How convincing are Clinical Results with Recombinant Pollen Allergens Immunotherapy ? Pr G. Pauli Hôpitaux Universitaires de Strasbourg Bischenberg - September 24th, 2007

Immunotherapy with recombinant allergens *Can clinicians reasonably hope for  Future authorized allergy vaccines : one or a few recombinant allergen molecules ?  Genetically engineered recombinant allergen molecules with optimized safety profiles ?

*The starting point cloning of allergenic proteins from the main allergen sources.  Demonstration of individual allergic responses to allergenic molecules  concept of component-resolved immunotherapy (Valenta, Clin Exp Allergy 1999).  Development of several methods for producing hypoallergenic engineered allergens vaccines  in order to produce « secondary generation molecules » with characteristics specifically designed to achieve more specific and safer approaches to immunotherapy.

 Concerning these new molecules : Study *of T cell reactivity (human T cell clones or T cell lines). *of allergenic potential (studies in humans). *of immunogenicity: induction of allergen-reactive IgG For the clinician it is essential to have these studies confirmed using these molecules in immunotherapy trials in allergic patients : « gold standard »

*Full length allergen with reduced IgE binding activity site-directed mutants *Allergen fragments *Allergen oligomers *Deletion mutants *Allergen chimeres *Folded molecules *Conjugated molecules *DNA shuffling and screening  HYPOALLERGENIC DERIVATIVES

Les essais cliniques(1) *rec Bet v 1 frag.(n=38) rec Bet v 1 trimer (n=38) Niederberger et al., PNAS 2004 placebo (49)  Augmentation des IgG1 et des IgG4 *Bet v1 folding variant (25) Klimeck et al., A CI Immunol 2005 Bouleau (26)  Augmentation des IgG 1 et des IgG4 Dans les 2 études les modifications des Scores Med. Et Symptôme n’étaient pas statistiquement significaves.Des effets secondaires étaient observés.

V. Niederberger et al., PNAS 2004 ; 101, Sup.2

Fig. 1:Number of patients (ordinate) according to the ratio (abscissa) of positive skin prick test concentration of rBet v 1 dimer (D), trimer (T), fragment 1 (F1) and fragment 2 (F2) with rBet v 1 wild type. Skin tests with hypoallergens (polymers and fragments from Bet v 1) G. Pauli, Clin Exp Allergy 2000

Les essais cliniques *Phl p1,Phl p2,Phl p5a,Phl p5b,Phl p6 (29) Placebo (28) Jutel, JACI 2005 Diminution du Score Symp. (p=0.15) Diminution duscore combiné(p=0.051)  Amb a 1 conj.CpG (14) Placebo (11) Creticos, N Engl J Med 2006 Diminution du Score nasal (p=0.02)

Immunotherapie avec des allergènes recombinants de la Phleole un Cocktail de 5 allergènes majeurs en concentrations équimolaires Essai en double-aveugle contre placebo. Janvier 2002 à Aout 2003 Phl p 1 10 µg Phl p 2 5 µg Phl p 5a 10 µg Phl p 5b 10 µg Phl p 6 5 µg Phl p 1 10 µg Phl p 2 5 µg Phl p 5a 10 µg Phl p 5b 10 µg Phl p 6 5 µg Jutel et al. JACI 2005;116:608

active (n=24) placebo (n=25) SMS 38.9% Scores symptomes et medicamenteux

Grass pollen specific IgG1 (µg/L) Blood sample no Grass pollen specific IgG4 (µg/L) Blood sample no. IgG1IgG4 Immunotherapy with recombinant grass allergens Jutel et al. JACI 2005;116:608 IgG anti-pollen de graminées IgG anti-pollen de graminées

solid-phase: nBet v 1 Serumpool A Serumpool B nBet v 1 rBet v 1 rBet v 1-FV Inhibitor [µg/ml] Inhibition [%] (solid-phase nBet v 1) inhibitor (µg/ml) Bet v 1-FV: un variant hypoallergenique de Bet v 1

Birch pollen specific IgG4Bet v 1 specific IgG4 rBet v 1-FV Comparator Pfaar O et al, submitted Phase II rBet v 1-FV versus bouleau (Novo Helisen)

Symptom medication score Bet v 1-FV Comparator (NHD) Score symptomes-Medicaments: Amélioration en 2 années Phase II rBet v 1-FV versus Novo Helisen Depot Kettner J et al. EAACI Göteborg 2007

Included n=228 Active n=116 received Active n=108 Placebo n=112 received Placebo n=103 Phase III rBet v1-FV FAS * n=98 *reached individual maintenance dose + efficacy data available Randomisation 9 drop-outs8 drop-outs FAS * n= Year - Baseline 2. & 3. Years immunotherapy

effect très marqué sur les IgG4 anti-bouleau A: Screening B: Baseline C: before treatment, D: maintenance dose (8 injections) E: birch pollen peak 2005 F: after 1 year treatment, autumn 2005 G: before pollen season 2006 H: birch pollen peak 2006 Phase III rBet v1-FV Kettner J et al. EAACI Göteborg 2007

Immunotherapy with Bet v 1

A randomized, double-blind, placebo-controlled study with recombinant Bet v1, natural Bet v1 and birch pollen extract G.Pauli,TH.Larsen,S.Rak,F.Horak,E.Pastorello, LK.Poulsen,R.Valenta all(submitted) Strasbourg(France),Copenhagen(Danemark), Gothenborg(Sweden),Vienna(Austria), Milan(Italy)

Primary efficacy analysis (ITT Population) Total symptom score of rhinoconjunctivitis (RC) Daily average total score of RC by treatment group Pollinic peak 2004 Daily average total score of RC by treatment group Pollinic peak 2005

Rescue Medication Score: pollen peak of 2004 TREATMENT EFFECT Differences LSMeans* 95% CI* p-value* Pbo - n Bet v [0.43;3.51] Pbo - r Bet v [0.29;3.30] Pbo - Birch 1.92 [0.37;3.46] Other pairwise comparisons : NS Results are confirmed: - with PP Population (p<0.05) - for pollen season 2004 (p<0.01) Pbo = Placebo *Analysis of variance with treatment and centre as factors (ANOVA on the ranks provide p-values) and Bonferroni adjustment against multiplicity. Statistical tests were two-sided and performed at an alpha level of 5%.

Skin prick tests with recombinant bet v1 (50µg/ml) Changes from baseline of wheal diameters after 1 and 2 years of treatment Comparison of each active group versus placebo: p<0.0001

*Essai pivot démontrant que l’immunothérapie avec un seul allergene recombinant est aussi efficace que l’extrait naturel. *Les allergènesrecombinants peuvent être produits avec un haut degré de pureté,de manière reproductible et pourrait remplacer les extraits traditionnels pour l’ immunotherapie. *Abscence d’effets secondaires notables(dose de maintenance:15 µg). *Apparition de nouvelles sensibilisations dans le groupe traité avec l’extrait de bouleau (Anticorps specifiques anti- Bet v 2 dans 3 cas). Pas de nouvelles sensibilisations dans les groupes traités par Bet v 1 et n Bet v 1.