Precision engineering for localization, validation, and modification of allergenic epitopes

Abstract

The allergen-IgE interaction is essential for the genesis of allergic responses, yet investigation of the molecular basis of these interactions is in its infancy. Precision engineering has unveiled the molecular features of allergen-antibody interactions at the atomic level. High-resolution technologies, including x-ray crystallography, nuclear magnetic resonance spectroscopy, and cryo-electron microscopy, determine allergen-antibody structures. X-ray crystallography of an allergen-antibody complex localizes in detail amino acid residues and interactions that define the epitope-paratope interface. Multiple structures involving murine IgG mAbs have recently been resolved. The number of amino acids forming the epitope broadly correlates with the epitope area. The production of human IgE mAbs from B cells of allergic subjects is an exciting recent development that has for the first time enabled an actual IgE epitope to be defined. The biologic activity of defined IgE epitopes can be validated in vivo in animal models or by measuring mediator release from engineered basophilic cell lines. Finally, gene-editing approaches using the Clustered Regularly Interspaced Short Palindromic Repeats technology to either remove allergen genes or make targeted epitope engineering at the source are on the horizon. This review presents an overview of the identification and validation of allergenic epitopes by precision engineering.

Keywords: Allergens; CRISPR; IgE monoclonal antibody; allergen engineering; allergenic epitopes; nuclear magnetic resonance; x-ray crystallography.

Figure 1.

A. Crystal structure (PDB: 8DB4) of Ara h 2 in complex with two Fab fragments of antibodies 13T1 and 22S1. B. Cryo-EM structure (PDB: 7MXL) of Bet v 1 in complex with three different Fab fragments of antibodies REGN5713, REGN5714 and REGN5715. Molecules are shown in space filling representation. Heavy chains are marked using darker colors. The figure shows that there is a limited number of antibodies that can simultaneously bind to an allergen due to steric restrictions.

Figure 2.

A. Crystal structure (PDB: 7MLH) of Der p 2 in complex with Fab of the IgE mAb 2F10. Der p 2 is shown in space filling representation with 2F10 epitope colored in pink. The Fab is presented using ribbon representation. Water molecules mediating the allergen antibody interactions are shown as red spheres. B. Close view of the allergen-antibody interface. Der p 2 is shown in ribbon representation with residues forming the epitope presented in stick representation and colored pink.

Figure 3.

Plot showing epitope area versus number of residues in the epitopes from structures in Table II. There is a statistically significant correlation between both variables (r = 0.68, p < 0.001). Stars indicate that there are two different experimental models for a particular allergen-antibody pair (Bet v 1 (REGN5713) and Bet v 1 (REGN5713)* correspond to the PDB structures 7N0U and 7MXL, respectively; Bet v 1 (REGN5715) and Bet v 1 (REGN5715)* correspond to the PDB structures 7N0V and 7MXL, respectively; Der p 1 (4C1) and Der p 1 (4C1)* correspond to the PDB structures 5VPG and 5VPH, respectively; Gal d 4 (VH H04) and Gal d 4 (VH H04)* correspond to the PDB structures 4UX3 and 4PGJ, respectively, and Hev b 8 (2F5) and Hev b 8 (2F5)* correspond to the PDB structures 7SBD and 7SBG, respectively). IgE epitopes are highlighted in color (green: murine IgE mAb; blue: human IgE mAb derived from combinatorial libraries; and yellow: human IgE mAb generated by hybridoma technology).

Figure 4.

CRISPR engineering a genetic target using (top) traditional CRISPR-Cas9, resulting in gene knockout following DNA double strand break; or (bottom) CRISPR base or prime editors, which enable precision editing of a single target DNA nucleotide with the assistance of associated deaminase or reverse transcriptase enzymes. To date, CRISPR engineering has been applied to edit allergen genes in cat, goat’s milk, soybean, peanut, hen’s egg, and wheat. BLG: β-lactoglobulin. (© InBio, 2023)