An interaction between S•tag and S•protein derived from human ribonuclease 1 allows site-specific conjugation of an enzyme to an antibody for targeted drug delivery

We have previously demonstrated that an antibody-avidin fusion protein could be used to deliver biotinylated enzymes to tumor cells for antibody-directed enzyme prodrug therapy. However, the presence of the chicken protein avidin suggests that immunogenicity may be a problem. To address this concern, we developed a new delivery system consisting of human proteins. The amino-terminal 15-amino-acid peptide derived from human ribonuclease 1 (human S
- tag) can bind with high affinity to human S
- protein (residues 21-124 of the same ribonuclease). We constructed an antibody-S
- protein fusion protein in which S
- protein was genetically linked to an anti-rat transferrin receptor IgG3 at the carboxyl terminus of the heavy chain. We also constructed an enzyme-S
- tag fusion protein in which S
- tag was genetically linked to the carboxyl terminus of Escherichia coli purine nucleoside phosphorylase (PNP). When these two fusion proteins were mixed, S
- tag and S
- protein interacted specifically and produced homogeneous antibody/PNP complexes that retained the ability to bind antigen. Furthermore, in the presence of the prodrug 2-fluoro-2′-deoxyadenosine in vitro, the complex efficiently killed rat myeloma cells overexpressing the transferrin receptor. These results suggest that human ribonuclease-based site-specific conjugation can be used in vivo for targeted chemotherapy of cancer.