以甲胎蛋白-MHC复合物为靶向的CAR T细胞治疗肝癌的效果评估

摘要/Abstract

摘要： 目的评估以甲胎蛋白(AFP)-MHC复合物为靶向的CAR T细胞治疗肝癌的效果。方法使用EasySep人类T细胞分离试剂盒分离T细胞。双特异性抗体ET1402L1设计成包含CD28/CD3共刺激域的第二代CAR,将CAR构建体克隆到慢病毒载体中,转导原代人T细胞。通过HepG2细胞构建小鼠异种肝癌肿瘤模型。将细胞分为HepG2组和AFP-CAR T细胞组,小鼠分为肿瘤模型组20只和AFP-CAR T靶向治疗组20只。通过CCK-8试剂盒检测细胞增殖。通过细胞LDH释放测定法测量T细胞毒性。PCR检测细胞因子TNFα、IFNγ、IL2和IL10的mRNA表达。ELISA分析肿瘤小鼠AFP水平。采用碘海醇对小鼠脏器组织造影成像。结果第1天两组细胞增殖比较没有差异(P>0.05),第3、5和7天,AFP-CAR T细胞组细胞增殖较HepG2组降低(P<0.05)。HepG2组细胞裂解率为(8.24±2.33)%,AFP-CAR T细胞组细胞裂解率为(72.19±8.32)%(P<0.05)。AFP-CAR T细胞组TNFα、IFNγ、IL-2和IL-10 mRNA分别为1.95±0.17、2.01±0.19、1.96±0.16、1.99±0.18,HepG2组分别为1.04±0.02、1.13±0.06、1.07±0.05、1.03±0.04(P<0.05)。第0天两组小鼠肿瘤大小比较差异无统计学意义(P>0.05),第10、20、30、40天,AFP-CAR T靶向治疗组肿瘤大小较肿瘤模型组减小(P<0.05)。肿瘤模型组AFP水平为(2564.18±265.19)μg/mL,AFP-CAR T靶向治疗组为(837.49±63.22)μg/mL(P<0.05)。结论以AFP-MHC复合物为靶向的CAR-T细胞靶向细胞内分泌的肿瘤抗原产物是可行有效的。

关键词: 甲胎蛋白-MHC复合物, 细胞免疫疗法, CAR-T, 肝癌, 异种移植

Abstract: Objective To investigate the effect of targeting alpha fetoprotein-major histocompatibility complex (AFP-MHC) with CAR T cell therapy for liver cancer.Methods The human liver cancer cell line HepG2 was purchased from Guangzhou Zhenniao Biotechnology Co.Ltd. Healthy human donor peripheral blood leukocytes were obtained from Pacific Blood Center, and EasySep Human T Cell Isolation Kit was used to separate T cells. The bispecific antibody ET1402L1 was designed as a second-generation CAR containing the CD28/CD3 costimulatory domain. The CAR construct was cloned into a lentiviral vector for the transduction of primary human T cells. Forty male BALB/c nude mice (weight 21-25 g; 4-6 weeks) were raised under the condition without specific pathogens. HepG2 cells were used to construct the xenogeneic liver cancer model. The cells were divided into HepG2 group and AFP-CAR T cell group. The mice were divided into tumor model group and AFP-CAR T targeted therapy group. The cell proliferation was detected by CCK-8 kit. T cell toxicity was evaluated by the cellular LDH release assay. The mRNA expression levels of cytokines tumor necrosis factor-α (TNFα), interferon gamma(IFNγ), interleukin-2 (IL2) and IL10 were detected by PCR. The size of xenograft tumor was measured by calipers. The AFP level of tumor mice was detected by ELISA. The contrast of mouse viscera was measured by clinical iodohyalcohol (GE, Omnipaque 350 mg I/ mL).Results There was no significant difference in cell proliferation between the 2 groups at the first day (P>0.05). The cell proliferation in AFP-CAR T cell group at day 3, 5 and 7 was significant lower than that in the HepG2 group (P<0.05). The cell lysis rate of HepG2 group and AFP-CAR T cell group were 8.24±2.33% and 72.19±8.32%, the difference was statistically significant (P<0.05). TNFα, IFNγ, IL2 and IL10 mRNA expression levels in AFP-CAR T cell group were higher than HepG2 group (TNFα: 1.95 ± 0.17 vs 1.04 ± 0.02; IFNγ: 2.01 ± 0.19 vs 1.13 ± 0.06; IL2: 1.96 ± 0.16 vs 1.07 ± 0.05; IL10: 1.99 ± 0.18 vs 1.03 ± 0.04) (P<0.05). There was no difference in tumor size between the 2 groups on day 0 (P>0.05). On day 10d, 20d, 30d and 40d, the tumor size of THE AFP-CAR T-targeted therapy group was decreased compared with that of the tumor model group (P<0.05). AFP levels of tumor model group and AFP-CAR T-targeted therapy group were 2564.18±265.19 μg/ mL and 837.49±63.22 μg/ml, respectively. Serum AFP level in the AFP-CAR T-targeted therapy group was lower than that in the tumor model group (P<0.05). The tumor size of the AFP-CAR T-targeted therapy group was lower than that of the tumor model group (P<0.05).Conclusion The clinical effect of targeting alpha-fetoprotein (AFP)-MHC complex with CAR T cell therapy for liver cancer is satisfactory.

Key words: Alpha-fetoprotein-major histocompatibility complex, Cellular immunotherapy, CAR-T, Liver cancer, Xenotransplantation

王玮, 沙钧平, 丁锋, 王新明. 以甲胎蛋白-MHC复合物为靶向的CAR T细胞治疗肝癌的效果评估[J]. 肝脏, 2022, 27(11): 1175-1179.

WANG Wei, SHA Jun-ping, DING Feng, WANG Xin-ming. Targeting AFP-MHC complex with CAR T cell therapy for liver cancer[J]. Chinese Hepatolgy, 2022, 27(11): 1175-1179.

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