新型PLAC1/CP1抗原致敏DC疫苗联合CIK对小鼠结肠癌模型免疫治疗作用和分子免疫学调节机制研究

Cancer immunotherapy has been receiving more and more attention in recent years, and has become a hot research topic. It plays a very important role in cancer assistant treatment. I'm personally in charge of this project. I have identified and reported PLAC1/CP1, a new class of colorectal cancer specific antigens in the international area, which induced a specific immune response in colorectal carcinoma patients. A positive correlation between this novel tumor antigen induced a specific immune response, and improved the survival rate of patients with colorectal carcinoma. It is an ideal target for CRC immunotherapy. I have identified four HLA-A*0201 restricted CD8 T cell epitopoes. This project through animal experimentation, and applying the new PLAC1/CP1 antigen pulsed DC vaccines to treat mice models with CRC. This project will focus our attention on studying the PLAC1/CP1 peptide pulsed DC vaccines, which united CIK, and the entire protein vaccine. We also detected the proliferation of activated T cell subsets, Treg and tumor cell apoptosis, through a variety of molecular immunology and molecular biology methods in order to deeply study immunotherapy mechanisms, and develop the best possible treatment programs. It has practical significance to provide a new safe and effective immunotherapy method, for improving a patient’s survival. At the same time, this study will have scientific significance for other types of malignant tumors... The research papers have been published in "Gastroenterology", "Immunogenetics", "Journal of gastroenterology" magazines, and other Chinese publications.

免疫治疗在恶性肿瘤辅助治疗方面的重要作用已引起国际上的密切关注，但尚无结肠癌有效免疫治疗的报道。本项目申请人在国际上首次鉴定和报告了PLAC1/CP1是一类新的肿瘤特异性抗原，前期体外研究证明该基因是结直肠癌免疫治疗的理想靶基因，并鉴定出四段HLA-A*0201 多肽表位作为候选治疗靶点，但PLAC1/CP1抗原疫苗对于体内实体瘤的疗效和分子免疫学作用机制有待阐明。本项目通过建立人免疫重建的小鼠肠癌模型，应用新型PLAC1/CP1抗原疫苗进行免疫治疗，重点比较和研究单纯多肽致敏DC疫苗组和多肽致敏DC疫苗组联合CIK方案的差别，同时分析包括T淋巴细胞亚群增殖活化、Treg和肿瘤细胞凋亡等多项细胞与分子标志，深入研究免疫治疗的调节机制，实现为临床选择最佳免疫治疗方案的目标，对发展具有我国自主知识产权的肿瘤免疫治疗具有重要科学意义，对提高患者的生存率和生存质量具有临床实际意义。

个体化治疗是恶性肿瘤治疗的重要策略。免疫治疗是近年来国际上的研究热点，但尚无结肠癌有效免疫治疗的报道。本项目申请人在国际上首次鉴定和报告了PLAC1/CP1是一类新的肿瘤特异性抗原，前期体外研究证明该基因是结直肠癌免疫治疗较理想的靶基因，并鉴定出四段HLA-A*0201 多肽表位作为候选治疗靶点，但PLAC1/CP1抗原疫苗对于体内实体瘤的疗效和有关作用机制仍不明确。本项目的研究内容主要包括：建立人免疫重建的SCID小鼠肠癌模型，应用新型PLAC1/CP1抗原疫苗进行免疫治疗，比较研究单肽致敏DC疫苗组和多肽致敏DC疫苗组的差别，分析包括T淋巴细胞亚群增殖活化、血液中调节性T细胞（Treg）的分泌细胞因子以及肿瘤微环境中T淋巴细胞亚群的鉴定。研究结果显示，经流式细胞术和PCR-SSP和PCR-SBP分型方法得到4份具有HLA-A*0201纯合型；完成合成、纯化四段PLAC1/CP1 HLA-A*0201抗原多肽，经PLAC1/CP1 树突状细胞（DC）负载抗原多肽刺激后，通过细胞计数法发现，活化的T淋巴细胞数量显著增生（67.74%，21/31），流式细胞术显示增值百分比（9.78%-11.73%~26.09%-51.88%）；应用胞内细胞因子染色技术显示胞HLA-A*0201标本DC负载PLAC1/CP1多肽刺激前后，CD8+T细胞I类细胞因子IFN-γ分泌量明显增加（0.11%-0.23%~0.45%-3.30%）； CD4+ T淋巴细胞经DC负载PLAC1/CP1多肽刺激后分泌Ⅱ类细胞因子IL-10的量增加不明显（0.18%-0.27%）。培养的DC表达CD86，CD80和HLA-DR。免疫重建后，用ELISA法检测小鼠血清中人IgG含量，结果显示IgG浓度在标准曲线内，重建成功。经检测，肿瘤组织中的淋巴细胞具有CD4CD25Foxp3 Treg细胞的表达。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的治疗。多肽+CIK治疗组生存期>多肽治疗组>无关肽治疗组，肿瘤大小多肽+CIK组<多肽治疗组<无关肽治疗组。PLAC1/CP1抗原多肽对于结直肠癌SCID小鼠免疫治疗及相关机制的研究，对推动临床免疫治疗，提高患者的生存率和生存质量具有实际意义。