CHARACTERIZATION OF CYTOTOXIC ACTIVITY OF SAPORIN ANTI-GP185 HER-2 IMMUNOTOXINS/

The oncogene HER-2/neu encodes a trans-membrane receptor of 185 kDa wi th tyrosine-kinase activity. Over-expression of this molecule has been reported in a significant proportion of human breast and ovarian carc inomas, characterized by a poor clinical prognosis. Two monoclonal ant ibodies (MAbs), recognizing distinct epitopes of the gp 185 extracellu lar domain, have been utilized in the present study for the production of immunotoxins (ITs) by conjugation to the type-1 RIP (ribosome-inac tivating protein) plant toxin saporin 6 (SAP). These ITs have been sho wn to retain tumor-specificity and specifically to inhibit protein syn thesis in the gp185HER-2(+) SK-BR-3 breast-carcinoma cell line with IC 50 values lower then 1 nM. Kinetics of the cytotoxic activity of the I Ts are characterized by a slow rate, since incubation times ranging fr om 24 to 60 hr, depending on the different degree of expression of the receptor, are required to determine > 90% inhibition in the incorpora tion of radiolabeled leucine. However, the cytotoxic activity of these ITs, as evaluated by a more sensitive clonogenic assay, appears highl y potent, since we have observed that 3 to 4 logs of cells are killed upon exposure to the ITs for short times at concentrations ranging fro m 1 to 5 x 10(-8) M. (C) 1993 Wiley-Liss, Inc.