Reprogramming of T cells for the Treatment of Melanoma

描述(由申请人提供)：最近的研究令人信服地表明，T细胞渗透的程度在许多癌症的自然发展中起着关键作用。兰德马克的一项研究发现，与目前用于癌症分期的组织病理学方法相比，肿瘤内细胞毒性T细胞的类型、密度和位置能够更好地预测患者的生存。然而，肿瘤微环境强烈抑制肿瘤特异性T细胞的扩增和效应功能。该项目的目标是通过精确定位肿瘤浸润性T细胞中限制其效应器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细胞介导的免疫反应具有根除癌症的潜力。为了实现这个目标，我们提出了一种新的体内shRN发现方法，该方法能够识别相关微环境中的关键基因和途径。我们的假设是，靶向功能失调T细胞内关键抑制物的shRNAs可以对它们进行重新编程，使其在肿瘤中经历实质性的扩张。T 细胞将用shRNA池进行基因改造，然后转移到荷瘤小鼠中，这样肿瘤内特定shRNA的丰富就可以通过 ShRNA盒的Illumina测序。这种体内方法还将使我们能够解决肿瘤学中的第二个相关问题，即确定在确定的细胞通路上以高度协同的方式发挥作用的联合疗法。我们将使用带有两个shRNA克隆位点的慢病毒载体来解决这个问题，这样就可以测试活性shRNA与一组shRNA之间的协同作用。我们将确定哪些shRNA组合在增殖、细胞因子产生和抗肿瘤细胞毒作用方面优化了体内T细胞的活性。这些治疗方法将在小鼠模型中进行测试，在该模型中，黑色素瘤根据人类疾病中发现的遗传损伤自发发展。这项工作的一个中心目标是通过与具有过继T细胞治疗专业知识的临床研究人员合作，将这些发现转化为临床应用。表达嵌合抗原受体(CARS)的T细胞过继转移是一种很有前途的方法，因为CARs的胞外区与肿瘤细胞表面分子具有高亲和力，而胞内区则诱导T细胞的激活。通过相同的慢病毒载体将shRNAs和CARS共表达到T细胞中，可显著提高T细胞在肿瘤内的存活和扩增。这一方法将在携带人类黑色素瘤的小鼠身上使用人类T细胞进行测试，作为临床转化的重要一步。

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.