肿瘤细胞治疗研究
英文原题：Cytotoxic activity of anti-mucin 1 chimeric antigen receptor T cells expressing PD-1-CD28 switch receptor against cholangiocarcinoma cells.
M.CAR/SR T细胞的细胞毒性功能较M.CAR T细胞增强，有望进一步用于CCA治疗的测试。
胆管癌（CCA）是一种致死性的胆管癌症，目前的标准化治疗手段难以对其进行有效治疗。这一缺陷促使我们开发针对CCA的过继性T细胞疗法，而该疗法需要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细胞中过表达的蛋白，是CAR T细胞开发的潜在靶抗原。然而，MUC1过表达也与程序性死亡配体1（PD-L1）的上调相关，PD-L1是一种免疫检查点蛋白，可抑制T细胞的抗肿瘤功能，可能导致CCA患者总生存期较差。
为克服这一问题，我们开发了含有PD-1-CD28转换受体（SR）的抗MUC1-CAR T细胞，即M.CAR/SR T细胞，以靶向MUC1并将PD-1/PD-L1相互作用的抑制信号转换为激活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CD3的序列，通过P2A与PD-1和CD28连接。
最初，在CCA细胞系中证实了MUC1和PD-L1蛋白的上调。M.CAR和SR在53.53 13.89%的转导T细胞中共表达，主要为CD8+ T细胞（85.7 0.75%，P<0.0001），具有效应记忆表型（59.22 16.31%，P < 0.01）。M.CAR/SR T细胞在表达MUC1的CCA细胞激活下产生高水平的细胞内肿瘤坏死因子-和干扰素-，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M.CAR T细胞相比，M.CAR/SR T细胞对表达PD-L1的KKU-213A细胞的细胞毒功能显著增强（分别为70.69 14.38%对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%对43.2 8.95%；P = 0.0402）。此外，还观察到与M.CAR T细胞相比，M.CAR/SR T细胞对KKU-213A球体的破坏显著增强（P = 0.0027）。
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 M.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 CD3 , linked with P2A, PD-1 and CD28. RESULTS: Initially, the upregulations of MUC1 and PD-L1 proteins were confirmed in CCA cell lines. M.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). M.CAR/SR T cells produced high levels of intracellular tumor necrosis factor- and interferon- 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 M.CAR/SR T cells against KKU-213A cells expressing PD-L1 was significantly enhanced compared with the M.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 M.CAR/SR T cells (P = 0.0027), compared with M.CAR T cells, was also observed. CONCLUSION: Taken together, the cytotoxic function of M.CAR/SR T cells was enhanced over the M.CAR T cells, which are potential to be further tested for CCA treatment.
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