Human IgE monoclonal antibodies define two unusual epitopes trapping dog allergen Can f 1 in different conformations

Abstract

Molecular analysis of interactions between IgE antibody and allergen allows the structural basis of IgE recognition to be defined. Human IgE (hIgE) epitopes of respiratory lipocalin allergens, including Can f 1, remain elusive due to a lack of IgE-allergen complexes. This study aims to map the structure of allergenic epitopes on Can f 1. The fragment antigen-binding (Fab) regions of Can f 1 specific human IgE monoclonal antibodies (hIgE mAb) were used to determine the structures of IgE epitopes. Epitope mutants were designed to target Can f 1 epitopes. Immunoassays and a human FcεRIα transgenic mouse model of passive anaphylaxis in vivo were used to assess the functional activity of epitope mutants. Crystal structures of natural or recombinant Can f 1 complexed with two hIgE mAb 1J11 and 12F3 Fabs, respectively, were determined. The hIgE mAb bound to two partially overlapping epitopes and recognized two different Can f 1 conformations. The hIgE mAb 12F3 showed an unusual mode of binding by protruding its heavy chain CDR3 inside the Can f 1 calyx. Epitope mutants generated based on the structural analyses displayed a 64%-89% reduction in IgE antibody binding and failed to induce passive anaphylaxis in a human FcεRIα transgenic mouse model. In summary, the structures of Can f 1-hIgE Fab complexes revealed two unique and partially overlapping epitopes on Can f 1. The modification of the identified IgE epitopes provides a pathway for the design of hypoallergens to treat dog allergies.

Keywords: Can f 1; IgE/allergen complexes; conformational epitope; dog allergen; epitope mutants; human IgE antibody; lipocalins.

FIGURE 1

X‐ray crystal structures of Can f 1‐hIgE mAb Fab complexes. (a) Cartoon and space‐filling representations of the structure of nCan f 1(magenta) bound to hIgE mAb 1J11 Fab (heavy chain in blue and light chain teal green, respectively). (b) Interface between nCan f 1 and the 1J11 Fab showing the epitope‐forming residues of nCan f 1 (magenta) interacting with the CDRs of the heavy chain (blue) and light chain (teal) of 1J11 Fab in stick diagram. H‐bonds in the interface are shown in black dash. (c) Cartoon and space‐filling representations of the structure of rCan f 1 bound to hIgE mAb 12F3 Fab (heavy and light chains are shown in green and gray, respectively). (d) Interface of 12F3‐rCan f 1 showing epitope forming residues of rCan f 1 (magenta) and 12F3 antibody (green for heavy chain and gray for light chain) in stick diagram representation. Water molecules present in the interface are shown in red sphere and H‐bond interactions are shown in black dashes. (e) The epitopes of 1J11 (blue), 12F3 (green), and partially overlapping epitope for both IgE mAbs (pale cyan) are shown on the surface of Can f 1 and (f) Can f 1 residues forming epitopes of both IgE mAbs are shown in a Venn diagram.

FIGURE 2

hIgE recognizes different conformational Can f 1N‐terminus. HIgE 1J11 binding to nCan f 1 (a) and hIgE 12F3 binding to rCan f 1 (b) shows the changed orientation of N‐terminus (shown in magenta) in each structure. (c) Superimposition of hIgE mAb 12F3 and 1J11 complexes with Can f 1 shows the orientation of the N‐termini and the distance between the residue Val11 in Can f 1 within respective complexes with hIgE mAb 12F3 and 1J11 Fab. (d) The 12F3 H‐CDR3 insertion inside the hydrophobic cavity of rCan f 1. (e) Superimposition of nCan f 1 in the 12F3‐rCan f 1 structure shows that the N‐terminus conformation as observed in nCan f 1‐1J11 interface would hinder the 12F3‐HCDR‐3 binding inside the Can f 1 cavity. (f) 1J11 H‐CDR3 binding orientation in nCan f 1 interface. (g) Superimposition of rCan f 1 in the 1J11‐nCan f 1 structure shows that the N‐terminus conformation of rCan f 1 from 12F3‐rCan f 1 hinder the 1J11 H‐CDR3 binding to Can f 1.

FIGURE 3

Inhibition Immunoassays of double epitope mutants. Inhibition immunoassays of 1J11‐12F3 double epitope mutants show the reduced ability of epitope mutants to inhibit (a) hIgE mAb 12F3 and (b) hIgE mAb 1J11 binding to wildtype Can f 1. The least inhibition of mAb binding to rCan f 1 was observed for mutant AKAA, followed by AKGA and AKA‐AAA as compared to wildtype Can f 1. The lowest percentage of inhibition exhibited by mutant AKAA implies poor interaction of this mutant with both antibodies arising from disrupted epitopes specific to these hIgE mAb.

FIGURE 4

Inhibition of polyclonal antibody (pAb) IgE binding to rCan f 1 by wildtype and mutant rCan f 1. The inhibition of polyclonal IgE from 10 dog allergic patient sera (represented by “PL”) by wildtype and mutant Can f 1. Lowered inhibition of pAb binding to rCan f 1 is shown for PL‐15 and PL‐28 for 1J11 mutant KA, and PL‐21, PL‐28, and PL‐59 for 1J11‐12F3 mutant AKAA. Data are averages of duplicates ± SD.

FIGURE 5

Murine model of passive systemic anaphylaxis. (a) Human FcεRIα transgenic mice sensitized with 100 μg total of Can f 1 specific IgE mAb 1J11 + 4F5 or 10G1 + 4F5 were challenged with 50 μg of rCan f 1 (Can f 1 WT) or 1J11 single epitope mutant KA (1J11 MUT). (b) Mice sensitized with 100 μg total of Can f 1 specific IgE mAb 1J11 + 4F5 + 12F3, 10G1 + 4F5, 12F3 + 4F5 or 1J11 + 4F5, and challenged with 1J11‐12F3 double epitope mutant AKAA (1J11/12F3 MUT) or 1J11 single epitope mutant KA (1J11 MUT), respectively. Anaphylaxis was monitored using an implanted temperature probe for 90 min following the 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 to anaphylaxis is indicated by an asterisk.