Relative cytotoxic activity of immunotoxins reactive with different epitopes on the extracellular domain of the c-erbB-2 (HER-2/neu) gene product p185

Different epitopes on the extracellular domain of the HER-2 receptor can se rve as distinct targets for immunotoxins. To determine the optimal epitope target for immunotoxin therapy, 7 anti-HER-2 ricin A chain murine monoclona l immunotoxins, each reactive with different epitopes of HER-2 receptor, we re tested for cytotoxic activity. The immunotoxins produced 1.2-4.6 logs of cytotoxicity in limiting dilution clonogenic assays with 2 breast cancer c ell lines that overexpressed HER-2. Cytotoxicity did not correlate with imm unoglobulin isotype, binding affinity, relative position of epitopes or int ernalization of the anti-HER-2 immunotoxins. Interestingly, the most and le ast effective immunotoxins bound to epitopes in very close proximity. Compe titive binding assays with unconjugated antibodies have previously indicate d that our antibodies recognized epitopes that are arranged in a linear arr ay. To orient this relative epitope map deletions were prepared in the HER- 2/neu gene and these mutant constructs were expressed in NIH3T3 cells. Epit ope expression was determined by antibody binding and radioimmunoassay. Epi topes targeted by the PB3, 454C11 and NB3 antibodies are localized N-termin al to the epitopes recognized by ID5, BD5, 741F8 and 520C9 antibodies. The 2 non-conformational epitopes PB3 and NB3 were localized to regions corresp onding to amino acides 78-242 of the p185(HER-2) protein. (C) 1999 Wiley-Li ss, Inc.