Synthetic RIG-I-like receptor agonist

Abstract

To provide synthetic RIG-I-like receptor agonists.SOLUTION: The present disclosure relates to, inter alia, RNA molecules (e.g., RNA hairpin agonists) that bind to and agonize RIG-I-like receptors (RLRs), and to use of the molecules in methods for treating or ameliorating one or more symptoms of a disorder (e.g., cancer). In some embodiments the RLR agonists of the disclosure comprise a sequence motif selected from the group consisting of: (i) a GT-repeat motif; (ii) a GA-repeat motif; (iii) an AUCG-repeat motif; (iv) an AU-repeat motif; (v) a dipyrimidine motif; (vi) a dipurine motif; (vii) a pyrimidine triplet motif; (viii) a purine triplet motif; (ix) a palindromic sequence motif; and (x) a combination of any of (i)-(ix).SELECTED DRAWING: None

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefit of U.S. Provisional Application No. 62 / 659,999, filed on April 19, 2018. The entire contents of the aforementioned application are incorporated herein by this reference. [Background technology]

[0002] Exogenous nucleic acids, particularly viral nucleic acids, that enter cells induce an innate immune response, triggering events such as interferon (IFN) production and cell death. Upon sensing viral RNA, the RIG-I-like receptor induces type I interferon (IFN) secretion, leading to upregulation of antiviral IFN-inducing proteins in infected and adjacent cells, and suppressing viral replication. Further downstream events attract immune cells, triggering an adaptive immune response. In addition, RIG-I ligands have been reported to induce apoptosis in many different types of tumor cells, but not in normal cells.

[0003] Further improved compositions and methods for modulating the activity of immunomodulatory proteins are still needed. Such agents can be used in cancer immunotherapy and the treatment of other conditions (e.g., chronic infections). It is necessary to develop improved RIG-I-like receptor ligands for diverse therapeutic immunomodulatory applications. [Overview of the project] [Means for solving the problem]

[0004] This disclosure is based, at least in part, on the discovery of synthetic RNA molecules that function as RIG-I-like receptor agonists.

[0005] In some embodiments, the Disclosure provides a synthetic RIG-I-like receptor (RLR) agonist that specifically binds to a RIG-I-like receptor (RLR), wherein the agonist comprises a blunt-terminated hairpin RNA containing a first polynucleotide linked to a second polynucleotide by a linker, the first polynucleotide being sufficiently complementary to the second polynucleotide and forming a double helix, the double helix containing fewer than 19 base pairs, the 5' terminal nucleotide of the first oligonucleotide comprising a 5' diphosphate or tripphosphate moiety, or a derivative or analog thereof, the agonist comprising a sequence motif, the sequence motif providing at least one improved RLR-mediated bioactivity compared to an agonist without the sequence motif. In some embodiments, the first polynucleotide comprises a sequence motif.

[0006] In some embodiments, the RLR agonist of this disclosure is as follows: (i) GT repeat motif, (ii) GA repeat motif, (iii) AUCG repeat motif, (iv) AU repeat motif, (v) Dipyrimidine motif, (vi) Ziplin motif, (vii) Pyrimidine triplet motif, (viii) Printed riplet motif, (ix) Palindromic arrangement motifs, and It includes an array motif selected from the group consisting of any combination of (x)(i)~(ix).

[0007] In some embodiments, the RLR agonists of this disclosure include a combination of sequence motifs. In some embodiments, the combination of sequence motifs is a GT repeat motif and a print repeat motif. In some embodiments, the combination of sequence motifs is an AUCG repeat motif and a dipyrimidine motif. In some embodiments, the combination of sequence motifs is an AUCC repeat motif and a diprine motif.

[0008] In some embodiments, the RLR agonists of the present disclosure include a sequence motif, which, compared to agonists that do not include this sequence motif, provides at least one improved RLR-mediated bioactivity, and the at least one improved bioactivity is (i) Increased cytokine production mediated by RLR, (ii) Increased expression of interferon-inducible genes mediated by RLR, (iii) Increased intracellular signaling mediated by RLRs, (iv) Increased binding affinity to RLR, and (v)(i)~(iv) is selected from any combination.

[0009] In some embodiments, the RLR agonists of this disclosure include a sequence motif that increases RLR-mediated type I interferon (e.g., IFN-α, IFN-β) production compared to agonists that do not include this sequence motif. In some embodiments, the RLR agonists of this disclosure include a sequence motif that increases RLR-mediated IL-1β production compared to agonists that do not include this sequence motif. In some embodiments, the RLR agonists of this disclosure include a sequence motif that increases RLR-mediated IP10 production compared to agonists that do not include this sequence motif. In some embodiments, the RLR agonists of this disclosure include a sequence motif that increases RLR-mediated IL-6, IL-12p70, MCP-1, and / or MIP-1β production compared to agonists that do not include this sequence motif.

[0010] In some embodiments, the RLR agonist of the Disclosure comprises a sequence motif, which is a GT repeat motif (e.g., GTGTGT) comprising a sequence of less than 19, about 15-18, about 15, about 10-15, about 10, about 5-10, about 5, about 4, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or 4 guanine and thymine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GT repeat motif comprising a sequence of less than 19 guanine and thymine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GT repeat motif comprising a sequence of about 15-18 guanine and thymine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GT repeat motif comprising a sequence of about 15 guanine and thymine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GT repeat motif containing about 10 to less than 15 sequences of guanine and thymine nucleotides or their derivatives or analogs. In some embodiments, the sequence motif is a GT repeat motif containing about 10 sequences of guanine and thymine nucleotides or their derivatives or analogs. In some embodiments, the sequence motif is a GT repeat motif containing about 5 to 10 sequences of guanine and thymine nucleotides or their derivatives or analogs. In some embodiments, the sequence motif is a GT repeat motif containing about 5 sequences of guanine and thymine nucleotides or their derivatives or analogs. In some embodiments, the sequence motif is a GT repeat motif containing about 4 sequences of guanine and thymine nucleotides or their derivatives or analogs.In some embodiments, GT repeat motifs confer enhanced bioactivity in RLR agonists, which include increased RLR-mediated cytokine production, increased RLR-mediated expression of interferon-inducible genes, increased RLR-mediated intracellular signaling, increased binding affinity to RLRs, and any combination thereof.

[0011] In some embodiments, the RLR agonist of this disclosure comprises a sequence motif, which is a GT repeat motif comprising a sequence of 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or 4 guanine and thymine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GT repeat motif comprising a sequence of 18 guanine and thymine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GT repeat motif comprising a sequence of 16 guanine and thymine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GT repeat motif comprising a sequence of 14 guanine and thymine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GT repeat motif comprising a sequence of 12 guanine and thymine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GT repeat motif comprising a sequence of 10 guanine and thymine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GT repeat motif comprising a sequence of eight guanine and thymine nucleotides or derivatives or analogs thereof. In some embodiments, the sequence motif is a GT repeat motif comprising a sequence of six guanine and thymine nucleotides or derivatives or analogs thereof. In some embodiments, the sequence motif is a GT repeat motif comprising a sequence of four guanine and thymine nucleotides or derivatives or analogs thereof. In some embodiments, the RLR agonist of the present disclosure comprises a sequence motif, which is a GT repeat motif, and the GT repeat motif is [GT] nThe formula is such that n = 2-9, 3-7, or 4-8. In some embodiments, the GT repeat motif confers enhanced bioactivity in RLR agonists, which is enhanced by increased RLR-mediated cytokine production, increased RLR-mediated expression of interferon-inducible genes, increased RLR-mediated intracellular signaling, increased binding affinity to RLR, and any combination of the above.

[0012] In some embodiments, the Disclosure provides an RLR agonist that specifically binds to an RLR, wherein the agonist comprises a blunt-terminated hairpin RNA comprising a first polynucleotide linked (operably ligated) to a second polynucleotide by a linker, the first polynucleotide being sufficiently complementary to the second polynucleotide and forming a double helix, the double helix comprising fewer than 19 base pairs, the 5' terminal nucleotide of the first oligonucleotide comprising a 5' diphosphate or tripphosphate moiety, or a derivative or analog thereof, the agonist comprising a sequence motif, the sequence motif conferring at least one improved RLR-mediated bioactivity compared to an agonist without the sequence motif, the first polynucleotide comprising the sequence motif, the sequence motif being a GT repeat motif comprising a sequence of about 14 guanine and thymine nucleotides. In some embodiments, the sequence motif is a GT repeat motif, and the GT repeat motif is [GT]7. In some embodiments, improved bioactivity includes increased RLR-mediated cytokine production, increased RLR-mediated expression of interferon-inducible genes, increased RLR-mediated intracellular signaling, increased binding affinity to RLRs, and any combination of the above.

[0013] In some embodiments, the Disclosure provides an RLR agonist that specifically binds to an RLR, wherein the agonist comprises a blunt-terminated hairpin RNA comprising a first polynucleotide linked to a second polynucleotide by a linker, the first polynucleotide being sufficiently complementary to the second polynucleotide and forming a double helix, the double helix comprising fewer than 19 base pairs, the 5' terminal nucleotide of the first oligonucleotide comprising a 5' diphosphate or tripphosphate moiety, or a derivative or analog thereof, the agonist comprising a sequence motif, the sequence motif conferring at least one improved RLR-mediated bioactivity compared to an agonist without the sequence motif, the first polynucleotide comprising the sequence motif, the sequence motif being a GT repeat motif comprising a sequence of six guanine and thymine nucleotides. In some embodiments, the sequence motif is a GT repeat motif, and the GT repeat motif is [GT]3. In some embodiments, the sequence motif is a GT repeat motif, which is [GT]3, followed by a print riplet motif and UCG, respectively. In some embodiments, the print riplet is GGA. In some embodiments, the improved bioactivity is increased RLR-mediated cytokine production, increased RLR-mediated expression of interferon-inducing genes, increased RLR-mediated intracellular signaling, increased binding affinity to RLR, and any combination of the above.

[0014] In some embodiments, the RLR agonist of this disclosure comprises a sequence motif, which is a GA repeat motif (e.g., GAGAGA) comprising a sequence of less than 19, about 15-18, about 15, about 10-15, about 10, about 5-10, about 5, about 4, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or 4 guanine and adenine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GA repeat motif comprising a sequence of less than 19 guanine and adenine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GA repeat motif comprising a sequence of about 15-18 guanine and adenine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GA repeat motif comprising a sequence of about 15 guanine and adenine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GA repeat motif containing about 10 to less than 15 sequences of guanine and adenine nucleotides or their derivatives or analogs. In some embodiments, the sequence motif is a GA repeat motif containing about 10 sequences of guanine and adenine nucleotides or their derivatives or analogs. In some embodiments, the sequence motif is a GA repeat motif containing about 5 to 10 sequences of guanine and adenine nucleotides or their derivatives or analogs. In some embodiments, the sequence motif is a GA repeat motif containing about 5 sequences of guanine and adenine nucleotides or their derivatives or analogs. In some embodiments, the sequence motif is a GA repeat motif containing about 4 sequences of guanine and adenine nucleotides or their derivatives or analogs.In some embodiments, GA repeat motifs confer enhanced bioactivity in RLR agonists, which include increased RLR-mediated cytokine production, increased RLR-mediated expression of interferon-inducible genes, increased RLR-mediated intracellular signaling, increased binding affinity to RLRs, and any combination thereof.

[0015] In some embodiments, the RLR agonist of this disclosure comprises a sequence motif, which is a GA repeat motif comprising sequences of 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or 4 guanine and adenine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GA repeat motif comprising sequences of 18 guanine and adenine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GA repeat motif comprising sequences of 16 guanine and adenine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GA repeat motif comprising sequences of 14 guanine and adenine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GA repeat motif comprising sequences of 12 guanine and adenine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GA repeat motif comprising sequences of 8 guanine and adenine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GA repeat motif comprising a sequence of six guanine and adenine nucleotides or their derivatives or analogs. In some embodiments, the sequence motif is a GA repeat motif comprising a sequence of four guanine and adenine nucleotides or their derivatives or analogs.

[0016] In some embodiments, the RLR agonist of the present disclosure comprises a sequence motif, which is a GA repeat motif, and the GA repeat motif is [GA] n The formula is such that n = 2-9, 3-7, or 4-8. In some embodiments, the GA repeat motif confers enhanced bioactivity in RLR agonists, which is enhanced by increased RLR-mediated cytokine production, increased RLR-mediated expression of interferon-inducible genes, increased RLR-mediated intracellular signaling, increased binding affinity to RLR, and any combination of the above.

[0017] In some embodiments, the Disclosure provides an RLR agonist that specifically binds to an RLR, wherein the agonist comprises a blunt-terminated hairpin RNA comprising a first polynucleotide linked to a second polynucleotide by a linker, the first polynucleotide being sufficiently complementary to the second polynucleotide and forming a double helix, the double helix comprising fewer than 19 base pairs, the 5' terminal nucleotide of the first oligonucleotide comprising a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof, the agonist comprising a sequence motif, the sequence motif conferring at least one improved RLR-mediated bioactivity compared to an agonist without the sequence motif, the first polynucleotide comprising the sequence motif, the sequence motif being a GA repeat motif comprising a sequence of about 14 guanine and adenine nucleotides. In some embodiments, the sequence motif is a GA repeat motif, and the GA repeat motif is [GA]7. In some embodiments, GA repeat motifs confer enhanced bioactivity in RLR agonists, which include increased RLR-mediated cytokine production, increased RLR-mediated expression of interferon-inducible genes, increased RLR-mediated intracellular signaling, increased binding affinity to RLRs, and any combination thereof.

[0018] In some embodiments, the RLR agonist of this disclosure comprises a sequence motif, which is an AUCG repeat motif (e.g., AUCGAUCG) comprising sequences of less than 19, about 16, about 12-16, about 12, about 8-12, about 6, 16, 12, and 8 adenine, uracil, cytosine, and guanine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is an AUCG repeat motif comprising sequences of less than 19 adenine, uracil, cytosine, and guanine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is an AUCG repeat motif comprising sequences of about 16 adenine, uracil, cytosine, and guanine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is an AUCG repeat motif comprising sequences of about 12-16 adenine, uracil, cytosine, and guanine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is an AUCG repeat motif containing a sequence of 12 adenine, uracil, cytosine, and guanine nucleotides, or a sequence of derivatives or analogs thereof. In some embodiments, the sequence motif is an AUCG repeat motif containing about 8 to 12 adenine, uracil, cytosine, and guanine nucleotides, or a sequence of derivatives or analogs thereof. In some embodiments, the sequence motif is an AUCG repeat motif containing about 6 adenine, uracil, cytosine, and guanine nucleotides, or a sequence of derivatives or analogs thereof. In some embodiments, the sequence motif is an AUCG repeat motif containing a sequence of 16 adenine, uracil, cytosine, and guanine nucleotides, or a sequence of derivatives or analogs thereof. In some embodiments, the sequence motif is an AUCG repeat motif containing a sequence of 12 adenine, uracil, cytosine, and guanine nucleotides, or a sequence of derivatives or analogs thereof.In some embodiments, the sequence motif is an AUCG repeat motif comprising a sequence of eight adenine, uracil, cytosine, and guanine nucleotides, or derivatives or analogs thereof. In some embodiments, the AUCG repeat motif confers enhanced bioactivity in the RLR agonist, which includes increased RLR-mediated cytokine production, increased RLR-mediated expression of interferon-inducible genes, increased RLR-mediated intracellular signaling, increased binding affinity to RLR, and any combination thereof.

[0019] In some embodiments, the RLR agonist of the present disclosure includes an array motif, which is an AUCG repeat motif, and the AUCG repeat motif is [AUCG] n The formula is such that n=2 to 4 or 2, 3 or 4. In some embodiments, the AUCG repeat motif confers enhanced bioactivity in RLR agonists, which is enhanced by increased RLR-mediated cytokine production, increased RLR-mediated expression of interferon-inducible genes, increased RLR-mediated intracellular signaling, increased binding affinity to RLR, and any combination of the above.

[0020] In some embodiments, the Disclosure provides an RLR agonist that specifically binds to an RLR, wherein the agonist comprises a blunt-terminated hairpin RNA comprising a first polynucleotide linked to a second polynucleotide by a linker, the first polynucleotide being sufficiently complementary to the second polynucleotide and forming a double helix, the double helix comprising fewer than 19 base pairs, the 5' terminal nucleotide of the first oligonucleotide comprising a 5' diphosphate or tripphosphate moiety, or a derivative or analog thereof, the agonist comprising a sequence motif, the sequence motif conferring at least one improved RLR-mediated bioactivity compared to an agonist not comprising the sequence motif, the first polynucleotide comprising the sequence motif, the sequence motif being an AUCG repeat motif comprising a sequence of about 12 guanine and adenine nucleotides. In some embodiments, the AUCG repeat motif is [AUCG]3. In some embodiments, the AUCG repeat motif confers enhanced bioactivity in RLR agonists, which includes increased RLR-mediated cytokine production, increased RLR-mediated expression of interferon-inducible genes, increased RLR-mediated intracellular signaling, increased binding affinity to RLRs, and any combination thereof.

[0021] In some embodiments, the RLR agonist of the present disclosure includes an AUCG repeat motif, preceded by a CG or dipyrimidine motif. In some embodiments, a CG motif precedes the AUCG repeat motif. In some embodiments, the AUCG repeat motif is [AUCG]3, preceded by a CG motif. In some embodiments, the AUCG repeat motif is [AUCG]3, preceded by a dipyrimidine motif CC.

[0022] In some embodiments, the RLR agonist of the present disclosure includes an AUCG repeat motif, preceded by a Ziplin motif. In some embodiments, the Ziplin motif is GA. In some embodiments, the AUCG repeat motif is [AUCG]3, preceded by a Ziplin motif GA. In some embodiments, the Ziplin motif II precedes the AUCG repeat motif.

[0023] In some embodiments, the RLR agonist of this disclosure comprises an AUCG repeat motif, in which one or more uridine nucleosides (U) are substituted with a modified nucleoside. In some embodiments, the modified nucleoside is ribothymidine (T). In some embodiments, the AUGC repeat motif is [AUCG]3, in which one or more uridine nucleosides (U) constituting the AUCG repeat motif are substituted with a modified nucleoside, the modified nucleoside being ribothymidine (T). In some embodiments, the AUGC repeat motif is [AUCG]3, in which one or more uridine nucleosides (U) constituting the AUCG repeat motif are substituted with a modified nucleoside, the modified nucleoside being ribothymidine (T), and GG is present before the AUGC repeat motif.

[0024] In some embodiments, the RLR agonist of this disclosure comprises an AUCG repeat motif, in which one or more guanosine nucleosides (G) are substituted with a modified nucleoside. In some embodiments, the modified nucleoside is inosine (I). In some embodiments, the AUGC repeat motif is [AUCG]3, in which one or more guanosine nucleosides (G) constituting the AUCG repeat motif are substituted with a modified nucleoside, the modified nucleoside being ribothymidine (T), and GG is present before the AUGC repeat motif.

[0025] In some embodiments, the RLR agonist of the present disclosure includes an AUCG repeat motif, with an IG preceding this motif. In some embodiments, the AUCG repeat motif is [AUCG]3, with an IG preceding this motif.

[0026] In some embodiments, the RLR agonist of the Disclosure comprises an AUCG repeat, in which one or more guanosine nucleosides (G) are substituted with inosine (I), and inosine (I) is present before the AUCG repeat. In some embodiments, the guanosine nucleosides (G) constituting the AUCG repeat are substituted with inosine (I), inosine (I) is present before the AUCG repeat, and the 5' terminal nucleotide of the first polynucleotide comprises inosine (I).

[0027] In some embodiments, the RLR agonist of the present disclosure includes an AUCG repeat motif, where the AUCG repeat motif is [AUCG]2. In some embodiments, the sequence motif is an AUCG repeat motif, where the AUCG repeat motif is [AUCG]2, and a ziplin motif precedes the AUCG repeat motif. In some embodiments, the sequence motif is an AUCG repeat motif, where the AUCG repeat motif is [AUCG]2, and a ziplin motif precedes the AUCG repeat motif, where the ziplin motif is GG.

[0028] In some embodiments, the RLR agonist of the present disclosure includes an AUCG repeat motif, where the AUCG repeat motif is [AUCG]2, and a print repeat motif precedes the AUCG repeat motif. In some embodiments, the print repeat motif is GGG. In some embodiments, the sequence motif is an AUCG repeat motif, where the AUCG repeat motif is [AUCG]2, and a print repeat motif precedes the AUCG repeat motif, where the print repeat motif is GGG. In some embodiments, the sequence motif is an AUCG repeat motif, where the AUCG repeat motif is [AUCG]2, and CCCCCG precedes the AUCG repeat motif. In some embodiments, the sequence motif is an AUCG repeat motif, where the AUCG repeat motif is [AUCG]2, and TCGUCG precedes the AUCG repeat motif.

[0029] In some embodiments, the RLR agonist of the Disclosure comprises a sequence motif, which comprises a sequence of fewer than 19, about 15-18, about 15, about 10-15, about 10, about 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or 4 nucleotides, or derivatives or analogs thereof, and is a palindromic sequence that is concatenated in any order to produce a palindrome. In some embodiments, the sequence motif comprises a sequence of fewer than 19 nucleotides, or derivatives or analogs thereof, and is a palindromic sequence that is concatenated in any order to produce a palindrome. In some embodiments, the sequence motif comprises a sequence of about 15-18 nucleotides, or derivatives or analogs thereof, and is a palindromic sequence that is concatenated in any order to produce a palindrome. In some embodiments, the sequence motif is a palindromic sequence comprising approximately 10 to 15 nucleotides, or their derivatives or analogs, which can be linked in any order to generate a palindrome. In some embodiments, the sequence motif is a palindromic sequence comprising approximately 10 nucleotides, or their derivatives or analogs, which can be linked in any order to generate a palindrome. In some embodiments, the sequence motif is a palindromic sequence comprising 18 nucleotides, or their derivatives or analogs, which can be linked in any order to generate a palindrome. In some embodiments, the sequence motif is a palindromic sequence comprising 17 nucleotides, or their derivatives or analogs, which can be linked in any order to generate a palindrome. In some embodiments, the sequence motif is a palindromic sequence comprising 16 nucleotides, or their derivatives or analogs, which can be linked in any order to generate a palindrome. In some embodiments, the sequence motif is a palindromic sequence comprising 15 nucleotides, or their derivatives or analogs, which can be linked in any order to generate a palindrome.In some embodiments, the sequence motif is a palindromic sequence comprising a sequence of 14 nucleotides, or derivatives or analogs thereof, which can be linked in any order to generate a palindrome. In some embodiments, the sequence motif is a palindromic sequence comprising a sequence of 13 nucleotides, or derivatives or analogs thereof, which can be linked in any order to generate a palindrome. In some embodiments, the sequence motif is a palindromic sequence comprising a sequence of 12 nucleotides, or derivatives or analogs thereof, which can be linked in any order to generate a palindrome. In some embodiments, the sequence motif is a palindromic sequence comprising a sequence of 11 nucleotides, or derivatives or analogs thereof, which can be linked in any order to generate a palindrome. In some embodiments, the sequence motif is a palindromic sequence comprising a sequence of 10 nucleotides, or derivatives or analogs thereof, which can be linked in any order to generate a palindrome. In some embodiments, the sequence motif is a palindromic sequence comprising a sequence of 9 nucleotides, or derivatives or analogs thereof, which can be linked in any order to generate a palindrome. In some embodiments, the sequence motif is a palindromic sequence comprising a sequence of 8 nucleotides, or derivatives or analogs thereof, which can be linked in any order to generate a palindrome. In some embodiments, the sequence motif is a palindromic sequence comprising a sequence of 7 nucleotides, or derivatives or analogs thereof, which can be linked in any order to generate a palindrome. In some embodiments, the sequence motif is a palindromic sequence comprising a sequence of 6 nucleotides, or derivatives or analogs thereof, which can be linked in any order to generate a palindrome. In some embodiments, the sequence motif is a palindromic sequence comprising a sequence of 5 nucleotides, or derivatives or analogs thereof, which can be linked in any order to generate a palindrome. In some embodiments, the sequence motif is a palindromic sequence comprising a sequence of 4 nucleotides, or derivatives or analogs thereof, which can be linked in any order to generate a palindrome.

[0030] In some embodiments, the RLR agonist of the present disclosure includes a linker, which is adjacent to the AU. In some embodiments, the linker is adjacent to the AU repeat motif, which is [AU] n The formula is such that n = 2 to 3. In some embodiments, the AU repeat motif is [AU]2.

[0031] In some embodiments, the present disclosure provides an RLR agonist that specifically binds to RLR, wherein the agonist is given by the following formula: It contains blunt-ended hairpin RNA including 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', in the formula, (i)(N1-N2-X1) comprises a first polynucleotide containing linked nucleotides N1, N2 and X1, (ii)(X2-N3-N4) comprises a second polynucleotide containing linked nucleotides X2, N3 and N4, (iii) N1, N2, N3, and N4 each contain a single nucleotide comprising a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine. (iv)N1 forms a base pair with N4, (v)N2 forms a base pair with N3, (vi)N1 comprises a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof. (vii)X1 and X2 are oligonucleotides containing a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine, respectively. (viii) X1 is complementary to X2, (ix) X1 and X2 are each the same length, ranging from 12 to 16 nucleotides in length. (x)L is a linker that operably links the first polynucleotide and the second polynucleotide. In the formula, at least one of N1, N2, N3, and N4 is inosine, and / or at least one of X1 and / or X2 comprises at least one inosine nucleoside, the inosine nucleoside base-pairing with cytidine in the hairpin RNA. In some embodiments, the RLR agonists of the present disclosure have enhanced bioactivity, which is increased RLR-mediated cytokine production, increased RLR-mediated expression of interferon-inducible genes, increased RLR-mediated intracellular signaling, increased binding affinity to RLR, and any combination thereof.

[0032] In some embodiments, the present disclosure provides an RLR agonist that specifically binds to RLR, the agonist comprising a blunt-terminated hairpin RNA containing a non-nucleotide linker, and the agonist has the following formula: The formula includes 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', and in the formula, (i)(N1-N2-X1) comprises a first polynucleotide containing linked nucleotides N1, N2 and X1, (ii)(X2-N3-N4) comprises a second polynucleotide containing linked nucleotides X2, N3 and N4, (iii) N1, N2, N3, and N4 each contain a single nucleotide comprising a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine. (iv)N1 forms a base pair with N4, (v)N2 forms a base pair with N3, (vi)N1 comprises a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof. (vii)X1 and X2 are oligonucleotides containing a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine, respectively. (viii) X1 is complementary to X2, (ix) X1 and X2 are each the same length, ranging from 12 to 16 nucleotides in length. (x)L is a non-nucleotide linker that covalently bonds the first polynucleotide and the second polynucleotide. Inosine, if present, forms a base pair with cytidine. In some embodiments, the RLR agonists of this disclosure have enhanced bioactivity, which includes increased RLR-mediated cytokine production, increased RLR-mediated expression of interferon-inducible genes, increased RLR-mediated intracellular signaling, increased binding affinity to RLR, and any combination thereof.

[0033] In some embodiments, N1 contains inosine and N4 contains cytidine. In some embodiments, N1 contains inosine and N4 contains cytidine, and X1 and X2 are each 12 nucleotides long. In some embodiments, N1 contains cytidine and N4 contains inosine. In some embodiments, N2 contains inosine and N3 contains cytidine. In some embodiments, N2 contains cytidine and N3 contains inosine. In some embodiments, N1 contains guanosine. In some embodiments, N2 contains guanosine. In some embodiments, N1 contains cytidine. In some embodiments, N2 contains cytidine. In some embodiments, N1 and N2 contain guanosine and N3 and N4 contain cytidine. In some embodiments, N1 and N2 contain cytidine and N3 and N4 contain guanosine. In some embodiments, N1 and N2 contain inosine and N3 and N4 contain cytidine. In some embodiments, N1 and N2 contain cytidine, and N3 and N4 contain inosine.

[0034] In some embodiments, the RLR agonist of this disclosure is given by the following formula: The formula includes 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', and in the formula, (i)(N1-N2-X1) comprises a first polynucleotide containing linked nucleotides N1, N2 and X1, (ii)(X2-N3-N4) comprises a second polynucleotide containing linked nucleotides X2, N3 and N4, (iii) N1, N2, N3, and N4 each contain a single nucleotide comprising a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine. (iv)N1 forms a base pair with N4, (v)N2 forms a base pair with N3, (vi)N1 comprises a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof. (vii)X1 and X2 are oligonucleotides containing a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine, respectively. (viii) X1 is complementary to X2, (ix) X1 and X2 are each the same length, ranging from 12 to 16 nucleotides in length. (x)L is a non-nucleotide linker that covalently bonds the first polynucleotide and the second polynucleotide. If present, inosine forms a base pair with cytidine, with N1 containing inosine, N4 containing cytidine, and X1 and / or X2 each containing at least one inosine. In some embodiments, N2 contains inosine, N3 contains cytidine, and X1 and / or X2 each contain at least one inosine. In some embodiments, N1 and N2 contain guanosine, N3 and N4 contain cytidine, and X1 and / or X2 each contain at least one inosine. In some embodiments, N1 and N2 contain guanosine, N3 and N4 contain cytidine, and X1 and X2 each contain at least one inosine. In some embodiments, N1 and N2 contain guanosine, N3 and N4 contain cytidine, and X1 and X2 each contain at least one inosine, with X1 and X2 each being 12 nucleotides long. In some embodiments, N1 and N2 contain cytidine, N3 and N4 contain guanosine, and X1 and X2 each contain at least one inosine. In some embodiments, N1 and N2 contain guanosine, N3 and N4 contain cytidine, and X1 and X2 each contain inosine, but do not contain guanosine nucleosides. In some embodiments, N1 and N2 contain guanosine, N3 and N4 contain cytidine, and X1 and X2 each contain at least one inosine, and X1 and X2 each have a length of 12 nucleotides. In some embodiments, N1 and N2 contain cytidine, N3 and N4 contain guanosine, and X1 and X2 each contain inosine, but do not contain guanosine nucleosides. In some embodiments, the RLR agonists of the present disclosure have enhanced bioactivity, which includes increased RLR-mediated cytokine production, increased RLR-mediated expression of interferon-inducible genes, increased RLR-mediated intracellular signaling, increased binding affinity to RLR, and any combination thereof.

[0035] In some embodiments, the RLR agonist of this disclosure is given by the following formula: The formula includes 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', and in the formula, (i)(N1-N2-X1) comprises a first polynucleotide containing linked nucleotides N1, N2 and X1, (ii)(X2-N3-N4) comprises a second polynucleotide containing linked nucleotides X2, N3 and N4, (iii) N1, N2, N3, and N4 each contain a single nucleotide comprising a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine. (iv)N1 forms a base pair with N4, (v)N2 forms a base pair with N3, (vi)N1 comprises a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof. (vii)X1 and X2 are oligonucleotides containing a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine, respectively. (viii) X1 is complementary to X2, (ix) X1 and X2 are each the same length, ranging from 12 to 16 nucleotides in length. (x)L is a non-nucleotide linker that covalently bonds the first polynucleotide and the second polynucleotide. Inosine, if present, forms a base pair with cytidine, N1 and N2 contain inosine, N3 and N4 contain cytidine, and X1 and / or X2 each contain at least one inosine. In some embodiments, N1 and N2 contain inosine, N3 and N4 contain cytidine, and X1 and X2 each contain at least one inosine, with X1 and X2 each being 12 nucleotides long. In some embodiments, N1 and N2 contain inosine, N3 and N4 contain cytidine, and X1 and X2 each contain at least one inosine. In some embodiments, N1 and N2 contain inosine, N3 and N4 contain cytidine, and X1 and X2 each contain at least one inosine, with X1 and X2 each being 12 nucleotides long. In some embodiments, N1 and N2 contain cytidine, N3 and N4 contain inosine, and X1 and / or X2 each contain at least one inosine. In some embodiments, N1 and N2 contain inosine, N3 and N4 contain cytidine, and X1 and X2 contain inosine but do not contain guanosine nucleoside. In some embodiments, N1 and N2 contain cytidine, N3 and N4 contain inosine, and X1 and X2 contain inosine but do not contain guanosine nucleoside. In some embodiments, the RLR agonists of the present disclosure have enhanced bioactivity, which is enhanced bioactivity including increased RLR-mediated cytokine production, increased RLR-mediated expression of interferon-inducible genes, increased RLR-mediated intracellular signaling, increased binding affinity to RLR, and any combination thereof.

[0036] In some embodiments, the RLR agonist of this disclosure is given by the following formula: The formula comprises 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', where X1 and X2 are each 12 nucleotides and contain 1, 2, 3, or 4 inosine nucleosides. In some embodiments, X1 and X2 are each 13 nucleotides and contain 1, 2, 3, 4, or 5 inosine nucleosides. In some embodiments, X1 and X2 are each 14 nucleotides and contain 1, 2, 3, 4, 5, or 6 inosine nucleosides. In some embodiments, X1 and X2 are each 15 nucleotides and contain 1, 2, 3, 4, 5, 6, or 7 inosine nucleosides. In some embodiments, X1 and X2 are each 16 nucleotides and contain 1, 2, 3, 4, 5, 6, 7, or 8 inosine nucleosides. In some embodiments, X1 and X2 are each 12 nucleotides and contain at least 10%, 20%, 30%, or 40% inosine nucleosides. In some embodiments, the RLR agonists of the present disclosure have enhanced bioactivity, which includes increased RLR-mediated cytokine production, increased RLR-mediated expression of interferon-inducible genes, increased RLR-mediated intracellular signaling, increased binding affinity to RLR, and any combination thereof.

[0037] In some embodiments, the present disclosure provides an RLR agonist that specifically binds to RLR, wherein the agonist is given by the following formula: It contains blunt-ended hairpin RNA including 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', in the formula, (i)(N1-N2-X1) comprises a first polynucleotide containing linked nucleotides N1, N2 and X1, (ii)(X2-N3-N4) comprises a second polynucleotide containing linked nucleotides X2, N3 and N4, (iii) N1, N2, N3, and N4 each contain a single nucleotide comprising a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine. (iv)N1 forms a base pair with N4, (v)N2 forms a base pair with N3, (vi)N1 comprises a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof. (vii)X1 is the array motif [AUCN5] x The formula includes, where N5 contains guanosine or inosine, and x is an integer whose value indicates the number of sequence motifs, where x = 3 or 4. (viii)X2 is the array motif [CN6AU] y The formula includes, where N6 contains guanosine or inosine, and y is an integer whose value indicates the number of sequence motifs, where y = 3 or 4. (ix)L is a linker that operably links the first polynucleotide and the second polynucleotide. In some cases, at least one of N1, N2, N3, and N4 is inosine, and the inosine nucleoside forms a base pair with cytidine in the hairpin RNA.

[0038] In some embodiments, N5 contains inosine and N6 contains inosine. In some embodiments, N5 contains guanosine and N6 contains inosine. In some embodiments, N5 contains inosine and N6 contains guanosine. In some embodiments, N5 contains guanosine (G) and N6 contains guanosine (G). In some embodiments, x=3 and y=3. In some embodiments, x=4 and y=4. In some embodiments, N1 contains inosine (I) and N4 contains cytidine (C). In some embodiments, N2 contains inosine (I) and N3 contains cytidine (C). In some embodiments, N3 contains inosine (I) and N2 contains cytidine (C). In some embodiments, N4 contains inosine (I) and N1 contains cytidine (C). In some embodiments, N1 contains guanosine (G). In some embodiments, N2 contains guanosine (G). In some embodiments, N1 comprises cytidine (C). In some embodiments, N2 comprises cytidine (C). In some embodiments, N1 and N2 comprises guanosine (G), and N3 and N4 comprises cytidine (C). In some embodiments, N1 and N2 comprises cytidine (C), and N3 and N4 comprises guanosine (G). In some embodiments, N1 and N2 comprises inosine (I), and N3 and N4 comprises cytidine (C). In some embodiments, N1 and N2 comprises cytidine (C), and N3 and N4 comprises inosine (I). In some embodiments, the RLR agonists of this disclosure have enhanced bioactivity, which is increased RLR-mediated cytokine production, increased RLR-mediated expression of interferon-inducible genes, increased RLR-mediated intracellular signaling, increased binding affinity to RLR, and any combination thereof.

[0039] In some embodiments of the RLR agonists of this disclosure, a linker is included, and the linker is either a nucleotide linker or a non-nucleotide linker. In some embodiments, the linker is a non-nucleotide linker. In some embodiments, the linker is a nucleotide linker. In some embodiments, the nucleotide linker includes a tetraloop, the nucleotide sequence of the tetraloop is as follows: (a) UNCG (wherein N = A, C, G, or U), (b) GNRA (wherein N = A, C, G or U, and R = A or G), (c) ANYA (wherein N = A, C, G or U, and Y = C or T), (d) CUYG (where Y = C or T), (e)UMAC (where M=A or C), and (f) Select from the group consisting of CUUG.

[0040] In some embodiments, the tetraloop arrangement is UUCG. In some embodiments, the tetraloop arrangement is GAUC.

[0041] In some embodiments, the RLR agonist of the present disclosure comprises a nucleotide linker comprising the nucleotide sequence UUUGAU or UGUUU. In some embodiments, the nucleotide linker comprises the nucleotide sequence UUUGAU. In some embodiments, the nucleotide linker comprises the nucleotide sequence UGUUU.

[0042] In some embodiments, the RLR agonists of this disclosure include a non-nucleotide linker, the non-nucleotide linker being: (a) Ethylene glycol linker, and (b) Selected from the group consisting of alkyl linkers.

[0043] In some embodiments, the non-nucleotide linker is a hexaethylene glycol linker. In some embodiments, the non-nucleotide linker is a C9 alkyl linker.

[0044] In some embodiments, the RLR agonist of the present disclosure comprises a 5'-diphosphate moiety, or a derivative or analog thereof. In some embodiments, the agonist comprises a 5'-triphosphate moiety, or a derivative or analog thereof. In some embodiments, the derivative or analog comprises a phosphate bioequivalent, which is selected from phosphonates, thiophosphonates, phosphorothioates, sulfates, sulfonates, sulfamates, thiazolidinones, carboxylates, malonates, boronic acids, benzoxabolols, boranophosphates, and squalamides.

[0045] In some embodiments, the agonist comprises a modified nucleotide, a modified nucleoside, or a modified nucleic acid base, or a combination thereof. In some embodiments, the agonist comprises modifications to internucleotide bonds or polynucleotide backbones.

[0046] In some embodiments, the RLR agonist of this disclosure has the following characteristics: (a) Specifically binds to one or more RLRs (e.g., RIG-1, MDA5 and / or LGP2), (b) Increase cytokine production mediated by RLR, (c) Increase the RLR-mediated expression of interferon-inducible genes (ISGs), (d) Increases RLR-dependent intracellular signaling, (e) To increase the stability of the double helix, (f) Increase binding affinity to RLR, (g) Reduce extratarget binding, (h) To lengthen the biological half-life, (i) To improve in vivo distribution and bioavailability, (j) Increase and / or enhance uptake into cells and / or tissues, (k) Reduce immunogenicity, and (l)(a)~(k) must be one or more of the combinations shown.

[0047] In some embodiments, the present disclosure provides an RLR agonist that specifically binds to RLR, wherein the agonist is given by the following formula: It contains blunt-ended hairpin RNA including 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', in the formula, (i)(N1-N2-X1) comprises a first polynucleotide containing linked nucleotides N1, N2 and X1, (ii)(X2-N3-N4) comprises a second polynucleotide containing linked nucleotides X2, N3 and N4, (iii) N1, N2, N3, and N4 each contain a single nucleotide comprising a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine. (iv)N1 forms a base pair with N4, (v)N2 forms a base pair with N3, (vi)N1 comprises a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof. (vii)X1 and X2 are oligonucleotides containing a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine, respectively. (viii) X1 is complementary to X2, (ix) X1 and X2 are each the same length, ranging from 12 to 16 nucleotides in length. (x)L is a linker that operably links the first polynucleotide and the second polynucleotide. N1 and N2 each contain guanosine, N3 and N4 each contain cytidine, X1 and X2 are each 12 nucleotides long, each contain at least one inosine nucleoside which forms a base pair with cytidine in the hairpin RNA, and L contains a nucleotide linker containing a tetraloop, the nucleotide sequence of the tetraloop is UUCG.

[0048] In some embodiments, the present disclosure provides an RLR agonist that specifically binds to RLR, wherein the agonist is given by the following formula: It contains blunt-ended hairpin RNA including 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', in the formula, (i)(N1-N2-X1) comprises a first polynucleotide containing linked nucleotides N1, N2 and X1, (ii)(X2-N3-N4) comprises a second polynucleotide containing linked nucleotides X2, N3 and N4, (iii) N1, N2, N3, and N4 each contain a single nucleotide comprising a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine. (iv)N1 forms a base pair with N4, (v)N2 forms a base pair with N3, (vi)N1 comprises a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof. (vii)X1 and X2 are oligonucleotides containing a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine, respectively. (viii) X1 is complementary to X2, (ix) X1 and X2 are each the same length, ranging from 12 to 16 nucleotides in length. (x)L is a linker that operably links the first polynucleotide and the second polynucleotide. N1 contains inosine, N2 contains guanosine, N3 and N4 each contain cytidine, X1 and X2 are each 12 nucleotides long, each contain at least one inosine nucleoside, the inosine nucleoside forms a base pair with cytidine in the hairpin RNA, L contains a nucleotide linker containing a tetraloop, the nucleotide sequence of the tetraloop is UUCG.

[0049] In some embodiments, the present disclosure provides an RLR agonist that specifically binds to RLR, wherein the agonist is given by the following formula: It contains blunt-ended hairpin RNA including 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', in the formula, (i)(N1-N2-X1) comprises a first polynucleotide containing linked nucleotides N1, N2 and X1, (ii)(X2-N3-N4) comprises a second polynucleotide containing linked nucleotides X2, N3 and N4, (iii) N1, N2, N3, and N4 each contain a single nucleotide comprising a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine. (iv)N1 forms a base pair with N4, (v)N2 forms a base pair with N3, (vi)N1 comprises a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof. (vii)X1 and X2 are oligonucleotides containing a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine, respectively. (viii) X1 is complementary to X2, (ix) X1 and X2 are each the same length, ranging from 12 to 16 nucleotides in length. (x)L is a linker that operably links the first polynucleotide and the second polynucleotide. N1 and N2 contain inosine, N3 and N4 contain cytidine, X1 and X2 are each 12 nucleotides long, each containing at least one inosine nucleoside which forms a base pair with cytidine in the hairpin RNA, and L contains a nucleotide linker containing a tetraloop, the nucleotide sequence of the tetraloop is UUCG.

[0050] In some embodiments, the present disclosure provides an RLR agonist that specifically binds to RLR, the agonist comprising a blunt-terminated hairpin RNA containing a non-nucleotide linker, and the agonist has the following formula: The formula includes 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', and in the formula, (i)(N1-N2-X1) comprises a first polynucleotide containing linked nucleotides N1, N2 and X1, (ii)(X2-N3-N4) comprises a second polynucleotide containing linked nucleotides X2, N3 and N4, (iii) N1, N2, N3, and N4 each contain a single nucleotide comprising a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine. (iv)N1 forms a base pair with N4, (v)N2 forms a base pair with N3, (vi)N1 comprises a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof. (vii)X1 and X2 are oligonucleotides containing a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine, respectively. (viii) X1 is complementary to X2, (ix) X1 and X2 are each the same length, ranging from 12 to 16 nucleotides in length. (x)L is a non-nucleotide linker that covalently bonds the first polynucleotide and the second polynucleotide. N1 and N2 contain guanosine, N3 and N4 contain cytidine, X1 and X2 are each 12 nucleotides long, and the non-nucleotide linker is a C9 alkyl linker.

[0051] In some embodiments, the present disclosure provides an RLR agonist that specifically binds to RLR, the agonist comprising a blunt-terminated hairpin RNA containing a non-nucleotide linker, and the agonist has the following formula: The formula includes 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', and in the formula, (i)(N1-N2-X1) comprises a first polynucleotide containing linked nucleotides N1, N2 and X1, (ii)(X2-N3-N4) comprises a second polynucleotide containing linked nucleotides X2, N3 and N4, (iii) N1, N2, N3, and N4 each contain a single nucleotide comprising a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine. (iv)N1 forms a base pair with N4, (v)N2 forms a base pair with N3, (vi)N1 comprises a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof. (vii)X1 and X2 are oligonucleotides containing a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine, respectively. (viii) X1 is complementary to X2, (ix) X1 and X2 are each the same length, ranging from 12 to 16 nucleotides in length. (x)L is a non-nucleotide linker that covalently bonds the first polynucleotide and the second polynucleotide. N1 and N2 contain guanosine, N3 and N4 contain cytidine, X1 and X2 are each 12 nucleotides long, and the non-nucleotide linker is a hexaethylene glycol linker.

[0052] In some embodiments, the Disclosure provides an RLR agonist that specifically binds to RLR, wherein the 5' terminal nucleotide of the agonist comprises a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof, and the agonist comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, and 36.

[0053] In some embodiments, the Disclosure provides an RLR agonist that specifically binds to an RLR, the agonist comprising a blunt-terminated hairpin RNA containing a first polynucleotide linked to a second polynucleotide by a linker, the first polynucleotide being sufficiently complementary to the second polynucleotide and forming a double helix, the double helix containing fewer than 19 base pairs, the 5' terminal nucleotide of the first oligonucleotide comprising a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof, the agonist comprising a sequence motif, the sequence motif conferring at least one improved RLR-mediated bioactivity compared to an agonist without this sequence motif, the first polynucleotide and the second polynucleotide being: (i) Sequence IDs 37 and 68, respectively (ii) Sequence IDs 38 and 69, respectively (iii) Sequence IDs 39 and 70, respectively (iv) Sequence IDs 40 and 71, respectively (v) Sequence IDs 41 and 72, respectively (vi) Sequence IDs 42 and 73, respectively (vii) Sequence numbers 43 and 74, respectively (viii) Sequence IDs 44 and 75, respectively (ix) Sequence numbers 45 and 76, respectively (x) Sequence numbers 46 and 77, respectively (xi) Sequence IDs 47 and 78, respectively (xii) Sequence IDs 48 and 79, respectively (xiii) Sequence IDs 49 and 80, respectively (xiv) Sequence IDs 50 and 81, respectively (xv) Sequence IDs 51 and 82, respectively (xvi) Sequence numbers 52 and 83, respectively (xvii) Sequence IDs 53 and 84, respectively (xviii) Sequence IDs 54 and 85, respectively (xix) Sequence numbers 55 and 86, respectively. (xx) Sequence numbers 56 and 87, respectively. (xxi) Sequence numbers 57 and 88, respectively. (xxii) Sequence IDs 58 and 89, respectively. (xxiii) Sequence numbers 59 and 89, respectively (xxiv) Sequence IDs 60 and 90, respectively (xxv) Sequence numbers 61 and 91, respectively. (xxvi) Sequence IDs 62 and 92, respectively (xxvii) Sequence numbers 63 and 91, respectively. (xxviii) Sequence numbers 64 and 93, respectively. (xxix) Sequence numbers 65 and 94, respectively. (xxx) Sequence numbers 66 and 95, respectively. (xxxi) Sequence numbers 67 and 96, respectively, and (xxxii) Each contains a nucleotide sequence selected from the group consisting of SEQ ID NOs. 63 and 97.

[0054] In some embodiments, the Disclosure provides RLR agonists that specifically bind to RLRs, wherein the agonist comprises a blunt-terminated hairpin RNA comprising at least one nucleotide including an inosine that forms a base pair with cytidine, and the agonist comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 22, 23, and 25.

[0055] In some embodiments, the Disclosure provides an RLR agonist that specifically binds to RLR, the agonist comprising a blunt-terminated hairpin RNA comprising at least one nucleotide containing inosine that forms a base pair with cytidine, the agonist comprising the formula 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', where (N1-N2-X1) comprises a first polynucleotide, (X2-N3-N4) comprises a second polynucleotide, and the first and second polynucleotides are as follows: (i) Sequence IDs 58 and 89, respectively (ii) Sequence IDs 59 and 89, respectively, and (iii) Each contains a nucleotide sequence selected from the group consisting of SEQ ID NOs. 61 and 91.

[0056] In some embodiments, the Disclosure provides an RLR agonist that specifically binds to RLR, the agonist comprising a blunt-terminated hairpin RNA containing a non-nucleotide linker, the agonist comprising the formula 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', where (N1-N2-X1) comprises a first polynucleotide, (X2-N3-N4) comprises a second polynucleotide, and the first and second polynucleotides are as follows: (i) Sequence IDs 37 and 68, respectively (ii) Sequence IDs 38 and 69, respectively (iii) Sequence IDs 39 and 70, respectively (iv) Sequence IDs 40 and 71, respectively (v) Sequence IDs 41 and 72, respectively (vi) Sequence IDs 42 and 73, respectively (vii) Sequence numbers 43 and 74, respectively (viii) Sequence IDs 44 and 75, respectively (ix) Sequence numbers 45 and 76, respectively (x) Sequence numbers 46 and 77, respectively (xi) Sequence IDs 47 and 78, respectively (xii) Sequence IDs 48 and 79, respectively (xiii) Sequence IDs 49 and 80, respectively (xiv) Sequence IDs 50 and 81, respectively (xv) Sequence IDs 51 and 82, respectively (xvi) Sequence numbers 52 and 83, respectively (xvii) Sequence IDs 53 and 84, respectively (xviii) Sequence IDs 54 and 85, respectively (xix) Sequence numbers 55 and 86, respectively. (xx) Sequence numbers 56 and 87, respectively. (xxi) Sequence numbers 57 and 88, respectively. (xxii) Sequence IDs 58 and 89, respectively. (xxiii) Sequence numbers 59 and 89, respectively (xxiv) Sequence IDs 60 and 90, respectively (xxv) Sequence numbers 61 and 91, respectively. (xxvi) Sequence IDs 62 and 92, respectively (xxvii) Sequence numbers 63 and 91, respectively. (xxviii) Sequence numbers 64 and 93, respectively. (xxix) Sequence numbers 65 and 94, respectively. (xxx) Sequence numbers 66 and 95, respectively. (xxxi) Sequence numbers 67 and 96, respectively, and (xxxii) Each contains a nucleotide sequence selected from the group consisting of SEQ ID NOs. 63 and 97.

[0057] In some embodiments of the RLR agonists provided by this disclosure, the nucleotide sequence containing the RLR agonist is not complementary to the genomic DNA sequence or mRNA sequence, the RLR agonist does not participate in RNA interference, and the RLR agonist does not silence gene expression.

[0058] In some embodiments, the Disclosure provides pharmaceutical compositions for stimulating an immune response to treat or slow the progression of cancer, or reduce or inhibit tumor growth in subjects where such treatment is required, comprising an RLR agonist provided by the Disclosure and a pharmaceutically acceptable carrier. In some embodiments, the RLR agonist is formulated in a polyethyleneimine (PEI) carrier. In some embodiments, the PEI carrier is JetPEI®.

[0059] In some embodiments, the Disclosure provides a method for increasing the RLR-mediated production of one or more cytokines in cells, the method comprising contacting cells with an RLR agonist provided by the Disclosure, wherein the RLR agonist increases the RLR-mediated cytokine production in cells. In some embodiments, the RLR agonist increases the RLR-mediated production of type I interferons (e.g., IFN-α, IFN-β) in cells. In some embodiments, the RLR agonist increases the RLR-mediated production of IL-1β in cells. In some embodiments, the RLR agonist increases the RLR-mediated production of IP-10 in cells. In some embodiments, the RLR agonist increases the RLR-mediated production of IL-6, IL-12p70, MCP-1, and / or MIP-1β in cells.

[0060] In some embodiments, the present disclosure provides a method for increasing the RLR-mediated expression of one or more interferon-inducible genes in cells, the method comprising contacting cells with an RLR agonist provided by the present disclosure, wherein the agonist increases the RLR-mediated expression of one or more interferon-inducible genes in cells.

[0061] In some embodiments, the present disclosure provides a method for increasing RLR-dependent intracellular signaling in cells, the method comprising contacting cells with an RLR agonist provided by the present disclosure, wherein the agonist increases RLR-dependent intracellular signaling.

[0062] In some embodiments, the Disclosure provides a method for stimulating an immune response in a subject, the method comprising administering to the subject an effective amount of an RLR agonist or pharmaceutical composition provided by the Disclosure.

[0063] In some embodiments, the Disclosure provides a method for treating or slowing the progression of cancer in a subject, the method comprising administering to the subject an effective amount of an RLR agonist or pharmaceutical composition provided by the Disclosure.

[0064] In some embodiments, the Disclosure provides a method for doing so in a subject where it is necessary to reduce or inhibit tumor growth, the method comprising administering to the subject an effective amount of an RLR agonist or pharmaceutical composition provided by the Disclosure.

[0065] In some embodiments, the present disclosure provides a method for stimulating an immune response in subjects that require treatment of cancer, treatment of cancer progression, or inhibition of tumor growth, the method comprising administering to a subject an effective amount of an RLR agonist or pharmaceutical composition provided by the present disclosure, wherein the agonist or pharmaceutical composition increases the RLR-mediated production of one or more cytokines in cells, increases the RLR-mediated expression of one or more interferon-inducible genes in cells, and / or increases RLR-dependent intracellular signaling in cells, thereby stimulating an immune response, treatment of cancer, treatment of cancer progression, or inhibition of tumor growth.

[0066] In some embodiments of the methods provided herein, the RLR agonist or pharmaceutical composition provided herein is administered in combination with one or more further therapeutic agents, the one or more further therapeutic agents being selected from the group consisting of chemotherapy, targeted anticancer therapy, oncolytic agents, cell death inducers, opsonizing agents (e.g., opsonized antibodies), cytotoxic agents, immunotherapy, cytokines, activators or agonists of costimulatory molecules, inhibitors of inhibitory molecules, vaccines, cellular immunotherapy, or combinations thereof.

[0067] In some embodiments, the RLR agonist or pharmaceutical composition provided herein is administered before or after the administration of one or more further therapeutic agents, or one or more further therapeutic agents are administered concurrently with, before, or after the administration of the agonist or pharmaceutical composition.

[0068] In some embodiments, one or more further therapeutic agents are PD-1 / PD-L1 antagonists, TIM-3 antagonists, VISTA antagonists, adenosine A2AR antagonists, B7-H3 antagonists, B7-H4 antagonists, BTLA antagonists, CTLA-4 antagonists, IDO antagonists, KIR antagonists, LAG-3 antagonists, Toll-like receptor 3 (TLR3) agonists, Toll-like receptor 7 (TLR7) agonists, and Toll-like receptor 9 (TLR9) agonists.

[0069] In some embodiments, one or more further therapeutic agents are agonists comprising polypeptides (e.g., antibodies, or their antigen-binding moieties) that specifically bind to CD137(4-1BB).

[0070] In some embodiments, one or more further therapeutic agents are agonists comprising polypeptides (e.g., antibodies, or their antigen-binding moieties) that specifically bind to CD134(OX40).

[0071] In some embodiments, one or more additional therapeutic agents are PD-1 / PD-L1 antagonists. In some embodiments, the PD-1 / PD-L1 antagonist is selected from the group consisting of PDR001, KEYTRUDA® (pembrolizumab), OPDIVO® (nivolumab), pidilizumab, MEDI0680, REGN2810, TSR-042, PF-06801591, and AMP-224. In some embodiments, the PD-1 / PD-L1 antagonist is selected from the group consisting of FAZ053, TENCENTRIQ® (atezolizumab), BAVENCIO® (avelumab), IMFINZI® (durvalumab), and BMS-936559.

[0072] In some embodiments, one or more additional therapeutic agents are TIM-3 antagonists.

[0073] In some embodiments, one or more additional therapeutic agents are VISTA antagonists.

[0074] In some embodiments, one or more further therapeutic agents are adenosine A2AR antagonists.

[0075] In some embodiments, one or more additional therapeutic agents are B7-H3 antagonists.

[0076] In some embodiments, one or more additional therapeutic agents are B7-H4 antagonists.

[0077] In some embodiments, one or more further therapeutic agents are BTLA antagonists.

[0078] In some embodiments, one or more additional therapeutic agents are CTLA-4 antagonists.

[0079] In some embodiments, one or more further therapeutic agents are IDO antagonists.

[0080] In some embodiments, one or more additional therapeutic agents are KIR antagonists.

[0081] In some embodiments, one or more additional therapeutic agents are LAG-3 antagonists.

[0082] In some embodiments, one or more further therapeutic agents are Toll-like receptor 3 (TLR3) agonists. In some embodiments, the TLR3 agonist is polyinosinic acid:polycytidylic acid (Poly I:C). In some embodiments, the TLR3 agonist is HILTONOL® (Poly ICLC). In some embodiments, the TLR3 agonist is polyadenylic acid-polyuridylic acid (Poly A:U). In some embodiments, the TLR3 agonist is RIBOXXIM® (RGIC® 100). In some embodiments, the TLR3 agonist is RIBOXXON® (RGIC® 50 bioconjugate). In some embodiments, the TLR3 agonist is RIBOXXOL® (RGIC® 50).

[0083] In some embodiments, one or more further therapeutic agents are Toll-like receptor 7 (TLR7) agonists. In some embodiments, the TLR7 agonist is GS-9620 (besatrimod). In some embodiments, the TLR7 agonist is imiquimod (ALDARA®). In some embodiments, the TLR7 agonist is reximod (R-848).

[0084] In some embodiments, one or more further therapeutic agents are Toll-like receptor 9 (TLR9) agonists. In some embodiments, the TLR9 agonist is a CpG oligodeoxynucleotide (CpG ODN). In some embodiments, the CpG ODN is a class A CpG ODN (CpG-A ODN). In some embodiments, the CpG ODN is a class B CpG ODN (CpG-B ODN). In some embodiments, the CpG ODN is a class C CpG ODN (CpG-C ODN).

[0085] In some embodiments, the Disclosure provides the use of RLR agonists or pharmaceutical compositions provided herein for use in combination, optionally with one or more further therapeutic agents, to stimulate an immune response and to treat or slow the progression of cancer or inhibit tumor growth in subjects where such treatment is required.

[0086] In some embodiments, the Disclosure provides the use of RLR agonists or pharmaceutical compositions provided herein in the manufacture of pharmaceuticals for use in combination with one or more further therapeutic agents, to stimulate an immune response and to do so in subjects where it is necessary to treat cancer, slow its progression, or inhibit tumor growth.

[0087] In some embodiments, the Disclosure provides a kit comprising an RLR agonist or pharmaceutical composition provided herein, instructions for use in stimulating an immune response in a subject, treating or slowing the progression of cancer, or inhibiting tumor growth in a subject, and optionally instructions for use in combination with one or more further therapeutic agents.

[0088] In some embodiments of the uses or kits provided herein, the RLR agonist or pharmaceutical composition provided herein is administered in combination with one or more further therapeutic agents, one or more of which are selected from the group consisting of chemotherapy, targeted anticancer therapy, oncolytic agents, cell death inducers, opsonizing agents (e.g., opsonized antibodies), cytotoxic agents, immunotherapy, cytokines, activators of costimulatory molecules, inhibitors of inhibitory molecules, vaccines, cellular immunotherapy, or combinations thereof.

[0089] In some embodiments of the use or kit provided by this disclosure, the RLR agonist or pharmaceutical composition provided by this disclosure is administered before or after the administration of one or more further therapeutic agents, or one or more further therapeutic agents are administered concurrently with, before or after the administration of the agonist or pharmaceutical composition.

[0090] In some embodiments of the uses or kits provided by this disclosure, one or more further therapeutic agents are PD-1 / PD-L1 antagonists, TIM-3 antagonists, VISTA antagonists, adenosine A2AR antagonists, B7-H3 antagonists, B7-H4 antagonists, BTLA antagonists, CTLA-4 antagonists, IDO antagonists, KIR antagonists, LAG-3 antagonists, Toll-like receptor 3 (TLR3) agonists, Toll-like receptor 7 (TLR7) agonists, and Toll-like receptor 9 (TLR9) agonists.

[0091] In some embodiments of the uses or kits provided by this disclosure, one or more further therapeutic agents are agonists comprising a polypeptide (e.g., an antibody, or its antigen-binding moiety) that specifically binds to CD137(4-1BB).

[0092] In some embodiments of the uses or kits provided herein, one or more further therapeutic agents of the herein are agonists comprising a polypeptide (e.g., an antibody, or its antigen-binding portion) that specifically binds to CD134(OX40). A patent or application file must include at least one drawing made in color. A copy of this patent or patent application publication, including the color drawing, will be provided by the Office upon request and payment of the necessary fees. The present invention provides, for example, the following items. (Item 1) A synthetic RIG-I-like receptor (RLR) agonist that specifically binds to a RIG-I-like receptor (RLR), wherein the agonist comprises a blunt-terminated hairpin RNA having a first polynucleotide linked to a second polynucleotide by a linker, the first polynucleotide being sufficiently complementary to the second polynucleotide and forming a double helix, the double helix containing fewer than 19 base pairs, the 5' terminal nucleotide of the first oligonucleotide comprising a 5' diphosphate or tripphosphate moiety, or a derivative or analog thereof, the agonist comprising a sequence motif, the sequence motif providing at least one improved bioactivity mediated by the RLR compared to an agonist without the sequence motif. (Item 2) The aforementioned array motif, (i) GT repeat motif, (ii) GA repeat motif, (iii) AUCG repeat motif, (iv) AU repeat motif, (v) Dipyrimidine motif, (vi) Ziplin motif, (vii) Pyrimidine triplet motif, (viii) Printed riplet motif, (ix) Palindromic arrangement motifs, and An agonist selected from the group consisting of any combination of (x)(i)~(ix), as described in item 1. (Item 3) The at least one improved bioactivity described above is (i) Increased cytokine production mediated by RLR, (ii) Increased expression of interferon-inducible genes mediated by RLR, (iii) Increased intracellular signaling mediated by RLRs, (iv) Increased binding affinity to RLR, and (v)(i)~(iv) A combination of the agonists listed in items 1 and 2. (Item 4) The agonists described in items 1-3, wherein the sequence motif is a GT repeat motif comprising a sequence of less than 19, approximately 15-18, approximately 15, approximately 10-15, approximately 10, approximately 5-10, approximately 5, approximately 4, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or 4 guanine and thymine nucleotides, or derivatives or analogs thereof. (Item 5) The aforementioned GT repeat motif is [GT] n The agonist described in item 4, where n=2 to 9 in the formula. (Item 6) The agonists described in items 4 and 5, wherein the aforementioned GT repeat motif is [GT]7. (Item 7) The agonist described in items 4 and 5, wherein the GT repeat motif is [GT]3, and the GT repeat motif is followed by a print repeat motif and a UCG, respectively. (Item 8) The agonist described in item 7, wherein the aforementioned print riplet motif is GGA. (Item 9) The agonists described in items 1-3, wherein the sequence motif is a GA repeat motif comprising a sequence of less than 19, approximately 15-18, approximately 15, approximately 10-15, approximately 10, approximately 5-10, approximately 5, approximately 4, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or 4 guanine and adenine nucleotides, or derivatives or analogs thereof. (Item 10) The aforementioned GA repeat motif is [GA] n The agonist described in item 9, where n=2 to 9 in the formula. (Item 11) The agonist described in item 10, wherein the aforementioned GA repeat motif is [GA]7. (Item 12) The agonist according to any one of items 1 to 3, wherein the array motif comprises an AUCG repeat motif of less than 19, about 16, about 12 to 16, about 12, about 8 to 12, about 6, 16, 12, 8 adenine, uracil, cytosine and guanine nucleotides, or a sequence of derivatives or analogs thereof. (Item 13) The AUCG repeat motif is [AUCG] n and, in the formula, n = 2 to 4, the agonist according to item 12. (Item 14) The agonist according to item 13, wherein the AUCG repeat motif is [AUCG]3. (Item 15) The agonist according to any one of items 12 to 14, wherein a CG or dipyrimidine motif is present before the AUCG repeat motif. (Item 16) The agonist according to item 15, wherein a CG is present before the AUCG repeat motif. (Item 17) The agonist according to item 15, wherein the dipyrimidine motif is CC. (Item 18) The agonist according to any one of items 12 to 14, wherein a di-purine motif is present before the AUCG repeat motif. (Item 19) The agonist according to item 18, wherein the di-purine motif is GA. (Item 20) The agonist according to item 18, wherein the di-purine motif is II. (Item 21) The agonist according to any one of items 12 to 20, wherein the U constituting the AUCG repeat motif is replaced with a modified nucleoside. (Item 22) The agonist according to item 21, wherein the modified nucleoside is ribothymidine (T). (Item 23) The agonist according to any one of items 12 to 20, wherein the G constituting the AUCG repeat motif is replaced with a modified nucleoside. (Item 24) The agonist described in item 23, wherein the modified nucleoside is inosine(I). (Item 25) Agonists as described in items 12-14, where IG exists before the aforementioned AUGG repeat motif. (Item 26) The agonists described in items 12-14, wherein the G constituting the AUCG repeat motif is replaced with inosine (I), and inosine (I) is present before the AUCG repeat. (Item 27) The agonist described in item 26, wherein the 5' terminal nucleotide of the first polynucleotide is inosine (I). (Item 28) The agonists described in items 12 and 13, wherein the aforementioned AUCG repeat motif is [AUCG]2. (Item 29) The agonist described in item 28, in which a Ziplin motif precedes the aforementioned AUCG repeat motif. (Item 30) The agonist described in item 29, wherein the aforementioned Ziplin motif is GG. (Item 31) The agonist described in item 28, in which a printed replet precedes the aforementioned AUCG repeat motif. (Item 32) The agonist described in item 31, wherein the aforementioned print replet is GGG. (Item 33) The agonist described in item 28, in which CCCCCG is present before the aforementioned AUCG repeat motif. (Item 34) The agonist described in item 28, in which TCGUCG is present before the aforementioned AUCG repeat motif. (Item 35) The agonists described in items 1 to 3, wherein the sequence motif comprises a sequence of fewer than 19, approximately 15 to 18, approximately 15, approximately 10 to 15, approximately 10, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or 4 nucleotides, or derivatives or analogs thereof, and is a palindromic sequence formed by concatenating these in any order that generates a palindrome. (Item 36) An agonist according to any one of items 1 to 35, wherein the linker is adjacent to the AU. (Item 37) The linker is adjacent to the AU repeat motif, and the AU repeat motif is [AU] n The agonist is one of the items 1 to 36, where n = 2 to 3 in the formula. (Item 38) The agonist described in item 37, wherein the aforementioned AU repeat motif is [AU]2. (Item 39) A synthetic RIG-I-like receptor (RLR) agonist that specifically binds to RIG-I-like receptors (RLRs), wherein the agonist has the following formula: It contains blunt-ended hairpin RNA including 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', in the formula, (i)(N1-N2-X1) comprises a first polynucleotide containing linked nucleotides N1, N2 and X1, (ii)(X2-N3-N4) comprises a second polynucleotide containing linked nucleotides X2, N3 and N4, (iii) N1, N2, N3, and N4 each contain a single nucleotide comprising a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine. (iv)N1 forms a base pair with N4, (v)N2 forms a base pair with N3, (vi)N1 comprises a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof. (vii)X1 and X2 are oligonucleotides containing a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine, respectively. (viii) X1 is complementary to X2, (ix) X1 and X2 are each the same length, ranging from 12 to 16 nucleotides in length. (x)L is a linker that operably links the first polynucleotide and the second polynucleotide, A synthetic RIG-I-like receptor (RLR) agonist in which at least one of N1, N2, N3, and N4 is inosine, and / or at least one of X1 and / or X2 comprises at least one inosine nucleoside, the inosine nucleoside forming a base pair with cytidine in the hairpin RNA. (Item 40) An agonist as described in item 39, wherein N1 contains inosine and N4 contains cytidine. (Item 41) An agonist as described in item 39, wherein N1 contains cytidine and N4 contains inosine. (Item 42) An agonist as described in item 39, wherein N2 contains inosine and N3 contains cytidine. (Item 43) An agonist as described in item 39, wherein N2 contains cytidine and N3 contains inosine. (Item 44) N1 is an agonist containing guanosine, as described in item 39. (Item 45) N2 is an agonist containing guanosine, as described in item 39. (Item 46) N1 is an agonist containing cytidine, as described in item 39. (Item 47) N2 is an agonist containing cytidine, as described in item 39. (Item 48) The agonist described in item 39, wherein N1 and N2 contain guanosine, and N3 and N4 contain cytidine. (Item 49) The agonist described in item 39, wherein N1 and N2 contain cytidine, and N3 and N4 contain guanosine. (Item 50) The agonist described in item 39, wherein N1 and N2 contain inosine, and N3 and N4 contain cytidine. (Item 51) The agonist described in item 39, wherein N1 and N2 contain cytidine, and N3 and N4 contain inosine. (Item 52) An agonist as described in item 39, wherein N1 contains inosine, N4 contains cytidine, and X1 and / or X2 each contain at least one inosine. (Item 53) The agonist described in item 39, wherein N2 contains inosine, N3 contains cytidine, and X1 and / or X2 each contain at least one inosine. (Item 54) An agonist as described in item 39, wherein N1 and N2 contain guanosine, N3 and N4 contain cytidine, and X1 and / or X2 each contain at least one inosine. (Item 55) The agonist described in item 39, wherein N1 and N2 contain guanosine, N3 and N4 contain cytidine, and X1 and X2 each contain at least one inosine. (Item 56) The agonist described in item 39, wherein N1 and N2 contain cytidine, N3 and N4 contain guanosine, and X1 and X2 each contain at least one inosine. (Item 57) An agonist as described in item 39, wherein N1 and N2 contain guanosine, N3 and N4 contain cytidine, and X1 and X2 each contain inosine, and no guanosine nucleoside is present. (Item 58) An agonist as described in item 39, wherein N1 and N2 contain cytidine, N3 and N4 contain guanosine, and X1 and X2 each contain inosine, and no guanosine nucleoside is present. (Item 59) The agonist described in item 39, wherein N1 and N2 contain inosine, N3 and N4 contain cytidine, and X1 and / or X2 each contain at least one inosine. (Item 60) The agonist described in item 39, wherein N1 and N2 contain inosine, N3 and N4 contain cytidine, and X1 and X2 each contain at least one inosine. (Item 61) The agonist described in item 39, wherein N1 and N2 contain cytidine, N3 and N4 contain inosine, and X1 and / or X2 each contain at least one inosine. (Item 62) An agonist as described in item 39, wherein N1 and N2 contain inosine, N3 and N4 contain cytidine, X1 and X2 contain inosine, and do not contain guanosine nucleoside. (Item 63) An agonist as described in item 39, wherein N1 and N2 contain cytidine, N3 and N4 contain inosine, X1 and X2 contain inosine, and no guanosine nucleoside is present. (Item 64) An agonist as described in item 39, wherein X1 and X2 each consist of 12 nucleotides and contain 1, 2, 3, or 4 inosine nucleosides. (Item 65) An agonist as described in item 39, wherein X1 and X2 each consist of 13 nucleotides and contain 1, 2, 3, 4, or 5 inosine nucleosides. (Item 66) An agonist as described in item 39, wherein X1 and X2 each consist of 14 nucleotides and contain 1, 2, 3, 4, 5, or 6 inosine nucleosides. (Item 67) An agonist as described in item 39, wherein X1 and X2 each consist of 15 nucleotides and contain 1, 2, 3, 4, 5, 6, or 7 inosine nucleosides. (Item 68) An agonist as described in item 39, wherein X1 and X2 each consist of 16 nucleotides, each containing 1, 2, 3, 4, 5, 6, 7, or 8 inosine nucleosides. (Item 69) An agonist as described in item 39, wherein X1 and X2 each consist of 12 nucleotides and contain at least 10%, 20%, 30%, or 40% inosine nucleosides. (Item 70) A synthetic RIG-I-like receptor (RLR) agonist that specifically binds to RIG-I-like receptors (RLRs), wherein the agonist has the following formula: It contains blunt-ended hairpin RNA including 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', in the formula, (i)(N1-N2-X1) comprises a first polynucleotide containing linked nucleotides N1, N2 and X1, (ii)(X2-N3-N4) comprises a second polynucleotide containing linked nucleotides X2, N3 and N4, (iii) N1, N2, N3, and N4 each contain a single nucleotide comprising a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine. (iv)N1 forms a base pair with N4, (v)N2 forms a base pair with N3, (vi)N1 comprises a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof. (vii)X1 is the array motif [AUCN5] x The formula includes, where N5 contains guanosine or inosine, and x is an integer whose value indicates the number of sequence motifs, where x = 3 or 4. (viii)X2 is the array motif [CN6AU] y The formula includes, where N6 contains guanosine or inosine, and y is an integer whose value indicates the number of sequence motifs, where y = 3 or 4. (ix)L is a linker that operably links the first polynucleotide and the second polynucleotide, A synthetic RIG-I-like receptor (RLR) agonist in which, in some cases, at least one of N1, N2, N3, and N4 is inosine, and the inosine nucleoside forms a base pair with cytidine in the hairpin RNA. (Item 71) The agonists listed in item 70, N5 containing inosine and N6 containing inosine. (Item 72) An agonist as described in item 71, wherein N5 contains guanosine and N6 contains inosine. (Item 73) An agonist as described in item 72, wherein N5 contains inosine and N6 contains guanosine. (Item 74) An agonist as described in item 73, wherein N5 contains guanosine (G) and N6 contains guanosine (G). (Item 75) An agonist described in any one of items 70-74, where x=3 and y=3. (Item 76) An agonist described in any one of items 70-74, where x=4 and y=4. (Item 77) An agonist as described in any one of items 70-74, wherein N1 contains inosine (I) and N4 contains cytidine (C). (Item 78) An agonist as described in any one of items 70-74, wherein N2 contains inosine (I) and N3 contains cytidine (C). (Item 79) An agonist as described in any one of items 70-74, wherein N3 contains inosine (I) and N2 contains cytidine (C). (Item 80) An agonist as described in any one of items 70-74, wherein N4 contains inosine (I) and N1 contains cytidine (C). (Item 81) N1 is an agonist containing guanosine (G), as described in any one of items 70-74. (Item 82) N2 is an agonist containing guanosine (G), as described in any one of items 70-74. (Item 83) An agonist according to any one of items 70-74, wherein N1 contains cytidine (C). (Item 84) N2 is an agonist containing cytidine (C), as described in any one of items 70-74. (Item 85) An agonist according to any one of items 70 to 74, wherein N1 and N2 contain guanosine (G) and N3 and N4 contain cytidine (C). (Item 86) An agonist according to any one of items 70 to 74, wherein N1 and N2 contain cytidine (C) and N3 and N4 contain guanosine (G). (Item 87) An agonist according to any one of items 70 to 74, wherein N1 and N2 contain inosine (I) and N3 and N4 contain cytidine (C). (Item 88) An agonist according to any one of items 70 to 74, wherein N1 and N2 contain cytidine (C) and N3 and N4 contain inosine (I). (Item 89) A synthetic RIG-I-like receptor (RLR) agonist that specifically binds to a RIG-I-like receptor (RLR), wherein the agonist comprises a blunt-terminated hairpin RNA containing a non-nucleotide linker, and the agonist has the following formula: The formula includes 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', and in the formula, (i)(N1-N2-X1) comprises a first polynucleotide containing linked nucleotides N1, N2 and X1, (ii)(X2-N3-N4) comprises a second polynucleotide containing linked nucleotides X2, N3 and N4, (iii) N1, N2, N3, and N4 each contain a single nucleotide comprising a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine. (iv)N1 forms a base pair with N4, (v)N2 forms a base pair with N3, (vi)N1 comprises a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof. (vii)X1 and X2 are oligonucleotides containing a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine, respectively. (viii) X1 is complementary to X2, (ix) X1 and X2 are each the same length, ranging from 12 to 16 nucleotides in length. (x)L is a non-nucleotide linker that covalently bonds the first polynucleotide and the second polynucleotide, Inosine is a synthetic RIG-I-like receptor (RLR) agonist that forms a base pair with cytidine when present. (Item 90) An agonist as described in item 89, wherein N1 contains inosine and N4 contains cytidine. (Item 91) An agonist as described in item 89, wherein N1 contains cytidine and N4 contains inosine. (Item 92) An agonist as described in item 89, wherein N2 contains inosine and N3 contains cytidine. (Item 93) An agonist as described in item 89, wherein N2 contains cytidine and N3 contains inosine. (Item 94) N1 is an agonist containing guanosine, as described in item 89. (Item 95) N2 is an agonist containing guanosine, as described in item 89. (Item 96) N1 is an agonist containing cytidine, as described in item 89. (Item 97) N2 is an agonist containing cytidine, as described in item 89. (Item 98) The agonist described in item 89, wherein N1 and N2 contain guanosine, and N3 and N4 contain cytidine. (Item 99) The agonist described in item 89, wherein N1 and N2 contain cytidine, and N3 and N4 contain guanosine. (Item 100) The agonist described in item 89, wherein N1 and N2 contain inosine, and N3 and N4 contain cytidine. (Item 101) The agonist described in item 89, wherein N1 and N2 contain cytidine, and N3 and N4 contain inosine. (Item 102) An agonist as described in item 89, wherein N1 contains inosine, N4 contains cytidine, and X1 and / or X2 each contain at least one inosine. (Item 103) The agonist described in item 89, wherein N2 contains inosine, N3 contains cytidine, and X1 and / or X2 each contain at least one inosine. (Item 104) An agonist as described in item 89, wherein N1 and N2 contain guanosine, N3 and N4 contain cytidine, and X1 and / or X2 each contain at least one inosine. (Item 105) The agonist described in item 89, wherein N1 and N2 contain guanosine, N3 and N4 contain cytidine, and X1 and X2 each contain at least one inosine. (Item 106) The agonist described in item 89, wherein N1 and N2 contain cytidine, N3 and N4 contain guanosine, and X1 and X2 each contain at least one inosine. (Item 107) An agonist as described in item 89, wherein N1 and N2 contain guanosine, N3 and N4 contain cytidine, and X1 and X2 each contain inosine, and no guanosine nucleoside is present. (Item 108) An agonist as described in item 89, wherein N1 and N2 contain cytidine, N3 and N4 contain guanosine, and X1 and X2 each contain inosine, and no guanosine nucleoside is present. (Item 109) The agonist according to item 89, wherein N1 and N2 contain inosine, N3 and N4 contain cytidine, and X1 and / or X2 each contain at least one inosine. (Item 110) The agonist according to item 89, wherein N1 and N2 contain inosine, N3 and N4 contain cytidine, and X1 and X2 each contain at least one inosine. (Item 111) The agonist according to item 89, wherein N1 and N2 contain cytidine, N3 and N4 contain inosine, and X1 and / or X2 each contain at least one inosine. (Item 112) The agonist according to item 89, wherein N1 and N2 contain inosine, N3 and N4 contain cytidine, X1 and X2 contain inosine and do not contain guanosine nucleoside. (Item 113) The agonist according to item 89, wherein N1 and N2 contain cytidine, N3 and N4 contain inosine, X1 and X2 contain inosine and do not contain guanosine nucleoside. (Item 114) The agonist according to item 89, wherein X1 and X2 are each 12 nucleotides and contain 1, 2, 3 or 4 inosine nucleosides. (Item 115) The agonist according to item 89, wherein X1 and X2 are each 13 nucleotides and contain 1, 2, 3, 4 or 5 inosine nucleosides. (Item 116) The agonist according to item 89, wherein X1 and X2 are each 14 nucleotides and contain 1, 2, 3, 4, 5 or 6 inosine nucleosides. (Item 117) The agonist according to item 89, wherein X1 and X2 are each 15 nucleotides and contain 1, 2, 3, 4, 5, 6 or 7 inosine nucleosides. (Item 118) The agonist according to item 89, wherein X1 and X2 are each 16 nucleotides and each contain 1, 2, 3, 4, 5, 6, 7 or 8 inosine nucleosides. (Item 119) An agonist as described in item 89, wherein X1 and X2 each consist of 12 nucleotides and contain at least 10%, 20%, 30%, or 40% inosine nucleosides. (Item 120) An agonist according to any one of items 1 to 88, wherein the linker is a nucleotide linker or a non-nucleotide linker. (Item 121) The agonist described in item 120, wherein the linker is a non-nucleotide linker. (Item 122) The agonist described in item 120, wherein the linker is a nucleotide linker. (Item 123) The nucleotide linker comprises a tetraloop, and the nucleotide sequence of the tetraloop is as follows: (a) UNCG (wherein N = A, C, G, or U), (b) GNRA (wherein N = A, C, G or U, and R = A or G), (c) ANYA (wherein N = A, C, G or U, and Y = C or T), (d) CUYG (where Y = C or T), (e)UMAC (where M=A or C), and (f) An agonist selected from the group consisting of CUUG, as described in item 122. (Item 124) The agonist according to item 122, wherein the nucleotide linker comprises the nucleotide sequence UUUGAU or UGUUU. (Item 125) The agonist described in item 123, wherein the sequence of the tetraloop is UUCG. (Item 126) The agonist described in item 123, wherein the sequence of the tetraloop is GAUC. (Item 127) The agonist described in item 124, wherein the nucleotide linker comprises the nucleotide sequence UUUGAU. (Item 128) The agonist according to item 124, wherein the nucleotide linker comprises the nucleotide sequence UGUUU. (Item 129) The aforementioned non-nucleotide linker, (a) Ethylene glycol linker, and (b) An agonist selected from the group consisting of alkyl linkers, as described in item 121 or any one of items 89-119. (Item 130) The agonist according to item 129, wherein the non-nucleotide linker is a hexaethylene glycol linker. (Item 131) The agonist according to item 129, wherein the non-nucleotide linker is a C9 alkyl linker. (Item 132) The agonist according to any one of items 1 to 131, wherein the agonist comprises a 5'-diphosphate moiety, or a derivative or analog thereof. (Item 133) The agonist according to any one of items 1 to 131, wherein the agonist comprises a 5'-triphosphate moiety, or a derivative or analog thereof. (Item 134) The agonist according to item 132 or 133, wherein the derivative or analog thereof comprises a phosphate bioequivalent, the phosphate bioequivalent being selected from phosphonates, thiophosphonates, phosphorothioates, sulfates, sulfonates, sulfamates, thiazolidinones, carboxylates, malonates, boronic acids, benzoxabolols, boranophosphates, and squalamides. (Item 135) The agonist described in any one of items 1 to 134 comprises a modified nucleotide, a modified nucleoside, or a modified nucleic acid base, or a combination thereof. (Item 136) The agonist described in any one of items 1 to 135, wherein the agonist includes modifications to internucleotide bonds or the polynucleotide backbone. (Item 137) The agonist has the following characteristics: (a) Specifically binds to one or more RLRs (e.g., RIG-1, MDA5, and / or LGP2), (b) Increases cytokine production mediated by RLR, (c) Increases the RLR-mediated expression of interferon-induced genes (ISGs), (d) Increases RLR-dependent intracellular signaling, (e) Enhances double-strand stability, (f) Increases binding affinity for RLR, (g) Reduces off-target binding, (h) Increases the biological half-life, (i) Improves in vivo distribution and bioavailability, (j) Increases and / or enhances uptake into cells and / or tissues, (k) Reduces immunogenicity, and (l) An agonist according to any one of Items 1 to 136, showing at least one or more of any combination of (a) to (k). (Item 138) A synthetic RIG-I-like receptor (RLR) agonist that specifically binds to a RIG-I-like receptor (RLR), wherein the 5'-terminal nucleotide of the agonist contains a 5'-diphosphate or triphosphate moiety, or a derivative or analog thereof, and the agonist contains a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, and 36. (Item 139) A synthetic RIG-I-like receptor (RLR) agonist that specifically binds to a RIG-I-like receptor (RLR), wherein the agonist comprises a blunt-terminated hairpin RNA containing a first polynucleotide linked to a second polynucleotide by a linker, the first polynucleotide being sufficiently complementary to the second polynucleotide and forming a double helix, the double helix containing fewer than 19 base pairs, the 5' terminal nucleotide of the first oligonucleotide comprising a 5' diphosphate or tripphosphate moiety, or a derivative or analog thereof, the agonist comprising a sequence motif, the sequence motif providing at least one improved bioactivity mediated by the RLR compared to an agonist without the sequence motif, and the first and second polynucleotides are as follows: (i) Sequence IDs 37 and 68, respectively (ii) Sequence IDs 38 and 69, respectively (iii) Sequence IDs 39 and 70, respectively (iv) Sequence IDs 40 and 71, respectively (v) Sequence IDs 41 and 72, respectively (vi) Sequence IDs 42 and 73, respectively (vii) Sequence numbers 43 and 74, respectively (viii) Sequence IDs 44 and 75, respectively (ix) Sequence numbers 45 and 76, respectively (x) Sequence numbers 46 and 77, respectively (xi) Sequence IDs 47 and 78, respectively (xii) Sequence IDs 48 and 79, respectively (xiii) Sequence IDs 49 and 80, respectively (xiv) Sequence IDs 50 and 81, respectively (xv) Sequence IDs 51 and 82, respectively (xvi) Sequence numbers 52 and 83, respectively (xvii) Sequence IDs 53 and 84, respectively (xviii) Sequence IDs 54 and 85, respectively (xix) Sequence numbers 55 and 86, respectively. (xx) Sequence numbers 56 and 87, respectively. (xxi) Sequence numbers 57 and 88, respectively. (xxii) Sequence IDs 58 and 89, respectively. (xxiii) Sequence numbers 59 and 89, respectively (xxiv) Sequence IDs 60 and 90, respectively (xxv) Sequence numbers 61 and 91, respectively. (xxvi) Sequence IDs 62 and 92, respectively (xxvii) Sequence numbers 63 and 91, respectively. (xxviii) Sequence numbers 64 and 93, respectively. (xxix) Sequence numbers 65 and 94, respectively. (xxx) Sequence numbers 66 and 95, respectively. (xxxi) Sequence numbers 67 and 96, respectively, and (xxxii) A synthetic RIG-I-like receptor (RLR) agonist containing a nucleotide sequence selected from the group consisting of SEQ ID NOs. 63 and 97, respectively. (Item 140) A synthetic RIG-I-like receptor (RLR) agonist that specifically binds to a RIG-I-like receptor (RLR), wherein the agonist comprises a blunt-terminated hairpin RNA containing at least one nucleotide including inosine that forms a base pair with cytidine, and the agonist comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 22, 23, and 25. (Item 141) A synthetic RIG-I-like receptor (RLR) agonist that specifically binds to a RIG-I-like receptor (RLR), wherein the agonist comprises a blunt-terminated hairpin RNA containing at least one nucleotide including inosine which forms a base pair with cytidine, and the agonist comprises the formula 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', where (N1-N2-X1) comprises a first polynucleotide, (X2-N3-N4) comprises a second polynucleotide, and the first and second polynucleotides are as follows: (i) Sequence IDs 58 and 89, respectively (ii) Sequence IDs 59 and 89, respectively, and (iii) A synthetic RIG-I-like receptor (RLR) agonist comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs. 61 and 91, respectively. (Item 142) A synthetic RIG-I-like receptor (RLR) agonist that specifically binds to a RIG-I-like receptor (RLR), wherein the agonist comprises a blunt-terminated hairpin RNA containing a non-nucleotide linker, and the agonist comprises the formula 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', where (N1-N2-X1) comprises a first polynucleotide, and (X2-N3-N4) comprises a second polynucleotide, and the first and second polynucleotides are as follows: (i) Sequence IDs 37 and 68, respectively (ii) Sequence IDs 38 and 69, respectively (iii) Sequence IDs 39 and 70, respectively (iv) Sequence IDs 40 and 71, respectively (v) Sequence IDs 41 and 72, respectively (vi) Sequence IDs 42 and 73, respectively (vii) Sequence numbers 43 and 74, respectively (viii) Sequence IDs 44 and 75, respectively (ix) Sequence numbers 45 and 76, respectively (x) Sequence numbers 46 and 77, respectively (xi) Sequence IDs 47 and 78, respectively (xii) Sequence IDs 48 and 79, respectively (xiii) Sequence IDs 49 and 80, respectively (xiv) Sequence IDs 50 and 81, respectively (xv) Sequence IDs 51 and 82, respectively (xvi) Sequence numbers 52 and 83, respectively (xvii) Sequence IDs 53 and 84, respectively (xviii) Sequence IDs 54 and 85, respectively (xix) Sequence numbers 55 and 86, respectively. (xx) Sequence numbers 56 and 87, respectively. (xxi) Sequence numbers 57 and 88, respectively. (xxii) Sequence IDs 58 and 89, respectively. (xxiii) Sequence numbers 59 and 89, respectively (xxiv) Sequence IDs 60 and 90, respectively (xxv) Sequence numbers 61 and 91, respectively. (xxvi) Sequence IDs 62 and 92, respectively (xxvii) Sequence numbers 63 and 91, respectively. (xxviii) Sequence numbers 64 and 93, respectively. (xxix) Sequence numbers 65 and 94, respectively. (xxx) Sequence numbers 66 and 95, respectively. (xxxi) Sequence numbers 67 and 96, respectively, and (xxxii) A synthetic RIG-I-like receptor (RLR) agonist containing a nucleotide sequence selected from the group consisting of SEQ ID NOs. 63 and 97, respectively. (Item 143) The agonist is an agonist according to any one of items 1 to 142, wherein the nucleotide sequence containing the agonist is not complementary to the genomic DNA sequence or mRNA sequence, the RLR agonist does not participate in RNA interference, and the RLR agonist does not silence gene expression. (Item 144) A pharmaceutical composition for stimulating an immune response to treat or slow the progression of cancer, or reduce or inhibit tumor growth in a subject where such treatment is required, comprising an agonist described in any one of items 1 to 143 and a pharmaceutically acceptable carrier. (Item 145) The pharmaceutical composition according to item 144, wherein the agonist is formulated in a polyethyleneimine (PEI) carrier. (Item 146) The pharmaceutical composition according to item 145, wherein the PEI carrier is JetPEI (registered trademark). (Item 147) A method for increasing the RLR-mediated production of one or more cytokines in a cell, the method comprising contacting the cell with an agonist described in any one of items 1 to 143, wherein the agonist increases the RLR-mediated cytokine production in the cell. (Item 148) A method for increasing the RLR-mediated expression of one or more interferon-inducible genes in a cell, the method comprising contacting the cell with an agonist described in any one of items 1 to 143, wherein the agonist increases the RLR-mediated expression of one or more interferon-inducible genes in the cell. (Item 149) A method for increasing RLR-dependent intracellular signaling in cells, the method comprising contacting the cells with an agonist described in any one of items 1 to 143, wherein the agonist increases RLR-dependent intracellular signaling. (Item 150) A method for stimulating an immune response in a subject, the method comprising administering to the subject an effective amount of an agonist according to any one of items 1 to 143 or a pharmaceutical composition according to any one of items 144 to 146. (Item 151) A method for treating or slowing the progression of cancer in a subject, wherein the method comprises administering to the subject an effective amount of an agonist according to any one of items 1 to 143 or a pharmaceutical composition according to any one of items 144 to 146. (Item 152) A method for reducing or inhibiting tumor growth in a subject that requires such reduction, the method comprising administering to the subject an effective amount of an agonist according to any one of items 1 to 143 or a pharmaceutical composition according to any one of items 144 to 146. (Item 153) A method for stimulating an immune response in a subject that requires treatment of cancer, slowing its progression, or inhibiting tumor growth, the method comprising administering to the subject an effective amount of an agonist according to any one of items 1 to 143 or a pharmaceutical composition according to any one of items 144 to 146, wherein the agonist or the pharmaceutical composition increases the RLR-mediated production of one or more cytokines in a cell, increases the RLR-mediated expression of one or more interferon-inducing genes in a cell, and / or increases RLR-dependent intracellular signaling in a cell, thereby stimulating the immune response, treating the cancer, slowing its progression, or inhibiting tumor growth. (Item 154) The method according to any one of items 150 to 153, wherein the agonist or pharmaceutical composition is administered in combination with one or more further therapeutic agents, the one or more further therapeutic agents being selected from the group consisting of chemotherapy, targeted anticancer therapy, oncolytic agents, cell death inducers, opsonizing agents (e.g., opsonized antibodies), cytotoxic agents, immunotherapy, cytokines, activators or agonists of costimulatory molecules, inhibitors of inhibitory molecules, vaccines, cellular immunotherapy, or combinations thereof. (Item 155) The method according to item 154, wherein the agonist or pharmaceutical composition is administered before or after the administration of the one or more further therapeutic agents, or the one or more further therapeutic agents are administered simultaneously with, before or after, the administration of the agonist or pharmaceutical composition. (Item 156) The method according to item 154 or 155, wherein the one or more additional therapeutic agents are a PD-1 / PD-L1 antagonist, a TIM-3 antagonist, a VISTA antagonist, an adenosine A2AR antagonist, a B7-H3 antagonist, a B7-H4 antagonist, a BTLA antagonist, a CTLA-4 antagonist, an IDO antagonist, a KIR antagonist, a LAG-3 antagonist, a Toll-like receptor 3 (TLR3) agonist, a Toll-like receptor 7 (TLR7) agonist, or a Toll-like receptor 9 (TLR9) agonist. (Item 157) The method according to item 154 or 155, wherein the one or more further therapeutic agents are agonists comprising a polypeptide (e.g., an antibody, or its antigen-binding portion) that specifically binds to CD137(4-1BB). (Item 158) The method according to item 154 or 155, wherein the one or more further therapeutic agents are agonists comprising a polypeptide (e.g., an antibody, or its antigen-binding portion) that specifically binds to CD134(OX40). (Item 159) The method according to item 156, wherein one or more of the additional therapeutic agents are PD-1 / PD-L1 antagonists. (Item 160) The method according to item 159, wherein the PD-1 / PD-L1 antagonist is selected from the group consisting of PDR001, KEYTRUDA® (pembrolizumab), OPDIVO® (nivolumab), pidilizumab, MEDI0680, REGN2810, TSR-042, PF-06801591, and AMP-224. (Item 161) The method according to item 159, wherein the PD-1 / PD-L1 antagonist is selected from the group consisting of FAZ053, TENCENTRIQ® (atezolizumab), BAVENCIO® (avelumab), IMFINZI® (durvalumab), and BMS-936559. (Item 162) The method according to item 156, wherein one or more of the aforementioned further therapeutic agents are TIM-3 antagonists. (Item 163) The method according to item 156, wherein one or more of the aforementioned further therapeutic agents are VISTA antagonists. (Item 164) The method according to item 156, wherein one or more of the aforementioned further therapeutic agents are adenosine A2AR antagonists. (Item 165) The method according to item 156, wherein one or more of the aforementioned further therapeutic agents are B7-H3 antagonists. (Item 166) The method according to item 156, wherein one or more of the aforementioned further therapeutic agents are B7-H4 antagonists. (Item 167) The method according to item 156, wherein one or more of the aforementioned further therapeutic agents are BTLA antagonists. (Item 168) The method according to item 156, wherein one or more of the aforementioned further therapeutic agents are CTLA-4 antagonists. (Item 169) The method according to item 156, wherein one or more of the aforementioned further therapeutic agents are IDO antagonists. (Item 170) The method according to item 156, wherein one or more of the aforementioned further therapeutic agents are KIR antagonists. (Item 171) The method according to item 156, wherein one or more of the aforementioned further therapeutic agents are LAG-3 antagonists. (Item 172) The method according to item 156, wherein one or more of the aforementioned further therapeutic agents are Toll-like receptor 3 (TLR3) agonists. (Item 173) The method according to item 172, wherein the TLR3 agonist is polyinosinic acid:polycytidylic acid (poly I:C). (Item 174) The TLR3 agonist is HILTONOL® (PolyICLC), as described in item 172. (Item 175) The method according to item 172, wherein the TLR3 agonist is polyadenylate-polyuridylate (poly A:U). (Item 176) The method described in item 172, wherein the TLR3 agonist is RIBOXXIM(registered trademark) (RGIC(registered trademark) 100). (Item 177) The method described in item 172, wherein the TLR3 agonist is RIBOXXON® (RGIC® 50 bioconjugate). (Item 178) The method described in item 172, wherein the TLR3 agonist is RIBOXXOL(registered trademark) (RGIC(registered trademark) 50). (Item 179) The method according to item 156, wherein one or more of the aforementioned further therapeutic agents are Toll-like receptor 7 (TLR7) agonists. (Item 180) The TLR7 agonist is GS-9620 (besatrimod), as described in item 179. (Item 181) The TLR7 agonist is imiquimod (ALDARA®), as described in item 179. (Item 182) The TLR7 agonist is reciquimod (R-848), as described in item 179. (Item 183) The method according to item 156, wherein one or more of the aforementioned further therapeutic agents are Toll-like receptor 9 (TLR9) agonists. (Item 184) The method described in item 183, wherein the TLR9 agonist is a CpG oligodeoxynucleotide (CpG ODN). (Item 185) The method according to item 184, wherein the CpG ODN is a Class A CpG ODN (CpG-A ODN). (Item 186) The method according to item 184, wherein the CpG ODN is a Class B CpG ODN (CpG-B ODN). (Item 187) The method according to item 184, wherein the CpG ODN is a class C CpG ODN (CpG-C ODN). (Item 188) Use of an agonist as described in any one of items 1 to 143 or a pharmaceutical composition as described in any one of items 144 to 146, for use in a subject where it is necessary to stimulate an immune response to treat cancer, slow its progression, or inhibit tumor growth, and to do so, possibly in combination with one or more further therapeutic agents. (Item 189) Use of an agonist as described in any one of items 1 to 143 or a pharmaceutical composition as described in any one of items 144 to 146 in the manufacture of a pharmaceutical product for use in combination with one or more further therapeutic agents, in cases where it is necessary to stimulate an immune response to treat cancer, slow its progression, or inhibit tumor growth. (Item 190) A kit comprising an agonist as described in any one of items 1 to 143 or a pharmaceutical composition as described in any one of items 144 to 146, instructions for use in stimulating an immune response in a subject, treating or slowing the progression of cancer, or inhibiting tumor growth in a subject, and, optionally, instructions for use in combination with one or more additional therapeutic agents. (Item 191) The agonist or pharmaceutical composition is administered in combination with one or more further therapeutic agents, the one or more further therapeutic agents being selected from the group consisting of chemotherapy, targeted anticancer therapy, oncolytic agents, cell death inducers, opsonizing agents (e.g., opsonized antibodies), cytotoxic agents, immunotherapy, cytokines, activators of costimulatory molecules, inhibitors of inhibitory molecules, vaccines, cellular immunotherapy, or combinations thereof, as described in item 188 or the kit described in item 190. (Item 192) The use or kit according to item 191, wherein the agonist or pharmaceutical composition is administered before or after the administration of the one or more further therapeutic agents, or the one or more further therapeutic agents are administered simultaneously with, before or after the administration of the agonist or pharmaceutical composition. (Item 193) The use described in any one of items 188, 189, 191, or 192 or the kit described in items 190-192, wherein the one or more additional therapeutic agents are a PD-1 / PD-L1 antagonist, a TIM-3 antagonist, a VISTA antagonist, an adenosine A2AR antagonist, a B7-H3 antagonist, a B7-H4 antagonist, a BTLA antagonist, a CTLA-4 antagonist, an IDO antagonist, a KIR antagonist, a LAG-3 antagonist, a Toll-like receptor 3 (TLR3) agonist, a Toll-like receptor 7 (TLR7) agonist, or a Toll-like receptor 9 (TLR9) agonist. (Item 194) The one or more further therapeutic agents are agonists comprising a polypeptide (e.g., an antibody, or its antigen-binding portion) that specifically binds to CD137(4-1BB), as described in any one of items 188, 189, 191, or 192, or the kits described in items 190-192. (Item 195) The one or more further therapeutic agents are agonists comprising a polypeptide (e.g., an antibody, or its antigen-binding portion) that specifically binds to CD134(OX40), as described in any one of items 188, 189, 191, or 192, or the kits described in items 190-192. [Brief explanation of the drawing]

[0093] [Figure 1] This provides a bar graph showing the quantification of IFN-α secretion from human PBMCs treated with RIG-I-like receptor agonists, including various modifications at three different concentrations. [Modes for carrying out the invention]

[0094] overview RIG-I-like receptors (RLRs) are a family of cytoplasmic pattern recognition receptors essential for detecting viral RNA and initiating the innate immune response. The RLR family includes retinoic acid-inducible gene I. These receptors include three members: I (RIG-I), Melanoma differentiation-associated gene 5 (MDA5), and Laboratory of Genetics and Physiology 2 (LGP2). These receptors are expressed in both immune and non-immune cell types and modulate signaling pathways that promote IRF3-dependent and IRF7-dependent expression of type I and type III interferons (IFNs), as well as NF-κB-dependent expression of inflammatory cytokines.

[0095] All three RLR family receptors possess a DExD / H box RNA helicase domain with ATPase activity. This domain, along with the adjacent C-terminal domain, is necessary for RNA binding. In addition, the C-terminal domains of RIG-I and LGP2 have been shown to act as repressive domains, ensuring that these receptors maintain an inactive configuration until they bind to activated RNA.

[0096] This disclosure provides RLR agonists comprising synthetic RNA molecules that fold to form double-stranded dsRNA and contain one or more sequence motifs that give one or more enhanced bioactivity.

[0097] definition Unless otherwise specified, the terms used in the claims and specification are defined as follows. In the event of any direct conflict with the terms used in the parent provisional patent application, the terms used in this application shall prevail.

[0098] Where used herein and in the appended claims, the singular forms “a,” “an,” and “the” should be noted to include plural objects unless it is explicitly indicated that their content has a different meaning. Furthermore, unless the context specifically requires otherwise, singular terms should include plural forms, and plural terms should include singular forms.

[0099] Where used herein, the term “about” (or “approximately”) is understood by those skilled in the art and varies to some extent depending on the context in which it is used. When using a term that is not clear to those skilled in the art, taking into account the context in which it is used, “about” means within ±10% of a particular value.

[0100] Agonist: As used herein, the term “agonist” is used in its broadest sense and encompasses any molecule or compound that partially or completely promotes, induces, increases, and / or activates the bioactivity of the natural polypeptides disclosed herein. The agonist molecules of this disclosure may include nucleic acids (e.g., oligonucleotides, polynucleotides), antibodies or antigen-binding fragments, fragments or amino acid sequence variants of natural polypeptides, peptides, oligonucleotides, lipids, carbohydrates, and low-molecular-weight organic molecules. In some embodiments, activation in the presence of an agonist is observed in a dose-dependent manner. In some embodiments, the measured signal (e.g., bioactivity) is at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% greater than the signal measured using a negative control under equivalent conditions. Methods for identifying agonists suitable for use in the methods of this disclosure are also disclosed herein. These methods include, but are not limited to, enzyme-linked immunosorbent assays (ELISA), Forte Bio® systems, fluorescence polarization (FP) assays, and radioimmunoassays (RIA). These assays determine the ability of an agonist to bind to a target polypeptide (e.g., a receptor or ligand) and thus demonstrate the agonist's ability to promote, increase, or activate the activity of that polypeptide. The efficacy of an agonist can also be determined using functional assays (e.g., the agonist's ability to activate or promote the function of a polypeptide). For example, a functional assay may involve contacting a polypeptide with a candidate agonist molecule and measuring a detectable change in one or more bioactive activities typically associated with that polypeptide. The potency of an agonist is usually determined by its EC (Effective Computation).50 Defined by the value (the concentration required to activate 50% of the agonist response). EC 50 The smaller the value, the greater the agonist's potency, and the lower the concentration required to activate the maximum biological response.

[0101] To improve: As used herein, the term “ameliorating” means any beneficial outcome in the treatment of a disease condition (e.g., cancer), including prevention, reduction of severity or progression, remission or cure.

[0102] Amino Acids: As used herein, the term “amino acids” refers not only to naturally occurring and synthetic amino acids, but also to amino acid analogs and amino acid mimics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids include not only those encoded by the genetic code, but also those subsequently modified, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as naturally occurring amino acids (i.e., carbon bonded to hydrogen, carboxyl group, amino group, and R group), such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs may have a modified R group (e.g., norleucine) or a modified peptide skeleton, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimics refer to chemical compounds that have a different structure from the general chemical structure of amino acids, but function in a manner similar to naturally occurring amino acids.

[0103] Amino acids may be referred herein by their commonly known three-letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Similarly, nucleotides may be referred by their commonly accepted one-letter codes.

[0104] Amino acid substitution: As used herein, “amino acid substitution” means replacing at least one existing amino acid residue in a given amino acid sequence (the amino acid sequence of the start polypeptide) with a second, different “substitute” amino acid residue. “Amino acid insertion” means incorporating at least one additional amino acid into a given amino acid sequence. Insertions typically consist of the insertion of one or two amino acid residues, but larger “peptide insertions,” e.g., insertions of about 3 to about 5, or up to about 10, 15, or 20 amino acid residues, are also possible. The inserted residues may be naturally occurring or non-natural, as disclosed above. “Amino acid deletion” means the removal of at least one additional amino acid from a given amino acid sequence.

[0105] Base composition: As used herein, the term “base composition” refers to the proportion of all nucleotides in a nucleic acid (e.g., RNA) consisting of guanine (or hypoxanthine) + cytosine and / or uracil (or thymine) + adenine nucleic acid bases.

[0106] Base pairing: As used herein, the term “base pairing” refers to two nucleic acid bases on opposing, complementary polynucleotide chains, or regions on the same chain, that interact through the formation of a specific hydrogen bond. As used herein, the term “Watson-Crick base pairing” is used interchangeably with “complementary base pairing” and refers to a set of base pairing rules, such that in DNA molecules, purines always bind to pyrimidines, as the nucleic acid base adenine (A) forms a complementary base pair with thymine (T), and guanine (G) forms a complementary base pair with cytosine (C). In RNA molecules, thymine is replaced by uracil (U), and uracil (U) forms a complementary base pair with adenine (A), as well as thymine (T). Complementary base pairs are linked by hydrogen bonds, and the number of hydrogen bonds differs for each base pair. As is known in the art, guanine (G)-cytosine (C) base pairs are linked by three hydrogen bonds, and adenine (A)-thymine (T) or uracil (U) base pairs are linked by two hydrogen bonds.

[0107] Base pairing interactions that do not follow these rules can result in natural, unnatural, and synthetic nucleic acids, which are referred to herein as “non-Watson-Crick base pairing” or, in this specification, “non-canonical base pairing.” “Fluctuating base pairing” refers to pairing between two nucleic acid bases in an RNA molecule that does not follow the Watson-Crick base pairing rules. For example, inosine is a nucleoside structurally similar to guanosine but lacks a 2-amino group. Inosine can form two hydrogen bonds with each of the four nucleic acid bases mentioned above (Oda et al., (1991) Nucleic Acids Res 19:5263-5267), and is often used by researchers as a “universal” base, meaning that it can base pair with all naturally occurring or canonical bases. The four main types of fluctuating base pairs are guanine-uracil (GU), hypoxanthine-uracil (IU), hypoxanthine-adenine (IA), and hypoxanthine-cytosine (IC). To maintain consistency in nucleic acid nomenclature, since hypoxanthine is the nucleic acid base of inosine, "I" is used for hypoxanthine, and the other nomenclature follows the names of the nucleic acid bases and their corresponding nucleosides (for example, not only guanine and guanosine, but also deoxyguanosine is "G"). The thermodynamic stability of fluctuating base pairs is equivalent to that of Watson-Crick base pairs. Fluctuating base pairs play a role in the formation of the secondary structure of RNA molecules.

[0108] In one embodiment, the present disclosure provides a synthetic RNA molecule that agonizes or activates one or more RIG-I-like receptors (RLRs), wherein inosine can be inserted only at a position that forms a base pair with cytidine (IC base pair), that is, inosine can substitute for guanosine but not for any other nucleoside.

[0109] Biologically active: As used herein, the phrase “biologically active” refers to the characteristic of any substance that is active in a biological system and / or organism. For example, a substance that, when administered to an organism, has a biological effect on that organism and is therefore “bioactive” is considered biologically active. In a particular embodiment, if a nucleic acid is biologically active, the portion of that nucleic acid that shares at least one bioactive property of the whole nucleic acid is typically referred to as the “biologically active” portion.

[0110] Covalently bonded: As used herein, the term “covalently bonded” (or, “conjugated,” “linked,” “connected,” “fused,” or “tethered”) means, when used in reference to two or more parts, that parts physically associate or connect to one another, either directly or through one or more further parts acting as linkers, by any means including chemical conjugation, recombinant technology, or enzymatic activity, to form a structure that is sufficiently stable so that the parts remain physically associated under the conditions in which the structure is used (e.g., physiological conditions).

[0111] Complementary: As used herein, the terms “complementary” or “complementarity” refer to the relationship between a sequence of nucleotides comprising two polynucleotide chains, or multiple regions of the same polynucleotide chain, and the formation of a double-stranded structure comprising that chain or region, such that the degree of consecutive base-pairing between the two chains or regions is sufficient for the formation of a double-stranded structure. Adenine (A) is known to form certain hydrogen bonds or “base pairs” with thymine (T) or uracil (U). Similarly, cytosine (C) is known to form base pairs with guanine (G). Non-canonical nucleic acid bases (e.g., inosine) are also known to be able to form hydrogen bonds with native bases. A sequence of nucleotides containing a first strand, region, or part or fragment thereof of a polynucleotide is said to be "fully complementary" if the first and second strands are aligned in an antiparallel manner with respect to a sequence of nucleotides containing a second strand, region, or part or fragment thereof of the same or different nucleic acid, and the degree of base pairing between these two strands is such that it maintains the double-stranded structure under the conditions in which this double-stranded structure is used (e.g., physiological conditions in cells). It should be understood that a complementary strand or region of a polynucleotide may contain some non-complementary base pairs. Complementarity may be "partial," in which case only some of the nucleic acid bases containing the polynucleotide are matched according to the base pairing rules. Alternatively, there may be "complete" or "whole" complementarity between nucleic acids. The degree of complementarity between polynucleotide strands or regions greatly affects the efficiency and strength of hybridization between those strands or regions, but the two complementary polynucleotides do not need to form base pairs at all nucleotide positions. In some embodiments, the first polynucleotide is 100% or "completely" complementary to the second polynucleotide and therefore forms base pairs at all nucleotide positions. In some embodiments, the first polynucleotide is not 100% complementary (e.g., 90%, or 80%, or 70%) and contains mismatched nucleotides at one or more nucleotide positions.While complete complementarity is often desired, some embodiments may include one or more mismatches, preferably 6, 5, 4, 3, 2, or 1.

[0112] Contact: As used herein, the term “contact” means to establish a physical connection between two or more entities. For example, contacting a cell with a drug (e.g., RNA, a lipid nanoparticle composition, or another pharmaceutical composition of the Disclosure) means that the cell and the drug are made to share a physical connection. Methods of contacting cells with external entities, in vivo, in vitro, and ex vivo, are well known in the field of biology. In exemplary embodiments of the Disclosure, the step of contacting a mammalian cell with a composition (e.g., isolated RNA, nanoparticles, or a pharmaceutical composition of the Disclosure) is performed in vivo. For example, contacting a lipid nanoparticle composition with a cell that may be placed within an organism (e.g., a mammal) (e.g., a mammalian cell) may be performed by any suitable route of administration (e.g., parenteral administration to an organism, including intravenous, intramuscular, intradermal, and subcutaneous administration). For cells present in vitro, a certain composition (e.g., lipid nanoparticles or isolated RNA) and the cells may be brought into contact, for example, by adding the composition to the cell culture medium, which may involve transfection or result in transfection. Furthermore, two or more cells may come into contact with the drug.

[0113] Denaturation: As used herein, the term “denaturation” refers to the process by which hydrogen bonds between nucleotides that have formed base pairs in a nucleic acid are broken, resulting in the loss of secondary and / or tertiary nucleic acid structure (e.g., separation of already annealed strands). Denaturation may occur as a result of the application of foreign substances, energy, or biochemical processes to nucleic acids.

[0114] Antigen-presenting cells: The term "antigen-presenting cells" or "APCs" refers to cells that present foreign antigens on their surface, forming complexes with MHC cells. T cells recognize these complexes using T cell receptors (TCRs). Examples of APCs include, but are not limited to, dendritic cells (DCs), peripheral blood mononuclear cells (PBMCs), monocytes (e.g., THP-1), B lymphoblasts (e.g., C1R.A2, 1518 B-LCL), and monocyte-derived dendritic cells (DCs). Some APCs internalize antigens by phagocytosis or by receptor-mediated endocytosis.

[0115] Apoptosis: As used herein, the term “apoptosis” refers to the programmed process of cell death that occurs in multicellular organisms (e.g., humans). The highly controlled biochemical and molecular events that trigger apoptosis may result in observable and characteristic morphological changes in cells, including membrane bleb formation, reduction in cell volume, condensation and fragmentation of chromosomal DNA, and mRNA breakdown. A common method for identifying apoptotic cells (including T cells) is to expose them to a protein conjugated with a fluorophore (annexin V). Annexin V is commonly used to detect apoptotic cells by its ability to bind to phosphatidylserine on the outer layer of the plasma membrane and is an early indicator that a cell is undergoing the process of apoptosis.

[0116] Blunt End: As used herein, the terms “blunt end” and “blunt-ended” refer to the terminal structure of a nucleic acid that is doubled or forms a double helix, in which both complementary strands, including the double helix, are terminated by a base pair at at least one end. Thus, neither strand of the double helix extends further from the end than the other.

[0117] Cancer antigen: As used herein, “cancer antigen” means (i) tumor-specific antigens, e.g., neoantigens; (ii) tumor-associated antigens; (iii) cells expressing tumor-specific antigens; (iv) cells expressing tumor-associated antigens; (v) embryonic antigens on tumors; (vi) autologous tumor cells; (vii) tumor-specific membrane antigens; (viii) tumor-associated membrane antigens; (ix) growth factor receptors; (x) growth factor ligands; and (xi) any other type of antigen or antigen-presenting cell or substance associated with cancer.

[0118] Carcinoma: As used herein, the term “carcinoma” refers to malignant tumors of epithelial or endocrine tissue, including, but is recognized in the art, respiratory carcinomas, gastrointestinal carcinomas, genitourinary carcinomas, testicular carcinomas, breast carcinomas, prostate carcinomas, endocrine carcinomas, and melanomas. RIG-I-like receptor (RLR) agonists described herein can be used to treat patients who have, are suspected of having, or are at high risk of developing any type of cancer, including renal carcinoma or melanoma. Exemplary carcinomas include those formed from the tissues of the cervix, lungs, prostate, breast, head and neck, colon, and ovaries. The term also includes carcinosarcoma, which includes malignant tumors composed of cancerous and sarcomatous tissues. “Adenocarcinoma” refers to carcinomas originating from glandular tissue, or those in which tumor cells form recognizable glandular lumen structures.

[0119] Cytotoxic T lymphocyte (CTL) response: As used herein, the term “cytotoxic T lymphocyte (CTL) response” refers to an immune response induced by cytotoxic T cells. The CTL response is primarily mediated by CD8+ T cells.

[0120] Double-stranded: As used herein, the term “double-stranded” refers to a structure formed by the complementary strand of a double-stranded polynucleotide, or by the complementary region of a single-stranded polynucleotide that folds itself. The double-stranded structure of nucleic acids arises as a result of complementary nucleotide sequences joining together or hybridizing through base-pair interactions.

[0121] EC50 :When used herein, "EC 50 The term "maximum response" refers to the concentration of the agonist that is 50% of the maximum response, i.e., midway between the maximum response and baseline, in either an in vitro or in vivo assay.

[0122] Effective dose: As used herein, the term “effective dose” or “effective dosage” is defined as the amount sufficient to achieve, or at least partially achieve, the desired effect.

[0123] Hairpin RNA: As used herein, the terms “hairpin RNA” or “RNA hairpin” refer to self-complementary RNA comprising a double-stranded RNA (dsRNA) stem consisting of a complementary nucleotide chain that forms a double helix by base-pairing at one end of a nucleotide linker containing an unpaired nucleotide loop (e.g., a tetraloop) containing an unpaired nucleotide, or by ending with a non-nucleotide linker containing a flexible chemical moiety (e.g., ethylene glycol), either of which connects to a complementary nucleotide chain. RNA hairpins may differ in stem length, loop and / or linker size and / or composition, number of base-pair mismatches in the stem, and actual nucleotide sequence. RNA hairpins may be given one or more functions, including, but not limited to, guiding the overall folding of the RNA molecule containing the hairpin, determining interactions in ribozymes, protecting messenger RNA (e.g., mRNA) from denaturation, functioning as a recognition motif or structure for RNA-binding proteins, and acting as a substrate for enzymatic reactions. Further descriptions of RNA hairpin structure and function can be found in Svoboda and Di Cara (2006) Cell Mol Life Sci 63(7-8):901-908 and the references contained herein. In some embodiments, the stem region of the hairpin RNA containing the RLR agonist provided herein terminates with a blunt end having a 5' triphosphate or diphosphate.

[0124] Need: As used herein, “needs prevention,” “needs treatment,” or “needs” refers to a subject who, in the judgment of an appropriate healthcare professional (e.g., a physician, nurse, or caregiver in the case of a human, or a veterinarian in the case of a non-human mammal), would reasonably benefit from a given treatment (e.g., treatment with a composition comprising a RIG-I-like receptor agonist).

[0125] Linker: As used herein, the term “linker” (or, instead, “tether” or “spacer”) refers to a portion that covalently bonds, attaches to, or couples two polynucleotide chains or regions together. As used herein, a linker containing a nucleotide is called a “nucleotide linker” (e.g., a tetraloop). As used herein, the term “non-nucleotide linker” refers to a linker that contains a chemical moiety but does not contain a nucleotide. Non-limiting examples of non-nucleotide linkers include linkers containing ethylene glycol (e.g., hexaethylene glycol), alkyl chains (e.g., C9 alkyl linkers), and stilbenz ethers. Further descriptions of linkers can be found in Paredes et al., (2011) Methods 54:251–259, which is incorporated herein by reference in its entirety.

[0126] LGP2: As used herein, the term "LGP2" refers to the Laboratory of Genetics and Physiology 2 polypeptide, a specific member of the RIG-I-like receptor family, encoded in humans by the DHX58 gene. In the art, alternative names and acronyms for LGP2 include DHX58, D11LGP2, D11Igp2e, and RLR-3. An exemplary amino acid sequence of full-length human LGP2 is shown in Table 4 (SEQ ID NO: 100) and herein. MELRSYQWEVIMPALEGKNIIIWLPTGAGKTRAAAYVAKRHLETVDGAKVVVVLVNRVHLVTQHGEEFRRMLDGRWTVTTLSGDMGPRAGFGHLARCHDLLICTAELLQMALTSPEEEEHVELTVFSLIVVDECHHTHKDTVYNVIMSQYLELKLQRAQPLPQVLGLTAS PGTGGASKLDGAINHVLQLCANLDTWCIMSPQNCCPQLQEHSQQPCKQYNLCHRRSQDPFGDLLKKLMDQIHDHLEMPELSRKFGTQMYEQQVVKLSEAAALAGLQEQRVYALHLRRYNDALLIHDTVRAVDALAALQDFYHREHVTKTQILCAERRLALLFDDRKNELA HLATHGPENPKLEMLEKILQRQFSSSNSPRGIIFTRTRQSAHSLLLWLQQQQGLQTVDIRAQLLIGAGNSSQSTHMTQRDQQEVIQKFQDGTLNLLVATSVAEEGLDIPHCNVVVRYGLLTNEISMVQARGRARADQSVYAFVATEGSRELKRELINEALETLMEQAVA AVQKMDQAEYQAKIRDLQQAALTKRAAQAAQRENQRQQFPVEHVQLLCINCMVAVGHGSDLRKVEGTHHVNVNPNFSNYYNVSRDPVVINKVFKDWKPGGVISCRNCGEVWGLQMIYKSVKLPVLKVRSMLLETPQGRIQAKKWSRVPFSVPDFDFLQHCAENLSDLSLD (NCBP deposit number: NP_077024.2).

[0127] Local administration: As used herein, “local administration” or “local delivery” refers to delivery that does not rely on transporting the composition or drug to its intended target tissue or site via the vascular system. For example, a composition may be delivered by injection or implantation of the composition or drug, or by injection or implantation of a device containing the composition or drug. After local administration near the target tissue or site, the composition or drug, or one or more of its components, may diffuse into the intended target tissue or site.

[0128] MDA5: As used herein, the term “MDA5” refers to the melanoma differentiation-associated protein 5 polypeptide, a specific member of the RIG-I-like receptor family, encoded in humans by the IFIH1 gene. In the art, alternative names and acronyms for MDA5 include AGS7, Hlcd, IDDM19, MDA-5, RLR-2, SGMRT1, and helicase C-domain 1-induced interferon. An exemplary amino acid sequence of full-length human MDA5 is shown in Table 4 (SEQ ID NO: 99) and herein. (NCBI deposit number: NP_071451.2).

[0129] Modification: As used herein, “modified” or “modified” refers to a structurally altered state or change resulting from modification of a polynucleotide (e.g., RNA). Polynucleotides may be modified in a variety of ways, including chemically, structurally, and / or functionally. For example, the RNA molecules of this disclosure may be modified by incorporating non-natural bases or sequence motifs that confer bioactivity, including functional sequences or secondary structures. In one embodiment, RNA is modified, for example, by introducing non-natural bases or chemically modified bases, nucleosides, and / or nucleotides, such as those related to natural ribonucleotides A, U, G, and C.

[0130] Naturally occurring: As used herein, the term “naturally occurring” as applied to a substance refers to the fact that the substance can be found in nature. For example, polypeptides or polynucleotide sequences, or their components, such as amino acids or nucleotides, that are present in organisms (including viruses) that can be isolated from natural sources and have not been artificially and intentionally modified in a laboratory are naturally occurring.

[0131] Nucleic acids: As used herein, the term “nucleic acids” refers to deoxyribonucleotides or ribonucleotides in either single-stranded or double-stranded forms, and their polymers or oligomers. Unless otherwise specified, the term encompasses nucleic acids including known analogs of natural nucleotides that have similar binding properties to a reference nucleic acid and are metabolized in a similar manner to naturally occurring nucleotides. Polymers of nucleotides are called “polynucleotides.” Examples of nucleic acids or polynucleotides in this disclosure include, but are not limited to, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), DNA-RNA hybrids, RNAi inducers, RNAi agents, siRNA, shRNA, miRNA, antisense RNA, ribozymes, catalytic DNA, RNA that induces triple helix formation, threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA, LNA having a β-D-ribo configuration, α-LNA having an α-L-ribo configuration (a diastereomer of LNA), 2'-amino-LNA having a 2'-amino functional group, and 2'-amino-α-LNA having a 2'-amino functional group), or hybrids thereof.

[0132] The polynucleotides used herein may consist of any polyribonucleotide or polydeoxyribonucleotide, and may be unmodified RNA or DNA, or modified RNA or DNA. For example, a polynucleotide may consist of single-stranded and double-stranded DNA, DNA which is a mixture of single-stranded and double-stranded regions, single-stranded and double-stranded RNA, RNA which is a mixture of single-stranded and double-stranded regions, or a hybrid molecule of DNA and RNA which may be single-stranded, or more typically double-stranded, or a mixture of single-stranded and double-stranded regions. In addition, a polynucleotide may consist of RNA or DNA, or a triple-stranded region which contains both RNA and DNA. A polynucleotide may also contain one or more modified bases or DNA or RNA backbone that have been modified for stability or other reasons. Examples of “modified” bases include tritylated bases. Examples of “modified nucleosides” include inosine and thymine, the latter of which thymine is found in or constitutes RNA. Various modifications may be made to DNA and RNA, and therefore, “polynucleotides” encompass chemically, enzymatically, or metabolically modified forms.

[0133] Nucleic acid structure: As used herein, the term “nucleic acid structure” refers to the arrangement or organization of atoms, chemical components, elements, motifs and / or sequences of nucleic acid bases, including nucleic acids (e.g., RNA), and / or the two-dimensional or three-dimensional state of nucleic acids. Thus, the term “RNA structure” refers to the arrangement or organization of atoms, chemical components, elements, motifs and / or sequences of nucleic acid bases, including RNA molecules (e.g., mRNA), and / or the two-dimensional and / or three-dimensional state of RNA molecules. Nucleic acid structures can be further divided into four organizational categories, as referred herein, “molecular structure,” “primary structure,” “secondary structure,” and “tertiary structure,” based on the increasing complexity of the organization.

[0134] Nucleic acid bases: As used herein, the term “nucleic acid base” (or “nucleotide base” or “nitrogen base”) refers to purine and pyrimidine heterocyclic compounds found in nucleic acids, including any derivatives or analogues of naturally occurring purines and pyrimidines that confer improved properties (e.g., binding affinity, nuclease resistance, chemical stability) to nucleic acids or any part or segment thereof. Adenine, cytosine, guanine, thymine, and uracil are the major or standard nucleic acid bases primarily found in natural nucleic acids. Other natural, unnatural, non-canonical, and / or synthetic nucleic acid bases may be incorporated into nucleic acids (e.g., those disclosed herein).

[0135] Nucleoside / Nucleotide: As used herein, the term “nucleoside” refers to a compound containing a sugar molecule (e.g., ribose in RNA or deoxyribose in DNA) or a derivative or analog thereof, which is covalently bonded to a nucleic acid base (e.g., purine or pyrimidine) or a derivative or analog thereof (also referred herein as “nucleoic acid base”). As used herein, the term “nucleotide” refers to a nucleoside covalently bonded to a phosphate group. As used herein, the term “ribonucleoside” refers to a compound containing ribose and a nucleic acid base (e.g., adenosine (A), cytidine (C), guanosine (G), 5-methyluridine (m)). 5 This refers to nucleosides containing uridine (U) or inosine (I).

[0136] "Operatively linked": As used herein, a nucleic acid, or a fragment or part thereof, for example, a polynucleotide or oligonucleotide, is "operably linked" if it is functionally related to another nucleic acid sequence, or a fragment or part thereof.

[0137] Polynucleotides / Oligoniloids: As used herein, the terms “polynucleotide” and “oligonucleotide” are interchangeable and refer to single- or double-stranded polymers or oligomers of nucleotides or nucleoside monomers consisting of naturally occurring bases, sugars, and intersugar (skeletal) bonds. The terms “polynucleotide” and “oligonucleotide” also include polymers and oligomers, or parts thereof, that contain bases, sugars, and intersugar (skeletal) bonds that do not exist naturally, and that function similarly. Because the term “polynucleotide” encompasses polymeric forms of nucleotides of any length, polynucleotides are not limited to any particular length of nucleotide sequence. Short polynucleotides are typically referred to as “oligonucleotides” in the art. In the scope of this disclosure, such modified or substituted polynucleotides and oligonucleotides are often preferred over their natural forms because the modifications enhance one or more desirable or beneficial bioproperties or activities, including, but not limited to, increased cytokine production, improved cellular uptake, and / or increased stability in the presence of nucleases. In some embodiments, the agonists of the present disclosure include polynucleotides and oligonucleotides comprising at least one region of a modified nucleotide that confers one or more beneficial properties or enhances bioactivity (e.g., increased nuclease resistance, increased cellular uptake, increased double-strand stability, increased binding affinity to a target polypeptide).

[0138] Palindromic Sequence: As used herein, the term “palindromic sequence” (or “palindrome”) refers to a sequence of nucleotides that are self-complementary. A sequence of nucleotides read from 5' to 3' is the same as a sequence of nucleotides containing a complementary chain when read from 5' to 3'. For example, the sequence 5'-ACCTAGGT-3' is a palindromic sequence because its complementary sequence 3'-TGGATCCA-5' is the same as the original sequence when read from 5' to 3'. In contrast, the sequence 5'-AGTGGCTG-3' is not a palindromic sequence because its complementary sequence 3'-TCACCGAC-5' is not the same as the original sequence when read from 5' to 3'.

[0139] In one embodiment, the agonist is composed of a first oligonucleotide, the sequence of the first oligonucleotide being a palindromic sequence. In another embodiment, the agonist is composed of a first oligonucleotide, the first oligonucleotide containing a palindromic sequence.

[0140] The palindromic sequence in the preferred oligonucleotide of the present invention preferably includes both the 5' end and the 3' end of the oligonucleotide, thus forming a blunt end. In one embodiment of the present invention, the oligonucleotide comprises a single palindromic sequence, and in another more preferred embodiment of the present invention, the oligonucleotide comprises two complementary palindromes interrupted by an intervening sequence, spacer, or linker that connects two palindromes in one or two different oligonucleotides to form a hairpin double with a blunt end.

[0141] Parenteral administration: As used herein, “parenteral administration,” “administered parenterally,” and other grammatically equivalent terms refer to, but are not limited to, other modes of administration other than enteral and topical administration, usually by injection, including injections and infusions into veins, nasal cavity, eye, muscle, artery, spinal cavity, capsule, orbit, heart, skin, abdominal cavity, trachea, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, spinal cord, epidural, brain, skull, carotid artery, and sternal regions.

[0142] Patient: As used herein, the term “patient” includes human and other mammalian subjects receiving prophylactic or therapeutic treatment.

[0143] Identity Percentage: As used herein, the term “identity percentage” means, in the context of two or more nucleic acid or polypeptide sequences, two or more sequences or subsequences that, when denucleated for maximum match and aligned, have a specified percentage of identical nucleotide or amino acid residues, measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to those skilled in the art) or by visual inspection. Depending on its application, the “identity percentage” may exist across regions of the sequences being compared, for example, across functional domains, or alternatively, across the entire length of the two sequences being compared. For sequence comparison, typically one sequence acts as a reference sequence, and the test sequence is compared against this sequence. When using a sequence comparison algorithm, the test sequence and reference sequence are entered into a computer, and subsequence coordinates and sequence algorithm program parameters are specified, if necessary. The sequence comparison algorithm then calculates the sequence identity percentage of the test sequence relative to the reference sequence based on the specified program parameters. The percentage of identity between two sequences is a function of the number of identical positions shared by these sequences, taking into account the number of gaps required to introduce the optimal alignment of the two sequences and the length of each gap (i.e., homology % = number of identical positions / total number of positions × 100). Sequence comparison and determination of the percentage of identity between two sequences can be achieved using mathematical algorithms, as described in the non-restrictive examples below.

[0144] The optimal alignment of sequences for comparison may be performed, for example, by the local homology algorithm of Smith & Waterman, Adv.Appl.Math.2:482 (1981), by the homologial alignment algorithm of Needleman & Wunsch, J.Mol.Biol.48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc.Nat'l.Acad.Sci.USA 85:2444 (1988), by computer implementation of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual observation (generally, see Ausubel et al., below).

[0145] One example of a suitable algorithm for determining sequence identity percentage and sequence similarity is the BLAST algorithm, described in Altschul et al., J.Mol.Biol.215:403-410 (1990). Software for performing BLAST analysis is publicly available from the National Center for Biotechnology Information website. The identity percentage between two nucleotides can be determined using the GAP program in the GCG software package, with the NWSgapdna.CMP matrix, gap weightings of 40, 50, 60, 70, or 80, and length weightings of 1, 2, 3, 4, 5, or 6 (available at http: / / www.gcg.com). The identity percentage between two nucleotides or amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4:11-17 (1989)), incorporated into the ALIGN algorithm (version 2.0), with the PAM120 weighted residue table, gap length penalty 12, and gap penalty 4. In addition, the percentage of identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. (48): 444-453 (1970)) algorithm incorporated into the GAP program in the GCG software package (available at http: / / www.gcg.com), using a Blossum 62 matrix or PAM250 matrix, gap weightings of 16, 14, 12, 10, 8, 6, or 4, and length weightings of 1, 2, 3, 4, 5, or 6.

[0146] The nucleic acid and protein sequences of this disclosure can further be used as “query sequences” for performing searches against public databases to identify, for example, relevant sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al. (1990) J.Mol.Biol.215:403-10. A BLAST nucleotide search can be performed using the NBLAST program with a score of 100 and a word length of 12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the present invention. A BLAST protein search can be performed using the XBLAST program with a score of 50 and a word length of 3 to obtain amino acid sequences homologous to the protein molecules of the present invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be used as described in Altschul et al., (1997) Nucleic Acids Res.25(17):3389-3402. When using the BLAST and Gapped BLAST programs, you can use the default parameters for each program (e.g., XBLAST and NBLAST). See http: / / www.ncbi.nlm.nih.gov.

[0147] Pharmacologically acceptable: As used herein, “pharmaceutically acceptable” means a compound, material, composition and / or dosage form that, to the extent reasonable medical judgment, is suitable for use in contact with human and animal tissues, organs and / or bodily fluids, without excessive toxicity, irritation, allergic reactions or other problems or complications commensurate with a reasonable benefit-risk ratio.

[0148] pharmaceutically acceptable carriers: As used herein, the term “pharmaceutically acceptable carriers” includes, but is not limited to, any physiologically compatible solvents, dispersions, coatings, antimicrobial and antifungal agents, isotonic agents and absorption retarders. The composition may also contain pharmaceutically acceptable salts, such as acid addition salts or base addition salts (see, for example, Berge et al. (1977) J Pharm Sci 66:1-19).

[0149] Phosphate: As used herein, the term “phosphate” means a salt or ester of phosphoric acid. A polyphosphate is a salt or ester of a polymer oxyanion formed from tetrahedral PO4(phosphate) structural units linked together by sharing an oxygen atom. As used herein, the term “diphosphate” refers to a polyphosphate containing two phosphate structural units. As used herein, the term “triphosphate” refers to a polyphosphate containing three phosphate structural units. In some embodiments, the disclosure provides RIG-I-like receptor agonists comprising a diphosphate moiety linked to the 5' end, or a derivative or analog thereof. In some embodiments, the disclosure provides RIG-I-like receptor agonists comprising a triphosphate moiety linked to the 5' end, or a derivative or analog thereof. In some embodiments, the derivative or analog thereof is a phosphate bioequivalent.

[0150] Phosphate Bioequivalents: As used herein, the term “phosphate bioequivalent” (or “phosphate mimetic”) refers to a chemical substituent or group that has physical or chemical properties similar to a phosphate, including biphosphate and triphosphate moieties, and provides bioproperties broadly similar to a phosphate. In drug design, the purpose of substituting one bioequivalent with another is to improve the desired bioproperties or physical properties of a compound without making significant changes to its chemical structure. The use of bioequivalents is widespread in drug development and is used, for example, to reduce the toxicity of a parent or lead compound, alter its bioavailability, or change its activity or metabolism (see, e.g., Rye and Baell (2005) Curr Med Chem 12(26):3127-3141; Elliot et al., (2012) MedChemCom 3(7):735-751, which is incorporated herein by reference in its entirety).

[0151] Polypeptides: As used herein, the terms “polypeptides,” “peptides,” and “proteins” are used interchangeably to refer to polymers of amino acid residues. The term applies to amino acid polymers, in which one or more amino acid residues are artificial chemical mimics of corresponding naturally occurring amino acids, as well as to naturally occurring and non-naturally occurring amino acid polymers.

[0152] To prevent: As used herein, and as used in relation to a condition, the term “to prevent” means the administration of a composition that reduces the frequency of a particular medical condition or delays the onset of symptoms in a subject compared to a subject who does not ingest the composition.

[0153] Purified: As used herein, when applied to any of the proteins (antibodies or fragments) described herein, the terms “purified” or “isolated” refer to peptides isolated or purified from naturally occurring components (e.g., proteins or other naturally occurring biomolecules or organic molecules), such as other proteins, lipids, and nucleic acids in the prokaryotes expressing that protein. Typically, a polypeptide is purified when it constitutes at least 60% by weight (e.g., at least 65, 70, 75, 80, 85, 90, 92, 95, 97, or 99) of the total protein in the sample.

[0154] Reference Ligand: As used herein, the terms “reference ligand” (used interchangeably with “reference agonist”) or “reference molecule” refer to a RIG-I-like receptor ligand used to establish a relationship between itself and one or more distinct RIG-I-like receptor ligands, the relationship being the relative agonist effect of the reference ligand and one or more distinct RIG-I-like receptor ligands. As used herein, this term implies a RIG-I-like receptor ligand or agonist useful as a competitor in a test or assay such as those described herein (e.g., an IFN induction assay), which is useful for the discovery, identification, or development of one or more distinct agonists that bind to the RIG-I-like receptor.

[0155] RIG-I: As used herein, the term "RIG-I" refers to the retinoic acid-inducible gene I polypeptide, a specific member of the RIG-I-like receptor family, encoded in humans by the DDX58 gene. In the art, alternative names and acronyms for RIG-I include DEAD-box polypeptide 58, RIGI, RLR-1, SGMRT2, and DEXD / H-box helicase 58. An exemplary amino acid sequence of full-length human RIG-I is shown in Table 4 (SEQ ID NO: 98) and herein. MTTEQRRSLQAFQDYIRKTLDPTYILSYMAPWFREEEVQYIQAEKNNKGPMEAATLFLKFLLELQEEGWFRGFLDALDHAGYSGLYEAIESWDFKKIEKLEEYRLLLKRLQPEFKTRIIPTDIISDLSECLINQECEEILQICSTKGMMAGAEKLVECLLRSDKENWPKTLKLALEKERNKFSELWIVEKGIKDVETEDLEDKMETSDIQIFYQEDPECQNLSENSCPPSEVSDTNLYSPFKPRNYQLELALPAMKGKNTIICAPTGCGKTFVSLLICEHHLKKFPQGQKGKVVFFANQIPVYEQQKSVFSKYFERHGYRVTGISGATAENVPVEQIVENNDIIILTPQILVNNLKKGTIPSLSIFTLMIFDECHNTSKQHPYNMIMFNYLDQKLGGSSGPLPQVIGLTASVGVGDAKNTDEALDYICKLCASLDASVIATVKHNLEELEQVVYKPQKFFRKVESRISDKFKYIIAQLMRDTESLAKRICKDLENLSQIQNREFGTQKYEQWIVTVQKACMVFQMPDKDEESRICKALFLYTSHLRKYNDALIISEHARMKDALDYLKDFFSNVRAAGFDEIEQDLTQRFEEKLQELESVSRDPSNENPKLEDLCFILQEEYHLNPETITILFVKTRALVDALKNWIEGNPKLSFLKPGILTGRGKTNQNTGMTLPAQKCILDAFKASGDHNILIATSVADEGIDIAQCNLVILYEYVGNVIKMIQTRGRGRARGSKCFLLTSNAGVIEKEQINMYKEKMMNDSILRLQTWDEAVFREKILHIQTHEKFIRDSQEKPKPVPDKENKKLLCRKCKALACYTADVRVIEECHYTVLGDAFKECFVSRPHPKPKQFSSFEKRAKIFCARQNCSHDWGIHVKYKTFEIPVIKIESFVVEDIATGVQTLYSKWKDFHFEKIPFDPAEMSK (NCBI Deposit Number: NP_055129.2).

[0156] RIG-I-like receptors: As used herein, the term “RIG-I-like receptors” (abbreviated as “RLR”) refers to any member of the family of DExD / H box RNA helicases that function as cytoplasmic pattern recognition sensors for pathogen-associated molecular patterns (PAMPs) typically found in viral RNA. Upon ligand binding, RLRs signal the activation of downstream transcription factors, driving type I interferon (IFN) production and antiviral gene expression, which trigger intracellular immune responses to control viral infection. Three RLR members have been identified: RIG-I (retinoic acid-induced gene I), MDA5 (melanoma differentiation-associated gene 5), and LGP2 (Laboratory of Genetics and Physiology 2 and a homolog of mouse D11lgp2) (Loo and Gale (2011) Immunity 34(5):680-692).

[0157] RIG-I-like receptor agonists: As used herein, the term "RIG-I-like receptor agonist" (which is used interchangeably with the term "RLR agonist") refers to nucleic acids (e.g., RNA) that bind to RIG-I-like receptors (RLRs) and partially or completely promote, induce, increase, and / or activate downstream pathways mediated by RLR signaling, or other RLR-mediated functions. Examples of RIG-I receptor agonists are provided herein.

[0158] Stable RNA secondary structures: As used herein, the term “stable RNA secondary structure” refers to a structure, fold, or conformation adopted by an RNA molecule, or a local segment or portion thereof, that is sustainably maintained under physiological conditions and characterized by a low free energy state. Typical examples of stable RNA secondary structures include double helix, hairpin, and stem-loop. Stable RNA secondary structures are known in the art to exhibit various bioactivities. As used with reference to polynucleotide double helix, the term “stable” means that the double helix remains essentially exclusively hybridized, structured, or annealed in double helix form under physiological conditions or under typical salt and temperature conditions used in the diagnostic or therapeutic applications of nucleic acids.

[0159] Subjects: As used herein, the term “subjects” includes any human or non-human animal. For example, the methods and compositions of the present invention can be used to treat subjects with immune disorders. The term “non-human animals” includes all vertebrates, e.g., mammals and non-mammals, e.g., non-human primates, sheep, dogs, cattle, chickens, amphibians, reptiles, etc.

[0160] T cells: The term "T cells" refers to a type of white blood cell that can be distinguished from other white blood cells by the presence of T cell receptors on its cell surface. T cells comprise several subsets, though not limited to them, including T helper cells (also known as TH cells or CD4+ T cells) and subtypes, including TH1, TH2, TH3, TH17, TH9, and TFH cells; cytotoxic T cells (TC cells, CD8+ T cells, cytotoxic T lymphocytes, T-killer cells, killer T cells); memory T cells and subtypes, including central memory T cells (TCM cells), effector memory T cells (TEM and TEMRA cells), and resident memory T cells (TRM cells); regulatory T cells (also known as Treg cells or suppressor T cells) and subtypes, including CD4+FOXP3+Treg cells, CD4+FOXP3-Treg cells, Tr1 cells, Th3 cells, and Treg17 cells; natural killer T cells (also known as NKT cells); mucosa-associated invariant T cells (MAIT); and gamma delta T cells (γδ T cells), Vγ9 / Vδ2 T cells. One or more of the T cells described above or not described may be the target cell type for the method of use of the present invention.

[0161] T cell activation: As used herein, the term “T cell activation” refers to a cellular process in which mature T cells expressing an antigen-specific T cell receptor on their surface recognize their congener antigen, enter the cell cycle, secrete cytokines or lytic enzymes, initiate cell-based effector functioning, or become competitors in response. T cell activation requires at least two signals for complete activation. The first signal occurs after the involvement of the T cell antigen-specific receptor (TCR) by the major histocompatibility complex (MHC), and the second signal occurs through the subsequent involvement of a co-stimulatory molecule (e.g., CD28). These signals are transmitted to the nucleus, resulting in T cell clonal proliferation, upregulation of activation markers on the cell surface, differentiation into effector cells, induction of cytotoxicity or cytokine secretion, induction of apoptosis, or a combination thereof.

[0162] T cell-mediated responses: As used herein, the term “T cell-mediated responses” refers to any response mediated by T cells, including, but not limited to, effector T cells (e.g., CD8+ cells) and helper T cells (e.g., CD4+ cells). Examples of T cell-mediated responses include, for example, T cell cytotoxicity and proliferation.

[0163] Tetraloop: As used herein, the term "tetraloop" refers to a type of four-base loop motif found in hairpin or stem-loop RNA secondary structures that caps a double helix at one end, linking the two strands that make up the double helix and providing stability to the hairpin structure.

[0164] Therapeutic agents: As used herein, the term “therapeutic agent” means any agent that, when administered to a subject, has a therapeutic, diagnostic and / or preventive effect and / or induces a desired biological and / or pharmacological effect.

[0165] Therapeutic dose: As used herein, the terms “therapeutic dose,” “therapeutic dosage,” or similar terms as used herein are intended to mean the amount of a drug (e.g., a synthetic RIG-I-like receptor agonist) that induces a desired biological or medical response, for example, in a patient already suffering from a disease, that cures or at least partially suppresses a condition or disease and its complications (e.g., improvement of one or more symptoms of cancer). The dose effective for this use depends on the severity of the disease being treated and the overall state of the patient’s own immune system.

[0166] To treat: As used herein, the terms “to treat,” “to treat,” and “treatment” refer to the therapeutic or preventive measures described herein. A “treatment” method involves administering the human antibodies of this disclosure to a subject in need of such treatment, for example, a subject in need of an improved immune response to a particular antigen, or a subject in which such a disorder may eventually be acquired, in order to prevent, cure, delay, reduce the severity of or improve one or more symptoms of a disorder or recurrent disorder, or to extend the subject’s survival beyond what would be expected in the absence of such treatment.

[0167] Tumor Microenvironment: As used herein, the term “tumor microenvironment” (or “cancer environment,” abbreviated as TME) refers to the cellular environment, or surrounding conditions in which a tumor or neoplasm exists, including, but not limited to, the surrounding blood vessels and noncancerous cells, including immunoadipocytes, fibroblasts, myeloid-derived inflammatory cells, and lymphocytes. Signaling molecules and the extracellular matrix also constitute the TME. The tumor and the surrounding microenvironment are closely related and constantly interacting. Tumors can influence the microenvironment by releasing extracellular signals, promoting tumor angiogenesis, and inducing peripheral immune tolerance, while immune cells in the microenvironment can influence the growth and evolution of tumor cells.

[0168] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to whom this disclosure relates. Preferred methods and materials are described below, but similar or equivalent methods and materials may also be used in the implementation or testing of methods and compositions currently disclosed. All publications, patent applications, patents and other references referenced herein are incorporated in their entirety by reference.

[0169] Equivalents and range Those skilled in the art will recognize many equivalents to the specific embodiments described herein, or can confirm this through experiments not exceeding routine experimentation. The scope of this disclosure is not limited to the foregoing description, but is set forth in the appended claims.

[0170] In the claims, articles such as “a,” “an,” and “the” may mean one or more unless shown to be contradictory or is not evident from the context. A claim or description containing “or” between one or more members of a group is deemed satisfied unless shown to be contradictory or is not evident from the context if one, more or all of the members of that group are present in, used in, or otherwise related to a given product or process. This disclosure includes embodiments in which exactly one member of that group is present in, used in, or otherwise related to a given product or process. This disclosure includes embodiments in which more or all of the members of that group are present in, used in, or otherwise related to a given product or process. Furthermore, it should be understood that this disclosure encompasses all variations, combinations, and substitutions in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the enumerated claims are introduced into another claim. For example, any claim dependent on another claim may be modified to include one or more limitations found in any other claim dependent on the same basic claim. Furthermore, where a claim references a composition, it should be understood that, unless otherwise indicated or unless it is obvious to a person skilled in the art that such reference would result in a contradiction or inconsistency, it includes a method of using that composition for any of the purposes disclosed herein and a method of producing that composition according to any of the manufacturing methods disclosed herein or other methods known in the art.

[0171] When elements are presented as a list, for example in Markush group format, it should be understood that each subgroup of those elements is also disclosed, and any element can be removed from that group. Generally, when the present invention or an aspect of the present invention is referred to as including certain elements, features, etc., it should be understood that a particular embodiment of the present invention or an aspect of the present invention consists of or is essentially derived from such elements, features, etc. For the sake of simplification, these embodiments are not specifically described herein in these terms.

[0172] Furthermore, it should be noted that the term “comprising” is intended to be inclusive and, while not required, may include additional elements or processes. Therefore, wherever the term “comprising” is used herein, the term “consisting of” is also included and disclosed.

[0173] Where a range is given, boundary values ​​are included. Furthermore, unless otherwise specifically indicated or is obvious from the content and the understanding of those skilled in the art, values ​​expressed as a range may be assumed to be any specific value or subrange within the ranges described in different embodiments of the invention, up to one-tenth of the lower limit unit of that range, unless the content clearly indicates otherwise.

[0174] In addition, it should be understood that any particular embodiment of the present invention contained in the prior art may be expressly excluded from any one or more claims. Since such embodiments are considered to be known to those skilled in the art, they may be excluded even if their exclusion is not expressly stated herein. Any particular embodiment of the composition of the present invention (e.g., any nucleic acid or protein encoded thereby, any method of production, any method of use, etc.) may be excluded from any one or more claims for any reason, whether or not it relates to the existence of the prior art.

[0175] All sources cited herein, such as references, publications, databases, database entries, and technology, are incorporated by reference to this application, even if not explicitly stated in the citation. In the event of any conflict between the cited sources and the descriptions in this application, the descriptions in this application shall prevail.

[0176] RIG-I-like receptors and their ligands This disclosure provides synthetic RNA ligands (RLR agonists) that specifically bind to and agonize RLRs, which are RRG-I-like receptors (RLRs). In some embodiments, this disclosure provides RLR agonists useful for the treatment of cancer. In some embodiments, this disclosure provides RLR agonists useful for the treatment of infectious diseases. In some embodiments, the RLR agonists induce cytokine production. In some embodiments, the RLR agonists increase the number of CD8+ T cells in the tumor microenvironment. In some embodiments, the RLR agonists induce protective anti-tumor immunity.

[0177] RIG-I-like receptors (RLRs) include a family of DExD / H-box RNA helicases that function as cytoplasmic pattern recognition receptors (PRRs) that sense the presence of pathogenic drugs by recognizing pathogen-associated molecular patterns (PAMPs). In particular, the intracellular presence of non-self (e.g., viral) RNA is detected by infected cells through RNA binding to RLRs, leading to the initiation and regulation of antiviral immunity. Like most viral RNAs, endogenous mRNA and RNA polymerase III transcripts are 5'-triphosphorylated, but eukaryotic mRNA has a 5' cap structure linked to N7-methylated guanosine, which prevents RIG-I activation. These structural differences between viral RNA and self RNA, along with differences in intracellular localization, are thought to enable the effective function of RIG-I as a defense against viral infection by preferentially detecting viral RNA. Molecular recognition and binding of non-self RNA ligands to RLRs drives specific intracellular signaling events, leading to the activation of transcription factors that drive type I interferon (IFN) production and antiviral gene expression. RLR-mediated induction of IFNs and production of inflammatory cytokines, as well as antiviral gene expression, trigger an immune response that controls viral infection (Yoneyama et al., (2015) Curr Opin Immunol 32:48-53).

[0178] Three RLR family members have been identified as the initial members and best characterize the RLR family: RIG-I (retinoic acid-induced gene I), MDA5 (melanoma differentiation-related gene 5), and LGP2 (Laboratory of Genetics and Physiology 2 and a homolog of mouse D11lgp2). RIG-I is an important component of the innate immune system and plays a crucial role in defense against RNA virus infections. In contrast to Toll-like receptors TLR3, TLR7, TLR8, and TLR9, which detect nucleic acids in some endosomes of immune cells, RIG-I is a cytoplasmic innate immune receptor expressed in all cell types (Kato et al., (2006) Nature 441(7089):101-105; Loo et al., (2008) J Virol 82(1):335-345). Two early studies independently established that RIG-I specifically detects viral RNA and is activated by viral RNA (Hornung et al., (2006) Science 314(5801):994-997; Pichlmair et al., (2006) Science 314(5801):997-1001).

[0179] High-resolution structures of the RIG-I / ligand complex provide molecular details of the binding of RIG-I to RNA ligands, particularly the activating ligand, double-stranded 5'-triphosphorylated RNA (ppp-dsRNA) (Civril et al., (2011) EMBO Reports 12(11):1127-1134; Jiang et al., (2011) Nature 479(7373):423-427; Kowalinski et al., (2011) Cell 147(2):423-435; Lu et al., (2010) Structure 18(8):1032-1043; Luo et al., (2011) Cell 147(2)409-422; Wang et al., (2010) Nature Structural & Molecular Biology). 17(7):781-787; Hornung et al., (2006) Science 314(5801):994-997; Pichlmair et al., (2006) Science 314(5801):997-1001; Schlee et al., (2009) Immunity 31(1):25-34). The crystal structure of the RIG-I / RNA complex shows protein binding to the backbone rather than the bases, which suggests that the RNA sequence may not affect RIG-I binding, or that the RNA sequence may exhibit effects or activities that have not yet been characterized. To date, evidence of sequence-dependent distinct interactions or affinities with RIG-I-like receptors and activation of RIG-I-like receptors has not been described in the art (Schlee and Hartmann (2010) Molecular Therapy 18(7):1254-1262).

[0180] Accordingly, the Disclosure provides synthetic RIG-I-like receptor (RLR) agonists comprising synthetic and / or engineered RLR RNA ligands that do not exist in nature. In some embodiments, the Disclosure provides RLR agonists that specifically bind to a RIG-I-like receptor (RLR), wherein the agonist comprises a blunt-terminated hairpin RNA comprising a first polynucleotide linked to a second polynucleotide by a linker, the first polynucleotide being sufficiently complementary to the second polynucleotide and forming a double helix, the double helix comprising fewer than 19 base pairs, the 5' terminal nucleotide of the first oligonucleotide comprising a 5' diphosphate or tripphosphate moiety, or a derivative or analog thereof, the agonist comprising a sequence motif, the sequence motif conferring at least one improved RLR-mediated bioactivity compared to agonists that do not contain this sequence motif.

[0181] In some embodiments, the RLR agonist of this disclosure includes a sequence motif, the sequence motif being: (i) GT repeat motif, (ii) GA repeat motif, (iii) AUCG repeat motif, (iv) AU repeat motif, (v) Dipyrimidine motif, (vi) Ziplin motif, (vii) Pyrimidine triplet motif, (viii) Printed riplet motif, (ix) Palindromic arrangement motifs, and The group is selected from any combination of (x)(i) to (ix).

[0182] In some embodiments, the RLR agonists of this disclosure include at least one improved bioactivity, the improved bioactivity being: (i) Increased cytokine production mediated by RLR, (ii) Increased expression of interferon-inducible genes mediated by RLR, (iii) Increased intracellular signaling mediated by RLRs, (iv) Increased binding affinity to RLR, and (v)(i)~(iv) is selected from any combination.

[0183] In some embodiments, the RLR agonists of the present disclosure comprise a sequence motif, which is a GT repeat motif comprising a sequence of less than 19, about 15-18, about 15, about 10-15, about 10, about 5-10, about 5, about 4, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or 4 guanine and thymine nucleotides, or derivatives or analogs thereof. In some embodiments, the GT repeat motif is [GT] n The formula is such that n = 2 to 9. In some embodiments, the GT repeat motif is [GT]7. In some embodiments, the GT repeat motif is [GT]3, followed by a print repeat and a UCG, respectively. In some embodiments, the print repeat is GGA.

[0184] In some embodiments, the sequence motif is a GA repeat motif comprising a sequence of less than 19, about 15-18, about 15, about 10-15, about 10, about 5-10, about 5, about 4, about 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or 4 guanine and adenine nucleotides, or derivatives or analogs thereof. In some embodiments, the GA repeat motif is [GA] n The formula is such that n = 2 to 9. In some embodiments, the GA repeat motif is [GA]7.

[0185] In some embodiments, the RLR agonists of the present disclosure comprise a sequence motif, which is an AUCG repeat motif comprising sequences of less than 19, about 16, about 12-16, about 12, about 8-12, about 6, about 16, 12, and 8 adenine, uracil, cytosine, and guanine nucleotides, or derivatives or analogs thereof.

[0186] In some embodiments, the AUCG repeat motif is [AUCG] n The formula is such that n = 2 to 4. In some embodiments, the AUCG repeat motif is [AUCG]3.

[0187] In some embodiments, a CG or dipyrimidine motif is present before the AUCG repeat motif. In some embodiments, a CG is present before the AUCG repeat motif. In some embodiments, the dipyrimidine motif is CC. In some embodiments, a diprine motif is present before the AUCG repeat motif. In some embodiments, the diprine motif is GA. In some embodiments, the diprine motif is GG.

[0188] In some embodiments, the RLR agonist of this disclosure comprises an AUCG repeat motif, in which one or more uridine nucleosides (U) are substituted with a modified nucleoside. In some embodiments, the modified nucleoside is ribothymidine (T). In some embodiments, the AUGC repeat motif is [AUCG]3, in which one or more uridine nucleosides (U) constituting the AUCG repeat motif are substituted with a modified nucleoside, the modified nucleoside being ribothymidine (T). In some embodiments, the AUGC repeat motif is [AUCG]3, in which one or more uridine nucleosides (U) constituting the AUCG repeat motif are substituted with a modified nucleoside, the modified nucleoside being ribothymidine (T), and GG is present before the AUGC repeat motif.

[0189] In some embodiments, the RLR agonist of this disclosure comprises an AUCG repeat motif, in which one or more guanosine nucleosides (G) are substituted with a modified nucleoside. In some embodiments, the modified nucleoside is inosine (I). In some embodiments, the AUGC repeat motif is [AUCG]3, in which one or more guanosine nucleosides (G) constituting the AUCG repeat motif are substituted with a modified nucleoside, the modified nucleoside being ribothymidine (T), and GG is present before the AUGC repeat motif.

[0190] In some embodiments, the RLR agonist of the present disclosure includes an AUCG repeat motif, with an IG preceding this motif. In some embodiments, the AUCG repeat motif is [AUCG]3, with an IG preceding this motif.

[0191] In some embodiments, the RLR agonist of the Disclosure comprises an AUCG repeat, in which one or more guanosine nucleosides (G) are substituted with inosine (I), and inosine (I) is present before the AUCG repeat. In some embodiments, the guanosine nucleosides (G) constituting the AUCG repeat are substituted with inosine (I), inosine (I) is present before the AUCG repeat, and the 5' terminal nucleotide of the first polynucleotide comprises inosine (I).

[0192] In some embodiments, the 5' terminal nucleotide of the first oligonucleotide contains inosine (I).

[0193] In some embodiments, the RLR agonist of the present disclosure includes an AUCG repeat sequence motif, where the AUCG repeat motif is [AUCG]2. In some embodiments, a ziplin motif precedes the AUCG repeat motif. In some embodiments, the ziplin motif is GG. In some embodiments, a print replet precedes the AUCG repeat motif. In some embodiments, the print replet is GGG. In some embodiments, CCCCCG precedes the AUCG repeat motif. In some embodiments, TCGUCG precedes the AUCG repeat motif.

[0194] In some embodiments, the RLR agonists of the Disclosure comprise a palindromic sequence comprising a sequence of fewer than 19, about 15-18, about 15, about 10-15, about 10, about 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or 4 nucleotides, or derivatives or analogs thereof, which are concatenated in any order to generate a palindrome.

[0195] In some embodiments, the linker is adjacent to the AU. In some embodiments, the linker is adjacent to the AU repeat motif, and the AU repeat motif is [AU] n The formula is such that n = 2 to 3. In some embodiments, the AU repeat motif is [AU]2.

[0196] In some embodiments, the Disclosure provides an RLR agonist that specifically binds to RLR, wherein the agonist comprises a blunt-terminated hairpin RNA containing at least one nucleotide, including an inosine that forms a base pair with cytidine, and the agonist has the following formula: The formula includes 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', and in the formula, (i)(N1-N2-X1) comprises a first polynucleotide containing linked nucleotides N1, N2 and X1, (ii)(X2-N3-N4) comprises a second polynucleotide containing linked nucleotides X2, N3 and N4, (iii) N1, N2, N3, and N4 each contain a single nucleotide comprising a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine. (iv)N1 forms a base pair with N4, (v)N2 forms a base pair with N3, (vi)N1 comprises a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof. (vii)X1 and X2 are oligonucleotides containing a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine, respectively. (viii) X1 is complementary to X2, (ix) X1 and X2 are each the same length, ranging from 12 to 16 nucleotides in length. (x)L is a linker that covalently bonds the first polynucleotide to the second polynucleotide.

[0197] In another embodiment, the Disclosure provides a synthetic RIG-I-like receptor (RLR) agonist that specifically binds to a RIG-I-like receptor (RLR), wherein the agonist comprises a blunt-terminated hairpin RNA containing a non-nucleotide linker, and the agonist has the following formula: The formula includes 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', and in the formula, (i)(N1-N2-X1) comprises a first polynucleotide containing linked nucleotides N1, N2 and X1, (ii)(X2-N3-N4) comprises a second polynucleotide containing linked nucleotides X2, N3 and N4, (iii) N1, N2, N3, and N4 each contain a single nucleotide comprising a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine. (iv)N1 forms a base pair with N4, (v)N2 forms a base pair with N3, (vi)N1 comprises a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof. (vii)X1 and X2 are oligonucleotides containing a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine, respectively. (viii) X1 is complementary to X2, (ix) X1 and X2 are each the same length, ranging from 12 to 16 nucleotides in length. (x)L is a non-nucleotide linker that covalently bonds the first polynucleotide to the second polynucleotide.

[0198] In some embodiments, inosine forms a base pair with cytidine when present in the RLR agonist.

[0199] In some embodiments, the linker (L) is a nucleotide linker or a non-nucleotide linker.

[0200] In some embodiments, the disclosure provides RLR agonists that specifically bind to RLRs, wherein the agonist comprises a blunt-terminated hairpin RNA containing a nucleotide linker or a non-nucleotide linker. RNA hairpins are one of the most common RNA secondary structure elements, and the hybridized portion or "stem" of the hairpin is often covered by an RNA tetraloop. An RNA tetraloop consists of four characteristic loop nucleotides that form a compressed, stable structure. While RNA tetraloops can be formed by many different nucleotide sequences, UNCG (N=A, C, G or U), GNRA (R=A or G), and CUUG tetraloops are the most frequently found. Tetraloops typically help initiate the RNA folding process, providing sites for intra-RNA or inter-RNA tertiary contacts and sites for protein binding, thereby facilitating the assembly of ribonucleoprotein particles. Further descriptions of tetraloops can be found in Cheong, H., Kim, N., and Cheong, C. (2015). RNA Structure: Tetraloops. In eLS, John Wiley & Sons, Ltd (eds.), which is incorporated herein by reference in its entirety.

[0201] Therefore, in some embodiments, the RLR agonist of this disclosure comprises a nucleotide linker including a tetraloop. In some embodiments, the nucleotide sequence of the tetraloop is as follows: (a) UNCG (wherein N = A, C, G, or U), (b) GNRA (wherein N = A, C, G or U, and R = A or G), (c) ANYA (wherein N = A, C, G or U, and Y = C or T), (d) CUYG (where Y = C or T), (e)UMAC (where M=A or C), and (f) Select from the group consisting of CUUG.

[0202] In some embodiments, the nucleotide linker contains the nucleotide sequence UUUGAU or UGUUU. In some embodiments, the tetraloop sequence is UUCG. In some embodiments, the tetraloop sequence is GAUC. In some embodiments, the nucleotide linker contains the nucleotide sequence UUUGAU. In some embodiments, the nucleotide linker contains the nucleotide sequence UGUUU.

[0203] In other embodiments, the RLR agonists of this disclosure include non-nucleotide linkers. As described herein, nucleic acid loops (e.g., tetraloops) are common elements found in nucleic acid secondary structures. Nucleotide loops arise in folded domains that occur in intra-chain double helix. Synthetic nucleic acids designed to include hairpin loops containing non-nucleotide linkers (e.g., non-nucleotide linkers) can replace several nucleotides that bridge the folded double-stranded structure. Non-nucleotide groups have also been used as linkers in unfolded structures. Such linkers can be useful substitutes for natural nucleotide linkers (e.g., tetraloops). For example, such linkers can shorten the synthesis of nucleic acids with the desired secondary structure by several steps, by replacing one relatively long non-nucleotide linker with several individual nucleotides that could naturally constitute a loop. Such non-natural loops or linkers (e.g., non-nucleotide linkers) can confer resistance to degradation by nucleases that normally act on natural loop structures in biological situations (e.g., in target cells at administration or in circulation). Non-nucleotide linking groups may also provide more stable folded structures than those resulting from nucleotide loops and / or linkers. Further descriptions of non-nucleotide linkers can be found in Rumney and Kool (1995) J Am Chem Soc 117:5635-5646, which is incorporated herein by reference in its entirety.

[0204] Therefore, in some embodiments, the RLR agonist of this disclosure is as follows: (a) Ethylene glycol linker, and (b) A non-nucleotide linker selected from the group consisting of alkyl linkers.

[0205] In some embodiments, the non-nucleotide linker is a hexaethylene glycol linker. In some embodiments, the non-nucleotide linker is a C9 alkyl linker.

[0206] In some embodiments, the RLR agonist comprises a 5'-diphosphate moiety, or a derivative or analog thereof. In some embodiments, the agonist comprises a 5'-triphosphate moiety, or a derivative or analog thereof. In some embodiments, the derivative or analog comprises a phosphate bioequivalent, which is selected from phosphonates, thiophosphonates, phosphorothioates, sulfates, sulfonates, sulfamates, thiazolidinones, carboxylates, malonates, boronic acids, benzoxabolols, boranophosphates, and squalamides.

[0207] In some embodiments, the agonist comprises a modified nucleotide, a modified nucleoside, or a modified nucleic acid base, or a combination thereof. In some embodiments, the agonist comprises modifications to internucleotide bonds or polynucleotide backbones.

[0208] In some embodiments, the RLR agonist of this disclosure has the following characteristics: (a) Specifically binds to one or more RLRs (e.g., RIG-1, MDA5 and / or LGP2), (b) Increase cytokine production mediated by RLR, (c) Increase the RLR-mediated expression of interferon-inducible genes (ISGs), (d) Increases RLR-dependent intracellular signaling, (e) To increase the stability of the double helix, (f) Increase binding affinity to RLR, (g) Reduce extratarget binding, (h) To lengthen the biological half-life, (i) To improve in vivo distribution and bioavailability, (j) Increase and / or enhance uptake into cells and / or tissues, (k) Reduce immunogenicity, and (l)(a)~(k) must be shown as at least one of the combinations.

[0209] In some embodiments, the present disclosure provides synthetic RIG-I-like receptor (RLR) agonists that specifically bind to RIG-I-like receptors (RLRs), wherein the agonist has the following formula: It contains blunt-ended hairpin RNA including 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', in the formula, (i)(N1-N2-X1) comprises a first polynucleotide containing linked nucleotides N1, N2 and X1, (ii)(X2-N3-N4) comprises a second polynucleotide containing linked nucleotides X2, N3 and N4, (iii) N1, N2, N3, and N4 each contain a single nucleotide comprising a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine. (iv)N1 forms a base pair with N4, (v)N2 forms a base pair with N3, (vi)N1 comprises a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof. (vii)X1 and X2 are oligonucleotides containing a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine, respectively. (viii) X1 is complementary to X2, (ix) X1 and X2 are each the same length, ranging from 12 to 16 nucleotides in length. (x)L is a linker that operably links the first polynucleotide and the second polynucleotide. In the formula, at least one of N1, N2, N3, and N4 is inosine, and / or at least one of X1 and / or X2 contains at least one inosine nucleoside, which forms a base pair with cytidine in the hairpin RNA.

[0210] In some embodiments, N1 contains inosine and N4 contains cytidine. In some embodiments, N1 contains cytidine and N4 contains inosine. In some embodiments, N2 contains inosine and N3 contains cytidine. In some embodiments, N2 contains cytidine and N3 contains inosine. In some embodiments, N1 contains guanosine. In some embodiments, N2 contains guanosine. In some embodiments, N1 contains cytidine. In some embodiments, N2 contains cytidine. In some embodiments, N1 and N2 contain guanosine and N3 and N4 contain cytidine. In some embodiments, N1 and N2 contain cytidine and N3 and N4 contain guanosine. In some embodiments, N1 and N2 contain inosine and N3 and N4 contain cytidine. In some embodiments, N1 and N2 contain cytidine and N3 and N4 contain inosine. In some embodiments, N1 contains inosine, N4 contains cytidine, and X1 and / or X2 each contain at least one inosine. In some embodiments, N2 contains inosine, N3 contains cytidine, and X1 and / or X2 each contain at least one inosine. In some embodiments, N1 and N2 contain guanosine, N3 and N4 contain cytidine, and X1 and / or X2 each contain at least one inosine. In some embodiments, N1 and N2 contain guanosine, N3 and N4 contain cytidine, and X1 and X2 each contain at least one inosine. In some embodiments, N1 and N2 contain cytidine, N3 and N4 contain guanosine, and X1 and X2 each contain at least one inosine. In some embodiments, N1 and N2 contain guanosine, N3 and N4 contain cytidine, and X1 and X2 each contain inosine, but do not contain guanosine nucleoside. In some embodiments, N1 and N2 contain cytidine, N3 and N4 contain guanosine, and X1 and X2 each contain inosine, but do not contain guanosine nucleoside.In some embodiments, N1 and N2 contain inosine, N3 and N4 contain cytidine, and X1 and / or X2 each contain at least one inosine. In some embodiments, N1 and N2 contain inosine, N3 and N4 contain cytidine, and X1 and X2 each contain at least one inosine. In some embodiments, N1 and N2 contain cytidine, N3 and N4 contain inosine, and X1 and / or X2 each contain at least one inosine. In some embodiments, N1 and N2 contain inosine, N3 and N4 contain cytidine, and X1 and X2 contain inosine, but do not contain guanosine nucleoside. In some embodiments, N1 and N2 contain cytidine, N3 and N4 contain inosine, and X1 and X2 contain inosine, but do not contain guanosine nucleoside. In some embodiments, X1 and X2 each have 12 nucleotides and contain 1, 2, 3, or 4 inosine nucleosides. In some embodiments, X1 and X2 each have 13 nucleotides and contain 1, 2, 3, 4, or 5 inosine nucleosides. In some embodiments, X1 and X2 each have 14 nucleotides and contain 1, 2, 3, 4, 5, or 6 inosine nucleosides. In some embodiments, X1 and X2 each have 15 nucleotides and contain 1, 2, 3, 4, 5, 6, or 7 inosine nucleosides. In some embodiments, X1 and X2 each have 16 nucleotides and contain 1, 2, 3, 4, 5, 6, 7, or 8 inosine nucleosides. In some embodiments, X1 and X2 each have 12 nucleotides and contain at least 10%, 20%, 30%, or 40% inosine nucleosides.

[0211] In some embodiments, the present disclosure provides synthetic RIG-I-like receptor (RLR) agonists that specifically bind to RIG-I-like receptors (RLRs), wherein the agonist has the following formula: It contains blunt-ended hairpin RNA including 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', in the formula, (i)(N1-N2-X1) comprises a first polynucleotide containing linked nucleotides N1, N2 and X1, (ii)(X2-N3-N4) comprises a second polynucleotide containing linked nucleotides X2, N3 and N4, (iii) N1, N2, N3, and N4 each contain a single nucleotide comprising a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine. (iv)N1 forms a base pair with N4, (v)N2 forms a base pair with N3, (vi)N1 comprises a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof. (vii)X1 is the array motif [AUCN5] x This includes, where N5 contains guanosine or inosine, and x is an integer whose value indicates the number of sequence motifs, such that x = 3 or 4. (viii)X2 is the array motif [CN6AU] y The formula includes, where N6 contains guanosine or inosine, and y is an integer whose value indicates the number of sequence motifs, where y = 3 or 4. (ix)L is a linker that operably links the first polynucleotide and the second polynucleotide. In some embodiments, at least one of N1, N2, N3, and N4 is inosine, and the inosine nucleoside forms a base pair with cytidine in the hairpin RNA. In some embodiments, N5 contains inosine and N6 contains inosine. In some embodiments, N5 contains guanosine and N6 contains inosine. In some embodiments, N5 contains inosine and N6 contains guanosine. In some embodiments, N5 contains guanosine (G) and N6 contains guanosine (G). In some embodiments, x=3 and y=3. In some embodiments, x=4 and y=4. In some embodiments, N1 contains inosine (I) and N4 contains cytidine (C). In some embodiments, N2 contains inosine (I) and N3 contains cytidine (C). In some embodiments, N3 contains inosine (I) and N2 contains cytidine (C). In some embodiments, N4 contains inosine (I) and N1 contains cytidine (C). In some embodiments, N1 contains guanosine (G). In some embodiments, N2 contains guanosine (G). In some embodiments, N1 contains cytidine (C). In some embodiments, N2 contains cytidine (C). In some embodiments, N1 and N2 contain guanosine (G) and N3 and N4 contain cytidine (C). In some embodiments, N1 and N2 contain cytidine (C) and N3 and N4 contain guanosine (G). In some embodiments, N1 and N2 contain inosine (I) and N3 and N4 contain cytidine (C). In some embodiments, N1 and N2 contain cytidine (C) and N3 and N4 contain inosine (I).

[0212] In some embodiments, linker(L) is a nucleotide linker or a non-nucleotide linker. In some embodiments, linker(L) is a nucleotide linker containing a tetraloop, the nucleotide sequence of the tetraloop being as follows: (a) UNCG (wherein N = A, C, G, or U), (b) GNRA (wherein N = A, C, G or U, and R = A or G), (c) ANYA (wherein N = A, C, G or U, and Y = C or T), (d) CUYG (where Y = C or T), (e)UMAC (where M=A or C), and (f) Select from the group consisting of CUUG.

[0213] In some embodiments, linker(L) is a nucleotide linker comprising the nucleotide sequence UUUGAU or UGUUU. In some embodiments, the nucleotide linker comprises the nucleotide sequence UUUGAU. In some embodiments, the nucleotide linker comprises the nucleotide sequence UGUUU.

[0214] In some embodiments, linker(L) is a nucleotide linker containing a tetraloop, the sequence of the tetraloop being UUCG. In some embodiments, the sequence of the tetraloop is GAUC.

[0215] In some embodiments, the linker (L) is as follows: (a) Ethylene glycol linker, and (b) A non-nucleotide linker selected from the group consisting of alkyl linkers.

[0216] In some embodiments, the non-nucleotide linker is a hexaethylene glycol linker. In some embodiments, the non-nucleotide linker is a C9 alkyl linker.

[0217] In some embodiments, the RLR agonist comprises a 5'-diphosphate moiety, or a derivative or analog thereof. In some embodiments, the agonist comprises a 5'-triphosphate moiety, or a derivative or analog thereof. In some embodiments, the derivative or analog comprises a phosphate bioequivalent, which is selected from phosphonates, thiophosphonates, phosphorothioates, sulfates, sulfonates, sulfamates, thiazolidinones, carboxylates, malonates, boronic acids, benzoxabolols, boranophosphates, and squalamides.

[0218] In some embodiments, the RLR agonist comprises a modified nucleotide, a modified nucleoside, or a modified nucleic acid base, or a combination thereof. In some embodiments, the agonist comprises modifications to internucleotide bonds or polynucleotide backbones.

[0219] In some embodiments, the RLR agonist of this disclosure has the following characteristics: (a) Specifically binds to one or more RLRs (e.g., RIG-1, MDA5 and / or LGP2), (b) Increase cytokine production mediated by RLR, (c) Increase the RLR-mediated expression of interferon-inducible genes (ISGs), (d) Increases RLR-dependent intracellular signaling, (e) To increase the stability of the double helix, (f) Increase binding affinity to RLR, (g) Reduce extratarget binding, (h) To lengthen the biological half-life, (i) To improve in vivo distribution and bioavailability, (j) Increase and / or enhance uptake into cells and / or tissues, (k) Reduce immunogenicity, and (l)(a)~(k) must be shown as at least one of the combinations.

[0220] In some embodiments, the Disclosure relates to a synthetic RIG-I-like receptor (RLR) agonist that specifically binds to a RIG-I-like receptor (RLR), wherein the agonist comprises a blunt-terminated hairpin RNA containing a first polynucleotide linked to a second polynucleotide by a linker, the first polynucleotide being sufficiently complementary to the second polynucleotide and forming a double helix, the double helix containing fewer than 19 base pairs, and the 5' terminal nucleotide of the first oligonucleotide being a 5' diphosphate or triphosphate moiety, or a derivative thereof. The invention provides a synthetic RIG-I-like receptor (RLR) agonist comprising a sequence motif, which, compared to an agonist without this sequence motif, provides at least one improved RLR-mediated bioactivity, wherein the agonist comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 and 36.

[0221] In some embodiments, the Disclosure provides a synthetic RIG-I-like receptor (RLR) agonist that specifically binds to a RIG-I-like receptor (RLR), wherein the agonist comprises a blunt-terminated hairpin RNA containing a first polynucleotide linked to a second polynucleotide by a linker, the first polynucleotide being sufficiently complementary to the second polynucleotide and forming a double helix, the double helix containing fewer than 19 base pairs, the 5' terminal nucleotide of the first oligonucleotide comprising a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof, the agonist comprising a sequence motif, the sequence motif conferring at least one improved RLR-mediated bioactivity compared to an agonist without the sequence motif, and the first polynucleotide and the second polynucleotide are as follows: (i) Sequence IDs 37 and 68, respectively (ii) Sequence IDs 38 and 69, respectively (iii) Sequence IDs 39 and 70, respectively (iv) Sequence IDs 40 and 71, respectively (v) Sequence IDs 41 and 72, respectively (vi) Sequence IDs 42 and 73, respectively (vii) Sequence numbers 43 and 74, respectively (viii) Sequence IDs 44 and 75, respectively (ix) Sequence numbers 45 and 76, respectively (x) Sequence numbers 46 and 77, respectively (xi) Sequence IDs 47 and 78, respectively (xii) Sequence IDs 48 and 79, respectively (xiii) Sequence IDs 49 and 80, respectively (xiv) Sequence IDs 50 and 81, respectively (xv) Sequence IDs 51 and 82, respectively (xvi) Sequence numbers 52 and 83, respectively (xvii) Sequence IDs 53 and 84, respectively (xviii) Sequence IDs 54 and 85, respectively (xix) Sequence numbers 55 and 86, respectively. (xx) Sequence numbers 56 and 87, respectively. (xxi) Sequence numbers 57 and 88, respectively. (xxii) Sequence IDs 58 and 89, respectively. (xxiii) Sequence numbers 59 and 89, respectively (xxiv) Sequence IDs 60 and 90, respectively (xxv) Sequence numbers 61 and 91, respectively. (xxvi) Sequence IDs 62 and 92, respectively (xxvii) Sequence numbers 63 and 91, respectively. (xxviii) Sequence numbers 64 and 93, respectively. (xxix) Sequence numbers 65 and 94, respectively. (xxx) Sequence numbers 66 and 95, respectively. (xxxi) Sequence numbers 67 and 96, respectively, and (xxxii) Each contains a nucleotide sequence selected from the group consisting of SEQ ID NOs. 63 and 97.

[0222] In some embodiments, the Disclosure provides synthetic RIG-I-like receptor (RLR) agonists that specifically bind to RIG-I-like receptors (RLRs), wherein the agonist comprises a blunt-terminated hairpin RNA containing at least one nucleotide including inosine that forms a base pair with cytidine, and the agonist comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 22, 23, and 25.

[0223] In some embodiments, the Disclosure provides a synthetic RIG-I-like receptor (RLR) agonist that specifically binds to a RIG-I-like receptor (RLR), the agonist comprising a blunt-terminated hairpin RNA comprising at least one nucleotide containing inosine that forms a base pair with cytidine, the agonist comprising the formula 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', where (N1-N2-X1) comprises a first polynucleotide, (X2-N3-N4) comprises a second polynucleotide, and the first and second polynucleotides are as follows: (i) Sequence IDs 58 and 89, respectively (ii) Sequence IDs 59 and 89, respectively, and (iii) Each contains a nucleotide sequence selected from the group consisting of SEQ ID NOs. 61 and 91.

[0224] In some embodiments, the present disclosure provides a synthetic RIG-I-like receptor (RLR) agonist that specifically binds to a RIG-I-like receptor (RLR), the agonist comprising a blunt-terminated hairpin RNA containing a non-nucleotide linker, the agonist comprising the formula 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', where (N1-N2-X1) comprises a first polynucleotide, (X2-N3-N4) comprises a second polynucleotide, and the first and second polynucleotides are as follows: (i) Sequence IDs 37 and 68, respectively (ii) Sequence IDs 38 and 69, respectively (iii) Sequence IDs 39 and 70, respectively (iv) Sequence IDs 40 and 71, respectively (v) Sequence IDs 41 and 72, respectively (vi) Sequence IDs 42 and 73, respectively (vii) Sequence numbers 43 and 74, respectively (viii) Sequence IDs 44 and 75, respectively (ix) Sequence numbers 45 and 76, respectively (x) Sequence numbers 46 and 77, respectively (xi) Sequence IDs 47 and 78, respectively (xii) Sequence IDs 48 and 79, respectively (xiii) Sequence IDs 49 and 80, respectively (xiv) Sequence IDs 50 and 81, respectively (xv) Sequence IDs 51 and 82, respectively (xvi) Sequence numbers 52 and 83, respectively (xvii) Sequence IDs 53 and 84, respectively (xviii) Sequence IDs 54 and 85, respectively (xix) Sequence numbers 55 and 86, respectively. (xx) Sequence numbers 56 and 87, respectively. (xxi) Sequence numbers 57 and 88, respectively. (xxii) Sequence IDs 58 and 89, respectively. (xxiii) Sequence numbers 59 and 89, respectively (xxiv) Sequence IDs 60 and 90, respectively (xxv) Sequence numbers 61 and 91, respectively. (xxvi) Sequence IDs 62 and 92, respectively (xxvii) Sequence numbers 63 and 91, respectively. (xxviii) Sequence numbers 64 and 93, respectively. (xxix) Sequence numbers 65 and 94, respectively. (xxx) Sequence numbers 66 and 95, respectively. (xxxi) Sequence numbers 67 and 96, respectively, and (xxxii) Each contains a nucleotide sequence selected from the group consisting of SEQ ID NOs. 63 and 97.

[0225] In some embodiments, the disclosure provides RLR agonists wherein the nucleotide sequence containing the agonist is not complementary to a genomic DNA sequence or mRNA sequence, the RLR agonist does not participate in RNA interference, and the RLR agonist does not silence gene expression.

[0226] RLR agonists containing modified nucleic acid bases, nucleosides, or nucleotides In some embodiments, the RLR agonists of this disclosure comprise one or more modified nucleic acid bases, nucleosides, or nucleotides. In some embodiments, the modified RLR agonists may possess useful properties, including improved stability, intracellular retention, improved target binding, and / or increased innate immune response, compared to a reference unmodified RLR agonist. Thus, the use of modified RLR agonists can not only have reduced immunogenicity but also enhance the efficiency of target binding and intracellular retention of nucleic acids. In one embodiment, the agonist provided by this disclosure comprises one or more oligonucleotides comprising at least one region modified to enhance target binding affinity. The affinity of an oligonucleotide to a target polypeptide (e.g., an RLR receptor) can be determined, for example, by measuring the degree of fluorescence polarization (FP) when a fluorescently labeled oligonucleotide binds to its target (Moerke (2009) Curr Protoc Chem Biol 1(1):1-15).

[0227] In another embodiment, the RLR agonist provided by this disclosure comprises at least one oligonucleotide comprising at least one region containing at least one modified nucleic acid base, nucleoside, or nucleotide that enhances the stability of the double helix. The stability of the double helix can be routinely determined by measuring the Tm of the double helix, which is the temperature at which the two oligonucleotide strands constituting the double helix dissociate, and the dissociation is detected by spectrophotometrics. The higher the Tm, the higher the stability of the double helix.

[0228] In one embodiment, the region of the oligonucleotide modified to enhance double-strand stability includes at least one nucleotide modified at the 2' position of the sugar, most preferably a 2'-O-alkyl, 2'-O-alkyl-O-alkyl, or 2'-fluoromodified nucleotide. In another embodiment, the oligonucleotide constituting the RLR agonist is also modified to improve nuclease resistance. Cells contain a variety of exonucleases and endonucleases that can degrade nucleic acids. Many nucleotide and nucleoside modifications have been shown to make the incorporated oligonucleotides more resistant to nuclease digestion than unmodified oligonucleotides. Nuclease resistance is routinely measured by incubating the oligonucleotide with a cell extract or isolated nuclease solution and measuring the amount of intact oligonucleotide remaining over time, usually by gel electrophoresis. Oligonucleotides modified to improve nuclease resistance survive intact for longer periods than unmodified oligonucleotides. Various oligonucleotide modifications that improve or confer nuclease resistance have been shown. Currently, oligonucleotides containing at least one phosphorothioate modification are more preferred. In some cases, oligonucleotide modifications that improve target binding affinity can also independently improve nuclease resistance (De Mesmaeker et al., 1995, Acc. Chem. Res. 28:366-374).

[0229] Some specific examples of preferred oligonucleotides envisioned in the present invention include, for example, those containing modified skeletons such as phosphorothioates, phosphotriesters, methylphosphonates, short-chain alkyl or cycloalkyl intersaccharide bonds, or short-chain heteroatoms or heterocyclic intersaccharide bonds. Most preferred are oligonucleotides having a phosphorothioate skeleton (including those synthesized in a stereospecific manner) and those having a heteroatom skeleton, particularly the CH2-NH-O-CH2, CH2-N(CH3)-O-CH2 [known as the methylene(methylimino) or MMI skeleton], CH2-ON(CH3)-CH2, CH2-N(CH3)-N(CH3)-CH2, and ON(CH3)-CH2-CH2 skeletons (wherein the natural phosphodiester skeleton is represented as OPO-CH2). The amide skeleton disclosed by De Mesmaeker et al. (1995, Acc. Chem. Res. 28:366-374) is also preferred. Oligonucleotides may also contain one or more substituted sugar moieties. Preferred oligonucleotides contain one of the following at the 2' position: OH, SH, SCH3, F, OCN, OCH3OCH3, OCH3O(CH2)nCH3, O(CH2)nNH2 or O(CH2)nCH3 (wherein n is 1 to about 10); C1-C10 lower alkyl, alkoxyalkoxy (also known in the art as O-alkyl-O-alkyl), substituted lower alkyl, alkaryl or aralkyl; Cl; Br; CN; CF3; OCF3; O-, S- or N-alkyl; O-, S- or N-alkenyl; SOCH3; SO2CH3; ONO2; NO2; N3; NH2; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl; RNA cleavage group; reporter group; intercalator; group for improving the pharmacokinetic properties of oligonucleotides; or group for improving the pharmacodynamic properties of oligonucleotides and other substituents having similar properties. A preferred modification is 2'-methoxyethoxy [also known as 2'-O-CH2CH2OCH3, 2'-O-(2-methoxyethyl), or 2'-MOE] (Martin et al., Helv. Chim. Acta, 1995, 78, 486).Other preferred modifications include 2'-methoxy(2'-O-CH3), 2'-propoxy(2'-OCH2CH2CH3), and 2'-fluoro(2'-F). Similar modifications may be made at other positions on the oligonucleotide, particularly at the 3' position of the sugar on the 3' terminal nucleotide and at the 5' position of the 5' terminal nucleotide. The oligonucleotide may also have sugar mimetic groups such as cyclobutyl instead of the pentofuranosyl group.

[0230] Oligonucleotides may also include, in addition to or instead of, modifications or substitutions of nucleic acid bases (often simply referred to as “bases” in the art). As used herein, “unmodified” or “natural” nucleic acid bases include adenine (A), guanine (G), thymine (T), cytosine (C), and uracil (U). Modified nucleic acid bases include those rarely or transiently found in natural nucleic acids, such as hypoxanthine, 6-methyladenine, 5-me pyrimidine, in particular 5-methylcytosine (also known as 5-methyl-2'-deoxycytosine, and often referred to as 5-me-C in this art), 5-hydroxymethylcytosine (HMC), glycosyl HMC, and gentobiosyl HMC, as well as synthetic nucleic acid bases, such as 2-aminoadenine, 2-thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N6(6-aminohexyl)adenine, and 2,6-diaminopurine. Kornberg, A., DNA Replication, WH Freeman & Co., San Francisco, 1980, pp. 75-77; Gebeyehu, G. et al., 1987, Nucl. Acids Res. 15:4513). “Universal” bases known in the art, such as inosine, may be included. 5-me-C substitutions have been shown to increase the stability of nucleic acid double helix by 0.6–1.2°C (Sanghvi, Y.S., Crooke, ST., and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276–278), and are currently preferred base substitutions.

[0231] Another modification of the oligonucleotide of the present invention involves chemically linking one or more moieties or conjugates to the oligonucleotide to improve its activity or uptake into cells. These parts, though not limited to, include lipid portions, such as cholesterol portions, cholesteryl portions (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053), thioethers, such as hexyl-S-tritylthiol (Manoharan et al., Ann. NYAcad. Sci., 1992, 660, 306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533), aliphatic chains, such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO Examples include phospholipids, polyamides or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969), or adamantane acetate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651). Oligonucleotides containing lipophilic moieties and methods for preparing such oligonucleotides are known in the art, for example, in U.S. Patents No. 5,138,045, No. 5,218,105 and No. 5,459,255.

[0232] The oligonucleotides of the present invention may also be provided as prodrugs, which generally consist of one or more moieties that are cleaved in the body to produce active oligonucleotides. An example of a prodrug method is described by Imbach et al. in WO Publication 94 / 26764.

[0233] It is not necessary for all positions in a given oligonucleotide to be uniformly modified; in fact, more than one of the above modifications may be incorporated into a single oligonucleotide, or even into a single nucleoside within a given oligonucleotide.

[0234] The oligonucleotides of the present invention are preferably about 8 to about 50 nucleotides in length. In the context of the present invention, this is understood to include the non-naturally occurring oligomers containing 8 to 50 monomers as described above herein.

[0235] Oligonucleotides used in accordance with the present invention may be prepared simply and routinely by well-known solid-phase synthesis techniques. Equipment for such synthesis is available from several suppliers, including Applied Biosystems. Any other means for such synthesis are also available, and the actual synthesis of oligonucleotides is well within the scope of the knowledge and ability of those skilled in the art. It is also well known that other oligonucleotides, such as phosphorothioates and alkylated derivatives, are prepared using similar techniques. It is also well known that fluorescently labeled, biotinylated, or other modified oligonucleotides, such as cholesterol-modified oligonucleotides, are synthesized using similar techniques and commercially available modified amidites and pore-controlled glass (CPG) products, e.g., biotin, fluorescein, acridine, or psoralen-modified amidites and / or CPGs (available from Glen Research, Stirling Va.).

[0236] In some embodiments, the RLR agonist comprises one or more (e.g., 1, 2, 3, or 4) different modified nucleic acid bases, nucleosides, or nucleotides. In some embodiments, the RLR agonist comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more) different modified nucleic acid bases, nucleosides, or nucleotides. In some embodiments, the modified RLR agonist may reduce degradation in the cells into which the RLR agonist is introduced compared to the corresponding unmodified RLR agonist.

[0237] In some embodiments, the modified nucleic acid base is modified uracil. Exemplary nucleic acid bases and nucleosides having modified uracil include pseudouridine (ψ), pyridine-4-onribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, and 2-thiouridine (s 2 U), 4-thio-uridine (s 4 U), 4-thiopsoiduridine, 2-thiopsoiduridine, 5-hydroxyuridine (ho 5 U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), 3-methyl-uridine (m 3 U), 5-methoxyuridine (mo 5 U), Uridine 5-oxyacetic acid (cmo 5 U), Uridine 5-oxyacetate methyl ester (mcmo 5 U), 5-carboxymethyl-uridine (cm 5 U), 1-carboxymethyl-psoidouridine, 5-carboxyhydroxymethyl-uridine (chm 5 U), 5-carboxyhydroxymethyl-uridinemethyl ester (mchm 5 U), 5-methoxycarbonylmethyluridine (mcm 5 U), 5-methoxycarbonylmethyl-2-thiouridine (mcm 5 s 2 U), 5-aminomethyl-2-thiouridine (nm5 s 2 U), 5-methylaminomethyluridine (mnm 5 U), 5-methylaminomethyl-2-thiouridine (mnm 5 s 2 U), 5-methylaminomethyl-2-seleno-uridine (mnm 5 se 2 U), 5-Carbamoylmethyluridine (ncm 5 U), 5-carboxymethylaminomethyluridine (cmnm 5 U), 5-carboxymethylaminomethyl-2-thiouridine (cmnm 5 s 2 U), 5-propynyl-uridine, 1-propynyl-psoidouridine, 5-taurinomethyl-uridine (τm 5 U), 1-taurinomethyl-psoidouridine, 5-taurinomethyl-2-thiouridine (τm 5 s 2 U), 1-taurinomethyl-4-thiopsoiduridine, 5-methyluridine (m 5 U, i.e., having the nucleic acid base deoxythymine), 1-methyl-psoiduridine (m 1 ψ), 5-methyl-2-thiouridine (m 5 s 2 U), 1-methyl-4-thio-psoidouridine (m 1 s 4 ψ), 4-thio-1-methyl-psoidouridine, 3-methyl-psoidouridine (m 3 ψ), 2-thio-1-methyl-psoidouridine, 1-methyl-1-deaz-psoidouridine, 2-thio-1-methyl-1-deaz-psoidouridine, dihydrouridine(D), dihydropsoidouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine(m 5 D) 2-thio-dihydrouridine, 2-thio-dihydropsoiduridine, 2-methoxy-uridine, 2-methoxy-4-thiouridine, 4-methoxy-psoiduridine, 4-methoxy-2-thiopsoiduridine, N1-methylpsoiduridine, 3-(3-amino-3-carboxypropyl)uridine (acp 3U), 1-methyl-3-(3-amino-3-carboxypropyl)psoidouridine (acp 3 ψ), 5-(isopentenylaminomethyl)uridine (inm 5 U), 5-(isopentenylaminomethyl)-2-thiouridine(inm 5 s 2 U), α-thio-uridine, 2'-O-methyl-uridine (Um), 5,2'-O-dimethyl-uridine (m 5 Um), 2'-O-methylpsoidouridine(ψm), 2-thio-2'-O-methyluridine(s 2 Um), 5-methoxycarbonylmethyl-2'-O-methyluridine (mcm 5 Um), 5-Carbamoylmethyl-2'-O-methyluridine (ncm 5 Um), 5-carboxymethylaminomethyl-2'-O-methyluridine (cmnm 5 Um), 3,2'-O-dimethyluridine (m 3 Um) and 5-(isopentenylaminomethyl)-2'-O-methyluridine(inm 5 Examples include Um), 1-thiouridine, deoxythymidine, 2'-F-alar-uridine, 2'-F-uridine, 2'-OH-alar-uridine, 5-(2-carbomethoxyvinyl)uridine, and 5-[3-(1-E-propenylamino)]uridine.

[0238] In some embodiments, the modified nucleic acid base is modified cytosine. Exemplary nucleic acid bases and nucleosides having modified cytosine include 5-azacytidine, 6-azacytidine, pseudoisocytidine, and 3-methylcytidine (m 3 C), N4-acetylcytidine (ac 4 C), 5-formyl-cytidine (f 5 C), N4-methylcytidine (m 4 C), 5-methylcytidine (m 5 C), 5-halo-cytidine (e.g., 5-iodocytidine), 5-hydroxymethylcytidine (hm 5C) 1-methyl-psoidisocytidine, pyrrolo-cytidine, pyrrolo-psoidisocytidine, 2-thio-cytidine (s 2 C), 2-thio-5-methylcytidine, 4-thio-psoidisocytidine, 4-thio-1-methyl-psoidisocytidine, 4-thio-1-methyl-1-deazapse-psoidisocytidine, 1-methyl-1-deazapse-psoidisocytidine, zebralin, 5-aza-zebralin, 5-methyl-zebralin, 5-aza-2-thio-zebralin, 2-thio-zebralin, 2-methoxycytidine, 2-methoxy-5-methylcytidine, 4-methoxy-psoidisocytidine, 4-methoxy-1-methyl-psoidisocytidine, lysidine (k2C), α-thiocytidine, 2'-O-methylcytidine (Cm), 5,2'-O-dimethylcytidine (m 5 Cm), N4-acetyl-2'-O-methylcytidine (ac 4 Cm), N4,2'-O-dimethylcytidine (m 4 Cm), 5-formyl-2'-O-methylcytidine (f 5 Cm), N4,N4,2'-O-trimethylcytidine (m 4 Examples include 2Cm), 1-thiocytidine, 2'-F-alacytidine, 2'-F-cytidine, and 2'-OH-alacytidine.

[0239] In some embodiments, the modified nucleic acid base is modified adenine. Exemplary nucleic acid bases and nucleosides having modified adenine include α-thio-adenosine, 2-amino-purine, 2,6-diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenosine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyl-adenosine (m 1 A) 2-methyl-adenine (m 2 A) N6-methyl-adenosine (m 6A), 2-Methylthio-N6-methyl-adenosine (ms 2 m 6 A), N6-Isopentenyl-adenosine (i 6 A), 2-Methylthio-N6-isopentenyl-adenosine (ms 2 i 6 A), N6-(cis-Hydroxyisopentenyl)adenosine (io 6 A), 2-Methylthio-N6-(cis-hydroxyisopentenyl)adenosine (ms 2 io 6 A), N6-Glycinylcarbamoyl-adenosine (g 6 A), N6-Threonylcarbamoyl-adenosine (t 6 A), N6-Methyl-N6-threonylcarbamoyl-adenosine (m 6 t 6 A), 2-Methylthio-N6-threonylcarbamoyl-adenosine (ms 2 g 6 A), N6,N6-Dimethyl-adenosine (m 6 2A), N6-Hydroxyvalylcarbamoyl-adenosine (hn 6 A), 2-Methylthio-N6-hydroxyvalylcarbamoyl-adenosine (ms 2 hn 6 A), N6-Acetyl-adenosine (ac 6 A), 7-Methyl-adenine, 2-Methylthio-adenine, 2-Methoxy-adenine, α-Thio-adenosine, 2’-O-Methyl-adenosine (Am), N6,2’-O-Dimethyl-adenosine (m 6 Am), N6,N6,2’-O-Trimethyl-adenosine (m 6 2Am), 1,2’-O-Dimethyl-adenosine (m 1 Am), 2’-O-Ribosyladenosine (phosphate) (Ar(p)), 2-Amino-N6-methyl-purine, 1-Thio-adenosine, 8-Azido-adenosine, 2’-F-Ara-adenosine, 2’-F-Adenosine, 2’-OH-Ara-adenosine and N6-(19-Amino-pentaoxanonadecyl)-adenosine are included.

[0240] In some embodiments, the modified nucleic acid base is a modified guanine. Exemplary nucleic acid bases and nucleosides having a modified guanine include α-thio-guanosine, inosine(I), and 1-methyl-inosine(M). 1 I) Wyosin (imG), Methyl Wyosin (mimG), 4-Demethyl Wyosin (imG-14), Iso Wyosin (imG2), Wybutosin (yW), Peroxywybutosin (o2yW), Hydroxywybutosin (OhyW), Unmodified Hydroxywybutosin (OhyW*), 7-Deaza-Guanosine, Quosin (Q), Epoxy Quosin (oQ), Galactosyl Quosin (galQ), Mannosyl Quosin (manQ), 7-Cyano-7-Deaza-Guanosine (preQ0), 7-Aminomethyl-7-Deaza-Guanosine (preQ1), Archeosin (G + ), 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine (m 7 G), 6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine, 1-methyl-guanosine (m 1 G), N2-methyl-guanosine (m 2 G), N2,N2-dimethyl-guanosine (m 2 2G), N2,7-dimethyl-guanosine (m 2,7 G), N2,N2,7-dimethyl-guanosine (m 2,2,7 G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine, α-thio-guanosine, 2'-O-methyl-guanosine (Gm), N2-methyl-2'-O-methyl-guanosine (m 2 Gm), N2,N2-dimethyl-2'-O-methyl-guanosine (m 2 2Gm), 1-methyl-2'-O-methyl-guanosine (m 1 Gm), N2,7-dimethyl-2'-O-methyl-guanosine (m 2,7Gm), 2'-O-methyl-inosine (Im), 1,2'-O-dimethyl-inosine (m 1 Examples include Im), 2'-O-ribosylguanosine (phosphate) (Gr(p)), 1-thio-guanosine, O6-methyl-guanosine, 2'-F-ala-guanosine, and 2'-F-guanosine.

[0241] In some embodiments, the RLR agonist of this disclosure comprises one or more combinations of the modified nucleic acid bases described above (for example, a combination of two, three, or four of the modified nucleic acid bases described above).

[0242] In certain embodiments, the RLR agonists of this disclosure are uniformly modified (i.e., completely modified and modified throughout the sequence) for a particular modification. For example, the RLR agonist is 5-methylcytidine (m 5 C) may be uniformly modified, which means that all cytosine residues in the mRNA sequence may be 5-methylcytidine (m 5 This means that it is replaced by C). Similarly, the RLR agonists of this disclosure may uniformly modify any type of nucleoside residue present in their sequence by replacing them with modified residues such as those described above.

[0243] Examples of nucleoside modifications and combinations that may exist in the RLR agonists of this disclosure include, but are not limited to, those described in PCT Patent Application Publications 2012 / 045075, 2014 / 081507, 2014 / 093924, 2014 / 164253 and 2014 / 159813.

[0244] The RLR agonists of this disclosure may include combinations of modifications to sugars, nucleic acid bases, and / or nucleoside bonds. These combinations may include any one or more modifications described herein.

[0245] Examples of modified nucleosides and combinations thereof are shown in Tables 1 and 2 below. These combinations of modified nucleotides can be used to form the RLR agonists of this disclosure. In certain embodiments, the modified nucleosides may partially or completely substitute for the natural nucleotides of the RLR agonists of this disclosure. As a non-limiting example, the natural nucleotide uridine may be substituted with a modified nucleoside described herein. In another non-limiting example, the natural nucleoside uridine may be partially substituted with at least one of the modified nucleosides disclosed herein (e.g., about 0.1%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99.9% of the natural uridine). [Table 1-1] [Table 1-2] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6]

[0246] According to this disclosure, the polynucleotides of this disclosure may be synthesized to include combinations or single modifications of Table 1 or Table 2.

[0247] When a single modification is listed, the listed nucleoside or nucleotide represents 100% of the modified A, U, G, or C nucleotide or nucleoside. When percentages are listed, they represent the proportion of a particular A, U, G, or C nucleic acid base tripphosphate to the total amount of A, U, G, or C tripphosphates present. For example, the following combination: 25% 5-aminoallyl-CTP + 75% CTP / 25% 5-methoxy-UTP + 75% UTP refers to a polynucleotide in which 25% of the cytosine tripphosphate is 5-aminoallyl-CTP, 75% of the cytosine is CTP, 25% of the uracil is 5-methoxy-UTP, and 75% of the uracil is UTP. When modified UTPs are not listed, naturally occurring ATP, UTP, GTP, and / or CTP are used for 100% of the nucleotide sites found in the polynucleotide. In this example, all GTP and ATP nucleotides remain unchanged.

[0248] How to manufacture an RLR agonist The RLR agonists of this disclosure may be produced by means available in the art, including, but not limited to, in vitro transcription (IVT) and synthesis methods. Enzymes (IVT), solid-phase, liquid-phase, combinations of synthesis methods, micro-region synthesis, and ligation methods may be utilized. In one embodiment, the RLR agonist is prepared using an IVT enzymatic synthesis method. Methods for producing polynucleotides by IVT are known in the art and are described in International Application No. PCT / US2013 / 30062, the contents of which are incorporated herein by reference in their entirety. Accordingly, this disclosure also includes polynucleotides, such as DNA, constructs, and vectors, that can be used to transcribe the RLR agonists described herein in vitro.

[0249] Non-naturally modified nucleic acid bases may be introduced into polynucleotides (e.g., RNA) during or after synthesis. In certain embodiments, the modification may be made to nucleoside bonds, purine or pyrimidine bases, or sugars. In certain embodiments, the modification may be introduced at the end of a polynucleotide chain or anywhere else in a polynucleotide chain by chemical synthesis or using polymerase enzymes. Examples of modified nucleic acids and their synthesis are disclosed in PCT application PCT / US2012 / 058519. The synthesis of modified polynucleotides is also described in Verma and Eckstein, Annual Review of Biochemistry, vol. 76, 99-134 (1998).

[0250] Polynucleotides or regions thereof may be conjugated with different functional moieties, such as targeting agents or delivery agents, fluorescent labels, lipids, or nanoparticles, using either an enzymatic or chemical ligation method. Conjugates of polynucleotides and modified polynucleotides are summarized in Goodchild, Bioconjugate Chemistry, vol.1(3), 165-187 (1990). The synthesis of oligonucleotides and polynucleotides, and their conjugations and ligations, are described in Taskova et al., (2017) Chembiochem 18(17):1671-1682; Gooding et al., (2016) Eur J Pharm. Biopharm 107:321-40;Menzi et al. (2015)Future Further details are found in Med Chem 7(13):1733-49; Winkler J., (2013) Ther Deliv.(7):791-809; Singh et al., (2010) Chem Soc Rev 39(6):2054-70; and Lu et al., (2010) Bioconjug Chem 21(2):187-202.

[0251] Pharmaceutical compositions and formulations In certain embodiments, the present invention provides a pharmaceutical composition comprising an RLR agonist together with a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative, and / or adjuvant.

[0252] In certain embodiments, the acceptable formulation materials are preferably non-toxic to the recipient at the dosage and concentration used. In certain embodiments, the formulation materials are for subcutaneous and / or intravenous administration. In certain embodiments, the pharmaceutical composition may include formulation materials for modifying, maintaining, or preserving, for example, the pH, osmotic pressure, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or osmosis of the composition.In certain embodiments, suitable formulation materials include, but are not limited to, amino acids (e.g., glycine, glutamine, asparagine, arginine, or lysine); antimicrobial agents; antioxidants (e.g., ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (e.g., borates, bicarbonates, Tris-HCl, citrates, phosphates, or other organic acids); fillers (e.g., mannitol or glycine); chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)); complexing agents (e.g., caffeine, polyvinylpyrrolidone, β-cyclodextrin, or hydroxypropyl-β-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (e.g., glucose, mannose, or dextrin); proteins (e.g., serum albumin, gelatin, or immunoglobulin); colorants, flavoring agents, and diluents; emulsifiers; hydrophilic polymers (e.g., polyvinylpyrrolidone); and low molecular weight polypeptides. Examples of additives include: counterions that form salts (e.g., sodium); preservatives (e.g., benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide); solvents (e.g., glycerin, propylene glycol, or polyethylene glycol); sugar alcohols (e.g., mannitol or sorbitol); suspending agents; surfactants or wetting agents (e.g., Pluronic®, PEG, sorbitan esters, polysorbates, e.g., polysorbate 20, polysorbate 80, Triton, tromethamine, lecithin, cholesterol, tyroxapal); stability enhancers (e.g., sucrose or sorbitol); tonicity enhancers (e.g., alkali metal halides, preferably sodium chloride or potassium chloride, mannitol, sorbitol); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants. (Remington's Pharmaceutical Sciences, 18th Edition, edited by ARGennaro, Mack Publishing Company (1995).In certain embodiments, the formulation comprises PBS; 20 mM NaOAC, pH 5.2, 50 mM NaCl; and / or 10 mM NAOAC, pH 5.2, 9% sucrose. In certain embodiments, the optimal pharmaceutical composition will be determined by those skilled in the art, for example, based on the intended route of administration, the form of delivery, and the desired dosage. See, for example, Remington's Pharmaceutical Sciences (cited above). In certain embodiments, such compositions may affect the physical state, stability, in vivo release rate, and / or in vivo excretion rate of the RLR agonist.

[0253] In certain embodiments, the primary vehicle or carrier in the pharmaceutical composition may be either aqueous or non-aqueous. For example, in certain embodiments, a suitable vehicle or carrier may be water for injection, saline solution, or artificial cerebrospinal fluid, and possibly other substances common to compositions for parenteral administration may be added. In certain embodiments, saline solution may include isotonic phosphate-buffered saline. In certain embodiments, neutral buffered saline or saline solution mixed with serum albumin are further exemplary vehicles. In certain embodiments, the pharmaceutical composition may include Tris buffer with a pH of about 7.0–8.5, or acetate buffer with a pH of about 4.0–5.5, which may further include sorbitol or a suitable substitute therefor. In certain embodiments, a composition containing an RLR agonist may be prepared for storage by mixing a selected composition having the desired purity with an optimal formulation agent (Remington's Pharmaceutical Sciences, cited above) in the form of a lyophilized cake or aqueous solution. Furthermore, in certain embodiments, the composition containing the RLR agonist may be formulated as a lyophilized product using a suitable excipient such as sucrose.

[0254] In certain embodiments, the pharmaceutical composition may be selected for parenteral delivery. In certain embodiments, the composition may be selected for delivery via the gastrointestinal tract, such as by inhalation or oral administration. The preparation of such pharmaceutically acceptable compositions is within the capabilities of those skilled in the art.

[0255] In certain embodiments, the formulation components are present at the administration site at an acceptable concentration. In certain embodiments, a buffer is used to maintain the composition at or slightly below the physiological pH, typically within a pH range of about 5 to about 8.

[0256] In certain embodiments, where parenteral administration is envisioned, the therapeutic composition may be in the form of a pyrogenically-free, parenterally acceptable aqueous solution containing an RLR agonist in a pharmaceutically acceptable vehicle. In certain embodiments, the vehicle for parenteral injection is sterile distilled water, in which the RLR agonist is formulated as a sterile isotonic solution and appropriately stored. In certain embodiments, the preparation may involve formulation of the desired molecule using a delivery vehicle or drug, such as injectable microspheres, biodegradable particles, polymer compounds (e.g., polylactic acid, polyglycolic acid, or polyethyleneimine (e.g., JetPEI®)), beads, or liposomes, which can provide controlled or sustained release of the product, which can then be delivered via depot injection. In certain embodiments, hyaluronic acid may also be used, which may have the effect of promoting duration in circulation. In certain embodiments, the desired molecule can be introduced using an implantable drug delivery device.

[0257] In certain embodiments, the pharmaceutical composition may be formulated for inhalation. In certain embodiments, the RLR agonist may be formulated as a dry powder for inhalation. In certain embodiments, the inhalation solution containing the RLR agonist may be formulated with a propellant for aerosol delivery. In certain embodiments, the solution may be sprayed. Lung delivery is further described in PCT application PCT / US94 / 001875, which describes the pulmonary delivery of chemically modified proteins.

[0258] In certain embodiments, the formulation may be administered orally. In certain embodiments, the RLR agonist administered in this manner may be formulated with or without carriers conventionally used in the formulation of solid dosage forms such as tablets and capsules. In certain embodiments, the capsule may be designed to release the active portion of the formulation at some point in the gastrointestinal tract when bioavailability is maximized and pre-systemic degradation is minimized. In certain embodiments, at least one further agent may be included to enhance the absorption of the RLR agonist. In certain embodiments, diluents, flavorings, low-melting-point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrants, and binders may also be used.

[0259] In certain embodiments, the pharmaceutical composition may contain an effective amount of RLR agonist in a mixture with a non-toxic excipient suitable for the manufacture of tablets. In certain embodiments, the solution may be prepared in unit dose form by dissolving the tablets in sterile water or another suitable vehicle. In certain embodiments, suitable excipients include, but are not limited to, inert diluents, e.g., calcium carbonate, sodium carbonate or sodium bicarbonate, lactose, or calcium phosphate; or binders, e.g., starch, gelatin, or acacia; or lubricants, e.g., magnesium stearate, stearic acid, or talc.

[0260] Further pharmaceutical compositions, including formulations containing RLR agonists in sustained-release or controlled-delivery formulations, will be apparent to those skilled in the art. In certain embodiments, techniques for formulating various other sustained-release or controlled-delivery means, e.g., liposome carriers, bio-erosive microparticles or porous beads and depot injections, are also known to those skilled in the art. See, for example, PCT application number PCT / US93 / 00829, which describes the controlled release of porous polymer microparticles for delivering pharmaceutical compositions. In certain embodiments, the sustained-release preparation may include a semipermeable polymer matrix in the form of a molded article, e.g., a film or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels, polylactides (US Patent No. 3,773,919 and EP058,481), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman et al., Biopolymers, 22:547-556 (1983)), poly(2-hydroxyethyl methacrylate) (Langer et al., J. Biomed. Mater. Res., 15:167-277 (1981) and Langer, Chem. Tech., 12:98-105 (1982)), ethylene vinyl acetate (Langer et al., previously mentioned), or poly-D(-)-3-hydroxybutyric acid (EP133,988). In certain embodiments, the sustained-release composition may also include liposomes, which can be prepared by any of several methods known in the art. For example, Eppstein et al., Proc. Natl. Acad. Sci. USA, 82:3688-3692 (1985); EP 036, 676; EP See 088,046 and EP 143,949.

[0261] Pharmaceutical compositions used for in vivo administration are typically sterile. In certain embodiments, this can be achieved by filtration through a sterile filtration membrane. In certain embodiments, if the composition is lyophilized, sterilization using this method can be performed either before or after lyophilization and reconstitution. In certain embodiments, compositions for parenteral administration can be stored in lyophilized form or in solution. In certain embodiments, parenteral compositions are generally placed in containers with sterile access ports, such as intravenous solution bags or vials with stoppers that can be penetrated by a subcutaneous needle.

[0262] In certain embodiments, once a pharmaceutical composition has been formulated, it can be stored in a sterile vial as a solution, suspension, gel, emulsion, solid, or as an aerosolized or lyophilized powder. In certain embodiments, such formulations can be stored either in a ready-to-use form or in a form that is reconstituted before administration (e.g., lyophilized).

[0263] In certain embodiments, a kit is provided for preparing single-dose medication units. In certain embodiments, the kit may include both a first container containing a dry protein and a second container containing an aqueous formulation. In certain embodiments, the kit includes pre-filled syringes of single-chamber and multi-chamber types (e.g., liquid syringes and lyosyringes).

[0264] In certain embodiments, the effective amount of a pharmaceutical composition containing an RLR agonist used therapeutically will depend, for example, on the nature and purpose of the treatment. Those skilled in the art will understand that, according to certain embodiments, the appropriate dosage level for treatment may vary in part depending on the molecule being delivered, the indication for which the RLR agonist is used, the route of administration, and the patient's physique (weight, body surface or organ size) and / or condition (age and overall health). In certain embodiments, a clinician may titrate the dosage and modify the route of administration to obtain the optimal therapeutic effect.

[0265] In certain embodiments, the dosing frequency will take into account the pharmacokinetic parameters of the RLR agonist in the formulation used. In certain embodiments, the clinician administers the composition until a dose is reached that achieves the desired effect. Thus, in certain embodiments, the composition may be administered over time as a single dose or two or more doses (which may or may not contain the same amount of the desired molecule), or as a continuous infusion via an implantable device or catheter. Further fine-tuning of the appropriate dosage is routinely performed by those skilled in the art and is within the scope of routine work performed by them. In certain embodiments, the appropriate dosage can be confirmed by using appropriate dose-response data.

[0266] In certain embodiments, the route of administration of the pharmaceutical composition is by known methods, such as orally, intravenously, intraperitoneally, intracerebral (intraparum), intraventricular, intramuscular, subcutaneously, intraocularly, intraarterially, intraportally, or intralesional route, by a sustained-release system, or by an implantable device. In certain embodiments, the composition may be administered by bolus injection, by continuous infusion, or by an implantable device. In certain embodiments, individual elements of a combination therapy may be administered by different routes.

[0267] In certain embodiments, the composition may be administered topically by implantation of a membrane, sponge, or other suitable material in which the desired molecule is absorbed or encapsulated. In certain embodiments, if an implantation device is used, the device may be implanted in any suitable tissue or organ, and the delivery of the desired molecule may be by diffusion, time-release bolus, or continuous administration. In certain embodiments, it may be desirable to use the pharmaceutical composition containing the RLR agonist ex vivo. In such examples, cells, tissues, and / or organs removed from a patient are exposed to the pharmaceutical composition containing the RLR agonist, and then the cells, tissues, and / or organs are subsequently returned to the patient by implantation.

[0268] In certain embodiments, the RLR agonist may be delivered by transplanting specific genetically engineered cells using a method such as that described herein to express and secrete the agonist. In certain embodiments, such cells may be animal or human cells and may be autologous, autologous xenogeneic, or heterogeneous. In certain embodiments, the cells may be immortalized. In certain embodiments, the cells may be encapsulated to reduce the opportunity for an immunological response and avoid invasion of surrounding tissue. In certain embodiments, the encapsulation material is typically a biocompatible, semipermeable polymer inclusion or membrane that allows for the release of protein products but prevents destruction of the cells by the patient's immune system or other harmful factors from surrounding tissue.

[0269] In some embodiments, the Disclosure provides pharmaceutical compositions for stimulating an immune response to treat or slow the progression of cancer, or reduce or inhibit tumor growth in subjects where such treatment is required, comprising an RLR agonist provided by the Disclosure and a pharmaceutically acceptable carrier. In some embodiments, the RLR agonist is formulated in a polyethyleneimine (PEI) carrier. In some embodiments, the PEI carrier is JetPEI®.

[0270] Purpose The compositions described herein can be used in diagnostic and therapeutic applications. For example, detectably labeled RLR agonists can be used in assays to detect the presence or amount of a target protein in a sample (e.g., a biological sample). The compositions can be used in in vitro assays to study the inhibition of a target function (e.g., RLR-mediated cellular signaling or response). In some embodiments, for example, if the composition binds to a target (e.g., a protein or polypeptide) and activates the target, the composition can be used as a positive control in assays designed to identify further novel compounds that also induce the activity of the target protein or polypeptide, and / or is otherwise useful in treating disorders associated with the target protein or polypeptide. For example, a composition that activates RLRs can be used as a positive control in assays to identify further compounds (e.g., small molecules, aptamers, or antibodies) that induce, increase, or stimulate RLR function. The compositions can also be used in therapeutic methods, as detailed below.

[0271] kit The kit may include the RLR agonist disclosed herein and instructions for use. The kit may also include, in a suitable container, the RLR agonist, one or more controls, and various buffers, reagents, enzymes, and other standard components well known in the art.

[0272] The container may include at least one vial, well, test tube, flask, bottle, syringe, or other container means in which the RLR agonist is placed and, if applicable, appropriately divided. If further components are given, the kit may include further containers capable of holding these components. The kit may also include means for containing the RLR agonist and any other reagent containers in a tightly sealed state for commercial sale. Such containers may include injection-molded or blow-molded plastic containers in which the desired vials are held. The container and / or kit may include labels with instructions and / or warnings for use.

[0273] In some embodiments, the Disclosure provides a kit comprising an RLR agonist or a pharmaceutical composition provided by the Disclosure, and instructions for use in stimulating an immune response in a subject, treating or slowing the progression of cancer, or inhibiting tumor growth in a subject, and optionally instructions for use in combination with one or more further therapeutic agents.

[0274] In some embodiments, the agonist or pharmaceutical composition is administered in combination with one or more further therapeutic agents, one or more of which are selected from the group consisting of chemotherapy, targeted anticancer therapy, oncolytic agents, cell death inducers, opsonizing agents (e.g., opsonized antibodies), cytotoxic agents, immunotherapy, cytokines, activators of costimulatory molecules, inhibitors of inhibitory molecules, vaccines, cellular immunotherapy, or combinations thereof.

[0275] In some embodiments, the RLR agonist or pharmaceutical composition is administered before or after the administration of one or more additional therapeutic agents, or one or more additional therapeutic agents are administered simultaneously with, before, or after the administration of the RLR agonist or pharmaceutical composition.

[0276] In some embodiments, one or more further therapeutic agents are PD-1 / PD-L1 antagonists, TIM-3 antagonists, VISTA antagonists, adenosine A2AR antagonists, B7-H3 antagonists, B7-H4 antagonists, BTLA antagonists, CTLA-4 antagonists, IDO antagonists, KIR antagonists, LAG-3 antagonists, Toll-like receptor 3 (TLR3) agonists, Toll-like receptor 7 (TLR7) agonists, and Toll-like receptor 9 (TLR9) agonists.

[0277] In some embodiments, one or more further therapeutic agents are agonists comprising polypeptides (e.g., antibodies, or their antigen-binding moieties) that specifically bind to CD137(4-1BB).

[0278] In some embodiments, one or more further therapeutic agents are agonists comprising polypeptides (e.g., antibodies, or their antigen-binding moieties) that specifically bind to CD134(OX40).

[0279] How to use The compositions of the present invention have numerous in vitro and in vivo applications, including detection and / or quantification of RLRs and / or agonism of RLR function.

[0280] The compositions described above are particularly useful in methods for treating or preventing various cancers or infections in subjects. These compositions can be administered to subjects (e.g., human subjects) using a variety of methods that are partially dependent on the route of administration. The routes may be, for example, intravenous injection or infusion (IV), subcutaneous injection (SC), intradermal injection (ID), intraperitoneal injection (IP), intramuscular injection (IM), intratumoral injection (IT), or intrathecal injection. The injection may be performed as a bolus or in a series of infusions.

[0281] Administration can be achieved, for example, by local injection, injection, or implantation. The implantation may be made of porous, non-porous, or gelatinous material, including membranes such as elastic membranes or fibers. The implantation may be configured for continuous or periodic release of the composition to a target. See, for example, U.S. Patent Application Publication No. 20080241223, U.S. Patent Nos. 5,501,856, 4,863,457, and 3,710,795, EP488401 and EP430539, the disclosures of which are incorporated herein by reference in their entirety. The composition can be delivered to a target by, for example, diffusion, erosion, or convection systems, such as osmotic pumps, biodegradable implants, electrodiffusion systems, electroosmotic systems, vapor pressure pumps, electrolytic pumps, foaming pumps, piezoelectric pumps, erosion-based systems, or implantable devices based on electromechanical systems.

[0282] In some embodiments, RLR agonists are therapeutically delivered to the subject by topical administration.

[0283] The appropriate dose of the RLR agonist described herein, which is effective in treating or preventing the target cancer, may be based on various factors, including, for example, the age, sex, and weight of the subject being treated, and the specific inhibitor compound used. Other factors that may influence the dose administered to a subject include, for example, the type or severity of the cancer or infection. For example, a subject with metastatic melanoma may require a different dose of RLR agonist than a subject with glioblastoma. Other factors may include, for example, other medical conditions currently or previously affected by the subject, the subject's overall health status, the subject's genetic predisposition, diet, administration time, excretion rate, drug combinations, and any other further therapies administered to the subject. It should also be understood that specific dosages and treatment regimens for any particular subject may also be based on the judgment of the treating healthcare professional (e.g., physician or nurse). The appropriate dosage is described herein.

[0284] A pharmaceutical composition may contain a therapeutically effective amount of the RLR agonist described herein. Such an effective amount can be readily determined by those skilled in the art, in part, based on the effect of the RLR agonist administered, or, if more than one agent is used, the combined effect of the RLR agonist and one or more additional active agents. The therapeutically effective amount of the RLR agonist described herein may vary depending on factors such as the individual's disease state, age, sex and weight, and the ability of the agonist (and one or more additional active agents) to induce a desired response in the individual, such as a reduction in tumor growth. For example, a therapeutically effective amount of RLR agonist can suppress (reduce the severity of or eliminate the onset of) and / or prevent any one of the symptoms of a particular disorder and / or a particular disorder known in the art or described herein. The therapeutically effective amount is also the amount in which the therapeutically beneficial effect outweighs the toxic or adverse effects of the composition.

[0285] The appropriate human dose of any of the RLR agonists described herein may be further evaluated, for example, in a Phase I dose-escalation study. For example, van Gurp et al. (2008) Am J Transplantation 8(8):1711-1718; Hanouska et al. (2007) Clin Cancer Res 13(2, part See 1):523-531; and Hetherington et al. (2006) Antimicrobial Agents and Chemotherapy 50(10):3499-3500.

[0286] In some embodiments, the composition comprises one of the RLR agonists described herein and one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11, or more) further therapeutic agents, so that the composition as a whole is therapeutically effective. For example, the composition may comprise an RLR agonist described herein and an alkylating agent, wherein the agonist and alkylating agent are at concentrations that, when combined, are effective for the treatment or prevention of cancer (e.g., melanoma) in a subject.

[0287] The toxicity and therapeutic efficacy of such compositions can be determined by known pharmaceutical procedures in cell cultures or experimental animals (e.g., animal models of any of the cancers described herein). These procedures may include, for example, LD 50 (A lethal dose for 50% of the aggregate) and ED 50 It may be used to determine the dose effective in 50% of the aggregate. The dose ratio between toxicity and therapeutic efficacy is the therapeutic index, and the ratio LD50. 50 / ED 50 It can be expressed as follows. RLR agonists exhibiting a high therapeutic index are preferred. Compositions exhibiting toxic side effects may be used, but care should be taken to design a delivery system in which such compounds target the site of the affected tissue, minimizing the possibility of damage to normal cells and thereby reducing side effects.

[0288] Data obtained from cell culture assays and animal studies can be used when considering the range of dosages for use in humans. For the RLR agonists described herein, the therapeutically effective dose can be initially estimated from cell culture assays. EC determined in cell cultures 50In animal models, a certain dose can be prescribed to achieve a circulating plasma concentration range that includes the agonist concentration that achieves the inhibition of the maximum half of the symptoms. Using such information, an effective dose in humans can be determined more accurately. Plasma levels can be measured, for example, by high-performance liquid chromatography. In some embodiments, for example, when local administration (e.g., administration to the eye or joint) is desired, cell culture or animal modeling can be used to determine the dose required to achieve a therapeutically effective concentration within the local site.

[0289] In some embodiments, this method can be carried out in conjunction with other therapies for cancer or infection. For example, the composition can be administered to a subject simultaneously with, before, or after, radiation, surgery, targeted or cytotoxic chemotherapy, chemoradiotherapy, hormone therapy, immunotherapy, gene therapy, cell transplantation therapy, precision medicine, genome editing therapy, or other drug therapies.

[0290] As described above, various cancers can be treated using the compositions described herein (e.g., RLR agonist compositions), which include, for example, but are not limited to, Kaposi's sarcoma, leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, myeloblastic promyelomocytic erythrocyte leukemia, chronic leukemia, chronic myeloid (granulocyte) leukemia, chronic lymphocytic leukemia, mantle cell lymphoma, primary central nervous system lymphoma, Burkitt lymphoma, marginal zone B-cell lymphoma, Polycythemia vera, Hodgkin's disease, non-Hodgkin's disease, multiple myeloma, Waldenström macroglobulinemia, heavy chain disease, solid tumors, sarcomas and carcinomas, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, osteosarcoma, chordoma, angiosarcoma, endosarcoma, lymphangiosarcoma, lymphangiosarcoma, synoviomas, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon sarcoma, colorectal carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, Sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonic carcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicular cancer, lung carcinoma, small cell lung carcinoma, non-small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal glandoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, retinoblastoma, nasopharyngeal carcinoma, esophageal carcinoma, These include basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, cancers of the brain and central nervous system (CNS), cervical cancer, choriocarcinoma, colorectal cancer, connective tissue cancer, cancers of the digestive system, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer, carcinoma in situ, kidney cancer, laryngeal cancer, liver cancer, lung cancer (small cell and large cell), melanoma, neuroblastoma, oral cancer (e.g., lips, tongue, mouth and pharynx), ovarian cancer, pancreatic cancer, rectal cancer, cancers of the respiratory system, sarcoma, skin cancer, gastric cancer, testicular cancer, thyroid cancer, uterine cancer, and cancers of the urinary system.

[0291] In some embodiments, the present disclosure provides a method for increasing the RLR-mediated production of one or more cytokines in cells, the method comprising contacting cells with an RLR agonist provided by the present disclosure, wherein the agonist increases the RLR-mediated cytokine production in the cells.

[0292] In some embodiments, the present disclosure provides a method for increasing the RLR-mediated expression of one or more interferon-inducible genes in cells, the method comprising contacting cells with an RLR agonist provided by the present disclosure, wherein the agonist increases the RLR-mediated expression of one or more interferon-inducible genes in cells.

[0293] In some embodiments, the present disclosure provides a method for increasing RLR-dependent intracellular signaling in cells, the method comprising contacting cells with an RLR agonist provided by the present disclosure, wherein the agonist increases RLR-dependent intracellular signaling.

[0294] In some embodiments, the Disclosure provides a method for stimulating an immune response in a subject, the method comprising administering to the subject an effective amount of an RLR agonist or a pharmaceutical composition provided by the Disclosure.

[0295] In some embodiments, the Disclosure provides a method for treating or slowing the progression of cancer in a subject, the method comprising administering to the subject an effective amount of an RLR agonist or a pharmaceutical composition provided by the Disclosure.

[0296] In some embodiments, the Disclosure provides a method for doing so in a subject where it is necessary to reduce or inhibit tumor growth, the method comprising administering to the subject an effective amount of an RLR agonist or a pharmaceutical composition provided by the Disclosure.

[0297] In some embodiments, the Disclosure provides a method for stimulating an immune response in subjects that require treatment of cancer, slowing its progression, or inhibiting tumor growth, the method comprising administering to a subject an effective amount of an RLR agonist or a pharmaceutical composition provided by the Disclosure, wherein the agonist or pharmaceutical composition increases the RLR-mediated production of one or more cytokines in cells, increases the RLR-mediated expression of one or more interferon-inducing genes in cells, and / or increases RLR-dependent intracellular signaling in cells, thereby stimulating an immune response, treatment of cancer, slowing its progression, or inhibiting tumor growth.

[0298] RLR agonists in combination with additional therapeutic agents In some embodiments, the RLR agonists described herein may be administered to a subject as monotherapy. Alternatively, the RLR agonists may be administered to a subject as combination therapy with another treatment, for example, another treatment for cancer. For example, combination therapy may involve administering to a subject (e.g., a human patient) one or more additional agents that provide a therapeutic benefit to a subject who has cancer or is at risk of developing cancer.

[0299] In some embodiments of the methods provided herein, an RLR agonist or pharmaceutical composition is administered in combination with one or more further therapeutic agents, the one or more further therapeutic agents being selected from the group consisting of chemotherapy, targeted anticancer therapy, oncolytic agents, cell death inducers, opsonizing agents (e.g., opsonized antibodies), cytotoxic agents, immunotherapy, cytokines, activators or agonists of costimulatory molecules, inhibitors of inhibitory molecules, vaccines, cellular immunotherapy, or combinations thereof.

[0300] In some embodiments, the RLR agonist or pharmaceutical composition is administered before or after the administration of one or more additional therapeutic agents, or one or more additional therapeutic agents are administered simultaneously with, before, or after the administration of the agonist or pharmaceutical composition.

[0301] In some embodiments, one or more further therapeutic agents are PD-1 / PD-L1 antagonists, TIM-3 antagonists, VISTA antagonists, adenosine A2AR antagonists, B7-H3 antagonists, B7-H4 antagonists, BTLA antagonists, CTLA-4 antagonists, IDO antagonists, KIR antagonists, LAG-3 antagonists, Toll-like receptor 3 (TLR3) agonists, Toll-like receptor 7 (TLR7) agonists, and Toll-like receptor 9 (TLR9) agonists.

[0302] Combination with chemotherapy drugs Examples of chemotherapeutic agents suitable for combination and / or co-administration with the compositions of the present invention include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyantrancendione, mitoxantrone, mitramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, as well as their analogues or homologues. Further drugs include, for example, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechloretamine, thioTEPA, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, cis-dichlorodiamine platinum(II) (DDP), procarbazolamine Examples include din, altoretamine, cisplatin, carboplatin, oxaliplatin, nedaplatin, satraplatin, or triplatin tetranitrate), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mitramycin, and anthramycin (AMC)), and antimitotic agents (e.g., vincristine and vinblastine) and temozolomide.

[0303] Combination with PD-1 / PD-L1 antagonist In some embodiments, the RLR agonists or pharmaceutically active compositions provided herein are combined with one or more PD-1 / PD-L1 antagonists that specifically bind to human PD-1 or PD-L1 and inhibit the bioactivity of PD-1 / PD-L1 and / or downstream pathways and / or cellular processes mediated by human PD-1 / PD-L1 signaling or other human PD-1 / PD-L1-mediated functions (e.g., administered in combination).

[0304] Accordingly, PD-1 / PD-L1 antagonists are provided herein that directly or allosterically block, antagonistize, repress, inhibit, or reduce PD-1 / PD-L1 bioactivity, including cellular processes mediated by downstream pathways and / or PD-1 / PD-L1 signaling, such as receptor binding to PD-1 / PD-L1 and / or induction of cellular responses thereto. PD-1 / PD-L1 antagonists that reduce the numerical value or amount of human PD-1 / PD-L1 produced by cells or subjects are also provided herein.

[0305] In some embodiments, the Disclosure provides PD-1 / PD-L1 antagonists that bind to human PD-1 and prevent, inhibit, or reduce the binding of PD-L1 to PD-1. In some embodiments, the PD-1 / PD-L1 antagonist binds to mRNA encoding PD-1 or PD-L1 and prevents translation. In some embodiments, the PD-1 / PD-L1 antagonist binds to mRNA encoding PD-1 or PD-L1 and causes degradation and / or turnover.

[0306] In some embodiments, PD-1 / PD-L1 antagonists inhibit PD-1 signaling or function. In some embodiments, PD-1 / PD-L1 antagonists block the binding of PD-1 to PD-L1, PD-L2, or both PD-L1 and PD-L2. In some embodiments, PD-1 / PD-L1 antagonists block the binding of PD-1 to PD-L1. In some embodiments, PD-1 / PD-L1 antagonists block the binding of PD-1 to PD-L2. In some embodiments, PD-1 / PD-L1 antagonists block the binding of PD-1 to both PD-L1 and PD-L2. In some embodiments, PD-1 / PD-L1 antagonists specifically bind to PD-1. In some embodiments, PD-1 / PD-L1 antagonists specifically bind to PD-L1. In some embodiments, PD-1 / PD-L1 antagonists specifically bind to PD-L2.

[0307] In some embodiments, PD-1 / PD-L1 antagonists inhibit the binding of PD-1 to its congener ligands. In some embodiments, PD-1 / PD-L1 antagonists inhibit the binding of PD-1 to PD-L1, to PD-L2, or to both PD-L1 and PD-L2. In some embodiments, PD-1 / PD-L1 antagonists do not inhibit the binding of PD-1 to its congener ligands.

[0308] In some embodiments, the PD-1 / PD-L1 antagonist is an isolated monoclonal antibody (mAb) or its antigen-binding fragment that specifically binds to PD-1 or PD-L1. In some embodiments, the PD-1 / PD-L1 antagonist is an antibody or its antigen-binding fragment that specifically binds to human PD-1. In some embodiments, the PD-1 / PD-L1 antagonist is an antibody or its antigen-binding fragment that specifically binds to human PD-L1. In some embodiments, the PD-1 / PD-L1 antagonist is an antibody or its antigen-binding fragment that binds to human PD-L1 and inhibits the binding of PD-L1 to PD-1. In some embodiments, the PD-1 / PD-L1 antagonist is an antibody or its antigen-binding fragment that binds to human PD-1 and inhibits the binding of PD-L1 to PD-1.

[0309] Several immune checkpoint antagonists that inhibit or disrupt the interaction between PD-1 and one or both of its ligands, PD-L1 and PD-L2, are either in clinical development or currently available to clinicians for treating cancer.

[0310] Examples of anti-human PD-1 monoclonal antibodies or their antigen-binding fragments, which may contain a PD-1 / PD-L1 antagonist in any of the compositions, methods, and uses provided herein, include, but are not limited to, KEYTRUDA® (pembrolizumab, MK-3475, h409A11; see US8952136, US8354509, US8900587 and EP2170959, all of which are included). However, the whole of these is included herein by reference; Merck), OPDIVO(registered trademark) (nivolumab, BMS-936558, MDX-1106, ONO-4538; see US7595048, US8728474, US9073994, US9067999, EP1537878, US8008449, US8779105 and EP2161336, all of which, the whole of these, is included herein by reference; Bristol Examples include Myers Squibb), MEDI0680 (AMP-514), BGB-A317 and BGB-108 (BeiGene), 244C8 and 388D4 (see WO2016 / 106159, the whole of which is incorporated herein by reference; Enumeral Biomedical), PDR001 (Novartis), and REGN2810 (Regeneron). Thus, in some embodiments, the PD-1 / PD-L1 antagonist is pembrolizumab. In some embodiments, the PD-1 / PD-L1 antagonist is nivolumab.

[0311] Examples of anti-human PD-L1 monoclonal antibodies, or antigen-binding fragments thereof, which may contain a PD-1 / PD-L1 antagonist in any of the compositions, methods, and uses provided herein, include, but are not limited to, BAVENCIO® (avelumab, MSB0010718C, see WO2013 / 79174, the whole of which is incorporated herein by reference; Merck / Pfizer), IMFINZI® (durvalumab, ME Examples include DI4736), TECENTRIQ® (atezolizumab, MPDL3280A, RG7446; see WO2010 / 077634, the whole thereof incorporated herein by reference; Roche), MDX-1105 (BMS-936559, 12A4; see US7943743 and WO2013 / 173223, both of which are incorporated herein by reference in their wholes; Medarex / BMS), and FAZ053 (Novartis). Thus, in some embodiments, the PD-1 / PD-L1 antagonist is avelumab. In some embodiments, the PD-1 / PD-L1 antagonist is durvalumab. In some embodiments, the PD-1 / PD-L1 antagonist is atezolizumab.

[0312] In some embodiments, the PD-1 / PD-L1 antagonist is an immunoadhesin that specifically binds to human PD-1 or human PD-L1, and is a fusion protein containing an extracellular or PD-1 binding portion of PD-L1 or PD-L2 that fuses to a constant region, such as the Fc region of an immunoglobulin molecule. Examples of immunoadhesin molecules that specifically bind to PD-1 are described in WO2010 / 027827 and WO2011 / 066342, both of which are incorporated herein by reference in their entirety. In some embodiments, the PD-1 / PD-L1 antagonist is AMP-224 (also known as B7-DCIg), a PD-L2-FC fusion protein that specifically binds to human PD-1.

[0313] Those skilled in the art will understand that any PD-1 / PD-L1 antagonist that binds to PD-1 or PD-L1 and disrupts the PD-1 / PD-L1 signaling pathway is suitable for the compositions, methods, and uses disclosed herein.

[0314] In some embodiments, PD-1 / PD-L1 antagonists are small molecules, nucleic acids, peptides, peptide mimes, proteins, carbohydrates, carbohydrate derivatives, or glycopolymers. Exemplary small molecule PD-1 inhibitors are described by Zhan et al., (2016) Drug Discov Today 21(6):1027-1036.

[0315] In some embodiments of the methods provided herein, the RLR agonist is combined with a PD-1 / PD-L1 antagonist, the PD-1 / PD-L1 antagonist being selected from the group consisting of PDR001, KEYTRUDA® (pembrolizumab), OPDIVO® (nivolumab), pidilizumab, MEDI0680, REGN2810, TSR-042, PF-06801591, and AMP-224. In some embodiments, the PD-1 / PD-L1 antagonist is selected from the group consisting of FAZ053, TENCENTRIQ® (atezolizumab), BAVENCIO® (avelumab), IMFINZI® (durvalumab), and BMS-936559.

[0316] Combination with TIM-3 Antagonist In some embodiments, the RLR agonists or pharmaceutical compositions provided by this disclosure are combined with (e.g., administered in combination with) a TIM-3 antagonist. The TIM-3 antagonist may be an antibody, its antigen-binding fragment, an immunoadhesin, a fusion protein, or an oligopeptide. In some embodiments, the TIM-3 antagonist is selected from MGB453 (Novartis), TSR-022 (Tesaro), or LY3321367 (Eli Lilly).

[0317] Combination with LAG-3 Antagonist In some embodiments, the RLR agonists or pharmaceutically active compositions provided herein are combined with (e.g., administered in combination with) a LAG-3 antagonist. The LAG-3 antagonist may be an antibody, its antigen-binding fragment, immunoadhesin, fusion protein, or oligopeptide. In some embodiments, the LAG-3 inhibitor is selected from LAG525 (Novartis), BMS-986016 (Bristol-Myers Squibb), TSR-033 (Tesaro), MK-4280 (Merck & Co), or REGN3767 (Regeneron).

[0318] Combination with Toll-like receptor (TLR) agonists In some embodiments, the RLR agonists or pharmaceutical compositions provided by this disclosure are combined with (for example, administered in combination with) TLR antagonists.

[0319] Toll-like receptors (TLRs) are a family of germline-encoded transmembrane proteins that facilitate pathogen recognition and activation of the innate immune system. (Hoffmann et al., (1999) Science 284:1313-1318; Rock et al., (1998) Proc Natl Acad Sci USA 95:588-593). TLRs are pattern recognition receptors (PRRs) expressed by cells of the innate immune system. Examples of known ligands for TLRs include Gram-positive bacteria (TLR-2), bacterial endotoxins (TLR-4), flagellin proteins (TLR-5), bacterial DNA (TLR-9), double-stranded RNA and poly(I:C) (TLR-3), and yeast (TLR-2). In vivo activation of TLRs initiates an innate immune response involving specific cytokines, chemokines, and growth factors. While all TLRs can activate specific intracellular signaling molecules, such as nuclear factor kappa beta (NF-κB) and mitogenic factor-activated protein kinase (MAP kinase), the specific set of cytokines and chemokines released appears to be unique to each TLR. TLR7, 8, and 9 comprise a subfamily of TLRs located in the endosomal or lysosomal compartments of immune cells, such as dendritic cells and monocytes. In contrast to TLR7 and 9, which are highly expressed in plasmacytoid dendritic cells (pDCs), TLR8 is primarily expressed in bone marrow dendritic cells (mDCs) and monocytes. This subfamily mediates the recognition of microbial nucleic acids, such as single-stranded RNA.

[0320] Small, low molecular weight (less than 400 daltons) synthetic imidazoquinoline compounds, similar to the purine nucleotides adenosine and guanosine, were the first TLR7 and TLR8 agonists identified. Many of these compounds exhibit antiviral and anticancer properties. For example, imiquimod (ALDARA®), a TLR7 agonist, has been approved by the U.S. Food and Drug Administration as a topical agent for the treatment of skin lesions caused by certain strains of human papillomavirus. Imiquimod may also be useful in the treatment of primary skin cancers and skin tumors, such as basal cell carcinoma, keratosinus acanthoma, actinic keratosis, and Bowen's disease. Reximod (R-848), a TLR7 / 8 agonist, is being evaluated as a topical agent for the treatment of human genital herpes.

[0321] The TLR agonists of this disclosure may be any TLR agonists. For example, TLR agonists may include natural or synthetic TLR ligands, mutaines or derivatives of TLR ligands, peptide mimics of TLR ligands, small molecules that mimic the biological function of TLR ligands, or antibodies that stimulate TLR receptors. A TLR ligand is any molecule that binds to a TLR.

[0322] In some embodiments, the RLR agonists or pharmaceutical compositions provided by this disclosure are combined with TLR agonists, the TLR agonists being selected from the group consisting of TLR1 agonists, TLR2 agonists, TLR3 agonists, TLR4 agonists, TLR5 agonists, TLR6 agonists, TLR7 agonists, TLR8 agonists, TLR9 agonists, TLR10 agonists and TLR11 agonists.

[0323] In some embodiments, the RLR agonists provided herein are combined with TLR3 agonists. TLR3 agonists are agonists that induce a signaling response via TLR3. Exemplary TLR3 agonists include, but are not limited to, polyinosinate:polycytidylic acid (Poly I:C), HILTONOL® (Poly ICLC), polyadenylic acid-polyuridylic acid (Poly A:U), RIBOXXIM® (RGIC® 100), RIBOXXON® (RGIC® 50 bioconjugate), and RIBOXXOL® (RGIC® 50).

[0324] In some embodiments, the RLR agonists provided by this disclosure are combined with polyinosinic acid:polycytidylic acid (Poly I:C). In some embodiments, the RLR agonists are combined with HILTONOL® (Poly ICLC). In some embodiments, the RLR agonists are combined with polyadenylic acid-polyuridylic acid (Poly A:U). In some embodiments, the RLR agonists are combined with RIBOXXIM® (RGIC® 100). In some embodiments, the RLR agonists are combined with RIBOXXON® (RGIC® 50 bioconjugate). In some embodiments, the RLR agonists are combined with RIBOXXOL® (RGIC® 50).

[0325] In some embodiments, the RLR agonists provided herein are combined with TLR7 agonists. TLR7 agonists are agonists that induce a signaling response via TLR7. Non-limiting examples of TLR7 agonists include single-stranded RNA (ssRNA), loxoribine (a guanosine analog derivatized at the N7 and C8 positions), imidazoquinoline compounds (e.g., imiquimod and reximod), or derivatives thereof. Further exemplary TLR7 agonists include, but are not limited to, GS-9620 (besatrimod), imiquimod (ALDARA®), and reximod (R-848).

[0326] In some embodiments, the RLR agonists provided by this disclosure are combined with GS-9620 (besatrimod). In some embodiments, the RLR agonists provided by this disclosure are combined with imiquimod (ALDARA®). In some embodiments, the RLR agonists are combined with rexiquimod (R-848).

[0327] In some embodiments, the RLR agonists provided herein are combined with TLR9 agonists. TLR9 agonists are agonists that induce a signaling response via TLR9. Examples of TLR9 agonists include, but are not limited to, CpG oligodeoxynucleotides (GpG ODNs). In some embodiments, the CpG ODNs are class A CpG ODNs (CpG-A ODNs), class B CpG ODNs (CpG-B ODNs), or class C CpG ODNs (CpG-C ODNs).

[0328] In some embodiments, the RLR agonists provided by this disclosure are combined with CpG oligodeoxynucleotides (CpG ODNs). In some embodiments, the CpG ODN is a class A CpG ODN (CpG-A ODN). In some embodiments, the CpG ODN is a class B CpG ODN (CpG-B ODN). In some embodiments, the CpG ODN is a class C CpG ODN (CpG-C ODN).

[0329] Other combinations In some embodiments, the RLR agonists or pharmaceutical compositions provided by this disclosure are combined with (for example, administered in combination with) VISTA antagonists, adenosine A2AR antagonists, B7-H3 antagonists, B7-H4 antagonists, BTLA antagonists, CTLA-4 antagonists, IDO antagonists, or KIR antagonists.

[0330] In some embodiments, the RLR agonists or pharmaceutical compositions provided by this disclosure are combined with an agonist comprising a polypeptide (e.g., an antibody or its antigen-binding moiety) that specifically binds to CD137(4-1BB) (e.g., administered in combination).

[0331] In some embodiments, the RLR agonists or pharmaceutical compositions provided by this disclosure are combined with an agonist comprising a polypeptide (e.g., an antibody or its antigen-binding moiety) that specifically binds to CD134(OX40) (e.g., administered in combination).

[0332] The RLR agonists described herein can replace or enhance previously or currently administered therapies. For example, treatment with an RLR agonist may discontinue or reduce the administration of one or more additional active agents, for example, by administering them at lower levels or doses. In some embodiments, administration of previous treatments can be maintained. In some embodiments, previous treatments are maintained until the level of the RLR agonist reaches a level sufficient to provide a therapeutic effect. The two therapies may be administered in combination.

[0333] Monitoring a subject (e.g., a human patient) for improvement in cancer means evaluating the subject for changes in disease parameters, such as a reduction in tumor growth, as defined herein. In some embodiments, the evaluation is performed at least one hour after administration, e.g., at least two, four, six, eight, twelve, 24, or forty-eight hours, or at least one day, two days, four days, ten days, thirteen days, twenty days, or longer, or at least one week, two weeks, four weeks, ten weeks, thirteen weeks, twenty weeks, or longer. The subject may be evaluated during one or more of the following periods: before treatment begins, during treatment, or after one or more elements of treatment have been administered. The evaluation may include evaluating the need for further treatment, e.g., whether the dosage, frequency, or duration of administration should be changed. It may also include evaluating the need to add or remove selected treatments, e.g., adding or removing any of the cancer treatments described herein.

[0334] In some embodiments, the RLR agonists described herein are administered to modulate a patient's T cell response, for example, by increasing T cell activation and / or proliferation. Improved T cell proliferation, IFN production and secretion, and / or T cell cytolytic activity may be beneficial for patients who require improved T cell proliferation, IFN production and secretion, and / or T cell cytolytic activity to treat a certain disease or condition. Therefore, in some embodiments, the RLR agonists of this disclosure are administered to patients who require these to induce or increase T cell activation, improve T cell proliferation, induce IFN production and / or secretion, and / or induce a cytolytic T cell response.

[0335] In some embodiments, the RLR agonists described herein can be used in methods for detecting and / or quantifying human RLR in biological samples. Thus, RLR agonists can be used to diagnose, predict, and / or determine the progression of a disease (e.g., cancer) in a patient, as described herein.

[0336] While this disclosure has been described with reference to its specific embodiments, it should be understood by those skilled in the art that various modifications may be made and equivalents may be substituted without departing from the true intent and scope of this disclosure. In addition, many modifications may be made to adapt specific circumstances, materials, substance compositions, processes, or one or more steps of a process to the purposes, intent and scope of this disclosure. All such modifications are intended to be within the scope of this disclosure. [Examples]

[0337] This disclosure will be better understood by reference to the following examples. However, the following examples should not be construed as limiting the scope of the disclosure. The examples and embodiments described herein are for illustrative purposes only, and various modifications or changes in that regard will be suggested to those skilled in the art and will be understood to be included in the spirit and scope of this application and the appended claims.

[0338] Example 1: Transfection of HuPBMCs with an RLR agonist induces cytokine production in vitro. To determine the effects of various modified RLR agonists on cytokine induction, the ability of RLR agonists to induce cytokine production was evaluated in vitro. Human peripheral blood mononuclear cells (huPBMCs) were prepared from two healthy donors and placed in 100 μL of RPMI1640 cell medium supplemented with fetal bovine serum (FCS), L-glutamine, and penicillin / streptavidin, in standard 96-well tissue culture plates, 2 × 10⁶ cells per well. 5Cells were seeded at a cell / well density. As shown in Figure 1, independent transfection of human PBMCs with RLR agonists was performed using Lipofectamine 2000 as the transfection reagent (except for G10 and ODN2216, for which direct incubation was applied and data are not shown). After incubating cells at 37°C for 24 hours in a humidified incubator, the cell culture supernatant was collected. The supernatant was immediately frozen and stored at -20°C. Using the U-Plex MSD platform, samples were thawed once for analysis of the cytokine IFN-α2a (Figure 1) and IL-1β, IP-10, IL-6, IL-12p70, MCP-1 and MIP-1β (data not shown), according to the manufacturer's instructions. Figure 1 shows dose-dependent induction of IFN-α secretion from human PBMCs treated with novel candidate RLR agonists containing various modifications and / or sequence motifs. RLR agonists were added at either 10 nM, 2 nM, or 0.4 nM concentrations. The amount of IFN-α2α released by cells in response to RLR agonist transfection is expressed in pg / mL.

[0339] Tables 3 and 4 show the sequences of each RLR agonist. Table 3 also shows the sequences and numbers corresponding to each compound tested in Figure 1. For example, compound X25224 in Figure 1 corresponds to "RIG7" and contains a first oligonucleotide containing SEQ ID NO: 42, which is linked to a second oligonucleotide containing SEQ ID NO: 73 via the linker "UUCG", and has a 5' diphosphate moiety. The sequence of RIG7 is also shown as sequence 6 in Table 4. [Table 3-1] [Table 3-2] [Table 4-1] [Table 4-2] Table 4-3

Claims

1. A synthetic RIG-I-like receptor (RLR) agonist that specifically binds to RIG-I-like receptors (RLRs), The agonist comprises a blunt-terminated hairpin RNA containing a first polynucleotide that is linked to a second polynucleotide by a linker, The first polynucleotide is sufficiently complementary to the second polynucleotide to form a double helix. The aforementioned double strand contains fewer than 19 base pairs, The 5' terminal nucleotide of the first polynucleotide comprises a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof. The derivative or analog thereof is a phosphate bioequivalent selected from phosphonates, thiophosphonates, phosphorothioates, sulfates, sulfonates, sulfamates, thiazolidinones, carboxylates, malonates, boronic acids, benzoxabolols, boranophosphates, and squalamides. A synthetic RIG-I-like receptor (RLR) agonist wherein the first polynucleotide comprises SEQ ID NO: 63 and the second polynucleotide comprises SEQ ID NO:

91.

2. A synthetic RIG-I-like receptor (RLR) agonist that specifically binds to RIG-I-like receptors (RLRs), The agonist comprises a blunt-terminated hairpin RNA containing a first polynucleotide that is linked to a second polynucleotide by a linker, The first polynucleotide is sufficiently complementary to the second polynucleotide to form a double helix. The aforementioned double strand contains fewer than 19 base pairs, The 5' terminal nucleotide of the first polynucleotide comprises a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof. The derivative or analog thereof is a phosphate bioequivalent selected from phosphonates, thiophosphonates, phosphorothioates, sulfates, sulfonates, sulfamates, thiazolidinones, carboxylates, malonates, boronic acids, benzoxabolols, boranophosphates, and squalamides. (i) The first polynucleotide contains SEQ ID NO: 38 and the second polynucleotide contains SEQ ID NO: 69, (ii) The first polynucleotide contains SEQ ID NO: 63 and the second polynucleotide contains SEQ ID NO: 91, or (iii) A synthetic RIG-I-like receptor (RLR) agonist in which the first polynucleotide comprises SEQ ID NO: 53 and the second polynucleotide comprises SEQ ID NO:

84.

3. The agonist according to claim 2, wherein the first polynucleotide comprises SEQ ID NO: 38 and the second polynucleotide comprises SEQ ID NO:

69.

4. A synthetic RIG-I-like receptor (RLR) agonist that specifically binds to RIG-I-like receptors (RLRs), The agonist comprises a blunt-terminated hairpin RNA containing a first polynucleotide that is linked to a second polynucleotide by a linker, The first polynucleotide is sufficiently complementary to the second polynucleotide to form a double helix. The aforementioned double strand contains fewer than 19 base pairs, The 5' terminal nucleotide of the first polynucleotide comprises a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof. The derivative or analog thereof is a phosphate bioequivalent selected from phosphonates, thiophosphonates, phosphorothioates, sulfates, sulfonates, sulfamates, thiazolidinones, carboxylates, malonates, boronic acids, benzoxabolols, boranophosphates, and squalamides. A synthetic RIG-I-like receptor (RLR) agonist wherein the first polynucleotide comprises SEQ ID NO: 48 and the second polynucleotide comprises SEQ ID NO:

79.

5. A synthetic RIG-I-like receptor (RLR) agonist that specifically binds to RIG-I-like receptors (RLRs), The agonist comprises a blunt-terminated hairpin RNA containing a first polynucleotide that is linked to a second polynucleotide by a linker, The first polynucleotide is sufficiently complementary to the second polynucleotide to form a double helix. The aforementioned double strand contains fewer than 19 base pairs, The 5' terminal nucleotide of the first polynucleotide comprises a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof. The derivative or analog thereof is a phosphate bioequivalent selected from phosphonates, thiophosphonates, phosphorothioates, sulfates, sulfonates, sulfamates, thiazolidinones, carboxylates, malonates, boronic acids, benzoxabolols, boranophosphates, and squalamides. A synthetic RIG-I-like receptor (RLR) agonist wherein the first polynucleotide comprises SEQ ID NO: 56 and the second polynucleotide comprises SEQ ID NO:

87.

6. The agonist according to any one of claims 1 to 5, wherein the linker is a nucleotide linker or a non-nucleotide linker.

7. The agonist according to claim 6, wherein the nucleotide linker comprises the sequence UUCG or UGUUU.

8. The agonist according to claim 6, wherein the non-nucleotide linker is selected from a hexaethylene glycol linker or a C9 alkyl linker.

9. A pharmaceutical composition comprising an agonist according to any one of claims 1 to 8 and a pharmaceutically acceptable carrier.

10. Use of an agonist according to any one of claims 1 to 8 in the manufacture of a pharmaceutical product for stimulating an immune response, treating or slowing the progression of cancer, or reducing or inhibiting tumor growth in a subject.

11. The aforementioned pharmaceutical is administered in combination with one or more further therapeutic agents, The use according to claim 10, wherein the one or more additional therapeutic agents are selected from the group consisting of chemotherapy, targeted anticancer therapy, oncolytic agents, cell death inducers, opsonizing agents, cytotoxic agents, immunotherapy, cytokines, activators or agonists of costimulatory molecules, inhibitors of inhibitory molecules, vaccines, cellular immunotherapy, and combinations thereof.

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