Hypoallergens for Allergen-specific Immunotherapy by Directed Molecular Evolution of Mite Group 2 Allergens

Abstract

Allergen-specific immunotherapy is the only treatment that provides long lasting relief of allergic symptoms. Currently, it is based on repeated administration of allergen extracts. To improve the safety and efficacy of allergen extract-based immunotherapy, application of hypoallergens, i.e. modified allergens with reduced IgE binding capacity but retained T-cell reactivity, has been proposed. It may, however, be difficult to predict how to modify an allergen to create a hypoallergen. Directed molecular evolution by DNA shuffling and screening provides a means by which to evolve proteins having novel or improved functional properties without knowledge of structure-function relationships of the target molecules. With the aim to generate hypoallergens we applied multigene DNA shuffling on three group 2 dust mite allergen genes, two isoforms of Lep d 2 and Gly d 2. DNA shuffling yielded a library of genes from which encoded shuffled allergens were expressed and screened. A positive selection was made for full-length, high-expressing clones, and screening for low binding to IgE from mite allergic patients was performed using an IgE bead-based binding assay. Nine selected shuffled allergens revealed 80-fold reduced to completely abolished IgE binding compared with the parental allergens in IgE binding competition experiments. Two hypoallergen candidates stimulated allergen-specific T-cell proliferation and cytokine production at comparable levels as the wildtype allergens in patient peripheral blood mononuclear cell cultures. The two candidates also induced blocking Lep d 2-specific IgG antibodies in immunized mice. We conclude that directed molecular evolution is a powerful approach to generate hypoallergens for potential use in allergen-specific immunotherapy.The large increase in allergic diseases observed during the past decades has urged the development of safe and efficient treatments of allergy. Many new concepts based on targeting the underlying immunological causes of IgE-mediated allergy have been suggested (1-5). However, the only treatment that causes a long-lasting relief of symptoms is allergen-specific immunotherapy (ASIT).3 Today ASIT is based on the repeated administration of allergen extracts prepared from the allergen source. Although successful clinical outcomes are well documented, several problems are associated with allergen extractbased ASIT, including the risk of inducing local and systemic side effects and induction of new sensitizations (6, 7).Large batches of well defined single allergen components can be produced using recombinant techniques, making it feasible to solve problems linked to allergen extract-based ASIT. The most severe side effects of ASIT are caused by the binding of injected allergen to allergen-specific IgE on high affinity Fc⑀RI receptor-bearing effector cells, leading to cross-linking of the Fc⑀RI receptors, degranulation, and release of anaphylactogenic mediators. Therefore, allergens with reduced IgE binding capacity have been proposed to improve the safety of ASIT (8...

Disruption of Allergenic Activity of the Major Grass Pollen Allergen Phl p 2 by Reassembly as a Mosaic Protein

“…For example, the use of recombinant allergen fragments or synthetic peptides requires that treatment is performed with a cocktail of several different molecules to provide the necessary epitopes for Ab induction and T cell tolerance, whereas our approach allows treatment with a single reassembled molecule, which should greatly ease the production of the vaccine. The recently described random reassembly of DNA fragments of different gene fragments (21,22) would be a similar approach but delivers a relatively large number of novel genes that have to be expressed and characterized regarding suitability. The induction of coincidently crossovers, deletions, insertions, inversions, and point mutations of genes through this molecular breeding technique or other mutational approaches may also lead to the loss of sequences necessary for the induction of Ab or T cell responses.…”

“…Mutants or deletion variants of allergens may lack T cell epitopes or important structures necessary to induce protective IgG responses. Randomly shuffled allergens can be relatively easily produced, but it may be quite time-consuming to select from a variety of mutants those with the most advantageous properties (21,22). To overcome these disadvantages, we have developed an alternative strategy for the conversion of an allergen into a hypoallergenic vaccine.…”

From Molecule Studies of Allergens to Development of Immunotherapy of Allergies

“…At T cell level, the ratio of Th1 to Th2 cytokines increases following immunotherapy, suggesting a restoration in Th1/Th2 balance [8]. Regulatory T cells are also stimulated in the course and are believed to play an important role in modifying the disease through the secretion of IL-10 and TGF-β [9] (Figure 2).…”

“…Although the precise mechanisms by which epitope-based immunotherapies mediating anti-inflammatory responses are not yet thoroughly defined, they generally share similar modulating abilities on T cell and B cell responses [7]. At T cell level, the ratio of Th1 to Th2 cytokines increases following immunotherapy, suggesting a restoration in Th1/Th2 balance [8]. Regulatory T cells are also stimulated in the course and are believed to play an important role in modifying the disease through the secretion of IL-10 and TGF-β [9] (Figure 2).…”

Construction of a chimeric aminopeptidase by a combination of gene shuffling and mutagenesis

“…However, recent investigations have demonstrated that genetic manipulation methods are useful to improve currently available natural enzymes. Such strategy includes directed molecular evolution methods such as recursive random DNA shuffling with simultaneous random mutagenesis and can improve the functions and properties of enzymes dramatically (Crameri et al 1998;Jiang et al 2007;Gafvelin et al 2007;Minshull and Stemmer 1999;Moore and Arnold 1996;Stemmer 1994). Enzymes that readily lend themselves to such approaches are those sharing highly homologous regions with other enzymes of different characteristics.…”