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

（2003）1.重组腺病毒（Ad）载体的产生。采用COS-TPC法构建表达人CEA的重组腺病毒载体（AxCACEA）。用COS-TPC法制备了重组AxCAGM-CSF和AxCALacZ。表达鼠IL-12的AxCAIL-12获自RIKEN BioResource Center。2.Ad载体介导的DC基因转染。使用离心方法用每种重组腺病毒（Ad）载体转染未成熟DC。我们的实验证明，在MOI为100的AxCACEA转染的DC中，大多数（67.5%）检测到CEA的表达。3. CEA转基因小鼠的出血。雄性和雌性CEA转基因小鼠[C57 BL/6 J-TgN（CEA Ge）18 FJP]（H-2 μ b）（CEA Tg）获自Dr.F.James Primus，并在和歌山医科大学的动物护理设施中饲养。通过对小鼠尾部DNA进行PCR分析，对出生的小鼠进行CEA转基因的筛选。（2004）1.脾细胞的细胞毒性活性， ...更多信息 用遗传修饰的DC免疫的小鼠。将小鼠通过皮下（s.c.）注射1×10^6个基因修饰的DC。在用DC免疫后14天分离脾细胞，然后将体内致敏的脾细胞合并并在具有rmIL-2 50 U/ml（BD PharMingen）的6孔板中培养。3天后，在4小时的Cr释放试验中测定脾细胞<51>。尽管用表达CEA的遗传修饰的DC免疫的小鼠中的脾细胞根本不显示针对野生型MC 38细胞的任何细胞毒性活性，但它们确实显示针对MC 38-CEA的细胞毒性（p&lt;0.0001）。DC-AxCACEA免疫的小鼠脾细胞的细胞毒活性通过与GM-CSF/IL-12基因共转导而显著增强（p&lt;0.05）。2.皮下肿瘤模型中遗传修饰的DC的治疗功效。将6至8周龄的CEA Tg用MC 38-CEA皮下接种于右侧腹。肿瘤接种（1×10^6 MC 38-CEA）后5天，在荷瘤小鼠的对侧皮下注射1×10^6个遗传修饰的DC。将这些小鼠随机分为以下六组（每组：n=5）：第1组：用PBS处理，第2组：用DC-AxCALacZ处理，第3组：用DC-AxCACEA处理，第4组：用DC-AxCACEA/IL-12处理，第5组：用DC-AxCACEA/GM-CSF处理，第6组：用DC-AxCACEA/GM-CSF/IL-12处理。皮下肿瘤的大小用以下公式估算：（短径）^2×长径×0.52。与使用DC-AxCALacZ或PBS的疫苗接种相比，使用DC-AxCACEA、DC-AxCACEA/IL-12、DC-AxCACEA/GM-CSF或DC-AxCACEA/GM-CSF/IL-12的单次疫苗接种显示出显著的治疗功效（第22天，p&lt;0.0001）。具体地，DC-AxCACEA/GM-CSF/IL-12的疫苗接种引起比其他组更有效的功效（第40天，p&lt;0.05），并且更重要的是，在肿瘤植入后第40天，在DC-AxCACEA/GM-CSF（2/5）和DC-AxCACEA/GM-CSF/IL-12（4/5）疫苗接种组中观察到无肿瘤小鼠。（2005年）1.用遗传修饰的DC接种的小鼠中的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存在大面积的增殖肿瘤细胞，几乎没有或没有淋巴细胞浸润，而在用DC-AxCACEA或DC-AxCACEA/GM-CSF/IL-12处理的小鼠的肿瘤组织中存在一些坏死和显著的淋巴细胞浸润。免疫荧光分析显示，尽管在接种DC-AxCAL.acZ的小鼠的肿瘤组织标本中没有检测到CD 8 ^+细胞和NK细胞，但在接种DC-AxCACEA的小鼠的肿瘤中有一些CD 8 ^+细胞浸润。重要的是，当用DC-AxCACEA/GM-CSF/IL-12治疗小鼠时，不仅CD 8 ^+细胞，而且NK细胞在CEA表达肿瘤周围大量浸润。2.用遗传修饰的DC免疫的小鼠中的毒性评估。在用DC-AxCACEA/GM-CSF/IL-12接种后，所有小鼠似乎保持健康而没有任何体重减轻（数据未显示）。此外，为了评估表达CEA/GM-CSF/IL-12的腺病毒转导的DC免疫的不良反应，当用1×10^6个基因修饰的DC免疫后14天处死小鼠时，收集小鼠血清样品以测定AST、ALT和Cr水平。血清AST、ALT和Cr水平均在正常范围内，与PBS对照组相似。少

(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