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J. Biol. Chem., Vol. 282, Issue 6, 3778-3787, February 9, 2007

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

Guro Gafvelin¹², Stephen Parmley¹, Theresa Neimert-Andersson, Ulrich Blank^¶||, Tove L. J. Eriksson, Marianne van Hage, and Juha Punnonen

From the Department of Medicine, Clinical Immunology and Allergy Unit, Karolinska Institutet, 17176 Stockholm, Sweden, Maxygen Inc., Redwood City, California 94063, ^¶Inserm U699, F-75018 Paris, France, and the ^||FacultédeMédecine, Site Xavier Bichat, Université Paris 7-Denis Diderot, F-75018 Paris, France

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.

Received for publication, August 18, 2006 , and in revised form, December 11, 2006.

^* This work was supported in part by grants from the Swedish Foundation for Health Care Sciences and Allergy Research, the Swedish Research Council, Swedish Asthma and Allergy Association's Research Foundation, the Swedish Cancer and Allergy Foundation, Hesselman's Foundation, Magnus Bergvall's Foundation, Konsul Th. C. Bergh's Foundation, Åke Wiberg's Foundation, the Stockholm County Council, and the Karolinska Institutet. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ These authors contributed equally to this work.

² To whom correspondence should be addressed: Karolinska Institutet, Dept. of Medicine, Clinical Immunology and Allergy Unit, Karolinska University Hospital, 171 76 Stockholm, Sweden. Tel.: 46-8-51776441; Fax: 46-9-335724; E-mail: guro.gafvelin{at}ki.se.

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