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

描述（由申请人提供）：近年来，已经报道了使用自体T细胞（过继转移）的成功离体免疫疗法用于多种癌症。然而，目前为这种疗法提供治疗性T细胞的努力涉及从患者外周血中分离细胞，体外扩增和抗原特异性“训练”，然后将训练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（B）体外扩增原代细胞的问题（c）与自体细胞治疗相关的发病率，以及最重要的（d）供体细胞的有限可用性。此外，“扩增”和“训练”用于过继治疗的患者分离的细胞所需的时间对于危重疾病来说通常被证明太长。因此，导致在合成的受控环境中从多能干细胞有效产生和扩增治疗性T细胞的技术可以为各种疾病应用提供可再生的、按需的和容易获得的细胞来源。尽管在过去几年中，我们在理解T细胞发育中涉及的分子信号方面取得了巨大进展，但干细胞衍生的T细胞的最终治疗适用性需要（a）定量理解微环境指导的造血祖细胞（HPC）分化为T细胞和（B）开发用于高通量产生功能性、特异性和非特异性造血干细胞的技术。适合于按需移植的抗原特异性T细胞。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），以了解受控Notch信号传导在T细胞发育中的作用，并以可扩展的方式将人类HPC引导至治疗性T细胞。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四聚体功能化磁性微珠（人工抗原呈递细胞），并评估如何脐带血来源的人CD 34 + CD 38-干细胞可以被定向为功能性的，治疗性的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，以研究如何将脐带血来源的造血祖细胞定向为T细胞谱系。具体来说，我们将研究通过这些人工基质细胞的有效notch信号传导如何触发notch特异性基因，并在存在来自小鼠或人基质细胞的旁分泌信号的情况下产生早期T细胞。我们还将研究通过磁性微珠（人工抗原呈递细胞）的四聚体信号传导是否可以进一步将这些干细胞衍生的早期T细胞分化为更成熟的CD 8+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.