A single-domain antibody library based on a stability-engineered human VH3 scaffold

Area of Science:

• Biotechnology
• Immunology
• Protein Engineering

Background:

• Conventional immunoglobulin G (IgG) molecules face challenges in therapeutic development due to large size and complexity.
• Single-domain antibodies (sdAbs) offer a smaller, functional alternative but human variable domains often exhibit poor stability as sdAbs.

Purpose of the Study:

• To engineer stable human single-domain antibody (sdAb) variants from the VH3-23 framework.
• To construct and validate a synthetic human VH sdAb library for therapeutic applications.

Main Methods:

• Phage display selection of a framework region 2-randomized human VH library under thermal challenge.
• Introduction of complementarity determining region diversity into a stable sdAb variant.
• Library validation through antigen panning and characterization of binders' affinity and biophysical properties.

Main Results:

• Identification of stable human VH3-23 sdAb variants with enhanced thermal stability, expression, and monomeric content.
• Successful construction and validation of a synthetic human VH sdAb library.
• Isolation and characterization of target-specific binders from the library with favorable affinity and biophysical profiles.

Conclusions:

• A stability-engineered human VH scaffold provides a facile source for generating high-quality sdAbs.
• Synthetic sdAb libraries based on engineered scaffolds are promising for diverse therapeutic applications.
• This approach overcomes limitations associated with conventional IgG and unstable human sdAbs.