Monomeric Beta-Lactoglobulin Mutants for Milk Allergy Immunotherapy

The patent applies parameter changes by modifying the quaternary structure of allergenic polypeptides from dimeric to monomeric form through site-directed mutagenesis. Specific amino acid substitutions (e.g., H146P, R148P, S150P in β-lactoglobulin) alter the molecular parameters to prevent dimer formation while preserving IgE-binding capability. This structural parameter change eliminates the crosslinking function that triggers allergic reactions while maintaining the ability to induce protective IgG antibodies.

The patent segments the functional properties of the allergen by separating IgE-binding activity from dimerization capability. The monomeric mutants retain the epitope recognition function (binding to IgE) but lose the oligomerization function (forming dimers). This functional segmentation allows the hypoallergen to compete with native allergens for IgE binding without triggering the crosslinking cascade that leads to mast cell degranulation and allergic symptoms.

The patent systematically changes the molecular parameters of allergens by introducing specific point mutations at dimer interface residues. These parameter changes (amino acid substitutions) destabilize the dimeric structure and shift the equilibrium toward monomeric form. The mutations are designed based on structural analysis of dimer interfaces, allowing rational development of hypoallergens with reduced allergenicity while maintaining immunogenicity.

The patent introduces asymmetry by creating monomeric variants from naturally symmetric dimeric structures. The site-directed mutagenesis creates asymmetric modifications at the dimer interface that prevent symmetric association. This structural asymmetry disrupts the homodimer formation required for crosslinking, thereby reducing allergenic activity while preserving the single-chain IgE-binding capability necessary for immunotherapy.

The patent applies local quality by making targeted modifications only at specific locations (dimer interface residues) while leaving the rest of the protein structure unchanged. Site-directed mutagenesis introduces amino acid substitutions at precisely defined positions (e.g., residues involved in hydrophobic or hydrogen-bonding interactions at the interface) to disrupt dimerization. The IgE-binding epitopes located away from the dimer interface remain intact, preserving binding capability while eliminating allergenicity locally at the interface region.

The patent uses molecular modeling and structural analysis as intermediaries to guide the mutagenesis process. Computational prediction of dimer interface residues and their contribution to oligomerization stability serves as an intermediary step between the goal of reducing allergenicity and the actual mutagenesis experiment. This intermediary analysis identifies optimal mutation sites that maximize dimer disruption while minimizing impact on IgE-binding function, thereby improving manufacturing precision.