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

MAbs. 2019 Jul;11(5):884-898. doi: 10.1080/19420862.2019.1618673. Epub 2019 Jun 9.

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.

MeSH terms

  • Animals
  • Antibodies, Monoclonal, Humanized / genetics*
  • Antibody Affinity*
  • Binding Sites, Antibody / genetics*
  • Binding Sites, Antibody / immunology
  • CHO Cells
  • CRISPR-Cas Systems
  • Complementarity Determining Regions / genetics
  • Cricetulus
  • Endodeoxyribonucleases
  • Flow Cytometry
  • Gene Editing
  • HEK293 Cells
  • Humans
  • Immunoglobulin Heavy Chains / genetics
  • Mutagenesis, Site-Directed
  • Programmed Cell Death 1 Receptor / immunology

Substances

  • Antibodies, Monoclonal, Humanized
  • Complementarity Determining Regions
  • Immunoglobulin Heavy Chains
  • Programmed Cell Death 1 Receptor
  • Endodeoxyribonucleases