[0001] This application claims priority to U.S. Provisional Patent Application filed Oct. 12, 2001 entitled “METHODS AND PRODUCTS FOR ENHANCING IMMUNE RESPONSES USING IMIDAZOQUINOLINE COMPOUNDS”, Serial No. 60/329,208, the contents of which are incorporated by reference herein in their entirety.
[0002] Cancer is the second leading cause of death, resulting in one out of every four deaths, in the United States. In 1997, the estimated total number of new diagnoses for lung, breast, prostate, colorectal and ovarian cancer was approximately two million. Due to the ever increasing aging population in the United States, it is reasonable to expect that rates of cancer incidence will continue to grow.
[0003] Asthma is a chronic inflammatory disease effecting 14-15 million persons in the United States alone.
[0004] Infectious disease is one of the leading causes of death throughout the world. In the United States alone the death rate due to infectious disease rose 58% between 1980 and 1992. During this time, the use of anti-infective therapies to combat infectious disease has grown significantly and is now a multi-billion dollar a year industry. Even with these increases in anti-infective agent use, the treatment and prevention of infectious disease remains a challenge to the medical community throughout the world.
[0005] The immunostimulatory capacity of a variety of immunostimulatory nucleic acids has been well documented. Depending upon their nature and composition and administration, immunostimulatory nucleic acids are capable of inducing T helper 1 (Th1) responses, of suppressing T helper 2 (Th2) responses, and in some instances, inducing Th2 responses.
[0006] Imidazoquinoline agents have similarly been reported to possess immunomodulatory activity, including the ability to activate B lymphocytes, induce interferon alpha (IFN-alpha) production, and upregulate tumor necrosis factor (TNF), interleukin 1 (IL-1) and interleukin 6 (IL-6). The utility of imidazoquinoline agents in the treatment of viral infections and tumors has also been suggested.
[0007] The invention is based, in part, on the finding that when imidazoquinoline agents are used in conjunction with other therapeutic agents, such as antibodies, immunostimulatory nucleic acids, antigens, C8-substituted guanosines, and disorder-specific medicaments, some unexpected and improved results are observed. For instance, the efficacy of the combination of imidazoquinoline agents and the other therapeutic agent is profoundly improved over the use of either compound alone.
[0008] The results are surprising, in part, because the imidazoquinoline agents and the other therapeutic agents in some instances act through different mechanisms and would not necessarily be expected to improve the efficacy of the other in a synergistic manner.
[0009] In one aspect, the invention provides a method for stimulating antibody-dependent cellular cytotoxicity (ADCC) in a subject. The method comprises administering an antibody and an agent selected from the group consisting of an imidazoquinoline agent and an C8-substituted guanosine to a subject in need of such treatment in an amount effective to stimulate antibody dependent cellular cytotoxicity in the subject. In some embodiments, the amount effective to stimulate antibody dependent cellular cytotoxicity is a synergistic amount.
[0010] In one embodiment, the imidazoquinoline agent is administered prior to the antibody. In another embodiment, the antibody is selected from the group consisting of an anti-cancer antibody, an anti-viral antibody, an anti-bacterial antibody, an anti-fungal antibody, an anti-allergen antibody, and an anti-self antigen antibody. In related embodiments, the subject has or is at risk of having a disorder selected from the group consisting of asthma/allergy, infectious disease, cancer and warts.
[0011] The following embodiments apply to this and other aspects of the invention.
[0012] In one embodiment, the agent is imidazoquinoline agent. In another embodiment, both the imidazoquinoline agent and the C8-substituted guanosine are administered to the subject. C8-substituted guanosines can be selected from the group consisting of 8- mercaptoguanosine, 8-bromoguanosine, 8-methylguanosine, 8-oxo-7,8-dihydroguanosine, C8-arylamino-2′-deoxyguanosine, C8-propynyl-guanosine, C8- and N7- substituted guanine ribonucleosides such as 7-allyl-8-oxoguanosine (loxoribine) and 7-methyl-8-oxoguanosine, 8-aminoguanosine, 8-hydroxy-2′-deoxyguanosine, and 8-hydroxyguanosine.
[0013] In some embodiments in which the imidazoquinoline agent is administered to the subject, the subject is further administered a poly-arginine. In other embodiments, interferon-alpha (e.g., Intron A) is administered to the subject.
[0014] In one embodiment, the imidazoquinoline agent is an imidazoquinoline amine. In another embodiment, the imidazoquinoline agent is selected from the group consisting of imiquimod/R-837, S-28463/R-848 (Resiquimod), imidazoquinoline amines, imidazopyridine amines, 6,7-fused cycloalkylimidazopyridine amines, 1,2 bridged imidazoquinoline amines, and 4-amino-2ethoxymethyl-alpha, alpha-dimethyl-1H-imidazo[4,5-c]quinolines-1-ethanol.
[0015] In still other embodiments, the method further comprises administering an immunostimulatory nucleic acid to the subject. In certain embodiments, the agent is administered prior to the immunostimulatory nucleic acid. The immunostimulatory nucleic acid may be selected from the group consisting of a CpG nucleic acid and a poly-G nucleic acid. In certain embodiments, the immunostimulatory nucleic acid is selected from the group consisting of a poly-T nucleic acid, a T-rich nucleic acid, a TG nucleic acid, a CpI nucleic acid and a methylated CpG nucleic acid. In some embodiments, the immunostimulatory nucleic acid has a backbone modification. The backbone modification may be selected from the group consisting of a phosphorothioate modification and a peptide modification (such as for example a morpholino backbone modification), but is not so limited. In one embodiment, the immunostimulatory nucleic acid has a backbone that is chimeric. In still another embodiment, the immunostimulatory nucleic acid is a nucleic acid that is free of CpG, T-rich or poly-G motifs. In some embodiments, the immunostimulatory nucleic acid with a phosphorothioate modified backbone is free of a CpG motif, a T-rich motif or a poly-G motif. The immunostimulatory nucleic acid may be a nucleic acid which stimulates a Th1 immune response. In some embodiments, the immunostimulatory nucleic acid which stimulates a Th1 immune response is not a CpG nucleic acid. In other embodiments, the immunostimulatory nucleic acid which stimulates a Th1 immune response is not a T-rich nucleic acid.
[0016] In another embodiment, the method further comprises exposing the subject to an antigen. The antigen may be selected from the group consisting of a tumor antigen, a viral antigen, a bacterial antigen, a parasitic antigen, and a fungal antigen.
[0017] In another aspect, the invention provides a method for modulating an immune response in a subject. The method comprises administering to a subject in need of such treatment an immunostimulatory nucleic acid and an agent selected from the group consisting of an imidazoquinoline agent and an C8-substituted guanosine in an amount effective to modulate the immune response. In one embodiment, the amount effective to modulate the immune response is a synergistic amount. In an important embodiment, the imidazoquinoline agent is administered prior to the immunostimulatory nucleic acid. In certain embodiments, the immunostimulatory nucleic acid is a CpG nucleic acid. In other embodiments, the immunostimulatory nucleic acid has a nucleotide sequence of (#2006) TCG TCG TTT TGT CGT TTT GTC GTT (SEQ ID NO:1).
[0018] In one embodiment, modulating an immune response means inducing a Th1 immune response. In another embodiment, the immune response is a Th1 immune response. In another embodiment, the immune response involves antibody dependent cellular cytotoxicity. In another embodiment, the immune response is an innate immune response. In some embodiments, the immune response is a local immune response, while in other embodiments, the immune response is a systemic immune response. In certain embodiments, the immune response is a mucosal immune response.
[0019] In this and other embodiments of the invention, the method further comprises administering a disorder-specific medicament to the subject. The disorder-order specific medicament may be selected from the group consisting of a cancer medicament, an asthma/allergy a medicament, an infectious disease medicament, and a wart medicament. The anti-microbial medicament may be selected from the group consisting of an anti-bacterial agent, an anti-viral agent, an anti-fungal agent, and an anti-parasitic agent. The cancer medicament may be selected from the group consisting of a chemotherapeutic agent, an immunotherapeutic agent and a cancer vaccine. The asthma/allergy medicament may be selected from the group consisting of steroids, immunomodulators, anti-inflammatory agents, bronchodilators, leukotriene modifiers, beta
[0020] In this and other aspects of the invention, the method is a method for treating or preventing a disorder in a subject having or at risk of having the disorder. The disorder may be selected from the group consisting of infectious disease, cancer and asthma or allergy. The subject may be an immunocompromised subject. In other embodiments, the subject is elderly or an infant.
[0021] The invention further provides compositions and kits. In one aspect, the invention provides a composition, comprising an imidazoquinoline agent, and an immunostimulatory nucleic acid. In one embodiment, the immunostimulatory nucleic acid is a CpG nucleic acid. In an important embodiment, the immunostimulatory nucleic acid has the nucleotide sequence (#2006) TCG TCG TTT TGT CGT TTT GTC GTT (SEQ ID NO:1).
[0022] The invention provides in another aspect another composition comprising an imidazoquinoline agent and an antibody. In one embodiment, the composition further comprises an immunostimulatory nucleic acid.
[0023] In still another aspect, the invention provides a composition comprising an imidazoquinoline agent and a disorder-specific medicament. The disorder-specific medicament may be selected from the group consisting of an asthma/allergy medicament, a cancer medicament, and an anti-microbial medicament. In one embodiment, the disorder-specific medicament is an anti-microbial medicament selected from the group consisting of an anti-bacterial agent, an anti-viral agent, an anti-fungal agent, and an anti-parasitic agent. In another embodiment, the disorder-specific medicament is a cancer medicament selected from the group consisting of a chemotherapeutic agent, an immunotherapeutic agent and a cancer vaccine. In still another embodiment, the disorder-specific medicament is an asthma/allergy medicament selected from the group consisting of steroids, immunomodulators, anti-inflammatory agents, bronchodilators, leukotriene modifiers, beta
[0024] The compositions may further comprise poly-arginine. In other embodiments, the compositions further comprise an antigen. In still another embodiments, the compositions further comprise an C8-substituted guanosine. In a preferred embodiment, the composition comprises an imidazoquinoline agent, an immunostimulatory nucleic acid, an antigen and poly-arginine. Optionally, the latter composition may also comprise an C8-substituted guanosine.
[0025] In another aspect, the invention provides a method for altering the dosage of a therapeutic agent required to prophylactically or therapeutically treat a subject having a disorder (e.g., infectious disease, cancer or asthma/allergy) by co-administering an imidazoquinoline agent with the therapeutic agent. The therapeutic agent may be selected from the group consisting of an antibody, an antigen, an immunostimulatory nucleic acid, an C8-substituted guanosine, and a disorder-specific medicament, but is not so limited. The invention provides a method for increasing the dose of the therapeutic agent that can be administered to a subject in need of such treatment. The method involves administering to a subject in need of such treatment a therapeutic agent in a dose which ordinarily induces side effects and administering to the subject an imidazoquinoline agent in an effective amount to inhibit the side effects. As an example, when the therapeutic agent is a disorder specific medicament such as an anti-cancer therapy (e.g., cancer medicament), common side effects include myelosuppression and microbial infections. Thus, in one embodiment, the side effect is myelosuppression and in another embodiment, the side effect is a microbial infection. In yet another embodiment, the side effect is an adverse allergic reaction.
[0026] In another aspect, the invention provides a method for decreasing the dose of a therapeutic agent which can be administered to a subject. The method involves administering to a subject in need of such treatment, a therapeutic agent in a sub-therapeutic dosage and an imidazoquinoline agent, wherein the combination of the sub-therapeutic dose of the therapeutic agent and the imidazoquinoline agent produces a therapeutic result. The method provides several advantages, including lower costs due to the decreased amount of therapeutic agent needed, and a reduced probability of inducing side effects resulting from the therapeutic agent because of the lower doses used.
[0027] According to other aspects, the invention involves methods for treating a subject having or at risk of having a disorder by administering an imidazoquinoline agent and a therapeutic agent in different dosing schedules. In one aspect, the invention is a method for treating a subject by administering to a subject in need of such treatment an effective amount of an imidazoquinoline agent, and subsequently administering to the subject a therapeutic agent. In a related aspect, the method involves administering a therapeutic agent to a subject, and subsequently administering an imidazoquinoline agent. In one embodiment, the imidazoquinoline agent is administered on a routine schedule. The routine schedule may be selected from the group consisting of a daily schedule, a weekly schedule, a monthly schedule, a bimonthly schedule, a quarterly schedule, and a semi-annual schedule. In another embodiment, the imidazoquinoline agent is administered on a variable schedule. The imidazoquinoline agent may be administered in a sustained release vehicle.
[0028] In other aspects, the invention is a method for treating a subject having a disorder by administering to a subject in need of such treatment a therapeutic agent in an effective amount for providing some symptomatic relief and subsequently administering an imidazoquinoline agent to the subject. In some embodiments, the imidazoquinoline agent is administered in an effective amount for upregulating, enhancing or activating an immune response. In some embodiments, the imidazoquinoline agent is administered in an effective amount for redirecting the immune response a Th1 immune response. In still other embodiments, a plurality of imidazoquinoline agents is administered.
[0029] In another aspect, the invention provides a method for treating a subject having or at risk of developing a disorder by administering to a subject in need of such treatment an imidazoquinoline agent and a therapeutic agent, wherein the imidazoquinoline agent is administered systemically and the therapeutic agent is administered locally.
[0030] In still another aspect, the invention provides a method for treating a subject having or at risk of developing a disorder by administering to the subject an imidazoquinoline agent on a routine schedule and a therapeutic agent. In other embodiments, the imidazoquinoline agent and/or the therapeutic agent are administered in two or more doses. Alternatively, the imidazoquinoline agent may be administered on a non-regular basis (e.g., at the start of symptoms).
[0031] According to another aspect, the invention provides a screening method for comparing Toll-like receptor (TLR) signaling activity of a test compound with TLR signaling activity of an imidazoquinoline. The method involves contacting a functional TLR selected from the group consisting of Toll-like receptor 7 (TLR7) and Toll-like receptor 8 (TLR8) with a reference imidazoquinoline and detecting a reference response mediated by a TLR signal transduction pathway; contacting a functional TLR selected from the group consisting of TLR7 and TLR8 with a test compound and detecting a test response mediated by a TLR signal transduction pathway; and comparing the test response with the reference response to compare the TLR signaling activity of the test compound with the imidazoquinoline. In a preferred embodiment the functional TLR is TLR8. In another preferred embodiment the functional TLR is TLR7.
[0032] In certain embodiments the functional TLR is contacted with the reference imidazoquinoline and the test compound independently. In a preferred embodiment the screening method is a method for identifying an imidazoquinoline mimic, wherein when the test response is similar to the reference response the test compound is an imidazoquinoline mimic.
[0033] In certain other embodiments the functional TLR is contacted with the reference imidazoquinoline and the test compound concurrently to produce a test-reference response mediated by a TLR signal transduction pathway; the test-reference response may be compared to the reference response. In a preferred embodiment the screening method is a method for identifying an imidazoquinoline agonist, wherein when the test-reference response is greater than the reference response the test compound is an imidazoquinoline agonist. In a preferred embodiment the screening method is a method for identifying an imidazoquinoline antagonist, wherein when the test-reference response is less than the reference response the test compound is an imidazoquinoline antagonist.
[0034] In certain embodiments the functional TLR is expressed in a cell. Preferably, the cell is an isolated mammalian cell that naturally expresses functional TLR8. In another preferred embodiment the cell is an isolated mammalian cell that naturally expresses functional TLR7. To facilitate practice of the method, in certain embodiments the cell expressing the functional TLR7 or functional TLR8 includes an expression vector comprising an isolated nucleic acid which encodes a reporter construct selected from the group consisting of interleukin 8 (IL-8), p40 subunit of interleukin 12 (IL-12 p40), nuclear factor kappa B-luciferase (NF-kappa B-luc), p40 subunit of interleukin 12-luciferase (IL-12 p40-luc), and tumor necrosis factor-luciferase (TNF-luc).
[0035] In certain other embodiments the functional TLR is part of a cell-free system.
[0036] In some embodiments the functional TLR is part of a complex with another TLR, including, for example, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, or TLR10. The complex can include two or more TLRs.
[0037] In certain embodiments the functional TLR is part of a complex with a non-TLR protein selected from the group consisting of myeloid differentiation factor 88 (MyD88), IL-1 receptor-associated kinase (IRAK), tumor necrosis factor receptor-associated factor 6 (TRAF6), I kappa B, NF-kappa B, and functional homologs and derivatives thereof.
[0038] In a preferred embodiments the reference imidazoquinoline is R-848 (Resiquimod). In another preferred embodiment the reference imidazoquinoline is R-847 (Imiquimod).
[0039] In certain embodiments the test compound is not a nucleic acid molecule. For example, in one embodiment the test compound is a polypeptide. In a preferred embodiment the test compound is an imidazoquinoline other than R-848 or R-847.
[0040] In certain embodiments the test compound is a part of a combinatorial library of compounds.
[0041] Each of the limitations of the invention can encompass various embodiments of the invention. It is, therefore, anticipated that each of the limitations of the invention involving any one element or combinations of elements can be included in each aspect of the invention.
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[0068] It is to be understood that the Figures are not required to enable the invention.
[0069] SEQ ID NO:1 is the nucleotide sequence of an immunostimulatory CpG nucleic acid (#2006).
[0070] SEQ ID NO:2 is the nucleotide sequence of an immunostimulatory T-rich nucleic acid (#2183).
[0071] SEQ ID NO:3 is the nucleotide sequence of a control non-CpG nucleic acid (#1982).
[0072] SEQ ID NO:4 is the nucleotide sequence of an immunostimulatory CpG nucleic acid (#8954).
[0073] SEQ ID NO:5 is the nucleotide sequence of a negative control nucleic acid (#5177).
[0074] SEQ ID NO:6 is the nucleotide sequence of human TLR9 cDNA (GenBank Accession No. AF245704).
[0075] SEQ ID NO:7 is the amino acid sequence of human TLR9 protein (GenBank Accession No. AAF78037).
[0076] SEQ ID NO:8 is the nucleotide sequence of murine TLR9 cDNA (GenBank Accession No. AF348140).
[0077] SEQ ID NO:9 is the amino acid sequence of murine TLR9 protein (GenBank Accession No. AAK29625).
[0078] SEQ ID NO:10 is the nucleotide sequence of a control GpC nucleic acid (#2006-GC).
[0079] SEQ ID NO:11 is the nucleotide sequence of a methylated CpG nucleic acid (#2006 methylated).
[0080] SEQ ID NO:12 is the nucleotide sequence of an immunostimulatory nucleic acid (#1668).
[0081] SEQ ID NO:13 is the nucleotide sequence of a GpC nucleic acid (#1668-GC).
[0082] SEQ ID NO:14 is the nucleotide sequence of a methylated CpG nucleic acid (#1668 methylated).
[0083] SEQ ID NO:15 is the nucleotide sequence of a first primer used to amplify human TLR7 cDNA.
[0084] SEQ ID NO:16 is the nucleotide sequence of a second primer used to amplify human TLR7 cDNA.
[0085] SEQ ID NO:17 is the nucleotide sequence of human TLR7 cDNA.
[0086] SEQ ID NO:18 is the amino acid sequence of human TLR7 protein.
[0087] SEQ ID NO:19 is the nucleotide sequence of a first primer used to amplify murine TLR7 cDNA.
[0088] SEQ ID NO:20 is the nucleotide sequence of a second primer used to amplify murine TLR7 cDNA.
[0089] SEQ ID NO:21 is the nucleotide sequence of murine TLR7 cDNA.
[0090] SEQ ID NO:22 is the amino acid sequence of murine TLR7 cDNA.
[0091] SEQ ID NO:23 is the nucleotide sequence of a first primer used to amplify human TLR8 cDNA.
[0092] SEQ ID NO:24 is the nucleotide sequence of a second primer used to amplify human TLR8 cDNA.
[0093] SEQ ID NO:25 is the nucleotide sequence of human TLR8 cDNA.
[0094] SEQ ID NO:26 is the amino acid sequence of human TLR8 cDNA.
[0095] SEQ ID NO:27 is the amino acid sequence of an N-terminal insertion in human TLR8 corresponding to GenBank Accession No. AF246971.
[0096] SEQ ID NO:28 is the nucleotide sequence of a first primer used to amplify murine TLR8 cDNA.
[0097] SEQ ID NO:29 is the nucleotide sequence of a second primer used to amplify murine TLR8 cDNA.
[0098] SEQ ID NO:30 is the nucleotide sequence of murine TLR8 cDNA.
[0099] SEQ ID NO:31 is the amino acid sequence of murine TLR8 protein.
[0100] The invention is based, in part, on the surprising discovery that administration of an imidazoquinoline agent and an antibody to a subject enhances antibody-dependent cellular cytoxicity (ADCC). Accordingly, in one aspect, the invention provides methods for treating humans and animals with imidazoquinoline agents in a dose sufficient to induce systemic activation of ADCC. Although not intending to be bound by any particular theory, it is postulated that imidazoquinoline agents enhance systemic ADCC by upregulating expression of Fc receptors and improving the functional activity of effector cells such as monocytes and macrophages. When a therapeutic antibody is co-administered to a subject with an imidazoquinoline agent, the enhanced ADCC activity will lead to a dramatic increase in therapeutic effect.
[0101] Imidazoquinolines are immune response modifiers thought to induce expression of several cytokines including interferons (e.g., IFN-alpha and IFN-alpha), TNF-alpha and some interleukins (e.g., IL-1, IL-6 and IL-12). Imidazoquinolines are capable of stimulating a Th1 immune response, as evidenced in part by their ability to induce increases in IgG2a levels. Imidazoquinoline agents reportedly are also capable of inhibiting production of Th2 cytokines such as IL-4, IL-5, and IL-13. Some of the cytokines induced by imidazoquinolines are produced by macrophages and dendritic cells. Some species of imidazoquinolines have been reported to increase NK cell lytic activity and to stimulate B cells proliferation and differentiation, thereby inducing antibody production and secretion.
[0102] As used herein, an imidazoquinoline agent includes imidazoquinoline amines, imidazopyridine amines, 6,7-fused cycloalkylimidazopyridine amines, and 1,2 bridged imidazoquinoline amines. These compounds have been described in U.S. Pat. Nos. 4,689,338, 4,929,624, 5,238,944, 5,266,575, 5,268,376, 5,346,905, 5,352,784, 5,389,640, 5,395,937, 5,494,916, 5,482,936, 5,525,612, 6,039,969 and 6,110,929. Particular species of imidazoquinoline agents include R-848 (S-28463); 4-amino-2ethoxymethyl-α,α-dimethyl-1H-imidazo[4,5-c]quinol ines-1-ethanol; and 1-(2-methylpropyl)-1H-imidazo[4,5-c]quinolin-4-amine (R-837 or Imiquimod). Imiquimod is currently used in the topical treatment of warts such as genital and anal warts and has also been tested in the topical treatment of basal cell carcinoma.
[0103] Antibodies useful in the invention include monoclonal antibodies, polyclonal antibodies, murine antibodies, human antibodies, chimeric murine-human antibodies, and the like. In some embodiments, antibody fragments can be used provided such fragments possess both an Fc and at least one Fab portion.
[0104] In some embodiments, the imidazoquinoline is administered at the same time as the antibody, while in other embodiments, it is administered prior to following antibody administration. If delivered prior to the administration of the antibody, the imidazoquinoline agent can be administered 1, 2, 3, 4, 5, 6, 7, or more days prior to the administration of antibody. If administered after the administration of the antibody, the imidazoquinoline agent can be administered 1, 2, 3, 4, 5, 6, 7, or more days after the administration of the antibody. In some preferred embodiments, the imidazoquinoline agent is administered within 48 hours, within 36 hours, within 24 hours, within 12 hours, within 6 hours, or within 4 hours of antibody administration, regardless of whether the antibody is administered prior to or following the imidazoquinoline agent.
[0105] Therapeutic antibodies useful in the invention may be specific for microbial antigens (e.g., bacterial, viral, parasitic or fungal antigens), cancer or tumor-associated antigens and self antigens. Preferred antibodies are those that recognize and bind to antigens present on or in a cell. Examples of suitable antibodies include but are not limited to Rituxan™ (rituximab, anti-CD20 antibody), Herceptin (trastuzumab), Quadramet, Panorex, IDEC-Y2B8, BEC2, C225, Oncolym, SMART M195, ATRAGEN, Ovarex, Bexxar, LDP-03, ior t6, MDX-210, MDX-11, MDX-22, OV103, 3622W94, anti-VEGF, Zenapax, MDX-220, MDX-447, MELIMMUNE-2, MELIMMUNE-1, CEACIDE, Pretarget, NovoMAb-G2, TNT, Gliomab-H, GNI-250, EMD-72000, LymphoCide, CMA 676, Monopharm-C, 4B5, ior egf.r3, ior c5, BABS, anti-FLK-2, MDX-260, ANA Ab, SMART ID10Ab, SMART ABL 364 Ab, CC49 (mAb B72.3), ImmuRAIT-CEA, anti-IL-4 antibody, an anti-IL-5 antibody, an anti-IL-9 antibody, an anti-Ig antibody, an anti-IgE antibody, serum-derived hepatitis B antibodies, recombinant hepatitis B antibodies, and the like.
[0106] Other antibodies similarly useful for the invention include alemtuzumab (B cell chronic lymphocytic leukemia), gemtuzumab ozogamicin (CD33+acute myeloid leukemia), hP67.6 (CD33+acute myeloid leukemia), infliximab (inflammatory bowel disease and rheumatoid arthritis), etanercept (rheumatoid arthritis), tositumomab, MDX-210, oregovomab, anti-EGF receptor mAb, MDX-447, anti-tissue factor protein (TF), (Sunol); ior-c5, c5, edrecolomab, ibritumomab tiuxetan, anti-idiotypic mAb mimic of ganglioside GD3 epitope, anti-HLA-Dr10 mAb, anti-CD33 humanized mAb, anti-CD52 humAb, anti-CD1 mAb (ior t6), MDX-22, celogovab, anti-17-1A mAb, bevacizumab, daclizumab, anti-TAG-72 (MDX-220), anti-idiotypic mAb mimic of high molecular weight proteoglycan (I-Mel-1), anti-idiotypic mAb mimic of high molecular weight proteoglycan (I-Mel-2), anti-CEA Ab, hmAbH11, anti-DNA or DNA-associated proteins (histones) mAb, Gliomab-H mAb, GNI-250 mAb, anti-CD22, CMA 676), anti-idiotypic human mAb to GD2 ganglioside, ior egf/r3, anti-ior c2 glycoprotein mAb, ior c5, anti-FLK-2/FLT-3 mAb, anti-GD-2 bispecific mAb, antinuclear autoantibodies, anti-HLA-DR Ab, anti-CEA mAb, palivizumab, bevacizumab, alemtuzumab, BLyS-mAb, anti-VEGF2, anti-Trail receptor; B3 mAb, mAb BR96, breast cancer; and Abx-Cb1 mAb.
[0107] Also included are antibodies such as the following, all of which are commercially available:
[0108] Apoptosis Antibodies: BAX Antibodies: Anti-Human Bax Antibodies (Monoclonal),Anti-Human Bax Antibodies (Polyclonal), Anti-Murine Bax Antibodies (Monoclonal), Anti-Murine Bax Antibodies (Polyclonal); Fas/Fas Ligand Antibodies: Anti-Human Fas/Fas Ligand Antibodies, Anti-Murine Fas/Fas Ligand Antibodies Granzyme Antibodies Granzyme B Antibodies; BCL Antibodies: Anti Cytochrome C Antibodies, Anti-Human BCL Antibodies (Monoclonal), Anti-Human bcl Antibodies (Polyclonal), Anti-Murine bcl Antibodies (Monoclonal), Anti-Murine bcl Antibodies (Polyclonal);
[0109] Miscellaneous Apoptosis Antibodies: Anti TRADD, TRAIL, TRAFF, DR3 Antibodies Anti-Human Fas/Fas Ligand Antibodies Anti-Murine Fas/Fas Ligand Antibodies;
[0110] Miscellaneous Apoptosis Related Antibodies: BIM Antibodies: Anti Human, Murine bim Antibodies (Polyclonal), Anti-Human, Murine bim Antibodies (Monoclonal);
[0111] PARP Antibodies Anti-Human PARP Antibodies (Monoclonal) Anti-Human PARP Antibodies(Polyclonal) Anti-Murine PARP Antibodies;
[0112] Caspase Antibodies: Anti-Human Caspase Antibodies (Monoclonal), Anti-Murine Caspase Antibodies;
[0113] Anti-CD Antibodies: Anti-CD29, PL18-5 PanVera, Anti-CD29, PL4-3 PanVera, Anti-CD41a, PT25-2 PanVera, Anti-CD42b, PL52-4 PanVera, Anti-CD42b, GUR20-5 PanVera, Anti-CD42b, WGA-3 PanVeraAnti-CD43, 1D4 PanVera, Anti-CD46, MCP75-6 PanVera, Anti-CD61, PL11-7 PanVera, Anti-CD61, PL8-5 PanVera, Anti-CD62/P-slctn, PL7-6 PanVera, Anti-CD62/P-slctn, WGA-1 PanVera, Anti-CD154, 5F3 PanVera;
[0114] Human Chemokine Antibodies: Human CNTF Antibodies, Human Eotaxin Antibodies, Human Epithelial Neutrophil Activating Peptide-78, Human Exodus Antibodies, Human GRO Antibodies, Human HCC-1 Antibodies, Human I-309 Antibodies, Human IP-10 Antibodies, Human I-TAC Antibodies, Human LIF Antibodies, Human Liver-Expressed Chemokine Antibodies, Human Lymphotaxin Antibodies, Human MCP Antibodies, Human MIP Antibodies, Human Monokine Induced by IFN-gamma Antibodies, Human NAP-2 Antibodies, Human NP-1 Antibodies, Human Platelet Factor-4 Antibodies, Human RANTES Antibodies, Human SDF Antibodies, Human TECK Antibodies;
[0115] Murine Chemokine Antibodies: Human B-Cell Attracting Murine Chemokine Antibodies, Chemokine-1 Antibodies, Murine Eotaxin Antibodies, Murine Exodus Antibodies, Murine GCP-2 Antibodies, Murine KC Antibodies, Murine MCP Antibodies, Murine MIP Antibodies, Murine RANTES Antibodies, Rat Chemokine Antibodies, Rat Chemokine Antibodies, Rat CNTF Antibodies, Rat GRO Antibodies, Rat MCP Antibodies, Rat MIP Antibodies, Rat RANTES Antibodies;
[0116] Cytokine/Cytokine Receptor Antibodies: Human Biotinylated Cytokine/Cytokine Receptor Antibodies, Human IFN Antibodies, Human IL Antibodies, Human Leptin Antibodies, Human Oncostatin Antibodies, Human TNF Antibodies, Human TNF Receptor Family Antibodies, Murine Biotinylated Cytokine/Cytokine Receptor Antibodies, Murine IFN Antibodies, Murine IL Antibodies, Murine TNF Antibodies, Murine TNF Receptor Antibodies;
[0117] Rat Cytokine/Cytokine Receptor Antibodies: Rat Biotinylated Cytokine/Cytokine Receptor Antibodies, Rat IFN Antibodies, Rat IL Antibodies, Rat TNF Antibodies;
[0118] ECM Antibodies: Collagen/Procollagen, Laminin, Collagen (Human), Laminin (Human), Procollagen (Human), Vitronectin/Vitronectin Receptor, Vitronectin (Human), Vitronectin Receptor (Human), Fibronectin/Fibronectin Receptor, Fibronectin (Human), Fibronectin Receptor (Human);
[0119] Growth Factor Antibodies: Human Growth Factor Antibodies, Murine Growth Factor Antibodies, Porcine Growth Factor Antibodies;
[0120] Miscellaneous Antibodies: Baculovirus Antibodies, Cadherin Antibodies, Complement Antibodies, C1q Antibodies, VonWillebrand Factor Antibodies, Cre Antibodies, HIV Antibodies, Influenza Antibodies, Human Leptin Antibodies, Murine Leptin Antibodies, Murine CTLA-4 Antibodies, P450 Antibodies, RNA Polymerase Antibodies;
[0121] Neurobio Antibodies: Amyloid Antibodies, GFAP Antibodies, Human NGF Antibodies, Human NT-3 Antibodies, Human NT-4 Antibodies.
[0122] Still other antibodies can be used in the invention and these include antibodies listed in references such as the MSRS Catalog of Primary Antibodies, and Linscott's Directory.
[0123] The imidazoquinoline agents can also be used with normal and hyper-immune globulin therapy. Normal immune globulin therapy utilizes a antibody product which is prepared from the serum of normal blood donors and pooled. This pooled product contains low titers of antibody to a wide range of antigens such as those of infectious pathogens (e.g., bacteria, viruses such as hepatitis A, parvovirus, enterovirus, fungi and parasites). Hyper-immune globulin therapy utilizes antibodies which are prepared from the serum of individuals who have high titers of an antibody to a particular antigen. Examples of hyper-immune globulins include zoster immune globulin (useful for the prevention of varicella in immunocompromised children and neonates), human rabies immunoglobulin (useful in the post-exposure prophylaxis of a subject bitten by a rabid animal), hepatitis B immune globulin (useful in the prevention of hepatitis B virus, especially in a subject exposed to the virus), and RSV immune globulin (useful in the treatment of respiratory syncitial virus infections).
[0124] Some commercially available anti-cancer antibodies are listed below along with their commercial source.
Cancer Immunotherapies in Development or on the Market MARKETER BRAND NAME (GENERIC NAME) INDICATION IDEC/Genentech, Rituxan ™ (rituximab, Mabthera) (IDEC- non-Hodgkin's lymphoma Inc./Hoffmann-LaRoche (first C2B8, chimeric murine/human anti-CD20 monoclonal antibody licensed for MAb) the treatment of cancer in the U.S.) Genentech/Hoffmann-La Roche Herceptin, anti-Her2 hMAb Breast/ovarian Cytogen Corp. Quadramet (CYT-424) radiotherapeutic Bone metastases agent Centocor/Glaxo/Ajinomoto Panorex ® (17-1A) (murine monoclonal Adjuvant therapy for antibody) colorectal (Dukes-C) Centocor/Ajinomoto Panorex ® (17-1A) (chimeric murine Pancreatic, lung, breast, monoclonal antibody) ovary IDEC IDEC-Y2B8 (murine, anti-CD20 MAb non-Hodgkin's lymhoma labeled with Yttrium-90) ImClone Systems BEC2 (anti-idiotypic MAb, mimics the GD Small cell lung epitope) (with BCG) ImClone Systems C225 (chimeric monoclonal antibody to Renal cell epidermal growth factor receptor (EGFr)) Techniclone International/Alpha Oncolym (Lym-1 monoclonal antibody non-Hodgkin's lymphoma Therapeutics linked to 131 iodine) Protein Design Labs SMART M195 Ab, humanized Acute myleoid leukemia Techniclone non-Hodgkin's lymphoma Corporation/Cambridge Antibody Technology Aronex Pharmaceuticals, Inc. ATRAGEN ® Acute promyelocytic leukemia ImClone Systems C225 (chimeric anti-EGFr monoclonal Head & neck, non-small antibody) + cisplatin or radiation cell lung cancer Altarex, Canada Ovarex (B43.13, anti-idiotypic CA125, Ovarian mouse MAb) Coulter Pharma (Clinical results Bexxar (anti-CD20 Mab labeled with non-Hodgkin's lymphoma have been positive, but the drug has been associated with significant bone marrow toxicity) Aronex Pharmaceuticals, Inc. ATRAGEN ® Kaposi's sarcoma IDEC Pharmaceuticals Rituxan ™ (MAb against CD20) pan-B Ab in B cell lymphoma Corp./Genentech combo. with chemotherapy LeukoSite/Ilex Oncology LDP-03, huMAb to the leukocyte antigen Chronic lymphocytic CAMPATH leukemia (CLL) Center of Molecular Immunology ior t6 (anti CD6, murine MAb) CTCL Cancer Medarex/Novartis MDX-210 (humanized anti-HER-2 bispecific Breast, ovarian antibody) Medarex/Novartis MDX-210 (humanized anti-HER-2 bispecific Prostate, non-small cell antibody) lung, pancreatic, breast Medarex MDX-11 (complement activating receptor Acute myelogenous (CAR) monoclonal antibody) leukemia (AML) Medarex/Novartis MDX-210 (humanized anti-HER-2 bispecific Renal and colon antibody) Medarex MDX-11 (complement activating receptor Ex vivo bone marrow (CAR) monoclonal antibody) purging in acute myelogenous leukemia (AML) Medarex MDX-22 (humanized bispecific antibody, Acute myleoid leukemia MAb-conjugates) (complement cascade activators) Cytogen OV103 (Yttrium-90 labelled antibody) Ovarian Cytogen OV103 (Yttrium-90 labelled antibody) Prostate Aronex Pharmaceuticals, Inc. ATRAGEN ® non-Hodgkin's lymphoma Glaxo Wellcome plc 3622W94 MAb that binds to EGP40 (17-1A) non-small cell lung, pancarcinoma antigen on adenocarcinomas prostate (adjuvant) Genentech Anti-VEGF, RhuMAb (inhibits Lung, breast, prostate, angiogenesis) colorectal Protein Design Labs Zenapax (SMART Anti-Tac (IL-2 receptor) Leukemia, lymphoma Ab, humanized) Protein Design Labs SMART M195 Ab, humanized Acute promyelocytic leukemia ImClone Systems C225 (chimeric anti-EGFr monoclonal Breast antibody) + taxol ImClone Systems (licensed from C225 (chimeric anti-EGFr monoclonal prostate RPR) antibody) + doxorubicin ImClone Systems C225 (chimeric anti-EGFr monoclonal prostate antibody) + adriamycin ImClone Systems BEC2 (anti-idiotypic MAb, mimics the GD Melanoma epitope) Medarex MDX-210 (humanized anti-HER-2 bispecific Cancer antibody) Medarex MDX-220 (bispecific for tumors that express Lung, colon, prostate, TAG-72) ovarian, endometrial, pancreatic and gastric Medarex/Novartis MDX-210 (humanized anti-HER-2 bispecific Prostate antibody) Medarex/Merck KgaA MDX-447 (humanized anti-EGF receptor EGF receptor cancers bispecific antibody) (head & neck, prostate, lung, bladder, cervical, ovarian) Medarex/Novartis MDX-210 (humanized anti-HER-2 bispecific Comb. Therapy with G- antibody) CSF for various cancers, esp. breast IDEC MELIMMUNE-2 (murine monoclonal Melanoma antibody therapeutic vaccine) IDEC MELIMMUNE-1 (murine monoclonal Melanoma antibody therapeutic vaccine) Immunomedics, Inc. CEACIDE ™ (I-131) Colorectal and other NeoRx Pretarget ™ radioactive antibodies non-Hodgkin's B cell lymphoma Novopharm Biotech, Inc. NovoMAb-G2 (pancarcinoma specific Ab) Cancer Techniclone Corporation/ TNT (chimeric MAb to histone antigens) Brain Cambridge Antibody Technology Techniclone International/ TNT (chimeric MAb to histone antigens) Brain Cambridge Antibody Technology Novopharm Gliomab-H (Monoclonals-Humanized Abs) Brain, melanomas, neuroblastomas Genetics Institute/AHP GNI-250 Mab Colorectal Merck KgaA EMD-72000 (chimeric-EGF antagonist) Cancer Immunomedics LymphoCide (humanized LL2 antibody) non-Hodgkin's B-cell lymphoma Immunex/AHP CMA 676 (monoclonal antibody conjugate) Acute myelogenous leukemia Novopharm Biotech, Inc. Monopharm-C Colon, lung, pancreatic Novopharm Biotech, Inc. 4B5 anti-idiotype Ab Melanoma, small-cell lung Center of Molecular Immunology ior egf/r3 (anti EGF-R humanized Ab) Radioimmunotherapy Center of Molecular Immunology ior c5 (murine MAb colorectal) for Colorectal radioimmunotherapy Creative BioMolecules/ BABS (biosynthetic antibody binding site) Breast cancer Chiron Proteins ImClone Systems/Chugai FLK-2 (monoclonal antibody to fetal liver Tumor-associated kinase-2 (FLK-2)) angiogenesis ImmunoGen, Inc. Humanized MAb/small-drug conjugate Small-cell lung Medarex, Inc. MDX-260 bispecific, targets GD-2 Melanoma, glioma, neuroblastoma Procyon Biopharma, Inc. ANA Ab Cancer Protein Design Labs SMART 1D10 Ab B-cell lymphoma Protein Design Labs/Novartis SMART ABL 364 Ab Breast, lung, colon Immunomedics, Inc. ImmuRAIT-CEA Colorectal
[0125] The invention is further based, in part, on the surprising discovery that administration of an imidazoquinoline agent and a therapeutic agent has unexpected benefit over the administration of either compound alone. Of particular importance is the use of immunostimulatory nucleic acids, C8-substituted guanosines, antigens, and disorder specific medicaments as therapeutic agents. In one important embodiment, compositions comprising imidazoquinoline agents, immunostimulatory nucleic acids, antigen and a polymer rich in arginine (e.g., poly-arginine), and optionally C8-substituted guanosine are used in the immunomodulatory methods of the invention.
[0126] The imidazoquinoline agents are also useful for redirecting an immune response to a Th1 immune response. Redirection of an immune response to a Th1 immune response can be assessed by measuring the levels of cytokines produced in response to the nucleic acid (e.g., by inducing monocytic cells and other cells to produce Th1 cytokines, including IL-12, IFN-alpha and GM-CSF). The redirection or rebalance of the immune response to a Th1 response is particularly useful for the treatment or prevention of asthma. For instance, an effective amount for treating asthma can be that amount useful for redirecting a Th2 type of immune response that is associated with asthma to a Th1 type of response. 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 isotype switching, eosinophil chemotaxis and activation and mast cell growth. Th1 cytokines, especially IFN-alpha and IL-12, can suppress the formation of Th2 clones and production of Th2 cytokines. The imidazoquinoline agents of the invention cause an increase in Th1 cytokines which helps to rebalance the immune system, preventing or reducing the adverse effects associated with a predominately Th2 immune response. The redirection of a Th2 to a Th1 immune response may result in a balanced expression of Th1 and Th2 cytokines or it may result in the induction of more Th1 cytokines than Th2 cytokines.
[0127] The invention also includes a method for inducing antigen non-specific innate immune activation and broad spectrum resistance to infectious challenge using the imidazoquinoline agents. The term antigen non-specific innate immune activation as used herein refers to the activation of immune cells other than B cells and for instance can include the activation of NK cells, T cells or other immune cells that can respond in an antigen independent fashion or some combination of these cells. A broad spectrum resistance to infectious challenge is induced because the immune cells are in active form and are primed to respond to any invading compound or microorganism. The cells do not have to be specifically primed against a particular antigen. This is particularly useful in biowarfare, and the other circumstances described above such as travelers.
[0128] The stimulation index of a particular imidazoquinoline agent can be tested in various immune cell assays. Preferably, the stimulation index of the imidazoquinoline agent with regard to B cell proliferation is at least about 5, preferably at least about 10, more preferably at least about 15 and most preferably at least about 20 as determined by incorporation of
[0129] Currently, some treatment protocols for certain disorders (e.g., cancer) call for the use of IFN-alpha. In one embodiment, the methods of the invention use imidazoquinoline agents as a replacement to the use of alpha-interferon (IFN-alpha) therapy in the treatment of certain disorders. Imidazoquinoline agents can be used to generate IFN-alpha endogenously. In yet other embodiments, the imidazoquinoline agents may be administered along with IFN-alpha. In some embodiments, the targeting agent of the invention or a disorder-specific medicament can also be administered to the subject along with the imidazoquinoline agent and IFN-alpha.
[0130] The invention embraces the administration of C8-substituted guanosines either in place of or along with the imidazoquinoline agents in the methods of the invention. C8-substituted guanosines are known to activate both natural killer (NK) cells and macrophages. Guanine ribonucleotides substituted at the C8 position with either a bromine or a thiol group are B cell mitogens and may act as B cell differentiation factors. (Feldbush et al. 1985
[0131] Certain methods and compositions of the invention comprise the administration or addition of poly-arginine. As used herein, poly-arginine is a homogenous polymer of arginine monomers. Poly-arginine may be of varying length, and may have a peptide backbone but is not so limited. In other embodiments, a polymer rich in arginine can also be used in place of the homogenous polymer of arginine. A polymer rich in arginine can be a polymer that has at least 2 contiguous arginines, at least 3 contiguous arginines, at least 4 contiguous arginines, and at least 5 contiguous arginines, or alternatively it may be a polymer in which at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of its monomers are arginine residues. It is to be understood, accordingly, that poly-arginine is also a polymer rich in arginine. Because of the positive charge of arginine, polymers rich in arginine (including poly-arginine) serve to neutralize the negative charge associated with some imidazoquinoline agents and the immunostimulatory nucleic acids.
[0132] An “immunostimulatory nucleic acid” as used herein is any nucleic acid containing an immunostimulatory motif or backbone that induces an immune response. The immune response may be characterized as, but is not limited to, a Th1-type immune response or a Th2-type immune response. Such immune responses are defined by cytokine and antibody production profiles which are elicited by the activated immune cells. In one preferred embodiment, pan activating immunostimulatory nucleic acids such as #2006 (TCG TCG TTT TGT CGT TTT GTC GTT) are used in combination with the imidazoquinoline agents in the methods of the invention.
[0133] Helper (CD4
[0134] The terms “nucleic acid” and “oligonucleotide” are used interchangeably to mean multiple nucleotides (i.e. molecules 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), thymidine (T) or uracil (U)) or a substituted purine (e.g. adenine (A) or guanine (G)). As used herein, the terms refer to oligoribonucleotides as well as oligodeoxyribonucleotides. The terms shall also include polynucleosides (i.e. a polynucleotide minus the phosphate) and any other organic base containing polymer. Nucleic acid molecules can be obtained from existing nucleic acid sources (e.g., genomic or cDNA), but are preferably synthetic (e.g. produced by nucleic acid synthesis).
[0135] Immunostimulatory nucleic acids may possess immunostimulatory motifs such as CpG, poly-G, poly-T, TG, methylated CpG, CpI, and T-rich motifs. In some embodiments of the invention, any nucleic acid, regardless of whether it possesses an identifiable motif, can be used in the combination therapy to modulate an immune response. Immunostimulatory backbones include, but are not limited to, phosphate modified backbones, such as phosphorothioate backbones. Immunostimulatory nucleic acids have been described extensively in the prior art and a brief summary of these nucleic acids is presented below.
[0136] In some embodiments, a CpG immunostimulatory nucleic acid is used in the methods of the invention. A CpG immunostimulatory nucleic acid is a nucleic acid which contains a CG dinucleotide, the C residue of which is unmethylated. The effects of CpG nucleic acids on immune modulation have been described extensively in U.S. Patent such as U.S. Pat. No. 6,194,388 B1, U.S. Pat. No. 6,207,646 B1, U.S. Pat. No. 6,239,116 B1 and U.S. Pat. No. 6,218,371 B1, and published patent applications, such as PCT/US98/03678, PCT/US98/10408, PCT/US98/04703, and PCT/US99/09863. The entire contents of each of these patents and patent applications is hereby incorporated by reference.
[0137] The terms CpG nucleic acid or CpG oligonucleotide as used herein refer to an immunostimulatory CpG nucleic acid unless otherwise indicated. The entire immunostimulatory nucleic acid can be unmethylated or portions may be unmethylated but at least the C of the 5′ CG 3′ must be unmethylated.
[0138] The CpG nucleic acid sequences of the invention include those broadly described above as well as disclosed in issued U.S. Pat. Nos. 6,207,646 B1 and 6,239,116 B1.
[0139] In other embodiments of the invention, a non-CpG immunostimulatory nucleic acid is used. A non-CpG immunostimulatory nucleic acid is a nucleic acid which either does not have a CpG motif in its sequence, or has a CpG motif which contains a methylated C residue. In some instances, chimeric oligonucleotides which lack a CpG motif are immunostimulatory and have many of the same prophylactic and therapeutic activities as a CpG oligonucleotide. Non-CpG immunostimulatory nucleic acids may induce Th1 or Th2 immune responses, depending upon their sequence, their mode of delivery and the dose at which they are administered.
[0140] Other immunostimulatory nucleic acids that are useful in the invention as targeting agents are Py-rich nucleic acids. Py-rich nucleic acids have similar immune stimulatory properties to CpG oligonucleotides regardless of whether a CpG motif is present. A Py-rich nucleic acid is a T-rich or C-rich immunostimulatory nucleic acid.
[0141] An important subset of non-CpG immunostimulatory nucleic acids are T-rich immunostimulatory nucleic acids. The T-rich immunostimulatory nucleic acids of the invention include those disclosed in published PCT patent application PCT/US00/26383, the entire contents of which are incorporated herein by reference. In some embodiments, T-rich nucleic acids 24 bases in length are used. A T-rich nucleic acid is a nucleic acid which includes at least one poly T sequence and/or which has a nucleotide composition of greater than 25% T nucleotide residues. A nucleic acid having a poly-T sequence includes at least four Ts in a row, such as 5′TTTT3′. Preferably the T-rich nucleic acid includes more than one poly T sequence. In preferred embodiments the T-rich nucleic acid may have 2, 3, 4, etc poly T sequences, such as oligonucleotide #2006 (TCG TCG TTT TGT CGT TTT GTC GTT) (SEQ ID NO:1). One of the most highly immunostimulatory T-rich oligonucleotides discovered according to the invention is a nucleic acid composed entirely of T nucleotide residues, e.g., oligonucleotide #2183 (TTT TTT TTT TTT TTT TTT TTT TTT) (SEQ ID NO:2). Other T-rich nucleic acids according to the invention have a nucleotide composition of greater than 25% T nucleotide residues, but do not necessarily include a poly T sequence. In these T-rich nucleic acids the T nucleotide resides may be separated from one another by other types of nucleotide residues, i.e., G, C, and A. In some embodiments, the T-rich nucleic acids have a nucleotide composition of greater than 35%, 40%, 50%, 60%, 70%, 80%, 90%, and 99%, T nucleotide residues and every integer % in between. Preferably the T-rich nucleic acids have at least one poly T sequence and a nucleotide composition of greater than 25% T nucleotide residues.
[0142] A C-rich nucleic acid is a nucleic acid molecule having at least one or preferably at least two poly-C regions or which is composed of at least 50% C nucleotides. A poly-C region is at least four C residues in a row. Thus a poly-C region is encompassed by the formula 5′CCCC 3′. In some embodiments it is preferred that the poly-C region have the formula 5′CCCCCC 3′. Other C-rich nucleic acids according to the invention have a nucleotide composition of greater than 50% C nucleotide residues, but do not necessarily include a poly C sequence. In these C-rich nucleic acids the C nucleotide residues may be separated from one another by other types of nucleotide residues, i.e., G, T, and A. In some embodiments the C-rich nucleic acids have a nucleotide composition of greater than 60%, 70%, 80%, 90%, and 99%, C nucleotide residues and every integer % in between. Preferably the C-rich nucleic acids have at least one poly C sequence and a nucleotide composition of greater than 50% C nucleotide residues, and in some embodiments are also T-rich.
[0143] TG nucleic acids can also be used in conjunction with the imidazoquinoline agents of the invention for modulating the immune system. Suitable TG nucleic acids are described in published PCT patent application PCT/US00/26383. A “TG nucleic acid” as used herein is a nucleic acid containing at least one TpG dinucleotide (thymidine-guanine dinucleotide sequence, i.e. “TG DNA” or DNA containing a 5′ thymidine followed by 3′ guanosine and linked by a phosphate bond) and activates a component of the immune system.
[0144] It has been shown that TG nucleic acids ranging in length from 15 to 25 nucleotides in length can exhibit an increased immune stimulation. Thus, in one aspect, the invention provides an oligonucleotide that is 15-27 nucleotides in length (i.e., an oligonucleotide that is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27 nucleotides in length) that may be a T-rich nucleic acid or may be a TG nucleic acid, or may be both a T-rich and a TG nucleic acid. Preferably, the TG oligonucleotides range in size from 15 to 25 nucleotides.
[0145] Another important subset of non-CpG immunostimulatory nucleic acids are poly-G immunostimulatory nucleic acids. A variety of references, including Pisetsky and Reich, 1993
[0146] Poly-G nucleic acids preferably are nucleic acids having the following formulas:
[0147] wherein X
[0148] The immunostimulatory nucleic acids of the invention can also be those which do not possess CpG, poly-G, or T-rich motifs.
[0149] Addition of a poly-A tail to an immunostimulatory nucleic acid can enhance the activity of the nucleic acid. It was discovered that when a highly immunostimulatory CpG nucleic acid (TCG TCG TTT TGT CGT TTT GTC GTT) (SEQ ID NO:1) was modified with the addition of a poly-A tail (AAAAAA) or a poly-T tail (TTTTTT), the resultant oligonucleotides increased in immune stimulatory activity. The ability of the poly-A tail and the poly-T tail to increase the immunostimulating properties of the oligonucleotide was very similar. The highly immunostimulatory CpG nucleic acid described above is a T-rich oligonucleotide. It is likely that if poly-A and poly-T tails are added to a nucleic acid which is not T-rich, it would have a more significant impact on the immunostimulating capability of the nucleic acid. Since the poly-T tail was added to a nucleic acid that was already highly T-rich the immune stimulating properties of the poly-T addition was diluted somewhat, although not completely. This finding has important implications for the use of poly-A regions. Thus in some embodiments the immunostimulatory nucleic acids include a poly-A region and in other embodiments they do not.
[0150] Exemplary immunostimulatory nucleic acid sequences include but are not limited to those immunostimulatory sequences described and listed in U.S. Non-Provisional patent application Ser. No. 09/669,187, filed on Sep. 25, 2000, and in corresponding published PCT patent application PCT/US00/26383.
[0151] The immunostimulatory 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. In certain embodiments, while the nucleic acid is single stranded, it is capable of forming secondary and tertiary structures (e.g., by folding back on itself, or by hybridizing with itself either throughout its entirety or at select segments along its length). Accordingly, while the primary structure of such a nucleic acid may be single stranded, its higher order structures may be double or triple stranded.
[0152] 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 immunostimulatory nucleic acid is in the range of between 8 and 100 and in some embodiments between 8 and 50 or 8 and 30 nucleotides in size.
[0153] Nucleic acids having modified backbones, such as phosphorothioate backbones, also fall within the class of immunostimulatory nucleic acids. U.S. Pat. Nos. 5,723,335 and 5,663,153 issued to Hutcherson, et al. and related PCT publication WO95/26204 describe immune stimulation using phosphorothioate oligonucleotide analogues. These patents describe the ability of the phosphorothioate backbone to stimulate an immune response in a non-sequence specific manner.
[0154] In the case when the immunostimulatory nucleic acid is administered in conjunction with a nucleic acid vector, such as a vector encoding an antigen, it is preferred that the backbone of the immunostimulatory nucleic acid be a chimeric combination of phosphodiester and phosphorothioate (or other phosphate modification). This is because the uptake of the plasmid vector by the cell may be hindered by 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 or phosphorothioate and that the plasmid be 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.
[0155] The terms nucleic acid and oligonucleotide also encompass nucleic acids or oligonucleotides with substitutions or modifications, such as in the bases and/or sugars. For example, they include nucleic acids having backbone sugars which are covalently attached to low molecular weight organic groups other than a hydroxyl group at the 3′ position and other than a phosphate group at the 5′ position. Thus modified nucleic acids may include a 2′-O-alkylated ribose group. In addition, modified nucleic acids may include sugars such as arabinose instead of ribose. Thus the nucleic acids may be heterogeneous in backbone composition thereby containing any possible combination of polymer units linked together such as peptide nucleic acids (which have amino acid backbone with nucleic acid bases). In some embodiments, the nucleic acids are homogeneous in backbone composition. Nucleic acids also include substituted purines and pyrimidines such as C-5 propyne modified bases (Wagner et al.,
[0156] For use in the instant invention, the nucleic acids of the invention can be synthesized de novo using any of a number of procedures well known in the art. For example, the beta-cyanoethyl phosphoramidite method (Beaucage, S. L., and Caruthers, M. H.,
[0157] For use in vivo, the nucleic acids may optionally be 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 endo-nuclease). Stabilization can be a function of length or secondary structure. Nucleic acids that are tens to hundreds of kbs long are relatively resistant to in vivo degradation. For shorter nucleic acids, secondary structure can stabilize and increase their effect. For example, if the 3′ end of an 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.
[0158] Alternatively, nucleic acid stabilization can be accomplished via phosphate 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 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 the 5′ end of the oligonucleotide and multiple (preferably five) phosphorothioate linkages at the 3′ end, can in some circumstances provide maximal activity and protect the nucleic acid from degradation by intracellular exo- and endonucleases. Other modified nucleic acids include phosphodiester-modified nucleic acids, combinations of phosphodiester and phosphorothioate nucleic acids, alkylphosphonate and arylphosphonate, alkylphosphorothioate and arylphosphorothioate, methylphosphonate, methylphosphorothioate, phosphorodithioate, p-ethoxy, morpholino, and combinations thereof. Nucleic acids having phosphorothioate linkages provide maximal activity and protect the nucleic acid from degradation by intracellular exo- and endo-nucleases. and combinations thereof. Each of these combinations and their particular effects on immune cells is discussed in more detail with respect to CpG nucleic acids in issued U.S. Pat. Nos. 6,207,646 B1 and 6,239,116 B1, the entire contents of which are hereby incorporated by reference. It is believed that these modified nucleic acids may show more stimulatory activity due to enhanced nuclease resistance, increased cellular uptake, increased protein binding, and/or altered intracellular localization.
[0159] The compositions of the invention may optionally be chimeric oligonucleotides. The chimeric oligonucleotides are oligonucleotides having a formula: 5′ Y
[0160] The center nucleotides (N
[0161] Modified backbones such as phosphorothioates may be synthesized using automated techniques employing either phosphoramidate or H-phosphonate chemistries. 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.,
[0162] 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), phosphodiester 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.
[0163] 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 than phosphodiester backbone immunostimulatory nucleic acids. For example, 2 μg/ml of the phosphorothioate has been shown to effect the same immune stimulation as a 90 μg/ml of the phosphodiester.
[0164] Another type of modified backbone, useful according to the invention, is a peptide nucleic acid. The backbone is composed of aminoethylglycine and supports bases which provide the DNA character. The backbone does not include any phosphate and thus may optionally have no net charge. The lack of charge allows for stronger DNA-DNA binding because the charge repulsion between the two strands does not exist. Additionally, because the backbone has an extra methylene group, the oligonucleotides are enzyme/protease resistant. Peptide nucleic acids can be purchased from various commercial sources, e.g., Perkin Elmer, or synthesized de novo.
[0165] 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 et al.,
[0166] 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, thymidine, uracil, and inosine. Other representative heterocyclic bases are disclosed in U.S. Pat. No. 3,687,808, issued to Merigan, et al. The terms “purines” or “pyrimidines” or “bases” are used herein to refer to both naturally-occurring or synthetic purines, pyrimidines or bases.
[0167] The immunostimulatory nucleic acids having backbone modifications useful according to the invention in some embodiments are S- or R-chiral immunostimulatory nucleic acids. An “S chiral immunostimulatory nucleic acid” as used herein is an immunostimulatory nucleic acid wherein at least two nucleotides have a backbone modification forming a chiral center and wherein a plurality of the chiral centers have S chirality. An “R chiral immunostimulatory nucleic acid” as used herein is an immunostimulatory nucleic acid wherein at least two nucleotides have a backbone modification forming a chiral center and wherein a plurality of the chiral centers have R chirality. The backbone modification may be any type of modification that forms a chiral center. The modifications include but are not limited to phosphorothioate, methylphosphonate, methylphosphorothioate, phosphorodithioate, 2′-Ome and combinations thereof.
[0168] The chiral immunostimulatory nucleic acids must have at least two nucleotides within the nucleic acid that have a backbone modification. All or less than all of the nucleotides in the nucleic acid, however, may have a modified backbone. Of the nucleotides having a modified backbone (referred to as chiral centers), a plurality have a single chirality, S or R. A “plurality” as used herein refers to an amount greater than 75%. Thus, less than all of the chiral centers may have S or R chirality as long as a plurality of the chiral centers have S or R chirality. In some embodiments at least 80,%, 85%, 90%, 95%, or 100% of the chiral centers have S or R chirality. In other embodiments at least 80%, 85%, 90%, 95%, or 100% of the nucleotides have backbone modifications.
[0169] The S- and R- chiral immunostimulatory nucleic acids may be prepared by any method known in the art for producing chirally pure oligonucleotides. Stec et al teach methods for producing stereopure phosphorothioate oligodeoxynucleotides using an oxathiaphospholane. Stec W J et al. (1995)
[0170] One or more immunostimulatory nucleic acids which may or may not differ in terms of their profile, sequence, backbone modifications and biological effect may be administered to a subject. As an example, CpG nucleic acids and T-rich nucleic acids may be administered to a single subject along with an imidazoquinoline agent. In another example, a plurality of CpG nucleic acids which differ in nucleotide sequence may also be administered to a subject along with the imidazoquinoline agent.
[0171] The immunostimulatory nucleic acids may be delivered to the subject in the form of a plasmid vector. In some embodiments, one plasmid vector could include both the immunostimulatory nucleic acid and a nucleic acid encoding a disorder-specific medicament and/or an antigen if either can be encoded by a nucleic acid. In still other embodiments, the plasmid may encode proteins or polypeptides involved in the stimulation or regulation of an immune response such as IFN-alpha, CD80, and the like. The immunostimulatory nucleic acid may be present in the coding sequences of the plasmid, however, their location is not so limited. In other embodiments, separate plasmids could be used. In yet other embodiments, no plasmids could be used.
[0172] The therapeutic agents described herein including imidazoquinoline agents, antigens, immunostimulatory nucleic acids, antibodies, C8-substituted guanosines, as well as the polymers rich in arginine can be physically combined without the need for covalent bonding between their substituents when used in the methods of the invention. Alternatively, they may also be conjugated in various combinations either directly or indirectly using linking molecules, as described below.
[0173] Examples of suitable linking molecules which can be used include bifunctional crosslinker molecules. The bifunctional crosslinker molecules may be homobifunctional or heterobifunctional, depending upon the nature of the molecules to be conjugated. Homobifunctional crosslinkers have two identical reactive groups. Heterobifunctional crosslinkers are defined as having two different reactive groups that allow for sequential conjugation reaction. Various types of commercially available crosslinkers are reactive with one or more of the following groups: primary amines, secondary amines, sulphydryls, carboxyls, carbonyls and carbohydrates. Examples of amine-specific crosslinkers are bis(sulfosuccinimidyl) suberate, bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone, disuccinimidyl suberate, disuccinimidyl tartarate, dimethyl adipimate.2 HCl, dimethyl pimelimidate.2 HCl, dimethyl suberimidate.2 HCl, and ethylene glycolbis-[succinimidyl-[succinate]]. Crosslinkers reactive with sulfhydryl groups include bismaleimidohexane, 1,4-di-[3′-(2′-pyridyldithio)-propionamido)]butane, 1-[p-azidosalicylamido]-4-[iodoacetamido]butane, and N-[4-(p-azidosalicylamido) butyl]-3′-[2′-pyridyldithio]propionamide. Cr