Novel Anti-Cancer Drug Composed of Human Proteins

1. 哺乳动物胰腺机器核糖核酸酶（RNase）通过二硫键与人表皮生长因子（EGF）化学偶联。人egf -人RNase偶联物对egf受体缺乏的小细胞肺癌细胞系H69无细胞毒性作用。但该偶联物对鳞状癌细胞系和乳腺癌细胞系表现出剂量依赖性的细胞毒性，并且该偶联物的细胞毒性与每个细胞系的EGFR表达水平呈正相关。未结合的EGF和RNase的混合物对任何细胞系的作用都不大于单独的RNase。重组人rna酶1 （RNase1）化学连接到重组人EGF。EGF- rnase偶联物对过表达EGF受体的细胞系表现出剂量依赖性的细胞毒性。未结合的EGF和RNase的混合物并不比单独的RNase有更大的作用。该偶联物对EGF受体缺乏的小细胞肺癌细胞无明显的细胞毒性。编码人胰腺RNase1的基因与编码人EGF或人IL-2的基因融合。这些DNA在大肠杆菌中表达，在包涵体中产生大量重组人蛋白，在体外溶解、折叠、恢复活性并纯化至均质。RNase-EGF偶联物对几种鳞状癌细胞具有细胞毒性（egf受体数量依赖）。游离EGF和游离RNase1的混合物没有比单独RNase1更大的作用，并且对受体缺乏的对照细胞没有检测到细胞毒性。

1. Mammalian pancreatic robonuclease (RNase) was conjugated chemically via a disulfide bond to human epidermal growth factor (EGF). The human EGF-mam malian RNase conjugate had no cytotoxic effect against the EGFreceptor-deficient small-cell lung cancer cell line H69. But this conjugate showed dose-dependent cytotoxicity against squamous carcinoma cell lines and breast cancer cell lines and the cytotoxicity of the conjugate correlated positively with the level of expression of EGFR by each cell line. An unconjugated mixture of EGF and RNase had no greater effect than RNase alone on any cell lines.2. Recombinant human RNase 1 (RNase1) was chemically linked to recombinant human EGF.The EGF-RNase conjugate showed dose-dependent cytotoxicity for EGF receptor-overexpressing cell lines. An unconjugated mixture of EGF and RNase had no greater effect than RNase alone. the conjugate showed no detectable cytotoxicity atainst EGF receptor-deficient small cell lung cancer cell.3. The gene coding for human pancreatic RNase1, was fused with a gene coding for human EGF or human IL-2. These DNA were expressed in E.Coli, efficient amounts of the recombinant human protein were produced in inclusion bodies, solubilized, refolded in vitro, regained activity and purified to homogeneity.4. The RNase-EGF conjugate was cytotoxic, EGF-receptor number dependently, to several squamous carcinoma cell lines. A mixture of free EGF and free RNase1 had no greater effect than RNase1 alone and no cytotoxicity was detectable for receptor deficient control cells.