Reprogramming of T cells for the Treatment of Melanoma

描述（由申请人提供）：最近的工作令人信服地表明，T细胞浸润的程度在许多癌症的自然进展中起着关键作用。 Landmak的一项研究发现，肿瘤内细胞毒性T细胞的类型、密度和位置能够比目前用于癌症分期的组织病理学方法更好地预测患者的生存期。 然而，肿瘤微环境强烈抑制肿瘤特异性T细胞的扩增和效应子功能。 该项目的目标是通过精确定位肿瘤浸润性T细胞内抑制其效应功能的关键遗传和生化缺陷来确定新的治疗靶点。 这种治疗靶标的鉴定在肿瘤学中具有广泛的相关性，因为T细胞介导的免疫应答具有根除癌症的潜力。 为了实现这一目标，我们提出了一种新的体内shRNA发现方法，使相关微环境中的关键基因和途径的识别。 我们的假设是，靶向功能失调的T细胞内的关键抑制剂的shRNA可以重新编程它们，使其在肿瘤中大量扩增。 不 细胞将用shRNA库进行遗传修饰，然后转移到荷瘤小鼠中，使得肿瘤内特定shRNA的富集可以通过以下方法定量： shRNA盒的Illumina测序。 这种体内方法也将使我们能够解决肿瘤学中的第二个相关问题，即确定以高度协同方式作用于特定细胞通路的联合疗法。 我们将使用具有两个shRNA克隆位点的慢病毒载体来解决这个问题，以便可以测试活性shRNA对shRNA库的协同作用。 我们将确定哪种shRNA组合在增殖、细胞因子产生和抗肿瘤细胞毒性作用方面优化体内T细胞活性。 这些治疗方法将在小鼠模型中进行测试，其中黑色素瘤基于人类疾病中发现的遗传病变自发发展。 这项工作的中心目标是通过与具有过继性T细胞治疗专业知识的临床研究者合作，将这些发现转化为临床应用。 表达“嵌合抗原受体”（汽车）的T细胞的连续转移已经成为一种有前景的方法，因为CAR的抗体样细胞外结构域以高亲和力结合肿瘤细胞上的表面分子，而细胞质结构域诱导T细胞活化。 通过相同的慢病毒载体将shRNA和汽车共表达到T细胞中可以大大增强T细胞在肿瘤内的存活和扩增。 这种方法将在携带人类黑色素瘤的小鼠中使用人类T细胞进行测试，作为临床转化的重要一步。 公共卫生相关性：该项目的目标是使用新的shRNA发现方法在相关的体内组织微环境中发现用于黑素瘤免疫治疗的新靶点。 这样的shRNA可用于增强过继性T细胞疗法对黑素瘤的功效。 一些已鉴定的基因产物也可以使用其他方法进行靶向，例如小分子和单克隆抗体。 我们的方法可能广泛适用于发现各种其他人类疾病中的T细胞功能调节因子。

DESCRIPTION (provided by applicant): Recent work has convincingly shown that the degree of T cell infiltration plays a critical role in the natural progression of many cancers. A landmak study found that the type, density, and location of cytotoxic T cells within tumors enabled better prediction of patient survival than histopathological methods currently used to stage cancer. However, the tumor microenvironment strongly inhibits expansion and effector functions of tumor-specific T cells. The goal of this project is to identify novel targes for therapy by pinpointing the key genetic and biochemical defects within tumor-infiltrating T cells that restrain their effector function. The identification of such therapeutic targts is of broad relevance in oncology because T cell mediated immune responses have the potential to eradicate cancers. Towards this goal, we propose a novel in vivo shRN discovery approach that enables identification of critical genes and pathways in the relevant microenvironment. Our hypothesis is that shRNAs which target critical inhibitors within dysfunctional T cells can reprogram them to undergo substantial expansion in tumors. T cells will be genetically modified with shRNA pools and then transferred into tumor-bearing mice so that enrichment of particular shRNAs within tumors can be quantified by Illumina sequencing of the shRNA cassette. This in vivo approach will also allow us to address a second related problem in oncology, the identification of combination therapies that act in a highly synergistic manner on defined cellular pathways. We will approach this issue using a lentiviral vector with two shRNA cloning sites, so that an active shRNA can be tested for synergy against a pool of shRNAs. We will determine which shRNA combinations optimize T cell activityin vivo in terms of proliferation, cytokine production and anti-tumor cytotoxic actio. These therapeutic approaches will be tested in a mouse model in which melanomas spontaneously develop based on genetic lesions found in the human disease. A central goal of this effort is to translate these discoveries into clinical application through collaboration with a clinical investigator with expertise in adoptive T cell therapy. Adoptive transfer of T cells that express 'chimeric antigen receptors' (CARs) has emerged as a promising approach because the antibody-like extracellular domain of a CARbinds with high affinity to a surface molecule on tumor cells, while the cytoplasmic domain induces T cell activation. Co-expression of shRNAs and CARs through the same lentiviral vector into T cells could greatly enhance T cell survival and expansion within tumors. This approach will be tested using human T cells in mice bearing human melanomas, as an important step towards clinical translation. PUBLIC HEALTH RELEVANCE: The goal of this project is to discover novel targets for immunotherapy of melanoma in the relevant in vivo tissue microenvironment using a novel shRNA discovery approach. Such shRNAs may be useful for enhancing the efficacy of adoptive T cell therapy for melanoma. Some of the identified gene products could also be targeted using other approaches, such as small molecules and monoclonal antibodies. Our approach may be widely applicable for the discovery of regulators of T cell function in a variety of other human diseases.