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A cell-free nanobody engineering platform rapidly generates SARS-CoV-2 neutralizing nanobodies

Xun Chen (), Matteo Gentili, Nir Hacohen and Aviv Regev ()
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Xun Chen: Broad Institute of MIT and Harvard
Matteo Gentili: Broad Institute of MIT and Harvard
Nir Hacohen: Broad Institute of MIT and Harvard
Aviv Regev: Broad Institute of MIT and Harvard

Nature Communications, 2021, vol. 12, issue 1, 1-14

Abstract: Abstract Antibody engineering technologies face increasing demands for speed, reliability and scale. We develop CeVICA, a cell-free nanobody engineering platform that uses ribosome display for in vitro selection of nanobodies from a library of 1011 randomized sequences. We apply CeVICA to engineer nanobodies against the Receptor Binding Domain (RBD) of SARS-CoV-2 spike protein and identify >800 binder families using a computational pipeline based on CDR-directed clustering. Among 38 experimentally-tested families, 30 are true RBD binders and 11 inhibit SARS-CoV-2 pseudotyped virus infection. Affinity maturation and multivalency engineering increase nanobody binding affinity and yield a virus neutralizer with picomolar IC50. Furthermore, the capability of CeVICA for comprehensive binder prediction allows us to validate the fitness of our nanobody library. CeVICA offers an integrated solution for rapid generation of divergent synthetic nanobodies with tunable affinities in vitro and may serve as the basis for automated and highly parallel nanobody engineering.

Date: 2021
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DOI: 10.1038/s41467-021-25777-z

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