基于HER2靶标的抗体偶联药物T-DM1内吞机制研究

Endocytic Mechanisms of the Antibody-Drug Conjugate Trastuzumab-DM1 Targeting HER2-Positive Breast Cancer SKBR3

目的:研究抗体偶联药物T-DM1与乳腺癌细胞SKBR3表面人表皮生长因子受体2(HER2)的相互作用及内吞机制。方法:采用基于表面等离子共振(SPR)原理的Biacore方法测试配体T-DM1与受体HER2的相互作用,通过流式细胞仪测定药物在体外内吞清除率,利用激光扫描共聚焦显微镜原位检测药物在细胞中的内吞过程。结果:T-DM1与HER2具有高亲和力,ka为6.234×10~6mol/(L·s),kd为3.077×10^(-4)/s,KD为4.936×10^(-11)mol/L,流式细胞实验证明受体与配体的结合具有饱和性;利用免疫荧光方法检测到T-DM1在SKBR3细胞内的消除呈时间-剂量依赖关系,实验表明4 h内荧光强度减低32%,48 h时降低约90%;共聚焦显微镜实验表明T-DM1先与SKBR3细胞表面的HER2受体结合,进入细胞内,反应1 h到达溶酶体,48 h时在显微镜下已消失。结论:T-DM1与HER2具有高亲和力及结合饱和性,在1 h内通过HER2介导内吞进入细胞,随后定位于溶酶体,最后于48 h裂解。

Objective：To study the binding properties of the antibody-drug conjugates T-DM1 with human epidermalgrowth factor receptor-2（HER2） on the surface of breast cancer cells SKBR3 and the endocytics mechanism.Methods：The interaction between ligand T-DM1 and receptor HER2 was tested by SPR-based Biacore,in vitro drug clearance was measured by monitoring cellular internalization using flow cytometery,and in situ drug endocytic process was observed using laser scanning confocal microscope.Results：Biacore200 experiments identified association constants of T-DM1 and HER2 with ka value of 6.234×106 mol/（L·s）,kd value of 3.077×10-4/s,KD value of 4.936×10-11 mol/L;flow cytometry experiments observed a saturated binding curves between the receptor and the ligand;the clearance of T-DM1 in SKBR3 followed time-dose dependent manner,with the association decreasing by 32％ after 4 hours,by 90％ after 48 hour;confocal microscopy experiments showed that T-DM1 first bound HER2 receptor on cell surface,then internalized into the cell,traveled to the lysosome after 1 hour reaction,and disappeared after 48 hours.Conclusion：T-DM1 targeted to lysosome through a HER2-mediated internalization and were cleared within the cell in a time-dependent manner.