A TARGET-SPECIFIC CHIMERIC TOXIN COMPOSED OF EPIDERMAL GROWTH-FACTOR AND PSEUDOMONAS EXOTOXIN A WITH A DELETION IN ITS TOXIN-BINDING DOMAIN

We have fused the epidermal growth factor (EGF) to the amino terminus of Pseudomonas exotoxin A (PE) to create a cytotoxic agent, designated EGF-PE, which preferentially kills EGF-receptor-bearing cells. In thi s study, we analyzed the effect of the Ia domain, the binding domain, of PE on the cytotoxicity of ECF-PE towards EGF-receptor-bearing cells and tried to develop a more potent EGF-receptor-targeting toxin. EGF- PE molecules with sequential deletions at the amino terminus of PE wer e constructed and expressed in E. coli strain BL21(DE3). The cytotoxic ity of these chimeric toxins was then examined. Our results show that the amino-terminal and carboxy-terminal regions of the Ia domain of PE are important for the cytotoxicity of a PE-based targeting toxin. To design a more potent PE-based EGF-receptor-targeting toxin, a chimeric toxin, named EGF-PE(Delta 34-220), which had most of the Ia domain de leted but retained amino acid residues 1-33 and 221-252 of this domain , was constructed. EGF-PE(Delta 34-220) has EGF-receptor-binding activ ity but does not show PE-receptor-binding activity and is mildly cytot oxic to EGF-receptor-deficient NR6 cells. As expected, EGF-PE(Delta 34 -220) is a more potent cytotoxic agent towards EGF-receptor-bearing ce lls than EGF-PE(Delta 1-252), where the entire Ia domain of PE was del eted. In addition, EGF-PE(Delta 34-220) was shown to be extremely cyto toxic to EGF-receptor-bearing cancer cells, such as A431, CE81T/VGH, a nd KB-3-1 cells. We also found that EGF-PE(Delta 34-220) was highly ex pressed in BL21(DE3) and could be easily purified by urea extraction. Thus, EGF-PE(Delta 34-220) can be a useful cytotoxic agent towards EGF -receptor-bearing cells.