Synthetic biology toolkit for precise tuning of T cell activity

项目摘要/摘要。嵌合抗原受体(CAR)T细胞为基础的免疫治疗 在血液系统恶性肿瘤患者中显示出治疗潜力。然而，它面临着重大的安全问题 (例如，细胞因子释放综合征和神经毒性)和由紧张性信号和T细胞引起的疗效丧失 疲惫不堪。这些不受欢迎的特征或多或少在淋巴细胞激活的远端被解码 由基质组成的双组分钙释放激活钙通道(CRAC)途径 相互作用分子(STIM)和ORAI在T细胞中形成主要的钙离子进入途径，控制T细胞的激活。 在T细胞受体(TCR)结合后，内质网(ER)中的钙离子耗竭是由基质感受到的 相互作用分子1(STIM1)启动一系列构象变化，最终激活 CRAC通道的成孔亚基ORAI1。钙离子内流诱导T细胞内一系列过程，包括 细胞溶解颗粒的分泌和钙调神经磷酸酶、CaMKII等钙依赖酶的激活 和ERK1/2，以及主要转录因子，如核转录因子κB和活化T细胞的核因子， 对适应性免疫来说是必不可少的。最重要的是，核转移的NFAT以不同的方式与其 结合伙伴促进各种T细胞的激活、分化、无能/衰竭和效应功能 细胞子集。值得注意的是，在CAR-T细胞治疗中观察到的紧张性信号和疲惫与 高活性的钙/NFAT信号转导。到目前为止，还没有FDA批准的CRAC通道阻滞剂来调节 用于治疗应用的CRAC通道。因此，仍然存在开发小说的迫切需要 靶向T细胞远端CRAC通道的介入性方法。与大多数现有研究不同的是 关于CAR本身或调控CAR-T细胞激活途径的近端信号成分，本项目 专注于在不对嵌合体进行任何修改的情况下对淋巴细胞激活途径的远端进行工程 抗原受体或近端TCR信号。M-PI计划开发一套基因编码的 可由光或药物精确控制的CREC通道阻滞剂(LiCRAB for Aim 1，和 分别用于AIM 2的DiCRAB)，从而赋予对T细胞活性的严格控制，以微调T细胞的功效和 减轻CAR-T细胞紧张性信号和/或衰竭。该项目的成功实施将探索 创新的免疫工程方法加速人类智能细胞疗法的设计 疾病。从机制上讲，这些工具可以用来探讨钙/NFAT信号转导的动力学需求。 CAR-T细胞激活。从翻译的角度来看，我们将产生广泛适用的基因编码 治疗性T细胞功能调整的工具，它很有希望克服紧张性信号/疲惫， 并遏制与现有FDA批准的CAR T细胞疗法相关的细胞因子风暴。

Project Summary / Abstract. Chimeric antigen receptor (CAR) T cell-based immunotherapy has shown curative potential in patients with haematological malignancies. However, it faces significant safety issues (e.g., cytokine release syndrome and neurotoxicity) and efficacy loss arising from tonic signaling and T cell exhaustion. These undesireable features are more or less decoded in the distal end of lymphocyte activation pathway, the two-component Calcium Release-Activated Calcium (CRAC) channel composed of stromal interaction molecule (STIM) and ORAI to form a major Ca2+ entry route in T cells and control T cell activation. Upon T-cell receptor (TCR) engagement, Ca2+ depletion in the endoplasmic reticulum (ER) is sensed by stromal interaction molecule 1 (STIM1) to initiate a series of conformational changes, culminating in the activation of the pore-forming subunit of the CRAC channel, ORAI1. Ca2+ influx induces a series of processes in T cells, including the secretion of cytolytic granules and the activation of Ca2+-dependent enzymes, including calcineurin, CaMKII and Erk1/2, as well as master transcription factors, such as NF-κB and nuclear factor of activated-T cells (NFAT), that are essential for adaptive immunity. Most importantly, nuclear translocated NFAT differentially engages its binding partners to promote the activation, differentiation, anergy/exhaustion, and effector functions of various T cell subsets. Notably, tonic signaling and exhaustion observed in CAR-T cell therapy are associated with hyperactive Ca2+/NFAT signaling. Till now, no FDA-approved CRAC channel blockers are in hand to modulate the CRAC channel for therapeutic applications. There remains, therefore, a critical need to exploit novel interventional approaches by targeting the distal CRAC channels in T cells. Unlike most existing studies centered on CAR per se or the proximal signaling components in modulating CAR-T cell activation pathway, this project focuses on engineering the distal end of lymphocyte activation pathway without any modifications to the chimeric antigen receptor or proximal TCR signaling. The m-PIs propose to to develop a suite of genetically-encoded CRAC channel Blockers (CRAB) that can be precisely controlled by light or drugs (LiCRAB for Aim 1, and DiCRAB for Aim 2, respectively), thereby conferring tight control of T cell activity to fine-tune T cell efficacy and mitigate CAR-T cell tonic signaling and/or exhaustion. The successful execution of this project will explore innovative immunoengineering approaches to accelerate the design of intelligent cell-based therapies for human disease. Mechanistically, the tools can be utilized to probe the kinetic requirement of Ca2+/NFAT signaling during CAR-T cell activation. From a translational perspective, we will generate broadly-applicable genetically-encoded tools for therapeutic T cell functional tuning, which hold great promise to overcome tonic signaling / exhaustion, and curtail cytokine storm associated with existing FDA-approved CAR T-cell therapies.