Bi-paratopic and multivalent VH domains block ACE2 binding and neutralize SARS-CoV-2

QCRG Structural Biology Consortium; CryoEM grid freezing/collection team; CryoEM data processing team; Mammalian cell expression team; Protein purification team; Crystallography team; Bacterial expression team; Infrastructure team; Leadership team

doi:10.1038/s41589-020-00679-1

Bi-paratopic and multivalent VH domains block ACE2 binding and neutralize SARS-CoV-2

QCRG Structural Biology Consortium, CryoEM grid freezing/collection team, CryoEM data processing team, Mammalian cell expression team, Protein purification team, Crystallography team, Bacterial expression team, Infrastructure team, Leadership team

Pharmacology

Research output: Contribution to journal › Article › peer-review

69 Scopus citations

Abstract

Neutralizing agents against SARS-CoV-2 are urgently needed for the treatment and prophylaxis of COVID-19. Here, we present a strategy to rapidly identify and assemble synthetic human variable heavy (VH) domains toward neutralizing epitopes. We constructed a VH-phage library and targeted the angiotensin-converting enzyme 2 (ACE2) binding interface of the SARS-CoV-2 Spike receptor-binding domain (Spike-RBD). Using a masked selection approach, we identified VH binders to two non-overlapping epitopes and further assembled these into multivalent and bi-paratopic formats. These VH constructs showed increased affinity to Spike (up to 600-fold) and neutralization potency (up to 1,400-fold) on pseudotyped SARS-CoV-2 virus when compared to standalone VH domains. The most potent binder, a trivalent VH, neutralized authentic SARS-CoV-2 with a half-maximal inhibitory concentration (IC₅₀) of 4.0 nM (180 ng ml⁻¹). A cryo-EM structure of the trivalent VH bound to Spike shows each VH domain engaging an RBD at the ACE2 binding site, confirming our original design strategy. [Figure not available: see fulltext.]

Original language	English (US)
Pages (from-to)	113-121
Number of pages	9
Journal	Nature Chemical Biology
Volume	17
Issue number	1
DOIs	https://doi.org/10.1038/s41589-020-00679-1
State	Published - Jan 2021

Funding

We thank members of the Wells Lab, particularly those working on COVID-19 projects, for their efforts and contributions. Specifically, we would like to thank J. Gramespacher for purifying binders and M. Nix (UCSF) for guidance on pseudotyped virus neutralization assays. Additionally, we thank the entire QCRG for its rapid large-scale collaborative effort. Specifically, we thank C. Puchades and C. Azumaya for their efforts in the optimization of cryo-EM grid freezing and data collection, T. Owens for assistance in protein purification, D. Diwanji for expression of Secto and A. Manglik (UCSF) for advice on cryo-EM experiments and protein purification. We also thank the laboratory of P. Kim (Stanford University) for providing plasmids for pseudotyped virus production. Finally, we thank M. Wilson, C. Chiu and R. Loudermilk (UCSF), as well as the patients, for providing convalescent sera. J.A.W. is supported by generous grants from NCI (R35 GM122451-01); the Chan-Zuckerberg Biohub, UCSF Program for Breakthrough Biomedical Research (PBBR); Fast Grants from Emergent Ventures at the Mercatus Center, George Mason University (#2154); and funding from The Harrington Discovery Institute (GA33116). S.A.L. is a Merck Fellow of the Helen Hay Whitney Foundation. K.S. is a Fellow of the Helen Hay Whitney Foundation. X.X.Z. is a Merck Fellow of the Damon Runyon Cancer Research Foundation, DRG-2297-17. J.R.B. and J.Z. are supported by the National Institutes of Health National Cancer Institute (F32 5F32CA239417 (J.R.B.) and 5F32CA236151-02 (J.Z.)). B.S.Z. is supported by the National Institutes of Health National Cancer Institute (T32, HL007185). N.J.R. and I.L. are supported by the National Science Foundation (GRFP). D.P.N. was the Connie and Bob Lurie Fellow of the Damon Runyon Cancer Research Foundation (DRG-2204-14) and supported by a UCSF-PBBR Postdoctoral Independent Research Award, which is partially funded by the Sandler Foundation. QCRG Structural Biology Consortium. In addition to those listed explicitly in the Author contributions, the structural biology portion of this work was performed by the QCRG (Quantitative Biosciences Institute Coronavirus Research Group) Structural Biology Consortium. Listed below are the contributing members of the consortium listed by teams in order of team relevance to the published work. The team leads (responsible for organization of each team and for the experimental design utilized within each team) are as follows: Cryo-EM grid freezing/collection team—Caleigh M. Azumaya, Cristina Puchades, Ming Sun; Cryo-EM data processing team—Miles Sasha Dickinson, Henry C. Nguyen; Mammalian cell expression team—Christian Billesboelle, Melody G. Campbell, Devan Diwanji, Carlos Nowotny, Amber M. Smith, Jianhua Zhao; Protein purification team—Daniel Asarnow, Michelle Moritz, Tristan W. Owens, Sergei Pourmal; Crystallography team—Nadia Herrera, Huong T. Kratochvil, Ursula Schulze-Gahmen, Iris D. Young; Bacterial expression team—Amy Diallo, Meghna Gupta, Erron W. Titus; Leadership team—Oren S. Rosenberg, Kliment A Verba. The QCRG Structural Biology Consortium has received support from the Quantitative Biosciences Institute, the Defense Advanced Research Projects Agency HR0011-19-2-0020 (to D.A.A. and K.A.V.; B. Shoichet PI), a FastGrants COVID19 grant (K.A. Verba PI), the Laboratory for Genomics Research (O.S. Rosenberg PI) and the Laboratory For Genomics Research (R.M. Stroud PI).

ASJC Scopus subject areas

Molecular Biology
Cell Biology

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10.1038/s41589-020-00679-1

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QCRG Structural Biology Consortium, CryoEM grid freezing/collection team, CryoEM data processing team, Mammalian cell expression team, Protein purification team, Crystallography team, Bacterial expression team, Infrastructure team, & Leadership team (2021). Bi-paratopic and multivalent VH domains block ACE2 binding and neutralize SARS-CoV-2. Nature Chemical Biology, 17(1), 113-121. https://doi.org/10.1038/s41589-020-00679-1

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abstract = "Neutralizing agents against SARS-CoV-2 are urgently needed for the treatment and prophylaxis of COVID-19. Here, we present a strategy to rapidly identify and assemble synthetic human variable heavy (VH) domains toward neutralizing epitopes. We constructed a VH-phage library and targeted the angiotensin-converting enzyme 2 (ACE2) binding interface of the SARS-CoV-2 Spike receptor-binding domain (Spike-RBD). Using a masked selection approach, we identified VH binders to two non-overlapping epitopes and further assembled these into multivalent and bi-paratopic formats. These VH constructs showed increased affinity to Spike (up to 600-fold) and neutralization potency (up to 1,400-fold) on pseudotyped SARS-CoV-2 virus when compared to standalone VH domains. The most potent binder, a trivalent VH, neutralized authentic SARS-CoV-2 with a half-maximal inhibitory concentration (IC50) of 4.0 nM (180 ng ml−1). A cryo-EM structure of the trivalent VH bound to Spike shows each VH domain engaging an RBD at the ACE2 binding site, confirming our original design strategy. [Figure not available: see fulltext.]",

author = "{QCRG Structural Biology Consortium} and {CryoEM grid freezing/collection team} and {CryoEM data processing team} and {Mammalian cell expression team} and {Protein purification team} and {Crystallography team} and {Bacterial expression team} and {Infrastructure team} and {Leadership team} and Bracken, {Colton J.} and Lim, {Shion A.} and Paige Solomon and Rettko, {Nicholas J.} and Nguyen, {Duy P.} and Zha, {Beth Shoshana} and Kaitlin Schaefer and Byrnes, {James R.} and Jie Zhou and Irene Lui and Jia Liu and Katarina Pance and Azumaya, {Caleigh M.} and Braxton, {Julian R.} and Brilot, {Axel F.} and Meghna Gupta and Fei Li and Lopez, {Kyle E.} and Arthur Melo and Merz, {Gregory E.} and Frank Moss and Joana Paulino and Pospiech, {Thomas H.} and Sergei Pourmal and Cristina Puchades and Rizo, {Alexandrea N.} and Smith, {Amber M.} and Ming Sun and Thomas, {Paul V.} and Feng Wang and Zanlin Yu and Daniel Asarnow and Braxton, {Julian R.} and Campbell, {Melody G.} and Chio, {Cynthia M.} and Chio, {Un Seng} and Dickinson, {Miles Sasha} and Devan Diwanji and Bryan Faust and Nick Hoppe and Mingliang Jin and Fei Li and Junrui Li and Yanxin Liu and Merz, {Gregory E.} and Nguyen, {Henry C.} and Joana Paulino and Pospiech, {Thomas H.} and Sergei Pourmal and Smriti Sangwan",

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year = "2021",

month = jan,

doi = "10.1038/s41589-020-00679-1",

language = "English (US)",

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QCRG Structural Biology Consortium, CryoEM grid freezing/collection team, CryoEM data processing team, Mammalian cell expression team, Protein purification team, Crystallography team, Bacterial expression team, Infrastructure team & Leadership team 2021, 'Bi-paratopic and multivalent VH domains block ACE2 binding and neutralize SARS-CoV-2', Nature Chemical Biology, vol. 17, no. 1, pp. 113-121. https://doi.org/10.1038/s41589-020-00679-1

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N2 - Neutralizing agents against SARS-CoV-2 are urgently needed for the treatment and prophylaxis of COVID-19. Here, we present a strategy to rapidly identify and assemble synthetic human variable heavy (VH) domains toward neutralizing epitopes. We constructed a VH-phage library and targeted the angiotensin-converting enzyme 2 (ACE2) binding interface of the SARS-CoV-2 Spike receptor-binding domain (Spike-RBD). Using a masked selection approach, we identified VH binders to two non-overlapping epitopes and further assembled these into multivalent and bi-paratopic formats. These VH constructs showed increased affinity to Spike (up to 600-fold) and neutralization potency (up to 1,400-fold) on pseudotyped SARS-CoV-2 virus when compared to standalone VH domains. The most potent binder, a trivalent VH, neutralized authentic SARS-CoV-2 with a half-maximal inhibitory concentration (IC50) of 4.0 nM (180 ng ml−1). A cryo-EM structure of the trivalent VH bound to Spike shows each VH domain engaging an RBD at the ACE2 binding site, confirming our original design strategy. [Figure not available: see fulltext.]

AB - Neutralizing agents against SARS-CoV-2 are urgently needed for the treatment and prophylaxis of COVID-19. Here, we present a strategy to rapidly identify and assemble synthetic human variable heavy (VH) domains toward neutralizing epitopes. We constructed a VH-phage library and targeted the angiotensin-converting enzyme 2 (ACE2) binding interface of the SARS-CoV-2 Spike receptor-binding domain (Spike-RBD). Using a masked selection approach, we identified VH binders to two non-overlapping epitopes and further assembled these into multivalent and bi-paratopic formats. These VH constructs showed increased affinity to Spike (up to 600-fold) and neutralization potency (up to 1,400-fold) on pseudotyped SARS-CoV-2 virus when compared to standalone VH domains. The most potent binder, a trivalent VH, neutralized authentic SARS-CoV-2 with a half-maximal inhibitory concentration (IC50) of 4.0 nM (180 ng ml−1). A cryo-EM structure of the trivalent VH bound to Spike shows each VH domain engaging an RBD at the ACE2 binding site, confirming our original design strategy. [Figure not available: see fulltext.]

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Bi-paratopic and multivalent VH domains block ACE2 binding and neutralize SARS-CoV-2

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