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: 10323430
• 负责人: Bryan J Venters
• 金额: $ 30.65万
• 依托单位: EPICYPHER, INC.
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-01 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10323430
• 关键词: Antibodies; Antibody Specificity; Bar Codes; Binding; Biological Assay; Biology; Cells; Chromatin; Chromium; DNA; Data; Development; Diagnostic; Disease; Elements; Enzyme-Linked Immunosorbent Assay; Epigenetic Process; Epitopes; Exhibits; Future; G-substrate; Gene Activation; Gene Expression; Genomics; Goals; Gold; Histones; Hyperactivity; In Situ; Maps; Market Research; Methods; Noise; Nucleosomes; Output; Peptides; Phase; Play; Post-Translational Protein Processing; Proteins; Recombinants; Recovery; Research; Resolution; Role; S-nitro-N-acetylpenicillamine; Signal Transduction; Site; Site-Directed Mutagenesis; Specificity; System; Technology; Testing; Therapeutic; Tn5 transposase; Validation; Work; base; chromatin protein; drug development; drug discovery; epigenetic drug; epigenomics; improved; innovation; method development; multiplex assay; novel; novel strategies; research and development; screening; single cell analysis

PROJECT SUMMARY Single cell (SC) epigenomics is a rapidly emerging field, driven by recent technology advances and the diverse roles that epigenetic features play in controlling gene activation. Histone post-translational modifications (PTMs) represent some of the most relevant and widely studied epigenomic factors in disease. Recent development of scCUT&Tag (SC Cleavage Under Targets and Tagmentation) provides the first tractable approach to map histone PTMs at SC resolution. This approach uses antibodies to bind chromatin proteins in situ, and then tethers a protein A-protein G and hyperactive Tn5 transposase (pAG-Tn5) fusion to these sites for tagmentation. Targeted DNA is then amplified and sequenced, delivering a streamlined, ultrasensitive assay for histone PTM mapping. Despite this progress, SC applications still present a unique challenge, as they generate incredibly sparse data (i.e. relatively few reads / SC) and require antibodies to exhibit high on-target epitope binding (i.e. efficiency) with minimal off-target binding (i.e. specificity). We envision that ultra-efficient antibodies could provide a new class of “SC-grade” antibodies that display highly efficient on-target recovery, which will dramatically improve assay sensitivity and reliability. However, the development of antibodies for SC applications is lacking, and pipelines that screen antibody candidate clones for high binding efficiency on nucleosomes do not exist. Here, EpiCypher is developing a novel antibody screening method to generate ultra-efficient “SC-grade” antibodies to leverage the full potential of scCUT&Tag technology for the epigenetic drug discovery research market. The innovation of our strategy is the application of recombinant modified designer nucleosome (dNuc) technology during antibody development. First, candidate clones are screened by ELISA for on-target binding using a biotinylated dNuc carrying the PTM target. Second, candidate clones are screened using a high-throughput multiplex assay (NucleoPlex™) wherein dNucs carrying on- and off- target PTMs are conjugated to barcoded Luminex xMAP beads. Third, successful candidates are purified and further analyzed for antibody specificity and efficiency using DNA-barcoded SNAP-ChIP® spike-in controls. In preliminary studies, we used our novel approach to select and validate ultra-efficient antibodies for several PTM targets, developing antibodies that exhibit a >5-10x increase in nucleosome capture efficiency; these high efficiency antibodies generated far superior signal-to-noise (S/N) in CUT&Tag assays (vs. current best-in-class antibodies). In Phase I, our goal is to demonstrate that ultra-efficient antibodies improve 1) S/N in low input bulk CUT&Tag assays, 2) the number of unique reads / cell in scCUT&Tag assays and 3) enable analysis of high value, low abundance marks (e.g. H3K4me3). In Phase II, we will use this novel pipeline to expand development of ultra-efficient histone PTM antibodies against a broad range of high value targets. These antibodies will also be used to develop low input and SC CUT&Tag assay kits to further our understanding of chromatin biology and develop improved therapeutics and diagnostics.

项目摘要 单细胞（SC）表观基因组学是一个迅速新兴的领域，在最近的技术进步和 表观遗传特征在控制基因激活中起着多种作用。组蛋白翻译后修饰 （PTMS）代表了疾病中一些最相关，最广泛研究的表观基因组因子。最近的 SCCUT＆TAG的开发（目标和标记下的SC分裂）提供了第一个可拖动的 以SC分辨率绘制组蛋白PTM的方法。这种方法使用抗体结合染色质蛋白 原位，然后将蛋白A-蛋白质G和多活跃TN5转座酶（PAG-TN5）融合到这些位点上 用于标记。然后对靶向DNA进行放大和测序，并提供简化的超敏化测定 用于组蛋白PTM映射。尽管取得了这些进展，SC应用程序仍然带来了独特的挑战 生成令人难以置信的稀疏数据（即相对较少的读取 / sc），并且需要抗体才能暴露高目标 表位结合（即效率）具有最小的脱靶结合（即特异性）。我们设想超高效率 抗体可以提供一类新的“ SC级”抗体，这些抗体表现出高效的目标 恢复，这将显着提高测定敏感性和可靠性。但是，发展 缺乏SC应用的抗体，并且筛选抗体候选克隆的管道用于高结合 不存在对核小体的效率。在这里，Epicypher正在开发一种新型的抗体筛选方法 生成高效的“ SC级”抗体，以利用SCCUT和TAG技术的全部潜力 表观遗传药物发现研究市场。我们策略的创新是重组的应用 在抗体开发过程中，改良的设计师核小体（DNUC）技术。首先，候选克隆 使用带有PTM靶的生物素化DNUC筛选ELISA进行靶向结合。第二， 使用高通量多重分析（NucleOplex™）筛选候选克隆，其中携带的DNUCS 在靶标PTM和脱离靶标的PTM与条形码Luminex Xmap珠共轭。第三，成功的候选人是 使用DNA-Barcoded Snap-Chip®Spike-In纯化并进一步分析了抗体的特异性和效率 控件。在初步研究中，我们使用了新方法来选择和验证超有效的抗体 几个PTM靶标，产生抗体，暴露于核小体捕获> 5-10倍的抗体 效率;这些高效率抗体在切割和标签测定中产生了远大的信噪比（S/N）（VS。 在第一阶段，我们的目标是证明超有效的抗体改善1） 低输入批量切割和标签测定中的S / N，2）SCCUT和TAG分析中唯一的读取 /单元格的数量和3）启用 分析高价值，低抽象标记（例如H3K4me3）。在第二阶段，我们将使用这种新颖的管道 扩大针对广泛的高价值靶标的超高效率组蛋白PTM抗体的开发。这些 抗体还将用于开发低输入和SC剪切和标记测定套件，以进一步了解我们对 编时生物学并发展了改进的治疗和诊断。