Abstract
While antibody libraries are traditionally screened in phage, bacterial, or yeast display formats, they are produced in large scale for pharmaceutical and commercial use in mammalian cell lines. The simpler organisms used for screening have significantly different folding and glycosylation machinery than mammalian cells; consequently, clones resulting from these libraries may require further optimization for mammalian cell expression. To streamline the antibody discovery process, we developed a Chinese hamster ovary (CHO) cell-based selection system that allows for long-term display of antibody Fab fragments. This system is facilitated by a semi-stable Epi-CHO episomal platform to maintain antibody expression for up to 2 months and is compatible with standard PCR-based mutagenesis strategies. This protocol describes the simple and accessible use of CHO display coupled with flow cytometry to enrich for antibody variants with increased ligand-binding affinity from large libraries of ~106 variants, using HER2-binding antibodies as an example.
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1 Electronic Supplementary Materials
Supplementary Fig. 1
4D5 and bD1 Fab display coding sequences. The DNA and amino acid (AA) sequences of both 4D5 and bD1 are shown with dashes where sequence is absent and dots for identical DNA or amino acids. Helpful cloning sites are highlighted in grey. Regions of interest are labeled above and underlined with beginning and end of the region indicated by arrows. Three dots (...) indicate identical DNA sequence to 4D5, and one dot (.) indicates identical amino acid sequence to 4D5. Double underline indicates amino acids targeted for mutagenesis (DOCX 31 kb)
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Nguyen, A.W., Le, K., Maynard, J.A. (2020). Engineering Antibodies on the Surface of CHO Cells. In: Zielonka, S., Krah, S. (eds) Genotype Phenotype Coupling. Methods in Molecular Biology, vol 2070. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9853-1_22
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DOI: https://doi.org/10.1007/978-1-4939-9853-1_22
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