FAST: towards safe and effective subcutaneous immunotherapy of persistent life-threatening food allergies

Although reliable figures are still largely unavailable, IgE-mediated food hypersensitivity (hereafter referred to as food allergy) is thought to affect around 1-2% of adults and 4-8% of children, i.e. roughly around 10 million EU inhabitants (reviewed in [1, 2]). Recent studies within the FP6-funded project EuroPrevall [3] showed tree nuts (hazelnut and walnut), fruits (apple, peach and kiwi) and peanut are the most common plant foods causing food allergy, followed by vegetables like carrot, tomato and celery. After milk and egg, fish and shrimp are most frequently causing food allergy to animal-derived foods (pers. comm. M. Fernandez-Rivas).

The clinical presentation of food allergy varies from mild local symptoms of the oral cavity, usually referred to as the oral allergy syndrome (OAS), to severe systemic reactions which can include life-threatening anaphylaxis. In the U.S., food-induced anaphylaxis is estimated to cause about 120,000 emergency room visits and 3000 hospitalizations each year [4].

The only available treatment for food allergy is avoidance, in conjunction with rescue medication in case of accidental exposure. However, hidden allergens in composite foods, unwanted contaminations and occasional poor adherence to dietary restrictions make avoidance difficult and ineffective. Therefore there is an urgent need to develop a treatment for food allergy that lowers the threshold significantly and makes avoidance less stringent. Allergen-specific immunotherapy (SIT) is the only treatment available that targets the immunological cause of the disease. It has proven successful in treatment of insect venom allergies and for respiratory allergies such as rhino-conjunctivitis and asthma to pollen and house dust mite [5‐7], but due to the duration and invasiveness (i.e. 3-5 years of monthly subcutaneous injections) and the risk of anaphylactic side-effects, SIT is a niche treatment compared to symptomatic drugs, though new alternative routes have been recently successfully explored [8].

Over the past decades, major inhalant and food allergens have been identified, purified, cloned and produced as recombinant proteins. The use of recombinant allergens to replace biological extracts will contribute to enhance the efficacy of SIT by better control over the dosage and elimination of some of the disadvantages (variability in product quality, difficulty in standardization of extracts, sensitization to new allergens) inherent to biological extracts (reviewed in [9]). The first clinical trials using recombinant allergens of birch, grass and ragweed pollen have demonstrated that single recombinant proteins can effectively replace extracts [10, 11].

For the development of immunotherapy for food allergy, most attention has so far been given to peanut egg and milk, as these foods are important causes of severe food allergy, mainly in children. Oral immunotherapy approaches for several foods (milk, egg, peanut) show desensitization but no tolerance and commonly have side-effects (well-reviewed in [8]). As children with transient milk or egg allergy seem to have IgE primarily directed to conformational epitopes, sensitive to heat or processing [12, 13], two clinical trials focused on investigating tolerance to heated milk and egg products in this population [14, 15]. Preliminary studies suggested accelerated tolerance induction, so a follow-up study is ongoing. Subcutaneous allergen-specific immunotherapy (SCIT) as a treatment for peanut allergy has been evaluated using aqueous peanut extract. Although a significant level of efficacy was demonstrated, anaphylactic side-effects, caused by IgE-binding to the injected allergen, were too frequent and the project was abandoned [16, 17]. In recent years, sublingual therapy has gained a considerable share of the market for the treatment of respiratory allergies in the form of extract-based drops or tablets. Side-effects are reported to be minimal and efficacy has been demonstrated. The first reports of SLIT with food allergens, date from 2003, in kiwi [18, 19]. More recently, SLIT using hazelnut [20, 21] and peanut extract [22] has been reported for the treatment of hazelnut and peanut allergy and peach peel extract enriched for LTP was used in a SLIT trial to treat peach allergy [23, 24]. These treatments resulted in a significant but moderately increased tolerated dose and systemic side-effects have so far rarely been reported. Despite these quite promising results, in FAST we have decided to target SCIT, the main reason for this being the expected higher efficacy and better compliance and safety, facilitated by performing treatment in an outpatient clinical environment.

To increase safety and develop effective SCIT for the treatment of food allergy, the allergen can be modified in such way that it exhibits significantly decreased IgE-binding potency, i.e. that it becomes hypoallergenic, but retains T-cell reactivity. In addition, these hypoallergens can be absorbed to aluminium hydroxide, which increases safety due to its depot effect and furthermore increases efficacy by its adjuvant effect.

There is still some disagreement concerning the immunological basis of the beneficial effect of immunotherapy. Allergic patients can typically be distinguished from healthy subjects by the presence of allergen-specific IgE antibodies, but a (usually not observed) decrease in specific IgE can not explain the beneficial effect of SIT. The current knowledge on the characteristics of the allergic immune response and its modulation by SIT has developed dramatically beyond the level of serum IgE antibodies, in particular knowledge on other isotypes such as IgG₄ and IgA, on various subsets of helper T-cells (Th-cells) and on the role of innate antigen presenting cells (like dendritic cells (DCs). Essentially there are two extremes for explaining the beneficial effect of SIT: inhibition of allergic reactions by blocking IgG₄ and IgA antibodies or by a shift from Th2 to Th1/Treg. FAST aims at induction of both using hypoallergenic but immunogenic recombinant allergens. Although double-blind placebo-controlled food challenge (DBPCFC) will always remain the primary read-out for establishing efficacy, it is not an appropriate tool to use at many (early) time-points during treatment. However, reliable early (composite) biomarkers for efficacy that correlate with the outcome of the DBPCFC are not (yet) available. To improve and identify relevant (composite) biomarkers for monitoring efficacy of immunotherapy it is important to further unravel the mechanism of protection in patients responding favorably to immunotherapy. In depth monitoring of humoral and cellular immune parameters will help identify such (early) biomarkers for efficacy.

Within this context it is the objective of the EU-funded collaborative project FAST to develop a safe and effective immunotherapy against persistent and life-threatening food allergies. The project includes 15 partners from 11 different countries. To address all the main objectives indicated above, the partnership first focuses on producing and testing a number of different mutant (hypo-) allergens (and wild-type allergens for comparison). Additionally, there are three companies, two partners from the pharmaceutical industry (BIAL, Spain and HAL Allergy, The Netherlands) and one biotech company (BIOMAY, Austria) that will focus on the production of the chosen hypoallergens under good manufacturing practice (GMP) for the clinical trials. In the consortium there are six clinical centers participating in six countries, chosen on the basis of expertise and geographic background. Lastly, allergen-specific MHC class II tertramers/multimers will be developed as well as mouse models for immunotherapy with hypoallergens.

Ninety percent of all food allergies are caused by ± 10 foods. Allergy to some of these foods i.e. milk and egg, are outgrown in the vast majority (milk) or up to 50% (egg) of children before the age of five. Although these are certainly relevant, persistent food allergies that stay throughout adulthood perhaps represent a more important target for developing immunotherapy (IT). Most attention for the development of IT for food allergy has so far been given to peanut and despite being usually outgrown, also to egg and milk. Apart from prevalence, the main reason for this focus is high risk of severe reactions induced by these foods. Allergy to fruits, like peach, and to tree nuts, fish and shrimp are also high on the list of candidates to develop novel therapies: allergy against these foods is prevalent, persistent and potentially life-threatening and avoidance negatively affects a healthy diet. For the development of a new concept for the treatment of severe persistent food allergies based on recombinant hypoallergens, the best prospects for reaching clinical testing at Phase II^b level within the life-time of an EU project are treatments targeting food allergies that are dominated by a single major allergen. As described above, in the case of severe allergies to peanut but also to tree nuts there are at least three major allergens, so multiple recombinant allergens would be needed. Therefore, in the FAST project we target two foods, one from animal origin, fish, and one from plant origin, peach. Severe reactions to fish and peach are both linked to a single dominant allergen, parvalbumin (Cyp c 1/Gad c 1) and lipid transfer protein (Pru p 3) respectively. Peach was chosen as a representative of all fruit allergies linked to lipid transfer protein. Both the natural and recombinant wildtype (WT) are compared to hypoallergenic variants produced in FAST.

Two ways of rendering allergens hypoallergenic are used in FAST. Recombinant technology allows modification by site-directed mutagenesis and with this method hypoallergenic mutants have been successfully developed for several food allergens. These include the major peanut allergens Ara h 1, 2 and 3 [25], the major fish allergen Cyp c 1 [26, 27] and the major fruit allergen Pru p 3 ([28] and own observation). Another approach is chemical modification. Since the 1970s, allergen extracts are treated with glutaraldehyde resulting in cross-linked proteins with reduced allergenicity, (also known as allergoïds) and hypoallergenicity can also be induced by reduction/alkylation in allergen molecules containing disulfide bridges. Here for the first time, we will apply this concept to recombinant food allergens.

In the consortium there are six participating clinical centers in six countries: Odense (OUH, Denmark), Łódź (MUL, Poland), Reykjavik (LSH, Iceland), Madrid (HCSC, Spain), Athens (NKUA, Greece) and Rome (IDI, Italy). These centers have been chosen on the basis of their specific expertise in food allergy and DBPCFC and their geographic background.

The methodology will be identical to those of the clinical studies performed within the EU-funded integrated project EuroPrevall [3] that were coordinated by the partner from Madrid (including standardized case record forms (CRFs), methods for skin prick testing (SPT) and double-blind placebo-controlled food challenges (DBPCFCs). From the clinical studies of EuroPrevall, we have concluded that fish allergy is observed across Europe but it is especially frequent in Iceland, Spain, Greece, Italy and Poland. Moreover, the Danish groups have published studies with 30+ codfish allergic patients [36, 37]. Peach allergy caused by LTP is frequently found in Spain, Italy and Greece. Together, these centers therefore provide the necessary expertise and cover some of the most important areas for fish and peach allergy in Europe. All clinical studies and clinical trials will be performed with the approval of the local ethics committees, and according to the national and European regulations.

For adequate monitoring and future improvement of immunotherapy for food allergy, it is necessary to establish which immune mechanisms are protective. First, this will be studied in a mouse model of food allergy immunotherapy. Later, comprehensive immunological studies during Phase II^b clinical trials will be carried out.

The FAST project will increase our understanding on how to bring the treatment of food allergy to a higher level, i.e. by adding an alternative to the spectrum of treatment modalities for food allergy that is currently restricted to avoidance and rescue medication, and SLIT or OIT. Overall, allergy is recognized as a major disease affecting around 30-40% of the population. Food allergy is estimated to affect about 5% of the population, but additionally it also is a major disease because of its great impact on the quality of life. Food allergy is potentially life-threatening and the risk of accidental intake causes great fear and ultimately leads to social isolation. The FAST project aims at significantly lowering thresholds and consequently improving quality of life of food allergy sufferers.

FAST investigates the development of novel therapies for the treatment of food allergy by combining the principles of traditional allergen-specific immunotherapy with biotechnology (hypoallergenic recombinant allergens). Standardization of allergen extracts has been a major challenge over the past decades. The introduction of highly purified products will end the situation where standardization of variable biological products puts an increasing burden on quality control departments and regulatory authorities.

The research proposed in FAST aims at the development of treatment for diseases that start in early childhood, using top clinical, biotechnological and immunological research. The strategy chosen will not involve children up to the stage of Phase II^b clinical trials. Phase I/II^a clinical trials will be carried out in adults only. Moreover, children enrolled in Phase II^b clinical trials will not be under 12. FAST aims to develop a novel therapy for food allergy that will have a positive impact on the diet of food allergic patients improve their quality of life, allowing them to stop avoiding fish and fruit which are important components of a healthy "non-obese" diet.

Once more the importance of child health is addressed. As mentioned food allergy is a disease that in most cases starts in very young children. The FAST project aims to develop a therapy for food allergy that in the future can also be safely used in children.

Obviously, the approach chosen by FAST is applicable to the treatment of other food but also respiratory allergies. Moreover, successful therapy is the first step towards allergen-specific preventive immunotherapy or vaccination. FAST targets a chronic disease that is potentially life-threatening by anaphylactic shock. Food allergy is currently untreatable, avoidance being the only remedy. It will do so by using the established (subcutaneous) route of administration. It will use established (chemical modification) and emerging (mutants) methods to achieve hypoallergenicity, aiming at increased safety of the treatment. It aims at replacing extracts by highly purified recombinant allergens. This will allow more accurate administration of active ingredients, which will hopefully improve efficacy. The mechanism of action will be investigated with a major focus on the potential role of allergen-specific regulatory T-cell. To be really able to study these cells at the level of specific epitopes, food allergen specific tetramers will be developed. Overall, the project aims at developing a novel strategy to replace avoidance and rescue medications as the only way to treat food allergy: allergen-specific immunotherapy with biotech hypoallergens.