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

目的：开发一种针对生长因子受体、人核糖核酸酶融合蛋白和抗生长因子受体或生长因子的单抗的新型导弹疗法。将人胰腺核糖核酸酶1(RNase1)和人白细胞介素2(IL-2)基因融合，获得融合蛋白，从包涵体中分离、复性，纯化为均一蛋白。HpRNase1-hIL-2可抑制HTLV-1感染的恶性T淋巴细胞高表达高亲和力IL-2受体的蛋白质合成，其IC50为2×10~(-8)M，而对受体缺陷的对照细胞则无抑制作用或hpRNase1单独作用的IL_(50)接近10~(-3)M。过量的hIL-2对蛋白质合成的抑制作用呈剂量依赖性。在人类混合淋巴细胞培养中，hpRNase1-hIL-2抑制反应细胞的增殖，其效力与环孢素A相当，而最小剂量的FK506显著提高了其效力。与体外IC50相当的浓度可以在动物身上安全地实现，并且可以在没有任何毒副作用的情况下升级。抗人表皮生长因子受体(EGFR)的鼠单抗(528)通过SPDP和2-IT与哺乳动物胰腺核糖核酸酶(RNase)偶联。该结合物对产生EGFR的鳞癌细胞显示出剂量依赖性的细胞毒性，对EGFR缺乏的小细胞肺癌细胞没有检测到的细胞毒性。结合物的细胞毒性与各细胞系的EGFR数呈正相关。向A431细胞中加入过量的528抗体后，A431细胞不再具有结合物的细胞毒性。该免疫结合物可用于高表达EGFR的鳞癌的靶向治疗。

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.