重组人源化抗人HGF单克隆抗体鉴定及其抗肿瘤活性研究

发布时间：2018-08-21 07:54

【摘要】：目的对人源化抗肝细胞生长因子(hepatocyte growth factor,HGF)单克隆抗体进行初步鉴定,并对其体内、外抗肿瘤活性及药代动力学特征进行研究。方法将基因工程技术制备的2株抗HGF人源化单克隆抗体HE19-12和HD19-27,通过UV、SDS-PAGE方法对其质量浓度、相对分子质量及纯度进行鉴定;MTT法评价抗体对人脑胶质瘤U87MG细胞的增殖抑制活性,并比较抗体人源化前后活性差异;采用ELISA法评价抗体结合HGF及竞争抑制HGF与受体结合的能力;Transwell法研究抗体对HGF促进PC-3细胞迁移的抑制能力;通过构建U87MG裸鼠移植瘤模型,比较抗体在体内的抗肿瘤作用;抗体通过静脉给予正常小鼠,分析其药代动力学特征。结果 2株抗体单体纯度均达到90%以上,均可抑制U87MG肿瘤细胞增殖,且人源化前后抗体活性差异不大。2株抗体均与HGF具有较强的结合能力,且可竞争抑制HGF与其受体C-Met的结合。细胞迁移实验显示,2株抗体具有抑制HGF促肿瘤细胞迁移的能力。裸鼠移植瘤试验显示HGF抗体可有效抑制肿瘤的生长,其中HE19-12在试验中显示更好的抗肿瘤活性。小鼠药代结果显示,HE19-12和HD19-27的半衰期分别为423.3 h和342.5 h。结论成功对制备出的2株人源化单克隆抗体进行初步鉴定及一系列抗肿瘤活性研究,证明具有较强抗肿瘤活性的HGF中和抗体,且具有良好的药代动力学特征,为HGF/C-Met靶点抗肿瘤药物的开发奠定基础。
[Abstract]:Objective to identify the anti-hepatocyte growth factor (hepatocyte growth) monoclonal antibody and to study its antitumor activity and pharmacokinetics in vitro and in vivo. Methods HE19-12 and HD19-27 monoclonal antibodies against HGF were prepared by genetic engineering technique. The mass concentration, relative molecular weight and purity of the two monoclonal antibodies against human glioma U87MG cells were evaluated by UVX SDS-PAGE method to evaluate the inhibitory activity of the antibodies on the proliferation of human glioma U87MG cells. The activity of antibody before and after humanization was compared, and the ability of antibody binding to HGF and competitive inhibition of HGF binding to receptor was evaluated by ELISA method. Transwell method was used to study the inhibitory effect of antibody on the migration of PC-3 cells induced by HGF. The antitumor effect of antibody in vivo was compared, and the pharmacokinetic characteristics of the antibody were analyzed by intravenous administration of the antibody to normal mice. Results the purity of the two antibody monomers was over 90%, which could inhibit the proliferation of U87MG tumor cells. The antibody activity of the two strains had no significant difference before and after humanization. The antibody activity of both strains had strong binding ability to HGF, and could compete to inhibit the binding of HGF to its receptor C-Met. Cell migration assay showed that two strains of antibodies could inhibit the migration of tumor cells induced by HGF. Tumor transplantation test in nude mice showed that HGF antibody could effectively inhibit tumor growth, and HE19-12 showed better antitumor activity in the experiment. The pharmacokinetic results showed that the half-lives of HE19-12 and HD19-27 were 423.3 h and 342.5 h, respectively. Conclusion two humanized monoclonal antibodies were successfully identified and a series of antitumor activities were studied. The results showed that HGF neutralizing antibodies had strong anti-tumor activity and had good pharmacokinetic characteristics. To lay a foundation for the development of anti-tumor drugs targeting HGF/C-Met.
【作者单位】：北京工业大学生命科学与生物工程学院;神州细胞工程有限公司;
【分类号】：R392.11

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