建立黑色素瘤抗原特異性癌症免疫療法及於小鼠黑色素瘤模式中評估抗腫瘤效果

Abstract

黑色素瘤細胞會表現多種分化抗原，包括Melan-A、Tyrosinas及GP 100，而這些分化抗原可當作免疫療法之標靶抗原，引起特異性之免疫反應。本實驗之研究目的為架構出可表現黑色素瘤抗原之質體，作為治療犬黑色素瘤之免疫療法。首先，以PCR方式來合成及增幅包含GP100及Tyrosinase之多抗原結合位序列，作為標靶抗原序列，再將其與可表現綠色螢光蛋白之真核表現質體連結。以螢光顯微鏡及西方墨點法來確認此合成之質體可於真核細胞中表現出抗原及螢光蛋白。而此合成之多抗原結合位質體以三種不同劑量(5, 50, 250 μg)分別以肌肉注射之方式，於C57BL/6J小鼠進行安全性及急性毒性評估，於七天觀察期中並無出現明顯之臨床症狀及副作用。於小鼠黑色素瘤模式中以治療性試驗來評估多抗原結合位質體之抗腫瘤效果，經腫瘤接種後第24天，多抗原結合位質體治療組之腫瘤體積生長受到顯著抑制(＊p<0.05)。因此，此多抗原結合位質體可能具有使黑色素瘤消退或增加病患存活時間之潛在效力，但仍需更深入的研究來確認應用於犬黑色素瘤上的效力。

Melanoma cells selectively express a number of differentiation antigens including melan-A, gp100, and tyrosinase that can serve as targets for an immunotherapy strategy. The aim of our study was to construct a melanoma specific antigen expression plasmid as an immunotherapy for canine melanoma. A multiepitope sequence, which modified from a previous study, was generated by annealing of a set of 8 primers by PCR reaction. A plasmid in which expression of the multiepitope sequence of melanoma antigens or fused with the GFP (green fluorescence protein) was constructed. The transient expression of multiepitope-GFP in eukaryotic cells (HEK-293T) was confirmed by fluorescent microscopy and western blot analysis. Evaluation of safety and acute toxicity was performed, and adverse effect was not significant in C57BL6/J mice intramuscularly injected with multiepitope plasmid at three different doses (5, 50, 250 μg) after 7 days of observation. Furthermore, a melanoma model was successfully established in C57BL/6J mice and the therapeutic effect of the plasmid was evaluated on these tumor bearing mice. The growth of tumor volume in the therapeutic group was significant inhibited than the mock group (＊p<0.05) and control group (＊p<0.05) on day 24. The multiepitope plasmid might be a potential therapy to regress the melanoma or improve survival time of patients and further investigation of multiepitope plasmid on canine melanoma is necessary.