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

项目摘要2：默克尔细胞癌(MCC)是一种罕见但往往致命的皮肤癌，由默克尔 细胞多瘤病毒(MCPyV)在80%的病例中。MCPyV T抗原癌蛋白在病毒中持续表达- 在病毒阴性者中，检测到阳性的MCC(VP-MCC)，而紫外线诱导的新抗原数量显著增加 MCCS(VN-MCC)，提示两个MCC亚群都含有免疫原性表位。基于我们早期对 MCC的免疫反应，我们的团队领导了多项临床试验，最近改变了对这一疾病的护理标准 癌症。与其他实体瘤相比，PD-1阻断在MCC中产生了较高的应答率，并且显著 与细胞毒性化疗相比，结果有所改善，细胞毒性化疗是晚期MCC的唯一优先选择。不幸的是， 40%的MCC患者最初没有从PD-1阻断中受益，约20%的应答者后来发展为获得性 抵抗。因此，迫切需要确定具有治疗靶向性的耐药机制。 在这项建议中，我们试图通过分析三种机制来确定PD-1阻断的可逆机制 整个项目的关键成分：目标1：MCC特异性T淋巴细胞。我们将确定T细胞 肿瘤的浸润方式、T细胞克隆多样性或功能障碍状态与对 PD-1阻断治疗。目的2：MCC肿瘤细胞。我们将确定MCC细胞的固有免疫特性 包括额外检查点分子的表达或抗原性受损与 对PD-1阻滞剂的反应/抵抗。目的3：MCC先天免疫。我们将研究它们之间的相关性 肿瘤内两种天然免疫细胞类型(巨噬细胞和NK细胞)的侵袭和MCC预后的确定 先天免疫刺激的治疗干预能否增强MCC获得性免疫。 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，这些进展将大大增加 我们识别和测试介导抵抗PD-1阻断的免疫机制的功能相关性的能力。 这些改进包括：(1)scRNAseq工作流程，可对少量(1个核心活检)进行 冷冻保存的材料，使仔细收集和选择高度信息量的患者样本和改进 分析深度；(2)患者来源的异种移植(PDX)小鼠中MCC肿瘤的扩展，用于后续的功能性 分析方法包括切片培养/外植体系统。最初将采用包括scRNAseq在内的公正的方法 为了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 从我们第一次提交的时候起。在我们外部和内部咨询委员会的指导下，我们将 选择与PD-1阻断反应最相关的通路。这些免疫逃避途径将是 在更多的患者中进行研究，将确定它们的功能意义/可逆性。 我们与免疫肿瘤学专家已建立的合作将极大地推动这一项目 机制包括马丁·麦克·契弗博士(FHCRC)、克里斯蒂安·辛里希(NCI)、德鲁·帕多尔(霍普金斯大学)、约翰 汤普森大学(UW)、苏珊娜·托帕里安(Suzanne Topian)(霍普金斯大学)、约翰·惠里(John Wherry)(宾州大学)和凯瑟琳·吴(Catherine Wu)(哈佛大学)。

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).