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.

摘要 嵌合抗原受体工程T细胞(CAR T细胞)为个体化癌症提供突破 心理治疗。在这种方法中，编码针对肿瘤抗原的CAR的基因被输送到患者的T细胞中，例如 体内使用慢病毒载体，然后将细胞重新注入患者体内。基因工程的CAR T细胞在患者体内扩增 并攻击和摧毁肿瘤抗原阳性的癌细胞。CAR T细胞的临床反应很强 治疗一些白血病，如儿童急性淋巴细胞白血病(ALL)，但对 成人ALL、慢性淋巴细胞白血病(CLL)和多发性骨髓瘤。汽车一体化的一个后果是- 在患者T细胞中编码慢病毒载体是宿主基因组的局部破坏。我们最近发表了一篇 导致插入突变支持成功治疗的例子--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 这与增强的T细胞功能和持久的缓解有关。在这里，我们利用 来自患者CAR T细胞插入突变的数据，以确定特别重要的基因和途径 有效的抗肿瘤活性。TGFBR2提供了插入突变发生在哪里的第二个例子 与CAR T细胞的扩张有关，单独的研究也表明这一功能降低 与改善购物车功能相关的基因。目前正在加紧努力，以调节这两个因素 提高治疗成功率的途径。我们已经完成了整合场地的纵向分析 在40名接受CAR T治疗的受试者中的分布，针对ALL和CLL，并发现了大量克隆性的例子 患者的扩增成功对治疗有反应，为了解CAR T细胞功能提供了独特的窗口。 我们手头还有另外266名受试者的样本，其中一些是长期应答者。 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的检测方法 调节靶基因的活性，表征CAR T细胞的增殖和抗肿瘤活性。因此，我们 建议详细研究这些基因和途径，并开发临床操作方法。 我们提出了以下具体目标：目的1.阐明高级CAR T细胞增殖的规律 以及利用慢病毒整合作为插入诱变剂的持久性。目标2.实施职能 分析CAR T细胞载体驱动相关基因以确定CAR重要的蛋白质和途径 T细胞增殖性、持久性和抗肿瘤活性。这个项目的输出将是操纵的方法 对有效的CAR T增殖和功能至关重要的基因和途径，然后将直接进行 进入临床开发阶段。