Single Vesicle Phenotyping of Circulating Exosomes Carrying Immunomodulatory Markers for Therapy Selection and Monitoring.

摘要 在过去的十年中，免疫疗法已经招募了患者自己的免疫系统来对抗免疫系统。 肿瘤特别是，成功阻断程序性细胞死亡-1（PD-1）及其受体之间的相互作用， 配体PD-L1为开发新的治疗方法铺平了道路， 癌的在癌症如非小细胞肺癌（NSCLC）中，PD-L1的上调，无论是在肿瘤细胞中还是在肿瘤细胞中， 在巨噬细胞中，降低宿主的免疫反应，从而增强肿瘤的侵袭性。而 PD-1/PD-L1检查点阻断免疫疗法已成为许多癌症患者的真实的“游戏规则改变者”， 反应率仍然相对较低，从15%到40%不等，这取决于癌症的类型和阶段。 这种疾病 目前对患者是否应接受PD-1/PD-L1阻断治疗的评估是基于 PD-L1的表达水平和肿瘤浸润免疫细胞的程度，使用免疫组织化学（IHC）。 这种方法有明显的缺点：1）样本是通过手术获得的，会带来额外的风险， 作为原发性肿瘤穿孔的结果，潜在地触发增强的转移;以及2）这些措施是 在预测缓解疗效方面不够敏感或特异，容易产生假阳性 结果此外，IHC缺乏明确的定量定义，即定义PD-L1阳性的阈值 肿瘤研究表明，尽管有一些患者接受了检查点阻断免疫疗法， 使用IHC检测PD-L1表达呈阳性，相反，大量患者反应良好 尽管在基于IHC的测试中对PD-1/PD-L1检测呈阴性。使患者免于无效的治疗， 限制那些暴露于潜在的自身免疫副作用的人数，寻找一个可靠的预测 允许患者选择将从抗PD-1/PD-L1治疗中获益的生物标志物已成为 比以往任何时候都更加紧迫。 在本提案中，我们将使用我们的ExoView技术创建一种能够识别和量化循环的检测方法， 携带PD-L1的外泌体。外泌体已被证明在许多病理学中起关键作用， 最近在抗肿瘤免疫方面。此外，外泌体的囊泡性质允许鉴定蛋白质 这些特征又可以揭示关于亲本细胞类型的有价值的信息。目标1将建立一个 本发明提供了一种抗体，其能够识别含有PD-L1的外泌体，并验证这种抗体的特异性和灵敏度。 人血浆中的抗体。目的2将定量源自免疫细胞的PD-L1阳性外泌体 vs.肿瘤细胞使用各种细胞标志物将能够鉴定表达PD-L1的外泌体的来源。 目标3将重点关注NSCLC血浆中免疫细胞与肿瘤细胞囊泡中PD-L1的定量 样本，并与PD-L1 IHC评分和抗PD-1/PD-L1治疗结果相关。

ABSTRACT Over the past decade, immunotherapy has recruited the patients’ own immune system in the fight against the tumor. In particular, the successful blocking of the interaction between programmed cell death-1 (PD-1) and its ligand PD-L1 has paved the way for the development of new treatments for several of the most devastating cancers. In cancers such as non-small cell lung cancer (NSCLC), upregulation of PD-L1, both in the tumor cells and in macrophages, reduces the host’s immune response, thereby enhancing tumor aggressiveness. While PD-1/PD-L1 checkpoint-blockade immunotherapy has become a real ‘game-changer’ for many cancer patients, response rates remain relatively low, ranging from 15 to 40%, depending on the type of cancer and the stage of the disease. The current assessment of whether a patient should be treated with PD-1/PD-L1 blockade therapy is based on expression levels of PD-L1 and degree of tumor-infiltrating immune cells using immunohistochemistry (IHC). This approach has significant downsides: 1) samples are surgically obtained, posing additional risk, and potentially triggering enhanced metastasis as a result of primary tumor perforation; and 2) these measures are rarely sensitive or specific enough in predicting response efficacy, being prone to generating false-positive results. In addition, IHC lacks a clean and quantitative definition of what threshold defines a PD-L1 positive tumor. It was shown that subsets of patients do not benefit from checkpoint-blockade immunotherapy despite testing positive for PD-L1 expression using IHC, and conversely, a significant number of patients responded well to it, despite testing negative for PD-1/PD-L1 in IHC-based tests. To spare patients from ineffective therapy and limit the number of those exposed to potential autoimmune side effects, the search for a reliable predictive biomarker allowing patient selection of those who would benefit from anti-PD-1/PD-L1 therapy has become more urgent than ever. In this proposal, we will use our ExoView technology to create an assay able to identify and quantify circulating exosomes that carry PD-L1. Exosomes have been shown to play a critical role in many pathologies and more recently in anti-tumor immunity. Furthermore, the vesicular nature of exosomes allows the identification of protein signatures which in turn may reveal valuable information about the parent cell type. Aim 1 will establish an antibody able to immobilize exosomes containing PD-L1 and validate the specificity and sensitivity of such antibodies in human plasma. Aim 2 will quantify PD-L1 positive exosomes originating from immune cells vs. tumor cells. Using various cellular markers will enable identifying the origin of PD-L1-expressing exosomes. Aim 3 will focus on Quantification of PD-L1 in vesicles from immune vs tumor cells in NSCLC plasma samples and correlate to PD-L1 IHC score and outcome of anti-PD-1/PD-L1 therapy.