Multivalent designed proteins protect against SARS-CoV-2 variants of concern

Hunt, Andrew C.; Case, James Brett; Park, Young-Jun; Cao, Longxing; Wu, Kejia; Walls, Alexandra C.; Liu, Zhuoming; Bowen, John E.; Yeh, Hsien-Wei; Saini, Shally; Helms, Louisa; Zhao, Yan Ting; Hsiang, Tien-Ying; Starr, Tyler N.; Goreshnik, Inna; Kozodoy, Lisa; Carter, Lauren; Ravichandran, Rashmi; Green, Lydia B.; Matochko, Wadim L.; Thomson, Christy A.; Vögeli, Bastain; Krüger-Gericke, Antje; VanBlargan, Laura A.; Chen, Rita E.; Ying, Baoling; Bailey, Adam L.; Kafai, Natasha M.; Boyken, Scott; Ljubetič, Ajasja; Edman, Natasha; Ueda, George; Chow, Cameron; Addetia, Amin; Panpradist, Nuttada; Gale, Michael; Freedman, Benjamin S.; Lutz, Barry R.; Bloom, Jesse D.; Ruohola-Baker, Hannele; Whelan, Sean P. J.; Stewart, Lance; Diamond, Michael S.; Veesler, David; Jewett, Michael C.; Baker, David

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 IC(50) 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.