Diphtheria-related peptide hormone gene fusions: a molecular genetic approach to chimeric toxin development.

Abstract

There has been considerable effort in chemically conjugating a variety of plant and bacterial toxins to monoclonal antibodies that are directed to surface antigens on target cells. Coupling has been mediated through disulfide linkage, and the resulting conjugates are known generically as immunotoxins. In general, there are a few shortfalls to this approach. For example, since it is clear that not all surface antigens are internalized, one cannot predict the fate of a given IT once bound to its determinant on the surface of a target cell. In addition, in most instances one must activate the amino moiety of lysine residues with a heterobifunctional reagent in order to form disulfide linkage between the ligand and toxophore components. Since the number of reactive groups may be large, the disulfide linked conjugate molecules most likely represent a family of isomeric molecules rather than a defined protein. As a result, one cannot readily manipulate the fine structure of an IT in order to probe the mechanism of toxophore entry into the target cell. The approach that our group has taken toward the development of targeted cytotoxins, however, differs in a fundamental way: Rather than chemically coupling the ligand with toxophore through a disulfide bond, we have turned to genetic engineering in order to create gene fusions whose chimeric products are joined through a peptide bond. Thus, we have genetically constructed a family of fusion genes in which the receptor binding domain of diphtheria toxin has been deleted and replaced with DNAs encoding either alpha-MSH or IL-2. In each instance, it was known that the polypeptide ligand component of the fusion protein bound to specific receptors on target cells and was internalized by receptor mediated endocytosis. We reasoned, therefore, that the substitution of the diphtheria toxin receptor binding domain by these ligands should result in the formation of 'new' toxins whose action should be targeted toward selected eukaryotic cells that expressed either the alpha-MSH or IL-2 receptor. As along as the ligand component was exposed on the surface of the chimeric toxin, the molecule should bind to its receptor and be drawn into the cell by receptor-mediated endocytosis. Since the toxin-related/peptide hormone fusion protein is the product of a chimeric gene, it is a single molecular species. This has allowed us to begin to probe by site-directed mutagenesis the structure of fragment B sequences that are required to facilitate the translocation of fragment A across the target cell membrane.(ABSTRACT TRUNCATED AT 400 WORDS)