WO2014201245A1 - Agoniste de tlr-9 comportant un agoniste de tlr-7 et/ou de tlr-8 pour traiter des tumeurs - Google Patents

Agoniste de tlr-9 comportant un agoniste de tlr-7 et/ou de tlr-8 pour traiter des tumeurs Download PDF

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WO2014201245A1
WO2014201245A1 PCT/US2014/042117 US2014042117W WO2014201245A1 WO 2014201245 A1 WO2014201245 A1 WO 2014201245A1 US 2014042117 W US2014042117 W US 2014042117W WO 2014201245 A1 WO2014201245 A1 WO 2014201245A1
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tumor
cpg
seq
cpg odn
group
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PCT/US2014/042117
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Dennis M. Klinman
Gan Zhao
John Vasilakos
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The United States Of America, As Represented By The Secretary, Department Of Health And Human Services
3M Innovative Properties Company
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7125—Nucleic acids or oligonucleotides having modified internucleoside linkage, i.e. other than 3'-5' phosphodiesters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants

Definitions

  • TLR-9 AGONIST WITH TLR-7 AND/OR TLR-8 AGONIST FOR TREATING TUMORS
  • This relates to the field of tumor biology, specifically to methods for treating tumors that include the use of a Toll-like receptor (TLR)-9 agonist and a TLR 7/TLR-8 agonist, such as with an imidazoquinoline compound and a CpG oligodeoxynucleotide.
  • TLR Toll-like receptor
  • TLR 7/TLR-8 agonist such as with an imidazoquinoline compound and a CpG oligodeoxynucleotide.
  • TLR-7 and TLR-8 are structurally related members of the TLR family. At least 13 different TLRs have been identified in mammals, with TLRs 7, 8, and 9 being similar in their recognition of nucleic acid motifs and expression within endosomal compartments (Janeway and Medzhitov, Annu Rev Immunol 20: 197-216.: 197-216, 2002; Hemmi et al., Nature 408:740-745, 2000; Akira et al., Cell 124:783-801, 2006).
  • TLR-7 and TLR-8 have anti-tumor activity.
  • the lead compound of this family is imiquimod, that is marketed as a topical agent.
  • the imidazoquinoline compounds are used alone or in combination with other therapies (such as anti-virals, anti-bacterials, other immune modulators or in therapeutic vaccine antigens) for treatment of chronic infections such as those caused by the human immunodeficiency virus (HIV), the hepatitis C virus (HCV), the hepatitis B virus (HBV), the herpes simplex virus (HSV), and H. pylori.
  • HCV human immunodeficiency virus
  • HCV hepatitis C virus
  • HBV hepatitis B virus
  • H. pylori herpes simplex virus
  • Imiquimod (R837, l-(2- methylpropyl)-l H-imidazo[4,5-c]quinolin-4-amine], and Resiquimod (R848, 4-amino-a,oc- dimethyl-2-ethoxymethyl-lH-imidazo[4,5-c]quinoline-l-ethanol) are selective ligands for TLR-7 and TLR-8, and are also used to treat many types of skin cancer and cutaneous metastases.
  • Cutaneous tumors that have responded well to topical treatment with imiquimod include basal cell carcinomas, keratoacanthomas, actinic keratosis, Bowen's disease, melanoma and cutaneous T-cell lymphomas (reviewed in Schon and Schon, Oncogene 27: 190-9, 2008).
  • Cancer is the second leading cause of human death next to coronary disease in the United States. Worldwide, millions of people die from cancer every year. In the United States alone, as reported by the American Cancer Society, cancer causes the death of well over a half-million people annually, with over 1.2 million new cases diagnosed per year. Death from cancer is increasing; cancer is soon predicted to become the leading cause of death in the United States. A need remains for therapeutic agents, such as combination therapies, that are effective for the treatment of cancer.
  • Methods for treating a tumor are disclosed herein.
  • These methods include administering to the subject a therapeutically effective amount of a TLR-9 agonist, such as a CpG ODN, and a therapeutically effective amount of a TLR-7 and/or TLR-8 agonist, such as an imidazoquinoline compound, for example a lipophilic imidazoquinoline compound (for example, wherein the imidazoquinoline compound comprises an aliphatic tail having from 1 to 23 carbon atoms, such as 11-23 carbon atoms, 15-23 carbon atoms, or 15-19 carbon atoms).
  • the CpG ODN and the imidazoquinoline compound for example a lipophilic imidazoquinoline compound, can be administered intratumorally. In some embodiments, these compounds act synergistically to treat the tumor in the subject.
  • methods for treating a subject with a tumor that include administering to the subject a therapeutically effective amount of a imidazoquinoline compound and an immunostimutlatory CpG oligodeoxynucleotide.
  • the methods can include administering to the subject a therapeutically effective amount of a imidazoquinoline compound having a formula:
  • R 1 is selected from hydrogen, C12-24 alkyl, Cn -24 heteroalkyl, substituted Ci-ioheteroalkyl, carboxyl, and hydroxyl
  • R 2 is selected from hydrogen, Ci-io alkyl, and substituted Ci-io alkyl
  • R 3 is selected from hydrogen, amine, substituted amine, hydroxyl, and Ci-10 alkoxy.
  • the method includes administering a K-type CpG ODN, a D-type CpG ODN, and/or a C-type CpG ODN.
  • one or more K-type CpG ODNs is administered to the subject.
  • the imidazoquinoline compound is N-(4- ⁇ [4-amino-2-butyl-lH-imidazo[4,5-c]quinolin-l-yl]oxy ⁇ butyl) octadecanamide, also known as 3M-052.
  • the CpG ODN is a K-type CpG ODN.
  • Fig. 1 A set of line graphs showing the effect of TLR agonists on the growth of small tumors. 10 5 CT26 colon carcinoma cells were implanted into the flank of syngeneic BALB/c mice. When the tumors reached -200 mm 3 in volume they were injected with 100 ⁇ g of CpG or control ODN and/or 50 ⁇ g of 3M-052 or 3M control. Each treatment was repeated 2 days later. Data show the change in tumor volume (mean + SE) of 6-8 mice from 2 independent experiments. *, p ⁇ 0.05; **, p ⁇ 0.01 compared with the control group
  • Fig. 2 A set of bar graphs showing the effect of TLR agonists on the frequency of tumor infiltrating MDSC, NK and CD8 T cells. Mice were treated as described in Fig. 1. The frequency of tumor-infiltrating MDSC, NK and CD8 + T cells was determined one day after the second treatment. Results show the mean + SD of each cell type as a percentage of total CD45 + tumor infiltrating cells analyzed independently in 6 mice from 2 independent experiments. *, p ⁇ 0.05; **, p ⁇ 0.01
  • Fig. 3 A set of bar graphs showing the effect of TLR agonists on tumor- specific CTL.
  • CT26 tumors were implanted into BALB/c mice as described in Fig 1.
  • Spleen cells and tumor infiltrating lymphocytes were isolated one day after the second treatment, stimulated ex vivo with AH-1 peptide, and monitored for IFNy secretion by ELIspot assay.
  • Results represent the mean + SD of 6 mice from 2 independent experiments. *, p ⁇ 0.05; **, p ⁇ 0.01
  • FIG. 4 A line graph showing the effect of depleting CD8 T cells on immune mediated protection.
  • CT26 tumors were implanted into BALB/c mice and treated with 100 ⁇ g of CpG and 50 ⁇ g of 3M as described in Fig 1.
  • the effect of depleting CD4 or CD8 T cells on tumor growth was determined as described in the methods section.
  • Data show the change in tumor volume (mean + SE) of 5 mice/group. *, p ⁇ 0.05; **, p ⁇ 0.01 compared with the control group Fig. 5.
  • Fig. 6 A set of line graphs showing the effect of TLR agonists on large established B 16- F10 tumors. 10 5 B 16-F10 melanoma cancer cells were implanted into the flank of syngeneic C57B1/6 mice. When the tumors reached -500 mm 3 in volume they were injected with 200 ⁇ g of CpG or control ODN and/or 100 ⁇ g of 3M-052 or 3M control twice weekly for one month. The change in tumor volume of 8 mice/group is shown (mean + SE).
  • Figs. 7A-7B TLR agonist therapy induces persistent immunity. 10 5 CT26 cells were implanted into the flank of syngeneic BALB/c mice. Large established tumors (-800 mm 3 in volume) were treated as described in Fig 5. Fig. 7A) Cells from the tumor draining LN were isolated one day after the third treatment, stimulated ex vivo with AH- 1 peptide, and monitored for IFNy secretion by ELIspot assay. Results represent the mean + SD of four independently studied mice/group. Fig.
  • Fig. 8 A line graph showing the in vivo persistence of 3M-052. Serum levels of 3M-05 and Resiquimod were measured at multiple time points after subcutaneous administration of 1 mg/kg of each agent. Blood was collected before and at various time post-delivery. % maximal serum concentration was calculated by the formula: serum level / maximum serum level X 100%. Results represent the mean of 5 independently studied animals/group.
  • Fig. 9. A set of plots showing the gating strategy used to identify the immune cells. Single cell suspensions were prepared as described in the methods section. Live cells isolated by density gradient centrifugation were stained and analyzed using an LSR-II flow cytometer. The gates used to identify specific cell subpopulations are shown.
  • Fig. 10. A bar graph showing that TLR agonist therapy does not affect T reg frequency. Mice were treated as described in Fig. 1. The frequency of tumor- infiltrating T reg was determined one day after the second treatment by staining for Foxp3 + cells. Results show the mean + SD of as a percentage of total CD45 + tumor infiltrating cells analyzed independently in 6 mice from 2 independent experiments.
  • Figs. 11A-11B A set of survival plots showing the effect of TLR agonists on large established tumors. Survival curves are provided for mice challenged with CT26 colon cancer cells (A) or B16-F10 melanoma cancer cells (B) and treated with 200 ⁇ g of CpG or control ODN and/or 100 ⁇ g of 3M-052 or 3M control twice weekly for one month as described in Figs 4, 5. **; p ⁇ .01 vs all 3 control groups.
  • nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand.
  • sequence Listing is submitted as an ASCII text file [4239-90644-02_Sequence_Listing.txt, June 12, 2014, 15.2 KB], which is incorporated by reference herein.
  • SEQ ID NO: 1 is a D-type CpG oligodeoxynucleotide (ODN).
  • SEQ ID Nos: 2-34 are K-type CpG ODNs.
  • SEQ ID Nos: 35-36 are control ODNs.
  • SEQ ID Nos: 37-63 are D-type CpG ODN.
  • Methods for treating a tumor are disclosed herein.
  • the methods are also of use to decrease the size and/or number of metastases.
  • the methods include administering to the subject a therapeutically effective amount of a TLR-9 agonist, such as a CpG ODN, and a therapeutically effective amount of a TLR-7 and/or TLR-8 agonist, such as an imidazoquinoline compound, such as a lipophilic imidazoquinoline compound.
  • a TLR-9 agonist such as a CpG ODN
  • a TLR-7 and/or TLR-8 agonist such as an imidazoquinoline compound, such as a lipophilic imidazoquinoline compound.
  • methods for treating a subject with a tumor that include administering to the subject a therapeutically effective amount of an imidazoquinoline compound and an
  • the methods include administering one or more K-type and/or D-type CpG oligodeoxynucleotides (ODNs).
  • ODNs D-type CpG oligodeoxynucleotides
  • the imidazoquinoline compound can be N-(4- ⁇ [4-amino-2-butyl-lH- imidazo[4,5-c]quinolin-l-yl]oxy ⁇ butyl) octadecanamide, also known as 3M-052.
  • the CpG ODN is a K-type CpG ODN.
  • the imidazoquinoline compound and an immunostimutlatory CpG oligodeoxynucleotide are administered intratumorally.
  • Alkyl A saturated or unsaturated monovalent hydrocarbon radical having a number of carbon atoms ranging from one to 30 (e.g., Ci-30 alkyl), which is derived from removing one hydrogen atom from one carbon atom of a parent compound (e.g., alkane, alkene, alkyne).
  • An alkyl group may be branched, straight-chain, or cyclic.
  • Alkenyl An unsaturated monovalent hydrocarbon radical having a number of carbon atoms ranging from two to 30 (e.g., C2-30 alkenyl), which has at least one carbon-carbon double bond and is derived from removing one hydrogen atom from one carbon atom of a parent alkene.
  • An alkenyl group may be branched, straight-chain, cyclic, cis, or trans.
  • Alkynyl A unsaturated monovalent hydrocarbon radical having a number of carbon atoms ranging from two to 30 (e.g., C2-30 alkynyl), which has at least one carbon-carbon triple bond and is derived from removing one hydrogen atom from one carbon atom of a parent alkyne.
  • An alkynyl group may be branched, straight-chain, or cyclic.
  • Animal Living multi-cellular vertebrate organisms, a category that includes, for example, mammals and birds.
  • mammal includes both human and non-human mammals.
  • subject includes both human and veterinary subjects.
  • C class oligodeoxynucleotides ODNs that resemble K ODNs and are composed of only phosphorothiote nucleotides.
  • C class ODNs have a TCGTCG motif at the 5' end and have a CpG motif imbedded in a palindromic sequence.
  • Backbone modifications like 2'-0-methyl modifications especially in the 5' part of the ODN influence IFN-alpha-producing capacity of these ODN.
  • C class ODNs have combined properties of D- and K-type CpG ODNs.
  • This class of ODNs stimulates B cells to secrete IL-6 and stimulates plasmacytoid dendritic cells to produce interferon- ⁇ .
  • C class ODNs also induce IP- 10 production and strong NK activation.
  • CpG or CpG motif A nucleic acid having a cytosine followed by a guanine linked by a phosphate bond in which the pyrimidine ring of the cytosine is unmethylated.
  • methylated CpG refers to the methylation of the cytosine on the pyrimidine ring, usually occurring at the 5-position of the pyrimidine ring.
  • a CpG motif is a pattern of bases that include an unmethylated central CpG surrounded by at least one base flanking (on the 3' and the 5' side of) the central CpG. Without being bound by theory, the bases flanking the CpG confer a significant part of the activity to the CpG oligodeoxynucleotide.
  • a CpG oligodeoxynucleotide is an
  • oligodeoxynucleotide that is at least about ten nucleotides in length and includes an unmethylated CpG.
  • CpG oligodeoxynucleotides include both D and K-type oligodeoxynucleotides (see below).
  • CpG oligodeoxynucleotides are single-stranded. The entire CpG oligodeoxynucleotide can be unmethylated or portions may be unmethylated. In one embodiment, at least the C of the 5' CG 3' is unmethylated.
  • thyroid cancer is a malignant tumor that arises in or from thyroid tissue
  • breast cancer is a malignant tumor that arises in or from breast tissue (such as a ductal carcinoma).
  • Residual cancer is cancer that remains in a subject after any form of treatment given to the subject to reduce or eradicate the cancer.
  • Metastatic cancer is a tumor at one or more sites in the body other than the site of origin of the original (primary) cancer from which the metastatic cancer is derived. Cancer includes, but is not limited to, solid tumors.
  • Chemotherapy; chemotherapeutic agents any chemical agent with therapeutic usefulness in the treatment of diseases characterized by abnormal cell growth. Such diseases include tumors, neoplasms, and cancer as well as diseases characterized by hyperplastic growth such as psoriasis.
  • a chemotherapeutic agent is an agent of use in treating neoplasms such as solid tumors.
  • a chemotherapeutic agent is radioactive molecule.
  • chemotherapeutic agent of use e.g. see Slapak and Kufe, Principles of Cancer Therapy, Chapter 86 in Harrison's Principles of Internal Medicine, 14th edition; Perry et al., Chemotherapy, Ch.
  • Chemotherapeutic agents include those known by those skilled in the art, including but not limited to: 5-fluorouracil (5-FU), azathioprine, cyclophosphamide, antimetabolites (such as Fludarabine), antineoplastics (such as Etoposide, Doxorubicin, methotrexate, and Vincristine), carboplatin, cis- platinum and the taxanes, such as taxol. Rapamycin has also been used as a chemotherapeutic.
  • Colon cancer Cancers, also called large bowel cancers, that include cancerous growths in the colon, rectum and appendix. With 655,000 deaths worldwide per year, it is the third most common form of cancer and the second leading cause of cancer-related death in the Western world. Many colorectal cancers are thought to arise from adenomatous polyps in the colon. These mushroom-like growths are usually benign, but some may develop into cancer over time. The majority of the time, the diagnosis of localized colon cancer is through colonoscopy. Therapy is usually through surgery, which in many cases is followed by chemotherapy. The first symptoms of colon cancer are usually vague, such as bleeding, weight loss, and fatigue (tiredness). Local (bowel) symptoms are rare until the tumor has grown to a large size. Generally, the nearer the tumor is to the anus, the more bowel symptoms are present.
  • Cytokine Proteins made by cells that affect the behavior of other cells, such as lymphocytes.
  • a cytokine is a chemokine, a molecule that affects cellular trafficking.
  • cytokines are interferon (IFN)y, IL-6, and IL-10.
  • D ODN D-type Oligodeoxynucleotide
  • D-type oligodeoxynucleotides include an unmethylated CpG dinucleotide. Inversion, replacement or methylation of the CpG reduces or abrogates the activity of the D
  • a D-type CpG ODN is at least about 16 nucleotides in length and includes a sequence represented by Formula ⁇ :
  • D ODNs can stimulate a cellular response.
  • D ODNs can be up to 30, 35, 40, 45 or 50 nucleotides in length.
  • D ODNs stimulate natural killer cells and the maturation of dendritic cells.
  • Heteroalkyl/Heteroalkenyl/Heteroalkynyl An alkyl, alkenyl, or alkynyl group, respectively, wherein one or more of the carbon atoms are each independently replaced with the one or more heteroatoms selected from oxygen, sulfur, and nitrogen.
  • Immune response A response of a cell of the immune system, such as a B cell or T cell to a stimulus.
  • the response is specific for a particular antigen (an "antigen- specific response”).
  • a "parameter of an immune response” is any particular measurable aspect of an immune response, including, but not limited to, cytokine secretion (IL-6, IL-10, IFNy, etc.), immunoglobulin production, dendritic cell maturation, and proliferation of a cell of the immune system.
  • cytokine secretion IL-6, IL-10, IFNy, etc.
  • immunoglobulin production IL-6, IL-10, IFNy, etc.
  • dendritic cell maturation a cell of the immune system.
  • a "substantial" increase in a parameter of the immune response is a significant increase in this parameter as compared to a control.
  • Specific, non- limiting examples of a substantial increase are at least about a 50% increase, at least about a 75% increase, at least about a 90% increase, at least about a 100% increase, at least about a 200% increase, at least about a 300% increase, and at least about a 500% increase.
  • One of skill in the art can readily identify a significant increase using known statistical methods.
  • a statistical test used to assess a substantial increase is the use of a Z test to compare the percent of samples that respond to an imidazoquinoline compound and a K-type CpG ODN as compared to the percent of samples that respond using the another type of ODN, such as a D-type CpG ODN, or as compared to the K-type CpG ODN alone (without the imidazoquinoline compound).
  • a non-parametric ANOVA can be used to compare differences in the magnitude of the response induced by a imidazoquinoline compound a K-type CpG ODN as compared to the percent of samples that respond using the K-type CpG ODN alone or the imidazoquinoline compound alone.
  • p ⁇ _0.05 is significant, and indicates a substantial increase in the parameter.
  • One of skill in the art can readily identify other statistical assays of use.
  • Isolated An "isolated" biological component (such as a nucleic acid, peptide or protein) has been substantially separated, produced apart from, or purified away from other biological components in the cell of the organism in which the component naturally occurs, i.e., other chromosomal and extrachromosomal DNA and RNA, and proteins. Nucleic acids, peptides and proteins which have been “isolated” thus include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids, peptides and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids.
  • K ODN An oligodeoxynucleotide including an unmethylated CpG motif that has a sequence represented by the formula:
  • K-type CpG ODNs can stimulate a humoral response.
  • K-type CpG ODNs stimulate the production of immunoglobulins, such as IgM and IgG.
  • K-type CpG ODNs can also stimulate proliferation of peripheral blood mononuclear cells and increase expression of IL-6 and/or IL-12, amongst other activities.
  • ad K-type CpG ODN is up to 30, 35, 40, 45, or 50 nucleotides in length.
  • Mammal This term includes both human and non-human mammals. Similarly, the term “subject” includes both human and veterinary subjects.
  • Melanoma A form of cancer that originates in melanocytes (cells that make the pigment melanin). Melanocytes are found primarily in the skin, but are also present in the bowel and eye. Melanoma in the skin includes superficial spreading melanoma, nodular melanoma, acral lentiginous melanoma, and lentigo maligna (melanoma). Any of the above types may produce melanin or can be amelanotic. Similarly, any subtype may show desmoplasia (dense fibrous reaction with neurotropism) which is a marker of aggressive behavior and a tendency to local recurrence. Other melanomas include clear cell sarcoma, mucosal melanoma, and uveal melanoma.
  • prognosis tumor thickness in millimeters (Breslow's depth), depth related to skin structures (Clark level), type of melanoma, presence of ulceration, presence of lymphatic/perineural invasion, presence of tumor infiltrating lymphocytes (if present, prognosis is better), location of lesion, presence of satellite lesions, and presence of regional or distant metastasis.
  • Nucleic acid A deoxyribonucleotide or ribonucleotide polymer in either single or double stranded form, and unless otherwise limited, encompasses known analogues of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides.
  • Oligonucleotide or "oligo” 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 (Py) (e.g. cytosine (C), thymine (T) or uracil (U)) or a substituted purine (Pu) (e.g. adenine (A) or guanine (G)).
  • oligonucleotide refers to both oligoribonucleotides (ORNs) and oligodeoxynucleotides (ODNs).
  • oligonucleotide also includes oligonucleosides (i.e. an oligonucleotide minus the phosphate) and any other organic base polymer. Oligonucleotides can be obtained from existing nucleic acid sources (e.g. genomic or cDNA), but are preferably synthetic (e.g. produced by oligonucleotide synthesis).
  • a “stabilized oligonucleotide” is an oligonucleotide that is relatively resistant to in vivo degradation (for example via an exo- or endo-nuclease).
  • a stabilized oligonucleotide has a modified phosphate backbone.
  • One specific, non-limiting example of a stabilized oligonucleotide has a phosphorothioate modified phosphate backbone (wherein at least one of the phosphate oxygens is replaced by sulfur).
  • oligonucleotides include: nonionic DNA analogs, such as alkyl- and aryl- phosphonates (in which the charged phosphonate oxygen is replaced by an alkyl or aryl group), phophodiester and alkylphosphotriesters, in which the charged oxygen moiety is alkylated. Oligonucleotides which contain a diol, such as
  • tetraethyleneglycol or hexaethyleneglycol at either or both termini have also been shown to be substantially resistant to nuclease degradation.
  • an “immunostimulatory oligodeoxynucleotide,” “immunostimulatory CpG containing oligodeoxynucleotide,” “CpG ODN,” refers to an oligodeoxynucleotide, which contains a cytosine, guanine dinucleotide sequence and (e.g. has a mitogenic effect or induces cytokine production) vertebrate immune cells.
  • an immunostimulatory CpG ODN stimulates a parameter of an immune response in a subject.
  • the cytosine, guanine is unmethylated.
  • oligonucleotide delivery complex is an oligonucleotide associated with (e.g. ionically or covalently bound to or encapsulated within) a targeting agent (e.g. a molecule that results in a higher affinity binding to a target cell (e.g. B-cell or natural killer (NK) cell) surface and/or increased cellular uptake by target cells).
  • a targeting agent e.g. a molecule that results in a higher affinity binding to a target cell (e.g. B-cell or natural killer (NK) cell) surface and/or increased cellular uptake by target cells.
  • oligonucleotide delivery complexes include oligonucleotides associated with: a sterol (e.g. cholesterol), a lipid (e.g. cationic lipid, virosome or liposome), or a target cell specific binding agent (e.g. a ligand recognized by a target cell specific receptor).
  • Preferred complexes must be sufficiently stable in vivo to prevent significant uncoupling prior to internalization by the target cell.
  • the complex should be cleavable or otherwise accessible under appropriate conditions within the cell so that the oligonucleotide is functional. (Gursel, /. Immunol. 167:3324, 2001).
  • Pharmaceutical agent or drug A chemical compound or composition capable of inducing a desired therapeutic or prophylactic effect when properly administered to a subject.
  • Pharmaceutical agents include, but are not limited to, chemotherapeutic agents and anti-infective agents.
  • Pharmaceutically acceptable carriers The pharmaceutically acceptable carriers useful in the methods and compositions disclosed herein are conventional. Remington 's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 15th Edition (1975), describes compositions and formulations suitable for pharmaceutical delivery of the fusion proteins herein disclosed.
  • parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
  • pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
  • physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like
  • solid compositions ⁇ e.g. , powder, pill, tablet, or capsule forms
  • conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate.
  • compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • non-toxic auxiliary substances such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • Preventing or treating a disease refers to inhibiting the full development of a disease, for example in a person who is known to have a predisposition to a disease such as a cancer.
  • An example of a person with a known predisposition is someone with a history of breast cancer in the family, or who has been exposed to factors that predispose the subject to a condition, such as melanoma.
  • Treatment refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop.
  • treatment refers to a reduction in size of a tumor, a decrease in the number and/or size of metastases, or a decrease in a symptom of the tumor.
  • purified does not require absolute purity; rather, it is intended as a relative term.
  • a purified peptide preparation is one in which the peptide or protein is more enriched than the peptide or protein is in its natural environment within a cell.
  • the protein or peptide represents at least 50% of the total peptide or protein content of the preparation.
  • the oligodeoxynucleotide represents at least 50% of the total nucleic acid content of the preparation.
  • Self-complementary nucleic acid sequence A nucleic acid sequence that can form
  • a nucleic acid sequence includes two or more bases in sequence that can form hydrogen bonds with two or more other bases in the same nucleic acid sequence, then the nucleic acid includes a self- complementary sequence.
  • a self-complementary nucleic acid sequence includes 3, 4, 5, 6 or more bases that could form hydrogen bonds with 3, 4, 5, 6 or more bases, respectively, of the same nucleic acid sequence.
  • the binding that occurs is typically electrostatic, hydrogen-bonding, or the result of lipophilic interactions. Accordingly, "specific binding" occurs between a paired species where there is interaction between the two that produces a bound complex having the characteristics of an antibody/antigen or enzyme/substrate interaction.
  • the specific binding is characterized by the binding of one member of a pair to a particular species and to no other species within the family of compounds to which the corresponding member of the binding member belongs.
  • an antibody preferably binds to a single epitope and to no other epitope within the family of proteins.
  • a TLR-9 agonist binds to TLR-9 and not to other TLRs, such as TLR-7 or TLR-8.
  • a TLR-7 agonist binds to TLR-7 and not to other TLRs, such as TLR-9.
  • a fundamental compound such as an aryl or aliphatic compound, or a radical thereof, having coupled thereto, typically in place of a hydrogen atom, a second substituent.
  • substituted aryl compounds or substituents may have an aliphatic group coupled to the closed ring of the aryl base, such as with toluene.
  • a long-chain hydrocarbon may have a substituent bonded thereto, such as an aryl group, a cyclic group, a heteroaryl group or a heterocyclic group.
  • Therapeutically effective dose A dose sufficient to prevent advancement, or to cause regression of a disease, or which is capable of relieving symptoms caused by a disease, such as pain.
  • Tumor An abnormal growth of cells, which can be benign or malignant. Cancer is a malignant tumor, which is characterized by abnormal or uncontrolled cell growth. Other features often associated with malignancy include metastasis, interference with the normal functioning of neighboring cells, release of cytokines or other secretory products at abnormal levels and suppression or aggravation of inflammatory or immunological response, invasion of surrounding or distant tissues or organs, such as lymph nodes, etc.
  • Methodastatic disease refers to cancer cells that have left the original tumor site and migrate to other parts of the body for example via the bloodstream or lymph system.
  • the amount of a tumor in an individual is the "tumor burden" which can be measured as the number, volume, or weight of the tumor.
  • a tumor that does not metastasize is referred to as "benign.”
  • a tumor that invades the surrounding tissue and/or can metastasize is referred to as "malignant.”
  • hematological tumors include leukemias, including acute leukemias (such as l lq23-positive acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia and myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia), chronic leukemias (such as chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma,
  • Hodgkin's disease non-Hodgkin's lymphoma (indolent and high grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia.
  • solid tumors such as sarcomas and carcinomas
  • solid tumors include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer (including basal breast carcinoma, ductal carcinoma and lobular breast carcinoma), lung cancers, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma,
  • An “established” or “existing” tumor is an existing tumor that can be discerned by diagnostic tests.
  • and established tumor can be palpated.
  • and "established tumor” is at least 500 mm 3 , such as at least 600 mm 3 , at least 700 mm 3 , or at least 800 mm 3 in size.
  • the tumor is at least 1 cm long.
  • a solid tumor and established tumor generally has an robust blood supply, and has induced Tregs and myeloid derviced suppressor cells (MDSC). Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.
  • ODNs CpG Oligodeoxynucleotides
  • CpG ODN Several types of CpG ODN are known in the art and can be used in the methods disclosed herein. These include K-type CpG ODN, D-type CpG ODN, and C ODN.
  • Combinations of ODNs can also be used, including multiple K-type CpG ODN, multiple D-type CpG ODN, and multiple C-type ODN.
  • more than one K-type CpG ODN are administered to the subject.
  • more than one D-type CpG ODN are administered to administered to the subject.
  • more than one C-type ODN are administered to the subject.
  • at least one K-type CpG ODN and at least one C- type ODNs are administered to the subject.
  • a K-type CpG ODN is not administered in combination with a D-type CpG ODN.
  • K-type CpG ODN or a mixture of K-type CpG ODNs is utilized in the methods disclosed herein.
  • K-type CpG ODN nucleic acid sequences useful in the methods disclosed herein are represented by the formula:
  • N is any nucleotide and Ni + N 2 is from about 0-26 bases.
  • Ni and N2 do not contain a CCGG quadmer or more than one CGG trimer; and the nucleic acid sequence is from about 8-30 bases in length, such as about 10 to 30 nucleotides in length.
  • nucleic acids of any size can be used in the methods disclosed herein if CpGs are present.
  • synthetic oligonucleotides of use do not include a CCGG quadmer or more than one CCG or CGG trimer at or near the 5' or 3' terminals and/or the consensus mitogenic CpG motif is not a palindrome.
  • a "palindromic sequence” or “palindrome” means an inverted repeat (i.e., a sequence such as ABCDEE'D'C'B'A', in which A and A' are bases capable of forming the usual Watson-Crick base pairs).
  • the methods include the use of an ODN which contains a CpG motif represented by the formula:
  • RD is selected from the group consisting of GpT, GpG, GpA, ApT and ApA
  • YT is selected from the group consisting of TpT or CpT
  • N is any nucleotide and Ni + N 2 is from about 0-26 bases, such that the ODN is about 8 to 30 nucleotides in length.
  • the methods disclosed herein include the use of an effective amount of at least one K-type CpG ODN, wherein the K-type CpG ODNs include an unmethylated
  • D is T, G or A
  • W is A or T
  • Ni, N 2 , N3, N 4 , N5, and N 6 are any nucleotides.
  • D is a T.
  • the K ODN(s) can be 10 to 30 nucleotides in length.
  • a K ODN can include multiple CpG motifs.
  • at least one nucleotide separates consecutive CpGs; N3D is selected from the group consisting of GpT, GpG, GpA, ApT and ApA; WN 4 is selected from the group consisting of TpT or CpT; N is any nucleotide and Ni + N 2 is from about 0-26 bases
  • Ni, and N 2 do not contain a CCGG quadmer or more than one CCG or CGG trimer.
  • CpG ODN are also in the range of 8 to 50 bases in length, such as 8 to 30 bases in length, but may be of any size (even many kb long) if sufficient motifs are present.
  • the K-type CpG ODN is 10 to 20 nucleotides in length, such as 12 to 18 nucleotides in length.
  • synthetic ODNs of this formula do not include a CCGG quadmer or more than one CCG or CGG trimer at or near the 5' and/or 3' terminals and/or the consensus CpG motif is not a palindrome.
  • CpG ODNs can be assayed for efficacy using methods described herein. It should be noted that exemplary K-type CpG ODNs are known in the art, and have been fully described, for example in PCT Publication No. WO 98/18810A1, and WO 01/22972, which are incorporated herein by reference. The K type OD can be stabilized.
  • a single K-type CpG ODN can be used in the methods disclosed herein.
  • the K-type CpG ODN comprises or consists of the nucleic acid sequence set forth as one of SEQ ID NO: 3-34.
  • the K-type CpG ODN can be any ODN listed above, including but not limited to K1555 or K3. However, it is also possible to use mixtures of K-type CpG ODNs having more than one K-type CpG ODN and an imidazoquinoline compound.
  • Exemplary combinations that can be used include 1) K3, K19, K110; 2) K19, K23, K123; K3, 3) K110, K123;4) K3, K23, K123; 5) K3, K19, K123; and 6) K19, K110, K123.
  • Additional exemplary combinations include at least two different K-type CpG ODNs, wherein one of the K-type CpG ODNs is K1555, and/or wherein one of the K-type CpG ODNs is K3.
  • D-type CpG ODNs also can be used in the method disclosed herein.
  • D-type CpG ODNs (also known as "A" class ODNs) differ both in structure and activity from K-type CpG ODNs (also known as “B” class ODNs) and a third type of ODNs, known as "C” class ODNs.
  • D-type CpG ODNs stimulate the release of cytokines from cells of the immune system, and induce the maturation of dendritic cells.
  • D-type CpG ODNs stimulate the release or production of interferon inducible protein (IP)- 10 and IFN-a by monocytes and/or plasmacytoid dendritic cells.
  • IP interferon inducible protein
  • a CpG motif in a D-type CpG ODN has been described by the formula: 5' RY-CpG-RY 3'
  • D-type oligonucleotides include an unmethylated CpG dinucleotide. Inversion, replacement or methylation of the CpG reduces or abrogates the activity of the D oligonucleotide.
  • a D-type CpG ODN is at least about 16 nucleotides in length and includes a sequence represented by the formula:
  • the region Pui Py 2 CpG Pu3 Py 4 is termed the CpG motif.
  • the region X 1X2X3 is termed the 5' flanking region, and the region X 4 XsX 6 is termed the 3' flanking region. If nucleotides are included 5' of X1X2X3 in the D ODN, these nucleotides are termed the 5' far-flanking region. Nucleotides 3' of ⁇ ⁇ 5 ⁇ 6 ⁇ the D ODN are termed the 3' far- flanking region.
  • Py 2 is a cytosine.
  • Pu3 is a guanidine.
  • Py 2 is a thymidine and Pu3 is an adenine.
  • Pui is an adenine and Py 2 is a tyrosine.
  • Pu3 is an adenine and Py 4 is a tyrosine.
  • N is from about 4 to about 8. In another specific, non-limiting example, N is about 6.
  • the D-type CpG ODN is at least about 16 nucleotides in length.
  • the D-type CpG ODNs can be from about 16 to about 50 nucleotides in length, or from about 18 to about 50 nucleotides in length, or from about 18 to about 40 nucleotides in length, or from about 18 to about 30 nucleotides in length.
  • Exemplary D-type CpG ODNs are disclosed below.
  • D-type CpG ODNs can include modified nucleotides and/or can be stabilized.
  • modified nucleotides can be included to increase the stability of a D-type CpG ODN.
  • CpG ODNs are "stabilized" by incorporating phosphothioate- modified nucleotides.
  • the CpG dinucleotide motif and its immediate flanking regions include phosphodiester rather than phosphothioate nucleotides.
  • the sequence Pui Py 2 CpG Pu3 Py 4 includes phosphodiester bases. In another specific, non-limiting example, all of the bases in the sequence Pui Py 2 CpG Pu3 Py 4 are phosphodiester bases.
  • X1X2X3 and X4XSX6(W)M (G)N include phosphodiester bases.
  • X1X2X3 Pui Py 2 CpG Pu3 Py 4 X 4 XSX6(W)M (G)N include phosphodiester bases.
  • sequence X 1X2X3 includes at most one or at most two phosphothioate bases and/or the sequence X4X5X6 includes at most one or at most two phosphothioate bases.
  • X 4 XSX6(W)M (G)N includes at least 1, at least 2, at least 3, at least 4, or at least 5 phosphothioate bases.
  • a D ODN can be a phosphothioate/phosphodiester chimera.
  • any suitable modification can be used to render a CpG ODN resistant to degradation in vivo (for example, via an exo- or endo-nuclease).
  • a modification that renders the oligodeoxynucleotide less susceptible to degradation is the inclusion of nontraditional bases such as inosine and quesine, as well as acetyl-, thio- and similarly modified forms of adenine, cytidine, guanine, thymine, and uridine.
  • modified nucleotides include nonionic DNA analogs, such as alkyl or aryl phosphonates (i.e., the charged phosphonate oxygen is replaced with an alkyl or aryl group, as set forth in U.S. Patent No. 4,469,863), phosphodiesters and alkylphosphotriesters (i.e., the charged oxygen moiety is alkylated, as set forth in U.S. Patent No. 5,023,243 and European Patent No. 0 092 574).
  • Oligonucleotides containing a diol, such as tetraethyleneglycol or hexaethyleneglycol, at either or both termini, have also been shown to be more resistant to degradation.
  • the CpG ODNs can also be modified to contain a secondary structure (e.g., stem-loop structure).
  • a secondary structure e.g., stem-loop structure
  • Pui Py 2 and Pu3 Py 4 are self-complementary.
  • XiX 2 X3 andX4XsX6 are self-complementary.
  • XiX 2 X3 Pui Py 2 and Pu3 Py 4 X4X5X6 are self-complementary.
  • Specific non- limiting examples of a D-type CpG ODN wherein Pui Py 2 and Pu3 Py 4 are self-complementary include, but are not limited to,
  • the D-type CpG ODN includes the motif XiX 2 X3 ACCGGT X4X5X6 (SEQ ID NO: 37), wherein XiX 2 X 3 AT and AT X4X5X6 are self-complementary.
  • the D-type CpG ODN includes the motif X1X2X3 ATCGAT X4X5X6 (SEQ ID NO: 38), wherein X1X2X3 AT and AT X4X5X6 are self-complementary.
  • the self-complementary base sequences can help to form a stem-loop structure with the CpG dinucleotide at the apex to facilitate immunostimulatory functions.
  • D-type CpG ODNs wherein Pui Py 2 and Pu3 Py 4 are self-complementary induce higher levels of IFN- ⁇ production from a cell of the immune system.
  • the self-complementarity need not be limited to Pui Py 2 and Pu3 Py 4 .
  • additional bases on each side of the three bases on each side of the CpG-containing hexamer form a self-complementary sequence (see above).
  • This oligodeoxynucleotide has a far-flanking region that is not self-complementary and induces high levels of IFN- ⁇ and IFN-a.
  • D-type CpG ODN is:
  • This D-type CpG ODN is of use for inducing production and/or release of cytokines from immune cells, although it lacks a self-complementary motif.
  • the D-type CpG ODNs are at least about 16 nucleotides in length. In a second embodiment, a D-type CpG ODN is at least about 18 nucleotides in length. In another embodiment, a D-type CpG ODN is from about 16 nucleotides in length to about 100 nucleotides in length. In yet another embodiment, a D-type CpG ODN is from about 16 nucleotides in length to about 50 nucleotides in length. In a further embodiment, a D-type CpG ODN is from about 18 nucleotides in length to about 30 nucleotides in length.
  • the D-type CpG ODN is at least 18 nucleotides in length, and at least two G's are included at the 5' end of the molecule, such that the oligodeoxynucleotide includes a sequence represented by the formula:
  • the D-type CpG ODN can include additional G's at the 5' end of the oligodeoxynucleotide.
  • about 1 or about 2 G's are included at the 5' end of an
  • oligodeoxynucleotide including a sequence as set forth as the above formula.
  • Examples of a D-type CpG ODN include, but are not limited to the sequence shown in the following table: ODN SEQUENCE SEQUENCE IDENTIFIER
  • D-type CpG ODN examples include, but are not limited to:
  • N is any base, or is no base at all.
  • N is a G.
  • the D-type CpG ODN includes, or consists of, the nucleic acid sequence set forth as one of SEQ ID NO: 39-63.
  • the D-type CpG ODN can be any ODN listed above, including but not limited to DV35, DV19, DV28 or DV29.
  • D-type CpG ODN can be used in combination.
  • multiple D-type CpG ODNs can be utilized in the methods disclosed herein.
  • two, three, four, five or more D-type CpG ODNs can be utilized to induce an immune response.
  • a single ODN can be generated that includes the two or more D-type CpG motifs disclosed herein.
  • DV35, DV19, DV28, DV29 or DV113, or two, three, four or five of these ODNs can be used in combination.
  • DV35, DV29 and DV19 can be used in combination.
  • Additional exemplary combinations include at least two different D-type CpG ODNs, wherein one of the D-type CpG ODNs is DV35, and/or wherein one of the D-type CpG ODNs is DV28.
  • D-type and K-type CpG ODNs can also be used in combination.
  • C-type ODNs also can be utilized in the methods disclosed herein.
  • C class ODNs have a TCGTCG motif at the 5 ' end and have a CpG motif imbedded in a palindromic sequence.
  • M362 is an exemplary C-type CpG ODN that contains a 5'-end 'TCGTCG-motif and a 'GTCGTT- motif .
  • C-type ODNs resemble K-type as they are composed entirely of phosphorothioate nucleotides, but resemble D-type in containing palindromic CpG motifs.
  • This class of ODNs stimulates B cells to secrete IL-6 and pDCs to produce IFN-a (see Hartmann et al., Eur.
  • a palindromic sequence of at least 8 nucleotides increases activity, for example a palindrome of at least 12, such as 14, 16, 18 or 20 nucleotides, increases activity.
  • the CpG-C ODNs include one to two TCG trinucleotides at or close to the 5' end of the ODN and a palindromic region of at least 10-12 bases, which contains at least two additional CG dinucleotides preferably spaced zero to three bases apart.
  • the CG dinucleotides in the palindrome are preferably spaced 1 , 2, or 3 nucleotides apart, although sequences with four nucleotide spacings retained low levels of IFN-a-inducing activity (see Marshall et al., J. Leukocyte Biol. 73: 781-792, 2003, incorporated herein by reference).
  • C-type ODNs are present in both early and late endosomes, and thus express properties in common with both K- and D-type CpG ODNs.
  • C-type CpG ODNs include ODN2216, ODN M362, ODN 1668, and ODN2395, which are available from Invivogen and C274, see also Marshall et al., supra.
  • the stabilized oligodeoxyonucleotide has a modified phosphate backbone.
  • a stabilized oligonucleotide has a phophorothioate modified phosphate backbone (wherein at least one of the phosphate oxygens is replaced by sulfur).
  • oligonucleotides include: nonionic DNA analogs, such as alkyl- and aryl- phosphonates (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. Oligonucleotides which contain a diol, such as tetraethyleneglycol or hexaethyleneglycol, at either or both termini have also been shown to be substantially resistant to nuclease degradation.
  • CpG ODN can be synthesized de novo using any of a number of procedures well known in the art.
  • the oligodeoxynucleotides can be synthesized as set forth in U.S. Patent No. 6,194,388, which is herein incorporated by reference in its entirety.
  • ODNs can be synthesized using any methods known to those of skill in the art. Automated synthesis of ODNs is routine. An ODN can be synthesized using, for example, the B-cyanoethyl phosphoramidite method or nucleoside H-phosphonate method. These chemistries can be performed by a variety of automated oligonucleotide synthesizers available in the market.
  • oligodeoxynucleotides can be prepared from existing nucleic acid sequences (e.g. genomic or cDNA) using known techniques, such as employing restriction enzymes, exonucleases or endonucleases, although this method is less efficient than direct synthesis.
  • Imidazoquinolines are of use in the methods disclosed herein. Imidazoquinolines are synthetic immunomodulatory drugs that act by binding toll-like receptors 7 and 8 (TLR7/TLR8) on dendritic cells, structurally mimicking these receptors' natural ligand, viral single-stranded RNA. Imidazoquinolines are heterocyclic compounds comprising a fused quinoline-imidazole skeleton. Derivatives, salts (including hydrates, solvates, and N-oxides), and prodrugs thereof also are contemplated by the present disclosure. Particular imidazoquinoline compounds are known in the art, see for example, U.S. Patent No. 6,518,265; and U.S. Patent No. 4,689,338. In some embodiments, the imidazoquinoline compound is not imiquimod and/or is not resiquimod. In additional embodiments, the imidazoquinoline compound is a lipophilic imidazoquinoline compound.
  • R 1 may be selected from hydrogen, C12-24 alkyl, Cn -24 heteroalkyl, substituted Ci-10 heteroalkyl, carboxyl, and hydroxyl
  • R 2 may be selected from hydrogen, Ci-10 alkyl, Ci-io heteroalkyl, and substituted Ci-io alkyl
  • R 3 may be selected from hydrogen, amino, aminoacyl, hydroxyl, and Ci-io alkoxy.
  • R 1 may be Cn-24 heteroalkyl, such as Cn-24 alkoxy (e.g., -OCn-24 alkyl), Cn-24 thioalkyl (e.g., -SCn -24 alkyl), and Cn-24 aminoalkyl (e.g., -NR 5 Cn-24 alkyl, wherein R 5 is selected from hydrogen, alkyl, cycloalkyl, aryl, and the like).
  • Cn-24 alkoxy e.g., -OCn-24 alkyl
  • Cn-24 thioalkyl e.g., -SCn -24 alkyl
  • Cn-24 aminoalkyl e.g., -NR 5 Cn-24 alkyl, wherein R 5 is selected from hydrogen, alkyl, cycloalkyl, aryl, and the like.
  • R 1 may be Ci-io heteroalkyl substituted with one or more substituents selected from amino (e.g., N(R 5 )2, wherein each R 5 independently is selected from hydrogen, alkyl, cycloalkyl, aryl, and the like), aminoacyl (e.g., -NC(0)Ci-3o alkyl, -NC(0)Ci -2 3 alkyl; such as -NC(0)Cn -2 3 alkyl; -NC(0)Ci5-23 alkyl; -NC(0)Ci5-i9 alkyl, and -NC(0)Ci7 alkyl), aminocarbonylamino (e.g., -NC(0)NR 5 C 1-30 alkyl, -NC(0)NR 5 Ci- 2 3 alkyl, such as -NC(0)NR 5 C 11-23 alkyl, -NC(0)NR 5 C 15-23 alkyl, -NC(0)NR 5 Ci 5 -i 9 alkyl, and -NC(0)NR 5 Ci 7 alkyl
  • R 1 is -X-Y-Z-R 6 wherein X is selected from the group consisting of a bond, -0-, and -NH-; Y is selected from the group consisting of Ci-ioalkyl and Ci- salkylOCi-salkyl; Z is selected from the group consisting of -NHC(O)-, -NHS(0) 2 -, -NHC(0)NH-; and R 6 is -Cn-23alkyl.
  • Y is C 2 -ioalkyl or Y is C 2 - 4 alkylOC 2 - 4alkyl, with certain embodiments being C 2 -salkyl.
  • X is selected from a group consisting of a bond and -O- in certain embodiments, with particular disclosed embodiments having X as -0-.
  • Z is -NHC(O)-.
  • Particular disclosed compounds may have an R 6 substituent that is Ci5-23alkyl, more typically R 6 is Ci5-Ci9alkyl.
  • R 1 is selected from the group consisting of - CH 2 CH 2 CH 2 CH 2 CH 2 NHC(0)Cn- 23 alkyl, -OCH 2 CH 2 CH 2 CH 2 NHC(0)Cn- 23 alkyl, - NHCH 2 CH 2 CH 2 CH 2 NHC(0)Cn- 23 alkyl, -CH 2 CH 2 OCH 2 CH 2 NHC(0)Cn- 23 alkyl. More typically, R 1 is selected from the group consisting of
  • the imidazoquinoline compound has the formula:
  • R 1 is selected from -Ci 2 - 24 alkyl, -OCn- 24 alkyl, -NHCn- 24 alkyl, and -X-Y-Z-R 6 , wherein X is selected from the group consisting of a bond, -0-, and -NH-;
  • Ci-ioalkyl and C1-5 alkylOCi- salkyl Z is selected from the group consisting of:
  • R 6 is -Cn- 23 alkyl
  • R 2 is selected from the group consisting of hydrogen, alkyl, alkylaminoalkyl, alkoxyalkyl, and hydroxyalkyl.
  • R 2 may be Ci-10 alkyl, such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, and the like.
  • R 2 may be Ci-10 alkyl substituted with one or more substituents selected from halogen (e.g., chloro, iodo, bromo, fluoro), trihaloalkyl (e.g., trifluoromethyl), aryl (e.g., C 6 -i4 aryl), heteroaryl (e.g., C 6 -i4 aryl wherein one or more carbon atoms is replaced with a heteroatom selected from oxygen, sulfur, and nitrogen), amino (e.g., -N(R 5 ) 2 , wherein each R 5 independently is selected from hydrogen, alkyl, cycloalkyl, aryl, and the like), aminoacyl (e.g., -NC
  • R 2 is selected from the group consisting of hydrogen, alkyl, alkylaminoalkyl, alkoxyalkyl, and hydroxyalkyl.
  • R 2 may be selected from hydrogen, methyl, ethyl, propyl, butyl, ethoxymethyl, methoxymethyl, 2-methoxyethyl, hydroxymethyl, and 2- hydroxyethyl.
  • R 2 is selected from the group consisting of ethyl, propyl, butyl, methoxyethyl, and ethoxymethyl.
  • R 2 is selected from the group consisting of butyl and ethoxymethyl.
  • R 3 may be amine (-NH 2 ), amino (e.g., -N(R 5 ) 2 , wherein each R 5 independently is selected from hydrogen, alkyl, cycloalkyl, aryl, heterocyclyl, heteroaryl, and the like), or aminoacyl (e.g., -NC(0)R 6 wherein R 6 is selected from Ci-io alkyl, Ci-io haloalkyl, hydrogen, C 6 -i4 aryl, and the like).
  • the imidazoquinoline compound has a formula:
  • n ranges from zero to 10; and R 1 and R 3 may be selected from any of the particular groups recited above for Formula I.
  • the imid ound has a formula:
  • R 2 and R 3 may be selected from any of the particular groups recited above for Formula I, and R 4 may be selected from Ci-io alkyl substituted with one or more of the substituents provided for substituted heteroalkyl, such as those provided for R 1 in Formula I, above.
  • imidazoquinoline compounds include the following:
  • This imidazoquinoline compound can be used in any of its pharmaceutically acceptable forms including solid, semi-solid, solvate (e.g., hydrate), wholly or partially dissolved (such as in a pharmaceutical composition), a prodrug, or dispersed in a pharmaceutically acceptable carrier.
  • Any pharmaceutically acceptable salt of the imidazoquinoline compound can also be used, see PCT Publication No. WO 2012/024284, which is incorporated herein by reference. Additional compounds are disclosed for example, in U.S. Patent No. 7,799,800, which is incorporated herein by reference.
  • the pharmaceutically acceptable salt may be selected from any suitable salt known in the art, such as (but not limited to) salts of organic and inorganic counter ions and salts of organic or inorganic acids.
  • the pharmaceutically acceptable salt may be a hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acetate, lactate, salicylate, citrate, tartrate, bitartrate, ascorbate, succinate, ammonium, potassium, sodium, calcium, magnesium, maleate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, mesylate, tosylate, or besylate salt.
  • One or more of the functional groups provided in any one of Formulas ⁇ - ⁇ may be manipulated to provide the desired pharmaceutically acceptable salt. Methods of forming pharmaceutically acceptable salts are readily recognized by a person of ordinary skill in the art.
  • prodrugs of the disclosed compounds include, but are not limited to, carbonates, esters, amides, carbamates, oximes, imines, phosphates, and ethers. Methods of forming prodrugs are readily recognized by a person of ordinary skill in the art. Pharmaceutical Compositions and Methods of Use
  • Methods are disclosed herein for producing an immune response to a tumor in a subject. Methods are also provided for treating a tumor in a subject. In some embodiments, the methods include treating an existing tumor in a subject. In additional embodiments, methods are disclosed herein for preventing conversion of a benign to a malignant lesion, or preventing metastasis in a subject. In some examples, the methods reduce a symptom of the tumor in the subject. In additional examples, the tumor is a solid tumor.
  • the methods include selecting a subject having a tumor, such as a benign or malignant tumor, and administering to the subject a therapeutically effective amount of (1) an imidazoquinoline compound and (2) a CpG ODN, such as K-type CpG ODN, D-type CpG ODN, C-type CpG ODN, or a combination thereof.
  • the methods are of use for treating the tumor, preventing metastasis and/or preventing the conversion of a benign to a malignant tumor.
  • the administration can be local.
  • the imidazoquinoline compound and the CpG ODN are administered intratumorally.
  • the embodiments disclosed herein the
  • imidazoquinoline compound can be a lipophilic imidazoquinoline compound, such as 3M-052.
  • the CpG ODN can be one of SEQ ID NOs: 3-34 or 39-65; combinations thereof can also be used. Exemplary methods are disclosed below.
  • the methods disclosed herein include selecting a subject in need of treatment, such as a subject with a tumor, and administering to the subject a therapeutically effective amount of both (1) the imidazoquinoline compound and (2) the one or more CpG ODN, such as K-type CpG ODN, D- type CpG ODN, C-type CpG ODN, or a combination thereof.
  • the one or more CpG ODN such as K-type CpG ODN, D- type CpG ODN, C-type CpG ODN, or a combination thereof.
  • more than one CpG ODN is utilized, such as two, three, four or five CpG ODN.
  • These ODNs can be of the same type or can be different types.
  • Additional agents can also be administered to the subject of interest, such as, but not limited to, chemotherapeutic agents. Additional treatments can also be
  • chemotherapeutic agents can also be administered to the subject.
  • the tumor can be benign or malignant.
  • the tumor can be any tumor of interest, including, but not limited to, melanoma and colorectal cancer.
  • the tumor is a lymphoma, breast cancer, lung cancer and colon cancer. Additional examples are skin tumors, breast tumors, brain tumors, cervical carcinomas, testicular carcinomas, head and neck tumors, gastrointestinal tract tumors, genitourinary system tumors, gynecological system tumors, breast, endocrine system tumors, skin tumors, a sarcoma of the soft tissue and bone, a mesothelioma, a melanoma, a neoplasm of the central nervous system, or a leukemia.
  • the tumor is a head and neck tumor, such as tumors of the nasal cavity, paranasal sinuses, nasopharynx, oral cavity, oropharynx, larynx, hypopharynx, salivary glands and paragangliomas.
  • the tumor is a lung tumor, such as a non-small cell lung cancer or a small cell lung cancer.
  • the tumor can be a tumor of the gastrointestinal tract, such as cancer of the esophagus, stomach, pancreas, liver, biliary tree, small intestine, colon, rectum and anal region.
  • the tumor can be a tumor of the genitourinary system, such as cancer of the kidney, urethra, bladder, prostate, urethra, penis and testis.
  • the tumor is a gynecologic tumor, such as cancer of the cervix, vagina, vulva, uterine body, gestational trophoblastic diseases, ovarian, fallopian tube, peritoneal, or breast.
  • the tumor is an endocrine system tumor, such as a thyroid tumor, parathyroid tumor, adrenal cortex tumor, pancreatic endocrine tumor, carcinoid tumor and carcinoid syndrome.
  • the tumor can be a sarcoma of the soft tissue and bone, a mesothelioma, a cancer of the skin, a melanoma, comprising cutaneous melanomas and intraocular melanomas, a neoplasm of the central nervous system, a cancer of the childhood, comprising retinoblastoma, Wilm's tumor,
  • the tumor can be a lymphoma, comprising non-Hodgkin's lymphomas, cutaneous T-cell lymphomas, primary central nervous system lymphoma, and Hodgkin's disease.
  • the tumor can be a leukemia, such as acute leukemia, chronic myelogenous leukemia and lymphocytic leukemia.
  • the tumor can be plasma cell neoplasms, a cancer of unknown primary site, a peritoneal carcinomastosis, a Kaposi's sarcoma, AIDS-associated lymphomas, AIDS-associated primary central nervous system lymphoma, AIDS-associated Hodgkin's disease and AIDS-associated anogenital cancers, a metastatic cancer to the liver, metastatic cancer to the bone, malignant pleural and pericardial effusions and malignant ascites.
  • the tumor is melanoma or colon cancer.
  • Treatment of the tumor is generally initiated after the diagnosis of the tumor, or after the initiation of a precursor condition (such as dysplasia or development of a benign tumor). Treatment can be initiated at the early stages of cancer, for instance, can be initiated before a subject manifests symptoms of a condition, such as during a stage I diagnosis or at the time dysplasia is diagnosed. However, treatment can be initiated during any stage of the disease, such as but not limited to stage I, stage ⁇ , stage ⁇ and stage IV cancers. In some examples, treatment is administered to these subjects with a benign tumor that can convert into a malignant or even metastatic tumor.
  • the presence of a tumor can be determined by methods known in the art, and typically include cytological and morphological evaluation.
  • the tumor can be an established tumor.
  • the cells can be in vivo or ex vivo, including cells obtained from a biopsy.
  • Treatment initiated after the development of a condition may result in decreasing the severity of the symptoms of one of the conditions, or completely removing the symptoms, or reducing metastasis, tumor volume or number of tumors.
  • the tumor becomes undetectable following treatment.
  • Treatment can also include increasing the immune response to the tumor, such as by increasing the humoral response, or cytokines, NK cells, activated CTLs, such as CD9+ T cells, or MDSCs, such as mMDSCs.
  • the formation of tumors is delayed, prevented or decreased.
  • the size of the primary tumor is decreased.
  • a symptom of the tumor is decreased.
  • tumor volume is decreased.
  • the methods are for the treatment of a subject with a tumor.
  • a therapeutically effective amount of (1) an imidazoquinoline compound, such as a lipophilic imidazoquinoline compound, for example, 3M052, and (2) one or more CpG ODN, such as K-type CpG ODN, D-type CpG ODN, C-type CpG ODN, or a combination thereof, is administered to the subject.
  • an imidazoquinoline compound such as a lipophilic imidazoquinoline compound, for example, 3M052
  • CpG ODN such as K-type CpG ODN, D-type CpG ODN, C-type CpG ODN, or a combination thereof.
  • Exemplary K-type CpG ODN, D-type CpG ODN and C-type ODN are listed above, see for example, SEQ ID NOs: 3-34 and 39-63.
  • the administration can be directly to the tumor.
  • An immune response can be measured, tumor volume can be measured, the number of metastatic lesions can be measured, or a symptom of a tumor can be measured.
  • a therapeutically effective dose can increase the immune response, decrease tumor volume, decrease the number and/or size of metastases, and/or decrease one or more symptoms of the tumor.
  • Treatment prior to the development of the condition is referred to herein as treatment of a subject that is "at risk" of developing the condition.
  • administration of a composition can be performed during or after the occurrence of the conditions described herein.
  • compositions can include (1) an imidazoquinoline compound and (2) one or more CpG ODN, such as K-type CpG ODN, D-type CpG ODN, C-type CpG ODN, or a combination thereof as active ingredients. These compositions can also include an additional agent, such as an additional chemotherapeutic agent.
  • the compositions described herein include both (1) the imidazoquinoline compound, such as a lipophilic imidazoquinoline compound (for example, 3M-052) and (2) the one or more CpG ODN, such as K-type CpG ODN, D-type CpG ODN, C-type CpG ODN, or a combination thereof. These compositions are of use for threating a tumor.
  • the composition includes 3M-052. These compositions can be formulated in a variety of ways for administration to a subject to induce an immune response to a tumor, or to delay, prevent, reduce the risk of developing, or treat, any tumor of interest.
  • the compositions described herein can also be formulated for application such that they prevent metastasis of an initial lesion.
  • the compositions are formulated for local administration, such as intratumoral administration. Pharmaceutical compositions are thus provided for both local use and for systemic use, formulated for use in human or veterinary medicine.
  • compositions will typically be used to treat human subjects they may also be used to treat similar or identical diseases in other vertebrates, such as other primates, dogs, cats, horses, and cows.
  • a suitable administration format may best be determined by a medical practitioner for each subject individually.
  • Various pharmaceutically acceptable carriers and their formulation are described in standard formulation treatises, e.g.,
  • the (1) the imidazoquinoline compound, such as a lipophilic imidazoquinoline compound (for example, 3M-052) and (2) the one or more CpG ODN, such as K-type CpG ODN, D-type CpG ODN, C-type CpG ODN, or a combination thereof can be administered in a composition that contains a synthetic or natural hydrophilic polymer as the carrier.
  • a synthetic or natural hydrophilic polymer examples include hydroxypropyl cellulose and polyethylene glycol.
  • the active ingredients can be mixed with a hydrophilic polymer in an appropriate solvent.
  • hydrophilic polymers keep well as they have a low water-content. At the time of use, they absorb water, becoming gels that also store well.
  • the firmness can be adjusted by mixing a polyhydric alcohol with a hydrophilic polymer similar to those above, such as cellulose, starch and its derivatives, or synthetic polymeric compounds. Hydrophilic sheets thus formed can be used.
  • compositions or pharmaceutical compositions including (1) the imidazoquinoline compound, such as a lipophilic imidazoquinoline compound (for example, 3M-052) and (2) the one or more CpG ODN, such as K-type CpG ODN, D-type CpG ODN, C-type CpG ODN, or a combination thereof, can be administered by any route, including parenteral administration, for example, intravenous, intraperitoneal, intramuscular, intraperitoneal, intrasternal, or intraarticular injection or infusion, or by sublingual, oral, topical, intranasal, or transmucosal administration, or by pulmonary inhalation.
  • parenteral administration for example, intravenous, intraperitoneal, intramuscular, intraperitoneal, intrasternal, or intraarticular injection or infusion, or by sublingual, oral, topical, intranasal, or transmucosal administration, or by pulmonary inhalation.
  • the imidazoquinoline compound and the one are more CpG ODN are administered to a tissue wherein the tumor is located, or directly into the tumor.
  • ODNs are provided as parenteral compositions, e.g. for injection or infusion, they are generally suspended in an aqueous carrier, for example, in an isotonic buffer solution at a pH of about 3.0 to about 8.0, preferably at a pH of about 3.5 to about 7.4, 3.5 to 6.0, or 3.5 to about 5.0.
  • Useful buffers include sodium citrate-citric acid and sodium phosphate-phosphoric acid, and sodium acetate-acetic acid buffers.
  • a form of repository or "depot" slow release preparation may be used so that therapeutically effective amounts of the preparation are delivered into the bloodstream over many hours or days following transdermal injection or delivery.
  • compositions including (1) the imidazoquinoline compound, such as a lipophilic imidazoquinoline compound (for example, 3M-052) and (2) the one or more CpG ODN, such as Retype CpG ODN, D-type CpG ODN, C-type CpG ODN, or a combination thereof are also suitably administered by sustained-release systems.
  • imidazoquinoline compound such as a lipophilic imidazoquinoline compound (for example, 3M-052)
  • CpG ODN such as Retype CpG ODN, D-type CpG ODN, C-type CpG ODN, or a combination thereof are also suitably administered by sustained-release systems.
  • sustained-release compositions include suitable polymeric materials (such as, for example, semi-permeable polymer matrices in the form of shaped articles, e.g., films, or mirocapsules), suitable hydrophobic materials (such as, for example, an emulsion in an acceptable oil) or ion exchange resins, and sparingly soluble derivatives (such as, for example, a sparingly soluble salt).
  • suitable polymeric materials such as, for example, semi-permeable polymer matrices in the form of shaped articles, e.g., films, or mirocapsules
  • suitable hydrophobic materials such as, for example, an emulsion in an acceptable oil
  • ion exchange resins such as, for example, an emulsion in an acceptable oil
  • sparingly soluble derivatives such as, for example, a sparingly soluble salt.
  • Preparations for administration can be suitably formulated to give controlled release of (1) the imidazoquinoline compound, such as a lipophilic imidazoquinoline compound (for example, 3M-052) and (2) the one or more CpG ODN, such as K-type CpG ODN, D-type CpG ODN, C-type CpG ODN, or a combination thereof over an extended period of time.
  • the pharmaceutical compositions may be in the form of particles comprising a biodegradable polymer and/or a polysaccharide jellifying and/or bioadhesive polymer, an amphiphilic polymer, an agent modifying the interface properties of the particles and a pharmacologically active substance. These compositions exhibit certain biocompatibility features which allow a controlled release of the active substance. See U.S. Patent No. 5,700,486.
  • parenteral formulations usually comprise injectable fluids that are pharmaceutically and physiologically acceptable fluid vehicles such as water, physiological saline, other balanced salt solutions, aqueous dextrose, glycerol or the like.
  • Excipients that can be included are, for instance, proteins, such as human serum albumin or plasma preparations.
  • the pharmaceutical composition to be administered may also contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in the art.
  • the formulations are prepared by contacting (1) the imidazoquinoline compound, such as a lipophilic imidazoquinoline compound (for example, 3M-052) and (2) the one or more CpG ODN, such as K-type CpG ODN, D-type CpG ODN, C-type CpG ODN, or a combination thereof each uniformly and intimately with liquid carriers or finely divided solid carriers or both. Then, if necessary, the product is shaped into the desired formulation.
  • the carrier is a parenteral carrier, and in some embodiments it is a solution that isotonic with the blood of the recipient. Examples of such carrier vehicles include water, saline, Ringer's solution, and dextrose solution. Non-aqueous vehicles such as fixed oils and ethyl oleate are also useful herein, as well as liposomes.
  • compositions that comprise (1) the imidazoquinoline compound, such as a lipophilic imidazoquinoline compound (for example, 3M-052) and (2) the one or more CpG ODN, such as K-type CpG ODN, D-type CpG ODN, C-type CpG ODN, or a combination thereof can be formulated in unit dosage form, suitable for individual administration of precise dosages.
  • the amount of active compound(s) administered will be dependent on the subject being treated, the severity of the affliction, and the manner of administration, and is best left to the judgment of the prescribing clinician. Within these bounds, the formulation to be administered will contain a quantity of the active component(s) in amounts effective to achieve the desired effect in the subject being treated.
  • therapeutically effective amounts of (1) the imidazoquinoline compound, such as a lipophilic imidazoquinoline compound (for example, 3M-052) and (2) the one or more CpG ODN, such as K-type CpG ODN, D-type CpG ODN, C-type CpG ODN, or a combination thereof, are of use for inducing an immune response to the tumor cells, treating a tumor, and/or preventing conversion of a benign to a malignant lesion, or preventing metastasis.
  • Administration may begin whenever the suppression or prevention of disease is desired, for example, at a certain age of a subject, or prior to an environmental exposure.
  • the therapeutically effective amount of (1) the imidazoquinoline compound, such as a lipophilic imidazoquinoline compound (for example, 3M-052) and (2) the one or more CpG ODN, such as K-type CpG ODN, D-type CpG ODN, C-type CpG ODN, or a combination thereof, will be dependent on the CpG ODN(s) utilized, the subject being treated, the severity and type of the affliction, and the manner of administration.
  • the exact dose is readily determined by one of skill in the art based on the potency of the specific compound (such as the ODN utilized and the imidazoquinoline compound), the age, weight, sex and physiological condition of the subject.
  • Suitable concentrations include, but are not limited to, about 1 to about 100 ⁇ g/gm K-type CpG ODN, such as about 5 to about 50 ⁇ g/gm, such as about 50 ⁇ g/gm CpG ODN. Additional suitable concentrations include 1 to 100 mg/kg, such as about 5 to about 50 mg/kg, such as about 10 mg/kg. Suitable does also include about 0.1 to about 2 mg/kg in humans. In other embodiments about 0.1 to about 10 mg/kg of the imidazoquinoline compound is administered, such as about 0.1 to about 1 mg/kg.
  • compositions that include a therapeutically effective amount of one or more CpG ODNs and an imidazoquinoline compound, such as a lipophilic imidazoquinoline compound (for example, 3M- 052).
  • an imidazoquinoline compound such as a lipophilic imidazoquinoline compound (for example, 3M- 052).
  • Additional agents can be administered, such as a cytokine, a chemokine, or a
  • chemotherapeutic agent can be included in the disclosed pharmaceutical compositions.
  • a cytokine can be administered, such as interleukin-2 (IL-2), granulocyte macrophage colony stimulating factor (GM-CSF), or interferon, such as interferon (IFN) ⁇ .
  • IL-2 interleukin-2
  • GM-CSF granulocyte macrophage colony stimulating factor
  • IFN interferon
  • surgical treatment can be administered to the subject. In one example, this administration is sequential. In other examples, this administration is simultaneous.
  • chemotherapeutic agents are alkylating agents, antimetabolites, natural products, or hormones and their antagonists.
  • alkylating agents include nitrogen mustards (such as mechlorethamine, cyclophosphamide, melphalan, uracil mustard or
  • chlorambucil alkyl sulfonates (such as busulfan), nitrosoureas (such as carmustine, lomustine, semustine, streptozocin, or dacarbazine).
  • antimetabolites include folic acid analogs (such as methotrexate), pyrimidine analogs (such as 5-FU or cytarabine), and purine analogs, such as mercaptopurine or thioguanine.
  • Examples of natural products include vinca alkaloids (such as vinblastine, vincristine, or vindesine), epipodophyllotoxins (such as etoposide or teniposide), antibiotics (such as dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin, or mitocycin C), and enzymes (such as L-asparaginase).
  • vinca alkaloids such as vinblastine, vincristine, or vindesine
  • epipodophyllotoxins such as etoposide or teniposide
  • antibiotics such as dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin, or mitocycin C
  • enzymes such as L-asparaginase
  • miscellaneous agents include platinum coordination complexes (such as cis-diamine-dichloroplatinum II also known as cisplatin), substituted ureas (such as hydroxyurea), methyl hydrazine derivatives (such as procarbazine), and adrenocrotical suppressants (such as mitotane and aminoglutethimide).
  • platinum coordination complexes such as cis-diamine-dichloroplatinum II also known as cisplatin
  • substituted ureas such as hydroxyurea
  • methyl hydrazine derivatives such as procarbazine
  • adrenocrotical suppressants such as mitotane and aminoglutethimide
  • hormones and antagonists include adrenocorticosteroids (such as prednisone), progestins (such as
  • hydroxyprogesterone caproate medroxyprogesterone acetate, and magestrol acetate
  • estrogens such as diethylstilbestrol and ethinyl estradiol
  • antiestrogens such as tamoxifen
  • androgens such as testerone proprionate and fluoxymesterone.
  • chemotherapy drugs include Adriamycin, Alkeran, Ara-C, BiCNU, Busulfan, CCNU,
  • Non-limiting examples of immunomodulators that can be used include AS- 101 (Wyeth- Ayerst Labs.), bropirimine (Upjohn), gamma interferon (Genentech), GM-CSF (granulocyte macrophage colony stimulating factor; Genetics Institute), IL-2 (Cetus or Hoffman-LaRoche), human immune globulin (Cutter Biological), EVIREG (from Imreg of New Jersey, La.), SK&F 106528, and TNF (tumor necrosis factor; Genentech).
  • mMDSC myeloid derived suppressor cells
  • TLRs 7, 8 and 9 myeloid derived suppressor cells
  • 3M-052 was supplied by 3M Drug Delivery System with 4mg/ml stock solution in ethanol. Endotoxin free phosphorothioate ODN were synthesized at the Core Facility of the Center for Biologies Evaluation and Research, Food and Drug Administration (Bethesda, MD) with the following sequence, CpG ODN 1555 (5 ' -GCTAGACGTTAGCGT-3 ' , SEQ ID NO: 30) and control ODN 1612 (5 ' -GCTAGAGCTTAGCGT-3 ' , SEQ ID NO: 35). All ODN were dissolved in phosphate buffered saline (PBS) at a concentration of 4mg/ml.
  • PBS phosphate buffered saline
  • mice and tumor cell lines Six to eight weeks wild type B ALB/c and C57BL/6 mice were utilized.
  • the CT26 colon cancer cell line, and the B16-F10 cell lines purchased from American Type Culture collection (Manassas, VA), were utilized in the experiments disclosed below.
  • Tumor cell lines were maintained in RPMI 1640 medium supplemented with 10% FCS, 100 U/ml penicillin, lOOug/ml streptomycin, 25 mM HEPES, 1.0 mM sodium pyruvate, nonessential amino acids, and 0.0035% 2-ME.
  • Tumor experiments For tumor induction, Balb/c mice were injected subcutaneously (s.c.) with 10 5 CT26 tumor cells and C57BL/6 mice with 10 5 B16-F10 tumor cells on the right flank. Treatment was initiated on week 2 or 3 by intratumoral inject 100/200 ug ODN, 50/100 ug 3M-052 or same volume of control 3M. Tumor size was calculated by the formula: (length x width x width)/2 and the mice with tumor exceeded a diameter of 2.0 cm were immediately euthanized. Tumor growth curves were generated from five mice per group, and all results were derived by combining data from two to three independent experiments. Two treatment regimens were used. For small tumors ( ⁇ 300 mm 3 ), two doses of 100 ⁇ g of
  • mice were injected intraperitoneally (i.p.) with 25 ul ascites of rat anti-mouse CD4 (L3/T4) or mouse anti-mouse CD8 (Ly2.2) Abs from Cedarlane labs (Burlington, NC) on day -2, 0, 3 and 6 post-tumor implantation.
  • Leukocytic infiltrates of the tumor site were prepared by surgical removal of tumor tissue followed by homogenization using a GentleMACS Dissociator (Miltenyi Biotec) and then digestion in RPMI containing 5% fetal calf serum, 250 U/mL type IV collagenase (Invitrogen) and 100 mg/mL DNase I (Roche Molecular Biochemicals) at 37° C for 30 minutes.
  • the resulting single cell suspension was passed through a 70 um cell strainer (BD Biosciences, Bedford, MA), and washed twice with RPMI.
  • Live cells were isolated by density gradient centrifugation (Histopaque-1077, Sigma- Aldrich), washed, and stained using the following Abs from BD Pharmingen (clone names provided in parentheses).
  • CDl lb Ml/70
  • Gr-1 RB6- 8C5
  • CD3 145-2C11
  • CD8 53-6.7
  • CD49b DX5
  • CD45 (30-F11) was used as a leukocyte marker.
  • Stained cells were analyzed using an LSR-II flow cytometer (Becton Dickinson).
  • Enzyme linked immunosorbent spot (ELISpot) assay Single cell suspensions were prepared from whole spleen or tumor drain lymph node and 1.5 - 3.0 x 10 5 cells/well were stimulated for 12 hours with AH-1 peptide (1 ⁇ g/ml) in 96 well EVIMULONTM II plates (Millipore, Billerica, MA) which coated with the monoclonal antibody (mAb) anti-interferon (IFN)g Ab (R4- 6A2) (BD Biosciences). The plates were washed and treated with biotinylated polyclonal goat anti- IFN- ⁇ Ab (R & D systems, MN) followed by streptavidin alkaline phosphatase.
  • mAb monoclonal antibody
  • IFN anti-interferon
  • Interleukin (IL)-12p40, IFNy, Granzyme B, arginase (Arg)l, nitric oxide synthetase (Nos)2, cytotoxic T-lymphocyte antigen (CTLA)4 and transforming growth factor (TGF)b mRNA levels were examined by using TAQMAN® Gene Expression Master Mix and Applied Biosystems STEPONETM reverse transcription polymerase chain reaction (RT-PCR) system. All primer sets were from the gene expression assay set (Applied Biosystems, Foster City, CA). Gene expression was normalized to the level of the glyceraldehyde 3 -phosphate dehydrogenase (GAPDH) housekeeping gene. Data were analyzed by STEPONETM software (Applied Biosystems) and expressed as a fold change in mRNA expression relative to control values. Ct values for all genes studied fell in the range of 22 - 35.
  • GPDH glyceraldehyde 3 -phosphate dehydrogenase
  • CT26 colon cancer cells were implanted subcutaneously into the flank of syngeneic BALB/c mice. When these tumors reached -200 mm3 in volume, 100 ⁇ g of ODN and/or 50 ⁇ g of 3M-052 was delivered intra-tumorally. This procedure was repeated two days later. Tumors in untreated mice proliferated rapidly (Fig. 1). The rate of proliferation was significantly reduced by treatment with either CpG ODN (CpG ODN K1555, see above) or 3M-052 although the tumors persisted. In contrast, mice treated with the combination of CpG ODN plus 3M-052 completely rejected their tumors (p ⁇ 0.01; Fig. 1).
  • mice were isolated from mice three days after treatment was initiated. These were stimulated in vitro with the CT26-derived AH-1 tumor peptide and interferon (IFN)y secretion (a surrogate for cytotoxic T lymphocyte (CTL) activity) monitored.
  • IFN interferon
  • CTL cytotoxic T lymphocyte
  • Immune cells in the tumor microenvironment profoundly influence the success of immunotherapy.
  • a single cell suspension was prepared from tumor samples, and the frequency of various immune subsets evaluated by FACS (Fig. 9).
  • the number of mMDSC is considered an important marker of immune suppression, as these cells suppress the tumoricidal activity of CTL and NK cells.
  • the frequency of Grl + CD1 lb + mMDSC was significantly elevated in mice bearing CT26 tumors (Fig 2).
  • Treatment with either CpG ODN or 3M-052 alone reduced the number of mMDSC infiltrating the tumor site by -50% (p. ⁇ 0.05).
  • the combination of these two agonists resulted in a nearly 90% reduction in mMDSC frequency (p. ⁇ 0.01, Fig 2). This effect was detectable by 1 day after the second treatment.
  • Splenocytes from mice in each group were isolated and stimulated ex vivo with the CT26-derived AH-1 tumor peptide.
  • the frequency of IFNy secreting cells was determined by ELIspot assay. Consistent with changes in the frequency of CD8 T cells noted above, the number of cells stimulated by AH-1 peptide to produce IFNy was >8-fold higher in mice treated with CpG ODN plus 3M-052 than in controls by 3 days post treatment (p ⁇ 0.001, Fig. 3).
  • mice that had been challenged with tumor and treated with the combination CpG ODN plus 3M-052 were injected with anti-CD8 Abs. As shown in Fig. 4, protection was abrogated by depletion of CD8 + but not CD4 + T cells, indicating that tumor-specific CD8 T cells were critical mediators of tumor immunity.
  • CpG ODN and 3M-052 combination treatment significantly increased Thl cytokines and CTL associated protein expression in the TIL, including IL-12 (Fig. 3), IFN- ⁇ (Fig. 3), Granzyme B (Fig. 3C) as compared with CpG ODN and 3M-052 alone treatment groups.
  • CpG ODN or 3M-052 alone only slightly induced production of these cytokines, as compared with the group treated with phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • CD8 T cells Treatment with CpG ODN and/or 3M-052 led to a significant changes in the frequency of CD8 T cells, NK cells and MDSC (Fig. 2).
  • the genes selected to evaluate CD8 and NK cell responses were IL-12 and IFNg (which contribute to the induction and maintenance of) and granzyme B (which mediates their cytotoxicity) (Trinchieri et al., Blood 1994, 84:4008-4027; Komita et al., J Hepatol 45:662-672, 2006; Packard et al., J Immunol 179:3812- 3820, 2007).
  • mice treated with either CpG ODN or 3M-052 had higher levels of expression of IL-12, IFNy and granzyme B than tumor infiltrating cells from untreated mice (p. ⁇ .05).
  • mRNA levels were significantly higher when compared to either agonist alone (see Table legend). This effect was additive for IL-12 and IFNy and supra-additive for Granzyme B.
  • mice were treated as described in Fig. 1.
  • mRNA was isolated from tumor infiltrating cells one day after the second treatment and analyzed by T-PC . Each point represents the mean + SD fold difference in cells from treated vs untreated tumor bearing mice derived from independently studying 6 mice/group in 2 independent experiments. *, p ⁇ 0.05; **, p ⁇ 0.01, ***, p ⁇ 0.001 when compared to PBS treated controls. Note: the level of expression of all genes from mice treated with CpG ODN plus 3M-052 was also significantly different (p ⁇ .01 - 0.05) from that of mice treated with CpG ODN alone or 3M-052 alone.
  • TGFb TGFb, Argl and Nos2 (which mediate the inhibitory activity of MDSC) and CTLA-4 (which acts as a negative regulator of T cell activation).
  • Immune suppression in the tumor microenvironment can take many forms.
  • One metric of the down-regulation of CTL activity is the expression of CTLA-4 by T cells and another is the production of the immunoinhibitory molecule TGFB.
  • CTLA-4 a homologue and antagonist of CD28 (Li et al., Int J Cancer 131:2584-2595, 2012; Walunas et al., Immunity 1 :405-413, 1994); and acts as negative regulator of T cell activation by depriving them of CD28-mediated co- stimulation (Walunas et al., supra ; Maasteller et al., J Immunol, 164:5319-5327, 2000).
  • TGF suppresses both innate and adaptive immune responses in the tumor
  • CT26 cancer cells were implanted as described above and treatment initiated only after the resultant tumors reached -800 mm 3 in volume. Mice were then injected intra-tumorally twice weekly for one month with 200 ⁇ g of CpG ODN and/or 100 ⁇ g of 3M-052. Tumors in untreated mice proliferated rapidly over this period, reaching a volume of >2,000 mm 3 within 10 days (mandating their sacrifice as per ACUC guidelines, Fig 5). While both CpG ODN and 3M-052 therapy slowed tumor growth and prolonged survival, tumors in all animals reached the 2,000 mm 3 by 3 weeks after the initiation of treatment (Fig 5). In contrast, 87% (13/15) of the mice treated with the combination of CpG ODN plus 3M-052 in 3 independent experiments completely rejected their tumors (p ⁇ 0.01 ; Fig. 5).
  • lymphocytes were isolated from the draining lymph node (LN) of mice challenged with CT26 tumors one week after the initiation of therapy. These cells were then stimulated in vitro with AH-1 peptide and their production of IFNy monitored. As in Fig. 3, T cells from mice treated with the combination of CpG ODN plus 3M-052 generated significantly stronger tumor specific responses that did any of the controls (p. ⁇ .001, Fig. 7 A).
  • TLR 7/8 agonists were water soluble and proved ineffective when co-administered with CpG ODN.
  • a relatively new TLR 7/8 agonist was identified that contains a modified tail allowing it to persist in vivo after being injected into the tumor (3M-052) (Smirnov et al., Vaccine 29:5434-5442, 2011). Further studies therefore evaluated the activity of locally administered 3M-052 in combination with CpG ODN.
  • combination therapy was initially examined under conditions where a single TLR agonist only delayed tumor growth (Fig. 1). Large tumors were then studied in which the combination of CpG ODN plus 3M-052 proved highly successful against both CT26 colon cancer and B10-F16 melanomas. Whereas each agonist alone barely delayed the progression of these large tumors, cure rates on the order of 80 - 90% were achieved by combination therapy. Indeed, as weeping of the injected material from the tumor site was sometimes observed, it is possible that even higher success rates might be achieved by technical improvements in TLR agonist delivery. Successful therapy of large tumors required twice- weekly treatment with CpG ODN plus 3M-052 over the course of ⁇ 1 month. A single dose had no detectable effect on the growth of large tumors while 1 - 2 weeks of combination therapy resulted in only short-lived tumor regression. Systemic treatment was uniformly unsuccessful.
  • CD28 is a co-stimulatory molecule that enhances the proliferation, cytokine production and survival of TCR-activated T cells. This process is antagonized by CTLA-4, a surface receptor that is up-regulated when T cells become activated (Walunas, Immunity 1:405-413, 1994; Masteller et al., J Immunol 164:5319-5327, 2000). It was observed that the level of mRNA encoding CTLA-4 was significantly reduced in mice receiving combination therapy (Table 1). This down-modulation of CTLA-4 may help explain the improved activity of tumor- specific T cells found in the current work (Fig. 3).
  • TGF is produced by tumor cells and Gr-1+ CD1 lb+ MDSC in the tumor microenvironment and serves to suppress both innate and adaptive arms of the immune system (Li et al., Int J Cancer 2012, 131:2584-2595; Bierie et al., Nat Rev Cancer 6:506-520, 2006; Flavell et al., Nat Rev Immunol 10:554-567, 2012).
  • TGF signaling can enhance tumor elimination by improving CTL activity (Gorelik and Flavell, Nat Med 1: 111 ⁇ -1122, 2001; Nam et al. Cancer Res 68:3915-3923, 2008).
  • CpG ODN A number of clinical trials have explored the activity of CpG ODN in cancer patients.
  • CpG treatment induces a dose-related increase in serum levels of IP-10, IFNa, MIP-la, and IL-12p40 (Kreig, Curr Oncol Rep 2004, 6:88-95, 2004;Offersen et al., Hum Vaccin Immunother 2012, 8: 1042-1047, 2001).
  • anti-tumor activity was observed in several phase II trials (Kreig et al., J Immunother 27:460-471, 2004) this finding was not reproduced in a definitive phase III study (Hirsh et al., J Clin Oncol 29:2667-2674, 2011; Manegold et al, Ann Oncol 23:72-77, 2012).
  • combination TLR 7/8/9 agonists can be critical for improving the host's anti-tumor response by acting on multiple cell types in the tumor microenvironment, including mMDSC, CD8 T lymphocytes and NK cells.
  • MDSC express receptors for both agonists and play a vital role protecting tumors from immune aggression by inhibiting T and NK cell activity (see, for example, Li et al., J Immunol 182:240-249, 2009).
  • the findings disclosed herein demonstrate that the combination of CpG ODN plus 3M-052 reduced mMDSC frequency by 10-fold when compared to untreated mice and 3-5 fold when compared to either agonist alone (Fig. 2).

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Abstract

L'invention concerne des procédés pour traiter une tumeur telle qu'une tumeur bénigne ou maligne. Les procédés comprennent l'administration au sujet d'une quantité thérapeutiquement efficace d'un agoniste de TLR-9, tel qu'un CpG ODN, et d'une quantité thérapeutiquement efficace d'un agoniste de TLR-7 et/ou de TLR-8, tel qu'un composé d'imidazoquinoléine. Ces composés agissent de façon synergique pour traiter la tumeur chez le sujet. Les procédés sont également utilisés pour réduire la dimension et/ou le nombre de métastases.
PCT/US2014/042117 2013-06-12 2014-06-12 Agoniste de tlr-9 comportant un agoniste de tlr-7 et/ou de tlr-8 pour traiter des tumeurs WO2014201245A1 (fr)

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EP3174538A4 (fr) * 2014-08-01 2018-02-28 3M Innovative Properties Company Méthodes et combinaisons thérapeutiques de traitement de tumeurs
WO2018060513A1 (fr) * 2016-09-30 2018-04-05 Galderma Research & Development Méthodes et compositions pour le traitement de lésions précancéreuses ou d'un cancer comprenant des agonistes de tlr/tlr ou tlr/clr
WO2018132539A1 (fr) * 2017-01-12 2018-07-19 Medimmune, Llc Sécurité et activité pharmacodynamique d'un agoniste du récepteur 7/8 de type toll
WO2020160054A1 (fr) 2019-02-01 2020-08-06 Canwell Biotech Limited Dérivés d'imidazoquinoline amine, composition pharmaceutique, utilisation de ceux-ci
CN111511740A (zh) * 2017-12-20 2020-08-07 3M创新有限公司 用作免疫应答调节剂的带有支链连接基团的酰胺取代的咪唑并[4,5-c]喹啉化合物
US10837018B2 (en) 2013-07-25 2020-11-17 Exicure, Inc. Spherical nucleic acid-based constructs as immunostimulatory agents for prophylactic and therapeutic use
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