A modular IgG-scFv bispecific antibody topology

Area of Science:

• Biotechnology
• Immunology
• Protein Engineering

Background:

• Bispecific antibodies (bsAbs) offer enhanced therapeutic potential by engaging multiple targets simultaneously.
• Developing stable and functional bsAb formats remains a key challenge in antibody engineering.

Purpose of the Study:

• To present a novel IgG-scFv bifunctional antibody format.
• To evaluate the impact of fusing a disulfide-stabilized single-chain variable fragment (scFv) to the C-terminus of the IgG light chain.
• To establish a standardized platform for constructing functional bsAbs.

Main Methods:

• Construction of the IgG-scFv bifunctional antibody format.
• Expression in mammalian cells and purification via one-step Protein A chromatography.
• Characterization of binding affinities, stability, and in vivo properties (tumor targeting, blood clearance).

Main Results:

• The IgG-scFv bsAb format retains parental binding affinities of both domains.
• The bsAb exhibits IgG-like stability and in vivo tumor targeting and blood clearance.
• Extension of the light chain with an scFv did not compromise IgG function or stability, despite potential disruption of disulfide bond formation.

Conclusions:

• The C-terminus of the IgG light chain can be extended with an scFv without affecting overall IgG function and stability.
• This IgG-scFv format provides a standardized and robust platform for developing bispecific antibodies.
• The findings facilitate the engineering of novel bifunctional antibodies for therapeutic applications.