HK1143741B - Rna sequence motifs in the context of defined internucleotide linkages inducing specific immune modulatory profiles - Google Patents

Description

RNA sequence motifs inducing specific immunomodulatory properties in a defined internucleotide linkage environment

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority from U.S. provisional application No. 60/964,448 entitled "sequence motifs inducing specific immunoregulatory conditions in the context of defined internucleotide linkages" filed on 8/13/2007 according to 35u.s.c. § 119(e), the entire contents of which are incorporated herein by reference.

Background

Toll-like receptors (TLRs) are a highly conserved family of Pattern Recognition Receptor (PRR) polypeptides that recognize pathogen-associated molecular patterns (PAMPs) and play a crucial role in innate immunity in mammals. At present, at least 10 family members have been identified, designated TLR1 to TLR 10. The cytoplasmic domain of various TLRs is characterized by the Toll-interleukin 1 receptor (TIR) domain (Medzhitov R et al, (1998) Mol Cell 2: 253-8). The recognition of microbial invasion by TLRs triggers activation of signaling cascades that are retained in evolution in drosophila and mammals. The adapter protein MyD88 containing a TIR domain has been reported to associate with TLRs and bind leukocytesInterleukin 1 receptor-related kinase (IRAK) and Tumor Necrosis Factor (TNF) receptor-related factor 6(TRAF6) are recruited to TLRs. It is believed that the MyD 88-dependent signaling pathway leads to NF-B transcription factors and c-Jun NH₂Terminal kinases (Jnk) mitogens activate the activation of protein kinases (MAPKs), which are key steps in immune activation and the production of inflammatory cytokines. For a review see Aderem A et al (2000) Nature 406: 782-87, and Akira S et al (2004) Nat RevImmunol 4: 499-511.

A number of specific TLR ligands have been identified. Ligands for TLR2 include peptidoglycans and lipopeptides (Yoshimura A et al (1999) J Immunol 163: 1-5; Yoshimura A et al (1999) JImmunol 163: 1-5; Alipirantis AO et al (1999) Science 285: 736-9). Lipoglycan (LPS) is a ligand for TLR4 (Poltorak A et al (1998) Science 282: 2085-8; HoshinoK et al (1999) J Immunol 162: 3749-52). Bacterial flagellins are ligands of TLR5 (Hayashi F et al (2001) Nature 410: 1099-1103). Peptidoglycan has been reported to be a ligand not only for TLR2 but also for TLR6 (Ozinsky A et al (2000) Proc Natl Acad SciUSA 97: 13766-71; Takeuchi O et al (2001) Int Immunol 13: 933-40). Recently, certain low molecular weight synthetic compounds, imidazoquinolines (imiquimod (R-837) and Rasimetide (R-848)), have been reported to be ligands for TLR7 and TLR8 (Hemmi H et al (2002) NatImmunol 3: 196-200; Jurk M et al (2002) Nat Immunol 3: 499).

Starting from the discovery that unmethylated bacterial DNA and its synthetic analogs (CpG DNA) are ligands for TLR9 (Hemmi H et al (2000) Nature 408: 740-5; Bauer S et al (2001) Proc Natl Acad Sci USA 98, 9237-42), it has been reported that ligands for certain TLRs include certain nucleic acid molecules. Recently, certain types of RNA have been reported to be immunostimulatory in a sequence-independent or sequence-dependent manner. In addition, these different immunostimulatory RNAs have been reported to stimulate TLR3, TLR7, and TLR 8.

Disclosure of Invention

The present invention relates broadly to immunostimulatory polymers containing certain immunostimulatory sequence motifs, and also to immunostimulatory compositions containing such immunostimulatory polymers and methods of using these immunostimulatory polymers and compositions. In certain aspects of the invention, the immunostimulatory polymer is an immunostimulatory Oligoribonucleotide (ORN). The immunostimulatory polymers of the invention may be used in any application or application where it is desirable to stimulate or enhance an immune response. As disclosed below, the immunostimulatory polymers of the invention are particularly useful in the preparation of pharmaceutical compositions comprising adjuvants, vaccines and other drugs for the treatment of a variety of conditions including infection, cancer, allergy and asthma. Thus, in certain aspects, the invention relates to compositions comprising the immunostimulatory polymers of the invention and methods of their use.

As disclosed in detail below, the immunostimulatory polymers of the invention are characterized in that they comprise at least one sequence-dependent immunostimulatory motif sequence. Sequence-dependent immunostimulatory motif sequences and polymers containing these motifs are disclosed to be potent inducers of TLR 7-related cytokine interferon alpha (IFN- α).

In one aspect, the invention provides a composition comprising rN₁-rC-rU-rC-rA-rN₂A single chain polymer of 4 to 100 units in length, wherein the polymer does not contain a U outside the motif rC-rU-rC-rA, wherein the polymer comprises a phosphodiester backbone, wherein N is₁And N₂Is not A₇And wherein rN₁-rC-rU-rC-rA-rN₂Not GCUCAA. In one embodiment, the composition further comprises a delivery vehicle, wherein the delivery vehicle is a liposome, a non-ionic vesicle (noisesome), a lipid complex (lipoplexe), a polymer complex (polyplexe), a lipopolymer complex (lipoplexe), a water-in-oil (W/O) emulsion, an oil-in-water (O/W) emulsion, a water-in-oil-in-water (W/O/W) multiple emulsion, a microemulsion, a nanoemulsion, a micelle, a dendrimer, a viral particle, a viroid, a polymeric nanoparticle (such as a nanosphere or a nanocapsule), or a polymeric microparticle (such as a microsphere or a microcapsule), and wherein the composition is free of lipofectin. In one embodiment, N₁Comprising at least one A. In another embodiment, N₁Comprising at least one C. In yet another embodiment, N₁Comprising at least one G. In yet another embodiment, N₁Comprising at least one T. In one embodiment, N₂Comprising at least one A. In another embodiment, N₂Comprising at least one C. In yet another embodiment, N₂Comprising at least one G. In yet another embodiment, N₂Comprising at least one T.

In another aspect, the invention provides a composition comprising an rN comprising an immunostimulatory RNA motif₃-rX₂-rC-rU-rC-rA-rX₃-rN₄The length of (1) is 4 to 100 units, wherein X₂And X₃Independently of one another are absent or are nucleotides selected from the group consisting of nucleotides consisting of C, G and A and nucleotide analogs thereof, wherein N is₃And N₄Independently of each other, is absent or is one or more nucleotides, wherein the polymer comprises a phosphodiester backbone and does not comprise two A' s₇Motif, and wherein rN₃-rX₂-rC-rU-rC-rA-rX₃-rN₄Is not GCUCAA or UUAUCGUAX₁CUCAC (SEQ ID NO: 34), wherein X₁Is A or C. In one embodiment, the composition further comprises a delivery vehicle, wherein the delivery vehicle is a liposome, a non-ionic vesicle, a lipid complex, a polymer complex, a lipopolymer complex, a water-in-oil (W/O) emulsion, an oil-in-water (O/W) emulsion, a water-in-oil-in-water (W/O/W) multiple emulsion, a microemulsion, a nanoemulsion, a micelle, a dendrimer, a viral particle, a viroid, a polymeric nanoparticle (such as a nanosphere or nanocapsule), or a polymeric microparticle (such as a microsphere or microcapsule), and wherein the composition is free of lipofectin. In one embodiment, X₂Is A. In one embodiment, X₂Is C. In another embodiment, X₂Is G. In one embodiment, X₃Is A. In another embodiment, X₃Is C. In yet another embodiment, X₃Is G. In one embodiment, N₃Comprising at least one A. In another embodiment, N₃Comprising at least one C. In yet another embodiment, N₃Comprising at least one G. In yet another embodiment, N₃Comprising at least one U. In one embodiment, N₄Comprising at least one A. In another embodiment, N₄Comprising at least one C. In yet another embodiment, N₄Comprising at least one G. In yet another embodiment, N₄Comprising at least one U.

The above compositions may contain other elements or modifications of the polymer. For example, in one embodiment, the composition further comprises an antigen. In one embodiment, the antigen is conjugated to the polymer. In one embodiment, rN₁-rC-rU-rC-rA-rN₂Or rN₃-rX₂-rC-rU-rC-rA-rX₃-rN₄The motif comprises a modified nucleobase selected from the group consisting of hypoxanthine, inosine, 8-oxoadenine and 7-substituted derivatives thereof, dihydrouracil, pseudouracil, 2-thiouracil, 4-thiouracil, 5-aminouracil, 5- (C)₁-C₆) Alkyl uracils, 5-methyl uracils, 5- (C)₂-C₆) Alkenyl uracils, 5- (C)₂-C₆) Alkynyl uracil, 5-hydroxymethyl uracil, 5-chlorouracil, 5-fluorouracil, 5-bromouracil, 5-hydroxycytosine, 5- (C)₁-C₆) Alkylcytosine, 5-methylcytosine, 5- (C)₂-C₆) Alkenyl cytosine, 5- (C)₂-C₆) Alkynylcytosine, 5-chlorocytosine, 5-fluorocytosine, 5-bromocytosine, 2-aminopurine, 2-amino-6-chloropurine, 2, 4-diaminopurine, 2, 6-diaminopurine, 8-azapurine, substituted 7-deazapurine, 7-deaza-7-substituted purine, 7-deaza-8-substituted purine, hydrogen (no basic residue), and any combination thereof. In another embodiment, the polymer is in rN₁-rC-rU-rC-rA-rN₂The motif can further comprise at least one modified nucleobase, wherein the modified nucleobase is selected from the group consisting of hypoxanthine, inosine, 8-oxoadeninePurine and 7-substituted derivatives thereof, dihydrouracil, pseudouracil, 2-thiouracil, 4-thiouracil, 5-aminouracil, 5- (C)₁-C₆) Alkyl uracils, 5-methyl uracils, 5- (C)₂-C₆) Alkenyl uracils, 5- (C)₂-C₆) Alkynyl uracil, 5-hydroxymethyl uracil, 5-chlorouracil, 5-fluorouracil, 5-bromouracil, 5-hydroxycytosine, 5- (C)₁-C₆) Alkylcytosine, 5-methylcytosine, 5- (C)₂-C₆) Alkenyl cytosine, 5- (C)₂-C₆) Alkynyl cytosine, 5-chlorocytosine, 5-fluorocytosine, 5-bromocytosine, N²-dimethylguanine, 7-deazaguanine, 8-azaguanine, 7-deaza-7-substituted guanine, 7-deaza-7- (C)₂-C₆) Alkynylguanine, 7-deaza-8-substituted guanine, 8-hydroxyguanine, 6-thioguanine, 8-oxoguanine, 2-aminopurine, 2-amino-6-chloropurine, 2, 4-diaminopurine, 2, 6-diaminopurine, 8-azapurine, substituted 7-deazapurine, 7-deaza-7-substituted purine, 7-deaza-8-substituted purine, hydrogen (no basic residue) and any combination thereof.

In another embodiment, the composition further comprises a lipophilic moiety covalently attached to the polymer. In one embodiment, the lipophilic moiety is selected from the group consisting of cholesteryl, palmitoyl, and fatty acyl. In another embodiment, the polymer does not contain CpG motifs. In yet another embodiment, the polymer does not comprise CCGAGCCGAGCUCACC (SEQ ID NO: 35). In one embodiment, the polymer is 4 to 20 units in length. In another embodiment, the polymer has a length of 10 to 25 units. In yet another embodiment, the polymer is 15 to 19 units in length. In one embodiment, each polymer unit is a ribonucleotide. In another embodiment, the polymer unit is a mixture of ribonucleotides and deoxyribonucleotides. In yet another embodiment, the deoxyribonucleotide comprises a TCG motif at the 5' end of the polymer. In yet another embodiment, at least one unit of the polymer is an amino acid. In one embodiment, the polymer is linked to a TLR9 agonist. In another embodiment, the TLR9 agonist is a small molecule. In yet another embodiment, the polymer is linked to a TLR7 agonist. In yet another embodiment, the polymer is linked to a TLR8 agonist.

In another aspect, the invention provides a method comprising contacting an immune cell capable of producing IFN- α with a single-stranded polymer of 4 to 100 units in length in an amount effective to induce the production of a therapeutically significant amount of IFN- α, wherein the polymer comprises an immunostimulatory RNA motif rN₁-rC-rU-rC-rA-rN₂And no U outside the motif rC-rU-rC-rA, wherein the polymer comprises a phosphodiester backbone, wherein N is₁And N₂Is not A₇And wherein rN₁-rC-rU-rC-rA-rN₂Is not GCUCAA or UUAUCGUAX₁CUCAC (SEQ ID NO: 34), wherein X₁Is A or C, wherein the polymer is not formulated with lipofectin. In one embodiment, the polymer comprises at least one stabilizing linkage outside of the immunostimulatory motif. In another embodiment, the polymer comprises 1-5 stabilizing linkages outside of the immunostimulatory motif. In one embodiment, the stabilizing bond is at the 5 'end and/or the 3' end.

In yet another aspect, the invention provides a method comprising contacting an immune cell capable of producing interferon alpha (IFN- α) with a single-stranded polymer of 4 to 100 units in length in an amount effective to induce the production of a therapeutically significant amount of IFN- α, wherein the polymer comprises an immunostimulatory RNA motif rN₃-rX₂-rC-rU-rC-rA-rX₃-rN₄Wherein X is₂And X₃Independently of one another are absent or are nucleotides selected from the group consisting of nucleotides consisting of C, G and A and nucleotide analogs thereof, wherein N is₃And N₄Independently of one another absent or one or more nucleotides, wherein the polymer comprises a phosphodiester backbone anddoes not contain two A₇Motif, and wherein rN₃-rX₂-rC-rU-rC-rA-rX₃-rN₄Is not GCUCAA or UUAUCGUAX₁CUCAC (SEQ ID NO: 34), wherein X₁Is A or C, wherein the polymer is not formulated with lipofectin.

In one embodiment, the above method does not result in immune cells producing significant amounts of tumor necrosis factor alpha (TNF- α), interferon gamma (TNF- γ), or interleukin 12(IL-12) in response to the polymer. In certain embodiments, the method is performed in vivo. In certain embodiments, the polymer is administered in the form of any of the compositions described above.

In another aspect, the invention provides a method of treating asthma, the method comprising administering to a subject suffering from asthma a therapeutically effective amount of a single-stranded polymer of 4-100 units in length, wherein the polymer comprises an immunostimulatory RNA motif rN₁-rC-rU-rC-rA-rN₂And no U outside the motif rC-rU-rC-rA, wherein the polymer comprises a phosphodiester backbone, wherein N is₁And N₂Is not A₇And wherein rN₁-rC-rU-rC-rA-rN₂Is not GCUCAA or UUAUCGUAX₁CUCAC (SEQ ID NO: 34), wherein X₁Is A or C.

In a further aspect, the invention provides a method of treating asthma, the method comprising administering to a subject suffering from asthma a therapeutically effective amount of a single-stranded polymer of 4-100 units in length, wherein the polymer comprises an immunostimulatory RNA motif rN₃-rX₂-rC-rU-rC-rA-rX₃-rN₄Wherein X is₂And X₃Independently of one another are absent or are nucleotides selected from the group consisting of nucleotides consisting of C, G and A and nucleotide analogs thereof, wherein N is₃And N₄Independently of each other, is absent or is one or more nucleotides, wherein the polymer comprises a phosphodiester backbone and does not comprise two A' s₇Motif, and wherein rN₃-rX₂-rC-rU-rC-rA-rX₃-rN₄Is not GCUCAA or UUAUCGUAX₁CUCAC (SEQ ID NO: 34), wherein X₁Is A or C.

In one embodiment, any of the above methods of treating asthma may further comprise administering to the subject an allergen. In one embodiment, the polymer is conjugated to the allergen. In certain embodiments, the polymer is administered in the form of any of the compositions described above.

In another aspect, the invention provides a method of treating an allergic condition, the method comprising administering to a subject suffering from an allergic condition a therapeutically effective amount of a single-stranded polymer of 4 to 100 units in length, wherein the polymer comprises an immunostimulatory RNA motif rN₁-rC-rU-rC-rA-rN₂And no U outside the motif rC-rU-rC-rA, wherein the polymer comprises a phosphodiester backbone, wherein N is₁And N₂Is not A₇And wherein rN₁-rC-rU-rC-rA-rN₂Is not GCUCAA or UUAUCGUAX₁CUCAC (SEQ ID NO: 34), wherein X₁Is A or C.

In a further aspect, the invention provides a method of treatment of an allergic condition, the method comprising administering to a subject suffering from an allergic condition a therapeutically effective amount of a single-stranded polymer of 4 to 100 units in length, wherein the polymer comprises an immunostimulatory RNA motif rN₃-rX₂-rC-rU-rC-rA-rX₃-rN₄Wherein X is₂And X₃Independently of one another are absent or are nucleotides selected from the group consisting of nucleotides consisting of C, G and A and nucleotide analogs thereof, wherein N is₃And N4 are independently absent or one or more nucleotides, wherein the polymer comprises a phosphodiester backbone and does not comprise two a' s₇Motif, and wherein rN₃-rX₂-rC-rU-rC-rA-rX₃-rN₄Is not GCUCAA or UUAUCGUAX₁CUCAC (SEQ ID NO: 34), wherein X₁Is A or C.

In one embodiment, any of the above methods of treating an allergic condition may further comprise administering an allergen to the subject. In one embodiment, the polymer is conjugated to the allergen. In certain embodiments, the polymer is administered in the form of any of the compositions described above.

In another aspect, the invention provides a method of treating cancer, the method comprising administering to a subject having cancer a cancer therapeutically effective amount of a single-stranded polymer 4 to 100 units in length, wherein the polymer comprises an immunostimulatory RNA motif rN₁-rC-rU-rC-rA-rN₂And no U outside the motif rC-rU-rC-rA, wherein the polymer comprises a phosphodiester backbone, wherein N is₁And N₂Is not A₇And wherein rN₁-rC-rU-rC-rA-rN₂Is not GCUCAA or UUAUCGUAX₁CUCAC (SEQ ID NO: 34), wherein X₁Is A or C.

In a further aspect, the invention provides a method of treating cancer, the method comprising administering to a subject having cancer a cancer therapeutically effective amount of a single-stranded polymer of 4-100 units in length, wherein the polymer comprises an immunostimulatory RNA motif rN₃-rX₂-rC-rU-rC-rA-rX₃-rN₄Wherein X is₂And X₃Independently of one another are absent or are nucleotides selected from the group consisting of nucleotides consisting of C, G and A and nucleotide analogs thereof, wherein N is₃And N₄Independently of each other, is absent or is one or more nucleotides, wherein the polymer comprises a phosphodiester backbone and does not comprise two A' s₇Motif, and wherein rN₃-rX₂-rC-rU-rC-rA-rX₃-rN₄Is not GCUCAA or UUAUCGUAX₁CUCAC (SEQ ID NO: 34), wherein X₁Is A or C.

In one embodiment, any of the above methods of treating cancer may further comprise administering a cancer antigen to the subject. In one embodiment, the polymer is conjugated to the antigen. In other embodiments, the method further comprises administering a second cancer medicament to the subject. In one embodiment, the cancer drug is one or more of carboplatin, paclitaxel (paclitaxel), cisplatin, 5-fluorouracil, doxorubicin, taxol, and gemcitabine (gemcitabine). In certain embodiments, the polymer is administered in the form of any of the compositions described above.

In another aspect, the invention provides a method of treating an infectious disease, the method comprising administering to a subject having an infectious disease a therapeutically effective amount of a single-stranded polymer 4-100 units in length, wherein the polymer comprises an immunostimulatory RNA motif rN₁-rC-rU-rC-rA-rN₂And no U outside the motif rC-rU-rC-rA, wherein the polymer comprises a phosphodiester backbone, wherein N is₁And N₂Is not A₇And wherein rN₁-rC-rU-rC-rA-rN₂Is not GCUCAA or UUAUCGUAX₁CUCAC (SEQ ID NO: 34), wherein X₁Is A or C.

In yet another aspect, the invention provides a method of treating an infectious disease, the method comprising administering to a subject having an infectious disease a therapeutically effective amount of a single-stranded polymer 4-100 units in length, wherein the polymer comprises an immunostimulatory RNA motif rN₃-rX₂-rC-rU-rC-rA-rX₃-rN₄Wherein X is₂And X₃Independently of one another are absent or are nucleotides selected from the group consisting of nucleotides consisting of C, G and A and nucleotide analogs thereof, wherein N is₃And N₄Independently of each other, is absent or is one or more nucleotides, wherein the polymer comprises a phosphodiester backbone and does not comprise two A' s₇Motif, and wherein rN₃-rX₂-rC-rU-rC-rA-rX₃-rN₄Is not GCUCAA or UUAUCGUAX₁CUCAC (SEQ ID NO: 34), wherein X₁Is A or C.

In one embodiment, any of the above methods of treating infectious diseases may further comprise administering a microbial antigen to the subject. In one embodiment, the polymer is conjugated to the antigen. In certain embodiments, the polymer is administered in the form of any of the compositions described above.

In another aspect, the invention provides a method for inducing a T helper type 1 (Th1) -like immune response in a subject, the method comprising administering to the subject an effective amount of a single stranded polymer of 4-100 units in length, wherein the polymer comprises an immunostimulatory RNA motif, rN₁-rC-rU-rC-rA-rN₂And no U outside the motif rC-rU-rC-rA, wherein the polymer comprises a phosphodiester backbone, wherein N is₁And N₂Is not A₇And wherein rN₁-rC-rU-rC-rA-rN₂Is not GCUCAA or UUAUCGUAX₁CUCAC (SEQ ID NO: 34), wherein X₁Is A or C.

In a further aspect, the invention provides a method for inducing a T helper type 1 (Th1) -like immune response in a subject, the method comprising administering to the subject an effective amount of a single stranded polymer of 4-100 units in length, wherein the polymer comprises an immunostimulatory RNA motif, rN₃-rX₂-rC-rU-rC-rA-rX₃-rN₄Wherein X is₂And X₃Independently of one another are absent or are nucleotides selected from the group consisting of nucleotides consisting of C, G and A and nucleotide analogs thereof, wherein N is₃And N₄Independently of each other, is absent or is one or more nucleotides, wherein the polymer comprises a phosphodiester backbone and does not comprise two A' s₇Motif, and wherein rN₃-rX₂-rC-rU-rC-rA-rX₃-rN₄Is not GCUCAA or UUAUCGUAX₁CUCAC (SEQ ID NO: 34), wherein X₁Is A or C.

In one embodiment, any of the methods of inducing a Th 1-like immune response described above may further comprise administering an antigen to the subject. In one embodiment, the polymer is conjugated to the antigen. In certain embodiments, the polymer is administered in the form of any of the compositions described above.

The invention is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments and of being practiced or of being carried out in various ways. Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of "including," "comprising," or "having," "containing," "involving," and variations thereof herein, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items.

Drawings

FIG. 1 is a graph showing the induction of IFN-. alpha.production in human Peripheral Blood Mononuclear Cells (PBMC) after contacting the cells with Oligoribonucleotides (ORN). ORN (starting concentration: 2. mu.M + 50. mu.g/ml DOTAP (N- [1- (2, 3-dioleoyloxy) propyl-1 ] -N, N, N-trimethylammoniummethane)) was incubated with human PBMC and the supernatants were tested for IFN-. alpha.by enzyme-linked immunosorbent assay (ELISA) after 24 hours. Shown are the positive control (CCGUCUGUUGUGUGACUC; SEQ ID NO: 1) and 4 test sequences (SEQ ID NO: 2-5, see Table 1). Mean ± SEM of 3 donors are shown. The y-axis shows the IFN-. alpha.concentration in pg/ml and the x-axis shows the log ORN concentration in. mu.M.

FIG. 2 is two graphs showing induction of IFN-. alpha.by ORN (FIG. 2A) and IL-12p40 (FIG. 2B). Shown are ORNs (SEQ ID NO: 1) known to induce both TLR 7-related and TLR 8-related cytokines and ORNs (SEQ ID NO: 3) that induce more TLR 7-related cytokines than TLR 8-related cytokines. Human PBMC were incubated with ORN in the presence of DOTAP (2. mu.M ORN and 50. mu.g/ml DOTAP) and supernatants were tested for IFN-. alpha.and IL-12p40 by ELISA after 24 hours. Mean ± SEM of 3 donors are shown. The y-axis is the IFN-. alpha.concentration in pg/ml (FIG. 2A) and the IL-12p40 concentration (FIG. 2B), and the x-axis shows the log ORN concentration in. mu.M.

FIG. 3 is a graph showing IFN- α production induced in human PBMC cells following exposure of the cells to Oligoribonucleotides (ORNs). ORN (starting concentration: 2. mu.M + 25. mu.g/ml) was incubated with human PBMC and supernatants were tested for IFN-. alpha.by ELISA after 24 hours. Shown is SEQ ID NO: 3 and 4 ORNs with subtle sequence variations (SEQ ID NOS: 6, 7, 11 and 12, see Table 2). Mean ± SEM of 3 donors are shown. The y-axis shows the IFN-. alpha.concentration in pg/ml and the x-axis shows the log ORN concentration in. mu.M.

FIG. 4 is two graphs showing induction of IFN-. alpha.by ORN (FIG. 4A) and IL-12p40 (FIG. 4B). Shown is SEQ ID NO: 3 and 3 other ORNs with subtle sequence variations (SEQ ID NOS: 8-10, see Table 3). Human PBMC were incubated with ORN in the presence of DOTAP (2. mu. MORN and 25. mu.g/ml DOTAP) and supernatants were tested for IFN-. alpha.and IL-12p40 by ELISA after 24 hours. Mean ± SEM of 3 donors are shown. The y-axis is the IFN-. alpha.concentration in pg/ml (FIG. 4A) and the IL-12p40 concentration (FIG. 4B), and the x-axis shows the log ORN concentration in. mu.M.

FIG. 5 is two graphs showing induction of IFN-. alpha.by ORN (FIG. 5A) and IL-12p40 (FIG. 5B). Shown is SEQ ID NO: 3 and 3 other ORNs with subtle sequence variations (SEQ ID NOS: 13-15, see Table 4). Human PBMC were incubated with ORN in the presence of DOTAP (2. mu. MORN and 25. mu.g/ml DOTAP) and supernatants were tested for IFN-. alpha.and IL-12p40 by ELISA after 24 hours. Mean ± SEM of 3 donors are shown. The y-axis is the IFN-. alpha.concentration in pg/ml (FIG. 5A) and the IL-12p40 concentration (FIG. 5B), and the x-axis shows the log ORN concentration in. mu.M.

FIG. 6 is two graphs showing induction of IFN-. alpha.by ORN (FIG. 6A) and IL-12p40 (FIG. 6B). Shown is SEQ ID NO: 3 and 3 other ORNs with subtle sequence variations (SEQ ID NOS: 19-21, see Table 5). Human PBMC were incubated with ORN in the presence of DOTAP (2. mu. MORN and 25. mu.g/ml DOTAP) and supernatants were tested for IFN-. alpha.and IL-12p40 by ELISA after 24 hours. Mean ± SEM of 3 donors are shown. The y-axis is the IFN-. alpha.concentration in pg/ml (FIG. 6A) and the IL-12p40 concentration (FIG. 6B), and the x-axis shows the log ORN concentration in. mu.M.

FIG. 7 is two graphs showing the induction of IFN-. alpha.by ORN (FIGS. 7A and 7B) and IL-12p40 (FIGS. 7C and 7D). Shown is SEQ ID NO: 3 and 6 other ORNs with subtle sequence variations (SEQ ID NOS: 16-18 and SEQ ID NOS: 22-24, see Table 3). Human PBMC were incubated with ORN in the presence of DOTAP (2. mu.M ORN and 25. mu.g/ml DOTAP) and supernatants were tested for IFN-. alpha.and IL-12p40 by ELISA after 24 hours. Mean ± SEM of 3 donors are shown. The y-axis is the IFN-. alpha.concentration in pg/ml (FIGS. 7A and 7B) and the IL-12p40 concentration (FIGS. 7C and 7D), and the x-axis shows the log ORN concentration in. mu.M.

FIG. 8 is a 4-panel graph comparing in vitro cytokine induction by ORN (GACACACACACUCACACACACACA; SEQ ID NO: 27) with an immunostimulatory UCA motif. SEQ ID NO: 27 did not induce significant IL-12 (FIG. 8A), IL-6 (FIG. 8B), IL-2R (FIG. 8C) or IL-7 (FIG. 8D). The results were compared to negative control (GACACACACACACACACACACACA; SEQ ID NO: 25), non-UCA ORN with a U-rich 3' end (GACACACACACACACACACACUUU; SEQ ID NO: 26) and positive control (UUGUUGUUGUUGUUGUUGUU (both phosphorothioate); SEQ ID NO: 28). Human PBMCs from 3 healthy donors were incubated with up to 2 μ M ORN in the presence of DOTAP for 24 hours. Supernatants were collected and assayed for cytokine or chemokine concentrations by ELISA. The y-axis is the concentration of cytokine or chemokine in pg/ml and the x-axis shows the log ORN concentration in μ M.

FIG. 9 is 4 graphs comparing in vitro cytokine induction by ORN with immunostimulatory UCA motifs (SEQ ID NO: 27). SEQ ID NO: 27 did not induce significant IL-10 (FIG. 9A), IL-15 (FIG. 9B), IL-12p40 (FIG. 9C) or TNF- α (FIG. 9D). The results were compared to negative controls (SEQ ID NO: 25), non-UCA ORNs with a U-rich 3' end (SEQ ID NO: 26)) and positive controls (SEQ ID NO: 28) a comparison is made. Human PBMCs from 3 healthy donors were incubated with up to 2 μ M ORN in the presence of DOTAP for 24 hours. Supernatants were collected and assayed for cytokine or chemokine concentrations by ELISA. The y-axis is the concentration of cytokine or chemokine in pg/ml and the x-axis shows the log ORN concentration in μ M.

FIG. 10 is a 4-piece graph comparing in vitro cytokine induction by ORN with immunostimulatory UCA motifs (SEQ ID NO: 27). SEQ ID NO: 27 did not induce significant MIP-1 α (FIG. 10B), IFN- γ (FIG. 10C) or MIP-1 β (FIG. 10D), but induced IFN- α (FIG. 10A). The results were compared to negative control (SEQ ID NO: 25), non-UCAORN with U-rich 3' end (SEQ ID NO: 26)) and positive control (SEQ ID NO: 28) a comparison is made. Human PBMCs from 3 healthy donors were incubated with up to 2 μ M ORN in the presence of DOTAP for 24 hours. Supernatants were collected and assayed for cytokine or chemokine concentrations by ELISA. The y-axis is the concentration of cytokine or chemokine in pg/ml and the x-axis shows the log ORN concentration in μ M.

FIG. 11 is a 3-piece graph comparing in vitro cytokine induction by ORN with immunostimulatory UCA motifs (SEQ ID NO: 27). SEQ ID NO: 27 did not induce significant MIG (FIG. 11C), but induced IP-10 (FIG. 11A) and MCP-1 (FIG. 11B). The results were compared to negative controls (SEQ ID NO: 25), non-UCA ORNs with a U-rich 3' end (SEQ ID NO: 26)) and positive controls (SEQ ID NO: 28) a comparison is made. Human PBMCs from 3 healthy donors were incubated with up to 2 μ M ORN in the presence of DOTAP for 24 hours. Supernatants were collected and assayed for cytokine or chemokine concentrations by ELISA. The y-axis is the concentration of cytokine or chemokine in pg/ml and the x-axis shows the log ORN concentration in μ M.

FIG. 12 is 4 graphs comparing in vivo cytokine induction by ORN with immunostimulatory UCA motifs (SEQ ID NO: 3). SEQ ID NO: 3 IFN-. alpha.was induced at 3-and 24-hour time points (FIGS. 12A and 12C) and IP-10 (FIGS. 12B and 12D). Converting SEQ ID NO: 3 was compared to the activity of 2 ORN (GACACACACACACACACACACAUU; SEQ ID NO: 30; and UUAUUAUUAUUAUUAUUAUU (phosphorothioate backbone); SEQ ID NO: 33) that induced both TLR 7-related and TLR 8-related cytokines and 2 ORN (UUGUUGUUGUUGUUGUUGUU; SEQ ID NO: 31; and UUAUUAUUAUUAUUAUUAUU (phosphorothioate backbone); SEQ ID NO: 32) that induced primarily TLR 8-related cytokines. The x-axis shows the ORN used (including saline and DOTAP as negative controls) and the y-axis shows cytokine concentration in pg/ml. HBS: buffered saline.

FIG. 13 is a 5-panel graph comparing in vivo cytokine induction by ORN with immunostimulatory UCA motif (SEQ ID NO: 3). SEQ ID NO: 3 did not induce significant amounts of TNF- α, IL-2, IL-12, IL-6 or IL-10 at the 3 hour time point (FIGS. 13A-E, respectively). Converting SEQ ID NO: 3 was compared with the activity of 2 ORN (SEQ ID NOs: 30 and 33) inducing both TLR 7-related and TLR 8-related cytokines and 2 ORN (SEQ ID NOs: 31 and 32) inducing mainly TLR 8-related cytokines. The x-axis shows the ORN used (including saline and DOTAP as negative controls) and the y-axis shows cytokine concentration in pg/ml. HBS: buffered saline.

FIG. 14 is a4 bar graph showing in vivo splenocyte activation by an ORN (SEQ ID NO: 3) bearing the immunostimulatory UCA motif. SEQ ID NO: 3 activating spleen CD3⁺T cells (FIGS. 14A and 14B) and DX5⁺B cells (fig. 14C and 14D). Cells were isolated from the spleen and isolated by FACS analysis. The x-axis shows the ORN used (including saline and DOTAP as negative controls), and the y-axis shows CD69⁺Cell% (A and C) or IL-12R⁺Cell% (B and D). HBS: buffered saline.

Figure 15 is a graph depicting SEQ ID NO: 27, graph of the induction of IFN- α compared to other ORN with up to 3U but no UCA motif. The y-axis is the IFN- α concentration in pg/ml and the x-axis shows the log ORN concentration in μ M.

FIG. 16 is 4 bar graphs depicting the induction of IFN- α, IP-10, IL-12 and IL-6 in TLR9 knockout (TLR9KO) mice, TLR7 knockout (TLR7KO) mice and control C57BL/6 mice in response to a designated ORN or CpG ODN 1826(SEQ ID NO: 34). HBS: buffered saline.

FIG. 17 is a bar graph depicting IFN- α and IP-10 induction in MyD88 knockout (MyD88KO) and control C57BL/6 mice. HBS: buffered saline.

Detailed Description

Immunostimulatory Oligoribonucleotides (ORN) have been shown to stimulate the human immune system in a TLR 7-dependent and/or TLR 8-dependent manner. For example, ORN containing motifs rich in GU and CU but lacking the poly G terminus may act on TLR7 and TLR 8. ORN with AU-rich but lacking poly G termini may act only on TLR 8. ORN containing immunostimulatory RNA motifs flanked by poly G motifs stimulate an immune response, probably through TLR7 rather than TLR 8. These produce significant amounts of IFN- α in the presence of cationic liposomal formulations such as, for example, DOTAP. This effect appears to be mediated by TLR7, since IFN- α producing plasmacytoid dendritic cells (pdcs) express TLR7 but not TLR 8. Other cytokines observed (e.g., TNF- α, IL-12, and IFN- γ) may be mediated through TLR 8. For example, activation of monocytes is likely to be an effect mediated directly through TLR8, as monocytes have been shown to express TLR8 but not TLR7 and to secrete TNF- α upon ssRNA stimulation. Recently, ORN have been identified that bear distinct immunological properties (immune profile) and have defined motifs for activating RNA-mediated responses. Some of these ORNs do not induce IFN- α production by human PBMCs, but do induce significant amounts of TNF- α, IL-12 and IFN- γ, which are directed to stimulation of TLR8 rather than TLR 7.

The present invention relates to the discovery of a class of polymers containing specific RNA motifs that induce RNA-mediated immune responses known as TLR 7-mediated responses (e.g. IFN- α production by pDC) without inducing a significant amount of TLR 8-mediated immune activation (i.e. production of cytokines produced by TLR 8-expressing cells, such as TNF- α production by monocytes). As used herein, "significant amount" refers to an amount that is different from the amount produced by other immunostimulatory ORN. By "does not induce significant amounts of TLR 8-mediated immune activation" is meant: immune activation (e.g., the level of factors associated with TLR activation induced) is minimal when compared to the levels induced by ORN described above, which contain ORN rich in GU and ORN rich in CU and lacking the poly G terminus, or other ORN that may stimulate TLR 8. Thus, the ORN of the invention induces less of the cytokines typical for RNA TLR8 or TLR7/8 ligands (e.g., the pro-inflammatory cytokines TNF- α, IL-6). In certain embodiments, a significant amount is a "significant amount". The class of polymers described herein are single-stranded, have a phosphodiester backbone, and have an immunostimulatory RNA motif with a CUCA sequence.

This class is associated with immune characteristics characterized by activation of a nearly exclusive TLR 7-like immune response. For example, as shown in fig. 4, the polymer of the invention SEQ ID NO: 3 induced a significant amount of IFN- α without significantly inducing IL-12p40 when formulated with DOTAP. In contrast, a polymer having a similar sequence but lacking the immunostimulatory CUCA motif, SEQ ID NO: 8-10 induced a large amount of IL-12p 40.

This novel immunostimulatory motif has been found to be immunostimulatory only in the context of a phosphodiester backbone. Interestingly, it has been found that immunostimulatory polymers of the invention containing this motif produce a strong IFN- α response but do not stimulate other typical cytokines (e.g., those induced in response to TLR8 stimulation). Thus, in one aspect, the invention provides an immunostimulatory polymer comprising an immunostimulatory motif that induces predominantly a TLR 7-associated cytokine and having a phosphodiester backbone.

In one aspect of the invention, the immunostimulatory RNA motif is rN₁-rC-rU-rC-rA-rN₂Wherein the polymer does not contain U outside of the motif rC-rU-rC-rA, wherein the polymer comprises a phosphodiester backbone, wherein N is₁And N₂Is not A₇(rA-rA-rA-rA-rA-rA-rA-rA) and wherein rN₁-rC-rU-rC-rA-rN₂Is not GCUCAA or UUAUCGUAX₁CUCAC (SEQ ID NO: 34), wherein X₁Is A or C. In certain embodiments, N₁Comprising at least one A, one C or one G. In other embodiments, N₁Comprising at least one T, e.g., in RNA-DNA chimeras. In certain embodiments, N₂Comprising at least one A, one C, one G or one T.

In another aspect of the invention, the immunostimulatory RNA motif is rN₃-rX₂-rC-rU-rC-rA-rX₃-rN₄Wherein X is₂And X₃Absent or a nucleotide selected from the group consisting of nucleotides and nucleotide analogs consisting of C, G and A, wherein N is₃And N₄Absent or one or more nucleotides, wherein the polymer comprises a phosphodiester backbone and does not comprise two A' s₇Motif, and wherein rN₃-rX₂-rC-rU-rC-rA-rX₃-rN₄Is not GCUCAA or UUAUCGUAX₁CUCAC (SEQ ID NO: 34), wherein X₁Is A or C. In certain embodiments, the polymer does not contain CCGAGCCGAGCUCACC (SEQ ID NO: 35). In certain embodiments of the invention, N₃And N₄Each independently containing at least one A, C, G or U.

In certain embodiments, the immunostimulatory polymer is 4 to 100 units in length. In other embodiments, the immunostimulatory polymer is 4 to 20 units in length. In other embodiments, the immunostimulatory polymer is 10 to 25 units in length. In other embodiments, the immunostimulatory polymer is 15 to 19 units in length.

As discussed in more detail in the examples below, CUCA for the immunostimulatory motif was found to be important for TLR 7-like immune responses. Surprisingly, the immunostimulatory effect is specific for the phosphodiester backbone, but not for the phosphorothioate backbone. In certain embodiments, the immunostimulatory polymer of the invention has more than one immunostimulatory motif.

The immunostimulatory polymers of the invention are single-stranded. According to the methods of the invention, the polymer is not designed to comprise a sequence complementary to a coding sequence in a human cell, and thus is not considered to be an antisense ORN or silencing rna (sirna). A "non-complementary" polymer is a polymer that does not contain sequences that strongly hybridize (e.g., do not hybridize under stringent conditions) to a particular coding region in a target cell. Thus, administration of non-complementary polymers does not result in gene silencing, particularly because the polymers of the invention are single stranded as compared to the double stranded molecules used for gene silencing.

The polymers of the invention are capable of inducing an immune response that induces a significant amount of IFN- α or IFN- α related molecules relative to background. IFN-alpha related molecules are cytokines or factors involved in the expression of IFN-alpha. These molecules include, but are not limited to, MIP 1-beta, IP-10, and MIP 1-alpha.

The present invention relates generally to immunostimulatory polymers comprising immunostimulatory RNA motifs, immunostimulatory compositions containing one or more immunostimulatory polymers of the invention, and methods of using the immunostimulatory polymers and immunostimulatory compositions of the invention. As used herein, the term "RNA" refers to two or more ribonucleotides (i.e., molecules each comprising a ribose and a purine or pyrimidine nucleoside base (e.g., guanine, adenine, cytosine, or uracil) linked to a phosphate group) covalently linked by a3 '-5' phosphodiester linkage.

As mentioned above, RNA refers to a polymer of ribonucleotides linked by 3 '-5' phosphodiester bonds. In certain embodiments, the immunostimulatory polymer of the invention is RNA. In other embodiments, the invention provides immunostimulatory compositions comprising chimeric DNA-RNA molecules comprising the immunostimulatory RNA motifs of the invention. In one embodiment of the invention, the deoxyribonucleotide residues of a DNA: RNA molecule comprise a TCG motif at the 5' end of the polymer. In one embodiment, the DNA component of the RNA molecule comprises a CpG nucleic acid, i.e., a TLR9 agonist. In one embodiment, the DNA and RNA portions of the RNA molecule are covalently linked by internucleotide phosphate linkages. In another embodiment, the DNA and RNA portions of the RNA molecule are covalently linked by a linker (e.g., a non-nucleotide linker).

In another embodiment, at least one unit of the polymer is an amino acid.

In certain embodiments, the immunostimulatory polymers of the invention are linked to a TLR9 agonist that is not a CpG nucleic acid. In certain embodiments, the immunostimulatory polymer of the invention is linked to a TLR7 agonist or a TLR9 agonist. The agonist may be a deoxyribonucleotide or a ribonucleotide, or it may be a peptide or a small molecule. The immunostimulatory polymers may be linked directly to the agonist or they may be linked via a linker.

Immunostimulatory polymers of the invention can encompass a variety of chemical modifications and substitutions compared to native RNA and DNA involving internucleotide phosphodiester linkages, β -D-ribose units, and/or native nucleotide bases (adenine, guanine, cytosine, thymine, uracil). Examples of chemical modifications are known to those skilled in the art and are described in the following references: for example, Uhlmann E et al (1990) Chem Rev 90: 543; "Protocols for Oligonucleotides and analogues" Synthesis and Properties & Synthesis and Analytical Techniques, S.Agrawal, Ed, Humana Press, Totowa, USA 1993; crook ST et al (1996) Annu Rev Pharmacol Toxicol 36: 107-129; and Hunziker J et al (1995) Mod Synth Methods 7: 331-417. The oligonucleotides of the invention may have one or more modifications, wherein each modification is located at a specific internucleotide phosphodiester bond and/or a specific β -D-ribose unit and/or a specific natural nucleotide base position compared to an oligonucleotide of the same sequence consisting of natural DNA or RNA.

For example, the invention relates to oligonucleotides that may comprise one or more modifications, wherein each modification is independently selected from:

a) replacing the internucleotide phosphodiester bond at the 3 'end and/or the 5' end of the nucleotide with a modified internucleotide linkage,

b) the phosphodiester bond at the 3 'end and/or the 5' end of the nucleotide is replaced by a dephosphorizing bond,

c) replacing the phosphorylated sugar units in the phosphorylated sugar backbone with another unit,

d) replacement of the beta-D-ribose unit with a modified sugar unit, and

e) the natural nucleotide base is replaced with a modified nucleotide base.

More detailed examples of chemical modifications of oligonucleotides are as follows.

In one embodiment, the ORN may have at least one stabilized internucleotide linkage. Typically, the linkage is at or near the 5 'end or the 3' end and is not within the immunostimulatory motif. The internucleotide phosphodiester bond at the 3 'end and/or the 5' end of the nucleotide may be replaced by at least one modified internucleotide linkage, wherein the modified internucleotide linkage is for example selected from the group consisting of phosphorothioate, phosphorodithioate, NR¹R²Phosphoramidates, boranophosphates (boranophosphates), alpha-hydroxybenzylphosphonates, phosphoric acid (C)₁～C₂₁) -O-alkyl esters, phosphoric acid [ (C)₆～C₁₂) Aryl radical- (C)₁～C₂₁) -O-alkyl]Ester, (C)₁～C₈) Alkyl phosphonate and/or (C)₆～C₁₂) Aryl phosphonate bond, (C)₇～C₁₂) -alpha-hydroxymethylaryl (e.g. as disclosed in WO 95/01363), wherein (C)₆～C₁₂) Aryl group, (C)₆～C₂₀) Aryl and (C)₆～C₁₄) Aryl is optionally substituted with halogen, alkyl, alkoxy, nitro, cyano, and wherein R is¹And R²Independently of one another, hydrogen, (C)₁～C₁₈) Alkyl, (C)₆～C₂₀) Aryl group, (C)₆～C₁₄) Aryl radical- (C)₁～C₈) Alkyl, preferably hydrogen, (C)₁～C₈) Alkyl, preferably (C)₁-C₄) Alkyl and/or methoxyethyl, or R¹And R²Together with the nitrogen bearing them, form a five-to six-membered heterocyclic ring which may additionally contain further heteroatoms from O, S and N. In one embodiment, the ORN has1 to 5 stabilizing bonds.

The phosphodiester bond at the 3 ' end and/or the 5 ' end of the nucleotide can be replaced by a dephosphorizing bond (dephosphorizing bonds are described, for example, in "Methods in Molecular Biology", volume 20, "Protocols for Oligonucleotides and Analogs", s.agrawal, ed., Humana Press, totawa 1993, chapter 16, page 355 onward) which is selected, for example, from the group consisting of dephosphorizing bond methylal, 3 ' -thiometal, methylhydroxylamine, oxime, methylenedimethylhydrazinium, dimethylene sulfone and/or silyl groups.

The phosphorylated sugar units (i.e.the β -D-ribose and internucleotide phosphodiester linkages together form a phosphorylated sugar unit) from the phosphorylated sugar backbone (i.e.the phosphorylated sugar backbone consisting of phosphorylated sugar units) may be replaced by another unit which is e.g.suitable for the construction of "morpholino derivative" oligomers (e.g.as described in Stirchak EP et al (1989) Nucleic Acids Res 17: 6129-41), i.e.e.e.by morpholino derivative units; or the further unit is suitable for the construction of polyamide nucleic acids ("PNA"; as described for example by Nielsen PE et al (1994) bioconjugateg Chem 5: 3-7), i.e.for example by PNA backbone units, for example by 2-aminoethylglycine.

The beta-ribose unit or the beta-D-2 '-deoxyribose unit may be replaced by a modified sugar unit, wherein the modified sugar unit is, for example, beta-D-ribose, alpha-D-2-deoxyribose, L-2' -deoxyribose, 2 '-F-arabinose,' -O- (C-O-)₁～C₆) Alkylribose (2' -O- (C)₁～C₆) The alkylribose is preferably 2 '-O-methylribose)' -O- (C₂～C₆) Alkenyl ribose, 2' - [ O- (C)₁～C₆) alkyl-O- (C)₁～C₆) Alkyl radical]Ribose, 2' -NH₂-2' -deoxyribose,. beta. -D-xylofuranose,. alpha. -arabinofuranose, 2, 4-dideoxy-. beta. -D-erythropyranose and carbocyclic sugar analogs (e.g. as described in Froehler J (1992) Am Chem Soc 114: 8320) and/or open-chain sugar analogs (e.g. as described by Vandedrissche et al (1993) Te)tetrahedron 49: 7223) and/or bicyclic carbohydrate analogs (e.g., Tarkov M et al (1993) Helv Chim Acta 76: 481).

Nucleic acids also include substituted purines and pyrimidines, e.g., C-5 propynopyrimidine and 7-deaza-7-substituted purines and like modified bases (Wagner RW et al (1996) Nat Biotechnol 14: 840-4). Purines and pyrimidines include, but are not limited to, adenine, cytosine, guanine and thymine, as well as other naturally occurring and non-naturally occurring nucleobases, aromatic moieties with and without substituents.

In one embodiment, the immunostimulatory polymer of the invention may comprise one or more modified nucleobases, i.e., derivatives of A, C, G, T and U, in addition to the immunostimulatory motif. Specific embodiments of these modified nucleobases include, but are not limited to, cytosines substituted at the 5-position (e.g., 5-methylcytosine, 5-fluorocytosine, 5-chlorocytosine, 5-bromocytosine, 5-iodocytosine, 5-hydroxycytosine, 5-hydroxymethylcytosine, 5-difluoromethylcytosine, and unsubstituted or substituted 5-alkynylcytosine), cytosines substituted at the 6-position, N4-substituted cytosines (e.g., N4-ethylcytosine), 5-aza-cytosine, 2-mercaptocytosine, isocytosine, pseudoisocytosine, cytosine analogs with fused ring systems (e.g., N, N' -propynocyanine or phenoxazine), and uracils and derivatives thereof (e.g., 5-fluorouracil, 5-bromouracil, 5-bromovinyluracil, 4-thiouracil, 5-propynyluracil), thymine derivatives (e.g., 2-thiothymine, 4-thiothymine, 6-substituted thymine), guanosine derivatives (e.g., 7-deazaguanine, 7-deaza-7-substituted guanine (e.g., 7-deaza-7- (C)₂～C₆) Alkynylguanine), 7-deaza-8-substituted guanine, hypoxanthine, N2-substituted guanine (e.g., N2-methylguanine), 8-substituted guanine (e.g., 8-hydroxyguanine and 8-bromoguanine), and 6-thioguanine), or adenosine derivative (5-amino-3-methyl-3H, 6H-thiazolo [4, 5-d)]Pyrimidine-2, 7-diones, 2, 6-diaminopurine, 2-aminopurine, purines, indoles, adenine, substitutedAdenine (e.g., N6-methyladenine, 8-oxoadenine)). The bases may also consist of universal bases (e.g.4-methylindole, 5-nitroindole, 3-nitropyrrole, P-base and K-base), aromatic ring systems (e.g.benzimidazole or dichlorobenzimidazole, 1-methyl-1H- [1, 2, 4 ]]Triazole-3-carboxamides), aromatic ring systems (e.g. fluorobenzene or difluorobenzene) or hydrogen atoms (dspacers). Modified U nucleobases are, for example, dihydrouracil, pseudouracil, 2-thiouracil, 4-thiouracil, 5-aminouracil, 5- (C)₁-C₆) Alkyl uracils, 5-methyl uracils, 5- (C)₂-C₆) Alkenyl uracils, 5- (C)₂-C₆) Uracil derivatives such as alkynyl uracil, 5-hydroxymethyl uracil, 5-chlorouracil, 5-fluorouracil and 5-bromouracil. The aforementioned modified nucleobases and their responsive nucleosides are available from commercial suppliers. This list is intended to be illustrative and should not be construed as limiting.

The compositions of the present invention encompass polymers with or without secondary or higher order. For example, in one embodiment, the polymer comprises a sequence of a nucleoside, nucleoside analog, or combination of nucleoside and nucleoside analog that is capable of forming a secondary structure provided by at least two adjacent hydrogen bonded base pairs. In one embodiment, the at least two adjacent hydrogen-bonded base pairs involve at least 3 consecutive bases per group in both groups. The nature of the continuity of the bases involved is thermodynamically favourable for the formation of a so-called clamp (clamp). However, contiguous bases may not be necessary, particularly when having a high GC content and/or a longer sequence. Typically, there are 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 base pairs in the sequence. In one embodiment, the hydrogen bonded base pairs may be classical Watson-Crick base pairs, i.e., G-C, A-U or A-T. In other embodiments, the hydrogen bonded base pairs may be non-canonical base pairs, e.g., G-U, G-G, G-A or U-U. In other embodiments, the hydrogen bonded base pairs may be Hoogsteen base pairs or other base pairs.

In one embodiment, the secondary structure is a stem loop (b)stem-loop) secondary structure. The stem-loop secondary structure or hairpin secondary structure may be created by intramolecular hydrogen bonding base pairing between complementary or at least partially complementary sequences. The complementary sequence or the at least partially complementary sequence represents a fully inverted repeat sequence or an interrupted inverted repeat sequence, respectively. For example, having a structure represented by 5' -X₁-X₂-X₃...X₃′-X₂′-X₁'-3' (wherein X is a nucleotide sequence)₁And X₁′、X₂And X₂' and X₃And X₃Each pair of' can form hydrogen-bonded base pairs) can contain inverted repeat segments, complete or intermittent, and have the potential to fold upon itself and form stem-loop secondary structures. When more than two inverted repeat segments are present, the individual polymers can also interact to form not only dimeric intermolecular complexes but also higher order intermolecular complexes or structures. One skilled in the art will recognize that conditions and/or sequences may be selected to favor formation of one secondary structure type over another.

In one aspect, the invention provides conjugates of the immunostimulatory polymers of the invention with lipophilic moieties. In certain embodiments, the immunostimulatory polymer is covalently linked to a lipophilic moiety. Lipophilic moieties will typically be present at more than one end of an immunostimulatory ORN with a free end, but in certain embodiments, lipophilic moieties may be present elsewhere in the immunostimulatory polymer and thus do not require that the immunostimulatory ORN have a free end. In one embodiment, the immunostimulatory polymer has a3 'end and a lipophilic moiety is covalently attached to the 3' end. The lipophilic group may typically be cholesteryl, modified cholesteryl, cholesterol derivatives, reduced cholesterol, cholesterol with substituents, modified cholesterol, cholestane, C16 alkyl chain, bile acid, cholic acid, taurocholic acid, deoxycholate, oleyl lithocholic acid, oleoyl cholic acid, glycolipid, phospholipid, sphingolipid, isoprenoid such as steroid, vitamin E, saturated fatty acid, unsaturated fatty acids, fatty acid esters such as triglycerides, pyrene, porphyrin, Texaphyrine, adamantane, acridine, biotin, coumarin, fluorescein, rhodamine, Texas Red (Texas-Red), digoxin, dimethoxytrityl, t-butyldimethylsilyl, t-butyldiphenylsilyl, cyanine dyes (e.g., Cy3 or Cy5), Hoechst 33258 dyes, psoralen, or ibuprofen. In certain embodiments, the lipophilic moiety is selected from cholesteryl, palmitoyl, and fatty acyl. It is believed that the inclusion of one or more such lipophilic moieties in the immunostimulatory ORN of the invention imparts additional stability against nuclease degradation. When more than two lipophilic moieties are present in a single immunostimulatory polymer of the invention, each lipophilic moiety may be selected independently of the other.

In one embodiment, the lipophilic group is attached to the 2' -position of the nucleotide of the immunostimulatory polymer. The lipophilic group may alternatively or additionally be linked to the heterocyclic nucleobase of the nucleotide of the immunostimulatory polymer. The lipophilic moiety may be covalently linked to the immunostimulatory polymer via any suitable direct or indirect linkage. In one embodiment, the linkage is direct and is an ester or amide bond. In one embodiment, the linkage is an indirect linkage and includes a spacer moiety, for example, one or more abasic nucleotide residues, an oligoethylene glycol such as a tripeptide polyethylene glycol (spacer 9) or a hexa polyethylene glycol (spacer 18), or an alkyl diol such as butanediol.

In one embodiment, the immunostimulatory polymer of the invention is mixed with a cationic lipid. In one embodiment, the cationic lipid is DOTAP (N- [1- (2, 3-dioleoyloxy) propyl-1]-N, N-trimethylammonium methosulfate). It is believed that DOTAP transports the polymer into the cell and specifically to the endosomal compartment where it can be released in a pH-dependent manner. Once inside the endosomal lumen, the polymer can interact with certain intracellular TLRs, thereby triggering LTR-mediated signaling pathways involved in generating an immune response. Other agents with similar properties (including transport into the lumen) may be used in place of or in addition to DOTAP. OthersLipid formulations include, for example,(non-liposomal lipids with specific DNA condensation enhancers) and(novel action dendrimer technology),(charge-reversible particles that can become positively charged upon crossing cell membranes) and Stable Nucleic Acid Lipid Particles (SNALP) employing lipid bilayers. Liposomes are commercially available from Gibco BRL, for example,and LIPOFECTAACE^TMThey are prepared from, for example, N- [1- (2, 3-dioleoyloxy) -propyl]Cationic lipids such as-N, N, N-trimethylammonium chloride (DOTMA) and dimethyldioctadecyl ammonium bromide (DDAB). The preparation of liposomes is well known in the art and has been described in numerous publications. Furthermore, Gregoriadis G (1985) Trends Biotechnol 3: 235-241 have also been reviewed for liposomes. In other embodiments, the immunostimulatory polymers of the invention are conjugated to microparticles, cyclodextrins, nanoparticles, non-ionic vesicles, dendrimers, polycationic peptides, viral and viroid particles orAnd (4) mixing.

In one embodiment, the immunostimulatory polymer of the invention is in the form of a covalently closed dumbbell molecule with a primary and a secondary structure. As described below, in one embodiment, such oligoribonucleotides comprise two single-stranded loops joined by an intervening double-stranded segment. In one embodiment, at least one single stranded loop comprises an immunostimulatory RNA motif of the invention. Other covalently closed dumbbell molecules of the invention include chimeric DNA RNA molecules in which, for example, the double-stranded fragments are at least partially DNA (e.g., homodimeric dsDNA or heterodimeric DNA RNA) and at least one single-stranded loop comprises an immunostimulatory RNA motif of the invention. Alternatively, the double-stranded fragment of the chimeric molecule is RNA.

In certain embodiments, the immunostimulatory polymer is isolated. An isolated molecule is a molecule that is substantially pure and free of other substances with which it is typically found in natural or in vivo systems to an extent that is practical and appropriate for its intended use. In particular, the immunostimulatory polymers are sufficiently pure and carry sufficiently little other cellular biological components to be useful, for example, for the preparation of pharmaceutical preparations. Since the isolated immunostimulatory polymer of the invention may be blended with a pharmaceutically acceptable carrier in a pharmaceutical formulation, the immunostimulatory polymer may constitute only a minor weight percentage of the formulation. However, immunostimulatory polymers are generally pure in that they have been separated from materials that may be associated with them in biological systems.

For use in the present invention, any of a variety of methods known in the art can be used or modified to synthesize the immunostimulatory polymers of the invention de novo. For example, the β -cyanoethylphosphorimide method (Beaucage SL et al (1981) Tetrahedron Lett 22: 1859); the nucleoside H-phosphonate method (Garegg P et al (1986) Tetrahedron Lett 27: 4051-4; Froehler BC et al (1986) Nucl Acid Res 14: 5399-407; Garegg P et al (1986) Tetrahedron Lett 27: 4055-8; Gaffney BL et al (1988) Tetrahedron Lett 29: 2619-22). These chemical methods can be performed by various automatic nucleic acid synthesizers which are commercially available. Other synthetic methods useful in the present invention are disclosed in Uhlmann E et al (1990) Chem Rev 90: 544-84 and Goodchild J (1990) Bioconjugate Chem 1: 165.

Oligoribonucleotide synthesis can be performed in solution or on a solid phase matrix. In solution, block coupling reactions (dimers, trimers, tetramers, etc.) are preferred, while solid phase synthesis is preferably performed in a stepwise manner using monomer building blocks. Various chemical methods such as the phosphotriester method, the H-phosphonate method and the phosphoramidite method have been described (Eckstein F (1991) Oligonucleotides and nucleotides, A Practical Approach, IRL Press, Oxford). Although the reactive phosphorus group is in the + V oxidation state in the phosphotriester process, the more reactive phosphorus + III derivatives are used in the coupling reaction of the phosphoramidite process and the H-phosphate process. In the latter two methods, phosphorus is oxidized after the coupling step to yield stable p (v) derivatives. If the oxidizing agent is iodine/water/base, a phosphodiester is obtained after deprotection. Conversely, if the oxidizing agent is a sulfurizing agent such as Beaucage's reagent, a phosphorothioate is obtained after deprotection.

An efficient method of oligoribonucleotide synthesis is a combination of solid phase synthesis using phosphoramidite chemistry, which was originally described by Matteucci and Caruthers et al for oligodeoxynucleotides (Matteucci et al (1981) J Am Chem Soc 103: 3185).

Oligoribonucleotides are synthesized analogously to oligodeoxynucleotides, with the difference that the 2' -hydroxyl group present in oligoribonucleotides has to be protected by a suitable hydroxyl protecting group. The monomers in the RNA monomer building block can be protected, for example, by a 2' -O-tert-butyldimethylsilyl (TBDMS) group. However, it has been reported that the use of a compound containing a 2' -O-Triisopropylsiloxymethyl (TOM) group (TOM protecting group)^TM) The RNA synthesis of monomers of (a) will result in higher coupling efficiency, since the TOM protecting group shows lower steric hindrance than the TBDMS group. The TBDMS protecting group is removed by using fluoride, whereas for the TOM group rapid deprotection can be achieved using ethanol/water solution of methylamine at room temperature. In the oligo (ribonucleotide) nucleotide synthesis, a strand extension from the 3 'end to the 5' end is preferred, which is achieved by coupling a ribonucleotide unit having a3 '-phosphorus (III) group or an activated derivative thereof to the free 5' -hydroxy group of another nucleotide unit.

The synthesis can be conveniently performed using an automated DNA/RNA synthesizer. Thus, the synthesis cycle recommended by the synthesizer supplier can be used. The coupling time is longer for the ribonucleoside phosphoramidite monomer (e.g., 400 seconds) than for the deoxynucleoside monomer. As a solid matrix, canUse ofA Controlled Pore Glass (CPG) matrix or an organic polymer matrix such as a primer matrix PS200 (Amersham). The solid matrix typically contains a first nucleoside, for example, 5 '-O-dimethoxytrityl-N-6-benzyloxyadenosine, attached through its 3' end. After the cleavage of the 5 '-O-dimethoxytrityl group with trichloroacetic acid, for example, 5' -O-dimethoxytrityl-N protected 2 '-O-tert-butyldimethylsilyl nucleoside-3' -O-phosphoramidite is used. After successive repeated cycles, the completed oligoribonucleotides were excised from the matrix and deprotected by treatment with concentrated ammonia/ethanol (3: 1 by volume) at 30 ℃ for 24 hours. Finally, the TBDMS protecting group is cleaved off using triethylamine/HF. The crude oligoribonucleotides can be purified by ion exchange High Pressure Liquid Chromatography (HPLC), ion-pair reverse phase HPLC or polyacrylamide gel electrophoresis (PAGE) and characterized by mass spectrometry.

The synthesis of the 5 '-conjugate proceeds directly by coupling the phosphoramidite of the molecule to be linked to the 5' -hydroxyl of the terminal nucleotide in solid phase synthesis. Various phosphoramidite derivatives of ligands such as cholesterol, acridine, biotin, psoralen, ethylene glycol or aminoalkyl residues are commercially available. Alternatively, aminoalkyl functionality may be introduced during solid phase synthesis, which allows post-synthetic derivatization by activating linker molecules such as active esters, isothiocyanates, or iodoacetamides.

Synthesis of the 3' end conjugate is typically accomplished by using a correspondingly modified solid matrix (e.g., a commercially available cholesterol derivatized solid matrix). However, conjugation can also be performed at internucleotide linkages, nucleobases, or ribose residues (e.g., the 2' -position of ribose).

For cyclic oligoribonucleotides, extension of the oligonucleotide strand can be performed on a nucleotide PS solid substrate (Glen Research) using standard phosphoramidite chemistry methods. The cyclization reaction is then carried out on a solid substrate using phosphotriester coupling (Alazzouzi et al (1997) Nucleotides 16: 1513-14). After final deprotection with ammonium hydroxide, essentially the only product obtained from the solution is the desired cyclic oligonucleotide.

The cyclic oligoribonucleotides of the invention include RNA in closed circular form and may also include single-stranded RNA with or without double-stranded RNA. For example, in one embodiment, the cyclic oligoribonucleotide comprises a double stranded RNA and assumes a dumbbell conformation with two single stranded loops joined by an intervening double stranded fragment. Covalently closed dumbbell-shaped CpG oligodeoxynucleotides have been described in U.S. Pat. No. 6,849,725. In another embodiment, the cyclic oligoribonucleotide comprises a double stranded RNA and assumes a conformation with more than three single stranded loops connected by intervening double stranded fragments. In one embodiment, the immunostimulatory RNA motif is located in one or more single-stranded fragments.

The immunostimulatory polymers of the invention may be used alone or in combination with other agents such as adjuvants. An adjuvant as used herein refers to a substance other than an antigen that enhances the activation of immune cells in response to the antigen (e.g., a humoral immune response and/or a cellular immune response). Adjuvants promote the accumulation and/or activation of helper cells to enhance antigen-specific immune responses. Adjuvants are used to increase the efficacy of vaccines (i.e., antigen-containing compositions for inducing protective immunity against antigens).

Adjuvants can work by two general mechanisms, and a given adjuvant or adjuvant formulation can act by one or two mechanisms. The first mechanism is to physically influence the distribution of the antigen at the cells or sites that generate the antigen-specific immune response, and this mechanism can be a delivery vehicle that alters the biodistribution of the antigen (including targeting a particular area or cell type) or establishes a storage effect so that the antigen is slowly released in the body to prolong the immune cells' contact with the antigen.

Such adjuvants include, but are not limited to alum (aluminum hydroxide, aluminum phosphate); emulsion-type preparations including water-in-oil or oil-in-water emulsions made of mineral or non-mineral oils. These adjuvants may be oil-in-water emulsions, for example, Montanide ISA 720(Seppic, airliquid, paris, france); MF-59 (squalene-in-water emulsion stabilized with Span 85 and Tween 80; Chiron Corporation, Emeryville, Calif.); and PROVAX (stabilizing detergents and micelle-forming agents; IDEC Pharmaceuticals Corporation, San Diego, Calif.). These adjuvants may also be water-in-oil emulsions, for example Montanide ISA50 (an oily composition of mannide oleate and mineral oil, Seppic) or Montanide ISA 206 (an oily composition of mannide oleate and mineral oil, Seppic).

The second adjuvant mechanism is as an immune response modifier or immunostimulant. These lead to the activation of immune cells to better present, recognize or respond to antigens and thus improve the kinetics, magnitude, phenotype or memory (memory) of antigen-specific responses. Immune response modifiers generally act through specific receptors such as Toll-like receptors or one of several other non-TLR pathways (e.g., RIG-I), however pathways are not known for certain immune response modifiers. Such adjuvants include, but are not limited to, saponins purified from the bark of the quillaja (q. saponaria tree) (e.g., QS21, a glycolipid that elutes at peak 21 when fractionated by HPLC; antipenigenics, inc., Worcester, ma), poly [ di (carboxyphenoxy) ] phosphazene (PCPP polymer; Virus Research Institute, USA), Flt3 ligand, and leishmania elongation factor (purified leishmania protein; Corixa Corporation, Seattle, washington).

There are many adjuvants that act through TLRs. Adjuvants that act through TLR4 include lipopolysaccharide derivatives such as monophosphoryl lipid A (MPL; Ribi ImmunoChem Research, Inc., Hamilton, Calif.) and muramyl dipeptide (MDP; Ribi); OM-174 (glucosamine disaccharide related to lipid A; OM Pharma SA, Meyrin, Switzerland). Flagellin is an adjuvant that acts through TLR 5. Double stranded RNA functions through TLR 3. Adjuvants that act through TLR7 and/or TLR8 include single stranded RNA or Oligoribonucleotides (ORN) and synthetic small molecule weight compounds that recognize and activate TLRs including imidazoquinoline amines (e.g., imiquimod, ranisimmod; 3M). Adjuvants that act through TLR9 include viral or bacterial derived DNA or synthetic Oligodeoxynucleotides (ODNs) such as CpG ODN.

Adjuvants that have both a physical effect and an immunostimulating effect are those compounds that have both of the above functions. Such adjuvants include, but are not limited to, ISCOMS (an immunostimulatory complex containing mixed saponins, lipids and forming virus-sized particles with pores capable of accommodating antigens; CSL, Melbourne, Australia), Pam3Cys, SB-AS2(SmithKline Beecham adjuvant System #2, an oil-in-water emulsion containing MPL and QS 21; SmithKline Beecham Biologicals [ SBB ], Rixendart, Belgium), SB-AS4(SmithKline Beecham adjuvant System #4, containing alum and MPL; SBB, Belgium), micelle-forming nonionic block copolymers such AS CRL and the like (these copolymers contain linear hydrophobic polyoxypropylene flanked by polyoxyethylene chains, Vaxcel, Inc., Norcross, Zodiac) and Syntex adjuvant formulations (Tween 1005, Tween 80 and nonionic block copolymers; SAF oil emulsions, SAF, oil-in-water emulsions, Biochemical, colloidal immune stimulating agent based nanoparticles such AS NanoLauter, Inc, Na-Ionic adjuvants 1312, seppic) and many of the transport vehicles described below.

Furthermore, the invention provides a composition comprising an immunostimulatory polymer of the invention and a further adjuvant, wherein the further adjuvant is a cationic polysaccharide such as chitosan or a cationic peptide such as protamine, a polyester, polylactic acid, polyglycolic acid or a copolymer of one or more of the above.

In addition, the present invention also provides a composition comprising the immunostimulatory polymer of the invention and another adjuvant, wherein the other adjuvant is a cytokine. In one embodiment, the composition is a conjugate of an immunostimulatory polymer of the invention and a cytokine.

Cytokines are soluble proteins and glycoproteins produced by many cell types that mediate inflammatory and immune responses. Cytokines mediate communication between cells of the immune system, acting locally as well as systemically to recruit cells and regulate their function and proliferation. Classes of cytokines include mediators and modulators of innate immunity, mediators and modulators of adaptive immunity, and hematopoietic stimulants. Included among the cytokines are interleukins (e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, and interleukins 19-32 (IL-19-L-32), etc.), chemokines (e.g., IP-10, RANTES, MIP-1, MIP-3, MCP-1, MCP-2, MCP-3, MCP-4, eotaxin, I-TAC, and BCA-1, etc.), and type 1 interferons (e.g., IFN- α and IFN- β), type 2 interferons (e.g., IFN-gamma), tumor necrosis factor alpha (TNF-alpha), transforming growth factor beta (TGF-beta), and other cytokines of various colony stimulating factors, including GM-CSF, G-CSF, and M-CSF.

In addition, the present invention provides compositions comprising an immunostimulatory polymer of the invention and an immunostimulatory CpG nucleic acid. In one embodiment, the composition is a conjugate of an immunostimulatory polymer of the invention and a CpG nucleic acid, e.g., an RNA to DNA conjugate.

Immunostimulatory CpG nucleic acids, as used herein, refer to natural or synthetic DNA sequences that contain CpG motifs and stimulate activation or proliferation of cells of the immune system. Immunostimulatory CpG nucleic acids have been described in a number of issued patents, published patent applications, and other publications, including U.S. Pat. nos. 6,194,388, 6,207,646, 6,214,806, 6,218,371, 6,239,116, and 6,339,068. In one embodiment, the immunostimulatory CpG nucleic acid is a CpG oligodeoxynucleotide (CpG ODN) of 6 to 100 nucleotides in length. In one embodiment, the immunostimulatory CpG nucleic acid is a CpG oligodeoxynucleotide (CpG ODN) of 8 to 40 nucleotides in length.

In certain embodiments, the polymer comprises CG dinucleotides. In other embodiments, the polymer is free of CG dinucleotides.

Immunostimulatory CpG nucleic acids include different classes of CpG nucleic acids. A family of CpG nucleic acids can potently activate B cells but are relatively weak for inducing IFN- α and NK cell activation; this family has been referred to as B family. The B-class CpG nucleic acids are generally fully stabilized and contain unmethylated CpG dinucleotides in certain preferred base contexts. See, for example, U.S. Pat. nos. 6,194,388, 6,207,646, 6,214,806, 6,218,371, 6,239,116, and 6,339,068. Another family of CpG nucleic acids can strongly induce IFN- α and NK cell activation but are relatively weak for stimulating B cells; this family has been referred to as family a. The a-group CpG nucleic acids typically have a palindromic sequence of at least 6 nucleotides containing a phosphodiester CpG dinucleotide and have a stabilized poly G sequence at their 5 'and/or 3' ends. See, for example, published international patent application WO 01/22990. There is also a family of CpG nucleic acids that activate B cells and NK cells and induce IFN- α; this family has been referred to as family C. The first characterized C-group CpG nucleic acids are generally fully stabilized, containing a B-group type sequence and a GC-rich palindromic or near-palindromic sequence. This family of CpG nucleic acids is described in published U.S. patent application 2003/0148976, which is incorporated herein by reference in its entirety.

Immunostimulatory CpG nucleic acids also include so-called soft CpG nucleic acids and semi-soft CpG nucleic acids as disclosed in published U.S. patent application 2003/0148976, the entire contents of which are incorporated herein by reference. These soft and semi-soft immunostimulatory CpG nucleic acids contain nuclease-resistant and nuclease-sensitive internucleotide linkages, wherein the different types of linkages are arranged according to specific rules.

In one aspect, the invention provides a vaccine comprising an antigen and an immunostimulatory polymer of the invention. "antigen" as used herein refers to any molecule that is recognized by a T cell antigen receptor or B cell antigen receptor. The term broadly includes any type of molecule that is recognized by the host immune system as a foreign object. Antigens generally include, but are not limited to, cells, cell extracts, proteins, polypeptides, peptides, polysaccharides, polysaccharide conjugates, peptidomimetics and non-peptidomimetics of polysaccharides and other molecules, small molecules, lipids, glycolipids, polysaccharides, sugars, viruses and viral extracts, and multicellular organisms such as parasites, as well as allergens. For antigens that are proteins, polypeptides, or peptides, the antigens may comprise nucleic acid molecules encoding the antigens. More specifically, antigens include, but are not limited to: a cancer antigen comprising a cancer cell and a molecule expressed in or on the cancer cell; microbial antigens including microorganisms and molecules expressed in or on microorganisms; allergens and other disease-associated molecules such as autoreactive T cells. Thus, in certain embodiments, the invention provides vaccines for cancer, infectious disease, allergy, addiction, disease caused by aberrantly folded proteins, autoimmune disease and cholesterol management.

Vaccines against infectious diseases may be prophylactic or therapeutic. The antigen in the vaccine may be whole live (attenuated), whole killed/inactivated, recombinant inactivated, subunit purified, subunit recombinant or a peptide. The vaccine may also comprise additional adjuvants or combinations of adjuvants. The additional adjuvant may be an adjuvant with a storage effect (e.g., alum) and an immunomodulatory agent (e.g., another TLR agonist or an immunomodulatory agent that acts through a non-TLR pathway) or as an immunostimulatory complex: () And the like having both effects. Adjuvants will be described in more detail below.

Vaccines against cancer may also be prophylactic or therapeutic. The cancer antigen may be whole cells (individual DC vaccines) or one or more polypeptides or peptides. These cancer antigens are typically attached to a carrier molecule. The vaccine may also comprise additional adjuvants or combinations of adjuvants such as those described above. Cancer antigens will be described in more detail below.

For vaccines used to treat allergy, the antigen is an allergen or a portion of an allergen. The allergen may be contained within or attached to the delivery vehicle. The allergen may be linked to an immunostimulatory polymer. The allergens will be described in more detail below.

Vaccines for the treatment of addiction may be used to treat addiction, for example nicotine addiction, cocaine addiction, methamphetamine or heroin addiction. In these cases, the addictive molecule is a proto-molecule or a hapten. The "antigen" for inclusion in a vaccine against an addictive disorder is typically a small molecule and may be conjugated to a carrier protein or other carrier particle, or may be incorporated into a viroid particle.

Vaccines for the treatment of diseases caused by abnormally folded proteins may be used for the treatment of diseases such as transmissible spongiform encephalopathies (variants of Creutzfeld-Jakob disease). In this case, the "antigen" is an itch prion (scrapie virus), which may be attached to a carrier protein or a live attenuated vector. An example of a vaccine against alzheimer's disease is, for example, a vaccine targeting beta-amyloid peptide or protein.

Vaccines for treating autoimmune diseases are also provided. These vaccines are useful in the treatment of autoimmune diseases in which molecules recognized by autoimmune cells have been identified. For example, vaccines against autoreactive T cells in response to myelin can be used to treat multiple sclerosis.

Vaccines useful for treating cardiovascular diseases and conditions are also provided. The vaccine can target molecules known to contribute to the disease development, such as lipoproteins, cholesterol and molecules involved in cholesterol metabolism. Vaccines for managing cholesterol may comprise, for example, Cholesterol Ester Transfer Protein (CETP) as an antigen. CETP promotes the exchange of cholesterol from anti-arteriosclerotic apolipoprotein a-I containing HDL particles to arteriosclerotic apolipoprotein B containing VLDL and LDL. Such vaccines can be used to treat high cholesterol or slow the progression of arteriosclerosis. The vaccines can be used to treat other cardiovascular diseases and conditions in which the target molecule is known.

In one aspect, the invention provides the use of an immunostimulatory polymer of the invention in the manufacture of a medicament for immunising a subject.

In one aspect, the invention provides a method of making a vaccine. The method comprises bringing into close association an immunostimulatory polymer of the invention with an antigen and optionally a pharmaceutically acceptable carrier.

In certain embodiments, the immunostimulatory polymer is conjugated to an antigen or allergen. The antigen and the immunostimulatory polymer may be directly conjugated or they may be indirectly conjugated through a linker.

As used herein, "microbial antigens" are antigens of microorganisms and include, but are not limited to, viruses, bacteria, parasites, and fungi. Such antigens include non-invasive microorganisms as well as natural isolates and fragments or derivatives thereof, as well as synthetic compounds that are the same as or similar to natural microbial antigens and induce an immune response specific for the microorganism. A compound is similar to a native microbial antigen if it induces an immune response (humoral and/or cellular) to the native microbial antigen. Such antigens are routinely used in the art and are well known to those of ordinary skill in the art.

Viruses are small infectious agents that typically comprise a nucleic acid core and a protein capsid, but are not independent organisms. The virus can also take the form of an infectious nucleic acid lacking the protein. The virus cannot survive in the absence of viable cells in which it can replicate. Viruses enter specific living cells by endocytosis or direct DNA (phage) injection and multiply resulting in disease. The propagated virus can then be released and infect other cells. Some viruses are DNA-containing viruses and others are RNA-containing viruses. In certain aspects, the invention is also intended to treat diseases in which the disease progression involves a prion, for example, bovine spongiform encephalopathy (i.e., mad cow disease, BSE) or pruritic infections in animals or creutzfeldt-jakob disease in humans.

Viruses include, but are not limited to, enteroviruses (including, but not limited to, picornaviridae, e.g., poliovirus, coxsackievirus, echovirus), rotaviruses, adenoviruses, and hepaciviruses (e.g., hepatitis a, hepatitis b, hepatitis c, hepatitis d, and hepatitis e). Specific examples of viruses that have been found in humans include, but are not limited to: retroviridae (e.g., human immunodeficiency viruses such as HIV-1 (also known as HTLV-III, LAV or HTLV-III/LAV or HIV-III) and other isolated strains such as HIV-LP); picornaviridae (e.g., poliovirus, hepatitis a virus; enterovirus, human coxsackievirus, rhinovirus, echovirus); caliciviridae (e.g., strains responsible for gastroenteritis); togaviridae (e.g., equine encephalitis virus, rubella virus); flaviviridae (e.g., dengue virus, encephalitis virus, yellow fever virus); coronaviridae (e.g., coronaviruses); rhabdoviridae (e.g., vesicular stomatitis virus, rabies virus); filoviridae (e.g., ebola virus); paramyxoviridae (e.g., parainfluenza virus, mumps virus, measles virus, respiratory syncytial virus); orthomyxoviridae (e.g., influenza virus); bunyaviridae (e.g., hantavirus, bunyavirus, phlebovirus, and nairovirus); arenaviridae (hemorrhagic fever virus); reoviridae (e.g., reoviruses, circoviruses, and rotaviruses); binuclear glyconucleoviridae; hepadnaviridae (hepatitis b virus); parvoviridae (parvoviruses); papovaviridae (papillomavirus, polyomavirus); adenoviridae (most adenoviruses); herpesviridae (herpes simplex virus (HSV)1 and 2, herpes zoster virus, Cytomegalovirus (CMV)); poxviridae (variola virus, vaccinia virus, poxvirus); iridoviridae (e.g., african swine fever virus); and other viruses such as acute laryngotracheobronchitis virus, alphavirus, Kaposi sarcoma-associated herpesvirus, Newcastle disease virus, Nipah virus (Nipah virus), Norwalk virus, papilloma virus, parainfluenza virus, avian influenza virus, SARS virus, West Nile virus.

Bacteria are unicellular organisms that reproduce asexually by binary division. Cells are classified and named based on their morphology, staining reactions, nutrient and metabolic requirements, antigenic structure, chemical composition, and genetic homology. Cells can be divided into three groups based on their morphological form, i.e., coccoid (cocci), straight rod (bacilli) and rod-shaped or helical (vibrio, campylobacter, spirochete and spirochete). It is more common to characterize cells as two classes of organisms, gram positive and gram negative, based on their staining reactions. Gram (Gram) refers to a staining method that is commonly performed in microbiological laboratories. Gram-positive organisms remain stained and appear dark purple after the staining step. Gram-negative organisms do not remain stained but take up a counterstain (counter-stain) and thus appear pink.

Infectious bacteria include, but are not limited to, gram-negative and gram-positive bacteria. Gram-positive bacteria include, but are not limited to, the genera pasteurella, staphylococcus, and streptococcus. Gram-negative bacteria include, but are not limited to, Escherichia coli, Pseudomonas and Salmonella. Specific examples of infectious bacteria include, but are not limited to: helicobacter pylori, legionella pneumophila, mycobacteria (e.g., mycobacterium tuberculosis, mycobacterium avium, mycobacterium intracellulare, mycobacterium kansasii, mycobacterium gordonae), staphylococcus aureus, neisseria gonorrhoeae, neisseria meningitidis, listeria monocytogenes, streptococcus pyogenes (group a streptococcus), streptococcus agalactiae (group B streptococcus), streptococcus (grass green), streptococcus faecalis, streptococcus bovis, streptococcus (anaerobe), streptococcus pneumoniae, pathogenic campylobacter, enterococcus, haemophilus influenzae, bacillus anthracis, corynebacterium diphtheriae, corynebacterium erysipelas, clostridium perfringens, clostridium tetani, enterobacter aerogenes, klebsiella pneumoniae, pasteurella multocida, bacteroides, fusobacterium nucleatum, streptococcus moniliformis, mycobacterium tuberculosis, mycobacterium, streptococcus pyogenes, streptococcus pneumoniae, streptococcus sobrinus, streptococcus kaempferi, Treponema pallidum, Treponema paucicostatum, Leptospira leptospira, rickettsia and Actinomyces israeli.

Parasites are organisms that depend on other organisms for survival, and therefore they must enter or infect another organism to continue their life cycle. The infected organism (i.e., the host) provides nutrients and habitat for the parasites. Although the term parasite in its broadest sense may include all infectious agents (i.e., bacteria, viruses, fungi, protozoa, and worms), in general, the term is used to refer only to protozoa, worms, and ectoparasitic arthropods (e.g., ticks, mites, etc.). Protozoa are unicellular organisms that replicate both intracellularly and extracellularly, particularly in the extracellular matrix of blood, intestinal tract or tissues. Helminths are multicellular organisms that are almost always extracellular (one exception being trichina). A worm typically needs to leave the original host and propagate to a secondary host for replication. In contrast to these types, ectoparasitic arthropods form a parasitic relationship with the exterior surface of the host body.

Parasites include intracellular parasites and obligate intracellular parasites. Examples of parasites include, but are not limited to, Plasmodium falciparum, Plasmodium ovale, Plasmodium malariae, Plasmodium vivax, Plasmodium knowlesi, Paecilomyces minutissima, Paecilomyces divergens, Trypanosoma cruzi, Toxoplasma gondii, Trichinella spiralis, Leishmania major, Leishmania donovani, Leishmania brasiliensis, Leishmania tropicalis, Trypanosoma gambiae, Trypanosoma rhodesiense, and Schistosoma mansoni.

Fungi are eukaryotic organisms, and only a few fungi cause infections in vertebrate mammals. Since fungi are eukaryotic organisms, they differ significantly from prokaryotic bacteria in size, structural composition, life cycle and reproduction machinery. Fungi are generally classified based on their morphological characteristics, mode of propagation and culture characteristics. Although fungi may cause different types of diseases in a subject (e.g., respiratory allergies caused by inhalation of fungal antigens, fungal poisoning caused by ingestion of toxic substances such as the toxins of the virulent mushrooms, Amanita phaseoloides and muscarinic peptides, and aflatoxins produced by aspergillus species), not all fungi cause infectious diseases.

Infectious fungi can cause systemic or superficial infections. Primary systemic infections may occur in normal healthy subjects, while opportunistic infections are most commonly seen in immunocompromised subjects. The most common fungal species responsible for primary systemic infections include blastomycosis, coccidioidomycosis and histoplasmosis. Common fungi that cause opportunistic infections in immunocompromised or immunosuppressed subjects include, but are not limited to, candida albicans, cryptococcus neoformans, and various aspergillus species. Systemic fungal infections are invasive infections of the internal organs. Fungal organisms typically enter the body via the lungs, gastrointestinal tract, or intravenous catheters. These types of infections can be caused by primary pathogenic fungi or opportunistic fungi.

Superficial fungal infections involve fungal growth on the external surface without invading internal tissues. Typical superficial fungal infections include cutaneous fungal infections involving the skin, hair or nails.

Diseases associated with fungal infections include aspergillosis, blastomycosis, candidiasis, chromoblastomycosis, coccidioidomycosis, cryptomycosis, fungal eye infections, fungal hair, nail and skin infections, histoplasmosis, lobomycosis, mycetoma locoregionium, mycosis fungoides, paracoccidiomycosis, disseminated penicilliosis marneffei, hyphomycosis, nosediomycosis, sporotrichosis, and zygomycosis.

Other medically relevant microorganisms have been extensively described in the literature, see, for example, c.g. a. thomas, Medical Microbiology, baillire Tindall, uk 1983, the entire contents of which are incorporated herein by reference. The foregoing lists are illustrative and not intended to be limiting.

As used herein, the terms "cancer antigen" and "tumor antigen" are used interchangeably to refer to a compound, such as a peptide, protein or glycoprotein, associated with a tumor cell or cancer cell that is capable of eliciting an immune response in the context of a Major Histocompatibility Complex (MHC) molecule when expressed on the surface of an antigen presenting cell. Cancer antigens that are differentially expressed by cancer cells can thus be exploited to target cancer cells. Cancer antigens are potentially capable of stimulating a significant tumor-specific immune response. Certain cancer antigens, while not necessarily expressed by normal cells, are encoded by normal cells. These antigens can be characterized as antigens that are normally silenced (i.e., not expressed) in normal cells, antigens that are expressed only at certain differentiation stages, and antigens that are expressed at certain times, such as embryonic and fetal antigens. Other cancer antigens are encoded by mutated cellular genes such as oncogenes (e.g., activated ras oncogene), suppressor genes (e.g., mutant p53), fusion proteins resulting from internal deletions or chromosomal translocations, and the like. Still other cancer antigens may be encoded by viral genes such as those carried on RNA and DNA tumor viruses.

Cancer antigens can be prepared from Cancer cells by preparing crude extracts of Cancer cells (e.g., as described in Cohen PA et al (1994) Cancer Res 54: 1055-8), by partially purifying the antigen, by recombinant techniques, or by de novo synthesis of known antigens. Cancer antigens include, but are not limited to, recombinantly expressed antigens, immunogenic portions thereof or the entire tumor or cancer or cells thereof. These antigens may be isolated or prepared recombinantly or by any other method known in the art.

Examples of tumor antigens include MAGE, MART-1/Melan-A, gp100, dipeptidyl peptidase IV (DPPIV), adenosine deaminase binding protein (ADAbp), cyclophilin b, large intestine-associated antigen (CRC) - - -C017-1A/GA733, carcinoembryonic antigen (CEA) and its immunogenic epitopes CAP-1 and CAP-2, etv6, aml1, Prostate Specific Antigen (PSA) and its immunogenic epitopes PSA-1, PSA-2 and PSA-3, Prostate Specific Membrane Antigen (PSMA), T cell receptor/CD 3-zeta chain, the MAGE tumor antigen family (e.g., MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, and fragment thereof, MAGE-Xp2(MAGE-B2), MAGE-Xp3(MAGE-B3), MAGE-Xp4(MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5), the GAGE tumor antigen family (e.g., GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9), BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, tyrosinase, p53, MUC family, HER2/neu, p21ras, RCAS1, alpha-fetoprotein, E-cadherin, alpha-catenin, beta-catenin and gamma-catenin, ctn 120, gp100, and so on^Pmel117PRAME, NY-ESO-1, cdc27, adenomatous polyposis coli protein (APC), fodrin, Connexin 37(Connexin 37), Ig idiotypes, P15, gp75, GM2 and GD2 gangliosides, viral products such as human papillomavirus proteins, the Smad tumor antigen family, lmp-1, P1A, EBV encoded nuclear antigen (EBNA) -1, brain glycogen phosphorylase, SSX-1, SSX-2(HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1 and CT-7, and c-erbB-2. This list is not intended to be limiting.

As used herein, an "allergen" is a molecule capable of eliciting an immune response characterized by the production of IgE. An allergen is also a substance that can induce an allergic or asthmatic response in a susceptible subject. Thus, in the context of the present invention, the term allergen refers to a specific type of antigen capable of eliciting an allergic response mediated by IgE antibodies.

The list of allergens is large and can include pollen, insect venom, animal dander, fungal spores and drugs (e.g., penicillin). Examples of natural animal and plant allergens include proteins specific to the following genera: canis (family canines); dermatophagoides (e.g., dust mites); fel (domestic cat); ragweed (ambrosia americana); lolium (e.g., perennial ryegrass and lolium multiflorum); genus cryptomeria (cryptomeria japonica); alternaria (Alternaria alternata)); alder genus; alnus japonica (Alnus gultinosa); betula (betula verrucosa); quercus (quercus alba); trifolium (olive); artemisia (artemisia); psyllium (e.g., Plantago lanceolata); pellitorium (e.g., Parietaria officinalis (Parietaria officinalis) and yarrow); cockroaches (e.g., german cockroach); genus Apis (e.g., honeybee (Apis multiflorum)); cypress (e.g., cypress, green dried cypress, and arborvitae); sabina (e.g., Juniperus sabinoides, selaginella, juniper, and Juniperus ashei), thuja (e.g., eastern Thuja orientalis), sabina (e.g., Japanese cypress), periplaneta (e.g., Periplaneta americana), agropyron (e.g., creeping wheatgrass), rye (e.g., rye), triticum (e.g., wheat), cocksfoot (e.g., dactylis glomerata), fescue (e.g., oxtail grass), Poa (e.g., Poa pratensis and Poa canadensis), Avena (e.g., oat), Nagrota (e.g., Naphalaris), Cyanota (e.g., Naemarrhiza villosa), Cymbopogon (e.g., Cymbopogon citratus), Avena (e.g., Ovatia), Avena (e.g., Cymbopogon citratus), Avena (e) (e.g., Oryza canadensis), Oenoma (e), Oenothera (e) (e.g., Tribute), sorghum halepense) and bromus (e.g., bromus formosanus).

In one aspect, the invention provides conjugates of the immunostimulatory polymer of the invention and an antigen. In one embodiment, the immunostimulatory polymer of the invention is covalently linked to an antigen. The covalent linkage between the immunostimulatory polymer and the antigen (covalent linkage) may be any suitable type of covalent linkage, provided that the immunostimulatory polymer and the antigen retain a measurable functional activity of the individual components when so linked. In one embodiment, the covalent linkage is a direct linkage. In another embodiment, the covalent linkage is an indirect linkage, e.g., through a linker moiety. The covalently linked immunostimulatory polymer and antigen may be treated intracellularly to release one from the other. In this way, delivery of individual components to cells may be improved compared to delivery if administered as separate formulations or separate components. In one embodiment, the antigen is the antigen itself, i.e., it is a preformed antigen.

In one aspect, the present invention provides a pharmaceutical composition comprising a composition of the present invention and a delivery vehicle. In various embodiments, the delivery vehicle may be selected from the group consisting of cationic lipids, liposomes, lipid helices (cochleates), virosomes, immunostimulatory complexes (c: (a) (a))) Microparticles, microspheres, nanospheres, unilamellar vesicles (LUVs), multilamellar vesicles, emulsified particles (emulsomes), and polycationic peptides, lipid complexes, polymer complexes, lipopolymer complexes, water-in-oil (W/O) emulsions, oil-in-water (O/W) emulsions, water-in-oil-in-water (W/O/W) multiple emulsions, microemulsions, nanoemulsions, micelles, dendrimers, virosomes, viroids, polymeric nanoparticles (e.g., nanospheres or nanocapsules)) Polymeric microparticles (e.g. microspheres or microcapsules), chitosan, cyclodextrins, non-ionic vesicles orAnd optionally a pharmaceutically acceptable carrier.

The pharmaceutically acceptable carrier is discussed below. The pharmaceutical composition of the present invention may further comprise an antigen, if necessary. The compositions of the invention and the antigen (when present) are combined with the delivery vehicle using any suitable method. The immunostimulatory composition may be contained within the delivery vehicle or may be presented or attached to the surface of the delivery vehicle that is exposed to the solvent. In one embodiment, the immunostimulatory polymer is presented or attached to the solvent-exposed surface of the delivery vehicle, while the antigen (when present) is contained within the delivery vehicle. In another embodiment, both the immunostimulatory polymer and the antigen are presented or associated with the solvent-exposed surface of the delivery vehicle. In yet another embodiment, the antigen is presented or attached to the solvent-exposed surface of the delivery vehicle, and the immunostimulatory polymer is contained within the delivery vehicle. In yet another embodiment, both the immunostimulatory polymer and the antigen (if included) are contained within a delivery vehicle.

The invention also provides methods of using the immunostimulatory compositions of the invention. In one aspect, the invention provides methods of activating immune cells. The method according to this aspect of the invention comprises contacting an immune cell in vitro or in vivo with an effective amount of a composition of the invention in order to activate the immune cell. The composition of the present invention may contain an antigen, if necessary. As used herein, "immune cell" refers to any bone marrow-derived cell that is capable of participating in a innate immune response or adaptive immune response. Cells of the immune system include, but are not limited to, Dendritic Cells (DCs), Natural Killer (NK) cells, monocytes, macrophages, granulocytes, B lymphocytes, plasma cells, T lymphocytes, and their precursor cells. In one embodiment, the immune cell is an immune cell capable of producing IFN- α, e.g., a plasmacytoid dendritic cell (pDC). In certain embodiments, the immune cell is a TLR7 expressing cell. In the context of the present invention, the method does not include formulation with lipofectin in an amount effective to induce therapeutically significant IFN- α production. In one embodiment, the immune cells do not produce therapeutically significant amounts of TNF-a in response to the polymer.

As used herein, the term "effective amount" refers to an amount of a substance necessary or sufficient to produce a desired biological effect. An effective amount may be, but is not necessarily limited to, an amount administered in a single administration.

In one embodiment, the compositions of the present invention can be used to activate immune cells by inducing the cells to enter an activated state that correlates with immune response. Activating immune cells refers to both eliciting and enhancing immune response. As used herein, the term "immune response" refers to any aspect of a innate immune response or adaptive immune response that reflects the proliferation, execution of effector immune functions, or production of immune cells involved in the activation of immune response gene products. Gene products involved in immune responses may include secreted products (e.g., antibodies, cytokines, and chemokines), as well as intracellular and cell-surface molecules characteristic of immune function (e.g., certain antigen Clusters of Differentiation (CDs), transcription factors, and gene transcripts). The term "immune response" may be used for a single cell or a population of cells.

Cytokine production can be assessed by any of several methods known in the art, including biological response assays, enzyme-linked immunosorbent assays (ELISAs), intracellular fluorescence-activated cell sorting (FACS) analysis, and reverse transcriptase/polymerase chain reaction (RT-PCR). In one embodiment, the immune response involves the production of IFN- α.

In one embodiment, the immune response involves the upregulation of cell surface markers (e.g., CD25, CD80, CD86, and CD154) activated by immune cells. Methods for determining cell surface expression of such markers are well known in the art and include FACS analysis.

To determine an immune response in a cell or population of cells, in one embodiment, the cell or population of cells expresses TLR 7. The cell may naturally express the TLR, or the cell may be manipulated to express the TLR by introducing into the cell a suitable expression vector for the TLR. In one embodiment, the cell or population of cells is taken as Peripheral Blood Mononuclear Cells (PBMCs). In one embodiment, the cell or population of cells is obtained as a cell line expressing the TLR. In one embodiment, the cell or population of cells is obtained as a transient transfectant that expresses the TLR. In one embodiment, the cell or population of cells is obtained as a stable transfectant that expresses the TLR.

Furthermore, for use in determining an immune response within a cell or population of cells, it is convenient to introduce into the cell or population of cells a reporter (reporter) construct responsive to intracellular signaling of a TLR. In one embodiment, such a reporter is a gene placed under the control of the NF-. kappa.B promoter. In one embodiment, the gene placed under the control of the promoter is luciferase. Under appropriate activation conditions, the luciferase reporter construct is expressed and emits a detectable light signal that can be quantified using a luminometer. Such reporter constructs and other suitable reporter constructs are commercially available.

The invention also contemplates the use of a cell-free TLR activation assay.

In certain aspects, the invention relates to compositions and methods for use in therapy. The immunostimulatory compositions of the invention may be used alone or in combination with other therapeutic agents. The immunostimulatory composition may be administered simultaneously or sequentially with the other therapeutic agent. When the immunostimulatory composition and other therapeutic agent are administered concurrently, they may be administered in the same or different formulations, but at the same time. In addition, when the immunostimulatory composition and the other therapeutic agent are administered simultaneously, they may be administered by the same or different routes of administration, but at the same time. When the administration of the immunostimulatory composition of the invention is separated in time from the administration of the other therapeutic agent, the immunostimulatory composition of the invention is administered sequentially with the other therapeutic agent. The time interval between administration of these compounds may be in minutes or may be longer. In one embodiment, the immunostimulatory composition of the invention is administered prior to administration of the other therapeutic agent. In one embodiment, the immunostimulatory composition of the invention is administered after administration of the additional therapeutic agent. In addition, when the immunostimulatory composition of the invention is administered sequentially with other therapeutic agents, it may be administered by the same or different route of administration. Other therapeutic agents include, but are not limited to, adjuvants, antigens, vaccines, and drugs that can be used to treat infections, cancer, allergies, and asthma.

In one aspect, the invention provides a method of vaccinating a subject. The method according to this aspect of the invention comprises administering to the subject an antigen and a composition of the invention. In one embodiment, administration of the antigen comprises administration of a nucleic acid encoding the antigen.

As used herein, a "subject" refers to a vertebrate. In various embodiments, the subject is a human, non-human primate, or other mammal. In certain embodiments, the subject is a mouse, rat, guinea pig, rabbit, cat, dog, pig, sheep, goat, cow, or horse.

For use in vaccination of a subject, in one embodiment, the composition of the invention comprises an antigen. The antigen may be isolated from the polymer of the invention or covalently linked thereto. In one embodiment, the composition of the invention does not itself comprise the antigen. In this embodiment, the antigen may be administered to the subject separately from the composition of the invention or co-administered with the composition of the invention. Separate administration includes temporal separation, separation of the site or route of administration, or separation of both the site or route of administration. When the composition of the invention is administered separately in time from the antigen, the antigen may be administered before or after the composition of the invention. In one embodiment, the antigen is administered 48 hours to 4 weeks after administration of the composition of the invention. The methods also contemplate administration of one or more booster doses of the antigen alone, the composition alone, or both after initial administration of the antigen and composition.

The invention also contemplates preparing a subject for future encounter with an unknown antigen by administering to the subject a composition of the invention, wherein the composition does not comprise an antigen. According to this embodiment, the subject is prepared to produce a more robust response to antigens that the subject later encounters (e.g., by environmental or occupational exposure). Such methods may be used, for example, by travelers, medical workers, and soldiers who are susceptible to microbial exposure.

In one aspect, the invention provides a method of treating a subject having an infection. The method according to this aspect of the invention comprises administering to a subject having an infection an effective amount of a composition of the invention and an infectious agent to treat the subject.

In one aspect, the invention provides the use of an immunostimulatory polymer of the invention in the manufacture of a medicament for treating an infection in a subject.

In one aspect, the invention provides compositions useful for treating infections. The composition according to this aspect comprises the immunostimulatory polymer of the invention and an infectious agent.

As used herein, the term "treating" with respect to a subject having a disease or condition shall mean preventing, ameliorating, or eliminating at least one sign or symptom of the disease or condition in the subject.

A subject with an infectious disease is a subject with a condition resulting from superficial, local or systemic invasion by its infectious microbe. The infectious microorganism may be a virus, bacterium, fungus or parasite as described above.

Infectious agents include, but are not limited to, antibacterial agents, antiviral agents, antifungal agents, and antiparasitic agents. Such as "anti-infective agents", "antibiotics", "antibacterial agents", "antiviral agents", "antifungal agents", "antiparasitic agents" and "parasiticides" have well-established meanings to those of ordinary skill in the art and are defined in standard medical texts. Briefly, antibacterial agents kill or inhibit bacteria and include antibiotics and other synthetic or natural compounds with similar functions. Antiviral agents may be isolated from natural sources or synthesized and may be used to kill or inhibit viruses. Antifungal agents are useful in the treatment of superficial fungal infections as well as opportunistic and primary systemic fungal infections. Antiparasitic agents kill or inhibit parasites. Many antibiotics are low molecular weight molecules produced by cells such as microorganisms as secondary metabolites (secondary metabolites). Typically, the antibiotic interferes with one or more functions or structures that are specific to the microorganism but not present in the host cell.

One of the problems with anti-infective therapy is the side effects that occur in the host treated with the anti-infective agent. For example, many anti-infective agents can kill or inhibit a broad spectrum of microorganisms and are not specific to a particular species type. Treatment with these types of anti-infective agents results in the killing of the normal microbial population living in the host as well as infectious microorganisms. Loss of these microbiota can lead to disease complications and predispose the host to infection by other pathogens because the microbiota competes with and acts as a barrier to infectious pathogens. Other side effects may result from the specific or non-specific effects of these chemicals on non-microbial cells or tissues of the host.

Another problem with the widespread use of anti-infective agents is the development of antibiotic-resistant strains of microorganisms. At present, vancomycin-resistant enterococci, penicillin-resistant pneumococcus, multi-resistant staphylococcus aureus, and multi-resistant tuberculosis strains have been developed, and are becoming major clinical problems. The widespread use of anti-infective agents will likely produce many antibiotic-resistant bacterial strains. As a result, new anti-infective strategies will have to combat these microorganisms.

Antibacterial microorganisms that are effective in killing or inhibiting a wide range of bacteria are known as broad spectrum antibiotics. Other types of antibacterial antibiotics are primarily effective against gram-positive or gram-negative species of bacteria. These types of antibiotics are called narrow spectrum antibiotics. Other antibiotics that are effective against a single organism or disease but not against other types of bacteria are called limited-spectrum (limited-spectrum) antibiotics.

Antibacterial agents are sometimes classified based on their primary mode of action. Typically, the antimicrobial agent is an inhibitor of cell wall synthesis, an inhibitor of cell membrane synthesis, an inhibitor of protein synthesis, an inhibitor of nucleic acid synthesis or function, and a competitive inhibitor. Cell wall synthesis inhibitors inhibit one step in the cell wall synthesis process and are typically one step in the synthesis of bacterial peptidoglycans. Cell wall synthesis inhibitors include beta-lactam antibiotics, natural penicillins, semi-synthetic penicillins, ampicillin, clavulanic acid, cephalosporins and bacitracin.

Beta-lactams are quaternary beta-lactam ring-containing antibiotics that inhibit the last step of peptidoglycan synthesis. The beta-lactam antibiotic may be synthetic or natural. The beta-lactam antibiotic produced by penicillium is a natural penicillin such as penicillin G or penicillin V. They are produced by fermentation of penicillium chrysogenum. Natural penicillins have a narrow spectrum of activity and are generally effective against streptococci, gonococci and staphylococci. Other types of natural penicillins that are also effective against gram-positive bacteria include penicillins F, X, K and O.

Semi-synthetic penicillins are typically modifications of the molecule 6-aminopenicillanic acid produced by the mold. 6-aminopenicillanic acid can be modified by the addition of side chains that yield penicillins with a broader spectrum of activity than the native penicillin or other advantageous properties. Certain types of semi-synthetic penicillins have a broad spectrum of resistance to gram-positive and gram-negative bacteria, but are inactivated by penicillinase. These semi-synthetic penicillins include ampicillin, carbenicillin, oxacillin, mezlocillin and piperacillin. Other types of semi-synthetic penicillins have a more narrow activity against gram-positive bacteria, but have improved properties such that they are not inactivated by penicillinase. These semi-synthetic penicillins include, for example, methicillin, dicloxacillin, and nafcillin. Certain broad-spectrum semisynthetic penicillins may be used in combination with beta-lactamase inhibitors such as clavulanic acid and sulbactam. Beta-lactamase inhibition has no antimicrobial effect but it acts to inhibit penicillinase, thus protecting the semi-synthetic penicillin from degradation.

Another class of beta-lactam antibiotics is the cephalosporins. Cephalosporins are sensitive to degradation by bacterial beta-lactamases and are not always effective alone. However, cephalosporins are resistant to penicillinase. They are effective against a wide variety of gram-positive and gram-negative bacteria. Cephalosporins include, but are not limited to, cephalothin, cephalexin, cefamandole, cefaclor, cefazolin, cefuroxime, cefoxitin, cefotaxime, cefsulodin, ceftamex, cefixime, ceftriaxone, cefoperazone, ceftazidime and moxalactam.

Bacitracins are another class of antibiotics that inhibit cell wall synthesis by inhibiting the release of the celiac peptide subunit or peptidoglycan from molecules that transport the subunit outside the membrane. Although bacitracin is effective against gram-positive bacteria, its use is generally limited to topical administration due to its high toxicity.

Carbapenems (carbapenem) are another broad-spectrum β -lactam antibiotic capable of inhibiting cell wall synthesis. Examples of carbapenems include, but are not limited to, imipenem (imipenem). Monobactam is also a broad spectrum of beta-lactam antibiotics and includes euztrenon. An antibiotic, vancomycin, produced by streptomyces is also effective against gram positive bacteria by inhibiting cell membrane synthesis.

Another class of antibacterial agents are those that act as inhibitors of cell membranes. These compounds disrupt the structure of the bacterial cell membrane or inhibit its function. One problem with membrane inhibitor based antimicrobials is that they are effective in both eukaryotic cells and bacteria due to the similarity of phospholipids in bacterial and eukaryotic membranes. Thus, these compounds are rarely specific enough to allow these compounds to be used systemically and avoid high dose use for topical administration.

One clinically useful cell membrane inhibitor is polymyxin. Polymyxin interferes with membrane function by binding to membrane phospholipids. Polymyxin is mainly effective against gram-negative bacteria and is commonly used in severe pseudomonas infections or pseudomonas infections that are resistant to less toxic antibiotics. Serious side effects associated with systemic administration of the compound include damage to the kidneys and other organs.

Other cell membrane inhibitors include the antifungal agents amphotericin B and nystatin, which are used primarily for the treatment of systemic fungal infections and candida yeast infections. Imidazoles are another class of membrane inhibitor antibiotics. Imidazoles are used as antibacterial as well as antifungal agents, for example, for the treatment of yeast infections, parasitic skin infections, and systemic fungal infections. Imidazoles include, but are not limited to, clotrimazole, miconazole, ketoconazole, itraconazole, and fluconazole.

Many antibacterial agents are protein synthesis inhibitors. These compounds prevent the synthesis of structural proteins and enzymes by bacteria and thus cause inhibition of bacterial cell growth or function or cell death. Typically, these compounds interfere with the transcriptional or translational processes. Antibacterial agents that block transcription include, but are not limited to, rifampin and ethambutol. Rifampicin, which inhibits RNA polymerase, has a broad spectrum of activity and is effective against gram positive and gram negative bacteria as well as Mycobacterium tuberculosis. Ethambutol is effective against mycobacterium tuberculosis.

Antibacterial agents that block translation interfere with bacterial ribosomes to prevent translation of mRNA into protein. Such compounds typically include, but are not limited to, tetracyclines, chloramphenicol, macrolides (e.g., erythromycin), and aminoglycosides (e.g., streptomycin).

Aminoglycosides are a class of antibiotics produced by bacteria of the genus streptomyces, for example, streptomycin, kanamycin, tobramycin, amikacin, and gentamicin. Aminoglycosides have been used to combat a wide variety of bacterial infections caused by gram-positive and gram-negative bacteria. Streptomycin is widely used as a major drug for the treatment of tuberculosis. Gentamicin, particularly when used in combination with tobramycin, is used against many gram-positive and gram-negative bacterial strains, including pseudomonas infections. Kanamycin is used against many gram-positive bacteria including penicillin-resistant staphylococci. One side effect that limits their clinical use is that at the doses necessary to achieve efficacy, their prolonged use has shown impairment of renal function and damage to the auditory nerve leading to hearing loss.

Another type of translation inhibitor antibacterial agent is the tetracyclines. Tetracyclines are a class of broad-spectrum antibiotics that are effective against a variety of gram-positive and gram-negative bacteria. Examples of tetracyclines include tetracycline, minocycline, doxycycline and chlortetracycline. They are important for the treatment of many types of bacteria, but are particularly important for the treatment of lyme disease. Tetracyclines have been overused and abused by the medical community resulting in a number of problems due to their low toxicity and minimal direct side effects. For example, overuse thereof tends to lead to the development of a wide range of resistance.

Antibacterial agents such as macrolides reversibly bind to the 50S ribosomal subunit and inhibit protein elongation by peptidyl transferases and/or prevent release of uncharged tRNA from the bacterial ribosome. These compounds include erythromycin, roxithromycin, clarithromycin, oleandomycin, and azithromycin. Erythromycin is active against most gram-positive bacteria (gonococci, legionella and haemophilus), but not against enterobacteria. Lincomycin and clindamycin, which block peptide bond formation during protein synthesis, are used against gram-positive bacteria.

Another type of translation inhibitor is chloramphenicol. Chloramphenicol binds to the 70S ribosome, which inhibits the bacterial enzyme peptidyl transferase, thereby preventing polypeptide chain growth during protein synthesis. One serious side effect associated with chloramphenicol is aplastic anemia. Aplastic anemia develops at doses of chloramphenicol that are effective in treating a small proportion of patients (1/50,000). Chloramphenicol, once a widely used prescription antibiotic, is now less commonly used due to death from anemia. But because of its effectiveness, it is still used in life-threatening situations (e.g., typhoid fever).

Certain antibacterial agents interfere with nucleic acid synthesis or function, e.g., bind to DNA or RNA so that information cannot be read. These include, but are not limited to, quinolones and co-trimoxazole (both synthetic chemicals) as well as the natural or semi-synthetic chemical rifamycin. Quinolones block bacterial DNA replication by inhibiting DNA gyrase, an enzyme required by bacteria to produce their circular DNA. Quinolones are broad spectrum, examples of which include norfloxacin, ciprofloxacin, enoxacin, nalidixic acid and temafloxacin. Nalidixic acid is a bactericidal agent that binds to DNA gyrase (topoisomerase) and thereby inhibits DNA gyrase activity, which is critical for DNA replication and allows supercoils to relax and reform. The primary use of nalidixic acid is in the treatment of lower Urinary Tract Infections (UTIs) as it is effective against several types of gram-negative bacteria (e.g., escherichia coli, enterobacter aerogenes, klebsiella pneumoniae, and proteus species) that are common causes of UTIs. Sulfamethoxazole is a combination of sulfamethoxazole and trimethoprim that blocks the bacterial synthesis of folic acid required for the formation of DNA nucleotides. Rifamycins are derivatives of rifamycins that have activity against gram-positive bacteria (including mycobacterium tuberculosis and meningitis caused by neisseria meningitidis) and certain gram-negative bacteria. Rifampicin binds to the beta subunit of polymerase and blocks the addition of the first nucleotide necessary to activate the polymerase, thereby blocking mRNA synthesis.

Another class of antibacterial agents are compounds that act as competitive inhibitors of bacterial enzymes. Most of the competitive inhibitors are similar in structure to bacterial growth factors and compete for binding but do not perform metabolic functions in the cell. These compounds include sulfonamides and chemically modified forms of sulfonamides having greater and broader antibacterial activity. Sulfonamides (e.g., sulfisoxazole and trimethoprim) are useful for the treatment of streptococcus pneumoniae, β -hemolytic streptococcus and escherichia coli, and have been used for the treatment of simple Urinary Tract Infections (UTI) caused by escherichia coli and for the treatment of meningococcal meningitis.

Antiviral agents are compounds that prevent infection of cells by viruses or replication of viruses within cells. Existing antiviral agents are much less effective than antibacterial agents because the viral replication process is so closely related to DNA replication in the host cell that nonspecific antiviral agents tend to be toxic to the host. Several stages in the viral infection process can be blocked or inhibited by antiviral agents. These phases include: attachment of the virus to the host cell (immunoglobulin or binding peptide), uncoating of the virus (e.g., amantadine), synthesis or translation of viral mRNA (e.g., interferon), replication of viral RNA or DNA (e.g., nucleoside analogs), maturation of new viral proteins (e.g., protease inhibitors), and budding and release of the virus.

Another class of antiviral agents are nucleoside analogs. Nucleoside analogs are synthetic compounds similar to nucleosides, but with incomplete or abnormal deoxyribose or ribose groups. Once nucleoside analogs enter the cell, they are phosphorylated, thereby producing a triphosphate form that competes with normal nucleotides for incorporation into viral DNA or RNA. Once the triphosphate form of the nucleoside analog is incorporated into the growing nucleic acid strand, it results in irreversible binding to the viral polymerase and thus chain termination. Nucleoside analogs include, but are not limited to, acyclovir (for treatment of herpes simplex virus and varicella zoster virus), ganciclovir (for treatment of cytomegalovirus), idoxuridine, ribavirin (for treatment of respiratory syncytial virus), dideoxyinosine, dideoxycytidine, and zidovudine (azidothymidine).

Another class of antiviral agents includes cytokines such as interferons. Interferons are cytokines secreted by virus-infected cells as well as immune cells. Interferons function by binding to specific receptors on cells adjacent to the infected cell, resulting in intracellular changes that protect the cell from viral infection. Interferon-alpha and interferon-beta also induce the expression of MHC class I and class II molecules on the surface of infected cells, resulting in an increase in antigen presentation for recognition by host immune cells. Alpha-interferon and beta-interferon are available as recombinant forms and have been used to treat chronic hepatitis B and C infections. At doses effective for antiviral therapy, interferons have serious side effects such as fever, malaise and weight loss.

Immunoglobulin treatment is used to prevent viral infections. Immunoglobulin therapy for viral infections differs from that for bacterial infections in that immunoglobulin therapy functions by binding to extracellular virions and preventing their attachment and entry into cells susceptible to viral infection, rather than being antigen-specific. The treatment may be used to avoid viral infection for a period of time during which the antibody is present in the host. Generally, there are two classes of immunoglobulin treatment, normal immunoglobulin treatment and hyper-immunoglobulin treatment. Normal immunoglobulin therapy utilizes antibody products prepared and pooled from sera of normal donors. The pooled antibody product contains low titers of antibodies against a large range of human viruses (e.g., hepatitis a, parvovirus, enterovirus (especially in newborns)). Hyperimmune globulin therapy utilizes antibodies prepared from the serum of individuals with high titers of antibodies to a particular virus. These antibodies are then used against a particular virus. Examples of hyperimmune globulin include herpes zoster immunoglobulin (useful for preventing varicella in immunocompromised children and newborns), human rabies immunoglobulin (useful for post exposure prevention in subjects bitten by rabies), hepatitis b immunoglobulin (useful for preventing hepatitis b virus, particularly in subjects exposed to the virus), and RSV immunoglobulin (useful for treating respiratory syncytial virus infection).

Antifungal agents are useful for the treatment and prevention of infectious fungi. Antifungal agents are sometimes classified by their mechanism of action. Certain antifungal agents act as cell wall inhibitors by inhibiting glucose synthase. Such antifungal agents include, but are not limited to basiungin/ECB. Other antifungal agents work by destabilizing the membrane as a whole. These antifungal agents include, but are not limited to, imidazoles (such as clotrimazole, sertaconazole, fluconazole, itraconazole, miconazole, ketoconazole, and voriconazole) as well as FK 463, amphotericin B, BAY 38-9502, MK 991, pradimicin, UK 292, butenafine, and terbinafine. Other antifungal agents work by destroying chitin (e.g., chitinase) or immunosuppression (501 cream).

Parasiticides are agents that kill parasites directly. These compounds are known in the art and are generally commercially available. Examples of parasiticides that may be used for human administration include, but are not limited to, albendazole, amphotericin B, benznidazole, thiochlorophene, chloroquine hydrochloride, chloroquine phosphate, clindamycin, dehydroimidine, diethylcarbamazine, dichloronitrfurfuryl acid ester, eflornithine, furazolidone, glucocorticoids, halofantrine, iodoquinol (iodoquinol), ivermectin, mebendazole, mefloquine, meglumine antimonate, merrsonamide, metrazine, metronidazole, niclosamide, nifolimus, hydroxyaminoquine, paromomycin, pentoxyethanamidine, piperazine, praziquantel, primaquine phosphate, proguanil, pyrantel pamoate, pyrimethamine-sulfonamide, pyrimethamine-sulfadoxine, aclonidine hydrochloride, quinidine sulfate, quinidine gluconate, spiramycin, sodium antimoniate (sodium gluconate), suramin, Tetracycline, doxycycline, thiabendazole, tinidazole, trimethoprim-sulfamethoxazole, and trypanospermine.

The polymers may also be used to inhibit a Th 2-like immune response in a subject. Th 2-type immune responses are characterized at least in part by the Th2 cytokines IL-4 and IL-5 and the isotype of antibodies that switch to IgE. Thus, inhibition of a Th 2-like response refers to a reduction in Th2 cytokine production and a reduction in other Th2 effects. The polymers can also be used to induce a Th 1-like immune response. Th1 and Th2 immune responses are counter-regulated to each other, such that biasing the immune response toward a Th 1-type immune response may avoid or improve a Th 2-type immune response.

The polymers are useful in the treatment and prevention of autoimmune diseases. Autoimmune diseases are a class of diseases: wherein the patient's autoantibodies react with host tissue or wherein the immune effector T cells are autoreactive to endogenous auto-peptides and result in destruction of the tissue. Thus, an immune response is elicited against a subject's own antigen (referred to as a self-antigen). Autoimmune diseases include, but are not limited to, rheumatoid arthritis, Crohn's disease, multiple sclerosis, Systemic Lupus Erythematosus (SLE), autoimmune encephalomyelitis, Myasthenia Gravis (MG), Hashimoto's thyroiditis, goodpasture's syndrome, pemphigus (e.g., pemphigus vulgaris), hyperthyroidism, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, scleroderma with anti-collagen antibodies, mixed connective tissue disease, polymyositis, pernicious anemia, idiopathic Addison's disease, autoimmune infertility, glomerulonephritis (e.g., crescent nephritis, proliferative glomerulonephritis), bullous pemphigoid, Shegan's syndrome(s) (Bu & gt & lt & gtsyndrome), insulin resistance, and autoimmune diabetes.

Autoantigen refers to an antigen of normal host tissue. Normal host tissue does not include cancer cells. Thus, in the context of autoimmune diseases, an immune response elicited against an autoantigen is an undesirable immune response and promotes destruction and damage of normal tissues, while an immune response elicited against a cancer antigen is a desirable immune response and promotes destruction of tumors or cancers. Thus, in certain aspects, the invention is directed to the treatment of autoimmune disorders, and it is not recommended that the polymer be administered with autoantigens, particularly those autoantigens that are targets of autoimmune disorders.

In other cases, the polymer may be delivered with a low dose of autoantigen. Many animal studies have shown that mucosal administration of low doses of antigen can result in immune hyporesponsiveness or "tolerance". The mechanism of activity may be a cytokine-mediated immune bias that dominates the Th2 and Th3 responses from Th1 (i.e., governed by TGF- β). Inhibition of activity with low dose antigen delivery also suppresses irrelevant immune responses (collateral inhibition) which are of considerable interest in the treatment of autoimmune diseases (e.g., rheumatoid arthritis and SLE). The bystander suppression (bystander suppression) involves the secretion of Th1 counter-regulated suppressor cytokines in the local environment, where pro-inflammatory Th1 cytokines are released in an antigen-specific or antigen-non-specific manner. As used herein, "tolerance" is used to refer to this phenomenon. In fact, oral tolerance has been effective in treating a variety of autoimmune diseases in animals, including: experimental Autoimmune Encephalomyelitis (EAE), experimental autoimmune myasthenia gravis, collagen-induced arthritis (CIA) and insulin-dependent diabetes. In these models, prevention and suppression of autoimmune disease and antigen-specific humoral and cellular responses shifted from a Th1 response to a Th2/Th3 response.

The compositions and methods of the invention can be used alone or in combination with other kit methods useful for treating cancer. In one aspect, the invention provides a method of treating a subject having cancer. The method according to this aspect of the invention comprises the step of administering to a subject having cancer an effective amount of a composition of the invention to treat the subject.

In one aspect, the invention provides a method of treating a subject having cancer. The method according to this aspect of the invention comprises the step of administering to a subject having cancer an effective amount of a composition of the invention and an anti-cancer treatment to treat the subject.

In one aspect, the invention provides the use of an immunostimulatory polymer of the invention in the manufacture of a medicament for treating cancer in a subject.

In one aspect, the invention provides compositions useful for treating cancer. The composition according to this aspect comprises the immunostimulatory polymer of the invention and a cancer medicament.

A subject with cancer is a subject with detectable cancerous cells. The cancer may be malignant or non-malignant. As used herein, "cancer" refers to uncontrolled cell growth that interferes with the normal functional role of body organs and systems. Cancer that migrates from its original location and is transplanted into an important organ may eventually lead to death of the subject by deterioration of the function of the affected organ. Hematopoietic cancers, such as leukemia, are able to beat normal hematopoietic compartments in a subject, thereby causing hematopoietic damage (in the form of anemia, thrombocytopenia, and neutropenia) that ultimately leads to death.

Metastases (metastasis) are areas of cancer cells that arise from the spread of cancer cells from the primary tumor to other parts of the body, at a different location than the primary tumor. In diagnosing a primary tumor mass, the subject can be monitored for the presence of metastases. In addition to monitoring specific symptoms, metastases are most commonly detected by using Magnetic Resonance Imaging (MRI) scans, Computed Tomography (CT), blood and platelet counts, liver function examinations, chest X-rays, and bone scans, alone or in combination.

Cancers include, but are not limited to, basal cell carcinoma, cholangiocarcinoma, bladder carcinoma, bone carcinoma, brain and Central Nervous System (CNS) carcinoma, breast carcinoma, cervical carcinoma, choriocarcinoma, colon and rectal carcinoma, connective tissue carcinoma, cancer of the digestive system, endometrial carcinoma, esophageal carcinoma, eye carcinoma, head and neck carcinoma, intraepithelial tumors, kidney carcinoma, laryngeal carcinoma, leukemia, liver carcinoma, lung carcinoma (e.g., small cell lung carcinoma and non-small cell lung carcinoma), lymphoma (including hodgkin's lymphoma and non-hodgkin's lymphoma), melanoma, myeloma, neuroblastoma, oral cancer (e.g., lip cancer, tongue cancer, oral cancer and pharyngeal cancer), ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, cancer of the respiratory system, sarcoma, skin cancer, gastric cancer, testicular cancer, thyroid cancer, uterine cancer, cancer of the urinary system, and other cancers, Adenocarcinoma and sarcoma.

The immunostimulatory compositions of the invention may also be administered in combination with an anti-cancer therapy. Anticancer therapy includes cancer drugs, radiation, and surgery. As used herein, "cancer drug" refers to an agent that is administered to a subject for the purpose of treating cancer. As used herein, "treating cancer" refers to preventing the development of cancer, reducing the symptoms of cancer, and/or inhibiting the growth of established cancer. In other aspects, a cancer medicament is administered to a subject at risk of developing cancer for the purpose of reducing the risk of developing cancer. Various types of drugs for cancer therapy are described herein. For the purposes of this specification, cancer drugs are classified as chemotherapeutic agents, immunotherapeutic agents, cancer vaccines, hormonal therapy, and biological response modifiers.

The chemotherapeutic agent may be selected from, but is not limited to: methotrexate, vincristine, doxorubicin, cisplatin, sugar-free chloroethylnitrosurea, 5-fluorouracil, mitomycin C, bleomycin, doxorubicin, dacarbazine, Taxol, fragamine GLA, valrubicin, carmustine and polifeprosan, MMI270, BAY 12-9566, RAS farnesyltransferase inhibitors, MMP, MTA/LY231514, LY 264618/lometrexol, Glamolec, CI-994, TNP-470, and metin/topotecan, PKC412, valsalva/PSC, 833, novolaks/mitoxantrone, Metaret/suramin, batimastat, E7070, BCH-4556, CS-682, 9-AC, AG3340, AG33, Incel/VX-710, VX-853, ZD 010641, 34641, ODN 698, Marsta 6/BB, Marsta 6/2516, CDP 2516/25100, and polifep, D2163, PD183805, DX8951f, LemonalDP 2202, FK 317, streptolysin preparation (Picibanil)/OK 432, AD 32/valrubicin, Metatarone/strontium derivative, Thalax/temozolomide, Evacet/Adriamycin liposomes, Yewtaxan/paclitaxel, taxol/paclitaxel, Hiroda/Capecitabine, Fluoroxylon/deoxyfluorouridine, Cyclinax/oral paclitaxel, oral paclitaxel (taxoid), SPU-077/cisplatin, HMR 1275/Fragiline (Flavopiridol), CP-358(774)/EGFR, CP-609(754)/RAS oncogene inhibitor, levo-182751/oral platinum, UFT (tegafur/uracil), Ergamiosol/levamisole, uracil/776C 85/5FU enhancer, Kemptol/imidazole, Camptor/BMS/Li, Tumodex/Raltitrexed (Raltitrexed), Leustatin/cladribine, Paxex/paclitaxel, Doxil/doxorubicin liposomes, Clitor/doxorubicin liposomes, Fudawawa/fludarabine, famicin/epirubicin, liposomal cytarabine (Depocyt), ZD1839, LU 79553/dinaphthalenedicarboximide, LU 103793/dolastatin, Caetyx/doxorubicin liposomes, Geraniol/gemcitabine, ZD 0473/Anormed, YM 116, iodide, CDK4 and CDK2 inhibitors, PARP inhibitors, D4809/Dexifosamide, Ifes/mesna (Mesnex)/ifosfamide, Weigemon/teniposide, carboplatin, Plantinol/cisplatin, etoposide/etoposide, ZD 9331, Taxotere/Taxotere (Taxol), guanosine, taxane prodrugs, arabinoside, taxane analogs, Nitrosureas, alkylating agents such as melphalam (melphalan) and cyclophosphamide, aminoglutethimide, asparaginase, busulfan, carboplatin, chlorambucil, cytarabine hydrochloride, dactinomycin, daunorubicin hydrochloride, estramustine sodium phosphate, etoposide (VP16-213), floxuridine, fluorouracil (5-FU), flutamide, hydroxyurea (hydroxyurea), ifosfamide, interferon alpha-2 a, interferon alpha-2 b, leuprolide acetate (LHRH-releasing factor analogue), lomustine (CCNU), dichloromethyldiethylamine hydrochloride (mechlorethamine), mercaptopurine, mesna, mitotane (o.p' -DDD), mitoxantrone hydrochloride, octreotide, plicamycin, procarbazine hydrochloride, streptozotocin, tamoxifen citrate, thioguanine, thiotepa, vinblastine sulfate, amsacrine (m-AMSA), Azacitidine, erythropoietin, altretamine (HMM), interleukin 2, propimidrazone (methyl-GAG, methylglyoxal diamondrazone, MGBG), pentostatin (2' desoxyintermodamycin), semustine (methyl-CCNU), teniposide (VM-26) and vindesine sulfate.

The immunotherapeutic agent may be selected from, but is not limited to: 3622W94, 4B5, ANA Ab, anti-FLK-2, anti-VEGF, ATRAGEN, AVASTIN (Bevacizumab; Genencoch), BABS, BEC2, BEXXAR (Toxicomab; GlaxoSmithKline), C225, CAMPATH (alemtuzumab; Genzyme Corp.), CEACID, CMA 676, EMD-72000, ERBITIX (Cetuximab; Imclone Systems, Inc.), Gliomab-H, GNI-250, HERCEPTIN (trastuzumab; Genencoch), IDEC-Y2B 8, IMMURARARAIT-CEA, Imior C5, ior egf. r3, ior t6, LDP-03, LymphoCide, MDX-11, MDX-22, MDX-210, MDX-220, MDX-260, MDX-447, MELIMMONE-1, MELIMMONE-2, Monopharmam-C, NovoMAb-G2, Oncolym, OV103, Ovarex, Panorex, Pretarget, Quadramet, Ributaxin, RITUXAN (Rituzumab; Genentech), SMART 1D10Ab, SMARTABL 364Ab, SMARTM195, TNT, and ZENAPAX (Dalizumab; Roche).

Cancer vaccines can be selected from, but are not limited to: EGF, anti-idiotype cancer vaccine, Gp75 antigen, GMK melanoma vaccine, MGV ganglioside conjugate vaccine, Her2/neu, Ovarex, M-Vax, O-Vax, L-Vax, STn-KHL cancer vaccine (thermoape), BLP25(MUC-1), liposome idiotype vaccine, melanoma vaccine, peptide antigen vaccine, toxin/antigen vaccine, MVA-based vaccine, PACIS, BCG vaccine, TA-HPV, TA-CIN, DISC-virus, and ImmuCyst/Theracs.

The compositions and methods of the present invention may be used alone or in combination with other agents and methods useful in the treatment of allergies. In one aspect, the invention provides a method of treating a subject having an allergic condition. The method according to this aspect of the invention comprises the step of administering to a subject suffering from an allergic condition an effective amount of a composition of the invention to treat the subject.

In one aspect, the invention provides a method of treating a subject having an allergic condition. The method according to this aspect of the invention comprises the step of administering to a subject suffering from an allergic condition an effective amount of a composition of the invention and an anti-allergic treatment to treat the subject.

In one aspect, the invention provides the use of an immunostimulatory polymer of the invention in the manufacture of a medicament for treating an allergic condition in a subject.

In one aspect, the invention provides compositions useful for treating allergic conditions. The composition according to this aspect comprises the immunostimulatory polymer of the invention and an allergy medication.

"subject having an allergic condition" shall mean a subject who is experiencing or has previously experienced an allergic reaction in response to an allergen. "allergic condition" or "allergy" refers to acquired hypersensitivity to a substance (allergen). Allergic conditions include, but are not limited to, eczema, allergic rhinitis or nasal cold, hay fever, allergic conjunctivitis, bronchial asthma, urticaria and food allergies, other specific conditions including atopic dermatitis, anaphylaxis (anaphylaxis), drug allergies and angioedema.

Allergy is typically an epitopic condition associated with the production of antibodies against allergens from a particular class of immunoglobulin (IgE). The production of an IgE-mediated response to common aeroallergens is also a factor suggesting susceptibility to asthma. If an allergen encounters specific IgE bound to the surface of an IgEFc receptor (fcer) on basophils (circulating in the blood) or mast cells (dispersed throughout solid tissue), the cells are activated, resulting in the production and release of mediators such as histamine, 5-hydroxytryptamine, and lipid mediators.

Allergic reactions occur when tissue-sensitizing immunoglobulins of the IgE type react with foreign allergens. IgE antibodies bind to mast cells and/or basophils, and these specialized cells then release the chemical mediator of the allergic reaction (vasoactive amine) when stimulated by the allergen that bridges the ends of the antibody molecule. Histamine, platelet activating factor, arachidonic acid metabolites, and 5-hydroxytryptamine are well known mediators of human allergic reactions. Histamine and other vasoactive amines are typically stored in mast cells and basophils. Mast cells are dispersed throughout animal tissue, while basophils circulate within the vascular system. These cells produce and store histamine intracellularly unless a sequence of specialized events involving IgE binding occurs to trigger its release.

The symptoms of allergic reactions vary depending on the in vivo location of the IgE reacting with the antigen. If the response occurs along the respiratory epithelium, the symptoms are usually sneezing, coughing and asthmatic reactions. If the reaction occurs in the digestive tract, as in the case of food allergies, it is usually abdominal pain and diarrhea. Systemic allergic reactions (e.g., after bee stings or administration of penicillin to allergic subjects) can be severe and often life threatening.

Allergy is associated with a Th2 type immune response (characterized at least in part by the Th2 cytokines IL-4 and IL-5) and the conversion of antibody isotypes to IgE. The immunostimulatory polymers of the invention are themselves useful in treating a subject suffering from an allergic condition, as the immunostimulatory polymers may bias the immune response towards a Th 1-type immune response. Alternatively or in addition, the immunostimulatory polymers of the invention can be used in combination with an allergen to treat a subject suffering from an allergic condition.

The immunostimulatory compositions of the invention may also be administered in combination with an anti-allergy therapy. Traditional methods of treating or preventing allergy involve allergy medication or desensitization therapy. Some of the ongoing therapies for treating or preventing allergy include the use of neutralizing anti-IgE antibodies. Antihistamines and other drugs that block the effects of chemical mediators of an allergic reaction help regulate the severity of the allergic symptoms but do not prevent the allergic reaction and have no effect on the subsequent allergic response. Desensitization therapy is usually performed by subcutaneous injection by administering a small dose of the allergen in order to induce an IgG-type response against the allergen. It is believed that the presence of IgG antibodies helps to neutralize the production of mediators induced by IgE antibodies. Initially, subjects were treated with very low doses of allergen to avoid causing severe reactions, and then the dose was slowly increased. Such treatment is dangerous because the subject is actually administered a compound that causes an allergic response, and severe allergic reactions may occur.

Allergic drugs include, but are not limited to, antihistamines, glucocorticoids, and prostaglandin inducers. Antihistamines are compounds that neutralize histamine released by mast cells or basophils. These compounds are well known in the art and are commonly used in allergy treatment. Antihistamines include, but are not limited to, acrivastine, astemizole, azatadine, azelastine, betadastine, brompheniramine, buclizine, cetirizine analogs, chlorpheniramine, clemastine, CS 560, cyproheptadine, desloratadine, dexchlorpheniramine, ebastine, epinastine, fexofenadine, HSR 609, hydroxyzine, levocabastine, loratadine, scopolamine, mizolastine, norastemizole, phenindamine, promethazine, pyrilamine, terfenadine, and tranilast.

Glucocorticoids include, but are not limited to, methylprednisolone, prednisolone, prednisone, beclomethasone, budesonide, dexamethasone, flunisolide, fluticasone propionate, and triamcinolone. Although dexamethasone is an anti-inflammatory glucocorticoid, it is not commonly used for the treatment of allergies or for the treatment of asthma in inhaled form because of its high absorption and long-term inhibitory side effects at effective doses. However, dexamethasone can be used according to the invention for the treatment of allergy or asthma, since it can be administered in low doses to reduce said side effects when it is administered in combination with the composition of the invention. Some of the side effects associated with glucocorticoid use include cough, difficulty sounding, canker sores (candidiasis), and systemic side effects at high doses, such as adrenal suppression, glucose intolerance, osteoporosis, aseptic osteonecrosis, cataract formation, growth inhibition, hypertension, muscle weakness, skin thinning, and susceptibility to bruising (Barnes & Peterson (1993) Am Rev Respir Dis 148: S1-S26; and Kamada AK et al (1996) Am JRespir Crit Care Med 153: 1739-48).

The compositions and methods of the present invention may be used alone or in combination with other agents and methods useful in the treatment of asthma. In one aspect, the invention provides a method of treating a subject suffering from asthma. The method according to this aspect of the invention comprises the step of administering to a subject suffering from asthma an effective amount of a composition of the invention to treat the subject.

In one aspect, the invention provides a method of treating a subject suffering from asthma. The method according to this aspect of the invention comprises the step of administering to a subject suffering from asthma an effective amount of a composition of the invention and an anti-asthma treatment to treat the subject.

In one aspect, the invention provides the use of an immunostimulatory polymer of the invention in the manufacture of a medicament for treating asthma in a subject.

In one aspect, the present invention provides compositions useful for treating asthma. The composition according to this aspect comprises the immunostimulatory polymer of the invention and an asthma medicament.

As used herein, "asthma" refers to a respiratory condition characterized by inflammation and narrowing of the airways and increased responsiveness of the airways to inhalants. Asthma is often (but not exclusively) associated with atopic conditions or allergic conditions. Symptoms of asthma include recurrent episodes of wheezing, shortness of breath, chest tightness, and coughing due to airflow obstruction. Airway inflammation associated with asthma can be detected by observing a series of physiological changes such as ablation of airway epithelium, subfilamental collagen deposition, edema, mast cell activation, infiltration of inflammatory cells (including neutrophils, eosinophils, and lymphocytes). As a result of airway inflammation, asthmatics often experience airway hyperresponsiveness, airflow limitation, respiratory symptoms, and disease chronization. Airflow limitation includes acute bronchoconstriction, airway edema, mucosal obstruction formation and airway remodeling, which are features that often lead to bronchial obstruction. In some asthma cases, fibrosis of the basement membrane subunit may occur, resulting in persistent abnormalities in lung function.

Research over the past few years has revealed that asthma may be caused by complex interactions between inflammatory cells, mediators, and other cells and tissues located in the airways. Mast cells, eosinophils, epithelial cells, macrophages and activated T cells all play an important role in the inflammatory processes associated with asthma (Djukukanovic R et al (1990) Am Rev Respir Dis 142: 434-457). It is believed that these cells can affect airway function by secreting preformed and newly synthesized mediators that act directly or indirectly on local tissues. Furthermore, it has been recognized that a subset of T lymphocytes (Th2) play an important role in the regulation of allergic inflammation in the airways by the release of selective cytokines and the resulting chronization of the disease (Robinson DS et al (1992) N Engl J Med 326: 298-.

Asthma is a complex disorder that occurs at different stages of development and can be classified as acute, subacute or chronic based on the degree of symptoms. The acute inflammatory response is associated with early cell recruitment within the airways. A subacute inflammatory response is associated with recruitment of cells and local cellular activation that contribute to a more persistent inflammatory condition. The chronic inflammatory response is characterized by a level of persistent cellular damage and ongoing repair processes that may cause permanent abnormalities in the airways.

A "subject with asthma" is a subject with a respiratory disorder characterized by inflammation and narrowing of the airways and increased responsiveness of the airways to inhalants. Factors associated with asthma triggering include, but are not limited to, allergens, hypothermia, exercise, viral infections, and SO₂。

As noted above, asthma may be associated with a Th2 type immune response (characterized at least in part by the Th2 cytokines IL-4 and IL-5) and the conversion of antibody isotypes to IgE. The Th1 and Th2 immune responses are counter-regulated to each other, such that an immune response biased towards a Th 1-type immune response prevents or alleviates a Th 2-type immune response, including allergy. Thus, the modified oligoribonucleotide analogues of the present invention are useful in themselves for treating a subject suffering from asthma, since said analogues may predispose the immune response to a Th 1-type immune response. Alternatively or in addition, the modified oligoribonucleotide analogues of the present invention can be used in combination with an allergen for the treatment of a subject suffering from asthma.

The immunostimulatory compositions of the invention may also be administered in combination with asthma treatment. Traditional approaches to treating or preventing asthma have involved the use of anti-allergic therapies (as described above) and many other agents including inhalants.

Drugs used to treat asthma are generally divided into two categories, fast-remitting drugs and long-term control drugs. Asthma (asthma)Patients take long-term control medications daily to achieve and maintain control of persistent asthma. Long-term control drugs include anti-inflammatory agents such as glucocorticoids, cromolyn sodium and nedocromil; such as long-acting beta₂Long-acting bronchodilators such as agonists and methylxanthines; and a leukotriene modifier. Fast-acting relief medications include short-acting beta₂Agonists, anticholinergics (anti-cholenergic) and systemic glucocorticoids. Each of these drugs is associated with a number of side effects, and none of the drugs, alone or in combination, prevents or cures asthma.

Asthma drugs include, but are not limited to, PDE-4 inhibitors, bronchodilators/beta-2 agonists, K + channel openers, VLA-4 antagonists, allylisoureide antagonists, inhibitors of thromboxane A2(TXA2) synthesis, xanthines, arachidonic acid antagonists, 5 lipoxygenase inhibitors, TXA2 receptor antagonists, TXA2 antagonists, 5-lipox activator protein inhibitors, and protease inhibitors.

Bronchodilators/beta₂Agonists are a class of compounds that cause bronchodilation or smooth muscle relaxation. Bronchodilators/beta₂Agonists include, but are not limited to, salmeterol, salbutamol, terbutaline, D2522/formoterol, fenoterol, bitolterol, pirbuterol methylxanthine, and metaproterenol. Long acting beta₂Agonists and bronchodilators are compounds used for long-term prevention of symptoms in addition to the anti-inflammatory treatment. Long acting beta₂Agonists include, but are not limited to, salmeterol and salbutamol. These compounds are often used in combination with glucocorticoids and are not usually used without any treatment of inflammation. They are associated with side effects such as tachycardia, skeletal muscle shivering, hypokalemia and prolongation of the QTc gap in excess.

Methylxanthines, including for example theophylline, are used for long term control and prevention of symptoms. These compounds cause bronchodilation and possibly adenosine antagonism due to phosphodiesterase inhibition. Dose-related acute toxicity is an important issue for such compounds. Therefore, routine serum concentrations must be monitored in order to account forToxicity due to individual differences in metabolic clearance and a narrow therapeutic window. Side effects include tachycardia, tachyarrhythmia, nausea, vomiting, central nervous system irritation, headache, seizures, hematemesis, hyperglycemia, and hypokalemia. Short-acting beta₂Agonists include, but are not limited to, salbutamol, bitolterol, pirbuterol, and terbutaline. And short-term effect beta₂Some side effects associated with the administration of agonists include tachycardia, skeletal muscle shivering, hypokalemia, increased lactate, headache and hyperglycemia.

Sodium tryptophan and nedocromil are used as long-acting control drugs mainly for preventing asthma symptoms caused by exercise or allergy symptoms caused by allergens. It is believed that such compounds can block early and late phase reactions with allergens by interfering with chloride channel function. They also stabilize mast cell membranes and inhibit activation and release of mediators from eosinophils and epithelial cells. A four to six week administration period is typically required to achieve maximum effect.

Anticholinergic agents are commonly used to relieve acute bronchospasm. It is believed that these compounds may function by competitive inhibition of muscarinic cholenergic receptors. Anticholinergics include, but are not limited to, ipratropium bromide. These compounds only moderate cholinergically mediated bronchospasm but do not alter any response to the antigen. Side effects include dry mouth and respiratory secretions, increased wheezing in some individuals, and blurred vision if sprayed into the eyes.

The immunostimulatory polymers of the invention are also useful in the treatment of airway remodeling. Airway remodeling results from smooth muscle cell proliferation and/or submucosal thickening within the airway, and ultimately leads to airway narrowing resulting in restricted airflow. The immunostimulatory polymers of the invention may prevent further remodeling and may even reduce tissue accumulation resulting from the remodeling process.

The immunostimulatory polymers of the invention are also useful for improving the survival, differentiation, activation and maturation of dendritic cells. The immunostimulatory oligoribonucleotides have the unique ability to promote cell survival, differentiation, activation and maturation of dendritic cells.

The immunostimulatory polymers of the invention also increase the lytic activity of natural killer cells and antibody-dependent cellular cytotoxicity (ADCC). ADCC can be performed using the immunostimulatory polymer in combination with antibodies specific for cellular targets such as cancer cells. When the immunostimulatory polymer is administered to a subject in combination with an antibody, the subject's immune system is induced to kill tumor cells. Antibodies useful in ADCC methods include antibodies that interact with cells in vivo. Many such antibodies specific for cellular targets have been described in the art and many are commercially available. In one embodiment, the antibody is an IgG antibody.

In certain aspects, the invention provides methods of enhancing epitope spreading. As used herein, "epitope spreading" refers to the epitope-specific diversification of a dominant epitope-specific immune response from an initial focus against a self or foreign protein to subdominant and/or recessive epitopes on that protein (intramolecular spreading) or other proteins (intermolecular spreading). Epitope spreading results in a multiple epitope-specific immune response.

The immune response consists of an initial amplification phase, which may be either harmful (e.g., autoimmune disease) or beneficial (e.g., vaccination), and a later, down-regulation phase that returns the immune system to homeostasis and produces memory. Epitope spreading may be an important component of both phases. In the context of tumors, enhancement of epitope spreading allows the subject's immune system to identify additional target epitopes that were not initially recognized by the immune system in response to the original treatment regimen, while reducing the likelihood of escape variants in the tumor population and thus affecting disease progression.

The oligoribonucleotides of the invention are useful for promoting epitope spreading in therapeutically beneficial markers such as cancer, viral and bacterial infections and allergy. In one embodiment, the method comprises the step of administering to a subject a vaccine comprising an antigen and an adjuvant and then administering to the subject at least two doses of an immunostimulatory polymer of the invention in an amount effective to induce a multiple epitope-specific immune response. In one embodiment, the method comprises the step of administering to a subject a vaccine comprising a tumor antigen and an adjuvant and then administering to the subject at least two doses of an immunostimulatory polymer of the invention in an amount effective to induce a multiplex epitope-specific immune response. In one embodiment, the method involves the application of a treatment regimen that causes exposure of immune system antigens in a subject, followed by at least two administrations of the immunostimulatory oligoribonucleotides of the invention to induce multiple epitope-specific immune responses (i.e., to promote epitope spreading). In various embodiments, the treatment regimen is surgery, radiation therapy, chemotherapy, other cancer drugs, vaccines, or cancer vaccines.

The treatment regimen may be administered in combination with an immunostimulant, in addition to subsequent immunostimulant treatments. For example, when the treatment regimen is a vaccine, it can be administered in combination with an adjuvant. The combination of vaccine and adjuvant may be a mixture or administered separately, i.e. injected (i.e. the same priming zone). The administration need not be simultaneous. If non-simultaneous injections are used, the timing may involve pre-injection of the adjuvant prior to use of the vaccine formulation.

After the treatment regimen is administered, an immunostimulatory monotherapy is initiated. The optimal frequency, duration and site of administration will depend on the target and other factors, but may be monthly or every other month, for example over a period of six months to two years. Alternatively, administration may be on a daily, weekly, or weekly basis, or may be multiple administrations over one day, week, or month. In certain instances, the period of administration may depend on the course of treatment, for example it may end after one week, one month, one year or more. In other cases, the monotherapy may be continuous as with an intravenous drip. The immunostimulant may be applied to the drainage zone commonly used for targets.

For use in therapy, different dosages may have to be employed for treatment of a subject depending on the activity of the compound, the mode of administration, the immunological purpose (i.e. prophylactic or therapeutic), the nature and severity of the condition, the age and weight of the subject. Administration of a given dose may be by a single administration in the form of a single dosage unit or several smaller dosage units. Multiple doses administered at specific intervals of weeks or months are commonly used to boost antigen-specific immune responses.

By selecting and measuring factors such as potency, relative bioavailability, patient weight, severity of side effects and preferred mode of administration from among the various active compounds in conjunction with the teachings provided herein, an effective prophylactic or therapeutic treatment regimen can be designed that does not result in significant toxicity but is entirely effective in treating a particular subject. An effective amount for any particular application may vary depending on factors such as the disease or condition to be treated, the particular therapeutic agent being administered, the size of the subject, or the severity of the disease or condition. One of ordinary skill in the art can empirically determine the effective amount of a particular nucleic acid and/or other therapeutic agent without undue experimentation.

Typical subject doses of the compounds described herein range from about 0.1 μ g/day to 10,000 mg/day, more typically from about 1 μ g/day to 8000 mg/day, and most typically from about 10 mg/day to 100 mg/day. Typical doses are about 0.1 μ g/kg/week to 20 mg/kg/week, more typically about 1 mg/kg/week to 10 mg/kg/week, and most typically about 1 mg/kg/week to 5 mg/kg/week, with respect to the subject's body weight.

Pharmaceutical compositions containing nucleic acids and/or other compounds may be administered by any suitable route of pharmaceutical administration. A variety of routes of administration may be utilized. The particular mode selected will, of course, depend on the particular agent or agents selected, the particular condition to be treated and the dosage required for therapeutic efficacy. In general, the methods of the invention may be practiced using any mode of administration that is medically acceptable, meaning any mode that produces an effective level of immune response without causing clinically unacceptable side effects. Preferred modes of administration are discussed herein. For use in therapy, an effective amount of the agent can be administered to a subject by any mode of delivery of a nucleic acid and/or other therapeutic agent to a desired surface (e.g., mucosal surface, systemic surface).

Administration of the pharmaceutical compositions of the present invention may be accomplished by any method known to those skilled in the art. Routes of administration include, but are not limited to, oral, parenteral, intravenous, intramuscular, intraperitoneal, intranasal, sublingual, intratracheal, inhalation, subcutaneous, ocular, vaginal and rectal routes. For the treatment or prevention of asthma or allergy, the compounds are preferably administered by inhalation, swallowing or by systemic route. Systemic routes include oral and parenteral routes. In certain embodiments, primarily in asthmatic patients, inhaled drugs are preferred for their direct delivery to the lungs (site of inflammation). Several types of devices are commonly used for inhalation administration. These device types include Metered Dose Inhalers (MDIs), breath-triggered MDIs, Dry Powder Inhalers (DPIs), spacing/holding chambers in combination with MDIs, and nebulizers.

The therapeutic agents of the present invention may be delivered to a particular tissue, cell type and/or immune system with the aid of a carrier. In the broadest sense, a "carrier" is any vehicle that can assist in transferring a composition to a target cell. The carrier generally transports immunostimulatory nucleic acids, antibodies, antigens, and/or disorder-specific drugs to the target cell with reduced degradation relative to the degree of degradation caused in the absence of the carrier.

In general, vectors useful in the present invention fall into two categories: biological carriers and chemical/physical carriers. Biological and chemical/physical carriers are useful for delivery and/or uptake of the therapeutic agents of the present invention.

Most biological vectors are used for delivery of nucleic acids, and this is most suitable for delivery of therapeutic agents that are themselves or comprise immunostimulatory nucleic acids.

In addition to the biological vectors discussed herein, chemical/physical vectors can also be used to deliver therapeutic agents including immunostimulatory nucleic acids, antibodies, antigens, and disorder-specific drugs. As used herein, "chemical/physical vector" refers to other natural or synthetic molecules, other than molecules derived from bacteria or viruses, capable of delivering nucleic acids and/or other drugs.

The preferred chemical/physical carrier of the present invention is a colloidal dispersion. Colloidal dispersion systems include lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. The preferred colloidal system of the present invention is a liposome. Liposomes are artificial membrane containers that can be used as delivery vehicles in vivo or in vitro. It has been shown that large lamellar vesicles (LUVs) with sizes ranging from 0.2 μm to 4.0 μm can encapsulate larger macromolecules. RNA, DNA and whole virions can be encapsulated in an aqueous interior and delivered to cells in a biologically active form (Fraley et al (1981) Trends Biochem Sci 6: 77).

Liposomes can be targeted to specific tissues by coupling the liposome to a specific ligand such as a monoclonal antibody, a sugar, a glycolipid, or a protein. Ligands that may be used to target liposomes to immune cells include, but are not limited to: intact molecules or fragments that interact with receptors and molecules specific for immune cells, e.g., antibodies that interact with cell surface markers of immune cells. Such ligands can be readily identified by binding assays well known to those skilled in the art. In other embodiments, the liposome can be targeted to the cancer by coupling the liposome to one of the immunotherapeutic antibodies discussed above. Alternatively, the vector may be coupled to a nuclear targeting peptide which directs the vector to the host cell nucleus.

Lipid preparations for transfection are commercially available from QIAGEN, e.g., EFFECTENE^TM(non-liposomal lipids with specific DNA condensation enhancers) and SUPERFECT^TM(novel dendrimer technology).

Liposomes are commercially available from Gibco BRL, for example, from N- [1- (2, 3-dioleoyloxy) -propyl]LIPOFECTIN comprising cationic lipid such as N, N, N-trimethyl ammonium chloride (DOTMA) and Dimethyl Dioctadecyl Ammonium Bromide (DDAB)^TMAnd LIPOFECTAACE^TM. Methods for preparing liposomes are well known in the art and have been performed in numerous publicationsA description is given. In Gregoriadis G (1985) Trends Biotechnol 3: liposomes were also reviewed in 235-241.

Certain cationic lipids, including in particular N- [1- (2, 3-dioleoyloxy) -propyl ] -N, N, N-trimethylammoniummethylsulfate (DOTAP), appear to be particularly advantageous when mixed with the modified oligoribonucleotide analogues of the present invention.

In one embodiment, the carrier is a biocompatible microparticle or implant suitable for implantation or administration to a mammalian recipient. Examples of bioerodible (bioerodible) implants that can be used according to this method are described in PCT International application No. PCT/US/03307 (publication No. WO95/24929, entitled "Polymeric Gene Delivery System"). PCT/US/03307 describes a biocompatible, preferably biodegradable, polymer matrix which can be used to load exogenous genes under the control of appropriate promoters.

The preferred form of the polymer matrix is a particulate form such as microspheres (where the nucleic acid and/or other therapeutic agent is dispersed throughout the solid polymer matrix) or microcapsules (where the nucleic acid and/or other therapeutic agent is stored in the core of the polymer shell). Other forms of polymer matrices for loading therapeutic agents include films, coatings, gels, implants and stents. The size and composition of the polymer matrix device is selected so as to produce favorable release kinetics within the tissue into which the matrix is introduced. Furthermore, the size of the polymer matrix may also be selected according to the method of delivery to be used (typically injection into tissue or administration of a suspension to the nose and/or lungs by aerosol). When the aerosol route is used, it is preferred that the polymer matrix and the nucleic acid and/or therapeutic agent are encapsulated within a surfactant vehicle. The polymer matrix composition may be selected to have both an advantageous degradation rate and to be formed from a bioadhesive material, thereby further increasing the effectiveness of delivery when the matrix is applied to a damaged nasal surface and/or pulmonary surface. Furthermore, the matrix composition may also be selected such that it is not degraded but released over an extended period of time by diffusion. In certain preferred embodiments, the nucleic acid is administered to the subject via an implant while the other therapeutic agent is administered acutely. Biocompatible microspheres suitable for delivery (e.g., oral or mucosal delivery) are disclosed in Chickering et al (1996) Biotech bioenng 52: 96-101 and Mathiowitz E et al (1997) Nature 386: 410-414 and PCT patent application WO 97/03702.

Both non-biodegradable and biodegradable polymer matrices can be used to deliver nucleic acids and/or therapeutic agents to a subject. A biocompatible matrix is preferred. These polymers may be natural polymers or synthetic polymers. The polymer is selected based on the period of time required for release (typically on the order of hours to more than a year). In general, particularly for nucleic acid agents, it is most desirable to release over a period of several hours to 3 months to 12 months. The polymer is in the form of a hydrogel that can absorb up to about 90% of its weight in water, if necessary, and is crosslinked with multivalent ions or other polymers, if necessary.

Bioadhesive polymers of particular interest include h.s.sawhney, c.p.pathak and j.a.hubell in Macromolecules, (1993) 26: 581-587, the teachings of which are incorporated herein. These polymers include hyaluronic acid, casein, gelatin (glutin), polyanhydrides, polyacrylic acid, alginates, chitosan, polymethyl methacrylate, polyethyl methacrylate, polybutyl methacrylate, polyisobutyl methacrylate, polyhexamethyl methacrylate, polyisodecyl methacrylate, polydodecyl methacrylate, polyphenyl methacrylate, polymethyl acrylate, isopropyl polyacrylate, isobutyl polyacrylate, and octadecyl polyacrylate.

If the therapeutic agent is a nucleic acid, the use of a compression agent may also be desirable. The compressing agent may also be used alone or in combination with a biological carrier or a chemical/physical carrier. As used herein, "compressing agent" refers to an agent such as histamine that neutralizes the negative charge on a nucleic acid and thereby allows the nucleic acid to be compressed into small particles. The compression of the nucleic acid facilitates the uptake of the nucleic acid by the target cells. The compressing agent may be used alone (i.e., to deliver the nucleic acid in a form that is more efficiently taken up by the cells), or more preferably in combination with one or more of the above-described vectors.

Other exemplary compositions that can be used to facilitate uptake of nucleic acids include calcium phosphate and other chemical mediators of intracellular trafficking, microinjection compositions, electroporation, and homologous recombination compositions (e.g., for integration of nucleic acids into preselected locations within the target cell chromosome).

As noted above, the polymers of the present invention can be co-formulated with a delivery vehicle. For example, the following transport vehicles have been described: a spiral body,Live bacterial vectors (e.g., salmonella, escherichia coli, bcg, shigella, lactobacillus), live viral vectors (e.g., vaccinia, adenovirus, herpes simplex virus), microspheres, nucleic acid vaccines, polymers (e.g., carboxymethylcellulose, chitosan), polymer rings, proteosomes, sodium fluoride, transgenic plants. In certain embodiments of the invention, the delivery vehicle is a liposome, a non-ionic vesicle, a lipid complex, a polymer complex, a lipopolymer complex, a water-in-oil (W/O) emulsion, an oil-in-water (O/W) emulsion, a water-in-oil-in-water (W/O/W) multiple emulsion, a microemulsion, a nanoemulsion, a micelle, a dendrimer, a viral particle, a viroid, a polymeric nanoparticle such as a nanosphere or nanocapsule, a polymeric microparticle such as a microsphere or microcapsule.

The formulations of the invention are administered in pharmaceutically acceptable solutions which may conventionally contain pharmaceutically acceptable concentrations of salts, buffers, preservatives, compatible carriers, adjuvants and optionally other therapeutic ingredients. In certain embodiments, the composition is sterile.

The term "pharmaceutically acceptable carrier" refers to one or more compatible solid or liquid fillers, diluents, or encapsulating substances suitable for administration to humans or other vertebrates. The term carrier refers to a natural or synthetic organic or inorganic ingredient mixed with an active ingredient to aid in application. The components of the pharmaceutical composition can also be blended with one another with the compounds of the present invention in a manner that does not present interactions that would significantly impair the desired pharmaceutical efficacy.

For oral administration, the compounds (i.e., nucleic acids, antigens, antibodies, and other therapeutic agents) can be readily formulated by mixing the active compounds with pharmaceutically acceptable carriers well known in the art. These carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a subject to be treated. Pharmaceutical preparations for oral use can be obtained as solid excipients, optionally grinding the resulting mixture, adding suitable auxiliaries if desired, and thereafter processing the mixture of granules to obtain tablets or dragee cores. Suitable excipients are in particular fillers such as sugars including lactose, sucrose, mannitol or sorbitol; cellulose preparations such as corn starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, for example, cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof (e.g., sodium alginate). The oral formulation may also be formulated in normal saline or buffer for neutralizing internal acidic conditions, if necessary, or the oral formulation may be administered without any carrier.

Suitable coatings are provided for the cores of the dragees. Concentrated sugar solutions may be used for this purpose, optionally containing gum arabic, talc, polyvinyl pyrrolidone, carbopol gum, polyethylene glycol and/or titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyes or pigments may be added to the tablets or dragee coatings for identifying or characterizing different combinations of active compound doses.

Pharmaceutical preparations which can be used orally include press-fit (push-fit) capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. Press-fit capsules can contain the active ingredients in admixture with fillers such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. In addition, microspheres formulated for oral administration may also be used. Such microspheres have been well defined in the art. All formulations for oral administration should be in dosages suitable for such administration.

For oral administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

For administration by inhalation, the compounds used in the present invention may be conveniently delivered in an aerosol spray-like form from pressurized packs or nebulizers using suitable propellants, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gases. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve for metering the delivery meter. Capsules and cartridges (e.g., of gelatin) for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

When the compound is intended for systemic delivery, it may be formulated for parenteral administration by injection (e.g., bolus injection) or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, synthetic fatty acid esters such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. If desired, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds so that highly concentrated solutions can be prepared.

Alternatively, the active compound may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use.

In addition, the compounds may also be formulated, for example, as rectal or vaginal compositions in suppositories or retention enemas containing conventional suppository bases (e.g., cocoa butter or other glycerides).

In addition to the foregoing formulations, the compounds may be formulated as depot preparations (depotpreperation). Such depot formulations may be co-formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives (e.g., a sparingly soluble salt).

The pharmaceutical composition may also comprise suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycol.

Suitable liquid or solid pharmaceutical preparation forms are, for example, aqueous or saline solutions for inhalation, microencapsulated forms, helix-encapsulated forms, coated on micro-gold particles, contained in liposomes, aerosolized forms, aerosols, pellets for implantation into the skin or forms which dry on sharp objects to be scraped into the skin. The pharmaceutical compositions may also comprise granules, powders, tablets, coated tablets, (micro) capsules, suppositories, syrups, emulsifiers, suspensions, creams, drops or preparations with delayed release of the active compound, in which excipients such as disintegrants, binders, coating agents, swelling agents, lubricants, taste enhancers, sweeteners or solubilizers and additives and/or auxiliaries are usually used as described above. The pharmaceutical compositions are suitable for use in a number of drug delivery systems. For a short review on drug delivery methods, see LangerR (1990) Science 249: 1527 and 1533, the contents of which are incorporated herein by reference.

The nucleic acid and optionally other therapeutic agents and/or antigens may be administered per se (neat administration) or in the form of a pharmaceutically acceptable salt. When used in medicine, the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically acceptable salts thereof. Such salts include, but are not limited to, salts prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, p-toluenesulfonic, tartaric, citric, methanesulfonic, formic, malonic, succinic, naphthalene-2-sulfonic, and benzenesulfonic acids. In addition, such salts may also be prepared as alkali metal or alkaline earth metal salts, such as sodium, potassium or calcium carboxy salts.

Suitable buffers include: acetic acid and salts (1% to 2% w/v); citric acid and salts (1% to 3% weight/volume); boric acid and salts (0.5% to 2.5% weight/volume); and phosphoric acid and salts (0.8% to 2% weight/volume). Suitable preservatives include benzalkonium chloride (0.003% to 0.03% w/v); chlorobutanol (0.3% to 0.9% weight/volume); p-hydroxybenzoic acid (0.01% -0.25% w/v) and Thimersol (0.004% -0.02% w/v).

The compositions may be conveniently presented in unit dosage form and prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the compound into association with a carrier that includes one or more accessory ingredients. Generally, the compositions are prepared by uniformly and intimately bringing into association the compound with liquid carriers and/or finely divided solid carriers, and then, if necessary, shaping the product. The liquid dosage unit is a bottle or ampoule. Solid dosage units are tablets, capsules and suppositories.

Other delivery systems may include time-release, delayed-release, or sustained-release delivery systems. These systems avoid repeated administration of the compound and are more convenient to the subject and the physician. Many types of delivery systems are known and available to those of ordinary skill in the art. They include polymer-based systems such as polylactide glycolides, copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and polyanhydrides. Microcapsules containing the above polymers of drugs are described, for example, in U.S. Pat. No. 5,075,109. The delivery system also includes non-polymeric systems: lipids including sterols (such as cholesterol and cholesterol esters) and fatty acids or neutral fats (such as monoglycerides, diglycerides, and triglycerides); a hydrogel release system; a silicone rubber system; a peptide-based system; coating with wax; compressed tablets using conventional binders and excipients; as well as partially fused implants, and the like. Specific examples include, but are not limited to: (a) aggressive systems in which the agents of the invention are contained in a form within a matrix, such as the systems described in U.S. Pat. nos. 4,452,775, 4,675,189, and 5,736,152; and (b) diffusive systems in which the active ingredient exudes from the polymer at a controlled rate, such as the systems described in U.S. Pat. nos. 3,854,480, 5,133,974, and 5,407,686. Additionally, pump-like hardware delivery systems may be used, some of which are adapted for implantation.

The invention is further illustrated by the following examples which should not be construed as further limiting the invention. All references, including literature references, issued patents, published patent applications, and pending patent applications, cited in this application are hereby expressly incorporated by reference in their entirety.

Examples

TLR7 and TLR8 recognize single stranded RNA or short Oligoribonucleotides (ORN). Incubation of immune cells expressing TLR7 and/or TLR8 results in the induction of cytokine production. Because of the different expression patterns of these two receptors, it is postulated that TLR 7-mediated signaling stimulates production of IFN- α by human pDC, whereas TLR8 primarily causes activation of mdcs and monocytes that produce IL-12, TNF- α and IFN- γ. The presence of RNA motifs that specifically induce TLR8 and TLR7/8 activity has been demonstrated. The following example shows the identification of additional motifs specific to the backbone. An important finding is that polymers containing this RNA motif induce primarily IFN- α, supporting a potent immunostimulatory motif that does not stimulate significant levels of proinflammatory cytokines.

Method of producing a composite material

And (3) ELISA (enzyme-Linked immunosorbent assay):

human PBMC were incubated with serial dilutions of ORN in the presence of DOTAP (starting from 2. mu.M ORN and 25. mu.g/ml DOTAP), and after 24 hours the supernatants were assayed for IFN-. alpha.and IL-12p40 by ELISA. Mean ± SEM of 3 donors are shown.

Cytokine detection

Human PBMC at approximately 5X 10⁶The concentration of cells/mL was resuspended and added to a round bottom 96-well plate (250 μ l/well). PBMCs were incubated with serially diluted ORN in the presence of DOTAP (starting from 2. mu. MORN and 25. mu.g/ml DOTAP) and culture Supernatants (SN) were collected after 24 hours. If not used immediately, SN is stored at-20 ℃ until needed.

The amount of cytokines in the SN was assessed using a commercially available ELISA kit for IL-12p40 (from BD Biosciences, Heidelberg, Germany) or an in-house ELISA kit developed with commercially available antibodies for IFN- α (PBL, New Brunswick, NJ, USA).

For the analysis of a wide range of cytokines and chemokines, a Multiplex analysis (Multiplex analysis) was performed using the luminex system from Bio-Rad (Munich, Germany) and the Multiplex kit from Biosource (Solingen, Germany).

Example 1: identification of immunostimulatory polymers that induce TLR 7-mediated, but not TLR-8-mediated cytokines

4 ORNs and positive controls were tested for their ability to induce IFN-. alpha.s (see Table 1 for sequence). ORN were incubated with human PBMC and supernatants were assayed for IFN- α by ELISA. ORN SEQ ID NO: 3 and SEQ ID NO: 5, but SEQ id no: 5 has a Phosphorothioate (PS) backbone, and ORN SEQ ID NO: 3 has a Phosphodiester (PO) backbone. Surprisingly, in contrast to the positive control ODN SEQ ID NO: 1 compared to SEQ id no: 5 induced only background levels of IFN- α, whereas SEQ ID NO: 3 induced the maximum IFN- α levels, SEQ ID NO: 1 has optimized GU-rich motifs within the phosphorothioate backbone (fig. 1). These data indicate that for SEQ ID NO: 3 and SEQ ID NO: 5, the PO backbone produced significant IFN- α induction.

In addition, two other ORNs with a PO backbone (SEQ ID NO: 2 and SEQ ID NO: 4) were also tested for their ability to induce IFN- α, and were shown to induce only very low levels of IFN- α. As shown in SEQ ID NO: 2 and SEQ ID NO: 3, the presence of one uridine (U) greatly enhances IFN- α induction. Uridine nucleotides are embedded within specific sequence motifs. The presence of a U outside this motif significantly reduced IFN- α production, as shown in SEQ id no: 3 and SEQ ID NO: 4 in sequence comparison.

ORN SEQ ID NO: 3 mainly induces IFN- α production (likely mediated by TLR 7) (see also FIG. 2A), but induces few other (TLR 8-mediated) cytokines, e.g., IL-12 (FIG. 2B), TNF- α (not shown), or IFN- γ (not shown).

TABLE 1

SEQ ID NO：1	rC*rC*rG*rU*rC*rU*rG*rU*rU*rG*rU*rG*rU*rG*rA*rC*rU*rC
		SEQ IDNO：2	rA-rA-rA-rC-rG-rC-rA-rC-rA-rG-rC-rC-rA-rA-rA-rG-rC-rA-rG
SEQ IDNO：3	rA-rA-rA-rC-rG-rC-rU-rC-rA-rG-rC-rC-rA-rA-rA-rG-rC-rA-rG
		SEQ ID NO：4	rA-rA-rA-rA-rA-rA-rA-rA-rU-rA-rA-rA-rA-rA-rA-rA-rA-rA
SEQ IDNO：5	rA*rA*rA*rC*rG*rC*rU*rC*rA*rG*rC*rC*rA*rA*rA*rG*rC*rA*rG

An ORN backbone: "-" indicates a phosphodiester and "-" indicates a phosphorothioate.

Example 2: novel immunostimulatory RNA motifs: identification of CUCA

To determine the optimal backbone-specific immunostimulatory motif, ORN were designed and tested for its ability to induce IFN- α. In contrast to the previously identified RNA motifs, the novel motifs identified were unique and specific to ORN with phosphodiester backbones (fig. 3). Human PBMC were incubated with ORN and supernatants were assayed for IFN- α by ELISA. A surprising short base sequence UCA was identified. The importance of the presence of adenine bases within this sequence is shown. For SEQ ID NO: 7 and SEQ ID NO: comparison of 6 (see table 2) shows that the optimal motif comprises one base 3' to UC. For SEQ ID NO: 11 and SEQ ID NO: 12 corroborates the importance of cytidine on the 3' side of this uridine.

TABLE 2

SEQ ID NO：3	rA-rA-rA-rC-rG-rC-rU-rC-rA-rG-rC-rC-rA-rA-rA-rG-rC-rA-rG
		SEQ ID NO：6	rA-rA-rA-rC-rG-rC-rA-rC-rA-rG-rC-rC-rA-rA-rA-rG-rC-rU-rC
SEQ ID NO：7	rA-rC-rG-rC-rA-rC-rA-rG-rC-rC-rA-rA-rA-rG-rC-rU-rC-rA-rG
		SEQ ID NO：11	rG-rC-rC-rA-rC-rC-rG-rA-rG-rC-rU-rG-rA-rA-rG-rG-rC-rA-rC-rC
SEQ ID NO：12	rG-rC-rC-rA-rC-rC-rG-rA-rG-rC-rU-rC-rA-rA-rG-rG-rC-rA-rC-rC

The exchange of the central C of the proposed minimal motif, which in the case of UGA (SEQ ID NO: 8) and UAA (SEQ ID NO: 9) leads to a loss of IFN-. alpha.inducing activity in the context of these ORNs, and in the case of UUA (SEQ ID NO: 10) leads to a reduction of the IFN-. alpha.response in the context of these ORNs (see FIG. 4). SEQ ID NO: 10 was most likely due to the presence of the motif GCUU containing two U.

These ORNs also show that the newly identified minimal motif is very specific for induction of IFN- α biased immune responses, since all 3 PO ORNs without a C in the motif (SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10) are more specific than the parent ORN containing the UCA motif SEQ ID NO: 3 induced much higher IL-12 levels (see Table 3).

TABLE 3

SEQ ID NO：3	rA-rA-rA-rC-rG-rC-rU-rC-rA-rG-rC-rC-rA-rA-rA-rG-rC-rA-rG
		SEQ ID NO：8	rA-rA-rA-rC-rG-rC-rU-rG-rA-rG-rC-rC-rA-rA-rA-rG-rC-rA-rG
SEQ ID NO：9	rA-rA-rA-rC-rG-rC-rU-rA-rA-rG-rC-rC-rA-rA-rA-rG-rC-rA-rG
		SEQ ID NO：10	rA-rA-rA-rC-rG-rC-rU-rU-rA-rG-rC-rC-rA-rA-rA-rG-rC-rA-rG

Further testing of sequence variation resulted in data showing that the adenosine portion of the UCA motif is important for IFN- α activity (fig. 5, sequence in table 4). Human PBMC were incubated with ORN and supernatants were tested for IFN-. alpha.and IL-12p40 by ELISA. Replacement of the adenosine within the UCA motif with either C or G (SEQ ID NO: 13, SEQ ID NO: 14) resulted in an ORN that induced only background levels of IFN- α. SEQ ID NO: 15(UCU) induced a modest amount of IFN- α, but less than the amount induced by the amino acid sequence of SEQ ID NO: 3 in terms of the amount of induction. This induction is likely due to the generation of additional sequence UCUG. Likewise, the polypeptide represented by SEQ ID NO: 3 induced IL-12 compared to SEQ id no: 14 and SEQ ID NO: the 15 phase ratio is very low.

TABLE 4

SEQ ID NO：3	rA-rA-rA-rC-rG-rC-rU-rC-rA-rG-rC-rC-rA-rA-rA-rG-rC-rA-rG
		SEQ ID NO：13	rA-rA-rA-rC-rG-rC-rU-rC-rC-rG-rC-rC-rA-rA-rA-rG-rC-rA-rG
SEQ ID NO：14	rA-rA-rA-rC-rG-rC-rU-rC-rG-rG-rC-rC-rA-rA-rA-rG-rC-rA-rG
		SEQ ID NO：15	rA-rA-rA-rC-rG-rC-rU-rC-rU-rG-rC-rC-rA-rA-rA-rG-rC-rA-rG

To further assess the necessity of additional nucleotides around the minimal motif, IFN- α inducing activity of UCA motifs with nucleotide modifications at 3 'and 5' was tested in comparison to IL-12 (sequences shown in Table 5). As shown in FIG. 6, the presence of only cytidine (CUCA, SEQ ID NO: 3) or uridine nucleotides (UUCA, SEQ ID NO: 21) in the 5' region of the minimal motif UCA produced ORNs with high IFN-. alpha.inducing properties. However, in this position only in the absence of uridine was observed the ORN induced a large amount of IFN-alpha and not a large amount of IL-12p40 activity, because the UUCA motif leads to a large production of both IFN-alpha and IL-12.

TABLE 5

SEQ ID NO：3	rA-rA-rA-rC-rG-rC-rU-rC-rA-rG-rC-rC-rA-rA-rA-rG-rC-rA-rG
		SEQ ID NO：19	rA-rA-rA-rC-rG-rG-rU-rC-rA-rG-rC-rC-rA-rA-rA-rG-rC-rA-rG
SEQ ID NO：20	rA-rA-rA-rC-rG-rA-rU-rC-rA-rG-rC-rC-rA-rA-rA-rG-rC-rA-rG
		SEQ ID NO：21	rA-rA-rA-rC-rG-rU-rU-rC-rA-rG-rC-rC-rA-rA-rA-rG-rC-rA-rG

Other sequence analyses showed that the nucleotide positions adjacent to the 4-mer motif CUCA did not appear to be as important and did not affect the ability of ORN to induce IFN- α production (table 6). However, these nucleotides did affect IL-12 production, as shown in figure 7. Nucleotides, whether on the 5 'side of the CUCA motif (FIG. 7A, SEQ ID NOS: 22, 23 and 24) or on its 3' side (FIG. 7B, SEQ ID NOS: 16, 17 and 18), did not appear to affect IFN- α inducing activity. These nucleotides appear to affect only IL-12 induction. Thus, for optimal TLR 7-specific induction properties, these positions should be as set forth in SEQ ID NO: 24 and SEQ ID NO: 18, as these ORN showed relatively strong IL-12 induction (fig. 7C and 7D).

TABLE 6

SEQ ID NO：3	rA-rA-rA-rC-rG-rC-rU-rC-rA-rG-rC-rC-rA-rA-rA-rG-rC-rA-rG
		SEQ ID NO：16	rA-rA-rA-rC-rG-rC-rU-rC-rA-rA-rC-rC-rA-rA-rA-rG-rC-rA-rG
SEQ ID NO：17	rA-rA-rA-rC-rG-rC-rU-rC-rA-rC-rC-rC-rA-rA-rA-rG-rC-rA-rG
		SEQ ID NO：18	rA-rA-rA-rC-rG-rC-rU-rC-rA-rU-rC-rC-rA-rA-rA-rG-rC-rA-rG
SEQ ID NO：22	rA-rA-rA-rC-rA-rC-rU-rC-rA-rG-rC-rC-rA-rA-rA-rG-rC-rA-rG
		SEQ ID NO：23	rA-rA-rA-rC-rC-rC-rU-rC-rA-rG-rC-rC-rA-rA-rA-rG-rC-rA-rG
SEQ ID NO：24	rA-rA-rA-rC-rU-rC-rU-rC-rA-rG-rC-rC-rA-rA-rA-rG-rC-rA-rG

Example 3: cytokine profile of the CUCA motif ORN

Immunostimulatory CUCA motif ORN SEQ ID NO: 27(GACACACACACUCACACACACACA) ability to induce various cytokines. Comparison of ORN SEQ ID NO: 27 with negative control (GACACACACACACACACACACACA; SEQ ID NO: 25), non-UCA ORN with a U-rich 3' terminus (GACACACACACACACACACACUUU; SEQ ID NO: 26) and positive control (GACACACACACUCACACACACACA; SEQ ID NO: 28). Human PBMC from 3 healthy donors were incubated with serial dilutions of ORN in the presence of DOTAP (starting from 2. mu.M ORN and 25. mu.g/ml DOTAP) for 24 h. SN was collected and cytokine concentrations or chemokine concentrations of various cytokines or chemokines were determined by ELISA. SEQ ID NO: 27 induced IFN- α and to a lesser extent IFN- α related molecules IP-10 and MCP-1.

To analyze larger sets of cytokines and chemokines, Multiplex assays were performed using the Luminex Multiplex kit. Two donors were used in the initial non-quantitative evaluation of Luminex data. The results are summarized in table 7 below. Likewise, SEQ ID NO: 27 induced only IFN-. alpha.and IP-10 and to a lesser extent MIP-1.

TABLE 7

	SEQ IDNO：25	SEQ IDNO：26	SEQ IDNO：28	SEQ IDNO：27	DOTAP
						IL-1B	-	+	+++	-	-
IL-1Ra	-	-	-	-	-
						IL-2	-	-	-	-	-
IL-2R	-	+	+++	-	-
						IL-4	n.d.	n.d.	n.d.	n.d.	n.d.
IL-5	-	-	-
						IL-6	-	+	+++	-	-
IL-7	-	++	+++	-	+
						IL-8	-	+	+	-	+
IL-10	-	-	+++	-	-
						IL-12p40	-	-	+++	-	-
IL-13	n.d.	n.d.	n.d.	n.d.	n.d.
						IL-15	-	+	+++	-	-
IL-17	n.d.	n.d.	n.d.	n.d.	n.d.
						TNF-α	-	-	+++	-	-
IFN-α	-	-	+++	++	-
						IFN-γ	-	-	+++	-	-
GMCSF	n.d.	n.d.	n.d.	n.d.	n.d.

	SEQ IDNO：25	SEQ IDNO：26	SEQ IDNO：28	SEQ IDNO：27	DOTAP
						MIP-1a	-	+	+++	-	-
MIP-1b	-	+	+++	-	-
						IP-10	-	-	+	++	+
MIG	-	+	+++	-	-
						Eotaxin	n.d.	n.d.	n.d.	n.d.	n.d.
Rantes	-	-	+	-	+
						MCP-1	-	+	++	+	+

n.d.: not detected

Example 4: significant Th1 cytokine and chemokine induction of ORN with UCA motif

ORN with UCA motif were tested for cytokine and chemokine induction in vivo. BALB/c mice were divided into two groups of 5 mice each and administered intravenously with ORN, DOTAP or buffered saline (HBS). The first group of animals was bled 3 hours after injection and serum levels of IP-10, IFN- α, TNF- α, IL-2, IL-12, IL-6 and IL-10 were determined using an appropriate cytokine specific ELISA. A second group of animals was bled 24 hours after injection and serum levels of IFN- α and IL-10 were determined. UCA ORN SEQ ID NO: 3 ability to induce cytokines and chemokines compared to two ORN (GACACACACACACACACACACAUU; SEQ id NO: 30; and UUAUUAUUAUUAUUAUUAUU (phosphorothioate backbone); SEQ id NO: 33)) that induce both TLR 7-related cytokines and TLR 8-related cytokines and two ORN (UUGUUGUUGUUGUUGUUGUU; SEQ ID NO: 31; and UUAUUAUUAUUAUUAUUAUU (phosphodiester backbone); SEQ ID NO: 32) a comparison is made. In addition, CpG ODN 1826 (TCCATGACGTTCCTGACGTT; SEQ ID NO: 36) was also used as a control. SEQ ID NO: 3 induced IFN- α (FIGS. 12A and 12C) and IP-10 (FIGS. 12B and 12D) at both the 3 hour and 24 hour time points, but did not induce significant amounts of TNF- α, IL-2, IL-12, IL-6, or IL-10 (FIGS. 13A-E, respectively) at the 3 hour time point. Thus, SEQ ID NO: 3 induced only IFN-alpha and IFN-alpha associated cytokines.

In addition, UCA ORN was shown to activate B and T cells from the spleen. SEQ ID NO: 3 activating spleen CD3⁺T cells (FIGS. 14A and 14B) and DX5⁺B cells (fig. 14C and 14D).

Example 5: the presence of UCA motifs rather than only U is important for IFN-alpha induction

In this experiment, it was shown that the presence of 1-3 uridines was not sufficient to induce IFN- α. Human PBMCs from 3 healthy donors were incubated with varying amounts of ORN (SEQ ID NO: 26-30, Table 8) for 24h in the presence of DOTAP (starting concentration: 2. mu.M ORN + 25. mu.g/ml DOTAP, 1: 3 serial dilutions in PBS). IFN- α was measured in the supernatant using an appropriate ELISA.

TABLE 8

SEQ ID NO：26	rG-rA-rC-rA-rC-rA-rC-rA-rC-rA-rC-rA-rC-rA-rC-rA-rC-rA-rC-rA-rC-rU-rU-rU
		SEQ ID NO：27	rG-rA-rC-rA-rC-rA-rC-rA-rC-rA-rC-rU-rC-rA-rC-rA-rC-rA-rC-rA-rC-rA-rC-rA
SEQ ID NO：28	rU*rU*rG*rU*rU*rG*rU*rU*rG*rU*rU*rG*rU*rU*rG*rU*rU*rG*rU*rU
		SEQ ID NO：29	rG-rA-rC-rA-rC-rA-rC-rA-rC-rA-rC-rA-rC-rA-rC-rA-rC-rA-rC-rA-rC-rA-rC-rU
SEQ ID NO：30	rG-rA-rC-rA-rC-rA-rC-rA-rC-rA-rC-rA-rC-rA-rC-rA-rC-rA-rC-rA-rC-rA-rU-rU

The results are shown in FIG. 15. Although in SEQ ID NO: 26. up to 3 uridines were present in 29 and 30, and these ORN without UCA motif did not induce IFN- α. In contrast, SEQ ID NO: 27 induced significant amounts of IFN- α.

Example 6: in vivo cytokine induction has strong TLR-7 dependence

In this experiment, TLR-7 deficient mice were shown not to respond to the ORN of the invention. TLR9 knockout (TLR9KO) and TLR7 knockout (TLR7KO) mice were back crossed (backscross) with C57BL/6 background mice and C57BL/6 control mice (n ═ 4 per group), followed by intravenous injection with 100 μ g of the selected ORN (SEQ ID NO: 3, 28 or 31) or CpG ODN 1826(SEQ ID NO: 36) at 1: 2 in dotap (sigma) as a positive control or buffered saline (HBS) as a negative control. 3 hours after injection, plasma was obtained and analyzed for IFN-. alpha.IP-10, IL-12 and IL-6 using appropriate Luminex and ELISA.

The results are shown in FIG. 16. TLR7KO mice respond to SEQ ID NO: 3. at 28 or 31, showed substantially no induction of IFN- α, IP-10, IL-12 or IL-6, but strong induction of these same cytokines in response to CpG ODN. In contrast, TLR9KO mice respond to SEQ ID NO: 3. 28 or 31 showed different degrees of IFN-. alpha.IP-10, IL-12 and IL-6 induction, but not these same cytokines in response to CpG ODN. Control C57BL/6 mice respond to SEQ ID NO: 3. 28 or 31 show different degrees of IFN-alpha, IP-10, IL-12 and IL-6 induction, and in response to CpG ODN show strong induction of these same cytokines.

Example 7: in vivo cytokine induction has strong MyD88 dependence

In this experiment, MyD88 deficient mice were shown not to react with the ORN of the invention. MyD88 knockout (MyD88KO) mice were backcrossed with C57BL/6 background mice and C57BL/6 control mice (n ═ 4 per group) followed by intravenous injection with 100 μ g of the selected ORN (SEQ ID NO: 3, 28, or 31) or CpG ODN 1826(SEQ ID NO: 36) at 1: 2 in DOTAP (Sigma) as a positive control or buffered saline (HBS) as a negative control. 3 hours after injection, plasma was obtained and analyzed for IFN-. alpha.and IP-10 using appropriate Luminex and ELISA.

The results are shown in FIG. 17. MyD88KO mice respond to SEQ ID NO: 3. at 28 or 31, there was essentially no induction of IFN-. alpha.or IP-10, but similar in response to CpG ODN. In contrast, control C57BL/6 mice respond to SEQ ID NO: 3. both 28 or 31 and in response to CpG ODN showed potent IFN-. alpha.and IP-10 induction.

In summary, the present inventors have identified a novel backbone-specific motif responsible for inducing IFN- α (likely TLR 7-mediated) with little activation of other cytokines (likely TLR 8-mediated) such as IL-12, IFN- γ or TNF- α. The smallest motif that determines these properties is rU-rC-rA. The backbone is a phosphodiester. The optimal motif based on this data is rN-rC-rU-rC-rA-rN, where N is C, A, G but not U (for minimal TLR8 mediated cytokine response).

Having thus described several aspects of at least one embodiment of this invention, it is to be appreciated various alterations, modifications, and improvements will readily occur to those skilled in the art. Such alterations, modifications, and improvements are intended to be part of this disclosure, and are intended to be within the spirit and scope of the invention. Accordingly, the foregoing description and drawings are by way of example only.

Sequence listing

<110> Kohler pharmaceuticals, Inc

<120> RNA sequence motifs inducing specific immunomodulating properties in a defined internucleotide linkage environment

<130>PC19793A

<150>US 60/964,448

<151>2007-08-13

<160>36

<170>PatentIn version 3.4

<210>1

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ccgucuguug ugugacuc 18

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aaacgcacag ccaaagcag 19

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aaacgcucag ccaaagcag 19

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aaaaaaaaua aaaaaaaa 18

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aaacgcucag ccaaagcag 19

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aaacgcacag ccaaagcuc 19

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acgcacagcc aaagcucag 19

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aaacgcugag ccaaagcag 19

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aaacgcuaag ccaaagcag 19

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aaacgcuuag ccaaagcag 19

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gccaccgagc ugaaggcacc 20

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gccaccgagc ucaaggcacc 20

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aaacgcuccg ccaaagcag 19

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aaacgcucgg ccaaagcag 19

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aaacgcucug ccaaagcag 19

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aaacgcucaa ccaaagcag 19

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aaacgcucac ccaaagcag 19

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aaacgcucau ccaaagcag 19

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aaacggucag ccaaagcag 19

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aaacccucag ccaaagcag 19

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aaacucucag ccaaagcag 19

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gacacacaca cacacacaca caca 24

<210>26

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gacacacaca cacacacaca cuuu 24

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gacacacaca cucacacaca caca 24

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uuguuguugu uguuguuguu 20

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gacacacaca cacacacaca cacu 24

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gacacacaca cacacacaca cauu 24

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uuguuguugu uguuguuguu 20

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uuauuauuau uauuauuauu 20

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uuauuauuau uauuauuauu 20

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uuaucguamc ucac 14

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ccgagccgag cucacc 16

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tccatgacgt tcctgacgtt 20

Claims (13)

1. A composition comprising an immunostimulatory RNA motif rN₁-rC-rU-rC-rA-rN₂The single-chain polymer of (1), wherein the polymer is A-A-A-C-G-C-U-C-A-G-C-C-A-A-G-C-A-G; the composition further comprises a delivery vehicle, wherein the delivery vehicle is a liposome, a non-ionic vesicle, a lipid complex, a polymer complex, a lipid-polymer complex, a water-in-oil emulsion, an oil-in-water emulsion, a water-in-oil-in-water multiple emulsion, a microemulsion, a nanoemulsion, a micelle, a dendrimer, a viral particle, a viroid, a polymeric nanoparticle(ii) a particle or a polymer microparticle, with the proviso that the liposome is not a lipofectin, and wherein "-" denotes a phosphodiester backbone.

2. The composition of claim 1, further comprising an antigen.

3. The composition of claim 2, wherein the antigen is conjugated to a polymer.

4. The composition of claim 1, further comprising a lipophilic moiety covalently attached to the polymer.

5. The composition of claim 4, wherein the lipophilic moiety is selected from the group consisting of cholesteryl, palmitoyl, and fatty acyl.

6. The composition of claim 1, wherein the polymer is linked to a CpG nucleic acid.

7. Use of a single chain immunostimulatory polymer a-C-G-C-U-C-a-G-C-a-G-C-a-G for the preparation of a medicament for inducing the production of interferon alpha in an immune cell, wherein "-" denotes a phosphodiester backbone.

8. The use of claim 7, wherein the immune cells respond to the polymer without producing significant amounts of tumor necrosis factor alpha.

9. The use of claim 7, wherein the immune cells respond to the polymer without producing a significant amount of interferon gamma.

10. The use of claim 7, wherein the immune cells respond to the polymer without producing significant amounts of interleukin 12.

11. Use according to claim 7, wherein the polymer is administered in the form of a composition according to any one of claims 1 to 7.

12. Use of a single chain immunostimulatory polymer a-C-G-C-U-C-a-G-C-a-G-C-a-G for the preparation of a medicament for inducing a TLR7 response, wherein "-" denotes a phosphodiester backbone.

13. The use of claim 12, wherein the TLR7 response is to induce production of interferon alpha in an immune cell.