The overall goal is to facilitate specific intracellular delivery of cytotoxic agents to human T-cell leukemia using antibody- directed liposomes. The approach will begin with in vitro optimization of endocytosis and cytotoxicity followed by in vivo studies of efficacy in a nude mouse model. We have already demonstrated efficacy of antibody-directed liposomes containing cytotoxic drug against murine T-cell leukemia in vitro and for elimination of leukemia cells from bone marrow. Recent studies show the ability to selectively inhibit growth in vitro of human T-leukemia cell lines. Liposomal drug delivery to the T-leukemia cells will be enhanced in vitro by: 1) The proper combination of antibodies to provide optimal cell binding and endocytosis will be selected. Ligands which target internalizing receptors such as antibodies against receptors for transferrin, interleukin 2, low density lipoprotein and major histocompatibility complex Class 1 will be compared to surface T-cell antigens such as the CD2, CD3, CD4, CD5, and CD7. Double targeting with the two different types of antibodies will also be used as another way to enhance specificity and efficacy. 2) Drug release will be improved through formulation of pH- sensitive antibody-targeted liposomes which will fuse with the plasma membrane and release their contents into the cytoplasm when they encounter the acidic environment of the endosome. 3) New techniques for trapping drugs using pH gradients will be used to increase encapsulation efficiency and decrease leakage. Thus allowing testing of more potent cytotoxic agents. 4) The optimal liposome formulation will be selected on the results of new fluorescent assays of binding and endocytosis, electron microscopy using colloidal gold as a marker of liposomal contents, and by growth inhibition and limiting dilution assays for efficacy against the T-leukemia. The in vivo efficacy will be tested against human T-cell leukemia lines established in athymic nude mice. The specificity and efficacy of the targeted liposomes containing cytotoxic agents for tumor growth inhibition will be compared to free drug, antibody alone, and non-specific liposomes. Toxicity to normal human hematopoietic progenitor cells will be examined in colony assays for BFU-E and CFU-C. Antibody-targeted liposomes, with their attributes of flexibility in selection of cytotoxic agent, multiple antibody-targeting, and pharmacokinetic advantages provide a promising new approach to treatment of T-cell leukemia and lymphoma in adult and pediatric cancers, as well as to many other disorders involving T-cells, such as acquired immunodeficiency syndrome, graft versus host disease, and autoimmune disorders.
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