恶性肿瘤目前仍然是造成人类死亡的主要原因之一，传统肿瘤治疗手段包括手术切除、化疗、放疗等，肿瘤免疫疗法的出现为肿瘤的治疗带来了一场革新。然而，目前的肿瘤免疫疗法仍存在诸多问题，需要对原有的肿瘤免疫疗法进行改进或与其它肿瘤治疗手段联合使用，进一步提高肿瘤免疫治疗的临床疗效和促进其广泛应用。因此，本课题提出构建环境响应型疫苗/药物微纳米递送载体用于肿瘤免疫治疗研究，增强疫苗/药物递送系统的抗肿瘤免疫效应，从而最大限度地利用机体自身的免疫防御能力抑制肿瘤生长。

       第一部分：设计构建pH响应型有机-无机杂化微颗粒肿瘤疫苗用于小鼠荷瘤模型的抗肿瘤免疫治疗研究：基于模型抗原卵清白蛋白（Ovalbumin, OVA）和硫酸铜（II）的有机-无机杂化颗粒疫苗通过自然结晶的方式形成。体外研究结果表明，该蛋白-无机杂化微结构可提高肿瘤疫苗的室温稳定性，有利于疫苗的贮存和运输。该微颗粒疫苗冻干粉在室温下放置一个月后，同-20℃放置的微颗粒疫苗冻干粉相比，具有相同的促进树突状细胞（Dendritic cells，DCs）成熟和活化作用。此外，该特殊结构的疫苗颗粒具有pH响应性，可以实现抗原的胞浆递送，促进MHC-Ⅰ抗原提呈。E.G7-OVA荷瘤小鼠抗肿瘤研究结果表明，该微颗粒疫苗可以有效抑制肿瘤生长，提高淋巴结内CD8⁺ T淋巴细胞的分化以及脾脏内CD8⁺ T淋巴细胞和抗原特异性T淋巴细胞的比例，并能显著提高荷瘤小鼠血清中细胞因子和抗体的分泌水平。

       第二部分：设计并构建了基质金属蛋白酶（Matrix metalloproteinase, MMP）响应型聚乙二醇/^DPPA多肽-阿霉素共负载自组装纳米药物（PEG/^DPPA-MMP-DOX NPs），用于化疗和PD-L1免疫检查点阻断的联合抗肿瘤研究，该纳米药物在肿瘤微环境MMP作用下，可从起始140 nm的大颗粒解组装释放小粒径DOX纳米粒（<30 nm），有效增强DOX纳米粒的肿瘤渗透性。在MMP作用下，释放的DOX纳米粒可以对肿瘤细胞进行杀伤并诱导其发生免疫原性死亡，引发抗肿瘤免疫应答；同时，经MMP响应性连接肽断裂而释放的PD-L1抗体拮抗剂^DPPA多肽，可与肿瘤细胞表面的PD-L1受体结合，阻断PD-1/PD-L1免疫检查点抑制通路，进而实现化疗联合免疫检查点阻断的协同抗肿瘤作用。荷瘤小鼠体内抗肿瘤实验结果表明，该纳米药物可以有效抑制肿瘤生长并降低DOX对小鼠的毒副作用，增强CD8⁺ T细胞在肿瘤组织内的比例，并降低调节性T细胞（Regulatory T cells, Tregs）的免疫抑制作用，促进细胞因子干扰素-γ（Interferon, IFN-γ）的产生。此外，该纳米药物介导的化疗联合免疫检查点阻断的抗肿瘤疗法可诱导长期记忆性抗肿瘤免疫          反应，有效抑制肿瘤的复发和转移。

综上所述，我们构建了两种环境响应型微纳米疫苗/药物递送系统，用于增强肿瘤免疫治疗疗效。一种是在原有肿瘤免疫疗法基础上改进，制备了pH响应型有机-无机杂化微颗粒肿瘤疫苗，该体系可以提高疫苗的室温稳定性并实现抗原的胞浆递送，促进MHC-Ⅰ抗原提呈从而增强CD8⁺ T淋巴细胞杀伤作用提高抗肿瘤疗效；另一种则是将肿瘤免疫疗法与化疗联合，制备了MMP响应型PEG/^DPPA-MMP- DOX NPs，该纳米药物实现了化疗杀伤与免疫检查点阻断的协同抗肿瘤作用，在增强纳米药物在肿瘤组织中的渗透能力的同时，进一步增强抗肿瘤疗效。

  Cancer is still one of the main causes of death. Traditional tumor treatment approaches include surgery, chemotherapy and radiotherapy. Cancer immunotherapies have demonstrated promising clinical responses in recent years and provide a new strategy against cancer. However, there are still many challenging concerns that remain to be addressed with current cancer immunotherapy and it is necessary to further improve the efficacy of cancer immunotherapy. In this study, we developed two novel formulations of tumor microenvironment responsive particles as drug delivery system for cancer immunotherapy, which may have a great potential for the more effective therapy of cancers.

  Part I: Here, a pH-responsive antigen-inorganic hybrid micro particle as a novel vaccine carrier was developed. Model antigen ovalbumin (OVA)-copper (II) sulfate hybrid vaccines (OVA-Cu-HVs) were mildly and facilely constructed through a biomimetic mineralization process. OVA-Cu-HVs can release OVA in a pH-responsive behavior and promote cytosolic release of antigen to enhance antigen cross-presentation. Immunization with OVA-Cu-HVs promoted the maturation of dendritic cells in draining lymph nodes, induced robust antigen-specific T lymphocyte response, and inhibited tumor growth in vivo. In addition, OVA-Cu-HVs were efficacious after being stored for 4 weeks at room temperature and are expected to simplify vaccine storage and lower the cost of cold storage for transportation.

  Part II: Here, a tumor microenvironment-responsive therapeutic peptide-conjugated prodrug nanoparticle was developed for enhanced tumor penetration and synergistic antitumor effects of chemotherapy and immune checkpoint blockade therapy. The prodrug nanoparticle (~140 nm) is composed of a short D-peptide antagonist of PD-L1 (^DPPA) conjugated doxorubicin (DOX) prodrug and a PEGylated DOX prodrug, which can dissociate into small DOX nanoparticles (< 30 nm) and release ^DPPA antagonist in tumor microenvironment. It is demonstrated that co-delivery of DOX and ^DPPA antagonist directly killed tumor cells, promoted the tumor-infiltrating cytotoxic T lymphocytes, reduced the tumor-infiltrating regulatory T cells and elicited a long-term immune memory effect to prevent tumor recurrence and metastasis. This tumor microenvironment-responsive prodrug nanoparticle holds promise as a co-delivery nanoplatform for the improved chemoimmunotherapy of solid tumors.

In summary, we have developed two novel formulations of tumor microenvironment responsive particles as drug delivery system for improving cancer immunotherapy. OVA-Cu-HVs can improve the room temperature stability of the vaccine and achieve the cytoplasmic delivery of the antigen, promote the MHC-Ⅰ antigen presentation to enhance the killing effect of CD8⁺ T lymphocytes and improve the anti-tumor efficacy. The tumor microenvironment-responsive PEG/^DPPA peptide-doxorubicin co-loaded nanoparticles offers a new strategy for future applications of nanomedicine to achieve deeper tumor penetration and better chemo-immune checkpoint blockade therapeutic effect.