Leukemia Specific Splice Isoforms as Neo-Antigens for T-Cell Immunotherapy

白血病特异性剪接亚型作为 T 细胞免疫治疗的新抗原

基本信息

批准号：
9208124
负责人：
Paul Michael Armistead
金额：
$ 50.04万
依托单位：
UNIV OF NORTH CAROLINA CHAPEL HILL
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-02-01 至 2021-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9208124
关键词：
Acute Myelocytic Leukemia Acute leukemia American Analytical Chemistry Antigen Presentation Antigens Autologous Bioinformatics Biological CD8-Positive T-Lymphocytes CD8B1 gene CRISPR/Cas technology Cell Line Cell model Cell-Mediated Cytolysis Cells Cessation of life Chemicals Chemistry Chronic Lymphocytic Leukemia Clinical Clinical Protocols Collaborations Complex Cytotoxic T-Lymphocytes Dana-Farber Cancer Institute Data Development Epitopes Funding Generations Genes Genetic Transcription Genomics Grant Hematologic Neoplasms Human Hybrids Immune Immune system Immunotherapy Laboratories Length Lymphocyte Malignant Neoplasms Manuscripts Mass Spectrum Analysis Measures Messenger RNA Methodology Methods Mutate Mutation North Carolina Pathway interactions Patients Peptides Population Process Production Protein Isoforms Protein Splicing Proteins Proteomics Publishing RNA Splicing Recurrence Research Research Proposals Role Sampling Somatic Mutation Source Spliceosomes Syndrome System T cell response T-Lymphocyte Techniques Testing Therapeutic Therapy trial Time Translations Universities Vaccine Therapy Viral Tumor Antigens chronic leukemia comparative computational chemistry computer science differential expression exome sequencing in vivo ion mobility killings leukemia leukemic stem cell mRNA Precursor mass spectrometer mortality novel prototype public health relevance tandem mass spectrometry therapeutic target transcriptome transcriptome sequencing transcriptomics

项目摘要

DESCRIPTION (provided by applicant): Chronic and acute leukemias are responsible for ~25,000 deaths annually in the US - a mortality rate that represents ~5% of all cancer related deaths. While leukemias are susceptible to antigen-specific T-cell mediated cytotoxicity, leukemia antigen (LA) discovery methods are complex and time consuming. Most LAs are derived from proteins that contain a somatic mutation or are aberrantly expressed or localized in the leukemia cell; however, another potential source of many LAs are antigenic peptides derived from leukemia specific protein splice isoforms. Our laboratory performed deep RNA-Sequencing of 8 acute myeloid leukemia (AML) samples, which did not contain spliceosome mutations, and identified >300,000 previously unannotated (i.e. novel) splice junctions. The central hypothesis of this project is that leukemia-specific splice junctions yield peptide epitopes that can be presented by class I HLA and targeted by CD8+ cytotoxic T-cells. Roughly 15% of AML and chronic lymphocytic leukemia (CLL) samples contain spliceosome mutations: primarily U2AF1 mutations in AML and SF3B1 mutations in CLL. In Aims 1 and 2, we will investigate the effect of these mutations on mRNA splicing, protein isoform generation and neo-antigen presentation in model cell lines modified to contain common U2AF1 and SF3B1 mutations (Aim 1) and then in human AML and CLL samples with or without a U2AF1 or SF3B1 mutation (Aim 2). In collaboration with Dr. Jan Prins, of the UNC Department of Computer Science, we will perform hybrid RNA-Sequencing (RNA-Seq) that combines short-read RNA-Seq data for splice junction discovery with long-read RNA-Seq to determine full mRNA isoform sequences. Following isoform sequencing we will test for protein translation of the isoform by probing for splice junction specific peptides using a prototype differential ion mobility spectrometer - mass spectrometer (DIMS-MS) that we have used with Dr. Gary Glish (UNC Department of Chemistry) on peptide discovery projects before. Aim 3 of this research proposal will apply these methods, as well as whole exome sequencing and comprehensive peptide epitope prediction performed by the lab of Dr. Catherine Wu, in a clinical protocol where we will genetically and computational predict LAs from AML or CLL patient samples, confirm their expression by DIMS-MS, and generate LA-specific T-cells from autologous patient-derived lymphocytes. At the completion of this project we will have characterized the effects of spliceosome mutations on mRNA splicing and its resultant effect on protein isoform production and epitope presentation. More significantly, we will have developed a methodology for personalized LA discovery, where we will identify and confirm patient-derived LA that are presented by the patient's HLA, and produce patient-derived LA-specific T-cells. The combined sequencing, bioinformatics and proteomics methods could be applied for the development of patient-specific T-cell therapeutics.

描述（由申请人提供）：在美国，慢性和急性白血病每年导致约 25,000 人死亡，死亡率约占所有癌症相关死亡的 5%，而白血病容易受到抗原特异性 T 细胞介导的细胞毒性的影响。白血病抗原 (LA) 的发现方法复杂且耗时，大多数 LA 来源于白血病细胞中含有体细胞突变或异常表达或定位的蛋白质；然而，许多 LA 的另一个潜在来源是源自白血病特异性蛋白剪接亚型的抗原肽，我们的实验室对 8 个急性髓系白血病 (AML) 样本进行了深度 RNA 测序，这些样本不包含剪接体突变，并鉴定了超过 300,000 个先前未注释的样本。该项目的中心假设是白血病特异性剪接点产生可以呈递的肽表位。大约 15% 的 AML 和慢性淋巴细胞白血病 (CLL) 样本含有剪接体突变：主要是 AML 中的 U2AF1 突变和 CLL 中的 SF3B1 突变。这些突变对含有常见 U2AF1 和SF3B1 突变（目标 1），然后在有或没有 U2AF1 或 SF3B1 突变的人类 AML 和 CLL 样本中（目标 2），我们将与北卡罗来纳大学计算机科学系的 Jan Prins 博士合作，进行混合 RNA 测序。 (RNA-Seq) 将用于剪接点发现的短读长 RNA-Seq 数据与长读长 RNA-Seq 相结合，以确定完整的 mRNA 同工型序列。在同工型测序之后，我们将测试蛋白质翻译。通过使用我们之前与 Gary Glish 博士（北卡罗来纳大学化学系）在肽发现项目的目标 3 中使用的原型微分离子迁移谱仪 - 质谱仪 (DIMS-MS) 来探测剪接点特异性肽，来分析异构体。研究计划将应用这些方法，以及 Catherine Wu 博士实验室进行的全外显子组测序和全面的肽表位预测，在临床方案中，我们将通过基因和计算方法预测 AML 或CLL 患者样本，通过 DIMS-MS 确认其表达，并从患者来源的自体淋巴细胞生成 LA 特异性 T 细胞。在该项目完成时，我们将表征剪接体突变对 mRNA 剪接的影响及其对 mRNA 剪接的影响。更重要的是，我们将开发一种个性化 LA 发现的方法，其中我们将识别和确认由患者 HLA 呈现的患者来源的 LA，并产生患者来源的 LA 特异性。 T 细胞。结合测序、生物信息学和蛋白质组学方法可用于开发患者特异性 T 细胞疗法。