title: Recombinant single-chain antibody fusion construct targeting human melanoma cells and containing tumor necrosis factor.
abstract: Fusion constructs targeting tumor cells have significant potential applications against both solid tumors and hematologic malignancies. We developed a fusion construct of tumor necrosis factor (TNF) and a single-chain antibody (scFvMEL) recognizing the gp240 antigen on human melanoma cells. The scFvMEL/TNF construct, like TNF itself, was found to exist in solution primarily as a trimer of 45 kDa monomers (trimeric molecular weight = 135 kDa). The fusion construct bound specifically to gp240 antigen-positive but not to antigen-negative cells. The TNF component of the construct was biologically active (specific activity = 1 x 10(7) U/mg) compared with free TNF (specific activity = 2.6 x 10(7) U/mg) and was more cytotoxic to antigen-positive A375-M melanoma cells (IC(50) = 100 pM) than TNF alone (IC(50) = 1,000 pM) and, additionally, was active against AAB-527 melanoma cells (IC(50) = 20 nM) resistant to TNF itself (IC(50) > 1,000 nM). The augmented cytotoxicity was mediated by antibody-specific binding to the cell surface. Both A375-M and AAB-527 cells were shown to express TNFR1 and TNFR2 on the cell surface. The TNF moiety of the fusion construct was efficiently delivered into cells in time-dependent increase in cytosol as assessed by immunofluorescent staining of human melanoma cells. Radiolabeled scFvMEL/TNF localized effectively in human melanoma xenografts in nude (nu/nu) mice with a tumor:blood ratio of approximately 8 at 72 hr after administration. Our studies suggest that because of its unique biologic activity and low antigenic potential, scFvMEL/TNF makes an excellent cytotoxic protein for potential clinical treatment of human melanoma.
authors.affiliation: Immunopharmacology and Targeted Therapy Section, Department of Bioimmunotherapy, University of Texas M.D. Anderson Cancer Center, Houston, TX 77030, USA.
authors.forename: Yuying
authors.initials: Y
authors.lastname: Liu
authors.forename: Lawrence H
authors.initials: LH
authors.lastname: Cheung
authors.forename: John W
authors.initials: JW
authors.lastname: Marks
authors.forename: Michael G
authors.initials: MG
authors.lastname: Rosenblum
mesh_terms: D000818:Animals; D000912:Antibodies, Neoplasm / Q000235:genetics*; D000951:Antigens, Neoplasm / Q000276:immunology; D016503:Drug Delivery Systems; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics; D000922:Immunotoxins / Q000008:administration & dosage* / Q000302:isolation & purification; D008546:Melanoma, Experimental / Q000188:drug therapy* / Q000276:immunology; D051379:Mice; D008819:Mice, Nude; D011509:Proteoglycans / Q000276:immunology*; D018124:Receptors, Tumor Necrosis Factor / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000008:administration & dosage* / Q000302:isolation & purification; D014018:Tissue Distribution; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics*
chemical_list: D000912:Antibodies, Neoplasm; D000951:Antigens, Neoplasm; D007128:Immunoglobulin Fragments; D000922:Immunotoxins; D011509:Proteoglycans; D018124:Receptors, Tumor Necrosis Factor; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; C094441:gp240 glycoprotein, human; C055966:immunoglobulin Fv