A Regulator of Innate Immune Responses

(19) TZZ ¥_T (11) EP 2 942 357 A1 (12) EUROPEAN PATENT APPLICATION (43) Date of publication: (51) Int Cl.: 11.11.2015 Bulletin 2015/46 C07K 14/47 (2006.01) A61K 38/00 (2006.01) C12N 15/113 (2010.01) (21) Application number: 15169327.2 (22) Date of filing: 04.08.2009 (84) Designated Contracting States: (72) Inventor: Barber, Glen N. AT BE BG CH CY CZ DE DK EE ES FI FR GB GR Palmetto Bay, FL 33157 (US) HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO SE SI SK SM TR (74) Representative: Inspicos A/S Kogle Allé 2 (30) Priority: 04.08.2008 US 129975 P P.O. Box 45 2970 Hørsholm (DK) (62) Document number(s) of the earlier application(s) in accordance with Art. 76 EPC: Remarks: 09805473.7 / 2 324 044 This application was filed on 27-05-2015 as a divisional application to the application mentioned (71) Applicant: Barber, Glen N. under INID code 62. Palmetto Bay, FL 33157 (US) (54) STING (STIMULATOR OF INTEFERON GENES), A REGULATOR OF INNATE IMMUNE RESPONSES (57) Novel molecules termed STING which include STING compositions are useful for the treatment of an nucleic acids, polynucleotides, oligonucleotides, pep- immune-related disorder, including treating and prevent- tides, mutants, variants and active fragments thereof, ing infection by modulating immunity. modulate innate and adaptive immunity in a subject. EP 2 942 357 A1 Printed by Jouve, 75001 PARIS (FR) EP 2 942 357 A1 Description RELATED APPLICATIONS 5 [0001] This application claims priority under 35 USC § 119 to U.S. Provisional Patent Application No. 61/129,975 filed August 4, 2008, the disclosure of which is incorporated by reference in its entirety. FIELD OF THE INVENTION 10 [0002] Embodiments of the invention relate to compositions and methods for modulating innate and adaptive immunity in a subject and/or for the treatment of an immune-related disorder, cancer, autoimmunity, treating and preventing infections. BACKGROUND 15 [0003] Cellular host defense responses to pathogen invasion principally involves the detection of pathogen associated molecular patterns (PAMPs) such as viral nucleic acid or bacterial cell wall components including lipopolysaccharide or flagellar proteins that results in the induction of anti-pathogen genes. For example, viral RNA can be detected by mem- brane bound Toll-like receptors (TLR’s) present in the endoplasmic reticulum (ER) and/or endosomes (e.g. TLR 3 and 20 7/8) or by TLR-independent intracellular DExD/H box RNA helicases referred to as retinoic acid inducible gene 1 (RIG- I)or melanoma differentiation associated antigen 5 (MDA5,also referred to as IFIH1 andhelicard). Theseevents culminate in the activation of downstream signaling events, much of which remains unknown, leading to the transcription of NF- κB and IRF3/7- dependent genes, including type IIFN. 25 SUMMARY [0004] This Summary is provided to present a summary of the invention to briefly indicate the nature and substance of the invention. It is submitted with the understanding that it will not be used to interpret or limit the scope or meaning of the claims. 30 [0005] STING molecules (Stimulator of Interferon Genes) modulate the immune system, in particular the innate immune system. Compositions comprising STING and/or other agents which modulate STING expression, activity and/or func- tions treat diseases such as cancer, infections, autoimmune diseases or disorders, inflammation and the like are ad- ministered to patients at risk of developing or for the treatment of patients afflicted with such diseases. [0006] Embodiments of the invention are also directed to molecules and pathways which directly or indirectly interact 35 or associate with STING. [0007] Other aspects of the invention are described infra. BRIEF DESCRIPTION OF THE DRAWINGS 40 [0008] Figure 1A shows the amino acid sequence of human and mouse STING (SEQ ID NOS: 1 and 2 respectively). Figure 1B shows a schematic representation of hSTING indicating TM and leucine rich regions. Figure 1C shows a Northern blot analysis of human STING. Figure 1D shows an immunoblot analysis of STING in HEK 293 cells. RNAi to STING 45 (hSTING) or control RNAi (NS) was used to confirm specificity of the antibody. Figure 1E shows a confocal analysis of HEK 293 cells transfected with hSTING tagged at the carboxyl end with HA. Transfected cells were also analyzed using ER-dsRed, Mitotracker or Golgi-dsRed. Figure 1F shows that fractionation experiments confirm that STING resides in the ER. Control antibodies indicate accuracy of fractionation (Calreticulin- ER, COX IV-mitochondria, beta actin- cytosol). 50 Figure 2A: 293T cells were transfected with 50ng p110-Luc plasmid and 10ng pRL-TK normalization plasmid with increasing amounts of (50ng, 150ng, 250ng) human hSTING, murine mSTING or control ΔRIG-I. Luciferase assays indicating IFNβ promoter activity were taken 36 hours post-transfection. 293T cells transfected as in (Figure 2A) with either PRDIII-I-Luc (Figure 2B), NF-κB-Luc (Figure 2C) or ISRE-Luc (Figure 2D) responsive plasmids were analyzed similarly. Figure 2E: 293T cells were transfected with 250 ng of vector alone or STING, IPS-1 or TBK-1 55 expressing plasmids for 24hrs and lysed cells analyzed by native gel electrophoresis and by subsequent immunoblot using antibody to detect IRF3 dimerization. Figure 2F: MEF’s were transfected with vector alone or hSTING or mSTING or IPS-1 expressing plasmid (500 ng) and mRNA retrieved after 24hrs post-transfection. IFN β mRNA was analyzed by qRT-PCR. Figure 2G: Medium from transfected MEFs was analyzed for IFN β protein by ELISA. Figure 2 EP 2 942 357 A1 2H: 293T cells were transfected with 250ng of control or hSTING expressing plasmid and mRNA retrieved after 36 hrs for analysis by DNA microarray. Figure 2I: MEF’s were transfected with vector alone or hSTING or mSTING or ΔRIG-I or IPS-1 expressing plasmid (500 ng) and after 36 hrs post-transfection were infected at an MOI of 1 with VSV-GFP. Figure 2J: Viral replication from experiment(Figure 2H) was measured by plaque assay. Figure 2K: 5 Normal or TBK-1 deficient MEFs were transfected with vector alone, hSTING, mSTING or TBK-1 expressing plasmids (500ng) and 100 ng murine IFN β-Luc reporter plasmid with 10ng PRL-TK for 24 hrs and luciferase measured. Figure 2L: Normal or FADD -/- MEFs were treated as in (Figure 2K) and luciferase measured. Figure 2M: Schematic of hSTING variants. Figure 2N: 293T cells were transfected as in (Figure 2A) with hSTING full-length or variants and luciferase measured. Figure 2O: 293T cells were transfected with 100ng full length STING and increasing amounts 10 of hSTING-Full, hSTING-N or hSTING-C (0ng, 150ng, 250ng) with luciferase plasmids as in (Figure 2A). Luciferase was measured after 36 hrs. Asterisks indicate significant difference (P < 0.05) as determined by Student’s t-test. Figure 3A: MEFs (C57/BL6) were treated with RNAi to mSTING and knockdown confirmed after 72 hours by immunoblot using anti-STING rabbit antiserum. Figure 3B: Fluorescence microscopy (GFP) of MEFs treated with RNAi to mSTING following 24 hrs infection with VSV-GFP (MOI 1). Figure 3C: RNAi treated cells were infected with 15 VSVGFP (MOI 1) for 16 hrs and IFNβ mRNA measured using quantitative RT-PCR. Figure 3D: Viral titers taken from RNAi treated or untreated MEFs after 24 hours. Figure 3E schematic diagram depicting targeted homologous recombination strategy of STING in ES cells. Figure 3F: quantitative RT-PCR analysis of STING mRNA in STING -/-or control litter mate MEFs. Figure 3G: Immunoblot of STING -/- cells or control MEFs using antiserum as in (Figure 3A). Figure 3H: Fluorescence microscopy (GFP) of STING -/-or control MEFs following 12 hrs infection with 20 VSV-GFP (MOI 0.1). Figure 3I: Viral titers taken from STING -/- or control MEFs following infection with VSV-GFP after 24 hours. Figure 3J: Viral titers taken from STING -/- or control MEFs following infection with VSV ΔM after 24 hours. Figure 3K: Endogenous IFN β levels measured from STING -/- or control MEFs infected with VSV-GFP (MOI 1) or Sendai Virus (SeV MOI 1) after 24 hours. Figure 3L: STING-/- MEF’s or controls were treated with transfected (Lipo 2000 [3ml/ml]) poly dA-dT for 24 hrs and IFN β measured by ELISA. Figure 3M: Time course analysis of DNA 25 transfected MEFs. Figure 3N: BMDM were treated with exogenous poly I:C (10 mg/ml) or LPS (10 mg/ml)or transfected poly dA-dT (as in Figure 3L; 10mg/ml) and IFNβ after 24 hours by ELISA. Figure 3O: GM-DC’s were treated as in Figure 3N. Asterisks indicate significant difference (P < 0.05) as determined by Student’s t-test. Figure 4A: 293T cells were co-transfected with HA-tagged STING and FLAG-tagged RIG-I or MDA5 for 24 hours. Cells were infected with SeV (MOI 1) for 12 hours. Cells were lysed and co-immunoprecipitated with anti-FLAG 30 antibody and after immunoblotting analyzed using antibody to HA. Figure 4B: HUVECs were lysed and immunoprecipitated using an antibody to endogenous hSTING. Washed precipitates were immunoblotted using an antibody to endogenous RIG-I. Figure 4C: 293T cells were co-transfected with HA-tagged STING and FLAG-tagged RIG-I, ΔRIG-I (aa1-284) or RIG-I-C (aa218-925) for 24 hours. Cells were lysed and co-immunoprecipitated with anti-FLAG antibody and after immunoblotting analyzed using antibody to HA. Figure 4D: 293T cells were co-transfected with 35 control vector (-) or increasing amounts of full-length, amino (aa1-230) or carboxyl (aa173-379) STING (0, 150ng and 250 ng) together with 150ng of ΔRIG-I.

Load more