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）通过单链抗体和 激活T细胞细胞毒性机制和共刺激。在临床研究中，CD28和 已整合到CD19特异性汽车中的4-1BB内分域已被证明在引起同样有效 肿瘤回归。但是，CD28和4-1BB的共刺激对动力学，代谢有所不同 CAR-T细胞的持久性以及管理这些差异的机制尚未完全理解。在 这项研究，我们已经确定，共核能募集到编码CD28的汽车突触中的LCK 导致抗原非依赖性的CAR-CD3ζ内域域的光磷酸化，并在T细胞激活上烙印 抗原参与。相反，编码4-1BB的汽车形成的突触招募了Themis-Shp1 磷酸酶复合物可减弱CAR-CD3ζ内域磷酸化和T细胞活化。我们有 还证明可以设计汽车突触以调整CD28共刺激的活动或 调整4-1BB共刺激的活动。该发现最近发表在癌细胞中。 值得注意的是，我们观察到LCK介导的CAR-CD3ζ在4-1BB--中的组成型磷酸化 在异种移植型模型中，CORTIMITIOM的CAR-TS不会导致CAR-TS过早耗尽。所以， 我们假设LCK介导的4-1BB的烙印构成了CORTAMER CAR-TS导致独特而关键 汽车-TS中的信号通路。 此外，除了近端信号传导外，汽车还深刻影响下游T细胞信号传导。我们发现 NF-κB活性受汽车共刺激类型的影响。确切地说，4-1BB影响更明显 NF-κB活性比CD28在CAR-TS中。 4-1BB中的NF-κB多动症不是由NF-κB过表达引起的， 而是通过减少A20活动。因此，我们假设4-1BB隔离a20减少了其 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 多级别，我们假设对NF-κB/A20进行调节可能会提高效率，持久性和安全性 汽车-TS。我们将开发两个具体目标： 目标1：机械地评估LCK介导的4-1BB的烙印如何促进速度 抗肿瘤活性而不会引起T细胞耗尽。我们将评估LCK是否在4-1BB中的过表达 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的影响以及如何操纵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中受到差异调节 加工的汽车TS，我们建议了解这种相互作用的功能并开发药理学 以及瞬时或永久调节NF-κB活性以提高安全性，持久性的遗传干预措施 和易于使用。