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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。
Objective To preliminary identify humanized anti-hepatocyte growth factor (HGF) monoclonal antibody and study its in vitro and in vivo anti-tumor activity and pharmacokinetic characteristics. Methods Two anti-HGF humanized monoclonal antibodies HE19-12 and HD19-27, which were prepared by genetic engineering, were identified by UV, SDS-PAGE and their quality, molecular weight and purity were evaluated. The anti-HGF antibody Human glioma U87MG cells proliferation inhibitory activity, and compared antibody humanized before and after the difference in activity; using ELISA method to evaluate the antibody binding to HGF and competitive inhibition of HGF and receptor binding capacity; Transwell method to study the antibody to HGF promote PC-3 The anti-tumor effect of the antibody in vivo was compared by constructing the U87MG xenograft model in nude mice. The antibody was intravenously administered to normal mice and its pharmacokinetic characteristics were analyzed. Results The purity of the two antibodies reached more than 90%, both inhibited the proliferation of U87MG tumor cells, and there was no significant difference in antibody activity before and after humanization. Both antibodies have strong binding capacity with HGF and can competitively inhibit the binding of HGF to its receptor C-Met. Cell migration experiments showed that the two antibodies have the ability to inhibit migration of HGF-promoting tumor cells. Nude mice xenograft assay showed that HGF antibody effectively inhibited tumor growth, HE19-12 showed better anti-tumor activity in the experiment. The results of mouse pharmacokinetics showed that the half-lives of HE19-12 and HD19-27 were 423.3 h and 342.5 h, respectively. Conclusion The preliminary identification of a series of humanized monoclonal antibodies and a series of antitumor activities were successfully carried out. The neutralizing antibodies against HGF with strong anti-tumor activity were proved to have good pharmacokinetic characteristics and HGF / C-Met target antitumor drug development to lay the foundation.