基于HER2靶标的抗体偶联药物T-DM1内吞机制研究

发布时间：2018-04-26 12:27

本文选题：抗体偶联药物 + T-DM　；参考：《生物技术通讯》2016年03期

【摘要】：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。
[Abstract]:Aim: to study the interaction and endocytosis mechanism between T-DM1 and human epidermal growth factor receptor 2 (ER2) on human breast cancer cell line SKBR3. Methods: the interaction between ligand T-DM1 and receptor HER2 was measured by Biacore method based on the principle of surface plasmon resonance (SPR), and the in vitro endocytosis clearance rate was determined by flow cytometry. The process of endocytosis of drugs in cells was detected by laser scanning confocal microscope in situ. Results the ratio of high affinity between HER2 and T-DM1 was 6.234 脳 10~6mol/(L / kd = 3.077 脳 10 ~ (-10) -4 / s ~ (-1) K ~ (-1) = 4.936 脳 10 ~ (-10) ~ (-1) mol / L ~ (-1). Flow cytometry showed that the binding of T-DM1 with ligand was saturated, and the elimination of T-DM1 in SKBR3 cells was time-dose dependent by immunofluorescence. The results showed that the fluorescence intensity decreased by 32 and 48 hours within 4 h, and the confocal microscope showed that T-DM1 binds to the HER2 receptor on the surface of SKBR3 cells and enters into the cells, and the reaction disappears under the microscope when the reaction reaches the lysosome for 48 h. ConclusionThree T-DM1 has high affinity and saturation to HER2, endocytosis is mediated by HER2 within 1 h, then located in lysosome, and finally cleavage at 48 h.
【作者单位】：军事医学科学院微生物流行病研究所;
【分类号】：R96

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