Bacterial cytoplasmic display platform Retained Display (ReD) identifies stable human germline antibody frameworks

Area of Science:

• Biotechnology
• Immunology
• Molecular Biology

Background:

• Conventional antibody surface display methods face challenges with protein export, limiting production yield and scalability.
• Efficient antibody discovery requires overcoming cellular membrane barriers for protein expression and folding.

Purpose of the Study:

• To develop a novel cytoplasmic antibody display platform, Retained Display (ReD), for screening soluble antibody fragments.
• To identify highly soluble and stable human single-chain variable fragment (scFv) frameworks for cytoplasmic expression.
• To demonstrate the utility of ReD for antibody discovery against exogenous targets.

Main Methods:

• Development of the Retained Display (ReD) platform utilizing detergent-permeabilized Escherichia coli to retain high-molecular weight complexes.
• Expression of human light chain families (λ and κ) fused to IGHV3-23 for cytoplasmic screening.
• Screening for soluble scFv frameworks and subsequent CDR3 diversification for target binding.

Main Results:

• ReD successfully enabled cytoplasmic presentation of exogenous targets for antibody screening.
• Specific human λ subfamilies (1, 3, and 6) were identified as soluble cytoplasmic partners with IGHV3-23.
• Identified antibody frameworks retained germline sequences in framework, CDR1, and CDR2 regions, differing from previous in vivo screens.
• A soluble scFv scaffold was identified, demonstrating tolerance to CDR3 diversification and successfully isolating an antibody that binds an exogenous protein target.

Conclusions:

• The Retained Display (ReD) platform offers a viable alternative to conventional surface display for antibody discovery.
• ReD facilitates the rapid identification of stable, soluble antibody frameworks suitable for cytoplasmic expression.
• This technology holds significant potential for developing antibodies against emerging infectious diseases and bioterror agents.