Hybrid toxins are constructed by covalently linking a toxic protein with a receptor binding protein for specific killing of target cells. Recently, hybrid toxins have been shown to be useful for in vitro purging of unwanted cells before bone marrow transplantation for treatment of various forms of leukemia and lymphomas, and have the potential for treating autoimmune diseases and AIDS. However, the receptor-mediated entry mechanism(s) through which the hybrids effect cell intoxication has not been actively studied. In this proposal, the effects of various variables involved in the receptor-mediated endocytosis on cell intoxication by various hybrid toxins will be studied. The effects of various metabolic inhibitors and low temperatures on internalization, diacytosis, degradation, subunit dissociation and intracellular distribution of hybrids of diphtheria toxin (DT) and its fragment A (DTA) conjugated to asialoorosomucoid (ASOR) or its reduced and carboxymethylated cyanogen bromide fragment I (RC-ASCNBr-I) will be studied in rat, rabbit and guinea pig hepatocytes, as well as in human hepatoma Hep G2 cells. The amount of the intracellular hybrid can be released by diacytosis and retained by the cell, as well as their molecular forms with respect to the size and receptor-binding activity also will be determined. Then the distribution of active DTA-containing molecules in the subcellular particles will be determined by fractionation in Percoll and sucrose density gradients. Subsequently hybrid toxins of DTA, DT and a translocation-competent DT mutant protein (CRM9) to asialoovine submaxillary glycoprotein, human transferrin or bovine insulin will be synthesized and similarly studied in rat hepatocytes and Hep G2 cells to determine the effect of varying the receptor-binding affinity, the mode of endocytosis and the target cell type on hybrid toxicity. Various endosomal particles preloaded with active DTA-containing molecules will be isolated under optimal conditions and then studied for release of DTA in a cell-free system to identify the endosomal compartment from which DTA moiety is translocated into the cytosol. Finally, hybrids of a murine anti- transferrin receptor monoclonal antibody will be conjugated to DTA, DT or CRM9 and the above studies will be repeated in various human cancer cells. The results of the proposed studies will lead us to know whether or not there are common and specific variables affecting toxicity of different hybrids utilizing different receptors and modes of receptor-mediated endocytosis for cell killing. This information will be very useful for future formulation of more efficient therapeutic hybrid toxins.
