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[Preprint]. 2021 Jul 7:2021.07.07.451375.

doi: 10.1101/2021.07.07.451375.

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

Andrew C Hunt^#^{1

2}, James Brett Case^#³, Young-Jun Park^#⁴, Longxing Cao^#^{4

5}, Kejia Wu^#^{4

5}, Alexandra C Walls^#⁴, Zhuoming Liu⁶, John E Bowen⁴, Hsien-Wei Yeh^{4

5}, Shally Saini^{4

7}, Louisa Helms^{8

7}, Yan Ting Zhao^{4

7

9}, Tien-Ying Hsiang¹⁰, Tyler N Starr¹¹, Inna Goreshnik^{4

5}, Lisa Kozodoy^{4

5}, Lauren Carter^{4

5}, Rashmi Ravichandran^{4

5}, Lydia B Green¹², Wadim L Matochko¹², Christy A Thomson¹², Bastain Vögeli^{1

2}, Antje Krüger-Gericke^{1

2}, Laura A VanBlargan³, Rita E Chen^{3

13}, Baoling Ying³, Adam L Bailey¹³, Natasha M Kafai^{3

13}, Scott Boyken^{4

5}, Ajasja Ljubetič^{4

5}, Natasha Edman^{4

5}, George Ueda^{4

5}, Cameron Chow^{4

5}, Amin Addetia^{4

14}, Nuttada Panpradist¹⁵, Michael Gale Jr¹⁰, Benjamin S Freedman^{8

7}, Barry R Lutz¹⁵, Jesse D Bloom^{11

16

17}, Hannele Ruohola-Baker^{4

7}, Sean P J Whelan⁶, Lance Stewart^{4

5}, Michael S Diamond^{3

13

6

18}, David Veesler⁴, Michael C Jewett^{1

2

19

20}, David Baker^{4

5

16}

Affiliations

¹ Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL, 60208, USA.
² Center for Synthetic Biology, Northwestern University, Evanston, IL, 60208, USA.
³ Department of Medicine, Washington University School of Medicine, St. Louis, MO, 63110, USA.
⁴ Department of Biochemistry, University of Washington, Seattle, WA, 98195, USA.
⁵ Institute for Protein Design, University of Washington, Seattle, WA, 98195, USA.
⁶ Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO, 63110, USA.
⁷ Institute for Stem Cell and Regenerative Medicine, University of Washington School of Medicine, Seattle, WA, 98109, USA.
⁸ Division of Nephrology and Kidney Research Institute, Department of Medicine, University of Washington School of Medicine, Seattle, WA, 98109, USA.
⁹ Oral Health Sciences, School of Dentistry, University of Washington, Seattle, WA, 98195, USA.
¹⁰ Department of Immunology, Center for Innate Immunity and Immune Disease, University of Washington, Seattle, WA, 98195, USA.
¹¹ Basic Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, WA, 98109, USA.
¹² Amgen Research, Biologic Discovery, Burnaby, BC, Canada.
¹³ Department of Pathology & Immunology, Washington University School of Medicine, St. Louis, MO, 63110, USA.
¹⁴ The Molecular and Cellular Biology Program, University of Washington, Seattle, WA, 98195, USA.
¹⁵ Department of Bioengineering, University of Washington, Seattle, WA, 98195, USA.
¹⁶ Howard Hughes Medical Institute, University of Washington, Seattle, WA, 98195, USA.
¹⁷ Department of Genome Sciences, University of Washington, Seattle, WA, 98195, USA.
¹⁸ Andrew M. and Jane M. Bursky Center for Human Immunology and Immunotherapy Programs, Washington University School of Medicine, St. Louis, MO, 63110, USA.
¹⁹ Chemistry of Life Processes Institute, Northwestern University, Evanston, IL, 60208, USA.
²⁰ Robert H. Lurie Comprehensive Cancer Center, Northwestern University, Chicago, IL, 60611, USA.

^# Contributed equally.

PMID: 34268509
PMCID: PMC8282097
DOI: 10.1101/2021.07.07.451375

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

Andrew C Hunt et al. bioRxiv. 2021.

[Preprint]. 2021 Jul 7:2021.07.07.451375.

doi: 10.1101/2021.07.07.451375.

Authors

Affiliations

¹ Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL, 60208, USA.
² Center for Synthetic Biology, Northwestern University, Evanston, IL, 60208, USA.
³ Department of Medicine, Washington University School of Medicine, St. Louis, MO, 63110, USA.
⁴ Department of Biochemistry, University of Washington, Seattle, WA, 98195, USA.
⁵ Institute for Protein Design, University of Washington, Seattle, WA, 98195, USA.
⁶ Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO, 63110, USA.
⁷ Institute for Stem Cell and Regenerative Medicine, University of Washington School of Medicine, Seattle, WA, 98109, USA.
⁸ Division of Nephrology and Kidney Research Institute, Department of Medicine, University of Washington School of Medicine, Seattle, WA, 98109, USA.
⁹ Oral Health Sciences, School of Dentistry, University of Washington, Seattle, WA, 98195, USA.
¹⁰ Department of Immunology, Center for Innate Immunity and Immune Disease, University of Washington, Seattle, WA, 98195, USA.
¹¹ Basic Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, WA, 98109, USA.
¹² Amgen Research, Biologic Discovery, Burnaby, BC, Canada.
¹³ Department of Pathology & Immunology, Washington University School of Medicine, St. Louis, MO, 63110, USA.
¹⁴ The Molecular and Cellular Biology Program, University of Washington, Seattle, WA, 98195, USA.
¹⁵ Department of Bioengineering, University of Washington, Seattle, WA, 98195, USA.
¹⁶ Howard Hughes Medical Institute, University of Washington, Seattle, WA, 98195, USA.
¹⁷ Department of Genome Sciences, University of Washington, Seattle, WA, 98195, USA.
¹⁸ Andrew M. and Jane M. Bursky Center for Human Immunology and Immunotherapy Programs, Washington University School of Medicine, St. Louis, MO, 63110, USA.
¹⁹ Chemistry of Life Processes Institute, Northwestern University, Evanston, IL, 60208, USA.
²⁰ Robert H. Lurie Comprehensive Cancer Center, Northwestern University, Chicago, IL, 60611, USA.

^# Contributed equally.

PMID: 34268509
PMCID: PMC8282097
DOI: 10.1101/2021.07.07.451375

Update in

Multivalent designed proteins neutralize SARS-CoV-2 variants of concern and confer protection against infection in mice.
Hunt AC, Case JB, Park YJ, Cao L, Wu K, Walls AC, Liu Z, Bowen JE, Yeh HW, Saini S, Helms L, Zhao YT, Hsiang TY, Starr TN, Goreshnik I, Kozodoy L, Carter L, Ravichandran R, Green LB, Matochko WL, Thomson CA, Vögeli B, Krüger A, VanBlargan LA, Chen RE, Ying B, Bailey AL, Kafai NM, Boyken SE, Ljubetič A, Edman N, Ueda G, Chow CM, Johnson M, Addetia A, Navarro MJ, Panpradist N, Gale M Jr, Freedman BS, Bloom JD, Ruohola-Baker H, Whelan SPJ, Stewart L, Diamond MS, Veesler D, Jewett MC, Baker D. Hunt AC, et al. Sci Transl Med. 2022 May 25;14(646):eabn1252. doi: 10.1126/scitranslmed.abn1252. Epub 2022 May 25. Sci Transl Med. 2022. PMID: 35412328 Free PMC article.

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 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.

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.

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Figures

**Fig. 1.. Multivalent minibinders exhibit unusually slow dissociation rates upon binding to the prefusion SARS-CoV-2-S glycoprotein.**
(**A, B**) Dissociation of the minibinder construct complexed with either the receptor binding domain (RBD) (A) or S trimer (S6P) (B) was monitored via competition with 100-fold molar excess of untagged MON1 using AlphaLISA (Mean ± SEM, n = 3 replicates from a single experiment).

**Fig. 2.. CryoEM structures of multivalent minibinders in complex with the SARS-CoV-2 S6P glycoprotein.**
(A) CryoEM map of TRI2–2 in complex with the S6P in two orthogonal orientations. (B) Zoomed-in view of the TRI2–2 and S6P complex interface obtained using local refinement of the RBD and TRI2–2. The RBD and MON2 built at 2.9 Å resolution are shown in yellow and blue, respectively. (C) CryoEM map of FUS31-G10 in complex with two RBDs. (D) CryoEM map of FUS231-P24 in complex with three RBDs. (E) Negative-stain EM map of FUS231-G10 in complex with S6P. The S trimer and minibinder models are placed in the whole map by rigid body fitting. EM density is shown as a transparent gray surface with a fitted atomic model. Spike protomers (PDB 7JZL) are shown in yellow, blue, and pink. Minibinders (PDB 7JZU, 7JZM, and MON2 atomic structure in this study) are shown in orange.

**Fig. 3.. FUS231-P12 enables detection of SARS-CoV-2 S trimer via BRET.**
(A) Schematic representation of the BRET sensor, teluc-FUS231-P12-mCyRFP3, to detect S trimer. (B) Luminescence emission spectra and image of the BRET sensor (100 pM) in the presence (orange trace, 100 pM) and absence (blue trace) of S2P. Emission color change was observed using a mobile phone camera (inset top right). (C) Titration of S2P with 100 pM sensor protein (Mean ± SEM, n = 3 replicates from a single experiment).

**Fig. 4.. Multivalency enhances both the breadth and potency of neutralization against SARS-CoV-2 variants by minibinders.**
(A) Dissociation of minibinder constructs from S6P variants after 24 hours was measured via competition with untagged TRI2–1 using AlphaLISA (mean, n = 3 replicates from a single experiment). Cells containing an X indicate insufficient signal in the no competitor condition to quantify the fraction of protein bound. (B) Competition of minibinder constructs with ACE2 for S6P measured via ELISA (mean, n = 2). (C) Neutralization of SARS-CoV-2 pseudovirus variants by minibinder constructs (mean, n = 2). (D) Neutralization of authentic SARS-CoV-2 by minibinder constructs (mean, n = 2). (E) Table summarizing neutralization potencies of multivalent minibinder constructs against SARS-CoV-2 pseudovirus variants. N/A indicates an IC₅₀ value above the tested concentration range and an IC₅₀ greater than 50,000 pM. (F) Table summarizing neutralization potencies of multivalent minibinder constructs against authentic SARS-CoV-2 variants. (G) Neutralization of B.1.351 SARS-CoV-2 variant by minibinder constructs (0.3 μM) in human kidney organoids (n = 3 to 12: Kruskal-Wallis test: ** P < 0.01, *** P < 0.001). (H) Relative gene expression of SARS-CoV-2 envelope protein (SARS-CoV2-E) in kidney organoids post viral infection with and without multivalent mini binders (0.3 μM) (n = 3 to 15: Kruskal-Wallis test: * P < 0.05, *** P < 0.001).

**Fig. 5.. Top multivalent minibinder candidates are escape resistant and protect mice from SARS-CoV-2 infection via pre- and post-exposure intranasal administration.**
(A) Plaque assays were performed to isolate VSV-SARS-CoV-2 chimera virus escape mutants against a control neutralizing antibody (2B04) and the FUS231-P12 and TRI2–2 multivalent minibinders. Images are representative of 36 replicate wells per multivalent minibinder. Large plaques, highlighted by black arrows, are indicative of escape. (B) Table summarizing the results of the viral escape screen. (**C-E**) K18-hACE2-transgenic mice (n = 6/timepoint) were dosed with 50 μg of the indicated minibinder by i.n. administration (50 μl total) 24 h prior (D-1) to infection with 10³ focus forming units of SARS-CoV-2 variants B.1.1.7, Wash-B.1.351, or Wash-P.1 i.n. on Day 0. (D) Daily weight-change following inoculation (mean ± SEM; n = 6, two-way ANOVA with Sidak’s post-test: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001). (E) At 6 days post infection (6 dpi) animals (n = 6/timepoint) were sacrificed and analyzed for the presence of SARS-CoV-2 viral RNA by RT-qPCR in the lung, heart, spleen, brain, or nasal wash (n = 6: Kruskal-Wallis test: ns, not significant, * P < 0.05, ** P < 0.01, *** P < 0.001). (**F-H**) K18-hACE2-transgenic mice (n = 6/timepoint) were dosed with 50 μg of the indicated minibinder by i.n. administration (50 μl total) 24 h after (D+1) infection with 10³ focus forming units of the SARS-CoV-2 Wash-B.1.351 variant on Day 0. (G) Daily weight-change following inoculation (mean ± SEM; n = 6, two-way ANOVA with Sidak’s post-test: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001). (H) At 6 dpi, animals (n = 6/timepoint) were sacrificed and analyzed for the presence of SARS-CoV-2 viral RNA by RT-qPCR in the lung, heart, spleen, brain, or nasal wash (n = 6: Mann-Whitney test: ns, not significant, * P < 0.05, ** P < 0.01, *** P < 0.001).

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This is a preprint.

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

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

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