Immunotoxins (IT-As) synthesized by conjugating cytotoxic plant proteins (e.g. ricin A chain [RTA]) to monoclonal antibodies (MAbs) recognizing tumor-associated antigens represent a promising approach to cancer therapy. To kill target cells, such IT-As must be internalized after binding to surface antigens, and the RTA moiety must be delivered to cytosolic ribosomes where the 60S subunit is irreversibly inactivated. Early clinical trials with IT-As have demonstrated partial tumor regressions in some patients, but efficacy has been modest with currently available constructs. Many factors are believed to limit the efficacy of IT-As including rapid serum clearance, inadequate tumor penetration, heterogeneity of target antigen expression, and inadequate delivery of RTA to ribosomes. The overall objective of this project is to investigate methods of manipulating the endocytosis, intracellular routing, translocation, and metabolism of IT-As, so that more potent cytotoxicity can be achieved in a tumor cell-specific manner. Particular emphasis will be placed on delineating the site and mechanisms of translocation of RTA (and similar toxins), since kinetic studies suggest that this is the rate limiting step in cell intoxication. Four specific goals are enunciated. First, we will contrast the magnitude of intracellular trafficking of effective and ineffective IT-As to the trans Golgi region of wild type and translocation-defective mutant cell lines by monitoring covalent modifications of the oligosaccharide side chains of RTA by Golgi-specific enzymes. These experiments are of interest since it is widely postulated that translocation occurs from the trans Golgi region of cells. Second, we will study differences in the intracellular routing and cytotoxicity of wild type and mutant RTA constructs genetically engineered to express amino acid sequences targeting the toxin to specific intracellular compartments (e.g. trans Golgi, endoplasmic reticulum lumen (ER), ER membrane, or lysosomes). It is predicted that enhanced trafficking to putative translocation-competent organelles (e.g. Golgi/ER) will augment cytotoxicity, whereas targeting to lysosomes will diminish toxicity compared with wild type RTA. Third, the translocation-competence of specific organelle membranes will be tested in two different cell-free assay systems after purification of endosomes, Golgi, ER, and lysosomes on sucrose and Percoll density gradients. Fourth, membrane glycoproteins involved in translocating RTA and other toxins across organelle membranes will be identified and characterized by a """"""""nearest neighbor"""""""" crosslinking strategy. The importance of translocation-associated membrane proteins identified by cross-linking protocols will be verified by membrane glycoprotein depletion and reconstitution experiments. A complete comprehension of the events involved in intracellular trafficking and translocation of RTA should permit synthesis of a new generation of more potent IT-As with preferential localization in translocation-competent subcellular compartments. Furthermore, the studies outlined in this grant are of general interest since similar plant and bacterial toxins mediate many important medical syndromes (Diphtheria, Shigellosis, Cholera, Pseudomonas shock) and because the basic mechanisms underlying protein translocation across biological membranes remain poorly understood.
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