CD133在包括肝细胞癌（HCC）在内的多种肿瘤中被认为是癌干细胞（CSCs）的标记物。针对CD133阳性CSCs的嵌合抗原受体特异性T细胞（CAR-T细胞）已经成为HCC临床治疗的一种工具，但免疫原性、临床级重组病毒载体高成本和潜在插入性突变等因素限制了它们的临床应用。本研究使用睡眠美人鱼转座子系统从微圈技术构建了分泌PD-1阻断剂单链抗体（CD133 CAR-T和PD-1s细胞），并在体外和体内分析了CD133 CAR-T和PD-1s细胞的抗肿瘤效力。单因素分析显示，在晚期（II和III期）的男性患者中，CD133的表达与较差的无进展生存期（PFS）（P = 0.0057）和总体生存（OS）（P = 0.015）显著关联，多因素分析显示一种向较差OS的趋势（P = 0.041）。HCC晚期男性患者的PD-L1联合阳性评分（CPS）约为早期HCC患者的20倍。我们成功产生了能够分泌PD-1阻断剂单链抗体的CD133 CAR-T和PD-1s细胞，并基于睡眠美人鱼系统和微圈载体，体外和异种移植小鼠模型中显著展现了对HCC的抗肿瘤活性。因此，CD133 CAR-T和PD-1s细胞可能是治疗晚期HCC男性患者靶向CD133阳性CSCs的可行策略。我们的研究提供了一种基于睡眠美人鱼系统和微圈载体构建CAR-T细胞并分泌检查点阻断抑制剂的非病毒策略，并揭示了这种策略对具有高CD133表达（免疫组织化学评分>2.284）的晚期HCC男性患者的潜在好处。© 2023. BioMed Central Ltd.，Springer Nature的一部分。

CD133 is considered a marker for cancer stem cells (CSCs) in several types of tumours, including hepatocellular carcinoma (HCC). Chimeric antigen receptor-specific T (CAR-T) cells targeting CD133-positive CSCs have emerged as a tool for the clinical treatment of HCC, but immunogenicity, the high cost of clinical-grade recombinant viral vectors and potential insertional mutagenesis limit their clinical application.CD133-specific CAR-T cells secreting PD-1 blocking scFv (CD133 CAR-T and PD-1 s cells) were constructed using a sleeping beauty transposon system from minicircle technology, and the antitumour efficacy of CD133 CAR-T and PD-1 s cells was analysed in vitro and in vivo.A univariate analysis showed that CD133 expression in male patients at the late stage (II and III) was significantly associated with worse progression-free survival (PFS) (P = 0.0057) and overall survival (OS) (P = 0.015), and a multivariate analysis showed a trend toward worse OS (P = 0.041). Male patients with advanced HCC exhibited an approximately 20-fold higher PD-L1 combined positive score (CPS) compared with those with HCC at an early stage. We successfully generated CD133 CAR-T and PD-1 s cells that could secrete PD-1 blocking scFv based on a sleeping beauty system involving minicircle vectors. CD133 CAR-T and PD-1 s cells exhibited significant antitumour activity against HCC in vitro and in xenograft mouse models. Thus, CD133 CAR-T and PD-1 s cells may be a therapeutically tractable strategy for targeting CD133-positive CSCs in male patients with advanced HCC.Our study provides a nonviral strategy for constructing CAR-T cells that could also secrete checkpoint blockade inhibitors based on a Sleeping Beauty system from minicircle vectors and revealed a potential benefit of this strategy for male patients with advanced HCC and high CD133 expression (median immunohistochemistry score > 2.284).© 2023. BioMed Central Ltd., part of Springer Nature.