Development of ultra-efficient antibodies for single cell mapping applications

PROJECT SUMMARYSingle 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 scCUTandamp;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 scCUTandamp;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 andgt;5-10x increase in nucleosome capture efficiency; these high efficiency antibodies generated far superior signal-to-noise (S/N) in CUTandamp;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 CUTandamp;Tag assays, 2) the number of unique reads / cell in scCUTandamp;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 CUTandamp;Tag assay kits to further our understanding of chromatin biology and develop improved therapeutics and diagnostics.PROJECT NARRATIVESingle cell (SC) epigenomics is a rapidly emerging field, driven by recent technology advances and an increased appreciation for the diverse roles that epigenetic features play in controlling gene expression. SC mapping of chromatin elements represents a unique challenge, as assays generate sparse data and thus require antibodies to exhibit ultra-high on-target epitope binding (i.e. efficiency) with minimal off-target binding (i.e. specificity). Here, EpiCypher is developing a novel antibody development method to generate ultra-efficient antibodies, which will enable reliable SC histone post-translational modification (PTM) mapping for the drug discovery research market.