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

Award Information
Agency: Department of Health and Human Services
Branch: National Institutes of Health
Contract: 1R44GM145007-01
Agency Tracking Number: R44GM145007
Amount: $1,024,329.00
Phase: Phase II
Program: SBIR
Solicitation Topic Code: 400
Solicitation Number: PA20-260
Solicitation Year: 2020
Award Year: 2022
Award Start Date (Proposal Award Date): 2022-01-20
Award End Date (Contract End Date): 2023-12-31
Small Business Information
Chapel Hill, NC 27514-4617
United States
DUNS: 078882699
HUBZone Owned: No
Woman Owned: No
Socially and Economically Disadvantaged: No
Principal Investigator
 (919) 448-7268
Business Contact
Phone: (434) 996-8107
Research Institution

PROJECT SUMMARYHistone 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 CUTandRUN (Cleavage Under Targets and 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 CUTandRUN, demonstrating its utility and reliability for PTM mapping. In Phase II, we will
develop a total of six Super Readers and validate them in CUTandRUN 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 CUTandRUN 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.

* Information listed above is at the time of submission. *

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