64Cu-NOTA-Herceptin的设计、 活性测定及肿瘤靶向分子显像研究

doi:10.7503/cjcu20160593

高等学校化学学报 ›› 2016, Vol. 37 ›› Issue (12): 2132.doi: 10.7503/cjcu20160593

• 研究论文: 无机化学 • 上一篇下一篇

⁶⁴Cu-NOTA-Herceptin的设计、活性测定及肿瘤靶向分子显像研究

朱华¹, 李一林², 赵传科³, 解清华^1,⁴, 刘菲¹, 韩雪迪¹, 高静¹, 夏传琴⁴, 沈琳²(), 杨志¹()

1. 北京大学肿瘤医院暨北京市肿瘤防治研究所核医学科, 2. 消化肿瘤内科,3. 生化与分子生物学研究室, 恶性肿瘤发病机制及转化研究教育部重点实验室, 北京 100142
4. 四川大学化学学院, 成都 610041

收稿日期:2016-08-22 出版日期:2016-12-10 发布日期:2016-11-22
作者简介:联系人简介: 杨志, 男, 博士, 研究员. 主要从事放射性药物研究. E-mail:pekyz@163.com; 沈琳, 女, 博士, 主任医师, 主要从事消化系统肿瘤研究. E-mail:linshenpku@163.com
基金资助:
国家自然科学基金(批准号: 81371592, 81401467, 81501519, 81301323, 81571705)和北京市自然科学基金(批准号: 7154188, 7162041)资助

Design and Bio-evaluation of ⁶⁴Cu-NOTA-Herceptin for Tumor Targeted Micro-PET Imaging^†

ZHU Hua¹, LI Yilin², ZHAO Chuanke³, XIE Qinghua^1,⁴, LIU Fei¹, HAN Xuedi¹, GAO Jing², XIA Chuanqin⁴, SHEN Lin^2,^*(), YANG Zhi^1,^*()

1. Department of Nuclear Medicine, 2. Department of Gastrointestinal Oncology,3. Departments of Biochemistry and Molecular Biology, Key Laboratory of Carcinogenesis and Translational Research(Ministry of Education), Peking University Cancer Hospital & Institute,Beijing 100142, China
4. College of Chemistry, Sichuan University, Chengdu 610041, China

Received:2016-08-22 Online:2016-12-10 Published:2016-11-22
Contact: SHEN Lin,YANG Zhi E-mail:linshenpku@163.com;pekyz@163.com
Supported by:
† Supported by the National Natural Science Foundation of China(Nos.81371592, 81401467, 81501519, 81301323, 81571705) and the Natural Science Foundation of Beijing, China(Nos.7154185, 7162041)

摘要/Abstract

摘要：

利用双功能螯合剂2-[(4-异硫氰基苯基)甲基]-1,4,7-三氮杂环九烷-1,4,7-三乙酸(NCS-Bz-NOTA)对Herceptin单抗表面的氨基进行修饰获得了NOTA-Herceptin, 通过基质辅助激光解吸电离飞行时间质谱(MALDI-TOF)对该偶联物进行了表征. 利用酶联免疫吸附测定了偶联前后Herceptin抗体效价的改变. 利用新型正电子核素⁶⁴Cu标记, 获得可用于肿瘤放射靶向精准诊疗的⁶⁴Cu-NOTA-Herceptin探针, 其标记率为90%, 放化纯度>98%, 比活度185 MBq/nmol. 分别进行了该探针在HER2过表达胃癌细胞NCI-N87及HER2低表达胃癌细胞BGC823等肿瘤细胞中的摄取实验, 测定了该探针的肿瘤特异性. 建立了荷人胃癌BGC823裸鼠模型, 通过微型正电子断层显像(Micro-PET)设备观察了探针在模型动物体内的代谢情况: 在静脉注射 7.4 MBq ⁶⁴Cu-NOTA-Herceptin探针后, 分别于4和60 h 进行正电子断层显像(PET)的显像, 观察到其在肿瘤部位的摄取有所富集, 且随着代谢时间的延长, 肝脏部位摄取得到明显降低. 研究结果表明, ⁶⁴Cu-NOTA-Herceptin探针有望应用于肿瘤放射性靶向诊疗.

关键词: 曲妥珠单抗, 64Cu, 免疫活性, 分子显像, 肿瘤靶向诊疗

Abstract:

The precursor compound 2-(p-thiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid-Herceptin(NOTA-Herceptin) was synthesized by the nucleophilic addition reaction of Herceptin and bi-functional chelator NCS-Bz-NOTA. The original Herceptin and NOTA-Herceptin conjugate were investigated by UV-Vis and MALDI-TOF mass spectra. The average number of chelators per Herceptin for the conjugated used in this study was 5.4. The enzyme-linked immunosorbent assay(ELISA) was conducted to compare the biological activity of original Herceptin and NOTA-Herceptin. NOTA-Herceptin kept high immunoreactivity towards HER2 antigen. Then, the PET radio-nuclide ⁶⁴Cu(T_1/2=12.7 h) was labeled to got the novel tumor Immuno-Theranostics probe ⁶⁴Cu-NOTA-Herceptin. The labeling efficiency of ⁶⁴Cu-NOTA-Herceptin was tested by Radio-TLC/HPLC. The radiolabeling yield was over 90%, the radio chemical purity was over 98% after PD-10 column purification and the specific activity was 185 MBq/nmol. The immune reactivity and specific activity of radiolabeled Herceptin with HER2 antigen were performed by HER2 positive NCI-N87 cell line and HER2 negative BGC823 cell line. Micro-PET imaging of BGC823 tumor-bearing nude mice revealed that tumor uptake of ⁶⁴Cu-NOTA-Herceptin got a gradual accumulation from 4 h to 60 h after intravenous injection of 7.4 MBq radiolabeled Herceptin. Uptake in the tumor was clearly visualized by emission computed tomography. ⁶⁴Cu-NOTA-Herceptin owns a great potential for molecular imaging of PET for diagnosis and follow-up of HER2 expression.

Key words: Herceptin, 64Cu, Immunoreactivity, Molecular imaging, Tumor targeting therapy

中图分类号:

TrendMD:

朱华, 李一林, 赵传科, 解清华, 刘菲, 韩雪迪, 高静, 夏传琴, 沈琳, 杨志. ⁶⁴Cu-NOTA-Herceptin的设计、活性测定及肿瘤靶向分子显像研究. 高等学校化学学报, 2016, 37(12): 2132.

ZHU Hua, LI Yilin, ZHAO Chuanke, XIE Qinghua, LIU Fei, HAN Xuedi, GAO Jing, XIA Chuanqin, SHEN Lin, YANG Zhi. Design and Bio-evaluation of ⁶⁴Cu-NOTA-Herceptin for Tumor Targeted Micro-PET Imaging^†. Chem. J. Chinese Universities, 2016, 37(12): 2132.

图/表 6

Fig.1 Modification of Herceptin antibody and radiolabeling methodology of 64Cu radionuclide

Fig.2 TMALDI-TOF mass spectrum analysis of Herceptin(A) and NOTA-Herceptin(B)

Fig.3 Biological activity of Herceptin before(a) and after(b) NOTA modification tested by ELISA

Fig.4 64Cu-NOTA-Herceptin cell uptake in HER2 negative BCG823(A) and HER2 positive NCl-N87(B)(A) Uptake time/min: a. 0; b. 5; c. 30; d. 60; (B) uptake time/min: a. 0; b. 5; c. 30; d. 60; e. 120; f.120(block group).

Fig.5 Micro-PET imaging of BGC823 tumor-bearing nude miceRed arrow indicates tumors. (A) 4 h PET imaging; (B) 60 h PET imaging.

Fig.6 Micro-PET imaging of human gastric BGC823 tumor-bearing nude mice(A) Sagittal, 4 h; (B) coronal, 4 h; (C) transverse, 4 h; (D) sagittal, 60 h; (E) coronal, 60 h; (F) transverse, 60 h.

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⁶⁴Cu-NOTA-Herceptin的设计、活性测定及肿瘤靶向分子显像研究

Design and Bio-evaluation of ⁶⁴Cu-NOTA-Herceptin for Tumor Targeted Micro-PET Imaging^†

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