Synthetic RIG-i-like receptor agonists

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 TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62 / 659,999, filed April 19, 2018. The entire contents of the above-referenced application are incorporated herein by this reference. [Background technology]

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

[0003] There is still a need for further improved compositions and methods for regulating the activity of immunomodulatory proteins. Such agents can be used in cancer immunotherapy and the treatment of other conditions (e.g., chronic infections). There is a need to develop improved RIG-I-like receptor ligands for various therapeutic immunomodulatory applications. Summary of the Invention [Means for solving the problem]

[0004] The present 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 present disclosure provides a synthetic RIG-I-like receptor (RLR) agonist that specifically binds to an RIG-I-like receptor (RLR), the agonist comprising a blunt-ended hairpin RNA comprising a first polynucleotide connected to a second polynucleotide by a linker, the first polynucleotide being sufficiently complementary to the second polynucleotide to form a duplex, the duplex comprising fewer than 19 base pairs, the 5'-most nucleotide of the first oligonucleotide comprising a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof, and the agonist comprising a sequence motif that confers at least one improved biological activity mediated by the RLR compared to an agonist that does not comprise the sequence motif. In some embodiments, the first polynucleotide comprises the sequence motif.

[0006] In some embodiments, the RLR agonist of the present disclosure is: (i) GT repeat motif, (ii) GA repeat motif; (iii) AUCG repeat motif; (iv) AU repeat motif; (v) dipyrimidine motif, (vi) ziprine motif, (vii) pyrimidine triplet motif, (viii) Printed triplet motif, (ix) palindromic sequence motifs, and (x) Contains a sequence motif selected from the group consisting of any combination of (i) to (ix).

[0007] In some embodiments, the RLR agonists of the present disclosure comprise a combination of sequence motifs. In some embodiments, the combination of sequence motifs is a GT repeat motif and a purine triplet 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 AUGC repeat motif and a dipurine motif.

[0008] In some embodiments, the RLR agonists of the present disclosure comprise a sequence motif that confers at least one improved biological activity mediated by an RLR compared to an agonist that does not comprise the sequence motif, the at least one improved biological activity being (i) Increased cytokine production mediated by RLRs; (ii) increased RLR-mediated expression of interferon-inducible genes; (iii) increased intracellular signaling mediated by RLRs; (iv) increased binding affinity to RLRs, and (v) A combination of any one of (i) to (iv) is selected.

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

[0010] In some embodiments, the RLR agonists of the present disclosure comprise a sequence motif, wherein the sequence motif is a GT repeat motif (e.g., GTGTGT) comprising a sequence of fewer 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 fewer 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 comprising a sequence of less than about 10-15 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 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 about 5-10 guanine and thymine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GT repeat motif comprising a sequence of about 5 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 4 guanine and thymine nucleotides, or derivatives or analogs thereof.In some embodiments, the GT repeat motif confers improved biological activity in the RLR agonist, the improved biological activity being increased RLR-mediated cytokine production, increased RLR-mediated expression of interferon-inducible genes, increased RLR-mediated intracellular signaling, increased binding affinity for RLRs, and any combination of the above.

[0011] In some embodiments, the RLR agonist of the present disclosure comprises a sequence motif, wherein the sequence motif 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 agonists of the present disclosure comprise a sequence motif, the sequence motif being a GT repeat motif, the GT repeat motif being [GT] nwhere n=2 to 9, 3 to 7, or 4 to 8. In some embodiments, the GT repeat motif confers improved biological activity in an RLR agonist, the improved biological activity being increased RLR-mediated cytokine production, increased RLR-mediated expression of interferon-inducible genes, increased RLR-mediated intracellular signaling, increased binding affinity for an RLR, and any combination of the above.

[0012] In some embodiments, the present disclosure provides an RLR agonist that specifically binds to an RLR, the agonist comprising a blunt-ended hairpin RNA comprising a first polynucleotide connected (operably linked) to a second polynucleotide by a linker, the first polynucleotide being sufficiently complementary to the second polynucleotide to form a duplex, the duplex comprising fewer than 19 base pairs, the 5'-most nucleotide of the first oligonucleotide comprising a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof, the agonist comprising a sequence motif that confers at least one improved biological activity mediated by an RLR compared to an agonist that does not comprise this sequence motif, the first polynucleotide comprising this sequence motif being a GT repeat motif comprising a sequence of approximately 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, the improved biological activity is 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 present disclosure provides an RLR agonist that specifically binds to an RLR, the agonist comprising a blunt-ended hairpin RNA comprising a first polynucleotide connected to a second polynucleotide by a linker, the first polynucleotide being sufficiently complementary to the second polynucleotide to form a duplex, the duplex comprising fewer than 19 base pairs, the 5'-most nucleotide of the first oligonucleotide comprising a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof, the agonist comprising a sequence motif that confers at least one improved biological activity mediated by an RLR compared to an agonist that does not comprise this sequence motif, the first polynucleotide comprising this 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, the GT repeat motif is [GT]3, and the GT repeat motif is followed by a print triplet motif and UCG, respectively. In some embodiments, the print triplet is GGA. In some aspects, the improved biological activity is increased RLR-mediated cytokine production, increased RLR-mediated expression of interferon-inducible genes, increased RLR-mediated intracellular signaling, increased binding affinity for RLRs, and any combination of the above.

[0014] In some embodiments, the RLR agonists of the present disclosure comprise a sequence motif, wherein the sequence motif is a GA repeat motif (e.g., GAGAGA) comprising a sequence of fewer 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 fewer 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 comprising a sequence of less than about 10-15 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 10 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 5-10 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 5 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 4 guanine and adenine nucleotides or derivatives or analogs thereof.In some embodiments, the GA repeat motif confers improved biological activity in RLR agonists, the improved biological activity being increased RLR-mediated cytokine production, increased RLR-mediated expression of interferon-inducible genes, increased RLR-mediated intracellular signaling, increased binding affinity for RLRs, and any combination of the above.

[0015] In some embodiments, the RLR agonist of the present disclosure comprises a sequence motif, wherein the sequence motif is a GA repeat motif comprising a sequence 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 a sequence of 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 16 guanine and adenine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GA repeat motif comprising a sequence of 14 guanine and adenine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GA repeat motif comprising a sequence of 12 guanine and adenine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GA repeat motif comprising a sequence 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 derivatives or analogs thereof, hi some embodiments, the sequence motif is a GA repeat motif comprising a sequence of four guanine and adenine nucleotides or derivatives or analogs thereof.

[0016] In some embodiments, the RLR agonists of the present disclosure comprise a sequence motif, which is a GA repeat motif, which is [GA] n where n=2 to 9, 3 to 7, or 4 to 8. In some embodiments, the GA repeat motif confers improved biological activity in an RLR agonist, the improved biological activity being increased RLR-mediated cytokine production, increased RLR-mediated expression of interferon-inducible genes, increased RLR-mediated intracellular signaling, increased binding affinity for an RLR, and any combination of the above.

[0017] In some embodiments, the present disclosure provides an RLR agonist that specifically binds to an RLR, the agonist comprising a blunt-ended hairpin RNA comprising a first polynucleotide connected to a second polynucleotide by a linker, the first polynucleotide being sufficiently complementary to the second polynucleotide to form a duplex, the duplex comprising fewer than 19 base pairs, the 5'-most nucleotide of the first oligonucleotide comprising a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof, the agonist comprising a sequence motif that confers at least one improved RLR-mediated biological activity compared to an agonist that does not comprise this sequence motif, the first polynucleotide comprising this sequence motif, the sequence motif being a GA repeat motif comprising a sequence of approximately 14 guanine and adenine nucleotides, the GA repeat motif being [GA]7. In some embodiments, the sequence motif is a GA repeat motif, and the GA repeat motif is [GA]7. In some embodiments, the GA repeat motif confers improved biological activity in RLR agonists, the improved biological activity being increased RLR-mediated cytokine production, increased RLR-mediated expression of interferon-inducible genes, increased RLR-mediated intracellular signaling, increased binding affinity for RLRs, and any combination of the above.

[0018] In some embodiments, the RLR agonists of the present disclosure comprise a sequence motif, wherein the sequence motif is an AUCG repeat motif (e.g., AUCGAUCG) comprising a sequence of fewer than 19, about 16, about 12-16, about 12, about 8-12, about 6, 16, 12, or 8 adenine, uracil, cytosine, and guanine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is an AUCG repeat motif comprising a sequence of fewer 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 a sequence 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 a sequence 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 comprising a sequence of 12 adenine, uracil, cytosine, and guanine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is an AUCG repeat motif comprising a sequence of about 8-12 adenine, uracil, cytosine, and guanine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is an AUCG repeat motif comprising a sequence of about 6 adenine, uracil, cytosine, and guanine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is an AUCG repeat motif comprising a sequence of 16 adenine, uracil, cytosine, and guanine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is an AUCG repeat motif comprising a sequence of 12 adenine, uracil, cytosine, and guanine nucleotides, or 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 improved biological activity on RLR agonists, such as 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.

[0019] In some embodiments, the RLR agonists of the present disclosure comprise a sequence motif, which is an AUCG repeat motif, which is [AUCG] n where n=2 to 4 or 2, 3, or 4. In some embodiments, the AUCG repeat motif confers improved biological activity in an RLR agonist, the improved biological activity being increased RLR-mediated cytokine production, increased RLR-mediated expression of interferon-inducible genes, increased RLR-mediated intracellular signaling, increased binding affinity for an RLR, and any combination of the above.

[0020] In some embodiments, the present disclosure provides an RLR agonist that specifically binds to an RLR, the agonist comprising a blunt-ended hairpin RNA comprising a first polynucleotide connected to a second polynucleotide by a linker, the first polynucleotide being sufficiently complementary to the second polynucleotide to form a duplex, the duplex comprising fewer than 19 base pairs, the 5'-most nucleotide of the first oligonucleotide comprising a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof, the agonist comprising a sequence motif that confers at least one improved RLR-mediated biological activity compared to an agonist that does not comprise this sequence motif, the first polynucleotide comprising this sequence motif being an AUCG repeat motif comprising a sequence of approximately 12 guanine and adenine nucleotides. In some embodiments, the AUCG repeat motif is [AUCG]3. In some embodiments, the AUCG repeat motif confers improved biological activity in an RLR agonist, the improved biological activity being increased RLR-mediated cytokine production, increased RLR-mediated expression of interferon-inducible genes, increased RLR-mediated intracellular signaling, increased binding affinity for an RLR, and any combination of the above.

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

[0022] In some embodiments, the RLR agonists of the present disclosure comprise an AUCG repeat motif, which is preceded by a dipurine motif. In some embodiments, the dipurine motif is GA. In some embodiments, the AUCG repeat motif is [AUCG]3, which is preceded by the dipurine motif GA. In some embodiments, the AUCG repeat motif is preceded by the dipurine motif II.

[0023] In some embodiments, the RLR agonists of the present disclosure comprise an AUGC repeat motif, wherein one or more uridine nucleosides (U) are replaced with a modified nucleoside. In some embodiments, the modified nucleoside is ribothymidine (T). In some embodiments, the AUGC repeat motif is [AUCG]3, wherein one or more uridine nucleosides (U) comprising the AUCG repeat motif are replaced with a modified nucleoside, wherein the modified nucleoside is ribothymidine (T). In some embodiments, the AUGC repeat motif is [AUCG]3, wherein one or more uridine nucleosides (U) comprising the AUCG repeat motif are replaced with a modified nucleoside, wherein the modified nucleoside is ribothymidine (T), and wherein the AUGC repeat motif is preceded by GG.

[0024] In some embodiments, the RLR agonists of the present disclosure comprise an AUGC repeat motif, in which one or more guanosine nucleosides (G) are replaced 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) comprising the AUCG repeat motif are replaced with a modified nucleoside, in which the modified nucleoside is ribothymidine (T), and in which GG precedes the AUGC repeat motif.

[0025] In some embodiments, the RLR agonists of the present disclosure comprise an AUCG repeat motif preceded by an IG, hi some embodiments, the AUCG repeat motif is [AUCG]3, preceded by an IG.

[0026] In some embodiments, the RLR agonist of the present disclosure comprises an AUCG repeat, wherein one or more guanosine nucleosides (G) are substituted with inosine (I), and wherein the AUCG repeat is preceded by an inosine (I). In some embodiments, the guanosine nucleosides (G) comprising the AUCG repeat are substituted with inosine (I), and wherein the AUCG repeat is preceded by an inosine (I), and wherein the 5'-most nucleotide of the first polynucleotide comprises an inosine (I).

[0027] In some embodiments, the RLR agonists of the present disclosure comprise an AUCG repeat motif, wherein the AUCG repeat motif is [AUCG]2. In some embodiments, the sequence motif is an AUCG repeat motif, wherein the AUCG repeat motif is [AUCG]2, and wherein the AUCG repeat motif is preceded by a dipurine motif. In some embodiments, the sequence motif is an AUCG repeat motif, wherein the AUCG repeat motif is [AUCG]2, and wherein the AUCG repeat motif is preceded by a dipurine motif, wherein the dipurine motif is GG.

[0028] In some embodiments, the RLR agonists of the present disclosure comprise an AUCG repeat motif, wherein the AUCG repeat motif is [AUCG]2 and is preceded by a print triplet motif. In some embodiments, the print triplet motif is GGG. In some embodiments, the sequence motif is an AUCG repeat motif, wherein the AUCG repeat motif is [AUCG]2 and is preceded by a print triplet motif, wherein the AUCG repeat motif is GGG. In some embodiments, the sequence motif is an AUCG repeat motif, wherein the AUCG repeat motif is [AUCG]2 and is preceded by CCCCCG. In some embodiments, the sequence motif is an AUCG repeat motif, wherein the AUCG repeat motif is [AUCG]2 and is preceded by TCGUCG.

[0029] In some embodiments, the RLR agonists of the present disclosure comprise a sequence motif, which is a palindromic sequence comprising less 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, linked in any order to form a palindrome. In some embodiments, the sequence motif is a palindromic sequence comprising less than 19 nucleotides, or derivatives or analogs thereof, linked in any order to form a palindrome. In some embodiments, the sequence motif is a palindromic sequence comprising about 15-18 nucleotides, or derivatives or analogs thereof, linked in any order to form a palindrome. In some embodiments, the sequence motif is a palindromic sequence comprising about 15 nucleotides, or derivatives or analogs thereof, linked in any order to form a palindrome. In some embodiments, the sequence motif is a palindromic sequence comprising a sequence of about 10-15 nucleotides, or derivatives or analogs thereof, which can be linked in any order to form a palindrome. In some embodiments, the sequence motif is a palindromic sequence comprising a sequence of about 10 nucleotides, or derivatives or analogs thereof, which can be linked in any order to form a palindrome. In some embodiments, the sequence motif is a palindromic sequence comprising a sequence of 18 nucleotides, or derivatives or analogs thereof, which can be linked in any order to form a palindrome. In some embodiments, the sequence motif is a palindromic sequence comprising a sequence of 17 nucleotides, or derivatives or analogs thereof, which can be linked in any order to form a palindrome. In some embodiments, the sequence motif is a palindromic sequence comprising a sequence of 16 nucleotides, or derivatives or analogs thereof, which can be linked in any order to form a palindrome. In some embodiments, the sequence motif is a palindromic sequence comprising a sequence of 15 nucleotides, or derivatives or analogs thereof, which can be linked in any order to form a palindrome.In some embodiments, the sequence motif is a palindromic sequence comprising a sequence of 14 nucleotides, or derivatives or analogs thereof, 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, 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, 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, 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, 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, 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, linked in any order to generate a palindrome. In some embodiments, the sequence motif is a palindromic sequence comprising a sequence of seven nucleotides, or derivatives or analogs thereof, which can be linked in any order to produce a palindrome. In some embodiments, the sequence motif is a palindromic sequence comprising a sequence of six nucleotides, or derivatives or analogs thereof, which can be linked in any order to produce a palindrome. In some embodiments, the sequence motif is a palindromic sequence comprising a sequence of five nucleotides, or derivatives or analogs thereof, which can be linked in any order to produce a palindrome. In some embodiments, the sequence motif is a palindromic sequence comprising a sequence of four nucleotides, or derivatives or analogs thereof, which can be linked in any order to produce a palindrome.

[0030] In some embodiments, the RLR agonist of the present disclosure comprises a linker, wherein the linker is flanked by AU. In some embodiments, the linker is flanked by AU repeat motifs, wherein the AU repeat motif is [AU] n wherein 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 an RLR, wherein the agonist has the following formula: 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', wherein (i) (N1-N2-X1) comprises a first polynucleotide comprising linked nucleotides N1, N2 and X1; (ii) (X2-N3-N4) comprises a second polynucleotide comprising linked nucleotides X2, N3 and N4; (iii) N1, N2, N3, and N4 each comprise 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 each an oligonucleotide containing a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine; (viii) X1 is complementary to X2; (ix) X1 and X2 are each 12 to 16 nucleotides in length and are the same length; (x) L is a linker that operably links the first polynucleotide and the second polynucleotide; wherein 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, which base pairs with a cytidine in the hairpin RNA. In some embodiments, the RLR agonist of the present disclosure has improved biological activity, 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 an RLR, wherein the agonist comprises a blunt-ended hairpin RNA comprising a non-nucleotide linker, and the agonist has the following formula: 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', wherein (i) (N1-N2-X1) comprises a first polynucleotide comprising linked nucleotides N1, N2 and X1; (ii) (X2-N3-N4) comprises a second polynucleotide comprising linked nucleotides X2, N3 and N4; (iii) N1, N2, N3, and N4 each comprise 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 each an oligonucleotide containing a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine; (viii) X1 is complementary to X2; (ix) X1 and X2 are each 12 to 16 nucleotides in length and are the same length; (x) L is a non-nucleotide linker that covalently links the first polynucleotide and the second polynucleotide; When present, inosine forms a base pair with cytidine. In some embodiments, the RLR agonist of the present disclosure has improved biological activity, and this improved biological activity is increased RLR-mediated cytokine production, increased RLR-mediated expression of interferon-induced genes, increased RLR-mediated intracellular signaling, increased binding affinity to RLR, and any combination of the above.

[0033] In some embodiments, N1 comprises inosine and N4 comprises cytidine. In some embodiments, N1 comprises inosine and N4 comprises cytidine, and X1 and X2 are each 12 nucleotides in length. In some embodiments, N1 comprises cytidine and N4 comprises inosine. In some embodiments, N2 comprises inosine and N3 comprises cytidine. In some embodiments, N2 comprises cytidine and N3 comprises inosine. In some embodiments, N1 comprises guanosine. In some embodiments, N2 comprises guanosine. In some embodiments, N1 comprises cytidine. In some embodiments, N2 comprises cytidine. In some embodiments, N1 and N2 comprise guanosine, and N3 and N4 comprise cytidine. In some embodiments, N1 and N2 comprise cytidine, and N3 and N4 comprise guanosine. In some embodiments, N1 and N2 comprise inosine, and N3 and N4 comprise cytidine. In some embodiments, N1 and N2 comprise cytidine and N3 and N4 comprise inosine.

[0034] In some embodiments, the RLR agonists of the present disclosure have the following formula: 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', wherein (i) (N1-N2-X1) comprises a first polynucleotide comprising linked nucleotides N1, N2 and X1; (ii) (X2-N3-N4) comprises a second polynucleotide comprising linked nucleotides X2, N3 and N4; (iii) N1, N2, N3, and N4 each comprise 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 each an oligonucleotide containing a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine; (viii) X1 is complementary to X2; (ix) X1 and X2 are each 12 to 16 nucleotides in length and are the same length; (x) L is a non-nucleotide linker that covalently links the first polynucleotide and the second polynucleotide; When present, inosine forms base pairs with cytidine, wherein N1 comprises inosine, N4 comprises cytidine, and X1 and / or X2 each comprise at least one inosine. In some embodiments, N2 comprises inosine, N3 comprises cytidine, and X1 and / or X2 each comprise at least one inosine. In some embodiments, N1 and N2 comprise guanosine, N3 and N4 comprise cytidine, and X1 and / or X2 each comprise at least one inosine. In some embodiments, N1 and N2 comprise guanosine, N3 and N4 comprise cytidine, and X1 and X2 each comprise at least one inosine. In some embodiments, N1 and N2 comprise guanosine, N3 and N4 comprise cytidine, and X1 and X2 each comprise at least one inosine. In some embodiments, N1 and N2 comprise guanosine, N3 and N4 comprise cytidine, and X1 and X2 each comprise at least one inosine, and X1 and X2 are each 12 nucleotides in length. In some embodiments, N1 and N2 comprise cytidine, N3 and N4 comprise guanosine, and X1 and X2 each comprise at least one inosine. In some embodiments, N1 and N2 comprise guanosine, N3 and N4 comprise cytidine, and X1 and X2 each comprise inosine and do not comprise a guanosine nucleoside. In some embodiments, N1 and N2 comprise guanosine, N3 and N4 comprise cytidine, and X1 and X2 each comprise at least one inosine, and X1 and X2 are each 12 nucleotides in length. In some embodiments, N1 and N2 comprise cytidine, N3 and N4 comprise guanosine, and X1 and X2 each comprise inosine and do not comprise a guanosine nucleoside. In some embodiments, the RLR agonists of the present disclosure have improved biological activity, which is increased RLR-mediated cytokine production, increased RLR-mediated expression of interferon-inducible genes, increased RLR-mediated intracellular signaling, increased binding affinity for RLRs, and any combination of the above.

[0035] In some embodiments, the RLR agonists of the present disclosure have the following formula: 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', wherein (i) (N1-N2-X1) comprises a first polynucleotide comprising linked nucleotides N1, N2 and X1; (ii) (X2-N3-N4) comprises a second polynucleotide comprising linked nucleotides X2, N3 and N4; (iii) N1, N2, N3, and N4 each comprise 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 each an oligonucleotide containing a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine; (viii) X1 is complementary to X2; (ix) X1 and X2 are each 12 to 16 nucleotides in length and are the same length; (x) L is a non-nucleotide linker that covalently links the first polynucleotide and the second polynucleotide; When present, inosine forms base pairs with cytidine, and N1 and N2 comprise inosine, N3 and N4 comprise cytidine, and X1 and / or X2 each comprise at least one inosine. In some embodiments, N1 and N2 comprise inosine, N3 and N4 comprise cytidine, and X1 and X2 each comprise at least one inosine, and X1 and X2 each are 12 nucleotides in length. In some embodiments, N1 and N2 comprise inosine, N3 and N4 comprise cytidine, and X1 and X2 each comprise at least one inosine. In some embodiments, N1 and N2 comprise inosine, N3 and N4 comprise cytidine, and X1 and X2 each comprise at least one inosine, and X1 and X2 each are 12 nucleotides in length. In some embodiments, N1 and N2 comprise cytidine, N3 and N4 comprise inosine, and X1 and X2 each comprise at least one inosine. In some embodiments, N1 and N2 comprise inosine, N3 and N4 comprise cytidine, and X1 and X2 comprise inosine and do not comprise guanosine nucleosides. In some embodiments, N1 and N2 comprise cytidine, N3 and N4 comprise inosine, and X1 and X2 comprise inosine and do not comprise guanosine nucleosides. In some embodiments, the RLR agonists of the present disclosure have improved biological activity, which is increased RLR-mediated cytokine production, increased RLR-mediated expression of interferon-inducible genes, increased RLR-mediated intracellular signaling, increased binding affinity for RLRs, and any combination of the above.

[0036] In some embodiments, the RLR agonists of the present disclosure have the following formula: 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', wherein X1 and X2 are each 12 nucleotides and comprise 1, 2, 3, or 4 inosine nucleosides. In some embodiments, X1 and X2 are each 13 nucleotides and comprise 1, 2, 3, 4, or 5 inosine nucleosides. In some embodiments, X1 and X2 are each 14 nucleotides and comprise 1, 2, 3, 4, 5, or 6 inosine nucleosides. In some embodiments, X1 and X2 are each 15 nucleotides and comprise 1, 2, 3, 4, 5, 6, or 7 inosine nucleosides. In some embodiments, X1 and X2 are each 16 nucleotides and comprise 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. In some embodiments, the RLR agonists of the present disclosure have improved biological activity, which is increased RLR-mediated cytokine production, increased RLR-mediated expression of interferon-inducible genes, increased RLR-mediated intracellular signaling, increased binding affinity for RLRs, and any combination of the above.

[0037] In some embodiments, the present disclosure provides an RLR agonist that specifically binds to an RLR, wherein the agonist has the following formula: 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', wherein (i) (N1-N2-X1) comprises a first polynucleotide comprising linked nucleotides N1, N2 and X1; (ii) (X2-N3-N4) comprises a second polynucleotide comprising linked nucleotides X2, N3 and N4; (iii) N1, N2, N3, and N4 each comprise 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 has the sequence motif [AUCN5] x wherein N5 comprises guanosine or inosine, x is an integer whose value indicates the number of sequence motifs, and x=3 or 4; (viii) X2 has the sequence motif [CN6AU] y wherein N6 comprises guanosine or inosine, y is an integer whose value indicates the number of sequence motifs, and y=3 or 4; (ix) L is a linker that operably links the first polynucleotide and the second polynucleotide; Optionally, at least one of N1, N2, N3, and N4 is inosine, and the inosine nucleoside base pairs with a cytidine in the hairpin RNA.

[0038] In some embodiments, N5 comprises inosine and N6 comprises inosine. In some embodiments, N5 comprises guanosine and N6 comprises inosine. In some embodiments, N5 comprises inosine and N6 comprises guanosine. In some embodiments, N5 comprises guanosine (G) and N6 comprises guanosine (G). In some embodiments, x=3 and y=3. In some embodiments, x=4 and y=4. In some embodiments, N1 comprises inosine (I) and N4 comprises cytidine (C). In some embodiments, N2 comprises inosine (I) and N3 comprises cytidine (C). In some embodiments, N3 comprises inosine (I) and N2 comprises cytidine (C). In some embodiments, N4 comprises inosine (I) and N1 comprises cytidine (C). In some embodiments, N1 comprises guanosine (G). In some embodiments, N2 comprises guanosine (G). In some embodiments, N1 comprises cytidine (C). In some embodiments, N2 comprises cytidine (C). In some embodiments, N1 and N2 comprise guanosine (G) and N3 and N4 comprise cytidine (C). In some embodiments, N1 and N2 comprise cytidine (C) and N3 and N4 comprise guanosine (G). In some embodiments, N1 and N2 comprise inosine (I) and N3 and N4 comprise cytidine (C). In some embodiments, N1 and N2 comprise cytidine (C) and N3 and N4 comprise inosine (I). In some aspects, the RLR agonists of the present disclosure have improved biological activity, which is increased RLR-mediated cytokine production, increased RLR-mediated expression of interferon-inducible genes, increased RLR-mediated intracellular signaling, increased binding affinity for RLRs, and any combination of the above.

[0039] In some embodiments of the RLR agonist of the present disclosure, a linker is included, and the linker is 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, and the nucleotide sequence of the tetraloop is as follows: (a) UNCG, where N=A, C, G, or U; (b) GNRA, where 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 (wherein Y=C or T); (e) UMAC, where M=A or C, and (f) selected from the group consisting of CUUG.

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

[0041] In some embodiments, the RLR agonists of the present disclosure comprise a nucleotide linker, wherein the nucleotide linker comprises 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 the present disclosure comprise a non-nucleotide linker, wherein the non-nucleotide linker is one of the following: (a) an ethylene glycol linker, and (b) an alkyl linker.

[0043] In some embodiments, the non-nucleotidic linker is a hexaethylene glycol linker. In some embodiments, the non-nucleotidic 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 bioisostere, wherein the phosphate bioisostere is selected from phosphonate, thiophosphonate, phosphorothioate, sulfate, sulfonate, sulfamate, thiazolidinone, carboxylate, malonate, boronic acid, benzoxaborole, boranophosphate, and squaramide.

[0045] In some embodiments, the agonist comprises a modified nucleotide, modified nucleoside, or modified nucleobase, or a combination thereof. In some embodiments, the agonist comprises a modification to the internucleotide linkage or polynucleotide backbone.

[0046] In some embodiments, the RLR agonists of the present disclosure have the following properties: (a) specifically binds to one or more RLRs (e.g., RIG-1, MDA5, and / or LGP2); (b) increase RLR-mediated cytokine production; (c) increasing RLR-mediated expression of interferon-inducible genes (ISGs); (d) increasing RLR-dependent intracellular signaling; (e) increasing the stability of the double strand; (f) increasing binding affinity to RLRs; (g) reducing off-target binding; (h) increasing biological half-life; (i) enhance biodistribution and bioavailability; (j) increasing and / or enhancing uptake into cells and / or tissues; (k) reducing immunogenicity, and (l) Shows one or more combinations of any of (a) to (k).

[0047] In some embodiments, the present disclosure provides an RLR agonist that specifically binds to an RLR, wherein the agonist has the following formula: 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', wherein (i) (N1-N2-X1) comprises a first polynucleotide comprising linked nucleotides N1, N2 and X1; (ii) (X2-N3-N4) comprises a second polynucleotide comprising linked nucleotides X2, N3 and N4; (iii) N1, N2, N3, and N4 each comprise 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 each an oligonucleotide containing a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine; (viii) X1 is complementary to X2; (ix) X1 and X2 are each 12 to 16 nucleotides in length and are the same length; (x) L is a linker that operably links the first polynucleotide and the second polynucleotide; N1 and N2 each contain a guanosine, N3 and N4 each contain a cytidine, X1 and X2 each are 12 nucleotides in length, X1 and X2 each contain at least one inosine nucleoside which base pairs with a cytidine in the hairpin RNA, L contains a nucleotide linker comprising a tetraloop, and the nucleotide sequence of the tetraloop is UUCG.

[0048] In some embodiments, the present disclosure provides an RLR agonist that specifically binds to an RLR, wherein the agonist has the following formula: 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', wherein (i) (N1-N2-X1) comprises a first polynucleotide comprising linked nucleotides N1, N2 and X1; (ii) (X2-N3-N4) comprises a second polynucleotide comprising linked nucleotides X2, N3 and N4; (iii) N1, N2, N3, and N4 each comprise 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 each an oligonucleotide containing a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine; (viii) X1 is complementary to X2; (ix) X1 and X2 are each 12 to 16 nucleotides in length and are the same length; (x) L is a linker that operably links the first polynucleotide and the second polynucleotide; N1 comprises an inosine, N2 comprises a guanosine, N3 and N4 each comprise a cytidine, X1 and X2 each are 12 nucleotides in length, X1 and X2 each comprise at least one inosine nucleoside, which base pairs with a cytidine in the hairpin RNA, L comprises a nucleotide linker comprising a tetraloop, and the nucleotide sequence of the tetraloop is UUCG.

[0049] In some embodiments, the present disclosure provides an RLR agonist that specifically binds to an RLR, wherein the agonist has the following formula: 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', wherein (i) (N1-N2-X1) comprises a first polynucleotide comprising linked nucleotides N1, N2 and X1; (ii) (X2-N3-N4) comprises a second polynucleotide comprising linked nucleotides X2, N3 and N4; (iii) N1, N2, N3, and N4 each comprise 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 each an oligonucleotide containing a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine; (viii) X1 is complementary to X2; (ix) X1 and X2 are each 12 to 16 nucleotides in length and are the same 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 in length, X1 and X2 each contain at least one inosine nucleoside, which base pairs with a cytidine in the hairpin RNA, and L contains a nucleotide linker containing a tetraloop, the nucleotide sequence of the tetraloop being UUCG.

[0050] In some embodiments, the present disclosure provides an RLR agonist that specifically binds to an RLR, wherein the agonist comprises a blunt-ended hairpin RNA comprising a non-nucleotide linker, and the agonist has the following formula: 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', wherein (i) (N1-N2-X1) comprises a first polynucleotide comprising linked nucleotides N1, N2 and X1; (ii) (X2-N3-N4) comprises a second polynucleotide comprising linked nucleotides X2, N3 and N4; (iii) N1, N2, N3, and N4 each comprise 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 each an oligonucleotide containing a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine; (viii) X1 is complementary to X2; (ix) X1 and X2 are each 12 to 16 nucleotides in length and are the same length; (x) L is a non-nucleotide linker that covalently links the first polynucleotide and the second polynucleotide; N1 and N2 contain guanosine, N3 and N4 contain cytidine, X1 and X2 are each 12 nucleotides in length, and the non-nucleotidic linker is a C9 alkyl linker.

[0051] In some embodiments, the present disclosure provides an RLR agonist that specifically binds to an RLR, wherein the agonist comprises a blunt-ended hairpin RNA comprising a non-nucleotide linker, and the agonist has the following formula: 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', wherein (i) (N1-N2-X1) comprises a first polynucleotide comprising linked nucleotides N1, N2 and X1; (ii) (X2-N3-N4) comprises a second polynucleotide comprising linked nucleotides X2, N3 and N4; (iii) N1, N2, N3, and N4 each comprise 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 each an oligonucleotide containing a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine; (viii) X1 is complementary to X2; (ix) X1 and X2 are each 12 to 16 nucleotides in length and are the same length; (x) L is a non-nucleotide linker that covalently links the first polynucleotide and the second polynucleotide; N1 and N2 contain guanosine, N3 and N4 contain cytidine, X1 and X2 are each 12 nucleotides in length, and the non-nucleotide linker is a hexaethylene glycol linker.

[0052] In some embodiments, the present disclosure provides an RLR agonist that specifically binds to an RLR, wherein the 5'-most 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 present disclosure provides an RLR agonist that specifically binds to an RLR, wherein the agonist comprises a blunt-ended hairpin RNA comprising a first polynucleotide connected to a second polynucleotide by a linker, wherein the first polynucleotide is sufficiently complementary to the second polynucleotide to form a duplex, wherein the duplex comprises fewer than 19 base pairs, wherein the 5'-most nucleotide of the first oligonucleotide comprises a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof, wherein the agonist comprises a sequence motif that confers at least one improved biological activity mediated by an RLR compared to an agonist that does not comprise the sequence motif, and wherein the first polynucleotide and the second polynucleotide are selected from the group consisting of: (i) SEQ ID NOs: 37 and 68, respectively; (ii) SEQ ID NOs: 38 and 69, respectively; (iii) SEQ ID NOs: 39 and 70, respectively; (iv) SEQ ID NOs: 40 and 71, respectively; (v) SEQ ID NOs: 41 and 72, respectively; (vi) SEQ ID NOs: 42 and 73, respectively; (vii) SEQ ID NOs: 43 and 74, respectively; (viii) SEQ ID NOs: 44 and 75, respectively; (ix) SEQ ID NOs: 45 and 76, respectively; (x) SEQ ID NOs: 46 and 77, respectively; (xi) SEQ ID NOs: 47 and 78, respectively; (xii) SEQ ID NOs: 48 and 79, respectively; (xiii) SEQ ID NOs: 49 and 80, respectively; (xiv) SEQ ID NOs: 50 and 81, respectively; (xv) SEQ ID NOs: 51 and 82, respectively; (xvi) SEQ ID NOs: 52 and 83, respectively; (xvii) SEQ ID NOs: 53 and 84, respectively; (xviii) SEQ ID NOs: 54 and 85, respectively; (xix) SEQ ID NOs: 55 and 86, respectively; (xx) SEQ ID NOs: 56 and 87, respectively; (xxi) SEQ ID NOs: 57 and 88, respectively; (xxii) SEQ ID NOs: 58 and 89, respectively; (xxiii) SEQ ID NOs: 59 and 89, respectively; (xxiv) SEQ ID NOs: 60 and 90, respectively; (xxv) SEQ ID NOs: 61 and 91, respectively; (xxvi) SEQ ID NOs: 62 and 92, respectively; (xxvii) SEQ ID NOs: 63 and 91, respectively; (xxviii) SEQ ID NOs: 64 and 93, respectively; (xxix) SEQ ID NOs: 65 and 94, respectively; (xxx) SEQ ID NOs: 66 and 95, respectively; (xxxi) SEQ ID NOs: 67 and 96, respectively, and (xxxii) each comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 63 and 97.

[0054] In some embodiments, the present disclosure provides an RLR agonist that specifically binds to an RLR, wherein the agonist comprises a blunt-ended hairpin RNA comprising at least one or more nucleotides including an inosine that base pairs with a cytidine, and wherein the agonist comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 22, 23, and 25.

[0055] In some embodiments, the present disclosure provides an RLR agonist that specifically binds to an RLR, wherein the agonist comprises a blunt-ended hairpin RNA comprising at least one or more nucleotides including an inosine that base pairs with a cytidine, wherein the agonist comprises the formula 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', wherein (N1-N2-X1) comprises a first polynucleotide and (X2-N3-N4) comprises a second polynucleotide, and wherein the first polynucleotide and the second polynucleotide are selected from the following: (i) SEQ ID NOs: 58 and 89, respectively; (ii) SEQ ID NOs: 59 and 89, respectively; and (iii) comprise a nucleotide sequence selected from the group consisting of SEQ ID NOs: 61 and 91, respectively.

[0056] In some embodiments, the present disclosure provides an RLR agonist that specifically binds to an RLR, wherein the agonist comprises a blunt-ended hairpin RNA comprising a non-nucleotide linker, wherein the agonist comprises the formula 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', wherein (N1-N2-X1) comprises a first polynucleotide and (X2-N3-N4) comprises a second polynucleotide, and wherein the first polynucleotide and the second polynucleotide are selected from the following: (i) SEQ ID NOs: 37 and 68, respectively; (ii) SEQ ID NOs: 38 and 69, respectively; (iii) SEQ ID NOs: 39 and 70, respectively; (iv) SEQ ID NOs: 40 and 71, respectively; (v) SEQ ID NOs: 41 and 72, respectively; (vi) SEQ ID NOs: 42 and 73, respectively; (vii) SEQ ID NOs: 43 and 74, respectively; (viii) SEQ ID NOs: 44 and 75, respectively; (ix) SEQ ID NOs: 45 and 76, respectively; (x) SEQ ID NOs: 46 and 77, respectively; (xi) SEQ ID NOs: 47 and 78, respectively; (xii) SEQ ID NOs: 48 and 79, respectively; (xiii) SEQ ID NOs: 49 and 80, respectively; (xiv) SEQ ID NOs: 50 and 81, respectively; (xv) SEQ ID NOs: 51 and 82, respectively; (xvi) SEQ ID NOs: 52 and 83, respectively; (xvii) SEQ ID NOs: 53 and 84, respectively; (xviii) SEQ ID NOs: 54 and 85, respectively; (xix) SEQ ID NOs: 55 and 86, respectively; (xx) SEQ ID NOs: 56 and 87, respectively; (xxi) SEQ ID NOs: 57 and 88, respectively; (xxii) SEQ ID NOs: 58 and 89, respectively; (xxiii) SEQ ID NOs: 59 and 89, respectively; (xxiv) SEQ ID NOs: 60 and 90, respectively; (xxv) SEQ ID NOs: 61 and 91, respectively; (xxvi) SEQ ID NOs: 62 and 92, respectively; (xxvii) SEQ ID NOs: 63 and 91, respectively; (xxviii) SEQ ID NOs: 64 and 93, respectively; (xxix) SEQ ID NOs: 65 and 94, respectively; (xxx) SEQ ID NOs: 66 and 95, respectively; (xxxi) SEQ ID NOs: 67 and 96, respectively, and (xxxii) each comprising 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 the present disclosure, the nucleotide sequence comprising the RLR agonist is not complementary to a genomic DNA sequence or an 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 present disclosure provides a pharmaceutical composition for stimulating an immune response, treating or delaying the progression of cancer, or reducing or inhibiting tumor growth in a subject in need thereof, the pharmaceutical composition comprising an RLR agonist provided by the present 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 present disclosure provides a method for increasing RLR-mediated production of one or more cytokines in a cell, the method comprising contacting a cell with an RLR agonist provided by the present disclosure, wherein the RLR agonist increases RLR-mediated cytokine production in the cell. In some embodiments, the RLR agonist increases RLR-mediated type I interferon (e.g., IFN-α, IFN-β) production in the cell. In some embodiments, the RLR agonist increases RLR-mediated IL-1β production in the cell. In some embodiments, the RLR agonist increases RLR-mediated IP-10 production in the cell. In some embodiments, the RLR agonist increases RLR-mediated IL-6, IL-12p70, MCP-1, and / or MIP-1β production in the cell.

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

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

[0062] In some embodiments, the present disclosure provides a method of 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 present disclosure.

[0063] In some embodiments, the present disclosure provides a method of treating or delaying 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 present disclosure.

[0064] In some embodiments, the present disclosure provides a method of reducing or inhibiting tumor growth in a subject in need thereof, the method comprising administering to the subject an effective amount of an RLR agonist or pharmaceutical composition provided by the present disclosure.

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

[0066] In some embodiments of the methods provided by the present disclosure, the RLR agonist or pharmaceutical composition provided by the present disclosure is administered in combination with one or more additional therapeutic agents, wherein the one or more additional therapeutic agents are selected from the group consisting of chemotherapy, targeted anti-cancer therapy, oncolytic agent, cell death inducer, opsonizing agent (e.g., opsonizing antibody), cytotoxic agent, immune system therapy, cytokine, activator or agonist of costimulatory molecule, inhibitor of inhibitory molecule, vaccine, cellular immunotherapy, or combinations thereof.

[0067] In some embodiments, an RLR agonist or pharmaceutical composition provided by the present disclosure is administered before or after administration of one or more additional therapeutic agents, or one or more additional therapeutic agents are administered simultaneously with, before, or after administration of the agonist or pharmaceutical composition.

[0068] In some embodiments, 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.

[0069] In some embodiments, the one or more additional therapeutic agents is an agonist comprising a polypeptide (eg, an antibody, or antigen-binding portion thereof) that specifically binds to CD137 (4-1BB).

[0070] In some embodiments, the one or more additional therapeutic agents is an agonist comprising a polypeptide (eg, an antibody, or antigen-binding portion thereof) that specifically binds to CD134 (OX40).

[0071] In some embodiments, the 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, the one or more additional therapeutic agents is a TIM-3 antagonist.

[0073] In some embodiments, the one or more additional therapeutic agents is a VISTA antagonist.

[0074] In some embodiments, the one or more additional therapeutic agents is an adenosine A2AR antagonist.

[0075] In some embodiments, the one or more additional therapeutic agents is a B7-H3 antagonist.

[0076] In some embodiments, the one or more additional therapeutic agents is a B7-H4 antagonist.

[0077] In some embodiments, the one or more additional therapeutic agents is a BTLA antagonist.

[0078] In some embodiments, the one or more additional therapeutic agents is a CTLA-4 antagonist.

[0079] In some embodiments, the one or more additional therapeutic agents is an IDO antagonist.

[0080] In some embodiments, the one or more additional therapeutic agents is a KIR antagonist.

[0081] In some embodiments, the one or more additional therapeutic agents is a LAG-3 antagonist.

[0082] In some embodiments, the one or more additional therapeutic agents are Toll-like receptor 3 (TLR3) agonists. In some embodiments, the TLR3 agonist is polyinosinic:polycytidylic acid (poly I:C). In some embodiments, the TLR3 agonist is HILTONOL® (poly ICLC). In some embodiments, the TLR3 agonist is polyadenylic-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, the one or more additional therapeutic agents are Toll-like receptor 7 (TLR7) agonists. In some embodiments, the TLR7 agonist is GS-9620 (vesatolimod). In some embodiments, the TLR7 agonist is imiquimod (ALDARA™). In some embodiments, the TLR7 agonist is resiquimod (R-848).

[0084] In some embodiments, the one or more additional 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 present disclosure provides for the use of an RLR agonist or pharmaceutical composition provided by the present disclosure to stimulate an immune response, treat or delay the progression of cancer, or inhibit tumor growth in a subject in need thereof, optionally in combination with one or more additional therapeutic agents.

[0086] In some embodiments, the present disclosure provides for the use of an RLR agonist or pharmaceutical composition provided by the present disclosure in the manufacture of a medicament for stimulating an immune response, treating or slowing the progression of cancer, or inhibiting tumor growth in a subject in need thereof, optionally in combination with one or more additional therapeutic agents.

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

[0088] In some embodiments of the uses or kits provided by the present disclosure, the RLR agonist or pharmaceutical composition provided by the present disclosure is administered in combination with one or more additional therapeutic agents, wherein the one or more additional therapeutic agents are selected from the group consisting of chemotherapy, targeted anti-cancer therapy, oncolytic agent, cell death inducer, opsonizing agent (e.g., opsonizing antibody), cytotoxic agent, immune system therapy, cytokine, activator of costimulatory molecule, inhibitor of inhibitory molecule, vaccine, cellular immunotherapy, or combinations thereof.

[0089] In some embodiments of the uses or kits provided by the present disclosure, the RLR agonist or pharmaceutical composition provided by the present disclosure is administered before or after the administration of one or more additional therapeutic agents, or the one or more additional therapeutic agents are administered simultaneously with, before, or after the administration of the agonist or pharmaceutical composition.

[0090] In some embodiments of the uses or kits provided by the present disclosure, the one or more additional therapeutic agents is 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.

[0091] In some embodiments of the uses or kits provided by the present disclosure, the one or more additional therapeutic agents is an agonist comprising a polypeptide (e.g., an antibody, or an antigen-binding portion thereof) that specifically binds to CD137 (4-1BB).

[0092] In some embodiments of the uses or kits provided by the present disclosure, the one or more additional therapeutic agents of the present disclosure is an agonist comprising a polypeptide (e.g., an antibody, or an antigen-binding portion thereof) that specifically binds to CD134 (OX40). The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. The present invention provides, for example, the following items. (Item 1) 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-ended hairpin RNA comprising a first polynucleotide connected to a second polynucleotide by a linker, the first polynucleotide being sufficiently complementary to the second polynucleotide to form a duplex, the duplex comprising fewer than 19 base pairs, the 5'-most nucleotide of the first oligonucleotide comprising a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof, and the agonist comprises a sequence motif that confers at least one improved biological activity mediated by the RLR compared to an agonist that does not comprise the sequence motif. (Item 2) The sequence motif is (i) GT repeat motif, (ii) GA repeat motif; (iii) AUCG repeat motif; (iv) AU repeat motif; (v) dipyrimidine motif, (vi) ziprine motif, (vii) pyrimidine triplet motif, (viii) Printed triplet motif, (ix) palindromic sequence motifs, and (x) The agonist according to item 1, selected from the group consisting of any combination of (i) to (ix). (Item 3) The at least one improved biological activity is (i) Increased cytokine production mediated by RLRs; (ii) increased RLR-mediated expression of interferon-inducible genes; (iii) increased intracellular signaling mediated by RLRs; (iv) increased binding affinity to RLRs, and (v) The agonist according to items 1 and 2, selected from any combination of (i) to (iv). (Item 4) 4. The agonist according to items 1 to 3, wherein the sequence motif 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. (Item 5) The GT repeat motif is [GT] n 5. The agonist according to item 4, wherein n=2 to 9. (Item 6) The agonist according to items 4 and 5, wherein the GT repeat motif is [GT]7. (Item 7) 6. The agonist according to items 4 and 5, wherein the GT repeat motif is [GT]3 and the GT repeat motif is followed by a print triplet motif and a UCG, respectively. (Item 8) 8. The agonist according to item 7, wherein the print triplet motif is GGA. (Item 9) 4. The agonist according to items 1 to 3, wherein 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, 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 GA repeat motif is [GA] n 10. The agonist according to item 9, wherein n=2 to 9. (Item 11) 11. The agonist of item 10, wherein the GA repeat motif is [GA]7. (Item 12) 4. The agonist according to items 1 to 3, wherein the sequence motif is an AUCG repeat motif comprising a sequence of less than 19, about 16, about 12-16, about 12, about 8-12, about 6, 16, 12, 8 adenine, uracil, cytosine and guanine nucleotides, or derivatives or analogs thereof. (Item 13) The AUCG repeat motif is [AUCG] n 13. The agonist according to item 12, wherein n=2 to 4. (Item 14) 14. The agonist of item 13, wherein the AUCG repeat motif is [AUCG]3. (Item 15) 15. The agonist according to items 12 to 14, wherein the AUCG repeat motif is preceded by a CG or dipyrimidine motif. (Item 16) The agonist according to Item 15, wherein the AUCG repeat motif is preceded by a CG. 16. The agonist according to item 15, wherein the dipyrimidine motif is CC. (Item 18) 15. The agonist according to items 12 to 14, wherein the AUCG repeat motif is preceded by a dipurine motif. (Item 19) 19. The agonist according to item 18, wherein the dipurine motif is GA. (Item 20) 19. The agonist according to item 18, wherein the dipurine motif is II. (Item 21) 21. The agonist according to any one of items 12 to 20, wherein U constituting the AUCG repeat motif is substituted with a modified nucleoside. (Item 22) 22. The agonist of item 21, wherein the modified nucleoside is ribothymidine (T). (Item 23) 21. The agonist according to any one of items 12 to 20, wherein G constituting the AUCG repeat motif is substituted with a modified nucleoside. (Item 24) 24. The agonist according to item 23, wherein the modified nucleoside is inosine (I). (Item 25) 15. The agonist according to items 12 to 14, wherein the AUCG repeat motif is preceded by an IG. (Item 26) 15. The agonist according to items 12 to 14, wherein G constituting the AUCG repeat motif is replaced with inosine (I) and an inosine (I) is present before the AUCG repeat. (Item 27) 27. The agonist of item 26, wherein the 5'-most nucleotide of the first polynucleotide is inosine (I). (Item 28) 14. The agonist according to items 12 and 13, wherein the AUCG repeat motif is [AUCG]2. (Item 29) 29. The agonist according to item 28, wherein the AUCG repeat motif is preceded by a dipurine motif. (Item 30) 30. The agonist of item 29, wherein the dipurine motif is GG. (Item 31) 29. The agonist according to item 28, wherein the AUCG repeat motif is preceded by a print triplet. (Item 32) 32. The agonist according to item 31, wherein the printlet is GGG. (Item 33) 29. The agonist of item 28, wherein the AUCG repeat motif is preceded by CCCCCG. (Item 34) 29. The agonist of item 28, wherein the AUCG repeat motif is preceded by TCGUCG. (Item 35) 4. The agonist according to items 1 to 3, wherein the sequence motif is a palindromic sequence comprising a sequence of less than 19, about 15-18, about 15, about 10-15, about 10, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or 4 nucleotides, or derivatives or analogs thereof, linked in any order to generate a palindrome. (Item 36) 36. The agonist according to any one of items 1 to 35, wherein the linker is flanked by AUs. (Item 37) The linker is adjacent to an AU repeat motif, and the AU repeat motif is [AU] n 37. The agonist according to any one of items 1 to 36, wherein n=2 to 3. (Item 38) 38. The agonist of item 37, wherein the AU repeat motif is [AU]2. (Item 39) 1. A synthetic RIG-I-like receptor (RLR) agonist that specifically binds to a RIG-I-like receptor (RLR), the agonist having the following formula: 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', wherein (i) (N1-N2-X1) comprises a first polynucleotide comprising linked nucleotides N1, N2 and X1; (ii) (X2-N3-N4) comprises a second polynucleotide comprising linked nucleotides X2, N3 and N4; (iii) N1, N2, N3, and N4 each comprise 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 each an oligonucleotide containing a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine; (viii) X1 is complementary to X2; (ix) X1 and X2 are each 12 to 16 nucleotides in length and are the same length; (x) L is a linker that operably links the first polynucleotide and the second polynucleotide; wherein 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, said inosine nucleoside base-pairing with a cytidine in said hairpin RNA. (Item 40) 39. The agonist according to item 39, wherein N1 comprises inosine and N4 comprises cytidine. (Item 41) 39. The agonist according to item 39, wherein N1 comprises cytidine and N4 comprises inosine. (Item 42) 40. The agonist according to item 39, wherein N2 comprises inosine and N3 comprises cytidine. (Item 43) 39. The agonist according to item 38, wherein N2 comprises cytidine and N3 comprises inosine. (Item 44) 39. The agonist according to item 38, wherein N1 comprises guanosine. (Item 45) 39. The agonist of item 38, wherein N2 comprises guanosine. (Item 46) 39. The agonist according to item 38, wherein N1 comprises cytidine. (Item 47) 39. The agonist of item 38, wherein N2 comprises cytidine. (Item 48) 40. The agonist according to item 39, wherein N1 and N2 comprise guanosine and N3 and N4 comprise cytidine. (Item 49) 39. The agonist according to item 39, wherein N1 and N2 comprise cytidine and N3 and N4 comprise guanosine. (Item 50) 39. The agonist according to item 39, wherein N1 and N2 comprise inosine and N3 and N4 comprise cytidine. (Item 51) 39. The agonist according to item 39, wherein N1 and N2 comprise cytidine and N3 and N4 comprise inosine. (Item 52) 39. The agonist according to item 39, wherein N1 comprises an inosine, N4 comprises a cytidine, and X1 and / or X2 each comprise at least one inosine. (Item 53) 40. The agonist according to item 39, wherein N2 comprises an inosine, N3 comprises a cytidine, and X1 and / or X2 each comprise at least one inosine. (Item 54) The agonist according to 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) 40. The agonist according to item 39, wherein N1 and N2 comprise guanosine, N3 and N4 comprise cytidine, and X1 and X2 each comprise at least one inosine. (Item 56) 40. The agonist according to item 39, wherein N1 and N2 comprise cytidine, N3 and N4 comprise guanosine, and X1 and X2 each comprise at least one inosine. (Item 57) 39. The agonist according to item 39, wherein N1 and N2 comprise guanosine, N3 and N4 comprise cytidine, and X1 and X2 each comprise inosine, and do not comprise a guanosine nucleoside. (Item 58) 39. The agonist according to item 39, wherein N1 and N2 comprise cytidine, N3 and N4 comprise guanosine, and X1 and X2 each comprise inosine, and do not comprise a guanosine nucleoside. (Item 59) 40. The agonist according to item 39, wherein N1 and N2 comprise inosine, N3 and N4 comprise cytidine, and X1 and / or X2 each comprise at least one inosine. (Item 60) 39. The agonist according to item 39, wherein N1 and N2 comprise inosine, N3 and N4 comprise cytidine, and X1 and X2 each comprise at least one inosine. (Item 61) 40. The agonist according to item 39, wherein N1 and N2 comprise cytidine, N3 and N4 comprise inosine, and X1 and / or X2 each comprise at least one inosine. (Item 62) The agonist according to Item 39, wherein N1 and N2 contain inosine, N3 and N4 contain cytidine, and X1 and X2 contain inosine but do not contain guanosine nucleosides. (Item 63) The agonist according to Item 39, wherein N1 and N2 contain cytidine, N3 and N4 contain inosine, and X1 and X2 contain inosine, and the agonist does not contain a guanosine nucleoside. (Item 64) 39. The agonist according to item 39, wherein X1 and X2 are each 12 nucleotides and contain 1, 2, 3 or 4 inosine nucleosides. (Item 65) 39. The agonist according to item 38, wherein X1 and X2 are each 13 nucleotides and contain 1, 2, 3, 4 or 5 inosine nucleosides. (Item 66) 39. The agonist according to item 38, wherein X1 and X2 are each 14 nucleotides and contain 1, 2, 3, 4, 5 or 6 inosine nucleosides. (Item 67) 39. The agonist according to item 38, wherein X1 and X2 are each 15 nucleotides and contain 1, 2, 3, 4, 5, 6 or 7 inosine nucleosides. (Item 68) 40. The agonist of item 39, wherein X1 and X2 are each 16 nucleotides and each contain 1, 2, 3, 4, 5, 6, 7 or 8 inosine nucleosides. (Item 69) 40. The agonist according to item 39, wherein X1 and X2 are each 12 nucleotides and contain at least 10%, 20%, 30% or 40% inosine nucleosides. (Item 70) 1. A synthetic RIG-I-like receptor (RLR) agonist that specifically binds to a RIG-I-like receptor (RLR), said agonist having the following formula: 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', wherein (i) (N1-N2-X1) comprises a first polynucleotide comprising linked nucleotides N1, N2 and X1; (ii) (X2-N3-N4) comprises a second polynucleotide comprising linked nucleotides X2, N3 and N4; (iii) N1, N2, N3, and N4 each comprise 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 has the sequence motif [AUCN5] x wherein N5 comprises guanosine or inosine, x is an integer whose value indicates the number of sequence motifs, and x=3 or 4; (viii) X2 has the sequence motif [CN6AU] y wherein N6 comprises guanosine or inosine, y is an integer whose value indicates the number of sequence motifs, and y=3 or 4; (ix) L is a linker that operably links the first polynucleotide and the second polynucleotide; Optionally, the synthetic RIG-I-like receptor (RLR) agonist, wherein at least one of N1, N2, N3, and N4 is inosine, and the inosine nucleoside forms a base pair with a cytidine in the hairpin RNA. (Item 71) 71. The agonist according to item 70, wherein N5 comprises inosine and N6 comprises inosine. (Item 72) 72. The agonist according to item 71, wherein N5 comprises guanosine and N6 comprises inosine. (Item 73) 73. The agonist according to item 72, wherein N5 comprises inosine and N6 comprises guanosine. (Item 74) 74. The agonist according to item 73, wherein N5 comprises guanosine (G) and N6 comprises guanosine (G). (Item 75) 75. The agonist according to any one of items 70 to 74, wherein x=3 and y=3. (Item 76) 75. The agonist according to any one of items 70 to 74, wherein x=4 and y=4. (Item 77) 75. The agonist according to any one of items 70 to 74, wherein N1 comprises inosine (I) and N4 comprises cytidine (C). (Item 78) 75. The agonist according to any one of items 70 to 74, wherein N2 comprises inosine (I) and N3 comprises cytidine (C). (Item 79) 75. The agonist according to any one of items 70 to 74, wherein N3 comprises inosine (I) and N2 comprises cytidine (C). (Item 80) 75. The agonist according to any one of items 70 to 74, wherein N4 comprises inosine (I) and N1 comprises cytidine (C). (Item 81) The agonist according to any one of Items 70 to 74, wherein N1 comprises guanosine (G). (Item 82) The agonist according to any one of Items 70 to 74, wherein N2 comprises guanosine (G). (Item 83) 75. The agonist according to any one of items 70 to 74, wherein N1 comprises cytidine (C). (Item 84) 75. The agonist according to any one of items 70 to 74, wherein N2 comprises cytidine (C). (Item 85) 75. The agonist of any one of items 70 to 74, wherein N1 and N2 comprise guanosine (G) and N3 and N4 comprise cytidine (C). (Item 86) 75. The agonist of any one of items 70 to 74, wherein N1 and N2 comprise cytidine (C) and N3 and N4 comprise guanosine (G). (Item 87) 75. The agonist of any one of items 70 to 74, wherein N1 and N2 comprise inosine (I) and N3 and N4 comprise cytidine (C). (Item 88) 75. The agonist of any one of items 70 to 74, wherein N1 and N2 comprise cytidine (C) and N3 and N4 comprise inosine (I). (Item 89) 1. A synthetic RIG-I-like receptor (RLR) agonist that specifically binds to a RIG-I-like receptor (RLR), the agonist comprising a blunt-ended hairpin RNA comprising a non-nucleotide linker, the agonist having the following formula: 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', wherein (i) (N1-N2-X1) comprises a first polynucleotide comprising linked nucleotides N1, N2 and X1; (ii) (X2-N3-N4) comprises a second polynucleotide comprising linked nucleotides X2, N3 and N4; (iii) N1, N2, N3, and N4 each comprise 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 each an oligonucleotide containing a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine; (viii) X1 is complementary to X2; (ix) X1 and X2 are each 12 to 16 nucleotides in length and are the same length; (x) L is a non-nucleotide linker that covalently links the first polynucleotide and the second polynucleotide; Inosine, when present, base pairs with cytidine, a synthetic RIG-I-like receptor (RLR) agonist. (Item 90) 89. The agonist according to item 89, wherein N1 comprises inosine and N4 comprises cytidine. (Item 91) 89. The agonist according to item 89, wherein N1 comprises cytidine and N4 comprises inosine. (Item 92) 89. The agonist according to item 89, wherein N2 comprises inosine and N3 comprises cytidine. (Item 93) 89. The agonist according to item 89, wherein N2 comprises cytidine and N3 comprises inosine. (Item 94) 89. The agonist according to item 89, wherein N1 comprises guanosine. (Item 95) 89. The agonist according to item 89, wherein N2 comprises guanosine. (Item 96) 89. The agonist according to item 89, wherein N1 comprises cytidine. (Item 97) 89. The agonist according to item 89, wherein N2 comprises cytidine. (Item 98) 89. The agonist according to item 89, wherein N1 and N2 comprise guanosine and N3 and N4 comprise cytidine. (Item 99) 89. The agonist according to item 89, wherein N1 and N2 comprise cytidine and N3 and N4 comprise guanosine. (Item 100) 89. The agonist according to item 89, wherein N1 and N2 comprise inosine and N3 and N4 comprise cytidine. (Item 101) 89. The agonist according to item 89, wherein N1 and N2 comprise cytidine and N3 and N4 comprise inosine. (Item 102) 89. The agonist according to item 89, wherein N1 comprises an inosine, N4 comprises a cytidine, and X1 and / or X2 each comprise at least one inosine. (Item 103) 89. The agonist according to item 89, wherein N2 comprises an inosine, N3 comprises a cytidine, and X1 and / or X2 each comprise at least one inosine. (Item 104) The agonist according to 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) 89. The agonist according to item 89, wherein N1 and N2 comprise guanosine, N3 and N4 comprise cytidine, and X1 and X2 each comprise at least one inosine. (Item 106) 89. The agonist according to item 89, wherein N1 and N2 comprise cytidine, N3 and N4 comprise guanosine, and X1 and X2 each comprise at least one inosine. (Item 107) 89. The agonist according to item 89, wherein N1 and N2 comprise guanosine, N3 and N4 comprise cytidine, and X1 and X2 each comprise inosine, and do not comprise a guanosine nucleoside. (Item 108) 89. The agonist according to item 89, wherein N1 and N2 comprise cytidine, N3 and N4 comprise guanosine, and X1 and X2 each comprise inosine, and do not comprise guanosine nucleosides. (Item 109) 89. The agonist according to item 89, wherein N1 and N2 comprise inosine, N3 and N4 comprise cytidine, and X1 and / or X2 each comprise at least one inosine. (Item 110) 89. The agonist according to item 89, wherein N1 and N2 comprise inosine, N3 and N4 comprise cytidine, and X1 and X2 each comprise at least one inosine. (Item 111) 89. The agonist according to item 89, wherein N1 and N2 comprise cytidine, N3 and N4 comprise inosine, and X1 and / or X2 each comprise at least one inosine. (Item 112) The agonist according to Item 89, wherein N1 and N2 comprise inosine, N3 and N4 comprise cytidine, and X1 and X2 comprise inosine but do not comprise guanosine nucleosides. (Item 113) The agonist according to Item 89, wherein N1 and N2 comprise cytidine, N3 and N4 comprise inosine, and X1 and X2 comprise inosine, and the agonist does not comprise a guanosine nucleoside. (Item 114) 89. 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) 89. 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) 89. 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) 89. 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) 89. The agonist of 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) 89. The agonist according to item 89, wherein X1 and X2 are each 12 nucleotides and contain at least 10%, 20%, 30% or 40% inosine nucleosides. (Item 120) 89. The agonist according to any one of items 1 to 88, wherein the linker is a nucleotide linker or a non-nucleotide linker. (Item 121) 121. The agonist according to item 120, wherein the linker is a non-nucleotidic linker. (Item 122) 121. The agonist according to item 120, wherein the linker is a nucleotide linker. (Item 123) The nucleotide linker comprises a tetraloop, the nucleotide sequence of the tetraloop being: (a) UNCG, where N=A, C, G, or U; (b) GNRA, where 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 (wherein Y=C or T); (e) UMAC, where M=A or C, and (f) The agonist according to item 122, selected from the group consisting of CUUG. (Item 124) 123. The agonist according to item 122, wherein the nucleotide linker comprises the nucleotide sequence UUUGAU or UGUUU. (Item 125) 124. The agonist according to item 123, wherein the sequence of the tetraloop is UUCG. (Item 126) 124. The agonist according to item 123, wherein the sequence of the tetraloop is GAUC. (Item 127) 125. The agonist according to item 124, wherein the nucleotide linker comprises the nucleotide sequence UUUGAU. (Item 128) 125. The agonist according to item 124, wherein the nucleotide linker comprises the nucleotide sequence UGUUU. (Item 129) the non-nucleotide linker (a) an ethylene glycol linker, and (b) an alkyl linker; (Item 130) 130. The agonist of item 129, wherein the non-nucleotidic linker is a hexaethylene glycol linker. (Item 131) 130. The agonist according to item 129, wherein the non-nucleotidic linker is a C9 alkyl linker. (Item 132) 132. The agonist according to any one of items 1 to 131, wherein the agonist comprises a 5' diphosphate moiety, or a derivative or analogue thereof. (Item 133) 132. The agonist according to any one of items 1 to 131, wherein the agonist comprises a 5' triphosphate moiety, or a derivative or analogue thereof. (Item 134) 134. The agonist according to item 132 or 133, wherein the derivative or analog thereof comprises a phosphate bioisostere, wherein the phosphate bioisostere is selected from phosphonate, thiophosphonate, phosphorothioate, sulfate, sulfonate, sulfamate, thiazolidinone, carboxylate, malonate, boronic acid, benzoxaborole, boranophosphate, squaramide. (Item 135) 135. The agonist according to any one of items 1 to 134, wherein the agonist comprises a modified nucleotide, a modified nucleoside or a modified nucleobase, or a combination thereof. (Item 136) 136. The agonist according to any one of items 1 to 135, wherein the agonist comprises a modification to the internucleotide bond or 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) increase RLR-mediated cytokine production; (c) increasing RLR-mediated expression of interferon-inducible genes (ISGs); (d) increasing RLR-dependent intracellular signaling; (e) increasing duplex stability; (f) increasing binding affinity to RLRs; (g) reducing off-target binding; (h) increasing biological half-life; (i) enhance biodistribution and bioavailability; (j) increasing and / or enhancing uptake into cells and / or tissues; (k) reducing immunogenicity, and (1) The agonist according to any one of items 1 to 136, which exhibits at least one or more of any combination of (a) to (k). (Item 138) 1. A synthetic RIG-I-like receptor (RLR) agonist that specifically binds to a RIG-I-like receptor (RLR), wherein the 5'-most nucleotide of the agonist comprises a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof, and 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. (Item 139) 1. A synthetic RIG-I-like receptor (RLR) agonist that specifically binds to a RIG-I-like receptor (RLR), the agonist comprising a blunt-ended hairpin RNA comprising a first polynucleotide connected to a second polynucleotide by a linker, the first polynucleotide being sufficiently complementary to the second polynucleotide to form a duplex, the duplex comprising fewer than 19 base pairs, the 5'-most nucleotide of the first oligonucleotide comprising a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof, the agonist comprising a sequence motif that confers at least one improved biological activity mediated by the RLR compared to an agonist that does not comprise the sequence motif, and the first polynucleotide and the second polynucleotide are selected from the group consisting of: (i) SEQ ID NOs: 37 and 68, respectively; (ii) SEQ ID NOs: 38 and 69, respectively; (iii) SEQ ID NOs: 39 and 70, respectively; (iv) SEQ ID NOs: 40 and 71, respectively; (v) SEQ ID NOs: 41 and 72, respectively; (vi) SEQ ID NOs: 42 and 73, respectively; (vii) SEQ ID NOs: 43 and 74, respectively; (viii) SEQ ID NOs: 44 and 75, respectively; (ix) SEQ ID NOs: 45 and 76, respectively; (x) SEQ ID NOs: 46 and 77, respectively; (xi) SEQ ID NOs: 47 and 78, respectively; (xii) SEQ ID NOs: 48 and 79, respectively; (xiii) SEQ ID NOs: 49 and 80, respectively; (xiv) SEQ ID NOs: 50 and 81, respectively; (xv) SEQ ID NOs: 51 and 82, respectively; (xvi) SEQ ID NOs: 52 and 83, respectively; (xvii) SEQ ID NOs: 53 and 84, respectively; (xviii) SEQ ID NOs: 54 and 85, respectively; (xix) SEQ ID NOs: 55 and 86, respectively; (xx) SEQ ID NOs: 56 and 87, respectively; (xxi) SEQ ID NOs: 57 and 88, respectively; (xxii) SEQ ID NOs: 58 and 89, respectively; (xxiii) SEQ ID NOs: 59 and 89, respectively; (xxiv) SEQ ID NOs: 60 and 90, respectively; (xxv) SEQ ID NOs: 61 and 91, respectively; (xxvi) SEQ ID NOs: 62 and 92, respectively; (xxvii) SEQ ID NOs: 63 and 91, respectively; (xxviii) SEQ ID NOs: 64 and 93, respectively; (xxix) SEQ ID NOs: 65 and 94, respectively; (xxx) SEQ ID NOs: 66 and 95, respectively; (xxxi) SEQ ID NOs: 67 and 96, respectively, and (xxxii) A synthetic RIG-I-like receptor (RLR) agonist comprising 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-ended hairpin RNA comprising at least one nucleotide including an inosine that base pairs with a 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), the agonist comprising a blunt-ended hairpin RNA comprising at least one nucleotide including an inosine that base pairs with a cytidine, the agonist comprising the formula 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', wherein (N1-N2-X1) comprises a first polynucleotide and (X2-N3-N4) comprises a second polynucleotide, the first polynucleotide and the second polynucleotide being selected from the following: (i) SEQ ID NOs: 58 and 89, respectively; (ii) SEQ ID NOs: 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) 1. A synthetic RIG-I-like receptor (RLR) agonist that specifically binds to a RIG-I-like receptor (RLR), the agonist comprising a blunt-ended hairpin RNA comprising a non-nucleotide linker, the agonist comprising the formula 5′-(N1-N2-X1)-L-(X2-N3-N4)-3′, wherein (N1-N2-X1) comprises a first polynucleotide and (X2-N3-N4) comprises a second polynucleotide, the first polynucleotide and the second polynucleotide being selected from the following: (i) SEQ ID NOs: 37 and 68, respectively; (ii) SEQ ID NOs: 38 and 69, respectively; (iii) SEQ ID NOs: 39 and 70, respectively; (iv) SEQ ID NOs: 40 and 71, respectively; (v) SEQ ID NOs: 41 and 72, respectively; (vi) SEQ ID NOs: 42 and 73, respectively; (vii) SEQ ID NOs: 43 and 74, respectively; (viii) SEQ ID NOs: 44 and 75, respectively; (ix) SEQ ID NOs: 45 and 76, respectively; (x) SEQ ID NOs: 46 and 77, respectively; (xi) SEQ ID NOs: 47 and 78, respectively; (xii) SEQ ID NOs: 48 and 79, respectively; (xiii) SEQ ID NOs: 49 and 80, respectively; (xiv) SEQ ID NOs: 50 and 81, respectively; (xv) SEQ ID NOs: 51 and 82, respectively; (xvi) SEQ ID NOs: 52 and 83, respectively; (xvii) SEQ ID NOs: 53 and 84, respectively; (xviii) SEQ ID NOs: 54 and 85, respectively; (xix) SEQ ID NOs: 55 and 86, respectively; (xx) SEQ ID NOs: 56 and 87, respectively; (xxi) SEQ ID NOs: 57 and 88, respectively; (xxii) SEQ ID NOs: 58 and 89, respectively; (xxiii) SEQ ID NOs: 59 and 89, respectively; (xxiv) SEQ ID NOs: 60 and 90, respectively; (xxv) SEQ ID NOs: 61 and 91, respectively; (xxvi) SEQ ID NOs: 62 and 92, respectively; (xxvii) SEQ ID NOs: 63 and 91, respectively; (xxviii) SEQ ID NOs: 64 and 93, respectively; (xxix) SEQ ID NOs: 65 and 94, respectively; (xxx) SEQ ID NOs: 66 and 95, respectively; (xxxi) SEQ ID NOs: 67 and 96, respectively, and (xxxii) A synthetic RIG-I-like receptor (RLR) agonist comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 63 and 97, respectively. (Item 143) 143. The agonist of any one of items 1 to 142, wherein the nucleotide sequence comprising the agonist is not complementary to a genomic DNA sequence or an mRNA sequence, the RLR agonist does not participate in RNA interference, and the RLR agonist does not silence gene expression. (Item 144) 144. A pharmaceutical composition for stimulating an immune response, treating or slowing the progression of cancer, or reducing or inhibiting tumor growth in a subject in need thereof, comprising an agonist according to any one of items 1 to 143 and a pharmaceutically acceptable carrier. (Item 145) 145. The pharmaceutical composition of claim 144, wherein the agonist is formulated in a polyethyleneimine (PEI) carrier. (Item 146) Item 146. The pharmaceutical composition of item 145, wherein the PEI carrier is JetPEI®. (Item 147) 144. A method for increasing RLR-mediated production of one or more cytokines in a cell, the method comprising contacting the cell with an agonist according to any one of items 1 to 143, wherein the agonist increases RLR-mediated cytokine production in the cell. (Item 148) 144. A method for increasing RLR-mediated expression of one or more interferon-inducible genes in a cell, the method comprising contacting the cell with an agonist according to any one of items 1 to 143, wherein the agonist increases RLR-mediated expression of one or more interferon-inducible genes in the cell. (Item 149) 144. A method for increasing RLR-dependent intracellular signaling in a cell, the method comprising contacting the cell with an agonist according to any one of items 1 to 143, wherein the agonist increases RLR-dependent intracellular signaling. (Item 150) 147. A method for stimulating an immune response in a subject, said method comprising administering to said subject an effective amount of the agonist according to any one of items 1 to 143 or the pharmaceutical composition according to any one of items 144 to 146. (Item 151) 147. A method of treating or delaying the progression of cancer in a subject, said method comprising administering to said subject an effective amount of the agonist of any one of items 1 to 143 or the pharmaceutical composition of any one of items 144 to 146. (Item 152) 147. A method of reducing or inhibiting tumor growth in a subject in need thereof, said method comprising administering to said 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) 14. A method of stimulating an immune response, treating or delaying the progression of cancer, or inhibiting tumor growth in a subject in need thereof, said method comprising administering to said subject an effective amount of the agonist according to any one of items 1 to 143 or the pharmaceutical composition according to any one of items 144 to 146, wherein said agonist or said pharmaceutical composition increases RLR-mediated production of one or more cytokines in a cell, increases RLR-mediated expression of one or more interferon-inducible genes in a cell, and / or increases RLR-dependent intracellular signaling in a cell, thereby stimulating said immune response, treating or delaying the progression of said cancer, or inhibiting the growth of said tumor. (Item 154) 154. The method of any one of items 150 to 153, wherein the agonist or pharmaceutical composition is administered in combination with one or more additional therapeutic agents, wherein the one or more additional therapeutic agents are selected from the group consisting of chemotherapy, targeted anti-cancer therapy, oncolytic agent, cell death inducer, opsonizing agent (e.g., opsonizing antibody), cytotoxic agent, immune system therapy, cytokine, activator or agonist of costimulatory molecule, inhibitor of inhibitory molecule, vaccine, cellular immunotherapy, or combination thereof. (Item 155) 155. The method of claim 154, wherein the agonist or pharmaceutical composition is administered before or after the administration of the one or more additional therapeutic agents, or the one or more additional therapeutic agents are administered simultaneously with, before, or after the administration of the agonist or pharmaceutical composition. (Item 156) 156. The method of item 154 or 155, wherein the one or more additional therapeutic agents is 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) 156. The method of claim 154 or 155, wherein the one or more additional therapeutic agents is an agonist comprising a polypeptide (e.g., an antibody, or an antigen-binding portion thereof) that specifically binds to CD137 (4-1BB). (Item 158) 156. The method of claim 154 or 155, wherein the one or more additional therapeutic agents is an agonist comprising a polypeptide (e.g., an antibody, or an antigen-binding portion thereof) that specifically binds to CD134 (OX40). (Item 159) 157. The method of claim 156, wherein the one or more additional therapeutic agents is a PD-1 / PD-L1 antagonist. (Item 160) 160. The method of 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) 160. The method of 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) 157. The method of claim 156, wherein the one or more additional therapeutic agents is a TIM-3 antagonist. (Item 163) 157. The method of claim 156, wherein the one or more additional therapeutic agents is a VISTA antagonist. (Item 164) Item 157. The method of item 156, wherein the one or more additional therapeutic agents is an adenosine A2AR antagonist. (Item 165) 157. The method of claim 156, wherein the one or more additional therapeutic agents is a B7-H3 antagonist. (Item 166) 157. The method of claim 156, wherein the one or more additional therapeutic agents is a B7-H4 antagonist. (Item 167) Item 157. The method of item 156, wherein the one or more additional therapeutic agents is a BTLA antagonist. (Item 168) 157. The method of claim 156, wherein the one or more additional therapeutic agents is a CTLA-4 antagonist. (Item 169) 157. The method of claim 156, wherein the one or more additional therapeutic agents is an IDO antagonist. (Item 170) 157. The method of claim 156, wherein the one or more additional therapeutic agents is a KIR antagonist. (Item 171) 157. The method of claim 156, wherein the one or more additional therapeutic agents is a LAG-3 antagonist. (Item 172) 157. The method of claim 156, wherein the one or more additional therapeutic agents is a Toll-like receptor 3 (TLR3) agonist. (Item 173) 173. The method of item 172, wherein the TLR3 agonist is polyinosinic:polycytidylic acid (poly I:C). (Item 174) Item 173. The method of item 172, wherein the TLR3 agonist is HILTONOL® (Poly ICLC). (Item 175) 173. The method of claim 172, wherein the TLR3 agonist is polyadenylic acid-polyuridylic acid (polyA:U). (Item 176) Item 173. The method of item 172, wherein the TLR3 agonist is RIBOXXIM® (RGIC® 100). (Item 177) Item 173. The method of item 172, wherein the TLR3 agonist is RIBOXXON® (RGIC® 50 bioconjugate). (Item 178) Item 173. The method of item 172, wherein the TLR3 agonist is RIBOXXOL® (RGIC® 50). (Item 179) 157. The method of claim 156, wherein the one or more additional therapeutic agents is a Toll-like receptor 7 (TLR7) agonist. (Item 180) Item 179. The method of item 179, wherein the TLR7 agonist is GS-9620 (vesatolimod). (Item 181) Item 179. The method of item 179, wherein the TLR7 agonist is imiquimod (ALDARA™). (Item 182) Item 179. The method of item 179, wherein the TLR7 agonist is resiquimod (R-848). (Item 183) 157. The method of claim 156, wherein the one or more additional therapeutic agents is a Toll-like receptor 9 (TLR9) agonist. (Item 184) 184. The method of claim 183, wherein the TLR9 agonist is a CpG oligodeoxynucleotide (CpG ODN). (Item 185) Item 185. The method of item 184, wherein the CpG ODN is a class A CpG ODN (CpG-A ODN). (Item 186) Item 185. The method of item 184, wherein the CpG ODN is a class B CpG ODN (CpG-B ODN). (Item 187) Item 185. The method of item 184, wherein the CpG ODN is a class C CpG ODN (CpG-C ODN). (Item 188) Use of the agonist according to any one of items 1 to 143 or the pharmaceutical composition according to any one of items 144 to 146 for stimulating an immune response, treating or delaying the progression of cancer or inhibiting tumor growth in a subject in need thereof, optionally in combination with one or more further therapeutic agents. (Item 189) Use 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 in the manufacture of a medicament for stimulating an immune response, treating or delaying the progression of cancer, or inhibiting tumor growth in a subject in need thereof, optionally in combination with one or more further therapeutic agents. (Item 190) A kit comprising an agonist according to any one of items 1 to 143 or a pharmaceutical composition according to any one of items 144 to 146 and instructions for use in stimulating an immune response in a subject, or treating or delaying the progression of cancer, or inhibiting tumor growth in a subject, optionally in combination with one or more further therapeutic agents. (Item 191) 191. The use of item 188 or the kit of item 190, wherein the agonist or pharmaceutical composition is administered in combination with one or more additional therapeutic agents, wherein the one or more additional therapeutic agents are selected from the group consisting of chemotherapy, targeted anti-cancer therapy, oncolytic agent, cell death inducer, opsonizing agent (e.g., opsonizing antibody), cytotoxic agent, immune system therapy, cytokine, activator of costimulatory molecules, inhibitor of inhibitory molecules, vaccine, cellular immunotherapy, or combinations thereof. (Item 192) 192. The use or kit of item 191, wherein the agonist or pharmaceutical composition is administered before or after the administration of the one or more additional therapeutic agents, or the one or more additional therapeutic agents are administered simultaneously with, before or after the administration of the agonist or pharmaceutical composition. (Item 193) 193. The use of any one of items 188, 189, 191 or 192 or the kit of items 190 to 192, wherein the one or more further 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, a Toll-like receptor 9 (TLR9) agonist. (Item 194) 193. The use of any one of items 188, 189, 191 or 192 or the kit of items 190 to 192, wherein the one or more additional therapeutic agents is an agonist comprising a polypeptide (e.g., an antibody, or an antigen-binding portion thereof) that specifically binds to CD137(4-1BB). (Item 195) 193. The use of any one of items 188, 189, 191 or 192 or the kit of items 190 to 192, wherein the one or more additional therapeutic agents is an agonist comprising a polypeptide (e.g., an antibody, or an antigen-binding portion thereof) that specifically binds to CD134 (OX40). [Brief explanation of the drawings]

[0093] [Figure 1] 1 provides a bar graph showing the quantification of IFN-α secretion from human PBMCs treated with three different concentrations of RIG-I-like receptor agonists containing various modifications. DETAILED DESCRIPTION OF THE INVENTION

[0094] overview RIG-I-like receptors (RLRs) are a family of cytoplasmic pattern recognition receptors essential for detecting viral RNA and initiating innate immune responses. The RLR family includes the retinoic acid-inducible gene I (REI) and the RIG-I-like receptor β (RLR). The three members of the IL-1 receptor family are RIG-I (receptor interferon-1), melanoma differentiation-associated gene 5 (MDA5), and Laboratory of Genetics and Physiology 2 (LGP2). These receptors are expressed on both immune and non-immune cell types and regulate signaling pathways that promote IRF3-dependent expression of type I and III interferons (IFNs), IRF7-dependent expression, and NF-κB-dependent expression of proinflammatory cytokines.

[0095] All three RLR family receptors contain a DExD / H-box RNA helicase domain with ATPase activity, which, together with the adjacent C-terminal domain, is required for RNA binding. In addition, the C-terminal domain of RIG-I and LGP2 has been shown to act as a repression domain, ensuring that the receptor remains in an inactive conformation until it binds to an activating RNA.

[0096] The present disclosure provides RLR agonists comprising synthetic RNA molecules that fold to form double-stranded dsRNA and contain one or more sequence motifs that confer one or more improved biological activities.

[0097] definition Terms used in the claims and specification are defined as set forth below unless otherwise specified. In the event of a direct conflict with a term used in the parent provisional patent application, the term used in this application controls.

[0098] It must be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Further, unless otherwise required by context, singular terms shall include plurals and plural terms shall include the singular.

[0099] About: As used herein, the term "about" (or "approximately") will be understood by those of ordinary skill in the art and will vary to some extent depending on the context in which it is used. When using a term that is not clear to those of ordinary skill in the art given the context in which it is used, "about" will mean up to ±10% of the 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 fully promotes, induces, increases, and / or activates the biological activity of a native polypeptide disclosed herein. Agonist molecules of the present disclosure may include nucleic acids (e.g., oligonucleotides, polynucleotides), antibodies or antigen-binding fragments, fragments or amino acid sequence variants of native polypeptides, peptides, oligonucleotides, lipids, carbohydrates, and small 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 comparable conditions. Also disclosed herein are methods for identifying agonists suitable for use in the disclosed methods. For example, these methods include, but are not limited to, binding assays such as enzyme-linked immunosorbent assay (ELISA), Forte Bio© system, fluorescence polarization (FP) assay, and radioimmunoassay (RIA). These assays determine the ability of an agonist to bind to a polypeptide of interest (e.g., a receptor or ligand), and thus indicate the ability of the agonist to promote, increase, or activate the activity of that polypeptide. The effectiveness of an agonist can also be determined using a functional assay (e.g., the ability of the agonist 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 biological activities normally associated with the polypeptide. The potency of an agonist is usually determined by its EC50 The EC value is defined as the concentration required to activate 50% of the agonist response. 50 The lower the value, the more potent the agonist and the lower the concentration required to activate the maximal biological response.

[0101] Ameliorating: As used herein, the term "ameliorating" refers to any therapeutically beneficial result in the treatment of a disease state (e.g., cancer), including prevention, reducing severity or progression, remission, or cure.

[0102] Amino acid: As used herein, the term "amino acid" refers not only to naturally occurring amino acids and synthetic amino acids, but also to amino acid analogs and amino acid mimetics 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 amino acids that are 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., carbons bonded to hydrogen, carboxyl groups, amino groups, and R groups), such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimetics refer to chemical compounds that have a structure that is different 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 to herein by either 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 to by their commonly accepted single-letter codes.

[0104] Amino acid substitution: As used herein, "amino acid substitution" refers to the replacement of at least one existing amino acid residue in a predetermined amino acid sequence (the amino acid sequence of the starting polypeptide) with a second, different, "replacement" amino acid residue. "Amino acid insertion" refers to the incorporation of at least one additional amino acid into a predetermined amino acid sequence. Insertions usually consist of the insertion of one or two amino acid residues, although larger "peptide insertions," e.g., insertions of about 3 to about 5, or up to about 10, 15, or 20 amino acid residues, can also be made. The inserted residues can be naturally occurring or non-naturally occurring, as disclosed above. "Amino acid deletion" refers to the removal of at least one additional amino acid from a predetermined amino acid sequence.

[0105] Base composition: As used herein, the term "base composition" refers to the percentage of total nucleotides of a nucleic acid (e.g., RNA) that consist of guanine (or hypoxanthine) + cytosine and / or uracil (or thymine) + adenine nucleobases.

[0106] Base pair: As used herein, the term "base pair" refers to two nucleic acid bases on opposing complementary polynucleotide strands, or regions of the same strand, that interact through the formation of specific hydrogen bonds. 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, whereby purines are always bonded to pyrimidines, such that in DNA molecules, 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), which, like thymine (T), forms a complementary base pair with adenine (A). Complementary base pairs are held together by hydrogen bonds, and the number of hydrogen bonds varies from base pair to base pair. As is known in the art, guanine (G)-cytosine (C) base pairs are joined by three hydrogen bonds, and adenine (A)-thymine (T) or uracil (U) base pairs are joined by two hydrogen bonds.

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

[0108] In one aspect, the present disclosure provides synthetic RNA molecules that agonize or activate one or more RIG-I-like receptors (RLRs), wherein inosine can only be inserted at positions that form base pairs with cytidine (IC base pairs), i.e., inosine can substitute for guanosine but cannot substitute for other nucleosides.

[0109] Biologically active: As used herein, the phrase "biologically active" refers to any characteristic of a substance that has activity in a biological system and / or organism. For example, a substance that, when administered to an organism, has a biological effect on the organism and thus has "bioactivity" is considered to be biologically active. In certain embodiments, when a nucleic acid is biologically active, a portion of that nucleic acid that shares at least one biological activity of the entire nucleic acid is typically referred to as a "biologically active" portion.

[0110] Covalently linked: As used herein, the term "covalently linked" (alternatively, "conjugated," "linked," "connected," "fused," or "tethered"), when used in reference to two or more moieties, means that the moieties are physically associated or connected to one another by any means, including chemical conjugation, recombinant techniques, or enzymatic activity, either directly or through one or more additional moieties that function as linking agents, to form a structure that is sufficiently stable so that the moieties remain physically associated under the conditions in which the structure will be used (e.g., physiological conditions).

[0111] Complementary: As used herein, the term "complementary" or "complementarity" refers to the relationship between the sequences of nucleotides comprising two polynucleotide strands, or regions of the same polynucleotide strand, and the formation of a double-stranded structure comprising the strands or regions, where the degree of continuous base pairing between the two strands or regions is sufficient to form a double-stranded structure. Adenine (A) is known to form specific 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 nucleobases (e.g., inosine) are also known to be able to hydrogen bond with natural bases. A sequence of nucleotides comprising a first strand, or a region, or a portion or fragment thereof, of a polynucleotide is said to be "sufficiently complementary" to a sequence of nucleotides comprising a second strand, or a region, or a portion or fragment thereof, of the same or a different nucleic acid, if the first and second strands are aligned in an antiparallel manner and the degree of base pairing between the two strands is such that the double-stranded structure is maintained under the conditions in which the double-stranded structure is used (e.g., physiological conditions in a cell). It should be understood that complementary strands or regions of a polynucleotide may contain some non-complementary base pairs. Complementarity may be "partial," in which only a portion of the nucleic acid bases comprising the polynucleotide are matched according to the base-pairing rules. Alternatively, there may be "complete" or "total" complementarity between the nucleic acids. The degree of complementarity between polynucleotide strands or regions significantly affects the efficiency and strength of hybridization between the strands or regions, but two complementary polynucleotides need not base-pair at every nucleotide position. In some embodiments, the first polynucleotide is 100% or "fully" complementary to the second polynucleotide, and thus forms base pairs at every nucleotide position. In some embodiments, the first polynucleotide is not 100% complementary (e.g., 90%, or 80%, or 70% complementary) and contains mismatched nucleotides at one or more nucleotide positions.While perfect complementarity is often desired, some embodiments may contain one or more, but preferably 6, 5, 4, 3, 2 or 1, mismatches.

[0112] Contacting: As used herein, the term "contacting" means establishing a physical connection between two or more entities. For example, contacting a cell with an agent (e.g., RNA, lipid nanoparticle composition, or other pharmaceutical composition of the present disclosure) means that the cell and the agent are made to share a physical connection. Methods for contacting cells with external entities, in vivo, in vitro, and ex vivo, are all well known in the field of biology. In exemplary embodiments of the present disclosure, the step of contacting mammalian cells with a composition (e.g., isolated RNA, nanoparticles, or pharmaceutical composition of the present disclosure) is performed in vivo. For example, contacting a lipid nanoparticle composition with cells (e.g., mammalian cells) that may be placed in an organism (e.g., a mammal) may be performed by any suitable administration route (e.g., parenteral administration to the organism, including intravenous, intramuscular, intradermal, and subcutaneous administration). For cells present in vitro, a composition (e.g., lipid nanoparticles or isolated RNA) may be contacted with the cells, for example, by adding the composition to the cell's culture medium, which may include or result in transfection. Additionally, more than one cell may be contacted with the agent.

[0113] Denaturation: As used herein, the term "denaturation" refers to a process in which hydrogen bonds between base-paired nucleotides in nucleic acids are broken and secondary and / or tertiary nucleic acid structure is lost (e.g., separation of previously annealed strands). Denaturation can occur through the application of foreign substances, energy, or biochemical processes to nucleic acids.

[0114] Antigen-presenting cell: The term "antigen-presenting cell" or "APC" refers to a cell that presents foreign antigens complexed with MHC on its surface. T cells recognize this complex using the T cell receptor (TCR). Examples of APCs include, but are not limited to, dendritic cells (DCs), peripheral blood mononuclear cells (PBMCs), monocytes (e.g., THP-1), B lymphoblastoid cells (e.g., C1R.A2, 1518 B-LCL), and monocyte-derived dendritic cells (DCs). Some APCs internalize antigens by phagocytosis or receptor-mediated endocytosis.

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

[0116] Blunt end: As used herein, the terms "blunt end" and "blunt-ended" refer to the structure of the ends of a duplex or double-stranded nucleic acid in which both complementary strands comprising the duplex terminate in base pairs at at least one end, such that neither strand of the duplex extends further from its end than the other.

[0117] Cancer antigen: As used herein, "cancer antigen" refers to (i) tumor-specific antigens, e.g., neoantigens, (ii) tumor-associated antigens, (iii) cells expressing tumor-specific antigens, (iv) cells expressing tumor-associated antigens, (v) fetal 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" is art-recognized and refers to malignant tumors of epithelial or endocrine tissues, including respiratory system carcinomas, digestive system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostate carcinomas, endocrine system carcinomas, and melanomas. The RIG-I-like receptor (RLR) agonists described herein can be used to treat patients who have, are suspected of having, or may be at high risk for developing any type of cancer, including renal carcinoma or melanoma. Exemplary carcinomas include those formed from tissues of the cervix, lung, prostate, breast, head and neck, colon, and ovary. The term also includes carcinosarcoma, which includes malignant tumors composed of carcinomatous and sarcomatous tissues. "Adenocarcinoma" refers to carcinomas derived from glandular tissue or in which tumor cells form recognizable glandular 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. CTL responses are primarily mediated by CD8+ T cells.

[0120] Double-stranded: As used herein, the term "duplex" refers to the structure created by the complementary strands of a double-stranded polynucleotide or the complementary regions of a single-stranded polynucleotide that fold back on themselves. A nucleic acid double-stranded structure occurs as a result of complementary nucleotide sequences binding together or hybridizing through base-pairing interactions.

[0121] EC50 As used herein, "EC 50 The term "agonist concentration" refers to the concentration of agonist that is 50% of the maximal response, i.e., halfway between the maximal 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 an amount sufficient to achieve or at least partially achieve a desired effect.

[0123] Hairpin RNA: As used herein, the term "hairpin RNA" or "RNA hairpin" refers to a self-complementary RNA comprising a double-stranded RNA (dsRNA) stem composed of complementary nucleotide strands that base-pair and terminate at one end with a nucleotide linker containing unpaired nucleotides, a loop of unpaired nucleotides (e.g., a tetraloop), or a non-nucleotide linker containing a flexible chemical moiety (e.g., ethylene glycol), either of which connects to the complementary nucleotide strand to form a duplex. RNA hairpins can vary in stem length, loop and / or linker size and / or composition, number of base pair mismatches within the stem, and actual nucleotide sequence. RNA hairpins may impart one or more functions, including, but not limited to, directing the overall folding of an RNA molecule containing the hairpin, determining interactions in ribozymes, protecting messenger RNA (e.g., mRNA) from denaturation, serving as a recognition motif or structure for an RNA-binding protein, and acting as a substrate for an enzymatic reaction. Further description 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 references contained therein. In some embodiments, the stem region of the hairpin RNA comprising an RLR agonist provided by the present disclosure terminates in a blunt end with a 5' triphosphate or diphosphate.

[0124] In need: As used herein, a subject "in need of prevention," "in need of treatment," or "in need of" refers to a subject who, according to the judgment of an appropriate medical practitioner (e.g., a doctor, nurse, or nursing professional 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" (alternatively, "tether" or "spacer") refers to a moiety that covalently attaches, attaches, or couples two polynucleotide strands or regions together. As used herein, a linker containing a nucleotide is referred to as a "nucleotide linker" (e.g., a tetraloop). As used herein, the term "non-nucleotide linker" refers to a linker that contains a chemical moiety and 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 stilbene diethers. Further description 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 Laboratory of Genetics and Physiology 2 polypeptide, a specific member of the RIG-I-like receptor family, which in humans is encoded by the DHX58 gene. Alternative names and acronyms for LGP2 in the art 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 carrying a composition or agent 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 agent, or by injection or implantation of a device containing the composition or agent. After local administration near the target tissue or site, the composition or agent, or one or more components thereof, may diffuse to 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, which in humans is encoded by the IFIH1 gene. Alternative names and acronyms for MDA5 in the art 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] Modified: As used herein, "modified" or "modification" refers to an altered state or change in structure 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, RNA molecules of the present disclosure may be modified by the incorporation of unnatural bases or sequence motifs that confer biological activity, including functional sequences or secondary structures. In one embodiment, RNA is modified by the introduction of unnatural or chemically modified bases, nucleosides, and / or nucleotides, e.g., relative to the natural ribonucleotides A, U, G, and C.

[0130] Naturally-occurring: As used herein, the term "naturally-occurring" as applied to an entity refers to the fact that an entity can be found in nature. For example, a polypeptide or polynucleotide sequence, or components thereof, e.g., amino acids or nucleotides, that are present in organisms (including viruses) that can be isolated from natural sources and have not been intentionally modified by man in a laboratory, is naturally-occurring.

[0131] Nucleic Acid: As used herein, the term "nucleic acid" refers to deoxyribonucleotides or ribonucleotides, and polymers or oligomers thereof, in either single- or double-stranded form. Unless otherwise specified, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Polymers of nucleotides are referred to as "polynucleotides." Exemplary nucleic acids or polynucleotides of the present 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 with a β-D-ribo configuration, α-LNA with an α-L-ribo configuration (a diastereomer of LNA), 2′-amino-LNA with a 2′-amino functional group, and 2′-amino-α-LNA with a 2′-amino functional group), or hybrids thereof.

[0132] As used herein, a polynucleotide may be composed of any polyribonucleotide or polydeoxyribonucleotide, and may be unmodified RNA or DNA, or modified RNA or DNA. For example, a polynucleotide may be composed of single- and double-stranded DNA, a mixture of single- and double-stranded regions of DNA, single- and double-stranded RNA, a mixture of single- and double-stranded regions of RNA, or a hybrid molecule containing DNA and RNA, which may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, a polynucleotide may be composed of triple-stranded regions containing RNA or DNA, or both RNA and DNA. A polynucleotide may also contain one or more modified bases or DNA or RNA backbones modified for stability or other reasons. "Modified" bases include, for example, tritylated bases. "Modified nucleosides" include, for example, inosine and thymine, the latter of which is found in or comprises RNA. A variety of modifications can be made to DNA and RNA; thus, "polynucleotide" embraces 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 the atoms, chemical components, elements, motifs, and / or sequences of nucleobases comprising a nucleic acid (e.g., RNA), and / or may refer to the two-dimensional or three-dimensional state of a nucleic acid. Accordingly, the term "RNA structure" refers to the arrangement or organization of the atoms, chemical components, elements, motifs, and / or sequences of nucleobases comprising an RNA molecule (e.g., mRNA), and / or may refer to the two-dimensional and / or three-dimensional state of an RNA molecule. Nucleic acid structures can be further divided into four organizational categories, referred to herein as "molecular structure," "primary structure," "secondary structure," and "tertiary structure," based on increasing organizational complexity.

[0134] Nucleobase: As used herein, the term "nucleobase" (alternatively, "nucleotide base" or "nitrogenous base") refers to the purine and pyrimidine heterocyclic compounds found in nucleic acids, including any derivatives or analogs of naturally occurring purines and pyrimidines, that confer improved properties (e.g., binding affinity, nuclease resistance, chemical stability) to a nucleic acid or a portion or segment thereof. Adenine, cytosine, guanine, thymine, and uracil are the major or standard nucleobases found predominantly in natural nucleic acids. Other natural, unnatural, non-canonical, and / or synthetic nucleobases can be incorporated into nucleic acids (e.g., those disclosed herein).

[0135] Nucleoside / Nucleotide: As used herein, the term "nucleoside" refers to a compound comprising a sugar molecule (e.g., ribose in RNA or deoxyribose in DNA), or a derivative or analog thereof, covalently linked to a nucleobase (e.g., a purine or pyrimidine), or a derivative or analog thereof (also referred to herein as a "nucleobase"). As used herein, the term "nucleotide" refers to a nucleoside covalently linked to a phosphate group. As used herein, the term "ribonucleoside" refers to a nucleoside comprising a ribose and a nucleobase (e.g., adenosine (A), cytidine (C), guanosine (G), 5-methyluridine (m), methyluridine (M ... 5 It refers to nucleosides containing uridine (U), inosine (I), or uridine (U).

[0136] Operably linked: As used herein, a nucleic acid, or fragment or portion thereof, e.g., a polynucleotide or oligonucleotide, is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence, or fragment or portion thereof.

[0137] Polynucleotide / Oligonucleotide: As used herein, the terms "polynucleotide" and "oligonucleotide" are used interchangeably and refer to single- or double-stranded polymers or oligomers of nucleotide or nucleoside monomers composed of naturally occurring bases, sugars, and intersugar (backbone) linkages. The terms "polynucleotide" and "oligonucleotide" also include polymers and oligomers, or portions thereof, that function similarly and contain non-naturally occurring bases, sugars, and intersugar (backbone) linkages. The term "polynucleotide" encompasses polymeric forms of nucleotides of any length; therefore, a polynucleotide is not limited to a nucleotide sequence of any particular length. Short polynucleotides are typically referred to in the art as "oligonucleotides." In the context of the present disclosure, such modified or substituted polynucleotides and oligonucleotides are often preferred over their native forms because the modifications enhance one or more desirable or beneficial biological properties 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, agonists of the present disclosure include polynucleotides and oligonucleotides that contain at least one region of modified nucleotides that impart one or more beneficial properties or enhance biological activity (e.g., increased nuclease resistance, increased cellular uptake, increased duplex stability, increased binding affinity for a target polypeptide).

[0138] Palindromic sequence: As used herein, the term "palindromic sequence" (or "palindrome") refers to a sequence of nucleotides that is self-complementary. The sequence of nucleotides in the 5' to 3' direction is the same as the sequence of nucleotides comprising the complementary strand 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 comprised of a first oligonucleotide, and the sequence of the first oligonucleotide is a palindromic sequence. In another embodiment, the agonist is comprised of a first oligonucleotide, and the first oligonucleotide comprises a palindromic sequence.

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

[0141] Parenteral administration: As used herein, the terms "parenteral administration," "administered parenterally," and other grammatically equivalent phrases refer to modes of administration other than enteral and topical administration, usually by injection, including, but not limited to, intravenous, intranasal, intraocular, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intracerebral, intracranial, intracarotid, and intrasternal injection and infusion.

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

[0143] Percent identity: As used herein, the term "percent identity" in the context of two or more nucleic acid or polypeptide sequences refers to two or more sequences or subsequences that have a specified percentage of the same nucleotide or amino acid residues when aligned for maximum correspondence 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 the application, the "percent identity" may be across a region of the sequences being compared, such as across a functional domain, or alternatively across the entire length of the two sequences being compared. For sequence comparison, typically, one sequence serves as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, the test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the designated program parameters. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap necessary to introduce optimal alignment of the two sequences (i.e., % homology = number of identical positions / total number of positions x 100). The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting examples below.

[0144] 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 homology 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 computerized implementations 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 inspection (see generally, Ausubel et al., infra).

[0145] One example of an algorithm suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available from the website of the National Center for Biotechnology Information. The percent identity between two nucleotides can be determined using the GAP program in the GCG software package, using a NWSgapdna.CMP matrix, a gap weight of 40, 50, 60, 70, or 80, and a length weight of 1, 2, 3, 4, 5, or 6 (available at http: / / www.gcg.com). The percent identity between two nucleotide 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), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Additionally, percent 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 a PAM250 matrix, gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0146] The nucleic acid and protein sequences of the present disclosure can also be used as a "query sequence" to perform searches against public databases, for example, to identify related 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. BLAST nucleotide searches 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. BLAST protein searches 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 utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http: / / www.ncbi.nlm.nih.gov.

[0147] Pharmaceutically acceptable: As used herein, the term "pharmaceutically acceptable" refers to compounds, materials, compositions and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues, organs and / or body fluids of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit-risk ratio.

[0148] Pharmaceutically acceptable carrier: As used herein, the term "pharmaceutically acceptable carrier" refers to and includes any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. The compositions may also include pharmaceutically acceptable salts, e.g., acid addition salts or base addition salts (see, e.g., Berge et al. (1977) J Pharm Sci 66:1-19).

[0149] Phosphate: As used herein, the term "phosphate" refers to a salt or ester of phosphoric acid. Polyphosphate is a salt or ester of a polymeric oxyanion formed from tetrahedral PO (phosphate) structural units linked together by sharing oxygen atoms. 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 present disclosure provides RIG-I-like receptor agonists comprising a diphosphate moiety linked to the 5'-terminus, or a derivative or analog thereof. In some embodiments, the present disclosure provides RIG-I-like receptor agonists comprising a triphosphate moiety linked to the 5'-terminus, or a derivative or analog thereof. In some embodiments, the derivative or analog is a phosphate bioisostere.

[0150] Phosphate bioisostere: As used herein, the term "phosphate bioisostere" (or "phosphate mimic") refers to a chemical substituent or group that has similar physical or chemical properties to phosphate, including biphosphate and triphosphate moieties, and provides broadly similar biological properties to phosphate. In drug design, the purpose of replacing one bioisostere with another is to improve the desired biological or physical properties of a compound without making significant changes to its chemical structure. The use of bioisosteres is widely used in pharmaceutical development, for example, to reduce the toxicity, alter the bioavailability, or modify the activity or metabolism of parent or lead compounds (see, e.g., Rye and Baell (2005) Curr Med Chem 12(26):3127-3141; Elliot et al. (2012) MedChemCom 3(7):735-751, the entire contents of which are incorporated herein by reference).

[0151] Polypeptide: As used herein, the terms "polypeptide," "peptide," and "protein" are used interchangeably to refer to a polymer of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of a corresponding naturally occurring amino acid, as well as to naturally occurring and non-naturally occurring amino acid polymers.

[0152] Prevent: As used herein, when used in reference to a condition, the term "prevent" refers to the administration of a composition that reduces the frequency of or delays the onset of symptoms of a medical condition in a subject compared to a subject who does not take 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 a peptide that has been separated or purified from components that naturally accompany it (e.g., proteins or other naturally occurring biomolecules or organic molecules), e.g., other proteins, lipids, and nucleic acids in the prokaryotic organism that expresses the protein. Typically, a polypeptide is purified when it constitutes at least 60% (e.g., at least 65, 70, 75, 80, 85, 90, 92, 95, 97, or 99%) by weight of the total protein in a sample.

[0154] Reference Ligand: As used herein, the term "reference ligand" (used interchangeably with "reference agonist") or "reference molecule" refers to a RIG-I-like receptor ligand that is used to establish a relationship between itself and one or more distinct RIG-I-like receptor ligands, the relationship being the relative agonistic effect of the reference ligand and one or more distinct RIG-I-like receptor ligands. As used herein, the term refers to a RIG-I-like receptor ligand or agonist that is useful as a competitor in tests or assays such as those described herein (e.g., IFN induction assays), which assays are useful for discovering, identifying, or developing one or more distinct agonists that bind to RIG-I-like receptors.

[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, which in humans is encoded by the DDX58 gene. Alternative names and acronyms for RIG-I in the art 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 receptor: As used herein, the term "RIG-I-like receptor" (abbreviated "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 1 interferon (IFN) production and antiviral gene expression, which elicit an intracellular immune response to control viral infection. Three RLR members have been identified: RIG-I (retinoic acid-inducible 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 agonist: As used herein, the term "RIG-I-like receptor agonist" (used interchangeably with the term "RLR agonist") refers to a nucleic acid (e.g., RNA) that binds to a RIG-I-like receptor (RLR) and partially or fully promotes, induces, increases, and / or activates biological activity, responses, and / or downstream pathways mediated by RLR signaling, or other RLR-mediated functions. Examples of RIG-I receptor agonists are provided herein.

[0158] Stable RNA secondary structure: 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 persistently maintained under physiological conditions and characterized by a low free energy state. Typical examples of stable RNA secondary structures include duplexes, hairpins, and stem-loops. Stable RNA secondary structures are known in the art to exhibit various biological activities. The term "stable," when used in reference to a polynucleotide duplex, means that the duplex remains essentially exclusively hybridized, structured, or annealed in a double-stranded form under physiological conditions or under typical salt and temperature conditions used in diagnostic or therapeutic applications of nucleic acids.

[0159] Subject: As used herein, the term "subject" includes any human or non-human animal. For example, the methods and compositions of the present invention can be used to treat a subject having an immune disorder. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cows, chickens, amphibians, reptiles, etc.

[0160] T cell: The term "T cell" 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 the cell surface. There are several subsets of T cells, including, but not limited to, T helper cells (also known as TH cells or CD4+ T cells) and subtypes, including TH1, TH2, TH3, TH17, TH9 and T H 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), mucosal-associated invariant T cells (MAIT), and gamma delta T cells (γδ T cells), Vγ9 / Vδ2 T cells. Any one or more of the T cells mentioned above or not mentioned may be the target cell type for the methods of use of the present invention.

[0161] T cell activation: As used herein, the term "T cell activation" or "T cell activation" refers to the cellular process by which mature T cells expressing antigen-specific T cell receptors on their surface recognize their cognate antigen and respond by entering the cell cycle, secreting cytokines or lytic enzymes, and initiating cell-based effector functions or becoming competitors. T cell activation requires at least two signals for full activation: one signal occurs after engagement of the T cell antigen-specific receptor (TCR) by the antigen major histocompatibility complex (MHC), and a second signal occurs with the subsequent engagement of costimulatory molecules (e.g., CD28). These signals are transduced to the nucleus, resulting in T cell clonal expansion, upregulation of activation markers on the cell surface, differentiation into effector cells, induction of cytotoxicity or cytokine secretion, induction of apoptosis, or a combination of these.

[0162] T cell-mediated response: As used herein, the term "T cell-mediated response" 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). 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 the duplex at one end, connects the two strands that make up the duplex, and provides stability to the hairpin structure.

[0164] Therapeutic Agent: As used herein, the term "therapeutic agent" refers to any agent that has a therapeutic, diagnostic and / or prophylactic effect and / or induces a desired biological and / or pharmacological effect when administered to a subject.

[0165] Therapeutically effective amount: As used herein, the terms "therapeutically effective amount," "therapeutically effective dose," or similar terms used herein, are intended to mean an amount of an agent (e.g., a synthetic RIG-I-like receptor agonist) that elicits a desired biological or medical response, e.g., a cure or at least partial arrest of a condition or disease and its complications (e.g., amelioration of one or more symptoms of cancer) in a patient already suffering from the disease. The amount effective for this use will depend on the severity of the disease being treated and the overall state of the patient's own immune system.

[0166] Treat: As used herein, the terms "treat," "treating," and "treatment" refer to therapeutic or prophylactic measures as described herein. Methods of "treatment" employ administering a human antibody of the present disclosure to a subject in need of such treatment, for example, to a subject in need of an improved immune response to a particular antigen, or to a subject who may ultimately acquire such a disorder, to prevent, cure, delay, lessen the severity, or ameliorate one or more symptoms of the disorder or recurring disorder, or to prolong the subject's survival beyond that 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 circumstances, in which a tumor or neoplasm resides, including the surrounding blood vessels and non-cancerous cells, including, but not limited to, immune cells, fibroblasts, bone marrow-derived inflammatory cells, and lymphocytes. Signaling molecules and the extracellular matrix also comprise the TME. The tumor and surrounding microenvironment are closely related and constantly interact. 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 commonly understood by those skilled in the art to which this disclosure pertains.Preferred methods and materials are described below, but similar or equivalent methods and materials to those described herein can also be used in the implementation or testing of the presently disclosed methods and compositions.All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety.

[0169] Equivalents and Scope Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein. The scope of the present disclosure is not limited to the above description, but is as set forth in the appended claims.

[0170] In the claims, articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or clear from the context. A claim or description containing “or” between one or more members of a group is deemed satisfied when one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process, unless indicated to the contrary or clear from the context. The present disclosure includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The present disclosure includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process. Furthermore, it should be understood that the present disclosure encompasses all variations, combinations, and substitutions of one or more limitations, elements, clauses, descriptive terms, etc. from one or more of the recited claims introduced into another claim. For example, any claim that is dependent on another claim may be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Furthermore, when a claim recites a composition, it should be understood that unless otherwise indicated or unless a contradiction or inconsistency would be apparent to one of ordinary skill in the art, it includes methods of using that composition for any of the purposes disclosed herein, and includes methods of making that composition, whether in accordance with any of the manufacturing methods disclosed herein or other methods known in the art.

[0171] Where elements are presented as a list, e.g., in Markush group format, it is to be understood that each subgroup of the elements is also disclosed, and that any element can be deleted from the group. In general, when the invention or aspects of the invention are referred to as comprising particular elements, features, etc., it is to be understood that particular embodiments of the invention or aspects of the invention consist of or consist essentially of such elements, features, etc. For simplicity, these embodiments have not been specifically described in these terms herein.

[0172] It should also be noted that the term "comprising" is intended to be inclusive and allows for the inclusion of additional elements or steps, although these are not required. Thus, when the term "comprising" is used herein, the term "consisting of" is also included and disclosed.

[0173] Where ranges are given, the boundaries are inclusive. Furthermore, unless otherwise indicated or apparent from the context and the understanding of one of ordinary skill in the art, it should be understood that values expressed as ranges can assume any particular value or subrange within the ranges set forth in different embodiments of the present invention, down to one-tenth of the unit of the lower limit of that range, unless the context clearly dictates otherwise.

[0174] In addition, it should be understood that any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the claims. Because such embodiments are deemed known to those of skill in the art, they may be excluded even if the exclusion is not explicitly 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 of the claims for any reason, whether related to the existence of prior art or not.

[0175] All sources cited herein, e.g., references, publications, databases, database entries, and techniques, are incorporated by reference into this application, even if not explicitly stated in the citation. In the event of a conflict between a cited source and the statement in this application, the statement in this application shall control.

[0176] RIG-I-like receptors and their ligands The present disclosure provides synthetic RNA ligands (RLR agonists) that specifically bind to and agonize RIG-I-like receptors (RLRs). In some embodiments, the present disclosure provides RLR agonists useful for treating cancer. In some embodiments, the present disclosure provides RLR agonists useful for treating 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) comprise a family of DExD / H-box RNA helicases that function as cytoplasmic pattern recognition receptors (PRRs) that detect the presence of pathogenic agents through the recognition of 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, initiating and regulating antiviral immunity. Like most viral RNAs, endogenous mRNAs and RNA polymerase III transcripts are 5'-triphosphorylated, whereas eukaryotic mRNAs possess a 5' cap structure linked to an N7-methylated guanosine, which prevents RIG-I activation. These structural differences between viral and self RNAs, along with their differential subcellular localization, may enable RIG-I to effectively function as a defense against viral infection by preferentially detecting viral RNA. Molecular recognition and binding of non-self RNA ligands to RLRs initiates specific intracellular signaling events culminating in the activation of transcription factors that drive type 1 interferon (IFN) production and antiviral gene expression. RLR-mediated induction of IFN and proinflammatory cytokine production, as well as antiviral gene expression, triggers immune responses that control viral infection (Yoneyama et al., (2015) Curr Opin Immunol 32:48-53).

[0178] Three RLR family members have been identified: RIG-I (retinoic acid-inducible gene I), MDA5 (melanoma differentiation-associated gene 5), and LGP2 (Laboratory of Genetics and Physiology 2 and its homolog of mouse D11lgp2). RIG-I is a key component of the innate immune system and plays a crucial role in defense against infection by RNA viruses. In contrast to the Toll-like receptors TLR3, TLR7, TLR8, and TLR9, which detect nucleic acids in the endosomes of some 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 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 RIG-I / ligand complexes have provided molecular details of RIG-I binding 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 RIG-I / RNA complex shows protein binding to the backbone, not the base, suggesting that RNA sequence may not affect RIG-I binding, or that RNA sequence may exhibit effects or activities that have not yet been characterized. To date, evidence of sequence-dependent different 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] Thus, the present disclosure provides synthetic RIG-I-like receptor (RLR) agonists, including non-naturally occurring synthetic and / or engineered RLR RNA ligands. In some embodiments, the present disclosure provides RLR agonists that specifically bind to RIG-I-like receptors (RLRs), wherein the agonist comprises a blunt-ended hairpin RNA comprising a first polynucleotide connected to a second polynucleotide by a linker, the first polynucleotide being sufficiently complementary to the second polynucleotide to form a duplex, the duplex comprising fewer than 19 base pairs, the 5'-most nucleotide of the first oligonucleotide comprising a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof, and the agonist comprises a sequence motif that confers at least one improved biological activity mediated by the RLR compared to an agonist that does not comprise the sequence motif.

[0181] In some embodiments, the RLR agonists of the present disclosure comprise a sequence motif, wherein the sequence motif is: (i) GT repeat motif, (ii) GA repeat motif; (iii) AUCG repeat motif; (iv) AU repeat motif; (v) dipyrimidine motif, (vi) ziprine motif, (vii) pyrimidine triplet motif, (viii) Printed triplet motif, (ix) palindromic sequence motifs, and (x) is selected from the group consisting of any combination of (i) to (ix).

[0182] In some embodiments, the RLR agonists of the present disclosure comprise at least one improved biological activity, wherein the improved biological activity is one of the following: (i) Increased cytokine production mediated by RLRs; (ii) increased RLR-mediated expression of interferon-inducible genes; (iii) increased intracellular signaling mediated by RLRs; (iv) increased binding affinity to RLRs, and (v) A combination of any one of (i) to (iv) is selected.

[0183] In some embodiments, the RLR agonists of the present disclosure comprise a sequence motif that 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 where n=2 to 9. In some embodiments, the GT repeat motif is [GT]. In some embodiments, the GT repeat motif is [GT], and the GT repeat motif is followed by a print triplet and a UCG, respectively. In some embodiments, the print triplet 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 where 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 that is an AUCG repeat motif comprising a sequence of less than 19, about 16, about 12-16, about 12, about 8-12, about 6, about 16, 12, or 8 adenine, uracil, cytosine, and guanine nucleotides, or derivatives or analogs thereof.

[0186] In some embodiments, the AUCG repeat motif is [AUCG] n where n=2 to 4. In some embodiments, the AUCG repeat motif is [AUCG]3.

[0187] In some embodiments, the AUCG repeat motif is preceded by a CG or dipyrimidine motif. In some embodiments, the AUCG repeat motif is preceded by a CG. In some embodiments, the dipyrimidine motif is CC. In some embodiments, the AUCG repeat motif is preceded by a dipurine motif. In some embodiments, the dipurine motif is GA. In some embodiments, the dipurine motif is GG.

[0188] In some embodiments, the RLR agonists of the present disclosure comprise an AUGC repeat motif, wherein one or more uridine nucleosides (U) are replaced with a modified nucleoside. In some embodiments, the modified nucleoside is ribothymidine (T). In some embodiments, the AUGC repeat motif is [AUCG]3, wherein one or more uridine nucleosides (U) comprising the AUCG repeat motif are replaced with a modified nucleoside, wherein the modified nucleoside is ribothymidine (T). In some embodiments, the AUGC repeat motif is [AUCG]3, wherein one or more uridine nucleosides (U) comprising the AUCG repeat motif are replaced with a modified nucleoside, wherein the modified nucleoside is ribothymidine (T), and wherein the AUGC repeat motif is preceded by GG.

[0189] In some embodiments, the RLR agonists of the present disclosure comprise an AUGC repeat motif, in which one or more guanosine nucleosides (G) are replaced 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) comprising the AUCG repeat motif are replaced with a modified nucleoside, in which the modified nucleoside is ribothymidine (T), and in which GG precedes the AUGC repeat motif.

[0190] In some embodiments, the RLR agonists of the present disclosure comprise an AUCG repeat motif preceded by an IG, hi some embodiments, the AUCG repeat motif is [AUCG]3, preceded by an IG.

[0191] In some embodiments, the RLR agonist of the present disclosure comprises an AUCG repeat, wherein one or more guanosine nucleosides (G) are substituted with inosine (I), and wherein the AUCG repeat is preceded by an inosine (I). In some embodiments, the guanosine nucleosides (G) comprising the AUCG repeat are substituted with inosine (I), and wherein the AUCG repeat is preceded by an inosine (I), and wherein the 5'-most nucleotide of the first polynucleotide comprises an inosine (I).

[0192] In some embodiments, the 5'-most nucleotide of the first oligonucleotide comprises an inosine (I).

[0193] In some embodiments, the RLR agonist of the present disclosure comprises an AUCG repeat sequence motif, wherein the AUCG repeat motif is [AUCG]2. In some embodiments, the AUCG repeat motif is preceded by a dipurine motif. In some embodiments, the dipurine motif is GG. In some embodiments, the AUCG repeat motif is preceded by a print triplet. In some embodiments, the print triplet is GGG. In some embodiments, the AUCG repeat motif is preceded by CCCCCG. In some embodiments, the AUCG repeat motif is preceded by TCGUCG.

[0194] In some embodiments, the RLR agonists of the present disclosure comprise a palindromic sequence, the palindromic sequence comprising a sequence of less 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, linked in any order to generate the palindrome.

[0195] In some embodiments, the linker is flanked by AU repeat motifs, and the AU repeat motifs are [AU] n wherein n=2 to 3. In some embodiments, the AU repeat motif is [AU]2.

[0196] In some embodiments, the present disclosure provides an RLR agonist that specifically binds to an RLR, wherein the agonist comprises a blunt-ended hairpin RNA comprising at least one or more nucleotides including an inosine that base pairs with a cytidine, and the agonist has the following formula: 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', wherein (i) (N1-N2-X1) comprises a first polynucleotide comprising linked nucleotides N1, N2 and X1; (ii) (X2-N3-N4) comprises a second polynucleotide comprising linked nucleotides X2, N3 and N4; (iii) N1, N2, N3, and N4 each comprise 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 each an oligonucleotide containing a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine; (viii) X1 is complementary to X2; (ix) X1 and X2 are each 12 to 16 nucleotides in length and are the same length; (x)L is a linker that covalently connects the first polynucleotide and the second polynucleotide.

[0197] In other aspects, 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-ended hairpin RNA comprising a non-nucleotide linker, the agonist having the following formula: 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', wherein (i) (N1-N2-X1) comprises a first polynucleotide comprising linked nucleotides N1, N2 and X1; (ii) (X2-N3-N4) comprises a second polynucleotide comprising linked nucleotides X2, N3 and N4; (iii) N1, N2, N3, and N4 each comprise 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 each an oligonucleotide containing a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine; (viii) X1 is complementary to X2; (ix) X1 and X2 are each 12 to 16 nucleotides in length and are the same length; (x)L is a non-nucleotide linker that covalently connects the first polynucleotide and the second polynucleotide.

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

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

[0200] In some embodiments, the present disclosure provides an RLR agonist that specifically binds to an RLR, the agonist comprising a blunt-ended hairpin RNA containing a nucleotide or non-nucleotide linker. RNA hairpins are one of the most common RNA secondary structural elements, and the hybridized portion or "stem" of the hairpin is often covered by an RNA tetraloop. RNA tetraloops are composed of four characteristic loop nucleotides that form a compact, stable structure. RNA tetraloops can be formed by many different nucleotide sequences, but UNCG (N=A, C, G, or U), GNRA (R=A or G), and CUUG tetraloops are most frequently found. Tetraloops usually help initiate the RNA folding process and provide sites for intra- and inter-RNA tertiary contacts and protein binding, thereby promoting the assembly of ribonucleoprotein particles. Further description of tetraloops can be found in Cheong, H., Kim, N. and Cheong, C. (2015). RNA Structure: Tetraloops. In eLS, John Wiley & Sons, Ltd (ed.), which is incorporated herein by reference in its entirety.

[0201] Thus, in some embodiments, the RLR agonists of the present disclosure comprise a nucleotide linker comprising a tetraloop. In some embodiments, the nucleotide sequence of the tetraloop is as follows: (a) UNCG, where N=A, C, G, or U; (b) GNRA, where 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 (wherein Y=C or T); (e) UMAC, where M=A or C, and (f) selected from the group consisting of CUUG.

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

[0203] In other embodiments, the RLR agonists of the present disclosure comprise a non-nucleotide linker. As described herein, nucleic acid loops (e.g., tetraloops) are common elements found in nucleic acid secondary structures. Nucleotide loops occur in folded domains that occur in intrastrand duplexes. Synthetic nucleic acids designed to contain hairpin loops containing non-nucleotide linking groups (e.g., non-nucleotide linkers) can replace several nucleotides that bridge folded double-stranded structures. Non-nucleotide groups have also been used as linkers in unfolded structures. Such linking groups can be useful replacements for natural nucleotide linkers (e.g., tetraloops). For example, such linking groups can shorten the synthesis of nucleic acids with desired secondary structures by several steps, by replacing a single relatively long non-nucleotide linking group with several individual nucleotides that would 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 the cells of a subject upon administration or in the circulation). Non-nucleotide linking groups also have the potential to provide more stable folded structures than those resulting from the use of nucleotide loops and / or linkers. Further description 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] Thus, in some embodiments, the RLR agonists of the present disclosure are: (a) an ethylene glycol linker, and (b) comprises a non-nucleotidic linker selected from the group consisting of alkyl linkers.

[0205] In some embodiments, the non-nucleotidic linker is a hexaethylene glycol linker. In some embodiments, the non-nucleotidic 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 bioisostere, wherein the phosphate bioisostere is selected from phosphonate, thiophosphonate, phosphorothioate, sulfate, sulfonate, sulfamate, thiazolidinone, carboxylate, malonate, boronic acid, benzoxaborole, boranophosphate, and squaramide.

[0207] In some embodiments, the agonist comprises a modified nucleotide, modified nucleoside, or modified nucleobase, or a combination thereof. In some embodiments, the agonist comprises a modification to the internucleotide linkage or polynucleotide backbone.

[0208] In some embodiments, the RLR agonists of the present disclosure have the following properties: (a) specifically binds to one or more RLRs (e.g., RIG-1, MDA5, and / or LGP2); (b) increase RLR-mediated cytokine production; (c) increasing RLR-mediated expression of interferon-inducible genes (ISGs); (d) increasing RLR-dependent intracellular signaling; (e) increasing duplex stability; (f) increasing binding affinity to RLRs; (g) reducing off-target binding; (h) increasing biological half-life; (i) enhance biodistribution and bioavailability; (j) increasing and / or enhancing uptake into cells and / or tissues; (k) reducing immunogenicity, and (l) At least one of the combinations of (a) to (k) is shown.

[0209] 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), wherein the agonist has the following formula: 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', wherein (i) (N1-N2-X1) comprises a first polynucleotide comprising linked nucleotides N1, N2 and X1; (ii) (X2-N3-N4) comprises a second polynucleotide comprising linked nucleotides X2, N3 and N4; (iii) N1, N2, N3, and N4 each comprise 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 each an oligonucleotide containing a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine; (viii) X1 is complementary to X2; (ix) X1 and X2 are each 12 to 16 nucleotides in length and are the same length; (x) L is a linker that operably links the first polynucleotide and the second polynucleotide; wherein 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 base pairs with a cytidine in the hairpin RNA.

[0210] In some embodiments, N1 comprises inosine and N4 comprises cytidine. In some embodiments, N1 comprises cytidine and N4 comprises inosine. In some embodiments, N2 comprises inosine and N3 comprises cytidine. In some embodiments, N2 comprises cytidine and N3 comprises inosine. In some embodiments, N1 comprises guanosine. In some embodiments, N2 comprises guanosine. In some embodiments, N1 comprises cytidine. In some embodiments, N2 comprises cytidine. In some embodiments, N1 and N2 comprise guanosine and N3 and N4 comprise cytidine. In some embodiments, N1 and N2 comprise cytidine and N3 and N4 comprise guanosine. In some embodiments, N1 and N2 comprise inosine and N3 and N4 comprise cytidine. In some embodiments, N1 and N2 comprise cytidine and N3 and N4 comprise inosine. In some embodiments, N1 comprises an inosine, N4 comprises a cytidine, and X1 and / or X2 each comprise at least one inosine. In some embodiments, N2 comprises an inosine, N3 comprises a cytidine, and X1 and / or X2 each comprise at least one inosine. In some embodiments, N1 and N2 comprise a guanosine, N3 and N4 comprise a cytidine, and X1 and / or X2 each comprise at least one inosine. In some embodiments, N1 and N2 comprise a guanosine, N3 and N4 comprise a cytidine, and X1 and X2 each comprise at least one inosine. In some embodiments, N1 and N2 comprise a cytidine, N3 and N4 comprise a guanosine, and X1 and X2 each comprise at least one inosine. In some embodiments, N1 and N2 comprise a guanosine, N3 and N4 comprise a cytidine, and X1 and X2 each comprise at least one inosine. In some embodiments, N1 and N2 comprise a guanosine, N3 and N4 comprise a cytidine, and X1 and X2 each comprise at least one inosine. In some embodiments, N1 and N2 comprise cytidine, N3 and N4 comprise guanosine, and X1 and X2 each comprise inosine and no guanosine nucleosides.In some embodiments, N1 and N2 comprise inosine, N3 and N4 comprise cytidine, and X1 and / or X2 each comprise at least one inosine. In some embodiments, N1 and N2 comprise inosine, N3 and N4 comprise cytidine, and X1 and X2 each comprise at least one inosine. In some embodiments, N1 and N2 comprise cytidine, N3 and N4 comprise inosine, and X1 and / or X2 each comprise at least one inosine. In some embodiments, N1 and N2 comprise inosine, N3 and N4 comprise cytidine, and X1 and X2 comprise inosine, and do not include a guanosine nucleoside. In some embodiments, N1 and N2 comprise cytidine, N3 and N4 comprise inosine, and X1 and X2 comprise inosine, and do not include a guanosine nucleoside. In some embodiments, 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.

[0211] 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), wherein the agonist has the following formula: 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', wherein (i) (N1-N2-X1) comprises a first polynucleotide comprising linked nucleotides N1, N2 and X1; (ii) (X2-N3-N4) comprises a second polynucleotide comprising linked nucleotides X2, N3 and N4; (iii) N1, N2, N3, and N4 each comprise 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 has the sequence motif [AUCN5] x wherein N5 comprises guanosine or inosine, x is an integer whose value indicates the number of sequence motifs, and x=3 or 4; (viii) X2 has the sequence motif [CN6AU] y wherein N6 comprises guanosine or inosine, y is an integer whose value indicates the number of sequence motifs, and y=3 or 4; (ix) L is a linker that operably links the first polynucleotide and the second polynucleotide; Optionally, at least one of N1, N2, N3, and N4 is inosine, and the inosine nucleoside base pairs with a cytidine in the hairpin RNA. In some embodiments, N5 comprises inosine, and N6 comprises inosine. In some embodiments, N5 comprises guanosine, and N6 comprises inosine. In some embodiments, N5 comprises inosine, and N6 comprises guanosine. In some embodiments, N5 comprises guanosine (G), and N6 comprises guanosine (G). In some embodiments, x=3 and y=3. In some embodiments, x=4 and y=4. In some embodiments, N1 comprises inosine (I), and N4 comprises cytidine (C). In some embodiments, N2 comprises inosine (I), and N3 comprises cytidine (C). In some embodiments, N3 comprises inosine (I), and N2 comprises cytidine (C). In some embodiments, N4 comprises inosine (I) and N1 comprises cytidine (C). In some embodiments, N1 comprises guanosine (G). In some embodiments, N2 comprises guanosine (G). In some embodiments, N1 comprises cytidine (C). In some embodiments, N2 comprises cytidine (C). In some embodiments, N1 and N2 comprise guanosine (G) and N3 and N4 comprise cytidine (C). In some embodiments, N1 and N2 comprise cytidine (C) and N3 and N4 comprise guanosine (G). In some embodiments, N1 and N2 comprise inosine (I) and N3 and N4 comprise cytidine (C). In some embodiments, N1 and N2 comprise cytidine (C) and N3 and N4 comprise inosine (I).

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

[0213] In some embodiments, the 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, the linker (L) is a nucleotide linker comprising a tetraloop, and the sequence of the tetraloop is UUCG. In some embodiments, the sequence of the tetraloop is GAUC.

[0215] In some embodiments, the linker (L) is: (a) an ethylene glycol linker, and (b) a non-nucleotidic linker selected from the group consisting of alkyl linkers.

[0216] In some embodiments, the non-nucleotidic linker is a hexaethylene glycol linker. In some embodiments, the non-nucleotidic 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 bioisostere, wherein the phosphate bioisostere is selected from phosphonate, thiophosphonate, phosphorothioate, sulfate, sulfonate, sulfamate, thiazolidinone, carboxylate, malonate, boronic acid, benzoxaborole, boranophosphate, and squaramide.

[0218] In some embodiments, the RLR agonist comprises a modified nucleotide, modified nucleoside, or modified nucleobase, or a combination thereof. In some embodiments, the agonist comprises a modification to the internucleotide linkage or polynucleotide backbone.

[0219] In some embodiments, the RLR agonists of the present disclosure have the following properties: (a) specifically binds to one or more RLRs (e.g., RIG-1, MDA5, and / or LGP2); (b) increase RLR-mediated cytokine production; (c) increasing RLR-mediated expression of interferon-inducible genes (ISGs); (d) increasing RLR-dependent intracellular signaling; (e) increasing duplex stability; (f) increasing binding affinity to RLRs; (g) reducing off-target binding; (h) increasing biological half-life; (i) enhance biodistribution and bioavailability; (j) increasing and / or enhancing uptake into cells and / or tissues; (k) reducing immunogenicity, and (l) At least one of the combinations of (a) to (k) is shown.

[0220] 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-ended hairpin RNA comprising a first polynucleotide connected to a second polynucleotide by a linker, the first polynucleotide being sufficiently complementary to the second polynucleotide to form a duplex, the duplex comprising fewer than 19 base pairs, and the 5'-most nucleotide of the first oligonucleotide having a 5' diphosphate or triphosphate moiety, or a derivative thereof. or analogs thereof, wherein the agonist comprises a sequence motif that confers at least one improved biological activity mediated by an RLR compared to an agonist that does not comprise the sequence motif, 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.

[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-ended hairpin RNA comprising a first polynucleotide connected to a second polynucleotide by a linker, wherein the first polynucleotide is sufficiently complementary to the second polynucleotide to form a duplex, wherein the duplex comprises fewer than 19 base pairs, wherein the 5'-most nucleotide of the first oligonucleotide comprises a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof, and wherein the agonist comprises a sequence motif that confers at least one improved biological activity mediated by an RLR compared to an agonist that does not comprise the sequence motif, and wherein the first polynucleotide and the second polynucleotide are selected from the group consisting of: (i) SEQ ID NOs: 37 and 68, respectively; (ii) SEQ ID NOs: 38 and 69, respectively; (iii) SEQ ID NOs: 39 and 70, respectively; (iv) SEQ ID NOs: 40 and 71, respectively; (v) SEQ ID NOs: 41 and 72, respectively; (vi) SEQ ID NOs: 42 and 73, respectively; (vii) SEQ ID NOs: 43 and 74, respectively; (viii) SEQ ID NOs: 44 and 75, respectively; (ix) SEQ ID NOs: 45 and 76, respectively; (x) SEQ ID NOs: 46 and 77, respectively; (xi) SEQ ID NOs: 47 and 78, respectively; (xii) SEQ ID NOs: 48 and 79, respectively; (xiii) SEQ ID NOs: 49 and 80, respectively; (xiv) SEQ ID NOs: 50 and 81, respectively; (xv) SEQ ID NOs: 51 and 82, respectively; (xvi) SEQ ID NOs: 52 and 83, respectively; (xvii) SEQ ID NOs: 53 and 84, respectively; (xviii) SEQ ID NOs: 54 and 85, respectively; (xix) SEQ ID NOs: 55 and 86, respectively; (xx) SEQ ID NOs: 56 and 87, respectively; (xxi) SEQ ID NOs: 57 and 88, respectively; (xxii) SEQ ID NOs: 58 and 89, respectively; (xxiii) SEQ ID NOs: 59 and 89, respectively; (xxiv) SEQ ID NOs: 60 and 90, respectively; (xxv) SEQ ID NOs: 61 and 91, respectively; (xxvi) SEQ ID NOs: 62 and 92, respectively; (xxvii) SEQ ID NOs: 63 and 91, respectively; (xxviii) SEQ ID NOs: 64 and 93, respectively; (xxix) SEQ ID NOs: 65 and 94, respectively; (xxx) SEQ ID NOs: 66 and 95, respectively; (xxxi) SEQ ID NOs: 67 and 96, respectively, and (xxxii) each comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 63 and 97.

[0222] 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), wherein the agonist comprises a blunt-ended hairpin RNA comprising at least one or more nucleotides including an inosine that base pairs with a cytidine, and wherein the agonist comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 22, 23, and 25.

[0223] 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), wherein the agonist comprises a blunt-ended hairpin RNA comprising at least one or more nucleotides including an inosine that base pairs with a cytidine, wherein the agonist comprises the formula 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', wherein (N1-N2-X1) comprises a first polynucleotide and (X2-N3-N4) comprises a second polynucleotide, and wherein the first polynucleotide and the second polynucleotide are selected from the following: (i) SEQ ID NOs: 58 and 89, respectively; (ii) SEQ ID NOs: 59 and 89, respectively; and (iii) comprise a nucleotide sequence selected from the group consisting of SEQ ID NOs: 61 and 91, respectively.

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

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

[0226] RLR agonists containing modified nucleobases, nucleosides or nucleotides In some embodiments, the RLR agonists of the present disclosure comprise one or more modified nucleic acid bases, nucleosides, or nucleotides. In some embodiments, modified RLR agonists may have useful properties, including improved stability, intracellular retention, improved target binding, and / or increased innate immune responses in cells into which the RLR agonist is introduced, compared to a reference unmodified RLR agonist. Thus, the use of modified RLR agonists may not only have reduced immunogenicity, but also enhance the efficiency of target binding and intracellular retention of nucleic acids. In one embodiment, the agonists provided by the present disclosure are comprised of one or more oligonucleotides containing at least one region modified to enhance target binding affinity. The affinity of an oligonucleotide for 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 the present disclosure is composed of at least one oligonucleotide, comprising at least one region that comprises at least one modified nucleobase, nucleoside or nucleotide that enhances the stability of duplex.The stability of duplex can be routinely determined by measuring the Tm of duplex, which is the temperature at which the two oligonucleotide strands that constitute duplex dissociate, and dissociation is detected by spectrophotometry.The higher the Tm, the higher the stability of duplex.

[0228] In one embodiment, the region of the oligonucleotide modified to enhance duplex stability contains 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'-fluoro modified nucleotide. In another embodiment, the oligonucleotide constituting the RLR agonist is also modified to enhance nuclease resistance. Cells contain a variety of exonucleases and endonucleases that can degrade nucleic acids. Many nucleotide and nucleoside modifications have been shown to render incorporated oligonucleotides more resistant to nuclease digestion than unmodified oligonucleotides. Nuclease resistance is routinely measured by incubating oligonucleotides with cell extracts or isolated nuclease solutions and measuring the extent of intact oligonucleotide remaining over time, usually by gel electrophoresis. Oligonucleotides modified to enhance nuclease resistance remain intact for longer periods of time than unmodified oligonucleotides. Various oligonucleotide modifications have been shown to enhance or confer nuclease resistance. 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 preferred oligonucleotides contemplated for the present invention include those containing modified backbones, such as phosphorothioates, phosphotriesters, methylphosphonates, short alkyl or cycloalkyl intersugar linkages, or short heteroatom or heterocyclic intersugar linkages. Most preferred are oligonucleotides with phosphorothioate backbones (including those synthesized in a stereospecific manner) and heteroatom backbones, particularly CH2-NH-O-CH2, CH2-N(CH3)-O-CH2 (known as methylene(methylimino) or MMI backbones), CH2-ON(CH3)-CH2, CH2-N(CH3)-N(CH3)-CH2, and ON(CH3)-CH2-CH2 backbones (where the natural phosphodiester backbone is represented as OPO-CH2). The amide backbones disclosed by De Mesmaeker et al. (1995, Acc. Chem. Res. 28:366-374) are 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, OCHOCH3, OCHO(CH2)nCH3, O(CH2)nNH2 or O(CH2)nCH3 (where 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 cleaving group; reporter group; intercalator; group for improving the pharmacokinetic properties of oligonucleotides; or group for improving the pharmacodynamic properties of oligonucleotides and other substituents with similar properties. A preferred modification includes 2'-methoxyethoxy [2'-O-CH2CH2OCH3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE] (Martin et al., Helv. Chim. Acta, 1995, 78, 486).Other preferred modifications include 2'-methoxy (2'-O-CH), 2'-propoxy (2'-OCHCHCH), and 2'-fluoro (2'-F). Similar modifications can also be made at other positions on the oligonucleotide, particularly the 3' position of the sugar on the 3'-terminal nucleotide and the 5' position of the 5'-terminal nucleotide. Oligonucleotides can also have sugar mimetics such as cyclobutyls in place of the pentofuranosyl group.

[0230] Oligonucleotides may also, or alternatively, contain nucleobase (often referred to in the art simply as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include adenine (A), guanine (G), thymine (T), cytosine (C), and uracil (U). Modified nucleobases include nucleobases that occur rarely or only occasionally in natural nucleic acids, such as hypoxanthine, 6-methyladenine, 5-me pyrimidines, particularly 5-methylcytosine (also known as 5-methyl-2'-deoxycytosine, often referred to in the art as 5-me-C), 5-hydroxymethylcytosine (HMC), glycosyl HMC, and gentobiosyl HMC, as well as synthetic nucleobases 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, W.H. 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 also be included. The 5-me-C substitution has been shown to increase nucleic acid duplex stability by 0.6-1.2°C (Sanghvi, Y.S., Crooke, S.T., and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278), and is currently the preferred base substitution.

[0231] Another modification of the oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates that improve the activity or cellular uptake of the oligonucleotide. Such moieties include, but are not limited to, lipid moieties such as cholesterol moieties, cholesteryl moieties (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. NY Acad. 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 J., 1991, 10, 111; Kabanov et al., FEBS Lett., 1990, 259, 327; Svinarchuk et al., Biochimie, 1993, 75, 49), phospholipid, polyamide or polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969), or adamantane acetic acid (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. Patent Nos. 5,138,045, 5,218,105 and 5,459,255.

[0232] The oligonucleotides of the present invention may be provided as prodrugs, which generally contain one or more moieties that are cleaved in the body to produce the active oligonucleotide. One example of the prodrug approach is described by Imbach et al. in WO Publication No. 94 / 26764.

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

[0234] Oligonucleotides of the invention are preferably from about 8 to about 50 nucleotides in length, which in the context of the present invention is understood to encompass non-naturally occurring oligomers comprising 8 to 50 monomers, as described herein above.

[0235] The oligonucleotides used in accordance with the present invention may be conveniently and routinely produced by the well-known technique of solid-phase synthesis. Equipment for such synthesis is sold by several suppliers, including Applied Biosystems. Any other means for such synthesis may also be used, and the actual synthesis of oligonucleotides is well within the knowledge and ability of those skilled in the art. It is also well known to use similar techniques to prepare other oligonucleotides, such as phosphorothioates and alkylated derivatives. It is also well known to synthesize fluorescently labeled, biotinylated, or other modified oligonucleotides, such as cholesterol-modified oligonucleotides, using similar techniques and commercially available modified amidites and controlled pore glass (CPG) products, such as biotin, fluorescein, acridine, or psoralen-modified amidites and / or CPGs (available from Glen Research, Sterling, Va.).

[0236] In some embodiments, the RLR agonist comprises one or more (e.g., 1, 2, 3, or 4) different modified nucleobases, 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 nucleobases, nucleosides, or nucleotides. In some embodiments, the modified RLR agonist may exhibit reduced degradation in cells into which the RLR agonist is introduced, compared to the corresponding unmodified RLR agonist.

[0237] In some embodiments, the modified nucleobase is a modified uracil. Exemplary nucleobases and nucleosides having modified uracil include pseudouridine (ψ), pyridin-4-one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s 2 U), 4-thio-uridine (s 4 U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (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-methoxy-uridine (mo 5 U), uridine 5-hydroxyacetic acid (cmo 5 U), uridine 5-hydroxyacetic acid methyl ester (mcmo 5 U), 5-carboxymethyl-uridine (cm 5 U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm 5 U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm 5 U), 5-methoxycarbonylmethyl-uridine (mcm 5 U), 5-methoxycarbonylmethyl-2-thiouridine (mcm 5 s 2 U), 5-aminomethyl-2-thio-uridine (nm5 s 2 U), 5-methylaminomethyl-uridine (mnm 5 U), 5-methylaminomethyl-2-thiouridine (mnm 5 s 2 U), 5-methylaminomethyl-2-seleno-uridine (mnm 5 se 2 U), 5-carbamoylmethyl-uridine (ncm 5 U), 5-carboxymethylaminomethyl-uridine (cmnm 5 U), 5-carboxymethylaminomethyl-2-thiouridine (cmnm 5 s 2 U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (τm 5 U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thiouridine (τm 5 s 2 U), 1-taurinomethyl-4-thio-pseudouridine, 5-methyl-uridine (m 5 U, i.e., having the nucleobase deoxythymine), 1-methyl-pseudouridine (m 1 ψ), 5-methyl-2-thiouridine (m 5 s 2 U), 1-methyl-4-thio-pseudouridine (m 1 s 4 ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m 3 ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m 5 D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp 3U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (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-methyl-pseudouridine (ψm), 2-thio-2'-O-methyl-uridine (s 2 Um), 5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcm 5 Um), 5-carbamoylmethyl-2'-O-methyl-uridine (ncm 5 Um), 5-carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm 5 Um), 3,2'-O-dimethyl-uridine (m 3 Um) and 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm 5 Um), 1-thio-uridine, deoxythymidine, 2'-F-ara-uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5-(2-carbomethoxyvinyl)uridine, and 5-[3-(1-E-propenylamino)]uridine.

[0238] In some embodiments, the modified nucleobase is a modified cytosine. Exemplary nucleobases and nucleosides having modified cytosines include 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine (m 3 C), N4-acetyl-cytidine (ac 4 C), 5-formyl-cytidine (f 5 C), N4-methyl-cytidine (m 4 C), 5-methyl-cytidine (m 5 C), 5-halo-cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm 5C), 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine (s 2 C), 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, lysidine (k2C), α-thio-cytidine, 2'-O-methyl-cytidine (Cm), 5,2'-O-dimethyl-cytidine (m 5 Cm), N4-acetyl-2'-O-methyl-cytidine (ac 4 Cm), N4,2'-O-dimethyl-cytidine (m 4 Cm), 5-formyl-2'-O-methyl-cytidine (f 5 Cm), N4,N4,2'-O-trimethyl-cytidine (m 4 2Cm), 1-thio-cytidine, 2'-F-ara-cytidine, 2'-F-cytidine and 2'-OH-ara-cytidine.

[0239] In some embodiments, the modified nucleobase is a modified adenine. Exemplary nucleobases and nucleosides having modified adenines 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-hydroxynovalylcarbamoyl-adenosine (hn 6 A), 2-methylthio-N6-hydroxynovalylcarbamoyl-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.

[0240] In some embodiments, the modified nucleobase is a modified guanine. Exemplary nucleobases and nucleosides having modified guanines include α-thio-guanosine, inosine (I), 1-methyl-inosine (m), 1 I), wyosine (imG), methylwyosine (mimG), 4-demethyl-wyosine (imG-14), isowyosine (imG2), wyobutosine (yW), peroxywyobutosine (o2yW), hydroxywyobutosine (OhyW), unmodified hydroxywyobutosine (OhyW*), 7-deaza-guanosine, quosine (Q), epoxyquosine (oQ), galactosyl-quosine (galQ), mannosyl-quosine (manQ), 7-cyano-7-deaza-guanosine (preQ0), 7-aminomethyl-7-deaza-guanosine (preQ1), and alkeosine (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 Im), 2'-O-ribosylguanosine(phosphate) (Gr(p)), 1-thio-guanosine, O6-methyl-guanosine, 2'-F-ara-guanosine and 2'-F-guanosine.

[0241] In some embodiments, the RLR agonists of the present disclosure comprise a combination of one or more of the above-described modified nucleobases (eg, a combination of two, three, or four of the above-described modified nucleobases).

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

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

[0244] The RLR agonists of the present disclosure may include combinations of modifications to the sugar, nucleobase, and / or internucleoside linkage, which may include any one or more of the modifications described herein.

[0245] Examples of modified nucleosides and combinations of modified nucleosides are shown in Tables 1 and 2 below. These combinations of modified nucleotides can be used to form the RLR agonists of the present disclosure. In certain embodiments, modified nucleosides can partially or completely replace natural nucleotides in the RLR agonists of the present disclosure. As a non-limiting example, the natural nucleotide uridine can be replaced with a modified nucleoside described herein. In another non-limiting example, the natural nucleoside uridine can be partially replaced 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] In accordance with the present disclosure, polynucleotides of the present disclosure may be synthesized to contain any combination or single modification of Table 1 or Table 2.

[0247] When a single modification is listed, the listed nucleoside or nucleotide represents 100% modified A, U, G, or C nucleotide or nucleoside.When percentages are listed, they represent the percentage of a specific A, U, G, or C nucleobase triphosphate relative to the total amount of A, U, G, or C triphosphates 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 cytosine triphosphates are 5-aminoallyl-CTP, while 75% of cytosines are CTP, while 25% of uracils are 5-methoxy-UTP, while 75% of uracils are UTP.When no modified UTP is listed, naturally occurring ATP, UTP, GTP, and / or CTP are used at 100% of the nucleotide positions found in the polynucleotide. In this example, all of the GTP and ATP nucleotides remain unmodified.

[0248] Methods for producing RLR agonists The RLR agonist of the present disclosure may be produced by means available in the art, including but not limited to in vitro transcription (IVT) and synthetic methods. Enzyme (IVT), solid phase, liquid phase, combined synthetic methods, small-area synthesis and ligation methods may be utilized. In one embodiment, the RLR agonist is produced 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. Therefore, the present disclosure also includes polynucleotides, such as DNA, constructs and vectors, that can be used to in vitro transcribe the RLR agonists described herein.

[0249] Non-naturally occurring modified nucleobases may be introduced into polynucleotides (e.g., RNA) during or after synthesis. In certain embodiments, modifications may be made to internucleoside linkages, purine or pyrimidine bases, or sugars. In certain embodiments, modifications may be introduced at the termini of a polynucleotide chain or elsewhere in a polynucleotide chain using chemical synthesis or polymerase enzymes. Examples of modified nucleic acids and their synthesis are disclosed in PCT Application No. 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] Either enzymatic or chemical ligation methods may be used to conjugate polynucleotides or regions thereof with different functional moieties, such as targeting or delivery agents, fluorescent labels, lipids, nanoparticles, etc. Conjugates of polynucleotides and modified polynucleotides are summarized in Goodchild, Bioconjugate Chemistry, vol. 1(3), 165-187 (1990). The synthesis of oligonucleotides, polynucleotides, and their conjugation and ligation 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 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 pharmaceutical compositions comprising an RLR agonist together with a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and / or adjuvant.

[0252] In certain embodiments, acceptable formulation materials are preferably nontoxic to recipients at the dosages and concentrations used. In certain embodiments, the formulation materials are for subcutaneous and / or intravenous administration. In certain embodiments, pharmaceutical compositions may contain formulation materials to modify, maintain, or preserve, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption, or penetration 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., borate, bicarbonate, Tris-HCl, citrate, phosphate, other organic acids); bulking agents (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 immunoglobulins); colorants, flavoring agents, and diluents; emulsifiers; hydrophilic polymers (e.g., polyvinylpyrrolidone); low molecular weight polypeptides. 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 such as polysorbate 20, polysorbate 80, Triton, tromethamine, lecithin, cholesterol, tyloxapal); 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 one skilled in the art based on, for example, the intended route of administration, delivery format, and desired dosage. See, e.g., Remington's Pharmaceutical Sciences, supra. 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 a pharmaceutical composition may be either aqueous or non-aqueous in nature. For example, in certain embodiments, a suitable vehicle or carrier may be water for injection, saline solution, or artificial cerebrospinal fluid, possibly supplemented with other substances common in compositions for parenteral administration. In certain embodiments, saline includes isotonic phosphate-buffered saline. In certain embodiments, neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. In certain embodiments, the pharmaceutical composition comprises Tris buffer at about pH 7.0-8.5 or acetate buffer at about pH 4.0-5.5, which may further contain sorbitol or a suitable substitute therefor. In certain embodiments, compositions containing an RLR agonist can be prepared for storage by combining a selected composition having the desired purity with an optimal formulation agent (Remington's Pharmaceutical Sciences, supra) in the form of a lyophilized cake or aqueous solution. Additionally, in certain embodiments, compositions comprising an RLR agonist may be formulated as a lyophilizate using appropriate excipients such as sucrose.

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

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

[0256] In certain embodiments, when parenteral administration is contemplated, the therapeutic composition may be in the form of a pyrogen-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, preparation may involve formulating the desired molecule using a delivery vehicle or agent, such as injectable microspheres, biodegradable particles, polymeric compounds (e.g., polylactic acid, polyglycolic acid, or polyethyleneimine (e.g., JetPEI®)), beads, or liposomes, which can then provide controlled or sustained release of the deliverable product via depot injection. In certain embodiments, hyaluronic acid may also be used, which may have the effect of promoting sustained duration in the circulation. In certain embodiments, the desired molecule may be introduced using an implantable drug delivery device.

[0257] In certain embodiments, pharmaceutical compositions can be formulated for inhalation.In certain embodiments, RLR agonist can be formulated as dry powder for inhalation.In certain embodiments, the inhalation solution comprising RLR agonist can be formulated with a propellant for aerosol delivery.In certain embodiments, the solution can be nebulized.Pulmonary administration is further described in PCT Application No. PCT / US94 / 001875, which describes the pulmonary delivery of chemically modified proteins.

[0258] In certain embodiments, it is envisioned that formulations can be administered orally.In certain embodiments, the RLR agonist administered in this manner can be formulated with or without the carrier that is commonly used in the formulation of solid dosage forms such as tablets and capsules.In certain embodiments, capsules can be designed to release the active part of the formulation at a certain time in the gastrointestinal tract when bioavailability is maximized and pre-systemic degradation is minimized.In certain embodiments, at least one additional agent can be included to promote the absorption of RLR agonists.In certain embodiments, diluents, flavorings, low-melting waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents and binders can also be used.

[0259] In certain embodiments, pharmaceutical compositions can contain an effective amount of RLR agonist in the mixture with non-toxic excipients suitable for tablet manufacture.In certain embodiments, tablet can be dissolved in sterile water or other suitable vehicle to prepare solution in unit dose form.In certain embodiments, suitable excipients include but are not limited to inert diluents such as calcium carbonate, sodium carbonate or sodium bicarbonate, lactose or calcium phosphate; or binders such as starch, gelatin or acacia; or lubricants such as magnesium stearate, stearic acid or talc.

[0260] Further pharmaceutical compositions will be clear to those skilled in the art, including formulations that contain RLR agonists in sustained release or controlled delivery formulations.In certain embodiments, the techniques for formulating various other sustained release or controlled delivery means, such as liposome carriers, bioerodible microparticles or porous beads and depot injections, are also known to those skilled in the art.For example, see PCT Application No. PCT / US93 / 00829, which describes the controlled release of porous polymer microparticles for delivering pharmaceutical compositions.In certain embodiments, sustained release preparations can comprise semipermeable polymer matrices in the form of shaped articles, such as films or microcapsules. Sustained-release matrices can include polyesters, hydrogels, polylactides (U.S. Pat. No. 3,773,919 and EP 058,481), 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., supra), or poly-D(-)-3-hydroxybutyrate (EP 133,988). In certain embodiments, sustained-release compositions may also contain liposomes, which can be prepared by any of several methods known in the art. See, e.g., Eppstein et al., Proc. Natl. Acad. Sci. USA, 82:3688-3692 (1985); EP 036,676; EP See EP 143,949 and EP 088,046.

[0261] The pharmaceutical composition used for in vivo administration is typically sterile.In certain embodiments, this can be achieved by filtration through sterile filtration membrane.In certain embodiments, when composition is lyophilized, sterilization using this method can be carried out either before or after lyophilization and reconstitution.In certain embodiments, the composition for parenteral administration can be stored in lyophilized form or solution.In certain embodiments, parenteral compositions are generally placed into a container with sterile access port, for example, an intravenous solution bag or vial with a stopper that can be pierced by a hypodermic injection needle.

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

[0263] In certain embodiments, a kit for producing a single-dose dosage unit is provided.In certain embodiments, the kit may include both a first container with a dried protein and a second container with an aqueous formulation.In certain embodiments, the kit includes a single-chamber and multi-chamber pre-filled syringe (for example, a liquid syringe and a lyosyringe).

[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 size (weight, body surface, or organ size) and / or condition (age and overall health). In certain embodiments, clinicians can titrate the dosage and modify the route of administration to achieve optimal therapeutic effects.

[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 dosage that achieves the desired effect is reached. Thus, in certain embodiments, the composition may be administered as a single dose or two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion via an implanted device or catheter. Further refinement of the appropriate dosage is routinely performed by those skilled in the art and is within the scope of routinely performed tasks. In certain embodiments, the appropriate dosage can be confirmed using appropriate dose-response data.

[0266] In certain embodiments, the pharmaceutical composition is administered by known methods, such as by oral administration, intravenous, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, subcutaneous, intraocular, intraarterial, intraportal or intralesional injection, by sustained release system, or by implantation device. In certain embodiments, the composition can be administered by bolus injection, or continuously by infusion, or by implantation device. In certain embodiments, the individual components of the combination therapy can be administered by different routes.

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

[0268] In certain embodiments, RLR agonists may be delivered by implanting specific cells genetically engineered to express and secrete the agonist using methods described herein. In certain embodiments, such cells may be animal or human cells, and may be autologous, autologous xenogeneic, or xenogeneic. In certain embodiments, the cells may be immortalized. In certain embodiments, to reduce the chance of an immunological response, the cells may be encapsulated to avoid infiltration of surrounding tissues. In certain embodiments, the encapsulating material is typically a biocompatible, semipermeable polymeric inclusion or membrane that allows release of the protein product but prevents destruction of the cells by the patient's immune system or other harmful factors from the surrounding tissues.

[0269] In some embodiments, the present disclosure provides a pharmaceutical composition for stimulating an immune response, treating or delaying the progression of cancer, or reducing or inhibiting tumor growth in a subject in need thereof, the pharmaceutical composition comprising an RLR agonist provided by the present 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 target function (e.g., RLR-mediated cell signaling or responses). In some embodiments, for example, if the composition binds to and activates a target (e.g., a protein or polypeptide), the composition can be used as a positive control in assays designed to identify additional novel compounds that also induce the activity of the target protein or polypeptide and / or are otherwise useful for treating disorders associated with the target protein or polypeptide. For example, a composition that activates an RLR can be used as a positive control in assays to identify additional 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 described in more detail below.

[0271] kit The kit may include an RLR agonist disclosed herein and instructions for use. The kit may also include, in suitable containers, the RLR agonist, one or more controls, 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, into which the RLR agonist is placed, and in some cases, may be appropriately aliquoted. If additional components are provided, the kit may include additional containers into which these components can be placed. The kit may also include a means for containing the RLR agonist and any other reagent containers in close confinement for commercial sale. Such containers may include injection-molded or blow-molded plastic containers into which the desired vials are held. The container and / or kit may include a label with instructions and / or warnings for use.

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

[0274] In some embodiments, the agonist or pharmaceutical composition is administered in combination with one or more additional therapeutic agents, wherein the one or more additional therapeutic agents are selected from the group consisting of chemotherapy, targeted anti-cancer therapy, oncolytic agent, cell death inducer, opsonizing agent (e.g., opsonizing antibody), cytotoxic agent, immune system therapy, cytokine, activator of costimulatory molecules, inhibitor of inhibitory molecules, vaccine, 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 the 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, 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.

[0277] In some embodiments, the one or more additional therapeutic agents is an agonist comprising a polypeptide (eg, an antibody, or antigen-binding portion thereof) that specifically binds to CD137 (4-1BB).

[0278] In some embodiments, the one or more additional therapeutic agents is an agonist comprising a polypeptide (eg, an antibody, or antigen-binding portion thereof) that specifically binds to CD134 (OX40).

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

[0280] The above-described compositions are particularly useful in methods for treating or preventing various cancers or infectious diseases in subjects. The compositions can be administered to a subject (e.g., a human subject) using a variety of methods, depending in part on the route of administration. The route may be, for example, intravenous injection or infusion (IV), subcutaneous injection (SC), intradermal injection (ID), intraperitoneal (IP) injection, intramuscular injection (IM), intratumoral injection (IT), or intrathecal injection. The injection may be performed as a bolus or continuous infusion.

[0281] Administration can be achieved, for example, by local infusion, injection, or implant. The implant can be made of porous, non-porous, or gelatinous materials, including membranes such as elastic membranes, or fibers. The implant can be configured for sustained or periodic release of the composition into a subject. 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 each of which are incorporated herein by reference in their entirety. The composition can be delivered to a subject, for example, by a diffusion, erosion, or convection system, such as an osmotic pump, a biodegradable implant, an electrodiffusion system, an electroosmotic system, a vapor pressure pump, an electrolytic pump, an effervescent pump, a piezoelectric pump, an erosion-based system, or an implantable device based on an electromechanical system.

[0282] In some embodiments, the RLR agonist is therapeutically delivered to the subject by local administration.

[0283] The appropriate dose of the RLR agonist described herein that can treat or prevent cancer in a subject may depend on various factors, including, for example, the age, sex, and weight of the subject being treated, as well as the specific inhibitor compound used.Other factors that affect the dose administered to a subject include, for example, the type or severity of cancer or infection.For example, a subject with metastatic melanoma may require a different dosage of RLR agonist than a subject with glioblastoma.Other factors may include, for example, other medical disorders that the subject is currently suffering from or has previously suffered from, the subject's overall health, the subject's genetic makeup, diet, administration time, excretion rate, drug combinations, and any other additional therapy administered to the subject.It should also be understood that the specific dosage and treatment regimen for any particular subject will also be based on the judgment of the treating medical professional (e.g., doctor or nurse).Appropriate dosages are described herein.

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

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

[0286] In some embodiments, the composition comprises any 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) additional therapeutic agents such that the composition as a whole is therapeutically effective. For example, a composition may comprise an RLR agonist described herein and an alkylating agent, each at concentrations that, when combined, are therapeutically effective for treating or preventing 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 culture or experimental animals (e.g., animal models of any of the cancers described herein). These procedures can be performed, for example, using LD 50 (lethal dose in 50% of the population) and ED 50 The dose ratio between toxic and therapeutic efficacy is the therapeutic index, and the ratio LD 50 / ED 50 RLR agonists that exhibit a high therapeutic index are preferred. Compositions that exhibit toxic side effects may be used, but care should be taken to design a delivery system that targets such compounds to the site of affected tissue, minimizing the potential for damage to normal cells and thereby reducing side effects.

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

[0289] In some embodiments, the method can be performed in conjunction with other therapies for cancer or infectious diseases. 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 therapy.

[0290] As discussed above, the compositions (e.g., RLR agonist compositions) described herein can be used to treat a variety of cancers, including, but not limited to, Kaposi's sarcoma, leukemia, acute lymphocytic leukemia, acute myeloid leukemia, myeloblastic promyelocytic myelomonocytic erythroid leukemia, chronic leukemia, chronic myeloid (granulocytic) leukemia, chronic lymphocytic leukemia, mantle cell lymphoma, primary central nervous system lymphoma, Burkitt's lymphoma, marginal zone B-cell lymphoma, Polycythemia vera, Hodgkin's disease, Non-Hodgkin's disease, Multiple myeloma, Waldenstrom's macroglobulinemia, Heavy chain disease, Solid tumors, Sarcomas and carcinomas, Fibrosarcoma, Myxosarcoma, Liposarcoma, Chondrosarcoma, Osteogenic sarcoma, Osteosarcoma, Chordoma, Angiosarcoma, Endosarcoma, Lymphangiosarcoma, Lymphangioendothelial sarcoma, Synovioma, 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, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, non-small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, retinoblastoma, nasopharyngeal carcinoma, esophageal carcinoma, These include basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, cancer of the brain and central nervous system (CNS), cervical cancer, choriocarcinoma, colon cancer, connective tissue cancer, cancer of the digestive system, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer, intraepithelial neoplasia, kidney cancer, laryngeal cancer, liver cancer, lung cancer (small cell, large cell), melanoma, neuroblastoma, oral cancer (e.g., lip, tongue, mouth, and pharynx), ovarian cancer, pancreatic cancer, rectal cancer, cancer of the respiratory system, sarcoma, skin cancer, stomach cancer, testicular cancer, thyroid cancer, uterine cancer, and cancer of the urinary system.

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

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

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

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

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

[0296] In some embodiments, the present disclosure provides a method of reducing or inhibiting tumor growth in a subject in need thereof, the method comprising administering to the subject an effective amount of an RLR agonist provided by the present disclosure or a pharmaceutical composition provided by the present disclosure.

[0297] In some embodiments, the present disclosure provides a method of stimulating an immune response, treating or slowing the progression of cancer, or inhibiting tumor growth in a subject in need thereof, the method comprising administering to the subject an effective amount of an RLR agonist provided by the present disclosure or a pharmaceutical composition provided by the present disclosure, wherein the agonist or pharmaceutical composition increases RLR-mediated production of one or more cytokines in a cell, increases RLR-mediated expression of one or more interferon-inducible genes in the cell, and / or increases RLR-dependent intracellular signaling in the cell, thereby stimulating the immune response, treating or slowing the progression of cancer, or inhibiting tumor growth.

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

[0299] In some embodiments of the methods provided by the present disclosure, the RLR agonist or pharmaceutical composition is administered in combination with one or more additional therapeutic agents, wherein the one or more additional therapeutic agents are selected from the group consisting of chemotherapy, targeted anti-cancer therapy, oncolytic agent, cell death inducer, opsonizing agent (e.g., opsonizing antibody), cytotoxic agent, immune system therapy, cytokine, activator or agonist of costimulatory molecule, inhibitor of inhibitory molecule, vaccine, 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 the 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, 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.

[0302] Combination with chemotherapy drugs Chemotherapeutic agents suitable for combination and / or co-administration with the compositions of the invention include, for example, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyantransinedione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, as well as analogs or homologs thereof. Additional agents include, for example, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioTEPA, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, cis-dichlorodiamineplatinum(II) (DDP), procarbazine, Anti-inflammatory drugs include cisplatin, altretamine, cisplatin, carboplatin, oxaliplatin, nedaplatin, satraplatin, or triplatin tetranitrate), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and antimitotic agents (e.g., vincristine and vinblastine) and temozolomide.

[0303] Combination with PD-1 / PD-L1 antagonists In some embodiments, an RLR agonist or pharmaceutical composition thereof provided by the present disclosure is combined (e.g., administered in combination) with one or more PD-1 / PD-L1 antagonists that specifically bind to human PD-1 or PD-L1 and inhibit the biological activity 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.

[0304] Accordingly, provided herein are PD-1 / PD-L1 antagonists that directly or allosterically block, antagonize, inhibit, inhibit, or reduce PD-1 / PD-L1 biological activity, including downstream pathways and / or cellular processes mediated by PD-1 / PD-L1 signaling, e.g., receptor binding to and / or eliciting a cellular response thereto. Also provided herein are PD-1 / PD-L1 antagonists that reduce the level or amount of human PD-1 / PD-L1 produced by a cell or a subject.

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

[0306] In some embodiments, the PD-1 / PD-L1 antagonist inhibits PD-1 signaling or function. In some embodiments, the PD-1 / PD-L1 antagonist blocks PD-1 binding to PD-L1, PD-L2, or both PD-L1 and PD-L2. In some embodiments, the PD-1 / PD-L1 antagonist blocks PD-1 binding to PD-L1. In some embodiments, the PD-1 / PD-L1 antagonist blocks PD-1 binding to PD-L2. In some embodiments, the PD-1 / PD-L1 antagonist blocks PD-1 binding to PD-L1 and PD-L2. In some embodiments, the PD-1 / PD-L1 antagonist specifically binds to PD-1. In some embodiments, the PD-1 / PD-L1 antagonist specifically binds to PD-L1. In some embodiments, the PD-1 / PD-L1 antagonist specifically binds to PD-L2.

[0307] In some embodiments, the PD-1 / PD-L1 antagonist inhibits binding of PD-1 to its cognate ligand. In some embodiments, the PD-1 / PD-L1 antagonist inhibits binding of PD-1 to PD-L1, PD-L2, or both PD-L1 and PD-L2. In some embodiments, the PD-1 / PD-L1 antagonist does not inhibit binding of PD-1 to its cognate ligand.

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

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

[0310] Examples of anti-human PD-1 monoclonal antibodies, or antigen-binding fragments thereof, that may comprise PD-1 / PD-L1 antagonists in any of the compositions, methods, and uses provided by the present disclosure include, but are not limited to, KEYTRUDA® (pembrolizumab, MK-3475, h409A11; see US8952136, US8354509, US8900587, and EP2170959, all of which are incorporated herein by reference). and other products, all of which are incorporated herein by reference in their entireties; Merck), OPDIVO® (nivolumab, BMS-936558, MDX-1106, ONO-4538; see US 7595048, US 8728474, US 9073994, US 9067999, EP 1537878, US 8008449, US 8779105 and EP 2161336, all of which are incorporated herein by reference in their entireties; Bristol Myers Squibb), MEDI0680 (AMP-514), BGB-A317 and BGB-108 (BeiGene), 244C8 and 388D4 (see WO2016 / 106159, incorporated herein by reference in its entirety; 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, that may comprise PD-1 / PD-L1 antagonists in any of the compositions, methods, and uses provided by the present disclosure include, but are not limited to, BAVENCIO® (avelumab, MSB0010718C, see WO2013 / 79174, incorporated herein by reference in its entirety; Merck / Pfizer), IMFINZI® (durvalumab, ME DI4736), TECENTRIQ® (atezolizumab, MPDL3280A, RG7446; see WO2010 / 077634, incorporated herein by reference in its entirety; Roche), MDX-1105 (BMS-936559, 12A4; see US7943743 and WO2013 / 173223, both of which are incorporated herein by reference in their entireties; 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, e.g., a fusion protein comprising the extracellular or PD-1-binding portion of PD-L1 or PD-L2 fused 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 by reference in their entireties. 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] It will be understood by one of skill in the art 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, the PD-1 / PD-L1 antagonist is a small molecule, nucleic acid, peptide, peptidomimetic, protein, carbohydrate, carbohydrate derivative, or glycopolymer. Exemplary small molecule PD-1 inhibitors are described in Zhan et al. (2016) Drug Discov Today 21(6):1027-1036.

[0315] In some embodiments of the methods provided by the present disclosure, the RLR agonist is combined with a PD-1 / PD-L1 antagonist, and 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.

[0316] Combination with TIM-3 antagonists In some embodiments, the RLR agonist or pharmaceutical composition thereof provided by the present disclosure is combined with (e.g., administered in combination with) a TIM-3 antagonist. The TIM-3 antagonist may be an antibody, an antigen-binding fragment thereof, 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 antagonists In some embodiments, the RLR agonist or pharmaceutical composition provided by the present disclosure is combined (e.g., administered in combination) with a LAG-3 antagonist. The LAG-3 antagonist may be an antibody, an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an 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, an RLR agonist or pharmaceutical composition thereof provided by the present disclosure is combined (eg, administered in combination) with a TLR antagonist.

[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 endotoxin (TLR-4), flagellin protein (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. Although all TLRs can activate specific intracellular signaling molecules, such as nuclear factor kappa beta (NF-κB) and mitogen-activated protein kinases (MAP kinases), the specific set of cytokines and chemokines released appears to be unique for each TLR. TLRs 7, 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 TLRs 7 and 9, which are highly expressed in plasmacytoid dendritic cells (pDCs), TLR8 is primarily expressed in myeloid DCs (mDCs) and monocytes. This subfamily mediates the recognition of microbial nucleic acids, such as single-stranded RNA.

[0320] Small, low-molecular-weight (<400 daltons) synthetic imidazoquinoline compounds, which resemble 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, the TLR7 agonist imiquimod (ALDARA™) 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, keratoacanthoma, actinic keratosis, and Bowen's disease. The TLR7 / 8 agonist resiquimod (R-848) is being evaluated as a topical agent for the treatment of human genital herpes.

[0321] The TLR agonist of the present disclosure may be any TLR agonist. For example, the TLR agonist may include a natural or synthetic TLR ligand, a mutein or derivative of a TLR ligand, a peptide mimetic of a TLR ligand, a small molecule that mimics the biological function of a TLR ligand, or an antibody that stimulates a TLR receptor. A TLR ligand is any molecule that binds to a TLR.

[0322] In some embodiments, an RLR agonist or pharmaceutical composition thereof provided by the present disclosure is combined with a TLR agonist, wherein the TLR agonist is selected from the group consisting of a TLR1 agonist, a TLR2 agonist, a TLR3 agonist, a TLR4 agonist, a TLR5 agonist, a TLR6 agonist, a TLR7 agonist, a TLR8 agonist, a TLR9 agonist, a TLR10 agonist, and a TLR11 agonist.

[0323] In some embodiments, the RLR agonist provided by the present disclosure is combined with a TLR3 agonist. A TLR3 agonist is an agonist that triggers a signaling response through TLR3. Exemplary TLR3 agonists include, but are not limited to, polyinosinic acid: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, an RLR agonist provided by the present disclosure is combined with polyinosinic:polycytidylic acid (poly I:C). In some embodiments, an RLR agonist is combined with HILTONOL® (poly ICLC). In some embodiments, an RLR agonist is combined with polyadenylic-polyuridylic acid (poly A:U). In some embodiments, an RLR agonist is combined with RIBOXXIM® (RGIC® 100). In some embodiments, an RLR agonist is combined with RIBOXXON® (RGIC® 50 bioconjugate). In some embodiments, an RLR agonist is combined with RIBOXXOL® (RGIC® 50).

[0325] In some embodiments, the RLR agonist provided by the present disclosure is combined with a TLR7 agonist. A TLR7 agonist is an agonist that causes a signaling response through TLR7. Non-limiting examples of TLR7 agonists include single-stranded RNA (ssRNA), loxoribine (a guanosine analog derivatized at N7 and C8 positions), imidazoquinoline compounds (e.g., imiquimod and resiquimod), or derivatives thereof. Further exemplary TLR7 agonists include, but are not limited to, GS-9620 (vesatolimod), imiquimod (ALDARA®) and resiquimod (R-848).

[0326] In some embodiments, an RLR agonist provided by the present disclosure is combined with GS-9620 (vesatolimod). In some embodiments, an RLR agonist provided by the present disclosure is combined with imiquimod (ALDARA™). In some embodiments, an RLR agonist is combined with resiquimod (R-848).

[0327] In some embodiments, the RLR agonist provided by the present disclosure is combined with a TLR9 agonist. The TLR9 agonist is an agonist that induces a signaling response through TLR9. Exemplary TLR9 agonists include, but are not limited to, CpG oligodeoxynucleotides (GpG ODN). In some embodiments, the CpG ODN is a class A CpG ODN (CpG-A ODN), a class B CpG ODN (CpG-B ODN), or a class C CpG ODN (CpG-C ODN).

[0328] In some embodiments, the RLR agonist provided by the present disclosure is combined with 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).

[0329] Other combinations In some embodiments, an RLR agonist or pharmaceutical composition thereof provided by the present disclosure is combined (e.g., administered in combination) with 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, or a KIR antagonist.

[0330] In some embodiments, an RLR agonist or pharmaceutical composition thereof provided by the present disclosure is combined (e.g., administered in combination) with an agonist comprising a polypeptide (e.g., an antibody, or antigen-binding portion thereof) that specifically binds to CD137 (4-1BB).

[0331] In some embodiments, an RLR agonist or pharmaceutical composition thereof provided by the present disclosure is combined (e.g., administered in combination) with an agonist comprising a polypeptide (e.g., an antibody, or an antigen-binding portion thereof) that specifically binds to CD134 (OX40).

[0332] The RLR agonist described herein can replace or strengthen previous or current therapy.For example, when treating with RLR agonist, the administration of one or more additional active agents can be stopped or reduced, for example, administered at a lower level or dosage.In some embodiments, the administration of previous therapy can be maintained.In some embodiments, the previous therapy is maintained until the level of RLR agonist reaches a level sufficient to provide therapeutic effect.Two therapies can also be administered in combination.

[0333] Monitoring a subject (e.g., a human patient) for cancer improvement, as defined herein, means evaluating the subject for changes in disease parameters, e.g., a decrease in tumor growth. In some embodiments, the evaluation is performed at least 1 hour, e.g., at least 2, 4, 6, 8, 12, 24, or 48 hours after administration, or at least 1, 2, 4, 10, 13, 20 days, or longer, or at least 1, 2, 4, 10, 13, 20 weeks, or longer. The subject can be evaluated at one or more of the following time periods: before treatment begins, during treatment, or after one or more elements of the treatment are administered. Evaluation may include assessing the need for further treatment, e.g., whether the dosage, frequency, or duration of treatment should be changed. It may also include assessing the need to add or remove a selected therapy, e.g., adding or removing any of the cancer treatments described herein.

[0334] In some embodiments, the RLR agonists described herein are administered to regulate T cell responses in patients, for example, by increasing T cell activation and / or proliferation. Improving T cell proliferation, IFN production and secretion, and / or T cell cytolytic activity can be beneficial to patients in need of such enhancement for the treatment of certain diseases or conditions. Thus, in some embodiments, the RLR agonists of the present disclosure are administered to patients in need thereof 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 RLRs in biological samples. Thus, the RLR agonists, as described herein, are used to determine the diagnosis, prognosis, and / or progression of a disease (e.g., cancer) in a patient.

[0336] While the present disclosure has been described with reference to specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the present disclosure. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present disclosure. All such modifications are intended to be within the scope of the present disclosure. [Example]

[0337] The present disclosure will be more fully understood by reference to the following examples. However, the following examples should not be construed as limiting the scope of the disclosure. It is understood that the examples and embodiments described herein are merely illustrative, and that various modifications or changes in this respect will be suggested to those skilled in the art and are within the spirit and scope of the present application and the appended claims.

[0338] Example 1: Transfection of HuPBMCs with RLR agonists induces cytokine production in vitro. To determine the effect of RLR agonists with various modifications on cytokine induction, the ability of RLR agonists to induce cytokine production was assessed in vitro. Human peripheral blood mononuclear cells (huPBMCs) were prepared from two healthy donors and plated at 2 × 10 cells / well in a standard 96-well tissue culture plate in 100 μL of RPMI 1640 cell medium supplemented with fetal calf serum (FCS), L-glutamine, and penicillin / streptavidin. 5Cells were seeded at a density of 1000 cells / well. As shown in Figure 1, independent transfections of huPBMCs with RLR agonists were 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 24 h of incubation at 37 °C in a humidified incubator, the cell culture supernatants were collected. The supernatants were immediately frozen and stored at -20 °C. Once thawed, samples were analyzed for the cytokines IFN-α2a (Figure 1), as well as IL-1β, IP-10, IL-6, IL-12p70, MCP-1, and MIP-1β (data not shown) using the U-Plex MSD platform according to the manufacturer's instructions. Figure 1 shows the 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. The amount of IFN-α2a released by cells in response to transfection of RLR agonists is expressed in pg / mL.

[0339] Tables 3 and 4 show the sequences of each RLR agonist. Table 3 also shows the sequence and number 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 connected via the linker "UUCG" to a second oligonucleotide containing SEQ ID NO: 73, and has a 5' diphosphate moiety. The sequence of RIG7 is also shown in Table 4 as sequence 6. [Table 3-1] [Table 3-2] [Table 4-1] [Table 4-2]

Table 4-3

Claims

[Claim 1] The invention described in this specification.

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