Pymm, PhillipRedmond, Samuel J.Dolezal, OlanMordant, FrancescaLopez, EsterCooney, James P.Davidson, Kathryn C.Haycroft, Ebene R.Tan, Chee WahSeneviratna, RebeccaGrimley, Samantha L.Purcell, Damian F.J.Kent, Stephen J.Wheatley, Adam K.Wang, Lin FaLeis, AndrewGlukhova, AlisaPellegrini, MarcChung, Amy W.Subbarao, KantaUldrich, Adam P.Tham, Wai HongGodfrey, Dale I.Gherardin, Nicholas A.2025-05-312025-05-31ORCID:/0000-0001-7950-8699/work/218987923http://www.scopus.com/inward/record.url?scp=85140254512&partnerID=8YFLogxKhttps://hdl.handle.net/1885/733756202The development of therapeutics to prevent or treat COVID-19 remains an area of intense focus. Protein biologics, including monoclonal antibodies and nanobodies that neutralize virus, have potential for the treatment of active disease. Here, we have used yeast display of a synthetic nanobody library to isolate nanobodies that bind the receptor-binding domain (RBD) of SARS-CoV-2 and neutralize the virus. We show that combining two clones with distinct binding epitopes within the RBD into a single protein construct to generate biparatopic reagents dramatically enhances their neutralizing capacity. Furthermore, the biparatopic nanobodies exhibit enhanced control over clinically relevant RBD variants that escaped recognition by the individual nanobodies. Structural analysis of biparatopic binding to spike (S) protein revealed a unique binding mode whereby the two nanobody paratopes bridge RBDs encoded by distinct S trimers. Accordingly, biparatopic nanobodies offer a way to rapidly generate powerful viral neutralizers with enhanced ability to control viral escape mutants.We thank staff from the flow cytometry facilities at the Department of Microbiology and Immunology at the Peter Doherty Institute. We thank Prof. Andrew C. Kruse (Harvard University, Boston, USA) for provision of the yeast nanobody display library. The authors acknowledge use of the Bio21 Advanced Microscopy Facility, and WEHI Information Technology Services and the WEHI Research Computing Platform for providing facilities and support that contributed to this work. This work was supported by the Jack Ma Foundation , the Ramsay Foundation , The Victorian State Government , Australia, the Medical Research Future Fund ( MRFF GNT2002073 ), the Australian Research Council (ARC; CE140100011 ) and the National Health and Medical Research Council , Australia (NHMRC; 1113293 and 1145373 ) and computational resources provided by the Australian Government through MASSIVE HPC facility ( www.massive.org.au ) under the National Computational Merit Allocation Scheme. N.A.G was supported by an ARC DECRA Fellowship ( DE210100705 ) and D.I.G was supported by an NHMRC Senior Principal Research Fellowship ( 1117766 ) and subsequently by an NHMRC investigator grant ( 2008913 ). W-H.T. is a Howard Hughes Medical Institute\u2013Wellcome Trust International Research Scholar (208693/Z/17/Z) and is support by an NHMRC Fellowship ( 1154937 ). M.P. is supported by an NHMRC Investigator Grant ( GNT1175011 ). A.G. is a CSL Centenary Fellow. K.S. is supported by an NHMRC Investigator Grant. The Melbourne WHO Collaborating Centre for Reference and Research on Influenza is supported by the Australian Government Department of Health . The work at Duke-NUS is supported by grants from Singapore National Medical Research Council ( STPRG-FY19-001 , COVID19RF-003 , COVID19RF-060 and OFLCG19May-0034 ). We thank staff from the flow cytometry facilities at the Department of Microbiology and Immunology at the Peter Doherty Institute. We thank Prof. Andrew C. Kruse (Harvard University, Boston, USA) for provision of the yeast nanobody display library. The authors acknowledge use of the Bio21 Advanced Microscopy Facility, and WEHI Information Technology Services and the WEHI Research Computing Platform for providing facilities and support that contributed to this work. This work was supported by the Jack Ma Foundation, the Ramsay Foundation, The Victorian State Government, Australia, the Medical Research Future Fund (MRFF GNT2002073), the Australian Research Council (ARC; CE140100011) and the National Health and Medical Research Council, Australia (NHMRC; 1113293 and 1145373) and computational resources provided by the Australian Government through MASSIVE HPC facility (www.massive.org.au) under the National Computational Merit Allocation Scheme. N.A.G was supported by an ARC DECRA Fellowship (DE210100705) and D.I.G was supported by an NHMRC Senior Principal Research Fellowship (1117766) and subsequently by an NHMRC investigator grant (2008913). W-H.T. is a Howard Hughes Medical Institute\u2013Wellcome Trust International Research Scholar (208693/Z/17/Z) and is support by an NHMRC Fellowship (1154937). M.P. is supported by an NHMRC Investigator Grant (GNT1175011). A.G. is a CSL Centenary Fellow. K.S. is supported by an NHMRC Investigator Grant. The Melbourne WHO Collaborating Centre for Reference and Research on Influenza is supported by the Australian Government Department of Health. The work at Duke-NUS is supported by grants from Singapore National Medical Research Council (STPRG-FY19-001, COVID19RF-003, COVID19RF-060 and OFLCG19May-0034). Conceptualization, N.A.G. and D.I.G.; methodology, N.A.G. P.P. O.D. F.M. E.L. J.P.C. K.C.D. C.W.T, L-F.W, A.L. A.G. M.P. A.W.C. and K.S.; formal analysis, N.A.G.; P.P. S.J.R. O.D. F.M. E.L. J.P.C. E.H. C.W.T. and S.L.G.; investigation, N.A.G.; P.P. S.J.R, A.G. O.D. F.M. E.L. J.P.C. E.H. C.W.T. R.S. and S.L.G.; resources, N.A.G. DIG, P.P. W-H.T. A.P.U. K.S. A.W.C. M.P. A.G. A.L. L-F.W. A.K.W. S.J.K. D.F.J.P. K.C.D. and O.D.; data curation, N.A.G. P.P. A.G. A.L. S.L.G. C.W.T. E.H. J.P.C. E.L. F.M. O.D. and S.J.R.; Writing \u2013 Original Draft, N.A.G. P.P. and D.I.G.; writing \u2013 review & editing, all authors; visualization, N.A.G. and P.P.; supervision, N.A.G, D.I.G. W-H.T, K.S. A.W.C. M.P. A.G. and D.J.P.; project administration, N.A.G. and D.I.G.; funding acquisition, N.A.G. D.I.G. W.H.T. A.W.C. A.K.W. and S.J.K. Lin-Fa Wang and Chee Wah Tan are co-inventors of the surrogate virus neutralization test commercialized under the tradename cPass by GenScript Biotech. We support inclusive, diverse, and equitable conduct of research.27enPublisher Copyright: © 2022 The Author(s)Immunologymicrobiologystructural biology2022-11-1810.1016/j.isci.2022.10525985140254512