多功能PLGA微粒式人工抗原提呈细胞对肿瘤的主动免疫治疗

Priming and amplifying antigen-specific T lymphocytes in vivo is one of the fundamental strategies for the tumor immunotherapy. Based on our previous works about latex bead-based artificial antigen-presenting cells, in this project, a novel artificial antigen-presenting cell system (aAPCs) will be developed by co-coupling MHC/peptide complexes, anti-CD28, anti-4-1BB, anti-CD2 and CD47 molecules onto the biodegradable poly (lactic-co-glycolic acid) (PLGA) microparticles and encapsulating the mixture of IL-2, IL-15, CCL21, blockade of both CTLA-4 and PD-1 inside. With the presentation of surface-bound ligands and paracrine delivery of soluble factors, the aAPCs are expected to possess multifunctional activities in vivo, such as recruiting antitumor effectors, targeting, priming and expanding tumor antigen-specific T cells by presenting antigen (signal 1) and costimulation (signal 2), releasing cytokines (signal 3) and providing adhesion molecules. Simultaneously, the aAPCs will be able to promote the expansion of specific T cells as memory T cells, block inhibitory pathways to enhance T cell proliferation and reduce the frequencies of suppressor cells. In addition, surface-bound CD47, as a marker of self, is expected to inhibit phagocytosis of PLGA particles by monocytes and macrophages to prolong their circulation time. In this study, the novel aAPCs will be administrated into B6 mice for the active immunotherapy of B16 melanoma pulmonary metastases and subcutaneous tumor. Antigen-specific T cell responses and tumor growth will be monitored. The optimal therapy regimens, precise mechanisms by which aAPCs achieve their therapeutic effects, spatial and temporal location，fate after injection, side effects and organ toxicity as well as effects on intact immune function will be elucidated. A series of modern techniques will be utilized, such as small animal optical imaging and tracking assay with double-fluorescence quantum dots, nuclear magnetic resonance, laser scanning confocal assay, electron microscopy, tissue sections and immunohistochemical staining, multicolor immunophenotyping by flow cytometry, and functional analysis for immune cells. This approach will provide initially the mechanistic insights in animal tests and pave the path to clinical trials for the novel active immunotherapy of cancers or persistent infections.

在前项国家自然基金中，我们完成了胶乳微球式人工抗原提呈细胞在体内靶向扩增肿瘤抗原特异性T细胞的观察性研究。本项目拟接着进行机制性探讨，并全面改造实验系统：用可生物降解的聚乳酸-羟基乙酸（PLGA）微粒做载体，表面负载MHC/peptide、共刺激分子、粘附分子和抗吞噬分子，内部包裹细胞因子、趋化因子和抑制性信号通路封闭剂，制备新型多功能人工抗原提呈细胞，在黑色素瘤肺转移鼠和皮下瘤鼠体内，通过表面提呈和旁分泌缓释方式募集和粘附T细胞，提供第一、二、三信号靶向活化和扩增肿瘤抗原特异性T细胞，同时封闭抑制性信号通路，扩增记忆T细胞，降低抑制性细胞浸润，并防止单核巨噬细胞对微粒的吞噬。利用活体量子点荧光成像和磁共振示踪、激光共聚焦、电镜、免疫组化、流式分析及细胞功能性检测等手段，研究该系统的更有效治疗方案、体内作用机制、时空运行、毒副作用及对整体免疫功能的影响，促使这一新免疫疗法早日走近临床研究。

在仿生微球或纳米球等载体上加载抗原和共刺激分子，制备人工抗原提呈细胞靶向结合和激活抗原特异性T细胞是实行肿瘤主动免疫疗法的新策略之一。本项目制备细胞大小的PLGA微球，在其内部包裹IL-2、IL-15、CCL21以及CTLA-4和PD-1的封闭性抗体，在其表面包被H-2Kb/TRP2180-188 二聚体、H-2Db/gp10025-33二聚体、CD28和4-1BB单抗、CD2单抗以及CD47-Fc等效应分子，制备携带11种免疫分子的多功能人工抗原提呈细胞（MaAPCs），研究其在体外和黑色素瘤鼠体内扩增抗原特异性T细胞并抑制肿瘤生长的效应、作用机制和时空分布规律等。. 主要结果如下：负载11种免疫分子的MaAPCs在体外比其它MPs具有更强大的、促进抗原特异性T细胞扩增的能力，能大幅提升抗原特异性T细胞的杀瘤细胞活性；在黑色素瘤鼠体内经尾静脉循环分布于各脏器以及脾脏和淋巴结等免疫器官，与CD8+ T细胞直接接触，同时较少被吞噬细胞所吞噬，在体内滞留36 小时；比其它MPs更能大幅提升治疗鼠体内TRP2180–188和gp10025-33特异性CD8+ T细胞的频率，增加肿瘤组织局部特异性CD8+ T细胞的浸润；大幅提高特异性CTL中活化和记忆性细胞的比例，增强其脱颗粒能力和对肿瘤细胞的特异性杀伤能力；极大地抑制了CD4+ 和CD8+调节性T细胞的增殖，抑制了特异性CTL的凋亡，提高了特异性CTL的体外增殖能力，促进炎症性细胞因子分泌，降低抑制性细胞因子的分泌。结果证实MaAPCs通过以上多种细胞机制激活机体抗肿瘤的细胞免疫反应，从而达到抑制肿瘤生长的目的，是一种高效的全新的特异性主动免疫策略。. 本研究还完成了以下工作：1、制备携带4种免疫分子的MaAPCs，验证了其抑制黑色素瘤生长的效应。2、制备球形和椭圆形并表面修饰PEG和CD47分子的纳米级aAPCs（222nm），验证了其体外抗吞噬能力和体内抗肿瘤能力。3、建立EAE小鼠模型，弄清MOG抗原特异性CD4+ 和CD8+ T细胞反应的动态变化规律。4、制备携带6种免疫分子的细胞大小的和纳米级的致耐受性人工抗原提呈细胞（TaAPCs和tNPs），验证了其治疗EAE的潜能、作用机制、时空分布和副作用等。本研究为肿瘤和自身免疫病提供了一种全新的特异性主动免疫治疗策略。