抗黏蛋白 1 嵌合抗原受体 T 细胞表达 PD-1-CD28 开关受体对胆管癌细胞的细胞毒性活性。
Cytotoxic activity of anti-mucin 1 chimeric antigen receptor T cells expressing PD-1-CD28 switch receptor against cholangiocarcinoma cells.
机构信息
Siriraj Center of Research Excellence for Cancer Immunotherapy (SiCORE-CIT), Research Department, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand; Division of Molecular Medicine, Research Department, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand.
Siriraj Center of Research Excellence for Cancer Immunotherapy (SiCORE-CIT), Research Department, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand; Division of Molecular Medicine, Research Department, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand.
出版信息
Cytotherapy. 2023 Feb;25(2):148-161. doi: 10.1016/j.jcyt.2022.10.006. Epub 2022 Nov 14.
BACKGROUND AIMS
Cholangiocarcinoma (CCA) is a lethal bile-duct cancer that is difficult to treat by current standard procedures. This drawback has prompted us to develop adoptive T-cell therapy for CCA, which requires an appropriate target antigen for binding of chimeric antigen receptor (CAR) T cells. Mucin 1 (MUC1), an overexpressed protein in CCA cells, is a potential target antigen for the CAR T-cell development. However, MUC1 overexpression also is associated with the upregulation of programmed death-ligand 1 (PD-L1), an immune checkpoint protein that prohibits anti-tumor functions of T cells, probably causing poor overall survival of patients with CCA.
METHODS
To overcome this problem, we developed anti-MUC1-CAR T cells containing PD-1-CD28 switch receptor (SR), namely αM.CAR/SR T cells, to target MUC1 and switch on the inhibitory signal of PD-1/PD-L1 interaction to activate CD28 signaling. Our lentiviral construct contains the sequences that encode anti-MUC1-single chain variable fragment, CD137 and CD3ζ, linked with P2A, PD-1 and CD28.
RESULTS
Initially, the upregulations of MUC1 and PD-L1 proteins were confirmed in CCA cell lines. αM.CAR and SR were co-expressed in 53.53 ± 13.89% of transduced T cells, mainly CD8 T cells (85.7 ± 0.75%, P<0.0001) with the effector memory phenotype (59.22 ± 16.31%, P < 0.01). αM.CAR/SR T cells produced high levels of intracellular tumor necrosis factor-α and interferon-γ in response to the activation by CCA cells expressing MUC1, including KKU-055 (27.18 ± 4.38% and 27.33 ± 5.55%, respectively, P < 0.05) and KKU-213A (47.37 ± 12.67% and 54.55 ± 8.66%, respectively, P < 0.01). Remarkably, the cytotoxic function of αM.CAR/SR T cells against KKU-213A cells expressing PD-L1 was significantly enhanced compared with the αM.CAR T cells (70.69 ± 14.38% versus 47.15 ± 8.413%, respectively; P = 0.0301), correlated with increased granzyme B production (60.6 ± 9.89% versus 43.2 ± 8.95%, respectively; P = 0.0402). Moreover, the significantly enhanced disruption of KKU-213A spheroids by αM.CAR/SR T cells (P = 0.0027), compared with αM.CAR T cells, was also observed.
CONCLUSION
Taken together, the cytotoxic function of αM.CAR/SR T cells was enhanced over the αM.CAR T cells, which are potential to be further tested for CCA treatment.
背景目的
胆管癌（CCA）是一种致命的胆管癌，目前的标准治疗方法难以治疗。这一缺点促使我们开发了用于 CCA 的过继性 T 细胞疗法，该疗法需要一种合适的靶抗原来结合嵌合抗原受体（CAR）T 细胞。黏蛋白 1（MUC1）是 CCA 细胞中过度表达的蛋白，是 CAR T 细胞开发的潜在靶抗原。然而，MUC1 的过度表达也与程序性死亡配体 1（PD-L1）的上调有关，PD-L1 是一种免疫检查点蛋白，可阻止 T 细胞的抗肿瘤功能，可能导致 CCA 患者的总体生存率较差。
方法
为了克服这个问题，我们开发了含有 PD-1-CD28 开关受体（SR）的抗 MUC1-CAR T 细胞，即αM.CAR/SR T 细胞，以靶向 MUC1 并开启 PD-1/PD-L1 相互作用的抑制信号，激活 CD28 信号。我们的慢病毒构建体包含编码抗 MUC1-单链可变片段、CD137 和 CD3ζ 的序列，与 P2A、PD-1 和 CD28 相连。
结果
最初，在 CCA 细胞系中证实了 MUC1 和 PD-L1 蛋白的上调。αM.CAR 和 SR 在转导的 T 细胞中主要是 CD8 T 细胞（85.7±0.75%，P<0.0001）中共同表达 53.53±13.89%，表现为效应记忆表型（59.22±16.31%，P<0.01）。αM.CAR/SR T 细胞在被表达 MUC1 的 CCA 细胞激活后产生高水平的细胞内肿瘤坏死因子-α和干扰素-γ，包括 KKU-055（分别为 27.18±4.38%和 27.33±5.55%，P<0.05）和 KKU-213A（分别为 47.37±12.67%和 54.55±8.66%，P<0.01）。值得注意的是，与 αM.CAR T 细胞相比，αM.CAR/SR T 细胞对表达 PD-L1 的 KKU-213A 细胞的细胞毒性功能显著增强（分别为 70.69±14.38%和 47.15±8.413%，P=0.0301），与颗粒酶 B 产生增加相关（分别为 60.6±9.89%和 43.2±8.95%，P=0.0402）。此外，还观察到与 αM.CAR T 细胞相比，αM.CAR/SR T 细胞显著增强了对 KKU-213A 球体的破坏（P=0.0027）。
结论
综上所述，αM.CAR/SR T 细胞的细胞毒性功能增强，有望进一步用于 CCA 的治疗。
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