PLAC1/CP1 HLA-A*0201限制性多肽致敏DC疫苗治疗原发和转移性结肠癌动物模型的实验研究

Immunotherapy is an effective adjuvant treatment programs for the malignant tumor. The tumor-associated antigen pulsed DC（dendritic cell, DC）vaccine strategy has been developed rapidly in recent years. Highly immunogenic tumor antigens are prerequisite for and key to the immunotherapy. The person in charge of this project identified a novel tumor antigen PLAC1/CP1 with higher immunogenicity in colorectal cancer. A positive correlation between this novel tumor antigen induced specific immune response and improved survival of patients with colorectal cancer. The antigen has the promise to play an important role especially in the treatment of advanced and metastatic colorectal cancer patients. We have identified PLAC1/CP1 specific MHCⅠmolecules of peptides which could induce the specific immune response in colorectal carcinoma patients. But its therapy to tumor in vivo is still unknown. The main study object of this project is to explore the therapy function of PLAC1/CP1 peptide vaccines. The detail study contents are: 1. Human immune reconstitution in immunodeficiency SCID mice; 2. Established colon cancer animal model on the basis of immunodeficiency SCID mice with human immune reconstitution; 3. Established colon metastatic liver cancer animal model on the basis of immunodeficiency SCID mice with human immune reconstitution; 4. Synthesize and purify these peptides. 5. Further study of the treatment of colorectal cancer animal models applied to PLAC1/CP1 peptide vaccines. It has practical significance to provide a new safe and effective immunotherapy way for improving patient's survival.

肿瘤抗原疫苗的免疫治疗策略中，选择高免疫原性的肿瘤抗原作为靶点是免疫治疗的关键。本课题申请人已在国际上首次报告了胎盘特异性基因PLAC1/CP1是一类新的肿瘤-胎盘特异性抗原，在结直肠癌中高表达，其诱导的特异性免疫应答与结直肠癌患者提高的生存期明显相关，是结直肠癌免疫治疗的理想靶点。我们在前期体外研究工作中已鉴定出PLAC1/CP1特异性MHCⅠ类分子结合的多肽在体外可诱导结直肠癌患者特异性免疫应答，但其对体内生长实体瘤的治疗效果尚不清楚，本项目将建立模拟人体环境的动物实体瘤模型，探讨应用PLAC1/CP1多价肽疫苗治疗肠癌的途径，对提高患者的生存期具有现实意义。具体研究计划：（1）免疫缺陷SCID小鼠的人免疫重建，（2）免疫重建SCID小鼠结肠癌，（3）免疫重建SCID小鼠肝转移癌动物模型的建立，（4）合成和纯化PLAC1/CP1多肽，（5）应用PLAC1/CP1多价肽致敏DC疫苗治疗结肠癌动物模型。

肿瘤免疫治疗策略的关键问题包括：1.高免疫原性抗原，2.多价肽疫苗，3.疫苗的安全、有效性。本项目前期工作对前两个问题进行了系统研究，在国际上首次鉴定和报告了PLAC1/CP1是一类新的结直肠癌相关抗原，其诱导的特异性免疫应答与结直肠癌患者生存期密切相关，提示其可成为结直肠癌免疫治疗的靶基因。但PLAC1/CP1抗原疫苗对体内实体瘤的免疫治疗作用尚不清楚。因此，本项目通过应用新的PLAC1/CP1抗原肽疫苗治疗小鼠大肠癌模型，探讨该治疗方案的安全性及有效性，为进一步临床试验的开展奠定基础。本项目所选用实验动物是严重联合免疫缺陷SCID（severe combined immune deficiency，SCID）小鼠，该小鼠无自身免疫系统，其主要特性是胸腺、脾脏、淋巴结中的T淋巴细胞和B淋巴细胞大大减少，细胞免疫和体液免疫功能缺陷，比较适合应用于异种免疫功能重建和肿瘤学的研究。具体研究内容包括:（1）合成、纯化已鉴定的PLAC1/CP1四段HLA-A*0201限制性多肽。（2）收集健康人外周血标本，进行外周血单核细胞的分离、培养、计数。（3）提取健康人外周血DNA并进行质量鉴定。（4）DNA分型鉴定，筛选HLA-A*0201亚型外周血标本备用。（5）树突状细胞DC的培养和表型鉴定。（6）外周血CD4+、CD8+T淋巴细胞的分离、活化和增殖能力检测。（7）制备HLA-A*0201限制性PLAC1/CP1多肽致敏DC疫苗。（8）免疫缺陷SCID小鼠的人免疫系统重建。（9）建立免疫系统重建后的SCID小鼠结肠癌模型及肝转移模型。（10）研究PLAC1/CP1多肽致敏DC疫苗对免疫重建SCID小鼠结肠癌皮下种植瘤和肝转移瘤模型的治疗。（11）解剖成瘤组织，临床病理学分析、鉴定。经初步研究发现，应用PLAC1/CP1多肽疫苗治疗后，小鼠肿瘤生长速度减慢较明显，解剖小鼠发现肿瘤伴有大片坏死，观察84天后，小鼠仍然存活；而未经免疫治疗的对照组小鼠肿瘤生长迅速，全部死于肿瘤。该结果提示，PLAC1/CP1多肽疫苗在结直肠癌的免疫治疗中将起着重要作用，具有临床应用前景。