Fast isolation of sub-nanomolar affinity alpaca nanobody against the Spike RBD of SARS-CoV-2 by combining bacterial display and a simple single-step density gradient selection
Valenzuela Nieto, G. E.; Jara, R.; Himelreichs, J.; Salinas, C.; Pinto, T.; Cheuquemilla, Y.; Margolles, Y.; Lopez Gonzalez del Rey, N.; Miranda Chacon, Z.; Cuevas, A.; Berking, A.; Deride, C.; Gonzalez-Moraga, S.; Mancilla, H.; Maturana, D.; Langer, A.; Toledo, J. P.; Müller, A.; Uberti, B.; Krall, P.; Ehrenfeld, P.; Blesa, J.; Chana-Cuevas, P.; Rehren, G.; Fernandez, L. A.; Rojas-Fernandez, A.
Show abstract
Despite unprecedented global efforts to rapidly develop SARS-CoV-2 treatments, in order to reduce the burden placed on health systems, the situation remains critical. Effective diagnosis, treatment, and prophylactic measures are urgently required to meet global demand: recombinant antibodies fulfill these requirements and have marked clinical potential. Here, we describe the fast-tracked development of an alpaca Nanobody specific for the receptor-binding-domain (RBD) of the SARS-CoV-2 Spike protein with therapeutic potential applicability. We present a rapid method for nanobody isolation that includes an optimized immunization regimen coupled with VHH library E. coli surface display, which allows single-step selection of high-affinity nanobodies using a simple density gradient centrifugation of the bacterial library. The selected single and monomeric Nanobody, W25, binds to the SARS-CoV-2 S RBD with sub-nanomolar affinity and efficiently competes with ACE-2 receptor binding. Furthermore, W25 potently neutralizes SARS-CoV-2 wild type and the D614G variant with IC50 values in the nanomolar range, demonstrating its potential as antiviral agent.
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