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

(2003) 1。重组腺病毒载体的生成。采用COS-TPC法合成了表达人CEA的重组腺病毒载体（AxCACEA）。重组蛋白AxCAGM-CSF和AxCALacZ也通过COS-TPC法生成。表达小鼠IL-12的AxCAIL-12从日本理化学研究所生物资源中心获得。Ad载体介导的基因转移到dc。采用离心法转染各重组腺病毒（Ad）载体的未成熟树突状细胞。我们的实验表明，在转染了AxCACEA的dc中，大多数（67.5%）检测到CEA的表达，MOI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）由f.j james Primus博士获得，在和歌山医科大学动物护理设施饲养。通过对小鼠尾部DNA进行PCR分析，筛选出生小鼠CEA转基因基因。(2004) 1。转基因dc免疫小鼠脾细胞的细胞毒活性小鼠曾通过皮下注射1×10^6转基因dc免疫。dc免疫14 d后分离脾细胞，将体内引物的脾细胞集中培养于含rmIL-2 50 U/ml （BD PharMingen）的6孔板中。3天后，用4 h ^<51>Cr释放法检测脾细胞。虽然用表达CEA的转基因dc免疫小鼠的脾脏细胞对野生型MC38细胞没有任何细胞毒性活性，但它们对MC38-CEA确实有细胞毒性（p<0.0001）。DC-AxCACEA可与GM-CSF/IL-12基因共转导，显著增强小鼠脾细胞的细胞毒活性（p<0.05）。2.转基因dc对皮下肿瘤模型的治疗效果。6 ~ 8周龄CEA Tg皮下接种MC38-CEA。肿瘤接种（1×10^6 MC38-CEA） 5天后，在荷瘤小鼠的对侧皮下注射1×10^6转基因dc。将小鼠随机分为6组（每组n=5）： 1组PBS， 2组DC-AxCALacZ， 3组DC-AxCACEA， 4组DC-AxCACEA/IL-12, 5组DC-AxCACEA/GM-CSF， 6组DC-AxCACEA/GM-CSF/IL-12。使用以下公式估计s.c.c肿瘤的大小：（短直径）^2×long diameter×0.52。单次接种DC-AxCACEA、DC-AxCACEA/IL-12、DC-AxCACEA/GM-CSF或DC-AxCACEA/GM-CSF/IL-12与接种DC-AxCALacZ或PBS相比，具有显著的治疗效果（第22天，p<0.0001）。其中，DC-AxCACEA/GM-CSF/IL-12接种组的疗效明显高于其他各组（第40天，p<0.05），更重要的是，DC-AxCACEA/GM-CSF（2/5）和DC-AxCACEA/GM-CSF/IL-12（4/5）接种组的小鼠在肿瘤植入后第40天均无肿瘤。(2005) 1。转基因dc疫苗接种小鼠肿瘤组织的组织学分析。DC-AxCALacZ处理的小鼠肿瘤组织中肿瘤细胞大面积增生，淋巴细胞浸润很少或没有，而DC-AxCACEA或DC-AxCACEA/GM-CSF/IL-12处理的小鼠肿瘤组织中有一些坏死和明显的淋巴细胞浸润。免疫荧光分析显示，DC-AxCAL接种小鼠肿瘤组织标本中未检测到CD8^+细胞和NK细胞。在DC-AxCACEA疫苗接种小鼠的肿瘤中，一些CD8^+细胞浸润。重要的是，当用DC-AxCACEA/GM-CSF/IL-12处理小鼠时，不仅CD8^+细胞，而且NK细胞在表达cea的肿瘤周围大量浸润。2. 转基因dc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，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，在1×10^6转基因dc免疫后14天，将小鼠的血清样本收集到AST、ALT和Cr水平。血清谷丙转氨酶、谷丙转氨酶和谷丙转氨酶水平均在正常范围内，与PBS治疗对照组基本一致。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