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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要组蛋白翻译后修饰 (PTM) 是研究最广泛的表观基因组因素之一，组蛋白 PTM 丰度/分布的改变与许多疾病病因有关。有限细胞群或单细胞 (SC) 中组蛋白 PTM 的表观基因组图谱将提供有机会研究稀有和异质细胞群的表观遗传景观并具有高度的能力用于药物发现研究的抗体介导的基因组作图方法的最新发展。 CUT&Tag（目标下切割和标记）允许研究精选的丰富组蛋白 PTM 尽管取得了这些进展，但超低输入和 SC CUT&Tag 测定仍然存在。一个独特的挑战，尚未成功应用于许多具有挑战性的目标，例如更少事实上，为了最大限度地提高每个细胞的数据产量，需要超低输入和 SC CUT&Tag 检测。抗体表现出高靶标表位结合和最小的脱靶结合，这是大多数商业化的我们设想一种新型“SC 级”抗体，可提供超高效的组蛋白 PTM。结合可显着提高 CUT&Tag 检测灵敏度、提高可靠性并提供新的途径在这里，EpiCypher 将利用新型抗体开发管道来解决目前难以解决的目标。生成超高效的“SC 级”抗体，以释放基因组作图技术的潜力：与使用组蛋白的传统抗体开发流程不同。用于筛选的肽，我们战略的核心创新是重组修饰的实施在抗体开发过程中设计核小体技术。在第一阶段等效研究中，我们使用了我们的新颖技术。针对两个关键组蛋白 PTM 靶标（H3K4me1 和 H3K4me1）选择和验证超高效抗体的方法 H3K4me3)，生成的抗体与当前相比，核小体捕获效率提高 >5-10 倍重要的是，这些超高效的抗体产生了显着更强的信号。采用低细胞输入的基因组作图测定中存在噪音，这为我们的研究提供了强有力的概念验证在第二阶段，我们将利用这一经过验证的抗体开发管道来开发一套超强抗体。高效的抗体，并使用这些试剂开发低输入和 SC CUT&Tag 检测以实现突破为了实现这一目标，我们将首先开发和筛选高价值组蛋白PTM的抗体。然后，我们将扩大生产规模并使用 CUT&Tag 检测严格验证抗体批次。最后，我们将测试下一代超高效的应用。抗体以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进行 beta 测试（目标 3）。一类新的组蛋白 PTM 抗体，将用于提高 CUT&Tag 检测的实用性突破性的染色质研究和药物发现。