Recombinant single-chain antibody fusion construct targeting human melanoma cells and containing tumor necrosis factor

Int J Cancer. 2004 Feb 10;108(4):549-57. doi: 10.1002/ijc.11524.


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.

Publication types

  • Comparative Study

MeSH terms

  • Animals
  • Antibodies, Neoplasm / genetics*
  • Antigens, Neoplasm / immunology
  • Drug Delivery Systems
  • Electrophoresis, Polyacrylamide Gel
  • Escherichia coli / genetics
  • Gene Expression
  • Humans
  • Immunoglobulin Fragments / genetics
  • Immunotoxins / administration & dosage*
  • Immunotoxins / isolation & purification
  • Melanoma, Experimental / drug therapy*
  • Melanoma, Experimental / immunology
  • Mice
  • Mice, Nude
  • Proteoglycans / immunology*
  • Receptors, Tumor Necrosis Factor / metabolism
  • Recombinant Fusion Proteins / administration & dosage*
  • Recombinant Fusion Proteins / isolation & purification
  • Tissue Distribution
  • Tumor Cells, Cultured
  • Tumor Necrosis Factor-alpha / genetics*


  • Antibodies, Neoplasm
  • Antigens, Neoplasm
  • Immunoglobulin Fragments
  • Immunotoxins
  • Proteoglycans
  • Receptors, Tumor Necrosis Factor
  • Recombinant Fusion Proteins
  • Tumor Necrosis Factor-alpha
  • gp240 glycoprotein, human
  • immunoglobulin Fv