| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2005: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 2004: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 2003: ¥2,100,000 (Direct Cost: ¥2,100,000)
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| Research Abstract |
(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
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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
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