The invention relates to new immunomodulatory compositions which comprise a cationic condensing agent, an immunomodulatory compound, and a stabilizing agent. The compositions of the invention typically form particles which have increased immunomodulatory activity as compared to immunomodulatory compounds not formulated in the compositions of the invention. Also provided are methods of making the compositions and methods for therapeutic use of the compositions.

Description of the Invention

SUMMARY OF THE INVENTION

The invention relates to new compositions and methods for modulating immune responses in individuals, particularly human individuals. Applicants have discovered new immunomodulatory compositions having substantially improved immunomodulatory activity.

In one aspect, the invention relates to compositions comprising a cationic condensing agent, an immunomodulatory compound (IMC), and a stabilizing agent. In another aspect, the invention relates to particulate compositions comprising a cationic condensing agent, an immunomodulatory compound (IMC), and a stabilizing agent. Also included are pharmaceutical compositions comprising an immunomodulatory composition of the invention and a pharmaceutically acceptable excipient. In certain embodiments, the compositions of the invention may further include, or optionally exclude, additional components, including fatty acids and/or antigen molecules.

In another aspect, the invention relates to methods of modulating an immune response in an individual, comprising administering to an individual an immunomodulatory composition of the invention in an amount sufficient to modulate an immune response in said individual. Immunomodulation according to the methods of the invention may be practiced on individuals including those suffering from a disorder associated with a Th2-type immune response (e.g., allergies or allergy-induced asthma), individuals receiving vaccines such as therapeutic vaccines (e.g., vaccines comprising an allergy epitope, a mycobacterial epitope, or a tumor associated epitope) or prophylactic vaccines, individuals with cancer, individuals having an infectious disease and individuals at risk of exposure to an infectious agent.

In a further aspect, the invention relates to methods of increasing interferon-gamma (IFN-.gamma.) in an individual, comprising administering an effective amount of an immunomodulatory composition of the invention to the individual. Administration of an immunomodulatory composition of the invention increases IFN-.gamma. in the individual. Suitable subjects for these methods include those individuals having idiopathic pulmonary fibrosis (IPF), scleroderma, cutaneous radiation-induced fibrosis, hepatic fibrosis including schistosomiasis-induced hepatic fibrosis, renal fibrosis as well as other conditions which may be improved by administration of IFN-.gamma..

In another aspect, the invention relates to methods of increasing IFN-.alpha. in an individual, comprising administering an effective amount of an immunomodulatory composition of the invention to the individual. Administration of an immunomodulatory composition of the invention increases IFN-.alpha. levels in the individual. Suitable subjects for these methods include those individuals having disorders which respond to the administration of IFN-.alpha., including viral infections and cancer.

In another aspect, the invention relates to methods of ameliorating one or more symptoms of an infectious disease, comprising administering an effective amount of an immunomodulatory composition of the invention to an individual having an infectious disease. Administration of an immunomodulatory composition of the invention ameliorates one or more symptoms of the infectious disease. The infectious diseases which may be treated in accordance with the invention include infectious diseases caused by a cellular pathogen (e.g., a mycobacterial disease, malaria, leishmaniasis, toxoplasmosis, schistosomiasis or clonorchiasis), and may include or exclude viral diseases.

The invention further relates to kits for carrying out the methods of the invention. The kits of the invention generally comprise a container comprising an immunomodulatory composition of the invention (or as described herein, materials for production of an immunomodulatory composition of the invention) and optionally include instructions for use of immunomodulatory composition for the immunomodulation of an individual, for example when the individual suffers from a disorder associated with a Th2-type immune response (e.g., allergies or allergy-induced asthma), is receiving vaccines such as therapeutic vaccines (e.g., vaccines comprising an allergy epitope, a mycobacterial epitope, or a tumor associated epitope) or prophylactic vaccines, suffers from cancer, suffers from an infectious disease or is at risk of exposure to an infectious agent.

The invention also provides methods of making the formulations of the invention, comprising combining an immunomodulatory compound (IMC), a stabilizing agent and a cationic condensing agent to make a mixture. In some embodiments, methods of making the formulations of the invention comprise combining an immunomodulatory compound (IMC) and a stabilizing agent to make an initial mixture, then adding a cationic condensing agent to the initial mixture, resulting in the immunomodulatory composition. The invention further provides immunomodulatory formulations made by these processes.

DETAILED DESCRIPTION OF THE INVENTION

The inventors have discovered new compositions and methods for modulating immune responses in individuals, including and particularly humans. The compositions of the invention comprise a particulate complex of a cationic condensing agent, an immunomodulatory compound (IMC), and a stabilizing agent. Surprisingly, the inventors have found that IMCs formulated with a cationic condensing agent and a stabilizing agent have substantially increased immunomodulatory activity compared to immunomodulatory compounds given alone.

General Techniques

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as, Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989); Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Animal Cell Culture (R. I. Freshney, ed., 1987); Handbook of Experimental Immunology (D. M. Weir & C. C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M. Miller & M. P. Calos, eds., 1987); Current Protocols in Molecular Biology (F. M. Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (J. E. Coligan et al., eds., 1991); The Immunoassay Handbook (D. Wild, ed., Stockton Press NY, 1994); Bioconjugate Techniques (Greg T. Hermanson, ed., Academic Press, 1996); and Methods of Immunological Analysis (R. Masseyeff, W. H. Albert, and N. A. Staines, eds., Weinheim: VCH Verlags gesellschaft mbH, 1993).

Compositions

The invention provides new compositions for modulating immune response in individuals. The new compositions are formulations comprising a cationic condensing agent, an IMC, and a stabilizing agent. In some embodiments, the compositions of the invention may also comprise an antigen and/or a fatty acid.

The compositions of the invention are typically in particulate form. As will be apparent to those of skill in the art, particulate compositions of the invention will consist of a population of particles of different sizes. Due to this naturally arising variability, the "size" of the particles in the compositions of the invention may be described in ranges or as a maximum or minimum diameter. Particles are considered to be a particular size if at least 95% of the particles (by mass) meet the specified dimension (e.g., if at least 97% of the particles are less than 20 .mu.m in diameter, then the composition is considered to consist of particles of less than 20 .mu.m in diameter). Particle size may be measured by any convenient method known in the art, including filtration (e.g., use of a "depth" filter to capture particles greater than a cutoff size), dynamic light scattering, electron microscopy, including TEM (particularly in combination with freeze-fracture processing) and SEM, and the like.

Preferably, the compositions of the invention comprise particles which are less than about 50 .mu.m in diameter, more preferably less than about 20 .mu.m in diameter, although in some embodiments the particles will be less than about 3, 2 or 1 .mu.m in diameter. Preferred particle size ranges include about 0.01 .mu.m to 50 .mu.m, 0.02 to 20 .mu.m, 0.05 to 5 .mu.m, and 0.05 to 3 .mu.m in diameter.

The components of the compositions of the invention may be present in various ratios/quantities in the compositions, although it is contemplated that the amounts of the stabilizing agent(s) and optional components such as fatty acids and antigen will remain relatively invariant, with stabilizing agents generally ranging from about 0.1% to 0.5% (v/v), fatty acids ranging from about 0 to 0.5%, and antigen concentrations ranging from about 0.1 to about 100 .mu.g/mL, preferably about 1 to about 100 .mu.g/mL, more preferably about 10 to 50 .mu.g/mL. The amounts and ratios of the IMC and the cationic condensing agent are subject to a greater range of variation in the compositions of the invention. The amount of IMC will vary to a certain extent as a function of the molecular weight of the IMC, and generally ranges from about 50 .mu.g/mL to about 2 mg/mL, preferably about 100 .mu.g/mL to 1 mg/mL. The cationic condensing agent is generally present in excess (in terms of mass) over the IMC, generally in ratios of about 1:2 (IMC:cationic condensing agent) to about 1:6, more preferably about 2:5 to 1:5.

Particle size in the compositions of the invention is a function of a number of variables. The inventors have found that the size distribution of particles in the compositions can be modulated by altering the ratio of cationic condensing agent to IMC. For example, altering the ratio of cationic condensing agent to IMC in the exemplary +ISS/0.4% Tween 85/0.4% oleate/polymyxin B compositions can alter mean particle size from about 1.5 .mu.m at cationic condensing agent:IMC=1 to about 45 .mu.m at cationic condensing agent:IMC=10.

In certain embodiments, the compositions comprise a cationic condensing agent, an IMC and a stabilizing agent that is a nonionic detergent. In other embodiments, the compositions comprise a membrane disrupting cationic lipopeptide (preferably a polymyxin, more preferably polymyxin B (PMXB)), an IMC and a stabilizing agent. In some embodiments the stabilizing agent is not a serum protein (particularly not a bovine serum protein). An exemplary composition of this class of embodiments utilizes a polyoxyethylene ether detergent such as Tween 80 or Tween 85 as the stabilizing agent, with oleate as an optional additional stabilizing agent.

In some embodiments, compositions of the invention comprise immunomodulatory particles, wherein the particles are made by the process of combining a cationic condensing agent, an IMC and a stabilizing agent that is a nonionic detergent. In other embodiments, compositions of the invention comprise immunomodulatory particles, wherein the particles are made by the process of combining a membrane disrupting cationic lipopeptide (preferably a polymyxin, more preferably PMBX), an IMC and a stabilizing agent. In some embodiments the stabilizing agent is not a serum protein (particularly not a bovine serum protein).

In some embodiments, compositions of the invention comprise immunomodulatory particles, wherein the particles are formed by the process of combining an IMC and a stabilizing agent that is a nonionic detergent, thereby forming an IMC/stabilizing agent mixture, and combining a cationic condensing agent with the IMC/stabilizing agent mixture. In other embodiments, compositions of the invention comprise immunomodulatory particles, wherein the particles are formed by the process of combining an IMC and a stabilizing agent, thereby forming an IMC/stabilizing agent mixture, and combining a membrane disrupting cationic lipopeptide (preferably a polymyxin, more preferably PMXB) with the IMC/stabilizing agent mixture. In some embodiments the stabilizing agent is not a serum protein (particularly not a bovine serum protein).

In some embodiments, compositions of the invention comprise immunomodulatory particles, wherein the particles comprise a cationic condensing agent, an IMC and a stabilizing agent that is a nonionic detergent. In other embodiments, compositions of the invention comprise immunomodulatory particles, wherein the particles comprise a membrane disrupting cationic lipopeptide (preferably a polymyxin, more preferably PMXB), an IMC and a stabilizing agent. In some embodiments the stabilizing agent is not a serum protein (particularly not a bovine serum protein).

Cationic Condensing Agents

Cationic condensing agents useful in the compositions and methods of the invention are molecules which are positively charged at physiological pH (i.e., pH of about 7.0 to about 7.5). Preferably, cationic condensing agents used in the instant invention are not zwitterionic and are polycationic, that is, having more than one positive charge per molecule. Cationic condensing agents useful in the instant invention include hydrophilic or amphipathic polycations.

Preferred cationic condensing agents include: (a) membrane disrupting cationic lipopeptides including, but not limited to polymyxins including polymyxin A, polymyxin B (including polymyxin B.sub.1 and polymyxin B.sub.2), polymyxin C, polymyxin D, polymyxin E (also known as colistin), polymyxin K, polymyxin M, polymyxin P, polymyxin S and polymyxin T, circulins including circulin A, circulin B, circulin C, circulin D, circulin E and circulin F, octapeptin, amphotericins including amphotericin B, and acylated peptides including octanoyl-KFFKFFKFF (SEQ ID NO:42) and acyl KALA (octanoyl-WEAKLAKALAKALAKHLAKALAKALEACEA (SEQ ID NO:43); (b) membrane disrupting cationic peptides including, but not limited to polymyxin B (PMXB) nonapeptide, cecropins including cecropin A, cecropin B and cecropin P1, KFFKFFKFF (SEQ ID NO:42) and KALA (WEAKLAKALAKALAKHLAKALAKALKACEA) (SEQ ID NO:44); (c) single chain cationic surfactants including, but not limited to cetyltrimethylammonium bromide (CTAB), benzyl-dimethyl-ammonium bromide (BDAB), CpyrB (cetyl-pyridinium bromide), CimB (cetyl imidazolium bromide), and polycationic polymers, including, but not limited to, poly-L-lysine (PLL) and polyethyleneimine (PEI). In certain embodiments, the cationic condensing agent is a membrane disrupting cationic lipopeptide, preferably a polymyxin, more preferably PMXB. In some embodiments, cationic condensing agents may exclude fatty acid esters (i.e., lipids) and double chain cationic surfactants.

Immunomodulatory Compounds

In accordance with the present invention, the immunomodulatory compound (IMC) is a molecule which has immunomodulatory activity and which comprises a nucleic acid moiety comprising the sequence 5'-CG-3'. The IMC may be a polynucleotide or it may be a compound which incorporates nucleic acid moieties, at least one of which comprises the sequence 5'-CG-3', such as a CIC. When the IMC is a polynucleotide, the polynucleotide may contain at least one ISS, and can contain multiple ISSs. The ISSs can be adjacent within the polynucleotide, or they can be separated by additional nucleotide bases within the polynucleotide. In certain embodiments, the IMC consists of an ISS. Accordingly, a polynucleotide IMC may contain combinations of any one or more ISS described herein. Alternately, the IMC may be a CIC comprising at least two nucleic acid moieties, at least one of which comprises the sequence 5'-CG-3', covalently linked to a spacer moiety. The IMC affects a measurable immune response, as measured in vitro, in vivo and/or ex vivo, when administered as a component of an immunomodulatory composition of the invention.

A polynucleotide IMC can be of any length greater than 6 bases or base pairs and generally comprises the sequence 5'-cytosine, guanine-3', preferably greater than 15 bases or base pairs, more preferably greater than 20 bases or base pairs in length. As is well-known in the art, the cytosine of the 5'-cytosine, guanine-3' sequence is generally unmethylated, especially at the C-5 position, although in certain embodiments, methylation of the cytosine may be permitted, for example, at the N-4 position. A polynucleotide IMC may also comprise the sequence 5'-purine, purine, C, G, pyrimidine, pyrimidine, C, G-3'. A polynucleotide IMC may also comprise the sequence 5'-purine, purine, C, G, pyrimidine, pyrimidine, C, C-3'. As indicated in polynucleotide sequences below, a polynucleotide IMC may comprise (i.e., contain one or more of) the sequence 5'-T, C, G-3' or 5'-T, C, G, A-3'. Accordingly, a polynucleotide IMC may comprise 5'-CG-3' and/or 5'-TCG-3' and/or 5'-TCGA-3', and in certain embodiments uracil (U) may be substituted for thymine. In some embodiments, a polynucleotide IMC may comprise the sequence 5'-C, G, pyrimidine, pyrimidine, C, G-3' (such as 5'-CGTTCG-3'). In some embodiments, a polynucleotide IMC may comprise the sequence 5'-C, G, pyrimidine, pyrimidine, C, G, purine, purine-3'. In some embodiments, a polynucleotide IMC comprises the sequence 5'-purine, purine, C, G, pyrimidine, pyrimidine-3' (such as 5'-AACGTT-3'). As further discussed below, a polynucleotide IMC may contain 5-bromocytosine in place of the "C" in the 5'-CG-3',5'-TCG-3',5'-TCGA-3',5'-UCG-3', or 5'-UCGA-3' of the ISS.

In some embodiments, a polynucleotide IMC may comprise the sequence 5'-purine, T, C, G, pyrimidine, pyrimidine-3'.

In some embodiments, the polynucleotide IMC comprises any of the following sequences: GACGCTCC; GACGTCCC; GACGTTCC; GACGCCCC; AGCGTTCC; AGCGCTCC; AGCGTCCC; AGCGCCCC; AACGTCCC; AACGCCCC; AACGTTCC; AACGCTCC; GGCGTTCC; GGCGCTCC; GGCGTCCC; GGCGCCCC; GACGCTCG; GACGTCCG; GACGCCCG; GACGTTCG; AGCGCTCG; AGCGTTCG; AGCGTCCG; AGCGCCCG; AACGTCCG; AACGCCCG; AACGTTCG; AACGCTCG; GGCGTTCG; GGCGCTCG; GGCGTCCG; GGCGCCCG.

In some embodiments, the polynucleotide IMC comprises any of the following sequences: GACGCT; GACGTC; GACGTT; GACGCC; GACGCU; GACGUC; GACGUU; GACGUT; GACGTU; AGCGTT; AGCGCT; AGCGTC; AGCGCC; AGCGUU; AGCGCU; AGCGUC; AGCGUT; AGCGTU; AACGTC; AACGCC; AACGTT; AACGCT; AACGUC; AACGUU; AACGCU; AACGUT; AACGTU; GGCGTT; GGCGCT; GGCGTC; GGCGCC; GGCGUU; GGCGCU; GGCGUC; GGCGUT; GGCGTU.

In some embodiments, the polynucleotide IMC comprises any of the following sequences: GABGCT; GABGTC; GABGTT; GABGCC; GABGCU; GABGUC; GABGUU; GABGUT; GABGTU; AGBGTT; AGBGCT; AGBGTC; AGBGCC; AGBGUU; AGBGCU; AGBGUC; AGBGUT; AGBGTU; AABGTC; AABGCC; AABGTT; AABGCT; AABGUC; AABGUU; AABGCU; AABGUT; AABGTU; GGBGTT; GGBGCT; GGBGTC; GGBGCC; GGBGUU; GGBGCU; GGBGUC; GGBGUT; GGBGTU, where B is 5-bromocytosine.

In some embodiments, the polynucleotide IMC comprises any of the following sequences: GABGCTCC; GABGTCCC; GABGTTCC; GABGCCCC; AGBGTTCC; AGBGCTCC; AGBGTCCC; AGBGCCCC; AABGTCCC; AABGCCCC; AABGTTCC; AABGCTCC; GGBGTTCC; GGBGCTCC; GGBGTCCC; GGBGCCCC; GABGCTCG; GABGTCCG; GABGCCCG; GABGTTCG; AGBGCTCG; AGBGTTCG; AGBGTCCG; AGBGCCCG; AABGTCCG; AABGCCCG; AABGTTCG; AABGCTCG; GGBGTTCG; GGBGCTCG; GGBGTCCG; GGBGCCCG; GABGCTBG; GABGTCBG; GABGCCBG; GABGTTBG; AGBGCTBG; AGBGTTBG; AGBGTCBG; AGBGCCBG; AABGTCBG; AABGCCBG; AABGTTBG; AABGCTBG; GGBGTTBG; GGBGCTBG; GGBGTCBG; GGBGCCBG, where B is 5-bromocytosine.

In some embodiments, the polynucleotide IMC comprises any of the following sequences: GABGCUCC; GABGUCCC; GABGUTCC; GABGTUCC; GABGUUCC; AGBGUUCC; AGBGTUCC; AGBGUTCC; AGBGCUCC; AGBGUCCC; AABGUCCC; AABGUUCC; AABGUTCC; AABGTUCC; AABGCUCC; GGBGUUCC; GGBGUTCC; GGBGUUCC; GGBGCUCC; GGBGUCCC; GABGCUCG; GABGUCCG; GABGUUCG; GABGUTCG; GABGTUCG; AGBGCUCG; AGBGUUCG; AGBGUTCG; AGBGTUCG; AGBGUCCG; AABGUCCG; AABGUUCG; AABGUTCG; AABGTUCG; AABGCUCG; GGBGUUCG; GGBGUTCG; GGBGTUCG; GGBGCUCG; GGBGUCCG; GABGCUBG; GABGUCBG; GABGUUBG; GABGUTBG; GABGTUBG; AGBGCUBG; AGBGUUBG; AGBGUCBG; AGBGUTBG; AGBGTUBG; AABGUCBG; AABGUUBG; AABGUTBG; AABGTUBG; AABGCUBG; GGBGUUBG; GGBGUTBG; GGBGTUBG; GGBGCUBG; GGBGUCBG, where B is 5-bromocytosine.

In some embodiments, the immunomodulatory compound comprises the sequence 5'-TGACTGTGAACGTTCGAGATGA-3' (SEQ ID NO:1). In other embodiments, the polynucleotide IMC comprises any of the sequences -- see Original Patent.

In some embodiments, a polynucleotide IMC can be 7 bases (or base pairs) or more. Accordingly, in some embodiments, the polynucleotide IMC comprises or consists of a sequence according to the formula 5'-TCGX.sub.1X.sub.2X.sub.3X.sub.4-3' or 5'-UCGX.sub.1X.sub.2X.sub.3X.sub.4-3' wherein X.sub.1, X.sub.2, X.sub.3, X.sub.4 are nucleotides. In some embodiments, the polynucleotide IMC comprises any of the following sequences: 5'-TCGTTTT-3'; 5'-TCGAAAA-3'; 5'-TCGCCCC-3'; 5'-TCGGGGG-3'; 5'-TCGUUUU-3'; 5'-TCGIIII-3'; 5'-UCGTTTT-3'; 5'-UCGAAAA-3'; 5'-UCGCCCC-3'; 5'-UCGGGGG-3'; 5'-UCGUUUU-3'; 5'-UCGIIII-3'. In some embodiments, the polynucleotide IMC comprises or consists of a sequence according to the formula 5'-X.sub.1TCGX.sub.2X.sub.3X.sub.4-3' or 5'-X.sub.1UCGX.sub.2X.sub.3X.sub.4-3', wherein X.sub.1, X.sub.2, X.sub.3, X.sub.4 are nucleotides. In some embodiments, the polynucleotide IMC comprises any of the following sequences: 5'-TTCGTTT-3'; 5'-ATCGATT-3'; 5'-TUCGTTT-3'; 5'-AUCGATT-3'. In other embodiments, the polynucleotide IMC comprises or consist of a sequence according to the formula 5'-X.sub.1X.sub.2TCGX.sub.3X.sub.4-3' or 5'-X.sub.1X.sub.2UCGX.sub.3X.sub.4-3', wherein X.sub.1, X.sub.2, X.sub.3, X.sub.4 are nucleotides. In some embodiments, the polynucleotide IMC comprises any of the following sequences: 5'-TTTCGTT-3'; 5'-AATCGAT-3'; 5'-TTUCGTT-3'; 5'-AAUCGAT-3'.

In certain embodiments, the polynucleotide IMC comprises a sequence of the formula 5'-X.sub.1X.sub.2 A X.sub.3 C G X.sub.4 T C G-3' (SEQ ID NO:21) wherein X.sub.1 is T, G, C or B (B=5-bromocytosine), wherein X.sub.2 is T, G, A or U, wherein X.sub.3 is T, A or C, wherein X.sub.4 is T, G or U. In certain embodiments, polynucleotide IMCs according to this formula exclude 5'-TGAACGTTCG-3' (SEQ ID NO:22) or 5'-GGAACGTTCG-3' (SEQ ID NO:23).

In other embodiments, the polynucleotide IMC comprises a sequence of the formula 5'-X.sub.1 X.sub.2 A X.sub.3 B G X.sub.4 T C G-3' (SEQ ID NO:24) wherein B is 5-bromocytosine, wherein X.sub.1 is T, G, C or B (B=5-bromocytosine), wherein X.sub.2 is T, G, A or U, wherein X.sub.3 is T, A or C, wherein X.sub.4 is T, G or U. In certain embodiments, polynucleotide IMCs according to this formula exclude 5'-TGAABGTTCG-3' (SEQ ID NO:25) (B=5-bromocytosine).

In some embodiments, the polynucleotide IMC comprises the sequence 5'-TCGTCGX.sub.1-3', wherein X.sub.1 is a nucleotide. In other embodiments, the polynucleotide IMC comprises any of the following sequences: 5'-TCGTCGA-3'; 5'-TCGTCGC-3'; 5'-TCGTCGG-3'; 5'-TCGTCGT-3'; 5'-TCGTCGU-3'; 5'-TCGTCGI-3'. In some embodiments, the polynucleotide IMC comprises the sequence 5'-TCGUCGX.sub.1-3',5'-UCGTCGX.sub.1-3', or 5'-UCGUCGX.sub.1-3', wherein X.sub.1 is a nucleotide. In some embodiments, the polynucleotide IMC comprises any of the following sequences: 5'-TCGUCGA-3'; 5'-TCGUCGC-3'; 5'-TCGUCGG-3'; 5'-TCGUCGT-3'; 5'-TCGUCGU-3'; 5'-TCGUCGI-3'; 5'-UCGTCGA-3'; 5'-UCGTCGC-3'; 5'-UCGTCGG-3'; 5'-UCGTCGT-3'; 5'-UCGTCGU-3'; 5'-UCGTCGI-3'; 5'-UCGUCGA-3'; 5'-UCGUCGC-3'; 5'-UCGUCGG-3'; 5'-UCGUCGT-3'; 5'-UCGUCGU-3'; 5'-UCGUCGI-3'.

In some embodiments, the polynucleotide IMC comprises a sequence according to the formula 5'-T mC GX.sub.1X.sub.2X.sub.3X.sub.4-3' or 5'-U mC GX.sub.1X.sub.2X.sub.3X.sub.4-3', wherein X.sub.1, X.sub.2, X.sub.3, X.sub.4 are nucleotides and wherein mC is a modified cytosine as described herein. In some embodiments, the polynucleotide IMC comprises a sequence according to the formula 5'-X.sub.1T mC GX.sub.2X.sub.3X.sub.4-3' or 5'-X.sub.1U mC GX.sub.2X.sub.3X.sub.4-3', wherein X.sub.1, X.sub.2, X.sub.3, X.sub.4 are nucleotides. In some embodiments, the ISS comprises a sequence according to the formula 5'-X.sub.1X.sub.2T mC GX.sub.3X.sub.4-3' or 5'-X.sub.1X.sub.2U mC GX.sub.3X.sub.4-3', wherein X.sub.1, X.sub.2, X.sub.3, X.sub.4 are nucleotides.

In some embodiments, the polynucleotide IMC comprises a sequence according to the formula 5'-T mC GTCGX.sub.1-3',5'-T mC GUCGX.sub.1-3',5'-U mC GTCGX.sub.1-3' or 5'-U mC GUCGX.sub.1-3', wherein X.sub.1 is a nucleotide. In some embodiments, the polynucleotide IMC comprises a sequence according to the formula 5'-TCGT mC GX.sub.1-3',5'-UCGT mC GX.sub.1-3'. 5'-TCGU mC GX.sub.1-3' or 5'-UCGU mC GX.sub.1-3', wherein X.sub.1 is a nucleotide. In other embodiments, the polynucleotide IMC comprises a sequence according to the formula 5'-T mC GT mC GX.sub.1-3', 5'-U mC GT mC GX.sub.1-3',5'-T mC GU mC GX.sub.1-3' or 5'-U mC GU mC GX.sub.1-3', wherein X.sub.1 is a nucleotide. As described herein, a modified cytosine (mC) includes addition of an electron-withdrawing moiety to C-5 and/or C-6 of a cytosine, including, but not limited to, C-5 halogenated cytosine, such as 5-bromocytosine.

Thus, in some embodiments, the polynucleotide IMC comprises any of the following sequences: 5'-TBGTTTT-3'; 5'-TBGAAAA-3'; 5'-TBGCCCC-3'; 5'-TBGGGGG-3'; 5'-TBGUUUU-3'; 5'-TBGIIII-3'; 5'-TBGTCGA-3'; 5'-TBGTCGC-3'; 5'-TBGTCGG-3'; 5'-TBGTCGT-3'; 5'-TBGTCGU-3'; 5'-TBGTCGI-3'; 5'-TCGTBGA-3'; 5'-TCGTBGC-3'; 5'-TCGTBGG-3'; 5'-TCGTBGT-3'; 5'-TCGTBGU-3'; 5'-TCGTBGI-3'; 5'-TBGTBGA-3'; 5'-TBGTBGC-3'; 5'-TBGTBGG-3'; 5'-TBGTBGT-3',5'-TBGTBGU-3'; 5'-TBGTBGI-3'; where B is 5-bromocytosine.

In some embodiments, the polynucleotide IMC consists of the sequence 5'-TCGTCGX.sub.1-3', wherein X.sub.1 is a nucleotide. In certain embodiments, the polynucleotide IMC consists of the sequence 5'-TCGTCGA-3'; 5'-TCGTCGC-3'; 5'-TCGTCGG-3'; 5'-TCGTCGT-3'; 5'-TCGTCGU-3'; 5'-TCGTCGI-3'. In some embodiments, the polynucleotide IMC consists of the sequence 5'-TCGUCGX.sub.1-3',5'-UCGTCGX.sub.1-3', or 5'-UCGUCGX.sub.1-3', wherein X.sub.1 is a nucleotide. In some embodiments, the polynucleotide IMC consists of any of the following sequences: 5'-TCGUCGA-3'; 5'-TCGUCGC-3'; 5'-TCGUCGG-3'; 5'-TCGUCGT-3'; 5'-TCGUCGU-3'; 5'-TCGUCGI-3'; 5'-UCGTCGA-3'; 5'-UCGTCGC-3'; 5'-UCGTCGG-3'; 5'-UCGTCGT-3'; 5'-UCGTCGU-3'; 5'-UCGTCGI-3'; 5'-UCGUCGA-3'; 5'-UCGUCGC-3'; 5'-UCGUCGG-3'; 5'-UCGUCGT-3'; 5'-UCGUCGU-3'; 5'-UCGUCGI-3'.

In some embodiments, the polynucleotide IMC consists of a sequence according to the formula 5'-T mC GX.sub.1X.sub.2X.sub.3X.sub.4-3' or 5'-U mC GX.sub.1X.sub.2X.sub.3X.sub.4-3', wherein X.sub.1, X.sub.2, X.sub.3, X.sub.4 are nucleotides and wherein mC is a modified cytosine as described herein. In some embodiments, the polynucleotide IMC consists of a sequence according to the formula 5'-X.sub.1T mC GX.sub.2X.sub.3X.sub.4-3' or 5'-X.sub.1U mC GX.sub.2X.sub.3X.sub.4-3', wherein X.sub.1, X.sub.2, X.sub.3, X.sub.4 are nucleotides. In some embodiments, the polynucleotide IMC consists of a sequence according to the formula 5'-X.sub.1X.sub.2T mC GX.sub.3X.sub.4-3' or 5'-X.sub.1X.sub.2U mC GX.sub.3X.sub.4-3', wherein X.sub.1, X.sub.2, X.sub.3, X.sub.4 are nucleotides.

In some embodiments, the polynucleotide IMC consists of a sequence according to the formula 5'-T mC GTCGX.sub.1-3',5'-T mC GUCGX.sub.1-3',5'-U mC GTCGX.sub.1-3' or 5'-U mC GUCGX.sub.1-3', wherein X.sub.1 is a nucleotide. In some embodiments, the polynucleotide IMC consists of a sequence according to the formula 5'-TCGT mC GX.sub.1-3',5'-UCGT mC GX.sub.1-3'. 5'-TCGU mC GX.sub.1-3' or 5'-UCGU mC GX.sub.1-3', wherein X.sub.1 is a nucleotide. In some embodiments, the polynucleotide IMC consists of a sequence according to the formula 5'-T mC GT mC GX.sub.1-3',5'-U mC GT mC GX.sub.1-3',5'-T mC GU mC GX.sub.1-3' or 5'-U mC GU mC GX.sub.1-3', wherein X.sub.1 is a nucleotide. As described herein, a modified cytosine (mC) includes addition of an electron-withdrawing moiety to C-5 and/or C-6 of a cytosine, including, but not limited to, C-5 halogenated cytosine, such as 5-bromocytosine.

Thus, in some embodiments, the polynucleotide IMC consists of any of the following sequences: 5'-TBGTTTT-3'; 5'-TBGAAAA-3'; 5'-TBGCCCC-3'; 5'-TBGGGGG-3'; 5'-TBGUUUU-3'; 5'-TBGIIII-3'; 5'-TBGTCGA-3'; 5'-TBGTCGC-3'; 5'-TBGTCGG-3'; 5'-TBGTCGT-3'; 5'-TBGTCGU-3'; 5'-TBGTCGI-3'; 5'-TCGTBGA-3'; 5'-TCGTBGC-3'; 5'-TCGTBGG-3'; 5'-TCGTBGT-3'; 5'-TCGTBGU-3'; 5'-TCGTBGI-3'; 5'-TBGTBGA-3'; 5'-TBGTBGC-3'; 5'-TBGTBGG-3'; 5'-TBGTBGT-3',5'-TBGTBGU-3'; 5'-TBGTBGI-3'; where B is 5-bromocytosine.

As described herein, a polynucleotide IMC is any length greater than 6 bases or base pairs. Accordingly, in some embodiments, a polynucleotide IMC is less than about any of the following lengths (in bases or base pairs): 10,000; 5,000; 2500; 2000; 1500; 1250; 1000; 750; 500; 300; 250; 200; 175; 150; 125; 100; 75; 50; 25; 20; 18; 16; 14. In some embodiments, a polynucleotide IMC is greater than about any of the following lengths (in bases or base pairs): 6, 7; 8; 10; 12; 14; 15; 20; 25; 30; 40; 50; 60; 75; 100; 125; 150; 175; 200; 250; 300; 350; 400; 500; 750; 1000; 2000; 5000; 7500; 10000; 20000; 50000. Alternately, the polynucleotide IMC can be any of a range of sizes having an upper limit of 10,000; 5,000; 2500; 2000; 1500; 1250; 1000; 750; 500; 300; 250; 200; 175; 150; 125; 100; 75; 50; 25; 20; 18; or 16 and an independently selected lower limit of 7; 8; 10; 12; 14; 15; 20; 25; 30; 40; 50; 60; 75; 100; 125; 150; 175; 200; 250; 300; 350; 400; 500; 750; 1000; 2000; 5000; 7500, wherein the lower limit is less than the upper limit.

A polynucleotide moiety of an IMC may contain modifications such as are known in the art including, but are not limited to, modifications of the 3'OH or 5'OH group, modifications of the nucleotide base, modifications of the sugar component, and modifications of the phosphate group. Various such modifications are described below.

It is preferred that cytosines present in the nucleotide moieties of the IMC are not methylated, however, in certain embodiments the IMC may contain on or more methylated cytosines. In such embodiments it is preferred that the cytosine of the 5'-CG-3' of the nucleotide moiety is not methylated at the C-5 position. However, methylation at position N-4 is contemplated in those IMCs with methylated cytosines.

A nucleotide moiety of an IMC may be single stranded or double stranded DNA, as well as single or double-stranded RNA or other modified polynucleotides. An IMC nucleotide moiety may or may not include one or more palindromic regions, which may be present in the motifs described above or may extend beyond the motif, and may comprise additional flanking sequences, some of which are described herein.

A nucleotide moiety of an IMC may contain naturally-occurring or modified, non-naturally occurring bases, and may contain modified sugar, phosphate, and/or termini. For example, phosphate modifications include, but are not limited to, methyl phosphonate, phosphorothioate, phosphoramidate (bridging or non-bridging), phosphotriester and phosphorodithioate and may be used in any combination. See, for example, IMCs listed in Table 8. Other non-phosphate linkages may also be used. Preferably, oligonucleotides of the present invention comprise phosphodiester and/or phosphorothioate backbones. Sugar modifications known in the field, such as 2'-alkoxy-RNA analogs, 2'-amino-RNA analogs and 2'-alkoxy- or amino-RNA/DNA chimeras and others described herein, may also be made and combined with any phosphate modification. Examples of base modifications include, but are not limited to, addition of an electron-withdrawing moiety to C-5 and/or C-6 of a cytosine of the ISS (e.g., 5-bromocytosine, 5-chlorocytosine, 5-fluorocytosine, 5-iodocytosine). See, for example, International Patent Application No. WO 99/62923.

The nucleotide moieties of the IMC can be synthesized using techniques and nucleic acid synthesis equipment which are well known in the art including, but not limited to, enzymatic methods, chemical methods, and the degradation of larger oligonucleotide sequences. See, for example, Ausubel et al. (1987); and Sambrook et al. (1989). When assembled enzymatically, the individual units can be ligated, for example, with a ligase such as T4 DNA or RNA ligase. U.S. Pat. No. 5,124,246. Oligonucleotide degradation can be accomplished through the exposure of an oligonucleotide to a nuclease, as exemplified in U.S. Pat. No. 4,650,675.

Nucleotide moieties of the IMC can also be isolated using conventional polynucleotide isolation procedures. Such procedures include, but are not limited to, hybridization of probes to genomic or cDNA libraries to detect shared nucleotide sequences, antibody screening of expression libraries to detect shared structural features and synthesis of particular native sequences by the polymerase chain reaction.

Circular polynucleotide IMC moieties can be isolated, synthesized through recombinant methods, or chemically synthesized. Where the circular polynucleotide IMC moiety is obtained through isolation or through recombinant methods, it will preferably be a plasmid. The chemical synthesis of smaller circular oligonucleotides can be performed using any method described in the literature. See, for instance, Gao et al. (1995) Nucleic Acids Res. 23:2025-2029; and Wang et al. (1994) Nucleic Acids Res. 22:2326-2333.

The techniques for making oligonucleotides and modified oligonucleotides are known in the art. Naturally occurring DNA or RNA, containing phosphodiester linkages, is generally synthesized by sequentially coupling the appropriate nucleoside phosphoramidite to the 5'-hydroxy group of the growing oligonucleotide attached to a solid support at the 3'-end, followed by oxidation of the intermediate phosphite triester to a phosphate triester. Once the desired oligonucleotide sequence has been synthesized, the oligonucleotide is removed from the support, the phosphate triester groups are deprotected to phosphate diesters and the nucleoside bases are deprotected using aqueous ammonia or other bases. See, for example, Beaucage (1993) "Oligodeoxyribonucleotide Synthesis" in Protocols for Oligonucleotides and Analogs, Synthesis and Properties (Agrawal, ed.) Humana Press, Totowa, N.J.; Warner et al. (1984) DNA 3:401 and U.S. Pat. No. 4,458,066.

The IMC nucleotide moiety can also contain phosphate-modified oligonucleotides. Synthesis of polynucleotides containing modified phosphate linkages or non-phosphate linkages is also know in the art. For a review, see Matteucci (1997) "Oligonucleotide Analogs: an Overview" in Oligonucleotides as Therapeutic Agents, (D. J. Chadwick and G. Cardew, ed.) John Wiley and Sons, New York, N.Y. The phosphorous derivative (or modified phosphate group) which can be attached to the sugar or sugar analog moiety in the oligonucleotides of the present invention can be a monophosphate, diphosphate, triphosphate, alkylphosphonate, phosphorothioate, phosphorodithioate or the like. The preparation of the above-noted phosphate analogs, and their incorporation into nucleotides, modified nucleotides and oligonucleotides, per se, is also known and need not be described here in detail. Peyrottes et al. (1996) Nucleic Acids Res. 24:1841-1848; Chaturvedi et al. (1996) Nucleic Acids Res. 24:2318-2323; and Schultz et al. (1996) Nucleic Acids Res. 24:2966-2973. For example, synthesis of phosphorothioate oligonucleotides is similar to that described above for naturally occurring oligonucleotides except that the oxidation step is replaced by a sulfurization step (Zon (1993) "Oligonucleoside Phosphorothioates" in Protocols for Oligonucleotides and Analogs, Synthesis and Properties (Agrawal, ed.) Humana Press, pp. 165-190). Similarly the synthesis of other phosphate analogs, such as phosphotriester (Miller et al. (1971) JACS 93:6657-6665), non-bridging phosphoramidates (Jager et al. (1988) Biochem. 27:7247-7246), N3' to P5' phosphoramidiates (Nelson et al. (1997) JOC 62:7278-7287) and phosphorodithioates (U.S. Pat. No. 5,453,496) has also been described. Other non-phosphorous based modified oligonucleotides can also be used (Stirchak et al. (1989) Nucleic Acids Res. 17:6129-6141). Oligonucleotides with phosphorothioate backbones can be more immunogenic than those with phosphodiester backbones and appear to be more resistant to degradation after injection into the host. Braun et al., (1988) J. Immunol. 141:2084-2089; and Latimer et al. (1995) Mol. Immunol. 32:1057-1064.

Nucleotide moieties of IMCs used in the compositions of the invention can comprise ribonucleotides (containing ribose as the only or principal sugar component), deoxyribonucleotides (containing deoxyribose as the principal sugar component), or, as is known in the art, modified sugars or sugar analogs can be incorporated in the nucleotide moiety. Thus, in addition to ribose and deoxyribose, the sugar moiety can be pentose, deoxypentose, hexose, deoxyhexose, glucose, arabinose, xylose, lyxose, and a sugar "analog" cyclopentyl group. The sugar can be in pyranosyl or in a furanosyl form. The sugar moiety of IMC nucleotide moieties is preferably the furanoside of ribose, deoxyribose, arabinose or 2'-0-alkylribose, and the sugar can be attached to the respective heterocyclic bases either in .alpha. or .beta. anomeric configuration. Sugar modifications include, but are not limited to, 2'-alkoxy-RNA analogs, 2'-amino-RNA analogs and 2'-alkoxy- or amino-RNA/DNA chimeras. The preparation of these sugars or sugar analogs and the respective "nucleosides" wherein such sugars or analogs are attached to a heterocyclic base (nucleic acid base) per se is known, and need not be described here, except to the extent such preparation can pertain to any specific example. Sugar modifications may also be made and combined with any phosphate modification in the preparation of a nucleotide moiety of a IMC.

The heterocyclic bases, or nucleic acid bases, which are incorporated in the nucleotide moiety of the IMC can be the naturally-occurring principal purine and pyrimidine bases, (namely uracil, thymine, cytosine, inosine, adenine and guanine, as mentioned above), as well as naturally-occurring and synthetic modifications of said principal bases.

Those skilled in the art will recognize that a large number of "synthetic" non-natural nucleosides comprising various heterocyclic bases and various sugar moieties (and sugar analogs) are available in the art, and that as long as other criteria of the present invention are satisfied, the nucleotide moiety of the IMC can include one or several heterocyclic bases other than the principal five base components of naturally-occurring nucleic acids. Preferably, however, the heterocyclic base in the nucleotide moiety includes, but is not limited to, uracil-5-yl, cytosin-5-yl, adenin-7-yl, adenin-8-yl, guanin-7-yl, guanin-8-yl, 4-aminopyrrolo[2,3-d]pyrimidin-5-yl, 2-amino-4-oxopyrolo[2,3-d]pyrimidin-5-yl, 2-amino-4-oxopyrrolo[2,3-d]pyrimidin-3-yl groups, where the purines are attached to the sugar moiety of the ISS via the 9-position, the pyrimidines via the 1-position, the pyrrolopyrimidines via the 7-position and the pyrazolopyrimidines via the 1-position.

The nucleotide moiety of the IMC may comprise at least one modified base as described, for example, in the commonly owned international application WO 99/62923. As used herein, the term "modified base" is synonymous with "base analog", for example, "modified cytosine" is synonymous with "cytosine analog." Similarly, "modified" nucleosides or nucleotides are herein defined as being synonymous with nucleoside or nucleotide "analogs." Examples of base modifications include, but are not limited to, addition of an electron-withdrawing moiety to C-5 and/or C-6 of a cytosine of the ISS. Preferably, the electron-withdrawing moiety is a halogen. Modified cytosines can include, but are not limited to, azacytosine, 5-bromocytosine, 5-chlorocytosine, chlorinated cytosine, cyclocytosine, cytosine arabinoside, 5-fluorocytosine, fluoropyrimidine, 5,6-dihydrocytosine, 5-iodocytosine, hydroxyurea, 5-nitrocytosine, 5-hydroxycytosine and any other pyrimidine analog or modified pyrimidine. Preferred modified uracils are modified at C-5 and/or C-6, preferably with a halogen, and include, but are not limited to, bromouracil such as 5-bromouracil, chlorouracil such as 5-chlorouracil, fluorouracil such as 5-fluorouracil, iodouracil such as 5-iodouracil, and hydroxyuracil. Also see, Kandimalla et al., 2001, Bioorg. Med. Chem. 9:807-813. See, for example, International Patent Application No. WO 99/62923. Other examples of base modifications include the addition of one or more thiol groups to the base including, but not limited to, 6-thio-guanine, 4-thio-thymine and 4-thio-uracil. Additionally, some nucleotide moieties may comprise modified bases such as 7-deazaguanosine in place of any guanosine residue, or a modified cytosine selected from N-4-ethylcytosine or N-4-methylcytosine in place of any cytosine residue, including the cytosine of the 5'-CG-3'.

The preparation of base-modified nucleosides, and the synthesis of modified oligonucleotides using said base-modified nucleosides as precursors, has been described, for example, in U.S. Pat. Nos. 4,910,300, 4,948,882, and 5,093,232. These base-modified nucleosides have been designed so that they can be incorporated by chemical synthesis into either terminal or internal positions of an oligonucleotide. Such base-modified nucleosides, present at either terminal or internal positions of an oligonucleotide, can serve as sites for attachment of a peptide or other antigen. Nucleosides modified in their sugar moiety have also been described (including, but not limited to, e.g., U.S. Pat. Nos. 4,849,513, 5,015,733, 5,118,800, 5,118,802) and can be used similarly.

In certain embodiments, the IMC is a chimeric immunomodulatory compound ("CIC"), such as those described in co-owned U.S. patent application Ser. Nos. 10/176,883 and 10/177,826, filed Jun. 21, 2002, which are fully incorporated by reference herein.

CICs contain one or more nucleic acid moieties and one or more non-nucleic acid spacer moieties. CICs conforming to a variety of structural formulas are contemplated for use as IMCs, including the core structures described in formulas I-VII, below. Formulas I-III show core sequences for "linear CICs." Formulas IV-VI show core sequences for "branched CICs." Formula VII shows a core structure for "single-spacer CICs."

In each formula provided herein, "N" designates a nucleic acid moiety (oriented in either a 5'.fwdarw.3' or 3'.fwdarw.5' orientation) and "S" designates a non-nucleic acid spacer moiety. A dash ("-") designates a covalent bond between a nucleic acid moiety and a non-nucleic acid spacer moiety. A double dash ("--") designates covalent bonds between a non-nucleic acid spacer moiety and at least 2 nucleic acid moieties. A triple dash ("---") designates covalent bonds between a non-nucleic acid spacer moiety and multiple (i.e., at least 3) nucleic acid moieties. Subscripts are used to designate differently positioned nucleic acid or non-nucleic acid spacer moieties. However, the use of subscripts to distinguish different nucleic acid moieties is not intended to indicate that the moieties necessarily have a different structure or sequence. Similarly, the use of subscripts to distinguish different spacer moieties is not intended to indicate that the moieties necessarily have different structures. For example, in formula II, infra, the nucleic acid moieties designated N.sub.1 and N.sub.2 can have the same or different sequences, and the spacer moieties designated S.sub.1 and S.sub.2 can have the same or different structures. Further, it is contemplated that additional chemical moieties (e.g., phosphate, mononucleotide, additional nucleic acid moieties, alkyl, amino, thio or disulfide groups or linking groups, and/or spacer moieties) may be covalently bound at the termini of the core structures.

Linear CICs have structures in which the non-nucleic acid spacer moieties in the core structure are covalently bound to no more than two nucleic acid moieties. Exemplary linear CICs conform to the following formulas -- see Original Patent.

Exemplary linear CICs include -- see Original Patent.

Preferred linear CICs include 5'-TCGTCGA-3'-HEG-5'-ACGTTCG-3'-HEG-5'-AGATGAT-3'(SEQ ID NO:54), 5'-TCGTCG-HEG-AACGTT-HEG-AGATGAT-3'(SEQ ID NO:55), and 5'-TCGTCGA-C3-ACGTTCG-C3-AGATGAT-3' (SEQ ID NO:56).

Branched CICs comprise a multivalent spacer moiety (S.sub.p) covalently bound to at least three (3) nucleic acid moieties. Exemplary branched CICs are described according to the following formulas -- see Original Patent.

Exemplary branched CICs include -- see Original Patent.

Preferred branched CICs include (5'-TCGACGT-3'-HEG).sub.2-glycerol-HEG-5'-TCGACGT-3'(SEQ ID NO:61) and (5'-TCGTCGA-3'-HEG).sub.2-glycerol-HEG-5'-AACGTTC-3'(SEQ ID NO:62).

Single spacer CICs comprise a structure in which there is a single nucleic acid moiety covalently conjugated to a single spacer moiety, i.e., -- see Original Patent.

Exemplary single spacer CICs include -- see Original Patent.

In certain embodiments, the terminal structures of the CIC are covalently joined (e.g., nucleic acid moiety-to-nucleic acid moiety; spacer moiety-to-spacer moiety, or nucleic acid moiety-to-spacer moiety), resulting in a circular conformation.

CICs for use as IMCs in the immunomodulatory compositions of the invention include at least one nucleic acid moiety. The term "nucleic acid moiety," as used herein, refers to a nucleotide monomer (i.e., a mononucleotide) or polymer (i.e., comprising at least 2 contiguous nucleotides). As used herein, a nucleotide comprises (1) a purine or pyrimidine base linked to a sugar that is in an ester linkage to a phosphate group, or (2) an analog in which the base and/or sugar and/or phosphate ester are replaced by analogs, e.g., as described infra. In a CIC comprising more than one nucleic acid moiety, the nucleic acid moieties may be the same or different.

Nucleic acid moieties used in CICs incorporated in the immunomodulatory compositions may comprise any of the polynucleotide IMC sequences disclosed herein, and may additionally be sequences of six base pairs or less. It is contemplated that in a CIC comprising multiple nucleic acid moieties, the nucleic acid moieties can be the same or different lengths. Nucleic acid moieties of six or fewer nucleotides preferably include the sequence 5'-CG-3', or optionally 5'-TCG-3' or 5'-ACG-3', although in certain embodiments where the CIC comprises more than one nucleic acid moiety, only one of the moieties need comprise the sequence 5'-CG-3'.

It is contemplated that in a CIC comprising multiple nucleic acid moieties, the nucleic acid moieties can be the same or different. Accordingly, in various embodiments, CICs incorporated into the immunomodulatory compositions comprise (a) nucleic acid moieties with the same sequence, (b) more than one iteration of a nucleic acid moiety, or (c) two or more different nucleic acid moieties. Additionally, a single nucleic acid moiety may comprise more than one ISS, which may be adjacent, overlapping, or separated by additional nucleotide bases within the nucleic acid moiety. In an embodiment, a nucleic acid moiety includes one or more palindromic regions. In the context of single-stranded oligonucleotides, the term "palindromic" refers to a sequence that would be palindromic if the oligonucleotide were complexed with a complementary sequence to form a double-stranded molecule. In another embodiment, one nucleic acid moiety has a sequence that is palindromic or partially palindromic in relation to a second nucleic acid moiety in the CIC. In an embodiment of the invention, the sequence of one or more of the nucleic acid moieties of a CIC is not palindromic. In another embodiment of the invention, the sequence of one or more of the nucleic acid moieties of a CIC does not include a palindromic sequence greater than four bases, optionally greater than 6 bases.

The CICs incorporated into the immunomodulatory compositions comprise one or more non-nucleic acid spacer moieties covalently bound to the nucleic acid moieties. For convenience, non-nucleic acid spacer moieties are sometimes referred to herein simply as "spacers" or "spacer moieties." Spacers are generally of molecular weight about 50 to about 50,000, typically from about 75 to about 5000, most often from about 75 to about 500, which are covalently bound, in various embodiments, to one, two, three, or more than three nucleic acid moieties. A variety of agents are suitable for connecting nucleic acid moieties. For example, a variety of compounds referred to in the scientific literature as "non-nucleic acid linkers," "non-nucleotidic linkers," or "valency platform molecules" may be used as spacers in a CIC. In certain embodiments, a spacer comprises multiple covalently connected subunits and may have a homopolymeric or heteropolymeric structure. It will be appreciated that mononucleotides and polynucleotides are not included in the definition of non-nucleic acid spacers, without which exclusion there would be no difference between nucleic acid moiety and an adjacent non-nucleic acid spacer moiety.

In certain embodiments, a spacer may comprise one or more abasic nucleotides (i.e., lacking a nucleotide base, but having the sugar and phosphate portions). Exemplary abasic nucleotides include 1'2'-dideoxyribose, 1'-deoxyribose, 1'-deoxyarabinose and polymers thereof.

Other suitable spacers comprise optionally substituted alkyl, optionally substituted polyglycol, optionally substituted polyamine, optionally substituted polyalcohol, optionally substituted polyamide, optionally substituted polyether, optionally substituted polyimine, optionally substituted polyphosphodiester (such as poly(1-phospho-3-propanol), and the like. Optional substituents include alcohol, alkoxy (such as methoxy, ethoxy, and propoxy), straight or branched chain alkyl (such as C1-C12 alkyl), amine, aminoalkyl (such as amino C1-C12 alkyl), phosphoramidite, phosphate, thiophosphate, hydrazide, hydrazine, halogen, (such as F, Cl, Br, or I), amide, alkylamide (such as amide C1-C12 alkyl), carboxylic acid, carboxylic ester, carboxylic anhydride, carboxylic acid halide, sulfonyl halide, imidate ester, isocyanate, isothiocyanate, haloformate, carbodiimide adduct, aldehydes, ketone, sulfhydryl, haloacetyl, alkyl halide, alkyl sulfonate, NR1R2 wherein R1R2 is --C(.dbd.O)CH.dbd.CHC(.dbd.O)(maleimide), thioether, cyano, sugar (such as mannose, galactose, and glucose), .alpha.,.beta.-unsaturated carbonyl, alkyl mercurial, .alpha.,.beta.-unsaturated sulfone.

Suitable spacers may comprise polycyclic molecules, such as those containing phenyl or cyclohexyl rings. The spacer may be a polyether such as polyphosphopropanediol, polyethyleneglycol, polypropylene glycol, a bifunctional polycyclic molecule such as a bifunctional pentalene, indene, naphthalene, azulene, heptalene, biphenylene, asymindacene, sym-indacene, acenaphthylene, fluorene, phenalene, phenanthrene, anthracene, fluoranthene, acephenathrylene, aceanthrylene, triphenylene, pyrene, chrysene, naphthacene, thianthrene, isobenzofuran, chromene, xanthene, phenoxathiin, which may be substituted or modified, or a combination of the polyethers and the polycyclic molecules. The polycyclic molecule may be substituted or polysubstituted with C1-C5 alkyl, C6 alkyl, alkenyl, hydroxyalkyl, halogen or haloalkyl group. Nitrogen-containing polyheterocyclic molecules (e.g., indolizine) are typically not suitable spacers. The spacer may also be a polyalcohol, such as glycerol or pentaerythritol. In one embodiment, the spacer comprises 1-phosphopropane).sub.3-phosphate or 1-phosphopropane).sub.4-phosphate (also called tetraphosphopropanediol and pentaphosphopropanediol). In one embodiment, the spacer comprises derivatized 2,2'-ethylenedioxydiethylamine (EDDA).

Specific examples of non-nucleic acid spacers useful in CICs include "linkers" described by Cload and Schepartz, J. Am. Chem. Soc. (1991), 113:6324; Richardson and Schepartz, J. Am. Chem. Soc. (1991), 113:5109; Ma et al., Nucleic Acids Research (1993), 21:2585; Ma et al., Biochemistry (1993), 32:1751; McCurdy et al., Nucleosides & Nucleotides (1991), 10:287; Jaschke et al., Tetrahedron Lett. (1993), 34:301; Ono et al., Biochemistry (1991), 30:9914; and Arnold et al., International Publication No. WO 89/02439 entitled "Non-nucleic acid Linking Reagents for Nucleotide Probes."

Other suitable spacers include linkers described by Salunkhe et al., J. Am. Chem. Soc. (1992), 114:8768; Nelson et al., Biochemistry 35:5339-5344 (1996); Bartley et al., Biochemistry 36:14502-511 (1997); Dagneaux et al., Nucleic Acids Research 24:4506-12 (1996); Durand et al., Nucleic Acids Research 18:6353-59 (1990); Reynolds et al., Nucleic Acids Research, 24:760-65 (1996); Hendry et al. Biochemica et Biophysica Acta, 1219:405-12 (1994); Altmann et al., Nucleic Acids Research, 23:4827-35 (1995). Still other suitable spacers are described in European Patent No. EP0313219B1 (Arnold et al.), "Non-nucleic acid linking reagents for nucleotide probes" and U.S. Pat. No. 6,117,657 (Usman et al.).

Exemplary non-nucleic acid spacers comprise oligo-ethylene glycol (e.g., triethylene glycol, tetraethylene glycol, hexaethylene glycol spacers, and other polymers comprising up to about 10, about 20, about 40, about 50, about 100 or about 200 ethylene glycol units), alkyl spacers (e.g., propyl, butyl, hexyl, and other C2-C12 alkyl spacers, e.g., usually C2-C10 alkyl, most often C2-C6 alkyl), abasic nucleotide spacers, symmetric or asymmetric spacers derived from glycerol, pentaerythritol or 1,3,5-trihydroxycyclohexane (e.g., symmetrical doubler and trebler spacer moieties described herein). Spacers can also comprise heteromeric or homomeric oligomers and polymers of the aforementioned compounds (e.g., linked by an amide, ester, ether, thioether, disulfide, phosphodiester, phosphorothioate, phosphoramidate, phosphotriester, phosphorodithioate, methyl phosphonate or other linkage).

Suitable spacer moieties can contribute charge and/or hydrophobicity to the CIC, contribute favorable pharmacokinetic properties (e.g., improved stability, longer residence time in blood) to the CIC, and/or result in targeting of the CIC to particular cells or organs. Spacer moieties can be selected or modified to tailor the CIC for desired pharmacokinetic properties or suitability for desired modes of administration (e.g., oral administration). It will be appreciated by the reader that, for convenience, a spacer (or spacer component) is sometimes referred to by the chemical name of the compound from which the spacer component is derived (e.g., hexaethylene glycol), with the understanding that the CIC actually comprises the conjugate of the compound and adjacent nucleic acid moieties or other spacer moiety components.

In a CIC comprising more than one spacer moiety, the spacers may be the same or different. Thus, in one embodiment all of the non-nucleic acid spacer moieties in a CIC have the same structure. In one embodiment, a CIC comprises non-nucleic acid spacer moieties with at least 2, at least 3, at least 4, at least 5, or at least 6 or more different structures.

In some contemplated embodiments of the invention, the spacer moiety of a CIC is defined to exclude certain structures. Thus, in some embodiments of the invention, a spacer is other than an abasic nucleotide or polymer of abasic nucleotides. In some embodiments of the invention, a spacer is other than a oligo(ethyleneglycol)(e.g., HEG, TEG and the like) or poly(ethyleneglycol). In some embodiments a spacer is other than a C3 alkyl spacer. In some embodiments, a spacer is other than a polypeptide. Thus, in some embodiments, an immunogenic molecule, e.g., a protein or polypeptide, is not suitable as a component of spacer moieties. However, as discussed infra, it is contemplated that in certain embodiments, a CIC is a "proteinaceous CIC" i.e., comprising a spacer moiety comprising a polypeptide. For example, as discussed infra, in some embodiments, a polypeptide antigen can be used as a platform (multivalent spacer) to which a plurality of nucleic acid moieties are conjugated. However, in some embodiments, the spacer moiety is not proteinaceous and/or is not an antigen (i.e., the spacer moiety, if isolated from the CIC, is not an antigen).

Suitable spacer moieties do not render the CIC of which they are a component insoluble in an aqueous solution (e.g., PBS, pH 7.0). Thus, the definition of spacers excludes microcarriers or nanocarriers. In addition, a spacer moiety that has low solubility, such as a dodecyl spacer (solubility <5 mg/ml when measured as dialcohol precursor 1,12-dihydroxydodecane) is not preferred because it can reduce the hydrophilicity and activity of the CIC. Preferably, spacer moieties have solubility much greater than 5 mg/ml (e.g., .gtoreq.20 mg/ml, .gtoreq.50 mg/ml or .gtoreq.100 mg/ml) when measured as dialcohol precursors.

The charge of a CIC may be contributed by phosphate, thiophosphate, or other groups in the nucleic acid moieties as well as groups in non-nucleic acid spacer moieties. In some embodiments of the invention, a non-nucleic acid spacer moiety carries a net charge (e.g., a net positive charge or net negative charge when measured at pH 7). In one useful embodiment, the CIC has a net negative charge. In some embodiments, the negative charge of a spacer moiety in a CIC is increased by derivatizing a spacer subunit described herein to increase its charge. For example, glycerol can be covalently bound to two nucleic acid moieties and the remaining alcohol can be reacted with an activated phosphoramidite, followed by oxidation or sulfurization to form a phosphate or thiophosphate, respectively. In certain embodiments the negative charge contributed by the non-nucleic acid spacer moieties in a CIC (i.e., the sum of the charges when there is more than one spacer) is greater than the negative charge contributed by the nucleic acid moieties of the CIC. Charge can be calculated based on molecular formula, or determined experimentally, e.g., by capillary electrophoresis (Li, ed., 1992, Capillary electrophoresis, Principles, Practice and Application Elsevier Science Publishers, Amsterdam, The Netherlands, pp 202-206).

As is noted supra, suitable spacers can be polymers of smaller non-nucleic acid (e.g., non-nucleotide) compounds, such as those described herein, that are themselves useful as spacers, including compounds commonly referred to as non-nucleotide "linkers." Such polymers (i.e., "multiunit spacers") may be heteromeric or homomeric, and often comprise monomeric units (e.g., HEG, TEG, glycerol, 1'2'-dideoxyribose, and the like) linked by an ester linkage (e.g., phosphodiester or phosphorothioate ester). Thus, in one embodiment the spacer comprises a polymeric (e.g., heteropolymeric) structure of non-nucleotide units (e.g., from 2 to about 100 units, alternatively 2 to about 50, e.g., 2 to about 5, alternatively e.g., about 5 to about 50, e.g., about 5 to about 20).

 

Claim 1 of 37 Claims

1. An immunostimulatory particulate composition comprising a cationic condensing agent, an immunostimulatory compound, and a nonionic detergent; wherein the cationic condensing agent is a cationic lipopeptide; and wherein the immunostimulatory compound comprises an immunostimulatory sequence (ISS), wherein the ISS comprises nucleotide sequence 5'-CG-3', wherein the cytosine of the nucleotide sequence 5'-CG-3' is unmethylated and the ISS is greater than about 6 base pairs in length and is less than about 100 base pairs in length.

 

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