Engineered super-affinity reagents for detection of histone post-translational modifications

用于检测组蛋白翻译后修饰的工程超亲和力试剂

• 批准号: 10553250
• 负责人: Zu-Wen Sun
• 金额: $ 102.43万
• 依托单位: EPICYPHER, INC.
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-01-20 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10553250
• 关键词: Address; Affinity; Antibodies; Automobile Driving; Avidity; Bar Codes; Binding; Biochemical; Biological Assay; Cell Count; Cell Line; Cell physiology; Cells; Chromatin; Clinical; Clinical Research; DNA; DNA Damage; DNA Repair; Detection; Development; Engineering; Environment; Epigenetic Process; Epitopes; Exhibits; Gene Expression; Genomics; High Pressure Liquid Chromatography; Histones; Laboratories; Libraries; Maps; Methods; Noise; Nucleosomes; Performance; Phase; Physiological; Population; Post-Translational Protein Processing; Preparation; Reader; Reagent; Recombinants; Research; Research Personnel; Role; Sampling; Services; Signal Transduction; Site-Directed Mutagenesis; Specificity; Technology; Testing; Time; Validation; Western Blotting; Work; clinical biomarkers; commercial launch; commercialization; drug development; epigenomics; improved; innovation; manufacture; manufacturing scale-up; novel; nuclease; repaired; response; stability testing; tool

PROJECT SUMMARY Histone post-translational modifications (PTMs) are widely studied due to their diverse roles in controlling gene expression. The study of PTMs largely relies on antibodies; however, we have shown that the majority of commercial histone PTM antibodies are plagued by low efficiency and/or low specificity. Antibody quality is of special concern for ultra-low cell input approaches, which require detection reagents to exhibit high on-target epitope binding (i.e. efficiency) with minimal off-target binding (i.e. specificity) to maximize yields from limited inputs (e.g. rare cell populations) and improve assay sensitivity. EpiCypher has begun to address this current roadblock by developing libraries of recombinant designer nucleosome (dNuc) substrates carrying diverse histone PTMs and leveraging this technology to rigorously assess antibody binding specificity and target enrichment. However, some PTM targets (e.g. H4K20me2) and unmodified states (e.g. H3K4me0) remain intractable (i.e. no good antibodies exist), and/or are not suitable for low input studies due to low target enrichment. Thus, new detection reagents are needed to access historically challenging targets and to enable ultra-low cell input approaches, opening new avenues to effectively study biologically relevant PTMs and advance clinical biomarker / drug development. Here, EpiCypher is developing Super Readers™: ultra-sensitive, first-in-class recombinant detection reagents that exhibit increased specificity and affinity (vs. antibodies) to enable 1) detection of historically challenging PTM targets and 2) ultra-low input PTM mapping. Natural chromatin reader domains have high specificity but often display low affinity when isolated. The innovation of this approach is the multimerization of chromatin reader domains, leveraging the avidity effects of multivalent interactions to create high-performance detection reagents. These breakthrough tools will be developed using EpiCypher’s proprietary DNA-barcoded dNuc technology to rigorously validate specificity on physiologically relevant substrates, and to enable cross- sample comparisons in CUT&RUN (Cleavage Under Targets & Release Using Nuclease), a novel ultrasensitive chromatin profiling approach. For proof-of-concept, we developed a Super Reader, confirmed its specificity on dNucs, and applied it in CUT&RUN, demonstrating its utility and reliability for PTM mapping. In Phase II, we will develop a total of six Super Readers and validate them in CUT&RUN under a range of conditions, including ultra- low cell inputs. Further, we will apply a Super Reader to profile a PTM with no quality antibodies available (H4K20me2), thus creating the first ever accurate genomic map for this mark and characterizing its role in DNA damage repair. Finally, we will optimize manufacturing of the six Super Readers, assemble beta-kits, perform external validation, and integrate these tools for in-house CUT&RUN assay services. Together, this work will lead to the development and commercialization of a new class of high-performance detection reagents that will provide first-time access to historically challenging PTMs and enable ultra-low cell input PTM mapping, thus driving novel epigenetics research and clinical advances that were previously unachievable.

项目摘要 组蛋白的翻译后修饰（PTMS）由于其在控制中的作用而被广泛研究 基因表达。 PTM的研究主要依赖于抗体。但是，我们已经证明了大多数 商业组蛋白PTM抗体受到低效率和/或低特异性的困扰。抗体质量是 对超低单元输入方法的特别关注，这些方法需要检测试剂才能暴露高目标 具有最小脱靶结合（即特异性）的表位结合（即效率），以最大程度地从有限 输入（例如稀有细胞群体）并提高测定敏感性。 Epicypher已经开始解决此电流 通过开发重组设计师核小体（DNUC）底物的库，携带多样 Hisstone PTM并利用该技术严格评估抗体结合特异性和靶标 丰富。但是，某些PTM靶标（例如H4K20ME2）和未修饰的状态（例如H3K4ME0） 棘手（即不存在良好抗体）和/或由于低靶标而不适合低输入研究 丰富。这是需要新的检测试剂来访问历史上的挑战目标并启用 超低的细胞输入方法，开辟了新的途径，以有效研究生物学相关的PTM和 提前临床生物标志物 /药物开发。 在这里，Epicypher正在开发Super Readers™：超敏感的第一类重组检测 暴露了提高特异性和亲和力（与抗体）以实现的试剂1）历史上检测 具有挑战性的PTM目标和2）超低输入PTM映射。天然染色质读取器领域高 特异性，但在隔离时通常显示出低亲和力。这种方法的创新是 染色质读取器域，利用多价相互作用的亲和力效应来创造高性能 检测试剂。这些突破工具将使用Epicypher的专有DNA-Barcoded开发 DNUC技术可以严格验证对物理相关底物的特异性，并使交叉 切割和运行中的样品比较（在目标下进行切割和使用核酸酶释放），一种新型的超敏感 染色质分析方法。为了概念证明，我们开发了一个超级读者，确认了它的特殊性 DNUCS并将其应用于剪切和运行中，证明了其对PTM映射的效用和可靠性。在第二阶段，我们将 在一系列条件下，包括超级读者的六个超级读者，并在包括Ultra- 低细胞输入。此外，我们将应用一名超级读者来介绍没有可用质量抗体的PTM （H4K20me2），因此为此标记创建了第一个准确的基因组图，并表征其在DNA中的作用 损坏维修。最后，我们将优化六个超级读者的制造，组装beta套，表演 外部验证，并将这些工具集成到内部切割和运行测定服务。在一起，这项工作将 导致一类新的高性能检测试剂的开发和商业化，这些试剂将 首次访问历史上挑战PTM并启用超低单元输入PTM映射，因此 驾驶以前无法实现的新型表观遗传学研究和临床进步。