Nanobodies against SARS-CoV-2 RBD from a Two-step Phage Screening of Universal and Focused Synthetic Libraries

Abstract Coronavirus disease 2019 (COVID-19) is an evolving global pandemic, and nanobody (Nb) is recognized as a potential diagnostic and therapeutic tool for infectious disease. Here, we designed and synthesized a humanized and highly diverse phage Nbs library hsNb-U (Humanized synthetic Nbs Library - Universal). We expressed and purified the SARS-CoV-2 receptor-binding domain (RBD), and screened this univeral library against the RBD protein target. Then, the CDR1 and CDR2 sequences of five leads obtained from the hsNb-U phage panning were combined with randomly mutated CDR3 to construct a targeted (focused) phage display library, hsNb-RBD, for subsequent phage panning and screening. From the obtained sequences, we expressed 45 unique anti-RBD candidate Nbs. Among the selected Nbs, eight were found to be highly expressed, and five of these show high-affinity to RBD (EC 50 less than 100nM). Finally, we found that Nb39 can compete with angiotensin converting enzyme 2 (ACE2) for binding to RBD. Overall, this two-step strategy of synthetic phage display libraries enables rapid selection of SARS-CoV-2 RBD nanobodies with potential therapeutic activity, and this two-step strategy can potentially be used for rapid discovery of Nbs against other targets.