CA2463798C - Modulation of immunostimulatory properties of oligonucleotide-based compounds by optimal presentation of 5' ends - Google Patents

Abstract

The invention relates to the therapeutic use of oligonucleotides as immunostimulatory agents in immunotherapy applications. More particularly, the invention provides immunomers for use in methods for generating an immune response or for treating a patient in need of immunostimulation. The immunomers of the invention 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

MODULATION OF IMMUNOSTIMULATORY PROPERTIES OF
OLIGONUCLEOTIDE-BASED COMPOUNDS BY OPTIMAL, PRESENTATION OF 5' ENDS
(Attorney Docket No. HYB-007US2) BACKGROUND OF THE INVENTION
Field of the Invention The invention relates to immunology and immunotherapy applications using oligonucleotides as immunostimulatory agents.
Summa 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 P'CR to antisense inhibition of gene expression and immunotherapy applications. This widespread use of oligonueleotides 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., 199 I ); and Uhlmann and Peymanõsupra; Agrawal and lyer, C.urr.
op. in Biotech 6:12 (1995); and Aniisense 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., .1.
Moiec. 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,

2 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 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. Sct (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 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 at, J. Clin. Invest. 98:1119-1129 (1996) discloses that such oligonucleotides activate human B cells. Moldoveanu etal., 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.

3 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; Zhao et at., 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 at., 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 enhance the immune response caused by immunostimulatory oligonucleotides.

4 BRIEF SUMMARY OF THE INVENTION
The invention provides methods for enhancing the immune response caused by oligonucleotide compounds. The methods according to the invention enable increasing the immunostimulatory effect of immunostimulatory oligonucleotides for immunotherapy applications. The present inventors have surprisingly discovered that modification of an immunostimulatory oligonucleotide to optimally present its

5' end dramatically enhances its immunostimulatory capability. Such an oligonucleotide is referred to herein as an "immunomer."
In a first aspect, therefore, the invention provides immunomers comprising at least two oligonucleotides linked at their 3' ends, an internuceotide linkage, or a functionalized nucleobase or sugar via a non-nucleotidic linker, at least one of the oligonucleotides being an immunostimulatory oligonucleotide and having an accessible 5' end.
In one embodiment, the immunomer comprises an immunostimulatory dinucleotide of 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.
In another embodiment, the immunomer comprises an immunostimulatory dinucleotide selected from the group consisting of CpG, C*pG, CpG*, and C*pG*, wherein C is cytidine or 2'-deoxycytidine, C* is 2'-deoxythymidine.
arabinocytidine, 2'-deoxy-2'-substitutedarabinocytidine, 2'-0-substitutedarabinocytidine, 2'-deoxy-5-hydroxycytidine, 2'-deoxy-N4-alkyl-cytidine, 2'-deoxy-4-thiouridine or other non-natural 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'-0-substituted-arabinoguanosine, 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.

In yet another embodiment, the immunostimulatory oligonucleotide comprises an immunostimulatory domain of formula (///):
5'-Nn-N I -Y-Z-N I -Nn-3' (///) wherein:
5 Y is cytidine, 2'-deoxythymidine, 2' deoxycytidine, arabinocytidine, 2%
deoxythymidine, 2'-deoxy-2'-substitutedarabinocytidine, 2'-0-substitutedambinocytidine, 2'-deoxy-5-hydroxycytidine, 2'-deoxy-N4-alkyl-cytidine, 2'-deoxy-4-thiouridine or other non-natural pyrimidine nucleoside;
Z is guanosine or 2'-deoxyguanosine, G* is 2' deoxy-7-deazaguanosine, 2'-deoxy-6-thioguanosine, arabinoguanosine, 2'-deoxy-2'substituted-arabinoguanosine, 2'-0-substituted-arabinoguanosine, 2'- deoxyinosine, or other non-natural purine nucleoside NI, 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, cc-deoxyribonucleosides, P¨L-deoxyribonucleosides, and nucleosides linked by a phosphodiester or modified intemucleoside linkage to the adjacent nucleoside on the 3' side, the modified intemucleotide linkage being selected from, without limitation, a linker having a length of from about 2 angstroms to about 200 angstroms, C2-C18 alkyl linker, poly(ethylene glycol) linker, 2-aminobuty1-1,3-propanediol linker, glyceryl linker, 2'-5' intemucleoside linkage, and phosphorothioate, phosphorodithioate, or methylphosphonate intemucleoside linkage;
Nn, at each occurrence, is a naturally occurring nucleoside or an immunostimulatory moiety, preferably selected from the group consisting of abasic nucleosides, arabinonucleosides, 2'-deoxyuridine, a-deoxyribonucleosides, 2'-0-substituted ribonucleosides, and nucleosides linked by a modified intemucleoside

6 linkage to the adjacent nucleoside on the 3' side, the modified internucleotide linkage being selected from the group consisting of amino linker, 2'-5' internucleoside linkage, and methylphosphonate intemucleoside linkage;
provided that at least one NI or Nn is an immunostimulatory moiety;
wherein n is a number from 0-30;
wherein the 3'end , an internucleotide linkage, or a functionalized nucleobase or sugar is linked directly or via a non-nucleotidic linker to another oligonucleotide, which may or may not be immunostimulatory.
In a second aspect, the invention provides immunomer conjugates, comprising an immunomer, as described above, and an antigen conjugated to the immunomer at a position other than the accessible 5' end.
In a third aspect, the invention provides pharmaceutical formulation comprising an immunomer or an immunomer conjugate according to the invention and a physiologically acceptable carrier.
In a fourth aspect, the invention provides methods for generating an immune response in a vertebrate, such methods comprising administering to the vertebrate an immunomer or immunomer conjugate according to the invention. In some embodiments, the vertebrate is a mammal.
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 immunomer or immunomer conjugate according to the invention. In various embodiments, the disease or disorder to be treated is cancer, an autoimmune disorder, airway inflammation, asthma, allergy, or a disease caused by a pathogen.

7 BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a schematic representation of representative immunomers of the invention.
Figure 2 depicts several representative immunomers of the invention.
Figure 3 depicts a group of representative small molecule linkers suitable for linear synthesis of immumomers of the invention.
Figure 4 depicts a group of representative small molecule linkers suitable for parallel synthesis of immunomers of the invention.
Figure 5 is a synthetic scheme for the linear synthesis of immunomers of the invention. DMTr = 4,4'-dimethoxytrityl; CE = cyanoethyl.
Figure 6 is a synthetic scheme for the parallel synthesis of immunomers of the invention. DMTr = 4,4'-dimethoxytrityl; CE = cyanoethyl.
Figure 7A is a graphic representation of the induction of IL-12 by immunomers 1-3 in BALB/c mouse spleen cell cultures. These data suggest that Immunomer 2, which has accessible 5'-ends, is a stronger inducer of IL-12 than monomeric Oligo I, and that Immunomer 3, which does not have accessible 5'-ends, has equal or weaker ability to produce immune stimulation compared with oligo 1.
Figure 7B is a graphic representation of the induction of IL-6 (top to bottom, respectively) by Immunomers 1-3 in BALB/c mouse spleen cells cultures. These data suggest that Immunomer 2, which has accessible 5'-ends, is a stronger inducer of IL-6 than monomeric Oligo 1, and that Immunomer 3, which does not have accessible 5'-ends, has equal or weaker ability to induce immune stimulation compared with Oligo 1.
Figure 7C is a graphic representation of the induction of 1L-10 by Immunomers 1-3 (top to bottom, respectively) in BALB/c mouse spleen cell cultures.

8 Figure 8A is a graphic representation of the induction of BALB/c mouse spleen cell proliferation in cell cultures by different concentrations of Immunomers 5 and 6, which have inaccessible and accessible 5'-ends, respectively.
Figure 8B is a graphic representation of BALB/c mouse spleen enlargement by Immunomers 4-6, which have an immunogenic chemical modification in the 5'-flanking sequence of the CpG motif. Again, the immunomer, which has accessible 5'-ends (6), has a greater ability to increase spleen enlargement compared with Immunomer 5, which does not have accessible 5'-end and with monomeric Oligo 4.
Figure 9A is a graphic representation of induction of IL-12 by different concentrations of Oligo 4 and Immunomers 7 and 8 in BALB/c mouse spleen cell cultures.
Figure 9B is a graphic representation of induction of IL-6 by different concentrations of Oligo 4 and Immunomers 7 and 8 in BALB/c mouse spleen cell cultures.
Figure 9C is a graphic representation of induction of IL-10 by different concentrations of Oligo 4 and Immunomers 7 and 8 in BALB/c mouse spleen cell cultures.
Figure 10A is a graphic representation of the induction of cell proliferation by Immunomers 14, 15, and 16 in BALB/c mouse spleen cell cultures.
Figure 10B is a graphic representation of the induction of cell proliferation by 1L-12 by different concentrations of Immunomers 14 and 16 in BALB/c mouse spleen cell cultures.
Figure IOC is a graphic representation of the induction of cell proliferation by IL-6 by different concentrations of Immunomers 14 and 16 in BALB/c mouse spleen cell cultures.

9 Figure 11 A is a graphic representation of the induction of cell proliferation by Oligo 4 and 17 and Immunomers 19 and 20 in BALB/c mouse spleen cell cultures.
Figure 11B is a graphic representation of the induction of cell proliferation IL-12 by different concentrations of Oligo 4 and 17 and Immunomers 19 and 20 in BALB/c mouse spleen cell cultures.
Figure 11C is a graphic representation of the induction of cell proliferation IL-6 by different concentrations of Oligo 4 and 17 and Immunomers 19 and 20 in BALB/c mouse spleen cell cultures.
Figure 12 is a graphic representation of BALB/c mouse spleen enlargement using oligonucleotides 4 and immunomers 14, 23, and 24.
Figure 13 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 14 shows the chemical substitutions used in Example 13.
Figure 15 shows cytokine profiles obtained using the modified oligonucleotides of Example 13.
Figure 16 shows relative cytokine induction for glycerol linkers compared with amino linkers.
Figure 17 shows relative cytokine induction for various linkers and linker combinations.
Figures 18 A-E shows relative nuclease resistance for various PS and PO
immunomers and oligonucleotides.
Figure 19 shows relative cytokine induction for PO immunomers compared with PS immunomers in BALB/c mouse spleen cell cultures.

Figure 20 shows relative cytokine induction for PO immunomers compared with PS immunomers in C3H/Hej mouse spleen cell cultures.
Figure 21 shows reduced tumor mass after administration of immunomer 157 compared to saline control.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The invention relates to the therapeutic use of oligonucleotides as immunostimulatory agents for immunotherapy applications.
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 immunostimulatoty effect for immunotherapy and methods for making and using such compounds. In addition, immunomers of the invention are useful as adjuvants in combination with DNA vaccines, antibodies, allergens, chemotherapeutic agents, and 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. Such an oligonucleotide is referred to herein as an "immunomer."
In a first aspect, the invention provides immunomers comprising at least two oligonucleotides linked at their 3' ends, or an intemucleoside 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 immunomers 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. 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.
For purposes of the invention, the term "immunomer" refers to any compound comprising at least two oligonucleotides linked at their 3' ends or internucleoside linkages, or functionalized nucleobase or sugar directly or via a non-nucleotidic linker, at least one of the oligonucleotides (in the context of the immunomer) being an immunostimulatory oligonucleotide and having an accessible 5' end, wherein the compound induces an immune response when administered to a vertebrate. In some embodiments, the vertebrate is a mammal, including a human.
In some embodiments, the immunomer comprises two or more immunostimulatory oligonucleotides, (in the context of the immunomer) which may be the same or different. Preferably, each such immunostimulatory oligonucleotide has at least one accessible 5' end.
In certain embodiments, in addition to the immunostimulatory oligonucleotide(s), the immunomer also comprises at least one oligonucleotide that is complementary to a gene. As used herein, the term "complementary to" means that the oligonucleotide hybridizes under physiological conditions to a region of the gene.
In some embodiments, the oligonucleotide downregulates expression of a gene.
Such downregulatory oligonucleotides preferably are selected from the group consisting of antisense oligonucleotides, ribozyme oligonucleotides, small inhibitory RNAs and decoy oligonucleotides. As used herein, the term "downregulate a gene" means to inhibit the transcription of a gene or translation of a gene product. Thus, the immunomers according to these embodiments of the invention can be used to target one or more specific disease targets, while also stimulating the immune system.

In certain embodiments, the immunomer includes 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 in 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 comprising poly(I)-poly(dC). In certain embodiments, at least one set of Nn includes a string of 3 to 10 dGs and/or Gs or 2'-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'-substitutedarabinose, 2'-0-substitutedarabinose or hexose sugar group. The nucleoside residues can be coupled to each other by any of the numerous known intemucleoside linkages. Such intemucleoside linkages include, without limitation, phosphodiester, phosphorothioate, phosphorodithioate, alkylphosphonate, alkylphosphonothioate, phosphotriester, phosphoramidate, siloxane, carbonate, carboalkoxy, acetamidate, carbamate, morpholino, borano, thioether, bridged phosphommidate, bridged methylene phosphonate, bridged phosphorothioate, and sulfone intemucleoside linkages. The term "oligonucleotide"
also encompasses polynucleosides having one or more stereospecific intemucleoside 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 intemucleoside linkage, whether or not the linkage comprises 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 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.
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'-fluoroarabinoside.

For purposes of the invention, the term "2'-substituted ribonucleoside"
includes ribonucleosides in which the hydroxyl group at the 2' position of the pentose moiety is substituted to produce a 2'-0-substituted ribonucleoside.
Preferably, such substitution is with a lower alkyl group containing 1-6 saturated or unsaturated carbon 5 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 such 2'-0-substituted ribonucleosides include, without limitation 2'-0-methylribonucleosides and 2'-0-methoxyethylribonucleosides.

10 The term "2'-substituted ribonucleoside" also includes ribonucleosides in which the 2'-hydroxyl group is replaced with a lower alkyl group containing 1-saturated or unsaturated carbon atoms, or with an amino or halo group.
Examples of such 2'-substituted ribonucleosides include, without limitation, 2'-amino, 2'-fluoro, 2'-allyl, and 2'-propargyl ribonucleosides.
15 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 etal. 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, dogs, horses, cattle, cows, pigs, 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 01/55370, published August 2, 2001. -Preferably, the immunostimulatory oligonucleotide comprises at least one phosphodiester, phosphorothioate, or phosphordithioate intemucleoside 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.
As used herein, the term "pyrimidine nucleoside" refers tot nucleoside wherein the base component of the nucleoside is a pyrimidine base. Similarly, the term "purine nucleoside" refers to a nucleoside wherein the base component of the nucleoside is a purine base. 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 (!):
' -cc (I) 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 S' 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-, -N H2, -SH
and -OH. Preferred hydrogen bond acceptors include, without limitation, C=S, and the ring nitrogen atoms of an aromatic heterocycle, e.g., N3 of cytosine.
In some embodiments, the base moiety in (/) 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 (/) 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 a preferred sugar moieties.
Preferred purine nucleoside analogs according to the invention have the structure (//):
A
/L-j\D
L
--N)D1 S' (11) 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 selected from the group consisting of C, 0, N and S;
and S' 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=0, C=S, -NO2 and the ring nitrogen atoms of an aromatic heterocycle, e.g., NI of guanine.
In some embodiments, the base moiety in (//) 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 (//) is a naturally occurring sugar moiety, as described above for structure (/).
In preferred embodiments, the immunostimulatory dinucleotide is selected from the group consisting of CpG, C*pG, CpG*, and C*pG*, wherein C is cytidine or 2'-deoxycytidine, C* is 2'-deoxythymidine, arabinocytidine, 2'-deoxythymidine, 2'-deoxy-2*-substitutedarabinocytidine, 2'-0-substitutedarabinocytidine, 2'-deoxy-hydroxycytidine, 2'-deoxy-N4-alkyl-cytidine, 2'-deoxy-4-thiouridine or other non-natural 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'-0-substituted-arabinoguanosine, 2'-deoxyinosine, 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 in immunostimulatory domain of structure (///):

5'-Nn-N I -Y-Z-N I -Nn-3' (///) wherein:
Y is cytidine, 2'deoxythymidine, 2' deoxycytidine arabinocytidine, 2'-deoxy-2'-substitutedarabinocytidine, 2'-deoxythymidine, 2'-0-substitutedarabinocytidine, 5 2'-deoxy-5-hydroxycytidine, 2'-deoxy-N4-alkyl-cytidine, 2'-deoxy-4-thiouridine or other non-natural pyrimidine nucleoside;
Z is guanosine or 2'-deoxyguanosine, G* is 2' deoxy-7-deazaguanosine, 2'-deoxy-6-thioguanosine, arabinoguanosine, 2'-deoxy-2'substituted-arabinoguanosine, 2'-0-substituted-arabinoguanosine, 2'deoxyinosine, or other non-natural purine 10 nucleoside;
N I, 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, a-deoxyribonucleosides, (3¨L-deoxyribonucleosides, and nucleosides linked by a phosphodiester or modified 15 internucleoside linkage to the adjacent nucleoside on the 3' side, the modified intemucleotide linkage being selected from, without limitation, a linker having a length of from about 2 angstroms to about 200 angstroms, C2-C18 alkyl linker, poly(ethylene glycol) linker, 2-aminobuty1-1,3-propanediol linker, glyceryl linker, 2'-5' intemucleoside linkage, and phosphorothioate, phosphorodithioate, or 20 methylphosphonate intemucleoside linkage;
Nn, at each occurrence, is preferably a naturally occurring nucleoside or an immunostimulatory moiety selected from the group consisting of abasic nucleosides, arabinonucleosides, 2'-deoxyuridine, a-deoxyribonucleosides, 2'-0-substituted ribonucleosides, and nucleosides linked by a modified internucleoside linkage to the adjacent nucleoside on the 3' side, the modified intemucleotide linkage preferably being selected from the group consisting of amino linker, 2'-5' intemucleoside linkage, and methylphosphonate intemucleoside linkage;

provided that at least one NI or Nn is an immunostimulatory moiety;
wherein n is a number from 0 to 30; and wherein the 3'end, an intemucleoside 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 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 stereospeciflc 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'-0-methylribose, 2'-0-methoxyethylribose, 2'-0-propargylribose, and 2'-deoxy-2'-fluororibose;
3'-substituted pentose sugars, including, without limitation, 3'-0-methylribose;
I ',2'-dideoxyribose; arabinose; substituted arabinose sugars, including, without limitation, I '-methylarabinose, 3'-hydroxymethylarabinose, 4'-hydroxymethyl-arabinose, 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'-0-substituted ribonucleoside, the immunostimulatory moiety is attached to the adjacent nucleoside by way of a 2'-5' intemucleoside linkage.

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 -0-(CH2-CH2-0-)õ (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,I3-L-deoxyribonucleosides and a¨deoxyribonucleosides. Preferred immunostimulatory moieties according to the invention incorporate 3' modifications, and further include nucleosides having unnatural intemucleoside 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-thioumcil, 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 (///), a methylphosphonate intemucleoside linkage at position N1 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 X 1 is an immunostimulatory moiety, and a 13-L-deoxyribonucleoside at position XI 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 intemucleoside linkage between that nucleoside residue and the adjacent nucleoside on the 3' side. Similarly, reference to a modified intemucleoside 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 - Position TYPICAL IMMUNOSTIMULATORY MOIETIES
NI Naturally-occurring nucleosides, abasic nucleoside, arabinonucleoside, 2'-deoxyuridine, fl-L-deoxyribonucleoside C2-C18 alkyl linker, poly(ethylene glycol) linkage, 2-aminobuty1-1,3-propanediol linker (amino linker), 2'-5' intemucleoside linkage, methylphosphonate intemucleoside linkage Nn Naturally-occurring nucleosides, abasic nucleoside, arabinonucleosides, 2'-deoxyuridine, 2'-0-substituted ribonucleoside, 2'-5' intemucleoside linkage, methylphosphonate intemucleoside linkage, provided that NI
and N2 cannot both be abasic linkages 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-(CH2CH2-0)õ-, wherein n is 3. The term "Spacer 18"
refers to a poly(ethylene glycol) linker of formula -0-(CH2CH2-0)õ-, wherein n is 6.

24 , As used herein, the term "C2-C18 alkyl linker refers to a linker of formula -0-(CH2)q-0-, where q is an integer from 2 to 18. Accordingly, the terms "C3-linker"
and "C3-alkyl linker" refer to a linker of formula -0-(CH2)3-0-. 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.
Table 2 Position TYPICAL IMMUNOSTIMULATORY MOIETY
5' N2 Naturally-occurring nucleosides, 2-aminobuty1-1,3-propanediol linker 5' NI Naturally-occurring nucleosides, [3-L-deoxyribonucleoside, C2-C18 alkyl linker, poly(ethylene glycol), abasic linker, 2-am inobuty1-1,3-propanediol linker 3' Ni Naturally-occurring nucleosides, l',2'-dideoxyribose, 2'-0-methyl-ribonucleoside, C2-C18 alkyl linker, Spacer 9, Spacer 18 3' N2 Naturally-occurring nucleosides, l',2'-dideoxyribose, 3'-deoxyribonucleoside,f3-L-deoxyribonucleoside, 2'-0-propargyl-ribonucleoside, C2-C18 alkyl linker, Spacer 9, Spacer 18, methylphosphonate intemucleoside linkage 3' N 3 Naturally-occurring nucleosides, l',2'-dideoxyribose, C2-C18 alkyl linker, Spacer 9, Spacer 18, methylphosphonate intemucleoside linkage, 2'-5' intemucleoside linkage, d(G)n, polyl-polydC
3'N 2+ 3'N 3 1',2'-dideoxyribose, P-L-deoxyribonucleoside, C2-C18 alkyl linker, d(G)n, polyl-polydC
3'N3+ 3' N 4 2'-0-methoxyethyl-ribonucleoside, methylphosphonate intemucleoside linkage, d(G)n, polyl-polydC
3'N5+ 3' N 6 1',2'-dideoxyribose, C2-C18 alkyl linker, d(G)n, polyl-polydC
5'N I+ 3' N 3 1',2'-dideoxyribose, d(G)n, polyl-polydC
Table 3 shows representative positions and structures of immunostimulatory moieties within an immunostimulatory oligonucleotide having a downstream potentiation domain.

Table 3 Position TYPICAL IMMUNOSTIMULATORY MOIETY
5' N2 mcthylphosphonate internucleoside linkage 5' NI methylphosphonate internucleoside linkage 3' NI l',2'-dideoxyribose, methylphosphonate internucleoside linkage, 2.-0-methyl 3' N2 1%2.-dideoxyribose. 13-L-deoxyribonucleoside. C2-C18 alkyl linker. Spacer 9. Spacer 18, 2-aminobuty1-1.3-propanediol linker, methylphosphonate internucleoside linkage, 2.-0-methyl 3' N3 3.-deoxyribonucleoside. 3.-0-substituted ribonucleoside, 2%0-propargyl-ribonucleoside 3'N2 + 3' N3 l',2'-dideoxyribose, 8-L-deoxyribonucleoside The immunomers according to the invention comprise at least two oligonucleotides linked at their 3' ends or intemucleoside linkage or a functionalized nucleobase or sugar via a non-nucleotidic linker. For purposes of the invention, a 5 "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 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, it-stacking interactions, and 10 hydrogen bonding. The term "non-nucleotidic linker" is not meant to refer to an intemucleoside 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 is considered to be a "nucleotidic linkage."
15 In some embodiments, the non-nucleotidic linker is a metal, including, without limitation, gold particles. In 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 20 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 13. 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.
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.
In some embodiments, the small molecule linker is glycerol or a glycerol homolog of the formula HO-(CH2)0-CH(OH)-(CH2)p-OH, wherein o and p independently are integers from I to about 6, from I to about 4, or from I to about 3.
In some other embodiments, the small molecule linker is a derivative of 1,3-diamino-2-hydroxypropane. Some such derivatives have the formula H0-(CH2),,,-C(0)NH-CH2-CH(OH)-CH2-NHC(0)-(CH2),õ-OH, wherein m is an integer from 0 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, as schematically depicted in Figure 1. For example, the small molecule linker glycerol has three hydroxyl groups to which oligonucleotides may be covalently attached. Some immunomers according to the invention, therefore, comprise more than two oligonucleotides linked at their 3' ends to a non-nucleotidic linker. Some such immunomers comprise at least two immunostimulatory oligonucleotides, each having an accessible 5' end.
The immunomers of the invention may conveniently be synthesized using an automated synthesizer and phosphoramidite approach as schematically depicted in Figures 5 and 6, and further described in the Examples. In some embodiments, the immunomers are synthesized by a linear synthesis approach (see Figure 5). As used herein, the term "linear synthesis" refers to a synthesis that starts at one end of the immunomer 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 immunomers.
An alternative mode of synthesis is "parallel synthesis", in which synthesis proceeds outward from a central linker moiety (see Figure 6). 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 immunomers has several advantages over linear synthesis: (I) 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 immunomer product.
At the end of the synthesis by either linear synthesis or parallel synthesis protocols, the immunomers may conveniently be deprotected with concentrated ammonia solution or as recommended by the phosphoramidite supplier, if a modified nucleoside is incorporated. The product immunomer is preferably purified by reversed phase HPLC, detritylated, desalted and dialyzed.
Table 4 shows representative immunomers according to the invention.
Additional immunomers are found described in the Examples.
Table 4. Examples of Immunomer Sequences Oligo or Sequences and Modification (5'-3') lmmunomer No.
1 5'-GAGAACGCTCGACCTT-3' 2 5'-GAGAACGCTCGACCTT-3'-3.-TTCCAGCTCGCAAGAG-5' 3 3'-TTCCAGCTCGCAAGAG-5'-5'-GAGAACGCTCGACCTT-3' 4 5'-CTATCTGACGTTCTCTGT-3' 5'-T-3' HNCO-C41-18-5.-CTATLTGACGTICTCTGT-3' HNCO-C4H8-5.-CTATLTGACGTTCTCTGT-3' 6 5LCTATLTGACGTTCTCTGT-3'-C4H8-CONH
3'5 6-CTATLTGACGTICTCTGT-3.-C4H8-CONH -C-' 7 5'-CTATCTGACGTTCTCTGT-3.-C4H8-CONH
5-CTATCTGACGTTCTCTGT-3'-C4H8-CONH
5'-CTATCTGACGTICTCT GT- 3' j_ 9 5'-CTATCTGAYGTTC TCTGT-3'3_ 3.-C-5' 5'-CTATCTGAYGTTCTCTGT-3' 5'-CTATCTGACRTTCTCTGT-3' 5'-CTATCTGACRTTCTCTGT-3' IF

11 5'-CTALCTGAYGTTCTCTGT-3' 5'-CTALCTGAYGTTCTCTGT-3' IF

12 5'-CTALCTGACRTTCTCTGT-3'3_ 3.-C-5' 5'-CTALCTGACRTTCTCTGT-3'

13 5'-CTGACGTICTCTGT-3'

14 5'-CTGACGTTCTCTGT-3' 5'-CTGACGTTCTCTGT-3' 51-CTGAYGTTCTCTGT-3' ' 5'-CTGAYGTTCTCTGT 3'-C-5 -3' 16 5'-CTGACRTTCTCTGT-3' 5'-CTGACRTTCTCTGT-3*
¨ -- ¨ --17 5'-XXTGACGTTCTCTGT-3' _ 3'-C-5' 5'-)0(XTGACGTTCTCTGT-3' -- ¨
19 5'-)0(XTGAYGT TCTCTGT-3' I ¨
3-C-5' 5- XXXTGAYGT TCTCTGT-3' 20 5'40(XTGACRT TCTCTGT-3' I . _ 3. -C-5 5- XXXTGACRT TCTCTGT-3 _ 21 5'-TCTGACGTTCT-3' 22 T-XXXTCTGACGTTCT-3'} . .
5'40CXTCTGAC GTTCT-3' 3 -C-5 23 5'400(TCTGAYGTTCT-3' I . .

51-)OCXTCTGAYG1TCT-3' 24 51-)0(XTCTGAC RTTCT-3' i .
3 -C-5.
5.40(XTCTGAC RTTCT-3' _TNHCOC4H8- _ = Symmetric longer branches; ¨ = Symmetric glycerol (short) branches L NHCOC41-18- _ L = C3-alkyl linker; X = 1',2'-dideoxyriboside; Y = 5 H dC; R = 7-deaza-dG
In a second aspect, the invention provides immunomer conjugates, comprising an immunomer, as described above, and an antigen conjugated to the immunomer 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 with" 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 immunomer 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 immunomer and antigen. Non limiting examples of such operative associations 5 include being part of the same liposome or other such delivery vehicle or reagent. In embodiments wherein the immunomer is covalently linked to the antigen, such covalent linkage preferably is at any position on the immunomer 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-10 nucleotidic linker. Alternatively, the antigen may itself be the non-nucleotidic linker.
In a third aspect, the invention provides pharmaceutical formulations comprising an immunomer or immunomer 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

15 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.
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 20 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.
25 In a fourth aspect, the invention provides methods for generating an immune response in a vertebrate, such methods comprising administering to the vertebrate an immunomer or immunomer 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. In preferred embodiments, the immunomer or immunomer conjugate is administered to a vertebrate in need of immunostimulation.
In the methods according to this aspect of the invention, administration of immunomers 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 immunomers 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 immunomer 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 immunomer 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, immunomers 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 immunomers of the invention can variously act as adjuvants and/or produce direct immunostimulatory effects.
Either the immunomer or the vaccine, or both, may optionally be linked to an immunogenic protein, such as keyhole limpet hemocyanin (KLH), 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 immunomer 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 immunomer, and/or independently the vaccine, and/or independently the adjuvant. The administration of the immunomer 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 immunomer or immunomer 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 immunomer or immunomer 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 immunomer. For example, in the treatment of cancer, it is contemplated that the immunomer or immunomer conjugate may be administered in combination with a chemotherapeutic compound.
While the invention has been described in connection with specific embodiments thereof, it will be understood that the scope of the claims should not be limited by the preferred embodiments set forth in the examples, but should be given the broadest interpretation consistent with the description as a whole.
EXAMPLES
Example 1: Synthesis of Oligonueleotides Containing Immunomodulatory Moieties Oligonucleotides were synthesized on a I gmol scale using an automated DNA synthesizer (Expedite 8909; PerSeptive Biosystems, Framingham, MA), following the linear synthesis or parallel synthesis procedures outlined in Figures 5 and 6.
Deoxyribonucleoside phosphoramidites were obtained from Applied Biosystems (Foster City, CA). I ',2'-dideoxyribose phosphoramidite, propyl- I -phosphoramidite, 2-deoxyuridine phosphoramidite, I,3-bis-[5-(4,4'-dimethoxytrityl)pentylamidy11-2-propanol phosphoramidite and methyl phosponamidite were obtained from Glen Research (Sterling, VA). ii-L-2'-deoxyribonucleoside phosphoramidite, ct-2'-deoxyribonucleoside phosphoramidite, mono-DMT-glycerol phosphoramidite and di-DMT-glycerol phosphoramidite were obtained from ChemGenes (Ashland, MA). (4-AminobutyI)-I,3-propanediol phosphoramidite was obtained from Clontech (Palo Alto, CA). Arabinocytidine phosphoramidite, arabinoguanosine, arabinothymidine and arabinouridine were obtained from Reliable Pharmaceutical (St. Louis, MO). Arabinoguanosine phosphoramidite, arabinothymidine phosphoramidite and arabinouridine phosphoramidite were synthesized at Hybridon, Inc. (Cambridge, MA) (Noronha et al. (2000) Biochem., 39:7050-7062).
All nucleoside phosphoramidites were characterized by 31P and 1H NMR
spectra. Modified nucleosides were incorporated at specific sites using normal coupling cycles. After synthesis, oligonucleotides were deprotected using concentrated ammonium hydroxide and purified by reverse phase HPLC, followed by dialysis. Purified oligonucleotides as sodium salt form were lyophilized prior to use.
Purity was tested by CGE and MALDI-TOF MS.
Example 2: Analysis of Spleen Cell Proliferation In vitro analysis of splenocyte proliferation was carried out using standard procedures as described previously (see, e.g., Zhao etal., Biochem Pharma 51:173-182 (1996)). The results are shown in Figure 8A. These results demonstrate that at the higher concentrations, lmmunomer 6, having two accessible 5' ends results in greater splenocyte proliferation than does Immunomer 5, having no accessible 5' end or Oligonucleotide 4, with a single accessible 5' end. Immunomer 6 also causes greater splenocyte proliferation than the LPS positive control.
Example 3: In vivo Splenomegaly Assays To test the applicability of the in vitro results to an in vivo model, selected oligonucleotides were administered to mice and the degree of splenomegaly was measured as an indicator of the level of immunostimulatory activity. A single dose of 5 mg/kg was administered to BALB/c mice (female, 4-6 weeks old, Harlan Sprague Dawley Inc, Baltic, CT) intraperitoneally. The mice were sacrificed 72 hours after oligonucleotide administration, and spleens were harvested and weighed. The results are shown in Figure 8B. These results demonstrate that Immunomer 6, having two accessible 5' ends, has a far greater immunostimulatory effect than do Oligonucleotide 4 or Immunomer 5.

Example 4: Cytoldne Analysis The secretion of IL-12 and IL-6 in vertebrate cells, preferably BALB/c mouse spleen cells or human PBMC, was measured by sandwich ELISA. The required reagents including cytokine antibodies and cytokine standards were purchased form 5 PharMingen, San Diego, CA. ELISA plates (Costar) were incubated with appropriate antibodies at 5 trg/mL in PBSN buffer (PBS/0.05% sodium azide, pH 9.6) overnight at 4 C and then blocked with PBS/I% BSA at 37 C for 30 minutes. Cell culture supernatants and cytokine standards were appropriately diluted with PBS/I 0%
FBS, added to the plates in triplicate, and incubated at 25 C for 2 hours. Plates were 10 overlaid with 1 pg/mL appropriate biotinylated antibody and incubated at 25 C for 1.5 hours. The plates were then washed extensively with PBS-T Buffer (PBS/0.05%
Tween 20) and further incubated at 25 C for 1.5 hours after adding streptavidin conjugated peroxidase (Sigma, St. Louis, MO). The plates were developed with Sure BIuCTM (Kirkegaard and Perry) chromogenic reagent and the reaction was terminated 15 by adding Stop Solution (Kirkegaard and Perry). The color change was measured on 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 20 (Histopaque-I077, Sigma, St. Louis, MO). Briefly, heparinized blood was layered onto the Histopaque-I077 (equal volume) in a conical centrifuge and centrifuged at 400 x g for 30 minutes at room temperature. The buffy coat, containing the mononuclear cells, was removed carefully and washed twice with isotonic phosphate buffered saline (PBS) by centrifugation at 250 x g for 10 minutes. The resulting cell 25 pellet was then resuspended in RPMI 1640 medium containing L-glutamine (MediaTech, Inc., Herndon, VA) and supplemented with 10% heat inactivated FCS
and penicillin-streptomycin (100U/m1). Cells were cultured in 24 well plates for different time periods at 1 X 106 cells/ml/well in the presence or absence of oligonucleotides. At the end of the incubation period, supernatants were harvested and *Trade -mark stored frozen at ¨70 C until assayed for various cytokines including IL-6 (BD

Pharmingen, San Diego, CA), 1L-10 (BD Pharmingen), IL-12 (BioSource International, Camarillo, CA), IFN-a (BioSource International) and -y (BD
Pharmingen) and TNF-a (BD Pharmingen) by sandwich ELISA. The results are shown in Table 5 below.
In all instances, the levels of IL-12 and IL-6 in the cell culture supernatants were calculated from the standard curve constructed under the same experimental conditions for IL-12 and IL-6, respectively. The levels of IL-10, 1FN-gamma and TNF-a in the cell culture supernatants were calculated from the standard curve constructed under the same experimental conditions for IL-10, IFN-gamma and TNF-a, respectively.
Table 5. lmmunomer Structure and Immunostimulatory Activity in Human PBMC
Cultures Oligo Sequences and Modification (5'-3') Oligo Length/ IL-12 (pg/mL) IL-6 (pg/mL) No.
or Each Chain D1 D2 D1 D2 25 5'-CTATCTGTCGTTCTCTGT-3' 18mer (PS) 184 332 3077 5369 26 5'-TCTGTCR1TTCT-3 \ llmer (PS) 237 352 3724 4892 Xi 51-TCTGTCR1TTCT-3' /
Oligo Sequences and Modification (5'-3') Oligo Length/ IL-10 (pg/mL) IFN-y (pg/mL) No.
__________________________________ or Each Chain D1 02 D1 __ 02 25 5'-CTATCTGTCGTTCTCTGT-3' 18mer (PS) 37 88 125 84 26 5.-TCTGTCR1TTCT-31 \ 11mer (PS) 48 139 251 40 Xi 5'-TCTGTCR1TTCT-31/
Oligo Sequences and Modification (5'-3') Oligo Length/ TNF-a(pg/mL) No.
or Each Chain D1 D2 5'-CTATCTGTCGTTCTCTGT-3' 18mer (PS) 537 nt 26 5'-TCTGTCR11TCT-3' \ llmer (PS) 681 nt 5-TCTGTCR1TTCT-3' / Xi -D1 and 02 are donors 1 and 2.

Table 5A. Immunomer Structure and Immunostimulatory Activity in BALB/c Mouse Spleen Cell Cultures -Olijo Sequences and Modification Oligo Length/ IL-12 (pg/mL) IL-6 (pg/rriL) No.
or Each Chain 3 _gmL 101.1g/rpL
26 5'-TCTGTCR1TTCT-3' \ 11mer (PS) 870 10670 51-TCTGICR1TTCT-3' 27 5'-TCTGTCR2TTCT-3' \x 11mer (PS) 1441 7664 - -5'-TCTGTCR21TCT-3' / 1 28 5'-TCTGTY2R2TTCT-3' \ llmer (PS) 1208 1021 5'-TCTGTY2R2TTCT-3' ¨29 ¨ 5'400-CTGTCRITTCT-3 \ 11mer (PS) 162 1013 Xi 5'-XXTCTGTCR1TTCT-3' 30 5'-CTGTCR2TTCTCTGT-3' 14mer (PO) 264 251 5.-CTGTCR21TCTCTGT-3' 31 ¨ 5I-CTGTY2R2TTCTCTGT-3' 14mer (PO) 149 119 Xi 5'-CTGTY2R2TTCTCTGT-3'/
32 5'-TCTGACR1TTCT-3' \ llmer (PS) 2520 9699 Xi 5-TCTGACRITTCT-3' 33 5'-XXTCTGACRITTCT-3' \ llmer (PS) 2214 16881 XI
5'-XXTCTGACR1TTCT-3' 34 5'-TCTGACR2TTCT-3' \ 11mer PS) 3945 10766 Xi 5-TCTGACR2TTCT-3' 35 5'-TCTGAY2R2TTCT-3' \ llmer (PS) 2573 19411 Xi 5'-TCTGAY2R2TTCT-3' 36 5.-CTGAY2GTTCTCTGT-3'N 14mer (PO) 2699 408 Xi 5'-CTGAY2GTTCTCTGT-3Y
37 5'-CTGACR2TTCTCTGT-3 '\ 14mer (PO) 839 85 Xi 51-CTGACR2TTCTCTGT-3'/
38 5'-CTGAY2R2TTCTCTGT-3'\ 14mer (PO) 143 160 51-CTGAY2R2TTCTCTGT-3''1 Normal phase represents a phosphorothioate linkage; Italic phase represents a phosphodiester linkage.
dG7-deaza 0 AraG<CJL 0 NH NH

R1 = R2 =
OH
0õ0 e GS" S"
Ara C NH2 N

OH
Y2 =
0õ0 e P-S"

II
X = 0õ0 =
e P-so /0 I 0 OH th.
In addition, the results shown in Figures 7A-C demonstrate that Oligonucleotide 2, with two accessible 5' ends elevates IL-12 and IL-6, but not IL-10 at lower concentrations than Oligonucleotides 1 or 3, with one or zero accessible 5' ends, respectively.

Example 5: Effect of Chain Length on Immunostimulatory Activity of Immunomers In order to study the effect of length of the oligonucleotide chains, immunomers containing 18, 14, 11, and 8 nucleotides in each chain were synthesized and tested for immunostimulatory activity, as measured by their ability to induce secretion of the cytokines IL-12 and IL-6 in BALB/c mouse spleen cell cultures (Tables 6-8). In this, and all subsequent examples, cytokine assays were carried out in BALB/c spleen cell cultures as described in Example 4.
Table 6. Immunomer Structure and Immunostimulatory Activity No. Sequences and Modification (5'-3) Oligo Length/ IL-12 (pg/mL) IL-6 (pg/mL) or Each Chain @ 0.3 ug/mL (g) 0.3 ug/mL
4 5'-CTATCTGACGTTCTCTGT-3 18mer 1802 176 39 5'-CTATCTGACGTTCTCTGT-3'i_ 18mer 1221 148 5.-CTATCTGACGTTCTCTGT-3' 40 5'-CTGACGTTCTCTGT-3'} 14mer 2107 548 5.-CTGACGTTCTCTGT-3' 41 5'-TCTGACG1TCT-3' 11mer 3838 1191 3'-T-5' 51-TCTGACGTTCT-3'}
42 5'-GACGTTCT-3'} 8mer 567 52 5'-GACGTTCT-3' Table 7. Immunomer Structure and Immunostimulatory Activity No. Sequences and Modification (5'-3') Oligo Length/ IL-12 (pg/mL) IL-6 (pg/mL) or Each Chain 1 g/mL 1 ug/mL
25 5'-CTATCTGTCGTTCTCTGT-3 18mer 291 65 43 5e-CTATCTGTCGTTCTCTGT-3' 1 18mer 430 39 3.-T-5.
5'-CTATCTGTCGTTCTCTGT-3' ¨
44 5.-C TGT CGTTCTCTGT-3' I . 14mer 813 59 3-T-5.
5'-C TGTCGTTCTCTGT-3' 5'-CTGTCGTTCTCT- 3' ]-3 -T-5. . 12mer 1533 123 5'-CTGTCGTTCTCT- 3' 46 5*-TCTGTC GTTCT-3'I . 11mer 2933 505 3. -T-5 5'-TCT GTC GTT CT-3' 47 5'-GTCGTTCT-3' . . 8mer 1086 26 i-3-T-5 5'-GTCGTICT-3' 48 5'-GTCGTTC-3'" . 7mer 585 34 3-T-5.
5'-GTCGTTC-3' 49 51-GTCGTT-3'1 3 -T-5 . . 6mer 764 76 5-GTCGTT-3' W-TCGTT-3'1 3 .-T-5 . 5mer 28 29 5e-TCGTT-3' Table 8. Immunomer Structure and Immunostimulatory Activity No. Sequences and Modification (5'-3') Oligo Length/ IL-12 (pg/mL) IL-6 (pg/mL) or Each Chain Tiig/m-L- 1 ug/mL
51 5'-CTCACTTTCGTTCTCTGT-3' 18mer 91 73 52 5'-CTCACTTTCGTTCTCTGT-3'} 18mer 502 99 3'-T-5' 5'-CTCACTTTCGTTCTCTGT-3' 53 5'-CMCGTTCTCTGT-3'} 14mer 683 119 5-CTTTCGTTCTCTGT-3' 54 5'-CTTTCGTTCTCT-3'} 12mer 633 102 5'-CTTTCGTTCTC T-3' ___________________________________________ ¨
55 5'-TTCGTTCT-3'} 8mer 687 243 6-TTCGTTCT-3' 56 5.-TCGTTCT-3'I 7mer 592 1252 6-TCGTTCT-3' The results suggest that the immunostimulatory activity of immunomers increased as the length of the oligonucleotide chains is decreased from 18-mers to 7-mers. lmmunomers having oligonucleotide chain lengths as short as 6-mers or 5-mers showed immunostimulatory activity comparable to that of the 18-mer oligonucleotide with a single 5' end. However, immunomers having oligonucleotide chain lengths as short as 6-mers or 5-mers have increased immunostimulatory activity when the linker is in the length of from about 2 angstroms to about 200 angstroms.
Example 6: Immunostimulatory Activity of Immunomers Containing A Non-Natural Pyrimidine or Non-Natural Purine Nucleoside As shown in Tables 9-11, immunostimulatory activity was maintained for immunomers of various lengths having a non-natural pyrimidine nucleoside or non-natural purine nucleoside in the immunostimulatory dinucleotide motif.

Table 9. Immunomer Structure and Immunostimulatory Activity No. Sequences and Modification (5'-3') Oligo Length/ IL-12 (pg/mL) IL-6 (pg/mL) or Each Chain CO 3 g/mL 3 g/mL
51 5'-CTCACTTTCGTTCTCTGT-3' 18mer 404 348 57 5s-TCTTTYGTTCT-3'} . 11mer 591 365 3. -T-5 5'-TCTTTYGTTCT-3' 58 5'-TCTTTC RTTCT- 3' 11mer 303 283 I
3. -T-5.
5'-TCTTTCRTTCT-3' 59 5'-TTYGTTCT-3 } 8mer 55 . 66 3-T-5..
5'-TTYGTTCT-3' 60 5.-TTCRTTCT-3' 1 8mer 242 143 3-T-5. .
5'-TTCRTTCT-3' NH2 =
CD
i N l 1 V 0 '8 I 11H
Y = 0 .)(2)/N1)4'0 R = N NH2 0......0 0,4.0 0 r%
? es/ r% 0 I

Table 10. Immunomer Structure and Immunostimulatory Activity No. Sequences and Modification (5'-3') Oligo Length/ IL-12 (pg/mL) IL-6 (pg/mL) or Each Chain 3 ug/mL 3 ug/mL
25 5'-CTATCTGTCGTTCTCTGT-3' 18mer 379 339 61 5.-3'-31_ 11mer 1127 470 ____ ¨
5'-3'-3' 3'-T-5' __________________________________________________ ¨ _ 62 5'-TCTGTCRTTCT-3'} 11mer 787 296 3.-T-5' 5'-TCTGTCRTTCT-3' 63 5'-GTYGTTCT-3'} 3-T-5. . 8mer 64 126 5e-GTYGTTCT-31 64 5'-GTCRTTCT-3'}3-T-5 , . 8mer 246 113 5.-GTCRTTCT-3' NH2 fs . I RI NH
V V

V = R = 0 I . I)440 I N NH2 O. 0 0 l oe es'Fic? s' o Table 11. Immunomer Structure and Immunostimulatory Activity No. Sequences and Modification (5'-3') Oligo Length/ IL-12 (pg/mL) IL-6 (pg/mL) or Each Chain 3 ug/mL 3 ug/mL
4 5'-CTATCTGACGTTCTCTGT-3 18mer 1176 1892 65 5-CTATCTGAYGTICTCTGT-3'1_ , 18mer 443 192 5-CTATCTGAYGTTCTCTGT-3' 665-CTATCTGACRTTCTCTGT-3'I 18mer 627 464 5-CTATCTGACRTTCTCTGT-3' 67 5'-CTGAYGTTCTCTGT-3'} 3.-T-5. 14mer 548 152 5'-CTGAYGTTCTCTGT-3' 68 5'-CTGACRTTCTCTGT-3'} . 14mer 1052 1020 3-T-5.
5'-CTGACRTTCTCTGT-3' 69 5'-TCTGAYGTTCT-3'} . . 11mer 2050 2724 5'-TCTGAYGTTCT-3' 70 5'-TCTGACRTTCT-3'} 3-T-5. . 11mer 1780 1741 --5t-TCTGACRTTCT-3' 71 5'-GAYGTrCT-3'} 8mer 189 55 3-T-5.' 5'-GAYGTTCT-3' 72 5'-GACRTTCT-3'} . 8mer 397 212 3-T-5.
5'-GACRTTCT-3' NH2 =
CD
i N i la 1 1 ,NkH

Y = R = N NH2 0.,n.0 0.,,.0 0 r-s=
%() Example 7: Effect of the Linker on Immunostimulatory Activity In order to examine the effect of the length of the linker connecting the two oligonucleotides, immunomers that contained the same oligonucleotides, but different linkers were synthesized and tested for immunostimulatory activity. The results shown in Table 12 suggest that linker length plays a role in the immunostimulatory activity of immunomers. The best immunostimulatory effect was achieved with C3-to C6-alkyl linkers or abasic linkers having interspersed phosphate charges.

Table 12. Immunomer Structure and Immunostimulatory Activity No. Sequences and Modification (5'-3') Oligo Length/ IL-12 (pg/mL) IL-6 (pg/mL) or Each Chain 0.3 ug/mL 1 g/mL
4 5'-CTATCTGACGTTCTCTGT-3' 18mer 257 635 73 5-CTGACGTTCT-5 10mer 697 1454 5-CTGACGTTCT-3' -74 5'-CTGACGTTCT-3'N 10mer 1162 669 5'-CTGACGTTCT-5 A
75 51-CTGACGTTCT-5 N 10mer 1074 1375 5e-CTGACGTTCT-3' 76 5'-CTGACGTTCT-3' N. 10mer 563 705 5-CTGACGTTCT-3' "'X4 77 5-CTGACGTICT-5 N 10mer 264 543 5-CTGACGTICT-5 , 78 5'-CTGACGTTCT-3'..x 10mer 1750 2258 5'-CTGACGTTCT-3' '''' 6 79 5'-CTGACGTICT-3' \ 10mer 2255 2034 5'-CTGACGTTCT-5 ".(X3PsX3) 80 5-CTGACGTTCT-3'N. 10mer 1493 1197 5-CTGACGTTCT-3' "..9(3PsVsX2 81 5'-CTGACGTTCT-3 10mer 3625 2642 \
5'_cTGACGTTCT-3'..,..(X6psX6) 82 5'-CTGACGTTCT-5 10mer 4248 2988 \
5-CTGACGTTCT-5 (X6psX6psXE
83 5-CTGACGTTCT-3'N. 10mer 1241 1964 5-CTGACGTTCT-3'.-,P03S

xi = s0"..rr"OH x2 = 0 N ./1NA 0.,..
C44. 2 2 4 , .
¨0-bX4 =".`0- (CH2)12 -- X6 = 11.4.0CIti' X6 = 2 6 ¨0 1 Example 8: Effect of Oligonucleotide Backbone on Immunostimulatory Activity In general, immunostimulatory oligonucleotides that contain natural phosphodiester backbones are less immunostimulatory than are the same length oligonucleotides with a phosphorothioate backbones. This lower degree of immunostimulatory activity could be due in part to the rapid degradation of phosphodiester oligonucleotides under experimental conditions. Degradation of oligonucleotides is primarily the result of 3'-exonucleases, which digest the oligonucleotides from the 3' end. The immunomers of this example do not contain a free 3' end. Thus, immunomers with phosphodiester backbones should have a longer half life under experimental conditions than the corresponding monomeric oligonucleotides, and should therefore exhibit improved immunostimulatory activity.
The results presented in Table 13 demonstrate this effect, with lmmunomers 84 and 85 exhibiting immunostimulatory activity as determined by cytokine induction in BALB/c mouse spleen cell cultures.
Table 13. Immunomer Structure and Immunostimulatory Activity No. Sequences and Modification (5'-3') Oligo Length/ --1C-1-2 (pg/mL) IL-6 (pg/mL) or Each Chain 0.3 ug/mL 1 pg/mL
4 5'-CTATCTGACGTTCTCTGT-3' 18mer 225 1462 84 51-CTGACGTICTCTGT-31._ 3 -T-5 (PO) 14mer 1551 159 ' 5.-CTGACGTTCTCTGT-3' 85 5'-LLCTGACGTTCTCTGT-3 14mer 466 ¨467 (P
5'-LLCTGACGTTCTCTGT-3' L = C3-Linker Example 9: Synthesis of Immunomers 73-92 Oligonucleotides were synthesized on 1 mol 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 ChemGenes (Ashland, MA). Modified nucleosides were incorporated into the oligonucleotides at specific site using normal coupling cycles. After the synthesis, oligonucleotides were deprotected using concentrated ammonium hydroxide and purified by reversed-phase HPLC, followed by dialysis. Purified oligonucleotides as sodium salt form were lyophilized prior to use. Purity of oligonucleotides was checked by CGE and MALDI-TOF MS (Bruker Proflex III MALDI-TOF Mass spectrometer).
Example 11 Immunomer Stability Oligonucleotides were incubated in PBS containing 10% bovine serum at 37 C for 4, 24 or 48 hours. Intact oligonucleotide was determined by capillary gel electrophoresis. The results are shown in Table 14.
Table 14. Digestion of Oligonucleotides in 10 % Bovine Serum PBS Solution Oligo Sequences and Modification (5'- CE analysis of oligos (% intact No. 3') oligo remained after digestion) after 4h After 24h after 48h 4 5-CTATCTGACGTTOTCTGT- 90.9 71.8 54.7 3'/PS
26 (5'-TCTGTCGTTCT)2S/PS 97.1 91.0 88.1 (G=dGdeaza) 86 (5'-CTGTCGTTCTCTGT)2S/P0 37.8 22.5 87 (5'- 73.1 56.8 36.8 XXCTGTCGTTCTCTGT)2S/P0 88 (5'-UCTGTCGTTCTCTGT)2S/P0 48.4 ' 36.7 X = C3-Linker, U, C = 2'-0Me-ribonucleoside Example 12: Effect of accessible 5' ends on immunostimulatory activity.
BALB/c mouse (4-8 weeks) spleen cells were cultured in RPM1 complete medium. Murine macrophage-like cells, J774 (American Type Culture Collection, Rockville, MD) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) FCS and antibiotics (100 1U/mL of penicillin G/streptomycin).
All other culture reagents were purchased from Mediatech (Gaithersburg, MD).
ELISAs for IL-12 and IL-6. BALB/c mouse spleen or J774 cells were plated in 24-well dishes at a density of 5x106 or 1x106cells/mL, respectively. The CpG
DNA dissolved in TE buffer (10 mM Tris-HCI, pH 7.5, 1 mM EDTA) was added to a final concentration of 0.03, 0.1, 0.3, 1.0, 3.0, or 10.0 g/mL to mouse spleen cell cultures and 1.0, 3.0, or 10.0 ilg/mL to J774 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 two or three times for each CpG DNA in triplicate for each concentration.
The secretion of IL-12 and IL-6 was measured by sandwich ELISA. The required reagents, including cytokine antibodies and standards were purchased from PharMingen. ELISA plates (Costar) were incubated with appropriate antibodies at 5 gg/mL in PBSN buffer (PBS/0.05% sodium azide, pH 9.6) overnight at 4 C and then blocked with PBS/1% BSA at 37 C for 30 min. Cell culture supernatants and cytokine standards were appropriately diluted with PBS/1% BSA, added to the plates in triplicate, and incubated at 25 C for 2 hr. Plates were washed and incubated with 1 itg/mL of appropriate biotinylated antibody and incubated at 25 C for 1.5 hr.
The plates were washed extensively with PBS/0.05% Tween 20 and then further incubated at 25 C for 1.5 hr after the addition of streptavidine-conjugated peroxidase (Sigma).
The plates were developed with Sure BIueTM (Kirkegaard and Perry) chromogenic reagent and the reaction was terminated by adding Stop Solution (Kirkegaard and Perry). The color change was measured on a Ceres 900 HD1 Spectrophotometer (Bio-Tek Instruments) at 450 nm. The levels of IL-12 and IL-6 in the cell culture supernatants were calculated from the standard curve constructed under the same experimental conditions for IL-12 and IL-6, respectively.
The results are shown in Table 15.
Table 15: Phosphorothioate CpG DNA sequences and modifications used in the 5 std/' CpG Sequence Length 5'-end 3'-end DNA #
89 5'-TCCATGACGTTCCTGATGC-3' 19-mer 1 1 90 5'-TCCATGACGTTCCTGATGC-3'-b 19-mer I blocked 91 5'-TCCATGACGTTCCTGATGC-3'-3'-g-5' 20-mer 2 blocked 92 5'-TCCATGACGTTCCTGATGC-3'-3'-h-5' 23-mer 2 blocked 93 5'-TCCATGACGTTCCTGATGC-3'-3'-i-5' 27-mer 2 blocked 94 5'-TCCATGACGTTCCTGATGC-3'-3'-j-5' 38-mer 2 blocked 95 b-5'-TCCATGACGTTCCTGATGC-3' 19-mer blocked 1 96 3'-c-5'-5'-TCCATGACGTTCCTGATGC-3' 20-mer blocked 2 97 3'-d-5'-5'-TCCATGACGTTCCTGATGC-3' 23-mer blocked 2 98 3 '-e-5 '-5'-TCCATGACGTTCCTGA
TGC-3 ' 27-mer blocked 2 99 3'-f-5 '-5'-TCCATGACGTT'CCTGATGC-3 ' 38-mer blocked 2 100 5'-TCCATGACGTTCCTGATGC-3'-k I 9-mer 1 blocked 101 1-5 '-TCCATGACGTT'CCTGATGC-3 ' 19-mer blocked 1 a: See Chart I for chemical structures b-1; 5'-CG-3' dinucleotide is shown underlined.

Chart 1 5' R-0--Dij.

i 0=P-0-- CCATGACG1TCCTGATG-0-ico 5' HOicom j 5cATG- C
I C)-IcOnj S- I: R, R' = a 3'0-R' 03 03 0=P 0 -S' (g) I
¨H
=P-S' (h) 0=P-S' I I
I
(a) OH
(b) 5.-TCCATGACGITCCTGATG-0 0 C
S. S' I 5' I 5' 5'-CCTGATG-0 C
-P-Oic.:0JT -P-0-v. J - 0 3 1(112ij ' ll II 0 0=113-S' (i) 03' 0 I
(c) HI 01' , I 0=P-S' (1) 3' CCA- I
HO is 0 0 OH
S' S-I 5' I 5' ¨p-o-lcor ¨p-o-icior o ii II
o o H . 0 0 0 (k): X
=CH2OH;
(e) I(n I x 0 (I): x = H.
CCATGAC-3' CCATGACGTTCCTGATGC-3' Table 16. Induction of IL-12 and IL-6 secretion by CpG DNA-conjugates in BALB/c mice spleen cell cultures CpG IL-12 (pg/mL)- SD IL-6 (pg/mL)=ESD
DNA tia 0.112g/mL 0.3 g/mL 1.0 g/mL 3.0 g/mL 10.0 g/mL 0.1 g/mL 0.314/mL 1.0 g/mL 3.0 g/mL 10.01.tg/mL
89 991+121 1820+224 2391+175 3507+127 2615+279 652+48 2858+180 13320+960 18625+1504 17229+1750 90 526+32 2100+175 1499+191 3019+35 3489+162 1387+152 1426+124 5420+370 19096+484 19381+2313 91 1030+11 1348+102 2060+130 3330+130 3582+259 923+22 2542+81 9054+120 14114+179 13693+264 92 1119+159 1726+207 2434+100 2966+204 3215+464 870+146 1905+56 7841+350 17146+1246 15713+693 93 1175+-68 2246+124 1812+75 2388+320 2545+202 1152+238 3499+116 7142+467 14064+167 13566+477 94 1087+121 1705+163 1797+141 2522+195 3054+103 1039+105 2043+157 4848+288 15527+224 21021+1427 95 1173+107 2170+155 2132+58 2812+203 3689+94 807+0.5 927+0.5 3344+0.5 10233+0.5 9213+0.5 96 866+51 1564+63 1525+63 2666+97 4030+165 750+63 1643+30 5559+415 11549+251 11060+651 97 227+3 495+96 1007+68 897+15 1355+97 302+18 374+22 1000+68 9106+271 13077+381 98 139+18 211+12 452+22 458+29 1178+237 220+23 235+18 383+35 1706+33 11530+254 UJ
99 181+85 282+105 846+165 2082+185 3185+63 467+122 437+85 1697+283 9781+13 11213+294 u, Medium 86 6 60+12 a: See Table 1 for sequences.

Taken together, the current results suggest that an accessible 5'-end of CpG
DNA is required for its optimal immunostimulatory activity and smaller groups such as a phosphorothioate, a mononucleotide, or a dinucleotide do not effectively block the accessibility of the 5'-end of CpG DNA to receptors or factors involved in the immunostimulatory pathway. However, the conjugation of molecules as large as fluorescein or larger at the 5'-end of CpG DNA could abrogate immunostimulatory activity. These results have a direct impact on the studies of immunostimulatory activity of CpG DNA-antigen/vaccine/monoclonal antibody (mAb) conjugates. The conjugation of large molecules such as vaccines or mAbs at the 5'-end of a CpG DNA could lead to suboptimal immunostimulatory activity of CpG 1-d DNA. The conjugation of functional ligands at the 3'-end of CpG DNA not only contributes to increased nuclease stability but also increased immunostimulatory potency of CpG DNA in vivo.

Example 13: Effect of linkers on cytokine secretion The following oligonucleotides were synthesized for this study. Each of these modified oligonucleotides can be incorporated into an immunomer.
Table 17. Sequences of CpG DNA showing the position of substitution.
CpG DNA Sequence Number 118 CTATC2GACGT1'CTCTGT
119 CTA2CTGACG11'CTCTGT

132 44TGACGT'TCTCTGT

142 CTATCT8ACGT1'CTCTOT

149 CTATCTGACG11'C8CTGT
150 CTATCTG9CGT1'CTCTGT
151 CTATCT9ACGT1'CTCTGT

a: See Figure 14 for the chemical structures of substitutions 1-9. All CpG DNAs are phosphorothioate backbone modified.
To evaluate the optimal linker size for potentiation of immunostimulatory activity, we measured IL-12 and IL-6 secretion induced by modified CpG DNAs in BALB/c mouse spleen cell cultures. All CpG DNAs induced concentration-dependent IL-12 and IL-6 secretion. Figure 15 shows data obtained at 1 g/mL
concentration of selected CpG DNAs, 116, 119, 126, 130, and 134, which had a linker at the fifth nucleotide position in the 5'-flanking sequence to the CpG
dinucleotide compared with the parent CpG DNA. The CpG DNAs, which contained C2- (1), C3-(2), and C4-linkers (3), induced secretion of IL-12 production similar to that of the parent CpG DNA 4. The CpG DNA that contained C6 and C9-linkers (4 and 5) at the fifth nucleotide position from CpG dinucleotide in the 5'-flanking sequence induced lower levels of IL-12 secretion than did the parent CpG DNA (Fig. 15), suggesting that substitution of linkers longer than a C4-linker results in the induction of lower levels of IL-12. All five CpG DNAs, which had linkers, induced two to three times higher IL-6 secretion than did the parent CpG DNA. The presence of a linker in these CpG DNAs showed a significant effect on the induction of IL-6 compared with CpG

DNAs that did not have a linker. However, we did not observe length-dependent linker effect on IL-6 secretion.
To examine the effect on immunostimulatory activity of CpG DNA containing ethylenegylcol-linkers, we synthesized CpG DNAs 137 and 138, in which a 5 triethyleneglycol-linker (6) is incorporated at the fifth nucleotide position in the 5'-and at the fourth nucleotide position in the 3'-flanking sequences to the CpG
dinucleotide, respectively. Similarly, CpG DNAs 139 and 140 contained a hexaethyleneglycol-linker (7) in the 5'- or the 3'-flanking sequence to the CpG
dinucleotide, respectively. All four modified CpG DNAs (137-140) were tested in 10 BALB/c mouse spleen cell cultures for cytokine induction (IL-12, 1L-6, and 1L-10) in comparison with parent CpG DNA 4. All CpG DNAs induced concentration-dependent cytokine production over the concentration range tested (0.03-10.0 ptg/mL) (data not shown). The levels of cytokines induced at 0.3 ptg/mL concentration of CpG
DNAs 137-140 are shown in Table 18. CpG DNAs 137 and 139, which had an 15 ethyleneglycol-linker in the 5'-flanking sequence induced higher levels of IL-12 (21061143 and 20661153 pg/mL) and IL-6 (2362 166 and 2507166 pg/mL) secretion than did parent CpG DNA 4 (Table 18). At the same concentration, 137 and 139 induced slightly lower levels of IL-10 secretion than did the parent CpG DNA
(Table 18). CpG DNA 138, which had a shorter ethyleneglycol-linker (6) in the 3'-flanking 20 sequence induced IL-12 secretion similar to that of the parent CpG DNA, but significantly lower levels of IL-6 and IL-10 (Table 18). CpG DNA 140, which had a longer ethyleneglycol-linker (7) induced significantly lower levels of all three cytokines tested compared with the parent CpG DNA (Table 18).
Though triethyleneglycol-linker (6) had a chain length similar to that of C9-25 linker (5), the CpG DNA containing triethyleneglycol-linker had better immunostimulatory activity than did CpG DNA containing C9-linker, as determined by induction of cytokine secretion in spleen cell cultures. These results suggest that the lower immunostimulatory activity observed with CpG DNA containing longer alkyl-linkers (4 and 5) may not be related to their increased length but to their hydrophobic characteristics. This observation prompted us to examine substitution of branched alkyl-linkers containing hydrophilic functional groups on immunostimulatory activity.
Table 18. Cytokine secretion induced by CpG DNAs containing an ethyleneglycol-linker in BALB/c mice spleen cell cultures.
CpG Cytokine, pg/mL
DNA
Number _________________________________ 138 1888 259 1081+25 47 14 Medium 84 13 33 6 2 1 To test the effect on immunostimulatory activity of CpG DNA containing branched alkyl-linkers, two branched alkyl-linkers containing a hydroxyl (8) or an amine (9) functional group were incorporated in parent CpG DNA 4 and the effects on immunostimulatory activity of the resulting modified CpG DNAs (150-154-Table 19) were examined. The data obtained with CpG DNAs 150-154, containing amino-linker 9 at different nucleotide positions, in BALB/c mouse spleen cell cultures (proliferation) and in vivo (splenomegaly) are shown in Table 19.
Table 19. Spleen cell proliferation induced by CpG DNA containing an aminobutyryl propanediol-linker in BALB/c mice spleen cell cultures and splenomegaly in BALB/c mice.

Parent CpG DNA 4 showed a CpG Spleen cell Spleen proliferation index of 3.7 0.8 at a DNA proliferation weight Numbera (PI)h (me concentration of 0.1 pig/mL. At the same 4 3.7 -0.8 121 16 concentration, modified CpG DNAs 151-154 150 2.5 0.6 107 11 containing amino-linker 9 at different 151 9.20E7 169 16 152 220 8 positions caused higher spleen cell 153 7.6 0.7 127 24 proliferation than did the parent CpG DNA
154 7.8 0.04 177 12 (Table 19). As observed with other linkers, M/V 1.2 0.3 102 8 when the substitution was placed adjacent to LPS 2.8 0.5 ND 10 CpG dinucleotide (150), a lower proliferation index was noted compared with parent CpG DNA (Table 19), further confirming that the placement of a linker substitution adjacent to CpG dinucleotide has a detrimental effect on immunostimulatory activity. In general, substitution of an amino-linker for 2'-deoxyribonucleoside in the 5'-flanking sequence (151 and 152) resulted in higher spleen cell proliferation than found with the substitution in the 3'-flanking sequence (153 and 154). Similar results were observed in the splenomegaly assay (Table 19), confirming the results observed in spleen cell cultures. Modified CpG DNAs containing glycerol-linker (8) showed immunostimulatory activity similar to or slightly higher that that observed with modified CpG DNA containing amino-linker (9) (data not shown).
In order to compare the immunostimulatory effects of CpG DNA containing linkers 8 and 9, we selected CpG DNAs 145 and 152, which had substitution in the 5'-flanking sequence and assayed their ability to induce cytokines IL-12 and secretion in BALB/c mouse spleen cell cultures. Both CpG DNAs 145 and 152 induced concentration-dependent cytokine secretion. Figure 4 shows the levels of IL-12 and IL-6 induced by 145 and 152 in mouse spleen cell cultures at 0.3n/mL
concentration compared with parent CpG DNA 4. Both CpG DNAs induced higher levels of 1L-12 and IL-6 than did parent CpG DNA 4. CpG DNA containing glycerol-linker (8) induced slightly higher levels of cytokines (especially IL-12) than did CpG

DNA containing amino-linker (9) (Figure 16). These results further confirm that the linkers containing hydrophilic groups are more favorable for immunostimulatory activity of CpG DNA.
We examined two different aspects of multiple linker substitutions in CpG
DNA. In one set of experiments, we kept the length of nucleotide sequence to 13-mer and incorporated one to five C3-linker (2) substitutions at the 5'-end (120-124).
These modified CpG DNAs permitted us to study the effect of an increase in the length of linkers without causing solubility problems. In the second set of experiments, we incorporated two of the same linker substitutions (3, 4, or 5) in adjacent positions in the 5'-flanking sequence to the CpG dinucleotide to study if there would be any additive effect on immunostimulatory activity.
Modified CpG DNAs were studied for their ability to induce cytokine production in BALB/c mouse spleen cell cultures in comparison with parent CpG
DNA 4. All CpG DNAs induced concentration-dependent cytokine production. The data obtained at 1.0 g/mL concentration of CpG DNAs is shown in Table 20. In this assay, parent CpG DNA 4 induced 967 28 pg/mL of IL-12, 1593 94.pg/mL of IL-6, and 14 6 pg/mL of IL-10 secretion at 1 p.g/mL of concentration. The data presented in Table 20 suggest that as the number of linker substitutions decreased IL-12 induction decreased. However, the induction of lower levels of IL-12 secretion by CpG DNAs 123 and 124 could be the result of the shorter length of CpG DNAs.
Our studies with unmodified CpG DNA shorter than 15-nucleotides showed insignificant immunostimulatory activity (data not shown). Neither length nor the number of linker substitutions have a lesser effect on IL-6 secretion. Though IL-10 secretion increased with linker substitutions, the overall IL-10 secretion by these CpG
DNAs was minimal.
CpG DNAs containing two linker substitutions (linker 3 - 127; linker-4 - 131;
linker-5 - 135) at the fourth and fifth positions in the 5'-flanking sequences to the CpG dinucleotide and the corresponding 5'-truncated versions 128, 132, and 136, respectively, were tested for their ability to induce cytokine secretion in BALB/c mouse spleen cell cultures. The levels of IL-12 and IL-6 secreted at 1.0 pg/mL

concentration are shown in Figure 17. The results presented in Figure 17 suggest that the immunostimulatory activity is dependent on the nature of the linker incorporated.
The substitution of the fourth and fifth nucleosides with C4-linker 3 (CpG DNA
127) had an insignificant effect on cytokine secretion compared with parent CpG DNA
4, suggesting that the nucleobase and sugar ring at these positions are not required for receptor recognition and/or binding. The deletion of the nucleotides beyond the linker substitutions (CpG DNA 128) caused higher IL-12 and IL-6 secretion than that found with CpG DNAs 4 and 127. As expected, the substitution of two C6-linkers (4) resulted in IL-12 secretion lower than and IL-6 secretion similar to that induced by parent CpG DNA 4. The 5'-truncated CpG DNA 132 induced higher cytokine secretion than did CpG DNA 131. The CpG DNAs 135 and 136, which had two C9-linkers (5), induced insignificant cytokine secretion, confirming the results obtained with mono-substituted CpG DNA containing the same linker as described above.
Example 14: Effect of Phosphodiester Linkages on Cytokine Induction To test the effect of phosphodiester linkages on immunomer-induced cytokine induction, the following molecules were synthesized.
Table 21 PO-Immunomer sequences and analytical data CpG Sequence Backbone Molecular Weight iµA.
-DNA Calculated Found' 04-0 4 5'-CTATCTGACGTTC1CTGT-3' PS 5702 5704 OH
155 5'-CTATCTGACGTTCTCTGT-3' PO 5432 5428 _____ 156 5'-CTG ACGTTCTCTGT-X-TGTCTCTTGCAGTC-5' PO 8656 8649 `".0t..5:õ..,, 9 157 5'-Y YCTGACGTTCTCTGT-X-TGTCTCTTGCAGTCYY-5' PO 9208 9214 0-P-I 0-'Arrows indicate 5.-3 directionality of CpG dinucleot ide in each DNA molecule and structures of X and Y are shown in boxes.
PS and PO stand for phosphorothioate and phosphodiester backbones, respectively.
vAs determined by MALDI-TOF mass spectrometry.

PS-CpG DNA 4 (Table 21) was found to induce an immune response in mice (data not shown) with PO-CpG DNA 155 serving as a control. PO-immunomers 156 and 157 each contain two identical, truncated copies of the parent CpG DNA 155 joined through their 3'-ends via a glyceryl linker, X (Table 21). While 156 and 157 5 each contain the same oligonucleotide segments of 14 bases, the 5'-ends of 157 were modified by the addition of two C3-linkers, Y (Table 21). All oligonucleotides 4, 155-157 contain a 'GACGIT hexameric motif known to activate the mouse immune system.
The stability of PO-immunomers against nucleases was assessed by incubating 10 CpG DNAs 4, 155-157 in cell culture medium containing 10% fetal bovine serum (FBS) (non-heat-inactivated) at 37 C for 4, 24, and 48 hr. Intact CpG DNA
remaining in the reaction mixtures were then determined by CGE. Figure 18 A-D
shows the nuclease digestion profiles of CpG DNAs 4, 155-157 incubated in 10%
FBS for 24 hr. The amount of full-length CpG DNA remaining at each time point is 15 shown in Figure 18 E. As expected, the parent PS-CpG DNA 4 is the most resistant to serum nucleases. About 55% of 18-mer 4 remained undegraded after 48 hr incubation. In contrast, only about 5% of full-length PO-immunomer 155 remained after 4 hr under the same experimental conditions confirming that DNA
containing phosphodiester linkages undergoes rapid degradation. As expected, both P0-20 immunomers 156 and 157 were more resistant than 155 to serum nucleases.
After 4 hr, about 62% and 73% of 156 and 157 respectively were intact compared with about 5% of 155 (Fig.18 E). Even after 48 hr, about 23% and 37% of 156 and 157, respectively, remained undegraded. As well as showing that 3'-3'-linked PO-immunomers are more stable against serum nucleases, these studies indicate that 25 chemical modifications at the 5'-end can further increase nuclease stability.
The immunostimulatory activity of CpG DNAs was studied in BALB/c and C3H/HeJ mice spleen cell cultures by measuring levels of cytokines IL-12 and secreted. All CpG DNAs induced a concentration-dependent cytokine secretion in BALB/c mouse spleen cell cultures (Fig. 19). At 3 g/mL, PS-CpG DNA 4 induced 26561256 and 1223411180 pg/mL of IL-12 and IL-6 respectively. The parent PO-CpG DNA 155 did not raise cytokine levels above background except at a concentration of 10 gg/mL. This observation is consistent with the nuclease stability assay results. In contrast, PO-immunomers 156 and 157 induced both IL-12 and secretion in BALB/c mouse spleen cell cultures.
The results presented in Figure 19 show a clear distinction in cytokine induction profiles of PS- and PO-CpG DNAs. PO-immunomers 156 and 157 induced higher levels of IL-12 than did PS-CpG DNA 4 in BALB/c mouse spleen cell cultures (Fig. 19A). In contrast, at concentrations up to 3 lig/mL, they produced negligible amounts of IL-6 (Fig. 19B). Even at the highest concentration (10 gg/mL), PO-immunomer 156 induced significantly less IL-6 than did PS-CpG DNA 4. The presence of C3 linkers at the 5'-terminus of PO-immunomer 157 resulted in slightly higher levels of IL-6 secretion compared with 156. However, importantly, the levels of IL-6 produced by PO-immunomer 157 are much lower than those induced by PS
CpG DNA 4. The inset of Figure 19A shows the ratio of IL-12 to IL-6 secreted at 3 1.1g/mL concentration. In addition to increasing IL-12 secretion, PO-immunomers 156 and 157 induced higher levels of IFN-y than did PS-CpG DNA 4 in BALB/c mouse spleen cell cultures (data not shown).
The different cytokine profiles induced by PO- and PS-CpG DNAs in BALB/c mouse spleen cell cultures prompted us to study the pattern of cytokine induction of CpG DNAs in C3H/HeJ mouse spleen cell cultures (an LPS lower-responsive strain).
All three CpG DNAs tested in this assay induced concentration-dependent cytokine secretion (Fig. 20A and B). Since PO-CpG DNA 155 failed to induce cytokine secretion in BALB/c mouse spleen cell cultures, it was not further tested in C3H/HeJ
spleen cell cultures. Both PO-immunomers 156 and 157 induced higher IL-12 production than did PS-CpG DNA 4 (Fig. 20A). However, at concentrations up to 1.1g/mL, neither induced IL-6 production. At the highest concentration tested (10 lig/mL), both induced significantly less IL-6 than did PS-CpG DNA 4 (Fig.
20B).

The ratio of IL-12 to IL-6 secreted is calculated to distinguish cytokine secretion profiles of PS and PO CpG DNAs (Fig. 20A inset). In addition, the C3H/HeJ
spleen cell culture results suggest that the responses observed with CpG DNAs are not due to LPS contamination.
PS-CpG DNAs have been shown to induce potent antitumor activity in vivo.
Since PO-CpG DNAs exhibited greater nuclease stability and induced higher levels of IL-12 and 1FN-y secretion in in vitro assays, we were interested to see if these desirable properties of PO-immunomers improve the antitumor activity in vivo.
We administered PO-immunomer 157 subcutaneously at a dose of 0.5 mg/kg every other day to nude mice bearing tumor xenografts of MCF-7 breast cancer cells that express wild-type p53, or DU-145 prostate cancer cells that express mutated p53. PO-immunomer 157 gave 57% growth inhibition of MCF-7 tumors on day 15 compared with the saline control (Fig. 21A). It also produced 52% growth inhibition of tumors on day 34 (Fig. 21B). These antitumor studies suggest that PO-immunomers of the proposed design exhibit potent antitumor activity in vivo.
Example 22: Short immunomers To test the effects of short immunomers on cytokine induction the following immunomers were used. These results show that immunomers as short as 5 nucleotides per segment are effective in inducing cytokine production.
.Table 22. lmmunomer Structure and lmmunostimulatory Activity in BABL/C Mouse Spleen Cell Cultures Oligo No. Sequences and Modification (5'-3') Oligo Length/ IL-12 (pg./mL) IL-6 (pg/mL) or Each Chain 10 g/mL 10 p_g/mL __ 4 5'-CTATCTGACGTTCTCTGT-3' 18mer 2731 4547 5'-CTATCTGTCGTTCTCTGT-3' 18mer 795 789 158 5'-TCTGACGTTCT-3' N llmer 3490 5319--5'-TCTGACGTTCT-3I
159¨ 5'-TCTGTCGTTCT-3 11mer 3265 - 4625 -Xi 5'-TCTGTCGTTCT-3' 160 5'-TCGTTG-3' \ 6mer 2085 2961 Xi ___________ 5.-TCGTTG-3 ' 161 5'-TCGTTG-3.XX \ 6mer 3169 5194 Xi 5'-TCGTTG-3'XX / ____________________________________________________ _ 162 5'-TCGTTG-31 \ 6mer 1015 705 Xi ___________ 5-TCGTTG-3'X' 163 6-TCGTT-3% \ 5mer 2623 3619 Xi 5'-TCGTT-3'X ' 164 51-ACGTTG-3'X \ 6mer 564 845 Xi ___________ 5.-ACGTTG-3'X ' _______________________________________ _ 165 5'-GCGTTG-3'X \ 6mer 196 0 Xi 5-GCGTTG-3'X /
166 5e-CCGTTG-3'X \ 6mer 219 0 Xi 5'-CCG11G-3'X' 167 5'-GTCGTT-3X \ 6mer 1441 5056 5'-GTCGTT-3% / Xi 168 5'-TGTCGT-3'X \ X 6mer 198 0 5'-TGTCGT-3'X / i 169 5-TCGTTG-3'X. 6mer 2410 4657 X1¨X3.-GTTGCT-;
51-TCGTTG-3'X' Normal phase represents a phosphorothioate linkage.
,õ0........õ.....¨,1 x= 0õ0 S" oC:i ' _________________________________________________________ II II
X1 =

, ____________________________________________________________ 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.

64a SEQUENCE LISTING
<110> Hybridon, Inc.
<120> Modulation of Immunostimulatory Properties of Oligonucleotide-Based Compounds By Optimal Presentation of 5' Ends <130> 15653-43CA FC/gc <140> 2,463,798 <141> 2002-10-24 <150> US 60/344,767 <151> 2001-10-24 <160> 115 <170> FastSEQ for Windows Version 4.0 <210> 1 <211> 16 <212> DNA
<213> Artificial Sequence <220>
<223> immunomer sequence <400> 1 gagaacgctc gacctt 16 <210> 2 <211> 16 <212> DNA
<213> Artificial Sequence <220>
<223> immunomer sequence <400> 2 ttccagctcg caagag 16 <210> 3 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> immunomer sequence <400> 3 ctatctgacg ttctctgt 18 64b <210> 4 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> immunomer sequence <221> modified_base <222> 5 <223> C3-alkyl linker <400> 4 ctatntgacg ttctctgt 18 <210> 5 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> immunomer sequence <221> modified_base <222> 9 <223> 50HdC
<400> 5 ctatctgayg ttctctgt 18 <210> 6 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> immunomer sequence <221> modified_base <222> 10 <223> 7-deaza-dG
<400> 6 ctatctgacr ttctctgt 18 <210> 7 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> immunomer sequence <221> modified_base 64c <222> 4 <223> C3-alkyl linker <221> modified_base <222> 9 <223> 50HdC
<400> 7 ctanctgayg ttctctgt 18 <210> 8 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> immunomer sequence <221> modified_base <222> 4 <223> C3-alkyl linker <221> modified_base <222> 10 <223> 7-deaza-dG
<400> 8 ctanctgacr ttctctgt 18 <210> 9 <211> 14 <212> DNA
<213> Artificial Sequence <220>
<223> immunomer sequence <400> 9 ctgacgttct ctgt 14 <210> 10 <211> 14 <212> DNA
<213> Artificial Sequence <220>
<223> immunomer sequence <221> modified_base <222> 5 <223> 50HdC
<400> 10 ctgaygttct ctgt 14 64d <210> 11 <211> 14 <212> DNA
<213> Artificial Sequence <220>
<223> immunomer sequence <221> modified_base <222> 6 <223> 7-deaza-dG
<400> 11 ctgacrttct ctgt 14 <210> 12 <211> 15 <212> DNA
<213> Artificial Sequence <220>
<223> immunomer sequence <221> modified_base <222> (1)...(2) <223> 1',2'-dideoxyriboside <400> 12 nntgacgttc tctgt 15 <210> 13 <211> 16 <212> DNA
<213> Artificial Sequence <220>
<223> immunomer sequence <221> modified_base <222> (1)...(3) <223> 1',2'-dideoxyriboside <400> 13 nnntgacgtt ctctgt 16 <210> 14 <211> 16 <212> DNA
<213> Artificial Sequence <220>
<223> immunomer sequence 64e <221> modified_base <222> (1)...(3) <223> 1',2'-dideoxyriboside <221> modified_base <222> 7 <223> 50HdC
<400> 14 nnntgaygtt ctctgt 16 <210> 15 <211> 16 <212> DNA
<213> Artificial Sequence <220>
<223> immunomer sequence <221> modified_base <222> (1)...(3) <223> 1',2'-dideoxyriboside <221> modified_base <222> 8 <223> 7-deaza-dG
<400> 15 nnntgacrtt ctctgt 16 <210> 16 <211> 11 <212> DNA
<213> Artificial Sequence <220>
<223> immunomer sequence <400> 16 tctgacgttc t 11 <210> 17 <211> 14 <212> DNA
<213> Artificial Sequence <220>
<223> immunomer sequence <221> modified_base <222> (1)...(3) <223> 1',2'-dideoxyriboside 64f <400> 17 nnntctgacg ttct 14 <210> 18 <211> 14 <212> DNA
<213> Artificial Sequence <220>
<223> immunomer sequence <221> modified base <222> (1)...(3) <223> 1',2'-dideoxyriboside <221> modified base <222> 9 <223> 50HdC
<400> 18 nnntctgayg ttct 14 <210> 19 <211> 14 <212> DNA
<213> Artificial Sequence <220>
<223> immunomer sequence <221> modified base <222> (1)...(3) <223> 1',2'-dideoxyriboside <221> modified_base <222> 10 <223> 7-deaza-dG
<400> 19 nnntctgacr ttct 14 <210> 20 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> immunomer sequence <400> 20 ctatctgtcg ttctctgt 18 64g <210> 21 <211> 11 <212> DNA
<213> Artificial Sequence <220>
<223> immunomer sequence <221> modified_base <222> 7 <223> 7-deazaguanosine <400> 21 tctgtcrttc t 11 <210> 22 <211> 11 <212> DNA
<213> Artificial Sequence <220>
<223> immunomer sequence <221> modified_base <222> 7 <223> deoxyarabinoguanosine <400> 22 tctgtcrttc t 11 <210> 23 <211> 11 <212> DNA
<213> Artificial Sequence <220>
<223> immunomer sequence <221> modified_base <222> 7 <223> arabinocytosine <221> modified_base <222> 8 <223> deoxyarabinoguanosine <400> 23 tctgtyrttc t 11 <210> 24 <211> 13 <212> DNA
<213> Artificial Sequence 64h <220>
<223> immunomer sequence <221> modified_base <222> (1)...(2) <223> propoxyphosphorothioate <221> modified_base <222> 9 <223> 7-deazaguanosine <400> 24 nntctgtcrt tct 13 <210> 25 <211> 14 <212> DNA
<213> Artificial Sequence <220>
<223> immunomer sequence <221> modified_base <222> 6 <223> dexoyarabinoguanosine <400> 25 ctgtcrttct ctgt 14 <210> 26 <211> 14 <212> DNA
<213> Artificial Sequence <220>
<223> immunomer sequence <221> modified_base <222> 5 <223> arabinocytosine <221> modified_base <222> 6 <223> deoxyarabinoguanosine <400> 26 ctgtyrttct ctgt 14 <210> 27 <211> 13 <212> DNA
<213> Artificial Sequence 64i <220>
=
<223> immunomer sequence <221> modified_base <222> (1)...(2) <223> propoxyphosphorothioate <221> modified_base <222> 9 <223> 7-deazaguanosine <400> 27 nntctgacrt tct 13 <210> 28 <211> 11 <212> DNA
<213> Artificial Sequence <220>
<223> immunomer sequence <221> modified_base <222> 7 <223> deoxyarabinoguanosine <400> 28 tctgacrttc t 11 <210> 29 <211> 11 <212> DNA
<213> Artificial Sequence <220>
<223> immunomer sequence <221> modified_base <222> 6 <223> arabinocytosine <221> modified_base <222> 7 <223> deoxyarabinoguanosine <400> 29 tctgayrttc t 11 <210> 30 <211> 14 <212> DNA
<213> Artificial Sequence 64j <220>
<223> immunomer sequence <221> modified_base <222> 5 <223> arabinocytosine <400> 30 ctgaygttct ctgt 14 <210> 31 <211> 14 <212> DNA
<213> Artificial Sequence <220>
<223> immunomer sequence <221> modified_base <222> 6 <223> deoxyarabinoguanosine <400> 31 ctgacrttct ctgt 14 <210> 32 <211> 14 <212> DNA
<213> Artificial Sequence <220>
<223> immunomer sequence <221> modified_base <222> 5 <223> arabinocytosine <221> modified_base <222> 6 <223> deoxyarabinoguanosine <400> 32 ctgayrttct ctgt 14 <210> 33 <211> 14 <212> DNA
<213> Artificial Sequence <220>
<223> immunomer sequence <400> 33 ctgtcgttct ctgt 14 64k <210> 34 <211> 12 <212> DNA
<213> Artificial Sequence <220>
<223> immunomer sequence <400> 34 ctgtcgttct ct 12 <210> 35 <211> 11 <212> DNA
<213> Artificial Sequence <220>
<223> immunomer sequence <400> 35 tctgtcgttc t 11 <210> 36 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> immunomer sequence <400> 36 ctcactttcg ttctctgt 18 <210> 37 <211> 14 <212> DNA
<213> Artificial Sequence <220>
<223> immunomer sequence <400> 37 ctttcgttct ctgt 14 <210> 38 <211> 12 <212> DNA
<213> Artificial Sequence <220>
<223> immunomer sequence <400> 38 ctttcgttct ct 12 <210> 39 <211> 11 <212> DNA
<213> Artificial Sequence <220>
<223> immunomer sequence <221> modified_base <222> 6 <223> 5-hydroxycytosine <400> 39 tctttygttc t 11 <210> 40 <211> 11 <212> DNA
<213> Artificial Sequence <220>
<223> immunomer sequence <221> modified_base <222> 7 <223> 7-deazaguanosine <400> 40 tctttcrttc t 11 <210> 41 <211> 11 <212> DNA
<213> Artificial Sequence <220>
<223> immunomer sequence <221> modified_base <222> 6 <223> 5-hydroxycytosine <400> 41 tctgtygttc t 11 <210> 42 <211> 11 <212> DNA
<213> Artificial Sequence 64m <220>
<223> immunomer sequence <221> modified_base <222> 7 <223> 7-deazaguanosine <400> 42 tctgtcrttc t 11 <210> 43 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> immunomer sequence <221> modified_base <222> 9 <223> 5-hydroxycytosine <400> 43 ctatctgayg ttctctgt 18 <210> 44 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> immunomer sequence <221> modified_base <222> 10 <223> 7-deazaguanosine <400> 44 ctatctgacr ttctctgt 18 <210> 45 <211> 14 <212> DNA
<213> Artificial Sequence <220>
<223> immunomer sequence <221> modified_base <222> 5 <223> 5-hydroxycytosine 64n <400> 45 ctgaygttct ctgt 14 <210> 46 <211> 14 <212> DNA
<213> Artificial Sequence <220>
<223> immunomer sequence <221> modified_base <222> 6 <223> 7-deazaguanosine <400> 46 ctgacrttct ctgt 14 <210> 47 <211> 11 <212> DNA
<213> Artificial Sequence <220>
<223> immunomer sequence <221> modified_base <222> 6 <223> 5-hydroxycytosine <400> 47 tctgaygttc t 11 <210> 48 <211> 11 <212> DNA
<213> Artificial Sequence <220>
<223> immunomer sequence <221> modified_base <222> 7 <223> 7-deazaguanosine <400> 48 tctgacrttc t 11 <210> 49 <211> 10 <212> DNA
<213> Artificial Sequence <220>
<223> immunomer sequence <400> 49 ctgacgttct 10 <210> 50 <211> 16 <212> DNA
<213> Artificial Sequence <220>
<223> immunomer sequence <221> modified_base <222> (1)...(2) <223> C3-linker <400> 50 nnctgacgtt ctctgt 16 <210> 51 <211> 11 <212> DNA
<213> Artificial Sequence <220>
<223> oligonucleotides in 10% Bovine Serum PBS solution <221> modified_base <222> 7 <223> dGdeaza <400> 51 tctgtcgttc t 11 <210> 52 <211> 16 <212> DNA
<213> Artificial Sequence <220>
<223> oligonucleotides in 10% Bovine Serum PBS solution <221> modified_base <222> (1)...(2) <223> C3-linker <400> 52 nnctgtcgtt ctctgt 16 <210> 53 <211> 15 64p <212> DNA
<213> Artificial Sequence <220>
<223> oligonucleotides in 10% Bovine Serum PBS solution <221> modified base <222> 1 <223> uridine <221> modified base <222> 2 <223> 21-0Me-ribonucleoside <400> 53 uctgtcgttc tctgt 15 <210> 54 <211> 19 <212> DNA
<213> Artificial Sequence <220>
<223> phosphorothioate CpG DNA sequence <400> 54 tccatgacgt tcctgatgc 19 <210> 55 <211> 17 <212> DNA
<213> Artificial Sequence <220>
<223> phosphorothioate CpG DNA sequence <400> 55 ccatgacgtt cctgatg 17 <210> 56 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> phosphorothioate CpG DNA sequence <400> 56 tccatgacgt tcctgatg 18 64q <210> 57 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> phosphorothioate CpG DNA sequence <400> 57 ccatgacgtt cctgatgc 18 <210> 58 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> CpG DNA sequence <400> 58 cctactagcg ttctcatc 18 <210> 59 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> CpG DNA sequence <221> misc_feature <222> 10 <223> alkyl linker 2 of Figure 14 <400> 59 cctactagcn ttctcatc 18 <210> 60 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> CpG DNA sequence <221> misc_feature <222> 7 <223> alkyl linker 2 of Figure 14 <400> 60 cctactngcg ttctcatc 18 64r <210> 61 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> CpG DNA sequence <221> misc_feature <222> 5 <223> alkyl linker 2 of Figure 14 <400> 61 cctantagcg ttctcatc 18 <210> 62 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> CpG DNA sequence <221> misc_feature <222> (4)...(5) <223> alkyl linker 2 of Figure 14 <400> 62 cctnntagcg ttctcatc 18 <210> 63 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> CpG DNA sequence <221> misc_feature <222> (1)...(2) <223> alkyl linker 2 of Figure 14 <400> 63 nntactagcg ttctcatc 18 <210> 64 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> CpG DNA sequence 64s <221> misc_feature <222> 12 <223> alkyl linker 2 of Figure 14 <400> 64 cctactagcg tnctcatc 18 <210> 65 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> CpG DNA sequence <221> misc_feature <222> 14 <223> alkyl linker 2 of Figure 14 <400> 65 cctactagcg ttcncatc 18 <210> 66 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> CpG DNA sequence <221> misc_feature <222> (14)...(15) <223> alkyl linker 2 of Figure 14 <400> 66 cctactagcg ttcnnatc 18 <210> 67 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> CpG DNA sequence <221> misc_feature <222> 4 <223> ethylene-glycol linker 6 of Figure 14 <400> 67 cctnctagcg ttctcatc 18 64t <210> 68 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> CpG DNA sequence <221> misc_feature <222> 14 <223> ethylene-glycol linker 6 of Figure 14 <400> 68 cctactagcg ttcncatc 18 <210> 69 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> CpG DNA sequence <221> misc_feature <222> 4 <223> ethylene-glycol linker 7 of Figure 14 <400> 69 cctnctagcg ttctcatc 18 <210> 70 <211> 19 <212> DNA
<213> Artificial Sequence <220>
<223> CpG DNA sequence <221> misc_feature <222> 14 <223> ethylene-glycol linker 7 of Figure 14 <400> 70 cctactagcg ttctncatc 19 <210> 71 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> CpG DNA sequence <221> misc_feature 64u <222> 5 <223> alkyl linker 1 of Figure 14 <400> 71 ctatntgacg ttctctgt 18 <210> 72 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> CpG DNA sequence <221> misc_feature <222> 4 <223> alkyl linker 1 of Figure 14 <400> 72 ctanctgacg ttctctgt 18 <210> 73 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> CpG DNA sequence <221> misc_feature <222> 8 <223> alkyl linker 2 of Figure 14 <400> 73 ctatctgncg ttctctgt 18 <210> 74 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> CpG DNA sequence <221> misc_feature <222> 6 <223> alkyl linker 2 of Figure 14 <400> 74 ctatcngacg ttctctgt 18 <210> 75 <211> 18 64v <212> DNA
<213> Artificial Sequence <220>
<223> CpG DNA sequence <221> misc_feature <222> 4 <223> alkyl linker 2 of Figure 14 <400> 75 ctanctgacg ttctctgt 18 <210> 76 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> CpG DNA sequence <221> misc_feature <222> (1)...(5) <223> alkyl linker 2 of Figure 14 <400> 76 nnnnntgacg ttctctgt 18 <210> 77 <211> 17 <212> DNA
<213> Artificial Sequence <220>
<223> CpG DNA sequence <221> misc_feature <222> (1)...(4) <223> alkyl linker 2 of Figure 14 <400> 77 nnnntgacgt tctctgt 17 <210> 78 <211> 16 <212> DNA
<213> Artificial Sequence <220>
<223> CpG DNA sequence <221> misc_feature <222> (1)...(3) <223> alkyl linker 2 of Figure 14 64w <400> 78 nnntgacgtt ctctgt 16 <210> 79 <211> 15 <212> DNA
<213> Artificial Sequence <220>
<223> CpG DNA sequence <221> misc_feature <222> (1)...(2) <223> alkyl linker 2 of Figure 14 <400> 79 nntgacgttc tctgt 15 <210> 80 <211> 14 <212> DNA
<213> Artificial Sequence <220>
<223> CpG DNA sequence <221> misc_feature <222> 1 <223> alkyl linker 2 of Figure 14 <400> 80 ntgacgttct ctgt 14 <210> 81 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> CpG DNA sequence <221> misc_feature <222> 5 <223> alkyl linker 3 of Figure 14 <400> 81 ctatntgacg ttctctgt 18 <210> 82 <211> 18 <212> DNA
<213> Artificial Sequence 64x <220>
<223> CpG DNA sequence <221> misc_feature <222> 4 <223> alkyl linker 3 of Figure 14 <400> 82 ctanctgacg ttctctgt 18 <210> 83 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> CpG DNA sequence <221> misc_feature <222> (4)...(5) <223> alkyl linker 3 of Figure 14 <400> 83 ctanntgacg ttctctgt 18 <210> 84 <211> 15 <212> DNA
<213> Artificial Sequence <220>
<223> CpG DNA sequence <221> misc_feature <222> (1)...(2) <223> alkyl linker 3 of Figure 14 <400> 84 nntgacgttc tctgt 15 <210> 85 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> CpG DNA sequence <221> misc_feature <222> 5 <223> alkyl linker 4 of Figure 14 <400> 85 ctatntgacg ttctctgt 18 64y <210> 86 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> CpG DNA sequence <221> misc_feature <222> 4 <223> alkyl linker 4 of Figure 14 <400> 86 ctanctgacg ttctctgt 18 <210> 87 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> CpG DNA sequence <221> misc_difference <222> (4)...(5) <223> alkyl linker 4 of Figure 14 <400> 87 ctanntgacg ttctctgt 18 <210> 88 <211> 15 <212> DNA -<213> Artificial Sequence <220>
<223> CpG DNA sequence <221> misc_feature <222> (1)...(2) <223> alkyl linker 4 of Figure 14 <400> 88 nntgacgttc tctgt 15 <210> 89 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> CpG DNA sequence <221> misc_feature 64z <222> 5 <223> alkyl linker 5 of Figure 14 <400> 89 ctatntgacg ttctctgt 18 <210> 90 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> CpG DNA sequence <221> misc_feature <222> 4 <223> alkyl linker 5 of Figure 14 <400> 90 ctanctgacg ttctctgt 18 <210> 91 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> CpG DNA sequence <221> misc_feature <222> (4)...(5) <223> alkyl linker 5 of Figure 14 <400> 91 ctanntgacg ttctctgt 18 <210> 92 <211> 15 <212> DNA
<213> Artificial Sequence <220>
<223> CpG DNA sequence <221> misc_feature <222> (1)...(2) <223> alkyl linker 5 of Figure 14 <400> 92 nntgacgttc tctgt 15 <210> 93 <211> 18 64aa <212> DNA
<213> Artificial Sequence <220>
<223> CpG DNA sequence <221> misc_feature <222> 4 <223> ethylene-glycol linker 6 of Figure 14 <400> 93 ctanctgacg ttctctgt 18 <210> 94 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> CpG DNA sequence <221> misc_difference <222> 14 <223> ethylene-glycol linker 6 of Figure 14 <400> 94 ctatctgacg ttcnctgt 18 <210> 95 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> CpG DNA sequence <221> misc_feature <222> 4 <223> ethylene-glycol linker 7 of Figure 14 <400> 95 ctanctgacg ttctctgt 18 <210> 96 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> CpG DNA sequence <221> misc_feature <222> 14 <223> ethylene-glycol linker 7 of Figure 14 64bb <400> 96 ctatctgacg ttcnctgt 18 <210> 97 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> CpG DNA sequence <221> misc_difference <222> 8 <223> branched alkyl linker 8 of Figure 14 <400> 97 ctatctgncg ttctctgt 18 <210> 98 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> CpG DNA sequence <221> miso_feature <222> 7 <223> branched alkyl linker 8 of Figure 14 <400> 98 ctatctnacg ttctctgt 18 <210> 99 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> CpG DNA sequence <221> misc_feature <222> 6 <223> branched alkyl linker 8 of Figure 14 <400> 99 ctatcngacg ttctctgt 18 <210> 100 <211> 18 <212> DNA
<213> Artificial Sequence 64cc <220>
<223> CpG DNA sequence <221> misc_feature <222> 5 <223> branched alkyl linker 8 of Figure 14 <400> 100 ctatntgacg ttctctgt 18 <210> 101 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> CpG DNA sequence <221> misc_feature <222> 4 <223> branched alkyl linker 8 of Figure 14 <400> 101 ctanctgacg ttctctgt 18 <210> 102 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> CpG DNA sequence <221> misc_feature <222> 11 <223> branched alkyl linker 8 of Figure 14 <400> 102 ctatctgacg ntctctgt 18 <210> 103 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> CpG DNA sequence <221> misc_feature <222> 12 <223> branched alkyl linker 8 of Figure 14 <400> 103 ctatctgacg tnctctgt 18 64dd <210> 104 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> CpG DNA sequence <221> misc_feature <222> 13 <223> branched alkyl llinker 8 of Figure 14 <400> 104 ctatctgacg ttntctgt 18 <210> 105 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> CpG DNA sequence <221> misc_feature <222> 14 <223> branched alkyl linker 8 of Figure 14 <400> 105 ctatctgacg ttcnctgt 18 <210> 106 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> CpG DNA sequence <221> misc_feature <222> 8 <223> branched alkyl linker 9 of Figure 14 <400> 106 ctatctgncg ttctctgt 18 <210> 107 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> CpG DNA sequence <221> misc_feature 64ee <222> 7 <223> branched alkyl linker 9 of Figure 14 <400> 107 ctatctnacg ttctctgt 18 <210> 108 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> CpG DNA sequence <221> misc_feature <222> 4 <223> branched alkyl linker 9 of Figure 14 <400> 108 ctanctgacg ttctctgt 18 <210> 109 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> CpG DNA sequence <221> misc_difference <222> 12 <223> branched alkyl linker 9 of Figure 14 <400> 109 ctatctgacg tnctctgt 18 <210> 110 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> CpG DNA sequence <221> misc_feature <222> 14 <223> branched alkyl linker 9 of Figure 14 <400> 110 ctatctgacg ttcnctgt 18 64ff <210> 111 <211> 29 <212> DNA
<213> Artificial Sequence <220>
<223> immunomer sequence <221> modified base <222> 15 <223> glyceryldiphosphodiester <400> 111 ctgacgttct ctgtntgtct cttgcagtc 29 <210> 112 <211> 33 <212> DNA
<213> Artificial Sequence <220>
<223> immunomer sequence <221> modified base <222> 1,2,32,33 <223> propoxyphosphodiester <221> modified_base <222> 17 <223> glyceryldiphosphodiester <400> 112 yyctgacgtt ctctgtntgt ctcttgcagt cyy 33 <210> 113 <211> 11 <212> DNA
<213> Artificial Sequence <220>
<223> immunomer sequence <400> 113 tctgacgttc t 11 <210> 114 <211> 11 <212> DNA
<213> Artificial Sequence <220>
<223> immunomer sequence 64gg <400> 114 tctgtcgttc t 11 <210> 115 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> immunomer sequence <400> 115 tgtctcttgc agtctatc 18

Claims (24)

65

1. An immunomer comprising at least two oligonucleotides linked at their 3 ends or internucleoside linkages or a functionalized nucleobase or sugar to a non-nucleotidic linker, wherein at least one of the oligonucleotides is an immunostimulatory oligonucleotide having an accessible 5' end and comprising an immunostimulatory dinucleotide selected from the group consisting of C*pG, CpG*, and C*pG*, wherein C is cytidine or 2'-deoxycytidine, 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 non-natural 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 non-natural purine nucleoside, and p is an internucleoside linkage selected from the group consisting of phosphodiester, phosphorothioate, and phosphorodithioate.

2. The immunomer according to claim 1, wherein the at least two oligonucleotides are each from 3 to 35 nucleotides in length.

3. The immunomer according to claim 1 having the structure 5'-TCTGTCRTTCT-3'-X-3'-TCTTRCTGTCT-5', wherein X is glycerol and R is 2'-deoxy-7-deazaguanosine.

4. The immunomer according to claim 1, wherein the non-naturally occurring pyrimidine has the structure (I):
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, excluding bromine;
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 S is a pentose or hexose sugar ring or a non-naturally occurring sugar.

5. The immunomer according to claim 4, wherein the sugar ring is derivatized with a phosphate moiety, modified phosphate moiety, or other non-nucleotidic linker moiety for linking the pyrimidine nucleoside to another nucleoside or nucleoside analog.

6. The immunomer according to claim 4, wherein the hydrogen bond donors are selected from the group consisting of -NH-, -NH2, -SH and -OH.

7. The immunomer according to claim 4, wherein the hydrogen bond acceptors are selected from the group consisting of C=O, C=S, and the ring nitrogen atoms of an aromatic heterocycle.

8. The immunomer according to claim 4, wherein the non-naturally occurring pyrimidine base is selected from the group consisting of 5-hydroxycytosine, 5-hydroxymethylcytosine, N4-alkylcytosine, N4-ethylcytosine, and 4-thiouracil.

9. The immunomer according to claim 4, wherein the non-naturally occurring sugar is selected from arabinose and arabinose analogs.

10. The immunomer according to claim 1, wherein the purine nucleoside has the structure (H):
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 selected from the group consisting of C, 0, N and S;
and S' is a pentose or hexose sugar ring, or a non-naturally occurring sugar.

11. The immunomer according to claim 10, wherein the sugar ring is derivatized with a phosphate moiety, modified phosphate moiety, or other linker moiety for linking the pyrimidine nucleoside to another nucleoside or nucleoside analog.

12. The immunomer according to claim 10, wherein the hydrogen bond donors are selected from the group consisting of -NH-, -NH2, -SH and -OH.

13. The immunomer according to claim 10, wherein the hydrogen bond acceptors are selected from the group consisting of C=O, C=S, -N= and the ring nitrogen atoms of an aromatic heterocycle.

14. The immunomer according to claim 10, wherein the non-naturally occurring purine is 6-thioguanine or 7-deazaguanine.

15. The immunomer according to claim 1, wherein the non-nucleotidic linker is selected from the group consisting of a linker from 2 angstroms to 200 angstroms in length, a metal, a soluble or insoluble biodegradable polymer bead, an organic moiety having functional groups that permit attachment to the 3'-terminal nucleoside of the oligonucleotide, a biomolecule, a cyclic or acyclic small molecule, amino acid, carbohydrate, cyclodextrin, adamantane, cholesterol, hapten, antibiotic, 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 6, a derivative of 1,3-diamino-2-hydroxypropane and 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.

16. An immunomer conjugate comprising an immunomer according to claim 1 and an antigen conjugated to the immunomer at a position other than the accessible 5' end.

17. The immunomer according to claim 1, wherein the immunomodulatory dinucleotide is C*pG*, wherein C* is selected from the group consisting of arabinocytosine and 2'-deoxy-2-substituted arabincytosine and wherein G* is selected from the group consisting of arabinoguanosine, 2'-deoxy-2'-substituted arabinguanosine, 2'-deoxy-7-deazaguanosine, 2'-deoxy-6-thioguanosine and 2'-deoxyinosine.

18. The immunomer according to claim 1, wherein the immunostimulatory dinucleotide is CpG*, wherein C is cytidine or 2'-deoxycytidine and wherein G*
is selected from the group consisting of arabinoguanosine, 2'-deoxy-2'-substituted arabinguanosine, 2'-deoxy-7-deazaguanosine, 2'-deoxy-6-thioguanosine, and 2'-deoxyinosine.

19. The immunomer according to claim 1, wherein the immunostimulatory dinucleotide is C*pG, wherein C* is selected from the group consisting of arabinocytosine and 2'-cleoxy-2-substituted arabincytosine and wherein G is guanosine or 2'-deoxyguanosine.

20. A pharmaceutical formulation comprising an immunomer according to any one of claims 1 to 15, 17 or 18 or the immunomer conjugate of claim 16 and a physiologically acceptable carrier.

21. The pharmaceutical formulation according to claim 20, further comprising an additional component selected from the group consisting of vaccines, antibodies, cytotoxic agents, allergens, antibiotics, chemotherapeutic agents, antisense oligonucleotides, peptides, proteins, gene therapy vectors and adjuvants.

22. The pharmaceutical formulation according to claim 21, wherein vaccines are DNA vaccines.

23. Use of an immunomer according to any one of claims 1 to 15, 17 or 18 or the immunomer conjugate of claim 16 in the manufacture of a medicament for the generation of an immune response in a vertebrate.

24. Use of an immunomer according to any one of claims 1 to 15, 17 or 18 or the immunomer conjugate of claim 16 for the generation of an immune response in a vertebrate.