Directed evolution of potent neutralizing nanobodies against SARS-CoV-2 using CDR-swapping mutagenesis

Jennifer M Zupancic; Alec A Desai; John S Schardt; Ghasidit Pornnoppadol; Emily K Makowski; Matthew D Smith; Andrew A Kennedy; Mayara Garcia de Mattos Barbosa; Marilia Cascalho; Thomas M Lanigan; Andrew W Tai; Peter M Tessier

doi:10.1016/j.chembiol.2021.05.019

Directed evolution of potent neutralizing nanobodies against SARS-CoV-2 using CDR-swapping mutagenesis

Cell Chem Biol. 2021 Sep 16;28(9):1379-1388.e7. doi: 10.1016/j.chembiol.2021.05.019. Epub 2021 Jun 24.

Affiliations

¹ Department of Chemical Engineering, University of Michigan, North Campus Research Complex, 2800 Plymouth Road, Ann Arbor, MI 48109, USA; Biointerfaces Institute, University of Michigan, Ann Arbor, MI 48109, USA.
² Department of Chemical Engineering, University of Michigan, North Campus Research Complex, 2800 Plymouth Road, Ann Arbor, MI 48109, USA; Department of Pharmaceutical Sciences, University of Michigan, Ann Arbor, MI 48109, USA; Biointerfaces Institute, University of Michigan, Ann Arbor, MI 48109, USA.
³ Department of Pharmaceutical Sciences, University of Michigan, Ann Arbor, MI 48109, USA; Biointerfaces Institute, University of Michigan, Ann Arbor, MI 48109, USA.
⁴ Division of Gastroenterology, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI, USA.
⁵ Department of Surgery, University of Michigan, Ann Arbor, MI 48109, USA.
⁶ Department of Surgery, University of Michigan, Ann Arbor, MI 48109, USA; Department of Microbiology and Immunology, University of Michigan, Ann Arbor, MI 48109, USA.
⁷ Division of Rheumatology, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI, USA.
⁸ Division of Gastroenterology, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI, USA; Department of Microbiology and Immunology, University of Michigan, Ann Arbor, MI 48109, USA; Medicine Service, Ann Arbor Veterans Administration Health System, Ann Arbor, MI, USA.
⁹ Department of Chemical Engineering, University of Michigan, North Campus Research Complex, 2800 Plymouth Road, Ann Arbor, MI 48109, USA; Department of Pharmaceutical Sciences, University of Michigan, Ann Arbor, MI 48109, USA; Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI 48109, USA; Biointerfaces Institute, University of Michigan, Ann Arbor, MI 48109, USA. Electronic address: ptessier@umich.edu.

Abstract

There is widespread interest in facile methods for generating potent neutralizing antibodies, nanobodies, and other affinity proteins against SARS-CoV-2 and related viruses to address current and future pandemics. While isolating antibodies from animals and humans are proven approaches, these methods are limited to the affinities, specificities, and functional activities of antibodies generated by the immune system. Here we report a surprisingly simple directed evolution method for generating nanobodies with high affinities and neutralization activities against SARS-CoV-2. We demonstrate that complementarity-determining region swapping between low-affinity lead nanobodies, which we discovered unintentionally but find is simple to implement systematically, results in matured nanobodies with unusually large increases in affinity. Importantly, the matured nanobodies potently neutralize both SARS-CoV-2 pseudovirus and live virus, and possess drug-like biophysical properties. We expect that our methods will improve in vitro nanobody discovery and accelerate the generation of potent neutralizing nanobodies against diverse coronaviruses.

Keywords: ACE2; COVID-19; RBD; affinity; maturation; nanobody; neutralization; receptor-binding domain; spike; yeast.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

Animals
Antibodies, Neutralizing / chemistry
Antibodies, Neutralizing / genetics*
Chlorocebus aethiops
Complementarity Determining Regions / genetics*
Epitopes
HEK293 Cells
Humans
Mutagenesis
SARS-CoV-2
Saccharomyces cerevisiae
Single-Domain Antibodies / chemistry
Single-Domain Antibodies / genetics*
Spike Glycoprotein, Coronavirus / chemistry
Vero Cells

Substances

Antibodies, Neutralizing
Complementarity Determining Regions
Epitopes
Single-Domain Antibodies
Spike Glycoprotein, Coronavirus
spike protein, SARS-CoV-2

Directed evolution of potent neutralizing nanobodies against SARS-CoV-2 using CDR-swapping mutagenesis

Authors

Affiliations

Abstract

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MeSH terms

Substances

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