An antibody with a variable-region coiled-coil "knob" domain.

Area of Science:

• Protein engineering
• Immunology
• Structural biology

Background:

• Antibodies possess unique structures in their complementarity determining regions (CDRs) that dictate antigen binding.
• Ultralong CDR3 regions, particularly in bovine antibodies, can adopt complex conformations.
• The heavy chain CDR3 (CDR3H) of bovine antibody BLV1H12 features a β-strand stalk and a disulfide-linked knob domain.

Purpose of the Study:

• To engineer a novel antibody by replacing the native β-strand stalk of the CDR3H with a designed coiled-coil motif.
• To assess the stability and expression of the engineered antibody.
• To evaluate the potential of this engineered antibody as a scaffold for fusion proteins with pharmacological applications.

Main Methods:

• X-ray crystallography to determine the structure of the parent antibody.
• Protein engineering to substitute the β-strand stalk with an antiparallel heterodimeric coiled-coil motif.
• Mass spectrometry (MS) analysis of hydrogen-deuterium (H-D) exchange to confirm coiled-coil structure.
• Expression in mammalian cells and stability assays.
• Functional assays involving fusion of the engineered antibody with bovine granulocyte colony-stimulating factor (bGCSF).

Main Results:

• The engineered antibody (Ab-coil) successfully expressed in mammalian cells with stability comparable to the parent antibody.
• MS H-D exchange analysis confirmed the formation of the designed coiled-coil structure.
• Fusion of Ab-coil with bGCSF resulted in a chimeric antibody that stably expressed.
• The chimeric antibody demonstrated potent proliferation of mouse NFS-60 cells, comparable to native bGCSF.

Conclusions:

• A novel coiled-coil CDR3 motif can be successfully engineered into antibodies.
• This motif provides a stable and versatile platform for creating antibody fusion proteins.
• Engineered antibody fusion proteins exhibit potent pharmacological activity, demonstrating utility in therapeutic applications.