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

基本信息

批准号：
10348150
负责人：
Alec Baker Wilkens
金额：
$ 5.18万
依托单位：
UNIVERSITY OF WASHINGTON
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：
2021-03-16 至 2024-03-15
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10348150
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 Data Reporting 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 Pharmacology Phenotype Physiological Production Publishing Receptor Activation Receptor Inhibition Receptor Signaling 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 cell 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 programs receptor single-cell RNA sequencing transcription factor transcriptomics tumor tumor xenograft

项目摘要

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.

项目摘要用于癌症免疫疗法的工程T细胞的当前方法使用激动剂抗CD3和-CD28 单克隆抗体（mAb）激活T细胞，这会诱导它们的增殖，但不会概括其他由抗原呈递细胞传递的命运信号。一个这样的信号轴，Notch途径，控制CD4+ T细胞效应子函数的获取并强烈影响行为。研究Notch信号传导在嵌合抗原受体（CAR）T细胞产生期间，我们使用抗CD3/CD28开发了培养系统 mab涂层的珠子和板涂的激动剂Notch1特异性mAb以诱导同时的T细胞激活和Notch信令。当转移到带有CD19+ Raji淋巴瘤的NSG小鼠中时，CD19特异性 NOTCH1型（N1）CD4+ CAR T细胞显示出明显的增殖优势（IgG）细胞。含有IgG CD8+ CAR T细胞和N1 CD4+ CAR T细胞的肿瘤小鼠证明了与给定IgG CD8+和IgG CD4+ CAR T细胞相比导致肿瘤的快速清除和免受肿瘤再挑战的保护。这些数据表明 Notch1激动剂可能代表了养子T细胞疗法的重大改善，但是机制目前尚不清楚Notch信号传导改善T细胞抗肿瘤功能的。 Notch是已知的诱导芳基烃受体（AHR）转录活性。 AHR活性的药理抑制在N1 CD4+ CAR T细胞培养中，N1和IgG细胞之间的特征差异降低了表面体外重新刺激后的表型，细胞因子产生和增殖。我假设Notch1 激动剂组织依赖AHR的转录组变化，从而增加增殖性细胞因子在CD4+ CAR T细胞中产生，促进CD4依赖性增殖，从而改善CAR T细胞功效。为了检验这一假设，我将评估N1中AHR抑制和激活的功能效应 IgG CD4+ CAR T细胞体外和体内，表征N1中的命运确定的转录事件 AHR抑制了N1 CD4+ CAR T细胞，并使用大量和单细胞RNA测序。总的来说，这些实验将研究Notch和AHR信号的实用性，在上级T细胞的产生中产品并建立Notch1激动剂提高CAR T细胞治疗功效的机制，为将这种有前途的生物医学前进转化为免疫疗法诊所的基础。