HLA-A2限制性人食管癌相关抗原COX-2表位的鉴定和改造

Identification and Optimization of HLA-A2 Restricted COX-2 Derived Antigen Peptides

目的筛选和鉴定食管癌广泛高表达蛋白COX-2的HLA-A2限制性CTL表位。方法运用生物信息学的方法,以SYFPEITHI法初步预测,结合MHCPred 2.0和NetChop3.0 Server在线分析,设计出五条新的抗原肽。通过标准Fmoc固相合成法合成抗原肽,以流式细胞仪对其与HLA-A2分子的结合力和稳定性进行分析,并对结合力较低的P66(FI<0.5)的原始序列进行改造,将其序列第一位的苯丙氨酸替换成酪氨酸(P66Y1),以MTT实验检测抗原肽诱导的特异性CTL对肿瘤细胞的杀伤作用。结果P321对HLA-A2分子有较高的结合力(FI=1.93),同时P66Y1与HLA-A2分子的结合力显著增强(FI=1.48),并且它们与HLA-A2分子的稳定性较好(DC50>2h)。P321和P66Y1对表达COX-2的EC-9706、EC-1的杀伤率分别明显高于P66。结论源于食管癌广泛高表达抗原COX-2的抗原肽P321和P66Y1能分别有效激发HLA-A2限制性CTL的免疫应答并杀伤肿瘤细胞。

Objective To identify the HLA-A2 restricted CTL epitopes of over-expressed COX-2 in esophageal carcinoma （EC）. Methods Five novel HLA-A2 restricted peptides of COX-2-derived antigen were predicted by SYFPEITHI prediction method combined with MHCPred and NetChop3. 0 Server. The candidate peptides were synthesized by standard Fmoc chemistry and their binding affinity and stability to HLA-A2 molecule were evaluated by flow cytometry. Otherwise the weak binding peptide P66 （FI〈0. 5） was optimized by substituting its phenylalanine with tyrosine, and the cytotoxic activities against the EC cells were determined by MTT assay. Results P321 showed higher affinity （FI = 1.93） for HLA-A2 molecule compared to other candidate peptides and P66Y1 exhibited remarkable affinity for HLA-A2 molecule （FI = 1.48）. Furthermore, these two peptides could bind stably with HLA-A2 molecule （DC50）2）. The MTT assay reflected that P321 and P66Y1 could specifically lyse EC-1 and EC-9706 cells which expressed COX-2 compared to P66. Conclusion P321 and P66Y1 derived from over-expressed COX-2 in esophageal carcinoma are potential epitopes which are capable of inducing HLA-A2-restricted CTLs and killing HLA-matched EC cells.