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.

Phage Display Derived Monoclonal Antibodies: From Bench to Bedside

“…For instance, in addition to their ability to produce glycosylated proteins, yeast and mammalian antibody libraries can be constructed to display full-length antibodies as well as antibody fragments, such as scFvs and fragment antigen-binding region (Fabs) ( 117 – 126 ), allowing the isolation of high affinity antibodies with definitive biological characteristics ( 122 , 123 ). For example, Parthiban et al has developed mammalian libraries that display around 10 million clones in IgG-format on the surface of HEK293 cells using CRISPR/Cas9 or transcription activator-like effector nucleases (TALENs) ( 127 ). These libraries can act as a quality filter for different antibody developability aspects, and to provide a very early insight into developability problems, such as aggregation and cross-reactivity.…”

Antibody discovery and engineering by enhanced CRISPR-Cas9 integration of variable gene cassette libraries in mammalian cells

“…Since correct folding, stability and post-translational modifications are all important for drug development properties, the possibility to engineer antibodies in a mammalian host offers a substantial advantage in terms of developability. 7 However, the exponentially lower throughput of mammalian transfection or genomic integration, 8 when compared to microbial transformation, still represents an intrinsic limitation of mammalian screening platforms.…”

Beyond affinity: selection of antibody variants with optimal biophysical properties and reduced immunogenicity from mammalian display libraries

“…Nuclease-mediated integration of antibody genes into a single genomic locus has previously been used to generate libraries displaying IgG-formatted antibodies in mammalian cells. 7 This was used for both de novo antibody discovery from naive libraries and for affinity maturation of antibodies. In addition to generating novel binders, we now demonstrate that mammalian display can be used to select variants with improved biophysical properties based on an inherent ability of mammalian display to differentiate between antibodies with different biophysical properties.…”

“…We have recently described the use of nuclease-directed integration in the construction of large libraries of binders displayed on the surface of mammalian cells. 7 The efficiency of nuclease-directed integration creates the opportunity to introduce diverse repertoires of millions of complete antibody genes into the same genomic locus within a population of cells, combining accurate integration into a pre-defined site with an efficiency that allows construction of large libraries. Targeting individual antibody genes to a single locus within the population has the additional advantage of effecting transcriptional normalization across the population.…”