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AAAAI 61st Annual Meeting 18-22 March 2005, San Antonio, Texas

Dear Specialist We welcome you to San Antonio and the 61st Meeting of the American Academy of Allergy, Asthma & Immunology. An impressive amount of original, scientific documentation will be presented and we have selected some of the abstracts which we hope will be of interest to you. This booklet contains 11 abstracts within the fields of immunological mechanisms in allergy, allergen characterisation and specific allergy vaccination. The abstracts demonstrate the collaboration between allergy specialists and ALK-Abelló and illustrate our long-standing tradition of research in cooperation with the international academic community. Please join us at ALK-Abelló stand no. 30 for further interesting discussions. We look forward to meeting you during AAAAI 2005.

Yours sincerely ALK-Abelló A/S

Henrik Jacobi Executive Vice President Research & Development

Scientific Contribution

Grass pollen tablets for sublingual immunotherapy in Seasonal Allergic Rhinitis (SAR) MA Calderón Sublingual Immunotherapy (SLIT) in sensitised mice induces mucosal IgA antibodies J Kildsgaard Clinical safety of a grass pollen allergen tablet for specific immunotherapy J Kleine-Tebbe Role of Penicillium molds in three cases of food allergy M Lombardero Safety and immunological changes during tablet based specific immunotherapy H-J Malling Tolerability of cluster immunotherapy with an alum adsorbed mite extract M Mauro Analysis of the crystal structure of rproDer p 1 with emphasis on differences in antibody binding of pro/mature Der p 1 K Meno Kinetics of cytokine production in human T-cell cultures AH Millner Major allergen content of standardised mite and grass allergenic extracts G Plunkett Identification of nDer p 3 as the serine protease activity associated with purified nDer p 1 from house dust mite extract J Sauer The optimal recombinant allergen for treatment of birch allergy should contain characteristics of both Bet v 1 and Mal d 1, the major apple allergen PA Würtzen












Grass pollen tablets for sublingual immunotherapy in Seasonal Allergic Rhinitis (SAR)

MA Calderón1, S Rak2, SR Durham1 1Upper Respiratory Medicine, Imperial College, London, United Kingdom 2Section of Allergy, Sahlgrenska University Hospital, Gothenburg, Sweden

Rationale Specific allergen immunotherapy is the only treatment modality for allergic diseases that has the potential to alter the basic pathophysiological mechanism. Sublingual immunotherapy has been developed to ease the access to specific immunotherapy and to minimise the risk of serious systemic reactions. The objective of this trial was to investigate the efficacy and safety of ALK grass tablet in individuals with SAR. Methods A randomised, double-blind, placebo-controlled trial was conducted during the spring grass pollen season 2003. 55 centres in 8 countries included 855 adults with grass pollen induced SAR, positive skin prick test and specific IgE to Phleum pratense. Subjects were randomised to 2;500, 25;000, 75;000 SQ-T or placebo for sublingual administration once daily with no initial updosing phase. The mean duration of treatment was 18 weeks. Additional anti-allergic symptomatic medication was given as required. Results Average rhinoconjunctivitis scores showed reductions of both symptom (16%) and medication (28%) for the 75,000 SQ-T group (p=0.071 and p=0.047). With the recommended pre-seasonal treatment of at least 8 weeks prior to the grass pollen season, the reductions were increased to 21% and 29%, respectively (p=0.002 and p=0.012). Statistically significant better rhinoconjunctivitis quality of life was obtained as well as an increased number of well days. The tablet was well tolerated and no safety concerns were observed. Conclusion A clear clinical proof of concept was established for the ALK grass tablet. Further studies are required to document the optimal dose and duration of treatment.


Sublingual Immunotherapy (SLIT) in sensitised mice induces mucosal IgA antibodies

J Kildsgaard, H Jacobi, K Lund, J Brimnes, Department of Experimental Immunology, ALK-Abelló A/S, Hørsholm, Denmark

Rationale In order to study the immunological response induced by SLIT, sensitised mice were given SLIT treatment with an extract of Phleum pratense (Phl p). Methods Mice were sensitised by ip injections of alum adsorped Phl p. Sensitised mice received SLIT for 2, 4 or 6 weeks at 3 different concentrations, including a buffer control. Serum samples were analysed for Phl p specific IgE, IgG1, IgG2a and total IgG. Specific IgA was measured in washes of the lungs (BAL) and the nasal passages (NAL). Results SLIT treatment of sensitised mice resulted in a 5 to 30 times increase of antigen specific IgA levels in BAL and NAL fluid compared to buffer treated mice. On the contrary, antigen specific IgE was undetectable in BAL and NAL fluid of SLIT as well as buffer treated animals. The IgA levels were proportional to the duration as well as to the dose of SLIT administration. Furthermore, secretory IgA from BAL or NAL was able to block the binding of serum IgE to a biotinylated extract of Phl p. In contrast to IgA, no changes in serum levels of IgE and IgGs were observed upon SLIT treatment of sensitised mice. Conclusion High levels of secreted IgA in BAL and NAL in mice having received SLIT demonstrate that SLIT induces a mucosal, non-allergic immunological response in mice.


Clinical safety of a grass pollen allergen tablet for specific immunotherapy

J Kleine-Tebbe1, DA Herold1, M Ribel2 1Allergy & Asthma Center Westend, Berlin, Germany, 2ALK-Abelló A/S, Hørsholm, Denmark

Rationale Identification of a safe dose range for the ALK grass tablet, developed for once-daily, self-administered sublingual immunotherapy. Methods Groups of 12 subjects with grass pollen induced rhinoconjunctivitis, positive skin prick test and specific IgE to Phleum pratense were allocated to 7 dosage groups (25,000; 75,000; 150,000; 300,000; 500,000; 750,000 and 1,000,000 SQ-T) for a double-blind, placebo-controlled phase I trial. After randomisation (3:1), subjects received a once-daily sublingual dose of active treatment or placebo for 28 days outside the grass pollen season. Groups began treatment at staggered intervals for intermittent safety reviews. Results The ALK grass tablet did not cause any serious or systemic adverse events (AEs). Overall incidence of AEs was 74% (1013 AEs), all being mild or moderate in severity and most considered treatment related with a clear dose dependency. Typical AEs were itching sensations in the mouth and throat, with an indication of lighter symptoms over time for recurrent AEs. Objective oral findings were also dose dependent (incidence increased from 0-56%), lasting longer and recurring more often at the higher doses. Findings included small edema under the tongue/lower lip or erythema on the soft palate. Rescue medication was required for 13 subjects; none withdrew due to AEs. No clinically significant changes in laboratory measurements, vital signs or ECG were found. Conclusion ALK grass tablet at sublingual doses up to 1,000,000 SQ-T daily caused no serious or systemic adverse events displaying a safety profile that allows further investigation as once-daily self-medication.


Role of Penicillium molds in three cases of food allergy

M Lombardero1, C Díaz-Donado2, B Añibarro3, Á Núñez4, D Barber1 1ALK-Abelló, S.A., Madrid, Spain, 2Hospital Comarcal del Bierzo, Ponferrada, Spain, 3Hospital Severo Ochoa, Leganés, Spain, 4Hospital Central de la Defensa, Madrid, Spain

Rationale Molds are present as additives or even pollutants in several foods. We described 3 cases of food allergy probably caused by Penicillium molds. Methods Patients referred allergic symptoms after ingestion of dry sausage (fuet) or ham (patient 1), after eating fuet or a specific rice (patient 2) or after ingestion of fuet, sheep cheese or pork (patient 3). A microbiological study of samples of white dust from the sausage casings and the rice that caused the allergic reaction was carried out. Skin prick tests (SPT) were performed with a standard battery of allergens, and also with sausage casings, the specific rice (patient 2), and Penicillium nalgiovense mycelium extract. IgE immunoblotting was performed with the patients' serum after SDS-PAGE. Results All patients had a positive SPT response to sausage casings and P. nalgiovense extract. Patient 2 also had a positive response to the rice that caused the symptoms, but not to other rice. The microbiological study disclosed the presence of the mold P. nalgiovense in the samples of the sausage casings, and several Aspergillus and mainly Penicillium species in the rice that caused the symptoms to patient 2. IgE immunoblotting of P. nalgiovense extract revealed the presence of serum IgE against several protein bands (range 15-100 kDa) in patients 1 and 3, and against a band of about 55 kDa in patient 2. Conclusion Penicillium molds used as additives in highly consumed products (i.e. processed cold meat) or present as pollutants in some foods could act as hidden allergens and explain some cases of food allergy.


Safety and immunological changes during tablet based specific immunotherapy

H-J Malling1, L Lund2, H Ipsen2, LK Poulsen1 1Allergy Clinic, National University Hospital, Copenhagen, Denmark 2Research Department, ALK-Abelló A/S, Hørsholm, Denmark

Rationale The ALK grass tablet is a new preparation for sublingual immunotherapy of grass pollen allergy. We aimed at determining a safe dose range, and subsequently confirming the safety during daily sublingual administration. Simultaneously, immunological changes were measured. Methods A phase I trial with a randomised, double-blind, placebo-controlled design, with dosing in a stepwise dose escalation fashion during the dose finding period, and afterwards with daily dosing 8 weeks prior to and 15 weeks during the grass pollen season (2,500; 25,000; 75,000 SQ-T or placebo). 52 subjects with grass pollen induced rhinoconjunctivitis, a positive skin prick test and specific IgE to Phleum pratense entered the trial. Results During the single dose treatment period, 65% of the subjects reported adverse events; primarily local reactions in the throat and mouth. The dose escalation was stopped at 375,000 SQ-T. Throughout the daily dose treatment periods, 67% of the subjects reported adverse events. The most frequent ones were itching in mouth, eyes or throat and rhinitis. The majority of adverse events were mild in severity and resolved within 1 day. 2 participants withdrew due to adverse events. Time and dose dependent increases of Phleum pratense specific IgG, IgA, IgE and IgE competing components were found in serum during the first 8 weeks of daily dosing, indicating that the treatment had a significant effect on the immune system in an allergen specific manner. Conclusion The ALK grass tablet administered on a daily basis was well tolerated and had the ability to affect the immune system.


Tolerability of cluster immunotherapy with an alum adsorbed mite extract

M Mauro1, M Russello1, G Gazzola1, R Alesina2, V Sillano3, A Dama4 1S. Anna Hospital, Como, Italy, 2S. Matteo Hospital, Pavia, Italy, 3Abbiategrasso Hospital, Milano, Italy, 4Borgo Trento Hospital, Verona, Italy

Rationale Cluster protocols of immunotherapy (IT) have advantages in terms of time saving and patient compliance, but are currently rarely used for routine treatment of patients with respiratory allergy. This study was aimed at evaluating the tolerability of a cluster schedule for IT with a house dust mite extract. Methods 19 subjects (10 males, 9 females, mean age 28.8 years, range 14-48 years) suffering from rhinoconjunctivitis and/or asthma caused by dust mites were included in the study. They were treated with an alum adsorbed dust mite extract standardised in BU (Pangramin® Plus, ALK-Abelló) with a clustered schedule of 4 days with weekly intervals and a top dose of 8 BU, then used as maintenance dose with monthly intervals. Results In the build-up phase, the patients received an overall number of 152 injections, with 8 (5%) local reactions and 1 (0.6%) systemic reaction. Local reactions were all of the late type and did not require any treatment, although it was necessary to repeat the dose in only 1 case. The systemic reaction occurred on the last day of the schedule and was characterised by faintness. In the maintenance phase, there was only 1 reaction with subjective symptoms (dizziness, malaise). Conclusion These data indicate a good tolerability of this cluster schedule limited to 1 month, instead of the common 3 months, the time needed to reach the maintenance dose in IT with dust mite allergen.


Analysis of the crystal structure of rproDer p 1 with emphasis on differences in antibody binding of pro/mature Der p 1

K Meno1, PB Thorsted1, H Ipsen1, O Kristensen2, M Gajhede2, K Lund1 1Research Department, ALK-Abelló A/S, Hørsholm, Denmark, 2The Danish University of Pharmaceutical Sciences, Copenhagen, Denmark

Rationale Recombinant Der p 1 as a tool for allergy diagnosis and/or a vaccine candidate is an attractive prospect, but the proteolytic actions of an active cysteine protease or presence of the pro peptide on the immature protein have been of concern for some time. Methods An enzymatically inactive rproDer p 1 variant has been crystallised and the structure was solved by X-ray crystallography. The antibody binding properties of nDer p 1, rDer p 1 and rproDer p 1 were assessed by in situ crossed line immune electrophoresis (CLIE). Results Here we present the production and crystallisation of a stable recombinant proDer p 1 variant. The mature region adopts a conformation similar to the mature form of other cysteine proteases suggesting that no major structural changes are induced by maturation. The pro region adopts a unique fold, which interacts with the active site cleft and a substantial area adjacent to this on the mature region. From the fermentation culture, we have furthermore succeeded in purifying a fraction of the recombinant molecules which have spontaneously matured. This enzymatically inactive mature rDer p 1 variant showed antibody binding properties indistinguishable from nDer p 1 in CLIE. Conclusion For the first time, we show here the crystal structure of rproDer p 1. The pro peptide is placed in a position where it shields certain B-cell epitopes, compared to rDer p 1 or nDer p 1. Furthermore, it was proven possible to purify a mature but inactive variant of rDer p 1 from the culture medium.


Kinetics of cytokine production in human T-cell cultures

AH Millner, J Kildsgaard, PB Thorsted, PA Würtzen Research Department, ALK-Abelló A/S, Hørsholm, Denmark

Rationale In in vitro culture systems, cytokines are used as hallmarks of T-cell responses and to distinguish between Th1, Th2 and Treg responses. Recently, detection systems that detect several cytokines in one sample have been introduced. This calls for a re-evaluation of the kinetics of T-cell cytokine production in culture systems to ensure that all cytokines measured are detectable at the time of sample collection and that cytokine profiles measured reflect the complete response. Methods T-cell lines were generated towards grass and mite allergens and subsequently challenged with the allergens or mitogens. Cytokines released were measured at different time points by Cytokine Beads Array (CBA, BD) system. Similar experiments were performed with PBMC stimulated with allergens and recall antigens. All cytokine responses were related to T-cell proliferation. Real-time PCR was performed on all samples and included mRNA detection of IFN-g, IL-13, IL-10, IL-5 and IL-4. Results In allergen specific T-cell lines, IL-5 and IFN-g were detectable from 16 hours of stimulation onwards and increased throughout the culture period. IL-10 was detectable from 24 hours with stagnation from 48 hours onwards. IL-4 and TNF-a were at maximum levels between 24 and 48 hours and declined thereafter. In mitogen stimulations, all cytokines were detectable from 16 hours onwards. In PBMC assays, the cytokine production was highly dependant on the strength of the stimuli. The mRNA levels were slightly ahead of the protein production. Conclusion This suggests that the optimal time point for sample collection in in vitro culture systems is after 6 days in PBMC cultures and after 36 hours when T-cell lines are stimulated with allergens if both Th1 (IFN-g), Th2 (IL-5) and Treg (IL-10) cytokines are measured simultaneously.


Major allergen content of standardised mite and grass allergenic extracts

G Plunkett1, N Noieam1, J Gaswint1, R Lankow2 1ALK-Abelló, Inc., Round Rock, TX, USA, 2ALK-Abelló, Inc., Port Washington, NY, USA

Rationale Regulations in the US require standardised mite and grass extracts to be labeled for potency in Allergy Units (AU) and Bioequivalent Allergy Units (BAU). Potency is determined by competition ELISA, which compares the ability of a sample to inhibit binding of IgE to immobilised extract with that of an FDA issued reference extract. Extracts are adjusted by dilution to be statistically equal to the FDA reference. The equivalence range is typically 2-fold relative potency. The purpose of this study was to evaluate the major allergen content in these standardised extracts. Methods Group 1 and 2 major allergens in Dermatophagoides farinae and D. pteronyssinus mite extracts, Cyn d 1 in Bermuda, and Group 5 in Northern grasses were measured in standardised lots produced over the last 6 years. Immunoassays developed by ALK-Abelló, Denmark and Spain, and validated in our laboratory were used. Results The mite major allergen range was 3-fold, demonstrating a good relationship between these proteins and the competition ELISA. D. pteronyssinus lots contain 30% less major allergen since 2001, a drop that may correspond to a change in the FDA reference. Bermuda grass Cyn d 1 levels varied by 5.5-fold over the testing period. Northern grass lots show good consistency with average variability in Group 5 allergen of 2.5-fold. Conclusion IgE ELISA is considered a total potency assay. The determination of major allergen proteins can provide additional information for quality control of allergenic extracts. Knowledge of the major allergen content of extracts may help prevent drift in consecutive analytical references.


Identification of nDer p 3 as the serine protease activity associated with purified nDer p 1 from house dust mite extract

J Sauer1, PB Thorsted1, K Mutenda2, H Ipsen1, K Lund1 1Research Department, ALK-Abelló A/S, Hørsholm, Denmark, 2University of Southern Denmark, Odense, Denmark

Rationale Several papers have reported a proteolytic activity susceptible to inhibition with SBTI (a serine protease inhibitor) in purified preparations of the cysteine protease nDer p 1 from Dermatophagoides pteronyssinus. nDer p 1 has even been proposed to exhibit a mixed cysteine-serine proteinase activity (Hewitt et al., Clin Exp Allergy, 1997, 27:201-207). Methods nDer p 1* was purified to apparent homogeneity from extracts of D. pteronyssinus house dust mites by a combination of HIC, AIX and gel filtration (fraction 1). The purified nDer p 1* preparation was separated by Soybean Trypsin Inhibitor (SBTI) affinity chromatography into 2 fractions; bound serine protease and unbound nDer p 1, respectively. Proteolytic activities in the 3 samples were assessed and proteins present were identified by MS. Results Proteolytic assays, MS-peptide mapping and MS-peptide sequencing unambiguously identified the serine protease activity in our nDer p 1* preparation as originating from trace amounts of nDer p 3. Furthermore, in resemblance with natural Der p 1 the cysteine protease activity of recombinant Der p 1 was not inhibited by SBTI, whereas the cysteine protease inhibitor E64 blocked the activity. Conclusion We have demonstrated that nDer p 1 only possesses cysteine protease activity and that the observed closely associated serine protease activity originates from trace amounts of contaminating nDer p 3 with a very high proteolytic activity.


The optimal recombinant allergen for treatment of birch allergy should contain characteristics of both Bet v 1 and Mal d 1, the major apple allergen

PA Würtzen, L Lund, L Hansen, A Millner, M Ferreras, J Holm, H Ipsen Department of Experimental Immunology, ALK-Abelló A/S, Hørsholm, Denmark

Introduction Modified recombinant allergens have been suggested for SIT to reduce the risk of side effects and obtain a completely standardised allergy vaccine. Mal d 1 can be considered a naturally modified Bet v 1 allergen with similar three-dimensional structure. However, it probably has too low amino acid homology to Bet v 1 to be optimal for SIT. Aim To investigate the immunological activity of different rBet v 1 and rMal d 1 allergens that have been modified by point mutations. Results A graded binding to each modified allergen between the high binding to Bet v 1 and the low or undetectable binding to Mal d 1 was found in IgE binding experiments. A gradual reduction was also detectable in histamine release assay and serum facilitated allergen presentation assays (S-FAP) demonstrating that these changes are clinically and biologically relevant. T-cell reactivity towards rMal d 1 or rMal d 1 mutants was only observed in selected birch allergic patients. Immunisation of mice with single wt (wild type) or mutant rMal d 1 molecules resulted in an antibody response without capacity to block IgE binding to Bet v 1. Conclusion IgE binding and clinical and biological responses to major allergens may be specifically adjusted by point mutations to obtain safe vaccine candidates. In contrast, the immunogenicity the degree of homology needed to modulate the existing Bet v 1 specific allergic response seem to be more dramatically influenced by modifications within these molecules as seen from T-cell cross reactivity and blocking capacity of murine antibodies.



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