In vitro Fab display: a cell-free system for IgG discovery

Area of Science:

• Biotechnology
• Molecular Biology
• Immunology

Background:

• Ribosome display is a powerful in vitro selection technology for discovering novel antibody fragments.
• Current ribosome display methods are limited to selecting single-chain protein fragments due to the open nature of cell-free reactions.

Purpose of the Study:

• To develop a simplified ribosome display approach for selecting multi-chain protein fragments, specifically antibody Fab fragments.
• To demonstrate the feasibility of generating functional antibody Fab fragments using this novel cell-free selection method.

Main Methods:

• A modified ribosome display system was developed to tether either the heavy or light chain of a two-chain antibody Fab fragment to the ribosome.
• Synthetic Fab heavy and light chain libraries were constructed and selected against carcinoembryonic antigen (CEA) and vascular endothelial growth factor (VEGF).
• Selected Fab fragments were reformatted into full-length immunoglobulin Gs (IgGs) and expressed in an optimized cell-free system for characterization.

Main Results:

• The developed method successfully displayed functional antibody Fab fragments, retaining antigen-binding capabilities.
• Novel antibody Fab fragments targeting CEA and VEGF were identified through selection against these antigens.
• The selected antibody fragments were successfully converted into full-length IgGs and expressed in a cell-free system, enabling rapid screening and characterization.

Conclusions:

• This cell-free platform offers a streamlined approach for the selection and discovery of multi-chain antibody fragments, including Fab fragments.
• The method facilitates the rapid generation of novel antibody therapeutics, such as IgGs, targeting specific antigens like CEA and VEGF.
• This technique expands the utility of ribosome display for antibody engineering and drug discovery.