Next Generation Cancer Immunotherapies to Defeat Melanoma

项目总结/摘要 这是第2类续期申请，旨在建立在最初资助期的进展基础上， 黑色素瘤患者的新疗法，并研究对 黑色素瘤免疫疗法。我们在初始资金的三个科学领域取得了重大进展 期我们定义了原发性和获得性免疫检查点阻断（ICB）的遗传机制， 抗原呈递机制和干扰素-γ（IFN-g）信号传导中的功能突变的丧失 这使我们成功地在小鼠模型中测试了联合疗法，并将它们带回到 正在进行临床试验的患者。在免疫疗法和免疫治疗的组合方法的科学领域， 靶向治疗，我们的小鼠模型数据提供了三个三联疗法（BRAF加MEK）的理由 抑制剂和抗PD-1或-PD-L1）临床项目，其中一项获得FDA批准。在科学 推进基因工程过继细胞转移（ACT）疗法的主题，我们已经进行了三个临床 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，并开发为体内T细胞扩增提供上级细胞因子支持的方法。 新的R35提案有三个主题。主题1集中在癌细胞如何被识别或逃脱T 细胞，专注于抗原呈递机制。它将使用已经收集的1，031个活检样本库 将使用新开发分离、测序和生物信息学分析方法来分析。主题 2是基于IFN-g在形成抗肿瘤免疫应答中的关键作用;认识到这种细胞因子 将癌细胞转化为抗肿瘤免疫反应的启动子， 提高免疫疗法的知识。这将通过CRISPR和药物筛选在一个小组中实现。 黑色素瘤细胞系，其次是途径分析和机制研究，希望导致新的 药理学靶点，以改善IFN-γ抗肿瘤活性。主题3重点关注T细胞功能和靶点 识别，特别是避免重复抗原暴露后的T细胞功能障碍，这是一个主要的免疫缺陷。 限制长期抗肿瘤反应。该项目将在T细胞中使用CRISPR筛选， 抗原暴露，以发现基因，当敲除时，导致T细胞维持其上级能力的基因。 在重复TCR接合时的功能性。此外，我们将分析转录因子，指导 过继转移的T细胞的上级体内扩增和抗肿瘤活性。 总之，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- 分析大量患者活检的最新方法使用CRISPR筛选进行机制分析， 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