D1.38 - A Generalizable High-Throughput Nanobody Discovery Platform for Airborne Allergens

Structural dissection of allergen–antibody interactions is essential for understanding IgE recognition and cross-reactivity. However, conventional antibody formats, including IgG- and IgE-derived Fab fragments, often face intrinsic limitations when applied to small, conformationally complex airborne allergens, including steric hindrance, limited epitope resolution, and poor suitability for high-resolution structural studies. Nanobodies (VHHs), owing to their single-domain architecture and high conformational adaptability, represent promising tools for allergen research but remain underexplored. Here, we aim to establish and validate a generalizable, high-throughput nanobody discovery and characterization platform for epitope-resolved analysis of structurally diverse airborne allergens.

An integrated and parallelized workflow was established encompassing recombinant allergen production, alpaca immunization, immune nanobody library construction, phage display selection, and systematic biochemical and biophysical characterization. Nanobody candidates were screened in parallel for allergen binding and complex stability using enzyme-linked immunosorbent assay and size-exclusion chromatography. Three clinically relevant airborne allergens with distinct molecular architectures—Der p 4 (house dust mite α-amylase), Alt a 1 (fungal major allergen), and Fel d 2 (feline serum albumin)—were employed as representative model systems to assess platform robustness and transferability.

For each allergen, a large and distinct set of nanobody binders was identified, yielding approximately 100 unique clones for Der p 4, 156 for Fel d 2, and 150 for Alt a 1, with no overlap observed between allergen-specific nanobody repertoires. Cluster analysis revealed a high degree of sequence diversity, with multiple nanobody families identified for each allergen. Distinct nanobody families exhibited diverse binding behaviors, consistent with recognition of multiple non-overlapping epitopes. From these repertoires, 5 (Der p 4), 3 (Fel d 2), and 6 (Alt a 1) representative nanobodies were selected for recombinant expression and characterization. All selected nanobodies specifically recognized the native allergen and formed stable, monodisperse complexes. Structural modeling suggested epitope-level diversity, supporting broad coverage of spatially distinct regions on the allergen surface.

This study establishes a robust, transferable, and high-throughput nanobody discovery platform for airborne allergens. By enabling systematic generation of stable, epitope-specific binders across allergens with divergent molecular sizes, folds, and disulfide architectures, this approach demonstrates broad applicability without reliance on allergen-specific optimization. Nanobodies thus provide versatile molecular probes for mechanistic and structural allergology, with broad potential applications in molecular diagnostics and allergy research.