双功能抗体抗CD3/抗CD19介导T细胞对靶细胞的杀伤作用

摘要

Background and purpose: Bispecific antibody is a special pattern of genetic engineering antibody. It possesses 2 antigen binding sites, one directed at the effector cells and the other at the target cells which are always tumor cells. This enables the effector cells to recruit to the targets efficiently and exert cytotoxic effect.This study aimed to research on the biological activity of antiCD3/antiCD19 diabody. Methods: A non-radioactive cytotoxicity assay was performed to evaluate the efficacy of the Diabody in mediating T-cell cytotoxicity by fluorescent dyes Calcein-AM. Human peripheral blood lymphocytes (PBL) were isolated using Ficoll-Hypaque density-gradient centrifugation from heparinized blood of healthy volunteers and depleted of monocytes by adherence to plastic flasks.Then T cells, CD4+ and CD8+ T cells were selected by FACS. In vitro cytotoxity test was performed by using Raji cells as targets and T cells, CD4+ and CD8+ T cells as effectors firstly. The cytotoxity of effectors mediated by Diabody in different concentration and E/T ratio was compared to PBS, antiCD3scFv+antiCD19scFv and antiCD3/antiPGP.Additionally, CD19 negtive cells K562 was set up as unrelated target. The secretion of IL-2 by activated T cells in groups of E/T 25: 1, concentration of antibody 500 ng/mL were analyzed by ELISA. Also releasing of IL-3, IFN-γ and TNF-α expressing the membrane molecular of these T cells were analyzed by Real-time PCR respectively. Results: Diabody increased the cytotoxicity of T cells in vitro apparently than the contrast groups (P＜0.05). So the secretion of IL-2, IL-3, IFN-γ and TNF-α the release of expressed on activated T cells were increased much more than the other groups. Conclusion: The antiCD3/antiCDl9 bispecific antibody is a potent agent for targeting human T cells in vivo.%背景与目的:双功能抗体有2个抗原结合臂,一方面具有肿瘤相关抗原,另一方面具有和免疫效应细胞表面分子标记结合的能力,并能有效地使效应细胞靶向杀火肿瘤细胞的作用.本实验探讨抗CD3/抗CD19双功能抗体介导T细胞杀伤靶细胞的作用.方法:利用非放射性荧光染料Calcein-AM进行抗体介导的体外特异性杀伤活性榆测,Ficoll-Hypaque法分离外周血淋巴细胞(peripheral blood lymphocyte,PBL),流式细胞术从中分选T淋巴细胞及CD4+T淋巴细胞和CD8+T淋巴细胞作为效应细胞.首先以Raji细胞为靶细胞,按不同效靶比,不同抗体浓度分组,以PBS、抗CD3scFv+、抗CD3scFv抗体及非相关抗体抗CD3/抗PGP为对照,另设CD19-K562细胞为非相关靶细胞组,比较双功能抗体介导特异性体外杀伤活性.取上述杀伤实验效靶比25:1,抗体浓度500 ng/mL各组,ELISA法比较激活的T细胞分泌IL-2的量,Real-time PcR法检测T细胞因子IL-3、IFN-γ、TNF-α激活后的表达变化.结果:体外介导T细胞杀伤靶细胞Raji的效果比对照组明显增强(P＜0.05),激活T细胞分泌IL-2、IL-3、IFN-γ及TNF-α的表达均明显高于对照组.结论:双功能抗体抗CD3/抗CD19在体外介导T细胞对靶细胞具有较强的杀伤作用.