一种筛选中和活性抗人TNF单抗细胞模型的建立及其应用

Establishment and application of a cell model for screening neutralizing monoclonal antibody against human TNF

目的:建立一种筛选中和活性抗人肿瘤坏死因子(tumornecrosisfactor,TNF)mAb细胞模型,并从一组抗人TNFmAb中筛选出具有相对较高中和活性的mAb.方法:用含100mL/LFCS的DMEM细胞培养液将L929细胞的浓度调整到1×108/L,按每孔100μL,加入至96孔细胞培养板中,培养过夜.弃细胞培养上清后,将200μL的实验液转入培养板中,继续培养16h.活细胞用结晶紫染色,脱色后,于570nm波长测定A值.结果:浓度在0.3～0.5mg/L范围内的放线菌素D(antinomycinD,ActD)对L929细胞的生长抑制作用比较稳定;TNF的浓度高于20kU/L时,能够使95%以上的实验细胞发生凋亡;浓度在12.5～50.0μg/L范围的mAb对TNF的中和作用具有明显的剂量依赖关系;在ActD,TNF和mAb的浓度分别为0.35mg/L,20kU/L和25.0μg/L的条件下,发现D2的中和活性与阳性对照抗体无显著性差异(P>0.05),但高于阴性对照抗体,E6,D12,E11(P<0.01).与阴性对照抗体比较,E6,E11也具有一定的中和活性(P<0.05),而D12无中和活性(P>0.05).结论:本研究建立的mAb抑制TNF诱导L929细胞凋亡的细胞模型可用于筛选中和活性抗TNFmAb;D2的中和活性相对较高,可作为构建治疗性嵌合抗体的制剂.

AIM: To establish and apply a cell model to screen neutralizing monoclonal antibody against human TNF. METHODS: L929 cells were seeded into flat bottom of 96-well microtiter plates at a density of 1×10 8/L in 100 μL DMEM containing 100 mL/L FCS and cultured at 37℃ for 16 h. Spent medium was removed and 200 μL of experimental solutions was added to each well. The plates were similarly reincubated for 16 h, followed by removal of culture supernatants and stained with crystal violet. The absorbance at 570 nm was determined. RESULTS: In the range from 0.3 mg/L to 0.5 mg/L, antinomycin D (ActD) had stable inhibitory effects on L929 cell growth. In the concentration over 20 kU/L, TNF could induce over 95% of experimental cells apoptosis. In the range from 12.5 μg/L to 50.0 μg/L, monoclonal antibody could neutralize TNF in a dose dependent manner. Under the condition that the concentrations of ActD, TNF and monoclonal antibody were 0.35 mg/L, 20 kU/L, and 25.0 μg/L, respectively, no significant difference was found between D2 and positive control antibody in aspect of neutralizing activity (P>0.05), but the neutralizing activity of D2 was significantly higher than those of E6, E11, D12 and negative control antibody (P<0.01). Compared with negative control antibody, E6 and E11 also had neutralizing activity (P<0.05), but D12 did not show any neutralizing activity (P>0.05). CONCLUSION: The cell model established and optimized in our study can be employed in screening neutralizing monoclonal antibodies against human TNF. By employing this model, we have identified that D2 is of high neutralizing activity, which can be used as a reagent for the construction of therapeutic chimeric antibody.