et al. Allergy (216);71(suppl.12): 45(abstract 87). The secondary efficacy parameters consistently supported this finding (Pfaar et al. Allergy 216;71(12):45 (abstract 19). Conclusion The liquid birch pollen extract has been dloped according to the current regulatory requirements. The optimal dose identified in phase II was mirrored by a significant and clinically relevant improvement in CSMS in phase III. The liquid birch pollen extract is an interesting new option for SLIT in patients with ARC with or without asthma. Session number, date and time: PDS3, Tuesday 29 May 218; 15:3-17: Session title: Efficacy and safety of novel immunotherapy approaches 6 EAACI, 26-3 May 218, Munich, Germany

7 Figure 1: Phase II study: Decrease in Lebel score compared to placebo after 5 months of treatment with the Liquid-Birch-Extract (mitt) -,5 p= p=.8-1,5 p< ,5 Figure 2: Phase II study: Increase in PNIF compared to placebo after 5 months of treatment with the Liquid-Birch-Extract Figure 3: Phase III study: Mean CSMS during the birch pollen season for placebo and Liquid- Birch-Extract 4, AUN/mL treated patients (ITT) Table 1: Phase III study: Clinical results p-value Liquid-Birch- Extract 4, AUN/mL - Placebo Difference (95 % CI) Mean (SE) Liquid-Birch- Extract 4, AUN/mL (N=179) Mean (SE) Placebo (N=178) <,1 -,46 (-,66, -,26) 1, (,8) 1,45 (,8) Primary endpoint Combined symptom- and medication-score (CSMS) during pollen season (PS) Secondary endpoints <,1 -,6 (-,87, -,33),96 (,1) 1,56 (,1) CSMS during peak PS <,1 -,37 (-,51, -,23),53 (,5),9 (,5) Symptom-score during peak PS,8 -,23 (-,4, -,6),43 (,6),66 (,6) Medication-score during peak PS Treatment group 4, AUN/ml 2, AUN/ml 1, AUN/ml 3,333 AUN/ml LS mean change from baseline in Lebel score compared to placebo p<.1 p=.476 p=.416 p= Clinical program of a liquid birch pollen extract for SLIT: concordance with current regulatory requirements of EMA O. Pfaar 1,2, L. Klimek 2, D. Boot 3, P.J. de Kam 3, A. Narkus 4, D. Yu 5, A. Larionov 5, D.J. Opstelten 5 1 Department of Otorhinolaryngology, Head and Neck Surgery, Universitätsmedizin Mannheim, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany. 2 Center for Rhinology and Allergology, Wiesbaden, Germany. 3 At time of clinical trial HAL Allergy BV, Leiden, The Netherlands, 4 MC Narkus, Bleckede, Germany, 5 HAL Allergy BV, Leiden, The Netherlands Background & Aim: According to the current EMA guideline (CHMP/EWP/1854/26) a dose-response relationship for clinical efficacy with different doses in sral study-arms should be established e.g. by nasal challenge tests. Subsequently, confirmatory trials should be performed using a randomized placebo-controlled design with a combined symptom and medication score as primary endpoint to show a predefined justified clinically relevant difference in the primary endpoint between test and control population. Here we report on the clinical dlopment program of a sublingual birch immunotherapy product LS mean change from baseline in PNIF (L/min) compared to placebo Methods: ,333 AUN/ml 1, AUN/ml 2, AUN/ml 4, AUN/ml Treatment group CSMS in pollen season (ITT) -32% p <,1 In a first randomized, double-blind, placebo-controlled (DBPC), multi-center, dose tolerability, dose-range-finding trial, patients with birch pollen-induced allergic rhinitis/rhinoconjunctivitis (ARC) with or without concomitant asthma (n=269, age: 18-6 years of age) were treated for 2 weeks with 4 doses of the liquid birch extract (3,333 AUN/ml; 1, AUN/ml; 2, AUN/ml; 4, AUN/ml) or placebo (ClinicalTrials. gov: NCT ). In a subsequent randomized, DBPC, parallel-group study, patients with birch pollen induced ARC with or without asthma (n=46, years of age) were treated with the liquid birch pollen extract (4, AUN/mL) or placebo pre- and co-seasonally (ClinicalTrials. gov NCT223137). 1,6 1,4 1,2 Results: 1,8,6 LS-Mean CSMS,4,2 Placebo SB In phase II the primary efficacy results demonstrated a clear dose response curve in the titrated nasal challenge (primary endpoint), with all active doses responding better than placebo and the 4, AUN/ml dose proved to be the most effective dose. These results were supported by the secondary efficacy parameters 1. In phase III a clinically relevant (3 2%) and statistically significant (p<.1) reduction in the CSMS (primary endpoint) in patients treated with the liquid birch extract (4, AUN/mL) compared to placebo were observed 2. The secondary efficacy parameters consistently supported this finding 3. Conclusion: The liquid birch pollen extract has been dloped according to the current regulatory requirements. The optimal dose identified in phase II was mirrored by a significant and clinically relevant improvement in CSMS in phase III. The liquid birch pollen extract is an interesting new option for SLIT in patients with ARC with or without asthma.,1 15,26 (7,64, 22,89) 67,53 (3,31) % well days during PS 52,26 (3,32),2-5,43 (-8,89, -1,97) 4,4 (1,32) % sre days during PS 9,47 (1,32) <,1 -,55 (-,77, -,33),55 (,9) 1,1 (,9) Rhinoconjunctivitis quality of life questionnaire-score (RQLQ-S) during PS,25 4,77 (1,68, 7,86) -3,95 (1,27) -8,72 (1,27) Euro Quality of Life Visual Analogue Scale (EQ-VAS) during PS References: (1) Pfaar et al. Allergy 216;71(1):99-17 (2) Pfaar et al. Allergy 216;71(12):45(abstract 87) (3) Pfaar et al. Allergy 216;71(12):45(abstract 19) In relation to this presentation, I declare the following, real or perceived conflict of interest. The authors are employees of HAL Allergy or were contracted for the study by HAL Allergy. EAACI Congress 218

8 Sublingual Immunotherapy with a liquid Phleum pratense extract is effective in grass pollen allergic patients Results of a phase II allergen exposure chamber (AEC) trial PDS3 - Efficacy and safety of novel immunotherapy approaches Clinical program of a liquid birch pollen extract for SLIT: concordance with current regulatory requirements of EMA E. Mantikou¹, P. Couroux², A. Salapatek², O. Pfaar 3,4, A. Narkus⁵, D. Yu¹, A. Larionov¹, D.J. Opstelten¹, P.J. de Kam¹ 1 HAL Allergy BV, Leiden, The Netherlands; 2 Inflamax Research Inc., Mississauga, ON Canada; 3 Department of Otorhinolaryngology, Head and Neck Surgery, Universitätsmedizin Mannheim, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany; 4 Center for Rhinology and Allergology, Wiesbaden, Germany; 5 MC Narkus, Bleckede, Germany Background According to the EMA guideline on the clinical dlopment of products for specific immunotherapy products should be tested in phase II at different doses in sral study-arms to establish a dose-response relationship for clinical efficacy before confirmatory trials can be initiated. Allergen exposure in an AEC may be used as primary endpoint. Methods The study was a single-center, randomized, double blind, placebo-controlled, phase II trial, treatment duration 1 months. 168 grass pollen allergic patients (18-65 years of age) with seasonal rhinitis/rhinoconjunctivitis (ARC) with (mild, GINA I) or without concomitant asthma were randomized to three different dosages of a liquid Phleum pratense extract (1, AUN/ ml; 4, AUN/mL; 8, AUN/mL) or placebo for sublingual administration (maximum dose 5 drops, up-dosing in 4 days). The primary efficacy endpoint was the mean Total Symptom Score (TSS: sum of individual scores for eight nasal and non-nasal symptoms, rated on a scale of -3) at the end of the trial. For TSS assessment a validated AEC was used with a standardized controlled grass pollen exposure at an average concentration of 35 ± 5 ppm 3 for 6 hours ensuring aeroallergen exposure under controlled and reproducible conditions. Results According to the pre-specified Emax model a borderline significant dose-response relationship for TSS was observed (p=.5 per protocol population,.57 ITT). At the end of the trial all patients (ITT) treated with the different doses of the liquid Phleum Pratense extract showed an improvement in the TSS compared to placebo (LS Means (SE): placebo 1.1 points (.8), 1. AUN/mL 8.6 (.85), 4. AUN/mL 8.19 (.82) and 8, AUN/mL 7.69 (.83), respectively). The TSS reduction compared to placebo ranged from 18.2% to 23.2%. The treatment emergent adverse nts (TEAE) were primarily local reactions, of mild intensity with a dose-response relationship. The incidence of patients dloping at least one systemic reaction (all Grade I), were similar between the active treatment groups and the placebo group. Conclusion A consistent improvement of ARC symptoms was observed with all doses of the liquid Phleum pratense extract, including the current marketed dose of 1, AUN/mL, compared to placebo in this AEC model. The safety profile was comparable to other sublingual immunotherapy (SLIT) products. Session number, date and time: TPS16, Sunday 27 May 218; 12:-13:3 Session title: Immunotherapy in the clinic 1 EAACI, 26-3 May 218, Munich, Germany 7 8 EAACI, 26-3 May 218, Munich, Germany

9 Sublingual Immunotherapy with a liquid Phleum pratense extract is effective in grass pollen allergic patients - Results of a phase II allergen exposure chamber (AEC) trial E. Mantikou 1, P. Couroux 2, A. Salapatek 2, O. Pfaar 3,4, A. Narkus 5, D. Yu 1, A. Larionov 1, D.J. Opstelten 1, P.J. de Kam 1 1 HAL Allergy BV, Leiden, The Netherlands; 2 Inflamax Research Inc., Mississauga, ON Canada; 3 Department of Otorhinolaryngology, Head and Neck Surgery, Universitätsmedizin Mannheim, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany; 4 Center for Rhinology and Allergology, Wiesbaden, Germany; 5 MC Narkus, Bleckede, Germany Background & Aim: Figure 1: Calculation of the primary endpoint, TSS Mild Moderate Sre According to the EMA guideline on the clinical dlopment of products for specific immunotherapy products should be tested in phase II at different doses in sral study-arms to establish a dose-response relationship for clinical efficacy before confirmatory trials can be initiated. Allergen exposure in an AEC may be used as a model for environmental exposure for efficacy assessment of SLIT. Daily Symptom Score (dss) red eyes watery eyes itchy/burning eyes itchy ear/palate Methods: The study was a single-center, randomized, double blind, placebocontrolled, phase II trial, treatment duration 1 months. 168 grass pollen allergic patients (18-65 years of age) with seasonal rhinitis/rhinoconjunctivitis (ARC) with (mild, GINA I) or without concomitant asthma were randomized to three different dosages of a liquid Phleum pratense extract (1, AUN/mL; 4, AUN/mL; 8, AUN/mL) or placebo for sublingual administration (maximum dose 5 drops, up-dosing in 4 days). The primary efficacy endpoint was the mean Total Symptom Score (TSS: sum of individual scores for eight nasal and non-nasal symptoms, rated on a scale of -3) at the end of the trial. For TSS assessment a validated for grass pollen AEC was used with a standardized controlled grass pollen exposure at an average concentration of 35 ± 5 ppm3 for 6 hours ensuring aeroallergen exposure under controlled and reproducible conditions. Results: According to the pre-specified Emax model a borderline significant dose-response relationship for TSS was observed (p=.5 per protocol population,.57 ITT). At the end of the trial all patients (ITT) treated with the different doses of the liquid Phleum Pratense extract showed an improvement in the TSS compared to placebo (LS Means (SE): placebo 1.1 points (.8), 1. AUN/mL 8.6 (.85), 4. AUN/mL 8.19 (.82) and 8, AUN/mL 7.69 (.83), respectively). The TSS reduction compared to placebo ranged from 18.2% to 23.2%. The treatment emergent adverse nts (TEAE) were primarily local reactions, of mild intensity with a doseresponse relationship. The incidence of patients dloping at least one systemic reaction (all Grade I), were similar between the active treatment groups and the placebo group. runny nose congestion itchy nose Nasal Non -Nasal Figure 2: Borderline significant TSS dose-response using the pre-specified Emax model (ITT population) sneezing Adjusted Mean TSS 8. 2 p*=.57 emax Table 1: Mean TSS at Visit 6 (ITT population) DOSE Parameter Statistics Placebo SP SP SP (N=4) 1, AUN/mL 4, AUN/mL 8, AUN/mL (N=35) (N=38) (N=37) Baseline N (missing) 4 () 35 () 38 () 37 () Mean (SD) (4.29) (4.6) (3.68) (4.11) Median Visit 6 N (missing) 4 () 35 () 38 () 37 () Mean (SD) 1.29 (5.43) 7.92 (5.59) 8.6 (4.46) 7.64 (5.53) Median Conclusion: A consistent improvement of ARC symptoms was observed with all doses of the liquid Phleum pratense extract, including the current marketed dose of 1, AUN/mL, compared to placebo in this AEC model. The safety profile was comparable to other sublingual immunotherapy (SLIT) products. Change N (missing) 4 () 35 () 38 () 37 () between Mean (SD) (6.19) (6.9) -5.6 (4.22) (4.78) Visit 6 and Median Baseline Trt Eff. (SE) (1.17) (1.14) (1.15) 9% CI [-3.89 to -.3] [-3.72 to.6] [-4.23 to -.43] Rel. Diff. (%) *The model included the mean TSS at visit 6 as a dependent variable, treatment as fixed factor and Baseline Mean TSS as covariate Trt Eff.= Treatment effect, Rel. Diff.= Relative difference. In relation to this presentation, I declare the following, real or perceived conflict of interest. The presenter is an employee of HAL Allergy. EAACI Congress 218

10 Dose response and onset of action of sublingual Immunotherapy with a liquid Phleum pratense extract in grass pollen allergic patients TPS16 - Immunotherapy in the clinic 1 Sublingual Immunotherapy with a liquid Phleum pratense extract is effective in grass pollen allergic patients Results of a phase II allergen exposure chamber (AEC) trial E. Mantikou¹, P. Couroux², A. Salapatek², O. Pfaar 3,4, P.J. de Kam¹, D. Yu¹, A. Narkus⁵, A. Larionov¹, D.J. Opstelten¹ 1 HAL Allergy BV, Leiden, The Netherlands; 2 Inflamax Research Inc., Mississauga, ON Canada; ³Department of Otorhinolaryngology, Head and Neck Surgery, Universitätsmedizin Mannheim, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany; ⁴Center for Rhinology and Allergology, Wiesbaden, Germany; ⁵MC Narkus, Bleckede, Germany. Background After establishing a tolerated dose range, studies should be performed to establish a dose-response relationship for clinical efficacy. Such studies may comprise a short-term treatment (e.g. 2-4 month) with different doses in sral study-arms. Allergen exposure in allergen exposure chambers (AEC) may be used as primary endpoint. Method In a single-center, randomized, double blind, placebo-controlled, phase II trial with a treatment duration of 1 months a total of 168 grass pollen allergic patients (18-65 years of age) with seasonal rhinitis/rhinoconjunctivitis (ARC) with or without concomitant asthma, were randomized to sublingual immunotherapy (SLIT) with three different dosages of a liquid Phleum pratense extract (1, AUN/mL; 4, AUN/mL; 8, AUN/mL) or placebo. An efficacy endpoint was the mean total symptom score (TSS: sum of individual scores for eight nasal and non-nasal symptoms, rated on a scale of -3) at baseline, after 5 months and 1 months of treatment. For TSS assessment a validated AEC was used with a standardized controlled grass pollen exposure at an average concentration of 35 ± 5 ppm 3 for 6 hours ensuring aeroallergen exposure under controlled and reproducible conditions. Results A statistically significant improvement of the TSS after 5 months compared to baseline was shown for all groups, including placebo. The results showed a dose-response curve reaching a plateau at 4, AUN/mL. After 1 months of treatment with the liquid Phleum pratense extract a further improvement in TSS was seen compared to placebo. Howr, clear differences between the various doses of the liquid Phleum pratense extract were no longer seen at this time point. Conclusion The results demonstrate that the allergen exposure chamber provides a suitable model to identify a clear dose response effect of treatment with a liquid Phleum pratense extract. The treatment duration was found to be of critical importance to identify differences between the various doses, suggesting that the dose may not only impact the effect-size but also the time of onset of effect. Session number, date and time: PDS3, Tuesday 29 May 218; 15:3-17: Session title: Efficacy and safety of novel immunotherapy approaches EAACI, 26-3 May 218, Munich, Germany 9 1 EAACI, 26-3 May 218, Munich, Germany

11 Dose response and onset of action of sublingual Immunotherapy with a liquid Phleum pratense extract in grass pollen allergic patients E. Mantikou 1, P. Couroux 2, A. Salapatek 2, O. Pfaar 3,4, P.J. de Kam 1, D. Yu 1, A. Narkus 5, A. Larionov 1, D.J. Opstelten 1 1 HAL Allergy BV, Leiden, The Netherlands; 2 Inflamax Research Inc., Mississauga, ON Canada; 3 Department of Otorhinolaryngology, Head and Neck Surgery, Universitätsmedizin Mannheim, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany; 4Center for Rhinology and Allergology, Wiesbaden, Germany; 5 MC Narkus, Bleckede, Germany Background & Aim: After establishing a tolerated dose range, studies should be performed to establish a dose-response relationship for clinical efficacy. Such studies may comprise a short-term treatment (e.g. 2-4 month) with different doses in sral study-arms. Allergen exposure in allergen exposure chambers (AEC) may be used as a model for environmental exposure for efficacy assessment of SLIT. Methods: In a single-center, randomized, double blind, placebo-controlled, phase II trial with a treatment duration of 1 months a total of 168 grass pollen allergic patients (18-65 years of age) with seasonal rhinitis/rhinoconjunctivitis (ARC) with or without concomitant asthma, were randomized to sublingual immunotherapy (SLIT) with three different dosages of a liquid Phleum pratense extract (1, AUN/mL; 4, AUN/mL; 8, AUN/mL) or placebo. An efficacy endpoint was the mean total symptom score (TSS: sum of individual scores for eight nasal and non-nasal symptoms, rated on a scale of -3) at baseline, after 5 months and 1 months of treatment. For TSS assessment a validated AEC was used with a standardized controlled grass pollen exposure at an average concentration of 35 ± 5 ppm3 for 6 hours ensuring aeroallergen exposure under controlled and reproducible conditions. Figure 1: Graphical representation of the change in the mean TSS from baseline to visit 4 (a) and visit 6 (b) including the standard error of the mean, per treatment group (ITT population) a. b Change of Mean TSS from Baseline to Visit 4 as compared to placebo Placebo SP 1,AUN/mL SP 4,AUN/mL SP 8,AUN/mL LS Mean TSS p=,291 p=,42 p=,5 Change of Mean TSS from Baseline to Visit 6 as compared to placebo Placebo SP 1,AUN/mL SP 4,AUN/mL SP 8,AUN/mL LS Mean TSS Results: A statistically significant improvement of the TSS after 5 months (visit 4) compared to baseline was shown for all groups, including placebo. The results showed a dose-response curve reaching a plateau at 4, AUN/mL. After 1 months (visit 6) of treatment with the liquid Phleum pratense extract a further improvement in TSS was seen compared to placebo. Howr, clear differences between the various doses of the liquid Phleum pratense extract were no longer seen at this time point. Conclusion: The results demonstrate that the allergen exposure chamber provides a suitable model to identify a clear dose response effect of treatment with a liquid Phleum pratense extract. The treatment duration was found to be of critical importance to identify differences between the various doses, suggesting that the dose may not only impact the effect-size but also the time of onset of effect p=,51 p=,59 p=,24 Table 1: Change from Baseline in Mean TSS at Visits 4 and 6, per treatment group (ITT Population) Parameter Statistics Placebo SP SP SP (N=4) 1, AUN/mL 4, AUN/mL 8, AUN/mL (N=35) (N=38) (N=37) Baseline N (missing) 4 () 35 () 38 () 37 () Mean (SD) (4.29) (4.6) (3.68) (4.11) Median Visit 4 N (missing) 4 () 35 () 38 () 35 (2) Mean (SD) (4.89) 1.29 (5.31) 9.6 (4.23) 9.13 (5.66) Median Change between N (missing) 4 () 35 () 38 () 35 (2) Visit 4 and Mean (SD) (4.81) (6.91) -4.7 (4.5) -4.7 (4.21) Baseline Median Visit 6 N (missing) 4 () 35 () 38 () 37 () Mean (SD) 1.29 (5.43) 7.92 (5.59) 8.6 (4.46) 7.64 (5.53) Median Change between N (missing) 4 () 35 () 38 () 37 () Visit 6 and Mean (SD) (6.19) (6.9) -5.6 (4.22) (4.78) Baseline Median In relation to this presentation, I declare the following, real or perceived conflict of interest.: the authors are employees of HAL Allergy or were contracted for the study by HAL Allergy. EAACI Congress 218

12 Efficacy of SLIT with different doses of a liquid phleum pratense extract was shown during the grass pollen season and in an Allergen Exposure Chamber (AEC) PDS3 - Efficacy and safety of novel immunotherapy approaches Dose response and onset of action of sublingual Immunotherapy with a liquid Phleum pratense extract in grass pollen allergic patients E. Mantikou¹, P. Couroux², A. Salapatek², O. Pfaar 3,4, P.J. de Kam¹, A. Narkus⁵, D. Yu¹, A. Larionov¹, D.J. Opstelten¹ 1 HAL Allergy BV, Leiden, The Netherlands; 2 Inflamax Research Inc., Mississauga, ON Canada; ³Department of Otorhinolaryngology, Head and Neck Surgery, Universitätsmedizin Mannheim, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany; ⁴Center for Rhinology and Allergology, Wiesbaden, Germany; ⁵MC Narkus, Bleckede, Germany. Background In Phase III allergen immunotherapy trials, Combined Symptom Medication Score (CSMS) is the recommended clinical outcome measure. In phase II studies, Total Symptom Score (TSS) in an AEC is accepted as primary endpoint. This study was performed both in the AEC and the field to examine whether TSS is consistent with CSMS. Methods In a single-center, randomized, double blind, placebo-controlled, dose range finding trial, 168 grass pollen allergic patients (18-65 years of age) with seasonal rhinitis/rhinoconjunctivitis with or without concomitant asthma, were randomized to three different dosages of a liquid phleum pratense extract (1, AUN/mL, 4, AUN/mL, 8, AUN/mL) or placebo for 1 months of sublingual immunotherapy (SLIT). The total symptom score (TSS: sum of individual scores for eight nasal and nonnasal symptoms, rated on a scale of -3) in a validated AEC was assessed at baseline and at the end of the trial, the combined symptom medication score (CSMS: 6 nasal and non-nasal symptoms and medication score, score can range from -6) during the grass pollen season at month 8-1. Results When comparing the change from baseline in TSS for each active treatment group with placebo, a statistically significant improvement in TSS was found for the 8, AUN/mL (p=.24) and a trend for a reduction for both the 1, AUN/mL group (p=.51) and 4, AUN/mL group (p=.59), at the end of the trial. The reduction in CSMS compared to placebo was substantial for all doses of SLIT with the liquid phleum pratense extract, ranging from 27% to 38% improvement and reached statistical significance for the 1, AUN/mL (p=.28) and 4, AUN/mL (p=.18) groups (8, AUN/mL group p=.63). Conclusions Efficacy of SLIT with different doses of a liquid phleum pratense extract was shown using different clinical symptom scores and clinical settings. TSS during controlled exposure to grass pollen in an AEC is consistent with CSMS measured during grass pollen season. Importantly, the clinically relevant improvement in CSMS achid at all investigated phleum pratense dose lls, provides a strong efficacy basis for the next phase of clinical dlopment. Session number, date and time: PDS3, Tuesday 29 May 218; 15:3-17: Session title: Efficacy and safety of novel immunotherapy approaches EAACI, 26-3 May 218, Munich, Germany EAACI, 26-3 May 218, Munich, Germany

13 Efficacy of SLIT with different doses of a liquid phleum pratense extract was shown during the grass pollen season and in an Allergen Exposure Chamber (AEC) E. Mantikou 1, P. Couroux 2, A. Salapatek 2, O. Pfaar 3,4, P.J. de Kam 1, A. Narkus 5, D. Yu 1, A. Larionov 1, D.J. Opstelten 1 1 HAL Allergy BV, Leiden, The Netherlands; 2 Inflamax Research Inc., Mississauga, ON Canada; 3 Department of Otorhinolaryngology, Head and Neck Surgery, Universitätsmedizin Mannheim, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany; 4 Center for Rhinology and Allergology, Wiesbaden, Germany; 5 MC Narkus, Bleckede, Germany Background & Aim: In Phase III allergen immunotherapy trials, a Combined Symptom Medication Score (CSMS) is the recommended clinical outcome measure. In phase II studies, Total Symptom Score (TSS) in an AEC is accepted as primary endpoint. This study was performed both in the AEC and the field to examine whether TSS is consistent with CSMS. Methods: In a single-center, randomized, double blind, placebo-controlled, dose range finding trial, 168 grass pollen allergic patients (18-65 years of age) with seasonal rhinitis/rhinoconjunctivitis with or without concomitant asthma, were randomized to three different dosages of a liquid phleum pratense extract (1, AUN/mL, 4, AUN/mL, 8, AUN/mL) or placebo for 1 months of sublingual immunotherapy (SLIT). The total symptom score (TSS: sum of individual scores for eight nasal and non-nasal symptoms, rated on a scale of -3) in a validated AEC was assessed at baseline and at the end of the trial, the combined symptom medication score (CSMS: 6 nasal and non-nasal symptoms and medication score, score can range from -6) during the grass pollen season at month 8-1. Results: When comparing the change from baseline in TSS for each active treatment group with placebo, a statistically significant improvement in TSS was found for the 8, AUN/mL (p=.24) and a trend for a reduction for both the 1, AUN/mL group (p=.51) and 4, AUN/mL group (p=.59), at the end of the trial. The reduction in CSMS compared to placebo was substantial for all doses of SLIT with the liquid phleum pratense extract, ranging from 27% to 38% improvement and reached statistical significance for the 1, AUN/mL (p=.28) and 4, AUN/mL (p=.18) groups (8, AUN/mL group p=.63). Conclusion: Efficacy of SLIT with different doses of a liquid phleum pratense extract was shown using different clinical symptom scores and clinical settings. TSS during controlled exposure to grass pollen in an AEC is consistent with CSMS measured during grass pollen season. Importantly, the clinically relevant improvement in CSMS achid at all investigated phleum pratense dose lls, provides a strong efficacy basis for the next phase of clinical dlopment. Figure 1: Graphical representation of the change in the mean TSS from baseline to visit 6 including the standard error of the mean, per treatment group (ITT population) Figure 2: Graphical representation of the mean CSMS including the standard error of the mean, per treatment group (ITT population) Table 1: Mean CSMS during the pollen season, per treatment group (ITT Population) Parameter Statistics Placebo SP SP SP (N=4) 1, AUN/mL 4, AUN/mL 8, AUN/mL (N=35) (N=38) (N=37) Summary n Statistics Mean (SD) 1.3 (.81).68 (.66).64 (.61).75 (.68) Median Trt Eff. (SE) (.183) (.186) -.28 (.182) 9% CI [-.657 to -.5] [-.72 to -.86] [-.581 to.21] P-value# Rel. Diff. (%) *The model included the mean CSMS as a dependent variable, treatment as fixed factor #One-sided test Change of Mean TSS from Baseline to Visit 6 as compared to placebo LS Mean TSS 1,4 1,2 1,8,6,4, Placebo SP 1,AUN/mL SP 4,AUN/mL SP 8,AUN/mL Mean CSMS during the pollen season p=,28 p=,51 p=,59 LS Mean CSMS Placebo SP 1,AUN/mL SP 4,AUN/mL SP 8,AUN/mL Subjects completed =75% of their diary data during the pollen season are included for the analysis Trt. Eff.= Treatment effect, Rel. Diff.= Relative difference. p=,18 p=,63 p=,24 In relation to this presentation, I declare the following, real or perceived conflict of interest: the authors are employees of HAL Allergy or were contracted for the study by HAL Allergy. EAACI Congress 218

14 Tolerability of a two week Rush up-dosing with modified allergens in pollen allergic subjects in the day-to-day practice PDS3 - Efficacy and safety of novel immunotherapy approaches Efficacy of SLIT with different doses of a liquid phleum pratense extract was shown during the grass pollen season and in an Allergen Exposure Chamber (AEC) A. Distler¹, H. van Schijndel², D. Yu², D. Pappelendam², M. Leineweber¹, N. van Os¹ 1 HAL Allergie GmbH, Düsseldorf, Germany; ²HAL Allergy BV, Leiden, The Netherlands. Background & Aim In two Phase IV studies with Grass 1 and Birch 2 pollen preparations the tolerability of a subcutaneous Rush up-dosing (three injections in two weeks) has been tested and proven to be save in adults. In the course of a non-interventional study (NIS) the tolerability of this treatment scheme was tested in the day-to-day practice. Data from all included patients who received either one or two therapies in parallel were analysed. Methods The prospective NIS was performed in 116 doctor s offices in Germany. The Rush up-dosing (Fig. 1) with 3 injections ( ml) was documented in patients getting a subcutaneous immunotherapy with modified tree or modified grass pollen allergens or a mixture of those. After each injection of initial treatment with PURETHAL (HAL Allergy BV, Leiden, The Netherlands) the patients recorded any side effects within 24 hours after the injection in a diary. By using an electronic case report form (ecrf) ry participating doctor s office documented four injections per patient, three injections of initial treatment and one injection of maintenance treatment. Results In total data from 169 patients were collected. 529 of the patients were male (49.5%) and the mean age of the patients was 38 years (Tab. 1). 97.9% (n = 972) of the patients with a complete documentation in the ecrf (n = 993) could reach the highest dose of.5 ml. Data from 148 patients were evaluable for early phase reactions. Early local reactions were reported in 15% (n = 157) and early systemic reactions in 1.1% (n = 12) of these patients. Late phase reaction data were available for 135 patients. Late local reactions occurred in 31.6% (n = 327) and late systemic reactions in 7.8% (n = 81) of these patients (Fig. 2). No sre systemic reactions grade III and IV were observed. 34.7% of the patients with available data on side effects were mono-sensitized and 65.3% of these patients were polysensitized. Only with regard to early local reactions a difference was determined between these two groups (12.4% vs 16.4%) (Fig. 3). 77.5% of the patients with available data on side effects suffered from rhino-conjunctivitis and 22.5% of these patients suffered from rhino-conjunctivitis and allergic asthma. Between these two groups a difference could be observed in early local reactions (13.2% vs 21.3%), late local reactions (3.4% vs 35.6%) and late systemic reactions (6.4% vs 12.9%) (Fig. 4). A subgroup of 29 patients (2.7%) out of the whole patient population (n = 169) was treated with two therapies (e.g. Trees 1% and Grasses 1%) in parallel. Early local reactions were observed in 13.8% (n = 4), late local reactions in 1.3% (n = 3) and late systemic reactions in 3.4% (n = 1) of these patients (Fig. 2). No early systemic reactions occurred. The maintenance dose could be administered to all patients. Conclusion More than 97% of the patients reached the maintenance dose of.5 ml and the overall tolerability was very good. In the context of this non-interventional study the data from daily practice confirmed the data that were previously obtained by two Phase IV studies. Session number, date and time: TPS16, Sunday 27 May 218; 12:-13:3 Session title: Immunotherapy in the clinic 1 EAACI, 26-3 May 218, Munich, Germany EAACI, 26-3 May 218, Munich, Germany

15 Tolerability of a two week Rush up-dosing with modified allergens in pollen allergic subjects in the day-to-day practice. A. Distler 1, H. van Schijndel 2, D. Yu 2, D. Pappelendam 2, M. Leineweber 1, N. van Os 1 1 HAL Allergie GmbH, Düsseldorf, Germany, 2 HAL Allergy BV, Leiden, The Netherlands Background & Aim: In two Phase IV studies with Grass 1 and Birch 2 pollen preparations the tolerability of a subcutaneous Rush up-dosing (three injections in two weeks) has been tested and proven to be save in adults. In the course of a non-interventional study (NIS) the tolerability of this treatment scheme was tested in the day-to-day practice. Data from all included patients who received either one or two therapies in parallel were analysed. Methods: The prospective NIS was performed in 116 doctor s offices in Germany. The Rush up-dosing (Fig. 1) with 3 injections ( ml) was documented in patients getting a subcutaneous immunotherapy with modified tree or modified grass pollen allergens or a mixture of those. After each injection of initial treatment with PURETHAL (HAL Allergy BV, Leiden, The Netherlands) the patients recorded any side effects within 24 hours after the injection in a diary. By using an electronic case report form (ecrf) ry participating doctor s office documented four injections per patient, three injections of initial treatment and one injection of maintenance treatment. Results: In total data from 169 patients were collected. 529 of the patients were male (49.5%) and the mean age of the patients was 38 years (Tab. 1). 97.9% (n = 972) of the patients with a complete documenttation in the ecrf (n = 993) could reach the highest dose of.5 ml. Data from 148 patients were evaluable for early phase reactions. Early local reactions were reported in 15% (n = 157) and early systemic reactions in 1.1% (n =12)of these patients. Late phase reaction data were available for 135 patients. Late local reactions occurred in 31.6% (n = 327) and late systemic reactions in 7.8% (n = 81) of these patients (Fig. 2). No sre systemic reactions grade III and IV were observed. 34.7% of the patients with available data on side effects were monosensitized and 65.3% of these patients were poly-sensitized. Only with regard to early local reactions a difference was determined between these two groups (12.4% vs 16.4%) (Fig. 3). 77.5% of the patients with available data on side effects suffered from rhino-conjunctivitis and 22.5% of these patients suffered from rhino-conjunctivitis and allergic asthma. Between these two groups a difference could be observed in early local reactions (13.2% vs 21.3%), late local reactions (3.4% vs 35.6%) and late systemic reactions (6.4% vs 12.9%) (Fig. 4). A subgroup of 29 patients (2.7%) out of the whole patient population (n = 169) was treated with two therapies (e.g. Trees 1% and Grasses 1%) in parallel. Early local reactions were observed in 13.8% (n = 4), late local reactions in 1.3% (n = 3) and late systemic reactions in 3.4% (n = 1) of these patients (Fig. 2). No early systemic reactions occurred. The maintenance dose could be administered to all patients. Conclusion: More than 97% of the patients reached the maintenance dose of.5 ml and the overall tolerability was very good. In the context of this non-interventional study the data from daily practice confirmed the data that were previously obtained by two Phase IV studies. References: (1) Pfaar et al.: Accelerated Up-Dosing of Subcutaneous Immunotherapy with a Registered Allergoid Grass Pollen Preparation. Int Arch Allergy Immunol 213; 16: (2) Buczylko et al.: Accelerated Up-Dosing of Subcutaneous Immunotherapy with a Registered Allergoid Birch Pollen Preparation. Int Arch Allergy Immunol 217; 172: Figure 1: Rush up-dosing scheme Table 1: Patient distribution Gender Male 49.5% Female 5.5% Age years old 66.4% > 45 years old 33.6% Sensitization Mono-sensitized 34.3% Poly-sensitized 65.7% Symptoms Allergic rhino-conjunctivitis 77.3% Allergic rhino-conjunctivitis and allergic asthma 22.7% 4% 35% 3% 25% 2% 15% 1% 5% % 4% 35% 3% 25% 2% 15% 1% 5% % 4% 35% 3% 25% 2% 15% 1% 5% % Initial treatment.1.3 early local reaction late local reaction Figure 2: Tolerability of Rush up-dosing early local reaction late local reaction early systemic reaction late systemic reaction early local reaction late local reaction Maintenance treatment Injection intervals: 4±2 weeks Weeks all patients subgroup two therapies early systemic reaction late systemic reaction Figure 3: Tolerability in mono-sensitized versus poly-sensitized patients mono-sensitized patients poly-sensitized patients rhino-conjunctivitis rhinoconjunctivitis + asthma early systemic reaction late systemic reaction Figure 4: Tolerability in patients with rhino-conjunctivitis versus patients with rhinoconjunctivitis and allergic asthma In relation to this presentation, I declare the following, real or perceived conflict of interest: the authors are employees of HAL Allergy. EAACI Congress 218

16 Tolerability of a two week Rush up-dosing with modified Trees, modified Grasses or modified Grasses / Trees mixture in pollen allergic subjects in the day-to-day practice TPS16 - Immunotherapy in the clinic 1 Tolerability of a two week Rush up-dosing with modified allergens in pollen allergic subjects in the day-to-day practice A. Distler 1, H. van Schijndel 2, D. Yu 2, D. Pappelendam 2, M. Leineweber 1, N. van Os 1 ¹HAL Allergie GmbH, Düsseldorf, Germany; 2 HAL Allergy BV, Leiden, The Netherlands. Background & Aim Two Phase IV studies with Grass1 and Birch² pollen preparations, respectively, have led to the authorization of a subcutaneous Rush up-dosing scheme (3 injections in 2 weeks for adults). In the Phase IV study with modified grass pollen allergens 92.8% of the patients and in the study with modified birch pollen allergens 98.4% of the patients reached the maintenance dose of.5 ml. In order to test the tolerability of the Rush up-dosing scheme in the day-to-day practice this non-interventional study was performed. Only data from patients who received one subcutaneous immunotherapy were analysed. Methods The prospective NIS was performed in 116 doctor s offices in Germany. The Rush up-dosing (Fig. 1) with 3 injections ( ml) in two weeks was documented in patients getting a subcutaneous immunotherapy with tree or grass pollen or a mixture of those. After each injection in the initial treatment with PURETHAL (HAL Allergy BV, Leiden, The Netherlands) the patients documented in a diary any side effects within 24 hours. By using an electronic case report form (ecrf) ry participating doctor s office documented a total of four injections per patient, three injections of initial treatment and one injection of maintenance treatment. Results Data from 14 patients undergoing one therapy were evaluable. Thereof 449 (43.2%) received Trees, 435 (41.8%) Grasses and 156 (15%) a mixture of Grasses and Trees (Fig. 2). 964 patients were completely documented in the ecrf. The maintenance dose of.5 ml was reached by 98.8% (n = 414) of the 419 patients treated with Trees, by 97.5 % (n = 385) of the 395 patients treated with Grasses and by 96% (n = 144) of the 15 patients treated with the mixture of Grasses and Trees (Fig. 3). The tolerability was analysed according to allergens and with regard to early and late local reactions (ELR, LLR) as well as early and late systemic reactions (ESR, LSR). Data from 119 patients were available for early phase reactions and data from 16 patients for late phase reactions. Based on these data the breakdown shown in Figure 4 was made and the percentage of patients with at least one side effect is depicted. After injection of Grasses/Trees mixture early local reactions occurred a little e frequently compared to single allergen preparations (2.9% vs Trees 13.7% and Grasses 14.3%). The frequency of late local reactions was similar for all allergen preparations (Trees 33.8%, Grasses 31.3%, Grasses/Trees 3.3%). The same was observed for early systemic reactions (Trees 1.6%, Grasses 1.2%, GraGrasses/Trees %) and late systemic reactions (Trees 9.4%, Grasses 6.3%, Grasses/Trees 8.6%). Sre systemic reactions (grade III and IV) did not occur. In total 15% 32.2% 1.2% 8.% Conclusion In total 97.8% of the patients reached the maintenance dose of.5 ml. The data of this non-interventional study show that the tolerability of the single allergen preparations Trees and Grasses and the tolerability of the Grasses/Trees mixture were similar. Due to the overall good tolerability the Rush up-dosing has proven itself in day-to-day practice. ELR LLR ESR LSR Trees 13.7% 33.8% 1.6% 9.4% Grasses 14.3% 31.3% 1.2% 6.3% Mixture Grasses / Trees 2.9% 3.3% % 8.6% Session number, date and time: TPS35, Monday 28 May 218; 12:15-13:45 Session title: Immunotherapy in the clinic 3 EAACI, 26-3 May 218, Munich, Germany EAACI, 26-3 May 218, Munich, Germany

17 Tolerability of a two week Rush up-dosing with modified Trees, modified Grasses or modified Grasses/Trees mixture in pollen allergic subjects in the day-to-day practice. A. Distler 1, H. van Schijndel 2, D. Yu 2, D. Pappelendam 2, M. Leineweber 1, N. van Os 1 1 HAL Allergie GmbH, Düsseldorf, Germany, 2 HAL Allergy BV, Leiden, The Netherlands Background & Aim: Two Phase IV studies with Grass 1 and Birch 2 pollen preparations, respectively, have led to the authorization of a subcutaneous Rush updosing scheme (3 injections in 2 weeks for adults). In the Phase IV study with modified grass pollen allergens 92.8% of the patients and in the study with modified birch pollen allergens 98.4% of the patients reached the maintenance dose of.5 ml. In order to test the tolerability of the Rush up-dosing scheme in the day-to-day practice this non-interventional study was performed. Only data from patients who received one subcutaneous immunotherapy were analysed. Methods: The prospective NIS was performed in 116 doctor s offices in Germany. The Rush up-dosing (Fig. 1) with 3 injections ( ml) in two weeks was documented in patients getting a subcutaneous immunotherapy with tree or grass pollen or a mixture of those. After each injection in the initial treatment with PURETHAL (HAL Allergy BV, Leiden, The Netherlands) the patients documented in a diary any side effects within 24 hours. By using an electronic case report form (ecrf) ry participating doctor s office documented a total of four injections per patient, three injections of initial treatment and one injection of maintenance treatment. Results: Data from 14 patients undergoing one therapy were evaluable. Thereof 449 (43.2%) received Trees, 435 (41.8%) Grasses and 156 (15%) a mixture of Grasses and Trees (Fig. 2). Initial treatment.1 41,8%.3 Figure 1: Rush up-dosing scheme 15% Maintenance treatment Injection intervals: 4±2 weeks Weeks ,2% Trees Grasses Grasses/Trees 964 patients were completely documented in the ecrf. The maintenance dose of.5 ml was reached by 98.8% (n = 414) of the 419 patients treated with Trees, by 97.5 % (n = 385) of the 395 patients treated with Grasses and by 96% (n = 144) of the 15 patients treated with the mixture of Grasses and Trees (Fig. 3). The tolerability was analysed according to allergens and with regard to early and late local reactions (ELR, LLR) as well as early and late systemic reactions (ESR, LSR). Data from 119 patients were available for early phase reactions and data from 16 patients for late phase reactions. Based on these data the breakdown shown in Figure 4 was made and the percentage of patients with at least one side effect is depicted. After injection of Grasses/Trees mixture early local reactions occurred a little e frequently compared to single allergen preparations (2.9% vs Trees 13.7% and Grasses 14.3%). The frequency of late local reactions was similar for all allergen preparations (Trees 33.8%, Grasses 31.3%, Grasses/Trees 3.3%). The same was observed for early systemic reactions (Trees 1.6%, Grasses 1.2%, Grasses/Trees %) and late systemic reactions (Trees 9.4%, Grasses 6.3%, Grasses/Trees 8.6%). Sre systemic reactions (grade III and IV) did not occur. Conclusion: In total 97.8% of the patients reached the maintenance dose of.5 ml. The data of this non-interventional study show that the tolerability of the single allergen preparations Trees and Grasses and the tolerability of the Grasses/Trees mixture were similar. Due to the overall good tolerability the Rush up-dosing has proven itself in day-today practice. Figure 2: Distribution of treatment with different allergen preparations 1% 8% 6% 4% 2% 98,8% 97,5% 96% % Trees Grasses Grasses/Trees Figure 3: Percentage of patients who reached the highest dose 4% 35% 3% 25% 2% 15% 1% 5% % Trees Grasses Grasses/Trees in total early local reaction late local reaction early systemic reaction late systemic reaction Figure 4: Tolerability according to different allergen preparations References: (1) Pfaar et al.: Accelerated Up-Dosing of Subcutaneous Immunotherapy with a Registered Allergoid Grass Pollen Preparation. Int Arch Allergy Immunol 213; 16: (2) Buczylko et al.: Accelerated Up-Dosing of Subcutaneous Immunotherapy with a Registered Allergoid Birch Pollen Preparation. Int Arch Allergy Immunol 217; 172: In relation to this presentation, I declare the following, real or perceived conflict of interest: the authors are employees of HAL Allergy. EAACI Congress 218

18 ielisa as a tool to measure IgE binding towards single modified peanut allergens TPS35 - Immunotherapy in the clinic 3 H. van Schijndel 1, N. Pantelic 1, R. van den Hout 1, J. Meijlis 1, H. van der Kleij 1, 1 HAL Allergy BV, Leiden, The Netherlands. Tolerability of a two week Rush up-dosing with modified Trees, modified Grasses or modified Grasses / Trees mixture in pollen allergic subjects in the day-to-day practice Background Immunotherapy has shown to be a potential treatment for food allergies but needs further research to improve safety. Modification of peanut allergens to reduce their allergenicity is a promising approach to dlop a safe and effective immunotherapy as shown by the successful completion of a first-in human safety and tolerability study using HAL-MPE1 in adult patients with peanut allergy (EudraCT ). In order to assess the impact of modification on individual peanut allergens and to assess its impact on IgE binding by individual patient sera, we have dloped peanut allergenspecific inhibition ELISAs. With this methodology we are able to identify patients with residual IgE binding to modified peanut allergens. Method IgE inhibition ELISAs (ielisas) were dloped and performed to test IgE binding towards purified Ara h2 and Ara h6 and their reduced and alkylated (modified) versions, using the individual responses of single patient sera. Results Ara h6-specific ielisas showed that modification of Ara h6 results in >95% reduction in IgE-binding for all individual sera tested. Ara h2-specific ielisas showed that modification of Ara h2 also results in >95% reduced IgE-binding for most of the sera, but some sera were identified which showed residual, 1%-2% IgE binding to mara h 2. In some of the latter sera, the presence of IgE binding to a linear hydroxyproline-containing peptide could be confirmed as a possible source for the residual IgE binding to mara h2. Conclusion We have dloped a methodology to assess residual IgE binding to modified peanut allergens. The sensitivity of the allergenspecific ielisas allowed us to discriminate between patient sera in which IgE binding to mara h2 and to mara h6 was virtually completely absent and sera in which 1-2% residual IgE binding to Ara h2 was observed. The clinical importance of these observations is yet unknown. Future clinical studies will need to ral whether the patient-specific IgE binding profiles to individual modified peanut allergens do correlate with the adverse nts profile of immunotherapy with modified peanut extract. Session number, date and time: TPS16, Sunday 27 May 218; 12:-13.3 Session title: Immunotherapy in the clinic 1 EAACI, 26-3 May 218, Munich, Germany EAACI, 26-3 May 218, Munich, Germany

19 ielisa as a tool to measure IgE binding towards single modified peanut allergens H. van Schijndel 1, N. Pantelic 1, R. van den Hout 1, J. Meijlis 1, H. van der Kleij 1 1 HAL Allergy BV, Leiden, The Netherlands Background & Aim: Immunotherapy has shown to be a potential treatment for food allergies but needs further research to improve safety. Modification of peanut allergens to reduce their allergenicity is a promising approach to dlop a safe immunotherapy as shown by the successful completion of a first-in human safety and tolerability study using HAL-MPE1 in adult patients with peanut allergy (EudraCT ). In order to assess the impact of modification on IgE binding by individual patient sera, peanut allergen-specific inhibition ELISAs have been dloped. With this methodology patients with residual IgE binding to modified peanut allergens can be identified. RP 5%mAra h2:.35 Serum D19 Serum D44 1, 9, 8, 7, 6, 5, % inhibition 4, 3, 2, 1,, -2, -1,5-1, -,5,,5 1, 1,5 2, 2,5 Log µg/ml Ara h 2 mara h 2 RP 5%mAra h2 :.9 1, 9, 8, 7, 6, 5, % inhibition 4, 3, 2, 1,, -3, -2,5-2, -1,5-1, -,5,,5 1, 1,5 2, 2,5 Log µg/ml Ara h 2 mara h 2 Methods: IgE inhibition ELISAs (ielisas) were dloped and performed to test IgE binding towards purified Ara h2 and Ara h6 and their reduced and alkylated (modified) versions and towards a linear peptide (PEP1), using 2 sera from peanut-allergic patients. Plates were coated with the antigen to be tested (Ara h2, mara h2, Ara h6, mara h6, PEP1). Eight serial dilutions of all samples were made. The serum pool was diluted according to previously determined Emax obtained in a checkerboard titration for each Ara coat. Detection was carried out by a secondary mouse anti human IgEhorseradish peroxidise (HRP). Plates were coloured with TMB and the colour reaction was stopped by adding.5m sulphuric acid. Absorption was measured at 45nm. A peanut extract in-house reference (IHRP) was used as a control and standard. Results: Ara h6-specific ielisas showed that modification of Ara h6 results in >95% reduction in IgE-binding for all individual sera tested (table 1). Ara h2-specific ielisas showed that modification of Ara h2 also results in >95% reduced IgE-binding for most of the sera, but some sera were identified which showed residual, 1%-2% IgE binding to mara h 2 (table 1). In some of the sera showing residual binding to mara h2, the presence of IgE binding to a linear hydroxyproline-containing peptide (PEP1) could be confirmed as a possible source for the residual IgE binding to mara h2 (fig.2). Conclusion: We have dloped a methodology to assess residual IgE binding to modified peanut allergens. The sensitivity of the allergenspecific ielisas allowed us to discriminate between patient sera in which IgE binding to mara h2 and to mara h6 was virtually completely absent and sera in which 1-2% residual IgE binding to Ara h2 was observed. The clinical importance of these observations is yet unknown. Future clinical studies will need to ral whether the patient-specific IgE binding profiles to individual modified peanut allergens do correlate with the adverse nts profile of immunotherapy with modified peanut extract. Figure 1: Typical ielisa result of (m)ara h2 for serum D19 and D44. Figure represents the inhibition curves calculated using the logarithms of the concentration (µg/ml) on the x-axis and the inhibition (%) on the y-axis. Ara h2 was used as a reference for the potency of mara h2.,3,25,2,15,1,5 Serum sample Reduction in IgE binding towards mara h2 (%) Reduction in IgE binding towards mara h6 (%) D D D D D D D D D D D7 1 1 D D D D D D D D D Table 1. Reduction in IgE binding towards mara h2 and mara h6 for 2 sera from peanut allergic patients. RP 5% D19 D6 D64 D69 D74 D77 D13 Figure 2: Relative IgE-potency of the PEP1 at 5% inhibition (RP5%) for serum D19, D6, D64, D69, D74, D77 and D13. In relation to this presentation, I declare the following, real or perceived conflict of interest: the authors are employees of HAL Allergy. EAACI Congress 218

20 An IgG inhibition ELISA to measure the stability of alum-adsorbed grass pollen allergoids TPS16 - Immunotherapy in the clinic 1 ielisa as a tool to measure IgE binding towards single modified peanut allergens C. Franso 1, N. Sinnige 1, A. Segaar 1, L. Boukich 1, J. Meijlis 1 1 HAL Allergy BV, Leiden, The Netherlands. Background An IgG inhibition ELISA was dloped for the stability determination of alum-adsorbed grass pollen allergoids. Despite the complex nature of allergoid products, this assay allows to study the stability of the allergoid Drug Product by means of allergoid specific IgG. Methods IgG inhibition ELISA: Rabbit IgG antibodies specific for grass allergen allergoids are pre-incubated with different concentrations of alum-adsorbed grass pollen allergoid. The mix is added to an allergoid coated microtiter plate. Unbound IgG will bind to the allergoid coat and is subsequently incubated with anti-igg HRP labeled conjugate and stained with TMB. Results are expressed as percentage inhibition relative to the uninhibited value. The concentration of alum-adsorbed allergoid that is required to inhibit 5% IgG is used as read-out. Circular Dichroism: Far-UV CD spectra (19 26 nm) were recorded on a J-815 Spectropolarimeter. A cuvette with a stirring compartment was used to keep the suspension homogeneous during measurement. Results The IgG inhibition ELISA assay is specific for grass pollen allergoids (not for other allergen allergoids), has a good inter- and intra-assay precision and is robust for assay variation. Thermally stressed alum-adsorbed grass pollen allergoids were used to show that the IgG inhibition assay can be used as a stability indicating method. Sre thermal stressing resulted in a higher 5% inhibition value, indicating a loss of IgG epitopes. Furthere, far-uv CD analyses showed that there is a close relation between the decreasing IgG binding capacity (5% inhibition values) and the loss secondary protein structures by unfolding (CD-ratio 27/222 nm values). Conclusion The IgG inhibition assay was demonstrated to be a valuable method to determine the stability of alum-adsorbed grass pollen allergoid preparations. In addition, a relation was shown between the IgG binding capacity and the change in secondary protein structures. Session number, date and time: TPS34, Monday 28 May 218; 12:15-13:45 Session title: Immunotherapy in the clinic 2 EAACI, 26-3 May 218, Munich, Germany 19 2 EAACI, 26-3 May 218, Munich, Germany

21 An IgG inhibition ELISA to measure the stability of Alum-adsorbed grass pollen allergoids C. Franso 1, N. Sinnige 1, A. Segaar 1, L. Boukich 1, J. Meijlis 1 1 HAL Allergy BV, Leiden, The Netherlands Background & Aim: IgE based potency assays for allergens are essential tests for monitoring stability in extracts. Howr, these IgE potency assays can not be used for potency determination of allergoid-based products, due to their reduced IgE reactivity. Therefore, a stability assay has been dloped based on the inhibition of IgG by grass pollen allergoids (Drug Substance) and alum-adsorbed allergoids (Drug Product). The feasibility of the IgG inhibition assay as a stability indicating assay was studied via thermal stressing of alumadsorbed allergoids in combination with monitoring the structural changes with Circular Dichroism spectroscopy. Methods: IgG inhibition assay: The dloped IgG inhibition assay uses IgG antibodies that were raised (in rabbits) against a mixed grass pollen allergoid. IgG antibodies specific for grass allergen allergoids are pre-incubated with different concentrations of alum-adsorbed grass pollen allergoid. The mix is added to an allergoid coated microtiter plate. Unbound IgG will bind to the allergoid coat and is subsequently incubated with anti-igg HRP labeled conjugate and stained with TMB. Results are expressed as percentage inhibition relative to the uninhibited value. The concentration of alumadsorbed allergoid that is required to inhibit 5% IgG is used as read-out. Circular Dichroism: Far-UV CD spectra (19 26 nm) were recorded on a J-815 Spectropolarimeter. A cuvette with a stirring compartment was used to keep the suspension homogeneous during measurement. Results: Specificity: The IgG serum raised against mixed grass pollen allergoid on immunoblot detected proteins in all the ten individual grass pollen extracts (Figure 1). Major allergen grass group 1 was detected in the IgG profile of all tested grass species. Furthere, the IgG serum proves to be specific in the IgG ELISA for allergoids of grass species showing a typical dose-response curve whereas allergoids from mite preparations do not inhibit IgG binding (Figure 2). Grass allergoids adsorbed onto aluminium show a similar dose response curve but with a reduced affinity due to its adsorbed state M M Figure 1: Immunoblot allergen profiles of extracts prepared from different grass pollen species (lane 2-11), grass pollen mixture (lane 1) and grass pollen allergoid (lane 12). Grass group 1 allergen is indicated with an arrow. Alum-adsorbed mixed grasses allergoid Alum-adsorbed mites allergoid Sample Lane Description 1 Ref. mixed grasses 2 Phleum pratense 3 Lolium perenne 4 Poa pratensis 5 Secale cereale 6 Agrostis stolonifera 7 Arrhenatheum elatius 8 Anthoxanthum odoratum 9 Festuca rubra 1 Dactylis glomerata 11 Holcus lanatus 12 Mixed grasses allergoid M Marker Mixed grass allergoid Alum-adsorbed mixed grasses allergoid 5% IgG inhibition value 27 AUeq/mL 85 AUeq/mL Figure 2: Dose response curve of mixed grasses allergoid, alum-adsorbed mixed grasses allergoid and a alum-adsorbed mite allergoid Stability indicating: Sre thermal stressing conditions were applied to monitor stability of allergoids. Exposure to 9 C for 4 and 8 hours resulted in a higher 5% inhibition value, indicating a loss of IgG epitopes (Figure 3). Far-UV CD spectra showed that at these temperatures the ellipticity around 2-21 nm increased which is indicative for the presence of e random coil structures (unfolding). Far-UV CD analyses showed that there is a close relation between the decreasing IgG binding capacity (increasing 5% inhibition values) and the loss secondary protein structures by unfolding (increased CD-ratio 27/222 nm values) (Figure 4). Conclusion: The IgG inhibition assay was demonstrated to be a valuable method to determine the stability of alum-adsorbed grass pollen allergoid preparations. In addition, a relation was established between the IgG binding capacity and the change in secondary protein structures via thermal stressing. Furthere, the data show that alum-adsorbed allergoids are resistant to higher temperatures. Figure 3: IgG inhibition curves (left) and far-uv CD spectra (right) of alum-adsorbed mixed grasses allergoid stored at 5 C (Blue) and exposed to 9 C for 4 hours (orange) and 8 hours (red) R² =,8314 5% IgG inhibition (AUeq/mL),5,7,9 1,1 1,3 1,5 CD-ratio 27/222 nm Figure 4: Relation between the decreased IgG binding capacity and the change of secondary structures (unfolding) of 3 batches (Circle, triangle, diamond) alum-adsorbed mixed grasses allergoids exposed to 5 C (blue), 9 C for 4 hours (orange) and 9 C for 8 hours (red). In relation to this presentation, I declare the following, real or perceived conflict of interest: the authors are employees of HAL Allergy. EAACI Congress 218

22 Glycosylation of rbet v 1.11 by Chinese Hamster Ovary cells affects proteolytic stability towards endo/lysosomal degradation TPS34 - Immunotherapy in the clinic 2 An IgG inhibition ELISA to measure the stability of alum-adsorbed grass pollen allergoids H.J.M. Warmenhoven 1,3,O.E. McKenna 4, J.W. van Schijndel 3, G. Gadermaier 4, P. Briza 4, R. van Ree 1,2 ¹Department of Experimental Immunology, ²Otorhinolaryngology, AMC, Amsterdam, The Netherlands, 3 HAL Allergy BV, Leiden, The Netherlands, ⁴Department of Molecular Biology, University of Salzburg, Austria. Background Many pharmaceutical biologicals are produced in Chinese Hamster Ovary cells (CHO) because they mimic human-like posttranslational modifications such as glycosylation. A CHO cell line stably expressing the major birch pollen allergen Bet v 1.11 produced glycosylated (glybet v 1 CHO) and non-glycosylated Bet v 1.11 (Bet v 1 CHO). In this study we investigated the effect of glycosylation of Bet v 1.11 on IgE binding, secondary structural organization and proteolytic susceptibility. Methods Bet v 1 CHO and glybet v 1 CHO were purified from CHO cell supernatant by concanavalin A affinity and immunoaffinity. As controls we included purified glybet v 1 digested with PNGase F to generate deglycosylated Bet v 1 (dgbet v 1 CHO) and Bet v 1 E. coli. IgE binding capacity of Bet v 1 CHO, glybet v 1 CHO and dgbet v 1 CHO was tested with ImmunoCAP IgE inhibition using a pool of birch pollen allergic patients sera. The secondary structure of the CHO derived Bet v 1 proteins was analyzed with far UV circular dichroism (CD) spectrometry. Proteolytic susceptibility was assessed by digesting the proteins with endolysosomal extracts of JAWS II cells. SDS-PAGE and mass spectrometry (MS) were used to analyze the degradation kinetics and peptide patterns. Results IgE inhibition curves and CD spectra of all Bet v 1 proteins were highly similar. In contrast, glybet v 1 CHO was degraded rapidly compared to Bet v 1 E. coli and Bet v 1 CHO which was partially restored after deglycosylation. MS raled the presence of an N-terminal aspartate amino acid and delayed degradation of a stretch amino acids upstream of the N-linked glycosylation site (Asn83). Conclusion Glycosylation affected the proteolytic stability of Bet v 1 by dendritic cell derived endo/lysosomal proteases which in turn may affect the display of peptides complexed with MHCII molecules leading to differential T helper cell polarization. Thus, the choice of expression host plays an important role in shaping posttranslational modifications which may alter the stability and possibly immunogenicity of the target protein. Session number, date and time: PDS21, Tuesday 29 May 218; 1:3-12: Session title: Innate immune cell activation in type-2 inflammation EAACI, 26-3 May 218, Munich, Germany EAACI, 26-3 May 218, Munich, Germany

23 Glycosylation of rbet v 1.11 by Chinese Hamster Ovary cells affects proteolytic stability towards endo-/lysosomal degradation H. Warmenhoven 1,3, O. McKenna 4, H. van Schijndel 3, G. Gadermaier 4, P. Briza 4, R. van Ree 1,2 1 Department of Experimental Immunology, 2 Otorhinolaryngology, AMC, Amsterdam, The Netherlands, 3 HAL Allergy BV, Leiden, The Netherlands, 4 Department of Biosciences, University of Salzburg, Austria Background & Aim: Many pharmaceutical biologicals are produced in Chinese Hamster Ovary cells (CHO) because they mimic human-like posttranslational modifications such as glycosylation. A CHO cell line stably expressing the major birch pollen allergen Bet v 1.11 produced glycosylated (glybet v 1 CHO) and non-glycosylated Bet v 1.11 (Bet v 1 CHO). In this study we investigated the effect of glycosylation of Bet v 1.11 on IgE binding, secondary structural organization and proteolytic susceptibility. 1 Figure 1 left: Inhibition of IgE binding to rbet v 1 caps (t215) by Bet v 1 E. coli, Bet v 1 CHO, glybet v 1 CHO and dgbet v 1 CHO. Figure 1 right: Far UV CD spectra of Bet v 1 E. coli, Bet v 1 CHO, glybet v 1 CHO and dgbet v 1 CHO Molar ellipticity [ θ]mrw (1 3 deg cm2 dmol-1) 5 Wavelength (nm) Bet v 1 CHO glybet v 1 CHO dgbet v 1 CHO Bet v 1 E. coli Methods: IgE binding: IgE binding capacity of Bet v 1 CHO, glybet v 1 CHO, dgbet v 1 CHO and Bet v 1 E. coli was tested with ImmunoCAP IgE inhibition using a pool of birch pollen allergic patients sera and rbet v 1 caps (t215) Bet v 1 CHO 5% deg. = 3-5 hours glybet v 1 CHO 5% deg. = -.5 hours Proteolytic degradation (%), Structure and protease digestion: The secondary structure of the CHO derived Bet v 1 proteins was analyzed with far UV circular dichroism (CD) spectrometry. Proteolytic susceptibility was assessed by digesting the proteins with endo-/lysosomal extracts of JAWS II cells. SDS-PAGE and mass spectrometry (MS) were used to analyze the degradation kinetics and peptide patterns 1. Results: IgE binding and CD spectra of all Bet v 1 proteins were highly similar (Figure 1). In contrast, glybet v 1 CHO was degraded rapidly compared to Bet v 1 E. coli and Bet v 1 CHO which was partially restored after deglycosylation (Figure 2). MS raled the presence of an N-terminal aspartate amino acid ( ) and delayed degradation of a stretch amino acids upstream of the N-linked glycosylation site (, Figure 3) Time (hrs) 5% deg. = 1-3 hours Proteolytic degradation (%) Proteolytic degradation (%) dgbet v 1 CHO, , Time (hrs) Figure 2: Degradation of Bet v 1 E. coli, Bet v 1 CHO, glybet v 1 CHO and dgbet v 1 CHO by endo/lysosomal proteases, visualized by SDS PAGE. Bet v 1 CHO Time (hrs) Proteolytic degradation (%) Inhibition (%),1,1,1 1 1 inhibitor (µm) Bet v 1 E. coli 5% deg. = 5 hours, Time (hrs) Conclusion: Glycosylation decreased the proteolytic stability of Bet v 1 exposed to dendritic cell derived endo-/lysosomal proteases which in turn may affect the display of peptides complexed with MHCII molecules leading to differential T helper cell polarization 2. Thus, the choice of expression host plays an important role in shaping posttranslational modifications which may alter the stability and, consequently, immunogenicity of the target protein. glybet v 1 CHO dgbet v 1 CHO References: (1) Wildner, Elsasser et al. 217 (2) Delamarre et al. 25 Figure 3: Peptide patterns of degraded Bet v 1 CHO (top), glybet v 1 CHO (dle) and dgbet v 1 CHO (bottom), analyzed with MS. Box indicates protease resistant stretch of amino acids. In relation to this presentation, I declare the following, real or perceived conflict of interest: H. Warmenhoven and H. van Schijndel are employees of HAL Allergy. EAACI Congress 218

24 Towards personalized pollen exposure measurements using hand held pollen samplers PDS21 - Innate immune cell activation in type-2 inflammation Glycosylation of rbet v 1.11 by Chinese Hamster Ovary cells affects proteolytic stability towards endo/lysosomal degradation L.A. de Weger 1, K. de Raat 1,2, J. de Haan 1,2, M. Mostert 2,3, W. van Leeuwen 2,3, R.Schelland 2,3, F. Molster 4, P.S. Hiemstra 1 ¹Department of Pulmonology, Leiden University Medical Center, ²University of Applied Sciences, ³Generade, ⁴Leidse Instrumentmakers School, Leiden, the Netherlands Background Allergic rhinitis caused by pollen is one of the most common allergic diseases. The presence of pollen in the air is currently centrally monitored at roof top lls, and not in the direct living environment of sensitized subjects. In the current project we aimed to dlop a handheld pollen sampler, called pollensniffer, that can collect pollen in the living environment of the allergic subjects. As a first step this device was validated against the standard Burkard pollen sampler and used to monitor local pollen concentrations at street ll in the city of Leiden. Methods Rooftop ll pollen were monitored routinely by a Hirst type pollen sampler (Burkard, UK). The pollensniffer (6x14 cm) consists of a conical inlet and a ventilator powered by a commercial powerbank. The pollen were collected on a cellulose strip. For the validation of the pollensniffer, the device was mounted on top of the Burkard sampler. All pollen collected on the strip from the Burkard and the pollensniffer were counted microscopically. Street ll pollen were monitored once ry week (April-June) at three locations in the city of Leiden, during the ning, day and ning. Statistical analyses were performed using the software package STATA 14. (StataCorp,TX). Results The correlation between the different pollen types collected by the pollensniffer and the Burkard sampler was high (correlation coefficient [CC] >.8). During the validation experiments the pollensniffer appeared to collect on average 7 times e pollen than the Burkard sampler. Street ll (pollensniffer) and roof top ll pollen counts (Burkard sampler) showed a very good correlation (CC>.85). Local street ll measurements in the city of Leiden showed that plane trees in one park produced pollen a week before the plane trees in another park. Grass pollen were observed at street ll 3 weeks before the pollen were observed at roof top ll. Conclusion Pollen numbers collected by the pollensniffer and the Burkard correlate well, but the pollensniffer collected on average 7 times e pollen than the Burkard sampler. Street ll measurements showed differences in pollen loads between locations. Furthere, street ll grass pollen were detected 3 weeks before they were observed at rooftop ll. These findings suggest that the pollensniffer is well suited for the measurement of pollen (and maybe other allergens) in the living environment of sensitized subjects. Acknowledgment This study was supported in part by a grant from Generade and RAAK Publiek (SIA). Session number, date and time: TPS44, Tuesday 29 May 218; 12:-13:3 Session title: Pollen and other aeroallergen exposition EAACI, 26-3 May 218, Munich, Germany EAACI, 26-3 May 218, Munich, Germany

25 Pollen counts daysum at street ll City Center Platanus 17 Huig Kweek 18 2 POP CUP ALN 4 1apr217 1jun217 date days with streetll measurements daysum streetll (pollensniffer) 1may217 Citycenter Church - Huigpark pollencounts BURKARD (log) SAL FRAX BET 2 ULM HIP CAR 4 6 ULM SAL POP HIP FRAX CUP CAR BET Pollen ALN CC.77 Figure 2: Map of the city of Leiden indicating the location of the pollensampler on the roof of the LUMC and the three locations of the streetll measurements. The insert shows the location of the city of Leiden in the Netherlands 19 Rumex Salix Betula Platanus Quercus Pinaceae Log pollencount (rooftop ll, Burkard) Plantago Poaceae 1 5 PLANT RUM POA PLAT QUE PIN SAL BET Pollen CC,38,85,83,16,93,96,7, aug217 rooftopll (Burkard) 1jul City Center Poaceae Huig Kweek EAACI Congress 218 Acknowledgements: This study was supported by a grant from Generade (call 216-5) and SIA (project number: RAAK.PUB3.45) Figure 9: Comparison of the daily grass pollen counts at rooftop ll (red line) and the daysum of the streetll measurements (blue bars). Green arrows indicate days that grass pollen were collected at streetll while no pollen were collected in the Burkard pollensampler at rooftop ll during the weeks to which these days belong. Figure 8: Streetll Poaceae pollencounts on three locations in Leiden. Rainclouds indicate days with rain Graphs by location Figure 6: Pollen counts (log) from rooftop ll (Burkard) and streetll (pollensniffer ). In the table the correlation coefficients between these counts are presented Figure 7: Streetll Platanus pollencounts on three locations in Leiden. Platanus pollen are collected in Huigpark one week before they were collected in Kweeklust park. 15 Graphs by location Buckard LUMC Figure 4: Simultaneous pollen counts (log) from the Burkard pollensampler and the pollensniffer mounted on the rain cover. In the table the correlation coefficients between these counts are presented. 2 In relation to this presentation, I declare the following, real or perceived conflict of interest: none of the authors have conflicts of interest. Pollen numbers collected by the pollensniffer and the Burkard correlate well, but the pollensniffer collected on average 6.5 times e pollen than the Burkard sampler. Street ll measurements showed differences in pollen loads between locations. Furthere, street ll grass pollen were detected 3 weeks before they were observed at rooftop ll.these findings suggest that the pollensniffer is well suited for the measurement of pollen (and maybe other allergens) in the living environment of sensitized subjects. Conclusion: Street ll (pollensniffer) and roof top ll pollen counts (Burkard sampler) showed a very good correlation (Fig.6). Local street ll measurements in the city of Leiden showed that plane trees in one park produced pollen a week before the plane trees in another park (Fig. 7). Grass pollen were observed at street ll 3 weeks before the pollen were observed at roof top ll (Fig. 8 and 9). Figure 5: Streetll (pollensniffer, left) and rooftop ll (Burkard right) pollen monitoring. Graphs by pollentype The correlation between the different pollen types collected by the pollensniffer and the Burkard sampler was high (Fig. 4). During these validation experiments the pollensniffer appeared to collect on average 6.5 times e pollen than the Burkard sampler. Results: Figure 3: Burkard pollen sampler on the roof of the LUMC with the pollensniffer mounted on the rain cover. Rooftop ll pollen were monitored routinely by a Hirst type pollen sampler (Burkard, UK). For the validation of the pollensniffer (Fig. 1) the device was mounted on top of the Burkard sampler (Fig. 3). All pollen collected on the strip from the Burkard and the pollensniffer were counted microscopically. Street ll pollen were monitored once ry week (April-July) at three locations in the city of Leiden (Fig. 2), during the ning, day and ning. Methods: Pollencounts POLLENSNIFFER (log) Figure 1: Pollensniffer connected to the powerbank Pollencounts POLLENSNIFFER (log) log pollencount (streetll, pollensniffer) Allergic rhinitis caused by pollen is one of the most common allergic diseases. The presence of pollen in the air is currently centrally monitored at rooftop lls, and not in the direct living environment of sensitized subjects. In the current project we aimed to dlop a handheld pollen sampler, called pollensniffer, that can collect pollen in the living environment of the allergic subjects. As a first step this device was validated against the standard Burkard pollen sampler and used to monitor local pollen concentrations at street ll in the city of Leiden Kweeklust pollen count Background & Aim: of Pulmonology, Leiden University Medical Center, 2University of Applied Sciences, 3Generade, 4Leidse Instrumentmakers School, Leiden, the Netherlands 1Department L.A. de Weger1, K. de Raat1,2, J. de Haan1,2, M. Mostert2,3, W. van Leeuwen2,3 F. Molster4, P.S. Hiemstra1 Towards personalized pollen exposure measurements using handheld pollensamplers po llen count log pollencount (streetll, pollensniffer) Pollen counts daysum at street ll daily counts at rooftop ll

26 Abbreviated leaflet texts. TPS44 - Pollen and other aeroallergen exposition Towards personalized pollen exposure measurements using hand held pollen samplers Basic information for PURETHAL Pollen Composition: Suspensions for subcutaneous injection, containing 2, AUM/ml allergenic substances chemically modified with glutaraldehyde from Grasses, Birch, Trees, Weeds pollen or mixtures of them and absorbed onto aluminium hydroxide. Excipients: sodium chloride, phenol, aluminium hydroxide, water for injections. Indication: treatment of immediate type allergic disorders (IgE-mediated), such as allergic rhinitis, allergic conjunctivitis and allergic bronchial asthma, which are triggered by sensitization to allergenic substances from pollen in adults, adolescents and children not below the age of 5 years. Dosage and administration: Treatment starts with a subcutaneous injection of.5 ml, which is subsequently increased with weekly doses (conventional or rush scheme) up to a monthly maintenance dose of.5 ml. Treatment should be carried out over a period of 3-5 successive years. Contraindications: Acute inflammatory diseases/frish infection at the target organ, secondary changes of the target organ (emphysema, bronchi-ectasia and others), autoimmune disorders, immune deficiencies and use of immuno-suppressants, sre uncontrolled asthma (particularly with a FEV1 persistently below 7% of the predicted value), cardiovascular failure with increased risk if using adrenaline, use of ß-blockers (including ß-blocker containing eye preparations), clinical active malignant tumour, initiation of therapy during pregnancy, or hypersensitivity to any of the excipients. Undesirable effects: Reactions generally arise within 3 minutes after receiving the injection. Howr, n sral hours after the injection side effects can occur. After injection local reactions at the injection site may occur. In addition systemic reactions may occur which can vary from mild sneezing to life-threatening anaphylactic shock. The most commonly reported local reactions after injection are: Swelling, oedema, hypersensitivity, erythema, urticaria, rash, pruritus and granuloma of the injection site. The most commonly reported systemic reactions are: Sneezing, cough, (allergic) rhinitis, throat irritation, asthma, dyspnoea, oropharyngeal pain, nasal congestion, nasal discomfort, nasal oedema, rhinorrhoea, nasopharyngitis, conjunctivitis, allergic rhino conjunctivitis, eye irritation, eye pruritus, lacrimation increased, angioedema, atopic dermatitis, urticaria, pruritus, erythema, swelling, fr, asthenia, nausea, abdominal pain, diarrhoea, dizziness, headache, somnolence, impaired concentration. The complete product information is available on request. HAL Allergy BV, Postbus 124, 232 BE Leiden, the Netherlands. Date: February 215 Note: PURETHAL Pollen is available as registered product or named patient prescription. In case of a registered product the locally approved product information can slightly differ from above. Basic information for PURETHAL Mites Composition: Suspension for subcutaneous injection containing 2, AUeq/ml allergenic substances chemically modified with glutaraldehyde from Dermatophagoides pteronyssinus (5%) and Dermatophagoides farina (5%) and absorbed onto aluminium hydroxide. Excipients: sodium chloride, phenol, aluminium hydroxide, water for injections. Indication: Treatment of immediate type allergic disorders (IgE-mediated), such as allergic rhinitis, allergic conjunctivitis and allergic bronchial asthma, which are triggered by sensitization to allergenic substances from house dust mites in adults, adolescents and children not below the age of 5 years. Dosage and administration: Treatment starts with a subcutaneous injection of.5 ml, which is subsequently increased with weekly doses up to a monthly maintenance dose of.5 ml. Treatment should be carried out over a period of 3-5 successive years. Contraindications: Acute inflammatory diseases/frish infection at the target organ, secondary changes of the target organ (emphysema, bronchi-ectasia and others), autoimmune disorders, immune deficiencies and use of immuno-suppressants, sre uncontrolled asthma (particularly with a FEV1 persistently below 7% of the predicted value), cardiovascular disorders with increased risk if using adrenaline, use of ß-blockers (including ß-blocker containing eye preparations), clinical active malignant tumour, initiation of therapy during pregnancy, or hypersensitivity to any of the excipients. Undesirable effects: Reactions generally arise within 3 minutes after receiving the injection. Howr, n sral hours after the injection side effects can be observed. After injection of PURETHAL Mites mixture, local reactions at the injection site may occur. In addition systemic reactions may occur. These can vary from mild sneezing to life-threatening anaphylactic shock. The most commonly reported local reactions after injection are swelling, erythema, warmth, paraesthesia, pain, induration, haerhage, and pruritus of the injection site. The most commonly reported systemic reactions are: Hypersensitivity, headache, malaise, fatigue, nausea, conjunctivitis, eye pruritus, lacrimation increased, dyspnoea, nasal congestion, sneezing, allergic rhinitis, rhinorrhoea, erythema, pruritus, swelling, atopic eczema, angioedema. The complete product information is available on request. HAL Allergy BV, Postbus 124, 232 BE Leiden, The Netherlands. Date: August 215 Note: PURETHAL Mites mixture is available as named patient prescription. Basic information for SUBLIVAC and SUBLIVAC FIX Composition: Sublingual drops, containing per ml 1, AU, AUN or PUN allergen extract, prepared according to the individual doctor s prescription. Excipients: glycerol, aminocaproic acid, disodium phosphate hydrate, sodium dihydrogen phosphate dihydrate, peppermint oil, purified water. Indication: Treatment of immediate type allergic disorders (IgE-mediated) such as allergic rhinitis, allergic conjunctivitis and allergic bronchial asthma, caused by allergens. Dosage and administration: The first day of the initial treatment starts with one drop. This dose is increased ry day with one drop until the highest daily dose of five drops is reached. The treatment is continued with five drops. The drops have to be administered under the tongue for at least 1 minute (preferably 2-3 minutes) before swallowing. A spoon may be used to administer the drops. It is advised to clean the dropper after use, for instance with a wet tissue. The treatment should be continued for 3 to 5 years. Contraindications: Partly or uncontrolled bronchial asthma with a FEV1 below 7%, sre autoimmune diseases, immune deficiencies and immunosuppression, malign neoplastic diseases with current symptoms, initiation of treatment during pregnancy, sre inflammation of the oral mucosa, hypersensitivity to any of the excipients. Undesirable effects: Local reactions in the mouth and throat, swelling of the lips or tongue. Reappearance of the patient specific allergic symptoms such as mild systemic reactions (itching eyes, sneezing, coughing, atopic eczema). In rare cases, intensified systemic reactions, like shortness of breath, generalized urticaria, or Quincke s oedema, can occur. After intake, the patient might experience diarrhoea and abdominal pain. These symptoms generally arise within 3 minutes after intake of the drops, howr can occur sral hours after. In individual cases, anaphylactic shock has been reported. The complete product information is available upon request at HAL Allergy BV, P.O. Box 124, 232 BE Leiden, The Netherlands. Date: October 216 Note: SUBLIVAC and SUBLIVAC FIX are available as named patient prescriptions. EAACI, 26-3 May 218, Munich, Germany EAACI, 26-3 May 218, Munich, Germany

27 Basisinformationen PURETHAL Bezeichnungen und Zusammensetzung: Suspensionen zur subkutanen Injektion, enthalten an Aluminiumhydroxid adsorbierte, mit Glutaraldehyd chemisch modifizierte allergene Substanzen aus Pollen (2. AUM/ml) oder Milben (2. AUeq/ml): PURETHAL Gräser: Pollen von 1 Gräsern (inkl. Roggen); PURETHAL Gräser + Getreide: Pollen von 9 Gräsern (45%), Roggen (5%) und Weizen (5%); PURETHAL Birke: Birkenpollen; PURETHAL Bäume: Pollen von Birke, Erle, Hasel zu gleichen Teilen; PURETHAL Gräser + Birke: Pollen von 1 Gräsern (5%) und Birke (5%); PURETHAL Gräser + Bäume: Pollen von 1 Gräsern (5%) und 3 Bäumen (Birke, Erle, Hasel mit insgesamt 5%); PURETHAL Beifuß: Beifußpollen; PURETHAL Milbenmischung: Derm. pteronyssinus (5%) und Derm. farinae (5%). Sonstige Bestandteile: NaCl, Phenol, Aluminiumhydroxid, Wasser zur Injektion. Anwendungsgebiete: Spezifische Immuntherapie allergischer Erkrankungen vom Soforttyp (IgE-vermittelt), wie Heuschnupfen (allergische Rhinitis), allergische Bindehautentzündung (Konjunktivitis) und allergisches Asthma bronchiale, ausgelöst durch eine Sensibilisierung gegenüber den enthaltenen allergenen Substanzen. Gegenanzeigen, absolute: Akute Entzündungsprozesse/Infektionskrankheiten am Reaktionsorgan; Sekundärveränderungen am Reaktionsorgan (z.b. Emphysem, Bronchiektasen); Autoimmunerkrankungen; Immundefekte; gleichzeitige Anwendung von Immunsuppressiva; schweres, unkontrollierbares Asthma bronchiale, insbesondere bei einem persistierenden FEV1 unter 7% Sollwert; Erkrankungen mit Kontraindikationen gegen die Anwendung von Adrenalin; gleichzeitige Behandlung mit ß-Blockern (auch ß-Blocker enthaltende Augentropfen); maligne Tuerkrankungen mit aktuellem Krankheitswert; Einleitung der Behandlung nicht während der Schwangerschaft; Sensibilisierung gegenüber einem der sonstigen Bestandteile; relative: Schwangerschaft und Stillzeit; akute allergische Beschwerden; nicht für Kinder unter 5 Jahren. Zum zeitlichen Intervall zu Schutzimpfungen und für weiterführende Informationen siehe Fachinformation. Nebenwirkungen: Allergische Lokalund/oder Allgemeinreaktionen. Überempfindlichkeit, anaphylaktischer Schock. Grippe, Otitis externa, Sinobronchitis, Sinusitis, Staphylokokkenpharyngitis. Essstörung. Kopfschmerz, Schwindelgefühl, Schläfrigkeit, Parästhesie, Geschmacksstörung, Aufmerksamkeitsstörungen. Schwellung des Auges, Konjunktivitis, Rhinokonjunktivitis, Augenreizung, Augenjucken, Tränensekretion verstärkt. Schwindel, Ohrschwellung, Ohrenjucken. Arrhythmie, Tachykardie. Kreislaufkollaps, Hitzegefühl, Hämatom. Rhinitis, Nasenverstopfung, Nasenödem, Rhinorrhoe, Niesen, Asthma, Atemnot, Husten, Bronchitis, Halstrockenheit, Rachenreizung, Rachenschmerzen, Kehlkopfirritation, Nasopharyngitis, Nasenbluten. Abdominalschmerzen, Gastritis, Übelkeit, Diarrhoe. Angioödem, Urtikaria, Erythem, Juckreiz, Ekzem, atopische Dermatitis, Ausschlag, Akne, Hautreizung. Muskuloskelettale Beschwerden. Ödem, Schwellung, Ermüdung, Schwäche, Brustschmerz, Blässe, Fieber. An der Injektionstelle: meist vorübergehende Granulome, Verhärtung, Schwellung, Urtikaria, Erythem, Überempfindlichkeit, Juckreiz, Schmerz. Patienten nach der Injektion mindestens 3 Minuten überwachen, eine Schockapotheke muss bereitgestellt sein. Nebenreaktionen können auch noch zu einem späteren Zeitpunkt auftreten. Für weiterführende Informationen und zur Behandlung von Nebenwirkungen siehe Fachinformation. In seltenen Fällen kann nach der Injektion leichte Müdigkeit auftreten, was beim Führen von Kraftfahrzeugen oder beim Bedienen von Maschinen zu berücksichtigen ist. Hinweis: Rezept- und apothekenpflichtig. Weitere Angaben zu Warnhinweisen und Vorsichtsmaßnahmen für die Anwendung, Wechselwirkungen mit anderen Arzneimitteln und sonstigen Wechselwirkungen, Schwangerschaft und Stillzeit und Nebenwirkungen sind der veröffentlichten Fachinformation zu entnehmen. (Stand: 4/218) Basisinformationen SUBLIVAC FIX / SUBLIVAC Pollenpräparate Bezeichnungen und Zusammensetzung: Allergenlösungen zur sublingualen Immuntherapie. 1 ml enthält 1. AUN oder PUN. SUBLIVAC FIX Bäume: Pollen von Birke, Erle, Hasel zu gleichen Teilen; SUBLIVAC FIX Birke: Birkenpollen; SUBLIVAC FIX Gräser: Pollen von 3 Gräsern; SUBLIVAC FIX Gräser+Roggen: Pollen von 3 Gräsern (5%) und Roggen (5%); SUBLIVAC FIX Lieschgras: Pollen aus Lieschgras. SUBLIVAC enthält Allergenextrakte nach individueller ärztlicher Rezeptur. Bezeichnung und Stärke der Allergene s. Etikett. Sonstige Bestandteile: Glycerol, Wasser, 6-Aminohexansäure (ε-amino-capronsäure/eaca), Dinatrium-hydrogenphosphat, Natrium dihydrogenphosphat, Pfefferminzöl. Anwendungsgebiete: Spezifische Immuntherapie allergischer Erkrankungen vom Soforttyp (IgE-vermittelt). Gegenanzeigen: akute Entzündungsprozesse/Infektionskrankheiten am Reaktionsorgan; Schwere Autoimmunerkrankungen; Immundefekte (auch durch Immunsuppressiva induziert); Krebserkrankungen mit aktuellem Krankheitswert; schweres oder unzureichend behandeltes Asthma (FEV1 < 7% vom Sollwert); Überempfindlichkeit gegenüber einem der sonstigen Bestandteile; Infektionen des Mund-/Rachenraumes; nach zahnärztlicher Behandlung (z.b. Zahnentfernung). Eine Hyposensibilisierungsbehandlung soll nicht während der Schwangerschaft begonnen werden. Zum zeitlichen Intervall zu Schutzimpfungen und für weiterführende Informationen siehe Fachinformation. Nebenwirkungen: Lokale Beschwerden, Schwellungen oder Juckreiz des Mundes, der Lippen und der Zunge, Mundtrockenheit, Geschwüre im Mund, Aphthen. Allergische Reaktionen wie Niesen, laufende oder verstopfte Nase, Nasenbeschwerden; Juckreiz oder Beschwerden des Ohrs; Juckreiz, Rötung, Tränen oder Brennen der Augen, Augenentzündung; Halsreizungen, Husten, Atemnot. Schwierigkeiten beim Schlucken, Magenbeschwerden, Erbrechen, Übelkeit, Bauchschmerzen, Appetitlosigkeit, Verdauungsstörungen. Ekzeme, Hautausschlag, Nesselsucht, schnelle oder starke Schwellung der Haut. In sehr seltenen Fällen anaphylaktische Reaktionen. Hinweis: Rezept- und apothekenpflichtig. Weitere Angaben zu Warnhinweisen und Vorsichtsmaßnahmen für die Anwendung, Wechselwirkungen mit anderen Arzneimitteln und sonstigen Wechselwirkungen, Schwangerschaft und Stillzeit und Nebenwirkungen sind der veröffentlichten Fachinformation zu entnehmen. (Stand: 4/218) HAL Allergie GmbH, Poststraße 5 6, D-4213 Düsseldorf HAL Allergy Handelsgesellschaft mbh, Johnstraße 4, A-115 Wien EAACI, 26-3 May 218, Munich, Germany 27

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30 Notes

31 International Offices Headquarters The Netherlands HAL Allergy Group J.H.Oortweg CH Leiden P.O. Box BE Leiden Tel.: +31-() Offices Distributors Germany HAL Allergie GmbH Poststraße Düsseldorf Tel.: +49-() Austria HAL Allergy Handels-GmbH Johnstraße Vienna Tel.: +43 () Poland HAL Allergy Sp. z o.o. Ul. Rumiana Warsaw Tel.: +48-() Spain HAL Allergy S.L.U Parque Empresarial Mas Blau II Avda. Les Garrigues El Prat de Llobregat Barcelona Tel.: Italy HAL Allergy s.r.l. Piazzale Asia, 21 Scala B, Piano 4, Interno 21, 144 Roma Tel: Belgium / Benelux HAL Allergy Benelux B.V. J.H. Oortweg CH Leiden The Netherlands Tel.: +31-() Estonia Balti Intermed OÜ Tartu mnt 84d 1112 Tallinn Tel.: Greece Alfamedica S.A. Sofokleous 1b Elliniko Tel.: /19 Portugal A.M.D. Passos, Lda. Rue Cidade de Coimbra no. 15A 1, Parede, Lisbon Tel.: Hungary Hungaropharma Zrt. 161 Budapest Király u.12 Tel.: Slovenia IRIS Mednarodna Trgovina D.O.O. Cesta v Gorice 8 1 Ljubljana Tel.:

32 Rising to the c hal lenge of improving allergy treatments HAL Allergy 218 MAB4894/1