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| 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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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.
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Received: 22 February 2020
Published: 13 August 2020
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Corresponding Authors:
Zhi-qian ZHANG
E-mail: zlzqzhang@bjmu.edu.cn
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