一种新型囊泡（exosome）介导HER2/neu靶向的乳腺癌T细胞瘤苗

One of the major obstacles in trastuzumab targeted therapy (passive immunotherapy) for human epeidermal growth factor receptor (HER2)-overexpressing breast cancer is the development of trastuzumab acquired resistance，warranting the search for other therapeutic strategies such as anti-HER2 cancer vaccine-based active immunotherapy. However, tumor immune tolerance and immunosuppressive microenvironment is currently an important challenge for cancer vaccine. Antigen-loaded mature dentritic cell (DC)-derived exosome (EXO)-targeted T cell-based vaccine (T-EXO) can efficiently stimulate antigen-specific CD8+ cytotoxic T lymphocyte (CTL) responses and long-term CTL memory via exosomal peptide/major histocompatibility complex (pMHC)-I，CD80 and endogenous IL-2 and CD40L signalling, suggesting a novel and promising cancer vaccine capable of potentially breaking self-tolerance to tumor-associated antigens (TAAs). Therefore, in this project, we will firstly culture bone marrow-derived DC (BM-DC) from HLA-A2.1 transgenic and FVB/NJ mice and prepare HER2/neu-specfic T-EXOHER2 and T-EXOneu anti-HER2/neu vaccine using adenoviral vectors (AdVHER2 expressing HER2 and AdVneu expressing neu)-infected BM-DC (DCHER2 and DCneu)-secreted EXOs (EXOHER2 and EXOneu). Then we assess the stimulatory effect of T-EXOHER2 on in vivo HER2-specfic CD8+ CTL responses by tetramer assay and CTL-mediated preventive and therapeutic antitumor immunity using HLA-A2.1/HER2 double gene-transfected B16HLA-A2.1/HER2 mouse melanoma cells in HER2/HLA-A2.1 double transgenic mice expressing human HER2 and human MHC class I molecule HLA-A2.1 with HLA-A2.1-restricted self-tolerance to human HER2; and also assess the stimulatory effect of in vivo T-EXOneu on neu-specfic CD8+ CTL responses by tetramer assay and CTL-mediated preventive and therapeutic antitumor immunity using neu-expressing Tg1-1 breast cancer cells in FVBneuN transgenic mice expressing rat neu with self-tolerance to rat neu and spontaneous development of mammary tumors. Lastly, we in vitro amplify HER2/neu-specific CD8+ CTLs (CTLHER2 and CTLneu) and investigate their killing activity to B16HLA-A2.1/HER2 and Tg1-1 tumor target cells in vitro, respectively. More importantly, we further evaluate the therapeutic effect of CTLHER2 on trastuzumab-resistant BT474HLA-A2.1 as well as trastuzumab-sensitive MCF-7 human breast cancer cells in vitro and in vivo in 7-day BT474HLA-A2.1 and MCF-7 xenograft tumors implanted in mammary fat pads of BALB/c nude mice. This project will provide a new therapeutic alternative for women with HER2-postive breast cancer, especially for trastuzumab-resistant breast cancer patients, and lay the experimental foundation for the application of novel exosome-targeted T cell-based vaccine in cancer immunotherapy.

获得性抗性是曲妥株单抗治疗HER2+乳腺癌的主要障碍，因此值得寻找其他治疗策略如抗HER2瘤苗。然而，肿瘤免疫耐受是当今瘤苗免疫治疗的重要挑战。成熟树突状细胞囊泡靶向的T细胞疫苗（T-EXO）能够有效刺激CD8+ CTL和免疫记忆，提示其是一种有潜力打破肿瘤自身耐受的新型瘤苗。本课题利用人HER2和HLA-A2.1双转基因小鼠HER2/HLA-A2.1和大鼠neu转基因小鼠FVBneuN，研究T-EXOHER2/neu诱导HER2/neu特异性CTL反应及保护性抗肿瘤免疫效能；并体外扩增HLA-A2.1和H-2Kq限制性的HER2/neu特异性CTLs，研究其对肿瘤靶细胞的体外直接杀伤效应，和对曲妥株单抗敏感和抗性的人乳腺癌裸鼠原位移植瘤的体内治疗功效。该课题将为HER2+尤其是曲妥株单抗抗性的乳腺癌病人提供一种新的治疗选择，并且为T-EXO疫苗在肿瘤免疫治疗中的应用提供实验依据。

获得性耐药是曲妥株单抗治疗HER2+乳腺癌的主要障碍，因此需要寻找其他如抗HER2瘤苗治疗策略。成熟树突状细胞（DC）来源囊泡（EXO）靶向的T细胞疫苗（Texo）能有效刺激CD8+ CTL 和免疫记忆，提示Texo是一种有潜力打破肿瘤自身免疫耐受的新型瘤苗。本项目通过表达大鼠neu、人HER2的腺病毒AdVneu、AdVHER2感染DC分离上清EXOneu、EXOHER2，并与CD4+ T细胞共孵育，制备了neu、HER2囊泡靶向的CD4+ T细胞瘤苗neu-Texo、HER2-Texo，在neu转基因FVBneuN（H-2Kq）小鼠、HLA-A2/HER2（HLA-A2）双转基因小鼠中研究了免疫刺激效应和抗肿瘤免疫能力。发现，neu-Texo能在neu自身免疫耐受FVBneuN小鼠中有效刺激neu特异性CD8+ CTL及诱导产生对neu+小鼠乳腺癌细胞Tg1-1的完全保护性免疫。HER2-Texo也能在HER2自身免疫耐受HLA-A2/HER2小鼠中刺激HLA-A2限制性、HER2特异性CD8+ CTL及诱导产生对HLA-A2+HER2+ BL6-10A2/HER2黑色素瘤的部分保护性免疫。为增强HER2-Texo免疫刺激和抗肿瘤免疫的效应，又构建了表达人HER21-407aa和大鼠neu408-690aa融合蛋白（HuRt）的腺病毒AdVHuRt，并制备了HuRt特异性Texo（HuRt-Texo）瘤苗。证实，HuRt-Texo较HER2-Texo在BALB/c（H-2Kd）小鼠中刺激了更强的CD4+ T、HER2特异性CD8+ CTL反应及HER2特异性抗体；HuRt-Texo能完全抑制已建立的HER2+ 4T1HER2小鼠乳腺癌肺转移的生长。在HLA-A2/HER2小鼠中，HuRt-Texo也能诱导产生对HLA-A2+HER2+ BL6-10A2/HER2黑色素瘤的完全保护性免疫。经体外扩增的HER2特异性CD8+ CTLs体外、裸鼠体内对HLA-A2+HER2+ BT474A2人乳腺癌细胞呈明显的HLA-A2限制性、HER2特异性的杀伤活性，能根除已建立的3-4mm大小的BT474A2乳腺癌移植瘤的生长。因此，新型的HER2特异性T细胞瘤苗HER2-Texo、HuRt-Texo可能为曲妥株单抗耐药的HER2+乳腺癌病人提供一种新的治疗选择。