Generation of therapeutic T cells from cord blood-derived stem cells

描述（由申请人提供）：近年来，自体T细胞体外免疫治疗（过继性转移）成功地治疗了多种癌症。然而，目前为这种治疗提供治疗性T细胞的努力涉及从患者外周血中分离细胞，扩增和抗原特异性“训练”，然后将训练后的细胞返回患者体内。这些方法受到以下因素的严重限制：(a)患者细胞分离的困难和低效率；(b)原代细胞体外扩增的问题；(c)自体细胞治疗相关的发病率，最重要的是(d)供体细胞的可用性有限。此外，“扩大”和“训练”用于过继治疗的患者分离细胞所需的时间对于危重疾病往往太长。因此，在合成的、受控的环境中，从多能干细胞高效地产生和扩增治疗性T细胞的技术可以为各种疾病的应用提供可再生的、按需的、现成的细胞来源。尽管在过去几年中，我们对T细胞发育过程中涉及的分子信号的理解取得了巨大的进步，但干细胞衍生的T细胞的最终治疗适用性需要(a)对微环境导向的造血祖细胞（HPC）分化为T细胞的定量理解，以及(b)开发适合按需移植的功能性抗原特异性T细胞的高通量生产技术。我们的目标是设计人工T细胞发育小生境（合成胸腺样微环境），以了解控制Notch信号在T细胞发育中的作用，并以可扩展的方式将人类HPCs引导到治疗性T细胞中。具体来说，我们建议合成缺口配体功能化（人工胸腺基质细胞）和HLA四聚体功能化磁微珠（人工抗原提呈细胞），并评估脐带血来源的人CD34+CD38干细胞如何被定向到功能性治疗性T细胞。公共卫生相关性：这个为期两年的项目的目标是开发模拟胸腺微环境条件的合成微珠，以研究脐带血来源的造血祖细胞如何被引导到T细胞谱系。具体来说，我们将研究通过这些人造基质细胞的notch信号如何有效地触发notch特异性基因，以及在来自小鼠或人类基质细胞信号的旁分泌信号的存在下如何产生早期T细胞。我们还将研究通过磁微珠（人工抗原提呈细胞）传递的四聚体信号是否能进一步将这些干细胞衍生的早期T细胞分化为更成熟的CD8+抗原特异性T细胞。

DESCRIPTION (provided by applicant): In recent years, successful ex-vivo immunotherapy with autologous T cells (adoptive transfer) has been reported for a variety of cancers. However, current efforts to provide therapeutic T cells for such therapy involve isolation of cells from the patient's peripheral blood, expansion and antigen specific "training" ex-vivo followed by return of the trained cells into the patient. These methods are severely constrained by (a) the difficulties and inefficiency of patient cell isolation (b) problems with expansion of primary cells in vitro (c) the morbidity associated with autologous cell therapy and above all (d) the limited availability of donor cells. In addition, the time required to "expand" and "train" patient-isolated cells for adoptive therapy can often prove to be too long for critical diseases. Therefore, technologies leading to efficient generation and expansion of therapeutic T cells from multipotent stem cells in a synthetic, controlled environment could provide a renewable, on-demand and readily available cell source for a variety of disease applications. Despite tremendous advances in the past few years in our understanding of the molecular signals involved in T cell development, the ultimate therapeutic applicability of stem cell-derived T cells require (a) Quantitative understanding of microenvironment- directed hematopoietic progenitor cell (HPC) differentiation into T cells and (b) Development of technologies for high-throughput production of functional, antigen- specific T cells suitable for on-demand transplantation. Our goal here is to engineer artificial T cell development niches (synthetic thymus-like microenvironment) to understand the effects of controlled Notch signaling in T cell development and direct human HPCs into therapeutic T cells in a scalable manner. Specifically, we propose to synthesize notch-ligand functionalized (artificial thymic stromal cells) and HLA tetramer functionalized magnetic microbeads (artificial antigen presenting cells), and evaluate how cord blood-derived human CD34+CD38- stem cells could be directed to functional, therapeutic T cells. PUBLIC HEALTH RELEVANCE: The goal of this two year project is to develop synthetic microbeads that mimic the micro-environmental conditions of the thymus in order to study how cord blood- derived hematopoietic progenitor cells can be directed to the T cell lineage. Specifically we would investigate how efficient notch signaling through these artificial stromal cells could trigger notch specific genes and in the presence of paracrine signals from mouse or human stromal cell signals generate early T cells. We would also investigate if tetramer signaling through magnetic microbeads (artificial antigen presenting cells) can further differentiate these stem cell-derived early T cells into more mature, CD8+ antigen specific T cells.