Treatment of allergies

Abstract

The present invention relates to modified allergens for use in the prevention, reduction, or treatment of an allergy in a human said use comprises exposing said human to an initial dose of said allergen of less than 625 AUeq modified allergen followed by multiple increasing dosages up to 50,000 AUeq modified allergen. Suitable modification according to the present invention are glutaraldehyde crosslinking and reduction and alkylation. The present invention further relates to methods for prevention, reduction, or treatment of an allergy in a human and to compositions comprising a modified allergen suitable for use in the prevention, reduction, or treatment of an allergy in a human.

Description

[0001] TREATMENT OF ALLERGIES

[0002] Description

[0003] The present invention relates to modified allergens, such as crosslinked ragweed modified allergens, for use in the prevention, reduction, or treatment of an allergy in a human said use comprises exposing said human to an initial dose of said allergen of less than 625 AUeq modified allergen followed by multiple increasing dosages up to 50,000 AUeq modified allergen. The present invention further relates to methods for prevention, reduction, or treatment of an allergy in a human and to compositions comprising a modified allergen suitable for use in the prevention, reduction, or treatment of an allergy in a human.

[0004] Exposure to ragweed Ambrosia artemisiifolia) pollen is one of the most common causes of type I allergic reactions in late summer and autumn in North America. It is most prevalently associated with allergic rhinitis / rhinoconjunctivitis (ARC). ARC is an upper airway inflammatory disease resulting from an IgE-mediated immunological response following allergen exposure. Symptoms of ARC include rhinorrhoea, sneezing, nasal itching, congestion as well as non-nasal symptoms such as conjunctivitis, itching of the eyes and tearing. In extreme cases, ARC symptoms can develop into asthma.

[0005] Approximately 26% of Americans between the ages of 6 and 59 are sensitized to ragweed pollen. Ragweed sensitization rates vary vastly in Europe: with sensitization rates of up to 60% in Hungary and 2.4% in Finland. Prediction models anticipate that ragweed prevalence will spread further and become domestic in a large part of Europe in the future. Globalization, as well as environmental factors such as temperature and carbon dioxide concentrations, are accountable for the expected increase in world-wide ragweed presence. Pollen season durations and pollen concentrations are also anticipated to rise. All together these changes will increase ragweed sensitization rates as well as allergy symptom severity.

[0006] Although ARC is usually not a severe disease, it is associated with a substantial economic burden as well as impairment in quality of life. Patients often suffer from decreased quality of sleep and cognitive function, leading to a reduced performance at school and work. In addition to the substantial direct medical costs of ARC and associated comorbidities, like sinusitis and asthma, the indirect costs of ARC due to loss of work productivity are also considerable. A survey asking 2500 full-time working Americans suffering from ARC if nasal allergies interfered with their work in the past year, revealed: 10% had missed work due to nasal allergies, 22% recognized that allergies interfered with their ability to work, and 20% found nasal allergies interfered with their work and lead to missed work. The survey also identified that workers rated their productivity 23% lower when suffering from allergy symptoms as opposed to when they were asymptomatic. Allergen-induced ARC treatment approaches include pharmacotherapy and specific allergen immunotherapy (AIT). Pharmacotherapy treatments such as nasal corticosteroids, antihistamines and decongestants focus on symptomatic relief. While such symptomatic medications are certified by clinical trials, they manage to control allergy symptoms in less than 50% of ragweed allergic subjects. AIT is the only disease-modifying treatment for IgE-mediated allergies with long-lasting effects post-treatment.

[0007] AIT is the practice of administering gradually increasing doses of allergens in order to reduce allergic symptoms and the need for medication. It works by directing T- lymphocytes to a state of tolerance. Allergic subjects have a deregulated immune system that has an excessive shift of T helper-precursor lymphocytes in the direction of the Th2 cells. Th2 cells help induce the formation of IgE which mediates the release of histamine and other pro- inflammatory signaling molecules, leading to allergen hypersensitivity. AIT counters this allergic reaction by increasing IgG formation. An elevation in IgG levels leads to a decrease in specific IgE levels as well as histamine release and consequently reduces allergic symptoms. Successful AIT leads to a decrease in both immediate- and late -phase allergic reactions after allergen exposure. AIT has been effectively used for the treatment of insect venom allergies as well as respiratory allergies such as allergic rhinitis to pollen and house dust mite.

[0008] Traditional AIT approaches for the treatment of ragweed allergy use aqueous protein extracts. While subcutaneous administration of aqueous allergens has been proven efficacious, it is associated with a slow up-dosing schedule as well as an increased concern for severe local and / or systemic allergic reactions. The uncertainty around the presence of contaminants as well as the exact amount of allergen in the aqueous extracts, makes it difficult to anticipate any allergic reactions and to predict the immunogenicity of the product. Consequently, approaches that improve the safety profile of AIT such as alternate routes of administration as well as the use of adjuvants and chemically modified extracts have been explored.

[0009] Sublingual immunotherapy (SLIT) is one method that has gained interest due to the potential for fewer severe side effects. Ragwitek® is a SLIT for the treatment of ragweed- induced ARC that should be taken once daily from twelve weeks before, through to the end of the ragweed pollen season. Ragwitek® has been demonstrated to be well-tolerated and effective. Studies, however, suggest compliance and efficacy may be lower for SLIT than for subcutaneous immunotherapy (SCIT) due to home administration of the SLIT products.

[0010] The safety and efficacy profile of SCIT has improved with the introduction of adjuvants and chemical extract modifications. Adjuvants like aluminum, polyethylene, glycol, tyrosine etc., are used to create depot formulations. These formulations slow down the rate of protein extract release into the tissue, and hence limit the chances of massive mast cell degranulation, reducing the risk of adverse events. Chemical extract modification of the allergen prevents specific IgE antibodies from recognizing the protein and hence limits the cascade of events that lead to an allergic reaction. Compared to non-modified allergen extracts, modified extracts allow higher doses to be administered as well as fewer up dosing steps without compromising immunogenicity. Pollinex R® is a SCIT for the treatment of ragweed allergy that is composed of a modified ragweed allergen extract adsorbed to tyrosine. Studies have demonstrated that pre-seasonal treatment with four weekly Pollinex R® injections is efficacious and safe.

[0011] Considering the above, it is an object of the present invention, amongst other, objects to provide more effective means for the treatment of allergies and specifically ragweed allergy.

[0012] This object of the present invention, amongst other objects, is met as outlined in the appended claims.

[0013] Specifically, this object of the present invention, amongst other objects is, according to a first aspect, met by modified allergens for use in the prevention, reduction, or treatment of allergy in a human wherein said use comprises exposing said human to an initial dose of said allergen of less than 625 AUeq modified allergen followed by multiple increasing dosages up to 50,000 AUeq modified allergen.

[0014] Within the context of the present invention 1 AUeq is defined the IgG potency after modification, where lAUeq / mL in IgG potency equals lAU / mL in IgE potency measured before modification.

[0015] According to the present invention, the initial dose is less than 625 AUeq modified allergen such as less than 500 AUeq, less than 315 AUeq, or less than 155 AUeq.

[0016] According to the present invention, the multiple increasing dosages are up to 50,000 AUeq modified allergen, such as up to 40,000 AUeq, up to 30,000 AUeq, up to 20,000 AUeq, up to 10,000 AUeq or up to 5,000 AUeq.

[0017] According to a preferred embodiment, the present modified allergen: is a crosslinked modified allergen, preferably a glutaraldehyde crosslinked modified allergen; or is a reduced and subsequently alkylated monomeric allergoid.

[0018] Glutaraldehyde modification prevents specific IgE-antibodies from recognizing the allergen without interfering with the sequential T-cell epitopes; consequently, the product allergenicity is reduced without affecting immunogenicity. The reduction in allergenicity of MRE versus unmodified ragweed extract is demonstrated by in vitro studies that show a significant reduction in mediator release. In vivo studies confirm that with crosslinked allergens such as crosslinked ragweed immunized animals had an increase in allergen specific IgG levels and an induction of cytokine release. Reduction and alkylation can, for example, be provided by a dithiothreitol (DTT) treatment of a monomeric allergoid for reduction, followed by a iodoacetamide (IAA) treatment for alkylation. Reduction and alkylation break up the disulfide bridges of a monomeric allergoid resulting in loss of conformation and consequently in significant reduction of allergenicity thereby resulting in a more favourable safety profile. This approach showed modulation of allergenicity and immunogenicity for Pru p 3, the major peach allergen and was previously investigated on the isolated peanut allergens Ara h 2 and 6.

[0019] According to another preferred embodiment, the present modified, preferably crosslinked, allergen is ragweed protein, and the present allergy is ragweed allergy, more specifically ragweed pollen allergy.

[0020] According to yet another preferred embodiment, the present and reduced and subsequently alkylated monomeric allergoid are selected from the group consisting of:

[0021] Common ragweed Ambrosia elatior) and Amb a 1 , Amb a 2 and / or Amb a l l proteins;

[0022] Rot fungus {Altemaria altemata) and Alt a 1 protein;

[0023] Mugwort Artemisia vulgaris) and Art v 1 protein;

[0024] Aspergillus fumigatus and Asp f 1 and / or Asp f 2 proteins

[0025] Bee venom {Apis Mellifera) and Api m 1, Api m 2 and / or Api m 5 proteins;

[0026] European beech {Fagus sylvatica) and Fag s 1 and / or Fag s 2 proteins;

[0027] European ash {Fraxinus excelsior) and Fra e 1 protein;

[0028] Bentgrass (Agrostis stolonifera) and Agr g 1 protein;

[0029] Sweet vernal grass {Anthoxanthum odoratum) and Ant o 1 protein;

[0030] Cocksfoot {Dactylis glomerata) and Dac g 1 v protein;

[0031] Perennial ryegrass {Lolium perenne) and Fol p 1 protein;

[0032] Tall oat grass {Arrhenatherum elatius) and Arr e 1 protein;

[0033] Red fescue {Festuca rubra) and Fes r 1 protein;

[0034] Kentucky blue grass {Poa pratensis) and Poa p 1 protein;

[0035] Cultivated rye {Secale c er eale) and Sec c 1 protein;

[0036] Velvet grass {Holcus lanatus) and Hol 1 1 protein;

[0037] Timothy {Phleum pratense) and Phi p 1 and / or Phi p 5 protein;

[0038] Cultivated wheat {Triticum aestivum) and Tri a 1 protein;

[0039] Dog {Canis familiaris) and Can f 1 protein;

[0040] Cat {Felis domesticus) and Fel d 1 protein;

[0041] House dust mite {Dermatophagoides pteronyssinus) and Der p 1 , Der p 2 and / or Der p 23 proteins; House dust mite (Dermatophagoides farinae) and Der f 1 and / or Der f 2 proteins;

[0042] Olive Olea europea) and Ole e 1 and / or Ole e 9 proteins;

[0043] Wall pellitory (Parietariajudaica) and Par j 1 and / or Par j 2 proteins;

[0044] English plantain (Plantago lanceolata) and Pla 1 1 protein;

[0045] Alder (Abuts glutinosa) and Ain g 1 protein;

[0046] Birch (Betula verrucosa) and Bet v 1 protein;

[0047] Hazel (Corylus avellana) and Cor a 1 protein; and

[0048] Common wasp venom (Vespula vulgaris) and Ves v 1, Ves v 2 and / or Ves v 5 proteins.

[0049] According to an especially preferred embodiment, the present modified allergen, preferably the modification is crosslinking, is complexed with an adjuvant, preferably with aluminium, more preferably aluminium hydroxide (A1(OH)3) or the present modified allergen is complexed and saturated with an adjuvant, preferably with aluminium, more preferably aluminium hydroxide (A1(OH)3). Adsorption of the modified allergens to aluminium hydroxide further reduces the allergenicity by reducing the rate of protein release into the body.

[0050] An example of a modified allergen according to the present invention is a modified allergen designated herein as HAL-MRE1, a glutaraldehyde-modified, aluminum hydroxide adsorbed ragweed extract. Glutaraldehyde modification prevents specific IgE-antibodies from recognizing the allergen without interfering with the sequential T-cell epitopes.

[0051] According to the present invention, the above treatment of allergy comprises, preferably, an initial dose of said modified allergen of less than 625 AUeq followed by multiple increasing dosages, preferably weekly, up to 30,000 AUeq modified food allergen such as up to up to 20,000 AUeq, up to 10,000 AUeq or up to 5,000 AUeq.

[0052] According to the present invention, the present modified allergen is, preferably, presented to the immune system by parenteral administration, subcutaneous administration, oral administration, intralymphatic administration, mucosal administration, intestinal administration, rectal administration, vaginal administration, epicutaneous administration, dermal administration, sublingual administration, ophthalmic administration, nasal administration, or combinations thereof.

[0053] Suitable formulations of the present modified allergen are in the form a pharmaceutical composition, a beverage, a food product, a toothpaste, a skin patch, a band aid, a mouth wash, a candy, a skin creme, a tablet, a lozenge, a food supplement, a foodstuff, or combinations thereof.

[0054] Considering the above, according to a second aspect, the present invention relates to methods for prevention, reduction, or treatment of allergy in a human 10. Method for prevention, reduction, or treatment of allergy in a human wherein the methods comprise the steps of: a) exposing said human to an initial dose of a modified allergen as defined above of less than 625 AUeq; b) subsequently exposing said human to multiple increasing dosages of said modified allergen up to 50,000 AUeq.

[0055] In the present method, the modified allergen is preferably complexed with an adjuvant, preferably with aluminium, more preferably aluminium hydroxide (A1(OH)3), preferably wherein said modified allergen is complexed and saturated with an adjuvant, preferably with aluminium, more preferably aluminium hydroxide (A1(OH)3).

[0056] The present allergy and reduced and subsequently alkylated monomeric allergoid are, preferqbly, selected from the group consisting of:

[0057] Common ragweed Ambrosia elatior) and Amb a 1 , Amb a 2 and / or Amb a l l proteins;

[0058] Rot fungus {Altemaria altemata) and Alt a 1 protein;

[0059] Mugwort Artemisia vulgaris) and Art v 1 protein;

[0060] Aspergillus fumigatus and Asp f 1 and / or Asp f 2 proteins

[0061] Bee venom {Apis Mellifera) and Api m 1, Api m 2 and / or Api m 5 proteins;

[0062] European beech {Fagus sylvatica) and Fag s 1 and / or Fag s 2 proteins;

[0063] European ash {Fraxinus excelsior) and Fra e 1 protein;

[0064] Bentgrass (Agrostis stolonifera) and Agr g 1 protein;

[0065] Sweet vernal grass {Anthoxanthum odoratum) and Ant o 1 protein;

[0066] Cocksfoot {Dactylis glomerata) and Dac g 1 v protein;

[0067] Perennial ryegrass {Lolium perenne) and Fol p 1 protein;

[0068] Tall oat grass {Arrhenatherum elatius) and Arr e 1 protein;

[0069] Red fescue {Festuca rubra) and Fes r 1 protein;

[0070] Kentucky blue grass {Poa pratensis) and Poa p 1 protein;

[0071] Cultivated rye {Secale c er eale) and Sec c 1 protein;

[0072] Velvet grass {Holcus lanatus) and Hol 1 1 protein;

[0073] Timothy {Phleum pratense) and Phi p 1 and / or Phi p 5 protein;

[0074] Cultivated wheat {Triticum aestivum) and Tri a 1 protein;

[0075] Dog {Canis familiaris) and Can f 1 protein;

[0076] Cat {Felis domesticus) and Fel d 1 protein;

[0077] House dust mite {Dermatophagoides pteronyssinus) and Der p 1 , Der p 2 and / or Der p 23 proteins; House dust mite (Dermatophagoides farinae) and Der f 1 and / or Der f 2 proteins;

[0078] Olive Olea europea) and Ole e 1 and / or Ole e 9 proteins;

[0079] Wall pellitory (Parietariajudaica) and Par j 1 and / or Par j 2 proteins;

[0080] English plantain (Plantago lanceolata) and Pla 1 1 protein;

[0081] Alder (Abuts glutinosa) and Ain g 1 protein;

[0082] Birch (Betula verrucosa) and Bet v 1 protein;

[0083] Hazel (Corylus avellana) and Cor a 1 protein; and

[0084] Common wasp venom (Vespula vulgaris) and Ves v 1, Ves v 2 and / or Ves v 5 proteins.

[0085] In the method according to present invention, a human is preferably exposed to the present modified allergen by parenteral administration, subcutaneous administration, oral administration, intralymphatic administration, mucosal administration, intestinal administration, rectal administration, vaginal administration, epicutaneous administration, dermal administration, sublingual administration, ophthalmic administration, nasal administration or combinations thereof formulated, where appropriate, as a pharmaceutical composition, a beverage, a food product, a toothpaste, a skin patch, a band aid, a mouth wash, a candy, a skin creme, a tablet, a lozenge, a food supplement, a foodstuff, or combinations thereof.

[0086] According to a third aspect, the present invention relates to compositions comprising a modified allergen as defined above suitable for the prevention, reduction, or treatment of allergy.

[0087] Suitable combinations of the present invention of allergies and compositions comprising modified, preferably crosslinked or reduced and alkylated, allergenic proteins are:

[0088] Common ragweed (Ambrosia elatior) and Amb a 1 , Amb a 2 and / or Amb a l l proteins;

[0089] Rot fungus (Altemaria altemata) and Alt a 1 protein;

[0090] Mugwort (Artemisia vulgaris) and Art v 1 protein;

[0091] Aspergillus fumigatus and Asp f 1 and / or Asp f 2 proteins

[0092] Bee venom (Apis Mellifera) and Api m 1, Api m 2 and / or Api m 5 proteins;

[0093] European beech (Fagus sylvatica) and Fag s 1 and / or Fag s 2 proteins;

[0094] European ash (Fraxinus excelsior) and Fra e 1 protein;

[0095] Bentgrass (Agrostis stolonifera) and Agr g 1 protein;

[0096] Sweet vernal grass (Anthoxanthum odoratum) and Ant o 1 protein;

[0097] Cocksfoot (Dactylis glomerata) and Dac g 1 v protein;

[0098] Perennial ryegrass (Folium perenne) and Lol p 1 protein;

[0099] Tall oat grass (Arrhenatherum elatius) and Arr e 1 protein; Red fescue (Festuca rubra) and Fes r 1 protein;

[0100] Kentucky blue grass (Poa pratensis) and Poa p 1 protein;

[0101] Cultivated rye Secale c er eale and Sec c 1 protein;

[0102] Velvet grass (Hol cits lanatus) and Hol 1 1 protein;

[0103] Timothy (Phleum pratense) and Phi p 1 and / or Phi p 5 protein;

[0104] Cultivated wheat (Triticum aestivum) and Tri a 1 protein;

[0105] Dog (Canis familiaris) and Can f 1 protein;

[0106] Cat (Fells domesticus) and Fel d 1 protein;

[0107] House dust mite (Dermatophagoides pteronyssinus) and Der p 1 , Der p 2 and / or Der p 23 proteins;

[0108] House dust mite (Dermatophagoides farinae) and Der f 1 and / or Der f 2 proteins;

[0109] Olive (Olea europea) and Ole e 1 and / or Ole e 9 proteins;

[0110] Wall pellitory (Parietariajudaica) and Par j 1 and / or Par j 2 proteins;

[0111] English plantain (Plantago lanceolata) and Pla 1 1 protein;

[0112] Alder (Alnus glutinosa) and Ain g 1 protein;

[0113] Birch (Betula verrucosa) and Bet v 1 protein;

[0114] Hazel (Corylus avellana) and Cor a 1 protein; and

[0115] Common wasp venom (Vespula vulgaris) and Ves v 1, Ves v 2 and / or Ves v 5 proteins.

[0116] According to an especially preferred embodiment, the allergy is ragweed pollen allergy, and the composition comprises a glutaraldehyde-modified, aluminum hydroxide adsorbed ragweed extract designated herein as HAL-MRE1.

[0117] The present invention will be further detailed in the examples below. In the examples, reference is made to figures wherein:

[0118] Figure 1: shows increase in RE, MRE, and Amb a 1 -specific IgG levels in serum of

[0119] WISTAR rats after repeated SC injections with HAL-MRE1. RE (A), MRE (B) and Amb a 1 (C)-specific IgG levels in serum of Wistar rats (n=20 per group) after repeated SC injections with Placebo (Plac) or HAL-MRE1 (MRE). Serum IgG was measured prior to treatment and after treatment at day 78. Statistical analysis was carried out using the Mann-Whitney U test. Significant differences are denoted by (**) or (***) for p<0.01 and 0.001, respectively.

[0120] Figure 2: Summary of up-dosing regimen EXAMPLES

[0121] Example 1: Non- clinical studies

[0122] Introduction

[0123] Ragweed-induced allergic rhinitis / rhinoconjunctivitis (ARC) is an upper airway inflammatory disease resulting from an IgE-mediated immunological response following allergen exposure. Symptoms include: rhinorrhea, sneezing, nasal itching, congestion, conjunctivitis, as well as itching and tearing of the eyes.

[0124] Materials

[0125] HAL-MRE1 is an AIT for subcutaneous administration containing A1(OH)3 adsorbed, glutaraldehyde modified allergens extracted from ragweed Ambrosia artemisiifolia). Unlike the traditional AIT treatment approaches, which uses aqueous allergen extracts, HAL- MRE1 is composed of a modified allergen extract adsorbed to A1(OH)3. The advantage of A1(OH)3 adsorption is that proteins are slowly released into the tissue over an extended period of time giving rise to fewer local reactions. Chemical modification of allergens aims to reduce binding of IgE antibodies (so-called allergenicity), which leads to a reduction of allergic side effects. However, in order to induce immune system tolerance towards the allergen, the modified allergen should still be able to trigger the immune system (so-called immunogenicity).

[0126] The allergenicity of modified ragweed extract (MRE) versus unmodified ragweed extract (RE) was investigated by mediator release experiments. The in vitro experiments demonstrated that RE induces mediator release at lower concentrations than MRE and the maximal release was stronger when the cells were stimulated with RE. Therefore, under these test conditions, chemical modification of RE resulted in a product candidate with an improved safety profile.

[0127] The immunogenic potency of MRE was evaluated by measuring the induction of RE-specific IgG in mice. The in vivo mouse and rat immunogenicity models demonstrated that immunizations with MRE / HAL-MRE1 lead to the induction of an IgGl antibody response against RE. This indicates that MRE is a strong inducer of antibodies that are cross-reactive with unmodified ragweed pollen proteins.

[0128] Further, immunogenic potencies of reduced and alkylated and glutaraldehyde crosslinked modified ragweed extracts were determined. Reduction was carried out by the incubation of ragweed extracts at 60°C with IM of DTT (5 pl / mg protein) during 1 hour. After this, samples were cooled below 40 °C for the alkylation and were incubated for 90 minutes with 0.5M of IAA (20 pl / mg protein). Glutaraldehyde modification was carried out by the addition of 8.8 pl / mg protein of 50% glutaraldehyde at 4 °C and overnight incubation. Subsequently, 52.8 pl / mg protein glycine (100 mg / ml) was added to block the remaining non-reacted aldehyde groups.

[0129] Using Phadia 250 and the ImmunoCAP technology, a potency assay was carried out using the modified ragweed extracts by determining the capacity of each extract to bind to antibody IgE of sensitized patients.

[0130] Regarding toxicology, two genotoxicity studies (a bacterial reverse mutation test and an in vitro micronucleus assay) and a repeat dose toxicity study in rats with HAL-MRE1 were performed. HAL-MRE1 did not exert any mutagenic activity in the bacterial reverse mutation test. The micronucleus test demonstrated that HAL-MRE1 did not have any clastogenic or aneugenic potential. Results from the repeat-dose in vivo rat study show that under the study conditions, and compared to HAL-MRE1 placebo, no evidence of systemic toxicity were noted at the tested dose of 20,000 AUeq / injection HAL-MRE1.

[0131] HAL-MRE1 (Table 1) is supplied in a multi-dose, colourless 6R Ph. Eur. Type I glass vial with a bromobutyl rubber stopper, aluminum cap with polypropylene flip-off seal, with a target filling volume of 3.6 mL of HAL-MRE1.

[0132] Table I: The composition of 1 mL of HAL-MRE1

[0133] 120,000 AU eq / mL used for the clinical trial, 40,000 AUeq / mL used for the toxicity studies

[0134] 2Amount of aluminum hydroxide is based on 1.0 mg / mL Aluminum

[0135] The production of HAL-MRE1 consists of several steps. First, an extract of ragweed is prepared. The extract is separated from the solid residue by filtration steps and analyzed for several parameters such as the allergenic activity and protein profile. After modification, glutaraldehyde is removed by diafiltration. Finally, the modified ragweed allergens in the extract are adsorbed on to Al(0H)3. Al(0H)3 acts as an adsorbent that effectively binds the (modified) ragweed allergens. The amount of Al(0H)3 is small and the intake is far less than that received from diet or medications such as some antacids.

[0136] Eleven short ragweed pollen allergens belonging to eight protein families have been recorded in the International Union of Immunological Societies (IUIS) allergen database (Table 2).

[0137] Table 2: Ragweed pollen allergens recorded in the IUIS allergen database

[0138] The major allergen in common ragweed is Amb a 1. It has 5 known isoallergen forms and a sensitization frequency greater than 90%. Clinical data strongly suggest that Amb a 1 represents the most abundant and important allergen in ragweed. Amb a 1 is composed of a mixture of 5 isoforms. Isoforms 01, 02 and 03 bind most of ragweed-specific IgE and isoforms 01 and 03 are the most potent stimulators of T cells.

[0139] Pharmacology studies

[0140] In order to avoid potentially dangerous side-effects resulting from the administration of ragweed allergens, a 2-step safety approach is followed. First, the RE preparation is chemically modified to reduce the allergen-specific IgE binding potential and subsequent IgE- mediated allergic responses. The second approach includes the binding of MRE to A1(OH)3 which provides a depot function increasing the safety of the preparation. A set of pharmacology studies were performed to investigate the safety and immunogenic responses of RE and MRE in an immunogenicity model in mouse and rat. An overview of these studies with their noteworthy findings is provided in Table 3. Table 3; Overview of pharmacology studies performed with MRE / HAL-MRE1

[0141] Admin.: Administration; F: Female; M: male; MRE: Modified Ragweed Extract; RE: Ragweed Extract SC: subcutaneously

[0142] Sera from ragweed-allergic patients Sera were obtained from 12 Austrian subjects with a convincing history of ragweed allergy as determined by clinical history, positive skin prick test and immunoblot analysis using ragweed pollen extracts. Subjects’ clinical and serologic characteristics are summarized in Table 4. Table 4: Clinical and serological characteristics of ragweed allergic patients 'RAST: Blood test to detect specific IgE antibodies. The RAST is scored on a scale from 0-6 with score 4, 5 and 6: Very high, ultra-high and extremely high levels of allergen-specific IgE

[0143] Immunoblot

[0144] IgE binding was assessed in sera of 12 Austrian patients by Immunoblot. Of the 12 ragweed-allergic sera, 10 showed IgE reactivity to Amb a 1 on immunoblot, while 2 donors (patients 11 and 12) did not react to Amb a 1. It was decided to include 2 sera of patients without Amb a 1 reactivity to be able to study the difference between these patients and patients with a strong response to Amb a 1. On immunoblot, the serum of patient 12 showed IgE reactivity with a small (below 15 kDa) protein band, which could account for the ragweed profilin Amb a 8. The serum of patient 11 reacted with a very diffuse band in the size range of approximately 25 kDa, which could correspond to Amb a 4, the ragweed homolog of the major allergen from mugwort Art v 1. As Amb a 4 is a glycoprotein.

[0145] Mass spectrometry analysis of the extracts

[0146] To characterize RE and MRE used for the pharmacology studies, mass spectrometry (MS) was performed. Clinical data strongly suggest that Amb a 1 represents the most abundant and important allergen in ragweed. Amb a 1 is composed of a mixture of 5 isoforms. Isoforms 01, 02 and 03 bind most of the patients’ ragweed-specific IgE and isoforms 01 and 03 are the most potent stimulators of patients’ T cells. MS analyses of RE and MRE revealed that after modification, all allergens present in the natural extract are still detectable (Table 5).

[0147] Table 5: Summary of MS-analysis of RE and MRE (Samples were double digested by trypsin / chymotrypsin, measured, and analysed with PEAKS Studio 8.5 based on all Amb a isoforms available in the IUIS allergen nomenclature database.)

[0148] Humanized rat basophil leukemia cell mediator-release assay (huRBL) The allergenicity of RE versus MRE was assessed by mediator release experiments using sera from ragweed allergic patients (see Table 4 for patient characteristics). Mediator release assays were performed using huRBL cells carrying the human Fcs I alpha chain (high affinity IgE receptor). HuRBL cells were passively sensitized with individual patient sera. Different doses of RE or MRE were used to trigger B-hexosaminidase release of huRBL cells. In all patients, RE induced mediator release at lower concentrations compared to

[0149] MRE. When calculating the protein amount which was necessary to trigger 25% of mediator release, on average, 100-1000 fold more MRE was required compared to RE (Table 6A). When the sera of the patients 11 and 12 (both Amb a 1 non-responders on immunoblot) were used, activation with MRE did not lead to any mediator release whereas activation with RE triggered substantial activation (due to reactivity to other allergens than Amb a 1 present in RE).

[0150] Summarized, MRE activated basophils at a significantly higher concentration compared to RE and led to a significantly lower maximum release of B-hexosaminidase. These data strongly suggest that modification is able to reduce allergenicity.

[0151] Table 6A: Potency reduction of MRE compared to RE using serum of 12 patients with established ragweed allergy

[0152] The immunogenic potencies of reduced and alkylated (Pl and P2 ) and glutaraldehyde crosslinked (P3 and P4) modified ragweed extracts are shown in Table 6B: Table 6B: Comparison of immunogenic potencies of two types of modification Mediator release assay in summary

[0153] In rat basophils, humanized with the high affinity human IgE receptor and sensitized with individual patient sera, basophil activation was reduced in all tested sera comparing exposure to MRE and RE. Overall the degranulation studies showed that the response to MRE is 100-1000 fold lower than the response to RE. Differences between patient sera may be explained by lower IgE antibody affinity or a reduced number of recognized IgE-binding epitopes. These data demonstrate that chemical modification impairs the capacity of RE to activate basophils, probably due to a reduced capability to cross-link the IgE receptor. MRE activated basophils at a significantly higher concentration compared to RE and led to a significantly lower maximum release of histamine, demonstrating the ability of MRE to reduce allergenicity. From a safety point of view, MRE is candidate for the development of immunotherapy for ragweed allergy.

[0154] ELISpot assay; splenocyte stimulation from RE and MRE- immunized mice

[0155] ELISpot assays were performed using splenocytes of RE / MRE-immunized mice harvested one week after the final immunization and stimulated with either MRE or tissue culture medium to identify Thl, Th2, and regulatory T cell activation following different immunization regimens. RE stimulation of splenocytes from mice immunized with either RE or MRE, both formulated with Al(0H)3, did not induce IL- 10 producing cells but only cells secreting IL-4, IL-5, and IFN-Y- Moreover, RE stimulation of splenocytes from mice immunized with matrix did not lead to the induction of cytokine-producing cells. According to this assay, both immunizations, with MRE as well as RE, led to the induction of splenocytes reactive to RE. This supports the idea to use MRE as immunotherapeutic candidate for the treatment of ragweed pollen allergies.

[0156] Immunogenicity of HAL-MRE1 in BALB / c mice

[0157] The immunogenic potency of MRE was evaluated by measuring the induction of RE-specific IgG in mice. Animal experiments were performed according to the guidelines of the Austrian Federal Ministry of Science, Research, and Economy.

[0158] In a murine immunogenicity model, animals immunized with RE and MRE both reacted equally strong to coated RE after two immunizations with formulated RE and MRE. This is reflected by the mean IgGl titers in sera of MRE and RE immunized mice (6.7* 104and 10.2* 104) compared to a mean titer of 0.1*104in sham immunized mice.

[0159] After 4 immunizations with RE and MRE, IgGl levels against coated RE , coated MRE or coated Amb a 1 were significantly increased in all treated groups compared to sham immunized animals. The level of Amb a 1 -specific IgG was measured to confirm that modified Amb a 1 in HAL-MRE1 is able to induce antibodies that can recognize the unmodified Amb a 1. The study showed that rats indeed developed antibodies that were able to recognize Amb a 1.

[0160] MRE immunized mice reacted significantly stronger to coated MRE compared to RE immunized animals. There was no difference in IgGl binding of MRE or RE immunized mice to coated RE. This indicates that MRE is a strong inducer of antibodies that are cross-reactive with unmodified ragweed pollen proteins.

[0161] Immunogenicity of HAL-MRE1 in Wistar Rats

[0162] The immunogenicity of HAL-MRE1 was repeated in Wistar rats (report HAL_002), the species used for the toxicology studies. Wistar rats were immunized subcutaneously with 0.5 mL 40,000 AUeq / mL HAL-MRE1 or RE, according to the same regime as used in the mouse immunogenicity study. Both RE and HAL-MRE1 immunizations show an increase in IgGl antibody production against RE, MRE and Amb a 1. These data again indicate that HAL-MRE1 is a strong inducer of antibodies, which are cross-reactive with unmodified ragweed pollen proteins.

[0163] Conclusion

[0164] Overall, the non-clinical pharmacology studies showed:

[0165] 1. Reduced mediator release by MRE from rat basophils loaded with IgE from ragweed- allergic patients;

[0166] 2. The induction of cytokines by RE stimulation of splenocytes of both RE as well as MRE-immunized mice;

[0167] 3. An IgG response in BALB / c mice immunized with MRE that was cross-reactive with the native RE preparation;

[0168] 4. An IgG response in Wistar rats immunized with MRE that was cross-reactive with the native RE preparation.

[0169] Toxicology

[0170] HAL-MRE1 was tested in 2 genotoxicity studies (a bacterial reverse mutation test and an in vitro micronucleus assay) and in a repeat-dose (11 weeks) toxicity study in Wistar rats. According to the “Guidelines on the nonclinical evaluation of vaccine adjuvants and adjuvanted vaccines”, only one relevant (i.e., immunologically responsive) animal species is required for toxicological testing of vaccines. These same principles can be applied to immunotherapies such as HAL-MRE1. The safety of HAL-MRE1 was evaluated in rats because they produce an immunological response to HAL-MRE1 treatment. The pivotal rat toxicology study and genotoxicity testing were performed according to Good Laboratory Practice (GLP). The testing strategy followed the following guidelines:

[0171] • ICH M3 (R2) - Non-clinical safety studies for the conduct of human clinical trials and marketing authorization for pharmaceuticals.

[0172] • WHO / BS / 2013.2214 - Guidelines in the nonclinical evaluation of vaccine adjuvants and adjuvanted vaccines

[0173] • CPMP / SWP / 465 / 95 - Note for Guidance on preclinical pharmacological and toxicological testing of vaccines

[0174] The GLP toxicology studies were conducted with a non-GMP produced development batch of HAL-MRE1, which was produced in a similar way as the final HAL-MRE1 drug product that will be used in the clinical trials.

[0175] Drug product HAL-MRE1: bacterial reverse mutation assay using a modified treat and plate methodology

[0176] HAL-MRE1 was assayed for mutations in four histidine -requiring strains (TA98, TA100, TA1535 and TA1537) of Salmonella typhimurium, and one tryptophan-requiring strain (WP2 uvrA pKMIOl) of Escherichia coli (Table 7), both in the absence and presence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9). A 'treat and plate' methodology was used for all treatments in this study because Error! Reference source not found, histidine, which may cause artefacts through growth stimulation in a standard plateincorporation test.

[0177] The bacterial reverse mutation assay has been used to investigate the mutagenic effect of many chemicals. A large database is available confirming the assay's ability to detect genetically active compounds of most chemical classes with around 80 to 90% sensitivity and specificity. When the bacterial strains are exposed to a mutagen, some of the bacteria in the treated population, through chemical interaction with the compound, undergo genetic changes which cause them to revert to a prototrophic state and thus grow in the absence of exogenous amino acids. Different tester strains were used because each strain is mutated by a particular class of chemical compound. A compound that is mutagenic in one strain may not be mutagenic in another. Table 7: The bacterial strains used in the bacterial reverse mutation assay'.

[0178] Organism Strain Type of Mutation Mutant Gene

[0179] S. typhimurium TA98 frame-shift Histidine

[0180] S. typhimurium TA 100 base-pair substitution Histidine

[0181] S. typhimurium TA1535 base-pair substitution Histidine

[0182] S. typhimurium TA1537 frame-shift Histidine

[0183] E. coli WP2 uvrA pKMIOl base-pair substitution Tryptophan

[0184] Treat and plate assays, in which the agent is incubated with the microbial cells prior to plating, are recommended in preference to standard plate-incorporation tests for compounds which may cause artefacts due to growth stimulation. This method allows the separation of treatment and selective systems.

[0185] To provide a robust and thorough assessment of the mutagenicity of HAL-MRE1, treatments were performed up to the maximum achievable treatment concentration by adding the maximum tolerated volume (0.5 mL per 2 mL treatment mix) of HAL-MRE1, as supplied, to the assay system. The maximum treatment concentration was therefore 300 pg protein / mL. The maximum concentration can also be expressed as 250 pL HAL-MREl / mL, which exceeds the maximum recommended concentration according to current regulatory guidelines (OECD, 1997).

[0186] Negative (vehicle and untreated) and positive control treatments were included for all strains in the mutation experiment. The mean numbers of revertant colonies all fell within acceptable ranges for vehicle control treatments and were consistent with those for the untreated controls, and were elevated by positive control treatments. Mutation experiment treatments of all the tester strains were performed in the absence and in the presence of S-9, using final concentrations of HAL-MRE1 at 0.9, 3, 9, 30, 90, and 300 pg protein / mL. Following these treatments, no evidence of toxicity was observed on any of the test plates.

[0187] Following HAL-MRE1 treatments of all the test strains in the absence and presence of S-9, the only notable increase occurred in strain TA1537 in the presence of S-9. This increase was relatively small but did just achieve the 3-fold threshold level over the concurrent vehicle control counts. However, this increase was not concentration-related, occurring as it did at a single intermediate concentration. Accordingly, this increase was not considered to be a true compound-related or mutagenic effect and was most likely due to normal biological variability in combination with relatively low vehicle control counts.

[0188] As no other treatments provided any increases in revertant numbers that were >2- fold (in strains TA98, TA100 and WP2 uvrA pKMIOl) or >3-fold (in strains TA1535 and TA1537) the concurrent vehicle control, this study provided no clear evidence of any HAL-MRE1 mutagenic activity in this assay system.

[0189] It was concluded that HAL-MRE1 did not induce mutation in four histidine - requiring strains (TA98, TA100, TA1535 and TA1537) of Salmonella typhimurium, and one tryptophan-requiring strain (WP2 uvrA pKMIOl) of Escherichia coli when tested under the conditions of this study. These conditions included treatments at concentrations up to at least 300 pg protein / mL (the maximum achievable concentration, and also equivalent to 250 pL HAL- MREl / mL which exceeds the maximum recommended concentration according to current regulatory guidelines) in the absence and in the presence of S-9.

[0190] HAL-MRE1: In vitro micronucleus study

[0191] In the micronucleus test, the clastogenic and aneugenic potential of HAL-MRE1 was evaluated by analysing its effects on the frequency of micronuclei in Chinese hamster ovary (CHO) cells treated in the absence and presence of S-9. Cells were treated with our product candidate without phenol. It was decided to test the drug product without phenol because there are a number of articles that suggest phenol causes significant increases in micronucleus frequency (EC, 2006. European Union Risk Assessment Report). When testing the drug product without phenol, the IMP is still effectively tested.

[0192] Treatment of cells with HAL-MRE1 (without Phenol) in the absence and presence of S-9 resulted in frequencies of micronucleated binucleate (MNBN) cells which were similar to and not significantly higher (at the p<0.05 level) than those observed in concurrent vehicle controls for all concentrations analysed. The MNBN cell frequency of all HAL-MRE1 (without Phenol) treated cultures (3+21 hour treatment, 24+0 hour treatment) fell within the 95% confidence interval of the current observed historical vehicle control (normal) ranges.

[0193] In conclusion, no increases in MNBN cell frequency were observed when tested under the conditions of this study.

[0194] Repeat dose toxicity studies: 11 week subcutaneous administration toxicity study with HAL MRE1 in rats with a 2 week recovery phase

[0195] The objective of this study was to evaluate the toxicity of the test article, HAL- MRE1, when administered twice weekly (22 doses) via subcutaneous injection to rats for at least 11 weeks and to assess the reversibility, persistence, or delayed occurrence of any effects after a 2-week recovery period.

[0196] Treatment groups and dosing schedule Male and female RccHan®:WISTAR rats were assigned to three groups, and doses were administered as indicated in Table 8. Animals were dosed via subcutaneous injection in the dorsal region twice weekly for 11 weeks at a volume of 0.5 mL / injection. The vehicle control article was HAL-MRE1 Placebo.

[0197] Table 8: Dosing scheme in repeat dose toxicity study

[0198] No. of Animals0Dose LevelNcDose Concentration^

[0199] GroupaMale Female (AUeq) (AUeq / mL)

[0200] 1 (Saline Control) 10 10 0 0

[0201] 2 (Vehicle Control)d,e 15 15 0 0

[0202] 3 (Clinical Dose)e15 15 20,000 40,000 a Group 1 was administered saline control article and Group 2 was administered vehicle control article only. b Dose levels and concentrations were expressed as active ingredient of test article. c Animals were dosed at a volume of 0.5 mL / injection. d Vehicle control article contains aluminum hydroxide and liquefied phenol. e Animals designated for recovery sacrifice (5 animals / sex in Groups 2 and 3) underwent 2 weeks of recovery following dose administration.

[0203] Toxicological findings

[0204] Assessment of toxicity was based on mortality, clinical observations, body weight changes, food consumption, ophthalmic observations, body temperatures, and clinical as well as anatomic pathology. Blood samples were collected for serology analysis.

[0205] No HAL-MRE1 -related mortality occurred; all animals survived until the scheduled sacrifice. Injection site swelling, which remained through the day of the recovery sacrifice, was observed for all animals administered HAL-MRE1 or vehicle control article, but not for animals administered saline control article. Differences in body temperature between groups were attributable, in part, to differences present prior to dosing initiation and could not be related directly to the administration of HAL-MRE1 or vehicle control article. No HAL-MRE1- or vehicle control article -related changes in body weight, food consumption, or ophthalmic examinations were observed.

[0206] Changes noted in animals administered HAL-MRE1 were minimally higher reticulocyte counts, moderately to mildly higher white blood cell counts, moderately to mildly higher neutrophil counts, and minimally higher monocyte counts on Day 78 of the dosing phase. Mildly to minimally higher circulating white blood cell counts and minimally higher neutrophil counts were also noted on Day 15 of the recovery phase, illustrating lessened but persistent evidence of inflammation in these animals.

[0207] Higher reticulocyte counts were supportive of a regenerative response likely due to hemorrhage noted in the subcutaneous injection site upon microscopic assessment of individual animals. Increased circulating white blood cell counts were due to higher neutrophil and monocyte counts and were consistent with an inflammatory response. These hematological changes correlated with higher fibrinogen concentrations for animals administered HAL-MRE1; however, fibrinogen concentrations were also higher in vehicle controls on Day 78 of the dosing phase. These changes correlated with lower albumin concentrations as a negative acute phase protein on Day 78 of the dosing phase in both sexes administered HAL-MRE1 and vehicle control females.

[0208] Additionally, globulin concentrations were higher on Day 78 of the dosing phase in animals administered HAL-MRE1, and higher values also occurred on Day 15 of the recovery phase in vehicle controls and animals administered HAL-MRE1. Higher globulin concentrations were supportive of antigenic / immune stimulation. These changes also correlated with inflammation, observed in the subcutaneous injection site upon microscopic assessment, which occurred in vehicle controls and animals administered HAL-MRE1.

[0209] Vehicle control article- or HAL-MRE1 -related hematology changes in animals on Day 15 of the recovery phase included minimally higher measures of red blood cell mass (hematocrit, hemoglobin concentration, and red blood cell counts) and higher reticulocyte counts. Higher red blood cell mass values were suggestive of minimal dehydration. Higher reticulocyte counts were consistent with a regenerative response. No vehicle control article- or HAL-MRE1- related urinalysis changes occurred.

[0210] HAL-MRE1 -related spleen weight increases occurred during the dosing phase in animals administered HAL-MRE1. This change persisted in the recovery phase and, as a result, increased weights were noted for recovery sacrifice animals previously administered HAL-MRE1. Vehicle control article- or HAL-MRE1 -related macroscopic observations were noted for both subcutaneous injection sites A and B used in this study (Dose site A being the left dorsal region and dose site B being the right dorsal region). Incidences of these findings were similar between the two groups during the dosing and recovery phases. Injection site observations included discolored (entire section or subcutis) and / or thickened (entire site or subcutis) for animals during the dosing phase and mass (few or multiple) and / or thickened (entire site or subcutis) at the end of the recovery phase. These differences in macroscopic observations were consistent with changes in the character of the inflammation which became more organized at the end of the recovery phase. These macroscopic observations correlated with microscopic changes of hemorrhage, fibroplasia, granulomatous inflammation, and / or necrosis of the subcutaneous injection sites. Vehicle control article- or HAL-MRE1 -related microscopic changes occurred during the dosing phase at subcutaneous injection sites A and B of animals administered vehicle control article or HAL-MRE1; these changes included granulomatous inflammation, hemorrhage, fibroplasia, and necrosis of the subcutis. Inflammation, fibroplasia, and necrosis changes were often noted at higher incidences with higher severities in animals administered HAL-MRE1, compared with vehicle controls. Vehicle controls had the same microscopic changes, but at higher incidences, with lower severity grades compared with animals administered HAL-MRE1.

[0211] Hemorrhage occurred at low incidence and low severity grade for both groups. During the recovery phase, vehicle control article- or HAL-MRE1 -related microscopic changes at Subcutaneous Injection Sites A and B of animals administered vehicle control article or HAL- MRE1 included granulomatous and lymphocytic inflammation, fibroplasia, and necrosis of the subcutis. Inflammation, fibroplasia, and necrosis were often noted with similar / comparable incidences and at similar severity grades for animals vaccinated with HAL-MRE1, compared with vehicle controls. Hemorrhage was not noted at the injection site of recovery animals indicating a start of recovery after not being treated for 2 weeks.

[0212] Immunogenicity evaluations

[0213] Immunogenicity of HAL-MRE1 was analyzed by measuring RE- and MRE- specific IgG titers in serum from rats in the repeat-dose toxicity study. IgG titers were determined at 2 time points: pre-dose and at sacrifice at day 78 for rats from the HAL-MRE1 group and the placebo group. Samples acquired from the repeated dose toxicity study were shipped from Covance (USA) to Salzburg University, Austria, for analysis. RE- and MRE-specific IgG was determined by ELISA.

[0214] RE-, MRE-, and Amb a 1 -specific IgG levels were determined prior to treatment and at the end of the 11 -week in life study. Sera of recovery animals were not measured for IgG induction.

[0215] Male and female WISTAR rats showed (Figure 1) an immunogenic response upon repeated administration with HAL-MRE1. HAL-MRE1 treatment induced a large increase in both MRE-specific and RE-specific IgG responses, compared to the HAL-MRE1 placebo group. Also Amb a 1 -specific IgG was induced after repeated injection of rats with HAL-MRE1. The level of Amb a 1 -specific IgG was measured to confirm that modified Amb a 1 in HAL-MRE1 is able to induce antibodies that can recognize the unmodified Amb a 1. The study showed that rats indeed developed antibodies that were able to recognize Amb a 1.

[0216] Summary In summary, no evidence of systemic toxicity was noted following subcutaneous administration of 20,000 AUeq / injection HAL-MRE1, 2 times per week for 11 weeks (total of 22 administrations; 0.5 mL / inj ection). HAL-MRE1 -related effects included changes at the injection sites (edema, induration, subcutaneous masses, nodular infiltration of enlarged granular basophilic macrophages, infiltration of lymphocytes / plasma cells, fibroplasia / fibrosis and necrosis) and in the spleen (increased weight, increased size and number of lymphoid follicles, increased evidence of extramedullary hematopoiesis), which were consistent with the expected immune reactions. These changes, with the exception of hemorrhage, were generally still present at the end of the treatment free period.

[0217] Overall, adverse findings associated with HAL-MRE1 were also present following administration of vehicle control article. Some findings were more pronounced in animals administered HAL-MRE1, compared with vehicle control article, but outcomes did not differ significantly between the groups. HAL-MRE1 treatment induced a large increase in both RE and MRE specific IgG responses, as well as Amb a 1 specific IgG responses, compared to the HAL- MRE1 placebo group, providing evidence of an immunogenic response in this repeat-dose toxicity study.

[0218] Conclusion

[0219] The adverse findings at the injection sites can mainly be attributed to the presence of A1(OH)3 in the composition of HAL-MRE1. In the repeat-dose toxicity study, the exposure to A1(OH)3 is far above the maximum exposure of patients in the proposed safety study. The exposure of rats to Al(0H)3 in the toxicity study is 11 mg (22 injections of 0.5 mg). In the first-in-human safety study, patients were exposed to 0.7 mg (cohort 1), 1.5 mg (cohort 2) or 3.0 mg (cohort 3) and HAL-MRE1 could demonstrate its safety.

[0220] Example 2: Effects in humans

[0221] The first-in-human phase I study was performed in Canada (study HAL- MRE1 / 0051). This study aimed to assess the safety and tolerability of HAL-MRE1 SCIT in adult subjects suffering from ragweed pollen-induced ARC with or without asthma by establishing a safe starting dose, followed by safe dose escalation schedule and a maximum tolerated dose.

[0222] Outpatients of Cliantha Research in Mississauga, Ontario, Canada fulfilling the inclusion and exclusion criteria were invited and selected to participate in the study. Inclusion criteria included a well-documented medical history of ARC to ragweed pollen, a positive nasal provocation test (NPT) to ragweed pollen (30 pg / mL Amb a 1), positive skin prick test (SPT) to ragweed pollen (mean wheal diameter > 3mm) and positive serum specific IgE test for ragweed allergen (IgE level >0.7 U / mL).

[0223] In total, 45 subjects with ragweed pollen-induced ARC were randomized into 3 study cohorts. None of the randomized subjects had asthma. In this study, different starting and target doses were investigated. Fifteen subjects of cohort 1 started their HAL-MRE1 or placebo treatment with a dose of 155 AUeq which was escalated to a maximum dose of 5,000 AUeq or placebo. Each subsequent cohort (15 subjects each) started with the 2nd dose of the previous cohort (or placebo), i.e. the starting dose of cohort 2 and 3 was 315 AUeq and 625 AUeq (or placebo) respectively, whereas the maximum dose of these cohorts was 10,000 AUeq or 20,000 AUeq (or placebo) respectively (see Figure 2). Study medication was administered once weekly with increasing doses for a period of 5 weeks up to the maximum dose, which was repeated once as final injection.

[0224] Injections of the study drug were administered weekly under controlled conditions at the investigational site and administered in an alternating manner on the extensor surfaces of the upper arms, a hand width above the elbow, up to the middle of the upper arm.

[0225] The safety and tolerability of HAL-MRE1 versus placebo treatment was assessed by evaluating the number, intensity and seriousness of early (< 30 minutes after injection), delayed (> 30 minutes to < 3 hours after injection) and late (> 3 hours to < 24 hours after injection) local and systemic reactions and the occurrence of treatment emergent adverse events (TEAEs). The severity of systemic reactions was assessed according to the World Allergy Organization (WAO) Subcutaneous Immunotherapy Systemic Reaction Grading System.

[0226] Additionally, safety laboratory parameters, urinalysis, vital signs, ECG, physical examination, pregnancy test and lung function tests were assessed. The safety population included all subjects who were randomized and received at least one dose of the study drug. Exploratory efficacy parameters were investigated by assessing the change in serum levels of ragweed specific immunoglobulins (IgE, IgG and IgG4) and the change in wheal size of the SPT with ragweed extract.

[0227] Subjects were randomized in 3 cohorts of 15 subjects each to HAL-MRE1 treatment or placebo in a 2:1 ratio. The actual number of subjects assigned to HAL-MRE1 and placebo in cohort 1 was 11 and 4 subjects respectively. In cohorts 2 and 3 the number of subjects assigned to HAL-MRE1 and placebo was 10 and 5 subjects, respectively.

[0228] The overall Safety population consisted of 60.0% (n=27) female subjects and 40.0% (n=18) male subjects. The mean age range of all subjects (all population) in the study was 47.6 (24-62) years; 52.3, 44.7 and 46.0 years in cohorts 1, 2, and 3 respectively. The majority of subjects were of Caucasian (42.2%) or African American (37.8%) origin.

[0229] Two subjects (one from placebo and one from HAL-MRE1 group) discontinued after randomization and start of study treatment. A summarized listing of these discontinuations is provided in Table .

[0230] Table 9: Summary of discontinued subjects (Safety Population)

[0231] 43 / 45 subjects completed the study and reached the maximum dose per cohort. 12 additional injections were given to 8 subjects in the HAL-MRE1 treatment group (up to 2 additional doses per subject were allowed by the Protocol); 1 additional injection in cohort 1, 4 in cohort 2 and 7 in cohort 3. Two additional injections in the placebo group in cohort 1 and 2 were due to out of window visits. All systemic and local reactions were assessed as treatment-related as per protocol requirements.

[0232] Local Reactions (primary endpoint):

[0233] There were 7 swellings > 8 cm at the site of injection reported by five subjects (16.1%) in the HAL-MRE1 group, and none in the placebo group, see Table 10 below. All swellings occurred as late reactions (> 3 h and < 24 h after injection) and were assessed as mild or moderate and drug-related. Systemic Reactions (primary endpoint):

[0234] In total, 10 systemic reactions were reported in 4 subjects in the HAL-MRE1 group and no systemic reactions were reported in the placebo group (3 systemic reactions were reported later than 24 hours and therefore were not included into Table 10 below). 7 Systemic reactions were reported within 24 hours after IMP administration (primary endpoint), see Table 10. All systemic reactions were assessed as study treatment related; 3 subjects (6 events) experienced a grade 1 systemic reaction, 1 subject (4 events) experienced a grade 2 systemic reaction. No Grade 3, 4 or 5 systemic reactions according to the WAO grading system were reported in any of the treatment groups. All 7 systemic reactions were reported within 24 hours by a total of 3 subjects in Cohort 2 and 3.

[0235] There was no relationship between the dose and the occurrence of systemic reactions in the HAL-MRE1 group detected, no systemic reactions were observed in the maximum dose per cohort, e.g. there were no dose-limiting symptoms detected in this study. No SAE was reported during the study.

[0236] Table 10: Summary of local (Swelling >8 cm) and systemic reactions within 24 hrs after injection

[0237] - primary endpoint (Safety population) in HAL-MRE1 / 0051

[0238] *Only mild and moderate local reactions reported

[0239] ** Only grade 1 and 2 systemic reactions reported

[0240] Abbreviations: E = No of events; n corresponds to number of subjects that experienced event; N = number of subjects in specified treatment group.

[0241] Note: Percentages are based on the number of subjects in specified treatment group.

[0242] It was observed that HAL-MRE1, despite the short treatment period in this study, was able to induce statistically significant changes in immune parameters (dose-dependent increase of ragweed-specific serum IgE, IgG and IgG4 levels compared to placebo) see Table 11 and to demonstrate a trend in decreasing skin reactivity to ragweed allergen. The mean change in wheal size between baseline and End of Study visit showed a dose dependent decrease in the HAL-MRE1 treatment groups, however, the clinical significance of this efficacy surrogate parameter has to be investigated in future trials.

[0243] Table 11. Output of ANCOVA: Summary of Short-term immunologic effect at baseline and EoS (ITT population - Combined cohort)

[0244] Abbreviation: N = number of subjects in specified treatment group; EOS / ET = end of study / early terminatio n.

[0245] Note: ANCOVA model include change from baseline as dependent variable, treatment as a factor and the ba seline as a covariate.

[0246] Safety and efficacy

[0247] The safety and tolerability of HAL-MRE1 versus placebo treatment were assessed by evaluating the number, intensity and seriousness of early (< 30 minutes after injection), delayed (> 30 minutes to < 3 hours after injection) and late (> 3 hours to < 24 hours after injection) local and systemic reactions and the occurrence of treatment emergent adverse events (TEAEs). The severity of systemic reactions was assessed according to the World Allergy Organization (WAO) Subcutaneous Immunotherapy Systemic Reaction Grading System. Additionally, safety laboratory parameters, urinalysis, vital signs, ECG, physical examination, pregnancy test and lung function test were assessed. The safety population included all subjects who were randomized and received at least one dose of the study drug.

[0248] Local Reactions Overall local reactions were reported more frequently in the HAL-MRE1 group compared to the placebo group (80.6% versus 64.3% of subjects per group), see Table 12. Most local reactions were reported as late events, occurring 3-24 hours after injection. Most local reactions were assessed as mild; only 2 / 155 reactions in the HAL-MRE1 group were reported as moderate.

[0249] The most common reported drug-related TEAEs were local reactions at the site of injection: pruritus (57.8%), injection site pain (44.4%), bruising (20.0%), erythema (15.6%) and large swellings > 8 cm (11.1% of subjects), see Table 12. The occurrence of pruritus, erythema and large swellings >8 cm was higher in the HAL-MRE1 treatment group (77.4%, 22.6% and 11.1%) compared to the placebo group (14.3%, 0.0% and 0.0% accordingly). Overall 32 subjects (71.1% of safety population) reported 218 swellings >0 and <8 cm within 3 hours after injection, with 204 swellings in 28 (90.3%) subjects in the combined HAL-MRE1 group and 14 swellings in 4 subjects (28.6%) in the placebo group.

[0250] In the same period 27 subjects in the HAL-MRE1 group reported 245 redness local reactions >0 to <8 cm and 3 subjects in the placebo group reporting 7 redness reactions, see table 13. The number of subjects who experienced swellings >0 and <8 cm developed in time: within 30 minutes after injection 19 subjects in the HAL-MRE1 group reported 42 swellings, whereas in the subsequent period of >30 minutes to <3 hours, 28 subjects reported 162 swellings; a similar time dependence was observed for the development of redness local reactions. Five subjects (16.1%) in the combined HAL-MRE1 group reported seven swellings >8 cm at the injection site and none in the combined placebo. Six swellings >8 cm were reported as mild, one as moderate. All swellings >8 cm were assessed as drug product related and occurred as late reactions (>3 hours and <24 hours).

[0251] Table 12. Summary of number and % of subjects with at least one local reaction (0-3 hrs) by type (swelling / redness) (Safety population - Combined cohort) Cohort 1 - one swelling was reported by one subject at maximum dose of 5,000 AUeq

[0252] Cohort 2 - one swelling was reported by one subject at dose 1 ,250 AUeq

[0253] Cohort 3 - two swellings were reported by one subject at the first up-dosing (625 AUeq), a second subject reported swellings at the first and second up-dosing (625 AUeq and 1,250 AUeq), and moreover one swelling was reported by a third subject at the second up-dosing (1,250 AUeq). No swelling >8 cm was reported at the maximum doses in cohorts 2 and 3. The number of swellings in cohort 3 was higher compared to the number in cohort 1 and cohort 2.

[0254] Table 13. Summary of all Local TEAEs by Intensity and Timing per Treatment group (Safety population - Combined Cohort)

[0255] Systemic reactions

[0256] Three subjects (9.7%) in the combined HAL-MRE1 group reported seven systemic reactions and none of the subjects in the combined placebo group reported systemic reactions within 24 hours after injection, see Table 10).

[0257] One subject (3.2%) experienced three delayed systemic reactions, allergic rhinitis (two events) and allergic conjunctivitis (one event); all three reactions were assessed as grade 1. Two subjects (6.5%) experienced four late systemic reactions. One subject reported feeling hot and urticaria - both reactions were assessed as grade 1 systemic reactions - and another subject reported nausea and vomiting which were assessed as grade 2. Furthermore, two subjects in the HAL-MRE1 group experienced three systemic reactions >24 hours after injection: nausea, vomiting (both grade 2) and urticaria (grade 1). Due to their very late occurrence, more than 24 hr after injection, these systemic reactions were not taken into account for the evaluation of the primary endpoint. No early (<30 minutes after injection) systemic reactions were reported in the study. No grade 3, 4 or 5 systemic reactions were reported in any of the treatment groups.

[0258] Overall, 35 subjects (77.8% of safety population) reported 194 local TEAEs within 24 hours after the IMP administration (including five subjects reporting seven swellings >eight cm), with 155 events in 26 (80.6%) subjects in the combined HAL-MRE1 group (83.8%) and 39 events in nine subjects in the combined placebo group (64.3%). All reported local TEAEs were related to the study drug. Because 10 TEAEs were reported beyond 24 hours, they were not included into the summary of TEAE, Table 14. Table 14: Summary of number and percentage of subjects with at least one related TEAE by SOC and PT (Safety population - Combined cohort)

[0259] The vast majority of local TEAEs was assessed as mild, 153 / 155 in the HAL- MRE1 group and 39 / 39 in the placebo group. Only 2 local reactions, reported by one subject, were assessed by the investigator as moderate in the HAL-MRE1 group, none in the placebo group. No severe local reaction was reported. In 13 subjects (34 events) in the HAL-MRE1 group and one subject (one event) in the placebo group an early (< 30 minutes after injection) local TEAE was reported. One subject in the HAL-MRE1 group in cohort 1 reported a moderate early local TEAE, all other events were assessed as mild.

[0260] In 14 subjects (20 events) in the HAL-MRE1 group and one subject (one event) in the placebo group a delayed (> 30 minutes after to < 3 hours after injection) local event was reported. All delayed reactions were assessed as mild. In 24 subjects (90 events) in the HAL- MRE1 group and eight subjects (31 events) in the placebo group a late (> three hours to < 24 hours after injection) local TEAE was reported. The same subject in the HAL-MRE1 group who reported a moderate early reaction, also reported a late local reaction; all other late events were assessed as mild. As shown in Table 15 local TEAEs were equally distributed across cohorts 1 , 2 and 3 in the HAL-MRE1 treatment group.

[0261] Table 15. Occurrence of local TEAEs per treatment group per cohort (Safety population)

[0262] Abbreviations: N = number of subjects in specified treatment group.

[0263] Note: Percentages are based on the number of subjects in specified treatment group.

[0264] Laboratory parameters (laboratory, chemistry and urinalysis) were similar among the different groups. No significant changes from baseline were observed during the study in any of the groups.

[0265] Vital signs were measured at each visit pre- and post-dose (30 min. + 5 min. after injection). All parameters were similar in the HAL-MRE1 treatment group compared to the placebo group at all time points measured. No significant changes were observed between the pre- and post-dose values at each visit and between baseline and end of study. No subject reported any AE related to vital signs during the study.

[0266] The ECG was measured at baseline only. Subjects with clinically significant abnormalities in ECG were excluded from the study.

[0267] There were three subjects with seven adverse events found after physical examination at a post baseline visit: right hand laceration, left jaw bruising, pruritus palate, tooth pain and left ear tingling. All of them were reported as not drug-related AEs. All of these subjects were in the HAL-MRE1 treatment group.

[0268] Since there were no asthmatic subjects included in the study, lung function was measured by FEV1 at screening and EoS / ETV for all subjects. Also, no ACT was used in the study as per protocol. No difference was observed in lung function between screening and EoS / ETV among the treatment groups. Summary

[0269] Preclinical experience

[0270] In vitro and in vivo data demonstrated that chemical modification reduces the IgE binding potential of RE, but the capability to trigger the induction of IgG antibodies is retained. Studies to investigate the safety of MRE demonstrated that mediator release from loaded basophils is reduced upon chemical modification of RE. In immunogenicity studies in mice and rats, MRE induces a strong RE-specific IgG response that is comparable to the response induced by RE. This indicates that MRE is a strong inducer of antibodies, which are cross-reactive with unmodified ragweed pollen proteins. Re-stimulation by RE of splenocytes from RE- and MRE-immunized mice lead to the induction of IL-4, IL-5 and IFN-y, independent of the immunization by RE or MRE, again demonstrating cross-reactivity. These preclinical studies demonstrated that MRE is equally immunogenic compared to RE. Moreover, MRE is safer than RE and Safety may be improved further by adsorption onto A1(OH)3.

[0271] No mutagenic activity of HAL-MRE1 was observed in both the bacterial reverse mutation test as well as the in vitro micronucleus test. The in vivo repeat-dose toxicology study showed that under the study conditions, and compared to HAL-MRE1 placebo, no evidence of systemic toxicity was observed at the tested dose of HAL-MRE1 drug product. All changes attributed to HAL-MRE1 were considered to be related to the expected immune reactions at the injection sites.

[0272] Dosage and Administration

[0273] HAL-MRE1 is a liquid suspension for subcutaneous administration containing modified ragweed extract adsorbed to aluminum hydroxide. For the present study, the investigational product was supplied in a multi-dose vial at a stock concentration of 20,000 AUeq / mL and contained other excipients. The composition of the diluent is the same as that of HAL-MRE1 except for the modified ragweed extract and aluminum hydroxide.

[0274] In the present study adverse event development was monitored closely. Following study drug administration, subjects remained on site for a minimum of 3 hours to monitor vital signs, possible early and delayed local and / or systemic reactions. Only if no relevant safety issues were observed, subjects were discharged. Since there were no significant safety concerns observed, the observation period was not extended at the discretion of the investigator. Depending on the occurrence and intensity of swelling at the injection site and grade of the systemic reaction, the dosage schedule had to be adjusted - repeated or decreased.

[0275] In vitro toxicity studies did not reveal HAL-MRE1 to induce any mutations or have any clastogenic or aneugenic potential. In vivo studies with rats treated with 20,000 AUeq (0.5mL of 40,000 AUeq) HAL-MRE1 per day twice a week for a 11 weeks did not reveal any systemic toxicity; only some changes at the injection site and in spleen weight as well as size were observed but this is expected based on the immune reactions.

[0276] In the HAL-MRE1 safety and tolerability study, treatment commenced with a low dose and was escalated slowly (increase factor 2 to 2.5) during weekly up-dosing visits.

Claims

CLAIMS1. Modified allergen for use in the prevention, reduction, or treatment of allergy in a human wherein said use comprises exposing said human to an initial dose of said modified allergen of less than 625 AUeq followed by multiple increasing dosages of said modified allergen up to 50,000 AUeq.

2. Modified allergen for use according to claim 1 , wherein said modified allergen is a crosslinked modified allergen, preferably a glutaraldehyde crosslinked modified allergen; or said modified allergen is a reduced and subsequently alkylated monomeric allergoid.

3. Modified allergen for use according to claim 1 or claim 2, wherein said modified allergen is ragweed protein and said allergy is ragweed allergy.

4. Modified allergen for use according to claim 2, wherein said allergy and said reduced and subsequently alkylated monomeric allergoid are selected from the group consisting of:Common ragweed Ambrosia elatior) and Amb a 1, Amb a 2 and / or Amb a l l proteins;Rot fungus {Altemaria altemata) and Alt a 1 protein;Mugwort Artemisia vulgaris) and Art v 1 protein;Aspergillus fumigatus and Asp f 1 and / or Asp f 2 proteinsBee venom {Apis Mellifera) and Api m 1, Api m 2 and / or Api m 5 proteins;European beech {Fagus sylvatica) and Fag s 1 and / or Fag s 2 proteins;European ash {Fraxinus excelsior) and Fra e 1 protein;Bentgrass (Agrostis stolonifera)and Agr g 1 protein;Sweet vernal grass {Anthoxanthum odoratum) and Ant o 1 protein;Cocksfoot {Dactylis glome rata) end Dac g 1 v protein;Perennial ryegrass {Lolium perenne) and Fol p 1 protein;Tall oat grass {Arrhenatherum elatius) and Arr e 1 protein;Red fescue {Festuca rubra) and Fes r 1 protein;Kentucky blue grass {Poa pratensis) and Poa p 1 protein;Cultivated rye {Secale c er eale) and Sec c 1 protein;Velvet grass {Holcus lanatus) and Hol 1 1 protein;Timothy {Phleum pratense) and Phi p 1 and / or Phi p 5 protein;Cultivated wheat (Triticum aestivum) and Tri a 1 protein;Dog (Canis familiaris) and Can f 1 protein;Cat (Fe ts domesticus) and Fel d 1 protein;House dust mite (Dermatophagoides pteronyssinus) and Der p 1 , Der p 2 and / or Der p 23 proteins;House dust mite (Dermatophagoides farinae) and Der f 1 and / or Der f 2 proteins;Olive (Olea europea) and Ole e 1 and / or Ole e 9 proteins;Wall pellitory (Parietariajudaica) and Par j 1 and / or Par j 2 proteins;English plantain (Plantago lanceolata) and Pla 1 1 protein;Alder (Alnus glutinosa) and Ain g 1 protein;Birch (Betula verrucosa) and Bet v 1 protein;Hazel (Corylus avellana) and Cor a 1 protein; andCommon wasp venom (Vespula vulgaris) and Ves v 1, Ves v 2 and / or Ves v 5 proteins.

5. Modified allergen for use according to any one of the claims 1 to 4, wherein said modified allergen is complexed with an adjuvant, preferably with aluminium, more preferably A1(OH)3.

6. Modified allergen for use according to claim 5, wherein said modified allergen is complexed and saturated with an adjuvant, preferably with aluminium, more preferably A1(OH)3.

7. Modified allergen for use according to any one of the claims 1 to 6, wherein said treatment comprises an initial dose of said modified allergen of less than 625 AUeq followed by multiple increasing dosages of said modified allergen, preferably weekly, up to 30,000 AUeq.

8. Modified allergen for use according to any one of the claims 1 to 7, wherein said human is exposed to said modified allergen by parenteral administration, subcutaneous administration, oral administration, intralymphatic administration, mucosal administration, intestinal administration, rectal administration, vaginal administration, epicutaneous administration, dermal administration, sublingual administration, ophthalmic administration, nasal administration, or combinations thereof.

9. Modified allergen for use according to any one of the claims 1 to 8, wherein said modified allergen is formulated as a pharmaceutical composition, a beverage, a food product, a toothpaste, a skin patch, a band aid, a mouth wash, a candy, a skin creme, a tablet, a lozenge, a food supplement, a foodstuff, or combinations thereof.

10. Method for prevention, reduction, or treatment of allergy in a human wherein the method comprises the steps of: a) exposing said human to an initial dose of a modified allergen as defined in any one of the claims 1 to 4 of less than 625 AUeq; b) subsequently exposing said human to multiple increasing dosages of said modified allergen up to 50,000 AUeq.

11. Method according to claim 10, wherein said modified allergen is complexed with an adjuvant, preferably with aluminium, more preferably Al(0H)3 or wherein said modified allergen is complexed and saturated with an adjuvant, preferably with aluminium, more preferably A1(OH)3.

12. Method according to claim 10 or claim 11, wherein said allergy and modified allergen are selected from the group consisting of:Common ragweed Ambrosia elatior) and Amb a 1, Amb a 2 and / or Amb a l l proteins;Rot fungus {Altemaria altemata) and Alt a 1 protein;Mugwort Artemisia vulgaris) and Art v 1 protein;Aspergillus fumigatus and Asp f 1 and / or Asp f 2 proteinsBee venom {Apis Mellifera) and Api m 1, Api m 2 and / or Api m 5 proteins;European beech {Fagus sylvatica) and Fag s 1 and / or Fag s 2 proteins;European ash {Fraxinus excelsior) and Fra e 1 protein;Bentgrass (Agrostis stolonifera)and Agr g 1 protein;Sweet vernal grass {Anthoxanthum odoratum) and Ant o 1 protein;Cocksfoot {Dactylis glome rata) end Dac g 1 v protein;Perennial ryegrass {Lolium perenne) and Fol p 1 protein;Tall oat grass {Arrhenatherum elatius) and Arr e 1 protein;Red fescue {Festuca rubra) and Fes r 1 protein;Kentucky blue grass {Poa pratensis) and Poa p 1 protein;Cultivated rye {Secale c er eale) and Sec c 1 protein;Velvet grass {Holcus lanatus) and Hol 1 1 protein;Timothy {Phleum pratense) and Phi p 1 and / or Phi p 5 protein;Cultivated wheat {Triticum aestivum) and Tri a 1 protein;Dog {Canis familiaris) and Can f 1 protein;Cat {Felis domesticus) and Fel d 1 protein;House dust mite (Dermatophagoides pteronyssinus) and Der p 1 , Der p 2 and / or Der p 23 proteins;House dust mite (Dermatophagoides farinae) and Der f 1 and / or Der f 2 proteins;Olive (Olea europea) and Ole e 1 and / or Ole e 9 proteins;Wall pellitory (Parietariajudaica) and Par j 1 and / or Par j 2 proteins;English plantain (Plantago lanceolata) and Pla 1 1 protein;Alder (Abuts glutinosa) and Ain g 1 protein;Birch (Betula verrucosa) and Bet v 1 protein;Hazel (Corylus avellana) and Cor a 1 protein; andCommon wasp venom (Vespula vulgaris) and Ves v 1, Ves v 2 and / or Ves v 5 proteins.

13. Method according to any one of the claims 10 to 12 wherein said human is exposed to said modified allergen by parenteral administration, subcutaneous administration, oral administration, intralymphatic administration, mucosal administration, intestinal administration, rectal administration, vaginal administration, epicutaneous administration, dermal administration, sublingual administration, ophthalmic administration, nasal administration or combinations thereof.

14. Method according to any one of the claims 10 to 13, wherein said modified allergen is formulated as a pharmaceutical composition, a beverage, a food product, a toothpaste, a skin patch, a band aid, a mouth wash, a candy, a skin creme, a tablet, a lozenge, a food supplement, a foodstuff, or combinations thereof.

15. Composition as defined in claim 9 comprising a modified allergen as defined in any one of the claims 1 to 6 suitable for the prevention, reduction, or treatment of allergy as defined in any one of the claims 1 to 4 in a human.

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