JP2015516989A - Modulation of immune response - Google Patents

Abstract

STING modulators can up-regulate or down-regulate immune responses. Administration of such modulators can be used to treat a disease or other undesirable condition in a subject, either directly or in combination with other agents.

Description

Government Rights The invention described herein was supported by the US Government under Grant No. R01A1079336 awarded by the National Institutes of Health. The United States government has certain rights in this invention.

  Embodiments of the invention are compositions for treating and preventing infections for modulating innate and adaptive immunity and / or for treating immune related disorders, cancer, autoimmunity in a subject. Things and methods.

  Cell host defense reactions against pathogen invasion mainly require detection of pathogen-associated molecular patterns (PAMPs) such as viral nucleic acids and bacterial cell wall components, including lipopolysaccharide or flagellar proteins, that lead to induction of anti-pathogen genes. For example, viral RNA is referenced by the membrane-bound Toll-like receptor (TLR) present in the endoplasmic reticulum (ER) and / or endosomes (eg, TLR3 and 7/8) or as retinoic acid-inducible gene 1 (RIG-I) TLR-independent intracellular DExD / H box RNA helicase or melanoma differentiation associated antigen 5 (IFIH1 and MDA5, also referred to as helicard). These events result in activation of downstream signaling events, many of which remain unknown, resulting in transcription of NF-κB-dependent and IRF3 / 7-dependent genes, including type I IFN.

The molecule STING (interferon gene stimulator), which plays an important role in the innate immune response, contains five putative transmembrane (TM) regions present mainly in the endoplasmic reticulum (ER) and contains NF-κB and IRF3. Both transcriptional pathways can be activated to induce type I IFN and exert a strong antiviral state after expression. See US Patent Application No. 13 / 057,662 and International Application No. PCT / US2009 / 052767. Loss of STING reduces the ability of Poly IC to activate type I IFN, and target homologous recombination produces murine embryonic fibroblasts ( ^{− / −} MEF) lacking STING, resulting in vesicular stomatitis virus ( VSV) became more susceptible to infections. SING can play an important role in recognizing DNA from viruses, bacteria, and other pathogens capable of infecting cells, because the DNA-mediated type I IFN response was inhibited in the absence of STING Was suggested. As a result of yeast two-hybrid and co-immunoprecipitation studies, STING is a member of the translocon-related protein (TRAP) complex Ssr2 that interacts with RIG-I and is required for protein translation across the ER membrane after translation It was suggested that it interacts with / TRAPβ. RNAi disruption of TRAPβ inhibited STING function and prevented production of type I IFN in response to poly IC.

  As a result of further experiments, STING binds to nucleic acids, including single-stranded and double-stranded DNA such as pathogen-derived DNA and apoptotic DNA, and is a pro-inflammatory gene in inflammatory pathologies such as DNA-mediated arthritis and cancer. It has been shown to play a central role in regulating expression. Described herein are various new methods and compositions for upregulating STING expression or function, along with further characterization of other cellular molecules that interact with STING. These discoveries allow the design of new adjuvants, vaccines, and therapies that regulate the immune and other systems.

  Described herein are methods for modulating an immune response in a subject having a disease or disorder associated with abnormal STING function. The method comprises administering to a subject a pharmaceutical composition comprising an agent that modulates STING function and a pharmaceutically acceptable carrier in an amount that is effective for the pharmaceutical composition to improve the subject's abnormal STING function. Administering. Can include the step of administering. The agent can be a small molecule that increases or decreases STING function or a nucleic acid molecule that binds to STING under intracellular conditions. STING binding nucleic acid molecules can be 40-150 base pair long single stranded DNA or 40-150, 60-120, 80-100, or 85-95 base pair long double stranded DNA or longer. . A STING binding nucleic acid molecule can be nuclease resistant. For example, it can be composed of nuclease resistant nucleotides. It can also be related to molecules that facilitate transmembrane transport. In this method, the disease or disorder can be a DNA-dependent inflammatory disease.

  Also described herein are methods of treating cancer in a subject having a cancerous tumor infiltrated with inflammatory immune cells. The method comprises a pharmaceutical composition comprising an agent that down-regulates STING function and expression and a pharmaceutically acceptable carrier, wherein the pharmaceutical composition determines the number of inflammatory immune cells infiltrating a cancerous tumor. Test in an amount effective to reduce at least 50% (eg, at least 50, 60, 70, 80, or 90%, or until the reduction in inflammatory cell infiltration is detectably reduced by histology or scanning) Administering to the body.

1A-G show STING-dependent innate immune signaling. FIG. 1A: Human telomerase fibroblasts (hTERT-BJ1) were transfected with various nucleotides (3 μg / mL) for 16 hours. Endogenous IFNβ levels were measured. To ensure efficient transfection, hTERT-BJ1 cells were transfected with FITC conjugated dsDNA90 and examined by fluorescence microscopy. FIG. 1B: hTERT-BJ1 cells were transfected with mock, random, or two independent human STING siRNA (siRNA 3 or 4) for 3 days, followed by dsDNA90 transfection (3 μg / mL) for 16 hours. Endogenous IFNβ levels were measured. β-actin was used as a control to demonstrate hSTING protein silencing by immunoblotting. FIG. 36C: Primary STING ^{+ / +} , STING ^{− / −} , STAT1 ^{+ / +} , or STAT1 ^{− / −} MEFs were transfected with or without dsDNA90 (3 μg / mL) for 3 hours. Total RNA was purified and examined for gene expression by the Illumina Sentrix BeadChip Array (Mouse WG6 version 2). FIG. 1D: hTERT-BJ1 cells were treated with NS or STING siRNA. Three days later, cells were treated with dsDNA90, ssDNA90, or ssDNA45 (3 μg / mL). After 16 hours, IFNβ mRNA levels were measured by real-time RT-PCR. FIG. 1E: hTERT-BJ1 cells were treated with NS or STING siRNA. Cells were treated with dsDNA90, ssDNA90, or ssDNA45 (3 μg / mL) for 3 days. After 16 hours, endogenous IFNβ levels were measured. FIG. 1F: Primary STING ^{+ / +} or STING ^{− / −} MEFs were transfected with or without dsDNA90 (3 μg / mL). After 3 hours, the same procedure as in FIG. 1C was performed. FIG. 1G: hTERT-BJ1 cells were treated with or without dsDNA90 (3 μg / mL) for 3 hours and stained with anti-HA antibody and calreticulin as an ER marker. Figures 2A-J show that STING binds to DNA. Figure 2A: 293T cells were transfected with the indicated plasmid. Cell lysates were analyzed by precipitation with biotin-dsDNA90 agarose and immunoblotting with anti-HA antibody. FIG. 2B: Schematic of STING mutant. FIG. 2C: Same as FIG. 2A. FIG. 2D: same as FIG. 2A. STING mutants that cannot bind to DNA are displayed in red. FIG. 2E: hTERT-BJ1 cells were transfected with biotin-conjugated dsDNA90 (B-dsDNA90, 3 μg / mL) for 6 hours and treated with DSS. Lysates were precipitated using streptavidin agarose and analyzed by immunoblotting with anti-HA antibody. FIG. 2F: STING is expressed in 293T cells, and after 36 hours, lysates are incubated with dsDNA90 agarose in the presence of competitors dsDNA90, poly (I: C), B-DNA, or ssDNA90 and using anti-HA antibody And analyzed by immunoblotting. FIG. 2G: 293T cells were transfected with HA-tagged STING, GFP, or TREX1. Cells were lysed and biotin-labeled ssDNA or dsDNA was added along with streptavidin agarose beads. The precipitate was analyzed by immunoblotting with anti-HA antibody. FIG. 2H: 293T cells were transfected with IFNβ-luciferase and STING mutant and luciferase activity was measured. FIG. 2I: hTERT-BJ1 cells were transfected with dsDNA90 and cross-linked with formaldehyde. STING was precipitated and bound DNA was detected by PCR using dsDNA90 specific primers. NC: negative control. PC: positive control, dsDNA90. FIG. 2J: STING ^{+ / +} or STING ^{− / −} MEFs were transfected with dsDNA90 and then performed identically to FIG. Error bars represent standard deviation. Data correspond to at least two independent experiments. Figures 3A-3H show that TREX1 is a negative regulator of STING signaling. FIG. 3A: Immunoblot confirming STING and / or TREX1 knockdown in siRNA treated hTERT-BJ1 cells. FIG. 3B: siRNA treated hTERT-BJ1 cells were transfected with dsDNA90 (3 μg / mL). After 16 hours, endogenous IFNβ levels were measured. ^* P <0.05. FIG. 3C: siRNA-treated hTERT-BJ1 cells were infected with HSV-1 (multiplicity of infection = 1), and the virus titer was measured. ^* P <0.05. FIG. 38D: siRNA-treated hTERT-BJ1 cells were infected with γ34.5-deficient HSV, and the virus titer was measured. FIG. 3E: Immunoblotting of NS or STING siRNA treated TREX1 ^{+ / +} or TREX1 ^{− / −} MEF confirming STING knockdown. FIG. 3F: siRNA treated TREX1 ^{+ / +} or TREX1 ^{− / −} MEFs were treated with dsDNA90 and IFNβ levels were measured after 16 hours. ^* P <0.05. FIG. 3G: siRNA treated TREX1 ^{+ / +} or TREX1 ^{− / −} MEFs were infected with HSV-1 (multiplicity of infection = 1) and virus titers were measured. ^* P <0.05. FIG. 3H: Immunofluorescence analysis of hTERT-BJ1 cells transfected with or without dsDNA90 using anti-TREX1 or anti-STING antibodies. ^* P <0.05, Student's t test. The standard deviation is represented by error bars. Data correspond to at least two independent experiments. FIGS. 4A-J show that TREX1 is associated with the oligosaccharyl transferase complex. FIG. 4A shows a schematic diagram of TREX1. Red represents the RPN1 binding site. FIG. 4B shows a schematic diagram of STING. Red represents the DAD1 binding site. FIG. 4C: RPN1 interacts with TREX1 in yeast two-hybrid analysis (1.pGBKT7, 2.pGBKT7-NFAR M9, 3.pGBKT7-TREX1, 4.pGBKT7-STING full length, 5.pGBKT7-STING C-terminus). FIG. 4D: 293T cells were co-transfected with TREX1-tGFP and RPN1-Myc. Lysates were immunoprecipitated using anti-Myc antibody and analyzed by immunoblotting. FIG. 4H: hTERT-BJ1 cells were treated with or without dsDNA90 (3 μg / mL). Six hours after transfection, cells were examined by immunofluorescence using anti-STING antibody or anti-DAD1 antibody. FIG. 41: Immunoblot analysis of the microsomal fraction after sucrose gradient centrifugation with the indicated antibody. I: Input. FIG. 4J: hTERT-BJ1 cells were treated with TREX1, Sec61A1, TRAPβ, NS, or STING siRNA. After 72 hours, cells were treated with dsDNA90 (3 μg / mL) for 16 hours and then endogenous IFNβ levels were measured. ^* P <0.05, Student's t test. The standard deviation is represented by error bars. Data correspond to at least two independent experiments. FIGS. 5A-G show that cytoplasmic DNA induces STING-dependent genes in hTERT-BJ1 cells. FIG. 5A: hTERT-BJ1 cells were treated with STING siRNA. After 3 days, the cells were treated with or without dsDNA90 for 3 hours. Total RNA was purified and examined for gene expression using Human HT-12_V4_Bead Chip. 5B-G: hTERT-BJ1 cells were treated as in FIG. 5A. Total RNA was examined by real-time PCR for IFNβ (FIG. 5B), PMAIP1 (FIG. 5C), IFIT1 (FIG. 5D), IFIT2 (FIG. 5E), IFIT3 (FIG. 5F), and PTGER4 (FIG. 5G). Real-time PCR was performed using TaqMan gene Expression Assay (Applied Biosystem). ^* P <0.05, Student's t test. The standard deviation is represented by error bars. Data correspond to at least two independent experiments. 6A-H show that STING-dependent genes are induced by cytoplasmic DNA at MEF. Primary STAT1 ^{+ / +} or STAT1 ^{− / −} MEFs were treated with dsDNA90, IFNα, or dsDNA90 and IFNα. Total RNA was purified and IFNβ (Figure 6A), IFIT1 (Figure 6B), IFIT2 (Figure 6C), IFIT3 (Figure 6D), CXCL10 (Figure 6E), GBP1 (Figure 6F), RSAD2 (Figure 6G), and CCL5 (Figure 6H) was examined by real-time PCR. ^* P <0.05, Student's t test. The standard deviation is represented by error bars. Data correspond to at least two independent experiments. FIGS. 7A-H show that cytoplasmic DNA induces a STING-dependent gene with MEF. In FIGS. 7A-H, STING ^{+ / +} or STING ^{− / −} MEFs were treated with or without dsDNA45, dsDNA90, ssDNA45, or ssDNA90 for 3 hours. Total RNA was purified and IFNβ (Figure 7A), IFIT1 (Figure 7B), IFIT2 (Figure 7C), IFIT3 (Figure 7D), CCL5 (Figure 7E), CXCL10 (Figure 7F), RSAD2 (Figure 7G), or GBP1 (Figure 7H) was examined by real-time PCR. ^* P <0.05, Student's t test. The standard deviation is represented by error bars. Data correspond to at least two independent experiments. 8A-D show STING localization and dimerization. FIG. 8A: MEFs stably expressing STING-HA were treated with ssDNA45, dsDNA45, ssDNA90, or dsDNA90. After 3 hours, cells were stained with anti-HA antibody or anti-calreticulin antibody. FIG. 8B: 293T cells were transfected with STING-HA and Myc-STING. Lysates were analyzed by precipitation with anti-Myc antibody and immunoblotting with anti-HA antibody. FIG. 8C. hTERT-BJ1 cells were treated with or without the crosslinker DSS. Cell lysates were subjected to immunoblotting using anti-STING antibody. FIG. 8D: 293T cells were transfected with the indicated plasmid and treated with DSS. Cell lysates were analyzed by immunoblotting with anti-HA antibody. FIGS. 9A-I show that DNA viruses induce STING-dependent genes with MEF. FIG. 9A: MEFs were infected with HSV deficient HSV for 3 hours (multiplicity of infection = 1). Total RNA was purified and examined for gene expression using an Illumina Centric Bead Chip array (Mouse WGS version 2). 9B-I: STING ^{+ / +} , STING ^{− / −} , or STAT ^{− / −} / STING ^{+ / +} MEFs were treated with or without dsDNA90, HSV, or γ34.5 deleted HSV for 3 hours. Total RNA was purified and IFNβ (Figure 9B), IFIT1 (Figure 9C), IFIT2 (Figure 9D), IFIT3 (Figure 9E), CCL5 (Figure 9F), CXCL10 (Figure 9G), RSAD2 (Figure 9H), or GBP1 (Figure 9I) was examined by real-time PCR. Error bars represent standard deviation. 10A-F show that STING interacts with IRF3 / 7 and NFκ-B. FIG. 10A: IRF7 binding site of promoter region of STING-dependent dsDNA90 stimulating gene. 10B-D: Nuclear extracts were isolated from mock-treated or dsDNA90-treated STING ^{+ / +} or STING ^{− / −} MEFs, IRF3 (FIG. 10B), IRF7 (FIG. 10C), and NF-κB (FIG. 10D) Were examined according to the manufacturer's instructions. The nuclear extract kit, TransAM IRF3, TransAM IRF7, and TransAMNFκB family Elisa kit were from Active Motif. ^* P <0.05, Student's t test. The standard deviation is represented by error bars. Data correspond to at least two independent experiments. FIG. 10E: STING ^{+ / +} or STING ^{− / −} MEFs were treated with poly (I: C), dsDNA90, or HSV1 and cells were stained with anti-IRF3 antibody. FIG. 10F. STING ^{+ / +} or STING ^{− / −} MEFs were treated with dsDNA90 or HSV1 and cells were stained with anti-p65 antibody. The loss of STING did not affect poly (I: C) -mediated innate immune signaling. FIGS. 11A-F show that STING binds to DNA in vitro. FIG. 11A: In vitro translation products were precipitated with biotin conjugated dsDNA90 and immunoblotted with anti-HA antibody. FIG. 11B: Schematic of STING mutant. 11C, 11D: Same as FIG. 11A. The STING mutant lacking aa242-341 (red) failed to bind to DNA. FIGS. 11E-F: In vitro translation products were precipitated with biotin-conjugated ssDNA90 and immunoblotted with anti-HA antibody. Figures 12A-G show that STING binds to DNA in vivo and in vitro. hTERT BJ1 cells were transfected with biotin-dsDNA90 and cross-linked by UV. Cells were lysed, precipitated with streptavidin agarose and analyzed by immunoblotting. FIGS. 12B-C hTERT-BJ1 cells were treated with IFI16 (FIG. 12B) or STING (FIG. 12C) siRNA and then performed identically to FIG. 12A. FIG. 12D: STING ^{+ / +} or STING ^{− / −} MEFs were processed as in FIG. 12A. FIG. 12E: STING-Flag expressing 293T cells were treated with or without biotin-dsDNA90 and cross-linked by DSS. Lysates were sedimented and analyzed by immunoblotting. FIG. 12F: 293T cells were transfected with dsDNA90 or ssDNA90, cross-linked by UV or DSS, then sedimented and analyzed by immunoblotting. FIG. 12G: STING-Flag expressing 293T cells were lysed and incubated with ds0NA90 or poly (I: C) and biotin-dsDNA90 agarose and then performed identically to FIG. 12E. Figures 13A-C show that STING binds to viral DNA. FIG. 13A: HSV, cytomegalovirus (CMV), or adenovirus (ADV) oligonucleotide sequences. Figures 13B-C: 293T cells were transfected with the indicated plasmid. Cell lysates were analyzed by precipitation with biolin-dsDNA90, biotin-HSV DNA120mer, biotin-ADV DNA120mer, or biotin-CMV DNA120mer agarose and immunoblotted with anti-HA antibody. 14A-C show that STING binds to DNA. FIG. 14A: Schematic of STING ELISA. FIG. 14B: Process according to one embodiment of a STING ELISA. FIG. 14C: dsDNA90 binding ability was measured by ELISA. ^* P <0.05, Student's t test. The standard deviation is represented by error bars. Data correspond to at least two independent experiments. 15A-D show that TREX1 is a negative regulator of STING signaling. FIG. 15A: hTERT-BJ1 cells were treated with TREX1, STING, or STING and TREX1 siRNA. Three days later, cells were infected with HSV-Luc for 48 hours (multiplicity of infection = 1). Lysates were measured for luciferase activity. FIG. 15B: Primary TREX1 ^{+ / +} or TREX ^{− / −} MEFs were transfected with NS or STING siRNA. Three days later, cells were infected with HSV-Luc for 48 hours (multiplicity of infection = 1). The lysate was measured. FIG. 15C: Primary TREX1 ^{+ / +} or TREX1 ^{− / −} MEFs were transfected with wild type TREX1. After 48 hours, cells were infected with HSV and HSV replication was measured. TREX1 expression was examined by immunoblotting. ^* P <0.05, Student's t test. The standard deviation is represented by error bars. Data correspond to at least two independent experiments. FIG. 15D: STING ^{+ / +} or STING ^{− / −} MEFs were treated with or without dsDNA90 for 3 hours. Total RNA was purified and examined for gene expression by Illumina Sentrix Bead Chip array (Mouse WG6 version 2). FIG. 16 shows that STING regulates ssDNA90-mediated IFNβ production with TREX ^{− / −} MEF. siRNA-treated TREX1 ^{+ / +} or TREX1 ^{− / −} MEFs were treated with ssDNA45 or ssDNA90, and IFNβ levels were measured 16 hours later. ^* P <0.05, Student's t test. The standard deviation is represented by error bars. Data correspond to at least two independent experiments. FIGS. 17A-H show that TREX1 is not a negative regulator of STING-dependent genes. FIG. 17A: TREX1 ^{+ / +} or TREX1 ^{− / −} MEFs were treated with HSV1, IFNα, dsDNA90, triphosphate RNA (TPRNA), and VSV. TPRNA and VSV weakly activated IFN-induced genes. Total RNA was purified and examined for gene expression by Illumina Sentrix Bead Chip array (Mouse WG6 version 2). Figures 17B-H: Total RNA for IFNβ (Figure 17B), IFIT1 (Figure 17C), IFIT2 (Figure 17D), IFIT3 (Figure 17E), CCL5 (Figure 17F), CXCL10 (Figure 17G), RSAD2 (Figure 17H) Tested by RT-PCR. No significant differences in IFN-induced genes were observed in TREX1-deficient cells. ^* P <0.05, Student's t test. The standard deviation is represented by error bars. Data correspond to at least two independent experiments. 18A-D show that TREX1 is associated with the oligosaccharyltransferase complex. A yeast two-hybrid library (AH109) was developed using an IFN-treated hTERT cDNA library. The library was screened using full-length TREX1 as a bait. Approximately 5 million cDNA expressing yeasts were screened (Clontech). 44 clones were isolated based on 3 independent yeast conjugation procedures. RPN1 was isolated a total of 8 times (3 times on screen 1, 2 times on screen 2, and 3 times on screen 3). Most of the clones other than RPN1 failed to interact with IREX1 after retesting. Of these 8 RPN1 isolated clones, 4 clones encoded aa258-397, 2 clones aa220-390, and 2 clones encoded aa240-367. TREX1 mutants were generated and the interaction between TREX1 (aa241 to 369) and RPN1 (aa256 to 397) was mapped. To isolate DAD1, the same library was screened using the C-terminal region of STING (aa 173-379). Twenty-four isolated clones (full length DADI) were found twice. Most of the clones other than DAD1 did not interact with STING after the reconfirmation test. Full length DAD1 was associated with STING regions 242-310. FIG. 18A: Schematic of TREX1 mutant. FIG. 18B: GAL4 binding domain fused to TREX1 or TREX1-4 interacts with RPN1 fused to GAL4 activation domain in a yeast two-hybrid screen. FIG. 18C: Schematic of STING mutant. FIG. 18D. The GAL4 binding domain fused to the STING-C terminus or STING-C2 interacts with DAD1 fused to the GAL4 activation domain in a yeast two-hybrid screen. FIGS. 19A-C show that TREX1 and STING are associated with the oligosaccharyl transferase complex. FIG. 19A: 293T cells were co-transfected with TREX1-tGFP and RPN1-Myc. Lysates were analyzed by immunoprecipitation with anti-tGFP antibody or IgG control and immunoblotting with the indicated antibody. FIG. 19B: 293T cells were co-transfected with Myc-STING or GFP-DAD1 to lyse the cells. Lysates were immunoprecipitated with anti-Myc antibody or IgG control and immunoblotted with anti-GFP antibody. FIG. 19C: 293T cells were co-transfected with TREX1-tGFP and RPN1-Myc, GFP-DAD1, or STING-HA. Lysates were analyzed by immunoprecipitation with anti-tGFP antibody or IgG control and immunoblotting with anti-tGFP antibody, anti-Myc antibody, anti-GFP antibody, or anti-HA antibody. FIG. 20 shows that TREX1 localizes to the endoplasmic reticulum. hTERT-BJ1 cells were transfected with RPN1-Myc. After 48 hours, cells were examined by immunofluorescence using anti-TREX1 antibody (red), anti-Myc antibody (green), or anti-calreticulin antibody (blue) as an endoplasmic reticulum marker. FIGS. 21A-H show that STING exogenously expressed in 293T cells reconstitutes the dsDNA90 response. FIG. 21A: 293T cells were infected with control lentivirus or hSTING lentivirus. One day after infection, the cells were treated with dsDNA90. After 6 hours, cells were stained with anti-STING antibody or anti-calreticulin antibody. FIG. 21B: Cell lysates (from FIG. 56A) were subjected to immunoblotting using anti-STING antibody. 21C-D: Lentiviral infected 293T cells were stimulated with dsDNA90 for 6 hours. Total RNA was purified and examined by real-time PCR for IFNβ (FIG. 21C) or IFIT2 (FIG. 21D). FIG. 21E: 293T cells stably transduced with control or hSTING lentivirus were subjected to brefeldin A (BFA) experiments as shown in the flow diagram. FIG. 21F: Cell lysates (from FIG. 21E) were subjected to immunoblotting using anti-STING antibody. FIG. 21G: Cell lysates (from FIG. 21E) were measured for IFNβ-luciferase activity. FIG. 21H: Primary STING ^{− / −} MEFs were stably transduced with control or mSTING. Cells were treated with dsDNA90 and endogenous IFNβ levels were measured by ELISA. ^* P <0.05, Student's t test. The standard deviation is represented by error bars. Data correspond to at least two independent experiments. FIG. 22 shows that translocon members regulate HSV1 replication. hTERT-BJ1 cells were treated with TREX1, Sec61A1, TRAPβ, NS, or STING siRNA. FIGS. 23A-C show that IFI16 is not required for IFN production by dsDNA90 in hTER-BJ1 cells. FIG. 23A: hTERT-BJt cells were treated with NS, IFI16, STING, or TREX1 siRNA. After 3 days, the cells were lysed and examined by immunoblotting for expression levels. FIG. 23B: siRNA treated hTERT-BJ1 cells were stimulated with dsDNA90 and IFNβ production was measured by ELISA. FIG. 23C: siRNA-treated hTERT-BJ1 cells were infected with HSV-luciferase (multiplicity of infection = 0.1). Two days after infection, the cells were lysed and luciferase activity was measured. ^* P <0.05, Student's t test. The standard deviation is represented by error bars. Data correspond to at least two independent experiments. FIG. 24 shows one embodiment of a STING cell based assay. FIG. 25 shows that drug “A” induces STING transport. FIG. 26 shows that drug “X” inhibits IFNβ mRNA production. FIG. 27 is a schematic diagram showing that STING is phosphorylated in response to cytoplasmic DNA. hTERT-BJ1 cells were transfected with 4 μg / mL ISD for 6 hours. Cell lysates were prepared in TNE buffer, subjected to immunoprecipitation with anti-STING antibody, and then subjected to SDS-PAGE. The gel was stained with CBB and then the band containing STING was analyzed by mLC / MS / MS on a Harvard Mass Spectrometry and Proteomics Resource Laboratory. Phosphorylation sites identified by alignment and mass spectrometry of STING amino acid sequences from different species. Serines 345, 358, 366, and 379 were identified by mass spectrometry. Serine 358 and S366 are important for STING function. FIG. 28 shows that STING serine 366 (S366) is important for IFNβ production in the cytoplasmic DNA pathway. 293T cells were transfected with a plasmid encoding mutant STING and a reporter plasmid. After 36 hours, luciferase activity was measured. STING ^{− / −} MEF cells were reconstituted with mutant STING and then the amount of IFNβ in the culture medium was measured by ELISA. S366 is important for IFN production by STING, and S358 probably also plays an important role. Figures 29A-D show that STING deficient mice are resistant to DMBA-induced inflammation and skin tumor formation. STING ^{+ / +} and STING ^{− / −} mice were treated with either acetone or 10 μg DMBA once weekly for 20 weeks on the shaved back. FIG. 29A: STING deficient animals are resistant to DNA damaging agents that cause skin cancer. The percentage of mice without skin tumors is shown on the Kaplan-Meier curve. FIG. 29B: Pictures of representative mice in each treatment group are shown. FIG. 29C: Histopathological examination was performed by H & E staining on mock- or DMBA-treated skin / skin tumor biopsy samples. Images were obtained at 20x magnification. FIG. 29D: Cytokine upregulation in STING expressing mice exposed to carcinogens. RNA extracted from mock- or DMBA-treated biopsy samples of skin / skin tumors was analyzed in a duplicated manner by the Illumina Sentry BeadChip Array (Mouse WG6 version 2). Total gene expression was analyzed. The most variable gene was selected. Rows represent individual genes and columns represent individual samples. The pseudo color represents whether the transfer level is less than, equal to, or greater than average (green, black, and red, respectively). The rate of change of gene expression is in the range of -5 to 5 on the log10 scale. Cytokines were not observed in the skin of STING deficient animals.

  Described herein are methods and compositions for modulating an immune response in a subject having a disease or disorder associated with abnormal STING function. The preferred embodiments described below illustrate the application of this composition and method. Nevertheless, from the description of this embodiment, other aspects of the invention can be made and / or implemented based on the description provided below.

  Methods and compositions for modulating an immune response in a subject (eg, human, dog, cat, horse, cow, goat, pig, etc.) having a disease or disorder associated with abnormal STING function modulate STING function There is a need for a pharmaceutical composition comprising an agent and a pharmaceutically acceptable carrier. However, the amount is an amount that is effective for the pharmaceutical composition to improve abnormal STING function in the subject.

  A disease or disorder associated with abnormal STING function can be either a cell that is deficient in STING function and expression causes or exacerbates the physical symptoms of the disease or disorder. Usually such disease or disorder, eg inflammatory condition, autoimmune condition, cancer (eg breast cancer, colorectal cancer, prostate cancer, ovarian cancer, leukemia cancer, lung cancer, endometrial cancer or liver cancer), atheroma Sclerosis, arthritis (eg osteoarthritis or rheumatoid arthritis), inflammatory bowel disease (eg ulcerative colitis or Crohn's disease), peripheral vascular disease, cerebrovascular accident (seizure), chronic inflammation present, inflammation Characterized by lesions with sexual cell infiltration, amyloid plaques in the brain (eg, Alzheimer's disease), Aicardi Gutierre syndrome, juvenile arthritis, osteoporosis, amyotrophic lateral sclerosis, or multiple Sclerosis is mediated by immune system cells.

  Agents are small molecules that increase or decrease STING function and expression (ie, organic or inorganic molecules having a molecular weight of less than 500, 1000, or 2000 Daltons) or intracellular conditions (ie, STING is normally located). Nucleic acid molecules that bind to STING under intracellular conditions). The agent can also be a STING binding nucleic acid molecule, which can be single stranded (ss) or double stranded (ds) RNA or DNA. Preferably, the nucleic acid is 40 to 150, 60 to 120, 80 to 100, or 85 to 95 base pairs in length or longer. A STING binding nucleic acid molecule can be nuclease resistant. For example, it can be composed of nuclease resistant nucleotides or can be in the form of a cyclic dinucleotide. It can also be related to molecules that facilitate transmembrane transport.

  Methods and compositions for treating cancer in a subject having a cancerous tumor infiltrated with inflammatory immune cells comprise an agent that down regulates STING function and expression and a pharmaceutically acceptable carrier. A pharmaceutical composition comprising is required. However, the amount is at least 50% of the number of inflammatory immune cells that infiltrate the cancerous tumor (eg, at least 50, 60, 70, 80, or 90%, or reduced inflammatory cell infiltration). Effective amount to reduce (until it is detectably reduced by histology or scanning).

  The compositions described herein are for oral, rectal, vaginal, topical, transdermal, subcutaneous, intravenous, intramuscular, insufflation, intrathecal, and intranasal administration. It can be included with one or more pharmaceutically acceptable carriers or excipients to make a pharmaceutical composition that can be administered by a variety of routes including the following. Formulations suitable for use in the present invention include Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa. , 17th ed. (1985).

  The active ingredient can be mixed with or diluted with the excipient and / or encapsulated in a carrier that can take the form of a capsule, sachet, paper, or other container It is. Where the excipient functions as a diluent, it can be a solid, semi-solid, or liquid material that acts as a medium, carrier, or medium for the active ingredient. Compositions include tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as solids or in liquid media), ointments, It can take the form of soft and hard gelatin capsules, suppositories, sterile injectable solutions, sterile solutions for intranasal administration (eg, spray devices), or sterile packaged powders. Formulations additionally contain lubricants such as talc, magnesium stearate, and mineral oils, wetting agents, emulsifiers and suspending agents, preservatives such as methyl and propyl hydroxybenzoates, sweeteners, and flavoring agents. May be included. Compositions according to the present invention can be formulated to provide rapid, sustained or delayed release of the active ingredient after administration to a patient by utilizing procedures known in the art.

  To prepare a solid formulation such as a tablet, the composition can be mixed with pharmaceutical excipients to form a solid pre-formulation composition containing a homogeneous mixture of compounds. Tablets or pills can be coated or otherwise compounded to provide a formulation that provides the benefit of prolonged action. For example, a tablet or pill may comprise an inner dosage component and an outer dosage component, the latter taking the form of an enclosure over the former. It is possible to separate the two components by an enteric layer that functions to resist disintegration in the stomach and to deliver the inner component intact into the duodenum or delay its release. A variety of materials can be used for such enteric layers or enteric coatings, including several polymeric acids and polymeric acids and shellac, cetyl alcohol, cellulose acetate. And a mixture with such materials.

  Liquid form preparations include suspensions and emulsions. To increase serum half-life, the formulation can be encapsulated, introduced into the lumen of a liposome, prepared as a colloid, or incorporated into a layer of liposomes. For example, Szoka et al., U.S. Pat. Nos. 4,235,871, 4,501,728, and 4,837,028, each incorporated herein by reference. Various methods are available for preparing liposomes, as described in.

  The composition is preferably formulated in the form of a unit dosage form of the active ingredient. The dosage to a patient will vary depending on what is being administered, the purpose of administration (eg, prophylaxis or treatment), the condition of the patient, the mode of administration, and the like. They are all within the skills of certified physicians and certified pharmacists. For therapeutic use, the composition is administered to a patient already suffering from the disease in an amount sufficient to cure the disease and its complications or at least partially cease its symptoms. Effective amounts for this use will depend on the disease state being treated. Furthermore, the attending clinician will be judged depending on such factors as the severity of the symptoms, the age, weight and general condition of the patient.

  All documents cited herein are hereby incorporated by reference. All publications and patent documents cited in this application are incorporated by reference for all purposes to the same extent as if each individual publication or patent document was presented separately. By citing various references in this document, Applicants do not admit that any particular reference is "prior art" to the present invention. Embodiments of inventive compositions and methods are illustrated in the following examples.

Example 1: Translocon-related STING complex with cytoplasmic DNA that regulates innate immunity Type I IFN reacts with cytoplasmic dsDNA as well as DNA viruses and intracellular bacteria in fibroblasts, macrophages, and dendritic cells (DC) The isolation of a new transmembrane component of the endoplasmic reticulum (ER), referred to as STING (interferon gene stimulator), which has been demonstrated to be essential for the production of ER, has already been described (US Patent Application No. 13 / 057,662 and International Application No. PCT / US2009 / 052767). The minimum size of dsDNA required to activate STING-dependent type I IFN signaling in murine cells was confirmed to be approximately 45 base pairs in murine cells. However, it has been observed that in normal human cells (hTERT), approximately 90 base pair dsDNA (referred to herein as interferon-stimulated dsDNA90) is required to fully activate type I IFN. It was. Using the RNAi knockdown procedure, it was further confirmed that STING is actually essential for the production of type I IFN at hTERT (FIG. 1B). Further analysis using a microarray procedure to measure mRNA expression suggested that cytoplasmic dsDNA can induce a wide array of innate immune genes in addition to type I IFN in hTERT cells (FIGS. 5A-G). ). The induction of these innate molecules, including members of the IFIT family, appeared to be STING-dependent because RNAi knockdown of STING at hTERT greatly eliminated stimulation by cytoplasmic dsDNA (FIGS. 5A-G). . It has been confirmed that cytoplasmic dsDNA induces various innate immunity genes in a STING-dependent manner using STING ^{+ / +} or ^{− / −} murine embryonic fibroblasts (MEF) (FIG. 1C). In order to confirm that the induction of these mRNAs is a STING-dependent gene (SDG) and is not stimulated by type I IFN-dependent autocrine or paracrine signaling, the type I IFN signaling deficient STAT1 ^{− / −} MEF was similarly treated with dsDNA. It was verified that the state in which the production of SDG was not affected by the treatment with Fig. 1C was maintained (Fig. 1C). Array results were confirmed by reverse transcriptase (RT) PCR analysis (FIGS. 6A-H and 7A-H). It was confirmed that 45 nucleotide ssDNA (ssDNA45) induces innate immune gene production weakly in hTERTS and weaker in MEF. However, transfected ssDNA containing 90 nucleotides (ssDNA90) was observed to more robustly activate a series of genes, including type I IFN, in hTERT cells (FIGS. 1D, 1E). It was similarly confirmed that cytoplasmic ssDNA90 induced innate immune gene production in a STING-dependent manner using STING ^{+ / +} or ^{− / −} murine embryonic fibroblasts (MEF) (FIGS. 1F and 7A- H). STING probably exists as a homodimer in the ER of both human and murine cells and activates type I IFN-dependent transcription factors by translocating from the ER to the perinuclear region in the presence of cytoplasmic ssDNA or dsDNA ligands To be observed (FIGS. 1G and 8A-E). HSV1 was also observed to activate innate immune gene production in a STING-dependent manner (FIGS. 9A-I). Many of the SDGs were confirmed to contain IRF7 binding sites in their promoter regions (FIGS. 10A-F). Thus, cytoplasmic ssDNA or dsDNA, including transfected plasmid DNA, can strongly induce transcription of a wide range of innate immunity-related genes that depend on STING.

To further evaluate the possibility that STING itself can associate with DNA species, 293T cells were transfected with STING, and after cell lysis, the C-terminal region (aa181 to 349) of STING was precipitated using biotin-labeled dsDNA90. Observing was observed (FIG. 2A). The STING N-terminal region (aa1-195) and three similarly HA-tagged controls (GFP, NFAR1, and IPS1) did not associate with dsDNA90. The DNA binding exonuclease TREX1 served as a positive control. A further series of extensive studies suggested that the amino acid region 242-241 of STING is probably involved in binding to dsDNA because STING variants lacking this region could not associate with nucleic acids (FIG. 2B). ~ D). In vitro expressed STING also bound to dsDNA under high salt and high detergent conditions (also excluding mutants lacking regions 242-341) (FIGS. 11A-F). Further evidence that STING may form a complex with dsDNA, possibly as a dimer, by transfecting biotin-labeled dsDNA90 into hTERT and treating such cells with a reversible cross-linking reagent (DSS) or UV light. Got. In both treatments, STING was observed to maintain association to DNA after cell lysis (FIG. 2E and FIGS. 12A-G). When STING RNAi knockdown was performed in hTERT cells, the observed binding was eliminated, and STING-DNA complexes were similarly observed only in wild-type MEF ( ^{+ / +} ), and STING-deficient MEFs ( ⁻ // ^- ) Was not observed (FIGS. 12A to G). From competition experiments with dsDNA related to HSV1, cytomegalovirus (CMV) and even adenovirus (ADV), STING can also bind not only to dsDNA but also to ssDNA (ssDNA90) but can bind to dsRNA. It was similarly confirmed that there was no suggestion (FIG. 2F). This was confirmed by expressing STING in vitro and observing the association with ssDNA90 (FIG. 2G). All analyzed STING mutants lacked the ability to activate the IFN-type promoter in 293T cells (FIG. 2H). DsDNA was also transfected into hTERT or MEF cells and treated with formaldehyde to crosslink cellular proteins to nucleic acids. Subsequent CHIP analysis following the STING pull-down further confirmed that the transfected DNA was able to associate directly with STING as determined using dsDNA90 specific primers (FIGS. 2I and 2J). It was observed that STING can bind to biotin-labeled DNA in an ELISA assay (FIGS. 14A-C). The data suggests that ssDNA and dsDNA-mediated innate signaling events are dependent on STING, and further evidence suggests that STING itself can be complexed to these nucleic acid structures to help trigger these events It was done.

The 3 ′ → 5 ′ DNA exonuclease TREX1 is also an ER-related molecule and is important for the degradation of checkpoint activated ssDNA species that otherwise could activate the immune system. RNAi used to silence TREX1 in hTERT cells significantly increased STING-dependent production of type I IFN by dsDNA90 (FIGS. 3A and 3B). Concomitantly, dsDNA viral HSV1 replication was significantly reduced in hTERT cells lacking TREX1 probably due to increased production of type I IFN and antiviral IFN stimulating gene (ISG) (FIGS. 3C and D). ). Luciferase expression from recombinant HSV expressing the luciferase gene was also significantly reduced in hTERT infected cells treated with RNAi to silence TREX1 (FIGS. 15A-D). These observations were extended using TREX1-deficient MEFs, suggesting that cytoplasmic dsDNA-dependent gene induction is greatly increased in the absence of TREX1 and that HSV1 replication is significantly reduced. (FIGS. 3E-G). To determine if STING was involved in the increased production of type I IFN observed in the absence of TREX1, silencing STING with TREX1 deficient hTERT or TREX1 ^{− / −} MEFs and cytosolic dsDNA Alternatively, it was treated with HSV1. Since the results suggested a significant reduction in type I IFN production in TRING1 deficient cells (both hTERT and MEF) lacking STING, the increased level of type I IFN observed in the absence of TREX1 is It is suggested to be STING dependent (FIGS. 3A-F). ^{Similarly, TREX -} / - be subjected to RNAi knockdown of STING in MEF, ssDNA90 mediated type I IFN production and congenital gene stimulation was confirmed to be eliminated (Fig. 16). It was confirmed from confocal analysis that TREX1 and STING were colocalized in the ER (FIG. 3H). However, cytoplasmic dsDNA, like STING, did not strongly induce TREX1 transport from the ER to the perinuclear region (FIG. 3H). Thus, it was observed that STING and TREX1 do not interact robustly as determined by coimmunoprecipitation analysis. TREX1 ^{+ / +} or ^{− / −} MEF under unstimulated conditions did not confirm dramatic differences in STING-dependent gene expression (FIGS. 17A-H). However, TREX1 is a dsDNA-inducible gene, which has been confirmed and can be upregulated in a STING-dependent manner (FIGS. 17A-H). Thus, dsDNA species complex with STING and accessory molecules to activate downstream signaling that activates transport events and transcription factors IRF3 / 7 and NF-κB involved in the induction of primary innate immune genes including TREX1 There is a high possibility of mediating events. Evidence suggests that STING-activated TREX1 is present in the ER region and degrades activator dsDNA and suppresses cytoplasmic dsDNA signaling in the form of negative feedback. Therefore, TREX1 is a negative regulator of STING.

  It was demonstrated from the data herein that STING is present in the ER as part of the translocon complex and associates with the translocon-related protein β (TRAPβ). The translocon complex includes Sec61α, β, and γ bound to TRAPα, β, γ, and δ and is capable of binding to ribosomes. Secreted and membrane proteins are transferred into the ER for proper folding and glycosylation before being exported. To identify the TREX1 binding partner, full-length TREX1 was used as a bait on a two-hybrid yeast screen. The results suggest that TREX1 interacts repetitively with a protein referred to as ribophorin I (RPN1), a 68 kDa type I transmembrane protein and a member of the oligosaccharyltransferase (OST) complex ( 4A-E, FIGS. 18A-D). When the OST complex enters the ER via a translocon, it catalyzes the transfer of the mannose oligosaccharide to the asparagine residue of the nascent polypeptide. At least seven proteins, including RPN1, RPN2, OST48, OST4, STT3A / B, TUSC3, and DAD1, contain the OST complex. Importantly, a similar screen using STING as a bait confirmed that STING could also associate with DAD1, a 16 kDa transmembrane protein (defender for apoptotic cell death) (FIGS. 14F-H). . Further analysis of these associations using the yeast two-hybrid method suggested that the C-terminal region of TREX1 (amino acids 241 to 369), including the transmembrane region, was involved in the binding of RPN1 to amino acids 258 to 397 ( 18A-D). In addition, amino acids 242-210 of STING were involved in the association with full-length DAD1 (FIGS. 18A-D). Co-immunoprecipitation studies confirmed the interaction of these molecules (FIGS. 4D and 4G and FIGS. 29A-C). Further co-immunoprecipitation experiments suggested an association of TREX1 and DAD1 (FIGS. 19A-C). Confocal analysis confirmed that TREX1 and RPN1 colocalized within the cell were not transported in response to cytoplasmic dsDNA (FIGS. 4E and 20). Similarly, STING and DAD1 were colocalized within the ER of the cell, but the latter molecule did not entrain STING into the endosomal compartment in the presence of cytoplasmic dsDNA (FIG. 4H). Cellular microsomal compartments containing ER were isolated by fractionation and examined by sucrose gradient analysis. TREX1 and STING are co-fractionated with ER markers RPN1 and RPN2, DAD1, and calreticulin, but this study suggests that they are not co-fractionated with nuclear histone H3. Was confirmed to be indistinguishable from the components of the translocon / OST complex (FIG. 4I). Thus, TREX1 is targeted to the ER OST / translocon complex containing STING, and this association occurs via association with RPN1, while the TREX1 TM region is responsible for TREX1 localization to the ER. I found it involved. To identify whether members of the OST, TRAP, or SRP (signal recognition peptide) complex affect dsDNA-dependent signaling, RNAi screens were performed to silence expression of these components. However, except for suppression of STING (essential for DNA-mediated type I IFN production, FIGS. 21A-H) and TREX1 suppression (which greatly increases type I IFN production), only Sec61α and TRAPβ silencing is responsible for signaling and HSV1 replication. The role of these translocon members in the control of this pathway was demonstrated (Figure 4J and Figure 22). It was observed that silencing of IFI16, which is also involved in cytoplasmic DNA sensing, did not robustly suppress dsDNA-dependent signaling, at least in hTERT cells (FIGS. 23A-C). Loss of IFI16 rescue expanded IFN production by dsDNA in the absence of TREX1, similar to the loss of STING (FIGS. 23A-C). However, reducing IFI16 allowed more effective HSV1 gene expression, confirming the important role of this molecule that affects viral replication. Silencing RPN1 or 2 also resulted in increased HSV1 gene expression, but did not significantly affect type I IFN production, so these components of OST are primarily N-glycosylated. It has been demonstrated that it can be involved in

  STING is capable of complexing with cytoplasmic intracellular ssDNA and dsDNA, which can include plasmid-based DNA and gene therapy vectors, and a wide range of innate immune genes such as type I IFN, IFIT family, and antiviral The data demonstrate that the induction of various chemokines important for the initiation of active and adaptive immune responses can be modulated. STING activation promotes the escort of TBK1 to the clathrin-coated endosomal compartment and is likely to activate IRF3 / 7 by a mechanism that has not yet been fully elucidated. TREX1 appears to be present at low levels in the cell and can itself be induced by STING. After translation, TREX1 localizes to the OST complex in close proximity to non-activated STING (also present in the OST / translocon complex), where it is probably DNA that can induce STING action without it. Decompose seeds. At this point, components of the translocon / OST complex, including STING and TREX1, regulate cytoplasmic ssDNA and dsDNA-mediated innate immune signaling. Since loss of TREX1 manifests an autoimmune disorder through increased type I IFN production, the disease may be induced through STING activity.

Example 2: STING Modulator A drug library was screened to identify agents that modulate STING expression, function, activity, and the like. FIG. 24 shows the steps of the STING cell-based assay.

  Libraries include BioMol ICCB known bioactive agent library (500 targets), LOPAC1280 ™ library of pharmacologically active compounds, Enzo Life Sciences, Screen-Well ™ phosphatase inhibitor library (33 known) Phosphatase inhibitor).

  MicroSource Spectrum Collection 2000 components, 50% drug component, 30% natural product, 20% other bioactive components, EMD: InhibitorSelect ™ 96 well protein kinase inhibitor library I, InhibitorSelect ™ 96 Well protein kinase inhibitor library II, InhibitorSelect ™ 96 well protein kinase inhibitor library IIIa, kinase library B kinase TrueClone collection, kinase deficient TrueClone collection.

Results showed that one drug (designated “Drug A”) induces STING transport (FIG. 60). Another drug (designated “Drug X”) inhibited IFNβ mRNA production (FIG. 61).
Table 2: The following were identified as STING inhibitors:

  STING activators included dihydroouabain and BNTX maleate salt hydrate.

Example 3: STING develops a self-DNA-dependent inflammatory disease Bone marrow-derived macrophages (BMDM) are obtained from Sting ^{+ / +} and Sting ^{− / −} mice, and 90 base pair dsDNA (dsDNA90) activates the STING pathway They were transfected with apoptotic DNA (aDNA) derived from or from dexamethasone (Dex) treated thymocytes. It was observed that both types of DNA strongly induced IFNβ production in BMDM and normal dendritic cells (BMDC) in a STING-dependent manner. DNA microarray experiments confirmed that aDNA induces a STING-dependent production of a broad array of innate immune cytokines and inflammation-related cytokines such as TNFα and even IFNβ in BMDM (Table 3). These data were confirmed by measuring cytokine production with Sting ^{+ / +} or Sting ^{− / −} BMDM treated with aDNA. Thus, STING can promote apoptotic DNA-mediated pro-inflammatory gene production in BMDM and even BMDC.

Table 3 shows the gene expression of genes that were more highly expressed in BMDM treated with apoptotic DNA (aDNA).

To determine if STING plays a role in DNase II-related inflammatory disease, knocking down STING and / or DNase II in THP1 cells or BMDM using RNAi resulted in loss of DNase II It was found that upregulation of cytokines including type I IFN is promoted in a STING-dependent manner in response to aDNA. Since DNase II ^{− / −} mice usually die before birth, DNase II ^{− / −} , Sting ^{− / −} , or Sting ^{− / −} DNase II ^{− / −} 17 day embryos of DKO (day E17) Was analyzed. Genotypic analysis, including RT-PCR and immunoblot, confirmed that embryos lacked Sting, DNase II, or both functional genes. DNase II ^{− / −} embryos, as described above, were observed to present anemia and were significantly different from Sting ^{− / −} DNase II ^{− / −} DKO embryos or controls lacking this phenotype significantly. Showed the difference. Lethal anemia has been reported to result from type I IFN inhibition of erythropoiesis at development. Subsequently, it was observed by hematoxylin and eosin staining that the liver of DNase II ^{− / −} embryos contained numerous infiltrating macrophages filled with phagocytic apoptotic cells involved in the production of high levels of cytokines. . In contrast to control mice, Sting-/-DNase II-/-embryo livers exhibited a similar phenotype. From analysis of fetal livers by TUNEL (terminal deoxynucleotide transferase-mediated dUTP biotinic end labeling), Sting ^{− / −} DNase II ^{− / −} embryos and DNase II deficient but non-wild type fetal livers have a large number of large It was confirmed to contain improperly digested dead cells. In vitro analysis suggested that macrophages of wild-type or DNase II ^{− / −} mouse embryos phagocytose apoptotic cells sufficiently. However, phagocytic apoptotic cell DNA is efficiently degraded in lysosomes of wild-type macrophages, but DNase II ^{− / −} macrophages cannot accumulate phagocytic nuclei and digest DNA. This event results in the stimulation of innate immune signaling pathways and the production of autoimmune related cytokines. In view of this, the ability of embryonic liver-derived macrophages lacking both DNase II and STING to be evaluated for phagocytosis of apoptotic cells and DNA digestion was evaluated. Sting ^{- /} - DN DNase II ^{- / -} macrophages, DN DNase II ^{- / -} as with macrophages, wild-type mice or Sting ^{- / -} as compared to the control macrophages harvested from mice, dexamethasone treated apoptotic thymocytes It was confirmed that phagocytic nuclei from cells could not be digested. Thus, macrophages harvested from the liver of Sting ^{− / −} DNase II ^{− / −} embryonic mice show similar ability to DNase II ^{− / −} macrophages as well as the ability to digest phagocytic apoptotic cells. Absent.

The above analysis was complemented by analyzing mRNA expression levels in the liver of embryonic mice. This study suggested negligible inflammatory gene production in the livers of wild type or Sting − / − embryos. However, it was observed that the DNase II − / − embryonic liver contained abnormally high levels of cytokine-associated mRNA. Importantly, the level of innate immunity gene expression activity was dramatically reduced in the liver of Sting − / − DNase II − / − mice compared to DNase II − / − mice. These results were confirmed by analyzing the mRNA expression level of a predetermined innate immune gene in the embryonic liver by RT-PCR. For example, IFNβ production was reduced by a fraction of Sting − / − DNase II − / − mice compared to DNase II − / − mice. Production of interferon-stimulated genes (ISG) such as 2′-5 ′ oligoadenylate synthetase (OAS), interferon-inducing protein with tetratricopeptide repeat (IFIT), interferon-inducing protein 27 (IFI27), ubiquitin-like modifier (ISG15) Was also dramatically reduced. Pro-inflammatory cytokines such as TNFα and IL1β were also reduced in Sting ^{− / −} and Sting ^{− / −} DNase II ^{− / −} embryonic liver compared to DNase II ^{− / −} mice. Innate immune gene production was dramatically suppressed in the absence of STING, but the presence of some genes was low in Sting ^{− / −} DNase II ^{− / −} mice as determined by array analysis However, it maintained a slightly increased state. This may be due to fluctuations in mRNA expression between the analyzed animals or possibly due to stimulation of other pathways. Many of these genes are regulated by the NF-κB pathway and the interferon regulatory factor (IRF) pathway. Therefore, the function of these transcription factors was evaluated in Sting ^{− / −} DNase II ^{− / −} or control murine embryonic fibroblasts (MEFs) generated from 14 day embryos (E14 days). In most cases, deficiency of NF-κB activity (p65 phosphorylation) was observed in Sting ^{− / −} DNase II ^{− / −} MEF when exposed to cytoplasmic DNA. The same deficiency was obtained with Sting ^{− / −} DNase II ^{− / −} BMDM when exposed to apoptotic DNA as well as cytoplasmic DNA. This was confirmed by noting that NF-κB and also IRF3, after exposure to dsDNA, could not be transferred with Sting ^{− / −} DNase II ^{− / −} MEF but could be transferred with control MEF. Thus, STING is probably involved in the control of self DNA-induced inflammatory cytokine production that is involved in the induction of lethal embryonic erythropoiesis.

To highlight the importance of STING in mediating lethal erythropoiesis promoted by self DNA, we evaluated whether DNase II ^{− / −} mice can be born in the absence of STING. Importantly, it was observed that DNase II ^{− / −} mice were born at an apparent Mendelian frequency when mated to the Sting − / − background. PCR genotyping, Northern blot analysis, RT-PCR analysis, and immunoblot analysis confirmed DNase II and STING deficiency in progeny mice. Sting ^{− / −} DNase II ^{− / −} double knockout mice (DKO) appeared to grow normally and exhibited similar size and weight compared to control mice, but Sting ^{− / −} mice It was still confirmed that it was slightly larger for unknown reasons. From preliminary immunological assessments, Sting ^{− / −} DNase II ^{− / −} DKO animals shared similar CD4 ⁺ / CD8 ⁺ profiles, similar to Sting ^{− / −} and wild type mice. It was suggested that DKO has onset of splenomegaly with aging. Splenomegaly has also been observed in surviving DNase II-deficient mice lacking type I IFN signaling (DNase II ^{− / −} Ifnar1 ^{− / −} mice) and has been reported to be due to swelling of the red pulp. However, the analysis of Sting ^{− / −} DNase II ^{− / −} mice sera produced low levels of common measurable cytokines, so that detectable abnormalities compared to 8 week old control mice Cytokine production was not suggested. Through these studies, the in vitro, Sting ^{- /} - DN DNase II ^{- / -} compared with control macrophages collected from mice ^- macrophages, DN DNase II ^{- / -} as with macrophages, wild-type mice or Sting ^{- /} It was confirmed that phagocytic nuclei from apoptotic thymocytes (Dex ⁺ ) could not be digested. When WT thymocytes were used as a target (Dex ⁻ ), the accumulation of undigested DNA in DNase II ^{− / −} Sting ^{− / −} macrophages was less pronounced. Thus, BMDM derived from Sting ^{− / −} DNase II ^{− / −} mice also cannot digest DNA of apoptotic cells, but in contrast to DNase II ^{− / −} BMDM, does not produce an inflammatory cytokine response.

Although it is possible to avoid DNase II-mediated embryonic lethality by mating DNase II ^+/− mice with type I IFN-deficient Ifnar1 ^{− / −} mice, the progeny obtained is that the undigested DNA is congenital Severe polyarthritis (arthritis score 2) occurs about 8 weeks after birth because it activates immune signaling pathways and induces the production of inflammatory cytokines such as TNFα. Importantly, Sting ^{− / −} DNase II ^{− / −} mice were confirmed to show no signs of polyarthritis after birth. The arthritis score was maintained at about 0 (no score) until 12 months of age in Sting ^{− / −} DNase II ^{− / −} , in contrast to the reported DNase II ^{− / −} Ifnar1 ^{− / -} mice, after similar period, exhibited arthritic score of up to 7. DNase II ^{− / −} Sting ^{− / −} H & E staining and TUNEL staining of spleen and thymus tissue of mice showed the presence of infiltrating macrophages that also contained apoptotic DNA, but 6 month old Sting ^{− / −} DN Histological diagnosis of the joints of ase II ^{− / −} mice showed no evidence of pannus infiltration in the joint structure, and exhibited normal bone (B) synovial joint (S) and cartilage (C) structures. Levels of TNFα, IL1β, and IL6 from Sting ^{− / −} DNase II ^{− / −} mouse sera remained at low levels, as expected from our array analysis in BMDM lacking STING. (Table 3). There was no evidence of CD4, CD68, or TRAP positive cell infiltration in the joints of Sting ^{− / −} DNase II ^{− / −} mice. Also, analysis of Sting ^{− / −} DNase II ^{− / −} mice sera did not suggest elevated levels of rheumatoid factor (RF), anti-dsDNA antibodies, or MMP3. Thus, loss of STING avoids pro-inflammatory cytokine production involved in self DNA-mediated polyarthritis.

  STING is involved in inflammatory diseases such as, for example, Aicardi Gutierre syndrome (AGS). AGS is a genetically determined encephalopathy characterized by calcification and demyelination of the basal ganglia and white matter. High levels of lymphocytes and type I IFN in the cerebrospinal fluid. The characteristics are very similar to chronic infections. Serum levels of type I IFN are also elevated in autoimmune syndrome systemic lupus erythematosus (SLE). AGS is caused by mutations in the 3'-5 'DNA exonuclease TREX1. When TREX1 function is lost, DNA species accumulate in the ER of the cell and activate the cytoplasmic DNA sensor (STING). TREX1 digests this DNA source to prevent innate immune gene activation (housekeeping function).

Given that STING appears to be involved in inflammatory diseases in apoptosis-deficient mice, it was next evaluated whether other types of self-DNA-induced diseases occur via activation of the STING pathway. For example, a 3 ′ repair exonuclease 1 (Trex1) deficient patient suffers from Aicardi Gutierre syndrome (AGS) causing lethal encephalitis characterized by the presence of high levels of type I IFN production in the cerebrospinal fluid To do. Because Trex1-deficient mice activate intracellular DNA sensors, autologous DNA that has not yet been characterized to induce cytokine production and induce lethal inflammatory malignant myocarditis (probably normally digested by Trex1) It exhibits a median life of about 10 weeks. Although loss of STING can extend the lifespan of Trex1 ^{− / −} mice, recent data suggest that the cause is unknown. These studies were expanded to confirm that Trex1-deficient BMDCs exposed to dsDNA90 (Trex1 ^{− / −} BMDC) slightly increased the level of type I IFN production. Importantly, loss of STING (Sting ^{− / −} Trex1 ^{− / −} BMDC) eliminated the ability of DNA to increase type I IFN production in Trex1 deficient BMDM. Interestingly, a decrease in the size of the Sting ^{− / −} Trex1 ^{− / −} and Sting ^{− / +} Trex1 ^{− / −} hearts was observed compared to Trex1 ^{− / −} mice. It was also confirmed that the evidence of myocarditis was dramatically reduced with Sting ^{− / −} Trex1 ^{− / −} compared to Trex1 ^{− / −} alone. In addition, Trex1 ^{- / -} antinuclear autoantibodies It was observed that quite prevalent in the sera of mice (ANA) ^{is, Sting - / - Trex1 - /} - almost completely absent in the sera of mice Met. Microarray analysis demonstrates that the levels of pro-inflammatory genes in the heart of Sting ^{− / −} Trex1 ^{− / −} , Sting ^{− / +} Trex1 ^{− / −} are dramatically reduced compared to Trex1 ^{− / −} mice It was done. Taken together, these data suggest that STING is involved in proinflammatory gene induction in Trex1-deficient mice and possibly AGS.

Example 4: Screening of kinases for STING S366 The activity of 217 protein kinase targets was evaluated against two peptides (A366 and S366) as substrates. Protein kinases were mixed with each peptide and then ³³ P-ATP and activity (CPM) was measured. The following kinases were identified as phosphorylating S366 in STING. The identification of a serine-targeting kinase opens the way for drug discovery. Agents that target this association can inhibit STING activity. It can also be used to inhibit STING activity for therapeutic purposes. STING overactivity can lead to inflammatory diseases that can exacerbate cancer.

Table 4: Screening of kinases for STING S366

Example 5: STING is involved in inflammation-related cancer When STING WT and STING ^{− / −} animals were treated with DNA damaging agents, mice lacking STING were resistant to tumor formation. This is because infiltrating immune cells such as dendritic cells, macrophages phagocytose damaged cells that have undergone necrosis or apoptosis, and DNA or other ligands of such cells produce STING and cytokines that promote tumorigenesis. Is to activate. STING may be involved in promoting tumor progression in a wide variety of other cancers.

Figures 29A-D show that STING deficient mice are resistant to DMBA-induced inflammation and skin tumor formation. STING ^{+ / +} and STING ^{− / −} mice were treated with either acetone or 10 μg DMBA once weekly for 20 weeks on the shaved back. FIG. 29A: STING deficient animals are resistant to DNA damaging agents that cause skin cancer. The percentage of mice without skin tumors is shown on the Kaplan-Meier curve. FIG. 29B: Pictures of representative mice in each treatment group are shown. FIG. 29C: Histopathological examination was performed by H & E staining on mock- or DMBA-treated skin / skin tumor biopsy samples. Images were obtained at 20x magnification. FIG. 29D: Cytokine upregulation in STING expressing mice exposed to carcinogens. RNA extracted from mock- or DMBA-treated biopsy samples of skin / skin tumors was analyzed in a duplicated manner by the Illumina Sentry BeadChip Array (Mouse WG6 version 2). Total gene expression was analyzed. The most variable gene was selected. Rows represent individual genes and columns represent individual samples. The pseudo color represents whether the transfer level is less than, equal to, or greater than the average. The rate of change of gene expression is in the range of -5 to 5 on the log10 scale. Cytokines were not observed in the skin of STING deficient animals.

Claims (13)

  In a method of modulating the immune response of a subject having a disease or disorder associated with abnormal STING function, the pharmaceutical composition comprises an agent that modulates STING function and a pharmaceutically acceptable carrier. Administering to said subject in an amount effective to ameliorate abnormal STING function of said subject.   2. The method of claim 1, wherein the agent is a small molecule that increases STING function.   2. The method of claim 1, wherein the agent is a small molecule that decreases STING function.   2. The method of claim 1, wherein the agent is a nucleic acid molecule that binds to STING under intracellular conditions.   6. The method according to claim 5, wherein the nucleic acid molecule is a single-stranded DNA having a length of 40 to 150 base pairs.   6. The method according to claim 5, wherein the nucleic acid molecule is a double-stranded DNA having a length of 40 to 150 base pairs.   6. The method according to claim 5, wherein the nucleic acid molecule is a double-stranded DNA having a length of 60 to 120 base pairs.   6. The method according to claim 5, wherein the nucleic acid molecule is a double-stranded DNA having a length of 80 to 100 base pairs.   6. The method according to claim 5, wherein the nucleic acid molecule is a double-stranded DNA having a length of 85 to 95 base pairs.   5. The method of claim 4, wherein the nucleic acid molecule comprises a nuclease resistant nucleotide.   5. The method of claim 4, wherein the nucleic acid molecule is associated with a molecule that facilitates transmembrane transport of the nucleic acid molecule.   2. The method of claim 1, wherein the disease or disorder is a DNA-dependent inflammatory disease. In a method of treating cancer in a subject having a cancerous tumor infiltrated with inflammatory immune cells, a pharmaceutical composition comprising an agent that down-regulates the function and expression of STING and a pharmaceutically acceptable carrier And administering the pharmaceutical composition to the subject in an amount effective to reduce the number of inflammatory immune cells infiltrating the cancerous tumor by at least 50%.

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