Multiple Site-Specific in Vitro Labeling of Single-Chain Antibody

For multiple site-specific conjugations of bioactive molecules to a single-chain antibody (scFv) molecule, we have constructed a human anti HER2 receptor, scFv, with a C-terminal fusion polypeptide containing 1, 3, or 17 threonine (Thr) residues. The C-terminal extended fusion polypeptides of these recombinant scFv fusion proteins are used as the acceptor substrate for human polypeptide-alpha-N-acetylgalactosaminyltransferase II (h-ppGalNAc-T2) that transfers either GalNAc or 2-keto-Gal. a modified galactose with a chemical handle. from their respective UDP-sugars to the side-chain hydroxyl group of the Thr residue(s). The recombinant scFv fusion proteins are expressed in E. coli as inclusion bodies and in vitro refolded and glycosylated with h-ppGaINAc-T2. Upon protease cleavage. the MALDI-TOF spectra of the glycosylated C-terminal fusion polypeptides showed that the glycosylated scFv fusion protein with a single Thr residue is fully glycosylated with a single 2-keto-Gal, whereas the glycosylated scFv fusion protein with 3 and 17 Thr residues is found as all equal mixture of 2-3 and 5-8 2-keto-Gal glycosylated fusion proteins, respectively. These fusion scFv proteins with the modified galactose are then conjugated with a fluorescence probe, Alexa488, that carries an orthogonal reactive group. The fluorescence labeled scFv proteins bind specifically to a human breast. cancer cell line (SK-BR-3) that overexpresses the HER2 receptor, indicating that the in vitro folded scFv fusion proteins are biologically active and the presence of conjugated multiple Alexa488 probes in their C-terminal end does not interfere with their binding to the antigen.

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