@article {Hunt2021.07.07.451375, author = {Andrew C. Hunt and James Brett Case and Young-Jun Park and Longxing Cao and Kejia Wu and Alexandra C. Walls and Zhuoming Liu and John E. Bowen and Hsien-Wei Yeh and Shally Saini and Louisa Helms and Yan Ting Zhao and Tien-Ying Hsiang and Tyler N. Starr and Inna Goreshnik and Lisa Kozodoy and Lauren Carter and Rashmi Ravichandran and Lydia B. Green and Wadim L. Matochko and Christy A. Thomson and Bastain V{\"o}geli and Antje Kr{\"u}ger-Gericke and Laura A. VanBlargan and Rita E. Chen and Baoling Ying and Adam L. Bailey and Natasha M. Kafai and Scott Boyken and Ajasja Ljubeti{\v c} and Natasha Edman and George Ueda and Cameron Chow and Amin Addetia and Nuttada Panpradist and Michael Gale, Jr and Benjamin S. Freedman and Barry R. Lutz and Jesse D. Bloom and Hannele Ruohola-Baker and Sean P. J. Whelan and Lance Stewart and Michael S. Diamond and David Veesler and Michael C. Jewett and David Baker}, title = {Multivalent designed proteins protect against SARS-CoV-2 variants of concern}, elocation-id = {2021.07.07.451375}, year = {2021}, doi = {10.1101/2021.07.07.451375}, publisher = {Cold Spring Harbor Laboratory}, abstract = {Escape variants of SARS-CoV-2 are threatening to prolong the COVID-19 pandemic. To address this challenge, we developed multivalent protein-based minibinders as potential prophylactic and therapeutic agents. Homotrimers of single minibinders and fusions of three distinct minibinders were designed to geometrically match the SARS-CoV-2 spike (S) trimer architecture and were optimized by cell-free expression and found to exhibit virtually no measurable dissociation upon binding. Cryo-electron microscopy (cryoEM) showed that these trivalent minibinders engage all three receptor binding domains on a single S trimer. The top candidates neutralize SARS-CoV-2 variants of concern with IC50 values in the low pM range, resist viral escape, and provide protection in highly vulnerable human ACE2-expressing transgenic mice, both prophylactically and therapeutically. Our integrated workflow promises to accelerate the design of mutationally resilient therapeutics for pandemic preparedness.One-Sentence Summary We designed, developed, and characterized potent, trivalent miniprotein binders that provide prophylactic and therapeutic protection against emerging SARS-CoV-2 variants of concern.Competing Interest StatementEach contributor attests that they have no competing interests relating to the subject contribution, except as disclosed. A.C.H, L.C., I.G., J.B.C., L.M., L.K., L.C., K.W., J.B., N.E., A.C.W., B.V., R.R. Y-J.P, H-W.Y., L.S., D.V., M.D., M.C.J. and D.B. are co-inventors on provisional patent applications that incorporate discoveries described in this manuscript. D.B. is a cofounder of Neoleukin Therapeutics. M.S.D. is a consultant for Inbios, Vir Biotechnology, and NGM Biopharmaceuticals and is on the Scientific Advisory Board of Moderna. H.R-B. is a Scientific Advisor of CuriBio. D.V. is a consultant for Vir Biotechnology. The Veesler lab received an unrelated sponsored research agreement from Vir Biotechnology. M.C.J. is a cofounder of SwiftScale Biologics, Stemloop, Inc., Design Pharmaceuticals, and Pearl Bio. The interests of M.C.J. are reviewed and managed by Northwestern University in accordance with their conflict of interest policies. B.S.F. is an inventor on patent applications related to kidney organoid differentiation and application. J.D.B. consults for Moderna on viral evolution and epidemiology and Flagship Labs 77 on deep mutational scanning. J.D.B. may receive a share of IP revenue as an inventor on a Fred Hutchinson Cancer Research Center-optined technology/patent (application WO2020006494) related to deep mutational scanning of viral proteins. L.G., W.M. and C.T. are current employees of Amgen and own Amgen stock. Z.L. and S.P.J.W. received unrelated sponsored research agreements from VIR Biotechnology, AbbVie, and SAB therapeutics.}, URL = {https://www.biorxiv.org/content/early/2021/07/07/2021.07.07.451375}, eprint = {https://www.biorxiv.org/content/early/2021/07/07/2021.07.07.451375.full.pdf}, journal = {bioRxiv} }