本實驗的目標是期望發展一種能有效活化毒殺型T細胞之佐劑組合，此組合將結合疫苗遞送系統以及免疫刺激型的佐劑。PELC是種乳狀疫苗遞送系統，由生物可吸收式高分子PEG-b-PLACL、Span 85和角鯊烯(Squalene)這三種物質組成。由近期的研究中指出PELC與TLR9之配體CpG(PELC/CpG)之組合在H5N1疫苗中可以增強體液性免疫去對抗H5N1的病毒，為了想知道這種組合是否也可以增強細胞性免疫去治療子宮頸癌，因此在本實驗中，利用此組合在重組蛋白和胜肽免疫療法觀察其效用。
胜肽RAHYNIVTF (RAH) 是段抗原決定位，為HPV16E7第49-57之胺基酸序列，會被H-2Db之毒殺型T細胞給專一辨識，在本實驗中，RAH混合PELC/CpG希望能活化毒殺型T細胞並應用在治療型子宮頸癌疫苗中。將這疫苗組合免疫C57BL/6小鼠，可發現比起只加單一佐劑的組別相比，RAH和PELC/CpG的組合能引起較高量的細胞分泌IFN-γ且能活化較多的CD107a+CD8+的毒殺型T細胞，且有較多專一辨識RAH的CD8T細胞。之後又更一部應用在TC-1子宮頸癌模型中，將RAH和PELC/CpG免疫已注射過TC-1癌細胞的小鼠，同時加入RAH和PELC/CpG的組別可以顯著的抑制腫瘤生長，比起RAH單加PELC或CpG的組別。
另外為了測PELC/CpG的組合是否也是用在重組蛋白免疫療法上，因此改用重組修飾後的 E7 蛋白 (rE7m)為抗原與PELC/CpG的組合去做小鼠免疫的測試，可發現PELC/CpG這組合可以引起Th1免疫力去對抗腫瘤。
本論文發現合併抗原遞送系統的PELC佐劑與免疫刺激型的佐劑(TLR9之配體CpG)有助於活化毒殺型T細胞去對抗腫瘤，為癌症免疫療法提供新的方向。
A rational approach towards the development of strong CTL-inducing adjuvant formulation is to combine vaccine delivery system and immune-stimulating reagents. PELC is an emulsion-type vaccine delivery system which contains a bioresorbable polymer PEG-b-PLACL, Span 85 and squalene. Our previous report has been shown that PELC formulated with TLR9 ligand (CpG) (PELC/CpG) in H5N1 vaccine could enhance humoral immunity to against H5N1 virus. In order to determine whether PELC/CpG can also enhance cellular immunity to treat cervical cancer, both protein and CTL epitope-based immunotherapies were used in this study.
Peptide RAHYNIVTF (RAH) is an H-2Db-restricted CTL epitope that derived from HPV16 E7 (amino acid 49-57). We formulated this peptide with PELC/CpG to induce CTL for developing therapeutic cervical cancer vaccine. The RAH formulated with PELC/CpG, immunization with C57BL/6 mice could induce higher number of IFN-γ-secreting cells and CD107a+CD8+ cytotoxic T cells than RAH formulated with PELC or CpG only. RAH formulated with PELC/CpG could also induce higher number of RAH-specific CD8+ T cells that determined using PE conjugated RAH/H-2Kb tetramer. Furthermore, RAH formulated with PELC/CpG to immunize TC-1 tumor-bearing mice once could induce strong inhibition of tumor growth compare to formulate with PELC or CpG only. In order to test whether this formulation could be used for protein antigen, recombinant mutant E7 protein (rE7m) was formulated with PELC/CpG. We found that rE7m formulated with PELC/CpG could induce a Th1-bias immunity against tumor.
Our results demonstrate that antigen formulated with both delivery system and immune-stimulating reagent (TLR9 ligand) could induce strong CTL responses against cancer.
