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的抗体已经彻底改变了癌症治疗方法 导致许多以前认为不可治愈的患者的持久缓解。然而，大多数患者 仍然具有耐药性，癌症类型的反应率可能差异很大。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， 研究人员在基础，转化和临床科学方面具有互补的专业知识， 目标是找到新的方法来分类和个性化癌症治疗。最近的结果从 研究人员认为，免疫检查点蛋白在外泌体中的包装在免疫系统中起着重要作用。 患者对免疫检查点抑制剂反应的差异。具体而言，不同量的PD-L1 可以被运输到外泌体，外泌体反过来可以在一定距离处起作用以抑制抗肿瘤T细胞功能， 肿瘤进展，甚至在抗PD-L1治疗耐药的模型中。本提案的目的是建立 通过关注外泌体PD-L1包装、作用和对PD-L1耐药的机制， 治疗性抗体特别是，该提案将测试肿瘤细胞可以选择性地 将PD-L1包装到外泌体中，外泌体以不同于免疫调节的方式抑制T细胞在远端的启动。 PD-L1和PD-1的细胞-细胞相互作用通常见于肿瘤床。这一假设是在广泛的 使用体外和体内模型的初步数据显示，包装在 外泌体相对于保留在细胞中在细胞系之间变化，外泌体PD-L1的抑制可导致 长期全身性抗肿瘤免疫，并且外泌体PD-L1对抗PD-L1抗体具有抗性。测试 总体假设，提出了以下目标：1）揭示机制的选择性 2）评价外泌体PD-L1对系统性免疫应答的影响的机制基础， 3）剖析外泌体PD-L1如何与其靶细胞相互作用并调节其靶细胞。在目标1中，结构/职能 和蛋白质组学方法被提出来鉴定PD-L1包装到外泌体中的调节剂。 此外，这些因子的表达和对治疗的抵抗之间的关联将使用 主要患者样本。在目标2中，将使用基于小鼠的免疫学方法和患者样本来 确定外泌体PD-L1在免疫轴中的功能。未被发现的见解将用于开发 一种增强抗肿瘤免疫应答的新治疗方法。在目标3中，显微镜和功能性 体外模型研究沿着与患者样本相关性研究将用于确定 外泌体PD-L1与其靶细胞相互作用，可能解释其对抗体的抗性。该提案 这是非常重要的，因为它预计将提供新的基础知识，可用于确定和 治疗大部分对当前免疫疗法有抵抗力的患者。虽然该提案侧重于一个子集， 特别强调前列腺癌的癌症模型中，所揭示的范例预计将是 与大多数（如果不是所有）肿瘤类型相关。

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.