Tuning CAR-T cell function

摘要 T细胞表达的嵌合抗原受体(CAR)通过单链抗体识别肿瘤细胞 激活T细胞的细胞毒机制和共刺激作用。在临床研究中，CD28和CD28介导的共刺激作用 整合到CD19特异性CAR中的4-1BB内域已被证明在导致 肿瘤消退。然而，CD28和4-1BB共刺激不同地调节动力学、新陈代谢 和CAR-T细胞的持久性，以及控制这些差异的机制还不完全清楚。在……里面 本研究中，我们发现LCK被共受体募集到编码CD28的CAR突触中 导致抗原非依赖性的CAR-CD3ζ内域磷酸化，并印记T细胞在 抗原结合。相反，编码4-1BB的CAR形成的突触招募THEMIS-SHP1 减弱CAR-CD3ζ内域磷酸化和T细胞激活的磷酸酶复合体。我们有 也证明了汽车突触可以被设计来调节CD28共刺激的活性或 调节4-1BB共刺激的活性。这一发现最近发表在《癌症细胞》杂志上。 值得注意的是，我们观察到LCK介导了4-1BB-Car-CD3ζ的结构性磷酸化。 在异种移植模型中，共刺激的CAR-T不会导致CAR-T的过早耗竭。因此， 我们假设在4-1BB共刺激的CAR中LCK介导的印迹导致唯一和关键的 汽车中的信号通路。 此外，除了近端信号外，CAR还对下游T细胞信号产生深远影响。我们发现 核因子-κB的活性受CAR共刺激类型的影响。准确地说，4-1BB诱导的更明显 核因子κB活性明显高于CD2 8。4-1BB中的核因子-κB高活性不是由核因子-κB的过度表达引起的， 而是通过减少A20的活性。因此，我们假设4-1BB隔离A20减少其 对核因子-κB的抑制作用此外，由于核因子-κB/A20的相互作用在控制T细胞功能方面起关键作用。 多个水平，我们假设调节NF-κB/A20可能增强治疗的有效性、持久性和安全性 小汽车。我们将制定两个具体目标： 目的1：机械评估LCK介导的4-1BB共刺激CAR印迹如何促进快速 具有抗肿瘤活性，且不会导致T细胞耗尽。我们将评估LCK在4-1BB中的过度表达 CAR激活独特的磷酸化、转录组和代谢途径。 目标2：机械评估4-1BB如何影响以及如何操纵NF-κB/A20相互作用 调节CAR-T细胞功能。由于A20/NF-κB的相互作用在4-1BB和CD28中受到不同的调节 共刺激细胞，我们建议了解这种相互作用是如何发挥作用的，并开发药理学 和基因干预，以瞬时或永久地调节NF-κB活性，以增强安全性、持久性 和CAR-T的疗效。

Abstract Chimeric antigen receptors (CAR) expressed by T cells recognize tumor cells via single chains antibodies and activate T cell cytotoxic machinery and costimulation. In clinical studies, costimulation mediated by CD28 and 4-1BB endodomains integrated into the CD19-specific CAR has been shown to be equally effective in causing tumor regression. However, CD28 and 4-1BB costimulation differentially modulates the kinetic, metabolism and persistence of CAR-T cells, and the mechanisms governing these differences are not fully understood. In this study, we have identified that LCK recruited by co-receptors into the synapse of the CAR encoding CD28 leads to antigen-independent CAR-CD3ζ endodomain phosphorylation and imprints T cell activation upon antigen engagement. In contrast, the synapse formed by the CAR encoding 4-1BB recruits the THEMIS-SHP1 phosphatase complex that attenuates CAR-CD3ζ endodomain phosphorylation and T cell activation. We have also proved that the CAR synapse can be engineered to tune down the activity of CD28 costimulation or to tune up the activity of the 4-1BB costimulation. This discovery has been recently published in Cancer Cell. Remarkably, we observed that LCK mediated constitutive phosphorylation of CAR-CD3ζ in 4-1BB- costimulated CAR-Ts does not lead to premature exhaustion of CAR-Ts in xenotransplant models. Therefore, we hypothesize that the LCK-mediated imprinting in 4-1BB costimulated CAR-Ts leads to unique and critical signaling pathways in CAR-Ts. Furthermore, in addition to proximal signaling, CARs profoundly affect downstream T cell signaling. We found that NF-κB activity is influenced by the type of CAR costimulation. Precisely, 4-1BB induces more pronounced NF-κB activity than CD28 in CAR-Ts. NF-κB hyperactivity in 4-1BB is not caused by NF-κB overexpression, but rather by reduced A20 activity. Therefore, we hypothesize that 4-1BB sequesters A20 reducing its inhibitory effects on NF-κB. Furthermore, since NF-κB/A20 interplay is critical in controlling T cell function at multiple levels, we hypothesize that regulating NF-κB/A20 may enhance efficacy, persistence and safety of CAR-Ts. We will develop two specific aims: Aim 1: To mechanistically assess how LCK-mediated imprinting of 4-1BB costimulated CAR-Ts promotes rapid antitumor activity without causing T cell exhaustion. We will assess whether LCK overexpression in the 4-1BB CAR activates unique phosphorylation, transcriptome and metabolic pathways. Aim 2: To mechanistically assess how 4-1BB affect and how the NF-κB/A20 interplay can be manipulated to modulate CAR-T cell functions. Since A20/NF-κB interplay is differentially regulated in 4-1BB vs. CD28 costimulated CAR-Ts, we propose to understand how this interplay functions and to develop pharmacologic and genetic interventions to transiently or permanently modulate NF-κB activity to enhance safety, persistence and efficacy of CAR-Ts.