“功能鸡尾酒”模式重组融合蛋白疫苗FEH-ACpG的抗HCC作用及机制研究

Prophylaxis and treatment of Hepatocellular Carcinoma (HCC) is a huge challenge worldwide. China is the worst-hit area of HCC, for the annual number of new cases and the death cases of HCC reached 55% and 45% of the world's total, respectively. With a high malignant degree, rapid progression, and high possibility of relapse and metastasis, HCC is known as "the king of cancer." So far, effective treatment for cure of HCC is still lacked. While, the breakthrough of cellular immunotherapy in recent years brought the new light on HCC treatment. However, the manufacturing processes and quality control remain difficult during DCs maturation with multiple stimulations in vitro. The objective of this project is to construct a new type therapeutical fusion protein vaccine, which is composed of three functional components, and achieved a "cocktail"-like function on individual key steps of the immunotherapy, including "antigen presentation", "induction of tumour-specific CTL", and "overcoming the immune suppression in tumor microenvironment (TME) ". The core component of the fusion protein is a 3-in-1 mixed-epitopes from tumor associate antigen (TAA) of incipient, medium and advanced stages of HCC, AFP, GPC3 and HPA, respectively, for providing the targets to the immune attacks launched by the vaccine. The sencond part of the fusion protein is a heat shock protein (HSP) 65, which is utilized to warn the antigen presentation cells (APCs), trigger MHC I type antigen presentation, and induce a cellular immune response. The last component in the protein is an anti-CD25 scFv, which is recruited to reverse the immunosuppression in the organism caused by blocking CD4+ CD25+ regulatory T cells. In addition, a MDSCs targeted aptamer coupling CpG was used as adjuvant, which can targetingly enter the TME, overcome the immunosuppression status and stimulate the immune response to attract CTL to kill cancer cells in TME. Following the construction and preparation of the therapeutical vaccine, the immunity effect and the anti-cancer efficacy of it will be evaluated thoroughly in molecular, cellular and systemic models. Further, the detailed mechanism of action will also be deeply investigated. This project will provide new ideas and methods to the immunotherapy of HCC, and provide a solid basis for clinical practice in the future.

肝癌（HCC）恶性度高、病程迅速、复发和转移性强，有"癌中之王"之称，至今仍缺乏有效治疗手段。细胞免疫治疗的兴起为其治疗带来了新希望，但由于肿瘤免疫耐受机制复杂，DCs需在体外成熟、需引入多种刺激因素等问题，导致其在规模化制备、质量控制与临床应用间的矛盾。本项目拟构建一种功能上具"鸡尾酒"性质的新型HCC治疗性融合蛋白疫苗，兼顾"抗原提呈"、"诱导肿瘤特异CTL"及"克服肿瘤微环境（TME）免疫抑制"三个关键环节，实现其免疫治疗的有效性。同时以重组蛋白模式解决当前肿瘤免疫治疗策略的质量控制难题。融合蛋白疫苗功能组分包含混合串联表位（免疫治疗靶标）、HSP65（触发细胞免疫）、CD25 scFv(拮抗Treg抑制）；并以适配子偶联CpG为佐剂靶向TME，全面激活免疫反应。采用多种模型并结合转化医学手段，在分子、细胞与整体水平系统考察其抗HCC作用及免疫机制，为肿瘤免疫治疗提供新的思路和手段。

肿瘤免疫治疗策略代表未来肿瘤治疗的重要方向之一，抗肿瘤免疫治疗成功需要“扶正祛邪”，一方面激活抗原提呈细胞向T细胞递呈抗原的功能，诱导T细胞活化产生保护性免疫反应；另一方面要克服肿瘤微环境的免疫抑制与免疫逃逸。本研究基于上述两种策略，提出“鸡尾酒”模式免疫疗法，通过激发机体特异性免疫功能，逆转肿瘤微环境的免疫抑制，达到有效进行肿瘤免疫治疗目的。构建疫苗核心组分，即混合肿瘤抗原表位肽，选择甲胎蛋白（AFP）、磷脂酰肌醇蛋白-3（GPC3）、肝素酶（HPA）表位肽，通过AFP3GPC34HPA2与热休克蛋白（HSP）、抗CD25scFv单链抗体（scFv）直接融合，该EFH融合蛋白更符合撷抗免疫逃逸机制，有效进行抗原提呈，发挥T细胞杀伤功能。此外，我们采用课题组前期筛选的CpG为佐剂，通过适配体靶向递送策略，采用体外逆转录法制备寡核苷酸适配子并对其进行化学修饰；脂质体包裹CpG ODN后，通过特异反应，利用后插入法将适配子插入脂质体，形成适配子-脂质体-CpG ODN复合物。使用粒度仪分析测定该复合物的平均粒径为95±15nm，Zeta电位分布为-15±5mV。透射电子显微镜观察复合物呈现球形或者类球形，粒径在100nm左右。通过荧光显微镜观察适配子-脂质体-FAM-CpG ODN复合物的入胞效率显著高于脂质体-CpG ODN复合物。体内研究中，对荷瘤C57小鼠进行尾静脉注射适配子-脂质体-FAM-CpG ODN，注射后2 h收集肿瘤组织，进行荧光检测，适配子可以明显增加肿瘤部位FAM-CpG的富集，证明该系统可以有效将CpG导入肿瘤内。体内药效实验中，当瘤体积达到100mm3时，尾静脉给药，CpG ODN的给药量为3.06mg/kg，2周监控瘤体的增长量来确定瘤体积的增长趋势。结果表明，给药组均具有抑瘤效果，且 IL-4Rα适配子复合物的抑瘤作用好于非靶向性适配子复合物。瘤组织CD31免疫组化表明，该复合物可以显著减少血管生成。此外，该复合物显著抑制组织中Treg细胞，显著降低肿瘤组织中MDSCs，另外骨髓和脾脏中的MDSCs也得到抑制，该递送系统对外周血中的MDSCs无显著影响。证明该递送系统有助于改善肿瘤微环境，逆转肿瘤的免疫状态。不同给药组肿瘤组织中，与血管生成和转移相关基因VEGF，MMP9显著降低，TNF-α，IL-2，IL-12为Th1型细胞因