Kandimalla, Ekambar R. (Southboro, MA, US)
Zhu, Fu-gang (Dorchester, MA, US)
2. An immunomodulatory composition comprising the immunomodulatory oligonucleotide immunomer according to claim 1; and further comprising a co-stimulatory molecule selected from the group consisting of cytokines, chemokines, protein ligands, a trans-activating factors, peptides, and peptides comprising a modified amino acid.
3. The immunomodulatory composition of claim 2, wherein the co-stimulatory molecule is conjugated to the immunomodulatory oligonucleotide immunomer.
4. The immunomodulatory composition of claim 2, further comprising an adjuvant.
5. The immunomodulatory composition of claim 2, further comprising a pharmaceutically acceptable carrier.
6. An immunomodulatory composition comprising the immunomodulatory oligonucleotide immunomer according to claim 1; and further comprising an antigen.
7. The immunomodulatory composition of claim 6, wherein the antigen is selected from the group consisting of peptides, glycoproteins, lipoproteins polysaccharides, and lipids.
8. The immunomodulatory composition according to claim 6, wherein the antigen is an allergen.
9. The immunomodulatory composition of claim 6, further comprising an adjuvant.
10. The immunomodulatory composition of claim 6, further comprising a pharmaceutically acceptable carrier.
11. An immunostimulatory oligonucleotide immunomer comprising at least two oligonucleotides linked by a non-nucleotide linker and having more than one 5′ end, wherein at least of the oligonucleotides has the sequence of SEQ ID NO 72.
12. An immunomodulatory composition comprising the immunomodulatory oligonucleotide immunomer according to claim 11; and further comprising a co-stimulatory molecule selected from the group consisting of cytokines, chemokines, protein ligands, a trans-activating factors, peptides, and peptides comprising a modified amino acid.
13. The immunomodulatory composition of claim 12, wherein the co-stimulatory molecule is conjugated to the immunomodulatory oligonucleotide immunomer.
14. The immunomodulatory composition of claim 12, further comprising an adjuvant.
15. The immunomodulatory composition of claim 12, further comprising a pharmaceutically acceptable carrier.
16. An immunomodulatory composition comprising the immunomodulatory oligonucleotide immunomer according to claim 11; and further comprising an antigen.
17. The immunomodulatory composition of claim 16, wherein the antigen is selected from the group consisting of peptides, glycoproteins, lipoproteins polysaccharides, and lipids.
18. The immunomodulatory composition according to claim 16, wherein the antigen is an allergen.
19. The immunomodulatory composition of claim 16, further comprising an adjuvant.
20. The immunomodulatory composition of claim 16, further comprising a pharmaceutically acceptable carrier.
21. A method for therapeutically treating a patient having airway inflammation, inflammatory disorders, allergy, or asthma, such method comprising administering to the patient an immunomer.
22. The method according to claim 21, wherein the immunomer comprises at least two oligonucleotides linked by a non-nucleotidic linker and having more than one 5′ end, wherein at least one of the oligonucleotides is an immunostimulatory oligonucleotide having an accessible 5′ end and comprises an immunostimulatory dinucleotide.
23. The method according to claim 21, wherein 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, 1-(2′-deoxy-β-D-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine, 2′-deoxy-2′-substitutedarabinocytidine, 2′-O-substitutedarabinocytidine, 2′-deoxy-5-hydroxycytidine, 2′-deoxy-N-4-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′-deoxyinosine, 2′-deoxy-2′substituted-arabinoguanosine, 2′-O-substituted-arabinoguanosine or other non-natural purine nucleoside.
24. The method according to claim 21, wherein the immunomer comprises at least two oligonucleotides linked by a non-nucleotide linker and having more than one 5′ end, wherein at least of the oligonucleotides has the sequence of SEQ ID NO 76.
25. The method according to claim 21, wherein the immunomer comprises at least two oligonucleotides linked by a non-nucleotide linker and having more than one 5′ end, wherein at least of the oligonucleotides has the sequence of SEQ ID NO 72.
26. The method according to claim 21, wherein the immunomer comprises at least two oligonucleotides linked by a non-nucleotide linker and having more than one 5′ end, wherein at least of the oligonucleotides has the sequence of SEQ ID NO 18.
27. The method according to claim 21, wherein the immunomer comprises at least two oligonucleotides linked by a non-nucleotide linker and having more than one 5′ end, wherein at least of the oligonucleotides has the sequence of SEQ ID NO 77.
28. The method according to claim 21, further comprising administering an antigen associated with said disease or disorder.
29. The method according to claim 21, wherein the immunomer or the antigen, or both, are linked to an immunogenic protein or non-immunogenic protein.
30. The method according to claim 21, further comprising administering an adjuvant.
31. A method for modulating an immune response in a patient having airway inflammation, inflammatory disorders, allergy, or asthma comprising administering to the patient an immunomer.
32. The method according to claim 31, wherein the immune response is a Th1 immune response.
33. The method according to claim 31, wherein the immune response is a Th2 immune response.
34. The method according to claim 31, wherein the immunomer comprises at least two oligonucleotides linked by a non-nucleotidic linker and having more than one 5′ end, wherein at least one of the oligonucleotides is an immunostimulatory oligonucleotide having an accessible 5′ end and comprises an immunostimulatory dinucleotide.
35. The method according to claim 31, wherein 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, 1-(2′-deoxy-β-D-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine, 2′-deoxy-2′-substitutedarabinocytidine, 2′-O-substitutedarabinocytidine, 2′-deoxy-5-hydroxycytidine, 2′-deoxy-N-4-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′-deoxyinosine, 2′-deoxy-2′substituted-arabinoguanosine, 2′-O-substituted-arabinoguanosine.
36. The method according to claim 31, wherein the immunomer comprises at least two oligonucleotides linked by a non-nucleotide linker and having more than one 5′ end, wherein at least of the oligonucleotides has the sequence of SEQ ID NO 76.
37. The method according to claim 31, wherein the immunomer comprises at least two oligonucleotides linked by a non-nucleotide linker and having more than one 5′ end, wherein at least of the oligonucleotides has the sequence of SEQ ID NO 72.
38. The method according to claim 31, wherein the immunomer comprises at least two oligonucleotides linked by a non-nucleotide linker and having more than one 5′ end, wherein at least of the oligonucleotides has the sequence of SEQ ID NO 18.
39. The method according to claim 31, wherein the immunomer comprises at least two oligonucleotides linked by a non-nucleotide linker and having more than one 5′ end, wherein at least of the oligonucleotides has the sequence of SEQ ID NO 77.
40. The method according to claim 31, further comprising administering an antigen associated with said disease or disorder.
41. The method according to claim 31, wherein the immunomer or the antigen, or both, are linked to an immunogenic protein or non-immunogenic protein.
42. The method according to claim 31, further comprising administering an adjuvant.
RELATED APPLICATION
This application claims the benefit of U.S. Provisional Application No. 60/528,277, filed Dec. 5, 2003, which is incorporated by reference in its entirety.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to immunology and immunotherapy applications using oligonucleotides as immunostimulatory agents.
2. Summary of the Related Art
Oligonucleotides have become indispensable tools in modern molecular biology, being used in a wide variety of techniques, ranging from diagnostic probing methods to PCR to antisense inhibition of gene expression and immunotherapy applications. This widespread use of oligonucleotides has led to an increasing demand for rapid, inexpensive and efficient methods for synthesizing oligonucleotides.
The synthesis of oligonucleotides for antisense and diagnostic applications can now be routinely accomplished. See, e.g., Methods in Molecular Biology , Vol. 20: Protocols for Oligonucleotides and Analogs pp. 165-189 (S. Agrawal, ed., Humana Press, 1993); Oligonucleotides and Analogues, A Practical Approach , pp. 87-108 (F. Eckstein, ed., 1991); and Uhlmann and Peyman, supra; Agrawal and lyer, Curr. Op. in Biotech. 6:12 (1995); and Antisense Research and Applications (Crooke and Lebleu, eds., CRC Press, Boca Raton, 1993). Early synthetic approaches included phosphodiester and phosphotriester chemistries. For example, Khorana et al., J. Molec. Biol. 72:209 (1972) discloses phosphodiester chemistry for oligonucleotide synthesis. Reese, Tetrahedron Lett. 34:3143-3179 (1978), discloses phosphotriester chemistry for synthesis of oligonucleotides and polynucleotides. These early approaches have largely given way to the more efficient phosphoramidite and H-phosphonate approaches to synthesis. For example, Beaucage and Caruthers, Tetrahedron Lett. 22:1859-1862 (1981), discloses the use of deoxyribonucleoside phosphoramidites in polynucleotide synthesis. Agrawal and Zamecnik, U.S. Pat. No. 5,149,798 (1992), discloses optimized synthesis of oligonucleotides by the H-phosphonate approach. Both of these modem 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. Sci . (USA) 85:7079-7083 (1988), discloses synthesis of oligonucleotide phosphoramidates and phosphorothioates using H-phosphonate chemistry.
More recently, several researchers have demonstrated the validity of the use of oligonucleotides as immunostimulatory agents in immunotherapy applications. The observation that phosphodiester and phosphorothioate oligonucleotides can induce immune stimulation has created interest in developing this side effect as a therapeutic tool. These efforts have focused on phosphorothioate oligonucleotides containing the dinucleotide natural CpG. Kuramoto et al., Jpn. J Cancer Res. 83:1128-1131 (1992) teaches that phosphodiester oligonucleotides containing a palindrome that includes a CpG dinucleotide can induce interferon-alpha and gamma synthesis and enhance natural killer activity. Krieg et al., Nature 371:546-549 (1995) discloses that phosphorothioate CpG-containing oligonucleotides are immunostimulatory. Liang et al., J. Clin. Invest. 98:1119-1129 (1996) discloses that such oligonucleotides activate human B cells. Moldoveanu et al., Vaccine 16:1216-124 (1998) teaches that CpG-containing phosphorothioate oligonucleotides enhance immune response against influenza virus. McCluskie and Davis, J. Immunol. 161:4463-4466 (1998) teaches that CpG-containing oligonucleotides act as potent adjuvants, enhancing immune response against hepatitis B surface antigen.
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 al., Antisense Nucleic Acid Drug Dev . (1997) 7:495-502; Zhao et al., Bioorg. Med Chem. Lett . (1999) 9:3453-3458; Zhao et al., Bioorg. Med Chem. Lett . (2000) 10:1051-1054; Yu et al., Bioorg. Med. Chem. Lett . (2000) 10:2585-2588; Yu et al., Bioorg. Med. Chem. Lett . (2001) 11:2263-2267; and Kandimalla et al., Bioorg. Med. Chem . (2001) 9:807-813.
One response that CpG-containing oligonucleotides may modulate is asthma. An allergic asthma response is characterized by activation of T-helper type 2 (Th2) lymphocytes. The responses induced by Th2 lymphocytes play a major role in the pathogenesis and propagation of allergic inflammation in asthma. The Th2 cytokine IL-5 increases the generation and survival of eosinophils, leading to increased airway eosinophilia. Other Th2 cytokines (IL-4, IL-9, and IL-13) also play critical roles in allergic inflammation by inducing production of allergen-specific IgE, mast-cell proliferation, endothelial-cell adhesion-molecule expression, and airway hyper-responsiveness. Corticosteroids are currently the only widely used treatment for allergic asthma. Steroid treatment is effective only in minimizing the manifestations of inflammation, however, and does not cure the disease. Continuous therapy is required to prevent the progression of allergic asthma.
These reports make clear that there remains a need to be able to enhance and modify the immune response caused by immunostimulatory oligonucleotides.
BRIEF SUMMARY OF THE INVENTION
The invention provides methods for enhancing and modifying 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 immunostimulatory oligonucleotide immunomer comprises the sequence of SEQ ID NO 76.
In a second aspect, the invention provides an immunomodulatory composition comprising the immunomodulatory oligonucleotide immunomer comprising the sequence of SEQ ID NO 76; and further comprising a co-stimulatory molecule selected from the group consisting of cytokines, chemokines, protein ligands, a trans-activating factors, peptides, and peptides comprising a modified amino acid. In this aspect of the invention, the co-stimulatory molecule is, optionally, conjugated to the immunomodulatory oligonucleotide immunomer, and the immunomodulatory composition further, optionally, comprises an adjuvant and/or a pharmaceutically acceptable carrier.
In a third aspect, the invention provides an immunomodulatory composition comprising the immunomodulatory oligonucleotide immunomer comprising the sequence of SEQ ID NO 76; and further comprising an antigen, wherein the antigen is selected from the group consisting of peptides, glycoproteins, lipoproteins, polysaccharides, and lipids, or wherein the antigen is an allergen. In this aspect of the invention, the immunomodulatory composition further, optionally, comprises an adjuvant and/or a pharmaceutically acceptable carrier.
In another embodiment, the immunostimulatory oligonucleotide immunomer comprises the sequence of SEQ ID NO 72.
In a fourth aspect, the invention provides an immunomodulatory composition comprising the immunomodulatory oligonucleotide immunomer comprising the sequence of SEQ ID NO 72; and further comprising a co-stimulatory molecule selected from the group consisting of cytokines, chemokines, protein ligands, a trans-activating factors, peptides, and peptides comprising a modified amino acid. In this aspect of the invention, the co-stimulatory molecule is, optionally, conjugated to the immunomodulatory oligonucleotide immunomer, and the immunomodulatory composition further, optionally, comprises an adjuvant and/or a pharmaceutically acceptable carrier.
In a fifth aspect, the invention provides an immunomodulatory composition comprising the immunomodulatory oligonucleotide immunomer comprising the sequence of SEQ ID NO 72; and further comprising an antigen, wherein the antigen is selected from the group consisting of peptides, glycoproteins, lipoproteins, polysaccharides, and lipids, or wherein the antigen is an allergen. In this aspect of the invention, the immunomodulatory composition further, optionally, comprises an adjuvant and/or a pharmaceutically acceptable carrier.
In another embodiment, the invention provides a method for therapeutically treating a patient having airway inflammation, inflammatory disorders, allergy, or asthma, such method comprising administering to the patient an immunomer.
In a sixth aspect, the invention provides a method for therapeutically treating a patient wherein the immunomer comprises at least two oligonucleotides linked by a non-nucleotidic linker and having more than one 5′ end, wherein at least one of the oligonucleotides is an immunostimulatory oligonucleotide having an accessible 5′ end and comprises an immunostimulatory dinucleotide. 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, 1-(2′-deoxy-β-D-ribofuranosyl)-2-oxo-7-deaza-8-methyl-pur ine, 2′-deoxy-2′-substitutedarabinocytidine, 2′-O-substitutedarabinocytidine, 2′-deoxy-5-hydroxycytidine, 2′-deoxy-N-4-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′-deoxyinosine, 2′-deoxy-2′substituted-arabinoguanosine, 2′-O-substituted-arabinoguanosine.
In a seventh aspect, the invention provides a method for therapeutically treating a patient, wherein the immunomer comprises the sequence of SEQ ID NO 76, or the sequence of SEQ ID NO 72, or the sequence of SEQ ID NO 18, or the sequence of SEQ ID NO 73.
In an eighth aspect, the invention provides a method for therapeutically treating a patient further comprising administering an antigen associated with said disease or disorder, wherein the immunomer or the antigen, or both, are linked to an immunogenic protein or non-immunogenic protein, and/or further comprising administering an adjuvant.
In another embodiment, the invention provides a method for modulating an immune response in a patient having airway inflammation, inflammatory disorders, allergy, or asthma comprising administering to the patient an immunomer, wherein the immune response is a Th1 and/or a Th2 immune response.
In a ninth aspect, the invention provides a method for modulating an immune response in a patient, wherein the immunomer comprises at least two oligonucleotides linked by a non-nucleotidic linker and having more than one 5′ end, wherein at least one of the oligonucleotides is an immunostimulatory oligonucleotide having an accessible 5′ end and comprises an immunostimulatory dinucleotide. 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, 1-(2′-deoxy-β-D-ribofuranosyl)-2-oxo-7-deaza-8-methyl-pur ine, 2′-deoxy-2′-substitutedarabinocytidine, 2′-O-substitutedarabinocytidine, 2′-deoxy-5-hydroxycytidine, 2′-deoxy-N-4-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′-deoxyinosine, 2′-deoxy-2′substituted-arabinoguanosine, 2′-O-substituted-arabinoguanosine.
In a tenth aspect the invention provides a method for modulating an immune response in a patient wherein the immunomer comprises the sequence of SEQ ID NO 76, or the sequence of SEQ ID NO 72, or the sequence of SEQ ID NO 18, or the sequence of SEQ ID NO 73.
In an eleventh aspect, the invention provides a method for therapeutically treating a patient further comprising administering an antigen associated with said disease or disorder, wherein the immunomer or the antigen, or both, are linked to an immunogenic protein or non-immunogenic protein, and/or further comprising administering an adjuvant.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic representation of representative immunomers of the invention.
FIG. 2 depicts several representative immunomers of the invention (All oligonucleotides shown are disclosed as SEQ ID NO: 2).
FIG. 3 depicts a group of representative small molecule linkers suitable for linear synthesis of immumomers of the invention.
FIG. 4 depicts a group of representative small molecule linkers suitable for parallel synthesis of immunomers of the invention.
FIG. 5 is a synthetic scheme for the linear synthesis of immunomers of the invention. DMTr=4,4′-dimethoxytrityl; CE=cyanoethyl.
FIG. 6 is a synthetic scheme for the parallel synthesis of immunomers of the invention. DMTr=4,4′-dimethoxytrityl; CE=cyanoethyl.
FIG. 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 1, and that Immunomer 3, which does not have accessible 5′-ends, has equal or weaker ability to produce immune stimulation compared with oligo 1.
FIG. 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.
FIG. 7C is a graphic representation of the induction of IL-10 by Immunomers 1-3 (top to bottom, respectively) in BALB/c mouse spleen cell cultures.
FIG. 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.
FIG. 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.
FIG. 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.
FIG. 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.
FIG. 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.
FIG. 10A is a graphic representation of the induction of cell proliferation by Immunomers 14, 15, and 16 in BALB/c mouse spleen cell cultures.
FIG. 10B is a graphic representation of the induction of cell proliferation by IL-12 by different concentrations of Immunomers 14 and 16 in BALB/c mouse spleen cell cultures.
FIG. 10C 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.
FIG. 11A 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.
FIG. 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.
FIG. 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.
FIG. 12 is a graphic representation of BALB/c mouse spleen enlargement using oligonucleotides 4 and immunomers 14, 23, and 24.
FIG. 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.
FIG. 14 shows the chemical substitutions used in Example 13.
FIG. 15 shows cytokine profiles obtained using the modified oligonucleotides of Example 13.
FIG. 16 shows relative cytokine induction for glycerol linkers compared with amino linkers.
FIG. 17 shows relative cytokine induction for various linkers and linker combinations.
FIGS. 18 A-E shows relative nuclease resistance for various PS and PO immunomers and oligonucleotides.
FIG. 19 shows relative cytokine induction for PO immunomers compared with PS immunomers in BALB/c mouse spleen cell cultures.
FIG. 20 shows relative cytokine induction for PO immunomers compared with PS immunomers in C3H/Hej mouse spleen cell cultures.
FIG. 21 shows relative cytokine induction for PO immunomers compared with PS immunomers in C3H/Hej mouse spleen cell cultures at high concentrations of immunomers.
FIG. 22 shows sequences and chemical modifications of immunomers (IMOs) (SEQ ID NOS 76, 72, 18, 77, 73, and 8, respectively in order of appearance).
FIGS. 23A and 23B show IMO prevention of OVA-induced Th2 immune response in mice as demonstrated by cytokine responses in spleen cell cultures.
FIGS. 24A and 24B show IMO prevention of OVA-induced Th2 immune response in mice as demonstrated by serum antibody responses.
FIG. 25 shows dose-dependent effects of IMOs 1 and 2 on established OVA-induced allergic asthma in mice. Cytokine secretion is in spleen cell cultures in OVA-recall response. Both IMOs 1 and 2 significantly inhibited IL-5 secretion in a dose-dependent fashion. IL-13 was significantly inhibited by both IMO compounds. Both IMO compounds induced dose-dependent IFN-g secretion.
FIG. 26 shows serum antigen-specific and total antibodies. IMOs 1 and 2 produced dose-dependent reduction of OVA-specific IgE and increase of OVA-specific IgG2a.
FIG. 27 shows the effect of a single high dose vs multiple lower doses of IMO compounds on local and systemic Th1 cytokine levels in naive mice. A single dose of 100 mg induced higher levels of systemic cytokine responses. On the contrary three smaller doses (3×33 mg) induced higher local (BALF) cytokine responses.
FIG. 28 shows dose-dependent effects of low multiple administrations of IMO compounds on local and systemic cytokine levels in naïve mice. IMO 1 increased local (BALF) cytokine levels but not systemic cytokine levels in mice when administered multiple times in small doses. This effect is dose-dependent.
FIG. 29 compares the effects of IMO and corticosteriod in vitro. Both IMO 1 and budesonide suppressed OVA-induced Th2 cytokine secretion. However, only IMO 1 showed strong Th1 cytokine induction.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The invention relates to the therapeutic use of oligonucleotides as immunostimulatory agents for immunotherapy applications. The issued patents, patent applications, and references that are cited herein are hereby incorporated by reference to the same extent as if each was specifically and individually indicated to be incorporated by reference. In the event of inconsistencies between any teaching of any reference cited herein and the present specification, the latter shall prevail for purposes of the invention.
The invention provides methods for enhancing and modifying 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. Allergic asthma is a particularly preferred condition for treatment by the present methods and compounds. Thus, the invention further provides compounds having optimal levels of immunostimulatory 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 internucleoside linkage or a functional ized 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(1)-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′-O-substitutedarabinose or hexose sugar group. The nucleoside residues can be coupled to each other by any of the numerous known internucleoside linkages. Such internucleoside linkages include, without limitation, phosphodiester, phosphorothioate, phosphorodithioate, alkylphosphonate, alkylphosphonothioate, phosphotriester, phosphoramidate, siloxane, carbonate, carboalkoxy, acetamidate, carbamate, morpholino, borano, thioether, bridged phosphoramidate, bridged methylene phosphonate, bridged phosphorothioate, and sulfone internucleoside linkages. The term “oligonucleotide” also encompasses polynucleosides having one or more stereospecific internucleoside linkage (e.g., (R P )- or (S P )-phosphorothioate, alkylphosphonate, or phosphotriester linkages). As used herein, the terms “oligonucleotide” and “dinucleotide” are expressly intended to include polynucleosides and dinucleosides having any such internucleoside linkage, whether or not the linkage comprises 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.
As used herein, the term “secondary structure” refers to intramolecular and intermolecular hydrogen bonding. Intramolecular hydrogen bonding results in the formation of a stem-loop structure. Intermolecular hydrogen bonding results in the formation of a duplexed nucleic acid molecule.
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′-O-substituted ribonucleoside. Preferably, such substitution is with a lower alkyl group containing 1-6 saturated or unsaturated carbon atoms, or with an aryl group having 6-10 carbon atoms, wherein such alkyl, or aryl group may be unsubstituted or may be substituted, e.g., with halo, hydroxy, trifluoromethyl, cyano, nitro, acyl, acyloxy, alkoxy, carboxyl, carboalkoxy, or amino groups. Examples of such 2′-O-substituted ribonucleosides include, without limitation 2′-O-methylribonucleosides and 2′-O-methoxyethylribonucleosides.
The term “2′-substituted ribonucleoside” also includes ribonucleosides in which the 2′-hydroxyl group is replaced with a lower alkyl group containing 1-6 saturated or unsaturated carbon atoms, or with an amino or halo group. Examples of such 2′-substituted ribonucleosides include, without limitation, 2′-amino, 2′-fluoro, 2′-allyl, and 2′-propargyl ribonucleosides.
The term “oligonucleotide” includes hybrid and chimeric oligonucleotides. A “chimeric oligonucleotide” is an oligonucleotide having more than one type of internucleoside linkage. One preferred example of such a chimeric oligonucleotide is a chimeric oligonucleotide comprising a phosphorothioate, phosphodiester or phosphorodithioate region and non-ionic linkages such as alkylphosphonate or alkylphosphonothioate linkages (see e.g., Pederson et al. U.S. Pat. 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. Pat. Nos. 5,652,355, 6,683,167, 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 Nov. 5, 1998; WO 01/12804, published Feb. 22, 2001; WO 01/55370, published Aug. 2, 2001; PCT/US01/13682, filed Apr. 30, 2001; and PCT/US01/30137, filed Sep. 26, 2001. Preferably, the immunostimulatory oligonucleotide comprises at least one phosphodiester, phosphorothioate, or phosphordithioate internucleoside linkage.
In some embodiments, the immunostimulatory oligonucleotide comprises an immunostimulatory dinucleotide of formula 5′-Pyr-Pur-3′, wherein Pyr is a natural or synthetic pyrimidine nucleoside and Pur is a natural or synthetic purine nucleoside. As used herein, the term “pyrimidine nucleoside” refers to a 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 (I):
(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—, —NH 2 , —SH and —OH. Preferred hydrogen bond acceptors include, without limitation, C═O, C═S, and the ring nitrogen atoms of an aromatic heterocycle, e.g., N3 of cytosine.
In some embodiments, the base moiety in (I) is a non-naturally occurring pyrimidine base. Examples of preferred non-naturally occurring pyrimidine bases include, without limitation, 5-hydroxycytosine, 5-hydroxymethylcytosine, N4-alkylcytosine, preferably N4-ethylcytosine, and 4-thiouracil. However, in some embodiments 5-bromocytosine is specifically excluded.
In some embodiments, the sugar moiety S′ in (1) 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 (II):
-
- (ii) 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, O, 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—, —NH 2 , —SH and —OH. Preferred hydrogen bond acceptors include, without limitation, C═O, C═S, —NO 2 and the ring nitrogen atoms of an aromatic heterocycle, e.g., N1 of guanine.
In some embodiments, the base moiety in (II) is a non-naturally occurring purine base. Examples of preferred non-naturally occurring purine bases include, without limitation, 6-thioguanine and 7-deazaguanine. In some embodiments, the sugar moiety S′ in (II) is a naturally occurring sugar moiety, as described above for structure (I).
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, 1-(2′-deoxy-β-D-ribofuranosyl)-2-oxo-7-deaza-8-methyl-pur ine, arabinocytidine, 2′-deoxythymidine, 2′-deoxy-2′-substitutedarabinocytidine, 2′-O-substitutedarabinocytidine, 2′-deoxy-5-hydroxycytidine, 2′-deoxy-N-4-alkyl-cytidine, 2′-deoxy-4-thiouridine or other non-natural pyrimidine nucleoside or rarely occurring 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 or rarely occurring 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 (III):
5′-Nn-N1-Y-Z-N1-Nn-3′ (III)
-
- wherein:
- Y is cytidine, 2′deoxythymidine, 2′ deoxycytidine arabinocytidine, 2′-deoxy-2′-substitutedarabinocytidine, 2′-deoxythymidine, 2′-O-substitutedarabinocytidine, 2′-deoxy-5-hydroxycytidine, 2′-deoxy-N-4-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′-O-substituted-arabinoguanosine, 2′deoxyinosine, or other non-natural purine nucleoside;
- N1, at each occurrence, is preferably a naturally occurring or a synthetic nucleoside or an immunostimulatory moiety selected from the group consisting of abasic nucleosides, arabinonucleosides, 2′-deoxyuridine, α-deoxyribonucleosides, β-L-deoxyribonucleosides, and nucleosides linked by a phosphodiester or modified internucleoside linkage to the adjacent nucleoside on the 3′ side, the modified internucleotide linkage being selected from, without limitation, a linker having a length of from about 2 angstroms to about 200 angstroms, C2-C18 alkyl linker, poly(ethylene glycol) linker, 2-aminobutyl-1,3-propanediol linker, glyceryl linker, 2′-5′ internucleoside linkage, and phosphorothioate, phosphorodithioate, or methylphosphonate internucleoside linkage;
- 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, α-deoxyribonucleosides, 2′-O-substituted ribonucleosides, and nucleosides linked by a modified internucleoside linkage to the adjacent nucleoside on the 3′ side, the modified internucleotide linkage preferably being selected from the group consisting of amino linker, 2′-5′ internucleoside linkage, and methylphosphonate internucleoside linkage;
- provided that at least one N1 or Nn is an immunostimulatory moiety;
- wherein n is a number from 0 to 30; and
- wherein the 3′end, an internucleoside linker, or a derivatized nucleobase or sugar is linked directly or via a non-nucleotidic linker to another oligonucleotide, which may or may not be immunostimulatory.
In some preferred embodiments, YZ is arabinocytidine or 2′-deoxy-2′-substituted arabinocytidine and arabinoguanosine or 2′deoxy-2′-substituted arabinoguanosine. Preferred immunostimulatory moieties include modifications in the phosphate backbones, including, without limitation, methylphosphonates, methylphosphonothioates, phosphotriesters, phosphothiotriesters, phosphorothioates, phosphorodithioates, triester prodrugs, sulfones, sulfonamides, sulfamates, formacetal, N-methylhydroxylamine, carbonate, carbamate, morpholino, boranophosphonate, phosphoramidates, especially primary amino-phosphoramidates, N3 phosphoramidates and N5 phosphoramidates, and stereospecific linkages (e.g., (R P )- or (S P )-phosphorothioate, alkylphosphonate, or phosphotriester linkages).
Preferred immunostimulatory moieties according to the invention further include nucleosides having sugar modifications, including, without limitation, 2′-substituted pentose sugars including, without limitation, 2′-O-methylribose, 2′-O-methoxyethylribose, 2′-O-propargylribose, and 2′-deoxy-2′-fluororibose; 3′-substituted pentose sugars, including, without limitation, 3′-O-methylribose; 1′,2′-dideoxyribose; arabinose; substituted arabinose sugars, including, without limitation, 1′-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′-O-substituted ribonucleoside, the immunostimulatory moiety is attached to the adjacent nucleoside by way of a 2′-5′ internucleoside 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 —O—(CH 2 —CH 2 —O—) n (n=1-9). Some other functionalized alkyl linkers are peptides or amino acids.
Preferred immunostimulatory moieties according to the invention further include DNA isoforms, including, without limitation, P-L-deoxyribonucleosides and a-deoxyribonucleosides. Preferred immunostimulatory moieties according to the invention incorporate 3′ modifications, and further include nucleosides having unnatural internucleoside linkage positions, including, without limitation, 2′-5′,2′-2′, 3′-3′ and 5′-5′ linkages.
Preferred immunostimulatory moieties according to the invention further include nucleosides having modified heterocyclic bases, including, without limitation, 5-hydroxycytosine, 5-hydroxymethylcytosine, N4-alkylcytosine, preferably N4-ethylcytosine, 4-thiouracil, 6-thioguanine, 7-deazaguanine, inosine, nitropyrrole, C5-propynylpyrimidine, and diaminopurines, including, without limitation, 2,6-diaminopurine.
By way of specific illustration and not by way of limitation, for example, in the immunostimulatory domain of structure (III), a methylphosphonate internucleoside linkage at position 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 X1 is an immunostimulatory moiety, and a β-L-deoxyribonucleoside at position X1 is an immunostimulatory moiety. See Table 1 below for representative positions and structures of immunostimulatory moieties. It is to be understood that reference to a linker as the immunostimulatory moiety at a specified position means that the nucleoside residue at that position is substituted at its 3′-hydroxyl with the indicated linker, thereby creating a modified internucleoside linkage between that nucleoside residue and the adjacent nucleoside on the 3′ side. Similarly, reference to a modified internucleoside linkage as the immunostimulatory moiety at a specified position means that the nucleoside residue at that position is linked to the adjacent nucleoside on the 3′ side by way of the recited linkage.
| TABLE 1 | |
| Position | TYPICAL IMMUNOSTIMULATORY MOIETIES |
| N1 | Naturally-occurring nucleosides, abasic nucleoside, |
| arabinonucleoside, 2′-deoxyuridine, β-L- | |
| deoxyribonucleoside C2-C18 alkyl linker, poly(ethylene glycol) | |
| linkage, 2-aminobutyl-1,3-propanediol linker | |
| (amino linker), 2′-5′ internucleoside linkage, methylphosphonate | |
| internucleoside linkage | |
| Nn | Naturally-occurring nucleosides, abasic nucleoside, |
| arabinonucleosides, 2′-deoxyuridine, 2′-O-substituted | |
| ribonucleoside, 2′-5′ internucleoside linkage, | |
| methylphosphonate internucleoside linkage, provided that N1 | |
| 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 —O—(CH 2 CH 2 —O) n —, wherein n is 3. The term “Spacer 18” refers to a poly(ethylene glycol) linker of formula —O—(CH 2 CH 2 —O) n —, wherein n is 6. As used herein, the term “C2-C18 alkyl linker refers to a linker of formula —O—(CH 2 ) q —O—, where q is an integer from 2 to 18. Accordingly, the terms “C3-linker” and “C3-alkyl linker” refer to a linker of formula —O—(CH 2 ) 3 —O—. For each of Spacer 9, Spacer 18, and C2-C18 alkyl linker, the linker is connected to the adjacent nucleosides by way of phosphodiester, phosphorothioate, or phosphorodithioate linkages.
| TABLE 2 | |
| Position | TYPICAL IMMUNOSTIMULATORY MOIETY |
| 5′ N2 | Naturally-occurring nucleosides, 2-aminobutyl-1,3-propanediol linker |
| 5′ N1 | Naturally-occurring nucleosides, β-L-deoxyribonucleoside, C2-C18 alkyl |
| linker, poly(ethylene glycol), abasic linker, 2-aminobutyl-1,3-propanediol | |
| linker | |
| 3′ N1 | Naturally-occurring nucleosides, 1′,2′-dideoxyribose, 2′-O-methyl- |
| ribonucleoside, C2-C18 alkyl linker, Spacer 9, Spacer 18 | |
| 3′ N2 | Naturally-occurring nucleosides, 1′,2′-dideoxyribose, 3′- |
| deoxyribonucleoside, β-L-deoxyribonucleoside, 2′-O-propargyl- | |
| ribonucleoside, C2-C18 alkyl linker, Spacer 9, Spacer 18, | |
| methylphosphonate internucleoside linkage | |
| 3′ N 3 | Naturally-occurring nucleosides, 1′,2′-dideoxyribose, C2-C18 alkyl |
| linker, Spacer 9, Spacer 18, methylphosphonate internucleoside linkage, | |
| 2′-5′ internucleoside linkage, d(G)n, polyI-polydC | |
| 3′N 2 + 3′N 3 | 1′,2′-dideoxyribose, β-L-deoxyribonucleoside, C2-C18 alkyl linker, |
| d(G)n, polyI-polydC | |
| 3′N3 + 3′ N 4 | 2′-O-methoxyethyl-ribonucleoside, methylphosphonate internucleoside |
| linkage, d(G)n, polyI-polydC | |
| 3′N5 + 3′ N 6 | 1′,2′-dideoxyribose, C2-C18 alkyl linker, d(G)n, polyI-polydC |
| 5′N1 + 3′ N 3 | 1′,2′-dideoxyribose, d(G)n, polyI-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 | methylphosphonate internucleoside linkage |
| 5′ N1 | methylphosphonate internucleoside linkage |
| 3′ N1 | 1′,2′-dideoxyribose, methylphosphonate internucleoside |
| linkage, 2′-O-methyl | |
| 3′ N2 | 1′,2′-dideoxyribose, β-L-deoxyribonucleoside, C2-C18 |
| alkyl linker, Spacer 9, Spacer 18, 2-aminobutyl-1,3- | |
| propanediol linker, methylphosphonate internucleoside | |
| linkage, 2′-O-methyl | |
| 3′ N3 | 3′-deoxyribonucleoside. 3′-O-substituted ribonucleoside, |
| 2′-O-propargyl-ribonucleoside | |
| 3′ N2 + 3′ N3 | 1′,2′-dideoxyribose, β-L-deoxyribonucleoside |
The immunomers according to the invention comprise at least two oligonucleotides linked at their 3′ ends or internucleoside linkage or a functionalized nucleobase or sugar via a non-nucleotidic linker. For purposes of the invention, a “non-nucleotidic linker” is any moiety that can be linked to the oligonucleotides by way of covalent or non-covalent linkages. Preferably such linker is from about 2 angstroms to about 200 angstroms in length. Several examples of preferred linkers are set forth below. Non-covalent linkages include, but are not limited to, electrostatic interaction, hydrophobic interactions, π-stacking interactions, and hydrogen bonding. The term “non-nucleotidic linker” is not meant to refer to an internucleoside linkage, as described above, e.g., a phosphodiester, phosphorothioate, or phosphorodithioate functional group, that directly connects the 3′-hydroxyl groups of two nucleosides. For purposes of this invention, such a direct 3′-3′ linkage is considered to be a “nucleotidic linkage.”
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 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 FIG. 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—(CH 2 ) o —CH(OH)—(CH 2 ) p —OH, wherein o and p independently are integers from 1 to about 6, from 1 to about 4, or from 1 to about 3. In some other embodiments, the small molecule linker is a derivative of 1,3-diamino-2-hydroxypropane. Some such derivatives have the formula HO—(CH 2 ) n —C(O)NH—CH 2 —CH(OH)—CH 2 —NHC(O)—(CH 2 ) n —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 FIG. 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 FIGS. 5 and 6, and further described in the Examples. In some embodiments, the immunomers are synthesized by a linear synthesis approach (see FIG. 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 FIG. 6). A solid support attached linker can be used for parallel synthesis, as is described in U.S. Pat. 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: (1) parallel synthesis permits the incorporation of identical monomeric units; (2) unlike in linear synthesis, both (or all) the monomeric units are synthesized at the same time, thereby the number of synthetic steps and the time required for the synthesis is the same as that of a monomeric unit; and (3) the reduction in synthetic steps improves purity and yield of the final 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.
Tables 4A and 4B show representative immunomers according to the invention. Additional immunomers are found described in the Examples.
| TABLE 4 | ||
| Examples of Immunomer Sequences | ||
| OLIGO/ | SEQ | |
| IMMUNOMER # | ID NO. | Sequences and Modification (5′-3′) |
| 1 | 1 | 5′-GAGAACGCTCGACCTT-3′ |
| 2 | 1 | 5′-GAGAACGCTCGACCTT-3′-3′-TTCCAGCTCGCAAGAG-5′ |
| 3 | 1 | 3′-TTCCAGCTCGCAAGAG-5′-5′-GCGAACGCTCGACCTT-3′ |
| 4 | 2 | 5′-CTATCTGACGTTCTCTGT-3′ |
| 5 | 78 and 3 |
|
| 6 | 78 and 3 |
|
| 7 | 2 |
|
| 8 | 2 |
|
| 9 | 4 |
|
| 10 | 5 |
|
| 11 | 79 and 6 |
|
| 12 | 79 and 7 |
|
| 13 | 8 | 5′-CTGACGTTCTCTGT-3′ |
| 14 | 8 |
|
| 15 | 6 |
|
| 16 | 7 |
|
| 17 | 9 | 5′-XXTGACGTTCTCTGT-3′ |
| 18 | 10 |
|
| 19 | 11 |
|
| 20 | 12 |
|
| 21 | 13 | 5′-TCTGACGTTCT-3′ |
| 22 | 14 |
|
| 23 | 15 |
|
| 24 | 16 |
|
|
| ||
|
| ||
| L = C3-alkyl linker; X = 1′,2′-dideoxyriboside; Y = 50H dC; R = 7-deaza-dG | ||
| TABLE 4B | ||
| SEQ ID | ||
| NO. | Sequences and Modification (5′-3′) | |
| 76 | 5′-CTGTC R TTCTC- X -CTCTT R CTGTC-5′ | |
| 72 | 5′-TC R TC R TTG- X -GTT R CT R CT-5′ | |
| 18 | 5′-TCTGTC R TTCT- X -TCTT R CTGTCT-5′ | |
| 77 | 5′-TCTGT R′ GTTCT- X -TCTTG R′ TGTCT-5′ | |
| 73 | 5′-TC R TC R TTG- X -GTT R CT R CT-5′ | |
| 8 | 5′- YY CTGACGTTCTCTGT- X -TGTCTCTTGCAGTC YY -5′ | |
| X = glycerol linker; | ||
| R = Arabinoguanosine; | ||
| R = 2′-deoxy-7′-deazaguanosine, | ||
| R′ = 1-(2′-deoxy-b-D-ribofuranosyl)-2-oxo-7-deaza-8-methyl-puri ne; | ||
| Y = C3-linker | ||
In another aspect the invention provides an immunostimulatory nucleic acid comprising at least two oligonucleotides, wherein the immunostimulatory nucleic acid has a secondary structure. In this aspect, immunostimulatory nucleic acid comprises a structure as detailed in formula (I).
Domain A-Domain B-Domain C (I)
Domains may be from about 2 to about 12 nucleotides in length. Domain A may be 5′-3′ or 3′-5′ or 2′-5′ DNA, RNA, RNA-DNA, DNA-RNA having or not having a palindromic or self-complementary domain containing or not containing at least one dinucleotide selected from the group consisting of CpG, YpG, YpR, CpR, R*pG and R*pR, wherein C is cytidine or 2′deoxycitidine, G is guanosine or 2′ deoxyguanosine, Y is cytidine, 2′-deoxythymidine, 2′-deoxycytidine, 2′ dideoxy-5-halocytosine, 2′ dideoxy-5-nitrocytosine, arabinocytidine, 2′-deoxy-2′-substitutedarabinocytidine, 2′-O-substituted arabinocytidine, 2′-deoxy-5-hydroxycytidine, 2′-deoxy-N-4-alkyl-cytidine, 2′-deoxy-4-thiouridine, other non-natural pyrimidine nucleosides, R* is 1-(2′-deoxy-β-D-ribofuranosyl)-2-oxo-7-deaza-8-methyl-pur ine; R is guanosine or 2′ deoxyguanosine, 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. In certain preferred embodiments, the immunostimulatory dinucleotide is not CpG.
In certain embodiments, Domain A will have more than one dinucleotide selected from the group consisting of CpG, YpG, YpR, CpR, R*pG and R*pR located in the 5′-end of the Domain A oligonucleotide.
Domain B is a linker joining Domains A and C that may be a 3′-‘5′ linkage, a 2′-5′ linkage, a 3′-3′ linkage, a phosphate group, a nucleoside, or a non-nucleoside linker that may be aliphatic, aromatic, aryl, cyclic, chiral, achiral, a peptide, a carbohydrate, a lipid, a fatty acid, mono- tri- or hexapolyethylene glycol, or a heterocyclic moiety.
Domain C may be 5′-3′ or 3′-5′,2′-5′ DNA, RNA, RNA-DNA, DNA-RNA, Poly 1-Poly C having or not having a palindromic or self-complementary sequence, which can or cannot have a dinucleotide selected from the group consisting of CpG, YpG, YpR, CpR, R*pG and R*pR, wherein C is cytidine or 2′deoxycitidine, G is guanosine or 2′ deoxyguanosine, Y is cytidine, 2′-deoxythymidine, 2′-deoxycytidine, 2′ dideoxy-5-halocytosine, 2′ dideoxy-5-halocytosine, arabinocytidine, 2′-deoxy-2′-substitutedarabinocytidine, 2′-O-substituted arabinocytidine, 2′-deoxy-5-hydroxycytidine, 2′-deoxy-N-4-alkyl-cytidine, 2′-deoxy-4-thiouridine, other non-natural pyrimidine nucleosides, R* is 1-(2′-deoxy-1-D-ribofuranosyl)-2-oxo-7-deaza-8-methyl-puri ne; R is guanosine or 2′ deoxyguanosine, 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. In certain preferred embodiments, the immunostimulatory dinucleotide is not CpG. In some embodiments, Domain B is preferably a non-nucloetidic linker connecting oligonucleotides of Domain A and Domain C, which are referred to as “immunomers.” In certain preferred embodiments, Domain C does not have the dinucleotide CpG, YpG, YpR, CpR, R*pG or R*pR.
In some embodiments, the oligonucleotides of contained in formula (I) are from about 12 to about 50 nucleotides in length. In certain embodiments the oligonucleotides of contained in formula (I) are from about 12 to about 26 nucleotides in length.
By way of non-limiting example, in certain embodiments of this aspect the immunostimulatory nucleic acid will have a structure as detailed in formula (II).
As one skilled in the art would recognize, there is a secondary structure element in the 3′ end of the molecule in the form of an intramolecular stem-loop.
By way of non-limiting example, in certain embodiments of this aspect the immunostimulatory nucleic acid will have a structure as detailed in formula (III)
The structure depicted in formula (III) is referred to herein as a “terminal dimer,” since the 3′ ends of the two molecules are blocked because the sequences of the two 3′ ends are complementary allowing for intermolecular hydrogen bonding. In addition, domains A and A′ may or may not be identical, domains B and B′ may or may not be identical and domains C and C′ may or may not be identical.
By way of non-limiting example, in certain embodiments of this aspect the immunostimulatory nucleic acid will have a structure as detailed in formula (IV).
As would be recognized by one skilled in the art, the 3′ end of the depicted molecule has a secondary structure because the complementary sequence of its 3′ end is hydrogen bonded to this region. In certain embodiments, a molecule such as a ligand may be attached to the 3′-end in order to facilitate cellular uptake or improve stability of the molecule.
Non-limiting examples of some nucleic acid molecules of the invention are presented in Tables 24B-C and 25B-C (see below).
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, or wherein the antigen is an allergen. For purposes of the invention, the term “associated with” means that the antigen is present when the pathogen, cancer, auto-immune disorder, food allergy, skin 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, skin 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. Nonlimiting examples of such operative associations 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-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 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 in pharmaceutical formulations. It will be understood that the characteristics of the carrier, excipient, or diluent will depend on the route of administration for a particular application. The preparation of pharmaceutically acceptable formulations containing these materials is described in, e.g., Remington's Pharmaceutical Sciences, 18th Edition, ed. A. Gennaro, Mack Publishing Co., Easton, Pa., 1990.
In a fourth aspect, the invention provides methods for generating an immune response in a vertebrate, such methods comprising administering to the vertebrate an 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, intramuscular, intrperitonal, subcutaneous, intradermal, 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 or nonimmunogenic carrier protein. Any of the plethora of adjuvants may be used including, without limitation, Freund's complete adjuvant, Freund's incomplete adjuvant, KLH, monophosphoryl lipid A (MPL), alum, and saponins, including QS-21, imiquimod, R848, or combinations thereof.
Toll-like receptors (TLRs) function as sensors of infection and induce the activation of innate and adaptive immune responses. TLRs recognize a wide variety of ligands, called pathogen-associated molecular patterns (PAMPs). Upon recognizing conserved pathogen-associated molecular products, TLRs activate host defense responses through their intracellular signalling domain, the Toll/interleukin-1 receptor (TIR) domain, and the downstream adaptor protein MyD88. Dendritic cells and macrophages normally respond to Toll-like receptor (TLR) ligands and cytokines (for example, interleukin-1β; IL-6 and tumour necrosis factor, TNF), which they also produce; natural killer (NK) cells and T cells are also involved in the pro-inflammatory circuit. After TLR stimulation by bacterial compounds, innate immune cells release a range of cytokines. Some examples of TLR ligands include, but are not limited to, lipoproteins; peptidoglycan, zymosan (TLR2), double-stranded RNA, polyl:polyC (TLR3), lipopolysaccharide, heat shock proteins, taxol (TLR4), flagellin (TLR5), and imidazoquinolines-R848, resiquimod, imiquimod; ssRNA (TLR7/8).
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 atopic dermatitis, allergic rhinitis (also known as hay fever), allergic conjunctivitis, urticaria (also known as hives), respiratory allergies and allergic reactions to other substances such as latex, medications and insect stings or problems commonly resulting from allergic rhinitis-sinusitis and otitis media. The term “airway inflammation” includes, without limitation, asthma. Specific examples of asthma include, but are not limited to, allergic asthma, non-allergic asthma, exercised-induced asthma, occupational asthma, and nocturnal asthma.
Allergic asthma is characterized by airway obstruction associated with allergies and triggered by substances called allergens. Triggers of allergic asthma include, but are not limited to, airborne pollens, molds, animal dander, house dust mites and cockroach droppings. Non-allergic asthma is caused by viral infections, certain medications or irritants found in the air, which aggravate the nose and airways. Triggers of non-allergic asthma include, but are not limited to, airborne particles (e.g., coal, chalk dust), air pollutants (e.g., tobacco smoke, wood smoke), strong odors or sprays (e.g., perfumes, household cleaners, cooking fumes, paints or varnishes), viral infections (e.g., colds, viral pneumonia, sinusitis, nasal polyps), aspirin-sensitivity, and gastroesophageal reflux disease (GERD). Exercise-induced asthma (EIA) is triggered by vigorous physical activity. Symptoms of EIA occur to varying degrees in a majority of asthma sufferers and are likely to be triggered as a result of breathing cold, dry air while exercising. Triggers of EIA include, but are not limited to, breathing airborne pollens during exercise, breathing air pollutants during exercise, exercising with viral respiratory tract infections and exercising in cold, dry air. Occupational asthma is directly related to inhaling irritants and other potentially harmful substances found in the workplace. Triggers of occupational asthma include, but are not limited to, fumes, chemicals, gases, resins, metals, dusts, vapors and insecticides.
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.
Without wishing to be bound to any particular theory, decreased exposure to bacteria may be partially responsible for the increased incidence of, severity of, and mortality due to allergic diseases such as asthma, atopic dermatitis, and rhinitis in the developed countries. This hypothesis is supported by evidence that bacterial infections or products can inhibit the development of allergic disorders in experimental animal models and clinical studies. Bacterial DNA or synthetic oligodeoxynucleotides containing unmethylated CpG dinucleotides in certain sequence contexts (CpG DNA) potently stimulate innate immune responses and thereby acquired immunity. The immune response to CpG DNA includes activation of innate immune cells, proliferation of B cells, induction of Th1 cytokine secretion, and production of immunoglobulins (Ig). The activation of immune cells by CpG DNA occurs via Toll-like receptor 9 (TLR9), a molecular pattern recognition receptor. CpG DNAs induce strong Th1-dominant immune responses characterized by secretion of IL-12 and IFN-γ. Immunomers (IMOs) alone or as allergen conjugates decrease production of IL-4, IL-5, and IgE and reduce eosinophilia in mouse models of allergic asthma. IMO compounds also effectively reverse established atopic eosinophilic airway disease by converting a Th2 response to a Th1 response.
OVA with alum is commonly used to establish a Th2-dominant immune response in various mouse and rat models. The Th2 immune response includes increased IL-4, IL-5, and IL-13 production, elevated serum levels of total and antigen-specific IgE, IgG 1, and lower levels of IgG2a. IMO compounds prevent and reverse established Th2-dominant immune responses in mice. The co-administration of IMO compounds with OVA/alum to mice reduces IL-4, IL-5, and IL-13 production and induces IFN-γ production in spleen-cell cultures subjected to antigen re-stimulation. Furthermore, IMO compounds inhibit antigen-specific and total IgE and enhance IgG2a production in these mice.
Injection of OVA/alum and IMO compounds induces a lymphocyte antigen-recall response (Th1-type) in mice characterized by low levels of Th2-associated cytokines, IgE and IgG1, and high levels of Th 1-associated cytokines and IgG2a. Co-administration of IMO compounds with other kinds of antigens, such as S. masoni egg and hen egg lysozyme, also result in reversal of the Th2-response to a Th1-dominant response in in vitro and in vivo studies. As described herein, IMO compounds effectively prevent development of a Th2 immune response and allow a strong Th1 response.
While Th2 cytokines trigger an Ig isotype switch towards production of IgE and IgG 1, the Th I cytokine IFN-γ induces production of IgG2a by B-lymphocytes. Mice injected with OVA/alum and IMO compounds produce lower levels of IL-4, IL-5, and IL-13 and higher levels of IFN-γ, accompanied by lower IgE and IgG1 and higher IgG2a levels, than mice injected with OVA/alum alone. This suggests the existence of a close link between Th1-cytokine induction and immunoglobulin isotype switch in mice that receive antigen and IMO compounds.
Serum antigen-specific and total IgE levels are significantly lower in mice receiving OVA/alum and IMO compounds than in mice receiving OVA/alum alone. In contrast, OVA-specific IgG1 levels are insignificantly changed and total IgG1 levels are only slightly decreased compared with mice injected with OVA/alum alone (data not shown). The different response may result from different mechanisms involved in the control of IgE and IgG1 class switch, though both isotypes are influenced by IL-4 and IL-13. For example, IL-6 promotes B lymphocytes to synthesize IgG1 in the presence of IL-4.
In any of the methods according to 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 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 an agent in any order, including simultaneous administration, as well as any temporally spaced order, for example, from sequentially with one immediately following the other to up to several days apart. Such combination treatment may also include more than a single administration of the immunomer, and independently the agent. The administration of the immunomer and agent may be by the same or different routes.
In any of the methods according to the invention, the agent useful for treating the disease or condition includes, but is not limited to, antigen, allegen, or co-stimulatory molecules such as cytokines, chemokines, protein ligands, trans-activating factors, peptides and peptides comprising modified amino acids. Additionally, the agent can include DNA vectors encoding for antigen or allegen.
The invention provides a kit comprising a immunostimulatory oligonucleotides and/or immunomers, the latter comprising at least two oligonucleotides linked together, such that the immunomer has more than one accessible 5′ end, wherein at least one of the oligonucleotides is an immunostimulatory oligonucleotide. In another aspect, the kit comprises an immunostimulatory oligonucleotide and/or immunostimulatory oligonucleotide conjugate and/or immunomer or immunomer conjugate according to the invention and a physiologically acceptable carrier. The kit will generally also include a set of instructions for use.
The examples below are intended to further illustrate certain preferred embodiments of the invention, and are not intended to limit the scope of the invention.
EXAMPLES
Example 1
Synthesis of Oligonucleotides Containing Immunomodulatory Moieties
Oligonucleotides were synthesized on a 1 μmol scale using an automated DNA synthesizer (Expedite 8909; PerSeptive Biosystems, Framingham, Mass.), following the linear synthesis or parallel synthesis procedures outlined in FIGS. 5 and 6.
Deoxyribonucleoside phosphoramidites were obtained from Applied Biosystems (Foster City, Calif.). 1′,2′-dideoxyribose phosphoramidite, propyl-1-phosphoramidite, 2-deoxyuridine phosphoramidite, 1,3-bis-[5-(4,4′-dimethoxytrityl)pentylamidyl]-2-propanol phosphoramidite and methyl phosponamidite were obtained from Glen Research (Sterling, Va.). β-L-2′-deoxyribonucleoside phosphoramidite, α-2′-deoxyribonucleoside phosphoramidite, mono-DMT-glycerol phosphoramidite and di-DMT-glycerol phosphoramidite were obtained from ChemGenes (Ashland, Mass.). (4-Aminobutyl)-1,3-propanediol phosphoramidite was obtained from Clontech (Palo Alto, Calif.). 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, Mass.) (Noronha et al. (2000) Biochem., 39:7050-7062).
All nucleoside phosphoramidites were characterized by 31 P and 1 H 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 et al., Biochem Pharma 51:173-182 (1996)). The results are shown in FIG. 8A. These results demonstrate that at the higher concentrations, Immunomer 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, Conn.) intraperitoneally. The mice were sacrificed 72 hours after oligonucleotide administration, and spleens were harvested and weighed. The results are shown in FIG. 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
Cytokine 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 PharMingen, San Diego, Calif. ELISA plates (Costar) were incubated with appropriate antibodies at 5 μg/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 minutes. Cell culture supernatants and cytokine standards were appropriately diluted with PBS/10% FBS, added to the plates in triplicate, and incubated at 25° C. for 2 hours. Plates were overlaid with 1 μg/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, Blue™ (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 HDI Spectrophotometer (Bio-Tek Instruments). The results are shown in Table 5A below.
Human peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood of healthy volunteers by Ficoll-Paque density gradient centrifugation (Histopaque-1077, Sigma, St. Louis, Mo.). Briefly, heparinized blood was layered onto the Histopaque-1077 (equal volume) in a conical centrifuge and centrifuged at 400×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×g for 10 minutes. The resulting cell 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 (100 U/ml). Cells were cultured in 24 well plates for different time periods at 1×10 6 cells/ml/well in the presence or absence of oligonucleotides. At the end of the incubation period, supernatants were harvested and stored frozen at −70° C. until assayed for various cytokines including IL-6 (BD Pharmingen, San Diego, Calif.), IL-10 (BD Pharmingen), IL-12 (BioSource International, Camarillo, Calif.), IFN-α (BioSource International) and -γ (BD Pharmingen) and TNF-α (BD Pharmingen) by sandwich ELISA. The results are shown in Tables 5 and 5A 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, IFN-gamma and TNF-α in the cell culture supernatants were calculated from the standard curve constructed under the same experimental conditions for IL-10, IFN-gamma and TNF-α, respectively.
| TABLE 5 | ||||||
| Immunomer Structure and Immunostimulatory Activity in Human PBMC Cultures | ||||||
| Oligo Length/ | IL-12 (pg/mL) | IL-6 (pg/mL) | ||||
| Oligo No. | Sequences and Modification (5′-3′) | or Each Chain | D1 | D2 | D1 | D2 |
| 25 | 5′-CTATCTGTCGTTCTCTGT-3′ | 18mer (PS) | 184 | 332 | 3077 | 5369 |
| SEQ ID NO: 17 | ||||||
| 26 SEQ ID NO: 18 |
| 11mer (PS) | 237 | 352 | 3724 | 4892 |
| Oligo Length/ | IL-10 (pg/mL) | ILN-γ (pg/mL) | ||||
| Oligo No. | Sequences and Modification (5′-3′) | or Each Chain | D1 | D2 | D1 | D2 |
| 25 | 5′-CTATCTGTCGTTCTCTGT-3′ | 18mer (PS) | 37 | 88 | 125 | 84 |
| SEQ ID NO: 17 | ||||||
| 26 SEQ ID NO: 18 |
| 11mer (PS) | 48 | 139 | 251 | 40 |
| Oligo Length/ | TNF-α (pg/mL) | |||||
| Oligo No. | Sequences and Modification (5′ | |||||