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Molecular targeting against growth factor receptors by a novel drug compord of human fusion proteins

人类融合蛋白的新型药物组合物针对生长因子受体的分子靶向

基本信息

批准号：
09470258
负责人：
UEDA Masakazu
金额：
$ 9.22万
依托单位：
Keio University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1999
项目状态：
已结题

项目摘要

To develop a new Missile therapy targeted at growth factor receptor, fusion proteins with a human Rnase and monoclonal antibody against growth factor receptors or growth factor. A hybrid human protein was engineered by fusing the genes encoding human pancreatic Rnase 1 and human IL-2, was isolated and refolded from E.coli inclusion bodies, and was purified to homogeneity. The hpRNase1-hIL-2 inhibited protein synthesis in HTLV-1 infected, malignant T lymphocytes, hyperproducing high affinity IL-2 receptors, with an IC50 of 2x10-8M, whereas no inhibition was detectable for receptor deficient control cells or hpRNase 1 alone had an IL50 of almost 10-3M.. A molar excess of hIL-2 locked the protein synthesis inhibition dose dependently. In a human mixed lymphocyte culture, hpRNase 1-hIL-2 inhibited the proliferation of responder cells with potency comparable to that of cyclosporine, while minimal doses of FK506 importantly improved its potency. Concentrations comparable to the IC50 in vitro could be safely achieved in animals, and could be escalated without any toxic side effects. The murine monoclonal antibody (528) against the human epidermal growth factor receptor (EGFR) was conjugated with mammalian pancreatic Rnase via SPDP and 2-IT. The conjugate showed dose-dependent cytotoxicity against EGFR-producing squamous cancer cells and no detectable cytotoxicity against EGFR-deficient small cell lung cancer cells. The cytotoxicity of the conjugate was positively correlated with the EGFR numbers of each cell line. The addition of excess 528 antibody to the medium rotected A431 cells from the conjugate cytotoxicity. This immunoconjugate might be useful for targeted treatment of squamous cell carcinomas hyperexpressing EGFR.

目的：研制一种以生长因子受体为靶点的新的导弹疗法，将人核糖核酸酶与生长因子受体或生长因子单克隆抗体融合。通过融合编码人胰腺RNA酶1和人IL-2的基因来工程化杂合人蛋白，从大肠杆菌包涵体中分离和重折叠，并纯化至均一。hpRNase 1-hIL-2抑制HTLV-1感染的恶性T淋巴细胞中的蛋白质合成，过度产生高亲和力IL-2受体，IC 50为2 × 10 - 8 M，而对于受体缺陷对照细胞没有检测到抑制作用，或者hpRNase 1单独具有几乎10- 3 M的IL 50。摩尔过量的hIL-2剂量依赖性地锁定蛋白质合成抑制。在人混合淋巴细胞培养物中，hpRNase 1-hIL-2抑制应答细胞增殖的效力与环孢素相当，而最小剂量的FK 506显著提高了其效力。在动物中可以安全地达到与体外IC 50相当的浓度，并且可以在没有任何毒副作用的情况下逐步增加。抗人表皮生长因子受体（EGFR）的鼠单克隆抗体（528）与哺乳动物胰腺RNA酶通过SPDP和2-IT偶联，偶联物对产生EGFR的鳞状细胞癌细胞显示出剂量依赖性的细胞毒作用，而对EGFR缺陷的小细胞肺癌细胞没有检测到细胞毒作用。偶联物的细胞毒性与各细胞系的EGFR数量呈正相关。向培养基中加入过量的528抗体可保护A431细胞免受偶联物的细胞毒性。这种免疫偶联物可能用于靶向治疗EGFR高表达的鳞状细胞癌。