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本研究尝试探讨抗CD3×CD87单链双特异抗体介导的单核细胞对CD87+前列腺癌细胞的杀伤作用.首先,利用基因工程手段,构建了pET24a/抗CD3×CD87单链双特异抗体原核表达载体;将重组载体转化BL-21(DE3)感受态细胞,用IPTG诱导蛋白表达;获得的工程菌经超声破碎,先在变性条件下亲和纯化目的蛋白,再采用稀释复性方法对scBsAb进行复性.其次,利用流式细胞术检测表达纯化的scBsAb与两种相应抗原的结合情况.最后,通过CCK-8法检测外周血单个核细胞(PBMCs)对CD87阳性前列腺癌细胞PC-3的杀伤效应.结果表明,利用基因工程手段获得了较高纯度的scBsAb;scBsAb能与PC-3细胞表面CD87特异性结合,并且能与CD3特异性结合;scBsAb的浓度及效靶比影响scBsAb介导的杀伤作用.在效靶比为10∶1,scBsAb浓度为100μg/mL时最大杀伤率达到40.86%;在scBsAb浓度为100μg/mL,效靶比为40∶1时达到最大杀伤率60.9%.此外,ELISA检测表明,在scBsAb介导的PBMCs靶向CD87阳性肿瘤细胞杀伤过程中IFN-γ的水平显著增高.结果证明,scBsAb能够在体外有效介导效应细胞杀伤CD87阳性肿瘤细胞.
This study was designed to investigate the anti-CD3 × CD87 single-chain bispecific antibody-mediated killing effect of monocytes on CD87 + prostate cancer cells.First, the prokaryotic expression of pET24a / anti-CD3 × CD87 single-chain bispecific antibody was constructed by genetic engineering The recombinant vector was transformed into BL-21 (DE3) competent cells and the protein was induced by IPTG. The obtained engineering bacteria were sonicated and screened. The target protein was affinity-purified under denaturing conditions and scFsAb Refolding.Secondly, flow cytometry was used to detect the binding of purified scBsAb to two corresponding antigens.Finally, the expression of PC-3 in CD87 positive prostate cancer cells (PBMCs) was detected by CCK-8 assay The results showed that scBsAb was obtained by genetic engineering. ScBsAb could specifically bind to CD87 on the surface of PC-3 cells and could specifically bind to CD3. The scBsAb-mediated The killing rate reached 40.86% when the target ratio was 10: 1 and the scBsAb concentration was 100μg / mL. The maximum killing rate was 60.9% when the scBsAb concentration was 100μg / mL and the target ratio was 40:1. In addition, ELISA tests Out in PBMCs scBsAb mediated targeting of CD87-positive tumor cell killing during the IFN-γ levels were significantly higher. The results demonstrate, in vitro scBsAb can be efficiently mediate effector cell killing CD87 positive tumor cells.