Department of Respiratory Medicine, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan.
Department of Biochemistry, Keio University School of Medicine, Tokyo, Japan.
Front Immunol. 2020 Jul 21;11:1405. doi: 10.3389/fimmu.2020.01405. eCollection 2020.
Increasing attention has been paid to human γδ T cells expressing Vγ2Vδ2 T cell receptor (also termed Vγ9Vδ2) in the field of cancer immunotherapy. We have previously demonstrated that a novel bisphosphonate prodrug, tetrakis-pivaloyloxymethyl 2-(thiazole-2-ylamino)ethylidene-1,1-bisphosphonate (PTA), efficiently expands peripheral blood Vγ2Vδ2 T cells to purities up to 95-99% in 10-11 days. In the present study, we first examined the effect of PTA on farnesyl diphosphate synthase (FDPS) using liquid chromatography mass spectrometry (LC-MS) to analyze the mechanism underlying the PTA-mediated expansion of Vγ2Vδ2 T cells. We find that the prodrug induced the accumulation of both isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), direct upstream metabolites of FDPS. This indicates that not only IPP but also DMAPP plays an important role in PTA-mediated stimulation of Vγ2Vδ2 T cells. We next analyzed TCR-independent cytotoxicity of Vγ2Vδ2 T cells. When human lung cancer cell lines were challenged by Vγ2Vδ2 T cells, no detectable cytotoxicity was observed in 40 min. The lung cancer cell lines were, however, significantly killed by Vγ2Vδ2 T cells after 4-16 h in an effector-to-target ratio-dependent manner, demonstrating that Vγ2Vδ2 T cell-based cell therapy required a large number of cells and longer time when tumor cells were not sensitized. By contrast, pulsing tumor cell lines with 10-30 nM of PTA induced significant lysis of tumor cells by Vγ2Vδ2 T cells even in 40 min. Similar levels of cytotoxicity were elicited by ZOL at concentrations of 100-300 μM, which were much higher than blood levels of ZOL after infusion (1-2 μM), suggesting that standard 4 mg infusion of ZOL was not enough to sensitize lung cancer cells in clinical settings. In addition, Vγ2Vδ2 T cells secreted interferon-γ (IFN-γ) when challenged by lung cancer cell lines pulsed with PTA in a dose-dependent manner. Taken together, PTA could be utilized for both expansion of Vγ2Vδ2 T cells and sensitization of tumor cells in Vγ2Vδ2 T cell-based cancer immunotherapy. For use in patients, further studies on drug delivery are essential because of the hydrophobic nature of the prodrug.
在癌症免疫治疗领域，人们越来越关注表达 γδ T 细胞受体 Vγ2Vδ2（也称为 Vγ9Vδ2）的人 γδ T 细胞。我们之前已经证明，一种新型双膦酸盐前药，四（特戊酰氧甲基）2-（噻唑-2-基氨基）乙叉-1,1-双膦酸（PTA），可在 10-11 天内有效地将外周血 Vγ2Vδ2 T 细胞扩增至纯度高达 95-99%。在本研究中，我们首先使用液相色谱-质谱（LC-MS）分析 PTA 对法呢基二磷酸合酶（FDPS）的作用，以研究 PTA 介导的 Vγ2Vδ2 T 细胞扩增的机制。我们发现该前药诱导异戊烯二磷酸（IPP）和二甲基烯丙基二磷酸（DMAPP）的积累，FDPS 的直接上游代谢物。这表明不仅 IPP，而且 DMAPP 在 PTA 介导的 Vγ2Vδ2 T 细胞刺激中发挥重要作用。我们接下来分析了 Vγ2Vδ2 T 细胞的 TCR 非依赖性细胞毒性。当人肺癌细胞系受到 Vγ2Vδ2 T 细胞的攻击时，在 40 分钟内未检测到细胞毒性。然而，在效应物与靶细胞的比例依赖性方式下，Vγ2Vδ2 T 细胞在 4-16 小时后显著杀死肺癌细胞系，表明 Vγ2Vδ2 T 细胞为基础的细胞治疗需要大量细胞并需要更长时间，因为肿瘤细胞未被致敏。相比之下，用 10-30 nM 的 PTA 脉冲肿瘤细胞系甚至在 40 分钟内即可诱导 Vγ2Vδ2 T 细胞对肿瘤细胞的显著溶解。用浓度为 100-300 μM 的 ZOL 可引起相似水平的细胞毒性，这远高于输注后 ZOL 的血液水平（1-2 μM），表明在临床环境中，标准的 4 mg ZOL 输注不足以使肺癌细胞致敏。此外，当用 PTA 脉冲的肺癌细胞系攻击时，Vγ2Vδ2 T 细胞以剂量依赖性方式分泌干扰素-γ（IFN-γ）。总之，PTA 可用于 Vγ2Vδ2 T 细胞的扩增和 Vγ2Vδ2 T 细胞为基础的癌症免疫治疗中的肿瘤细胞的致敏。为了在患者中使用，由于前药的疏水性，还需要进一步研究药物输送。
