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

 描述(申请人提供)：慢性和急性白血病在美国每年造成约25,000人死亡--死亡率占所有癌症相关死亡人数的约5%。虽然白血病容易受到抗原特异性T细胞介导的细胞毒作用的影响，但白血病抗原(LA)的发现方法复杂且耗时。大多数LAS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LAS的另一个潜在来源是来自白血病特异性蛋白剪接异构体的抗原肽。我们的实验室对8个不包含剪接体突变的急性髓系白血病(AML)样本进行了深度RNA测序，发现了300,000个以前未注释的(即新的)剪接连接。该项目的中心假设是，白血病特异性剪接连接产生的多肽表位可以由I类人类白细胞抗原提呈，并被CD8+细胞毒T细胞靶向。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。在目标1和目标2中，我们将研究这些突变对含有共同U2AF1和SF3B1突变的模型细胞系(目标1)以及带有或不带有U2AF1或SF3B1突变的人类AML和CLL样本(目标2)的mRNA剪接、蛋白质异构体生成和新抗原呈递的影响。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)，将短读RNA-Seq数据用于剪接连接发现，与长读RNA-Seq相结合，以确定完整的mRNA异构体序列。在异构体测序之后，我们将使用我们之前在多肽发现项目中与Gary Glish博士一起使用的原型差分离子迁移率光谱仪-质谱仪(DIMS-MS)，通过探测剪接连接特定多肽来测试异构体的蛋白质翻译。这项研究计划的目标3将应用这些方法，以及Catherine Wu博士的实验室进行的完整外显子组测序和全面的多肽表位预测，在临床方案中，我们将从AML或CLL患者样本中通过遗传和计算预测LAS，通过DIMS-MS确认它们的表达，并从自体患者来源的淋巴细胞中产生LA特异性T细胞。在这个项目完成时，我们将表征剪接体突变对信使核糖核酸剪接的影响，以及它对蛋白质异构体的产生和表位呈现的影响。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患者来源的LA，这些LA由患者的HLA呈现，并产生患者来源的LA特异性T细胞。测序、生物信息学和蛋白质组学相结合的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.