Abstract
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 IC50 in the low picomolar range.
Highlights
In addition to conventional antibodies, camelids produce heavy chain-only antibodies, which lack the light chain [1]
We reported the development of an alpaca-derived nanobody, Ty1, which binds to the SARS-CoV-2 spike receptor binding domain (RBD), potently neutralizing the virus [27]
We evaluated the different constructs in SARS-CoV-2 pseudovirus neutralization assays as well as infectious SARS-CoV-2 plaque reduction neutralization tests (PRNTs)
Summary
In addition to conventional antibodies, camelids produce heavy chain-only antibodies, which lack the light chain [1]. The antigen-binding region of these antibodies is composed of a single domain which can be expressed independently as a ~15 kDa antibody fragment and is referred to as a VHH or nanobody [2,3]. Despite being only 10% of the size of a conventional IgG antibody, and despite their monomeric nature, nanobodies can bind their target antigen with high specificity and affinity. Nanobodies are biochemically well-behaved proteins and can be expressed to high levels in yeast, E. coli, or mammalian cells [4,5]. Because of their small size, they are relatively easy to functionalize using genetic, chemical, or enzymatic strategies. Nanobodies are increasingly popular in molecular biology research, molecular imaging, immunology, as diagnostic tools or for therapeutic applications [6,7,8,9,10,11]
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