Development of ultra-efficient antibodies for single cell mapping applications

项目摘要 单细胞（SC）表观基因组学是一个快速新兴的领域，由最近的技术进步和生物技术的发展所驱动。 表观遗传特征在控制基因激活中发挥的不同作用。组蛋白翻译后修饰 PTMs代表了疾病中一些最相关和广泛研究的表观基因组因素。最近 scCUT&Tag（SC靶下切割和标签化）的开发提供了第一个易于处理的 以SC分辨率定位组蛋白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G和高活性Tn 5转座酶（pAG-Tn 5）融合物拴系到这些位点 用于标签化。然后对靶向DNA进行扩增和测序，提供一种流线型的超灵敏检测方法 用于组蛋白PTM作图。尽管取得了这一进展，但SC应用仍然面临着独特的挑战，因为它们 产生令人难以置信的稀疏数据（即相对较少的读数/ SC），并需要抗体表现出高的靶向 表位结合（即效率）与最小的脱靶结合（即特异性）。我们设想超高效 抗体可以提供一类新的“SC级”抗体， 回收率，这将大大提高测定灵敏度和可靠性。但发展 缺乏用于SC应用的抗体，以及筛选高结合抗体候选克隆的管道 对核小体的效率不存在。在这里，EpiCypher正在开发一种新的抗体筛选方法， 生产超高效的“SC级”抗体，充分利用scCUT&Tag技术的潜力， 表观遗传药物发现研究市场。我们战略的创新之处在于重组技术的应用 在抗体开发过程中使用修饰的设计者核小体（dNuc）技术。第一，候选克隆 使用携带PTM靶标的生物素化dNuc通过ELISA筛选靶向结合。第二、 使用高通量多重测定法（Nucleotide ™）筛选候选克隆，其中携带 靶上和脱靶PTM与条形码化的LuminexxMAP珠缀合。第三，成功的候选人是 纯化并使用DNA条形码化的SNAP-ChIP®掺入进一步分析抗体特异性和效率。 对照在初步研究中，我们使用我们的新方法来选择和验证超高效抗体， 几个PTM靶标，开发出在核小体捕获方面表现出>5- 10倍增加的抗体 效率;这些高效抗体在CUT&Tag测定中产生远优于上级信噪比（S/N）（相对于 当前同类最佳抗体）。在第一阶段，我们的目标是证明超高效抗体改善1） 低输入批量CUT&Tag测定中的S/N，2）scCUT&Tag测定中的独特读数/细胞的数量，以及3）使 分析高值、低丰度标记（如H3 K4 me 3）。在第二阶段，我们将利用这一新的管道， 扩大针对广泛的高价值靶标的超高效组蛋白PTM抗体的开发。这些 抗体也将用于开发低输入和SC CUT&Tag检测试剂盒，以进一步了解 染色质生物学和开发改进的治疗和诊断。

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.