预警素对新型生物纳米抗原载体介导的肝癌免疫治疗作用研究

Hepatocellular carcinoma (HCC) is one of the most lethal malignancies worldwide, and currently there is no effective treatment available in the clinic. Recently, exosomes, membranous nanovesicles secreted by most cells, show great promise as a surrogate for Dendritic Cell (DC) and a novel form of noncellular vaccine for tumor immunotherapy. In our previous studies, we demonstrated that exosomes derived from HCC-specific antigen- alpha-fetoprotein (AFP)-expressing DCs, namely DEXAFP, effectively inhibited tumor growth in HCC mouse models. However, vaccines with stronger immunogenicity and long-lasting immunotherapeutic effects are critical and urgently needed for HCC patients with more complicated immune microenvironment. Our preliminary data showed that alarmin HMGN1, a novel immunoadjuvant which can promote DC maturation and recruit DCs in response to external injuries, could significantly improve the immunogenicity and long-lasting antitumor effects of DEXAFP when HMGN1 functional peptide was loaded on DEXAFP, suggesting that HMGN1 functional peptide can potentiate the immunogenicity of DEXAFP. Therefore, in this proposal, we aim to explore the feasibility and potential of the long-lasting anti-tumor immune response mediated by HMGN1 functional peptide modified DEXAFP in autochthonous hepatocellular carcinoma, which was equivalent to the highly complex and heterogeneous pathologies and immune microenvironment observed in HCC patients. Further investigation on the underlying anti-tumor mechanism will shed light on the role of HMGN1 in promoting DEXAFP immunogenicity. In summary, we wish to provide a novel and effective immunotherapeutic strategy and approach for HCC treatment.

肝癌严重危害人类健康，但目前临床缺乏有效治疗手段。近年来，一种细胞分泌的纳米囊泡结构exosome，作为新型非细胞疫苗在肿瘤免疫治疗中取得可喜进展。本课题组前期工作证明：负载肝癌特异性抗原（AFP）的树突状细胞来源exosome（DEXAFP）可介导肝癌有效抑瘤作用，但如何进一步提高其免疫原性及持久免疫治疗效果，对于免疫微环境复杂的肝癌病人治疗极为重要。前期初步研究中，发现新型免疫佐剂-预警素HMGN1，可显著提高DEXAFP免疫原性并能对肝癌产生更为持久免疫抑制效果，说明预警素可以增强DEXAFP的免疫效能。因此，本项目拟在复杂性与异质性与病人极为相似的原发性肝癌模型上，对HMGN1功能短肽修饰的DEXAFP的抗肝癌免疫应答能力和肿瘤微环境的改善作用及应用潜力展开深入、系统的研究，并详细阐释其作用途径和机制，以期为肝癌免疫治疗提供一种新的途径和策略。

肝癌是死亡率仅次于肺癌的第二大恶性肿瘤，但目前临床缺乏有效治疗手段。尽管以DC为基础的肝癌免疫治疗具有一定效果，但肝癌病人免疫功能处于抑制状态，癌组织中浸润DC的功能受抑。DC 来源的exosomes (DEXs)，具有其独特的优势，可携带大量DC 细胞来源的功能蛋白分子及免疫协同刺激因子，可取代DC 疫苗直接或间接激活免疫应答。前期实验已证明负载AFP的树突状细胞来源exosome（DEXAFP）可介导一定抑瘤作用，但未能显著改善肿瘤微环境尤其是肿瘤组织中的DC细胞和肿瘤特异性T细胞的活化和浸润，因而缺乏对肿瘤抗原（尤其是新生抗原）的有效提呈和识别。本项目通过exosome锚定肽CP05将新型预警素HMGN1功能短肽高效修饰在DEXAFP表面，获得新型生物纳米疫苗DEXAFP-N1ND，并在小鼠肝癌皮下瘤模型上初步测试结果显示，相对于DEXAFP治疗组可明显抑制肿瘤生长，为后续研究其介导抗肝癌免疫应答能力及体内作用途径和机制的阐释奠定了实验基础。随后在肝癌原位大瘤模型中系统测试其抑瘤效果有限，且外周血及淋巴器官中DC和T细胞活化效果不佳。因而进一步将肝癌肿瘤特异靶向肽P47高效修饰在该疫苗表面，获得多功能修饰的DEX疫苗（DEXP&A&N）,可显著提高肿瘤靶向效率10倍以上。除此之外，该疫苗可在肝癌原位大瘤模型中显著抑制肿瘤生长，且明显增加肿瘤特异性T细胞的浸润，主要通过招募和活化具有较强抗原交叉提呈功能的DC亚群，进而激活肿瘤特异性免疫应答。进一步阐释了DEXP&A&N疫苗体内作用途径和介导免疫应答的作用机制，以及系统评估其长期介导抗肿瘤免疫应答效果。综上，针对肝癌特殊的免疫抑制微环境，进一步提高DEXAFP的免疫原性和肿瘤组织靶向性，有效改善了中晚期肿瘤免疫微环境及抑瘤效果，详细阐释其作用途径和机制，以期为肝癌免疫治疗提供一种新的途径和策略。