Molecular Engineering to Retarget Human Cytokines
Abstract
Overexpression of the HER2/neu oncogene in breast cancer cells blocks the biological activity of the cytokine TNF and allows these cells to escape immune surveillance mechanisms. We have developed a fusion construct composed of a single-chain antibody recognizing HER2/neu and TNF. Against cells over-expressing HER2/neu and resistant to TNF, this scfv23/TNF fusion construct was shown to have impressive cytotoxic properties (I.C.50 = 35-50 nM) . Signal transduction studies indicate that the cytotoxic effects of the scfv23/TNF construct are not mediated by changes in TNFR-1, TRADD, TRAF2, NfkB, AKT, or caspase 3 or 7. In contrast, we found activation of caspase 8 and 6 to be the primary pathway for activation of apoptosis by scfv23/TNF. We also found that treatment of SKBR3 cells with scfv23/TNF resulted in inhibition of MMP-9. This is in contrast to treatment of cells with TNF, which results in activation of MMP-9. In preparation for animal model studies, we have also developed a bacterial expression system for generation of the requisite large amounts of protein needed for the planned in vivo studies.
Document Details
- Document Type
- Technical Report
- Publication Date
- Jul 01, 2001
- Accession Number
- ADA398551
Entities
People
- Michael G. Rosenblum
Organizations
- The University of Texas MD Anderson Cancer Center