Discovery of autoimmune antigens from non-coding sequences

从非编码序列发现自身免疫抗原

• 批准号: 10286063
• 负责人: Miriam Isaaca Rosenberg
• 金额: $ 18.08万
• 依托单位: HEBREW UNIVERSITY OF JERUSALEM
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-13 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10286063
• 关键词: Acute; Address; Adult; Affinity; Antibodies; Antigens; Autoantibodies; Autoantigens; Autoimmune; Autoimmune Diseases; Autoimmunity; Bacteriophage T7; Bacteriophages; Base Sequence; Binding; Biological Assay; Biological Markers; Brain; Cells; Cerebrospinal Fluid; Characteristics; Childhood; Clinical; Complex; DNA Transposable Elements; Diagnosis; Disease; Disease Marker; Embryo; Encephalitis; Epitopes; Genes; Genome; Goals; HLA Antigens; Human; Immune Targeting; Immune system; Immunoprecipitation; Immunosuppression; Knowledge; Lead; Length; Libraries; Light; Link; Mass Spectrum Analysis; Methods; Molecular; Monitor; Multiple Sclerosis; N-Methyl-D-Aspartate Receptors; Neuroimmune; Neuromyelitis Optica; Neurons; Oligonucleotides; Open Reading Frames; Opsoclonus-Myoclonus Syndromes; Orphan; Pathway interactions; Patients; Peptide Library; Peptides; Phage Display; Phage ImmunoPrecipitation Sequencing; Positioning Attribute; Process; Proteins; Proteome; Random Peptide Libraries; Reporting; Risk; Sampling; Serum; Source; Specificity; Testing; Translating; Translations; Tumor Antigens; Untranslated RNA; Untranslated Regions; Variant; Virion; Work; adaptive immunity; antigen diagnostic; base; cohort; combinatorial; design; expression vector; high risk; immunogenic; improved; mimetics; neglect; next generation sequencing; novel diagnostics; patient screening; pediatric patients; population based; screening; specific biomarkers; success; tool; tumor

7. Project Summary. This project addresses an acute clinical need for the many neuroimmune diseases with no known causative antigen, and those with variation that remains unexplained by single defining antigens. Antibodies in NMDA-receptor encephalitis (α-NMDAR) have revolutionized diagnosis and treatment of that paraneoplastic disease (PND), while other PNDs, like Opsoclonus Myoclonus Syndrome (OMS), await these groundbreaking discoveries. Several methods, such as Phage immunoprecipitation and sequencing (PhIP-seq) have been used successfully to pan for biomarkers and defining autoantigens. However, for some diseases, these methods have failed. One possibility is that defining antigens have been missed by conventional screens because of unconventional origins. We hypothesize that some important autoantigens may reside within “non-coding” sequences. These include the vast landscape of small open reading frames (sORFs), or “micropeptides”, that originate in regions of the genome, like UTRs and long “non-coding” RNAs, that may have been erroneously overlooked in past screens. Importantly for this work, their proven translation is temporally and spatially regulated, they can be tumor specific antigens and are presented to the immune system. A second source of untested proteins in these assays is transposable elements. The proteins they naturally encode, and those that arise from their transposition into active genes, are also potential sources of immunogenic material. While few are active, their expression in embryos, adult brain and in tumor suggest their potential relevance for paraneoplastic autoimmunity. Cryptic peptides generated by a host of mechanisms remain a further challenge to screens that rely on defined target reference peptidomes. Our study aims to improve upon previous PhIPseq searches, by designing and building new libraries that cover this unconventional set of peptides. In Aim 1, we will design and build two new T7 PhIP-seq libraries covering this defined “non-coding” peptidome: one with targets represented as arrays of oligos encoding overlapping peptides, and a second in which full-length targets are expressed, to mitigate risk of disrupting epitopes by sequence fragmentation. In Aim 2, we will screen samples from pediatric neuroimmune disease patients (and controls) against these gene-specific libraries, to identify distinctive signatures of reactivity. To capture information about cryptic antigenic peptides, Aim 3 will test the same samples against an extant random peptide library, which reports on convergent feature specificity of patient samples from each disorder. This high-risk approach, if successful, will lead quickly to revolutionary new diagnostic paradigms for the neuroimmune diseases tested, as it did for NMDAR encephalitis, with paradigm-shifting potential for other autoimmune diseases, as well.

7. 项目概要。 该项目解决了许多未知的神经免疫疾病的迫切临床需求 致病抗原，以及那些具有单一定义抗原无法解释的变异的抗原。 NMDA 受体脑炎 (α-NMDAR) 的抗体彻底改变了以下疾病的诊断和治疗 副肿瘤性疾病 (PND)，而其他 PND，如眼阵挛性肌阵挛综合征 (OMS)， 等待这些突破性的发现。多种方法，例如噬菌体免疫沉淀法和 测序 (PhIP-seq) 已成功用于寻找生物标志物和定义自身抗原。 然而，对于某些疾病，这些方法却失败了。一种可能性是定义抗原 由于非传统的起源而被传统屏幕所错过。我们假设 一些重要的自身抗原可能存在于“非编码”序列中。其中包括广大的 小型开放阅读框（sORF）或“微肽”的景观，起源于 基因组，如 UTR 和长“非编码”RNA，过去可能被错误地忽视 屏幕。对于这项工作来说重要的是，他们经过验证的翻译是在时间和空间上受到调节的，他们 可以是肿瘤特异性抗原并被呈递给免疫系统。第二个来源 这些测定中未经测试的蛋白质是转座元件。它们天然编码的蛋白质，以及 那些因转座成活性基因而产生的，也是免疫原性的潜在来源 材料。虽然很少有活跃的，但它们在胚胎、成人大脑和肿瘤中的表达表明它们 与副肿瘤自身免疫的潜在相关性。由许多宿主产生的隐性肽 对于依赖定义的目标参考肽组的筛选来说，机制仍然是一个进一步的挑战。 我们的研究旨在通过设计和构建新的库来改进以前的 PhIPseq 搜索 涵盖了这组非常规的肽。在目标 1 中，我们将设计并构建两个新的 T7 PhIP-seq 涵盖这一定义的“非编码”肽组的库：其目标表示为数组 编码重叠肽的寡核苷酸，以及表达全长靶标的第二个寡核苷酸，以 降低序列断裂破坏表位的风险。在目标 2 中，我们将筛选来自 儿科神经免疫疾病患者（和对照）针对这些基因特异性库，以确定 独特的反应特征。为了捕获有关神秘抗原肽的信息，Aim 3 将 针对现有的随机肽库测试相同的样本，该库报告收敛特征 每种疾病的患者样本的特异性。这种高风险的方法如果成功，将导致 快速为所测试的神经免疫疾病提供革命性的新诊断范式，就像它所做的那样 NMDAR 脑炎也具有改变其他自身免疫性疾病范式的潜力。