JP5005878B2 - Regulation of immunostimulation of oligonucleotide compounds by optimal display of the 5 'end - Google Patents

Regulation of immunostimulation of oligonucleotide compounds by optimal display of the 5 'end Download PDF

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JP5005878B2
JP5005878B2 JP2003558124A JP2003558124A JP5005878B2 JP 5005878 B2 JP5005878 B2 JP 5005878B2 JP 2003558124 A JP2003558124 A JP 2003558124A JP 2003558124 A JP2003558124 A JP 2003558124A JP 5005878 B2 JP5005878 B2 JP 5005878B2
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nucleoside
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oligonucleotide
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サディール アグラワル,
エカンバー エム. カンディマッラ,
ラクシュミ バガット,
ドン ユー,
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イデラ ファーマシューティカルズ インコーポレイテッドIdera Pharmaceuticals, Inc.
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Description

Detailed Description of the Invention

Background of the Invention
FIELD OF THE INVENTION This invention relates to immunology and immunotherapy applications using oligonucleotides as immunostimulants.

Summary of Related Art Oligonucleotides have become an indispensable tool in modern molecular biology, and are used in a wide range of technologies from probe-based diagnosis using PCR to antisense inhibition of gene expression and immunotherapy applications. This wide range of uses for oligonucleotides has led to an increasing demand for fast, cheap and efficient methods of synthesizing oligonucleotides.

  Synthesis of oligonucleotides for antisense and diagnostic applications is now routine. For example, Methods in Molecular Bioloy, Vol. 20: Prolocols 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); Uhlmann and Peyman, supra; Agrawal andlyer, Curr. Op. In Biotech. 6: 12 (1995); Antisense Research and Applications (Crooke and Lebleu, eds., CRC Press, Boca Raton, 1993).

  Early synthetic approaches included phosphodiester and phosphotriester chemistry. 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 oligonucleotide and polynucleotide synthesis. These early approaches have greatly shifted to more efficient phosphoramidite and H-phosphonate synthesis. For example, Beaucage and Caruthers, Tetrahedron Lett. 22: 1859-1862 (1981) discloses the use of deoxyribonucleoside phosphoramidites in polynucleotide synthesis. Agrawal and Zamecnik, U.S. Patent No. 5,149, 798 (1992) discloses optimized oligonucleotide synthesis by the H-phosphonate approach.

  All these modern approaches have been used to synthesize oligonucleotides with various modified internucleotide linkages. Agrawal and Goodchild, Tetrahedron Lett. 28: 3539-3542 (1987) shows oligonucleotide methylphosphonate synthesis using phosphoramidite chemistry. Connolly etal., Biochem. 23: 3443 (1984) discloses the synthesis of oligonucleotide phosphorothioates using phosphoramidite chemistry. Jager et al., Biochem. 27: 7237 (1988) discloses the synthesis of oligonucleotide phosphoramidates using phosphoramidite chemistry. Agrawal etal., Proc. Natl. Acad. Sci. (USA) 85: 7079-7083 (1988) discloses the synthesis of oligonucleotide phosphoramidates and phosphorothioates using H-phosphonate chemistry.

  More recently, several researchers have demonstrated the effectiveness of using oligonucleotides as immunostimulators in immunotherapy applications. The finding that phosphodiester and phosphorothioate oligonucleotides can cause immunostimulation has generated interest in developing this side effect as a therapeutic tool. These attempts focus on phosphorothioate oligonucleotides containing the natural CpG dinucleotide.

  Kuramoto et al., Jpn. J. Cancer Res. 83: 1128-1131 (1992) states that phosphodiester oligonucleotides containing palindrome containing CpG dinucleotides induce the synthesis of interferon-alpha and gamma, It shows that the activity can be enhanced. Krieg etal., Nature 371: 546-549 (1995) discloses that oligonucleotides containing phosphorothioate CpG 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) show that phosphorothioate oligonucleotides containing CpG enhance immune responses against influenza viruses. McCluskie and Davis, J. Immunol. 161: 4463-4466 (1998) show that oligonucleotides containing CpG act as potent adjuvants and enhance the immune response to hepatitis B surface antigens.

  Other modifications to CpG-containing phosphorothioate oligonucleotides can affect the ability to act as a modulator of immune responses. For example, Zhao et al., Biochem. Pharmacol. (1996) 51: 173-182; Zhao et al., Biochem Pharmacol. (1996) 52: 1537-1544; 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. Letl. (2000) 10: 2585-2588; Yu et al., Bioorg. Med Chem. Lett. (2001) 11: 2263-2267; Kandimalla et al., Bioorg. Med Chem. (2001) 9: 807-813.

  These reports reveal that there is still a need to be able to enhance the immune response caused by immunostimulatory oligonucleotides.

BRIEF SUMMARY OF THE INVENTION The present invention provides a method for enhancing the immune response caused by oligonucleotide compounds. The method according to the present invention can increase the immunostimulatory effect of immunostimulatory oligonucleotides for immunotherapy applications. The present inventors have surprisingly discovered that a modification of an immunostimulatory oligonucleotide that optimally presents its 5 'end dramatically enhances its immunostimulatory activity. Such oligonucleotides are referred to herein as “immunomers”.

  Thus, in a first aspect, the present invention comprises at least two oligonucleotides linked to their 3 ′ end, internucleoside linkages, or functionalized nucleobases or sugars via a non-nucleotide linker, At least one of the oligonucleotides is an immunostimulatory oligonucleotide and has an accessible 5 'end.

  In one embodiment, the immunomer has an immunostimulatory formula represented by the formula 5′-Pyr-Pur-3 ′, wherein Pyr is a natural or non-natural pyrimidine nucleoside and Pur is a natural or non-natural purine nucleoside. Sex dinucleotides.

In other embodiments, the immunomer is wherein C is cytidine or 2'-deoxycytidine and C * is 2'-deoxythymidine, arabinocytidine, 2'-deoxy-2'-substituted arabinocytidine. 2'-O-substituted arabinocytidine, 2'-deoxy-5-hydroxycytidine, 2'-deoxy-N4-alkyl-cytidine, 2'-deoxy-4-thiouridine or other unnatural pyrimidine nucleosides, G is guanosine or 2'-deoxyguanosine, and G * is 2'-deoxy-7-deazaguanosine, 2'-deoxy-6-thioguanosine, arabinoguanosine, 2'-deoxy-2'-substituted- Arabinoguanosine, 2'-O-substituted-arabinoguanosine, or other non-natural purine nucleoside, p is an internucleoside selected from the group consisting of phosphodiester, phosphorothioate and phosphorodithioate Bond is, in represented, including CpG, C * pG, CpG *, and C * pG * immunostimulatory dinucleotide selected from the group consisting of. In a preferred embodiment, the immunostimulatory dinucleotide is not CpG.

In yet another embodiment, the immunostimulatory oligonucleotide has the formula (III):
5'-Nn-N1-YZ-N1-Nn-3 '(III)

In the formula:
Y is cytidine, 2′-deoxythymidine, 2′-deoxycytidine, arabinocytidine, 2′-deoxy-2′-substituted arabinocytidine, 2′-O-substituted arabinocytidine, 2′-deoxy-5- Hydroxycytidine, 2′-deoxy-N4-alkyl-cytidine, 2′-deoxy-4-thiouridine or other unnatural pyrimidine nucleoside;

Z is guanosine or 2'-deoxyguanosine, G * is 2'-deoxy-7-deazaguanosine, 2'-deoxy-6-thioguanosine, arabinoguanosine, 2'-deoxy-2'-substituted-arabino Guanosine, 2′-O-substituted-arabinoguanosine, 2′-deoxyinosine or other non-natural purine nucleoside,

N1 is in each case preferably a naturally occurring or synthetic nucleoside, or an abasic nucleoside, an arabino nucleoside, a 2′-deoxyuridine, an α-deoxyribonucleoside, a β-L-deoxyribonucleoside, and An immunostimulatory moiety selected from the group consisting of a phosphodiester or a nucleoside linked to a 3 'adjacent nucleoside by a modified internucleoside linkage, wherein the modified internucleoside linkage has a length of about Linkers with lengths from 2 angstroms to about 200 angstroms, C2-C18 alkyl linkers, poly (ethylene glycol) linkers, 2-aminobutyl-1,3-propanediol linkers, glyceryl linkers, 2'-5 'internucleosides Bond and phosphorothio Over bets, selected without limitation from phosphorodithioate or methylphosphonate internucleoside linkage;

Nn in each case is a naturally occurring nucleoside, or preferably an abasic nucleoside, an arabino nucleoside, a 2'-deoxyuridine, an α-deoxyribonucleoside, a 2'-O-substituted ribonucleoside, and a modified internucleoside. An immunostimulatory moiety selected from the group consisting of a nucleoside linked to a 3 'adjacent nucleoside by a nucleoside bond, wherein the modified internucleoside bond comprises an amino linker, a 2'-5' internucleoside bond, And selected from the group consisting of methylphosphonate internucleoside linkages;
At least one N1 or Nn is an immunostimulatory moiety;
Where n is a number from 0-30;

Wherein the 3 ′ end, internucleoside linkage, or functionalized nucleobase or sugar binds to another oligonucleotide, with or without immunostimulatory, directly or via a non-nucleotide linker. ing,
An immunostimulatory region represented by

  In a second aspect, the present invention provides an immunomer complex comprising the above immunomer and an antigen bound to the immunomer at a position other than the accessible 5 ′ end.

  In a third aspect, the present invention provides a pharmaceutical formulation comprising an immunomer or immunomer complex according to the present invention and a physiologically acceptable carrier.

  In a fourth aspect, the present invention provides a method for eliciting an immune response in a vertebrate, said method comprising administering an immunomer to a vertebrate or an immunomer complex according to the present invention. Including. In some embodiments, the vertebrate is a mammal.

  In a fifth aspect, the present invention provides a method for therapeutically treating a patient suffering from a disease or disorder, said method comprising a patient of an immunomer or immunomer complex according to the present invention Administration. In various embodiments, the disease or disorder to be treated is a cancer, autoimmune disease, airway inflammation, asthma, allergy, or a disease caused by a pathogen.

Detailed Description of the Preferred Embodiments The present invention relates to the therapeutic use of oligonucleotides as immunostimulators for immunotherapy applications. The patents, patent applications and references cited and registered herein are hereby incorporated by reference to the same extent as if each was clearly and individually indicated. In case of conflict between the teachings of the references cited herein and the present specification, the present specification shall control for the purposes of the present invention.

  The present invention is not limited to cancer, autoimmune diseases, asthma, respiratory allergies, food allergies, bacteria, parasites, and viral infections, and is applicable to immunotherapy applications in adults, children, and animals. Methods are provided for enhancing the immune response caused by the immunostimulatory compound used. Accordingly, the present invention further provides compounds having an optimal level of immunostimulatory effect for immunotherapy and methods for making and using such compounds. In addition, the immunomers of the present invention are also useful as adjuvants in combination with DNA vaccines, antibodies, allergens, chemotherapeutic agents and antisense oligonucleotides.

The inventors have surprisingly discovered that modifications of immunostimulatory oligonucleotides that optimally present their 5 ′ ends dramatically affect their immunostimulatory properties. Such oligonucleotides are referred to herein as immunomers.

  In a first aspect, the present invention comprises at least two oligonucleotides whose 3 ′ end, internucleoside linkage, or functionalized nucleobase or sugar is attached to a non-nucleotide linker, wherein at least the oligonucleotide of One of them is an immunostimulatory oligonucleotide with an accessible 5 'end.

  The term “accessible 5 ′ end” as used herein means that the 5 ′ end of the oligonucleotide can be used sufficiently so that it can recognize and bind to the immunomer and access factors that stimulate the immune system. To do. In oligonucleotides with accessible 5 ′ ends, the 5′OH position of the terminal sugar is not covalently linked to two or more nucleoside residues. Optionally, the 5′OH may be linked to a phosphate, phosphorothioate or phosphorodithioate moiety, an aromatic or aliphatic linker, cholesterol or other component that does not interfere with accessibility.

  In the present invention, “immunomer” includes at least two oligonucleotides bound to its 3 ′ end, internucleoside bond, or functionalized nucleobase or sugar, directly or via a non-nucleotide linker, In this context, at least one of the oligonucleotides is an immunostimulatory oligonucleotide and refers to any compound having an accessible 5 'end, said compound being an immune response when administered to a vertebrate Is something that provokes. In some embodiments, the vertebrate is a mammal, including a human.

  In certain embodiments, the immunomer comprises two or more immunostimulatory oligonucleotides, which (in the context of the immunomer) can be the same or different. Preferably, each said immunostimulatory oligonucleotide has at least one accessible 5 'end.

  In some embodiments, the immunomer comprises at least one oligonucleotide complementary to the gene in addition to the immunostimulatory oligonucleotide. As used herein, “complementary” means that an oligonucleotide hybridizes with a region of a gene under physiological conditions. In some embodiments, the oligonucleotide down-regulates gene expression. Such down-regulating oligonucleotides are preferably selected from the group consisting of antisense oligonucleotides, ribozyme oligonucleotides, inhibitory small RNAs and decoy oligonucleotides. As used herein, “down-regulation of a gene” refers to inhibiting gene transcription or translation of a gene product. Thus, according to these aspects of the invention, immunomers can be used to target the target of one or more specific diseases while stimulating the immune system.

  In some embodiments, the immunomer comprises a ribozyme or a decoy oligonucleotide. As used herein, “ribozyme” refers to an oligonucleotide having catalytic activity. Preferably, the ribozyme binds with a specific nucleic acid as a target and cleaves the target. The “decoy oligonucleotide” used herein binds to a transcription factor in a sequence-specific manner and stops transcription activity. Preferably, the ribozyme or decoy oligonucleotide exhibits a secondary structure including without limitation a stem loop or hairpin structure. In some embodiments, at least one oligonucleotide comprises poly (I) -poly (dC). In some embodiments, at least one set of Nn comprises 3 to 10 dG and / or G or 2′-substituted ribo or arabino G 2.

  For the purposes of the present invention, “oligonucleotide” refers to a polynucleoside consisting of a plurality of linked nucleoside units. Said oligonucleotides can be obtained from existing nucleic acid sources including genomic DNA or cDNA, but are preferably produced by synthetic methods. In preferred embodiments, each nucleoside unit comprises a heterocyclic base and pentofuranosyl, trehalose, arabinose, 2'-deoxy-2'-substituted arabinose, 2'-O-substituted arabinose or a hexasaccharide. Nucleoside residues can be linked together by a number of known internucleoside linkages.

  The internucleoside bond is not limited, but is phosphodiester, phosphorothioate, phosphorodithioate, alkyl phosphonate, alkyl phosphorothioate, phosphotriester, phosphoramidate, siloxane, carbonate, carboalkoxy, acetamidate, carbamate, morpholino , Borano, thioether, bridged phosphoramidate, bridged methylene phosphonate, bridged phosphorothioate, and sulfone internucleoside linkages.

  “Oligonucleotides” also include polynucleosides having one or more stereospecific internucleoside linkages (eg, (Rp)-or (Sp) -phosphorothioate, alkylphosphonate, or phosphotriester linkages). As used herein, “oligonucleotide” and “dinucleotide” particularly mean any of the aforementioned polynucleoside and dinucleoside having an internucleoside bond, whether or not the bond includes a phosphate group. In certain preferred embodiments, these internucleoside linkages may be phosphodiester, phosphorothioate, or phosphodithioate linkages, or combinations thereof.

  In some embodiments, each oligonucleotide has 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 oligonucleotide has from about 5 to about 18, alternatively from about 5 to about 14 nucleoside residues. “About” here means that the exact number is not important. Thus, the number of nucleoside residues in an oligonucleotide is not critical, and oligonucleotides having 1 or 2 fewer nucleoside residues, or 1 to several additional nucleoside residues, are equivalent in each of the above embodiments. It means that there is. In some embodiments, the one or more oligonucleotides have 11 nucleosides.

  “Oligonucleotide” also encompasses polynucleosides having additional substituents including, without limitation, protein groups, lipophilic groups, intercalating agents, diamines, folic acid, cholesterol, and adamantane. “Oligonucleotide” also includes peptide nucleic acids (PNA), peptide nucleic acids linked to phosphate groups (PHONA), locked nucleic acids (LNA), morpholino backbone oligonucleotides and oligonucleotides having backbone portions with alkyl linkers and amino linkers, It includes any other nucleobase including polymers.

  The oligonucleotides of the invention include naturally occurring nucleosides, modified nucleosides or mixtures thereof. As used herein, “modified nucleoside” refers to 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 described herein. In some embodiments, the modified nucleoside is a 2'-substituted ribonucleoside, an arabino nucleoside, or a 2'-deoxy-2'-fluoro arabinoside.

  For the purposes of the present invention, “2′-substituted ribonucleosides” include ribonucleosides in which the hydroxyl group at the 2 ′ position of the pentose monosaccharide moiety is substituted to become a 2′-O-substituted ribonucleoside. Preferably, such substitution has a lower alkyl group containing 1-6 saturated or unsaturated carbon atoms, or an aryl group containing 6-10 carbon atoms, and such alkyl or aryl groups May be unsubstituted or substituted with a halo, hydroxy, trifluoromethyl, cyano, nitro, acyl, acyloxy, alkoxy, carboxyl, carboalkoxy, or amino group. Examples of such 2′-O-substituted ribonucleosides include, without limitation, 2′-O-methyl ribonucleosides and 2′-O-methoxyethyl ribonucleosides.

  “2′-substituted ribonucleosides” also include ribonucleosides in which the 2 ′ hydroxyl group is substituted with a lower alkyl group containing 1-6 saturated or unsaturated carbon atoms, or an amino or halo group. Examples of such 2'-substituted ribonucleosides include, without limitation, 2'-amino, 2'-fluoro, 2'-allyl, and 2'-propargyl ribonucleoside.

  “Oligonucleotide” includes hybrid or chimeric oligonucleotides. A “chimeric oligonucleotide” is an oligonucleotide having one or more internucleoside linkages. Preferred examples of such chimeric oligonucleotides include phosphorothioate, phosphodiester, or phosphorodithioate moieties and nonionic linkages such as alkylphosphonate or alkylphosphonothionate linkages. (See, for example, Pederson etal. U.S. Patent Nos. 5, 635,377 and 5, 366, 878)

  A “hybrid oligonucleotide” is an oligonucleotide having one or more nucleosides. Preferred examples of such hybrid oligonucleotides include ribonucleotides or 2′-substituted ribonucleotide sites, and deoxyribonucleotide sites (see, eg, Metelev and Agrawal, U.S. Patent Nos. 5,652,355, 6,346,614 and 6,143,881).

  For the purposes of the present invention, an “immunostimulatory oligonucleotide” refers to an oligonucleotide as described above that elicits an immune response when administered to vertebrates such as fish, birds, or mammals. The “mammal” herein includes, without limitation, rats, mice, cats, dogs, horses, livestock, cows, pigs, rabbits, primates excluding humans, humans, and the like. Useful immunostimulatory oligonucleotides are published in Agrawal et al., WO 98/49288, November 5,1998; published in WO01 / 12804, February 22,2001; published in WO01 / 55370, August2, 2001; PCT / USO1 / 13682, April. 30,2001 application; and PCT / USOI / 30137, September 26,2001 application can be found. Preferably, the immunostimulatory oligonucleotide comprises at least one phosphodiester, phosphorothioate or phosphorodithioate internucleoside linkage.

  In some embodiments, the immunostimulatory oligonucleotide has an immunostimulatory activity represented by the formula 5′-Pyr-Pur-3 ′, wherein Pyr is a natural or synthetic pyrimidine nucleoside and Pur is a natural or synthetic purine nucleoside. Contains dinucleotides. As used herein, “pyrimidine nucleoside” refers to a nucleoside in which the base component of the nucleoside is a pyrimidine base. Similarly, “purine nucleoside” refers to a nucleoside in which the base component of the nucleoside is a purine base. For purposes of the present 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 are according to the invention (I):
In the formula:
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, 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, electron donating group;
X is carbon or nitrogen;
S ′ is a pentose or hexose ring, or a non-naturally occurring saccharide,
It has the structure represented by these.

  Preferably, the sugar ring is derivatized with a phosphate moiety, a modified phosphate moiety, or other linker moiety suitable for attaching pyrimidine nucleosides to other nucleosides or nucleoside analogs.

Preferred hydrogen bond donors include, without limitation, —NH—, —NH 2 , —SH and —OH. Preferred hydrogen bond acceptors include, but are not limited to, C = O, C = S, and the ring nitrogen atom of an aromatic heterocyclic ring , such as 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 the purposes of the present invention, a “naturally occurring sugar moiety” is a sugar moiety that is present as part of a nucleic acid, eg, ribose and 2′-deoxyribose, and “a non-naturally occurring sugar moiety” All sugar moieties that do not exist as part of the nucleic acid, but are used as backbones for oligonucleotides, such as hexasaccharides. Arabinose and arabinose derivatives are examples of preferred sugar moieties.

Preferred purine nucleoside analogues are according to the invention (II):
In the formula:
D is a hydrogen bond donor;
D ′ is selected from the group consisting of hydrogen, hydrogen bond donor, hydrogen bond acceptor, hydrophilic group;
A is a hydrogen bond acceptor or a hydrophilic group;
X is carbon or nitrogen;
Each L is independently selected from the group consisting of C, O, N and S;
And
S ′ is a pentose or hexose ring, or a non-naturally occurring saccharide,
It has the structure represented by these.

  Preferably, the sugar ring is derivatized with a phosphate moiety, a modified phosphate moiety, or other linker moiety suitable for attachment of a pyrimidine nucleoside to another nucleoside or nucleoside analog.

Preferred hydrogen bond donors include, without limitation, —NH—, —NH 2 , —SH and —OH. Preferred hydrogen bond acceptors include, but are not limited to, C═O, C═S, —NO 2 and the ring nitrogen atom of an aromatic heterocycle , such as N1 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, 6-thioguanine and 7-deazaguanine. In some embodiments, the sugar moiety S ′ in (II) is a sugar moiety as described above for non-naturally occurring structure (I).

In a preferred embodiment, the immunostimulatory dinucleotide is selected from the group consisting of CpG, C * pG, CpG * and C * pG * , where C is cytidine or 2′-deoxycytidine and C * is 2'-deoxythymidine, arabinocytidine, 2'-deoxy-2'-substituted arabinocytidine, 2'-O-substituted arabinocytidine, 2'-deoxy-5-hydroxycytidine, 2'-deoxy-N4- Alkyl-cytidine, 2'-deoxy-4-thiouridine or other unnatural pyrimidine nucleoside, G is guanosine or 2'-deoxyguanosine, G * is 2'-deoxy-7-deazaguanosine, 2 ' -Deoxy-6-thioguanosine, arabinoguanosine, 2'-deoxy-2'-substituted-arabinoguanosine, 2'-O-substituted-arabinoguanosine, 2'-deoxyinosine or other unnatural purine nucleosides P is phosphodiester, phos It is an internucleoside bond selected from the group consisting of holothioate and phosphorodithioate. In certain preferred embodiments, the immunostimulatory dinucleotide is not CpG.

The immunostimulatory oligonucleotide may include an immunostimulatory moiety on one or both sides of the immunostimulatory dinucleotide. Thus, in some embodiments, the immunostimulatory oligonucleotide has a structure (III):
5'-Nn-N 1 -YZN 1 -Nn-3 '(III)
Where:

Y is cytidine, 2'-deoxythymidine, 2'-deoxycytidine, arabinocytidine, 2'-deoxy-2'-substituted arabinocytidine, 2'-O-substituted arabinocytidine, 2'-deoxy-5-hydroxycytidine 2'-deoxy-N4-alkyl-cytidine, 2'-deoxy-4-thiouridine or other unnatural pyrimidine nucleoside;
Z is guanosine or 2'-deoxyguanosine, G * is 2'-deoxy-7-deazaguanosine, 2'-deoxy-6-thioguanosine, arabinoguanosine, 2'-deoxy-2'-substituted-arabino Guanosine, 2′-O-substituted-arabinoguanosine, 2′-deoxyinosine or other non-natural purine nucleoside,

N1 is preferably in each case a naturally occurring or synthetic nucleoside, or an abasic nucleoside, arabino nucleoside, 2′-deoxyuridine, α-deoxyribonucleoside, β-L-deoxyribonucleoside, and phospho An immunostimulatory moiety selected from the group consisting of a diester or a nucleoside linked to the 3 'adjacent nucleoside by a modified internucleoside bond, wherein the modified internucleoside bond is not limited A linker having a length from about 2 angstroms to about 200 angstroms in length, C2-C18 alkyl linker, polyethylene glycol linker, 2-aminobutyl-1,3-propanediol linker, glyceryl linker, 2'-5 ' An internucleoside bond, and Phosphorothioate, phosphorodithioate or methylphosphonate internucleoside linkage is selected from;

Nn is preferably in each case a naturally occurring nucleoside or abasic nucleoside, arabino nucleoside, 2′-deoxyuridine, α-deoxyribonucleoside, 2′-O-substituted ribonucleoside, and modified An immunostimulatory moiety selected from the group consisting of a nucleoside linked to a 3 'adjacent nucleoside by an internucleoside bond, wherein the modified internucleoside bond is an amino linker, a 2'-5' internucleoside bond And selected from the group consisting of methylphosphonate internucleoside linkages;
At least one N1 or Nn has an immunostimulatory moiety;
Where n is a number from 0-30; and

In the formula, the 3 ′ end, internucleoside linkage, or functionalized nucleobase or sugar binds to another oligonucleotide, with or without immunostimulatory, directly or via a non-nucleotide linker. ing,
An immunostimulatory region represented by

  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, but are not limited to, methylphosphonate, methylphosphonothionate, phosphotriester, phosphothiotriester, phosphorothioate, phosphorodithioate, triester prodrug, sulfone, sulfonamide, sulfamate, Formacetal, N-methylhydroxylamine, carbonate, carbamate, morpholino, boranophosphonate, phosphoramidate, especially primary aminophosphoramidate, N3 phosphoramidate and N5 phosphoramidate, and stereospecific binding ( For example, modifications in the phosphate backbone including (Rp)-or (Sp) -phosphorothioate, alkylphosphonate, or phosphotriester linkages).

Preferred immunostimulatory moieties are further according to the present invention without limitation, 2′-O-methyl ribose, 2′-O-methoxyethyl ribose, 2′-O-propargyl ribose, and 2′-deoxy. including 2'-fluoro ribose, 2'-substituted pentose; including 3'-O- methyl ribose without limitation 3'-substituted pentose; 1 ', 2'-dideoxyribose; arabinose Substituted arabinose sugars including, but not limited to, 1′-methylrabinose, 3′-hydroxymethylarabinose, 4′-hydroxymethylarabinose and 2′-substituted arabinose sugars; 1,5-anhydro without limitation Hexatrisaccharides including hexitol; and nucleosides with sugar modifications including but not limited to alpha-anomers.

  In embodiments where the modified sugar is a 3'-deoxyribonucleoside or a 3'-O-substituted ribonucleoside, the immunostimulatory moiety is linked to an adjacent nucleoside via a 2'-5 'internucleoside linkage. .

  Preferred immunostimulatory moieties are further according to the present invention include peptide nucleic acids (PNA), peptide nucleic acids linked to phosphate groups (PHONA), locked nucleic acids (LNA), morpholino backbone oligonucleotides and without limitation alkyl linkers or amino acids. Includes oligonucleotides with other carbohydrate backbone modifications and substitutions, including oligonucleotides having linker moieties with a length from about 2 angstroms to about 200 angstroms including linkers.

  The alkyl linker may be branched or unbranched, may be substituted or unsubstituted, and may be 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 contain one or more functional groups selected from the group consisting of hydroxy, amino, thiol, ether, amide, thioamide, ester, urea and thioether. Some such functionalized alkyl linkers are polyethylene glycol linkers of the formula —O— (CH 2 —CH 2 —O—) n (n = 1-9). Some other functionalized alkyl linkers are peptides or amino acids.

  Preferred immunostimulatory moieties further include, according to the present invention, DNA isoforms that include, without limitation, β-L-deoxyribonucleosides and α-deoxyribonucleosides. Preferred immunostimulatory moieties incorporate 3 ′ modifications according to the present invention, and further include, but are not limited to, 2′-5 ′, 2′-2 ′, 3′-3 ′ and 5′-5 ′ linkages. Including nucleosides having unnatural internucleoside binding positions.

  Preferred immunostimulatory moieties are further according to the invention, without limitation, 5-hydroxycytosine, 5-hydroxymethylcytosine, N4-ethylcytosine, 4-thiouracil, 6-thioguanine, 7-deazaguanine, inosine, nitro Includes nucleosides with modified heterocyclic bases including pyrrole, C5-propynylpyrimidine, and diaminopurines including, but not limited to 2,6-diaminopurine.

As a specific example , but not limited to, for example, in the immunostimulatory region of structure (II I) , a methylphosphonate internucleoside linkage at the N1 or Nn position is an immunostimulatory moiety and ranges from about 2 angstroms to about 200 A linker having an angstrom length, a C2-C18 alkyl linker at the X1 position is an immunostimulatory moiety, and a β-L-deoxyribonucleoside at the X1 position is an immunostimulatory moiety.

  Table 1 shows typical positions and structures of immunostimulatory portions. A reference to a linker as an immunostimulatory moiety at a particular position is that the nucleoside residue at that position is replaced with the linker indicated at its 3'-hydroxyl position, so that the nucleoside residue and the 3 ' It is understood to mean creating a modified internucleoside bond between adjacent nucleosides.

  Similarly, a reference to a modified internucleoside linkage as an immunostimulatory moiety at a particular position is where the nucleoside residue at that position is linked to the 3 'adjacent nucleoside via the listed linkage. Means that.

Table 2 shows representative positions and structures of immunostimulatory moieties in immunostimulatory oligonucleotides having upstream enhancement regions. As used herein, “spacer 9” refers to a polyethylene glycol linker represented by the formula —O— (CH 2 —CH 2 —O—) n —, wherein n is 3. “Spacer 18” refers to a polyethylene glycol linker represented by the formula —O— (CH 2 —CH 2 —O—) n —, where n is 6.

The “C2-C18 alkyl linker” as used herein is a linker represented by the formula —O— (CH 2 ) q —O—, wherein q is an integer from 2 to 18. Accordingly, “C3 linker” and “C3 alkyl linker” refer to a linker represented by the formula —O— (CH 2 ) 3 —O—. In spacer 9, spacer 18, and C2-C18 alkyl linker, respectively, the linker is attached to the adjacent nucleoside via a phosphodiester, phosphorothioate, or phosphorodithioate linkage.

  Table 3 shows representative positions and immunostimulatory moiety structures in immunostimulatory oligonucleotides with downstream enhancement regions.

  The immunomer, according to the present invention, comprises at least two oligonucleotides linked via a non-nucleotide linker to its 3 'end, an internucleoside linkage, or a functionalized nucleobase or sugar. For the purposes of the present invention, a “non-nucleotide linker” is any moiety that can be attached to an oligonucleotide via a covalent or non-covalent bond. Preferably such linkers are from about 2 angstroms to about 200 angstroms in length. Some examples of preferred linkers are the following four sets: Non-covalent bonds include but are not limited to electrostatic interactions, hydrophobic interactions, π-stacking interactions and hydrogen bonds.

  “Non-nucleotide linker” does not mean an internucleoside linkage as described above, such as, for example, a phosphodiester, phosphorothioate, or phosphorodithioate functional group that binds directly to the 3′-hydroxyl group of two nucleosides. For the purposes of the present invention, such direct 3′-3 ′ linkages are considered “nucleotide linkages”.

  In some embodiments, the non-nucleotide linker is a metal including but not limited to gold particles. In some other embodiments, the non-nucleotide linker is a soluble or insoluble biodegradable polymer bead.

  In yet other embodiments, the non-nucleotide linker is an organic moiety having a functional group that is allowed to bind to the oligonucleotide. Such a bond is preferably by a stable covalent bond. As a non-limiting example, the linker may be attached to any suitable position of the nucleoside, as depicted in FIG. In some preferred embodiments, the linker is attached to the 3′-hydroxyl. In such embodiments, preferably the linker comprises a hydroxyl functional group that is preferably attached to the 3′-hydroxyl by a phosphodiester, phosphorothioate, phosphorodithioate, or non-phosphate linkage.

  In some embodiments, the non-nucleotide linker is a biomolecule including but not limited to polypeptides, antibodies, lipids, antigens, allergens, and oligosaccharides. In some embodiments, the non-nucleotide linker is a small molecule. For the purposes of the present invention, a small molecule is an organic moiety having a molecular weight of less than 1000 Da. In some embodiments, the small molecule has a molecular weight less than 750 Da.

  In some embodiments, the small molecule is an aliphatic or aromatic hydrocarbon, either optionally, linear, attached to or attached to the oligonucleotide, hydroxy, amino, It may contain one or more functional groups selected from the group consisting of thiol, thioether, ether, amide, thioamide, ester, urea and thiourea. Small molecules 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, in non-nucleotide linkers, “small molecule” is not meant to include nucleosides.

In some embodiments, the small molecule linker is a glycerol or glycerol homologue HO- (CH 2 ) o -CH (OH)-(CH 2 ) p -OH, wherein o and p are independently from 1 to about A glycerol or glycerol homolog represented by 6, 1 to about 4, or an integer from 1 to about 3. In some embodiments, the small molecule linker is a derivative of 1,3-diamino-2-hydroxypropane. Some such derivatives have the formula HO- (CH 2 ) m -C (O, wherein m is an integer from 0 to about 10, 0 to about 6, 2 to about 6 or 2 to about 4. ) NH-CH 2 -CH (OH ) -CH 2 -NHC (O) - with a (CH 2) m -OH.

  Some non-nucleotide linkers allow the attachment of two or more oligonucleotides according to the present invention, as schematically depicted in FIG. For example, glycerol, a small molecule linker, has three hydroxyl groups to which oligonucleotides may be covalently linked. Therefore, some immunomers comprise two or more oligonucleotides with non-nucleotide linkers attached to their 3 ′ ends according to the present invention. Some such immunomers include at least two immunostimulatory oligonucleotides each having an accessible 5 'end.

  The immunomer of the present invention may be synthesized for convenience using an automatic synthesizer or a phosphoramidite method as schematically illustrated in FIGS. 5 and 6 and further as described in the examples. . In some embodiments, the immunomers are synthesized by a linear synthesis method (see FIG. 5). As used herein, “linear synthesis” refers to a synthesis that starts from one end of an immunomer and proceeds linearly to the other end. Linear synthesis is permitted to incorporate any monomer units that are not identical or identical (in terms of length, base composition, and / or chemical modification involved) into the immunomer.

  An alternative to synthesis is “parallel synthesis” where synthesis proceeds outward from the central linker moiety (see FIG. 6). As described in U.S. Patent No. 5,912,332, a solid support attached to a linker is used for parallel synthesis. A common solid support (such as a phosphate bound to a gap glass support) can be used.

  Immunomer parallel synthesis has several advantages over linear synthesis: (1) Parallel synthesis allows the incorporation of identical monomer units; (2) Both (or all), unlike linear synthesis Since the monomer units are synthesized simultaneously, the number of synthesis steps and the time required for synthesis are the same as that of the monomer units; and (3) the reduction of the synthesis steps improves the purity and yield of the final immunomer product.

  As the phosphoramidite supplier recommends when the modified nucleoside is incorporated, the immunomer may be conveniently deprotected with concentrated ammonia solution at the end of the synthesis by either linear or parallel synthesis protocol. The immunomeric product is preferably purified by reverse phase HPLC, detritylated, desalted and dialyzed.

  Table 4 shows representative immunomers according to the present invention. Other immunomers are described in the examples.

  In a second aspect, the present invention provides an immunomer complex comprising an immunomer as described above and an antigen bound to the immunomer at a position other than the accessible 5 ′ end. In some embodiments, the non-nucleotide linker comprises an antigen attached to an oligonucleotide. In some other embodiments, the antigen is bound 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 pathogens, antigens associated with cancer, antigens associated with autoimmune diseases, antigens associated with other diseases such as, but not limited to, animal and child diseases . For the purposes of the present invention, “relevant” refers to the presence of a pathogen, cancer, autoimmune disease, food allergy, respiratory allergy, asthma or other illness, or pathogen In the absence of cancer, autoimmune disease, food allergies, respiratory allergies, asthma or other illnesses, it means that the antigen is either absent or present in reduced amounts.

  The immunomer is either covalently bound to the antigen or otherwise operably bound. As used herein, “operably linked” refers to all bonds that maintain the activity of both the immunomer and the antigen. Non-limiting examples of such operable linkages include those that are part of the same liposome or such delivery vehicle or delivery agent. In embodiments where the immunomer is covalently bound to the antigen, such covalent linkage is preferably at all positions of the immunomer except at the accessible 5 ′ end of the immunostimulatory oligonucleotide. For example, the antigen may be bound to an internucleoside bond and may be bound to a non-nucleotide linker. Alternatively, the antigen may itself be a non-nucleotide linker.

  In a third aspect, the present invention provides a pharmaceutical formulation comprising an immunomer or immunomer complex according to the present invention and a physiologically acceptable carrier. As used herein, “physiologically acceptable” refers to a substance that does not inhibit the effect of an immunomer 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.

  “Carrier” as used herein refers to all excipients, diluents, fillers, salts, buffers, stabilizers, solubilizers, lipids, or other well known in the art of application in pharmaceutical formulations. Contains substances. It will be understood that the characteristics of the carrier, such as excipients or diluents, will depend on the method of administration for a particular application. The preparation of pharmaceutically acceptable formulations containing these substances is described in Remington's Pharmaceutical. Sciences, 18th Edition, ed. A. Gennaro, Mack Publishing Co., Easton, PA, 1990.

  In a fourth aspect, the present invention provides a method for eliciting an immune response in a vertebrate, such a method comprising the step of adding the immunomer or immunomer complex according to the present invention to a vertebrate. Including administration. In some embodiments, the vertebrate is a mammal. For the purposes of the present invention, “mammal” is specifically meant to include humans. In a preferred embodiment, the immunomer or immunomer complex is administered to a vertebrate in need of immunostimulation.

  In the method according to this aspect of the invention, the administration of the immunomer is, without limitation, parenteral, oral, sublingual, transdermal, topical, intranasal, aerosol, intraocular, intratracheal, rectal, intravaginal, Or by a gene gun or skin patch, or in the form of eye drops or mouthwash. Administration of the immunomer therapeutic composition can be performed using known methods at dosages and for periods of time effective to reduce disease symptoms or surrogate markers. For systemic administration, the therapeutic composition is preferably administered at a sufficient concentration to achieve a blood level of immunomer of from about 0.0001 micromolar to about 10 micromolar. For topical administration, it may be effective at lower concentrations than this, and may be tolerated at higher concentrations. Preferably, the total dose range of the immunomer ranges from about 0.001 mg per patient per day to 200 mg per day body weight. For the treatment of a single illness in a person, it is desirable to administer one or more of the therapeutic compositions of the present invention simultaneously or sequentially in a therapeutically effective concentration.

  In certain preferred embodiments, the immunomers of the invention comprise vaccines, antibodies, cytotoxic agents, allergens, antibiotics, antisense oligonucleotides, peptides, proteins, gene therapy vectors, DNA vaccines, and / or the specificity of immune responses or Administered in combination with an adjuvant to increase size. In these embodiments, the immunomers of the present invention can variously act as adjuvants and / or produce direct immunostimulatory effects.

  The immunomer and / or vaccine may optionally be conjugated to an immunogenic protein such as keyhole limpet hemocyanin (KLH), cholera toxin B subunit, or other immunogenic carrier protein. Any of a variety of adjuvants can be used including, but not limited to, Freund's complete adjuvant, KLH, monophospholipid A (MPL), alum, and saponins including QS-21, imiquimod, R848 or combinations thereof.

  For the purposes of this aspect of the invention, “in combination” means during the treatment of the same disease in the same patient, and the immunomer and / or vaccine and / or adjuvant is temporally up to a few days apart. It includes administration in any order, including simultaneous administration as well as through intervals. Such combined therapy may also include other than immunomer administration alone and / or vaccine alone, and / or adjuvant alone. Administration of immunomers and / or vaccines and / or adjuvants may be by the same or different routes.

  The method according to this aspect of the invention is useful for model studies of the immune system. The method is also useful for prophylactic or therapeutic treatment of humans or animals. For example, the method is useful for pediatric and vertebrate vaccine applications.

  In a fifth aspect, the present invention provides a method for therapeutically treating a patient suffering from a disease or disorder, said method comprising an immunomer or a patient of an immunomer complex according to the present invention. Administration. In various embodiments, the disease or disorder to be treated is cancer, autoimmune disease, airway inflammation, inflammatory disease, allergy, asthma, or a disease caused by a pathogen. Pathogens include bacteria, parasites, fungi, viruses, viroids, and prions. Administration is carried out as described in the fourth aspect of the invention.

  For the purposes of the present invention, “allergy” includes, without limitation, food allergies and airway allergies. Airway inflammation includes but is not limited to asthma. As used herein, “autoimmune disease” refers to a disease in which “self” proteins are attacked by the immune system. Such terms include autoimmune asthma.

  In any of the methods according to this aspect of the invention, the immunomer or immunomer complex can be administered in combination with any other drug or condition that reduces the immunostimulatory effect of the immunomer effective to treat the disease. For example, in the treatment of cancer, it means that an immunomer or immunomer complex can be administered in combination with a chemotherapeutic compound.

The following examples are intended to further describe preferred embodiments of the present invention and are not intended to limit the scope of the invention.
Example

Example 1 Synthesis of Oligonucleotides Containing Immunomodifying Portions Oligonucleotides were synthesized on an 1 μmol scale using an automated DNA synthesizer according to the linear or parallel synthesis method summarized in FIG. (Expedite 8909; PerSeptive Biosystems, Framingham, MA).

  Deoxyribonucleoside phosphoramidites were obtained from Applied Biosystem (Foster City, CA). 1 ', 2'-dideoxyribose phosphoramidite, propyl-I-phosphoramidite, 2-deoxyuridine phosphoramidite, 1,3-bis- [5- (4,4'-dimethoxytrityl) pentylamidyl] 2-Propanol phosphoramidite and methyl phosphoramidite were obtained from Glen Research (Sterling, VA). β-L-2'-deoxyribonucleoside phosphoramidite, α-2'-deoxyribonucleoside phosphoramidite, mono-DMT-glycerol phosphoramidite and di-DMT-glycerol phosphoramidite were obtained from ChemGenes (Ashland, MA) It was. (4-Aminobutyl) -1,3-propanediol phosphoramidite was obtained from Clontech (Palo Alto, CA). Arabinocytidine phosphoramidites, arabinoguanosine, arabinothymidine and arabinouridine are reliable pharmaceuticals (St. Louis, MO). Arabinoguanosine phosphoramidites, arabinothymidine phosphoramidites and arabinouridine phosphors Amidites were synthesized by hybridone (Cambridge, MA) (Noronha et al. (2000) Biochem., 39: 7050-7062).

All nucleoside phosphoramidites are characterized by 31 P and 1 H NMR spectra. The modified nucleoside was incorporated at a specific position using a normal coupling cycle. After synthesis, the oligonucleotide was deprotected with concentrated ammonium hydroxide and purified by reverse phase HPLC followed by dialysis. Oligonucleotides purified as sodium salt form were lyophilized before use. Purity was tested by CGE and MALDI-TOF MS.

Example 2: Analysis of spleen cell proliferation In vitro analysis of spleen cell proliferation was performed by standard methods previously described. (See, for example, Zhao et al., Biochem Pharma 51: 173-182 (1996)). The results are shown in FIG. 8A. These results show that at high concentrations, an immunomer 6 with two accessible 5 ′ ends is less than an immunomer 5 without an accessible 5 ′ end or an oligonucleotide 4 with one accessible 5 ′ end. , Indicating that it has resulted in greater splenocyte proliferation. Immunomer 6 also causes greater splenocyte proliferation than the positive control LPS.

Example 3 In Vivo Splenomegaly Assay To test the applicability of in vitro results to an in vivo model, selected oligonucleotides were administered to mice and the extent of splenomegaly was measured using the level of immunostimulatory activity as an indicator. . A dose of 5 mg / kg was administered intraperitoneally to BALB / c mice (female, 4-6 weeks old, Harlan Sprague Dawley Inc, Baltic, CT). The mice were sacrificed 72 hours after oligonucleotide administration and the spleen was removed and weighed. The result is shown in FIG. 8B. These results indicate that immunomer 6 with two accessible 5 ′ ends has a much greater immunostimulatory effect than oligonucleotide 4 or immunomer 5.

Example 4: Cytokine analysis IL-12 and IL-6 secretion in vertebrate cells, preferably spleen cells of BALB / c mice or human PBMC, was measured by sandwich ELISA. Necessary reagents including cytokine antibodies and cytokine standards were purchased from Farmingen (San Diego, CA). ELISA plates (Costar) are incubated overnight at 4 ° C with the appropriate antibody at a concentration of 5 μg / mL in PBSN buffer (PBS / 0.05% sodium azide, pH 9.6) and then at 37 ° C with PBS / 1% BSA. Blocked for 30 minutes. Cell culture supernatant and cytokine standards were diluted appropriately with PBS / 10% FBS, added to the plate in triplicate, and incubated at 25 ° C. for 2 hours.

  Plates were overlaid with the appropriate biotin-labeled antibody at a concentration of 1 μg / mL and incubated at 25 ° C. for 1.5 hours. The plate is then washed thoroughly with PBS-T buffer (PBS / 0.05% Tween 20), streptavidin-conjugated peroxidase (Sigma, St. Louis, MO) is added, and further incubated at 25 ° C. for 1.5 hours. did. The plate was developed using Sure Blue ™ (Kirkegaard and Perry) chromogenic reagent and the reaction was terminated by adding stop solution (Kirkegaard and Perry). The color change was measured with a Ceres 900 HDI spectrophotometer (Bio-Tek Instruments). The results are shown in Table 5A below.

Human peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood of healthy volunteers by Ficoll-Paque density gradient centrifugation (Histopaque-1077, Sigma, St. Louis, MO). Briefly, heparinized blood was layered on Histopaque-1077 (equal volume) in a conical centrifuge tube and centrifuged at 400 × g for 30 minutes at room temperature. The soft layer containing mononuclear cells was carefully removed and washed twice with isotonic phosphate buffer (PBS) by centrifugation at 250 xg for 10 minutes. The resulting cell pellet is then resuspended in RPMI 1640 medium (Mediatech, Herndon, VA) containing L-glutamine and supplemented with 10% heat-inactivated FCS and penicillin-streptomycin (100 U / ml). did. The cells were cultured in 24-well plates at 1 × 10 6 cells / ml / well for different times in the presence or absence of oligonucleotides. At the end of the culture, the supernatant was collected and IL-6 (BD Pharmingen, San Diego, CA), IL-10 (BD Pharmingen), IL-12 (BioSource International, Camarillo, CA), IFN-α (BioSource International ) And -γ (BD Pharmingen) and various cytokines including TNF-α (BD Pharmingen) were stored frozen at -70 ° C until analysis. The results are shown in Table 5 below.

  In all cases, the concentration of IL-12 and IL-6 in the cell culture supernatant was calculated from a standard curve written under the same experimental conditions as IL-12 and IL-6, respectively. The concentrations of IL-10, IFN-γ and TNF-α in the cell culture supernatant were calculated from calibration curves written under the same experimental conditions as IL-10, IFN-γ and TNF-α, respectively.

Normal font represents phosphorothioate linkage; italic font represents phosphodiester linkage.

  Furthermore, the results shown in FIGS. 7A-C show that oligonucleotide 2 with two accessible 5 ′ ends is lower than oligonucleotide 1 or 3 with or without one accessible 5 ′ end, respectively. Concentrations increase IL-12 and IL-6, but not IL-10.

Example 5: Effect of chain length on immunostimulatory activity of immunomer To examine the effect of oligonucleotide chain length, an immunomer containing 18, 14, 11 and 8 nucleotides in each chain was synthesized and BALB / c Immunostimulatory activity was examined by measuring the ability to induce secretion of cytokines IL-12 and IL-6 in mouse spleen cell cultures (Table 6-8). In this example and in all subsequent examples, cytokine analysis was performed in spleen cell cultures of BALB / c mice as described in Example 4.

  These results suggest that the immunostimulatory activity of the immunomer increases as the length of the oligonucleotide chain decreases from 18-mer to 7-mer. Immunomers with 6-mer or 5-mer oligonucleotide chain length showed comparable immunostimulatory activity compared to 18-mer oligonucleotides with one 5 'end. However, immunomers with 6-mer or 5-mer oligonucleotide chain lengths have increased immunostimulatory activity when the linker is from about 2 angstroms to 200 angstroms in length.

Example 6: Immunostimulatory Activity of Immunomers Containing Non-Natural Pyrimidines or Non-Natural Purine Nucleosides As shown in Table 9-11, immunostimulatory activity can be achieved by non-natural pyrimidine nucleosides or non-natural purine nucleosides in the immunostimulatory dinucleotide motif. Have been maintained for various lengths of immunomers.

Example 7: Effect of linker on immunostimulatory activity To examine the effect of linker length attached to two oligonucleotides, immunomers containing the same oligonucleotide but containing different linkers were synthesized and tested for immunostimulatory activity. . The results shown in Table 12 suggest that the linker length plays a role in the immunostimulatory activity of the immunomer. The best immunostimulatory effect was obtained with C3- to C6-alkyl linkers or abasic linkers with dispersed phosphate charges.

Example 8: Effect of oligonucleotide backbone on immunostimulatory activity In general, immunostimulatory oligonucleotides containing a natural phosphodiester backbone are less immunostimulatory than oligonucleotides of the same length having a phosphorothioate backbone. This is due in part to the rapid degradation of phosphodiester oligonucleotides under experimental conditions. Oligonucleotide degradation is first by 3'-exonuclease, which cleaves the oligonucleotide from the 3 'end. The immunomer of this example does not contain a free 3 'end. Thus, an immunomer with a phosphodiester backbone will have a longer half-life than its corresponding monomeric oligonucleotide under experimental conditions and thus will exhibit improved immunostimulatory activity. The results shown in Table 13 show this effect, with immunomers 84 and 85 showing the immunostimulatory activity determined by cytokine induction in BALB / c mouse spleen cell cultures.

Example 9 Synthesis of Immunomer 73-92 Oligonucleotides were synthesized on an 1 μmole scale using an automated DNA synthesizer (Expedite 8909 PerSeptive Biosystems). Deoxynucleoside phosphoramidites were obtained from Applied Biosystems (Foster City, CA). 7-deaza-2'-deoxyguanosine phosphoramidite was obtained from Glen Research (Sterling Virginia). 1,3-bis-DMT-glycerol-CPG was obtained from Chemgene (Ashland, MA). The modified nucleoside was incorporated at a specific position using a normal coupling cycle. After synthesis, the oligonucleotides were deprotected with concentrated ammonium hydroxide and purified by reverse phase HPLC followed by dialysis. Oligonucleotides purified as sodium salt form were lyophilized before use. Oligonucleotide purity was checked by CGE and MALDI-TOF MS (BrukerProflex 111 MALDI-TOF Mass spectrometer).

Example 10 : Immunomer stability Oligonucleotides were incubated in PBS containing 10% bovine serum at 37 ° C for 4, 24 or 48 hours. Intact oligonucleotides were measured by capillary gel electrophoresis. The results are shown in Table 14.

Example 11 : Effect of accessible 5 'end on immunostimulatory activity
Spleen cells of BALB / c mice (4-8 weeks old) were cultured in RPMI complete medium. Mouse macrophage-like cells, J774 (American Type Culture Collection, Rockville, MD) are cultured in Dulbecco's modified Eagle's medium supplemented with 10% (v / v) FCS and antibiotics (penicillin G / streptomycin 100 IU / mL) did. All other culture reagents were purchased from Mediatech (Gaithersburg, MD). .

IL-12 and IL-6 ELISA. The BALB / c mouse spleen cells or J774 cells, in 24-well dishes, respectively, were seeded at a density of 5x10 6 or 1X 10 6 cells / mL. Dissolve CpG DNA in TE buffer (10 mM Tris-HCI, pH 7.5, 1 mM EDTA) to a final concentration of 0.03, 0.1, 0.3, 1.0, 3.0, or 10.0 μg / mL. In addition to spleen cell cultures and to J774 cell cultures at 1.0, 3.0 or 10.0 μg / mL. The cells were then cultured at 37 ° C. for 24 hours and the supernatant was collected for ELISA analysis. Experiments were performed in triplicate for each concentration two or three times.

IL-12 and IL-6 secretion were measured by sandwich ELISA. Necessary reagents including cytokine antibodies and cytokine standards were purchased from Farmingen (San Diego, CA). ELISA plates (Costar) are incubated overnight at 4 ° C with the appropriate antibody at a concentration of 5 μg / mL in PBSN buffer (PBS / 0.05% sodium azide, pH 9.6) and then at 37 ° C with PBS / 1% BSA. Blocked for 30 minutes. Cell culture supernatant and cytokine standards were diluted appropriately with PBS / 1% BSA, added to the plate in triplicate, and incubated at 25 ° C. for 2 hours. The plate was washed and incubated with the appropriate biotin-labeled antibody at a concentration of 1 μg / mL and incubated at 25 ° C. for 1.5 hours. The plate was thoroughly washed with PBS / 0.05% Tween 20, streptavidin-bound peroxidase (Sigma) was added, and further incubated at 25 ° C. for 1.5 hours. The plate was developed using Sure Blue ™ (Kirkegaard and Perry) chromogenic reagent and the reaction was terminated by adding stop solution (Kirkegaard and Perry). The color change was measured with a Ceres 900 HDI spectrophotometer (Bio-Tek Instruments). The concentrations of IL-12 and IL-6 in the cell culture were calculated by a calibration curve drawn under the same experimental conditions as IL-12 and IL-6, respectively.
The results are shown in Table 15.

Taken together, this result shows that the accessible 5 'end of CpG DNA is required for optimal immunostimulatory activity, and small groups such as phosphorothioate, mononucleotide, or dinucleotide can be used for receptors involved in the immunostimulatory pathway and This suggests that it does not efficiently inhibit the accessibility of the CpG DNA 5 ′ end to the factor. However, binding of molecules equal to or larger than fluorescein to the 5 ′ end of CpG DNA can suppress immunostimulatory activity. These results have a direct impact on the study of the immunostimulatory activity of CpG DNA-antibody / vaccine / monoclonal antibody (mAb) complexes. Binding of large molecules, such as vaccines or mAbs, to the 5 ′ end of CpG DNA can make the immunostimulatory activity of CpG DNA suboptimal. The binding of functional ligands to the 3 ′ end of CpG DNA contributes not only to increased nuclease stability but also to increased immunostimulation in vivo.

Example 12 : Effect of linker on cytokine secretion The following oligonucleotides were synthesized for this study. Each modified oligonucleotide can be incorporated into an immunomer.

  To evaluate the optimal linker size for enhanced immunostimulatory activity, IL-12 and IL-6 secretion by modified CpG DNA in BALB / c mouse spleen cell cultures was measured. All CpG DNA caused concentration-dependent secretion of IL-12 and IL-6. Table 15 was selected as 116, 119, 126, 130 and 134 having a linker at the 5th nucleotide position to the CpG dinucleotide in the 5'-flanking sequence compared to the original CpG DNA. Data obtained at a concentration of 1 μg / mL of CpG DNA is shown. CpG DNA containing C2- (1), C3- (2), and C4-linker (3) caused secretion of IL-12 production similar to that of the original CpG DNA 4. CpG DNA containing C6 and C9-linkers (4 and 5) at the 5th nucleotide position from the CpG dinucleotide in the 5'-flanking sequence has a lower level of IL-12 secretion than the original CpG DNA (FIG. 15) suggests that longer linker substitutions than the C4-linker result in lower levels of induction of IL-12. All five CpG DNAs with linkers caused IL-6 secretion 2 or 3 times higher than the original CpG DNA. The presence of the linker in these CpG DNAs showed a significant effect on the induction of IL-6 compared to CpG DNA without the linker. However, we could not observe a length-dependent linker effect on IL-6 secretion.

  To examine the effect on the immunostimulatory activity of CpG DNA containing an ethylene glycol-linker, we determined the position of the 5th nucleotide in the 5'-flanking sequence to the CpG dinucleotide and 3'-flanking, respectively. CpG DNA137 and 138 were synthesized in which triethylene glycol-linker (6) was incorporated at the position of the fourth nucleotide in the sequence. Similarly, CpG DNAs 139 and 140 each contain a hexaethylene glycol-linker (7) in the 5'- or 3'-flanking sequence to the CpG dinucleotide. All four modified CpG DNA (137-140) caused concentration-dependent cytokine production over the range of concentrations investigated (0.03-10.0 μg / mL) (data not shown).

  The concentrations of cytokines induced at a concentration of 0.3 μg / mL of CpG DNAs137-140 are shown in Table 18. CpG DNA137 and 139 with an ethylene glycol-linker in the 5 'flanking sequence are more IL-12 (2106 ± 143 and 2066 ± 153 pg / mL) and IL-6 (2362 ± 166) than the original CpG DNA4. And 2507 ± 66 pg / mL) caused a higher concentration of secretion (Table 18). At the same concentration, 137 and 139 caused secretion of IL-10 at a slightly lower concentration than the original CpG DNA (Table 18). CpG DNA138 with a shorter ethylene glycol-linker (6) in the 3 'flanking sequence caused IL-12 secretion similar to the original CpG DNA, but the concentrations of IL-6 and IL-10 were significant (Table 18). CpG DNA 140 with a longer ethylene glycol linker (7) was induced at significantly lower concentrations for all three cytokines tested compared to the original CpG DNA (Table 18).

  Triethylene glycol-linker (6) has a chain length similar to that of C9-linker (5), but CpG DNA containing triethylene glycol-linker was determined by induction of cytokine secretion in spleen cell cultures. -Better immunostimulatory activity than CpG DNA containing linker. These results indicate that the lower immunostimulatory activity observed with CpG DNA containing longer alkyl-linkers (4 and 5) may be related to its hydrophobicity rather than its increased length. Suggests. This result led us to investigate the substitution of branched alkyl-linkers containing hydrophobic functional groups in immunostimulatory activity.

  To investigate the effect of CpG DNA containing branched alkyl linkers on immunostimulatory activity, two branched alkyl linkers containing hydroxyl (8) or amine (9) functional groups can be incorporated into the original CpG DNA4 and the result The effect of the modified CpG DNA (150-154-Table 19) on the immunostimulatory activity was examined. Data obtained with CpG DNA150-154 containing amino linker 9 at different nucleotide positions in spleen cell culture (proliferation) and in vivo (splenomegaly) of BALB / c mice are shown in Table 19.

  The original CpG DNA 4 showed a growth rate of 3.7 ± 0.8 at a concentration of 0.1 μg / mL. At the same concentration, modified CpG DNA 151-154 containing amino linker 9 at different positions caused higher spleen cell proliferation than the original CpG DNA (Table 19). As observed with other linkers, when the substitution is placed adjacent to the CpG dinucleotide (150), lower growth is observed compared to the original CpG DNA, and the linker adjacent to the CpG dinucleotide. It is further confirmed that the arrangement of substitutions has a detrimental effect on immunostimulatory activity. In general, substitution of 2'-deoxyribonucleosides in the 5'-flanking sequence with an amino linker (151 and 152) results in a higher spleen than is found with substitutions in the 3'-flanking sequence (153 and 154) Causes cell proliferation. Similar results have been observed in splenomegaly analysis (Table 19), confirming the results observed in spleen cell cultures. Modified CpG DNA (8) containing a glycerol linker showed the same or slightly higher immunostimulatory activity as observed with modified CpG DNA containing an amino linker (9) (data not shown) .

To compare the immunostimulatory effects of CpG DNA containing linkers 8 and 9, we selected CpG DNA145 and 152 with substitutions in the 5 'flanking sequence and selected IL-12 and cytokine IL-12 in spleen cell cultures of BALB / c mice. The inducibility of IL-6 was analyzed. Both CpG DNA145 and 152 caused concentration-dependent cytokine secretion. FIG. 16 shows the IL-12 and IL-6 concentrations induced by 145 and 152 at a concentration of 0.3 μg / mL in mouse spleen cell cultures compared to the original CpG DNA4. Both CpG DNAs induced higher concentrations of IL-12 and IL-6 than CpG DNA4. CpG DNA containing a glycerol linker (8) induced a slightly higher concentration of cytokine (particularly IL-12) than CpG DNA containing an amino linker (9) (FIG. 16). These results further confirmed that a linker containing a hydrophobic group is more preferable for the immunostimulatory activity of CpG DNA.

  We investigated two different aspects of substitution of multiple linkers in CpG DNA. In a series of experiments we fixed the length of the nucleotide sequence to the 13-mer and incorporated 1 to 5 C3-linker (2) substituents at the 5 'end (120-124). With these modified CpG DNA we were able to study the effect on increasing linker length without causing solubility problems. In the second series of experiments, we investigated whether there was an additive effect on immunostimulatory activity by looking at the same linker substituent (3, 3) at the position adjacent to the CpG dinucleotide in the 5 ′ flanking sequence. Two of 4 or 5) were incorporated.

The modified CpG DNA was examined for its ability to induce cytokine production in BALB / c mouse spleen cell cultures compared to the original CpG DNA 4. All CpG DNA induced cytokine production in a concentration-dependent manner . Analysis of this, the original CpG DNA 4 at a concentration of 1 [mu] g / mL, 967 of ± 28 pg / mL IL-12 , 1593 of ± 94.pg / mL IL-6, and 14 ± 6 pg / mL Induced lL-10 secretion . Data is as it reduces the number of linker substituent, induction of IL-12 suggests that the decrease. However, induction of lower concentrations of IL-12 secretion by CpG DNA123 and 124 can result in shorter CpG DNA length. Our study with CpG DNA shorter than unmodified 15-nucleotides showed slight immunostimulatory activity (not shown in the data). Neither the length nor the number of linker substituents has any effect on IL-6 secretion. Although IL-10 secretion was increased by linker substituents, overall IL-10 secretion by these CpG DNAs was minimal.

  CpG DNA containing two linker substituents (linker 3-127; linker-4-131; linker-5-135) at the 4th and 5th positions in the 5'-flanking sequence, and the corresponding 5 ' Short cuts, 128, 132 and 136, respectively, were examined for their ability to induce cytokine secretion in cultured BALB / c mouse spleen cells. The concentrations of IL-12 and IL-6 at a concentration of 1.0 μg / mL are shown in FIG. The results presented in FIG. 17 suggest that the immunostimulatory activity depends on the type of linker incorporated. Substitution of the 4th and 5th nucleotides with C4-linker 3 (CpG DNA 127) has a minor effect on cytokine secretion compared to the original CpG DNA 4, and the nucleobase and sugar at these positions. It suggests that the ring is not required for receptor recognition and / or binding.

  Deletion of nucleotides beyond the linker substituent (CpG DNA 128) causes higher IL-12 and IL-6 secretion than CpG DNA4 and 127. As expected, the two C6-linker (4) substitutions were induced by lower secretion of IL-12 and by the original CpG DNA4 than by the original CpG DNA4. The result was similar secretion of IL-6. CpG DNA132 truncated at 5 'induced higher cytokine secretion than CpG DNA131. CpG DNA 135 and 136 with two C9-linkers (5) induced slight cytokine secretion, confirming the results obtained with mono-substituted CpG DNA containing the same linker as described above.

Example 13 : Effect of phosphodiester binding on cytokine induction In order to examine the effect of phosphodiester binding on cytokine induction induced by immunomers, the following molecules were synthesized.

  PS-CpG DNA 4 (Table 21) was found to elicit an immune response in mice using PO-CpG DNA 155 as a control (data not shown). PO-immunomers 156 and 157 each contain two identical, short-cut copies of the original CpG DNA 155 attached to its 3 ′ end via the glyceryl linker X (Table 21). 156 and 157 each contain the same oligonucleotide segment of 14 bases, but the 5 ′ end of 157 is modified by the addition of two C3-linkers Y (Table 21). All oligonucleotides from 4,155-157 contain a hexameric motif called 'GACGTT' which is known to activate the mouse immune system.

  The stability of PO-immunomers to nucleases is as follows: CpG DNA 4, 155-157 in cell culture medium containing 10% bovine serum (FBS) (not heat-inactivated) for 4, 24 and 48 hours at 37 ° C. Evaluated by incubating. Intact CpG DNA remaining in the reaction solution was measured by CGE. Figures 18A-D show nuclease cleavage profiles of CpG DNA4, 155-157 incubated for 24 hours in 10% FBS. The amount of full length CpG DNA remaining at each time is shown in FIG. 18E. As expected, the original PS-CpG DNA 4 was the most resistant to serum nucleases. Approximately 55% of the 4 18-mers remained intact after 48 hours of incubation.

  In contrast, after 4 hours under the same experimental conditions, only about 5% of the full-length PO-immunomer 155 remained, confirming that DNA containing phosphodiester bonds was rapidly degraded. As expected, both PO-immunomers 156 and 157 were more resistant to serum nucleases than 155. After 4 hours, 155 and 157 were about 62% and 73% intact, respectively, compared to about 5% intact (FIG. 18E). Even after 48 hours, approximately 23% and 37% of 156 and 157, respectively, remained intact. Just as 3'-3'-linked PO-immunomers have been shown to be more stable against serum nucleases, these studies suggest that chemical modification at the 5 'end can further increase stability against nucleases It is shown that.

  The immunostimulatory activity of CpG DNA has been studied by measuring the concentrations of secreted cytokines IL-12 and IL-6 in spleen cell cultures of BALB / c and C3H / HeJ mice. All CpG DNAs induced concentration-dependent cytokine secretion in spleen cell cultures of BALB / c mice (FIG. 19). At 3 μg / mL, PS-CpG DNA 4 induced 2656 ± 256 and 12234 ± 1180 pg / mL IL-12 and IL-6, respectively. The original PO-CpG DNA155 did not increase the cytokine concentration over background except at a concentration of 10 μg / mL. This result is consistent with the results of nuclease stability analysis. On the other hand, PO-immunomers 156 and 157 caused secretion of both IL-12 and IL-6 in spleen cell cultures of BALB / c mice.

  The results presented in FIG. 19 show a clear difference in the cytokine induction profiles of PS- and PO-CpG DNA. PO-immunomers 156 and 157 induced higher concentrations of IL-12 than PS-CpG DNA 4 in spleen cell cultures of BALB / c mice (FIG. 19A). On the other hand, they produced negligible amounts of IL-6 up to a concentration of 3 μg / mL (FIG. 19B). Even at high concentrations (10 μg / mL), PO-immunomer 156 induced IL-6 significantly less than PS-CpG DNA4. The presence of the C3 linker at the 5 ′ end of PO-immunomer 157 resulted in a slightly higher concentration of IL-6 secretion compared to 156.

  Importantly, however, the concentration of IL-6 produced by PO-immunomer 157 was much lower than that induced by PS CpG DNA 4. The inset of FIG. 19A shows the ratio of IL-12 to IL-6 secreted at a concentration of 3 μg / mL. In addition to increased secretion of IL-12, PO-immunomers 156 and 157 induced higher concentrations of IFN-γ in spleen cell cultures of BALB / c mice compared to PS-CpG DNA 4 (data Is not shown).

  Different cytokine profiles induced by PO- and PS-CpG DNA in spleen cell cultures of BALB / c mice show us the pattern of cytokine induction of CpG DNA in C3H / HeJ mouse spleen cell cultures (LPS insensitive strains) I decided to study. All three CpG DNAs examined in this analysis elicited concentration-dependent cytokine secretion (FIGS. 20A and B). Since PO-CpG DNA 155 did not elicit cytokine secretion in spleen cell cultures of BALB / c mice, it was not further investigated in C3H / HeJ spleen cell cultures. PO-immunomers 156 and 157 induced higher IL-12 production than PS-CpG DNA 4 (FIG. 20A). However, none induced IL-6 production up to a concentration of 3 μg / mL. At the highest concentration examined (10 μg / mL), all induced significantly lower IL-6 than PS-CpG DNA 4 (FIG. 20B). The ratio of IL-12 to secreted IL-6 has been calculated to distinguish the cytokine secretion profiles of PS and PO CpG DNA (Fig. 20A inset). Furthermore, C3H / HeJ spleen cell culture results suggest that the effects observed with CpG DNA are not due to LPS contamination.

  PS-CpG DNA has been shown to cause potent anticancer activity in vivo. Since PO-CpG DNA exhibits better nuclease stability in in vitro analysis and elicits higher concentrations of IL-12 and IFN-γ secretion, we have found that these favorable properties of PO-immunomers are in vivo I was interested in investigating whether to improve anticancer activity. We injected PO-immunomer 157 at 0.5 mg / day every other day in nude mice that had received MCF-7 breast cancer cells expressing wild-type p53 or DU-145 prostate cancer cells expressing mutant p53. It was administered subcutaneously at a dose of kg. PO-immunomer 157 inhibited the growth of MCF-7 tumors by 57% on day 15 compared to controls administered saline (FIG. 21A). On day 34, DU-145 tumor growth was also inhibited by 52% (FIG. 21B). These anti-cancer studies suggest that the proposed designed PO-immunomer exhibits potent anti-cancer activity in vivo.

Example 14 : Short immunomer To examine the effect of a short immunomer on cytokine induction, the following immunomer was used. These results indicate that immunomers as short as 5 nucleotides per segment are effective in inducing cytokine production.

While the invention has been described in some detail for purposes of clarity and understanding, it will be understood that various changes in form and detail may be made without departing from the actual scope of the invention and the appended claims. Those of ordinary skill in the art will understand upon reading this disclosure.

FIG. 2 schematically shows a typical immunomer of the present invention. Several representative immunomers of the present invention are represented. Figure 2 represents a series of representative small molecule linkers suitable for linear synthesis of immunomers of the invention. Figure 2 represents a series of representative small molecule linkers suitable for parallel synthesis of immunomers of the invention. It is a synthetic | combination schematic diagram of the linear synthesis | combination of the immunomer of this invention. DMTr = 4, 4'-dimethoxytrityl; CE = cyanoethyl. It is a synthetic | combination schematic diagram of the parallel synthesis | combination of the immunomer of this invention. DMTr = 4, 4'-dimethoxytrityl; CE = cyanoethyl. It is a graph figure of the induction | guidance | derivation of IL-12 by immunomer 1-3 in the culture | cultivation of the spleen cell of a BALB / c mouse | mouth. These data show that immunomer 2 with an accessible 5 'end induces IL-12 more strongly than monomeric oligo 1, and immunomer 3 without an accessible 5' end has oligo 1 and This suggests that only equal or weaker immune stimuli can occur. It is a graph figure of the induction | guidance | derivation of IL-6 by immunomer 1-3 in the culture | cultivation of the spleen cell of a BALB / c mouse | mouth (from the top to the bottom). These data show that immunomer 2 with an accessible 5 'end induces IL-6 more strongly than monomeric oligo 1, and immunomer 3 without an accessible 5' end has oligo 1 and This suggests that only equal or weaker immune stimuli can occur. It is a graph figure of the induction | guidance | derivation of IL-10 by immunomer 1-3 in the culture | cultivation of the spleen cell of a BALB / c mouse | mouth (from the top to the bottom). FIG. 4 is a graphical representation of BALB / c mouse spleen cell proliferation induction in culture systems by different concentrations of immunomers 5 and 6, each having an unreachable 5 ′ end and an accessible 5 ′ end. FIG. 4 is a graph of spleen expansion in BALB / c mice by immunomer 4-6 having an immunogenic chemical modification in the 5 ′ flanking sequence of the CpG motif. Again, immunomer 6 with an accessible 5 ′ end can increase spleen expansion more than immunomer 5 and monomeric oligo 4 without an accessible 5 ′ end. FIG. 4 is a graph of IL-12 induction by different concentrations of oligo 4 and immunomers 7 and 8 in culture of spleen cells of BALB / c mice. FIG. 6 is a graph of IL-6 induction by different concentrations of oligo 4 and immunomers 7 and 8 in culture of spleen cells of BALB / c mice. FIG. 2 is a graph of IL-10 induction by different concentrations of oligo 4 and immunomers 7 and 8 in culture of spleen cells of BALB / c mice. FIG. 2 is a graph of cell proliferation induction by immunomers 14, 15 and 16 in spleen cell culture of BALB / c mice. In the spleen cell cultures of BALB / c mice is a graphic representation of the induction of IL-12 by immunomers 14, 15 and 16 of different concentrations. In the spleen cell cultures of BALB / c mice is a graphic representation of the induction of IL-6 by immunomers 14, 15 and 16 of different concentrations. FIG. 4 is a graph showing cell proliferation induction by oligos 4 and 17 and immunomers 19 and 20 in culture of BALB / c mouse spleen cells. In cultures of BALB / c mouse spleen cells is a graphic representation of the induction of IL-12 by oligo 4 and 17 different concentrations of the immunomer 19 and 20. In cultures of BALB / c mouse spleen cells is a graphic representation of the induction of IL-6 by oligo 4 and 17 different concentrations of the immunomer 19 and 20. FIG. 4 is a graph of BALB / c mouse spleen expansion using oligonucleotide 4 and immunomers 14, 23 and 24. FIG. 3 is a schematic diagram of the 3 ′ terminal nucleoside of an oligonucleotide, showing that a non-nucleotide linkage can be attached to the nucleoside at the nucleobase, 3 ′ or 2 ′ position. The chemical substituents used in Example 13 are shown. FIG. 5 shows the cytokine profile obtained with the modified oligonucleotide in Example 13. FIG. Shows the induction of relative cytokines by a glycerol linker compared to an amino linker. The relative cytokine induction by various linkers and linker combinations is shown. A-E shows the relative nuclease resistance of various PS and PO immunomers and oligonucleotides. Figure 6 shows the relative cytokine induction of PO immunomer compared to PS immunomer in cultures of BALB / c mouse spleen cells. Figure 3 shows the relative cytokine induction of PO immunomer compared to PS immunomer in culture of C3H / Hej mouse spleen cells. Figure 2 shows the antitumor activity of PO immunomeric compounds in vivo.

Claims (22)

  1. Even without least an immunomer (immunomer) comprising two oligonucleotides, wherein the oligonucleotide is the 3 'end, an internucleoside linkage, or in functionalized nucleobase or sugar, is bound to a non-nucleotide linker , At least one of the oligonucleotides is an immunostimulatory oligonucleotide having a 5 ′ end accessible by a receptor or factor involved in the immunostimulatory pathway and comprising an immunostimulatory dinucleotide, Wherein the immunostimulatory dinucleotide is selected from the group consisting of C * pG, CpG * and C * pG * , wherein C is cytidine or 2′-deoxycytidine and C * is 2′- Deoxythymidine, arabinocytidine, 2'-deoxy-2'-substituted arabinocytidine, 2'-O-substituted arabinocytidine, 2'-deoxy-5-hydroxycytidine 2'-deoxy -N4- alkyl - cytidine or 2'-deoxy-4-thiouridine, G is guanosine or 2'-deoxyguanosine, G * is 2'-deoxy-7-deazaguanosine, 2 ' -Deoxy-6-thioguanosine, arabinoguanosine, 2'-deoxy-2'-substituted-arabinoguanosine, 2'-O-substituted-arabinoguanosine or 2'-deoxyinosine, and p is a phosphodiester Wherein said immunomer is an internucleoside bond selected from the group consisting of phosphorothioate and phosphorodithioate.
  2. At least one of the oligonucleotides has a structure
    5'-Nn-N1-YZ-N1-Nn-3 '(III)
    Where:
    YZ is an immunostimulatory dinucleotide,
    N1 is in each case a naturally occurring or synthetic nucleoside or an immunostimulatory moiety;
    Nn is in each case a naturally occurring or synthetic nucleoside or an immunostimulatory moiety;
    Each immunostimulatory moiety is independently abasic nucleoside, arabino nucleoside, 2'-deoxyuridine, α-deoxyribonucleoside, β-L-deoxyribonucleoside, and 3 'adjacent nucleoside Wherein the modified internucleoside linkage is selected from the group consisting of a C2-C18 alkyl linker, a poly (ethylene glycol) linkage, 2-aminobutyl-1,3-propane. Diol linker, glyceryl linker, 2'-5 'internucleoside bond, methylphosphonate internucleoside bond; methylphosphonothioate, phosphotriester, phosphothiotriester, phosphorothioate, phosphorodithioate, triester prodrug, sulfone, sulfo The nucleoside is selected from the group consisting of amide, sulfamate, formacetal, N-methylhydroxylamine, carbonate, carbamate, morpholino, boranophosphonate, phosphoramidate, and stereospecific bonds, without limitation 2'-substituted pentasaccharides, including 2'-O-methyl ribose, 2'-O-methoxyethyl ribose, 2'-O-propargyl ribose, and 2'-deoxy-2'-fluororibose;3'- 3′-substituted pentasaccharides including O-methylribose; 1 ′, 2′-dideoxyribose; arabinose; substituted arabinose sugars; nucleosides with hexasaccharide and alpha-anomeric sugar modifications, peptide nucleic acids (PNA), phosphates Group-bound peptide nucleic acids (PHONA), locked nucleic acids (LNA), morpholino nucleic acids, and alkylphosphorus 2 to 200 angstroms long -Oligonucleotides having backbone linker moieties with amino linkers, DNA isoforms, β-L-deoxyribonucleosides, α-deoxyribonucleosides, nucleosides with unnatural internucleoside binding positions, and nucleosides with modified heterocyclic bases Including:
    The immunomer according to claim 1, which has a structure represented by n being a number of 0-30.
  3. The immunomer according to claim 2 , wherein the phosphoramidate is a primary aminophosphoramidate, N3 phosphoramidate or N5 phosphoramidate.
  4. Nn in each case is a naturally occurring nucleoside or abasic nucleoside, arabino nucleoside, 2'-deoxyuridine, α-deoxyribonucleoside, 2'-O-substituted or 2'-substituted ribonucleoside, and modifications An immunostimulatory moiety selected from the group consisting of a nucleoside linked to a nucleoside adjacent to the 3 ′ side by a modified internucleoside bond, wherein the modified internucleoside bond comprises an amino linker, 2′-5 ′ Selected from the group consisting of a nucleoside bond and a methylphosphonate internucleoside bond;
    At least one N1 or Nn is an immunostimulatory moiety and 5′N1 comprises a nucleobase;
    A 3 ′ end, an internucleoside linkage, or a functionalized nucleobase or sugar is attached to another oligonucleotide via a non-nucleotide linker,
    The immunomer according to claim 2 or 3.
  5. Construction
    The immunomer according to any one of claims 1 to 4 , which has
  6. A non-nucleotidic linker, a linker from 2 angstroms to 200 angstroms in length, a metal, a soluble or insoluble biodegradable polymer bead, an organic moiety having a functional group that allows binding to the 3 'terminal nucleoside of the oligonucleotide, Molecules, cyclic or non-cyclic small molecules, aliphatic or aromatic hydrocarbons, any of which are optionally attached to the oligonucleotide or attached to the oligonucleotide in a straight chain, hydroxy, amino, thiol Selected from the group consisting of thioether, ether, amide, thioamide, ester, urea and thiourea, which may contain one or more functional groups, amino acid, carbohydrate, cyclodextrin, adamantane, cholesterol, hapten, antibiotic, o and p is the formula represented by an integer from 1 independently to 6 HO- (CH 2) o- CH (OH) - (CH 2) glycerol or glycerol homolog of the p-OH and 1,3-diamino -2 The immunomer according to any one of claims 1 to 5 , which is selected from the group consisting of -hydroxypropane derivatives.
  7. The immunomer according to any one of claims 1 to 6 , wherein the internucleoside bond comprises a phosphorothioate bond.
  8. C * is a arabino cytidine or 2'-deoxy-2'-substituted arabino cytidine, and, G * is arabinoguanosine or 2'-deoxy-2'-substituted - arabinoguanosine, 2'-deoxy - The immunomer according to any one of claims 1 to 4 , 6 to 7 , which is 7 -deazaguanosine, 2'-deoxy-6-thioguanosine, or 2'-deoxyinosine.
  9. A pharmaceutical preparation comprising the immunomer according to any one of claims 1 to 8 and a physiologically acceptable carrier.
  10. 10. The pharmaceutical of claim 9, further comprising an antigen , vaccine, antibody, cytotoxic agent, allergen, antibiotic, antisense oligonucleotide, chemotherapeutic agent, peptide, protein, gene therapy vector, DNA vaccine and / or adjuvant. Formulation.
  11. The pharmaceutical preparation according to claim 9 or 10 , for eliciting an immune response in a vertebrate.
  12. A pharmaceutical preparation for therapeutically treating a patient having a disease or disorder, comprising the immunomer according to any one of claims 1 to 8 .
  13. 13. The pharmaceutical formulation of claim 12 , wherein the disease or disorder to be treated is cancer, autoimmune disease, airway inflammation, inflammatory disease, skin disease, allergy, asthma, or a disease caused by a pathogen.
  14. 14. The pharmaceutical preparation according to claim 12 or 13 , wherein the disease or disorder to be treated is cancer, an autoimmune disease, airway inflammation, allergy, asthma, or a disease caused by a pathogen.
  15. 15. Any of claims 9-14, administered in combination with a vaccine , antibody, cytotoxic agent, allergen, antibiotic, antisense oligonucleotide, chemotherapeutic agent, peptide, protein, gene therapy vector, DNA vaccine and / or antigen. A pharmaceutical preparation according to claim 1.
  16. The pharmaceutical formulation according to any one of claims 9 to 15 , which is administered in combination with an adjuvant.
  17. Of Imunoma over according to any one of claims 1-8, for the manufacture of a pharmaceutical formulation for eliciting an immune response in a vertebrate.
  18. Claims Imunoma over according to any one of claim 1-8, for the manufacture of a pharmaceutical formulation for treating therapeutically a patient having a disease or disorder.
  19. 19. Use according to claim 18 , wherein the disease or disorder to be treated is cancer, autoimmune disease, airway inflammation, inflammatory disease, skin disease, allergy, asthma or a disease caused by a pathogen.
  20. 20. Use according to claim 18 or 19 , wherein the disease or disorder to be treated is cancer, autoimmune disease, airway inflammation, allergy, asthma, or a disease caused by a pathogen.
  21. The pharmaceutical formulation is to be administered in combination with a vaccine , antibody, cytotoxic agent, allergen, antibiotic, antisense oligonucleotide, chemotherapeutic agent, peptide, protein, gene therapy vector, DNA vaccine and / or antigen 21. Use according to any of claims 17-20.
  22. The use according to any of claims 17 to 21 , wherein the pharmaceutical preparation is to be administered in combination with an adjuvant.
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