title: Design of a bifunctional fusion protein for ovarian cancer drug delivery: single-chain anti-CA125 core-streptavidin fusion protein.
abstract: We have developed a universal ovarian cancer cell targeting vehicle that can deliver biotinylated therapeutic drugs. A single-chain antibody variable domain (scFv) that recognizes the CA125 antigen of ovarian cancer cells was fused with a core-streptavidin domain (core-streptavidin-VL-VH and VL-VH-core-streptavidin orientations) using recombinant DNA technology and then expressed in Escherichia coli using the T7 expression system. The bifunctional fusion protein (bfFp) was expressed in a shaker flask culture, extracted from the periplasmic soluble protein, and affinity purified using an IMAC column. The two distinct activities (biotin binding and anti-CA125) of the bfFp were demonstrated using ELISA, Western blot and confocal laser-scanning microscopy (CLSM). The ELISA method utilized human NIH OVCAR-3 cells along with biotinylated bovine serum albumin (B-BSA) or biotinylated liposomes, whereas, the Western blot involved probing with B-BSA. The CLSM study has shown specificity in binding to the OVCAR-3 cell-line. ELISA and Western blot studies have confirmed the bifunctional activity and specificity. In the presence of bfFp, there was enhanced binding of biotinylated antigen and liposome to OVCAR-3 cells. In contrast, the control EMT6 cells, which do not express the CA125 antigen, showed minimal binding of the bfFp. Consequently, bfFp based targeting of biotinylated therapeutic drugs, proteins, liposomes, or nanoparticles could be an alternative, convenient method to deliver effective therapy to ovarian cancer patients. Peritoneal infusion of the bfFp-therapeutic complex could also be effective in locally targeting the most common site of metastatic spread.
authors.affiliation: Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, AB, Canada.
authors.forename: Welson Wen-Shang
authors.initials: WW
authors.lastname: Wang
authors.forename: Dipankar
authors.initials: D
authors.lastname: Das
authors.forename: Stephen A
authors.initials: SA
authors.lastname: McQuarrie
authors.forename: Mavanur R
authors.initials: MR
authors.lastname: Suresh
mesh_terms: D000818:Animals; D000918:Antibody Specificity; D000970:Antineoplastic Agents / Q000378:metabolism*; D001665:Binding Sites; D001710:Biotin / Q000031:analogs & derivatives / Q000378:metabolism*; D015153:Blotting, Western; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D004337:Drug Carriers; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins; D051379:Mice; D018613:Microscopy, Confocal; D010051:Ovarian Neoplasms / Q000276:immunology / Q000378:metabolism*; D011506:Proteins / Q000276:immunology / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D019809:Streptavidin / Q000235:genetics / Q000378:metabolism*
chemical_list: D000970:Antineoplastic Agents; D004337:Drug Carriers; D007135:Immunoglobulin Variable Region; D047908:Intracellular Signaling Peptides and Proteins; C549756:NBR1 protein, human; D011506:Proteins; D011993:Recombinant Fusion Proteins; D001710:Biotin; D019809:Streptavidin