An engineered bivalent single-chain antibody fragment that increases antigen binding activity

Area of Science:

• Biotechnology
• Protein Engineering
• Immunology

Background:

• Single chain variable fragment (scFv) antibodies are valuable tools in research and therapeutics.
• Engineering scFvs for enhanced stability and binding affinity is crucial for their applications.
• Disulfide-bonded dimers can improve the functional properties of antibody fragments.

Purpose of the Study:

• To construct and characterize bivalent single chain Fv (scFv) antibody fragments with C-terminal cysteine extensions.
• To evaluate the impact of cysteine modifications on scFv dimerization and antigen-binding activity.
• To compare the performance of scFv constructs with varying numbers of cysteines.

Main Methods:

• Construction of scFv variants with one (scFv-1Cys) or two (scFv-2Cys) cysteines fused to the C-terminus.
• Expression and secretion of scFv proteins in a recombinant Pichia pastoris system.
• Western blot analysis under non-reducing conditions to assess dimerization.
• Competitive radioimmunoassay to measure antigen-binding activity.

Main Results:

• The scFv protein was successfully expressed and secreted as a dimer with a C-terminal disulfide bridge.
• The scFv-1Cys construct showed a significantly higher dimer/monomer ratio compared to the scFv-2Cys construct.
• scFv-1Cys exhibited antigen binding activity comparable to the parental monoclonal antibody (MAb) and significantly higher than control scFv monomer and scFv-2Cys.

Conclusions:

• C-terminal cysteine fusion is an effective strategy for generating disulfide-bonded scFv dimers.
• The scFv-1Cys variant demonstrates enhanced dimerization and superior antigen-binding activity.
• This engineered bivalent scFv holds promise for improved antibody-based applications.