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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  2020, Vol. 40 Issue (7): 9-14    DOI: 10.13523/j.cb.2001045
研究报告     
抗α2δ1/CD3双特异性抗体的制备和功能的初步研究 *
杨笑莹1,李梦2,赵威2,唐敏1,张志谦1,**()
1 重庆医科大学检验医学院 临床检验诊断学教育部重点实验室 重庆 400016
2 北京大学肿瘤医院细胞生物室 北京 100142
Preparation and Preliminary Characterization of Anti-α2δ1/CD3 Bispecific Antibody
YANG Xiao-ying1,LI Meng2,ZHAO Wei2,TANG Min1,ZHANG Zhi-qian1,**()
1 Key Laboratory of Clinical Laboratory Diagnostics of Ministry Education, Faculty of Laboratory Medicine, Chongqing Medicine University, Chongqing 400016, China
2 Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Department of Cell Biology,Peking University Cancer Hospital and Institute, Beijing 100142, China
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摘要：

目的:构建抗α2δ1和CD3的双特异性抗体,并在体外初步评价其杀伤肝癌细胞的功能。 方法:通过基因工程技术,构建BiTE形式的anti-α2δ1/CD3双特异性抗体(BsAb),转染Expi 293F细胞96h后,使用镍离子亲和色谱纯化出双特异性抗体,使用流式细胞术检测anti-α2δ1/CD3 BsAb对α2δ1和CD3的结合性质,使用Perkin Elmer Operetta 高内涵成像仪测定anti-α2δ1/CD3 BsAb介导细胞毒性T淋巴细胞(CTLs)对高表达α2δ1的人肝癌细胞Hep-12的杀伤效应,ELISA法检测杀伤过程中CTLs分泌hIL-2和hIFN-γ的变化。结果:anti-α2δ1/CD3 BsAb可以特异性结合α2δ1和CD3,anti-α2δ1/CD3 BsAb可以有效介导CTLs靶向杀伤高表达α2δ1的人肝癌细胞Hep-12,其介导杀伤Hep-12细胞的EC50为8pmol/L,对于低表达α2δ1的人肝癌细胞Hep-11, anti-α2δ1/CD3 BsAb不能介导CTLs发挥杀伤作用,并且在杀伤过程中Hep-12细胞组CTLs释放的hIL-2和hIFN-γ比Hep11细胞组显著增多(P<0.05)。结论:anti-α2δ1/CD3 BsAb能有效介导CTLs体外杀伤高表达α2δ1的人肝癌细胞Hep-12,为双特异性抗体的肝癌免疫治疗奠定了一定的基础。
关键词： 双特异性抗体;  BiTE;  α2δ1;  CD3    
Abstract:

Objective: The voltage-gated calcium channel α2δ1 (isoform 5) subunit has been identified as a surface marker and therapeutic target for the tumor-initiating cells(TICs) of hepatocellular carcinoma(HCC). The anti-α2δ1 monoclonal antibody 1B50-1 could attenuate the growth of HCC in vivo by eradicating TICs. Hence, it is essential to construct the anti-α2δ1/CD3 bispecific antibody (BsAb) and evaluate its ability to kill liver cancer cells in vitro. Methods: The anti-α2δ1 scFv and anti-CD3 scFv were constructed by overlap PCR. Then the anti-α2δ1 scFv and anti-CD3 scFv were connected by (G4S1)3 linker and the bispecific antibody fragment was cloned into eukaryotic expression vector. After transfection of the plasmid into Expi 293F cells for 96 hours, the bispecific antibody was purified using nickel ion affinity chromatography. Flow cytometry was used to determine the binding properties of the BsAb for α2δ1 and CD3. Perkin Elmer Operetta High Content Imager was used to determine the ability of the BsAb directing cytotoxic T lymphocytes (CTLs) to kill Hep-12 liver cancer cell line which expresses high level of α2δ1. Enzyme-linked immunosorbent assay (ELISA) was used to detect the changes of hIL-2 and hIFN-γ secreted by CTLs during killing. Results: SDS-PAGE results show that the molecular weight of the anti-α2δ1/CD3 BsAb is consistent with theoretical value and the purified anti-α2δ1/CD3 BsAb is of great purity. Flow cytometry analysis reveals that the anti-α2δ1/CD3 BsAb binds specifically to the cells expressing α2δ1 or CD3. Cytotoxicity assay demonstrates that the BsAb can effectively mediate lysis of the α2δ+ Hep-12 cells (EC50=8pmol/L), while minimal cell lysis is observed for Hep-11 cells which express little α2δ1. Furthermore, the hIL-2 and hIFN-γ released by CTLs in the Hep-12 cell group during the killing process are higher than Hep-11 cell group (P<0.05). Conclusion: The anti-α2δ1/CD3 BsAb can effectively direct CTLs to kill the α2δ1+ Hep-12 cells in vitro, providing an alternative candidate of immunotherapy drug of liver cancer with bispecific antibodies.

Key words: Bispecific antibody    BiTE    α2δ1    CD3
收稿日期: 2020-02-22 出版日期: 2020-08-13
ZTFLH:  Q51  
基金资助: * 国家重点研发计划(2016YFC1303400);国家自然科学基金资助项目(81730075)
通讯作者: 张志谦     E-mail: zlzqzhang@bjmu.edu.cn
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引用本文:

杨笑莹,李梦,赵威,唐敏,张志谦. 抗α2δ1/CD3双特异性抗体的制备和功能的初步研究 *[J]. 中国生物工程杂志, 2020, 40(7): 9-14.

YANG Xiao-ying,LI Meng,ZHAO Wei,TANG Min,ZHANG Zhi-qian. Preparation and Preliminary Characterization of Anti-α2δ1/CD3 Bispecific Antibody. China Biotechnology, 2020, 40(7): 9-14.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2001045    或    https://manu60.magtech.com.cn/biotech/CN/Y2020/V40/I7/9

图1  Anti-α2δ1/CD3 BsAb的结构示意图及纯化结果
图2  Anti-α2δ1/CD3 BsAb的结合性质
图3  Anti-α2δ1/CD3 BsAb介导CTLs体外杀伤肝癌细胞的效果
图4  Anti-α2δ1/CD3 BsAb诱导CTLs释放hIL-2、hIFN-γ的结果
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