Next Generation Cancer Immunotherapies to Defeat Melanoma

项目摘要/摘要 这是一种类型2续订申请，旨在建立在最初资金期限内开发的进展 黑色素瘤患者的新疗法及对其应答和抵抗的机制研究 黑色素瘤免疫疗法。我们在最初资金的三个科学领域取得了重大进展 句号。我们定义了原发和获得性免疫检查点阻断(ICB)的遗传机制 抗原提呈机制功能突变与干扰素-γ信号转导 途径，这使我们成功地在小鼠模型上测试了联合疗法，并将它们带回 正在进行临床试验的患者。在免疫疗法的组合方法的科学领域 靶向治疗，我们的小鼠模型数据为三种三联疗法(BRAF加MEK)提供了依据 抑制剂和抗PD-1或-PD-L1)临床计划，其中一项已获得FDA批准。在科学上 以推进基因工程过继细胞转移(ACT)治疗为主题，我们已经进行了三次临床 试验，开发了分离T细胞受体(TCR)的优化方法，帮助开发了一种非病毒的方法 T细胞基因编辑，并开发方法，为体内T细胞扩增提供优越的细胞因子支持。 新的R35提案有三个主题。主题1集中在癌细胞是如何识别或逃避T细胞的 细胞，专注于抗原呈递机制。它将使用一个包含1031份已经收集的活检组织的资料库 将使用新开发的分离、测序和生物信息学分析方法对其进行分析。主旋律 2是基于干扰素-g在形成抗肿瘤免疫反应中的关键作用；认识到这种细胞因子 将癌细胞转变为抗肿瘤免疫反应的使能器提供了利用 提高免疫治疗水平的知识。这将使用CRISPR和一个小组中的药物筛选来实现 黑色素瘤细胞系，随后进行路径分析和机制研究，有望导致新的 提高干扰素-g抗肿瘤活性的药理靶点。主题3集中在T细胞的功能和靶点上 识别，特别是在避免反复暴露抗原时T细胞功能障碍方面，这是一个主要的 对长期抗肿瘤反应的限制。该项目将在T细胞中使用CRISPR筛查 抗原暴露寻找基因，当被敲除时，导致T细胞保持其自身的优越能力 重复TCR参与时的功能。此外，我们将分析指导我们的转录因子 过继转移的T细胞具有良好的体内扩增和抗肿瘤活性。 总而言之，新的R35提案建立在当前内审办的科学基础上。我们建议使用状态- 分析大量患者活检组织的ART方法，使用CRISPR筛查进行机械分析 并使用模型系统来改善对癌症免疫疗法的反应和治疗耐药性。 1

PROJECT SUMMARY / ABSTRACT This is a Type 2 renewal application aimed at building on the progress over the initial funding period to develop new therapies for patients with melanoma and studying the mechanistic basis of response and resistance to melanoma immunotherapies. We made significant progress in the three scientific areas of the initial funding period. We defined genetic mechanisms of primary and acquired to immune checkpoint blockade (ICB) through loss of function mutations in the antigen presenting machinery and the interferon-gamma (IFN-g) signaling pathway, which led us to successfully test combination therapies in mouse models and bring them back to patients in ongoing clinical trials. In the scientific area of combinatorial approaches of immunotherapy and targeted therapy, our mouse model data provided the justification for three triple therapy (BRAF plus MEK inhibitors and anti-PD-1 or -PD-L1) clinical programs, one of them resulting in an FDA approval. In the scientific theme of advancing gene engineered adoptive cell transfer (ACT) therapies, we have conducted three clinical trials, developed optimized means to isolate T cell receptors (TCRs), helped develop an approach for non-viral T cell gene editing, and develop approaches to provide superior cytokine support for in vivo T cell expansion. The new R35 proposal has three themes. Theme 1 is centered on how cancer cells are recognized or escape T cells, focusing on the antigen presentation machinery. It will use a repository of 1,031 already collected biopsies that will be analyze using newly developed methods of isolation, sequencing and bioinformatics analyses. Theme 2 is based on the key role of IFN-g in shaping an antitumor immune response; the realization that this cytokine turns the cancer cells into enablers of the antitumor immune response provides opportunities for exploiting the knowledge to improve immunotherapies. This will be achieved using CRISPR and drug screens in a panel of melanoma cell lines, followed by pathway analyses and mechanistic studies hopefully leading to new pharmacological targets to improve IFN-g antitumor activity. Theme 3 is focused on the T cell function and target recognition, and in particular in avoiding T cell dysfunction upon repeated antigen exposure, which is a major limitation to long term antitumor responses. This project will use CRISPR screens in T cells undergoing repeated antigen exposure to find genes that, when knocked-out, result in superior ability of T cells to maintain their functionality upon repeated TCR engagement. Furthermore, we will analyze the transcription factors that guide a superior in vivo expansion and antitumor activity of adoptively transferred T cells. In conclusion, the new R35 proposal builds on the science from the current OIA. We propose to use state-of- the-art approaches to analyze large numbers of patient biopsies, use CRISPR screens for mechanistic analyses and use model systems to improve responses and treat resistance to cancer immunotherapies. 1