A novel recombinant fusion toxin targeting HER-2/NEU-over-expressing cellsand 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 responsi ble for cellular progression of the neoplastic phenotype. Transfection of m ammalian cells and over-expression of HER2/neu appears to result in reduced sensitivity to the cytotoxic effects of tumor necrosis factor (TNF) and re duced 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 c oli, The fusion protein was expressed and purified by ion-exchange chromato graphy, 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 tes ted for TNF activity against L-929 cells and found to have biological activ ity 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-negat ive) cells. Cytotoxicity studies against log-phase human breast carcinoma c ells (SKBR-3-HP) over-expressing HER2/neu demonstrate that the sFv23/TNF fu sion construct was 1,000-fold more active than free TNF. Tumor cells expres sing higher levels of HER2/neu (SKBR-3-LP) were relatively resistant to bot h the fusion construct and native TNF, These studies suggest that fusion co nstructs targeting the HER2/neu surface domain and containing TNF are more effective cytotoxic agents in vitro than native TNF and may be effective ag ainst tumor cells expressing intermediate, but not high, levels of HER2/neu , Int. J. Cancer 88:267-273, 2000, (C) 2000 Wiley-Liss, Inc.