title: A human biotin acceptor domain allows site-specific conjugation of an enzyme to an antibody-avidin fusion protein for targeted drug delivery.
abstract: We have previously constructed an antibody-avidin (Av) fusion protein, anti-transferrin receptor (TfR) IgG3-Av, which can deliver biotinylated molecules to cells expressing the TfR. We now describe the use of the fusion protein for antibody-directed enzyme prodrug therapy (ADEPT). The 67 amino acid carboxyl-terminal domain (P67) of human propionyl-CoA carboxylase alpha subunit can be metabolically biotinylated at a fixed lysine residue. We genetically fused P67 to the carboxyl terminus of the yeast enzyme FCU1, a derivative of cytosine deaminase that can convert the non-toxic prodrug 5-fluorocytosine to the cytotoxic agent 5-fluorouracil. When produced in Escherichia coli cells overexpressing a biotin protein ligase, the FCU1-P67 fusion protein was efficiently mono-biotinylated. In the presence of 5-fluorocytosine, the biotinylated fusion protein conjugated to anti-rat TfR IgG3-Av efficiently killed rat Y3-Ag1.2.3 myeloma cells in vitro, while the same protein conjugated to an irrelevant (anti-dansyl) antibody fused to Av showed no cytotoxic effect. Efficient tumor cell killing was also observed when E. coli purine nucleoside phosphorylase was similarly targeted to the tumor cells in the presence of the prodrug 2-fluoro-2'-deoxyadenosine. These results suggest that when combined with P67-based biotinylation, anti-TfR IgG3-Av could serve as a universal delivery vector for targeted chemotherapy of cancer.
authors.affiliation: Department of Microbiology, Molecular Genetics and Molecular Biology Institute, University of California Los Angeles, CA 90095, USA.
authors.forename: Tsuneaki
authors.initials: T
authors.lastname: Asai
authors.forename: Ryan
authors.initials: R
authors.lastname: Trinh
authors.forename: Patrick P
authors.initials: PP
authors.lastname: Ng
authors.forename: Manuel L
authors.initials: ML
authors.lastname: Penichet
authors.forename: Letitia A
authors.initials: LA
authors.lastname: Wims
authors.forename: Sherie L
authors.initials: SL
authors.lastname: Morrison
mesh_terms: D000818:Animals; D000918:Antibody Specificity / Q000235:genetics; D000970:Antineoplastic Agents / Q000008:administration & dosage; D001360:Avidin / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D001710:Biotin / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D045744:Cell Line, Tumor; D016503:Drug Delivery Systems / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D007074:Immunoglobulin G / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D009101:Multiple Myeloma / Q000188:drug therapy*; D011355:Prodrugs / Q000008:administration & dosage* / Q000378:metabolism; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods*; D017434:Protein Structure, Tertiary; D011683:Purine-Nucleoside Phosphorylase / Q000008:administration & dosage* / Q000235:genetics* / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000008:administration & dosage / Q000378:metabolism; D016896:Treatment Outcome
chemical_list: D000970:Antineoplastic Agents; D007074:Immunoglobulin G; D011355:Prodrugs; D011993:Recombinant Fusion Proteins; D001360:Avidin; D001710:Biotin; D011683:Purine-Nucleoside Phosphorylase