A target-specific chimeric toxin composed of epidermal growth factor and Pseudomonas exotoxin A with a deletion in its toxin-binding domain

Abstract

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 this study, we analyzed the effect of the Ia domain, the binding domain of PE on the cytotoxicity of EGF-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 were constructed and expressed in E. coli strain BL21(DE3). The cytotoxicity 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 deleted but retained amino acid residues 1-33 and 221-252 of this domain, was constructed. EGF-PE(delta 34-220) has EGF-receptor-binding activity but does not show PE-receptor-binding activity and is mildly cytotoxic to EGF-receptor-deficient NR6 cells. As expected, EGF-PE(delta 34-220) is a more potent cytotoxic agent towards EGF-receptor-bearing cells than EGF-PE(delta 1-252), where the entire Ia domain of PE was deleted. In addition, EGF-PE(delta 34-220) was shown to be extremely cytotoxic to EGF-receptor-bearing cancer cells, such as A431, CE81T/VGH, and KB-3-1 cells. We also found that EGF-PE(delta 34-220) was highly expressed 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.

Characterization of monoclonal antibody B7, which neutralizes the cytotoxicity of Pseudomonas aeruginosa exotoxin A

“…Plasmids and bacterial strain. The plasmids encoding mutants of PE with sequential deletions at the amino and carboxyl termini were constructed as described previously (5,19). Simplified maps of these two sets of PE deletion mutants are shown diagrammatically in Fig.…”

Fusion Protein Vaccine by Domains of Bacterial Exotoxin Linked with a Tumor Antigen Generates Potent Immunologic Responses and Antitumor Effects

“…The PCR products were purified using 5% polyacrylamide gel; then, the bands corresponding to each product were cut out for elution. The E7 PCR fragment was inserted into pET and pPE(DIII) vectors as detailed in our previous reports (25,26). The E7 fragment was inserted into the EcoRI/ XhoI cloning site of the pET15 or pET15-PE(DIII) plasmid to generate the plasmid encoding E7 or PE(DIII)-E7 (named pE7 and pETA-E7, respectively).…”

“…The bound proteins were then eluted with different buffers (pH 8.0, 7.0, 6.5, 6.0, 5.0, 4.0, and 3.5) containing 4 mol/L urea, 0.3 mol/L NaCl, 20 mmol/L Tris-HCl, and 20 mmol/L phosphate buffer. After purification, the protein elution fractions were analyzed for purity and quantification by SDS-PAGE analysis as described previously (26).…”

Targeting Phthalocyanines to Tumor Cells Using Epidermal Growth Factor Conjugates

“…Their conjugates with growth factors, oncofetal proteins and antibodies against various cell surface receptors are now widely employed for targeted delivery of antitumor drugs to neoplastic tissues [1][2][3]. The large variety of neoplastic tissues carries a multitude of specific receptors for vector molecules which enables the selected delivery of cytostatic agents conjugated to protein vectors to tumor cells [4][5][6].…”