It is now clear that recombinant DNA and protein engineering methodologies can be applied to the development of fusion toxins in which the receptor binding domain of the ADP-ribosyltransferase bacterial protein toxins (diphtheria and Pseudomonase exotoxin A) can be replaced with a variety of eukaryotic cell receptor specific ligands. The fusion toxins have been shown to be exceedingly potent and selective both in vitro and in vivo. This research proposal is focused on the genetic construction, expression, and structure/functional analysis of a new class of fusion toxin which is based upon the ribosome inactivating protein Shiga-like toxin. The cytotoxicity of Shiga-like toxin is based upon the enzymatic activity of the A1 subunit to cleave adenine 2325 from the 28S ribosomal RNA and thereby inhibit protein synthesis. Recent studies have shown that a Shiga- like A1 subunit:IL-2 fusion toxin can be genetically constructed, expressed, and purified. The Shiga-like A subunit-IL-2 fusion toxin is a highly potent and selective cytotoxic agent for IL-2 receptor bearing target cells. In addition to structure function analysis, both in vitro and in vivo studies are proposed to compare the diphtheria toxin-based IL-2 fusion toxin with the Shiga-like toxin based IL-2 fusion toxin. Since these two agents inhibit protein synthesis in target cells by different mechanisms, studies to examine the additive or synergistic action of these two classes fusion toxin are proposed. The diphtheria toxin-based fusion toxins are cytotoxic by virtue of their ability to ADP-ribosylate elongation factor 2 in the cytosol of target cells. Importantly, only EF-2 that cycles off the ribosome is sensitive to ADP-ribosylation, and as a result, rapidly proliferating cells are more sensitive than quiescent cells. In contrast, the ribosome inhibiting proteins have been shown to cleave adenine 2325 from the 28S ribosomal RNA regardless of the basal rate of protein synthesis, and therefore should have a higher apparent activity against slow growing cells. This difference suggests that the Shiga-like toxin-based fusion toxins would have increased efficacy in the treatment of more indolent tumors. Research proposed in this application will provide a new class of fusion toxins, increase our understanding of the protein engineering of fusion toxins, and provide additional experimental therapeutic agents for the treatment of both hematologic and solid tumors.
| vanderSpek, J; Cosenza, L; Woodworth, T et al. (1994) Diphtheria toxin-related cytokine fusion proteins: elongation factor 2 as a target for the treatment of neoplastic disease. Mol Cell Biochem 138:151-6 |
