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

目的：开发一种新的靶向生长因子受体的导弹治疗方法，结合人Rnase融合蛋白和抗生长因子受体或生长因子的单克隆抗体。将编码人胰腺rna酶1和人IL-2的基因融合，构建了一种人类杂交蛋白，从大肠杆菌包涵体中分离、折叠，并进行纯化。hpRNase -1 - IL-2抑制htlm -1感染的恶性T淋巴细胞的蛋白质合成，高亲和力IL-2受体产生，IC50为2x10-8M，而受体缺陷对照细胞没有检测到抑制作用，或者单独hpRNase1的IL50几乎为10-3M。摩尔过量的hIL-2以剂量依赖性锁定蛋白合成抑制。在人混合淋巴细胞培养中，hpRNase 1-hIL-2抑制应答细胞的增殖，其效力与环孢素相当，而小剂量的FK506显著提高了其效力。在动物体内可以安全地达到与体外IC50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。通过SPDP和2-IT将抗人表皮生长因子受体（EGFR）的小鼠单克隆抗体（528）与哺乳动物胰腺rna酶偶联。该偶联物对产生egfr的鳞状癌细胞具有剂量依赖性的细胞毒性，而对缺乏egfr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。在培养基中加入过量的528抗体可以保护A431细胞免受共轭细胞毒性的影响。该免疫偶联物可用于靶向治疗高表达EGFR的鳞状细胞癌。

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.