The SARS-CoV-2 virus is more transmissible than previous coronaviruses and causes a more serious illness than influenza. The SARS-CoV-2 receptor binding domain (RBD) of the spike protein binds to the human angiotensin-converting enzyme 2 (ACE2) receptor as a prelude to viral entry into the cell. Using a naive llama single-domain antibody library and PCR-based maturation, we have produced two closely related nanobodies, H11-D4 and H11-H4, that bind RBD (KD of 39 and 12 nM, respectively) and block its interaction with ACE2. Single-particle cryo-EM revealed that both nanobodies bind to all three RBDs in the spike trimer. Crystal structures of each nanobody–RBD complex revealed how both nanobodies recognize the same epitope, which partly overlaps with the ACE2 binding surface, explaining the blocking of the RBD–ACE2 interaction. Nanobody-Fc fusions showed neutralizing activity against SARS-CoV-2 (4–6 nM for H11-H4, 18 nM for H11-D4) and additive neutralization with the SARS-CoV-1/2 antibody CR3022.
Bibliographical noteFunding Information:
This work was supported by the Rosalind Franklin Institute, funding delivery partner EPSRC. PPUK is funded by the Rosalind Franklin Institute EPSRC grant no. EP/ S025243/1. J.H.N., A.L.B., P.J.H., M.W. and P.W. are supported by Wellcome Trust (100209/Z/12/Z). X-ray data were obtained using Diamond Light Source COVID-19 Rapid Access time on Beamline I03 (proposal MX27031). T.M. is supported by Cancer Research UK grants C20724/A14414 and C20724/A26752 (to C. Siebold). J.G.-J. is funded by the National Secretariat of Education (Senescyt–IFTH), Ecuador. H.M.E.D. and J. Ren are supported by the Wellcome Trust (101122/Z/13/Z) and D.I.S. by the UK Medical Research Council (MR/N00065X/1). D.I.S., P.R. and A.R.T. are funded by the Chinese Academy of Medical Sciences (CAMS) Innovation Fund for Medical Science (CIFMS), China (grant no. 2018-I2M-2-002). T.K.T. is funded by the EPA Cephalosporin Fund and The Townsend–Jeantet Charitable Trust (charity no. 1011770). The core virus neutralization facility is supported by gifts to the Oxford COVID-19 Research Response Fund. M.L.K. is supported by the Biotechnology and Biological Sciences Research Council (BBSRC; BB/M011224/1). EM results were obtained at the cryo-EM facility (OPIC) in the Division of Structural Biology, University of Oxford, part of the UK Centre of Instruct-ERIC, and the national EM facility at Diamond, eBIC, through rapid access proposal BI27051. We thank colleagues at the SGC (Oxford) mass spectrometry service for their assistance.