Research of new recombinant anticancer drug against epidermal growth factor receptor as a molecular target

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Study 1 : To minimize the side effects, we made a new anticancer drug which consisted of only physiologically active materials. We have made a conjugate of epidermal growth factor (EGF) and eosinophil cationic protein (ECP) and studied its cytotoxic effects on cancer cells. EGF and ECP were conjugated with 2-iminothiolane and SPDP. Cytotoxic effects of the conjugate were determined on BT-20 cells which overexpressed EGFR and H69 cells which did not expressed EGFR using a MTT assay. The conjugate killed BT-20 cells dose-dependently, but it did not kill H69 cells. Additional EGF bloked the cytotxic effects. These results suggested that the new anticancer drug which consisted of only physiologically active materials is promising and useful as a targeting tool.Study 2 : We fused a pancreatic-type ribonuclease (RNase) gene to human basic fibroblast growth factor (bFGF), and studied the inhibitory effect of the fused protein (CL-RFN89) on angiogenesis in vitro and in vivo. CL-RFN89 binding was assayed using a fluorescence labeled method. Inhibitory effects on in vitro angiogenesis were evaluated by an assay using a collagen gel system. Inhibitory effects on in vivo angiogenesis were evaluated by an mouse dorsal air sac assay. Fluorescence labeled CL-RFN89 was detected on cell membranes and its binding was inhibited by excess amount of FGF. Tube-formation of HUVEC was inhibited by CL-RFN89 dose-dependently and 53% inhibition was shown in 2uM of CL-RFN89. As for in vivo effects, 0.8 new vessels were observed in 2uM of CL-RFN89, but 4.4 new vessels were observed in none of CL-RFN89. These results suggested that CL-RFN89 bound to cell thgrough FGF receptor and that CL-RFN89 had anti-angiogenetic effects both in vitro and in vivo.