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標題: | 利用DNA初免/腺病毒加強策略來增強腫瘤新抗原疫苗的免疫性 Enhancement of Immunogenicity for Neoantigen Vaccine by DNA Prime/Adenovirus Boost Strategy |
作者: | Haitao Jiang 蔣海濤 |
指導教授: | 陶秘華(Mi-Hua Tao) |
關鍵字: | 癌症免疫治療,腫瘤新抗原疫苗,DNA疫苗,放射性療法,合併治療,腫瘤新抗原預測,腫瘤新抗原專一性T細胞, Cancer immunotherapy,Cancer neoantigen vaccine,DNA vaccine,Radiation therapy,Combination therapy,Cancer neoantigen prediction,Neoantigen-specific T cell, |
出版年 : | 2020 |
學位: | 碩士 |
摘要: | 癌症免疫療法最近取得了重要的進展: 腫瘤新生抗原疫苗(Cancer neoantigen vaccines)。免疫療法要起作用,靠的是免疫細胞尤其是T細胞去辨認並殺死腫瘤細胞,並且有研究證實腫瘤內存在由基因突變產生的腫瘤新生抗原(cancer neoantigen),這種新生抗原是腫瘤專一性的,且大量文獻指出其可以引發腫瘤專一性的免疫反應,因此是非常好的腫瘤疫苗抗原。目前大量的研究都是用長片段勝肽疫苗或mRNA疫苗,雖然取得了一定的成效,但是也發現這些疫苗引發了大量的腫瘤新生抗原專一性CD4+ T細胞。儘管如此,目前的免疫學理論都是認爲CD8+ T細胞才是真正殺死腫瘤的細胞,因此尋找一個可以引發腫瘤專一性CD8+ T細胞的疫苗平臺就非常重要。DNA疫苗目前在腫瘤新生抗原疫苗中的研究並不多,而且相比於其他疫苗還可以在DNA 疫苗的序列中攜帶細胞激素作爲佐劑來加強疫苗的效果。因此將腫瘤新生抗原和DNA疫苗結合是一個很有吸引力的研究方向。 我們使用B16F10細胞作爲腫瘤模型,根據在文獻中所報告的B16F10腫瘤細胞的突變,我們用軟體預測出21條能和第一型主要組織相容性複合物 (MHC class I)有較好結合能力的腫瘤新生抗原胜肽片段,並把他們的DNA序列全部接在一起後用選殖的方式接到表現質體和腺病毒載體上來作為疫苗的載體,同時也設置一組可以同時表現小鼠 GM-CSF作爲佐劑的組別用來測試其是否可以增強疫苗的效果。 實驗室先前的數據顯示,使用長片段胜肽疫苗免疫,在ELISPOT實驗中發現,其引發了大量的CD4+ T細胞,而其引發的CD8+ T細胞僅可以辨認1條腫瘤新生抗原胜肽片段。若使用DNA疫苗進行免疫,其引發的CD8+ T細胞反應可以辨認3條腫瘤新生抗原胜肽片段。同時,在有攜帶GM-CSF做佐劑的組別,其引發的腫瘤新生抗原專一性T細胞數量比沒有佐劑的組別有顯著提高。 接下來使用B16F10腫瘤模型以預防性免疫的方式進行疫苗保護效果測試,發現可以延緩腫瘤的生長速度,但有GM-CSF的組別和沒有GM-CSF的組別沒有顯著差別。最後,為了盡可能發揮疫苗的作用,我們將疫苗和放射性治療合併來測試對B16F10的治療效果,發現可以顯著延緩腫瘤生長,且有GM-CSF的組別治療效果顯著好過沒有GM-CSF的組別。 相比於比勝肽疫苗,DNA疫苗能夠引起更多腫瘤新生抗原專一性的CD8+ T細胞,而且以GM-CSF做佐劑可以進一步增強免疫反應的強度。使用預防性免疫的方式看到腫瘤新生抗原疫苗對腫瘤有抑制效果,但如果結合放射性治療可以看到更顯著的抑制效果,且有GM-CSF的組別比沒有的組別效果更明顯,也符合之前在ELISPOT中看到的結果。以上資料證明了DNA疫苗結合GM-CSF做佐劑的潛力,並且強調了疫苗和放射治療的合併使用在抑制腫瘤生長中的作用。 Cancer neoantigen vaccine is a recent breakthrough for cancer immunotherapy. The basis of immunotherapy is the recognition of cancer cells by immune cells, especially T cells, and a lot of research has pointed out that cancer cells possess neoantigens, which derive from gene mutations and are the essential component for T cell recognition. Cancer neoantigens can induce cancer-specific immune responses, and therefore, it is a perfect cancer vaccine candidate. Currently, most studies on neoantigen vaccine development are working on long peptide vaccines and mRNA vaccines. Though these studies presented some positive results, instead of inducing CD8+ T cells these vaccines induced strong CD4+ T cell responses. It is known that CD8+ T cells are the most powerful immune cells for destroying cancer cells, so finding a vaccine platform that can induce dominant CD8+ T cell responses is strongly needed. DNA vaccine is a powerful vaccine platform to induce antigen-specific CD8+ T cells. The efficacy of DNA vaccines can be enhanced by co-delivery of cytokine genes as a biological adjuvant. So, DNA vaccines that produce both neoantigens and cytokines would be an attractive approach to develop potent cancer vaccines. However, compared to other vaccine platforms, few research of neoantigen DNA vaccines has been reported. B16F10 is the tumor model used in this study. We predicted 21 neoantigen peptides of B16F10 that have high binding affinity with MHC Class I, then linked the DNA sequences of all the peptides in string, cloned into an expression plasmid and an adenoviral vector. We also constructed plasmid and adenoviral vectors that express both neoantigen and mouse GM-CSF to investigate whether it can improve the response of neoantigen vaccines. Mice immunized with plasmid DNA priming and adenoviral vector boosting, strong CD8+ T cell responses specific for 3 neoantigen peptides were induced. In the group with GM-CSF, the response of neoantigen-specific T cells was significantly enhanced compared with those without the adjuvant. The result is different from long peptide vaccination, which induced robust CD4+ T cell responses but much fewer neoantigen-specific CD8+ T cells. In both prophylactic and therapeutic settings, DNA priming and adenoviral vector boosting of neoantigen vaccines significantly delayed tumor growth, and vaccines with GM-CSF adjuvant showed better therapeutic effect than those without the adjuvant. We demonstrated with vaccines with GM-CSF induced stronger neoantigen-specific T cells responses. Our results demonstrate the potential use of DNA priming and adenoviral vector boosting strategy in developing cancer neoantigen vaccines. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/51542 |
DOI: | 10.6342/NTU202002738 |
全文授權: | 有償授權 |
顯示於系所單位: | 醫學檢驗暨生物技術學系 |
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