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A novel recombinant fusion toxin targeting HER-2/NEU-over-expressing cells and containing human tumor necrosis factor
M G Rosenblum1, S A Horn, L H Cheung
1Immunopharmacology and Targeted Therapy Section, Department of Bioimmunotherapy, University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030, USA. mrosenbl@notes.mdacc.tmc.edu
Abstract:
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.
Insights
A novel fusion protein combining an antibody targeting HER2/neu with tumor necrosis factor (TNF) shows enhanced cancer cell killing. This HER2-targeted TNF therapy is more effective than native TNF against breast cancer cells with intermediate HER2/neu expression.
Area of Science:
- Oncology
- Molecular Biology
- Immunotherapy
Background:
- Over-expression of the proto-oncogene HER2/neu drives cancer progression and reduces sensitivity to tumor necrosis factor (TNF).
- Targeting HER2/neu offers a potential strategy to overcome treatment resistance in HER2/neu-expressing tumors.
Purpose of the Study:
- To develop and evaluate a novel fusion protein construct combining a HER2/neu-specific antibody with TNF for targeted cancer therapy.
- To assess the efficacy of the sFv23/TNF fusion protein against HER2/neu-positive breast cancer cells in vitro.
Main Methods:
- A single-chain antibody (sFv23) targeting HER2/neu was genetically fused to human TNF.
- The sFv23/TNF fusion protein was expressed in E. coli, purified, and characterized using SDS-PAGE and Western analysis.
- Biological activity and cytotoxicity were evaluated against various cancer cell lines, including HER2-positive SK-BR-3 cells.
Main Results:
- The purified sFv23/TNF fusion protein exhibited comparable TNF activity to native TNF and specifically bound to HER2-positive cells.
- The fusion construct demonstrated a 1,000-fold increase in cytotoxic activity against HER2/neu-overexpressing breast cancer cells compared to free TNF.
- Cells with very high HER2/neu expression showed relative resistance to both native TNF and the fusion construct.
Conclusions:
- Fusion constructs targeting HER2/neu with TNF represent a potent in vitro cytotoxic agent.
- This targeted therapy shows promise against tumors with intermediate HER2/neu expression, offering a potential therapeutic advantage over native TNF.