A Human IgE Antibody Binding Site on Der p 2 for the Design of a Recombinant Allergen for Immunotherapy

Abstract

Der p 2 is one of the most important allergens from the house dust mite Dermatophagoides pteronyssinus. Identification of human IgE Ab binding epitopes can be used for rational design of allergens with reduced IgE reactivity for therapy. Antigenic analysis of Der p 2 was performed by site-directed mutagenesis based on the x-ray crystal structure of the allergen in complex with a Fab from the murine IgG mAb 7A1 that binds an epitope overlapping with human IgE binding sites. Conformational changes upon Ab binding were confirmed by nuclear magnetic resonance using a 7A1-single-chain variable fragment. In addition, a human IgE Ab construct that interferes with mAb 7A1 binding was isolated from a combinatorial phage-display library constructed from a mite-allergic patient and expressed as two recombinant forms (single-chain Fab in Pichia pastoris and Fab in Escherichia coli). These two IgE Ab constructs and the mAb 7A1 failed to recognize two Der p 2 epitope double mutants designed to abolish the allergen-Ab interaction while preserving the fold necessary to bind Abs at other sites of the allergen surface. A 10-100-fold reduction in binding of IgE from allergic subjects to the mutants additionally showed that the residues mutated were involved in IgE Ab binding. In summary, mutagenesis of a Der p 2 epitope defined by x-ray crystallography revealed an IgE Ab binding site that will be considered for the design of hypoallergens for immunotherapy.

Structural Aspects of the Allergen-Antibody Interaction

“…Analysis of the allergen-antibody interfaces at the amino acid level shows that paratopes are formed by 18–28 residues that interact with epitopes composed of a similar number of amino acids (12–25 in the set of 16 complexes analyzed here) ( Figure 4B , Table 2 ) ( 45 – 50 , 52 , 53 , 56 , 58 , 60 – 64 ). In addition, it is possible to examine the distribution of various amino acids in the epitope and paratope areas.…”

“…The X-ray crystallographic structures of allergen-antibody complexes were first determined for egg lysozyme with fragments of murine IgG monoclonal antibodies (mAb) ( 42 – 47 ). Subsequently, other structures were reported for other allergens, where murine IgG mAb were selected as surrogates for human IgE, due to their capacity to inhibit binding of human IgE antibody to the allergen ( Table 2 ) ( 48 – 53 , 56 , 58 , 60 – 64 ). These studies involved the purification of an allergen either from the natural source or from in vitro cultures expressing recombinant allergens.…”

“…Once the IgG epitope was identified, site-directed mutagenesis of the allergen residues involved in antibody recognition was performed, followed by IgE antibody binding analysis of the mutants, to identify IgE antibody binding sites. This approach resulted in the design and production of allergen mutants with decreased capacity to bind IgE, which are being investigated as future candidates for immunotherapy ( 51 , 58 , 67 , 73 ).…”

“…An analysis of 16 allergen-antibody structures selected from Table 2 revealed a detailed description of the interface formed by epitopes and paratopes ( 45 – 50 , 52 , 53 , 56 , 58 , 60 – 64 ). Typically, the interface area falls in the 650–920 Å 2 range (an average of 813 Å 2 ; Figure 4A ) ( 91 ).…”

“…This type of interaction is relatively common in antigen-antibody interfaces, as it is formed by aromatic residues (e.g., Phe, Tyr, Trp) and side chains of Arg or Lys, which are over-represented in epitopes and paratopes. For example, cation-π interactions were observed in interfaces formed between Bla g 2 and mAb 7C11, Der p 1 and 5H8, as well as between Der p 2 and mAb 7A1 ( 50 , 56 , 58 ). Side chains of aromatic residues may be also involved in various π-π interactions ( 56 , 105 ).…”