發展細胞型佐劑及腫瘤疫苗

Development of cell-based adjuvant and tumor vaccine

江姿瑩(Tzu-Ying Chiang)

指導教授 ： 林綉茹

高雄醫學大學/醫學院/醫學遺傳學研究所/碩士(2006年)

摘要

我們將老鼠之細胞激素(白介素2、白介素18、顆粒球巨嗜細胞聚落刺激因子)送入Balb/c mice同源之3T3細胞(小鼠纖維母細胞)膜表面，作為細胞型佐劑免疫小鼠，藉由細胞激素能夠增強免疫反應促使產生專一性針對標的蛋白的抗體。我們證實膜式細胞激素能夠高表現於Balb/3T3細胞膜上，並以西方點墨法確認細胞激素分子量。之後進行脾臟細胞增生試驗，將表現膜式細胞激素的3T3細胞與Balb/c小鼠脾臟細胞共同培養，發現膜式細胞激素能夠刺激脾臟細胞增生，證實膜式細胞激素具有生物活性，能夠刺激脾臟細胞活化增生。我們所選用的標的蛋白為EVI2B，是人類第一型膜蛋白，被證實高表現於大腸癌細胞。我們將EVI2B短暫表現於Balb/3T3, Balb/3T3-mIL-2, mGM-CSF and mIL-18，將這些細胞免疫Balb/c mice，結果顯示membrane IL-2為最佳之免疫刺激佐劑，能夠產生最好的抗EVI2B抗體效價。因此未來我們能夠利用3T3 IL-2為細胞型免疫刺激佐劑，將我們的目標膜蛋白表現於3T3 IL-2細胞膜上，來產生針對標的蛋白的抗體，便利基礎研究或是臨床上之應用。
另一方面，藉由細胞激素能夠刺激免疫反應活化的特性，將細胞激素表現於腫瘤細胞(CT-26, 小鼠大腸癌細胞)膜上，建構膜式細胞激素腫瘤疫苗免疫小鼠，促使免疫細胞記憶並且攻擊毒殺腫瘤，達到預防及治療腫瘤的效果。我們的結果證實膜式細胞激素(mIL-18、IL-2、GM-CSF)確實高表現於CT-26細胞膜表面。因此我們將表現膜式細胞激素的腫瘤細胞作為疫苗皮下注射小鼠，結果顯示膜式細胞激素能夠改變腫瘤細胞在活體內的生長能力(tumorgenicity)，mIL-2抑制腫瘤細胞生長的效果最好。10天後於對側打入母體腫瘤細胞，發現mIL-18+GM-CSF能夠誘發最佳免疫記憶性，抑制re-challenge CT-26 腫瘤細胞的生長。未來將膜式細胞激素腫瘤疫苗免疫長有腫瘤之小鼠，期望免疫細胞活化後毒殺存在活體內或轉移之腫瘤，達到治療腫瘤之療效。

關鍵字

佐劑 ； 腫瘤疫苗

並列摘要

we are developing a novel cell adjuvant for naïve
targeted-protein immunization. Endogenous Balb/3T3 mouse
fibroblast cell was engineered to express mouse membrane IL-2,GM-CSF and IL-18 as cell adjuvents for immunization. We have demonstrated that functional membrane IL-2, GM-CSF and IL-18 can highly express on cell surface to stimulate the proliferation of splenocytes. The endogenous Balb/3T3
cytokines can＇t induce detectable antibody response after
immunization. Based on these results, EVI2B, a human type I
membrane protein, has been proved as oncogene and
overexpression in human colorectal cancer will be as
membrane-targeted protein for the development of antibody drug.The EVI2B has been cloned into pLNCX plasmid and successfully expressed on the Balb/3T3 cell surface. pLHCX-EVI2B will be transiently transfected into Balb/3T3, or Balb/3T3-mIL2, mGM-CSF and mIL18, respectively, and then immunized into Balb/c mice to determine the titer of anti-EVI2B antibodies. Our data showed that mIL2 was the best immune stimulator, Balb/3T3-mIL2 EVI2B immunization can produced the highest anti-EVI2B antibody titer. In the future we can use mIL2 as cell based adjuvant to provide a valuable tool for membrane targeted protein immunization.
In another way, utilize the characteristic of cytokines
which can stimulate immune cells to activate and proliferate, we hope to develop a membrane cytokine tumor vaccine by expressing membrane form IL-18、IL-2 and GM-CSF in tumor to induce synergistic anti-tumor effects. To this aim, we have successfully used retrovirus transduction to stably high express membrane IL-18 、IL-2 、 GM-CSF in CT-26 (murine colorectal carcinoma cells), respectively. We also found that CT-26/mIL-2 tumor vaccine can change tumorgenicity to inhibit the tumor growth in vivo. Importantly, immunization of CT-26/IL-18+GMCSF can induce good memory immunity to prevent the growth of parental CT-26 re-challenge cells. In future, we will estimate the therapeutic efficacy of membrane cytokine tumor vaccine in CT-26 tumor bearing Balb/c mice, positive result will provide a novel method for cancer immunotherapy.

並列關鍵字

IL-18 ； Il-2 ； GM-CSF ； EVI2B

參考文獻

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