Intrapleural immunotherapeutic nanoparticles for MPE treatment

摘要 继发于非小细胞肺癌(NSCLC)的恶性胸腔积液(MPE)是一种重要的 临床病人管理中的挑战。MPE通常是晚期恶性肿瘤和预后的指标。 MPE的发生率极低，中位生存期在4-9个月之间。MPE的存在通常排除了 手术干预，许多MPE患者由于极度贫困而不适合化疗 条件。目前MPE的护理治疗标准主要是姑息治疗。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，并伴有丰富的肿瘤-- 促进免疫细胞表型，对抗肿瘤免疫产生负面影响。之前的尝试是 改善胸膜腔内注射免疫疗法的时间在一定程度上导致了 功效。最近出现的免疫检查点阻断(ICB)免疫疗法重新引起了人们的注意 有兴趣寻求一种有效的策略来缓解免疫感冒MPE，以加强ICB的免疫治疗。 干扰素基因刺激物(STING)途径最近被发现在多种疾病中起重要作用 诱导抗肿瘤免疫。作为一种有效的刺激剂，cGAMP结合胞浆中的刺激物来激活I型 干扰素(IFN)的产生。然而，cGAMP的固有性质使其容易被 存在于许多组织中的磷酸二酯酶，在恶性肿瘤中发现该酶的水平较高。 积液。此外，以前的研究表明，在肿瘤驻留的抗原呈递中，STIN的激活 细胞(APC)是诱导肿瘤特异性CD8+T细胞免疫的必要条件。我们最近开发了一种 APC靶向递送cGAMP(LNP-STING)的新纳米技术策略。我们组装LNP- 在脂质体外层用磷脂酰丝氨酸刺痛，以促进其识别和摄取，最好是 通过APC，并负载cGAMP与磷酸钙复合，提高了负载效率和 CGAMP释放到胞浆，在那里它与刺痛结合。在这个项目中，我们建议建立一个最优的LNP- 胸腔内给药和测试LNP-STING是否能将免疫感冒转化为 促炎热MPE。我们的中心假设是胸膜腔内注射LNP能够缓解 免疫抑制MPE，从而为抗PD-L1 ICB的良好反应奠定了基础。我们将测试 在MPE小鼠模型和NSCLC患者的MPE样本中进行联合免疫治疗。我们建议 建立胸膜腔内APC靶向递送刺激剂(Aim1)的最佳LNP-STING。到时候我们会的 确定胸腔内注射LNP是否有效缓解免疫性寒战MPE(AIM2)。我们将最后确定是否 胸腔内注射LNP-STING可提高ICB免疫疗法(Aim3)的疗效。更重要的是，我们将受益于 胸膜腔内注射LNP联合抗PD-L1 ICB的生物学机制 可能同时参与了先天和获得性抗癌免疫。这项工作意义重大，因为它可能具有 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.