Mechanisms of Exosome Driven Immunoregulation of Cancer Progression

项目摘要： 针对免疫检查点蛋白PD-L1和PD-1的抗体使癌症治疗发生了革命性的变化 导致许多以前被认为是不可治愈的患者的持久缓解。然而，大多数患者 保持耐药性，癌症类型的应答率可能会有很大差异。因此，迫切需要 了解反应的变化，以改善癌症护理。这项提案汇集了一个团队 在基础、翻译和临床科学方面具有互补专业知识的研究人员，他们共享长期 目标是找到新的方法对癌症治疗进行分类和个性化。最近的结果来自 研究人员认为，免疫检查点蛋白在支持该病毒的外体中的包装起着重要作用 患者对免疫检查点抑制剂的反应存在差异。具体地说，不同数量的Pd-L1 可以被运输到外体，而外体又可以在一定距离内作用于抑制抗肿瘤T细胞功能，从而使 肿瘤进展，即使在抗PD-L1治疗耐药的模型中也是如此。这项提议的目标是建立 通过重点研究外体PD-L1的包装、作用和耐药性的机制来了解这些发现 治疗性抗体。特别是，该提案将检验肿瘤细胞可以选择性地 将PD-L1包装到外体中，以一种与外体不同的方式抑制远端视野的T细胞启动 PD-L1和PD-1的细胞间相互作用通常见于肿瘤床。这一假设的前提是广泛的 体外和体内模型的初步数据显示，包装在体内的PD-L1的相对比例 不同细胞系的外切体与保留在细胞中的比例不同，抑制外切体PD-L1可导致 长期的全身性抗肿瘤免疫，以及胞外体PD-L1对抗PD-L1抗体具有抵抗力。为了测试 在总体假设中，提出了以下目标：1)揭示选择性心力衰竭的潜在机制 将PD-L1包装成外体，2)评价外体PD-L1‘S对系统影响的机制基础 免疫，3)剖析外体PD-L1如何与其靶细胞相互作用和调节。目标1，结构/功能 并提出了蛋白质组学方法来鉴定PD-L1包装成外体的调控因子。 此外，这些因子的表达与治疗耐药性之间的关联将使用以下方法进行评估 初级病人样本。在目标2中，将使用基于老鼠的免疫学方法和患者样本来 确定胞外体PD-L1在免疫轴中的作用。未发现的洞察力随后将用于开发 一种增强抗肿瘤免疫反应的新的治疗方法。在AIM 3中，显微镜和功能 体外模型研究以及与患者样本的关联研究将被用来确定如何 外体PD-L1与其靶细胞相互作用，可能解释其对抗体的抵抗力。该提案是 非常重要的是，它预计将提供新的基本知识，可用于识别和 治疗对当前免疫疗法有抵抗力的大部分患者。虽然该提案侧重于一个子集 在特别强调前列腺癌的癌症模型中，发现的范例预计是 与大多数肿瘤类型相关，如果不是所有类型的话。

PROJECT ABSTRACT: Antibodies against the immune checkpoint proteins PD-L1 and PD-1 have revolutionized cancer therapeutics resulting in durable remissions in many patients previous considered incurable. However, a majority of patients remain resistant and cancer types can vary greatly in their response rates. Thus, there is an urgent need to understand the variation in response in order to improve cancer care. This proposal brings together a team of investigators with complementary expertise in basic, translational, and clinical science who share the long-term goal of finding novel approaches to categorize and personalize cancer treatments. Recent results from the investigators suggest a major role for the packaging of immune checkpoint proteins in exosomes underlying the variation in responses to immune checkpoint inhibitors among patients. Specifically, varying amounts of PD-L1 can be trafficked to exosomes, which in turn can act at a distance to suppress anti-tumor T cell function, enabling tumor progression, even in models resistant to anti-PD-L1 treatments. The objective of this proposal is to build on these findings by focusing on the mechanism of exosomal PD-L1 packaging, action, and resistance to therapeutic antibodies. In particular, the proposal will test the overall hypothesis that tumor cells can selectively package PD-L1 into exosomes that suppress T cell priming at distal sights in a fashion that is distinct from the cell-cell interactions of PD-L1 and PD-1 normally seen in the tumor bed. The hypothesis is premised on extensive preliminary data using in vitro and in vivo models showing that the relative fraction of PD-L1 packaged in exosomes versus retained in cells varies between cell lines, that suppression of exosomal PD-L1 can result in long-term systemic anti-tumor immunity, and that exosomal PD-L1 is resistant to anti-PD-L1 antibodies. To test the overall hypothesis, the following aims are proposed: 1) Uncover mechanisms underlying the selective packaging of PD-L1 into exosomes, 2) Evaluate the mechanistic basis of exosomal PD-L1’s impact on systemic immunity, 3) Dissect how exosomal PD-L1 interacts with and regulates its target cells. In aim 1, structure/function and proteomic approaches are proposed to identify the regulators of PD-L1 packaging into exosomes. Furthermore, associations between expression of these factors and resistance to therapy will be evaluated using primary patient samples. In aim 2, mouse-based immunological approaches and patient samples will be used to determine where in the immune axis exosomal PD-L1 functions. Uncovered insights will then be used to develop a novel therapeutic approach to enhance the anti-tumor immune response. In aim 3, microscopy and functional studies in in vitro models along with association studies with patient samples will be used to determine how exosomal PD-L1 interacts with its target cells potentially explaining its resistance to antibodies. The proposal is highly significant in that it is expected to provide new fundamental knowledge that can be used to identify and treat the large fraction of patients resistant to current immune therapies. While this proposal focuses on a subset of cancer models with a particular emphasis on prostate cancer, the paradigms uncovered are expected to be relevant across most if not all tumor types.