融合一段SARS片段至腫瘤來源的胜肽可提高免疫反應

Abstract

腫瘤細胞會表現低免疫反應之腫瘤相關抗原 (TAA)。一段癌胚抗原 (CEA) 在CT26腫瘤模式中被用來模擬低免疫反應之腫瘤相關抗原。我們藉由SARS冠狀病毒的片段修飾之胜肽疫苗提高在Balb/c老鼠動物模式裡的CEA專一療效。並且藉由提高SARS片段上抗原決定位 (epitope) 與主要組織適應性複合體第一型 (MHC class I) 之結合力來確保SARS片段之免疫反應效果。利用網路軟體 (http://www.syfpeithi.de/) 分析顯示，突變越多的抗原決定位表示其與主要組織適應性的結合力越好。這些質體設計分別轉殖入沙門氏桿菌 (Salmonella typhimurium) 藉以免疫Balb/c老鼠。細胞生理細胞激素測試(in vitro cytokine assay)顯示只帶有CEA的組別只引起介白質IL-4的分泌，而其他多帶有 SARS片段的組別，不論有無突變點，都能有效的引起腫瘤壞死因子-α (TNF-α) 及介白質IL-10的分泌。活體細胞激素測試 (in vivo cytokine assay) 則顯示單獨的CEA不能引起Th1與Th2的反應，但多加的SARS片段則可。老鼠在經過免疫後接種腫瘤大小上的表現也以有SARS片段者來得小，甚者其存活率與無腫瘤率也以SARS組較佳，顯示SARS片段，不論有無突變，對CT26有較好的抑制效果。在治療上面，CEA表現無法有效的抑制腫瘤生長，但SARS則可。 綜合以上結果，低免疫反應之腫瘤相關抗原，如CEA，是無法在動物模式上面帶來有效的保護效果，而在我們建立的系統裡，一段普遍的抗原—SARS片段—則可以加強原低免疫反應的腫瘤相關抗原的反應，進而提高DNA疫苗的效果。此外，我們也建立了一個平台，可以利用電腦預測的方式提高佐藥疫苗的療效。

Tumor cells express tumor-associated antigens (TAAs) that are usually in low immunogenicity. A fragment of carcinoembryonic antigen (CEA) was utilized to simulate the low immunogenous TAA on colon carcinoma, CT26. To enhance the efficiency of our DNA vaccine, it was fused with an exogenous SARS-CoV fragment which is high immunogenous and is expected to induce and enhance the immune response in our animal model, Balb/c mice. The SARS fragment was mutated based on the affinity prediction between the epitope and MHC molecules (H2-Kd) by Internet software (http://www.syfpeithi.de/). The more mutations there are in a construct, the higher the affinity is. These constructs were then transformed into Salmonella typhimurium and orally fed to immunize Balb/c mice. In vitro cytokine profile reveals that CEA alone induces only IL-4 secretion whereas constructs with an additional SARS fragments, whether mutated or not, can significantly induce TNF-αand IL-10 secretion. In vivo cytokine profile shows that CEA alone can not induce any cytokine secretion but an additional SARS fragment fused to CEA can induce both Th1 and Th2 responses. Mice in the protection assay also had smaller tumor volume than those with CEA alone. The efficiency of the CEA-SARS immunization (both non-mutated and mutated) is reflected on their survival rate and tumor-free rate, which are both higher than the CEA alone group. Moreover, the SARS fragment fused with CEA effectively slowed tumor growth in the therapeutic assay. In conclusion, low immunogenous TAAs, such as CEA, can not effectively induce the immune response of animal models. We have set up a system in which the foreign parental or mutated SARS fragments could enhance the anti-tumor efficacy of the tumor vaccine against endogenous tumor antigens. Furthermore, we provide a platform to enhance the adjuvant effect of the foreign peptide by computer prediction.