The invention provides methods for modulating the immune response caused by CpG dinucleotide-containing compounds. The methods according to the invention enables both decreasing the immunostimulatory effect for antisense applications, as well as increasing the immunostimulatory effect for immunotherapy applications.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention relates to the therapeutic use of oligonucleotides, both in the antisense approach, and as immunostimulatory agents. The patents and publications cited herein reflect the level of knowledge in the field and are hereby incorporated by reference in their entirety. In the event of conflict between any teaching of any reference cited herein and the present specification, the latter shall prevail, for purposes of the invention.

The invention provides methods for modulating the immune response caused by CpG dinucleotide-containing compoundss. The methods according to the invention enable both decreasing the immunostimulatory effect for antisense applications, as well as increasing the immunostimulatory effect for immunotherapy applications. Thus, the invention further provides oligonucleotides having optimal levels of immunostimulatory effect for either application and methods for making and using such oligonucleotides.

The present inventor has surprisingly discovered that positional modification of CpG-containing oligonucleotides dramatically affects their immunostimulatory capabilities. In particular, 2' or 3' sugar or base modifications of oligonucleotides, or introduction of a modified internucleoside linkage, at particular positions 5' or 3' to the CpG dinucleotide, including 5' or 3' end modifications, or combinations thereof, either enhances or reduces their immunostimulatory effect in a reproducible and predictable manner.

In a first aspect, the invention provides a method for modulating the immunostimulatory effect of a CpG dinucleotide containing compound by introducing an immunomodulatory moiety at a position either 5' to or 3' to the CpG dinucleotide. Preferred compounds according to this aspect of the invention generally include additional oligonucleotide sequences.

In certain preferred embodiments the method is used to make an oligonucleotide that is complementary to a gene or gene transcript. In certain preferred embodiments, the oligonucleotide has antisense activity. In some preferred embodiments, only one immunomodulatory moiety is introduced into the oligonucleotide for each CpG dinucleotide present in the oligonucleotide. In some preferred embodiments, only one immunomodulatory moiety is introduced into the oligonucleotide.

In certain preferred embodiments, the oligonucleotide made according to this aspect of the invention does not have antisense activity and/or is not complementary to a gene.

As herein, the term "complementary" means having the ability to hybridize to a genomic region, a gene, or an RNA transcript thereof under physiological conditions. Such hybridization is ordinarily the result of base-specific hydrogen bonding between complementary strands, preferably to form Watson-Crick or Hoogsteen base pairs, although other modes of hydrogen bonding, as well as base stacking can also lead to hybridization. As a practical matter, such hybridization can be inferred from the observation of specific gene expression inhibition.

As used herein, "antisense activity" means that the oligonucleotide, when introduced into a cell or an animal, causes a reduction in the expression of the gene to which it is complementary.

The method according to this aspect of the invention can be conveniently carried out using any of the well-known synthesis techniques by simply using an appropriate immunomodulatory moiety monomer synthon in the synthesis process in a cycle following, immediately or otherwise the incorporation of the CpG dinucleotide. Preferred monomers include phosphoramidites, phosphotriesters and H-phosphonates.

For purposes of the invention, "introducing an immunomodulatory moiety into position Y2" simply means synthesizing an oligonucleotide that has an immunomodulatory moiety at such a position, with reference to the following structure: 5'-Yn . . . Y6-Y5-Y4-Y3-Y2-Y1-CG-X1-X2-X3-X4-X5-X6-X7-X8-X9 . . . Xm-3',

wherein C is cytosine, G is guanosine, a substituted guanosine, including inosine and 7-deazaguanosine, and each X and Y is independently a nucleoside or an immunomodulatory moiety, and n is a number from -9 to +20, and m is a number from -6 to +20.

Procedures for synthesis of oligonucleotides are well known in the art.

In a second aspect, the invention provides compounds having increased or reduced immunostimulatory effect, the compounds comprising a CpG dinucleotide and an immunomodulatory moiety, wherein the increased or reduced immunomodulatory effect is relative to a similar compound lacking the immunomodulatory moiety. Preferred compounds according to this aspect of the invention generally include additional oligonucleotide sequences. Preferably, such oligonucleotide sequences will have from about 6 to about 50 nucleotides, most preferably from about 12 to about 35 nucleotides.

Certain preferred compounds according to the invention have the structure: 5'-Yn . . . Y6-Y5-Y4-Y3-Y2-Y1-CG-X1-X2-X3-X4-X5-X6-X7-X8-X9 . . . Xm-3',

wherein C is cytosine, G is guanosine, a substituted guanosine, including inosine and 7-deazaguanosine, and each X and Y is independently a nucleoside or an immunomodulatory moiety, and n is a number from -9 to +20, and m is a number from -6 to +20.

In particularly preferred embodiments, the base sequence that is modified to provide the compound is 5'-CTATCTGACGTTCTCTGT-3' (SEQ ID NO: 1) or 5'-CCTACTAGCGTTCTCATC-3' (SEQ ID NO: 4) Preferred immunomodulatory moieties include one or more abasic nucleoside, 1,3-propanediol linker (substituted or unsubstituted), and/or modified base-containing nucleosides, including Hexa (ethylene glycol), nitropyrrole, nitroindole, deoxyuridine, inosine, isoguanosine, 2-aminopurine, nebularine, 7-deazaguanosine, 4-thiodeoxyuridine, 4-thiothymidine, d-isoguanosine, d-iso-5-methylcytosine, P-base, and 3'--3' linkage. As a general rule, introduction of an immunomodulatory moiety at position Y6, Y5, Y4, or Y3, or a combination thereof, increases the immunostimulatory effect of the oligonucleotide. Generally, introduction of an immunomodulatory moiety at position Y2 maintains immunostimulatory effect. Generally, introduction of an immunomodulatory moiety at position Y1 maintains or reduces immunostimulatory effect. Generally, introduction of an immunomodulatory moiety at position C abolishes immunostimulatory effect. Generally, introduction of an immunomodulatory moiety at position G abolishes immunostimulatory effect, except for 7-deazaguanosine, which maintains immunostimulatory effect. Generally, introduction of an immunomodulatory moiety at position X1 maintains or reduces immunostimulatory effect. Generally, introduction of an immunomodulatory moiety at position X2 has little impact on immunostimulatory effect. Generally, introduction of an immunomodulatory moiety at position X3 maintains or increases immunostimulatory effect. Generally, introduction of an immunomodulatory moiety at position X4, X5, X6, X7-Xm, or any combination thereof, increases immunostimulatory effect.

Certain preferred oligonucleotides according to this aspect of the invention are complementary to a gene or gene transcript. More preferably, such oligonucleotides have antisense activity. In some preferred embodiments, the oligonucleotide has only one immunomodulatory moiety for each CpG dinucleotide present in the oligonucleotide. In some preferred embodiments, the oligonucleotide has only one immunomodulatory moiety. In other preferred embodiments, the compounds according to this aspect of the invention do not have antisense activity and/or are not complementary to a gene.

In a third aspect, the invention provides a method for obtaining an antisense-specific reduction in the expression of a gene in a mammal, including a human, the method comprising administering to the mammal an oligonucleotide that is complementary to the gene and which comprises a CpG dinucleotide and an immunomodulatory moiety, wherein the oligonucleotide has less immunostimulatory effect than a similar oligonucleotide lacking the immunomodulatory moiety.

In some preferred embodiments, the oligonucleotide has only one immunomodulatory moiety for each CpG dinucleotide present in the oligonucleotide. In some preferred embodiments, the oligonucleotide has only one immunomodulatory moiety.

In the methods according to this aspect of the invention, preferably, administration of antisense oligonucleotides should be parenteral, oral, sublingual, transdermal, topical, intranasal, intrtracheal, or intrarectal. Administration of the therapeutic compositions can be carried out using known procedures at dosages and for periods of time effective to reduce symptoms or surrogate markers of the disease. When administered systemically, the therapeutic composition is preferably administered at a sufficient dosage to attain a blood level of oligonucleotide from about 0.001 micromolar to about 10 micromolar. For localized administration, much lower concentrations than this may be effective, and much higher concentrations may be tolerated. Preferably, a total dosage of oligonucleotide will range from about 0.1 mg oligonucleotide per patient per day to about 200 mg oligonucleotide per kg body weight per day. It may be desirable to administer simultaneously, or sequentially a therapeutically effective amount of one or more of the therapeutic compositions of the invention to an individual as a single treatment episode. In a preferred embodiment, after the composition of matter is administered, one or more measurement is taken of biological effects selected from the group consisting of complement activation, mitogenesis and inhibition of thrombin clot formation.

The method according to this aspect of the invention is useful in animal models of disease or gene expression, and is further useful for the therapeutic treatment of human or animal disease.

In a fourth aspect, the invention provides a method for inducing an immune response in a mammal, including a human, the method comprising administering to the mammal a compound comprising a CpG dinucleotide and and an immunomodulatory moiety, wherein the compound has greater immunostimulatory effect than a similar compound lacking the immunomodulatory moiety.

In the methods according to this aspect of the invention, preferably, administration of compounds should be parenteral, oral, sublingual, transdermal, topical, intranasal, intratracheal, or intrarectal. Administration of the therapeutic compositions can be carried out using known procedures at dosages and for periods of time effective to reduce symptoms or surrogate markers of the disease. When administered systemically, the therapeutic composition is preferably administered at a sufficient dosage to attain a blood level of oligonucleotide from about 0.001 micromolar to about 10 micromolar. For localized administration, much lower concentrations than this may be effective, and much higher concentrations may be tolerated. Preferably, a total dosage of oligonucleotide will range from about 0.1 mg oligonucleotide per patient per day to about 200 mg oligonucleotide per kg body weight per day. It may be desirable to administer simultaneously, or sequentially a therapeutically effective amount of one or more of the therapeutic compositions of the invention to an individual as a single treatment episode. In a preferred embodiment, after the composition of matter is administered, one or more measurement is taken of biological effects selected from the group consisting of complement activation, mitogenesis and inhibition of thrombin clot formation.

In certain preferred embodiments, compounds according to the invention are administered in combination with vaccines, antibodies, cytotoxics, antisense oligonucleotides, gene therapy vectors, DNA vaccines and/or adjuvants to enhance the specificity or magnitude of the immune response. Either the compound or the vaccine, or both may optionally be linked to an immunogenic protein, such as keyhole limpet hemocyanin, cholera toxin B subunit, or any other immunogenic carrier protein. Any of the plethora of adjuvants may be used, including, without limitation, Freund's complete adjuvant. For purposes of this aspect "in combination with" means in the course of treating the same disease in the same patient, and includes administering the oligonucleotide and/or the vaccine and/or the adjuvant in any order, including simultaneous administration, as well as temporally spaced order of up to several days apart. Such combination treatment may also include more than a single administration of the oligonucleotide, and/or independently the vaccine, and/or independently the adjuvant. The administration of the oligonucleotide and/or vaccine and/or adjuvant may be by the same or different routes.

The method according to this aspect of the invention is useful for model studies of the immune system, and is further useful for the therapeutic treatment of human or animal disease.

For purposes of all aspects of the invention, the term "oligonucleotide" includes polymers of two or more deoxyribonucleotides, or any modified nucleoside, including 2'-halo-nucleosides, 2' or 3' substituted, 2' or 3'-O-substituted ribonucleosides, deazanucleosides or any combination thereof. Such monomers may be coupled to each other by any of the numerous known internucleoside linkages. In certain preferred embodiments, these internucleoside linkages may be phosphodiester, phosphotriester, phosphorothioate, or phosphoramidate linkages, 2' 5' linkages of any of the forgoing, or combinations thereof. The term oligonucleotide also encompasses such polymers having chemically modified bases or sugars and/or having additional substituents, including without limitation lipophilic groups, intercalating agents, diamines and adamantane. The term oligonucleotide also encompasses PNA, LNA and oligonucleotides comprising non-pentose sugar (e.g. hexose) backbones or backbone sections. For purposes of the invention the terms "2'-O-substituted" and "3'-O-substituted" mean (respectively) substitution of the 2' (or 3') position of the pentose moiety with a halogen (preferably Cl, Br, or F), or an -O-lower alkyl group containing 1 6 saturated or unsaturated carbon atoms, or with an -O-aryl or allyl group having 2 6 carbon atoms, wherein such alkyl, aryl or allyl group may be unsubstituted or may be substituted, e.g., with halo, hydroxy, trifluoromethyl, cyano, nitro, acyl, acyloxy, alkoxy, carboxyl, carbalkoxyl, or amino groups; or such 2' substitution may be with a hydroxy group (to produce a ribonucleoside), an amino or a halo group, but not with a 2' (or 3') H group. For purposes of all aspects of the invention, the terms "CpG" or "CpG dinucleotide" means the dinucleotide 5'-deoxycytidine-deoxyguanidine or deoxyguanidine analog-3', wherein p is an internucleotide linkage, and wherein the sugar backbone of the dinucleotide may be ribose, deoxyribose, or 2' substituted ribose, or combinations thereof. In preferred embodiments of the first three aspects of the invention, p is selected from phosphodiester, phosphorothioate, alkylphosphonate, phosphotriester, stereospecific (Rp or Sp) phosphorothioate or alkylphosphonate, and 2' 5' covalent linkages of any of the above. The non-phosphodiester, non-phosphorothioate embodiments will further reduce immunostimulatory effects. In preferred embodiments of the last three aspects of the invention, p is selected from phosphodiester, phosphorothioate and phosphordithioate.
 

Claim 1 of 3 Claims

1. A method for modulating the immunostimulatroy effect of a 5'-CpG-3' dinucleotide containing compound, wherein C is cytosine and G is guanosine or a substituted guanosine, by introducing an immunomodulatory moiety at a position either 5' or 3' to the CpG dinucleotide, wherein the immunomodulatory moiety is selected from the group consisting of abasic nucleoside, 1,3-propanediol linker, nitropyrrole, nitroindole, deoxyuridine, inosine, isoguanosine, 2-aminopurine, nebularine, 7-deazaguanosine. 4-thiodeoxyuridine, 4-thiothymidine, d-isoguanosine, d-iso-5-methylcytosine, P-base, and 3'--3' linkage, and wherein the compound is 12 to 35 nucleotides in length and wherein the compound does not have antisense activity.

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