Engineered super-affinity reagents for detection of histone post-translational modifications

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PROJECT SUMMARY Histone post-translational modifications (PTMs) are widely studied due to their diverse roles in controlling gene expression. The study of PTMs largely relies on antibodies; however, we have shown that the majority of commercial histone PTM antibodies are plagued by low efficiency and/or low specificity. Antibody quality is of special concern for ultra-low cell input approaches, which require detection reagents to exhibit high on-target epitope binding (i.e. efficiency) with minimal off-target binding (i.e. specificity) to maximize yields from limited inputs (e.g. rare cell populations) and improve assay sensitivity. EpiCypher has begun to address this current roadblock by developing libraries of recombinant designer nucleosome (dNuc) substrates carrying diverse histone PTMs and leveraging this technology to rigorously assess antibody binding specificity and target enrichment. However, some PTM targets (e.g. H4K20me2) and unmodified states (e.g. H3K4me0) remain intractable (i.e. no good antibodies exist), and/or are not suitable for low input studies due to low target enrichment. Thus, new detection reagents are needed to access historically challenging targets and to enable ultra-low cell input approaches, opening new avenues to effectively study biologically relevant PTMs and advance clinical biomarker / drug development. Here, EpiCypher is developing Super Readers™: ultra-sensitive, first-in-class recombinant detection reagents that exhibit increased specificity and affinity (vs. antibodies) to enable 1) detection of historically challenging PTM targets and 2) ultra-low input PTM mapping. Natural chromatin reader domains have high specificity but often display low affinity when isolated. The innovation of this approach is the multimerization of chromatin reader domains, leveraging the avidity effects of multivalent interactions to create high-performance detection reagents. These breakthrough tools will be developed using EpiCypher’s proprietary DNA-barcoded dNuc technology to rigorously validate specificity on physiologically relevant substrates, and to enable cross- sample comparisons in CUT&RUN (Cleavage Under Targets & Release Using Nuclease), a novel ultrasensitive chromatin profiling approach. For proof-of-concept, we developed a Super Reader, confirmed its specificity on dNucs, and applied it in CUT&RUN, demonstrating its utility and reliability for PTM mapping. In Phase II, we will develop a total of six Super Readers and validate them in CUT&RUN under a range of conditions, including ultra- low cell inputs. Further, we will apply a Super Reader to profile a PTM with no quality antibodies available (H4K20me2), thus creating the first ever accurate genomic map for this mark and characterizing its role in DNA damage repair. Finally, we will optimize manufacturing of the six Super Readers, assemble beta-kits, perform external validation, and integrate these tools for in-house CUT&RUN assay services. Together, this work will lead to the development and commercialization of a new class of high-performance detection reagents that will provide first-time access to historically challenging PTMs and enable ultra-low cell input PTM mapping, thus driving novel epigenetics research and clinical advances that were previously unachievable.