Department of Liver Surgery and Transplantation, Liver Cancer Institute, Zhongshan Hospital, Fudan University, Shanghai, 200032, China.
Key Laboratory of Carcinogenesis and Cancer Invasion, Ministry of Education, Fudan University, Shanghai, 200032, China.
BMC Cancer. 2022 Jul 25;22(1):814. doi: 10.1186/s12885-022-09861-1.
Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and the adoptive immunotherapy of which is worth studying. CD133, a kind of cancer stem cell (CSC) antigen, together with glypican-3 (GPC3) has been proved to be highly expressed in HCC cells and both of them are used as targets to generate chimeric antigen receptor (CAR) T cells. But there are limitations like "off-target" toxicity, low transfection efficacy and weak antitumor ability in CAR T cells treatment.
The peripheral blood was acquired from healthy donors and T cells were separated by density-gradient centrifugation. We used an electroporation system to deliver anti-CD133 and anti-GPC3 single chain Fragment variable (scFv) structures as target genes into the T cells. The cell membrane was opened by the momentary electric current effect, and the target gene was delivered into the cell by non-viral minicircle DNA (mcDNA) vector. The flow cytometry and western blot assays were used to detect whether the two scFv were simultaneously transfected and the transfection efficacy of this bispecific CAR T cell generation method. We respectively detected the in vitro and in vivo tumor-suppression efficacy of CAR T cells through the CCK-8 assays and the HCC xenograft mice models. The CoG133-CAR T cells containing both CD133 and GPC3 antigen recognition sites were the effector cells. CD133-CAR T cells and GPC3-CAR T cells were defined as single-targeted control groups, normal T and mock T cells were defined as blank control groups.
The mcDNA vector accommodated two target gene structures successfully transfected to generate bispecific CAR T cells. The detection methods on gene level and protein level confirmed that CoG133-CAR T cells had considerable transfection efficiency and exhibited both antigen-binding capacity of CD133 and GPC3. Compared to single-targeted CAR T cells or control T cells, CoG133-CAR T cells performed enhanced eliminated efficacy against CD133 and GPC3 double-positive HCC cell line in vitro and HCC xenograft mice in vivo. Hematoxylin and eosin (H&E) staining indicated no fatal "off-target" combination existed on CoG133-CAR T cells and major organs.
Our study suggests that it is with higher efficiency and more safety to prepare bispecific CAR T cells through non-viral mcDNA vectors. CoG133-CAR T cells have enhanced tumor-suppression capacity through dual antigen recognition and internal activation. It provides an innovative strategy for CAR T therapy of HCC, even solid tumors.
肝细胞癌（HCC）是全球最常见的癌症之一，其过继免疫治疗值得研究。CD133 是一种癌症干细胞（CSC）抗原，与磷脂酰聚糖-3（GPC3）一起在 HCC 细胞中高表达，两者均被用作产生嵌合抗原受体（CAR）T 细胞的靶标。但是，CAR T 细胞治疗存在“脱靶”毒性、低转染效率和弱抗肿瘤能力等局限性。
从健康供体采集外周血，通过密度梯度离心分离 T 细胞。我们使用电穿孔系统将抗 CD133 和抗 GPC3 单链片段可变（scFv）结构作为靶基因导入 T 细胞。瞬时电流效应打开细胞膜，通过非病毒微小环 DNA（mcDNA）载体将靶基因递送入细胞。流式细胞术和 Western blot 检测同时转染两个 scFv 的情况，以及这种双特异性 CAR T 细胞生成方法的转染效率。通过 CCK-8 检测和 HCC 异种移植小鼠模型，分别检测 CAR T 细胞的体外和体内肿瘤抑制效力。含有 CD133 和 GPC3 抗原识别位点的 CoG133-CAR T 细胞为效应细胞。CD133-CAR T 细胞和 GPC3-CAR T 细胞为单靶向对照组，正常 T 细胞和 mock T 细胞为空白对照组。
mcDNA 载体成功容纳两个靶基因结构，转染生成双特异性 CAR T 细胞。基因水平和蛋白水平的检测方法证实，CoG133-CAR T 细胞具有相当高的转染效率，并表现出 CD133 和 GPC3 的抗原结合能力。与单靶向 CAR T 细胞或对照 T 细胞相比，CoG133-CAR T 细胞在体外对 CD133 和 GPC3 双阳性 HCC 细胞系和体内 HCC 异种移植小鼠具有增强的消除效力。苏木精和伊红（H&E）染色表明 CoG133-CAR T 细胞和主要器官上不存在致命的“脱靶”结合。
本研究表明，使用非病毒 mcDNA 载体制备双特异性 CAR T 细胞具有更高的效率和安全性。CoG133-CAR T 细胞通过双重抗原识别和内部激活增强了肿瘤抑制能力。为 HCC 甚至实体瘤的 CAR T 治疗提供了一种创新策略。
