NY-ESO-1/MAGEA3/SSX2重组耻垢分枝杆菌的构建及其抗肿瘤作用的研究

Although much evidence has been gathered demonstrating that immune effectors can play a significant role in controlling tumor growth under natural conditions or in response to therapeutic manipulation, it is clear that malignant cells do evade immune surveillance in most cases. Considering that anticancer active specific immunotherapy seems to have reached a plateau of results and that currently no vaccination regimen is indicated as a standard anticancer therapy, the dissection of the molecular events underlying tumor immune escape is the necessary condition to make anticancer vaccines a therapeutic weapon effective enough to be implemented in the routine clinical setting.M.S antigens can be presented at the cell surfaces and antigen-presenting cells in the context of major histocompatibility complex (MHC) class II, stimulating CD4+ T cells and inducing a primarily T helper type (Th) 1 immune response . This complex and robust immune reaction evoked by M.S is evidenced by a massive transient secretion of cytokines , including interleukin (IL)-1, IL-2, IL-5, IL-6, IL-8, IL-10, IL-12, IL-15, IL-18, tumor necrosis factor (TNF)-α, granulocyte-monocyte colony stimulating factor (GMCSF), and interferon (IFN)-γ ,thus could correct the tumor patients low immunity status.CTA antigens ,which are specifically expressed in the normal testis and show aberrant expression in various malignancies, is the focus of an intense wave of research, both as putative biomarkers and for their potential to constitute convenient targets for immunotherapeutic strategies.This study we planed to construct the recombinant Mycobacterium Smegmatis which can express the fused protein of two or three CTA antigens(NY-ESO-1/MAGE A3/SSX2).At first, we screen of the recombinant M.S with high immunity efficacy through BALB/c mice experiment.then we used the rM.S immunity the bearing tumor NOD-SCID-HU mice, examined tumor characteristics, clinical and pathologic response to treatment, associated morbidities, local and distant recurrence, overall survival and vaccine,s safety.Thus we could screened the rM.S which can be used for the tumor prevention and treatment, and lay a foundation for tumor vaccine and the specific targeting treatment.

肿瘤免疫治疗的重点集中在如何提高肿瘤抗原的免疫原性及如何避免肿瘤细胞的免疫逃逸，针对这个问题，我们利用耻垢分枝杆菌杆菌可非特异性地激发肿瘤患者的细胞及体液免疫应答，从而纠正患者免疫低下状态，同时再利用肿瘤睾丸抗原(CTA)可特异性的提高机体对于肿瘤的细胞毒杀伤作用，构建一种可表达CTA抗原的重组耻垢分枝杆菌菌苗。该菌苗可表达两到三种即能刺激机体产生细胞免疫应答又能产生体液免疫应答的睾丸肿瘤抗原（NY-ESO-1/MAGE A3/SSX2），我们将不同排列组合的rM.S通过普通动物实验（BALB/c）筛选到具有较强免疫效力的重组耻垢分枝杆菌，然后再通过NOD-SCID-HU小鼠实验验证其在荷瘤小鼠模型中控制肿瘤生长及延长小鼠生存时间，以及其在免疫小鼠后抑制肿瘤生长及延长小鼠生存时间及疫苗的安全性，从而筛选到可用于肿瘤预防及治疗的重组耻垢分枝杆菌菌株，为肿瘤疫苗的研制及特异性靶向治疗奠定基础。

通过克隆技术构建得到SSX2、NY-eso-1及MAGEA3两两结合的融合蛋白，即SSX2--NY-eso-1（SN）、SSX2—MAGEA3（SM）、NY-eso-1—SSX2（NS）、NY-eso-1—MAGEA3（NM）、MAGEA3—SSX2（MS）及MAGEA3—NY-eso-1（MN）组合的6种融合蛋白。通过对比蛋白表达量及western blot结果选取SN、NS、MS及SM，通过电转的方法将连接有融合蛋白的载体转入耻垢分枝杆菌中得到重组耻垢分枝杆菌（rM.S）。将成功转入融合片段的耻垢分枝杆菌进行扩大培养制备标准rM.S菌株对Balb/c小鼠进行皮下注射。通过在正常Balb/c小鼠体内实验验证了重组耻垢分枝杆菌可以刺激机体使其产生SSX2、NY-eso-1及MAGEA3三种抗体的产生，同时CD4+T细胞亚群的比例增加，血清中IL-2、IFN-r及TNF-a等细胞因子水平较免疫前明显提高。在荷瘤的NOD-SCID-Hu动物模型中继续实验，免疫重组耻垢分枝杆菌后瘤体生产速度受到抑制，但流式细胞技术实验中发现CD4+T细胞亚群并未明显增加，同时脾细胞增值效果也不好，故调整方案，用重组耻垢分枝杆菌免疫正常Balb/c小鼠，在抗体效价达到最高后取血清通过尾静脉注射到荷瘤裸鼠体内进行治疗， 40天后检测瘤体体积及细胞因子表达，实验初步达到预期结果，但仍需要进一步优化实验方案。