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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2023, Vol. 43 Issue (10): 10-19    DOI: 10.13523/j.cb.2305006
研究报告     
抗EpCAM免疫毒素制备与体外活性研究*
刘玉萍1,邓昌平2,马兴元2,刘秋丽1,鲍雯1,郑文云1,**()
1 华东理工大学药学院 上海市新药设计重点实验室 上海 200237
2 华东理工大学生物工程学院 生物反应器工程国家重点实验室 上海 200237
Preparation and in vitro Activity of Anti-EpCAM Immunotoxin
LIU Yu-ping1,DENG Chang-ping2,MA Xing-yuan2,LIU Qiu-li1,BAO Wen1,ZHENG Wen-yun1,**()
1 Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China
2 State Key Laboratory of Bioreactor Engineering, School of Biological Engineering, East China University of Science and Technology, Shanghai 200237, China
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摘要：

目的:上皮细胞粘附分子(epithelial cell adhesion molecule,EpCAM)在正常细胞中的表达局限于上皮基底外侧细胞,且具有隐蔽性,而在包括膀胱癌在内的多种上皮性癌症中过表达且处于易结合的状态,是抗肿瘤治疗的特异性有效靶标之一;以EpCAM作为膀胱癌靶向治疗的靶点,探索高效、安全的膀胱癌新型治疗方法。方法:在分子水平利用柔性肽GGGGS将EpCAM单链抗体4D5MOCB与毒素PE38KDEL连接设计制备免疫毒素,在大肠杆菌BL21(DE3)中进行表达,并研究其与膀胱癌细胞的结合和对癌细胞生长的抑制作用。结果:4D5MOCB介导的免疫毒素具有良好的选择性,与EpCAM高表达的膀胱癌细胞5637结合良好,其结合率约为阴性细胞结合率的169倍,并能有效抑制癌细胞生长,IC50最低可达0.5 pmol/L,同时能够抑制细胞克隆形成和细胞迁移,诱导细胞凋亡;免疫毒素与EpCAM阴性细胞HeLa不结合,也不具有抑制细胞活性的作用。结论:制备的基因工程免疫毒素具有较好的选择性,能够有效抑制阳性癌细胞的增殖和转移,且比相同抗原的抗体药物偶联物(antibody-drug conjugates,ADC)的制备更简单,均一性更好,为免疫毒素应用于实体瘤的治疗提供了实验基础。
关键词： 重组免疫毒素;  EpCAM;  绿脓杆菌外毒素A;  单链抗体;  靶向治疗    
Abstract:

Objective: Epithelial cell adhesion molecule(EpCAM) is one of the specific and effective targets of anti-tumor therapy, because the expression of EpCAM is limited to the basolateral epithelial cells in normal cells and is concealed. However, EpCAM is overexpressed in a variety of epithelial cancers, including bladder cancer, and is in a state of easy binding. Since immunotoxins targeting EpCAM have not been applied to the treatment of bladder cancer, new treatments for bladder cancer will be explored. Methods: The immunotoxin was constructed by linking the single-chain antibody of EpCAM 4D5MOCB with toxin PE38KDEL by flexible peptide GGGGS at the molecular level for design and preparation, and expressed in Escherichia coli BL21 (DE3). The binding effect of the immunotoxin on bladder cancer cells and its inhibitory effect on the growth of bladder cancer cells were studied. Results: The quantity of 4D5MOCB-mediated immunotoxin selectively binding to positive cells is about 169 times that binding to negative cells. The immunotoxin could effectively inhibit the growth of 5637, SW780 and RT4 with an IC50 up to 0.5 pmol/L. At the same time, it could inhibit cell colony formation and cell migration and induce cell apoptosis. The immunotoxin did not bind to EpCAM-negative HeLa cells and had no inhibitory activity. Conclusion: The immunotoxin prepared in this study has good selectivity and can effectively inhibit the proliferation and metastasis of positive cancer cells. The preparation of immunotoxin is simpler than that of antibody-drug conjugates (ADCs) and it is more homogeneous than ADCs. This study also provides an experimental basis for the application of immunotoxin in the treatment of solid tumors.
Key words: Recombinant immunotoxin    EpCAM    Pseudomonas aeruginosa exotoxin A    Single chain antibody    Targeted therapy
收稿日期: 2023-05-04 出版日期: 2023-11-02
ZTFLH:  Q78  
基金资助: *国家重点研发计划(2018YFA0902804)
通讯作者: **电子信箱:zwy@ecust.edu.cn   
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刘玉萍
邓昌平
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刘秋丽
鲍雯
郑文云

引用本文:

刘玉萍, 邓昌平, 马兴元, 刘秋丽, 鲍雯, 郑文云. 抗EpCAM免疫毒素制备与体外活性研究*[J]. 中国生物工程杂志, 2023, 43(10): 10-19.

LIU Yu-ping, DENG Chang-ping, MA Xing-yuan, LIU Qiu-li, BAO Wen, ZHENG Wen-yun. Preparation and in vitro Activity of Anti-EpCAM Immunotoxin. China Biotechnology, 2023, 43(10): 10-19.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2305006    或    https://manu60.magtech.com.cn/biotech/CN/Y2023/V43/I10/10

图1  重组免疫毒素载体的构建与蛋白制备
图2  EpCAM在不同癌细胞中的表达情况及免疫毒素与细胞的结合
图3  免疫毒素、抗体及毒素对不同细胞的MTT实验结果
细胞类别 细胞名称 作用时间 DP/(nmol·L-1) 4D5MOCB/(nmol·L-1) PE38KDEL/(nmol·L-1)
EpCAM阴性细胞 HeLa 48 h 147.077 - -
72 h 128.411 783.356 -
T24 48 h 183.434 - -
72 h 179.295 - -
UMUC-3 48 h 228.133 - -
72 h 130.944 620.053 -
EpCAM阳性细胞 5637 48 h 0.949 785.591 -
72 h 0.065 150.838 671.097
SW780 48 h 3.922 - -
72 h 0.079 109.544 -
RT4 48 h 0.001 - -
72 h 0.000 05 293.423 114.900
表1  免疫毒素DP作用各细胞系的IC50值
图4  免疫毒素对细胞克隆形成与细胞迁移的影响
图5  免疫毒素对5637和HeLa细胞诱导凋亡作用
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