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

项目总结 染色质结构的改变与许多人类疾病有关，通常表现为 组蛋白翻译后修饰(PTM)的变化。组蛋白PTM通常通过染色质进行分析 免疫沉淀(CHIP)，依靠抗体来丰富染色质亚群。然而，其精确度 这类抗体在生物医学领域日益受到关注，阻碍了染色质科学的进步。 以及相关的药物/诊断。EpiCypher®是使用重组 设计核小体(DNucs)，早期专注于PTM抗体特异性测试。使用EpiCypher的 颠覆性定量芯片平台(SNAP-CHIP®；样本归一化和抗体分析)，我们发现 80%的商业化PTM抗体表现出惊人的脱靶结合和低结合 效率。这些结果突显了在抗组蛋白抗体的选择和验证方面存在的重大问题。 PTMS，目前利用组蛋白多肽进行候选选择。理想情况下，我们会将SNAP-CHIP 在抗体开发的早期进行检测；然而，SNAP-CHIP是低通量、劳动密集型的，因此不 适合于抗体的开发。 在这个直接转到第二阶段的计划中，我们将使用NucleoPlex™来解决这些缺陷，它将 将dNucs修饰为Luminex®xMAP®微珠，用于多路高通量抗体筛查。创新 这项建议的一项建议是创建dNuc-xMAP珠面板(即NucleoPlex面板)，使开启和关闭- 单次反应中的靶向抗体分析。在第一阶段等价概念验证研究中，我们将 条形码的xMAP珠子到含有组蛋白甲基赖氨酸PTM的dNucs上，并使用这个面板询问 结合&gt；50商用芯片级PTM抗体。NucleoPlex数据表现强劲 与SNAP-CHIP的一致性，时间和成本只有SNAP-CHIP的一小部分，表明NucleoPlex提供了准确和 核小体背景下PTM抗体的快速特异性筛选。 EpiCypher准备在NucleoPlex平台上取得重大突破，重新定义 组蛋白PTM抗体的开发、筛选和验证。这项第二阶段研究的一个主要目标是 将NucleoPlex集成到重组抗体开发流水线中，并演示该方法如何 增加高特异性抗体的产生。为此，我们将首先验证和扩展制造 针对不同的组蛋白PTM家族的NucleoPlex面板(目标1)，然后将NucleoPlex应用于开发 重组抗体(目标2)。最后，我们将开发组合修饰的核小体来审问 在相邻的PTM的背景下的抗体结合特异性，已被证明影响抗体结合 (目标3)。NucleoPlex作为低成本多路抗体筛查平台的发展将加快 高特异组蛋白PTM靶向试剂的产生，这将对 表观遗传学领域，每年节省数百万美元，浪费在低质量的检测试剂上。

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.