Intrapleural immunotherapeutic nanoparticles for MPE treatment

摘要 恶性胸腔积液（MPE）继发于非小细胞肺癌（NSCLC）， 临床患者管理的挑战。MPE通常提示晚期恶性肿瘤和预后 MPE的预后极差，中位生存期为4-9个月。MPE的存在通常会导致 手术干预，许多MPE患者因化疗效果极差而不适合化疗 条件目前MPE的标准治疗主要是姑息性的。重要的临床证据表明 MPE的肿瘤免疫微环境（TIME）具有深刻的免疫抑制作用， 促进免疫细胞的表型，这对抗肿瘤免疫产生负面影响。的先前尝试 改善涉及免疫治疗剂胸膜内给药的时间已经导致了一定程度的 功效最近出现的免疫治疗与免疫检查点阻断（ICB）引起了新的 有兴趣寻求一种有效减轻免疫冷MPE的策略，以增强ICB免疫治疗。 干扰素基因刺激因子（STING）通路最近已被确定在干扰素基因表达中发挥重要作用。 诱导抗肿瘤免疫。作为一种有效的STING激动剂，cGAMP在胞质溶胶中连接STING以激活I型 干扰素（IFN）的产生。然而，cGAMP的固有性质使其易于被微生物降解。 磷酸二酯酶，存在于许多组织中，并且在恶性肿瘤中鉴定出更高水平的酶。 积液此外，先前的研究表明，肿瘤驻留抗原呈递细胞内STING的激活， 细胞（APC）是诱导肿瘤特异性CD 8 +T细胞免疫所必需的。我们最近开发了一种 用于cGAMP（LNP-STING）的APC靶向递送的新型纳米技术策略。我们召集利比里亚国家警察- 在脂质体外层用磷脂酰丝氨酸进行STING，以促进其识别和摄取， 通过APC，并负载与磷酸钙复合的cGAMP，以提高负载效率和细胞增殖。 cGAMP释放到胞质溶胶，在那里它结合STING。在这个项目中，我们建议建立一个最佳的LNP- 用于胸膜内施用的STING，并测试胸膜内LNP-STING是否将免疫冷转化为 促炎性热MPE。我们的中心假设是胸膜内LNP-STING能够减轻 免疫抑制MPE，从而为对抗PD-L1 ICB的有利应答奠定基础。我们将测试 在MPE小鼠模型和NSCLC患者的MPE样品中的联合免疫疗法。我们建议 建立用于胸膜内APC靶向递送STING激动剂（Aim 1）最佳LNP-STING。然后我们将 确定胸膜内LNP-STING是否有效减轻免疫冷MPE（Aim 2）。我们将最后确定， 胸膜内LNP-STING增强ICB免疫疗法的功效（Aim 3）。更重要的是， 深入了解胸膜内LNP-STING联合抗PD-L1 ICB的生物学机制， 可能同时参与先天性和适应性抗癌免疫。这项工作是重要的，因为它可能有一个 有可能对MPE免疫疗法产生影响。

Abstract Malignant pleural effusion (MPE) secondary to non-small cell lung cancer (NSCLC) represents a significant challenge in clinical patient management. MPE is commonly indicative of late stage malignancy and prognosis of MPE is extremely poor, with a median survival between 4-9 months. The presence of MPE often precludes surgical intervention, and many patients with MPE are not fit for chemotherapy due to the extremely poor condition. Current standard of care treatment for MPE is largely palliative. Significant clinical evidence suggests that the tumor immune microenvironment (TIME) of MPE is profoundly immunosuppressive with abundant tumor- promoting phenotype of immune cells, which impacts negatively on antitumor immunity. Previous attempts to improve the TIME involving intrapleural administration of immunotherapeutics have led to some degree of efficacy. The recent advent of immunotherapy with immune checkpoint blockade (ICB) has aroused renewed interest in seeking an effective strategy to mitigate the immune cold MPE to enhance the ICB immunotherapy. Stimulators of interferon genes (STING) pathway has recently been identified to play an important role on induction of antitumor immunity. As a potent STING agonist, cGAMP ligates STING in cytosol to activate type I interferons (IFNs) production. However, intrinsic property of cGAMP makes it susceptible to degradation by a phosphodiesterase that exists in many tissues, and higher levels of the enzyme are identified in malignant effusions. Moreover, previous studies indicate that activation of STING within tumor–resident antigen-presenting cells (APCs) is necessary for induction of tumor-specific CD8+T cell immunity. We have recently developed a novel nanotechonological strategy for APC-targeted delivery of cGAMP (LNP-STING). We assemble LNP- STING with phosphatidylserine on the outer layer of liposome to facilitate its recognition and uptake preferably by APCs, and load cGAMP complexed with calcium phosphate to enhance both the loading efficiency and the release of cGAMP to cytosol, where it binds to STING. In this project, we propose to establish an optimal LNP- STING for intrapleural administration and test if intrapleural LNP-STING converts the immune cold into proinflammatory hot MPE. Our central hypothesis is that intrapleural LNP-STING enables to mitigate the immunosuppressive MPE, thereby setting the stage for favorable response to anti-PD-L1 ICB. We will test the combination immunotherapy in both MPE mouse models and NSCLC patients’ MPE samples. We propose to establish an optimal LNP-STING for intrapleural APC-targeted delivery of STING agonist (Aim1). We will then determine if intrapleural LNP-STING effectively mitigate the immune cold MPE (Aim2). We will last determine if intrapleural LNP-STING enhances efficacy of the ICB immunotherapy (Aim3). More importantly, we will gain insights into the biological mechanisms of intrapleural LNP-STING in combination with anti-PD-L1 ICB, which likely engage both the innate and adaptive anticancer immunity. This work is significant because it may have a potential to make an impact on the MPE immunotherapy.