A potent SARS-CoV-2 neutralising nanobody shows therapeutic efficacy in the Syrian golden hamster model of COVID-19

Abstract

SARS-CoV-2 remains a global threat to human health particularly as escape mutants emerge. There is an unmet need for effective treatments against COVID-19 for which neutralizing single domain antibodies (nanobodies) have significant potential. Their small size and stability mean that nanobodies are compatible with respiratory administration. We report four nanobodies (C5, H3, C1, F2) engineered as homotrimers with pmolar affinity for the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. Crystal structures show C5 and H3 overlap the ACE2 epitope, whilst C1 and F2 bind to a different epitope. Cryo Electron Microscopy shows C5 binding results in an all down arrangement of the Spike protein. C1, H3 and C5 all neutralize the Victoria strain, and the highly transmissible Alpha (B.1.1.7 first identified in Kent, UK) strain and C1 also neutralizes the Beta (B.1.35, first identified in South Africa). Administration of C5-trimer via the respiratory route showed potent therapeutic efficacy in the Syrian hamster model of COVID-19 and separately, effective prophylaxis. The molecule was similarly potent by intraperitoneal injection.

The use of nanobodies in a sensitive ELISA test for SARS-CoV-2 Spike 1 protein

“…The use of the nanobody pairs thus gave an ELISA that is simple to use as a laboratory tool to monitor the heterologous production of Spike protein. We have shown that C5 has a potent therapeutic effect when administered topically or intraperitoneally to hamsters [14]. This, combined with our previous observation of additive neutralization between non-overlapping neutralizing agents [13], suggests that C1 or F2 could be effective in combination with C5.…”

“…The two non-overlapping epitopes used for the ELISA. In yellow space fill is a nanobody that recognizes the ACE2 RBD epitope [13,14] known as cluster 2 [15] (group 1 [18]) epitope and in blue a second nanobody [14] which recognizes a different RBD epitope, first described in the CR3022 structure [16,17], known as cluster 1 [15] (group 4 [18]) epitope.…”

“…One of these, H4, was shown to neutralize SARS-CoV-2 with nM potency [13]. We have recently reported another set of nanobodies with pM binding affinity (agents C1, F2, C5) [14]. Structural analysis of nanobodies revealed that one group (cluster 2 antibodies [15]) (H4, C5) bound to an epitope on the RBD that overlapped with the ACE2 binding epitope [13,14].…”

“…We have recently reported another set of nanobodies with pM binding affinity (agents C1, F2, C5) [14]. Structural analysis of nanobodies revealed that one group (cluster 2 antibodies [15]) (H4, C5) bound to an epitope on the RBD that overlapped with the ACE2 binding epitope [13,14]. A second group (cluster 1 antibodies [15]) (C1, F2) bound to a non-overlapping epitope at a different location on RBD [14].…”

“…Structural analysis of nanobodies revealed that one group (cluster 2 antibodies [15]) (H4, C5) bound to an epitope on the RBD that overlapped with the ACE2 binding epitope [13,14]. A second group (cluster 1 antibodies [15]) (C1, F2) bound to a non-overlapping epitope at a different location on RBD [14]. This second site was first disclosed by the potent neutralizing human antibody CR3022 [16,17].…”