The Study of Recombinant BCG and Interleukin-12 DNAVaccines for Immunogene therapy of Bladder Cancer

• Main Authors: Chi-Feng Lee, 李奇峰
• Other Authors: Sun-Yran Chang
• Format: Others
• Language: zh-TW
• Published: 2003
• Online Access: http://ndltd.ncl.edu.tw/handle/18256985228943852909

博士 === 國防醫學院 === 生命科學研究所 === 92 === Intravesical immunotherapy with live Mycobacterium bovis bacillus Calmette—Guérin (BCG) is the treatment of choice for superficial bladder cancers. Nevertheless, a significant proportion of patients do not respond to this therapy, and adverse effects are common. Here we evaluate the antitumor effects of multiple recombinant BCG DNA (multi-rBCG) and murine interleukin-12 DNA (mIL-12) vaccines on xenografted murine bladder tumor models (MBT-2) and demonstrate the ability of multicomponent and multisubunit DNA vaccines to enhance Th1-polarized responses, effector cell responses as well as suppression of bladder tumor growth in mice. In this study, four mycobacteria candidate genes (Ag85A, Ag85B, Mpt64, and PstS3) were cloned, fused with ESAT6, and ligated into eukaryotic expression vectors. To construct the mIL-12 plasmid (pIRES-p40p35), light (p35) and heavy (p40) chain cDNAs were amplified from a cDNA library from C3H/HeN mouse spleen cells and together inserted into the pIRES vector. Treatment with combined multi-rBCG and mIL-12 vaccines was examined in syngeneic C3H/HeN mice and athymic nude mice. The delivery efficiency of multi-rBCG expression was detected by western blotting, flow cytometry and semi-quantitative reverse transcription polymerase chain reactions. Splenocytes from immunized groups of mice were re-stimulated in vitro and examined for cytotoxicity against MBT-2 cells. Inhibition of tumor growth was monitored and antitumor effects were evaluated after one dose of electroporative immunogene therapy, with measurement of systemic cytokine responses and phenotyping of infiltrating lymphocytes in tumors. Our results showed that transfected human and murine bladder cancer cells demonstrated high in vitro expressions of the recombinant subcomponents. In vivo expression of multi-rBCG was efficient and reached a maximum on day 7 after electroporation. Mice with tumors injected with multi-rBCG plus mIL-12 increased production of serum Th1-type cytokines including interferon-gamma (IFN-g) and IL-12 significantly within twenty-one days, but no significant elevations of tumor necrosis factor-a (TNF-a), interleukin-2 (IL-2), interleukin-4 (IL-4 or interleukin-5 (IL-5) were found. Treatment with multi-rBCG and/or mIL-12 in C3H/HeN mice induced infiltration of CD4+/CD8+ T cells and expansion of NK cells within tumors. Increased immune cell infiltration and induction of apoptosis were also noted after treatment with multi-rBCG alone, with the mIL-12 vaccine alone, and─most significantly─with the multi-rBCG plus mIL-12 vaccine combination. On day twenty-eight after electroporation, the growth of MBT-2 implants treated with multi-rBCG, mIL-12 or multi-rBCG plus mIL-12 was significantly inhibited. The cumulative survival of mice treated with multi-rBCG plus mIL-12 was significantly higher than the other three groups. Electroporation of multi-rBCGplusmIL-12 resulted in complete tumor eradication in seven of eight mice (P < 0.01) within 28 days. By contrast, however, athymic nude mice treated in the same way showed no significant immune cells within tumors, and died from the fast growing tumors. In conclusion, an electroporation method of recombinant BCG gene therapy has been effectively demonstrated in subcutaneous xenografted bladder tumor models. The induction of antitumor activity required a thymus-dependent immune response. The antitumor effects correlate with the eliciting of Th1 cytokine, T-cell lymphocyte and NK cell-mediated cytotoxic immune responses. Currently, our data were published in the Urology (Lee CF et al., 2003), Cancer Gene Therapy (Lee CF et al., 2003) and Journal of Urology (Lee CF et al., 2003), respectively. These results present new possibilities for the treatment of bladder cancer using recombinant BCG DNA vaccines.