Synthetic rig-i-like receptor agonists

Abstract

The present disclosure relates, inter alia, to RNA molecules (e.g., RNA hairpin agonists) that bind to and agonize RIG-I-like receptor (RLR), and to the use of the molecules in methods for treating a condition (e.g., cancer) or ameliorating one or more symptoms of the condition.

Description

[0001] This application is a divisional application of the invention patent application with the application date of April 19, 2019, application number 201980026849.7, and invention name “Synthetic RIG-I-like receptor agonist”.

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 659,999, filed on April 19, 2018. The entire contents of the above application are incorporated herein by this reference. Background Art

[0003] Exogenous nucleic acids (especially viral nucleic acids) introduced into cells induce innate immune responses, leading to events such as interferon (IFN) production and cell death. Upon sensing viral RNA, RIG-I-like receptors induce type I interferon (IFN) secretion, resulting in upregulation of antiviral IFN-induced proteins in infected cells and adjacent cells, which inhibits viral replication. Additional 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.

[0004] Still need additional and improved compositions and methods to modulate the activity of immunomodulatory proteins. Such agents can be used for cancer immunotherapy and the treatment of other conditions (such as chronic infections). Need to develop improved RIG-I-like receptor ligands for a variety of therapeutic immunomodulatory applications. Summary of the invention

[0005] The present disclosure is based, at least in part, on the discovery of synthetic RNA molecules that act as RIG-I-like receptor agonists.

[0006] In some aspects, 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, the blunt-ended hairpin RNA comprises a first polynucleotide connected to a second polynucleotide by a joint, wherein the first polynucleotide is fully complementary to the second polynucleotide to form a duplex, wherein the duplex comprises less than 19 base pairs, wherein the nucleotide closest to the 5' of the first oligonucleotide comprises a 5' diphosphate or triphosphate portion or a derivative or analog thereof, and wherein the agonist comprises a sequence motif that provides at least one increased biological activity mediated by the RLR relative to an agonist that does not comprise the sequence motif. In some embodiments, the first polynucleotide comprises the sequence motif.

[0007] In some embodiments, the RLR agonists of the present disclosure comprise a sequence motif selected from the group consisting of:

[0008] (i) GT repeat motif;

[0009] (ii) GA repeat motif;

[0010] (iii) AUCG repeat motif;

[0011] (iv) AU repeat motif;

[0012] (v) dipyrimidine motif;

[0013] (vi) dipurine motif;

[0014] (vii) pyrimidine triplet motif;

[0015] (viii) purine triad motif;

[0016] (ix) a palindromic sequence motif; and

[0017] (x) A combination of any one of (i) to (ix).

[0018] In some embodiments, the RLR agonists disclosed herein include 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.

[0019] In some embodiments, the RLR agonists of the present disclosure include a sequence motif that provides at least one increased biological activity mediated by the RLR relative to an agonist that does not include the sequence motif, wherein the at least one increased biological activity is selected from:

[0020] (i) RLR-mediated increase in cytokine production;

[0021] (ii) increase in RLR-mediated expression of interferon-stimulated genes;

[0022] (iii) increase in RLR-mediated intracellular signaling;

[0023] (iv) increase in binding affinity to RLR; and

[0024] (v) A combination of any of (i) to (iv).

[0025] In some embodiments, the RLR agonist of the present invention includes the sequence motif that increases the production of type I interferon (e.g., IFN-α, IFN-β) mediated by RLR relative to an agonist that does not include a sequence motif. In some embodiments, the RLR agonist of the present invention includes the sequence motif that increases the production of IL-1β mediated by RLR relative to an agonist that does not include a sequence motif. In some embodiments, the RLR agonist of the present invention includes the sequence motif that increases the production of IP-10 mediated by RLR relative to an agonist that does not include a sequence motif. In some embodiments, the RLR agonist of the present invention includes the sequence motif that increases the production of IL-6, IL-12p70, MCP-1 and / or MIP-1β mediated by RLR relative to an agonist that does not include a sequence motif.

[0026] In some embodiments, the RLR agonists of the present disclosure include sequence motifs, wherein the sequence motifs are GT-repeat motifs (e.g., GTGTGT), the GT-repeat motifs include <19, about 15 to 18, about 15, about 10 to 15, about 10, about 5 to 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 their derivatives or analogs. In some embodiments, the sequence motifs are GT-repeat motifs, the GT-repeat motifs include <19 guanine and thymine nucleotides or their derivatives or analogs. In some embodiments, the sequence motifs are GT-repeat motifs, the GT-repeat motifs include about 15 to 18 guanine and thymine nucleotides or their derivatives or analogs. 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 about 10 to 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 to 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 aspects, the GT repeat motif provides enhanced biological activity in the RLR agonist, wherein the enhanced biological activity is an increase in RLR-mediated cytokine production; an increase in RLR-mediated expression of interferon-stimulated genes; an increase in RLR-mediated intracellular signaling; an increase in binding affinity to RLR; and a combination of any of the foregoing.

[0027] In some embodiments, the RLR agonist of the present disclosure comprises a sequence motif, wherein the sequence motif is a GT-repeat motif, and the GT-repeat motif comprises a sequence of 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 or 4 guanine and thymine nucleotides or their derivatives or analogs. In some embodiments, the sequence motif is a GT-repeat motif, and the GT-repeat motif comprises a sequence of 18 guanine and thymine nucleotides or their derivatives or analogs. In some embodiments, the sequence motif is a GT-repeat motif, and the GT-repeat motif comprises a sequence of 16 guanine and thymine nucleotides or their derivatives or analogs. In some embodiments, the sequence motif is a GT-repeat motif, and the GT-repeat motif comprises a sequence of 14 guanine and thymine nucleotides or their derivatives or analogs. In some embodiments, the sequence motif is a GT-repeat motif, and the GT-repeat motif includes a sequence of 12 guanine and thymine nucleotides or their derivatives or analogs. In some embodiments, the sequence motif is a GT-repeat motif, and the GT-repeat motif includes a sequence of 10 guanine and thymine nucleotides or their derivatives or analogs. In some embodiments, the sequence motif is a GT-repeat motif, and the GT-repeat motif includes a sequence of 8 guanine and thymine nucleotides or their derivatives or analogs. In some embodiments, the sequence motif is a GT-repeat motif, and the GT-repeat motif includes a sequence of 6 guanine and thymine nucleotides or their derivatives or analogs. In some embodiments, the sequence motif is a GT-repeat motif, and the GT-repeat motif includes a sequence of 4 guanine and thymine nucleotides or their derivatives or analogs. In some embodiments, the RLR agonist of the present disclosure comprises a sequence motif, wherein the sequence motif is a GT-repeat motif, wherein the GT repeat motif is [GT] n , wherein n=2 to 9, 3 to 7 or 4 to 8. In some aspects, the GT repeat motif provides an increased biological activity in the RLR agonist, wherein the increased biological activity is an increase in RLR-mediated cytokine production; an increase in RLR-mediated expression of interferon-stimulated genes; an increase in RLR-mediated intracellular signaling; an increase in binding affinity to RLR; and a combination of any of the foregoing.

[0028] In some aspects, the present disclosure provides a RLR agonist that specifically binds to RLR, wherein the agonist comprises a blunt-ended hairpin RNA, the blunt-ended hairpin RNA comprising a first polynucleotide connected (operably 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 less than 19 base pairs, wherein the nucleotide closest to 5' of the first oligonucleotide comprises a 5' diphosphate or triphosphate portion or a derivative or analog thereof, wherein the agonist comprises a sequence motif that provides at least one improved biological activity mediated by the RLR relative to an agonist that does not include the sequence motif, wherein the first polynucleotide comprises the sequence motif, and wherein the sequence motif is a GT repeat motif, wherein the GT repeat motif comprises a sequence of about 14 guanine and thymine nucleotides. In some embodiments, the sequence motif is a GT repeat motif, wherein the GT repeat motif is [GT]7. In some aspects, the increased biological activity is an increase in RLR-mediated cytokine production; an increase in RLR-mediated expression of interferon-stimulated genes; an increase in RLR-mediated intracellular signaling; an increase in binding affinity to RLR; and a combination of any of the foregoing.

[0029] In some aspects, the present disclosure provides a RLR agonist that specifically binds to RLR, wherein the agonist comprises a blunt-ended hairpin RNA, the 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 less than 19 base pairs, wherein the nucleotide closest to 5' of the first oligonucleotide comprises a 5' diphosphate or triphosphate portion or a derivative or analog thereof, wherein the agonist comprises a sequence motif that provides at least one improved biological activity mediated by the RLR relative to an agonist that does not include the sequence motif, wherein the first polynucleotide comprises the sequence motif, and wherein the sequence motif is a GT repeat motif, wherein the GT repeat motif comprises a sequence of 6 guanine and thymine nucleotides. In some embodiments, the sequence motif is a GT repeat motif, wherein the GT repeat motif is [GT]3. In some embodiments, the sequence motif is a GT repeat motif, wherein the GT repeat motif is [GT]3, and wherein the GT repeat sequence is followed by a purine triplet and UCG, respectively. In some embodiments, the purine triplet is GGA. In some aspects, the improved biological activity is an increase in RLR-mediated cytokine production; an increase in RLR-mediated expression of interferon-stimulated genes; an increase in RLR-mediated intracellular signaling; an increase in binding affinity to RLR; and a combination of any of the foregoing.

[0030] In some embodiments, the RLR agonists of the present disclosure include a sequence motif, wherein the sequence motif is a GA repeat motif (e.g., GAGAGA), the GA repeat motif includes a sequence of <19, about 15 to 18, about 15, about 10 to 15, about 10, about 5 to 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, the GA repeat motif includes a sequence of <19 guanine and adenine nucleotides or derivatives or analogs thereof. In some embodiments, the sequence motif is a GA repeat motif, the GA repeat motif includes a sequence of about 15 to 18 guanine and adenine nucleotides or derivatives or analogs thereof. In some embodiments, the sequence motif is a GA repeating motif, and the GA repeating motif includes a sequence of about 15 guanine and adenine nucleotides or their derivatives or analogs. In some embodiments, the sequence motif is a GA repeating motif, and the GA repeating motif includes a sequence of about 10 to 15 guanine and adenine nucleotides or their derivatives or analogs. In some embodiments, the sequence motif is a GA repeating motif, and the GA repeating motif includes a sequence of about 10 guanine and adenine nucleotides or their derivatives or analogs. In some embodiments, the sequence motif is a GA repeating motif, and the GA repeating motif includes a sequence of about 5 to 10 guanine and adenine nucleotides or their derivatives or analogs. In some embodiments, the sequence motif is a GA repeating motif, and the GA repeating motif includes a sequence of about 5 guanine and adenine nucleotides or their derivatives or analogs. In some embodiments, the sequence motif is a GA repeating motif, and the GA repeating motif includes a sequence of about 4 guanine and adenine nucleotides or their derivatives or analogs. In some aspects, the GA repeat motif provides enhanced biological activity in the RLR agonist, wherein the enhanced biological activity is an increase in RLR-mediated cytokine production; an increase in RLR-mediated expression of interferon-stimulated genes; an increase in RLR-mediated intracellular signaling; an increase in binding affinity to RLR; and a combination of any of the foregoing.

[0031] In some embodiments, the RLR agonists of the present disclosure include a sequence motif, the sequence motif is a GA repeat motif, and the GA repeat motif includes 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, and the GA repeat motif includes a sequence of 18 guanine and adenine nucleotides or derivatives or analogs thereof. In some embodiments, the sequence motif is a GA repeat motif, and the GA repeat motif includes a sequence of 16 guanine and adenine nucleotides or derivatives or analogs thereof. In some embodiments, the sequence motif is a GA repeat motif, and the GA repeat motif includes a sequence of 14 guanine and adenine nucleotides or derivatives or analogs thereof. In some embodiments, the sequence motif is a GA repeat motif, and the GA repeat motif includes a sequence of 12 guanine and adenine nucleotides or derivatives or analogs thereof. In some embodiments, the sequence motif is a GA repeat motif, and the GA repeat motif includes a sequence of 8 guanine and adenine nucleotides or their derivatives or analogs. In some embodiments, the sequence motif is a GA repeat motif, and the GA repeat motif includes a sequence of 6 guanine and adenine nucleotides or their derivatives or analogs. In some embodiments, the sequence motif is a GA repeat motif, and the GA repeat motif includes a sequence of 4 guanine and adenine nucleotides or their derivatives or analogs.

[0032] In some embodiments, the RLR agonist of the present disclosure comprises a sequence motif, wherein the sequence motif is a GA repeat motif, wherein the GA repeat motif is [GA] n , wherein n=2 to 9, 3 to 7 or 4 to 8. In some aspects, the GA repeat motif provides an increased biological activity in the RLR agonist, wherein the increased biological activity is an increase in RLR-mediated cytokine production; an increase in RLR-mediated expression of interferon-stimulated genes; an increase in RLR-mediated intracellular signaling; an increase in binding affinity to RLR; and a combination of any of the foregoing.

[0033] In some aspects, the present disclosure provides a RLR agonist that specifically binds to RLR, wherein the agonist comprises a blunt-ended hairpin RNA, the blunt-ended hairpin RNA comprises a first polynucleotide connected to a second polynucleotide by a linker, wherein the first polynucleotide is fully complementary to the second polynucleotide to form a duplex, wherein the duplex comprises less than 19 base pairs, wherein the nucleotide closest to 5' 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 provides at least one improved biological activity mediated by the RLR relative to an agonist that does not comprise the sequence motif, wherein the first polynucleotide comprises the sequence motif, and wherein the sequence motif is a GA repeat motif, wherein the GA repeat motif comprises a sequence of about 14 guanine and adenine nucleotides. In some embodiments, the sequence motif is a GA repeat motif, wherein the GA repeat motif is [GA]7. In some aspects, the GA repeat motif provides enhanced biological activity in the RLR agonist, wherein the enhanced biological activity is an increase in RLR-mediated cytokine production; an increase in RLR-mediated expression of interferon-stimulated genes; an increase in RLR-mediated intracellular signaling; an increase in binding affinity to RLR; and a combination of any of the foregoing.

[0034] In some embodiments, the RLR agonists of the present disclosure include a sequence motif, wherein the sequence motif is an AUCG repeat motif (e.g., AUCGAUCG), and the AUCG repeat motif includes a sequence of <19, about 16, about 12 to about 16, about 12, about 8 to 12, about 6, about 16, 12, 8 adenine, uracil, cytosine and guanine nucleotides or derivatives or analogs thereof. In some embodiments, the sequence motif is an AUCG repeat motif, and the AUCG repeat motif includes a sequence of <19 adenine, uracil, cytosine and guanine nucleotides or derivatives or analogs thereof. In some embodiments, the sequence motif is an AUCG repeat motif, and the AUCG repeat motif includes 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, and the AUCG repeat motif includes a sequence of about 12 to 16 adenine, uracil, cytosine and guanine nucleotides or their derivatives or analogs. In some embodiments, the sequence motif is an AUCG repeat motif, and the AUCG repeat motif includes a sequence of about 12 adenine, uracil, cytosine and guanine nucleotides or their derivatives or analogs. In some embodiments, the sequence motif is an AUCG repeat motif, and the AUCG repeat motif includes a sequence of about 8 to 12 adenine, uracil, cytosine and guanine nucleotides or their derivatives or analogs. In some embodiments, the sequence motif is an AUCG repeat motif, and the AUCG repeat motif includes a sequence of about 6 adenine, uracil, cytosine and guanine nucleotides or their derivatives or analogs. In some embodiments, the sequence motif is an AUCG repeat motif, and the AUCG repeat motif includes a sequence of 16 adenine, uracil, cytosine and guanine nucleotides or their derivatives or analogs. In some embodiments, the sequence motif is an AUCG repeat motif, and the AUCG repeat motif includes a sequence of 12 adenine, uracil, cytosine and guanine nucleotides or their derivatives or analogs. In some embodiments, the sequence motif is an AUCG repeat motif, and the AUCG repeat motif includes a sequence of 8 adenine, uracil, cytosine and guanine nucleotides or their derivatives or analogs. In some aspects, the AUCG repeat motif provides an improved biological activity in the RLR agonist, wherein the improved biological activity is an increase in RLR-mediated cytokine production; an increase in RLR-mediated expression of interferon-stimulated genes; an increase in RLR-mediated intracellular signaling; an increase in binding affinity to RLR; and a combination of any of the foregoing.

[0035] In some embodiments, the RLR agonist of the present disclosure comprises a sequence motif, wherein the sequence motif is an AUCG repeat motif, wherein the AUCG repeat motif is [AUCG] n , wherein n=2 to 4 or 2, 3 or 4. In some aspects, the AUCG repeat motif provides an increased biological activity in the RLR agonist, wherein the increased biological activity is an increase in RLR-mediated cytokine production; an increase in RLR-mediated expression of interferon-stimulated genes; an increase in RLR-mediated intracellular signaling; an increase in binding affinity to RLR; and a combination of any of the foregoing.

[0036] In some aspects, the present disclosure provides a RLR agonist that specifically binds to RLR, wherein the agonist comprises a blunt-ended hairpin RNA, the blunt-ended hairpin RNA comprises 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 less than 19 base pairs, wherein the nucleotide closest to 5' 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 provides at least one increased biological activity mediated by the RLR relative to an agonist that does not comprise the sequence motif, wherein the first polynucleotide comprises the sequence motif, and wherein the sequence motif is an AUCG repeat motif that comprises a sequence of about 12 guanine and adenine nucleotides. In some embodiments, the AUCG repeat motif is [AUCG]3. In some aspects, the AUCG repeat motif provides enhanced biological activity in the RLR agonist, wherein the enhanced biological activity is an increase in RLR-mediated cytokine production; an increase in RLR-mediated expression of interferon-stimulated genes; an increase in RLR-mediated intracellular signaling; an increase in binding affinity to RLR; and a combination of any of the foregoing.

[0037] In some embodiments, the RLR agonists of the present disclosure include an AUCG repeat motif, wherein the motif is preceded by a CG or a dipyrimidine motif. In some embodiments, the AUCG repeat motif is preceded by a CG. In some embodiments, the AUCG repeat motif is [AUCG]3 and is preceded by a CG. In some embodiments, the AUCG repeat motif is [AUCG]3 and is preceded by the dipyrimidine motif CC.

[0038] In some embodiments, the RLR agonists of the present disclosure include an AUCG repeating motif, wherein the motif is preceded by a dipurine motif. In some embodiments, the dipurine motif is GA. In some embodiments, the AUCG repeating motif is [AUCG]3 and is preceded by the dipurine motif GA. In some embodiments, the AUCG repeating motif is preceded by the dipurine motif II.

[0039] In some embodiments, the RLR agonist of the present disclosure includes an AUCG repeating motif, wherein one or more uridine nucleosides (U) are replaced by modified nucleosides. In some embodiments, wherein the modified nucleosides are ribothymidine (T). In some embodiments, the AUGC repeating motif is [AUCG]3, wherein the one or more uridine nucleosides (U) including the AUGC repeating motif are replaced by modified nucleosides, wherein the modified nucleosides are ribothymidine (T). In some embodiments, the AUGC repeating motif is [AUCG]3, wherein the one or more uridine nucleosides (U) including the AUCG repeating motif are replaced by modified nucleosides, wherein the modified nucleosides are ribothymidine (T), and wherein the AUGC repeating motif is preceded by GG.

[0040] In some embodiments, the RLR agonist of the present disclosure includes an AUCG repeating motif, wherein one or more guanosine nucleosides (G) are replaced by modified nucleosides. In some embodiments, the modified nucleosides are inosine (I). In some embodiments, the AUGC repeating motif is [AUCG]3, wherein the one or more guanosine nucleosides (G) including the AUCG repeating motif are replaced by modified nucleosides, wherein the modified nucleosides are ribothymidine (T), and wherein the AUGC repeating motif is preceded by GG.

[0041] In some embodiments, the RLR agonists of the present disclosure include an AUCG repeat motif, wherein the motif is preceded by IG. In some embodiments, the AUCG repeat motif is [AUCG]3 and is preceded by IG.

[0042] In some embodiments, the RLR agonists of the present disclosure include AUCG repeats, wherein one or more guanosine nucleosides (G) are replaced by inosine (I), wherein the AUCG repeats are preceded by inosine (I). In some embodiments, the guanosine nucleosides (G) comprising the AUCG repeats are replaced by inosine (I), wherein the AUCG repeats are preceded by inosine (I), wherein the nucleotide closest to 5' of the first polynucleotide includes inosine (I).

[0043] In some embodiments, the RLR agonists of the present disclosure include 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, wherein the AUCG repeat motif is preceded by a dipurine motif, and wherein the dipurine motif is GG.

[0044] In some embodiments, the RLR agonist of the present disclosure comprises an AUCG repeat motif, wherein the AUCG repeat motif is [AUCG]2, and wherein the AUCG repeat motif is preceded by a purine triplet motif. In some embodiments, the purine triplet motif is GGG. In some embodiments, the sequence motif is an AUCG repeat motif, wherein the AUCG repeat motif is [AUCG]2, wherein the AUCG repeat motif is preceded by a purine triplet motif, and wherein the purine triplet motif is GGG. 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 CCCCCG. 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 TCGUCG.

[0045] In some embodiments, the RLR agonist of the present disclosure includes a sequence motif, wherein the sequence motif is a palindromic sequence, and the palindromic sequence includes a sequence of <19, about 15 to 18, about 15, about 10 to 15, about 10, about 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 or 4 nucleotides or their derivatives or analogs connected in any order to produce a palindrome. In some embodiments, the sequence motif is a palindromic sequence, and the palindromic sequence includes a sequence of <19 nucleotides or their derivatives or analogs connected in any order to produce a palindrome. In some embodiments, the sequence motif is a palindromic sequence, and the palindromic sequence includes a sequence of about 15-18 nucleotides or their derivatives or analogs connected in any order to produce a palindrome. In some embodiments, the sequence motif is a palindromic sequence, and the palindromic sequence includes a sequence of about 15 nucleotides or their derivatives or analogs connected in any order to produce 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 connected in any order to produce a palindrome. In some embodiments, the sequence motif is a palindromic sequence, comprising a sequence of about 10 nucleotides or derivatives or analogs thereof connected in any order to produce a palindrome. In some embodiments, the sequence motif is a palindromic sequence, comprising a sequence of 18 nucleotides or derivatives or analogs thereof connected in any order to produce a palindrome. In some embodiments, the sequence motif is a palindromic sequence, comprising a sequence of 17 nucleotides or derivatives or analogs thereof connected in any order to produce a palindrome. In some embodiments, the sequence motif is a palindromic sequence, comprising a sequence of 16 nucleotides or derivatives or analogs thereof connected in any order to produce a palindrome. In some embodiments, the sequence motif is a palindromic sequence, comprising a sequence of 15 nucleotides or derivatives or analogs thereof connected in any order to produce a palindrome. In some embodiments, the sequence motif is a palindromic sequence, comprising a sequence of 14 nucleotides or derivatives or analogs thereof connected in any order to produce a palindrome. In some embodiments, the sequence motif is a palindromic sequence, comprising a sequence of 13 nucleotides or derivatives or analogs thereof connected in any order to produce a palindrome. In some embodiments, the sequence motif is a palindromic sequence, comprising a sequence of 12 nucleotides or derivatives or analogs thereof connected in any order to produce a palindrome. In some embodiments, the sequence motif is a palindromic sequence, comprising a sequence of 11 nucleotides or derivatives or analogs thereof connected in any order to produce a palindrome.In some embodiments, the sequence motif is a palindromic sequence, comprising a sequence of 10 nucleotides or derivatives or analogs thereof connected in any order to produce a palindrome. In some embodiments, the sequence motif is a palindromic sequence, comprising a sequence of 9 nucleotides or derivatives or analogs thereof connected in any order to produce a palindrome. In some embodiments, the sequence motif is a palindromic sequence, comprising a sequence of 8 nucleotides or derivatives or analogs thereof connected in any order to produce a palindrome. In some embodiments, the sequence motif is a palindromic sequence, comprising a sequence of 7 nucleotides or derivatives or analogs thereof connected in any order to produce a palindrome. In some embodiments, the sequence motif is a palindromic sequence, comprising a sequence of 6 nucleotides or derivatives or analogs thereof connected in any order to produce a palindrome. In some embodiments, the sequence motif is a palindromic sequence, comprising a sequence of 5 nucleotides or derivatives or analogs thereof connected in any order to produce a palindrome. In some embodiments, the sequence motif is a palindromic sequence, comprising a sequence of 4 nucleotides or derivatives or analogs thereof connected in any order to produce a palindrome.

[0046] In some embodiments, the RLR agonists of the present disclosure include a linker, wherein the linker is flanked by AU. In some embodiments, the linker is flanked by an AU repeat motif, wherein the AU repeat motif is [AU] n , wherein n=2 to 3. In some embodiments, the AU repeat motif is [AU]2.

[0047] In some aspects, the present disclosure provides an RLR agonist that specifically binds to RLR, wherein the agonist comprises a blunt-ended hairpin RNA, wherein the blunt-ended hairpin RNA comprises the formula:

[0048] 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', where

[0049] (i) (N1-N2-X1) comprises a first polynucleotide comprising linked nucleotides N1, N2 and X1;

[0050] (ii) (X2-N3-N4) comprises a second polynucleotide comprising linked nucleotides X2, N3 and N4;

[0051] (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;

[0052] (iv) N1 base pairs with N4;

[0053] (v) N2 base pairs with N3;

[0054] (vi) N1 comprises a 5' diphosphate or triphosphate moiety or a derivative or analog thereof;

[0055] (vii) X1 and X2 are each an oligonucleotide comprising a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine and inosine;

[0056] (viii) X1 and X2 are complementary;

[0057] (ix) X1 and X2 are each 12 nucleotides to 16 nucleotides in length and are the same length; and

[0058] (x) L is a linker that operably connects the first polynucleotide and the second polynucleotide,

[0059] Wherein at least one of N1, N2, N3 and N4 is inosine, and / or at least one of X1 and / or X2 includes at least one inosine nucleoside, and wherein the inosine nucleoside is paired with the cytidine base in the hairpin RNA. In some aspects, the RLR agonist of the present disclosure has an increased biological activity, wherein the increased biological activity is an increase in RLR-mediated cytokine production; an increase in RLR-mediated expression of interferon-stimulated genes; an increase in RLR-mediated intracellular signaling; an increase in binding affinity to RLR; and a combination of any of the foregoing.

[0060] In some aspects, the present disclosure provides an RLR agonist that specifically binds to RLR, wherein the agonist comprises a blunt-ended hairpin RNA, the blunt-ended hairpin RNA comprises a non-nucleotide linker, and wherein the agonist comprises the formula:

[0061] 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', where

[0062] (i) (N1-N2-X1) comprises a first polynucleotide comprising linked nucleotides N1, N2 and X1;

[0063] (ii) (X2-N3-N4) comprises a second polynucleotide comprising linked nucleotides X2, N3 and N4;

[0064] (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;

[0065] (iv) N1 base pairs with N4;

[0066] (v) N2 base pairs with N3;

[0067] (vi) N1 comprises a 5' diphosphate or triphosphate moiety or a derivative or analog thereof;

[0068] (vii) X1 and X2 are each an oligonucleotide comprising a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine and inosine;

[0069] (viii) X1 and X2 are complementary;

[0070] (ix) X1 and X2 are each 12 nucleotides to 16 nucleotides in length and are the same length; and

[0071] (x) L is a non-nucleotide linker that covalently links the first polynucleotide and the second polynucleotide,

[0072] Wherein, if present, inosine is base paired with cytidine. In some aspects, the RLR agonists of the present disclosure have an increased biological activity, wherein the increased biological activity is an increase in RLR-mediated cytokine production; an increase in RLR-mediated expression of interferon-stimulated genes; an increase in RLR-mediated intracellular signaling; an increase in binding affinity to RLR; and a combination of any of the foregoing.

[0073] In some embodiments, N1 includes inosine and N4 includes cytidine. In some embodiments, N1 includes inosine and N4 includes cytidine, and X1 and X2 are each 12 nucleotides in length. In some embodiments, N1 includes cytidine and N4 includes inosine. In some embodiments, N2 includes inosine and N3 includes cytidine. In some embodiments, N2 includes cytidine and N3 includes inosine. In some embodiments, N1 includes guanosine. In some embodiments, N2 includes guanosine. In some embodiments, N1 includes cytidine. In some embodiments, N2 includes cytidine. In some embodiments, N2 includes cytidine. In some embodiments, N1 and N2 include guanosine, and N3 and N4 include cytidine. In some embodiments, N1 and N2 include cytidine, and N3 and N4 include guanosine. In some embodiments, N1 and N2 include inosine, and N3 and N4 include cytidine. In some embodiments, N1 and N2 include cytidine, and N3 and N4 include guanosine. In some embodiments, N1 and N2 include inosine, and N3 and N4 include cytidine. In some embodiments, N1 and N2 include cytidine, and N3 and N4 include inosine.

[0074] In some embodiments, the RLR agonists of the present disclosure include the formula:

[0075] 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', where

[0076] (i) (N1-N2-X1) comprises a first polynucleotide comprising linked nucleotides N1, N2 and X1;

[0077] (ii) (X2-N3-N4) comprises a second polynucleotide comprising linked nucleotides X2, N3 and N4;

[0078] (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;

[0079] (iv) N1 base pairs with N4;

[0080] (v) N2 base pairs with N3;

[0081] (vi) N1 comprises a 5' diphosphate or triphosphate moiety or a derivative or analog thereof;

[0082] (vii) X1 and X2 are each an oligonucleotide comprising a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine and inosine;

[0083] (viii) X1 and X2 are complementary;

[0084] (ix) X1 and X2 are each 12 nucleotides to 16 nucleotides in length and are the same length; and

[0085] (x) L is a non-nucleotide linker that covalently links the first polynucleotide and the second polynucleotide,

[0086] Wherein, if present, inosine is base paired with cytidine, and wherein N1 comprises inosine, and N4 comprises cytidine, and X1 and / or X2 each comprises at least one inosine. In some embodiments, N2 comprises inosine, and N3 comprises cytidine, and X1 and / or X2 each comprises 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, and N3 and N4 comprise cytidine, and X1 and X2 each comprise at least one inosine. In some embodiments, N1 and N2 comprise guanosine, and N3 and N4 comprise cytidine, and X1 and X2 each comprise at least one inosine. In some embodiments, N1 and N2 comprise guanosine, and 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, and N3 and N4 comprise guanosine, and X1 and X2 each comprise at least one inosine. In some embodiments, N1 and N2 include guanosine, and N3 and N4 include cytidine, and X1 and X2 each include inosine and do not include guanosine nucleosides. In some embodiments, N1 and N2 include guanosine, and N3 and N4 include cytidine, X1 and X2 each include at least one inosine, and X1 and X2 are each 12 nucleotides in length. In some embodiments, N1 and N2 include cytidine, and N3 and N4 include guanosine, and X1 and X2 each include inosine and do not include guanosine nucleosides. In some aspects, the RLR agonist of the present disclosure has an increased biological activity, wherein the increased biological activity is an increase in RLR-mediated cytokine production; an increase in RLR-mediated expression of interferon-stimulated genes; an increase in RLR-mediated intracellular signaling; an increase in binding affinity to RLR; and a combination of any of the foregoing.

[0087] In some embodiments, the RLR agonists of the present disclosure include the formula:

[0088] 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', where

[0089] (i) (N1-N2-X1) comprises a first polynucleotide comprising linked nucleotides N1, N2 and X1;

[0090] (ii) (X2-N3-N4) comprises a second polynucleotide comprising linked nucleotides X2, N3 and N4;

[0091] (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;

[0092] (iv) N1 base pairs with N4;

[0093] (v) N2 base pairs with N3;

[0094] (vi) N1 comprises a 5' diphosphate or triphosphate moiety or a derivative or analog thereof;

[0095] (vii) X1 and X2 are each an oligonucleotide comprising a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine and inosine;

[0096] (viii) X1 and X2 are complementary;

[0097] (ix) X1 and X2 are each 12 nucleotides to 16 nucleotides in length and are the same length; and

[0098] (x) L is a non-nucleotide linker that covalently links the first polynucleotide and the second polynucleotide,

[0099] Wherein, if present, inosine and cytidine base pair, and wherein N1 and N2 include inosine, and N3 and N4 include cytidine, and X1 and / or X2 each include at least one inosine. In some embodiments, N1 and N2 include inosine, and N3 and N4 include cytidine, and X1 and X2 each include at least one inosine, and X1 and X2 are each 12 nucleotides in length. In some embodiments, N1 and N2 include inosine, and N3 and N4 include cytidine, and X1 and X2 each include at least one inosine. In some embodiments, N1 and N2 include inosine, and N3 and N4 include cytidine, and X1 and X2 each include at least one inosine, and X1 and X2 are each 12 nucleotides in length. In some embodiments, N1 and N2 include cytidine, and N3 and N4 include inosine, and X1 and X2 each include at least one inosine, and X1 and X2 are each 12 nucleotides in length. In some embodiments, N1 and N2 include inosine, and N3 and N4 include cytidine, and X1 and X2 include inosine and do not include guanosine nucleosides. In some embodiments, N1 and N2 include cytidine, and N3 and N4 include inosine, and X1 and X2 include inosine and do not include guanosine nucleosides. In some aspects, the RLR agonists of the present disclosure have improved biological activity, wherein the improved biological activity is an increase in RLR-mediated cytokine production; an increase in RLR-mediated expression of interferon-stimulated genes; an increase in RLR-mediated intracellular signaling; an increase in binding affinity to RLR; and a combination of any of the foregoing.

[0100] In some embodiments, the RLR agonists of the present disclosure include the formula:

[0101] 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', wherein X1 and X2 are each 12 nucleotides and include 1, 2, 3 or 4 inosine nucleosides. In certain embodiments, X1 and X2 are each 13 nucleotides and include 1, 2, 3, 4 or 5 inosine nucleosides. In certain embodiments, X1 and X2 are each 14 nucleotides and include 1, 2, 3, 4, 5 or 6 inosine nucleosides. In certain embodiments, X1 and X2 are each 15 nucleotides and include 1, 2, 3, 4, 5, 6 or 7 inosine nucleosides. In certain embodiments, X1 and X2 are each 16 nucleotides and include 1, 2, 3, 4, 5, 6, 7 or 8 inosine nucleosides. In some embodiments, X1 and X2 are each 12 nucleotides and include at least 10%, 20%, 30% or 40% inosine nucleosides. In some aspects, the RLR agonists of the present disclosure have an increased biological activity, wherein the increased biological activity is an increase in RLR-mediated cytokine production; an increase in RLR-mediated expression of interferon-stimulated genes; an increase in RLR-mediated intracellular signaling; an increase in binding affinity to RLR; and a combination of any of the foregoing.

[0102] In some aspects, the present disclosure provides an RLR agonist that specifically binds to RLR, wherein the agonist comprises a blunt-ended hairpin RNA, wherein the blunt-ended hairpin RNA comprises the formula:

[0103] 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', where

[0104] (i) (N1-N2-X1) comprises a first polynucleotide comprising linked nucleotides N1, N2 and X1;

[0105] (ii) (X2-N3-N4) comprises a second polynucleotide comprising linked nucleotides X2, N3 and N4;

[0106] (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;

[0107] (iv) N1 base pairs with N4;

[0108] (v) N2 base pairs with N3;

[0109] (vi) N1 comprises a 5' diphosphate or triphosphate moiety or a derivative or analog thereof;

[0110] (vii) X1 includes the sequence motif [AUCN5] x , wherein N5 is comprised of guanosine or inosine, wherein x is an integer whose value indicates the number of sequence motifs, and wherein x=3 or 4;

[0111] (viii) X2 includes the sequence motif [CN6AU] y , wherein N6 comprises guanosine or inosine, wherein y is an integer whose value indicates the number of sequence motifs, and wherein y=3 or 4;

[0112] (ix) L is a linker that operably connects the first polynucleotide and the second polynucleotide,

[0113] Optionally wherein at least one of N1, N2, N3 and N4 is inosine, and wherein the inosine nucleoside base pairs with a cytidine in the hairpin RNA.

[0114] In some embodiments, N5 includes inosine and N6 includes inosine. In some embodiments, N5 includes guanosine and N6 includes inosine. In some embodiments, N5 includes inosine and N6 includes guanosine. In some embodiments, N5 includes guanosine (G) and N6 includes guanosine (G). In some embodiments, x=3 and y=3. In some embodiments, x=4 and y=4. In some embodiments, N1 includes inosine (I) and N4 includes cytidine (C). In some embodiments, N2 includes inosine (I) and N3 includes cytidine (C). In some embodiments, N3 includes inosine (I) and N2 includes cytidine (C). In some embodiments, N4 includes inosine (I) and N1 includes cytidine (C). In some embodiments, N1 includes guanosine (G). In some embodiments, N2 includes guanosine (G). In some embodiments, N1 includes cytidine (C). In some embodiments, N2 includes cytidine (C). In some embodiments, N1 and N2 include guanosine (G) and N3 and N4 include cytidine (C). In some embodiments, N1 and N2 include cytidine (C), and N3 and N4 include guanosine (G). In some embodiments, N1 and N2 include inosine (I), and N3 and N4 include cytidine (C). In some embodiments, N1 and N2 include cytidine (C), and N3 and N4 include inosine (I). In some aspects, the RLR agonists of the present disclosure have improved biological activity, wherein the improved biological activity is an increase in RLR-mediated cytokine production; an increase in RLR-mediated expression of interferon-stimulated genes; an increase in RLR-mediated intracellular signaling; an increase in binding affinity to RLR; and a combination of any of the foregoing.

[0115] In some embodiments, the RLR agonist of the present disclosure comprises a linker, wherein 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 comprises a four-membered ring, wherein the nucleotide sequence of the four-membered ring is selected from the group consisting of:

[0116] (a) UNCG, where N = A, C, G or U;

[0117] (b) GNRA, wherein N = A, C, G or U, and wherein R = A or G;

[0118] (c) ANYA, wherein N = A, C, G or U, and wherein Y = C or T;

[0119] (d) CUYG, where Y = C or T;

[0120] (e) UMAC, where M = A or C; and

[0121] (f)CUUG.

[0122] In some embodiments, the sequence of the four-membered loop is UUCG. In some embodiments, the sequence of the four-membered loop is GAUC.

[0123] In some embodiments, the RLR agonist of the present disclosure includes a nucleotide linker, wherein the nucleotide linker includes the nucleotide sequence UUUGAU or UGUUU. In some embodiments, the nucleotide linker includes the nucleotide sequence UUUGAU. In some embodiments, the nucleotide linker includes the nucleotide sequence UGUUU.

[0124] In some embodiments, the RLR agonist of the present disclosure comprises a non-nucleotide linker, wherein the non-nucleotide linker is selected from the group consisting of:

[0125] (a) a glycol connector; and

[0126] (b) Alkyl linker.

[0127] In some embodiments, the non-nucleotide linker is a hexaethylene glycol linker. In some embodiments, the non-nucleotide linker is a C9 alkyl linker.

[0128] In some embodiments, the RLR agonists of the present disclosure include a 5' diphosphate moiety or a derivative or analog thereof. In some embodiments, the agonists include a 5' triphosphate moiety or a derivative or analog thereof. In some embodiments, the derivatives or analogs thereof include phosphate bioisosteres selected from the following: phosphonates, phosphonothioates, phosphorothioates, sulfates, sulfonates, sulfamates, thiazolidinones, carboxylates, malonates, boric acid, benzoborazole, borophosphates, and squaramides.

[0129] In some embodiments, the agonist comprises a modified nucleotide, a modified nucleoside, or a modified nucleobase, or a combination thereof. In some embodiments, the agonist comprises a modification of an internucleotide bond or of a polynucleotide backbone.

[0130] In some embodiments, the RLR agonists of the present disclosure exhibit one or more of the following properties:

[0131] (a) specifically binds to one or more RLRs (e.g., RIG-1, MDA5 and / or LGP2);

[0132] (b) increase RLR-mediated cytokine production;

[0133] (c) increase RLR-mediated expression of interferon-stimulated genes (ISGs);

[0134] (d) increase RLR-dependent intracellular signaling;

[0135] (e) increasing the stability of the duplex;

[0136] (f) increase binding affinity to RLR;

[0137] (g) reduce off-target binding;

[0138] (h) Increase biological half-life;

[0139] (i) Increase biodistribution and bioavailability;

[0140] (j) increasing and / or enhancing uptake into cells and / or tissues;

[0141] (k) reducing immunogenicity; and

[0142] (l) A combination of any one of (a) to (k).

[0143] In some aspects, the present disclosure provides an RLR agonist that specifically binds to RLR, wherein the agonist comprises a blunt-ended hairpin RNA, wherein the blunt-ended hairpin RNA comprises the formula:

[0144] 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', where

[0145] (i) (N1-N2-X1) comprises a first polynucleotide comprising linked nucleotides N1, N2 and X1;

[0146] (ii) (X2-N3-N4) comprises a second polynucleotide comprising linked nucleotides X2, N3 and N4;

[0147] (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;

[0148] (iv) N1 base pairs with N4;

[0149] (v) N2 base pairs with N3;

[0150] (vi) N1 comprises a 5' diphosphate or triphosphate moiety or a derivative or analog thereof;

[0151] (vii) X1 and X2 are each an oligonucleotide comprising a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine and inosine;

[0152] (viii) X1 and X2 are complementary;

[0153] (ix) X1 and X2 are each 12 nucleotides to 16 nucleotides in length and are the same length; and

[0154] (x) L is a linker that operably connects the first polynucleotide and the second polynucleotide,

[0155] wherein N1 and N2 each comprise guanosine, wherein N3 and N4 each comprise cytidine, wherein X1 and X2 are each 12 nucleotides in length, wherein X1 and X2 each comprise at least one inosine nucleoside, wherein the inosine nucleoside base pairs with the cytidine in the hairpin RNA, and wherein L comprises a nucleotide linker, wherein the nucleotide linker comprises a four-membered ring, wherein the nucleotide sequence of the four-membered ring is UUCG.

[0156] In some aspects, the present disclosure provides an RLR agonist that specifically binds to RLR, wherein the agonist comprises a blunt-ended hairpin RNA, wherein the blunt-ended hairpin RNA comprises the formula:

[0157] 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', where

[0158] (i) (N1-N2-X1) comprises a first polynucleotide comprising linked nucleotides N1, N2 and X1;

[0159] (ii) (X2-N3-N4) comprises a second polynucleotide comprising linked nucleotides X2, N3 and N4;

[0160] (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;

[0161] (iv) N1 base pairs with N4;

[0162] (v) N2 base pairs with N3;

[0163] (vi) N1 comprises a 5' diphosphate or triphosphate moiety or a derivative or analog thereof;

[0164] (vii) X1 and X2 are each an oligonucleotide comprising a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine and inosine;

[0165] (viii) X1 and X2 are complementary;

[0166] (ix) X1 and X2 are each 12 nucleotides to 16 nucleotides in length and are the same length; and

[0167] (x) L is a linker that operably connects the first polynucleotide and the second polynucleotide,

[0168] wherein N1 comprises inosine and N2 comprises guanosine, wherein N3 and N4 each comprise cytidine, wherein X1 and X2 are each 12 nucleotides in length, wherein X1 and X2 each comprise at least one inosine nucleoside, wherein the inosine nucleoside base pairs with the cytidine in the hairpin RNA, and wherein L comprises a nucleotide linker, wherein the nucleotide linker comprises a four-membered ring, wherein the nucleotide sequence of the four-membered ring is UUCG.

[0169] In some aspects, the present disclosure provides an RLR agonist that specifically binds to RLR, wherein the agonist comprises a blunt-ended hairpin RNA, wherein the blunt-ended hairpin RNA comprises the formula:

[0170] 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', where

[0171] (i) (N1-N2-X1) comprises a first polynucleotide comprising linked nucleotides N1, N2 and X1;

[0172] (ii) (X2-N3-N4) comprises a second polynucleotide comprising linked nucleotides X2, N3 and N4;

[0173] (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;

[0174] (iv) N1 base pairs with N4;

[0175] (v) N2 base pairs with N3;

[0176] (vi) N1 comprises a 5' diphosphate or triphosphate moiety or a derivative or analog thereof;

[0177] (vii) X1 and X2 are each an oligonucleotide comprising a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine and inosine;

[0178] (viii) X1 and X2 are complementary;

[0179] (ix) X1 and X2 are each 12 nucleotides to 16 nucleotides in length and are the same length; and

[0180] (x) L is a linker that operably connects the first polynucleotide and the second polynucleotide,

[0181] wherein N1 and N2 comprise inosine, and N3 and N4 comprise cytidine, wherein X1 and X2 are each 12 nucleotides in length, wherein X1 and X2 each comprise at least one inosine nucleoside, wherein the inosine nucleoside base pairs with the cytidine in the hairpin RNA, and wherein L comprises a nucleotide linker, wherein the nucleotide linker comprises a four-membered ring, wherein the nucleotide sequence of the four-membered ring is UUCG.

[0182] In some aspects, the present disclosure provides an RLR agonist that specifically binds to RLR, wherein the agonist comprises a blunt-ended hairpin RNA, the blunt-ended hairpin RNA comprises a non-nucleotide linker, and wherein the agonist comprises the formula:

[0183] 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', where

[0184] (i) (N1-N2-X1) comprises a first polynucleotide comprising linked nucleotides N1, N2 and X1;

[0185] (ii) (X2-N3-N4) comprises a second polynucleotide comprising linked nucleotides X2, N3 and N4;

[0186] (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;

[0187] (iv) N1 base pairs with N4;

[0188] (v) N2 base pairs with N3;

[0189] (vi) N1 comprises a 5' diphosphate or triphosphate moiety or a derivative or analog thereof;

[0190] (vii) X1 and X2 are each an oligonucleotide comprising a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine and inosine;

[0191] (viii) X1 and X2 are complementary;

[0192] (ix) X1 and X2 are each 12 nucleotides to 16 nucleotides in length and are the same length; and

[0193] (x) L is a non-nucleotide linker that covalently links the first polynucleotide and the second polynucleotide,

[0194] wherein N1 and N2 comprise guanosine, wherein N3 and N4 comprise cytidine, wherein X1 and X2 are each 12 nucleotides in length, and wherein the non-nucleotide linker is a C9 alkyl linker.

[0195] In some aspects, the present disclosure provides an RLR agonist that specifically binds to RLR, wherein the agonist comprises a blunt-ended hairpin RNA, the blunt-ended hairpin RNA comprises a non-nucleotide linker, and wherein the agonist comprises the formula:

[0196] 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', where

[0197] (i) (N1-N2-X1) comprises a first polynucleotide comprising linked nucleotides N1, N2 and X1;

[0198] (ii) (X2-N3-N4) comprises a second polynucleotide comprising linked nucleotides X2, N3 and N4;

[0199] (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;

[0200] (iv) N1 base pairs with N4;

[0201] (v) N2 base pairs with N3;

[0202] (vi) N1 comprises a 5' diphosphate or triphosphate moiety or a derivative or analog thereof;

[0203] (vii) X1 and X2 are each an oligonucleotide comprising a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine and inosine;

[0204] (viii) X1 and X2 are complementary;

[0205] (ix) X1 and X2 are each 12 nucleotides to 16 nucleotides in length and are the same length; and

[0206] (x) L is a non-nucleotide linker that covalently links the first polynucleotide and the second polynucleotide,

[0207] wherein N1 and N2 comprise guanosine, wherein N3 and N4 comprise cytidine, wherein X1 and X2 are each 12 nucleotides in length, and wherein the non-nucleotide linker is a hexaethylene glycol linker.

[0208] In some aspects, the present disclosure provides an RLR agonist that specifically binds to RLR, wherein the 5'-most nucleotide of the agonist comprises a 5' diphosphate or triphosphate portion or a derivative or analog thereof, and wherein the agonist comprises a nucleotide sequence selected from the group consisting of: SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 and 36.

[0209] In some aspects, the present disclosure provides an RLR agonist that specifically binds to an RLR, wherein the agonist comprises a blunt-ended hairpin RNA, the blunt-ended hairpin RNA comprising a first polynucleotide connected to a second polynucleotide via a linker, wherein the first polynucleotide is sufficiently complementary to the second polynucleotide to form a duplex, wherein the duplex comprises less 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 provides at least one increased biological activity mediated by the RLR relative to an agonist that does not comprise the sequence motif, and wherein the first polynucleotide and the second polynucleotide comprise a nucleotide sequence selected from the group consisting of:

[0210] (i) SEQ ID NOs: 37 and 68, respectively;

[0211] (ii) SEQ ID NOs: 38 and 69, respectively;

[0212] (iii) SEQ ID NOs: 39 and 70, respectively;

[0213] (iv) SEQ ID NOs: 40 and 71, respectively;

[0214] (v) SEQ ID NOs: 41 and 72, respectively;

[0215] (vi) SEQ ID NOs: 42 and 73, respectively;

[0216] (vii) SEQ ID NOs: 43 and 74, respectively;

[0217] (viii) SEQ ID NOs: 44 and 75, respectively;

[0218] (ix) SEQ ID NOs: 45 and 76, respectively;

[0219] (x) are SEQ ID NOs: 46 and 77, respectively;

[0220] (xi) SEQ ID NOs: 47 and 78, respectively;

[0221] (xii) SEQ ID NOs: 48 and 79, respectively;

[0222] (xiii) SEQ ID NOs: 49 and 80, respectively;

[0223] (xiv) SEQ ID NOs: 50 and 81, respectively;

[0224] (xv) are SEQ ID NOs: 51 and 82, respectively;

[0225] (xvi) are SEQ ID NOs: 52 and 83, respectively;

[0226] (xvii) SEQ ID NOs: 53 and 84, respectively;

[0227] (xviii) are SEQ ID NOs: 54 and 85, respectively;

[0228] (xix) are SEQ ID NOs: 55 and 86, respectively;

[0229] (xx) are SEQ ID NOs: 56 and 87, respectively;

[0230] (xxi) are SEQ ID NOs: 57 and 88, respectively;

[0231] (xxii) SEQ ID NOs: 58 and 89, respectively;

[0232] (xxiii) are SEQ ID NOs: 59 and 89, respectively;

[0233] (xxiv) SEQ ID NOs: 60 and 90, respectively;

[0234] (xxv) are SEQ ID NOs: 61 and 91, respectively;

[0235] (xxvi) are SEQ ID NOs: 62 and 92, respectively;

[0236] (xxvii) are SEQ ID NOs: 63 and 91, respectively;

[0237] (xxviii) are SEQ ID NOs: 64 and 93, respectively;

[0238] (xxix) are SEQ ID NOs: 65 and 94, respectively;

[0239] (xxx) are SEQ ID NOs: 66 and 95, respectively;

[0240] (xxxi) are SEQ ID NOs: 67 and 96, respectively; and

[0241] (xxxii) are SEQ ID NOs: 63 and 97, respectively.

[0242] In some aspects, the present disclosure provides an RLR agonist that specifically binds to RLR, wherein the agonist comprises a blunt-ended hairpin RNA, the blunt-ended hairpin RNA comprises at least one or more nucleotides, the at least one or more nucleotides comprise inosine that is base-paired with cytidine, and wherein the agonist comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 22, 23, and 25.

[0243] In some aspects, the present disclosure provides an RLR agonist that specifically binds to RLR, wherein the agonist comprises a blunt-ended hairpin RNA, the blunt-ended hairpin RNA comprises at least one or more nucleotides, the at least one or more nucleotides comprising inosine base-paired with 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 comprise a nucleotide sequence selected from the group consisting of:

[0244] (i) SEQ ID NOs: 58 and 89, respectively;

[0245] (ii) SEQ ID NOs: 59 and 89, respectively; and

[0246] (iii) SEQ ID NO: 61 and 91, respectively.

[0247] In some aspects, the present disclosure provides an RLR agonist that specifically binds to RLR, wherein the agonist comprises a blunt-ended hairpin RNA, the blunt-ended hairpin RNA comprises 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 comprise a nucleotide sequence selected from the group consisting of:

[0248] (i) SEQ ID NOs: 37 and 68, respectively;

[0249] (ii) SEQ ID NOs: 38 and 69, respectively;

[0250] (iii) SEQ ID NOs: 39 and 70, respectively;

[0251] (iv) SEQ ID NOs: 40 and 71, respectively;

[0252] (v) SEQ ID NOs: 41 and 72, respectively;

[0253] (vi) SEQ ID NOs: 42 and 73, respectively;

[0254] (vii) SEQ ID NOs: 43 and 74, respectively;

[0255] (viii) SEQ ID NOs: 44 and 75, respectively;

[0256] (ix) SEQ ID NOs: 45 and 76, respectively;

[0257] (x) are SEQ ID NOs: 46 and 77, respectively;

[0258] (xi) SEQ ID NOs: 47 and 78, respectively;

[0259] (xii) SEQ ID NOs: 48 and 79, respectively;

[0260] (xiii) SEQ ID NOs: 49 and 80, respectively;

[0261] (xiv) SEQ ID NOs: 50 and 81, respectively;

[0262] (xv) are SEQ ID NOs: 51 and 82, respectively;

[0263] (xvi) are SEQ ID NOs: 52 and 83, respectively;

[0264] (xvii) SEQ ID NOs: 53 and 84, respectively;

[0265] (xviii) are SEQ ID NOs: 54 and 85, respectively;

[0266] (xix) are SEQ ID NOs: 55 and 86, respectively;

[0267] (xx) are SEQ ID NOs: 56 and 87, respectively;

[0268] (xxi) are SEQ ID NOs: 57 and 88, respectively;

[0269] (xxii) SEQ ID NOs: 58 and 89, respectively;

[0270] (xxiii) are SEQ ID NOs: 59 and 89, respectively;

[0271] (xxiv) SEQ ID NOs: 60 and 90, respectively;

[0272] (xxv) are SEQ ID NOs: 61 and 91, respectively;

[0273] (xxvi) are SEQ ID NOs: 62 and 92, respectively;

[0274] (xxvii) are SEQ ID NOs: 63 and 91, respectively;

[0275] (xxviii) are SEQ ID NOs: 64 and 93, respectively;

[0276] (xxix) are SEQ ID NOs: 65 and 94, respectively;

[0277] (xxx) are SEQ ID NOs: 66 and 95, respectively;

[0278] (xxxi) are SEQ ID NOs: 67 and 96, respectively; and

[0279] (xxxii) are SEQ ID NOs: 63 and 97, respectively.

[0280] In some embodiments of the RLR agonist provided by the present disclosure, the nucleotide sequence constituting the RLR 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.

[0281] In some aspects, the present disclosure provides a pharmaceutical composition for stimulating an immune response in a subject in need, treating a cancer in the subject, or delaying the progression of the cancer, or reducing or inhibiting the tumor growth in the subject, 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

[0282] In some aspects, the present disclosure provides a method for increasing the 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 RLR agonist increases the production of RLR-mediated cytokines in the cell. In some embodiments, the RLR agonist increases the production of type I interferons (e.g., IFN-α, IFN-β) mediated by RLR in the cell. In some embodiments, the RLR agonist increases the production of IL-1β mediated by RLR in the cell. In some embodiments, the RLR agonist increases the production of IP-10 mediated by RLR in the cell. In some embodiments, the RLR agonist increases the production of IL-6, IL-12p70, MCP-1 and / or MIP-1β mediated by RLR in the cell.

[0283] In some aspects, the present disclosure provides a method for increasing RLR-mediated expression of one or more interferon-stimulated 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-stimulated genes in the cell.

[0284] In some aspects, 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.

[0285] In some aspects, 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 a pharmaceutical composition provided by the present disclosure.

[0286] In some aspects, the present disclosure provides a method of treating or delaying 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.

[0287] In some aspects, 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.

[0288] In some aspects, the present disclosure provides a method for stimulating an immune response in a subject in need thereof, treating cancer in the subject, or delaying the progression of the cancer, or inhibiting tumor growth in the subject, the method comprising administering to the subject an effective amount of an RLR agonist or a pharmaceutical composition provided by the present disclosure, wherein the agonist or the pharmaceutical composition increases RLR-mediated production of one or more cytokines in a cell, increases RLR-mediated expression of one or more interferon-stimulated genes in a cell, and / or increases RLR-dependent intracellular signaling in a cell, thereby stimulating the immune response, treating the cancer, or delaying the progression of the cancer, or inhibiting tumor growth.

[0289] 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 drugs, cell death inducers, modulators (e.g., modulatory antibodies), cytotoxic agents, immune-based therapies, cytokines, activators or agonists of co-stimulatory molecules, inhibitors of inhibitory molecules, vaccines, cellular immunotherapy, or a combination thereof.

[0290] In some embodiments, the RLR agonist or pharmaceutical composition provided herein is administered before or after administration of the one or more additional therapeutic agents, or wherein the one or more additional therapeutic agents are administered simultaneously with, before, or after administration of the agonist or the pharmaceutical composition.

[0291] In some embodiments, the one or more additional therapeutic agents are PD-1 / PD-L1 antagonists, TIM-3 antagonists, VISTA antagonists, adenosine A2AR antagonists, B7-H3 antagonists, B7-H4 antagonists, BTLA antagonists, CTLA-4 antagonists, IDO antagonists, KIR antagonists, LAG-3 antagonists, Toll-like receptor 3 (TLR3) agonists, Toll-like receptor 7 (TLR7) agonists, Toll-like receptor 9 (TLR9) agonists.

[0292] 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).

[0293] 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).

[0294] In some embodiments, the one or more additional therapeutic agents are PD-1 / PD-L1 antagonists. In some embodiments, the PD-1 / PD-L1 antagonists are selected from the group consisting of: PDR001, (pembrolizumab), (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, (atezolizumab), (avelumab), (durvalumab) and BMS-936559.

[0295] In some embodiments, the one or more additional therapeutic agents is a TIM-3 antagonist.

[0296] In some embodiments, the one or more additional therapeutic agents is a VISTA antagonist.

[0297] In some embodiments, the one or more additional therapeutic agents is an adenosine A2AR antagonist.

[0298] In some embodiments, the one or more additional therapeutic agents is a B7-H3 antagonist.

[0299] In some embodiments, the one or more additional therapeutic agents is a B7-H4 antagonist.

[0300] In some embodiments, the one or more additional therapeutic agents is a BTLA antagonist.

[0301] In some embodiments, the one or more additional therapeutic agents is a CTLA-4 antagonist.

[0302] In some embodiments, the one or more additional therapeutic agents is an IDO antagonist.

[0303] In some embodiments, the one or more additional therapeutic agents is a KIR antagonist.

[0304] In some embodiments, the one or more additional therapeutic agents is a LAG-3 antagonist.

[0305] 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 acid:polycytidylic acid (poly I:C). In some embodiments, the TLR3 agonist is (polyICLC). In some embodiments, the TLR3 agonist is polyadenylic acid-polyuridylic acid (poly A:U). In some embodiments, the TLR3 agonist is In some embodiments, the TLR3 agonist is ( In some embodiments, the TLR3 agonist is

[0306] 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 TM In some embodiments, the TLR7 agonist is resiquimod (R-848).

[0307] 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).

[0308] In some aspects, the present disclosure provides a use of an RLR agonist or a pharmaceutical composition provided by the present disclosure for stimulating an immune response in a subject in need thereof, treating cancer in the subject or delaying the progression of the cancer or inhibiting tumor growth in the subject, wherein the agonist or the pharmaceutical composition is optionally used in combination with one or more additional therapeutic agents.

[0309] In some aspects, the present disclosure provides a use of an RLR agonist or a pharmaceutical composition provided by the present disclosure in the manufacture of a medicament for stimulating an immune response in a subject in need thereof, treating cancer in the subject or delaying the progression of the cancer, or inhibiting tumor growth in the subject, wherein the agonist or the pharmaceutical composition is optionally used in combination with one or more additional therapeutic agents.

[0310] In some aspects, the present disclosure provides a kit comprising an RLR agonist or pharmaceutical composition provided by the present disclosure and instructions, wherein the kit is used to stimulate an immune response in a subject, or to treat cancer in the subject, or to delay the progression of the cancer, or to inhibit tumor growth in the subject, and the kit optionally has instructions for use in combination with one or more additional therapeutic agents.

[0311] 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 drugs, cell death inducers, modulators (e.g., modulatory antibodies), cytotoxic agents, immune-based therapies, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules, vaccines, cellular immunotherapy, or a combination thereof.

[0312] 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 the one or more additional therapeutic agents, or wherein the one or more additional therapeutic agents are administered at the same time, before or after the administration of the agonist or the pharmaceutical composition.

[0313] In some embodiments of the uses or kits provided by the present disclosure, the one or more additional therapeutic agents are PD-1 / PD-L1 antagonists, TIM-3 antagonists, VISTA antagonists, adenosine A2AR antagonists, B7-H3 antagonists, B7-H4 antagonists, BTLA antagonists, CTLA-4 antagonists, IDO antagonists, KIR antagonists, LAG-3 antagonists, Toll-like receptor 3 (TLR3) agonists, Toll-like receptor 7 (TLR7) agonists, Toll-like receptor 9 (TLR9) agonists.

[0314] In some embodiments of the uses or kits provided by the present disclosure, the one or more additional therapeutic agents are agonists comprising a polypeptide (eg, an antibody or an antigen-binding portion thereof) that specifically binds to CD137 (4-1BB).

[0315] In some embodiments of the uses or kits provided by the present disclosure, the one or more additional therapeutic agents described herein are agonists comprising a polypeptide (eg, an antibody or an antigen-binding portion thereof) that specifically binds to CD134 (OX40). BRIEF DESCRIPTION OF THE DRAWINGS

[0316] The patent or application file contains at least one drawing executed in color. Copies of the patent or patent application publication with one or more color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0317] Figure 1 Provided is a bar graph depicting the quantification of IFN-a secretion from human PBMCs treated with 3 different concentrations of RIG-I-like receptor agonists including various modifications. DETAILED DESCRIPTION

[0318] Overview

[0319] RIG-I-like receptors (RLRs) are a family of cytoplasmic pattern recognition receptors that are essential for detecting viral RNA and initiating innate immune responses. The RLR family includes three members: retinoic acid-inducible gene I (RIG-I), melanoma differentiation-associated gene 5 (MDA5), and genetics and physiology laboratory protein 2 (LGP2). These receptors are expressed in both immune cell types and non-immune cell types and regulate the signal transduction pathways that promote the IRF3, IRF7-dependent expression of type I and type III interferons (IFNs) and the NF-κB-dependent expression of proinflammatory cytokines.

[0320] All three RLR family receptors have a DExD / H box RNA helicase domain with ATPase activity. This domain, together with the adjacent C-terminal domain, is essential for RNA binding. In addition, the C-terminal domains of RIG-I and LGP2 have been shown to act as repressor domains, thereby ensuring that the receptor remains in an inactive conformation until it is bound by an activating RNA.

[0321] The present disclosure provides RLR agonists including synthetic RNA molecules that fold to form double-stranded dsRNA and include one or more sequence motifs that provide one or more enhanced biological activities.

[0322] definition

[0323] Terms used in the claims and specification are defined as described below unless otherwise indicated. In the event of a direct conflict with terms used in the parent provisional patent application, the terms used in this application shall control.

[0324] 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 context clearly dictates otherwise. Additionally, unless the context requires otherwise, singular terms shall include pluralities and plural terms shall include the singular.

[0325] About: As used herein, the term "about" (alternatively "approximately") will be understood by those of ordinary skill and will vary to some extent depending on the context in which it is used. If there is a use of the term that is unclear to those of ordinary skill, "about" will mean up to ±10% of the particular value, given the context in which the term is used.

[0326] Agonist: As used herein, the term "agonist" is used in its broadest sense and encompasses any molecule or compound that partially or completely promotes, induces, increases and / or activates the biological activity of the natural polypeptides disclosed herein. Agonist molecules according to the present disclosure may include nucleic acids (e.g., oligonucleotides, polynucleotides), antibodies or antigen-binding fragments, fragments or amino acid sequence variants of natural polypeptides, peptides, oligonucleotides, lipids, carbohydrates and 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., biological activity) 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 with a negative control under comparable conditions. Also disclosed herein are methods of identifying agonists suitable for use in the methods of the present disclosure. For example, these methods include, but are not limited to, binding assays such as enzyme-linked immunosorbent assays (ELISAs), Forte Systems, fluorescence polarization (FP) assays, and radioimmunoassays (RIA). These assays determine the ability of an agonist to bind to a polypeptide of interest (e.g., a receptor or ligand), and are therefore indicative of the ability of the agonist to promote, increase, or activate the activity of the polypeptide. Functional assays can also be used to determine the efficacy of an agonist, such as the ability of an agonist to activate or promote the function of a polypeptide. For example, a functional assay can include contacting a polypeptide with a candidate agonist molecule and measuring a detectable change in one or more biological activities typically associated with the polypeptide. The potency of an agonist is typically measured by its EC 50 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 is and the lower the concentration required to activate a maximal biological response.

[0327] Amelioration: As used herein, the term "amelioration" refers to any therapeutically beneficial result in the treatment of a disease state (eg, cancer), including prevention, lessening of severity or progression, remission, or cure.

[0328] Amino Acids: As used herein, the term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as naturally occurring amino acids, i.e., carbon bound to hydrogen, carboxyl, amino, and R groups, e.g., homoserine, norleucine, methionine sulfoxide, 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 an amino acid but function in a manner similar to naturally occurring amino acids.

[0329] 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. Likewise, nucleotides may be referred to by their commonly accepted single-letter codes.

[0330] 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. Although insertions will typically consist of the insertion of one or two amino acid residues, larger "peptide insertions" may also be performed, such as insertions of about three to about five or even up to about ten, fifteen or twenty amino acid residues. As disclosed above, the inserted one or more residues may be naturally occurring or non-naturally occurring. "Amino acid deletion" refers to the removal of at least one amino acid residue from a predetermined amino acid sequence.

[0331] Base composition: As used herein, the term "base composition" refers to the proportion of the total nucleotides of a nucleic acid (eg, RNA) consisting of guanine (or hypoxanthine) + cytosine and / or uracil (or thymine) + adenine nucleobases.

[0332] Base pair: As used herein, the term "base pair" refers to two nucleobases on a relatively complementary polynucleotide chain, or a region of the same chain that interacts through the formation of specific hydrogen bonds. As used herein, the term "Watson-Crick base pairing", which is used interchangeably with "complementary base pairing", refers to a set of base pairing rules in which purines are always combined with pyrimidines, so that the nucleobases adenine (A) form complementary base pairs with thymine (T), and guanine (G) forms complementary base pairs with cytosine (C) in DNA molecules. In RNA molecules, thymine is replaced by uracil (U), which is similar to thymine (T) and forms complementary base pairs with adenine (A). Complementary base pairs are bound together by hydrogen bonds, and the number of hydrogen bonds varies between base pairs. As is known in the art, guanine (G)-cytosine (C) base pairs are bound by three (3) hydrogen bonds, and adenine (A)-thymine (T) or uracil (U) base pairs are bound by two (2) hydrogen bonds.

[0333] Base pairing interactions that do not follow these rules can occur in natural, non-natural and synthetic nucleic acids, and are referred to herein as "non-Watson-Crick base pairing" or alternatively "atypical base pairing". "Wobble base pairing" 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 2-amino. Inosine can form two hydrogen bonds with each of the four natural nucleobases (Oda et al., (1991) Nucleic Acids Res 19:5263-5267), and researchers generally use it as a "universal" base, which means that it can be paired with all naturally occurring or typical bases. The four main wobble base pairs are guanine-uracil (GU) base pair, hypoxanthine-uracil (IU) base pair, hypoxanthine-adenine (IA) base pair and hypoxanthine-cytosine (IC) base pair. In order to maintain consistency in nucleic acid nomenclature, "I" is used for hypoxanthine, since hypoxanthine is the nucleobase of inosine; the nomenclature otherwise follows the name of the nucleobase and its corresponding nucleoside (e.g., "G" is used for both guanine and guanosine and for deoxyguanosine). The thermodynamic stability of the wobble base pairs is comparable to that of the Watson-Crick base pairs. The wobble base pairs play a role in the formation of secondary structure in RNA molecules.

[0334] 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 where it will base pair with cytidine (IC base pair); that is, inosine can replace guanosine, but not other nucleosides.

[0335] Biologically active: As used herein, the phrase "biologically active" refers to the property of any substance that is active in a biological system and / or organism. For example, a substance that has a biological effect on an organism when administered to the organism is considered to be biologically active, and therefore has "biological activity." In specific embodiments, when a nucleic acid is biologically active, a portion of the nucleic acid that shares at least one biological activity of the entire nucleic acid is generally referred to as a "biologically active" portion.

[0336] Covalently linked: As used herein, the term "covalently linked" (alternatively "conjugated," "linked," "attached," "fused," or "tethered") when used with respect to two or more moieties means that the moieties are physically associated or linked to each other, either directly or through one or more additional moieties that act as linkers, by any means including chemical conjugation, recombinant techniques, or enzymatic activity, to form a sufficiently stable structure such that the moieties remain physically associated under the conditions in which the structure is used (e.g., physiological conditions).

[0337] Complementary: As used herein, the term "complementary" or "complementarity" refers to the relationship between the sequence of nucleotides comprising two polynucleotide chains or a region of the same polynucleotide chain and the formation of a duplex comprising the chain or region, wherein the degree of continuous base pairing between the two chains or regions is sufficient to produce a duplex 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 base pair with guanine (G). It is also known that atypical nucleobases (e.g., inosine) can hydrogen bond with natural bases. It is said that if the degree of base pairing between the two chains maintains a duplex structure under conditions where a duplex structure is used (e.g., physiological conditions in a cell), the sequence of nucleotides comprising the first chain of a polynucleotide or a region, part or fragment thereof is "sufficiently complementary" to the sequence of nucleotides comprising the second chain of the same or different nucleic acid or a region, part or fragment thereof. It should be understood that the complementary strand or complementary region of polynucleotides can include some non-complementary base pairs.Complementarity can be "partial", wherein according to the base pairing rule, only some of the core bases in the core bases including polynucleotides are matched.Or, "completely" or "all" complementarity can be present between nucleic acids.Although the degree of complementarity between polynucleotide chains or regions has a significant impact on the efficiency and intensity of hybridization between chains or regions, it is not required that two complementary polynucleotides are all base-paired at each nucleotide position.In certain embodiments, the first polynucleotide is 100% or "completely" complementary to the second polynucleotide, and therefore forms a base pair at each nucleotide position.In certain 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.Although perfect complementarity is usually desired, some embodiments can include one or more mismatches but preferably 6, 5, 4, 3, 2 or 1 mismatches.

[0338] Contact: As used herein, the term "contact" means to establish 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 share a physical connection. Methods for contacting cells with external entities in vivo, in vitro and ex vivo are well known in the field of biology. In an exemplary embodiment of the present disclosure, the step of contacting a mammalian cell with a composition (e.g., RNA, nanoparticle or pharmaceutical composition isolated of the present disclosure) is performed in vivo. For example, contacting a lipid nanoparticle composition with a cell (e.g., a mammalian cell) that can be placed in an organism (e.g., a mammal) can be performed by any suitable route of administration (e.g., parenteral administration to an organism, including intravenous, intramuscular, intradermal and subcutaneous administration). For cells present in vitro, the composition (e.g., lipid nanoparticle or isolated RNA) and the cell can, for example, be contacted by adding the composition to the culture medium of the cell, and can involve or cause transfection. In addition, more than one cell can be contacted by an agent.

[0339] Denaturation: As used herein, the term "denaturation" refers to a process by which hydrogen bonding between base-paired nucleotides in a nucleic acid is disrupted, resulting in loss of secondary and / or tertiary nucleic acid structure (e.g., separation of previously annealed strands). Denaturation can occur by applying external substances, energy, or biochemical processes to the nucleic acid.

[0340] Antigen presenting cell: The term "antigen presenting cell" or "APC" is a cell that displays a foreign antigen complexed with an MHC on its surface. T cells recognize this complex using a T cell receptor (TCR). Examples of APCs include, but are not limited to, dendritic cells (DCs), peripheral blood mononuclear cells (PBMCs), monocytes (such as THP-1), B-type lymphoblasts (such as C1R.A2, 1518B-LCL), and monocyte-derived dendritic cells (DCs). Some APCs internalize antigens by phagocytosis or receptor-mediated endocytosis.

[0341] 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 lead to apoptosis may result in observable and characteristic morphological changes in cells, including cell membrane blebbing, cell volume shrinkage, chromosome DNA condensation and fragmentation, and mRNA decay. A common method for identifying cells undergoing apoptosis (including T cells) 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 on the outer leaflet of the plasma membrane, which is an early indicator that the cell is undergoing the apoptotic process.

[0342] Blunt end: As used herein, the terms "blunt end" and "blunt-ended" refer to the structure of a duplex nucleic acid or the end of a double-stranded nucleic acid, wherein the two complementary strands comprising the duplex terminate at a base pair at least at one end. Thus, neither strand comprising the duplex extends further from the end than the other strand.

[0343] Cancer antigen: As used herein, "cancer antigen" refers to (i) tumor-specific antigens, such as neoantigens, (ii) tumor-associated antigens, (iii) cells expressing tumor-specific antigens, (iv) cells expressing tumor-associated antigens, (v) embryonic antigens on tumors, (vi) autologous tumor cells, (vii) tumor-specific membrane antigens, (viii) tumor-associated membrane antigens, (ix) growth factor receptors, (x) growth factor ligands, and (xi) any other type of antigen or antigen-presenting cell or material associated with cancer.

[0344] Cancer: As used herein, the term "cancer" is art-recognized and refers to a malignant tumor of epithelial or endocrine tissue, including respiratory cancer, gastrointestinal cancer, urogenital cancer, testicular cancer, breast cancer, prostate cancer, endocrine cancer, and melanoma. The RIG-I-like receptor (RLR) agonists described herein can be used to treat patients who are suspected of having, or may have a high risk of having any type of cancer (including renal cancer or melanoma). Exemplary cancers include cancers formed in the cervix, lung, prostate, breast, head and neck, colon, and ovarian tissue. The term also includes carcinosarcoma, which includes a malignant tumor composed of cancerous tissue and sarcoma tissue. "Adenocarcinoma" refers to a cancer derived from glandular tissue or in which tumor cells form a recognizable glandular structure.

[0345] 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 mainly mediated by CD8+ T cells.

[0346] Duplex: As used herein, the term "duplex" refers to a structure formed by complementary strands of a double-stranded polynucleotide, or a complementary region of a single-stranded polynucleotide that folds back on itself. The duplex structure of nucleic acids is the result of complementary nucleotide sequences being bound together or hybridized through base pairing interactions.

[0347] EC 50 As used herein, the term "EC 50 ” refers to the concentration of an agonist that induces a response in an in vitro or in vivo assay that is 50% of the maximal response, ie, halfway between the maximal response and the baseline.

[0348] 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.

[0349] 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 chains that base pair to form a duplex that terminates at one end in a nucleotide linker that includes a loop of unpaired nucleotides (e.g., a four-membered ring) that includes unpaired nucleotides or terminates in a non-nucleotide linker that includes a flexible chemical moiety (e.g., ethylene glycol), any of which connects the complementary nucleotide chains. RNA hairpins can vary in the length of the stem, the size and / or composition of the loop and / or linker, the number of base pair mismatches within the stem, and the actual nucleotide sequence. RNA hairpins can provide one or more functions, including but not limited to directing the overall folding of the RNA molecule comprising the hairpin, determining interactions in ribozymes, protecting messenger RNA (e.g., mRNA) from degradation, serving as a recognition motif or structure for RNA binding proteins, and serving as a substrate for enzymatic reactions. 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 the RLR agonist provided by the present disclosure terminates in a blunt end with a 5' triphosphate or diphosphate.

[0350] In Need: As used herein, a subject "in need of prevention," "in need of treatment," or "in need thereof" is a subject who, in the judgment of an appropriate medical practitioner (e.g., a physician, nurse, or nurse practitioner in the case of humans; a veterinarian in the case of non-human mammals), would reasonably benefit from a given treatment (such as treatment with a composition comprising a RIG-I-like receptor agonist).

[0351] Linker: As used herein, the term "linker" (alternatively "tether" or "spacer") refers to a portion that covalently links, attaches, or couples two polynucleotide chains or regions together. As used herein, a linker comprising a nucleotide is referred to as a "nucleotide linker" (e.g., a four-membered ring). As used herein, the term "non-nucleotide linker" refers to a linker that includes a chemical moiety and does not include a nucleotide. Non-limiting examples of non-nucleotide linkers include linkers that include ethylene glycol (e.g., hexaethylene glycol), alkyl chains (e.g., C9 alkyl linkers), and distyryl 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.

[0352] LGP2: As used herein, the term "LGP2" refers to the genetic and physiological laboratory protein 2 LGP2 is a polypeptide (a specific member of the RIG-I-like receptor family) and is encoded by the DHX58 gene in humans. 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 listed in Table 4 (SEQ ID NO: 100) and herein:

[0353] MELRSYQWEVIMPALEGKNIIIWLPTGAGKTRAAAYVAKRHLETVDGAKVVVLVNRVHLVTQHGEEFRRMLDGRWTVTTLSGDMGPRAGFGHLARCHDLLICTAELLQMALTSPEEEEHVELTVFSLIVVDECHHTHKDTVYNVIMSQYLELKLQRAQPLPQVLGLTAS PGTGGASKLDGAINHVLQLCANLDTWCIMSPQNCCPQLQEHSQQPCKQYNLCHRRSQDPFGDLLKKLMDQIHDHLEMPELSRKFGTQMYEQQVVKLSEAAALAGLQEQRVYALHLRRYNDALLIHDTVRAVDALAALQDFYHREHVTKTQILCAERRLLALFDDRKNELA HLATHGPENPKLEMLEKILQRQFSSSNSPRGIIFTRTRQSAHSLLLWLQQQQGLQTVDIRAQLLIGAGNSSQSTHMTQRDQQEVIQKFQDGTLNLLVATSVAEEGLDIPHCNVVVRYGLLTNEISMVQARGRARADQSVYAFVATEGSRELKRELINEALETLMEQAVA AVQKMDQAEYQAKIRDLQQAALTKRAAQAAQRENQRQQFPVEHVQLLCINCMVAVGHGSDLRKVEGTHHVNVNPNFSNYYNVSRDPVVINKVFKDWKPGGVISCRNCGEVWGLQMIYKSVKLPVLKVRSMLLETPQGRIQAKKWSRVPFSVPDFDFLQHCAENLSDLSLD

[0354] (NCBP accession number: NP_077024.2)

[0355] Local administration: As used herein, "local administration" or "local delivery" refers to delivery that is independent of the transport of a composition or agent through the vascular system to its intended target tissue or site. For example, the composition can be delivered by injection or implantation of the composition or agent or by injection or implantation of a device containing the composition or agent. Following local administration near a target tissue or site, the composition or agent or one or more components thereof can diffuse to the intended target tissue or site.

[0356] MDA5: As used herein, the term "MDA5" refers to melanoma differentiation-associated protein 5 polypeptide (a specific member of the RIG-I-like receptor family) and is encoded by the IFIH1 gene in the human body. Alternative names and acronyms for MDA5 in this field include AGS7, Hlcd, IDDM19, MDA-5, RLR-2, SGMRT1, and interferon induced with helicase C domain 1. The exemplary amino acid sequence of full-length human MDA5 is listed in Table 4 (SEQ ID NO:99) and here:

[0357]

[0358] (NCBI accession number: NP_071451.2)

[0359] Modified: As used herein, "modified" or "modification" refers to an altered state or structural change caused by modification of a polynucleotide (e.g., RNA). Polynucleotides can be modified in various ways, including chemically, structurally, and / or functionally. For example, the RNA molecules of the present disclosure can be modified by incorporating non-natural bases or sequence motifs that provide biological activity, including functional sequences or secondary structures. In one embodiment, RNA is modified by the introduction of non-natural or chemically modified bases, nucleosides and / or nucleotides, for example, as it relates to natural ribonucleotides A, U, G and C.

[0360] Naturally occurring: As used herein, the term "naturally occurring" as applied to an object refers to the fact that the object can be found in nature. For example, a polypeptide or polynucleotide sequence or its components, such as amino acids or nucleotides, that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally occurring.

[0361] Nucleic acid: As used herein, the term "nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers or oligomers thereof in single-stranded or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties to 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 acids (RNA), deoxyribonucleic acids (DNA), DNA-RNA hybrids, RNAi inducers, RNAi agents, siRNA, shRNA, miRNA, antisense RNA, ribozymes, catalytic DNA, RNA that induces triple helix formation, threose nucleic acids (TNA), glycol nucleic acids (GNA), peptide nucleic acids (PNA), locked nucleic acids (LNA, including LNA with a β-D-ribose configuration, α-LNA with an α-L-ribose configuration (diastereomers of LNA), 2'-amino-LNA with 2'-amino functionalization, and 2'-amino-α-LNA with 2'-amino functionalization) or hybrids thereof.

[0362] The polynucleotide used herein can be composed of any polyribonucleotide or polydeoxyribonucleotide, which can be unmodified RNA or DNA or modified RNA or DNA. For example, the polynucleotide can be composed of single-stranded and double-stranded DNA, DNA as a mixture of single-stranded and double-stranded regions, single-stranded and double-stranded RNA, and RNA as a mixture of single-stranded and double-stranded regions, including hybrid molecules of DNA and RNA that can be single-stranded or more typically double-stranded or a mixture of single-stranded and double-stranded regions. In addition, the polynucleotide can be composed of a triple-stranded region including RNA or DNA or both RNA and DNA. The polynucleotide can also contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons. "Modified" bases include, for example, trityl-methylated bases. "Modified nucleosides" include, for example, inosine and thymine, when the latter is present in RNA or includes RNA. DNA and RNA can be modified in various ways; therefore, "polynucleotides" include forms modified by chemistry, enzymes, or metabolism.

[0363] Nucleic acid structure: As used herein, the term "nucleic acid structure" refers to the arrangement or organization of atoms, chemical compositions, elements, motifs and / or nucleobase sequences including nucleic acids (e.g., RNA), and / or may refer to a two-dimensional or three-dimensional state of a nucleic acid. Thus, the term "RNA structure" refers to the arrangement or organization of atoms, chemical compositions, elements, motifs and / or nucleobase sequences including RNA molecules (e.g., mRNA), and / or may refer to a two-dimensional and / or three-dimensional state of an RNA molecule. Nucleic acid structure can be further divided into four organizational categories based on increased organizational complexity, which are referred to herein as "molecular structure," "primary structure," "secondary structure," and "tertiary structure."

[0364] Nucleobase: As used herein, the term "nucleobase" (alternatively "nucleotide base" or "nitrogenous base") refers to a purine or pyrimidine heterocyclic compound found in nucleic acids, including any derivatives or analogs of naturally occurring purines and pyrimidines that impart enhanced properties (e.g., binding affinity, nuclease resistance, chemical stability) to nucleic acids or portions or fragments thereof. Adenine, cytosine, guanine, thymine, and uracil are the major or typical nucleobases found in natural nucleic acids. Other natural, non-natural, atypical, and / or synthetic nucleobases may also be incorporated into nucleic acids, such as those disclosed herein.

[0365] Nucleoside / Nucleotide: As used herein, the term "nucleoside" refers to a compound containing a sugar molecule (e.g., ribose in RNA or deoxyribose in DNA), or a derivative or analog thereof covalently linked to a nucleobase (e.g., 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 nucleotide group comprising a ribose sugar and a nucleobase (e.g., adenosine (A), cytidine (C), guanosine (G), 5-methyluridine (m-1), 5-methyluridine (m-2), 5-methyluridine (m-3), 5-methyluridine (m-4), 5-methyluridine (m-5), 5-methyluridine (m-6), 5-methyluridine (m-7), 5-methyluridine (m-8), 5-methyluridine (m-9), 5-methyluridine (m-10), 5-methyluridine (m-11), 5-methyluridine (m-12), 5-methyluridine (m-13), 5-methyluridine (m-14), 5-methyluridine (m-15), 5-methyluridine (m-16), 5-methyluridine (m-17), 5-methyluridine (m-18), 5-methyluridine (m-19), 5-methyluridine (m-21), 5-methyluridine (m-19), 5-methyluridine (m-22), 5-methyluridine (m-19), 5-methyluridine (m-23), 5-methyluridine (m-18), 5-methyluridine (m-19), 5-methyluridine (m-19), 5-methyluridine (m- 5 U), uridine (U), or inosine (I)).

[0366] Operably linked: As used herein, a nucleic acid, or a fragment or portion thereof (such as a polynucleotide or oligonucleotide) is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence, or a fragment or portion thereof.

[0367] Polynucleotide / oligonucleotide: As used herein, the terms "polynucleotide" and "oligonucleotide" are used interchangeably and refer to single-stranded or double-stranded polymers or oligomers of nucleotides or nucleoside monomers composed of naturally occurring bases, sugars, and inter-sugar (backbone) bonds. The terms "polynucleotide" and "oligonucleotide" also include polymers and oligomers that include non-naturally occurring bases, sugars, and inter-sugar (backbone) bonds or portions thereof that function in a similar manner. Polynucleotides are not limited to nucleotide sequences of any particular length, as the term "polynucleotide" encompasses polymeric forms of nucleotides of any length. Short polynucleotides are generally referred to as "oligonucleotides" in the art. In the context of the present disclosure, such modified or substituted polynucleotides and oligonucleotides are generally superior to natural forms because the modification increases one or more desired or beneficial biological properties or activities, including but not limited to increased cytokine production, enhanced cellular uptake, and / or increased stability in the presence of nucleases. In some embodiments, agonists of the present disclosure include polynucleotides and oligonucleotides containing at least one region of modified nucleotides that impart one or more beneficial properties or increase biological activity (e.g., increased nuclease resistance, increased uptake into cells, increased duplex stability, increased binding affinity for a target polypeptide).

[0368] Palindromic sequence: As used herein, the term "palindromic sequence" (alternatively "palindrome") refers to a self-complementary nucleotide sequence; wherein when reading from 5' to 3', the nucleotide sequence in the 5' to 3' direction is identical to the nucleotide sequence comprising the complementary strand. For example, the sequence 5'-ACCTAGGT-3' is a palindromic sequence because, when reading from 5' to 3', its complementary sequence 3'-TGGATCCA-5' is identical to the original sequence. In contrast, the sequence 5'-AGTGGCTG-3' is not a palindromic sequence because, when reading from 5' to 3', its complementary sequence 3'-TCACCGAC-5' is not identical to the original sequence.

[0369] In one embodiment, the agonist consists of a first oligonucleotide, wherein the sequence of the first oligonucleotide is a palindromic sequence. In another embodiment, the agonist consists of a first oligonucleotide, wherein the first oligonucleotide comprises a palindromic sequence.

[0370] The palindromic sequence in the preferred oligonucleotides of the present invention preferably comprises the 5' end of the oligonucleotide and the 3' end of the oligonucleotide, thereby forming a blunt end. In one embodiment of the present invention, the oligonucleotide comprises a single palindromic sequence, and in another more preferred embodiment of the present invention, the oligonucleotide comprises two complementary palindromes interrupted by an intervening sequence, spacer or linker connecting two palindromes within 1 or 2 different oligonucleotides, thereby forming a hairpin duplex with blunt ends.

[0371] Parenteral administration: As used herein, "parenteral administration," "parenterally administered," and other grammatically equivalent phrases refer to modes of administration other than enteral administration and topical administration, usually by injection, and include, but are not limited to, intravenous, intranasal, intraocular, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intracerebral, intracranial, intracarotid, and intrasternal injection and infusion.

[0372] Patient: As used herein, the term "patient" includes human and other mammalian subjects receiving prophylactic or therapeutic treatment.

[0373] Percentage of identity: As used herein, in the context of two or more nucleic acids or polypeptide sequences, the term "percentage of identity" refers to two or more sequences or subsequences with a specified percentage of identical nucleotides or amino acid residues when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms (e.g., BLASTP and BLASTN or other algorithms available to the skilled person) or by visual inspection. Depending on the application, "percentage of identity" can exist over the region of the sequence being compared, for example, over a functional domain, or alternatively, over the full length of the two sequences to be compared. In order to perform sequence comparison, a sequence usually serves as a reference sequence for comparison with a test sequence. When using a sequence comparison algorithm, the test sequence and the reference sequence are input to a computer, subsequence coordinates are specified (if necessary), and sequence algorithm program parameters are specified. Then, the sequence comparison algorithm calculates the sequence identity percentage of one or more test sequences relative to the reference sequence based on the specified program parameters. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology = # of identical positions / total # of positions x 100), taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap. The comparison of sequences between the two sequences and the determination of percent identity can be accomplished using a mathematical algorithm as described in the following non-limiting examples.

[0374] Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981); by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970); by the similarity search method of Pearson and 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 Avenue, Madison, Wisconsin); or by visual inspection (see generally, Ausubel et al., below).

[0375] An example of an algorithm suitable for determining sequence identity and sequence similarity percentages is the BLAST algorithm, which is described in Altschul et al., Journal of Mol. Biol. 215:403-410 (1990). Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information website. The percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package (available at http: / / www.gcg.com), using the NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70 or 80 and a length weight of 1, 2, 3, 4, 5 or 6. The percent identity between two nucleotide or amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Computers in Biosciences (CABIOS), 4: 11-17, 1989) incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Molecular Biology (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, a gap weight of 16, 14, 12, 10, 8, 6, or 4, and a length weight of 1, 2, 3, 4, 5, or 6.

[0376] The nucleic acid and protein sequences disclosed herein can be further used as "query sequences" to search 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, Journal of Molecular Biology 215: 403-10). BLAST nucleotide searches can be performed with the NBLAST program (score = 100, word length = 12) to obtain nucleotide sequences homologous to the nucleic acid molecules of the present invention. BLAST protein searches can be performed with the XBLAST program (score = 50, word length = 3) to obtain amino acid sequences homologous to the protein molecules of the present invention. In order to obtain a gapped alignment for comparison purposes, a gapped BLAST as described in Altschul et al. (1997) Nucleic Acids Research 25 (17): 3389-3402 can be used. When using BLAST programs and gapped BLAST programs, the default parameters of the corresponding programs (e.g., XBLAST and NBLAST) can be used. See http: / / www.ncbi.nlm.nih.gov.

[0377] Pharmaceutically acceptable: As used herein, the term "pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues, organs and / or body fluids of humans and animals without producing excessive toxicity, irritation, allergic response or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0378] Pharmaceutically acceptable carrier: As used herein, the term "pharmaceutically acceptable carrier" means and encompasses any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. that are physiologically compatible. The composition may contain a pharmaceutically acceptable salt, such as an acid addition salt or a base addition salt (see, e.g., Berge et al. (1977) J Pharm Sci 66: 1-19).

[0379] Phosphate: As used herein, the term "phosphate" means a salt or ester of phosphoric acid. Polyphosphates are salts or esters of polymeric oxyanions formed by tetrahedral PO4 (phosphate) structural units linked together by shared oxygen atoms. As used herein, the term "diphosphate" refers to a polyphosphate comprising two phosphate structural units. As used herein, the term "triphosphate" refers to a polyphosphate comprising three phosphate structural units. In some embodiments, the present disclosure provides a RIG-I-like receptor agonist comprising a diphosphate moiety or a derivative or analog thereof connected to the 5' end. In some embodiments, the present disclosure provides a RIG-I-like receptor agonist comprising a triphosphate moiety or a derivative or analog thereof connected to the 5' end. In some embodiments, the derivative or analog thereof is a phosphate bioisostere.

[0380] Phosphate bioisosteres: As used herein, the term "phosphate bioisostere" (alternatively "phosphate mimetic") refers to a chemical substituent or group that has physical or chemical properties similar to those of a phosphate and produces biological properties that are roughly similar to those of phosphates containing diphosphate and triphosphate moieties. In drug design, the purpose of exchanging one bioisostere for another is to enhance the desired biological or physical properties of a compound without significantly changing the chemical structure. Bioisosteres are widely used in drug development and are used, for example, to reduce toxicity, alter bioavailability, or modify the activity or metabolism of a parent or lead compound (see, e.g., Rye and Baell (2005) Curr Med Chem 12(26):3127-3141; Elliot et al., (2012) Med Chem Com 3(7):735-751, which are incorporated herein by reference in their entirety.

[0381] Polypeptide: As used herein, the terms "polypeptide," "peptide," and "protein" are used interchangeably to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues is an artificial chemical mimetic of the corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.

[0382] Prevention: As used herein, the term "prevention" when used with respect to a condition refers to the administration of a composition that reduces the frequency or delays the onset of symptoms of a medical condition in a subject relative to a subject not receiving the composition.

[0383] Purified: As used herein, the term "purified" or "isolated" when applied to any protein (antibody or fragment) described herein refers to a polypeptide that has been separated or purified from components (e.g., proteins or other naturally occurring biological or organic molecules) that naturally accompany it, such as other proteins, lipids, and nucleic acids in the prokaryotes that express 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.

[0384] 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 and is used to establish a relationship between itself and one or more different RIG-I-like receptor ligands, wherein the relationship is the relative agonistic effect of the reference ligand and one or more different RIG-I-like receptor ligands. As used herein, the term means a RIG-I-like receptor ligand or agonist used as a competitor in a test or assay, such as those described herein (e.g., an IFN induction assay), wherein the assay can be used to discover, identify or develop one or more different agonists that bind to a RIG-I-like receptor.

[0385] RIG-I: As used herein, the term "RIG-I" refers to retinoic acid-inducible gene I RIG-I is a polypeptide (a specific member of the RIG-I-like receptor family) and is encoded by the DDX58 gene in humans. 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 listed in Table 4 (SEQ ID NO: 98) and herein:

[0386] MTTEQRRSLQAFQDYIRKTLDPTYILSYMAPWFREEEVQYIQAEKNNKGPMEAATLFLKFLLELQEEGWFRGFLDALDHAGYSGLYEAIESWDFKKIEKLEEYRLLLKRLQPEFKTRIIPTDIISDLSECLINQECEEILQICSTKGMMAGAEKLVECLLRSDKENWPKTLKLALEKERNKFSELWIVEKGIKDVETEDLEDKMETSDIQIFYQEDPECQNLSENSCPPSEVSDTNLYSPFKPRNYQLELALPAMKGKNTIICAPTGCGKTFVSLLICEHHLKKFPQGQKGKVVFFANQIPVYEQQKSVFSKYFERHGYRVTGISGATAENVPVEQIVENNDIIILTPQILVNNLKKGTIPSLSIFTLMIFDECHNTSKQHPYNMIMFNYLDQKLGGSSGPLPQVIGLTASVGVGDAKNTDEALDYICKLCASLDASVIATVKHNLEELEQVVYKPQKFFRKVESRISDKFKYIIAQLMRDTESLAKRICKDLENLSQIQNREFGTQKYEQWIVTVQKACMVFQMPDKDEESRICKALFLYTSHLRKYNDALIISEHARMKDALDYLKDFFSNVRAAGFDEIEQDLTQRFEEKLQELESVSRDPSNENPKLEDLCFILQEEYHLNPETITILFVKTRALVDALKNWIEGNPKLSFLKPGILTGRGKTNQNTGMTLPAQKCILDAFKASGDHNILIATSVADEGIDIAQCNLVILYEYVGNVIKMIQTRGRGRARGSKCFLLTSNAGVIEKEQINMYKEKMMNDSILRLQTWDEAVFREKILHIQTHEKFIRDSQEKPKPVPDKENKKLLCRKCKALACYTADVRVIEECHYTVLGDAFKECFVSRPHPKPKQFSSFEKRAKIFCARQNCSHDWGIHVKYKTFEIPVIKIESFVVEDIATGVQTLYSKWKDFHFEKIPFDPAEMSK

[0387] (NCBI accession number: NP_055129.2)

[0388] RIG-I-like receptor: As used herein, the term "RIG-I-like receptor" (abbreviated "RLR") refers to any member of the DExD / H-box RNA helicase family that acts as a cytoplasmic pattern recognition sensor for pathogen-associated molecular patterns (PAMPs) commonly found in viral RNA. Upon ligand binding, RLRs signal downstream transcription factor activation to drive type 1 interferon (IFN) production and antiviral gene expression, which triggers an intracellular immune response to control viral infection. Three RLR members have been identified: RIG-I (retinoic acid-inducible gene 1), MDA5 (melanoma differentiation-associated factor 5), and LGP2 (homolog of Laboratory of Genetics and Physiology protein 2 and mouse D11lgp2) (Loo and Gale (2011) Immunity 34(5):680-692).

[0389] 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 completely promotes, induces, increases, and / or activates a biological activity, response, and / or one or more downstream pathways mediated by RLR signaling or other RLR-mediated functions. Examples of RIG-I receptor agonists are provided herein.

[0390] 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 fragment or portion thereof, which is continuously maintained under physiological conditions and characterized as a low free energy state. Typical examples of stable RNA secondary structures include duplexes, hairpins and stem loops. It is known in the art that stable RNA secondary structures exhibit various biological activities. The term "stable" as used with respect to polynucleotide duplexes means that the duplex remains hybridized, structured or annealed essentially exclusively in the form of a duplex under physiological conditions or under typical salt and temperature conditions used in nucleic acid diagnostic or therapeutic applications.

[0391] 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 with 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.

[0392] 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 a T cell receptor on the cell surface. There are several subpopulations 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 TFH cells), cytotoxic T cells (also known as 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 γδT cells (γδT cells) (including Vγ9 / Vδ2T cells). Any one or more of the aforementioned or unmentioned T cells may be a target cell type for the methods of use of the present invention.

[0393] T cell activation: As used herein, the term "T cell activation" or "activation of T cells" refers to a cellular process in which a mature T cell expressing an antigen-specific T cell receptor on its surface recognizes its cognate antigen and responds by entering the cell cycle, secreting cytokines or lytic enzymes, and initiating or becoming capable of performing cell-based effector functions. T cell activation requires at least two signals to become fully activated. The first signal occurs after the T cell antigen-specific receptor (TCR) engages through the antigen major histocompatibility complex (MHC), and the second signal occurs through the subsequent engagement of a co-stimulatory molecule (e.g., CD28). These signals are transmitted to the nucleus and result in clonal expansion of T cells, upregulation of activation markers on the cell surface, differentiation into effector cells, induction of cytotoxicity or cytokine secretion, induction of apoptosis, or a combination thereof.

[0394] 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.

[0395] Quadruplex: As used herein, the term "quadruplex" refers to a type of four-base ring motif found in hairpin or stem-loop RNA secondary structures that caps the duplex at one end, thereby connecting the two strands comprising the duplex and providing stability to the hairpin structure.

[0396] Therapeutic agent: As used herein, the term "therapeutic agent" refers to any agent that has a therapeutic, diagnostic, and / or prophylactic effect and / or elicits a desired biological and / or pharmacological effect when administered to a subject.

[0397] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" or "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 will elicit a desired biological or medical response, e.g., cure or at least partially inhibit a condition or disease in a patient already suffering from the disease and its complications (e.g., improvement of one or more symptoms of cancer). An amount effective for this use will depend on the severity of the condition being treated and the general state of the patient's own immune system.

[0398] Treatment: As used herein, the terms "treat," "treating," and "treatment" refer to therapeutic or preventive measures as described herein. The methods of "treatment" employ the administration of human antibodies of the present disclosure to a subject in need of such treatment, e.g., a subject in need of an enhanced immune response to a particular antigen or a subject who may eventually acquire such a disorder, to prevent, cure, delay, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or to prolong the survival of the subject beyond the expected survival in the absence of such treatment.

[0399] Tumor microenvironment: As used herein, the term "tumor microenvironment" (alternatively "cancer microenvironment"; abbreviated as TME) refers to the cellular environment or environment in which a tumor or neoplasm exists, including 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 extracellular matrix also include TME. Tumors and the surrounding microenvironment are closely related and constantly interact. Tumors can affect the microenvironment by releasing extracellular signals, promoting tumor angiogenesis, and inducing peripheral immune tolerance, and immune cells in the microenvironment can affect the growth and evolution of tumor cells.

[0400] Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art described in the present disclosure. Preferred methods and materials are described below, but methods and materials similar or equivalent to those described herein may also be used to practice or test the currently disclosed methods and compositions. All publications, patent applications, patents and other references mentioned herein are incorporated herein by reference in their entirety.

[0401] Equivalents and scope

[0402] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments of the invention described herein.The scope of the present disclosure is not intended to be limited to the above description, but rather is set forth in the appended claims.

[0403] In the claims, articles such as "a", "an", and "the" may mean one or more, unless indicated to the contrary by the context or otherwise obvious from the context. Claims or descriptions containing "or" between one or more members of a group are considered satisfied if one, more than one, or all of the members of the group are present in, used in, or otherwise relevant to a given product or process, unless indicated to the contrary by the context or otherwise obvious from the context. The present disclosure includes embodiments in which exactly one member of the group is present in, used in, or otherwise relevant to a given product or process. The present disclosure includes embodiments in which more than one or all of the members of the group are present in, used in, or otherwise relevant to a given product or process. In addition, it should be understood that the present disclosure covers all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the listed claims are introduced into another claim. For example, any claim attached to another claim may be amended to include one or more limitations found in any other claim attached to the same base claim. In addition, where the claims recite a composition, it should be understood that unless otherwise indicated or unless a contradiction or inconsistency would be obvious to one of ordinary skill in the art, methods of using the composition for any purpose disclosed herein are included, and methods of preparing the composition according to any preparation method disclosed herein or other methods known in the art are included.

[0404] Where elements are presented in a list format such as a Markush group format, it is understood that each subgroup of the elements is also disclosed, and any one or more elements may be removed from the group. It is understood that, in general, where the invention or aspects of the invention are referred to as including specific elements, features, etc., certain embodiments of the invention or certain aspects of the invention consist of or consist essentially of such elements, features, etc. For simplicity, these embodiments are not specifically described herein.

[0405] It should also be noted that the term "comprising" is intended to be open ended and allows but does not require the inclusion of additional elements or steps. Thus, when the term "comprising" is used herein, the term "consisting of" is also encompassed and disclosed.

[0406] When a range is given, the endpoints are included. In addition, it should be understood that unless otherwise indicated or otherwise becomes apparent from the context and understanding of one of ordinary skill in the art, the values ​​expressed as ranges may assume that any specific value or sub-range within the range described in different embodiments of the present invention is one-tenth of the lower limit unit of the range (unless otherwise clearly indicated in the context).

[0407] In addition, it should be understood that any specific 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 considered to be known to those of ordinary skill in the art, they may be excluded even if such exclusion is not explicitly proposed herein. Any specific embodiment of the composition of the present invention (e.g., any nucleic acid or protein encoded thereby; any production method; any method of use, etc.) may be excluded from any one or more claims for any reason, whether or not related to the existence of the prior art.

[0408] All cited sources, e.g., references, publications, databases, database entries, and techniques cited herein 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 description of this application, the description in this application shall prevail.

[0409] RIG-I-like receptors and their ligands

[0410] The present disclosure provides synthetic RNA ligands that specifically bind to RIG-I-like receptors (RLRs) and excite RLRs (RLR agonists). In some aspects, the present disclosure provides RLR agonists that can be used to treat cancer. In some aspects, the present disclosure provides RLR agonists that can be used to treat infectious diseases. In some embodiments, RLR agonists induce cytokine production. In some embodiments, RLR agonists increase the number of CD8+T cells in the tumor microenvironment. In some embodiments, RLR agonists induce protective anti-tumor immunity.

[0411] RIG-I-like receptors (RLRs) include a family of DExD / H-box RNA helicases that act as cytoplasmic pattern recognition receptors (PRRs) that sense the presence of pathogenic agents by recognizing pathogen-associated molecular patterns (PAMPs). In particular, the intracellular presence of non-self (e.g., viral) RNA is sensed by infected cells through the binding of RNA to RLRs, and can cause the initiation and regulation of antiviral immunity. Similar to most viral RNAs, endogenous mRNA and RNA polymerase III transcripts are also 5′-triphosphated, but eukaryotic mRNAs have a 5′-cap structure that prevents RIG-I activation by connecting to guanosine methylated at N7. These structural differences between viral RNA and self RNA, together with differences in intracellular localization, are considered to be able to achieve the effective function of RIG-I in resisting viral infection by preferentially detecting viral RNA. Molecular recognition and binding of non-self RNA ligands to RLRs propagate specific intracellular signaling events, ultimately activating transcription factors that drive type 1 interferon (IFN) production and antiviral gene expression. RLR-mediated induction of IFN and inflammatory cytokine production and antiviral gene expression initiates an immune response for controlling viral infection (Yoneyama et al. (2015) Curr Opin Immunol 32:48-53).

[0412] Three RLR family members have been identified: RIG-I (retinoic acid inducible gene 1) - the founding member and best characterized of the RLR family, MDA5 (melanoma differentiation-associated factor 5) and LGP2 (genetic and physiology laboratory protein 2 and homolog of mouse D11lgp2). RIG-I is an important component of the innate immune system and plays a key role in defending against infections caused by RNA viruses. In contrast to the Toll-like receptors TLR3, TLR7, TLR8 and TLR9 that detect nucleic acids in the endosomes of immune cell subsets, 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).

[0413] The high-resolution structure of the RIG-I / ligand complex provides molecular details of RIG-I binding to RNA ligands, particularly to the activating ligand, double-stranded 5'-triphosphated 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 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) Immunology 31(1):25-34). The crystal structure of the RIG-I / RNA complex shows that the protein binds to the backbone rather than the base, suggesting that the RNA sequence may not affect RIG-I binding or that the RNA sequence may exhibit an effect or activity that has not yet been characterized. To date, evidence for sequence-dependent differential interactions or affinities and activation of RIG-I-like receptors has not been described in the art (Schlee and Hartmann (2010) Molecular Therapy 18(7):1254-1262).

[0414] Therefore, the present disclosure provides RIG-I sample receptor (RLR) agonist including non-naturally occurring, synthetic and / or engineered RLR RNA ligands. In some respects, the present disclosure provides a kind of RLR agonist, it is specifically bound to RIG-I sample receptor (RLR), wherein the agonist includes a flat-ended hairpin RNA, the flat-ended hairpin RNA includes a first polynucleotide connected to a second polynucleotide by a joint, wherein the first polynucleotide is fully complementary to the second polynucleotide to form a duplex, wherein the duplex includes less than 19 base pairs, wherein the nucleotide closest to 5' of the first oligonucleotide includes a 5' diphosphate or triphosphate moiety or its derivative or analog, and wherein the agonist includes a certain sequence motif, and the sequence motif provides at least one biological activity of improvement mediated by the RLR relative to the agonist not including the sequence motif.

[0415] In some embodiments, the RLR agonists of the present disclosure comprise a sequence motif, wherein the sequence motif is selected from the group consisting of:

[0416] (i) GT repeat motif;

[0417] (ii) GA repeat motif;

[0418] (iii) AUCG repeat motif;

[0419] (iv) AU repeat motif;

[0420] (v) dipyrimidine motif;

[0421] (vi) dipurine motif;

[0422] (vii) pyrimidine triplet motif;

[0423] (viii) purine triad motif;

[0424] (ix) a palindromic sequence motif; and

[0425] (x) A combination of any one of (i) to (ix).

[0426] In some embodiments, the RLR agonists of the present disclosure comprise at least one increased biological activity, wherein the increased biological activity is selected from:

[0427] (i) RLR-mediated increase in cytokine production;

[0428] (ii) increase in RLR-mediated expression of interferon-stimulated genes;

[0429] (iii) increase in RLR-mediated intracellular signaling;

[0430] (iv) increase in binding affinity to RLR; and

[0431] (v) A combination of any of (i) to (iv).

[0432] In some embodiments, the RLR agonists of the present disclosure comprise a sequence motif, wherein the sequence motif is a GT repeat motif, the GT repeat motif comprising a sequence of <19, about 15 to 18, about 15, about 10 to 15, about 10, about 5 to 10, about 5, about 4, about 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 , wherein n=2 to 9. In some embodiments, the GT repeat motif is [GT]7. In some embodiments, the GT repeat motif is [GT]3, and wherein the GT repeat motif is followed by a purine triplet and UCG, respectively. In some embodiments, the purine triplet is GGA.

[0433] In some embodiments, the sequence motif is a GA repeat motif comprising a sequence of <19, about 15 to 18, about 15, about 10 to 15, about 10, about 5 to 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 their derivatives or analogs. In some embodiments, the GA repeat motif is [GA] n , wherein n = 2 to 9. In some embodiments, the GA repeat motif is [GA]7.

[0434] In some embodiments, the RLR agonists of the present disclosure comprise a sequence motif, wherein the sequence motif is an AUCG repeat motif, and the AUCG repeat motif comprises a sequence of <19, about 16, about 12 to about 16, about 12, about 8 to 12, about 6, about 16, 12, 8 adenine, uracil, cytosine and guanine nucleotides or their derivatives or analogs.

[0435] In some embodiments, the AUCG repeat motif is [AUCG] n , wherein n=2 to 4. In some embodiments, the AUCG repeat motif is [AUCG]3.

[0436] In some embodiments, the AUCG repeat motif is preceded by CG or a dipyrimidine motif. In some embodiments, the AUCG repeat motif is preceded by 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.

[0437] In some embodiments, the RLR agonist of the present disclosure includes an AUCG repeating motif, wherein one or more uridine nucleosides (U) are replaced by modified nucleosides. In some embodiments, wherein the modified nucleosides are ribothymidine (T). In some embodiments, the AUGC repeating motif is [AUCG]3, wherein the one or more uridine nucleosides (U) including the AUCG repeating motif are replaced by modified nucleosides, wherein the modified nucleosides are ribothymidine (T). In some embodiments, the AUGC repeating motif is [AUCG]3, wherein the one or more uridine nucleosides (U) including the AUCG repeating motif are replaced by modified nucleosides, wherein the modified nucleosides are ribothymidine (T), and wherein the AUGC repeating motif is preceded by GG.

[0438] In some embodiments, the RLR agonist of the present disclosure includes an AUCG repeating motif, wherein one or more guanosine nucleosides (G) are replaced by modified nucleosides. In some embodiments, the modified nucleosides are inosine (I). In some embodiments, the AUGC repeating motif is [AUCG]3, wherein the one or more guanosine nucleosides (G) including the AUCG repeating motif are replaced by modified nucleosides, wherein the modified nucleosides are ribothymidine (T), and wherein the AUGC repeating motif is preceded by GG.

[0439] In some embodiments, the RLR agonists of the present disclosure include an AUCG repeat motif, wherein the motif is preceded by IG. In some embodiments, the AUCG repeat motif is [AUCG]3 and is preceded by IG.

[0440] In some embodiments, the RLR agonists of the present disclosure include AUCG repeats, wherein one or more guanosine nucleosides (G) are replaced by inosine (I), wherein the AUCG repeats are preceded by inosine (I). In some embodiments, the guanosine nucleosides (G) comprising the AUCG repeats are replaced by inosine (I), wherein the AUCG repeats are preceded by inosine (I), wherein the nucleotide closest to 5' of the first polynucleotide includes inosine (I).

[0441] In some embodiments, the 5'-most nucleotide of the first oligonucleotide comprises inosine (I).

[0442] In some embodiments, the RLR agonists of the present disclosure include 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 purine triplet. In some embodiments, the purine 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.

[0443] In some embodiments, the RLR agonists of the present disclosure comprise a palindromic sequence, wherein the palindromic sequence comprises a sequence of <19, about 15 to 18, about 15, about 10 to 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 that produces a palindrome.

[0444] In some embodiments, the linker is flanked by AU. In some embodiments, the linker is flanked by an AU repeat motif, wherein the AU repeat motif is [AU] n , wherein n=2 to 3. In some embodiments, the AU repeat motif is [AU]2.

[0445] In some aspects, the present disclosure provides an RLR agonist that specifically binds to RLR, wherein the agonist comprises a blunt-ended hairpin RNA, the blunt-ended hairpin RNA comprises at least one or more nucleotides, the at least one or more nucleotides comprising inosine that is base-paired with cytidine, and wherein the agonist comprises the formula:

[0446] 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', where

[0447] (i) (N1-N2-X1) comprises a first polynucleotide comprising linked nucleotides N1, N2 and X1;

[0448] (ii) (X2-N3-N4) comprises a second polynucleotide comprising linked nucleotides X2, N3 and N4;

[0449] (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;

[0450] (iv) N1 base pairs with N4;

[0451] (v) N2 base pairs with N3;

[0452] (vi) N1 comprises a 5' diphosphate or triphosphate moiety or a derivative or analog thereof;

[0453] (vii) X1 and X2 are each an oligonucleotide comprising a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine and inosine;

[0454] (viii) X1 and X2 are complementary;

[0455] (ix) X1 and X2 are each 12 nucleotides to 16 nucleotides in length and are the same length; and

[0456] (x) L is a linker that covalently connects the first polynucleotide and the second polynucleotide.

[0457] In other aspects, 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, the blunt-ended hairpin RNA comprises a non-nucleotide linker, and wherein the agonist comprises the formula:

[0458] 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', where

[0459] (i) (N1-N2-X1) comprises a first polynucleotide comprising linked nucleotides N1, N2 and X1;

[0460] (ii) (X2-N3-N4) comprises a second polynucleotide comprising linked nucleotides X2, N3 and N4;

[0461] (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;

[0462] (iv) N1 base pairs with N4;

[0463] (v) N2 base pairs with N3;

[0464] (vi) N1 comprises a 5' diphosphate or triphosphate moiety or a derivative or analog thereof;

[0465] (vii) X1 and X2 are each an oligonucleotide comprising a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine and inosine;

[0466] (viii) X1 and X2 are complementary;

[0467] (ix) X1 and X2 are each 12 nucleotides to 16 nucleotides in length and are the same length; and

[0468] (x) L is a non-nucleotide linker that covalently links the first polynucleotide and the second polynucleotide.

[0469] In some embodiments, inosine (if present in the RLR agonist) base pairs with cytidine.

[0470] In some embodiments, the linker (L) is a nucleotide linker or a non-nucleotide linker.

[0471] In some aspects, the present disclosure provides a kind of RLR agonist, it specifically binds to RLR, wherein the agonist comprises a hairpin RNA with a flat end, and the hairpin RNA with a flat end comprises a nucleotide linker or a non-nucleotide linker. RNA hairpin is one of the most common RNA secondary structure elements, wherein the hybridized part or "stem" of the hairpin is often covered by RNA four-membered ring. RNA four-membered ring is composed of characteristic four-membered ring nucleotides that form a compact and stable structure. Although it can be formed by many different nucleotide sequences, UNCG (N = A, C, G or U), GNRA (R = A or G) and CUUG four-membered ring are the most common. The four-membered ring usually helps to start the RNA folding process, and provides a tertiary contact for or between RNAs and a site for protein binding, thereby promoting the assembly of ribonucleoprotein particles. Additional descriptions of tetraloops can be found in Cheong, H., Kim, N., and Cheong, C. (2015) RNA Structure: Tetraloops. Encyclopedia of Life Sciences (eLS) John Wiley & Sons, Ltd (Eds.), which is incorporated herein by reference in its entirety.

[0472] Therefore, in some embodiments, the RLR agonist of the present disclosure includes a nucleotide linker, and the nucleotide linker includes a four-membered ring. In some embodiments, the nucleotide sequence of the four-membered ring is selected from the group consisting of:

[0473] (a) UNCG, where N = A, C, G or U;

[0474] (b) GNRA, wherein N = A, C, G or U, and wherein R = A or G;

[0475] (c) ANYA, wherein N = A, C, G or U, and wherein Y = C or T;

[0476] (d) CUYG, where Y = C or T;

[0477] (e) UMAC, where M = A or C; and

[0478] (f)CUUG.

[0479] In some embodiments, the nucleotide linker comprises the nucleotide sequence UUUGAU or UGUUU. In some embodiments, the sequence of the four-membered ring is UUCG. In some embodiments, the sequence of the four-membered ring is GAUC. In some embodiments, the nucleotide linker comprises the nucleotide sequence UUUGAU. In some embodiments, the nucleotide linker comprises the nucleotide sequence UGUUU.

[0480] In other respects, RLR agonists of the present disclosure include non-nucleotide linkers. As described herein, nucleic acid loops (e.g., four-membered rings) are common elements found in nucleic acid secondary structures. Nucleotide loops appear in the domain of folding present in the intrachain duplex. Synthetic nucleic acids designed to contain hairpin loops including non-nucleotide linkers (e.g., non-nucleotide linkers) can replace several nucleotides of the duplex structure of bridge folding. Non-nucleotide groups have also been used as linkers in non-folding structures. Such linkers can be useful substitutes for natural nucleotide linkers (e.g., four-membered rings). For example, it can shorten the synthesis of nucleic acids with desired secondary structures by several steps, because a relatively long non-nucleotide linker replaces several single nucleotides that can usually constitute a ring. Such non-natural loops or linkers (e.g., non-nucleotide linkers) can impart resistance to degradation caused by nucleases, which usually act on natural loop structures in biological contexts (e.g., in cells or in the circulation of subjects when applied). Compared to nucleotide loops and / or linkers, non-nucleotide linkers are also likely to provide more stable folding structures. Additional description of non-nucleotidic 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.

[0481] Thus, in some embodiments, the RLR agonist of the present disclosure comprises a non-nucleotide linker selected from the group consisting of:

[0482] (a) a glycol connector; and

[0483] (b) Alkyl linker.

[0484] In some embodiments, the non-nucleotide linker is a hexaethylene glycol linker. In some embodiments, the non-nucleotide linker is a C9 alkyl linker.

[0485] 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 thereof comprises a phosphate bioisostere selected from the group consisting of phosphonates, phosphonothioates, phosphorothioates, sulfates, sulfonates, sulfamates, thiazolidinones, carboxylates, malonates, boric acid, benzoborazole, borophosphates, and squaramides.

[0486] In some embodiments, the agonist comprises a modified nucleotide, a modified nucleoside, or a modified nucleobase, or a combination thereof. In some embodiments, the agonist comprises a modification of an internucleotide bond or of a polynucleotide backbone.

[0487] In some aspects, the RLR agonists of the present disclosure exhibit at least one or more of the following properties:

[0488] (a) specifically binds to one or more RLRs (e.g., RIG-1, MDA5 and / or LGP2);

[0489] (b) increase RLR-mediated cytokine production;

[0490] (c) increase RLR-mediated expression of interferon-stimulated genes (ISGs);

[0491] (d) increase RLR-dependent intracellular signaling;

[0492] (e) increasing the stability of the duplex;

[0493] (f) increase binding affinity to RLR;

[0494] (g) reduce off-target binding;

[0495] (h) Increase biological half-life;

[0496] (i) Increase biodistribution and bioavailability;

[0497] (j) increasing and / or enhancing uptake into cells and / or tissues;

[0498] (k) reducing immunogenicity; and

[0499] (l) A combination of any one of (a) to (k).

[0500] In some aspects, 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 the formula:

[0501] 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', where

[0502] (i) (N1-N2-X1) comprises a first polynucleotide comprising linked nucleotides N1, N2 and X1;

[0503] (ii) (X2-N3-N4) comprises a second polynucleotide comprising linked nucleotides X2, N3 and N4;

[0504] (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;

[0505] (iv) N1 base pairs with N4;

[0506] (v) N2 base pairs with N3;

[0507] (vi) N1 comprises a 5' diphosphate or triphosphate moiety or a derivative or analog thereof;

[0508] (vii) X1 and X2 are each an oligonucleotide comprising a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine and inosine;

[0509] (viii) X1 and X2 are complementary;

[0510] (ix) X1 and X2 are each 12 nucleotides to 16 nucleotides in length and are the same length; and

[0511] (x) L is a linker that operably connects the first polynucleotide and the second polynucleotide,

[0512] 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, and wherein the inosine nucleoside base pairs with a cytidine in the hairpin RNA.

[0513] In some embodiments, N1 includes inosine and N4 includes cytidine. In some embodiments, N1 includes cytidine and N4 includes inosine. In some embodiments, N2 includes inosine and N3 includes cytidine. In some embodiments, N2 includes cytidine and N3 includes inosine. In some embodiments, N1 includes guanosine. In some embodiments, N2 includes guanosine. In some embodiments, N1 includes cytidine. In some embodiments, N2 includes cytidine. In some embodiments, N2 includes cytidine. In some embodiments, N1 and N2 include guanosine and N3 and N4 include cytidine. In some embodiments, N1 and N2 include cytidine and N3 and N4 include guanosine. In some embodiments, N1 and N2 include inosine and N3 and N4 include cytidine. In some embodiments, N1 and N2 include cytidine and N3 and N4 include guanosine. In some embodiments, N1 and N2 include inosine and N3 and N4 include cytidine. In some embodiments, N1 and N2 include cytidine and N3 and N4 include inosine. In some embodiments, N1 includes inosine and N4 includes cytidine, and X1 and / or X2 each include at least one inosine. In some embodiments, N2 includes inosine and N3 includes cytidine, and X1 and / or X2 each include at least one inosine. In some embodiments, N1 and N2 include guanosine, N3 and N4 include cytidine, and X1 and / or X2 each include at least one inosine. In some embodiments, N1 and N2 include guanosine, and N3 and N4 include cytidine, and X1 and X2 each include at least one inosine. In some embodiments, N1 and N2 include cytidine, and N3 and N4 include guanosine, and X1 and X2 each include at least one inosine. In some embodiments, N1 and N2 include guanosine, and N3 and N4 include cytidine, and X1 and X2 each include inosine and do not include guanosine nucleosides. In some embodiments, N1 and N2 include cytidine, and N3 and N4 include guanosine, and X1 and X2 each include inosine and do not include guanosine nucleosides. In some embodiments, N1 and N2 include inosine, and N3 and N4 include cytidine, and X1 and / or X2 each include at least one inosine. In some embodiments, N1 and N2 include inosine, and N3 and N4 include cytidine, and X1 and X2 each include at least one inosine. In certain embodiments, N1 and N2 include cytidine, and N3 and N4 include inosine, and X1 and / or X2 each include at least one inosine. In certain embodiments, N1 and N2 include inosine, and N3 and N4 include cytidine, and X1 and X2 include inosine and do not include guanosine nucleosides. In certain embodiments, N1 and N2 include cytidine, and N3 and N4 include inosine, and X1 and X2 include inosine and do not include guanosine nucleosides. In certain embodiments, X1 and X2 are each 12 nucleotides and include 1, 2, 3 or 4 inosine nucleosides. In certain embodiments, X1 and X2 are each 13 nucleotides and include 1, 2, 3, 4 or 5 inosine nucleosides. In certain embodiments, X1 and X2 are each 14 nucleotides and include 1, 2, 3, 4, 5 or 6 inosine nucleosides.In some embodiments, each of X1 and X2 is 15 nucleotides and includes 1, 2, 3, 4, 5, 6, or 7 inosine nucleosides. In some embodiments, each of X1 and X2 is 16 nucleotides and includes 1, 2, 3, 4, 5, 6, 7, or 8 inosine nucleosides. In some embodiments, each of X1 and X2 is 12 nucleotides and includes at least 10%, 20%, 30%, or 40% inosine nucleosides.

[0514] In some aspects, 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 the formula:

[0515] 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', where

[0516] (i) (N1-N2-X1) comprises a first polynucleotide comprising linked nucleotides N1, N2 and X1;

[0517] (ii) (X2-N3-N4) comprises a second polynucleotide comprising linked nucleotides X2, N3 and N4;

[0518] (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;

[0519] (iv) N1 base pairs with N4;

[0520] (v) N2 base pairs with N3;

[0521] (vi) N1 comprises a 5' diphosphate or triphosphate moiety or a derivative or analog thereof;

[0522] (vii) X1 includes the sequence motif [AUCN5] x , wherein N5 is comprised of guanosine or inosine, wherein x is an integer whose value indicates the number of sequence motifs, and wherein x=3 or 4;

[0523] (viii) X2 includes the sequence motif [CN6AU] y , wherein N6 comprises guanosine or inosine, wherein y is an integer whose value indicates the number of sequence motifs, and wherein y=3 or 4;

[0524] (ix) L is a linker that operably connects the first polynucleotide and the second polynucleotide,

[0525] Optionally, at least one of N1, N2, N3, and N4 is inosine, and wherein the inosine nucleoside is base paired 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 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 include guanosine (G), and N3 and N4 include cytidine (C). In some embodiments, N1 and N2 include cytidine (C), and N3 and N4 include guanosine (G). In some embodiments, N1 and N2 include inosine (I), and N3 and N4 include cytidine (C). In some embodiments, N1 and N2 include cytidine (C), and N3 and N4 include inosine (I).

[0526] In some embodiments, the linker (L) is a nucleotide linker or a non-nucleotide linker. In some embodiments, the linker (L) comprises a nucleotide linker of a four-membered ring, wherein the nucleotide sequence of the four-membered ring is selected from the group consisting of:

[0527] (a) UNCG, where N = A, C, G or U;

[0528] (b) GNRA, wherein N = A, C, G or U, and wherein R = A or G;

[0529] (c) ANYA, wherein N = A, C, G or U, and wherein Y = C or T;

[0530] (d) CUYG, where Y = C or T;

[0531] (e) UMAC, where M = A or C; and

[0532] (f)CUUG.

[0533] 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.

[0534] In some embodiments, the linker (L) is a nucleotide linker comprising a four-membered ring, wherein the sequence of the four-membered ring is UUCG. In some embodiments, the sequence of the four-membered ring is GAUC.

[0535] In some embodiments, the linker (L) is a non-nucleotidic linker selected from the group consisting of:

[0536] (a) a glycol connector; and

[0537] (b) Alkyl linker.

[0538] In some embodiments, the non-nucleotide linker is a hexaethylene glycol linker. In some embodiments, the non-nucleotide linker is a C9 alkyl linker.

[0539] 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 thereof comprises a phosphate bioisostere selected from the group consisting of phosphonates, phosphonothioates, phosphorothioates, sulfates, sulfonates, sulfamates, thiazolidinones, carboxylates, malonates, boric acid, benzoborazole, borophosphates, and squaramides.

[0540] In some embodiments, the RLR agonist comprises a modified nucleotide, a modified nucleoside, or a modified nucleobase, or a combination thereof. In some embodiments, the agonist comprises a modification of an internucleotide bond or a polynucleotide backbone.

[0541] In some aspects, the RLR agonist exhibits at least one or more of the following properties:

[0542] (a) specifically binds to one or more RLRs (e.g., RIG-1, MDA5 and / or LGP2);

[0543] (b) increase RLR-mediated cytokine production;

[0544] (c) increase RLR-mediated expression of interferon-stimulated genes (ISGs);

[0545] (d) increase RLR-dependent intracellular signaling;

[0546] (e) increasing the stability of the duplex;

[0547] (f) increase binding affinity to RLR;

[0548] (g) reduce off-target binding;

[0549] (h) Increase biological half-life;

[0550] (i) Increase biodistribution and bioavailability;

[0551] (j) increasing and / or enhancing uptake into cells and / or tissues;

[0552] (k) reducing immunogenicity; and

[0553] (l) A combination of any one of (a) to (k).

[0554] In some aspects, 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, the blunt-ended hairpin RNA comprising a first polynucleotide connected to a second polynucleotide via a linker, wherein the first polynucleotide is sufficiently complementary to the second polynucleotide to form a duplex, wherein the duplex comprises less 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 provides at least one increased biological activity mediated by the RLR relative to an agonist that does not comprise the sequence motif, and wherein the agonist comprises a nucleotide sequence selected from the group consisting of: SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 and 36.

[0555] In some aspects, 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, the 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 less 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 provides at least one increased biological activity mediated by the RLR relative to an agonist that does not comprise the sequence motif, and wherein the first polynucleotide and the second polynucleotide comprise a nucleotide sequence selected from the group consisting of:

[0556] (i) SEQ ID NOs: 37 and 68, respectively;

[0557] (ii) SEQ ID NOs: 38 and 69, respectively;

[0558] (iii) SEQ ID NOs: 39 and 70, respectively;

[0559] (iv) SEQ ID NOs: 40 and 71, respectively;

[0560] (v) SEQ ID NOs: 41 and 72, respectively;

[0561] (vi) SEQ ID NOs: 42 and 73, respectively;

[0562] (vii) SEQ ID NOs: 43 and 74, respectively;

[0563] (viii) SEQ ID NOs: 44 and 75, respectively;

[0564] (ix) SEQ ID NOs: 45 and 76, respectively;

[0565] (x) are SEQ ID NOs: 46 and 77, respectively;

[0566] (xi) SEQ ID NOs: 47 and 78, respectively;

[0567] (xii) SEQ ID NOs: 48 and 79, respectively;

[0568] (xiii) SEQ ID NOs: 49 and 80, respectively;

[0569] (xiv) SEQ ID NOs: 50 and 81, respectively;

[0570] (xv) are SEQ ID NOs: 51 and 82, respectively;

[0571] (xvi) are SEQ ID NOs: 52 and 83, respectively;

[0572] (xvii) SEQ ID NOs: 53 and 84, respectively;

[0573] (xviii) are SEQ ID NOs: 54 and 85, respectively;

[0574] (xix) are SEQ ID NOs: 55 and 86, respectively;

[0575] (xx) are SEQ ID NOs: 56 and 87, respectively;

[0576] (xxi) are SEQ ID NOs: 57 and 88, respectively;

[0577] (xxii) SEQ ID NOs: 58 and 89, respectively;

[0578] (xxiii) are SEQ ID NOs: 59 and 89, respectively;

[0579] (xxiv) SEQ ID NOs: 60 and 90, respectively;

[0580] (xxv) are SEQ ID NOs: 61 and 91, respectively;

[0581] (xxvi) are SEQ ID NOs: 62 and 92, respectively;

[0582] (xxvii) are SEQ ID NOs: 63 and 91, respectively;

[0583] (xxviii) are SEQ ID NOs: 64 and 93, respectively;

[0584] (xxix) are SEQ ID NOs: 65 and 94, respectively;

[0585] (xxx) are SEQ ID NOs: 66 and 95, respectively;

[0586] (xxxi) are SEQ ID NOs: 67 and 96, respectively; and

[0587] (xxxii) are SEQ ID NOs: 63 and 97, respectively.

[0588] In some aspects, 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, the blunt-ended hairpin RNA comprising at least one or more nucleotides, the at least one or more nucleotides comprising inosine base-paired with cytidine, and wherein the agonist comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 22, 23, and 25.

[0589] In some aspects, 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, the blunt-ended hairpin RNA comprises at least one or more nucleotides, the at least one or more nucleotides comprising inosine base-paired with 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 comprise a nucleotide sequence selected from the group consisting of:

[0590] (i) SEQ ID NOs: 58 and 89, respectively;

[0591] (ii) SEQ ID NOs: 59 and 89, respectively; and

[0592] (iii) SEQ ID NO: 61 and 91, respectively.

[0593] In some aspects, 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, the blunt-ended hairpin RNA comprises 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 comprise a nucleotide sequence selected from the group consisting of:

[0594] (i) SEQ ID NOs: 37 and 68, respectively;

[0595] (ii) SEQ ID NOs: 38 and 69, respectively;

[0596] (iii) SEQ ID NOs: 39 and 70, respectively;

[0597] (iv) SEQ ID NOs: 40 and 71, respectively;

[0598] (v) SEQ ID NOs: 41 and 72, respectively;

[0599] (vi) SEQ ID NOs: 42 and 73, respectively;

[0600] (vii) SEQ ID NOs: 43 and 74, respectively;

[0601] (viii) SEQ ID NOs: 44 and 75, respectively;

[0602] (ix) SEQ ID NOs: 45 and 76, respectively;

[0603] (x) are SEQ ID NOs: 46 and 77, respectively;

[0604] (xi) SEQ ID NOs: 47 and 78, respectively;

[0605] (xii) SEQ ID NOs: 48 and 79, respectively;

[0606] (xiii) SEQ ID NOs: 49 and 80, respectively;

[0607] (xiv) SEQ ID NOs: 50 and 81, respectively;

[0608] (xv) are SEQ ID NOs: 51 and 82, respectively;

[0609] (xvi) are SEQ ID NOs: 52 and 83, respectively;

[0610] (xvii) SEQ ID NOs: 53 and 84, respectively;

[0611] (xviii) are SEQ ID NOs: 54 and 85, respectively;

[0612] (xix) are SEQ ID NOs: 55 and 86, respectively;

[0613] (xx) are SEQ ID NOs: 56 and 87, respectively;

[0614] (xxi) are SEQ ID NOs: 57 and 88, respectively;

[0615] (xxii) SEQ ID NOs: 58 and 89, respectively;

[0616] (xxiii) are SEQ ID NOs: 59 and 89, respectively;

[0617] (xxiv) SEQ ID NOs: 60 and 90, respectively;

[0618] (xxv) are SEQ ID NOs: 61 and 91, respectively;

[0619] (xxvi) are SEQ ID NOs: 62 and 92, respectively;

[0620] (xxvii) are SEQ ID NOs: 63 and 91, respectively;

[0621] (xxviii) are SEQ ID NOs: 64 and 93, respectively;

[0622] (xxix) are SEQ ID NOs: 65 and 94, respectively;

[0623] (xxx) are SEQ ID NOs: 66 and 95, respectively;

[0624] (xxxi) are SEQ ID NOs: 67 and 96, respectively; and

[0625] (xxxii) are SEQ ID NOs: 63 and 97, respectively.

[0626] In some aspects, the disclosure provides RLR agonists, 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.

[0627] RLR agonists comprising modified nucleobases, nucleosides or nucleotides

[0628] In certain embodiments, the RLR agonist of the present disclosure includes one or more modified nucleobases, nucleosides or nucleotides. In certain embodiments, compared with the unmodified RLR agonist of reference, the modified RLR agonist can have useful properties, including enhanced stability, intracellular retention, enhanced target binding and / or the increase of the induction of the innate immune response of the cell introducing the RLR agonist. Therefore, the purposes of the modified RLR agonist can enhance the efficiency of target binding, the intracellular retention of nucleic acid and the immunogenicity with reduction. In one embodiment, the agonist provided by the present disclosure is composed of one or more oligonucleotides, and the one or more oligonucleotides include at least one district modified to increase target binding affinity. The affinity of oligonucleotide to its target polypeptide (such as RLR receptor) can be determined by, for example, measuring the degree of fluorescence polarization (FP) when the fluorescently labeled oligonucleotide is combined with its target (Moerke (2009) "Current Protocol of Chemical Biology (Curr Protoc Chem Biol)" 1 (1): 1-15).

[0629] In another embodiment, the RLR agonist provided by the present disclosure is composed of at least one oligonucleotide, the at least one oligonucleotide comprising at least one region, the at least one region comprising at least one modified nucleobase, nucleoside or nucleotide that increases the stability of the duplex. The stability of the duplex can be determined conventionally by measuring the Tm of the duplex, the Tm being the temperature at which the two oligonucleotide chains comprising the duplex dissociate; the dissociation is detected by spectrophotometry. The higher the Tm, the greater the stability of the duplex.

[0630] In one embodiment, the region of the oligonucleotide modified to increase duplex stability includes at least one nucleotide modified at the 2' position of the sugar, most preferably a nucleotide modified by 2'-O-alkyl, 2'-O-alkyl-O-alkyl or 2'-fluorine. In another embodiment, the oligonucleotide comprising an RLR agonist is also modified to enhance nuclease resistance. Cells contain a variety of exonucleases and endonucleases that can degrade nucleic acids. Many nucleotides and nucleosides have been shown to make the oligonucleotides incorporated therein more resistant to nuclease digestion than unmodified oligonucleotides. Nuclease resistance is routinely measured by incubating the oligonucleotide with a cell extract or an isolated nuclease solution, and the degree of retention of the complete oligonucleotide over time is usually measured by gel electrophoresis. Compared to unmodified oligonucleotides, the oligonucleotides modified to enhance their nuclease resistance survive longer in their entirety. A variety of oligonucleotide modifications have been shown to enhance or confer nuclease resistance. Oligonucleotides containing at least one phosphorothioate modification are currently more preferred. In some cases, oligonucleotide modifications that enhance target binding affinity can also independently enhance nuclease resistance (De Mesmaeker et al., 1995, Acc. Chem. Res. 28:366-374).

[0631] Specific examples of preferred oligonucleotides contemplated by the present invention include oligonucleotides containing modified backbones, for example, phosphorothioate, phosphotriester, methylphosphonate, short chain alkyl or cycloalkyl sugar inter-linkages or short chain heteroatom or heterocyclic sugar inter-linkages. Most preferred are oligonucleotides with phosphorothioate backbones (including oligonucleotides synthesized in a stereospecific manner) and oligonucleotides with heteroatom backbones, particularly CH2—NH—O—CH2, CH2—N(CH3)—O—CH2 [referred to as methylene (methylimino) or MMI backbones], CH2—O—N(CH3)—CH2, CH2—N(CH3)—N(CH3)—CH2 and O—N(CH3)—CH2—CH2 backbones, wherein the natural phosphodiester backbone is represented as O—P—O—CH2). Amide backbones disclosed by DeMesmaeker et al. (1995, Review of Chemical Research 28:366-374) are also preferred. Oligonucleotides may also contain one or more substituted sugar moieties. Preferred oligonucleotides include one of the following at the 2′ position: OH, SH, SCH3, F, OCN, OCH3OCH3, OCH3O(CH2)nCH3, O(CH2)nNH2 or O(CH2)nCH3, wherein n is 1 to about 10; C1 to C10 lower alkyl, alkoxyalkoxy (also referred to 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; heterocycloalkylaryl; aminoalkylamino; polyalkylamino; substituted silyl; RNA cleavage group; reporter group; intercalator; a group for improving the pharmacokinetic properties of the oligonucleotide; or a group for improving the pharmacodynamic properties of the oligonucleotide, as well as other substituents with similar properties. Preferred modifications include 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—CH3), 2′-propoxy (2′—OCH2CH2CH3) and 2′-fluoro (2′-F). Similar modifications can also be made at other positions on the oligonucleotide, particularly at the 3′ position on the 3′ terminal nucleotide of the sugar and at the 5′ position of the 5′ terminal nucleotide. Oligonucleotides can also have sugar mimetics, such as cyclobutyl replacing the pentofuranosyl group.

[0632] Additionally or alternatively, oligonucleotides may also include modifications or substitutions of nucleobases (commonly referred to in the art as "bases"). As used herein, "unmodified" or "natural" nucleobases include adenine (A), guanine (G), thymine (T), cytosine (C), and uracil (U). Modified nucleobases include nucleobases that are only rarely or transiently found in natural nucleic acids, for example, hypoxanthine, 6-methyladenine, 5-me pyrimidine (particularly 5-methylcytosine (also known as 5-methyl-2′ deoxycytosine and commonly referred to in the art as 5-me-C)), 5-hydroxymethylcytosine (HMC), glycosyl HMC and gentiobiose HMC, as well as synthetic nucleobases, for example, 2-aminoadenine, 2-thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N6 (6-aminohexyl) adenine and 2,6-diaminopurine. Kornberg, A., DNA Replication, WH Freeman & Co., San Francisco, 1980, pp. 75-77; Gebeyehu, G. et al., 1987, Nucleic Acids Research 15:45-13). "Universal" bases known in the art, such as inosine, may also be included. 5-me-C substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2°C. (Sanghvi, YS, Crooke, ST and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278), and are currently preferred base substitutions.

[0633] Another modification of the oligonucleotides of the invention involves chemically attaching to the oligonucleotide one or more moieties or conjugates that enhance 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., 1989, 86, 6553), bile acids (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053), thioethers, for example, hexyl-S-triphenylmethanethiol (Manoharan et al., Ann. NY Acad. Sci., 1994, 4, 1053), 92, 660, 306; Manoharan et al., Bioorganic and Medicinal Chemistry Letters, 1993, 3, 2765), thiocholesterol (Oberhauser et al., Nucleic Acids Research, 1992, 20, 533), aliphatic chains, for example, dodecanediol or undecyl residues (Saison-Behmoaras et al., Journal of the European Molecular Biology Association (EMBO J.), 1991, 10, 111; Kabanov et al., FEBS Letters, 1991, 10, 111). Lett., 1990, 259, 327; Svinarchuk et al., Biochimie, 1993, 75, 49), phospholipids, polyamines or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969) or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651). Oligonucleotides including lipid moieties and methods for preparing such oligonucleotides are known in the art, for example, U.S. Pat. Nos. 5,138,045, 5,218,105 and 5,459,255.

[0634] The oligonucleotides of the invention may be provided in a prodrug form, which includes one or more moieties that are typically cleaved in vivo to yield the active oligonucleotide. An example of a prodrug approach is described by Imbach et al. in WO Publication 94 / 26764.

[0635] 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 into a single oligonucleotide or even within a single nucleoside within an oligonucleotide.

[0636] The oligonucleotides according to the invention are preferably about 8 to about 50 nucleotides in length. In the context of the present invention, it is to be understood that this encompasses non-naturally occurring oligomers having 8 to 50 monomers as described above.

[0637] The oligonucleotide used according to the present invention can be conveniently and routinely prepared by well-known solid phase synthesis techniques. The equipment used for this synthesis is sold by several suppliers including Applied Biosystems. Any other device for this synthesis can also be used; the actual synthesis of oligonucleotides is completely within the knowledge and ability of those of ordinary skill in the art. It is also well known to use similar techniques to prepare other oligonucleotides (such as thiophosphates and alkylated derivatives). It is also well known that similar techniques and commercially available modified amido and controlled pore glass (CPG) products are used, such as amido and / or CPG (available from Glen Research, Sterling Va., Sterling, Virginia) modified by biotin, fluorescein, acridine or psoralen to synthesize fluorescently labeled, biotinylated or other modified oligonucleotides, such as cholesterol-modified oligonucleotides.

[0638] In certain embodiments, the RLR agonist comprises one or more (e.g., 1, 2, 3 or 4) different modified core bases, nucleosides or nucleotides. In certain 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 core bases, nucleosides or nucleotides. In certain embodiments, relative to the corresponding unmodified RLR agonist, the modified RLR agonist has a reduced degradation in the cell into which the RLR agonist is introduced.

[0639] 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 5U), uridine 5-oxyacetic acid (cmo 5 U), uridine 5-oxyacetate (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-thio-uridine (mcm 5 s 2 U), 5-aminomethyl-2-thio-uridine (nm 5 s 2 U), 5-methylaminomethyl-uridine (mnm 5 U), 5-methylaminomethyl-2-thio-uridine (mnm 5 s 2 U), 5-methylaminomethyl-2-seleno-uridine (mnm 5 s 2 U), 5-carbamoylmethyl-uridine (ncm 5 U), 5-carboxymethylaminomethyl-uridine (cmnm 5 U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm 5 s 2 U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinemethyl-uridine (τm 5 U), 1-taurine methyl-pseudouridine, 5-taurine methyl-2-thio-uridine (τm 5 s 2 U), 1-tauromethyl-4-thio-pseudouridine, 5-methyl-uridine (m 5 U, i.e., having the nucleobase deoxythymidine), 1-methyl-pseudouridine (m 1 ψ), 5-methyl-2-thio-uridine (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 5D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-thio-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp 3 U), 1-methyl-3-(3-amino-3-carboxypropyl) pseudouridine (acp 3 ψ), 5-(isopentenylaminomethyl)uridine (inm 5 U), 5-(isopentenylaminomethyl)-2-thio-uridine (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-arabino-uridine, 5-(2-carbonylmethoxyvinyl)uridine, and 5-[3-(1-E-propenylamino)]uridine.

[0640] In some embodiments, the modified nucleobase is a modified cytosine. Exemplary nucleobases and nucleosides having a modified cytosine include 5-azura-cytidine, 6-azura-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 5 C), 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine (s 2C), 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, zebulin, 5-aza-zebulin, 5-methyl-zebulin, 5-aza-2-thio-zebulin, 2-thio-zebulin, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, lysine (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-arabino-cytidine, 2'-F-cytidine and 2'-OH-arabino-cytidine.

[0641] In some embodiments, the modified nucleobase is a modified adenine. Exemplary nucleobases and nucleosides having a modified adenine include α-thio-adenosine, 2-amino-purine, 2,6-diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenosine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyl-adenosine (m 1 A), 2-methyl-adenine (m 2 A), N6-methyl-adenosine (m 6 A), 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-glycylcarbamoyl-adenosine (g 6A), 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-hydroxynorvalylcarbamoyl-adenosine (hn 6 A), 2-methylthio-N6-hydroxyvalylcarbamoyl-adenosine (ms 2 hn 6 A), N6-acetyl-adenosine (ac 6 A), 7-methyl-adenine, 2-methylthio-adenine, 2-methoxy-adenine, α-thio-adenosine, 2′-O-methyl-adenosine (Am), N6,2′-O-dimethyl-adenosine (m 6 Am), N6,N6,2′-O-trimethyl-adenosine (m 6 2Am), 1,2′-O-dimethyl-adenosine (m 1 Am), 2′-O-ribosyl adenosine (phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1-thio-adenosine, 8-azido-adenosine, 2′-F-arabino-adenosine, 2′-F-adenosine, 2′-OH-arabino-adenosine and N6-(19-amino-pentaoxahedronadecanyl)-adenosine.

[0642] In some embodiments, the modified nucleobase is a modified guanine. Exemplary nucleobases and nucleosides having a modified guanine include α-thio-guanosine, inosine (I), 1-methyl-inosine (m 1 I), wyoside (imG), methyl wyoside (mimG), 4-demethyl-wyoside (imG-14), iso-wyoside (imG2), wyoside (yW), peroxy wyoside (o2yW), hydroxy wyoside (OhyW), undermodified hydroxy wyoside (OhyW*), 7-deaza-guanosine, braided glycoside (Q), epoxy braided glycoside (oQ), galactosyl-braided glycoside (galQ), mannosyl-braided glycoside (manQ), 7-cyano-7-deaza-guanosine (preQ0), 7-aminomethyl-7-deaza-guanosine (preQ1), archaeoside (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 (m1 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-methylguanosine (m 2, 7 Gm), 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-arabino-guanosine and 2'-F-guanosine.

[0643] In some embodiments, the RLR agonists of the present disclosure comprise a combination of one or more of the aforementioned modified nucleobases (eg, a combination of 2, 3, or 4 of the aforementioned modified nucleobases).

[0644] In certain embodiments, the RLR agonists of the present disclosure are uniformly modified (i.e., fully modified, modified throughout the entire sequence) for specific modifications. For example, the RLR agonists can be modified with 5-methyl-cytidine (m 5 C) uniformly modified, meaning that all cytosine residues in the mRNA sequence are modified with 5-methyl-cytidine (m 5 C) Substitution. Similarly, the RLR agonists of the present disclosure can be modified for any type of nucleoside residue present in the sequence by substitution with modified residues such as those indicated above.

[0645] 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 Publications: WO2012045075, WO2014081507, WO2014093924, WO2014164253, and WO2014159813.

[0646] The RLR agonists of the present disclosure may comprise combinations of modifications to sugars, nucleobases and / or internucleoside bonds. These combinations may comprise any one or more modifications described herein.

[0647] Examples of modified nucleosides and modified nucleosides combinations are provided in Tables 1 and 2 below. These combinations of modified nucleotides can be used to form RLR agonists of the present disclosure. In certain embodiments, modified nucleosides can partially or completely replace the natural nucleotides of RLR agonists of the present disclosure. As a non-limiting example, the natural nucleotide uridine can be replaced by the modified nucleosides 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%).

[0648] Table 1. Combinations of nucleoside modifications

[0649]

[0650]

[0651] Table 2. Modified nucleosides and their combinations

[0652]

[0653]

[0654]

[0655]

[0656]

[0657] According to the present disclosure, the polynucleotides of the present disclosure can be synthesized to include the combined modifications of Table 1 or Table 2 or a single modification.

[0658] In the case of listing a single modification, the nucleosides or nucleotides listed represent 100% of the A, U, G or C nucleotides or nucleosides that have been modified. In the case of listing percentages, these percentages represent the percentage of specific A, U, G or C nucleobase triphosphates in the total amount of A, U, G or C triphosphates. For example, the combination: 25% 5-aminoallyl-CTP+75% CTP / 25% 5-methoxy-UTP+75% UTP refers to the following polynucleotides: wherein 25% of the cytosine triphosphates are 5-aminoallyl-CTP and 75% of the cytosines are CTP; however, 25% of the uracils are 5-methoxy UTP and 75% of the uracils are UTP. In the case of not listing modified UTPs, naturally occurring ATP, UTP, GTP and / or CTP are used at 100% of the sites of those nucleotides found in the polynucleotides. In this example, all GTP and ATP nucleotides remain unmodified.

[0659] Method for preparing RLR agonists

[0660] The RLR agonists of the present disclosure can be produced by methods available in the art, including but not limited to in vitro transcription (IVT) and synthesis methods. Enzymatic (IVT), solid phase, liquid phase, combinatorial synthesis methods, cell synthesis and connection methods can be used. In one embodiment, the RLR agonist is prepared using an IVT enzymatic synthesis method. Methods for preparing polynucleotides by IVT are known in the art and are described in International Application 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 for in vitro transcription of the RLR agonists described herein.

[0661] During or after synthesis, non-natural modified nucleobases can be introduced into polynucleotides, such as RNA. In certain embodiments, the modification can be on a nucleoside bond, a purine base or a pyrimidine base or a sugar. In a particular embodiment, the modification can be at the end of a polynucleotide chain or anywhere else in a polynucleotide chain; introduced by chemical synthesis or by 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).

[0662] Polynucleotides or regions thereof can be conjugated to various functional moieties such as targeting or delivery agents, fluorescent markers, liquids, nanoparticles, etc. using enzymatic methods or chemical ligation methods. Conjugates of polynucleotides and modified polynucleotides are reviewed in Goodchild, Bioconjugate Chemistry, Vol. 1(3), 165-187 (1990). The synthesis of oligonucleotides, polynucleotides, and conjugates and ligations thereof are further 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.

[0663] Pharmaceutical compositions and formulations

[0664] In certain embodiments, the present invention provides pharmaceutical compositions comprising an RLR agonist and a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and / or adjuvant.

[0665] In certain embodiments, acceptable formulation materials are preferably non-toxic to the recipient at the dosages and concentrations employed. In certain embodiments, one or more formulation materials are used for sc and / or IV administration. In certain embodiments, pharmaceutical compositions may contain formulation materials for modifying, maintaining or preserving, for example, the pH, osmotic pressure, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or permeability of the composition. In certain embodiments, suitable formulation materials include, but are not limited to: amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobial agents; antioxidants (such as ascorbic acid, sodium sulfite or sodium bisulfite); buffers (such as borates, bicarbonates, Tris-HCl, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, β-cyclodextrin or hydroxypropyl-β-cyclodextrin); bulking agents; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose or dextrin); proteins (such as serum albumin, gelatin or immunoglobulins); colorants, flavorings and diluents; emulsifiers; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptide; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenylethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerol, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, cholesterol, tyloxapol); stability enhancers (such as sucrose or sorbitol); tonicity enhancers (such as alkali metal halides, preferably sodium chloride or potassium chloride, mannitol sorbitol); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants. (Remington's Pharmaceutical Sciences, 18th ed., A.R. Gennaro, ed., Mack Publishing Company, 1995). In certain embodiments, the formulation comprises PBS; 20 mM NaOAC, 50 mM NaCl at pH 5.2; and / or 10 mM NAOAC, 9% sucrose at pH 5.2. 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 dose. See, e.g., Remington's Pharmaceutical Sciences, supra. In certain embodiments, such compositions may affect the physical state, stability, rate of in vivo release, and / or rate of in vivo clearance of the RLR agonist.

[0666] In certain embodiments, the main medium or carrier in the pharmaceutical composition can be aqueous or non-aqueous in nature. For example, in certain embodiments, suitable medium or carrier can be water for injection, physiological saline solution or artificial cerebrospinal fluid that may be supplemented with other materials common in the composition for parenteral administration. In certain embodiments, saline includes isotonic phosphate buffered saline. In certain embodiments, neutral buffered saline or saline mixed with serum albumin is another exemplary medium. In certain embodiments, the pharmaceutical composition includes a Tris buffer of about pH 7.0 to 8.5, or an acetate buffer of about pH 4.0 to 5.5, so the pharmaceutical composition can further include sorbitol or a suitable substitute. In certain embodiments, the composition including the RLR agonist can be prepared by mixing the selected composition with the desired purity level with the optional formulation (Remington Pharmaceutical Science, see above) in the form of a lyophilized cake or an aqueous solution for storage. Additionally, in certain embodiments, the composition including the RLR agonist can be formulated into a lyophilized product using suitable excipients such as sucrose.

[0667] In certain embodiments, pharmaceutical compositions can be selected for parenteral delivery. In certain embodiments, compositions can be selected for suction or for delivery by the digestive tract (such as oral). The preparation of such pharmaceutically acceptable compositions is within the ability of those skilled in the art.

[0668] In certain embodiments, the formulation components are present at concentrations 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 the pH range of about 5 to about 8.

[0669] In certain embodiments, when parenteral administration is envisioned, the therapeutic composition may be in the form of a pyrogen-free, parenterally acceptable aqueous solution comprising an RLR agonist in a pharmaceutically acceptable vehicle. In certain embodiments, the vehicle for parenteral injection is sterile distilled water, wherein the RLR agonist is formulated as a sterile isotonic solution and is appropriately stored. In certain embodiments, the preparation may involve combining the desired molecule with a delivery vehicle or agent (such as injectable microspheres, bioerodible particles, polymeric compounds (such as polylactic acid, polyglycolic acid or polyethyleneimine (e.g., )), beads or liposomes). In certain embodiments, hyaluronic acid can also be used, and the hyaluronic acid can have the effect of promoting the duration lasting in the circulation. In certain embodiments, an implantable drug delivery device can be used to introduce the desired molecule.

[0670] In certain embodiments, the pharmaceutical composition can be formulated for inhalation. In certain embodiments, the RLR agonist can be formulated as a dry powder for inhalation. In certain embodiments, an inhalation solution comprising the RLR agonist can be formulated with a propellant for aerosol delivery. In certain embodiments, the solution can be atomized. Pulmonary administration is further described in PCT application No. PCT / US94 / 001875, which describes pulmonary delivery of chemically modified proteins.

[0671] In certain embodiments, it is envisioned that the formulation can be administered orally. In certain embodiments, the RLR agonist used in this way can be formulated with or without those carriers that are customarily used for the compound of solid dosage forms such as tablets and capsules. In certain embodiments, the capsule can be designed to release the active part of the formulation at a certain point in the gastrointestinal tract when bioavailability is maximized and pre-systemic degradation (pre-systemic degradation) is minimized. In certain embodiments, at least one other reagent can be included to promote the absorption of the RLR agonist. In certain embodiments, diluents, flavorings, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrants and adhesives can also be used.

[0672] In certain embodiments, the pharmaceutical composition may involve an effective amount of an RLR agonist in a mixture with a non-toxic excipient suitable for the manufacture of tablets. In certain embodiments, the solution may be prepared in unit-dose form by dissolving the tablet in sterile water or another suitable vehicle. In certain embodiments, suitable excipients include but are not limited to: an inert diluent such as calcium carbonate, sodium carbonate or bicarbonate, lactose or calcium phosphate; or a binder such as starch, gelatin or gum arabic; or a lubricant such as magnesium stearate, stearic acid or talc.

[0673] Other pharmaceutical compositions will be apparent to those skilled in the art, and include formulations related to RLR agonists in the form of formulations that are sustained or controlled for delivery. In certain embodiments, the technology for preparing various other sustained or controlled delivery modes, such as liposome carriers, bioerodible microparticles or porous beads and long-acting injections, is also known to those skilled in the art. See, for example, PCT application No. PCT / US93 / 00829, which describes the controlled release of porous polymer microparticles for delivery of pharmaceutical compositions. In certain embodiments, sustained release formulations may include a semi-permeable polymer matrix in the form of a molded article (e.g., a film or microcapsule). The sustained release matrix may comprise polyesters, hydrogels, polylactides (U.S. Pat. No. 3,773,919 and EP 058,481), copolymers of L-glutamic acid and γ-ethyl-L-glutamic acid (Sidman et al., Biopolymers 22:547-556 (1983)), poly(2-hydroxyethyl-methacrylic acid) (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-hydroxybutyric acid (EP 133,988). In certain embodiments, the slow release composition may also comprise liposomes, which may be prepared by any of several methods known in the art. See, eg, Eppstein et al., Proc. Natl. Acad. Sci. USA, 82:3688-3692 (1985); EP 036,676; EP 088,046 and EP 143,949.

[0674] The pharmaceutical composition for in vivo administration is usually sterile. In certain embodiments, this can be accomplished by means of filtration through a sterile filtration membrane. In certain embodiments, when the composition is freeze-dried, the sterilization performed using this method can be performed before or after freeze-drying and reorganization. In certain embodiments, the composition for parenteral administration can be stored in a lyophilized form or in a solution form. In certain embodiments, parenteral compositions are usually placed in a container with a sterile access port, for example, an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle.

[0675] In certain embodiments, once the pharmaceutical composition has been formulated, it can be stored in a sterile vial in the form of a solution, suspension, gel, emulsion, solid, or in the form of a dehydrated or lyophilized powder. In certain embodiments, such formulations can be stored in a ready-to-use form or in a form that is reconstituted (e.g., lyophilized) prior to administration.

[0676] In certain embodiments, kits for producing single-dose administration units are provided. In certain embodiments, the kit can contain both a first container with a dried protein and a second container with an aqueous formulation. In certain embodiments, kits containing single-chamber and multi-chamber prefilled syringes (e.g., liquid syringes and lyosyringes) are included.

[0677] In certain embodiments, the effective amount of the pharmaceutical composition including RLR agonists to be used in treatment will depend on, for example, treatment context and treatment goals. It will be appreciated by those skilled in the art that the appropriate dosage level for treatment according to certain embodiments will therefore vary in part according to the size (body weight, body surface or organ size) and / or condition (age and general health) of the delivered molecule, the indication using the RLR agonists, the route of administration and the patient. In certain embodiments, the clinician can titrate the dosage and modify the route of administration to obtain the best therapeutic effect.

[0678] In certain embodiments, the frequency of administration will consider the pharmacokinetic parameters of the RLR agonist in the formulation used. In certain embodiments, the clinician will use the composition before reaching the dosage for achieving the desired effect. In certain embodiments, the composition can therefore be used over time in a single dose or in the form of two or more dosages (which may or may not contain the desired molecule of the same amount), or by implanting a device or catheter in the form of continuous infusion. The further refinement of appropriate dose is routinely performed by those of ordinary skill in the art, and is within the scope of the task routinely performed. In certain embodiments, appropriate dose can be determined by using appropriate dose-response data.

[0679] In certain embodiments, the route of administration of the pharmaceutical composition complies with known methods, such as oral administration, injection by: intravenous, intraperitoneal, intracerebral (intra-organ parenchyma), intracerebroventricular, intramuscular, subcutaneous, intraocular, intra-arterial, intra-portal or intralesional routes; by sustained release systems or implant devices. In certain embodiments, the composition can be administered by bolus injection or continuously by infusion, or by implant devices. In certain embodiments, the individual elements of the combination therapy can be administered by different routes.

[0680] In certain embodiments, compositions can be applied locally by implanting membrane, sponge or another suitable material that has absorbed or encapsulated desired molecule. In certain embodiments, when using implant device, the device can be implanted in any suitable tissue or organ, and the delivery of desired molecule can be by diffusion, timed release group or continuous application. In certain embodiments, it may be desirable to use the pharmaceutical composition comprising RLR agonist in an ex vivo manner. In such cases, cells, tissues and / or organs removed from the patient are exposed to the pharmaceutical composition comprising RLR agonist, after which cells, tissues and / or organs are subsequently implanted back into the patient.

[0681] In certain embodiments, RLR agonists can be delivered by implanting certain cells that have been genetically engineered to express and secrete agonists using methods as described herein. In certain embodiments, such cells can be animal cells or human cells, and can be autologous, heterologous or xenogeneic. In other embodiments, cells can be immortalized. In certain embodiments, in order to reduce the chance of immune response, cells can be encapsulated to avoid the infiltration of surrounding tissues. In certain embodiments, encapsulating materials are typically biocompatible, semi-permeable polymer shells or membranes that allow one or more protein products to release, but prevent cells from being destroyed by the patient's immune system or other harmful factors from surrounding tissues.

[0682] In some aspects, the present disclosure provides a pharmaceutical composition and a pharmaceutically acceptable carrier for stimulating an immune response in a subject in need, treating or delaying the progression of cancer in a subject in need, or reducing or inhibiting tumor growth in a subject in need, including an RLR agonist according to the present disclosure. In some embodiments, the RLR agonist is formulated in a polyethyleneimine (PEI) carrier. In some embodiments, the PEI carrier is

[0683] application

[0684] Compositions as described herein can be used for diagnostic and therapeutic applications. For example, detectably labeled RLR agonists can be used in assays to detect the presence or amount of target protein in a sample (e.g., a biological sample). The composition can be used in an in vitro assay to study the inhibition of target function (e.g., cell signaling or response mediated by RLR). In some embodiments, for example, where the composition is combined with a target (e.g., a protein or polypeptide) and activates the target, the composition can be used as a positive control in the assay, and the assay is designed to identify the activity of the target protein or polypeptide and / or otherwise used to treat other novel compounds of the disease associated with the target protein or polypeptide. For example, in the assay, the RLR activation composition can be used as a positive control to identify other compounds (e.g., small molecules, aptamers, or antibodies) that induce, increase, or stimulate RLR function. The composition can also be used in the methods of treatment described in detail below.

[0685] Reagent test kit

[0686] The kit may include an RLR agonist as disclosed herein and instructions for use. In a suitable container, the kit may include an RLR agonist, one or more controls, and various buffers, reagents, enzymes, and other standard ingredients well known in the art.

[0687] The container may include at least one vial, well, test tube, flask, bottle, syringe or other container device into which the RLR agonist may be placed and in some cases may be appropriately aliquoted. In the case where additional components are provided, the test kit may contain additional containers into which this component may be placed. The test kit may also include a device for accommodating the RLR agonist and a reagent container for commercial sale that is tightly constrained. Such containers may include injection or blow-molded plastic containers in which the desired vial is retained. The container and / or test kit may include a label with instructions for use and / or warnings.

[0688] In some aspects, the present disclosure provides a kit comprising an RLR agonist provided by the present disclosure, or comprising 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, and optionally instructions for use in combination with one or more additional therapeutic agents.

[0689] In some embodiments, the agonist or the 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 drugs, cell death inducers, modulators (e.g., modulatory antibodies), cytotoxic agents, immune-based therapies, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules, vaccines, cellular immunotherapy, or a combination thereof.

[0690] In some embodiments, the RLR agonist or the pharmaceutical composition is administered before or after the one or more additional therapeutic agents, or wherein the one or more additional therapeutic agents are administered simultaneously with, before, or after the administration of the RLR agonist or the pharmaceutical composition.

[0691] In some embodiments, the one or more additional therapeutic agents are PD-1 / PD-L1 antagonists, TIM-3 antagonists, VISTA antagonists, adenosine A2AR antagonists, B7-H3 antagonists, B7-H4 antagonists, BTLA antagonists, CTLA-4 antagonists, IDO antagonists, KIR antagonists, LAG-3 antagonists, toll-like receptor 3 (TLR3) agonists, toll-like receptor 7 (TLR7) agonists, toll-like receptor 9 (TLR9) agonists.

[0692] 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).

[0693] 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).

[0694] How to use

[0695] The compositions of the present invention have a variety of in vitro and in vivo utilities involving the detection and / or quantification of RLRs and / or agonism of RLR function.

[0696] The above composition is particularly useful for treating or preventing a variety of cancers or infectious diseases in a subject. The composition can be administered to a subject, such as a human subject, using a variety of methods, which may depend 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 injection (IP), intramuscular injection (IM), intratumoral injection (IT) or intrathecal injection. The injection may be in the form of a bolus or continuous infusion.

[0697] Administration can be achieved by, for example, local infusion, injection or by means of an implant. The implant can have a porous material, a non-porous material or a colloid material, and the material comprises a film (such as a silicone rubber film) or a fiber. The implant can be configured to release the composition continuously or periodically to the subject. See, for example, U.S. Patent Application Publication No. 20080241223; U.S. Patent No. 5,501,856; No. 4,863,457; and No. 3,710,795; No. EP488401; and No. EP 430539, the disclosure of each of which is incorporated herein by reference in its entirety. The composition can be delivered to the subject by means of an implantable device based on, for example, diffusion, erodibility or convection systems (e.g., osmotic pumps, biodegradable implants, electrical diffusion systems, electroosmotic systems, vapor pressure pumps, electrolytic pumps, effervescent pumps, piezoelectric pumps, systems based on erosion or electromechanical systems).

[0698] In some embodiments, the RLR agonist is therapeutically delivered to a subject by topical administration.

[0699] The RLR agonists described herein can treat or prevent the appropriate dosage of cancer in the subject's body and can depend on multiple factors, including the age, sex and weight of the subject to be treated and the specific inhibitor compound used. Other factors affecting the dosage applied to the subject include the type or severity of cancer or infectious diseases, for example. For example, a subject with metastatic melanoma may need to apply a different dose of RLR agonists compared to a subject with glioblastoma. Other factors may include, for example, other medical conditions that affect the subject simultaneously or previously, the subject's overall health, the subject's genetic predisposition, diet, application time, excretion rate, drug combination, and any other additional therapeutic agent applied to the subject. It should also be understood that the specific dosage and treatment regimen for any particular subject will also depend on the judgment of the treatment medical practitioner (e.g., doctor or nurse). Suitable dosage is described herein.

[0700] The pharmaceutical composition may include an RLR agonist of a therapeutically effective amount as described herein. Such an effective amount may be easily determined by a person of ordinary skill in the art based in part on the combined effect of the RLR agonist or RLR agonist and one or more other activating agents (if more than one medicament is used) administered. The therapeutically effective amount of an RLR agonist as described herein may also vary according to the following factors: such as the disease state, age, sex and body weight of an individual and the ability of the agonist (and one or more other activating agents) to induce a desired response (e.g., reduction in tumor growth) in an individual. For example, an RLR agonist of a therapeutically effective amount may suppress (mitigate its severity or eliminate its occurrence) and / or prevent a particular condition, and / or any one of the symptoms of a particular condition known in the art or described herein. A therapeutically effective amount is also an amount in which a beneficial effect of treatment exceeds any toxicity or deleterious effect of the composition.

[0701] Suitable human doses of any RLR agonist described herein can be further evaluated in, for example, a Phase I dose escalation study. See, for example, 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.

[0702] In some embodiments, the composition contains any RLR agonist as described herein and one or more (e.g., two, three, four, five, six, seven, eight, nine, 10 or 11 or more) other therapeutic agents, so that the composition as a whole is effective for treatment. For example, the composition can contain an RLR agonist as described herein and an alkylating agent, wherein the agonist and the medicament are each in a concentration that is effective for treatment or prevention of cancer (e.g., melanoma) in the subject when combined.

[0703] 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 used, for example, to determine LD 50 (a dose lethal to 50% of the population) and ED 50 (The dose that is therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as LD 50 / ED 50RLR agonists that exhibit high therapeutic indices are preferred. Although compositions that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue and serves to minimize potential damage to normal cells and thereby reduce side effects.

[0704] The data obtained from cell culture assays and animal studies can be used to formulate a range of dosages for use in humans. For the RLR agonists described herein, the therapeutically effective dose can be estimated initially from cell culture assays. The dose in the animal model can be formulated to achieve an EC as determined in cell culture. 50 (i.e., the concentration of the agonist that achieves half-maximal inhibition of symptoms). This information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography. In some embodiments, for example, where local administration (e.g., to the eye or joints) is desired, cell culture or animal models can be used to determine the dose required to achieve a therapeutically effective concentration within the local site.

[0705] In some embodiments, the method can be performed in combination 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.

[0706] As described above, the compositions described herein (e.g., RLR agonist compositions) can be used to treat a variety of cancers, such as, but not limited to, Kaposi's sarcoma, leukemia, acute lymphocytic leukemia, acute myeloid leukemia, myeloblastic promyelocytic myelomonocytic monocytic erythroleukemia, 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, 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, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, sarcoma of the colon, colorectal cancer, 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, bronchogenic carcinoma, renal cell carcinoma, hepatocarcinoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor tumor), cervical cancer, uterine cancer, testicular cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, nasopharyngeal cancer, esophageal cancer, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain and central nervous system cancer cancers of the ovarian, ovarian, pancreatic, rectal, respiratory, sarcoma, skin, stomach, testicular, thyroid, uterine, and urinary tract.

[0707] In some aspects, 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.

[0708] In some aspects, the present disclosure provides a method for increasing RLR-mediated expression of one or more interferon-stimulated 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-stimulated genes in the cell.

[0709] In some aspects, 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.

[0710] In some aspects, 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.

[0711] In some aspects, 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 provided by the present disclosure or a pharmaceutical composition provided by the present disclosure.

[0712] In some aspects, 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.

[0713] In some aspects, the present disclosure provides a method for stimulating an immune response in a subject in need thereof, treating cancer in the subject, or delaying the progression of the cancer, or inhibiting tumor growth in the 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, wherein the agonist or the pharmaceutical composition increases RLR-mediated production of one or more cytokines in a cell, increases RLR-mediated expression of one or more interferon-stimulated genes in a cell, and / or increases RLR-dependent intracellular signaling in a cell, thereby stimulating the immune response, treating the cancer, or delaying the progression of the cancer, or inhibiting tumor growth.

[0714] Combinations of RLR agonists with additional therapeutic agents

[0715] In some embodiments, the RLR agonists described herein can be administered to a subject in the form of a monotherapy. Alternatively, the RLR agonists can be administered to a subject in the form of a combination therapy with another treatment (e.g., another treatment for cancer). For example, a combination therapy can include administering to a subject (e.g., a human patient) one or more additional agents that provide a therapeutic benefit to a subject suffering from cancer or having a risk of cancer.

[0716] In some embodiments of the methods provided by the present disclosure, an 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 drugs, cell death inducers, modulators (e.g., modulatory antibodies), cytotoxic agents, immune-based therapies, cytokines, activators or agonists of co-stimulatory molecules, inhibitors of inhibitory molecules, vaccines, cellular immunotherapy, or a combination thereof.

[0717] In some embodiments, the RLR agonist or the pharmaceutical composition is administered before or after administration of the one or more additional therapeutic agents, or wherein the one or more additional therapeutic agents are administered simultaneously with, before, or after administration of the agonist or the pharmaceutical composition.

[0718] In some embodiments, the one or more additional therapeutic agents are PD-1 / PD-L1 antagonists, TIM-3 antagonists, VISTA antagonists, adenosine A2AR antagonists, B7-H3 antagonists, B7-H4 antagonists, BTLA antagonists, CTLA-4 antagonists, IDO antagonists, KIR antagonists, LAG-3 antagonists, toll-like receptor 3 (TLR3) agonists, toll-like receptor 7 (TLR7) agonists, toll-like receptor 9 (TLR9) agonists.

[0719] Combination with chemotherapeutic agents

[0720] Suitable chemotherapeutic agents for use in combination and / or co-administration with the compositions of the present invention include, for example, paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthrancindione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. Additional agents include, for example, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil dacarbazine), alkylating agents (e.g., nitrogen mustard, thiotepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, cis-dichlorodiaminoplatin (II) (DDP), procarbazine, hexamethylmelamine, 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)), as well as antimitotic agents (e.g., vincristine and vinblastine) and temozolomide.

[0721] Combination with PD-1 / PD-L1 antagonists

[0722] In some embodiments, the RLR agonists or pharmaceutical compositions thereof provided by the present disclosure are 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 PD-1 / PD-L1 biological activity and / or one or more downstream pathways and / or cellular processes or other human PD-1 / PD-L1-mediated functions mediated by human PD-1 / PD-L1 signaling.

[0723] Therefore, provided herein are PD-1 / PD-L1 antagonists that directly or allosterically block, antagonize, suppress, inhibit or reduce the biological activity of PD-1 / PD-L1, and the PD-1 / PD-L1 antagonists include downstream pathways and / or cellular processes mediated by PD-1 / PD-L1 signaling, such as receptor binding and / or initiation of cellular responses to PD-1 / PD-L1. Also provided herein are PD-1 / PD-L1 antagonists that reduce the number or amount of human PD-1 or PD-L1 produced by cells or subjects.

[0724] In some embodiments, the present disclosure provides a PD-1 / PD-L1 antagonist that binds to human PD-1 and prevents, inhibits, or reduces the binding of PD-L1 to PD-1. In some aspects, the PD-1 / PD-L1 antagonist binds to mRNA encoding PD-1 or PD-L1 and prevents translation. In some embodiments, the PD-1 / PD-L1 antagonist binds to mRNA encoding PD-1 or PD-L1 and causes degradation and / or conversion.

[0725] 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 the 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 blocks the binding of PD-1 to PD-L1. In some embodiments, the PD-1 / PD-L1 antagonist blocks the binding of PD-1 to PD-L2. In some embodiments, the PD-1 / PD-L1 antagonist blocks the binding of PD-1 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.

[0726] In some embodiments, the PD-1 / PD-L1 antagonist inhibits the binding of PD-1 to its cognate ligand. In some embodiments, the PD-1 / PD-L1 antagonist inhibits the binding of PD-1 to PD-L1, PD-1 to PD-L2, or PD-1 to both PD-L1 and PD-L2. In some embodiments, the PD-1 / PD-L1 antagonist does not inhibit the binding of PD-1 to its cognate ligand.

[0727] In some embodiments, the PD-1 / PD-L1 antagonist is an isolated monoclonal antibody (mAb) or an 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 an antigen-binding fragment thereof that specifically binds to human PD-1. In some embodiments, the PD-1 / PD-L1 antagonist is an antibody or an antigen-binding fragment thereof that specifically binds to human PD-L1. In some embodiments, the PD-1 / PD-L1 antagonist is an antibody or an antigen-binding fragment thereof that specifically binds to human PD-L1. In some embodiments, the PD-1 / PD-L1 antagonist is an antibody or an antigen-binding fragment that binds to human PD-L1 and inhibits the binding of PD-L1 to PD-1. In some embodiments, the PD-1 / PD-L1 antagonist is an antibody or an antigen-binding fragment that binds to human PD-1 and inhibits the binding of PD-L1 to PD-1.

[0728] Several immune checkpoint antagonists that inhibit or disrupt the interaction between PD-1 and one or both of its ligands, PD-L1 and PD-L2, are in clinical development or are currently available to clinicians for the treatment of cancer.

[0729] Examples of anti-human PD-1 monoclonal antibodies or antigen-binding fragments thereof that may include PD-1 / PD-L1 antagonists in any of the compositions, methods, and uses provided in the present disclosure include, but are not limited to: (pembrolizumab, MK-3475, h409A11; see US8952136, US8354509, US8900587 and EP2170959, all patent applications are incorporated herein by reference in their entirety; Merck), (nivolumab, BMS-936558, MDX-1106, ONO-4538; see US7595048, US8728474, US9073994, US9067999, EP1537878, US8008449, US8779105 and EP2161336, all of which are incorporated herein by reference in their entirety; Bristol-Myers Squibb), MEDI0680 (AMP-514), BGB-A317 and BGB-108 (BeiGene), 244C8 and 388D4 (see WO2016106159, which are incorporated herein by reference in their 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.

[0730] Examples of anti-human PD-L1 monoclonal antibodies or antigen-binding fragments thereof that may include PD-1 / PD-L1 antagonists in any of the compositions, methods, and uses provided by the present disclosure include, but are not limited to: (avelumab, MSB0010718C, see WO2013 / 79174, which is incorporated herein by reference in its entirety; Merck / Pfizer), (Durvalumab, MEDI4736), (atezumab, MPDL3280A, RG7446; see WO2010 / 077634, the patent application is incorporated herein by reference in its entirety; Roche), MDX-1105 (BMS-936559, 12A4; see US7943743 and WO2013 / 173223, both patent applications are incorporated herein by reference in their entirety; Medarex / BMS) and FAZ053 (Novartis). Therefore, 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.

[0731] In some embodiments, the PD-1 / PD-L1 antagonist is an immunoadhesin that specifically binds to human PD-1 or human PD-L1, for example, a fusion protein containing an 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 immunoadhesion molecules that specifically bind to PD-1 are described in WO2010 / 027827 and WO2011 / 066342, both of which are incorporated herein by reference in their entirety. In some embodiments, the PD-1 / PD-L1 antagonist is AMP-224 (also known as B7-DCIg), which is a PD-L2-FC fusion protein that specifically binds to human PD-1.

[0732] It will be understood by those of ordinary skill 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.

[0733] In some embodiments, the PD-1 / PD-L1 antagonist is a small molecule, a nucleic acid, a peptide, a peptide mimetic, a protein, a carbohydrate, a carbohydrate derivative, or a glycopolymer. Exemplary small molecule PD-1 inhibitors are described in Zhan et al. (2016) Drug Discovery Today 21(6):1027-1036.

[0734] In some embodiments of the methods provided by the present disclosure, the RLR agonist is combined with a PD-1 / PD-L1 antagonist, wherein the PD-1 / PD-L1 antagonist is selected from the group consisting of: PDR001, (pembrolizumab), (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, (atezolizumab), (Avelumab), (durvalumab) and BMS-936559.

[0735] Combination with TIM-3 antagonists

[0736] In some embodiments, the RLR agonist or its pharmaceutical composition provided by the present disclosure is combined with a TIM-3 antagonist (e.g., administered in combination). The TIM-3 antagonist can 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).

[0737] Combination with LAG-3 antagonists

[0738] In some embodiments, the RLR agonist or its pharmaceutical composition provided by the present disclosure is combined with a LAG-3 antagonist (e.g., administered in combination). The LAG-3 antagonist can 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).

[0739] Combination with Toll-like receptor (TLR) agonists

[0740] In some embodiments, an RLR agonist or a pharmaceutical composition thereof provided by the present disclosure is combined with (eg, administered in combination with) a TLR agonist.

[0741] Toll-like receptors (TLRs) are a family of germline-encoded transmembrane proteins that promote pathogen recognition and activation of the innate immune system. (Hoffmann et al., (1999) Science 284: 1313-1318; Rock et al., (1998) Proceedings of the National Academy of Sciences 95: 588-593). TLRs are pattern recognition receptors (PRRs) and are expressed by cells of the innate immune system. Examples of known ligands for TLRs include Gram-positive bacteria (TLR-2), bacterial endotoxins (TLR-4), flagellin proteins (TLR-5), bacterial DNA (TLR-9), double-stranded RNA and poly I:C (TLR-3) and yeast (TLR-2). In vivo activation of TLRs initiates innate immune responses involving specific cytokines, chemokines and growth factors. Although all TLRs can activate certain intracellular signal transduction molecules, such as nuclear factor κβ (NF-κB) and mitogen-activated protein kinase (MAP kinase), the specific set of cytokines and chemokines released seem to be unique for each TLR. TLR7, 8 and 9 include subfamilies of TLRs located in the endosomal compartments or lysosomal compartments of immune cells such as dendritic cells and monocytes. Contrary to TLR7 and 9, which are highly expressed on plasmacytoid dendritic cells (pDC), TLR8 is mainly expressed on myeloid DC (mDC) and monocytes. This subfamily mediates the recognition of microbial nucleic acids such as single-stranded RNA.

[0742] Small, low molecular weight (less than 400 Daltons) synthetic imidazoquinoline compounds, similar to the purine nucleotides adenosine and guanosine, were the first TLR7 and TLR8 agonists to be identified. Many of these compounds have demonstrated antiviral and anticancer properties. For example, the TLR7 agonist imiquimod (ALDARA TM ) is approved by the US Food and Drug Administration as a topical agent for the treatment of skin lesions caused by certain strains of human papillomavirus. Imiquimod can also be used to treat primary skin cancer 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 genital herpes in humans.

[0743] TLR agonists according to the present disclosure can be any TLR agonists. For example, TLR agonists can encompass natural or synthetic TLR ligands, mutant proteins or derivatives of TLR ligands, peptide mimics of TLR ligands, small molecules that simulate the biological functions of TLR ligands, or antibodies that stimulate TLR receptors. TLR ligands are any molecules that bind to TLRs.

[0744] In some embodiments, the RLR agonist or its pharmaceutical composition 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.

[0745] In some embodiments, the TLR agonists provided by the present disclosure are combined with TLR3 agonists. TLR3 agonists are agonists that cause signaling responses through TLR3. Exemplary TLR3 agonists include, but are not limited to, polyinosinic acid: polycytidylic acid (poly I:C), (polyICLC), polyadenylic acid-polyuridylic acid (poly A:U), ( Bioconjugates) and

[0746] In some embodiments, the RLR agonists provided by the present disclosure are combined with polycytidylic acid: polycytidylic acid (poly I:C). In some embodiments, the RLR agonists are combined with In some embodiments, the RLR agonist is combined with poly(A:U). In some embodiments, an RLR agonist is combined with ( In some embodiments, an RLR agonist is combined with combination.

[0747] In some embodiments, the TLR agonist provided by the present disclosure is combined with a TLR7 agonist. A TLR7 agonist is an agonist that causes a signal transduction response through TLR7. Non-limiting examples of TLR7 agonists include single-stranded RNA (ssRNA), loxoribine (a guanosine analog derived at positions N7 and C8), imidazoquinoline compounds (e.g., imiquimod and resiquimod) or derivatives thereof. Additional exemplary TLR7 agonists include, but are not limited to, GS-9620 (visamod), imiquimod (ALDARA TM ) and resiquimod (R-848).

[0748] In some embodiments, the RLR agonists provided herein are combined with GS-9620 (visamod). In some embodiments, the RLR agonists are combined with imiquimod (ALDARA TM). In some embodiments, the RLR agonist is combined with resiquimod (R-848).

[0749] In some embodiments, the RLR agonist provided by the present disclosure is combined with a TLR9 agonist. A TLR9 agonist is an agonist that causes a signal transduction response through TLR9. Exemplary TLR9 agonists include but are not limited to CpG oligodeoxynucleotides (GpG ODN). In some embodiments, CpG ODN is a Class A CpG ODN (CpG-AODN), a Class B CpG ODN (CpG-B ODN) or a Class C CpG ODN (CpG-B ODN).

[0750] 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).

[0751] Other combinations

[0752] In some embodiments, the RLR agonists or pharmaceutical compositions thereof provided by the present disclosure are 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.

[0753] In some embodiments, an RLR agonist or a pharmaceutical composition thereof provided by the present disclosure is combined (eg, administered in combination) with an agonist comprising a polypeptide (eg, an antibody or an antigen-binding portion thereof) that specifically binds to CD137 (4-1BB).

[0754] In some embodiments, an RLR agonist or pharmaceutical composition thereof provided by the present disclosure is combined (eg, administered in combination) with an agonist comprising a polypeptide (eg, an antibody or an antigen-binding portion thereof) that specifically binds to CD134 (OX40).

[0755] RLR agonists as described herein can replace or enhance the therapy previously or currently administered. For example, when treated with RLR agonists, the administration of one or more additional activating agents can be stopped or reduced, such as administered at a lower level or dosage. In certain embodiments, the administration of the previous therapy can be maintained. In certain embodiments, the previous therapy will be maintained until the level of the RLR agonist reaches a level sufficient to provide a therapeutic effect. The two therapies can be administered in combination.

[0756] As defined herein, monitoring a subject (e.g., a human patient) for improvement in cancer means assessing a change in a subject's disease parameter, such as a decrease in tumor growth. In some embodiments, the assessment is performed at least one (1) hour after administration, e.g., at least 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, or 48 hours, or at least 1 day, 2 days, 4 days, 10 days, 13 days, 20 days, or more days, or at least 1 week, 2 weeks, 4 weeks, 10 weeks, 13 weeks, 20 weeks, or more weeks. The subject may be assessed during one or more of the following time periods: before treatment begins; during treatment; or after one or more elements of a treatment that have been administered. The assessment may include assessing the need for further treatment, e.g., assessing whether the dosage, frequency of administration, or duration of treatment should be changed. It may also include assessing the need to add or reduce a selected treatment modality, e.g., adding or reducing any treatment for a cancer described herein.

[0757] In some embodiments, the RLR agonists described herein are used, for example, to regulate the T cell response in the patient by increasing T cell activation and / or proliferation. The enhancement of T cell proliferation, IFN production and secretion and / or the cytolytic activity of T cells may be beneficial for patients who need them to treat diseases or conditions. Therefore, in some embodiments, the RLR agonists disclosed herein are used to patients in need, to induce or increase T cell activation, enhance T cell proliferation, induce the production and / or secretion of IFN and / or induce cytolytic T cell responses.

[0758] In certain embodiments, RLR agonists as described herein can be used in methods for detecting and / or quantifying human RLR in biological samples.Therefore, RLR agonists as described herein are used for diagnosing, predicting and / or determining the progress of a disease (e.g., cancer) in a patient.

[0759] Although the present disclosure has been described with reference to specific embodiments, it will be appreciated 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 specific circumstances, materials, combinations of substances, processes, one or more processing steps to the purpose, spirit and scope of the present disclosure. All such modifications are intended to fall within the scope of the present disclosure.

[0760] Examples

[0761] By referring to the following examples, the present disclosure can be more fully understood. However, the examples should not be construed as limiting the scope of the present disclosure. It should be understood that the examples and embodiments described herein are only for illustrative purposes, and the various modifications or changes made therefrom will inspire those skilled in the art and will be included in the spirit and scope of the present application and in the scope of the appended claims.

[0762] Example 1: Transfection of HuPBMCs with RLR agonists induces cytokine production in vitro

[0763] To determine the effects of RLR agonists including various modifications on cytokine induction, the ability of RLR agonists to induce cytokine production in vitro was assessed. Human peripheral blood mononuclear cells (huPBMCs) were prepared from two healthy donors and cultured at 2 × 10 cells / mL in 100 μL of RPMI 1640 cell culture medium supplemented with fetal calf serum (FCS), L-glutamine, and Pen / Strep. 5 The cells were seeded at a density of 10 cells / well in a standard 96-well tissue culture plate. Lipofectamine 2000 was used as the transfection reagent (except for G10 and ODN 2216, where direct incubation was applied, data not shown). Figure 1 huPBMCs were transfected with RLR agonists as indicated in . Cells were incubated at 37°C in a humidified incubator for 24 hours and then cell culture supernatants were harvested. Supernatants were immediately frozen and stored at -20°C. Samples were thawed once for expression of the cytokine IFN-α2a ( Figure 1 ) as well as IL-1β, IP-10, IL-6, IL-12p70, MCP-1, and MIP-1β were analyzed (data not shown). Figure 1 Shown is a dose-dependent induction of IFN-α secretion by human PBMCs treated with novel candidate RLR agonists comprising various modifications and / or sequence motifs. RLR agonists were added at 10 nM, 2 nM or 0.4 nM. The amount of IFN-α2a released by cells in response to RLR agonist transfection is given in pg / mL.

[0764] Tables 3 and 4 show the sequence of each RLR agonist. Table 3 also shows Figure 1 The sequence and number corresponding to each compound tested in . For example, Figure 1Compound X25224 in corresponds to "RIG7", which includes a first oligonucleotide comprising SEQ ID NO: 42 connected to a second oligonucleotide comprising SEQ ID NO: 73 via a linker "UUCG" and has a 5' diphosphate moiety. The sequence of RIG7 is also listed as SEQ ID NO: 6 in Table 4.

[0765] Table 3: Oligonucleotide combination table

[0766]

[0767]

[0768] (-) indicates no 5' phosphate; (pp) indicates 5' diphosphate; (ppp) indicates 5' triphosphate

[0769] Table 4: Sequence Listing

[0770]

[0771]

[0772]

[0773]

[0774]

Claims

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, wherein the blunt-ended hairpin RNA comprises a first polynucleotide connected to a second polynucleotide via a linker, wherein the first polynucleotide is sufficiently complementary to the second polynucleotide to form a duplex, wherein the duplex comprises less 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 [AUCG] n Repeat motif wherein n=3 and wherein the AUCG repeat motif closest to the 5' is preceded by a CG.

2. The agonist of claim 1, wherein the first polynucleotide comprises SEQ ID NO:63 and the second polynucleotide comprises SEQ ID NO:

91.

3. 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, wherein the blunt-ended hairpin RNA comprises a first polynucleotide connected to a second polynucleotide via a linker, wherein the first polynucleotide is sufficiently complementary to the second polynucleotide to form a duplex, wherein the duplex comprises less 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 [AUCG] n Repeat motif wherein n=2 and wherein the AUCG repeat motif closest to the 5' is preceded by CG.

4. The agonist of claim 3, wherein the first polynucleotide comprises SEQ ID NO:38 and the second polynucleotide comprises SEQ ID NO:

69.

5. 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, wherein the blunt-ended hairpin RNA comprises a first polynucleotide connected to a second polynucleotide via a linker, wherein the first polynucleotide is sufficiently complementary to the second polynucleotide to form a duplex, wherein the duplex comprises less 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 [GA]7 motif.

6. The agonist of claim 5, wherein the first polynucleotide comprises SEQ ID NO:48 and the second polynucleotide comprises SEQ ID NO:

79.

7. 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, wherein the blunt-ended hairpin RNA comprises a first polynucleotide connected to a second polynucleotide via a linker, wherein the first polynucleotide is sufficiently complementary to the second polynucleotide to form a duplex, wherein the duplex comprises less 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, The agonist comprises a [GT]n motif, wherein n=3 or n=7.

8. The agonist of claim 7, wherein the first polynucleotide comprises SEQ ID NO:55 and the second polynucleotide comprises SEQ ID NO:

86.

9. The agonist of claim 7, wherein the first polynucleotide comprises SEQ ID NO:56 and the second polynucleotide comprises SEQ ID NO:

87.

10. The agonist according to any one of claims 1-9, wherein the linker is a nucleotide linker.

11. The agonist of any one of claims 1-9, wherein the linker is a non-nucleotidic linker.

12. The agonist of claim 10, wherein the nucleotide linker comprises a four-membered ring comprising the sequence UUCG.

13. The agonist according to claim 11, wherein the non-nucleotidic linker is selected from a hexaethylene glycol linker or a C9 alkyl linker.

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

15. Use of an agonist according to any one of claims 1 to 9 in the preparation of a pharmaceutical product for use in a subject in need thereof: (i) stimulate immune response; (ii) to treat or delay the progression of cancer; or (iii) reducing or inhibiting tumor growth.

16. The use according to claim 15, wherein the pharmaceutical product further comprises one or more additional therapeutic agents, wherein the one or more additional therapeutic agents are selected from the group consisting of: PD-1 antagonists, PD-L1 antagonists, TIM-3 antagonists, VISTA antagonists, adenosine A2AR antagonists, B7-H3 antagonists, B7-H4 antagonists, BTLA antagonists, CTLA-4 antagonists, IDO antagonists, KIR antagonists, LAG-3 antagonists, Toll-like receptor 3 agonists, Toll-like receptor 7 agonists and Toll-like receptor 9 agonists.

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