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.

项目总结