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)载体的构建。用COS-TPC法构建表达人CEA的重组腺病毒载体AxCACEA。用COS-TPC法获得重组AxCAGM-CSF和AxCALacZ。表达小鼠IL-12的AxCAIL-12由理研生物资源中心提供。2.腺病毒载体介导的DC基因转移。用离心法将每个重组腺病毒(Ad)载体导入未成熟DC。我们的实验表明，在MOI为100的大多数(67.5%)转染AxCACEA的DC中检测到CEA的表达。3.CEA转基因小鼠的出血情况。雄性和雌性CEA转基因小鼠[C57BL/6J-TGN(CEA GE)18FJP](H-2^b)(CEA Tg)由F.James Primus博士提供，在和歌山医科大学动物护理设施饲养。通过对小鼠尾部DNA的聚合酶链式反应分析，对出生的小鼠进行了CEA转基因的筛选。(2004年)1.…中脾细胞的细胞毒活性用转基因DC免疫更多的小鼠。这些小鼠曾经用皮下(S.C.)注射1×106个转基因树突状细胞。树突状细胞免疫后14天分离脾细胞，将体内免疫的脾细胞混合培养在含rmIL-2 50U/ml(Bd PharMingen)的6孔板中。3d后，取脾细胞进行4h、51h、51h铬释放试验。尽管表达CEA的转基因DC免疫的小鼠的脾细胞对野生型MC38细胞没有任何细胞毒活性，但它们确实对MC38-CEA表现出细胞毒活性(p&lt；0.0001)。共转导GM-CSF/IL-12基因可显著增强DC-AxCACEA免疫小鼠脾细胞的细胞毒活性(p&lt；0.05)。2.转基因DC对皮下肿瘤模型的治疗作用6~8周龄CEA-Tg右侧皮下接种MC38-CEA。肿瘤接种后5天(1×10~(-6)MC38-CEA)，对侧皮下注射1×10~(-6)转基因DC。将小鼠随机分为6组(每组5只)：1组：PBS组，2组：DC-AxCALacZ组，3组：DC-AxCACEA组，4组：DC-AxCACEA/IL-12组，5组：DC-AxCACEA/GM-CSF组，6组：DC-AxCACEA/GM-CSF/IL-12组。肿瘤大小的估算公式如下：(短径)^2×长径×0.52。DC-AxCACEA、DC-AxCACEA/IL-12、DC-AxCACEA/GM-CSF或DC-AxCACEA/GM-CSF/IL-12一次接种与DC-AxCALacZ或PBS接种相比，疗效显著(第22天，p&lt；0.0001)。特别是DC-AxCACEA/GM-CSF/IL-12免疫组的免疫效果优于其他组(第40天，p&lt；0.05)，更重要的是，DC-AxCACEA/GM-CSF(2/5)和DC-AxCACEA/GM-CSF/IL-12(4/5)免疫组在肿瘤移植后第40天观察到无瘤小鼠。(2005)1.转基因DC免疫小鼠肿瘤组织的组织学分析。DC-AxCALacZ治疗组小鼠肿瘤组织中有大量增殖的肿瘤细胞，淋巴细胞很少或根本没有，而DC-AxCACEA或DC-AxCACEA/GM-CSF/IL-12治疗组小鼠肿瘤组织中有一些坏死和显著的淋巴细胞浸润。免疫荧光分析显示，DC-AxCAL.acZ免疫组小鼠肿瘤组织中未检测到CD8~+细胞和NK细胞，但有CD8~+细胞渗入肿瘤组织。重要的是，当小鼠接受DC-AxCACEA/GM-CSF/IL-12治疗时，不仅CD8~+细胞而且NK细胞在CEA表达的肿瘤周围大量渗透。2.转基因树突状细胞免疫小鼠的毒性评价接种DC-AxCACEA/GM-CSF/IL-12后，所有小鼠似乎都保持健康，体重没有任何下降(数据未显示)。此外，为了评估腺病毒转导的表达CEA/GM-CSF/IL-12的DC免疫小鼠的不良反应，在1×10~(-6)转基因DC免疫后14天处死小鼠，采集血清标本，测定AST、ALT和Cr水平。血清AST、ALT、Cr水平均在正常范围内，与PBS对照组相近。较少

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.

Adenoviral-mediated gene transduction of the hepatocyte growth factor (HGF) antagonist, NK4, suppresses peritoneal metastases of gastric cancer in nude mice.

• DOI: 10.1016/j.ejca.2004.05.006
• 发表时间: 2004-09
• 期刊: European journal of cancer
• 影响因子: 8.4
• 作者: K. Ueda;M. Iwahashi;I. Matsuura;M. Nakamori;Masaki Nakamura;T. Ojima;T. Naka;Koichiro Ishida;Kunio Matsumoto;Toshikazu Nakamura;H. Yamaue

Dendritic cells transduced with tumor-associated antigen gene elicit potent therapeutic antitumor immunity: Comparison with immunodominant peptide-pulsed DCs

• DOI: 10.1159/000086770
• 发表时间: 2005-01-01
• 期刊: ONCOLOGY
• 影响因子: 3.5
• 作者: Nakamura, M;Iwahashi, M;Yamaue, H
• 通讯作者: Yamaue, H

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

Th1为主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.
• 通讯作者: Nakamori M et al.

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

结合两膜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.;Ueda K et al.;Nakamori M et al.;Nakamori M et al.
• 通讯作者: Nakamori M et al.