ANALYSIS OF ANTIGENICITY OF MUC-1 MUCIN AS A TUMOR ANTIGEN AND POSSIBILITY OF THIS MOLECULE FOR THE USE OF COMBINED ANTI-CANCER IMMUNOTHERAPY WITH GENE TRANSFER

MUC-1粘蛋白作为肿瘤抗原的抗原性分析以及该分子用于基因转移联合抗癌免疫治疗的可能性

• 批准号: 08671356
• 负责人: INOUE Shuhei
• 金额: $ 1.47万
• 依托单位: SHIGA UNIVERSITY OF MEDICAL SCIENCE
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08671356/
• 关键词: MUC-1 mucin; Tumor antigenicity; Anti-tumor immunity; Gene transfer; Anti-cancer immunotherapy; Preparation for clinical intervention

(1)Establishment of a MUC-1 transduced cancer cell line and analysis of its antigenicity as a tumor antigenAMUC-1 transduced cell line was established by transfection of MUC-1 cDNA into breast cancer cell line obtained from a cancer patient. To study the antigenicity of the tranduced cell line, cytotoxic activity of peripheral blood lymphocytes (PBL) against the cell line was examined. A strong lytic activity wa boserved.(2)Induction of cytotoxic T lymphocytes (CTL) specific for MUC-1 mucin and characterization of themMUC-1 specific CTL were induced by co-culture of PBL from cancer patients with the MUC-1 tranduced cell line in the presence of cytokines. The induced CTL showed CD3/CD4 surface markers and TCRalphabeta chain on the surface by FACS.(3)Analysis of antigenic determinants and specificity of the mucin molecule recognized by the CTLThe induced CTL were studied for the cytotoxic activity against the MUC-1 transduced cell line by cytotoxicity assay. Although a strong activity was seen against the MUC-1-expressing cell line, no activity was against the parent cell line. Adding anti-MUC-1 antibodies showed an inhibitory effect on the cytotoxicity observed.(4)Effect of B7 and IL-12 on enhancement of CTL functionApparent propagation of the CTL was induced via B7 molecule expressed on NZK-muc. On the other hand, CTL induced using stimulators without B7 expression showed negligible propagation. IL-12 supplement resulted in differentiation of CTL into CD8 positive T lymphocytes.(5)Targetting MUC-1 mucin on cancer cells for combined anti-cancer immunotherapy with gene transfer techniqueTo enhance the CTL function, transfection of cytokine gene into the CTL is in process. Retrovirus expression vectors inserted with TNF-alpha and interferon-beta cDNA have been prepared.

（1）MUC-1转导的癌细胞系的建立及其作为肿瘤抗原的抗原性分析通过将MUC-1 cDNA转染到从癌症患者获得的乳腺癌细胞系中，建立了AMUC-1转导的细胞系。为了研究转导细胞系的抗原性，检测了外周血淋巴细胞（PBL）对该细胞系的细胞毒活性。观察到强烈的裂解活性。（2）MUC-1特异性细胞毒性T淋巴细胞（CTL）的诱导及鉴定：在细胞因子存在下，将肿瘤患者外周血淋巴细胞（PBL）与MUC-1转导的细胞系共培养，诱导出MUC-1特异性CTL。经流式细胞仪检测，诱导的CTL细胞表面有CD 3/CD 4分子标记，并有TCR α链。（3）CTL识别的粘蛋白分子的抗原决定簇和特异性分析用细胞毒试验研究了诱导的CTL对MUC-1转导细胞系的细胞毒活性。尽管观察到针对表达MUC-1的细胞系的强活性，但没有针对亲本细胞系的活性。添加抗MUC-1抗体显示对观察到的细胞毒性的抑制作用。（4）B7和IL-12对CTL功能的增强作用通过在NZK-muc上表达的B7分子诱导CTL的明显增殖。另一方面，使用无B7表达的刺激物诱导的CTL显示可忽略的增殖。IL-12的补充导致CTL分化为CD 8阳性T淋巴细胞。（5）MUC-1粘蛋白在肿瘤细胞表面的靶向表达与基因转移技术联合应用的研究已经制备了插入有TNF-α和干扰素-β cDNA的逆转录病毒表达载体。