Recombinant single-chain antibody fusion construct targeting human melanoma cells and containing tumor necrosis factor.
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.
Liu
Yuying
Y
Immunopharmacology and Targeted Therapy Section, Department of Bioimmunotherapy, University of Texas M.D. Anderson Cancer Center, Houston, TX 77030, USA.
Cheung
Lawrence H
LH
Marks
John W
JW
Rosenblum
Michael G
MG
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*
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