| Objective:Bispecific antibodies（Bs Abs）have become a new method of tumor therapy in recent years,which can kill tumor cells by mediating T cells.The recurrence and metastasis of liver cancer are usually related to tumor initiating cells（TICs）.Our laboratory has previously found thatα2δ1（isoform 5）is a marker for tumor-initiating cells of liver cancer.1 B50-1 targeting this antigen can attenuate the growth of liver cancer by eradicating TICs.The purpose of this study was to construct bispecific antibody againstα2δ1 and CD3,and evaluate its ability to kill liver cancer stem cells in vitro and in vivo.Methods:Eukaryotic expression plasmids harboring the expression cassette of anti-α2δ1/CD3 Bs Abs in the form of bispecific T-cell engager（Bi TE）and dual-variable domain immunoglobulin（DVD-Ig）was constructed by using standard DNA recombinant techniques,and were transiently transfected into Expi 293F cells.The cell supernatants were colllected and purified for 1B50-1-g OKT3-7 Bi TE and 1B50-1-V9 Bi TE by using Ni2+sepharose on day 4after transfection,and for 1B50-1-g OKT3-7 DVD-Ig G1 on day 6 after transfection by using Protein G affinity chromatography.The purified1 B50-1-g OKT3-7 Bi TE,1 B50-1-V9 Bi TE and 1 B50-1-g OKT3-7 DVD-Ig G1 were identified and assessed the purity by SDS-PAGE protein electrophoresis and Coomassie brilliant blue staining.Superdex 200 was used to test the aggregation properties of 1B50-1-V9 Bi TE and 1B50-1-g OKT3-7 DVD-Ig G1.Flow Cytometry was used to determine the binding properties of 1B50-1-g OKT3-7 DVD-Ig G1 forα2δ1 and CD3.Perkin Elmer Operetta High Content Imager was used to determine the ability of1 B50-1-g OKT3-7 DVD-Ig G1 directing CTLs to kill Hep-12 liver cancer cell line which expresses high level ofα2δ1.Enzyme-linked immunosorbent assay（ELISA）was used to detect the release of TNF-αand IFNγsecreted by CTLs during the killing process.Hep-12 cells/PBMC co-transplantation model was used to determine the ability of 1B50-1-g OKT3-7 DVD-Ig G1 inhibiting the tumorigenic ability of Hep-12 cells in vivo.Results:The eukaryotic expression plasmids stably expressing 1B50-1-g OKT3-7 Bi TE,1B50-1-V9 Bi TE and 1B50-1-g OKT3-7 DVD-Ig G1were successfully constructed.The molecular weight of 1B50-1-g OKT3-7Bi TE,1B50-1-V9 Bi TE and 1B50-1-g OKT3-7 DVD-Ig G1 were in accordance with the theoretical molecular weight,and the purity of anti-α2δ1/CD3 Bs Abs was great.The yields of 1B50-1-g OKT3-7 Bi TE,1B50-1-V9 Bi TE,and 1B50-1-g OKT3-7 DVD-Ig G1 were 0.5-0.7 mg/L,6-7mg/L,and 2-3 mg/L,respectively.Size exclusion chromatography（SEC）showed that 1B50-1-V9 Bi TE contained more polymerized proteins,which did not meet expectation,while 1B50-1-g OKT3-7 DVD-Ig G1 contained more monomers,and the peak position was as expected.The yield of 1B50-1-g OKT3-7 DVD-Ig G1 with correct conformation was the highest and the quality met the requirements for bispecific antibodies,and hence was selected in the subsequent experiments.Flow Cytometry showed that1 B50-1-g OKT3-7 DVD-Ig G1 can specifically bind to cells expressingα2δ1 and CD3.Cytotoxicity assay demonstrated that the 1B50-1-g OKT3-7 DVD-Ig G1 can effectively mediate lysis of theα2δ1+Hep-12 cells(EC50=1.9 pmol/L),while minimal cell lysis was observed for Hep-11 cells which express littleα2δ1.During the killing process,the TNF-αand IFNγreleased by CTLs in Hep-12 cell group were higher than Hep-11 cell group（P<0.05）.Furthermore,1B50-1-g OKT3-7 DVD-Ig G1 could effectively inhibit the tumorgenicity ability of Hep-12 cells in vivo.Conclusion:1B50-1-g OKT3-7 DVD-Ig G1 can effectively mediate specific killing of theα2δ1+Hep-12 cells by T cells in vitro and in vivo,which provides an alternative candidate of immunotherapy drug of liver cancer with bispecific antibodies. |
