Selection, biophysical and structural analysis of synthetic nanobodies that effectively neutralize SARS-CoV-2

Custódio, Tânia F.; Das, Hrishikesh; Sheward, Daniel J; Hanke, Leo; Pazicky, Samuel; Pieprzyk, Joanna; Sorgenfrei, Michèle; Schroer, Martin; Gruzinov, Andrey; Jeffries, Cy; Graewert, Melissa; Svergun, Dmitri; Dobrev, Nikolay; Remans, Kim; Seeger, Markus A.; McInerney, Gerald M; Murrell, Ben; Hällberg, B. Martin; Löw, Christian

The coronavirus SARS-CoV-2 is the cause of the ongoing COVID-19 pandemic. Therapeutic neutralizing antibodies constitute a key short-to-medium term approach to tackle COVID-19. However, traditional antibody production is hampered by long development times and costly production. Here, we report the rapid isolation and characterization of nanobodies from a synthetic library, known as sybodies (Sb), that target the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. Several binders with low nanomolar affinities and efficient neutralization activity were identified of which Sb23 displayed high affinity and neutralized pseudovirus with an IC50 of 0.6 µg/ml. A cryo-EM structure of the spike bound to Sb23 showed that Sb23 binds competitively in the ACE2 binding site. Furthermore, the cryo-EM reconstruction revealed a novel conformation of the spike where two RBDs are in the ‘up’ ACE2-binding conformation. The combined approach represents an alternative, fast workflow to select binders with neutralizing activity against newly emerging viruses.