Fab is the most efficient format to express functional antibodies by yeast surface display

Area of Science:

• Biotechnology
• Immunology
• Molecular Engineering

Background:

• Yeast surface display is a common method for engineering monoclonal antibodies (mAbs).
• Various formats exist for mAb engineering, including single-chain variable fragments (scFv) and antigen-binding fragments (Fab).
• Expression efficiency and functionality can vary significantly between different engineered antibody formats.

Purpose of the Study:

• To compare the expression levels and antigen-binding efficiency of different mAb formats engineered via yeast surface display.
• To identify the most versatile and efficient format for generating functional antibodies.

Main Methods:

• Expressed six different mAbs (four anti-TNF, two anti-IpaD) in scFv, antigen-binding fragment (Fab), and single-chain Fab formats using yeast surface display.
• Quantified expression levels and assessed antigen-binding efficiency and affinity for each format.
• Analyzed the cause of expression variations, investigating light chain display and potential misfolded dimers.

Main Results:

• Single-chain variable fragment (scFv) and single-chain Fab formats showed poor or weak expression for some antibodies.
• All six antibodies expressed as antigen-binding fragments (Fab) exhibited strong binding and high affinity, comparable to soluble forms.
• Expression variations in Fab formats were attributed to light chain display, not misfolded dimers, and did not impact functionality.

Conclusions:

• The antigen-binding fragment (Fab) format is the most versatile for engineering monoclonal antibodies via yeast surface display.
• Fab fragments ensure efficient expression and high-affinity antigen binding, outperforming scFv and single-chain Fab formats.
• Yeast surface display engineering of mAbs benefits from selecting the appropriate format, with Fab proving most reliable.