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@#[摘 要] CAR-T是一种基因改造后的细胞免疫治疗手段， T细胞输入体内后可持续活化增殖，非限制性识别并杀伤肿瘤细胞。 嵌合CD19受体的CAR-T在急性淋巴细胞白血病中的平均有效率接近80%，但在实体肿瘤中却未观察到明显疗效。肿瘤抗原识 别不佳及肿瘤微环境抑制是影响疗效的主要原因，提升抗原的特异性既能增加疗效也能避免脱靶效应的发生。本文主要综述肿 瘤抗原的特点及其在CAR-T治疗实体瘤中的应用现状，重点分析神经节苷酯（disialoganglioside，GD2）、EGFRvⅢ、CEA抗原特点 及应用情况，探讨如何对抗原进行精准选择，其中基因突变后产生的新抗原最有成为特异性抗原的潜力。
ABSTRACT
@#[Abstract] Objective:To prepare the third generation CAR-T cells targeting EGFRvⅢ (EGFRvⅢCAR-T) and to detect its specific killing effect against EGFRvⅢ+ U87 cells in vitro and in vivo. Methods: Human CD3+ T cells were transfected with lentiviral EGFRv Ⅲ/3CAR, which was generated by calcium phosphate co-precipitation of three plasmids. The expression of EGFRvⅢ/3CAR in T cells was detected by Western blotting and flow cytometry. In vitro killing effect of EGFRvⅢ/3CAR-T cells on EGFRvⅢ+ U87 cells was detected by 51Cr release assay. The secretion of cytokine IFN-γ of EGFRvⅢ/3CAR-T cells was detected by ELISA. Nude mouse xenograft model was constructed to detect the in vivo cytotoxicity of EGFRvⅢ/3CAR-T cells on xenograft tumor. Results: The EGFRvⅢ/3CAR lentivirus was successfully packaged with an average titer of 5×106 TU/ml. Western blotting showed that a protein band of approximate 58 000 molecular weight was observed in EGFRvⅢ/3CAR-T cells but absent in untransfected T cells. Flow cytometry indicated the average transduction efficiency of EGFRvⅢ/3CAR was 52.3%. 51Cr release assay showed that the specific killing effect of EGFRvⅢ/ 3CAR-T cells was positively correlated with E/T ratio (E∶T=4∶1, 8∶1, 16∶1, 32∶1). ELISA showed that cytokine IFN-γ secretion was (1 836±148.2) pg/ml, which was significantly different from that of NTT and GFP+ T cells (P<0.01). The specific killing activity of EGFRvⅢ/3CAR-T cells and IFN-γ secretion were both dependent on the expression level of EGFRvⅢ in U87 cells. The tumor growth monitoring results showed that the tumor volume of EGFRvⅢ/3CAR-T cell group was significantly different from that of GFP+ T cell group and PBS group around 3 weeks after injection (P<0.01). Conclusion: EGFRvⅢ/3CAR-T cells demonstrated specific antitumor effectagainstEGFRvⅢ+U87cellsbothinvitro and in vivo, providing basis for immunotherapyofgliomainfuture clinical use.
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Background and purpose:PTEN mutation has been found in 20%-40% of malignant gliomas.The common mutant epidermal growth factor receptor(EGFR vⅢ)was reported to coexpress in PTEN-deficient EGFR-expressing tumor.PTEN has been shown to interact directly with FAK and reduce its tyrosine phosphorylation levels to inhibit cell invasion.The invasion of glioma cells with EGFRvⅢ expression and PTEN deficiency is increased.This study was to observe whether PTEN inhibits glioma cell invasion even in the presence of strong pro-invasive signals provided by constitutive EGFR activity.Methods:U87?EGFR cells were transfected with pcDNA3.1 constructs encoding PTEN and the cells invasion levels were detected by transwell invasion assay.The expression of FAK was detected by immunoblotting.FAK expression vector was transfected into U87?EGFR-wtPTEN cells and the change of cells invasion was documented.Results:PTEN and PTEN(G129E)could inhibit cell invasion induced by EGFRvⅢ.PTEN and PTEN(G129E)could decrease the FAK phosphorylation at Tyr397.Over expression of FAK in U87?EGFR-PTEN abrogated PTEN-induced down-regulation of the phosphorylation status of FAK and rescued cell invasion.Conclusions:PTEN could inhibit cell invasion induced by EGFRvⅢ by dephosphorylating FAK.
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Objective To detect expression of epidermal growth factor receptor variant Ⅲ (EGFRvⅢ) in human suprarenal epithelioma to explore its relation with the genesis and development of suprarenal epithelioma.Methods Immunohistochemistry and pathologic image analysis were used to semiquantitatively detect the expression of EGFRvⅢ protein in eighty human suprarenal epithelioma tissues and twenty-four normal renal tissues.Results Positive expression rate of EGFRvⅢ was 46% in human suprarenal epithelioma tissues and 8% in normal renal tissues.Their average gray scale values were 148.49?13.05 and 155.65?14.86,respectively,which showed a significant difference (t=2.13,P=0.04).There was no obvious difference between males and females (149.01?13.70 and 147.40?11.76,t=0.54,P=0.59).No significant association was observed between EGFRvⅢ expression and age (r=0.01,P=0.92).Conclusion Many human suprarenal epithelioma tissues express EGFRvⅢ.EGFRvⅢ may play certain role in the genesis and development of human suprarenal epithelioma.