SBIR-STTR Award

Cytoplasmic nucleic acids alert the immune system to invading pathogens and trigger a robust type I interferon IFN response via activation of the STING stimulator of interferon genes receptor The sensor for cytoplasmic nucleic acids was recently discovered to be a cyclic GMP AMP synthase which produces a unique cyclic dinucleotide second messenger cGAMP that serves as an agonist for the STING receptor Aberrant activation of the cGAS STING pathway is rapidly emerging as an important underlying cause of debilitating and sometimes fatal autoimmune disorders including systemic lupus erythematosus SLE scleroderma and Aicardi Goutieres Syndrome AGS cGAS is an obvious target for therapeutic intervention In addition very recent studies have indicated that stimulating the STING pathway may be an effective strategy for cancer immunotherapy Development of small molecule inhibitors and activators of cGAS are clearly therapeutic strategies that need to be explored However development of homogenous i e mix and read cGAS assays for high throughput screening HTS of small molecules is quite challenging as it requires specific detection of the cGAMP product in the presence of the substrates ATP and GTP Current assay methods involve chromatographic isolation of radioactive cGAMP produced from radiolabeled substrates From a broader perspective simple homogenous methods for detecting cGAMP in cell and tissue samples would be an extremely powerful tool for basic research drug discovery and translational studies targeting the cGAS STING pathway Our long term Phase I Phase II goal is to develop robust HTS compatible cGAS enzymatic assays and cellular cGAMP assays to accelerate discovery and clinical translation of compounds that modulate STING mediated immune responses In Phase I we will develop homogenous immunodetection methods for cGAMP with fluorescence polarization FP and time resolved F rster resonance energy transfer TR FRET signals and incorporate them into biochemical HTS assays for cGAS BellBrook has pioneered the development of HTS assays based on detection of nucleotides and our preliminary studies indicate that we will be successful in producing a monoclonal antibody mAb for cGAMP with the selectivity required for a cGAS enzymatic assay We will complete development and characterization of cGAMP mAbs synthesize fluorescent tracers that bind to the mAbs and develop the FP and TR FRET based competitive immunoassays We will then produce recombinant human cGAS using well defined E coli expression and affinity purification methods and optimize the cGAS enzymatic assays Lastly we will perform pilot screens with a LOPAC library of pharmacologically active compounds and a K diversity set to assess the level of assay interference and to demonstrate robust HTS performance The proposed assays will fulfill the key requirements for HTS including homogenous detection robustness good dynamic range low signal variability low levels of interference and outstanding reagent and signal stability In Phase II we will complete development of the biochemical cGAS HTS assay including full scale reproducible production of stable reagents for commercial assay kits We will also develop optimize and validate the reagents for the more challenging requirements of detecting cGAMP in cell lysates and tissue samples i e as an endpoint in phenotypic HTS assays and translational studies in animals Development of simple HTS homogenous methods for cGAMP in biological samples combined with cGAS enzyme assays will provide a powerful platform for discovering and characterizing compounds that modulate STING mediated immune responses and translating them into clinical candidates Narrative The ability to sense foreign nucleic acids and mount an immune response is an important weapon in the bodyandapos s armory against invading pathogens however when this system becomes aberrantly activated by self DNA it causes debilitating autoimmune disorders such as lupus and scleroderma We plan to develop simple rapid assays to detect a recently discovered signaling molecule that plays a central role in the immune response to nucleic acids the assays will enable scientists to search for drugs that prevent aberrant immune activation in autoimmune diseases

Cyclic GMP-AMP synthase (cGAS) is a recently discovered enzyme that acts as a foreign DNA sensor to elicit an immune response to pathogens via activation of the STING (stimulator of interferon genes) receptor. Though there are other DNA sensors (e.g., TLR9 ), the cGAS-cGAMP-STING pathway appears to be essential for DNA- mediated immune response irrespective of cell type or DNA sequence. DNA binds to a specific site on cGAS in a non-sequence-dependent manner and activates its catalytic activity, resulting in the production of a unique cyclic nucleotide G(2'-5')pA(3'-5')p (cGAMP) from ATP and GTP precursors. cGAMP binds to the STING receptor with nanomolar affinity and induces expression of type I interferons. Thus, cGAMP plays a fundamental role in human immunity, acting upstream of both T and B cells to trigger an innate immune response. Shortly after its discovery in 2013, aberrant activation of cGAS by self-DNA was shown to contribute to debilitating and sometimes fatal autoimmune diseases, such as systemic lupus erythematosus (SLE), and knocking out cGAS abrogates disease in animal models. The cGAS-STING pathway has also been shown to play a key role in the innate immune response to tumors, and intratumoral injection of cGAMP analogs is an emerging strategy for cancer immunotherapy. Modulating cGAS activity is autoimmunity and cancer. Tumor immunityATP GTP cGAS cGAMP Autoimmune diseases IRF Antimicrobial Immunity 3 STING 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 SLE. therefore a very compelling therapeutic strategy, both for Though basic research on the cGAS/STING pathway and efforts to target it therapeutically have expanded rapidly, the difficulty in detecting cGAMP has seriously hindered progress on both fronts. It functions at nanomolar concentrations, whereas other nucleotides, including its precursors ATP and GTP, are present at levels as much as 1000-fold higher, making specific detection of cGAMP in cell or tissue extracts extremely challenging. Currently, the only method used is methanol extraction followed by HPLC purification and LC/MS. Similarly, methods for detecting cGAS enzymatic activity involve chromatographic separation of radioactive cGAMP from ATP and GTP. Development of drugs that modulate cGAS will require sensitive, homogenous assay methods for detecting cGAMP with exquisite specificity that can be used for biochemical and cellular HTS assays, and eventually for biomarker assays to support translational research. In Phase I, we produced monoclonal antibodies that recognize cGAMP with more than 1000-fold selectivity vs. other nucleotides and used one of them to develop homogenous detection assays with fluorescent readouts (FP and TR-FRET). We validated the assays as a robust biochemical HTS platform using purified human cGAS; these assays will allow us to begin screening for cGAS modulators under separate grant applications. In Phase II, we will develop more sensitive cGAMP detection reagents and methods that can be used for cellular HTS and biomarker assays. Taking inspiration from recent examples of sandwich-based assays for small molecules, we will use cGAMP antigen design approaches and in vitro evolution to generate a pair of single chain Fv'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 assays. The effort will include contributions from two outstanding academic scientists to buttress BellBrook's assay development expertise. Dr. Karl Wittrup, Director of the Koch Institute at MIT and inventor of the yeast display system that we will use for scFv affinity maturation, will act as a consultant for the critical in vitro evolution step. Dr. Keith Elkon, Head of the Division of Rheumatology at University of Washington, one of the world's leading experts on the molecular and genetic basis for autoimmune diseases, who is pioneering efforts to elucidate the role of cGAS/STING in SLE, will collaborate on validation of the biomarker assay with samples from animal models and patients. The development of simple, quantitative cGAMP assays would have very significant scientific and medical impact. The biochemical and cellular cGAMP assays will enable BellBrook and collaborators to pursue HTS- driven efforts to develop first-in-class lead molecules targeting the cGAS/STING pathway for autoimmunity and cancer immunotherapy. More broadly, commercialization of the assays as kits will fill critical gaps in the tools needed for basic cellular and biochemical studies of the cGAS/STING pathway.

Thesaurus Terms:
Affinity; Analog; Animal Model; Antibodies; Antibody Affinity; Antigens; Antimicrobial; Applications Grants; Assay Development; Autoantibodies; Autoimmune Diseases; Autoimmunity; B-Cell Activation; B-Lymphocytes; Base; Basic Science; Binding; Biochemical; Biological; Biological Assay; Biological Markers; Biomarker Validation; Breathing; Cancer Immunotherapy; Cell Extracts; Cell Type; Cells; Cellular Assay; Clinical; Commercialization; Coupled; Cyclic Gmp; Cyclic Nucleotides; Design; Detection; Development; Dinucleoside Phosphates; Disease; Dna; Dna Binding; Dna Sequence; Drug Development; Drug Discovery; Engineering; Enzyme Activity; Enzyme-Linked Immunosorbent Assay; Enzymes; Epitopes; Evolution; Fluorescence Polarization; Fluorescence Resonance Energy Transfer; Fluorescence-Activated Cell Sorting; Gold; Guanosine Triphosphate; Head; Health; High Pressure Liquid Chromatography; High Throughput Screening; Human; Immune; Immune Response; Immune System; Immune System Function; Immunity; Immunoassay; In Vitro; Injections; Innate Immune Response; Innate Immune System; Innovation; Institutes; Interferon Activation; Interferon Type I; Interferons; Knock-Out; Lead; Liquid Chromatography Mass Spectrometry; Malignant Neoplasms; Mediating; Medical; Methanol; Methods; Microbial; Molecular Genetics; Monitor; Monoclonal Antibodies; Mouse Model; Nanomolar; Neoplastic Cell; Nucleotides; Pathogen; Pathway Interactions; Patients; Periodicity; Phase; Play; Production; Professor; Property; Pseudotoxoplasmosis Syndrome; Radioactive; Radiometry; Reagent; Receptor; Recruit; Research; Research Personnel; Response; Rheumatology; Role; Sampling; Scientist; Scleroderma; Screening; Second Messenger Systems; Sensor; Signaling Molecule; Site; Small Molecule; Specificity; Stimulator Of Interferon Genes; System; Systemic Lupus Erythematosus; T Cell Response; T-Lymphocyte; Targeted Treatment; Testing; Therapeutic; Therapeutic Intervention; Thin Layer Chromatography; Time; Tissue Extracts; Tissue Sample; Tissues; Tool; Tracer; Translational Research; Tumor; Tumor Dna; Tumor Microenvironment; Universities; Viral; Washington; Work; Yeasts;