HAL Allergy Abstractbook EAACI 2014

Scientific Contribution 2014 33 rd Congress of the European Academy of Allergy and Clinical Immunology EAACI, 7-11 June 2014, Copenhagen, Denmark

55 years in the allergy business

Dear reader, The European Academy of Allergy and Clinical Immunology (EAACI) is a major global platform for education, promotion of scientific excellence and advocacy relating to all aspects of allergy. HAL Allergy is one of Europe’s top players in the field of allergen vaccination and devotes a great deal of its resources and investments to the development of allergen specific immunotherapy. As a result, we are proud to present our research and clinical practice work during the annual EAACI congress in Copenhagen.

Dirk-Jan Opstelten, PhD Research & Development Director

In this booklet you will find all our abstracts dealing with HAL Allergy’s recent clinical and pre-clinical research and development activities.

HAL Allergy’s clinical development program aimed at meeting today’s requirements for registration of specific immunotherapy products is progressing successfully. We recently completed dose range finding studies with our subcutaneous house dust mite allergoid preparation and our sublingual birch pollen extract of which the results are presented in this booklet. In addition, we present clinical data on the use of our subcutaneous artemisia allergoid preparation in daily practice.

Our pre-clinical work focuses on unraveling the mechanisms of immunotherapy and on developing novel vaccine candidates with an excellent safety and efficacy profile. Examples of the presented work include studies on immunotherapy in a mouse model for birch pollen allergy and the recombinant expression of Bet v 1 in a mammalian cell line.

Diderik Boot, PhD Medical Director

In addition, we work continuously on improving the methodologies to characterize allergen preparations used for immunotherapy. We present our latest developments representing the state-of-the-art analysis of birch pollen, bee venom and house dust mite allergen preparations.

We hope you find this scientific contribution interesting. Please visit our website or ask your local sales representative should you require further information on our R&D program or on our products.

Kind regards,

Diderik Boot, PhD

Dirk-Jan Opstelten, PhD

Medical Director

Research & Development Director

Contents

Scientific Contribution

Clinical Dose finding study with a subcutaneous specific immunotherapy allergoid in patients with house dust mite induced allergic rhinitis/rhinoconjunctivitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

6

Determination of the optimal effective and safe dose of a birch pollen extract for the treatment of allergic rhinitis; results of a phase II study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Rush up-dosing regimen with an Artemisia vulgaris depot allergoid preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Efficacy and safety of immunotherapy in patients with different clinical characteristics . . . . . . . . . . . . . . . . . . . . . . . . . .

8 10 12

Pre-Clinical Characterisation of Recombinant Bet v1.0101 produced in Chinese Hamster Ovary cells . . . . . . . . . . . . . . . . . . . . . . . . .

14

Sequential effect of subcutaneous immunotherapy for birch pollen allergy on Th2 cell suppression and attenuation of airway hyperreactivity in a murine asthma model .. . . . . . . . . . . . . . . . . .

16

Development Physicochemical characterisation of bee venom . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Stress stability study on a birch pollen extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Maturation of a mite allergy vaccine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

18 20 22

Dose finding study with a subcutaneous specific immunotherapy allergoid in patients with house dust mite induced allergic rhinitis/rhinoconjunctivitis

O. Pfaar1, M.J. Nell2, J.D. Boot2, D.J.E. Opstelten2, S.A. Versteeg3, R. van Ree3, A. Roger4, H. Riechelmann5, J.N.G. Oude Elberink6, C. Bachert7 1

Center for Rhinology and Allergology Wiesbaden, Germany, Department of Otorhinolaryngology, Head and Neck Surgery, University

Hospital Mannheim, Germany, 2 HAL Allergy BV, Leiden, The Netherlands, 3 Experimental Immunology, Academic Medical Center, Amsterdam, The Netherlands, 4 Unitat d’Allèrgia, Hospital Universitari Germans Trias i Pujol, Badalona, Spain, 5 Universitätsklinik für Hals-, Nasen- und Ohrenheilkunde, Innsbruck, Austria, 6 Dept of Allergology, University Medical Center Groningen, University of Groningen, The Netherlands, 7 UZ Gent, Ear-, Nose and Throat Department, Gent, Belgium

Background Previously, the safety and tolerability of increasing doses of a subcutaneous specific immunotherapy allergoid were determined. The objective of the current dose finding study was to identify the optimal, i.e. safe and effective dose in patients with HDM induced allergic rhinitis/rhinoconjunctivitis (AR).

Methods A multinational, randomized, double-blind, placebo-controlled, parallel-group, phase II dose finding study was conducted.

290 adult patients suffering from AR (148 females; 142 males) were randomized to receive placebo, 6,667, 20,000, 50,000 or 100,000 AUeq/ml. Skin prick testing, specific IgE and a positive titrated nasal provocation test (TNPT) confirmed that allergy complaints were triggered by HDM. Patients were up-dosed weekly, followed by monthly maintenance dosing of 0.5 ml by subcutaneous injections. The primary outcome of the study was the TNPT after 12 months of treatment. Secondary outcomes included TNPT after 6 months, immunoglobulin analysis, and safety assessments.

Results A dose response curve was observed whereby doses of 20,000 AUeq/ml and higher showed significant improvement in the TNPT after one year of treatment (Table 1). After 6 months we could not yet demonstrate a significant difference.

Specific IgG and IgG4 levels were dose dependently increased in all dose groups, supporting the immunogenic effect of the allergoid Mites preparation. The highest dose group induced more treatment emergent adverse events compared to the lower dose groups.

Conclusion The efficacy and safety of SCIT allergoid for HDM induced AR was assessed. Doses of 20,000 AUeq/ml and higher demonstrated significant efficacy compared to placebo. The risk-benefit ratio favours the use of 20,000 AUeq/ml and 50,000 AUeq/ml.

Table 1: Mean change in symptom score after treatment

Placebo 6,667 20,000 50,000 100,000

AUeq/ml AUeq/ml AUeq/ml AUeq/ml Adjusted Least Square (LS) Mean End of Study - Baseline (±SE)

-1.74 ± 0.26

-2.28 ± 0.26

-2.52 ± 0.25

-2.57 ± 0.26

-2.47 ± 0.27

Difference LS Means (±SE)

-

-0.54 ± 0.37

-0.74 ± 0.36

-0.85 ± 0.37

-0.74 ± 0.37

P-Values

-

0.1405 0.0396 0.0217 0.0487

EAACI, 7-11 June 2014, Copenhagen, Denmark Abstract number: 953, Session number, date and time: PDS 17, Tuesday 10 June 2014; 15:30 - 17:00 Session title: AIT phase II and III clinical trials

6

PDS 17 - AIT phase II and III clinical trials

Dose finding study with a subcutaneous specific immunotherapy allergoid in patients with house dust mite induced allergic rhinitis/rhinoconjunctivitis

7

In relation to this presentation, I declare the following, real or perceived conflict of interest. The presenter is a consultant of HAL Allergy.

EAACI Congress 2014

Determination of the optimal effective and safe dose of a birch p ollen extract for the treatment of allergic rhinitis; results of a phase II study

O. Pfaar1, E. van Twuijver2, J.D. Boot2, D.J.E. Opstelten2, L. Klimek1, R. van Ree3, P. Kuna4, P. Panzner5.

1

Center for Rhinology and Allergology Wiesbaden, Germany, Department of Otorhinolaryngology, Head and Neck Surgery, University Hospital

Mannheim 2HAL Allergy BV, Leiden, The Netherlands, 3Department of Experimental Immunology, Academic Medical Center, University of Amsterdam, The Netherlands 4Barlicki University Hospital, Medical University of Lodz, Poland, 5Dept. of Allergology and Immunology, Medical Faculty in Plzen, Charles University Prague, Czech Republic.

Background A combined tolerability, dose finding study in adults with different dosages of a sublingual liquid birch pollen preparation (SB) was conducted to determine the optimal effective and safe dose.

Methods A randomized, double-blind, placebo-controlled, parallel-group, multi-centre, phase II study was conducted in Poland, Czech Republic and Germany. 269 adult subjects (134 females;135 males) with birch pollen induced allergic rhinitis were randomized to receive placebo, SB 3,333; 10,000; 20,000 or 40,000 AUN/ml. The primary endpoint was defined as the absolute difference in mean symptom score following TNPT between 5 months of treatment and baseline in the different SB dosage groups compared to placebo. Furthermore, safety, tolerability, birch pollen specific immunoglobulin (Ig) levels and Peak Nasal Inspiratory Flow (PNIF) were measured. All outcome measures were determined outside the birch pollen season.

Results In all treatment groups an improvement in symptom scores after 5 months of treatment compared to baseline was observed, with an additional stepwise improvement in the active treatment groups compared to placebo (table 1). The dose response curve observed after active treatment was significant. Furthermore, active treatment resulted in an increase in PNIF and serum IgG levels compared to placebo. The biggest improvements were found in the 40,000 AUN/ml treatment group. All active dosages resulted in more local and systemic adverse reactions compared to placebo, mainly mild and well-controlled.

Conclusion A multi-centre trial was conducted to evaluate the dose response and dose tolerability of SB. All active treatment groups showed better responses than placebo for both primary and secondary parameters. In the present study, the results indicate that SB 40,000 AUN/ml is the most optimal effective and safe dose.

Table 1: Mean change in symptom score after treatment with different dosages of SB compared to placebo Placebo Adjusted Least Square Mean

3,333

SB (AUN/ml) 10,000 20,000

-1.90

-2.29

-2.42

-2.90

-3.45

Difference of Least Square Means (SE)

-

-0.39 (0.37)

-0.52 (0.36)

-1.00 (0.37)

-1.54 (0.37)

P-Value

-

0.293

0.158

0.008

<0.001

EAACI, 7-11 June 2014, Copenhagen, Denmark Abstract number: 1426, Session number, date and time: PDS 17, Tuesday 10 June 2014; 15:30 - 17:00 Session title: AIT phase II and III clinical trials

8

40,000

PDS 17 - AIT phase II and III clinical trials

Determination of the optimal effective and safe dose of a birch p ollen extract for the treatment of allergic rhinitis; results of a phase II study

9

In relation to this presentation, I declare the following, real or perceived conflict of interest. The presenter is a consultant of HAL Allergy.

EAACI Congress 2014

Rush up-dosing regimen with an Artemisia vulgaris dep ot allergoid preparation

A. Distler1, E.Hippke2, J.D. Boot3 , N. Angelova3, P. van Osch3, U. Neumann4, 1

HAL Allergie GmbH, DĂźsseldorf, Germany, 2 ENT practice, Berlin, Germany, 3 HAL Allergy BV, Leiden, The Netherlands,

4

ENT practice, Wolmirstedt, Germany

Background Subcutaneous specific immunotherapy is up-dosed in a stepwise fashion. An accelerated up-dosing regimen with a grass depot allergoid preparation has been found as safe as the conventional up-dosing and is already approved. Data regarding the tolerability of a accelerated up-dosing regimen with Artemisia vulgaris (mugwort) were not available until now.

Methods 7 practitioners who had already treated patients allergic to Artemisia in autumn/winter 2012 with the rush up-dosing regimen (weekly injection (inj.) of 0.1â&#x20AC;&#x201C;0.3â&#x20AC;&#x201C;0.5 ml of depot allergoid preparation Artemisia vulgaris; HAL Allergy, Leiden, Netherlands) were asked to retrospectively document their experiences based on data in the patient charts. The documentation with a standardized questionnaire included the up-dosing and the maintenance treatment.

Results In total 31 patients (Mean age 42 years; 17 females) were included in the analysis. 196 inj. could be evaluated (100 in the up-dosing and 96 in the maintenance). 30 patients (96.7%) reached the maintenance dose of 0.5 ml. During up-dosing 2 patients (6.4%) required a dose adjustment due to a local reaction. During maintenance phase 1 patient (3.2%) required a dose adjustment due to a local reaction. Most often local reactions like swelling and redness were observed. These were reported following 56 inj. (28.6%) in 16 patients (51.6%). The mean swelling size was small: 2.1 cm (0.2-5.0 cm) during updosing and 1.8 cm (0.2-3.0 cm) during maintenance phase. Mild systemic reactions were observed following 10 inj. (5.1%) in 4 patients (12.9%). Very rarely cooling or an antihistamine (1.5% of inj.) was necessary for the treatment of an adverse reaction. 18 patients were very satisfied, 12 patients were satisfied and 1 patient was rather dissatisfied with the up-dosing regimen. Judgement of the physicians corresponded to this.

Conclusion The rush up-dosing regimen was proven to be well tolerated and might be used to up-dose patients within 2 weeks.

EAACI, 7-11 June 2014, Copenhagen, Denmark Abstract number: 1448, Session number, date and time: TPS 76, Tuesday 10 June 2014; 12:00 - 13:30 Session title: Vaccines

10

TPS 76 - Vaccines

Rush up-dosing regimen with an Artemisia vulgaris dep ot allergoid preparation

11

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 2014

Efficacy and safety of immunotherapy in patients with different clinical characteristics

M.J. Nell1, O. Pfaar 2, J.D. Boot1, E. van Twuijver1, P. van Osch1, C. Bachert3.

1

HAL Allergy BV, Leiden, The Netherlands, 2 Center for Rhinology and Allergology Wiesbaden, Germany, Department of Otorhinolaryngology,

Head and Neck Surgery, University Hospital Mannheim, Germany, 3UZ Gent, Ear-, Nose and Throat Department, Gent, Belgium.

Background Allergen specific immunotherapy (SIT) is indicated in patients with IgE mediated moderate to severe intermittent or persistent allergic rhinitis (AR). In guidelines several patient characteristics are listed as success factors for SIT treatment.

The efficacy of SIT for house dust mite (HDM) and birch pollen induced AR was recently examined. The aim of this additional analysis was to determine whether SIT is effective in subgroups of patients with different clinical characteristics.

Methods Adult patients with a positive medical history for HDM or birch pollen-induced AR, confirmed by a positive skin prick test (SPT) and specific IgE levels participated in two different multicentre phase II efficacy studies with SIT. The primary outcome of these studies was a titrated nasal provocation test (TNPT). The following subgroups were examined: with or without asthma, with or without perennial co-sensitisation, high or low SPT diameter, high or low IgE levels and age. The mean improvement in the TNPT score after treatment was compared between these subgroups.

Results SIT induced an improvement in the TNPT compared to placebo in both studies. In the HDM study we observed no differences in efficacy between all investigated subgroups. In the birch study the improvement in the subgroup without asthma was significantly higher (p=0.03) compared to the subgroup with asthma. No differences were observed between the other subgroups.

Conclusion For HDM SIT the subgroups showed similar efficacy profiles as in the overall study population with no differences between the subgroups. For birch SIT this was the same, with the exception of patients without asthma. This finding requires confirmation in larger clinical efficacy studies. Overall, the results support the use of SIT in IgE mediated moderate to severe intermittent or persistent AR, regardless of concomitant asthma, sensitization status, severity profile or age.

EAACI, 7-11 June 2014, Copenhagen, Denmark Abstract number: 1495, Session number, date and time: TPS 43, Monday 9 June 2014; 12:15 - 13:45 Session title: Immunotherapy - AIT clinics I

12

TPS 43 - Immunotherapy - AIT clinics I

Efficacy and safety of immunotherapy in patients with different clinical characteristics

13

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 2014

Characterisation of Recombinant Bet v1.0101 pro duced in Chinese Hamster O var y cells

H. Warmenhoven, M. Vigil Garcia, H. van Schijndel HAL Allergy BV, Leiden, The Netherlands

Background Chinese Hamster Ovary (CHO) cells are the most widely used expression system for the production of therapeutic proteins.

CHO cells contain the appropriate machinery, comparable to human cells, for the production of properly folded and posttranslationally modified proteins. In this study, we used CHO cells as a production platform for the recombinant Bet v1.0101 isoform, the immunodominant major allergen from birch pollen.

Methods CHO-S cells were cultured and transfected with the commercial expression vector pcDNA3.1 (Life Technologies) containing the strong human CMV promoter, the Bet v1.0101 gene and a G418 resistance cassette. Individual clones from stably transfected cell pools under selection pressure with G418 were isolated by limiting dilution, generating monoclonal cell lines. Bet v1.0101 from the harvested medium was purified using several chromatographic techniques.

Various immunological, biochemical and physicochemical assays were applied for characterization and comparison of the newly produced rBet v1.0101 to natural and other recombinant forms of Bet v1.

Results Stable CHO-S clones producing rBet v1.0101 were isolated. The purified rBet v1.0101 showed a high purity as determined

by SDS PAGE. The results from the assays used for characterisation and comparison showed a high degree of similarity between the different forms of Bet v1.

Conclusion In this study, we have demonstrated the possibility of producing, in CHO cells, rBet v1.0101 with similar biochemical and immunological characteristics compared to other available forms of rBet v1. This is, based on available literature data, the first plant-derived recombinant allergen expressed in a mammalian cell line.

EAACI, 7-11 June 2014, Copenhagen, Denmark Abstract number: 1447, Session number, date and time: TPS 46, Monday 09 June 2014; 12:15 - 13:45 Session title: Molecular allergology III

14

TPS 46 - Molecular allergology III

Characterisation of Recombinant Bet v1.0101 pro duced in Chinese Hamster O var y cells

15

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 2014

Sequential effect of subcutaneous immunotherapy for birch p ollen allergy on Th2 cell suppression and attenuation of airway hyperreactivity in a murine asthma mo del L. van Rijt1, A. Logiantara1, L. Utsch1, D. Canbaz1, D.J.E. Opstelten3, H. van der Kleij3, R. van Ree1,2,3 Dept Experimental Immunology, 2Dept Otorhinolaryngology, AMC, Amsterdam, the Netherlands, 3HAL Allergy BV, Leiden, The Netherlands

1

Background The suppression of Th2 cytokine production by allergen specific Th2 cells is considered to be critical for the suppression of allergic symptoms by subcutaneous immunotherapy (SCIT). The aim of this study was to develop a mouse model for birch pollen (BP) allergy that can be used as a preclinical model to improve allergen immunotherapy and to elucidate the underlying mechanisms that contribute to improvement of clinical symptoms.

Methods Mice with birch pollen induced allergic airway inflammation received 2, 4, 6 or 8 weekly subcutaneous immunotherapy injections with birch pollen extract adsorbed to alum. The effect of the number of injections and an increasing dose of birch pollen extract adsorbed to an equal concentration alum on the amelioration of airway inflammation, Th2 cytokine production, immunoglobulin production and airway hyperresponsiveness was determined.

Results After two injections, Th2 cytokine production, eosinophil recruitment, peribronchial inflammatory infiltrates were suppressed and BP specific immunoglobulins were induced but this did not prevent airway hyperresponsiveness. Only after eight injections, airway hyperresponsiveness was suppressed which was accompanied by an increasing titer of BP IgG2a while other immunological parameters were stably suppressed after 2 injections.

Conclusion This study showed that amelioration of airway hyperreactivity was delayed compared with suppression of Th2 mediated eosinophilic airway inflammation by BP SCIT. Induction of sufficient protective BP immunoglobulins might be involved.

EAACI, 7-11 June 2014, Copenhagen, Denmark Session number, date and time: OAS 30, Tuesday 10 June 2014; 15:30 - 17:00 Session title: Animal studies in asthma

16

OAS 30 - Animal studies in asthma

Sequential effect of subcutaneous immunotherapy for birch p ollen allergy on Th2 cell suppression and attenuation of airway hyperreactivity in a murine asthma mo del

17

In relation to this presentation, I declare no real or perceived conflict of interest. HAL Allergy sponsored the research.

EAACI Congress 2014

Physicochemical characterisation of bee venom

N. Sinnige, E. Schepens, S. Quaak, R. van den Hout, D. Luykx HAL Allergy BV, Leiden, The Netherlands

Background A physicochemical characterisation study was performed on bee venom (Apis mellifera) with respect to identity, content and protein structure.

Methods SDS-PAGE: Reduced samples were applied to 4-12% Bis-tris gels combined with silver staining. MS: Tryptic digests were prepared from bee venom and from SDS-PAGE bands. Peptides were separated via nano-HPLC before electrospray ionisation. Ionised peptides were fragmented revealing amino acid sequences. RP-HPLC: Reversed Phase-HPLC was performed on an YMC column, using purified Api m 1 as a standard. Protein: Lowry assay using BSA as a standard. CD: Far-UV CD spectra were recorded from 260-190 nm. Fluorescence: Emission spectra were recorded from 290-400 nm, with excitation at 280 nm.

Results The SDS-PAGE protein profile revealed a broad range of proteins between 3-100 kDa. Protein bands corresponding to major allergens Api m 1 (3 bands between 15-25 kDa), Api m 2 (45 kDa) and Api m 5 (90 kDa) were identified by MS. The abundant protein band at 3 kDa was identified by MS as Api m 4. Also Api m 3, 6, 7, 8, 9, 10 and 11 were identified in the bee venom using MS. The amount of Api m 1 was determined by RP-HPLC and was 12 % in relation to the total protein amount. The CD-spectrum of the bee venom indicated the presence of proteins with mainly Ă&#x;-structures and random coil structures.

The fluorescence spectrum showed an emission maximum value at 350 nm indicating that the aromatic amino acids are largely exposed to polar surroundings (hydrophilic). These structural data indicate the presence of a relatively high amount of small proteins.

Conclusion With the physicochemical assays applied it was possible to characterise bee venom. Major allergens Api m 1, Api m 2 and Api m 5, and various minor allergens were identified. The amount of Api m 1 in total protein (12 %) is confirmed by literature findings. The structural data can be explained by the presence of the two most abundant proteins Api m 1 and Api m 4.

EAACI, 7-11 June 2014, Copenhagen, Denmark Abstract number: 1415, Session number, date and time: PDS 1, Sunday 8 June 2014; 10:30 - 12:00 Session title: Open questions in hymenoptera venom allergy

18

PDS 1 - Open questions in hymenoptera venom allergy

Physicochemical characterisation of bee venom

19

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 2014

Stress stability study on a birch p ollen extract

S. Acharya, G. J. Stam, N. Sinnige, C. Franso, E. Kerkvliet, R. van den Hout, D. Luykx HAL Allergy BV, Leiden, The Netherlands

Background Stressed and non-stressed birch pollen extracts were investigated with several analytical assays to monitor identity (SDSPAGE, immunoblot), content (protein, major allergen Bet v 1), potency (IgE potency) and structure (CD, Fluorescence, HPSEC).

Methods Stressed conditions: Incubation at 45째C (12 days), 60째C (3 days) or 90째C (1 Hour), freeze-thawing (5 times) or shaking (15 min at 2600 rpm). SDS-PAGE: Reduced samples on 4-12% Bis-tris gels and stained with silver. Immunoblot: After SDS-PAGE, proteins were transferred to PVDF membrane and stained using pooled sera from birch allergic patients, HRP-conjugated antibody and TMB substrate. Protein: Bradford assay using BSA as a standard. Major allergen content: An ELISA was used to quantify Bet v 1 content. Potency: the allergenic activity was measured using an IgE-inhibition assay. CD: Spectra were recorded from 260-190 nm. Fluorescence: Emission spectra were recorded from 290-400 nm, excitation at 280 nm. HP-SEC: A Bio-Sec 3 size exclusion chromatography column was used with UV-detection.

Results SDS-PAGE showed disappearance of bands and appearance of higher molecular weight bands for the birch extract upon thermal stressing. Immunoblot showed reduced intensity of major allergen bands for the 90째C stressed sample. The protein content was not affected by stressing the birch extract. The Bet v 1 content and IgE potency decreased by temperature stressing. Temperature stressing induced unfolding of proteins according to CD and fluorescence spectroscopy. HP-SEC showed aggregation of birch proteins after thermal treatment. Freeze-thawing and shaking did not affect any of the investigated properties of the birch extract.

Conclusion Temperature stressing of a birch allergen extract affected the protein profile, Bet v 1 content and IgE potency. In parallel, protein unfolding and protein aggregation occurred.

EAACI, 7-11 June 2014, Copenhagen, Denmark Abstract number: 1417, Session number, date and time: TPS 76, Tuesday 10 June 2014; 12:00 - 13:30 Session title: Vaccines

20

TPS 76 - Vaccines

Stress stability study on a birch p ollen extract

21

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 2014

Maturation of a mite allergy vaccine

S. Acharya, J. de Bruijn, M. Busch, R. van den Hout, D. Luykx HAL Allergy BV, Leiden, The Netherlands

Background Ageing or maturation is a well-known phenomenon in protein adsorption onto aluminium hydroxide (AlO(OH)). Mite allergy vaccines used in this study contain mite extracts bound to AlO(OH). For safety and efficacy the vaccines are required to be stable. Both the protein and AlO(OH) can undergo physical and structural changes which in turn can affect the nature and binding strength of the partners. This study is aimed at revealing possible changes of these aspects in time. Fresh and old mite allergy vaccine preparations were monitored for a period of 7 months using various physicochemical assays.

Investigation was done on binding strength, protein structure, AlO(OH) structure and the appearance.

Methods Protein desorption behaviour: The amount of protein desorbed from the AlO(OH) treated with 0.5 M phosphate buffer was determined. Circular Dichroism (CD): Spectra were recorded from 190 to 260 nm. The CD ratio 207/222 nm was monitored. Analytical centrifugation: The percentage of sediment after analytical centrifugation at 400 rpm was monitored.

Light-scattering: The 90째 light-scattering intensity at 400 nm was monitored.

Results The binding of the mite extracts to the AlO(OH) in freshly prepared vaccines changed considerably in the first three months.

The desorption of protein with phosphate buffer gets more difficult in time indicating increase of the binding strength of the extracts. No changes were recorded with respect to (i) appearance of the suspension (ii) protein structure of the mite extracts as observed with CD or (iii) AlO(OH) structure as observed with analytical ultracentrifugation and light scattering for a period of 7 months.

Conclusion This study demonstrates that the binding strength of mite extracts to AlO(OH) in a mite allergy vaccine becomes stronger in time, especially the first 3 months after adsorption. The results obtained in this study reflect the effect of ageing or maturation.

EAACI, 7-11 June 2014, Copenhagen, Denmark Abstract number: 1420, Session number, date and time: TPS 76, Tuesday 10 June 2014; 12:00 - 13:30 Session title: Vaccines

22

TPS 76 - Vaccines

Maturation of a mite allergy vaccine

23

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 2014

SUBLIVAC

®

Composition: SUBLIVAC , sublingual drops, contains allergen extracts that are specifically prescribed for the patient on the basis of a diagnostic examination.

Indication: Treatment of IgE-mediated allergy in patients with symptoms of allergic rhinitis, allergic conjunctivitis and/or allergic asthma, caused by allergens.

Dosage and administration: The treatment is started with a daily dose of one drop. This dose is then increased every day with one drop until the highest daily dose of five drops is reached. The treatment is continued with five drops. The drops are to be kept under the tongue for at least 1 minute (preferably 2 - 3 minutes) before they are swallowed. The treatment should be continued for 3 to 5 years in order to remain as symptom-free as possible after the completion of the course of treatment. Contraindications: Acute infections of the eye, airways or organs involved in the immune system, secondary changes in the lungs(e.g. lung emphysema or bronchi-ectasie), severe immunopathological diseases or malignancies (e.g. autoimmune diseases of the kidneys, thyroid glands or the nervous system, rheumatism, tuberculosis and HIV), immunodeficiencies (including as a consequence of immunosuppressants), severe uncontrolled asthma with FEV 1 Warnings and precautions: If the treatment with pollen extracts is started during the pollen season, there is an increased risk of side effects. Take special care in case of pregnancy, inflammations in the mouth, after tooth extraction and in case of treatment with ß-blockers (including ß-blocker containing eye preparations). In the rare case that an anaphylactic shock occurs with the concomitant use of a ß-blocker, adrenaline as a rescue medication can be less efficacious and a higher dose of adrenaline may be required.

Severe systemic allergic reactions should be treated with Adrenalin. under 70%, or hypersensitivity to any of the excipients. Side effects: Local reactions in the mouth and throat, stomach upset and diarrhoea. Worsening of allergic reactions such as rhinitis, conjunctivitis, coughing and atopic eczema. Intensified systemic reactions (such as shortness of breath, generalised urticaria or Quincke’s oedema) can develop in rare cases. Intensified allergic reactions can particularly develop in very hypersensitive patients. These symptoms generally arise within 30 minutes after intake of the drops. In individual cases anaphylactic shock has been reported in patients with concomitant asthma. Typical warning signs are burning, itching and heat sensation up and under the tongue, throat and palm of the hand and sole of the foot. Conditions for storage: Store in a refrigerator (2°C - 8°C).When the product is in use, it can be stored for a maximum of 6 months below 25 °C. Package: A SUBLIVAC Initial and maintenance treatment set consists of one bottle with dropper containing 24 ml fluid. The complete product information is available upon request. HAL Allergy BV, P.O. Box 1204, 2302 BE Leiden, The Netherlands. Date: July 2013 ®

®

PURETHAL

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Composition: PURETHAL contains 20,000 AUM/ml modified pollen allergen extracts, 20,000 AUeq/ml of modified mites allergen extract respectively adsorbed onto aluminium hydroxide; suspension for subcutaneous injection. Indications: Treatment of immediate type allergic disorders (IgE-mediated), such as allergic rhinitis, allergic conjunctivitis and allergic bronchial asthma, which are triggered by allergenic substances. Dosage and administration: The therapy is started with a subcutaneous injection of 0.05 ml. After the first injection the dosage is increased stepwise to a maximum dose of 0.5 ml that is finally administered in monthly intervals. For PURETHAL grasses an accelerated stepwise increase in adults may be used (Rush scheme). Always check by aspiration that the injection needle has not entered a blood vessel. Do not exceed the maximum dose of 0.5 ml. It is advised to carry out the treatment over a period of 3 - 5 successive years. Contraindications: Acute inflammatory diseases/feverish infection at the target organ, secondary changes of the target organ (emphysema, bronchi-ectasia and others), autoimmune disorders (e.g. of the kidneys, thyroid gland, nervous system and rheumatic diseases), immune deficiencies (e.g. that caused by immuno-suppressants), severe uncontrolled asthma particularly with an FEV 1 persistency below 70%, cardiovascular failure with increased risk if using adrenalin, clinical active malignant tumor, hypersensitivity to any of the excipients. Special warnings and special precautions for use: Treatment with immunotherapy injections should only be performed by physicians qualified in allergology. Appropriate emergency treatment for shock must be immediately available during and after every injection. The patient must remain under medical supervision after the injection for 30 minutes. Special care should be taken in case of treatment with β-blockers, pregnancy and lactation, use of the product in children below the age of 5. Prophylactic immunization should be carried out no sooner than 7 days after the last injection. Side effects: Especially in the case of patients with a high degree of sensitization, intensified allergic reactions may occur. These symptoms generally arise within 30 minutes of receiving the injection. Intensified local reactions at the injection site. Reappearance of patient specific allergic symptoms as mild systemic reactions (itching of eyes, sneezing, coughing, atopic eczema), intensified systemic reactions (shortness of breath, generalized urticaria, Quincke’s oedema), in extreme rare cases also anaphylactic shock.

After use subcutaneous knots and swellings (granulomas) at the injection site may be observed. Using the Rush scheme more side effects, primarily mild by nature, can occur. Package: PURETHAL is delivered in a 6 ml multidose vial with stopper and sealed with an aluminium cap. The complete product information is available on request. HAL Allergy BV, P.O. Box 1204, 2302 BE Leiden, The Netherlands. Date: April 2013 ®

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VENOMENHAL

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Composition: VENOMENHAL Bee, active ingredient: bee venom. VENOMENHAL Wasp, active ingredient: wasp venom. Six injection bottles each containing 120 µg pure, freeze-dried bee or wasp venom. Excipients: HSA (human serum albumin) and mannitol. Solvent: 6 injection bottles each containing 1.2 ml of a solution of sodium chloride, phenol, HSA and water for injection. Indications: Dermal testing and specific immunotherapy for patients with IgE-mediated insect venom allergy, who experienced a systemic reaction after being stung by a bee or wasp. Contraindications: Acute inflammatory process/febrile infectious diseases; secondary disorders (e.g. emphysema, bronchiectasis); autoimmune disorders; immunodeficiency (including that induced by immunosuppressants); severe or inadequately controlled asthma (especially with FEV1 < 70% of reference value); cardiovascular disorders with increased risk when using adrenaline; treatment with ß-blockers (including ß-blocker-containing eye drops) and ACE-inhibitors; malignant tumours with current clinical significance; sensitivity to one of the excipients. These listed contraindications must be weighed against the danger to the patient from another insect sting. Do not start treatment when pregnant. Details about the interval to leave after vaccinations and further information, see Instructions for use and specialist information. In addition, the known contraindications to conducting dermal testing, e.g. skin disease near the test site (see specialist information). Adverse effects: Along with local reactions, systemic allergic reactions of varying severity can occur during dermal testing and therapy: anaphylactic shock, headache, flushing, coughing, dyspnoea, wheezing, diarrhoea, dyspepsia, nausea, vomiting, itching, rash, urticaria, angioedema, eczema, erythema, arthralgia, swelling or pruritus or urticaria at the injection site, feeling unwell, fatigue, malaise, pain, fever, swelling, irregular pulse, irregular blood pressure. In rare cases a slight fatigue may be evident after the injection, which should be considered when driving or operating machinery. Patients must be monitored at least 30 minutes after the injection, and a shock kit must be on hand. Adverse effects can also develop several hours after the injection. For more information and for the treatment of adverse effects, see specialist information. NB: prescription-only. The complete product information is available on request: HAL Allergy BV, P.O. Box 1204, 2302 BE Leiden, The Netherlands.

Date: April 2014 ®

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SUBLIVAC

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Basisinformationen SUBLIVAC FIX / SUBLIVAC . Zusammensetzung: Allergenlösungen zur sublingualen Immuntherapie. Genaue Bezeichnung und Stärke der Allergene s.

Etikett. SUBLIVAC enthält Allergenextrakte nach individueller ärztlicher Rezeptur. Sonstige Bestandteile: Glycerol, Wasser, 6-Aminohexansäure (e-Amino-Capronsäure/ EACA), Dinatrium-hydrogenphosphat, Natrium dihydrogenphosphat, Pfefferminzöl. Anwendungsgebiete: Spezifische Immuntherapie allergischer Erkrankungen vom Soforttyp (IgE-vermittelt). Gegenanzeigen: Sekundärveränderungen am Reaktionsorgan (z.B. Emphysem, Bronchiektasen); Schwere Autoimmunerkrankungen; Immundefekte (auch durch Immunsuppressiva induziert); Krebserkrankungen mit aktuellem Krankheitswert; schweres oder unzureichend behandeltes Asthma (FEV 1 < 70% vom Sollwert); Überempfindlichkeit gegenüber einem der sonstigen Bestandteile; akute Entzündungsprozesse/Infektionskrankheiten am Reaktionsorgan; 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: Lokalreaktionen leichter Art im Zungen-/Mundbereich wie Juckreiz, Brennen, Prickeln, Kribbeln oder Schwellung; milde allergische Allgemeinreaktion (Augenjucken, Fließschnupfen, Niesreiz, Husten, Verschlechterung eines atopischen Ekzems). In Einzelfällen gesteigerte Allgemeinreaktion wie Atemnot, generalisierte Urtikaria, oder Quincke-Ödem. Bauchschmerzen und Durchfall können mit zeitlicher Verzögerung vorkommen. In Einzelfällen ist ein anaphylaktischer Schock bei Patienten mit Asthma aufgetreten. Für weiterführende Informationen und zur Behandlung von Nebenwirkungen siehe Fachinformation. Hinweis: Verschreibungspflichtig. HAL Allergie GmbH, Poststraße 5-6, D-40213 Düsseldorf. Datum: April 2014 ®

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PURETHAL

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Basisinformationen PURETHAL . Zusammensetzung: Suspensionen zur subkutanen Injektion, enthalten an Aluminiumhydroxid adsorbierte, mit Glutaraldehyd chemisch modifizierte allergene Substanzen aus Pollen (20.000 AUM/ml) oder Milben (20.000 AUeq/ml). Sonstige Bestandteile: NaCl, Phenol, Aluminiumhydroxid, Wasser zur Injektion. Anwendungsgebiete: Spezifische Immuntherapie allergischer Erkrankungen vom Soforttyp (IgE-vermittelt). 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 FEV 1 unter 70% Sollwert; Erkrankungen mit Kontraindikationen gegen die Anwendung von Adrenalin; gleichzeitige Behandlung mit ß-Blockern (auch ß-Blocker enthaltende Augentropfen); maligne Tumorerkrankungen 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: Exazerbation der patientenspezifischen allergischen Symptomatik. Allergische Lokal- und/oder Allgemeinreaktionen bis hin zum anaphylaktischen Schock. In Einzelfällen Granulombildung am Injektionsort, Schwäche, Schwindel, Konzentrationsstörungen, Kopfschmerzen, Magen-Darm-Beschwerden, Gelenkschmerzen, Fieber. Patienten nach der Injektion mindestens 30 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: Verschreibungspflichtig. HAL Allergie GmbH, Poststraße 5-6, D-40213 Düsseldorf. Datum: April 2014 ®

VENOMENHAL

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Basisinformationen VENOMENHAL Biene / VENOMENHAL Wespe. Zusammensetzung: VENOMENHAL Biene, Wirkstoff: Bienengift. VENOMENHAL Wespe, Wirkstoff: Wespengift. 6 Durchstechflaschen enthalten jeweils 120 µg reines, gefriergetrocknetes Bienen- bzw. Wespengift. Sonstige Bestandteile: HSA (humanes Serum Albumin) und Mannitol. Lösungsmittel: 6 Durchstechflaschen enthalten jeweils 1,2 ml einer Lösung von Natriumchlorid, Phenol, HSA und Wasser zur Injektion.

Anwendungsgebiete: Hauttestung und spezifische Immuntherapie von Patienten mit IgE - vermittelter Insektengiftallergie, bei denen systemische Reaktionen nach Bienen- oder Wespenstich aufgetreten sind. Gegenanzeigen: Akute Entzündungsprozesse/fieberhafte Infektionskrankheiten; Sekundärveränderungen (z.B. Emphysem, Bronchiektasen); Autoimmunerkrankungen; Immundefekte (auch durch Immunsuppressiva induziert); schweres oder unzureichend behandeltes Asthma (insbesondere bei FEV 1 < 70% vom Sollwert); Herz- und Kreislauferkrankungen mit erhöhtem Risiko bei der Anwendung von Adrenalin; Behandlung mit ß-Blockern (auch ß-Blocker enthaltenden Augentropfen) und mit ACE-Hemmern; maligne Tumorerkrankungen mit aktuellem Krankheitswert; Sensibilisierung gegenüber einem der sonstigen Bestandteile. Die genannten Kontraindikationen sollten gegen die Gefährdung des Patienten durch einen erneuten Insektenstich abgewogen werden.

Einleitung der Behandlung nicht während der Schwangerschaft. Zum zeitlichen Intervall zu Schutzimpfungen und für weiterführende Informationen siehe Gebrauchsund Fachinformation. Ferner gelten die bekannten Gegenanzeigen für die Durchführung von Hauttestungen, wie z.B. Hautkrankheiten im Testbereich (siehe Fachinformation). Nebenwirkungen: Neben Lokalreaktionen können bei der Hauttestung und der Therapie systemische allergische Reaktionen unterschiedlicher Schweregrade auftreten: Anaphylaktischer Schock, Kopfschmerz, Gesichtsrötung (Flush), Husten, Dyspnoe, Giemen, Diarrhö, Dyspepsie, Übelkeit, Erbrechen, Juckreiz, Ausschlag, Urtikaria, Angioödem, Ekzem, Erythem, Arthralgie, Schwellung oder Pruritus oder Urtikaria an der Injektionsstelle, Unwohlsein, Müdigkeit, Krankheitsgefühl, Schmerzen, Fieber, Schwellung, Pulsanomalien, Blutdruckanomalien, In seltenen Fällen kann nach der Injektion leichte Müdigkeit auftreten, was beim Führen von Kraftfahrzeugen oder beim Bedienen von Maschinen zu berücksichtigen ist. Patienten nach der Injektion mindestens 30 Minuten überwachen, eine Schockapotheke muss bereitgestellt sein. Nebenwirkungen können auch mehrere Stunden nach der Injektion auftreten. Für weiterführende Informationen und zur Behandlung von Nebenwirkungen siehe Fachinformation. Hinweis: Verschreibungspflichtig. HAL Allergie GmbH, Poststraße 5-6, D-40213 Düsseldorf. Datum: April 2014 ®

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Notes Notes

Addresses

The international scope of HAL Allergy.

Headquarters

Austria HAL Allergy Handels-GmbH

Italy HAL Allergy s.r.l.

The Netherlands HAL Allergy Group

Johnstrasse 4-6 1150 Vienna Tel.: +43-(0)1-4893100 www.hal-allergy.at

Piazzale Asia, 21 Scala B, Piano 4, Interno 21 00144 Roma Tel.: +39-06-97243570 www.halallergy.it

J.H. Oortweg 15-17 2333 CH Leiden Tel: +31-(0)88-1959 000 www.hal-allergy.com

Benelux HAL Allergy Benelux BV J.H. Oortweg 15-17 2333 CH Leiden Tel. NL: +31-(0)88-1959 140 Tel. BE: +32-(0)2-5278380 www.hal-allergy.nl www.hal-allergy.be

Germany HAL Allergie GmbH PoststraĂ&#x;e 5-6 40213 DĂźsseldorf Tel.: +49-(0)211-977650 www.hal-allergie.de

Poland HAL Allergy Sp. z o.o.

Ul. Rumiana 65 02-956 Warschau Tel.: +48-22-8581614 www.hal-allergy.pl

Spain HAL Allergy S.L.U. Parque Empresarial Mas Blau II Avda. Les Garrigues, 46 08820-El Prat de Llobregat, Barcelona Tel.: +34-902-110-686 www.halallergy.es

HAL Allergy distributors Greece Alfamedica S.A. 22 Katechaki Street 11525 Athens Tel.: +30-210-6728318

Portugal A.M.D. Passos, Lda. Edif. República 2º Piso Escritório X Avenida da República 2645-143 Alcabideche Tel.: +351-21-4578087

Slovenia IRIS Mednarodna Trgovina D.O.O. Cesta v Gorice 8 1000 Ljubljana Tel.: +386-1-2006684 www.iris.si

Rising to the c hal lenge of improving allergy treatments

www.hal-allergy.com

Š HAL Allergy 2014 | MAB 48091-04