HAL Abstractbook EAACI Geneva 2012

Scientific Contribution

31 st Congress of the European Academy of Allergy and Clinical Immunology 2012, EAACI, 16 -20 June 2012, Geneva

Dear

Congress delegate,

Diderik Boot, PhD Medical Director

On behalf of HAL Allergy we would like to welcome you to the 31st EAACI congress in Geneva. The theme of this year’s congress is “At the crossroads of research, practice and education”. HAL Allergy is one of Europe’s top players in the field of allergen vaccination and devotes a major part of its resources and investments to the development of allergen specific immunotherapy. We are proud to present our ongoing research and clinical practice work during the biggest European congress on allergy and clinical immunology.

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

HAL Allergy’s clinical development program is committed to meeting today’s requirements for gaining registration for specific immunotherapy products.

The clinical development program is represented with two abstracts related to the dose range finding study with PURETHAL® Mites which was initiated in the fall of 2011 and will be completed in spring 2013. In addition we also present you with clinical data on the use of PURETHAL®, SUBLIVAC® and VENOMENHAL® in daily practice.

Dirk-Jan Opstelten, PhD Research & Development Director

At this year’s meeting there will be two oral presentations dealing with our latest pre-clinical work on a novel vaccine for peanut allergy. We are on track with our research program to develop a subcutaneous immunotherapy product to safely treat peanut-allergic patients and intend to complete all preclinical studies this year.

Furthermore, we present various abstracts on the characterisation and quantification of our allergen products, in particular our PURETHAL® range which involve chemically modified allergens, so-called allergoids. HAL Allergy is progressing with the development and implementation of new antibody based and physicochemical methodologies to characterise pharmaceutical allergoid preparations and to assure their quality.

We hope you will have a successful 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 Evaluation of the cytokine response induced by allergen-specific immunotherapy with a modified allergoid in patients with allergic rhinits with or without asthma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

6

Application of a standardised titrated nasal provocation test with HDM extract in a multicentre clinical study in allergic rhinitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

8

Patient knowledge, perceptions, expectations, and satisfaction, on subcutaneous and sublingual allergen-specific immunotherapy: a real life survey . . . . . . . . . . . . . . . . . . . . . . . . . . .

10

Influence of co-morbidities and co-medications on safety and tolerability in the buildup phase of venom immunotherapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

12

Design of dose range finding study with allergen specific immunotherapy in patients with house dust mite induced allergic rhinitis/rhinoconjunctivitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

14

Safety of pre- and coseasonal start of sublingual immunotherapy treatment in patients with pollen allergy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

16

Pre-clinical Chemical modification of a peanut extract decreases IgE binding while the immunogenicity is maintained . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

18

Allergenic potency of chemically modified Ara h2/h6 evaluated with basophil activation, RBL-SX38 activation, and solid phase IgE-immunoassay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

20

The content of allergens Ara h1, Ara h2, Ara h3 and Ara h6 in different peanut cultivars commonly consumed in Europe and the USA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

22

Development Comparison of antibody-based assay with physicochemical assays for monitoring the stability of alum-adsorbed mite allergoid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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

Characterisation of alum-adsorbed pollen allergoids with physicochemical techniques . . . . . . . . . . . . . . . . . . . . . . . . .

An inhibition ELISA method for the determination of relative potency of mite preparations . . . . . . . . . . . . . . . . . . . . .

An antibody-based technique for stability studies on alum adsorbed birch pollen allergoid preparations . . . . . . .

Biochemical characterisation of chemically modified Ara h2 isoforms; structure-function relationships . . . . . . . . . .

24 26 28 30 32 34

Evaluation of the cytokine resp onse induced by allergen-specific immunotherapy with a mo dified allergoid in patients with allergic rhinitis with or without asthma A. Malet-Casajuana (1), A. Roger (2), P. Amat-Par (1), N. Depreux (2), T. Gonzalez-Quevedo (3), M.J. Cruz (4).

(1) Alergocentre, Barcelona, Spain, (2) Allergy Unit. Germans Trias Pujol University Hospital. Badalona, Spain. (3) Allergy Unit. Virgen del Rocío University Hospital, Sevilla, Spain. (4) HAL Allergy, Barcelona, Spain.

Background Allergy is characterized by a T helper type 2 (Th2) cell-mediated immune response. It has been demonstrated that after allergen-specific immunotherapy (SIT) there is a change in the balance between Th1/Th2 responses and the induction of a more allergen tolerant state. The aim of the present study was to investigate in patients with house dust mite (HDM) induced allergic rhinitis/rhinoconjunctivitis the changes in Th1, Th2 and T regulatory (Treg) cytokines following SIT treatment

Methods Twenty-nine adult patients (14F/15M, 33 yrs range 16-65 years) with a history of HDM induced allergic rhinitis/rhinoconjunctivitis with or without clinically stable mild persistent asthma were treated with subcutaneous SIT, containing 20,000 AUeq/ml of modified mites allergen extract adsorbed onto aluminium hydroxide. Blood samples were collected before and 3 and 6 months after SIT treatment. In serum samples IFN-γ, IL-10, IL-4, IL-5, and Il-13 levels were determined by flow cytometry using a commercial multiplexing kit (Milliplex Human Cytokine Kit, Millipore Corporation) according to the instructions provided by the manufacturer. The samples were analyzed using a Fortessa flow cytometer.

Results Following SIT, a significant increase in the level of IFN-γ was found after 3 and 6 months of treatment (p = 0.004 and 0.005 respectively). This increase was also observed in the levels of IL-10 after 6 months of treatment (p = 0.019). Moreover, a tendency towards significant decrease of the levels of IL-5 were observed after 6 months, compared with the basal values (p = 0.088). No significant differences were found in the levels of IL-4 and IL13 after SIT.

Conclusions The results obtained in the present study suggest that SIT triggers a Th2 to Th1 shift and an induction of IL-10 secreting Treg cells. These events occur early and suggest a SIT tolerance induction in these patients.

EAACI, 16-20 June 2012, Geneva Abstract number: 2, Session date and time: Sunday 17 June; 10:30 - 12:00 Session title: OAS 1 - Allergen immunotherapy: new aspects in diagnostics and treatment.

6

OAS 1 - Allergen immunotherapy: new aspects in diagnostics and treatment.

Evaluation of the cytokine resp onse induced by allergen-specific immunotherapy with a mo dified allergoid in patients with allergic rhinitis with or without asthma.

7

Spain.

Unit. Virgen del Rocío University Hospital, Sevilla, Spain, 4HAL Allergy, Barcelona, Spain.

Barcelona, Spain, 2Allergy Unit. Germans Trias Pujol University Hospital. Badalona, Spain.

Background & Aim:

Mean age in years (range)

Patients

33 (16-65)

n = 29

n = 29antibodies Table 1: Demographic characteristics and values of specific Patients inGender the study population.

(M (%) / F (%)) 15(51.7%)/ 14(48.3%)

in the study population.

Twenty nine patients (Table 1, Figure 1) were included. SIT induced a significant increase in serum levels of Table 1: Demographic characteristics and values of specificwere antibodies decrease in IL-5 (Figure 2). No significant differences found in the levels of IL-4 and IL13 after SIT.

Twenty nine patients (Table 1, Figure 1) were included. SIT induced a significant increase in serum levels of decrease in IL-5 (Figure 2). No significant differences were found in the levels of IL-4 and IL13 after SIT. Results:

Rhinitis 54%

Rhinitis 54%

and IL-10 and a significant

and IL-10 and a significant

cytokine bound to the specific fluorescent bead. The reaction was detected by adding a streptavidin-phycoerythrin conjugate to the reaction Results:The samples were analyzed using a Fortessa flow cytometer. mixture.

Methods: Adult patients with a history of HDM induced allergic rhinitis/rhinoconjunctivitis with or without concomitant clinically stable mild persistent asthma Methods: were screened. Eligible patients were treated with subcutaneous SIT (PURETHAL®, HAL Allergy BV, Leiden, The Netherlands), containing Adult patients with a history of HDM induced allergic rhinitis/rhinoconjunctivitis with or without concomitant clinically stable mild persistent asthma 20,000 AUeq/ml of modified mites allergen extract adsorbed onto aluminium hydroxide. Blood samples were collected before, 3 and 6 months were screened. Eligible patients were treated with subcutaneous SIT (PURETHAL®, HAL Allergy BV, Leiden, The Netherlands), containing after SIT treatment. In serum samples IFN- , IL-10, IL-4, IL-5, and Il-13 levels were determined by flow cytometry using a commercial multiplexing 20,000 AUeq/ml of modified mites allergen extract adsorbed onto aluminium hydroxide.

Blood samples were collected before, 3 and 6 months kit (Milliplex Human Cytokine Kit, Millipore Corporation).

The detection system was a sandwich immunoassay performed on unique fluorescent after SITwhich treatment.

In serum samples IFN- , IL-10, IL-5, anddyes, Il-13 and levels determined by flow cytometry using to a commercial beads, contained a precise distribution of twoIL-4, fluorescent a were specific cytokine capture antibody linked its surface. multiplexing The kit (Milliplex Human Cytokine Kit, Millipore Corporation).

The detection system was a sandwich immunoassay performed onthe unique fluorescent fluorescent bead sets were incubated with a biological sample. A biotinylated cytokine reporter antibody was then used to identify captured beads, a precise distribution of two fluorescent dyes, by andadding a specific cytokine capture antibody linked to its surface. The cytokinewhich boundcontained to the specific fluorescent bead. The reaction was detected a streptavidin-phycoerythrin conjugate to the reaction fluorescent setswere wereanalyzed incubated with a biological A biotinylated cytokine reporter antibody was then used to identify the captured mixture. Thebead samples using a Fortessa flow sample. cytometer.

Allergy is characterized Background & Aim: by a T helper type 2 (Th2) cell-mediated immune response. It has been demonstrated that after allergen-specific immunotherapy (SIT) therebyis aa T modulation of allergen-specific antibody immune responses, a changeItinhas the balance between Th1/Th2 and Allergy is characterized helper type 2 (Th2) cell-mediated response. been demonstrated that responses after allergen-specific the induction of a more allergen tolerant state.

The induction of allergen specific serum IgG antibodies has previously been demonstrated.

The immunotherapy (SIT) there is a modulation of allergen-specific antibody responses, a change in the balance between Th1/Th2 responses and aim induction of the present to investigate the changes in Th1, Th2 and T regulatory following thepreviously initiation ofbeen SIT treatment in the of a study more was allergen tolerant state.

The induction of allergen specific (Treg) serum cytokines IgG antibodies has demonstrated.

The patients with house dust mite (HDM) induced allergic rhinitis/rhinoconjunctivitis aim of the present study was to investigate the changes in Th1, Th2 and T regulatory (Treg) cytokines following the initiation of SIT treatment in patients with house dust mite (HDM) induced allergic rhinitis/rhinoconjunctivitis

3 Allergy

3 Allergy Unit. Virgen del Rocío University 4HAL Allergy, Barcelona, Spain.

1 Alergocentre, Hospital, Sevilla, Spain, Barcelona, Spain, 2Allergy Unit.

Germans Trias Pujol University Hospital. Badalona,

1 Alergocentre,

Evaluation of induced by by allergen-specific 22 -- Evaluation ofthe thecytokine cytokineresponse response induced allergen-specific immunotherapy with a modified allergoid in patients with allergic immunotherapy with a modified allergoid in patients with allergic rhinitis with or without asthma rhinitis with or without asthma

N IF

γ

A

P = 0.004

N IF

γ

B

P = 0.005

P = 0.033

N IF

γ

C

0.1

1

10

100

1 IL

0

A

4.3 (3.0-6.7L)

3.7 (2.4-5.3L)

17.3 (1.3-57.5)

33 (16-65)

P = 0.025

10 IL

B

P = 0.019

P = 0.374

1 IL

0

C

0.01

0.1

1

10

100

5 IL

A

P = 0.271

5 IL

B

P = 0.088

P = 0.001

5 IL

C

Asthma + Rhinitis 23%

Figure 1: Distribution of respiratory disease.

Asthma 23%

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

probably related to the clinical efficacy of the treatment.

EAACI Congress 2012 EAACI Congress 2012

occur early and suggest a SIT tolerance induction in these patients. These changes are part of the mechanism of action of immunotherapy and is

The results obtained in the present study suggest that SIT triggers a Th2 to Th1 shift and an induction of IL-10 secreting Treg cells. These events

Conclusion:

Figure 2: Cytokine concentration before and after treatment with immunotherapy: A = before treatment; B = 3 months after treatment; C = 6 months after treatment

0.1

1

10

100

Median FVC1 (L) (range)

Median serum specific IgE (IU/ml) to mites (range) Median FEV1 (L) (range)

Mean age in years (range)

15(51.7%)/ 14(48.3%)

IL10 (pg/ml)

Gender (M (%) / F (%))

IL5 (pg/ml)

Application of a standardized titrated nasal provocation test with HDM extract in a multicentre clinical study in allergic rhinitis

C. Bachert (1), O. Pfaar (2), M.J. Nell (3), J.D. Boot (3), H. Nienhuis (3), Z. Diamant (4,5).

(1) UZ Gent, Ear-, Nose and Throat Department, Gent, Belgium, (2) Center for Rhinology and Allergology Wiesbaden, Germany, (3) HAL Allergy BV, Leiden, The Netherlands, (4) Skane University Hospital, Dept of Respir Med & Allergol, Lund, Sweden and (5) QPS-NL, Groningen, The Netherlands.

Background Titrated nasal provocation test (TNPT) can be used in the diagnosis of allergic rhinitis (AR) and as a disease model in clinical trials evaluating the efficacy of treatment. However, the techniques for provocation and readouts vary, requiring standardization to enable the use of TNPT as a tool in multicentre trials. We performed a standardized TNPT and documented the upper airway response by symptom scores and peak nasal inspiratory flow (PNIF) measurements. In addition, we compared the upper airway responses across the different allergen concentrations and investigated a possible correlation.

Methods Patients with suspected HDM-induced AR were screened prior to enrollment into a multicentre efficacy study with HDM immunotherapy. Patients with a positive medical history and a positive skin prick test to HDM underwent a standardized TNPT starting with the diluent and up to 3 serial concentrations of a standardized allergen extract (D. Pter 100, 1,000 and 10,000 AU/ml, 1 puff per nostril) at 20 minutes intervals. The upper airway response was quantified by a composite symptom score according to Lebel and a PNIF measurement 15 minutes after each administration. TNPT was considered positive once a score ≥6 was reached. Symptom scores and PNIF were repeated 1 hour after the provocative allergen concentration.

Results 150 patients were screened. 103 patients (54M/49F, mean age 32 years, mean wheal size HDM 6mm) had a positive TNPT, the most prominent symptoms being nasal blockage and rhinorrhoea, followed by pruritus and sneezing. A consistent dose-response relationship was found between serial allergen concentrations and increasing symptom scores, matching a gradual decrease in PNIF. One hour after the provocative allergen concentration, both measurements had not returned to baseline values (Table). There was a moderate though highly significant correlation between symptom scores and PNIF (r=0.54; p<0.01).

Conclusion When applied in a multicentre setting in patients with suspected HDM-induced AR, a standardized TNPT with a HDM extract induces a consistent gradual increase in allergic symptoms and signs that can be reliably quantified by well-defined composite symptom scores and PNIF.

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

Timepoint

Pre Diluent

Post Diluent 100 AU/ml 1,000 AU/ml 10,000 AU/ml Diluent

Lebel Score

0.8±0.8

0.9±1.0

3.7±2.1

5.5±2.2

6.5±2.9

3.2±1.7

PNIF (L/min)

135±45

132±47

113±48

93±44

66±42

109±43

EAACI, 16-20 June 2012, Geneva Abstract number: 37, Session date and time: Sunday 17 June; 13:30 - 15:00 Session title: OAS 7 - Diagnosis and treatment of allergic rhinoconjunctivitis: recent development

8

1 hour post TNPT

OAS 7 - Diagnosis and treatment of allergic rhinoconjunctivitis:

recent development

Application of a standardized titrated nasal provocation test with HDM extract in a multicentre clinical study in allergic rhinitis

9

Gent, Ear-, Nose and Throat Department, Gent, Belgium; 2Center for Rhinology and Allergology Wiesbaden, Germany; 1UZ Gent, Ear-, Nose and Throat Department, 2Center for Rhinology and Allergology Wiesbaden, Germany; Gent, Belgium; Allergy BV, Leiden, The Netherlands; 4Skane University Hospital, Dept of Respir Med & Allergol, Lund, Sweden 4Skane University Hospital, Dept of Respir Med & Allergol, Lund, Sweden 3HAL Allergy BV, Leiden, The Netherlands; and 5QPS-NL, Groningen, The Netherlands and 5QPS-NL, Groningen, The Netherlands

Results: Results: 150 patients were screened. 103 patients (Table 1) had a positive TNPT. The TNPT was well tolerated and none of the patients experienced any

Methods:Methods: Patients: Patients: M/F aged HDM-related AR, with without concomitant, clinically stablestable asthma (FEV1 >70% predicted) were M/F18-60 agedyears 18-60with years with HDM-related AR,orwith or without concomitant, clinically asthma (FEV1 >70% predicted) were screenedscreened prior to enrollment into a multicentre efficacyefficacy study with Patients with awith positive AR-history and and a positive skinskin prior to enrollment into a multicentre studyHDM with immunotherapy.

HDM immunotherapy.

Patients a positive AR-history a positive prick testprick to HDM, underwent a standardized TNPT. TNPT. test to HDM, underwent a standardized Methods:Methods: The TNPT thewith allergenâ&#x20AC;&#x2122;s diluent diluent followed by maximal 3 serial concentrations of a of standardized allergen extract (D. (D. Thestarted TNPT with started the allergenâ&#x20AC;&#x2122;s followed by maximal 3 serial concentrations a standardized allergen extract Pteronyssinus 100,and 1,000 and AU/ml, 10,000 AU/ml, HAL Allergy BV, Leiden, The Netherlands), administered a nasal spraying device (1 puff Pteronyssinus 100, 1,000 10,000 HAL Allergy BV, Leiden, The Netherlands), administered by a by nasal spraying device (1 puff per per at 20 minutes The airway upper airway response was quantified a composite symptom according to Lebel PNIF nostril) atnostril) 20 minutes intervals.intervals.

The upper response was quantified by a by composite symptom scorescore according to Lebel andand PNIF measurements of 3 measured

by In-check nasal inspiratory flow meter [Clement Clarke, Harlow, at baseline (pre-diluent), measurements (highest (highest value ofvalue 3 measured

by In-check nasal inspiratory flow meter [Clement Clarke, Harlow, UK]) UK]) at baseline (pre-diluent), 15 15 minutes after each administration and 60 minutes post-challenge.

Symptoms were recorded using the following scoring system: sneezes minutes after each administration and 60 minutes post-challenge. Symptoms were recorded using the following scoring system: sneezes 2 = 20,= 0, 5 = 3 points, rhinorrhoea = 1 point, posterior rhinorrhoea = 1 point, difficult breathing 1 point, = 1sneezes point, sneezes 5 = 3 points, anterioranterior rhinorrhoea = 1 point, posterior rhinorrhoea = 1 point, difficult breathing = 1 =point, 1 1 sneezes sneezes 3-4 = 1 3-4 point, blocked nostril = 2 points, 2 blocked nostrils = 3 points, nasal pruritus = 1 point, pruritus in palate or ear = 1 point, conjunctivitis = 1 point (total blocked nostril = 2 points, 2 blocked nostrils = 3 points, nasal pruritus = 1 point, pruritus in palate or ear = 1 point, conjunctivitis = 1 point (total The TNPT was considered positive a total 6 was reached. score range: 0-11 The points).

The pre-diluent not allowed 3. be The3.

TNPT was considered positive once once a total scorescore 6 was reached. score range: 0-11 points). pre-diluent was notwas allowed to be to

Background & Aim: Background & Aim: Titrated nasal provocation tests (TNPT) with a relevant allergen can be used in the diagnosis of allergic rhinitis (AR) and as a disease model in Titrated nasal provocation tests (TNPT) with a relevant allergen can be used in the diagnosis of allergic rhinitis (AR) and as a disease model in clinical trials evaluating the efficacy of treatment.

However, both the techniques used used for provocation and the of the evoked upper clinical trials evaluating the efficacy of treatment.

However, both the techniques for provocation and readouts the readouts of the evoked upper airway response vary, requiring standardization to enable the use of TNPT as a tool in multicentre trials.

We performed a standardized TNPT airway response vary, requiring standardization to enable the use of TNPT as a tool in multicentre trials. We performed a standardized TNPT and documented the upper airway by symptom scores scores and peak flow (PNIF) measurements.

In addition, we compared and documented the upperresponse airway response by symptom andnasal peak inspiratory nasal inspiratory flow (PNIF) measurements.

In addition, we compared the upperthe airway responses across the different allergen concentrations and investigated a possible correlation between both readouts. upper airway responses across the different allergen concentrations and investigated a possible correlation between both readouts.

3HAL

1UZ

C. Bachert1, O. Pfaar 2, M.J. Nell3, J.D. Boot3, H.E. Nienhuis3, Z. Diamant4,5 C. Bachert1, O. Pfaar 2, M.J. Nell3, J.D. Boot3, H.E. Nienhuis3, Z. Diamant4,5

37 - Application of a standardized titrated nasal 37 - Application of a standardized titrated nasal provocation test with HDM extract in a provocation test with HDM extract in a multicentre clinical study in allergic rhinitis multicentre clinical study in allergic rhinitis

n = 103

10 7

Positive response (n) Mean SPT-wheal size (mm)

34 6

1,000 AU/ml 59 5

10,000 AU/ml Figure 1: Concentration-response curves with mean ( 95%CI) changes in composite symptom scores (red circles) and PNIF measurements (blue circles) following TNPT with HDM allergen.

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

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

EAACI Congress 2012

EAACI Congress 2012

Conclusion: When applied in a multicentre setting in patients with suspected HDM-induced AR, a standardized TNPT with a HDM extract induces a consistent gradual increase in allergic symptoms and signs that can be reliably quantified by a well-defined composite symptom score and PNIF measurements.

100 AU/ml

Concentration

Table 2: Positive TNPT-response and corresponding SPT-wheal sizes

Gender (M (%) / F (%)) 54(52.4%)/ 49(47.6%) Mean age ± SD in years (range) 32 ± 11 (18-57) SPT to HDM extract: Mean wheal size in mm ± SD 6 ± 2 (3-12) (range)

Patients

Table 1: Patients’ demographic characteristics

clinically significant adverse events. The most prominent symptoms were nasal blockage and rhinorrhoea, followed by pruritus and sneezing. A consistent concentration-response relationship was found between serial allergen concentrations and increasing symptom scores, matching a gradual decrease in PNIF. One hour after the provocative allergen concentration, both parameters had not completely returned to baseline values (Figure 1). There was a moderate though highly significant correlation between symptom scores and PNIF measurements (r=0.54; p<0.01). The majority of patients had a positive TNPT-response following administration of the 10,000 AU/ml concentration (Table 2). Patients with a positive TNPT-response at a lower allergen concentration tended to have a larger wheal size although differences were not statistically significant (Table 2).

Patient Knowledge, Perceptions, Expectations, and Satisfaction, on Subcutaneous and Sublingual Allergen Specific Immunotherapy: a real life survey I. Baiardini (1), F. Puggioni (2), S. Menoni (3), J.D. Boot (4), Z. Diamant (5), F. Braido (6), G.W. Canonica (6).

(1) University of Genoa, Allergy and Respiratory Diseases, Department of Internal Medicine, Genoa, Italy, (2) IRCCS Istituto Clinico Humanitas, Allergy and respiratory Disease Department, Milano, Italy, (3) University of Genoa, Biostatistic Unit, Department of Health Science, Genoa, Italy, (4) HAL Allergy BV, Leiden, The Netherlands, (5) Skane University Hospital, Dept of Respir Med & Allergol, Lund, Sweden and (6) QPS-NL, Groningen, The Netherlands.

Background Assessing patient’s viewpoint on treatment provides useful information enabling customized therapeutic approach.

Presently, only a few small studies addressing the patient’s perspective on allergen specific immunotherapy (SIT) are available. The aim of this cross-sectional multicentre survey was to evaluate subjective viewpoints on SIT in a large cohort of patients treated with subcutaneous (SCIT) or sublingual (SLIT) immunotherapy.

Methods A novel survey of 28 questions assessing patient’s knowledge (6), perception (12), expectations (5) and satisfaction (5) was developed by an expert panel. Sixteen physicians from 15 allergology centres in North Italy asked 30 consecutive patients with allergic rhinitis with or without asthma treated with SIT to fill out the survey. The physicians were also asked to report for each patient their satisfaction level regarding SIT.

Results Fully completed surveys from 434 patients (55.3% male; 66.7% polysensitized, 74% SLIT) were analyzed. SIT consisted of grass pollen (37.1%), mites (26.6%), tree pollen (17%), flower pollen (9.2%), moulds (5.6%), epithelia (2%) and other (2.5%); mean duration of treatment was 2.5 years. Most patients acquired their SIT knowledge through their physician (95%).

Conversely, the physicians opinion in their choice to start with SIT was important. The majority of patients (70.6%) believed that SIT should be continued for more than two years. Most patients perceived SIT to be safe and easy to integrate in daily routine. The main motivation when starting SIT were its potential effect to alter the course of the disease (45.7%), less need of anti-allergy drugs (28.2%), and dissatisfaction with the current pharmacotherapy (19.3%). Both patient and physician satisfaction was high (VAS score 74/100 and 78/100, respectively) and a significant correlation between patients’ and physicians’ satisfaction scores was found in both groups (SCIT: r = 0.612, p <0.001; SLIT: r = 0.608, p <0.001). No major difference was found in patients answers based on the level of education. Compared to SCIT, SLIT was frequently considered easier to take (p=0.024) and without side effects (p=0.026)

Conclusion This real life, cross-sectional survey evaluated in a large cohort different aspects of patient’s perspective on SIT. Despite some gaps and misconceptions, the majority of patients had an adequate level of knowledge, perception, expectations and satisfaction about SIT, which corresponded well with the physician’s satisfaction level.

EAACI, 16-20 June 2012, Geneva Abstract number: 874, Session date and time: Monday 18 June; 12:15 - 13:30 Session title: Poster 32 - Update in the treatment of allergic rhinitis

10

Poster Session 32 - Update in the treatment of allergic rhinitis

Patient Knowledge, Perceptions, Expectations, and Satisfaction, on Subcutaneous and Sublingual Allergen Specific Immunotherapy: a real life survey

11

I. Baiardini1, F. Puggioni2, S. Menoni3, J.D. Boot4, Z. Diamant5, F. Braido1, G.W. Canonica1

®

®

5Skane

®

®

Gender

Survey Patients

Questions

Completely True

11.3% 88.7% 29.7% 54.5% 14.3% 1.5% 33.3% 66.7%

Mild Moderate 1 2 3 4 Monosensitized Polysensitized 37.1%

26.0%

SCIT

37.1% 26.6%

Mites

Don’t know Other

10.1%

9.2%

Flower pollen Partly False Completely False

Patient answers

17.0%

Tree pollen

10.1%

9.2% Grass pollen Other

Flower pollen

74.0%

17.0% SLIT

SCIT 26.6% Tree pollen

Mites

Grass pollen

SLIT

74.0%

66.7%

Polysensitized

14.3%

3

1.5%

54.5%

2

33.3%

29.7%

1

26.0%

59.4%

11.3% Persistent 88.7%

40.6%

4

Partly True

Administered allergen Table 2: Patients perception about SIT

Administered allergen

Sensitization

12 2.5 ± 1.3

31.5

238 Males / 192 Females

n = 434

Intermittent 59.4%

40.6%

Monosensitized

Asthma – GINA Classification

Asthma – GINA Classification

Sensitization

Mild

Persistent

Rhinitis – ARIA classification Moderate

Rhinitis – ARIA classification

12 2.5 ± 1.3

31.5

238 Males / 192 Females

and clinical data

n = 434

Mean duration of treatment ± SD in years Intermittent

Mean duration of treatment ± SD in years

Mean age ± SD in years

Mean age ± SD in years

Gender

Survey Patients

1: Patient Table 1: PatientTable demographics anddemographics clinical data

University Hospital, Dept of Respir Med & Allergol, Lund, Sweden and QPS-NL, Groningen, The Netherlands

of Genoa, Allergy and Respiratory Diseases, Department of Internal Medicine, Genoa, Italy, 2IRCCS Istituto Clinico Humanitas, Allergy and respiratory Disease 4HAL Allergy BV, Leiden, The Netherlands, 1UniversityDepartment, Milano, Italy, 3University Genoa, Biostatistic Unit, Department of Health Science, Genoa, Italy, of Genoa, Allergy and Respiratory Diseases,of Department of Internal Medicine, Genoa, Italy, 2IRCCS Istituto Clinico Humanitas, Allergy and respiratory Disease 4HAL Allergy BV, Leiden, The Netherlands, 5Skane Department, Milano, Italy, 3University of Genoa, Biostatistic Unit, Department of HealthMed Science, Genoa, Italy, University Hospital, Dept of Respir & Allergol, Lund, Sweden and QPS-NL, Groningen, The Netherlands

1University

I. Baiardini , F. Puggioni , S. Menoni , J.D. Boot , Z. Diamant , F. Braido , G.W. Canonica1

Allergen Specific Immunotherapy: a real life survey 1 2 3 4 5 1

874 - Patient Knowledge, Perceptions, Expectations and Satisfaction on Perceptions, Subcutaneous and Sublingual 874 - Patient Knowledge, Expectations and Satisfaction on Subcutaneous and Sublingual Allergen Specific Immunotherapy: a real life survey

:

satisfaction

42.7%

37.0% 35.2%

50.8% 56.0%

27.5%

47.1%

68.6%

37.4%

Partly True

-

1.0%

Did not specifiy

EAACI Congress 2012

EAACI Congress 2012

Figure 1: Correlation between patients’ and physicians’ satisfaction level (VAS score) on subcutaneous (left panel; r = 0.612, p <0.001) and sublingual (right panel r = 0.608, p <0.001) SIT

6.4%

22.6%

10.5%

Avoid worsening of the disease

Symptom reduction Less need of anti-allergy medication

38.2%

3.0%

9.2%

8.1%

3.5%

7.6%

Don’t know

Patient answers

5.1%

0.2%

0.2%

21.4%

0.7%

2.8%

1.8%

0.5%

Partly False Completely False 0.2% 0.2%

Patient answers

Improvement in health related quality of life

Recovery from allergy

Possible answers

Table 3: Patients expectation about SIT

SIT is handy to use in daily routine It is easy to remember to take SIT SIT allows to better control your allergy

SIT is easy to take

SIT is safe

Completely True 54.5%

In relation to this presentation, I declare the following, real or perceived conflicts of interest: the presenter has received a travel grant from HAL Allergy.

In relation to this presentation, I declare the following, real or perceived conflicts of interest: the presenter has received a travel grant from HAL Allergy.

expected

perceived

knowledge

Questions

Table 2: Patients perception about SIT

Inf luence of co-morbidities and co-medications on safety and tolerability in the buildup phase of venom immunotherapy

A. Bauer, K. Bernkopf, P. Spornraft-Ragaller.

Department of Dermatology, University Hospital Carl Gustav Carus, TU Dresden, Germany.

Background Safety and tolerability of specific venom immunotherapy (VIT) may be influenced by co-morbidities and co-medications.

In the current study safety and tolerability of a 3 day ultra-rush protocol in patients with co-morbidities and co-medications was assessed during the build-up phase.

Methods Safety and tolerability of VIT (wasp venom n=114; bee venom n=11) was analysed in patients with co-morbidities and comedications. Starting with 0.02 µg venom, the maintenance dose of 100 µg was reached in 15 dose increments within 3 days.

Local and systemic reactions were documented during the build-up phase.

Results 124 patients (67 female/57 male, 51 years range 18-81 years) with an established history of venom allergy were included.

55 (44%) patients suffered from cardiovascular co-morbidities, 22 (17.7%) from metabolic disorders, 24 (19.3%) from skin or respiratory atopy and 18 (14.5%) showed increased baseline tryptase levels >11.4 µg/l. During the build-up phase of VIT local reactions were seen in 46 (37.1%) patients and large local reactions in 69 (55.6%) patients. No significant differences were seen between the groups. Systemic reactions (SR) grade I-III were seen in 10 (8,1%) patients (SR I: 5 (4%) patients, SR II: 4 (3.2%), SR III: 1 (0.8%)). No SR grade IV were seen. Multivariate logistic regression analysis revealed ACE-inhibitor medication (OR 16.14; 95%CI 2.5-104.27), female sex (OR 9.17; 95%CI 1.34-62.97), increased baseline serum tryptase levels (OR 6.68; 95%CI 1.04-43.01) and therapy with bee venom (OR 30.99; 95%CI 3.18-302.34) to be relevant risk factor for SR during the buildup phase of VIT.

Conclusion Safety and tolerability of ultra rush VIT was not influenced by cardiovascular, metabolic or atopic co-morbidities. However, female sex, bee venom sensitization, increased baseline serum tryptase levels and medication with ACE-inhibitors were significantly associated with an increased risk to develop SR during the build-up phase of VIT.

EAACI, 16-20 June 2012, Geneva Abstract number: 1087, Session date and time: Monday 18 June; 12:15 - 13:30 Session title: Poster 46 - Advances in immunotherapy: latest studies

12

Poster Session 46 - Advances in immunotherapy: latest studies

Inf luence of co-morbidities and co-medications on safety and tolerability in the buildup phase of venom immunotherapy

13

Design of a dose range finding study with allergen specific immunotherapy in patients with house dust mite induced allergic rhinitis/rhinoconjunctivitis C. Bachert (1), O. Pfaar (2), A. Roger (3), H. Riechelmann (4), J.N.G. Oude Elberink (5), M.J. Nell (6), J.D. Boot (6).

(1) UZ Gent, Ear-, Nose and Throat Department, Gent, Belgium, (2) Center for Rhinology and Allergology Wiesbaden, Germany, (3) Unitat d’Allèrgia, Hospital Universitari Germans Trias i Pujol, Badalona, Spain, (4) Universitätsklinik für Hals-, Nasen- und Ohrenheilkunde, Innsbruck, Austria, (5) Dept of Allergology, University Medical Center Groningen, University of Groningen, The Netherlands, (6) HAL Allergy BV, Leiden, The Netherlands.

Background In order to comply with the 2008 EMA guidelines on the development of specific immunotherapy (SIT) products, a clinical development program was started to obtain full marketing authorization for a subcutaneous SIT allergoid for the treatment of house dust mite (HDM) allergy. Safety and tolerability of increasing doses have previously been determined in an openlabel clinical study. The next step is a dose range finding (DRF) study to identify the optimal, i.e. safe and effective dose in patients with HDM induced allergic rhinitis/rhinoconjunctivitis (AR). As there is a wide variety of study designs in terms of inclusion criteria, doses, study duration, end-points, analysis of data, and control of environmental variables in the evaluation of SIT products, this abstract highlights the design and rationale of the DRF study.

Method The current study is a 1 year, multicenter, randomized, double-blind, placebo-controlled, 5 arm parallel-group, DRF study (ClinicalTrials.gov NCT01438463). Approximately 250 patients (50 per arm) with persistent, symptomatic AR with or without concomitant asthma related to HDM will be randomized. Due to the need of at least 4 active study arms in a DRF study, the use of a classical endpoint for SIT (i.e. the symptom & medication score) as primary parameter was not feasible because of the necessity of a large sample size. We therefore selected the titrated nasal provocation test (TNPT) with HDM allergen as the primary parameter. The TNPT is a reproducible exacerbation model of allergic rhinitis often applied to evaluate the efficacy of anti-allergy medications. The allergen-induced upper airway response will be quantified by recording symptom scores (Lebel-Score) and nasal flow (Peak Nasal Inspiratory Flow) after each HDM challenge. To minimize seasonal influences the absolute difference in mean symptom score in the TNPT after one year of treatment and baseline will be used as primary endpoint. Patients will only be subjected to a TNPT in the absence of baseline symptoms and patients with seasonal allergies will only be challenged outside the corresponding pollen season. The study is performed in 40 clinical study centers in 5 European countries.

Results The first patient was recruited in October 2011 and results will be available in 2013.

Conclusion According to the EMA guidelines we designed a Phase II DRF study to identify the optimal dose of a subcutaneous SIT allergoid for patients with HDM induced AR.

EAACI, 16-20 June 2012, Geneva Abstract number: 1393, Session date and time: Tuesday 19 June; 12:00 - 13:30 Session title: Poster 68 - New clinical studies in immunotherapy

14

Poster Session 68 - New clinical studies in immunotherapy

Design of a dose range finding study with allergen specific immunotherapy in patients with house dust mite induced allergic rhinitis/rhinoconjunctivitis

15

2

3

4

5

6

6

Methods: Methods: The current study is a 1 year, multicenter, randomized, TNPT & Randomisation TNPT The current study is a 15 year, multicenter, DRF randomized, TNPT & Randomisation 12 months double-blind, placebo-controlled, arm parallel-group, 12 months 3,333 AUeq/dose double-blind, placebo-controlled, arm parallel-group, DRF study (ClinicalTrials.gov NCT01438463). 5Approximately 250 3,333 AUeq/dose 10,000 AUeq/dose study(50 (ClinicalTrials.gov NCT01438463).

Approximately 250 patients per arm) with persistent, symptomatic AR with 25,000 AUeq/dose10,000 AUeq/dose patientsconcomitant (50 per arm) with persistent, symptomatic or without asthma related to HDM will be AR with Screening 50,000 AUeq/dose25,000 AUeq/dose Screening randomized.

Inclusion criteria are presented in Table 1. will be or without concomitant asthma related to HDM Placebo 50,000 AUeq/dose Patients were treated for one year with are mitespresented allergoid (HAL randomized.

Inclusion criteria in Table 1.

TNPT

Background & Aim: Table 1: Inclusion criteria of the DRF study.

In order &toAim: comply with the 2008 EMA guidelines onTable the 1: Inclusion Background criteria of the DRF study.

Inclusion criteria development of specific immunotherapy (SIT) products, a In order to comply with the 2008 EMA guidelines on the Inclusion criteria Signed informed consent clinical development program was started to obtain full development of specific immunotherapy (SIT) products, a Signed informed consent Patients (male or female) must be 18 and 60 years at screening clinical development programforwas started to obtain marketing authorization a subcutaneous SIT full allergoid for or female) must be 18 and 60 years at screening marketing authorization for a dust subcutaneous SIT allergy allergoid1.for the treatment of house mite (HDM) Safety Patients and (malePatients with allergic rhinitis or rhinoconjunctivitis for at least 1 year; allergic 1. Safety and the treatment of house dust mite (HDM) allergy Patients with allergic rhinitis or rhinoconjunctivitis forwithout at least concomitant 1 year; allergicclinically stable symptoms related to HDM, with or tolerability of increasing doses have previously been symptoms related to HDM, with ormoderate without concomitant clinically stable tolerability of increasing doses have previously been controlled mild to asthma (according to GINA classification) determined in an open-label clinical study. The next stepcontrolled is a mild to moderate asthma (according to GINA classification) determined in an open-label clinical study. The next step is a with a history of concomitant asthma should have a FEV1 > 70% at dose range finding (DRF) study to identify the optimal,Patients i.e. with aPatients history of concomitant asthma should have a FEV1 > 70% at dose range finding (DRF) study to identify the optimal, i.e. inclusion. Patients without a history of asthma should have a FEV1 > 70% or a inclusion. Patients without a history of asthma should have a FEV1 > 70% or a effective in patients withinduced HDM induced PEF > 80% safesafe and and effective dose dose in patients with HDM PEF > 80% allergic rhinitis/rhinoconjunctivitis (AR). allergic rhinitis/rhinoconjunctivitis (AR). As thereAsis there a wideis a wide SPT HDMD.D.farpter and/or D.diameter far (mean3mm, wheal diameter 3mm, Positive SPT Positive to HDM D. pterto and/or (mean wheal variety of study designs in terms of inclusion criteria, doses, compared to negative control and should negative control should be negative) variety of study designs in terms of inclusion criteria, doses, compared to negative control and negative control be negative) study duration, end-points, analysis data, andofcontrol of study duration, end-points, analysis of data,ofand control Serum specific pter or (> D. far at screening (> Serum specific IgE-test (ssIgE)IgE-test level for (ssIgE) HDM D. level pter orfor D.HDM far at D. screening environmental variables in the in evaluation of SIT products, environmental variables the evaluation of SIT products, 0.7 U/ml) 0.7 U/ml) this this abstract highlights the design and rationale of the DRF abstract highlights the design and rationale of the DRF Positive TNPTPositive for HDMTNPT D. pterfor extract (Lebel 6 at (Lebel or below HDMatD.screening pter extract at score screening score 6 at or below study.

10,000 AU/ml) study.

10,000 AU/ml)

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

1 UZ Gent, Ear-, Nose and Throat Department, Gent, Belgium, 2 Center for Rhinology and Allergology Wiesbaden, Germany, 3 Unitat d’Allèrgia, Hospital 1 2 4 5 4 Universitätsklinik C. Bachert , O.Trias Pfaar , A. Badalona, Roger 3, H. Riechelmann , J.N.G. Elberink , M.J. Nell6, J.D. Boot6 Austria, 5 Dept of Allergology, für Oude Hals-, Nasenund Ohrenheilkunde, Innsbruck, Universitari Germans i Pujol, Spain, 6 University Medical Center Groningen, University of Groningen, The Netherlands, HAL Allergy BV, Leiden, The Netherlands

1

C. Bachert , O. Pfaar , A. Roger , H. Riechelmann , J.N.G. Oude Elberink , M.J. Nell , J.D. Boot HDM induced allergic rhinitis/rhinoconjunctivitis

1393 - Design of a Dose Range Finding study with allergen specific immunotherapy in patients with 1393 - Design of a Dose Range Finding study with allergen HDM induced allergic rhinitis/rhinoconjunctivitis specific immunotherapy in patients with

Pre Post 100 Diluent Diluent AU/ml (Baseline) 20min. after diluent -

Placebo

10,000 AU/ml

20min.

20min. after after previous previous

1,000 AU/ml

1h after last ch.

Post ch.

EAACI Congress 2012

EAACI Congress 2012

Figure 3: Performance of a nasal challenge (left) and PNIF (right).

Figure 2: Lebel and PNIF score sheet for the TNPT outcome.

PNIF score (highest of 3 measurements)

Nasal obstruction (Difficult nasal breathing=1, One nostril blocked=2, Both nostrils blocked=3) Pruritus (Itchy nose=1, Itchy palate and/or ear=1, Irritated eyes=1) Total LEBEL score

Runny Nose (Posterior=1, Anterior=1)

Sneezing (1-2=0, 3-4=1, 5 =3)

Time of measurement

Actual Time of application

Scheduled time for next challenge

Allergen

Figure 1: Design of the study

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.

References: 1 EMA Guideline on the development of products for specific immunotherapy for the treatment of allergic diseases. CHMP/EWP/18504/2006, London. 20 November 2008.

Conclusion: According to the EMA guidelines we designed a Phase II DRF study to identify the optimal dose of a subcutaneous SIT allergoid for patients with HDM induced AR.

Results: The first patient was recruited in October 2011 and the last patient was enrolled in March 2012. Results will be available in 2013.

Patients were treated for one year with mites allergoid (HAL Allergy, Leiden, The Netherlands) see Figure 1. Due to the need of at least 4 active study arms in a DRF study, the use of a classical endpoint for SIT (i.e. the symptom & medication score) as primary parameter was not feasible because of the necessity of a large sample size. Therefore, the titrated nasal provocation test (TNPT) with HDM allergen as the primary parameter was selected. The TNPT is a reproducible exacerbation model of allergic rhinitis often applied to evaluate the efficacy of anti-allergy medications.

The allergen-induced upper airway response will be quantified by recording symptom scores (Lebel score) and nasal flow (Peak Nasal Inspiratory Flow, PNIF) after each HDM challenge (see Figures 2 and 3). To minimize seasonal influences the absolute difference in mean symptom score in the TNPT after one year of treatment and baseline will be used as primary endpoint. Patients will only be subjected to a TNPT in the absence of baseline symptoms and patients with seasonal allergies will only be challenged outside the corresponding pollen season. The study is performed in 40 clinical study centers in 5 European countries.

Safety of pre- and coseasonal start of sublingual immunotherapy treatment in patients with p ollen allergy

A. Distler (1), H. Kietzmann (2), H. Brüning (2), A. Roger (3), N. Angelova (4), J.D. Boot (4).

(1) HAL Allergie GmbH, Medical Department, Düsseldorf, Germany, (2) CutiCon, Kiel, Germany, (3) Allergy Unit University Hospital Germans Trias Pujol. Badalona, Spain, (4) HAL Allergy BV, Leiden, Netherlands.

Background Start of treatment with sublingual allergen immunotherapy (SLIT) during the relevant pollen season (co-seasonal) may result in more and/or worse adverse events (AEs) compared to the start outside the pollen season (pre-seasonal). A post marketing surveillance study in adults and children treated with SLIT pollen was performed to evaluate safety and clinical efficacy during routine application at home.

Methods Subjects with allergic rhinoconjunctivitis with or without mild asthma due to pollen were treated with SLIT Pollen for approximately 1 year, according to onsite routine. Patients started with one sublingual drop and add one drop each consecutive day until on day 5 the maintenance dose is reached. At each visit, the number of daily drops and AEs were recorded. The patients filled in a dairy during the first 30 treatment days to monitor AEs. The treatment’s efficacy was evaluated after 1 year. The start of the respective pollen season was based on local pollen counts in Germany and Spain.

Results 129 pts (88 female, mean age 38 years) were included in the study: 43.4% received grasses, 8.5% birch, and 47.3% received a combination or other pollen antigens. In total 40.3% of the patients started SLIT co- and 59.7% pre-seasonally. 89.1% of patients reached maintenance dose within 5 days (88.5% co- vs 89.6% pre-seasonal). During up-dosing 37.9% of the patients (38.4% co- vs 37.6% pre-seasonal) experienced a total of 82 adverse drug reactions. 60 local reactions (most common were allergic reactions in the mouth and abdominal pain) and 22 systemic reactions (most common were pruritus of eyes and ears). No difference in the number and severity of adverse drug reactions was observed between the two groups.

Patients’ assessment of their own situation improved in 74.4%, remained equal in 11.6% and was unknown in 13.9% of the patients. No patient reported a worsening of their situation. The satisfaction of patients about the use of SLIT pollen was (very) satisfied in 75.2%, (very) unsatisfied in 11.6% and unknown in 12.4% of the patients.

Conclusion Co-seasonal start of treatment was found to be as safe as the pre-seasonal start of treatment with SLIT pollen. This is practical since it is difficult to up-dose before the season due to lengthening of the pollen season, new pollen allergens and a high number of patients with multiple pollen allergies. Subjects were generally satisfied with their treatment.

EAACI, 16-20 June 2012, Geneva Abstract number: 1394, Session date and time: Tuesday 19 June; 12:00 - 13:30 Session title: Poster 68 - New clinical studies in immunotherapy

16

Poster Session 68 - New clinical studies in immunotherapy

Safety of pre- and coseasonal start of sublingual immunotherapy treatment in patients with p ollen allergy

17

1HAL Allergie GmbH, Düsseldorf, Germany; 2CutiCon, Kiel, Germany; 3Allergy Unit University Hospital Germans Trias Pujol.

3Allergy Unit University Hospital Germans Trias Pujol.

Allergie GmbH, Düsseldorf, Germany; 2CutiCon, Kiel, Germany; Badalona, Spain; 4HAL Allergy BV, Leiden, Netherlands Badalona, Spain; 4HAL Allergy BV, Leiden, Netherlands

20 21 11 1

20 21 11 1

37 33 5 1

37 33 5 1

57 54 16 2

Methods: Methods: study performed 18 clinical centres in Germany ThisThis study waswas performed in 18inclinical centres in Germany Spain.Subjects Subjects a history allergic andandSpain. with with a history of of allergic rhinoconjunctivitis or without concomitant asthma rhinoconjunctivitis with with or without mild mild concomitant asthma ® ® to pollen treated pollen (SUBLIVAC duedue to pollen werewere treated with with SLITSLIT pollen (SUBLIVAC 2: Occurrence of adverse reactions TableTable 2: Occurrence of adverse reactions AllergyBV, BV,Leiden, Leiden, Netherlands) HALHALAllergy The The Netherlands) for for Co-seasonal Pre-seasonal Total approximately 1 year, according to onsite routine.

Patients Co-seasonal Pre-seasonal approximately 1 year, according to onsite routine.

Patients started sublingual added (pts)(pts) started withwith one one sublingual dropdrop and and added one one drop drop each consecutive day until on day 5 the maintenance Patients with adverse reactions 38.4% 38.4% each consecutive day until on day 5 the maintenance dosedose Patients with adverse reactions 37.6% 37.6% 37.9% reached.

Treatment continued during the pollen waswas reached.

Treatment was was continued during the pollen Patients withreactions local reactions Patients with local 34% 34% 34.2% 34.2% 34.1% season.

At each the number of daily drops season.

At each visit,visit, the number of daily drops and and AEs AEs recorded. patients a diary during the first Patients with systemic reactions 9.4% 9.4% werewere recorded.

The The patients filledfilled in a in diary during the first Patients with systemic reactions 13.2% 13.2% 11.6% 30 treatment to monitor 1 year of treatment 30 treatment daysdays to monitor AEs.AEs. AfterAfter 1 year of treatment patients scored allergy complaints compared to before patients scored theirtheir allergy complaints compared to before

- Grasses - Grasses - Trees- Trees - Parietaria sp.

- Parietaria sp.

Olea europea - Olea europea

57 54 16 2

11.6%

34.1%

37.9%

Total

Background & Aim: 1: Patient demographics and administered allergens Background & Aim: TableTable 1: Patient demographics and administered allergens Different updosing schedules for sublingual allergen Different updosing schedules for sublingual allergen Co-seasonalPre-seasonal Pre-seasonal Co-seasonal Total Total immunotherapy (SLIT) exist. of treatment during immunotherapy (SLIT) exist.

StartStart of treatment during the the (n = 129) (n = 129) relevant pollen season (co-seasonal) in more relevant pollen season (co-seasonal) may may resultresult in more and/or worse adverse reactions compared a start outside GenderGender (M / F) (M / F) 20 / 33 20 / 33 20 / 56 20 / 56 40 / 89 40 / 89 and/or worse adverse reactions compared to a to start outside pollen season (pre-seasonal).

To investigate a non the the pollen season (pre-seasonal).

To investigate this, this, a non Mean agein±years SD in years 36.3 ±15.3 36.3 ±15.3 38.3 ±15.3 38.3 ±15.3 37.8 ±15.2 37.8 ±15.2 Mean age ± SD interventional study in adults children treated interventional study in adults and and children treated with with SLITSLIT Adolescents 7 5 12 7 5 12 pollen performed to evaluate safety clinical efficacy Adolescents pollen waswas performed to evaluate safety and and clinical efficacy during routine application. during routine application.

Prescribed allergens 53 76 129 Prescribed allergens 53 76 129

1HAL

1 2 2 3 4 A. Distler , H. Kietzmann , H. Brüning , A. Roger , N. Angelova , J.D.4 Boot 4 1 2 2 3 4 A. Distler , H. Kietzmann , H. Brüning , A. Roger , N. Angelova , J.D. Boot

1394 - Safety preand co-seasonal start of sublingual 1394 - Safety of of preand co-seasonal start of sublingual immunotherapy treatment in patients with pollen allergy immunotherapy treatment in patients with pollen allergy

0% 35%

53%

14%

0%

Number of adverse reactions

33%

Worsened

Much improved Improved Equal

Number of patients [%]

EAACI Congress 2012

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Figure 1: Patientsâ&#x20AC;&#x2122; assessment of improvement in the co- (left) and preseasonal (right) group.

53%

12%

Swelling

Pruritus

Lip pruritus

Gingival pruritus

Dyspepsia

Throat irritation

Oral discomfort

Glossodynia

Ear pruritus

Lip swelling

Paraesthesia oral

Burning sensation

Abdominal pain

Oral pruritus

Swollen tongue

MedDRA Preferred Term

Table 3: Overview of most frequent adverse reactions

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

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

Conclusion: Co-seasonal start of treatment was found to be as safe as the pre-seasonal start of treatment with SLIT pollen. This is practical since it is difficult to up-dose before the season due to lengthening of the pollen season, new pollen allergens and a high number of patients with multiple pollen allergies. Subjects were generally satisfied with their treatment.

Results: 129 pts were included in the study (Table 1). At the time of the study all patients were only prescribed one type of pollen SLIT. The allergens are listed in Table 1.

In total 40.3% of the patients started SLIT in the season (co-seasonal) and 59.7% outside (pre-seasonal) the season. 89.1% of patients reached maintenance dose within 5 days (88.5% co- vs. 89.6% pre-seasonal).

During up-dosing a total of 82 adverse reactions in 49 pts were reported; 60 local reactions (most common were allergic reactions in the mouth and abdominal pain) and 22 systemic reactions (most common were pruritus of eyes and ears). No differences in the number and severity of adverse drug reactions were observed between the two groups (Table 2 and 3).

Almost all patients reported an improvement and no patient reported a worsening of their allergic complaints (Figure 1).

No difference was seen between the co- and pre-seasonal groups. Patients were generally satisfied about the use of SLIT pollen: 34.1% were very satisfied, 41.1% were satisfied, 9.3% were unsatisfied, 2.3% were very unsatisfied and 13.2% were unknown.

treatment. The start of the respective pollen season was based on local pollen counts in Germany and Spain. Based on this data patients were classified as co- or pre-seasonal.

Chemical mo dification of a peanut extract decreases IgE binding while the immunogenicity is maintained

H. van der Kleij (1), J. Smit (2), S.C. Dreskin (3), H. Warmenhoven (1), D. Verbart (1), E. Knol (4), A. Knulst (4), E. Kerkvliet (1), R. van den Hout (1), R. van Ree (5), R. Pieters (2), E. van Hoffen (4), S.J. Koppelman (1).

(1) HAL Allergy BV, Leiden, The Netherlands, (2) Institute for Risk Assessment Sciences, Immunotoxicology, Utrecht University, Utrecht, The Netherlands, (3) University of Colorado Denver School of Medicine, Aurora, CO, USA, (4) Dermatology/Allergology, University Medical Center Utrecht, Utrecht, The Netherlands, (5) Experimental Immunology, Academic Medical Centre, Amsterdam, The Netherlands.

Rationale Peanuts are 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. Peanut sIT could be an option but earlier attempts with aqueous peanut extract were hampered by safety issues. Therefore, a chemically modified peanut extract with improved safety characteristics is being investigated for its potential use in immunotherapy.

Methods A peanut extract (PE) was prepared and modified by reduction of the disulfide bonds and subsequent alkylation of the free Cys residues. The modified extract (mPE) was analysed for biochemical parameters using electrophoreses and far UV CD spectroscopy. Using a set of sera obtained from 20 US and 9 Dutch peanut allergic patients, solid-state IgE-binding assays and mediator release assays (MRA) using RBL SX38 cells and human basophils were performed. The potency ofPE and mPE to activate T-cells was evaluated using PE-specific T-cell lines from 9 peanut-allergic patients. The potency of PE and mPE to induce PE-specific IgG was evaluated in mice too. Furthermore, the in vivo safety of PE and mPE was tested by measuring body temperature upon subcutaneous challenges in mice allergic to PE.

Results The IgE-binding of mPE was for all patients tested lower than for PE (mean remaining potency: 7.2% Âą 5%), which was confirmed by IgE-immunoblotting. The potency of mPE in MRA was for all patients tested lower than for PE (mean reduction: 10 to100 fold, range 3 to >10,000 fold). PE-specific T-cell lines were activated equally well with mPE and PE. Mice sensitized with PE experienced a severe anaphylactic reaction upon subcutaneous challenge starting at 0.01 mg PE, while 0.3 mg mPE was tolerated without signs of an allergic reaction. PE and mPE were equally potent in inducing PE-specific IgG antibodies in mice.

Conclusions Using in vitro and in vivo models, we have shown that an allergoid preparation of peanut extract has a reduced allergenicity compared to its native counterpart, while the immunogenicity is maintained.

EAACI, 16-20 June 2012, Geneva Abstract number: 4, Session date and time: Sunday 17 June; 10:30 - 12:00 Session title:Â OAS 1 - Allergen immunotherapy: new aspects in diagnostics and treatment

18

OAS 1 - Allergen immunotherapy: new aspects in diagnostics and treatment

Chemical mo dification of a peanut extract decreases IgE binding while the immunogenicity is maintained

19

2

3

1

1

4

4

1

75

A.A. 100 100

25

12.5

6.25

3.13

PE RA-PE

100 PE PE RA-PE RA-PE

Proliferation (SI)

Proliferation (SI)

150

100 100

150 150

Proliferation (SI)

100

Proliferation (SI)

A.

25

12.5

6.25

3.13

PE RA-PE

100

PE PE RA-PE RA-PE

stimulation index

100 100

p=0.25 p=0.25

p=0.25

200

Proliferation (SI)

300 300

blanc 0

25

50

Proliferation (SI)

50

1.56

μ g/ml)6.25 3.13 1.56 0 conce blanc 50ntration 25 (12.5 blanc 50 25 12.5 6.25 3.13 1.56 conce ntration ( μ g/ml) conce ntration ( μ g/ml)

25

50

75

0

50

50

1.56

Specific IgG titres at day 63 after 5 immunizations (i.p.) with 10 or 100 µg

μ g/ml) 6.25 3.13 1.56 0 concentration blanc 50 25 (μ 12.5 blanc 50 25 12.5 6.25 3.13 1.56 concentration (μ μ g/ml) concentration (μ μ g/ml)

50

Figure 2: Specific IgG responses to native versus modified PE. Figure 2: Specific responses to native modified Specific IgG titres day 63IgG after immunizations (i.p.) versus with 10 or 100 µg PE. Figure 2:atSpecific IgG 5responses to native versus modified PE.

blanc 0

50

IgG (AU)

n (%)

75

IgG (AU) IgG (AU)

(%) )

75

75

PE-RA PE-RA

D48

D44

D19 D19 D44 D44

16,9

0,4

0,4

1,3

0,4

1,3

product.Specific Control IgG micetitres wereatinjected only. Dots represent theor 100 µg 200 day 63 with aftermatrix 5 immunizations (i.p.) with 10 The Peanut extract (PE) preparation isis 200 product.

Controlmice miceinwere injected with matrix only.

Dots represent the IgG titre of individual arbitrary units (AU) and bars represent the The Peanut extract (PE) preparation product. Control mice were injected with matrix only. Dots represent the chemically modified by reduction of the IgGtitre titreofofeach individual in arbitrary units (AU) and bars represent the median group.mice IgG titre of individual mice in arbitrary units (AU) and bars represent the chemically of the 100 chemicallymodified modifiedby byreduction reduction median titre of each group. median titre of each group.

100 intramolecular disulfide bonds of PE, of the 100 intramolecular disulfide bonds ofofPE, intramolecular disulfide bonds PE, and alkylation of the resulting free 0 and alkylation ofof the resulting free 10 μg 100 μ g 10 μg 100 μg con and alkylation the resulting free 0 0 sulfhydryl groups (RA). μgμg 100 μg 1010 μ gμ g 100 μ gμ g 1010 100 100 μg con con PE PE-RA sulfhydryl sulfhydrylgroups groups(RA).

(RA).

PEPE PE-RA PE-RA For immunogenicity, the allergoid was For Forimmunogenicity, immunogenicity,the theallergoid allergoidwas was A. 100 adsorbed to aluminium hydroxide.

PE Sample Realative potency (%) B. A.A.100100 adsorbed Realative potency (%) 1,3 B.B. D19 Sample PE-RAPEPE adsorbedtotoaluminium aluminiumhydroxide. hydroxide.

Sample Realative potency (%)

Methods: Methods: The Methods: Peanut extract (PE) preparation is

300

0

25

50

75

• •Peanuts are ofofthe 10 Peanuts areone one themost mostcommon common foods responsible for food-induced 10 foods responsible for food-induced 10 foods responsible for food-induced anaphylaxis in adults. anaphylaxis ininadults.

1 anaphylaxis adults.

• A curative treatment is not available.

PE-RA 1 PE • •AAcurative treatment isisnot available.

1 PE PE-RA curative treatment not available.

PE PE-RA • A modified peanut extract (allergoid) is Figure 1: Response of PE-specific TCLs (T cell lines) to native PE and RA-PE. • •AAmodified peanut extract (allergoid) is modified peanut (allergoid) isProliferation Figurewas 1: Response as of PE-specific lines) to native PE and RA-PE. divided by blanc). A. Representative stimulationTCLs index (T (SI,cell proliferation to allergen stimulation being investigated for itsextract potential use Figure 1:expressed Response the of PE-specific TCLs (Ti.e., cell lines) to native PE and RA-PE. Proliferation expressed as the stimulation index (SI, to9allergen being investigated for its potential use examples of a TCLwas of an allergic patient.

B. Immunogenicity ofi.e., PE proliferation and RA-PE in TCLs. stimulation divided by blanc). A. Representative Proliferation was expressed as the stimulation index (SI, i.e., proliferation to allergen stimulation divided by blanc). A. Representative being investigated for its potential use in immunotherapy. examples of a TCL of an allergic patient. B. Immunogenicity of PE and RA-PE in 9 TCLs. examples of a TCL of an allergic patient. B. Immunogenicity of PE and RA-PE in 9 TCLs. ininimmunotherapy. immunotherapy.

Background & Aim: Background &&Aim: Background Aim: • Peanuts are one of the most common

1HAL Allergy BV, Leiden, The Netherlands, 2Institute for Risk Assessment Sciences and Utrecht Centre for Food Allergy, Utrecht, 3 4Dep. Dermatology/Allergology, UMC Utrecht, Utrecht, Colorado School2of Medicine, Aurora, CO, USA, Sciences The Netherlands, 1HAL University Allergy BV,ofLeiden, TheDenver Netherlands, Institute for Risk Assessment and Utrecht Centre for Food Allergy, Utrecht, 1HALThe 2Institute 5Experimental Allergy BV, Leiden, The Netherlands, for RiskAurora, Assessment Sciences Utrecht Centre for Food Allergy, Utrecht, Netherlands, Immunology, Academic Medical Centre, Amsterdam, The Netherlands 3University 4Dep.and The Netherlands, of Colorado Denver School of Medicine, CO, USA, Dermatology/Allergology, UMC Utrecht, Utrecht, 3 4 University of Colorado Denver School of Medicine, Aurora, CO, USA, Dermatology/Allergology, The Netherlands, The 5Experimental Netherlands, Immunology, Academic Medical Centre,Dep. Amsterdam, The Netherlands UMC Utrecht, Utrecht, The Netherlands, 5Experimental Immunology, Academic Medical Centre, Amsterdam, The Netherlands

1, R. van Ree 4, S.J. 1 1, J. 2, S.C. 1, D. 1, E.Koppelman 4, E. Kerkvliet1, Pieters2, E. van Hoffen R. van den Hout H. van der Kleij Smit Dreskin3,5,H.R.Warmenhoven Verbart Knol4, A. Knulst 1, D. Verbart1, E. Knol4, A. Knulst4, E. Kerkvliet1, H. van der Kleij1, J. Smit2, S.C.1 Dreskin3, H.5 Warmenhoven 2, E. van 4, S.J. Koppelman1 R. van den Hout , R. van Ree , R. Pieters Hoffen R. van den Hout1, R. van Ree5, R. Pieters2, E. van Hoffen4, S.J. Koppelman1

1

4 - Chemical modification of a peanut extract decreases 44- -Chemical modification ofofaapeanut decreases Chemical modification peanutextract extract decreases IgE binding while the immunogenicity is maintained IgE binding while the immunogenicity is maintained IgE binding while the immunogenicity is maintained H. van der Kleij , J. Smit , S.C. Dreskin , H. Warmenhoven , D. Verbart , E. Knol , A. Knulst , E. Kerkvliet ,

stimulation index stimulation index

concentration (ng/ml) concentration (ng/ml)

13

PE PE PE-RA PE-RA

100 100

relative basophil activation (%)

relative basophil activation (%)

Inhibition Inhibition (

p<0.01 p<0.01

Day:Day: 0 01

12

2

D48 D48 D50 D50 D60 D60 D62 D62 D64 D64 D65 D65 D68 D68 D69 D69 D70 D70 D74 D74 D77D77

14 14

16,9 16,9 1,4 1,4 5,3 5,3 4,1 4,1 17,8 17,8 0,3 0,3 4,3 4,3 11,3 11,3 9,1 9,1 4,5 4,5 11,811,8

PE PE

RA-PE RA-PE

Challenge by by Challenge subcutaneous injection subcutaneous injection

42 42

32 32 30 30 28 28 0 010 10 20 20 30 30 40 40 50 50 60 60 70 70 80 80 90 90 100100 Time after challenge (minutes) Time after challenge (minutes)

30 30

28 28 0 010 10 20 20 30 30 40 40 50 50 60 60 70 70 80 80 90 90 100100 Time after challenge (minutes) Time after challenge (minutes)

3 mg PE-RA 3 mg PE-RA

0.6 0.6 mgmg PE-RA PE-RA

28 28

32 32

36 36

38 38

21 21

6 mg PE +PE 15+ 15 g Cholera Toxin by oral gavage 6 mg g Cholera Toxin by oral gavage

7

34 34

3 mg PE PE 3 mg

0.6 0.6 mgmg PE PE

0.010.01

7

34 34

36 36

100 100 10001000 310 10

1

0.1 0.1

1

10 10

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

EAACI Congress 2012

EAACI Congress 2012 EAACI Congress 2012

Figure 6: 6: Body temperature following i.p.i.p. challenge; Anaphylaxis to to thethe native preparation.

AnAn i.p.i.p. challenge with 0.60.6 mgmg PEPE perper Figure Body temperature following challenge; Anaphylaxis native preparation. challenge with mouse resulted in an anaphylactic shock response.

The mice in the groups challenged with modified PEPE showed small decreases in in mouse resulted in an anaphylactic shock response.

The mice in the groups challenged with modified showed small decreases temperature at the highest dose (3 (3 mgmg RA-PE).

Control mice diddid notnot respond to the challenge (with 3 mg of PE or or PE-RA). temperature at the highest dose RA-PE).

Control mice respond to the challenge (with 3 mg of PE PE-RA).

% CD63+ basophils

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

An An allergoid allergoid preparation preparation ofof peanut peanut extract extract has has a a reduced reduced allergenicity allergenicity compared comparedtotoitsitsnative nativecounterpart, counterpart,while while the theimmunogenicity immunogenicityisismaintained. maintained.

Conclusion: Conclusion:

38 38

0 concon 0.030.03 0.010.01 0.3 0.3 1

Temperature (Celcius) Temperature (Celcius)

0

25 25

% CD63+ basophils

50 50

25 25 Efficacy: Efficacy: 0 0 • •PE-specific PE-specifichuman humanT-cell T-celllines lineswere were 0.01 0.10.1 1 1 10 10 100100 0.01 inhibitor concentration (μ μg/ml) inhibitor concentration (μ μg/ml) activated activatedequally equallywell wellwith withPE PEand and Figure 3: 3: Inhibition ELISA. Figure Inhibition ELISA. D80D80 2,8 2,8 RA-PE. RA-PE. A. A. Inhibition curves of the native and modified PEPE of a serum Inhibition curves of the native and modified ofUS a US serum D81D81 8,2 8,2 pool of 20 patients.

Test samples compete with PE coated to ELISA pool of 20 patients. Test samples compete with PE coated to ELISA D98D98 8,2 8,2 • •InInmice, mice,PE PEand andRA-PE RA-PEwere wereequally equally plates forfor binding to serum IgE. Increasing concentrations of test D103 12,212,2 plates binding to serum IgE. Increasing concentrations of test D103 6,2 6,2 D105 sample result in inhibition of binding.

The amount of the IgEIgE binding D105 sample result in inhibition of binding.

The amount of the binding potent potentinininducing inducingPE-specific PE-specific D107 8,8 8,8 in presence of PE compared to that in absence of PE is calculated as D107 in presence of PE compared to that in absence of PE is calculated as D114 5,9 5,9 D114 percentage inhibition.

IgG percentage inhibition.

IgGantibodies antibodiesininmice. mice. pool 1,3 1,3 pool Safety: Safety: CPE determined in an IgEIgE inhibition ELISA using sera of 20 peanut allergic patients from B. B. IgEIgE potency of RA-CPE relative to native CPE determined in an inhibition ELISA using sera of 20 peanut allergic patients from potency of RA-CPE relative to native thethe US. An in-house reference (IHR) allergen preparation is tested to compare the inhibition curves by parallel line analyses.

The IgEIgE US. An in-house reference (IHR) allergen preparation is tested to compare the inhibition curves by parallel line analyses. The • •Modification Modificationlowered loweredthe theIgE-binding IgE-bindinginin binding efficiency is expressed as as relative potency (RP) compared to the IHR. binding efficiency is expressed relative potency (RP) compared to the IHR. allallpatients to a mean remaining potency patients to a mean remaining potency ofof7.2% 7.2% 5%.

5%.

• •The Themediator mediatorrelease releaseassay assay(MRA) (MRA) showed showeda areduced reducedpotency potencyofof10-100 10-100 fold fold(range (range3 3toto>10,000 >10,000fold). fold).

• •Mice Micesensitized sensitizedwith withPE PEexperienced experienceda a Figure 5: 5: Animal model.

Mice were sensitized with 6 mg PEPE Figure Animal model.

Mice were sensitized with 6 mg severe severeanaphylactic anaphylacticreaction reactionupon upon Figure 4: 4: The potency of of native PEPE and RA-PE Figure The potency native and RA-PE and 1515 µgµg Cholera Toxin viavia oral gavage and challenged (i.p.) and Cholera Toxin oral gavage and challenged (i.p.)

Representative example. in in thethe indirect MRA. A. A. Representative example. indirect MRA. with PE,PE, PEPE adsorbed to alum, RA-PE preparation and RA-PE with adsorbed to alum, RA-PE preparation and RA-PE subcutaneous subcutaneouschallenge challengewith with0.1 0.1mg mg B. B. Relative potency of native PEPE and RA-PE to activate basophils adsorbed to alum (n=6 perper group).

Relative potency of native and RA-PE to activate basophils adsorbed to alum (n=6 group).

PE, PE,while while3 3mg mgRA-PE RA-PEwas wasstill still 40 40 40 40 tolerated toleratedwithout withoutproblems. problems.

0.1 0.1 mgmg PE-RA PE-RA 0.1 0.1 mgmg PE PE

Results: Results:

50 50

Temperature (Celsius) Temperature (Celsius)

Allergenic p otency of chemically mo dified Ara h2/h6 evaluated with basophil activation, RBL-SX38 activation, and solid phase IgE-immunoassay H. van der Kleij (1), H. Warmenhoven (1), D. Verbart (1), Q. Wang (2), R. van Ree (3), S.C. Dreskin (2), S.J. Koppelman (1).

(1) HAL Allergy BV, Leiden, The Netherlands, (2) University of Colorado Denver School of Medicine, Aurora, CO, USA, (3) Experimental Immunology, Academic Medical Centre, Amsterdam, The Netherlands.

Rationale Peanuts are one of the most common foods responsible for food-induced anaphylaxis and there is no curative treatment for peanut allergy. Peanut sIT could be an option but earlier attempts with aqueous peanut extract were hampered by safety issues. Major peanut allergens Ara h2 and Ara h6 have been modified to make them suitable for immunotherapy.

This study evaluates the potency of modified Ara h2 and Ara h6 in mediator release assays in comparison with native, not modified Ara h2 and Ara h6.

Methods Ara h2 and Ara h6 were purified and chemically modified by means of reduction of disulfide bonds and subsequent alkylation of free Cys. Twenty patients from the greater Denver area were recruited and serum was collected. Total- and peanut-specific IgE was determined as well as IgE to individual peanut allergens. RBL SX38 cell expressing the human IgE receptor and basophils from healthy donors were loaded with the serum samples and were subsequently exposed to different concentrations of either native or modified Ara h2 / Ara h6. Mediator release was determine and expressed as percentage of maximum release. Potency of native and modified Ara h2 / Ara h6 was also evaluated applying a solid phase IgE- binding assay performed with individual patient sera.

Results For all tested patient sera it was observed that the potency of modified Ara h2 /Ara h 6 was reduced compared to native Ara h2 / Ara h6. Using the RBL-SX38 assay a mean reduction of 100- fold was found (range: 3 to <10,000 fold). Similar data were found with the basophil assay and the fold reduction observed per patient correlated between the two mediator release tests. For modified Ara h2 / Ara h6, IgE-binding as determined using the solid phase IgE-binding assay was for all patient sera reduced compared to native Ara h2 / Ara h6. Sera for which the modification had the largest effect on the activity in mediator release assays also showed the largest reduction in potency using the solid-phase IgE binding test.

Conclusions The allergenic potency of modified Ara h2/Ara h6 is about 100-fold lower that that of native Ara h2/Ara h6, making them more suitable candidates for subcutaneous immunotherapy.

EAACI, 16-20 June 2012, Geneva Abstract number: 62, Session date and time: Sunday 17 June; 15:30 - 17:00 Session title:Â OAS 11 - Molecules and multiplexing in allergy diagnosis

20

OAS 11 - Molecules and multiplexing in allergy diagnosis

Allergenic p otency of chemically mo dified Ara h2/h6 evaluated with basophil activation, RBL-SX38 activation, and solid phase IgE-immunoassay

21

Methods: • Ara h2 and Ara h6 were purified and Methods: chemically by means of and • Ara h2 andmodified Ara h6 were purified reduction (R) of disulfide bonds and chemically modified by means of subsequent alkylation (A) of free Cys. reduction (R) of disulfide bonds and Preparations were characterized by subsequent alkylation (A) of free Cys. SDS-PAGE and by far-UV CD Preparations were characterized by spectoscropy.

SDS-PAGE and by far-UV CD • Twenty patients from the greater spectoscropy.

Denver area were recruited and serum • Twenty patients from the greater was collected. area were and serum • Denver RBL SX38 cells andrecruited basophils from

Background & Aim: Background Aim: • Peanuts are & one of the most common • Peanuts are one most common foods responsible of forthe food-induced foods responsible for food-induced anaphylaxis in adults. in adults.

• anaphylaxis Peanut sIT with aqueous peanut extract has failed because of safety issues.

• Peanut sIT with aqueous peanut extract • has A modified preparation of the issues.

2 major failed because of safety peanut allergens Ara h2 of and Ara h6 is •A modified preparation the 2 major being investigated for its peanut allergens Ara h2 potential and Ara use h6 is in immunotherapy being investigated for its potential use in immunotherapy 1

2

-5

-15

-15 -10

-10

-5

0

5

0

10

15

5

10

200

200

D60

D50

D48

D70 D65 D68

D68 D62 D69 D64

D65

D64

D62

D60

D44

D19

73

37

306

533

138

1015

293

290 2803 1112 2421

293

2421

2803

37

306

533

138

D48 D50

73 Total IgE 1015

D19 Sample D44

500

52

180

0

0

0

0,8

3,4

13

591

0 Relative 0,2 potency (%) 0,4 0 0,3 0,2 0,5 0,4 0,3 0,3 0,8 0,5 0 0,3 0

Peanut65 Specific IgE 71 39 38 65 56 71 13 38 591 56 180 13 52

39

Total IgE PeanutRelative Table 1: Serology ofSpecific recruited patients IgE potency (%)

Sample

Table 1: Serology of recruited patients

210

220

240

230

230

220

240

250

100

0 0.01

25

50

75

100

native

native

RA

D103

0 0.01

RA

0 0.01

25

50

75

0.1 1 10 100 1000 Inhibitor concentration (μ μg/ml) D64 100

1 10 100 1000 250.1 Inhibitor concentration (μ μg/ml)

50

75

100

native

D105 RA

RA

0 0.01

0.1 1 10 100 1000 InhibitorD114 concentration (μ μg/ml)

0.1 1 10 100 1000 25 Inhibitor concentration (μ μg/ml)

50

75

native 100

100 Figure D103 2: Solid phase IgE-binding test

D105

B. Native conglutin shows a typical spectrum for proteins consisting mainly B. Native conglutin shows a typical Nativ e of alpha-helices (minimal ellipticity spectrum for proteins consisting mainly RA values at 208 and 222 nm) but also of alpha-helices (minimal ellipticity beta-structure. In contrast, RA shows a values at 208 and 222 nm) but also typical spectrum for unfolded protein.

260 beta-structure. In contrast, RA shows a Strong decrease of ellipticity at 222 nm typical spectrum for unfolded protein.

250 260 indicates loss of alpha-helices and Strong decrease of ellipticity at increase of random coil structure. This 222 nm indicates by loss offact alpha-helices and that the can be explained the increase random structure. This disulphide bridgesofhave beencoil cleaved. can be explained by the fact that the disulphide bridges have been cleaved.

Figure 2: Solid phase IgE-binding test

Wavelength (nm)

Wavelength (nm)

210

RA

Nativ e

15 B. The far-UV CD spectra of native and modified conglutin.

A. The native (1) and RA (2) Ara h2/h6 preparations were analyzed by SDS-PAGE. The proteins were A. The using nativeCoomassie-staining.

(1) and RA (2) Ara h2/h6 preparations visualized were analyzed by SDS-PAGE. The proteins were visualized using Coomassie-staining.

Mw

CD (mDeg)

A.

CD (mDeg)

Figure 1. Characteristics of peanut preparations B. The far-UV CD spectra of native and modified conglutin.

Mw 1 2

Inhibition (%)

A.

Figure 1. Characteristics of peanut preparations

HAL Allergy BV, Leiden, The Netherlands, 2 University of Colorado Denver School of Medicine, Aurora, CO, USA 3 Immunology, Academic Medical Centre, Amsterdam, The Netherlands HAL AllergyExperimental BV, Leiden, The Netherlands, 2 University of Colorado Denver School of Medicine, Aurora, CO, USA 3 Experimental Immunology, Academic Medical Centre, Amsterdam, The Netherlands

Inhibition (%)

1

Inhibition (%)

1

H. van der Kleij1, H. Warmenhoven1, D. Verbart1, Q. Wang2, R. van Ree3, S.C. Dreskin2, S.J. Koppelman1 H. van der Kleij1, H. Warmenhoven1, D. Verbart1, Q. Wang2, R. van Ree3, S.C. Dreskin2, S.J. Koppelman1

62 -- Allergenic Ara h2/h6 evaluated 62 Allergenic potency potencyof ofchemically chemicallymodified modified Ara h2/h6 evaluated with basophil activation, RBL-SX38 activation, and with basophil activation, RBL-SX38 activation, and solid phase IgE-immunoassay solid phase IgE-immunoassay

Inhibition (%)

D114

22

65

14

0 2,0

0,7

1

ng/ml

10

Serum pool

100

RA

native

1000

0

10

20

30

RA

0.1 1 10 100 1000 Inhibitor concentration (μ μ g/ml)

native

D64

0 0.01

25

50

75

100

RA

0.1 1 10 100 1000 Inhibitor concentration (μ μg/ml)

native

D114

10

ng/ml

D65

100

RA

native

1000

0

10

20

30

1

10

ng/ml

D80

100

RA

native

1000

IgE inhibition curves of the native and modified Ara h2/6 of individual patients.

Test samples compete with native Ara h2/6 coated to ELISA plates for binding to serum IgE. Increasing concentrations of test sample result in inhibition of binding. The amount of the IgE binding in presence of Ara h2/6 compared to that in absence of Ara h2/6 is calculated as percentage inhibition.

0 0.01

25

50

75

100

1

ng/ml

100

RA

native

D64

10000

0 0.01

10

20

30

40

50

60

1

native

100

ng/ml

D65

10000

RA

0.01

0

10

20

30

40

50

60

1

100

ng/ml

D68

10000

RA

native

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

EAACI Congress 2012

EAACI Congress 2012

In the indirect BAT, human basophils were sensitized with individual sera and triggered with native Ara h 2/6 or RA-Ara h 2/6. Data are shown as % release (in relation to total degranulation minus background).

0.01

0

10

20

30

40

50

60

release (%)

Figure 4: Degranulation of human basophils to native or modified Ara h2/6

RBL SX-38 cells were sensitized either with a serum pool (A) or individual sera (B and C) and triggered with native Ara h 2/6 or RA-Ara h 2/6.

Data are shown as “net” degranulation (total degranulation minus background).

0 0.1

10

20

30

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

Conclusion: The allergenic potency of modified Ara h2/6 is about 100-fold lower than that of native Ara h2/Ara h6, making them more suitable candidates for subcutaneous immunotherapy.

117

D107

6,4

0,4

9,4

9,7

1,5

0,5

IgE binding potency of the native and modified preparations were determined in 20 US patients with established clinical allergy for peanut. The IgE binding is expressed as relative potency (RP) compared to an in-house reference allergen preparation.

88 199

D105

787

285

49

29

0

3,4

Inhibition (%)

Figure 3: Degranulation of RBL SX-38 cells to native or modified Ara h2/6

net % degranulation release (%)

Results: All tested patient sera displayed a reduced response to RA-Ara h2/6: • A mean reduction of 100- fold was found (range: 3 to <10,000 fold) using both the RBL SX38 and the indirect basophil assay.

• The fold reduction observed per patient correlated between the two mediator release tests.

• Sera for which the modification had the largest effect on the activity in mediator release assays also showed the largest reduction in potency using the solidphase IgE binding test.

16

80

219

D81 1797

545

D80 D98

505

D77

D103

81

194

D74

13

D70

500

290 1112

D69

net degranulation release (%)

was collected.

• RBL SX38 cells and basophils from healthy donors were loaded with the serum samples and subsequently exposed to either native Ara h2/6 or modified Ara h2/6 (RA-Ara h2/6).

• Potency was also evaluated applying a solid phase IgE- binding assay performed with individual patient sera.

net degranulation

Inhibition (%)

The content of allergens Ara h1, Ara h2, Ara h3 and Ara h6 in different peanut cultivars commonly consumed in Europe and the USA

S.J. Koppelman(1,2), D. Apostolovic (1), H. Warmenhoven (1), D. Verbart (1), S. L. Taylor (2), T. Isleib (3), S. Maleki (3).

(1) HAL Allergy BV, Leiden, The Netherlands, (2) Food Allergy Research and Resources Programme, University of Nebraska, Lincoln NE, USA, (3) US Dept of Agriculture, New Orleans LA, USA.

Rationale Peanut allergens Ara h2 and Ara h6 are considered to be the most relevant peanut allergens, while Ara h1 and to a lesser extent Ara h3, may play an important role as well. There are four main peanut cultivars commonly consumed in the Western world and their major allergen content is only partially known. Also, the effect of roasting on the extractability of major allergens is not well known.

Methods Twelve peanuts samples comprising the four main cultivars Runner, Spanish, Valencia, and Virginia, were obtained and part of these sample were roasted (140 째C for 15 minutes in a preheated circulating hot air oven). Extracts were made and total protein content was measured using Bradford analysis. SDS-PAGE was used to visualize the protein bands patterns and the content of Ara h1, Ara h2, Ara h3, and Ara h6 was quantified using rp-HPLC. A solid phase IgE binding test was applied for evaluation of allergenic potency.

Results Protein concentration in varietal extracts varied from 13.9 to 21.2 mg/ml, and roasting reduced the extractability by 22 to 50%.

Band patterns indicated that the four allergens of interest were present in samples of all varieties. The ranges of individual allergens in the tested samples were as follows: Ara h1: 11-32%; Ara h2: 7-16%; Ara h3: 38-76%; and Ara h6: 4-14%. In extracts of roasted peanuts, the relative content of Ara h1 was lower while that of Ara h3 was higher compared to non-roasted. The relative content of Ara h2 and Ara h6 did not vary between raw and roasted. The allergenic potencies compared to inhouse reference extracts varied from 99 to 139% for the raw samples, and roasting increased the potency only marginally with an increase of 14 to 30% relative to non-roasted counterparts.

Conclusions The main peanut cultivars consumed in the Western world all contain the main peanut allergens Ara h1, Ara h2, Ara h3, and Ara h6 with a slight variation of the relative content of these allergens between the different cultivars. Roasting had minimal effects on allergenic potency with the sera used in this study.

EAACI, 16-20 June 2012, Geneva Abstract number: 1463, Session date and time: Tuesday 19 June; 12:00 - 13:30 Session title: Poster 73 - Molecular components in allergy diagnosis

22

Poster Session 73 - Molecular components in allergy diagnosis

The content of allergens Ara h1, Ara h2, Ara h3 and Ara h6 in different peanut cultivars commonly consumed in Europe and the USA

23

Methods: Methods: Twelve peanuts samples comprising the four Twelve peanuts samples comprising the four main cultivars Runner, Spanish, Valencia, main cultivars Runner, Spanish, Valencia, and Virginia, were obtained and part of and Virginia, wereroasted obtained C forpart 15 of these sample were (140 and C for these sample were roasted (140 hot minutes in a preheated circulating air 15 oven).

Extracts made and total protein minutes in a were preheated circulating hot air content was measured using Bradford oven). Extracts were made and total proteinA. analysis.

SDS-PAGE was used to visualize content was measured using Bradford A. the proteinSDS-PAGE bands patterns thetocontent analysis. wasand used visualize of h1, Ara h2, Ara h3, and Arathe h6 content was theAra protein bands patterns and quantified A solid IgEwas of Ara h1,using Ara rp-HPLC. h2, Ara h3, andphase Ara h6 binding test was applied for evaluation quantified using rp-HPLC. A solid phaseofIgE 800 allergenic potency. An extract from Virginia

well known.

Background Background&&Aim: Aim: Peanut allergens AraAra h6 h6 areareA. A. Peanut allergensAra Arah2h2and and considered peanut consideredtotobebethe themost mostrelevant relevant peanut allergens, while Ara h1 and to a allergens, while Ara h1 and to lesser a lesser extent anan important rolerole as as extentAra Arah3, h3,may mayplay play important well. There are four main peanut cultivars well. There are four main peanut cultivars commonly consumed in the Western world commonly consumed in the Western world and their major allergen content is only and their major allergen content is only partially known. Also, the effect of roasting partially known. Also, the allergens effect of is roasting on the extractability of major not B. on the extractability of major allergens is not B. well known.

A215 (mAU)

A215 (mAU)

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600

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used for band assignment (right margin).

Purified allergens Ara h1, Ara h2, Ara h3, and Ara h6 were used for band assignment (right margin).

Purified allergens Ara h1, Ara h2, Ara h3, and Ara h6 were

14. Virginia; Brantley Virginia (Raw)

A. Peanut sample analysed under reducing conditions.

A. Peanut sample analysed under reducing conditions.

B. Peanut sample analysed under non-reducing conditions.

B. Peanut sample analysed under non-reducing conditions.

1. Bio-Rad Molecular weight marker (kDa) 1. Bio-Rad Molecular weight marker (kDa) 2. Laemmli sample buffer 2. Laemmli sample buffer 3. Runner; GAO6G Runner (Raw) 3. Runner; GAO6G Runner (Raw) 4. Runner; Tiftguard Runner (Raw) 4. Runner; Tiftguard 5. Runner; Florunner 4/14 (Raw) Runner (Raw) 5. Runner; Florunner 4/14 (Raw) 6. Runner; Florunner 4/14 (Roasted) Runner; 7. Spanish; 6.

Olin SpanishFlorunner (Raw) 4/14 (Roasted) Spanish; Spanish (Raw) 8. Spanish; 7.

Olin SpanishOlin (Roasted) Spanish; Olin Spanish (Roasted) 9. Valencia;8.

Valencia A (Raw) 9.Valencia Valencia; 10. Valencia; A Valencia (Roasted)A (Raw) 11. Virginia;10.

Nc-v11 Virginia (Raw) A (Roasted) Valencia; Valencia 12. Virginia;11.

Nc-v11 Virginia (Roasted) Virginia; Nc-v11 Virginia (Raw) 13. Virginia;12.

Gregory Virginia (Raw) Virginia; Nc-v11 Virginia (Roasted) 14. Virginia;13.

Brantley Virginia (Raw)Virginia (Raw) Virginia; Gregory

Figure 1: SDS-PAGE of the various peanut samples Figure 1: SDS-PAGE of the various peanut samples and purified peanut allergen references and purified peanut allergen references

2Food Allergy 2Food Allergy Allergy BV, Leiden, The Netherlands, Research and Resources Program, Program, UniversityUniversity of HAL Allergy BV, Leiden, The Netherlands, Research and Resources of 3US Dept ofDept Agriculture, New Orleans USA LA, USA Nebraska, Lincoln, NE, USA, of Agriculture, New LA, Orleans Nebraska, Lincoln, NE,3US USA,

A215 (mAU)

1HAL 1

1,2, D. Apostolovic 1, H. Warmenhoven 1, D. Verbart 1, 1,2, D. 1, D. Verbart 1, Koppelman S.J.S.J. Koppelman Apostolovic1, H. Warmenhoven 2 3 3 2 3 3 Taylor , T. Isleib S. Maleki S.L. S.L. Taylor , T. Isleib and S.and Maleki

1463- The - The content allergens 1463 content of of allergens AraAra h1, h1, Ara Ara h2, h2, Arah3h3and andAra Ara in different peanut cultivars Ara h6h6 in different peanut cultivars commonly consumed in Europe the USA commonly consumed in Europe andand the USA

A215 (mAU)

5 Time (min)

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Inhibitor concentration (Âľg/ml)

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10,00

Extracts of the peanut extracts were diluted in a range of 0.01 to 10 g/ml. Examples of of Virginia peanut (Green: Brantley raw; Yellow: Gregory raw; Red: NC-v11 roasted; Blue: NC-v11 raw) are shown in comparison to the IHR. The plasma pool used consisted of 14 plasmas from patients, all clinically allergic to peanut, mean IgE to peanu 36,9 kU/L.

Figure 3: Potency determination of the various extracts using human IgE ELISA.

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

EAACI Congress 2012

EAACI Congress 2012

Table 1: Extractable protein, content of individual allergens and IgE-binding potency of the various peanut samples

0,0 0,01

25,0

50,0

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100,0

A. Example of chromatogram of samples of Virginia (Green: Brantley raw; Yellow: Gregory raw; Red: NC-v11 roasted; Blue: NC-v11 raw).

Individual curves were shifted vertically, range for each sample is 0-800 mAU. B. Chromatogram of purified peanut allergen references Ara h1: blue; Ara h2: red; Ara h3: yellow; Ara h6: green.

Column: X-Bridge BEH Phenyl; 3.5 m; 135Ă&#x2026;; [2.1 x 150 mm]. Temperature: 30 1 C. Sample volume: 0.02 ml, flow rate: 0.3 ml/min.

Eluent A: 0.1 % TFA in MQ Water; eluent B: 0.085 % TFA in MeOH . Absorbances were measured by diode array and recorded for 215 and 280 nm. Sample size for extracts: 20 l of 1 mg/ml solution.

Figure 2: Determination of peanut allergens by rp-HPLC.

0

800

In relation to this presentation, I declare the following, real or perceived conflicts of interest: the presenter was an employee of HAL Allergy during when this research was conducted

Conclusion: The main peanut cultivars consumed in the Western world all contain the main peanut allergens Ara h1, Ara h2, Ara h3, and Ara h6 with a slight variation of the relative content of these allergens between the different cultivars. Roasting had minimal effects on allergenic potency with the sera used in this study.

Results: Protein concentration in varietal extracts varied from 13.9 to 21.2 mg/ml, and roasting reduced the extractability by 22 to 50%.

Band patterns indicated that the four allergens of interest were present in samples of all varieties. The ranges of individual allergens in the tested samples were as follows: Ara h1: 11-32%; Ara h2: 716%; Ara h3: 38-76%; and Ara h6: 4-14%. In extracts of roasted peanuts, the relative content of Ara h1 was lower while that of Ara h3 was higher compared to non-roasted.

The relative content of Ara h2 and Ara h6 did not vary between raw and roasted. The allergenic potencies compared to in-house reference extracts varied from 99 to 139% for the raw samples, and roasting increased the potency only marginally with an increase of 14 to 30% relative to non-roasted counterparts.

binding test was applied for evaluation of allergenic potency. An extract from Virginia peanuts (raw, Greer, Lenoir NC, USA ) was used as in-house reference (IHR).

Inhibition (%)

Comparison of antibo dy-based assay with physicochemical assays for monitoring the stability of alum-adsorbed mite allergoid

D. Luykx, E. Kerkvliet, J. de Bruijn, N. Sinnige, R. van den Hout.

HAL Allergy BV, Leiden, The Netherlands.

Background An IgG inhibition assay has been developed previously to monitor the stability of alum-adsorbed mite allergoids in a mite allergy vaccine (ALU-M). In addition, two physicochemical methods have been developed to monitor the structural stability of the mite vaccine. These methods concern circular dichroism (CD) and front-face fluorescence spectroscopy (FF-fluorescence). In order to find out whether there is a relation between the antibody-based assay and physicochemical methods, temperature-stressed samples were analysed with both types of methods and the results were compared.

Methods ALU-M: D. pteronyssinus and D. farinae mite extracts were modified with glutaraldehyde and adsorbed onto aluminium hydroxide. After adsorption the species were mixed. IgG inhibition assay: Mite allergoid specific IgG antibodies were used to determine the 50% IgG inhibition value. CD: Far-UV CD spectra were recorded from 195 to 260 nm while stirring the sample suspension. FF-fluorescence: Fluorescence emission spectra were recorded from 290 to 400 nm (excitation at 280 nm).

Results Storage of the ALU-M vaccine at various elevated temperatures resulted in increased IgG inhibition values and spectral alterations with CD and FF-fluorescence, meaning structural alterations of the adsorbed protein. The increase of the 50% IgG inhibition value indicates a reduction of IgG binding to the epitopes. The spectral alterations observed with CD indicate partial loss of ι-helices and increase of β-structure at higher temperatures. The spectral alterations observed with FF-fluorescence indicate formation of a more rigid protein structure at higher temperatures. When the temperature stress stability data obtained with the three methods were compared, a relation was observed between the IgG inhibition assay and CD assay by means of the CD-ratio 207/222 nm. Samples stored at elevated temperatures showed a similar trend with respect to IgG inhibition and CD ratio; both values increased in time.

Conclusion By performing stress stability studies on ALU-M it was shown that the IgG inhibition assay and physicochemical assays were stability indicating. The results obtained with the IgG inhibition assay and CD assay relate well suggesting a relation between the IgG binding and secondary protein structure of the adsorbed mite allergoid.

EAACI, 16-20 June 2012, Geneva Abstract number: 278, Session date and time: Monday 18 June; 10:45 - 12:15 Session title: PDS 5 - Immunotherapy: vaccines and mechanisms

24

PDS 5 - Immunotherapy: vaccines and mechanisms

Comparison of antibo dy-based assay with physicochemical assays for monitoring the stability of alum-adsorbed mite allergoid

25

Background & Aim:

FF-fluorescence: Fluorescence emission spectra were recorded from 290 to 400 nm with excitation at 280 nm. The fluorescence intensity at

CD: Far-UV CD spectra were recorded from 195 to 260 nm and under FF-fluorescence: Fluorescence emission spectra were recorded from stirring conditions of the sample. The CD ratio 207/222 nm was 290 to 400 nm with excitation at 280 nm. The fluorescence intensity at monitored.

326 nm was monitored.

monitored.

CD: Far-UV CDassay: spectra were recorded from 195 260 nm and under IgG inhibition Mite allergoid specific IgGtoantibodies were used to stirring conditions of the sample.

The CD ratio 207/222 nm was determine the 50% IgG inhibition value.

IgG inhibition assay:ALU-M Mite allergoid specific were used to Stress conditions: was stored at IgG 5 or antibodies 37ºC for several months, at determine the 50%days, IgG inhibition value.

50ºC for several or at 90ºC for 6 hours.

modified with glutaraldehyde and at adsorbed aluminium hydroxide.

Stress conditions: ALU-M was stored 5 or 37ºConto for several months, at Afterwards the two species were 50ºC for several days, or at 90ºC formixed.

6 hours.

ALU-M: D. pteronyssinus and D. farinae mite extracts were separately modified with glutaraldehyde and adsorbed onto aluminium hydroxide.

Methods: Afterwards two species were ALU-M: D.the pteronyssinus and mixed.

D. farinae mite extracts were separately

Methods:

stability alum-adsorbed in a mite allergy vaccine (ALU- the An IgG ofinhibition assay mite has allergoids been developed previously to monitor M). In addition, two physicochemical methods developed stability of alum-adsorbed mite allergoids in have a mitebeen allergy vaccineto(ALUmonitor the structural stability of the mite vaccine: circular M).

In addition, two physicochemical methods have beendichroism developed to (CD) and front-face fluorescence spectroscopy (FF-fluorescence).

In monitor the structural stability of the mite vaccine: circular dichroism order to find out whether there is a relation between the antibody-based (CD) and front-face fluorescence spectroscopy (FF-fluorescence). In assay and physicochemical methods, temperature-stressed samples order to find out whether there is a relation between the antibody-based were analysed with both types of methods and the results were assay and physicochemical methods, temperature-stressed samples compared. were analysed with both types of methods and the results were compared.

An IgG inhibition assay has been developed previously to monitor the Background & Aim:

HAL Allergy BV, Leiden, The Netherlands

HAL Allergy BV, Leiden, The Netherlands

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D. Luykx, E. Kerkvliet, J. de Bruijn, N. Sinnige and R. van den Hout

D. Luykx, E. Kerkvliet, J. de Bruijn, N. Sinnige and R. van den Hout

9

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5°C Figure 1: (A) 50% IgG 37°C inhibition values, (B) CD ratio 207/222 nm and (C) FFfluorescence intensity of Figure (A) 50% IgG ALU-M stored at 5ºC1:(blue) inhibition values, (B) CD or 37ºC (green) for several

10

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278 -- Comparison assay withwith physicochemical assays 278 Comparisonofofantibody-based antibody-based assay physicochemical assays for the stability of alum-adsorbed mite mite allergoid formonitoring monitoring the stability of alum-adsorbed allergoid

50% IgG inhibition (AUeq/ml) CD ratio FF fluorescence (x1E7)

50% IgG inhibition (AUeq/ml) CD ratio luorescence (x1E7)

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

1000

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50% IgG Inhibition (AUeq/ml)

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EAACI Congress 2012

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Figure 3: 50% IgG inhibition data vs CD ratio 207/222 nm data obtained after storage of ALU-M at 5ºC (blue) 37ºC (green), 50ºC (red) and 90ºC (yellow).

Figure 2: (A) 50% IgG inhibition values, (B) CD ratio 207/222 nm and (C) FFfluorescence intensity of ALU-M stored at 50ºC (red) for several days.

Storage time in days

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

By performing stress stability studies on ALU-M it was shown that the IgG inhibition assay and physicochemical assays were stability indicating. The results obtained with the IgG inhibition assay and CD assay relate well suggesting a relation between the IgG binding and secondary protein structure of the adsorbed mite allergoid. Both assays were shown to be more sensitive than the FF-fluorescence assay.

Conclusion:

When the temperature stress stability data obtained with the three methods were compared, a relation is observed between the IgG inhibition assay and CD assay by means of the CD-ratio 207/222 nm (Fig. 3). Samples stored at elevated temperatures (i.e., 37, 50 and 90ºC) showed a similar trend with respect to IgG inhibition and CD ratio. The data of these assays support each other indicating that both assays can be used as stability indicating assay for ALU-M.

The stability of ALU-M was studied by monitoring the 50% IgG inhibition behaviour, CD-ratio 207/222 nm and FF-fluorescence intensity at 326 nm at various temperatures for different periods of time (Fig. 1 and 2).

The antibody-based assay and physicochemical assays did not show any changes when ALU-M was stored at 5ºC for a period of 1 year (Fig. 1). Storage of the ALU-M vaccine at 37ºC resulted in increased IgG inhibition values and spectral alterations with CD and FF-fluorescence (Fig. 1), meaning structural alterations of the adsorbed protein. The increase of the 50% IgG inhibition value indicates a reduction of IgG binding to the epitopes. The spectral alterations observed with CD indicate partial loss of -helices and increase of -structure at higher temperatures (increase of CD ratio 207/222 nm). The spectral alterations observed with FF-fluorescence indicate formation of a more rigid protein structure at higher temperatures (increase of fluorescence at 326 nm). Comparison of the results of the three different assays obtained at 37ºC indicate that the antibody-based and CD assay are more sensitive to detect structural changes for ALU-M than FFfluorescence (i.e., after 1 month). The same holds when the vaccine is stored at 50ºC for a few days (Fig. 2). In this case the fluorescence intensity first decreases in time before it increases again. Apparently, the conditions at 50ºC quench the fluorescence signal.

CD ratio

Results:

50 % IgG inhibition (AUeq/ml)

3.0

CD ratio

326 nm was monitored.

FF fluoresence (x1E7)

Characterisation of p ollen allergoids with physicochemical techniques

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

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

Background Birch and grass allergoids are drug substances for birch and grass allergy vaccines, respectively. 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, determination of the degree of polymerisation and cross-linking, and investigation of the protein structures. Several physicochemical techniques were tested for their suitability to analyse these characteristics in the allergoid and corresponding allergen extract: Mass spectrometry (MS), SDS-PAGE, HPLC-SEC, lysine determination and fluorescence spectroscopy.

Methods Allergoids: Allergen extracts from birch and grasses were treated with glutaraldehyde to obtain birch and grass allergoids, respectively. 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: GF250 and GF450 SEC columns were 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 Several isoforms of relevant birch allergen Bet v1 were identified in the birch allergoid with MS. In the grass allergoid relevant grass allergens group 1 and 5 were identified. SDS-PAGE showed for both allergoids the formation of various high molecular weight molecules including masses â&#x2030;Ľ250kDa. HPLC-SEC showed for both allergoids the formation of molecules â&#x2030;Ľ670 kDa. For both pollen extracts a minority of HPLC peaks (6 and 9%) was corresponding to proteins with a molecular mass >44 kDa. For the allergoids the majority of peaks (76 and 75%) was corresponding to proteins with a molecular mass (far) >44 kDa. Determination of free lysines showed that the majority of the lysines were modified in both pollen allergoids (73 and 74%). Fluorescence indicated hydrophobic tertiary protein structures after modification of the pollen extracts.

Conclusion Applying a combination of physicochemical techniques was shown to be a suitable approach to characterise the pollen allergoids well: Identification of the relevant allergens, determination of the degree of polymerisation and cross-linking, and investigation of the protein structures were accomplished.

EAACI, 16-20 June 2012, Geneva Abstract number: 287, Session date and time: Monday 18 June; 10:45 - 12:15 Session title: PDS 5 - Immunotherapy: vaccines and mechanisms

26

PDS 5 - Immunotherapy: vaccines and mechanisms

Characterisation of p ollen allergoids with physicochemical techniques

27

M

1

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Birch 4 5 6

4

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Grass

Allergoids Grass

Allergoids

Extracts Birch Birch Grass 1 2 3 Birch 4 5 6 Grass 1 2 3

Extracts

Lysine determination: Samples were hydrolysed into amino acids followed(SEC) A GF250 or GF450 size exclusion chromatography HPLC-SEC: by lysinewas determination with HPLC. By determining column used combined with UV-detection (215 the nm).amount of free lysines in the extract and allergoid, the amount of modified lysines in the Lysine determination: Samples were hydrolysed into amino acids followed allergoid can be calculated.

applied onto 10-20% polyacrylamide SDS-PAGE:AReduced GF250 orsamples GF450 were size exclusion chromatography (SEC) HPLC-SEC: gels and stained with Coomassie Blue. column was used combined with UV-detection (215 nm).

by lysine determination with HPLC. By determining the amount of free Fluorescence spectroscopy: Emission spectra were recorded from 290-400 lysines in the extract and allergoid, the amount of modified lysines in the

A2

A1

158 kDa

Birch 1

1.35 kDa

17 kDa

158 kDa670 kDa 1.35 kDa 44 kDa

B2

Standard

B1 158 kDa

158 kDa

670 kDa

Grass 1

1.35 kDa

17 kDa

1.35 kDa 44 kDa

Standard

grass670(4-6) extracts and corresponding allergoids (1-6). kDa 44 kDa 44 kDa Major birch allergen Bet v1 and B1 A1 grass allergens Standard Standard major group 1 and 5 are boxed.

17 kDa 670 kDa 17 kDa

grass (4-6) extracts and corresponding allergoids (1-6). Major birch allergen Bet v1 and the MS instrument via electrospray ionisation.

Ionised peptides were Reduced samples were applied onto 10-20% polyacrylamide SDS-PAGE: major grass allergens group 1 and 5 are boxed.

Figure 1: SDS-PAGE patterns of molecular weight marker (M), three birch (1-3) and three fragmented revealing amino acid sequences (i.e., identification). gels and stained with Coomassie Blue.

tryptic digests prepared from the samples.

Subsequently, the MS: with First glutaraldehyde to were obtain birch and grass allergoids, respectively. peptides were separated with nano-liquid chromatography before entering the via were electrospray ionisation. were the Firstinstrument tryptic digests prepared from the Ionised samples.peptides Subsequently, MS: MS Figure 1: SDS-PAGE patterns of molecular weight marker (M), three birch (1-3) and three fragmented revealing amino acid identification). before entering peptides were separated with sequences nano-liquid(i.e., chromatography

Allergoids: Allergen extracts from birch and mixed grass pollen were treated

Allergoids: Allergen extracts from birch and mixed grass pollen were treated Methods: with glutaraldehyde to obtain birch and grass allergoids, respectively.

Methods:

(MS), SDS-PAGE, HPLC-SEC, lysine determination and fluorescence spectroscopy.

M

2 Research International, Wageningen, The Netherlands Plant Plant Research International, Wageningen, The Netherlands

1 HAL 1 HAL Allergy BV, Leiden, The Netherlands Allergy BV, Leiden, The Netherlands

Background & Aim: Birch and grass allergoids are used as drug substances for birch and grass Birch and grass respectively. allergoids are used drugallergoids substances forbeen birchpoorly and grass allergy vaccines, Until nowasthese have allergy vaccines, respectively.

Until A now these allergoids have been poorly characterised due to their complexity. better characterisation is needed.

This includes identification the major Aallergens, determination ofis the characterised due to their of complexity. better characterisation needed. degree of polymerisation and cross-linking, andallergens, investigation of the proteinof the This includes identification of the major determination structures.

Several physicochemical techniquesand were applied to of analyse degree of polymerisation and cross-linking, investigation the protein these characteristics three successive, separately were produced birchtoand structures.

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

Background & Aim:

2

1, J. 1, J.1Cordewener 2, T. America 2 and R. 1 1, de 2 and D.D. Luykx Bruijn vanR. den Hout Luykx J. de Bruijn , J. Cordewener 2, T. America van den Hout1

287 --Characterisation Characterisation pollen allergoids physicochemical techniques 287 ofof pollen allergoids withwith physicochemical techniques

15 nm

A4

A3

A2

B4

B3

B2

Retention time (min)

Birch 3

Birch 2

Birch 1

Grass 3

Grass 2

Grass 1

311

Fluorescence emission maximum (nm)

311

79

74

Birch 2

73 329

Modified lysines (%) Fluorescence emission maximum (nm)

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

76

Grass 1 HPLC: Peak area above 44 kDa (%)

Characteristic

330

76

79

Grass 3

311

78

77

Birch 3

EAACI Congress 2012

EAACI Congress 2012

329

73

68

Grass 2

Table 2: Physicochemical characteristics of three batches of grass allergoids

74

75

Birch 1

Modified lysines (%)

HPLC: Peak area above 44 kDa (%)

Characteristic

Table 1: Physicochemical characteristics of three batches of birch allergoids

Figure 2: HPLC-SEC patterns (A215 nm) of calibration standard (A1 and B1), three birch extracts (blue line) and corresponding allergoids (red line) (A2-A4), and three grass extracts (blue line) and corresponding allergoids (red line) (B2-B4). The black vertical line in the chromatogram splits peaks corresponding to molecular masses above and below 44 kDa. A GF250 SEC column was applied for birch, a GF450 SEC column for grasses.

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

Applying a combination of physicochemical techniques was shown to be a suitable approach to characterise the pollen allergoids well: Identification of the major allergens, determination of the degree of polymerisation and cross-linking, and investigation of the protein structures were accomplished.

This study shows consistency for the modification of the pollen extracts.

Conclusion:

Fluorescence spectroscopy: Modification of the birch and grass extracts resulted in a shift of the emission maximum to a lower wavelength and a decrease of fluorescence intensity (data not shown). This indicates that the surroundings of the aromatic amino acids in the proteins change to a more hydrophobic environment which is indicative for protein polymerisation. The emission maximum values obtained for the pollen allergoids show consistency as well (Table 1-2).

Lysine determination: For the birch allergoids 74-79% of the lysines were modified and for the grass allergoids 73-76% of the lysines were modified (Table 1-2). This shows a consistent degree of protein cross-linking.

HPLC-SEC: For the three birch and grass extracts a minority of HPLC peaks is corresponding to proteins with a molecular mass above 44 kDa (Fig. 2). The chromatograms of the birch and grass allergoid batches showed the presence of a variety of high molecular weight molecules including molecules with molecular masses 670 kDa. Here, a consistent majority of peaks (74-77% for birch and 68-79% for grasses) is corresponding to proteins with a molecular mass (far) above 44 kDa showing a consistent degree of protein polymerisation (Table 1-2).

SDS-PAGE: For the three birch extracts, similar protein profiles were observed (Fig. 1). The band corresponding to 15-18 kDa indicates the presence of major allergen Bet v1. For the three grass extracts also similar protein profiles were observed. Here, the bands corresponding to 27-34 kDa indicate the presence of major allergens group 1 and 5. For all allergoids the formation of various high molecular weight molecules is shown via a smear and a band around 250 kDa (molecules 250 kDa) on gel.

MS: Several isoforms of the major birch allergen Bet v1 were identified in the three birch allergoids (a, b, c, d/h, f/i). Next to the major grass allergens group 1 and 5, also group 2, 3, 4 and 6 allergens were identified in the three grass allergoids (data not shown).

Results:

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

allergoid can be calculated.

Absorbance 215 nm

Characterisation of alum-adsorbed p ollen allergoids with physicochemical techniques

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

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

Background Several physicochemical techniques have been selected to investigate their suitability to characterise a birch allergy vaccine (ALU-B) and grass allergy vaccine (ALU-G). These techniques are mass spectrometry (MS), circular dichroism (CD), and front-face fluorescence (FF-fluorescence). They should be able to investigate the alum-adsorbed and modified allergens on all structural levels, i.e. primary structure (for protein identification), secondary and tertiary structure (for structural stability). Samples were temperature stressed to find out if these techniques are stability indicating.

Method Alum-adsorbed pollen allergoids: Allergen extracts from birch and grasses were modified with glutaraldehyde and adsorbed onto aluminium hydroxide to obtain ALU-B and ALU-G, respectively. MS: Preparation of tryptic digests and peptide separation via nano-LC before electrospray ionisation. Ionised peptides were fragmented revealing amino acid sequences. CD: Far-UV CD spectra were recorded from 195 to 260 nm while stirring the sample suspension. FF-fluorescence: Fluorescence emission spectra were recorded from 290 to 400 nm, with excitation at 280 nm.

Results Via MS several isoforms of relevant birch allergen Bet v1 were identified in ALU-B. In ALU-G relevant grass allergens group 1 and 5 were identified. The CD-spectrum of ALU-B and ALU-G represented protein structures consisting of mainly α-helices and ß-structure. The FF-fluorescence spectrum of both products showed an emission maximum at 326 nm suggesting hydrophobic surroundings for the aromatic amino acids present in both protein samples. Storage of ALU-B and ALU-G at several elevated temperatures resulted in spectral alterations meaning structural alterations of protein. CD spectra indicated partially loss of α-helices (increase of ratio 207/222 nm) while fluorescence spectra indicated formation of a less rigid structure (decrease of fluorescence at 326 nm).

Conclusion MS, CD and FF-fluorescence were able to characterise the complex birch and grass allergy vaccines on all structural levels, i.e. primary, secondary and tertiary structure. Via MS the relevant birch and grass allergens could be identified while via CD and FF-fluorescence the protein structures could be investigated. Moreover, CD and FF-fluorescence were shown to be promising stability indicating assays.

EAACI, 16-20 June 2012, Geneva Abstract number: 560, Session date and time: Sunday 17 June; 12:00 - 13:30 Session title: Poster 13 - Immunotherapy: allergens and allergen-preparations

28

Poster Session 13 - Immunotherapy: allergens and allergen-preparations

Characterisation of alum-adsorbed p ollen allergoids with physicochemical techniques

29

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Figure 1: Far-UV CD-spectrum of ALU-B (A) and ALU-G (B). The arrows indicate the -10 ellipticity at 207 and 222 nm.

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firstly involved of tryptic digests Figure 1: Far-UV CD-spectrum of ALU-B (A) and ALU-G (B). The arrows indicate the MS: The applied Stress conditions: ALU-Bprocedure and ALU-G were storedpreparation at 5 and 37ºC for 1-3 25 25 the sample.

Next, and the peptides were separated via 2D nano-liquid ellipticity at 207 and 222 nm. months,from at 50ºC for 12 hours, at 90ºC for 1 hour. chromatography before electrospray ionisation and entering the MS 20 20 MS: The applied procedure firstlyidentification, involved preparation of tryptic digestswere instrument.

For protein the ionised peptides 25 25 from thefragmented sample. Next, the peptides separated via 2D nano-liquid revealing the aminowere acid sequence.

15 15 chromatography before electrospray ionisation and entering the MS CD: Far-UV CD spectra were recorded from 195 to 260 nm and under 20 20 instrument. For protein identification, the ionised peptides were stirring conditions of the sample. The CD ratio 207/222 nm was 10 10 fragmented revealing the amino acid sequence.

Several physicochemical techniques have been selected to investigate their suitability characterise a birch allergy vaccine (ALU-B) and grass Background & toAim: vaccine (ALU-G).

These techniques are mass spectrometry Severalallergy physicochemical techniques have been selected to investigate (MS), circular dichroism (CD), andvaccine front-face fluorescence their suitability to characterise a birch allergy (ALU-B) and grass (FFThey These should techniques be able to investigate alum-adsorbed allergy fluorescence). vaccine (ALU-G). are mass the spectrometry modified allergens on all structural levels, i.e. primary structure (MS), circular dichroism (CD), and front-face fluorescence (FF- (for protein identification), secondary and tertiary structure (for structural fluorescence). They should be able to investigate the alum-adsorbed stability).

Samples temperature stressed to structure find out (for if these modified allergens on all were structural levels, i.e. primary techniques are stability indicating. protein identification), secondary and tertiary structure (for structural stability). Samples were temperature stressed to find out if these Methods: techniques are stability indicating.

Alum-adsorbed pollen allergoids: Allergen extracts from birch and mixed grass pollen were modified with glutaraldehyde, and adsorbed onto Methods: aluminium hydroxide to obtain ALU-B and ALU-G, respectively.

Alum-adsorbed pollen allergoids: Allergen extracts from birch and mixed ALU-B andglutaraldehyde, ALU-G were stored 5 and 37ºC for 1-3 Stress were conditions: grass pollen modified with and atadsorbed onto months, at 50ºC for 12 hours, and at 90ºC for 1 hour. aluminium hydroxide to obtain ALU-B and ALU-G, respectively.

Background & Aim:

E llip tic ity (m D eg )

Plant Research International, Wageningen, The Netherlands

Plant 1 Research International, Wageningen, The Netherlands HAL Allergy BV, Leiden, The Netherlands

E llip tic ity (m D eg )

2

2

HAL Allergy BV,2,Leiden, The Netherlands 2 and R. van den Hout1 D. Luykx1, J. de Bruijn1, J. Cordewener T. America

E llip tic ity (m D e g )

560 - Characterisation of alum-adsorbed pollen allergoids with 560 - Characterisation of alum-adsorbed pollen allergoids with physicochemical techniques 2, T. America2 and R. van den Hout1 D. Luykx1, physicochemical J. de Bruijn1, J. Cordewenertechniques

E llip tic ity (m D e g ) c e in te n s ity (1 0 6 x L U )

(1 0 6 x L U )

c e in te n s ity (1 0 6 x L U )

(1 0 6 x L U )

Wavelength (nm)

0 290 300 310 320 330 340 350 360 370 380 390 400

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

EAACI Congress 2012

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Figure 4: Fluorescence intensity at 326 nm of ALU-B (A) and ALU-G (B) stored at 5 and 37ºC for 1-3 months.

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Figure 3: CD-ratio 207/222 nm of ALU-B (A) and ALU-G (B) stored at 5 and 37ºC for 1-3 months.

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Figure 2: FF-fluorescence spectrum of ALU-B (A) and ALU-G (B). The arrow indicates the fluorescence intensity at 326 nm. The peak at 310 nm represents the water raman peak.

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

MS, CD and FF-fluorescence were able to characterise the complex birch and grass allergy vaccines on all structural levels, i.e. primary, secondary and tertiary structure. Via MS the major birch and grass allergens were identified while via CD and FF-fluorescence the protein structures were elucidated. CD and FF-fluorescence were shown to be promising stability indicating assays.

Conclusion:

By monitoring the CD-ratio 207/222 nm (i.e., loss of -helices) and fluorescence intensity at 326 nm (i.e., rigidity), the structural stability of ALU-B and ALU-G was followed at 5 and 37ºC for 1-3 months (Fig. 3-4).

Storage at 5ºC did not affect the secondary and tertiary protein structures of both vaccines. However, structural changes were observed for the pollen vaccines when stored at 37ºC. Storage at 50ºC for 12 hours did result in similar structural changes as was found after 1-2 months at 37ºC (data not shown). The protein structures of ALU-B and ALU-G were mostly effected when stored at 90ºC for 1 hour (data not shown). In this case the CD-ratio increased to 1.20 and 1.56 for ALU-B and ALU-G, respectively. With respect to the fluorescence, the intensities decreased for both vaccines to 9.5 x 106.

Storage of ALU-B and ALU-G at several elevated temperatures resulted in spectral alterations meaning structural alterations of protein. CD spectra indicated partially loss of -helices (increase of ratio 207/222 nm) while fluorescence spectra indicated formation of a less rigid structure (decrease of fluorescence at 326 nm).

The CD-spectrum of ALU-B and ALU-G represented protein structures consisting of mainly -helices (i.e., ellipticity at 208 and 222 nm) and structure (Fig. 1). The FF-fluorescence spectrum of both vaccines showed an emission maximum at 326 nm suggesting hydrophobic surroundings for the aromatic amino acids present in both protein samples (Fig. 2).

Via MS several isoforms of the major birch allergen Bet v1 were identified in ALU-B (a, b, c, d/h, f/i). In ALU-G the major grass allergens group 1 and 5 were identified (data not shown).

Results:

FF-fluorescence: Fluorescence emission spectra were recorded from 290 to 400 nm with excitation at 280 nm. The fluorescence intensity at 326 nm was monitored.

F lu o r e s c e

C D ra tio 2 0 7 /2 2 2 n m F lu o re s c e n c e in te n s ity (1 0 6 x L U )

monitored.

F lu o r e s c e

C D ra tio 2 0 7 /2 2 2 n m

F lu o re s c e n c e in te n s ity (1 0 6 x L U )

An Inhibition ELISA metho d for the determination of Relative Potency of Mite preparations

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

HAL Allergy BV, Leiden, The Netherlands.

Background We have developed and (pre-)validated a new method for the determination of IgE potency of mites. Advantages of the new method are i) all components are well characterised and verifiable, ii) the method is specific for different mite cultures and has a high precision, and iii) the costs of reagents are relatively low.

Method The assay starts with a pre-incubation of mite preparations with a sera pool obtained from patients with established clinical allergy for mite allergens. Thereafter, the mixture is transferred to 96-well plates coated with mite allergens from the mite species and mite source of interest. Unbound specific IgE binds to the coated wells and the total amount of IgE is quantitatively stained by using an anti-IgE streptavidine conjugate. The extent to which the IgE binds to the coat is compared with the maximum amount of IgE that was bound if the sera pool was not pre-incubated (E-max). The difference of the preincubated samples and E-max is indicated as percentage inhibition of the IgE preparation. As control, an in-house reference (IHR) is included with the assay to compare the inhibition curves by parallel line analyses. The IgE binding efficiency of an allergen preparation is expressed by relative potency (RP) compared to the IHR. The allergen preparations that were tested with the new method are different mite drug substances from Dermatophagoides pteronyssinus and Dermatophagoides farinae. The results were compared with results obtained with the ImmunoCap assay.

Results All mite preparations tested showed linear inhibition curves with high correlations (R2â&#x2030;Ľ0,99) and reproducible RP data (intermediate precision (CV) of 10%). Furthermore, RP data measured with the new method were comparable with the RP data measured with the ImmunoCap.

Conclusion The newly developed IgE inhibition ELISA is well suited for use of potency determination of different mite species from bodies and cultures. In comparison with the ImmunoCap assay, the newly developed assay has an improved precision and all reagents are well characterised.

EAACI, 16-20 June 2012, Geneva Abstract number: 571, Session date and time: Sunday 17 June; 12:00 - 13:30 Session title: Poster 13 - Immunotherapy: allergens and allergen-preparations

30

Poster Session 13 - Immunotherapy: allergens and allergen-preparations

An Inhibition ELISA metho d for the determination of Relative Potency of Mite preparations

31

A A

ME-B ME-B

ME-C ME-C

Coating of mite extract Coating of mite extract

B B

Competition between Competition between coat & mite sample coat & mite sample for for binding of IgE in the binding of IgE in the serum serum

Mixture of mite proteins Mixture of mite proteins patient serum andand patient serum is is added added

CC

Colour is detected Colour is detected at at 450nm 450nm

HRP is stained TMB HRP is stained withwith TMB

Bound is detected Bound IgE IgE is detected labeled Îą-IgE withwith HRPHRP labeled Îą-IgE

Wash

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IHR IHR mite extract mite extract

Linear curves computed Linear curves are are computed by using bound by using ratioratio (%) (%) bound IgE IgE vs E-max.

Potency vs E-max. Potency is is determined by using PLA. determined by using PLA.

iELISA: assay starts coating of micotiter plates using extracts of the source of interest 1, followed A), followed by incubation an incubation in the coated iELISA: TheThe assay starts withwith coating of micotiter plates using mitemite extracts of the mitemite source of interest (Fig(Fig 1, A), by an in the coated wells of mite preparations with a sera pool obtained from patients with established clinical allergy for mite allergens (Fig1, B).

Unbound specific IgE binds to the wells of mite preparations with a sera pool obtained from patients with established clinical allergy for mite allergens (Fig1, B). Unbound specific IgE binds to the coated wells and the total amount of IgE is quantitatively stained by using an HRP anti-IgE conjugate (Fig 1, C).

The extent to which IgE binds to the coat coated wells and the total amount of IgE is quantitatively stained by using an HRP anti-IgE conjugate (Fig 1, C). The extent to which IgE binds to the coat is is compared maximum amount of IgE bound if only serum incubated (E-max). difference of the pre-incubated samples E-max compared withwith the the maximum amount of IgE thatthat waswas bound if only serum waswas incubated (E-max).

TheThe difference of the pre-incubated samples andand E-max is is indicated as percentage inhibition of the IgE preparation.

As control, an in-house reference (IHR) is included to compare the inhibition curves by parallel indicated as percentage inhibition of the IgE preparation. As control, an in-house reference (IHR) is included to compare the inhibition curves by parallel line line analyses (PLA) 1, D). binding efficiency of allergen an allergen preparation is expressed by relative potency compared to the allergen analyses (PLA) (Fig(Fig 1, D).

TheThe IgE IgE binding efficiency of an preparation is expressed by relative potency (RP)(RP) compared to the IHR.IHR. TheThe allergen preparations tested method different extracts Dermatophagoides pteronyssinus Dermatophagoides farinae. preparations thatthat werewere tested withwith the the newnew method are are different mitemite extracts fromfrom Dermatophagoides pteronyssinus andand Dermatophagoides farinae.

TheThe results of the iELISA were compared with results obtained with the ImmunoCap assay results of the iELISA were compared with results obtained with the ImmunoCap assay

Methods: Methods:

potency assays specific reagents as biotinylated allergen extracts or allergen used in the ImmunoCap system relatively expensive IgE IgE potency assays withwith specific reagents suchsuch as biotinylated allergen extracts or allergen capscaps used in the ImmunoCap system are are relatively expensive andand contain reagents that can not be characterized.

In addition, the ImmunoCap assay for mites was not developed for potency testing of different mite cultures. contain reagents that can not be characterized. In addition, the ImmunoCap assay for mites was not developed for potency testing of different mite cultures.

Therefore, a new method for the determination of IgE potency of mites developed pre-validated Therefore, a new method for the determination of IgE potency of mites waswas developed andand pre-validated

Background and aim: Background and aim:

E. Kerkvliet, I. Peekel R. van Hout E. Kerkvliet, I. Peekel andand R. van denden Hout HAL Allergy BV, Leiden, The Netherlands HAL Allergy BV, Leiden, The Netherlands

571 - An Inhibition ELISA method the determination 571 - An Inhibition ELISA method forfor the determination ofof Relative Potency Mite preparations Relative Potency ofof Mite preparations

Wash

IgE)

Wash

(IgE)

Wash

= HRP-labeled conjugate

10

100

1000

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0.55

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0.68

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0.47

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Table 2. Intra-mediate precision of RP data and variation (CV%) of 4 different samples.

Intramediate precision is determined in three runs; these data represent the data from the run with the highest variation. The mean RP value is an average of 3 different sample dilutions analysed in one run.

D. farinae bodies D. pter bodies D. farinae cultures Mite mix cultures

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EAACI Congress 2012

EAACI Congress 2012

In comparison with the ImmunoCap assay, the newly developed assay has an improved precision and all reagents are well characterised.

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

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

20

46

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23

CV%

ImmunoCap

Table 3. RP data of 4 different samples tested with the iELISA and the ImmunoCap. The results of the iELISA are from the inter-assay precision determination. iELISA: average and CV% of 6 independent runs; ImmunoCap: average and CV% of 3 independent runs.

Mite mix cultures

1.15

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0.84

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D. farinae bodies

Test sample

iELISA

The newly developed IgE inhibition ELISA is well suited for use of potency determination of different mite species from bodies and cultures.

Conclusion:

Table 1. RP data and variation (CV%) from 4 different samples tested in two concentrations. The RP value is an average of 3 different runs. Each individual RP value is an average of an independent duplicate.

Mite mix cultures

D. farinae cultures

D. pter bodies

D. farinae bodies

Test sample

All mite preparations tested showed linear inhibition curves with high correlations (R2 0,99). Different mite species from both bodies and cultures were tested in two concentrations in triplicate (Table 1). Furthermore, intra-assay (Table 2) and inter-assay (Table 3) precision was determined for 4 different mite products and show a maximal day-to-day variation of 10% (n=6). No significant differences were found when RP data measured with the new method were compared with RP data measured with the ImmunoCap.

Results:

ImmunoCap: The ImmunoCap assay in brief: the assay starts with a pre-incubation of mite extract and serum, after which the mixture is transferred to the ImmunoCAP system (Phadia AB, Sweden) loaded with CAPs coated with mite allergens. Unbound IgE, which was unable to bind the product during preincubation, binds to the CAP and is detected by an anti-IgE conjugate. A fluorogenic substrate is added and the amount of IgE on the CAP is measured quantitatively by fluorescence. RP is determined by using PLA.

mite concentration in AU/ml

= IgE

1

= TMB

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IgE inhibitio

An antibo dy-based technique for stability studies on alum adsorbed birch p ollen allergoid preparations

E. Kerkvliet, C. Franso, A. Segaar, R. van den Hout.

HAL Allergy BV, Leiden, The Netherlands.

Background IgE based potency assays for allergens are essential tests for monitoring stability in extracts. However, these IgE potency assays can not be used for potency determination of allergoid-based products, since they essentially lack IgE reactivity.

Furthermore, aluminium can deteriorate binding of antibodies to proteins attached to a solid phase. We have developed and validated an IgG based inhibition assay that is suitable for determining IgG inhibition of an alum-adsorbed birch pollen allergoid (ALU-B). In order to find out whether the assay is stability indicating, temperature-stressed samples were analysed with the new method.

Method Rabbit IgG antibodies specific for birch allergoid are pre-incubated with various concentrations of ALU-B. The mix is then added to a birch allergoid coated microtiter plate to bind free allergoid-specific IgG, which is quantitatively measured by a donkey anti-rabbit HRP conjugate and stained with TMB. The extent to which the IgG antibodies bind the plate in presence of added inhibitor (ALU-B) is compared with the maximum amount of IgG antibodies that binds in absence of the inhibitor.

Results are finally expressed as percentage inhibition relative to the uninhibited value and the concentration at 50% inhibition is used as read out. An in house reference of birch allergoid is included as assay control and is used for factorizing the IgG inhibition values to reduce day to day variation.

Results Validation of the IgG inhibition assay showed that the assay is specific for birch preparations, linear, precise (CV=16%) and robust. Furthermore, the assay detected a two-fold decrease in IgG inhibition when two doses of ALU-B with a two-fold difference in strength were analysed. Storage of the ALU-B vaccine at various elevated temperatures resulted in increased 50% IgG inhibition values indicating a reduction of IgG binding to the epitopes.

Conclusion We have successfully developed and validated an assay that determines IgG inhibition of the alum-adsorbed birch pollen vaccine. Moreover, the sensitivity of the assay is sufficient to at least detect a two-fold reduction in potency. Although the assay is still under evaluation whether the IgG inhibition assay can be used for stability studies of ALU-B, preliminary results showed reduced IgG binding to temperature stressed ALU-B vaccine.

EAACI, 16-20 June 2012, Geneva Abstract number: 572, Session date and time: Sunday 17 June; 12:00 - 13:30 Session title: Poster 13 - Immunotherapy: allergens and allergen-preparations

32

Poster Session 13 - Immunotherapy: allergens and allergen-preparations

An antibo dy-based technique for stability studies on alum adsorbed birch p ollen allergoid preparations

33

The newly developed inhibitionthat assay uses IgG antibodies specific for birch pollenIgG allergoids were obtained by specific forof birch allergoids thatmodified were obtained immunization rabbitspollen with glutaraldehyde extracts by (allergoid) of birchofpollen.

Thewith assay starts with a pre-incubation immunization rabbits glutaraldehyde modified extracts of (allergoid) a mixture of anti-allergoid serum and different concentrations of birch pollen. The assay starts with a pre-incubation of ofalum-adsorbed birch pollen serum allergoid After prea mixture of anti-allergoid and(ALU-B). different concentrations incubation the mixture is added to an birch allergoid of alum-adsorbed birch pollen allergoid (ALU-B). coated After premicrotiter platethe to bind free allergoid-specific incubation mixture is added to an IgG. birchSubsequently, allergoid coated by an HRPIgG. conjugate and bound IgG isplate quantitatively measured microtiter to bind free allergoid-specific Subsequently, stained with TMB. The extent to which the IgG antibodies bind bound IgG is quantitatively measured by an HRP conjugate and to stained the plate presence of ALU-B is compared with the bind withinTMB. The extent to which the IgG antibodies maximum amount of IgG antibodies that binds in absence of the to the plate in presence of ALU-B is compared with

Methods: Methods: The newly developed IgG inhibition assay uses IgG antibodies

IgE based potency assays for for allergens are are essential teststests for for IgE based potency assays allergens essential monitoring stability in extracts.

However, these IgE potency monitoring stability in extracts. However, these IgE potency assays cancan notnot be be used for for potency determination of allergoidassays used potency determination of allergoidbased products, since they essentially lack IgE reactivity. based products, since they essentially lack IgE reactivity.

Furthermore, aluminium can deteriorate binding of antibodies to Furthermore, aluminium can deteriorate binding of antibodies to proteins attached to a solid phase. We previously developed an proteins attached to a solid phase. We previously developed an IgG based inhibition assay for monitoring stability of alum IgG based inhibition assay for monitoring stability of alum adsorbed mite preparations. A similar assay has now been adsorbedfor mite assay hasof now developed thepreparations. determinationA ofsimilar IgG inhibition alumbeen developed for the determination of IgG inhibition of alum adsorbed birch pollen allergoids. This assay has been studied pollen This the assay hashas been studied foradsorbed use as abirch stability testallergoids. and whether assay been for use as a stability test and whether the assay has sufficient sensitive to detect a 2 times reduction. Finally, thebeen sufficient sensitiveaccording to detect 2 times reduction. Finally, the assay was validated theacurrent guidelines. assay was validated according the current guidelines.

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Lane 1: Marker; Lane 2: Lane 1: Marker; Lane 2: Birch pollen extract; pollen extract; Lane 3-5: Birch 3 different Lane 3-5: Birch pollen allergoid3 different Birch pollen allergoid batches. The box batches. indicates the Bet v1The box indicates the Bet v1 band 4 5 band

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ALU-B batch A

Batch B

inhibition value

294 AUeq/ml 50% IgG CV inhibition value Batch B 50% IgG

½ Normal dose

Normal dose 128 AUeq/ml ½ Normal dose 294 AUeq/ml 2.0%

inhibition value 128 AUeq/ml 1.5%

50% IgG CV inhibition value Batch A 50% IgG Normal dose

Batch A

CV

2.0%

1.5%

CV

Figure 1A. Direct ELISA of the rabbit antiFigure 1B. Immunoblot of rabbit serum 1A.Birch Direct ELISA of the Figure of rabbit serumFigure tested on pollen extract andrabbit on antiimmunized with 1B. birchImmunoblot pollen allergoid.

The serum serum Birchdose-responses pollen extract and on immunized birchpollen pollen allergoid. The allergoid.

Thetested serumon shows anti-serum is tested with on birch extract to both preparations with higher for and on three different allergoid allergoid.

The serum showsaffinity dose-responses anti-serum is birch testedpollen on birch pollen extract the allergoid preparations.. to bothpreparation. preparations with higher affinity for and on three different birch pollen allergoid the allergoid preparation. preparations..

IgG inhibition (%)

Background and aim: Background and aim:

OD value (450 nm)

E. Kerkvliet, C. Franso, A. Segaar R. den van Hout den Hout E. Kerkvliet, C. Franso, A. Segaar and and R. van HAL Allergy BV, BV, Leiden, The The Netherlands HAL Allergy Leiden, Netherlands

572- An - Anantibody-based antibody-based Technique Stability Studies 572 Technique forfor Stability Studies on on AlumAdsorbed Adsorbed Birch pollen allergoid preparations Alum Birch pollen allergoid preparations

OD value (450 nm)

IgG inhibition (%)

1

10

100

1000

ALU-B in AUeq/ml

10000

100000

ALU-B in AUeq/ml

100000

148

140

137

61

64

66

CV = 15% CV = 16% Temperature conditions are crucial

Intra-assay precision Inter-assay precision Robustness

Table 3. Validation results of the 50% IgG inhibition assay for ALU-B batches.

EAACI Congress 2012

Normal distribution of points; anchor points approach upper and lower asymptote

Specific for ALU-B and not to other PURETHAL preparations

Model

Specificity

Results

Table 2. The 50% IgG inhibition values of each curve are presented in the table. The decrease of 50% IgG inhibition was calculated in relation to the ALU-B sample analysed at t0. CV of all measurements is below 10%

90°°C, 4 hours

90°°C, 1 hour

37°°C, 2 months

Validation parameters

Figure 3. The graph represents IgG inhibition curves of a PUR-B batch stored at 5°C, 25°C, 37 °C for 2 months and at 90°C for 1 and 4 hrs. The dotted line shows the 50% inhibition of the different curves.

-10 1

0

10

20

30

40

60

80

% recovery

112

90

50% IgG inhibition value in AUeq/ml

25°°C, 2 months

T0

Storage conditions

50

10000

90°C , 4 hrs

96

1000

90°C , 1 hr

94

100

37°C

5.5%

7.4%

5°°C, 2 months

10

25°C

286 AUeq/ml

128 AUeq/ml

Table 1. 50% IgG inhibition values of 2 batches ALU-B, also tested when 1:1 diluted in formulation buffer.

½ Normal dose

Normal dose

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90

100

5°C

Figure 2. IgG inhibition curves of 2 batches ALU-B (blue curves) and IgG inhibition curves measured in their supernatant (red and orange curves).

0

20

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

The newly developed and validated IgG inhibition assay is suitable for measuring IgG binding to PUR-B batches and is able to at least detect a two-fold reduction in potency.

The assay showed reduced IgG binding to temperature stressed ALU-B vaccine, suggesting that the assay is stability indicating.

Conclusion:

The birch pollen allergoid preparation induced high specific IgG titers in rabbits, which was demonstrated by ELISA and immunoblotting (Fig. 1). Immunoblotting shows that the rabbit anti serum recognises birch extract, birch allergoid preparations and the relevant birch allergen Bet v1 (Fig. 1B). Using different concentrations of ALU-B mixed with the rabbit serum resulted in dose-response curves on birch allergoid pollen coated microtiter plates. By testing supernatant of PUR-B batches it was proven that the dose-response curves were mainly caused by ALU-B and not by unbound material since supernatant contains a more than 100 times lower signal (Fig 2). Table 1 shows a significant increase in the 50% IgG inhibition value when two times diluted batches are analysed, indicating that the sensitivity of the assay is sufficient to detect a 50% reduction in activity. Storage of the ALU-B vaccine at various elevated temperatures resulted in increased 50% IgG inhibition values indicating a reduction of IgG binding to the epitopes (Fig 3, Table 2). Validation of the assay showed that the assay is specific for birch, has a normal distribution of points, shows a good precision and is robust (Table 3). The assay is still under evaluation to establish whether the IgG inhibition ELISA is suitable for longitudinal monitoring of stability of the adsorbed product

Results:

maximum amount of IgG antibodies that binds in absence of ALU-B (E-max). Results are finally expressed as percentage inhibition relative to the uninhibited value. The 50% inhibition value is used as read out. An in house birch allergoid reference sample is used for day-to-day correction and as assay control.

Suitability of the assay was determined with different PUR-B batches and batches stored at elevated temperatures.

IgG inhibition (%)

Biochemical characterization of chemically mo dified Ara h2 isoforms; structure-function relationships

D. Luykx (1) , D. Apostolovic (1), H. Warmenhoven (1), D. Verbart (1), G.A.H. de Jong (2), S.J. Koppelman (1).

(1) HAL Allergy BV, Leiden, The Netherlands, (2) TNO, Zeist, The Netherlands.

Background Modified forms of major peanut allergens Ara h2 and Ara h6 were shown to be less allergenic than their native counterparts, making them potentially suitable for peanut allergy immunotherapy. Two isoforms of Ara h2 are known: heavy isoform of appr. 20 kDa and a light isoform of appr. 17 kDa. Furthermore, Ara h6 is an allergen of appr. 15 kDa, homologous to Ara h2. This study aims to describe the biochemical characteristics and IgE-binding potency of the modified allergens Ara h2-heavy isoform, Ara h2-light isoform, and Ara h6 in comparison with their native counterparts.

Methods Ara h2-heavy isoform, Ara h2-light isoform, and Ara h6 were purified to homogeneity to obtain the native allergens. To obtain the modified allergens, the native allergens were modified by reduction of the disulfide bonds and subsequent alkylation of the free Cys residues. The modified allergens as well as their native counter parts were analysed for change in molecular weight, secondary structure, and allergenicity, using SDS-PAGE, far-UV CD spectroscopy, and an IgE-binding assay respectively.

Results The modification of the allergens was optimized by investigating the kinetics, resulting in a consistent procedure for modification. All three allergens showed an increase in apparent molecular weight and unfolding of protein structure upon reduction and alkylation. Consequently, the IgE-binding potency was decreased about a hundred-fold for each of the tested allergens.

Conclusions Understanding the biochemical nature of the modification of the major peanut allergens Ara h2 and Ara h6 allows to optimize the process to reduce their allergenicity.

EAACI, 16-20 June 2012, Geneva Abstract number: 1448, Session date and time: Tuesday 19 June; 12:00 - 13:30 Session title: Poster 71 - Clinical and experimental applications of molecular allergology

34

Poster Session 71 - Clinical and experimental applications of molecular

allergology

Biochemical characterization of chemically mo dified Ara h2 isoforms; structure-function relationships

35

peptides were fragmented to reveal the (modified) amino acid sequence Far-UV CD were recorded from 190 to 260 CD: for identity.

MSspectra was also applied for intact native andnm. modified allergens.

Fluorescence spectroscopy: Fluorescence spectra were recorded from

Mass Blue. spectrometry: Tryptic peptides obtained from the samples were separated via liquid chromatography before electrospray ionisation and Tryptic obtained from theThe samples Mass spectrometry: entering the LTQ Orbitrap XL peptides hybrid mass spectrometer. ionisedwere separated via liquid chromatography before electrospray ionisation peptides were fragmented to reveal the (modified) amino acid sequence and entering Orbitrap XLfor hybrid The ionised for identity.the MSLTQ was also applied intact mass native spectrometer. and modified allergens.

were applied onto 14% polyacrylamide gels and stained with Coomassie

iodoacetamide (50 mM). partially reduced and fully reduced samples Non-reduced, SDS-PAGE: were applied onto 14% polyacrylamide gels and stained with Coomassie SDS-PAGE: Non-reduced, partially reduced and fully reduced samples Blue.

Modification: Methods:Ara h2-heavy isoform, Ara h2-light isoform and Ara h6 were modified Ara by reduction the disulphide bonds with 0.5 or 5 mM Modification: h2-heavyof isoform, Ara h2-light isoform and Ara h6 DTT subsequent alkylation the free bonds Cys with residues were and modified by reduction of theofdisulphide 0.5 orwith 5 mM iodoacetamide (50 mM).

DTT and subsequent alkylation of the free Cys residues with

Methods:

binding assay.

Modified forms of major peanut allergens Ara h2 and Ara h6 were shown Modified forms of major peanut allergens Ara h2 and Ara h6 were shown to be less allergenic than their native counterparts. In this way they are to be less allergenic than their native counterparts. In this way they are potentially suitable for peanut allergy immunotherapy. Two isoforms of potentially suitable for peanut allergy immunotherapy. Two isoforms of Ara h2 are known: a heavy isoform of ~20 kDa and a light isoform of ~17 Ara h2 a heavy isoform ~20kDa kDaisand a light isoform of ~17 kDa. Araare h6 known: with a molecular weight ofof~15 homologous with Ara kDa. Ara h6 with a molecular weight of ~15 kDa is homologous with h2. This study aims to investigate the biochemical characteristics of Ara h2. Thisand study aims Ara to investigate biochemicalwith characteristics reduced alkylated h2 isoforms the in comparison their native of reduced and alkylated Ara h2 isoforms in comparison with theirthe native counterparts and to study the kinetics of the modification. Therefore, counterparts and to study the kinetics of the modification.

Therefore, three modified allergens were analyzed for a change in molecular the three modified allergens analyzed a SDS-PAGE, change in molecular weight, protein structure andwere allergenicity by for using mass weight, protein structure and allergenicity by using SDS-PAGE, mass spectrometry (MS), CD and fluorescence spectroscopy, and an IgEspectrometry binding assay. (MS), CD and fluorescence spectroscopy, and an IgE-

Background Background&&Aim: Aim:

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2 2 3 3 HAL Leiden, The Netherlands, TNO Quality of Life, Zeist, The Netherlands, University of Belgrade, Belgrade, Serbia Serbia HALAllergy AllergyBV, BV, Leiden, The Netherlands, TNO Quality of Life, Zeist, The Netherlands, University of Belgrade, Belgrade,

11

1 &1 D. Apostolovic1, D. 1, D.1 Verbart1, A. 1de Jong2, H.H.Warmenhoven Warmenhoven & D. Apostolovic1, Luykx D. Luykx , D. Verbart , A. de Jong2, 3, S. 1 3,Koppelman 1 T. T. Cirkovic Velickovic S. Koppelman Cirkovic Velickovic

1448-Biochemical Biochemicalcharacterization characterization chemical modification of Ara 1448of of chemical modification of Ara h2 h2 isoforms: structure-function relationships isoforms: structure-function relationships

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EAACI Congress 2012

EAACI Congress 2012

Figure 3: Intrinsic fluorescence spectra of native (black) and modified (red) Ara h2heavy isoform (A), Ara h2-light isoform (B) and Ara h6 (C).

0 290

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Figure 2: Far UV CD spectra of native (black) and modified (blue) Ara h2-heavy isoform (A), Ara h2-light isoform (B) and Ara h6 (C).

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Figure 1: SDS-PAGE patterns of Ara h2-heavy isoform (A), Ara h2-light isoform (B), and Ara h6 (C) after reducing the allergens with 0.5 or 5.0 mM DTT for different periods of time (0-60 min). Molecular weight marker (M).

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.

Reduction and alkylation of Ara h2 and Ara h6 results in complete modification of all isoforms resulting in unfolded protein structures.

Conclusion:

The kinetics of the peanut allergen modification was studied with SDSPAGE as an increase of molecular mass was expected during reduction and alkylation (Fig. 1). By using 0.5 mM DTT the three allergens were totally modified after 30 min whereas 5 min was needed for modification when using 5 mM DTT. According to the SDS-PAGE patterns the increase of the molecular mass for the modified allergens appears to be ~4 kDa. According to MS, a mass increase of 465.6 Da was determined for Ara h2. Considering the presence of 8 Cys residues in Ara h2 and the alkylation (which adds 57 Da per Cys residue), this latter mass should be correct. The overestimated mass increase for the modified allergens obtained with SDS-PAGE can be explained by their protein structures. Far-UV CD spectra indicate typical -helix structures for the native allergens (i.e., ellipticity at 208 and 222 nm) whereas typical defolded protein structures were observed for the modified allergens (Fig. 2). Defolded and therefore stretched proteins migrate relatively slow through the SDS-PAGE gel in comparison to folded proteins resulting in bands that correspond to relatively higher molecular masses than expected. The defolded protein structures of the modified allergens are supported by fluorescence spectroscopy (Fig. 3). After full reduction of the three allergens, the intrinsic fluorescence intensity decreases and a small shift of the fluorescence emission maximum (Emax) is observed for the Ara h2-light isoform and Ara h6 (i.e., 3 and 8 nm, respectively).

These findings indicate that the Trp and Tyr residues in Ara h2, and Tyr residues in Ara h6 (Ara h6 does not contain Trp) are getting more exposed to the buffer. This occurs during defolding of proteins. The defolded protein structures of the peanut allergens result in a considerable decrease of allergenicity as the relative rest potency (IgE) of modified Ara h2-heavy isoform, Ara h2-light isoform and Ara h6 was determined to 0.01, 0.02 and 0, respectively.

Results:

IgE inhibition assay: The modified allergens compete with their corresponding native form coated to ELISA plates for binding to serum IgE.

E llip t ic it y ( m D e g )

F lu o r e s c e n c e i n te n s ity (1 0 5 L U )

Fluorescence spectroscopy: Fluorescence spectra were recorded from 290 to 390 nm with excitation at 280 nm.

E llip t ic it y ( m D e g )

F lu o r e s c e n c e i n te n s ity (1 0 5 L U )

CD: Far-UV CD spectra were recorded from 190 to 260 nm.

E llip t ic it y ( m D e g )

F lu o r e s c e n c e in te n s ity (1 0 5 L U )

Summar y

Product information.

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. 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. 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: May 2011 ®

®

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 ®

®

36

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 Lokalund/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

®

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 ®

venomenHAL

®

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 ACEHemmern; 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 ®

®

®

®

®

®

Notes Notes

26

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.hal-allergy.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-allergy.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

Romania SC Diagnostic Terapie Halcis Alergie SRL Mendeleev 11 300392 Timisoara Romania Tel.: +40-256-221413 www.halcis.ro

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

www.hal-allergy.com

Š HAL Allergy 2012 | MAB 48091-02