Qingpu Branch of Zhongshan Hospital Affiliated to Fudan University, Shanghai, 201700, China.
Birth Defects and Regenerative Medicine Laboratory, Department of Biochemistry & Molecular Biology, Biomedicine and Health Graduate Education Innovation Center, Shanxi Medical University, No. 56, Xinjian South Road, Taiyuan, Shanxi, 030001, China.
BMC Gastroenterol. 2024 Aug 6;24(1):249. doi: 10.1186/s12876-024-03286-9.
To construct chimeric antigen receptor (CAR)-T cells targeting epithelial cell adhesion molecule (EpCAM) antigen (anti-EpCAM-CAR-T).
A third-generation CAR-T cell construct used a single-chain variable fragment derived from monoclonal antibody against human EpCAM. Peripheral blood mononuclear cells were extracted from volunteers. The proportion of cluster of differentiation 8 positive (CD8+) and CD4 + T cells was measured using flow cytometry. Western blot was used to detect the expression of EpCAM-CAR. The killing efficiency was detected using the MTT assay and transwell assay, and the secretion of killer cytokines tumour necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) was detected using the ELISA. The inhibitory effect of EpCAM-CAR-T on colorectal cancer in vivo was detected using xenografts.
It was found that T cells expanded greatly, and the proportion of CD3+, CD8 + and CD4 + T cells was more than 60%. Furthermore, EpCAM-CAR-T cells had a higher tumour inhibition rate in the EpCAM expression positive group than in the negative group (P < 0.05). The secretion of killer cytokines TNF-α and IFN-γ in the EpCAM expression positive cell group was higher than that in the negative group (P < 0.05). In the experimental group treated with EpCAM-CAR-T cells, the survival rate of nude mice was higher (P < 0.05), and the tumour was smaller than that in the blank and control groups (P < 0.05). The secretion of serum killer cytokines TNF-α and IFN-γ in tumour-bearing nude mice in the experimental group treated with EpCAM-CAR-T cells was higher than that in the blank and control groups (P < 0.05).
This study successfully constructed EpCAM-CAR cells and found that they can target and recognise EpCAM-positive tumour cells, secrete killer cytokines TNF-α and IFN-γ and better inhibit the growth and metastasis of colorectal cancer in vitro and in vivo than unmodified T cells.
构建靶向上皮细胞黏附分子（EpCAM）抗原的嵌合抗原受体（CAR）-T 细胞（抗-EpCAM-CAR-T）。
第三代 CAR-T 细胞构建物使用源自抗人 EpCAM 单克隆抗体的单链可变片段。从志愿者中提取外周血单个核细胞。使用流式细胞术测量 CD8+和 CD4+T 细胞的比例。使用 Western blot 检测 EpCAM-CAR 的表达。使用 MTT 测定和 Transwell 测定检测杀伤效率，并使用 ELISA 检测杀伤细胞因子肿瘤坏死因子-α（TNF-α）和干扰素-γ（IFN-γ）的分泌。使用异种移植检测 EpCAM-CAR-T 在体内对结直肠癌的抑制作用。
发现 T 细胞大量扩增，CD3+、CD8+和 CD4+T 细胞的比例超过 60%。此外，EpCAM-CAR-T 细胞在 EpCAM 表达阳性组的肿瘤抑制率高于阴性组（P<0.05）。EpCAM 表达阳性细胞组中杀伤细胞因子 TNF-α和 IFN-γ的分泌量高于阴性组（P<0.05）。在接受 EpCAM-CAR-T 细胞治疗的实验组中，裸鼠的存活率更高（P<0.05），肿瘤体积小于空白组和对照组（P<0.05）。接受 EpCAM-CAR-T 细胞治疗的实验组荷瘤裸鼠血清杀伤细胞因子 TNF-α和 IFN-γ的分泌量高于空白组和对照组（P<0.05）。
本研究成功构建了 EpCAM-CAR 细胞，发现其可靶向识别 EpCAM 阳性肿瘤细胞，分泌杀伤细胞因子 TNF-α和 IFN-γ，体外和体内均能更好地抑制结直肠癌的生长和转移，优于未经修饰的 T 细胞。
