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A comprehensive search of functional sequence space using large mammalian display libraries created by gene editing

The construction of large libraries in mammalian cells allows the direct screening of millions of molecular variants for binding properties in a cell type relevant for screening or production. We have created mammalian cell libraries of up to 10 million clones displaying a repertoire of IgG-formatte...

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Autores principales: Parthiban, Kothai, Perera, Rajika L., Sattar, Maheen, Huang, Yanchao, Mayle, Sophie, Masters, Edward, Griffiths, Daniel, Surade, Sachin, Leah, Rachael, Dyson, Michael R., McCafferty, John
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Taylor & Francis 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6601556/
https://www.ncbi.nlm.nih.gov/pubmed/31107136
http://dx.doi.org/10.1080/19420862.2019.1618673
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author Parthiban, Kothai
Perera, Rajika L.
Sattar, Maheen
Huang, Yanchao
Mayle, Sophie
Masters, Edward
Griffiths, Daniel
Surade, Sachin
Leah, Rachael
Dyson, Michael R.
McCafferty, John
author_facet Parthiban, Kothai
Perera, Rajika L.
Sattar, Maheen
Huang, Yanchao
Mayle, Sophie
Masters, Edward
Griffiths, Daniel
Surade, Sachin
Leah, Rachael
Dyson, Michael R.
McCafferty, John
author_sort Parthiban, Kothai
collection PubMed
description The construction of large libraries in mammalian cells allows the direct screening of millions of molecular variants for binding properties in a cell type relevant for screening or production. We have created mammalian cell libraries of up to 10 million clones displaying a repertoire of IgG-formatted antibodies on the cell surface. TALE nucleases or CRISPR/Cas9 were used to direct the integration of the antibody genes into a single genomic locus, thereby rapidly achieving stable expression and transcriptional normalization. The utility of the system is illustrated by the affinity maturation of a PD-1-blocking antibody through the systematic mutation and functional survey of 4-mer variants within a 16 amino acid paratope region. Mutating VH CDR3 only, we identified a dominant “solution” involving substitution of a central tyrosine to histidine. This appears to be a local affinity maximum, and this variant was surpassed by a lysine substitution when light chain variants were introduced. We achieve this comprehensive and quantitative interrogation of sequence space by combining high-throughput oligonucleotide synthesis with mammalian display and flow cytometry operating at the multi-million scale.
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spelling pubmed-66015562019-07-08 A comprehensive search of functional sequence space using large mammalian display libraries created by gene editing Parthiban, Kothai Perera, Rajika L. Sattar, Maheen Huang, Yanchao Mayle, Sophie Masters, Edward Griffiths, Daniel Surade, Sachin Leah, Rachael Dyson, Michael R. McCafferty, John MAbs Report The construction of large libraries in mammalian cells allows the direct screening of millions of molecular variants for binding properties in a cell type relevant for screening or production. We have created mammalian cell libraries of up to 10 million clones displaying a repertoire of IgG-formatted antibodies on the cell surface. TALE nucleases or CRISPR/Cas9 were used to direct the integration of the antibody genes into a single genomic locus, thereby rapidly achieving stable expression and transcriptional normalization. The utility of the system is illustrated by the affinity maturation of a PD-1-blocking antibody through the systematic mutation and functional survey of 4-mer variants within a 16 amino acid paratope region. Mutating VH CDR3 only, we identified a dominant “solution” involving substitution of a central tyrosine to histidine. This appears to be a local affinity maximum, and this variant was surpassed by a lysine substitution when light chain variants were introduced. We achieve this comprehensive and quantitative interrogation of sequence space by combining high-throughput oligonucleotide synthesis with mammalian display and flow cytometry operating at the multi-million scale. Taylor & Francis 2019-06-09 /pmc/articles/PMC6601556/ /pubmed/31107136 http://dx.doi.org/10.1080/19420862.2019.1618673 Text en © 2019 The Author(s). Published with license by Taylor & Francis Group, LLC. http://creativecommons.org/licenses/by-nc-nd/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License (http://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited, and is not altered, transformed, or built upon in any way.
spellingShingle Report
Parthiban, Kothai
Perera, Rajika L.
Sattar, Maheen
Huang, Yanchao
Mayle, Sophie
Masters, Edward
Griffiths, Daniel
Surade, Sachin
Leah, Rachael
Dyson, Michael R.
McCafferty, John
A comprehensive search of functional sequence space using large mammalian display libraries created by gene editing
title A comprehensive search of functional sequence space using large mammalian display libraries created by gene editing
title_full A comprehensive search of functional sequence space using large mammalian display libraries created by gene editing
title_fullStr A comprehensive search of functional sequence space using large mammalian display libraries created by gene editing
title_full_unstemmed A comprehensive search of functional sequence space using large mammalian display libraries created by gene editing
title_short A comprehensive search of functional sequence space using large mammalian display libraries created by gene editing
title_sort comprehensive search of functional sequence space using large mammalian display libraries created by gene editing
topic Report
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6601556/
https://www.ncbi.nlm.nih.gov/pubmed/31107136
http://dx.doi.org/10.1080/19420862.2019.1618673
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