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[Expression and immunological activity of an anti-CD3×VEGFR2 bispecific single-chain antibody]
Li-juan Dai1, Cheng-wei Yan, Shu-zhen Li
1Department of Pharmacy, College of Pharmacy, Harbin Medical University, Harbin 150081, China.
Aim:
To construct and express an anti-VEGFR2/anti-CD3 bispecific single-chain antibody (bscVEGFR2×CD3)and to identify its binding specificities to CD3 and VEGFR2.
Methods:
The gene encoding anti-VEGFR2/anti-CD3 bispecific single-chain antibody was designed and synthesized. Bispecific single-chain antibody (bsc-Ab) DNA was subcloned into a eukaryotic expression vector pcDNA3.1(+), then transfected into Chinese hamster ovary (CHO) cells and stable expression cell lines were selected. Expressed Bsc-Ab was purified by His-tag affinity chromatography and confirmed by 120 g/L SDS-PAGE and Western blotting. Antigen binding activity of the bsc-Ab was analyzed by FACS.
Results:
The plasmid DNA containing bispecific single-chain fragments were confirmed. BscVEGFR2×CD3 was secreted by CHO into the supernatant. Six stable expression cell lines were established. The molecular weight of bsc-Ab was correct indicated by SDS-PAGE and Western blotting. The bsc-Ab could specifically bind to CD3(+); jurkat cells and VEGFR2(+); A375 cells.
Conclusion:
An anti-VEGFR2/anti-CD3 bispecific single-chain antibody is successfully constructed and expressed, and the antibody has specific binding capacity to CD3 and VEGFR2.
