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

## TL;DR

Scientists engineered antibody fragments to attach efficiently to polysaccharides, improving their potential for medical use.

## Contribution

A universal method for site-specific conjugation of VHHs to polysaccharides using engineered cysteine residues.

## Key findings

- Genetically engineered cysteine residues enabled efficient conjugation of VHHs to maleimide-functionalized polysaccharides.
- Conjugation efficiency varied among VHHs due to structural differences, but was improved with a glycine-serine linker.
- Incorporating two free cysteine residues in the C-terminal tail further enhanced conjugation efficiency.

## Abstract

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.