Development of apoptosis-inducing gene therapy using prostate specific membrane antigen (PSMA) antibody

使用前列腺特异性膜抗原（PSMA）抗体开发细胞凋亡诱导基因疗法

• 批准号: 14571523
• 负责人: KOGA Shoji
• 金额: $ 2.24万
• 依托单位: Tokyo Medical University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14571523/
• 关键词: Prostate cancer; Prostate specific membrane antigen; Gene therapy; Antibody

Preparation of prostate cancer model with nude miceFor prostate cancer cell lines, LNCaP cells in which existence of the PSMA antigen has been confirmed, C4-2 cells, their subline, and PSMA-negative PC-3 cells were used. 1×10^6 LNCaP and C4-2 cells were subcutaneously injected, respectively. The C4-2 cells formed a tumor mass more easily (100%) than LNCaP cells (70%) showing appropriate as a model.In vitro study using PSMA antibody conjugated Caspase 8 plasmidAt the beginning, the hTERT promoter driven Capase 8 plasmid was prepared. First, the cloned hTERT promoter (384bp) was integrated into the pGL3 basic vector. Next, the luciferase gene of the pGL3 vector was replaced by the Caspase 8 gene. Then, the PSMA antibody and the vector prepared above or pGL3 control vector (SV4O promoter driven luciferase gene) were biotynilated. At the same time, the PSMA antibody was biotynilated. The PSMA antibody conjugated luciferase plasmid (PSMA antibody and pGL3 control vector) and PSMA antibody C … More aspase B DNA plasmid (PSMA antibody and hTERT promoter driven Caspase 8 plasmid) were prepared by conjugating the biotynilated vector and antibody with avidin, respectively. The luciferase assay was conducted by introducing the PSMA antibody conjugated luciferase plasmid into the LNCaP, C4-2 and PC-3 cells. The luciferase activities of the LNCaP and C4-2 cells were high, showing 10.3 and 28.4, respectively, when letting those of the PC-3 cells to be 1.0. In addition, the status of apoptosis in each cell line in administration of the PSMA antibody Caspase 8 DNA plasmid was confirmed by the FACS following the tunel staining. The results were 1.2% inPC3, 3.0% in LNCaP and 4.2% in C4-2, showing no significant difference.In vivo study using PSMA antibody conjugated Caspase 8 plasmid1, 10 and 100 μg of the PSMA antibody conjugated luciferase plasmid were administered to the homologous prostate cancer models(PC-3 and C4-2),respectively. The tumor was taken three days after administration and immunostained with the anti-luciferase antibody. The C4-2 tumor was partially stained in 10 and 100 μg administration, whereas the PC-3 tumor was not stained at all. Next, 100 μg of the PSMA antibody Caspase 8 DNA plasmid was administered daily when the tumor grew to the size of 10mm in diameter in the homologous prostate cancer models(PC-3 and C4-2),and its antiproliferative effects were examined. There was no significant difference though some antiproliferative effects were found, compared with the control group (the PSMA antibody conjugated luciferase plasmid administration group). Less

前列腺癌细胞系使用确认了PSMA抗原的存在的LNCaP细胞、C4-2细胞及其亚系、PSMA阴性的PC-3细胞。分别皮下注射1×10^6 LNCaP和C4-2细胞。C4-2细胞比LNCaP细胞（70%）更容易形成肿瘤块（100%），显示适合作为模型。首先，将克隆的hTERT启动子（384 bp）整合到pGL 3基础载体中。接着，将pGL 3载体的荧光素酶基因替换为胱天蛋白酶8基因。然后，将PSMA抗体和上述制备的载体或pGL 3对照载体（SV 4 O启动子驱动的荧光素酶基因）生物素化。同时，将PSMA抗体生物素化。PSMA抗体缀合的荧光素酶质粒（PSMA抗体和pGL 3对照载体）和PSMA抗体C ...更多信息 通过将生物素化载体和抗体分别与抗生物素蛋白缀合来制备天冬氨酸蛋白酶B DNA质粒（PSMA抗体和hTERT启动子驱动的Caspase 8质粒）。通过将PSMA抗体缀合的荧光素酶质粒引入LNCaP、C4-2和PC-3细胞中进行荧光素酶测定。LNCaP和C4-2细胞的荧光素酶活性高，当PC-3细胞的荧光素酶活性为1.0时，分别为10.3和28.4。此外，在TUNEL染色后，通过FACS确认在施用PSMA抗体胱天蛋白酶8 DNA质粒中每个细胞系中的细胞凋亡状态。结果在PC-3、LNCaP和C4 -2中分别为1.2%、3.0%和4.2%，显示无显著性差异。给药后3天取出肿瘤，用抗荧光素酶抗体免疫染色。C4-2肿瘤在10和100 μg给药中部分染色，而PC-3肿瘤完全不染色。接着，当同源前列腺癌模型（PC-3和C4-2）中肿瘤生长至直径10 mm大小时，每天施用100 μg的PSMA抗体胱天蛋白酶8 DNA质粒，并检查其抗增殖作用。与对照组（PSMA抗体结合的荧光素酶质粒给药组）相比，尽管发现了一些抗增殖作用，但没有显著差异。少

项目成果

Koga S, Hirohata S, Kondo Y, Komata T, Takakura M, Inoue M, Kyo S, Kondo S.: "FADD gene therapy using the human telomerase catalytic subunit (hTERT) gene promoter to restrict induction of apoptosis to tumors in vitro and in vivo"Anticancer Res.. 21(3B). 1

Koga S、Hirohata S、Kondo Y、Komata T、Takakura M、Inoue M、Kyo S、Kondo S.：“FADD 基因疗法使用人端粒酶催化亚基 (hTERT) 基因启动子来限制体外肿瘤细胞凋亡的诱导，

Komata T, Kondo Y, Kanzawa T, Hirohata S, Koga S, Sumiyoshi H, Srinivasula SM, Barna DP, Germane IM, Takakura M, Inoue M, Alnemri ES, Shay JW, Kyo S, Kondo S.: "Treatment of malignant glioma cells with. the transfer of constitutively activecaspase-6 using

Komata T，Kondo Y，Kanzawa T，Hirohata S，Koga S，Sumiyoshi H，Srinivasula SM，Barna DP，Germane IM，Takakura M，Inoue M，Alnemri ES，Shay JW，Kyo S，Kondo S.：“恶性癌症的治疗

obayashi H, Koga S, Novick AC, Toma H, Fairchild RL.: "T-cell mediated induction of allogeneic endothelial cell chemokine expression."Transplantation. 75(4). 529-536 (2003)

obayashi H、Koga S、Novick AC、Toma H、Fairchild RL.：“T 细胞介导的同种异体内皮细胞趋化因子表达的诱导。”移植。

Komata T, Koga S, Hirohata S, Takakura M, Germano IM, Inone M, Kyo S, Kondo S, Kondo Y.: "A novel treatment of human malignant gliomas in vitro and in vivo : FADD genetransfer under the control of the human telomerase reverse transcriptase genepromoter."I

Komata T、Koga S、Hirohata S、Takakura M、Germano IM、Inone M、Kyo S、Kondo S、Kondo Y.：“体外和体内人类恶性胶质瘤的一种新治疗方法：在人类控制下的 FADD 基因转移

Kondo Y, Komata T, Kondo S.: "Treatment of bladder cancer cells in vitro and in vivo with 2-5A antisensetelomerase RNA."Gene Ther. 8(8). 654-658 (2001)

Kondo Y、Komata T、Kondo S.：“用 2-5A 反义端粒酶 RNA 体外和体内治疗膀胱癌细胞。”Gene Ther。