Role of NOTCH1 Signaling in Engineering CD4+ T Cells for Cancer Immunotherapy

NOTCH1 信号转导在改造 CD4 T 细胞用于癌症免疫治疗中的作用

• 批准号: 10596984
• 负责人: Alec Baker Wilkens
• 金额: $ 5.27万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-03-16 至 2024-03-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10596984
• 关键词: Agonist; Antigen-Presenting Cells; Area; Aryl Hydrocarbon Receptor; Biomedical Research; CD19 gene; CD28 gene; CD3 Antigens; CD4 Positive T Lymphocytes; CD8-Positive T-Lymphocytes; CD8B1 gene; Cell Culture Techniques; Cell physiology; Cells; Characteristics; Clinic; Coculture Techniques; Communities; Data; Disease; Dissociation; Engineering; Event; Exhibits; Gene Expression; Gene Expression Regulation; Generations; Genes; Genetic Transcription; Hematology; Human; IL7 gene; Immunoglobulin G; Immunotherapy; In Vitro; Interleukin-2; Learning; Ligands; Ligation; Lymphoma; Malignant Neoplasms; Mediating; Methods; Monoclonal Antibodies; Mus; NOTCH1 gene; Pathway interactions; Phenotype; Physiological; Production; Proliferating; Publishing; Receptor Activation; Receptor Inhibition; Receptor Signaling; Reporting; Role; Signal Transduction; Solid; Stromal Cells; Surface; System; T cell differentiation; T cell therapy; T-Cell Activation; T-Cell Proliferation; T-Lymphocyte; Testing; Translating; Translations; Treatment Efficacy; Work; Xenograft Model; behavior influence; cancer immunotherapy; cell behavior; chimeric antigen receptor; chimeric antigen receptor T cells; cytokine; effector T cell; engineered T cells; experimental study; improved; in vivo; inhibitor; neglect; next generation; notch protein; novel; pharmacologic; programs; receptor; single-cell RNA sequencing; transcription factor; transcriptomic profiling; transcriptomics; tumor; tumor xenograft

PROJECT SUMMARY Current methods for engineering T cells for cancer immunotherapy use agonistic anti-CD3 and -CD28 monoclonal antibodies (mAb) to activate T cells, which induce their proliferation but do not recapitulate other fate-determining signals delivered by antigen-presenting cells. One such signaling axis, the NOTCH pathway, controls CD4+ T cell effector function acquisition and strongly influences behavior. To study NOTCH signaling during chimeric antigen receptor (CAR) T cell production, we developed a culture system using anti-CD3/CD28 mAb-coated beads and plate-coated agonistic NOTCH1-specific mAb to induce simultaneous T cell activation and NOTCH signaling. When transferred into NSG mice bearing CD19+ Raji lymphoma, CD19-specific NOTCH1-agonized (N1) CD4+ CAR T cells displayed a marked proliferative advantage over control (IgG) cells. Tumor-bearing mice given both IgG CD8+ CAR T cells and N1 CD4+ CAR T cells demonstrated superior expansion of both subsets compared to mice given IgG CD8+ and IgG CD4+ CAR T cells, resulting in rapid tumor clearance and protection from tumor re-challenge. These data demonstrate that NOTCH1 agonism could represent a significant improvement to adoptive T cell therapy, but the mechanisms by which NOTCH signaling improves T cell anti-tumor function are currently not understood. NOTCH is known to induce aryl hydrocarbon receptor (AhR) transcriptional activity. Pharmacologic inhibition of AhR activity during N1 CD4+ CAR T cell culture reduced characteristic differences between N1 and IgG cells in surface phenotype, cytokine production and proliferation upon in vitro restimulation. I hypothesize that NOTCH1 agonism organizes AhR-dependent transcriptomic changes that augment proliferative cytokine production in CD4+ CAR T cells, promoting CD4-dependent proliferation that improves CAR T cell efficacy. To test this hypothesis, I will assess the functional effects of AhR inhibition and activation in N1 and IgG CD4+ CAR T cells in vitro and in vivo, and characterize fate-determining transcriptional events in N1, IgG and AhR-inhibited N1 CD4+ CAR T cells using bulk and single-cell RNA sequencing. Collectively, these experiments will investigate the utility of NOTCH and AhR signaling in the generation of superior CAR T cell products and establish the mechanisms by which NOTCH1 agonism improves CAR T cell therapeutic efficacy, laying the groundwork for translation of this promising biomedical advance into immunotherapy clinics.

项目摘要 目前用于癌症免疫治疗的工程化T细胞的方法使用激动性抗-CD 3和-CD 28 单克隆抗体（mAb）激活T细胞，诱导其增殖，但不重演其他 由抗原呈递细胞传递的决定命运的信号。一个这样的信号传导轴，NOTCH途径， 控制CD 4 + T细胞效应功能的获得并强烈影响行为。研究NOTCH信号 在嵌合抗原受体（CAR）T细胞生产过程中，我们开发了一种使用抗CD 3/CD 28的培养系统， mAb包被的珠和板包被的激动性NOTCH 1特异性mAb诱导同时T细胞活化 和NOTCH信令。当转移到携带CD 19 + Raji淋巴瘤的NSG小鼠中时，CD 19特异性 NOTCH 1激动的（N1）CD 4 + CAR T细胞显示出优于对照（IgG）的显著增殖优势。 细胞给予IgG CD 8 + CAR T细胞和N1 CD 4 + CAR T细胞两者的荷瘤小鼠显示出 与给予IgG CD 8+和IgG CD 4 + CAR T细胞的小鼠相比，两个亚群的扩增均具有上级性， 从而导致快速的肿瘤清除和防止肿瘤再攻击。这些数据证明 NOTCH 1激动可能代表了过继性T细胞治疗的显著改善，但其机制可能与细胞毒性有关。 NOTCH信号传导通过何种途径改善T细胞抗肿瘤功能目前尚不清楚。Notch已知 以诱导芳烃受体（AhR）转录活性。AhR活性的药理学抑制 在N1期间，CD 4 + CAR T细胞培养减少了N1和IgG细胞之间在表面 表型、细胞因子产生和体外再刺激后的增殖。我假设NOTCH 1 激动作用组织AhR依赖性转录组学变化， 在CD 4 + CAR T细胞中产生，促进CD 4依赖性增殖，改善CAR T细胞 功效为了验证这一假设，我将评估AhR抑制和激活在N1中的功能效应， 体外和体内IgG CD 4 + CAR T细胞，并表征N1，IgG 和AhR抑制的N1 CD 4 + CAR T细胞。总的来说，这些 实验将研究NOTCH和AhR信号传导在产生上级CAR T细胞中的效用 并建立了NOTCH 1激动改善CAR T细胞治疗功效的机制， 为将这一有前途的生物医学进步转化为免疫治疗诊所奠定了基础。