Linking insertional mutagenesis and cell function to improve CAR T cell therapy

将插入突变与细胞功能联系起来以改善 CAR T 细胞疗法

基本信息

批准号：
10640072
负责人：
Frederic D Bushman
金额：
$ 59.21万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-06-01 至 2024-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10640072
关键词：
Acute Lymphocytic Leukemia Adult Acute Lymphocytic Leukemia Alleles Animal Model Antigens Apoptosis Automobile Driving B lymphoid malignancy Bioinformatics Biological Assay CAR T cell therapy CD19 gene Cancer Patient Cell Culture Techniques Cell physiology Cells Cessation of life Childhood Acute Lymphocytic Leukemia Chronic Lymphocytic Leukemia Clinical Clonal Expansion Clone Cells Clustered Regularly Interspaced Short Palindromic Repeats Custom Cyclic AMP DNA Data Data Set Disease remission Engineering Enzymes Event Failure Gene Modified Gene Targeting Genes Genome Goals Hand Human Human Genome Inositol Phosphates Insertional Mutagenesis Lentivirus Vector Link Malignant Neoplasms Methods Multiple Myeloma Mutagens Output Pathway interactions Patients Phosphotransferases Positioning Attribute Proliferating Proteins Publishing Reagent Reporter Genes Reporting Role Route Sampling Series Solid Neoplasm Specific qualifier value System T cell differentiation T cell therapy T-Cell Proliferation T-Lymphocyte TGFBR2 gene Testing Therapeutic Time Translating Tumor Antigens Validation cancer cell cancer immunotherapy cancer therapy cell growth chimeric antigen receptor clinical development cohort deep sequencing demethylation engineered T cells follow-up gene function genetic manipulation high risk improved improved outcome integration site kinase inhibitor lentiviral integration leukemia longitudinal analysis mouse model personalized cancer therapy receptor relapse risk response success tissue culture tumor vector

项目摘要

Summary Chimeric antigen receptor-engineered T cells (CAR T cells) provide a breakthrough for personalized cancer therapy. In this approach, a gene encoding a CAR targeting tumor antigens is delivered into patient T-cells ex vivo using a lentiviral vector, then cells reinfused into patients. The engineered CAR T cells expand in the patient and attack and destroy tumor antigen-positive cancer cells. Robust clinical responses are seen with CAR T cell therapy in some leukemias such as pediatric acute lymphocytic leukemia (ALL), but lower rates of response in adult ALL, chronic lymphocytic leukemia (CLL) and multiple myeloma. One consequence of integration of a CAR- encoding lentiviral vector in patient T cells is local disruption of the host genome. We recently published an example where the resulting insertional mutagenesis bolstered successful therapy--patient T-cell expansion was associated with an integration event in TET2, which encodes an enzyme involved in CpG demethylation, and this was mechanistically linked with enhanced T cell function and durable remission. Here we take advantage of data from insertional mutagenesis of patient CAR T cells to identify genes and pathways of particular importance for effective anti-tumor activity. TGFBR2 provides a second example of where insertional mutagenesis was associated with expansion of CAR T cells, and separate studies have also implicated reduced function of this gene as associated with improved CART function. Intense efforts are now under way to modulate both of these pathways to enhance therapeutic success. We have completed longitudinal analysis of integration site distributions in 40 CAR T-treated subjects, targeting both ALL and CLL, and find numerous examples of clonal expansions in patients successfully responding to therapy, providing a unique window on CAR T cell function. We have in hand samples from another 266 subjects, some of whom are responders showing long term persistence of CAR T cells. We have further devised a series of assays in cell culture and mouse models to modulate activity of targeted genes and characterize CAR T cell proliferation and anti-tumor activity. Thus we propose to investigate these genes and pathways in detail and develop means for manipulating them clinically. We propose the following Specific Aims: Aim 1. Elucidate the rules governing superior CAR T cell proliferation and persistence taking advantage of lentiviral integration as an insertional mutagen. Aim 2. Carry out functional analyses of genes implicated in vector driving of CAR T cells to identify proteins and pathways important for CAR T proliferation, persistence and anti-tumor activity. The output of this project will be methods for manipulating genes and pathways important for effective CAR T proliferation and function, which will then be taken directly into clinical development.

概括嵌合抗原受体工程T细胞（CAR T细胞）为个性化癌症提供了突破治疗。在这种方法中，编码靶向肿瘤抗原的汽车的基因被输送到患者T细胞中体内使用慢病毒载体，然后将细胞重新植入患者中。工程的汽车T细胞在患者中扩展并攻击并破坏肿瘤抗原阳性癌细胞。使用CAR T细胞可以看到强大的临床反应在某些白血病中的治疗，例如小儿急性淋巴细胞性白血病（全部），但较低的反应率成人全部，慢性淋巴细胞性白血病（CLL）和多发性骨髓瘤。汽车整合的结果之一在患者T细胞中编码慢病毒载体的是宿主基因组的局部破坏。我们最近发表了示例所得的插入诱变增强成功治疗的示例 - 患者T细胞扩张是与TET2中的集成事件相关，该事件编码涉及CpG脱甲基化的酶，并且这是与增强的T细胞功能和持久缓解相关的。在这里我们利用来自患者汽车T细胞插入诱变的数据，以识别具有特殊重要性的基因和途径有效的抗肿瘤活性。 TGFBR2提供了插入诱变的第二个例子与CAR T细胞的扩展以及单独的研究有关，也暗示了此功能的功能降低基因与改善的CART功能相关。现在正在努力调整这两个增强治疗成功的途径。我们已经完成了整合位点的纵向分析 40个由T型THEREAT受试者的分布，针对所有人和CLL，并找到许多克隆的例子成功反应治疗的患者的扩张，为CAR T细胞功能提供了独特的窗口。我们手头有266个受试者的样本，其中一些是响应者长期显示的汽车T细胞的持久性。我们进一步设计了一系列在细胞培养和小鼠模型中的测定调节靶向基因的活性并表征CAR T细胞增殖和抗肿瘤活性。因此我们建议详细研究这些基因和途径，并开发手段以在临床上操纵它们。我们提出以下特定目的：目标1。阐明有关上级C型细胞增殖的规则持久性利用慢病毒整合作为插入诱变。目标2。执行功能对与载体驱动CAR T细胞相关的基因分析，以鉴定对CAR重要的蛋白质和途径 t增殖，持久性和抗肿瘤活性。该项目的输出将是操纵的方法对于有效的汽车t增殖和功能很重要的基因和途径，然后将直接采用进入临床发展。