EP4599065A2 - Doppelsträngige oligonukleotide und verfahren zu ihrer verwendung - Google Patents

Doppelsträngige oligonukleotide und verfahren zu ihrer verwendung

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Publication number
EP4599065A2
EP4599065A2 EP23875846.0A EP23875846A EP4599065A2 EP 4599065 A2 EP4599065 A2 EP 4599065A2 EP 23875846 A EP23875846 A EP 23875846A EP 4599065 A2 EP4599065 A2 EP 4599065A2
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EP
European Patent Office
Prior art keywords
seq
dna oligonucleotide
constrained
nucleotides
nucleotide
Prior art date
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Pending
Application number
EP23875846.0A
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English (en)
French (fr)
Inventor
Marcin Tomasz KORTYLEWSKI
Chia-Yang Hung
Xiaowei Zhang
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City of Hope
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City of Hope
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Publication date
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Publication of EP4599065A2 publication Critical patent/EP4599065A2/de
Pending legal-status Critical Current

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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/117—Nucleic acids having immunomodulatory properties, e.g. containing CpG-motifs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/341—Gapmers, i.e. of the type ===---===
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed
    • C—CHEMISTRY; METALLURGY
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    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/31—Combination therapy
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    • C12N2320/00—Applications; Uses
    • C12N2320/50—Methods for regulating/modulating their activity
    • C12N2320/51—Methods for regulating/modulating their activity modulating the chemical stability, e.g. nuclease-resistance

Definitions

  • 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:
  • Class C CpG ODN “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.
  • Class C CpG ODNs include ODN 2395, ODN M362, and ODN D-SL03, the sequences of which are known in the art.
  • STAT6 refers to a “Signal transducer and activator of transcription 6"’ protein and homologs thereof.
  • STAT6 refers to the protein associated with Entrez Gene 6778, OMIM 601512, UniProt P42226, and/or RefSeq (protein) NP001 171549.
  • the reference numbers immediately above refer to the protein, and associated nucleic acids, known as of the date of filing of this application.
  • the term “linked” or “conjugated” when referring to two moieties means the two moieties are bonded, wherein the bond or bonds connecting the two moieties are covalent or non-covalent.
  • the two moieties are covalently bonded to each other (e.g. directly or through a linking group).
  • 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.
  • 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.
  • 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.
  • 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.
  • the intemucleotide linkages in DNA are phosphodiester, phosphodiester derivatives, or a combination of both.
  • Nucleic acids can include nonspecific sequences.
  • 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.
  • 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.
  • Unmodified nucleotide refers to a nucleotide that is not modified from its natural state.
  • 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.
  • a “constrained nucleotide” refers to a nucleotide that is modified to maintain a rigid backbone structure.
  • 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.
  • cMOE-modified nucleotide or “2 ’,4 ’-constrained 2’-O-methoxyethyl-modified nucleotide” refers to a nucleotide re: where Bx is the base.
  • the cMOE-modified nucleotide is a (7?)-cMOE-modified nucleotide.
  • the cMOE-modified nucleotide is a (S)-cMOE-modified nucleotide. See Pallan et al, Chem Commun (Camb), 48(66):8195-8197 (2012).
  • 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.
  • the spacer phosphoramidite is a C3 spacer phosphoramidite, a C6 spacer phosphoramidite, or a C 12 spacer phosphoramidite.
  • Abasic spacer or “dspacer” is a l’,2’-dideoxyribose without a nucleobase attached.
  • 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.
  • synthetic antisense nucleic acids e.g. oligonucleotides
  • synthetic antisense nucleic acids are generally between 15 and 25 bases in length.
  • antisense nucleic acids are capable of hybridizing to (e.g. selectively hybridizing to) a target nucleic acid.
  • the antisense nucleic acid hybridizes to the target nucleic acid in vitro.
  • the antisense nucleic acid hybridizes to the target nucleic acid in a cell.
  • the antisense nucleic acid hybridizes to the target nucleic acid in an organism.
  • the antisense nucleic acid hybridizes to the target nucleic acid under physiological conditions.
  • 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.
  • 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.
  • 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).
  • A adenine
  • C cytosine
  • G guanine
  • T thymine
  • U uracil
  • T thymine
  • 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 nucleo
  • Every' nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid.
  • 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
  • TGG which is ordinarily the only codon for try ptophan
  • complement 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.
  • 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.
  • 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).
  • 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.
  • a “protein gene product” is a protein expressed from a particular gene.
  • 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
  • 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.
  • 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.
  • solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein.
  • nucleic acid, virus, or protein 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.
  • Percentage of sequence identity is determined by comparing tw o optimally aligned sequences over a comparison window 7 , 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 identity 7 .
  • 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 identity 7 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).
  • 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.
  • the preferred algorithms can account for gaps and the like.
  • 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.
  • 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.
  • 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.
  • 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.
  • the control is used as a standard of comparison in evaluating experimental effects.
  • a control is the measurement of the activity 7 of a protein in the absence of a compound as described herein (including embodiments and examples).
  • 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.
  • 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).
  • 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.
  • An alkoxy is an alkyl attached to the remainder of the molecule via an oxygen linker (-O-).
  • 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-.
  • 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.
  • alkenylene by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from an alkene.
  • 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.
  • 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-CH 2 -S-CH 2 -CH 2 -, -O-CH2-CH2-NH-CH2-, -O-(CH 2 ) 3 -O-PO 3 -, -O-(CH 2 )-O-PO 3 -, -O-(CH2)2-O-PO 3 -, -O-(CH2)4-O-PO 3 -, and the like.
  • heteroatoms can also occupy either or both of the chain termini (e.g.. alkyleneoxy.
  • 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.
  • 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.
  • oxo means an oxygen that is double bonded to a carbon atom.
  • alkylsulfonyl 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”).
  • 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.
  • 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.
  • -NR'R includes, but is not limited to, 1 -pyrrolidinyl and 4- morpholinyl.
  • 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
  • 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.
  • the ring-forming substituents are attached to adjacent members of the base structure.
  • two ringforming substituents attached to adjacent members of a cyclic base structure create a fused ring structure.
  • the ring-forming substituents are attached to a single member of the base structure.
  • two ring-forming substituents attached to a single member of a cyclic base structure create a spirocyclic structure.
  • the ring-forming substituents are attached to non-adj acent members of the base structure.
  • Two of the substituents on adj acent atoms of the ary l or heteroaryl ring may optionally 7 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.
  • 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) 2 NR'-, 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.
  • 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'-.
  • 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.
  • heteroatom or “ring heteroatom” are meant to include, oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), and silicon (Si).
  • a “substituent group,” as used herein, means a group selected from the following moieties: [0083] (A) oxo, halogen, -CF 3 , -CN, -OH, -NH 2 , -COOH, -CONH 2 , -NO 2 , -SH, -SO 2 C1, -SO3H. -SO4H. -SO 2 NH 2 , -NHNH 2 .
  • 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-C 2 o 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 C 3 -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 heteroary
  • 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 un
  • 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.
  • 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
  • each substituted or unsubstituted heteroaryl is a substituted or unsubstituted 5 to 10 membered heteroaryl.
  • 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
  • each substituted or unsubstituted heteroarylene is a substituted or unsubstituted 5 to 10 membered heteroarylene.
  • 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
  • each substituted or unsubstituted heteroaryl is a substituted or unsubstituted 5 to 9 membered heteroaryl.
  • 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
  • each substituted or unsubstituted heteroarylene is a substituted or unsubstituted 5 to 9 membered heteroarylene.
  • the compound is a chemical species set forth in the Examples section below.
  • activation means positively affecting (e.g. increasing) the activity or function of the protein relative to the activity 7 or function of the protein in the absence of the activator.
  • 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.
  • 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
  • agonist refers 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.
  • inhibition 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.
  • 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.
  • inhibition refers to reduction of a disease or symptoms of disease. In embodiments, inhibition refers to a reduction in the activity 7 of a particular protein target.
  • 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.
  • 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).
  • 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).
  • inhibitor refers 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.
  • the first DNA oligonucleotide comprises from about 5 unmodified nucleotides to about 70 unmodified nucleotides
  • 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.
  • the first DNA oligonucleotide comprises from about 6 unmodified nucleotides to about 50 unmodified nucleotides
  • 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.
  • the first DNA oligonucleotide comprises from about 8 unmodified nucleotides to about 40 unmodified nucleotides
  • 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.
  • the first DNA oligonucleotide comprises from about 8 unmodified nucleotides to about 30 unmodified nucleotides
  • 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.
  • the first DNA oligonucleotide comprises from about 8 unmodified nucleotides to about 26 unmodified nucleotides
  • 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.
  • the first DNA oligonucleotide comprises from about 10 unmodified nucleotides to about 30 unmodified nucleotides
  • 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.
  • the first DNA oligonucleotide comprises from about 10 unmodified nucleotides to about 28 unmodified nucleotides
  • 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.
  • the first DNA oligonucleotide comprises from about 10 unmodified nucleotides to about 24 unmodified nucleotides
  • 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.
  • the first DNA oligonucleotide comprises from about 10 unmodified nucleotides to about 22 unmodified nucleotides
  • 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.
  • the first DNA oligonucleotide comprises from about 12 unmodified nucleotides to about 20 unmodified nucleotides
  • 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.
  • the first DNA oligonucleotide comprises from about 14 unmodified nucleotides to about 18 unmodified nucleotides
  • 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.
  • the constrained nucleotide is a LNA-modified nucleotide, a cMOE-modified nucleotide, or a cET-modified nucleotide.
  • the constrained nucleotide is a LNA- modified nucleotide.
  • 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.
  • the second DNA oligonucleotide comprises a nucleotide having a phosphorothioate modification.
  • the first DNA oligonucleotide comprises about 10 unmodified nucleotides
  • 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.
  • the first DNA oligonucleotide comprises about 11 unmodified nucleotides
  • 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.
  • the first DNA oligonucleotide comprises about 14 unmodified nucleotides
  • 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.
  • the first DNA oligonucleotide comprises about 15 unmodified nucleotides
  • 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.
  • the first DNA oligonucleotide comprises about 18 unmodified nucleotides
  • 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.
  • the first DNA oligonucleotide comprises about 19 unmodified nucleotides
  • 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.
  • the first DNA oligonucleotide comprises about 20 unmodified nucleotides
  • 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.
  • the first DNA oligonucleotide comprises about 21 unmodified nucleotides
  • 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.
  • the first DNA oligonucleotide comprises about 22 unmodified nucleotides
  • 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.
  • the constrained nucleotide is a LNA-modified nucleotide, a cMOE-modified nucleotide, or a cET-modified nucleotide.
  • the constrained nucleotide is a LNA- modified nucleotide.
  • 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.
  • the second DNA oligonucleotide comprises a nucleotide having a phosphorothioate modification.
  • 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.
  • 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.
  • the constrained nucleotide is a LNA-modified nucleotide, a cMOE- modified nucleotide, or a cET-modified nucleotide.
  • the constrained nucleotide is a LNA-modified nucleotide.
  • the constrained nucleotide is a cMOE-modified nucleotide.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the constrained nucleotide is a LNA-modified nucleotide, a cMOE-modified nucleotide, or a cET-modified nucleotide.
  • the constrained nucleotide is a LNA- modified nucleotide.
  • the constrained nucleotide is a cMOE-modified nucleotide.
  • the constrained nucleotide is a cET-modified nucleotide.
  • the constrained nucleotides in the second DNA oligonucleotide can be the same or different.
  • the constrained nucleotides on the 3 ’ end are different than the constrained nucleotides on the 5‘ end.
  • the constrained nucleotides on the 3’ end are the same as the constrained nucleotides on the 5’ end.
  • the second DNA oligonucleotide comprises a nucleotide having a phosphorothioate modification.
  • 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.
  • the constrained nucleotide is a LNA-modified nucleotide, a cMOE-modified nucleotide, or a cET-modified nucleotide.
  • the constrained nucleotide is a LNA-modified nucleotide.
  • 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.
  • the second DNA oligonucleotide comprises a nucleotide having a phosphorothioate modification.
  • the term “on the 3’ end’' means the terminal nucleotide(s) are adjacent the 3‘ end.
  • the term “on the 5' end” means the terminal nucleotide(s) are adjacent the 5’ end.
  • three contiguous constrained nucleotides on the 3’ end refers to the three contiguous nucleotides in lower case letter and underlined on the 3’ end.
  • two contiguous constrained nucleotides on the 3’ end refers to the two contiguous nucleotides in lower case letter and underlined on the 5’ end.
  • the term '‘contiguous” has the plain and ordinary meaning of “next to” or “together in sequence.”
  • the two or more nucleotides can be alternating.
  • alternating means that the terminal nucleotide is constrained, the next sequential nucleotide is not constrained, and the next sequential nucleotide is constrained.
  • two alternating constrained nucleotides on the 5’ end 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.
  • the first DNA oligonucleotide and the second DNA oligonucleotide do not have the same number of nucleotides.
  • the first DNA oligonucleotide can have 18 unmodified nucleotides and the second DNA oligonucleotide can have 16 nucleotides.
  • the first DNA oligonucleotide can have 16 unmodified nucleotides and the second DNA oligonucleotide can have 18 nucleotides.
  • the first DNA oligonucleotide and the second DNA oligonucleotide have the same number of nucleotides.
  • the first DNA oligonucleotide can have 16 unmodified nucleotides and the second DNA oligonucleotide can have 16 nucleotides.
  • 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.
  • 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.
  • 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.
  • the second DNA oligonucleotide further comprises a nucleotide having a modification.
  • the modification can be a spacer modification or a nucleotide modification.
  • the second DNA oligonucleotide further comprises a nucleotide having a modification to the base or sugar.
  • 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.
  • the second DNA oligonucleotide further comprises a nucleotide having a 2’O-methyl group.
  • 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.
  • 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.
  • the constrained nucleotide is a LNA-modified nucleotide, a cMOE-modified nucleotide, or a cET-modified nucleotide.
  • the constrained nucleotide is a LNA- modified nucleotide.
  • the constrained nucleotide is a cMOE-modified nucleotide.
  • the constrained nucleotide is a cET-modified nucleotide.
  • the second DNA oligonucleotide further comprises a spacer modification.
  • 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.
  • the spacer phosphoramidite is a C3 spacer phosphoramidite, a C6 spacer phosphoramidite, or a C 12 spacer phosphoramidite.
  • the second DNA oligonucleotide further comprises an abasic spacer modification.
  • the second DNA oligonucleotide further comprises a C3 spacer phosphoramidite.
  • 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.
  • 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.
  • the second DNA oligonucleotide is a STAT3 antisense oligonucleotide.
  • 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.
  • the STAT3 antisense oligonucleotide comprises SEQ ID NO:2.
  • the STAT3 antisense oligonucleotide comprises SEQ ID NO: 4.
  • 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
  • underline refers to a phosphorothioated nucleotide (phosphorothioated intemucleotide linkage); single quote (e g., G’) indicates a 2’0-methyl modification, X 1 is an abasic spacer (“/idSp/”), and X 2 is C3 spacer phosphoramidite (‘7iSpC3/”).
  • 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.
  • the first DNA oligonucleotide comprises SEQ ID NO: 1.
  • the first DNA oligonucleotide comprises SEQ ID NO:3.
  • the first DNA oligonucleotide comprises SEQ ID NO: 5.
  • the first DNA oligonucleotide comprises SEQ ID NO:7.
  • the first DNA oligonucleotide comprises SEQ ID NO:9.
  • the first DNA oligonucleotide comprises SEQ ID NO: 11.
  • the first DNA oligonucleotide comprises SEQ ID NO: 13. [0120] Table 2
  • 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.
  • 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.
  • 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.”
  • the compounds described herein comprise a phosphorothioated CpG oligodeoxynucleotide (ODN).
  • ODN phosphorothioated CpG oligodeoxynucleotide
  • the CpG ODN is a CpG-A ODN, a CpG-B ODN, a CpG-C ODN, or a combination of two or more thereof.
  • the CpG ODN is a CpG-A ODN.
  • the CpG ODN is a CpG-B ODN.
  • the CpG ODN is a CpG-C ODN.
  • 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.
  • the CpG ODN is CpG ODN 1585.
  • the CpG ODN is CpG ODN 2216.
  • the CpG ODN is CpG ODN 2336.
  • 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.
  • the phosphorothioated CpG oligodeoxynucleotide comprises SEQ ID NO:43.
  • the phosphorothioated CpG oligodeoxynucleotide comprises SEQ ID NO:43.
  • the phosphorothioated CpG oligodeoxynucleotide comprises SEQ ID NO:43, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18.
  • the phosphorothioated CpG oligodeoxynucleotide comprises SEQ ID NO:15, SEQ ID NO: 19, SEQ ID NO:20, or SEQ ID NO:21.
  • 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.
  • 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.
  • the phosphorothioated CpG oligodeoxynucleotide is covalently bonded to the first DNA oligonucleotide via the moiety of Formula (A).
  • 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.
  • 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.
  • the phosphorothioated CpG oligodeoxynucleotide is covalently bonded to the first DNA oligonucleotide via the moiety of Formula (A).
  • 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.
  • 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.
  • the phosphorothioated CpG oligodeoxynucleotide is covalently bonded to the first DNA oligonucleotide via the moiety of Formula (A).
  • 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.
  • 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.
  • the phosphorothioated CpG oligodeoxynucleotide is covalently bonded to the first DNA oligonucleotide via the moiety of Formula (A).
  • underline refers to a phosphorothioated nucleotide (phosphorothioated intemucleotide linkage).
  • the phosphorothioated CpG ODN is linked to the first DNA oligonucleotide by any linking group known in the art.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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’.
  • zl is an integer from 0 to 5.
  • zl is an integer from 2 to 4.
  • z2 is an integer from 0 to 5.
  • z2 is an integer from 2 to 4.
  • z3 is an integer from 0 to 5.
  • zl is an integer from 2 to 4.
  • z4 is an integer from 3 to 7.
  • z4 is an integer from 4 to 6.
  • each X is -OH.
  • 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.
  • the compound further comprises a detectable moiety-.
  • the phosphorothioated CpG ODN, the first DNA oligonucleotide, the second DNA oligonucleotide, or any combination thereof comprise a detectable moiety 7 .
  • the phosphorothioated CpG ODN comprises a detectable moiety .
  • the first DNA oligonucleotide comprises a detectable moiety-.
  • the second DNA oligonucleotide comprises a detectable moiety 7 .
  • 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.
  • 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.
  • the phosphorothioate nucleic acid or phosphorothioate polymer backbone includes a detectable agent, as disclosed herein and known in the art.
  • 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.
  • Exemplary 7 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
  • 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.
  • 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.
  • the compound (2) is any compound described herein, including all embodiments of the compounds described herein.
  • the constrained nucleotide is a LNA-modified nucleotide, a cMOE-modified nucleotide, or a cET-modified nucleotide.
  • the constrained nucleotide is a LNA- modified nucleotide.
  • the constrained nucleotide is a cMOE-modified nucleotide.
  • the constrained nucleotide is a cET-modified nucleotide.
  • the second DNA oligonucleotide comprises a nucleotide having a phosphorothioate modification.
  • 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.
  • 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.
  • 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.
  • Parenteral administration includes, e.g., intra-tumoral, intravenous, intramuscular, intra-arteriole, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial.
  • the compounds or pharmaceutical compositions described herein are parenterally administered to a patient.
  • 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.
  • the administering does not include administration of any active agent other than the recited active agent.
  • 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.
  • 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.
  • the compound is any compound described herein, including all embodiments of the compounds described herein.
  • the methods further comprise administering to the patient an effective amount of an immune checkpoint inhibitor.
  • the immune checkpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor.
  • the immune checkpoint inhibitor is a PD-L1 inhibitor.
  • the immune checkpoint inhibitor is a PD-1 inhibitor.
  • the methods further comprise administering to the patient an effective amount of an anti-cancer agent.
  • 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.
  • 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.
  • 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.
  • 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.
  • the compound is any compound described herein, including all embodiments of the compounds described herein.
  • the methods further comprise administering to the patient an effective amount of an immune checkpoint inhibitor.
  • the immune checkpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor.
  • the immune checkpoint inhibitor is a PD-L1 inhibitor.
  • the immune checkpoint inhibitor is a PD-1 inhibitor.
  • the methods further comprise administering to the patient an effective amount of an anti-cancer agent.
  • 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.
  • the cancer is glioma.
  • the glioma is astrocytoma, glioblastoma, or oligodendroglioma.
  • the glioma is astrocytoma.
  • the glioma is glioblastoma.
  • the glioma is oligodendroglioma.
  • the glioma is a brain stem glioma.
  • 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.
  • the cranial primitive neuroectodermal tumor is esthesioneuroblastoma.
  • the cancer is ependymal tumor.
  • the ependymal tumor is ependymoma, myxopapillary ependymoma, or subependymoma.
  • the ependymal tumor is ependymoma.
  • the ependymal tumor is myxopapillary ependymoma.
  • the ependymal tumor is subependymoma.
  • the cancer is hemangiopericytoma.
  • 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.
  • 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.
  • the compound is any compound described herein, including all embodiments of the compounds described herein.
  • the methods further comprise administering to the patient an effective amount of an immune checkpoint inhibitor.
  • the immune checkpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor.
  • the immune checkpoint inhibitor is a PD-L1 inhibitor.
  • the immune checkpoint inhibitor is a PD-1 inhibitor.
  • the methods further comprise administering to the patient an effective amount of an anti-cancer agent.
  • 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.
  • 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.
  • the compound is any compound described herein, including all embodiments of the compounds described herein.
  • the cancer is acute myeloid leukemia.
  • the methods further comprise administering to the patient an effective amount of an immune checkpoint inhibitor.
  • the immune checkpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor.
  • the immune checkpoint inhibitor is a PD-L1 inhibitor.
  • the immune checkpoint inhibitor is a PD-1 inhibitor.
  • the methods further comprise administering to the patient an effective amount of an anti-cancer agent.
  • 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.
  • 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.
  • the compound is any compound described herein, including all embodiments of the compounds described herein.
  • the methods further comprise administering to the patient an effective amount of an immune checkpoint inhibitor.
  • the immune checkpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor.
  • the immune checkpoint inhibitor is a PD-L1 inhibitor.
  • the immune checkpoint inhibitor is a PD-1 inhibitor.
  • the methods further comprise administering to the patient an effective amount of an anti-cancer agent.
  • 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.
  • 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.
  • the compound is any compound described herein, including all embodiments of the compounds described herein.
  • the constrained nucleotide is a LNA-modified nucleotide, a cMOE-modified nucleotide, or a cET-modified nucleotide.
  • the constrained nucleotide is a LNA-modified nucleotide.
  • the constrained nucleotide is a cMOE-modified nucleotide.
  • the constrained nucleotide is a cET-modified nucleotide.
  • the second DNA oligonucleotide comprises a nucleotide having a phosphorothioate modification.
  • the methods further comprise administering to the patient an effective amount of an immune checkpoint inhibitor.
  • the immune checkpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor.
  • the immune checkpoint inhibitor is a PD-L1 inhibitor.
  • the immune checkpoint inhibitor is a PD-1 inhibitor.
  • the methods further comprise administering to the patient an effective amount of an anti-cancer agent.
  • 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.
  • 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.
  • the epidermoid carcinoma is esophageal cancer.
  • the cancer is anal cancer.
  • the cancer is cervical cancer.
  • the cancer is head and neck cancer.
  • the cancer is vaginal cancer.
  • the PD-1 inhibitor is pembrolizumab, nivolumab. cemiplimab, dostarlimab, camrelizumab, sintilimab, tislelizumab, toripalimab, spartalizumab, retifanlimab, pimivalimab (JTX-4014), AMP-224, or MEDI0680 (AMP-514).
  • 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.
  • the PD-L1 inhibitor is atezolizumab, avelumab, durvalumab, envafolimab, cosibelimab, AUNP12, CA-170, or BMS-986189.
  • the PD-L1 inhibitor is atezolizumab, avelumab, durvalumab, envafolimab, cosibelimab, AUNP12, CA-170. or BMS-986189.
  • the PD-L1 inhibitor is atezolizumab, avelumab, durvalumab, envafolimab, or cosibelimab.
  • 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.
  • “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.
  • a patient is human.
  • 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.
  • an anti-cancer agent is a chemotherapeutic.
  • an anti-cancer agent is an agent identified herein having utility in methods of treating cancer.
  • 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.
  • nitrogen mustards e.g., mechloroethamine, cyclophosphamide, chlorambucil, meiphalan
  • ethylenimine and methylmelamines e.g., hexamethlymelamine, thi
  • 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.
  • Epothilones e.g. Epothilone A, Epothilone B, Epothilone C (i.e. desoxy epothilone A or dEpoA), Epothilone D (i.e. KOS-862
  • 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.
  • Eleutherobins such as Desmethyleleutherobin, Desaetyleleutherobin, Isoeleutherobin A, and Z- Eleutherobin
  • caribaeoside caribaeolin
  • Halichondrin B Diazonamide A
  • Taccalonolide A Diozostatin
  • (-)-Phenylahistin i.e.
  • NSCL-96F0357 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
  • anti-CD52, anti- HLA-DR, and anti-VEGF monoclonal antibodies include 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 ni In, 90 Y, or 131 L 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,
  • TAK-285 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Embodiment 9 The compound of any one of Embodiments 3 to 7, wherein the two constrained nucleotides are alternating.
  • Embodiment 12 The compound of Embodiment 6, 7, or 9, wherein the constrained nucleotides are alternating.
  • Embodiment 13 The compound of any one of Embodiments 1 to 12, wherein the constrained nucleotide is a LNA-modified nucleotide.
  • Embodiment 14 The compound of any one of Embodiments 1 to 12, wherein the constrained nucleotide is a cMOE-modified nucleotide.
  • Embodiment 15 The compound of any one of Embodiments 1 to 12, wherein the constrained nucleotide is a cET-modified nucleotide.
  • 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.
  • 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.
  • 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.
  • 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.
  • Embodiment 20 The compound of any one of Embodiments 1 to 19, wherein the second DNA oligonucleotide comprises a nucleotide having a phosphorothioate modification.
  • Embodiment 23 The compound of any one of Embodiments 1 to 22, wherein the second DNA oligonucleotide is an antisense oligonucleotide.
  • 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.
  • the glioma is astrocytoma, glioblastoma, or oligodendroglioma
  • the cranial primitive neuroectodermal tumor is medulloblastoma, cerebral neuroblastoma, pineoblastoma, or esthesioneuroblastoma
  • the ependymal tumor is ependymoma, myxopapillary
  • Embodiment 57 The method of Embodiment 49, wherein the cancer is prostate cancer.
  • Embodiment 58 The method of Embodiment 49, wherein the cancer is an epidermoid carcinoma.
  • Embodiment 63 The method of Embodiment 60, wherein the immune checkpoint inhibitor is a PD-Ll inhibitor.
  • Embodiment 64 The method of Embodiment 63, wherein the PD-L1 inhibitor is atezolizumab, avelumab. durvalumab, envafolimab. cosibelimab, AUNP12, CA-170, or BMS- 986189.
  • Embodiment 65 A pharmaceutical composition comprising the compound of any one of Embodiments 1 to 47 and an immune checkpoint inhibitor.
  • Embodiment 66 The pharmaceutical composition of Embodiment 65, wherein the immune checkpoint inhibitor is a PD-inhibitor or a PD-L1 inhibitor.
  • Embodiment 67 A STAT3 antisense oligonucleotide comprising SEQ ID NO: 4.
  • 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.
  • 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 nucleot
  • Embodiment N4 The compound of Embodiment N3, wherein the two or three constrained nucleotides are contiguous or alternating.
  • 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.
  • 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.
  • Embodiment N7 The compound of any one of Embodiments N1 to N6, wherein the second DNA oligonucleotide comprises a nucleotide having a phosphorothioate modification.
  • 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.
  • Embodiment N9 The compound of any one of Embodiments N1 to N8, wherein the second DNA oligonucleotide is an antisense oligonucleotide.
  • Embodiment N10 The compound of any one of Embodiments N1 to N8, wherein the second DNA oligonucleotide is a STAT3 antisense oligonucleotide.
  • 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.
  • 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; (a) the
  • 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.
  • 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.
  • 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.
  • 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 oli
  • Embodiment N A pharmaceutical composition comprising the compound of anyone of Embodiments N1 to N16 and a pharmaceutically acceptable excipient.
  • 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.
  • Embodiment N19 The method of Embodiment N18, wherein the cancer is glioma.
  • 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.
  • 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.
  • 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.
  • Embodiment N22 The method of Embodiment N21, wherein the immune checkpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor.
  • 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.
  • the PD-1 inhibitor is pembrolizumab, nivolumab, cemiplimab, dostarlimab, camrelizumab, sintilimab, tislelizumab, toripalimab, spart
  • 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).
  • 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).
  • CpG-STAT3ASOs 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).
  • LNA-modified CpG-STAT3dsASO treatment was combined with systemic administration of PD1 blocking antibodies.
  • the combination treatment produced long-term tumor free survival of 5/6 treated mice for over 150 days.
  • the locally injected LNA- modified CpG-STAT3dsASO showed efficacy also against RM9 prostate tumors in mice, with evidence of abscopal effects (FIG. 12).
  • 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.

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EP23875846.0A 2022-10-07 2023-10-06 Doppelsträngige oligonukleotide und verfahren zu ihrer verwendung Pending EP4599065A2 (de)

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