US20040162262A1 - Immunomodulatory oligonucleotides - Google Patents
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- US20040162262A1 US20040162262A1 US10/789,353 US78935304A US2004162262A1 US 20040162262 A1 US20040162262 A1 US 20040162262A1 US 78935304 A US78935304 A US 78935304A US 2004162262 A1 US2004162262 A1 US 2004162262A1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/47—Quinolines; Isoquinolines
- A61K31/4706—4-Aminoquinolines; 8-Aminoquinolines, e.g. chloroquine, primaquine
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants
Definitions
- oligodeoxyriboiiucleotides are able to enter cells in a saturable, sequence independent, and temperature and energy dependent fashion (reviewed in Jaroszewski, J. W., and J. S. Cohen. 1991.
- Lymphocyte ODN uptake has been shown to be regulated by cell activation.
- Spleen cells stimulated with the B cell mitogen LPS had dramatically enhanced ODN uptake in the B cell population, while spleen cells treated with the T cell mitogen Con A showed enhanced ODN uptake by T but not B cells (Krieg, A. M., F. Gmelig-Meyling, M. F. Gourley, W. J. Kisch, L. A. Chrisey, and A. D. Steinberg. 1991. “Uptake of oligodeoxbonucleotides by lymphoid cells is heterogeneous and inducible”. Antisense Research and Development 1:161).
- Guanine ribonucleotides substituted at the CS position with either a bromine or a thiol group are B cell mitogens and may replace “B cell differentiation factors” (Feldbush, T. L., and Z. K. Ballas. 1985. “Lymphokine-like activity of 8-mercaptoguanosine: induction of T and B cell differentiation”. J. Immunol. 134:3204; and Goodman, M. G. 1986. “Mechanism of synergy between T cell signals and CS-substituted guanine nucleosides in humoral immunity: B lymphotropic cytokines induce responsiveness to 8-mercaptoguanosine”. J. Immunol.
- 8-mercaptoguanosine and 8-bromoguanosine also can substitute for the cytokine requirement for the generation of MHC restricted CTI (Feldbush, T. L., 1985. cited supra), augment murine NK activity (Koo, G. C., M. E. Jewell, C. L. Manyak, N. H. Sigal, and L. S. Wicker. 1988. “Activation of murine natural killer cells and macrophages by 8-bromoguanosine”. J. Immunol. 140:3249), and synergize with IL-2 in inducing murine LAK generation (Thompson, R. A., and Z. K. Ballas. 1990.
- LAK lymphokine-activated killer cells. V. 8-Mercaptoguanosine as an IL-2-sparing agent in LAK generation”. J. Immunol. 145:3524).
- the NK and LAK augmenting activities of these C8-substituted guanosines appear to be due to their induction of IFN (Thompson, R. A., et al. 1990. cited supra).
- a 5′ triphosphorylated thymidine produced by a mycobacterium was found to be mitogenic for a subset of human ⁇ T cells (Constant, P., F. Davodeau, M. A. Peyrat, Y. Poquet, G. Puzo, M.
- CREB cAMP response element binding protein
- ATF activating transcription factor
- CREB/ATF family of transcription factors is a ubiquitously expressed class of transcription factors of which 11 members have so far been cloned (reviewed in de Groot, R. P., and P. Sassone-Corsi: “Hormonal control of gene expression: Multiplicity and versatility of cyclic adenosine 3′,5′-monophosphate-responsive nuclear regulators”. Mol. Endocrin. 7:145, 1993; Lee, K. A. W., and N. Masson: “Transcriptional regulation by CREB and its relatives”. Biochim. Biophys. Acta 1174:221, 1993.).
- bZip basic region/leucine zipper
- All cells appear to express one or more CREB/ATF proteins, but the members expressed and the regulation of mRNA splicing appear to be tissue-specific. Differential splicing of activation domains can determine whether a particular CREB/ATF protein will be a transcriptional inhibitor or activator. Many CREB/ATF proteins activate viral transcription, but some splicing variants which lack the activation domain are inhibitory.
- CREB/ATF proteins can bind DNA as homo- or hetero-dimers through the cAMP response element, the CRE, the consensus form of which is the unmethylated sequence TGACGTC (binding is abolished if the CpG is methylated) (Iguchi-Ariga, S. M. M., and W. Schaffner: “CpG methylation of the cAMP-responsive enhancer/promoter sequence TGACGTCA abolishes specific factor binding as well as transcriptional activation”. Genes & Develop. 3:612, 1989.).
- CREB/ATF proteins appear to regulate the expression of multiple genes through the CRE including immunologically important genes such as fos, jun B, Rb-1, IL-6, IL-1 (Tsukada, J., K Saito, W. R. Waterman, A. C. Webb, and P. E. Auron: “Transcription factors NF-IL6 and CREB recognize a common essential site in the human prointerleukin 1 ⁇ gene”. Mol.
- Ehrlich “Binding of AP-1/CREB proteins and of MDBP to contiguous sites downstream of the human TGF-B1 gene”. Biochim. Biophys. Acta 1219:55, 1994.), TGF- ⁇ 2, class II MHC (Cox, P. M., and C. R Goding: “An ATF/CREB binding motif is required for aberrant constitutive expression of the MHC class II DRa promoter and activation by SV40 T-antigen”. Nucl. Acids Res. 20:4881, 1992.), E-selectin, GM-CSF, CD-8 ⁇ , the germline Ig ⁇ constant region gene, the TCR V ⁇ gene, and the proliferating cell nuclear antigen (Huang, D., P. M.
- CBP basal transcription factor
- TFIIB basal transcription factor
- CREB also has been reported to interact with dTAFII 110, a TATA binding protein-associated factor whose binding may regulate transcription (Ferreri, K., G. Gill, and M. Montminy: “The cAMP-regulated transcription factor CREB interacts with a component of the TFIID complex”.
- CREB/ATF proteins can specifically bind multiple other nuclear factors (Hoeffler, J. P., J. W. Lustbader, and C.-Y. Chen: “Identification of multiple nuclear factors that interact with cyclic adenosine 3′,5′-monophosphate response element-binding protein and activating transcription factor-2 by protein-protein interactions”. Mol. Endocrinol 5:256, 1991) but the biologic significance of most of these interactions is unknown. CREB is normally thought to bind DNA either as a homodimer or as a heterodimer with several other proteins.
- CREB monomers constitutively activate transcription Krajewski, W., and K. A. W. Lee: “A monomeric derivative of the cellular transcription factor CREB functions as a constitutive activator”. Mol. Cell. Biol. 14:7204, 1994.).
- cytomegalovirus immediate early promoter one of the strongest known mammalian promoters, contains eleven copies of the CRE which are essential for promoter function (Chang, Y.-N., S. Crawford, J. Stall, D. R Rawlins, K.-T. Jeang, and G. S. Hayward: “The palindromic series I repeats in the simian cytomegalovirus major immediate-early promoter behave as both strong basal enhancers and cyclic AMP response elements”. J. Virol.
- HTLV-1 Human T lymphotropic virus-I
- Tax the retrovirus which binds to CREB/ATF proteins and redirects them from their normal cellular binding sites to different DNA sequences (flanked by G- and C-rich sequences) present within the HTLV transcriptional enhancer
- the instant invention is based on the finding that certain oligonucleotides containing unmethylated cytosine-guanine (CpG) dinucleotides activate lymphocytes as evidenced by in vitro and in vivo data Based on this finding, the invention features, in one aspect, novel immunostimulatory oligonucleotide compositions.
- CpG cytosine-guanine
- an immunostimulatory oligonucleotide is synthetic, between 2 to 100 base pairs in size and contains a consensus mitogenic CpG motif represented by the formula:
- X 1 , X 2 , X 3 and X 4 are nucleotides and a GCG trinucleotide sequence is not present at or near the 5′ and 3′ termini.
- CpG containing immunostimulatory oligonucleotides are preferably in the range of 8 to 40 base pairs in size.
- Prolonged immunostimulation can be obtained using stabilized oligonucleotides, particularly phosphorothioate stabilized oligonucleotides.
- Enhanced immunostimulatory activity has been observed where X 1 X 2 is the dinucleotide GpA and/or X 3 X 4 is the dinucleotide is most preferably TpC or also TpT. Further enhanced immunostimulatory activity has been observed where the consensus motif X 1 X 2 CGX 3 X 4 is preceded on the 5′ end by a T.
- lymphocytes can either be obtained from a subject and stimulated ex viva upon contact with an appropriate oligonucleotide; or a non-methylated CpG containing oligonucleotide can be administered to a subject to facilitate in vivo activation of a subject's lymphocytes.
- Activated lymphocytes stimulated by the methods described herein (e.g. either ex vivo or in vivo), can boost a subject's immune response.
- the immunostimulatory oligonucleotides can therefore be used to treat, prevent or ameliorate an immune system deficiency (e.g., a tumor or cancer or a viral, fungal, bacterial or parasitic infection in a subject.
- immunostimulatory oligonucleotides can also be administered as a vaccine adjuvant, to stimulate a subject's response to a vaccine.
- the ability of immunostimulatory cells to induce leukemic cells to enter the cell cycle suggests a utility for treating leukemia by increasing the sensitivity of chronic leukemia cells and then administering conventional ablative chemotherapy.
- the invention features neutral oligonucleotides (i.e. oligonucleotide that do not contain an unmethylated CpG or which contain a methylated CpG dinucleotide).
- a neutralizing oligonucleotide is complementary to an immunostimulatory sequence, but contains a methylated instead of an unmethylated CpG dinucleotide sequence and therefore can compete for binding with umethylated CpG containing oligonucleotides.
- the methylation occurs at one or more of the four carbons and two nitrogens comprising the cytosine six member ring or at one or more of the five carbons and four nitrogens comprising the guanine nine member double ring.
- 5′ methyl cytosine is a preferred methylated CpG.
- the invention features useful methods using the neutral oligonucleotides.
- in vivo administration of neutral oligonucleotides should prove useful for treating diseases such as systemic lupus erythematosus, sepsis and autoimmune diseases, which are caused or exacerbated by the presence of unmethylated CpG dimers in a subject
- methylation CpG containing antisense oligonucleotides or oligonucleotide probes would not initiate an immune reaction when administered to a subject in vivo and therefore would be safer than corresponding unmethylated oligonucleotides.
- the invention features immunoinhibitory oligonucleotides, which are capable of interfering with the activity of viral or cellular transcription factors.
- immunoinhibitory oligonucleotides are between 2 to 100 base pairs in size and contain a consensus immunoinhibitory CpG motif represented by the formula:
- X is a pyrimidine.
- immunoinhibitory oligonucleotides are preferably in the range of 8 to 40 base pairs in size. Prolonged immunostimulation can be obtained using stabilized oligonucleotides, particularly phosphorothioate stabilized oligonucleotides.
- the invention features various uses for immunoinhibitory oligonucleotides.
- Immunoinhibitory oligonucleotides have antiviral activity, independent of any antisense effect due to complementarity between the oligonucleotide and the viral sequence being targeted.
- oligonucleotide or “oligo” shall mean multiple nucleotides (i.e. molecules comprising a sugar (e.g. ribose or deoxyribose) linked to a phosphate group and to an exchangeable organic base, which is either a substituted pyrimidine (e.g. cytosine (C), thymine (T or uracil (U)) or a substituted purine (e.g. adenine (A) or guanine (G)).
- oligonucleotide refers to both oligoribonucleotides (ORNs) and oligodeoxyribonucleotides (ODNs).
- oligonucleotide shall also include oligonucleosides (i.e. an oligonucleotide minus the phosphate) and any other organic base containing polymer. Oligonucleotides can be obtained from existing nucleic acid sources (e.g. genomic or cDNA), but are preferably synthetic (e.g. produced by oligonucleotide synthesis).
- a “stabilized oligonuclectide” shall mean an oligonucleotide that is relatively resistant to in vivo degradation (e.g. via an exo- or endo-nuclease).
- Preferred stabilized oligonucleotides of the instant invention have a modified phosphate backbone.
- Especially preferred oligonucleotides have a phosphorothioate modified phosphate backbone (i.e. at least one of the phosphate oxygens is replaced by sulfur).
- oligonucleotides include: nonionic DNA analogs, such as alkyl- and aryl-phosphonates (in which the charged phosphonate oxygen is replaced by an alkyl or aryl group), phosphodiester and alkylphosphotriesters, in which the charged oxygen moiety is alkylated. Oligonucleotides which contain a diol, such as tetraethyleneglycol or hexaethyleneglycol, at either or both termini have also been shown to be substantially resistant to nuclease degradation.
- an “immunostimulatory oligonucleotide”, “immunostimulatory CpG containing oligonucleotide”, or “CpG. ODN” refer to an oligonucleotide, which contains a cytosine, guanine dinucleotide sequence and stimulates (e.g. has a mitogenic effect) on vertebrate lymphocyte.
- Preferred immunostimulatory oligonucleotides are between 2 to 100 base pairs in size and contain a consensus mitogenic CpG motif represented by the formula:
- X 1 , X 2 , X 3 and X 4 are nucleotides and a GCG trinucleotide sequence is not present at or near the 5′ and 3′ termini.
- the immunostimulatory oligonucleotides range between 8 to 40 base pairs in size.
- the immunostimulatory oligonucleotides are preferably stabilized oligonucleotides, particularly preferred are phosphorothioate stabilized oligonucleotides.
- X 1 X 2 is the dinucleotide GpA.
- X 3 X 4 is preferably the dinucleotide TpC or also TpT.
- the consensus motif X 1 X 2 CGX 3 X 4 is preceded on the 5′ end by a T. Particularly preferred consensus sequences are TGACGTT or TGACGTC.
- a “neutral oligonucleotide” refers to an oligonucleotide that does not contain an unmethylated CpG or an oligonucleotide which contains a methylated CpG dinucleotide.
- a neutralizing oligonucleotide is complementary to an immunostimulatory sequence, but contains a methylated instead of an unmethylated CpG dinucleotide sequence and therefore can compete for binding with unmethylated CpG containing oligonucleotides.
- the methylation occurs at one or more of the four carbons and two nitrogens comprising the cytosine six member ring or at one or more of the five carbons and four nitrogens comprising the guanine nine member double ring.
- 5′ methyl cytosine is a preferred methylated CpG.
- an “immunoinhibitory oligonucleotide” or “immunoinhibitory CpG containing oligonucleotide” is an oligonucleotide that.
- Preferable immunoinhibitory oligonucleotides are between 2 to 100 base pairs in size and can be represented by the formula:
- X is a pyrimidine.
- immunoinhibitory oligonucleotides are preferably in the range of 8 to 40 base pairs in size. Prolonged immunostimulation can be obtained using stabilized oligonucleotides, particularly phosphorothioate stabilized
- “Palindromic sequence” shall mean an inverted repeat (i.e. a sequence such as ABCDEE′D′C′B′A′ in which A and A′ are bases capable of forming the usual Watson-Crick base pairs. In vivo, such sequences may form double stranded structures.
- oligonucleotide delivery complex shall mean an oligonucleotide associated with (e.g. ionically or covalently bound to; or encapsulated within) a targeting means (e.g. a molecule that results in higher affinity binding to target cell (e.g. B-cel and natural killer (NK) cell) surfaces and/or increased cellular uptake by target cells).
- a targeting means e.g. a molecule that results in higher affinity binding to target cell (e.g. B-cel and natural killer (NK) cell) surfaces and/or increased cellular uptake by target cells.
- oligonucleotide delivery complexes include oligonucleotides associated with: a sterol (e.g. cholesterol), a lipid (e.g. a cationic lipid, virosome or liposome), or a target cell specific binding agent (e.g. a ligand recognized by target cell specific receptor).
- Preferred complexes must be sufficiently stable in vivo to prevent significant uncoupling prior to internalition by the target cell.
- the complex should be cleavable under appropriate conditions within the cell so that the oligonucleotide is released in a functional form.
- an “immune system deficiency” shall mean a disease or disorder in which the subject's immune system is not functioning in normal capacity or in which it would be useful to boost a subject's immune response for example to eliminate a tumor or cancer (e.g. tumors of the brain, lung (e.g. small cell and non-small cell), ovary, breast, prostate, colon, as well as other carcinomas and sarcomas) or a viral (e.g. HIV, herpes), fungal (e.g. Candida sp .), bacterial or parasitic (e.g. Leishmania, Toxoplasma) infection in a subject.
- a tumor or cancer e.g. tumors of the brain, lung (e.g. small cell and non-small cell), ovary, breast, prostate, colon, as well as other carcinomas and sarcomas) or a viral (e.g. HIV, herpes), fungal (e.g. Candida sp .), bacterial or parasitic (e.
- a “disease associated with immune system activation” shall mean a disease or condition caused or exacerbated by activation of the subject's immune system. Examples include systemic lupus erythematosus, sepsis and autoimmune diseases such as rheumatoid arthritis and multiple sclerosis.
- a “subject” shall mean a human or vertebrate animal including a dog, cat, horse, cow, pig, sheep, goat, chicken, monkey, rat, mouse, etc.
- ODN 1 and 2 were synthesized. These ODNs, including the two original “controls” (ODN 1 and 2) and two originally synthesized as “antisense” (ODN 3D and 3M; Krieg A. M. J. Immunol. 143:2448 (1989)), were then examined for in vitro effects on spleen cells (representative sequences are listed in Table 1).
- ODN that contained CpG dinucleotides induced B cell activation and IgM secretion; the magnitude of this stimulation typically could be increased by adding more CpG dinucleotides (Table 1; compare ODN 2 to 2a or 3D to 3Da and 3 Db). Stimulation did not appear to result from an antisense mechanism or impurity. ODN caused no detectable activation of ⁇ or other T cell populations.
- the optimal stimulatory motif was determined to consist of a CpG flanked by two 5′ purines (preferably a GpA dinucleotide) and two 3′ pyrimidines (preferably a TpT or TpC dinucleotide). Mutations of ODN to bring the CpG motif closer to this ideal improved stimulation (e.g. compare ODN 2 to 2e; 3M to 3Md) while mutations that disturbed the motif reduced stimulation (e.g. compare ODN 3D to 3Df; 4 to 4b, 4c and 4d). On the other hand, mutations outside the CpG motif did not reduce stimulation (e.g. compare ODN 1 to 1d; 3D to 3Dg; 3M to 3Me).
- ODNs shorter than 8 bases were non-stimulatory (e.g. ODN 4e).
- ODN 4e the most stimulatory sequence identified was TCAACGTT (ODN 4) which contains the self complementary “palindrome” AACGTT.
- ODN containing Gs at both ends showed increased stimulation, particularly if the the ODN were rendered nuclease resistant by phosphorothioate modification of the terminal internucleotide linkages.
- ODN 1585 (5′ GGGGTCAACGTTCAGGGGGG 3′ (SEQ ID NO:1)), in which the first two and last five internucleotide linkages are phosphorothioate modified caused an average 25.4 fold increase in mouse spleen cell proliferation compared to an average 3.2 fold increase in proliferation induced by ODN 1638, which has the same sequence as ODN 1585 except that the 10 Gs at the two ends are replaced by 10 As.
- the effect of the G-rich ends is cis; addition of an ODN with poly G ends but no CpG motif to cells along with 1638 gave no increased proliferation.
- CpG-ODN induced cycling in more than 95% of B cells (Table 2).
- Splenic B lymphocytes sorted by flow cytometry into CD23 ⁇ (marginal zone) and CD23+(follicular) subpopulations were equally responsive to ODN ⁇ induced stimulation, as were both resting and activated populations of B cells isolated by fractionation over Percoll gradients.
- CpG ODN peripheral blood mononuclear cells
- PBMCs peripheral blood mononuclear cells
- CLL chronic lymphocytic leukemia
- Control ODN containing no CpG dinucleotide sequence showed no effect on the basal proliferation of 442 cpm and 874 cpm (proliferation measured by 3 H thymidine incorporation) of the human cells.
- a phosphorothioate modified CpG ODN 3Md SEQ ID NO: 25
- a phosphorothioate modified CpG ODN 3Md induced increased proliferation of 7210 and 86795 cpm respectively in the two patients at a concentration of just 1 ⁇ M. Since these cells had been frozen, they may have been less responsive to the oligos than fresh cells in vivo.
- cells from CLL patients typically are non-proliferating, which is why traditional chemotherapy is not effective.
- Certain B cell lines such as WEHI-231 are induced to undergo growth arrest and/or apoptosis in response to crosslinking of their antigen receptor by anti-IgM (Jakway, J. P. et al., “Growth regulation of the B lymphoma cell line WEHI-231 by anti-immunoglobulin, lipopolysaccharide and other bacterial products” J. Immunol. 137: 2225 (1986); Tsubata, T., J. Wu and T. Honjo: B-cell apoptosis induced by antigen receptor crosslinking is blocked by a T-cell signal through CD40. “Nature 364: 645 (1993)).
- WEHI-231 cells are rescued from this growth arrest by certain stimuli such as LPS and by the CD40 ligand. ODN containing the CpG motif were also found to protect WEHI-231 from anti-IgM induced growth arrest, indicating that accessory cell populations are not required for the effect.
- CpG ODN levels of cytokines and prostaglandins in vitro and in vivo were measured. Unlike LPS, CpG ODN were not found to induce purified macrophages to produce prostaglandin PGE2. In fact, no apparent direct effect of CpG ODN was detected on either macrophages or T cells. In vivo or in whole spleen cells, no significant increase in the following interleukins: IL-2, IL-3, IL4, or IL-10 was detected within the first six hours. However, the level of IL-6 increased strikingly within 2 hours in the serum of mice injected with CpG ODN. Increased expression of IL-12 and interferon gamma (IFN- ⁇ ) by spleen cells was also detected within the first two hours.
- IFN- ⁇ interferon gamma
- mice were injected once intraperitoneally with PBS or phosphorothioate CpG or non-CpG ODN at a dose of 33 mg/kg (approximately 500 ⁇ g/mouse).
- Pharmacokinetic studies in mice indicate that this dose of phosphorothioate gives levels of approximately 10% g/g in spleen tissue (within the effective concentration range determined from the in vitro studie's described herein) for longer than twenty-four hours (Agrawal, S. et al. (1991) Proc. Natl. Acad. Sci. USA 91:7595).
- Spleen cells from mice were examined twenty-four hours after ODN injection for expression of B cells surface activation markers Ly-6A/E, Bla-1, and class II MHC using three color flow cytometry and for their spontaneous proliferation using 3 H thymidine: Expression of all three activation markers was significantly increased in B cells from mice injected with CpG ODN, but not from mice injected with PBS or non-CpG ODN. Spontaneous 3 H thymidine incorporation was increased by 2-6 fold in spleen cells from mice injected with the stimulatory ODN compared to PBS or non-CpG ODN-injected mice. After 4 days, serum IgM levels in mice injected with CpG ODN in vivo were increased by approximately 3-fold compared to controls. Consistent with the inability of these agents to activate T cells, there was minimal change in T cell expression of the IL-2R or CD-44.
- Example 4 As described in further detail in Example 4, experiments were conducted to determine whether CpG containing oligonucleotides stimulated the activity of natural killer (NK) cells in addition to B cells. As shown in Table 3, a marked induction of NK activity among spleen cells cultured with CpG ODN 1 and 3Dd was observed. In contrast, there %% as relatively no induction in effectors that had been treated with non-CpG control ODN.
- ODN containing CpG dinucleotides that are not in the stimulatory motif described above were found to block the stimulatory effect of other mitogenic CpG ODN.
- an a typical CpG motif consisting of a GCG near or at the 5′ and/or 3′ end of CpG ODN actually inhibited stimulation of proliferation by other CpG motifs.
- Methylation or substitution of the cytosine in a GCG motif reverses this effect.
- a GCG motif in an ODN has a modest mitogenic effect, though far lower than that seen with the preferred CpG motif.
- CpG-ODN did not induce any detectable Ca 2+ flux, changes in protein tyrosine phosphorylation, or IP 3 generation.
- Flow cytometry with FITC-conjugated ODN with or without a CpG motif was performed as described in Zhao, Q et al.,( Antisense Research and Development 3:53-66 (1993)), and showed equivalent membrane binding, cellular uptake, efflux, and intracellular localization. This suggests that there may not be cell membrane proteins specific for CpG ODN.
- TGACGIT/C The optimal CpG motif (TGACGIT/C is identical to the CRE (cyclic AMP response element). Like the mitogenic effects of CpG ODN, binding of CREB to the CRE is abolished if the central CpG is methylated. Electrophoretic mobility shift assays were used to determine whether CpG ODN, which are single stranded, could compete with the binding of B cell CREB/ATF proteins to their normal binding site, the doublestranded CRE. Competition assays demonstrated that single stranded ODN containing CpG motifs could completely compete the binding of CREB to its binding site, while ODN without CpG motifs could not.
- the stimulatory CpG motif is common in microbial genomic DNA, but quite rare in vertebrate DNA.
- bacterial DNA has been reported to induce B cell proliferation and immunoglobulin (Ig) production, while mammalian DNA does not (Messina, J. P. et al., J. Immunol. 147:1759 (1991)).
- Ig immunoglobulin
- Example 3 in which methylation of bacterial DNA with CpG methylase was found to abolish mitogenicity, demonstrates that the difference in CpG status is the cause of B cell stimulation by bacterial DNA. This data supports the following conclusion: that unmethylated CpG dinucleotides present within bacterial DNA are responsible for the stimulatory effects of bacterial DNA.
- lymphocyte activation by the CpG motif represents an immune defense mechanism that can thereby distinguish bacterial from host DNA.
- Host DNA would induce little or no lymphocyte activation due to it CpG suppression and methylation.
- Bacterial DNA would cause selective lymphocyte activation in infected tissues. Since the CpG pathway synergizes with B cell activation through the antigen receptor, B cells bearing antigen receptor specific for bacterial antigens would receive one activation signal through cell membrane Ig and a second signal from bacterial DNA, and would therefore tend to be preferentially activated. The interrelationship of this pathway with other pathways of B cell activation provide a physiologic mechanism employing a polyclonal antigen to induce antigen-specific responses.
- oligonucleotides can be synthesized de novo using any of a number of procedures well known in the art.
- P-cyanoethyl phosphoramidite method S. L. Beaucage and M. H. Caruthers, (1981) Tet. Let. 22:1859
- nucleoside H-phosphonate method Garegg et al., (1986) Tet. Let. 27: 4051-4054
- Froehler et al. (1986) Nucl. Acid Res. 14: 5399-5407
- Garegg et al. (1986) Tet.
- oligonucleotide synthesizers available in the market.
- oligonucleotides can be prepared from existing nucleic acid sequences (e.g. genomic or cDNA) using known techniques, such as those employing restriction enzymes, exonucleases or endonucleases.
- oligonucieotides are preferably relatively resistant to degradation (e.g. via endo- and exo-nucleases). Oligonucleotide stabilization can be accomplished via phosphate backbone modifications. A preferred stabilized oligonucleotide has a phosphorothioate modified backbone. The pharmacokinetics of phosphorothioate ODN show that they have a systemic half-life of forty-eight hours in rodents and suggest that they may be useful for in vivo applications (Agrawal, S. et al. (1991) Proc. Natl. Acad. Sci. USA 88:7595).
- Phosphorothioates may be synthesized using automated techniques employing either phosphoramidate or H phosphonate chemistries.
- Aryl- and alkyl-phosphonates can be made e.g. (as described in U.S. Pat. No. 4,469,863); and alkylphosphotriesters (in which the charged oxygen moiety is alkylated as described in U.S. Pat. No. 5,023,243 and European Patent No. 092,574) can be prepared by automated solid phase synthesis using commercially available reagents. Methods for making other DNA backbone modifications and substitutions have been described (Uhlmann, E. and Peyman, A. (1990) Chem. Rev. 90:544; Goodchild, J. (1990) Bioconjugate Chem. 1:165).
- oligonucleotides may be associated with a molecule that results in higher affinity binding to target cell (e.g. B-cell and natural killer (NK) cell) surfaces and/or increased cellular uptake by target cells to form an “oligonucleotide delivery complex”.
- Oligonucleotides can be ionically, or covalently associated with appropriate molecules using techniques which are well known in the art.
- a variety of coupling or crosslinling agents can be used e.g. protein A, carbodiimide, and N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP).
- Oligonucleotides can alternatively be encapsulated in liposomes or virosomes using well-known techniques.
- oligonucleotides containing at least one unmethylated CpG dinucleotide can be administered to a subject in vivo to treat an “immune system deficiency”.
- oligonucleotides containing at least one unmethylated CpG dinucleotide can be contacted with lymphocytes (e.g. B cells or NK cells) obtained from a subject having an immune system deficiency ex vivo and activated lymphocytes can then be reimplanted in the subject.
- lymphocytes e.g. B cells or NK cells
- Immunostimulatory oligonucleotides can also be administered to a subject in conjunction with a vaccine, as an adjuvant, to boost a subject's immune system to effect better response from the vaccine.
- a vaccine as an adjuvant
- the unmethylated CpG dinucleotide is administered slightly before or at the same time as the vaccine.
- Preceding chemotherapy with an immunostimulatory oligonucleotide should prove useful for increasing the responsiveness of the malignant cells to subsequent chemotherapy.
- CpG ODN also increased natural killer cell activity in both human and murine cells. Induction of NK activity may likewise be beneficial in cancer immunotherapy.
- Oligonucleotides that are complementary to certain target sequences can be synthesized and administered to a subject in vivo.
- antisense oligonucleotides hybridize to complementary mRNA, thereby preventing expression of a specific target gene.
- sequence-specific effects of antisense oligonucleotides have made them useful research tools for the investigation of protein function.
- Phase I/II human trials of systemic antisense therapy are now underway for acute myelogenous leukemia and HIV.
- oligonucleotide probes i.e. oligonucleotides with a detectable label
- In vivo administration and detection of oligonucleotide probes may be useful for diagnosing certain diseases that are caused or exacerbated by certain DNA sequences (e.g. systemic lupus erythematosus, sepsis and autoimmune diseases).
- Antisense oligonucleotides or oligonucleotide probes in which any or all CpG dinucleotide is methylated would not produce an immune reaction when administered to a subject in vivo and therefore would be safer than the corresponding non-methylated CpG containing oligonucleotide.
- an effective amount of an appropriate oligonucleotide alone or formulated as an oligonucleotide delivery complex can be administered to a subject by any mode allowing the oligonucleotide to be taken up by the appropriate target cells (.e.g. B-cells and NK cells).
- Preferred routes of administration include oral and transdermal (e.g. via a patch).
- Other routes of administration include injection (subcutaneous, intravenous, parenteral, intraperitoneal, intrathecal, etc.). The injection can be in a bolus or a continuous infusion.
- an oligonucleotide alone or as an oligonucleotide delivery complex can be administered in conjunction with a pharmaceutically acceptable carrier.
- pharmaceutically acceptable carrier is intended to include substances that can be coadministered with an oligonucleotide or an oligonucleotide delivery complex and allows the oligonucleotide to perform its intended function.
- examples of such carriers include solutions, solvents, dispersion media, delay agents, emulsions and the like. The use of such media for pharmaceutically active substances are well known in the art. Any other conventional carrier suitable for use with the oligonucleotides falls within the scope of the instant invention.
- an effective amount of an oligonucleotide refers to that amount necessary or sufficient to realize a desired biologic effect.
- an effective amount of an oligonucleotide containing at least one methylated CpG for treating an immune system deficiency could be that amount necessary to eliminate a tumor, cancer, or bacterial, viral or fungal infection.
- An effective amount for use as a vaccine adjuvant could be that amount useful for boosting a subject's immune response to a vaccine.
- An “effective amount” of an oligonucleotide lacking a non-methylated CpG for use in treating a disease associated with immune system activation could be that amount necessary to outcompete non-methylated CpG containing nucleotide sequences.
- the effective amount for any particular application can vary depending on such factors as the disease or condition being treated, the particular oligonucleotide being administered, the size of the subject, or the severity of the disease or condition.
- One of ordinary skill in the art can empirically determine the effective amount of a particular oligonucleotide without necessitating undue experimentation.
- autoimmune responses to self antigens would also tend to be preferentially triggered by bacterial infections, since autoantigens could also provide a second activation signal to autoreactive B cells triggered by bacterial DNA.
- autoantigens could also provide a second activation signal to autoreactive B cells triggered by bacterial DNA.
- the induction of autoimmunity by bacterial infections is a common clinical observance.
- the autoimmune disease systemic lupus erythematosus which is: i) characterized by the production of anti-DNA antibodies; ii) induced by drugs which inhibit DNA methyltransferase (Cornacchia, E. J. et al., J. Clin. Invest.
- sepsis which is characterized by high morbidity and mortality due to massive and nonspecific activation of the immune system may be initiated by bacterial DNA and other products released from dying bacteria that reach concentrations sufficient to directly activate many lymphocytes.
- Lupus, sepsis and other “diseases associated with immune system activation” may be treated, prevented or ameliorated by administering to a subject oligonucleotides lacking an unmethylated CpG dinucleotide (e.g. oligonucleotides that do not include a CpG motif or oligonucleotides in which the CpG motif is methylated) to block the binding of unmethylated CpG containing nucleic acid sequences.
- Oligonucleotides lacking an unmethylated CpG motif can be administered alone or in conjunction with compositions that block an immune cell's reponse to other mitogenic bacterial products (e.g. LPS).
- oligonucleotides containing an unmethylated CpG dinucleotide can treat, prevent or ameliorate the disease lupus.
- Lupus is commonly thought to be triggered by bacterial or viral infections. Such infections have been reported to stimulate the production of nonpathogenic antibodies to single stranded DNA. These antibodies likely recognize primarily bacterial sequences including unmethylated CpGs. As disease develops in lupus, the anti-DNA antibodies shift to pathogenic antibodies that are specific for double-stranded DNA. These antibodies would have increased binding for methylated CpG sequences and their production would result from a breakdown of tolerance in lupus.
- lupus may result when a patient's DNA becomes hypomethylated, thus allowing anti-DNA antibodies specific for unmethylated CpGs to bind to self DNA and trigger more widespread autoimmunity through the process referred to as “epitope spreading”.
- oligonucleotides containing GCG trinucleotide sequences at or near both termini have antiviral activity, independent of any antisense effect due to complementarity between the oligonucleotide and the viral sequence being targeted. Based on this activity, an effective amount of inhibitory oligonucleotides can be administered to a subject to treat or prevent a viral infection.
- B cells were purified from spleens obtained from 6-12 wk old specific pathogen free DBA/2 or BXSB mice bred in the University of Iowa animal care facility; no substantial strain differences were noted) that were depleted of T cells with anti-Thy-1.2 and complement and centrifugation over lympholyte M (Cedarlane Laboratories, Hornby, Ontario, Canada) (“B cells”). B cells contained fewer than 1% CD4+ or CD8+cells. 8 ⁇ 10 4 B cells were dispensed in triplicate into 96 well microtiter plates in 100 ⁇ l RPM! containin!g 10% FBS (heat inactivated to 65° C.
- PBMCs perpheral blood monocyte cells
- CLL chronic lymphocytic leukemia
- DBA/2 B cells were cultured with no DNA or 50 ⁇ g/ml of a) Micrococcus lysodeikticus; b) NZB/N mouse spleen; and c) NFS/N mouse spleen genomic DNAs for 48 hours, then pulsed with 3 H thymidine for 4 hours prior to cell harvest.
- Duplicate DNA samples were digested with DNAse I for 30 minutes at 37 C prior to addition to cell cultures.
- E coli DNA also induced an 8.8 told increase in the number of IgM secreting B cells by 48 hours using the ELISA-spot assay.
- DBA/2 B cells were cultured with either no additive, 50 ⁇ g/ml LPS or the ODN 1; 1a; 4; or 4a at 20 ⁇ M. Cells were cultured and harvested at 4, 8, 24 and 48 hours. BXSB cells were cultured as in Example 1 with 5, 10, 20, 40 or 80 FM of ODN 1; 1a; 4; or 4a or LPS. In this experiment, wells with no ODN had 3833 cpm. Each experiment was performed at least three times with similar results. Standard deviations of the triplicate wells were ⁇ 5%.
- Percent specific lysis was determined by calculating the ratio of the 51 Cr released in the presence of effector cells minus the 51 Cr released when the target cells are cultured alone, over the total counts released after cell lysis in 2% acetic acid minus the 51 Cr cpm released when the cells are cultured alone.
- mice were weighed and injected IP with 0.25 ml of sterile PBS or the indicated phosphorothioate ODN dissolved in PBS. Twenty four hours later, spleen cells were harvested, washed, and stained for flow cytometry using phycoerythrin conjugated 6B2 to gate on B cells in conjunction with biotin conjugated anti Ly-6A/E or anti-Ia d (Pharmingen, San Diego, Calif.) or anti-Bla-1 (Hardy, R. R. et al., J. Exp. Med. 159:1169 (1984). Two mice were studied for each condition and analyzed individually.
- B cells were cultured with phosphorothioate ODN with the sequence of control ODN 1a or the CpG ODN 1d and 3 Db and then either pulsed after 20 hr with 3 H uridine or after 44 hr with 3 H thymidine before harvesting and determining cpm.
- WEHI-231 cells (5 ⁇ 10 4 /well) were cultured for 1 hr. at 37 C in the presence or absence of LPS or the control ODN 1a or the CpG ODN 1d and 3 Db before addition of anti-IgM (1 ⁇ /ml). Cells were cultured for a further 20 hr. before a 4 hr. pulse with 2 ⁇ Ci/well 3 H thymidine. In this experiment, cells with no ODN or anti-IgM gave 90.4 ⁇ 10 3 by addition of anti-IgM.
- the phosphodiester ODN shown in Table 1 gave similar protection, though with some nonspecific suppression due to ODN degradation. Each experiment was repeated at least 3 times with similar results.
- mice DBA/2 female mice (2 mos. old) were injected IP with 500 ⁇ g CpG or control phosphorothioate ODN. At various time points after injection, the mice were bled. Two mice were studied for each time point. IL-6 was measured by Elisa, and IL-6 concentration was calculated by comparison to a standard curve generated using recombinant IL-6. The sensitivity of the assay was 10 pg/ml. Levels were undetectable after 8 hr.
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Abstract
Description
- [0001] The work resulting in this invention was supported in part by National Institute of Health Grant No. R29-AR42556-01. The U.S. Government may therefore be entitled to certain rights in the invention.
- DNA Binds to Cell Membrane and is Internalized
- In the 1970's, several investigators reported the binding of high molecular weight DNA to cell membranes (Lerner, R. A., W. Meinke, and D. A. Goldstein. 1971. “Membrane-associated DNA in the cytoplasm of diploid humanalymphocytes”.Proc. Natl. Acad. Sci. USA 68:1212; Agrawal, S. K, R. W. Wagner, P. K. McAllister, and B. Rosenberg. 1975. “Cell-surface-associated nucleic acid in tumorigenic cells made visible with platinum-pyrimidine complexes by electron microscopy”. Proc. Natl. Acad. Sci. USA 72:928). In 1985 Bennett et al. presented the first evidence that DNA binding to lymphocytes is similar to a ligand receptor interaction: binding is saturable, competitive, and leads to DNA endocytosis and degradation (Bennett, R. M., G. T. Gabor, and M. M. Merritt, 1985. “DNA binding to human leukocytes. Evidence for a receptor-mediated association, internalization, and degradation of DNA”. J. Clin Invest. 76:2182). Like DNA, oligodeoxyriboiiucleotides (ODNs) are able to enter cells in a saturable, sequence independent, and temperature and energy dependent fashion (reviewed in Jaroszewski, J. W., and J. S. Cohen. 1991. “Cellular uptake of antisense oligodeoxynucleotides”. Advanced Drug Delivery Reviews 6:235; Akhtar, S., Y. Shoji, and R. L. Juliano. 1992. “Pharmaceutical aspects of the biological stability and membrane transport characteristics of antisense oligonucleotides”. In: Gene Regulation Biology of Antisense RNA and DNA. R. P. Erickson, and J. G. Izant, eds. Raven Press, Ltd. New York, pp. 133; and Zhao, Q., T. Waldschimidt, E. Fisher, C. J. Herrera, and A. M. Krieg., 1994. “Stage specific oligonucleotide uptake in murine bone marrow B cell precursors”. Blood, 84:3660). No receptor for DNA or ODN uptake has yet been cloned, and it is not yet clear whether ODN binding and cell uptake occurs through the same or a different mechanism from that of high molecular weight DNA.
- Lymphocyte ODN uptake has been shown to be regulated by cell activation. Spleen cells stimulated with the B cell mitogen LPS had dramatically enhanced ODN uptake in the B cell population, while spleen cells treated with the T cell mitogen Con A showed enhanced ODN uptake by T but not B cells (Krieg, A. M., F. Gmelig-Meyling, M. F. Gourley, W. J. Kisch, L. A. Chrisey, and A. D. Steinberg. 1991. “Uptake of oligodeoxbonucleotides by lymphoid cells is heterogeneous and inducible”.Antisense Research and Development 1:161).
- Immune Effects of Nucleic Acids
- Several polynucleotides have been extensively evaluated as biological response modifiers. Perhaps the best example is poly (1, C) which is a potent inducer of IFN production as well as a macrophage activator and inducer of NK activity (Talmadge, J. E., J. Adams, H. Phillips, M. Collins, B. Lenz, M. Schneider, E. Schlick, R. Ruffinann, R. H. Wiltrout, and M. A. Chirigos. 1985. “Immunomodulatory effects in mice of polyinosinic-polycytidylic acid complexed with poly-L:-lysine and carboxymethylcellulose”.Cancer Res. 45:1058; Wiltrout, R. H., R. R. Salup, T. A. Twilley, and J. E. Talmadge. 1985. “Immunomodulation of natural killer activity by polyribonucleotides”. J. Biol. Resp. Mod 4:512; Krown, S. E. 1986. “Interferons and interferon inducers in cancer treatment”. Sem. Oncol. 13:207; and Ewel, C. H., S. J. Urba, W. C. Kopp, J. W. Smith II, R. G. Steis, J. L. Rossio, D. L. Longo, M. J. Jones, W. G. Alvord, C. M. Pinsky, J. M. Beveridge, K. L. McNitt, and S. P. Creekmore. 1992. “Polyinosinic-polycytidylic acid complexed with poly-L-lysine and carboxymethylcellulose in combination with interleukin 2 in patients with cancer: clinical and immunological effects”. Canc. Res. 52:3005). It appears that this murine NK activation may be due solely to induction of IFN β secretion (Ishikawa, R., and C. A. Biron. 1993. “IFN induction and associated changes in splenic leukocyte distribution”. J. Immunol. 150:3713). This activation was specific for the ribose sugar since deoxyribose was ineffective. Its potent in vitro antitumor activity led to several clinical trials using poly (I,C) complexed with poly-L-lysine and carboxymethylcellulose (to reduce degradation by RNAse) (Talmadge, J. E., et al., 1985. cited supra; Wiltrout, R. H., et al., 1985. cited supra); Krown, S. E., 1986. cited supra); and Ewel, C. H., et al., 1992. cited supra). Unfortunately, toxic side effects have thus far prevented poly (I,C) from becoming a useful therapeutic agent
- Guanine ribonucleotides substituted at the CS position with either a bromine or a thiol group are B cell mitogens and may replace “B cell differentiation factors” (Feldbush, T. L., and Z. K. Ballas. 1985. “Lymphokine-like activity of 8-mercaptoguanosine: induction of T and B cell differentiation”.J. Immunol. 134:3204; and Goodman, M. G. 1986. “Mechanism of synergy between T cell signals and CS-substituted guanine nucleosides in humoral immunity: B lymphotropic cytokines induce responsiveness to 8-mercaptoguanosine”. J. Immunol. 136:3335). 8-mercaptoguanosine and 8-bromoguanosine also can substitute for the cytokine requirement for the generation of MHC restricted CTI (Feldbush, T. L., 1985. cited supra), augment murine NK activity (Koo, G. C., M. E. Jewell, C. L. Manyak, N. H. Sigal, and L. S. Wicker. 1988. “Activation of murine natural killer cells and macrophages by 8-bromoguanosine”. J. Immunol. 140:3249), and synergize with IL-2 in inducing murine LAK generation (Thompson, R. A., and Z. K. Ballas. 1990. “Lymphokine-activated killer (LAK) cells. V. 8-Mercaptoguanosine as an IL-2-sparing agent in LAK generation”. J. Immunol. 145:3524). The NK and LAK augmenting activities of these C8-substituted guanosines appear to be due to their induction of IFN (Thompson, R. A., et al. 1990. cited supra). Recently, a 5′ triphosphorylated thymidine produced by a mycobacterium was found to be mitogenic for a subset of human γδ T cells (Constant, P., F. Davodeau, M. A. Peyrat, Y. Poquet, G. Puzo, M. Bonneville, and J. J. Fournie. 1994. “Stimulation of human γδ T cells by nonpeptidic mycobacterial ligands” Science 264:267). This report indicated the possibility that the immune system may have evolved ways to preferentially respond to microbial nucleic acids.
- Several observations suggest that certain DNA structures may also have the potential to activate lymphocytes. For example, Bell et al. reported that nucleosomal protein-DNA complexes (but not naked DNA) in spleen cell supernatants caused B cell proliferation and immunoglobulin secretion (Bell, D. A., B. Morrison, and P. VandenBygaart. 1990. “Immunogenic DNA-related factors”.J. Clin. Invest. 85:1487). In other cases, naked DNA has been reported to have immune effects. For example, Messina et al. have recently reported that 260 to 800 bp fragments of poly (dG)*(dC) and poly (dG*dC) were mitogenic for B cells (Messina, J. P., G. S. Gilkeson, and D. S. Pisetsky. 1993. “The influence of DNA structure on the in vitro stimulation of murine lymphocytes by natural and synthetic polynucleotide antigens”. Cell. Immunol 147:148). Tokunaga, et al. have reported that dG*dC induces γ-IFN and NK activity (Tokunaga, S. Yamamoto, and K Namba. 1988. “A synthetic single-stranded DNA, poly(dG, dC), induces interferon-α/β and −γ, augments natural killer activity, and suppresses tumor growth” Jpn. J. Cancer Res. 79:682). Aside from such artificial homopolymer sequences, Pisetsky et al. reported that pure mammalian DNA has no detectable immune effects, but that DNA from certain bacteria induces B cell activation and immunoglobulin secretion (Messina, J. P., G. S. Gilkeson, and D. S. Pisetsky. 1991. “Stimulation of in vitro murine lymphocyte proliferation by bacterial DNA”. J. Immunol. 147:1759). Assuming that these data did not result from some unusual contaminant, these studies suggested that a particular structure or other characteristic of bacterial DNA renders it capable of triggering B cell activation. Investigations of mycobacterial DNA sequences have demonstrated that ODN which contain certain palindrome sequences can activate NK cells (Yamamoto, S., T. Yamamoto, T. Kataoka, E. Kuramoto, O. Yano, and T. Tokunaga. 1992. “Unique palindromic sequences in synthetic oligonucleotides are required to induce INF and augment INF-mediated natural killer activity”. J. Immunol. 148:4072; Kuramoto, E., O. Yano, Y. Kimura, M. Baba, T. Makino, S. Yamamoto, T. Yamamnoto, T. Kataoka, and T. Tokunaga 1992. “Oligonucleotide sequences required for natural killer cell activation”. Jpn. J. Cancer Res. 83:1128).
- Several phosphorothioate modified ODN have been reported to induce in vitro or in vivo B cell stimulation (Tanaka, T., C. C. Chu, and W. E. Paul. 1992. “An antisense oligonucleotide complementary to a sequence in Iγ2b increases γ2b germline transcripts, stimulates B cell DNA synthesis, and inhibits immunoglobulin secretion”.J. Exp. Med. 175:597; Branda, R. F., A. L. Moore, L. Mathews, J. J. McCormack, and G. Zon. 1993. “Immune stimulation by an antisense oligomer complementary to the rev gene of HIV-1”. Biochem. Pharmacol 45:2037; McIntyre, K. W., K. Lombard-Gillooly, J. R Perez, C. Kunsch, U. M. Sarmiento, J. D. Larigan, K. T. Landreth, and R Narayanan. 1993. “A sense phosphorothioate oligonucleotide directed to the initiation codon of transcription factor NF-κ β T65 causes sequence-specific immune stimulation”. Antisense Res. Develop. 3:309; and Pisetsky, D. S., and C. F. Reich 1993. “Stimulation of murine lymphocyte proliferation by a phosphorothioate oligonucleotide with antisense activity for herpes simplex virs”. Life Sciences 54:101). These reports do not suggest a common structural motif or sequence element in these ODN that might explain their effects.
- The CREB/ATF family of transcription factors and their role in replication
- The cAMP response element binding protein (CREB) and activating transcription factor (ATF) or CREB/ATF family of transcription factors is a ubiquitously expressed class of transcription factors of which 11 members have so far been cloned (reviewed in de Groot, R. P., and P. Sassone-Corsi: “Hormonal control of gene expression: Multiplicity and versatility of cyclic adenosine 3′,5′-monophosphate-responsive nuclear regulators”.Mol. Endocrin. 7:145, 1993; Lee, K. A. W., and N. Masson: “Transcriptional regulation by CREB and its relatives”. Biochim. Biophys. Acta 1174:221, 1993.). They all belong to the basic region/leucine zipper (bZip) class of proteins. All cells appear to express one or more CREB/ATF proteins, but the members expressed and the regulation of mRNA splicing appear to be tissue-specific. Differential splicing of activation domains can determine whether a particular CREB/ATF protein will be a transcriptional inhibitor or activator. Many CREB/ATF proteins activate viral transcription, but some splicing variants which lack the activation domain are inhibitory. CREB/ATF proteins can bind DNA as homo- or hetero-dimers through the cAMP response element, the CRE, the consensus form of which is the unmethylated sequence TGACGTC (binding is abolished if the CpG is methylated) (Iguchi-Ariga, S. M. M., and W. Schaffner: “CpG methylation of the cAMP-responsive enhancer/promoter sequence TGACGTCA abolishes specific factor binding as well as transcriptional activation”. Genes & Develop. 3:612, 1989.).
- The transcriptional activity of the CRE is increased during B cell activation (Xie, H. T. C. Chiles, and T. L. Rothstein: “Induction of CREB activity via the surface Ig receptor of B cells”.J. Immunol. 151:880, 1993.). CREB/ATF proteins appear to regulate the expression of multiple genes through the CRE including immunologically important genes such as fos, jun B, Rb-1, IL-6, IL-1 (Tsukada, J., K Saito, W. R. Waterman, A. C. Webb, and P. E. Auron: “Transcription factors NF-IL6 and CREB recognize a common essential site in the human prointerleukin 1β gene”. Mol. Cell. Biol. 14:7285, 1994; Gray, G. D., O. M. Hernandez, D. Hebel, M. Root, J. M. Pow-Sang, and E. Wickstrom: “Antisense DNA inhibition of tumor growth induced by c-Ha-ras oncogene in nude mice”. Cancer Res. 53:577, 1993), IFN-β (Du, W., and T. Maniatis: “An ATF/CREB binding site protein is required for virus induction of the human interferon B gene”. Proc. Natl. Acad. Sci. USA 89:2150, 1992), TGF-β1 (Asiedu, C. K., L. Scott, R. K. Assoian, M. Ehrlich: “Binding of AP-1/CREB proteins and of MDBP to contiguous sites downstream of the human TGF-B1 gene”. Biochim. Biophys. Acta 1219:55, 1994.), TGF-β2, class II MHC (Cox, P. M., and C. R Goding: “An ATF/CREB binding motif is required for aberrant constitutive expression of the MHC class II DRa promoter and activation by SV40 T-antigen”. Nucl. Acids Res. 20:4881, 1992.), E-selectin, GM-CSF, CD-8α, the germline Igα constant region gene, the TCR Vβ gene, and the proliferating cell nuclear antigen (Huang, D., P. M. Shipman-Appasamy, D. J. Orten, S. H. Hinrichs, and M. B. Prystowsky: “Promoter activity of the proliferating-cell nuclear antigen gene is associated with inducible CRE-binding proteins in interleukin 2-stimulated T lymphocytes”. Mol. Cell. Biol. 14:4233, 1994.). In addition to activation through the cAMP pathway, CREB can also mediate transcriptional responses to changes in intracellular Ca++concentration (Sheng, M., G. McFadden, and M. E. Greenberg: “Membrane depolarization and calcium induce c-fos transcription via phosphorylation of transcription factor CREB”. Neuron 4:571, 1990).
- The role of protein-protein interactions in transcriptional activation by CREB/ATF proteins appears to be extremely important. Activation of CREB through the cyclic AMP pathway requires protein kinase A (PKA), which phosphorylates CREB341 on ser 133 and allows it to bind to a recently cloned protein, CBP (Kwok, R. P. S., J. R. Lundblad, J. C. Chrivia, J. P. Richards, H. P. Bachiinger, R. G. Brennan, S. G. E. Roberts, M. K Green, and RH. Goodman: “Nuclear protein CBP is a coactivator for the transcription factor CREB”. Nature 370:223, 1994; Arias, J., A. S. Alberts, P. Brindle, F. X. Claret, T. Smea, M. Karin, J. Feramisco, and M. Montrniny: “Activation of cAMP and mitogen responsive genes relies on a common nuclear factor”. Nature 370:226, 1994.). CBP in turn interacts with the basal transcription factor TFIIB causing increased transcription. CREB also has been reported to interact with dTAFII 110, a TATA binding protein-associated factor whose binding may regulate transcription (Ferreri, K., G. Gill, and M. Montminy: “The cAMP-regulated transcription factor CREB interacts with a component of the TFIID complex”. Proc. Natl. Acad. Sci. USA 91:1210, 1994.). In addition to these interactions, CREB/ATF proteins can specifically bind multiple other nuclear factors (Hoeffler, J. P., J. W. Lustbader, and C.-Y. Chen: “Identification of multiple nuclear factors that interact with cyclic adenosine 3′,5′-monophosphate response element-binding protein and activating transcription factor-2 by protein-protein interactions”. Mol. Endocrinol 5:256, 1991) but the biologic significance of most of these interactions is unknown. CREB is normally thought to bind DNA either as a homodimer or as a heterodimer with several other proteins. Surprisingly, CREB monomers constitutively activate transcription (Krajewski, W., and K. A. W. Lee: “A monomeric derivative of the cellular transcription factor CREB functions as a constitutive activator”. Mol. Cell. Biol. 14:7204, 1994.).
- Aside from their critical role in regulating cellular transcription, it has recently been shown that CREB/ATF proteins are subverted by some infectious viruses and retroviruses, which require them for viral replication. For example, the cytomegalovirus immediate early promoter, one of the strongest known mammalian promoters, contains eleven copies of the CRE which are essential for promoter function (Chang, Y.-N., S. Crawford, J. Stall, D. R Rawlins, K.-T. Jeang, and G. S. Hayward: “The palindromic series I repeats in the simian cytomegalovirus major immediate-early promoter behave as both strong basal enhancers and cyclic AMP response elements”.J. Virol. 64:264, 1990). At least some of the transcriptional activating effects of the adenovirus E1A protein, which induces many promoters, are due to its binding to the DNA binding domain of the CREB/ATF protein, ATF-2, which mediates E1A inducible transcription activation (Liu, F., and M. R. Green: “Promoter targeting by adenovirus E1a through interaction with different cellular DNA-binding domains”. Nature 368:520, 1994). It has also been suggested that E1A binds to the CREB-binding protein, CBP (Arany, Z., W. R. Sellers, D. M. Livingston, and R. Eckner: “E1A-associated p300 and CREB-associated CBP belong to a conserved family of coactivators”. Cell 77:799, 1994). Human T lymphotropic virus-I (HTLV-1), the retrovirus which causes human T cell leukemia and tropical spastic paresis, also requires CREB/ATF proteins for replication. In this case, the retrovirus produces a protein, Tax, which binds to CREB/ATF proteins and redirects them from their normal cellular binding sites to different DNA sequences (flanked by G- and C-rich sequences) present within the HTLV transcriptional enhancer (Paca-Uccaralertkun, S., L.-J. Zhao, N. Adya, J. V. Cross, B. R. Cullen, I. M. Boros, and C.-Z. Giam: “In vitro selection of DNA elements highly responsive to the human T-cell lymphotropic virus type I transcriptional activator, Tax”. Mol. Cell. Biol. 14:456, 1994; Adya, N., L.-J. Zhao, W. Huang, 1. Boros, and C.-Z. Giam: “Expansion of CREB's DNA recognition specificity by Tax results from interaction with Ala-Ala-Arg at positions 282-284 near the conserved DNA-binding domain of CREB”. Proc. Natl. Acad. Sci. USA 91:5642, 1994).
- The instant invention is based on the finding that certain oligonucleotides containing unmethylated cytosine-guanine (CpG) dinucleotides activate lymphocytes as evidenced by in vitro and in vivo data Based on this finding, the invention features, in one aspect, novel immunostimulatory oligonucleotide compositions.
- In a preferred embodiment, an immunostimulatory oligonucleotide is synthetic, between 2 to 100 base pairs in size and contains a consensus mitogenic CpG motif represented by the formula:
- 5′X1X2CGX3X4 3′
- wherein C and G are unmethylated, X1, X2, X3 and X4 are nucleotides and a GCG trinucleotide sequence is not present at or near the 5′ and 3′ termini.
- For facilitating uptake into cells, CpG containing immunostimulatory oligonucleotides are preferably in the range of 8 to 40 base pairs in size. Prolonged immunostimulation can be obtained using stabilized oligonucleotides, particularly phosphorothioate stabilized oligonucleotides. Enhanced immunostimulatory activity has been observed where X1X2 is the dinucleotide GpA and/or X3X4 is the dinucleotide is most preferably TpC or also TpT. Further enhanced immunostimulatory activity has been observed where the consensus motif X1X2CGX3X4 is preceded on the 5′ end by a T.
- In a second aspect, the invention features useful methods, which are based on the immunostimulatory activity of the oligonucleotides. For example, lymphocytes can either be obtained from a subject and stimulated ex viva upon contact with an appropriate oligonucleotide; or a non-methylated CpG containing oligonucleotide can be administered to a subject to facilitate in vivo activation of a subject's lymphocytes. Activated lymphocytes, stimulated by the methods described herein (e.g. either ex vivo or in vivo), can boost a subject's immune response. The immunostimulatory oligonucleotides can therefore be used to treat, prevent or ameliorate an immune system deficiency (e.g., a tumor or cancer or a viral, fungal, bacterial or parasitic infection in a subject. In addition, immunostimulatory oligonucleotides can also be administered as a vaccine adjuvant, to stimulate a subject's response to a vaccine. Further, the ability of immunostimulatory cells to induce leukemic cells to enter the cell cycle, suggests a utility for treating leukemia by increasing the sensitivity of chronic leukemia cells and then administering conventional ablative chemotherapy.
- In a third aspect, the invention features neutral oligonucleotides (i.e. oligonucleotide that do not contain an unmethylated CpG or which contain a methylated CpG dinucleotide). In a preferred embodiment, a neutralizing oligonucleotide is complementary to an immunostimulatory sequence, but contains a methylated instead of an unmethylated CpG dinucleotide sequence and therefore can compete for binding with umethylated CpG containing oligonucleotides. In a preferred embodiment, the methylation occurs at one or more of the four carbons and two nitrogens comprising the cytosine six member ring or at one or more of the five carbons and four nitrogens comprising the guanine nine member double ring. 5′ methyl cytosine is a preferred methylated CpG.
- In a fourth aspect, the invention features useful methods using the neutral oligonucleotides. For example, in vivo administration of neutral oligonucleotides should prove useful for treating diseases such as systemic lupus erythematosus, sepsis and autoimmune diseases, which are caused or exacerbated by the presence of unmethylated CpG dimers in a subject In addition, methylation CpG containing antisense oligonucleotides or oligonucleotide probes would not initiate an immune reaction when administered to a subject in vivo and therefore would be safer than corresponding unmethylated oligonucleotides.
- In a fifth aspect, the invention features immunoinhibitory oligonucleotides, which are capable of interfering with the activity of viral or cellular transcription factors. In a preferred embodiment, immunoinhibitory oligonucleotides are between 2 to 100 base pairs in size and contain a consensus immunoinhibitory CpG motif represented by the formula:
- 5′GCGXnGCG3′
- wherein X=a nucleotide and n=in the range of 0-50. In a preferred embodiment, X is a pyrimidine.
- For facilitating uptake into cells, immunoinhibitory oligonucleotides are preferably in the range of 8 to 40 base pairs in size. Prolonged immunostimulation can be obtained using stabilized oligonucleotides, particularly phosphorothioate stabilized oligonucleotides.
- In a sixth and final aspect, the invention features various uses for immunoinhibitory oligonucleotides. Immunoinhibitory oligonucleotides have antiviral activity, independent of any antisense effect due to complementarity between the oligonucleotide and the viral sequence being targeted.
- Other features and advantages of the invention will become more apparent from the following detailed description and claims.
- Definitions
- As used herein, the following terms and phrases shall have the meanings set forth below:
- An “oligonucleotide” or “oligo” shall mean multiple nucleotides (i.e. molecules comprising a sugar (e.g. ribose or deoxyribose) linked to a phosphate group and to an exchangeable organic base, which is either a substituted pyrimidine (e.g. cytosine (C), thymine (T or uracil (U)) or a substituted purine (e.g. adenine (A) or guanine (G)). The term “oligonucleotide” as used herein refers to both oligoribonucleotides (ORNs) and oligodeoxyribonucleotides (ODNs). The term “oligonucleotide” shall also include oligonucleosides (i.e. an oligonucleotide minus the phosphate) and any other organic base containing polymer. Oligonucleotides can be obtained from existing nucleic acid sources (e.g. genomic or cDNA), but are preferably synthetic (e.g. produced by oligonucleotide synthesis).
- A “stabilized oligonuclectide” shall mean an oligonucleotide that is relatively resistant to in vivo degradation (e.g. via an exo- or endo-nuclease). Preferred stabilized oligonucleotides of the instant invention have a modified phosphate backbone. Especially preferred oligonucleotides have a phosphorothioate modified phosphate backbone (i.e. at least one of the phosphate oxygens is replaced by sulfur). Other stabilized oligonucleotides include: nonionic DNA analogs, such as alkyl- and aryl-phosphonates (in which the charged phosphonate oxygen is replaced by an alkyl or aryl group), phosphodiester and alkylphosphotriesters, in which the charged oxygen moiety is alkylated. Oligonucleotides which contain a diol, such as tetraethyleneglycol or hexaethyleneglycol, at either or both termini have also been shown to be substantially resistant to nuclease degradation.
- An “immunostimulatory oligonucleotide”, “immunostimulatory CpG containing oligonucleotide”, or “CpG. ODN” refer to an oligonucleotide, which contains a cytosine, guanine dinucleotide sequence and stimulates (e.g. has a mitogenic effect) on vertebrate lymphocyte. Preferred immunostimulatory oligonucleotides are between 2 to 100 base pairs in size and contain a consensus mitogenic CpG motif represented by the formula:
- 5′X1X2CGX3X4 3′
- wherein C and G are unmethylated, X1, X2, X3 and X4 are nucleotides and a GCG trinucleotide sequence is not present at or near the 5′ and 3′ termini.
- Preferably the immunostimulatory oligonucleotides range between 8 to 40 base pairs in size. In addition, the immunostimulatory oligonucleotides are preferably stabilized oligonucleotides, particularly preferred are phosphorothioate stabilized oligonucleotides. In one preferred embodiment, X1X2 is the dinucleotide GpA. In another preferred embodiment, X3X4 is preferably the dinucleotide TpC or also TpT. In a particularly preferred embodiment, the consensus motif X1X2CGX3X4 is preceded on the 5′ end by a T. Particularly preferred consensus sequences are TGACGTT or TGACGTC.
- A “neutral oligonucleotide” refers to an oligonucleotide that does not contain an unmethylated CpG or an oligonucleotide which contains a methylated CpG dinucleotide. In a preferred embodiment, a neutralizing oligonucleotide is complementary to an immunostimulatory sequence, but contains a methylated instead of an unmethylated CpG dinucleotide sequence and therefore can compete for binding with unmethylated CpG containing oligonucleotides. In a preferred embodiment, the methylation occurs at one or more of the four carbons and two nitrogens comprising the cytosine six member ring or at one or more of the five carbons and four nitrogens comprising the guanine nine member double ring. 5′ methyl cytosine is a preferred methylated CpG.
- An “immunoinhibitory oligonucleotide” or “immunoinhibitory CpG containing oligonucleotide” is an oligonucleotide that. Preferable immunoinhibitory oligonucleotides are between 2 to 100 base pairs in size and can be represented by the formula:
- 5′GCGXnGCG3′
- wherein X=a nucleotide and n=in the range of 0-50. In a preferred embodiment, X is a pyrimidine.
- For facilitating uptake into cells, immunoinhibitory oligonucleotides are preferably in the range of 8 to 40 base pairs in size. Prolonged immunostimulation can be obtained using stabilized oligonucleotides, particularly phosphorothioate stabilized
- “Palindromic sequence” shall mean an inverted repeat (i.e. a sequence such as ABCDEE′D′C′B′A′ in which A and A′ are bases capable of forming the usual Watson-Crick base pairs. In vivo, such sequences may form double stranded structures.
- An “oligonucleotide delivery complex” shall mean an oligonucleotide associated with (e.g. ionically or covalently bound to; or encapsulated within) a targeting means (e.g. a molecule that results in higher affinity binding to target cell (e.g. B-cel and natural killer (NK) cell) surfaces and/or increased cellular uptake by target cells). Examples of oligonucleotide delivery complexes include oligonucleotides associated with: a sterol (e.g. cholesterol), a lipid (e.g. a cationic lipid, virosome or liposome), or a target cell specific binding agent (e.g. a ligand recognized by target cell specific receptor). Preferred complexes must be sufficiently stable in vivo to prevent significant uncoupling prior to internalition by the target cell. However, the complex should be cleavable under appropriate conditions within the cell so that the oligonucleotide is released in a functional form.
- An “immune system deficiency” shall mean a disease or disorder in which the subject's immune system is not functioning in normal capacity or in which it would be useful to boost a subject's immune response for example to eliminate a tumor or cancer (e.g. tumors of the brain, lung (e.g. small cell and non-small cell), ovary, breast, prostate, colon, as well as other carcinomas and sarcomas) or a viral (e.g. HIV, herpes), fungal (e.g.Candida sp.), bacterial or parasitic (e.g. Leishmania, Toxoplasma) infection in a subject.
- A “disease associated with immune system activation” shall mean a disease or condition caused or exacerbated by activation of the subject's immune system. Examples include systemic lupus erythematosus, sepsis and autoimmune diseases such as rheumatoid arthritis and multiple sclerosis.
- A “subject” shall mean a human or vertebrate animal including a dog, cat, horse, cow, pig, sheep, goat, chicken, monkey, rat, mouse, etc.
- Certain Unmethblated CpG Containing Oligos Have B Cell Stimulatorv Activity As Shown in vitro and in vivo
- In the course of investigating the lymphocyte stimulatory effects of two antisense oligonucleotides specific for endogenous retroviral sequences, using protocols described in the attached Examples 1 and 2, it was surprisingly found that two out of twenty-four “controls” (including various scrambled, sense, and mismatch controls for a panel of “antisense” ODN) also mediated B cell activation and IgM secretion, while the other “controls” had no effect.
- Two observations suggested that the mechanism of this B cell activation by the “control” ODN may not involve antisense effects 1) comparison of vertebrate DNA sequences listed in GenBank showed no greater homology than that seen with non-stimulatory ODN and 2) the two controls showed no hybridization to Northern blots with 10 μg of spleen poly A+ RNA. Resynthesis of these ODN on a different synthesizer or extensive purification by polyacrylamide gel electrophoresis or high pressure liquid chromatography gave identical stimulation, eliminating the possibility of an impurity. Similar stimulation was seen using B cells from C3H/HeJ mice, eliminating the possibility that lipopolysaccharide (LPS) contamination could account for the results.
- The fact that two “control” ODN caused B cell activation similar to that of the two “antisense” ODN raised the possibility that all four ODN were stimulating B cells through some non-antisense mechanism involving a sequence motif that was absent in all of the other nonstimulatory control ODN. In comparing these sequences, it was discovered that all of the four stimulatory ODN contained ODN dinucleotides that were in a different sequence context from the nonstimulatory control.
- To determine whether the CpG motif present in the stimulatory ODN was responsible for the observed stimulation, over 300 ODN ranging in length from 5 to 42 bases that contained methylated, unmethylated, or no CpG dinucleotides in various sequence contexts were synthesized. These ODNs, including the two original “controls” (ODN 1 and 2) and two originally synthesized as “antisense” (ODN 3D and 3M; Krieg A. M.J. Immunol. 143:2448 (1989)), were then examined for in vitro effects on spleen cells (representative sequences are listed in Table 1). Several ODN that contained CpG dinucleotides induced B cell activation and IgM secretion; the magnitude of this stimulation typically could be increased by adding more CpG dinucleotides (Table 1; compare ODN 2 to 2a or 3D to 3Da and 3 Db). Stimulation did not appear to result from an antisense mechanism or impurity. ODN caused no detectable activation of γδ or other T cell populations.
- Mitogenic ODN sequences uniformly became nonstimulatory if the CpG dinucleotide was mutated (Table 1; compare ODN 1 to 1a; 3D to. 3Dc; 3M to 3Ma; and 4 to 4a) or if the cytosine of the CpG dinucleotide was replaced by 5-methylcytosine (Table 1; ODN 1b, 2b, 2c, 3Dd, and 3 Mb). In contrast, methylation of other cytosines did not reduce ODN activity (ODN 1c, 2d, 3De and 3Mc). These data confirmed that a CpG motif is the essential element present in ODN that activate B cells.
- In the course of these studies, it became clear that the bases flanking the CpG dinucleotide played an important role in determining the B cell activation induced by an ODN. The optimal stimulatory motif was determined to consist of a CpG flanked by two 5′ purines (preferably a GpA dinucleotide) and two 3′ pyrimidines (preferably a TpT or TpC dinucleotide). Mutations of ODN to bring the CpG motif closer to this ideal improved stimulation (e.g. compare ODN 2 to 2e; 3M to 3Md) while mutations that disturbed the motif reduced stimulation (e.g. compare ODN 3D to 3Df; 4 to 4b, 4c and 4d). On the other hand, mutations outside the CpG motif did not reduce stimulation (e.g. compare ODN 1 to 1d; 3D to 3Dg; 3M to 3Me).
- Of those tested, ODNs shorter than 8 bases were non-stimulatory (e.g. ODN 4e). Among the forty-eight 8 base ODN tested, the most stimulatory sequence identified was TCAACGTT (ODN 4) which contains the self complementary “palindrome” AACGTT. In further optimizing this motif, it was found that ODN containing Gs at both ends showed increased stimulation, particularly if the the ODN were rendered nuclease resistant by phosphorothioate modification of the terminal internucleotide linkages. ODN 1585 (5′ GGGGTCAACGTTCAGGGGGG 3′ (SEQ ID NO:1)), in which the first two and last five internucleotide linkages are phosphorothioate modified caused an average 25.4 fold increase in mouse spleen cell proliferation compared to an average 3.2 fold increase in proliferation induced by ODN 1638, which has the same sequence as ODN 1585 except that the 10 Gs at the two ends are replaced by 10 As. The effect of the G-rich ends is cis; addition of an ODN with poly G ends but no CpG motif to cells along with 1638 gave no increased proliferation.
- Other octamer ODN containing a 6 base palindrome with a TpC dinucleotide at the 5′ end were also active if they were close to the optimal motif (e.g. ODN 4b, 4c). Other dinucleotides at the 5′ end gave reduced stimulation (eg ODN 4f; all sixteen possible dinucleotides were tested). The presence of a 3′ dinucleotide was insufficient to compensate for the lack of a 5′ dinucleotide (eg. ODN. 4g). Disruption of the palindrome eliminated stimulation in octamer ODN (eg., ODN 4h), but palindromes were not required in longer ODN.
TABLE 1 Oligonucleotide Stimulation of B Cells Stimulation Index′ ODN Sequence (5′ to 3′)† 3H Uridine IgM Production 1 (SEQ ID NO: 2) GCTAGACGTTAGCGT 6.1 ± 0.8 17.9 ± 3.6 1a (SEQ ID NO: 3) ......T........ 1.2 ± 0.2 1.7 ± 0.5 1b (SEQ ID NO: 4) ......Z........ 1.2 ± 0.1 1.8 ± 0.0 1c (SEQ ID NO: 5) ............Z.. 10.3 ± 4.4 9.5 ± 1.8 1d (SEQ ID NO: 6) ..AT......GAGC. 13.0 ± 2.3 18.3 ± 7.5 2 (SEQ ID NO: 7) ATGGAAGGTCCAGCGTTCTC 2.9 ± 0.2 13.6 ± 2.0 2a (SEQ ID NO: 8) ..C..CTC..G......... 7.7 ± 0.8 24.2 ± 3.2 2b (SEQ ID NO: 9) ..Z..CTC.ZG..Z...... 1.6 ± 0.5 2.8 ± 2.2 2c (SEQ ID NO: 10) ..Z..CTC..G......... 3.1 ± 0.6 7.3 ± 1.4 2d (SEQ ID NO: 11) ..C..CTC..G......Z.. 7.4 ± 1.4 27.7 ± 5.4 2e (SEQ ID NO: 12) ............A....... 5.6 ± 2.0 ND 3D (SEQ ID NO: 13) GAGAACGCTGGACCTTCCAT 4.9 ± 0.5 19.9 ± 3.6 3Da (SEQ ID NO: 14) .........C.......... 6.6 ± 1.5 33.9 ± 6.8 3Db (SEQ ID NO: 15) .........C.......G.. 10.1 ± 2.8 25.4 ± 0.8 3Dc (SEQ ID NO: 16) ...C.A.............. 1.0 ± 0.1 1.2 ± 0.5 3Dd (SEQ ID NO: 17) .....Z.............. 1.2 ± 0.2 1.0 ± 0.4 3De (SEQ ID NO: 18) .............Z...... 4.4 ± 1.2 18.8 ± 4.4 3Df (SEQ ID NO: 19) .......A............ 1.6 ± 0.1 7.7 ± 0.4 3Dg (SEQ ID NO: 20) .........CC.G.ACTG.. 6.1 ± 1.5 18.6 ± 1.5 3M (SEQ ID NO: 21) TCCATGTCGGTCCTGATGCT 4.1 ± 0.2 23.2 ± 4.9 3Ma (SEQ ID NO: 22) ......CT............ 0.9 ± 0.1 1.8 ± 0.5 3Mb (SEQ ID NO: 23) .......Z............ 1.3 ± 0.3 1.5 ± 0.6 3Mc (SEQ ID NO: 24) ...........Z........ 5.4 ± 1.5 8.5 ± 2.6 3Md (SEQ ID NO: 25) ......A..T.......... 17.2 ± 9.4 ND 3Me (SEQ ID NO: 26) ...............C..A. 3.6 ± 0.2 14.2 ± 5.2 4 TCAACGTT 6.1 ± 1.4 19.2 ± 5.2 4a ....GC.. 1.1 ± 0.2 1.5 ± 1.1 4b ...GCGC. 4.5 ± 0.2 9.6 ± 3.4 4c ...TCGA. 2.7 ± 1.0 ND 4d ..TT..AA 1.3 ± 0.2 ND 4e -....... 1.3 ± 0.2 1.1 ± 0.5 4f C....... 3.9 ± 1.4 ND 4g --......CT 1.4 ± 0.3 ND 4h .......C 1.2 ± 0.2 ND LPS 7.8 ± 2.5 4.8 ± 1.0 - The kinetics of lymphocyte activation were investigated using mouse spleen cells. When the cells were pulsed at the same time as ODN addition and harvested just four hours later, there was already a two-fold increase in3H uridine incorporation. Stimulation peaked at 12-48 hours and then decreased. After 24 hours, no intact ODN were detected, perhaps accounting for the subsequent fall in stimulation when purified B cells with or without anti-IgM (at a submitogenic dose) were cultured with CpG ODN, proliferation was found to synergistically increase about 10-fold by the two mitogens in combination after 48 hours. The magnitude of stimulation was concentration dependent and consistently exceeded that of LPS under optimal conditions for both. Oligonucleotides containing a nuclease resistant phosphorothioate backbone were approximately two hundred times more potent than unmodified oligonucleotides.
- Cell cycle analysis was used to determine the proportion of B cells activated by CpG-ODN. CpG-ODN induced cycling in more than 95% of B cells (Table 2). Splenic B lymphocytes sorted by flow cytometry into CD23−(marginal zone) and CD23+(follicular) subpopulations were equally responsive to ODN− induced stimulation, as were both resting and activated populations of B cells isolated by fractionation over Percoll gradients. These studies demonstrated that CpG-ODN induce essentially all B cells to enter the cell cycle.
TABLE 2 Cell Cycle Analysis with CpG ODN Percent of cells in Treatment G0 G1 SA + G2 + M Media 97.6 2.4 0.02 ODN 1a 95.2 4.8 0.04 ODN 1d 2.7 74.4 22.9 ODN 3Db 3.5 76.4 20.1 LPS (30 μg/ml) 17.3 70.5 12.2 - The mitogenic effects of CpG ODN on human cells, were tested on peripheral blood mononuclear cells (PBMCs) obtained from two patients with chronic lymphocytic leukemia (CLL), as described in Example 1. Control ODN containing no CpG dinucleotide sequence showed no effect on the basal proliferation of 442 cpm and 874 cpm (proliferation measured by3H thymidine incorporation) of the human cells. However, a phosphorothioate modified CpG ODN 3Md (SEQ ID NO: 25) induced increased proliferation of 7210 and 86795 cpm respectively in the two patients at a concentration of just 1 μM. Since these cells had been frozen, they may have been less responsive to the oligos than fresh cells in vivo. In addition, cells from CLL patients typically are non-proliferating, which is why traditional chemotherapy is not effective.
- Certain B cell lines such as WEHI-231 are induced to undergo growth arrest and/or apoptosis in response to crosslinking of their antigen receptor by anti-IgM (Jakway, J. P. et al., “Growth regulation of the B lymphoma cell line WEHI-231 by anti-immunoglobulin, lipopolysaccharide and other bacterial products” J. Immunol. 137: 2225 (1986); Tsubata, T., J. Wu and T. Honjo: B-cell apoptosis induced by antigen receptor crosslinking is blocked by a T-cell signal through CD40. “Nature 364: 645 (1993)). WEHI-231 cells are rescued from this growth arrest by certain stimuli such as LPS and by the CD40 ligand. ODN containing the CpG motif were also found to protect WEHI-231 from anti-IgM induced growth arrest, indicating that accessory cell populations are not required for the effect.
- To better understand the immune effects of unmethylated CpG ODN, the levels of cytokines and prostaglandins in vitro and in vivo were measured. Unlike LPS, CpG ODN were not found to induce purified macrophages to produce prostaglandin PGE2. In fact, no apparent direct effect of CpG ODN was detected on either macrophages or T cells. In vivo or in whole spleen cells, no significant increase in the following interleukins: IL-2, IL-3, IL4, or IL-10 was detected within the first six hours. However, the level of IL-6 increased strikingly within 2 hours in the serum of mice injected with CpG ODN. Increased expression of IL-12 and interferon gamma (IFN-γ) by spleen cells was also detected within the first two hours.
- To determine whether CpG ODN can cause in vivo immune stimulation, DBA/2 mice were injected once intraperitoneally with PBS or phosphorothioate CpG or non-CpG ODN at a dose of 33 mg/kg (approximately 500 μg/mouse). Pharmacokinetic studies in mice indicate that this dose of phosphorothioate gives levels of approximately 10% g/g in spleen tissue (within the effective concentration range determined from the in vitro studie's described herein) for longer than twenty-four hours (Agrawal, S. et al. (1991)Proc. Natl. Acad. Sci. USA 91:7595). Spleen cells from mice were examined twenty-four hours after ODN injection for expression of B cells surface activation markers Ly-6A/E, Bla-1, and class II MHC using three color flow cytometry and for their spontaneous proliferation using 3H thymidine: Expression of all three activation markers was significantly increased in B cells from mice injected with CpG ODN, but not from mice injected with PBS or non-CpG ODN. Spontaneous 3H thymidine incorporation was increased by 2-6 fold in spleen cells from mice injected with the stimulatory ODN compared to PBS or non-CpG ODN-injected mice. After 4 days, serum IgM levels in mice injected with CpG ODN in vivo were increased by approximately 3-fold compared to controls. Consistent with the inability of these agents to activate T cells, there was minimal change in T cell expression of the IL-2R or CD-44.
- Degradation of phophodiester ODN in serum is predominantly mediated by 3′ exonucleases, while intracellular ODN degradation is more complex, involving 5′ and 3′ exonucleases and endonucleases. Using a panel of ODN bearing the 3D sequence with varying numbers of phosphorothioate modified linkages at the 5′ and 3′ ends, it was empirically determined that two 5′ and five 3′ modified linkages are required to provide optimal stimulation with this CpG ODN.
- Unmethylated CpG Containing Oligas Have NK Cell Stimulatorv Activity
- As described in further detail in Example 4, experiments were conducted to determine whether CpG containing oligonucleotides stimulated the activity of natural killer (NK) cells in addition to B cells. As shown in Table 3, a marked induction of NK activity among spleen cells cultured with CpG ODN 1 and 3Dd was observed. In contrast, there %% as relatively no induction in effectors that had been treated with non-CpG control ODN.
TABLE 3 Induction Of NK Activity By CpG Oligodeoxynucleotides (ODN) % YAC-1 % 2C11 Specific Lysis* Specific Lysis Effector:Target Effector:Target ODN 50:1 100:1 50:1 100:1 None −1.1 −1.4 15.3 16.6 1 16.1 24.5 38.7 47.2 3Dd 17.1 27.0 37.0 40.0 non-CpG ODN −1.6 −1.7 14.8 15.4 - Neutralizing Activity of Methylated CpG Containing Oligos
- B cell mitogenicity of ODN in which cytosines in CpG motifs or elsewhere were replaced by 5-methylcytosine were tested as described in Example 1. As shown in Table 1 above, ODN containing methylated CpG motifs were non-mitogenic (Table 1; ODN 1c, 2f, 3De, and 3Mc). However, methylation of cytosines other than in a CpG dinucleotide retained their stimulatory properties (Table 1, ODN 1d, 2d, 3Df, and 3Md).
- Immunoinhibitory Activit” of Oligos Containing a GCG Trinucleotide Sequence at or near both termini
- In some cases, ODN containing CpG dinucleotides that are not in the stimulatory motif described above were found to block the stimulatory effect of other mitogenic CpG ODN. Specifically the addition of an a typical CpG motif consisting of a GCG near or at the 5′ and/or 3′ end of CpG ODN actually inhibited stimulation of proliferation by other CpG motifs. Methylation or substitution of the cytosine in a GCG motif reverses this effect. By itself, a GCG motif in an ODN has a modest mitogenic effect, though far lower than that seen with the preferred CpG motif.
- Proposed Mechanisms of Action of Immunostimulatory. Neutralizing and Immunoinhibitory Oligonucleotides
- Unlike antigens that trigger B cells through their surface Ig receptor, CpG-ODN did not induce any detectable Ca2+ flux, changes in protein tyrosine phosphorylation, or IP 3 generation. Flow cytometry with FITC-conjugated ODN with or without a CpG motif was performed as described in Zhao, Q et al.,(Antisense Research and Development 3:53-66 (1993)), and showed equivalent membrane binding, cellular uptake, efflux, and intracellular localization. This suggests that there may not be cell membrane proteins specific for CpG ODN. Rather than acting through the cell membrane, that data suggests that unmethylated CpG containing oligonucleotides require cell uptake for activity: ODN covalently linked to a solid Teflon support were nonstimulatory, as were biotinylated ODN immobilized on either avidin beads or avidin coated petri dishes. CpG ODN conjugated to either FITC or biotin retained full mitogenic properties, indicating no steric hindrance.
- The optimal CpG motif (TGACGIT/C is identical to the CRE (cyclic AMP response element). Like the mitogenic effects of CpG ODN, binding of CREB to the CRE is abolished if the central CpG is methylated. Electrophoretic mobility shift assays were used to determine whether CpG ODN, which are single stranded, could compete with the binding of B cell CREB/ATF proteins to their normal binding site, the doublestranded CRE. Competition assays demonstrated that single stranded ODN containing CpG motifs could completely compete the binding of CREB to its binding site, while ODN without CpG motifs could not. These data support the conclusion that CpG ODN exert their mitogenic effects through interacting with one or more B cell CREB/ATF proteins in some way. Conversely, the presence of GCG sequences or other a typical CPG motifs near the 5′ and/or 3′ ends of ODN likely interact with CREB/ATF proteins in away that does not cause activation, and may even prevent it.
- The stimulatory CpG motif is common in microbial genomic DNA, but quite rare in vertebrate DNA. In addition, bacterial DNA has been reported to induce B cell proliferation and immunoglobulin (Ig) production, while mammalian DNA does not (Messina, J. P. et al.,J. Immunol. 147:1759 (1991)). Experiments further described in Example 3, in which methylation of bacterial DNA with CpG methylase was found to abolish mitogenicity, demonstrates that the difference in CpG status is the cause of B cell stimulation by bacterial DNA. This data supports the following conclusion: that unmethylated CpG dinucleotides present within bacterial DNA are responsible for the stimulatory effects of bacterial DNA.
- Teleologically, it appears likely that lymphocyte activation by the CpG motif represents an immune defense mechanism that can thereby distinguish bacterial from host DNA. Host DNA would induce little or no lymphocyte activation due to it CpG suppression and methylation. Bacterial DNA would cause selective lymphocyte activation in infected tissues. Since the CpG pathway synergizes with B cell activation through the antigen receptor, B cells bearing antigen receptor specific for bacterial antigens would receive one activation signal through cell membrane Ig and a second signal from bacterial DNA, and would therefore tend to be preferentially activated. The interrelationship of this pathway with other pathways of B cell activation provide a physiologic mechanism employing a polyclonal antigen to induce antigen-specific responses.
- Method for Making Immunostimulatory Oligas
- For use in the instant invention, oligonucleotides can be synthesized de novo using any of a number of procedures well known in the art. For example, the P-cyanoethyl phosphoramidite method (S. L. Beaucage and M. H. Caruthers, (1981)Tet. Let. 22:1859); nucleoside H-phosphonate method (Garegg et al., (1986) Tet. Let. 27: 4051-4054; Froehler et al., (1986) Nucl. Acid Res. 14: 5399-5407; Garegg et al., (1986) Tet. Let. 27: 40554058, Gaffney et al., (1988) Tet. Let. 29:2619-2622). These chemistries can be performed by a variety of automated oligonucleotide synthesizers available in the market. Alternatively, oligonucleotides can be prepared from existing nucleic acid sequences (e.g. genomic or cDNA) using known techniques, such as those employing restriction enzymes, exonucleases or endonucleases.
- For use in vivo, oligonucieotides are preferably relatively resistant to degradation (e.g. via endo- and exo-nucleases). Oligonucleotide stabilization can be accomplished via phosphate backbone modifications. A preferred stabilized oligonucleotide has a phosphorothioate modified backbone. The pharmacokinetics of phosphorothioate ODN show that they have a systemic half-life of forty-eight hours in rodents and suggest that they may be useful for in vivo applications (Agrawal, S. et al. (1991) Proc. Natl. Acad. Sci. USA 88:7595). Phosphorothioates may be synthesized using automated techniques employing either phosphoramidate or H phosphonate chemistries. Aryl- and alkyl-phosphonates can be made e.g. (as described in U.S. Pat. No. 4,469,863); and alkylphosphotriesters (in which the charged oxygen moiety is alkylated as described in U.S. Pat. No. 5,023,243 and European Patent No. 092,574) can be prepared by automated solid phase synthesis using commercially available reagents. Methods for making other DNA backbone modifications and substitutions have been described (Uhlmann, E. and Peyman, A. (1990)Chem. Rev. 90:544; Goodchild, J. (1990) Bioconjugate Chem. 1:165).
- For administration in vivo, oligonucleotides may be associated with a molecule that results in higher affinity binding to target cell (e.g. B-cell and natural killer (NK) cell) surfaces and/or increased cellular uptake by target cells to form an “oligonucleotide delivery complex”. Oligonucleotides can be ionically, or covalently associated with appropriate molecules using techniques which are well known in the art. A variety of coupling or crosslinling agents can be used e.g. protein A, carbodiimide, and N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP). Oligonucleotides can alternatively be encapsulated in liposomes or virosomes using well-known techniques.
- The present invention is further illustrated by the following Examples which in no way should be construed as further limiting. The entire contents of all of the references (including literature references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby expressly incorporated by reference.
- Therapeutic Uses of Immunostimulatory Oligos
- Based on their immunostimulatory properties, oligonucleotides containing at least one unmethylated CpG dinucleotide can be administered to a subject in vivo to treat an “immune system deficiency”. Alternatively, oligonucleotides containing at least one unmethylated CpG dinucleotide can be contacted with lymphocytes (e.g. B cells or NK cells) obtained from a subject having an immune system deficiency ex vivo and activated lymphocytes can then be reimplanted in the subject.
- Immunostimulatory oligonucleotides can also be administered to a subject in conjunction with a vaccine, as an adjuvant, to boost a subject's immune system to effect better response from the vaccine. Preferably the unmethylated CpG dinucleotide is administered slightly before or at the same time as the vaccine.
- Preceding chemotherapy with an immunostimulatory oligonucleotide should prove useful for increasing the responsiveness of the malignant cells to subsequent chemotherapy. CpG ODN also increased natural killer cell activity in both human and murine cells. Induction of NK activity may likewise be beneficial in cancer immunotherapy.
- Therapeutic Uses for Neutral Oligonucleotides
- Oligonucleotides that are complementary to certain target sequences can be synthesized and administered to a subject in vivo. For example, antisense oligonucleotides hybridize to complementary mRNA, thereby preventing expression of a specific target gene. The sequence-specific effects of antisense oligonucleotides have made them useful research tools for the investigation of protein function. Phase I/II human trials of systemic antisense therapy are now underway for acute myelogenous leukemia and HIV.
- In addition, oligonucleotide probes (i.e. oligonucleotides with a detectable label) can be administered to a subject to detect the presence of a complementary sequence based on detection of bound label. In vivo administration and detection of oligonucleotide probes may be useful for diagnosing certain diseases that are caused or exacerbated by certain DNA sequences (e.g. systemic lupus erythematosus, sepsis and autoimmune diseases).
- Antisense oligonucleotides or oligonucleotide probes in which any or all CpG dinucleotide is methylated, would not produce an immune reaction when administered to a subject in vivo and therefore would be safer than the corresponding non-methylated CpG containing oligonucleotide.
- For use in therapy, an effective amount of an appropriate oligonucleotide alone or formulated as an oligonucleotide delivery complex can be administered to a subject by any mode allowing the oligonucleotide to be taken up by the appropriate target cells (.e.g. B-cells and NK cells). Preferred routes of administration include oral and transdermal (e.g. via a patch). Examples of other routes of administration include injection (subcutaneous, intravenous, parenteral, intraperitoneal, intrathecal, etc.). The injection can be in a bolus or a continuous infusion.
- An oligonucleotide alone or as an oligonucleotide delivery complex can be administered in conjunction with a pharmaceutically acceptable carrier. As used herein, the phrase “pharmaceutically acceptable carrier” is intended to include substances that can be coadministered with an oligonucleotide or an oligonucleotide delivery complex and allows the oligonucleotide to perform its intended function. Examples of such carriers include solutions, solvents, dispersion media, delay agents, emulsions and the like. The use of such media for pharmaceutically active substances are well known in the art. Any other conventional carrier suitable for use with the oligonucleotides falls within the scope of the instant invention.
- The language “effective amount” of an oligonucleotide refers to that amount necessary or sufficient to realize a desired biologic effect. For example, an effective amount of an oligonucleotide containing at least one methylated CpG for treating an immune system deficiency could be that amount necessary to eliminate a tumor, cancer, or bacterial, viral or fungal infection. An effective amount for use as a vaccine adjuvant could be that amount useful for boosting a subject's immune response to a vaccine. An “effective amount” of an oligonucleotide lacking a non-methylated CpG for use in treating a disease associated with immune system activation, could be that amount necessary to outcompete non-methylated CpG containing nucleotide sequences. The effective amount for any particular application can vary depending on such factors as the disease or condition being treated, the particular oligonucleotide being administered, the size of the subject, or the severity of the disease or condition. One of ordinary skill in the art can empirically determine the effective amount of a particular oligonucleotide without necessitating undue experimentation.
- The studies reported above indicate that unmethylated CpG containing oligonucleotides are directly mitogenic for lymphocytes (e.g. B cells and NK cells). Together, with the presence of these sequences in bacterial DNA, these results suggest that the underrepresentation of CpG dinucleotides in animal genomes, and the extensive methylation of cytosines present in such dinucleotides, may be explained by the existence of an immune defense mechanism that can distinguish bacterial from host DNA. Host DNA would commonly be present in many anatomic regions and areas of inflammation due to apoptosis (cell death), but generally induces little or no lymphocyte activation. However, the presence of bacterial DNA containing unmethylated CpG motifs can cause lymphocyte activation precisely in infected anatomic regions, where it is beneficial. This novel activation pathway provides a rapid alternative to T cell dependent antigen specific B cell activation. However, it is likely that B cell activation would not be totally nonspecific. B cells bearing antigen receptors specific for bacterial products could receive one activation signal through cell membrane Ig, and a second from bacterial DNA, thereby more vigorously triggering antigen specific immune responses.
- As with other immune defense mechanisms, the response to bacterial DNA could have undesirable consequences in some settings. For example, autoimmune responses to self antigens would also tend to be preferentially triggered by bacterial infections, since autoantigens could also provide a second activation signal to autoreactive B cells triggered by bacterial DNA. Indeed the induction of autoimmunity by bacterial infections is a common clinical observance. For example, the autoimmune disease systemic lupus erythematosus, which is: i) characterized by the production of anti-DNA antibodies; ii) induced by drugs which inhibit DNA methyltransferase (Cornacchia, E.J. et al., J. Clin. Invest. 92:38 (1993)); and iii) associated with reduced DNA methylation (Richardson, B., L. et al., Arth. Rheum 35:647 (1992)), is likely triggered at least in part by activation of DNA-specific B cells through stimulatory signals provided by CpG motifs, as well as by binding of bacterial DNA to antigen receptors.
- Further, sepsis, which is characterized by high morbidity and mortality due to massive and nonspecific activation of the immune system may be initiated by bacterial DNA and other products released from dying bacteria that reach concentrations sufficient to directly activate many lymphocytes.
- Lupus, sepsis and other “diseases associated with immune system activation” may be treated, prevented or ameliorated by administering to a subject oligonucleotides lacking an unmethylated CpG dinucleotide (e.g. oligonucleotides that do not include a CpG motif or oligonucleotides in which the CpG motif is methylated) to block the binding of unmethylated CpG containing nucleic acid sequences. Oligonucleotides lacking an unmethylated CpG motif can be administered alone or in conjunction with compositions that block an immune cell's reponse to other mitogenic bacterial products (e.g. LPS).
- The following serves to illustrate mechanistically how oligonucleotides containing an unmethylated CpG dinucleotide can treat, prevent or ameliorate the disease lupus. Lupus is commonly thought to be triggered by bacterial or viral infections. Such infections have been reported to stimulate the production of nonpathogenic antibodies to single stranded DNA. These antibodies likely recognize primarily bacterial sequences including unmethylated CpGs. As disease develops in lupus, the anti-DNA antibodies shift to pathogenic antibodies that are specific for double-stranded DNA. These antibodies would have increased binding for methylated CpG sequences and their production would result from a breakdown of tolerance in lupus. Alternatively, lupus may result when a patient's DNA becomes hypomethylated, thus allowing anti-DNA antibodies specific for unmethylated CpGs to bind to self DNA and trigger more widespread autoimmunity through the process referred to as “epitope spreading”.
- In either case, it may be possible to restore tolerance in lupus patients by coupling antigenic oligonucleotides to a protein carrier such as gamma globulin (IgG). Calf-thymus DNA complexed to gamma globulin has been reported to reduce anti-DNA antibody formation.
- Therapeutic Uses of Oligos Containing GCG Trinucleotide Sequences at or Near Both Termini
- Based on their interaction with CREB/ATF, oligonucleotides containing GCG trinucleotide sequences at or near both termini have antiviral activity, independent of any antisense effect due to complementarity between the oligonucleotide and the viral sequence being targeted. Based on this activity, an effective amount of inhibitory oligonucleotides can be administered to a subject to treat or prevent a viral infection.
- B cells were purified from spleens obtained from 6-12 wk old specific pathogen free DBA/2 or BXSB mice bred in the University of Iowa animal care facility; no substantial strain differences were noted) that were depleted of T cells with anti-Thy-1.2 and complement and centrifugation over lympholyte M (Cedarlane Laboratories, Hornby, Ontario, Canada) (“B cells”). B cells contained fewer than 1% CD4+ or CD8+cells. 8×104 B cells were dispensed in triplicate into 96 well microtiter plates in 100 μl RPM! containin!g 10% FBS (heat inactivated to 65° C. for 30 mL), 50 μM 2-mercaptoethanol, 100 U/ml penicillin, 100 ug/ml streptomycin, and 2 mM L-glutamate. 20 μM ODN were added at the start of culture for 20 h at 37° C., cells pulsed with 1 μCi of 3H uridine, and harvested and counted 4 hr later. Ig secreting B cells were enumerated using the ELISA spot assay after culture of whole spleen cells with ODN at 20 μM for 48 hr. Data, reported in Table 1, represent the stimulation index compared to cells cultured without ODN. Cells cultured without ODN gave 687 cpm, while cells cultured with 20 μg/ml LPS (determined by titration to be the optimal concentration) gave 99,699 cpm in this experiment. 3H thymidine incorporation assays showed similar results, but with some nonspecific inhibition by thymidine released from degraded ODN (Matson. S and A. M. Krieg (1992) Nonspecific suppression of 3H-thymidine incorporation by control oligonucleotides. Antisense Research and Development 2:325).
- For cell cycle analysis, 2×106 B cells were cultured for 48 hr. in 2 ml tissue culture medium alone, or with 30 μg/ml LPS or with the indicated phosphorothioate modified ODN at 1 μM. Cell cycle analysis was performed as described in (Darzynkiewicz, Z. et al., Proc. Natl. Acad. Sci. USA 78:2881(1981)).
- To test the mitogenic effects of CpG ODN on human cells, perpheral blood monocyte cells (PBMCs) were obtained from two patients with chronic lymphocytic leukemia (CLL), a disease in which the circulating cells are malignant B cells. Cells were cultured for 48 hrs and pulsed for 4 hours with tritiated thymidine as described above.
- Single cell suspensions from the spleens of freshly killed mice were treated with anti-Thyl, anti-CD4, and anti-CD8 and complement by the method of Leibson et al.,J. Exp. Med 154:1681 (1981)). Resting B cells (<,02% T cell contamination) were isolated from the 63-70% band of a discontinuous Percoll gradient by the procedure of DeFranco et al, J Exp. Med. 155:1523 (1982). These were cultured as described above in 30 μM ODN or 20 μg/1 ml LPS for 48 hr. The number of B cells actively secreting IgM was maximal at this time point, as determined by ELIspot assay (Klinman, D. M. et al. J. Immunol. 144:506 (1990)). In that assay, B cells were incubated for 6 hrs on anti-Ig coated microtiter plates. The Ig they produced (>99% IgM) was detected using phosphatase-labelled anti-Ig (Southern Biotechnology Associated, Birmingham, Ala.). The antibodies produced by individual B cells were visualized by addition of BCIP (Sigma Chemical Co., St. Louis Mo.) which forms an insoluble blue precipitate in the presence of phosphatase. The dilution of cells producing 20-40 spots/well was used to determine the total number of antibody-secreting B cells/sample. All assays were performed in triplicate. In some experiments, culture supernatants were assayed for IgM by ELISA, and showed similar increases in response to CpG-ODN.
- table 1
- DBA/2 B cells were cultured with no DNA or 50 μg/ml of a) Micrococcus lysodeikticus; b) NZB/N mouse spleen; and c) NFS/N mouse spleen genomic DNAs for 48 hours, then pulsed with3H thymidine for 4 hours prior to cell harvest. Duplicate DNA samples were digested with DNAse I for 30 minutes at 37 C prior to addition to cell cultures. E coli DNA also induced an 8.8 told increase in the number of IgM secreting B cells by 48 hours using the ELISA-spot assay.
- DBA/2 B cells were cultured with either no additive, 50 μg/ml LPS or the ODN 1; 1a; 4; or 4a at 20 μM. Cells were cultured and harvested at 4, 8, 24 and 48 hours. BXSB cells were cultured as in Example 1 with 5, 10, 20, 40 or 80 FM of ODN 1; 1a; 4; or 4a or LPS. In this experiment, wells with no ODN had 3833 cpm. Each experiment was performed at least three times with similar results. Standard deviations of the triplicate wells were <5%.
- 10×106 C57BL/6 spleen cells were cultured in two ml RPMI (supplemented as described for Example 1) with or without 40 μM CpG or non-CpG ODN for forty-eight hours. Cells were washed, and then used as effector cells in a short term 51 Cr release assay with YAC-1 and 2C11, two NK sensitive target cell lines (Ballas, Z. K. et al. (1993) J. Immunol. 150:17). Effector cells were added at various concentrations to 104 51Cr-labeled target cells in V-bottom microtiter plates in 0.2 ml, and incubated in 5% CO2 for 4 hr. at 37° C. Plates were then centrifuged, and an aliquot of the supernatant counted for radioactivity. Percent specific lysis was determined by calculating the ratio of the 51 Cr released in the presence of effector cells minus the 51Cr released when the target cells are cultured alone, over the total counts released after cell lysis in 2% acetic acid minus the 51Cr cpm released when the cells are cultured alone.
- Mice were weighed and injected IP with 0.25 ml of sterile PBS or the indicated phosphorothioate ODN dissolved in PBS. Twenty four hours later, spleen cells were harvested, washed, and stained for flow cytometry using phycoerythrin conjugated 6B2 to gate on B cells in conjunction with biotin conjugated anti Ly-6A/E or anti-Iad (Pharmingen, San Diego, Calif.) or anti-Bla-1 (Hardy, R. R. et al., J. Exp. Med. 159:1169 (1984). Two mice were studied for each condition and analyzed individually.
- B cells were cultured with phosphorothioate ODN with the sequence of control ODN 1a or the CpG ODN 1d and 3 Db and then either pulsed after 20 hr with3H uridine or after 44 hr with 3H thymidine before harvesting and determining cpm.
- WEHI-231 cells (5×104/well) were cultured for 1 hr. at 37 C in the presence or absence of LPS or the control ODN 1a or the CpG ODN 1d and 3 Db before addition of anti-IgM (1 μ/ml). Cells were cultured for a further 20 hr. before a 4 hr. pulse with 2 μCi/well 3H thymidine. In this experiment, cells with no ODN or anti-IgM gave 90.4×103 by addition of anti-IgM. The phosphodiester ODN shown in Table 1 gave similar protection, though with some nonspecific suppression due to ODN degradation. Each experiment was repeated at least 3 times with similar results.
- DBA/2 female mice (2 mos. old) were injected IP with 500μg CpG or control phosphorothioate ODN. At various time points after injection, the mice were bled. Two mice were studied for each time point. IL-6 was measured by Elisa, and IL-6 concentration was calculated by comparison to a standard curve generated using recombinant IL-6. The sensitivity of the assay was 10 pg/ml. Levels were undetectable after 8 hr.
- Whole cell extracts from CH12.LX B cells showed 2 retarded bands %% hen analyzed by EMSA with the CRE probe (free probe is off the bottom of the figure). The CREB/ATF protein(s) binding to the CRE were competed by the indicated amount of cold CRE, and by single-stranded CpG ODN, but not by non-CpG ODN.
- Equivalents
- Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents of the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.
-
1 27 1 20 DNA Artificial Sequence Synthetic oligonucleotide 1 ggggtcaacg ttcagggggg 20 2 15 DNA Artificial Sequence Synthetic oligonucleotide 2 gctagacgtt agcgt 15 3 15 DNA Artificial Sequence Synthetic oligonucleotide 3 gctagatgtt agcgt 15 4 15 DNA Artificial Sequence Synthetic oligonucleotide 4 gctagangtt agcgt 15 5 15 DNA Artificial Sequence Synthetic oligonucleotide 5 gctagacgtt agngt 15 6 15 DNA Artificial Sequence Synthetic oligonucleotide 6 gcatgacgtt gagct 15 7 20 DNA Artificial Sequence Synthetic oligonucleotide 7 atggaaggtc cagcgttctc 20 8 20 DNA Artificial Sequence Synthetic oligonucleotide 8 atcgactctc gagcgttctc 20 9 20 DNA Artificial Sequence Synthetic oligonucleotide 9 atngactctn gagngttctc 20 10 20 DNA Artificial Sequence Synthetic oligonucleotide 10 atngactctc gagcgttctc 20 11 20 DNA Artificial Sequence Synthetic oligonucleotide 11 atcgactctc gagcgttntc 20 12 20 DNA Artificial Sequence Synthetic oligonucleotide 12 atggaaggtc caacgttctc 20 13 20 DNA Artificial Sequence Synthetic oligonucleotide 13 gagaacgctg gaccttccat 20 14 20 DNA Artificial Sequence Synthetic oligonucleotide 14 gagaacgctc gaccttccat 20 15 20 DNA Artificial Sequence Synthetic oligonucleotide 15 gagaacgctc gaccttcgat 20 16 20 DNA Artificial Sequence Synthetic oligonucleotide 16 gagcaagctg gaccttccat 20 17 20 DNA Artificial Sequence Synthetic oligonucleotide 17 gagaangctg gaccttccat 20 18 20 DNA Artificial Sequence Synthetic oligonucleotide 18 gagaacgctg gacnttccat 20 19 20 DNA Artificial Sequence Synthetic oligonucleotide 19 gagaacgatg gaccttccat 20 20 20 DNA Artificial Sequence Synthetic oligonucleotide 20 gagaacgctc cagcactgat 20 21 20 DNA Artificial Sequence Synthetic oligonucleotide 21 tccatgtcgg tcctgatgct 20 22 20 DNA Artificial Sequence Synthetic oligonucleotide 22 tccatgctgg tcctgatgct 20 23 20 DNA Artificial Sequence Synthetic oligonucleotide 23 tccatgtngg tcctgatgct 20 24 20 DNA Artificial Sequence Synthetic oligonucleotide 24 tccatgtcgg tnctgatgct 20 25 20 DNA Artificial Sequence Synthetic oligonucleotide 25 tccatgacgt tcctgatgct 20 26 20 DNA Artificial Sequence Synthetic oligonucleotide 26 tccatgtcgg tcctgctgat 20 27 19 DNA Artificial Sequence Synthetic oligonucleotide 27 gggtcaagtc tgagggggg 19
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US6977245B2 (en) | 1999-04-12 | 2005-12-20 | The United States Of America As Represented By The Department Of Health And Human Services | Oligodeoxynucleotide and its use to induce an immune response |
EP1176966B1 (en) * | 1999-04-12 | 2013-04-03 | THE GOVERNMENT OF THE UNITED STATES OF AMERICA, as represented by THE SECRETARY, DEPARTMENT OF HEALTH AND HUMAN SERVICES | Oligodeoxynucleotide and its use to induce an immune response |
US20050002958A1 (en) * | 1999-06-29 | 2005-01-06 | Smithkline Beecham Biologicals Sa | Vaccines |
ATE378348T1 (en) * | 2000-01-14 | 2007-11-15 | Us Health | OLIGODEOXYNUCLEOTIDES AND THEIR USE FOR INDUCING AN IMMUNE RESPONSE |
EP1311288A1 (en) * | 2000-01-20 | 2003-05-21 | Ottawa Health Research Institute | Immunostimulatory nucleic acids for inducing a th2 immune response |
US7585847B2 (en) * | 2000-02-03 | 2009-09-08 | Coley Pharmaceutical Group, Inc. | Immunostimulatory nucleic acids for the treatment of asthma and allergy |
US20040131628A1 (en) * | 2000-03-08 | 2004-07-08 | Bratzler Robert L. | Nucleic acids for the treatment of disorders associated with microorganisms |
US20030129251A1 (en) | 2000-03-10 | 2003-07-10 | Gary Van Nest | Biodegradable immunomodulatory formulations and methods for use thereof |
US7129222B2 (en) * | 2000-03-10 | 2006-10-31 | Dynavax Technologies Corporation | Immunomodulatory formulations and methods for use thereof |
KR100917101B1 (en) * | 2000-08-04 | 2009-09-15 | 도요 보세키 가부시키가이샤 | Flexible metal laminate and production method thereof |
AU2001297693A1 (en) * | 2000-12-08 | 2002-09-12 | Coley Pharmaceutical Gmbh | Cpg-like nucleic acids and methods of use thereof |
CN100334228C (en) * | 2001-06-21 | 2007-08-29 | 戴纳瓦克斯技术公司 | Cimeric immunomodulatory compounds and methods of using the same |
US7666674B2 (en) | 2001-07-27 | 2010-02-23 | The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services | Use of sterically stabilized cationic liposomes to efficiently deliver CPG oligonucleotides in vivo |
WO2003020884A2 (en) * | 2001-08-14 | 2003-03-13 | The Government Of The United States Of America As Represented By The Secretary Of Health And Human Services | Method for rapid generation of mature dendritic cells |
DE60234375D1 (en) | 2001-09-14 | 2009-12-24 | Cytos Biotechnology Ag | PACKAGING IMMUNSTIMULATING CpG IN VIRUS LIKE PARTICLES: PREPARATION METHOD AND USE |
WO2003031573A2 (en) * | 2001-10-05 | 2003-04-17 | Coley Pharmaceutical Gmbh | Toll-like receptor 3 signaling agonists and antagonists |
AU2002366710A1 (en) | 2001-12-20 | 2003-07-09 | The Government Of The United States Of America As Represented By The Secretary Of The Department Of | USE OF CpG OLIGODEOXYNUCLEOTIDES TO INDUCE ANGIOGENESIS |
US8466116B2 (en) | 2001-12-20 | 2013-06-18 | The Unites States Of America As Represented By The Secretary Of The Department Of Health And Human Services | Use of CpG oligodeoxynucleotides to induce epithelial cell growth |
US7569553B2 (en) | 2002-07-03 | 2009-08-04 | Coley Pharmaceutical Group, Inc. | Nucleic acid compositions for stimulating immune responses |
US7576066B2 (en) | 2002-07-03 | 2009-08-18 | Coley Pharmaceutical Group, Inc. | Nucleic acid compositions for stimulating immune responses |
US7605138B2 (en) * | 2002-07-03 | 2009-10-20 | Coley Pharmaceutical Group, Inc. | Nucleic acid compositions for stimulating immune responses |
US8263091B2 (en) * | 2002-09-18 | 2012-09-11 | The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services | Method of treating and preventing infections in immunocompromised subjects with immunostimulatory CpG oligonucleotides |
US7537767B2 (en) * | 2003-03-26 | 2009-05-26 | Cytis Biotechnology Ag | Melan-A- carrier conjugates |
US20050013812A1 (en) * | 2003-07-14 | 2005-01-20 | Dow Steven W. | Vaccines using pattern recognition receptor-ligand:lipid complexes |
CA2536139A1 (en) * | 2003-09-25 | 2005-04-07 | Coley Pharmaceutical Group, Inc. | Nucleic acid-lipophilic conjugates |
US20050239733A1 (en) * | 2003-10-31 | 2005-10-27 | Coley Pharmaceutical Gmbh | Sequence requirements for inhibitory oligonucleotides |
WO2005047506A1 (en) | 2003-11-04 | 2005-05-26 | Geron Corporation | Rna amidates and thioamidates for rnai |
TW200533750A (en) * | 2004-02-19 | 2005-10-16 | Coley Pharm Group Inc | Immunostimulatory viral RNA oligonucleotides |
TWI235440B (en) * | 2004-03-31 | 2005-07-01 | Advanced Semiconductor Eng | Method for making leadless semiconductor package |
AU2005326144A1 (en) * | 2004-06-08 | 2006-08-03 | Coley Pharmaceutical Gmbh | Abasic oligonucleotide as carrier platform for antigen and immunostimulatory agonist and antagonist |
US20080009455A9 (en) * | 2005-02-24 | 2008-01-10 | Coley Pharmaceutical Group, Inc. | Immunostimulatory oligonucleotides |
AU2006235284A1 (en) * | 2005-04-08 | 2006-10-19 | Coley Pharmaceutical Group, Inc. | Methods for treating infectious disease exacerbated asthma |
NZ565311A (en) * | 2005-07-07 | 2009-10-30 | Pfizer | Anti-ctla-4 antibody and cpg-motif-containing synthetic oligodeoxynucleotide combination therapy for cancer treatment |
EP1924692A2 (en) * | 2005-09-16 | 2008-05-28 | Coley Pharmaceutical GmbH | Modulation of immunostimulatory properties of short interfering ribonucleic acid (sirna) by nucleotide modification |
EP1945766A2 (en) * | 2005-09-16 | 2008-07-23 | Coley Pharmaceutical GmbH | Immunostimulatory single-stranded ribonucleic acid with phosphodiester backbone |
WO2007092315A2 (en) * | 2006-02-03 | 2007-08-16 | The Regents Of The University Of California | Immunostimulation by cpg oligonucleotide-virus complexes |
JP5473336B2 (en) * | 2006-02-15 | 2014-04-16 | アディウタイド・ファーマスーティカルズ・ゲーエムベーハー | Compositions and methods relating to the formulation of oligonucleotides |
US8173219B2 (en) * | 2006-06-09 | 2012-05-08 | Georgia-Pacific Chemicals Llc | Porous fiberglass materials having reduced formaldehyde emissions |
US20090142362A1 (en) * | 2006-11-06 | 2009-06-04 | Avant Immunotherapeutics, Inc. | Peptide-based vaccine compositions to endogenous cholesteryl ester transfer protein (CETP) |
CN101878311A (en) * | 2007-08-01 | 2010-11-03 | 艾德拉药物股份有限公司 | Novel synthetic agonists of TLR9 |
EP2123758A1 (en) * | 2008-05-20 | 2009-11-25 | Wageningen Universiteit | Influenza cap-leader sequence |
TWI351288B (en) * | 2008-07-04 | 2011-11-01 | Univ Nat Pingtung Sci & Tech | Cpg dna adjuvant in avian vaccines |
GB0815675D0 (en) * | 2008-08-28 | 2008-10-08 | Mabtech Ab | Antibody secreting cell elispot |
US8552165B2 (en) * | 2008-12-09 | 2013-10-08 | Heather Davis | Immunostimulatory oligonucleotides |
BRPI0923341B8 (en) | 2008-12-09 | 2021-05-25 | Coley Pharm Group Inc | immunostimulatory oligonucleotide, vaccine comprising the same and its use |
EP2382474B1 (en) | 2009-01-20 | 2015-03-04 | Transgene SA | Soluble icam-1 as biomarker for prediction of therapeutic response |
SG2014014021A (en) | 2009-03-24 | 2014-07-30 | Transgene Sa | Biomarker for monitoring patients |
CA3033133C (en) | 2009-03-25 | 2021-11-09 | The Board Of Regents Of The University Of Texas System | Compositions for stimulation of mammalian innate immune resistance to pathogens |
NZ594896A (en) | 2009-04-17 | 2013-07-26 | Transgene Sa | Biomarker for monitoring patients |
EP2451974A2 (en) | 2009-07-08 | 2012-05-16 | Idera Pharmaceuticals, Inc. | Oligonucleotide-based compounds as inhibitors of toll-like receptors |
CA2767458A1 (en) | 2009-07-10 | 2011-01-13 | Bruce Acres | Biomarker for selecting patients and related methods |
WO2013007703A1 (en) * | 2011-07-08 | 2013-01-17 | Universität Zürich | CLASS A CpG OLIGONUCLEOTIDES FOR PREVENTION OF VIRAL INFECTION IN CATS |
EP2734210B1 (en) * | 2011-07-22 | 2017-11-22 | Abbott Laboratories | Galactooligosaccharides for preventing injury and/or promoting healing of the gastrointestinal tract |
CA2919268C (en) | 2013-07-25 | 2023-09-05 | Exicure, Inc. | Spherical nucleic acid-based constructs as immunostimulatory agents for prophylactic and therapeutic use |
PL3048170T3 (en) * | 2013-09-20 | 2020-01-31 | National Institutes Of Biomedical Innovation, Health And Nutrition | Complex containing oligonucleotide having immunopotentiating activity and use thereof |
CN112107693B (en) | 2013-12-03 | 2023-05-26 | 西北大学 | Liposome particles, method for preparing said liposome particles and use thereof |
EP3508198A1 (en) | 2014-06-04 | 2019-07-10 | Exicure, Inc. | Multivalent delivery of immune modulators by liposomal spherical nucleic acids for prophylactic or therapeutic applications |
WO2016044839A2 (en) | 2014-09-19 | 2016-03-24 | The Board Of Regents Of The University Of Texas System | Compositions and methods for treating viral infections through stimulated innate immunity in combination with antiviral compounds |
CA2968531A1 (en) | 2014-11-21 | 2016-05-26 | Northwestern University | The sequence-specific cellular uptake of spherical nucleic acid nanoparticle conjugates |
US11364304B2 (en) | 2016-08-25 | 2022-06-21 | Northwestern University | Crosslinked micellar spherical nucleic acids |
US11696954B2 (en) | 2017-04-28 | 2023-07-11 | Exicure Operating Company | Synthesis of spherical nucleic acids using lipophilic moieties |
KR20200028997A (en) | 2017-07-13 | 2020-03-17 | 노오쓰웨스턴 유니버시티 | General and direct method of preparing oligonucleotide-functionalized metal-organic framework nanoparticles |
KR102578060B1 (en) * | 2018-03-23 | 2023-09-13 | 한림대학교 산학협력단 | An anti-bacterial antibody and a use of the same |
CN111378622B (en) * | 2018-12-29 | 2022-12-02 | 华东师范大学 | Nucleic acid-encoded CAR-T cells and preparation method and application thereof |
EP4104830A1 (en) | 2021-06-16 | 2022-12-21 | Burghardt Wittig | Sequential innate and adaptive immune modulation for cancer treatment |
Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5194428A (en) * | 1986-05-23 | 1993-03-16 | Worcester Foundation For Experimental Biology | Inhibition of influenza virus replication by oligonucleotide phosphorothioates |
US5232829A (en) * | 1989-09-29 | 1993-08-03 | Hoffmann-La Roche Inc. | Detection of chlamydia trachomatis by polymerase chain reaction using biotin labelled lina primers and capture probes |
US5646126A (en) * | 1994-02-28 | 1997-07-08 | Epoch Pharmaceuticals | Sterol modified oligonucleotide duplexes having anticancer activity |
US5723335A (en) * | 1994-03-25 | 1998-03-03 | Isis Pharmaceuticals, Inc. | Immune stimulation by phosphorothioate oligonucleotide analogs |
US6030954A (en) * | 1991-09-05 | 2000-02-29 | University Of Connecticut | Targeted delivery of poly- or oligonucleotides to cells |
US6620805B1 (en) * | 1996-03-14 | 2003-09-16 | Yale University | Delivery of nucleic acids by porphyrins |
US20070224210A1 (en) * | 2002-08-19 | 2007-09-27 | Coley Pharmaceutical Group, Inc. | Immunostimulatory nucleic acids |
US20070232622A1 (en) * | 2003-06-20 | 2007-10-04 | Coley Pharmaceutical Gmbh | Small molecule toll-like receptor (TLR) antagonists |
Family Cites Families (319)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US2005A (en) * | 1841-03-16 | Improvement in the manner of constructing molds for casting butt-hinges | ||
US2006A (en) * | 1841-03-16 | Clamp for crimping leather | ||
US2004A (en) * | 1841-03-12 | Improvement in the manner of constructing and propelling steam-vessels | ||
US2007A (en) * | 1841-03-16 | Improvement in the mode of harvesting grain | ||
US2204A (en) * | 1841-07-30 | Improvement in the mode of propelling ships, boats, and other vessels | ||
US2003A (en) * | 1841-03-12 | Improvement in horizontal windivhlls | ||
US2002A (en) * | 1841-03-12 | Tor and planter for plowing | ||
US2215233A (en) | 1937-11-13 | 1940-09-17 | Simon L Ruskin | Iron compound of nucleotides and their organic hydrolytic decomposition products and method of making same |
US3627874A (en) | 1969-07-16 | 1971-12-14 | Merck & Co Inc | Vaccine preparation |
US3906092A (en) | 1971-11-26 | 1975-09-16 | Merck & Co Inc | Stimulation of antibody response |
US3911117A (en) | 1971-12-20 | 1975-10-07 | Fredrik Ender | Raw fish and iron chelated with glutamic or ribonucleic acid in a mink diet |
US3914450A (en) | 1973-04-09 | 1975-10-21 | Anheuser Busch | Concentrated extract of yeast and processes of making same |
DE2643213C2 (en) | 1976-09-25 | 1985-02-21 | Bayer Ag, 5090 Leverkusen | Process for attenuating or inactivating microorganisms |
US5023243A (en) * | 1981-10-23 | 1991-06-11 | Molecular Biosystems, Inc. | Oligonucleotide therapeutic agent and method of making same |
ES507187A0 (en) | 1981-11-16 | 1983-01-01 | Union Ind Y Agro Ganader S A U | PROCEDURE FOR OBTAINING AN ADDITIONAL HUMANIZED MILK OF NUCLEOTIDES FOR CHILD FEEDING. |
US5766920A (en) * | 1982-08-11 | 1998-06-16 | Cellcor, Inc. | Ex vivo activation of immune cells |
US4452775A (en) * | 1982-12-03 | 1984-06-05 | Syntex (U.S.A.) Inc. | Cholesterol matrix delivery system for sustained release of macromolecules |
JPS60126220A (en) * | 1983-12-09 | 1985-07-05 | Otsuka Pharmaceut Factory Inc | Nucleic acid component composition |
IT1178044B (en) | 1984-10-09 | 1987-09-03 | Polifarma Spa | PHARMACEUTICAL ACTIVE AGENT FOR THE TREATMENT OF CEREBRAL DISEASES BASED ON URIDINE |
US4741914A (en) * | 1984-11-13 | 1988-05-03 | Ajinomoto Co., Inc. | Flavor enhancing seasoning containing deodorized garlic extract and process |
US5308626A (en) * | 1985-06-28 | 1994-05-03 | Toni N. Mariani | Lymphokine activated effector cells for antibody-dependent cellular cytotoxicity (ADCC) treatment of cancer and other diseases |
JPH0669953B2 (en) | 1985-08-16 | 1994-09-07 | 日産化学工業株式会社 | Cerebrospinal system neurotrophic agent |
US4956296A (en) | 1987-06-19 | 1990-09-11 | Genex Corporation | Cloned streptococcal genes encoding protein G and their use to construct recombinant microorganisms to produce protein G |
JPS62275667A (en) * | 1986-05-22 | 1987-11-30 | Ajinomoto Co Inc | Production of seasoning with good body or food with enhanced good body |
US4806463A (en) * | 1986-05-23 | 1989-02-21 | Worcester Foundation For Experimental Biology | Inhibition of HTLV-III by exogenous oligonucleotides |
US5075109A (en) * | 1986-10-24 | 1991-12-24 | Southern Research Institute | Method of potentiating an immune response |
US5276019A (en) * | 1987-03-25 | 1994-01-04 | The United States Of America As Represented By The Department Of Health And Human Services | Inhibitors for replication of retroviruses and for the expression of oncogene products |
US6338853B1 (en) * | 1987-04-23 | 2002-01-15 | Jean-Claude Bystryn | Anti-cancer vaccine |
US4963387A (en) | 1987-05-20 | 1990-10-16 | Chugai Seiyaku Kabushiki Kaisha | Salt substitute and foodstuffs containing same |
ES2007350A6 (en) | 1987-05-29 | 1989-06-16 | Ganadera Union Ind Agro | Food products enriched with nucleosides and/or nucleotides and preparation thereof. |
CA1339596C (en) | 1987-08-07 | 1997-12-23 | New England Medical Center Hospitals, Inc. | Viral expression inhibitors |
GB2216416B (en) | 1988-03-11 | 1992-06-24 | Sandoz Ltd | Nucleobase source for the stimulation of the immune system |
US5268365A (en) | 1988-03-11 | 1993-12-07 | Rudolph Frederick B | Nucleotides, nucleosides, and nucleobases in immune function restoration enhancement or maintenance |
CA1336174C (en) * | 1988-07-22 | 1995-07-04 | Ronald Peter Potman | Method for the preparation of a yeast extract said yeast extract, its use as a food flavour and a food composition comprising the yeast extract |
NZ230747A (en) | 1988-09-30 | 1992-05-26 | Bror Morein | Immunomodulating matrix comprising a complex of at least one lipid and at least one saponin; certain glycosylated triterpenoid saponins derived from quillaja saponaria molina |
US5004810A (en) * | 1988-09-30 | 1991-04-02 | Schering Corporation | Antiviral oligomers |
US5231085A (en) * | 1988-10-31 | 1993-07-27 | Sandoz Ltd. | Compositions and methods for the enhancement of host defense mechanisms |
US5087617A (en) * | 1989-02-15 | 1992-02-11 | Board Of Regents, The University Of Texas System | Methods and compositions for treatment of cancer using oligonucleotides |
US6214804B1 (en) * | 1989-03-21 | 2001-04-10 | Vical Incorporated | Induction of a protective immune response in a mammal by injecting a DNA sequence |
US4958013A (en) | 1989-06-06 | 1990-09-18 | Northwestern University | Cholesteryl modified oligonucleotides |
US5399346A (en) * | 1989-06-14 | 1995-03-21 | The United States Of America As Represented By The Department Of Health And Human Services | Gene therapy |
US4981684A (en) * | 1989-10-24 | 1991-01-01 | Coopers Animal Health Limited | Formation of adjuvant complexes |
US5178860A (en) * | 1989-09-01 | 1993-01-12 | Coopers Animal Health Limited | Adjuvant complexes and vaccine made therefrom |
US5786189A (en) * | 1989-11-29 | 1998-07-28 | Smithkline Beecham Biologicals (S.A.) | Vaccine |
US5457189A (en) * | 1989-12-04 | 1995-10-10 | Isis Pharmaceuticals | Antisense oligonucleotide inhibition of papillomavirus |
US5506212A (en) * | 1990-01-11 | 1996-04-09 | Isis Pharmaceuticals, Inc. | Oligonucleotides with substantially chirally pure phosphorothioate linkages |
US5212295A (en) * | 1990-01-11 | 1993-05-18 | Isis Pharmaceuticals | Monomers for preparation of oligonucleotides having chiral phosphorus linkages |
US5514577A (en) * | 1990-02-26 | 1996-05-07 | Isis Pharmaceuticals, Inc. | Oligonucleotide therapies for modulating the effects of herpes viruses |
US5248670A (en) * | 1990-02-26 | 1993-09-28 | Isis Pharmaceuticals, Inc. | Antisense oligonucleotides for inhibiting herpesviruses |
EP0468520A3 (en) | 1990-07-27 | 1992-07-01 | Mitsui Toatsu Chemicals, Inc. | Immunostimulatory remedies containing palindromic dna sequences |
US5234811A (en) | 1991-09-27 | 1993-08-10 | The Scripps Research Institute | Assay for a new gaucher disease mutation |
US6042838A (en) * | 1991-02-15 | 2000-03-28 | Uab Research Foundation | immunogenic compositions for mucosal administration of pneumococcal surface protein A (PspA) |
US6022853A (en) * | 1991-08-30 | 2000-02-08 | Creative Biomolecules, Inc. | Morphogen-enriched dietary composition |
US5858784A (en) * | 1991-12-17 | 1999-01-12 | The Regents Of The University Of California | Expression of cloned genes in the lung by aerosol- and liposome-based delivery |
US5756353A (en) * | 1991-12-17 | 1998-05-26 | The Regents Of The University Of California | Expression of cloned genes in the lung by aerosol-and liposome-based delivery |
ES2174845T3 (en) | 1992-02-04 | 2002-11-16 | Chiron Corp | HEPATITIS THERAPEUTICS. |
US5643578A (en) * | 1992-03-23 | 1997-07-01 | University Of Massachusetts Medical Center | Immunization by inoculation of DNA transcription unit |
US6498147B2 (en) | 1992-05-22 | 2002-12-24 | The Scripps Research Institute | Suppression of nuclear factor-κb dependent processes using oligonucleotides |
JPH067114A (en) | 1992-06-24 | 1994-01-18 | Kuroda Munetada | Production of aqueous of nucleic acid |
EP0654077A4 (en) * | 1992-07-17 | 1996-03-13 | Ribozyme Pharm Inc | Method and reagent for treatment of animal diseases. |
US5585479A (en) | 1992-07-24 | 1996-12-17 | The United States Of America As Represented By The Secretary Of The Navy | Antisense oligonucleotides directed against human ELAM-I RNA |
RU95104940A (en) * | 1992-07-27 | 1997-01-10 | Хайбрайдон | Method of incorporation of alkylphosphonothioate or arylphosphonothioate internucleotide linkage in oligonucleotide, method of oligonucleotide synthesis, method of gene expression inhibition, treatment method |
US6107062A (en) * | 1992-07-30 | 2000-08-22 | Inpax, Inc. | Antisense viruses and antisense-ribozyme viruses |
HUT72400A (en) | 1992-10-05 | 1996-04-29 | Hybridon Inc | Therapeutic anti-hiv oligonucleotide, pharmaceutical compositions containing them and methods for use of compositions |
US5575613A (en) * | 1992-11-16 | 1996-11-19 | Mcneall Engineering Pty. Ltd. | Pallet dispenser |
US5593972A (en) * | 1993-01-26 | 1997-01-14 | The Wistar Institute | Genetic immunization |
US5650156A (en) * | 1993-02-22 | 1997-07-22 | Vivorx Pharmaceuticals, Inc. | Methods for in vivo delivery of nutriceuticals and compositions useful therefor |
DE69429337T3 (en) * | 1993-04-02 | 2012-08-30 | Anticancer Inc. | METHOD FOR THE ADMINISTRATION OF SPATIAL COMPOSITIONS TO HAIR FOLLICLES |
US5567604A (en) * | 1993-04-23 | 1996-10-22 | Aronex Pharmaceuticals, Inc. | Anti-viral guanosine-rich oligonucleotides |
SG54115A1 (en) * | 1993-04-27 | 1998-11-16 | Gerber Scient Products Inc | Thermal printing apparatus with improved power supply |
EP0705335A1 (en) * | 1993-06-23 | 1996-04-10 | Genesys Pharma Inc. | Antisense oligonucleotides and therapeutic use thereof in human immunodeficiency virus infection |
AU7319994A (en) * | 1993-06-30 | 1995-01-24 | Board Of Regents, The University Of Texas System | Nucleotide preparation and uses thereof in wound healing |
KR960704033A (en) * | 1993-07-19 | 1996-08-31 | 다니엘 엘. 캐시앙 | Oligonucleotides with Activity against Humand Immunodeficiency Virus |
US6004534A (en) | 1993-07-23 | 1999-12-21 | Massachusetts Institute Of Technology | Targeted polymerized liposomes for improved drug delivery |
US6605708B1 (en) | 1993-07-28 | 2003-08-12 | Hybridon, Inc. | Building blocks with carbamate internucleoside linkages and oligonucleotides derived therefrom |
US5985847A (en) | 1993-08-26 | 1999-11-16 | The Regents Of The University Of California | Devices for administration of naked polynucleotides which encode biologically active peptides |
US5804566A (en) | 1993-08-26 | 1998-09-08 | The Regents Of The University Of California | Methods and devices for immunizing a host through administration of naked polynucleotides with encode allergenic peptides |
US5849719A (en) | 1993-08-26 | 1998-12-15 | The Regents Of The University Of California | Method for treating allergic lung disease |
US5679647A (en) * | 1993-08-26 | 1997-10-21 | The Regents Of The University Of California | Methods and devices for immunizing a host against tumor-associated antigens through administration of naked polynucleotides which encode tumor-associated antigenic peptides |
US20030109469A1 (en) | 1993-08-26 | 2003-06-12 | Carson Dennis A. | Recombinant gene expression vectors and methods for use of same to enhance the immune response of a host to an antigen |
US5830877A (en) | 1993-08-26 | 1998-11-03 | The Regents Of The University Of California | Method, compositions and devices for administration of naked polynucleotides which encode antigens and immunostimulatory |
FR2711670B1 (en) * | 1993-10-22 | 1996-01-12 | Pasteur Institut | Nucleotide vector, composition containing it and vaccine for immunization against hepatitis. |
JP2906949B2 (en) * | 1993-10-27 | 1999-06-21 | 富士ゼロックス株式会社 | Hypertext device |
DE4338704A1 (en) * | 1993-11-12 | 1995-05-18 | Hoechst Ag | Stabilized oligonucleotides and their use |
US5595756A (en) * | 1993-12-22 | 1997-01-21 | Inex Pharmaceuticals Corporation | Liposomal compositions for enhanced retention of bioactive agents |
US5712384A (en) * | 1994-01-05 | 1998-01-27 | Gene Shears Pty Ltd. | Ribozymes targeting retroviral packaging sequence expression constructs and recombinant retroviruses containing such constructs |
US5700590A (en) | 1994-01-10 | 1997-12-23 | Abbott Laboratories | Nutritional formula with ribo-nucleotides |
US5602109A (en) * | 1994-01-10 | 1997-02-11 | Abbott Laboratories | Method to enhance the immune system of a human |
US5488039A (en) * | 1994-01-10 | 1996-01-30 | Abbott Laboratories | Method for the production of an enteral formula containing ribo-nucleotides |
US5492899A (en) * | 1994-01-10 | 1996-02-20 | Abbott Laboratories | Infant nutritional formula with ribo-nucleotides |
US5728518A (en) * | 1994-01-12 | 1998-03-17 | The Immune Response Corporation | Antiviral poly-and oligonucleotides |
CA2190121A1 (en) | 1994-03-15 | 1995-09-21 | Edith Mathiowitz | Polymeric gene delivery system |
US5596091A (en) * | 1994-03-18 | 1997-01-21 | The Regents Of The University Of California | Antisense oligonucleotides comprising 5-aminoalkyl pyrimidine nucleotides |
US6727230B1 (en) * | 1994-03-25 | 2004-04-27 | Coley Pharmaceutical Group, Inc. | Immune stimulation by phosphorothioate oligonucleotide analogs |
US5543152A (en) | 1994-06-20 | 1996-08-06 | Inex Pharmaceuticals Corporation | Sphingosomes for enhanced drug delivery |
US5741516A (en) | 1994-06-20 | 1998-04-21 | Inex Pharmaceuticals Corporation | Sphingosomes for enhanced drug delivery |
US20030050263A1 (en) * | 1994-07-15 | 2003-03-13 | The University Of Iowa Research Foundation | Methods and products for treating HIV infection |
US6207646B1 (en) * | 1994-07-15 | 2001-03-27 | University Of Iowa Research Foundation | Immunostimulatory nucleic acid molecules |
US6429199B1 (en) * | 1994-07-15 | 2002-08-06 | University Of Iowa Research Foundation | Immunostimulatory nucleic acid molecules for activating dendritic cells |
US6239116B1 (en) * | 1994-07-15 | 2001-05-29 | University Of Iowa Research Foundation | Immunostimulatory nucleic acid molecules |
ATE420171T1 (en) * | 1994-07-15 | 2009-01-15 | Univ Iowa Res Found | IMMUNOMODULATORY OLIGONUCLEOTIDES |
US7935675B1 (en) * | 1994-07-15 | 2011-05-03 | University Of Iowa Research Foundation | Immunostimulatory nucleic acid molecules |
US20030026782A1 (en) * | 1995-02-07 | 2003-02-06 | Arthur M. Krieg | Immunomodulatory oligonucleotides |
US5646262A (en) * | 1994-07-28 | 1997-07-08 | Georgetown University | Antisense oligonucleotides against hepatitis B viral replication |
US5753613A (en) * | 1994-09-30 | 1998-05-19 | Inex Pharmaceuticals Corporation | Compositions for the introduction of polyanionic materials into cells |
AU3559695A (en) * | 1994-09-30 | 1996-04-26 | Inex Pharmaceuticals Corp. | Glycosylated protein-liposome conjugates and methods for their preparation |
EP1179340A3 (en) * | 1994-09-30 | 2003-05-07 | INEX Pharmaceutical Corp. | Compositions for the introduction of polyanionic materials into cells |
US5591721A (en) * | 1994-10-25 | 1997-01-07 | Hybridon, Inc. | Method of down-regulating gene expression |
US6630455B1 (en) | 1995-01-13 | 2003-10-07 | Vanderbilt University | Methods for inducing mucosal immune responses |
DE19502912A1 (en) | 1995-01-31 | 1996-08-01 | Hoechst Ag | G-Cap Stabilized Oligonucleotides |
JP3580900B2 (en) | 1995-04-20 | 2004-10-27 | ホクレン農業協同組合連合会 | Food and feed containing, as an active ingredient, a composition mainly comprising a sugar containing an α-glucosidase inhibitor |
US5858987A (en) * | 1995-05-05 | 1999-01-12 | Mitotix, Inc. | E6AP antisense constructs and methods of use |
US5612060A (en) * | 1995-05-25 | 1997-03-18 | Alexander; J. Wesley | Enhancement of transplant graft survival through nutritional immunomodulation and immunosuppressive therapy |
US5955059A (en) * | 1995-06-06 | 1999-09-21 | Trustees Of Boston University | Use of locally applied DNA fragments |
IL122290A0 (en) | 1995-06-07 | 1998-04-05 | Inex Pharmaceuticals Corp | Lipid-nucleic acid complex its preparation and use |
US5976580A (en) | 1995-06-07 | 1999-11-02 | Novus International, Inc. | Nutrient formulation and process for enhancing the health, livability, cumulative weight gain or feed efficiency in poultry and other animals |
US5994315A (en) | 1995-06-07 | 1999-11-30 | East Carolina University | Low adenosine agent, composition, kit and method for treatment of airway disease |
US5981501A (en) | 1995-06-07 | 1999-11-09 | Inex Pharmaceuticals Corp. | Methods for encapsulating plasmids in lipid bilayers |
US5705385A (en) * | 1995-06-07 | 1998-01-06 | Inex Pharmaceuticals Corporation | Lipid-nucleic acid particles prepared via a hydrophobic lipid-nucleic acid complex intermediate and use for gene transfer |
US6248720B1 (en) * | 1996-07-03 | 2001-06-19 | Brown University Research Foundation | Method for gene therapy using nucleic acid loaded polymeric microparticles |
AU714584B2 (en) | 1995-07-21 | 2000-01-06 | Brown University Research Foundation | A method for gene therapy using nucleic acid loaded polymeric microparticles |
CN1141740A (en) | 1995-07-28 | 1997-02-05 | 伍梅四 | Roe powder serial health-care food |
US5968909A (en) | 1995-08-04 | 1999-10-19 | Hybridon, Inc. | Method of modulating gene expression with reduced immunostimulatory response |
EA199800272A1 (en) | 1995-10-04 | 1998-10-29 | Иммунекс Корпорейшн | FACTOR, STIMULATING DENDRIT CELLS |
US5736152A (en) * | 1995-10-27 | 1998-04-07 | Atrix Laboratories, Inc. | Non-polymeric sustained release delivery system |
US5780448A (en) * | 1995-11-07 | 1998-07-14 | Ottawa Civic Hospital Loeb Research | DNA-based vaccination of fish |
GB9525902D0 (en) | 1995-12-16 | 1996-02-21 | Zeneca Ltd | Fungus |
US20030078223A1 (en) * | 1996-01-30 | 2003-04-24 | Eyal Raz | Compositions and methods for modulating an immune response |
ATE437943T1 (en) | 1996-01-30 | 2009-08-15 | Univ California | EXPRESSION VECTORS THAT INDUCE AN ANTIGEN-SPECIFIC IMMUNE RESPONSE AND METHODS FOR THEIR USE. |
SE9600648D0 (en) * | 1996-02-21 | 1996-02-21 | Bror Morein | Receptor binding unit |
US5994316A (en) | 1996-02-21 | 1999-11-30 | The Immune Response Corporation | Method of preparing polynucleotide-carrier complexes for delivery to cells |
US6030955A (en) * | 1996-03-21 | 2000-02-29 | The Trustees Of Columbia University In The City Of New York And Imclone Systems, Inc. | Methods of affecting intracellular phosphorylation of tyrosine using phosphorothioate oligonucleotides, and antiangiogenic and antiproliferative uses thereof |
CN1151840C (en) * | 1996-05-09 | 2004-06-02 | 太平洋制药控股公司 | Stimulation of host defence mechanisms against tumors |
US6184037B1 (en) | 1996-05-17 | 2001-02-06 | Genemedicine, Inc. | Chitosan related compositions and methods for delivery of nucleic acids and oligonucleotides into a cell |
US5705109A (en) * | 1996-06-20 | 1998-01-06 | Westvaco Corporation | Ozone treatment for composite paperboard/polymer package |
US5895652A (en) * | 1996-07-29 | 1999-04-20 | Longevity Institute International | Method of metabolic adjuvanation and cellular repair |
EP0930893B1 (en) * | 1996-10-11 | 2005-04-13 | The Regents of The University of California | Immunostimulatory polynucleotide/immunomodulatory molecule conjugates |
EP0855184A1 (en) * | 1997-01-23 | 1998-07-29 | Grayson B. Dr. Lipford | Pharmaceutical composition comprising a polynucleotide and an antigen especially for vaccination |
US6214806B1 (en) * | 1997-02-28 | 2001-04-10 | University Of Iowa Research Foundation | Use of nucleic acids containing unmethylated CPC dinucleotide in the treatment of LPS-associated disorders |
US6406705B1 (en) * | 1997-03-10 | 2002-06-18 | University Of Iowa Research Foundation | Use of nucleic acids containing unmethylated CpG dinucleotide as an adjuvant |
US5965542A (en) | 1997-03-18 | 1999-10-12 | Inex Pharmaceuticals Corp. | Use of temperature to control the size of cationic liposome/plasmid DNA complexes |
US6426334B1 (en) * | 1997-04-30 | 2002-07-30 | Hybridon, Inc. | Oligonucleotide mediated specific cytokine induction and reduction of tumor growth in a mammal |
US6835395B1 (en) | 1997-05-14 | 2004-12-28 | The University Of British Columbia | Composition containing small multilamellar oligodeoxynucleotide-containing lipid vesicles |
US20030104044A1 (en) * | 1997-05-14 | 2003-06-05 | Semple Sean C. | Compositions for stimulating cytokine secretion and inducing an immune response |
CA2301575C (en) * | 1997-05-20 | 2003-12-23 | Ottawa Civic Hospital Loeb Research Institute | Vectors and methods for immunization or therapeutic protocols |
DE69819150T3 (en) * | 1997-06-06 | 2007-12-20 | Dynavax Technologies Corp., San Diego | IMMUNOSTIMULATING OLIGONUCLEOTIDES, COMPOSITIONS THEREOF, AND METHOD OF USE THEREOF |
US6589940B1 (en) * | 1997-06-06 | 2003-07-08 | Dynavax Technologies Corporation | Immunostimulatory oligonucleotides, compositions thereof and methods of use thereof |
US20040006034A1 (en) * | 1998-06-05 | 2004-01-08 | Eyal Raz | Immunostimulatory oligonucleotides, compositions thereof and methods of use thereof |
US5922766A (en) * | 1997-07-02 | 1999-07-13 | Acosta; Phyllis J. B. | Palatable elemental medical food |
EP1009440B1 (en) * | 1997-07-03 | 2008-09-17 | MacFarlane, Donald E. | Method for inhibiting immunostimulatory dna associated responses |
US6110745A (en) | 1997-07-24 | 2000-08-29 | Inex Pharmaceuticals Corp. | Preparation of lipid-nucleic acid particles using a solvent extraction and direct hydration method |
US5877309A (en) * | 1997-08-13 | 1999-03-02 | Isis Pharmaceuticals, Inc. | Antisense oligonucleotides against JNK |
DK1009413T3 (en) * | 1997-09-05 | 2007-06-11 | Univ California | Use of immunostimulatory oligonucleotides for the prevention or treatment of asthma |
US7393630B2 (en) * | 1997-12-16 | 2008-07-01 | Novartis Vaccines And Diagnostics, Inc. | Use of microparticles combined with submicron oil-in-water emulsions |
US20050031638A1 (en) * | 1997-12-24 | 2005-02-10 | Smithkline Beecham Biologicals S.A. | Vaccine |
JPH11209289A (en) * | 1998-01-22 | 1999-08-03 | Taisho Pharmaceut Co Ltd | Mucosal immunity inducer |
EP0933368A1 (en) * | 1998-02-02 | 1999-08-04 | SSP Co., Ltd. | Triazole derivative or salt thereof, preparation process thereof and pharmaceutical containing said compound as an effective ingredient (antimycotic) |
DE69935507T2 (en) * | 1998-04-03 | 2007-12-06 | University Of Iowa Research Foundation | METHOD AND PRODUCTS FOR STIMULATING THE IMMUNE SYSTEM BY IMMUNOTHERAPEUTIC OLIGONUCLEOTIDES AND CYTOKINES |
EP1077708A1 (en) | 1998-05-06 | 2001-02-28 | University Of Iowa Research Foundation | Methods for the prevention and treatment of parasitic infections and related diseases using cpg oligonucleotides |
CA2328406A1 (en) * | 1998-05-14 | 1999-11-18 | Hermann Wagner | Methods for regulating hematopoiesis using cpg-oligonucleotides |
SI1077722T1 (en) | 1998-05-22 | 2007-02-28 | Ottawa Health Research Inst | Methods and products for inducing mucosal immunity |
US6248329B1 (en) * | 1998-06-01 | 2001-06-19 | Ramaswamy Chandrashekar | Parasitic helminth cuticlin nucleic acid molecules and uses thereof |
US6562798B1 (en) | 1998-06-05 | 2003-05-13 | Dynavax Technologies Corp. | Immunostimulatory oligonucleotides with modified bases and methods of use thereof |
EP1089751A4 (en) | 1998-06-23 | 2001-07-25 | Univ Leland Stanford Junior | Adjuvant therapy |
US20030022854A1 (en) * | 1998-06-25 | 2003-01-30 | Dow Steven W. | Vaccines using nucleic acid-lipid complexes |
US20040247662A1 (en) | 1998-06-25 | 2004-12-09 | Dow Steven W. | Systemic immune activation method using nucleic acid-lipid complexes |
US6693086B1 (en) | 1998-06-25 | 2004-02-17 | National Jewish Medical And Research Center | Systemic immune activation method using nucleic acid-lipid complexes |
US6241086B1 (en) * | 1998-07-07 | 2001-06-05 | Case Logic, Inc. | Sleeve for holding digital video discs and graphics |
AU764532B2 (en) | 1998-07-27 | 2003-08-21 | University Of Iowa Research Foundation, The | Stereoisomers of CpG oligonucleotides and related methods |
EP1104306B1 (en) | 1998-08-10 | 2006-01-11 | Antigenics Inc. | Compositions of cpg and saponin adjuvants and methods of use thereof |
AU777225B2 (en) | 1998-09-03 | 2004-10-07 | Coley Pharmaceutical Gmbh | G-motif oligonucleotides and uses thereof |
US20020065236A1 (en) * | 1998-09-09 | 2002-05-30 | Yew Nelson S. | CpG reduced plasmids and viral vectors |
FR2783170B1 (en) * | 1998-09-11 | 2004-07-16 | Pasteur Merieux Serums Vacc | IMMUNOSTIMULATING EMULSION |
WO2000016804A1 (en) | 1998-09-18 | 2000-03-30 | Dynavax Technologies Corporation | METHODS OF TREATING IgE-ASSOCIATED DISORDERS AND COMPOSITIONS FOR USE THEREIN |
WO2000020039A1 (en) | 1998-10-05 | 2000-04-13 | The Regents Of The University Of California | Methods and adjuvants for stimulating mucosal immunity |
EP1117433A1 (en) | 1998-10-09 | 2001-07-25 | Dynavax Technologies Corporation | Anti hiv compositions comprising immunostimulatory polynucleotides and hiv antigens |
AU2870300A (en) | 1999-02-05 | 2000-08-25 | Genzyme Corporation | Use of cationic lipids to generate anti-tumor immunity |
US6207819B1 (en) * | 1999-02-12 | 2001-03-27 | Isis Pharmaceuticals, Inc. | Compounds, processes and intermediates for synthesis of mixed backbone oligomeric compounds |
EP1176966B1 (en) * | 1999-04-12 | 2013-04-03 | THE GOVERNMENT OF THE UNITED STATES OF AMERICA, as represented by THE SECRETARY, DEPARTMENT OF HEALTH AND HUMAN SERVICES | Oligodeoxynucleotide and its use to induce an immune response |
US6977245B2 (en) * | 1999-04-12 | 2005-12-20 | The United States Of America As Represented By The Department Of Health And Human Services | Oligodeoxynucleotide and its use to induce an immune response |
WO2000062787A1 (en) | 1999-04-15 | 2000-10-26 | The Regents Of The University Of California | Methods and compositions for use in potentiating antigen presentation by antigen presenting cells |
US6558670B1 (en) * | 1999-04-19 | 2003-05-06 | Smithkline Beechman Biologicals S.A. | Vaccine adjuvants |
IL145982A0 (en) * | 1999-04-19 | 2002-07-25 | Smithkline Beecham Biolog | Vaccines |
DE60013591T2 (en) | 1999-04-29 | 2005-02-03 | Coley Pharmaceutical Gmbh | SCREENING FOR MODULATORS OF THE FUNCTION OF IMMUNSTIMULATORY DNA |
US6737066B1 (en) * | 1999-05-06 | 2004-05-18 | The Immune Response Corporation | HIV immunogenic compositions and methods |
CA2412345A1 (en) * | 1999-06-16 | 2000-12-21 | University Of Iowa Research Foundation | Antagonism of immunostimulatory cpg-oligonucleotides by 4-aminoquinolines and other weak bases |
US6514948B1 (en) * | 1999-07-02 | 2003-02-04 | The Regents Of The University Of California | Method for enhancing an immune response |
DE19935756A1 (en) * | 1999-07-27 | 2001-02-08 | Mologen Forschungs Entwicklung | Covalently closed nucleic acid molecule for immune stimulation |
KR100651294B1 (en) | 1999-08-13 | 2006-11-28 | 이데라 파마슈티칼즈, 인코포레이티드 | MODULATION OF OLIGONUCLEOTIDE CpG-MEDIATED IMMUNE STIMULATION BY POSITIONAL MODIFICATION OF NUCLEOSIDES |
EP1204425B1 (en) | 1999-08-19 | 2009-01-07 | Dynavax Technologies Corporation | Methods of modulating an immune response using immunostimulatory sequences and compositions for use therein |
US20050249794A1 (en) | 1999-08-27 | 2005-11-10 | Semple Sean C | Compositions for stimulating cytokine secretion and inducing an immune response |
DK1221955T3 (en) | 1999-09-25 | 2006-01-30 | Univ Iowa Res Found | Immune-stimulating nucleic acid |
US6949520B1 (en) * | 1999-09-27 | 2005-09-27 | Coley Pharmaceutical Group, Inc. | Methods related to immunostimulatory nucleic acid-induced interferon |
US20010031262A1 (en) * | 1999-12-06 | 2001-10-18 | Michael Caplan | Controlled delivery of antigens |
ATE378348T1 (en) * | 2000-01-14 | 2007-11-15 | Us Health | OLIGODEOXYNUCLEOTIDES AND THEIR USE FOR INDUCING AN IMMUNE RESPONSE |
EP1311288A1 (en) | 2000-01-20 | 2003-05-21 | Ottawa Health Research Institute | Immunostimulatory nucleic acids for inducing a th2 immune response |
EP1253947A4 (en) | 2000-01-31 | 2005-01-05 | Univ California | Immunomodulatory polynucleotides in treatment of an infection by an intracellular pathogen |
US7585847B2 (en) * | 2000-02-03 | 2009-09-08 | Coley Pharmaceutical Group, Inc. | Immunostimulatory nucleic acids for the treatment of asthma and allergy |
WO2001062207A2 (en) | 2000-02-23 | 2001-08-30 | The Regents Of The University Of California | Method for treating inflammatory bowel disease and other forms of gastrointestinal inflammation |
US20030130217A1 (en) * | 2000-02-23 | 2003-07-10 | Eyal Raz | Method for treating inflammatory bowel disease and other forms of gastrointestinal inflammation |
US20020156033A1 (en) | 2000-03-03 | 2002-10-24 | Bratzler Robert L. | Immunostimulatory nucleic acids and cancer medicament combination therapy for the treatment of cancer |
US20040131628A1 (en) * | 2000-03-08 | 2004-07-08 | Bratzler Robert L. | Nucleic acids for the treatment of disorders associated with microorganisms |
US20020098199A1 (en) | 2000-03-10 | 2002-07-25 | Gary Van Nest | Methods of suppressing hepatitis virus infection using immunomodulatory polynucleotide sequences |
US20020028784A1 (en) | 2000-03-10 | 2002-03-07 | Nest Gary Van | Methods of preventing and treating viral infections using immunomodulatory polynucleotide sequences |
US20020107212A1 (en) | 2000-03-10 | 2002-08-08 | Nest Gary Van | Methods of reducing papillomavirus infection using immunomodulatory polynucleotide sequences |
US20010046967A1 (en) * | 2000-03-10 | 2001-11-29 | Gary Van Nest | Methods of preventing and treating respiratory viral infection using immunomodulatory polynucleotide |
US20030129251A1 (en) * | 2000-03-10 | 2003-07-10 | Gary Van Nest | Biodegradable immunomodulatory formulations and methods for use thereof |
US7129222B2 (en) * | 2000-03-10 | 2006-10-31 | Dynavax Technologies Corporation | Immunomodulatory formulations and methods for use thereof |
US7157437B2 (en) * | 2000-03-10 | 2007-01-02 | Dynavax Technologies Corporation | Methods of ameliorating symptoms of herpes infection using immunomodulatory polynucleotide sequences |
WO2001071885A1 (en) * | 2000-03-20 | 2001-09-27 | Alpha Technologies, Inc. | Uninterruptible power supplies employing fuel cells |
AU2001249609A1 (en) * | 2000-03-28 | 2001-10-08 | Department Of Veterans Affairs | Methods for increasing a cytotoxic T lymphocyte response in vivo |
US20020142978A1 (en) | 2000-04-07 | 2002-10-03 | Eyal Raz | Synergistic improvements to polynucleotide vaccines |
ATE299696T1 (en) * | 2000-05-12 | 2005-08-15 | Pharmacia & Upjohn Co Llc | VACCINE COMPOSITION AND METHOD FOR PRODUCING THE SAME AND METHOD FOR VACCINATION OF VERTEBRATE ANIMALS |
US6339630B1 (en) | 2000-05-18 | 2002-01-15 | The United States Of America As Represented By The United States Department Of Energy | Sealed drive screw operator |
PT1296714E (en) * | 2000-06-22 | 2009-10-15 | Coley Pharm Gmbh | Combination of cpg and antibodies directed against cd19,cd20, cd22 or cd40 for the treatment or prevention of cancer. |
US20020165178A1 (en) | 2000-06-28 | 2002-11-07 | Christian Schetter | Immunostimulatory nucleic acids for the treatment of anemia, thrombocytopenia, and neutropenia |
US20020198165A1 (en) | 2000-08-01 | 2002-12-26 | Bratzler Robert L. | Nucleic acids for the prevention and treatment of gastric ulcers |
AU2002212187A1 (en) * | 2000-09-01 | 2002-03-13 | Epigenomics Ag | Diagnosis of illnesses or predisposition to certain illnesses |
US20020091097A1 (en) * | 2000-09-07 | 2002-07-11 | Bratzler Robert L. | Nucleic acids for the prevention and treatment of sexually transmitted diseases |
JP2005500806A (en) * | 2000-09-15 | 2005-01-13 | コーリー ファーマシューティカル ゲーエムベーハー | Process for high-throughput screening of immune agonist / immunoantagonists based on CpG |
FR2814958B1 (en) * | 2000-10-06 | 2003-03-07 | Aventis Pasteur | VACCINE COMPOSITION |
GB0025577D0 (en) * | 2000-10-18 | 2000-12-06 | Smithkline Beecham Biolog | Vaccine |
AU2001297693A1 (en) | 2000-12-08 | 2002-09-12 | Coley Pharmaceutical Gmbh | Cpg-like nucleic acids and methods of use thereof |
WO2002053141A2 (en) * | 2000-12-14 | 2002-07-11 | Coley Pharmaceutical Group, Inc. | Inhibition of angiogenesis by nucleic acids |
CN1293192C (en) * | 2000-12-27 | 2007-01-03 | 戴纳瓦克斯技术公司 | Immunoregulation polynucleotide and using method thereof |
US20020193332A1 (en) * | 2001-02-12 | 2002-12-19 | Hedley Mary Lynne | Methods of treating bladder disorders |
US20030050268A1 (en) * | 2001-03-29 | 2003-03-13 | Krieg Arthur M. | Immunostimulatory nucleic acid for treatment of non-allergic inflammatory diseases |
US6969484B2 (en) * | 2001-06-18 | 2005-11-29 | Toray Industries, Inc. | Manufacturing method and device for electret processed product |
CN100334228C (en) | 2001-06-21 | 2007-08-29 | 戴纳瓦克斯技术公司 | Cimeric immunomodulatory compounds and methods of using the same |
US7785610B2 (en) * | 2001-06-21 | 2010-08-31 | Dynavax Technologies Corporation | Chimeric immunomodulatory compounds and methods of using the same—III |
WO2003000232A2 (en) | 2001-06-25 | 2003-01-03 | Yissum Research Development Company Of The Hebrew University Of Jerusalem | Method for preparation of vesicles loaded with immunostimulator y oligodeoxynucleotides |
US7666674B2 (en) * | 2001-07-27 | 2010-02-23 | The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services | Use of sterically stabilized cationic liposomes to efficiently deliver CPG oligonucleotides in vivo |
AU2002318944A1 (en) | 2001-08-01 | 2003-02-17 | Coley Pharmaceutical Gmbh | Methods and compositions relating to plasmacytoid dendritic cells |
JP4607452B2 (en) * | 2001-08-07 | 2011-01-05 | ダイナバックス テクノロジーズ コーポレイション | Immunomodulating composition, formulation and method of use thereof |
DE10138833A1 (en) * | 2001-08-14 | 2003-02-27 | Daimler Chrysler Ag | Device and method for remote diagnostics of vehicles |
WO2003020884A2 (en) * | 2001-08-14 | 2003-03-13 | The Government Of The United States Of America As Represented By The Secretary Of Health And Human Services | Method for rapid generation of mature dendritic cells |
DK1446162T3 (en) * | 2001-08-17 | 2008-12-08 | Coley Pharm Gmbh | Combination motif immunostimulatory oligonucleotides with enhanced activity |
WO2004012669A2 (en) * | 2002-08-01 | 2004-02-12 | The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services | Method of treating inflammatory arthropathies with suppressors of cpg oligonucleotides |
US7514414B2 (en) * | 2001-09-24 | 2009-04-07 | The United States Of America As Represented By The Department Of Health And Human Services | Suppressors of CpG oligonucleotides and methods of use |
US20030119774A1 (en) * | 2001-09-25 | 2003-06-26 | Marianna Foldvari | Compositions and methods for stimulating an immune response |
KR20040053136A (en) * | 2001-09-28 | 2004-06-23 | 펄듀 리서치 파운데이션 | Method of treatment using ligand-immunogen conjugate |
WO2003031573A2 (en) | 2001-10-05 | 2003-04-17 | Coley Pharmaceutical Gmbh | Toll-like receptor 3 signaling agonists and antagonists |
US20030139364A1 (en) | 2001-10-12 | 2003-07-24 | University Of Iowa Research Foundation | Methods and products for enhancing immune responses using imidazoquinoline compounds |
US7276489B2 (en) * | 2002-10-24 | 2007-10-02 | Idera Pharmaceuticals, Inc. | Modulation of immunostimulatory properties of oligonucleotide-based compounds by optimal presentation of 5′ ends |
EP1441763A2 (en) | 2001-11-07 | 2004-08-04 | Inex Pharmaceuticals Corp. | Mucosal adjuvants comprising an oligonucleotide and a cationic lipid |
CN1169434C (en) | 2001-11-20 | 2004-10-06 | 成都天友生物科技股份有限公司 | Temp-sensitive lethality male silkworm breeding method |
AU2002366710A1 (en) * | 2001-12-20 | 2003-07-09 | The Government Of The United States Of America As Represented By The Secretary Of The Department Of | USE OF CpG OLIGODEOXYNUCLEOTIDES TO INDUCE ANGIOGENESIS |
EP1474432A1 (en) * | 2002-02-04 | 2004-11-10 | Biomira Inc. | Immunostimulatory, covalently lipidated oligonucleotides |
US8088388B2 (en) * | 2002-02-14 | 2012-01-03 | United Biomedical, Inc. | Stabilized synthetic immunogen delivery system |
AU2003230806B2 (en) * | 2002-04-04 | 2009-05-07 | Zoetis Belgium S.A. | Immunostimulatory G,U-containing oligoribonucleotides |
JP2006515266A (en) * | 2002-04-22 | 2006-05-25 | バイオニケ ライフ サイエンシーズ インコーポレイテッド | Oligonucleotide compositions and their use for modulating immune responses |
WO2003103586A2 (en) * | 2002-06-05 | 2003-12-18 | Coley Pharmaceutical Group, Inc. | Method for treating autoimmune or inflammatory diseases with combinations of inhibitory oligonucleotides and small molecule antagonists of immunostimulatory cpg nucleic acids |
US7576066B2 (en) * | 2002-07-03 | 2009-08-18 | Coley Pharmaceutical Group, Inc. | Nucleic acid compositions for stimulating immune responses |
US20040053880A1 (en) | 2002-07-03 | 2004-03-18 | Coley Pharmaceutical Group, Inc. | Nucleic acid compositions for stimulating immune responses |
US7569553B2 (en) * | 2002-07-03 | 2009-08-04 | Coley Pharmaceutical Group, Inc. | Nucleic acid compositions for stimulating immune responses |
US7605138B2 (en) | 2002-07-03 | 2009-10-20 | Coley Pharmaceutical Group, Inc. | Nucleic acid compositions for stimulating immune responses |
US7807803B2 (en) * | 2002-07-03 | 2010-10-05 | Coley Pharmaceutical Group, Inc. | Nucleic acid compositions for stimulating immune responses |
WO2004004654A2 (en) * | 2002-07-03 | 2004-01-15 | Cambridge Scientific, Inc. | Vaccines to induce mucosal immunity |
WO2004007743A2 (en) | 2002-07-17 | 2004-01-22 | Coley Pharmaceutical Gmbh | Use of cpg nucleic acids in prion-disease |
KR20050052467A (en) * | 2002-08-12 | 2005-06-02 | 다이나박스 테크놀로지 코퍼레이션 | Immunomodulatory compositions, methods of making, and methods of use thereof |
DE10239495A1 (en) * | 2002-08-28 | 2004-03-11 | BSH Bosch und Siemens Hausgeräte GmbH | Sieve for dishwashing machine may be cleared by periodic reversals of current and side of sieve facing material to be filtered is faced with non-stick material |
US8263091B2 (en) * | 2002-09-18 | 2012-09-11 | The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services | Method of treating and preventing infections in immunocompromised subjects with immunostimulatory CpG oligonucleotides |
WO2004026888A2 (en) | 2002-09-19 | 2004-04-01 | Coley Pharmaceutical Gmbh | Toll-like receptor 9 (tlr9) from various mammalian species |
US8043622B2 (en) * | 2002-10-08 | 2011-10-25 | The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services | Method of treating inflammatory lung disease with suppressors of CpG oligonucleotides |
US7998492B2 (en) * | 2002-10-29 | 2011-08-16 | Coley Pharmaceutical Group, Inc. | Methods and products related to treatment and prevention of hepatitis C virus infection |
CA2504493C (en) * | 2002-11-01 | 2015-12-29 | The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services | Method of preventing infections from bioterrorism agents with immunostimulatory cpg oligonucleotides |
JP2006512927A (en) | 2002-12-11 | 2006-04-20 | コーリー ファーマシューティカル グループ,インコーポレイテッド | 5 'CPG nucleic acids and methods of use thereof |
KR100525321B1 (en) * | 2002-12-13 | 2005-11-02 | 안웅식 | Pharmaceutical composition for prophylaxis and treatment of papillomavirus-derived diseases comprising papillomavirus antigen protein and CpG-oligodeoxynucleotide |
US7517520B2 (en) * | 2003-03-26 | 2009-04-14 | Cytos Biotechnology Ag | Packaging of immunostimulatory oligonucleotides into virus-like particles: method of preparation and use |
AU2004226605A1 (en) | 2003-04-02 | 2004-10-14 | Coley Pharmaceutical Group, Ltd. | Immunostimulatory nucleic acid oil-in-water formulations for topical application |
WO2005016235A2 (en) * | 2003-04-14 | 2005-02-24 | The Regents Of The University Of California | Combined use of impdh inhibitors with toll-like receptor agonists |
WO2004094671A2 (en) | 2003-04-22 | 2004-11-04 | Coley Pharmaceutical Gmbh | Methods and products for identification and assessment of tlr ligands |
ATE476196T1 (en) * | 2003-06-17 | 2010-08-15 | Mannkind Corp | COMPOSITION FOR INITIATING, IMPROVEMENT AND MAINTAINING IMMUNE RESPONSES AGAINST MHC CLASS I RESTRICTED EPITOPES, FOR PROPHYLACTIC OR THERAPEUTIC PURPOSES |
US20060251623A1 (en) * | 2003-07-10 | 2006-11-09 | Caytos Biotechnology Ag | Packaged virus-like particles |
US20050013812A1 (en) * | 2003-07-14 | 2005-01-20 | Dow Steven W. | Vaccines using pattern recognition receptor-ligand:lipid complexes |
AU2004259204B2 (en) * | 2003-07-15 | 2010-08-19 | Idera Pharmaceuticals, Inc. | Synergistic stimulation of the immune system using immunostimulatory oligonucleotides and/or immunomer compounds in conjunction with cytokines and/or chemotherapeutic agents or radiation therapy |
BRPI0413906A (en) | 2003-08-28 | 2006-10-24 | Immune Response Corp Inc | HIV immunogenic compositions, kit and related methods |
OA13310A (en) * | 2003-09-05 | 2007-04-13 | Anadys Pharmaceuticals Inc | TLR7 ligands for the treatment of hepatitis C. |
CN1211443C (en) | 2003-09-10 | 2005-07-20 | 江苏鸿业涂料科技产业有限公司 | Resin emulsion for high penetrating powder cathod electrophoresis paint |
CA2536139A1 (en) | 2003-09-25 | 2005-04-07 | Coley Pharmaceutical Group, Inc. | Nucleic acid-lipophilic conjugates |
US20050215501A1 (en) * | 2003-10-24 | 2005-09-29 | Coley Pharmaceutical Group, Inc. | Methods and products for enhancing epitope spreading |
KR101107818B1 (en) * | 2003-10-30 | 2012-01-31 | 콜레이 파마시티컬 그룹, 인코포레이티드 | C-class oligonucleotide analogs with enhanced immunostimulatory potency |
US20050239733A1 (en) * | 2003-10-31 | 2005-10-27 | Coley Pharmaceutical Gmbh | Sequence requirements for inhibitory oligonucleotides |
US20050100983A1 (en) * | 2003-11-06 | 2005-05-12 | Coley Pharmaceutical Gmbh | Cell-free methods for identifying compounds that affect toll-like receptor 9 (TLR9) signaling |
EP2060269A3 (en) * | 2003-12-08 | 2009-08-19 | Hybridon, Inc. | Modulation of immunostimulatory properties by small oligonucleotide-based compounds |
US9090673B2 (en) | 2003-12-12 | 2015-07-28 | City Of Hope | Synthetic conjugate of CpG DNA and T-help/CTL peptide |
US20050181035A1 (en) | 2004-02-17 | 2005-08-18 | Dow Steven W. | Systemic immune activation method using non CpG nucleic acids |
TW200533750A (en) * | 2004-02-19 | 2005-10-16 | Coley Pharm Group Inc | Immunostimulatory viral RNA oligonucleotides |
EP1730281A2 (en) * | 2004-04-02 | 2006-12-13 | Coley Pharmaceutical Group, Inc. | Immunostimulatory nucleic acids for inducing il-10 responses |
US7883687B2 (en) * | 2004-05-03 | 2011-02-08 | Astellas Pharma Inc. | 15O-labeled monosaccharide and producing method thereof |
AU2005326144A1 (en) | 2004-06-08 | 2006-08-03 | Coley Pharmaceutical Gmbh | Abasic oligonucleotide as carrier platform for antigen and immunostimulatory agonist and antagonist |
BRPI0512110A (en) * | 2004-06-15 | 2007-10-23 | Idera Pharmaceutical Inc | immunostimulatory oligonucleotide multimers |
CA2572427A1 (en) * | 2004-07-18 | 2006-01-18 | Heather L. Davis | Immuno stimulating complex and oligonucleotide formulations for inducing enhanced interferon-gamma responses |
WO2006134423A2 (en) * | 2004-07-18 | 2006-12-21 | Coley Pharmaceutical Group, Ltd. | Methods and compositions for inducing innate immune responses |
US7897810B2 (en) * | 2004-09-02 | 2011-03-01 | Eastman Chemical Company | Optimized production of aromatic dicarboxylic acids |
MY159370A (en) | 2004-10-20 | 2016-12-30 | Coley Pharm Group Inc | Semi-soft-class immunostimulatory oligonucleotides |
US20080009455A9 (en) * | 2005-02-24 | 2008-01-10 | Coley Pharmaceutical Group, Inc. | Immunostimulatory oligonucleotides |
AU2006235284A1 (en) * | 2005-04-08 | 2006-10-19 | Coley Pharmaceutical Group, Inc. | Methods for treating infectious disease exacerbated asthma |
US20060241076A1 (en) * | 2005-04-26 | 2006-10-26 | Coley Pharmaceutical Gmbh | Modified oligoribonucleotide analogs with enhanced immunostimulatory activity |
NZ565311A (en) | 2005-07-07 | 2009-10-30 | Pfizer | Anti-ctla-4 antibody and cpg-motif-containing synthetic oligodeoxynucleotide combination therapy for cancer treatment |
AU2006284889B2 (en) * | 2005-08-31 | 2011-08-18 | The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services | Methods of altering an immune response induced by CpG oligodeoxynucleotides |
EP1924692A2 (en) | 2005-09-16 | 2008-05-28 | Coley Pharmaceutical GmbH | Modulation of immunostimulatory properties of short interfering ribonucleic acid (sirna) by nucleotide modification |
EP1945766A2 (en) | 2005-09-16 | 2008-07-23 | Coley Pharmaceutical GmbH | Immunostimulatory single-stranded ribonucleic acid with phosphodiester backbone |
KR20080072934A (en) * | 2005-11-25 | 2008-08-07 | 콜리 파마슈티칼 게엠베하 | Immunostimulatory oligoribonucleotides |
JP5473336B2 (en) | 2006-02-15 | 2014-04-16 | アディウタイド・ファーマスーティカルズ・ゲーエムベーハー | Compositions and methods relating to the formulation of oligonucleotides |
NZ571412A (en) * | 2006-04-14 | 2010-07-30 | Merck & Co Inc | Substituted imidazole 4-carboximides as cholecystokinin-1 receptor modulators |
US8027888B2 (en) | 2006-08-31 | 2011-09-27 | Experian Interactive Innovation Center, Llc | Online credit card prescreen systems and methods |
WO2008033432A2 (en) | 2006-09-12 | 2008-03-20 | Coley Pharmaceutical Group, Inc. | Immune modulation by chemically modified ribonucleosides and oligoribonucleotides |
MX2009003398A (en) | 2006-09-27 | 2009-08-12 | Coley Pharm Gmbh | Cpg oligonucleotide analogs containing hydrophobic t analogs with enhanced immunostimulatory activity. |
EP2068912A2 (en) | 2006-09-27 | 2009-06-17 | Coley Pharmaceutical Group, Inc. | Compositions of tlr ligands and antivirals |
KR20090058584A (en) | 2006-10-26 | 2009-06-09 | 콜리 파마슈티칼 게엠베하 | Oligoribonucleotides and uses thereof |
US20090142362A1 (en) | 2006-11-06 | 2009-06-04 | Avant Immunotherapeutics, Inc. | Peptide-based vaccine compositions to endogenous cholesteryl ester transfer protein (CETP) |
EP2089411A4 (en) * | 2006-12-04 | 2010-01-27 | Univ Illinois | Compositions and methods to treat cancer with cupredoxins and cpg rich dna |
CN101678098A (en) | 2007-05-17 | 2010-03-24 | 科勒制药集团公司 | Class a oligonucleotides with immunostimulatory potency |
CN101820908A (en) | 2007-10-09 | 2010-09-01 | 科利制药公司 | The immune stimulatory oligonucleotide analogs that comprises modified sugar moieties |
US8268492B2 (en) * | 2007-11-30 | 2012-09-18 | GM Global Technology Operations LLC | Fuel cell stack features for improved water management |
US8222225B2 (en) * | 2008-05-21 | 2012-07-17 | The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services | Method of treating pneumoconiosis with oligodeoxynucleotides |
TWI351288B (en) * | 2008-07-04 | 2011-11-01 | Univ Nat Pingtung Sci & Tech | Cpg dna adjuvant in avian vaccines |
US8053422B2 (en) * | 2008-12-04 | 2011-11-08 | The United States Of America As Represented By The Department Of Health And Human Services | Anti-cancer oligodeoxynucleotides |
-
1999
- 1999-10-08 US US09/415,142 patent/US20030026782A1/en not_active Abandoned
-
2003
- 2003-07-30 US US10/631,676 patent/US20040087534A1/en not_active Abandoned
- 2003-10-21 US US10/690,495 patent/US20040143112A1/en not_active Abandoned
-
2004
- 2004-01-30 US US10/769,626 patent/US20040162258A1/en not_active Abandoned
- 2004-02-26 US US10/789,536 patent/US8309527B2/en not_active Expired - Fee Related
- 2004-02-26 US US10/789,353 patent/US20040162262A1/en not_active Abandoned
- 2004-02-26 US US10/788,199 patent/US20040181045A1/en not_active Abandoned
- 2004-02-26 US US10/787,737 patent/US20040171150A1/en not_active Abandoned
- 2004-02-26 US US10/789,051 patent/US20040142469A1/en not_active Abandoned
- 2004-02-26 US US10/788,191 patent/US20040152656A1/en not_active Abandoned
- 2004-05-17 US US10/847,650 patent/US20050004062A1/en not_active Abandoned
- 2004-07-09 US US10/888,089 patent/US8148340B2/en not_active Expired - Fee Related
- 2004-07-09 US US10/888,885 patent/US20050009774A1/en not_active Abandoned
-
2005
- 2005-02-25 US US11/067,516 patent/US20050239736A1/en not_active Abandoned
- 2005-05-11 US US11/127,797 patent/US8114848B2/en not_active Expired - Fee Related
- 2005-05-11 US US11/127,803 patent/US20050244379A1/en not_active Abandoned
- 2005-05-11 US US11/128,127 patent/US20070009482A9/en not_active Abandoned
- 2005-12-07 US US11/296,644 patent/US20060094683A1/en not_active Abandoned
-
2006
- 2006-12-22 US US11/645,106 patent/US20070202128A1/en not_active Abandoned
Patent Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5194428A (en) * | 1986-05-23 | 1993-03-16 | Worcester Foundation For Experimental Biology | Inhibition of influenza virus replication by oligonucleotide phosphorothioates |
US5232829A (en) * | 1989-09-29 | 1993-08-03 | Hoffmann-La Roche Inc. | Detection of chlamydia trachomatis by polymerase chain reaction using biotin labelled lina primers and capture probes |
US6030954A (en) * | 1991-09-05 | 2000-02-29 | University Of Connecticut | Targeted delivery of poly- or oligonucleotides to cells |
US5646126A (en) * | 1994-02-28 | 1997-07-08 | Epoch Pharmaceuticals | Sterol modified oligonucleotide duplexes having anticancer activity |
US5723335A (en) * | 1994-03-25 | 1998-03-03 | Isis Pharmaceuticals, Inc. | Immune stimulation by phosphorothioate oligonucleotide analogs |
US6620805B1 (en) * | 1996-03-14 | 2003-09-16 | Yale University | Delivery of nucleic acids by porphyrins |
US20070224210A1 (en) * | 2002-08-19 | 2007-09-27 | Coley Pharmaceutical Group, Inc. | Immunostimulatory nucleic acids |
US20070232622A1 (en) * | 2003-06-20 | 2007-10-04 | Coley Pharmaceutical Gmbh | Small molecule toll-like receptor (TLR) antagonists |
Cited By (71)
Publication number | Priority date | Publication date | Assignee | Title |
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US8129351B2 (en) | 1994-07-15 | 2012-03-06 | The University Of Iowa Research Foundation | Immunostimulatory nucleic acid molecules |
US7935675B1 (en) | 1994-07-15 | 2011-05-03 | University Of Iowa Research Foundation | Immunostimulatory nucleic acid molecules |
US7674777B2 (en) | 1994-07-15 | 2010-03-09 | University Of Iowa Research Foundation | Immunostimulatory nucleic acid molecules |
US8309527B2 (en) | 1994-07-15 | 2012-11-13 | University Of Iowa Research Foundation | Immunomodulatory oligonucleotides |
US8258106B2 (en) | 1994-07-15 | 2012-09-04 | University Of Iowa Research Foundation | Immunostimulatory nucleic acid molecules |
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US8148340B2 (en) | 1994-07-15 | 2012-04-03 | The United States Of America As Represented By The Department Of Health And Human Services | Immunomodulatory oligonucleotides |
US20050148537A1 (en) * | 1994-07-15 | 2005-07-07 | University Of Iowa Research Foundation | Immunostimulatory nucleic acid molecules |
US20030191079A1 (en) * | 1994-07-15 | 2003-10-09 | University Of Iowa Research Foundation | Methods for treating and preventing infectious disease |
US7713529B2 (en) | 1994-07-15 | 2010-05-11 | University Of Iowa Research Foundation | Methods for treating and preventing infectious disease |
US20080026011A1 (en) * | 1994-07-15 | 2008-01-31 | University Of Iowa Research Foundation | Immunostimulatory nucleic acid molecules |
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Also Published As
Publication number | Publication date |
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US20050009774A1 (en) | 2005-01-13 |
US20070202128A1 (en) | 2007-08-30 |
US20050244380A1 (en) | 2005-11-03 |
US20050239736A1 (en) | 2005-10-27 |
US20040087534A1 (en) | 2004-05-06 |
US20040162258A1 (en) | 2004-08-19 |
US20040171150A1 (en) | 2004-09-02 |
US20030026782A1 (en) | 2003-02-06 |
US20070010470A9 (en) | 2007-01-11 |
US20050004062A1 (en) | 2005-01-06 |
US8114848B2 (en) | 2012-02-14 |
US20040152656A1 (en) | 2004-08-05 |
US20050244379A1 (en) | 2005-11-03 |
US8148340B2 (en) | 2012-04-03 |
US20040152657A1 (en) | 2004-08-05 |
US20050245477A1 (en) | 2005-11-03 |
US20070009482A9 (en) | 2007-01-11 |
US20060094683A1 (en) | 2006-05-04 |
US20040142469A1 (en) | 2004-07-22 |
US20040143112A1 (en) | 2004-07-22 |
US20050037403A1 (en) | 2005-02-17 |
US20040181045A1 (en) | 2004-09-16 |
US8309527B2 (en) | 2012-11-13 |
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