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

Guro Gafvelin,Stephen Parmley,Theresa Neimert-Andersson,Ulrich Blank,Tove L.J Eriksson,Marianne Van Hage,Juha Punnonen

doi:10.1074/jbc.m607938200

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 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.

Highlights

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 allergen-specific immunotherapy (ASIT)
Molecular modifications that change the overall three-dimensional structure of the protein can be performed. Such modifications include disruption of disulfide bonds (18 –20), oligomerization [21, 22], duplication of amino acid sequences [23], fragmentation [24, 25], and introduction of prolines [16] or point mutations in known functional protein domains [25]. These approaches have proven successful for many allergens, but it is often difficult to predict modifications that result in hypoallergenic properties while retaining
The two Lep d 2 isoforms, Lep d 2.01 and Lep d 2.02, share 89.6% identity at the amino acid level and Gly d 2 share 79.2% identity with Lep d 2.01 and 78.4% with Lep d 2.02. These three dust mite group 2 allergens are well suited as model genes to test the concept of directed molecular evolution for generating hypoallergens

Summary

Introduction

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. Compared with wildtype rLep d 2, all nine shuffled allergens selected from the screening exhibited strongly reduced binding to mite allergic patient IgE, corresponding to about 80-fold reduction to almost complete loss of binding.

Results

Conclusion