NOT-gated CAR T cells to overcome on-target, off-tumor toxicity in AML

项目总结 每年，美国有近2万名患者被诊断为急性髓系白血病(AML)， 尽管进行了高强度化疗，在许多情况下还使用了干细胞，但5年总存活率仍然令人沮丧 移植。嵌合抗原受体(CAR)T细胞治疗在B细胞恶性肿瘤患者中的成功 激发了人们对将这种革命性的免疫疗法应用于AML的浓厚兴趣。然而，临床上 翻译受到AML靶向表达在不可或缺的健康组织上的重叠的限制 增加他们对CAR T细胞介导的细胞毒的敏感性。内皮细胞的毒性尤其值得关注。 针对一些正在调查的AML目标。为了推进CAR T细胞治疗AML，至关重要的是 制定策略，在保持抗白血病效果的同时，避免正常组织进入靶点， 非肿瘤毒性。这一提议的中心假设是抗原特异性抑制性CARs(ICars)可以是 被设计成调制CAR T细胞激活信号，并将降低AML CAR的靶向和肿瘤外毒性 T细胞。在强大的初步数据的指导下，这一假设将以三个具体目标进行检验：1)确定 整合到含有iCar的非门控CAR T细胞的最佳抑制基序；2)确定机制 通过这种方式，iCars中的抑制基序可以使用高级蛋白质组来阻断CAR T细胞信号 技术；以及3)通过整合转录和转录因子确定内皮细胞特异性非门表面靶点 蛋白质组数据。这项提案的主要创新包括实施有针对性的筛查，以确定最佳- 可跨目标推广的类内ICAR，并应用质量细胞分析(CyTOF)进行讯问 CAR T细胞中的基本抑制性信号介体。虽然这项提案的直接焦点是设计 这些实验定义的原则将为应用这一点提供框架 技术广泛应用于其他类型的肿瘤。 拟议的研究活动是全面职业发展计划的一部分，将在过去的基础上再接再厉 申请人的专业知识，对她发展成为一名专注于 转化性免疫疗法。特别是，申请人将获得高维蛋白质组学方面的专业知识和 生物信息学：利用斯坦福大学世界级的科学和指导环境 大学。申请者将由克里斯托·麦考尔博士指导，她因在基本车方面的工作而闻名 T细胞免疫学和翻译免疫疗法，由拉维·马吉蒂博士共同指导，他是一位 急性髓系白血病干细胞的治疗靶向。在他们的指导下，以及集结的咨询委员会 (包括卡拉·戴维斯博士、安苏曼·萨特帕西博士和肯尼斯·温伯格博士)和具体的教育计划， 申请者将获得必要的支持和资源，以有效地实现拟议的目标 在K08训练期后过渡到独立。

PROJECT SUMMARY Each year, nearly 20,000 patients in the United States are diagnosed with acute myeloid leukemia (AML), and 5-year overall survival rates remain dismal despite high intensity chemotherapy and, in many cases, stem cell transplant. Success of chimeric antigen receptor (CAR) T cell therapy in patients with B cell malignancies has prompted intense interest in applying this revolutionary type of immunotherapy to AML. However, clinical translation has been limited by overlap of AML target expression on indispensable, healthy tissues, which increases their susceptibility to CAR T cell-mediated cytotoxicity. Endothelial cell toxicity is of particular concern for some AML targets under investigation. To advance CAR T cell therapy for AML, it is critically important to devise strategies to preserve anti-leukemic efficacy while simultaneously sparing normal tissue from on-target, off-tumor toxicity. The central hypothesis of this proposal is that antigen-specific inhibitory CARs (iCARs) can be engineered to modulate CAR T cell activation signals and will decrease on-target, off-tumor toxicity of AML CAR T cells. Guided by strong preliminary data, this hypothesis will be tested with three specific aims: 1) Identifying optimal inhibitory motifs to incorporate into iCAR-containing NOT-gated CAR T cells; 2) Defining mechanisms by which those inhibitory motifs within iCARs can interrupt CAR T cell signaling using advanced proteomic techniques; and 3) Determining endothelial-specific NOT-gate surface targets by integrating transcriptomic and proteomic data. Key innovations of this proposal include implementation of a targeted screen to identify a best- in-class iCAR that will be generalizable across targets and application of mass cytometry (CyTOF) to interrogate fundamental inhibitory signaling mediators in CAR T cells. While the immediate focus of this proposal is designing a NOT-gated CAR for AML, principles defined by these experiments will provide the framework for applying this technology broadly to other tumor types. The proposed research activities are part of a comprehensive career development plan that will build on past expertise of the applicant and are crucial to her development as an independent investigator focused on translational immunotherapy. In particular, the applicant will gain expertise in high dimensional proteomics and bioinformatics by taking advantage of the world class scientific and mentorship environment at Stanford University. The applicant will be mentored by Dr. Crystal Mackall, renowned for her work on fundamental CAR T cell immunology and translational immunotherapy, and co-mentored by Dr. Ravi Majeti, an expert on therapeutic targeting of AML stem cells. Under their guidance, along with the assembled advisory committee (including Drs. Kara Davis, Ansuman Satpathy, and Kenneth Weinberg) and specific educational plan, the applicant will receive the necessary support and resources to accomplish the proposed aims and efficiently transition to independence following the K08 training period.