Molecular Engineering to Retarget Human Cytokines
Abstract
Overexpression of HER2/neu in breast cancer is one mechanism responsible for cellular progression of the neoplastic phenotype. Overexpression 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 antibody sfv23 recognizes the cell-surface domain of HER2/neu. The cDNA was fused to the cDNA encoding human TNF and cloned into an expression plasmid. The fusion protein was expressed and purified by ion exchange chromatography. SD S-PAGE demonstrated a single band at the expected molecular weight (43 kDa). Western analysis confirmed the presence of both the antibody component and the TNF component. The construct was shown to have biological activity similar to that of authentic TNF (S.A. 420 nM) against L-929 cells. The scfv23/TNF construct bound to SKBR-3 (HER2 positive) but not to A-375 human melanoma (HER2 negative) cells. Cytotoxicity studies against breast carcinoma cells (SKBR-3-HP) overexpressing HER2/neu demonstrate that the sfv23/TNF fusion construct was 1000-fold more active than free 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 levels of HER2/neu.
Document Details
- Document Type
- Technical Report
- Publication Date
- Jul 01, 2000
- Accession Number
- ADA384140
Entities
People
- Michael Rosenblum
Organizations
- The University of Texas MD Anderson Cancer Center