Multiplex Evolution of Antibody Fragments Utilizing a Yeast Surface Display Platform

Area of Science:

• Synthetic biology
• Protein engineering
• Immunology

Background:

• CRISPR/Cas9 technology enables high-throughput assessment of mutations for desired phenotypes and genotype-phenotype correlations.
• Antibody engineering is crucial for therapeutic applications, focusing on enhancing binding affinity and stability.
• Understanding genotype-phenotype relationships is key to improving antibody function.

Purpose of the Study:

• To develop and present a novel method, Multiplex Navigation of Antibody Structure (MINAS), for comprehensive antibody engineering.
• To map the contribution of complementarity-determining and framework regions to antibody properties using a high-throughput approach.
• To identify specific antibody mutants with significantly enhanced binding affinities.

Main Methods:

• Utilized CRISPR/Cas9-based trackable editing for targeted mutations within antibody regions.
• Employed yeast-displayed libraries combined with fluorescent-activated cell sorting (FACS) for library screening.
• Designed and introduced mutations across all complementarity-determining and framework regions of a model scFv antibody.

Main Results:

• Identified specific antibody mutants exhibiting up to 100-fold higher binding affinities compared to the wild-type antibody.
• Successfully mapped the functional impact of mutations in different antibody regions on binding affinity and stability.
• Demonstrated the effectiveness of MINAS in identifying beneficial mutations for antibody enhancement.

Conclusions:

• MINAS provides a powerful platform for accelerating antibody protein engineering and optimizing therapeutic candidates.
• The integration of CRISPR/Cas9 editing with yeast surface display significantly expands the scope of trackable protein engineering.
• This approach facilitates a deeper understanding of structure-function relationships in antibodies, paving the way for novel biologics.