DETAILED DESCRIPTION

[0078] It was surprisingly discovered according to the invention that certain CpG-like nucleic acids are immunostimulatory nucleic acids despite their lack of unmethylated CpG dinulceotides that previously had been reported to be crucial to the immunostimulatory effects of CpG nucleic acids. The CpG-like nucleic acids are useful in any application for which CpG nucleic acids are effective for inducing an activating, stimulating, or proliferative biological response.

[0079] Unmethylated CpG sequences, while relatively rare in human DNA, are commonly found in the DNA of infectious organisms such as bacteria. The human immune system has apparently evolved to recognize unmethylated CpG sequences as an early warning sign of infection and to initiate an immediate and powerful immune response against invading pathogens. Thus unmethylated CpG-containing nucleic acids, relying on this innate immune defense mechanism, can utilize a unique and natural pathway for immune therapy without causing adverse reactions frequently seen with other immune stimulatory agents. The effects of unmethylated CpG nucleic acids on immune modulation have been described extensively in published patent applications, such as PCT US95/01570; PCT/US97/19791; PCT/US98/03678; PCT/US98/10408; PCT/US98/04703; PCT/US99/07335; and PCT/US99/09863. The entire contents of each of these patent applications is hereby incorporated by reference.

[0080] A “CpG nucleic acid” is a nucleic acid which includes at least one unmethylated CpG dinucleotide. A nucleic acid containing at least one unmethylated CpG dinucleotide is a nucleic acid molecule which contains an unmethylated cytosine in a cytosine-guanine dinucleotide sequence (i.e., “CpG DNA” or DNA containing a 5′cytosine followed by 3′ guanine and linked by a phosphate bond) and activates the immune system. The CpG nucleic acids can be double-stranded or single-stranded. Generally, double-stranded molecules are more stable in vivo, while single-stranded molecules have increased immune activity. Thus in some aspects of the invention it is preferred that the nucleic acid be single-stranded and in other aspects it is preferred that the nucleic acid be double-stranded. The terms CpG nucleic acid or CpG oligonucleotide as used herein refer to an immunostimulatory CpG nucleic acid or immunostimulatory CpG oligonucleotide unless otherwise indicated. The entire immunostimulatory CpG nucleic acid can be unmethylated or portions may be unmethylated, but at least the C of the 5′-CG-3′ dinucleotide must be unmethylated.

[0081] An “immunostimulatory nucleic acid” as used herein is any nucleic acid containing an immunostimulatory motif or backbone that induces an immune response. An immune response, as used herein, includes the stimulation of immune cells and of non-immune cells to secrete or express factors which participate in and/or characterize immune activation. This term thus includes, without limitation, stimulation of cytokine secretion by various types of cells including lymphocytes, professional antigen-presenting cells (APCs, including dendritic cells), and epithelial cells; stimulation of immunoglobulin secretion by B cells; and stimulation of cell surface molecule expression of costimulatory molecules and coreceptors on T cells, B cells, natural killer (NK) cells, monocytes, macrophages, and APCs.

[0082] An amount of an immunostimulatory nucleic acid effective to induce an immune response is an amount of an immunostimulatory nucleic acid that, when administered to a subject or contacted with cells of a subject, induces the stimulation of immune cells and/or non-immune cells to secrete or express factors which participate in and/or characterize immune activation. In certain embodiments the amount is effective for inducing cytokine secretion. Examples of cytokines are given below. Methods for determining cytokine induction and secretion are well known in the art and include enzyme-linked immunosorbent assay (ELISA), intracellular fluorescence-activated cell sorting (FACS), and bioassay. In certain other embodiments the amount is effective for stimulating NK cell activity. Methods for determining NK cell activity are well known in the art and include determination of target cell killing (cell lysis), cell surface expression of activation marker CD69 (e.g., by FACS), and secretion of interferon gamma (IFN-γ; e.g., by ELISA). Additional examples of specific cell surface markers of immune activation can include, without limitation, expression of CD11b, CD25, CD28, CD43, CD54, CD62L, CD71, CD80, CD86, CD95L, CD106, CD134, and CD134L.

[0083] The terms “nucleic acid” and “oligonucleotide” are used interchangeably to mean multiple nucleotides (i.e., molecules each comprising a sugar (e.g., ribose or deoxyribose) linked to a phosphate group and to an exchangeable organic base, which is either a substituted pyrimidine (e.g., cytosine (C), thymine (T) or uracil (U)) or a substituted purine (e.g., adenine (A) or guanine (G)). Also included are derivatives of C, including without limitation 2′,3′-dideoxycitidine (ddC), 5-bromo-ddC, 3′-amino-2,3-ddC, 5-iodo-2′-deoxycytidine (dC), and derivatives of G, including without limitation 3′-azido-2′,3′-dideoxyguanosine (ddG), 7-deaza-2′-deoxyguanosine (dG), 8-bromo-dG, 8-bromo-2′-dG, 06-methyl-2′-dG. As used herein, the terms “nucleic acid” and “oligonucleotide” refer to oligoribonucleotides as well as oligodeoxyribonucleotides (ODN). The terms shall also include polynucleosides (i.e., a polynucleotide minus the phosphate) and any other organic base-containing polymer. Nucleic acids include vectors, e.g., plasmids, as well as oligonucleotides. Nucleic acid molecules can be obtained from natural nucleic acid sources (e.g., genomic DNA or cDNA from prokaryotes including bacteria and from eukaryotes including yeast) and are referred to herein as “isolated,” but are preferably synthetic (e.g., produced by oligonucleotide synthesis).

[0084] The invention provides in one aspect a CpG-like nucleic acid. As used herein, a “CpG-like nucleic acid” refers to an immunostimulatory nucleic acid having all the characteristics of a CpG nucleic acid as described herein, with at least one of the following exceptions: (1) the cytosine base of the C of the at least one CpG dinucleotide is 5′ methylated (methylated CpG); (2) the G of the at least one CpG dinucleotide is replaced by an I (inosine; CpI nucleic acid); (3) the sugar of the C of the at least one CpG dinucleotide is modified to be a 2′-alkoxy cytosine; (4) the C of the at least one CpG dinucleotide is replaced by Z, where Z is selected from 2′-deoxyuridine (dU), 5-fluoro-2′-dU, and dSpacer, where dSpacer is a sugar moiety without a base; (5) the G of the at least one CpG dinucleotide is replaced by Y, where Y is selected from 2-aminopurine, xanthosine, N7-methyl-xanthosine, nebularine, and dSpacer, where dSpacer is a sugar moiety without a base. According to certain preferred embodiments the 2′-alkoxy cytosine is 2′-methoxy cytosine (2′-OMe-cytosine nucleic acid).

[0085] The CpG-like nucleic acids thus can incorporate certain modifications or substitutions involving either the bases or the sugar moieties of the backbone. Those with methylated CpG involve, in a preferred embodiment, methylation at the 5′ position of the base cytosine. Those with CpI involve use of a particular base, inosine. Those with 2′-OMe-cytosine involve substitution of a methoxy group (OCH ₃ ; OMe) for a hydroxy group (OH) at the 2′ position of the sugar moiety linked to the base cytosine.

[0086] In some embodiments a CpG-like nucleic acid can be based on a previously known CpG nucleic acid. In other embodiments a CpG-like nucleic acid can be different from a previously known CpG nucleic acid. Also according to some preferred embodiments the CpG-like nucleic acid has a modified backbone, including a phosphate modified backbone (see below).

[0087] According to a preferred embodiment of this aspect of the invention, the CpG-like nucleic acid is at least 18 nucleotides long and is not an antisense nucleic acid. As used herein, an “antisense nucleic acid” refers to a nucleic acid sequence that (1) either has sequence complementarity to a structural gene sequence of the host, or is specifically hybridizable, under stringent conditions, with a nucleic acid sequence of the treated host, and (2) when complexed to the structural gene or nucleic acid sequence causes a loss of function of the structural gene or nucleic acid sequence. An antisense nucleic acid is specifically hybridizable when binding of the compound to the target DNA or RNA molecule interferes with the normal function of the target DNA or RNA to cause a loss of utility, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and in the case of in vitro assays, under conditions in which the assays are performed. Mere complementarity to chromosomal DNA or messenger RNA may not define antisense, since not all sequences complementary to a particular gene or nucleic acid sequence cause a loss of function of the particular gene or nucleic acid sequence. Typically an antisense nucleic acid will be able to hybridize, under stringent conditions, to a sequence in or corresponding to the coding region of a gene, including and particularly involving an intron. Alternatively, an antisense nucleic acid typically will be able to hybridize, under stringent conditions, to sequence in a regulatory element associated with a gene, e.g., a promoter or enhancer. Those skilled in the art can readily determine, without undue experimentation, whether a given CpG-like nucleic acid is an antisense nucleic acid.

[0088] As used herein, a “methylated CpG nucleic acid” refers to an immunostimulatory nucleic acid having all the characteristics of a CpG nucleic acid as described herein, with the exception that the C of the at least one CpG dinucleotide is methylated. This finding was quite unexpected. It has been widely reported that when the C of a CpG dinucleotide was replaced with a 5-methylcytosine, immunostimulatory activity was lost. Surprisingly, then, it was found according to the invention that replacement of the C of a CpG dinucleotide with 5-methylcytosine did not cause this profound loss of activity. Such an immunostimulatory nucleic acid may include at least one methylated CpG dinucleotide and at least one unmethylated CpG dinucleotide. In an alternative embodiment, the entire immunostimulatory nucleic acid can be methylated, including all CpG dinucleotides present.

[0089] In one preferred embodiment the invention provides an immunostimulatory nucleic acid which is a methylated CpG nucleic acid represented by at least the formula:

5 ′X X ₂ CGX ₃ X ₄ 3′

[0090] wherein C is methylated and X ₁ , X ₂ , X ₃ , and X ₄ are nucleotides. In one embodiment X ₂ is adenine, guanine, cytosine, or thymine. In another embodiment X ₃ is adenine, guanine, cytosine, or thymine. In other embodiments X ₂ is guanine, adenine, or thymine and X ₃ is cytosine, adenine, or thymine.

[0091] In another embodiment the immunostimulatory nucleic acid is an isolated methylated CpG nucleic acid represented by at least the formula:

5 ′N ₁ X ₁ X ₂ CGX ₃ X ₄ N ₂ 3′

[0092] wherein C is methylated, X ₁ , X ₂ , X ₃ , and X ₄ are nucleotides, and N ₁ , and N ₂ are nucleic acid sequences composed of from about 0-25 nucleotides each. In one embodiment X ₁ X ₂ is a dinucleotide selected from the group consisting of: GpG, GpA, GpT, ApG, ApA, ApT, CpG, CpA, CpT, TpG, TpA, and TpT; and X ₃ X ₄ is a dinucleotide selected from the group consisting of: ApG, ApA, ApT, ApC, CpG, CpA, CpC, TpG, TpA, TpT, and TpC. Preferably X ₁ X ₂ is GpA or GpT and X ₃ X ₄ is TpT. In other embodiments X ₁ or X ₂ or both are purines and X ₃ or X ₄ or both are pyrimidines or X ₁ X ₂ is GpA and X ₃ or X ₄ or both are pyrimidines. In another preferred embodiment X ₁ X ₂ is a dinucleotide selected from the group consisting of: TpA, ApA, ApC, ApG, and GpG. In yet another embodiment X ₃ X ₄ is a dinucleotide selected from the group consisting of: ApG, ApA, ApC, TpG, TpA, TpT, and CpA. X ₁ X ₂ in another embodiment is a dinucleotide selected from the group consisting of: GpT, GpC, ApT, TpG, TpT, TpC, CpG, CpT, and CpC.

[0093] In another preferred embodiment the immunostimulatory nucleic acid has the sequence

5 ′TCNTX ₁ X ₂ CGX ₃ X ₄ 3′

[0094] wherein the C of the CG dinucleotide is methylated, X ₁ , X ₂ , X ₃ , and X ₄ are nucleotides, and N is a nucleic acid sequence composed of from about 0-25 nucleotides. The immunostimulatory nucleic acids of the invention in some embodiments include X ₁ X ₂ selected from the group consisting of GpG, GpA, GpT, and ApA and X ₃ X ₄ is selected from the group consisting of TpT, TpC, and CpT.

[0095] As used herein, a “CpI nucleic acid” refers to an immunostimulatory nucleic acid having all the characteristics of a CpG nucleic acid as described herein, with the exception that the G of the at least one CpG dinucleotide is replaced by an inosine (I). Inosine is a purine that represents a deaminated form of adenosine or guanosine in vertebrates. Stryer L, Biochemistry , 4th ed., W H Freeman, 1995. It was found according to this aspect of the invention that phosphorothioate ODN with inosine for guanosine exchanges are as effective as immunostimulatory nucleic acids as phosphorothioate CpG ODN. This finding was quite unexpected. Surprisingly, replacement of the G of a CpG dinucleotide with another purine, I, did not cause a loss of activity. Such an immunostimulatory nucleic acid may include at least one CpI dinucleotide and at least one unmethylated or methylated CpG dinucleotide. In an alternative embodiment, the immunostimulatory nucleic acid may have the same sequence as a CpG nucleic acid except that all the CpG dinucleotides are replaced by CpI dinucleotides. In a further alternative embodiment, the immunostimulatory nucleic acid may have the same sequence as a CpG nucleic acid except that all the guanosine bases are replaced by inosine bases.

[0096] In one preferred embodiment the invention provides an immunostimulatory nucleic acid which is a CpI nucleic acid represented by at least the formula:

5 ′X ₁ X ₂ CIX ₃ X ₄ 3′

[0097] wherein C is cytosine, I is inosine, and X ₁ , X ₂ , X ₃ , and X ₄ are nucleotides. In certain embodiments the C of the CpI dinucleotide in the formula above is unmethylated. In certain other embodiments the C of the CpI dinucleotide in the formula above is methylated. In one embodiment X ₂ is adenine, guanine, inosine, cytosine, or thymine. In another embodiment X ₃ is adenine, guanine, inosine, cytosine, or thymine. In other embodiments X ₂ is guanine, adenine, or thymine and X ₃ is cytosine, adenine, or thymine.

[0098] In another embodiment the immunostimulatory nucleic acid is an isolated CpI nucleic acid represented by at least the formula:

5 ′N ₁ X ₁ X ₂ CIX ₃ X ₄ N ₂ 3′

[0099] wherein C is cytosine, I is inosine, X ₁ , X ₂ , X ₃ , and X ₄ are nucleotides, and N ₁ , and N ₂ are nucleic acid sequences composed of from about 0-25 nucleotides each. In certain embodiments the C of the CpI dinucleotide in the formula above is unmethylated. In one embodiment X ₁ X ₂ is a dinucleotide selected from the group consisting of: GpG, GpA, GpT, ApG, IpI, IpG, GpI, IpA, IpT, ApI, ApA, ApT, CpG, CpI, CpA, CpT, TpG, TpI, TpA, and TpT; and X ₃ X ₄ is a dinucleotide selected from the group consisting of: ApG, ApI, ApA, ApT, ApC, CpG, CpI, CpA, CpC, TpG, TpI, TpA, TpT, and TpC. Preferably X ₁ X ₂ is GpA, GpT, IpA or IpT, and X ₃ X ₄ is TpT. In other embodiments X ₁ or X ₂ or both are purines and X ₃ or X ₄ or both are pyrimidines, or X ₁ X ₂ is GpA or IpA and X ₃ or X ₄ or both are pyrimidines. In another preferred embodiment X ₁ X ₂ is a dinucleotide selected from the group consisting of: TpA, ApA, ApC, ApG, ApI, IpI, IpG, GpI, and GpG. In yet another embodiment X ₃ X ₄ is a dinucleotide selected from the group consisting of: ApG, ApI, ApA, ApC, TpG, TpI, TpA, TpT, and CpA. X ₁ X ₂ in another embodiment is a dinucleotide selected from the group consisting of: GpT, GpC, IpT, IpC, ApT, TpG, TpI, TpT, TpC, CpG, CpI, CpT, and CpC.

[0100] In another preferred embodiment the immunostimulatory nucleic acid has the sequence

5 ′TCNTX ₁ X ₂ CIX ₃ X ₄ 3′

[0101] wherein C is cytosine, I is inosine, X ₁ , X ₂ , X ₃ , and X ₄ are nucleotides, and N is a nucleic acid sequence composed of from about 0-25 nucleotides. In certain embodiments the C of the CpI dinucleotide in the formula above is unmethylated. The immunostimulatory nucleic acids of the invention in some embodiments include X ₁ X ₂ selected from the group consisting of GpG, GpA, GpT, IpI, IpA, IpT, IpG, GpI, and ApA and X ₃ X ₄ is selected from the group consisting of TpT, TpC, and CpT.

[0102] As used herein, a “2′-alkoxy cytosine CpG nucleic acid” refers to an immunostimulatory nucleic acid having all the characteristics of a CpG nucleic acid as described herein, with the exception that the sugar moiety of the C of the at least one CpG dinucleotide is alkylated at the 2′ position, thereby substituting for C a 2′-O-alkyl cytosine. In a preferred embodiment the sugar moiety of the C of the at least one CpG dinucleotide is methylated at the 2′ position, thereby substituting for C a 2′-O-methyl cytosine (2′-OMe C). Such O-alkyl cytosine CpG nucleic acids may include at least one O-alkyl cytosine CpG dinucleotide and at least one unmodified CpG dinucleotide. In an alternative embodiment, every cytosine of the CpG-like nucleic acid can be a 2′-alkoxy cytosine, including all CpG dinucleotides present.

[0103] The finding that the 2′-alkoxy cytosine CpG nucleic acids of the invention are immunostimulatory was quite unexpected. Previous studies have reported that replacement of specific individual or pairs of deoxynucleosides flanking a CpG dinucleotide with corresponding 2′-methoxy deoxynucleosides affects immunostimulatory potential of the olionucleotide sequence in a highly position-dependent manner. Zhao Q et al. (1999) Bioorg Med Chem Lett 9:3453-8; Zhao Q et al. (2000) Bioorg Med Chem Lett 10:1051-4. According to those reports, the closer the substituted nucleosides occur relative to the CpG dinucleotide, the weaker the immunostimulatory potential of the oligonucleotide as a whole. Surprisingly, then, it was found according to the instant invention that substitution of 2′-alkoxy cytosine for cytosine had no dramatic influence on the immunostimulatory activity of the oligonucleotide.

[0104] In certain preferred embodiments the invention provides an immunostimulatory nucleic acid which is a 2′-alkoxy cytosine CpG nucleic acid represented by at least the formula:

5 ′X ₁ X ₂ CGX ₃ X ₄ 3′

[0105] wherein C is 2′-alkoxy cytosine and X ₁ , X ₂ , X ₃ , and X ₄ are nucleotides. In one embodiment X ₂ is adenine, guanine, cytosine, or thymine. In another embodiment X ₃ is adenine, guanine, cytosine, or thymine. In other embodiments X ₂ is guanine, adenine, or thymine and X ₃ is cytosine, adenine, or thymine.

[0106] In other embodiments the immunostimulatory nucleic acid is an isolated 2′-alkoxy cytosine CpG nucleic acid represented by at least the formula:

5 ′N ₁ X ₁ X ₂ CGX ₃ X ₄ N ₂ 3′

[0107] wherein C is 2′-alkoxy cytosine, X ₁ , X ₂ , X ₃ , and X ₄ are nucleotides, and N ₁ , and N ₂ are nucleic acid sequences composed of from about 0-25 nucleotides each. In one embodiment X ₁ X ₂ is a dinucleotide selected from the group consisting of: GpG, GpA, GpT, ApG, ApA, ApT, CpG, CpA, CpT, TpG, TpA, and TpT; and X ₃ X ₄ is a dinucleotide selected from the group consisting of: ApG, ApA, ApT, ApC, CpG, CpA, CpC, TpG, TpA, TpT, and TpC. Preferably X ₁ X ₂ is GpA or GpT and X ₃ X ₄ is TpT. In other embodiments X ₁ or X ₂ or both are purines and X ₃ or X ₄ or both are pyrimidines or X ₁ X ₂ is GpA and X ₃ or X ₄ or both are pyrimidines. In another preferred embodiment X ₁ X ₂ is a dinucleotide selected from the group consisting of: TpA, ApA, ApC, ApG, and GpG. In yet another embodiment X ₃ X ₄ is a dinucleotide selected from the group consisting of: ApG, ApA, ApC, TpG, TpA, TpT, and CpA. X ₁ X ₂ in another embodiment is a dinucleotide selected from the group consisting of: GpT, GpC, ApT, TpG, TpT, TpC, CpG, CpT, and CpC.

[0108] In other preferred embodiments the immunostimulatory nucleic acid has the sequence

5 ′TCNTX ₁ X ₂ CGX ₃ X ₄ 3′

[0109] wherein the C of the CG dinucleotide is 2′-alkoxy cytosine, X ₁ , X ₂ , X ₃ , and X ₄ are nucleotides, and N is a nucleic acid sequence composed of from about 0-25 nucleotides. The immunostimulatory nucleic acids of the invention in some embodiments include X ₁ X ₂ selected from the group consisting of GpG, GpA, GpT, and ApA and X ₃ X ₄ is selected from the group consisting of TpT, TpC, and CpT.

[0110] In yet further embodiments according to this aspect, the invention provides CpG-like nucleic acids which incorporate a combination of the types of modifications enumerated above. Thus, the invention also provides CpG-like nucleic acids incorporating a CpI dinucleotide and either a methylated C in the CpI dinucleotide or a 2′-alkoxy cytosine in the CpI dinucleotide. Likewise, the invention also embraces CpG-like nucleic acids incorporating both methylated cytosine in the CpG dinucleotide and a 2′-alkoxy cytosine in the CpI dinucleotide.

[0111] In another aspect the invention provides CpG-like immunostimulatory nucleic acids having a sequence including at least the formula

5 ′X ₁ X ₂ ZYX ₃ X ₄ 3′

[0112] wherein Z is selected from the group consisting of cytosine and dSpacer; Y is selected from the group consisting of inosine, 2-aminopurine, nebularine, and dSpacer; Z is not cytosine when Y is inosine; and X ₁ , X ₂ , X ₃ , and X ₄ are nucleotides. Such CpG-like immunostimulatory nucleic acids are referred to herein as ZpY oligonucleotides.

[0113] The term “dSpacer” as used herein refers to a sugar-phosphate moiety that is like a nucleotide lacking a base. This entity can be incorporated into a nucleic acid backbone by 5′-to-3′ type linkages that join nucleotides.

[0114] In embodiments where Z is cytosine, it can be methylated or unmethylated.

[0115] In some embodiments according to this aspect of the invention, the immunostimulatory nucleic acid has a sequence including at least the formula

5 ′TCNTX ₁ X ₂ ZYX ₃ X ₄ 3′

[0116] wherein N is a nucleic acid sequence composed of from about 0-25 nucleotides.

[0117] In some embodiments according to this aspect of the invention, the immunostimulatory nucleic acid is an isolated nucleic acid.

[0118] In some embodiments the invention provides a pharmaceutical composition which includes at least an amount of the immunostimulatory nucleic acid effective to induce an immune response and a pharmaceutically acceptable carrier. The pharmaceutical composition according to this embodiment of the invention can be prepared by placing an amount of the immunostimulatory nucleic acid effective to induce an immune response in a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier can in some embodiments include a sustained-release device. Other medicaments and agents may be included in the pharmaceutical composition, including an antigen, an antibiotic (antiviral, antibacterial, antifungal, and antiparasitic agents), an ulcer medicament, an allergy or asthma medicament, a medicament for the treatment of anemia, thrombocytopenia, or neutropenia, and a cytokine. In certain preferred embodiments which include a cytokine, the cytokine is selected from among IL-2, IL-3, IL-4, IL-18, IFN-α, IFN-γ, TNF-α, Flt3 ligand, G-CSF, and GM-CSF.

[0119] The pharmaceutical compositions containing an immunostimulatory nucleic acid according to this aspect of the invention will in some embodiments include a plurality of immunostimulatory nucleic acids having different sequences. In some embodiments the pharmaceutical composition can include a CpG nucleic acid having at least one unmethylated CpG motif.

[0120] In certain preferred embodiments the immunostimulatory nucleic acid has between 6 and 100 nucleotides, preferably between 8 and 40 nucleotides.

[0121] Also according to this aspect of the invention, in some embodiments wherein the immunostimulatory nucleic acid has a modified backbone. In certain preferred embodiments the the modified backbone is a phosphate modified backbone.

[0122] For facilitating uptake into cells, the immunostimulatory nucleic acids are preferably in the range of 6 to 100 bases in length. However, nucleic acids of any size greater than 6 nucleotides (even many kb long) are capable of inducing an immune response according to the invention if sufficient immunostimulatory motifs are present. Preferably the CpG-like nucleic acid is in the range of between 8 and 100 and in some embodiments between 8 and 40 or between 8 and 30 nucleotides in size.

[0123] The CpG-like nucleic acids can be formulated together with at least one other nucleic acid. In some instances the at least one other nucleic acid is an immunostimulatory nucleic acid, e.g., a CpG nucleic acid or another CpG-like nucleic acid. In other instances, the at least one other nucleic acid is a nucleic acid vector.

[0124] The CpG-like nucleic acids can be administered in conjunction with at least one other nucleic acid. In some instances the at least one other nucleic acid is an immunostimulatory nucleic acid, e.g., a CpG nucleic acid or another CpG-like nucleic acid. In other instances, the at least one other nucleic acid is a nucleic acid vector.

[0125] In the case when the immunostimulatory nucleic acid is administered in conjunction with a nucleic acid vector, under certain circumstances it is useful if the backbone of the immunostimulatory nucleic acid is a chimeric combination of phosphodiester and phosphorothioate (or other phosphate modification). A cell may have difficulty taking up a plasmid vector in the presence of completely phosphorothioate oligonucleotide. Thus when both a vector and an oligonucleotide are delivered to a subject, it is preferred that the oligonucleotide have a chimeric backbone, or, if the oligonucleotide has a completely phosphorothioate backbone, that the plasmid is associated with a vehicle that delivers it directly into the cell, thus avoiding the need for cellular uptake. Such vehicles are known in the art and include, for example, liposomes and gene guns.

[0126] In the case when more than one immunostimulatory nucleic acid is administered, either alone or in conjunction with a vector, the backbone of one immunostimulatory nucleic acid can be completely phosphorothioate and the backbone of another immunostimulatory nucleic acid completely phosphodiester. Thus, for example, a phosphorothioate ODN may be given together with a phosphodiester ODN.

[0127] For use in the instant invention, the immunostimulatory nucleic acids can be synthesized de novo using any of a number of procedures well known in the art. Such compounds are referred to as “synthetic nucleic acids.” These methods of synthesis include, for example, the β-cyanoethyl phosphoramidite method (Beaucage S L and Caruthers M H (1981) Tetrahedron Lett 22:1859), and the nucleoside H-phosphonate method (Garegg et al. (1986) Tetrahedron Lett 27:4051-4054; Froehier et al. (1986) Nucl Acid Res 14:5399-5407; Garegg et al. (1986) Tetrahedron Lett 27:4055-4058; Gaffney et al. (1988) Tetrahedron Lett 29:2619-2622). These chemistries can be performed by a variety of automated oligonucleotide synthesizers available in the market. These nucleic acids are referred to as synthetic nucleic acids. Alternatively, immunostimulatory nucleic acids can be produced on a large scale in plasmids (see Sambrook et al., Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratory Press, New York, 1989) and separated into smaller pieces or administered whole. Nucleic acids can be prepared from natural nucleic acid sequences (e.g., genomic DNA or cDNA) using known techniques, such as those employing restriction enzymes, exonucleases or endonucleases. Nucleic acids prepared in this manner are referred to as isolated nucleic acids. The term “immunostimulatory nucleic acid” encompasses both synthetic and isolated immunostimulatory nucleic acids.

[0128] For use in vivo, nucleic acids are preferably relatively resistant to degradation (e.g., are stabilized). A “stabilized nucleic acid molecule” shall mean a nucleic acid molecule that is relatively resistant to in vivo degradation (e.g., via an exo- or endonuclease). Stabilization can be a function of length or secondary structure. Immunostimulatory nucleic acids that are tens to hundreds of kbs long are relatively resistant to in vivo degradation. For shorter immunostimulatory nucleic acids, secondary structure can stabilize and increase their effect. For example, if the 3′ end of a nucleic acid has self-complementarity to an upstream region, so that it can fold back and form a sort of stem-loop structure, then the nucleic acid becomes stabilized and therefore exhibits more activity.

[0129] Alternatively, nucleic acid stabilization can be accomplished via backbone modifications. Preferred stabilized nucleic acids of the instant invention have a modified backbone. It has been demonstrated that modification of the nucleic acid backbone provides enhanced activity of the immunostimulatory nucleic acids when administered in vivo. One type of modified backbone is a phosphate backbone modification. Inclusion in immunostimulatory nucleic acids of at least two phosphorothioate linkages at or near the 5′ end of the oligonucleotide and multiple (preferably five) phosphorothioate linkages at or near the 3′ end, can in some circumstances provide maximal activity and protect the nucleic acid from degradation by intracellular exo- and endonucleases. Other phosphate-modified nucleic acids include phosphodiester-modified nucleic acids, combinations of phosphodiester and phosphorothioate nucleic acids, alklphosphonate and arylphosphonate, alklphosphorothioate and arylphosphorothioate, phosphorodithioate, and combinations thereof. Each of these combinations in CpG nucleic acids and their particular effects on immune cells is discussed in more detail in PCT Published Patent Applications PCT/US95/01570 and PCT/US97/19791, the entire contents of which are hereby incorporated by reference. Although Applicants are not bound by the theory, it is believed that these phosphate-modified nucleic acids may show more stimulatory activity due to enhanced nuclease resistance, increased cellular uptake, increased protein binding, and/or altered intracellular localization.

[0130] Modified backbones such as phosphorothioates may be synthesized using automated techniques employing either phosphoramidate or H-phosphonate chemistries as described above. Aryl- and alkyl-phosphonates can be made, e.g., as described in U.S. Pat. No. 4,469,863; and alkylphosphotriesters (in which the charged oxygen moiety is alkylated as described in U.S. Pat. No. 5,023,243 and European Patent No. 092,574) can be prepared by automated solid phase synthesis using commercially available reagents. Methods for making other DNA backbone modifications and substitutions have been described. Uhlmann E and Peyman A (1990) Chem Rev 90:544; Goodchild J (1990) Bioconjugate Chem 1:165.

[0131] Both phosphorothioate and phosphodiester nucleic acids containing immunostimulatory motifs are active in immune cells. However, based on the concentration needed to induce immunostimulatory nucleic acid-specific effects, the nuclease-resistant phosphorothioate backbone immunostimulatory nucleic acids are more potent. In certain in vitro assays, phosphorothioate CpG is two orders of magnitude more potent than phosphodiester CpG. Krieg A M et al. (1995) Nature 374:546-549.

[0132] Another class of backbone modifications include 2′-O-methylribonucleosides (2′-OMe). These types of substitutions are described extensively in the prior art and in particular with respect to their immunostimulating properties in Zhao Q et al. (1999) Bioorg Med Chem Lett 9:3453-8. Zhao et al. describes methods of preparing 2′-OMe modifications to nucleic acids.

[0133] The nucleic acid molecules of the invention may include naturally occurring or synthetic purine or pyrimidine heterocyclic bases as well as modified backbones. Purine or pyrimidine heterocyclic bases include, but are not limited to, adenine, guanine, cytosine, thymine, uracil, and inosine. Other representative heterocyclic bases are disclosed in U.S. Pat. No. 3,687,808, issued to Merigan, et al. The term purine or pyrimidine or bases are used herein to refer to both naturally occurring and synthetic purines, pyrimidines or bases.

[0134] Other stabilized nucleic acids include: nonionic DNA analogs, such as alkyl- and aryl-phosphates (in which the charged phosphonate oxygen is replaced by an alkyl or aryl group), alkylphosphodiester and alkylphosphotriesters, in which the charged oxygen moiety is alkylated. Nucleic acids which contain diol, such as tetraethyleneglycol or hexaethyleneglycol, at either or both termini have also been shown to be substantially resistant to nuclease degradation.

[0135] According to further aspects of the invention, the CpG-like nucleic acids are useful for treating a subject that has or is at risk of developing certain diseases, including an infectious disease due to an infection with an infectious agent, an allergy or asthma where the allergen or predisposition to asthma is known or suspected, or a cancer in which a specific cancer antigen has been identified. The CpG-like nucleic acids can also be given without the antigen or allergen for shorter term protection against infection, cancer, allergy or asthma, and in this case repeated doses will allow longer-term protection.

[0136] The term “treating” is defined as administering to a subject a therapeutically effective amount of a compound (e.g., an oligonucleotide of the invention) that is sufficient to prevent the onset of, alleviate the symptoms of, or stop the progression of a disorder or disease being treated.

[0137] A “subject” according to the invention shall mean a human or other vertebrate animal including but not limited to a dog, cat, horse, cow, pig, sheep, goat, rabbit, guinea pig, rat, mouse, non-human primate (e.g., monkey), chicken, and fish (aquaculture species, e.g., salmon).

[0138] A “subject at risk” as used herein is a subject that has a predisposition for having a disease or that has any identified or suspected risk of exposure to an agent or condition associated with the development of a disease. Generally, the risk of developing a particular disease will be higher for the subject at risk than for other individuals who are considered not to be at risk.

[0139] With reference to an infectious disease, a “subject at risk of developing an infectious disease” as used herein is a subject that has any identified or suspected risk of exposure to an infection-causing pathogen. For instance, a subject at risk of developing an infection may be a subject that is living in or planning to travel to an area where a particular type of infectious agent is found. A subject at risk may be a subject that through lifestyle, occupation, or a medical procedure is potentially exposed to bodily fluids or materials which may contain infectious organisms or agents. Alternatively, a subject at risk may be a subject that through lifestyle, occupation, or medical procedures is exposed to infectious organisms or agents. A subject at risk of developing infection also may be a subject at risk of biowarfare, e.g., military personnel or those living in areas at risk of terrorist attack. Subjects at risk of developing infection also include general populations to which a medical agency recommends vaccination against a particular infectious organism or agent.

[0140] With reference to allergy and asthma, a subject at risk as used herein is a subject that has any identified or suspected risk of exposure to an allergy- or asthma-inducing antigen. For instance, a subject at risk of developing allergy or asthma may be a subject that is living in or planning to travel to an area where a particular type of antigen or allergen is found. A subject at risk of developing allergy or asthma may be a subject that through lifestyle or employment activities is exposed directly to the antigen or allergen. Subjects at risk of developing allergy and asthma also include general populations to which a medical agency recommends vaccination with a particular antigen. If the antigen is an allergen and the subject develops seasonal allergic responses to that particular antigen, e.g., during pollen season, then the subject may be at risk of developing allergy during the season of exposure to the antigen. A subject at risk of developing an allergy or asthma includes those subjects that have been identified as having an allergy or asthma but that don′t have the active disease during the CpG-like nucleic acid treatment, as well as subjects that are considered to be at risk of developing these diseases because of genetic or environmental factors.

[0141] With reference to cancer, a “subject at risk of developing a cancer” is one that has an increased probability of having cancer. These subjects include, for instance, subjects having a genetic abnormality, the presence of which has been demonstrated to have a correlative relation to a higher likelihood of having a cancer, as well as subjects exposed to cancer-causing agents such as tobacco, asbestos, or other chemical toxins, or subjects that have previously been treated for cancer and are in apparent remission. When a subject at risk of developing a cancer is treated with an antigen specific for the type of cancer to which the subject is at risk of developing and a CpG-like nucleic acid, the subject may be able to kill the cancer cells as they develop. If a tumor begins to form in the subject, the subject will develop a specific immune response against the tumor antigen.

[0142] In addition to the use of the CpG-like nucleic acids for prophylactic treatment, the invention also encompasses the use of the CpG-like nucleic acids for the treatment of a subject having an infection, an allergy, asthma, or a cancer.

[0143] An infectious disease, as used herein, is a disease arising from the presence of a foreign microorganism or infectious pathogen in the body. A “subject that has an infectious disease” is a subject that has been exposed to an infectious pathogen and has acute or chronic detectable levels of the pathogen in the body. Infectious pathogens include viruses, bacteria, fungi, parasites, and other infectious agents. The CpG-like nucleic acids can be used with an antigen to mount an antigen-specific systemic or mucosal immune response that is capable of reducing the level of or eradicating the infectious pathogen.

[0144] A subject that has an allergy is a subject that has or is at risk of developing an allergic reaction in response to an allergen. An allergy refers to acquired hypersensitivity to a substance (allergen). Allergic conditions include but are not limited to eczema, urticaria (hives), allergic asthma, allergic rhinitis or coryza, hay fever, conjunctivitis, food allergies, and other atopic conditions.

[0145] Currently, allergic diseases are generally treated either symptomatically (see below) or definitively, the latter by the injection of small doses of antigen followed by subsequent increasing dosage of antigen. It is believed that this procedure induces tolerization to the allergen to prevent further allergic reactions. These methods, however, can take several years to be effective and are associated with the risk of side effects such as anaphylactic shock. The methods of the invention avoid these problems.

[0146] Allergies are generally characterized by IgE antibody generation against harmless allergens. The cytokines that are induced by systemic or mucosal administration of CpG-like nucleic acids are predominantly of a class called Th1 (examples are IL-12 and IFN-γ) and these induce both humoral (antibody) and cellular immune responses. The types of antibodies associated with a Th1 response are generally more protective because they have high neutralization and opsonization capabilities. The other major type of immune response, which is termed a Th2 immune response, is associated with the production of IL-4, IL-5 and IL-10 cytokines. Th2 responses involve predominately antibodies and these have less protective effect against infection and some Th2 isotypes (e.g., IgE) are associated with allergy. In general, it appears that allergic diseases are mediated by Th2 type immune responses while Th1 responses provide the best protection against infection, although excessive Th1 responses are associated with autoimmune disease. Based on the ability of the CpG-like nucleic acids to shift the immune response in a subject from a Th2 response (which is associated with production of IgE antibodies and allergy) to a Th1 response (which is protective against allergic reactions), an effective dose for inducing an immune response of a CpG-like nucleic acid can be administered to a subject to treat or prevent an allergy.

[0147] Thus, the CpG-like nucleic acids have significant therapeutic utility in the treatment of allergic and non-allergic conditions such as asthma. Th2 cytokines, especially IL-4 and IL-5, are elevated in the airways of asthmatic subjects. These cytokines promote important aspects of the asthmatic inflammatory response, including IgE isotope switching, eosinophil chemotaxis and activation, and mast cell degranulation. Th1 cytokines, especially IFN-γ and IL-12, can suppress the formation of Th2 clones and production of Th2 cytokines.

[0148] A subject that has asthma is a subject having a disorder of the respiratory system characterized by inflammation, narrowing of the airways, and increased reactivity of the airways to inhaled agents. Typically asthma is a chronic disease characterized by acute episodes of reversible, generalized airway narrowing. Thus a subject that has asthma need not require the subject currently be symptomatic, i.e., currently have an acute exacerbation of asthma. Methods of diagnosing asthma are well known in the art, and these can include measurement of reversible airway obstruction in response to challenge with a beta-adrenergic agonist, histamine, methacholine, or cold air. Asthma is frequently, although not exclusively associated with atopic or allergic symptoms.

[0149] A subject that has a cancer is a subject that has detectable cancerous cells. The c