SBIR/STTR Award attributes
Project Summary Cyclic GMP AMP synthasecGASis a recently discovered enzyme that acts as a foreign DNA sensor to elicit an immune response to pathogens via activation of the STINGstimulator of interferon genesreceptorThough there are other DNA sensorse gTLRthe cGAS cGAMP STING pathway appears to be essential for DNAmediated immune response irrespective of cell type or DNA sequenceDNA binds to a specific site on cGAS in a non sequence dependent manner and activates its catalytic activityresulting in the production of a unique cyclic nucleotide GandaposandapospAandaposandapospcGAMPfrom ATP and GTP precursorscGAMP binds to the STING receptor with nanomolar affinity and induces expression of type I interferonsThuscGAMP plays a fundamental role in human immunityacting upstream of both T and B cells to trigger an innate immune responseShortly after its discovery inaberrant activation of cGAS by self DNA was shown to contribute to debilitating and sometimes fatal autoimmune diseasessuch as systemic lupus erythematosusSLEand knocking out cGAS abrogates disease in animal modelsThe cGAS STING pathway has also been shown to play a key role in the innate immune response to tumorsand intratumoral injection of cGAMP analogs is an emerging strategy for cancer immunotherapyModulating cGAS activity is autoimmunity and cancerTumor immunityATP GTPcGAS cGAMP Autoimmune diseases IRF Antimicrobial ImmunitySTING NF B Type I IFNs Activation of cGAS by cytoplasmic DNA initiates activation of the innate immune response via induction of Type I IFNs which induce tumor cell specific T cell responses in cancer but induce autoantibodies and cause extensive tissue damage in autoimmune diseases such as SLEtherefore a very compelling therapeutic strategyboth for Though basic research on the cGAS STING pathway and efforts to target it therapeutically have expanded rapidlythe difficulty in detecting cGAMP has seriously hindered progress on both frontsIt functions at nanomolar concentrationswhereas other nucleotidesincluding its precursors ATP and GTPare present at levels as much asfold highermaking specific detection of cGAMP in cell or tissue extracts extremely challengingCurrentlythe only method used is methanol extraction followed by HPLC purification and LC MSSimilarlymethods for detecting cGAS enzymatic activity involve chromatographic separation of radioactive cGAMP from ATP and GTPDevelopment of drugs that modulate cGAS will require sensitivehomogenous assay methods for detecting cGAMP with exquisite specificity that can be used for biochemical and cellular HTS assaysand eventually for biomarker assays to support translational researchIn Phase Iwe produced monoclonal antibodies that recognize cGAMP with more thanfold selectivity vsother nucleotides and used one of them to develop homogenous detection assays with fluorescent readoutsFP and TR FRETWe validated the assays as a robust biochemical HTS platform using purified human cGASthese assays will allow us to begin screening for cGAS modulators under separate grant applicationsIn Phase IIwe will develop more sensitive cGAMP detection reagents and methods that can be used for cellular HTS and biomarker assaysTaking inspiration from recent examples of sandwich based assays for small moleculeswe will use cGAMP antigen design approaches and in vitro evolution to generate a pair of single chain Fvandapos s that bind cGAMP simultaneously with picomolar affinity to allow direct detection of cGAMP in cell and tissue lysates with TR FRET and or ELISA assaysThe effort will include contributions from two outstanding academic scientists to buttress BellBrookandapos s assay development expertiseDrKarl WittrupDirector of the Koch Institute at MIT and inventor of the yeast display system that we will use for scFv affinity maturationwill act as a consultant for the critical in vitro evolution stepDrKeith ElkonHead of the Division of Rheumatology at University of Washingtonone of the worldandapos s leading experts on the molecular and genetic basis for autoimmune diseaseswho is pioneering efforts to elucidate the role of cGAS STING in SLEwill collaborate on validation of the biomarker assay with samples from animal models and patientsThe development of simplequantitative cGAMP assays would have very significant scientific and medical impactThe biochemical and cellular cGAMP assays will enable BellBrook and collaborators to pursue HTSdriven efforts to develop first in class lead molecules targeting the cGAS STING pathway for autoimmunity and cancer immunotherapyMore broadlycommercialization of the assays as kits will fill critical gaps in the tools needed for basic cellular and biochemical studies of the cGAS STING pathway Narrative Cyclic GAMP is a recently discovered biomolecule that activates the immune system for defense against microbial pathogens and tumor cellsWe are developing simpleinexpensive methods for detecting cGAMP that will help researchers understand how the innate immune system functions in health and disease and accelerate development of targeted therapies for autoimmune diseases and cancer immunotherapy
