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Rapid generation of potent antibodies by autonomous hypermutation in yeast

View ORCID ProfileAlon Wellner, View ORCID ProfileConor McMahon, Morgan S. A. Gilman, Jonathan R. Clements, Sarah Clark, Kianna M. Nguyen, Ming H. Ho, View ORCID ProfileJung-Eun Shin, View ORCID ProfileJared Feldman, View ORCID ProfileBlake M. Hauser, View ORCID ProfileTimothy M. Caradonna, View ORCID ProfileLaura M. Wingler, View ORCID ProfileAaron G. Schmidt, View ORCID ProfileDebora S. Marks, Jonathan Abraham, View ORCID ProfileAndrew C. Kruse, View ORCID ProfileChang C. Liu
doi: https://doi.org/10.1101/2020.11.11.378778
Alon Wellner
1Department of Biomedical Engineering, University of California, Irvine, CA 92697, USA
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Conor McMahon
2Department of Biological Chemistry and Molecular Pharmacology, Blavatnik Institute, Harvard Medical School, Boston, MA 02115, USA
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Morgan S. A. Gilman
2Department of Biological Chemistry and Molecular Pharmacology, Blavatnik Institute, Harvard Medical School, Boston, MA 02115, USA
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Jonathan R. Clements
1Department of Biomedical Engineering, University of California, Irvine, CA 92697, USA
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Sarah Clark
3Department of Microbiology, Blavatnik Institute, Harvard Medical School, Boston, MA 02115, USA
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Kianna M. Nguyen
1Department of Biomedical Engineering, University of California, Irvine, CA 92697, USA
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Ming H. Ho
1Department of Biomedical Engineering, University of California, Irvine, CA 92697, USA
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Jung-Eun Shin
4Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA
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Jared Feldman
5Ragon Institute of MGH, MIT and Harvard, Cambridge, MA 02139, USA
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Blake M. Hauser
5Ragon Institute of MGH, MIT and Harvard, Cambridge, MA 02139, USA
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Timothy M. Caradonna
5Ragon Institute of MGH, MIT and Harvard, Cambridge, MA 02139, USA
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Laura M. Wingler
6Department of Medicine, Duke University Medical Center, Durham, NC 27710, USA; Howard Hughes Medical Institute, Duke University Medical Center, Durham, NC 27710, USA
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Aaron G. Schmidt
3Department of Microbiology, Blavatnik Institute, Harvard Medical School, Boston, MA 02115, USA
5Ragon Institute of MGH, MIT and Harvard, Cambridge, MA 02139, USA
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Debora S. Marks
4Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA
7Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA
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Jonathan Abraham
3Department of Microbiology, Blavatnik Institute, Harvard Medical School, Boston, MA 02115, USA
7Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA
8Department of Medicine, Division of Infectious Diseases, Brigham and Women’s Hospital, Boston, MA 02115, USA
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Andrew C. Kruse
2Department of Biological Chemistry and Molecular Pharmacology, Blavatnik Institute, Harvard Medical School, Boston, MA 02115, USA
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  • For correspondence: andrew_kruse@hms.harvard.edu ccl@uci.edu
Chang C. Liu
1Department of Biomedical Engineering, University of California, Irvine, CA 92697, USA
9Department of Chemistry, University of California, Irvine, CA 92697, USA
10Department of Molecular Biology & Biochemistry, University of California, Irvine, CA 92697, USA
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  • For correspondence: andrew_kruse@hms.harvard.edu ccl@uci.edu
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Abstract

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, has poor compatibility with certain antigens (e.g., integral membrane proteins), and suffers from self-tolerance and immunodominance, which limit the functional spectrum of antibodies that can be obtained. Here, we describe Autonomous Hypermutation yEast surfAce Display (AHEAD), a synthetic recombinant antibody generation technology that imitates somatic hypermutation inside engineered yeast. In AHEAD, antibody fragments are encoded on an error-prone orthogonal DNA replication system, resulting in Saccharomyces cerevisiae populations that continuously mutate surface-displayed antibody repertoires. Simple cycles of yeast culturing and enrichment for antigen binding drive the evolution of high-affinity antibody clones in a readily parallelizable process that takes as little as 2 weeks. We applied AHEAD to generate nanobodies against the SARS-CoV-2 S glycoprotein, a GPCR, and other targets. The SARS-CoV-2 nanobodies, concurrently evolved from an open-source naïve nanobody library in 8 independent experiments, reached subnanomolar affinities through the sequential fixation of multiple mutations over 3-8 AHEAD cycles that saw ∼580-fold and ∼925-fold improvements in binding affinities and pseudovirus neutralization potencies, respectively. These experiments highlight the defining speed, parallelizability, and effectiveness of AHEAD and provide a template for streamlined antibody generation at large with salient utility in rapid response to current and future viral outbreaks.

Competing Interest Statement

Provisional patents have been filed on this work, with A.W., C.M., A.C.K., and C.C.L. as co-inventors. A.C.K. is a co-founder and advisor of Tectonic Therapeutic, Inc., and of the Institute for Protein Innovation. C.C.L. is a co-founder of K2 Biotechnologies, Inc., which focuses on the use of continuous evolution technologies applied to antibody engineering.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.
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Rapid generation of potent antibodies by autonomous hypermutation in yeast
Alon Wellner, Conor McMahon, Morgan S. A. Gilman, Jonathan R. Clements, Sarah Clark, Kianna M. Nguyen, Ming H. Ho, Jung-Eun Shin, Jared Feldman, Blake M. Hauser, Timothy M. Caradonna, Laura M. Wingler, Aaron G. Schmidt, Debora S. Marks, Jonathan Abraham, Andrew C. Kruse, Chang C. Liu
bioRxiv 2020.11.11.378778; doi: https://doi.org/10.1101/2020.11.11.378778
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Rapid generation of potent antibodies by autonomous hypermutation in yeast
Alon Wellner, Conor McMahon, Morgan S. A. Gilman, Jonathan R. Clements, Sarah Clark, Kianna M. Nguyen, Ming H. Ho, Jung-Eun Shin, Jared Feldman, Blake M. Hauser, Timothy M. Caradonna, Laura M. Wingler, Aaron G. Schmidt, Debora S. Marks, Jonathan Abraham, Andrew C. Kruse, Chang C. Liu
bioRxiv 2020.11.11.378778; doi: https://doi.org/10.1101/2020.11.11.378778

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