Biotherapeutics Group, National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Hertfordshire EN6 3QG, UK.
Cytokine. 2011 Jul;55(1):141-51. doi: 10.1016/j.cyto.2011.03.019. Epub 2011 Apr 13.
The failure of preclinical testing to predict the severity of the cytokine storm experienced by the recipients of the superagonistic anti-CD28 monoclonal antibody (mAb) TGN1412 during its Phase 1 clinical trial prompted the development of new in vitro experimental approaches for mimicking in vivo cytokine release and lymphoproliferation. Peripheral blood mononuclear cells (PBMC) presented to TGN1412 immobilised on plastic has previously been shown to stimulate a pro-inflammatory cytokine response. The aim of the present study was to investigate a 'co-culture' model for the detection of TGN1412-like immunomodulatory activity in which TGN1412 was presented to PBMC in the presence of monolayers of endothelium-derived cells and other cell types, followed by measurement of cytokine levels in the culture supernatants and proliferation of PBMC. Culturing PBMC with TGN1412 over primary human umbilical vein endothelial cells (HUVEC) and HUVEC-derived cell lines retaining classic endothelial markers, but not cell lines of non-endothelial origin, mediated the specific release of IL-6, IL-8 and TNFα, and proliferation of PBMC. Low levels of IL-2 and IFNγ were also detected in supernatants with most donors of PBMC. An anti-CD28 mAb agonist, i.e., not a superagonist like TGN1412, did not stimulate cytokine release or proliferation of PBMC in co-cultures. In conclusion, co-culture experiments for TGN1412-specific cytokine release required cells of endothelial origin. However, the profile of released cytokines in co-cultures did not mirror that in the clinical trial participants or the responses from PBMC exposed to TGN1412 immobilised on plastic, suggesting that TGN1412 stimulation of PBMC can occur through more than one mechanism.
在 TGN1412 单克隆抗体（mAb）的 I 期临床试验中，临床前测试未能预测接受者细胞因子风暴的严重程度，这促使开发新的体外实验方法来模拟体内细胞因子释放和淋巴增殖。先前已经表明，固定在塑料上的 TGN1412 呈递外周血单核细胞（PBMC）会刺激促炎细胞因子反应。本研究的目的是研究一种“共培养”模型，用于检测 TGN1412 样免疫调节活性，其中 TGN1412 在单层内皮细胞衍生细胞和其他细胞类型存在的情况下呈递给 PBMC，然后测量培养上清液中的细胞因子水平和 PBMC 的增殖。与原发性人脐静脉内皮细胞（HUVEC）和保留经典内皮标志物的 HUVEC 衍生细胞系共培养 PBMC 与 TGN1412 一起培养，而不是非内皮来源的细胞系，介导了 IL-6、IL-8 和 TNFα 的特异性释放以及 PBMC 的增殖。大多数 PBMC 供体的上清液中还检测到低水平的 IL-2 和 IFNγ。一种抗 CD28 mAb 激动剂，即不是像 TGN1412 那样的超强激动剂，在共培养物中不会刺激 PBMC 释放细胞因子或增殖。总之，TGN1412 特异性细胞因子释放的共培养实验需要内皮来源的细胞。然而，共培养物中释放的细胞因子谱与临床试验参与者或暴露于固定在塑料上的 TGN1412 的 PBMC 的反应并不相同，这表明 TGN1412 刺激 PBMC 可能通过不止一种机制发生。
