Novel format single-domain antibodies using human VL domain as scaffold

Area of Science:

• Biotechnology
• Immunology
• Protein Engineering

Background:

• Single-domain antibodies (sdAbs) offer advantages like small size and stability.
• Current sdAb development often relies on animal immunization and subsequent humanization.
• The variable CDR3 region in sdAbs complicates library construction.

Purpose of the Study:

• To develop synthetic single-domain antibody (sdAb) libraries using a stability-engineered human light-chain variable domain (VL) scaffold.
• To evaluate the efficiency of generating functional sdAbs through limited randomization on non-CDR loops.
• To compare different panning strategies for sdAb discovery.

Main Methods:

• Construction of synthetic sdAb libraries utilizing a stability-optimized human VL scaffold.
• Limited randomization of three loops opposite the CDRs using degenerate codons.
• Ribosome display and phage pIX display for library construction and antigen panning.

Main Results:

• Synthetic sdAbs exhibited excellent secretory expression efficiency in eukaryotic systems.
• Both standalone ribosome display and combined ribosome display/phage pIX display yielded target-specific binders.
• The combined panning approach showed superior performance compared to standalone ribosome display.

Conclusions:

• Synthetic sdAb libraries based on a stability-optimized human VL scaffold offer a reliable method for generating functional sdAbs.
• This strategy simplifies sdAb development, reducing reliance on animal immunization.
• The developed platform holds potential for next-generation single-domain bispecific antibody development.