Tuning CAR-T cell function

调节 CAR-T 细胞功能

• 批准号: 10310510
• 负责人: Gianpietro Dotti
• 金额: $ 48.71万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-01-01 至 2025-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10310510
• 关键词: 1-Phosphatidylinositol 3-Kinase; Affect; Antibodies; Antigens; Attenuated; Automobile Driving; B-Lymphocytes; CD19 gene; CD28 gene; Cell Respiration; Cell physiology; Clinical; Clinical Research; Complex; Cytotoxic T-Lymphocytes; Data; Development; Engineering; Equilibrium; Event; Genes; Genetic Transcription; Human; Hyperactivity; Impairment; Kinetics; Lead; Malignant Neoplasms; Mediating; Metabolic Pathway; Metabolism; Monoclonal Antibodies; NR0B2 gene; Necrosis; Patients; Pharmacology; Phosphatidylinositols; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Pre-Clinical Model; Predisposition; Prevention; Protein Tyrosine Phosphatase; Proteins; Proteomics; Publishing; Reporting; Safety; Shapes; Side; Signal Pathway; Signal Transduction; Signaling Molecule; Solid Neoplasm; Speed; Synapses; T Chain; T cell therapy; T-Cell Activation; T-Cell Receptor; T-Lymphocyte; TNF Receptor-Associated Factors; TNF gene; Ubiquitin; Xenograft Model; Zinc Fingers; antigen binding; cancer cell; chimeric antigen receptor; chimeric antigen receptor T cells; cytokine; design; exhaustion; gene therapy; imprint; leukemia treatment; leukemia/lymphoma; metabolomics; neoplastic cell; novel; overexpression; premature; receptor; recruit; response; transcriptome; tumor

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.

抽象的 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并 激活 T 细胞的细胞毒性机制和共刺激。在临床研究中，CD28 介导的共刺激和 整合到 CD19 特异性 CAR 中的 4-1BB 内域已被证明在引起 肿瘤消退。然而，CD28 和 4-1BB 共刺激对动力学、代谢有不同的调节作用。 CAR-T 细胞的差异和持久性，以及控制这些差异的机制尚不完全清楚。在 在这项研究中，我们发现LCK被共受体招募到编码CD28的CAR的突触中 导致不依赖于抗原的 CAR-CD3ze 内域磷酸化并印记 T 细胞激活 抗原结合。相比之下，由编码 4-1BB 的 CAR 形成的突触招募 THEMIS-SHP1 磷酸酶复合物，可减弱 CAR-CD3ze 内域磷酸化和 T 细胞活化。我们有 还证明了 CAR 突触可以被设计来降低 CD28 共刺激的活性或 调整 4-1BB 共刺激的活动。这一发现最近发表在《Cancer Cell》杂志上。 值得注意的是，我们观察到 LCK 介导 4-1BB- 中 CAR-CD3z 的组成型磷酸化 在异种移植模型中，共刺激的 CAR-T 不会导致 CAR-T 过早耗尽。所以， 我们假设 4-1BB 共刺激 CAR-T 中 LCK 介导的印记会导致独特且关键的结果 CAR-T 中的信号通路。 此外，除了近端信号传导之外，CAR 还深刻影响下游 T 细胞信号传导。我们发现 NF-κB 活性受 CAR 共刺激类型的影响。准确地说，4-1BB 诱导更明显 CAR-T 中 NF-κB 活性高于 CD28。 4-1BB 中 NF-κB 过度活跃并非由 NF-κB 过度表达引起， 而是通过减少 A20 活性来实现。因此，我们假设 4-1BB 隔离 A20，从而降低其 对 NF-κB 的抑制作用。此外，由于 NF-κB/A20 相互作用对于控制 T 细胞功能至关重要 多个水平上，我们假设调节 NF-κB/A20 可能会增强疗效、持久性和安全性 CAR-T。我们将制定两个具体目标： 目标 1：从机制上评估 LCK 介导的 4-1BB 共刺激 CAR-T 的印记如何促进快速 抗肿瘤活性而不引起 T 细胞耗竭。我们将评估 4-1BB 中 LCK 是否过度表达 CAR 激活独特的磷酸化、转录组和代谢途径。 目标 2：从机制上评估 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 相互作用在 4-1BB 与 CD28 中受到不同的调节 共刺激 CAR-T，我们建议了解这种相互作用如何发挥作用并开发药理学 和基因干预可暂时或永久调节 NF-κB 活性，以增强安全性和持久性 以及 CAR-T 的功效。