New variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continue to arise and prolong the coronavirus disease 2019 (COVID-19) pandemic. Here, we used a cell-free expression workflow to rapidly screen and optimize constructs containing multiple computationally designed miniprotein inhibitors of SARS-CoV-2. We found the broadest efficacy was achieved with a homotrimeric version of the 75-residue angiotensin-converting enzyme 2 (ACE2) mimic AHB2 (TRI2-2) designed to geometrically match the trimeric spike architecture. Consistent with the design model, in the cryo-electron microscopy structure TRI2-2 forms a tripod at the apex of the spike protein that engaged all three receptor binding domains simultaneously. TRI2-2 neutralized Omicron (B.1.1.529), Delta (B.1.617.2), and all other variants tested with greater potency than the monoclonal antibodies used clinically for the treatment of COVID-19. TRI2-2 also conferred prophylactic and therapeutic protection against SARS-CoV-2 challenge when administered intranasally in mice. Designed miniprotein receptor mimics geometrically arrayed to match pathogen receptor binding sites could be a widely applicable antiviral therapeutic strategy with advantages over antibodies in greater resistance to viral escape and antigenic drift, and advantages over native receptor traps in lower chances of autoimmune responses.

Multivalent designed proteins neutralize SARS-CoV-2 variants of concern and confer protection against infection in mice

Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL, 60208, USA

Department of Biochemistry, University of Washington, Seattle, WA, 98195, USA

Institute for Protein Design, University of Washington, Seattle, WA, 98195, USA

Howard Hughes Medical Institute, University of Washington, Seattle, WA, 98195, USA

Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO, 63110, USA

Institute for Stem Cell and Regenerative Medicine, University of Washington School of Medicine, Seattle, WA, 98109, USA

Division of Nephrology, Department of Medicine, University of Washington School of Medicine, Seattle, WA, 98109, USA

Kidney Research Institute, University of Washington School of Medicine, Seattle, WA, 98109, USA

Department of Laboratory Medicine and Pathology, University of Washington School of Medicine, Seattle, WA, 98109, USA

Oral Health Sciences, School of Dentistry, University of Washington, Seattle, WA, 98195, USA

Department of Immunology, Center for Innate Immunity and Immune Disease, University of Washington, Seattle, WA, 98195, USA

Basic Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, WA, 98109, USA

Amgen Research, Biologic Discovery, Burnaby, V5A 1V7, BC, Canada

Invizyne Technologies Inc., Monrovia, CA, 91016, USA

Department of Pathology &amp; Immunology, Washington University School of Medicine, St. Louis, MO, 63110, USA

Department of Pathology &amp; Laboratory Medicine, University of Wisconsin – Madison, Madison, WI, 53705, USA

Department for Synthetic Biology and Immunology, National Institute of Chemistry, Ljubljana, SI-1000, Slovenia

Molecular and Cellular Biology Graduate Program, University of Washington, Seattle, WA, 98195, USA

USA Medical Scientist Training Program, University of Washington, Seattle, WA, 98195, USA

Neolukin Therapeutics Inc., Seattle, WA, 98102, USA

The Molecular and Cellular Biology Program, University of Washington, Seattle, WA, 98195, USA

Department of Bioengineering, University of Washington, Seattle, WA, 98195, USA

Department of Genome Sciences, University of Washington, Seattle, WA, 98195, USA

Andrew M. and Jane M. Bursky Center for Human Immunology and Immunotherapy Programs, Washington University School of Medicine, St. Louis, MO, 63110, USA

Chemistry of Life Processes Institute, Northwestern University, Evanston, IL, 60208, USA

Robert H. Lurie Comprehensive Cancer Center, Northwestern University, Chicago, IL, 60611, USA