Tuning CAR-T cell function

调节 CAR-T 细胞功能

基本信息

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
项目类别：
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

项目摘要

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，共刺激由 CD28 和整合到 CD19 特异性 CAR 中的 4-1BB 内域已被证明在引起然而，CD28 和 4-1BB 共刺激对肿瘤的动力学、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 细胞激活相反，由编码 4-1BB 的 CAR 形成的突触招募 THEMIS-SHP1。我们有减弱 CAR-CD3ze 内域磷酸化和 T 细胞激活的磷酸酶复合物。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 共刺激的活性这一发现最近发表在《Cancer Cell》上。值得注意的是，我们观察到 LCK 介导 4-1BB- 中 CAR-CD3z 的组成型磷酸化共刺激 CAR-T 不会导致异种移植模型中 CAR-T 过早耗尽。我们追求 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，我们发现，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。 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 中的 NF-κB 活性高于 CD28。4-1BB 中 NF-κB 的高活性不是由 NF-κB 过度表达引起的。而是通过减少 A20 活性，因此，我们认为 4-1BB 会隔离 A20，从而减少其活性。此外，由于 NF-κB/A20 相互作用对于控制 T 细胞功能至关重要。多个层面上，我们追求调节 NF-κB/A20 可以增强治疗的有效性、持久性和安全性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，因为 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。