The specific aims of this investigation are: (i) the modification of the DNA encoding fragment B at three restriction sites such that a cysteine codon is C-terminal and a unique Pstl site is introduced just upstream from the Cys codon; (ii) the assembly of hybrid toxins at the level of protein chemistry, using the sulfhydryl group of the C-terminal Cys to form disulfide linkage with modified thyrotropin releasing hormone and melanocyte stimulating hormone as the cell receptor-specific ligand; (iii) the assembly of hybrid toxin genes through fusion of a synthetic oligonucleotide encoding Alpha-MSH with varying amounts of the tox gene; (iv) expression and purification of the toxin related-hormone fusion polypeptides; and (v) the determination of specific toxicity and spectrum of cytotoxicity in both mouse and human cell lines. The long range objectives of this proposal are to construct a family of toxin-hormone hybrid genes and to test their respective protein fusion products for targeted cytotoxicity. The toxin-hormone hybrid genes will be assembled from those portions of the diphtheria toxin structural gene which include the enzymatically active fragment A and varying amounts of the DNA which encode the B fragment. Preliminary results have clearly shown that we have been able to genetically construct, express, and demonstrate selective cytotoxicity of one of the diphtheria toxin-related Alpha-MSH fusion proteins described in this application. The comparative cytotoxicities of the toxin-related Alpha-MSH hybrids that are assembled through both disulfide linkage and gene fusion should provide additional insight into those functional domains of diphtheria toxin fragment B which (i) are involved in the binding of diphtheria toxin to its receptor on the surface of sensitive eukaryotic cells, and (ii) are essential for the membrane translocation of fragment A. In addition, the genetic construction and expression of these gene fusions whose products have targeted cytotoxicity toward human melanoma cells raises the potential for the development of a new class of therapeutic biologicals for malignant melanoma.
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