Multiplex nucleosome-based profiling for the development of next-generation chromatin labeling reagents

基于多重核小体的分析，用于开发下一代染色质标记试剂

• 批准号: 10094217
• 负责人: Zu-Wen Sun
• 金额: $ 92.02万
• 依托单位: EPICYPHER, INC.
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-02-03 至 2023-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10094217
• 关键词: Acylation; Address; Antibodies; Antibody Specificity; Arginine; Bar Codes; Binding; Biological Assay; Biomedical Research; Chromatin; Chromatin Structure; Collaborations; Complement; Couples; Data; Detection; Development; Diagnostic; Epigenetic Process; Family; Generations; Genomics; Goals; Histones; K562 Cells; Label; Letters; Libraries; Licensing; Lysine; Methods; Methylation; Nucleosomes; Peptides; Performance; Pharmaceutical Preparations; Phase; Post-Translational Protein Processing; Production; Reaction; Reagent; Recombinant Antibody; Recombinants; S-nitro-N-acetylpenicillamine; Sampling; Science; Signal Transduction; Site-Directed Mutagenesis; Specificity; Technology; Testing; Time; Validation; Variant; Voice; base; candidate selection; chromatin immunoprecipitation; combinatorial; cost; cross reactivity; drug development; human disease; innovation; interest; next generation; phase 2 study; screening; success; technology development

PROJECT SUMMARY Alterations in chromatin structure are associated with many human diseases and often characterized by changes in histone post-translational modifications (PTMs). Histone PTMs are commonly analyzed by chromatin immunoprecipitation (ChIP), which relies on antibodies to enrich chromatin subsets. However, the accuracy of such antibodies is an increasing concern in the biomedical field, impeding advancements in chromatin science and related drugs / diagnostics. EpiCypher® is pioneering the development of technologies that use recombinant designer nucleosomes (dNucs), with an early focus on PTM antibody specificity testing. Using EpiCypher’s disruptive quantitative ChIP platform (SNAP-ChIP®; Sample Normalization and Antibody Profiling), we found that >80% of commercially-available PTM antibodies display a striking amount of off-target binding and low binding efficiency. These results underscore substantial problems in the selection and validation of antibodies to histone PTMs, which currently utilize histone peptides for candidate selection. Ideally, we would integrate SNAP-ChIP assays early in antibody development; however, SNAP-ChIP is low-throughput, labor intensive, and thus not suitable for antibody development. In this Direct-to-Phase II proposal we will address these deficiencies with NucleoPlex™, which couples modified dNucs to Luminex® xMAP® beads for multiplexed, high-throughput antibody screening. The innovation of this proposal is the creation of dNuc-xMAP bead panels (i.e. NucleoPlex panels) that enable both on- and off- target antibody profiling in a single reaction. In Phase I equivalent proof-of-concept studies, we conjugated barcoded xMAP beads to dNucs containing histone methyl-lysine PTMs, and used this panel to interrogate the binding of >50 commercially available ChIP-grade PTM antibodies. NucleoPlex data demonstrated strong concordance with SNAP-ChIP, at fraction of the time and cost, indicating that NucleoPlex provides accurate and rapid specificity screening of PTM antibodies in a nucleosomal context. EpiCypher is poised to make a significant breakthrough with the NucleoPlex platform, redefining histone PTM antibody development, screening, and validation. A major goal of this Phase II study is to integrate NucleoPlex into a recombinant antibody development pipeline, and demonstrate how this approach increases production of highly specific antibodies. To this end, we will first validate and scale manufacturing of NucleoPlex panels for distinct histone PTM families (Aim 1), and then apply NucleoPlex toward the development of recombinant antibodies (Aim 2). Finally, we will develop combinatorially-modified nucleosomes to interrogate antibody binding specificity in the context of adjacent PTMs, which have been shown to impact antibody binding (Aim 3). The development of NucleoPlex as a low-cost multiplexed antibody screening platform will accelerate the generation of highly specific histone PTM-targeting reagents, which will have a lasting impact on the epigenetics field and save millions of dollars annually that are wasted on low-quality detection reagents.

项目摘要 染色质结构的改变与许多人类疾病有关，并且通常以 组蛋白翻译后修饰（PTM）的变化。组蛋白PTM通常通过染色质 免疫沉淀（ChIP），其依赖于抗体来富集染色质亚群。然而， 这种抗体在生物医学领域越来越受到关注，阻碍了染色质科学的进步。 和相关药物/诊断。EpiCypher®是使用重组技术开发的先驱 设计者核小体（dNucs），早期专注于PTM抗体特异性测试。使用EpiCypher的 在破坏性定量ChIP平台（SNAP-ChIP®;样品归一化和抗体分析）上，我们发现， >80%的市售PTM抗体显示出惊人量的脱靶结合和低结合 效率这些结果凸显了组蛋白抗体选择和验证中的实质性问题 PTM目前利用组蛋白肽进行候选选择。理想情况下，我们将SNAP-ChIP SNAP-ChIP在抗体开发的早期进行检测;然而，SNAP-ChIP是低通量的，劳动密集型的，因此不适合用于抗体开发。 适合抗体开发。 在这个直接进入第二阶段的提案中，我们将通过NucleoStimulator ™解决这些缺陷， 将经修饰的dNucs与Luminex® xMAP®珠结合，用于多重、高通量抗体筛选。创新 该提议的一个重要方面是创建dNuc-xMAP珠面板（即NucleoDegradation面板）， 在单一反应中进行靶向抗体分析。在I期等效概念验证研究中， 将xMAP珠条形码化到含有组蛋白甲基-赖氨酸PTM的dNucs上，并使用该面板来询问 >50种市售ChIP级PTM抗体的结合。核素数据显示， 与SNAP-ChIP的一致性，在时间和成本的一小部分，表明Nucleotide提供了准确的， 核小体背景下PTM抗体的快速特异性筛选。 EpiCypher准备在NucleoCypher平台上取得重大突破， 组蛋白PTM抗体开发、筛选和验证。这项II期研究的主要目标是 将Nucleotide整合到重组抗体开发管道中，并演示这种方法如何 增加高度特异性抗体的产生。为此，我们将首先验证和规模化生产 不同组蛋白PTM家族的核苷酸组（Aim 1），然后将核苷酸组应用于开发 重组抗体（Aim 2）。最后，我们将开发组合修饰的核小体， 在相邻PTM的情况下的抗体结合特异性，已显示其影响抗体结合 (Aim 3）。Nucleotide作为低成本多重抗体筛选平台的发展将加速 高度特异性的组蛋白PTM靶向试剂的产生，这将对免疫系统产生持久的影响。 表观遗传学领域，并节省数百万美元，每年浪费在低质量的检测试剂。

项目成果

Complex-dependent histone acetyltransferase activity of KAT8 determines its role in transcription and cellular homeostasis.

• DOI: 10.1016/j.molcel.2021.02.012
• 发表时间: 2021-04-15
• 期刊: Molecular cell
• 影响因子: 16
• 作者: Radzisheuskaya A;Shliaha PV;Grinev VV;Shlyueva D;Damhofer H;Koche R;Gorshkov V;Kovalchuk S;Zhan Y;Rodriguez KL;Johnstone AL;Keogh MC;Hendrickson RC;Jensen ON;Helin K
• 通讯作者: Helin K

Quantification of citrullinated histones: Development of an improved assay to reliably quantify nucleosomal H3Cit in human plasma.

• DOI: 10.1111/jth.15003
• 发表时间: 2020-10
• 期刊: Journal of thrombosis and haemostasis : JTH
• 作者: Thålin C;Aguilera K;Hall NW;Marunde MR;Burg JM;Rosell A;Daleskog M;Månsson M;Hisada Y;Meiners MJ;Sun ZW;Whelihan MF;Cheek MA;Howard SA;Saxena-Beem S;Noubouossie DF;Key NS;Sheikh SZ;Keogh MC;Cowles MW;Lundström S;Mackman N;Wallén H;Johnstone AL
• 通讯作者: Johnstone AL