Hypoallergens for allergen-specific immunotherapy by directed molecular evolution of mite group 2 allergens.
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 wild-type 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.
Gafvelin
Guro
G
Department of Medicine, Clinical Immunology and Allergy Unit, Karolinska Institutet, 17176 Stockholm, Sweden. guro.gafvelin@ki.se
Parmley
Stephen
S
Neimert-Andersson
Theresa
T
Blank
Ulrich
U
Eriksson
Tove L J
TL
van Hage
Marianne
M
Punnonen
Juha
J
D000485:Allergens / Q000235:genetics* / Q000276:immunology / Q000627:therapeutic use*; D000818:Animals; D003888:Desensitization, Immunologic; D019020:Directed Molecular Evolution / Q000379:methods; D004391:Dust / Q000276:immunology; D005260:Female; D006801:Humans; D008213:Lymphocyte Activation; D051379:Mice; D008805:Mice, Inbred A; D008925:Mites / Q000235:genetics / Q000276:immunology*; D020033:Protein Isoforms / Q000235:genetics / Q000276:immunology / Q000627:therapeutic use; D011506:Proteins / Q000235:genetics / Q000276:immunology / Q000627:therapeutic use*; D051381:Rats; D011995:Recombination, Genetic
D000485:Allergens; D004391:Dust; C090039:Lep d I allergen; D020033:Protein Isoforms; D011506:Proteins