Bispecific nanobodies targeting Bet v 1 and ICAM-1 decrease respiratory epithelial penetration of Bet v 1.

BACKGROUND: The major birch pollen allergen Bet v 1 primarily affects the nose and eyes of allergic patients. After crossing the epithelial barrier, it causes IgE-mediated effector cell release, triggering inflammatory responses. We sought to develop bispecific nanobodies that bind Bet v 1 on mucosal surfaces to prevent allergen penetration and subsequent activation of effector cells. Intercellular adhesion molecule 1 (ICAM-1), highly upregulated on epithelial cells of allergic individuals, was chosen as an anchor. METHODS: Genetically fused nanobodies targeting Bet v 1 and ICAM-1 were generated and characterized for specificity, affinity, Bet v 1 immobilization on the surface of the human bronchial epithelial cell line, 16HBE14o-, and their potential to inhibit Bet v 1 penetration through a cell monolayer. RESULTS: Bispecific nanobodies recognized ICAM-1, Bet v 1 and related tree pollen allergens (Aln g 1, Cor a 1). While we observed a clear co-localization of nanobodies and Bet v 1 on the 16HBE14o-cell surface after 30 min, Bet v 1 was slightly internalized after 4h at 32 °C. Dissociation rate constants between bispecific nanobodies and antigens were 3-6 times faster at 32 °C compared to 25 °C. Bispecific nanobodies reduced Bet v 1 penetration and hence decreased basophil activation. CONCLUSION: We generated bivalent nanobodies specific for ICAM-1 and Bet v 1. These nanobodies diminished trans-epithelial transport of Bet v 1 by ICAM-1-mediated immobilization at the cell surface. Reduced Bet v 1 penetration resulted in decreased basophil activation, demonstrating the potential of local administration of nanobodies for the prevention of pollen allergy.