TY - JOUR
T1 - Genetic Engineering of VHH Antibody Fragments for Efficient Site-Specific Conjugation to Polysaccharides
AU - Zhong, Lin
AU - Morshuis, Lisanne C.M.
AU - Koerselman, Michelle
AU - Memelink, Angela
AU - Kolecka, Anna
AU - Heukers, Raimond
AU - Verrips, Theo
AU - Karperien, Marcel
AU - Zoetebier, Bram
N1 - Publisher Copyright:
© 2025 The Authors. Published by American Chemical Society.
PY - 2025/6/18
Y1 - 2025/6/18
N2 - 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.
AB - 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.
KW - UT-Hybrid-D
UR - https://www.scopus.com/pages/publications/105006707863
U2 - 10.1021/acs.bioconjchem.5c00167
DO - 10.1021/acs.bioconjchem.5c00167
M3 - Article
C2 - 40407350
AN - SCOPUS:105006707863
SN - 1043-1802
VL - 36
SP - 1319
EP - 1328
JO - Bioconjugate chemistry
JF - Bioconjugate chemistry
IS - 6
ER -