The long term objectives of this project include: (1) attaining maximum specific cytotoxicity possible from ricin A chain conjugates through a better understanding of internalization processes and (2) improving the pharmacology of these conjugates to permit sufficient in vivo targeting for systematic therapeutic usage. Past studies have shown that the specificity characteristics of many cell surface reactive antibodies and ligands can be conferred upon the cytolytic A chain subunit of the plant toxic ricin. The highly selective cytotoxic properties of such conjugates allow for their usage in vitro for complete destruction of target cells without affecting antigen negative """"""""bystander"""""""" cells. Establishing a large panel of well characterized A chain conjugates is one of the aims of this study. Such agents at present can be used for the selection of antigen and receptor varient cells for fundamental studies and for the ex vivo elimination of tumor cells from bone marrow before usage in autologous transplantation therapy. These two practical aspects will be pursued using structural biochemical techniques and clonogenic assays respectively. A comprehensive examination of conjugate internalization pathways and mechanisms as well as the inluence of known potenitating agents on these parameters will be undertaken since access of A chain to cytoplasmic ribosomes appears to be the rate limiting step to toxicity. Time lapse fluorescence microscopy will be used to follow the traverse of appropriately tagged conjugates from the cell surface into various compartments within living cells. The ability to manipulate release into the cytoplasm promises to make these cytotoxins even faster acting and more potent then they already are. Cell typetarget antigen combinations which have been resistent to the effect of a specific cytotoxin may be rendered susceptible to its action by developing additional internalization modifiers or vescle disrupting agents with improved qualities. Systemic in vivo applications thus far have been thwarted in part at least by the ability of animals to eliminate conjugates from the circulation before they localize at the tumor site. Cytotoxins with preferable pharmacological properties will be formed by chemical, enzymatic or biosynthetic alterations of the composite antibody and A chain halves. Blood levels of these improved molecules in animals will be monitored with radioimmunoassays while localization at target sites and therapeutic response will be tested using human tumor cells heterotransplanted into nude mice.
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