基于ECM1优势CTL表位双重修饰的多表位长肽-DC疫苗抗三阴性乳腺癌靶向效应与免疫学机制研究

The 5-year survival rate of Triple negative breast cancer (TNBC) is less than 30%, and currently there are no effective treatments. DC vaccines targeting antigens in TNBC become a promising therapeutic strategy for the treatment of TNBC. However, the lack of ideal antigenic peptides and carries is a major hurdle. We identified HLA-A2.1-restricted ECM1-preponderant CTL epitopes that specifically inhibited TNBC after induction. Multi-epitope long peptides were generated by amino acid substitution and connected by protease-sensitive linkers. We found that after cleaved by proteases, the multi-epitope long peptides induced stronger CTL effects than preponderant epitopes. Liposome nanoparticles containing the long peptides recognized DC, and promoted DC to endocytose the peptide, thus increasing the CTL effects of the peptide. The purpose of this project is to study the role of multi-epitope long peptides in inducing memory ability and specifically identifying the TNBC-targeted immune responses of CTL of ECM1, to explore the mechanisms by which multi-epitope long peptides produce HLA-A2.1-restricted CTL epitopes via DC cross-presentation, to investigate that DC -targeted liposome nanoparticles increase the immune responses of multi-epitope peptides to the TNBC, and to investigate the immunological effect of multi-epitope long peptide liposome nanoparticle DC vaccine on the treatment of TNBC. We believe that achieving these goals provides the theoretical and experimental basis for the development of novel TNBC- targeted DC vaccines.

三阴性乳腺癌（TNBC）5年生存率不足30%，缺少有效的治疗方式。靶向TNBC抗原肽DC疫苗有望成为实现有效治疗突破口，但缺少理想抗原肽及载体。课题组前期筛选出具有诱导靶向抑制TNBC作用的HLA-A2.1限制性ECM1优势CTL表位；经氨基酸替换、蛋白酶体敏感连接臂连接修获得多表位长肽，且发现其可被蛋白酶体自连接臂处酶切，诱导较优势表位更强CTL效应；脂质体纳米粒可靶向识别DC、增加多表位长肽DC内吞量，进而增强其诱导的CTL效应。本课题拟在明确多表位长肽诱导具有记忆功能、并特异识别ECM1的抗TNBC靶向CTL免疫效应基础上，阐明多表位长肽经DC交叉呈递产生HLA-A2.1-CTL表位肽复合物的机制，验证脂质体纳米粒靶向识别DC、提升多表位长肽靶向抗TNBC免疫效应，考察多表位长肽脂质体纳米粒DC疫苗抗TNBC靶向免疫效应。本研究为研发靶向抗TNBC治疗性DC疫苗奠定理论与实验基础。

肿瘤治疗性疫苗是以肿瘤组织/细胞裂解物、蛋白、多肽或者基因等不同形式“抗原”，导入患者体内，以实现诱导机体抗肿瘤免疫应答之目的，因其兼有良好的预防和治疗效果，并且安全性高，而成为肿瘤治疗领域的研究热点。传统的肿瘤治疗性疫苗多关注CD8+T细胞的活化，而NK细胞对肿瘤免疫也具有重要意义。. 本项目首次鉴定了三阴性乳腺癌相关抗原ECM1来源的HLA-A2.1限定性优势CTL表位肽LA和YL，为新型肿瘤治疗性疫苗提供了优秀的候选药物。LA和YL体外可经树突状细胞（DCs）活化CD8+T细胞，同时意外发现LA还可以经DCs活化NK细胞。本项目进一步阐明了单纯优势表位多肽LA和YL经液泡途径被DC交叉呈递，从而激活CTL特异抗肿瘤效应的作用机制；同时发现并阐明优势CTL表位肽LA经TLR4-p38 MAPK通路上调 DCs细胞MICA/B表达，进而激动NKG2D活化NK细胞的DC-NK cross-talk免疫活化机制。为了解决多肽进入体内后不稳定、半衰期短、免疫效应弱的缺点，设计并制备了能够主动识别DCs，并提升多肽体内稳定性的脂质体制剂YL-Lipo和LA-Lipo，从而使得LA和YL可以直接皮下注射使用，证实LA和YL在体内也能够产生有效的免疫应答和抗肿瘤功能。并且由于LA 能够在活化CD8+T细胞的同时，进一步活化NK细胞，因此产生的抗肿瘤效应明显强于YL。最后，检测发现，YL-Lipo或LA-Lipo皮下免疫小鼠，并未对小鼠体重、常见血液学各项指标、骨髓细胞、肝脏，肾脏，心脏，肺、胃、肠等重要组织器官的形态学、主要脏器指数和肝、肾功能相关血清生化指标等产生明显影响，表明在体内应用YL-Lipo或LA-Lipo对正常组织器官无明显免疫脱靶毒性。可见YL-Lipo和LA-Lipo是安全且有效的抗肿瘤疫苗。. 本项目的研究成果，尤其是多肽LA，作为新型肿瘤治疗性疫苗，明显优于传统的只注重提升CD8+T细胞功能的疫苗，开拓了肿瘤疫苗研发思路，提供了优秀的候选疫苗，为攻克肿瘤治疗难题提供了极有希望的工具。