Structural analysis of human IgE monoclonal antibody epitopes on dust mite allergen Der p 2

Abstract

Background: Human IgE (hIgE) mAbs against major mite allergen Der p 2 developed using human hybridoma technology were used for IgE epitope mapping and analysis of epitopes associated with the hIgE repertoire.

Objective: We sought to elucidate the new hIgE mAb 4C8 epitope on Der p 2 and compare it to the hIgE mAb 2F10 epitope in the context of the allergenic structure of Der p 2.

Methods: X-ray crystallography was used to determine the epitope of anti-Der p 2 hIgE mAb 4C8. Epitope mutants created by targeted mutagenesis were analyzed by immunoassays and in vivo using a human high-affinity IgE receptor (FcεRIα)-transgenic mouse model of passive systemic anaphylaxis.

Results: The structure of recombinant Der p 2 with hIgE mAb 4C8 Fab was determined at 3.05 Å. The newly identified epitope region does not overlap with the hIgE mAb 2F10 epitope or the region recognized by 3 overlapping hIgE mAbs (1B8, 5D10, and 2G1). Compared with wild-type Der p 2, single or double 4C8 and 2F10 epitope mutants bound less IgE antibodies from allergic patients by as much as 93%. Human FcεRIα-transgenic mice sensitized by hIgE mAbs, which were susceptible to anaphylaxis when challenged with wild-type Der p 2, could no longer cross-link FcεRI to induce anaphylaxis when challenged with the epitope mutants.

Conclusions: These data establish the structural basis of allergenicity of 2 hIgE mAb nonoverlapping epitopes on Der p 2, which appear to make important contributions to the hIgE repertoire against Der p 2 and provide molecular targets for future design of allergy therapeutics.

Keywords: IgE; anaphylaxis; antibody; epitope; house dust mite.

Figure 1:

A. Der p 2 is shown in magenta, and the Fab heavy chain and light chain are shown in blue and green, respectively. B. Epitope residues of Der p 2 and 4C8 Fab CDR residues of heavy and light chain are in stick representation. Residue interactions are shown by black dashes. C. X-ray crystal structure of hIgE 4C8 Fab, heavy chain is shown in blue and light chain in green, with each CDR represented by different colors. D. The heavy and light chain CDRs in a space-filling representation. E. The superimposition of structures of Der p 2 in complex with 4C8 (blue), 2F10 (grey) and 7A1 (green). F. Surface representation of rDer p 2 with epitope region of 4C8 (blue), 2F10 (grey), and 7A1 (green). The overlapping residues between 2F10 and 7A1 Fab are teal.

Figure 2:

A. Total interface area of complexes Der p 2–7A1 (PDB:6OY4), Der p 2–2F10 (PDB:7MLH) and Der p 2– 4C8 Fabs (PDB:8VK1). The areas contributed by the heavy and light chains are shown in blue and green, respectively. B. H-bonds formed by heavy chain (blue) and light chain (green) in the complexes of Der p 2 with 7A1, 2F10 and 4C8 antibodies, respectively.

Figure 3:

A. Inhibition of hIgE mAb 2F10 binding to rDer p 2 by rDer p 2, 2F10 epitope mutant, 4C8 epitope mutant, or 4C8–2F10 double epitope mutant. B. Inhibition of hIgE mAb 4C8 binding to rDer p 2 by rDer p 2, 2F10 epitope mutant, 4C8 epitope mutant, or 4C8–2F10 double epitope mutant.

Figure 4:

Dose-response curves of hIgE mAb binding to Der p 2 wild-type and 4C8 epitope mutant (AAS), 2F10 epitope mutant (KKD), and double 4C8–2F10 epitope mutant (AAS-KK). Data are averages of 2–3 experiments ± SD.

Figure 5:

Inhibition of polyclonal IgE Ab binding from mite-allergic subjects to Der p 2 by 4C8 epitope mutant (AAS), 2F10 epitope mutant (KKD), and double 4C8–2F10 epitope mutant (AAS-KK). Plasma from the mite-allergic subject from which hIgE mAb 4C8 was derived is designated as “S”. Plasma from the mite-allergic subjects is designated as “PL”. Data are averages of duplicates ± SD.

Figure 6:

Human FcεRIα–transgenic mice were sensitized with human Der p 2–specific IgE mAbs 2F10 + 4C8, 2F10 + 2G1, or 2F10 + 4C8 + 2G1. Animals were challenged with rDer p 2 wildtype or mutant antigen. Anaphylaxis was monitored using an implanted temperature probe for 90 min following challenge. Time points with calculated P values <0.05 are underscored with a colored bar. Data are means ± SD of each experimental group. The number of mice (n) for each experimental group is shown in parenthesis. The number of mice in each group that succumb from anaphylaxis are indicated by an asterisk.