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（PTM）是一些研究最广泛的表观基因组因子， 组蛋白PTM丰度/分布的改变与许多疾病病因学有关。 在有限的细胞群体或单细胞（SC）中组蛋白PTM的表观基因组作图将提供 有机会研究稀有和异质细胞群体的表观遗传景观， 用于药物发现研究。抗体介导的基因组作图方法的研究进展 CUT&Tag（Cleavage Under Targets and Tagmentation）允许研究选择的、丰富的组蛋白PTM 使用非常少的细胞甚至SC。尽管取得了这一进展，但超低输入和SC CUT&Tag检测仍然存在 这是一个独特的挑战，尚未成功地应用于许多具有挑战性的目标，如更少 丰富的组蛋白PTM。事实上，为了最大化每个细胞的数据产量，超低输入和SC CUT&Tag测定需要 抗体显示出高的中靶表位结合和最小的脱靶结合，这是大多数商业化的 抗体不能提供。我们设想了一类新的“SC级”抗体，其提供超高效的组蛋白PTM 结合显著提高CUT&Tag检测灵敏度，提高可靠性，并提供新的 这些目标目前还难以实现。在这里，EpiCypher将利用一种新的抗体开发管道， 产生超高效的“SC级”抗体，以释放基因组作图技术的潜力， 第二代超低输入/ SC应用与使用组蛋白的传统抗体开发管道不同， 肽筛选，我们的战略的一个中心创新是实施重组修饰 在抗体开发过程中设计核小体技术。在第一阶段的等效研究中，我们使用了我们的小说 一种选择和验证两个关键组蛋白PTM靶点（H3 K4 me 1和 H3 K4 me 3），产生相对于电流，核小体捕获效率增加>5- 10倍的抗体。 最好的抗体重要的是，这些超高效抗体产生了显著更大的信号- 噪声在基因组作图测定，采用低细胞输入，证明了我们的强有力的概念验证， approach.在第二阶段，我们将利用这一经过验证的抗体开发管道开发一套超 高效的抗体，并使用这些试剂开发低输入和SC CUT&Tag检测， 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1）。然后，我们将扩大生产规模，并在CUT&Tag检测中严格验证抗体批次， 低投入和SC工作流程（目标2）。最后，我们将测试我们的下一代超高效 抗体，使尖端的免疫学研究，并提供我们的抗体和验证的协议， 领先的表观遗传学实验室进行β测试（目标3）。该研究项目将导致开发 一类新的组蛋白PTM抗体，将用于增加CUT&Tag检测的实用性， 突破性的染色质研究和药物发现。