WO2007055682A2 - Immunostimulatory properties of oligonucleotide-based compounds comprising modified immunostimulatory dinucleotides - Google Patents

Immunostimulatory properties of oligonucleotide-based compounds comprising modified immunostimulatory dinucleotides Download PDF

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WO2007055682A2
WO2007055682A2 PCT/US2005/040215 US2005040215W WO2007055682A2 WO 2007055682 A2 WO2007055682 A2 WO 2007055682A2 US 2005040215 W US2005040215 W US 2005040215W WO 2007055682 A2 WO2007055682 A2 WO 2007055682A2
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immunostimulatory oligonucleotide
administering
tctgtr
oligonucleotide
immunostimulatory
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PCT/US2005/040215
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French (fr)
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WO2007055682A8 (en
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Sudhir Agrawal
Dong Yu
Ekambar R. Kandimalla
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Idera Pharmaceuticals
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Priority to PCT/US2005/040215 priority Critical patent/WO2007055682A2/en
Priority to EP05851389A priority patent/EP1945653A4/en
Priority to AU2005338050A priority patent/AU2005338050B2/en
Priority to CNA2005800524839A priority patent/CN101351469A/en
Priority to CA002630118A priority patent/CA2630118A1/en
Priority to MX2008006004A priority patent/MX2008006004A/en
Priority to JP2008538854A priority patent/JP2009514525A/en
Publication of WO2007055682A2 publication Critical patent/WO2007055682A2/en
Publication of WO2007055682A8 publication Critical patent/WO2007055682A8/en

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    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/117—Nucleic acids having immunomodulatory properties, e.g. containing CpG-motifs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/04—Drugs for disorders of the respiratory system for throat disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/17—Immunomodulatory nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/318—Chemical structure of the backbone where the PO2 is completely replaced, e.g. MMI or formacetal
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/321—2'-O-R Modification
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/33—Chemical structure of the base

Definitions

  • the invention relates to immunology and immunotherapy applications using oligonucleotides as immunostimulatory agents.
  • Oligonucleotides have become indispensable tools in modern molecular biology, being used in a wide variety of techniques, ranging from diagnostic probing methods to PCR to antisense inhibition of gene expression and immunotherapy applications. This widespread use of oligonucleotides has led to an increasing demand for rapid, inexpensive and efficient methods for synthesizing oligonucleotides.
  • oligonucleotides for antisense and diagnostic applications can now be routinely accomplished. See, e.g., Methods in Molecular Biology, Vol. 20: Protocols for Oligonucleotides and Analogs pp. 165-189 (S. Agrawal, ed., Humana Press, 1993); Oligonucleotides and Analogues, A Practical Approach, pp. 87-108 (F. Eckstein, ed., 1991); and Uhlmann and Peyman, supra; Agrawal and Iyer, Curr. Op. in Biotech. 6:12 (1995); and Antisense Research and Applications (Crooke and Lebleu, eds., CRC Press, Boca Raton, 1993).
  • oligonucleotides as immunostimulatory agents in immunotherapy applications.
  • the observation that phosphodiester and phosphorothioate oligonucleotides can induce immune stimulation has created interest in developing this side effect as a therapeutic tool.
  • These efforts have focused on phosphorothioate oligonucleotides containing the dinucleotide natural CpG. Kuramoto et al., Jpn. J. Cancer Res. 83:1128-1131 (1992) teaches that phosphodiester oligonucleotides containing a palindrome that includes a CpG dinucleotide can induce interferon-alpha and gamma synthesis and enhance natural killer activity.
  • CpG-containing phosphorothioate oligonucleotides can also affect their ability to act as modulators of immune response. See, e.g., Zhao et al., Biochem. Pharmacol. (1996) 51 :173-182; Zhao et al., Biochem Pharmacol. (1996) 52:1537-1544;
  • the invention provides methods for modulating the immune response caused by oligonucleotide compounds.
  • the methods according to the invention enable modifying the cytokine profile produced by immunostimulatory oligonucleotides for immunotherapy applications.
  • the present inventors have surprisingly discovered that modification of immunostimulatory dinucleotides allows flexibility in the nature of the immune response produced and that certain modifications overcome the species specificities observed to date of the immunostimulatory sequences.
  • the invention provides an immunostimulatory oligonucleotide having a structure from the group of 5'-TCTGTROTTCT-X-TCTTGRTGTCT-S'; 5'-
  • the invention provides pharmaceutical compositions. These compositions comprise any one of the compositions disclosed in the first aspect of the invention and a pharmaceutically acceptable carrier.
  • the invention provides a method for generating an immune response in a vertebrate, the method comprising administering to the vertebrate an immunostimulatory oligonucleotide having a structure from the group of 5 ' -TCTGTR' GTTCT-X- TCTTGR'TGTCT-5'; 5 5 -TCTGTR 5 GTTC 1 U 1 -X-U 1 C 1 TTGR 5 TGTCT-5 5 ; 5'- CTGTR'GTTCTC-X-CTCTTGR'TGTC-5'; 5 '-CTGTR 5 GTTCU 1 C 1 -X-
  • the invention provides a method for therapeutically treating a vertebrate having cancer, an autoimmune disorder, airway inflammation, inflammatory disorders, skin disorders, allergy, asthma or a disease caused by a pathogen, such method comprising administering to the patient an immunostimulatory oligonucleotide having a structure from the group of 5' -TCTGTR' GTTCT-X-TCTTGR 5 TGTCT-5 5 ; 5'-
  • the invention provides a method for preventing cancer, an autoimmune disorder, airway inflammation, inflammatory disorders, skin disorders, allergy, asthma or a disease caused by a pathogen in a vertebrate, such method comprising administering to the vertebrate an immunostimulatory oligonucleotide having a structure
  • Figure 1 depicts a group of representative small molecule linkers suitable for linear synthesis of immunostimulatory oligonucleotides of the invention.
  • Figure 2 depicts a group of representative small molecule linkers suitable for parallel synthesis of immunostimulatory oligonucleotides of the invention.
  • Figure 3 is a synthetic scheme for the linear synthesis of irnmunostimulatory oligonucleotides of the invention.
  • DMTr 4,4'-dimethoxytrityl
  • CE cyanoethyl.
  • Figure 4 is a synthetic scheme for the parallel synthesis of immunostimulatory oligonucleotides of the invention.
  • DMTr 4,4'-dimethoxytrityl
  • CE cyanoethyl.
  • Figure 5 is a schematic representation of the 3 '-terminal nucleoside of an oligonucleotide, showing that a non-nucleotidic linkage can be attached to the nucleoside at the nucleobase, at the 3' position, or at the 2' position.
  • Figure 6 shows IL- 12 induction in C57BL/6 mouse spleen cell cultures by immunostimulatory oligonucleotides of the invention.
  • Figure 7 shows IL-6induction in C57BL/6 mouse spleen cell cultures by immunostimulatory oligonucleotides of the invention.
  • Figure 8 shows IFN- ⁇ induction in human pDC cultures by immunosthnulatory oligonucleotides of the invention.
  • Figure 9 shows IFN- ⁇ induction in human PBMC cultures by immunostimulatory oligonucleotides of the invention.
  • Figure 10 shows Human B cell proliferation by immunostimulatory oligonucleotides of the invention.
  • the invention relates to the therapeutic use of oligonucleotides as immunostimulatory agents for immunotherapy applications.
  • the issued patents, patent applications, and references that are cited herein are hereby incorporated by reference to the same extent as if each was specifically and individually indicated to be incorporated by reference, hi the event of inconsistencies between any teaching of any reference cited herein and the present specification, the latter shall prevail for purposes of the invention.
  • the invention provides methods for enhancing the immune response caused by immunostimulatory compounds used for immunotherapy applications such as, but not limited to, treatment of cancer, autoimmune disorders, asthma, respiratory allergies, food allergies, and bacteria, parasitic, and viral infections in adult and pediatric human and veterinary applications.
  • the invention further provides compounds having optimal levels of immunostimulatory effect for immunotherapy and methods for making and using such compounds, hi addition, compounds of the invention are useful as adjuvants in combination with DNA vaccines, antibodies, and allergens; and in combination with chemotherapeutic agents and/or antisense oligonucleotides.
  • the present inventors have surprisingly discovered that modification of an immunostimulatory oligonucleotide to optimally present its 5' ends dramatically affects its immunostimulatory capabilities, hi addition, the present inventors have discovered that the cytokine profile and species specificity of an immune response can be modulated by using novel purine or pyrimidine structures as part of an immunostimulatory oligonucleotide.
  • the invention provides immunostimulatory oligonucleotides alone or comprising at least two oligonucleotides linked at their 3' ends, or an internucleoside linkage or a functionalized nucleobase or sugar to a non-nucleotidic linker, at least one of the oligonucleotides being an immunostimulatory oligonucleotide and having an accessible 5' end.
  • the term "accessible 5' end" means that the 5' end of the oligonucleotide is sufficiently available such that the factors that recognize and bind to oligonucleotide and stimulate the immune system have access to it.
  • the 5' OH position of the terminal sugar is not covalently linked to more than two nucleoside residues or any other moiety that interferes with interaction with
  • the 5' OH can be linked to a phosphate, phosphorothioate, or phosphorodithioate moiety, an aromatic or aliphatic linker, cholesterol, or another entity which does not interfere with accessibility.
  • the immunostimulatory oligonucleotides according to the invention preferably further comprise an immunostimulatory dinucleotide comprising a novel purine or pyrimidine.
  • the immunostimulatory oligonucleotides include a ribozyme or a decoy oligonucleotide.
  • ribozyme refers to an oligonucleotide that possesses catalytic activity.
  • the ribozyme binds to a specific nucleic acid target and cleaves the target.
  • decoy oligonucleotide refers to an oligonucleotide that binds to a transcription factor hi a sequence-specific manner and arrests transcription activity.
  • the ribozyme or decoy oligonucleotide exhibits secondary structure, including, without limitation, stem-loop or hairpin structures.
  • at least one oligonucleotide comprises poly (I)-poly (C).
  • at least one set of Nn includes a string of 3 to 10 dGs and/or Gs or T- substituted ribo or arabino Gs.
  • oligonucleotide refers to a polynucleoside formed from a plurality of linked nucleoside units. Such oligonucleotides can be obtained from existing nucleic acid sources, including genomic or cDNA, but are preferably produced by synthetic methods.
  • each nucleoside unit includes a heterocyclic base and a pentofuranosyl, trehalose, arabinose, 2'-deoxy-2'-substituted arabinose, 2'-O- substituted arabinose or hexose sugar group.
  • the nucleoside residues can be coupled to each other by any of the numerous known internucleoside linkages.
  • internucleoside linkages include, without limitation, phosphodiester, phosphorothioate, phosphorodithioate, alkylphosphonate, alkylphosphonothioate, phosphotriester, phosphoramidate, siloxane, carbonate, carboalkoxy, acetamidate, carbamate, morpholino, borano, thioether, bridged phosphoramidate, bridged methylene phosphonate, bridged phosphorothioate, and sulfone internucleoside linkages.
  • oligonucleotide also encompasses polynucleosides having one or more stereospecific internucleoside linkage (e.g., (Rp)- or (Sp)- phosphorothioate, alkylphosphonate, or phosphotriester linkages).
  • Rp stereospecific internucleoside linkage
  • Sp phosphorothioate
  • alkylphosphonate alkylphosphonate
  • phosphotriester linkages e.g., phosphotriester linkages
  • these internucleoside linkages may be phosphodiester, phosphorothioate, or phosphorodithioate linkages, or combinations thereof.
  • the oligonucleotides each have from about 3 to about 35 nucleoside residues, preferably from about 4 to about 30 nucleoside residues, more preferably from about 4 to about 20 nucleoside residues.
  • the immunostimulatory oligonucleotides comprise oligonucleotides have from about 5 to about 18, or from about 5 to about 14, nucleoside residues. As used herein, the term "about” implies that the exact number is not critical.
  • the number of nucleoside residues in the oligonucleotides is not critical, and oligonucleotides having one or two fewer nucleoside residues, or from one to several additional nucleoside residues are contemplated as equivalents of each of the embodiments described above.
  • one or more of the oligonucleotides have 11 nucleotides.
  • preferred embodiments have from about 13 to about 35 nucleotides, more preferably from about 13 to about 26 nucleotides.
  • oligonucleotide also encompasses polynucleosides having additional substituents including, without limitation, protein groups, lipophilic groups, intercalating agents, diamines, folic acid, cholesterol and adamantane.
  • oligonucleotide also encompasses any other nucleobase containing polymer, including, without limitation, peptide nucleic acids (PNA), peptide nucleic acids with phosphate groups (PHONA), locked nucleic acids (LNA), morpholino-backbone oligonucleotides, and oligonucleotides having backbone sections with alkyl linkers or amino linkers.
  • PNA peptide nucleic acids
  • PONA peptide nucleic acids with phosphate groups
  • LNA locked nucleic acids
  • morpholino-backbone oligonucleotides oligonucleotides having backbone sections with alkyl linkers or amino linkers.
  • the oligonucleotides of the invention can include naturally occurring nucleosides, modified nucleosides, or mixtures thereof.
  • modified nucleoside is a nucleoside that includes a modified heterocyclic base, a modified sugar moiety, or a combination thereof.
  • the modified nucleoside is a non-natural pyrimidine or purine nucleoside, as herein described.
  • the modified nucleoside is a 2'-substituted ribonucleoside an arabinonucleoside or a 2'-deoxy-2'- substituted-arabinoside.
  • the term "2'-substituted ribonucleoside” or "2'- substituted arabinoside” includes ribonucleosides or arabinonucleoside in which the hydroxyl
  • EV 488513839 US group at the 2' position of the pentose moiety is substituted to produce a 2 '-substituted or 2'- O-substituted ribonucleoside.
  • substitution is with a lower alkyl group containing 1-6 saturated or unsaturated carbon atoms, or with an aryl group having 6-10 carbon atoms, wherein such alkyl, or aryl group may be unsubstituted or may be substituted, e.g., with halo, hydroxy, trifluoromethyl, cyano, nitro, acyl, acyloxy, alkoxy, carboxyl, carboalkoxy, or amino groups.
  • 2'-0-substituted ribonucleosides or 2'-O- substituted-arabinosides include, without limitation 2'-O-methylribonucleosides or 2'-O- methylarabinosides and 2'-O-methoxyethylribonucleosides or 2'-O- methoxyethylarabinosides.
  • 2'-substituted ribonucleoside or "2'-substituted arabinoside” also includes ribonucleosides or arabinonucleosides in which the 2'-hydroxyl group is replaced with a lower alkyl group containing 1-6 saturated or unsaturated carbon atoms, or with an amino or halo group.
  • Examples of such 2' -substituted ribonucleosides or 2' -substituted arabinosides include, without limitation, 2'-amino, 2'-fluoro, 2'-allyl, and 2'-propargyl ribonucleosides or arabinosides.
  • oligonucleotide includes hybrid and chimeric oligonucleotides.
  • a "chimeric oligonucleotide” is an oligonucleotide having more than one type of internucleoside linkage.
  • One preferred example of such a chimeric oligonucleotide is a chimeric oligonucleotide comprising a phosphorothioate, phosphodiester or phosphorodithioate region and non-ionic linkages such as alkylphosphonate or alkylphosphonothioate linkages (see e.g., Pederson et al. U.S. Patent Nos. 5,635,377 and 5,366,878).
  • hybrid oligonucleotide is an oligonucleotide having more than one type of nucleoside.
  • One preferred example of such a hybrid oligonucleotide comprises a ribonucleotide or 2'-substituted ribonucleotide region, and a deoxyribonucleotide region (see, e.g., Metelev and Agrawal, U.S. Patent No. 5,652,355, 6,346,614 and 6,143,881).
  • the term "immunostimulatory oligonucleotide” refers to an oligonucleotide as described above that induces an immune response when administered to a vertebrate, such as a fish, fowl, or mammal.
  • a vertebrate such as a fish, fowl, or mammal.
  • mammal includes, without limitation rats, mice, cats, dog ⁇ 5 horses, cattle, cows, pigs,
  • the immunostimulatory oligonucleotide comprises at least one phosphodiester, phosphorothioate, or phosphorodithioate internucleoside linkage.
  • the immunostimulatory oligonucleotide comprises an immunostimulatory dinucleotide of formula 5'-Pyr-Pur-3', wherein Pyr is a natural or synthetic pyrimidine nucleoside and Pur is a natural or synthetic purine nucleoside.
  • the immunostimulatory oligonucleotide comprises an immunostimulatory dinucleotide of formula 5'-Pur*-Pur-3', wherein Pur* is a synthetic purine nucleoside and Pur is a natural or synthetic purine nucleoside, hi various places the dinucleotide is expressed as RpG, C*pG or YZ, in which case respectively, R, C* 5 or Y represents a synthetic purine.
  • a particularly preferred synthetic purine is 2-oxo-7-deaza-8- methyl-purine.
  • pyrimidine nucleoside refers to a nucleoside wherein the base component of the nucleoside is a monocyclic nucleobase.
  • purine nucleoside refers to a nucleoside wherein the base component of the nucleoside is a bicyclic nucleobase.
  • a “synthetic" pyrimidine or purine nucleoside includes a non-naturally occurring pyrimidine or purine base, a non-naturally occurring sugar moiety, or a combination thereof.
  • Preferred pyrimidine nucleosides according to the invention have the structure (7):
  • D is a hydrogen bond donor
  • D' is selected from the group consisting of hydrogen, hydrogen bond donor, hydrogen bond acceptor, hydrophilic group, hydrophobic group, electron withdrawing group and electron donating group;
  • A is a hydrogen bond acceptor or a hydrophilic group
  • A' is selected from the group consisting of hydrogen bond acceptor, hydrophilic group, hydrophobic group, electron withdrawing group and electron donating group;
  • X is carbon or nitrogen;
  • S 1 is a pentose or hexose sugar ring, or a non-naturally occurring sugar.
  • the sugar ring is derivatized with a phosphate moiety, modified phosphate moiety, or other linker moiety suitable for linking the pyrimidine nucleoside to another nucleoside or nucleoside analog.
  • Preferred hydrogen bond donors include, without limitation, -NH-, -NH 2 , -SH and
  • the base moiety in (i) is a non-naturally occurring pyrimidine base.
  • non-naturally occurring pyrimidine bases include, without limitation, 5-hydroxycytosine, 5-hydroxymethylcytosine, N4-alkylcytosine, preferably N4- ethylcytosine, and 4-thiouracil.
  • 5-bromocytosine is specifically excluded.
  • the sugar moiety S' in (i) is a non-naturally occurring sugar moiety.
  • a "naturally occurring sugar moiety” is a sugar moiety that occurs naturally as part of nucleic acid, e.g., ribose and 2'-deoxyribose
  • a "non-naturally occurring sugar moiety” is any sugar that does not occur naturally as part of a nucleic acid, but which can be used in the backbone for an oligonucleotide, e.g, hexose.
  • Arabinose and arabinose derivatives are examples of preferred sugar moieties.
  • D is a hydrogen bond donor;
  • D' is selected from the group consisting of hydrogen, hydrogen bond donor, and hydrophilic group;
  • A is a hydrogen bond acceptor or a hydrophilic group
  • X is carbon or nitrogen
  • each L is independently an atom selected from the group consisting of C, O, N and S;
  • S 1 is a pentose or hexose sugar ring, or a non-naturally occurring sugar.
  • the sugar ring is derivatized with a phosphate moiety, modified phosphate moiety, or other linker moiety suitable for linking the pyrimidine nucleoside to another nucleoside or nucleoside analog.
  • Preferred hydrogen bond donors include, without limitation, -NH-, -NH 2 , -SH and
  • the base moiety in (II) is a non-naturally occurring purine base.
  • preferred non-naturally occurring purine bases include, without limitation, 2-amino-6-thiopurine and 2-amino-6-oxo-7-deazapurine.
  • the sugar moiety S' in (II) is a naturally occurring sugar moiety, as described above for structure (I).
  • the immunostimulatory dinucleotide is selected from the group consisting of CpG, C*pG, CpG*, and C*pG*, wherein the base of C is cytosine, the base of C* is 2'-thymine, 5-hydroxycytosine, N4-alkyl-cytosine, 4-thiouracil or other non- natural pyrimidine, or 2-oxo-7-deaza-8-methylpurine, wherein when the base is 2-oxo-7- deaza-8-methyl-purine, it is preferably covalently bound to the 1 '-position of a pentose via the 1 position of the base; the base of G is guanosine, the base of G* is 2-amino-6-oxo-7- deazapurine, 2-oxo-7-deaza-8-methylpurine, 6-thioguanine, 6-oxopurine, or other non- natural purine nucleoside, and p is an internu
  • the immunostimulatory oligonucleotides may include immunostimulatory moieties on one or both sides of the immunostimulatory dinucleotide.
  • the immunostimulatory oligonucleotide comprises an immunostimulatory domain of structure (III): 5'-Nn-Nl-Y-Z-Nl-Nn-3' (III) wherein: the base of Y is cytosine, thymine, 5-hydroxycytosine, N4-alkyl-cytosine, 4- thiouracil or other non-natural pyrimidine nucleoside, or 2-oxo-7-deaza-8 methyl purine, wherein when the base is 2-oxo-7-deaza-8-methyl-purine, it is preferably covalently bound to the 1 '-position of a pentose via the 1 position of the base; the base of Z is guanine, 2-amino-6-oxo-7-deazapurine, 2-oxo-7deaza-8- methyl
  • Nl and Nn independent at each occurrence, is preferably a naturally occurring or a synthetic nucleoside or an immunostimulatory moiety selected from the group consisting of abasic nucleosides, arabinonucleosides, 2'-deoxyuridine, ⁇ -deoxyribonucleosides, ⁇ -L-deoxyribonucleosides, and nucleosides linked by a phosphodiester or modified internucleoside linkage to the adjacent nucleoside on the 3' side, the modified internucleotide linkage being selected from, without limitation, a linker having a length of from about 2
  • YZ is arabinocytidine or 2 '-deoxy-2' -substituted arabinocytidine and arabinoguanosine or 2 'deoxy-2 '-substituted arabinoguanosine.
  • Preferred immunostimulatory moieties include natural phosphodiester backbones and modifications in the phosphate backbones, including, without limitation, methylphosphonates, methylphosphonothioates, phosphotriesters, phosphothiotriesters, phosphorothioates, phosphorodithioates, triester prodrugs, sulfones, sulfonamides, sulfamates, formacetal, N- methylhydroxylamine, carbonate, carbamate, morpholino, boranophosphonate, phosphoramidates, especially primary amino-phosphoramidates, N3 phosphoramidates and N5 phosphoramidates, and stereospecific linkages (e.g., (Rp)- or (Sp)-phosphorothioate, alkylphosphonate, or phosphotriester linkages).
  • methylphosphonates methylphosphonothioates
  • phosphotriesters phosphothiotriesters
  • phosphorothioates phospho
  • Preferred immunostimulatory moieties according to the invention further include nucleosides having sugar modifications, including, without limitation, 2 '-substituted pentose sugars including, without limitation, 2'-O-methylribose, 2'-O-methoxyethylribose, 2'-O- propargylribose, and 2'-deoxy-2'-fluororibose; 3 '-substituted pentose sugars, including, without limitation, 3'-O-methylribose; r,2'-dideoxyribose; arabinose; substituted arabinose sugars, including, without limitation, l'-methylarabinose, 3'-hydroxymethylarabinose, 4'-hydroxymethylarabinose, 3'-hydroxyarabinose and 2'-substituted arabinose sugars; hexose sugars, including, without limitation, 1,5-anhydrohexitol; and alpha-anomers.
  • the immunostimulatory moiety is attached to the adjacent nucleoside by way of a 2'-5' internucleoside linkage.
  • EV 488513839 US Preferred immunostimulatory moieties according to the invention further include oligonucleotides having other carbohydrate backbone modifications and replacements, including peptide nucleic acids (PNA), peptide nucleic acids with phosphate groups (PHONA), locked nucleic acids (LNA), morpholino backbone oligonucleotides, and oligonucleotides having backbone linker sections having a length of from about 2 angstroms to about 200 angstroms, including without limitation, alkyl linkers or amino linkers.
  • the alkyl linker may be branched or unbranched, substituted or unsubstituted, and chirally pure or a racemic mixture.
  • alkyl linkers have from about 2 to about 18 carbon atoms. In some preferred embodiments such alkyl linkers have from about 3 to about 9 carbon atoms.
  • Some alkyl linkers include one or more functional groups selected from the group consisting of hydroxy, amino, thiol, thioether, ether, amide, thioamide, ester, urea, and thioether.
  • Some other functionalized alkyl linkers are peptides or amino acids.
  • Preferred immunostimulatory moieties according to the invention further include
  • DNA isoforms including, without limitation, ⁇ -L-deoxyribonucleosides and ⁇ -deoxyribonucleosides.
  • Preferred immunostimulatory moieties according to the invention incorporate 3' modifications, and further include nucleosides having unnatural internucleoside linkage positions, including, without limitation, 2 '-5', 2'-2', 3 '-3' and 5 '-5' linkages.
  • Preferred immunostimulatory moieties according to the invention further include nucleosides having modified heterocyclic bases, including, without limitation, 5-hydroxycytosine, 5-hydroxymethylcytosine, N4-alkylcytosine, preferably N4-ethylcytosine, 4-thiouracil, 6-thioguanine, 7-deazaguanine, inosine, nitropyrrole, C5-propynylpyrimidine, and diaminopurines, including, without limitation, 2,6-diaminopurine.
  • modified heterocyclic bases including, without limitation, 5-hydroxycytosine, 5-hydroxymethylcytosine, N4-alkylcytosine, preferably N4-ethylcytosine, 4-thiouracil, 6-thioguanine, 7-deazaguanine, inosine, nitropyrrole, C5-propynylpyrimidine, and diaminopurines, including, without limitation, 2,6-diaminopurine.
  • a methylphosphonate internucleoside linkage at position Nl or Nn is an immunostimulatory moiety
  • a linker having a length of from about 2 angstroms to about 200 angstroms, C2-C18 alkyl linker at position Xl is an immunostimulatory moiety
  • EV 488513839 US immunostimulatory moiety and a ⁇ -L-deoxyribonucleoside at position Xl is an immunostimulatory moiety. See Table 1 below for representative positions and structures of immunostimulatory moieties. It is to be understood that reference to a linker as the immunostimulatory moiety at a specified position means that the nucleoside residue at that position is substituted at its 3'-hydroxyl with the indicated linker, thereby creating a modified internucleoside linkage between that nucleoside residue and the adjacent nucleoside on the 3' side.
  • reference to a modified internucleoside linkage as the immunostimulatory moiety at a specified position means that the nucleoside residue at that position is linked to the adjacent nucleoside on the 3' side by way of the recited linkage.
  • Table 2 shows representative positions and structures of immunostimulatory moieties within an immunostimulatory oligonucleotide having an upstream potentiation domain.
  • Spacer 9 refers to a poly(ethylene glycol) linker of formula -0-(CH 2 CH 2 -O) n -, wherein n is 3.
  • Spacer 18 refers to a polyethylene glycol) linker of formula -0-(CH 2 CH 2 -O) n -, wherein n is 6.
  • C2-C18 alkyl linker refers to a linker of formula -O-(CH 2 ) ? -O-, where q is an integer from 2 to 18.
  • C3-linker and C3-alkyl linker refer to a linker of formula -O-(CH 2 ) 3 -O-.
  • the linker is connected to the adjacent nucleosides by way of phosphodiester, phosphorothioate, or phosphorodithioate linkages.
  • Table 3 shows representative positions and structures of immunostimulatory moieties within an immunostimulatory oligonucleotide having a downstream potentiation domain.
  • the immunostimulatory oligonucleotides according to the invention comprise at least two oligonucleotides linked at their 3' ends or internucleoside linkage or a functionalized nucleobase or sugar via a non-nucleotidic linker.
  • a "non- nucleotidic linker” is any moiety that can be linked to the oligonucleotides by way of covalent or non-covalent linkages.
  • linker is from about 2 angstroms to about 200 angstroms in length.
  • Non- covalent linkages include, but are not limited to, electrostatic interaction, hydrophobic interactions, ⁇ -stacking interactions, and hydrogen bonding.
  • the term "non-nucleotidic linker" is not meant to refer to an internucleoside linkage, as described above, e.g., a phosphodiester, phosphorothioate, or phosphorodithioate functional group, that directly connects the 3'-hydroxyl groups of two nucleosides.
  • a direct 3 -3' linkage is considered to be a "nucleotidic linkage.”
  • the non-nucleotidic linker is a metal, including, without limitation, gold particles, hi some other embodiments, the non-nucleotidic linker is a soluble or insoluble biodegradable polymer bead.
  • the non-nucleotidic linker is an organic moiety having functional groups that permit attachment to the oligonucleotide. Such attachment preferably is by any stable covalent linkage.
  • the linker may be attached to any suitable position on the nucleoside, as illustrated in Figure 5.
  • the linker is attached to the 3'-hydroxyl.
  • the linker preferably comprises a hydroxyl functional group, which preferably is attached to the 3'- hydroxyl by means of a phosphodiester, phosphorothioate, phosphorodithioate or non- phosphate-based linkages.
  • the non-nucleotidic linker is a biomolecule, including, without limitation, polypeptides, antibodies, lipids, antigens, allergens, and oligosaccharides.
  • the non-nucleotidic linker is a small molecule.
  • a small molecule is an organic moiety having a molecular weight of less than 1,000 Da. In some embodiments, the small molecule has a molecular weight of less than 750 Da.
  • the small molecule is an aliphatic or aromatic hydrocarbon, either of which optionally can include, either in the linear chain connecting the oligonucleotides or appended to it, one or more functional groups selected from the group consisting of hydroxy, amino, thiol, thioether, ether, amide, thioamide, ester, urea, and thiourea.
  • the small molecule can be cyclic or acyclic. Examples of small molecule linkers include, but are not limited to, amino acids, carbohydrates, cyclodextrins, adamantane, cholesterol, haptens and antibiotics.
  • the term "small molecule” is not intended to include a nucleoside.
  • the small molecule linker is glycerol or a glycerol homolog of the formula HO-(CH 2 ) o -CH(OH)-(CH 2 ) p -OH, wherein o and p independently are integers from 1 to about 6, from 1 to about 4, or from 1 to about 3.
  • the small molecule linker is a derivative of l,3-diamino-2-hydroxypropane.
  • Some such derivatives have the formula HO-(CH 2 ) m -C(O)NH-CH 2 -CH(OH)-CH 2 -NHC(O)-(CH 2 ) w -OH, wherein m is an integer from O to about 10, from 0 to about 6, from 2 to about 6, or from 2 to about 4.
  • Some non-nucleotidic linkers according to the invention permit attachment of more than two oligonucleotides.
  • the small molecule linker glycerol has three hydroxyl groups to which oligonucleotides may be covalently attached.
  • Some immunostimulatory oligonucleotides according to the invention therefore, comprise more than two oligonucleotides linked at their 3' ends to a non-nucleotidic linker.
  • the immunostimulatory oligonucleotides of the invention may conveniently be synthesized using an automated synthesizer and phosphoramidite approach as schematically depicted in Figures 3 and 4, and further described in the Examples, hi some embodiments, the immunostimulatory oligonucleotides are synthesized by a linear synthesis approach (see Figure 3).
  • linear synthesis refers to a synthesis that starts at one end of the immunostimulatory oligonucleotide and progresses linearly to the other end. Linear synthesis permits incorporation of either identical or un-identical (in terms of length, base composition and/or chemical modifications incorporated) monomeric units into the immunostimulatory oligonucleotides.
  • An alternative mode of synthesis is "parallel synthesis", in which synthesis proceeds outward from a central linker moiety (see Figure 4).
  • a solid support attached linker can be used for parallel synthesis, as is described in U.S. Patent No. 5,912,332.
  • a universal solid support such as phosphate attached controlled pore glass support can be used.
  • Parallel synthesis of immunostimulatory oligonucleotides has several advantages over linear synthesis: (1) parallel synthesis permits the incorporation of identical monomeric units; (2) unlike in linear synthesis, both (or all) the monomeric units are synthesized at the same time, thereby the number of synthetic steps and the time required for the synthesis is the same as that of a monomeric unit; and (3) the reduction in synthetic steps improves purity and yield of the final immunostimulatory oligonucleotide product.
  • the immunostimulatory oligonucleotides may conveniently be deprotected with concentrated ammonia solution or as recommended by the phosphoramidite supplier, if a modified nucleoside is incorporated.
  • the product immunostimulatory oligonucleotide is preferably purified by reversed phase HPLC, detritylated, desalted and dialyzed.
  • Table 4 shows representative immunostrmulatory oligonucleotides according to the invention.
  • R' l-(2'-deoxy- ⁇ -D-ribofuranosyl)-2-oxo-7-deaza-8-methyl- ⁇ urine;
  • a 1 ZC 1 ZG 1 ZU 1 2'-O- methyl-ribonucleotides;
  • R 2 5 -deoxy-7-deazaguanosine;
  • X glycerol linker.
  • the invention provides immunostimulatory oligonucleotide conjugates comprising an immunostimulatory oligonucleotide, as described above, and an antigen conjugated to the immunostimulatory oligonucleotide at a position other than the accessible 5' end.
  • the non-nucleotidic linker comprises an antigen, which is conjugated to the oligonucleotide.
  • the antigen is conjugated to the oligonucleotide at a position other than its 3' end.
  • the antigen produces a vaccine effect.
  • the antigen is preferably selected from the group consisting of antigens associated with a pathogen, antigens associated with a cancer, antigens associated with an auto-immune disorder, and antigens associated with other diseases such as, but not limited to, veterinary or pediatric diseases.
  • the term "associated with 55 means that the antigen is present when the pathogen, cancer, auto-immune disorder, food allergy, respiratory allergy, asthma or other disease is present, but either is not present, or is present in reduced amounts, when the pathogen, cancer, auto-immune disorder, food allergy, respiratory allergy, or disease is absent.
  • the immunostimulatory oligonucleotide is covalently linked to the antigen, or it is otherwise operatively associated with the antigen.
  • the term "operatively associated with” refers to any association that maintains the activity of both immunostimulatory oligonucleotide and antigen. Nonlimiting examples of such operative associations include being part of the same liposome or other such delivery vehicle or reagent.
  • such covalent linkage preferably is at any position on the
  • the antigen may be attached at an internucleoside linkage or may be attached to the non-nucleotidic linker. Alternatively, the antigen may itself be the non-nucleotidic linker.
  • the invention provides pharmaceutical formulations comprising an immunostimulatory oligonucleotide or immunostimulatory oligonucleotide conjugate according to the invention and a physiologically acceptable carrier.
  • physiologically acceptable refers to a material that does not interfere with the effectiveness of the immunostimulatory oligonucleotide and is compatible with a biological system such as a cell, cell culture, tissue, or organism.
  • a biological system such as a cell, cell culture, tissue, or organism.
  • the biological system is a living organism, such as a vertebrate.
  • carrier encompasses any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, or other material well known in the art for use in pharmaceutical formulations. It will be understood that the characteristics of the carrier, excipient, or diluent will depend on the route of administration for a particular application. The preparation of pharmaceutically acceptable formulations containing these materials is described in, e.g., Remington 's Pharmaceutical Sciences, 18th Edition, ed. A. Gennaro, Mack Publishing Co., Easton, PA, 1990.
  • the invention provides methods for generating an immune response in a vertebrate, such methods comprising administering to the vertebrate an immunostimulatory oligonucleotide or immunostimulatory oligonucleotide conjugate according to the invention.
  • the vertebrate is a mammal.
  • the term "mammal" is expressly intended to include humans, hi preferred embodiments, the immunostimulatory oligonucleotide or immunostimulatory oligonucleotide conjugate is administered to a vertebrate in need of immunostimulation.
  • administration of immunostimulatory oligonucleotide or immunostimulatory oligonucleotide conjugate can be by any suitable route, including, without limitation, parenteral, oral, sublingual, transdermal, topical, intranasal, aerosol, intraocular, intratracheal, intrarectal, vaginal, by gene gun, dermal patch or in eye drop or mouthwash form.
  • parenteral oral, sublingual, transdermal
  • topical intranasal
  • aerosol intraocular, intratracheal, intrarectal, vaginal
  • the therapeutic composition is preferably administered at a sufficient dosage to attain a blood level of immunostimulatory oligonucleotide from about 0.0001 micromolar to about 10 micromolar. For localized administration, much lower concentrations than this may be effective, and much higher concentrations may be tolerated.
  • a total dosage of immunostimulatory oligonucleotide ranges from about 0.001 mg per patient per day to about 200 mg per kg body weight per day. It may be desirable to administer simultaneously, or sequentially a therapeutically effective amount of one or more of the therapeutic compositions of the invention to an individual as a single treatment episode.
  • immunostimulatory oligonucleotide or immunostimulatory oligonucleotide conjugate according to the invention are administered in combination with vaccines, antibodies, cytotoxic agents, allergens, antibiotics, antisense oligonucleotides, peptides, proteins, gene therapy vectors, DNA vaccines and/or adjuvants to enhance the specificity or magnitude of the immune response.
  • the immunostimulatory oligonucleotides of the invention can variously act as adjuvants and/or produce direct immunostimulatory effects.
  • Either the immunostimulatory oligonucleotide or immunostimulatory oligonucleotide conjugate or the vaccine, or both, may optionally be linked to an immunogenic protein, such as keyhole limpet hemocyanin (KXH), cholera toxin B subunit, or any other immunogenic carrier protein.
  • an immunogenic protein such as keyhole limpet hemocyanin (KXH), cholera toxin B subunit, or any other immunogenic carrier protein.
  • KXH keyhole limpet hemocyanin
  • MPL monophosphoryl lipid A
  • alum and saponins, including QS-21, imiquimod, R848, or combinations thereof.
  • the term "in combination with” means in the course of treating the same disease in the same patient, and includes administering the immunostimulatory oligonucleotide and/or the vaccine and/or the adjuvant in any order, including simultaneous administration, as well as temporally spaced order of up to several days apart.
  • Such combination treatment may also include more than a single administration of the immunostimulatory oligonucleotide, and/or independently the vaccine, and/or
  • EV 488513839 US independently the adjuvant.
  • the administration of the immunostimulatory oligonucleotide and/or vaccine and/or adjuvant may be by the same or different routes.
  • the methods according to this aspect of the invention are useful for model studies of the immune system.
  • the methods are also useful for the prophylactic or therapeutic treatment of human or animal disease.
  • the methods are useful for pediatric and veterinary vaccine applications.
  • the invention provides methods for therapeutically treating a patient having a disease or disorder, such methods comprising administering to the patient an immunostimulatory oligonucleotide or immunostimulatory oligonucleotide conjugate according to the invention.
  • the disease or disorder to be treated is cancer, an autoimmune disorder, airway inflammation, inflammatory disorders, allergy, asthma or a disease caused by a pathogen.
  • Pathogens include bacteria, parasites, fungi, viruses, viroids and prions.
  • Administration is carried out as described for the fourth aspect of the invention.
  • allergy includes, without limitation, food allergies and respiratory allergies.
  • airway inflammation includes, without limitation, asthma.
  • the term "autoimmune disorder” refers to disorders in which "self proteins undergo attack by the immune system. Such term includes autoimmune asthma.
  • the immunostimulatory oligonucleotide or immunostimulatory oligonucleotide conjugate can be administered in combination with any other agent useful for treating the disease or condition that does not diminish the immunostimulatory effect of the immunostimulatory oligonucleotide.
  • the immunostimulatory oligonucleotide or immunostimulatory oligonucleotide conjugate may be administered in combination with a chemotherapeutic compound.
  • Oligonucleotides were synthesized on a 1 ⁇ mol to 0.1 mM scale using an automated DNA synthesizer (OligoPilot II, AKTA, (Amersham) and/or Expedite 8909 (Applied Biosystem)), following the linear synthesis or parallel synthesis procedures outlined in Figures 3 and 4.
  • Oligonucleotides were synthesized on a 1 ⁇ mol to 0.1 mM scale using an automated DNA synthesizer (OligoPilot II, AKTA, (Amersham) and/or Expedite 8909 (Applied Biosystem)), following the linear synthesis or parallel synthesis procedures outlined in Figures 3 and 4.
  • 5'-DMT dA, dG, dC and T phosphoramidites were purchased from Proligo (Boulder, CO). 5'-DMT 7-deaza-dG and araG phosphoramidites were obtained from Chemgenes (Wilmington, MA). DiDMT-glycerol linker solid support was obtained from Chemgenes. l-(2'-deoxy- ⁇ -D-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine amidite was obtained from Glen Research (Sterling, VA), 2'-O-methylribonuncleoside amidites were obtained from Promega (Obispo, CA).
  • oligonucleotides were phosphorothioate backbone modified. All nucleoside phosphoramidites were characterized by 31 P and 1 H NMR spectra. Modified nucleosides were incorporated at specific sites using normal coupling cycles recommended by the supplier. After synthesis, oligonucleotides were deprotected using concentrated ammonium hydroxide and purified by reverse phase HPLC, detritylation, followed by dialysis. Purified oligonucleotides as sodium salt form were lyophilized prior to use. Purity was tested by CGE and MALDI-TOF MS. Endotoxin levels were determined by LAL test and were below 1.0 EU/mg.
  • Example 2 Activity of short-immunostimulatory oligonucleotides in murine spleen cell cultures
  • C57/BL6 spleen cells were cultured with indicated concentrations of compounds. After 24 hours the supernatants were collected and the levels of IL- 12 and IL-6 were determined by ELISA. All immunostimulatory oligonucleotides showed a concentration- dependent induction of two typical cytokines, IL-12 and IL-6 ( Figures 6-7).
  • PBMCs Peripheral blood mononuclear cells
  • CBR Laboratories CBR Laboratories, Boston, MA
  • pDCs were isolated from PBMCs by positive selection using the BDCA4 cell isolation kits (Miltenyi Biotec) according to the manufacturer's instructions.
  • pDCs were plated in 96-well dishes using IXlO 6 cells/ml.
  • the IMOs dissolved in DPBS pH 7.4; Mediatech
  • the cells were then incubated at 37 0 C for 24 hr and the supernatants were collected for ELISA assays. The experiments were performed in triplicate wells. The levels of IFN- ⁇ were measured by sandwich ELISA.
  • Human PBMCs were plated in 48-well plates using 5X10 6 cells/ml.
  • the IMOs dissolved in DPBS (pH 7.4; Mediatech) were added to a final concentration of 10.0 ⁇ g/ml to the cell cultures.
  • the cells were then incubated at 37 °C for 24 hr and the supernatants were collected for ELISA assays.
  • the experiments were performed in triplicate wells.
  • the levels of IFN- ⁇ were measured by sandwich ELISA.
  • the required reagents, including cytokine antibodies and standards, were purchased from PharMingen.
  • the culture medium used for the assay consisted of RPMI 1640 medium supplemented with 1.5 mM glutamine, 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, 50 ⁇ M 2-mercaptoethanol, 100 IU/ml penicillin-streptomycin mix and 10% heat- inactivated fetal bovine serum.
  • a total of 0.5 X 10 6 B cells per ml i.e.l X lO 5 /200 ⁇ l/well) were stimulated in 96 well flat bottom plates with different concentrations of test oligonucleotides in triplicate for a total period of 72 hours.
  • EV 488513839 US cell harvester and radioactive incorporation was determined using standard liquid scintillation technique. The results are expressed either as mean cpm +/-SD or as proliferation index (cpm treated group/cpm medium control).

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Abstract

The invention relates to the therapeutic use of oligonucleotides as immunostimulatory agents in immunotherapy applications. More particularly, the invention provides an immunostimulatory oligonucleotides for use in methods for generating an immune response or for treating a patient in need of immunostimulation. The immunostimulatory oligonucleotides of the invention preferably comprise novel purines. The immunostimulatory oligonucleotides according to the invention further comprise at least two oligonucleotides linked at their 3' ends, internucleoside linkages or functionalized nucleobase or sugar to a non-nucleotidic linker, at least one of the oligonucleotides being an immunostimulatory oligonucleotide and having an accessible 5' end.

Description

IMMUNOSTIMULATORY PROPERTIES OF OLIGONUCLEOTIDE-BASED COMPOUNDS COMPRISING MODIFIED IMMUNOSTIMULATORY
DINUCLEOTIDES
(Attorney Docket No. IDR-039PC)
BACKGROUND OF THE INVENTION
Field of the Invention
The invention relates to immunology and immunotherapy applications using oligonucleotides as immunostimulatory agents.
Summary of the Related Art
Oligonucleotides have become indispensable tools in modern molecular biology, being used in a wide variety of techniques, ranging from diagnostic probing methods to PCR to antisense inhibition of gene expression and immunotherapy applications. This widespread use of oligonucleotides has led to an increasing demand for rapid, inexpensive and efficient methods for synthesizing oligonucleotides.
The synthesis of oligonucleotides for antisense and diagnostic applications can now be routinely accomplished. See, e.g., Methods in Molecular Biology, Vol. 20: Protocols for Oligonucleotides and Analogs pp. 165-189 (S. Agrawal, ed., Humana Press, 1993); Oligonucleotides and Analogues, A Practical Approach, pp. 87-108 (F. Eckstein, ed., 1991); and Uhlmann and Peyman, supra; Agrawal and Iyer, Curr. Op. in Biotech. 6:12 (1995); and Antisense Research and Applications (Crooke and Lebleu, eds., CRC Press, Boca Raton, 1993). Early synthetic approaches included phosphodiester and phosphotriester chemistries. For example, Khorana et al., J. Molec. Biol 72:209 (1972) discloses phosphodiester chemistry for oligonucleotide synthesis. Reese, Tetrahedron Lett. 34:3143-3179 (1978), discloses phosphotriester chemistry for synthesis of oligonucleotides and polynucleotides. These early approaches have largely given way to the more efficient phosphoramidite and H- phosphonate approaches to synthesis. For example, Beaucage and Caruthers, Tetrahedron Lett. 22:1859-1862 (1981), discloses the use of deoxyribonucleoside phosphoramidites in
EV 488513839 US polynucleotide synthesis. Agrawal and Zamecnik, U.S. Patent No. 5,149,798 (1992), discloses optimized synthesis of oligonucleotides by the H-phosphonate approach. Both of these modern approaches have been used to synthesize oligonucleotides having a variety of modified internucleotide linkages. Agrawal and Goodchild, Tetrahedron Lett. 28:3539-3542 (1987), teaches synthesis of oligonucleotide methylphosphonates using phosphoramidite chemistry. Connolly et al., Biochem. 23:3443 (1984), discloses synthesis of oligonucleotide phosphorothioates using phosphoramidite chemistry. Jager et al., Biochem. 27:7237 (1988), discloses synthesis of oligonucleotide phosphoramidates using phosphoramidite chemistry. Agrawal et al., Proc. Natl. Acad. Sd. (USA) 85:7079-7083 (1988), discloses synthesis of oligonucleotide phosphoramidates and phosphorothioates using H-phosphonate chemistry.
More recently, several researchers have demonstrated the validity of the use of oligonucleotides as immunostimulatory agents in immunotherapy applications. The observation that phosphodiester and phosphorothioate oligonucleotides can induce immune stimulation has created interest in developing this side effect as a therapeutic tool. These efforts have focused on phosphorothioate oligonucleotides containing the dinucleotide natural CpG. Kuramoto et al., Jpn. J. Cancer Res. 83:1128-1131 (1992) teaches that phosphodiester oligonucleotides containing a palindrome that includes a CpG dinucleotide can induce interferon-alpha and gamma synthesis and enhance natural killer activity. Krieg et al, Nature 371:546-549 (1995) discloses that phosphorothioate CpG-containing oligonucleotides are immunostimulatory. Liang et al. , J Clin. Invest. 98:1119-1129 (1996) discloses that such oligonucleotides activate human B cells. Moldoveanu et al, Vaccine 16:1216-124 (1998) teaches that CpG-containing phosphorothioate oligonucleotides enhance immune response against influenza virus. McCluskie and Davis, J Immunol. 161:4463-4466 (1998) teaches that CpG-containing oligonucleotides act as potent adjuvants, enhancing immune response against hepatitis B surface antigen. Hartman et al, J. Immunol 164: 1617- 1624 (2000) teaches that the immunostimulatory sequence is species specific, and different between mice and primates.
Other modifications of CpG-containing phosphorothioate oligonucleotides can also affect their ability to act as modulators of immune response. See, e.g., Zhao et al., Biochem. Pharmacol. (1996) 51 :173-182; Zhao et al., Biochem Pharmacol. (1996) 52:1537-1544;
-2-
EV 488513839 US Zhao et al, Antisense Nucleic Acid Drug Dev. (1997) 7:495-502; Zhao et al., Bioorg. Med. Chem. Lett. (1999) 9:3453-3458; Zhao et al., Bioorg. Med. Chem. Lett. (2000) 10:1051-1054; Yu et al., Bioorg. Med. Chem. Lett. (2000) 10:2585-2588; Yu et al., Bioorg. Med. Chem. Lett. (2001) 11:2263-2267; and Kandimalla et al., Bioorg. Med. Chem. (2001) 9:807-813.
These reports make clear that there remains a need to be able to modulate the immune response caused by immunostimulatory oligonucleotides and to overcome species specificity of the immunostmiulatory sequences.
-3-
EV 488513839 US BRIEF SUMMARY OF THE INVENTION
The invention provides methods for modulating the immune response caused by oligonucleotide compounds. The methods according to the invention enable modifying the cytokine profile produced by immunostimulatory oligonucleotides for immunotherapy applications. The present inventors have surprisingly discovered that modification of immunostimulatory dinucleotides allows flexibility in the nature of the immune response produced and that certain modifications overcome the species specificities observed to date of the immunostimulatory sequences.
In a first aspect the invention provides an immunostimulatory oligonucleotide having a structure from the group of 5'-TCTGTROTTCT-X-TCTTGRTGTCT-S'; 5'-
TCTGTR5GTTC1U1-X-U1C1TTGR5TGTCT-S'; 5 ' -CTGTR' GTTCTC-X-CTCTTGR' TGTC- 5'; 5'-CTGTR5GTTCU1C1-X-C1U1CTTGR5TGTC-S'; 5'-CTGTR5GTTC1U1C1-X- C1UIC1TTGR5TGTC-S5; S'-TCTGTR'GTTCT-X-CGTTCGAACGT-S'; 5'- TCTGTR5GACAG-X-GACAGR5TGTCT-55; 5'-TCTGTR5GACA1G1-X- G1A1CAGR5TGTCT-S5; 5' -TCAGTR' GTT AG-X-GATTGR5 TGACT-55; 5'- TCAGTR5GACTG-X-GTCAGR5TGACT-55; 5'-TR5GTR5GAR5GAT-X- TAGR'AGR'TGR'T-5'; 5'-TR5GTR5GTAGTA-X-ATGATGR5TGR5T-S'; 5'- TR5GAAR5GTTCT-X-TCTTGR5AAGR5T-55; 5'-TR5GTAR5GTACT-X- TCATGR5 ATGR'T-55 and 55-TCRAACRTTCR-X-RCTTRCAARCT-5^ wherein R5 = 1- (25-deoxy-β-D-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine; A1ZC1ZG1ZU1 = 2'-O-methyl- ribonucleotides; R = 25-deoxy-7-deazaguanosine and X = glycerol linker.
In a second aspect the invention provides pharmaceutical compositions. These compositions comprise any one of the compositions disclosed in the first aspect of the invention and a pharmaceutically acceptable carrier. In a third aspect the invention provides a method for generating an immune response in a vertebrate, the method comprising administering to the vertebrate an immunostimulatory oligonucleotide having a structure from the group of 5 ' -TCTGTR' GTTCT-X- TCTTGR'TGTCT-5'; 55-TCTGTR5GTTC1U1-X-U1C1TTGR5TGTCT-55; 5'- CTGTR'GTTCTC-X-CTCTTGR'TGTC-5'; 5 '-CTGTR5GTTCU1 C1-X-
-4-
EV 488513839 US C1U1CTTGR5TGTC-S'; 5'-CTGTROTTC1U1C1-X-CIU1C1TTGR5TGTC-S'; 55- TCTGTR'GTTCT-X-CGTTCGAACGT-5'; S'-TCTGTR'GACAG-X-GACAGR'TGTCT- 5'; 5'-TCTGTR'GACA1G1-X-G1A1CAGR'TGTCT-55; 5'-TCAGTR'GTTAG-X- GATTGR'TGACT-55; S'-TCAGTR'GACTG-X-GTCAGR'TGACT-S'; 5'- TR'GTR'GAR'GAT-X-TAGR'AGR'TGR'T-S'; 5 '-TR' GTR' GTAGTA-X- ATGATGR'TGR'T-5'; 55-TR5GAAR5GTTCT-X-TCTTGR5AAGR'T-55; 5'- TR'GTAR'GTACT-X-TCATGR'ATGR'T-S5 and 5'-TCRAACRTTCR-X- RCTTRCAARCT-5', wherein R' = l-(2'-deoxy-β-D-ribofuranosyl)-2-oxo-7-deaza-8- methyl-purine; A1ZC1ZG1AJ1 = 2'-O-methyl-ribonucleotides; R = 2'-deoxy-7-deazaguanosine and X = glycerol linker.
In a fourth aspect the invention provides a method for therapeutically treating a vertebrate having cancer, an autoimmune disorder, airway inflammation, inflammatory disorders, skin disorders, allergy, asthma or a disease caused by a pathogen, such method comprising administering to the patient an immunostimulatory oligonucleotide having a structure from the group of 5' -TCTGTR' GTTCT-X-TCTTGR5TGTCT-55; 5'-
TCTGTR5GTTC1U1-X-UIC1TTGR5TGTCT-S'; 5 '-CTGTR5 GTTCTC-X-CTCTTGR5 Tores'; S'-CTGTR'GTTCUiCrX-CiU^TTGR'TGTC-S'; 5'-CTGTR^^ C1U1C1TTGR5TGTC-S'; S'-TCTGTR'GTTCT-X-CGTTCGAACGT-S'; 5'- TCTGTR'GACAG-X-GACAGR'TGTCT-S'; 5'-TCTGTR5GACA1G1-X- G1A1CAGR5TGTCT-S5; 5'-TCAGTROTTAG-X-GATTGR5TGACT-S'; 5'- TCAGTR5GACTG-X-GTCAGR5TGACT-55; 5'-TR5GTR5GAR5GAT-X- TAGR'AGR'TGR5T-55; 55-TR5GTR5GTAGTA-X-ATGATGR5TGR5T-55; 55- TR'GAAR'GTTCT-X-TCTTGR'AAGR'T-S'; 5 '-TR' GTAR' GT ACT-X- TCATGR' ATGR'T-55 and 55-TCRAACRTTCR-X-RCTTRCAARCT-55, wherein R5 = 1- (25-deoxy-β-D-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine; AiZC1ZG1ZU1 = 25-O-methyl- ribonucleotides; R = 25-deoxy-7-deazaguanosine and X = glycerol linker.
In a fifth aspect the invention provides a method for preventing cancer, an autoimmune disorder, airway inflammation, inflammatory disorders, skin disorders, allergy, asthma or a disease caused by a pathogen in a vertebrate, such method comprising administering to the vertebrate an immunostimulatory oligonucleotide having a structure
-5-
EV 488513839 US from the group of 5'-TCTGTR5GTTCT-X-TCTTGRTGTCT-S'; 5'-TCTGTR5GTTC1U1-X- U1C1TTGR5TGTCT-S'; S'-CTGTR'GTTCTC-X-CTCTTGR'TGTC-S'; 5'- CTGTR5GTTCU1C1-X-C1U1CTTGRTGTC-S'; 5'-CTGTR5GTTC1U1C1-X- CIUIC1TTGR5TGTC-S5; S'-TCTGTR'GTTCT-X-CGTTCGAACGT-S'; 5'- TCTGTR'GACAG-X-GACAGR'TGTCT-S'; 5 '-TCTGTR5GAC A1 G1-X- G1A1CAGR5TGTCT-S'; S'-TCAGTR'GTTAG-X-GATTGR'TGACT-S'; 5'- TCAGTR'GACTG-X-GTCAGR'TGACT-S'; 5'-TR5GTR5GAR5GAT-X- TAGR'AGR'TGR'T-55; 5 '-TR5 GTR5 GT AGTA-X-ATGATGR5TGR5T-S5; 5'- TR'GAAR'GTTCT-X-TCTTGR'AAGR'T-S'; 5'-TR5GTAR5GTACT-X- TCATGR' ATGR'T-5' and S'-TCRAACRTTCR-X-RCTTRCAARCT-S', wherein R' = 1- (25-deoxy-β-D-riboruranosyl)-2-oxo-7-deaza-8-methyl-purine; A1ZC1ZGiAJ1 = 25-O-methyl- ribonucleotides; R = 25-deoxy-7-deazaguanosine and X = glycerol linker.
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EV 488513839 US BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 depicts a group of representative small molecule linkers suitable for linear synthesis of immunostimulatory oligonucleotides of the invention.
Figure 2 depicts a group of representative small molecule linkers suitable for parallel synthesis of immunostimulatory oligonucleotides of the invention.
Figure 3 is a synthetic scheme for the linear synthesis of irnmunostimulatory oligonucleotides of the invention. DMTr = 4,4'-dimethoxytrityl; CE = cyanoethyl.
Figure 4 is a synthetic scheme for the parallel synthesis of immunostimulatory oligonucleotides of the invention. DMTr = 4,4'-dimethoxytrityl; CE = cyanoethyl.
Figure 5 is a schematic representation of the 3 '-terminal nucleoside of an oligonucleotide, showing that a non-nucleotidic linkage can be attached to the nucleoside at the nucleobase, at the 3' position, or at the 2' position.
Figure 6 shows IL- 12 induction in C57BL/6 mouse spleen cell cultures by immunostimulatory oligonucleotides of the invention.
Figure 7 shows IL-6induction in C57BL/6 mouse spleen cell cultures by immunostimulatory oligonucleotides of the invention.
Figure 8 shows IFN-α induction in human pDC cultures by immunosthnulatory oligonucleotides of the invention.
Figure 9 shows IFN-α induction in human PBMC cultures by immunostimulatory oligonucleotides of the invention.
Figure 10 shows Human B cell proliferation by immunostimulatory oligonucleotides of the invention.
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EV 488513839 US DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The invention relates to the therapeutic use of oligonucleotides as immunostimulatory agents for immunotherapy applications. The issued patents, patent applications, and references that are cited herein are hereby incorporated by reference to the same extent as if each was specifically and individually indicated to be incorporated by reference, hi the event of inconsistencies between any teaching of any reference cited herein and the present specification, the latter shall prevail for purposes of the invention.
The invention provides methods for enhancing the immune response caused by immunostimulatory compounds used for immunotherapy applications such as, but not limited to, treatment of cancer, autoimmune disorders, asthma, respiratory allergies, food allergies, and bacteria, parasitic, and viral infections in adult and pediatric human and veterinary applications. Thus, the invention further provides compounds having optimal levels of immunostimulatory effect for immunotherapy and methods for making and using such compounds, hi addition, compounds of the invention are useful as adjuvants in combination with DNA vaccines, antibodies, and allergens; and in combination with chemotherapeutic agents and/or antisense oligonucleotides.
The present inventors have surprisingly discovered that modification of an immunostimulatory oligonucleotide to optimally present its 5' ends dramatically affects its immunostimulatory capabilities, hi addition, the present inventors have discovered that the cytokine profile and species specificity of an immune response can be modulated by using novel purine or pyrimidine structures as part of an immunostimulatory oligonucleotide.
In a first aspect, the invention provides immunostimulatory oligonucleotides alone or comprising at least two oligonucleotides linked at their 3' ends, or an internucleoside linkage or a functionalized nucleobase or sugar to a non-nucleotidic linker, at least one of the oligonucleotides being an immunostimulatory oligonucleotide and having an accessible 5' end. As used herein, the term "accessible 5' end" means that the 5' end of the oligonucleotide is sufficiently available such that the factors that recognize and bind to oligonucleotide and stimulate the immune system have access to it. In oligonucleotides having an accessible 5' end, the 5' OH position of the terminal sugar is not covalently linked to more than two nucleoside residues or any other moiety that interferes with interaction with
-8-
EV 488513839 US the 5' end. Optionally, the 5' OH can be linked to a phosphate, phosphorothioate, or phosphorodithioate moiety, an aromatic or aliphatic linker, cholesterol, or another entity which does not interfere with accessibility. The immunostimulatory oligonucleotides according to the invention preferably further comprise an immunostimulatory dinucleotide comprising a novel purine or pyrimidine.
In certain embodiments, the immunostimulatory oligonucleotides include a ribozyme or a decoy oligonucleotide. As used herein, the term "ribozyme" refers to an oligonucleotide that possesses catalytic activity. Preferably, the ribozyme binds to a specific nucleic acid target and cleaves the target. As used herein, the term "decoy oligonucleotide" refers to an oligonucleotide that binds to a transcription factor hi a sequence-specific manner and arrests transcription activity. Preferably, the ribozyme or decoy oligonucleotide exhibits secondary structure, including, without limitation, stem-loop or hairpin structures. In certain embodiments, at least one oligonucleotide comprises poly (I)-poly (C). In certain embodiments, at least one set of Nn includes a string of 3 to 10 dGs and/or Gs or T- substituted ribo or arabino Gs.
For purposes of the invention, the term "oligonucleotide" refers to a polynucleoside formed from a plurality of linked nucleoside units. Such oligonucleotides can be obtained from existing nucleic acid sources, including genomic or cDNA, but are preferably produced by synthetic methods. In preferred embodiments each nucleoside unit includes a heterocyclic base and a pentofuranosyl, trehalose, arabinose, 2'-deoxy-2'-substituted arabinose, 2'-O- substituted arabinose or hexose sugar group. The nucleoside residues can be coupled to each other by any of the numerous known internucleoside linkages. Such internucleoside linkages include, without limitation, phosphodiester, phosphorothioate, phosphorodithioate, alkylphosphonate, alkylphosphonothioate, phosphotriester, phosphoramidate, siloxane, carbonate, carboalkoxy, acetamidate, carbamate, morpholino, borano, thioether, bridged phosphoramidate, bridged methylene phosphonate, bridged phosphorothioate, and sulfone internucleoside linkages. The term "oligonucleotide" also encompasses polynucleosides having one or more stereospecific internucleoside linkage (e.g., (Rp)- or (Sp)- phosphorothioate, alkylphosphonate, or phosphotriester linkages). As used herein, the terms "oligonucleotide" and "dinucleotide" are expressly intended to include polynucleosides and dinucleosides having any such internucleoside linkage, whether or not the linkage comprises
-9-
EV 488513839 US a phosphate group. In certain preferred embodiments, these internucleoside linkages may be phosphodiester, phosphorothioate, or phosphorodithioate linkages, or combinations thereof.
In some embodiments, the oligonucleotides each have from about 3 to about 35 nucleoside residues, preferably from about 4 to about 30 nucleoside residues, more preferably from about 4 to about 20 nucleoside residues. In some embodiments, the immunostimulatory oligonucleotides comprise oligonucleotides have from about 5 to about 18, or from about 5 to about 14, nucleoside residues. As used herein, the term "about" implies that the exact number is not critical. Thus, the number of nucleoside residues in the oligonucleotides is not critical, and oligonucleotides having one or two fewer nucleoside residues, or from one to several additional nucleoside residues are contemplated as equivalents of each of the embodiments described above. In some embodiments, one or more of the oligonucleotides have 11 nucleotides. In the context of immunostimulatory oligonucleotides, preferred embodiments have from about 13 to about 35 nucleotides, more preferably from about 13 to about 26 nucleotides. The term "oligonucleotide" also encompasses polynucleosides having additional substituents including, without limitation, protein groups, lipophilic groups, intercalating agents, diamines, folic acid, cholesterol and adamantane. The term "oligonucleotide" also encompasses any other nucleobase containing polymer, including, without limitation, peptide nucleic acids (PNA), peptide nucleic acids with phosphate groups (PHONA), locked nucleic acids (LNA), morpholino-backbone oligonucleotides, and oligonucleotides having backbone sections with alkyl linkers or amino linkers.
The oligonucleotides of the invention can include naturally occurring nucleosides, modified nucleosides, or mixtures thereof. As used herein, the term "modified nucleoside" is a nucleoside that includes a modified heterocyclic base, a modified sugar moiety, or a combination thereof. In some embodiments, the modified nucleoside is a non-natural pyrimidine or purine nucleoside, as herein described. In some embodiments, the modified nucleoside is a 2'-substituted ribonucleoside an arabinonucleoside or a 2'-deoxy-2'- substituted-arabinoside.
For purposes of the invention, the term "2'-substituted ribonucleoside" or "2'- substituted arabinoside" includes ribonucleosides or arabinonucleoside in which the hydroxyl
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EV 488513839 US group at the 2' position of the pentose moiety is substituted to produce a 2 '-substituted or 2'- O-substituted ribonucleoside. Preferably, such substitution is with a lower alkyl group containing 1-6 saturated or unsaturated carbon atoms, or with an aryl group having 6-10 carbon atoms, wherein such alkyl, or aryl group may be unsubstituted or may be substituted, e.g., with halo, hydroxy, trifluoromethyl, cyano, nitro, acyl, acyloxy, alkoxy, carboxyl, carboalkoxy, or amino groups. Examples of 2'-0-substituted ribonucleosides or 2'-O- substituted-arabinosides include, without limitation 2'-O-methylribonucleosides or 2'-O- methylarabinosides and 2'-O-methoxyethylribonucleosides or 2'-O- methoxyethylarabinosides. The term "2'-substituted ribonucleoside" or "2'-substituted arabinoside" also includes ribonucleosides or arabinonucleosides in which the 2'-hydroxyl group is replaced with a lower alkyl group containing 1-6 saturated or unsaturated carbon atoms, or with an amino or halo group. Examples of such 2' -substituted ribonucleosides or 2' -substituted arabinosides include, without limitation, 2'-amino, 2'-fluoro, 2'-allyl, and 2'-propargyl ribonucleosides or arabinosides.
The term "oligonucleotide" includes hybrid and chimeric oligonucleotides. A "chimeric oligonucleotide" is an oligonucleotide having more than one type of internucleoside linkage. One preferred example of such a chimeric oligonucleotide is a chimeric oligonucleotide comprising a phosphorothioate, phosphodiester or phosphorodithioate region and non-ionic linkages such as alkylphosphonate or alkylphosphonothioate linkages (see e.g., Pederson et al. U.S. Patent Nos. 5,635,377 and 5,366,878).
A "hybrid oligonucleotide" is an oligonucleotide having more than one type of nucleoside. One preferred example of such a hybrid oligonucleotide comprises a ribonucleotide or 2'-substituted ribonucleotide region, and a deoxyribonucleotide region (see, e.g., Metelev and Agrawal, U.S. Patent No. 5,652,355, 6,346,614 and 6,143,881).
For purposes of the invention, the term "immunostimulatory oligonucleotide" refers to an oligonucleotide as described above that induces an immune response when administered to a vertebrate, such as a fish, fowl, or mammal. As used herein, the term "mammal" includes, without limitation rats, mice, cats, dogε5 horses, cattle, cows, pigs,
-1 1 -
EV 488513839 US rabbits, non-human primates, and humans. Useful immunostimulatory oligonucleotides can be found described in Agrawal et al., WO 98/49288, published November 5, 1998; WO 01/12804, published February 22, 2001; WO 01/5537O5 published August 2, 2001; PCT/USOl/13682, filed April 30, 2001; and PCT/USOl/30137, filed September 26, 2001. Preferably, the immunostimulatory oligonucleotide comprises at least one phosphodiester, phosphorothioate, or phosphorodithioate internucleoside linkage.
In some embodiments, the immunostimulatory oligonucleotide comprises an immunostimulatory dinucleotide of formula 5'-Pyr-Pur-3', wherein Pyr is a natural or synthetic pyrimidine nucleoside and Pur is a natural or synthetic purine nucleoside. In some preferred embodiments, the immunostimulatory oligonucleotide comprises an immunostimulatory dinucleotide of formula 5'-Pur*-Pur-3', wherein Pur* is a synthetic purine nucleoside and Pur is a natural or synthetic purine nucleoside, hi various places the dinucleotide is expressed as RpG, C*pG or YZ, in which case respectively, R, C*5 or Y represents a synthetic purine. A particularly preferred synthetic purine is 2-oxo-7-deaza-8- methyl-purine. When this synthetic purine is in the Pur* position of the dinucleotide, species-specificity (sequence dependence) of the immunostimulatory effect is overcome and cytokine profile is improved. As used herein, the term "pyrimidine nucleoside" refers to a nucleoside wherein the base component of the nucleoside is a monocyclic nucleobase. Similarly, the term "purine nucleoside" refers to a nucleoside wherein the base component of the nucleoside is a bicyclic nucleobase. For purposes of the invention, a "synthetic" pyrimidine or purine nucleoside includes a non-naturally occurring pyrimidine or purine base, a non-naturally occurring sugar moiety, or a combination thereof.
Preferred pyrimidine nucleosides according to the invention have the structure (7):
Figure imgf000013_0001
-12-
EV 488513839 US wherein:
D is a hydrogen bond donor;
D' is selected from the group consisting of hydrogen, hydrogen bond donor, hydrogen bond acceptor, hydrophilic group, hydrophobic group, electron withdrawing group and electron donating group;
A is a hydrogen bond acceptor or a hydrophilic group;
A' is selected from the group consisting of hydrogen bond acceptor, hydrophilic group, hydrophobic group, electron withdrawing group and electron donating group; X is carbon or nitrogen; and
S1 is a pentose or hexose sugar ring, or a non-naturally occurring sugar.
Preferably, the sugar ring is derivatized with a phosphate moiety, modified phosphate moiety, or other linker moiety suitable for linking the pyrimidine nucleoside to another nucleoside or nucleoside analog. Preferred hydrogen bond donors include, without limitation, -NH-, -NH2, -SH and
-OH. Preferred hydrogen bond acceptors include, without limitation, C=O, C=S, and the ring nitrogen atoms of an aromatic heterocycle, e.g., N3 of cytosine.
In some embodiments, the base moiety in (i) is a non-naturally occurring pyrimidine base. Examples of preferred non-naturally occurring pyrimidine bases include, without limitation, 5-hydroxycytosine, 5-hydroxymethylcytosine, N4-alkylcytosine, preferably N4- ethylcytosine, and 4-thiouracil. However, in some embodiments 5-bromocytosine is specifically excluded.
In some embodiments, the sugar moiety S' in (i) is a non-naturally occurring sugar moiety. For purposes of the present invention, a "naturally occurring sugar moiety" is a sugar moiety that occurs naturally as part of nucleic acid, e.g., ribose and 2'-deoxyribose, and a "non-naturally occurring sugar moiety" is any sugar that does not occur naturally as part of a nucleic acid, but which can be used in the backbone for an oligonucleotide, e.g, hexose. Arabinose and arabinose derivatives are examples of preferred sugar moieties.
-13-
EV 488513839 US Preferred purine nucleoside analogs according to the invention have the structure (II):
Figure imgf000015_0001
wherein:
D is a hydrogen bond donor; D' is selected from the group consisting of hydrogen, hydrogen bond donor, and hydrophilic group;
A is a hydrogen bond acceptor or a hydrophilic group; X is carbon or nitrogen; each L is independently an atom selected from the group consisting of C, O, N and S; and
S1 is a pentose or hexose sugar ring, or a non-naturally occurring sugar.
Preferably, the sugar ring is derivatized with a phosphate moiety, modified phosphate moiety, or other linker moiety suitable for linking the pyrimidine nucleoside to another nucleoside or nucleoside analog. Preferred hydrogen bond donors include, without limitation, -NH-, -NH2, -SH and
-OH. Preferred hydrogen bond acceptors include, without limitation, C=O, C=S, -NO2 and the ring nitrogen atoms of an aromatic heterocycle, e.g., Nl of guanine.
In some embodiments, the base moiety in (II) is a non-naturally occurring purine base. Examples of preferred non-naturally occurring purine bases include, without limitation, 2-amino-6-thiopurine and 2-amino-6-oxo-7-deazapurine. In some embodiments, the sugar moiety S' in (II) is a naturally occurring sugar moiety, as described above for structure (I).
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EV 488513839 US In preferred embodiments, the immunostimulatory dinucleotide is selected from the group consisting of CpG, C*pG, CpG*, and C*pG*, wherein the base of C is cytosine, the base of C* is 2'-thymine, 5-hydroxycytosine, N4-alkyl-cytosine, 4-thiouracil or other non- natural pyrimidine, or 2-oxo-7-deaza-8-methylpurine, wherein when the base is 2-oxo-7- deaza-8-methyl-purine, it is preferably covalently bound to the 1 '-position of a pentose via the 1 position of the base; the base of G is guanosine, the base of G* is 2-amino-6-oxo-7- deazapurine, 2-oxo-7-deaza-8-methylpurine, 6-thioguanine, 6-oxopurine, or other non- natural purine nucleoside, and p is an internucleoside linkage selected from the group consisting of phosphodiester, phosphorothioate, and phosphorodithioate. In certain preferred embodiments, the immunostimulatory dinucleotide is not CpG.
The immunostimulatory oligonucleotides may include immunostimulatory moieties on one or both sides of the immunostimulatory dinucleotide. Thus, in some embodiments, the immunostimulatory oligonucleotide comprises an immunostimulatory domain of structure (III): 5'-Nn-Nl-Y-Z-Nl-Nn-3' (III) wherein: the base of Y is cytosine, thymine, 5-hydroxycytosine, N4-alkyl-cytosine, 4- thiouracil or other non-natural pyrimidine nucleoside, or 2-oxo-7-deaza-8 methyl purine, wherein when the base is 2-oxo-7-deaza-8-methyl-purine, it is preferably covalently bound to the 1 '-position of a pentose via the 1 position of the base; the base of Z is guanine, 2-amino-6-oxo-7-deazapurine, 2-oxo-7deaza-8- methylpurine, 2-amino-6-thio-purine, 6-oxopurine or other non-natural purine nucleoside;
Nl and Nn, independent at each occurrence, is preferably a naturally occurring or a synthetic nucleoside or an immunostimulatory moiety selected from the group consisting of abasic nucleosides, arabinonucleosides, 2'-deoxyuridine, α-deoxyribonucleosides, β-L-deoxyribonucleosides, and nucleosides linked by a phosphodiester or modified internucleoside linkage to the adjacent nucleoside on the 3' side, the modified internucleotide linkage being selected from, without limitation, a linker having a length of from about 2
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EV 488513839 US angstroms to about 200 angstroms, C2-C18 alkyl linker. poly(ethylene glycol) linker, 2- aminobutyl- 1,3 -propanediol linker, glyceryl linker, 2'-5' internucleoside linkage, and phosphorothioate, phosphorodithioate, or methylphosphonate internucleoside linkage; provided that at least one Nl or Nn is optionally an immunostimulatory moiety; wherein n is a number from 0 to 30; and wherein the 3 'end, an internucleoside linker, or a derivatized nucleobase or sugar is linked directly or via a non-nucleotidic linker to another oligonucleotide, which may or may not be immunostimulatory.
In some preferred embodiments, YZ is arabinocytidine or 2 '-deoxy-2' -substituted arabinocytidine and arabinoguanosine or 2 'deoxy-2 '-substituted arabinoguanosine. Preferred immunostimulatory moieties include natural phosphodiester backbones and modifications in the phosphate backbones, including, without limitation, methylphosphonates, methylphosphonothioates, phosphotriesters, phosphothiotriesters, phosphorothioates, phosphorodithioates, triester prodrugs, sulfones, sulfonamides, sulfamates, formacetal, N- methylhydroxylamine, carbonate, carbamate, morpholino, boranophosphonate, phosphoramidates, especially primary amino-phosphoramidates, N3 phosphoramidates and N5 phosphoramidates, and stereospecific linkages (e.g., (Rp)- or (Sp)-phosphorothioate, alkylphosphonate, or phosphotriester linkages).
Preferred immunostimulatory moieties according to the invention further include nucleosides having sugar modifications, including, without limitation, 2 '-substituted pentose sugars including, without limitation, 2'-O-methylribose, 2'-O-methoxyethylribose, 2'-O- propargylribose, and 2'-deoxy-2'-fluororibose; 3 '-substituted pentose sugars, including, without limitation, 3'-O-methylribose; r,2'-dideoxyribose; arabinose; substituted arabinose sugars, including, without limitation, l'-methylarabinose, 3'-hydroxymethylarabinose, 4'-hydroxymethylarabinose, 3'-hydroxyarabinose and 2'-substituted arabinose sugars; hexose sugars, including, without limitation, 1,5-anhydrohexitol; and alpha-anomers. In embodiments in which the modified sugar is a 3'-deoxyribonucleoside or a 3'-O-substituted ribonucleoside, the immunostimulatory moiety is attached to the adjacent nucleoside by way of a 2'-5' internucleoside linkage.
-16-
EV 488513839 US Preferred immunostimulatory moieties according to the invention further include oligonucleotides having other carbohydrate backbone modifications and replacements, including peptide nucleic acids (PNA), peptide nucleic acids with phosphate groups (PHONA), locked nucleic acids (LNA), morpholino backbone oligonucleotides, and oligonucleotides having backbone linker sections having a length of from about 2 angstroms to about 200 angstroms, including without limitation, alkyl linkers or amino linkers. The alkyl linker may be branched or unbranched, substituted or unsubstituted, and chirally pure or a racemic mixture. Most preferably, such alkyl linkers have from about 2 to about 18 carbon atoms. In some preferred embodiments such alkyl linkers have from about 3 to about 9 carbon atoms. Some alkyl linkers include one or more functional groups selected from the group consisting of hydroxy, amino, thiol, thioether, ether, amide, thioamide, ester, urea, and thioether. Some such functionalized alkyl linkers are poly(ethylene glycol) linkers of formula -O-(CH2-CH2-O-)n (n = 1-9). Some other functionalized alkyl linkers are peptides or amino acids. Preferred immunostimulatory moieties according to the invention further include
DNA isoforms, including, without limitation, β-L-deoxyribonucleosides and α-deoxyribonucleosides. Preferred immunostimulatory moieties according to the invention incorporate 3' modifications, and further include nucleosides having unnatural internucleoside linkage positions, including, without limitation, 2 '-5', 2'-2', 3 '-3' and 5 '-5' linkages.
Preferred immunostimulatory moieties according to the invention further include nucleosides having modified heterocyclic bases, including, without limitation, 5-hydroxycytosine, 5-hydroxymethylcytosine, N4-alkylcytosine, preferably N4-ethylcytosine, 4-thiouracil, 6-thioguanine, 7-deazaguanine, inosine, nitropyrrole, C5-propynylpyrimidine, and diaminopurines, including, without limitation, 2,6-diaminopurine.
By way of specific illustration and not by way of limitation, for example, in the immunostimulatory domain of structure (III), a methylphosphonate internucleoside linkage at position Nl or Nn is an immunostimulatory moiety, a linker having a length of from about 2 angstroms to about 200 angstroms, C2-C18 alkyl linker at position Xl is an
-17-
EV 488513839 US immunostimulatory moiety, and a β-L-deoxyribonucleoside at position Xl is an immunostimulatory moiety. See Table 1 below for representative positions and structures of immunostimulatory moieties. It is to be understood that reference to a linker as the immunostimulatory moiety at a specified position means that the nucleoside residue at that position is substituted at its 3'-hydroxyl with the indicated linker, thereby creating a modified internucleoside linkage between that nucleoside residue and the adjacent nucleoside on the 3' side. Similarly, reference to a modified internucleoside linkage as the immunostimulatory moiety at a specified position means that the nucleoside residue at that position is linked to the adjacent nucleoside on the 3' side by way of the recited linkage. Table 1
Figure imgf000019_0001
Table 2 shows representative positions and structures of immunostimulatory moieties within an immunostimulatory oligonucleotide having an upstream potentiation domain. As used herein, the term "Spacer 9" refers to a poly(ethylene glycol) linker of formula -0-(CH2CH2-O)n-, wherein n is 3. The term "Spacer 18" refers to a polyethylene glycol) linker of formula -0-(CH2CH2-O)n-, wherein n is 6. As used herein, the term "C2-C18 alkyl linker refers to a linker of formula -O-(CH2)?-O-, where q is an integer from 2 to 18. Accordingly, the terms "C3-linker" and "C3-alkyl linker" refer to a linker of formula -O-(CH2)3-O-. For each of Spacer 9, Spacer 18, and C2-C18 alkyl linker, the linker is connected to the adjacent nucleosides by way of phosphodiester, phosphorothioate, or phosphorodithioate linkages.
-18-
EV 488513839 US Table 2
Figure imgf000020_0001
Table 3 shows representative positions and structures of immunostimulatory moieties within an immunostimulatory oligonucleotide having a downstream potentiation domain.
Table 3
Figure imgf000020_0002
- 19-
EV 488513839 US The immunostimulatory oligonucleotides according to the invention comprise at least two oligonucleotides linked at their 3' ends or internucleoside linkage or a functionalized nucleobase or sugar via a non-nucleotidic linker. For purposes of the invention, a "non- nucleotidic linker" is any moiety that can be linked to the oligonucleotides by way of covalent or non-covalent linkages. Preferably such linker is from about 2 angstroms to about 200 angstroms in length. Several examples of preferred linkers are set forth below. Non- covalent linkages include, but are not limited to, electrostatic interaction, hydrophobic interactions, π-stacking interactions, and hydrogen bonding. The term "non-nucleotidic linker" is not meant to refer to an internucleoside linkage, as described above, e.g., a phosphodiester, phosphorothioate, or phosphorodithioate functional group, that directly connects the 3'-hydroxyl groups of two nucleosides. For purposes of this invention, such a direct 3 -3' linkage (no linker involved) is considered to be a "nucleotidic linkage."
In some embodiments, the non-nucleotidic linker is a metal, including, without limitation, gold particles, hi some other embodiments, the non-nucleotidic linker is a soluble or insoluble biodegradable polymer bead.
In yet other embodiments, the non-nucleotidic linker is an organic moiety having functional groups that permit attachment to the oligonucleotide. Such attachment preferably is by any stable covalent linkage. As a non-limiting example, the linker may be attached to any suitable position on the nucleoside, as illustrated in Figure 5. In some preferred embodiments, the linker is attached to the 3'-hydroxyl. In such embodiments, the linker preferably comprises a hydroxyl functional group, which preferably is attached to the 3'- hydroxyl by means of a phosphodiester, phosphorothioate, phosphorodithioate or non- phosphate-based linkages.
In some embodiments, the non-nucleotidic linker is a biomolecule, including, without limitation, polypeptides, antibodies, lipids, antigens, allergens, and oligosaccharides. In some other embodiments, the non-nucleotidic linker is a small molecule. For purposes of the invention, a small molecule is an organic moiety having a molecular weight of less than 1,000 Da. In some embodiments, the small molecule has a molecular weight of less than 750 Da.
-20-
EV 488513839 US In some embodiments, the small molecule is an aliphatic or aromatic hydrocarbon, either of which optionally can include, either in the linear chain connecting the oligonucleotides or appended to it, one or more functional groups selected from the group consisting of hydroxy, amino, thiol, thioether, ether, amide, thioamide, ester, urea, and thiourea. The small molecule can be cyclic or acyclic. Examples of small molecule linkers include, but are not limited to, amino acids, carbohydrates, cyclodextrins, adamantane, cholesterol, haptens and antibiotics. However, for purposes of describing the non-nucleotidic linker, the term "small molecule" is not intended to include a nucleoside. hi some embodiments, the small molecule linker is glycerol or a glycerol homolog of the formula HO-(CH2)o-CH(OH)-(CH2)p-OH, wherein o and p independently are integers from 1 to about 6, from 1 to about 4, or from 1 to about 3. In some other embodiments, the small molecule linker is a derivative of l,3-diamino-2-hydroxypropane. Some such derivatives have the formula HO-(CH2)m-C(O)NH-CH2-CH(OH)-CH2-NHC(O)-(CH2)w-OH, wherein m is an integer from O to about 10, from 0 to about 6, from 2 to about 6, or from 2 to about 4.
Some non-nucleotidic linkers according to the invention permit attachment of more than two oligonucleotides. For example, the small molecule linker glycerol has three hydroxyl groups to which oligonucleotides may be covalently attached. Some immunostimulatory oligonucleotides according to the invention, therefore, comprise more than two oligonucleotides linked at their 3' ends to a non-nucleotidic linker.
The immunostimulatory oligonucleotides of the invention may conveniently be synthesized using an automated synthesizer and phosphoramidite approach as schematically depicted in Figures 3 and 4, and further described in the Examples, hi some embodiments, the immunostimulatory oligonucleotides are synthesized by a linear synthesis approach (see Figure 3). As used herein, the term "linear synthesis" refers to a synthesis that starts at one end of the immunostimulatory oligonucleotide and progresses linearly to the other end. Linear synthesis permits incorporation of either identical or un-identical (in terms of length, base composition and/or chemical modifications incorporated) monomeric units into the immunostimulatory oligonucleotides.
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EV 488513839 US An alternative mode of synthesis is "parallel synthesis", in which synthesis proceeds outward from a central linker moiety (see Figure 4). A solid support attached linker can be used for parallel synthesis, as is described in U.S. Patent No. 5,912,332. Alternatively, a universal solid support (such as phosphate attached controlled pore glass) support can be used.
Parallel synthesis of immunostimulatory oligonucleotides has several advantages over linear synthesis: (1) parallel synthesis permits the incorporation of identical monomeric units; (2) unlike in linear synthesis, both (or all) the monomeric units are synthesized at the same time, thereby the number of synthetic steps and the time required for the synthesis is the same as that of a monomeric unit; and (3) the reduction in synthetic steps improves purity and yield of the final immunostimulatory oligonucleotide product.
At the end of the synthesis by either linear synthesis or parallel synthesis protocols, the immunostimulatory oligonucleotides may conveniently be deprotected with concentrated ammonia solution or as recommended by the phosphoramidite supplier, if a modified nucleoside is incorporated. The product immunostimulatory oligonucleotide is preferably purified by reversed phase HPLC, detritylated, desalted and dialyzed.
Table 4 shows representative immunostrmulatory oligonucleotides according to the invention.
Table 4. Examples of Immunostimulatory Oligonucleotides Sequences
Figure imgf000023_0001
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EV 488513839 US
Figure imgf000024_0001
R' = l-(2'-deoxy-β-D-ribofuranosyl)-2-oxo-7-deaza-8-methyl-ρurine; A1ZC1ZG1ZU1 = 2'-O- methyl-ribonucleotides; R = 25-deoxy-7-deazaguanosine; X = glycerol linker.
In a second aspect, the invention provides immunostimulatory oligonucleotide conjugates comprising an immunostimulatory oligonucleotide, as described above, and an antigen conjugated to the immunostimulatory oligonucleotide at a position other than the accessible 5' end. In some embodiments, the non-nucleotidic linker comprises an antigen, which is conjugated to the oligonucleotide. In some other embodiments, the antigen is conjugated to the oligonucleotide at a position other than its 3' end. In some embodiments, the antigen produces a vaccine effect. The antigen is preferably selected from the group consisting of antigens associated with a pathogen, antigens associated with a cancer, antigens associated with an auto-immune disorder, and antigens associated with other diseases such as, but not limited to, veterinary or pediatric diseases. For purposes of the invention, the term "associated with55 means that the antigen is present when the pathogen, cancer, auto-immune disorder, food allergy, respiratory allergy, asthma or other disease is present, but either is not present, or is present in reduced amounts, when the pathogen, cancer, auto-immune disorder, food allergy, respiratory allergy, or disease is absent.
The immunostimulatory oligonucleotide is covalently linked to the antigen, or it is otherwise operatively associated with the antigen. As used herein, the term "operatively associated with" refers to any association that maintains the activity of both immunostimulatory oligonucleotide and antigen. Nonlimiting examples of such operative associations include being part of the same liposome or other such delivery vehicle or reagent. In embodiments wherein the immunostimulatory oligonucleotide is covalently linked to the antigen, such covalent linkage preferably is at any position on the
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EV 488513839 US immunostimulatory oligonucleotide other than an accessible 5' end of an immunostimulatory oligonucleotide. For example, the antigen may be attached at an internucleoside linkage or may be attached to the non-nucleotidic linker. Alternatively, the antigen may itself be the non-nucleotidic linker. In a third aspect, the invention provides pharmaceutical formulations comprising an immunostimulatory oligonucleotide or immunostimulatory oligonucleotide conjugate according to the invention and a physiologically acceptable carrier. As used herein, the term "physiologically acceptable" refers to a material that does not interfere with the effectiveness of the immunostimulatory oligonucleotide and is compatible with a biological system such as a cell, cell culture, tissue, or organism. Preferably, the biological system is a living organism, such as a vertebrate.
As used herein, the term "carrier" encompasses any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, or other material well known in the art for use in pharmaceutical formulations. It will be understood that the characteristics of the carrier, excipient, or diluent will depend on the route of administration for a particular application. The preparation of pharmaceutically acceptable formulations containing these materials is described in, e.g., Remington 's Pharmaceutical Sciences, 18th Edition, ed. A. Gennaro, Mack Publishing Co., Easton, PA, 1990.
In a fourth aspect, the invention provides methods for generating an immune response in a vertebrate, such methods comprising administering to the vertebrate an immunostimulatory oligonucleotide or immunostimulatory oligonucleotide conjugate according to the invention. In some embodiments, the vertebrate is a mammal. For purposes of this invention, the term "mammal" is expressly intended to include humans, hi preferred embodiments, the immunostimulatory oligonucleotide or immunostimulatory oligonucleotide conjugate is administered to a vertebrate in need of immunostimulation.
In the methods according to this aspect of the invention, administration of immunostimulatory oligonucleotide or immunostimulatory oligonucleotide conjugate can be by any suitable route, including, without limitation, parenteral, oral, sublingual, transdermal, topical, intranasal, aerosol, intraocular, intratracheal, intrarectal, vaginal, by gene gun, dermal patch or in eye drop or mouthwash form. Administration of the therapeutic compositions of
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EV 488513839 US immunostimulatory oligonucleotides can be carried out using known procedures at dosages and for periods of time effective to reduce symptoms or surrogate markers of the disease. When administered systemically, the therapeutic composition is preferably administered at a sufficient dosage to attain a blood level of immunostimulatory oligonucleotide from about 0.0001 micromolar to about 10 micromolar. For localized administration, much lower concentrations than this may be effective, and much higher concentrations may be tolerated. Preferably, a total dosage of immunostimulatory oligonucleotide ranges from about 0.001 mg per patient per day to about 200 mg per kg body weight per day. It may be desirable to administer simultaneously, or sequentially a therapeutically effective amount of one or more of the therapeutic compositions of the invention to an individual as a single treatment episode.
In certain preferred embodiments, immunostimulatory oligonucleotide or immunostimulatory oligonucleotide conjugate according to the invention are administered in combination with vaccines, antibodies, cytotoxic agents, allergens, antibiotics, antisense oligonucleotides, peptides, proteins, gene therapy vectors, DNA vaccines and/or adjuvants to enhance the specificity or magnitude of the immune response. In these embodiments, the immunostimulatory oligonucleotides of the invention can variously act as adjuvants and/or produce direct immunostimulatory effects.
Either the immunostimulatory oligonucleotide or immunostimulatory oligonucleotide conjugate or the vaccine, or both, may optionally be linked to an immunogenic protein, such as keyhole limpet hemocyanin (KXH), cholera toxin B subunit, or any other immunogenic carrier protein. Any of the plethora of adjuvants may be used including, without limitation, Freund's complete adjuvant, KLH, monophosphoryl lipid A (MPL), alum, and saponins, including QS-21, imiquimod, R848, or combinations thereof. For purposes of this aspect of the invention, the term "in combination with" means in the course of treating the same disease in the same patient, and includes administering the immunostimulatory oligonucleotide and/or the vaccine and/or the adjuvant in any order, including simultaneous administration, as well as temporally spaced order of up to several days apart. Such combination treatment may also include more than a single administration of the immunostimulatory oligonucleotide, and/or independently the vaccine, and/or
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EV 488513839 US independently the adjuvant. The administration of the immunostimulatory oligonucleotide and/or vaccine and/or adjuvant may be by the same or different routes.
The methods according to this aspect of the invention are useful for model studies of the immune system. The methods are also useful for the prophylactic or therapeutic treatment of human or animal disease. For example, the methods are useful for pediatric and veterinary vaccine applications.
In a fifth aspect, the invention provides methods for therapeutically treating a patient having a disease or disorder, such methods comprising administering to the patient an immunostimulatory oligonucleotide or immunostimulatory oligonucleotide conjugate according to the invention. In various embodiments, the disease or disorder to be treated is cancer, an autoimmune disorder, airway inflammation, inflammatory disorders, allergy, asthma or a disease caused by a pathogen. Pathogens include bacteria, parasites, fungi, viruses, viroids and prions. Administration is carried out as described for the fourth aspect of the invention. For purposes of the invention, the term "allergy" includes, without limitation, food allergies and respiratory allergies. The term "airway inflammation" includes, without limitation, asthma. As used herein, the term "autoimmune disorder" refers to disorders in which "self proteins undergo attack by the immune system. Such term includes autoimmune asthma. In any of the methods according to this aspect of the invention, the immunostimulatory oligonucleotide or immunostimulatory oligonucleotide conjugate can be administered in combination with any other agent useful for treating the disease or condition that does not diminish the immunostimulatory effect of the immunostimulatory oligonucleotide. For example, in the treatment of cancer, it is contemplated that the immunostimulatory oligonucleotide or immunostimulatory oligonucleotide conjugate may be administered in combination with a chemotherapeutic compound.
The examples below are intended to further illustrate certain preferred embodiments of the invention, and are not intended to limit the scope of the invention.
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EV 488513839 US EXAMPLES
Example 1 : Synthesis of Oligonucleotides Containing immunostimulatory Moieties
Oligonucleotides were synthesized on a 1 μmol to 0.1 mM scale using an automated DNA synthesizer (OligoPilot II, AKTA, (Amersham) and/or Expedite 8909 (Applied Biosystem)), following the linear synthesis or parallel synthesis procedures outlined in Figures 3 and 4.
5'-DMT dA, dG, dC and T phosphoramidites were purchased from Proligo (Boulder, CO). 5'-DMT 7-deaza-dG and araG phosphoramidites were obtained from Chemgenes (Wilmington, MA). DiDMT-glycerol linker solid support was obtained from Chemgenes. l-(2'-deoxy-β-D-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine amidite was obtained from Glen Research (Sterling, VA), 2'-O-methylribonuncleoside amidites were obtained from Promega (Obispo, CA). All oligonucleotides were phosphorothioate backbone modified. All nucleoside phosphoramidites were characterized by 31P and 1H NMR spectra. Modified nucleosides were incorporated at specific sites using normal coupling cycles recommended by the supplier. After synthesis, oligonucleotides were deprotected using concentrated ammonium hydroxide and purified by reverse phase HPLC, detritylation, followed by dialysis. Purified oligonucleotides as sodium salt form were lyophilized prior to use. Purity was tested by CGE and MALDI-TOF MS. Endotoxin levels were determined by LAL test and were below 1.0 EU/mg.
Example 2: Activity of short-immunostimulatory oligonucleotides in murine spleen cell cultures
C57/BL6 spleen cells were cultured with indicated concentrations of compounds. After 24 hours the supernatants were collected and the levels of IL- 12 and IL-6 were determined by ELISA. All immunostimulatory oligonucleotides showed a concentration- dependent induction of two typical cytokines, IL-12 and IL-6 (Figures 6-7).
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EV 488513839 US Example 3: (need protocol for IFN-alpha induction in human pDC)
Peripheral blood mononuclear cells (PBMCs) from freshly drawn healthy volunteer blood (CBR Laboratories, Boston, MA) were isolated by Ficoll density gradient centrifugation method (Histopaque-1077, Sigma). pDCs were isolated from PBMCs by positive selection using the BDCA4 cell isolation kits (Miltenyi Biotec) according to the manufacturer's instructions. pDCs were plated in 96-well dishes using IXlO6 cells/ml. The IMOs dissolved in DPBS (pH 7.4; Mediatech) were added to a final concentration of 10.0 μg/ml to the cell cultures. The cells were then incubated at 37 0C for 24 hr and the supernatants were collected for ELISA assays. The experiments were performed in triplicate wells. The levels of IFN-α were measured by sandwich ELISA. The required reagents, including cytokine antibodies and standards, were purchased from PharMingen.
Example 4: IFN-alpha induction in human FMBC
Human PBMCs were plated in 48-well plates using 5X106 cells/ml. The IMOs dissolved in DPBS (pH 7.4; Mediatech) were added to a final concentration of 10.0 μg/ml to the cell cultures. The cells were then incubated at 37 °C for 24 hr and the supernatants were collected for ELISA assays. The experiments were performed in triplicate wells. The levels of IFN-α were measured by sandwich ELISA. The required reagents, including cytokine antibodies and standards, were purchased from PharMingen.
Example 5: Human B-cell proliferation
The culture medium used for the assay consisted of RPMI 1640 medium supplemented with 1.5 mM glutamine, 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, 50 μM 2-mercaptoethanol, 100 IU/ml penicillin-streptomycin mix and 10% heat- inactivated fetal bovine serum. A total of 0.5 X 106 B cells per ml (i.e.l X lO5 /200 μl/well) were stimulated in 96 well flat bottom plates with different concentrations of test oligonucleotides in triplicate for a total period of 72 hours. After 66 h, cells were pulsed with 0.75 μCi of [3H] -thymidine (ICi = 37 GBq; Perkin Elmer Life Sciences) in 20 μl RPMI 1640 medium (no serum) per well and harvested 8h later. The plates were then, harvested using a
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EV 488513839 US cell harvester and radioactive incorporation was determined using standard liquid scintillation technique. The results are expressed either as mean cpm +/-SD or as proliferation index (cpm treated group/cpm medium control).
EQUIVALENTS While the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be appreciated by one skilled in the art from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the invention and appended claims.
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Claims

WHAT IS CLAIMED IS:
1. An immunostimulatory oligonucleotide having a structure from the group of 5'- TCTGTR' GTTC1U1-X-U1CITTGR5TGTCT-S' ; 5'-CTGTR5GTTCTC-X- CTCTTGR'TGTC-5'; 5'-CTGTROTTCUIC1-X-CIU1CTTGR5TGTC-S'; 5'- CTGTR5GTTC1U1CI-X-C1U1C1TTGRTGTC-S'; 5'-TCTGTR5GTTCT-X-
CGTTCGAACGT-5'; 5' -TCTGTR' GAC AG-X-GAC AGR'TGTCT-5'; 5'- TCTGTR5GACA1G1-X-GIA1CAGR5TGTCT-S'; 5'-TCAGTR5GTTAG-X- GATTGR'TGACT-5'; S'-TCAGTR'GACTG-X-GTCAGR'TGACT-S'; 5'- TR'GTR'GAR'GAT-X-TAGR'AGR'TGR'T-S5; 5'-TR'GTR'GTAGTA-X- ATGATGR'TGR'T-5'; 5'-TR5GAAR5GTTCT-X-TCTTGR5AAGR5T-S'; and 5'-
TR5GTAR5GTACT-X-TCATGR5ATGR'T-55, wherein R5 = l-(2'-deoxy-β-D- ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine; A1ZC1ZG1AJ1 = 25-O-methyl- ribonucleotides; R = 25-deoxy-7-deazaguanosine and X = glycerol linker.
2. An immunostimulatory oligonucleotide according to Claim 1 having the structure 55- TCTGTR5GTTC1Ul-X-U1C1TTGR5TGTCT-55.
3. An immunostimulatory oligonucleotide according to Claim 1 having the structure 55- CTGTR5 GTTCTC-X-CTCTTGR'TGTC-5' .
4. An immunostimulatory oligonucleotide according to Claim 1 having the structure 55- CTGTR5GTTCU1C1-X-C1UlCTTGR5TGTC-55.
5. An immunostimulatory oligonucleotide according to Claim 1 having the structure 55- CTGTROTTC1UICI-X-C1U1C1TTGR5TGTC-S'.
6. An immunostimulatory oligonucleotide according to Claim 1 having the structure 5'- TCTGTR'GTTCT-X-CGTTCGAACGT-5'.
7. An immunostimulatory oligonucleotide according to Claim 1 having the structure 5'- TCTGTR'GACAG-X-GACAGR'TGTCT-S' .
8. An immunostimulatory oligonucleotide according to Claim 1 having the structure 55- TCTGTR5GACA1G1-X-G1A1CAGR5TGTCT-S'.
9. An immunostimulatory oligonucleotide according to Claim 1 having the structure 5'- TCAGTR'GTTAG-X-GATTGR'TGAC f -5 ' .
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10. An immunostimulatory oligonucleotide according to Claim 1 having the structure 5'- TCAGTR'GACTG-X-GTCAGR'TGACT-S'.
11. An immunostimulatory oligonucleotide according to Claim 1 having the structure 5'- TR'GTR'GAR'GAT-X-TAGR'AGR'TGRT-5'.
12. An immunostimulatory oligonucleotide according to Claim 1 having the structure 5'- TR'GTR'GTAGTA-X-ATGATGR'TGRT-5'.
13. An immunostimulatory oligonucleotide according to Claim 1 having the structure 5'- TR'GAAR'GTTCT-X-TCTTGR'AAGR'T-S'.
14. An immunostimulatory oligonucleotide according to Claim 1 having the structure 5'- TR'GTAR'GTACT-X-TCATGR'ATGR'T-S'.
15. A pharmaceutical formulation comprising the oligonucleotide according to Claim 1 and a physiologically acceptable carrier.
16. A method for generating an immune response in a vertebrate, the method comprising administering to the vertebrate an immunostimulatory oligonucleotide having a structure from the group of 5'-TCTGTR5GTTCIU1-X-U1C1TTGR5TGTCT-S'; 5'-
CTGTR' GTTCTC-X-CTCTTGR'TGTC-5' ; 5'-CTGTR5GTTCU1C1-X- C1U1CTTGRTGTC-S'; 5'-CTGTR5GTTC1U1C1-X-C1U1C1TTGR5TGTC-S'; 5'- TCTGTR'GTTCT-X-CGTTCGAACGT-5'; 5'-TCTGTR5GACAG-X- GACAGR'TGTCT-5'; 5'-TCTGTR5GACAiGrX-G1A1CAGR5TGTCT-S'; 5'- TCAGTR'GTTAG-X-GATTGR'TGACT-S'; 5'-TCAGTR5GACTG-X-
GTCAGR'TGACT-55; 5 '-TR5 GTR' GAR5GAT-X-TAGR5AGR5TGR5T-S5; 5'- TR'GTR'GTAGTA-X-ATGATGR'TGR'T-S5; 5'-TR5GAAR5GTTCT-X- TCTTGR'AAGR'T-5'; and 5'-TR5GTAR5GTACT-X-TCATGR5ATGR'T-5\ wherein R5 = l-(25-deoxy-β-D-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine; At/Ci/Gi/Ui = 2'-O-methyl-ribonucleotides; R = 2'-deoxy-7-deazaguanosine and X
= glycerol linker.
17. The method according to Claim 16, wherein the route of administration is selected from parenteral, oral, sublingual, transdermal, topical, intranasal, aerosol,
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EV 488513839 US intraocular, intratracheal, intrarectal, vaginal, gene gun, dermal patch, eye drop and mouthwash.
18. The method according to Claim 16 comprising administering an mimunostimulatory oligonucleotide having the structure 5'-TCTGTR5GTTC1U1-X- U1C1TTGR5TGTCT-S'.
19. The method according to Claim 16 comprising administering an immunostimulatory oligonucleotide having the structure 5'-CTGTR5GTTCTC-X- CTCTTGR'TGTC-5'.
20. The method according to Claim 16 comprising administering an immunostimulatory oligonucleotide having the structure 5 ' -CTGTR5 GTTCU1 C i -X-
C1U1CTTGR5TGTC-S'.
21. The method according to Claim 16 comprising administering an immunostimulatory oligonucleotide having the structure 5'-CTGTR5GTTCIU1C1-X- C1U1C1TTGR5TGTC-S5.
22. The method according to Claim 16 comprising administering an immunostimulatory oligonucleotide having the structure 5 '-TCTGTR'GTTCT-X- CGTTCGAACGT-55.
23. The method according to Claim 16 comprising administering an immunostimulatory oligonucleotide having the structure 5'-TCTGTR5GACAG-X- GACAGR'TGTCT-S'.
24. The method according to Claim 16 comprising administering an immunostirnulatory oligonucleotide having the structure 5'-TCTGTR5GACA1G1-X- G1A1CAGR5TGTCT-S'.
25. The method according to Claim 16 comprising administering an immunostimulatory oligonucleotide having the structure 5 ' -TCAGTR5 GTT AG-X-
GATTGR'TGACT-55.
26. The method according to Claim 16 comprising administering an immunostimulatory oligonucleotide having the structure 55 -TCAGTR5 G ACTG-X- GTCAGR'TGACT-S5.
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27. The method according to Claim 16 comprising administering an immunostimulatory oligonucleotide having the structure 5'-TR5GTR5GAR5GAT-X- TAGR'AGR'TGR'T-5' .
28. The method according to Claim 16 comprising administering an
5 immunostimulatory oligonucleotide having the structure 55 -TR5 GTR' GTAGTA-X-
ATGATGR'TGR'T-5'.
29. The method according to Claim 16 comprising administering an immunostimulatory oligonucleotide having the structure 55-TR5GAAR5GTTCT-X- TCTTGR'AAGR'T-5'.
10 30. The method according to Claim 16 comprising administering an immunostimulatory oligonucleotide having the structure 5'-TROTAR5GTACT-X- TCATGR'ATGR'T-5'.
31. A method for therapeutically treating a vertebrate having cancer, an autoimmune disorder, airway inflammation, inflammatory disorders, skin disorders, allergy,
15 asthma or a disease caused by a pathogen, such method comprising administering to the patient an immunostimulatory oligonucleotide having a structure from the group OfS5TCTGTR5GTTC1U1-X-U1C1TTGRTGTCT-S5; 5'- CTGTR'GTTCTC-X-CTCTTGR'TGTC-5'; 5'-CTGTR5GTTCU1C1-X- C1U1CTTGR5TGTC-S'; S'-CTGTR'GTTdUid-X-dUiC^TGR'TGTC-S'; 5'-
20 TCTGTR'GTTCT-X-CGTTCGAACGT-5'; 55-TCTGTR5GACAG-X-
GACAGRTGTCT-55; 5'-TCTGTR5GACA1G1-X-G1AICAGRTGTCT-S'; 5'- TCAGTR'GTTAG-X-GATTGRTGACT-5 ' ; 5 ' -TCAGTR' GACTG-X- GTCAGRTGACT-5'; S'-TR'GTR'GAR'GAT-X-TAGR' AGRTGR'T-5'; 5'- TR'GTR'GTAGTA-X-ATGATGRTGRT-S5; 55-TR5GAAR5GTTCT-X-
25 TCTTGR5 AAGRT-5'; and S'-TR'GTAR'GTACT-X-TCATGRΑTGR'T-S', wherein R' = l-(2'-deoxy-β-D-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine; A1ZC1AJ1ZU1 = 2'-O-methyl-ribonucleotides; R = 2'-deoxy-7-deazaguanosine and X = glycerol linker.
32. The method according to Claim 31 , wherein the route of administration is selected
T A
JU from parenteral, oral, sublingual, transdermal, topical, intranasal, aerosol,
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EV 488513839 US intraocular, intratracheal, intrarectal, vaginal, gene gun, dermal patch, eye drop and mouthwash.
33. The method according to Claim 31 comprising administering an immunostimulatory oligonucleotide having the structure 5'-TCTGTR5GTTC1UI-X- U1C1TTGR5TGTCT-S'.
34. The method according to Claim 31 comprising administering an immunostimulatory oligonucleotide having the structure 5'-CTGTR5GTTCTC-X- CTCTTGR'TGTC-5'.
35. The method according to Claim 31 comprising administering an immunostimulatory oligonucleotide having the structure 5'-CTGTR5GTTCUIC1-X- dUiCTTGR'TGTC-55.
36. The method according to Claim 31 comprising administering an immunostimulatory oligonucleotide having the structure 5'-CTGTR5GTTCIU1C1-X- C1U1C1TTGR5TGTC-S'.
37. The method according to Claim 31 comprising administering an immunostimulatory oligonucleotide having the structure 5'-TCTGTR5GTTCT-X- CGTTCGAACGT-5'.
38. The method according to Claim 31 comprising administering an immunostimulatory oligonucleotide having the structure 5'-TCTGTR5GACAG-X- GACAGR'TGTCT-S5.
39. The method according to Claim 31 comprising administering an immunostimulatory oligonucleotide having the structure 5 '-TCTGTR5GAC A1 G1-X- G1A1CAGR5TGTCT-S'.
40. The method according to Claim 31 comprising administering an immunostimulatory oligonucleotide having the structure 5' -TCAGTR' GTTAG-X-
GATTGR'TGACT-5'.
41. The method according to Claim 31 comprising administering an immunostimulatory oligonucleotide having the structure 5'-TCAGTR5GACTG-X- GTCAGR IGACT-55.
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42. The method according to Claim 31 comprising administering an immunostimulatory oligonucleotide having the structure 5'-TR5GTR5GAR5GAT-X- TAGR'AGR'TGR'T-55.
43. The method according to Claim 31 comprising administering an immunostimulatory oligonucleotide having the structure 55-TR5GTR5GTAGTA-X-
ATGATGR'TGR'T-5'.
44. The method according to Claim 31 comprising administering an immunostimulatory oligonucleotide having the structure 55-TR5GAAR5GTTCT-X- TCTTGR'AAGR'T-55.
45. The method according to Claim 31 comprising administering an immunostimulatory oligonucleotide having the structure 55 -TR' GTAR' GTACT-X- TCATGR'ATGR'T-5'.
46. A method for preventing cancer, an autoimmune disorder, airway inflammation, inflammatory disorders, skin disorders, allergy, asthma or a disease caused by a pathogen in a vertebrate, such method comprising administering to the vertebrate an immunostimulatory oligonucleotide having a structure from the group of 5'- TCTGTR5GTTC1U1-X-U1C1TTGR5TGTCT-S' ; 5'-CTGTR5GTTCTC-X- CTCTTGR'TGTC-5'; 55-CTGTR5GTTCUlC1-X-C1U1CTTGR5TGTC-55; 5'- CTGTR' GTTC1U1C1-X-C1U1C1TTGR5TGTC-S5 ; 55-TCTGTR5GTTCT-X- CGTTCGAACGT-S5; 5 '-TCTGTR5 GACAG-X-GACAGR5 TGTCT-55; 5'-
TCTGTR5GACA1G1-X-G1A1CAGR5TGTCT-S'; 55-TCAGTR'GTTAG-X- GATTGR'TGACT-5'; 5 '-TCAGTR5 GACTG-X-GTC AGR5 TGACT-55; 5'- TR5GTR5GAR5GAT-X-TAGR5 AGR'TGR'T-5'; 5'-TR'GTR'GTAGTA-X- ATGATGR'TGR'T-55; 5 '-TR5 GAAR5 GTTCT-X-TCTTGR5AAGR5T-S5; and 5'- TR'GTAR'GTACT-X-TCATGR5ATGR5T-55, wherein R5 = l-(2'-deoxy-β-D- ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine; A1ZC1ZG1AJ1 = 2'-O-methyl- ribonucleotides; R = 2'-deoxy-7-deazaguanosine and X = glycerol linker.
47. The method according to Claim 46, wherein the route of administration is selected from parenteral, oral, sublingual, transdermal, topical, intranasal, aerosol,
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EV 488513839 US intraocular, intratracheal, intrarectal, vaginal, gene gun, dermal patch, eye drop and mouthwash.
48. The method according to Claim 46 comprising administering an immunostimulatory oligonucleotide having the structure 5'-TCTGTR5GTTC1U1-X- U1C1TTGR5TGTCT-S'.
49. The method according to Claim 46 comprising administering an immunostimulatory oligonucleotide having the structure 5'-CTGTR5GTTCTC-X- CTCTTGR'TGTC-5'.
50. The method according to Claim 46 comprising administering an immunostimulatory oligonucleotide having the structure 5'-CTGTR5GTTCU1C1-X-
C1U1CTTGR5TGTC-S'.
51. The method according to Claim 46 comprising adrninistering an immunostimulatory oligonucleotide having the structure 5'-CTGTR5GTTC1U1C1-X- C1U1C1TTGR5TGTC-S'.
52. The method according to Claim 46 comprising administering an immunostimulatory oligonucleotide having the structure 5'-TCTGTR5GTTCT-X- CGTTCGAACGT-5'.
53. The method according to Claim 46 comprising administering an immunostimulatory oligonucleotide having the structure 55-TCTGTR5GACAG-X- GACAGR'TGTCT-5'.
54. The method according to Claim 46 comprising administering an immunostimulatory oligonucleotide having the structure 5 '-TCTGTR5GACA1 G1-X- G1A1CAGR5TGTCT-S'.
55. The method according to Claim 46 comprising administering an immunostimulatory oligonucleotide having the structure 55 -TCAGTR' GTTAG-X-
GATTGR'TGACT-5'.
56. The method according to Claim 46 comprising administering an immunostimulatory oligonucleotide having the structure 5'-TCAGTR5GACTG-X- GTCAGR'TGACT-55.
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57. The method according to Claim 46 comprising administering an immunostimulatory oligonucleotide having the structure 5'-TR5GTR5GAR5GAT-X- TAGR'AGR'TGR'T-5'.
58. The method according to Claim 46 comprising administering an immunostimulatory oligonucleotide having the structure 5'-TR'GTR'GTAGTA-X-
ATGATGR'TGR'T-5'.
59. The method according to Claim 46 comprising administering an immunostimulatory oligonucleotide having the structure 5'-TR5GAAR5GTTCT-X- TCTTGR'AAGR'T-55.
60. The method according to Claim 46 comprising administering an immunostimulatory oligonucleotide having the structure 5'-TR5GTAR'GTACT-X- TCATGR'ATGR'T-5'.
61. The oligonucleotide according to Claim 1, further comprising an antibody, antisense oligonucleotide, protein, antigen, allergen, chemotherapeutic agent or adjuvant.
62. The pharmaceutical composition according to Claim 15, further comprising an antibody, antisense oligonucleotide, protein, antigen, allergen, chemotherapeutic agent or adjuvant.
63. The method according to Claim 16, further comprising administering an antibody, antisense oligonucleotide, protein, antigen, allergen, chemotherapeutic agent or adjuvant.
64. The method according to Claim 31 , further comprising administering an antibody, antisense oligonucleotide, protein, antigen, allergen, chemotherapeutic agent or adjuvant.
65. The method according to Claim 46, further comprising administering an antibody, antisense oligonucleotide, protein, antigen, allergen, chemotherapeutic agent or adjuvant.
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PCT/US2005/040215 2005-11-07 2005-11-07 Immunostimulatory properties of oligonucleotide-based compounds comprising modified immunostimulatory dinucleotides WO2007055682A2 (en)

Priority Applications (7)

Application Number Priority Date Filing Date Title
PCT/US2005/040215 WO2007055682A2 (en) 2005-11-07 2005-11-07 Immunostimulatory properties of oligonucleotide-based compounds comprising modified immunostimulatory dinucleotides
EP05851389A EP1945653A4 (en) 2005-11-07 2005-11-07 Immunostimulatory properties of oligonucleotide-based compounds comprising modified immunostimulatory dinucleotides
AU2005338050A AU2005338050B2 (en) 2005-11-07 2005-11-07 Immunostimulatory properties of oligonucleotide-based compounds comprising modified immunostimulatory dinucleotides
CNA2005800524839A CN101351469A (en) 2005-11-07 2005-11-07 Immune stimulation specialty of compound containing modified immune irritation dinucleotide base on oligonucleotide
CA002630118A CA2630118A1 (en) 2005-11-07 2005-11-07 Immunostimulatory properties of oligonucleotide-based compounds comprising modified immunostimulatory dinucleotides
MX2008006004A MX2008006004A (en) 2005-11-07 2005-11-07 Immunostimulatory properties of oligonucleotide-based compounds comprising modified immunostimulatory dinucleotides.
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US7723500B2 (en) 1994-07-15 2010-05-25 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US7812000B2 (en) * 2001-10-24 2010-10-12 Idera Pharmaceuticals, Inc. Modulation of immunostimulatory properties of oligonucleotide-based compounds by optimal presentation of 5′ ends
US7851454B2 (en) * 2003-02-07 2010-12-14 Idera Pharmaceutials, Inc. Short immunomodulatory oligonucleotides
US11123294B2 (en) 2014-06-04 2021-09-21 Exicure Operating Company Multivalent delivery of immune modulators by liposomal spherical nucleic acids for prophylactic or therapeutic applications
US11213593B2 (en) 2014-11-21 2022-01-04 Northwestern University 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
US11433131B2 (en) 2017-05-11 2022-09-06 Northwestern University Adoptive cell therapy using spherical nucleic acids (SNAs)
US11696954B2 (en) 2017-04-28 2023-07-11 Exicure Operating Company Synthesis of spherical nucleic acids using lipophilic moieties
US11866700B2 (en) 2016-05-06 2024-01-09 Exicure Operating Company Liposomal spherical nucleic acid (SNA) constructs presenting antisense oligonucleotides (ASO) for specific knockdown of interleukin 17 receptor mRNA

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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7723500B2 (en) 1994-07-15 2010-05-25 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US7812000B2 (en) * 2001-10-24 2010-10-12 Idera Pharmaceuticals, Inc. Modulation of immunostimulatory properties of oligonucleotide-based compounds by optimal presentation of 5′ ends
US7851454B2 (en) * 2003-02-07 2010-12-14 Idera Pharmaceutials, Inc. Short immunomodulatory oligonucleotides
US7470674B2 (en) * 2005-11-07 2008-12-30 Idera Pharmaceuticals, Inc. Immunostimulatory properties of oligonucleotide-based compounds comprising modified immunostimulatory dinucleotides
US11123294B2 (en) 2014-06-04 2021-09-21 Exicure Operating Company Multivalent delivery of immune modulators by liposomal spherical nucleic acids for prophylactic or therapeutic applications
US11957788B2 (en) 2014-06-04 2024-04-16 Exicure Operating Company Multivalent delivery of immune modulators by liposomal spherical nucleic acids for prophylactic or therapeutic applications
US11213593B2 (en) 2014-11-21 2022-01-04 Northwestern University Sequence-specific cellular uptake of spherical nucleic acid nanoparticle conjugates
US11866700B2 (en) 2016-05-06 2024-01-09 Exicure Operating Company Liposomal spherical nucleic acid (SNA) constructs presenting antisense oligonucleotides (ASO) for specific knockdown of interleukin 17 receptor mRNA
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
US11433131B2 (en) 2017-05-11 2022-09-06 Northwestern University Adoptive cell therapy using spherical nucleic acids (SNAs)

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MX2008006004A (en) 2008-10-01
AU2005338050A1 (en) 2007-05-18
CN101351469A (en) 2009-01-21
EP1945653A4 (en) 2012-03-14
CA2630118A1 (en) 2007-05-18
EP1945653A2 (en) 2008-07-23
WO2007055682A8 (en) 2007-08-02
AU2005338050A8 (en) 2008-07-03
JP2009514525A (en) 2009-04-09
AU2005338050B2 (en) 2012-02-02

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