Engineering detours around the biologic barriers to allogeneic, iPSC-derived CAR T immunotherapy

项目摘要/摘要 嵌合抗原受体(CAR)T细胞疗法在其他治疗中取得了显著效果- 难治性血液系统恶性肿瘤。目前，制造自体CAR T细胞的过程是 具有挑战性，因为需要重新生成每一种个体化治疗方法，以及 患者之间的T细胞生物学，导致不可预测和不一致的临床反应。因此，在那里 在体外从一种无限更新的人类同种异体来源中产生CAR T细胞的兴趣越来越大 多能干细胞(HPSC)。这种方法的一个优点是hPSCs高度服从于基因 编辑，提供多种途径来操纵最终T细胞产品的功能。 然而，从hPSC产生CAR T细胞存在几个生物障碍。第一，表达 在T细胞分化早期转CAR基因将发育转向先天淋巴途径 而不是传统的T血统。第二，去除内源性T细胞受体(TCR)以防止 同种异体反应导致发育受阻，因为成熟的T细胞不能再进行阳性选择。 这项提议寻求克服这些挑战，以产生非同种异体反应的干细胞来源的CAR T 通过部署创新的阶段特异性表达策略和一种新的体外方法来诱导 成熟的常规T细胞的分化，人工胸腺器官(ATO)系统。 ATO是一种一流的体外高效产生成熟、单一阳性(SP)CD8+的方法 以及来自多种干细胞来源的CD4+T细胞，包括hPSCs。在以前建立的基础上改进 系统，ATO可以有力地支持积极选择和成熟到SP阶段。我们的初步研究 已经证明，先天的命运转移可以通过实现延迟的、特定阶段的 仅限于成熟T细胞的CAR转基因表达。在本提案中，具体目标1将建立在 延迟CAR表达模型及内源性TCR干扰对CAR T细胞的影响 阿托斯群岛的发展。《特定目标2》将评估两种不同的外源性阳性传递方法 在缺乏内源TCR的情况下，选择信号拯救发育。这些方法包括 在生理上表达调性信号CAR或瞬时表达外源TCR 适宜的双阳性(DP)T细胞分期。本提案中制定的方法将使 产生hPSC衍生的非同种异体反应T细胞疗法，最终降低成本并增加 为更多有需要的患者提供治疗。

PROJECT SUMMARY/ABSTRACT Chimeric antigen receptor (CAR) T cell therapy has produced remarkable results in otherwise treatment- refractory hematological malignancies. Currently, the process of manufacturing autologous CAR T cells is challenging due to the need for de novo generation of each individualized therapy and the inherent variability in T cell biology between patients, leading to unpredictable and inconsistent clinical responses. As a result, there is a growing interest in generating CAR T cells in vitro from an infinitely renewing, allogeneic source of human pluripotent stem cells (hPSCs). One advantage of this approach is that hPSCs are highly amenable to genetic editing, providing multiple avenues to manipulate the function of the final T cell product. However, there are several biologic barriers to generating CAR T cells from hPSCs. First, expression of the CAR transgene early in T cell differentiation diverts development towards the innate lymphoid pathway instead of the conventional T lineage. Second, the removal of the endogenous T cell receptor (TCR) to prevent alloreactivity leads to a block in development, as maturing T cells can no longer undergo positive selection. This proposal seeks to overcome these challenges to generating non-alloreactive, stem cell-derived CAR T cells by deploying an innovative stage-specific expression strategy and a novel in vitro method to induce the differentiation of mature conventional T cells, the Artificial Thymic Organoid (ATO) system. The ATO is a first-in-class, in vitro method for efficiently generating mature, single positive (SP) CD8+ and CD4+ T cells from multiple stem cell sources, including hPSCs. Improving upon previously established systems, the ATO can robustly support positive selection and maturation to the SP stage. Our preliminary studies have already demonstrated that innate fate diversion can be overcome by achieving delayed, stage-specific expression of the CAR transgene that is limited to mature T cells. In this proposal, Specific Aim 1 will build upon the delayed CAR expression model and evaluate how the disruption of the endogenous TCR affects CAR T cell development in the ATOs. Specific Aim 2 will evaluate two different methods of delivering exogenous positive selection signals to rescue development in the absence of the endogenous TCR. These methods include expression of a tonically signaling CAR or transiently expressing an exogenous TCR at the physiologically appropriate double positive (DP) T cell stage. The approaches developed in this proposal will enable the generation of hPSC-derived, non-alloreactive T cell therapy, ultimately reducing the cost and increasing the access to treatment for more patients in need.