HAL Allergy Abstractbook EAACI 2013

Scientific Contribution 2013 32 nd Congress of the European Academy of Allergy and Clinical Immunology & World Allergy O rganization, World Allergy & Asthma Congress 2013 EAACI WAO, 22-26 June 2013, Milan, Italy

Dear

Congress delegate,

Diderik Boot, PhD Medical Director

On behalf of HAL Allergy we would like to welcome you to the EAACI & WAO World Allergy and Asthma congress in Milan. This joint congress of the European Academy of Allergy and Clinical Immunology and the World Allergy Organization promises to be the largest congress of allergists and related specialists in the world in 2013. HAL Allergy, being one of Europe’s top players in the field of allergen vaccination, is proud to be able to present our R&D work for such a large and international audience.

In this abstract book you will find all our abstracts dealing with clinical, preclinical and development work, including information on specific dates and times of presentations.

HAL Allergy’s clinical development program is progressing at full speed to meet today’s requirements for gaining registration for specific immunotherapy products. We have recently completed the clinical phase of three large scale dose range finding studies with PURETHAL® Mites, SUBLIVAC® Birch and SUBLIVAC® Phleum. The results are currently under analysis and this year we will present some baseline findings. In addition we will present clinical data on the use of PURETHAL® pollen in daily practice.

Dirk-Jan Opstelten, PhD Research & Development Director

At this year’s meeting we will present pre-clinical data in a mouse model proving the efficacy of our peanut SCIT product candidate. The mouse model data show that both modification of peanut allergen extract and adsorption to Al(OH)3 have a positive effect on the safety of the SCIT product whilst efficacy is retained. Based on these new data we intend to further pursue the development of the peanut SCIT product.

Furthermore, we will present various abstracts on the characterisation and quantification of our allergen products, including our chemically modified allergens, so-called allergoids. We have developed antibody based assays as well as a physicochemical method (HPLC) to measure the major allergen content in our VENOMENHAL® products and SUBLIVAC® grass pollen products.

We hope you will have a fruitful congress. Please visit our booth in the exhibition area 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 Safety of maintenance dose subcutaneous immunotherapy: the occurrence of local and systemic reactions in patients with seasonal allergic rhinitis during and outside the pollen season . . . . . . . . . . . . . . . . . . . . . . . . . .

6

Correlation of symptom score and peak nasal inspiratory flow following a standardized titrated nasal provocation test with a birch pollen extract in a multi-centre clinical study in allergic rhinitis. . . . . . . . . . . . . . . . . .

Nasal provocation, skin reactivity and allergen-specific serum IgE levels in allergic rhinitis . . . . . . . . . . . . . . . . . . . . . .

8 10

Pre-Clinical Safety and efficacy of immunotherapy with native or allergoid Ara h 2/6 in a peanut allergy mouse model .. . . . . . .

12 14

Alum adjuvant in birch pollen immunotherapy decreased body drop temperature but did not contribute to the adaptive immune response in mice. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

16

A novel peanut allergoid is safe and effective in a murine immunotherapy model for peanut allergy . . . . . . . . . . . . .

Development An Inhibition ELISA for the determination of major allergen group 1 in different grass species . . . . . . . . . . . . . . . . . . .

An Inhibition ELISA method for the determination of potency of tree pollen preparations . . . . . . . . . . . . . . . . . . . . . .

A quantitative ELISA for determining Antigen 5 content in wasp venom . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Quantification of bee allergen Api m 1 in bee venom by HPLC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Characterisation of Parietaria pollen allergoids with physicochemical techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

18 20 22 24 26

Safety of maintenance dose subcutaneous immunotherapy: the occurrence of local and systemic reactions in patients with seasonal allergic rhinitis during and outside the p ollen season A. Distler (1), N. van Os (1), P. Bubel (2), U. Neumann (3), N. Angelova (4), H. Nienhuis (4), J.D. Boot (4).

(1) HAL Allergie GmbH, Medical Department, Düsseldorf, Germany, (2) ENT Cooperative Sachsen-Anhalt, Eisleben, Germany, (3) ENT Cooperative Sachsen-Anhalt, Wolmirstedt, Germany, (4) HAL Allergy BV, Leiden, Netherlands.

Background During the pollen season an increase in the severity of local and systemic reactions due to the increase in allergen exposure has been documented. As a result, dose reduction during the pollen season is advised in the leaflet of many subcutaneous allergen immunotherapy (SCIT) products and is often applied in clinical practice. The product under investigation is a suspension of a glutaraldehyde-modified allergen extract from pollen adsorbed onto aluminum hydroxide (allergoid). Such preparations are considered safer than non-modified allergen extracts and as a result dose reduction is not required during the pollen season. The current non interventional study in adults and children aimed to investigate if there is a difference in local and systemic reactions during and outside the pollen season when the maintenance dose is kept unchanged.

Methods Subjects with a history of allergic rhinoconjunctivitis with or without mild concomitant asthma due to pollen were treated with SCIT pollen allergoids for approximately 1 year, according to onsite routine. Patients received SCIT with the highest recommended maintenance dose (0.5 ml). At each visit, local (wheal and flare size) and systemic reactions were recorded.

Patients filled in a diary after every injection. Before and after treatment the patient was asked to judge his/her allergy symptoms on a VAS scale. The duration of the respective pollen season was based on local pollen counts.

Results 233 patients (105 female, mean age 33 years, 44 children) were included in the study analysis. 44.5% received grasses, 43.2% birch or trees, and 12.3% received a combination of grasses and trees.

The number of injections during maintenance dose was 3.168 (617 during and 2.551 outside the pollen season). The average wheal size was 9.8 and 8.5 mm inside and outside the pollen season, respectively. The average flare size was 18.9 and 23.3 mm inside and outside the pollen season, respectively. 53% of the patients reported an adverse event (AE). The vast majority (92.9%) was injection site related. No severe systemic AE (grade 2 – 4) occurred. The decrease in overall, lung, eye and nose symptoms compared to baseline was – 45.3%, - 16.7%, – 48.7% and - 45.2%, respectively.

Conclusion Maintenance dose SCIT with pollen allergoids during the pollen season didn’t show more local and systemic reactions compared to injections outside the pollen season. In addition, 1 year of treatment resulted in improvements in allergic symptoms.

Topics: Immunotherapy vaccines Presentation preference: Oral presentation

EAACI WAO, 22-26 June 2013, Milan, Italy Abstract number: 1301, Session date and time: Monday 24 June; 12:15 - 13:45 Session title: Developments in clinical research in allergen-specific immunotherapy

6

Developments in clinical research in allergen-specific immunotherapy

Safety of maintenance dose subcutaneous immunotherapy: the occurrence of local and systemic reactions in patients with seasonal allergic rhinitis during and outside the p ollen season

7

Methods: Subjects with a history of allergic rhinoconjunctivitis with or without mild concomitant asthma due to pollen were treated with SCIT pollen allergoids (PURETHAL ®, HAL Allergy BV, Leiden) for approximately 1 year, according to onsite routine. Patients received SCIT with the highest

617 165 (26.7%) 148 (24.0%)

Number of observations (injections) Number of injections with flare size > 0 mm Number of injections with wheal size > 0 mm

In season

577 (22.6%)

690 (27%)

2,551

Outside season

Table 2: Number of observations and local reactions

236 105 (44.5%) 102 (43.2%) 29 (12.3%) Prescribed allergen - Grasses - Birch/Trees - Birch/Trees + Grasses

5.3 – 80.4

Minimum - Maximum

44

33.0 ± 16.6

Mean age ± SD in years

Children & Adolescents

128 / 105

Gender (M / F)

Total (n = 233)

Table 1: Patient demographics and administered allergens

Allergie GmbH, Medical Department, Düsseldorf, Germany; 2ENT-Association Sachsen-Anhalt, Germany; 3HAL Allergy BV, Leiden, Netherlands

Background & Aim: During the pollen season an increase in the severity of local and systemic reactions due to the increase in allergen exposure has been documented. As a result, dose reduction during the pollen season is advised in the leaflet of many subcutaneous allergen immunotherapy (SCIT) products and is often applied in clinical practice. The product under investigation is a suspension of a glutaraldehyde-modified allergen extract from pollen adsorbed onto aluminum hydroxide (allergoid). Such preparations are considered safer than non-modified allergen extracts and as a result dose reduction is not required during the pollen season. The current non interventional study in adults and children aimed to investigate if there is a difference in local and systemic reactions during and outside the pollen season when the maintenance dose is kept unchanged.

1HAL

A. Distler 1, N. van Os1, P. Bubel 2, U. Neumann 2, N. Angelova 3, H. Nienhuis 3, J.D. Boot 3

1301 - Safety of maintenance dose SCIT: the occurrence of local and systemic reactions in patients with seasonal allergic rhinitis during and outside the pollen season

725 (22.9%)

855 (26.9%)

3,168

Total

Overall

Eye

Nose

Lung

after therapy

before therapy

3 (1%) 5 (2%) 5 (2%) 1 (0.4%) 5 (2%) 2 (0.9%) 3 (1%) 5 (2%)

35 (15%) 11 (5%) 13 (6%) 20 (9%) 99 (42%) 30 (13%) 9 (4%) 29 (12%) 3 (1%)

Number of patients

EAACI WAO Congress 2013 EAACI Congress 2013

Figure 1: Patients’ assessment of symptoms during the pollen season before and after one year of SCIT treatment

-10

10

30

50

70

90

Local reactions Injection site erythema Injection site haematoma Injection site induration Injection site pain Injection site pruritus Injection site swelling Injection site urticaria Injection site warmth Oedema peripheral Systemic reactions Rhinitis Rhinorrhoea Sneezing Urticaria Eye pruritus Fatigue Paraesthesia Headache

MedDRA Preferred Term

Table 3: Overview of most frequent adverse reactions

In relation to this presentation, I declare the following, real or perceived conflicts of interest: the presenter is an employee of HAL Allergy.

In relation to this presentation, I declare the following, real or perceived conflicts of interest:

Conclusion: Maintenance dose SCIT with pollen allergoids (0.5 ml) during the pollen season didn’t show more local and systemic reactions compared to injections outside the pollen season. In addition, 1 year of treatment resulted in improvements in allergic symptoms.

Results: 233 patients were included in the study analysis (Table 1).

The prescribed pollen allergens are listed in Table 1.

The number of injections during maintenance dose was 3,168 (617 during and 2,551 outside the pollen season).

Average number of injections per patient was 14 4.5.

The average wheal size was similar inside and outside the pollen season (9.8 and 8.5 mm, respectively). The average flare size was also similar inside and outside the pollen season (18.9 and 23.3 mm, respectively).

53% of the patients reported an adverse event (AE, Table 3). The vast majority (92.9%) was injection site related. No severe systemic AEs (grade 2 – 4) were reported. Patients didn’t stop the SCIT treatment due to local or systemic AE. In rare cases the patient used an antihistamine tablet for treatment of AEs. Local treatment was done by cooling.

After 1 year of treatment the decrease in overall, eye, nose and lung symptoms compared to baseline was -45.3%, -48.7% and -45.2% and -16.7%, respectively (Figure 1).

recommended maintenance dose (0.5 ml). At each visit, local (wheal and flare size) and systemic reactions were recorded. Patients filled in a diary after every injection.

Before and after one year of treatment the patient was asked to judge his/her allergy symptoms on a visual analogue scale (VAS). The duration of the respective pollen season was based on local pollen counts.

VAS [mm] ± SD

Correlation of symptom score and peak nasal inspirator y f low following a standardized titrated nasal provocation test with a birch p ollen extract in a multi-centre clinical study in allergic rhinitis O. Pfaar (1), E. van Twuijver (2), J.D. Boot (2), L. Klimek (1), H. Nienhuis (2), Z. Diamant (3), P. Kuna (4), P. Panzner (5).

(1) Center for Rhinology and Allergology Wiesbaden, Department of Otorhinolaryngology, Head and Neck Surgery, University Hospital Mannheim, Germany, (2) HAL Allergy BV, Medical Department, Leiden, The Netherlands, (3) Skane University Hospital, Lund, Sweden and University Medical Centre Groningen, Groningen, The Netherlands, (4) Poradnia Alergologii i Chorób Płuc Uniwersytecki Szpital Kliniczny im.

N. Barlickiego, Lodz, Poland, (5) Dept. of Allergology and Immunology, Medical Faculty in Plzen, Charles University Prague, Czech Rep.

Background The titrated nasal provocation test (TNPT) can be used in the diagnosis of allergic rhinitis (AR) and as a primary endpoint for clinical efficacy in phase II trials. In a multi-centre dose tolerability/dose-range-finding (DT/DRF) study, a standardized TNPT was performed to assess the upper airway response to allergen-specific provocation. The upper airway response was determined by symptom scores combined with peak nasal inspiratory flow (PNIF) measurements.

Methods Patients with suspected birch pollen-induced AR were screened prior to enrollment into a multi-centre DT/DRF study with birch pollen sublingual immunotherapy. Patients with a positive skin prick test to birch pollen and positive serum specific anti-birch IgE-test underwent a standardized TNPT. Intranasal application of a diluent followed by up to 3 sequential concentrations of a standardized allergen extract (Betula verrucosa 100, 1,000 and 10,000 AU/ml, 1 puff per nostril) was performed at 20 minutes intervals. Fifteen minutes after each application the upper airway response was quantified by a composite symptom score according to Lebel in combination with a PNIF measurement. TNPT was considered positive if a symptom score ≥6 was reached. Symptom scores and PNIF were repeated 1 hour after the last allergen application.

Results A total of 317 patients was screened, 274 patients had a positive TNPT, and for 245 patients (120M/125F) both symptom scores and PNIF results were available. The most prominent symptoms were nasal obstruction and pruritus, followed by rhinorrhoea and sneezing. A consistent dose-response relationship was observed between sequential allergen concentrations and increasing symptom scores, which were associated with a decrease in PNIF. A significant correlation was demonstrated between symptom scores and PNIF(r=-0.51, p<0.0001).

Conclusion In a multi-centre trial, a standardized TNPT with a Betula verrucosa extract induced a consistent increase in allergic symptoms which is associated with a decrease in PNIF in patients with suspected birch pollen-induced AR. These results are in line with previous reported results with an HDM extract and indicate that the TNPT can be used with both seasonal and perennial allergens.

Table: Symptom scores and PNIF (mean ± SD) following TNPT with birch pollen allergen.

Timepoint

Pre Diluent

Post Diluent

100 AU/ml

1,000 AU/ml

10,000 AU/ml

1 hour post TNPT

Lebel Score

0.1 ± 0.3

0.3 ± 0.6

3.9 ± 2.3

7.0 ± 2.3

8.2 ± 1.1

2.5 ± 2.0

PNIF (L/min)

139 ± 47

136 ± 49

107 ± 45

80 ± 43

71 ± 44

111 ± 46

EAACI WAO, 22-26 June 2013, Milan, Italy Abstract number: 400, Session date and time: Tuesday 25 June; 13:30 - 15:00 Session title: Clinical rhinology

8

Clinical rhinology

Correlation of symptom score and peak nasal inspirator y f low following a standardized titrated nasal provocation test with a birch p ollen extract in a multi-centre clinical study in allergic rhinitis

9

Methods: Patients: M/F aged 18-60 years with birch pollen-induced AR, with or without clinically stable asthma (FEV 1 >70% predicted) were screened prior to enrollment into a multicentre DT/DRF study with birch pollen sublingual immunotherapy. Patients with a positive skin prick test to birch pollen and positive serum specific anti-birch IgE-test underwent a standardized TNPT. Methods: The TNPT started with the allergen’s diluent followed by maximally 3 sequential concentrations of a standardized allergen extract (Betula verrucosa 100, 1,000 and 10,000 AU/ml, HAL Allergy BV, Leiden, The

Background & Aim: The titrated nasal provocation test (TNPT) can be used in the diagnosis of allergic rhinitis (AR) and as an endpoint for efficacy in clinical trials. In a multi-centre dose tolerability/dose-range-finding (DT/DRF) study, a standardized TNPT was performed to assess the upper airway response to allergen-specific provocation. The upper airway response was determined by symptom scores combined with peak nasal inspiratory flow (PNIF) measurements.

7.9 ± 2.9 (3.0 - 20.0) 31.5 ± 27.4 (0.9 - 99.3) (n=111)

9.1 ± 3.5 (4.0 - 20.0) 32.8 ± 26.6 (1.0 – 99.6) (n=104)

SPT to birch pollen extract in mm ± SD (range) Birch pollen specific IgE in kU/l (range)

Runny Nose

Sneezing

245

242

Post Diluent

243

100 AU/ml

203

1,000 AU/ml

68

10,000 AU/ml

Total

0.2 ± 0.5

238

Post Challenge

(n=215)

32.1± 26.9 (0.9 - 99.6)

8.5 ± 3.3 (3.0 - 20.0)

36 ± 10 (19-59)

245 (100%)

Mean ± SD 0.0 ± 0.1 0.0 ± 0.2 0.7 ± 1.0 1.5 ± 1.2 1.8 ± 1.2

N

Pre Diluent

Timepoint

37 ± 10 (19-59)

35 ± 10 (19-59)

Mean age ± SD in years (range)

Table 2: Symptom scores (Lebel score)

125 (51.0%)

Female 120 (49.0%)

Number (%)

Male

Table 1: Age and gender distribution

for Rhinology and Allergology Wiesbaden, Department of Otorhinolaryngology, Head and Neck Surgery, University Hospital Mannheim, Germany, Allergy BV, Leiden, The Netherlands, 3Skane University Hospital, Lund, Sweden and University Medical Centre Groningen, Groningen, The Netherlands, 4Poradnia Alergologii i Chorób P uc Uniwersytecki Szpital Kliniczny im. N. Barlickiego, Lodz, Poland, 5Dept. of Allergology and Immunology, Medical Faculty in Plzen, Charles University Prague, Czech Rep.

2HAL

1Center

O. Pfaar1, E. van Twuijver2, J.D. Boot2, L. Klimek1, H. Nienhuis2, Z. Diamant3, P. Kuna4, P. Panzner5.

400 - Correlation of symptom score and peak nasal inspiratory flow following a standardized titrated nasal provocation test with a birch pollen extract in a multi-centre clinical study in allergic rhinitis

244

244

203

71

245

244

244

203

71

245

244

244

203

71

245

244

244

203

68

Post Diluent

50

100

150

200

2.5 ± 2.0

245

0.6 ± 0.8

245

1.0 ± 0.9

245

1,000 AU/ml 10,000 AU/ml Post Challenge

Provocation

100 AU/ml

Mean ± SD 0.1 ± 0.3 0.3 ± 0.6 3.9 ± 2.6 7.0 ± 2.3 8.2 ± 1.1

N

Mean ± SD 0.0 ± 0.2 0.0 ± 0.2 1.0 ± 0.9 1.7 ± 0.9 2.0 ± 0.9

N

Mean ± SD 0.0 ± 0.2 0.1 ± 0.3 1.2 ± 1.0 2.1 ± 0.9 2.4 ± 0.9

N

0.7 ± 0.7

245

EAACI WAO Congress 2013

Figure 1: Concentration response curves with mean (± 95% CI) changes in symptom scores (Lebel score, red dots) and PNIF measurements (blue squares) following TNPT with a birch pollen extract.

Pre Diluent

0

2

4

6

8

10

245

Mean ± SD 0.0 ± 0.1 0.1 ± 0.3 1.0 ± 0.7 1.6 ± 0.5 1.8 ± 0.5

N

In relation to this presentation, I declare the following, real or perceived conflicts of interest: the presenting author is a consultant of HAL Allergy.

Conclusion: In a multi-centre trial, a standardized TNPT with a Betula verrucosa extract induced a consistent increase in allergic symptoms which is associated with a decrease in PNIF in patients with suspected birch pollen-induced AR. These results are in line with previous reported results with an HDM extract and indicate that the TNPT can be used with both seasonal and perennial allergens.

Results: A total of 317 patients was screened, 274 patients had a positive TNPT, and for 245 patients (120M/125F; Table 1) both symptom scores and PNIF results were available. The most prominent symptoms were nasal obstruction and pruritus, followed by rhinorrhoea and sneezing (Table 2). A consistent dose-response relationship was observed between sequential allergen concentrations and increasing symptom scores, which were associated with a decrease in PNIF. A significant correlation was demonstrated between symptom scores and PNIF (Figure 1; r=-0.51, p<0.0001).

12

Total score

Pruritis

Nasal Obstruction

Total Lebel Score

Netherlands) administered by a nasal spraying device (1 puff per nostril) at 20 minutes intervals. The upper airway response was quantified by a composite symptom score according to Lebel and PNIF measurements (highest value of 3 measured by In-check nasal inspiratory flow meter [Clement Clarke, Harlow, UK] at baseline (pre-diluent), 15 minutes after each administration and 60 minutes postchallenge. Symptoms were recorded using the following scoring system: sneezes ≤2= 0, sneezes 3-4= 1 point, sneezes ≥5= 2 points; anterior rhinorrhea= 1 point, posterior rhinorrhea= 1 point; difficult breathing= 1 point, 1 blocked nostril= 2 points, 2 blocked nostril= 3 points; nasal pruritus= 1 point, pruritus in palate or ear= 1 point, conjunctivitis = 1 point (total score range: 0-11 points). The pre-diluent was not allowed to be ≥3. The TNPT was considered positive once a total score ≥6 was reached.

PNIF (L/min)

Nasal provocation, skin reactivity and allergenspecific serum IgE levels in allergic rhinitis

M.J. Nell (1), J.D. Boot (1), H.E. Nienhuis (1), E. van Twuijver (1), O. Pfaar (2), C. Bachert (3).

(1) HAL Allergy BV, Leiden, The Netherlands, (2) Center for Rhinology and Allergology Wiesbaden, Germany, (3) UZ Gent, Ear, Nose and Throat Department, Gent, Belgium.

Background The diagnosis of allergic rhinitis (AR) is normally based on the measurement of allergen-specific serum IgE levels, skin prick testing and/or provocation testing. In a couple of studies with grass or birch pollen allergic patients, the relation between these diagnostic parameters has been explored. The results were contradictory and no clear relationship between these parameters could be demonstrated. We examined a possible relationship between these parameters in birch or house dust mite (HDM) allergic patients.

Methods Patients with suspected birch or HDM-induced AR were screened prior to enrollment into two separate multicentre studies with birch or HDM immunotherapy. Patients with a positive medical history, a positive skin prick test (SPT) to birch pollen or HDM, and a specific serum IgE (sIgE) level to birch pollen or HDM of > 0.7U/ml underwent a standardized titrated nasal provocation test (TNPT) using 100, 1,000 and 10,000 AU/ml of a birch pollen or HDM allergen extract. The concentrations eliciting a positive response (i.e. provocative dose) were compared with the sIgE levels and the outcomes of the SPT.

Results 317 patients were screened for the birch pollen immunotherapy study and 425 for the HDM study. All three diagnostic parameters were positive for 244 patients (119M/125F, mean age 36 year) in the birch study and for 287 patients (142M/145F, mean age 31 years) in the HDM study. For the birch pollen allergic patients the SPT and sIgE results were comparable between the 3 provocation concentrations (see Table 1). The results in HDM allergic patients, on the other hand, showed a decrease in sIgE level at the provocative dose of 10,000 AU/ml. For the SPT outcome in HDM allergic patients no difference was observed.

Conclusion No apparent relationship could be demonstrated between the three diagnostic parameters for both birch pollenâ&#x20AC;&#x201C; and HDM allergic patients. From our results it appears that the outcome in neither the SPT nor the sIgE levels is predictive for the provocative dose in the TNPT.

Table 1: Results from the 3 diagnostic parameters in birch pollen and in HDM allergic patients.

Provocative dose (AU/ml) SlgE (U/ml) Median (range) SPT diameter (mm) Median (range)

Birch pollen (n=244)

100

1,000

10,000

100

1,000

10,000

28.4 (1.0-99.6)

23.0 (0.9-99.3)

22.8 (1.2-96.5)

29.1 (0.8-280)

32.5 (0.8-532)

17.6 (0.7-504)

8.0 (3-20)

8.0 (3-20)

8.0 (3-20)

8.0 (3-18)

7.0 (3-18)

7.0 (3-21)

EAACI WAO, 22-26 June 2013, Milan, Italy Abstract number: 541, Session date and time: Monday 24 June; 10:45 - 12:15 Session title:Â New developments in the treatment of allergic rhinitis

10

HDM (n=287)

New developments in the treatment of allergic rhinitis

Nasal provocation, skin reactivity and allergen-specific serum IgE levels in allergic rhinitis

11

Methods: Patients with suspected birch or HDM-induced AR were screened prior to enrollment into two separate multicentre studies with birch or HDM immunotherapy. Patients with a positive medical history, a positive skin prick test (SPT) to birch pollen or HDM, and a specific serum IgE (sIgE) level

Background & Aim: The diagnosis of allergic rhinitis (AR) is normally based on the patients medical history in combination with several diagnostic tests. The measurement of allergen-specific serum IgE levels and skin prick testing can confirm the presence of atopy. Nasal provocation testing can confirm the sensitivity to specific allergic triggers. In several studies with grass or birch pollen allergic rhinitis patients, the relation between these diagnostic parameters has been explored.

The results were contradictory and no clear relationship between these parameters could be demonstrated. We examined a possible relationship between these parameters in a large population of birch or house dust mite (HDM) allergic patients.

300 250 200 150 100 50

200 150 100 50

22.8 (1.2– 96.5) 8.0 (3–20)

23.0 (0.9– 99.3) 8.0 (3–20)

250

10,000

1,000

300

Provocative dose 100 (AU/ml) sIgE (U/ml) 28.4 Median (range) (1.0– 99.6) 8.0 SPT diameter (mm) (3–20) Median (range)

Birch pollen (n=244)

29.1 (0.8– 280) 8.0 (3–18)

100

32.5 (0.8– 532) 7.0 (3–18)

1,000

HDM (n=287)

17.6 (0.7– 504) 7.0 (3–21)

10,000

Table 1: Results from the 3 diagnostic parameters in birch pollen and HDM allergic patients

1 HAL Allergy BV, Leiden, The Netherlands, 2Center for Rhinology and Allergology Wiesbaden, Department of Otorhinolaryngology, Head and Neck Surgery, University Hospital Mannheim, Germany, 3UZ Gent, Ear, Nose and Throat Department, Gent, Belgium

M.J. Nell1, J.D. Boot1, H.E. Nienhuis1, E. van Twuijver1, O. Pfaar2, C. Bachert3

541 – Nasal provocation, skin reactivity and allergenspecific serum IgE levels in allergic rhinitis

IgE (U/ml)

study

Birch

1,000 AU/ml Provocation

10,000 AU/ml

0

100 AU/ml

4 3 2 1 0

3 2 1 0

Birch

5

4

study

6

5

study

Mites

1,000 AU/ml Provocation

10,000 AU/ml

study

Mites

1,000 AU/ml Provocation

10,000 AU/ml

7

8

9 10 11 12 13 14 15

0

1

2

4

5

6

7

Mites

9 10 11 12 13 14 15

Birch

8

Diameter SPT (mm) study

3

Provocation = 1,000 AU/ml

0

50

100

150

200

250

300

0

1

2

3

4

5

6

7

8

9 10 11 12 13 14 15

Diameter SPT (mm)

Provocation = 10,000 AU/ml

EAACI WAO Congress 2013

Figure 3: Relationship between sIgE levels and SPT diameter for the different provocative doses in the birch pollen and the HDM study

6

0 5

0

4

50

50

Diameter SPT (mm)

100

100

3

150

150

2

200

200

1

250

250

0

300

300

Provocation = 100 AU/ml

Figure 2: Relationship between SPT diameter and the provocative dose in the birch pollen and the HDM study ( represents the median)

100 AU/ml

7

6

10,000 AU/ml

8

7

1,000 AU/ml Provocation

9

8

100 AU/ml

11

9

12 10

12 11

13

13

10

15 14

15 14

In relation to this presentation, I declare the following, real or perceived conflicts of interest: the presenter is an employee of HAL Allergy.

Conclusion: This study was performed in a large well characterized study population and diagnostic tests were completely standardized. No apparent relationship could be demonstrated between the three diagnostic parameters for both birch pollen and HDM allergic patients. From our results it appears that the outcome in neither the SPT nor the sIgE levels is predictive for the provocative dose in the TNPT and confirms the importance of the TNPT to define the sensitivity of AR patients.

Results: 317 patients were screened for the birch pollen immunotherapy study and 425 for the HDM study. All three diagnostic parameters were positive for 244 patients (119M/125F, mean age 36 year) in the birch study and for 287 patients (142M/145F, mean age 31 years) in the HDM study. For the birch pollen allergic patients the SPT and sIgE results were comparable between the 3 provocation concentrations (see Table 1). The results in HDM allergic patients, on the other hand, showed a (non significant) decrease in sIgE level at the provocative dose of 10,000 AU/ml. For the SPT outcome in HDM allergic patients no difference was observed. In Figures 1 to 3 the different relationships are presented.

0

100 AU/ml

Figure 1: Relationship between sIgE levels and provocative dose in the birch pollen and the HDM study ( represents the median)

Diameter SPT (mm)

to birch pollen or HDM of > 0.7U/ml underwent a standardized titrated nasal provocation test (TNPT) using 100, 1,000 and 10,000 AU/ml of a birch pollen or HDM allergen extract. The concentrations eliciting a positive response (i.e. provocative dose) were compared with the sIgE levels and the outcomes of the SPT. A possible relationship was analysed with the Kendall's Tau test.

IgE (U/ml)

A novel peanut allergoid is safe and effective in a murine immunotherapy mo del for peanut allergy

H. van der Kleij (1), H. Warmenhoven (1), D. Opstelten (1), R. Pieters (2), Monique Visser (1), J. Smit (2) (1) HAL Allergy BV, Leiden, The Netherlands, (2) Institute for Risk Assessment Sciences, Immunotoxicology, Utrecht University, Utrecht, The Netherlands

Background Peanut allergy accounts for the majority of severe food-related allergic reactions. Since commonly used allergen-specific immunotherapy has not been successful due to the high risk of serious side-effects, there is a need for new treatment strategies. Chemically modified allergen extracts with improved safety characteristics are being investigated for its potential use in immunotherapy.

Methods Peanut extract (PE) from de-fatted peanut powder was modified by reduction of disulfide bonds and subsequent alkylation of the free Cys residues resulting in an allergoid PE (mPE) followed by adsorption to aluminum hydroxide (alum) if applicable. The potency of PE and mPE to induce PE-specific IgG was evaluated after i.p. injections in mice. In addition, mice were sensitized intra-gastrically for PE and either 1) s.c. challenged with different concentrations of (m)PE +/- various alum concentrations to assess the safety profile of these product candidates, or 2) de-sensitized by s.c. injections of either PE or mPE +/- alum for 3-6 weeks (immunotherapy), followed by oral and i.p. challenges to assess the efficacy profile of the preparations. Body temperature was measured after challenge as an objective parameter of an anaphylactic shock response.

In addition, during the course of immunotherapy, blood samples were taken for analysis of antibody responses and mast cell activation.

Results Mice sensitized for PE experienced severe anaphylactic symptoms upon s.c. challenge with PE. These effects were aborted after complete binding of PE to alum. Modified PE (no alum) also did not give rise to anaphylactic reactions, even when given up to 30 fold higher dosages. PE and mPE were equally potent in inducing PE-specific IgG antibodies in mice. Immunotherapy with both PE and mPE (+/- alum) resulted in a significant dose-dependent reduction of the anaphylactic response upon systemic challenge. In addition, both PE and mPE (+/- alum) were able to induce strong increases in the levels of PEspecific IgG1 and IgG2a compared to non-desensitized mice. Surprisingly, the mucosal mast cell response after challenge was decreased after immunotherapy with PE but not with mPE, independent of the binding to alum.

Conclusion Using in vivo mouse models, we have shown that a peanut allergoid preparation adsorbed to alum has a significantly improved safety profile compared to its native counterpart while retaining its immunogenicity and efficacy profile.

EAACI WAO, 22-26 June 2013, Milan, Italy Abstract number: 218, Session date and time: Sunday 23 June; 10:30 - 12:00 Session title:Â Novel mechanisms in allergen-specific immunotherapy: new molecules and new routes

12

Novel mechanisms in allergen-specific immunotherapy: new molecules and new routes

A novel peanut allergoid is safe and effective in a murine immunotherapy mo del for peanut allergy

13

Results:

The Peanut extract (PE) preparation is chemically modified by reduction of the intramolecular disulfide bonds of PE, and alkylation of the resulting free sulfhydryl groups (mPE). After modification, the allergoid was adsorbed to aluminum hydroxide (alum).

Methods:

• Peanut allergy accounts for the majority of severe food-related allergic reactions.

• A curative treatment is not available.

• Modified peanut extract (mPE) is being investigated for its potential use in immunotherapy.

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A. Immunogenicity model; BALB/c mice are immunized with 10 or 100 µg product in 200 µl every other week. Control mice were injected with matrix only. Blood samples were taken in time to determine the immune response to PE and mPE. B. In vivo IgG responses cross-reactive with native PE are detected in BALB/c mice immunized with PE and mPE in time. Connecting lines represent the mean IgG response (+/- sem) in different groups.

C. Specific IgG titres at day 70 after 5 immunizations (i.p.) with 10 or 100 µg product.

Dots represent the IgG titre of individual mice in arbitrary units (AU).

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Figure 3: Body temperature following s.c. challenge; Anaphylaxis to the native preparation.

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A. Mice were sensitized with 6 mg PE and 15 µg Cholera Toxin via oral gavage and challenged (i.p.) on day 42 with PE (+/- alum) and mPE (+/- alum)(n=6 per group). B. Mice were sensitized with 6 mg PE and 15 µg Cholera Toxin via oral gavage. SCIT injections, starting at day 42, were given 2x a week for a total of 6 weeks. Mice were challenged by gavage (12 mg PE) on day 91 followed by 2 i.p. challenges with 0.1 mg PE on days 98 and 112.

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Figure 2: Animal models for safety (A) and efficacy (B)

IgG (AU*106)

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Figure 1: Specific IgG responses to native versus modified PE

(Celcius)

Background & Aim:

HAL Allergy BV, Leiden, The Netherlands, Institute for Risk Assessment Sciences and Utrecht Centre for Food Allergy, Utrecht, The Netherlands

IgG (AU)

2

1

H. van der Kleij1, H. Warmenhoven1, D. Opstelten1, R. Pieters2, M. Visser1, J. Smit2

218 - A novel peanut allergoid is safe and effective in a murine immunotherapy model for peanut allergy

elsius)

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EAACI WAO Congress 2013

SCIT with both PE and mPE induced significant amounts of PE-specific antibodies compared to the allergic group of mice that did not receive immunotherapy. The mucosal mast cell response after challenge was decreased after SCIT with PE but not with mPE.

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Figure 6: Effect of SCIT on the immune response on day 99

After sensitization, mice were s.c. challenge with various concentrations PE and mPE (+/- alum). SCIT with PE is more effective in reducing the anaphylactic shock response (A). Adsorption of mPE to alum tends to increase the efficacy of IT (B). A clear dose-response relation is observed for mPE when adsorbed to alum (C).

Temperature

A.

Temperature

Figure 5: Effect of SCIT on body temperature following s.c. challenge

After sensitization, mice were s.c. challenge with 0.6 mg PE adsorbed to various amounts of alum (increasing the % of unbound PE).

Complete binding of PE to alum aborted the potency of PE to elicit an anaphylactic shock as measured by temperature (A) and symptom scores (B) assigned on a scale from 0 (non symptoms) to 5 (death). Partial binding resulted in a dose-dependent shock response.

A. Clinical score

Figure 4: The influence of PE binding to aluminum hydroxide on body temperature following s.c. challenge

Temperature

In relation to this presentation, I declare the following, real or perceived conflicts of interest: the presenter is an employee of HAL Allergy.

Modification of PE followed by alum adsorption resulted in a hypo-allergenic product while maintaining as immunogenic and efficacious as its native counterpart PE.

Conclusion:

0

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After sensitization, s.c. challenge with 0.6 mg PE per mouse resulted in an anaphylactic shock response (A), while 3 mg mPE was still tolerated without problems (B). Control mice did not respond to challenge (data not shown).

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Safety: • Mice sensitized with PE experienced a severe anaphylactic reaction upon subcutaneous (s.c.) challenge with 0.1 mg PE, while 3 mg mPE was still tolerated without problems.

• Complete binding of PE to alum aborted the anaphylactic symptoms upon s.c. challenge • The mucosal mast cell response after challenge was decreased after SCIT with PE but not with mPE. Efficacy: • PE and mPE were equally potent in inducing PE-specific IgG antibodies.

• After adsorption to alum, SCIT with both PE and mPE effectively reduced the shock response of mice to challenge.

• SCIT with both PE and mPE (+/- alum) induced strong increases in the levels of PE-specific IgE, IgG1 and IgG2a.

IgG2a (AU)

Safety and efficacy of immunotherapy with native or allergoid Ara h 2/6 in a peanut allergy mouse mo del

J. Smit (1), R. Pieters (1), M. van Roest (1), L. Kruijssen (1), S. Koppelman (2), D. Opstelten (2), H. van der Kleij (2).

(1) Institute for Risk Assessment Sciences, Immunotoxicology, Utrecht University, Utrecht, The Netherlands, (2) HAL Allergy BV, Leiden, The Netherlands.

Background Peanut is one of the most common foods responsible for food-induced anaphylaxis in adults. Until now , a curative treatment is not available for peanut-allergic patients. Allergen-specific immunotherapy has not been successful for the treatment of food-allergy because of the high risk of serious side-effects. Therefore, chemically modified allergen extracts with improved safety characteristics are developed for immunotherapy.

Methods The major peanut allergens Ara h 2 and Ara h 6 (nAra h 2/6) were co-purified by extraction from de-fatted peanut powder and modified by reduction of disulfide bonds and subsequent alkylation of the free Cys residues (mAra h 2/6). Mice were sensitized intra-gastrically for nAra h 2/6 and treated repeatedly with subcutaneous injections of either nAra h 2/6 or mAra h 2/6 (immunotherapy). The efficacy of immunotherapy was tested by means of measuring allergy parameters after a series of systemic or local challenges.

Results First, when injected with alum, nAra h 2/6 and mAra h 2/6 were equally potent in inducing IgG antibodies in mice, showing that the modification did not affect immunogenicity. Second, mice sensitized for nAra h 2/6 reacted with strong anaphylactic symptoms after subcutaneous challenge with nAra h 2/6. Modified Ara h 2/6 did not give rise to such reactions, even when given up to 100 fold higher dosages. Third, immunotherapy with nAra h 2/6 reduced the anaphylactic reaction upon systemic challenge, and this could also be demonstrated for immunotherapy with mAra h 2/6 , although at higher dosages only. In addition, immunotherapy with nAra h 2/6 resulted in a temporary increase in levels of Ara h 2/6-specific IgG1, followed by a reduction in IgE serum levels. There was no significant effect in these antibodies after immunotherapy treatment with mAra h 2/6.

Conclusion Using in vivo mouse models, we have shown that an allergoid preparation of the peanut allergens Ara h 2 and 6 has a significantly improved safety profile compared to its native counterpart. This preparation has retained its immunogenicity but is significantly less efficient in lowering clinical allergic responses than native protein. This study supports the usefulness of mouse models in testing safety, immunogenicity and efficacy of new immunotherapeutic preparations.

EAACI WAO, 22-26 June 2013, Milan, Italy Abstract number: 1829, Session date and time: Sunday 23 June; 10:30 - 12:00 Session title: Novel mechanisms in allergen-specific immunotherapy: new molecules and new routes

14

Novel mechanisms in allergen-specific immunotherapy: new molecules and new routes

Safety and efficacy of immunotherapy with native or allergoid Ara h 2/6 in a peanut allergy mouse mo del.

15

Alum adjuvant in birch p ollen immunotherapy decreased bo dy drop temperature but did not contribute to the adaptive immune resp onse in mice

L.S. van Rijt (1), A. Logiantara (1), R. van Ree(1,2), D. Opstelten (3), H. van der Kleij (3).

(1) Academic Medical Center, Experimental Immunology, Amsterdam, The Netherlands, (2) Academic Medical Center, Otorhinolaryngology, Amsterdam, The Netherlands, (3) HAL Allergy BV, Leiden, The Netherlands.

Background Aluminum hydroxide (alum) is widely used in subcutaneous immunotherapy (SCIT) for more than 50 years. While the prevailing theory has been that adsorption of allergens to alum induces slow release of allergens, reduces adverse effects and increases effectiveness of treatment, this was not proven formally.

Methods In a mouse model for birch pollen-mediated allergic airway inflammation, mice were de-sensitised by subsequent subcutaneous injections with birch pollen extract alone or adsorbed to alum. As an objective parameter of a shock response, body temperature was measured after each subcutaneous injection. After the de-sensitisation phase, mice were challenged intranasally with birch pollen extract. Subsequently, the antigen-specific T cell response was determined and antigen-specific antibody responses were analysed.

Results The adsorption of the birch pollen extract to alum resulted in a significant reduction of the shock response as measure by a decreased drop in body temperature. The drop in body temperature became gradually less with subsequent SCIT injections in both groups. SCIT decreased the production of Th2 cytokines IL-5 and IL-13 and increased birch pollen specific IgG1 drastically. These effects were independent of the presence of alum in the preparation.

Conclusion This study showed that alum did not suppress the antigen specific T cell response nor was required for the induction of the humoral response. Alum did increase the safety profile of the preparation during the immunotherapy phase. This study underscores the need for more detailed research concerning the use of alum in immunotherapeutic preparations.

Furthermore, alternative adjuvants to optimise SCIT need to be explored.

EAACI WAO, 22-26 June 2013, Milan, Italy Abstract number: 1294, Session date and time: Monday 24 June; 12:15 - 13:45 Session title:Â Molecular biomarkers for allergen-specific immunotherapy

16

Molecular biomarkers for allergen-specific immunotherapy

Alum adjuvant in birch p ollen immunotherapy decreased bo dy drop temperature but did not contribute to the adaptive immune resp onse in mice

17

An Inhibition ELISA for the determination of major allergen group 1 in different grass species

E. Kerkvliet (1), N. Sinnige (1), H. Warmenhoven (1), J. Akkerdaas (2), R. van Ree (2) and R. van den Hout (1) (1) HAL Allergy BV, Leiden, The Netherlands, (2) Academic Medical Center, Amsterdam, The Netherlands.

Background Allergy induced by grass pollen can be reduced by means of sublingual allergen-specific immunotherapy (IT). One of the major allergens in grass pollen is grass group 1. Two antibody-based methods, a sandwich-ELISA and an inhibition ELISA, were developed for the quantification of grass group 1 in grass pollen extracts. Both methods were compared in order to determine the most appropriate assay for use of quantification of group 1 in a grass pollen IT product that consists of 4 different grass pollen extracts.

Methods The sandwich ELISA uses two specific monoclonal antibodies (7E7 and 1B8) against Lol p1 major allergen, one on the solid phase and one as detecting antibody. The inhibition ELISA (iELISA) uses one specific monoclonal antibody (7E7) that is mixed with grass pollen extract and recombinant Phleum p1 allergen on the solid phase. The products tested were pollen extracts of four different grass species, i.e. Phleum pratense, Lolium perenne, Poa pratensis and Secale cereale. The standards used are well characterized grass group 1 allergens (Phl p1, Lol p1, Poa p1 and Sec c1) purified from grass pollen extracts.

Results The sandwich ELISA showed different curves (e.g. non-parallel lines, different OD maxima) when grass pollen extracts from different grass species were analysed, while the iELISA showed similar curves for all four grass species. Further development of the sandwich-ELISA would involve separate development of 4 methods with different conditions, while only one method could be used with the iELISA for these extracts of 4 grass species. Therefore, the iELISA was chosen for further development and qualification. Qualification showed that the iELISA generates reproducible results with low variation.

Conclusion Quantification of grass group 1 major allergen in four different grass species can be done with one method using an iELISA. The sandwich ELISA was less suitable since different test conditions were needed when testing different grass species. The newly developed iELISA can precisely quantify group 1 allergen in pollen extracts from Phleum pratense, Lolium perenne, Poa pratensis and Secale cereale.

EAACI WAO, 22-26 June 2013, Milan, Italy Abstract number: 768, Session date and time: Sunday 23 June; 12:00 - 13:30 Session title:Â Tools for improving laboratory and clinical allergy diagnosis

18

Tools for improving laboratory and clinical allergy diagnosis

An Inhibition ELISA for the determination of major allergen group 1 in different grass species

19

An Inhibition ELISA metho d for the determination of p otency of tree p ollen preparations

D. van Deursen, I. Peekel, R. van den Hout, E. Kerkvliet.

HAL Allergy BV, Leiden, The Netherlands.

Background Widely used potency assays with specific reagents such as biotinylated allergen extracts, or assays using the allergen cap system, are relatively expensive and contain reagents that cannot be characterized. We developed an IgE inhibition ELISA, using only well characterized components, which is able to determine the IgE potency of pollen preparations from separate as well as from mixed tree species.

Methods 96-well plates are coated with extracts from Betula verrucosa, Alnus glutinosa or Corylus avellana pollen, or with a mixture of these extracts. After blocking, different dilutions of pollen samples are added to the coated plates followed by adding a sera pool obtained from patients with established clinical allergy for spring tree allergens. The tree pollen specific IgE will bind to the coated allergens or to the tree pollen allergens in the solution (competition). After washing away all unbound antibodies and proteins, bound IgE is quantitatively stained with HRP-conjugated anti-IgE. An in-house reference preparation is included in the assay and parallel-line analysis is performed to calculate potency. The pollen extracts tested are drug substances from birch, hazel or alder, and drug products containing spring tree mixtures. The results were compared to potency data from commercially available assays using a biotinylated ligand or an allergen caps system.

Results All samples tested showed linear inhibition curves with high correlations (r>0.99) and reproducible potency data (CVâ&#x2030;¤10%).

Furthermore, the potency data were comparable with the potency data obtained with the commercially available assays (difference<20%).

Conclusion The developed IgE inhibition ELISA is well suited for the use of potency determination of drug substances and products containing single species tree pollen preparations as well mixtures. Compared with the commercially available assays, the newly developed assay produces similar results and uses better characterized components.

Thematic area: 16 Laboratory tests for allergy

EAACI WAO, 22-26 June 2013, Milan, Italy Abstract number: 1143, Session date and time: Monday 24 June; 12:15 - 13:45 Session title:Â Novel aspects in allergy diagnosis

20

Novel aspects in allergy diagnosis

An Inhibition ELISA metho d for the determination of p otency of tree p ollen preparations

21

Coating microtiter plate

Adding tree pollen

Tree specific human IgE

Bound human IgE is

Staining with TMB and

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96-well plates are coated with extracts from Betula verrucosa, Alnus glutinosa or Corylus avellana pollen, or with a mixture of these extracts.

After blocking, different dilutions of pollen samples are added to the coated plates followed by adding a human sera pool obtained from patients with established clinical allergy for spring tree allergens. The spring tree pollen specific human IgE binds to the coated allergens or to the tree pollen allergens in the solution (competition). After washing away all unbound antibodies and proteins, bound IgE is quantitatively detected with HRP-conjugated anti-IgE. The amount of bound IgE is compared with a control in which a maximum amount of IgE is bound, this by incubating the sera pool without the presence of the sample of interest (E-max). The difference of incubated samples and E-max is indicated as percentage inhibition of the IgE preparation. As a reference, an in-house reference preparation (IHRP) is included in the assay and parallel-line analysis (PLA) is performed to calculate the relative potency (RP) of the test preparation with respect to the reference.

Methods:

Vaccines against tree pollen allergies are often based on complex mixtures of tree pollen allergens from a single species or from different tree species. We have developed an IgE inhibition ELISA (iELISA), which is able to determine the allergenic activity (potency) of pollen preparations from single tree species Betula verrucosa, Alnus glutinosa or Corylus avellana, as well as from mixture of these tree species.

Widely used IgE potency assays use commercially available reagents such as biotinylated allergen extracts, or use the allergen cap system, which are relatively expensive and contain reagents that cannot be characterised. Our assay uses components which are all well characterised and verifiable. Furthermore, the method is specific for the mentioned tree pollen allergens and has a high precision, and the costs of reagents are relative low.

Background & Aim:

HAL Allergy BV, Leiden, The Netherlands

D. van Deursen, I. Peekel, R. van den Hout, E. Kerkvliet

1143 - An Inhibition ELISA method for the determination of potency of tree pollen preparations

IgE inhibition (%)

samples together with sera from patients

binds to the tree pollen allergens of coat or solution detected with HRPconjugated anti-IgE antibody

Figure 1: Schematic overview of the iELISA assay for testing the mixture.

with Spring tree extracts (Betula, Alnus, Corylus)

detection at 450 nm data of % IgE inhibition. The RP is determined by PLA

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6.9 6.1 8.2 4.0

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Table 1: RP data of 4 different samples (DP= drug product) tested with the iELISA showing the intraassay variation (CV%) and mean RP values. The results of the iELISA are mean values from 3 runs.

Betula verrucosa IE Betula batch A IE Betula batch B DP Betula batch A DP Betula batch B Alnus glutinosa IE Alnus batch A IE Alnus batch B DP Alnus batch A DP Alnus batch B Corylus avellana IE Corylus batch A IE Corylus batch B DP Corylus batch A DP Corylus batch B Spring Trees DP Spring Tree mix batch A DP Spring Tree mix batch B

Test sample

2.59 1.75 0.80 0.80 1.2 1.1

1.01 0.98

2.24 1.82 0.90 1.10

1.17 1.56 1.00 1.00

RP Commercial assay

2.06 1.77 0.83 0.83

2.13 1.92 1.08 1.09

1.38 1.46 0.82 0.85

RP iELISA

Table 2: RP data of 14 different samples (IE= intermediate extract, DP= drug product) tested with the iELISA compared with historical data obtained with commercially available assays. The results of the iELISA are the mean from 3-4 independent experiments.

In relation to this presentation, I declare the following, real or perceived conflicts of interest: the presenter is an employee of HAL Allergy.

In relation to this presentation, I declare the following, real or perceived conflicts of interest: the presenter is an employee of HAL Allergy

EAACI WAO Congress 2013

EAACI Congress 2013

Conclusion: The developed IgE inhibition ELISA is well suited for the use of IgE potency determination of drug products and intermediates containing single species tree pollen preparations and mixtures. Compared with the commercially available assays, the newly developed assay produces similar results, shows better precision, and uses better characterized components.

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Figure 2: Inhibition curves of the IHRP and two different test preparation of Betula verrucosa.

The RP were determined of intermediate extracts (IE) and drug product (DP) from birch, hazel or alder pollen extracts, and drug products containing spring tree mixtures. On basis of the potency of IE the DP is formulated. All samples tested showed linear inhibition curves (see example in figure 2) with high correlations (r>0.99) and reproducible potency data with low intra- and inter-assay variation (CV<9% and CV<11% respectively). Furthermore, the RP data were comparable with the historical potency data obtained with the commercially available assays, the difference was within assay variation (difference<20%). The assay shows better precision than the commercially available assays.

Results:

The results of the iELISA were compared to historical data obtained with commercially available assays using a biotinylated ligand (for Betula verrucosa and Alnus glutinosa) or an allergen cap system (for Corylus avellana and the spring tree mixture). The principle of the method using commercially available biotinylated ligands is the competition between the allergen of interest from the tree extracts with the biotin-labelled allergen. With the allergen cap system a mixture of spring tree extract pre-incubated with serum is added to a cap containing a cellulose polymer coated with the concerning tree pollen allergen. IgE not bound to the product during pre-incubation, binds to the cap and is detected.

IgE inhibition (%)

A quantitative ELISA for determining Antigen 5 content in wasp venom

D. van Deursen (1), R. van den Hout (1), R. van Ree (2), E. Kerkvliet (1).

(1) HAL Allergy BV, Leiden, The Netherlands, (2) Experimental Immunology, Academic Medical Center, Amsterdam, The Netherlands.

Background Allergy induced by wasp venom can be treated by means of allergen-specific immunotherapy. For wasp venom, the main allergen reported is the protein Antigen 5, known by the allergen nomenclature as Ves v 5 (for Vespula vulgaris) or Ves g 5 (for Vespula germanica). We developed a major allergen ELISA and investigated the Antigen 5 content in different wasp venom products.

Methods 96-well plates are coated with monoclonal antibodies directed against Ves g 5. After blocking, dilution ranges of wasp venom preparations are added and detection is done with a polyclonal antibody from rabbit directed against Ves g 5.

Subsequently, incubation is performed with an HRP-conjugated goat-anti-rabbit antibody. The 96-well plates are stained with TMB and the concentration Antigen 5 is calculated by the use of the standard curve. The new method uses the 4-parameter non-linear regression model for determining the Antigen 5 content in test preparations. The standards used in the ELISA is Antigen 5 protein purified from wasp venoms, which was characterized and quantified by amino acid analysis, FPLC, SDS-PAGE and protein determination. Antigen 5 concentration was determined in one drug product and three different suppliers of raw materials.

Results The standards showed good 4-parameter dose response curves with high correlations (r>0.99) and the samples tested showed high intra- and inter assay precision (CVâ&#x2030;¤ 8%). The Antigen 5 content of the wasp venom batches tested was: 1.8% (drug product; Vespula spp.), 1.8% (supplier A; Vespula spp.), 1.7% (supplier B; Vespula spp.), 1.9% (supplier C; Vespula vulgaris) and 2.0% (supplier C; Vespula germanica). This is in accordance with data from the literature.

Conclusion The ELISA method is a suitable technique to quantify Antigen 5 content in different wasp venom products (raw materials and drug product).

Thematic area: 16 Laboratory tests for allergy

EAACI WAO, 22-26 June 2013, Milan, Italy Abstract number: 786, Session date and time: Sunday 23 June; 12:00 - 13:30 Session title:Â Molecules, chips and cells: new tools in allergy diagnosis

22

Molecules, chips and cells: new tools in allergy diagnosis

A quantitative ELISA for determining Antigen 5 content in wasp venom

23

HAL Allergy BV, Leiden, The Netherlands, 2 Experimental Immunology, Academic Medical Center, Amsterdam, The Netherlands

Bound polyclonal is

= Rabbit anti-Ves g5

= Anti-Ves g5 monoclonal

= Wasp venom

= TMB

= HRP-conjugate

Methods: 96-well plates are coated with monoclonal antibodies directed against Ves g 5. After blocking, dilution ranges of wasp venom preparations are added and detection is done with a polyclonal rabbit antibody directed against Ves g 5. Subsequently, incubation is performed with an HRP-conjugated goat-anti-rabbit antibody. The 96-well plates are stained with TMB and the concentration Antigen 5 is calculated by the use of an Antigen 5 standard curve. The new method uses the 4-parameter non-linear regression model for determining the Antigen 5 content in test preparations. The standard is a purified Antigen 5 protein from wasp venoms that was characterized by amino acid analysis, FPLC, SDS-PAGE and protein determination, and quantified by amino acid analysis.

Background & Aim: Allergy induced by wasp venom can be treated by means of allergen-specific immunotherapy. For wasp venom, the main allergen reported is the protein Antigen 5, known by the allergen nomenclature as Ves v 5 (for Vespula vulgaris) or Ves g 5 (for Vespula germanica). We developed a major allergen ELISA and investigated the Antigen 5 content in different wasp venom products.

1

D. van Deursen1, S. Quaak1, R. van den Hout1, R. van Ree2, E. Kerkvliet1

786 - A quantitative ELISA for determining Antigen 5 content in wasp venom

Binding of allergen Antigen 5

Figure 1: Schematic overview of the assay.

Coating microtiter plate with anti-Ves g5 monoclonal Binding Rabbit anti-Ves g5 polyclonal

detected with HRPconjugated goat-antirabbit antibody Staining with TMB and determining Antigen 5 concentration

0.1

1

Concentration (ng/mL)

10

100

1000

A B C batch A C batch B C batch C C batch D

Vespula spp.

Vespula spp.

Vespula spp.

Vespula spp.

Vespula vulgaris Vespula vulgaris Vespula germanica Vespula germanica

Species

1.8 1.6 1.8 1.7 1.9 1.9 1.9 2.0

Content Ag5 (%)

Ag5: Antigen 5, DP: Drug Product, DS: Drug Substance Vespula spp.: mix of Vespula vulgaris and Vespula germanica

DP batch A DP batch B DS supplier DS supplier DS supplier DS supplier DS supplier DS supplier

Test preparation

Table 1: Antigen 5 content of the different wasp venom batches.

All batches were analysed in triplicate.

In relation to this presentation, I declare the following, real or perceived conflicts of interest: the presenter is a employee of HAL Allergy.

the presenter is an employee of HAL Allergy

In relation to this presentation, I declare the following, real or perceived conflicts of interest:

1

EAACI WAO Congress 2013

EAACI Congress 2013

Hoffman DR. J Allergy Clin Immunol. 1993; 92(5):707-16. 2 Lu G et al. J Immunol. 1993; 150(7):2823-30. 3 Winkler et al. Immunology. 2003; 110:376-385.4 King et al. J Allergy Clin Immunol 1985; 75:621-8.

Conclusion: The ELISA method is a suitable technique to quantify Antigen 5 content in different wasp venom products.

0

0.4

0.8

1.2

1.6

2

2.4

Figure 2: Dose response 4-Parameter curve of the Antigen 5 standard. The curve was performed in duplicate.

Results: The standard showed good 4-parameter dose response curves with high correlations (r>0.99, see figure 2 as example). The Antigen 5 concentration was determined in two different batches of drug product and in six different batches of drug substance obtained from three different suppliers. These test samples contained wasp venom from the species Vespula vulgaris or Vespula germanica, or a mix of both species, named here as Vespula spp. From each test sample three different concentrations in a linear range were analyzed from which a mean Antigen 5 concentration was calculated. The ELISA is suitable for detecting Ves g 5 and Ves v 5 because both are immunologically extremely cross-reactive due to their high degree of sequence identity 1, 2. All samples tested showed high assay precision (CVâ&#x2030;¤ 9%). As shown in table 1, the Antigen 5 content (% major allergen/total protein) is consistent between the venom batches of the different wasp species tested. The results are in accordance with data from the literature 3, 4.

OD (450 nm)

Quantification of bee allergen Api m 1 in bee venom

by HPLC

J. de Bruijn, R. van den Hout, S. Quaak and D. Luykx.

HAL Allergy BV, Leiden, The Netherlands.

Background Bee (Apis mellifera) venom is an important source for insect venom allergies. Compared to respiratory and food allergen sources, the protein composition of bee venom is relatively simple. One relevant allergen in bee venom is Api m 1 or more commonly known as phospholipase A2. A reversed-phase HPLC method was developed and tested for its suitability to quantify Api m 1.

Methods Samples: Api m 1 standard was purified from bee venom. Bee venom raw material and bee venom drug product (containing albumin) were tested along with the standard on HPLC. HPLC: Reversed-phase HPLC was performed on an Agilent 1200 system. The samples were analysed on an YMC column (YMC Co. Ltd. Kyoto, Japan) and XbridgeTM C8 column combined with UV detection at 210 nm. A gradient was applied in presence of acetonitrile and trifluoracetic acid.

Results The HPLC chromatogram of the Api m 1 standard showed two peaks. The first peak accounted for 85-90% of the total peak area. The two Api m 1 peaks were better separated with the YMC column than with the Xbridge column. The total peak area was related to the Api m 1 concentration. A linear relationship was observed in the protein concentration range of 12.5-600 μg/ml. The repeatability was shown to be good (CV≤5%). An HPLC chromatogram of bee venom raw material showed that the Api m 1 peaks were well separated from the other protein peaks. Based on the HPLC chromatograms, 12% of the total protein in bee venom was determined to be Api m 1 (w/w). Humane serum albumine which is added as a stabiliser to the bee venom drug product did not interfere with the Api m 1 peaks in the HPLC chromatogram.

Conclusion The developed reversed-phase HPLC method is a suitable method to quantify relevant allergen Api m 1 in bee venom raw material and drug product.

EAACI WAO, 22-26 June 2013, Milan, Italy Abstract number: 655, Session date and time: Sunday 23 June; 10:30 - 12:00 Session title: Diagnosis of venom allergy

24

Diagnosis of venom allergy

Quantification of bee allergen Api m 1 in bee venom

by HPLC

25

HPLC: Reversed-Phase High Performance Liquid Chromatography (RPLC) was performed on an Agilent 1200 system. The bee venom samples and Api m 1 standards in presence of Tween-20 were applied on an YMC column (250 x 3.0 mm, I.D.) (YMC Co. Ltd. Kyoto, Japan). This is a narrowbore column containing 5 µm particles with a pore diameter of 300Å. The RPLC column was equilibrated with acetonitrile in presence of trifluoracetic acid (TFA). The gradient elution consisted of an increase of acetonitrile. A flow rate of 0.4 ml/min at 35 ºC was applied in combination with UV-detection (210 nm). Drug Substance and Drug Product were tested along with the standard of Api m 1 on RPLC.

Samples: Api m 1 standard was purified from bee venom using ion exchange chromatography and gel filtration. Bee venom batches included formulated (Drug Product) and non-formulated bee venom (Drug Substance). Drug Product contained added albumin.

Methods:

Bee (Apis mellifera) venom is an important source of insect venom allergies. The venom has been used successfully for many decades in allergen-specific immunotherapy. Compared to respiratory and food allergen sources, the protein composition of bee venom is relatively simple. Bee venom allergens described amongst others are phospholipase A2 (Api m 1), hyaluronidase (Api m 2), acid phosphatase (Api m 3), mellitin (Api m 4), dipeptidyl peptidase (Api m 5), and an additional number of less common allergens. One of the major allergens described for bee venom is Api m 1. It accounts for around 10-12% of the total protein in bee venom1. A quantitative reversed-phase HPLC method was developed and tested for its suitability to quantify Api m 1 in bee venom and formulated bee venom. HPLC-fractions were collected and corresponding peaks were identified with mass spectrometry.

Background & Aim:

HAL Allergy BV, Leiden, The Netherlands

0

5

15

20

Retention time (min)

10

25

30

35

min

30000

40000

Figure 1: HPLC pattern of a purified Api m 1 standard. An YMC RPLC column was applied combined with UV-detection at 210 nm at a flow rate of 0.4 ml/min. 20 µl of 300 µg/ml Api m 1 was injected.

0

100

200

300

400

500

600

mAU

J. de Bruijn, E. Schepens, R. van den Hout, S. Quaak and D. Luykx

655 - Quantification of bee allergen Api m 1 in bee venom by HPLC

Absorbance 210 nm (mAU)

rea (µV/s)

12.0

12.1

12.3

12.1

11.7

0

50

100

200

250

300

350

400

450

Concentration Api m 1 (µg/ml)

150

500

550

600

0

0

5

5

10

10

Drug Product

Drug Substance

15

15

1

1

20

2

20

2

25

25

3

3

30

4

30

35

35

min

min

B

A

EAACI WAO Congress 2013

EAACI Congress 2013

Retention time (min) Figure 3: HPLC patterns of Drug Substance (A) and Drug Product (B). An YMC RPLC column was applied combined with UV-detection at 210 nm at a flow rate of 0.4 ml/min.

The numbered HPLC peaks represent Api m 1 (1,2), mellitin (3) and albumin (4).

0

100

200

300

400

500

600

mAU

0

100

200

300

400

500

600

mAU

Figure 2: Calibration curve of Api m1 standards (25-600 µg/ml) using linear regression; y=75.1x – 566.8, coefficient of determination (R²) = 0.999

0

10000

20000

In relation to this presentation, I declare the following, real or perceived conflicts of interest: the presenter is an employee of HAL Allergy.

In relation to this presentation, I declare the following, real or perceived conflicts of interest: the presenter is an employee of HAL Allergy

1

Leluk J et al. Comparative studies on the protein composition of hymenopteran venom reservoirs. Toxicon 1989;27:105-114

The developed RPLC method was shown to be a suitable method to quantify major allergen Api m 1 in bee venom. The amount of Api m 1 determined in Drug Substance and Drug Product batches was consistent with the amounts described in literature.

Conclusion:

Api m 1 (%)

Drug Product

Batch 1 Batch 2 Batch 3 Batch 1 Batch 2

Drug Substance

Table 1: Api m 1 content in three different Drug Substance and two Drug Product batches

The HPLC chromatogram of the Api m 1 standard showed two peaks well separated (Fig. 1). The first peak accounted for 85-90% of the total peak area. The total peak area was related to the Api m 1 concentration. A linear relationship was observed for the protein concentration range of 25600 µg/ml (Fig. 2). An HPLC chromatogram of the Drug Substance showed that the Api m 1 peaks were well separated from the other peaks corresponding to bee proteins (Fig. 3.A). Albumin which is added as a stabiliser to the Drug Product did not interfere with the Api m 1 peaks in the HPLC chromatogram (Fig. 3.B). The proteins present in the collected HPLC fractions were identified by MS. This confirmed the presence of Api m 1, mellitin or albumin (Fig. 3). The LOD value was determined to be <10 µg/ml and the LLOQ 25 µg/ml. The repeatability was shown to be good (CV ≤ 5%). The amount of Api m 1 in three Drug Substance and two Drug Product batches was determined to be 12.0 ± 0.3 % of the total protein content via this RPLC method (Table 1). This supports the findings in literature1.

Results:

MS: First tryptic digests were prepared from the samples. Subsequently, the peptides were separated with nano-liquid chromatography before entering the MS instrument via electrospray ionisation. Ionised peptides were fragmented revealing amino acid sequences (i.e., identification).

Peak Absorbance 210 nm(mAU)

Characterisation of Parietaria p ollen allergoids with physicochemical techniques

J. de Bruijn (1), R. van den Hout (1), J. Cordewener (2), T. America (2) and D. Luykx (1).

(1) HAL Allergy BV, Leiden, The Netherlands, (2) Plant Research International, Wageningen, The Netherlands.

Background Parietaria allergoids are drug substances for the Parietaria allergy vaccine. Until now these allergoids have been poorly characterised due to their complexity. Nevertheless, a better characterisation is needed. This includes identification of the relevant allergens and determination of the degree of polymerisation and cross-linking. Several physicochemical techniques were tested for their suitability to analyse these characteristics in the allergoid and corresponding allergen extract: Mass Spectrometry, SDS-PAGE, HPLC-SEC, lysine determination and fluorescence spectroscopy.

Methods Allergoids: Allergen extracts from Parietaria judaica and Parietaria officiniales pollen were treated with glutaraldehyde to obtain Parietaria allergoids. MS: Preparation of tryptic digests and peptide separation via nano-LC before electrospray ionisation. Ionised peptides were fragmented revealing sequences. SDS-PAGE: 10-20% gels with Coomassie Blue staining and reduced samples. HPLC: A GF250 SEC column was used combined with UV-detection. Lysine determination: Samples were hydrolysed into amino acids followed by free lysine determination with HPLC. Fluorescence: Emission spectra were recorded from 290-400 nm, with excitation at 280 nm.

Results Relevant Parietaria allergens Par j 1 and Par j 2 were identified in the Parietaria allergoids with MS. SDS-PAGE showed for the allergoids the formation of various high molecular weight molecules including masses ≥250 kDa. HPLC-SEC showed for the allergoids the formation of molecules ≥670 kDa. For the Parietaria extracts a minority of HPLC peaks were corresponding to proteins with a molecular mass >44 kDa (11%). For the corresponding allergoids the majority of peaks (52%) were corresponding to proteins with a molecular mass >44 kDa. Determination of free lysines showed that the majority of the lysines were modified in the Parietaria allergoids (76%). Fluorescence intensities obtained for the allergoids were very low indicating quenching.

Conclusion Applying a combination of physicochemical techniques was shown to be a suitable approach to characterise the Parietaria allergoids well: Identification of the relevant allergens and determination of the degree of polymerisation and crosslinking were accomplished. Fluorescence spectroscopy appeared to be not suitable to analyse the protein structures of the Parietaria allergoids.

EAACI WAO, 22-26 June 2013, Milan, Italy Abstract number: 471, Session date and time: Sunday 23 June; 12:00 - 13:30 Session title: New developments in allergen-specific immunotherapy

26

New developments in allergen-specific immunotherapy

Characterisation of Parietaria p ollen allergoids with physicochemical techniques

27

HAL Allergy BV, Leiden, The Netherlands

Plant Research International, Wageningen, The Netherlands

Lysine determination: Samples were hydrolysed into amino acids followed

HPLC-SEC: A GF250 size exclusion chromatography (SEC) column was used combined with UV-detection (215 nm).

SDS-PAGE: Reduced samples were applied onto 10-20% polyacrylamide gels and stained with Coomassie Blue.

MS: First tryptic digests were prepared from the samples. Subsequently, the peptides were separated with nano-liquid chromatography before entering the MS instrument via electrospray ionisation. Ionised peptides were fragmented revealing amino acid sequences (i.e., identification).

Allergoids: Allergen extracts from Parietaria pollen were treated with glutaraldehyde to obtain allergoids.

Methods:

Allergoids of Parietaria pollen are used as drug substance for Parietaria allergy vaccines. Until now these allergoids have been poorly characterised due to its complexity. A better characterisation is required. This includes identification of the major allergens, determination of the degree of polymerisation and cross-linking, and investigation of the protein structures.

Several physicochemical techniques were applied to analyse these characteristics in three successive, separately produced allergoids of Parietaria pollen and corresponding allergen extracts: Mass spectrometry (MS), SDS-PAGE, HPLC-SEC, lysine determination and fluorescence spectroscopy.

Background & Aim:

2

1

M

1

2

Extracts

3

4

5

6

Allergoids

150

175

mAU

A1

670 kDa

44 kDa 17 kDa

Standard

Figure 1: SDS-PAGE patterns of molecular weight marker (M), three parietaria (1-3) extracts and corresponding allergoids (4-6). Major parietaria allergens Par j 1 and Par j 2 are boxed.

Par j 2

Par j 1

J. de Bruijn1, R. van den Hout1, J. Cordewener2, T. America2 and D. Luykx1

471 - Characterisation of Parietaria pollen allergoids with physicochemical techniques

6

A4

6

A3

6

A2

6

8

8

8

8

10

10

10

10

158 kDa

12

12

12

12

16

16

16

16

18

18

18

18

20

20

20

20

22

22

24

24

24

Parietaria 3

22

24

Parietaria 2

22

Parietaria 1

Retention time (min)

14

14

14

14

1.35 kDa

52 74

Modified lysines (%)

Batch 1

HPLC: Peak area above 44 kDa (%)

Characteristic

In relation to this presentation, I declare the following, real or perceived conflicts of interest: the presenter is an employee of HAL Allergy.

Applying a combination of physicochemical techniques was shown to be a suitable approach to characterise the Parietaria pollen allergoids well: Identification of the major allergens, determination of the degree of polymerisation and determination of the degree of cross-linking were accomplished. Investigation of the tertiary protein structures was not successful. However, based on the other characterisation data, consistency for the modification of the extracts of Parietaria pollen was shown.

Conclusion:

min

min

min

min

Figure 2: HPLC-SEC patterns (A215 nm) of calibration standard (A1), three Parietaria pollen extracts (blue line) and corresponding allergoids (red line) (A2-A4). The black vertical line in the chromatogram splits peaks corresponding to molecular masses above and below 44 kDa. A GF250 SEC column was applied.

0

25

50

75

100

125

150

175

mAU

0

25

50

75

100

125

150

175

mAU

0

25

50

75

100

125

150

175

mAU

0

25

50

75

100

125

77

51

Batch 2

EAACI WAO Congress 2013

72

67

Batch 3

Fluorescence spectroscopy: Non-conclusive spectra were obtained for the modified Parietaria pollen extracts. The fluorescence intensities were too low. This is most probably due to quenching of the fluorescence signal by Table 1 Physiochemical characteristics of three batches of Parietaria pollen allergoids colouring ingredients in the matrix.

Lysine determination: For the Parietaria pollen allergoids 72-77% of the lysines were modified (Table 1). This shows a consistent degree of protein cross-linking.

HPLC-SEC: For the three Parietaria pollen extracts a minority of HPLC peaks is corresponding to proteins with a molecular mass above 44 kDa (Fig. 2). The chromatograms of the Parietaria pollen allergoid batches showed the presence of a variety of high molecular weight molecules including molecules with molecular masses â&#x2030;Ľ 670 kDa. Here, a consistent majority of peaks (51-67%) is corresponding to proteins with a molecular mass (far) above 44 kDa showing a consistent degree of protein polymerisation (Table 1).

SDS-PAGE: For the Parietaria pollen extracts, similar protein profiles were observed (Fig. 1). The bands corresponding to 10-15 kDa indicate the presence of major allergens Par j 1 and Par j 2. For all allergoids the formation of various high molecular weight molecules is shown via a smear and a band around 250 kDa (molecules â&#x2030;Ľ 250 kDa) on gel.

MS: Major allergens Par j 1 and Par j 2 were identified in the three Parietaria pollen allergoids. Five peptides were identified corresponding to Par j 1 and six peptides corresponding to Par j 2.

Results:

Fluorescence spectroscopy: Emission spectra were recorded from 290-400 nm with excitation at 280 nm.

by lysine determination with HPLC. By determining the amount of free lysines in the extract and allergoid, the amount of modified lysines in the allergoid can be calculated.

Absorbance 215 nm

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 under 70%, or hypersensitivity to any of the excipients. 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 and after removal teeth or molars. 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. 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: April 2011 ®

®

PURETHAL

®

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 ®

®

®

venomenHAL

®

Composition: VENOMENHAL contains 120 micrograms/vial freeze-dried bee or wasp venom; powder and solvent for solution for injection.Indications: Diagnostic use (prick-testing and intracutaneous testing) and for causal treatment of patients with IgE-mediated insect venom allergy who have suffered a systemic reaction after a bee or wasp sting. Dosage and administration: The 120 μg freeze-dried insect venom should be dissolved with 1.2 ml solvent to 100 μg/ml. The dilution series are prepared by diluting 0.5 ml dissolved venom with 4.5 ml solution. Skin testing: The skin tests are performed by means of end point titration. It always begins with the skin-prick test to determine the patient’s level of sensibility. The intracutaneous test determines the individual starting concentration for treatment. Immunotherapy: Usually the initial treatment is carried out in hospital by administering several subcutaneous injections with increasing doses daily, starting with 0.1 ml of 0.0001 μg/ml until after day 5 the maintenance dose of 1 ml of 100 μg/ml is reached. If in-patient treatment is not possible, immunotherapy may be performed in outpatients, with one injection every 7 days at slowly increasing doses and concentrations. Treatment with the maintenance dose is continued as an outpatient procedure, the injection interval being extended gradually from 7 to 14, 21 and finally 28 days. It is advised to carry out the treatment over a period of 3 - 5 successive years. Contraindications: Skin testing: Secondary infections of the skin, hypersensitivity to any of the excipients. Relative: Acute severe allergic reactions, treatment with ß-blockers, pregnancy, cardiovascular diseases with increased risk if using adrenalin. Immunotherapy: Acute inflammatory diseases/feverish infection at the target organ; active tuberculosis; secondary alterations of the target organ (emphysema, bronchi-ectasis and others); autoimmune disorders e.g. of the kidneys, thyroid gland, nervous system and rheumatic diseases); immune deficiencies (e.g. that caused by immunosuppresants); severe uncontrolled asthma, particularly with an FEV1 persistently below 70%; cardiovascular diseases with increased risk if using adrenalin; use of ß-blockers or ACE inhibitors, 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.

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. Occasionally delayed reactions occur (type III and IV). Package: A VENOMENHAL set consists of 6 vials with each 120 μg freezed-dried bee or wasp venom and 6 vials with each with 1.2 ml solvent. A DILUENT set contains 10 vials with each 4.5 ml diluent. The complete product information is available upon request.

HAL Allergy BV, P.O. Box 1204, 2302 BE Leiden, The Netherlands. Date: May 2011 ®

®

28

SUBLIVAC

®

Basisinformationen SUBLIVAC FIX / SUBLIVAC . Zusammensetzung: Lösungen zur sublingualen Immuntherapie. SUBLIVAC enthält Allergenextrakte nach individueller ärztlicher Rezeptur. 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, absolute: Akute Entzündungsprozesse/Infektionskrankheiten am Reaktionsorgan; Sekundärveränderungen am Reaktionsorgan (z.B. Emphysem, Bronchiektasen); schwere Autoimmunerkrankungen; maligne Tumorerkrankungen mit aktuellem Krankheitswert; Immundefekte (auch durch Immunsuppressiva induziert); schweres, unkontrollierbares Asthma bronchiale, insbesondere bei einem persistierenden FEV 1 unter 70% Sollwert; Sensibilisierung gegenüber einem der sonstigen Bestandteile; relative: Schwangerschaft und Stillzeit; Infektionen des Mund-/Rachenraumes; nach zahnärztlicher Behandlung (z.B. Zahnentfernung). Zum zeitlichen Intervall zu Schutzimpfungen und für weiterführende Informationen siehe Fachinformation. Nebenwirkungen: Exazerbation der patientenspezifischen allergischen Symptomatik.

Allergische Lokal- und/oder Allgemeinreaktionen; Bauchschmerzen und Durchfall; Exazerbation eines atopischen Ekzems. Für weiterführende Informationen und zur Behandlung von Nebenwirkungen siehe Fachinformation. Hinweise: Verschreibungspflichtig. Arzneimittel für Kinder unzugänglich aufbewahren. HAL Allergie GmbH, Poststraße 5-6, D-40213 Düsseldorf. Datum: Mai 2012 ®

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 (auch durch Immunsuppressiva induziert); schweres, unkontrollierbares Asthma bronchiale, insbesondere bei einem persistierenden FEV 1 unter 70% Sollwert; maligne Tumorerkrankungen mit aktuellem Krankheitswert; Erkrankungen mit Kontraindikationen gegen die Anwendung von Adrenalin; Sensibilisierung gegenüber einem der sonstigen Bestandteile; relative: Schwangerschaft und Stillzeit; nicht für Kinder unter 5 Jahren; Behandlung mit ß-Blockern. Zum zeitlichen Intervall zu Schutzimpfungen und für weiterführende Informationen siehe Fachinformation. Nebenwirkungen: Exazerbation der patientenspezifischen allergischen Symptomatik. 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. Hinweise: Verschreibungspflichtig. Arzneimittel für Kinder unzugänglich aufbewahren. In seltenen Fällen kann nach der Injektion leichte Müdigkeit auftreten, was beim Führen von Kraftfahrzeugen oder beim Bedienen von Maschinen zu berücksichtigen ist. Warnhinweise: Hyposensibilisierungsimpfstoffe zur Injektion dürfen nur durch allergologisch weitergebildete bzw. allergologisch erfahrene Ärzte verschrieben und angewendet werden. HAL Allergie GmbH, Poststraße 5-6, D-40213 Düsseldorf. Datum: Mai 2012 ®

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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 Therapie von Patienten mit IgE - vermittelter Insektengiftallergie, bei denen systemische Reaktionen nach Bienen- oder Wespenstich aufgetreten sind. Gegenanzeigen: Akute Entzündungsprozesse / Infektionskrankheiten am Reaktionsorgan; Sekundärveränderungen am Reaktionsorgan (z.B. Emphysem, Bronchiektasen); Autoimmunerkrankungen; aktive Tuberkulose; Immundefekte (auch durch Immunsuppressiva induziert); schweres, unkontrollierbares Asthma bronchiale, insbesondere bei einem persistierenden FEV 1 unter 70% Sollwert; Herz- und Kreislauferkrankungen mit erhöhtem Risiko bei der Anwendung von Adrenalin; Behandlung mit ß-Blockern und mit ACE-Hemmern; maligne Tumorerkrankungen mit aktuellem Krankheitswert; Sensibilisierung gegenüber einem der sonstigen Bestandteile des Arzneimittels. Während der Schwangerschaft ist von der Einleitung einer Behandlung mit VENOMENHAL Biene oder VENOMENHAL Wespe abzusehen. Zum zeitlichen Intervall zu Schutzimpfungen und für weiterführende Informationen siehe Gebrauchs- und Fachinformation. Ferner gelten die bekannten Gegenanzeigen für die Durchführung von Hauttestungen (siehe Gebrauchs- und Fachinformation). Die genannten Kontraindikationen sollten gegen die Gefährdung des Patienten durch einen eventuellen Insektenstich abgewogen werden. Nebenwirkungen: Lokal- und/oder Allgemeinreaktionen bis hin zum anaphylaktischen Schock. Patienten nach der Injektion mindestens 30 Minuten überwachen, eine Schockapotheke muss bereitgestellt sein. Gelegentlich treten auch Reaktionen vom verzögerten Typ (Typ III und IV nach Coombs und Gell) auf. Für weiterführende Informationen und zur Behandlung von Nebenwirkungen siehe Gebrauchs- und Fachinformation. Hinweise: Verschreibungspflichtig. Arzneimittel für Kinder unzugänglich aufbewahren. In seltenen Fällen kann nach der Injektion leichte Müdigkeit auftreten, was beim Führen von Kraftfahrzeugen oder beim Bedienen von Maschinen zu berücksichtigen ist. Warnhinweise: Hyposensibilisierungsimpfstoffe zur Injektion dürfen nur durch allergologisch weitergebildete bzw. allergologisch erfahrene Ärzte verschrieben und angewendet werden. HAL Allergie GmbH, Poststraße 5-6, D-40213 Düsseldorf. Datum: Mai 2012 ®

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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

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Š HAL Allergy 2013 | MAB 48091-03