Pre-clinical allergenicity assessment of IgE epitope-targeted Der p 2 mutants demonstrate potential as hypoallergenic AIT candidates

Glorismer Pena-Amelunxen¹Mohadeseh Asghari¹Kriti Khatri²Jill Glesner³Serge Versteeg^4,5Ronald van Ree^4,5Martin D Chapman³Scott Smith⁶Maksymilian Chruszcz²Anna Pomés³Lorenz Aglas^1,7,8*

• ¹Department of Biosciences and Medical Biology, University of Salzburg, Salzburg, Austria
• ²Michigan State University, East Lansing, MI, United States
• ³InBio, Charlottesville, VA, United States
• ⁴Department of Experimental Immunology, Amsterdam University Medical Centers, Amsterdam, Netherlands
• ⁵Department of Otorhinolaryngology, Amsterdam University Medical Centers, Amsterdam, Netherlands
• ⁶Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, United States
• ⁷Institute of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria
• ⁸Human Microbiome (HUMI) Research Program, Faculty of Medicine, University of Helsinki, Helsinki, Finland

Background: Advancements in hybridoma technology have enabled the production of human IgE monoclonal antibodies (hIgE-mAb) for successful IgE-epitope mapping of major allergens. Here, we assessed the hypoallergenicity of three IgE-epitope mutants (single 4C8 or 2F10, and double 4C8+2F10 epitope mutants) of house dust mite allergen (HDM) Der p 2. Methods: Humanized rat basophilic leukemia (huRBL) cells, passively sensitized overnight with either pairs of Der p 2 specific hIgE-mAb (2F10, 4C8 or 2G1) or HDM-allergic serum (n=8), were stimulated with either wildtype (WT) Der p 2 or an IgE-epitope mutant and mediator release was measured. Results: No degranulation was induced upon stimulation with all mutants, when cells were sensitized with pairs of hIgE-mAb specific for at least one mutated epitope. HIgE-mAb specific for non-mutated epitopes led to mediator release comparable to WT Der p 2, indicating that epitopes recognized by the three different hIgE-mAb are not overlapping and that the 3D-structure of the mutants is conserved. The double 4C8+2F10 epitope mutant had a significantly reduced maximal mediator release (48.3%) compared to WT, in cells sensitized with allergic donor serum. Overall, the area-under-the-curve of mediator release curves induced by the mutants was significantly lower (31-65%) compared to WT. When comparing the EC20, the double 4C8+2F10 epitope mutant required a 158-fold higher antigen concentration to induce the same extent of mediator release as WT Der p 2. Conclusion: Der p 2 epitope mutants display significantly reduced allergenicity. Particularly, the double 4C8+2F10 epitope mutant demonstrated a strong potential as a novel AIT vaccine candidate.