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治疗显示出有希望的 治疗结果，其中一些疗法被用作一线治疗患者， 转移性非小细胞肺癌。然而，并非所有患者都能从ICB中获益，一些患者最初对ICB有反应， 有时会产生后天抵抗力。多系统免疫相关不良事件。因此， 重要的是要准确地识别和预测肺癌患者谁将响应ICB之前和期间， 疗程在这里，我们提出了一种创新的筛查策略，旨在早期预测和实时 监测肺癌患者对ICB的应答者和非应答者。该方法基于 与抗癌免疫特异性相关循环外泌体的分离、检测和表征 而不是分析外泌体的总池。外泌体是分泌的纳米大小的颗粒， 对起源细胞特异的核酸、蛋白质和脂质货物，其被认为是亲本细胞的镜像。 细胞此外，它们可以很容易地从生物流体中提取，作为疾病状态的生物标志物的来源， 治疗反应。在此，我们寻求具体分析两个循环外泌体群体，包括 肿瘤源性外泌体（TEX）和PD-L1+外泌体，它们是肿瘤状态的宝贵决定因素。 肿瘤负荷和抗癌免疫活性。我们制定了两个具体目标， 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器， 用于定量分析TEX和PD-L1+外泌体的平台，随后进行TEX的分子分析， 探索与治疗结果相关的分子特征。为此，我们将通过基因工程 通过将生物发光蛋白与肿瘤特异性和PD-L1特异性 靶向分子。目的2将评估来自NSCLC患者血清的循环外泌体 用ICB治疗。目标1中开发的平台将用于定量分析纺织品， 治疗期间的PD-L1+外泌体，以及观察到的变化是否可用作预测 用于早期识别和监测ICB应答者和非应答者的标志物。流通的TEX 随后将被分离;它们的外泌体转录物和长非编码RNA的特征在于： 使用下一代测序技术探索与治疗结果相关的分子特征。我们 将应用计算方法进行数据整合，分析和解释的基础上， TEX、外泌体PD-L1的表达水平、TEX的分子特征和患者的临床特征。总之， 我们的项目是高度转化的，可能对肺癌患者产生重大影响。如果成功，这 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， 为ICB治疗无效的患者开发潜在的新疗法。

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.