Genetic Engineering of VHH Antibody Fragments for Efficient Site-Specific Conjugation to Polysaccharides

Site-selective modifications of proteins, without compromising their biological activity, are highly sought after due to their critical role in many biomedical applications. Here, we established a universal and efficient approach for site-selective conjugation of a variable domain of single-chain heavy-chain only antibody fragments (VHH) to polysaccharides using thiol–maleimide chemistry, known for its specificity and efficiency. This is achieved by genetically engineering an unpaired cysteine (Cys) residue in a C-terminal extension of VHHs. In this study, we synthesized two maleimide-functionalized polysaccharides, i.e., dextran-maleimide (Dex-Mal) and hyaluronic acid-maleimide (HA-Mal), for protein conjugation. Six distinct VHHs were selected and engineered with C-terminal extensions containing Cys residues for conjugation with Dex-Mal and HA-Mal. Conjugation efficiency varied among VHHs due to structural heterogeneity, which influenced the reactivity of the engineered Cys residues. One VHH, specific to TNFα (anti-TNFα-VHH), exhibited low conjugation efficiency (<20%); however, efficiency was fully restored when a flexible glycine-serine G₄S linker was introduced between the variable domain and the C-terminal Cys tag. Additionally, incorporation of two free Cys residues in the C-terminal tail further enhanced conjugation efficiency. This work establishes a robust and versatile approach for generating protein–polysaccharide conjugates, paving the way for therapeutic and diagnostic applications.