Chicken scFvs with an Artificial Cysteine for Site-Directed Conjugation

Area of Science:

• Bioconjugation Chemistry
• Protein Engineering
• Immunotechnology

Background:

• Site-directed conjugation is crucial for developing targeted therapeutics and diagnostics.
• Chicken single-chain variable fragments (scFv) offer advantages but require efficient modification strategies.
• Cysteine residues are key for specific chemical conjugation via their sulfhydryl groups.

Purpose of the Study:

• To identify amino acid residues in chicken scFv frameworks suitable for cysteine replacement.
• To generate and characterize artificial cysteine mutants of chicken scFv.
• To evaluate the feasibility of site-directed conjugation to these engineered cysteine residues.

Main Methods:

• Generated 157 artificial cysteine mutants by replacing framework residues in chicken scFv (VH and VL domains).
• Characterized mutants for retained binding activity.
• Engineered scFv-Ckappa fusion proteins with selected cysteine mutants (VL: L5, L6, L7; VH: H13, H16) for conjugation studies.
• Prepared charge-variant mutants near H13 to assess conjugation stability.

Main Results:

• Identified at least 27 VL and 37 VH residues amenable to cysteine substitution without compromising binding activity.
• Successfully achieved PEG-conjugation to the sulfhydryl group of artificial cysteines in five scFv mutants.
• Charge variants of H13 mutant retained binding activity and conjugation capability, demonstrating improved stability potential.

Conclusions:

• Established a method for generating chicken scFv mutants with engineered cysteine residues for site-directed conjugation.
• Demonstrated the successful conjugation of PEG to these mutants, preserving their binding affinity.
• The findings provide a versatile platform for developing novel antibody-based conjugates for therapeutic and diagnostic applications.