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伴随着免疫学、细胞生物学以及纳米科技的深入研究，肿瘤治疗性疫苗成为肿瘤治疗的新型治疗手段。其具有适用范围广以及副作用低的特点，因此发展潜力巨大。本论文针对肿瘤治疗性疫苗领域存在的生物安全性低、肿瘤抗原无法进行主要组织相容性复合体（major histocompatibility complex，MHC）I提呈、肿瘤微环境抑制免疫反应等关键问题，结合仿生领域最新理论突破和技术创新，分别提出和发展了“制备仿生型”抗原-碳酸钙疫苗体系和“功能仿生型”外泌体疫苗体系，均取得了理想的肿瘤治疗效果。论文具体开展的研究工作如下：1. 发展抗原诱导碳酸钙结晶的方法，制备粒径均一且具有高效抗原装载能力的抗原-碳酸钙纳米球，并对其树突状细胞（dendritic cell，DC）活化效果进行系统评价。模拟自然界的生物矿化过程，利用模型抗原鸡卵清白蛋白（Ovalbumin，OVA）对碳酸钙的结晶过程进行诱导，成功制备出粒径均一（～500 nm）、OVA装载率高达19.6%的球霰石晶型碳酸钙纳米球OVA@NP。体外研究结果表明，OVA@NP具有良好的生物安全性，可以高效递送抗原进入DC内部，充分激活DC，并实现高效的MHC I抗原提呈效果。2. 深入研究了OVA@NP诱导抗原发生MHC I交叉提呈的胞内机制，并基于小鼠肿瘤模型考察其在肿瘤治疗方面的表现。OVA@NP被DC摄取进入溶酶体后，依靠其独特的晶型结构，在弱酸性溶酶体内部发生快速酸解，释放大量二氧化碳（carbon dioxide，CO2），涨破溶酶体，促进MHC I交叉提呈。与此同时，物理性CO2气压胁迫细胞产生自噬，进一步增强MHC I交叉提呈效果。OVA@NP注射到小鼠体内后可以有效募集抗原提呈细胞（antigen presenting cell，APC）至注射部位摄取抗原，促进CD8 T细胞大量增殖并提升其在肿瘤组织的浸润，明显抑制了肿瘤的生长，显著延长了荷瘤小鼠的生存期。3. 提出一种具有免疫激活及肿瘤微环境双效调节能力的外泌体型仿生肿瘤疫苗构建新策略，成功制备该疫苗并确认了其淋巴结及肿瘤双靶向效果。通过巨噬细胞对肿瘤细胞核的内吞，实现了肿瘤抗原在巨噬细胞上的内源性表达；脂多糖（lipopolysaccharide，LPS）的协同刺激进一步激活了巨噬细胞表面共刺激分子的表达，并将巨噬细胞向具有抗肿瘤作用的M1型巨噬转化。通过差速离心法，获得了携带肿瘤抗原、MHC I分子、共刺激分子及激活型细胞因子的仿生APC杂合外泌体（nucleus-lps-exosome，nc-lps-exo）。通过荷瘤小鼠体内示踪实验，证明了nc-lps-exo良好的淋巴结和肿瘤双靶向能力。4．分别考察了nc-lps-exo在淋巴结内的免疫激活效果及在肿瘤部位的微环境改善能力。靶向到淋巴结的nc-lps-exo，一部分通过被APC摄取的方式激活APC，继而激活T淋巴细胞增殖；另一部分nc-lps-exo则结合在T细胞表面，直接激活T淋巴细胞增殖。靶向到肿瘤部位的nc-lps-exo约四分之一与肿瘤相关巨噬相互作用，促使其向M1型巨噬转化；同时nc-lps-exo降低了调节性T细胞（regulatory T cells，Treg）在肿瘤部位的浸润，提升了细胞毒性T淋巴细胞（cytotoxic T lymphocytes，CTL）的肿瘤浸润比例，有效地改善了免疫抑制性的肿瘤微环境。5. 确认了nc-lps-exo的生物安全性，并基于多种小鼠肿瘤模型，系统全面地评价了该杂合外泌体疫苗在肿瘤治疗方面的表现。体内实验结果证明，杂合外泌体疫苗的注射不会引发肿瘤产生及组织器官的损伤，具有良好的生物安全性。在抗肿瘤原位生长效果考察中发现，杂合外泌体疫苗对于E.G7淋巴瘤和Muc1-B16黑色素瘤均有良好的肿瘤抑制效果，显著延长了小鼠的生存期。杂合外泌体疫苗与程序性死亡因子-1（programmed death-1，PD-1）抗体联合使用后，对Muc1-B16黑色素瘤细胞在肺脏、心脏及肾脏的转移展现了良好的预防效果，并有效地防止了Muc1-B16黑色素瘤手术切除后原位的复发及肺转移情况。;With the progress of immunology and nanotechnology, therapeutic cancer vaccine has been developed as a new approach to inhibit tumor growth, metastasis, and recurrence. Therapeutic cancer vaccine holds great promise due to its wide applicability and low side effect. Despite these advantages, therapeutic cancer vaccine still has to face the following issues: biosecurity, tumor antigen major histocompatibility complex (MHC) I presentation, and immune-suppressive tumor microenvironment. By combining the latest biomimetic theoretical and technical breakthrough, “manufacture biomimetic” antigen-calcium carbonate vaccine and “functional biomimetic” exosome vaccine were proposed and developed.This thesis mainly included the following issues:1. OVA@calcium carbonate nanoparticles (OVA@NP) with uniform size and high antigen loading rate were prepared by developing novel antigen-induced crystallization, and the dendritic cell (DC) activation of OVA@NP was systematically evaluated in vitro. Imitating the biomineralization process, model antigen Ovalbumin (OVA) was utilized as soft template to guide the crystallization of CaCO3. After condition optimization, vaterite OVA-CaCO3 nanoparticles with uniform size (~ 500 nm) and high OVA loading rate (19.61 %) were successfully prepared. Further study in vitro revealed that OVA@NP could efficiently ferry antigen into DC, activate DC, and induce MHC I antigen cross presentation.2. The intracellular mechanism was further explored in details about how OVA@NP improved MHC I antigen cross-presentation, and the efficiency of OVA@NP in anticancer therapy was systematically investigated by employing murine tumor model. After endocytosed by DC, OVA@NP would be transported into lysosome. Depending on the unique vaterite crystallite and hierarchical structure, calcium carbonate dissolved rapidly in the acidic environment of lysosome. Accompanied by eruptible carbon dioxide (CO2) generation, the pressure in the lysosome dramatically increased, resulting in lysosome deconstruction and antigen lysosome escape, which would promote MHC I cross-presentation. Meanwhile, the mechanical stress sourced from generated CO2 induced autophagy, which would further promote MHC I cross-presentation. OVA@NP would recruit antigen presenting cell (APC) to injection site for the antigen uptake, and then enhance CD8 T cell proliferation and their infiltration in tumor tissue, and finally achieve potent anticancer effect in aspects of tumor suppression and lifetime extension.3. New strategy of constructing an exosome bionic vaccine with immunity-activation and tumor microenvironment regulation ability was presented. The vaccine was successfully prepared and its dual-targeting ability towards lymph node and tumor was confirmed. Through the uptake of tumor cell nucleus by macrophage, tumor antigen was endogenous expressed on macrophage. With the co-stimulation of lipopolysaccharide (LPS), enhanced costimulatory molecules presentation and the M1 macrophage transformation were achieved. After differential centrifugation, bionic APC hybrid exosomes (nucleus-lps-exosome, nc-lps-exo), which expressed tumor antigen, MHC I complex, costimulatory molecules and activator cytokines, were obtained. Its dual-targeting ability towards lymph node and tumor was confirmed through the in vivo tracking experiments on the tumor-bearing mice.4. The immune activation inside lymph node and the tumor microenvironment regulation of nc-lps-exo were separately investigated. Part of nc-lps-exo that inside lymph node activated APC through the uptake pathway and then promoted T cell proliferation. The other part of nc-lps-exo bound to the surface of T cell and directly induced T cell proliferation through the interaction between MHC molecules/costimulatory molecules and their corresponding ligands. Among the nc-lps-exo inside tumor tissue, there was about 1/4 nc-lps-exo that interacted with tumor-associated macrophage and promoted its transformation to M1 macrophage. In addition, nc-lps-exo greatly reduced regulatory T cells (Treg) infiltration and enhanced cytotoxic T lymphocytes (CTL) infiltration inside tumor tissue, effectively improving the immune-suppressive tumor microenvironment.5. The biosecurity of nc-lps-exo was confirmed. The efficiency of nc-lps-exo in anticancer therapy was systematically investigated by employing multiple murine tumor models. The in vivo results demonstrated that nc-lps-exo could not induce tumorigenesis or injury to visceral organs, indicating the excellent safety of the current vaccine formulation. The anticancer investigation showed that the usage of hybrid exosome achieved potent anticancer effects in aspects of in situ E.G7 lymphoma/Muc1-B16 melanoma suppression and lifetime extension. In combination with programmed death-1 (PD-1) antibody, the hybrid exosome vaccine even showed excellent performance in Muc1-B16 melanoma metastasis (lung, heart and kidney) prevention. Besides, this combination therapy effectively prevented the postoperative recurrence and the related pulmonary metastasis.
|王双. 仿生型肿瘤治疗性疫苗的构建和应用[D]. 中国科学院研究生院,2018.|
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