修饰的肿瘤抗原肽CEA_(610D)疫苗的体外抗肿瘤作用

In vitro anti-tumor activity of altered tumor-associated antigen CEA_(610D) peptide

目的:评价修饰的CEA610D抗原肽疫苗体外抗肿瘤免疫的有效性,为其临床应用提供依据。方法:多肽固相合成法合成HLA-A2CEA修饰肽(CEA610D)、HLA-A2天然肽CEA605-613(天然肽)和HLA-A2无关肽MAGE-3(无关肽),采用同种异体混合淋巴细胞与肽共孵育的方法,诱导肽特异性细胞毒性T淋巴细胞(CTL),利用MTT法检测CTL的增殖和杀伤活性;流式细胞术观察细胞表型;RT-PCR检测细胞穿孔素的表达;ELISA法检测CTL上清中IFN-γ的水平。结果:CEA610D疫苗产生肽特异性CTL的能力最强(P<0.05),其CD8+T细胞数也高于天然肽组和无关肽组;在效靶比为40:1时,CEA610D和天然肽所诱导的CTL对CEA+HLA-A2限制性人结肠癌细胞T84的杀伤活性可达(56.7±3.73)%和(51.2±1.86)%,而3种肽特异性CTL细胞对CEA+HLA-A2人结肠癌lovo细胞杀伤活性维持在本底水平;CEA610D组的CTL所释放的杀伤相关介质穿孔素和上清中IFN-γ的水平也显著高于其余两组(P<0.01)。结论:与天然肽相比,CEA610D疫苗可打破自身表位的免疫耐受,在体外抗肿瘤免疫中更具优势。

Objective ：To investigated the in vivo efficacy of altered CEA610D peptide vaccine against tumor, so as to provide a basis for its clinical use. Methods： Altered CEA peptide CEA610D, natural CEA peptide CEA605-613 and MAGE- 3 peptide were synthesized by polypeptide solid-phase synthesis system. The cytotoxicity T lymphocytes （CTLs） were induced by autologous mixed lymphocytes reaction implused with above peptides; proliferation and cytotoxicity of different CTLs were measured by MTT; phenotypes of the CTLs were detected by flow cytometry ; expression of perforin in different CTLs were assayed by RT-PCR; IFN-γ levels in the supematants of different CTLs were detected by ELISA. Results： CEA610D peptide more efficiently induced CTL than CEA605-613 and MAGE-3 peptide did （P 〈 0.05）. The number of CD8＋ T cells in CEA610D group was significantly larger than those in CEA605-613 and MAGE-3 groups （P 〈 0. 05）. When the E： T was 40： 1, the cytotoxicity of CTLs induced by CEA610D and CEA605-613against CEA ＋ HLA-A2 ＋ T84 cells were （56.7 ± 3.73） % and （51.2 ± 1.86）%, respectively. But the CTL cytotoxicities induced by the three peptides against CEA ＋ HLA-A2-Lovo cells were all at the background level. Perforin expression and IFN-γ level of CTLs in CEA61oD group were significantly higher than those in the other two groups （P 〈 0.01 ）. Conclusion： Compared with natural CEA605-613 peptide, altered CEA60D peptide can effectively break the immune tolerance to self peptide, and thus has a stronger anti-tumor activity in vitro.