Expression, Identification And Bioactivity Characterization Of An Anti-CD3/anti-CD20 Bispecific Single-Chain Antibody

BiTEs (bispeecific T cell engagers) is a kind of bispecific single-chain antibodies with the formation of V_LI-linker-V_H1-linker-V_H2-linker-V_L2. It not only can link T cells and pathogenic cells, but also can efficiently stimulate cytotoxic T cells and induce target cell destruction. The anti-CD3/anti-CD20 bispecific single-chain antibody is a kind of BiTEs .which has been thought as an potent and attractive candidate in B cell lymphoma therapy.The synthetic gene with 1640bp encoding for the anti-CD3/anti-CD20 bispecific single-chain antibody was designed and abtained by SOE (splicing by overlap extension) PCR. The cDNA was cloned into transient expression vector pcDNA3.1 and Flp-In~? expression vector pcDNA5/FRT, then separately transfeced into COS-7 cells for a transient expressin and Flp-In? CHO cells to generate a stable expression cell line with a capacity for expressing anti-CD3/anti-CD20 bispecific single-chain antibodies at 300μg/L. The protein, which had a molecular weight of 59.5KD, was purified by Ni-NTA affinity chromatography. Immunofluorescence assay and cellular rosetting showed that it can specific react on Jurkat (CD3+) and Ramous (CD20+) cells. Observed through the scanning electron microscope, the anti-CD3/anti-CD20 bispecific single-chain antibody can effectively lyse human B-lymphoma cells in the presence of T-enriched human peripheral blood lymphocytes (PBL). The potency of cytotoxicity is dependent on the ratios of effector cells to target cells used. Further, the antibody directs lysis of Ramous cells in a dose and time-dependent manner. The specific lysis can be 87.3% at antibody concentration of 5 μg/mL and effector to target cells ratio used at 10:1. Obvious opoptosis characters can be seen by AV/PI coloration and the changes of genes correlative with apoptosis were also detected by apoptosis gene arrays, which suggested that ATM-p53 pathway may be the main apoptosis way of Ramous cells induced by T cells in the presence of the anti-CD3/anti-CD20 bispecific single-chain antibody. All these results would set light on the further study of its use in B cell lymphoma clinical trial.