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


ABSTRACT: 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.

SUBMITTER: Parthiban K 

PROVIDER: S-EPMC6601556 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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

Parthiban Kothai K   Perera Rajika L RL   Sattar Maheen M   Huang Yanchao Y   Mayle Sophie S   Masters Edward E   Griffiths Daniel D   Surade Sachin S   Leah Rachael R   Dyson Michael R MR   McCafferty John J  

mAbs 20190609 5


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 t  ...[more]

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