The roles of EBV-specific T cells in response to checkpoint blockade immunotherapy of EBV-driven nasopharyngeal carcinoma

项目总结/摘要 鼻咽癌（NPC）是一种由EB病毒（EBV）驱动的恶性肿瘤， 东南亚地方性NPC的总体5年生存率仅为51%，这代表了未满足的临床 需要的虽然已知NPC肿瘤被EBV感染并表达PD-L1，但对PD-L1在NPC肿瘤中的作用知之甚少。 EBV特异性T细胞在控制NPC和抗PD-1治疗中显示出较低的疗效率（ORR ~20%）。 本研究的目的是更好地了解EB病毒特异性T细胞反应在NPC免疫病理学中的作用 和免疫反应。我们将测试的具体假设是：1。EBV特异性T细胞有助于 通过组合抗PD-1和抗CTLA-4检查点阻断免疫疗法引起的肿瘤控制，和2. 外周EBV特异性T细胞的表型和克隆特征/动力学可用作 NPC患者的临床结果指标。为了验证这些假设，我们将利用两个 新加坡NPC患者队列：队列1。51例新诊断鼻咽癌患者队列， PBMC和存档FFPE组织可用，队列2。一个50名患者的队列参与了一项II期研究， 试验（NCT 03097939-国家癌症中心新加坡）测试伊匹鲁单抗和 Nivolumimab（Ipi.+ Nivo.）使用纵向收集的PBMC和组织活检进行免疫治疗。最近 来自该临床试验的已发表的初步分析（AACR 2020）显示，Ipi.+尼沃治疗安全 并在复发和转移性NPC患者中获得持久的反应， 之间的联系。另外，对新诊断的鼻咽癌进行了初步分析， 来自队列1的患者外周血样本显示了CD 8 + T细胞的某些表型特征之间的关联。 T细胞和临床参数，如EBV-DNA水平。因此，在目标1中。我们会调查 这些初步鉴定的NPC相关CD 8 + T细胞表型的相关性。除了深入的单 细胞转录、这些细胞的功能性TCR序列分析、细胞成像、转录分析和 患者匹配肿瘤的大量TCR测序将允许发现外周T细胞与肿瘤细胞之间的新关联。 细胞和肿瘤微环境。在目标2中，我们将描述NPC中EBV特异性T细胞反应的特征， 在组合Ipi.+期间的外周和肿瘤微环境尼沃免疫疗法治疗以鉴定新的 EBV特异性T细胞表型特征与免疫应答之间的关系。在Aim中 3.,我们将研究T细胞克隆动力学与治疗诱导的NPC特异性 通过比较外周血和肿瘤活检样品中TCR克隆多样性， 治疗的不同阶段。总体而言，NPC外周中EBV特异性T细胞表型的表征 肿瘤采用多种尖端方法不仅会提高我们对NPC的认识， 免疫景观，而且还可能鉴定临床相关的EBV特异性T细胞表型， 在未来的NPC免疫治疗试验中进行测试。

Project Summary/Abstract Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus (EBV)-driven malignancy that is endemic to East and Southeast Asia. The overall 5-year survival rate for endemic NPC is only 51%, which represents an unmet clinical need. While NPC tumors are known to be infected with EBV and express PD-L1, little is known about the role of EBV-specific T cells in the control of NPC and anti-PD-1 therapy has shown a low rate of efficacy (ORR ~20%). The goal of this study is to better understand the role of EBV-specific T cell responses in NPC immunopathology and immunotherapeutic response. We will test the specific hypotheses that: 1. EBV-specific T cells contribute to tumor control elicited by combination anti-PD-1 and anti-CTLA-4 checkpoint blockade immunotherapy, and 2. that the phenotypic and clonal characteristics/dynamics of peripheral EBV-specific T cells can be useful as indicators of clinical outcomes for NPC patients. To test these hypotheses, we will leverage our access to two Singaporean NPC patient cohorts: Cohort 1. A 51-patient cohort of new-diagnosis NPC for which viably frozen PBMCs and archival FFPE tissues are available and Cohort 2. A 50-patient cohort participating in a phase II trial (NCT03097939 - National Cancer Centre Singapore) testing the combination of Ipilumimab and Nivolumimab (Ipi.+Nivo.) immunotherapy with longitudinally collected PBMCs and tissue biopsies. Recently published preliminary analysis (AACR 2020) from this clinical trial shows that combined Ipi.+Nivo. therapy is safe and achieved durable responses in recurrent and metastatic NPC patients and identified a negative association between circulating EBV-DNA levels and response. In addition, preliminary analysis of new-diagnosis NPC patient peripheral blood samples from Cohort 1 show associations between certain phenotypic profiles of CD8+ T cells and clinical parameters such as EBV-DNA levels. Therefore, in Aim 1. we will investigate the clinical relevance of these preliminarily identified NPC-associated CD8+ T cell phenotypes. In addition to in-depth single cell transcriptional, functional TCR sequence profiling of these cells, cellular imaging, transcriptional profiling and bulk TCR sequencing of patient-matched tumor will allow discovery of novel associations between peripheral T cells and the tumor microenvironment. In Aim 2., we will characterize EBV-specific T cell responses in the NPC periphery and tumor microenvironment during combination Ipi.+Nivo. immunotherapy treatment to identify novel associations between the phenotypic profiles of EBV-specific T cells and immunotherapeutic response. In Aim 3., we will investigate T cell clonal dynamics associated with treatment induced changes to the NPC-specific immune response by comparing the TCR clonal diversity in peripheral blood and tumor biopsy samples from different stages of treatment. Overall, characterization of EBV-specific T cells phenotypes in the NPC periphery and the tumor using multiple cutting-edge approaches will not only improve our understanding of the NPC immune landscape, but also potentially identify clinically relevant EBV-specific T cell phenotypes that could be tested in future NPC immunotherapy trials.