Project themes and work packages

This theme deals with "recombinant antigen production" as indicated in the call topic, and their immunological and physico-chemical evaluation. Replacement of extracts by recombinant major allergens specifically targets the aim to be novel and effective ("novel, (…..) efficient therapeutic strategies"). By introducing the concept of hypo-allergenicity it targets the aim to be novel with guarantee safety ("novel, safe (……) therapeutic strategies"). The main S&T challenge of Theme 1 is to produce recombinant allergens and hypo-allergenic derivatives of sufficient yield and quality. At the molecular level there are challenges relating to the expression of mutant molecules and their molecular characterization through comparisons with correctly-folded and fully immune-reactive wild-type molecules. There are also challenges in chemical modification, i.e. glutaraldehyde modification and reduction/alkylation to induce hypo-allergenicity. Here the molecular size distribution, tendencies to aggregate and immune-reactivity (in vitro T- and B-cell reactivity as well as performance in an in vivo mouse model) have to be compared to unmodified wild-type recombinant molecules. The stability of the mutant and chemically-modified preparations is also an important issue. Comparison of different hypo-allergenic approaches will lead to the selection of candidates for clinical interventions. Last but not least, a major S&T objective is the GMP-production of these selected hypo-allergens for application in Phase I and II clinical trials. In summary, the S&T objectives of Theme 1 are to obtain:
GMP-grade hypo-allergenic recombinant parvalbumin- and recombinant lipid transfer protein-based products for the treatment of fish and fruit allergy, respectively. These products will have to fulfill the following requirements:

• < 5% but preferably < 1% residual allergenicity compared to wild-type allergen as judged by IgE-binding, and basophil histamine release.
• Similar immunogenicity of hypo-allergenic products when compared to wild-type allergen as judged by their capacity to induce blocking IgG antibodies in laboratory animals and T-cell proliferation using PBMCs from fish/fruit-allergic patients.
• Effectiveness in a relevant mouse model of food allergy and immunotherapy.
• Acceptable production yield and physico-chemical profile with respect to homogeneity, molecular size distribution and stability.

This theme deals with the organization and evaluation of all the clinical and intervention studies to be undertaken during the course of this project. The theme will target "European capability in therapy of diseases which are currently untreatable, where life is at stake". It will "demonstrate proof of principle, and carry out early clinical trials" by performing Phase I and II clinical trials. Moreover, successful interventions will stimulate the development of similar products that not only target other foods but also respiratory allergies ("advanced therapies and technologies with broad potential application"). Thus it will "boost European biotechnology industry, especially the SME sector"). Clinical studies will also provide the basis for the studies on immune mechanisms and early biomarkers that are outlined in Theme 3 ("interplay between clinical investigation and understanding of underlying processes"). The main S&T objectives are to set up multi-center clinical studies that will fulfill regulatory and ethical requirements at both EU and national levels. At the same time, these studies need to provide sufficient power (sample size) to be able to draw reliable conclusions on the safety and efficacy of interventions. The design of the studies will also need to accommodate answering scientific (mechanisms and biomarkers) as well as clinical questions (safety, efficacy). Complexity of such studies requires well-designed database management and logistics of sample flows and storage. In summary, the S&T objectives of Theme 2 are to demonstrate:
Efficacy and safety of GMP-grade hypo-allergenic recombinant parvalbumin- and recombinant lipid transfer protein-based products for the treatment of fish and fruit allergy, respectively. These products will have to fulfill the following requirements:

• At least 30% improvement over placebo
• An acceptable level of local and systemic side-effects

This theme deals with the underlying immune mechanisms of protection against food allergy in successfully treated food allergic patients ("understanding of underlying processes"). The main S&T objectives of Theme 3 are to perform in depth humoral (IgE, IgG, IgA) and cellular immunology (T-cell subsets, epitope-specific T-cells, effector cells, APCs) studies within multi-center interventions studies. To compare studies, it is of great importance that immune assays are standardized throughout the consortium. Where possible, analyses will be centralized (e.g. serological analyses, immuno-histochemical analyses of biopsies, reading of ELISPOT, Luminex analyses on cell supernatants). Some immune assays need to be performed on freshly harvested cells (T-cell proliferation, direct basophil histamine release). This requires local analyses at each center. The challenge in this situation is to achieve optimal standardization of methods. In summary, the S&T objectives of Theme 3 are to elucidate:

• The mechanism(s) of protection in successfully treated patients, by performing for the first time comprehensive studies of both humoral and cellular immune responses against food allergens in multi-center clinical trials and standardized protocols.