IMMUNOTOXINS TO THE HER-2 ONCOGENE PRODUCT - FUNCTIONAL AND ULTRASTRUCTURAL ANALYSIS OF THEIR CYTOTOXIC ACTIVITY

Two immunotoxins were prepared using monoclonal antibodies (mAb) direc ted towards two distinct epitopes of the gp185(HER-2) extracellular do main, and the type I ribosome inactivating protein (RIP) plant toxin s aporin 6. Cell protein synthesis inhibition assay reveals that the imm unotoxins display a potent and specific cytotoxicity that is character ized by a slow rate, since the time required to inhibit incorporation of radiolabeled leucine completely ranges from 36 h to 60 h depending on the target cell line and the immunotoxin. Because this feature may hamper the immunotherapeutic use of these conjugates we analysed this further by studying the early phases of internalization of immunotoxin s by immunoelectron microscopy. The results of this study have demonst rated that the distribution pattern of the immunotoxins and of the unc onjugated mAb over the cell surface overlaps. Similarly the mAb and im munotoxins are internalized into the cell by two different pathways: v ia clathrin-coated pits or via smaller uncoated pits and vesicles. A h igher degree of internalization is achieved when the two immunotoxins are used in combination. Unlike the slow kinetics of cell intoxication the process of immunotoxin endocytosis is characterized by a rapid ra te of internalization (above 40% at 5 min in the SK-BR-3 cell line). A lthough these findings provide no clue to explain the mechanisms of th e slow rate of cytotoxicity of the two immunotoxins their rapid intern alization indicates that these reagents can be exploited in immunother apeutic approches to gp185(HER-2)-expressing malignancies.