ABSTRACT
COVID-19 continues to be a severe public health thread worldwide. The major challenge to control this pandemic is the rapid mutation rate of the SARS-Cov-2 virus, leading to the escape of the protection of vaccines and most of the neutralizing antibodies to date. Thus, it is pivotal to develop neutralizing antibodies with broad-spectrum activity targeting multiple SARS-Cov-2 variants. In this study, we first got a synthetic nanobody (named C5) which could interfere with ACE2 binding to SARS-Cov-2 spike protein and its RBD domain. The affinity matured format of C5 clone, named C5G2, has a single digit nanomolar affinity to RBD domain and inhibit its binding to ACE2 with an IC50 of 3.7 nM. Pseudovirus assay indicated that the monovalent C5G2 could protect the cells from the infection of SARS-Cov-2 wild type virus as well as most of the virus of concern, i.e. Alpha, Beta, Gamma and Omicron variants. Strikingly, C5G2 has the highest potency against omicron among all the variants with the IC50 of 4.9ng/ml (0.3 nM). We further solved the Cryo-EM structure of C5G2 in complex with the Spike trimer. The structure data showed that C5G2 bind to RBD mainly through its CDR3 at a vast region that not overlapping with the ACE2 binding surface. Surprisingly, C5G2 also bind to a distinct epitope residing in the NTD domain of spike protein through the same CDR3 loop, which may further increase its potency against the virus infection. Thus, this bi-paratopic nanobody may be served as an effective drug for the prophylaxis and therapy of the Omicron infection.