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

Area of Science:

• Biotechnology
• Molecular Biology
• Immunology

Background:

• Mammalian cell libraries offer a platform for screening molecular variants.
• Displaying antibodies on cell surfaces facilitates binding property assessment.

Purpose of the Study:

• To develop and demonstrate a high-throughput mammalian display system for antibody engineering.
• To perform affinity maturation of a PD-1 blocking antibody.

Main Methods:

• Construction of large-scale mammalian cell libraries (up to 10 million clones) displaying IgG antibodies.
• Utilizing TALE nucleases or CRISPR/Cas9 for targeted genomic integration and stable expression.
• Employing high-throughput oligonucleotide synthesis, mammalian display, and multi-million scale flow cytometry.

Main Results:

• Successful generation of stable mammalian cell libraries for antibody display.
• Demonstrated affinity maturation of a PD-1 blocking antibody through systematic paratope region mutation.
• Identified specific amino acid substitutions (Tyr to His, then Lys) enhancing antibody binding affinity.

Conclusions:

• Mammalian display libraries provide a powerful tool for comprehensive antibody sequence space interrogation.
• The developed system enables rapid and quantitative functional screening of antibody variants.
• This approach accelerates antibody discovery and optimization for therapeutic applications.