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MAbs. 2019 Jun 9:1-15. doi: 10.1080/19420862.2019.1618673. [Epub ahead of print]

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

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1
a IONTAS Ltd , Cambridge , UK.

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.

KEYWORDS:

CRISPR/Cas9; IgG antibody library; Mammalian display; TALE nuclease; affinity maturation; fluorescence-activated cell sorting; gene editing; gene targeting; human therapeutic antibody discovery; magnetic-activated cell sorting

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