Immunotoxins (IT-As) synthesized by conjugating cytotoxic plant proteins (e.g. ricin A chain) to monoclonal antibodies (MoAbs) recognizing tumor-associated antigens represent a promising new approach to cancer therapy. To kill target cells, such IT-As must be internalized after binding to surface antigens, and the ricin A chain moiety must be delivered to cytosolic ribosomes where the 60S subunit is irreversibly inactivated. Preclinical and phase I clinical trials with IT-As have demonstrated encouraging anti-tumor effects, but in most cases only partial tumor regressions have occurred. Many factors are believed to contribute to the modest efficacy of IT-As including rapid serum clearance, inadequate tumor penetration, heterogeneity of target antigen expression, and inadequate delivery of A-chain molecules to ribosomes. Other investigators have described approaches to the first 3 problems. In this project, we propose to study the endocytosis, intracellular routing, translocation, and metabolism of IT-As, using biochemical, morphologic, electrophoretic, chromatographic, autoradiographic, and cell fractionation techniques. Three informative model systems will be employed: 1) IT-As targeting different epitopes of the CD2 molecule which differ 100-fold in cytotoxicity for T cells, 2) parental and """"""""translocation-deficient"""""""" mutants of the AKR/A cell line which differ 300-fold in sensitivity to anti-Thy 1.1 IT-As, and 3) anti-CD19 and anti-CD3 IT-As which are currently in clinical use. To study differences in intracellular routing, we will utilize post-embedding electron immunomicroscopy, cellular radioimmunoassays, and Percoll gradient fractionation of cells. Specific radiolabeling of the MoAb and ricin A moieties will be employed to analyze the fates of these two components separately. Two novel assays will be used to rigorously test the prevailing hypothesis that IT-As are translocated to the cytosol from the trans Golgi region. First, covalent modifications of the oligosaccharide side chains of ricin A chain by Golgi-specific enzymes will be monitored following endocytosis. Secondly, undisrupted endosomes, Golgi, and lysosomes will be purified on sucrose gradients and tested in a cell-free, reconstitution assay to determine their capability of translocating A chain to a purified cytosolic fraction. Intracellular metabolism of IT-As will be determined by SDS-PAGE, HPLC, and thin layer chromatography. The impact of new cross-linking agents and potentiating agents on intracellular behavior will also be assessed. A complete comprehension of these events should suggest interventions which will maximize translocation of ricin A chain to ribosomes and should facilitate more effective design, selection, and utilization of immunotoxins. Furthermore, the studies outlined in this grant may be of interest to basic immunologists since the lymphocyte surface antigens studied mediate important recognition and/or cell activation functions, and delineation of the behavior of MoAbs following binding may serve as ligand models in circumstances where the natural ligands are unknown or difficult to employ.
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