Project 2: Characterizing and overcoming failure to respond to PD-1 blockade therapy

摘要项目2：默克尔细胞癌（MCC）是一种罕见的，但往往致命的皮肤癌引起的默克尔 细胞多瘤病毒（MCPyV）在80%的情况下。MCPyV T抗原癌蛋白在病毒中持续表达， 阳性MCC（VP-MCC），而在病毒阴性MCC中检测到显著高数量的UV诱导的新抗原。 MCCs（VN-MCC），表明两个MCC亚群都含有免疫原性表位。基于我们对 MCC的免疫反应，我们的小组领导了多项临床试验，最近改变了护理标准， 癌与其他实体瘤相比，PD-1阻断在MCC中产生了高应答率， 与晚期MCC的唯一首选细胞毒性化疗相比，结局改善。不幸的是， 40%的MCC患者最初并未从PD-1阻滞中受益，约20%的缓解者后来发展为获得性疾病 阻力因此，迫切需要确定耐药性的治疗靶向机制。 在这项提案中，我们试图通过分析三种机制来确定PD-1阻断的可逆机制。 本项目的关键组成部分：目标1：MCC特异性T淋巴细胞。我们将确定T细胞是否 肿瘤浸润模式、T细胞克隆多样性或功能障碍状态与免疫应答失败相关。 PD-1阻断治疗。目的2：MCC肿瘤细胞。我们将确定MCC细胞的内在免疫特性是否 包括额外检查点分子的表达或抗原性受损， 对PD-1阻断的应答/抗性。目标3：MCC先天免疫。我们将研究 两种先天性免疫细胞类型（巨噬细胞和NK细胞）的肿瘤内浸润和MCC结果，以确定 先天免疫刺激的治疗性干预是否可以增强MCC的适应性免疫。 值得注意的是，自2017年5月提交以来，我们取得了几项重大进展， 我们鉴定和测试介导对PD-1阻断的抗性的免疫机制的功能相关性的能力。 这些进展包括：（1）scRNAseq工作流程，可以对少量（1个核心活检）的 冷冻保存的材料，能够仔细收集和选择高度信息化的患者样本， （2）患者来源的异种移植物（PDX）小鼠中的MCC肿瘤扩增，用于随后的功能性研究; 包括切片培养/外植体系统的测定。最初将采用包括scRNAseq在内的无偏倚方法 优化我们识别新的和重要的免疫逃避机制的能力，正如我们已经证明的那样， 从我们第一次提交以来。在外部和内部咨询委员会的指导下，我们将 选择与PD-1阻断反应最密切相关的途径。这些免疫逃避途径将 在大量患者中进行研究，并确定其功能意义/可逆性。 这个项目将大大使我们建立的合作与专家解剖免疫肿瘤学 机制，包括博士马丁“Mac”奇弗（FHCRC），基督教Hinrichs（NCI），德鲁Pardoll（霍普金斯），约翰 汤普森（华盛顿大学）、苏珊娜·托帕利安（霍普金斯）、约翰·惠里（宾夕法尼亚大学）和凯瑟琳·吴（哈佛）。

SUMMARY PROJECT 2: Merkel cell carcinoma (MCC) is a rare but often deadly skin cancer caused by the Merkel cell polyomavirus (MCPyV) in 80% of cases. MCPyV T-antigen oncoproteins are persistently expressed in virus- positive MCCs (VP-MCC), while remarkably high numbers of UV-induced neoantigens are detected in virus-negative MCCs (VN-MCC), suggesting both MCC subsets harbor immunogenic epitopes. Based on our early studies of the immune response in MCC, our group led multiple clinical trials that have recently changed the standard of care for this cancer. PD-1 blockade has yielded high response rates in MCC as compared with other solid tumors, and markedly improved outcomes compared to cytotoxic chemotherapy, the only prior option for advanced MCC. Unfortunately, 40% of MCC patients do not initially benefit from PD-1 blockade and ~20% of responders later develop acquired resistance. Consequently, there is an urgent need to identify therapeutically targetable mechanisms of resistance. In this proposal, we seek to identify reversible mechanisms of resistance to PD-1 blockade by analyzing three critical components across this Project: Aim 1: MCC-specific T lymphocytes. We will determine whether T cell infiltration patterns into tumors, T cell clonal diversity, or dysfunctional status are associated with failure to respond to PD-1 blockade therapy. Aim 2: MCC tumor cells. We will determine whether MCC cell intrinsic immune characteristics including expression of additional checkpoint molecules or impaired antigenicity are associated with response/resistance to PD-1 blockade. Aim 3: MCC innate immunity. We will study the correlation between intratumoral infiltration of two innate immune cell types (macrophages and NK cells) and MCC outcome to determine whether therapeutic intervention with innate immune stimulation can augment MCC adaptive immunity. Notably, we have made several significant advances since our May 2017 submission which will greatly increase our ability to identify and test the functional relevance of immune mechanisms mediating resistance to PD-1 blockade. These advances include: (1) A scRNAseq workflow which can be performed on small amounts (1 core biopsy) of cryopreserved material, enabling careful collection and selection of highly informative patient samples and improved depth of analysis; (2) MCC tumor expansion in patient derived xenograft (PDX) mice for subsequent use in functional assays including a slice-culture/explant system. Unbiased approaches including scRNAseq, will be initially employed to optimize our capacity to identify novel and significant immune evasion mechanisms, as we have demonstrated since the time of our initial submission. Under the guidance of our External and Internal Advisory Boards, we will then select the pathways most strongly associated with PD-1 blockade response. These immune evasion pathways will be studied in a larger number of patients and their functional significance/reversibility will be determined. This project will be greatly enabled by our established collaborations with experts in dissecting immuno-oncology mechanisms including Drs. Martin “Mac” Cheever (FHCRC), Christian Hinrichs (NCI), Drew Pardoll (Hopkins), John Thompson (UW), Suzanne Topalian (Hopkins), John Wherry (UPenn) and Catherine Wu (Harvard).