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

• 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
• 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

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 嵌合抗原受体工程化 T 细胞（CAR T 细胞）为个性化癌症提供突破 治疗。在这种方法中，编码针对肿瘤抗原的 CAR 的基因被递送到患者 T 细胞中 使用慢病毒载体进行体内研究，然后将细胞回输到患者体内。工程化 CAR T 细胞在患者体内扩增 攻击并消灭肿瘤抗原阳性的癌细胞。 CAR T 细胞具有强劲的临床反应 某些白血病如小儿急性淋巴细胞白血病（ALL）的治疗，但在以下人群中的反应率较低 成人 ALL、慢性淋巴细胞白血病 (CLL) 和多发性骨髓瘤。 CAR 整合的结果之一是 在患者 T 细胞中编码慢病毒载体是对宿主基因组的局部破坏。我们最近发布了 所产生的插入诱变支持成功治疗的示例——患者 T 细胞扩增 与 TET2 中的整合事件相关，TET2 编码参与 CpG 去甲基化的酶，以及 这在机制上与增强的 T 细胞功能和持久缓解有关。在这里我们利用 来自患者 CAR T 细胞插入突变的数据，用于识别特别重要的基因和通路 以获得有效的抗肿瘤活性。 TGFBR2 提供了插入突变的第二个例子 与 CAR T 细胞的扩增有关，单独的研究也表明这种细胞的功能下降 与改善 CART 功能相关的基因。目前正在大力调整这两个方面 提高治疗成功率的途径。我们已经完成了整合站点的纵向分析 针对 ALL 和 CLL 的 40 名接受 CAR T 治疗的受试者中的分布，并发现了许多克隆的例子 扩大患者对治疗的成功反应，为了解 CAR T 细胞功能提供了独特的窗口。 我们手头有另外 266 名受试者的样本，其中一些是表现出长期反应的反应者 CAR T 细胞的持久性。我们进一步设计了一系列细胞培养和小鼠模型测定法 调节靶基因的活性并表征 CAR T 细胞增殖和抗肿瘤活性。因此我们 提议详细研究这些基因和途径，并开发临床操纵它们的方法。 我们提出以下具体目标： 目标 1. 阐明高级 CAR T 细胞增殖的规则 和利用慢病毒整合作为插入诱变剂的持久性。目标 2. 执行职能 分析与 CAR T 细胞载体驱动相关的基因，以确定对 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作方法 对于有效的 CAR T 增殖和功能很重要的基因和途径，然后将直接获取 进入临床开发。