C. Dilazzaro et al., IMMUNOTOXINS TO THE HER-2 ONCOGENE PRODUCT - FUNCTIONAL AND ULTRASTRUCTURAL ANALYSIS OF THEIR CYTOTOXIC ACTIVITY, Cancer immunology and immunotherapy, 39(5), 1994, pp. 318-324
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.