卵巢癌相关抗原TM4SF1 HLA-A2限制性CTL表位的预测、筛选及免疫反应性鉴定

Malignant tumor is the number one cause of death in Chinese resident. It causes heavy social and economic burden. Mortality rate of ovarian cancer ranks the first on the list among female genital tract malignant tumor. 5-year overall survival of stage Ⅲ～Ⅳ ovarian cancer treated with standard operation and chemotherapy is approximately 20～25%. Besides early diagnose and treat,tumor biological immune targeted therapy is another important breakthrough point to improve survival rate. The preliminary study had screened associated antigen gene TM4SF1 from cDNA library derived from ascites cancer cell of epithelial ovarian cancer patients by combined modified SEREX （serological analysis of recombinant cDNA expression libraries,SEREX）with SSH（suppression subtractive hybridization,SSH）. The preliminary study also showed TM4SF1 was overexpression in cancer tissues compared with normal tissues of ovary,breast,intestine. So, we propose a scientific hypothesis that TM4SF1（transmembrane 4 L6 family member 1, TM4SF1） is a tumor overexpression antigen of epithelial cancers. It has potentiality of immune targeted therapy. CTL induced by peptides derived from TM4SF1 can kill cancer cells which overexpression TM4SF1,then inhibit tumor growth. In this subject we will predict,screen and immunoreactively identify the HLA-A2 restricted CTL epitopes of TM4SF1.Our research will provide new important targets for ovarian cancer clinical treatment and provide theoretical basis for new cancer treatment strategies based on cancer specific cytotoxic T lymphocytes.

恶性肿瘤已成为中国居民的第一位死因，它带来了沉重的社会和经济负担。卵巢癌的死亡率在女性生殖道恶性肿瘤中居首位，在规范的手术及化学治疗下晚期病人的5年生存率仅为20-30%。生物免疫靶向治疗是早诊、早治之外提高生存率的另一个重要突破点。我们前期研究中从卵巢癌腹水肿瘤细胞cDNA文库中筛选出肿瘤相关抗原TM4SF1，并证实其在卵巢癌中高表达，而在正常卵巢组织中低表达或不表达。在本研究中我们提出一个科学假设：TM4SF1是在卵巢上皮性癌的高表达一种肿瘤抗原，它有可能具有免疫治疗靶点的潜能，来源于TM4SF1的HLA-A2限制性多肽可诱导肿瘤特异的CTL，从而杀灭表达TM4SF1的卵巢癌细胞，抑制肿瘤生长。此课题将对TM4SF1抗原的HLA-A2限制性CTL表位进行预测、筛选及鉴定。我们的研究成果有望为卵巢癌的临床治疗提供新的重要靶点，为以诱导肿瘤特异的CTL杀灭癌细胞的治疗策略提供理论依据。

在规范治疗的情况下，卵巢癌的5年生存率并没有明显提高约为30%左右，生物免疫靶向治疗成为手术、化疗之外的重要辅助治疗手段。本课题组前期筛选出的卵巢癌相关抗原TM4SF1在卵巢上皮性癌组织中的高表达、在卵巢良性肿瘤组织及正常卵巢组织中的低表达，且随临床期别的升高表达水平也增高，提示TM4SF1有免疫治疗靶点的潜能。. 本项目联合BIMAS、SYFPEITHI、IEDB、PROPREDⅠ4种表位肽预测法，获得10条TM4SF1 HLA-A2限制性的CTL候选表位肽（P1-P10）。经检测：P1、P2、P3、P4、P7、P8、P9、P10与HLA-A2分子的复合物具有良好绑定稳定性，P1、P5、P10具有较强的免疫原性；CCK8细胞毒性实验检测体外人卵巢癌细胞杀灭效果结果显示：P1、P5、P10均能够激发HLA-A2阳性卵巢癌病人外周血CTL的免疫反应,P1及P10的免疫原性更强。P10免疫的小鼠脾细胞诱导的CTL在100∶1效靶比时对人卵巢癌细胞株HO8910PM(HLA-A2+,TM4SF1+)的杀伤作用最强为(51.38±5.56) %，均显著高于P1、P5(P<0.05)，但对于沉默了TM4SF1的HO8910PM(HLA-A2+,TM4SF1-)的杀伤作用明显下降（16.50±5.56) %；负载P10的DCs 诱导的CTL在40∶1效靶比时对HO8910PM(HLA-A2+,TM4SF1+)的杀伤作用为(53.31 ±2.93 )% ,均显著高于P1、P5( P<0.05 ),而对于用抗HLA-A2抗体预处理过的HO8910PM (HLA-A2+,TM4SF1+)的杀伤作用明显下降 (28.54±2.04 )%；未负载多肽的DCs细胞刺激的CD8+T细胞对HO8910PM( HLA-A2+,TM4SF1+)没有杀伤作用。. TM4SF1的HLA-A2限制性CTL表位P10能诱导人外周血单核细胞的CTL反应, P10免疫的小鼠脾细胞诱导的CTL及负载P10的DCs 诱导的CTL及能特异性杀伤靶细胞，表明P10能够被自然呈递并表达于HLA-A2+及TM4SF1+肿瘤细胞的表面,有免疫治疗的潜力。. 本研究结果为卵巢癌的免疫治疗提供了新的HLA-A2+限制性CTL表位肽，有望为卵巢癌的免疫靶向提供新的思路，为下一步研究提供理论依据。