Noninvasive monitoring of therapeutic response to immune checkpoint inhibitors using circulating exosomes in non-small cell lung cancer

摘要/摘要：免疫检查点阻断(ICB)疗法的出现彻底改变了 治疗晚期癌症，包括肺癌。特别是，抗PD-1/PD-L1治疗显示出良好的前景 治疗结果，其中一些疗法被用作慢性阻塞性肺疾病患者的一线治疗 转移性非小细胞肺癌。然而，并不是所有患者都从ICB中受益，一些最初对ICB有反应的患者也是如此 产生后天抵抗力，有时。多系统免疫相关不良事件。因此，它是至关重要的 准确识别和预测肺癌患者在治疗前和治疗期间对ICB的反应很重要 疗程。在此，我们提出了一种针对早期预测和实时的创新筛选策略 肺癌患者ICB应答者和无应答者的监测。此方法基于 抗癌免疫相关循环外切体的分离、检测和鉴定 回应，而不是分析外切体的总库。Exosome是分泌的纳米大小的颗粒，含有 起源细胞特有的核酸、蛋白质和脂类物质，它们被认为是亲本细胞的镜子。 细胞。此外，它们可以很容易地从生物体液中提取出来，作为疾病状态和 治疗反应。在这里，我们试图具体分析两个循环外切体群体，包括 肿瘤来源的外切体(TES)和PD-L1外切体是该状态的宝贵决定因素 肿瘤负担和抗癌免疫活性。我们制定了两个具体目标，这两个目标是 关于开发技术以实现我们的目标：AIM1将开发一种高度敏感和选择性的传感 TES和PD-L1外切体定量分析平台及TES的分子分析 探索与治疗结果相关的分子签名。为此，我们将对小说进行基因工程 生物发光蛋白与肿瘤特异性和PD-L1特异性融合的生物发光探针 分别以分子为目标。目的2评估非小细胞肺癌患者血清中的循环外切体 采用ICB治疗。在AIM 1中开发的平台将用于TES和 PD-L1外切体在治疗过程中的变化，以及观察到的变化是否可以作为预测 用于早期识别和监测ICB应答者和非应答者的标记。流通中的纺织品 随后将被分离；它们的外体转录本和长的非编码RNA将被 使用下一代测序来探索与治疗结果相关的分子签名。我们 将基于以下数量应用计算方法进行数据集成、分析和解释 TES、胞外体PD-L1的表达水平、TES的分子特征以及患者的临床特征。总而言之， 我们的项目具有很高的翻译性，可能会对肺癌患者产生重大影响。如果成功，这将是 研究将帮助医生准确预测和识别将从ICB治疗中受益的患者，以及 为对ICB治疗无效的患者开发潜在的新治疗方法。

Summary/ Abstract: The emergence of immune checkpoint blockade (ICB) therapy has revolutionized the treatment for advanced cancers including lung cancer. Particularly, anti-PD-1/PD-L1 therapy shows promising therapeutic outcomes, and some of these therapies are employed as the first line treatment of patients with metastatic NSCLC. However, not all patients benefit from ICB and some of patients who initially respond to ICB develop acquired resistance and sometimes. multisystem immune-related adverse events. Thus, it is critically important to accurately identify and predict lung cancer patients who will respond to ICB before and during a course of treatment. Here, we propose an innovative screening strategy aiming at early prediction and real-time monitoring of responders and non-responders to ICB for lung cancer patients. This approach is based on the isolation, detection, and characterization of circulating exosomes specifically associated with anticancer immune response rather than analyzing total pool of exosomes. Exosomes are secreted nano-sized particles containing nucleic acid, protein, and lipid cargo specific to the cell of origin, which are considered as a mirror of the parental cells. Besides, they can be easily extracted from biofluids as a source of biomarkers of disease status and treatment response. Herein, we seek to specifically analyze two population of circulating exosomes including tumor-derived exosomes (TEXs) and PD-L1+ exosomes that serve as the invaluable determinants of the status of tumor burden and anticancer-immune activity, respectively. We have formulated two Specific Aims that hinge on developing technologies to achieve our goals: Aim1 will develop a highly sensitive and selective sensing platform for quantitative analysis of TEXs and PD-L1+ exosomes followed by molecular analysis of TEXs to explore the molecular signatures associated with treatment outcome. For that, we will genetically engineer novel bioluminescence-based probes by fusing bioluminescence proteins with tumor-specific and PD-L1-specific targeting molecules, respectively. Aim 2 will evaluate circulating exosomes from serum of NSCLC patients treated with ICB therapy. The platform developed in Aim 1 will be used for the quantitative analysis of TEXs and PD-L1+ exosomes during the treatment, and whether the observed changes can be utilized as a predictive marker for early identification and monitoring of responders and non-responders to ICB. The circulating TEXs will be subsequently isolated; their exosomal transcripts, and long non-coding RNAs will be characterized by using next-generation sequencing to explore the molecular signatures associated with therapeutic outcome. We will apply computational approaches for data integration, analysis, and interpretation based on the number of TEXs, expression level of exosomal PD-L1, molecular signatures of TEXs, and patient’s clinical features. In sum, our project is highly translational and could have a significant impact on lung cancer patients. If successful, this research will help physicians to accurately predict and identify patients who will benefit from ICB therapy, and develop potential novel treatments for patients who are non-responders to ICB therapy.