Synthetic biology toolkit for precise tuning of T cell activity

项目概要/摘要。基于嵌合抗原受体（CAR）T细胞的免疫疗法具有 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， (e.g.,细胞因子释放综合征和神经毒性）以及由紧张性信号传导和T细胞 疲惫不堪这些不受欢迎的特征或多或少在淋巴细胞活化的远端被解码 钙释放激活钙通道（CRAC）是一种双组分的钙释放激活钙通道， 相互作用分子（STIM）和奥赖在T细胞中形成主要的Ca 2+进入途径并控制T细胞活化。 在T细胞受体（TCR）接合后，内质网（ER）中的Ca 2+耗尽被基质细胞（ECM）感知。 相互作用分子1（STIM 1）引发一系列构象变化，最终激活了 CRAC通道的成孔亚基，ORAI 1。Ca 2+内流诱导T细胞中的一系列过程，包括 细胞溶解颗粒的分泌和Ca 2+依赖性酶的激活，包括钙调神经磷酸酶、CaMK II 和Erk 1/2，以及主转录因子，如NF-κB和活化T细胞核因子（NFAT）， 对适应性免疫至关重要。最重要的是，核转位的NFAT差异性地参与了其 结合配偶体以促进各种T细胞的活化、分化、无反应性/耗竭和效应子功能， 细胞亚群值得注意的是，在CAR-T细胞疗法中观察到的紧张性信号传导和耗竭与以下相关： 高活性的Ca 2 +/NFAT信号传导。到目前为止，没有FDA批准的CRAC通道阻滞剂在手，以调节 用于治疗应用的CRAC通道。因此，仍然迫切需要开发新的 通过靶向T细胞中的远端CRAC通道的介入方法。与大多数现有研究不同， 在CAR本身或调节CAR-T细胞活化途径的近端信号成分上，该项目 重点是工程化淋巴细胞活化途径的远端，而不对嵌合体 抗原受体或近端TCR信号传导。m-PI建议开发一套基因编码的 CRAC通道阻断剂（CRAB），其可以通过光或药物精确控制（LiCRAB用于Aim 1，和 DiCRAB分别用于Aim 2），从而赋予T细胞活性的严格控制以微调T细胞功效， 减轻CAR-T细胞紧张性信号传导和/或耗竭。该项目的成功实施将探索 创新的免疫工程方法，以加速人类智能细胞治疗的设计 疾病从机制上讲，这些工具可用于探测在细胞周期中Ca 2 +/NFAT信号传导的动力学要求。 CAR-T细胞活化。从翻译的角度来看，我们将产生广泛适用的基因编码的 用于治疗性T细胞功能调节的工具，其具有克服紧张性信号传导/衰竭的巨大希望， 并减少与现有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.