A novel bispecific DARPin targeting Fc[gamma]RIIB and Fc[epsi]RI-bound IgE inhibits allergic responses

• Published in: Allergy (Copenhagen) 2017-08, Vol.72 (8), p.1174
• Main Authors: Zellweger, F, Gasser, P, Brigger, D, Buschor, P, Vogel, M, Eggel, A
• Format: Article
• Language: English
• Subjects: Affinity; Allergens; Allergies; Anaphylaxis; Ankyrins; Binders; Cell activation; Cloning; Degranulation; Display devices; Effector cells; Enzyme-linked immunosorbent assay; Fc receptors; Flow cytometry; Hypersensitivity; Hypersensitivity (immediate); Immunoglobulin E; Immunoglobulin G; Leukocytes (basophilic); Mast cells; Proteins; T cell receptors
• ISSN: 0105-4538; 1398-9995
• DOI: 10.1111/all.13109

Background Binding of allergen-specific IgE to its high-affinity receptor Fc[epsi]RI on basophils and mast cells is a central event in the development of allergies. Exposure of these cells to allergens induces the release of soluble mediators causing allergic symptoms. The inhibitory low-affinity IgG Fc-receptor Fc[gamma]RIIB is co-expressed on allergic effector cells and has been implicated in negative regulation of immediate hypersensitivity responses. In order to harvest the inhibitory function of this receptor, we aimed to select specific binders against Fc[gamma]RIIB and to generate a bispecific molecule simultaneously targeting Fc[gamma]RIIB and Fc[epsi]RI-bound IgE on the surface of allergic effector cells. Methods We selected Fc[gamma]RIIB-specific binding molecules from a library of designed ankyrin repeat proteins using ribosome display technology. The bispecific binding modality was generated by molecular cloning and recombinant protein expression. We determined binding characteristics on molecular and cellular levels using SPR,ELISA, and flow cytometry. The inhibitory potential of the newly described molecules was assessed in different cellular degranulation assays ex vivo and in a mouse model of passive systemic anaphylaxis. Results We demonstrate that the selected DARPin proteins recognize Fc[gamma]RIIB with high affinity. Furthermore, the bispecific binding protein successfully interferes with allergen-induced cell degranulation and efficiently inhibits systemic anaphylaxis in vivo. Mechanistically, we report that Fc[gamma]RIIB-mediated inhibition of effector cell activation requires direct ligation to an activating Fc[epsi]RI receptor. Conclusion The described bispecific DARPin protein has the ability to co-ligate Fc[gamma]RIIB with Fc[epsi]RI-bound IgE on allergic effector cells and represents an efficient dual-modality to interfere with allergic hypersensitivity reactions.