CN104540945A - Modulating immune responses - Google Patents

Abstract

Modulators of STING are able to upregulate or down regulate immune responses. Administration of such modulators can be used to treat diseases or other undesireable conditions in a subject either directly or in combination with other agents.

Description

Immunity moderation is replied

Government rights

The present invention described here completes under the grant number R01A1079336 authorized by NIH under United States Government supports.United States Government enjoys some right in the present invention.

Invention field

Embodiments of the invention relate to for regulating the congenital of experimenter with acquired immunity and/or for immune related disorder, cancer, autoimmune treatment, treat and the composition of preventing infection and method.

Background

The cellular host defense response of pathogenic agent invasion is related generally to the detection of the pathogenic agent associated molecular pattern (PAMP) of the induction causing antipathogen gene, such as viral nucleic acid or bacterial cell wall components (comprising lipopolysaccharides or flagellin).Such as, viral RNA can be detected in conjunction with Toll-sample acceptor (TLR) or by DExD/H box DBPA (being called vitamin A acid induced gene 1 (RIG-I)) in TLR-dependent cell or Differentiation of Human Melanoma Cell Line related antigen 5 (MDA5, also referred to as IFIH1 and helicard) by the film be present in endoplasmic reticulum (ER) and/or endosome (such as TLR 3 and 7/8).These events reach a climax in the activation of downstream signaling events, and wherein major part is still unknown, thus cause transcribing of NF-κ B and IRF3/7-dependent gene (comprising I type IFN).

General introduction

Comprise as a kind of STING (stimulator of interferon gene) playing the molecule of keying action in innate immune response putative transmembrane (TM) district that 5 are mainly arranged in endoplasmic reticulum (ER), and NF-κ B and IRF3 transcription pathway can be activated thus inducing type I IFN and play effective antiviral state after expression.See U.S. Patent Application Serial Number 13/057,662 and PCT/US 2009/052767.The disappearance of STING reduce shortage STING that ability and making that poly-IC activates I type IFN produced by target homologous recombination ( ^-/-mEF) muroid embryo fibroblast is subject to vesicular stomatitis virus (VSV) and infects.When there is not STING, DNA mediation I type IFN reply suppressed, show STING may identify from virus, bacterium and other can cells infected pathogenic agent DNA in play a significant role.Yeast two-hybrid and co-immunoprecipitation research show that STING and RIG-I interacts and interacts with Ssr2/TRAP β, and Ssr2/TRAP β is the member that posttranslational protein matter crosses over translocon-associated protein (TRAP) complex body needed for the transposition of ER film.The RNAi of TRAP β melts and inhibits STING function and the generation hindered the I type IFN that poly-IC replys.

Experiment display in addition, STING self combines such as from nucleic acid (comprising strand and double-stranded DNA) and the apoptosis DNA of pathogenic agent, and plays central role in regulating the pro-inflammation genes in inflammatory conditions (sacroiliitis of such as DNA mediation and cancer) to express.There is described herein the various novel method and composition that raise STING expression or function, together with the other sign with STING other cellular elementss interactional.These find the design being allowed for the new auxiliary of immunity moderation system and other system, vaccine and therapy.

There is described herein the method for the immunne response for regulating the experimenter suffering from the relevant disease of a kind of and abnormal STING function or imbalance.These methods can comprise 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 improves the abnormal STING function of this experimenter effectively.This medicament can be a kind of small molecules increasing or reduce STING function, or a kind of nucleic acid molecule being bonded to STING under cellular conditions.This nucleic acid molecule in conjunction with STING can be that the single stranded DNA of a kind of length between 40 and 150 base pairs or a kind of length are between 40 and 150,60 and 120,80 and 100 or 85 and 95 base pairs or longer double-stranded DNA.The STING nucleic acid molecule of this combination can be Nuclease, such as, be made up of the Nucleotide of Nuclease.It can also associate with a kind of molecule of transmembrane transport of assisting.In these methods, this disease or imbalance can be a kind of DNA-dependency inflammatory diseasess.

Also describe treatment at this to suffer from by the method for the cancer of the experimenter of the cancerous tumour of inflammatory immune cell infiltrate.These methods can comprise 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, wherein the amount of this pharmaceutical composition can will infiltrate the reduced number at least 50% of the inflammatory immunocyte of cancerous tumour (such as effectively, at least 50%, 60%, 70%, 80% or 90%, or until the minimizing of inflammatory cell infiltration can be reduced by histology or scanning with detecting).

Accompanying drawing explanation

Figure 1A-G shows STING dependency innate immunity intracellular signaling.Figure 1A: by human telomerase reverse inoblast (hTERT-BJ1) different IPs thuja acid (3 μ g/ml) transfection 16h.Measure endogenous IFN β level.By dsDNA90 transfection conjugated for hTERT-BJ1 cell FITC, checked by fluorescent microscope, to guarantee effective transfection.Figure 1B: that hTERT-BJ1 cell is simulated, random or two independently mankind STING siRNA (siRNA 3 or 4) transfections 3 days, dsDNA90 transfection subsequently (3 μ g/ml) 16 hours.Measure endogenous IFN β level.The silence of hSTING albumen is shown, with beta-actin thing in contrast by immunoblotting.Figure 36 C: by elementary STING ^{+ /+}, STING ^-/-, STAT1 ^{+ /+}or STAT1 ^-/-mEF with or without dsDNA90 (3 μ g/ml) transfection 3 hours.Purifying total serum IgE and check genetic expression by Illumina Sentrix bead chip array (mouse WG6 version 2).Fig. 1 D: by hTERT-BJ1 cell NS or STING siRNA process.After 3 days, cell dsDNA90, ssDNA90 or ssDNA45 (3 μ g/ml) are processed.After 16 hours, measure IFN β mRNA level in-site by real-time RT-PCR.Fig. 1 E: by hTERT-BJ1 cell NS or STING siRNA process.At the 3rd day, cell dsDNA90, ssDNA90 or ssDNA45 (3 μ g/ml) are processed.After 16 hours, measure endogenous IFN β level.Fig. 1 F: by elementary STING ^{+ /+}or STING ^-/-mEF with or without dsDNA90 (3 μ g/ml) transfection.After 3h, identical with Fig. 1 C.Fig. 1 G: hTERT-BJ1 cell is used or processes 3 hours without dsDNA90 (3 μ g/ml) and dye with anti-HA antibody with as the calprotectin of ER marker.

Fig. 2 A-J shows STING and is bonded to DNA.Fig. 2 A: by the 293T cell plasmid transfection of specifying.Anti-HA antibody is used to be analyzed by immunoblotting cell lysate vitamin H-dsDNA90 sepharose precipitation.The schematic diagram of Fig. 2 B:STING mutant.Fig. 2 C: identical with Fig. 2 A.Fig. 2 D: identical with Fig. 2 A.Redness can not be labeled as in conjunction with the STING variant of DNA.Fig. 2 E: by dsDNA90 (B-dsDNA90 conjugated for hTERT-BJ1 cell vitamin H; 3 μ g/ml) transfection 6h with DSS process.Lysate used Streptavidin sepharose precipitation and use anti-HA antibody to be analyzed by immunoblotting.Fig. 2 F: at 293T cells STING and after 36 hours, under the existence of rival dsDNA90, poly-(I:C), b form dna or ssDNA90, hatch lysate with dsDNA90 agarose and use anti-HA antibody to be analyzed by immunoblotting.Fig. 2 G: STING, GFP or TREX1 transfection that 293T cell is marked with HA-.Biotin labeled ssDNA or dsDNA and Streptavidin agarose beads are added in lysis.Anti-HA antibody is used to be analyzed throw out by immunoblotting.Fig. 2 H: 293T cell IFN β-luciferase and the transfection of STING variant are measured uciferase activity.Fig. 2 I: by hTERT-BJ1 cell with dsDNA90 transfection and and formaldehyde crosslinking.Precipitate STING and use dsDNA90 Auele Specific Primer to be detected the DNA combined by PCR.NC: negative control.PC: positive control, dsDNA90.Fig. 2 J: by STING ^{+ /+}or STING ^-/-mEF dsDNA90 transfection and then identical with Fig. 2 I.Error bars represents standard deviation.Data representation at least two independent experiments.

Fig. 3 A-3H shows the down regulator that TREX1 is STING intracellular signaling.Fig. 3 A: immunoblotting confirms the striking low (knockdown) of STING and/or TREX1 in the hTERT-BJ1 cell of siRNA process.Fig. 3 B: by dsDNA90 (the 3 μ g/ml) transfection of the hTERT-BJ1 cell of siRNA process.After 16 hours, measure endogenous IFN β level.*P<0.05。Fig. 3 C: the hTERT-BJ1 cell HSV-1 (m.o.i=1) of siRNA process is infected and measures virus titer.*P<0.05。Figure 38 D: the HSV lacked by the hTERT-BJ1 cell γ 34.5 of siRNA process infects and measures virus titer.*P<0.05。The TREX1 of Fig. 3 E:NS or STING siRNA process ^{+ /+}or TREX1 ^-/-the immunoblotting of MEF confirms that STING strikes low.Fig. 3 F: by the TREX1 of siRNA process ^{+ /+}or TREX1 ^-/-mEF with dsDNA90 process and after 16 hours, measures IFN β level.*P<0.05。Fig. 3 G: by the TREX1 of siRNA process ^{+ /+}or TREX1 ^-/-mEF HSV-1 (m.o.i=1) infects and measures virus titer.*P<0.05。Fig. 3 H: the immunofluorescence analysis using or do not use the anti-TREX1 of the hTERT-BJ1 cell of dsDNA90 transfection or anti-STING antibody.* P<0.05, Si Shi t checks.Error bars represents standard deviation.Data representation at least two independent experiments.

Fig. 4 A-J: display TREX1 associates with oligosaccharyl transferase complex body.Fig. 4 A shows the schematic diagram of TREX1.Red expression RPN1 binding site.Fig. 4 B shows the schematic diagram of STING.Red expression DAD1 binding site.Fig. 4 C: in yeast two-hybrid analysis (1.pGBKT7,2.pGBKT7-NFAR M9,3.pGBKT7-TREX1,4.pGBKT7-STING total length, 5.pGBKT7-STING C-end), RPN1 and TREX1 interact.Fig. 4 D: by 293T cell TREX1-tGFP and RPN1-Myc cotransfection.Anti-Myc antibody mediated immunity is used by lysate to precipitate and analyzed by immunoblotting.Fig. 4 H: hTERT-BJ1 cell is used or processes without dsDNA90 (3 μ g/ml).6h after transfection, uses anti-STING or anti-DAD1 antibody by immunofluorescent cell.Fig. 4 I: after sucrose gradient centrifugation, uses the antibody of specifying to carry out immunoblotting assay to microsomal fraction.I: input.Fig. 4 J: by TREX1, Sec61A1, TRAP β, NS or STING siRNA process of hTERT-BJ1 cell.After 72h, cell dsDNA90 (3 μ g/ml) is processed 16h and then measures endogenous IFN β level.* P<0.05, Si Shi t checks.Error bars represents standard deviation.Data representation at least two independent experiments.

Fig. 5 A-G showed cell matter DNA induces STING-dependent gene in hTERT-BJ1 cell.Fig. 5 A: by the STING siRNA process of hTERT-BJ1 cell.After 3 days, by cell use or without dsDNA90 process 3h.Purifying total serum IgE and use mankind HT-12_V4_ bead chip check genetic expression.Fig. 5 B-G: hTERT-BJ 1 cell such as carrying out in fig. 5 is processed.The total serum IgE of IFN β (Fig. 5 B), PMAIP1 (Fig. 5 C), IFIT1 (Fig. 5 D), IFIT2 (Fig. 5 E), IFIT3 (Fig. 5 F) and PTGER4 (Fig. 5 G) is checked by PCR in real time.TaqMan determination of gene expression (Applied Biosystems, Inc. (Applied Biosystem)) is used to carry out PCR in real time.* P<0.05, Si Shi t checks.Error bars represents standard deviation.Data representation at least two independent experiments.

Fig. 6 A-H is presented in MEF, and STING-dependent gene is induced by cytoplasmic DNA.By elementary STAT1 ^{+ /+}or STAT1 ^-/-mEF dsDNA90, IFN α or the dsDNA90 process together with IFN α.Purifying total serum IgE and check IFN β (Fig. 6 A), IFIT1 (Fig. 6 B), IFIT2 (Fig. 6 C), IFIT3 (Fig. 6 D), CXCL 10 (Fig. 6 E), GBP1 (Fig. 6 F), RSAD2 (Fig. 6 G) and CCL5 (Fig. 6 H) by PCR in real time.* P<0.05, Si Shi t checks.Error bars represents standard deviation.Data representation at least two independent experiments.

Fig. 7 A-H shows, and cytoplasmic DNA induces STING-dependent gene in MEF.In Fig. 7 A-H, by STING ^{+ /+}or STING ^-/-mEF with or without dsDNA45, dsDNA90, ssDNA45 or ssDNA90 process 3h.Purifying total serum IgE and check IFN β (Fig. 7 A), IFIT1 (Fig. 7 B), IFIT2 (Fig. 7 C), IFIT3 (Fig. 7 D), CCL5 (Fig. 7 E), CXCL 10 (Fig. 7 F), RSAD2 (Fig. 7 G) or GBP1 (Fig. 7 H) by PCR in real time.* P<0.05, Si Shi t checks.Error bars represents standard deviation.Data representation at least two independent experiments.

Fig. 8 A-D shows STING location and dimerization.Fig. 8 A: by the MEF of stably express STING-HA ssDNA45, dsDNA45, ssDNA90 or dsDNA90 process.After 3h, use anti-HA or anti-calprotectin antibody by cell dyeing.Fig. 8 B: by 293T cell STING-HA and Myc-STING transfection.Lysate is precipitated by anti-Myc antibody and uses anti-HA antibody to be analyzed by immunoblotting.Fig. 8 C. is by hTERT-BJ 1 cell use or without cross liner DS S process.Anti-STING antibody is used to make cell lysate stand immunoblotting.Fig. 8 D: by 293T cell with the plasmid transfection of specifying and with DSS process.Anti-HA antibody is used to be analyzed cell lysate by immunoblotting.

Fig. 9 A-I shows, and DNA virus induces STING-dependent gene in MEF.Fig. 9 A: the HSV (m.o.i.=1) lacked by MEF γ 34.5 infects 3h.Purifying total serum IgE and use Illumina Sentric bead chip array (mouse WGS version 2) to check genetic expression.Fig. 9 B-I: by STING ^{+ /+}, STING ^-/-or STAT ^-/-/ STING ^{+ /+}the HSV process 3h that MEF uses or lacks without dsDNA90, HSV or γ 34.5.Purifying total serum IgE and check IFN β (Fig. 9 B), IFIT1 (Fig. 9 C), IFIT2 (Fig. 9 D), IFIT3 (Fig. 9 E), CCL5 (Fig. 9 F), CXCL 10 (Fig. 9 G), RSAD2 (Fig. 9 H) or GBP1 (Fig. 9 I) by PCR in real time.Error bars represents standard deviation.

Figure 10 A-F shows STING and IRF3/7 and NF κ-B and interacts.IRF7 binding site in the promoter region of Figure 10 A:STING dependency dsDNA90 stimulated gene.Figure 10 B-D: the instruction following manufacturers, from mock process or the STING of dsDNA90 process ^{+ /+}or STING ^-/-isolated cell nuclear extract in MEF and check that IRF3 (Figure 10 B), IRF7 (Figure 10 C) and NF-κ B (Figure 10 D) activate.Nuclear extraction kit, TransAM IRF3, TransAM IRF7 and TransAMNF κ B family Elisa test kit are from the high industrial company (Active Motif) of uncle.* P<0.05, Si Shi t checks.Error bars represents standard deviation.Data representation at least two independent experiments.Figure 10 E: by STING ^{+ /+}or STING ^-/-mEF poly-(I:C), dsDNA90 or HSV1 process and pass through anti-IRF3 antibody by cell dyeing.Figure 10 F. is by STING ^{+ /+}or STING ^-/-mEF dsDNA90 or HSV1 process and by anti-p65 antibody by cell dyeing.The disappearance of STING does not affect the innate immunity intracellular signaling that poly-(I:C) mediates.

Figure 11 A-F shows STING in vitro in conjunction with DNA.Figure 11 A: dsDNA90 conjugated for vitro translation product vitamin H is precipitated and carries out immunoblotting by anti-HA antibody.The schematic diagram of Figure 11 B:STING variant.Figure 11 C, 11D: identical with Figure 11 A.The STING variant (redness) lacking aa242-341 can not in conjunction with DNA.Figure 11 E-F: ssDNA90 conjugated for vitro translation product vitamin H is precipitated and carries out immunoblotting by anti-HA antibody.

Figure 12 A-G shows STING in vivo and in vitro in conjunction with DNA.Vitamin H-dsDNA90 the transfection of hTERT BJ1 cell is cross-linked by UV.Analyzed by immunoblotting by lysis and by Streptavidin sepharose precipitation.Figure 12 B-C is then identical with Figure 12 A by hTERT-BJ1 cell IFI16 (Figure 12 B) or STING (Figure 12 C) siRNA process.Figure 12 D: by STING ^{+ /+}or STING ^-/-mEF is as processed in fig. 12.Figure 12 E: by express STING-Flag 293T cell with or without vitamin H-dsDNA90 process and be cross-linked by DSS.Lysate is precipitated and is analyzed by immunoblotting.Figure 12 F: by 293T cell with or without dsDNA90 or ssDNA90 transfection and by UV or DSS crosslinked and then precipitation and being analyzed by immunoblotting.Figure 12 G: the 293T lysis of STING-Flag will be expressed and to hatch with vitamin H-dsDNA90 agarose with ds0NA90 or poly-(I:C) and then identical with Figure 12 E.

Figure 13 A-C shows STING in conjunction with viral DNA.The oligonucleotide sequence of Figure 13 A:HSV, cytomegalovirus (CMV) or adenovirus (ADV).Figure 13 B-C: by the 293T cell plasmid transfection of specifying.Anti-HA antibody is used to be analyzed by immunoblotting cell lysate vitamin H-dsDNA90, vitamin H-HSV DNA120mer, vitamin H-ADV DNA 120mer or vitamin H-CMV DNA 120mer sepharose precipitation.

Figure 14 A-C shows STING in conjunction with DNA.The schematic diagram of Figure 14 A:STING ELISA.The process of an embodiment of Figure 14 B:STING ELISA.Figure 14 C: the binding ability being measured dsDNA90 by ELISA.* P<0.05, Si Shi t checks.Error bars represents standard deviation.Data representation at least two independent experiments.

Figure 15 A-D shows the down regulator that TREX1 is a kind of STING intracellular signaling.Figure 15 A: by hTERT-BJ1 cell TREX1, STING or STING and TREX1siRNA process.After 3 days, cell HSV-Luc (m.o.i=1) is infected 48h.Measure lysate uciferase activity.Figure 15 B: by elementary TREX1 ^{+ /+}or TREX ^-/-mEF NS or STING siRNA transfection.After 3 days, cell HSV-Luc (m.o.i=1) is infected 48h.Measure lysate.Figure 15 C: by elementary TREX1 ^{+ /+}or TREX ^-/-mEF NS or STING siRNA transfection.After 48h, cell HSV is infected and measures HSV and copies.Check that TREX1 expresses by immunoblotting.* P<0.05, Si Shi t checks.Error bars represents standard deviation.Data representation at least two independent experiments.Figure 15 D: by STING ^{+ /+}or STING ^-/-mEF with or without dsDNA90 process 3h.Purifying total serum IgE and check genetic expression by Illumina Sentrix bead chip array (mouse WG6 version 2).

Figure 16 shows STING at TREX ^-/-the IFN β regulating ssDNA90 to mediate in MEF produces.By the TREX1 of siRNA process ^{+ /+}or TREX1 ^-/-mEF ssDNA45 or ssDNA90 process and after 16 hours, measure IFN β level.* P<0.05, Si Shi t checks.Error bars represents standard deviation.Data representation at least two independent experiments.

Figure 17 A-H shows the down regulator that TREX1 is not a kind of STING-dependent gene.Figure 17 A: by TREX1 ^{+ /+}or TREX1 ^-/-mEF HSV1, IFN α, dsDNA90, triphosphoric acid RNA (TPRNA) and VSV process.TPRNA and VSV faintly activates the gene of IFN induction.Purifying total serum IgE and check genetic expression by Illumina Sentrix bead chip array (mouse WG6 version 2).Figure 17 B-H: the total serum IgE being checked IFN β (Figure 17 B), IFIT1 (Figure 17 C), IFIT2 (Figure 17 D), IFIT3 (Figure 17 E), CCL5 (Figure 17 F), CXCL 10 (Figure 17 G), RSAD2 (Figure 17 H) by RT-PCR.The significant difference on the gene of IFN induction is not observed in the cell lacking TREX1.* P<0.05, Si Shi t checks.Error bars represents standard deviation.Data representation at least two independent experiments.

Figure 18 A-D shows TREX1 and associates with oligosaccharyl transferase complex body.The hTERT cDNA library of IFN process is used to produce a yeast two-hybrid library (AH109).Total length TREX1 is used as bait and screens this library.Screen the yeast (Clontech company) of about 5,000,000 expression cDNA.44 clones are separated from 3 independently yeast heterozygosis program.Altogether RPN1 is separated 8 times (in screening 1 three times, in screening 2 twice and in screening 3 three times).Great majority clone (except RPN1) can not interact with IREX1 after retesting.In the clone that these eight RPN1 are separated, four clones coding aa 258-397, two clones coding aa 220-390 and two clones coding aa 240-367.Produce TREX1 variant and mark and draw the interaction between TREX1 (aa.241-369) and RPN1 (aa 256-397).In order to be separated DAD1, the C-end region (aa 173-379) of STING is used for screening same library.Find twice 24 clones be separated, total length DADI.Great majority clone (except DAD1) can not interact with STING after reaffirming research.See that the region 242-310 of total length DAD1 and STING associates.The schematic diagram of Figure 18 A:TREX1 mutant.Figure 18 B: in the screening of yeast two hybrid body, the GAL4 binding domain merged to TREX1 or TREX1-4 interacts with merging to the RPN1 of GAL4 activation domain.The schematic diagram of Figure 18 C:STING mutant.Figure 18 D. is in the screening of yeast two hybrid body, and the GAL4 binding domain merged to STING-C-end or STING-C2 interacts with the DAD1 merged to GAL4 activation domain.

Figure 19 A-C shows TREX1 with STING and associates with oligosaccharyl transferase complex body.Figure 19 A: by 293T cell TREX1-tGFP and RPN1-Myc cotransfection.The antibody of specifying is used to be analyzed by immunoblotting with anti-tGFP antibody or IgG contrast immunoprecipitation lysate.Figure 19 B: by 293T cell and Myc-STING or GFP-DAD1 cotransfection and by lysis.Anti-GFP antibody is used to carry out immunoblotting with anti-Myc antibody or IgG contrast immunoprecipitation lysate.Figure 19 C: by 293T cell TREX1-tGFP and RPN1-Myc, GFP-DAD1 or STING-HA cotransfection.Lysate is contrasted immunoprecipitation by anti-tGFP antibody or IgG and used anti-tGFP, anti-Myc, anti-GFP or anti-HA antibody to be analyzed by immunoblotting.

Figure 20 shows TREX1 and is positioned in endoplasmic reticulum.By the RPN1-Myc transfection of hTERT-BJ1 cell.After 48h, use anti-TREX1 antibody (redness), anti-Myc antibody (green) or as the anti-calprotectin antibody (blueness) of endoplasmic reticulum marker thing by immunofluorescent cell.

The STING that Figure 21 A-H is presented at heterogenous expression in 293T cell rebuilds dsDNA90 response.Figure 21 A: by 293T cell contrast slow virus or hSTING slow virus infection.Infect after 1 day, by cell dsDNA90 process.After 6h, use anti-STING or anti-calprotectin antibody by cell dyeing.Figure 21 B: use anti-STING antibody to make cell lysate (from Figure 56 A) stand immunoblotting.Figure 21 C-D: the 293T cell dsDNA90 of slow virus infection is stimulated 6h.Purifying total serum IgE and check IFN β (Figure 21 C) or IFIT2 (Figure 21 D) by PCR in real time.Figure 21 E: the 293T cell using contrast or hSTING slow virus to stablize transduction stands brefeldin A (BFA) experiment, as illustrated in the flow.Figure 21 F: use anti-STING antibody to make cell lysate (from Figure 21 E) stand immunoblotting.Figure 21 G: the IFN β-uciferase activity measuring cell lysate (from Figure 21 E).Figure 21 H: by elementary STING ^-/-mEF contrast or mSTING stably transduce.By cell with dsDNA90 process and by ELISA measurement endogenous IFN β level.* P<0.05, Si Shi t checks.Error bars represents standard deviation.Data representation at least two independent experiments.

Figure 22 shows translocon member and regulates HSV1 to copy.By TREX1, Sec61A1, TRAP β, NS or STING siRNA process of hTERT-BJ1 cell.

Figure 23 A-C is presented in hTER-BJ1 cell to be produced by the IFN-of dsDNA90 does not need IFI16.Figure 23 A: by NS, IFI16, STING or TREX1siRNA process of hTERT-BJt cell.After 3 days, lysis is checked expression level by immunoblotting.Figure 23 B: by the hTERT-BJ1 cell of siRNA process with dsDNA90 process and by ELISA measurement IFN β generation.Figure 23 C: the hTERT-BJ1 cell of siRNA process HSV-luciferase (m.o.i=0.1) is infected.After infecting the 2nd day, uciferase activity is measured in lysis.* P<0.05, Si Shi t checks.Error bars represents standard deviation.Data representation at least two independent experiments.

Figure 24 shows an embodiment of the mensuration based on STING cell.

Figure 25 shows medicine " A " and induces STING to transport.

Figure 26 shows medicine " X " and suppresses IFN β mRNA to produce.

Figure 27 is a schematic diagram, display STING phosphorylation in the response of cytoplasmic DNA.By the ISD transfection 6h of hTERT-BJ1 cell with 4 μ g/ml.In TNE damping fluid, preparing cell lysate and then making it through benefiting from the immunoprecipitation of anti-STING antibody, standing SDS-PAGE subsequently.Gel CBB is dyeed and is then analyzed at Harvard's mass spectrum and proteomics Resource Facility (Harvard Mass Spectrometry and Proteomics Resource Laboratory) band comprising STING by mLC/MS/MS.Comparison from different plant species STING aminoacid sequence and identify phosphorylation site by mass spectroscopy.By mass spectroscopy qualification Serine 345,358,366 and 379.Serine 358 and S366 are important for STING function.

Figure 28 is presented in cytoplasmic DNA approach, and the Serine 366 (S366) of STING is important for IFN β produces.By 293T the cell plasmid of encode mutant STING and reporter plasmid transfection.After 36hr, measure uciferase activity.By STING ^-/-mEF cell is reconstructed with mutant STING and is then measured the amount of the IFN β in substratum by ELISA.S366 is important for the IFN by STING produces and S358 also seems to play a significant role.

Figure 29 A-D shows inflammation and the skin carcinoma generation of STING deficient mice tolerance DMBA induction: by STING ^{+ /+}and STING ^-/-mouse weekly with acetone simulation process or the DMBA process with 10 μ g, continues 20 weeks on the dorsal part shaving hair.The tolerance of Figure 29 A:STING deficient animals causes the DNA damage agent of skin carcinoma.Without the per-cent of the mouse of dermatoma shown in Kaplan-Meier (Kaplan – Meier) curve.Figure 29 B: the picture showing the representative mouse of each treatment group.Figure 29 C: carry out histopathological examination by H & E dyeing in the skin/dermatoma biopsy of mock or DMBA process.Image is taken under 20X amplifies.Figure 29 D: cytokine up regulation in the mouse being exposed to carcinogenic expression STING.The RNA of the skin/dermatoma biopsy of extracting from mock or DMBA process is analyzed in duplicate by Illumina Sentrix bead chip display (mouse WG6 version 2).Analyzing total genetic expression.Select most variable gene.The gene that line display is independent; Independent sample is shown in list.Pseudo-color show transcript level lower than, be equal to or higher than mean value (be respectively green, black and redness).Genetic expression; Multiple change log10 scale range is between-5 to 5.Cytokine is not observed in the skin of STING deficient animals.

Describe in detail

There is described herein the method and composition of the immunne response for regulating the experimenter suffering from the relevant disease of a kind of and abnormal STING function or imbalance.Preferred embodiment described below describes adjusting of these compositions and method.Even so, according to the explanation of these embodiments, can complete based on the following explanation provided and/or put into practice other aspects of the present invention.

For regulate suffer from the relevant disease of a kind of and abnormal STING function or imbalance experimenter (such as, people, dog, cat, horse, ox, sheep, pig etc.) the method and composition of immunne response relate to a kind of pharmaceutical composition, 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 the abnormal STING function of this experimenter.

The disease relevant to abnormal STING function or to lack of proper care can be that the cell of wherein tool defective STING function or expression causes or any disease of physical symptom of deteriorating condition or imbalance or imbalance.Commonly, this type of disease or imbalance are mediated by immune system cell, such as, inflammatory conditions, autoimmune conditions, cancer (such as, mammary cancer, colorectal carcinoma, prostate cancer, ovarian cancer, leukemia, lung cancer, carcinoma of endometrium or liver cancer), arteriosclerosis, sacroiliitis (such as, osteoarthritis or rheumatoid arthritis), inflammatory bowel (such as, ulcerative colitis or Crohn disease), peripheral vascular disorder, cerebrovascular accident (apoplexy), there is the disease of chronic inflammatory diseases, the disease characterized by the infringement with inflammatory cell infiltration, the disease of amyloid plaque is there is (such as in brain, alzheimer's disease), Ai Kadi-Gutierrez syndrome (Aicardi-Goutieres syndrome), juvenile arthritis, osteoporosis, amyotrophic lateral sclerosis or multiple sclerosis.

This medicament can be a kind of small molecules increasing or reduce STING function or expression (namely, a kind of there is the organic or inorganic molecule being less than 500,1000 or 2000 daltonian molecular weight) or one (that is, under the intracellular condition be normally positioned at wherein at STING) under cellular conditions be bonded to the nucleic acid molecule of STING.This medicament can also be a kind of nucleic acid molecule in conjunction with STING, and this nucleic acid molecule can be a kind of strand (ss) or double-strand (ds) RNA or DNA.Preferably, the length of this nucleic acid is between 40 and 150,60 and 120,80 and 100 or 85 and 95 base pairs or longer.This nucleic acid molecule in conjunction with STING can be Nuclease, such as, be made up of the Nucleotide of Nuclease or be in ring-type dinucleotides form.It can also associate with a kind of molecule of transmembrane transport of assisting.

Be used for the treatment of to suffer from and related to a kind of pharmaceutical composition by the method and composition of the cancer of the experimenter of the cancerous tumour of inflammatory immune cell infiltrate, this pharmaceutical composition comprises a kind of medicament and the pharmaceutically acceptable carrier of one of lowering STING function or expression, wherein the amount of this pharmaceutical composition can will infiltrate the reduced number at least 50% of the inflammatory immunocyte of cancerous tumour (such as effectively, at least 50%, 60%, 70%, 80% or 90%, or until the minimizing of inflammatory cell infiltration can be reduced by histology or scanning with detecting).

These compositions described here can be included together with one or more pharmaceutically acceptable carriers or vehicle, these carriers or vehicle are provided for and can give pharmaceutical composition by multiple path, these paths comprise oral, rectum, vagina, locally, in skin, subcutaneous, intravenously, intramuscular, suction, sheath and intranasal administration.The preparation be applicable to for using in the present invention is found in Lei Mingdun pharmaceutical science (Remington's Pharmaceutical Sciences), Mack Publishing Company (Mack Publishing Company), Philadelphia, Pennsylvania, in the 17th edition (1985).

This or these activeconstituentss can with a kind of mixed with excipients, diluted by a kind of vehicle, and/or be closed in a kind of carrier, this carrier can be in the form of capsule, pouch, paper or other containers.When this vehicle is as a kind of thinner, it can be a kind of solid, semisolid or fluent material, and this material can work as the vehicle of activeconstituents, carrier or medium.These compositions can be in tablet, pill, powder, lozenge, pouch, cachet, elixir, suspension agent, emulsion, solution, syrup, aerosol (as solid or be in liquid medium), ointment, soft gelatin capsule and hard gelatin capsule, suppository, sterile injectable solution, form for the sterile liquid (such as, a kind of spraying plant) of intranasal administration or the powder of sterile packed.These preparations can comprise in addition: lubricant, such as talcum, Magnesium Stearate and mineral oil; Wetting agent; Emulsifying agent and suspension agent; Sanitas, such as methyl benzoic acid ester and propylhydroxy benzoate; Sweeting agent; And seasonings.Can preparing composition of the present invention, so that by utilizing program as known in the art to give to patient, providing the quick, lasting of activeconstituents or delayed release.

For the preparation of solid formulation (such as tablet), said composition can be mixed with a kind of pharmaceutical excipient, to form a kind of solid preformulation composition of uniform mixture of inclusion compound.Can by tablet or coating of pill or chemical combination to otherwise, to provide the formulation of the advantage providing prolongation effect.Such as, tablet or pill can comprise a kind of interior dosage component and a kind of external dose component, and the latter is in the form of the coating on the former.These two kinds of components can by an enteric layer separately, this enteric layer be used for the disintegration of resisting in stomach and in allowing component intactly transmit and enter in duodenum or delayed release.Multiple material may be used for this type of enteric layer or dressing, and this type of material comprises the mixture of many polymeric acid and polymeric acid and the such as material of shellac, cetyl alcohol and rhodia.

The liquid form of preparation comprises suspension agent and emulsion.In order to strengthen serum half-life, by encapsulated for these preparations, can introduce in the tube chamber of liposome, be prepared as colloid, or mix in lipid layers.Multiple method can be used for preparing liposome, as described in the U.S. Patent number 4,235,871,4,501,728 and 4,837,028 people such as such as Szoka (cable clamp), will be combined in this each via quoting.

Preferably, these compositions are formulated as unit dosage that is this or these activeconstituentss.Giving to change depending on the medicine given, the object given (such as prevention or treatment), the state of patient, the mode that gives etc. to the amount of patient, being all positioned at qualified doctor and the technology of pharmacist.In treatment use, with the patient enough cured or suppress the symptom of disease and the amount of complication thereof to be given by composition to suffering from this disease at least in part.The amount being effective to this purposes will depend on the disease states for the treatment of and depend on following factor by the judgement of attending clinician, the severity of such as symptom, the age of patient, body weight and general status etc.

All Files is combined in this by reference as mentioned herein.For all objects, all publications of quoting in this application and patent document are combined in this by reference, and its combination degree is pointed out individually as each independent publication or patent document.By quoting of different reference in this document, ladies and gentlemen applicant does not admit, any concrete reference paper is relative to " prior art " of the present invention.The embodiment of the compositions and methods of the invention is described in the following example.

Example

Example 1: to be correlated with STING and cytoplasmic DNA complex body for regulating the translocon of innate immunity

Previously, describe the separation of the new cross-film component of a kind of endoplasmic reticulum (ER), this component is called STING (stimulator of interferon gene), STING is proved to be as producing I type IFN necessary (see U.S. Patent Application Serial Number 13/057,662 and PCT/US 2009/052767) in inoblast, scavenger cell and dendritic cell (DC) in response to tenuigenin dsDNA and DNA virus and Intracellular bacterial response.It is noted that the minimum size activating the dsDNA needed for STING-dependency I type IFN intracellular signaling in murine cells is about 45 base pairs in murine cells.But, in Normal human cells (hTERT), observe the dsDNA (stimulating dsDNA90 referred to here as Interferon, rabbit) activating I type IFN completely and need to have about 90 base pairs.Use RNAi to strike low program, confirm in addition in hTERT, STING is generation institute required (Figure 1B) of I type IFN really.The further analysis using microarray procedures to measure mrna expression shows except I type IFN, and tenuigenin dsDNA can also induce a series of gene of innate immunity widely (Fig. 5 A-G) in hTERT cell.Seemingly STING-is dependent in the induction of these intrinsic molecules (comprising the member of IFIT family), because the RNAi of the STING in hTERT strikes and lowly eliminates the stimulation (Fig. 5 A-G) of tenuigenin dsDNA to them to a great extent.Use STING ^{+ /+}or ^-/-muroid embryo fibroblast (MEF) confirms that tenuigenin dsDNA, and this tenuigenin dsDNA induces multiple innate immunity gene (Fig. 1 C) in a kind of STING-dependency mode.In order to confirm that the induction of these mRNA is STING-dependent gene (SDG) and is not stimulated, by the STAT1 of I type IFN-intracellular signaling defect by I type IFN dependency autocrine or paracrine intracellular signaling ^-/-mEF dsDNA processes similarly and confirms the generation of SDG unaffected (Fig. 1 C).Reversed transcriptive enzyme (RT) pcr analysis confirms array result (Fig. 6 A-H and 7A-H).Notice, the ssDNA (ssDNA45) with 45 Nucleotide faintly induces innate immunity gene to produce and fainter in MEF in hTERT.But the ssDNA (ssDNA90) observing the transfection comprising 90 Nucleotide more effectively activates series of genes (comprising I type IFN) (Fig. 1 D, 1E) in hTERT cell.Use STING ^{+ /+}or ^-/-muroid embryo fibroblast (MEF) confirms that tenuigenin ssDNA90 induces innate immunity gene generation (Fig. 1 F and 7A-H) in a kind of STING-dependency mode similarly.Observe, STING may be arranged in the ER of the mankind and murine cells as homodimer, and under the existence of tenuigenin ssDNA or dsDNA part, move to He Zhou district from ER, to activate the I type IFN-dependent transcription factor (Fig. 1 G and 8A-E).Observe HSV1 similarly and activate innate immunity gene generation (Fig. 9 A-I) in a kind of STING dependency mode.Confirm, many SDG comprise IRF7 binding site (Figure 10 A-F) in its promoter region.Therefore, tenuigenin ssDNA or dsDNA comprising the plasmid DNA of transfection effectively can induce transcribing of a series of genes involved of innate immunity widely relying on STING.

In order to assess further STING self can with the possibility of DNA class association, by the STING transfection of 293T cell and the C-end region (aa181-349) observing STING after cell lysis can use biotin labeled dsDNA90 and precipitate (Fig. 2 A).The contrast (GFP, NFAR1 and IPS1) of the N-end region (aa 1-195) of STING and three kinds of similar HA marks does not associate with dsDNA90.Using the exonuclease TREX1 in conjunction with DNA as positive control.A series of research widely shows in addition, and the amino acid region 242-341 of STING may be responsible in conjunction with dsDNA, and the STING variant in default of this region can not associate with nucleic acid (Fig. 2 B-D).The STING of vivoexpression is also bonded to dsDNA (lack similarly those variants of the region 242-341 except) (Figure 11 A-F) under high salt and high washing composition condition.By biotin labeled dsDNA90 transfection is entered to obtain with a kind of reversible crosslink reagent (DSS) or UV optical processing in hTERT and by this type of cell other about STING can with the evidence of dsDNA compound (may be a kind of dimer).Under two kinds of dispositions, all observe STING after cell lysis and retain its associating (Fig. 2 E and Figure 12 A-G) with DNA.The RNAi of STING in hTERT cell strikes and lowly eliminates the combination observed, and only wild-type MEF ( ^{+ /+}) in observe STING-DNA complex body, but lack STING MEF ( ^-/-) in do not observe (Figure 12 A-G).Confirm the relevant dsDNA of HSV1, cytomegalovirus (CMV) and adenovirus (ADV) similarly.Competitive assay illustrates, STING can also be bonded to ssDNA (ssDNA90) and dsDNA, but is not bonded to dsRNA (Fig. 2 F).This is by expressing STING in vitro and observing and associating of ssDNA90 and being confirmed (Fig. 2 G).The all STING variants analyzed lack the ability (Fig. 2 H) activating IFN type promotor in 293T cell.Also dsDNA transfection to be entered in hTERT or MEF cell and by formaldehyde treated, so that cell protein is cross-linked to nucleic acid.After STING catches (pull down), CHIP subsequently analyzes and confirms further, and the DNA of transfection can directly associate with STING, as use dsDNA90 Auele Specific Primer determined (Fig. 2 I and 2J).Observe in ELISA measures, STING can be bonded to biotin labeled DNA (Figure 14 A-C).Data show congenital intracellular signaling event dependent that ssDNA and dsDNA-mediate in STING and prove STING self can compound to these nucleic acid constructs, to help to trigger these events.

Associate molecule as the TREX1 of 3 '->5 ' DNA exonuclease or a kind of ER and be important for the ssDNA kind that degraded check point is activated, these ssDNA kinds can activating immune system to otherwise.The STING dependency of being undertaken by dsDNA90 that RNAi for TREX1 reticent in hTERT cell significantly increases I type IFN produces (Fig. 3 A and 3B).Concomitantly, being replicated in the hTERT cell lacking TREX1 of dsDNA virus HSV1 is reduced to a great extent, the generation (Fig. 3 C and D) of the rising of the gene (ISG) that may stimulate due to I type IFN and antiviral IFN.In the hTERT cells infected of the RNAi process with reticent TREX1, the luciferase expression from the recombinant chou HSV of expressing luciferase gene is also significantly lower (Figure 15 A-D).By using TREX1 defect MEF to extend these observationss, show that the induction of tenuigenin dsDNA-dependent gene is raised to a great extent when there is not TREX1 and HSV1 copies remarkable minimizing (Fig. 3 E-G) similarly.In order to determine the generation of the rising of the I type IFN whether STING is responsible for observing when there is not TREX1, hTERT or TREX1 of TREX1 will be lacked ^-/-sTING in MEF is reticent and with tenuigenin dsDNA or these cells of HSV1 process.These results show that I type IFN generation reduces to a great extent in the TREX1 deficient cells (hTERT and MEF) lacking STING, shows that the level of the rising of the I type IFN observed when there is not TREX1 is STING-dependent (Fig. 3 A-F).Similarly, notice that STING is at TREX ^-/-rNAi in MEF strikes the low I type IFN also eliminating ssDNA90-mediation and produces and congenital gene stimulation (Figure 16).Burnt analysis confirmation TREX1 and STING of copolymerization is positioned (Fig. 3 H) in ER altogether.But tenuigenin dsDNA does not induce the TREX1 being similar to STING to be transported to He Zhou district (Fig. 3 H) from ER effectively.Therefore, do not observe STING and TREX1 and effectively interact, as by co-immunoprecipitation analysis determined.Under non-stimulated condition, TREX1+ /+or ^-/-in MEF, not to be noted significant difference (Figure 17 A-H) in the expression of STING-dependent gene.But TREX1 is a kind of dsDNA-induced gene, confirms and can be raised (Figure 17 A-H) in a kind of STING-dependency mode.Therefore, dsDNA kind and STING and accessory molecule compound are seem rational with mediated transport and downstream signaling events, and these events activate transcription factor IRF3/7 and the NF-κ B of the induction being responsible for elementary innate immunity gene (comprising TREX1).This evidence shows, the TREX1 that STING-activates is arranged in ER district to degrade activator dsDNA and with a kind of negative feedback mode T suppression cell matter dsDNA intracellular signaling.Therefore, TREX1 is a kind of STING down regulator.

In this digital proof, STING is arranged in ER as a part for translocon complex body, associates with translocon-associated protein β (TRAP β).Translocon complex body comprises and Sec61 α β and γ that can be attached to ribosomal TRAP α β, γ and δ coupling.Secretory protein and membranin transposition enter in ER for correctly folding and carried out glycosylation before being output.In order to identify TREX1 binding partners, in dual-hybrid yeast screening, total length TREX1 is used as bait.Result shows, TREX1 is cyclically called the protein-interacting of ribophorin I (RPN1) with one, this albumen is a kind of 68kDa I type transmembrane protein and is member (Fig. 4 A-E of oligosaccharyl transferase (OST) complex body; Figure 18 A-D).When nascent polypeptide enters ER by translocon, this OST complex catalyzes mannose oligosaccharide is transferred on the asparagine residue of these nascent polypeptide.At least seven kinds of protein comprise OST complex body, comprise RPN1, RPN2, OST48, OST4, STT3A/B, TUSC3 and DAD1.Significantly, the similar screening that STING is used as bait determined, STING can also associate with DAD1 (resisting the defender of apoptotic cell death), and DAD1 is a kind of 16kDa transmembrane protein (Figure 14 F-H).Use yeast two-hybrid approach to analyze these associations further, show that the amino acid 258-397 (Figure 18 A-D) of RPN1 is responsible for being bonded in the C-end region (amino acid 241-369) of the TREX1 comprising its cross-film district.In addition, the amino acid 242-310 of STING is responsible for associating with total length DAD1 (Figure 18 A-D).Co-immunoprecipitation research confirms the interaction (Fig. 4 D and 4G and Figure 29 A-C) of these molecules.Other co-immunoprecipitation experiment indicates the association (Figure 19 A-C) of TREX1 and DAD1.Burnt analysis confirmation TREX1 and RPN1 of copolymerization to be positioned altogether in cell but not to transport (Fig. 4 E and Figure 20) in response to tenuigenin dsDNA.Similarly, STING and DAD1 is positioned in the ER of cell altogether, but the latter's molecule under the existence of tenuigenin dsDNA not with STING to endosome compartment (Fig. 4 H).Comprise the cytomicrosome compartment of ER by fractional separation and checked by Sucrose gradient analysis.This research shows that TREX1 and STING and ER marker RPN1 and RPN2, DAD1 and calprotectin are by common fraction, but uncommon with nucleohistone H3 fraction, the Subcellular Localization confirming them is unrecognizable (Fig. 4 I) between the component of translocon/OST complex body.Therefore, the OST/ translocon complex body comprising STING of TREX1 target ER, and this association occurs by associating with RPN1, although find that the TM district of TREX1 relates in the location of TREX1 to ER.In order to identify whether the member of OST, TRAP or SRP (signal recognition peptide) complex body affects the conduction of dsDNA-dependent signals, carry out RNAi screening with the expression of these components reticent.But, except suppressing the STING raising I type IFN generation to a great extent (by the I type IFN generation of DNA mediation is required; Figure 21 A-H) and TREX1 outside, only Sec61 α and TRAP β silence affects intracellular signaling significantly and HSV1 copies, proves that these transposons member plays certain effect (Fig. 4 J and Figure 22) in this approach of control.The silence (also involving in cytoplasmic DNA sensing) observing IFI16 does not effectively suppress the conduction of dsDNA-dependent signals, at least in hTERT cell (Figure 23 A-C) like this.Be similar to the disappearance of STING, the disappearance of IFI16 can not save IFN generation (Figure 23 A-C) of the increase caused by dsDNA when there is not TREX1.But the IFI16 of minimizing makes it possible to carry out more skilled HSV1 genetic expression, confirming this part is affecting the vital role in virus replication.The silence of RPN1 or 2 also causes the increase of HSV1 genetic expression, but also non-remarkably influenced I type IFN produces, and proves that these components of OST may relate generally to N-glycosylation.

Digital proof, STING can with ssDNA and dsDNA in cytoplasmic cell (DNA based on plasmid and gene therapy vector can be comprised) compound, can regulate the induction of a series of gene of innate immunity widely, these genes are such as I type IFN, IFIT family and multiple for antiviral activity with for initiation Acquired immune response and the chemokine wanted of overstating.STING activates and plausibly assists TBK1 to be escorted the endosome compartment to clathrin covering, activates IRF3/7 with the mechanism by illustrating completely not yet.TREX1 manifests and to be present in cell with low-level and self is derivable by STING.After translation, TREX1 is positioned to OST complex body near unactivated STING (being also arranged in OST/ translocon complex body), wherein its degraded can cause the DNA kind of STING effect to otherwise speculatively.The innate immunity intracellular signaling that the component (comprising STING and TREX1 now) of translocon/OST complex body regulates tenuigenin ssDNA and dsDNA-to mediate.Autoimmunization sexual maladjustment is shown, so be possible by these diseases of STING activity inducement because the disappearance of TREX1 is produced by the I type IFN raised.

Example 2:STING instrumentality

Screening of medicaments library, to identify the medicament regulating STING expression, function, activity etc.Figure 24 shows the step of the mensuration based on STING cell.

These libraries comprise the known biological activity library of BioMol ICCB, 500 targets; The LOPAC1280 of pharmaceutical active compounds ^tMlibrary; Grace assistant life science (Enzo LifeSciences), Screen-Well ^tMinhibitors of phosphatases library, 33 kinds of known inhibitors of phosphatases;

Micro-resource spectrum collecting center (MicroSource Spectrum Collection) 2000 kinds of components, 50% drug component, 30% natural product, 20% other biological active ingredient; EMD:InhibitorSelect ^tM96 porin kinase inhibitor library I, InhibitorSelect ^tM96 porin kinase inhibitor library II, InhibitorSelect ^tM96 porin kinase inhibitor library IIIa; Kinases library B kinases really clones (TrueClone) collecting center; Kinase deficiency really clones collecting center.

Result shows a kind of medicine (be called ' medicine A ') induction STING transport (Figure 60).Another kind of medicine (being called " medicine X ") suppresses IFN β mRNA to produce (Figure 61).

Table 2: be accredited as STING inhibitor below:

The activator induction dihydro unabain of STING and BNTX maleate salt hydrate.

Example 3:STING shows self-DNA-dependency inflammatory diseases

The scavenger cell (BMDM) of bone marrow derived obtains from Sting+ /+and Sting ^-/-mouse and used the transfection of 90 base pair dsDNA (dsDNA90) to activate STING approach, or with deriving from apoptosis DNA (aDNA) transfection of the thymocyte that dexamethasone (Dex) processes.Observe, the DNA of two types effectively induces the generation of IFN β in BMDM and conventional dendritic cells (BMDC) in a kind of STING dependency mode.DNA microarray experimental verification, the STING-dependency that aDNA triggers a series of innate immunity widely and inflammation-related cytokines (such as IFN β and TNF α) in BMDM produces (table 3).By measuring with the Sting+ of aDNA process /+or Sting ^-/-cytokine in BMDM produces and confirms these data.Therefore, the pro-inflammation genes that STING can assist the apoptosis DNA-in BMDM and BMDC to mediate produces.

Table 3 shows the genetic expression of gene in the BMDM processed with apoptosis DNA (aDNA) compared with high expression level.

In order to determine whether STING plays certain effect in DNase II related inflammatory diseases, RNAi is used to be struck by STING and/or the DNase II in THP1 cell or BMDM low and notice that the disappearance of DNase II is with the rise of a kind of STING-dependency mode helper cell factor (comprising I type IFN) in response to aDNA.Because DNase is II ^-/-mouse is usually in prenatal mortality, so analysis DNase II ^-/-, Sting ^-/-or Sting ^-/-dNase II ^-/-dKO 17 days embryos (E17 days).Genotypic analyses (comprising RT-PCR and immunoblotting) confirms that these embryos lack Sting, DNaseII or two kinds of functional genes.Observe DNase II as above ^-/-embryo shows anaemia, this and the Sting lacking this phenotype significantly ^-/-dNase II ^-/-dKO embryo or contrast form remarkable contrast.Report that lethal anaemia is because I type IFN suppresses erythropoiesis in growth course.Observed by h and E dyeing subsequently, DNase II ^-/-many infiltrating macrophages being full of the apoptotic cell engulfed being responsible for the high-caliber cytokine of production are comprised in the liver of embryo.With control mice by contrast, Sting ^-/-dNase II ^-/-the liver of embryo shows similar phenotype.Analyze tire liver by TUNEL (the dUTP biotin nick end mark of terminal deoxynucleotidyl transferase mediation) to confirm, Sting ^-/-dNase II ^-/-embryo and DNase II-defect but the tire liver of non-wild-type comprise the dying cell of great inappropriate digestion perhaps.Analyzed in vitro shows, from wild-type or DNase II ^-/-the scavenger cell of the embryo of mouse engulfs apoptotic cell fully.But, although the DNA of the apoptotic cell engulfed effectively is degraded in the lysosome of wild-type scavenger cell, DNase II ^-/-scavenger cell gathers the nucleus engulfed and can not DNA digestion.This event causes the stimulation of innate immunity signal transduction path and the generation of autoimmunization relevant cell factor.Given this, the capability evaluation of the scavenger cell of shortage DNase II and STING derived by embryo liver is whether they engulf apoptotic cell and DNA digestion.Notice and be similar to DNase II ^-/-scavenger cell, and takes from wild-type or Sting ^-/-the contrast scavenger cell of mouse is compared, Sting ^-/-dNase II ^-/-scavenger cell can not digest the nucleus engulfed of the apoptosis thymocyte from dexamethasone process.Therefore, DNase II is similar to ^-/-scavenger cell, gathers in the crops from Sting ^-/-dNase II ^-/-the scavenger cell of the liver of fetal mice shows the apoptotic cell that can not digest and engulf similarly.

Above analysis is supplemented by the mrna expression level analyzed in the liver of fetal mice.This research shows, considerably less proinflammatory gene produces at wild-type or Sting ^-/-in the liver of embryo.But, observe DNase II ^-/-the cytokine comprising unusual high levels in the liver of embryo is correlated with mRNA.Significantly, with DNase II ^-/-mouse is compared, Sting ^-/-dNase II ^-/-the liver of mouse has the innate immunity activity of gene expression of significantly reduce level.Selected by being analyzed in embryo liver by RT-PCR, the mrna expression level of innate immunity gene confirms these results.Such as, with DNaseII ^-/-mouse is compared, and the generation of IFN β is at Sting ^-/-dNase II ^-/-several times are reduced in mouse.The generation of crucial interferon-stimulated gene (ISG) is also significantly reduce, and these crucial interferon-stimulated genes are such as 2 '-5 ' oligoadenylate synthetase (OAS), there is interferon inducible protein (IFIT), interferon inducible protein 27 (IFI27) and ubiquitin sample properties-correcting agent (ISG15) that trilateral tetrapeptide (tetratricopeptide) repeats.With DNase II ^-/-mouse is compared, and pro-inflammatory cytokine (such as TNF α and IL1 β) is at Sting ^-/-and Sting ^-/-dNase II ^-/-embryo liver in be also reduce.Although the generation of congenital gene is significantly suppressed, at Sting when there is not STING ^-/-dNase II ^-/-still there are some genes in mouse slightly to raise, although be in low-level, as by array analysis determined, this may be the change of mrna expression between the animal owing to analyzing, or perhaps due to the stimulation of other approach.Many in these genes are conditioned by NF-κ B and interferon regulatory factor (IRF) approach.Therefore, grow from 14 days embryo (E14 days) Sting ^-/-dNase II ^-/-or in contrast muroid embryo fibroblast (MEF), assess the function of these transcription factors.Mainly, when being exposed to cytoplasmic DNA, at Sting ^-/-dNase II ^-/-the defect of NF-kB activity (p65 phosphorylation) is observed in MEF.When being exposed to apoptosis DNA and cytoplasmic DNA, at Sting ^-/-dNaseII ^-/-same defect is obtained in BMDM.This, by after being exposed to dsDNA, notices that NF-κ B and IRF3 can not at Sting ^-/-dNase II ^-/-in MEF, transposition still can be confirmed in contrast MEF in transposition.Therefore, the inflammatory cytokine that STING may be responsible for controlling self-DNA-induction produces, and this inflammatory cytokine produces and is responsible for causing lethal embryo's erythropoiesis.

In order to extend the importance of STING in the lethal erythropoiesis regulating self-DNA-to assist, assessment DNase II ^-/-whether mouse can be born when there is not STING.Significantly, when at Sting ^-/-when background is hybridized, DNase II ^-/-mouse is born, with obvious Menedelian frequency.Pcr gene somatotype, RNA trace, RT-PCR and immunoblotting assay confirm DNase II in progeny mice and STING defect.Compared with control mice, Sting ^-/-dNase II ^-/-two knock-out mice (DKO) is revealed as normal growth and shows similar size, but notices Sting ^-/-mouse is larger a little due to still unclear reason.Preliminary immunologic evaluation also shows, Sting ^-/-dNaseII ^-/-dKO animal is enjoyed and is similar to Sting ^-/-with the similar CD4 of wild-type mice ⁺/ CD8 ⁺spectrum, but notice that DKO's develops into splenomegaly with advancing age.Lacking survival DNase II deficient mice ((the DNase II of I type IFN intracellular signaling ^-/-ifnar1 ^-/-mouse) in also to observe splenomegaly and reported be amplification due to red pulp.But, when 8 week age, compared with control mice, from Sting ^-/-dNase II ^-/-the serum analysis of mouse shows do not have detectable abnormal cytokine to produce, because the level of cytokine produced is generally lower immeasurablel.By these research, notice in vitro, be similar to DNase II ^-/-scavenger cell, and takes from wild-type or Sting ^-/-the contrast scavenger cell of mouse is compared, Sting-/-DNase II ^-/-scavenger cell can not digest from apoptosis thymocyte (Dex ⁺) the nucleus engulfed.When WT thymocyte is used as target (Dex ^-) time, indigested DNA is at DNase II ^-/-sting ^-/-gathering in scavenger cell is distant.Therefore, Sting is derived from ^-/-dNase II ^-/-the BMDM of mouse can not digest the DNA from apoptotic cell, although with DNase II ^-/-bMDM compares and does not produce inflammatory cytokine response.

Although the embryonic lethal that DNase II mediates can pass through DNase II ^+/-mouse and I type IFN defect Ifnar1 ^-/-mouse hybrid and avoiding, but the birth of the gained afterwards offspring of about 8 weeks suffers serious polyarthritis (arthritic scores is 2), because indigested DNA activates innate immunity signal transduction path and the generation of inflammatory cytokines (such as TNF α).Significantly, Sting is noticed ^-/-dNase II ^-/-any polyarthritis symptom is not shown after mouse birth.Until 12 months, at Sting ^-/-dNase II ^-/-arthritic scores in mouse is still approximately in zero (without score), with the DNase II of report of arthritic scores shown after the similar time up to 7 ^-/-ifnar1 ^-/-mouse is formed and compares.Although DNase II ^-/-sting ^-/-the spleen of mouse and H & E and the TUNEL dyeing of thymic tissue describe the existence of the infiltrating macrophages also comprising apoptosis DNA, but from 6 monthly age Sting ^-/-dNase II ^-/-the histology in the joint of mouse shows normal bone (B) synovial joint (S) and cartilage (C) structure, does not have the sign that pannus infiltrates in articulation structure.From Sting ^-/-dNase II ^-/-tNF α, the IL1 β of the serum of mouse and the level of IL6 are still in lower level, as from we to lack STING BMDM array analysis in predict (table 3).At Sting ^-/-dNase II ^-/-the sign that the intraarticular of mouse does not have CD4, CD68 or TRAP positive cell to infiltrate.Sting ^-/-dNaseII ^-/-the serum analysis of mouse also shows that the level of Rheumatoid factors, polyclonal (RF), anti-dsDNA antibody or MMP3 does not raise.Therefore, the disappearance of STING eliminates the pro-inflammatory cytokine generation of the polyarthritis of responsible self-DNA mediation.

STING is responsible for inflammatory diseases, such as, as Ai Kadi-Gutierrez syndrome (AGS).AGS is confirmed as encephalopathic from genetics angle and is characterized by the calcification of Basal ganglia and white matter, demyelination.High-caliber lymphocyte in cerebrospinal fluid and I type IFN.The chronic infection of these characteristic simulations.The serum level of I type IFN is also raise in autoimmune syndromes systemic lupus erythematous (SLE).AGS is by 3 '-5 ' sudden change of DNA exonuclease TREX1 causes.The DNA kind of TREX1 afunction is gathered and activating cells matter DNA sensor (STING) in the ER of cell.TREX1 digests this DNA source (special function (housekeeping function)) to stop innate immunity gene activation.

In view of STING seems to be responsible for inflammatory diseases in Apoptosis defects mouse, whether the disease that next oneself-DNA of assessment other types triggers is occurred by the activation of STING approach.Such as, the patient of 3' repair exonuclease 1 (Trex1) defect suffers Ai Kadi-Gutierrez syndrome (AGS), and this syndrome promotes there is the fatal encephalitis that high-caliber I type IFN is produced as sign in cerebrospinal fluid.Trex1 deficient mice shows the median life of about 10 weeks, because can be speculated as normally digested by Trex1 be have activated DNA sensor in cell by the oneself-DNA characterized not yet so far, this trigger cytokine produce and cause the myocarditis that lethal inflammatory increases the weight of.Nearest data show that the disappearance of STING can extend Trex1 ^-/-the life-span of mouse, but reason is unknown.These researchs are extended and notice, are being exposed to the Trex1 defect BMDC (Trex1 of dsDNA90 ^-/-bMDC) in, I type IFN generation level slightly raises.Significantly, the disappearance (Sting of STING ^-/-trex1 ^-/-bMDC) ability that DNA strengthens I type IFN generation in Trex1 defective type BMDM is eliminated.Enjoyably, when with Trex1 ^-/-when mouse is compared, observe Sting ^-/-trex1 ^-/-, Sting ^-/+trex1 ^-/-heart size reduce.With independent Trex1 ^-/-compare, at Sting ^-/-trex1 ^-/-in also notice the sign that myocarditis significantly reduces.In addition, at Trex1 ^-/-very general anti-core autoantibody (ANA) is observed, at Sting in the serum of mouse ^-/-trex1 ^-/-almost anti-core autoantibody is there is not completely in the serum of mouse.Micro-array analysis shows and Trex1 ^-/-mouse is compared, at Sting ^-/-trex1 ^-/-, Sting ^-/+trex1 ^-/-heart in the pro-inflammation genes of remarkable minimizing level.In sum, these data show that STING is responsible for pro-inflammation genes induction and is plausibly responsible for AGS in Trex1 deficient mice.Example 4: the kinases for the S366 of STING screens

For 2 kinds of peptides (A366 and S366) as substrate, the activity of assessment 217 kinds of protein kinase targets.By protein kinase and often kind of peptide and ³³p-ATP mixes and then measures activity (CPM).In STING, following kinases is accredited as and makes S366 phosphorylation.The kinase whose qualification of this Serine of target opens the approach leading to drug discovery.The medicine of this association of target can suppress STING active and may be used for suppressing the therapeutic purpose of STING activity.STING crosses the inflammatory diseases that activity can cause worsening cancer.

Table 4: the kinases for the S366 of STING screens

Example 5:STING is responsible for inflammation associated cancer

By STING WT and STING ^-/-animal with DNA damage agent process and lack STING mouse tolerance tumour formed.This is because infiltrating immune cells (such as dendritic cell, scavenger cell etc.) is engulfed experience downright bad or apoptotic damaging cells, and from the DNA of this type of damaging cells and the generation of other ligand activation STING neoplastic cytokine and activation promotion is swollen.STING can relate in the tumour progression of assisting other cancers diversified.

Figure 29 A-D shows inflammation and the skin carcinoma generation of STING deficient mice tolerance DMBA induction: by STING ^{+ /+}and STING ^-/-mouse weekly with acetone simulation process or the DMBA process with 10 μ g, continues 20 weeks on the dorsal part shaving hair.The tolerance of Figure 29 A:STING deficient animals causes the DNA damage agent of skin carcinoma.Without the per-cent of the mouse of dermatoma shown in kaplan-Meier curve.Figure 29 B: the picture showing the representative mouse of each treatment group.Figure 29 C: carry out histopathological examination by H & E dyeing in the skin/dermatoma biopsy of mock or DMBA process.Image is taken under 20X amplifies.Figure 29 D: cytokine up regulation in the mouse being exposed to carcinogenic expression STING.The RNA of the skin/dermatoma biopsy of extracting from mock or DMBA process is analyzed in duplicate by Illumina Sentrix bead chip display (mouse WG6 version 2).Analyzing total genetic expression.Select most variable gene.The gene that line display is independent; Independent sample is shown in list.Pseudo-color show transcript level lower than, be equal to or higher than mean value.Genetic expression; Multiple change log10 scale range is between-5 to 5.Cytokine is 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.