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

描述（由申请人提供）：能够控制乙型肝炎病毒（HBV）和消除表达HBV抗原（Ag）的肝细胞癌（HCC）的病毒特异性T细胞在慢性HBV感染患者中缺失或功能失调。目前的抗病毒治疗针对病毒逆转录酶，很少建立对HBV复制的免疫控制。HBV特异性CD8+细胞毒性T淋巴细胞（CTL）的过继细胞转移（ACT）是一种非常有前途的治疗慢性HBV感染和HBV相关HCC的方法。幼稚或中枢记忆T细胞衍生的效应CTL -“正确的”或“高活性的”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，并且对稳态细胞因子有更高的反应能力。然而，由于难以从患者身上获得足够数量的高反应性CTL，这种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利用表达ag特异性T细胞受体（TCR）的多能干细胞（PSC）作为基于细胞治疗的高反应性CTL的来源的策略。本应用的目的是确定从经HBV特异性TCR基因修饰的诱导PSC（即iPSC-CTL）中获得的HBV特异性CTL的基本特性，用于潜在的针对慢性HBV感染和HBV相关HCC的免疫治疗开发。我们的中心假设是，我们的体外分化系统开发的hbv特异性iPSC-CTL是幼稚的单克隆hbv特异性CD8+ T细胞，可用于生成高度反应性的hbv特异性CTL，用于细胞治疗。在已发表的结果和申请人实验室的初步数据的指导下，将通过追求两个特定目标来验证这一假设：1)确定hbv特异性iPSC-CTL与其抗hbv活性相关的基本特性；2)检测hbv携带小鼠体内hbv特异性iPSC- CTL的抗hbv活性。在第一个目标下，将使用在申请人实验室中已经建立的可行的体外和体内方法。hbv特异性iPSC-CTL在表型、特异性和功能方面的基本特性将被定义。在第二个目标下，利用小鼠HBV感染和HBV相关肿瘤模型，将确定HBV特异性iPSC-CTL用于细胞治疗的疗效。该方法是创新的，因为HBV特异性iPSC-CTL基因修饰HBV特异性TCR用于基于细胞的慢性HBV感染和HBV相关HCC的治疗之前尚未被探索过。这项提议的研究意义重大，因为它将为产生高反应性病毒特异性PSC-T细胞提供新的见解和策略，从而推动治疗性T细胞用于感染性疾病和癌症的治疗。

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.