Treatment of peanut allergy

Abstract

The present invention relates to modified peanut protein extracts for use in the prevention, reduction, or treatment of peanut allergy in a human wherein said use comprises exposing said human to an initial dose of said peanut protein extract of less than 0.1 µg followed by multiple increasing dosages up to 750 µg peanut protein extract, and wherein said peanut protein extract protein extract preferably a reduced and subsequently alkylated peanut protein extract. The present modified peanut protein extracts can be 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 crème, a tablet, a lozenge, a food supplement, a foodstuff or combinations thereof.

Description

[0001] TREATMENT OF PEANUT ALLERGY

[0002] Description

[0003] The present invention relates to peanut protein extracts for use in the prevention, reduction, or treatment of peanut allergy in a human and to methods for prevention, reduction, or treatment of peanut allergy in a human.

[0004] Peanut allergy is an immunoglobulin E (IgE) mediated hypersensitivity reaction to peanut proteins. Peanut allergy symptoms can range from minor ones such as oral pruritus, to a life-threatening anaphylactic reaction. Allergic symptoms occur from within minutes to a few hours upon ingestion of even trace quantities of peanut and frequently multiple organs are involved, including cutaneous, cardiovascular, gastrointestinal and respiratory systems. Symptoms range from a relatively mild urticarial rash, vomiting, diarrhoea, wheezing, and dyspnoea, to severe throat angioedema, cardiovascular collapse or fulminant anaphylaxis.

[0005] Allergy to peanuts (Arachis hypogaea) is a serious public health problem that is estimated to affect between 0.6 and 1.0 % of the adult population and between 1 and 3% of children in developed countries. Current data suggests that prevalence has increased and is now between 0.1 and 4.5 % of the world- wide adult population, depending on geographic area.

[0006] Over the last few decades, an increase in the prevalence of peanut allergy amongst children and adolescents in the US has been observed with an increase from 0.64% in 1997 to 2.14% in 2007. Based on a specially developed survey administered to US households between 2015 and 2016, the prevalence of convincing peanut allergy was estimated to be 2.2% in children. In approximately 70% of the children with peanut allergy, the first allergic reaction occurs before the age of 2 and thereafter peanut allergy reactions rarely resolve. Despite the availability of Palforzia (Stallergenes Greer) in the US since the beginning of 2020, management of peanut allergy consists of avoidance of peanut allergens along with immediate access to rescue medication. This requires a profound lifelong change in dietary habits and, in more severe disease cases, the continuous carrying of an epinephrine auto-injector.

[0007] Peanut allergy has significant medical and psychosocial implications for peanut allergic subjects and their families. The quality of life perceived by peanut allergic subjects is reduced due to the constant fear of severe allergic reactions or even fatal reactions from accidental ingestions and peanut-traces in food as well as the necessity to adhere to a strict peanut-avoiding diet. Approximately 55% of peanut allergic patients have accidental ingestions, occurring at school, at home, and in restaurants. In addition, visits to physicians, hospitalizations and emergency department visits have a considerable economic impact on the health care system.

[0008] Accordingly, there is a medical need for a disease modifying treatment, such as immunotherapy, for peanut allergy. Development of a safe and an effective immunotherapy is especially important for children (aged 5-11 years) and adolescents (aged 12-17 years), since the probability of experiencing a severe reaction appears to increase during childhood.

[0009] Specific allergen immunotherapy (AIT) is the practice of administering gradually increasing doses of allergens to reduce allergic symptoms and the need for medication. The allergen-specific approaches under pre-clinical and clinical investigation comprise subcutaneous, oral, sublingual and epicutaneous immunotherapy with peanut extracts as well as applications of hypoallergenic peanut allergens or T-cell epitope peptides.

[0010] Subcutaneous immunotherapy (SCIT) has been successfully used for the treatment of insect venom allergies and for respiratory allergies such as allergic rhinitis to pollen and house dust mite. Early attempts with SCIT for peanut allergy using an aqueous, native peanut extract (PE) resulted in an unacceptably high rate of systemic reactions. Although the treatment resulted in increased tolerance to peanut, the administration of peanut SCIT resulted in severe systemic reactions often requiring treatment with epinephrine, which renders this form of therapy unacceptable. For a clinically applicable treatment, a safe peanut exposure protocol both with respect to the initial dosage and subsequent dosages is therefore deemed a prerequisite.

[0011] Presently, oral peanut allergen immunotherapy Palforzia was approved by the FDA and marketed in the US at the beginning of 2020. However, Palforzia contains a black box warning regarding potential anaphylaxis caused by the treatment. Other limitations of OIT should be taken into account, such as the use of OIT in patients with eosinophilic esophagitis, the risk of other gastrointestinal events caused by the treatment, and frequent loss of tolerance after finishing treatment. Currently, clinical development of other novel treatments for peanut allergy such as sublingual immunotherapy, epicutaneous immunotherapy and the use of anti-IgE, is mainly directed at the treatment of peanut allergic adolescents and children. Still, these treatments may be hampered by poor compliance, and less than optimal safety monitoring during dosing compared to subcutaneous administration.

[0012] It is an object of the present invention, amongst other objects, to provide safe peanut exposure protocols both with respect to the initial dosage and subsequent dosages.

[0013] The object of the present invention, amongst other objects, is met by the appended claims.

[0014] Specifically, the object of the present invention, amongst other objects, is, according to a first aspect, met by peanut protein extracts for use in the prevention, reduction, or treatment of peanut allergy in a human wherein said use comprises exposing said human to an initial dose of said peanut protein extracts of less than 0.1 pg followed by multiple increasing dosages up to 750 pg peanut protein extracts, and wherein said peanut protein extracts are modified peanut protein extracts, preferably reduced and subsequently alkylated peanut protein extracts. Initial dosages of less than 0.1 pg peanut extract were found by the present inventors as dosages not causing major safety issues in two human studies. It was further found by the present inventors that favourable pharmacological interventions in combination with a positive safety profile were most pronounced in paediatric, i.e., children and adolescents populations.

[0015] Currently 16 allergenic components have been identified in peanuts. Numerous worldwide studies have addressed the prevalence of allergen-specific IgE in peanut allergic patients’ sera. In those studies, Ara h 1, Ara h 2, Ara h 3 and Ara h 6 are regarded as major allergens based on their ability to bind IgE on Western blots, to interact with IgE in radioallergosorbent test (RAST), and to have allergenic activity as assessed by in vitro and / or in vivo functional assays. Ara h 2 and Ara h 6 are the most potent and clinically relevant peanut allergens.

[0016] Ara h 2 is the most important predictor of clinical peanut allergy, and is the allergen most often associated with severe reactions. Ara h 6 sensitization is associated with IgE antibodies that cross-react with Ara h 2. Rarely does sensitization to Ara h 6 occur in the absence of sensitization to Ara h 2. Moreover, IgE immuno-reactivity towards Ara h 2 often also correlates with positive IgE against Ara h 1 and Ara h 3. In the case that IgE antibodies to Ara h 2 and / or Ara h 1 / Ara h 3 are detected in patient sera, more than 95% of the corresponding patients will have clinical symptoms when ingesting peanuts. If only IgE immune reactivity towards Ara h 2 is detected, 87% of the subjects report clinical symptoms.

[0017] According to the present invention, the present peanut protein extracts are modified peanut protein extracts, preferably reduced and subsequently alkylated peanut protein extracts. Modified peanut extracts provides improved safety profiles because allergenicity of the peanut extract is reduced through chemical modification of the peanut proteins, more specifically the Ara h 2 and Ara h 6 allergens.

[0018] Chemical modification can be achieved using formaldehyde or glutaraldehyde. With a chemically modified allergen, the IgE-antibodies no longer recognize the allergen, and thereby prevent the allergic cascade leading to an allergic reaction. The chemical modifications do not affect T-cell epitopes, therefore resulting in decreased allergenicity with retained immunogenicity. Several studies have shown that modified allergen extracts for aeroallergens show clinical efficacy with a superior safety profile compared to non-modified allergen extracts. However, modification with glutaraldehyde of peanut proteins does not result in a substantial decrease in IgE-binding.

[0019] The compact stable structure of Ara h 2 is dependent on 4 intra-molecular disulfide bridges formed by the molecule’s 8 cysteine residues. To decrease the allergenicity, the peanut extract can be reduced with, for example, dithiothreitol (DTT) and subsequently treated with, for example, iodoacetamide (IAA) for alkylation. Reduction and alkylation break up the disulfide bridges of Ara h 2 (and Ara h 6), 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 (Toda et al., 2011) and was previously investigated on the isolated peanut allergens Ara h 2 and 6. Moreover, the combination of a) modification of peanut extract by reduction and alkylation, and b) preferably adsorption onto aluminium hydroxide, reduced the risk of allergic reactions by 30-fold in an allergic mouse model compared to non-modified, non-adsorbed peanut extract.

[0020] Considering the above, the present invention preferably relates to modified peanut protein extracts complexed with an adsorbant, preferably with aluminium, more preferably A1(OH)3 or / and to peanut protein extracts complexed and saturated with an adsorbant, preferably with aluminium, more preferably A1(OH)3.

[0021] Chemical modification of peanut protein extracts followed by adsorption onto A1(OH)3 was demonstrated to reduce the anaphylactic potency in in vivo models. Furthermore, it was demonstrated that modified peanut protein extract adsorbed onto A1(OH)3 was able to elicit a peanut-specific immune response in an animal model and, additionally, it was able to provide a dose-dependent therapeutic effect in a mouse model for immunotherapy of peanut allergy. Accordingly, treatment of peanut-allergic patients with chemically modified peanut protein extracts adsorped onto A1(OH)3 will result in an improved safety profile without loss of immunotherapeutic efficacy.

[0022] Suitable modified peanut protein extracts for use according to the present invention can be prepared as follows. First, peanut proteins extracted from (defatted) peanut powder are subjected to an extraction step, whereafter the insoluble fraction is removed by centrifugation and filtration. The remaining soluble fraction is diluted with buffer to a specified protein concentration yielding an intermediate product. Subsequently, the intermediate product is chemically modified by reduction and alkylation preferably using DTT and IAA. Then, the modified intermediate product is concentrated and diafiltrated to remove the excess of DTT and IAA and to exchange buffer followed by pre-filtration (0.2 pm) and sterile filtration (0.2 pm). After filtration, the modified peanut protein extract is diluted with phosphate buffer to a fixed protein concentration, mixed with phenol-containing phosphate buffer and subsequently mixed with aluminium hydroxide in a ratio of 0.75 mg peanut protein per mg A1(OH)3). The aluminium adsorbed product is formulated with a phosphate buffer containing sodium chloride to obtain the final product and stored at 2 - 8 °C.

[0023] According to the present invention, the present use comprises an initial dose of said peanut protein extract of less than 0.1 pg followed by weekly increasing dosages up to 300 pg to 500 pg peanut protein extract, more preferably an initial dose of the peanut protein extract of 0.05 pg or less, increasing weekly dosages of the peanut protein extract up to a maintenance dose of said peanut protein extract of 350 to 400 pg peanut protein extract.

[0024] The present use preferably comprises exposing a human to the peanut protein extract 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.

[0025] Preferably, the present peanut protein extracts are 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.

[0026] According to a second aspect, the present invention relates to methods for prevention, reduction, or treatment of peanut allergy in a human wherein the method comprises the steps of: a) exposing a human to an initial dose of said peanut protein extract of less than 0.1 pg; b) subsequently exposing a human to multiple increasing dosages up to 750 pg peanut protein extract.

[0027] In the present method, the protein extract is a modified peanut protein extract, preferably a reduced and subsequently alkylated peanut protein extract.

[0028] Further, in the present method, the peanut protein extract is complexed with an adjuvant, preferably with aluminium, more preferably Al(0H)3 or the peanut protein extract is complexed and saturated with an adjuvant, preferably with aluminium, more preferably A1(OH)3.

[0029] According to an embodiment, the present invention preferably relates to methods comprising the steps of: a) exposing said human to an initial dose of said peanut protein extract of less than 0.1 pg; b) subsequently exposing said human to weekly increasing dosages up to 300 pg to 500 pg peanut protein extract.

[0030] According to the present invention a human is preferably exposed to a peanut protein extract 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. The peanut protein extract is preferably 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.

[0031] The present invention will be further detailed in the examples below. In the examples, reference is made to figures wherein:

[0032] Figure 1 and 2: show body temperature drop in SCIT-treated allergic mice after PE challenge;

[0033] Figure 3: shows frequency (% of subjects) of drug related events;

[0034] Figure 4: shows rules for increase / reduction of dosages of MPE;

[0035] Example 1: Nonclinical studies

[0036] To avoid potentially dangerous side-effects resulting from the administration of peanut allergens, a 2-step safety approach was followed. First, the peanut extract (PE) preparation was chemically modified by reduction and alkylation to reduce the allergen-specific IgE binding potential and subsequent IgE-mediated allergic responses. The second approach included the binding of modified peanut extract (MPE) to A1(OH)3 which shields off IgE epitopes and provides further increased safety of the preparation. Overall, the nonclinical pharmacology studies showed:

[0037] 1. MPE induced proliferation of peanut specific T-cell lines derived from peripheral blood monocytes of patients with clinically established peanut allergy;

[0038] 2. Reduced IgE binding to MPE on Immunoblot using sera of peanut allergic patients;

[0039] 3. Reduced IgE binding to MPE in the IgE potency assay using sera from individual peanut allergic patients;

[0040] 4. Reduced mediator release by MPE from basophils loaded with IgE from peanut-allergic patients;

[0041] 5. An MPE-specific IgG response in immunized BALB / c mice that was cross-reactive with the native PE preparation;

[0042] 6. An MPE-specific IgG response in immunized rats that was cross-reactive with the native PE preparation;

[0043] 7. Lower anaphylactic potential of MPE in a mouse peanut allergy model;

[0044] 8. Lower anaphylactic potential of A1(OH)3 adsorbed PE in a mouse peanut allergy model;

[0045] 9. Diminished allergic responses after immunotherapy with MPE following peanut challenge in a mouse SCIT model. Pharmacokinetics and product metabolism in animals

[0046] Dedicated nonclinical pharmacokinetic studies are generally not needed for vaccines and immunotherapies. The safety concerns associated with immunotherapies are generally not related to pharmacokinetics but are related to the potential induction of an immune response. The evidence of exposure to modified peanut extracts can be measured through the induction of antibody responses. Following subcutaneous administration of modified peanut extract, IgG antibody responses to MPE have been shown in a murine immunogenicity model in BALB / c mice, Sprague-Dawley rats and in the GLP, 7-week toxicity study in rats.

[0047] In vitro pharmacology studies

[0048] An overview of all in vitro immunogenicity / allergenicity studies with their noteworthy findings are provided in Table 1 below.

[0049] Table 1; Overview of in vitro studies

[0050] PE: Peanut extract, MPE: Modified Peanut Extract, CPE: Crude Peanut Extract

[0051] Note: CPE and PE are similar extracts. During development the term CPE was replaced by PE

[0052] In vivo pharmacology studies A set of in vivo pharmacology studies have been performed comprising of the investigation of anaphylactic and immunogenic responses of PE and MPE in a mouse model for peanut allergy and a mouse immunotherapy model and immunogenicity models in mouse and rat (summarized in Table 2).

[0053] Table 2; Overview of in vivo immunogenicity studies

[0054] Admin.: Administration; F: Female; M: male; MPE: Modified Peanut Extract; PE: Peanut extract

[0055] Evaluation of effects on anaphylactic response in the mouse model for peanut allergy

[0056] A mouse model for peanut allergy was developed to evaluate the effects of PE and MPE on the anaphylactic response and to determine the impact of the modification and the adsorption to Al(0H)3. Mice (n=6, females) were sensitized intra-gastrically (i.g.) by administering 6 mg PE and 15 pg cholera toxin on days 0, 1, 2, 7, 14, 21, 28. Thereafter, mice were challenged subcutaneously (s.c.) in the neck on day 42 with 200 pl of different test preparations or their respective controls. Test preparations included 0.1 mg, 0.6 mg or 3 mg of PE or MPE and 0.6 mg of PE or MPE adsorbed to different concentrations of A1(OH)3. As an objective parameter of anaphylactic shock, body temperature was measured by means of rectal thermometry every 10-20 minutes for 90 minutes after subcutaneous challenge to assess the anaphylactic potency of the preparations.

[0057] Sensitized mice were challenged either with PE or MPE at three different doses (0.1 mg, 0.6 mg, and 3 mg). A challenge with PE resulted in a severe allergic reaction. Ten minutes after injection a rapid, dose-dependent drop in body temperature occurred. A noticeable drop in temperature already occurred at the lowest challenge dose (0.1 mg / mouse). At the highest dose of 3 mg / mouse of PE, the body temperature dropped to such an extent that 3 mice had to be sacrificed. In contrast, a challenge with MPE did not result in such a reaction, not even at the highest dose of 3 mg / mouse. Clinical scores (not shown) corroborated these results.

[0058] Adsorption of PE to Al( OH) 3 reduces the anaphylactic potential in the mouse model for peanut Allergy

[0059] The effects of adsorption to Al(0H)3 on the anaphylactic potential of PE was investigated in a mouse peanut allergy model. A subcutaneous injection with 0.6 mg PE per mouse resulted in a severe anaphylactic shock response in all mice as measured by decreased body temperature and clinical symptom score. In contrast, none of the mice showed any signs of immediate anaphylactic shock using the same amount of PE fully adsorbed to Al(0H)3. A delayed anaphylactic response was noted using a PE preparation in which 40 or 70% of the protein was adsorbed to Al(0H)3 . In the group challenged with a PE preparation in which 90% of the protein was adsorbed to Al(0H)3, the anaphylactic response was delayed and decreased in magnitude. Non-sensitized mice did not respond to any of the challenges. Since subcutaneous injection of MPE (0.1 and 0.6 mg) did not result in an anaphylactic response, the impact of adsorption of MPE to Al(0H)3 could not be investigated.

[0060] Complete binding of PE to Al(0H)3 inhibited the PE-induced anaphylactic responses, but anaphylactic responses were still observed when PE was partially bound to Al(0H)3. showing the importance of binding PE to Al(0H)3 in order to prevent anaphylactic shock in peanut allergic mice.

[0061] Mouse peanut immunotherapy model

[0062] A mouse model for peanut allergy immunotherapy was developed to compare PE and MPE preparations for efficacy and to assess the impact of the adsorption to Al(0H)3. All mice (female, n=6 per group) were sensitized by intragastric (i.g.)administration of 6 mg PE and 15 pg cholera toxin in 400 pl PBS per mouse on days 0, 1, 2, 7, 14, 21 and 28. For de-sensitization, mice received PE or MPE + / - Al(0H)3 by s.c. administration two times a week for a period of 6 weeks in the neck. Mice were i.g. challenged with 12 mg PE per mouse on day 91. In addition, all mice were intraperitoneal (i.p.) challenged with 0.1 mg PE per mouse on days 98 and 112, followed by assessment of anaphylaxis.

[0063] As an objective parameter of anaphylactic shock, body temperature was measured after each i.p. challenge by means of rectal thermometry every 10-20 minutes for 90 minutes. At several time points during the course of the experiment, blood was taken for the measurement of antibodies. Non-sensitized mice and PE-sensitized mice (no immunotherapy) were used as PBS control and PE control, respectively.

[0064] Immunotherapy with MPE increases Tolerability to Peanut in a Mouse Peanut Allergy Model

[0065] Immunotherapy with PE and MPE was assessed following s.c. administrations 2 times a week for 6 weeks. PE-sensitized mice were de-sensitized with either native PE (0.03 mg or 0.1 mg) or MPE (0.03, 0.1 or 1 mg) followed by i.g. and i.p. challenges with PE. The impact of immunotherapy with PE or MPE was determined by measuring allergic parameters (i.e. decreased body temperature, clinical scores including appearance, pilo-erection, and mast cell degranulation) after i.p. and i.g. challenges.

[0066] Immunotherapy with PE as well as MPE diminished the anaphylactic reaction upon i.p. challenges. Immunotherapy with either PE or MPE effectively reduced the temperature response after the i.p. challenge on day 112 compared to PE sensitized but untreated mice. Treatment with PE showed a better efficacy compared to MPE as indicated by a smaller drop in temperature (not significant) and better clinical scores. However, the dose of 0.1 mg PE used for treatment resulted in severe side-effects. In this model, MPE can be dosed at least thirty-fold higher than PE because of its improved safety profile.

[0067] PE-specific antibodies (IgE, IgGi and IgGza) were determined in serum during the course of the experiments. The treatment with PE and MPE resulted in similar antibody levels in the serum of PE sensitized mice.

[0068] IgE levels were higher in all groups compared to the negative PBS control. Immunotherapy with native PE and MPE did not result in a reduction of IgE serum levels, but both preparations prevented the increase in serum IgE after i.g. and i.p. challenges with native PE, an increase that is clearly shown in the sensitized group of mice that did not receive immunotherapy. Mice that received immunotherapy displayed strongly increased levels of IgGi as well as IgGza in the serum with comparable levels between groups.

[0069] Immunotherapy with modified peanut protein extract adsorbed onto Al( OH) 3 diminished anaphylactic responses in a dose-dependent way following peanut challenge in a mouse model for peanut allergy

[0070] After the i.p. challenge on day 112, the anaphylactic response as measured by the drop in temperature after the challenge was reduced in mice receiving immunotherapy with MPE adsorbed on to Al(0H)3. De-sensitization with different amounts of MPE adsorbed onto A1(OH)3 demonstrated a dose-dependent effect on the anaphylactic response as measured by temperature decrease (Figure 1). At the highest dose tested, the HAL-MPE1 preparation showed an improved efficacy profile compared to the non-adsorbed MPE preparation (Figure 2).

[0071] PE-specific antibodies (IgE, IgGi and IgGza) were determined in serum of all mice in the different treatment groups during the course of the experiment. As commonly seen in the clinic, immunotherapy caused an increase in the level of PE-specific antibodies. PE-specific IgE levels are significantly higher after 3 weeks of immunotherapy (day 58) compared to the levels after PE sensitization (day 35) demonstrating the boost of the immune system due to immunotherapy. At the next time point of measurement (day 84, after another 3 weeks of immunotherapy), IgE levels were significantly decreased in the groups treated with A1(OH)3- adsorbed MPE compared to the levels at day 58. Mice treated with Al(OH)3-adsorbed MPE displayed an increase in serum IgGi levels and an increase of PE-specific IgGza levels (comparable with IgGr in humans) during immunotherapy, in line with antibody level development in patients in the clinic receiving immunotherapy.

[0072] Toxicology Modified peanut extract was tested in 2 genotoxicity studies (a bacterial reverse mutation test and a mouse lymphoma test, MLA) and in repeat-dose (1 week and 7 week) toxicity studies in Sprague-Dawley rats. According to the “Guidelines on the nonclinical evaluation of vaccine adjuvants and adjuvanted vaccines” (WHO / BS / 2013 / 2214), 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-MPE1. Therefore, the safety of HAL-MPE1 was evaluated in rats, because they produce an immunological response to HAL- MPE1 treatment. The GLP toxicology studies were conducted with a non-GMP produced development batch of MPE, which was produced in a similar way as to the final MPE DP that will be used in the human clinical trial(s).

[0073] A study was conducted to evaluate the potential of modified peanut extract to induce reverse point mutations in 5 Salmonella typhimurium strains, TA1535, TA1537, TA98, TA100 and TA102 The study was performed to GLP and conducted in compliance with OECD Guideline for testing of chemicals 471 Genetic Toxicology: Bacterial Reverse Mutation Test (July 1997). The 5 strains were selected because they carry a mutation affecting one of the genes governing the synthesis of histidine. The mutagenic potential of the test article was assessed in the presence and absence of metabolic activation by liver homogenate (S9) obtained from rats.

[0074] All treatments in this study were performed using a sterile, ready to use, off-white suspension, composed of 750 pg / mL modified peanut proteins (extracted from crude peanut extract), 1 mg / mL aluminium hydroxide, 0.42% phenol in 3 mM KPO4 buffer and 0.9% NaCl. The test article was directly added to the test system. In addition, a placebo control without peanut proteins: a sterile ready to use, off-white suspension composed of 1 mg / mL aluminium, 0.51% phenol in 3 mM KPO4 buffer and 0.9% NaCl) was tested at a single dose level.

[0075] With regard to the phenol concentration, the concentration (0.42%) is the actual measured concentration. The concentrations mentioned in the toxicology report is the nominal concentration of 0.5% which is normally also on the label.

[0076] Experiment 1 (including the range finder experiment previously conducted in the strain TA100) was performed in the absence and in the presence of S9, using the plate incorporation method with dose levels of 1, 2, 3, 4 and 5 pL / plate, including the dose level of 0.5 pL for the strain TA100, and the placebo at 5 pL, the negative (vehicle) and positive controls. Following these treatments no evidence of toxicity was observed.

[0077] Experiment 2 was performed in all the tester strains in the absence and in the presence of S9 using the pre-incubation method. The maximum dose level of 5 pL / plate was retained for all strains. Extended dose level intervals were employed covering the range 0.75 - 5 pL / plate. Following these treatments no evidence of toxicity was noted for all the tester strains TA98, TA100 and TA102. For the strain TA1537 without S9 at the dose level of 5 pL toxicity was noted. For the strain TA1535 without S9 evidence of toxicity was observed in the form of a slight decrease in the number of revertant colonies at all dose levels. Consequently, an additional experiment was performed in strain TA1535 only, in absence of metabolic activation with an extended dose level interval (10 dose levels) covering the range 0.075 - 5 pL / plate. In this experiment no evidence of toxicity was observed.

[0078] In each experiment performed, the test article was completely soluble in the aqueous assay system at all dose levels treated. For all strains negative (vehicle) and positive control treatments were included. The mean numbers of revertant colonies all fell within acceptable ranges for negative control treatments and were significantly elevated by positive control treatments. In addition, the placebo gave similar results compared to the negative control. Following treatments of all the tester strains in the absence and presence of S9, no significant increases in revertant numbers were observed. Data were analyzed using the two-fold (TA98, TA100, TA102) or three-fold (TA1535 and TA1537) increase rule.

[0079] It was concluded that HAL-MPE1 was negative in the reverse mutation test conducted in 5 histidine -requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium when tested under the conditions of this study. These conditions included treatments using the plate incorporation and the pre-incubation methods at dose level of up to 5 pL / plate in the absence and in the presence of rat liver metabolic activation system (S9).

[0080] A study was conducted to evaluate the potential of modified peanut extract to induce forward mutation at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells using the cloning procedure in microwell plates (Report 8288-265). The study was performed according to GLP and conducted in compliance with OECD Guideline for testing of chemicals 476 Genetic Toxicology: In vitro mammalian cell gene mutation test (July 1997). The evaluations were conducted in the presence and absence of metabolic activation by liver homogenate (S9). The study consisted of a cytotoxicity range-finding experiment followed by three independent experiments, each conducted in the absence and presence of S9. Cells are exposed to the test article for 3 or 24 hours without S9-mix, and for 3 hours with S9-mix.

[0081] All treatments in this study were performed using a sterile, ready to use, off-white suspension, composed of 750 pg / mL modified peanut proteins (extracted from crude peanut extract, 1 mg / mL aluminium, 0.42% phenol (if present) in 3 rnM KPO4 buffer and 0.9% NaCl. The test article was directly added to the test system. All concentrations were expressed in terms of volume of modified peanut protein extract per volume of culture medium in the test system (pL / mL).

[0082] With regard to the phenol concentration, the concentration (0.42%) is the actual measured concentration. In the cytotoxicity range-finder experiment, no precipitate was observed at any concentration tested in the absence or presence of S9. No toxicity was noted in the absence of S9 (3 -hour and 24-hour treatments). Marked toxicity was observed in the presence of S9 (3 -hour treatment) at the highest concentration of 5 pL / mL for the test article and for the placebo (Relative Total Growth (RTG) was reduced to 33% and 40%, respectively).

[0083] In experiment 1 (3-hour treatment) no precipitate was observed and no cytotoxicity was noted in the absence of S9. Toxicity was noted at 2.5 and 5 pL / mL for the placebo (RTG = 54% and 31%, respectively) and at 1.25, 2.5 and 5 pl / mL for the modified peanut extract in the presence of S9 (RTG = 60% to 51%). In the presence of S9, the highest concentration the modified peanut extract and its placebo induced a significant increase in mutant frequency (MF) with Induced Mutant Frequency (IMF) values of 172.0 and 249.9, respectively. These values exceed the Global Evaluation Factor (GEF) of 126 mutants per 106 viable cells. Statistically significant linear trends were observed for the 3-hour treatments in the presence of S9 with the modified peanut extract and placebo.

[0084] In experiment 2 (24 h treatment) no cytotoxicity was found in the absence of S9. In the presence of S9, toxicity was noted at 3, 4 and 5 pL / mL for the placebo (RTG = 53% to 30%) and at 2, 3, 4 and 5 pL / mL for the modified peanut extract (RTG = 63% to 34%). the modified peanut extract and its placebo induced increases in MF at concentrations of 3, 4 and 5 pL / mL at which the IMF values exceeded the GEF of 126. Statistically significant linear trends were observed after 3-hour treatments with HAL-MPE1 and HAL-MPE1 placebo in the presence of S9. Suggestions were made that the increases in MF may be attributable to the matrix of the modified peanut extract. The matrix was composed of phosphate buffer, Al(0H)3, 0.9% NaCl and 0.51% phenol. Phenol is known to induce a positive response in the MLA test, suggesting that increases observed may be due to the phenol present in the matrix of the modified peanut extract. Published literature has shown that phenol has a weak mutagenic effect in the MLA test in the presence and absence of S9. Consequently, a third experiment using the placebo and the modified peanut extract with and without phenol was performed.

[0085] In Experiment 3, without phenol no precipitates or cytotoxicity (RTG close to, or higher than 70%) was found. In line with previous results, 3-hour treatment with the modified peanut extract containing 0.42% phenol in the presence of S9 resulted in IMF values exceeding the GEF of 126 (131.2 at 3 pl, 218.8 at 4 pl and 559.1 at 5 pl / mL, respectively). A significant linear trend was found. In contrast, no significant increases in MF or statistically significant linear trends were observed after treatment with the modified peanut extract without phenol and the placebo without phenol.

[0086] It was concluded that the placebo containing 0.42% phenol and the modified peanut extract containing 0.51% phenol induced mutation at the tk-locus of L5178Y mouse lymphoma cells when tested up to cytotoxic concentrations in 3 independent experiments, in the presence of a rat liver metabolic activation system (S9). In the same test system, the placebo without phenol and the modified peanut extract without phenol did not induce mutation at the tk- locus when tested up to 5 pL / mL (the maximum volume addition recommended by the test guidelines used for this study) in the presence of S9. A positive mutagenic effect of phenol in this assay in the presence and absence of S9 has been well-documented. Phenol is a common preservative in s.c. allergen immunotherapy products. Thus, it can be concluded that all mutagenic effects found in the presence of liver homogenate S9 were attributable to phenol.

[0087] Repeat dose toxicity studies

[0088] The objective of this preliminary study (not performed under GLP) was to determine the potential toxicity of the modified peanut protein extract when administered 3 times for 1 week to rats via the s.c. route (total of 3 administrations) to allow progression to a pivotal repeat-dose toxicity study

[0089] Sprague-Dawley rats (8 weeks of age on Day 1) received a suspension of the modified peanut protein extract in vehicle at 375 pg / day corresponding to 750 pg / mL by the s.c. route, 3 times per week for 1 week (total of 3 administrations). The dosing volume was 0.5 mL / animal / injection on 3 different dorsal injection sites. There were 3 animals per sex in the treated group.

[0090] Parameters evaluated daily included mortality and clinical signs. Body weights were recorded on day 4 during the pre-test period and on days 1 , 3 and 6 prior to treatment during the treatment period. Food consumption was measured on days 3 and 6 of the treatment period. Rats were euthanized and necropsied at the end of the treatment period on day 7. Macroscopic observation was performed for all animals, weights of selected organs were recorded, and representative tissue samples were collected and preserved in appropriate fixative. No microscopic examination was performed.

[0091] There were no mortalities and no systemic clinical signs throughout the study. No body weight losses were seen, and rats were eating normally.

[0092] The s.c. administration of the modified peanut protein extract three times for 1 week (3 administrations) to rats at doses of 375 pg / day resulted in induration at the injection sites after 4 days in both sexes and an increase in the absolute thymus organ weight in 1 / 3 males. Relevant macroscopic findings consisted of a thick aspect and / or red area at the injection sites in all animals and a large size thymus in 1 / 3 males. Due to the absence of clinical pathology data, microscopic data, and control animals, it is unclear if the inflammatory changes noted were related to the test article or to injection trauma. Sprague-Dawley rats (8 weeks of age on Day 1) received 375 pg / day modified peanut protein extract, placebo or salt solution by bolus s.c. injection, 3 times per week for 7 weeks (total of 22 administrations). The dosing volume was 0.5 mL / animal / inj ection on 6 different dorsal injection sites (identified SI to S6). There were 10 animals per sex per toxicity group (Groups 1, 2 and 3 - subgroup 1) and 5 additional animals per sex in the placebo group (group 1) and the modified peanut protein extract group (group 3) which served as recovery animal groups (subgroup 2). Recovery animals were maintained without compound administration for 2 weeks, after completion of 7 weeks of dosing.

[0093] Assessment of toxicity was based on mortality, clinical observations, body weights, food consumption, injection site irritation scores, body temperatures, ophthalmic examinations, and clinical and anatomic pathology. For the toxicology evaluation, the modified peanut protein extract was compared to the placebo (a matrix commonly used in s.c. immunotherapy with a proven safety record). Blood samples were also collected for immunogenicity evaluations.

[0094] All animals survived to the designated necropsy time. There were no modified peanut protein extract related adverse systemic clinical signs or effects on body weights, food consumption, body temperature, ophthalmic parameters, or urinalysis parameters. The modified peanut protein extract treatment induced a large increase in both PE-specific IgG responses, compared to the placebo group and the salt solution treated group, which plateaued on day 23 of treatment but was still present at the end of the treatment-free period. Although there were increases in the IgG responses, there were no body temperature changes indicative of an anaphylactic response.

[0095] The placebo or the modified peanut protein extract related effects at the injection sites consisted of edema and injection site indurations. Edema was noted in 1 male treated with placebo and in 8 animals treated with the modified peanut protein extract (8 / 30: 7 slight, 1 severe), on one site per animal, occasionally, between day 4 and day 18. Injection site indurations (3, exceptionally 4 on the same injection site) were noted in all animals treated with placebo or the modified peanut protein extract, on all injection sites throughout the dosing and recovery periods. The incidence of observations increased slightly with the number of doses administered per site (3 or 4 occasions per site). The incidence of observations was generally higher and the day of the first observation was similar or slightly earlier in the modified peanut protein extract group compared to the placebo group.

[0096] Compared to the placebo group, the modified peanut protein extract related hematological changes recorded on day 2 and / or at the end of the dosing phase (day 51) consisted of higher white blood cell count (WBC) in both sexes (males: +32% and +26% on days 2 and 51, respectively, females: +31% on day 51), significant higher neutrophil count on day 51 (males: +108%; females: +124%), higher mean monocyte count (males: +80% on day 2; females: + 45% on day 51), and in females only, slightly lower red blood cell count (RBC: 6%), hemoglobin (Hb: - 7%), packed cell volume (PCV: 7%), and slightly higher reticulocyte count (RABS: +16%). These changes were still noted at the end of the treatment-free period and correspond to the changes expected with an immunogenic agent. Overall, these changes were correlated with the main microscopic changes noted in animals euthanized on day 51 (subgroup 1) such as the moderate to marked local inflammatory changes noted at the injection sites but with a slightly higher severity in the modified peanut protein extract group. The minimal increased incidence and / or severity of extramedullary hematopoiesis and the minimal increased lymphoid follicles observed in the spleen for both sexes in the modified peanut protein extract group were considered to be related to the higher inflammatory changes recorded at the injection sites in this group.

[0097] The modified peanut protein extract related coagulation changes recorded at the end of the dosing period consisted of higher mean fibrinogen values (males +17%, females +29%), compared to the placebo group. No relevant test-article related changes in coagulation parameters were noted at the end of the treatment-free period. Fibrinogen is a surrogate marker of acute inflammation; thus, the increased fibrinogen values noted in the modified peanut protein extract group are likely related to the inflammatory reaction noted in the microscopic examination of the injection sites.

[0098] The modified peanut protein extract related clinical chemistry changes recorded at the end of dosing phase consisted of higher mean globulin (GLB; males +26%, females +33%) and lower albumin (ALB) and A / G ratio (males: -5% and -26%, females: 13% and 35%, respectively), compared to the placebo group. Changes were still present at the end of the treatment-free period. These changes correlated with the microscopic findings of moderate to marked local inflammatory changes that were noted at the injection sites in the modified peanut protein extract dosed animals euthanized on day 51.

[0099] The modified peanut protein extract related organ weight changes recorded at the end of the dosing phase consisted of increased spleen weights (males +34%, females +12%), compared to the placebo. At the end of the treatment-free period, higher adrenal gland (up to +41%) and spleen (up to +46%) weights were noted in the HAL-MPE1 male group and were accompanied by higher relative to terminal body weights (+22%). The slightly higher adrenal gland weights were considered to be fortuitous because there were no test article related findings (either at necropsy, organ weights or microscopically) at the end of the dosing period.

[0100] At the end of the dosing period, macroscopic findings were observed in 118 of the 120 injection sites (males and females combined) and consisted of the presence of s.c. nodules (size < 8 mm in diameter) and masses (size > 8 mm wide). Nodules were usually 1 to 3 in number (rarely 4) at the affected site, usually with a white or beige color (rarely cream) and firm. When compared to the placebo group, the incidence of masses was slightly higher in the modified peanut protein extract group while the incidence of nodules was similar in the two groups. Other noteworthy findings at the injection sites were the occasional occurrence of red areas and thickening in the groups. The only macroscopic finding observed at the injection sites in the salt solution group was the presence of red area in 2 / 120 injection sites. At the end of the 2-week treatment-free period, macroscopic findings were observed in 59 of the 60 injection sites (males and females combined). The main findings were still the presence of s.c. nodules and masses, the incidence being overall similar in males and females. Nodules were single or multiple (up to 5 in number) at the affected site, with a white or beige color and firm. The incidences of nodules and masses were overall similar in both groups.

[0101] At the end of the 7- week dosing period, microscopic findings were observed at the injection sites and in the spleen. The s.c. administration induced moderate to marked local inflammatory changes at the injection sites. These changes consisted of various combinations of mainly focal / multifocal nodular infiltration of macrophages, infiltration of lymphocytes / plasma cells, fibroplasia / fibrosis and necrosis. Polynuclear cells (mainly eosinophils) and focal / multifocal hemorrhage / hemosiderosis were additional noteworthy findings. The macrophages were enlarged with a slightly basophilic granular cytoplasm. They were usually restricted to the subcutis but extended occasionally into the superficial muscular layer, hypodermis and / or dermis.

[0102] The infiltration of lymphocyte and plasma cells, the fibroplasia (fibroblast proliferation) and the fibrosis (collagen deposition) were mainly oriented at the periphery of the nodular accumulation(s) of macrophages. Fibroplasia was usually associated with neovascularization, vascular congestion and edema. Necrosis was characterized by the presence of necrotic amorphous material with occasional mineralized debris in the central area of the nodular collections of macrophages. Infiltration of macrophages, fibroplasia / fibrosis and necrosis were considered to be the histological correlate of the nodules, masses and thickening observed at necropsy. Overall, the mean severity scores of lymphocytes / plasma cells, fibroplasia / fibrosis, eosinophils and necrosis were slightly higher in the modified peanut protein extract group when compared to the placebo group. The main findings observed at the injection site of the salt solution group were minimal fibroplasia / fibrosis, consistent with mechanical traumas induced by the repeated administrations. Following the 2-week treatment-free period, footprints of the treatment were still present at the injection sites. The s.c. inflammation was still mainly characterized by focal / multifocal nodular infiltration of macrophages, infiltration of lymphocytes / plasma cells, fibroplasia / fibrosis and necrosis, changes being similar in males and females. Overall, the mean severity scores of lymphocytes / plasma cells, fibroplasia / fibrosis and necrosis were slightly higher in the modified peanut protein extract group when compared to the placebo group. When compared to the end of dosing, the score of inflammatory parameters were overall similar at the end of the 2-week treatment free period except for a lower infiltration of eosinophils.

[0103] In the modified peanut protein extract group, there were minimally increased size and number of lymphoid follicles in the spleen for some males and females and, when compared to other groups, minimally increased incidence and / or severity of extramedullary hematopoiesis in both sexes. In the modified peanut protein extract group, there were still minimally increased size and number of lymphoid follicles in the spleen for some males and, when compared to the placebo group, minimally increased mean scores of extramedullary hematopoiesis in both sexes.

[0104] In conclusion, no evidence of systemic toxicity was noted following s.c. administration of 375 pg / day of the modified peanut protein extract, 3 times per week for 7 weeks (total of 22 administrations; 0.5 mL / inj ection), the modified peanut protein extract related effects included changes at the injection sites (edema, indurations, s.c. 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 were generally still present at the end of the treatment free period. The modified peanut protein extract treatment induced a large increase in both PE and MPE specific IgG responses, compared to the placebo group and the salt solution treated group. Although the modified peanut protein extract stimulated antigen-specific IgG responses, there were no body temperature changes indicative of an anaphylactic response.

[0105] Example 2: effects in humans

[0106] Two phase 1 clinical studies were performed (summarized in Table 3 below)

[0107] Table 3; Summary of performed clinical studies with modified peanut protein extract. Study HAL MPE1 / 0043, phase 1, FIH, Denmark

[0108] The FIH study with modified peanut extract (MPE) was performed in Denmark (Study HAL-MPE1 / 0043). The aim of this study was to investigate the safety and tolerability of SCIT-treatment following incremental doses of a MPE in adults with peanut allergy.

[0109] Outpatients of the Dermatology and Allergy Centre department of the Odense University Hospital fulfilling the inclusion and exclusion criteria were the source of subject selection. Inclusion criteria included a well-documented medical history of systemic reactions after ingestion of peanut, a positive food challenge at <1.5 gram peanut protein ingestion within the last 2 years, positive serum specific anti-peanut and Ara h 2 IgE-test (>0.7 kU / L) within the last 2 years and Forced Expiratory Volume in 1 sec (FEVl)>70% of predicted value.

[0110] Subjects were treated for a period of approximately 3-4 months during which at least 17 visits to the clinic were required. Subjects received weekly ascending doses of MPE (i.e. 0.005, 0.05, 0.1, 0.25, 0.5, 1, 2, 5, 10, 20, 38, 94, 188, 375 pg peanut protein). Injections of study drug were administered weekly in the clinic, 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.

[0111] Several procedures and tests were performed that included physical examination, blood sampling for hematology and blood chemistry parameters, Electrocardiogram (ECG), lung function test and vital signs. Furthermore, subjects were monitored in the clinic for the occurrence of early (< 4 hrs. after dosing) local and systemic reactions. Subjects were asked to measure and / or record late local and systemic reactions occurring > 4 hours after the injection in a diary.

[0112] After having signed the informed consent, a total of 17 subjects were screened and randomized, of which 11 subjects were assigned to MPE and 6 subjects to placebo treatment. Subjects received between 1 and 20 injections of study medication, with the range of 1-20 injections in the MPE group and 2-20 injections in the placebo group. The overall mean treatment duration was 98.0 days in the MPE group and 94.7 days in the placebo group.

[0113] In the MPE treatment group, 8 subjects completed the study and received the highest maintenance dose at least once. Three subjects discontinued the study prematurely. One subject discontinued due to withdrawal of consent, one due to a TEAEs (urticaria, asthma, rhinitis grade II) and one due to an SAE (hypersensitivity grade II, 25-year-old female). In the placebo group, 3 subjects completed the study, and 3 subjects discontinued the study due to withdrawal of consent. No notable differences in demographics and baseline characteristics were observed between the treatment groups.

[0114] All subjects were Caucasian with a mean age of 20.9 years and 53% were female. Demographic and baseline characteristics were comparable between groups and all subjects had a history of well controlled asthma (none of the subjects were on chronic inhaled steroids). A summary of the baseline peanut specific IgE is presented in Table 4 below. Table 4; HAL-MPE1 / 0043: Overview of peanut specific serum IgE

[0115] The documented medical histories were generally comparable between groups. A total of 88.2% of the subjects had a history of intermittent asthma and 41.2% had a history of atopic dermatitis. However, the MPE group had a higher percentage of subjects with a history of asthma (100% vs. 50.0% in the placebo group) and atopic dermatitis (54.5% vs. 16.7% in the placebo group). Furthermore, a total of 70.6% (12 / 17) of subjects had concomitant allergies, almost equally distributed between the MPE group (72.7%) and the placebo group (66.7%).

[0116] Study HAL MPE1 / 0049, phase 1, US and Canada

[0117] In addition to Study HAL-MPE 1 / 0043, a phase 1, multi-center study was performed in the US and Canada in order to evaluate the safety and tolerability of MPE in adults, adolescents and children suffering from peanut allergy (Study HAL-MPE1 / 0049).

[0118] The study was a randomized, double blind, placebo-controlled study (active : placebo ratio of 2:1) consisting of two parts. In the first part, 12 adults (18-50 years, Cohort 1) were randomized and in the second part, 15 adolescents (12-17 years, Cohort 2) and 15 children (5- 11 years, Cohort 3) were randomized.

[0119] The main inclusion criteria were: medical history of systemic reactions after ingestion of peanut, positive serum specific anti-peanut IgE (>5.0kU / L) and Ara h 2 IgE-test (>2.0 kU / L), skin prick test to peanut >3 mm in the last 2 years and FEVl>80% predicted (adults / adolescents) or Peak Expiratory Flow (PEF)>80% predicted (children).

[0120] The treatment schedule was very similar to the study in Denmark (except for the first dose of 0.005 pg peanut that was omitted). The study consisted of an up-dosing period of 12 weekly injections. The maintenance treatment started with a first dose of 375 pg and consisted in total of 3 injections, two of these maintenance injections were weekly injections and the last dose was administered following a 2-week interval to evaluate this as a potential schedule for future studies. Up to 5 additional injections for dose repeats were allowed per protocol.

[0121] All randomized subjects received the treatment according to their assigned treatment group. The number of injections given to subjects ranged from 4-20 injections in the HAL-MPE1 group and 15-19 injections in the placebo group. The overall mean treatment duration was 110.3 days in the MPE group and 120.1 days in the placebo group. All subjects randomized to the placebo group completed the treatment, while in the MPE group, 11 out of 27 subjects discontinued treatment prior to study completion. The most common reason for discontinuation in this group was based on the investigator’s decision (7 out of 11 subjects): 5 subjects of those had to discontinue because they had received all 5 additional injections (maximum allowed per protocol), and 2 subjects discontinued due to SAEs (both were adults). Four other subjects (1 adult, 1 adolescent and 2 children) who discontinued the study, withdrew consent.

[0122] Demographic characteristics per cohort are presented in Table 5. The baseline IgE levels against peanut and Ara h2 between the three cohorts were considerably different. In addition, there were differences in the IgE levels between the HAL-MPE1 and placebo group but they were not consistent among the three cohorts, e.g. in children mean levels of IgE were higher than in the placebo group while for the other cohorts, this seemed to be contrariwise. It can be assumed that the small number of subjects randomized to the treatment groups as well as the large individual variations in IgE baseline levels could be the reason for these differences. Table 5; HAL-MPE1 / 0049: Demographics

[0123] *Other: Asian and Caucasian, South-East Asian, African American and Asian, American Indian and Caucasian

[0124] Medical history results showed that nearly 90% (88.1%) of subjects for all cohorts combined (n=42) experienced a past or concomitant medical condition. The most common concomitant medical conditions were asthma and allergic rhinitis (50.0% and 47.6%, respectively). The prevalence of asthma was well balanced between the two treatment groups (51.9% in the HAL-MPE1 group; 46.7% in the placebo group), while allergic rhinitis was more frequently reported in the placebo group (66.7% in the MPE vs. 37.0% in the placebo group). Eczema, atopic dermatitis and allergic conjunctivitis were the next most common medical conditions recorded with a prevalence of 21.4%, 19.0% and 14.3% of the combined study population, respectively. More specifically, eczema prevailed in the placebo group (33.3% vs 14.8%), as well as allergic conjunctivitis (26,7% vs. 7.4%), while atopic dermatitis was present more in the MPE group with 22.2% of subjects versus the placebo group with 13.3% of subjects. In general, with regard to prevalence of medical conditions the observed differences per treatment group per cohort were not considered as clinically significant and may be due to the relatively small size of the treatment groups.

[0125] In total 32 (76%) out of total 42 subjects had other concomitant inhalant-, food- and drug allergies, of which 21 (77.7%) in the MPE group and 12 (80.0%) in the placebo group. The most common allergies other than peanut were: cat- (21.4%), dog- (11.9%) and trees allergy (11.9%).

[0126] Safety study HAL MPE1 / 0043, phase 1, FIH, Denmark

[0127] Safety and tolerability were assessed by determination of early and late local and systemic reactions (onset < 4hrs vs. > 4 hrs. after dosing), occurrence of adverse events, and assessment of laboratory values, vital signs and lung function. Local reactions were defined as any untoward responses to an IMP localized at the site of injection to any dose administered.

[0128] Early local reactions (LR) (i.e. onset within 4 hrs. after dosing) were more frequently observed in the active treatment. The most reported LR was redness at the injection site (90.9% in the active group vs. 16.7% in the placebo group). No wheal sizes larger than 5 cm were recorded. None of the early local reactions were considered as TEAEs.

[0129] A total of 20 late (i.e. starting after 4 hours following dosing) local reactions were reported by 8 subjects. In the HAL-MPE1 group, 7 subjects reported 18 late local reactions of which 17 were considered of mild and 1 (i.e. injection site swelling) of moderate intensity. In the placebo group, 1 subject experienced 2 mild, late local reactions (Table 6).

[0130] Table 6; HAL-MPE1 / 0043: Overview of late local reactions

[0131] Ml: Maintenance dose 1; N.A. Not applicable

[0132] Systemic reactions were defined as any untoward responses to the IMP distant from the injection site to any dose administered. Classification of systemic reactions was conform the World Allergy Organization Subcutaneous Immunotherapy Systemic Reaction Grading System.

[0133] Early systemic reactions (i.e. onset within 4 hrs. after dosing) were observed in 6 out of 11 subjects receiving active treatment, whereas no early systemic reactions were observed in the placebo group. The majority of the early systemic reactions were of mild and moderate severity, i.e. grade I and II (Table 7). In three subjects, drug-related asthma of moderate severity (grade II) was observed within 4 hours following study drug administration, occurring during the on-site observation period. Symptoms immediately resolved upon treatment, and the subjects were discharged within 4 hours after study drug administration.

[0134] Table 7; HAL-MPE1 / 0043: Overview of early systemic reactions (i.e. occurring within 4 hours after injection)

[0135] Note that these AEs were reported during the full duration of the presented ‘’Post- Dose Assessment’ ’ time-intervals.

[0136] The combination of flushing and stridor were assessed as a hypersensitivity reaction which was classified as drug related SAE. One subject (22 year-old female) experienced early grade II and III systemic reactions starting 2.5 hours after HAL-MPE1 injection no. 13, containing 188 pg MPE (i.e. throat irritation, flushing and stridor). The combination of these symptoms was considered a grade II hypersensitivity reaction and classified as a drug related SAE which led to the decision to discontinue study medication. The subject showed immediate improvement of the hypersensitivity symptoms following treatment with antihistamines, corticosteroids and inhalation adrenaline. The subject was fully recovered within 3 hours after onset of the event. No other grade III or SAEs and no grade IV reactions were reported in the study.

[0137] Late systemic reactions (i.e. occurring >4 hours after injection) were observed both in the active and placebo treatment groups. A total of 108 related (i.e., with at least a possible drug relationship) late systemic reactions were recorded in 88% of the subjects (Table 8). The majority (107 / 108) of the late systemic reactions was of grade I (mild) intensity. In the active treatment group, 68 late mild (all grade I) systemic reactions were reported by 7 of the 11 subjects (64%), while in the placebo group, 3 of the 6 subjects (50%) reported 40 late systemic reactions with an at least possible relationship to study medication, of which 39 reactions were of mild intensity (grade I) and 1 of moderate intensity (grade II). Table 8; HAL-MPE1 / 0043: Overview of late systemic reactions (i.e. occurring >4 hours after injection)

[0138] The late systemic reactions were mainly attributable to AEs in the Gastrointestinal disorders SOC, including abdominal pain and nausea. No relevant differences in the incidence of these gastrointestinal reactions were observed, since these reactions were also reported in the placebo group with similar incidence. A TEAE was defined as an event that first appeared during treatment or which worsened relative to the pre-treatment baseline. Among the 17 subjects who received study drug, 10 (91%) in the active group and 5 (83%) in the placebo group reported at least one TEAE (Table 9). In total, 201 TEAEs were reported of which 149 were assessed as drug-related. The majority of drug- related TEAEs (n=135) were of mild intensity. The frequency of drug-related TEAEs was higher in the active treatment group (91%) in comparison with the placebo group (50%).

[0139] Table 9. HAL-MPE1 / 0043: Overview of TEAEs

[0140] Regarding laboratory values, vital signs, ECG and lung function, no clinically relevant changes were observed. No AEs related to the elevated level of aluminum in blood and urine were reported.

[0141] For secondary endpoints, increases in IgG and IgGr levels specific for PE, Ara h 1, Ara h 2, Ara h 3, Ara h 6 were observed following active treatment as compared to placebo, whereas no changes in Ara h 8 and Ara h 9 specific IgG and IgGr levels were observed compared to placebo. IgE levels specific for PE and peanut allergens (Ara h 1, Ara h 2, Ara h 3, Ara h 6, Ara h 8, Ara h 9) did not show clear effects. However, a trend towards a difference in basophil histamine release between the placebo group and the active treatment group was observed. The overall result of the peanut specific titrated skin prick test showed a significant decrease in wheal size following treatment with HAL-MPE1 as compared to placebo.

[0142] Safety study HAL MPE1 / 0049, phase 1, US and Canada

[0143] Safety and tolerability were assessed similarly to the HAL-MPE1 / 0043 study by determination of immediate, early and late local and systemic reactions. In addition, the occurrence of Treatment Emergent Adverse Events (TEAEs), changes in laboratory values, vital signs, plasma and urine aluminum levels and lung function have been determined. Local and systemic reactions were distinguished in an onset time of <1 hour (immediate), 1-4 hours (early) and >4 hours (late) after the injection of MPE or placebo.

[0144] Local reactions ‘swelling’ and ‘erythema’ were not considered a TEAE if the diameter of the reaction was <5cm. Nonetheless, these particular reactions were observed within 4 hours after the dosing occurred (early reaction) and therefore registered separately. As expected, they occurred more frequently in the MPE group compared to the placebo group: erythema 63% vs 40% and swelling 70.4% vs 33.3%, respectively.

[0145] Local reactions, reported as TEAEs, were observed in 63.0% of subjects in the HAL-MPE1 group and in 46.7% of subjects in the placebo group (Table 10). Only a few local reactions with moderate severity were recorded in the MPE group for the PTs 'injection site pruritus' (3 subjects, 5 events), 'injection site urticaria' (2 subjects, 2 events), 'injection site erythema' (2 subjects, 3 events), and 'injection site pain' (1 subject, 1 event).

[0146] Overall, the most frequently reported PTs of local reactions as TEAEs in the HAL- MPE1 group were: 'injection site pruritus' (14 subjects, 51.9%; 42 events), 'injection site erythema' (10 subjects, 37.0%; 34 events), 'injection site urticaria / swelling(>5cm)' (13 subjects, 48.1%; 27 events) and 'injection site pain' (7 subjects, 25.9%; 25 events). Among these LRs, 'injection site urticaria / swelling(>5cm)' and 'injection site pruritus' were notably more frequently reported in the HAL-MPE1 group compared to the placebo group (pruritus: 51.9% vs 13.3%, urticaria / swelling: 48.1% vs 13,3%).

[0147] Occurrence of late local reactions was similar in both treatment groups, except for PT ‘injection site pruritus’ which was more frequently reported in the subjects in the MPE group compared to the placebo group (44.4% vs 6.7% of subjects, respectively). Table 10; HAL-MPE1 / 0049: Occurrence and intensity of local reactions (TEAEs only) per time of onset

[0148] *No severe local reactions occurred in the study.

[0149] Overall, 40.5% of the study subjects experienced at least one systemic reaction; 48.1% of subjects in the MPE group and 26.7% in the placebo group. All systemic reactions were classified in the grading range O-III in both treatment groups.

[0150] With respect to time of onset, most of the systemic reactions reported in the MPE group were early (12 subjects, 47 events) and immediate reactions (9 subjects, 20 events) whereas late reactions were observed in only 3 subjects (3 events). The number of immediate and early systemic reactions was considerably higher in the HAL-MPE1 group compared to the placebo group; the number of late reactions was similar in both treatment groups (Table 11).

[0151] Table 11; HAL-MPE1 / 0049: Occurrence and grading of systemic reactions per time of onset

[0152] The SOC in which the most (>10% of subjects overall) systemic reactions have been reported were 'Respiratory, thoracic and mediastinal disorders' (28.6%) and 'Skin and subcutaneous tissue disorders (26.2%), with a higher occurrence in the HAL-MPE1 group compared to placebo: 40.7% vs. 6.7% and 37% vs. 6.7%, respectively. Within these SOCs, the most common PTs with a notable difference between the MPE group vs. the placebo group were: 'urticaria' (37% vs 6.7%), 'allergic rhinitis' (18.5% vs 0%), 'wheezing' (14.8% vs 0%) and 'allergic cough' (14.8% vs 0%). Almost all grade II systemic reactions occurred in the MPE group: 'urticaria' (22.2%), 'bronchospasm' (7.4%) and 'wheezing' (7.4%). Only one grade II systemic reaction ‘vomiting’ (6.7%) occurred in the placebo group.

[0153] With respect to respiratory systemic reactions related to asthma (asthma, wheezing, bronchospasm, cough), these events occurred in all cohorts, except one (asthma, grade II) which occurred only in the MPE group (Table 12). Important to note is that there were no differences in occurrence of respiratory, asthma-related TEAEs identified with respect to the following parameters: with / without prescription of controller medication; with / without peanut allergy related respiratory symptoms in medical history, or the use of inhaled corticosteroids. Table 12; HAL-MPE1 / 0049: Listing of respiratory systemic reactions (asthma related) per subject occurrence of systemic reactions. No systemic reactions were observed at the doses <10 pg of peanut protein in the MPE group. All grade II reactions occurred at the doses of 20 pg and higher. A TEAE was defined as an adverse event that first appeared during treatment or which existed at baseline but worsened during the study treatment.

[0154] A summary of all Treatment Emergent Adverse Events (TEAEs) reported during the treatment period is presented in Table 13. The majority of subjects in both treatment groups experienced at least one TEAE. Almost all observed adverse events were graded as mild or moderate. Remarkably, the only subject who experienced a severe TEAE, as well as the 2 subjects who discontinued the study because of a TEAE and the 3 subjects who experienced SAEs were all adults (Cohort 1). Table 13; HAL-MPE1 / 0049: Overview of Treatment Emergent Adverse Events.

[0155] Percentages are based on the number of subjects in the Safety Population in each treatment group. Only data up to the follow up visit is included in this summary.

[0156] In total, 436 TEAEs were reported of which 297 were assessed as drug-related. The majority of drug-related TEAEs (n=243) were of mild intensity. The frequency of drug-related TEAEs was higher in the active treatment group (81.5%) in comparison with the placebo group (53.3%).

[0157] An overview of all TEAEs per MedDRA (v.19) System Organ Class (SOC) for the

[0158] 2 treatment groups is presented in Figure 3. More than 50% of subjects experienced at least one TEAEs from the following SOCs: 'General disorders and administration site conditions', 'Respiratory, thoracic and mediastinal disorders' and 'Skin and subcutaneous tissue disorders'. Within these 3 SOCs there were no significant differences noted between the age groups.

[0159] As specified in Figure 3, the following PTs were reported more frequently in the HAL-MPE1 group compared to the placebo group: ‘Injection site pruritus’, ‘Injection site urticaria’, ‘Urticaria’, ‘Rhinitis allergic', 'Wheezing', 'Allergic cough' and ‘Diarrhea’, which are known systemic reactions of SCIT.

[0160] Epinephrine was used three times during the study as rescue medication, all for grade II systemic reactions (‘Wheezing’, ‘Allergic cough’, ‘Anaphylactic reaction’). In all cases, only a single administration of epinephrine was required and symptoms resolved within 1 hour after administration. In addition, in all cases when epinephrine administration was required, subjects received it at the study site.

[0161] 50.0% of study subjects had a medical history of asthma. However, not only subjects with a medical history of asthma developed asthma related respiratory systemic reactions during the treatment with MPE. Regarding laboratory values, vital signs and lung function, there were 5 clinically relevant changes (AEs) in the HAL-MPE1 group and 2 in the placebo group, which belonged to the SOC 'Investigations'. Out of these AEs, 2 (2 / 42 subjects, 4.7%) were related to the increase in serum aluminum and 1 (1 / 42 subjects, 2.4%) event of urine aluminum increased, all in the HAL-MPE1 group. However, all these 3 AEs were of the mild severity and the Data and Safety Monitoring Board (DSMB) did not raise safety concerns regarding increased aluminum levels in blood and urine.

[0162] After study completion, safety follow-up visits were conducted to collect any updates on adverse events and concomitant medication. These visits were performed 1 month after the last dose (on-site) and by telephone at 2, 3, and 6 months after last study drug administration. Overall, 21 events were reported during the follow-up period, 14 in the MPE group (9 / 27 subjects, 33%) and 7 in the placebo group (5 / 15 subjects, 33%). All these events were assessed as not related to study drug treatment, except for one event in the placebo group ('bilateral upper arm itching' of mild intensity). Also, two events (starting at the same day) in one subject in the placebo group were reported as a SAE but were assessed as not related to the study drug ('asthma exacerbation' and 'rhinovirus test positive').

[0163] During the course of this study, no deaths occurred. Three SAEs were reported for 3 different subjects (all adults in the MPE group): a grade II anaphylactic reaction (after the dose of 38 pg, 25 year-old female), a grade II asthma exacerbation (after 3 doses of 375 pg, 23 year-old female) and a case of eosinophilic esophagitis (after 2 doses of 375 pg, 27 year-old male).

[0164] The first SAE of grade II anaphylaxis was comprised of chest tightness, abdominal pain, erythematous rash (urticaria to upper back / neck / hairline), facial flushing and edema of the ears. The symptoms were experienced after injection with 38 pg MPE and resolved after treatment with diphenhydramine and epinephrine. The events were assessed as possibly related to the study drug and the subject discontinued treatment per the investigator's decision.

[0165] The second SAE was a grade II asthma exacerbation, as part of a systemic allergic reaction after the last maintenance injection of MPE (375 pg). The systemic allergic reaction consisting of coughing, wheezing, urticaria localized to the neck, otalgia, rhinitis and sore throat, started to develop 12 minutes after dosing. Two inhalations of salbutamol were administered and this was repeated approximately 15 minutes later. This resulted in significant symptom resolution. Cetirizine was administered resulting in prompt improvement of symptoms other than asthma. The subject was discharged from the hospital on the same day and 12 day later confirmed that symptoms were resolved. The subject's medical history contained mild asthma.

[0166] The SAE of eosinophilic esophagitis is considered an atypical event in this trial. This subject had a pre-existing hiatal hernia, the maintenance treatment period was relatively short (only 3 doses of 375 pg had been received), and the symptoms didn’t disappear after treatment discontinuation. Causality was assessed by the investigator as possibly related to the study drug. The subject was followed for about 6 months after treatment discontinuation and eosinophilic esophagitis was still ongoing by that time. The subject had no complaints when following physician's advice and continued treatment with proton-pump inhibitors. The subject was then lost to follow up. In general, there was no major imbalance reported for the SOC “Gastrointestinal disorders” between the HAL-MPE1 and placebo groups in this trial. Moreover, eosinophilic esophagitis is not a condition that previously has been associated with SCIT, it is more known as a potential risk of SLIT or OIT.

[0167] The safety results of this study confirmed a similar outcome that was previously observed in adults in the Phase 1 study HAL-MPE 1 / 0043 in Denmark. Moreover, this study demonstrated that MPE is also safe and well tolerated in children and adolescents with peanut allergy.

[0168] Efficacy

[0169] The short-term immunological results showed an increase in IgG against peanut and major peanut allergens (Ara h 1, Ara h 2, Ara h 3 and Ara h 6) in all cohorts during treatment with MPE. The increase of IgG_i. the subset of IgG which has been associated with the development of tolerance to IgE-mediated allergies, was most pronounced in the adolescent's and children’s cohort. IgE against peanut and peanut allergens was overall only slightly increased after treatment with MPE (Table 14). Usually during immunotherapy studies, IgE tends to increase after the first months of treatment and then to decrease. The extent of this study may have been a limiting factor in demonstrating this effect and should therefore be further investigated in future studies with a longer treatment duration.

[0170] Overall, this study confirmed that MPE is generally safe and well tolerated in adults with peanut allergy. More importantly, for the first time it showed that this treatment is also safe and well tolerated in subjects between 5-17 years of age. Moreover, a trend was observed towards a more favorable immunological effect at the younger age groups, combined with a slightly better safety profile (no SAEs, no discontinuations due to an AE and no severe reactions in adolescents and children). Table 14; HAL-MPE1 / 0049: Ratio of geometric mean fold change (ANCOVA model) for PE specific IgG, IgG4and IgE serum levels, incl. P-Values (per cohort)

[0171] ** Maintenances / Early Termination visit

[0172] Summary

[0173] In vitro and in vivo data demonstrated that chemical modification reduces the IgE binding potential of peanut extract (PE), while the capability to trigger PE-specific T-cells is retained. Studies to investigate the safety of modified peanut extract (MPE) demonstrated that the in vitro IgE binding potential of PE is reduced upon chemical modification leading to a decrease in mediator release from basophils loaded with IgE from peanut allergic subjects. In addition, a challenge with MPE in the safety mouse model did not result in severe allergic reactions, even at the higher doses tested. The specific mouse model for peanut immunotherapy (efficacy model) showed that treatment with MPE is effective. Treatment with the non-modified PE had a better efficacy profile however it resulted in severe side effects, but treatment with MPE was shown to have a better safety profile. In addition, MPE adsorbed to A1(OH)3 appeared to be more effective and safe than non-adsorbed MPE in this model.

[0174] No mutagenic activity of MPE was observed in the bacterial reverse mutation test. A low mutagenicity for both MPE and placebo was observed in the MLA test. Phenol is present in both MPE and placebo and a positive mutagenic effect of phenol in the MLA test in the presence and absence of S9 has well been documented. The in vivo repeat-dose toxicology study has shown that under the study conditions, and compared to MPE placebo, no evidence of systemic toxicity were noted at the tested dose of MPE DP, and all changes attributed to MPE were considered to be related to the expected immune reaction at the injection sites.

[0175] MPE is a suspension for subcutaneous administration containing modified peanut allergens adsorbed onto A1(OH)3. Preparation of MPE dilutions for subcutaneous administration in humans is performed by qualified staff according to the detailed instructions shown at the pharmacy manual used in the clinical trials.

[0176] Study medication is administered at weekly (up-dosing) or bi-weekly (maintenance) intervals 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. The dosing interval for the maintenance period is under investigation and might be changed based on the results of future clinical trials. The dose schedule is shown in Table 15. Table 15; Up-dosing and maintenance injection schedule

[0177] *In the HAL-MPE1 / 0043 and HAL-MPE1 / 0043 : this dose was named as Maintenance dose 1.

[0178] **The final dosing interval and the treatment duration to be defined based on the future studies’ results.

[0179] Subcutaneous injections are performed at the study site by authorized and trained staff. A physician must always be at the study site and directly available. Trained site personnel must be present on call in the hospital during every injection, and it must be possible for them to reach the study site within a few minutes. Close monitoring of each subject at the study site is required after administration of the study medication, with an observation period of 2-4 hours following each administration. Prior to each administration, subjects should be asked about adverse reactions to the previous administration and the use of concomitant medication. During the up-dosing period, the decision to administer an increased, the same or a reduced dose should be based on the results observed after the previous dose as explained in Figure 4.

Claims

CLAIMS1. Peanut protein extract for use in the prevention, reduction, or treatment of peanut allergy in a human wherein said use comprises exposing said human to an initial dose of said peanut protein extract of less than 0.1 pg followed by multiple increasing dosages up to 750 pg peanut protein extract, and wherein said peanut protein extract is a modified peanut protein extract, preferably a reduced and subsequently alkylated peanut protein extract.

2. Peanut protein extract for use according to claim 1 , wherein said peanut protein extract is complexed with an adjuvant, preferably with aluminium, more preferably Al(0H)3.

3. Peanut protein extract for use according to claim 1 or claim 2, wherein said peanut protein extract is complexed and saturated with an adjuvant, preferably with aluminium, more preferably Al(0H)3.

4. Peanut protein extract for use according to any one of the claims 1 to 3, wherein said treatment comprises an initial dose of said peanut protein extract of less than 0.1 pg followed by weekly increasing dosages up to 300 pg to 500 pg peanut protein extract.

5. Peanut protein extract for use according to any one of the claims 1 to 4, wherein said human is exposed to said peanut protein extract 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.

6. Peanut protein extract for use according to any one of the claims 1 to 5, wherein said treatment comprises an initial dose of said peanut protein extract of 0.05 pg or less, increasing weekly dosages of said peanut protein extract up to a maintenance dose of said peanut protein extract of 350 to 400 pg peanut protein extract.

7. Peanut protein extract for use according to any one of the claims 1 to 6, wherein said peanut protein extract 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.

8. Method for prevention, reduction, or treatment of peanut allergy in a human wherein the method comprises the steps of: a) exposing said human to an initial dose of a peanut protein extract of less than 0.1 pg; b) subsequently exposing said human to multiple increasing dosages up to 750 pg peanut protein extract; wherein said peanut protein extract is a modified peanut protein extract, preferably a reduced and subsequently alkylated peanut protein extract.

9. Method according to claim 8, wherein said peanut protein extract is complexed with an adjuvant, preferably with aluminium, more preferably Al(0H)3.

10. Method according to claim 8 or claim 9, wherein said peanut protein extract is complexed and saturated with an adjuvant, preferably with aluminium, more preferably Al(0H)3.

11. Method according to any one of the claims 8 to 10, wherein said method comprises the steps of: a) exposing said human to an initial dose of said peanut protein extract of less than 0.1 pg; b) subsequently exposing said human to weekly increasing dosages up to 300 pg to 500 pg peanut protein extract.

12. Method according to claim 11, wherein said method comprises the steps of: a) exposing said human to an initial dose of said peanut protein extract of less than 0.05 pg; b) subsequently exposing said human to weekly increasing dosages up to 350 to 400 pg peanut protein extract.

13. Method according to any one of the claims 8 to 12 wherein said human is exposed to said peanut protein extract 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 8 to 13, wherein said peanut protein extract is formulated as 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.

Images