Cancer vaccine therapy using genetically modified dendritic cells expressing tumor-associated antigen and cytokines

使用表达肿瘤相关抗原和细胞因子的转基因树突状细胞进行癌症疫苗治疗

• 批准号: 15591354
• 负责人: IWAHASHI Makoto
• 金额: $ 2.24万
• 依托单位: Wakayama Medical University School of Medicine
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2005
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15591354/
• 关键词: Dendritic cells; Adenoviral vector; CEA; Transgenic mice; GM-CSF; IL-12; 樹状細胞(DC); adenovirus vector; transgenic mouse; 成熟化(maturation)

(2003) 1.Generation of recombinant adenoviral (Ad) vectors. The recombinant adenoviral vector which expresses human CEA (AxCACEA) was generated by the COS-TPC method. The recombinant AxCAGM-CSF and AxCALacZ were also generated by the COS-TPC method. AxCAIL-12 which expresses murine IL-12 were obtained from RIKEN BioResource Center. 2.Ad vector-mediated gene transfer into DCs. Immature DCs were transfected with each recombinant adenoviral (Ad) vector using a centrifugal method. Our experiments demonstrated that the expression of CEA was detected in the majority (67.5 %) of DCs transfected with AxCACEA at a MOI of 100. 3.The bleeding of CEA transgenic mice. Male and female CEA transgenic mice [C57BL/6J-TgN(CEA Ge)18FJP](H-2^b)(CEA Tg) were obtained from Dr.F.James Primus, and were bred at the animal care facility of Wakayama Medical University. The screening of born mice for the CEA transgene was carried out by PCR analysis on mouse tail DNA.(2004) 1.Cytotoxic activity of spleen cells in … More mice immunized with genetically modified DCs. The mice were once immunized by a subcutaneous (s.c.) injection of 1×10^6 genetically modified DCs. Spleen cells were isolated 14 days after immunization with DCs, and then the in vivo-primed spleen cells were pooled and cultured in a 6-well plate with rmIL-2 50 U/ml (BD PharMingen). After 3 days, spleen cells were assayed in a 4-h ^<51>Cr release assay. Although spleen cells in the mice immunized with genetically modified DCs expressing CEA did not show any cytotoxic activity against wild-type MC38 cells at all, they did show cytotoxicity against MC38-CEA (p<0.0001). The cytotoxic activity of spleen cells in mice immunized with DC-AxCACEA was significantly augmented by co-transduction with the GM-CSF/IL-12 gene (p<0.05). 2.Therapeutic efficacy of genetically modified DCs in subcutaneous tumor models. Six to 8-week old CEA Tg were inoculated subcutaneously in the right flank with MC38-CEA. Five days after tumor inoculation (1×10^6 MC38-CEA), tumor-bearing mice were injected subcutaneously in the opposite flank with 1×10^6 genetically modified DCs. These mice were randomly divided into the following six groups (each group : n=5) : group 1:treated with PBS, group 2:treated with DC-AxCALacZ, group 3:treated with DC-AxCACEA, group 4:treated with DC-AxCACEA/IL-12, group 5 : treated with DC-AxCACEA/GM-CSF, group 6:treated with DC-AxCACEA/GM-CSF/IL-12. The size of the s.c.tumor was estimated using the following formula : (short diameter)^2×long diameter×0.52. A single vaccination using DC-AxCACEA,DC-AxCACEA/IL-12,DC-AxCACEA/GM-CSF or DC-AxCACEA/GM-CSF/IL-12 showed remarkable therapeutic efficacy compared with a vaccination using DC-AxCALacZ or PBS (Day22,p<0.0001). In particular, the vaccination of DC-AxCACEA/GM-CSF/IL-12 elicited a more potent efficacy than that of the other groups (Day40, p<0.05), and more importantly, tumor-free mice were observed in the DC-AxCACEA/GM-CSF (2/5) and the DC-AxCACEA/GM-CSF/IL-12 (4/5) vaccination groups on day 40 after tumor implantation.(2005) 1. A histological analysis of tumor tissue in mice vaccinated with genetically modified DCs. There were large areas of proliferating tumor cells with little or no lymphocytic infiltration in tumor tissues from the mice treated with DC-AxCALacZ, while there were some necroses and remarkable lymphocyte infiltrations in tumor tissues from mice treated with DC-AxCACEA or DC-AxCACEA/GM-CSF/IL-12. An immunofluorescent analysis showed that although neither CD8^+ cells nor NK cells were detected in tumor tissue specimens from mice vaccinated with DC-AxCAL.acZ, some CD8^+ cells infiltrated into the tumors of the mice vaccinated with DC-AxCACEA. Importantly, when the mice were treated with DC-AxCACEA/GM-CSF/IL-12, not only CD8^+ cells but NK cells were heavily infiltrated around CEA-expressing tumors. 2. Evaluation of toxicity in mice immunized with genetically modified DCs. After vaccination with DC-AxCACEA/GM-CSF/IL-12, all mice seemed to remain healthy without any body weight loss (data not shown). Additionally, to evaluate the adverse effects by immunization of adenovirally transduced DCs expressing CEA/GM-CSF/IL-12, serum samples of mice were collected to AST, ALT and Cr levels when the mice were sacrificed at 14 days after immunization with 1×10^6 genetically modified DCs. The serum levels of AST, ALT and Cr were within the normal ranges respectively, and they were similar to that from the control mice treated with PBS. Less

（2003）1。重组腺病毒（AD）载体的产生。通过COS-TPC方法产生了表达人CEA（AXCACEA）的重组腺病毒载体。重组AXCAGM-CSF和Axcalacz也通过COS-TPC方法产生。表达鼠IL-12的Axcail-12是从Riken Bioresource中心获得的。 2。ad载体介导的基因转移到DC中。使用一个离心方法将未成熟的DC与每个重组腺病毒（AD）载体翻译。我们的实验表明，在大多数（67.5％）中检测到CEA的表达（67.5％）在100的MOI中转化为100。3。CEA转基因小鼠的出血。雄性和雌性CEA转基因小鼠[C57BL/6J-TGN（CEA GE）18FJP]（H-2^B）（CEA TG）获得了F.F.James Primus博士，并在Wakayama医科大学的动物护理设施中繁殖。通过PCR分析对小鼠尾部DNA进行了筛选CEA翻译的筛选。（2004）1。脾细胞的细胞毒性活性在……更多用遗传修饰的DC免疫的小鼠中。小鼠曾经通过皮下（S.C.）注射1×10^6转基因DC进行免疫。用DC免疫后14天分离脾细胞，然后将体内生化的袖细胞合并，并在具有RMIL-2 50 U/ml的6孔板中培养（BD Pharmingen）。 3天后，将囊细胞分配为4-H ^<51> Cr释放测定法。尽管用表达CEA的一般修饰的DC免疫的小鼠中的小鼠细胞完全没有显示出对野生型MC38细胞的细胞毒性活性，但它们确实显示出针对MC38-CEA的细胞毒性（P <0.0001）。通过与GM-CSF/IL-12基因共转染，用DC-轴免疫的小鼠中脾细胞的细胞毒性显着增强（P <0.05）。 2.皮下肿瘤模型中一般修饰的DC的治疗效率。 MC38-CEA在右侧右侧接种了六到8周的CEA TG。肿瘤接种（1×10^6 MC38-CEA）后五天，将承重肿瘤的小鼠皮下注射在相对的侧面，并以1×10^6的1×10^6通常修饰的DC。将这些小鼠随机分为以下六组（每组：n = 5）：第1组：与PBS趋向2组：与DC-AXCALACZ一起播出，第3组：与DC-Xakacacea趋向于DC-XAXCACEA，第4组：使用DC-axcacea/IL-12趋向于DC-akcacea/IL-12，第5组，用DC-axcacacea/gm-csfected，GM-CSFERDED，均为6号。 DC-轴/GM-CSF/IL-12。使用以下公式估算s.c. tumor的尺寸：（短直径）^2×长直径×0.52。使用DC-axcacea，DC-轴/IL-12，DC-axcacea/gm-CSF或DC-axcacacea/gm-csf/il-12进行单一的真空化，与使用DC-axcalacz或pbs（Day2222222，p <0.00001）相比，显示出显着的治疗效率。特别是，与其他组相比，DC-轴/GM-CSF/IL-12的疫苗与其他组（Day40，P <0.05）相比，引起了更大的潜在效率，并且更重要的是，在DC-AXCACACACACACACACACEA/GM-CSF（2/5）中观察到无肿瘤的小鼠，以及DC-CACCACEA/GMCACACACEA/GM-CSF（4/gm-caCCAUM/gm-csf）。肿瘤植入后。（2005）1。对一般修饰的DC疫苗接种的小鼠中肿瘤组织的组织学分析。在用DC-axcalacz处理的小鼠的肿瘤组织中，有很大的增殖肿瘤细胞，而肿瘤组织中的肿瘤细胞几乎没有淋巴细胞浸润，而在与DC- XCACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACEA或DC-XAXCACACACACACACACACACACACACACACACACACACACACACACEA或GMCACACEA/GMCACEA/GMCACEA/GM-CSF中的肿瘤组织中有一些坏死和明显的淋巴细胞浸润。免疫荧光分析表明，尽管在接种DC-axcal.acz的小鼠中均未检测到CD8^+细胞和NK细胞，但某些CD8^+细胞浸入了DC-Xaxcacea疫苗接种的小鼠的肿瘤中。重要的是，当将小鼠用DC-轴/GM-CSF/IL-12处理时，不仅CD8^+细胞，而且NK细胞在表达CEA的肿瘤周围大量浸润。 2。用转基因DC免疫的小鼠的毒性评估。在使用DC-axcacea/gm-CSF/IL-12接种疫苗后，所有小鼠似乎都健康，没有任何体重减轻（数据未显示）。此外，为了评估免疫能力的CEA/GM-CSF/IL-12的不良反应，当通过1×10^6基因修饰的DC在免疫后14天处死小鼠时，将小鼠的血清样品收集到AST，ALT和CR水平。 AST，ALT和CR的血清水平分别在正常范围内，它们与用PBS处理的对照小鼠相似。较少的

项目成果

Intensification of antitumor effect by Th1-dominant adoptive immunogene therapy for advanced orthotopic colon cancer

Th1 主导的过继免疫基因疗法对晚期原位结肠癌的抗肿瘤作用增强

• 发表时间: 2003
• 期刊: Clinical Cancer Research 9
• 作者: Ojima T et al.;Ojima T et al.;Ozawa S et al.;Nakamura M et al.;Ueda K et al.;Nakamori M et al.;Nakamori M et al.;Ueda K et al.;Nakamori M et al.
• 通讯作者: Nakamori M et al.

Improvement of carcinoembryonic antigen-specific prodrug gene therapy for experimental colon cancer

• DOI: 10.1067/msy.2003.73
• 发表时间: 2003-03-01
• 期刊: SURGERY
• 影响因子: 3.8
• 作者: Ueda, K;Iwahashi, M;Yamaue, HK
• 通讯作者: Yamaue, HK

Ueda K et al.: "Improvement of carcinoembryonic antigen-specific prodrug gene therapy for experimental colon cancer"Surgery. 133. 309-317 (2003)

Ueda K等人：“实验性结肠癌癌胚抗原特异性前药基因治疗的改进”手术。

Effective therapy of metastatic ovarian cancer with an oncolytic herpes simplex virus incorporating two menvrane fusion

使用融合两种 menvrane 融合的溶瘤单纯疱疹病毒有效治疗转移性卵巢癌

• 发表时间: 2003
• 期刊: Clinical Cancer Research 9
• 作者: Ojima T et al.;Ojima T et al.;Ozawa S et al.;Nakamura M et al.;Ueda K et al.;Nakamori M et al.;Nakamori M et al.
• 通讯作者: Nakamori M et al.

Nakamori M et al.: "Intensification of antitumor effect by Th1-dominant adoptive immunogene therapy for advanced orthotoplic colon cancer."Clin Cancer Res. 9. 2357-2365 (2003)

Nakamori M 等人：“通过 Th1 主导的过继免疫基因疗法增强晚期原位结肠癌的抗肿瘤效果。”Clin Cancer Res。