CN104540945A - Modulating immune responses - Google Patents

Abstract

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

Description

Regulate immune response

government rights

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

field of invention

Embodiments of the invention relate to compositions and methods for modulating innate and adaptive immunity in a subject and/or for treatment of immune-related disorders, cancer, autoimmunity, treatment and prevention of infection.

background

Cellular host defense responses to pathogen invasion primarily involve the detection of pathogen-associated molecular patterns (PAMPs), such as viral nucleic acids or bacterial cell wall components including lipopolysaccharide or flagellin, leading to the induction of antipathogenic genes. For example, viral RNA can pass through membrane-bound Toll-like receptors (TLRs) present in the endoplasmic reticulum (ER) and/or endosomes (such as TLR 3 and 7/8) or through TLR-dependent intracellular DExD/ H-box RNA helicase (known as retinoic acid-inducible gene 1 (RIG-I)) or melanoma differentiation-associated antigen 5 (MDA5, also known as IFIH1 and helicard) detection. These events culminate in the activation of downstream signaling events, most of which are still unknown, resulting in the transcription of NF-κΒ and IRF3/7-dependent genes, including type I IFN.

overview

STING (Stimulator of Interferon Genes), a molecule that plays a key role in the innate immune response, includes five putative transmembrane (TM) domains located primarily in the endoplasmic reticulum (ER) and is capable of activating NF- Both the κB and IRF3 transcriptional pathways thereby induce type I IFN and exert a potent antiviral state upon expression. See US Patent Application Serial No. 13/057,662 and PCT/US2009/052767. Loss of STING reduces the ability of polyIC to activate type I IFN and renders murine embryonic fibroblasts lacking STING ( ^-/- MEF) generated by targeted homologous recombination susceptible to vesicular stomatitis virus (VSV) infection. In the absence of STING, DNA-mediated type I IFN responses were suppressed, suggesting that STING may play an important role in recognizing DNA from viruses, bacteria, and other pathogens that can infect cells. Yeast two-hybrid and co-immunoprecipitation studies reveal that STING interacts with RIG-I and interacts with Ssr2/TRAPβ, a member of the translocon-associated protein (TRAP) complex required for translocation of post-translational proteins across ER membranes member. RNAi ablation of TRAPβ inhibits STING function and blocks type I IFN production in response to polyIC.

Additional experiments have shown that STING itself binds, for example, nucleic acids from pathogens (including single- and double-stranded DNA) and apoptotic DNA, and plays a role in modulating pro-inflammatory gene expression in inflammatory disorders such as DNA-mediated arthritis and cancer play a central role in. Various novel methods and compositions for upregulating STING expression or function are described herein, along with additional characterization of other cellular molecules that interact with STING. These findings allow for the design of new adjuvants, vaccines and therapies for modulating the immune system and other systems.

Methods for modulating an immune response in a subject suffering from a disease or disorder associated with abnormal STING function are described herein. The methods can include the step of administering to the subject an amount of a pharmaceutical composition comprising an agent that modulates STING function and a pharmaceutically acceptable carrier, wherein the amount of the pharmaceutical composition is effective Improve the subject's abnormal STING function. The agent can be a small molecule that increases or decreases the function of STING, or a nucleic acid molecule that binds to STING under intracellular conditions. The STING-binding nucleic acid molecule can be a single-stranded DNA between 40 and 150 base pairs in length or a 40 and 150, 60 and 120, 80 and 100, or 85 and 95 base pairs in length Between or longer double-stranded DNA. The bound STING nucleic acid molecule may be nuclease resistant, eg, consist of nuclease resistant nucleotides. It can also be associated with a molecule that facilitates transmembrane transport. In these methods, the disease or disorder can be a DNA-dependent inflammatory disease.

Also described herein are methods of treating cancer in a subject suffering from a cancerous tumor infiltrated by inflammatory immune cells. These methods may include the step of administering to the subject an amount of a pharmaceutical composition comprising an agent that down-regulates STING function or expression and a pharmaceutically acceptable carrier, wherein the amount of the pharmaceutical composition is effective to reduce the number of inflammatory immune cells infiltrating the cancerous tumor by at least 50% (e.g., at least 50%, 60%, 70%, 80%, or 90%), or until the reduction in inflammatory cell infiltration is determined by histological or scan detectably reduced).

Description of drawings

Figures 1A-G illustrate STING-dependent innate immune signaling. Figure 1A: Human telomerase fibroblasts (hTERT-BJ1) were transfected with different nucleotides (3 μg/ml) for 16 h. Endogenous IFNβ levels were measured. hTERT-BJ1 cells were transfected with FITC-conjugated dsDNA90 and examined by fluorescence microscopy to ensure efficient transfection. Figure IB: hTERT-BJ1 cells were transfected with mock, random or two independent human STING siRNAs (siRNA 3 or 4) for 3 days, followed by dsDNA90 transfection (3 μg/ml) for 16 hours. Endogenous IFNβ levels were measured. Silencing of hSTING protein was demonstrated by immunoblotting with β-actin as a control. Figure 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 gene expression was examined by Illumina Sentrix bead chip array (mouse WG6 version 2). Figure ID: hTERT-BJ1 cells were treated with NS or STING siRNA. After 3 days, cells were treated with dsDNA90, ssDNA90 or ssDNA45 (3 μg/ml). After 16 hours, IFNβ mRNA levels were measured by real-time RT-PCR. Figure 1E: hTERT-BJ1 cells were treated with NS or STING siRNA. On day 3, cells were treated with dsDNA90, ssDNA90 or ssDNA45 (3 μg/ml). After 16 hours, endogenous IFNβ levels were measured. Figure 1F: Primary STING ^+/+ or STING ^-/- MEFs were transfected with or without dsDNA90 (3 μg/ml). After 3h, the same as Figure 1C. Figure 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 ER marker.

Figures 2A-J show STING binding to DNA. Figure 2A: 293T cells were transfected with the indicated plasmids. Cell lysates were precipitated with biotin-dsDNA90 agarose and analyzed by immunoblotting using anti-HA antibody. Figure 2B: Schematic representation of STING mutants. Figure 2C: Same as Figure 2A. Figure 2D: Same as Figure 2A. STING variants that cannot bind DNA are marked in red. Figure 2E: hTERT-BJ1 cells were transfected with biotin-conjugated dsDNA90 (B-dsDNA90; 3 μg/ml) for 6 h and treated with DSS. Lysates were precipitated using streptavidin agarose and analyzed by immunoblotting using anti-HA antibody. Figure 2F: After expressing STING in 293T cells and 36 hours later, in the presence of competitor dsDNA90, poly(I:C), B-DNA or ssDNA90, the lysate was incubated with dsDNA90 agarose and immunized with anti-HA antibody. blot for analysis. Figure 2G: 293T cells were transfected with HA-tagged STING, GFP or TREX1. Cells were lysed and biotin-labeled ssDNA or dsDNA and streptavidin sepharose beads were added. Precipitates were analyzed by immunoblotting using anti-HA antibody. Figure 2H: 293T cells were transfected with IFNβ-luciferase and STING variants and luciferase activity was measured. Figure 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. Figure 2J: STING ^+/+ or STING ^-/- MEFs were transfected with dsDNA90 and then the same as in Figure 2I. Error bars represent standard deviation. Data represent at least two independent experiments.

Figures 3A-3H show that TREX1 is a negative regulator of STING signaling. Figure 3A: Immunoblot confirming knockdown of STING and/or TREX1 in siRNA-treated hTERT-BJ1 cells. Figure 3B: siRNA-treated hTERT-BJ1 cells were transfected with dsDNA90 (3 μg/ml). After 16 hours, endogenous IFNβ levels were measured. *P<0.05. Figure 3C: siRNA-treated hTERT-BJ1 cells were infected with HSV-1 (moi=1) and virus titers were measured. *P<0.05. Figure 38D: siRNA-treated hTERT-BJ1 cells were infected with γ34.5-deleted HSV and virus titers were measured. *P<0.05. Figure 3E: Immunoblot of NS or STING siRNA-treated TREX1 ^+/+ or TREX1 ^−/− MEFs confirms STING knockdown. Figure 3F: siRNA-treated TREX1 ^+/+ or TREX1 ^-/- MEFs were treated with dsDNA90 and 16 hours later, IFNβ levels were measured. *P<0.05. Figure 3G: siRNA-treated TREX1 ^+/+ or TREX1 ^-/- MEFs were infected with HSV-1 (moi=1) and virus titers were measured. *P<0.05. Figure 3H: Immunofluorescence analysis of hTERT-BJ1 cells transfected with or without dsDNA90 by anti-TREX1 or anti-STING antibodies. *P<0.05, Student's t-test. Error bars represent standard deviation. Data represent at least two independent experiments.

Figures 4A-J: Shows that TREX1 associates with the oligosaccharyltransferase complex. Figure 4A shows a schematic representation of TREX1. Red indicates RPN1 binding sites. Figure 4B shows a schematic diagram of STING. Red indicates DAD1 binding sites. Figure 4C: RPN1 interacts with TREX1 in yeast two-hybrid assays (1. pGBKT7, 2. pGBKT7-NFAR M9, 3. pGBKT7-TREX1, 4. pGBKT7-STING full-length, 5. pGBKT7-STING C-terminus) . Figure 4D: 293T cells were co-transfected with TREX1-tGFP and RPN1-Myc. Lysates were immunoprecipitated using anti-Myc antibody and analyzed by immunoblotting. Figure 4H: hTERT-BJ1 cells were treated with or without dsDNA90 (3 μg/ml). At 6 h after transfection, cells were examined by immunofluorescence using anti-STING or anti-DAD1 antibodies. Figure 4I: Immunoblot analysis of microsomal fractions using the indicated antibodies after sucrose gradient centrifugation. I: Enter. Figure 4J: hTERT-BJ1 cells were treated with TREX1, Sec61A1, TRAPβ, NS or STING siRNA. After 72 h, cells were treated with dsDNA90 (3 μg/ml) for 16 h and endogenous IFNβ levels were then measured. *P<0.05, Student's t-test. Error bars represent standard deviation. Data represent at least two independent experiments.

Figures 5A-G show that cytoplasmic DNA induces STING-dependent genes in hTERT-BJ1 cells. Figure 5A: hTERT-BJ1 cells were treated with STING siRNA. After 3 days, cells were treated with or without dsDNA90 for 3 h. Total RNA was purified and gene expression was checked using human HT-12_V4_bead chip. Figure 5B-G: hTERT-BJ1 cells were treated as in Figure 5A. Total RNA of IFNβ (Fig. 5B), PMAIP1 (Fig. 5C), IFIT1 (Fig. 5D), IFIT2 (Fig. 5E), IFIT3 (Fig. 5F) and PTGER4 (Fig. 5G) was examined by real-time PCR. Real-time PCR was performed using the TaqMan Gene Expression Assay (Applied Biosystem). *P<0.05, Student's t-test. Error bars represent standard deviation. Data represent at least two independent experiments.

Figures 6A-H show that in MEFs, STING-dependent genes are induced by cytoplasmic DNA. Primary STAT1 ^+/+ or STAT1 ^−/− MEFs were treated with dsDNA90, IFNα, or dsDNA90 with IFNα. Total RNA was purified and examined by real-time PCR for IFNβ (Fig. 6A), IFIT1 (Fig. 6B), IFIT2 (Fig. 6C), IFIT3 (Fig. 6D), CXCL10 (Fig. 6E), GBP1 (Fig. 6F), RSAD2 (Fig. 6G) and CCL5 (Fig. 6H). *P<0.05, Student's t-test. Error bars represent standard deviation. Data represent at least two independent experiments.

Figures 7A-H show that cytoplasmic DNA induces STING-dependent genes in MEFs. In Figure 7A-H, STING ^+/+ or STING ^−/− MEFs were treated with or without dsDNA45, dsDNA90, ssDNA45 or ssDNA90 for 3 h. Total RNA was purified and examined by real-time PCR for IFNβ (Fig. 7A), IFIT1 (Fig. 7B), IFIT2 (Fig. 7C), IFIT3 (Fig. 7D), CCL5 (Fig. 7E), CXCL10 (Fig. 7F), RSAD2 (Fig. 7G) or GBP1 (Fig. 7H). *P<0.05, Student's t-test. Error bars represent standard deviation. Data represent at least two independent experiments.

Figure 8A-D shows STING localization and dimerization. Figure 8A: MEFs stably expressing STING-HA were treated with ssDNA45, dsDNA45, ssDNA90 or dsDNA90. After 3 h, cells were stained using anti-HA or anti-calreticulin antibodies. Figure 8B: 293T cells were transfected with STING-HA and Myc-STING. Lysates were precipitated by anti-Myc antibody and analyzed by immunoblotting using anti-HA antibody. Figure 8C. hTERT-BJ1 cells were treated with or without the cross-linker DSS. Cell lysates were subjected to immunoblotting using anti-STING antibody. Figure 8D: 293T cells were transfected with the indicated plasmids and treated with DSS. Cell lysates were analyzed by immunoblotting using anti-HA antibody.

Figures 9A-I show that DNA viruses induce STING-dependent genes in MEFs. Figure 9A: MEFs were infected with γ34.5-deleted HSV (moi=1) for 3 h. Total RNA was purified and gene expression was examined using an Illumina Sentric bead chip array (mouse WGS version 2). Figure 9B-I: STING ^+/+ , STING ^-/- or STAT ^-/- /STING ^+/+ MEFs were treated with or without dsDNA90, HSV or γ34.5-deleted HSV for 3 h. Total RNA was purified and examined by real-time PCR for IFNβ (Fig. 9B), IFIT1 (Fig. 9C), IFIT2 (Fig. 9D), IFIT3 (Fig. 9E), CCL5 (Fig. 9F), CXCL10 (Fig. 9G), RSAD2 (Fig. 9H) or GBP1 (Fig. 9I). Error bars represent standard deviation.

Figures 10A-F show that STING interacts with IRF3/7 and NFκ-B. Figure 10A: IRF7 binding sites in the promoter regions of STING-dependent dsDNA90-stimulated genes. Figure 10B-D: Following the manufacturer's instructions, nuclear extracts were isolated from mock-treated or dsDNA90-treated STING ^+/+ or STING ^-/- MEFs and examined for IRF3 (Figure 10B), IRF7 (Figure 10C) and NF -κB (Fig. 10D) activation. The nucleus extraction kit, TransAM IRF3, TransAM IRF7 and TransAMNFκB family Elisa kit were from Active Motif. *P<0.05, Student's t-test. Error bars represent standard deviation. Data represent at least two independent experiments. FIG. 10E : STING ^+/+ or STING ^−/− MEFs were treated with poly(I:C), dsDNA90 or HSV1 and cells were stained by anti-IRF3 antibody. Figure 10F. STING ^+/+ or STING ^-/- MEFs were treated with dsDNA90 or HSV1 and cells were stained by anti-p65 antibody. Loss of STING does not affect poly(I:C)-mediated innate immune signaling.

Figures 11A-F show that STING binds DNA in vitro. Figure 11A: In vitro translation products were precipitated with biotin-conjugated dsDNA90 and immunoblotted by anti-HA antibody. Figure 1 IB: Schematic representation of STING variants. Fig. 11C, 11D: Same as Fig. 11A. The STING variant (red) lacking aa242-341 cannot bind DNA. Figures 11E-F: In vitro translation products were precipitated with biotin-conjugated ssDNA90 and immunoblotted by anti-HA antibody.

Figures 12A-G show that STING binds DNA in vivo and in vitro. hTERT BJ1 cells were transfected with biotin-dsDNA90 and crosslinked by UV. Cells were lysed and precipitated by streptavidin agarose and analyzed by immunoblotting. Figures 12B-C hTERT-BJ1 cells were treated with IFI16 (Figure 12B) or STING (Figure 12C) siRNA and then the same as in Figure 12A. Figure 12D: STING ^+/+ or STING ^-/- MEFs were processed as in Figure 12A. Figure 12E: 293T cells expressing STING-Flag were treated with or without biotin-dsDNA90 and cross-linked by DSS. Lysates were pelleted and analyzed by immunoblotting. Figure 12F: 293T cells were transfected with or without dsDNA90 or ssDNA90 and crosslinked by UV or DSS and then pelleted and analyzed by immunoblotting. Figure 12G: 293T cells expressing STING-Flag were lysed and incubated with dsONA90 or poly(I:C) and biotin-dsDNA90 agarose and then the same as Figure 12E.

Figures 13A-C show that STING binds viral DNA. Figure 13A: Oligonucleotide sequences of HSV, cytomegalovirus (CMV) or adenovirus (ADV). Figure 13B-C: 293T cells were transfected with the indicated plasmids. Cell lysates were precipitated with biotin-dsDNA90, biotin-HSV DNA120mer, biotin-ADV DNA 120mer or biotin-CMV DNA 120mer agarose and analyzed by immunoblotting using anti-HA antibody.

Figures 14A-C show that STING binds DNA. Figure 14A: Schematic representation of the STING ELISA. Figure 14B: Procedure of one embodiment of the STING ELISA. Figure 14C: Measurement of dsDNA90 binding ability by ELISA. *P<0.05, Student's t-test. Error bars represent standard deviation. Data represent at least two independent experiments.

Figures 15A-D show that TREX1 is a negative regulator of STING signaling. Figure 15A: hTERT-BJ1 cells were treated with TREX1, STING or STING and TREX1 siRNA. After 3 days, the cells were infected with HSV-Luc (moi=1) for 48h. Lysate luciferase activity was measured. Figure 15B: Primary TREX1 ^+/+ or TREX ^-/- MEFs were transfected with NS or STING siRNA. After 3 days, the cells were infected with HSV-Luc (moi=1) for 48h. Measure the lysate. Figure 15C: Primary TREX1 ^+/+ or TREX ^-/- MEFs were transfected with NS or STING siRNA. After 48 h, cells were infected with HSV and HSV replication was measured. TREX1 expression was examined by immunoblotting. *P<0.05, Student's t-test. Error bars represent standard deviation. Data represent at least two independent experiments. FIG. 15D : STING ^+/+ or STING ^−/− MEFs were treated with or without dsDNA90 for 3 h. Total RNA was purified and gene expression was examined by Illumina Sentrix bead chip array (mouse WG6 version 2).

Figure 16 shows that STING regulates ssDNA90-mediated IFNβ production in TREX ^-/- MEFs. siRNA-treated TREX1 ^+/+ or TREX1 ^−/− MEFs were treated with ssDNA45 or ssDNA90 and 16 hours later, IFNβ levels were measured. *P<0.05, Student's t-test. Error bars represent standard deviation. Data represent at least two independent experiments.

Figures 17A-H show that TREX1 is not a negative regulator of a STING-dependent gene. Figure 17A: TREX1 ^+/+ or TREX1 ^-/- MEFs were treated with HSV1, IFNα, dsDNA90, triphosphate RNA (TPRNA) and VSV. TPRNA and VSV weakly activate IFN-induced genes. Total RNA was purified and gene expression was examined by Illumina Sentrix bead chip array (mouse WG6 version 2). Figure 17B-H: Examination of IFNβ (Figure 17B), IFIT1 (Figure 17C), IFIT2 (Figure 17D), IFIT3 (Figure 17E), CCL5 (Figure 17F), CXCL 10 (Figure 17G), RSAD2 (Figure 17G) by RT-PCR 17H) total RNA. No significant differences in IFN-induced genes were observed in cells lacking TREX1. *P<0.05, Student's t-test. Error bars represent standard deviation. Data represent at least two independent experiments.

Figures 18A-D show that TREX1 associates with the oligosaccharyltransferase complex. A yeast two-hybrid library (AH109) was generated using the IFN-treated hTERT cDNA library. The library was screened using full length TREX1 as bait. About 5 million yeasts expressing cDNA (Clontech Company) were screened. Forty-four clones were isolated from 3 independent yeast hybridization programs. In total, RPN1 was isolated 8 times (three times in screen 1, two times in screen 2 and three times in screen 3). Most clones (except RPN1) were unable to interact with IREX1 after retesting. Among the eight RPN1 isolated clones, four clones encoded aa 258-397, two clones encoded aa 220-390 and two clones encoded aa 240-367. TREX1 variants were generated and the interaction between TREX1 (aa.241-369) and RPN1 (aa 256-397) was mapped. To isolate DAD1, the C-terminal region of STING (aa 173-379) was used to screen the same library. Two 24 isolated clones were found with full length DADI. Most clones (except DAD1) were unable to interact with STING after revalidation studies. See that full-length DAD1 associates with region 242-310 of STING. Figure 18A: Schematic representation of TREX1 mutants. Figure 18B: GAL4 binding domain fused to TREX1 or TREX1-4 interacts with RPN1 fused to GAL4 activation domain in yeast two-hybrid screen. Figure 18C: Schematic representation of STING mutants. Figure 18D. GAL4 binding domain fused to STING-C-terminus or STING-C2 interacts with DAD1 fused to GAL4 activation domain in yeast two-hybrid screen.

Figures 19A-C show that TREX1 and STING associate with the oligosaccharyltransferase complex. Figure 19A: 293T cells were co-transfected with TREX1-tGFP and RPN1-Myc. Lysates were immunoprecipitated with anti-tGFP antibody or IgG control and analyzed by immunoblotting using the indicated antibodies. Figure 19B: 293T cells were co-transfected with Myc-STING or GFP-DAD1 and cells were lysed. Lysates were immunoprecipitated with anti-Myc antibody or IgG control and immunoblotted with anti-GFP antibody. Figure 19C: 293T cells were co-transfected with TREX1-tGFP and RPN1-Myc, GFP-DAD1 or STING-HA. Lysates were immunoprecipitated by anti-tGFP antibody or IgG control and analyzed by immunoblotting using anti-tGFP, anti-Myc, anti-GFP or anti-HA antibodies.

Figure 20 shows that TREX1 is localized in the endoplasmic reticulum. hTERT-BJ1 cells were transfected with RPN1-Myc. After 48 h, cells were examined by immunofluorescence using anti-TREX1 antibody (red), anti-Myc antibody (green) or anti-calreticulin antibody (blue) as an endoplasmic reticulum marker.

Figures 21A-H show that exogenously expressed STING in 293T cells reconstitutes the dsDNA90 response. Figure 21A: 293T cells were infected with control lentivirus or hSTING lentivirus. One day after infection, cells were treated with dsDNA90. After 6 h, cells were stained using anti-STING or anti-calreticulin antibodies. Figure 21B: Cell lysates (from Figure 56A) were subjected to immunoblotting using anti-STING antibody. Figure 21C-D: Lentivirus-infected 293T cells were stimulated with dsDNA90 for 6h. Total RNA was purified and examined for IFNβ (Fig. 21C) or IFIT2 (Fig. 21D) by real-time PCR. FIG. 21E : 293T cells stably transduced with control or hSTING lentivirus were subjected to Brefeldin A (BFA) experiments, as shown in the flow chart. Figure 21F: Cell lysates (from Figure 21E) were subjected to immunoblotting using anti-STING antibody. Figure 21G: Measurement of IFN[beta]-luciferase activity of cell lysates (from Figure 21E). Figure 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. Error bars represent standard deviation. Data represent at least two independent experiments.

Figure 22 shows that translocon members regulate HSV1 replication. hTERT-BJ1 cells were treated with TREX1, Sec61A1, TRAPβ, NS or STING siRNA.

Figures 23A-C show that IFI16 is not required for IFN-production by dsDNA90 in hTER-BJ1 cells. Figure 23A: hTERT-BJt cells were treated with NS, IFI16, STING or TREX1 siRNA. After 3 days, cells were lysed and expression levels were examined by immunoblotting. Figure 23B: siRNA-treated hTERT-BJ1 cells were treated with dsDNA90 and IFNβ production was measured by ELISA. Figure 23C: siRNA-treated hTERT-BJ1 cells were infected with HSV-luciferase (m.o.i=0.1). On day 2 post infection, cells were lysed and luciferase activity was measured. *P<0.05, Student's t-test. Error bars represent standard deviation. Data represent at least two independent experiments.

Figure 24 shows an example of a STING cell-based assay.

Figure 25 shows that drug "A" induces STING trafficking.

Figure 26 shows that drug "X" inhibits IFN[beta] mRNA production.

Figure 27 is a schematic diagram showing phosphorylation of STING in response to cytoplasmic DNA. hTERT-BJ1 cells were transfected with 4 μg/ml ISD for 6h. Cell lysates were prepared in TNE buffer and then subjected to immunoprecipitation with anti-STING antibody followed by SDS-PAGE. The gel was stained with CBB and then analyzed for bands including STING by mLC/MS/MS at the Harvard Mass Spectrometry and Proteomics Resource Laboratory. STING amino acid sequences from different species were aligned and phosphorylation sites were identified by mass spectrometry. Serines 345, 358, 366 and 379 were identified by mass spectrometry. Serine 358 and S366 are important for STING function.

Figure 28 shows that in the cytoplasmic DNA pathway, Serine 366 (S366) of STING is important for IFNβ production. 293T cells were transfected with a plasmid encoding the mutant STING and a reporter plasmid. After 36 hrs, luciferase activity was measured. STING ^−/− MEF cells were reconstituted with mutant STING and then the amount of IFNβ in the medium was measured by ELISA. S366 is important for IFN production by STING and S358 also appears to play an important role.

Figure 29A-D shows that STING-deficient mice are resistant to DMBA-induced inflammation and skin carcinogenesis: STING ^+/+ and STING ^-/- mice were mock-treated weekly with acetone or with 10 μg of DMBA on the shaved dorsal side Treatment continued for 20 weeks. Figure 29A: STING deficient animals are resistant to DNA damaging agents that cause skin cancer. The percentage of mice free of skin tumors is shown in Kaplan-Meier curves. Figure 29B: Shows pictures of representative mice for each treatment group. Figure 29C: Histopathological examination by H&E staining on mock or DMBA treated skin/skin tumor biopsies. Images were taken at 20X magnification. Figure 29D: Cytokine upregulation in STING-expressing mice exposed to carcinogens. RNA extracted from mock or DMBA-treated skin/skin tumor biopsies was analyzed in duplicate by Illumina Sentrix bead chip arrays (mouse WG6 version 2). Analyze total gene expression. Select the most variable gene. Rows represent individual genes; columns represent individual samples. Pseudocolors indicate transcript levels below, equal to, or above the mean (green, black, and red, respectively). Gene expression; fold change log10 scale between -5 and 5. Cytokines were not observed in the skin of STING-deficient animals.

Detailed description

Methods and compositions for modulating the immune response in a subject suffering from a disease or disorder associated with abnormal STING function are described herein. The preferred examples described below illustrate adaptations of these compositions and methods. Nonetheless, based on the description of these embodiments, other aspects of the invention can be completed and/or practiced based on the description provided below.

Methods and compositions for modulating the immune response of a subject (e.g., human, dog, cat, horse, cow, sheep, pig, etc.) suffering from a disease or disorder associated with abnormal STING function involve a pharmaceutical combination A substance, the pharmaceutical composition includes an agent for regulating STING function and a pharmaceutically acceptable carrier, wherein the amount of the pharmaceutical composition is effective to improve the abnormal STING function of the subject.

A disease or disorder associated with abnormal STING function can be any disease or disorder in which cells with defective STING function or expression cause or worsen the physical symptoms of the disease or disorder. Commonly, such diseases or disorders are mediated by cells of the immune system, e.g., inflammatory disorders, autoimmune disorders, cancers (e.g., breast, colorectal, prostate, ovarian, leukemia, lung, endometrial cancer or liver cancer), arteriosclerosis, arthritis (eg, osteoarthritis or rheumatoid arthritis), inflammatory bowel disease (eg, ulcerative colitis or Crohn's disease), peripheral vascular disease, cerebrovascular accident (stroke), diseases with chronic inflammation, diseases characterized by lesions with inflammatory cell infiltration, diseases with amyloid plaques in the brain (eg, Alzheimer's disease), Ecardi-Gutierrez syndrome Aicardi-Goutieres syndrome, juvenile arthritis, osteoporosis, amyotrophic lateral sclerosis, or multiple sclerosis.

The agent can be a small molecule (i.e., an organic or inorganic molecule having a molecular weight of less than 500, 1000, or 2000 Daltons) that increases or decreases STING function or expression or a molecule that increases or decreases STING function or expression under intracellular conditions (i.e., A nucleic acid molecule that binds to STING under conditions within a cell in which the STING is normally located). The agent can also be a STING-binding nucleic acid molecule, which can be a single-stranded (ss) or double-stranded (ds) RNA or DNA. Preferably, the nucleic acid is between 40 and 150, 60 and 120, 80 and 100 or 85 and 95 base pairs or longer in length. Such STING-binding nucleic acid molecules may be nuclease-resistant, eg, consist of nuclease-resistant nucleotides or be in the form of a circular dinucleotide. It can also be associated with a molecule that facilitates transmembrane transport.

Methods and compositions for treating cancer in a subject suffering from a cancerous tumor infiltrated by inflammatory immune cells relate to a pharmaceutical composition comprising an agent that down-regulates the function or expression of STING and a pharmaceutical An acceptable carrier, wherein the amount of the pharmaceutical composition is effective to reduce the number of inflammatory immune cells infiltrating a cancerous tumor by 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).

The compositions described herein may be included together with one or more pharmaceutically acceptable carriers or excipients to allow administration of the pharmaceutical compositions by various routes, such as These include oral, rectal, vaginal, topical, transdermal, subcutaneous, intravenous, intramuscular, inhalational, intrathecal, and intranasal administration. Suitable formulations for use in the present invention are found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, PA, 17th Edition (1985).

The active ingredient(s) can be mixed with an excipient, diluted by an excipient, and/or enclosed within a carrier, which can be in the form of a capsule, sachet, paper or other container. When the excipient acts as a diluent, it can be a solid, semi-solid or liquid material which can function as a vehicle, carrier or medium for the active ingredient. These compositions can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as solid or in a liquid medium), ointments, soft gelatins In the form of capsules and hard gelatin capsules, suppositories, sterile injectable solutions, sterile liquids for intranasal administration (eg, a spray device) or sterile packaged powders. These formulations may additionally include: lubricating agents, such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preservatives, such as methylparabens and propylhydroxybenzoates; sweeteners; and flavoring agents. The compositions of the invention can be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to the patient using procedures known in the art.

For the preparation of solid formulations such as tablets, the composition can be mixed with a pharmaceutically acceptable excipient to form a solid preformulation composition comprising a homogeneous mixture of the compounds. Tablets or pills may be coated or otherwise compounded to provide a dosage form giving the advantage of prolonged action. For example, a tablet or pill may comprise an inner dosage component and an outer dosage component, the latter in the form of an envelope over the former. The two components may be separated by an enteric layer that acts to resist disintegration in the stomach and allows intact delivery of the inner component into the duodenum or delayed release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with materials such as shellac, cetyl alcohol and cellulose acetate.

Liquid forms of formulations include suspensions and emulsions. To enhance serum half-life, these formulations can be encapsulated, introduced into the lumen of liposomes, prepared as a colloid, or incorporated into the liposome layer. A variety of methods are available for preparing liposomes, as described in, eg, US Patent Nos. 4,235,871, 4,501,728, and 4,837,028 to Szoka et al., each of which is incorporated herein by reference.

Preferably, the compositions are formulated in unit dosage form of the active ingredient(s). The amount administered to a patient will vary depending on the drug administered, the purpose of the administration (eg, prophylaxis or treatment), the state of the patient, the mode of administration, etc., all within the skill of a qualified physician and pharmacist. In therapeutic applications, the compositions are administered to a patient already suffering from the disease in an amount sufficient to cure or at least partially suppress the symptoms of the disease and its complications. Amounts effective for this use will depend on the disease condition being treated and, by the judgment of the attending clinician, on factors such as severity of symptoms, age, weight, and general condition of the patient, and the like.

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

example

Example 1: Translocon-associated STING complexes with cytoplasmic DNA for regulation of innate immunity

Previously, the isolation of a novel transmembrane component of the endoplasmic reticulum (ER) called STING (Stimulator of Interferon Genes) was described, and STING was shown to respond to cytoplasmic dsDNA as well as DNA viruses and intracellular bacteria Required for the production of type I IFN in fibroblasts, macrophages and dendritic cells (DCs) in response (see US Patent Application Serial No. 13/057,662 and PCT/US2009/052767). Note that the minimum size of dsDNA required to activate STING-dependent type I IFN signaling in murine cells is approximately 45 base pairs in murine cells. However, in normal human cells (hTERT), it was observed that a dsDNA of approximately 90 base pairs is required for full activation of type I IFN (referred to herein as interferon-stimulated dsDNA90). Using the RNAi knockdown procedure, it was additionally confirmed that STING is indeed required for type I IFN production in hTERT (Fig. IB). Further analysis using a microarray procedure to measure mRNA expression revealed that, in addition to type I IFN, cytoplasmic dsDNA could induce a broad array of innate immune genes in hTERT cells (Fig. 5A-G). The induction of these intrinsic molecules, including members of the IFIT family, appears to be STING-dependent, as RNAi knockdown of STING in hTERT largely abolished their stimulation by cytoplasmic dsDNA (Fig. 5A-G). Using STING ^+/+ or ^-/- murine embryonic fibroblasts (MEFs), it was confirmed that cytoplasmic dsDNA induces multiple innate immune genes in a STING-dependent manner (Fig. 1C). 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, type I IFN-signaling-deficient STAT1 ^-/- MEFs were treated with dsDNA Treatment was similar and demonstrated that SDG production was not affected (Fig. 1C). Reverse transcriptase (RT) PCR analysis confirmed the array results (Figures 6A-H and 7A-H). Note that ssDNA with 45 nucleotides (ssDNA45) weakly induces innate immune gene production in hTERT and even weaker in MEFs. However, transfected ssDNA comprising 90 nucleotides (ssDNA90) was observed to more potently activate a range of genes, including type I IFN, in hTERT cells (Fig. ID, IE). That cytoplasmic ssDNA90 induces innate immune gene production in a STING-dependent manner was similarly confirmed using STING ^+/+ or ^-/- murine embryonic fibroblasts (MEFs) (Fig. 1F and 7A-H). observed that STING likely resides as a homodimer in the ER of both human and murine cells and migrates from the ER to the perinuclear region in the presence of cytoplasmic ssDNA or dsDNA ligands to activate type I IFN-dependent Transcription factors (Figures 1G and 8A-E). It was similarly observed that HSV1 activates innate immune gene production in a STING-dependent manner (Fig. 9A-I). It was confirmed that many SDGs contain IRF7 binding sites in their promoter regions (Fig. 10A-F). Thus, cytoplasmic ssDNA or dsDNA including transfected plasmid DNA can potently induce STING-dependent transcription of a broad array of innate immunity-related genes.

To further assess the possibility that STING itself could be associated with DNA species, 293T cells were transfected with STING and after cell lysis it was observed that the C-terminal region of STING (aa181-349) could be precipitated using biotinylated dsDNA90 (Fig. 2A). The N-terminal region of STING (aa 1-195) as well as three similar HA-tagged controls (GFP, NFAR1 and IPS1 ) did not associate with dsDNA90. The DNA-binding exonuclease TREX1 was used as a positive control. Another extensive series of studies suggested that the amino acid region 242–341 of STING may be responsible for binding dsDNA, as STING variants lacking this region were unable to associate with nucleic acids (Fig. 2B–D). STING expressed in vitro also bound to dsDNA under high salt and high detergent conditions (except for those variants that similarly lacked region 242-341) (Fig. 11A-F). Additional information about the ability of STING to complex with dsDNA (possibly as a dimer) was obtained by transfecting biotinylated dsDNA90 into hTERT and treating the cells with a reversible cross-linking reagent (DSS) or UV light evidence of. With both treatments, STING was observed to retain its association with DNA after cell lysis (Fig. 2E and Fig. 12A-G). RNAi knockdown of STING in hTERT cells abolished the observed binding, and STING-DNA complexes were also observed only in wild-type MEFs ( ^+/+ ), but not in STING-deficient MEFs ( ^-/- ). observed (Fig. 12A-G). HSV1, cytomegalovirus (CMV), and adenovirus (ADV)-associated dsDNA were similarly identified. Competition experiments demonstrated that STING could also bind to ssDNA (ssDNA90) as well as dsDNA, but not to dsRNA (Fig. 2F). This was confirmed by expressing STING in vitro and observing association with ssDNA90 (Fig. 2G). All STING variants analyzed lacked the ability to activate IFN-type promoters in 293T cells (Fig. 2H). The dsDNA was also transfected into hTERT or MEF cells and treated with formaldehyde to cross-link cellular proteins to nucleic acids. Subsequent CHIP analysis after STING pull down further confirmed that transfected DNA could directly associate with STING, as determined using dsDNA90-specific primers (Figures 2I and 2J). It was observed in an ELISA assay that STING could bind to biotin-labeled DNA (Fig. 14A-C). The data suggest that ssDNA and dsDNA-mediated innate signaling events are dependent on STING and demonstrate that STING itself is able to complex to these nucleic acid structures to help trigger these events.

TREX1, a 3'->5' DNA exonuclease, is also an ER-associated molecule and is important for degrading checkpoint-activated ssDNA species that can otherwise activate the immune system. RNAi for silencing TREX1 in hTERT cells significantly increased the STING-dependent production of type I IFN by dsDNA90 ( FIGS. 3A and 3B ). Concomitantly, replication of the dsDNA virus HSV1 was largely reduced in TREX1-deficient hTERT cells, likely due to elevated production of type I IFN and antiviral IFN-stimulated genes (ISGs) (Fig. 3C and D). Luciferase expression from recombinant HSV expressing the luciferase gene was also significantly lower in hTERT-infected cells treated with RNAi that silenced TREX1 (Fig. 15A-D). These observations were extended by using TREX1-deficient MEFs, similarly showing that cytoplasmic dsDNA-dependent gene induction was largely elevated and HSV1 replication was significantly reduced in the absence of TREX1 (Fig. 3E-G). To determine whether STING is responsible for the elevated production of type I IFN observed in the absence of TREX1, STING was silenced in TREX1-deficient hTERT or TREX1 ^−/− MEFs and these cells were treated with cytoplasmic dsDNA or HSV1. These results demonstrate that type I IFN production is largely reduced in STING-deficient TREX1-deficient cells (both hTERT and MEF), suggesting that the elevated levels of type I IFN observed in the absence of TREX1 are a consequence of STING - dependent (Fig. 3A-F). Similarly, it was noted that RNAi knockdown of STING in TREX ^−/− MEFs also abolished ssDNA90-mediated type I IFN production and innate gene stimulation ( FIG. 16 ). Confocal analysis confirmed that TREX1 and STING co-localized in the ER (Fig. 3H). However, cytoplasmic dsDNA did not efficiently induce STING-like trafficking of TREX1 from the ER to the perinuclear region (Fig. 3H). Therefore, STING and TREX1 were not observed to interact robustly, as determined by co-immunoprecipitation analysis. Under non-stimulatory conditions, no significant differences were noted in the expression of STING-dependent genes in TREX1+/+ or ^-/- MEFs (Fig. 17A-H). However, TREX1, a dsDNA-induced gene, was identified and could be upregulated in a STING-dependent manner (Fig. 17A-H). It is therefore plausible that dsDNA species complex with STING and accessory molecules to mediate trafficking and downstream signaling events that activate the transcription factors IRF3/7 and NF-κB responsible for the induction of primary innate immunity genes, including TREX1. This evidence suggests that STING-activated TREX1 localizes in the ER region to degrade activator dsDNA and inhibit cytoplasmic dsDNA signaling in a negative feedback manner. Thus, TREX1 is a negative regulator of STING.

The data here demonstrate that STING is localized in the ER as part of the translocon complex, associated with transloson-associated protein beta (TRAPβ). The translocon complex includes Sec61αβ and γ coupled to TRAPαβ, γ and δ that can attach to ribosomes. Secreted and membrane proteins translocate into the ER for proper folding and glycosylation before being exported. To identify TREX1-binding partners, full-length TREX1 was used as bait in a two-hybrid yeast screen. The results showed that TREX1 cyclically interacts with a protein called ribosome-binding protein I (RPN1), a 68 kDa type I transmembrane protein and a member of the oligosaccharyltransferase (OST) complex ( Figures 4A-E; Figures 18A-D). The OST complex catalyzes the transfer of mannose oligosaccharides to asparagine residues of nascent polypeptides as they enter the ER via translocons. At least seven proteins comprise the OST complex, including RPN1, RPN2, OST48, OST4, STT3A/B, TUSC3, and DAD1. Remarkably, a similar screen using STING as a bait determined that STING could also associate with DAD1, a defender against apoptotic cell death, a 16 kDa transmembrane protein (Fig. 14F-H). Further analysis of these associations using the yeast two-hybrid approach indicated that the C-terminal region of TREX1 (amino acids 241-369), including its transmembrane region, was responsible for binding to amino acids 258-397 of RPN1 (Figure 18A-D). In addition, amino acids 242-310 of STING are responsible for association with full-length DAD1 (Fig. 18A-D). Co-immunoprecipitation studies confirmed the interaction of these molecules (Figures 4D and 4G and Figures 29A-C). Additional co-immunoprecipitation experiments demonstrated the association of TREX1 with DAD1 (Fig. 19A-C). Confocal analysis confirmed that TREX1 and RPN1 co-localized in cells but were not trafficked in response to cytoplasmic dsDNA (Figure 4E and Figure 20). Similarly, STING and DAD1 co-localized in the ER of cells, but the latter molecule did not accompany STING to the endosomal compartment in the presence of cytoplasmic dsDNA (Fig. 4H). Microsomal compartments of cells including ER were fractionated and examined by sucrose gradient analysis. This study demonstrates that TREX1 and STING are co-fractionated with ER markers RPN1 and RPN2, DAD1, and calreticulin, but not with nuclear histone H3, confirming their subcellular localization at the translocon/OST complex The components were not identifiable (Fig. 4I). Thus, TREX1 targets the OST/translocon complex of ER including STING, and this association occurs through association with RPN1, although the TM region of TREX1 was found to be involved in the localization of TREX1 to ER. To identify whether members of the OST, TRAP or SRP (signal recognition peptide) complex affect dsDNA-dependent signaling, RNAi screens were performed to silence the expression of these components. However, only Sec61α and TRAPβ silencing significantly affected signaling and HSV1 replicated, demonstrating that these transposon members play a role in controlling this pathway (Figure 4J and Figure 22). It was observed that silencing of IFI16 (also implicated in cytoplasmic DNA sensing) did not robustly inhibit dsDNA-dependent signaling, at least in hTERT cells (Fig. 23A-C). Similar to deletion of STING, deletion of IFI16 also failed to rescue increased IFN production by dsDNA in the absence of TREX1 (Fig. 23A-C). However, reduced IFI16 enabled more proficient HSV1 gene expression, confirming the important role of this molecule in influencing viral replication. Silencing of RPN1 or 2 also resulted in increased HSV1 gene expression, but also did not significantly affect type I IFN production, demonstrating that these components of OST may be primarily involved in N-glycosylation.

Data demonstrate that STING, which can complex with cytoplasmic intracellular ssDNA and dsDNA (which can include plasmid-based DNA and gene therapy vectors), can modulate the induction of a broad array of innate immune genes such as type I IFN, the IFIT family, and Various chemokines important for antiviral activity and for eliciting adaptive immune responses. STING activation plausibly assists in escorting TBK1 to clathrin-coated endosomal compartments to activate IRF3/7 by a mechanism that is still not fully elucidated. TREX1 appears to be present in cells at low levels and is itself inducible by STING. Post-translationally, TREX1 localizes to the OST complex next to non-activated STING (also in the OST/translocon complex), where it presumably degrades DNA species that would otherwise cause STING to act. Components of the translocon/OST complex, now including STING and TREX1, regulate cytoplasmic ssDNA and dsDNA-mediated innate immune signaling. Because loss of TREX1 reveals autoimmune dysregulation through elevated type I IFN production, it is possible that these diseases are induced by STING activity.

Example 2: STING modulators

Screen drug libraries to identify agents that modulate STING expression, function, activity, etc. Figure 24 shows the steps of the STING cell-based assay.

These libraries include the BioMol ICCB Known Bioactive Library, 500 targets; the LOPAC1280 ^™ library of pharmaceutically active compounds; Enzo LifeSciences, Screen-Well ^™ Phosphatase Inhibitor Library, 33 known phosphatases Inhibitors;

MicroSource Spectrum Collection 2000 components, 50% drug components, 30% natural products, 20% other bioactive components; EMD: InhibitorSelect ^TM 96-well protein kinase inhibitor library I, InhibitorSelect ^TM 96-well protein kinase inhibitor library II, InhibitorSelect ^TM 96-well protein kinase inhibitor library IIIa; Kinase library B Kinase true clone (TrueClone) collection center; Kinase-deficient true clone collection center.

The results showed that one drug (called 'Drug A') induced STING trafficking (Figure 60). Another drug (designated "Drug X") inhibited IFN[beta] mRNA production (Figure 61).

Table 2: The following are identified as STING inhibitors:

Activators of STING induce dihydrooubain and BNTX maleate hydrate.

Example 3: STING reveals self-DNA-dependent inflammatory disease

Bone marrow-derived macrophages (BMDM) were obtained from Sting+/+ and Sting ^−/− mice and transfected with 90 base pair dsDNA (dsDNA90) to activate the STING pathway, or with dexamethasone (Dex Apoptotic DNA (aDNA) transfection of )-treated thymocytes. It was observed that both types of DNA potently induce IFNβ production in BMDM and conventional dendritic cells (BMDC) in a STING-dependent manner. DNA microarray experiments confirmed that aDNA triggers the STING-dependent production of a broad array of innate immune and inflammation-related cytokines such as IFNβ and TNFα in BMDM (Table 3). These data were confirmed by measuring cytokine production in Sting+/+ or Sting ^−/− BMDMs treated with aDNA. Thus, STING may assist apoptotic DNA-mediated pro-inflammatory gene production in BMDM as well as in BMDC.

Table 3 shows the gene expression of higher expressed genes in BMDM treated with apoptotic DNA (aDNA).

To determine whether STING plays a role in DNase II-associated inflammatory diseases, we knocked down STING and/or DNase II in THP1 cells or BMDM using RNAi and noted that loss of DNase II responds in a STING-dependent manner to aDNA to assist in the upregulation of cytokines, including type I IFN. Because DNase II ^−/− mice typically die before birth, DNase II ^−/− , Sting ^−/− or Sting ^−/− DNase II ^−/− DKO 17 day embryos (E17 days) were analyzed. Genotyping analysis (including RT-PCR and immunoblotting) confirmed that these embryos lacked Sting, DNase II, or both functional genes. It was observed that DNase II ^−/− embryos, as described above, exhibited anemia, in marked contrast to Sting ^−/− DNase II ^−/− DKO embryos or controls, which were markedly lacking this phenotype. Lethal anemia has been reported to be due to suppression of erythropoiesis by type I IFN during development. It was subsequently observed by hematoxylin and eosin staining that the liver of DNase II ^−/− embryos contained many infiltrating macrophages filled with phagocytic apoptotic cells responsible for the production of high levels of cytokines. Livers of Sting ^-/- DNase II ^-/- embryos exhibited similar phenotypes compared with control mice. Analysis of fetal livers by TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP biotin nick-end labeling) confirmed that Sting ^-/- DNase II ^-/- embryos and DNase II-deficient but non-wild-type fetal livers contained many large Improperly digested dying cells. In vitro analysis has shown that macrophages from embryos of wild-type or DNase II ^−/− mice substantially phagocytose apoptotic cells. However, while the DNA of phagocytosed apoptotic cells is efficiently degraded in the lysosomes of wild-type macrophages, DNase II ^-/- macrophages accumulate phagocytosed nuclei and are unable to digest DNA. This event leads to the stimulation of innate immune signaling pathways and the production of autoimmune-related cytokines. In light of this, the capacity of embryonic liver-derived macrophages lacking both DNase II and STING was assessed as whether they phagocytized apoptotic cells and digested DNA. Note that similar to DNase II ^-/- macrophages, Sting ^{- /-} DNase II ^-/- macrophages were unable to digest the Phagocytosis of nuclei in methazone-treated apoptotic thymocytes. Thus, like DNase II ^−/− macrophages, macrophages harvested from the liver of Sting ^−/− DNase II ^−/− embryonic mice similarly exhibit the inability to digest phagocytized apoptotic cells.

The above analysis was supplemented by analyzing mRNA expression levels in the liver of embryonic mice. This study showed that very few inflammatory genes were produced in the livers of wild-type or Sting ^-/- embryos. However, it was observed that the livers of DNase II ^-/- embryos contained abnormally high levels of cytokine-related mRNAs. Remarkably, livers of Sting ^−/− DNase II ^{−/− mice} had significantly reduced levels of innate immune gene expression activity compared to DNase II −/− mice. These results were confirmed by RT-PCR analysis of mRNA expression levels of selected innate immunity genes in embryonic livers. For example, IFNβ production was reduced several-fold in Sting ^{− /−} DNase II ^−/− mice compared to DNaseII −/− mice. Production of key interferon-stimulated genes (ISGs) such as 2'-5' oligoadenylate synthase (OAS), interferon-induced ISG with tetratricopeptide repeats was also significantly reduced. protein (IFIT), interferon-inducible protein 27 (IFI27), and ubiquitin-like modifier (ISG15). Pro-inflammatory cytokines such as TNFα and IL1β were also reduced in embryonic livers of Sting ^−/− and Sting ^−/− DNase II ^−/− mice compared with DNase II ⁻ /− mice. While the production of innate genes was significantly suppressed in the absence of STING, some genes remained slightly elevated, albeit at low levels, in Sting ^-/- DNase II ^-/- mice, as determined by array analysis , which could be due to variations in mRNA expression between the animals analyzed, or perhaps due to stimulation of other pathways. Many of these genes are regulated through the NF-κB and interferon regulatory factor (IRF) pathways. Therefore, the function of these transcription factors was assessed in Sting ^−/− DNase II ^−/− or control murine embryonic fibroblasts (MEFs) developed from day 14 embryos (E14 days). Primarily, a defect in NF-κB activity (p65 phosphorylation) was observed in Sting ^−/− DNase II ^−/− MEFs when exposed to cytoplasmic DNA. The same defect was obtained in Sting ^−/− DNaseII ^−/− BMDM when exposed to apoptotic DNA as well as cytoplasmic DNA. This was confirmed by noting that NF-κΒ as well as IRF3 also failed to translocate in Sting ^−/− DNase II ^−/− MEFs but could translocate in control MEFs after exposure to dsDNA. Thus, STING may be responsible for the control of auto-DNA-induced inflammatory cytokine production responsible for lethal embryonic erythropoiesis.

To extend the importance of STING in regulating self-DNA-assisted lethal erythropoiesis, it was assessed whether DNase II ^-/- mice could be born in the absence of STING. Remarkably, DNase II ^-/- mice were born with distinct Mendelian frequencies when crossed on Sting ^-/- backgrounds. PCR genotyping, Northern blot, RT-PCR, and immunoblot analysis confirmed DNase II and STING deficiencies in progeny mice. Sting ^-/- DNase II ^-/- double knockout mice (DKO) appeared to grow normally and exhibit a similar size compared to control mice, but it was noted that Sting ^-/- mice for reasons that remain unclear slightly larger. Preliminary immunological assessment also indicated that Sting ^-/- DNaseII ^-/- DKO animals shared a similar CD4 ⁺ /CD8 ⁺ profile to Sting ^-/- and wild-type mice, but noted the development of DKO with age For splenomegaly. Splenomegaly was also observed in surviving DNase II-deficient mice lacking type I IFN signaling ((DNase II ^-/- Ifnar1 ^-/- mice) and has been reported to be due to enlargement of the red pulp. However, at 8 weeks At 20 years of age, analysis of sera from Sting ^-/- DNase II ^-/- mice showed no detectable abnormal cytokine production compared to control mice, as the levels of cytokines produced were generally lower and unmeasurable From these studies, it was noted that in vitro, similarly to DNase II ^-/- macrophages, Sting-/- DNase II ^-/- compared to control macrophages taken from wild-type or Sting ^-/- mice Macrophages are unable to digest phagocytosed nuclei from apoptotic thymocytes (Dex ⁺ ). When WT thymocytes are used as targets (Dex ^- ), undigested DNA in DNase II ^-/- Sting ^-/- macrophages The accumulation in is less pronounced. Thus, BMDM derived from Sting ^-/- DNase II ^-/- mice are also unable to digest DNA from apoptotic cells, although they are not inflammatory compared to DNase II ^-/- BMDM Cytokine response.

Although DNase II-mediated embryonic lethality can be avoided by crossing DNase II ^+/- mice with type I IFN-deficient Ifnar1 ^-/- mice, the resulting offspring at approximately 8 weeks postnatal suffer severe polyarthritis (arthritis score of 2) because undigested DNA activates innate immune signaling pathways and triggers the production of inflammatory cytokines such as TNFα. Remarkably, it was noted that Sting ^−/− DNase II ^−/− mice did not show any signs of polyarthritis postnatally. Arthritis scores in Sting ^−/− DNase II ^−/− mice remained approximately at zero (no score) ^until 12 months, compared to reported DNase II −/− mice exhibiting arthritis scores as high as 7 after similar times. ^/- Ifnar1 ^-/- mouse formation compared. Although H&E and TUNEL staining of spleen and thymus tissue from DNase II ^-/- Sting ^-/- mice demonstrated the presence of infiltrating macrophages that also contained apoptotic DNA, samples from 6-month-old Sting ^-/- DNase II ^{-/ -} Histology of joints of mice exhibiting normal bony (B) synovial joint (S) and cartilage (C) architecture with no evidence of pannus infiltration in the joint structure. Levels of TNFα, IL1β and IL6 in sera from Sting ^−/− DNase II ^−/− mice remained low, as predicted from our array analysis of STING-deficient BMDMs (Table 3). There was no evidence of CD4, CD68 or TRAP positive cell infiltration in the joints of Sting ^-/- DNase II ^-/- mice. Serum analysis of Sting ^-/- DNaseII ^-/- mice also showed no elevated levels of rheumatoid factor (RF), anti-dsDNA antibodies, or MMP3. Thus, loss of STING abolishes pro-inflammatory cytokine production responsible for auto-DNA-mediated polyarthritis.

STING is responsible for inflammatory diseases such as Acardi-Gutierrez Syndrome (AGS), for example. AGS is genetically defined as an encephalopathy and is characterized by calcification and demyelination of the basal ganglia and white matter. High levels of lymphocytes and type I IFN in CSF. These features mimic chronic infection. Serum levels of type I IFN are also elevated in the autoimmune syndrome systemic lupus erythematosus (SLE). AGS is caused by mutations in the 3'-5' DNA exonuclease TREX1. TREX1 loss-of-function DNA species accumulate in the ER of cells and activate cytoplasmic DNA sensors (STING). TREX1 digests this source of DNA (housekeeping function) to prevent innate immune gene activation.

Given that STING appears to be responsible for inflammatory disease in apoptosis-deficient mice, we next assessed whether other types of self-DNA-triggered disease occur through activation of the STING pathway. For example, patients deficient in 3' repair exonuclease 1 (Trex1) suffer from Acardi-Gutierrez syndrome (AGS), which promotes lethality characterized by the presence of high levels of type I IFN production in the CSF Encephalitis. Trex1-deficient mice exhibit a median lifespan of approximately 10 weeks because as yet uncharacterized self-DNA presumably normally digested by Trex1 activates intracellular DNA sensors, which trigger cytokine production and lead to lethal inflammatory Exacerbated myocarditis. Recent data suggest that loss of STING extends lifespan in Trex1 ^-/- mice, but the reason is unknown. These studies were extended and noted slightly elevated levels of type I IFN production in Trex1 deficient BMDCs (Trex1 ^−/− BMDCs) exposed to dsDNA90. Remarkably, deletion of STING (Sting ^-/- Trex1 ^-/- BMDC) abrogates the ability of DNA to enhance type I IFN production in Trex1-deficient BMDM. Interestingly, a reduced size of the hearts of Sting ^−/− Trex1 ^−/− , Sting ^−/+ Trex1 ^−/− mice was observed when compared to Trex1 ^−/− mice. Significantly reduced signs of myocarditis were also noted in Sting ^-/- Trex1 ^-/- compared to Trex1 ^-/- alone. In addition, very prevalent antinuclear autoantibodies (ANA) were observed in the sera of Trex1 ^−/− mice, and antinuclear autoantibodies were almost completely absent in the sera of Sting ^−/− Trex1 ^−/− mice. Microarray analysis revealed significantly reduced levels of proinflammatory genes in hearts of Sting − ^{/ −} Trex1 ^−/− , Sting ^−/+ Trex1 ^−/− compared to Trex1 −/− mice. Taken together, these data suggest that STING is responsible for pro-inflammatory gene induction and, plausibly, AGS in Trex1-deficient mice. Example 4: Kinase screen against S366 of STING

The activity of 217 protein kinase targets was assessed against 2 peptides (A366 and S366) as substrates. Protein kinase was mixed with each peptide ^and33P -ATP and then the activity (CPM) was measured. In STING, the following kinases were identified as phosphorylating S366. The identification of kinases targeting this serine opens avenues towards drug discovery. Drugs targeting this association can inhibit STING activity and can be used for therapeutic purposes to inhibit STING activity. STING overactivity can lead to inflammatory diseases that can worsen cancer.

Table 4: Kinase screen against S366 of STING

Example 5: STING is responsible for inflammation-related cancers

STING WT and STING ^−/− animals were treated with DNA damaging agents and mice lacking STING were resistant to tumor formation. This is because infiltrating immune cells (such as dendritic cells, macrophages, etc.) engulf damaged cells that have undergone necrosis or apoptosis, and DNA and other ligands from such damaged cells activate STING and activate tumor-promoting Cytokine production. STING could be involved in assisting tumor progression in a variety of other cancers.

Figure 29A-D shows that STING-deficient mice are resistant to DMBA-induced inflammation and skin carcinogenesis: STING ^+/+ and STING ^-/- mice were mock-treated weekly with acetone or with 10 μg of DMBA on the shaved dorsal side Treatment continued for 20 weeks. Figure 29A: STING deficient animals are resistant to DNA damaging agents that cause skin cancer. The percentage of mice free of skin tumors is shown in Kaplan-Meier curves. Figure 29B: Shows pictures of representative mice for each treatment group. Figure 29C: Histopathological examination by H&E staining on mock or DMBA treated skin/skin tumor biopsies. Images were taken at 20X magnification. Figure 29D: Cytokine upregulation in STING-expressing mice exposed to carcinogens. RNA extracted from mock or DMBA-treated skin/skin tumor biopsies was analyzed in duplicate by Illumina Sentrix bead chip arrays (mouse WG6 version 2). Analyze total gene expression. Select the most variable gene. Rows represent individual genes; columns represent individual samples. Pseudocolors indicate transcript levels below, equal to, or above average. Gene expression; fold change log10 scale between -5 and 5. Cytokines were not observed in the skin of STING-deficient animals.

Claims (13)

1. one kind for regulating the method for the immunne response of the experimenter suffering from the relevant disease of a kind of and abnormal STING function or imbalance, the method comprises the step giving a certain amount of pharmaceutical composition to this experimenter, this pharmaceutical composition comprises and a kind ofly regulates the medicament of STING function and the pharmaceutically acceptable carrier of one, and wherein the amount of this pharmaceutical composition effectively improves this abnormal STING function of this experimenter.

2. the method for claim 1, wherein this medicament is a kind of small molecules increasing STING function.

3. the method for claim 1, wherein this medicament is a kind of small molecules reducing STING function.

4. the method for claim 1, wherein this medicament is a kind of nucleic acid molecule being bonded to STING under cellular conditions.

5. method as claimed in claim 5, wherein this nucleic acid molecule is the single stranded DNA of a kind of length between 40 and 150 base pairs.

6. method as claimed in claim 5, wherein this nucleic acid molecule is the double-stranded DNA of a kind of length between 40 and 150 base pairs.

7. method as claimed in claim 5, wherein this nucleic acid molecule is the double-stranded DNA of a kind of length between 60 and 120 base pairs.

8. method as claimed in claim 5, wherein this nucleic acid molecule is the double-stranded DNA of a kind of length between 80 and 100 base pairs.

9. method as claimed in claim 5, wherein this nucleic acid molecule is the double-stranded DNA of a kind of length between 85 and 95 base pairs.

10. method as claimed in claim 4, wherein this nucleic acid molecule comprises the Nucleotide of Nuclease.

11. methods as claimed in claim 4, wherein this nucleic acid molecule associates with a kind of molecule of the transmembrane transport of this nucleic acid molecule of assisting.

12. the method for claim 1, wherein this disease or imbalance are a kind of DNA-dependency inflammatory diseasess.

13. 1 kinds of treatments are suffered from by the method for the cancer of the experimenter of the cancerous tumour of inflammatory immune cell infiltrate, the method comprises the step giving a certain amount of pharmaceutical composition to this experimenter, this pharmaceutical composition comprises a kind of medicament and the pharmaceutically acceptable carrier of one of lowering STING function or expression, and wherein the amount of this pharmaceutical composition can will infiltrate the reduced number at least 50% of the inflammatory immunocyte of this cancerous tumour effectively.