DNAM1 and 2B4 Costimulatory Domains Enhance the Cytotoxicity of Anti-GPC3 Chimeric Antigen Receptor-Modified Natural Killer Cells Against Hepatocellular Cancer Cells in vitro
DNAM1 and 2B4 Costimulatory Domains Enhance the Cytotoxicity of Anti-GPC3 Chimeric Antigen Receptor-Modified Natural Killer Cells Against Hepatocellular Cancer Cells in vitro
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DOI：
10.2147/cmar.s253565
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发表时间：
2020-01-01
影响因子：
3.3
通讯作者：
Zeng, Jinhua
中科院分区：
文献类型：
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作者：
Huang, Yao;Zeng, Jianxing;Zeng, Jinhua
目的：肝细胞癌是世界范围内第六大常见癌症和第三大癌症相关死亡原因。继嵌合抗原受体(CAR)-T疗法在治疗B细胞恶性肿瘤方面取得成功后，针对GPC3的细胞免疫疗法最近被用于肝癌的治疗。然而，CAR-T细胞不是现成的，总是会引起细胞因子释放综合征，使用自然杀伤(NK)细胞作为效应细胞可以消除这种综合征。由于共刺激信号对CAR-T细胞的激活、持续或细胞毒作用是必需的，我们推测在肝癌治疗中CAR-NK细胞也需要共刺激信号。方法：构建了5个含有不同共刺激结构域的抗GPC3 CAR质粒。它们包括Z(只有CD3 Zeta结构域，无共刺激结构域)、CD28.Z(T细胞共刺激结构域CD28)、DNAM1/2B4.Z(NK细胞相关共刺激结构域DNAM1或2B4)和DNAM1/2B4.Z(两个NK细胞相关共刺激结构域)，分别产生各自的CAR-NK-92细胞。采用四甲基偶氮唑盐比色法检测不同共刺激区域对CAR-NK细胞增殖的影响。用细胞凋亡率和流式细胞仪分析其对持续时间的影响。体外实验研究了其对正常肝细胞和GPC3(+)恶性细胞的特异性细胞毒作用。用ELISA法检测CAR-NK-92细胞释放的细胞因子(肿瘤坏死因子-α和干扰素-γ)的浓度。结果：CAR-NK-92细胞需要NK细胞相关的共刺激信号。与CD28或没有共刺激信号相比，带有DNAM1和/或2B4的CAR-NK-92细胞扩增更快，并且持续存在较低的凋亡率。所有CAR-NK-92细胞在体外均表现出特殊的细胞毒作用。与不含任何共刺激结构域或同时含有DNAM1和2B4的T细胞共刺激结构域相比，具有NK细胞相关共刺激结构域的CAR-NK-92细胞具有更强的细胞毒活性。细胞因子释放实验结果与细胞毒实验结果一致。结论：首次提出了利用DNAM1和2B4共刺激结构域产生抗GPC3CAR-NK-92细胞的策略，该细胞在体外对肝癌细胞具有更强的杀伤活性。
Purpose: Hepatocellular cancer (HCC) is the sixth most prevalent cancer and the third leading cause of cancer-related death worldwide. Cellular immunotherapy against glypican 3 (GPC3) has recently been used in the treatment of HCC, following the success of chimeric antigen receptor (CAR)-T therapy in treatment of B cell malignancy. However, CAR-T cells are not "off-the-shelf" and always cause cytokine release syndrome, which can be eliminated by using natural killer (NK) cells as effector cells. Since a costimulatory signal is necessary for the activation, persistence, or cytotoxicity of CAR-T cells, we speculated that the costimulatory signal is also required for CAR-NK cells in HCC treatment.Methods: Five anti-GPC3 CAR plasmids containing different costimulatory domains were constructed. They included Z (only the CD3 zeta domain, no costimulatory domain), CD28.Z (T-cell costimulatory domain CD28), DNAM1/2B4.Z (NK-cell-associated costimulatory domain DNAM1 or 2B4), and DNAM1/2B4.Z (both NK-cell-associated costimulatory domains) Respective CAR-NK-92 cells were generated. The MTT viability assay was performed to evaluate the effect of the different costimulatory domains on CAR-NK-cell proliferation. The effect on persistence was analyzed using an apoptosis assay and flow cytometry. Special cytotoxicity against normal hepatocellular cells and GPC3(+) malignant cells was investigated in vitro. The concentra-tion of cytokines (TNF-alpha and IFN-gamma) released by CAR-NK-92 cells was also measured by ELISA.Results: NK-cell-associated costimulatory signal was necessary for CAR-NK-92 cells. CAR-NK-92 cells with DNAM1 and/or 2B4 expanded more quickly and persisted with a lower apoptotic ratio, compared to the presence of CD28 or no costimulatory signal. All CAR-NK -92 cells showed special cellular cytotoxicity in vitro. CAR-NK-92 cells with NK-cell-associated costimulatory domains exhibited higher cytotoxic ability compared with those without any costimulatory domain or with T-cell costimulatory domain CAR-NK-92 cells with both DNAM1 and 2B4 displayed the highest cytotoxicity. The cytokine release assay results were consistent with those of the cytotoxicity assay.Conclusion: We provided the first evidence supporting a strategy using DNAM1 and 2B4 costimulatory domains to generate anti-GPC3 CAR-NK-92 cells, which exhibits enhanced cytotoxicity against hepatocellular cancer cells in vitro.