Rapid generation of potent antibodies by autonomous hypermutation in yeast

The predominant approach for antibody generation remains animal immunization, which can yield exceptionally selective and potent antibody clones owing to the powerful evolutionary process of somatic hypermutation. However, animal immunization is inherently slow, not always accessible and poorly comp...

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Published inNature chemical biology Vol. 17; no. 10; pp. 1057 - 1064
Main Authors Wellner, Alon, McMahon, Conor, Gilman, Morgan S. A., Clements, Jonathan R., Clark, Sarah, Nguyen, Kianna M., Ho, Ming H., Hu, Vincent J., Shin, Jung-Eun, Feldman, Jared, Hauser, Blake M., Caradonna, Timothy M., Wingler, Laura M., Schmidt, Aaron G., Marks, Debora S., Abraham, Jonathan, Kruse, Andrew C., Liu, Chang C.
Format Journal Article
LanguageEnglish
Published New York Nature Publishing Group US 01.10.2021
Nature Publishing Group
Subjects
631/250/255
631/92/469
631/92/552
Antibodies
Antigens
Biochemical Engineering
Biochemistry
Bioorganic Chemistry
Cell Biology
Chemistry
Chemistry and Materials Science
Chemistry/Food Science
DNA biosynthesis
G protein-coupled receptors
Glycoproteins
Immunization
Nanobodies
Severe acute respiratory syndrome coronavirus 2
Somatic hypermutation
Yeast
Yeasts
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Summary:The predominant approach for antibody generation remains animal immunization, which can yield exceptionally selective and potent antibody clones owing to the powerful evolutionary process of somatic hypermutation. However, animal immunization is inherently slow, not always accessible and poorly compatible with many antigens. Here, we describe ‘autonomous hypermutation yeast surface display’ (AHEAD), a synthetic recombinant antibody generation technology that imitates somatic hypermutation inside engineered yeast. By encoding antibody fragments on an error-prone orthogonal DNA replication system, surface-displayed antibody repertoires continuously mutate through simple cycles of yeast culturing and enrichment for antigen binding to produce high-affinity clones in as little as two weeks. We applied AHEAD to generate potent nanobodies against the SARS-CoV-2 S glycoprotein, a G-protein-coupled receptor and other targets, offering a template for streamlined antibody generation at large. Autonomous hypermutation yeast surface display (AHEAD) mimics the process of somatic hypermutation in animals to enable the rapid in vitro evolution of antibodies, including nanobodies targeting the RBD of SARS-CoV-2.
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Present address: Department of Pharmacology and Cancer Biology, Duke University, Durham, NC 27710, USA.
Author contributions Statement
All authors contributed to experimental design and data analysis. A.W., C.M., A.C.K., and C.C.L. were responsible for the conception of AHEAD. A.W., M.H.H, V.J.H. and K.M.N. carried out experiments establishing the first-generation of AHEAD and made improvements to reach the second-generation AHEAD system. C.M. carried out AHEAD experiments for the evolution of anti-AT1R nanobodies and selected parent anti-SARS-CoV-2 for evolution using AHEAD. A.W., J.C., and M.H.H. carried out AHEAD experiments for the evolution of anti-GFP, anti-HSA, and anti-SARS-CoV-2 nanobodies. A.W., C.M., M.S.A.G., S.C., and L.M.W. characterized the activities of evolved nanobodies in binding assays (A.W., C.M., and L.M.W.), SPR measurements (C.M. and M.S.A.G.), neutralization assays (S.C.), and ACE2 competition assays (S.C.). J.F., B.M.H., T.M.C., and A.W. were responsible for the expression of RBD used throughout this study. A.W. and V.J.H. were responsible for the RBD mutational scanning experiments and NGS data analysis that mapped target epitopes and RBD escape mutations for anti-RBD nanobodies. J.-E.S. and D.S.M. were responsible for computational design aspects for the naïve ~200,000-member nanobody library and A.W. inserted that library into AHEAD. A.C.K. and C.C.L. oversaw all aspects of the project, D.S.M. supervised computational nanobody library design, J.A. supervised neutralization and ACE2 competition assays, and A.G.S. supervised the preparation of RBD. A.W carried out the DMS analysis. A.W., C.M., A.C.K., and C.C.L. wrote the manuscript with input and contributions from all authors.
Present address: Vertex Pharmaceuticals, Boston, MA 02210, USA.
ISSN:1552-4450
1552-4469
DOI:10.1038/s41589-021-00832-4