Immunotoxins synthesized by conjugating cytotoxic plant proteins to monoclonal antibodies recognizing tumor-associated antigens have appeared promising in pre-clinical studies but have shown only modest efficacy so far in clinical trials. To kill target cells, immunotoxins must be internalized after binding to surface antigens and a commonly used plant protein, ricin A-chain must be delivered to cytosolic ribosomes where the 60S subunit is irreversibly inactivated. The overall objective of this project is to investigate methods of manipulating the intracellular routing of immunotoxins, so that the therapeutic index can be improved. Emphasis will be placed on facilitating translocation of toxins to the cytosol since this is the rate-limiting step in cell intoxication. Four specific goals are enunciated: First, the investigator will identify and characterize the membrane proteins normally involved in ricin A-chain translocation by a """"""""nearest neighbor"""""""" cross-linking strategy. The importance of translocation-associated membranes identified by cross- linking will be verified by membrane glycoprotein depletion and reconstitution experiments. Second, the prevailing hypothesis will be tested that endoplasmic reticulum """"""""translocon"""""""" pores are critical for toxin translocation by selective immunodepletion of the Sec 61p and TRAM proteins which are known to be essential structural and functional components of the ER translocon channels. Third, mutant ricin A-chain constructs will be genetically engineered which express in tandem amino acid sequences targeting the toxin to translocation-competent intracellular compartments (e.g., GOLGI, ER) followed by sequences possessing novel """"""""pore-forming"""""""" domains (e.g., GALA) for membrane penetration. Fourth, the translocation efficiency of immunotoxins will be enhanced by chemical conjugation to polycarboxylic acids (e.g., polypropylacrylic acid) which undergo conformational changes upon transfer from the neutral extracellular compartment to the acidic environment of endosomal compartments. These conformational changes result in endosomal membrane disruption permitting rapid egress of ricin A-chain to the cytosome. It is believed in a more complete comprehension of the events involved in the intracellular trafficking and translocation of ricin A-chain should permit synthesis of more effective clinical reagents. Furthermore, the studies in this project are believed to be important independent of immunotoxin therapy since similar 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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