Multivalent antibody constructs have a broad range of clinical and biotechnological applications. Nanobodies are especially useful as components for multivalent constructs as they allow increased valency while maintaining a small molecule size. We here describe a novel, rapid method for the generation of bi- and multivalent nanobody constructs with oriented assembly by Cu-free strain promoted azide-alkyne click chemistry (SPAAC). We used sortase A for ligation of click chemistry functional groups site-specifically to the C-terminus of nanobodies before creating C-to-C-terminal nanobody fusions and 4-arm polyethylene glycol (PEG) tetrameric nanobody constructs. We demonstrated the viability of this approach by generating constructs with the SARS-CoV-2 neutralizing nanobody Ty1. We compared the ability of the different constructs to neutralize SARS-CoV-2 pseudotyped virus and infectious virus in neutralization assays. The generated dimers neutralized the virus similarly to a nanobody-Fc fusion variant, while a 4-arm PEG based tetrameric Ty1 construct dramatically enhanced neutralization of SARS-CoV-2, with an IC 50 in the low picomolar range.
【저자키워드】 SARS-CoV-2, neutralization, single-domain antibody fragment, nanobody, sortase A, click chemistry, PEG linker, multivalent, 【초록키워드】 variant, virus, nanobody, viability, nanobodies, Neutralizing, small molecule, group, fusion, ligation, Infectious virus, Polyethylene glycol, functional groups, Multivalent antibody, glycol, components, component, alkyne, azide, C-terminus, neutralization assays, SARS-CoV-2 pseudotyped virus, terminal, approach, neutralize, neutralized, functional, demonstrated, promoted, creating, the SARS-CoV-2,