Linsky, Thomas W. and Vergara, Renan and Codina, Nuria and Nelson, Jorgen W. and Walker, Matthew J. and Su, Wen and Barnes, Christopher O. and Hsiang, Tien-Ying and Esser-Nobis, Katharina and Yu, Kevin and Reneer, Z. Beau and Hou, Yixuan J. and Priya, Tanu and Mitsumoto, Masaya and Pong, Avery and Lau, Uland Y. and Mason, Marsha L. and Chen, Jerry and Chen, Alex and Berrocal, Tania and Peng, Hong and Clairmont, Nicole S. and Castellanos, Javier and Lin, Yu-Ru and Josephson-Day, Anna and Baric, Ralph S. and Fuller, Deborah H. and Walkey, Carl D. and Ross, Ted M. and Swanson, Ryan and Bjorkman, Pamela J. and Gale, Michael and Blancas-Mejia, Luis M. and Yen, Hui-Ling and Silva, Daniel-Adriano (2020) De novo design of potent and resilient hACE2 decoys to neutralize SARS-CoV-2. Science, 370 (6521). pp. 1208-1214. ISSN 0036-8075. PMCID PMC7920261. doi:10.1126/science.abe0075. https://resolver.caltech.edu/CaltechAUTHORS:20201109-112630767
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Abstract
We developed a de novo protein design strategy to swiftly engineer decoys for neutralizing pathogens that exploit extracellular host proteins to infect the cell. Our pipeline allowed the design, validation, and optimization of de novo hACE2 decoys to neutralize SARS-CoV-2. The best decoy, CTC-445.2, binds with low nanomolar affinity and high specificity to the RBD of the spike protein. Cryo-EM shows that the design is accurate and can simultaneously bind to all three RBDs of a single spike protein. Because the decoy replicates the spike protein target interface in hACE2, it is intrinsically resilient to viral mutational escape. A bivalent decoy, CTC-445.2d, shows ~10-fold improvement in binding. CTC-445.2d potently neutralizes SARS-CoV-2 infection of cells in vitro and a single intranasal prophylactic dose of decoy protected Syrian hamsters from a subsequent lethal SARS-CoV-2 challenge.
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| Additional Information: | © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. This is an open-access article distributed under the terms of the Creative Commons Attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Received 31 July 2020; accepted 29 October 2020. Published online 5 November 2020. We thank M. Dougan, L. Aberman, U. Ulge, J. Rathbun, and J. Drachman for useful discussions and comments on this manuscript; Neoleukin Therapeutics, Inc. (“Neoleukin”) for supporting this work; S. Chen and A. Malyutin (Caltech) for maintaining electron microscopes; and J. Vielmetter and the Protein Expression Center in the Beckman Institute at Caltech for expression assistance. All of the computational resources for the de novo protein design were provided by Neoleukin’s high-performance “Neo” computational cluster. Funding: This work was supported by NIH grants AI145296 and AI127463 and a Department of Defense grant subcontract to M.G.; NIH grant P50 8 P50 AI150464-13 and the Caltech Merkin Institute for Translational Research to P.J.B.; the Hanna Gray Fellowship Program from the Howard Hughes Medical Institute and the Post-doctoral Enrichment Program from the Burroughs Wellcome Fund to C.O.B; NIH NIAID grant HHSN272201400006C to H.-L.Y.; and NIH grant R01 AI089728 to R.S.B. Electron microscopy was performed at the Caltech Beckman Institute Resource Center for Transmission Electron Microscopy. This project was also supported by the North Carolina Policy Collaboratory at the University of North Carolina at Chapel Hill with funding from the North Carolina Coronavirus Relief Fund established and appropriated by the North Carolina General Assembly. T.M.R. is supported by the Georgia Research Alliance as an Eminent Scholar. “Neoleukin” is a trademark of Neoleukin Therapeutics, Inc. The views and opinions expressed in this article are those of the authors and do not necessarily reflect the position of Neoleukin. Author contributions: T.W.L. designed and coordinated the research, developed computational design methods, designed de novo protein decoys of ACE2, characterized designs, and wrote the manuscript. R.V. designed de novo proteins, performed molecular biology, characterized and optimized the designs, and wrote the manuscript. N.C. designed de novo proteins, characterized and optimized the designs, and wrote the manuscript. J.W.N. designed de novo proteins, characterized and optimized the designs, performed molecular biology, performed SSM experiments, and wrote the manuscript. M.J.W. designed de novo proteins, performed molecular biology, characterized and optimized the designs, and wrote the manuscript. W.S. performed neutralization assays with the live SARS-CoV-2 virus in Vero E6 cells and edited the manuscript. C.O.B. performed cryo-EM data collection and structure solutions and analyzed the structure together with P.J.B. T.-Y.H. performed cell-neutralization assays with the live SARS-CoV-2 NanoLuc virus in Calu-3 cells. K.E.-N. performed cell-neutralization assays with the live SARS-CoV-2 NanoLuc virus in Calu-3 cells. Y.J.H. developed the nLUC reporter virus. K.Y. designed and performed ACE2 competition assays and developed methods to quantify the de novo designs in tissue lysates. T.P. designed, purified, and characterized de novo proteins. M.M. designed de novo proteins. A.P. designed de novo proteins and performed binding characterizations. U.Y.L. designed de novo proteins. M.L.M. performed pharmacokinetic studies in mice, coordinated the research for cross-reactivity binding assay, and edited the manuscript. J.C. performed pharmacokinetic studies in mice. Z.B.R. and T.M.R. performed the SARS-CoV-2 viral protection studies in hamsters. A.C. performed the ACE2 enzymatic assay and cytotoxicity assays with VeroE6. T.B. purified and characterized de novo proteins. H.P. performed mass spectrometry. N.S.C. performed molecular biology. J.Ca. developed and implemented computational tools for collaborative de novo protein design. Y.-R.L. designed de novo proteins. A.J.-D. coordinated project operations and wrote the manuscript. R.S.B. coordinated the development of the nLUC reporter virus and edited the manuscript. C.D.W. coordinated the research for ACE2 competition assays and methods to quantify the de novo designs and edited the manuscript. R.S. coordinated the research for in vitro neutralization testing, in vivo viral challenge modeling, and in vivo pharmacokinetics of the de novo proteins and edited the manuscript. D.H.F. designed the in vivo experiments. M.G. coordinated and directed the research for in vitro NanoLuc SARS-CoV-2 neutralization and edited the manuscript. L.M.B.-M. designed de novo proteins, coordinated the purification and characterization of the de novo proteins, and edited the manuscript. H.-L.Y. coordinated the research for in vitro SARS-CoV-2 neutralization and edited the manuscript. D.-A.S. generated the original idea to design the de novo decoys to neutralize SARS-CoV-2, designed the research, developed computational design selection strategies, wrote the manuscript, and directed the effort. Competing interests: T.W.L., N.C., J.W.N., and D.-A.S. are inventors on provisional patent applications for the de novo decoys described in this work. D.-A.S. and C.D.W. are cofounders of Neoleukin Therapeutics. Neoleukin authors own options and/or stock in the company. Data and materials availability: PyRosetta code used to generate initial perturbations for mobile secondary structure elements is available in the supplemental materials, appendix A. The cryo-EM maps generated from cryo-EM studies of the CTC-445.2-S 6P complex (states 1 to 4) have been deposited at the Electron Microscopy Databank (EMDB 786 http://www.emdataresource.org/) under the following accession codes: EMD-22913 (state 1), EMD-22914 (state 2), EMD-22915 (state 3), and EMD-22916 (state 4). The atomic coordinates for the CTC-445.2-S 6P complex (state 4) have been deposited at the PDB (http://www.rcsb.org/) under the accession code 7KL9. Neoleukin materials may be made available to academic noncommercial researchers through a material transfer agreement upon request. | ||||||||||||||||||||||||||||||||||||||||||||||||||||
| Group: | Richard N. Merkin Institute for Translational Research, COVID-19 | ||||||||||||||||||||||||||||||||||||||||||||||||||||
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| Issue or Number: | 6521 | ||||||||||||||||||||||||||||||||||||||||||||||||||||
| PubMed Central ID: | PMC7920261 | ||||||||||||||||||||||||||||||||||||||||||||||||||||
| DOI: | 10.1126/science.abe0075 | ||||||||||||||||||||||||||||||||||||||||||||||||||||
| Record Number: | CaltechAUTHORS:20201109-112630767 | ||||||||||||||||||||||||||||||||||||||||||||||||||||
| Persistent URL: | https://resolver.caltech.edu/CaltechAUTHORS:20201109-112630767 | ||||||||||||||||||||||||||||||||||||||||||||||||||||
| Official Citation: | De novo design of potent and resilient hACE2 decoys to neutralize SARS-CoV-2. BY THOMAS W. LINSKY, RENAN VERGARA, NURIA CODINA, JORGEN W. NELSON, MATTHEW J. WALKER, WEN SU, CHRISTOPHER O. BARNES, TIEN-YING HSIANG, KATHARINA ESSER-NOBIS, KEVIN YU, Z. BEAU RENEER, YIXUAN J. HOU, TANU PRIYA, MASAYA MITSUMOTO, AVERY PONG, ULAND Y. LAU, MARSHA L. MASON, JERRY CHEN, ALEX CHEN, TANIA BERROCAL, HONG PENG, NICOLE S. CLAIRMONT, JAVIER CASTELLANOS, YU-RU LIN, ANNA JOSEPHSON-DAY, RALPH S. BARIC, DEBORAH H. FULLER, CARL D. WALKEY, TED M. ROSS, RYAN SWANSON, PAMELA J. BJORKMAN, MICHAEL GALE JR., LUIS M. BLANCAS-MEJIA, HUI-LING YEN, DANIEL-ADRIANO SILVA. SCIENCE 04 DEC 2020 : 1208-1214; DOI: 10.1126/science.abe0075 | ||||||||||||||||||||||||||||||||||||||||||||||||||||
| Usage Policy: | No commercial reproduction, distribution, display or performance rights in this work are provided. | ||||||||||||||||||||||||||||||||||||||||||||||||||||
| ID Code: | 106510 | ||||||||||||||||||||||||||||||||||||||||||||||||||||
| Collection: | CaltechAUTHORS | ||||||||||||||||||||||||||||||||||||||||||||||||||||
| Deposited By: | George Porter | ||||||||||||||||||||||||||||||||||||||||||||||||||||
| Deposited On: | 09 Nov 2020 20:09 | ||||||||||||||||||||||||||||||||||||||||||||||||||||
| Last Modified: | 16 Nov 2021 18:54 |
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