Epicypher, Inc. — Department of Health and Human Services SBIR Phase I: 400

Description

PROJECT SUMMARYNucleosomesthe basic repeating unit of chromatinare comprised of DNA wrapped around a histone octamer coreHistones are decorated with a variety of post translational modificationsPTMswhich interact with proteins to regulate diverse biological processes and diseasePTMs are a rapidly growing target in biomedical researchand are typically analyzed via antibody dependent chromatin immunoprecipitationChIPand enzyme linked immunosorbent assaysELISAsThusthe generation of highly specific antibodies is a top priority for the fieldboth for academic researchand for its application to diagnosticprognostic assays and therapeuticsEpiCypherpioneered the commercialization of recombinant designer nucleosomesdNucsfor PTM antibody specificity testing with our SNAP ChIPSample Normalization and Antibody ProfilingplatformUsing SNAP ChIPwe found that andgtof commercially available PTM antibodies display a striking amount of crossreactivitySuch widespread deficiencies are partly due to histone PTM antibody development protocolswhich rely on histone peptides for immunizationselection and validationWe found that histone peptide arrays fail to generate reliable antibody specificity profilesand that a nucleosome based substraterepresentative of the in vivo physiological contextis required for predicting PTM antibody performance in native chromatinHoweverthe size of conventional immunoglobulinskDahas also made it difficult to target PTMs in or near the histone globular domainadding another layer of complexity to PTM antibody developmentClearlynew technologies are required to generate highly specific reagents capable of defining PTMs in clinical assaysHereEpiCypher will develop recombinant NucleobodiesNucleosome binding single domain antibodiesto PTMs that have been challenging to target with conventional methodsH Kmeand H K meSingle domain antibodiessdAbsare derived from camelide gllamaheavy chain only antibodiesHcAbssdAbs are smallkDasolublestable in solutionand fully sequencedmaking them highly diverserenewable toolsThe innovation of our proposal is the development of highly specific sdAbs to PTMsNucleobodiesutilizing dNucs for immunization and as reliable physiological substrates for candidate selection and final functional validationIn Aimof this proposalwe will develop Nucleobodies to H Kmeand H K meusing dNucs for immunization and onand off target screeningIn Aimwe will identify ChIPgrade Nucleobodies with our proprietary SNAP ChIP technologyand validate reagents in quantitative ELISA and ChIP seqestablishing the utility of these tools in chromatin research and clinically relevant applicationsNucleobodies have the potential to dramatically expand the variety of PTMs available for biomedical researchand with their increased stabilityspecificitywill accelerate the application of PTMs as biomarkers and drug targets PROJECT NARRATIVEEpiCypherhas pioneered the commercialization of recombinant designer nucleosomesdNucsfor profiling the specificity of antibodies to histone post translational modificationsPTMsOur recently released SNAP ChIPSample Normalization and Antibody Profilingplatform revealed that andgtof ChIP grade antibodies display off target bindingFurthermoremany PTMse gH Kmeare difficult to target with conventional immunoglobulin antibodiesOverallthese results highlight significant problems underlying PTM antibody developmentand call for alternative production and screening strategiesHereEpiCypher will leverage their expertise in dNuc technology to develop llama derived NucleobodiesNucleosome binding single domain antibodiesagainst challenging histone PTMsdelivering highly specificrenewableand dynamic tools for chromatin research