Asai Tsuneaki, Wims Letitia A, Morrison Sherie L
Department of Microbiology, Immunology, and Molecular Genetics, University of California Los Angeles, 405 Hilgard Avenue, Los Angeles, CA 90095, USA.
J Immunol Methods. 2005 Apr;299(1-2):63-76. doi: 10.1016/j.jim.2005.01.020. Epub 2005 Apr 7.
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 Stag) can bind with high affinity to human Sprotein (residues 21-124 of the same ribonuclease). We constructed an antibody-Sprotein fusion protein in which Sprotein was genetically linked to an anti-rat transferrin receptor IgG3 at the carboxyl terminus of the heavy chain. We also constructed an enzyme-Stag fusion protein in which Stag was genetically linked to the carboxyl terminus of Escherichia coli purine nucleoside phosphorylase (PNP). When these two fusion proteins were mixed, Stag and Sprotein 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.
