Development of ultra-efficient antibodies for single cell mapping applications

开发用于单细胞作图应用的超高效抗体

• 批准号: 10601458
• 负责人: Andrea Lynn Johnstone
• 金额: $ 100.77万
• 依托单位: EPICYPHER, INC.
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-03-10 至 2026-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10601458
• 关键词: Antibodies; Antibody Specificity; Bar Codes; Binding; Biological Assay; Cells; Characteristics; Chromatin; Clinical; DNA; Data; Development; Disease; Enzyme-Linked Immunosorbent Assay; Epigenetic Process; Epitopes; Etiology; Exhibits; Feedback; Genomics; Goals; Histones; Immune; Immunization; Immunize; Immunologics; Immunology; Laboratories; Maps; Mediating; Noise; Nucleosomes; Oryctolagus cuniculus; Peptides; Phase; Population; Post-Translational Protein Processing; Protocols documentation; Reagent; Recombinant Antibody; Recombinants; Research; Research Project Grants; Sampling; Signal Transduction; Site-Directed Mutagenesis; Specificity; Technology; Testing; Validation; assay development; cross reactivity; drug discovery; epigenomics; improved; in vivo; innovation; manufacture; manufacturing scale-up; multiplex assay; next generation; novel; novel strategies; phase 2 study; screening; three dimensional structure; tool

PROJECT SUMMARY Histone post-translational modifications (PTMs) are some of the most widely studied epigenomic factors, and alterations in histone PTM abundance / distribution have been implicated in numerous disease etiologies. Epigenomic mapping of histone PTMs in limited cell populations or single cells (SCs) would provide the opportunity to study the epigenetic landscape of rare and heterogenous cell populations and be highly enabling for drug discovery research. The recent development of the antibody-mediated genomic mapping approach CUT&Tag (Cleavage Under Targets and Tagmentation) permits the study of select, abundant histone PTMs using very few cells and even SCs. Despite this progress, ultra-low input and SC CUT&Tag assays still present a unique challenge, and have not yet been successfully applied to many challenging targets, such as less abundant histone PTMs. Indeed, to maximize data yield per cell, ultra-low input and SC CUT&Tag assays require antibodies to exhibit high on-target epitope binding with minimal off-target binding, which most commercial antibodies do not offer. We envision a new class of “SC-grade” antibodies that deliver ultra-efficient histone PTM binding for dramatically increased CUT&Tag assay sensitivity, improving reliability and providing access to new targets that are currently intractable. Here, EpiCypher will leverage a novel antibody development pipeline to generate ultra-efficient, “SC-grade” antibodies to unlock the potential of genomic mapping technology for next- generation ultra-low input / SC applications. Unlike traditional antibody development pipelines that use histone peptides for screening, a central innovation of our strategy is the implementation of recombinant modified designer nucleosome technology during antibody development. In Phase I equivalent studies, we used our novel approach to select and validate ultra-efficient antibodies for two key histone PTM targets (H3K4me1 and H3K4me3), generating antibodies that exhibit a >5-10x increase in nucleosome capture efficiency vs. current best-in-class antibodies. Importantly, these ultra-efficient antibodies generated significantly greater signal-to- noise in genomic mapping assays that employ low cell inputs, demonstrating strong proof-of-concept for our approach. In Phase II, we will leverage this validated antibody development pipeline to develop a suite of ultra- efficient antibodies and use these reagents to develop low input and SC CUT&Tag assays for breakthrough immunology research. Toward this goal, we will first develop and screen antibodies for high-value histone PTM targets (Aim 1). We will then scale up production and rigorously validate antibody lots in CUT&Tag assays using both low input and SC workflows (Aim 2). Finally, we will test the application of our next-generation ultra-efficient antibodies to enable cutting-edge immunological research and provide our antibodies and validated protocols to leading epigenetics laboratories for beta testing (Aim 3). This research project will result in the development of a new class of histone PTM antibodies that will be used to increase the utility of CUT&Tag assays for breakthrough chromatin research and drug discovery.

项目摘要 组蛋白翻译后修饰（PTMS）是一些研究最广泛的表观基因组因子，即 在许多疾病病因中，组蛋白PTM抽象 /分布的改变已暗示。 在有限的细胞群或单个细胞（SC）中，组蛋白PTM的表观基因组映射将提供 研究罕见和异源细胞种群的表观遗传景观的机会，并具有高度支持 用于药物发现研究。抗体介导的基因组映射方法的最新发展 切割和标签（目标和标记下的裂解）允许研究精选的大型组蛋白PTMS 使用很少的细胞甚至SC。尽管取得了这种进展，但超低输入和SC剪切和标签测定仍存在 一个独特的挑战，尚未成功地应用于许多挑战目标，例如 丰富的组蛋白PTM。实际上，为了最大化每个单元的数据产量，超低输入和SC剪切和TAG分析需要 与最小脱靶结合结合的裸露高目标表位的抗体，大多数商业 抗体不提供。我们设想了一类新的“ SC级”抗体，可提供超高效率的组蛋白PTM 具有巨大提高的剪切和标记测定敏感性，提高可靠性并提供新的访问权限的约束力 目前棘手的目标。在这里，Epicypher将利用一种新型的抗体开发管道 产生超有效的“ SC级”抗体，以解锁基因组映射技术的潜力 生成超低输入 / SC应用。与使用组蛋白的传统抗体开发管道不同 用于筛查的肽，我们策略的核心创新是重组修改的实施 抗体开发过程中的设计师核小体技术。在第一阶段的同等研究中，我们使用了小说 选择和验证两个关键组蛋白PTM靶标（H3K4ME1和 H3K4me3），生成抗体，暴露于核小体捕获效率与电流> 5-10倍 一流的抗体。重要的是，这些超高效抗体产生了明显更大的信噪比 基因组映射测定中的噪声表明，员工低细胞输入，证明了我们的概念证明 方法。在第二阶段，我们将利用这一经过验证的抗体开发管道来开发一套超级 有效的抗体，并使用这些试剂来开发低输入和SC剪切和标记测定以突破 免疫学研究。为了实现这一目标，我们将首先开发和筛选高价值组蛋白PTM的抗体 目标（目标1）。然后，我们将使用剪切和标记测定中的抗体批次进行扩展，并使用 低输入和SC工作流程（AIM 2）。最后，我们将测试下一代超高效率的应用 抗体能够实现尖端免疫学研究并提供我们的抗体和经过验证的方案 领先的表观遗传学实验室用于Beta测试（AIM 3）。该研究项目将导致 一类新的Hisstone PTM抗体将用于增加剪切和标记测定的实用性 突破性染色质研究和药物发现。