WO2024077228A2 - Double-stranded oligonucleotides and methods of use - Google Patents

Abstract

The disclosure provides, inter alia, compounds comprising a phosphorothioated CpG oligodeoxynucleotide linked to a DNA oligonucleotide that is hybridized to another DNA oligonucleotide that contain a constrained nucleotide, pharmaceutical compositions containing the compounds, and methods of treating cancer using the compounds.

Description

DOUBLE-STRANDED OLIGONUCLEOTIDES AND METHODS OF USE

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to US Application No. 63/414,160 filed October 7, 2022, the disclosure of which is incorporated by reference herein in its entirety.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0002] This invention was made with government support under W81XWH-19-1-0852 awarded by the Medical Research and Development Command, and R01 CA215183 awarded by the National Institutes of Health. The government has certain rights in the invention.

REFERENCE TO A "SEQUENCE LISTING," A TABLE, OR A COMPUTER PROGRAM LISTING APPENDIX SUBMITTED AS AN ASCII FILE

[0003] The Sequence Listing entitled "‘048440-850001WO-Sequence Listing,’" written in XML format, having 92,140 bytes, created on October 3, 2023, is incorporated by reference.

BACKGROUND

[0004] The immune system can serve as extrinsic tumor suppressor. However, the microenvironment of established tumors is typically characterized by a paucity of tumor-specific CD8⁺ T cells together with an excess of suppressive regulatory T cells and myeloid-denved suppressor cells that promote tumor immune evasion. Myeloid cells and other immune cells in the tumor microenvironment also produce growth factors and angiogenic/metastatic factors critical for tumor progression. Signal Transducer and Activator of Transcription 3 (STAT3) is an important oncogenic molecule. The orchestration of these processes in the tumor microenvironment is highly dependent on the oncogenic transcription factor, STAT3. In particular, STAT3 plays a critical role in mediating tumor immune evasion. Activated STAT3 in myeloid cells inhibits expression of a large number of immunostimulatory molecules related to Thl-type responses, while promoting production of several key immunosuppressive factors as well as angiogenic factors. In addition, by mediating signaling of certain cytokines and grow th factors, notably IL-6, STAT3 activation in myeloid cells activates STAT3 in tumor cells, enhancing tumor cell proliferation and survival. There is a need in medicine for new compounds and methods for treating of cancer that are safe and effective. The disclosure is directed to these, as well as other, important ends. BRIEF SUMMARY

[0005] Provided herein are compounds comprising a phosphorothioated CpG oligodeoxynucleotide (ODN) linked to a first DNA oligonucleotide, wherein the first DNA oligonucleotide is hybridized to a second DNA oligonucleotide; wherein the first DNA oligonucleotide comprises from about 5 unmodified nucleotides to about 70 unmodified nucleotides, and the second DNA oligonucleotide comprises: (i) from about 5 nucleotides to about 70 nucleotides, and (ii) a constrained nucleotide on the 3‘ end and a constrained nucleotide on the 5’ end. In embodiments, the constrained nucleotide is a LNA-modified nucleotide, a cMOE-modified nucleotide, or a cET-modified nucleotide.

[0006] Provided herein are methods of treating cancer by administering to a patient an effective amount of the compounds described herein. In embodiments, the methods further comprise administering to the patient an effective amount of an immune checkpoint inhibitor, such as a PD-1 pathway inhibitor.

[0007] These and other embodiments and aspects of the disclosure are described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0008] FIGS. 1A-1D show serum stability and activity of the double-stranded antisense oligonucleotides. FIG. 1A: Double-stranded STAT3ASO shows greater serum stability than single-stranded oligonucleotides. Oligonucleotides before and after incubation in 50% human serum were resolved on 15% PAGE. FIGS. 1B-1C: Double-stranded STAT3- (FIGS. IB, ID) or androgen receptor (AR)-specific ASOs show comparable or improved knockdown of their respective targets compared to their respective single-stranded ASOs within 48h after transfection (FIGS. 1B-1C; 100 ng each) or spontaneous/gymnotic uptake (FIG. ID; 1 pM) in human LnCaP prostate cancer. A431 carcinoma, U118 glioma or mouse GL261 glioma cells. With reference to FIGS. 1A-1D, STAT3ASO is SEQ ID NO:4; STAT3 dsASO is SEQ ID NO:3 hybridized to SEQ ID NO:4; AR-ASO is 5’ accAAGTTTCTTCagc (SEQ ID NO:35, where bold-lower case indicates LNA-modified nucleotide) (described by Zhang et al, Nucleic Acids Research, 46(7):3612-3624 (2018); and ds-AR-ASO (SEQ ID NO:35) hybridized to 5’gctgaagaaacttggt (SEQ ID NO:36)

[0009] FIGS. 2A-2D show design, serum stability and in vitro activity of the CpG-conjugated double-stranded STAT3 antisense oligonucleotides. FIG. 2A: Schematic design of the doublestranded CpG-STAT3dsASO. FIG. 2B: Serum stability' of the double-stranded CpG- STAT3dsASO compared to each separate oligonucleotide strand (ASO alone and CpG- passenger strand). Oligonucleotides were incubated in 50% human serum at 37°C for the indicated times and then resolved on 15% PAGE. FIGS. 2C-2D: Double-stranded CpG- STAT3dsASO retains target knockdown potency and kinetics comparable with STAT3ASO alone. Human A431 (FIG. 2C) and mouse GL261 cells (FIG. 2D) were incubated for various times with 1 pM of the indicated oligonucleotides. Western blot assessment of STAT3 protein normalized to the level of P-actin used as a control. With reference to FIGS. 2A-2D, CpG- STAT3 dsASO is SEQ ID NO: 15 linked via the moiety of Formula (A) to SEQ ID NO:3, wherein SEQ ID NO:3 is hybridized to SEQ ID NO:4; CpG-passenger is SEQ ID NO: 15 linked via the moiety of Formula (A) to SEQ ID NO:3; and STAT3ASO is SEQ ID NO:4; wherein the moiety of Formula (A)(in this figure and throughout the application) is:

[0010] FIGS. 3A-3F show that CpG-STAT3dsASO shows better potency at target gene knockdown than single-stranded CpG-STAT3ASO variants in different human and mouse target cells. Human prostate cancer cells (DU145, LAPC4, and ENZR42D) (FIGS. 3A-3C), human (U251) (FIG. 3D) and mouse (GL261) glioma cells (FIG. 3E). and mouse macrophages (RAW264.7) (FIG. 3F) were incubated for 72 h with 1 pM of the indicated 2’-O-methyl- or LNA-modified oligonucleotides. Total STAT3 protein levels were quantified by Western blotting, with normalization of P-actin; shown in quantification of band intensities. With reference to FIGS. 3A-3F, CpG-STAT3ASO (2'OMe) is SEQ ID NO: 15 linked via the moiety of Formula (A) to SEQ ID NO:33; CpG-passenger (LNA) SEQ ID NO: 15 linked via the moiety of Formula (A) to SEQ ID NO: 3; STAT3ASO (LNA) is SEQ ID NO:4; CpG-STAT3 dsASO (LNA) is SEQ ID NO: 15 linked via the moiety of Formula (A) to SEQ ID NO:3, wherein SEQ ID NO:3 is hybridized to SEQ ID NO:4; and CpG-STAT3 ssASO (LNA) is SEQ ID NO: 15 linked via the moiety of Formula (A) to SEQ ID NO:4.

[0011] FIGS. 4A-4F provide a comparison of time- and dose-dependent ASO uptake by human and mouse target cells. Human (U251, LN229, T98G) (FIGS. 4A-4C) and mouse (GL261, K-luc) glioma cells (FIGS. 4D-4E) and mouse microglia cell line (BV2) (FIG. 4F) were incubated for 1 h or 4 h with fluorescently-labeled double-stranded CpG-STAT3dsASO^Cy3 or with single-stranded CpG-STAT3ssASO^Cy3 or unconjugated STAT3ssASO^Cy3 at 100 nM or 500 nM concentrations. The oligonucleotide internalization was assessed using flow cytometry. With reference to FIGS. 4A-3F, STAT3ASO is SEQ ID NO:4; CpG-STAT3 dsASO is SEQ ID NO: 15 linked via the moiety of Formula (A) to SEQ ID NO:3, wherein SEQ ID NO:3 is hybridized to SEQ ID NO:4; and CpG-STAT3 ssASO is SEQ ID NO: 15 linked via the moiety of Formula (A) to SEQ ID NO:4.

[0012] FIGS. 5A-5E show a comparison of time- and dose-dependent ASO uptake by primaryhuman immune cells. Human PBMCs were incubated for 1 h or 4 h with fluorescently-labeled double-stranded CpG-STAT3dsASO^Cy3 or with single-stranded CpG-STAT3ssASO^Cy3 or unconjugated STAT3ssASO^Cy3 at 100 nM or 500 nM concentrations. The oligonucleotide internalization was assessed using flow cytometry. Shown are histograms indicating oligonucleotide uptake by primary human CDlc⁺ myeloid dendritic cells (mDC) (FIG. 5A), CD303⁺plasmacytoid DC (pDC) (FIG. 5B), CD14⁺monocytes (FIG. 5C), CD19⁺ B cells (FIG. 5D), and CD3⁺T cells (FIG. 5E). With reference to FIGS. 5A-5E, STAT3ASO is SEQ ID NO:4; CpG-STAT3 dsASO is SEQ ID NO: 15 linked via the moiety of Formula (A) to SEQ ID NO:3, wherein SEQ ID NO:3 is hybridized to SEQ ID NO:4; and CpG-STAT3 ssASO is SEQ ID NO: 15 linked via the moiety of Formula (A) to SEQ ID NO:4.

[0013] FIGS. 6A-6D show a comparison of dose-dependent ASO uptake by primary mouse immune cells. Mouse splenocytes were incubated for 4 h with fluorescently-labeled doublestranded CpG-STAT3dsASO^Cy3 or with single-stranded CpG-STATSssASO¹^³ or unconjugated STAT3ssASO^Cy3 at 100 nM or 500 nM concentrations. The oligonucleotide internalization was assessed using flow cytometry. Shown are histograms indicating oligonucleotide uptake by CD1 lb⁺CDl lc- macrophages (FIG. 6A), CD1 lb⁺CDl lc⁺ dendritic cells (DCs) (FIG. 6B), CD19+ B cells (FIG. 6C), and CD3⁺ T cells (FIG. 6D). With reference to FIGS. 6A-6D, STAT3ASO is SEQ ID NO:4; CpG-STAT3 dsASO is SEQ ID NO: 15 linked via the moiety of Formula (A) to SEQ ID NO:3. wherein SEQ ID NO:3 is hybridized to SEQ ID NO:4; and CpG- STAT3 ssASO is SEQ ID NO: 15 linked via the moiety of Formula (A) to SEQ ID NO:4.

[0014] FIGS. 7A-7F show biodistribution of the intratumorally injected CpG-STAT3ASOs in mouse brain. C57/BL6 mice bearing intracranial GL261 glioma tumors were injected with 1 mg/kg of single- or double-stranded CpG-STAT3ASO^Cy3 or with unconjugated STAT3ASO^Cy3. Mice were euthanized 3 days or 5 days later to harvest brains. Percentages of Cy3⁺ Ml-like macrophages (CD1 lb'CD45^l"g^llCD86' ) (FIG. 7A). M2-like macrophages

(CD1 1 b⁺CD45^lug^hCD206⁺) (FIG. 7B), Ml-like microglia (CD11 b⁺CD45^lowCD86⁺) (FIG. 7C), M2-like microglia (CDl lb⁺CD45^hig^hCD206⁺) (FIG. 7D), DCs (CDllb⁺CDl lc⁺) (FIG. 7E), and MDSCs (CD1 lb⁺Grl⁺) (FIG. 7F) were assessed using flow cytometry in single-cell suspensions of non-tumor bearing and tumor bearing brain hemisphere. Results of experiments using 34 mice per each group; means+SEM. With reference to FIGS. 7A-7F, CpG-STAT3ASO (2'0Me) is SEQ ID NO: 15 linked via the moiety of Formula (A) to SEQ ID NO:33; STAT3ASO-h/m#3(LNA) is SEQ ID NO:4; and CpG-STAT3ASO h/m#3(LNA) is SEQ ID NO: 15 linked via the moiety of Formula (A) to SEQ ID NO:4.

[0015] FIGS. 8A-8F show that double-stranded CpG-STAT3dsASO is safer and better tolerated by naive mice than single-stranded CpG-STAT3ssASO. FIG. 8A: Determination of maximum tolerated dose (MTD) of CpG-STAT3ASO variants in naive mice. C57BL/6 mice were injected intracranially using 0.1, 0.3. or 1 mg/kg of CpG-STAT3 dsASO or CpG- STAT3ssASO twice a week and euthanized 2 weeks later. Body weight measurements for each dosing group; shown are means±SEM. FIGS. 8B-8C: Mouse behavior assessed using acute tolerability scoring system (ATSS) (FIG. 8B) is altered more significantly by low doses of the single-stranded but not by double-stranded CpG-STAT3ASO (FIG. 8C). The assessment of acute CNS toxicity based on the mice phenotypic behavior at I and 4 h after IC injection of CpG-STAT3ASOs. FIGS. 8D-8F: Single- but not double-stranded CpG-STAT3ASO induces significant changes in leukocytes and platelets in treated mice. Hematological parameters such as red blood cells (RBC), white blood cells (WBC) and platelets (PLT) assessed in blood collected from mice treated as described above. Grey area indicates the expected normal range for each parameter. Results of experiments using a total of 3-4 mice from each group; means±SEM. With reference to FIGS. 8A-8F, dsASO is SEQ ID NO: 15 linked via the moiety' of Formula (A) to SEQ ID NO:3, wherein SEQ ID NO:3 is hybridized to SEQ ID NO:4; and ssASO is SEQ ID NO: 15 linked via the moiety of Formula (A) to SEQ ID NO:4.

[0016] FIGS. 9A-9B show local administration of LNA-modified single- or double-stranded CpG-STAT3ASOs both lead to similar direct antitumor effects against intracranial human glioma xenotransplants in immunodeficient mice. Human U251 glioma-bearing NSG mice were injected intratumorally/IC using 1 mg/kg of 2’O-methyl- or LNA-modified CpG-STAT3ASOs every other day as indicated by red arrows. FIG. 9A: Tumor progression and occasionally extracranial spread was monitored using bioluminescent imaging (BLI). FIG. 9B: Both types of CpG-STAT3ASO^LNA significantly extended animal survival; shown are the Kaplan-Meier survival curves (n=5-6). With reference to FIGS. 9A-9B, CpG-STAT3ASO (2’0Me) is SEQ ID NO: 15 linked via the moiety of Formula (A) to SEQ ID NO: 34; CpG-STAT3dsASO (LNA) is SEQ ID NO:43 linked via the moiety of Formula (A) to SEQ ID NO: 1, wherein SEQ ID NO: 1 is hybridized to SEQ ID NO:2; and CpG-STAT3-ssASO (LNA) is SEQ ID NO:43 linked via the moiety of Formula (A) to SEQ ID NO:2. [0017] FIGS. 10A-10I show local administration of single- and double-stranded CpG- STAT3ASOs improves animal survival and triggers immune activation in immunocompetent mice. C57BL/6 mice bearing established GL261 gliomas were injected intratumorally/IC using 0.25 mg/kg of 2’-O-methyl-or LNA-modified CpG-STAT3ASOs twice weekly. FIG. 10A: CpG-STAT3ASO treatments significantly improved animal survival; shown are the Kaplan- Meier survival curves (n=10-l l). FIGS. 10B-10F: All three types of CpG-STAT3ASO injections induced maturation/activation of intratumoral DCs. macrophages and microglia, while reducing numbers of tumor-associated M2 macrophages and resting microglia as assessed using flow cytometry'. Show n are percentages of mature DCs (CD1 lb⁺CDl lc⁺MHCII⁺ CD80⁺), mature macrophages (CD1 lb⁺CDl lc’MHCII+CD80+MHCII⁺CD80⁺), M2 -like macrophages (CD 1 lb ¹ F4/80¹ CD206¹ ), activated microglia (CD 11 b ¹ CD45^lowMHCII ¹ CX3CR1 ¹ ) and resting microglia (CDl lb⁺CD45^lowMHCirCX3CRl⁺). FIGS. 10G-10I: CpG-STAT3ASO injections improve the ratio of intratumoral CD8 T cells to Tregs. Shown are percentages of CD8⁺ T cells (CD3⁺CD8⁺), regulatory T cells (CD3⁺CD4⁺FOXP3⁺), and their ratio. Data are presented as means±SEM; *P < 0.05, **P < 0.01, *** < 0.001 by one-way ANOVA with Tukey’s multiplecomparison post hoc test. With reference to FIGS. 10A-10I, CpG-STAT3ASO (2’OMe) is SEQ ID NO:15 linked via the moiety of Formula (A) to SEQ ID NO:33; CpG-STAT3dsASO (LNA) is SEQ ID NO: 15 linked via the moiety of Formula (A) to SEQ ID NO:3, which is hybridized to SEQ ID NO:4; and CpG-STAT3-ssASO (LNA) is SEQ ID NO: 15 linked via the moiety of Formula (A) to SEQ ID NO:4.

[0018] FIGS. 11A-11F: show the combination of CpG-STAT3dsASO with PD1 blockade results in glioma regression in the majority of treated mice. C57BL/6 mice bearing established GL261 gliomas were injected twice weekly using intraperitoneal injections of 200 pg of PD1- specific or control antibodies, using intratumoral/IC injections of 0.25 mg/kg of LNA-modified CpG-STAT3dsASO or both treatments combined. FIG. 11A-11E: Tumor progression was monitored using BLI. FIG. 11F: The combination CpG-STAT3ASO/anti-PDl treatments led to complete glioma regression in 5/6 treated mice; shown is the Kaplan-Meier survival curve (n=6- 7/group). With reference to FIGS. 11A-11F, IgG is polyclonal rat IgG (Bio X Cell, Cat: BE0094); Anti-PD-1 is anti-mouse PD-1 antibody (from Bio X Cell, Clone:29F. lA12, Cat: BE0273); and CpG-STAT3 dsASO (LNA) is SEQ ID NO: 15 linked via the moiety of Formula (A) to SEQ ID NO:3, which is hybridized to SEQ ID NO:4.

[0019] FIG. 12 shows that double-stranded CpG-STAT3dsASO (bottom right panel) has greater potency than single-stranded CpG-STAT3ssASO (top right panel) or CpG-psODN (CpG passenger strand only) (bottom left panel) against RM9 prostate tumors. C57BL/6 mice bearing established, dual RM9 tumors were treated using local intratumoral injection of indicated oligonucleotide. Tumor growth progression was monitored by caliper measurement at the secondary, not injected tumor site; (n=4-5/group).

[0020] FIG. 13 shows that local administration of LNA-modified single- or double-stranded CpG-STAT3ASOs improved survival in mice bearing syngeneic intracranial QPP8 glioma. C57BL/6 mice bearing established QPP8 gliomas (Qk/Tp53/Pten-deY) were injected intracranially /IC using 0.25mg/kg of LNA-modified CpG-STAT3ASOs twice weekly. CpG STAT3ASO treatments significantly improved animal survival; shown are Kaplan-Meier survival curves (n=6/group). The median survival for PBS was 49 days. The median survival for LNA-modified STAT3 ASO was 52 days. The median survival for LNA-modified CpG- STAT3-double stranded ASO (CpG-STAT3dsASO^LNA) was 66.5 days. The median survival for LNA-modified CpG-STAT3-single stranded ASO (CpG-STAT3ssASO^LNA) was 61.5 days.

[0021] FIG. 14 shows that the combination of CpG-STAT3dsASO ith PD1 blockade resulted in QPP8 glioma regression in the majority of treated mice. C57BL/6 mice bearing established QPP8 gliomas were injected twice weekly using Intraperitoneal/IP using 200pg of PDl-specific antibodies, using intracranially /IC of 0.25mg/kg of LNA-modified CpG- STAT3ASOs or both treatment combined. The combination of CpG-STAT3dsASO/anti-PDl led the glioma regression 4/5 treated mice; shown are Kaplan -Meier survival curves (n=5/group).

[0022] FIG. 15 shows that surviving mice were protected from rechallenge with the same tumor. Mice surviving after the combination anti-PDl/CpG-STAT3dsASO treatment were rechallenged using intracranial injection with 10⁴ GL261 cells and the tumor progression was monitored using bioluminescent imaging (BLI). Most of the previously treated survivor mice but not naive mice survived glioma rechallenge; shown are Kaplan-Meier survival curves (n=4- 5/group). The median survival for the control group w^?as 21 days. The median survival for the mice receiving the combination anti-PDl and LNA-modified CpG-STAT3dsASO treatment was more than 152 days. With reference to FIGS. 12-15, LNA-modified STAT3 ASO is SEQ ID NO:4; LNA-modified CpG-STAT3-double stranded ASO (CpG-STAT3dsASO^LNA) is SEQ ID NO: 15 linked via the moiety of Formula (A) to SEQ ID NO:3, wherein SEQ ID NO:3 is hybridized to SEQ ID NO:4; and LNA-modified CpG-STAT3-single stranded ASO (CpG- STAT3SSASO^LNA) is SEQ ID NO: 15 linked via the moiety of Formula (A) to SEQ ID NO:4. DETAILED DESCRIPTION

[0023] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary' skill in the art. See, e.g., Singleton et al., Dictionary of Microbiology and Molecular Biology. 2nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989). Any methods, devices and materials similar or equivalent to those described herein can be used in the practice of this disclosure. The following definitions are provided to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present disclosure.

[0024] The term “CpG oligodeoxynucleotide” or “CpG ODN” refers to a 5’ C nucleotide connected to a 3’ G nucleotide through a phosphodiester intemucleotide linkage or a phosphodiester derivative intemucleotide linkage. In embodiments, a CpG ODN includes a phosphodiester intemucleotide linkage. In embodiments, a CpG ODN includes a phosphodiester derivative intemucleotide linkage.

[0025] The term “Class A CpG ODN'’ or “A-class CpG ODN” or “D-type CpG ODN” or “Class A CpG DNA sequence” refers to a CpG motif including oligodeoxynucleotide including one or more of poly-G sequence at the 5’, 3’, or both ends; an internal palindrome sequence including CpG motif; or one or more phosphodiester derivatives linking deoxynucleotides. In embodiments, a Class A CpG ODN includes poly-G sequence at the 5’, 3’, or both ends; an internal palindrome sequence including CpG motif; and one or more phosphodiester derivatives linking deoxynucleotides. In embodiments, the phosphodiester derivative is phosphorothioate Examples of Class A CpG ODNs include ODN D19, ODN 1585, ODN 2216, and ODN 2336, the sequences of which are known in the art.

[0026] The term “Class B CpG ODN” or “B-class CpG ODN” or “K-type CpG ODN” or “Class B CpG DNA sequence” refers to a CpG motif including oligodeoxynucleotide including one or more of a 6mer motif including a CpG motif; phosphodiester derivatives linking all deoxynucleotides. In embodiments, a Class B CpG ODN includes one or more copies of a 6mer motif including a CpG motif and phosphodiester derivatives linking all deoxynucleotides. In embodiments, the phosphodiester derivative is phosphorothioate. In embodiments, a Class B CpG ODN includes one 6mer motif including a CpG motif. In embodiments, a Class B CpG ODN includes two copies of a 6mer motif including a CpG motif. In embodiments, a Class B CpG ODN includes three copies of a 6mer motif including a CpG motif. In embodiments, a Class B CpG ODN includes four copies of a 6mer motif including a CpG motif. Examples of Class B CpG ODNs include ODN 1668, ODN 1826, ODN 2006, ODN 2007, ODN BW006, and ODN D-SL01, the sequences of which are known in the art.

[0027] The term “Class C CpG ODN’' or “C-class CpG ODN” “ or “C-type CpG DNA sequence” refers to an oligodeoxynucleotide including a palindrome sequence including a CpG motif and phosphodiester derivatives (phosphorothioate) linking all deoxynucleotides. Examples of Class C CpG ODNs include ODN 2395, ODN M362, and ODN D-SL03, the sequences of which are known in the art.

[0028] The term “STAT” or “STAT transcription factor” refer to a “Signal transducer and activator of transcription” protein and homologs thereof (e.g. STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, STAT6, STAT7, STAT8, STAT7/8, STAT9). In embodiments, “STAT transcription factor” refers to a human protein. Included in the term “STAT transcription factor” are the w ildtype and mutant forms of the protein. In embodiments, “STAT transcription factor” refers to the wildtype protein. In embodiments, “STAT transcription factor” refers to a mutant protein. “Phosphorylated STAT” refers to a STAT protein that is phosphorylated and activated by the phosphorylation. In embodiments, activation of a STAT transcription factor means the STAT is capable of activating transcription.

[0029] A “STAT3” or “STAT3 protein” refers to any of the recombinant or naturally- occurring forms of the Signal transducer and activator of transcription 3 (STAT3) protein or variants or homologs thereof that maintain STAT3 protein activity (e g. within at least 50%, 80%. 90%. 95%. 96%. 97%. 98%. 99% or 100% activity compared to STAT3). In embodiments, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the w hole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring STAT3 polypeptide. In embodiments, the STAT3 protein is substantially identical to the protein identified by the NCBI reference number GI: 47458820, or a variant or homolog having substantial identity thereto. In embodiments, the STAT3 protein is substantially identical to the protein identified by the NCBI reference number GI: 1610577068, or a variant or homolog having substantial identity thereto. In embodiments, the STAT3 protein is substantially identical to the protein identified by the NCBI reference number GI: 1610577050. or a variant or homolog having substantial identity thereto. “Phosphorylated STAT3” refers to a STAT3 protein that is phosphorylated and activated by the phosphorylation. In embodiments, a phosphory lated STAT3 is phosphorylated on tyrosine 705 or the residue corresponding to tyrosine 705 in homologs. In embodiments, activation of STAT3 means the STAT3 is capable of activating transcription. [0030] The terms "STAT3 gene'’ or “STAT3 sequence” as used herein refer to the gene or variants thereof that code for an STAT3 polypeptide capable of maintaining the activity of the STAT3 polypeptide (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the STAT3 polypeptide). In embodiments, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity⁷ across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous nucleic acid portion) compared to the STAT3 sequence. In embodiments, STAT3 is substantially identical to the nucleic acid sequence identified by Accession No. NG007370 or a variant or homolog having substantial identity⁷ thereto.

[0031] The term “STAT4” refers to a ‘‘Signal transducer and activator of transcription 4” protein and homologs thereof. In embodiments, “STAT4” refers to the protein associated with Entrez Gene 6775, OMIM 600558, UniProt Q14765, and/or RefSeq (protein) NP001230764. In embodiments, the reference numbers immediately above refer to the protein, and associated nucleic acids, known as of the date of filing of this application.

[0032] The term “STAT5A” refers to a ‘'Signal transducer and activator of transcription 5A” protein and homologs thereof. In embodiments, “STAT5A” refers to the protein associated with Entrez Gene 6776, OMIM 601511, UniProt P42229, and/or RefSeq (protein) NP003143. In embodiments, the reference numbers immediately above refer to the protein, and associated nucleic acids, known as of the date of filing of this application.

[0033] The term “STAT5B” refers to a “Signal transducer and activator of transcription 5B” protein and homologs thereof. In embodiments, “STAT5B” refers to the protein associated with Entrez Gene 6777, OMIM 604260, UniProt P51692, and/or RefSeq (protein) NP036580. In embodiments, the reference numbers immediately above refer to the protein, and associated nucleic acids, known as of the date of filing of this application.

[0034] The term “STAT6” refers to a “Signal transducer and activator of transcription 6"’ protein and homologs thereof. In embodiments, “STAT6” refers to the protein associated with Entrez Gene 6778, OMIM 601512, UniProt P42226, and/or RefSeq (protein) NP001 171549. In embodiments, the reference numbers immediately above refer to the protein, and associated nucleic acids, known as of the date of filing of this application.

[0035] The term “linked” or “conjugated” when referring to two moieties (e.g., a phosphorothioated CpG ODN linked to a first DNA oligonucleotide) means the two moieties are bonded, wherein the bond or bonds connecting the two moieties are covalent or non-covalent. In embodiments, the two moieties are covalently bonded to each other (e.g. directly or through a linking group). Exemplary linking groups include a covalent bond, a nucleic acid sequence, a DNA sequence, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, substituted or unsubstituted heteroarylene, or combinations of two or more thereof.

[0036] “Nucleic acid” refers to nucleotides (e g., deoxy ribonucleotides or ribonucleotides) and polymers thereof in either single-, double- or multiple-stranded form, or complements thereof; or nucleosides (e.g., deoxyribonucleosides or ribonucleosides). In embodiments, “nucleic acid” does not include nucleosides. The terms “polynucleotide,” “oligonucleotide,” “oligo” or the like refer, in the usual and customary sense, to a linear sequence of nucleotides. The term “nucleoside” refers, in the usual and customary sense, to a glycosylamine including a nucleobase and a five-carbon sugar (ribose or deoxyribose). Non limiting examples, of nucleosides include, cytidine, uridine, adenosine, guanosine, thymidine and inosine. The term “nucleotide” refers, in the usual and customary sense, to a single unit of a polynucleotide, i.e., a monomer. Nucleotides can be ribonucleotides, deoxyribonucleotides, or modified versions thereof. Examples of polynucleotides contemplated herein include single and double stranded DNA. single and double stranded RNA, and hybrid molecules having mixtures of single and double stranded DNA and RNA. Examples of nucleic acid, e.g. polynucleotides, contemplated herein include any types of RNA, e.g. mRNA, siRNA, miRNA, and guide RNA and any types of DNA. genomic DNA. plasmid DNA. and minicircle DNA, and any fragments thereof. The term “duplex” in the context of polynucleotides refers, in the usual and customary sense, to double strandedness. Nucleic acids can be linear or branched. For example, nucleic acids can be a linear chain of nucleotides or the nucleic acids can be branched, e.g., such that the nucleic acids comprise one or more arms or branches of nucleotides. Optionally, the branched nucleic acids are repetitively branched to form higher ordered structures such as dendrimers and the like.

[0037] Nucleic acids, including e.g.. nucleic acids with a phosphorothioate backbone, can include one or more reactive moieties. As used herein, the term reactive moiety includes any group capable of reacting with another molecule, e.g., a nucleic acid or polypeptide through covalent, non-covalent or other interactions. By way of example, the nucleic acid can include an amino acid reactive moiety that reacts with an amio acid on a protein or polypeptide through a covalent, non-covalent or other interaction.

[0038] The terms also encompass nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non- naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphodiester derivatives including, e.g., phosphorami date, phosphorodiamidate, phosphorothioate (also known as phosphorothioate having double bonded sulfur replacing oxygen in the phosphate), phosphorodithioate, phosphonocarboxylic acids, phosphonocarboxylates, phosphonoacetic acid, phosphonoformic acid, methyl phosphonate. boron phosphonate, or O-methylphosphoroamidite linkages (see Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press) as well as modifications to the nucleotide bases such as, 2’0-methyl, 5 ’fluoro, 2'-deoxy-2'fluoro, 2'-deoxy, a universal base nucleotide, a 5-C methyl nucleotide, an inverted deoxybasic residue incorporation. 5-methyl cytidine, or pseudouridine; and peptide nucleic acid backbones and linkages. Other analog nucleic acids include those with positive backbones; non-ionic backbones, modified sugars, and non-ribose backbones (e.g. phosphorodiamidate morpholino oligos or locked nucleic acids (LNA) as known in the art). Nucleic acids containing one or more carbocyclic sugars are also included within one definition of nucleic acids. Modifications of the ribose-phosphate backbone may be done for a variety of reasons, e.g., to increase the stability and half-life of such molecules in physiological environments or as probes on a biochip. Mixtures of naturally occurring nucleic acids and analogs can be made; alternatively, mixtures of different nucleic acid analogs, and mixtures of naturally occurring nucleic acids and analogs may be made. In embodiments, the intemucleotide linkages in DNA are phosphodiester, phosphodiester derivatives, or a combination of both.

[0039] Nucleic acids can include nonspecific sequences. As used herein, the term ‘‘nonspecific sequence” refers to a nucleic acid sequence that contains a series of residues that are not designed to be complementary to or are only partially complementary to any other nucleic acid sequence. By way of example, a nonspecific nucleic acid sequence is a sequence of nucleic acid residues that does not function as an inhibitory nucleic acid when contacted with a cell or organism.

[0040] “Unmodified nucleotide” refers to a nucleotide that is not modified from its natural state.

[0041] “Modified nucleotide” refers to a nucleotide that is modified from its natural state. The modification to the nucleotide can be to the base, the sugar, the phosphate, or two or more thereof. Nucleotides can be modified, for example, to include 2'-O-aminopropyl group, a 2’-O- ethyl group, a 2’-fluoro group, a 2’-O-methyl group, 2’-deoxy-2’fluoro group, a 2’-O- methoxy ethyl group, a 2’-O-allyl group, a 2’-O-propyl group, a 2’-O-pentyl group, or a constrained nucleotide.

[0042] A “constrained nucleotide" refers to a nucleotide that is modified to maintain a rigid backbone structure. In embodiments, the constrained nucleotide refers to a nucleotide in which the pentose is modified to maintain a rigid backbone structure. Exemplary modifications to nucleotides that maintain a rigid structure include locked nucleic acids (e.g., LNA-modified nucleotides), cMOE-modified nucleotides, cEt-modified nucleotides, and the like. Constrained nucleotides are described, for example, by Pallan et al, Chem Commun (Camb), 48(66): 8195- 8197 (2012), the disclosure of which is incorporated by reference herein in its entirety.

[0043] “LNA-modified nucleotide” refers to a locked nucleotide or a bridged nucleotide in which the pentose moiety is modified with an extra bridge connecting the 2’ oxygen and 4’ carbon, as shown in the structure below.

where Bx is base. See Braasch et al, Chemistry & Biology. 8: 1-7 (2001).

[0044] “cMOE-modified nucleotide” or “2 ’,4 ’-constrained 2’-O-methoxyethyl-modified nucleotide” refers to a nucleotide re:

where Bx is the base. In embodiments, the cMOE-modified nucleotide is a (7?)-cMOE-modified nucleotide. In embodiments, the cMOE-modified nucleotide is a (S)-cMOE-modified nucleotide. See Pallan et al, Chem Commun (Camb), 48(66):8195-8197 (2012). [0045] “2’-O-ethyl-modified nucleotide” or “2’, 4’ -constrained 2'-O-ethyl-modified nucleotide” or ’cEt-modified nucleotide” refers to a nucleotide having the following structure:

where Bx is the base. In embodiments, the cEt-modified nucleotide is a (/ )-cEt-modified nucleotide. In embodiments, the cEt-modified nucleotide is a (5)-cEt-modified nucleotide. See Pallan et al, Chem Commun (Camb), 48(66):8195-8197 (2012).

[0046] A “spacer modification” refers to a moiety that does not include a nucleobase. Exemplary spacer modifications include an abasic spacer, a spacer phosphoramidite, abasic phosphoramidite, hexadecane phosphoramidite, octadecane phosphoramidite, a C6 disulfide phosphoramidite, and the like. In embodiments, the spacer phosphoramidite is a C3 spacer phosphoramidite, a C6 spacer phosphoramidite, or a C 12 spacer phosphoramidite.

[0047] A “C3 spacer phosphoramidite” is a spacer modification represented by the structure:

[0048] “Abasic spacer” or “dspacer” is a l’,2’-dideoxyribose without a nucleobase attached.

[0049] An “antisense nucleic acid” as referred to herein is a nucleic acid (e.g., DNA or RNA molecule) that is complementary’ to at least a portion of a specific target nucleic acid and is capable of reducing transcription of the target nucleic acid (e.g. mRNA from DNA), reducing the translation of the target nucleic acid (e.g. mRNA). altering transcript splicing (e.g. single stranded morpholino oligo), or interfering with the endogenous activity of the target nucleic acid. Typically, synthetic antisense nucleic acids (e.g. oligonucleotides) are generally between 15 and 25 bases in length. Thus, antisense nucleic acids are capable of hybridizing to (e.g. selectively hybridizing to) a target nucleic acid. In embodiments, the antisense nucleic acid hybridizes to the target nucleic acid in vitro. In embodiments, the antisense nucleic acid hybridizes to the target nucleic acid in a cell. In embodiments, the antisense nucleic acid hybridizes to the target nucleic acid in an organism. In embodiments, the antisense nucleic acid hybridizes to the target nucleic acid under physiological conditions. Antisense nucleic acids may comprise naturally occurring nucleotides or modified nucleotides such as, e.g., phosphorothioate, methylphosphonate, and anomeric sugar-phosphate, backbone-modified nucleotides.

[0050] In the cell, the antisense nucleic acids hybridize to the corresponding RNA forming a double-stranded molecule. The antisense nucleic acids interfere with the endogenous behavior of the RNA and inhibit its function relative to the absence of the antisense nucleic acid.

Furthermore, the double-stranded molecule may be degraded via the RNAi pathway. The use of antisense methods to inhibit the in vitro translation of genes is well known in the art. Further, antisense molecules which bind directly to the DNA may be used. Antisense nucleic acids may be single or double stranded nucleic acids. Non-limiting examples of antisense nucleic acids include siRNAs (including their derivatives or pre-cursors, such as nucleotide analogs), short hairpin RNAs (shRNA), micro RNAs (miRNA), saRNAs (small activating RNAs) and small nucleolar RNAs (snoRNA) or certain of their derivatives or pre-cursors.

[0051] A polynucleotide is typically composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); and thymine (T) (uracil (U) for thymine (T) when the polynucleotide is RNA). Thus, the term “polynucleotide sequence” is the alphabetical representation of a polynucleotide molecule; alternatively, the term may be applied to the polynucleotide molecule itself. This alphabetical representation can be input into databases in a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homology searching. Polynucleotides may optionally include one or more non-standard nucleotide(s), nucleotide analog(s) and/or modified nucleotides.

[0052] “Conservatively modified variants” applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, “conservatively modified variants” refers to those nucleic acids that encode identical or essentially identical amino acid sequences. Because of the degeneracy of the genetic code, a number of nucleic acid sequences will encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every' position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent variations,” which are one species of conservatively modified variations. Every' nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for try ptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.

[0053] The term “complement,” as used herein, refers to a nucleotide (e.g.. RNA or DNA) or a sequence of nucleotides capable of base pairing with a complementary nucleotide or sequence of nucleotides. As described herein and commonly know n in the art the complementary' (matching) nucleotide of adenosine is thymidine and the complementary (matching) nucleotide of guanosine is cytosine. Thus, a complement may include a sequence of nucleotides that base pair with corresponding complementary nucleotides of a second nucleic acid sequence. The nucleotides of a complement may partially or completely match the nucleotides of the second nucleic acid sequence. Where the nucleotides of the complement completely match each nucleotide of the second nucleic acid sequence, the complement forms base pairs with each nucleotide of the second nucleic acid sequence. Where the nucleotides of the complement partially match the nucleotides of the second nucleic acid sequence only some of the nucleotides of the complement form base pairs with nucleotides of the second nucleic acid sequence. Examples of complementary' sequences include coding and a non-coding sequences, wherein the non-coding sequence contains complementary nucleotides to the coding sequence and thus forms the complement of the coding sequence. A further example of complementary sequences are sense and antisense sequences, wherein the sense sequence contains complementary nucleotides to the antisense sequence and thus forms the complement of the antisense sequence. The complementarity of sequences may be partial, in which only some of the nucleic acids match according to base pairing, or complete, where all the nucleic acids match according to base pairing. Thus, two sequences that are complementary to each other, may have a specified percentage of nucleotides that are the same (i.e., about 60% identity', preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%. 97%. 98%, 99%. or higher identity' over a specified region).

[0054] The term “gene” means the segment of DNA involved in producing a protein; it includes regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons). The leader, the trailer as well as the introns include regulatory' elements that are necessary' during the transcription and the translation of a gene. Further, a “protein gene product” is a protein expressed from a particular gene.

[0055] The word “expression” or “expressed” as used herein in reference to a gene means the transcriptional and/or translational product of that gene. The level of expression of a DNA molecule in a cell may be determined on the basis of either the amount of corresponding mRNA that is present within the cell or the amount of protein encoded by that DNA produced by the cell. The level of expression of non-coding nucleic acid molecules (e.g., siRNA) may be detected by standard PCR or Northern blot methods well know n in the art.

[0056] The phrase “specifically (or selectively) binds” to an antibody or “specifically (or selectively) immunoreactive with,” when referring to a protein or peptide, refers to a binding reaction that is determinative of the presence of the protein, often in a heterogeneous population of proteins and other biologies. Thus, under designated immunoassay conditions, the specified antibodies bind to a particular protein at least two times the background and more typically more than 10 to 100 times background. Specific binding to an antibody under such conditions requires an antibody that is selected for its specificity for a particular protein. For example, polyclonal antibodies can be selected to obtain only a subset of antibodies that are specifically immunoreactive with the selected antigen and not with other proteins. This selection may be achieved by subtracting out antibodies that cross-react with other molecules. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein.

[0057] The terms “isolate” or “isolated” when applied to a nucleic acid, virus, or protein, denotes that the nucleic acid, virus, or protein is essentially free of other cellular components with which it is associated in the natural state. It can be, for example, in a homogeneous state and may be in either a dry or aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein that is the predominant species present in a preparation is substantially purified.

[0058] “Percentage of sequence identity" is determined by comparing tw o optimally aligned sequences over a comparison window⁷, w herein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity⁷.

[0059] The terms “identical” or percent “identity ,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity⁷ over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms or by manual alignment and visual inspection (see, e.g., http://www.ncbi.nlm.nih.gov/BLAST/ or the like). Such sequences are then said to be “substantially identical.” This definition also refers to, or may be applied to, the compliment of a test sequence. The definition also includes sequences that have deletions and/or additions, as well as those that have substitutions. As described below, the preferred algorithms can account for gaps and the like. Preferably⁷, identity exists over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is 50-100 amino acids or nucleotides in length.

[0060] An amino acid or nucleotide base “position” is denoted by a number that sequentially identifies each amino acid (or nucleotide base) in the reference sequence based on its position relative to the N-terminus (or 5'-end). Due to deletions, insertions, truncations, fusions, and the like that must be taken into account when determining an optimal alignment, in general the amino acid residue number in a test sequence determined by simply counting from the N- terminus will not necessarily be the same as the number of its corresponding position in the reference sequence. For example, in a case where a variant has a deletion relative to an aligned reference sequence, there will be no amino acid in the variant that corresponds to a position in the reference sequence at the site of deletion. Where there is an insertion in an aligned reference sequence, that insertion w ill not correspond to a numbered amino acid position in the reference sequence. In the case of truncations or fusions there can be stretches of amino acids in either the reference or aligned sequence that do not correspond to any amino acid in the corresponding sequence.

[0061] The terms “numbered with reference to” or “corresponding to,” when used in the context of the numbering of a given amino acid or polynucleotide sequence, refers to the numbering of the residues of a specified reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence.

[0062] The term “about” means a range of values including the specified value, which a person of ordinary skill in the art would consider reasonably similar to the specified value. In embodiments, about means within a standard deviation using measurements generally acceptable in the art. In embodiments, about means a range extending to +/- 10% of the specified value. In embodiments, about means a range extending to +/- 5% of the specified value. In embodiments, about means a range extending to +/- 1 of the specified value. In embodiments, about includes the specified value.

[0063] “Control” or “control experiment” is used in accordance with its plain ordinary' meaning and refers to an experiment in which the subjects or reagents of the experiment are treated as in a parallel experiment except for omission of a procedure, reagent, or variable of the experiment. In embodiments, the control is used as a standard of comparison in evaluating experimental effects. In embodiments, a control is the measurement of the activity⁷ of a protein in the absence of a compound as described herein (including embodiments and examples). One of skill in the art will understand which standard controls are most appropriate in a given situation and be able to analyze data based on comparisons to standard control values. Standard controls are also valuable for determining the significance (e.g. statistical significance) of data. For example, if values for a given parameter are widely variant in standard controls, variation in test samples will not be considered as significant.

[0064] Where substituent groups are specified by their conventional chemical formulae, written from left to right, they equally encompass the chemically identical substituents that would result from writing the structure from right to left, e g., -CH2O- is equivalent to -OCH2-

[0065] The term “alkyl,” by itself or as part of another substituent, means, unless otherwise stated, a straight (i.e., unbranched) or branched non-cyclic carbon chain (or carbon), or combination thereof, which may be fully saturated, mono- or polyunsaturated and can include di- and multivalent radicals, having the number of carbon atoms designated (i.e., C1-C10 means one to ten carbons). Examples of saturated hydrocarbon radicals include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, (cyclohexyl)methyl, homologs and isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. An unsaturated alky l group is one having one or more double bonds or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, viny l. 2-propenyl, croty l. 2- isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(l ,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3- butynyl, and the higher homologs and isomers. An alkoxy is an alkyl attached to the remainder of the molecule via an oxygen linker (-O-).

[0066] The term “alky lene,” by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from an alkyl, as exemplified, but not limited by, -CH2CH2CH2-. Typically, an alkyl (or alkylene) group will have from 1 to 24 carbon atoms, with those groups having 10 or fewer carbon atoms being preferred in the present invention. A “lower alkyl” or “lower alkylene” is a shorter chain alkyl or alkylene group, generally having eight or fewer carbon atoms. The term “alkenylene,” by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from an alkene.

[0067] The term “heteroalkyl,” by itself or in combination with another term, means, unless otherwise stated, a stable non-cyclic straight or branched chain, or combinations thereof, including at least one carbon atom and at least one heteroatom selected from the group consisting of O, N, P, Si, and S, and wherein the nitrogen and sulfur atoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quatemized. The heteroatom(s) O, N, P, S, and Si may be placed at any interior position of the heteroalkyl group or at the position at which the alkyl group is attached to the remainder of the molecule. Examples include, but are not limited to: -(CH₂)₂-O-CH₃, -(CH₂)₂-NH-CH₃, -(CH₂)₃-OH, -CH2-NH2, -CH2-NO2, -(CH₂)₂-N(CH₃)-CH₃, -CH₂-S-CH₂-CH₃, -S(O)-CH₃, -(CH₂)2-S(O)₂-CH₃, -CH=CH-O-CH₃, -Si(CH₃)₃, -CH₂-CH=N-OCH₃, -O-CH₃, -CH=CH-N(CH₃)-CH₃, -O-CH₂-CH₃, and -CN. Up to two or three heteroatoms may be consecutive, such as, for example, -CH2-NH-OCH₃ and -CH₂- O-Si(CH₃)₃.

[0068] The term “heteroalkylene,” by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from heteroalkyl, as exemplified, but not limited by, -CH2-CH₂-S-CH₂-CH₂-, -O-CH2-CH2-NH-CH2-, -O-(CH₂)₃-O-PO₃-, -O-(CH₂)-O-PO₃-, -O-(CH2)2-O-PO₃-, -O-(CH2)4-O-PO₃-, and the like. For heteroalkylene groups, heteroatoms can also occupy either or both of the chain termini (e.g.. alkyleneoxy. alkylenedioxy. alkyleneamino, alkylenediamino, and the like). Still further, for alkylene and heteroalkylene linking groups, no orientation of the linking group is implied by the direction in which the formula of the linking group is written. For example, the formula -C(O)2R'- represents both -C(O)2R'- and -R'C(O)₂-. As described above, heteroalkyl groups, as used herein, include those groups that are attached to the remainder of the molecule through a heteroatom, such as -C(O)R’, -C(O)NR’, -NR’R", -OR¹, -SR¹, and/or -SO2R'. Where “heteroalkyd” is recited, followed by recitations of specific heteroalky l groups, such as -NR'R" or the like, it will be understood that the terms heteroalky 1 and -NR'R" are not redundant or mutually exclusive. Rather, the specific heteroalkyl groups are recited to add clarity. Thus, the term “heteroalkyl” should not be interpreted as excluding specific heteroalkyl groups, such as -NR'R" or the like. [0069] The terms “cycloalkyd” and “heterocycloalkyl,” by themselves or in combination with other terms, mean, unless otherwise stated, cyclic non-aromatic versions of “alkyl” and “heteroalkyd,” respectively, wherein the carbons making up the ring or rings do not necessarily need to be bonded to a hydrogen due to all carbon valencies participating in bonds with nonhydrogen atoms. Additionally, for heterocycloalky 1, a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule. Examples of cycloalkyl include, but are not limited to. cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 1 -cyclohexenyl,

3-cyclohexenyl, cycloheptyl, and the like. Examples of heterocycloalkyl include, but are not limited to, l-(l,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl,

4-morpholinyl, 3-morpholinyl. tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl. 1 -piperazinyl, 2-piperazinyl. and the like. A “cycloalkylene” and a “heterocycloalkylene,” alone or as part of another substituent, means a divalent radical derived from a cycloalkyl and heterocycloalkyl, respectively.

[0070] The terms “halo” or “halogen,” by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom. Additionally, terms such as “haloalkyl” are meant to include monohaloalkyl and polyhaloalkyl. For example, the term “halo(Ci-C4)alkyl” includes, but is not limited to, fluoromethyl, difluoromethyl, trifluoromethyl. 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.

[0071] The term “acyl” means, unless otherwise stated, -C(O)R where R is a substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyd, substituted or unsubstituted heterocycloalkyd, substituted or unsubstituted ary l, or substituted or unsubstituted heteroaryl.

[0072] The term “ary 1” means, unless otherwise stated, a polyunsaturated, aromatic, hydrocarbon substituent, which can be a single ring or multiple rings (preferably from 1 to 3 rings) that are fused together (i.e., a fused ring aryl) or linked covalently (e.g., biphenyl). A fused ring aryl refers to multiple rings fused together wherein at least one of the fused rings is an ary l ring. The term “heteroaryl” refers to ary 1 groups (or rings) that contain at least one heteroatom such as N, O, or S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom(s) are optionally quatemized. Thus, the term “heteroaryl” includes fused ring heteroaryl groups (i.e., multiple rings fused together wherein at least one of the fused rings is a heteroaromatic ring). A 5,6-fused ring heteroary dene refers to tw o rings fused together, wherein one ring has 5 members and the other ring has 6 members, and w herein at least one ring is a heteroary! ring. Likewise, a 6,6-fused ring heteroarylene refers to two rings fused together, wherein one ring has 6 members and the other ring has 6 members, and wherein at least one ring is a heteroaryl ring. And a 6.5-fused ring heteroarylene refers to two rings fused together, wherein one ring has 6 members and the other ring has 5 members, and wherein at least one ring is a heteroaryl ring. A heteroaryl group can be attached to the remainder of the molecule through a carbon or heteroatom. Non-limiting examples of ary l and heteroaryl groups include phenyl,

1 -naphthyl, 2-naphthyl, 4-biphenyl, 1 -pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl. 4-imidazolyl, pyrazinyl, 2-oxazolyL 4-oxazolyl. 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2 -fury 1, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5 -benzothiazolyl, purinyl,

2-benzimidazolyl, 5-indolyl, 1 -isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl,

3-quinolyl. and 6-quinolyl. Substituents for each of the above noted aryl and heteroaryl ring systems are selected from the group of acceptable substituents described below. An ‘’arylene” and a “heteroarylene,” alone or as part of another substituent, mean a divalent radical derived from an aryl and heteroaryl, respectively. Non-limiting examples of heteroary l groups include pyridinyl, pyrimidinyl. thiophenyl, thienyl, furanyl, indolyl. benzoxadiazolyl, benzodioxolyl, benzodioxanyl, thianaphthanyl, pyrrolopyndinyl, indazolyL quinolinyl, quinoxalinyl, pyridopyrazinyl, quinazolinonyl, benzoisoxazolyl, imidazopyridinyl, benzofuranyl, benzothienyl, benzothiophenyl, phenyl, naphthyl, biphenyl, pyrrolyl, pyrazolyl, imidazolyl, py razinyl, oxazolyl, isoxazolyl, thiazolyl, furylthienyl, pyridyl, pyrimidyl, benzothiazolyl, purinyl, benzimidazolyl, isoquinolyl, thiadiazolyl. oxadiazolyl, pyrrolyl, diazolyl, triazolyl, tetrazolyl, benzothiadiazolyl, isothiazolyl, pyrazolopyrimidinyl, pyrrolopyrimidinyl, benzotriazolyl, benzoxazolyl, or quinolyl. The examples above may be substituted or unsubstituted and divalent radicals of each heteroary l example above are non-limiting examples of heteroarylene.

[0073] A fused ring heterocyloalkyl-aryl is an aryl fused to a helerocycloalk l. A fused ring heterocycloalkyl-heteroaryl is a heteroaryl fused to a heleroc cloalkvl. A fused ring heterocycloalkyl-cycloalkyl is a heterocycloalkyl fused to a cycloalkyl. A fused ring heterocycloalkyl-heterocycloalkyl is a heterocycloalkyd fused to another heterocycloalkyl. Fused ring heterocycloalkyl-ary 1, fused ring heterocycloalkyl-heteroaryl, fused ring heterocycloalky 1- cycloalky 1. or fused ring heterocycloalkyl-heterocycloalkyl may each independently be unsubstituted or substituted with one or more of the substitutents described herein.

[0074] The term “oxo” means an oxygen that is double bonded to a carbon atom.

[0075] The term “alkylsulfonyl,” as used herein, means a moiety having the formula -S(02)-R', where R' is a substituted or unsubstituted alkyl group as defined above. R' may have a specified number of carbons (e.g., “C1-C4 alkylsulfonyl”).

[0076] Each of the above terms (e g., ‘‘alkyl,’" “heteroalkyl,” “aryl,"’ and “heteroaryl"’) includes both substituted and unsubstituted forms of the indicated radical. Preferred substituents for each type of radical are provided below.

[0077] Substituents for the alkyl and heteroalkyl radicals (including those groups often referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) can be one or more of a variety of groups selected from, but not limited to, -OR'. =0, =NR', =N-0R', -NR'R", -SR', -halogen, -SiR'R'R'". -OC(O)R', -C(O)R’, -CO₂R'. -CONR'R", -OC(O)NR'R", -NR"C(0)R', -NR'-C(0)NR"R", -NR"C(O)₂R', -NR-C(NR'R"R"')=NR"", -NR-C(NR'R")=NR'", -S(O)R', -S(O)₂R', -S(O)₂NR'R", -NRSO2R', -NR'NR'R'", ONR'R", NR'C=(O)NR"NR"'R"", -CN, -NO2, in a number ranging from zero to (2m'+l), where m' is the total number of carbon atoms in such radical. R, R', R", R'", and R"" each preferably independently refer to hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl (e.g., aryl substituted with 1-3 halogens), substituted or unsubstituted heteroaryl, substituted or unsubstituted alkyl, alkoxy, or thioalkoxy groups, or arylalkyl groups. When a compound of the invention includes more than one R group, for example, each of the R groups is independently selected as are each R', R", R'", and R"" group when more than one of these groups is present. When R' and R" are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 4-, 5-, 6-, or 7- membered ring. For example, -NR'R" includes, but is not limited to, 1 -pyrrolidinyl and 4- morpholinyl. From the above discussion of substituents, one of skill in the art will understand that the term “alkyd” is meant to include groups including carbon atoms bound to groups other than hy drogen groups, such as haloalkyl (e.g., -CF3 and -CH2CF3) and acyl

(e.g., -C(O)CH₃, -C(O)CF₃, -C(O)CH₂OCH3, and the like).

[0078] Similar to the substituents described for the alkyl radical, substituents for the aryl and heteroaryl groups are varied and are selected from, for example: -OR', -NR'R", -SR', -halogen. -SiR'R'R'", -OC(O)R', -C(O)R’, -CO2R', -CONR'R", -OC(O)NR'R", -NR"C(O)R', -NR'-C(0)NR"R", -NR"C(O)₂R', -NR-C(NR'R"R'")=NR"", -NR-C(NR'R")=NR"', -S(O)R’, -S(O)₂R', -S(O)₂NR'R", -NRSO2R', -NR'NR'R'", -ONR’R", -NR'C=(O)NR"NR'"R"", -CN, -NO2, -R', -N3. -CH(Ph)2, fluoro(Ci-C4)alkoxy, and fluoro(Ci-C4)alkyl. in a number ranging from zero to the total number of open valences on the aromatic ring system; and where R', R", R'", and R"" are preferably independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl. When a compound of the invention includes more than one R group, for example, each of the R groups is independently selected as are each R', R", R'", and R"" groups when more than one of these groups is present.

[0079] Two or more substituents may optionally be joined to form ary l, heteroaryl, cycloalkyl, or heterocycloalkyl groups. Such so-called ring-forming substituents are typically, though not necessarily, found attached to a cyclic base structure. In embodiments, the ring-forming substituents are attached to adjacent members of the base structure. For example, two ringforming substituents attached to adjacent members of a cyclic base structure create a fused ring structure. In embodiments, the ring-forming substituents are attached to a single member of the base structure. For example, two ring-forming substituents attached to a single member of a cyclic base structure create a spirocyclic structure. In embodiments, the ring-forming substituents are attached to non-adj acent members of the base structure.

[0080] Two of the substituents on adj acent atoms of the ary l or heteroaryl ring may optionally⁷ form a ring of the formula -T-C(O)-(CRR')q-U-, wherein T and U are independently -NR-, -O-, -CRR'-, or a single bond, and q is an integer of from 0 to 3. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -A-(CH2)_r-B-, wherein A and B are independently -CRR'-, -O-, -NR-, -S-, -S(O) -, -S(O)₂-, -S(O)₂NR'-, or a single bond, and r is an integer of from 1 to 4. One of the single bonds of the new ring so formed may optionally be replaced with a double bond. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -(CRR')s-X'- (C"R"R"')d-, where s and d are independently integers of from 0 to 3, and X' is -O-, -NR'-, -S-, -S(O)-, -S(O)2-, or -S(O)2NR'-. The substituents R, R', R", and R'" are preferably independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl.

[0081] As used herein, the terms “heteroatom’’ or “ring heteroatom” are meant to include, oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), and silicon (Si).

[0082] A “substituent group,” as used herein, means a group selected from the following moieties: [0083] (A) oxo, halogen, -CF₃, -CN, -OH, -NH₂, -COOH, -CONH₂, -NO₂, -SH, -SO₂C1, -SO3H. -SO4H. -SO₂NH₂, -NHNH₂. -ONH₂, -NHC=(O)NHNH₂, -NHC=(O) NH₂, -NHSO₂H, -NHC= (O)H, -NHC(O)-OH, -NHOH, -OCF3, -OCHF₂, -NHSO₂CH₃, -N₃, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloal kyl. unsubstituted aryl, unsubstituted heteroaryl, and

[0084] (B) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, substituted with at least one substituent selected from:

[0085] (i) oxo, halogen, -CF₃, -CN, -OH, -NH₂, -COOH, -CONH₂, -NO₂, -SH, -SO₂C1, -SO₃H, -SO4H, -SO₂NH₂, -NHNH₂, -0NH₂, -NHC=(0)NHNH₂, -NHC=(O) NH₂, -NHSO₂H, -NHC= (O)H, -NHC(0)0H. -NHOH, -OCF₃, -OCHF₂, -NHSO₂CH₃, -N₃, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted ary l, unsubstituted heteroaryl, and

[0086] (ii) alk l, heteroalkyl, cycloalkyd, heterocycloalkyl, ary 1, heteroary 1, substituted with at least one substituent selected from:

[0087] (a) oxo. halogen, -CF₃, -CN, -OH, -NH₂. -COOH, -CONH₂. -NO₂, -SH. -SO₂C1, -SO₃H, -SO₄H, -SO₂NH₂, -NHNH₂, -ONH₂, -NHC=(0)NHNH₂, -NHC=(O) NH₂, -NHSO₂H, -NHC= (O)H, -NHC(0)-0H, -NHOH, -OCF₃, -OCHF₂, -NHSO₂CH₃, -N₃, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroary 1, and

[0088] (b) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, substituted with at least one substituent selected from: oxo, halogen, -CF₃, -CN, -OH, -NH₂, -COOH,

-CONH₂. -NO₂, -SH. -SO₂C1, -SO₃H, -SO4H, -SO₂NH₂, -NHNH₂, -0NH₂, -NHC=(0)NHNH₂, -NHC=(O) NH₂, -NHSO2H, -NHC= (O)H, -NHC(O)-OH, -NHOH, -OCF₃, -OCHF2, -NHSO₂CH₃, -N₃, unsubstituted alkyd, unsubstituted heteroalkyl, unsubstituted cycloalkyd, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl.

[0089] A “size-limited substituent” or “ size-limited substituent group,” as used herein, means a group selected from all of the substituents described above for a “substituent group,” wherein each substituted or unsubstituted alkyl is a substituted or unsubstituted Ci-C₂o alkyd, each substituted or unsubstituted heteroalky l is a substituted or unsubstituted 2 to 20 membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C₃-Cs cycloalkyd, each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 8 membered heterocycloalkyl, each substituted or unsubstituted ary l is a substituted or unsubstituted Ce-Cw aryl, and each substituted or unsubstituted heteroaryl is a substituted or unsubstituted 5 to 10 membered heteroaryl.

[0090] A “lower substituent’" or “ lower substituent group,” as used herein, means a group selected from all of the substituents described above for a “substituent group,” wherein each substituted or unsubstituted alkyl is a substituted or unsubstituted Ci-Cs alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8 membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C3-C7 cycloalkyl, each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 7 membered heterocycloalkyl, each substituted or unsubstituted aryl is a substituted or unsubstituted Ce-C aryl, and each substituted or unsubstituted heteroaryl is a substituted or unsubstituted 5 to 9 membered heteroaryl.

[0091] In embodiments, each substituted group described in the compounds herein is substituted with at least one substituent group. More specifically, in embodiments, each substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkyd ene, substituted cycloalky lene, substituted heterocycloalkylene, substituted ary lene, and/or substituted heteroarylene described in the compounds herein are substituted with at least one substituent group. In embodiments, at least one or all of these groups are substituted with at least one size-limited substituent group. In embodiments, at least one or all of these groups are substituted with at least one lower substituent group.

[0092] In aspects of the compounds herein, each substituted or unsubstituted alkyl may be a substituted or unsubstituted C1-C20 alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 20 membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted Cs-Cs cycloalkyl, each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 8 membered heterocycloalkyl, each substituted or unsubstituted aryl is a substituted or unsubstituted Ce-Cw aryl, and/or each substituted or unsubstituted heteroaryl is a substituted or unsubstituted 5 to 10 membered heteroaryl. In aspects of the compounds herein, each substituted or unsubstituted alkylene is a substituted or unsubstituted C1-C20 alkylene, each substituted or unsubstituted heteroalkylene is a substituted or unsubstituted 2 to 20 membered heteroalkylene, each substituted or unsubstituted cycloalkylene is a substituted or unsubstituted C3-C8 cycloalkylene, each substituted or unsubstituted heterocycloalkylene is a substituted or unsubstituted 3 to 8 membered heterocycloalkylene, each substituted or unsubstituted arylene is a substituted or unsubstituted Ce-Cw arylene, and/or each substituted or unsubstituted heteroarylene is a substituted or unsubstituted 5 to 10 membered heteroarylene.

[0093] In embodiments, each substituted or unsubstituted alkyl is a substituted or unsubstituted Ci-Cs alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8 membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C3-C7 cycloalkyl, each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 7 membered heterocycloalkyl, each substituted or unsubstituted aryl is a substituted or unsubstituted C6-C10 aryl, and/or each substituted or unsubstituted heteroaryl is a substituted or unsubstituted 5 to 9 membered heteroaryl. In embodiments, each substituted or unsubstituted alkylene is a substituted or unsubstituted Ci-Cs alkylene, each substituted or unsubstituted heteroalkylene is a substituted or unsubstituted 2 to 8 membered heteroalkylene, each substituted or unsubstituted cycloalkylene is a substituted or unsubstituted C3-C7 cycloalkylene, each substituted or unsubstituted heterocycloalkylene is a substituted or unsubstituted 3 to 7 membered heterocycloalkylene, each substituted or unsubstituted arylene is a substituted or unsubstituted Ce-Cio arylene, and/or each substituted or unsubstituted heteroarylene is a substituted or unsubstituted 5 to 9 membered heteroarylene. In embodiments, the compound is a chemical species set forth in the Examples section below.

[0094] The term “activation,” “activate,” “activating,” “activator” and the like in reference to a protein-inhibitor interaction means positively affecting (e.g. increasing) the activity or function of the protein relative to the activity⁷ or function of the protein in the absence of the activator. In aspects activation means positively affecting (e.g. increasing) the concentration or levels of the protein relative to the concentration or level of the protein in the absence of the activator. The terms may reference activation, or activating, sensitizing, or up-regulating signal transduction or enzymatic activity or the amount of a protein decreased in a disease. Thus, activation may include, at least in part, partially or totally increasing stimulation, increasing or enabling activation, or activating, sensitizing, or up-regulating signal transduction or enzymatic activity or the amount of a protein associated with a disease (e.g., a protein which is decreased in a disease relative to a non-diseased control). Activation may include, at least in part, partially or totally increasing stimulation, increasing or enabling activation, or activating, sensitizing, or up- regulating signal transduction or enzymatic activity or the amount of a protein

[0095] The terms “agonist,” “activator,” “upregulator,” etc. refer to a substance capable of detectably increasing the expression or activity of a given gene or protein. The agonist can increase expression or activity 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more in comparison to a control in the absence of the agonist. In certain instances, expression or activity is higher than the expression or activity in the absence of the agonist.

[0096] The term “inhibition’", “inhibit”, “inhibiting” and the like in reference to a proteininhibitor interaction means negatively affecting (e.g. decreasing) the activity or function of the protein relative to the activity or function of the protein in the absence of the inhibitor. In aspects inhibition means negatively affecting (e g. decreasing) the concentration or levels of the protein relative to the concentration or level of the protein in the absence of the inhibitor. In aspects inhibition refers to reduction of a disease or symptoms of disease. In embodiments, inhibition refers to a reduction in the activity⁷ of a particular protein target. Thus, inhibition includes, at least in part, partially or totally blocking stimulation, decreasing, preventing, or delaying activation, or inactivating, desensitizing, or down-regulating signal transduction or enzymatic activity or the amount of a protein. In embodiments, inhibition refers to a reduction of activity of a target protein resulting from a direct interaction (e.g. an inhibitor binds to the target protein). In embodiments, inhibition refers to a reduction of activity of a target protein from an indirect interaction (e.g. an inhibitor binds to a protein that activates the target protein, thereby preventing target protein activation).

[0097] The terms “inhibitor,” “repressor” or “antagonist” or “downregulator” interchangeably refer to a substance capable of detectably decreasing the expression or activity of a given gene or protein. The antagonist can decrease expression or activity 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more in comparison to a control in the absence of the antagonist. In embodiments, expression or activity is lower than the expression or activity in the absence of the antagonist.

[0098] Compounds

[0099] Provided herein are compounds comprising a first DNA oligonucleotide and a second DNA oligonucleotide. The first DNA oligonucleotide comprises from about 5 unmodified nucleotides to about 70 unmodified nucleotides, and the second DNA oligonucleotide comprises: (i) from about 5 nucleotides to about 70 nucleotides, and (ii) a constrained nucleotide on the 3’ end and a constrained nucleotide on the 5’ end. In embodiments, the first DNA oligonucleotide comprises from about 6 unmodified nucleotides to about 60 unmodified nucleotides, and the second DNA oligonucleotide comprises: (i) from about 6 nucleotides to about 60 nucleotides, and (ii) a constrained nucleotide on the 3’ end and a constrained nucleotide on the 5 ’ end. In embodiments, the first DNA oligonucleotide comprises from about 6 unmodified nucleotides to about 50 unmodified nucleotides, and the second DNA oligonucleotide comprises: (i) from about 6 nucleotides to about 50 nucleotides, and (ii) a constrained nucleotide on the 3’ end and a constrained nucleotide on the 5’ end. In embodiments, the first DNA oligonucleotide comprises from about 8 unmodified nucleotides to about 40 unmodified nucleotides, and the second DNA oligonucleotide comprises: (i) from about 8 nucleotides to about 40 nucleotides, and (ii) a constrained nucleotide on the 3’ end and a constrained nucleotide on the 5’ end. In embodiments, the first DNA oligonucleotide comprises from about 8 unmodified nucleotides to about 30 unmodified nucleotides, and the second DNA oligonucleotide comprises: (i) from about 8 nucleotides to about 30 nucleotides, and (ii) a constrained nucleotide on the 3’ end and a constrained nucleotide on the 5‘ end. In embodiments, the first DNA oligonucleotide comprises from about 8 unmodified nucleotides to about 26 unmodified nucleotides, and the second DNA oligonucleotide comprises: (i) from about 8 nucleotides to about 26 nucleotides, and (ii) a constrained nucleotide on the 3’ end and a constrained nucleotide on the 5’ end. In embodiments, the first DNA oligonucleotide comprises from about 10 unmodified nucleotides to about 30 unmodified nucleotides, and the second DNA oligonucleotide comprises: (i) from about 10 nucleotides to about 30 nucleotides, and (ii) a constrained nucleotide on the 3’ end and a constrained nucleotide on the 5’ end. In embodiments, the first DNA oligonucleotide comprises from about 10 unmodified nucleotides to about 28 unmodified nucleotides, and the second DNA oligonucleotide comprises: (i) from about 10 nucleotides to about 28 nucleotides, and (ii) a constrained nucleotide on the 3’ end and a constrained nucleotide on the 5’ end. In embodiments, the first DNA oligonucleotide comprises from about 10 unmodified nucleotides to about 24 unmodified nucleotides, and the second DNA oligonucleotide comprises: (i) from about 10 nucleotides to about 24 nucleotides, and (ii) a constrained nucleotide on the 3’ end and a constrained nucleotide on the 5’ end. In embodiments, the first DNA oligonucleotide comprises from about 10 unmodified nucleotides to about 22 unmodified nucleotides, and the second DNA oligonucleotide comprises: (i) from about 10 nucleotides to about 22 nucleotides, and (ii) a constrained nucleotide on the 3’ end and a constrained nucleotide on the 5' end. In embodiments, the first DNA oligonucleotide comprises from about 10 unmodified nucleotides to about 20 unmodified nucleotides, and the second DNA oligonucleotide comprises: (i) from about 10 nucleotides to about 20 nucleotides, and (ii) a constrained nucleotide on the 3’ end and a constrained nucleotide on the 5’ end. In embodiments, the first DNA oligonucleotide comprises from about 12 unmodified nucleotides to about 20 unmodified nucleotides, and the second DNA oligonucleotide comprises: (i) from about 12 nucleotides to about 20 nucleotides, and (ii) a constrained nucleotide on the 3’ end and a constrained nucleotide on the 5’ end. In embodiments, the first DNA oligonucleotide comprises from about 14 unmodified nucleotides to about 18 unmodified nucleotides, and the second DNA oligonucleotide comprises: (i) from about 14 nucleotides to about 18 nucleotides, and (ii) a constrained nucleotide on the 3’ end and a constrained nucleotide on the 5’ end. In embodiments, the constrained nucleotide is a LNA-modified nucleotide, a cMOE-modified nucleotide, or a cET-modified nucleotide. In embodiments, the constrained nucleotide is a LNA- modified nucleotide. In embodiments, the constrained nucleotide is a cMOE-modified nucleotide. In embodiments, the constrained nucleotide is a cET-modified nucleotide. The constrained nucleotides in the second DNA oligonucleotide can be the same or different. In embodiments, the constrained nucleotides on the 3 ’ end are different than the constrained nucleotides on the 5’ end. In embodiments, the constrained nucleotides on the 3’ end are the same as the constrained nucleotides on the 5’ end. In embodiments, the second DNA oligonucleotide comprises a nucleotide having a phosphorothioate modification.

[0100] In embodiments, the first DNA oligonucleotide comprises about 10 unmodified nucleotides, and the second DNA oligonucleotide comprises: (i) about 10 nucleotides, and (ii) a constrained nucleotide on the 3’ end and a constrained nucleotide on the 5' end. In embodiments, the first DNA oligonucleotide comprises about 11 unmodified nucleotides, and the second DNA oligonucleotide comprises: (i) about 11 nucleotides, and (ii) a constrained nucleotide on the 3’ end and a constrained nucleotide on the 5’ end. In embodiments, the first DNA oligonucleotide comprises about 12 unmodified nucleotides, and the second DNA oligonucleotide comprises: (i) about 12 nucleotides, and (ii) a constrained nucleotide on the 3’ end and a constrained nucleotide on the 5’ end. In embodiments, the first DNA oligonucleotide comprises about 13 unmodified nucleotides, and the second DNA oligonucleotide comprises: (i) about 13 nucleotides, and (ii) a constrained nucleotide on the 3‘ end and a constrained nucleotide on the 5’ end. In embodiments, the first DNA oligonucleotide comprises about 14 unmodified nucleotides, and the second DNA oligonucleotide comprises: (i) about 14 nucleotides, and (ii) a constrained nucleotide on the 3’ end and a constrained nucleotide on the 5’ end. In embodiments, the first DNA oligonucleotide comprises about 15 unmodified nucleotides, and the second DNA oligonucleotide comprises: (i) about 15 nucleotides, and (ii) a constrained nucleotide on the 3‘ end and a constrained nucleotide on the 5’ end. In embodiments, the first DNA oligonucleotide comprises about 16 unmodified nucleotides, and the second DNA oligonucleotide comprises: (i) about 1 nucleotides, and (ii) a constrained nucleotide on the 3’ end and a constrained nucleotide on the 5‘ end. In embodiments, the first DNA oligonucleotide comprises about 17 unmodified nucleotides, and the second DNA oligonucleotide comprises: (i) about 17 nucleotides, and (ii) a constrained nucleotide on the 3‘ end and a constrained nucleotide on the 5’ end. In embodiments, the first DNA oligonucleotide comprises about 18 unmodified nucleotides, and the second DNA oligonucleotide comprises: (i) about 18 nucleotides, and (ii) a constrained nucleotide on the 3’ end and a constrained nucleotide on the 5’ end. In embodiments, the first DNA oligonucleotide comprises about 19 unmodified nucleotides, and the second DNA oligonucleotide comprises: (i) about 19 nucleotides, and (ii) a constrained nucleotide on the 3’ end and a constrained nucleotide on the 5’ end. In embodiments, the first DNA oligonucleotide comprises about 20 unmodified nucleotides, and the second DNA oligonucleotide comprises: (i) about 20 nucleotides, and (ii) a constrained nucleotide on the 3’ end and a constrained nucleotide on the 5' end. In embodiments, the first DNA oligonucleotide comprises about 21 unmodified nucleotides, and the second DNA oligonucleotide comprises: (i) about 21 nucleotides, and (ii) a constrained nucleotide on the 3’ end and a constrained nucleotide on the 5’ end. In embodiments, the first DNA oligonucleotide comprises about 22 unmodified nucleotides, and the second DNA oligonucleotide comprises: (i) about 22 nucleotides, and (ii) a constrained nucleotide on the 3’ end and a constrained nucleotide on the 5' end. In embodiments, the constrained nucleotide is a LNA-modified nucleotide, a cMOE-modified nucleotide, or a cET-modified nucleotide. In embodiments, the constrained nucleotide is a LNA- modified nucleotide. In embodiments, the constrained nucleotide is a cMOE-modified nucleotide. In embodiments, the constrained nucleotide is a cET-modified nucleotide. The constrained nucleotides in the second DNA oligonucleotide can be the same or different. In embodiments, the constrained nucleotides on the 3 ’ end are different than the constrained nucleotides on the 5’ end. In embodiments, the constrained nucleotides on the 3’ end are the same as the constrained nucleotides on the 5’ end. In embodiments, the second DNA oligonucleotide comprises a nucleotide having a phosphorothioate modification.

[0101] In embodiments of the compounds described herein, the second DNA oligonucleotide comprises one constrained nucleotide on the 3’ end and one constrained nucleotide on the 5’ end. In embodiments, the second DNA oligonucleotide comprises two constrained nucleotide on the 3 ’ end and one constrained nucleotide on the 5 ’ end. In embodiments, the second DNA oligonucleotide comprises two constrained nucleotide on the 3 ' end and one constrained nucleotide on the 5‘ end. wherein the two constrained nucleotides are contiguous. In embodiments, the second DNA oligonucleotide comprises two constrained nucleotide on the 3’ end and one constrained nucleotide on the 5’ end, wherein the two constrained nucleotides are alternating. In embodiments, the second DNA oligonucleotide comprises one constrained nucleotide on the 3’ end and two constrained nucleotide on the 5' end. In embodiments, the second DNA oligonucleotide comprises one constrained nucleotide on the 3’ end and two constrained nucleotide on the 5’ end, wherein the two constrained nucleotides are contiguous. In embodiments, the second DNA oligonucleotide comprises one constrained nucleotide on the 3’ end and two constrained nucleotide on the 5’ end, wherein the two constrained nucleotides are alternating. In embodiments, the constrained nucleotide is a LNA-modified nucleotide, a cMOE- modified nucleotide, or a cET-modified nucleotide. In embodiments, the constrained nucleotide is a LNA-modified nucleotide. In embodiments, the constrained nucleotide is a cMOE-modified nucleotide. In embodiments, the constrained nucleotide is a cET-modified nucleotide. The constrained nucleotides in the second DNA oligonucleotide can be the same or different. In embodiments, the constrained nucleotides on the 3 ’ end are different than the constrained nucleotides on the 5’ end. In embodiments, the constrained nucleotides on the 3’ end are the same as the constrained nucleotides on the 5’ end. In embodiments, the second DNA oligonucleotide comprises a nucleotide having a phosphorothioate modification.

[0102] In embodiments, the second DNA oligonucleotide comprises one constrained nucleotide on the 3’ end and three constrained nucleotide on the 5’ end. In embodiments, the second DNA oligonucleotide comprises one constrained nucleotide on the 3‘ end and three constrained nucleotide on the 5’ end, wherein the three constrained nucleotides are contiguous. In embodiments, the second DNA oligonucleotide comprises one constrained nucleotide on the 3’ end and three constrained nucleotide on the 5’ end, wherein the three constrained nucleotides are alternating. In embodiments, the second DNA oligonucleotide comprises three constrained nucleotide on the 3' end and one constrained nucleotide on the 5’ end. In embodiments, the second DNA oligonucleotide comprises three constrained nucleotide on the 3’ end and one constrained nucleotide on the 5’ end, wherein the three constrained nucleotides are contiguous. In embodiments, the second DNA oligonucleotide comprises three constrained nucleotide on the 3’ end and one constrained nucleotide on the 5’ end, wherein the three constrained nucleotides are alternating. In embodiments, the constrained nucleotide is a LNA-modified nucleotide, a cMOE-modified nucleotide, or a cET-modified nucleotide. In embodiments, the constrained nucleotide is a LNA-modified nucleotide. In embodiments, the constrained nucleotide is a cMOE-modified nucleotide. In embodiments, the constrained nucleotide is a cET-modified nucleotide. The constrained nucleotides in the second DNA oligonucleotide can be the same or different. In embodiments, the constrained nucleotides on the 3’ end are different than the constrained nucleotides on the 5’ end. In embodiments, the constrained nucleotides on the 3’ end are the same as the constrained nucleotides on the 5’ end. In embodiments, the second DNA oligonucleotide comprises a nucleotide having a phosphorothioate modification. [0103] In embodiments, the second DNA oligonucleotide comprises two constrained nucleotides on the 3’ end and two constrained nucleotides on the 5' end. In embodiments, the second DNA oligonucleotide comprises two constrained nucleotides on the 3’ end and two constrained nucleotides on the 5’ end, wherein the two constrained nucleotides on the 3’ end are contiguous and the two constrained nucleotides on the 5’ end are contiguous. In embodiments, the second DNA oligonucleotide comprises two constrained nucleotides on the 3’ end and two constrained nucleotide on the 5’ end, wherein the two constrained nucleotides on the 3’ end are alternating and the two constrained nucleotides on the 5’ end are alternating. In embodiments, the second DNA oligonucleotide comprises two constrained nucleotides on the 3’ end and two constrained nucleotides on the 5’ end, wherein the two constrained nucleotides on the 3’ end are contiguous and the two constrained nucleotides on the 5’ end are alternating. In embodiments, the second DNA oligonucleotide comprises two constrained nucleotides on the 3’ end and two constrained nucleotides on the 5’ end, wherein the two constrained nucleotides on the 3’ end are alternating and the two constrained nucleotides on the 5’ end are contiguous. In embodiments, the constrained nucleotide is a LNA-modified nucleotide, a cMOE-modified nucleotide, or a cET-modified nucleotide. In embodiments, the constrained nucleotide is a LNA-modified nucleotide. In embodiments, the constrained nucleotide is a cMOE-modified nucleotide. In embodiments, the constrained nucleotide is a cET-modified nucleotide. The constrained nucleotides in the second DNA oligonucleotide can be the same or different. In embodiments, the constrained nucleotides on the 3’ end are different than the constrained nucleotides on the 5’ end. In embodiments, the constrained nucleotides on the 3’ end are the same as the constrained nucleotides on the 5’ end. In embodiments, the second DNA oligonucleotide comprises a nucleotide having a phosphorothioate modification.

[0104] In embodiments, the second DNA oligonucleotide comprises two constrained nucleotide on the 3’ end and three constrained nucleotide on the 5’ end. In embodiments, the second DNA oligonucleotide comprises two constrained nucleotides on the 3’ end and three constrained nucleotides on the 5’ end, wherein the two constrained nucleotides on the 3’ end are contiguous and the three constrained nucleotides on the 5’ end are contiguous. In embodiments, the second DNA oligonucleotide comprises two constrained nucleotides on the 3’ end and three constrained nucleotides on the 5" end, wherein the two constrained nucleotides on the 3’ end are alternating and the three constrained nucleotides on the 5’ end are alternating. In embodiments, the second DNA oligonucleotide comprises two constrained nucleotides on the 3’ end and three constrained nucleotides on the 5’ end, wherein the two constrained nucleotides on the 3’ end are contiguous and the three constrained nucleotides on the 5’ end are alternating. In embodiments, the second DNA oligonucleotide comprises two constrained nucleotides on the 3’ end and three constrained nucleotides on the 5’ end, wherein the two constrained nucleotides on the 3' end are alternating and the three constrained nucleotides on the 5’ end are contiguous. In embodiments, the constrained nucleotide is a LNA-modified nucleotide, a cMOE-modified nucleotide, or a cET-modified nucleotide. In embodiments, the constrained nucleotide is a LNA-modified nucleotide. In embodiments, the constrained nucleotide is a cMOE-modified nucleotide. In embodiments, the constrained nucleotide is a cET-modified nucleotide. The constrained nucleotides in the second DNA oligonucleotide can be the same or different. In embodiments, the constrained nucleotides on the 3’ end are different than the constrained nucleotides on the 5’ end. In embodiments, the constrained nucleotides on the 3’ end are the same as the constrained nucleotides on the 5’ end. In embodiments, the second DNA oligonucleotide comprises a nucleotide having a phosphorothioate modification.

[0105] In embodiments, the second DNA oligonucleotide comprises three constrained nucleotides on the 3’ end and two constrained nucleotides on the 5’ end. In embodiments, the second DNA oligonucleotide comprises three constrained nucleotides on the 3‘ end and two constrained nucleotides on the 5’ end, wherein the three constrained nucleotides on the 3’ end are contiguous and the two constrained nucleotides on the 5‘ end are contiguous. In embodiments, the second DNA oligonucleotide comprises three constrained nucleotides on the 3’ end and two constrained nucleotides on the 5’ end, wherein the three constrained nucleotides on the 3’ end are alternating and the two constrained nucleotides on the 5’ end are alternating. In embodiments, the second DNA oligonucleotide comprises three constrained nucleotides on the 3’ end and two constrained nucleotides on the 5’ end, wherein the three constrained nucleotides on the 3’ end are contiguous and the two constrained nucleotides on the 5’ end are alternating. In embodiments, the second DNA oligonucleotide comprises three constrained nucleotides on the 3’ end and two constrained nucleotides on the 5’ end, wherein the three constrained nucleotides on the 3’ end are alternating and the two constrained nucleotides on the 5’ end are contiguous. In embodiments, the constrained nucleotide is a LNA-modified nucleotide, a cMOE-modified nucleotide, or a cET-modified nucleotide. In embodiments, the constrained nucleotide is a LNA- modified nucleotide. In embodiments, the constrained nucleotide is a cMOE-modified nucleotide. In embodiments, the constrained nucleotide is a cET-modified nucleotide. The constrained nucleotides in the second DNA oligonucleotide can be the same or different. In embodiments, the constrained nucleotides on the 3 ’ end are different than the constrained nucleotides on the 5‘ end. In embodiments, the constrained nucleotides on the 3’ end are the same as the constrained nucleotides on the 5’ end. In embodiments, the second DNA oligonucleotide comprises a nucleotide having a phosphorothioate modification.

[0106] In embodiments, the second DNA oligonucleotide comprises three constrained nucleotides on the 3‘ end and three constrained nucleotides on the 5’ end. In embodiments, the second DNA oligonucleotide comprises three constrained nucleotides on the 3’ end and three constrained nucleotides on the 5’ end, wherein the three constrained nucleotides on the 3’ end are contiguous and the three constrained nucleotides on the 5’ end are contiguous. In embodiments, the second DNA oligonucleotide comprises three constrained nucleotides on the 3’ end and three constrained nucleotides on the 5’ end, wherein the three constrained nucleotides on the 3’ end are alternating and the three constrained nucleotides on the 5’ end are alternating. In embodiments, the second DNA oligonucleotide comprises three constrained nucleotides on the 3 ’ end and three constrained nucleotides on the 5 ’ end, wherein the three constrained nucleotides on the 3‘ end are contiguous and the three constrained nucleotides on the 5’ end are alternating. In embodiments, the second DNA oligonucleotide comprises three constrained nucleotides on the 3’ end and three constrained nucleotides on the 5’ end, wherein the three constrained nucleotides on the 3’ end are alternating and the three constrained nucleotides on the 5’ end are contiguous. In embodiments, the constrained nucleotide is a LNA-modified nucleotide, a cMOE-modified nucleotide, or a cET-modified nucleotide. In embodiments, the constrained nucleotide is a LNA-modified nucleotide. In embodiments, the constrained nucleotide is a cMOE-modified nucleotide. In embodiments, the constrained nucleotide is a cET-modified nucleotide. The constrained nucleotides in the second DNA oligonucleotide can be the same or different. In embodiments, the constrained nucleotides on the 3’ end are different than the constrained nucleotides on the 5’ end. In embodiments, the constrained nucleotides on the 3' end are the same as the constrained nucleotides on the 5’ end. In embodiments, the second DNA oligonucleotide comprises a nucleotide having a phosphorothioate modification.

[0107] In embodiments of the second DNA oligonucleotide described herein, the term “on the 3’ end’' means the terminal nucleotide(s) are adjacent the 3‘ end. Similarly, the term “on the 5' end” means the terminal nucleotide(s) are adjacent the 5’ end. For example, the phrase “a constrained nucleotide on the 3’ end” with reference to, for example, a nucleotide having the structure: 5’-CTATTTGGATGTCAGc-3’ (SEQ ID NO:37), refers to the terminal nucleotide in lower case letter and underlined on the 3’ end. Similarly, the phrase “a constrained nucleotide on the 5’ end” with reference to, for example, a nucleotide having the structure: 5'- cTATTTGGATGTCAGC-3’ (SEQ ID NO:38), refers to the terminal nucleotide in lower case letter and underlined on the 5’ end. [0108] When two or more constrained nucleotides are on the 3‘ end or the 5’ end, the two or more nucleotides can be contiguous. For example, three contiguous constrained nucleotides on the 3’ end, with reference to, for example, a nucleotide having the structure: 5’- CTATTTGGATGTCagc-3 ’ (SEQ ID NO:39), refers to the three contiguous nucleotides in lower case letter and underlined on the 3’ end. Similarly, two contiguous constrained nucleotides on the 3’ end, with reference to. for example, a nucleotide having the structure: 5‘- ctATTTGGATGTCAGC-3’ (SEQ ID NO:40), refers to the two contiguous nucleotides in lower case letter and underlined on the 5’ end. Thus, the term '‘contiguous” has the plain and ordinary meaning of “next to” or “together in sequence.”

[0109] When two or more constrained nucleotides are on the 3’ end or the 5’ end, the two or more nucleotides can be alternating. As used herein, the term “alternating” means that the terminal nucleotide is constrained, the next sequential nucleotide is not constrained, and the next sequential nucleotide is constrained. For example, two alternating constrained nucleotides on the 5’ end, with reference to, for example, a nucleotide having the structure: 5’- cTaTTTGGATGTCAGC-3’ (SEQ ID NO:41), refers to the two nucleotides in lower case letter and underlined on the 5’ end which alternate between the locked nucleic acid, a nucleic acid that does not have an LNA modification, and a locked nucleic acid. As another example, three alternating LNA- modified nucleotides on the 3’ end, with reference to, for example, a nucleotide having the structure: 5’-CTATTTGGATGTcaGc-3’ (SEQ ID NO:42), refers to the three nucleotides in lower case letter and underlined on the 3‘ end which alternate betw een the locked nucleic acid, a nucleic acid that does not have an LNA modification, and two locked nucleic acids. Thus, the term “alternating” can otherwise be described as contiguous, except that the second nucleotide from the 3’ end or 5’ end does not have a LNA modification.

[0110] In embodiments of the compounds described herein, the first DNA oligonucleotide and the second DNA oligonucleotide do not have the same number of nucleotides. For example, the first DNA oligonucleotide can have 18 unmodified nucleotides and the second DNA oligonucleotide can have 16 nucleotides. As another example, the first DNA oligonucleotide can have 16 unmodified nucleotides and the second DNA oligonucleotide can have 18 nucleotides.

[0111] In embodiments of the compounds described herein, the first DNA oligonucleotide and the second DNA oligonucleotide have the same number of nucleotides. For example, the first DNA oligonucleotide can have 16 unmodified nucleotides and the second DNA oligonucleotide can have 16 nucleotides.

[0112] In embodiments of the compounds described herein, the second DNA oligonucleotide comprises a nucleotide having a phosphorothioate modification. In embodiments, at least 10% of the nucleotides in the second DNA oligonucleotide have a phosphorothioate modification. In embodiments, at least 20% of the nucleotides in the second DNA oligonucleotide have a phosphorothioate modification. In embodiments, at least 30% of the nucleotides in the second DNA oligonucleotide have a phosphorothioate modification. In embodiments, at least 40% of the nucleotides in the second DNA oligonucleotide have a phosphorothioate modification. In embodiments, at least 50% of the nucleotides in the second DNA oligonucleotide have a phosphorothioate modification. In embodiments, at least 60% of the nucleotides in the second DNA oligonucleotide have a phosphorothioate modification. In embodiments, at least 70% of the nucleotides in the second DNA oligonucleotide have a phosphorothioate modification. In embodiments, at least 80% of the nucleotides in the second DNA oligonucleotide have a phosphorothioate modification. In embodiments, at least 90% of the nucleotides in the second DNA oligonucleotide have a phosphorothioate modification. In embodiments, all the nucleotides in the second DNA oligonucleotide have a phosphorothioate intemucleotide linkage.

[0113] In embodiments, the second DNA oligonucleotide further comprises a nucleotide having a modification. The modification can be a spacer modification or a nucleotide modification. In embodiments, the second DNA oligonucleotide further comprises a nucleotide having a modification to the base or sugar. In embodiments, the second DNA oligonucleotide comprises a nucleotide having a modification selected from the group consisting of 2’-O- aminopropyl group, a 2’-0-ethyl group, a 2'-fluoro group, a 2’-O-methyl group, 2'-deoxy- 2’fluoro group, a 2'-O-methoxyethyl group, a 2’-O-allyl group. a 2’-O-propyl group. a 2'-O- pentyl group, and a constrained nucleotide. In embodiments, the second DNA oligonucleotide further comprises a nucleotide having a 2’O-methyl group. In embodiments, the second DNA oligonucleotide further comprises a nucleotide having a 2’ fluoro group. In embodiments, the second DNA oligonucleotide further comprises a nucleotide having a 2’-deoxy-2’fluoro group. In embodiments, the second DNA oligonucleotide further comprises a nucleotide have a constrained nucleic acid modification, where this constrained nucleic acid modification is in addition to the constrained nucleotide on the 3’ end and 5’ end. In embodiments, this additional nucleotide have a constrained nucleic acid modification is contiguous with the constrained nucleotide on the 3' end or the 5‘ end. In embodiments, this additional this additional nucleotide have a constrained nucleic acid modification is separated from the constrained nucleotide on the 3’ end or the 5' end by one or more nucleotides that do not have a constrained modification. In embodiments, the constrained nucleotide is a LNA-modified nucleotide, a cMOE-modified nucleotide, or a cET-modified nucleotide. In embodiments, the constrained nucleotide is a LNA- modified nucleotide. In embodiments, the constrained nucleotide is a cMOE-modified nucleotide. In embodiments, the constrained nucleotide is a cET-modified nucleotide.

[0114] In embodiments, the second DNA oligonucleotide further comprises a spacer modification. In embodiments, the spacer modification is an abasic spacer, a spacer phosphoramidite, abasic phosphorami dite, hexadecane phosphoramidite, octadecane phosphoramidite, a C6 disulfide phosphoramidite, or a combination of two or more thereof. In embodiments, the spacer phosphoramidite is a C3 spacer phosphoramidite, a C6 spacer phosphoramidite, or a C 12 spacer phosphoramidite. In embodiments, the second DNA oligonucleotide further comprises an abasic spacer modification. In embodiments, the second DNA oligonucleotide further comprises a C3 spacer phosphoramidite.

[0115] In embodiments, the second DNA oligonucleotide is an antisense oligonucleotide. In embodiments, the second DNA oligonucleotide is a STAT antisense oligonucleotide. In embodiments, the second DNA oligonucleotide is a STAT1 antisense oligonucleotide. In embodiments, the second DNA oligonucleotide is a STAT2 antisense oligonucleotide. In embodiments, the second DNA oligonucleotide is a STAT3 antisense oligonucleotide. In embodiments, the second DNA oligonucleotide is a STAT4 antisense oligonucleotide. In embodiments, the second DNA oligonucleotide is a STAT5 antisense oligonucleotide. In embodiments, the second DNA oligonucleotide is a STAT6 antisense oligonucleotide.

[0116] In embodiments, the second DNA oligonucleotide is a STAT3 antisense oligonucleotide. In embodiments, the STAT3 antisense oligonucleotide comprises SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO:35, or SEQ ID NO:36. In embodiments, the STAT3 antisense oligonucleotide comprises SEQ ID NO:2. In embodiments, the STAT3 antisense oligonucleotide comprises SEQ ID NO: 4. In embodiments, the STAT3 antisense oligonucleotide comprises SEQ ID NO:6. In embodiments, the STAT3 antisense oligonucleotide comprises SEQ ID NO:8. In embodiments, the STAT3 antisense oligonucleotide comprises SEQ ID NO: 10. In embodiments, the STAT3 antisense oligonucleotide comprises SEQ ID NO: 12. In embodiments, the STAT3 antisense oligonucleotide comprises SEQ ID NO: 14. In embodiments, the STAT3 antisense oligonucleotide comprises SEQ ID NO:35. In embodiments, the STAT3 antisense oligonucleotide comprises SEQ ID NO:36. [0117] Table 1

[0118] In Table 1, bold lower case refers to a LNA-modified nucleotide: underline refers to a phosphorothioated nucleotide (phosphorothioated intemucleotide linkage); single quote (e g., G’) indicates a 2’0-methyl modification, X¹ is an abasic spacer (“/idSp/”), and X² is C3 spacer phosphoramidite (‘7iSpC3/”).

[0119] In embodiments, the first DNA oligonucleotide comprises SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, or SEQ ID NO: 13. In embodiments, the first DNA oligonucleotide comprises SEQ ID NO: 1. In embodiments, the first DNA oligonucleotide comprises SEQ ID NO:3. In embodiments, the first DNA oligonucleotide comprises SEQ ID NO: 5. In embodiments, the first DNA oligonucleotide comprises SEQ ID NO:7. In embodiments, the first DNA oligonucleotide comprises SEQ ID NO:9. In embodiments, the first DNA oligonucleotide comprises SEQ ID NO: 11. In embodiments, the first DNA oligonucleotide comprises SEQ ID NO: 13. [0120] Table 2

[0121] In Table 2. all the nucleotides are unmodified.

[0122] In embodiments, the first DNA oligonucleotide comprises SEQ ID NO: 1 and the second DNA oligonucleotide comprises SEQ ID NO:2. In embodiments, the first DNA oligonucleotide comprises SEQ ID NO:3 and the second DNA oligonucleotide comprises SEQ ID NO:4. In embodiments, the first DNA oligonucleotide comprises SEQ ID NO: 5 and the second DNA oligonucleotide comprises SEQ ID NO:6. In embodiments, the first DNA oligonucleotide comprises SEQ ID NO:7 and the second DNA oligonucleotide comprises SEQ ID NO:8. In embodiments, the first DNA oligonucleotide comprises SEQ ID NO:9 and the second DNA oligonucleotide comprises SEQ ID NOTO. In embodiments, the first DNA oligonucleotide comprises SEQ ID NO: 11 and the second DNA oligonucleotide comprises SEQ ID NO: 12. In embodiments, the first DNA oligonucleotide comprises SEQ ID NO: 13 and the second DNA oligonucleotide comprises SEQ ID NO: 14. In embodiments, the first DNA oligonucleotide comprises SEQ ID NOT and the second DNA oligonucleotide comprises SEQ ID NO:35. In embodiments, the first DNA oligonucleotide comprises SEQ ID NO: 1 and the second DNA oligonucleotide comprises SEQ ID NO:36.

[0123] The phrase “a phosphorothioated CpG oligodeoxynucleotide linked to a first DNA oligonucleotide that is hybridized to a second DNA oligonucleotide” is equivalent to the phrase “a phosphorothioated CpG oligodeoxynucleotide linked to a first DNA oligonucleotide, wherein the first DNA oligonucleotide is hybridized to a second DNA oligonucleotide.”

[0124] The compounds described herein comprise a phosphorothioated CpG oligodeoxynucleotide (ODN). In embodiments, the CpG ODN is a CpG-A ODN, a CpG-B ODN, a CpG-C ODN, or a combination of two or more thereof. In embodiments, the CpG ODN is a CpG-A ODN. In embodiments, the CpG ODN is a CpG-B ODN. In embodiments, the CpG ODN is a CpG-C ODN. In embodiments, the CpG ODN is CpG ODN 1585, CpG ODN 2216, CpG ODN 2336, CpG ODN 1668, CpG ODN 1826, CpG ODN 2006, CpG ODN 2007, CpG ODN BW006, CpG ODN D-SL01, CpG ODN 2395, CpG ODN M362. CpG ODN D-SL03, CpG ODN DI 9, or a combination of two or more thereof. In embodiments, the CpG ODN is CpG ODN 1585. In embodiments, the CpG ODN is CpG ODN 2216. In embodiments, the CpG ODN is CpG ODN 2336. In embodiments, the CpG ODN is CpG ODN 1668. In embodiments, the CpG ODN is CpG ODN 1826. In embodiments, the CpG ODN is CpG ODN 2006. In embodiments, the CpG ODN is CpG ODN 2007. In embodiments, the CpG ODN is CpG ODN BW006. In embodiments, the CpG ODN is CpG ODN D-SL01. In embodiments, the CpG ODN is CpG ODN 2395. In embodiments, the CpG ODN is CpG ODN CpG ODN M362. In embodiments, the CpG ODN is CpG ODN D-SL03. In embodiments, the CpG ODN is CpG ODN DI 9.

[0125] In embodiments, the phosphorothioated CpG oligodeoxynucleotide comprises SEQ ID NO:43. SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17. SEQ ID NO: 18, SEQ ID NO: 19. SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, or SEQ ID NO:32. In embodiments, the phosphorothioated CpG oligodeoxynucleotide comprises SEQ ID NO:43. SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, or SEQ ID NO:21. In embodiments, the phosphorothioated CpG oligodeoxynucleotide comprises SEQ ID NO:43, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18. In embodiments, the phosphorothioated CpG oligodeoxynucleotide comprises SEQ ID NO:15, SEQ ID NO: 19, SEQ ID NO:20, or SEQ ID NO:21. In embodiments, the CpG ODN is SEQ ID NO:43 or SEQ ID NO: 15. In embodiments, the CpG ODN is SEQ ID NO:43. In embodiments, the CpG ODN is SEQ ID NO: 15. In embodiments, the CpG ODN is SEQ ID NO:16. In embodiments, the CpG ODN is SEQ ID NO:17. In embodiments, the CpG ODN is SEQ ID NO: 18. In embodiments, the CpG ODN is SEQ ID NO: 19. In embodiments, the CpG ODN is SEQ ID NO:20. In embodiments, the CpG ODN is SEQ ID NO:21. In embodiments, the CpG ODN is SEQ ID NO:22. In embodiments, the CpG ODN is SEQ ID NO:23. In embodiments, the CpG ODN is SEQ ID NO:24. In embodiments, the CpG ODN is SEQ ID NO:25. In embodiments, the CpG ODN is SEQ ID NO:26. In embodiments, the CpG ODN is SEQ ID NO:27. In embodiments, the CpG ODN is SEQ ID NO:28. In embodiments, the CpG ODN is SEQ ID NO:29. In embodiments, the CpG ODN is SEQ ID NO:30. In embodiments, the CpG ODN is SEQ ID NO:31. In embodiments, the CpG ODN is SEQ ID NO:32.

[0126] In embodiments, the compounds described herein include a phosphorothioated CpG oligodeoxynucleotide comprising SEQ ID NO:43 covalently bonded to a first DNA oligonucleotide comprising SEQ ID NO: 1 , wherein SEQ ID NO: 1 is hybridized to a second DNA oligonucleotide comprising SEQ ID NO:2; wherein the 3’ end of the phosphorothioated CpG oligodeoxynucleotide is linked to the 5’ end of the first DNA oligonucleotide. In embodiments, the phosphorothioated CpG oligodeoxynucleotide comprising SEQ ID NO: 15 is covalently bonded to a first DNA oligonucleotide comprising SEQ ID NO: 1, wherein SEQ ID NO: 1 is hybridized to a second DNA oligonucleotide comprising SEQ ID NO:2; wherein the 3’ end of the phosphorothioated CpG oligodeoxynucleotide is linked to the 5’ end of the first DNA oligonucleotide. In embodiments, the phosphorothioated CpG oligodeoxynucleotide is covalently bonded to the first DNA oligonucleotide via the moiety of Formula (A).

[0127] In embodiments, the compounds described herein include a phosphorothioated CpG oligodeoxynucleotide comprising SEQ ID NO:43 covalently bonded to a first DNA oligonucleotide comprising SEQ ID NO: 1, wherein SEQ ID NO: 1 is hybridized to a second DNA oligonucleotide comprising SEQ ID NO:35; wherein the 3’ end of the phosphorothioated CpG oligodeoxynucleotide is linked to the 5’ end of the first DNA oligonucleotide. In embodiments, the phosphorothioated CpG oligodeoxynucleotide comprising SEQ ID NO: 15 is covalently bonded to a first DNA oligonucleotide comprising SEQ ID NO: 1, wherein SEQ ID NO: 1 is hybridized to a second DNA oligonucleotide comprising SEQ ID NO:35; wherein the 3’ end of the phosphorothioated CpG oligodeoxynucleotide is linked to the 5‘ end of the first DNA oligonucleotide. In embodiments, the phosphorothioated CpG oligodeoxynucleotide is covalently bonded to the first DNA oligonucleotide via the moiety of Formula (A).

[0128] In embodiments, the compounds described herein include a phosphorothioated CpG oligodeoxynucleotide comprising SEQ ID NO:43 covalently bonded to a first DNA oligonucleotide comprising SEQ ID NO: 1, wherein SEQ ID NO: 1 is hybridized to a second DNA oligonucleotide comprising SEQ ID NO:36; wherein the 3‘ end of the phosphorothioated CpG oligodeoxynucleotide is linked to the 5’ end of the first DNA oligonucleotide. In embodiments, the phosphorothioated CpG oligodeoxynucleotide comprising SEQ ID NO: 15 is covalently bonded to a first DNA oligonucleotide comprising SEQ ID NO: 1, wherein SEQ ID NO: 1 is hybridized to a second DNA oligonucleotide comprising SEQ ID NO:36; wherein the 3’ end of the phosphorothioated CpG oligodeoxynucleotide is linked to the 5’ end of the first DNA oligonucleotide. In embodiments, the phosphorothioated CpG oligodeoxynucleotide is covalently bonded to the first DNA oligonucleotide via the moiety of Formula (A).

[0129] In embodiments, the compounds described herein include a phosphorothioated CpG oligodeoxynucleotide comprising SEQ ID NO: 15 covalently bonded to a first DNA oligonucleotide comprising SEQ ID NO:3, wherein SEQ ID NO:3 is hybridized to a second DNA oligonucleotide comprising SEQ ID NO:4, wherein the 3’ end of the phosphorothioated CpG oligodeoxynucleotide is linked to the 5’ end of the first DNA oligonucleotide. In embodiments, the phosphorothioated CpG oligodeoxynucleotide comprising SEQ ID NO:43 is covalently bonded to a first DNA oligonucleotide comprising SEQ ID NO:3, wherein SEQ ID NO:3 is hybridized to a second DNA oligonucleotide comprising SEQ ID NO:4, wherein the 3’ end of the phosphorothioated CpG oligodeoxynucleotide is linked to the 5’ end of the first DNA oligonucleotide. In embodiments, the phosphorothioated CpG oligodeoxynucleotide is covalently bonded to the first DNA oligonucleotide via the moiety of Formula (A).

[0130] Table 3

[0131] In Table 3, underline refers to a phosphorothioated nucleotide (phosphorothioated intemucleotide linkage).

[0132] The phosphorothioated CpG ODN is linked to the first DNA oligonucleotide by any linking group known in the art. In embodiments, the linking group comprises a bond, a nucleic acid sequence, a DNA sequence, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, substituted or unsubstituted heteroarylene, or a combination of two or more thereof. In embodiments, the linking group comprises a bond, a nucleic acid sequence, unsubstituted alkylene, unsubstituted heteroalkydene, or a combination of two or more thereof. In embodiments, the linking group is a covalent bond. In embodiments, the linking group is a nucleic acid sequence. In embodiments, the linking group is a DNA sequence. In embodiments, the linking group comprises a nucleic acid sequence and a substituted or unsubstituted alkylene. In embodiments, the linking group comprises a nucleic acid sequence and an unsubstituted alkylene. In embodiments, the linking group comprises a nucleic acid sequence and a substituted or unsubstituted heteroalky lene. In embodiments, the linking group comprises a nucleic acid sequence and an unsubstituted heteroalkylene. In embodiments, the linking group comprises a substituted or unsubstituted heteroalkylene. In embodiments, the linking group comprises a substituted heteroalkylene.

[0133] In embodiments, the linking group comprises a substituted heteroalkydene. In embodiments, the linking group comprises a substituted 6 to 60 membered heteroalkylene. In embodiments, the linking group comprises a substituted 6 to 54 membered heteroalkylene. In embodiments, the linking group comprises a substituted 12 to 48 membered heteroalky lene. In embodiments, the linking group comprises a substituted 18 to 42 membered heteroalky lene. In embodiments, the linking group comprises a substituted 24 to 36 membered heteroalky lene. In embodiments, the linking group comprises a substituted 30 membered heteroalky lene. In embodiments, the heteroalkylene comprises an oxygen atom, a phosphorous atom, or a combination thereof. In embodiments, the substituents on the substituted heteroalkylene comprise oxo, -OH, -O', or a combination of two or more thereof. In embodiments, the linking group comprises a substituted 18 to 42 membered heteroalkylene; wherein the heteroalkylene comprises an oxygen atom, a phosphorous atom, or a combination thereof; and wherein the substituents are independently selected from the group consisting of oxo, -OH, and -O'.

[0134] In embodiments, the linking group comprises any one of the following structures:

wherein zl, z2, z3 and z4 are independently integers from 0 to 20; and each X is independently - OH or -O’. In embodiments, zl is an integer from 0 to 5. In embodiments, zl is an integer from 2 to 4. In embodiments, z2 is an integer from 0 to 5. In embodiments. z2 is an integer from 2 to 4. In embodiments, z3 is an integer from 0 to 5. In embodiments, zl is an integer from 2 to 4. In embodiments, z4 is an integer from 3 to 7. In embodiments, z4 is an integer from 4 to 6. In embodiments, each X is -OH.

[0135] In embodiments, the linking group comprises the structure:

wherein n is an integer from 1 to 10. In embodiments, n is an integer from 2 to 8. In embodiments, n is an integer from 3 to 7. In embodiments, n is an integer from 4 to 6. In embodiments, n is 1. In embodiments, n is 2. In embodiments, n is 3. In embodiments, n is 4. In embodiments, n is 5. In embodiments, n is 6. In embodiments, n is 7. In embodiments, n is 8. In embodiments, n is 9. In embodiments, n is 10.

[0136] In embodiments, the compound further comprises a detectable moiety-. In embodiments, the phosphorothioated CpG ODN, the first DNA oligonucleotide, the second DNA oligonucleotide, or any combination thereof comprise a detectable moiety⁷. In embodiments, the phosphorothioated CpG ODN comprises a detectable moiety . In embodiments, the first DNA oligonucleotide comprises a detectable moiety-. In embodiments, the second DNA oligonucleotide comprises a detectable moiety⁷.

[0137] A compound compnsing a detectable moiety' is one that is bound, either covalently, through a linker or a chemical bond, or noncovalently, through ionic, van der Waals, electrostatic, or hydrogen bonds to a detectable moiety' such that the presence of the nucleic acid may be detected by detecting the presence of the detectable moiety bound to the nucleic acid. Alternatively, a method using high affinity interactions may achieve the same results where one of a pair of binding partners binds to the other, e g., detectable moiety. In embodiments of the compounds described herein, the phosphorothioate nucleic acid or phosphorothioate polymer backbone includes a detectable agent, as disclosed herein and known in the art. [0138] A “detectable agent” or “detectable moiety” is a compound or composition detectable by appropriate means such as spectroscopic, photochemical, biochemical, immunochemical, chemical, magnetic resonance imaging, or other physical means. A detectable moiety is a monovalent detectable agent or a detectable agent bound (e.g. covalently and directly or via a linking group) with another compound, e.g., a nucleic acid. Exemplary⁷ detectable agents/moieties for use in the present disclosure include an antibody ligand, a peptide, a nucleic acid, radioisotopes, paramagnetic metal ions, fluorophore (e.g. fluorescent dyes), electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, a biotin-avidin complex, a biotin-streptavidin complex, digoxigenin, magnetic beads (e.g., DYNABEADS® by ThermoFisher, encompassing functionalized magnetic beads such as DYNABEADS® M-270 amine by ThermoFisher), paramagnetic molecules, paramagnetic nanoparticles, ultrasmall superparamagnetic iron oxide nanoparticles, ultrasmall superparamagnetic iron oxide nanoparticle aggregates, superparamagnetic iron oxide nanoparticles, superparamagnetic iron oxide nanoparticle aggregates, monocrystalline iron oxide nanoparticles, monocrystalline iron oxide, nanoparticle contrast agents, liposomes or other delivery vehicles containing Gadolinium chelate molecules, gadolinium, radionuclides (e.g. carbon-11, nitrogen-13, oxygen-15, fluorine- 18, rubidium-82), fluorodeoxyglucose (e.g. fluorine-18 labeled), any gamma ray emitting radionuclides, positron-emitting radionuclide, radiolabeled glucose, radiolabeled water, radiolabeled ammonia, biocolloids, microbubbles (e.g. including microbubble shells including albumin, galactose, lipid, and/or polymers; microbubble gas core including air. heavy gases, perfluorcarbon, nitrogen, octafluoropropane, perflexane lipid microsphere, perflutren, etc ), iodinated contrast agents (e.g. iohexol, iodixanol, ioversol, iopamidol, ioxilan, iopromide, diatrizoate, metrizoate. ioxaglate), barium sulfate, thorium dioxide, gold, gold nanoparticles, gold nanoparticle aggregates, fluorophores, two-photon fluorophores, or haptens and proteins or other entities which can be made detectable, e.g., by incorporating a radiolabel into a peptide or antibody specifically reactive with a target peptide.

[0139] Pharmaceutical Compositions

[0140] In embodiments, the disclosure provides pharmaceutical compositions comprising a compound described herein and a pharmaceutically acceptable excipient. “Pharmaceutically acceptable excipient” refers to a substance that aids the administration of an active agent to and absorption by a subject and can be included in the compositions of the present disclosure without causing a significant adverse toxicological effect on the patient. Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, normal saline solutions, lactated Ringer's, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavors, salt solutions (such as Ringer's solution), alcohols, oils, gelatins, carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethycellulose, polyvinyl pyrrolidine, and colors, and the like. Such preparations can be sterilized and, if desired, mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and/or aromatic substances and the like that do not deleteriously react with the compounds of the disclosure. One of skill in the art will recognize that other pharmaceutical excipients are useful in the present disclosure.

[0141] Provided herein are pharmaceutical compositions comprising: (1) an immune checkpoint inhibitor and (2) a compound which comprises a phosphorothioated CpG oligodeoxynucleotide linked to a first DNA oligonucleotide that is hybridized to a second DNA oligonucleotide; wherein the first DNA oligonucleotide comprises from about 5 unmodified nucleotides to about 70 unmodified nucleotides; and the second DNA oligonucleotide comprises (i) from about 5 nucleotides to about 70 nucleotides, and (ii) a constrained nucleotide on the 3’ end and a constrained nucleotide on the 5’ end. In embodiments, the compound (2) is any compound described herein, including all embodiments of the compounds described herein. In embodiments, the constrained nucleotide is a LNA-modified nucleotide, a cMOE-modified nucleotide, or a cET-modified nucleotide. In embodiments, the constrained nucleotide is a LNA- modified nucleotide. In embodiments, the constrained nucleotide is a cMOE-modified nucleotide. In embodiments, the constrained nucleotide is a cET-modified nucleotide. In embodiments, the second DNA oligonucleotide comprises a nucleotide having a phosphorothioate modification. In embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor. In embodiments, the immune checkpoint inhibitor is a PD-L1 inhibitor. In embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor.

[0142] A “effective amount” is an amount sufficient for a compound to accomplish a stated purpose relative to the absence of the compound (e.g. achieve the effect for which it is administered, treat a disease). An example of an “effective amount” is an amount sufficient to contribute to the treatment of a disease which could also be referred to as a “therapeutically effective amount.” A “reduction” of a symptom or symptoms (and grammatical equivalents of this phrase) means decreasing of the severity' or frequency of the symptoms or elimination of the symptoms. The exact amounts will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy. 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins). For any compound described herein, the therapeutically effective amount can be initially determined from cell culture assays. Target concentrations will be those concentrations of active compound that are capable of achieving the methods described herein, as measured using the methods described herein or known in the art. As is known in the art, therapeutically effective amounts for use in humans can also be determined from animal models. For example, a dose for humans can be formulated to achieve a concentration that has been found to be effective in animals. The dosage in humans can be adjusted by monitoring the effectiveness of the compounds or compositions described herein, and adjusting the dosage upwards or downwards. Adjusting the dose to achieve maximal efficacy in humans based on the methods described above and other methods is well within the capabilities of the ordinarily skilled artisan.

[0143] Dosages may be varied depending upon the requirements of the patient and the compound being employed. The dose administered to a patient, in the context of the present disclosure, should be sufficient to effect a beneficial therapeutic response in the patient over time. The size of the dose also will be determined by the existence, nature, and extent of any adverse side-effects. Determination of the proper dosage for a particular situation is within the skill of the practitioner. Generally, treatment is initiated with smaller dosages which are less than the optimum dose of the compound. Thereafter, the dosage is increased by small increments until the optimum effect under circumstances is reached. Dosage amounts and intervals can be adjusted individually to provide levels of the administered compound effective for the particular clinical indication being treated. This will provide a therapeutic regimen that is commensurate with the severity of the individual's disease state.

[0144] The term ’‘administering” means oral administration, administration as a suppository, topical contact, intravenous, parenteral, intraperitoneal, intramuscular, intralesional, intrathecal, intranasal or subcutaneous administration, or the implantation of a slow-release device, e.g., a mini-osmotic pump, to a subject. Administration is by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, e.g., intra-tumoral, intravenous, intramuscular, intra-arteriole, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial. In embodiments, the compounds or pharmaceutical compositions described herein are parenterally administered to a patient. In embodiments, the compounds or pharmaceutical compositions descnbed herein are administered intra-tumorally to a patient. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, etc. In embodiments, the administering does not include administration of any active agent other than the recited active agent.

[0145] Methods of Treatment

[0146] In embodiments, the disclosure provides methods of treating cancer in a patient in need thereof. The methods of treating cancer comprise administering to the patient in an effective amount of a compound comprising a phosphorothioated CpG oligodeoxynucleotide linked to a first DNA oligonucleotide that is hybridized to a second DNA oligonucleotide; wherein the first DNA oligonucleotide comprises from about 5 unmodified nucleotides to about 70 unmodified nucleotides; and the second DNA oligonucleotide comprises (i) from about 5 nucleotides to about 70 nucleotides, and (ii) a constrained nucleotide on the 3’ end and a constrained nucleotide on the 5’ end. In embodiments, the constrained nucleotide is a LNA-modified nucleotide, a cMOE-modified nucleotide, or a cET-modified nucleotide. In embodiments, the constrained nucleotide is a LNA-modified nucleotide. In embodiments, the constrained nucleotide is a cMOE-modified nucleotide. In embodiments, the constrained nucleotide is a cET-modified nucleotide. In embodiments, the second DNA oligonucleotide comprises a nucleotide having a phosphorothioate modification. In embodiments, the compound is any compound described herein, including all embodiments of the compounds described herein. In embodiments, the methods further comprise administering to the patient an effective amount of an immune checkpoint inhibitor. In embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor. In embodiments, the immune checkpoint inhibitor is a PD-L1 inhibitor. In embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor. In embodiments, the methods further comprise administering to the patient an effective amount of an anti-cancer agent. In embodiments, the cancer is a central nervous system cancer, leukemia, lymphoma, a solid tumor cancer, or an epidermoid carcinoma. In embodiments, the cancer is a central nervous system cancer. In embodiments, the cancer is a solid tumor cancer. In embodiments, the cancer is an epidermoid carcinoma. In embodiments, the cancer is leukemia. In embodiments, the cancer is lymphoma. In embodiments, the cancer is B cell lymphoma.

[0147] In embodiments, the disclosure provides methods of treating a central nervous system cancer in a patient in need thereof by administering to the patient in an effective amount of a compound comprising a phosphorothioated CpG oligodeoxynucleotide linked to a first DNA oligonucleotide that is hybridized to a second DNA oligonucleotide; wherein the first DNA oligonucleotide comprises from about 5 unmodified nucleotides to about 70 unmodified nucleotides; and the second DNA oligonucleotide comprises (i) from about 5 nucleotides to about 70 nucleotides, and (ii) a constrained nucleotide on the 3’ end and a constrained nucleotide on the 5’ end. In embodiments, the constrained nucleotide is a LNA-modified nucleotide, a cMOE-modified nucleotide, or a cET-modified nucleotide. In embodiments, the constrained nucleotide is a LNA-modified nucleotide. In embodiments, the constrained nucleotide is a cMOE-modified nucleotide. In embodiments, the constrained nucleotide is a cET-modified nucleotide. In embodiments, the second DNA oligonucleotide comprises a nucleotide having a phosphorothioate modification. In embodiments, the compound is any compound described herein, including all embodiments of the compounds described herein. In embodiments, the methods further comprise administering to the patient an effective amount of an immune checkpoint inhibitor. In embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor. In embodiments, the immune checkpoint inhibitor is a PD-L1 inhibitor. In embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor. In embodiments, the methods further comprise administering to the patient an effective amount of an anti-cancer agent. In embodiments, the central nervous system cancer is glioma, cranial primitive neuroectodermal tumor, ependymal tumor, hemangiopericytoma, germ cell tumor, pineal tumor, or primary central nervous system lymphoma. In embodiments, the cancer is glioma. In embodiments, the glioma is astrocytoma, glioblastoma, or oligodendroglioma. In embodiments, the glioma is astrocytoma. In embodiments, the glioma is glioblastoma. In embodiments, the glioma is oligodendroglioma. In embodiments, the glioma is a brain stem glioma. In embodiments, the glioma is a mixed glioma. In embodiments, the glioma is an optic pathway glioma. In embodiments, the cancer is cranial primitive neuroectodermal tumor. In embodiments, the cranial primitive neuroectodermal tumor is medulloblastoma, cerebral neuroblastoma, pineoblastoma, or esthesioneuroblastoma. In embodiments, the cranial primitive neuroectodermal tumor is medulloblastoma. In embodiments, the cranial primitive neuroectodermal tumor is cerebral neuroblastoma. In embodiments, the cranial primitive neuroectodermal tumor is pineoblastoma. In embodiments, the cranial primitive neuroectodermal tumor is esthesioneuroblastoma. In embodiments, the cancer is ependymal tumor. In embodiments, the ependymal tumor is ependymoma, myxopapillary ependymoma, or subependymoma. In embodiments, the ependymal tumor is ependymoma. In embodiments, the ependymal tumor is myxopapillary ependymoma. In embodiments, the ependymal tumor is subependymoma. In embodiments, the cancer is hemangiopericytoma. In embodiments, the cancer is germ cell tumor. In embodiments, the cancer is pineal tumor. In embodiments, the cancer is primary central nervous system lymphoma. [0148] In embodiments, the disclosure provides methods of treating a glioma in a patient in need thereof by administering to the patient in an effective amount of a compound comprising a phosphorothioated CpG oligodeoxynucleotide linked to a first DNA oligonucleotide that is hybridized to a second DNA oligonucleotide; wherein the first DNA oligonucleotide comprises from about 5 unmodified nucleotides to about 70 unmodified nucleotides; and the second DNA oligonucleotide comprises (i) from about 5 nucleotides to about 70 nucleotides, and (ii) a constrained nucleotide on the 3’ end and a constrained nucleotide on the 5’ end. In embodiments, the constrained nucleotide is a LNA-modified nucleotide, a cMOE-modified nucleotide, or a cET-modified nucleotide. In embodiments, the constrained nucleotide is a LNA- modified nucleotide. In embodiments, the constrained nucleotide is a cMOE-modified nucleotide. In embodiments, the constrained nucleotide is a cET-modified nucleotide. In embodiments, the second DNA oligonucleotide comprises a nucleotide having a phosphorothioate modification. In embodiments, the compound is any compound described herein, including all embodiments of the compounds described herein. In embodiments, the methods further comprise administering to the patient an effective amount of an immune checkpoint inhibitor. In embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor. In embodiments, the immune checkpoint inhibitor is a PD-L1 inhibitor. In embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor. In embodiments, the methods further comprise administering to the patient an effective amount of an anti-cancer agent.

[0149] In embodiments, the disclosure provides methods of treating leukemia in a patient in need thereof by administering to the patient in an effective amount of a compound compnsing a phosphorothioated CpG oligodeoxynucleotide linked to a first DNA oligonucleotide that is hybridized to a second DNA oligonucleotide; wherein the first DNA oligonucleotide comprises from about 5 unmodified nucleotides to about 70 unmodified nucleotides; and the second DNA oligonucleotide comprises (i) from about 5 nucleotides to about 70 nucleotides, and (ii) a constrained nucleotide on the 3’ end and a constrained nucleotide on the 5’ end. In embodiments, the constrained nucleotide is a LNA-modified nucleotide, a cMOE-modified nucleotide, or a cET-modified nucleotide. In embodiments, the constrained nucleotide is a LNA- modified nucleotide. In embodiments, the constrained nucleotide is a cMOE-modified nucleotide. In embodiments, the constrained nucleotide is a cET-modified nucleotide. In embodiments, the second DNA oligonucleotide comprises a nucleotide having a phosphorothioate modification. In embodiments, the compound is any compound described herein, including all embodiments of the compounds described herein. In embodiments, the cancer is acute myeloid leukemia. In embodiments, the methods further comprise administering to the patient an effective amount of an immune checkpoint inhibitor. In embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor. In embodiments, the immune checkpoint inhibitor is a PD-L1 inhibitor. In embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor. In embodiments, the methods further comprise administering to the patient an effective amount of an anti-cancer agent.

[0150] In embodiments, the disclosure provides methods of treating a solid tumor cancer in a patient in need thereof by administering to the patient in an effective amount of a compound comprising a phosphorothioated CpG oligodeoxynucleotide linked to a first DNA oligonucleotide that is hybridized to a second DNA oligonucleotide; wherein the first DNA oligonucleotide comprises from about 5 unmodified nucleotides to about 70 unmodified nucleotides; and the second DNA oligonucleotide comprises (i) from about 5 nucleotides to about 70 nucleotides, and (ii) a constrained nucleotide on the 3’ end and a constrained nucleotide on the 5’ end. In embodiments, the constrained nucleotide is a LNA-modified nucleotide, a cMOE- modified nucleotide, or a cET-modified nucleotide. In embodiments, the constrained nucleotide is a LNA-modified nucleotide. In embodiments, the constrained nucleotide is a cMOE-modified nucleotide. In embodiments, the constrained nucleotide is a cET-modified nucleotide. In embodiments, the second DNA oligonucleotide comprises a nucleotide having a phosphorothioate modification. In embodiments, the compound is any compound described herein, including all embodiments of the compounds described herein. In embodiments, the methods further comprise administering to the patient an effective amount of an immune checkpoint inhibitor. In embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor. In embodiments, the immune checkpoint inhibitor is a PD-L1 inhibitor. In embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor. In embodiments, the methods further comprise administering to the patient an effective amount of an anti-cancer agent. In embodiments, the solid tumor cancer is prostate cancer, breast cancer, colorectal cancer, bladder cancer, lung cancer, liver cancer, pancreatic caner, renal cancer, stomach cancer, or melanoma. In embodiments, the solid tumor cancer is prostate cancer. In embodiments, the solid tumor cancer is breast cancer. In embodiments, the solid tumor cancer is colorectal cancer. In embodiments, the solid tumor cancer is bladder cancer. In embodiments, the solid tumor cancer is lung cancer. In embodiments, the solid tumor cancer is liver cancer. In embodiments, the solid tumor cancer is pancreatic caner. In embodiments, the solid tumor cancer is renal cancer. In embodiments, the cancer is stomach cancer. In embodiments, the solid tumor cancer is melanoma. [0151] In embodiments, the disclosure provides methods of treating prostate cancer in a patient in need thereof by administering to the patient in an effective amount of a compound comprising a phosphorothioated CpG oligodeoxynucleotide linked to a first DNA oligonucleotide that is hybridized to a second DNA oligonucleotide; wherein the first DNA oligonucleotide comprises from about 5 unmodified nucleotides to about 70 unmodified nucleotides; and the second DNA oligonucleotide comprises (i) from about 5 nucleotides to about 70 nucleotides, and (ii) a constrained nucleotide on the 3’ end and a constrained nucleotide on the 5’ end. In embodiments, the compound is any compound described herein, including all embodiments of the compounds described herein. In embodiments, the constrained nucleotide is a LNA-modified nucleotide, a cMOE-modified nucleotide, or a cET-modified nucleotide. In embodiments, the constrained nucleotide is a LNA-modified nucleotide. In embodiments, the constrained nucleotide is a cMOE-modified nucleotide. In embodiments, the constrained nucleotide is a cET-modified nucleotide. In embodiments, the second DNA oligonucleotide comprises a nucleotide having a phosphorothioate modification. In embodiments, the methods further comprise administering to the patient an effective amount of an immune checkpoint inhibitor. In embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor. In embodiments, the immune checkpoint inhibitor is a PD-L1 inhibitor. In embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor. In embodiments, the methods further comprise administering to the patient an effective amount of an anti-cancer agent.

[0152] In embodiments, the disclosure provides methods of treating epidermoid carcinoma in a patient in need thereof by administering to the patient in an effective amount of a compound comprising a phosphorothioated CpG oligodeoxynucleotide linked to a first DNA oligonucleotide that is hybridized to a second DNA oligonucleotide; wherein the first DNA oligonucleotide comprises from about 5 unmodified nucleotides to about 70 unmodified nucleotides; and the second DNA oligonucleotide comprises (i) from about 5 nucleotides to about 70 nucleotides, and (ii) a constrained nucleotide on the 3’ end and a constrained nucleotide on the 5’ end. In embodiments, the constrained nucleotide is a LNA-modified nucleotide, a cMOE-modified nucleotide, or a cET-modified nucleotide. In embodiments, the constrained nucleotide is a LNA-modified nucleotide. In embodiments, the constrained nucleotide is a cMOE-modified nucleotide. In embodiments, the constrained nucleotide is a cET-modified nucleotide. In embodiments, the second DNA oligonucleotide comprises a nucleotide having a phosphorothioate modification. In embodiments, the compound is any compound described herein, including all embodiments of the compounds described herein. In embodiments, the methods further comprise administering to the patient an effective amount of an immune checkpoint inhibitor. In embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor. In embodiments, the immune checkpoint inhibitor is a PD-L1 inhibitor. In embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor. In embodiments, the methods further comprise administering to the patient an effective amount of an anti-cancer agent. In embodiments, the epidermoid carcinoma is thyroid cancer, esophageal cancer, vaginal cancer, anal cancer, cervical cancer, or head and neck cancer. In embodiments, the epidermoid carcinoma is thyroid cancer. In embodiments, the epidermoid carcinoma is esophageal cancer. In embodiments, the cancer is anal cancer. In embodiments, the cancer is cervical cancer. In embodiments, the cancer is head and neck cancer. In embodiments, the cancer is vaginal cancer.

[0153] In embodiments of the methods described herein, the PD-1 inhibitor is pembrolizumab, nivolumab. cemiplimab, dostarlimab, camrelizumab, sintilimab, tislelizumab, toripalimab, spartalizumab, retifanlimab, pimivalimab (JTX-4014), AMP-224, or MEDI0680 (AMP-514). In embodiments, the PD-1 inhibitor is pembrolizumab, nivolumab, cemiplimab, dostarlimab, camrelizumab, sintilimab, tislelizumab, toripalimab, retifanlimab, or spartalizumab. In embodiments, the PD-1 inhibitor is pembrolizumab. In embodiments, the PD-1 inhibitor is nivolumab. In embodiments, the PD-1 inhibitor is cemiplimab. In embodiments, the PD-1 inhibitor is dostarlimab. In embodiments, the PD-1 inhibitor is camrelizumab. In embodiments, the PD-1 inhibitor is sintilimab. In embodiments, the PD-1 inhibitor is tislelizumab. In embodiments, the PD-1 inhibitor is toripalimab. In embodiments, the PD-1 inhibitor is spartalizumab. In embodiments, the PD-1 inhibitor is pimivalimab. In embodiments, the PD-1 inhibitor is retifanlimab. In embodiments, the PD-1 inhibitor is AMP-224. In embodiments, the PD-1 inhibitor is MEDI0680.

[0154] In embodiments of the methods described herein, the PD-L1 inhibitor is atezolizumab, avelumab, durvalumab, envafolimab, cosibelimab, AUNP12, CA-170, or BMS-986189. In embodiments, the PD-L1 inhibitor is atezolizumab, avelumab, durvalumab, envafolimab, cosibelimab, AUNP12, CA-170. or BMS-986189. In embodiments, the PD-L1 inhibitor is atezolizumab, avelumab, durvalumab, envafolimab, or cosibelimab. In embodiments, the PD-L1 inhibitor is atezolizumab. In embodiments, the PD-L1 inhibitor is avelumab. In embodiments, the PD-L1 inhibitor is durvalumab. In embodiments, the PD-L1 inhibitor is envafolimab. In embodiments, the PD-L1 inhibitor is cosibelimab. In embodiments, the PD-L1 inhibitor is ALJNP12. In embodiments, the PD-L1 inhibitor is CA-170. In embodiments, the PD-L1 inhibitor is BMS-986189. [0155] The term “cancer” refers to all ty pes of cancer, neoplasm or malignant tumors found in mammals (e.g. humans), including leukemias, lymphomas, carcinomas and sarcomas.

Exemplary cancers that can be treated with the compounds and pharmaceutical compositions described herein include brain cancer, glioma, glioblastoma, neuroblastoma, prostate cancer, colorectal cancer, pancreatic cancer, medulloblastoma, melanoma, cervical cancer, gastric cancer, ovarian cancer, lung cancer, cancer of the head, Hodgkin's disease, and Non-Hodgkin's lymphomas. Other cancers that may be treated with the compounds and pharmaceutical compositions described herein include cancer of the thyroid, endocrine system, brain, breast, cervix, colon, head and neck, liver, kidney, lung, ovary, pancreas, rectum, stomach, and uterus. Additional examples of cancers that may be treated with the compounds and pharmaceutical compositions described herein include, thyroid carcinoma, cholangiocarcinoma, pancreatic adenocarcinoma, skin cutaneous melanoma, colon adenocarcinoma, rectum adenocarcinoma, stomach adenocarcinoma, esophageal carcinoma, head and neck squamous cell carcinoma, breast invasive carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, non-small cell lung carcinoma, mesothelioma, multiple myeloma, neuroblastoma, glioma, glioblastoma, ovarian cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, primary brain tumors, malignant pancreatic insulanoma, malignant carcinoid, urinary bladder cancer, premalignant skin lesions, testicular cancer, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, endometrial cancer, adrenal cortical cancer, neoplasms of the endocrine or exocrine pancreas, medullary thy roid cancer, medullary thyroid carcinoma, melanoma, colorectal cancer, papillary thyroid cancer, hepatocellular carcinoma, or prostate cancer.

[0156] The terms “treating” or “treatment” refers to any indicia of clinical success in the therapy or amelioration of a disease (e.g., cancer), including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the disease more tolerable to the patient; slowing in the rate of degeneration or decline; making the final point of degeneration less debilitating; improving a patient’s physical or mental well-being. The treatment or amelioration of symptoms can be based on objective or subjective parameters; including the results of a physical examination. The term “treating” does not include preventing.

[0157] “Patient” or “subject in need thereof’ refers to a living organism suffering from or prone to a disease n that can be treated by administration of a compound or pharmaceutical composition herein. Non-limiting examples include humans, other mammals, bovines, rats, mice, dogs, cats, monkeys, goat, sheep, cows, and other non-mammalian animals. In embodiments, a patient is human.

[0158] Cancer model organism, as used herein, is an organism exhibiting a phenoty pe indicative of cancer, or the activity of cancer causing elements, within the organism. The term cancer is defined above. A wide variety of organisms may serve as cancer model organisms, and include for example, cancer cells and mammalian organisms such as rodents (e.g. mouse or rat) and primates (such as humans). Cancer cell lines are widely understood by those skilled in the art as cells exhibiting phenotypes or genotypes similar to in vivo cancers. Cancer cell lines as used herein includes cell lines from animals (e.g. mice) and from humans.

[0159] The term “immune checkpoint inhibitor’ refers to a compound (e g., an antibody) that is capable of binding to an inhibitory receptor or capable of interfering with the interaction between an inhibitory receptor and its ligand, wherein the inhibitory receptor is essential to balance co-stimulatory receptor activity and limit T-cell activation. Thus, immune checkpoint inhibitors target immune system checkpoints such as the PD-1 pathway.

[0160] “PD-1 pathway inhibitor” refers to a substance capable of detectably lowering expression of or activity level of the PD-1 signaling pathway compared to a control. An “inhibitor” is a compound or small molecule that inhibits the PD-1 signaling pathway e.g.. by binding, partially or totally blocking stimulation of the PD-1 pathway, decrease, prevent, or delay activation of the PD-1 pathway, or inactivate, desensitize, or down-regulate signal transduction, gene expression or enzymatic activity of the PD-1 pathway. In embodiments, the PD-1 pathway inhibitor is a programmed death-ligand 1 (PD-L1) inhibitor or a PD-1 inhibitor. A PD-L1 inhibitor is a substance that, at least in part, partially or totally blocks stimulation, decreases, prevents, or delays activation, or inactivates, desensitizes, or down-regulates signal transduction of PD-1. A PD-1 inhibitor is a substance that, at least in part, partially or totally blocks stimulation, decreases, prevents, or delays activation, or inactivates, desensitizes, or down-regulates signal transduction of PD-1.

[0161] In embodiments of the methods of treating cancer described herein, the methods further comprise administering to the patient an effective amount of an anti-cancer agent. “Anticancer agent” is used in accordance with its plain ordinary meaning and refers to a composition (e.g. compound, drug, antagonist, inhibitor, modulator) having antineoplastic properties or the ability to inhibit the growth or proliferation of cells. Exemplary anti-cancer agents include antibodies, small molecules, large molecules, and combinations thereof. In embodiments, an anti-cancer agent is a chemotherapeutic. In embodiments, an anti-cancer agent is an agent identified herein having utility in methods of treating cancer. In embodiments, an anti-cancer agent is an agent approved by the FDA or similar regulatory agency of a country other than the USA. for treating cancer. Examples of anti-cancer agents include, but are not limited to, MEK (e.g. MEK1, MEK2, or MEK1 and MEK2) inhibitors (e.g. XL518, CI-1040, PD035901, selumetinib/ AZD6244, GSK1120212/ trametmib, GDC-0973, ARRY-162, ARRY-300, AZD8330, PD0325901, U0126, PD98059, TAK-733, PD318088, AS703026, BAY 869766), alkylating agents (e.g., cyclophosphamide, ifosfamide, chlorambucil, busulfan, melphalan. mechlorethamine, uramustine, thiotepa, nitrosoureas, nitrogen mustards (e.g., mechloroethamine, cyclophosphamide, chlorambucil, meiphalan), ethylenimine and methylmelamines (e.g., hexamethlymelamine, thiotepa), alkyl sulfonates (e.g., busulfan), nitrosoureas (e g., carmustine, lomusitne, semustine, streptozocin), triazenes (decarbazine)), anti -metabolites (e.g., 5- azathioprine. leucovorin, capeci tabine, fludarabine, gemcitabine, pemetrexed, raltitrexed, folic acid analog (e.g., methotrexate), or pyrimidine analogs (e.g., fluorouracil, floxouridine, Cytarabine), purine analogs (e.g., mercaptopurine, thioguanine, pentostatin), etc.), plant alkaloids (e.g., vincristine, vinblastine, vinorelbine, vindesine, podophyllotoxin. paclitaxel, docetaxel, etc.), topoisomerase inhibitors (e.g., irinotecan, topotecan, amsacrine, etoposide (VP 16), etoposide phosphate, teniposide, etc.), antitumor antibiotics (e.g., doxorubicin, adriamycin, daunorubicin, epirubicin, actinomycin, bleomycin, mitomycin, mitoxantrone, plicamycin, etc.), platinum-based compounds or platinum containing agents (e.g. cisplatin, oxaloplatin, carboplatin), anthracenedione (e.g., mitoxantrone), substituted urea (e.g., hydroxyurea), methyl hydrazine derivative (e.g., procarbazine), adrenocortical suppressant (e.g., mitotane, aminoglutethimide), epipodophyllotoxins (e.g., etoposide), antibiotics (e.g., daunorubicin, doxorubicin, bleomycin), enzymes (e.g., L-asparaginase), inhibitors of mitogen-activated protein kinase signaling (e.g. U0126, PD98059, PD184352, PD0325901, ARRY-142886, SB239063, SP600125, BAY 43-9006, wortmannin, or LY294002, Syk inhibitors, mTOR inhibitors, antibodies (e.g., rituxan), gossyphol, genasense, polyphenol E, Chlorofusin, all trans-retinoic acid (ATRA), bryostatin, tumor necrosis factor-related apoptosisinducing ligand (TRAIL), 5 -aza-2'-deoxy cytidine, all trans retinoic acid, doxorubicin, vincristine, etoposide, gemcitabine, imatinib (Gleevec.RTM.), geldanamycin, 17-N-Allylamino- 17-Demethoxygeldanamycin (17-AAG), flavopiridol, LY294002, bortezomib, trastuzumab, BAY 1 1-7082, PKC412, PD184352, 20-epi-l, 25 dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; aclarubicin; acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TK antagonists; altretamine; ambamustine; amidox; amifostine; aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole; andrographolide; angiogenesis inhibitors; antagonist D; antagonist G; antarelix; anti-dorsalizing morphogenetic protein- 1; antiandrogen, prostatic carcinoma; antiestrogen; antineoplaston; antisense oligonucleotides; aphidicolin glycinate; apoptosis gene modulators; apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA; arginine deaminase; asulacrine; atamestane; atrimustine; axinastatin 1; axinastatin 2; axinastatin 3; azasetron; azatoxin; azatyrosine; baccatin III derivatives; balanol; batimastat; BCR/ABL antagonists; benzochlorins; benzoylstaurosporine; beta lactam derivatives; beta-alethine; betaclamycin B; betulinic acid; bFGF inhibitor; bicalutamide; bisantrene; bisaziridinylspermine; bisnafide; bistratene A; bizelesin; breflate; bropirimine; budotitane; buthionine sulfoximine; calcipotriol; calphostin C; camptothecin derivatives; canarypox IL-2; capecitabine; carboxamide-amino- triazole; carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived inhibitor; carzelesin; casein kinase inhibitors (ICOS); castanospermine; cecropin B; cetrorelix; chlorins; chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin; cladribine; clomifene analogues; clotrimazole; collismycin A; collismycin B; combretastatin A4; combretastatin analogue; conagenin; crambescidin 816; crisnatol; cryptophy cin 8; cryptophycin A derivatives; curacin A; cyclopentanthraquinones; cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor; cytostatin; dacliximab; decitabine; dehydrodidemnin B; deslorelin; dexamethasone; dexifosfamide; dexrazoxane; dexverapamil; diaziquone; didemnin B; didox; diethylnorspermme; dihydro-5-azacytidine; 9-dioxamycin; diphenyl spiromustine; docosanol; dolasetron; doxifluridine; droloxifene; dronabinol; duocarmycin SA; ebselen; ecomustine; edelfosine; edrecolomab; eflomithine; elemene; emitefur; epirubicin; epristeride; estramustine analogue; estrogen agonists; estrogen antagonists; etanidazole; etoposide phosphate; exemestane; fadrozole; fazarabine; fenretinide; filgrastim; finasteride; flavopiridol; flezelastine; fluasterone; fludarabine; fluorodaunorunicin hydrochloride; forfenimex; formestane; fostriecin; fotemustine; gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix; gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam; heregulin; hexamethylene bisacetamide; hypericin; ibandronic acid; idarubicin; idoxifene; idramantone; ilmofosine; ilomastat; imidazoacridones; imiquimod; immunostimulant peptides; insulin-like growth factor-1 receptor inhibitor; interferon agonists; interferons; interleukins; iobenguane; iododoxorubicin; ipomeanol. 4-; iroplact; irsogladine; isobengazole; isohomohalicondrin B; itasetron; jasplakinolide; kahalalide F; lamellann-N triacetate; lanreotide; leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole; leukemia inhibiting factor; leukocyte alpha interferon; leuprolide+estrogen+progesterone; leuprorelin; levamisole; liarozole; linear polyamine analogue; lipophilic disaccharide peptide; lipophilic platinum compounds; lissoclinamide 7; lobaplatin; lombricine; lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine; lurtotecan; lutetium texaphyrin; tysofylline; lytic peptides; maitansine; mannostatin A; marimastat; masoprocol; maspin; matrilysin inhibitors; matrix metalloproteinase inhibitors; menogaril; merbarone; meterelin; methioninase; metoclopramide; MIF inhibitor; mifepristone; miltefosine; mirimostim; mismatched double stranded RNA; mitoguazone; mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growth factor-saporin; mitoxantrone; mofarotene; molgramostim; monoclonal antibody, human chorionic gonadotrophin; monophosphoryl lipid A+myobacterium cell wall sk; mopidamol; multiple drug resistance gene inhibitor; multiple tumor suppressor 1 -based therapy; mustard anti cancer agent; mycaperoxide B; mycobacterial cell wall extract; myriaporone; N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip; naloxone+pentazocine; napavin; naphterpin; nartograstim; nedaplatin; nemorubicin; neridronic acid; neutral endopeptidase; nilutamide; nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn; O6-benzylguanine; octreotide; okicenone; oligonucleotides; onapristone; ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin; osaterone; oxaliplatin; oxaunomycin; palauamine; palmitoylrhizoxin; pamidronic acid; panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase; peldesine; pentosan polysulfate sodium; pentostatin; pentrozole; perflubron; perfosfamide; perillyl alcohol; phenazinomycin; phenylacetate; phosphatase inhibitors; picibaml; pilocarpine hydrochloride; pirarubicin; piritrexim; placetin A; placetin B; plasminogen activator inhibitor; platinum complex; platinum compounds; platinumtriamine complex; porfimer sodium; porfiromycin; prednisone; propyl bis-acridone; prostaglandin J2: proteasome inhibitors; protein A-based immune modulator; protein kinase C inhibitor; protein kinase C inhibitors, microalgal; protein tyrosine phosphatase inhibitors; purine nucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine; pyridoxylated hemoglobin poly oxy ethylerie conjugate; raf antagonists; raltitrexed; ramosetron; ras famesyl protein transferase inhibitors; ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re 186 etidronate; rhizoxin; ribozymes; RII retinamide; rogletimide; rohitukine; romurtide; roquinimex; rubiginone Bl; ruboxyl; safingol; saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics; semustine; senescence derived inhibitor 1; sense oligonucleotides; signal transduction inhibitors; signal transduction modulators; single chain antigen-binding protein; sizofuran; sobuzoxane; sodium borocaptate; sodium phenylacetate; solverol; somatomedin binding protein; sonermin; sparfosic acid; spicamycin D; spiromustine; splenopentin; spongistatin 1; squalamine; stem cell inhibitor; stem-cell division inhibitors; stipiamide; stromelysin inhibitors; sulfinosine; superactive vasoactive intestinal peptide antagonist; suradista; suramin; swainsonine: synthetic glycosaminoglycans; tallimustine; tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium: tegafur; tellurapyrylium; telomerase inhibitors; temoporfin; temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine; thaliblastine; thiocoraline; thrombopoietin; thrombopoietin mimetic; thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroid stimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocene bichloride; topsentin; toremifene; totipotent stem cell factor; translation inhibitors; tretinoin; triacetyluridine; triciribine; trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinase inhibitors; tyrphostins; UBC inhibitors; ubenimex; urogenital sinus-derived grow th inhibitory factor; urokinase receptor antagonists; vapreotide; variolin B; vector system, erythrocyte gene therapy; velaresol; veramine; verdins; verteporfin; vinorelbine; vinxaltine; vitaxin; vorozole; zanoterone; zeniplatin; zilascorb; zinostatin stimalamer, Adriamycin, Dactinomycin, Bleomycin, Vinblastine, Cisplatin, acivicin; aclarubicin; acodazole hydrochloride; acronine; adozelesin; aldesleukin; altretamine; ambomycin; ametantrone acetate; aminoglutethimide; amsacrine; anastrozole; anthramycim asparaginase; asperlin; azacitidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide; bisantrene hydrochloride; bisnafide dimesylate; bizelesin; bleomycin sulfate; brequinar sodium; bropirimine; busulfan; cactinomycin; calusterone; caracemide; carbetimer; carboplatin; carmustine; carubicin hydrochloride; carzelesin; cedefingol; chlorambucil; cirolemycin; cladribine; crisnatol mesylate; cyclophosphamide; cytarabine; dacarbazine; daunorubicin hydrochloride; decitabine; dexormaplatin; dezaguanine; dezaguanine mesylate; diaziquone; doxorubicin; doxorubicin hydrochloride; droloxifene; droloxifene citrate; dromostanolone propionate; duazomycin; edatrexate; eflomithine hydrochloride; elsamitrucin; enloplatin; enpromate; epipropidine; epirubicin hydrochloride; erbulozole; esorubicin hydrochloride; estramustine; estramustine phosphate sodium; etanidazole; etoposide; etoposide phosphate; etoprine; fadrozole hydrochloride; fazarabine; fenretinide; floxuridine; fludarabine phosphate; fluorouracil; fluorocitabine; fosquidone; fostriecin sodium; gemcitabine; gemcitabine hy drochloride; hydroxyurea; idarubicin hydrochloride; ifosfamide; iimofosine; interleukin II (including recombinant interleukin II, or rlL2), interferon alfa-2a; interferon alfa-2b; interferon alfa-nl; interferon alfa-n3; interferon beta-la; interferon gamma-lb; iproplatin; irinotecan hydrochloride; lanreotide acetate; letrozole; leuprolide acetate; liarozole hydrochloride; lometrexol sodium; lomustine; losoxantrone hydrochloride; masoprocol; maytansine; mechlorethamine hydrochloride; megestrol acetate; melengestrol acetate; melphalan; menogaril; mercaptopurine; methotrexate; methotrexate sodium; metoprine; meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin; mitomalcin; mitomycin; mitosper; mitotane; mitoxantrone hydrochloride; mycophenolic acid; nocodazoie; nogalamycin; ormaplatin; oxisuran; pegaspargase; peliomycin; pentamustine; peplomycin sulfate; perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride; plicamycin; plomestane; porfimer sodium; porfiromycin; prednimustine; procarbazine hydrochloride; puromycin; puromycin hydrochloride; pyrazofurin; riboprine; rogletimide; safmgol; safmgol hydrochloride; semustine; simtrazene; sparfosate sodium; sparsomycin; spirogermanium hydrochloride; spiromustine; spiroplatin; streptonigrin; streptozocin; sulofenur; talisomycin; tecogalan sodium; tegafur; teloxantrone hydrochloride; temoporfm; teniposide; teroxirone; testolactone; thiamiprine; thioguanine; thiotepa; tiazofurin; tirapazamine; toremifene citrate; trestolone acetate; triciribine phosphate; trimetrexate; trimetrexate glucuronate; triptorelin; tubulozole hydrochloride; uracil mustard; uredepa; vapreotide; verteporfin; vinblastine sulfate; vincristine sulfate; vindesine; vindesine sulfate; vinepidine sulfate; vinglycinate sulfate; vinleurosine sulfate; vinorelbine tartrate; vinrosidine sulfate; vinzolidine sulfate; vorozole; zeniplatin; zinostatin; zorubicin hydrochloride, agents that arrest cells in the G2-M phases and/or modulate the formation or stability of microtubules, (e.g. Taxol.TM (i.e. paclitaxel), Taxotere.TM, compounds comprising the taxane skeleton, Erbulozole (i.e. R-55104), Dolastatin 10 (i.e. DLS-10 and NSC-376128), mivobulin isethionate (i.e. as CI-980), vincristine, NSC-639829, Discodermolide (i.e. as NVP- XX-A-296), ABT-751 (Abbott, i.e. E-7010), Altorhyrtins (e.g. Altorhyrtin A and Altorhyrtin C), Spongistatins (e.g. Spongistatin 1, Spongistatin 2, Spongistatin 3, Spongistatin 4, Spongistatin 5, Spongistatin 6, Spongistatin 7, Spongistatin 8, and Spongistatin 9), Cemadotin hydrochloride (i.e. LU-103793 and NSC-D-669356), Epothilones (e.g. Epothilone A, Epothilone B, Epothilone C (i.e. desoxy epothilone A or dEpoA), Epothilone D (i.e. KOS-862, dEpoB, and desoxyepothilone B), Epothilone E, Epothilone F. Epothilone B N-oxide. Epothilone A N-oxide, 16-aza-epothilone B, 21-aminoepothilone B (i.e. BMS-310705), 21 -hydroxy epothilone D (i.e. Desoxyepothilone F and dEpoF), 26-fluoroepothilone, Auristatin PE (i.e. NSC-654663), Soblidotin (i.e. TZT-1027), , Vincristine sulfate, Cryptophycin 52 (i.e. LY -355703), Vitilevuamide. Tubulysin A. Canadensol, Centaureidin (i.e. NSC-106969). Oncocidin Al (i.e. BTO-956 and DIME), Fijianolide B, Laulimalide, Narcosine (also known as NSC-5366), Nascapine, Hemiasterlin, Vanadocene acetylacetonate, Monsatrol, Inanocine (i.e. NSC-698666), Eleutherobins (such as Desmethyleleutherobin, Desaetyleleutherobin, Isoeleutherobin A, and Z- Eleutherobin), caribaeoside, caribaeolin, Halichondrin B, Diazonamide A, Taccalonolide A, Diozostatin, (-)-Phenylahistin (i.e. NSCL-96F037), Myoseverin B, Resverastatin phosphate sodium, steroids (e.g., dexamethasone), finasteride, aromatase inhibitors, gonadotropin-releasing hormone agonists (GnRH) such as goserelin or leuprolide, adrenocorticosteroids (e.g., prednisone), progestins (e.g., hydroxyprogesterone caproate, megestrol acetate, medroxyprogesterone acetate), estrogens (e.g., diethlystilbestrol, ethinyl estradiol), antiestrogen (e.g., tamoxifen), androgens (e.g., testosterone propionate, fluoxymesterone), antiandrogen (e.g., flutamide), immunostimulants (e.g., Bacillus Calmette-Guerin (BCG), levamisole, interleukin-2, alpha-interferon, etc.), monoclonal antibodies (e.g., anti-CD20, anti-HER2. anti-CD52, anti- HLA-DR, and anti-VEGF monoclonal antibodies), immunotoxins (e.g., anti-CD33 monoclonal antibody -calicheamicin conjugate, anti-CD22 monoclonal antibody-pseudomonas exotoxin conjugate, etc.), radioimmunotherapy (e.g., anti-CD20 monoclonal antibody conjugated to ⁿⁱIn, ⁹⁰Y, or ¹³¹L etc ), triptolide, homoharringtonine, dactinomycin, doxorubicin, epirubicin, topotecan, itraconazole, vindesine, cerivastatin, vincristine, deoxyadenosine, sertraline, pitavastatin, irinotecan, clofazimine, 5 -nonyl oxy tryptamine, vemurafenib, dabrafenib, erlotinib, gefitinib, EGFR inhibitors, epidermal growth factor receptor (EGFR)-targeted therapy or therapeutic (e.g. gefitinib, erlotinib, cetuximab, lapatinib, panitumumab, vandetanib, afatinib/BIBW2992, CI-1033/canertinib, neratinib/HKI-272, CP-724714. TAK-285, AST-1306, ARRY334543, ARRY-380, AG-1478, dacomitinib/PF299804, OSI-420/desmethyl erlotinib, AZD8931, AEE788, pelitinib/EKB-569, CUDC-101, WZ8040, WZ4002, WZ3146, AG-490, XL647, PD153035, BMS-599626), sorafenib, imatinib, sunitinib, dasatinib, hormonal therapies, or the like.

[0162] Embodiments

[0163] Embodiment 1. A compound comprising a phosphorothioated CpG oligodeoxynucleotide linked to a first DNA oligonucleotide that is hybridized to a second DNA oligonucleotide; wherein: (a) the first DNA oligonucleotide comprises from about 5 unmodified nucleotides to about 70 unmodified nucleotides; and (b) the second DNA oligonucleotide comprises: (i) from about 5 nucleotides to about 70 nucleotides, and (ii) a constrained nucleotide on the 3’ end and a constrained nucleotide on the 5‘ end.

[0164] Embodiment 2. The compound of Embodiment 1, wherein the second DNA oligonucleotide comprises one constrained nucleotide on the 3’ end and one constrained nucleotide on the 5’ end.

[0165] Embodiment 3. The compound of Embodiment 1, wherein the second DNA oligonucleotide comprises one constrained nucleotide on the 3’ end and two constrained nucleotides on the 5’ end.

[0166] Embodiment 4. The compound of Embodiment 1, wherein the second DNA oligonucleotide comprises two constrained nucleotides on the 3’ end and one constrained nucleotide on the 5’ end.

[0167] Embodiment 5. The compound of Embodiment 1, wherein the second DNA oligonucleotide comprises two constrained nucleotides on the 3’ end and two constrained nucleotide on the 5’ end.

[0168] Embodiment 6. The compound of Embodiment 1, wherein the second DNA oligonucleotide comprises two constrained nucleotides on the 3' end and three constrained nucleotides on the 5’ end.

[0169] Embodiment 7. The compound of Embodiment 1. wherein the second DNA oligonucleotide comprises three constrained nucleotides on the 3 ’ end and two constrained nucleotides on the 5’ end.

[0170] Embodiment 8. The compound of any one of Embodiments 3 to 7, wherein the two constrained nucleotides are contiguous.

[0171] Embodiment 9. The compound of any one of Embodiments 3 to 7, wherein the two constrained nucleotides are alternating.

[0172] Embodiment 10. The compound of Embodiment 1, wherein the second DNA oligonucleotide comprises three constrained nucleotides on the 3' end and three constrained nucleotides on the 5’ end.

[0173] Embodiment 11. The compound of Embodiment 6, 7, or 9, wherein the three constrained nucleotides are contiguous.

[0174] Embodiment 12. The compound of Embodiment 6, 7, or 9, wherein the constrained nucleotides are alternating.

[0175] Embodiment 13. The compound of any one of Embodiments 1 to 12, wherein the constrained nucleotide is a LNA-modified nucleotide.

[0176] Embodiment 14. The compound of any one of Embodiments 1 to 12, wherein the constrained nucleotide is a cMOE-modified nucleotide.

[0177] Embodiment 15. The compound of any one of Embodiments 1 to 12, wherein the constrained nucleotide is a cET-modified nucleotide.

[0178] Embodiment 16. The compound of any one of Embodiments 1 to 12, wherein the constrained nucleotide is a LNA-modified nucleotide, a cMOE-modified nucleotide, or a cET- modified nucleotide.

[0179] Embodiment 17. The compound of any one of Embodiments 1 to 16, wherein the first DNA oligonucleotide comprises from about 10 unmodified nucleotides to about 22 unmodified nucleotides; and the second DNA oligonucleotide comprises from about 10 nucleotides to about 22 nucleotides.

[0180] Embodiment 18. The compound of Embodiment 17. wherein the first DNA oligonucleotide comprises from about 14 unmodified nucleotides to about 18 unmodified nucleotides; and the second DNA oligonucleotide comprises from about 14 nucleotides to about 18 nucleotides.

[0181] Embodiment 19. The compound of any one of Embodiments 1 to 18, wherein the first DNA oligonucleotide and the second DNA oligonucleotide comprise the same number of nucleotides.

[0182] Embodiment 20. The compound of any one of Embodiments 1 to 19, wherein the second DNA oligonucleotide comprises a nucleotide having a phosphorothioate modification.

[0183] Embodiment 21. The compound of any one of Embodiments 1 to 20, wherein the second DNA oligonucleotide further comprises a nucleotide having a modification selected from the group consisting of 2’-O-aminopropyl group, a 2’-0-ethyl group, a 2’-fluoro group, a 2’-O- methyl group, 2’-deoxy-2’fluoro group, a 2’-O-methoxyethyl group, a 2’-O-allyl group, a 2’-O- propyl group, a 2’-O-pentyl group, and a constrained nucleotide.

[0184] Embodiment 22. The compound of any one of Embodiments 1 to 21, wherein the second DNA oligonucleotide further comprises a spacer modification.

[0185] Embodiment 23. The compound of any one of Embodiments 1 to 22, wherein the second DNA oligonucleotide is an antisense oligonucleotide.

[0186] Embodiment 24. The compound of Embodiment 23, wherein the antisense oligonucleotide is a STAT3 antisense oligonucleotide.

[0187] Embodiment 25. The compound of Embodiment 24, wherein the STAT3 antisense oligonucleotide comprises SEQ ID NO:2. SEQ ID NO:4. SEQ ID NO:6, SEQ ID NO:8, SEQ ID NOTO, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO:35, or SEQ ID NO:36.

[0188] Embodiment 26. The compound of Embodiment 24, wherein the first DNA oligonucleotide comprises SEQ ID NO: 1 and the STAT3 antisense oligonucleotide comprises SEQ ID NOT.

[0189] Embodiment 27. The compound of Embodiment 24, wherein the first DNA oligonucleotide comprises SEQ ID NOT and the STAT3 antisense oligonucleotide comprises SEQ ID NO:4. [0190] Embodiment 28. The compound of Embodiment 24, wherein the first DNA oligonucleotide comprises SEQ ID NO:5 and the STAT3 antisense oligonucleotide comprises SEQ ID NO:6.

[0191] Embodiment 29. The compound of Embodiment 24, wherein the first DNA oligonucleotide comprises SEQ ID NO:7 and the STAT3 antisense oligonucleotide comprises SEQ ID NO: 8.

[0192] Embodiment 30. The compound of Embodiment 24, wherein the first DNA oligonucleotide comprises SEQ ID NO:9 and the STAT3 antisense oligonucleotide comprises SEQ ID NOTO.

[0193] Embodiment 31. The compound of Embodiment 24, wherein the first DNA oligonucleotide comprises SEQ ID NO: 11 and the STAT3 antisense oligonucleotide comprises SEQ ID NO: 12.

[0194] Embodiment 32. The compound of Embodiment 24, wherein the first DNA oligonucleotide comprises SEQ ID NO: 13 and the STAT3 antisense oligonucleotide comprises SEQ ID NO: 14.

[0195] Embodiment 33. The compound of Embodiment 24, wherein the first DNA oligonucleotide comprises SEQ ID NOT and the STAT3 antisense oligonucleotide comprises SEQ ID NO:35.

[0196] Embodiment 34. The compound of Embodiment 24, wherein the first DNA oligonucleotide comprises SEQ ID NOT and the STAT3 antisense oligonucleotide comprises SEQ ID NO:36.

[0197] Embodiment 35. The compound of any one of Embodiments 1 to 34, wherein the phosphorothioated CpG oligodeoxynucleotide is Class A CpG oligodeoxynucleotide, a Class B CpG oligodeoxynucleotide, or a Class C CpG oligodeoxynucleotide.

[0198] Embodiment 36. The compound of any one of Embodiments 1 to 34, wherein the phosphorothioated CpG oligodeoxynucleotide comprises SEQ ID NO:43, SEQ ID NO: 15, SEQ ID NO:16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO 21, SEQ ID NO:22, SEQ ID NO:23. SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26. SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NOTO, SEQ ID NO:31, or SEQ ID NO:32.

[0199] Embodiment 37. The compound of Embodiment 36, wherein the phosphorothioated CpG oligodeoxynucleotide comprises SEQ ID NO:43. [0200] Embodiment 38. The compound of Embodiment 36, wherein the phosphoro thioated CpG oligodeoxynucleotide comprises SEQ ID NO: 15.

[0201] Embodiment 39. The compound of any one of Embodiments 1 to 38, wherein the first DNA oligonucleotide is a passenger strand and the second DNA oligonucleotide is a guide strand.

[0202] Embodiment 40. The compound of any one of Embodiments 1 to 39, wherein the 3’ end of the phosphorothioated CpG oligodeoxynucleotide is linked to the 5’ end of the first DNA oligonucleotide.

[0203] Embodiment 41. The compound of any one of Embodiments 1 to 39, wherein the 3’ end of the phosphorothioated CpG oligodeoxynucleotide is linked to the 3’ end of the first DNA oligonucleotide.

[0204] Embodiment 42. The compound of Embodiment 1, wherein the phosphorothioated CpG oligodeoxynucleotide comprises SEQ ID NO:43; the first DNA oligonucleotide comprises SEQ ID NO: 1; the second DNA oligonucleotide comprises SEQ ID NO:2; and the 3’ end of the phosphorothioated CpG oligodeoxynucleotide is linked to the 5’ end of the first DNA oligonucleotide.

[0205] Embodiment 43. The compound of Embodiment 1, wherein the phosphorothioated CpG oligodeoxynucleotide comprises SEQ ID NO: 15; the first DNA oligonucleotide comprises SEQ ID NO:3; the second DNA oligonucleotide comprises SEQ ID NO:4; and the 3’ end of the phosphorothioated CpG oligodeoxynucleotide is linked to the 5’ end of the first DNA oligonucleotide.

[0206] Embodiment 44. The compound of any one of Embodiments 1 to 43, wherein the linking group comprises a bond, a nucleic acid, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, substituted or unsubstituted heteroarylene, or a combination of two or more thereof.

[0207] Embodiment 45. The compound of Embodiment 44, wherein the linking group comprises substituted 6 to 60 membered heteroalkylene.

[0208] Embodiment 46. The compound of Embodiment 45, wherein the linking group comprises substituted heteroalkylene of the formula:

, wherein n is an integer from 1 to 10.

[0209] Embodiment 47. The compound of Embodiment 46, wherein n is 5.

[0210] Embodiment 48. A pharmaceutical composition comprising the compound of any one of Embodiments 1 to 47 and a pharmaceutically acceptable excipient.

[0211] Embodiment 49. A method of treating cancer in a patient in need thereof, the method comprising administering to the patient an effective amount of the compound of any one of Embodiments 1 to 47 or the pharmaceutical composition of Embodiment 48.

[0212] Embodiment 50. The method of Embodiment 49, wherein the cancer is glioma, cranial primitive neuroectodermal tumor, ependymal tumor, hemangiopericytoma, germ cell tumor, pineal tumor, or primary' central nervous system lymphoma.

[0213] Embodiment 51. The method of Embodiment 50, wherein the central nervous system cancer is glioma, cranial primitive neuroectodermal tumor, ependymal tumor, hemangiopericytoma, germ cell tumor, pineal tumor, or primary’ central nervous system lymphoma.

[0214] Embodiment 52. The method of Embodiment 51 , wherein the glioma is astrocytoma, glioblastoma, or oligodendroglioma; the cranial primitive neuroectodermal tumor is medulloblastoma, cerebral neuroblastoma, pineoblastoma, or esthesioneuroblastoma; and the ependymal tumor is ependymoma, myxopapillary ependy moma, or subependymoma.

[0215] Embodiment 53. The method of Embodiment 49, wherein the cancer is glioma.

[0216] Embodiment 54. The method of Embodiment 49, wherein the cancer is leukemia.

[0217] Embodiment 55. The method of Embodiment 49, wherein the cancer is a solid tumor cancer.

[0218] Embodiment 56. The method of Embodiment 49, wherein the cancer is prostate cancer, breast cancer, colorectal cancer, bladder cancer, lung cancer, liver cancer, pancreatic caner, renal cancer, stomach cancer, or melanoma.

[0219] Embodiment 57. The method of Embodiment 49, wherein the cancer is prostate cancer.

[0220] Embodiment 58. The method of Embodiment 49, wherein the cancer is an epidermoid carcinoma. [0221] Embodiment 59. The method of Embodiment 58, wherein the epidermoid carcinoma is thyroid cancer, esophageal cancer, vaginal cancer, anal cancer, cervical cancer, or head and neck cancer.

[0222] Embodiment 60. The method of any one of Embodiments 49 to 59, further comprising administering to the patient an effective amount of an immune checkpoint inhibitor.

[0223] Embodiment 61. The method of Embodiment 60, wherein the immune checkpoint inhibitor is a PD-1 inhibitor.

[0224] Embodiment 62. The method of Embodiment 61, wherein the PD-1 inhibitor is pembrolizumab, nivolumab, cemiplimab, dostarlimab, camrelizumab, sintilimab, tislelizumab, toripalimab, spartalizumab, retifanlimab, pimivalimab, AMP -224, or MEDI0680.

[0225] Embodiment 63. The method of Embodiment 60, wherein the immune checkpoint inhibitor is a PD-Ll inhibitor.

[0226] Embodiment 64. The method of Embodiment 63, wherein the PD-L1 inhibitor is atezolizumab, avelumab. durvalumab, envafolimab. cosibelimab, AUNP12, CA-170, or BMS- 986189.

[0227] Embodiment 65. A pharmaceutical composition comprising the compound of any one of Embodiments 1 to 47 and an immune checkpoint inhibitor.

[0228] Embodiment 66. The pharmaceutical composition of Embodiment 65, wherein the immune checkpoint inhibitor is a PD-inhibitor or a PD-L1 inhibitor.

[0229] Embodiment 67. A STAT3 antisense oligonucleotide comprising SEQ ID NO: 4.

[0230] Embodiments N1-N23

[0231] Embodiment Nl. A compound comprising a phosphorothioated CpG oligodeoxynucleotide linked to a first DNA oligonucleotide that is hybridized to a second DNA oligonucleotide; wherein: (a) the first DNA oligonucleotide comprises from about 5 unmodified nucleotides to about 70 unmodified nucleotides; and (b) the second DNA oligonucleotide comprises: (i) from about 5 nucleotides to about 70 nucleotides, and (ii) a constrained nucleotide on the 3’ end and a constrained nucleotide on the 5’ end.

[0232] Embodiment N2. The compound of Embodiment N 1 , wherein the second DNA oligonucleotide comprises one constrained nucleotide on the 3’ end and one constrained nucleotide on the 5’ end. [0233] Embodiment N3. The compound of Embodiment N 1 , wherein: (i) the second DNA oligonucleotide comprises one constrained nucleotide on the 3’ end and two constrained nucleotides on the 5’ end; (ii) the second DNA oligonucleotide comprises two constrained nucleotides on the 3’ end and one constrained nucleotide on the 5’ end; (iii) the second DNA oligonucleotide comprises two constrained nucleotides on the 3’ end and two constrained nucleotide on the 5’ end; (iv) the second DNA oligonucleotide comprises two constrained nucleotides on the 3’ end and three constrained nucleotides on the 5’ end; (v) the second DNA oligonucleotide comprises three constrained nucleotides on the 3’ end and two constrained nucleotides on the 5’ end; or (vi) the second DNA oligonucleotide comprises three constrained nucleotides on the 3‘ end and three constrained nucleotides on the 5’ end.

[0234] Embodiment N4. The compound of Embodiment N3, wherein the two or three constrained nucleotides are contiguous or alternating.

[0235] Embodiment N5. The compound of any one of Embodiments N1 to N4, wherein the constrained nucleotide is a LNA-modified nucleotide, a cMOE-modified nucleotide, or a cET- modified nucleotide.

[0236] Embodiment N6. The compound of any one of Embodiments N 1 to N5, wherein the first DNA oligonucleotide comprises from about 10 unmodified nucleotides to about 22 unmodified nucleotides; and the second DNA oligonucleotide comprises from about 10 nucleotides to about 22 nucleotides.

[0237] Embodiment N7. The compound of any one of Embodiments N1 to N6, wherein the second DNA oligonucleotide comprises a nucleotide having a phosphorothioate modification.

[0238] Embodiment N8. The compound of any one of Embodiments N1 to N7, wherein the second DNA oligonucleotide further comprises a nucleotide having a modification selected from the group consisting of 2’-O-aminopropyl group, a 2’-0-ethyl group, a 2’-fluoro group, a 2’-O- methyl group, 2’-deoxy-2’fluoro group, a 2’-O-methoxyethyl group, a 2’-O-allyl group, a 2’-O- propyl group, a 2’-O-pentyl group, and a constrained nucleotide.

[0239] Embodiment N9. The compound of any one of Embodiments N1 to N8, wherein the second DNA oligonucleotide is an antisense oligonucleotide.

[0240] Embodiment N10. The compound of any one of Embodiments N1 to N8, wherein the second DNA oligonucleotide is a STAT3 antisense oligonucleotide.

[0241] Embodiment N11. The compound of Embodiment N 10, wherein the STAT3 antisense oligonucleotide comprises SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO 35, or SEQ ID NO:36.

[0242] Embodiment N12. The compound of any one of Embodiments N1 to Ni l, wherein: (a) the first DNA oligonucleotide comprises SEQ ID NO: 1 and the STAT3 antisense oligonucleotide comprises SEQ ID NO:2; (b) the first DNA oligonucleotide comprises SEQ ID NO:3 and the STAT3 antisense oligonucleotide comprises SEQ ID NO:4; (c) the first DNA oligonucleotide comprises SEQ ID NO:5 and the STAT3 antisense oligonucleotide comprises SEQ ID NO:6; (d) the first DNA oligonucleotide comprises SEQ ID NO:7 and the STAT3 antisense oligonucleotide comprises SEQ ID NO: 8; (e) the first DNA oligonucleotide comprises SEQ ID NO:9 and the STAT3 antisense oligonucleotide comprises SEQ ID NOTO; (f) the first DNA oligonucleotide comprises SEQ ID NO: 11 and the STAT3 antisense oligonucleotide comprises SEQ ID NO: 12; (g) the first DNA oligonucleotide comprises SEQ ID NO: 13 and the STAT3 antisense oligonucleotide comprises SEQ ID NO: 14; (h) the first DNA oligonucleotide comprises SEQ ID NO: 1 and the STAT3 antisense oligonucleotide comprises SEQ ID NO: 35; or (i) the first DNA oligonucleotide comprises SEQ ID NO: 1 and the STAT3 antisense oligonucleotide comprises SEQ ID NO:36.

[0243] Embodiment N13. The compound of any one of Embodiments N1 to N12, wherein the phosphorothioated CpG oligodeoxynucleotide comprises SEQ ID NO:43, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO 21, SEQ ID NO:22, SEQ ID NO:23. SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26. SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO 30, SEQ ID NO:31, or SEQ ID NO:32.

[0244] Embodiment N 14. The compound of any one of Embodiments N1 to N13, wherein the first DNA oligonucleotide is a passenger strand and the second DNA oligonucleotide is a guide strand.

[0245] Embodiment N15. The compound of any one of Embodiments N1 to N14, wherein the 3’ end of the phosphorothioated CpG oligodeoxynucleotide is linked to the 5’ end of the first DNA oligonucleotide or wherein the 3’ end of the phosphorothioated CpG oligodeoxynucleotide is linked to the 3’ end of the first DNA oligonucleotide.

[0246] Embodiment N 16. The compound of Embodiment Nl, wherein: (a) the phosphorothioated CpG oligodeoxynucleotide comprises SEQ ID NO:43; the first DNA oligonucleotide comprises SEQ ID NO: 1; the second DNA oligonucleotide comprises SEQ ID NO:2; and the 3‘ end of the phosphorothioated CpG oligodeoxynucleotide is linked to the 5‘ end of the first DNA oligonucleotide; or (b) the phosphorothioated CpG oligodeoxynucleotide comprises SEQ ID NO: 15; the first DNA oligonucleotide comprises SEQ ID NO:3; the second DNA oligonucleotide comprises SEQ ID NO:4; and the 3’ end of the phosphorothioated CpG oligodeoxynucleotide is linked to the 5’ end of the first DNA oligonucleotide.

[0247] Embodiment N 17. A pharmaceutical composition comprising the compound of anyone of Embodiments N1 to N16 and a pharmaceutically acceptable excipient.

[0248] Embodiment N 18. A method of treating cancer in a patient in need thereof, the method comprising administering to the patient an effective amount of the compound of any one of Embodiments N1 to N16 or the pharmaceutical composition of Embodiment N17.

[0249] Embodiment N19. The method of Embodiment N18, wherein the cancer is glioma.

[0250] Embodiment N20. The method of Embodiment N18, wherein the cancer is a central nervous system cancer, leukemia, prostate cancer, breast cancer, colorectal cancer, bladder cancer, lung cancer, liver cancer, pancreatic caner, renal cancer, stomach cancer, melanoma, or an epidermoid carcinoma.

[0251] Embodiment N21. The method of any one of Embodiments N18 to N20, further comprising administering to the patient an effective amount of an immune checkpoint inhibitor.

[0252] Embodiment N22. The method of Embodiment N21, wherein the immune checkpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor.

[0253] Embodiment N23. The method of Embodiment N22, wherein: (i) the PD-1 inhibitor is pembrolizumab, nivolumab, cemiplimab, dostarlimab, camrelizumab, sintilimab, tislelizumab, toripalimab, spartalizumab, retifanlimab, pimivalimab, AMP-224, or MEDI0680, and (ii) the PD-L1 inhibitor is atezolizumab, avelumab, durvalumab, envafolimab, cosibelimab, AUNP12, CA-170, or BMS-986189.

EXAMPLES

[0254] It is understood that the examples described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and scope of this application and claims.

[0255] The application of immunostimulatory oligonucleotides against brain tumors is limited by potential immunotoxicities. Here, the inventors developed a TLR9-targeted double-stranded STAT3 antisense oligonucleotide (CpG-STAT3dsASO) with optimized stability and efficacy for cancer immunotherapy.

[0256] Example 1 [0257] To augment the STAT3 knockdown in glioma cells, locked-nucleic acid (LNA) chemistn’ was used within ASO part of the molecule. As shown in FIG. 1, double-stranded ASO molecules comprising a gapmer ASO hybridized to a complementary and not phosphorothioate DNA strand are stable in human serum (FIG. 1A) and result in target gene knock down comparable to a single-stranded ASO (FIGS. 1B-1D) in various cancer cells. Similar effects were observed for dsASO targeting STAT3 as well as dsASO specific to a different gene, androgen receptor in prostate cancer cells (FIG. 1C).

[0258] This design was used to create a TLR9-targeted CpG-dsASO conjugate (FIG. 2A). The conjugate showed over 2 days half-life in human serum and was still detectable after 5 days of incubation (FIG. 2B). The CpG-STAT3dsASO w as confirmed to have similar potency and kinetics of target gene knockdown as a single stranded STAT3ASO alone in cancer cells, such as prostate cancer, epidermoid carcinoma, glioma cells, and mouse macrophages (FIGS. 2C-2D, FIG. 3). All chemically-modified and fluorescently labeled CpG-STAT3ASO variants were rapidly internalized by human and mouse glioma cells and myeloid cells in vitro (FIGS. 4-6). None of the STAT3ASO variants w as internalized by human T cells, while mouse T cells showed low’ internalization of high oligonucleotide concentrations at longer incubation times only. Biodistribution experiments in mice with intracranial gliomas using intratumoral oligonucleotide injections confirmed very efficient uptake by a variety of myeloid cells in the brain including macrophages, microglia, DCs and MDSCs (FIG. 7).

[0259] Example 2

[0260] The potential toxicity of intracranial dosing of single-stranded CpG-STAT3 ASO and double-stranded CpG-STAT3ASO was compared (FIG. 8). The repeated four injections of CpG-STATdsASO (0. 1-1 mg/kg) were well tolerated with about 10% of body weight loss at the highest dose of 1 mg/kg. In contrast, already at 0.3 mg/kg dosing CpG-STAT3ssASO resulted in about 25% body weight loss in mice which is considered an adverse effect (FIG. 8A). The assessment of acute neural toxicity based on the mice phenotypic behavior confirmed that IC injection of CpG-STATdsASO was significantly better tolerated than standard single-stranded conjugate (FIG. 8B). These observations were also associated with significantly elevated levels of platelets in mice treated with single-stranded but not with double-stranded CpG-STAT3ASO conjugates (FIG. 8C).

[0261] Example 3

[0262] The antitumor efficacy of the benchmark 2’O-methyl-modified CpG-STAT3ASO was compared with single- and double-stranded LNA-modified CpG-STAT3ASO. As shown in FIG. 9, both LNA-modified CpG-STAT3ASO but not the benchmark molecule reduced progression of orthotopic human U251 glioma and improved survival of immunodeficient mice. In the absence of immune system, mice tolerated well 1 mg/kg injection of single- and doublestranded LNA-modified CpG-STAT3ASO. The antitumor efficacy of the treatments against syngeneic GL261 model in immunocompetent mice was assessed (FIG. 10A). Local administration of all tested single- and double-stranded CpG-STAT3ASOs improved animal survival and triggered immune activation in immunocompetent mice. All three types of CpG- STAT3ASO injections induced maturation/ activation of intratumoral DCs, macrophages and microglia, while reducing numbers of tumor-associated M2 macrophages and resting microglia as assessed using flow cytometry (FIG. 10B). Importantly, CpG-STAT3ASO injections improved the ratio of intratumoral CD8 T cells to Tregs (FIG. IOC).

[0263] Example 4

[0264] To further improve treatment efficacy. LNA-modified CpG-STAT3dsASO treatment was combined with systemic administration of PD1 blocking antibodies. As shown in FIG. 11, LNA-modified CpG-STAT3dsASO sensitized GL261 glioma-bearing mice to immune checkpoint inhibition resulting in synergistic affect and complete tumor regression in the majority of treated mice after 2-week treatment. The combination treatment produced long-term tumor free survival of 5/6 treated mice for over 150 days. In addition, the locally injected LNA- modified CpG-STAT3dsASO showed efficacy also against RM9 prostate tumors in mice, with evidence of abscopal effects (FIG. 12).

[0265] Example 5

[0266] LNA-modified CpG-STAT3dsASO activity was verified in the new model of immune checkpoint resistant QPP8 (Ok Tp53 len-de ) glioma derived from neural stem cells. As shown in FIG. 13. LNA-modified CpG-STAT3dsASO showed improved antitumor effect as a single agent compared to the single-stranded LNA-modified CpG-STAT3ASO. When combined with PDl-specific antibodies, LNA-modified CpG-STAT3dsASO resulted in complete regression of QPP8 gliomas in the majority of treated mice (FIG. 14). Neither anti-PDl alone nor LNA- modified CpG-STAT3dsASO resulted in glioma eradication but both improved mice survival. To verify that surviving mice develop protective antitumor immunity, we used mice that rejected tumor in the study in FIG. 11. The majority of these mice resisted the rechallenge with the same type of glioma, while all aged-matched naive mice developed tumors (FIG. 15). [0267] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents. cited in the application are expressly incorporated by reference herein in their entirety and for all purposes.

Claims

CLAIMS What is claimed is:

1. A compound comprising a phosphorothioated CpG oligodeoxynucleotide linked to a first DNA oligonucleotide that is hybridized to a second DNA oligonucleotide; wherein:

(a) the first DNA oligonucleotide comprises from about 5 unmodified nucleotides to about 70 unmodified nucleotides; and

(b) the second DNA oligonucleotide comprises:

(i) from about 5 nucleotides to about 70 nucleotides, and

(ii) a constrained nucleotide on the 3' end and a constrained nucleotide on the 5’ end.

2. The compound of claim 1, wherein the second DNA oligonucleotide comprises one constrained nucleotide on the 3‘ end and one constrained nucleotide on the 5’ end.

3. The compound of claim 1 , wherein:

(i) the second DNA oligonucleotide comprises one constrained nucleotide on the 3 ’ end and two constrained nucleotides on the 5 ’ end;

(ii) the second DNA oligonucleotide comprises two constrained nucleotides on the 3’ end and one constrained nucleotide on the 5’ end;

(iii) the second DNA oligonucleotide comprises two constrained nucleotides on the 3’ end and two constrained nucleotide on the 5’ end;

(iv) the second DNA oligonucleotide comprises two constrained nucleotides on the 3’ end and three constrained nucleotides on the 5’ end;

(v) the second DNA oligonucleotide comprises three constrained nucleotides on the 3’ end and two constrained nucleotides on the 5’ end; or

(vi) the second DNA oligonucleotide comprises three constrained nucleotides on the 3’ end and three constrained nucleotides on the 5’ end.

4. The compound of claim 3, wherein the two or three constrained nucleotides are contiguous or alternating.

5. The compound of claim 1, wherein the constrained nucleotide is a LNA-modified nucleotide, a cMOE-modified nucleotide, or a cET-modified nucleotide.

6. The compound of claim 1, wherein the first DNA oligonucleotide comprises from about 10 unmodified nucleotides to about 22 unmodified nucleotides; and the second DNA oligonucleotide comprises from about 10 nucleotides to about 22 nucleotides.

7. The compound of claim 1, wherein the second DNA oligonucleotide comprises a nucleotide having a phosphorothioate modification.

8. The compound of claim 1, wherein the second DNA oligonucleotide further comprises a nucleotide having a modification selected from the group consisting of 2’-O- aminopropyl group, a 2’-0-ethyl group, a 2'-fluoro group, a 2’-O-methyl group, 2'-deoxy- 2’fluoro group, a 2’-O-methoxyethyl group, a 2’-O-allyl group, a 2’-O-propyl group. a 2’-O- pentyl group, and a constrained nucleotide.

9. The compound of claim 1, wherein the second DNA oligonucleotide is an antisense oligonucleotide.

10. The compound of claim 1, wherein the second DNA oligonucleotide is a STAT3 antisense oligonucleotide.

11. The compound of claim 10, wherein the STAT3 antisense oligonucleotide comprises SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO:12, SEQ ID NO: 14, SEQ ID NO:35, or SEQ ID NO:36.

12. The compound of claim 1, wherein:

(a) the first DNA oligonucleotide comprises SEQ ID NO: 1 and the STAT3 antisense oligonucleotide comprises SEQ ID NO: 2;

(b) the first DNA oligonucleotide comprises SEQ ID NO:3 and the STAT3 antisense oligonucleotide comprises SEQ ID NO:4;

(c) the first DNA oligonucleotide comprises SEQ ID NO:5 and the STAT3 antisense oligonucleotide comprises SEQ ID NO: 6;

(d) the first DNA oligonucleotide comprises SEQ ID NO: 7 and the STAT3 antisense oligonucleotide comprises SEQ ID NO: 8;

(e) the first DNA oligonucleotide comprises SEQ ID NO:9 and the STAT3 antisense oligonucleotide comprises SEQ ID NO: 10;

(1) the first DNA oligonucleotide comprises SEQ ID NO: 11 and the STAT3 antisense oligonucleotide comprises SEQ ID NO: 12;

(g) the first DNA oligonucleotide comprises SEQ ID NO: 13 and the STAT3 antisense oligonucleotide comprises SEQ ID NO: 14;

(h) the first DNA oligonucleotide comprises SEQ ID NO: 1 and the STAT3 antisense oligonucleotide comprises SEQ ID NO:35; or

(i) the first DNA oligonucleotide comprises SEQ ID NO: 1 and the STAT3 antisense oligonucleotide comprises SEQ ID NO: 36.

13. The compound of claim 1, wherein the phosphorothioated CpG oligodeoxynucleotide comprises SEQ ID NO:43, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID N0:18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO 28, SEQ ID NO:29, SEQ ID NO:30. SEQ ID NO:31, or SEQ ID NO:32.

14. The compound of claim 1, wherein the first DNA oligonucleotide is a passenger strand and the second DNA oligonucleotide is a guide strand.

15. The compound of claim 1, wherein the 3’ end of the phosphorothioated CpG oligodeoxynucleotide is linked to the 5’ end of the first DNA oligonucleotide or wherein the 3’ end of the phosphorothioated CpG oligodeoxynucleotide is linked to the 3‘ end of the first DNA oligonucleotide.

16. The compound of claim 1 , wherein:

(a) the phosphorothioated CpG oligodeoxynucleotide comprises SEQ ID NO:43; the first DNA oligonucleotide comprises SEQ ID NO: 1; the second DNA oligonucleotide comprises SEQ ID NO:2; and the 3’ end of the phosphorothioated CpG oligodeoxynucleotide is linked to the 5 ’ end of the first DNA oligonucleotide; or

(b) the phosphorothioated CpG oligodeoxynucleotide comprises SEQ ID NO: 15; the first DNA oligonucleotide comprises SEQ ID NO:3; the second DNA oligonucleotide comprises SEQ ID NO:4; and the 3’ end of the phosphorothioated CpG oligodeoxynucleotide is linked to the 5 ’ end of the first DNA oligonucleotide.

17. A pharmaceutical composition comprising the compound of claim 1 and a pharmaceutically acceptable excipient.

18. A method of treating cancer in a patient in need thereof, the method comprising administering to the patient an effective amount of the compound of claim 1.

19. The method of claim 18, wherein the cancer is glioma..

20. The method of claim 18, wherein the cancer is a central nervous system cancer, leukemia, prostate cancer, breast cancer, colorectal cancer, bladder cancer, lung cancer, liver cancer, pancreatic caner, renal cancer, stomach cancer, melanoma, or an epidermoid carcinoma.

21. The method of claim 18, further comprising administering to the patient an effective amount of an immune checkpoint inhibitor.

22. The method of claim 21, wherein the immune checkpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor.

23. The method of claim 22, wherein:

(i) the PD-1 inhibitor is pembrolizumab, nivolumab, cemiplimab, dostarlimab, camrelizumab, sintilimab, tislelizumab, toripalimab, spartalizumab, retifanlimab, pimivalimab, AMP-224, or MEDI0680, and

(li) the PD-L1 inhibitor is atezolizumab, avelumab, durvalumab, envafohmab, cosibelimab, AUNP12, CA-170, or BMS-986189.

24. A STAT3 antisense oligonucleotide comprising SEQ ID NO:4.