D1.227 - Nanobody-based anti-FcεRI antibodies for therapeutic targeting of the IgE-FcεRI complex

The IgE-FcεRI complex on mast cells and basophils is key for executing the effector functions in IgE-mediated allergic diseases. Beyond anti-IgE antibodies, there is still room for novel therapeutic strategies to inhibit the IgE-FcεRI inflammatory axis. The direct targeting of the FcεRI, is an underexplored concept for therapeutic intervention in allergic diseases, since it is imperative to disable receptor-crosslinking and cellular activation. Thus, we aimed to generate nanobodies (nbs) against the FcεRI which could potentially block IgE binding, disrupt the IgE-FcεRI interaction or target the receptor for internalization.

A llama was immunized and yeast surface display immune libraries were generated and selected via MACS and FACS. The FcεRIα ectodomains were expressed in HEK293 cells. Enriched nanobody genes were fused to IgG1 Fc domains, expressed in mammalian cells, purified by affinity chromatography and assessed via ELISA and biolayer interferometry for reactivity and kinetics of binding.

The library of 10^8 transformants was subjected to screening via MACS and FACS, resulting in pronounced reactivity and enrichment. On-yeast staining of the single clones documented high level expression of nbs on the yeast and clear-cut binding to the FcεRI. Sequence analysis revealed several unique nanobody clones with divergent CDR3s with a length of up to 20 residues. On-yeast staining further assessed binding to the FcεRIα-IgE-Fc complex, suggesting that the majority of nbs targets epitopes independent of the FcεRI-IgE interaction site. Structural predictions of epitopes also supported these analyses.

The nanobodies were converted into nb-based human IgG1 antibodies and expressed in mammalian cells at high yields and in properly assembled form. The immunoreactivity of the antibodies was verified in ELISA and kinetic analysis via BLI revealed nanomolar affinities. Cellular analyses revealed retained biological activity of the nb-based human IgG1 antibodies.

Hence, the nanobody-based anti-FcεRI antibodies established here represent novel tools for dissecting and interfering with the IgE-FcεRI complex and promising targeting moieties for broadening treatment options in allergic diseases.