近年來，癌症免疫療法成為癌症治療中日益突出的議題。癌症免疫療法在通過選擇性地增加針對腫瘤特異性抗原的T細胞群來增強免疫系統，從而引發癌細胞死亡。而免疫療法的理想目標是腫瘤特異性突變，針對腫瘤特異性突變可以減緩癌症的進展並且具有較少的副作用。因此，迫切需要選擇腫瘤特異性突變和開發用於癌症進展的適當免疫治療策略。在此期間，全基因組定序的發展使得個人化免疫療法成為可能。儘管腫瘤細胞的突變在每個癌症患者中是特異性的，但癌症患者仍然存在2%的共同突變。因此，本篇研究目的為以合理的成本開發針對癌症患者的腫瘤特異性突變的個人化免疫療法。由於成本低，安全性高，DNA疫苗是一種經濟實惠的平台。為了研究DNA疫苗在個性化免疫治療中的潛力，我們想了解針對由腫瘤特異性突變編碼的腫瘤特異性新抗原的DNA疫苗是否可以延緩腫瘤進展。先前的研究已在B16F10黑色素瘤中鑑定了幾種免疫原性新表位。因此，我們基於鑑定的突變肽片段設計了多個新表位DNA疫苗。此外，利用密碼子使用偏好和tRNA拷貝數來優化DNA疫苗。為了在體外觀察DNA疫苗的表達，我們構建了帶有GFP-tag的DNA疫苗。我們通過西方點墨法和螢光顯微鏡觀察轉染到HEK293T和B16F10中的GFP-tag的DNA疫苗會表達出在疫苗上的蛋白片段。為了研究多表位DNA疫苗在B16F10黑色素瘤動物模型中的治療效果，我們通過皮下注射將B16F10細胞植入C57BL/6小鼠，並在腫瘤可觸知時用多表位DNA疫苗進行免疫治療。我們的數據顯示，DNA疫苗有效減弱了B16F10原位腫瘤模型中的腫瘤生長。在腫瘤組織免疫染色的結果分析顯示在經過DNA疫苗的治療後增加了CD4+和CD8+T細胞在腫瘤組織的浸潤，並且流式細胞術分析顯示樹突細胞群在淋巴結以及脾臟中增加。這些數據顯示DNA疫苗將通過樹突細胞引發抗腫瘤免疫反應並增加CD4+和CD8+T細胞。雖然仍需要進一步調查，但我們認為這樣的癌症疫苗新方法可能會對癌症治療方法帶來更多的可能性及潛在的希望。

In recent years, cancer immunotherapy plays an increasingly prominent role in the treatment of cancer. The cancer immunotherapy aims to boost the immune system by selectively increasing the population of T cells, which specifically targeted to the tumor-unique antigens, thereby initiating cancer cell death. Meanwhile, an ideal target for immunotherapy is tumor-specific mutations, and targeting tumor-specific mutation leads to reducing the progression of cancer and having less side effect. Consequently, there is an urgent need to select suitable tumor-specific mutation and development appropriate immunotherapeutic strategy for cancer progression. In the interval, the development of whole-genome sequencing makes the personalized immunotherapy possible. Although the mutations of tumor cells are specific in cancer patients, there are still 2% mutations shared in cancer patients. Therefore, the present study aimed to develop personalized immunotherapy against tumor-specific mutations for cancer patients at a reasonable cost. Until now, the DNA vaccine is an affordable platform due to the low cost and high safety. To study the potential of DNA vaccine in personalized immunotherapy, we investigate whether DNA vaccine targeting tumor-specific neo-antigens encoded by tumor-specific mutations can delay the tumor progression. Several immunogenic neo-epitopes were identified in B16 melanoma. Therefore, we designed multiple neo-epitope DNA vaccine based on the identified mutation peptide fragment. Furthermore, the codon usage bias and tRNA copy number were considered to optimize DNA vaccine. To observe the expression of the DNA vaccine in vitro, we constructed a GFP-tag DNA vaccine and observed the protein expression in HEK293T and B16F10 by western blotting and fluorescence microscope, respectively. To further investigate the therapeutic effect of multiple-epitope DNA vaccine in B16F10 melanoma animal model, we implanted B16F10 cells into C57BL/6 mice by subcutaneous injection and performed immunotherapy with multiple-epitope DNA vaccine when the tumor was palpable. Our data indicated that the DNA vaccine attenuated tumor growth in B16F10 orthotopic model. The analysis of immunohistochemistry showed that the infiltration of CD4+ and CD8+ T cells increased in tumor tissues. The flow cytometry analysis revealed that dendritic cell population increased in the lymph node and spleen. These data indicated that DNA vaccine would elicit antitumor immune responses via dendritic cell and increase CD4+ and CD8+ T cells. Although further investigations are still required, our new approach to discovering cancer vaccine may shed unique insight into the treatment with cancer.

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