A novel recombinant fusion toxin targeting HER-2/NEU-over-expressing cells and containing human tumor necrosis factor.
Over-expression of the proto-oncogene HER2/neu in breast cancer and certain other tumors appears to be a central mechanism that may be partly responsible for cellular progression of the neoplastic phenotype. Transfection of mammalian cells and over-expression of HER2/neu appears to result in reduced sensitivity to the cytotoxic effects of tumor necrosis factor (TNF) and reduced sensitivity to immune effector killing. The single-chain recombinant antibody sFv23 recognizes the cell-surface domain of HER2/neu. The cDNA for this antibody was fused to the cDNA encoding human TNF, and this sFv23/TNF fusion construct was cloned into a plasmid for expression in Escherichia coli. The fusion protein was expressed and purified by ion-exchange chromatography. SDS-PAGE demonstrated a single band at the expected m.w. (43 kDa). Western analysis confirmed the presence of both the antibody component and the TNF component in the final fusion product. The fusion construct was tested for TNF activity against L-929 cells and found to have biological activity similar to that of authentic TNF (SA 420 nM). The scFv23/TNF construct bound to SKBR-3 (HER2-positive) but not to A-375 human melanoma (HER2-negative) cells. Cytotoxicity studies against log-phase human breast carcinoma cells (SKBR-3-HP) over-expressing HER2/neu demonstrate that the sFv23/TNF fusion construct was 1, 000-fold more active than free TNF. Tumor cells expressing higher levels of HER2/neu (SKBR-3-LP) were relatively resistant to both the fusion construct and native TNF. These studies suggest that fusion constructs targeting the HER2/neu surface domain and containing TNF are more effective cytotoxic agents in vitro than native TNF and may be effective against tumor cells expressing intermediate, but not high, levels of HER2/neu.
Rosenblum
M G
MG
Immunopharmacology and Targeted Therapy Section, Department of Bioimmunotherapy, University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030, USA. mrosenbl@notes.mdacc.tmc.edu
Horn
S A
SA
Cheung
L H
LH
D000818:Animals; D001943:Breast Neoplasms; D002470:Cell Survival / Q000187:drug effects*; D003001:Cloning, Molecular; D004926:Escherichia coli; D005260:Female; D018734:Genes, erbB-2; D006801:Humans; D000922:Immunotoxins / Q000235:genetics / Q000493:pharmacokinetics / Q000633:toxicity*; D007700:Kinetics; D007739:L Cells; D008545:Melanoma; D051379:Mice; D000090063:Proto-Oncogene Mas; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000032:analysis / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000493:pharmacokinetics / Q000633:toxicity; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000493:pharmacokinetics / Q000633:toxicity*
D000922:Immunotoxins; D000090063:Proto-Oncogene Mas; D018719:Erb-b2 Receptor Tyrosine Kinases; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; C000715470:MAS1 protein, human