Improving Binding Breadth and Potency of NA-targeting Nanobodies by Multivalency

Abstract

Influenza A viruses (IAVs) pose a continuous threat to global health, causing high mortality and morbidity rates worldwide each year. Neuraminidase (NA), a surface glycoprotein of IAV that plays a critical role in viral release and spreading, poses as a promising target for antivirals. While antivirals targeting NA have already been developed, there is an increasing emergence of resistance. Nanobodies, derived from heavy chain only antibodies, offer a promising alternative. Their unique characteristics, including their ability to target cryptic epitopes, increased tissue penetration and adaptability, make them suitable as antivirals. This study characterized NA-targeting nanobodies, revealing their potential as antiviral agents. N1-targeting and N2-targeting nanobodies were isolated from llamas. These nanobodies exhibited broad-reactive binding to multiple N1 or N2 proteins with ELISA-based affinities in the nanomolar to picomolar range. Most of these nanobodies demonstrated NA inhibition, as assessed by ELLA. To enhance NA binding breadth and NA inhibition, we created multivalent nanobodies with two different formats. Bivalent nanobodies were generated by fusion with a human IgG1 Fc domain, while tetravalent nanobodies were generated by fusion with a GCN4 tetramerization domain. Multimerization was found to significantly enhance NA binding, reduce dissociation rate, and enhance NA inhibition. Interestingly, tetravalent nanobodies displayed greater NA inhibition than bivalent nanobodies, showcasing the benefits of tetravalency in NA targeting. These nanobodies show promise as novel antivirals against NA for combating IAV infections. Future structural studies, epitope mapping, and additional modifications of our nanobodies can guide the development of these nanobodies as potent antiviral agents.