The specific aims of this proposal are focused on the continuation of the development of diphtheria toxin-related / peptide hormone fusion proteins for eukaryotic cells receptor specific targeted therapy. We have demonstrated that diphtheria toxin receptor binding domain substitution with either alpha-melanocyte stimulating hormone, or interleukin-2 sequences results in the formation of unique toxins with highly specific target cell activity. Alpha-MSH-toxin has been show to selectively cytotoxic for alpha-MSH receptor bearing malignant melanoma cells in vitro; and essentially devoid of activity for cells lacking the receptor. In a similar fashion, IL-2-toxin has been shown to be selectively toxic for high affinity IL-2 receptor bearing cells both in vitro and in vivo. Moreover, the cytotoxic action of IL-2-toxin has been show to be mediated through the IL-2 receptor, to required passage through an acidification competent vesicle, and to ADP-ribosylate the intracellular target enzyme, elongation factor 2. The biologic action of these chimeric toxins are, as anticipated, unaffected by the presence of anti-diphtheria toxin antibodies in serum. the present proposal is directed towards the further protein engineering of alpha-MSH-toxin in order to generate derivative proteins which are resistant to proteolytic degradation. Studies directed toward the continue development of Sacchromyces cerevisiae dph mutant strains as cloning and expression vehicles is also proposed. Finally, I propose the genetic construction, expression, purification, reconstitution, and in vitro studies of a second generation chimeric toxin using the alpha-subunit of the alpha / beta gonadotrophin Leuteinizing Hormone as the ligand component of a new chimeric toxin. The long term goal of these studies are centered on the development of these genetically engineered chimeric toxins as prototypes in the development of new biologicals which are targeted at specific cell surface receptors that are characteristic for selected malignancies.
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