CD3/KIF20A双特异性抗体介导T淋巴细胞对KIF20A阳性胰腺癌PANC1细胞的抗肿瘤效应

CD3/KIF20A bispecific antibody mediated anti-tumor effects of T lymphocytes on KIF20A positive PANC1 cells

目的探讨胰腺癌双特异性抗体诱导特异性T淋巴细胞的效应及其对KIF20A阳性表达的胰腺癌PANC1细胞系的杀伤作用。方法采用化学交联的方法,经凝胶分子排阻Sephrose-G25层析纯化,制备CD3/KIF20A双特异性抗体(双抗)并浓缩。采集健康志愿者外周血,采用淋巴细胞分离液分离出单核细胞,再分别培养为树突样细胞(DC)及T细胞,并联合培养为DC-T细胞,同时应用维生素C干预DC-T细胞(vcDC-T细胞)。采用流式细胞仪检测各组T细胞亚群及培养上清中IFN-γ、IL-2、IL-4、IL-12水平。将10、50、100、300 ng双抗装载的1×105个T细胞、DC-T细胞、vcDC-T细胞与PANC1细胞(20∶1)共培养2、6、10 h后,明确杀伤率最高的双抗装载剂量;将DC-T细胞及装载杀伤率最高的双抗剂量的DC-T细胞、vcDC-T细胞与PANC1细胞(20∶1)共培养2、4、6、8、10、12 h,倒置显微镜下观察效应细胞对靶细胞的聚集效应,乳酸脱氢酶(LDH)法检测肿瘤细胞杀伤率。结果CD3/KIF20A双抗经SDS凝胶电泳鉴定,其分子质量为130000。与DC-T细胞比较,vcDC-T细胞的CD8+CD28+及CD40L亚群比例增加[(47.6±15.8)%比(38.2±7.6)%,(52.1±4.9)%比(44.7±3.2)%],负性调节细胞Treg比例降低[(4.3±0.8)%比(8.3±1.1)%],分泌的IL-2、IFN-γ、IL-12增加[(201.2±17.3)ng/L比(163.4±13.1)ng/L,(303.3±22.6)ng/L比(221.8±17.6)ng/L,(190.4±11.7)ng/L比(80.3±8.6)ng/L],差异均有统计学意义(P值均<0.05);显微镜下可观察到100 ng CD3/KIF20A双抗装载T细胞对KIF20A+胰腺癌PANC1细胞有明显的靶向性,有最强的杀伤力。效靶细胞比20∶1、共培养4 h时,双抗vcDC-T细胞对PANC1细胞的杀伤率为(88.6±2.6)%,显著高于双抗DC-T组的(68.4±3.4)%及DC-T组的(39.2±2.1)%,杀伤率分别提高20%及45%以上。结论CD3/KIF20A双特异性抗体装载的DC-T细胞能显著提高对KIF20A阳性表达的胰腺癌PANC1细胞的杀伤率,维生素C干预后更进一步增强对双抗装载T细胞的细胞杀伤能力。

Objective To investigate the induction of specific T lymphocyte by bispecific monoclonal antibody in pancreatic cancer and its killing effects on KIF20A positive pancreatic cancer PANC1 cell line.Methods CD3/KIF20A bispecific monoclonal antibody was prepared and concentrated by chemical cross-linking method and purified by Sephrose-25 gel chromatography.Peripheral blood samples of healthy volunteers were collected,and monocytes were isolated using lymphocyte separation solution,and then cultured as dendritic cells(DC)and T cells respectively,and then co-cultured as DC-T cells.Meanwhile vitamin C was used to treat DC-T cells(vcDC-T cells).The levels of IFN-γ,IL-2,IL-4 and IL-12 in the supernatants and T cell subsets were detected by flow cytometry.About 1×105 T cells,DC-T cells,and vcDC-T cells with 10,50,100 and 300 ng CD3/KIF20A antibody loaded were cocultured with PANC1 cells(20∶1)for 2,6 and 10 hours to determine the highest killing rate dosage of CD3/KIF20A antibody loaded cells.DC-T cells and DC-T cells,vcDC-T cells loaded with the highest killing rate dosage of CD3/KIF20A antibody were cocultured with PANC1 cells(20∶1)for 2,6,8,10 and 12 hours.The aggregation effect of effector cells on target cells was observed under inverted microscope,the killing rate of tumor cells was detected by LDH method.Results The molecular weight of CD3/KIF20A antibody was 130000 measured and validated by SDS gel electrophoresis.The ratio of CD8+CD28+and CD40L subsets of vcDC-T cells was increased[(47.6±15.8)%vs(38.2±7.6)%,(52.1±4.9)%vs(44.7±3.2)%]compared with that of DC-T cells,the ratio of negative regulatory cells(Treg)was decreased[(4.3±0.8)%vs(8.3±1.1)%];the release of IL-2,IFN-γand IL-12 was increased[(201.2±17.3)ng/L,(163.4±13.1)ng/L,(303.3±22.6)ng/L vs 221.8±17.6)ng/L,(190.4±11.7)ng/L vs(80.3±8.6)ng/L].All the differences were statistically significant(P<0.01).100 ng CD3/KIF20A loaded T cells were observed under microscope,which obviously targeted KIF20A+pancreatic cancer PANC1 cells and had a strongest killing power.At the killing cells to targeting cells ratio of 20∶1 with 4-hour coculture,the killing rate of CD3/KIF20A-vcDC-T cells on PANC1 cells was(88.6±2.6)%,which was significantly higher than(68.4±3.4)%and(39.2±2.1)%in the CD3/KIF20A-DC-T group and(39.2±2.1)%in the DC-T group,increasing by 20%and at lease 45%,respectively.Conclusions DC-T cells loaded with CD3/KIF20A antibody can significantly increase the killing rate of KIF20A positive pancreatic cancer PANC1 cells,and vitamin C intervention can further enhance the killing ability of antibody loaded T cells.