Directed differentiation of HBV-specific CTL from iPSC for cell-based therapies

描述(申请人提供)：在慢性乙肝感染患者中，能够控制乙肝病毒(乙肝病毒)和消除表达乙肝病毒抗原(Ag)的肝细胞癌(HCC)的病毒特异性T细胞被缺失或功能障碍。目前的抗病毒治疗以病毒逆转录酶为靶点，很少建立对乙肝病毒复制的免疫控制。乙肝病毒特异性CD8+细胞毒性T淋巴细胞(CTL)过继细胞转移(ACT)是治疗慢性乙肝病毒感染和乙肝病毒相关性肝细胞癌的一种很有前途的治疗方法。幼稚或中央记忆型T细胞衍生效应细胞CTL-“正确”或“高活性”CTL是基于ACT的免疫治疗的最佳群体，因为这些细胞具有高增殖潜力，比终末分化细胞更不容易凋亡，并且对动态平衡细胞因子有更高的反应能力。然而，由于很难从患者那里获得足够数量的高反应性CTL，这样的ACT通常是不可行的。我们研究计划的长期目标是开发和优化策略，利用表达抗原特异性T细胞受体(TCR)的多潜能干细胞(PSC)作为高活性CTL的来源，用于基于细胞的治疗。本应用的目的是确定从诱导的PSC(即IPSC-CTL)中获得的HBV特异性CTL的基本性质，为抗慢性HBV感染和HBV相关性肝细胞癌的潜在免疫治疗开发提供基础。中心假设是我们的体外分化系统开发的HBV特异性IPSC-CTL是单纯的单克隆性HBV特异性CD8+T细胞，可用于产生高反应性的HBV特异性CTL，用于基于细胞的治疗。在已发表的结果和来自申请人实验室的初步数据的指导下，这一假设将通过追求两个具体目标来验证：1)确定与其抗乙肝病毒活性相关的乙肝病毒特异性IPSC-CTL的基本性质；2)确定乙肝病毒特异性IPSC-CTL在携带乙肝病毒的小鼠中的抗乙肝病毒活性。在第一个目标下，将使用体外和体内方法，这些方法已经在申请人的实验室被确定为可行的。将定义乙肝病毒特异性IPSC-CTL的表型、特异性和功能的基本特性。在第二个目标下，利用乙肝病毒感染和乙肝病毒相关肿瘤的小鼠模型，将确定乙肝病毒特异性IPSC-CTL用于细胞治疗的疗效。这种方法是创新的，因为用乙肝病毒特异性TCR基因修饰的乙肝病毒特异性IPSC-CTL用于慢性乙肝感染和乙肝相关肝细胞癌的细胞治疗之前还没有被探索过。这项拟议的研究意义重大，因为它将为产生高活性的病毒特异性PSC-T细胞提供新的见解和策略，从而推动治疗性T细胞用于传染病和癌症的治疗。

DESCRIPTION (provided by applicant): Virus-specific T cells capable of controlling hepatitis B virus (HBV) and eliminating hepatocellular carcinoma (HCC) expressing HBV antigen (Ag) are deleted or dysfunctional in patients with chronic HBV infection. Current antiviral therapy targets the virus reverse transcriptase and rarely establishes immunological control over HBV replication. Adoptive cell transfer (ACT) of HBV-specific CD8+ cytotoxic T lymphocytes (CTL) is an highly promising treatment for chronic HBV infection and HBV-associated HCC. Naive or central memory T cell-derived effector CTL - the "right" or "highly reactive" CTL are the optimal populations for ACT-based immunotherapy, because these cells have a high proliferative potential, are less prone to apoptosis than terminally differentiated cells and have a higher ability to respond to homeostatic cytokines. However, such ACT is often not feasible due to difficulties in obtaining sufficient numbers of highly reactive CTL from patients. The long-term goal of our research program is to develop and optimize strategies for utilizing pluripotent stem cells (PSC) expressing Ag-specific T cell receptor (TCR) as a source of highly reactive CTL for cell-based therapies. The objective in this application is to determine the fundamental properties of HBV-specific CTL from induced PSC (i.e., iPSC-CTL) genetically modified with HBV-specific TCR, for potential immunotherapeutic development against chronic HBV infection and HBV-associated HCC. The central hypothesis is that HBV-specific iPSC-CTL developed by our in vitro differentiation system are naive monoclonal HBV-specific CD8+ T cells, which can be used to generate highly reactive HBV-specific CTL for cell-based therapies. Guided by published results and preliminary data from the applicant's laboratory, this hypothesis will be tested by pursuing two specific aims: 1) to define the fundamental properties of HBV-specific iPSC-CTL that relate to their anti-HBV activity; and 2) to determine the anti-HBV activity of HBV-specific iPSC- CTL in HBV-bearing mice. Under the first aim, in vitro and in vivo approaches, which have been established as feasible in the applicant's laboratory, will be used. The fundamental properties of HBV-specific iPSC-CTL regarding phenotype, specificity and function will be defined. Under the second aim, using murine models of HBV infection and HBV-associated tumor, the efficacy of HBV-specific iPSC-CTL for cell-based therapies will be determined. The approach is innovative, because HBV-specific iPSC-CTL genetically modified with HBV- specific TCR for cell-based therapies of chronic HBV infection and HBV-associated HCC has not been previously explored. The proposed research is significant, because it will provide new insight and strategies for generating highly reactive virus-specific PSC-T cells and in so doing drive forward use of therapeutic T cells for the treatments of infectious diseases and cancers.