High-Affinity IgE Recognition of a Conformational Epitope of the Major Respiratory Allergen Phl p 2 As Revealed by X-Ray Crystallography

Abstract

We report the three-dimensional structure of the complex between the major respiratory grass pollen allergen Phl p 2 and its specific human IgE-derived Fab. The Phl p 2-specific human IgE Fab has been isolated from a combinatorial library constructed from lymphocytes of a pollen allergic patient. When the variable domains of the IgE Fab were grafted onto human IgG1, the resulting Ab (huMab2) inhibited strongly the binding of allergic patients’ IgE to Phl p 2 as well as allergen-induced basophil degranulation. Analysis of the binding of the allergen to the Ab by surface plasmon resonance yielded a very low dissociation constant (KD = 1.1 × 10−10 M), which is similar to that between IgE and Fcε;RI. The structure of the Phl p 2/IgE Fab complex was determined by x-ray crystallography to 1.9 Å resolution revealing a conformational epitope (876 Å2) comprised of the planar surface of the four-stranded anti-parallel β-sheet of Phl p 2. The IgE-defined dominant epitope is discontinuous and formed by 21 residues located mostly within the β strands. Of the 21 residues, 9 interact directly with 5 of the 6 CDRs (L1, L3, H1, H2, H3) of the IgE Fab predominantly by hydrogen bonding and van der Waals interactions. Our results indicate that IgE Abs recognize conformational epitopes with high affinity and provide a structural basis for the highly efficient effector cell activation by allergen/IgE immune complexes.

Mapping of Conformational IgE Epitopes with Peptide-Specific Monoclonal Antibodies Reveals Simultaneous Binding of Different IgE Antibodies to a Surface Patch on the Major Birch Pollen Allergen, Bet v 1

“…The dissociation constant K D was calculated from corresponding k a and k d to be 8.35e-11 M for mAb2 and 1.05e-9 M for mAb12. The affinities of both mAbs are therefore comparable to those recorded for human allergen-specific IgE Abs (9,18,45).…”

“…In two recent studies, the three-dimensional structure of an allergen in complex with a corresponding human IgE Ab has been reported (9,18). These studies have indeed visualized the conformational recognition of allergens by IgE, but the technique of cocrystallization does not allow analyzing the repertoire of a polyclonal IgE response toward an allergen.…”

“…Second, it has been shown that IgE recognition of allergens can be affected by alterations of the allergen conformation (e.g., calcium depletion from calciumbinding allergens, induction of structural changes through sitedirected mutagenesis) (15)(16)(17). Two recently published structures of IgE allergen-immune complexes visualize the recognition of conformational epitopes by allergic patients IgE Abs (9,18).…”

Advances in allergen-microarray technology for diagnosis and monitoring of allergy: The MeDALL allergen-chip

“…The human monoclonal Phl p 2-specific IgG 1 was described [40,41]. Such as for IgE, a dilution series of 1 μg/ml, 500 ng/ml, 100, 10, 1, 0.4, 0.3, 0.2 and 0.1 ng/ml was prepared of the monoclonal Phl p 2-specific IgG 1 and measured in triplicates using the MeDALL allergen-chip.…”

From Molecule Studies of Allergens to Development of Immunotherapy of Allergies

“…Identification of B cell epitopes is therefore the cornerstone of a successful therapy. With the advancement of bioinformatics, B cell epitopes of most of the major allergens such as peach Pru p 3 [21], latex Hev b 2 [22], shrimp Pen a 1 [14], fish Sco j 1 [23], cow's milk α-lactalbumin [24], hazelnut and walnut 11S globulin [25], timothy grass pollen Phl p 2 [26], peanut Ara h 2 [27], and egg white ovalbumin [28] have been identified and "tailored" modifications of amino acid residues in the epitope resulting in reduced IgE affinity can easily be achieved by sitedirected mutagenesis. Today, based on the B cell epitope data, major efforts are directed to develop a safe and consistent therapeutic effect by manipulating these epitopes.…”