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

 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例死亡-死亡率占所有癌症相关死亡的约5%。虽然白血病对抗原特异性T细胞介导的细胞毒性敏感，但白血病抗原（LA）发现方法复杂且耗时。大多数LA来源于含有体细胞突变或在白血病细胞中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来源于白血病特异性蛋白质剪接同种型的抗原肽。我们的实验室对8个不含剪接体突变的急性髓性白血病（AML）样本进行了深度RNA测序，并鉴定了> 300，000个先前未注释的（即新的）剪接点。该项目的中心假设是白血病特异性剪接点产生可以由I类HLA呈递并由CD 8+细胞毒性T细胞靶向的肽表位。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：AML中主要是U2 AF 1突变，CLL中主要是SF 3B 1突变。在目的1和2中，我们将研究这些突变对经修饰以含有常见U2 AF 1和SF 3B 1突变的模型细胞系（目的1）以及随后在有或无U2 AF 1或SF 3B 1突变的人AML和CLL样本（目的2）中的mRNA剪接、蛋白质同种型生成和新抗原呈递的影响。与计算机科学系的Jan Prins博士合作，我们将进行混合RNA测序（RNA-Seq），将用于剪接点发现的短读RNA-Seq数据与长读RNA-Seq相结合，以确定完整的mRNA亚型序列。在亚型测序后，我们将通过使用原型差示离子迁移谱仪-质谱仪（DIMS-MS）探测剪接点特异性肽来测试亚型的蛋白质翻译，我们之前曾与加里Glish博士（化学系）在肽发现项目中使用过该仪器。本研究提案的目的3将在临床方案中应用这些方法，以及Catherine Wu博士实验室进行的全外显子组测序和综合肽表位预测，我们将从AML或CLL患者样本中遗传和计算预测LA，通过DIMS-MS确认其表达，并从自体患者来源的淋巴细胞中产生LA特异性T细胞。在这个项目完成后，我们将有特点的剪接体突变对mRNA剪接的影响，其对蛋白质异构体的生产和表位呈递的影响。更重要的是，我们将开发出一种个性化LA发现的方法，其中我们将鉴定和确认由患者HLA呈递的患者来源的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以应用于患者特异性T细胞疗法的开发。

 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.