U.S. patent number 10,907,161 [Application Number 16/389,432] was granted by the patent office on 2021-02-02 for synthetic rig-i-like receptor agonists.
This patent grant is currently assigned to Checkmate Pharmaceuticals, Inc.. The grantee listed for this patent is Checkmate Pharmaceuticals, Inc.. Invention is credited to Arthur M. Krieg, Aaron Jay Morris.
United States Patent |
10,907,161 |
Krieg , et al. |
February 2, 2021 |
Synthetic RIG-I-like receptor agonists
Abstract
The present disclosure relates to, inter alia, RNA molecules
(e.g., RNA hairpin agonists) that bind to and agonize RIG-I-like
receptors (RLRs), and to use of the molecules in methods for
treating, or ameliorating one or more symptoms of, a disorder
(e.g., cancer).
Inventors: |
Krieg; Arthur M. (Cambridge,
MA), Morris; Aaron Jay (Brighton, MA) |
Applicant: |
Name |
City |
State |
Country |
Type |
Checkmate Pharmaceuticals, Inc. |
Cambridge |
MA |
US |
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Assignee: |
Checkmate Pharmaceuticals, Inc.
(Cambridge, MA)
|
Family
ID: |
1000005335038 |
Appl.
No.: |
16/389,432 |
Filed: |
April 19, 2019 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20200063141 A1 |
Feb 27, 2020 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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62659999 |
Apr 19, 2018 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N
15/1138 (20130101); C12N 2310/531 (20130101); C12N
2310/331 (20130101) |
Current International
Class: |
C12N
15/113 (20100101) |
References Cited
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2012/130886 |
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Oct 2012 |
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2013/064584 |
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May 2013 |
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2013/097965 |
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Jul 2013 |
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2014/049079 |
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Apr 2014 |
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Aug 2014 |
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WO-2014159990 |
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Oct 2014 |
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2015/091578 |
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2016/011324 |
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2017/173427 |
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WO-2019126240 |
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Jun 2019 |
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WO |
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WO-2019246450 |
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Dec 2019 |
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WO |
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|
Primary Examiner: Whiteman; Brian
Attorney, Agent or Firm: Nelson Mullins Riley &
Scarborough LLP Mandragouras, Esq.; Amy E.
Parent Case Text
RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Application
No. 62/659,999, filed on Apr. 19, 2018. The entire content of the
aforementioned application is incorporated herein by this
reference.
Claims
The invention claimed is:
1. A synthetic RIG-I-like receptor (RLR) agonist that specifically
binds to a RIG-I-like receptor (RLR), wherein the agonist comprises
a blunt-ended, hairpin RNA comprising a first polynucleotide
connected to a second polynucleotide by a linker, 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 derivative or analog thereof; wherein the agonist comprises a
[AUCG]n repeat motif, wherein n=3, and wherein the 5' most AUCG
repeat motif is preceded by CG.
2. The agonist of claim 1, wherein the linker is a nucleotide
linker or non-nucleotide linker.
3. The agonist of claim 2, wherein the nucleotide linker comprises
a tetraloop comprising the sequence UUCG.
4. The agonist of claim 2, wherein the non-nucleotide linker is
selected from a hexaethylene glycol linker or a C9 alkyl
linker.
5. The agonist of claim 1, wherein the first polynucleotide
comprises SEQ ID NO: 50 and the second polynucleotide comprises SEQ
ID NO: 81.
6. The agonist of claim 5, wherein the linker is a nucleotide
linker or a non-nucleotide linker.
7. The agonist of claim 6, wherein the nucleotide linker comprises
a tetraloop comprising the sequence UUCG.
8. The agonist of claim 6, wherein the non-nucleotide linker is
selected from a hexaethylene glycol linker or a C9 alkyl
linker.
9. A pharmaceutical composition comprising the agonist of claim 1,
and a pharmaceutically acceptable carrier.
10. A pharmaceutical composition comprising the agonist of claim 5,
and a pharmaceutically acceptable carrier.
11. A pharmaceutical composition comprising the agonist of claim 7,
and a pharmaceutically acceptable carrier.
12. A synthetic RIG-I-like receptor (RLR) agonist that specifically
binds to a RIG-I-like receptor (RLR), wherein the agonist comprises
a blunt-ended, hairpin RNA comprising a first polynucleotide
connected to a second polynucleotide by a linker, wherein the first
polynucleotide is sufficiently complementary to the second
polynucleotide to form a duplex, wherein the duplex comprises less
than 19 base pairs, wherein the 5' most nucleotide of the first
oligonucleotide comprises a 5' diphosphate or triphosphate moiety,
or derivative or analog thereof; wherein the agonist comprises a
[AUCG]n repeat motif, wherein n=3, and wherein the 5' most AUCG
repeat motif is preceded by IG.
13. The agonist of claim 12, wherein the linker is a nucleotide
linker or a non-nucleotide linker.
14. The agonist of claim 13, wherein the nucleotide linker
comprises a tetraloop comprising the sequence UUCG.
15. The agonist of claim 13, wherein the non-nucleotide linker is
selected from a hexaethylene glycol linker or a C9 alkyl
linker.
16. The agonist of claim 12, wherein the first polynucleotide
comprises SEQ ID NO: 61 and the second polynucleotide comprises SEQ
ID NO: 91.
17. The agonist of claim 16, wherein the linker is a nucleotide
linker or a non-nucleotide linker.
18. The agonist of claim 17, wherein the nucleotide linker
comprises a tetraloop comprising the sequence UUCG.
19. The agonist of claim 17, wherein the non-nucleotide linker is
selected from a hexaethylene glycol linker or a C9 alkyl
linker.
20. A pharmaceutical composition comprising the agonist of claim
12, and a pharmaceutically acceptable carrier.
21. A pharmaceutical composition comprising the agonist of claim
16, and a pharmaceutically acceptable carrier.
22. A pharmaceutical composition comprising the agonist of claim
18, and a pharmaceutically acceptable carrier.
23. A synthetic RIG-I-like receptor (RLR) agonist that specifically
binds to a RIG-I-like receptor (RLR), wherein the agonist comprises
a blunt-ended, hairpin RNA comprising a first polynucleotide
connected to a second polynucleotide by a linker, wherein the first
polynucleotide is sufficiently complementary to the second
polynucleotide to form a duplex, wherein the duplex comprises less
than 19 base pairs, wherein the 5' most nucleotide of the first
oligonucleotide comprises a 5' diphosphate or triphosphate moiety,
or derivative or analog thereof; wherein the agonist comprises a
[AUCG]n repeat motif, wherein n=3, wherein the 5' most AUCG repeat
is preceded by GG, and wherein each G in the AUCG motif is
substituted by inosine.
24. The agonist of claim 23, wherein the linker is a nucleotide
linker or a non-nucleotide linker.
25. The agonist of claim 24, wherein the nucleotide linker
comprises a tetraloop comprising the sequence UUCG.
26. The agonist of claim 24, wherein the non-nucleotide linker is
selected from a hexaethylene glycol linker or a C9 alkyl
linker.
27. The agonist of claim 23, wherein the first polynucleotide
comprises SEQ ID NO: 59 and the second polynucleotide comprises SEQ
ID NO: 89.
28. The agonist of claim 27, wherein the linker is a nucleotide
linker or a non-nucleotide linker.
29. The agonist of claim 28, wherein the nucleotide linker
comprises a tetraloop comprising the sequence UUCG.
30. The agonist of claim 28, wherein the non-nucleotide linker is
selected from a hexaethylene glycol linker or a C9 alkyl
linker.
31. A pharmaceutical composition comprising the agonist of claim
23, and a pharmaceutically acceptable carrier.
32. A pharmaceutical composition comprising the agonist of claim
27, and a pharmaceutically acceptable carrier.
33. A pharmaceutical composition comprising the agonist of claim
29, and a pharmaceutically acceptable carrier.
Description
SEQUENCE LISTING
The instant application contains a Sequence Listing which has been
submitted electronically in ASCII format and is hereby incorporated
by reference in its entirety. Said ASCII copy, created Nov. 5,
2019, is named "CMN-002_SequenceListing" and is 46937 bytes in
size.
BACKGROUND
Exogenous nucleic acids, particularly viral nucleic acids,
introduced into cells induce an innate immune response, resulting
in, among other events, interferon (IFN) production and cell death.
Upon sensing viral RNA, RIG-I-like receptors induce type I
interferon (IFN) secretion leading to upregulation of antiviral
IFN-induced proteins in the infected and neighboring cells, which
inhibits virus replication. Further downstream events attract
immune cells and trigger the adaptive immune response. In addition,
RIG-I ligands have been reported to induce the apoptosis of many
different types of tumor cells, but not of normal cells.
There remains a need for additional and improved compositions and
methods to modulate the activity of immunomodulatory proteins. Such
agents can be used for cancer immunotherapy and treatment of other
conditions, such as chronic infection. There is a need to develop
improved RIG-I-like receptor ligands for diverse therapeutic
immunomodulatory applications.
SUMMARY OF THE DISCLOSURE
The present disclosure is based, at least in part, on the discovery
of synthetic RNA molecules that function as RIG-I-like receptor
agonists.
In some aspects, the disclosure provides a synthetic RIG-I-like
receptor (RLR) agonist that specifically binds to a RIG-I-like
receptor (RLR), wherein the agonist comprises a blunt-ended,
hairpin RNA comprising a 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 derivative or
analog thereof, and 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. In some embodiments, the first polynucleotide comprises the
sequence motif.
In some embodiments the RLR agonists of the disclosure comprise a
sequence motif selected from the group consisting of:
(i) a GT-repeat motif;
(ii) a GA-repeat motif;
(iii) a AUCG-repeat motif;
(iv) an AU-repeat motif;
(v) a dipyrimidine motif;
(vi) a dipurine motif;
(vii) a pyrimidine triplet motif;
(viii) a purine triplet motif;
(ix) a palindromic sequence motif; and
(x) a combination of any of (i)-(ix).
In some embodiments, the RLR agonists of the disclosure comprise a
combination of sequence motifs. In some embodiments the combination
of sequence motifs is a GT-repeat motif and a purine triplet motif.
In some embodiments, the combination of sequence motifs is an
AUCG-repeat motif and a dipyrimidine motif. In some embodiments,
the combination of sequence motifs is an AUGC-repeat motif and a
dipurine motif.
In some embodiments, the RLR agonists of the disclosure comprise 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 at least one improved
biological activity is selected from:
(i) an increase in RLR-mediated cytokine production;
(ii) an increase in RLR-mediated expression of
interferon-stimulated genes;
(iii) an increase in RLR-mediated intracellular signaling;
(iv) an increase in binding affinity to RLRs; and
(v) a combination of any of (i)-(iv).
In some embodiments, the RLR agonists of the disclosure comprise a
sequence motif that increases RLR-mediated type I interferon (e.g.,
IFN-.alpha., IFN-(3) production relative to an agonist that does
not comprise the sequence motif. In some embodiments, the RLR
agonists of the disclosure comprise a sequence motif that increases
RLR-mediated IL-1.beta. production relative to an agonist that does
not comprise the sequence motif. In some embodiments, the RLR
agonists of the disclosure comprise a sequence motif that increases
RLR-mediated IP-10 production relative to an agonist that does not
comprise the sequence motif. In some embodiments, the RLR agonists
of the disclosure comprise a sequence motif that increases
RLR-mediated IL-6, IL-12p70, MCP-1 and/or MIP-1.beta. production
relative to an agonist that does not comprise the sequence
motif.
In some embodiments, the RLR agonists of the disclosure comprise a
sequence motif, wherein the sequence motif is a GT-repeat motif
(e.g., GTGTGT) comprising a sequence of <19, about 15-18, about
15, about 10-15, about 10, about 5-10, about 5, about 4, 18, 17,
16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or 4 guanine and thymine
nucleotides, or derivatives or analogs thereof. In some
embodiments, the sequence motif is a GT-repeat motif comprising a
sequence of <19 guanine and thymine nucleotides, or derivatives
or analogs thereof. In some embodiments, the sequence motif is a
GT-repeat motif comprising a sequence of about 15-18 guanine and
thymine nucleotides, or derivatives or analogs thereof. In some
embodiments, the sequence motif is a GT-repeat motif comprising a
sequence of about 15 guanine and thymine nucleotides, or
derivatives or analogs thereof. In some embodiments, the sequence
motif is a GT-repeat motif comprising a sequence of about 10-15
guanine and thymine nucleotides, or derivatives or analogs thereof.
In some embodiments, the sequence motif is a GT-repeat motif
comprising a sequence of about 10 guanine and thymine nucleotides,
or derivatives or analogs thereof. In some embodiments, the
sequence motif is a GT-repeat motif comprising a sequence of about
5-10 guanine and thymine nucleotides, or derivatives or analogs
thereof. In some embodiments, the sequence motif is a GT-repeat
motif comprising a sequence of about 5 guanine and thymine
nucleotides, or derivatives or analogs thereof. In some
embodiments, the sequence motif is a GT-repeat motif comprising a
sequence of about 4 guanine and thymine nucleotides, or derivatives
or analogs thereof. In some aspects, the GT-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 RLRs;
and a combination of any of the foregoing.
In some embodiments, the RLR agonists of the disclosure comprise a
sequence motif, wherein the sequence motif is a GT-repeat motif
comprising a sequence of 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8,
7, 6, 5, or 4 guanine and thymine nucleotides, or derivatives or
analogs thereof. In some embodiments, the sequence motif is a
GT-repeat motif comprising a sequence of 18 guanine and thymine
nucleotides, or derivatives or analogs thereof. In some
embodiments, the sequence motif is a GT-repeat motif comprising a
sequence of 16 guanine and thymine nucleotides, or derivatives or
analogs thereof. In some embodiments, the sequence motif is a
GT-repeat motif comprising a sequence of 14 guanine and thymine
nucleotides, or derivatives or analogs thereof. In some
embodiments, the sequence motif is a GT-repeat motif comprising a
sequence of 12 guanine and thymine nucleotides, or derivatives or
analogs thereof. In some embodiments, the sequence motif is a
GT-repeat motif comprising a sequence of 10 guanine and thymine
nucleotides, or derivatives or analogs thereof. In some
embodiments, the sequence motif is a GT-repeat motif comprising a
sequence of 8 guanine and thymine nucleotides, or derivatives or
analogs thereof. In some embodiments, the sequence motif is a
GT-repeat motif comprising a sequence of 6 guanine and thymine
nucleotides, or derivatives or analogs thereof. In some
embodiments, the sequence motif is a GT-repeat motif comprising a
sequence of 4 guanine and thymine nucleotides, or derivatives or
analogs thereof. In some embodiments, the RLR agonists of the
disclosure comprise a sequence motif, wherein the sequence motif is
a GT-repeat motif, wherein the GT-repeat motif is [GT].sub.n,
wherein n=2 to 9, 3-7, or 4-8. In some aspects, the GT-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 RLRs; and a combination of any of the foregoing.
In some aspects, the disclosure provides an RLR agonist that
specifically binds to an RLR, wherein the agonist comprises a
blunt-ended, hairpin RNA comprising a first polynucleotide
connected to (operably linked 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 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 GT-repeat motif
comprising 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].sub.7. 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 RLRs;
and a combination of any of the foregoing.
In some aspects, the disclosure provides an RLR agonist that
specifically binds to an RLR, wherein the agonist comprises a
blunt-ended, hairpin RNA comprising a first polynucleotide
connected to a second polynucleotide by a linker, wherein the first
polynucleotide is sufficiently complementary to the second
polynucleotide to form a duplex, wherein the duplex comprises less
than 19 base pairs, wherein the 5' most nucleotide of the first
oligonucleotide comprises a 5' diphosphate or triphosphate moiety,
or 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
GT-repeat motif comprising 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].sub.3. In some
embodiments, the sequence motif is a GT-repeat motif, wherein the
GT-repeat motif is [GT].sub.3, and wherein the GT-repeat 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 RLRs;
and a combination of any of the foregoing.
In some embodiments, the RLR agonists of the disclosure comprise a
sequence motif, wherein the sequence motif is a GA-repeat motif
(e.g., GAGAGA) comprising a sequence of <19, about 15-18, about
15, about 10-15, about 10, about 5-10, about 5, about 4, 18, 17,
16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or 4 guanine and adenine
nucleotides, or derivatives or analogs thereof. In some
embodiments, the sequence motif is a GA-repeat motif comprising a
sequence of <19 guanine and adenine nucleotides, or derivatives
or analogs thereof. In some embodiments, the sequence motif is a
GA-repeat motif comprising a sequence of about 15-18 guanine and
adenine nucleotides, or derivatives or analogs thereof. In some
embodiments, the sequence motif is a GA-repeat motif comprising a
sequence of about 15 guanine and adenine nucleotides, or
derivatives or analogs thereof. In some embodiments, the sequence
motif is a GA-repeat motif comprising a sequence of about 10-15
guanine and adenine nucleotides, or derivatives or analogs thereof.
In some embodiments, the sequence motif is a GA-repeat motif
comprising a sequence of about 10 guanine and adenine nucleotides,
or derivatives or analogs thereof. In some embodiments, the
sequence motif is a GA-repeat motif comprising a sequence of about
5-10 guanine and adenine nucleotides, or derivatives or analogs
thereof. In some embodiments, the sequence motif is a GA-repeat
motif comprising a sequence of about 5 guanine and adenine
nucleotides, or derivatives or analogs thereof. In some
embodiments, the sequence motif is a GA-repeat motif comprising a
sequence of about 4 guanine and adenine nucleotides, or derivatives
or analogs thereof. In some aspects, the GA-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 RLRs;
and a combination of any of the foregoing.
In some embodiments, the RLR agonists of the disclosure comprise a
sequence motif is a GA-repeat motif comprising a sequence of 18,
17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or 4 guanine and
adenine nucleotides, or derivatives or analogs thereof. In some
embodiments, the sequence motif is a GA-repeat motif comprising a
sequence of 18 guanine and adenine nucleotides, or derivatives or
analogs thereof. In some embodiments, the sequence motif is a
GA-repeat motif comprising a sequence of 16 guanine and adenine
nucleotides, or derivatives or analogs thereof. In some
embodiments, the sequence motif is a GA-repeat motif comprising a
sequence of 14 guanine and adenine nucleotides, or derivatives or
analogs thereof. In some embodiments, the sequence motif is a
GA-repeat motif comprising a sequence of 12 guanine and adenine
nucleotides, or derivatives or analogs thereof. In some
embodiments, the sequence motif is a GA-repeat motif comprising a
sequence of 8 guanine and adenine nucleotides, or derivatives or
analogs thereof. In some embodiments, the sequence motif is a
GA-repeat motif comprising a sequence of 6 guanine and adenine
nucleotides, or derivatives or analogs thereof. In some
embodiments, the sequence motif is a GA-repeat motif comprising a
sequence of 4 guanine and adenine nucleotides, or derivatives or
analogs thereof.
In some embodiments, the RLR agonists of the disclosure comprise a
sequence motif, wherein the sequence motif is a GA-repeat motif,
wherein the GA-repeat motif is [GA].sub.n, where n=2 to 9, 3 to 7
or 4 to 8. In some aspects, the GA-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 RLRs;
and a combination of any of the foregoing.
In some aspects, the disclosure provides an RLR agonist that
specifically binds to an RLR, wherein the agonist comprises a
blunt-ended, hairpin RNA comprising a first polynucleotide
connected to a second polynucleotide by a linker, wherein the first
polynucleotide is sufficiently complementary to the second
polynucleotide to form a duplex, wherein the duplex comprises less
than 19 base pairs, wherein the 5' most nucleotide of the first
oligonucleotide comprises a 5' diphosphate or triphosphate moiety,
or 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 comprising 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].sub.7. In some
aspects, the GA-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 RLRs; and a combination of any of the
foregoing.
In some embodiments, the RLR agonists of the disclosure comprise a
sequence motif, wherein the sequence motif is a AUCG-repeat motif
(e.g., AUCGAUCG) comprising a sequence of <19, about 16, about
12-16, about 12, about 8-12, about 6, 16, 12, 8 adenine, uracil,
cytosine, and guanine nucleotides, or derivatives or analogs
thereof. In some embodiments, the sequence motif is a AUCG-repeat
motif comprising a sequence of <19 adenine, uracil, cytosine,
and guanine nucleotides, or derivatives or analogs thereof. In some
embodiments, the sequence motif is a AUCG-repeat motif comprising a
sequence of about 16 adenine, uracil, cytosine, and guanine
nucleotides, or derivatives or analogs thereof. In some
embodiments, the sequence motif is a AUCG-repeat motif comprising a
sequence of about 12-16 adenine, uracil, cytosine, and guanine
nucleotides, or derivatives or analogs thereof. In some
embodiments, the sequence motif is a AUCG-repeat motif comprising a
sequence of about 12 adenine, uracil, cytosine, and guanine
nucleotides, or derivatives or analogs thereof. In some
embodiments, the sequence motif is a AUCG-repeat motif comprising a
sequence of about 8-12 adenine, uracil, cytosine, and guanine
nucleotides, or derivatives or analogs thereof. In some
embodiments, the sequence motif is a AUCG-repeat motif comprising a
sequence of about 6 adenine, uracil, cytosine, and guanine
nucleotides, or derivatives or analogs thereof. In some
embodiments, the sequence motif is a AUCG-repeat motif comprising a
sequence of 16 adenine, uracil, cytosine, and guanine nucleotides,
or derivatives or analogs thereof. In some embodiments, the
sequence motif is a AUCG-repeat motif comprising a sequence of 12
adenine, uracil, cytosine, and guanine nucleotides, or derivatives
or analogs thereof. In some embodiments, the sequence motif is a
AUCG-repeat motif comprising a sequence of 8 adenine, uracil,
cytosine, and guanine nucleotides, or derivatives or analogs
thereof. 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 RLRs;
and a combination of any of the foregoing.
In some embodiments, the RLR agonists of the disclosure comprise a
sequence motif, wherein the sequence motif is an AUCG-repeat motif,
wherein the AUCG-repeat motif is [AUCG].sub.n, where n=2 to 4 or 2,
3 or 4. 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 RLRs;
and a combination of any of the foregoing.
In some aspects, the disclosure provides an RLR agonist that
specifically binds to an RLR, wherein the agonist comprises a
blunt-ended, hairpin RNA comprising a first polynucleotide
connected to a second polynucleotide by a linker, wherein the first
polynucleotide is sufficiently complementary to the second
polynucleotide to form a duplex, wherein the duplex comprises less
than 19 base pairs, wherein the 5' most nucleotide of the first
oligonucleotide comprises a 5' diphosphate or triphosphate moiety,
or 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
AUCG-repeat motif comprising a sequence of about 12 guanine and
adenine nucleotides. In some embodiments, the AUCG-repeat motif is
[AUCG].sub.3. 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 RLRs;
and a combination of any of the foregoing.
In some embodiments, the RLR agonists of the disclosure comprise a
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].sub.3 and is preceded by a CG. In some embodiments, the
AUCG-repeat motif is [AUCG].sub.3 and is preceded by the
dipyrimidine motif CC.
In some embodiments, the RLR agonists of the disclosure comprise an
AUCG-repeat motif, wherein the motif is preceded by a dipurine
motif. In some embodiments, the dipurine motif is GA. In some
embodiments, the AUCG-repeat motif is [AUCG].sub.3 and is preceded
by the dipurine motif GA. In some embodiments, the AUCG-repeat
motif is preceded by the dipurine motif II.
In some embodiments, the RLR agonists of the disclosure comprise an
AUCG-repeat motif, wherein one or more uridine nucleosides (U) are
substituted with a modified nucleoside. In some embodiments,
wherein the modified nucleoside is ribothymidine (T). In some
embodiments, the AUGC-repeat motif is [AUCG].sub.3, wherein the one
or more uridine nucleosides (U) comprising the AUCG-repeat motif
are substituted with a modified nucleoside, wherein the modified
nucleoside is ribothymidine (T). In some embodiments, the
AUGC-repeat motif is [AUCG].sub.3, wherein the one or more uridine
nucleosides (U) comprising the AUCG-repeat motif are substituted
with a modified nucleoside, wherein the modified nucleoside is
ribothymidine (T), and wherein the AUGC-repeat motif is preceded by
GG.
In some embodiments, the RLR agonists of the disclosure comprise an
AUCG-repeat motif, wherein one or more guanosine nucleosides (G)
are substituted with a modified nucleoside. In some embodiments,
the modified nucleoside is inosine (I). In some embodiments, the
AUGC-repeat motif is [AUCG].sub.3, wherein the one or more
guanosine nucleosides (G) comprising the AUCG-repeat motif are
substituted with a modified nucleoside, wherein the modified
nucleoside is ribothymidine (T), and wherein the AUGC-repeat motif
is preceded by GG.
In some embodiments, the RLR agonists of the disclosure comprise a
AUCG-repeat motif, wherein the motif is preceded by a IG. In some
embodiments, the AUCG-repeat motif is [AUCG].sub.3 and is preceded
by a IG.
In some embodiments, the RLR agonists of the disclosure comprise an
AUCG-repeat, wherein one or more guanosine nucleosides (G) are
substituted with an inosine (I), wherein the AUCG-repeat is
preceded by an inosine (I). In some embodiments, the guanosine
nucleosides (G) comprising the AUCG-repeat are substituted with an
inosine (I), wherein the AUCG-repeat is preceded by an inosine (I),
wherein the 5' most nucleotide of the first polynucleotide
comprises inosine (I).
In some embodiments, the RLR agonists of the disclosure comprise an
AUCG-repeat motif, wherein the AUCG-repeat motif is [AUCG].sub.2.
In some embodiments, the sequence motif is an AUCG-repeat motif,
wherein the AUCG-repeat motif is [AUCG].sub.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].sub.2, wherein the AUCG-repeat
motif is preceded by a dipurine motif, and wherein the dipurine
motif is GG.
In some embodiments, the RLR agonists of the disclosure comprise an
AUCG-repeat motif, wherein the AUCG-repeat motif is [AUCG].sub.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].sub.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].sub.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].sub.2,
and wherein the AUCG-repeat motif is preceded by TCGUCG.
In some embodiments, the RLR agonists of the disclosure comprise a
sequence motif, wherein the sequence motif is a palindromic
sequence comprising a sequence of <19, about 15-18, about 15,
about 10-15, about 10, about 18, 17, 16, 15, 14, 13, 12, 11, 10, 9,
8, 7, 6, 5, or 4 nucleotides, or derivatives or analogs thereof,
linked in any order that results in a palindrome. In some
embodiments, the sequence motif is a palindromic sequence
comprising a sequence of <19 nucleotides, or derivatives or
analogs thereof, linked in any order that results in a palindrome.
In some embodiments, the sequence motif is a palindromic sequence
comprising a sequence of about 15-18 nucleotides, or derivatives or
analogs thereof, linked in any order that results in a palindrome.
In some embodiments, the sequence motif is a palindromic sequence
comprising a sequence of about 15 nucleotides, or derivatives or
analogs thereof, linked in any order that results in 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, linked in any order that results in a palindrome.
In some embodiments, the sequence motif is a palindromic sequence
comprising a sequence of about 10 nucleotides, or derivatives or
analogs thereof, linked in any order that results in a palindrome.
In some embodiments, the sequence motif is a palindromic sequence
comprising a sequence of 18 nucleotides, or derivatives or analogs
thereof, linked in any order that results in a palindrome. In some
embodiments, the sequence motif is a palindromic sequence
comprising a sequence 17 nucleotides, or derivatives or analogs
thereof, linked in any order that results in a palindrome. In some
embodiments, the sequence motif is a palindromic sequence
comprising a sequence of 16 nucleotides, or derivatives or analogs
thereof, linked in any order that results in a palindrome. In some
embodiments, the sequence motif is a palindromic sequence
comprising a sequence of 15 nucleotides, or derivatives or analogs
thereof, linked in any order that results in a palindrome. In some
embodiments, the sequence motif is a palindromic sequence
comprising a sequence of 14 nucleotides, or derivatives or analogs
thereof, linked in any order that results in a palindrome. In some
embodiments, the sequence motif is a palindromic sequence
comprising a sequence of 13 nucleotides, or derivatives or analogs
thereof, linked in any order that results in a palindrome. In some
embodiments, the sequence motif is a palindromic sequence
comprising a sequence of 12 nucleotides, or derivatives or analogs
thereof, linked in any order that results in a palindrome. In some
embodiments, the sequence motif is a palindromic sequence
comprising a sequence of 11 nucleotides, or derivatives or analogs
thereof, linked in any order that results in a palindrome. In some
embodiments, the sequence motif is a palindromic sequence
comprising a sequence of 10 nucleotides, or derivatives or analogs
thereof, linked in any order that results in a palindrome. In some
embodiments, the sequence motif is a palindromic sequence
comprising a sequence of 9 nucleotides, or derivatives or analogs
thereof, linked in any order that results in a palindrome. In some
embodiments, the sequence motif is a palindromic sequence
comprising a sequence of 8 nucleotides, or derivatives or analogs
thereof, linked in any order that results in a palindrome. In some
embodiments, the sequence motif is a palindromic sequence
comprising a sequence of 7 nucleotides, or derivatives or analogs
thereof, linked in any order that results in a palindrome. In some
embodiments, the sequence motif is a palindromic sequence
comprising a sequence of 6 nucleotides, or derivatives or analogs
thereof, linked in any order that results in a palindrome. In some
embodiments, the sequence motif is a palindromic sequence
comprising a sequence of 5 nucleotides, or derivatives or analogs
thereof, linked in any order that results in a palindrome. In some
embodiments, the sequence motif is a palindromic sequence
comprising a sequence of 4 nucleotides, or derivatives or analogs
thereof, linked in any order that results in a palindrome.
In some embodiments, the RLR agonists of the disclosure comprise 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].sub.n, where n=2 to 3. In some embodiments, the
AU-repeat motif is [AU].sub.2.
In some aspects, the disclosure provides an RLR agonist that
specifically binds to RLRs, wherein the agonist comprises a
blunt-ended, hairpin RNA comprising the formula:
5'-(N.sub.1-N.sub.2-X.sub.1)-L-(X.sub.2-N.sub.3-N.sub.4)-3',
wherein (N.sub.1-N.sub.2-X.sub.1) comprises a first polynucleotide
comprising linked nucleotides N.sub.1, N.sub.2 and X.sub.1; (ii)
(X.sub.2-N.sub.3-N.sub.4) comprises a second polynucleotide
comprising linked nucleotides X.sub.2, N.sub.3 and N.sub.4; (iii)
N.sub.1, N.sub.2, N.sub.3 and N.sub.4 each comprise a single
nucleotide comprising a nucleoside selected from the group
consisting of: adenosine, guanosine, cytidine, 5-methyluridine,
uridine and inosine; (iv) N.sub.1 base pairs with N.sub.4; (v) N2
base pairs with N.sub.3; (vi) N.sub.1 comprises a 5' diphosphate or
triphosphate moiety, or derivative or analog thereof; (vii) X.sub.1
and X.sub.2 are each oligonucleotides comprising nucleosides
selected from the group consisting of: adenosine, guanosine,
cytidine, 5-methyluridine, uridine and inosine; (viii) X.sub.1 is
complementary to X.sub.2; (ix) X.sub.1 and X.sub.2 are each 12
nucleotides to 16 nucleotides in length and are the same length,
and; (x) L is a linker that operably links the first polynucleotide
and the second polynucleotide,
wherein at least one of N1, N2, N3, and N4 is inosine and/or at
least one of X1 and/or X2 comprises at least one inosine
nucleoside, and wherein the inosine nucleoside base pairs with
cytidine in the hairpin RNA. In some aspects, the RLR agonist of
the disclosure has an 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 RLRs;
and a combination of any of the foregoing.
In some aspects, the disclosure provides an RLR agonist that
specifically binds to RLRs, wherein the agonist comprises a
blunt-ended, hairpin RNA comprising a non-nucleotide linker, and
wherein the agonist comprises the formula:
5'-(N.sub.1-N.sub.2-X.sub.1)-L-(X.sub.2-N.sub.3-N.sub.4)-3',
wherein
(i) (N.sub.1-N.sub.2-X.sub.1) comprises a first polynucleotide
comprising linked nucleotides N.sub.1, N.sub.2 and X.sub.1;
(ii) (X2-N.sub.3-N.sub.4) comprises a second polynucleotide
comprising linked nucleotides X.sub.2, N.sub.3 and N.sub.4;
(iii) N.sub.1, N.sub.2, N.sub.3 and N.sub.4 each comprise a single
nucleotide comprising a nucleoside selected from the group
consisting of: adenosine, guanosine, cytidine, 5-methyluridine,
uridine and inosine;
(iv) N.sub.1 base pairs with N.sub.4;
(v) N.sub.2 base pairs with N.sub.3;
(vi) N.sub.1 comprises a 5' diphosphate or triphosphate moiety, or
derivative or analog thereof;
(vii) X.sub.1 and X.sub.2 are each oligonucleotides comprising
nucleosides selected from the group consisting of: adenosine,
guanosine, cytidine, 5-methyluridine, uridine and inosine;
(viii) X.sub.1 is complementary to X.sub.2;
(ix) X.sub.1 and X.sub.2 are each 12 nucleotides to 16 nucleotides
in length and are the same length, and;
(x) L is the non-nucleotide linker that covalently links the first
polynucleotide and the second polynucleotide,
wherein inosine, if present, base pairs with cytidine. In some
aspects, the RLR agonist of the disclosure has an 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 RLRs; and a combination of any of the foregoing.
In some embodiments, N1 comprises inosine and N4 comprises
cytidine. In some embodiments, N1 comprises inosine and N4
comprises cytidine and X1 and X2 are each 12 nucleotides in length.
In some embodiments, N1 comprises cytidine and N4 comprises
inosine. In some embodiments, N2 comprise inosine and N3 comprises
cytidine. In some embodiments, N2 comprises cytidine and N3
comprises inosine. In some embodiments, N1 comprises guanosine. In
some embodiments, N2 comprises guanosine. In some embodiments, N1
comprises cytidine. In some embodiments, N2 comprises cytidine. In
some embodiments, N1 and N2 comprise guanosine and N.sub.3 and
N.sub.4 comprise cytidine. In some embodiments, N1 and N2 comprise
cytidine and N.sub.3 and N.sub.4 comprise guanosine. In some
embodiments, N1 and N2 comprise inosine and N.sub.3 and N.sub.4
comprise cytidine. In some embodiments, N1 and N2 comprise cytidine
and N.sub.3 and N.sub.4 comprise inosine.
In some embodiments, the RLR agonists of the disclosure comprise
the formula:
5'-(N.sub.1-N.sub.2-X1)-L-(X.sub.2-N.sub.3-N.sub.4)-3', wherein
(i) (N.sub.1-N.sub.2-X.sub.1) comprises a first polynucleotide
comprising linked nucleotides N.sub.1, N.sub.2 and X.sub.1;
(ii) (X.sub.2-N.sub.3-N.sub.4) comprises a second polynucleotide
comprising linked nucleotides X.sub.2, N.sub.3 and N.sub.4;
(iii) N.sub.1, N.sub.2, N.sub.3 and N.sub.4 each comprise a single
nucleotide comprising a nucleoside selected from the group
consisting of: adenosine, guanosine, cytidine, 5-methyluridine,
uridine and inosine;
(iv) N.sub.1 base pairs with N.sub.4;
(v) N.sub.2 base pairs with N.sub.3;
(vi) N.sub.1 comprises a 5' diphosphate or triphosphate moiety, or
derivative or analog thereof;
(vii) X.sub.1 and X.sub.2 are each oligonucleotides comprising
nucleosides selected from the group consisting of: adenosine,
guanosine, cytidine, 5-methyluridine, uridine and inosine;
(viii) X.sub.1 is complementary to X.sub.2;
(ix) X.sub.1 and X.sub.2 are each 12 nucleotides to 16 nucleotides
in length and are the same length, and;
(x) L is the non-nucleotide linker that covalently links the first
polynucleotide and the second polynucleotide,
wherein inosine, if present, base pairs with cytidine, and wherein
N1 comprises inosine and N4 comprises cytidine, and X1 and/or X2
each comprise at least one inosine. In some embodiments, N2
comprises inosine and N3 comprises cytidine, and X1 and/or X2 each
comprise at least one inosine. In some embodiments, N1 and N2
comprise guanosine N.sub.3 and N.sub.4 comprise cytidine, and X1
and/or X2 each comprise at least one inosine. In some embodiments,
N1 and N2 comprise guanosine and N.sub.3 and N.sub.4 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, 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 comprise guanosine and N3 and N4 comprise cytidine, and X1 and
X2 each comprise inosine and no guanosine nucleosides. In some
embodiments, N1 and N2 comprise guanosine and N3 and N4 comprise
cytidine, 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 inosine and no guanosine nucleosides. In some
aspects, the RLR agonist of the disclosure has an 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 RLRs; and a combination of any of the foregoing.
In some embodiments, the RLR agonists of the disclosure comprise
the formula:
5'-(N.sub.1-N.sub.2-X.sub.1)-L-(X.sub.2-N.sub.3-N.sub.4)-3',
wherein
(i) (N.sub.1-N.sub.2-X.sub.1) comprises a first polynucleotide
comprising linked nucleotides N.sub.1, N.sub.2 and X.sub.1;
(ii) (X.sub.2-N.sub.3-N.sub.4) comprises a second polynucleotide
comprising linked nucleotides X.sub.2, N.sub.3 and N.sub.4;
(iii) N.sub.1, N.sub.2, N.sub.3 and N.sub.4 each comprise a single
nucleotide comprising a nucleoside selected from the group
consisting of: adenosine, guanosine, cytidine, 5-methyluridine,
uridine and inosine;
(iv) N.sub.1 base pairs with N.sub.4;
(v) N.sub.2 base pairs with N.sub.3;
(vi) N.sub.1 comprises a 5' diphosphate or triphosphate moiety, or
derivative or analog thereof;
(vii) X.sub.1 and X.sub.2 are each oligonucleotides comprising
nucleosides selected from the group consisting of: adenosine,
guanosine, cytidine, 5-methyluridine, uridine and inosine;
(viii) X.sub.1 is complementary to X.sub.2;
(ix) X.sub.1 and X.sub.2 are each 12 nucleotides to 16 nucleotides
in length and are the same length, and;
(x) L is the non-nucleotide linker that covalently links the first
polynucleotide and the second polynucleotide,
wherein inosine, if present, base pairs with cytidine, and wherein
N1 and N2 comprise inosine and N.sub.3 and N.sub.4 comprise
cytidine, and X1 and/or X2 each comprise at least one inosine. In
some embodiments, N1 and N2 comprise inosine and N.sub.3 and
N.sub.4 comprise cytidine, 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 inosine and N.sub.3 and N.sub.4
comprise cytidine, and X1 and X2 each comprise at least one
inosine. In some embodiments, N1 and N2 comprise inosine and N3 and
N4 comprise cytidine, 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
inosine, and X1 and/or X2 each comprise at least one inosine. In
some embodiments, N1 and N2 comprise inosine and N.sub.3 and
N.sub.4 comprise cytidine, and X1 and X2 comprise inosine and no
guanosine nucleosides. In some embodiments, N1 and N2 comprise
cytidine and N.sub.3 and N.sub.4 comprise inosine, and X1 and X2
comprise inosine and no guanosine nucleosides. In some aspects, the
RLR agonist of the disclosure has an 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 RLRs; and a combination of any of the foregoing.
In some embodiments, the RLR agonists of the disclosure comprise
the formula:
5'-(N.sub.1-N.sub.2-X.sub.1)-L-(X.sub.2-N.sub.3-N.sub.4)-3',
wherein X1 and X2 are each 12 nucleotides and comprise 1, 2, 3 or 4
inosine nucleosides. In some embodiments, X1 and X2 are each 13
nucleotides and comprise 1, 2, 3, 4 or 5 inosine nucleosides. In
some embodiments, X1 and X2 are each 14 nucleotides and comprise 1,
2, 3, 4, 5 or 6 inosine nucleosides. In some embodiments, X1 and X2
are each 15 nucleotides and comprise 1, 2, 3, 4, 5, 6, or 7 inosine
nucleosides. In some embodiments, X1 and X2 are each 16 nucleotides
and each comprise 1, 2, 3, 4, 5, 6, 7, or 8 inosine nucleosides. In
some embodiments, X1 and X2 are each 12 nucleotides and comprise at
least 10%, 20%, 30% or 40% inosine nucleosides. In some aspects,
the RLR agonist of the disclosure has an 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 RLRs; and a combination of any of the foregoing.
In some aspects, the disclosure provides an RLR agonist that
specifically binds to RLRs, wherein the agonist comprises a
blunt-ended, hairpin RNA comprising the formula:
5'-(N.sub.1-N.sub.2-X.sub.1)-L-(X.sub.2-N.sub.3-N.sub.4)-3',
wherein (N.sub.1-N.sub.2-X.sub.1) comprises a first polynucleotide
comprising linked nucleotides N.sub.1, N.sub.2 and X.sub.1, (ii)
(X.sub.2-N.sub.3-N.sub.4) comprises a second polynucleotide
comprising linked nucleotides X.sub.2, N.sub.3 and N.sub.4; (iii)
N.sub.1, N.sub.2, N.sub.3 and N.sub.4 each comprise a single
nucleotide comprising a nucleoside selected from the group
consisting of: adenosine, guanosine, cytidine, 5-methyluridine,
uridine and inosine; (iv) N.sub.1 base pairs with N.sub.4; (v)
N.sub.2 base pairs with N.sub.3; (vi) N.sub.2 comprises a 5'
diphosphate or triphosphate moiety, or derivative or analog
thereof; (vii) X.sub.1 comprises a sequence motif
[AUCN.sub.5].sub.x, wherein N.sub.5 is comprises guanosine or
inosine, wherein x is an integer whose value indicates the number
of sequence motifs, and wherein x=3 or 4; (viii) X.sub.2 comprises
a sequence motif [CN.sub.6AU].sub.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; (ix) L is a linker
that operably links the first polynucleotide and the second
polynucleotide,
optionally, wherein at least one of N1, N2, N3, and N4 is inosine,
and wherein the inosine nucleoside base pairs with cytidine in the
hairpin RNA.
In some embodiments, N5 comprises inosine and N6 comprises inosine.
In some embodiments, N5 comprises guanosine and N6 comprises
inosine. In some embodiments, N5 comprises inosine and N6 comprises
guanosine. In some embodiments, N5 comprises guanosine (G) and N6
comprises guanosine (G). In some embodiments, x=3 and y=3. In some
embodiments, x=4 and y=4. In some embodiments, N1 comprises inosine
(I) and N4 comprises cytidine (C). In some embodiments, N2
comprises inosine (I) and N3 comprises cytidine (C). In some
embodiments, N3 comprises inosine (I) and N2 comprises cytidine
(C). In some embodiments, N4 comprises inosine (I) and N1 comprises
cytidine (C). In some embodiments, N1 comprises guanosine (G). In
some embodiments, N2 comprises guanosine (G). In some embodiments,
N1 comprises cytidine (C). In some embodiments, N2 comprises
cytidine (C). In some embodiments, N1 and N2 comprise guanosine (G)
and N.sub.3 and N.sub.4 comprise cytidine (C). In some embodiments,
N1 and N2 comprise cytidine (C) and N.sub.3 and N.sub.4 comprise
guanosine (G). In some embodiments, N1 and N2 comprise inosine (I)
and N.sub.3 and N.sub.4 comprise cytidine (C). In some embodiments,
N1 and N2 comprise cytidine (C) and N.sub.3 and N.sub.4 comprise
inosine (I). In some aspects, the RLR agonist of the disclosure has
an 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 RLRs; and a combination of any of the
foregoing.
In some embodiments of the RLR agonist of the 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 tetraloop, wherein the nucleotide sequence of the
tetraloop is selected from the group consisting of: (a) UNCG,
wherein N=A, C, G, or U; (b) GNRA, wherein N=A, C, G, or U, and
wherein R=A or G; (c) ANYA, wherein N=A, C, G, or U, and wherein
Y=C or T; (d) CUYG, wherein Y=C or T; (e) UMAC, wherein M=A or C;
and (f) CUUG.
In some embodiments, the sequence of the tetraloop is UUCG. In some
embodiments, the sequence of the tetraloop is GAUC.
In some embodiments, the RLR agonist of the disclosure comprises a
nucleotide linker, wherein the nucleotide linker comprises the
nucleotide sequence UUUGAU or UGUUU. In some embodiments, the
nucleotide linker comprises the nucleotide sequence UUUGAU. In some
embodiments, the nucleotide linker comprises the nucleotide
sequence UGUUU.
In some embodiments, the RLR agonist of the disclosure comprises a
non-nucleotide linker, wherein the non-nucleotide linker is
selected from the group consisting of: (a) an ethylene glycol
linker; and (b) an alkyl linker.
In some embodiments, the non-nucleotide linker is a hexaethylene
glycol linker. In some embodiments, the non-nucleotide linker is a
C9 alkyl linker.
In some embodiments, the RLR agonist of the disclosure comprises a
5' diphosphate moiety, or a derivative or analog thereof. In some
embodiments, the agonist comprises a 5' triphosphate moiety, or a
derivative or analog thereof. In some embodiments, the derivative
or analog thereof comprises a phosphate bioisostere is selected
from: a phosphonate, a thiophosphonate, a phosphorothioate, a
sulfate, a sulfonate, a sulfamate, a thiazolidinone, a carboxylate,
a malonate, a boronic acid, a benzoxaborole, a boranophosphate, a
squaramide.
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
to the internucleotide linkages or to the polynucleotide
backbone.
In some embodiments, the RLR agonist of the disclosure exhibits one
or more of the following properties: (a) specifically binds to one
or more RLRs (e.g. RIG-1, MDA5 and/or LGP2); (b) increases
RLR-mediated cytokine production; (c) increases RLR-mediated
expression of interferon-stimulated genes (ISGs); (d) increases
RLR-dependent intracellular signaling; (e) increases stability of
the duplex; (f) increases binding affinity to RLRs; (g) decreases
off-target binding; (h) increases biological half-life; (i)
increases biodistribution and bioavailability; (j) increases and/or
enhances uptake into cells and/or tissues; (k) decreases
immunogenicity; and (l) a combination of any of (a)-(k).
In some aspects, the disclosure provides an RLR agonist that
specifically binds to RLRs, wherein the agonist comprises a
blunt-ended, hairpin RNA comprising the formula:
5'-(N.sub.1-N.sub.2-X.sub.1)-L-(X.sub.2-N.sub.3-N.sub.4)-3',
wherein
(i) (N.sub.1-N.sub.2-X.sub.1) comprises a first polynucleotide
comprising linked nucleotides N.sub.1, N.sub.2 and X.sub.1,
(ii) (X2-N.sub.3-N.sub.4) comprises a second polynucleotide
comprising linked nucleotides X.sub.2, N.sub.3 and N.sub.4;
(iii) N.sub.1, N.sub.2, N.sub.3 and N.sub.4 each comprise a single
nucleotide comprising a nucleoside selected from the group
consisting of: adenosine, guanosine, cytidine, 5-methyluridine,
uridine and inosine;
(iv) N.sub.1 base pairs with N.sub.4;
(v) N.sub.2 base pairs with N.sub.3;
(vi) N.sub.1 comprises a 5' diphosphate or triphosphate moiety, or
derivative or analog thereof;
(vii) X.sub.1 and X.sub.2 are each oligonucleotides comprising
nucleosides selected from the group consisting of: adenosine,
guanosine, cytidine, 5-methyluridine, uridine and inosine;
(viii) X.sub.1 is complementary to X.sub.2;
(ix) X.sub.1 and X.sub.2 are each 12 nucleotides to 16 nucleotides
in length and are the same length, and;
(x) L is a linker that operably links the first polynucleotide and
the second polynucleotide,
wherein N1 and N2 each comprise guanosine, wherein N.sub.3 and
N.sub.4 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
cytidine in the hairpin RNA, and wherein L comprises a nucleotide
linker comprising a tetraloop, wherein the nucleotide sequence of
the tetraloop is UUCG.
In some aspects, the disclosure provides an RLR agonist that
specifically binds to RLRs, wherein the agonist comprises a
blunt-ended, hairpin RNA comprising the formula:
5'-(N.sub.1-N.sub.2-X.sub.1)-L-(X.sub.2-N.sub.3-N.sub.4)-3',
wherein (i) (N.sub.1-N.sub.2-X.sub.1) comprises a first
polynucleotide comprising linked nucleotides N.sub.1, N.sub.2 and
X.sub.1;
(ii) (X.sub.2-N.sub.3-N.sub.4) comprises a second polynucleotide
comprising linked nucleotides X.sub.2, N.sub.3 and N.sub.4;
(iii) N.sub.1, N.sub.2, N.sub.3 and N.sub.4 each comprise a single
nucleotide comprising a nucleoside selected from the group
consisting of: adenosine, guanosine, cytidine, 5-methyluridine,
uridine and inosine;
(iv) N.sub.1 base pairs with N.sub.4;
(v) N.sub.2 base pairs with N.sub.3;
(vi) N.sub.1 comprises a 5' diphosphate or triphosphate moiety, or
derivative or analog thereof;
(vii) X.sub.1 and X.sub.2 are each oligonucleotides comprising
nucleosides selected from the group consisting of: adenosine,
guanosine, cytidine, 5-methyluridine, uridine and inosine;
(viii) X.sub.1 is complementary to X.sub.2;
(ix) X.sub.1 and X.sub.2 are each 12 nucleotides to 16 nucleotides
in length and are the same length, and;
(x) L is a linker that operably links the first polynucleotide and
the second polynucleotide,
wherein N1 comprises inosine and N2 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
cytidine in the hairpin RNA, and wherein L comprises a nucleotide
linker comprising a tetraloop, wherein the nucleotide sequence of
the tetraloop is UUCG.
In some aspects, the disclosure provides an RLR agonist that
specifically binds to RLRs, wherein the agonist comprises a
blunt-ended, hairpin RNA comprising the formula:
5'-(N.sub.1-N.sub.2-X.sub.1)-L-(X.sub.2-N.sub.3-N.sub.4)-3',
wherein
(i) (N.sub.1-N.sub.2-X.sub.1) comprises a first polynucleotide
comprising linked nucleotides N.sub.1, N.sub.2 and
(ii) (X.sub.2-N.sub.3-N.sub.4) comprises a second polynucleotide
comprising linked nucleotides X.sub.2, N.sub.3 and N.sub.4;
(iii) N.sub.1, N.sub.2, N.sub.3 and N.sub.4 each comprise a single
nucleotide comprising a nucleoside selected from the group
consisting of: adenosine, guanosine, cytidine, 5-methyluridine,
uridine and inosine;
(iv) N.sub.1 base pairs with N.sub.4;
(v) N.sub.2 base pairs with N.sub.3;
(vi) N.sub.1 comprises a 5' diphosphate or triphosphate moiety, or
derivative or analog thereof;
(vii) X.sub.1 and X.sub.2 are each oligonucleotides comprising
nucleosides selected from the group consisting of: adenosine,
guanosine, cytidine, 5-methyluridine, uridine and inosine;
(viii) X.sub.1 is complementary to X.sub.2;
(ix) X.sub.1 and X.sub.2 are each 12 nucleotides to 16 nucleotides
in length and are the same length, and;
(x) L is a linker that operably links the first polynucleotide and
the second polynucleotide,
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 cytidine in the hairpin RNA, and
wherein L comprises a nucleotide linker comprising a tetraloop,
wherein the nucleotide sequence of the tetraloop is UUCG.
In some aspects, the disclosure provides an RLR agonist that
specifically binds to RLRs, wherein the agonist comprises a
blunt-ended, hairpin RNA comprising a non-nucleotide linker, and
wherein the agonist comprises the formula:
5'-(N.sub.1-N.sub.2-X.sub.1)-L-(X.sub.2-N.sub.3-N.sub.4)-3',
wherein
(N.sub.1-N.sub.2-X.sub.1) comprises a first polynucleotide
comprising linked nucleotides N.sub.1, N.sub.2 and X.sub.1;
(ii) (X.sub.2-N.sub.3-N.sub.4) comprises a second polynucleotide
comprising linked nucleotides X.sub.2, N.sub.3 and N.sub.4;
(iii) N.sub.1, N.sub.2, N.sub.3 and N.sub.4 each comprise a single
nucleotide comprising a nucleoside selected from the group
consisting of: adenosine, guanosine, cytidine, 5-methyluridine,
uridine and inosine;
(iv) N.sub.1 base pairs with N.sub.4;
(v) N.sub.2 base pairs with N.sub.3;
(vi) N.sub.1 comprises a 5' diphosphate or triphosphate moiety, or
derivative or analog thereof;
(vii) X.sub.1 and X.sub.2 are each oligonucleotides comprising
nucleosides selected from the group consisting of: adenosine,
guanosine, cytidine, 5-methyluridine, uridine and inosine;
(viii) X.sub.1 is complementary to X.sub.2;
(ix) X.sub.1 and X.sub.2 are each 12 nucleotides to 16 nucleotides
in length and are the same length, and;
(x) L is the non-nucleotide linker that covalently links the first
polynucleotide and the second polynucleotide,
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.
In some aspects, the disclosure provides an RLR agonist that
specifically binds to RLRs, wherein the agonist comprises a
blunt-ended, hairpin RNA comprising a non-nucleotide linker, and
wherein the agonist comprises the formula:
5'-(N.sub.1-N.sub.2-X.sub.1)-L-(X.sub.2-N.sub.3-N.sub.4)-3',
wherein
(i) (N.sub.1-N.sub.2-X.sub.1) comprises a first polynucleotide
comprising linked nucleotides N.sub.1, N.sub.2 and X.sub.1;
(ii) (X.sub.2-N.sub.3-N.sub.4) comprises a second polynucleotide
comprising linked nucleotides X.sub.2, N.sub.3 and N.sub.4;
(iii) N.sub.1, N.sub.2, N.sub.3 and N.sub.4 each comprise a single
nucleotide comprising a nucleoside selected from the group
consisting of: adenosine, guanosine, cytidine, 5-methyluridine,
uridine and inosine;
(iv) N.sub.1 base pairs with N.sub.4;
(v) N.sub.2 base pairs with N.sub.3;
(vi) N.sub.1 comprises a 5' diphosphate or triphosphate moiety, or
derivative or analog thereof;
(vii) X.sub.1 and X.sub.2 are each oligonucleotides comprising
nucleosides selected from the group consisting of: adenosine,
guanosine, cytidine, 5-methyluridine, uridine and inosine;
(viii) X.sub.1 is complementary to X.sub.2;
(ix) X.sub.1 and X.sub.2 are each 12 nucleotides to 16 nucleotides
in length and are the same length, and;
(x) L is the non-nucleotide linker that covalently links the first
polynucleotide and the second polynucleotide,
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.
In some aspects, the disclosure provides an RLR agonist that
specifically binds to RLR, wherein the 5' most nucleotide of the
agonist comprises a 5' diphosphate or triphosphate moiety, or
derivative or analog thereof, and wherein the agonist comprises the
nucleotide sequence selected from the group consisting of SEQ ID
NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35
and 36.
In some aspects, the disclosure provides an RLR agonist that
specifically binds to RLRs, wherein the agonist comprises a
blunt-ended, hairpin RNA comprising a first polynucleotide
connected to a second polynucleotide by a linker, wherein the first
polynucleotide is sufficiently complementary to the second
polynucleotide to form a duplex, wherein the duplex comprises less
than 19 base pairs, wherein the 5' most nucleotide of the first
oligonucleotide comprises a 5' diphosphate or triphosphate moiety,
or 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, and wherein the first polynucleotide
and the second polynucleotide comprise the nucleotide sequences
selected from the group consisting of:
(i) SEQ ID NO: 37 and 68, respectively;
(ii) SEQ ID NO: 38 and 69, respectively;
(iii) SEQ ID NO: 39 and 70, respectively;
(iv) SEQ ID NO: 40 and 71, respectively;
(v) SEQ ID NO: 41 and 72, respectively;
(vi) SEQ ID NO: 42 and 73, respectively;
(vii) SEQ ID NO: 43 and 74, respectively;
(viii) SEQ ID NO: 44 and 75, respectively;
(ix) SEQ ID NO: 45 and 76, respectively;
(x) SEQ ID NO: 46 and 77, respectively;
(xi) SEQ ID NO: 47 and 78, respectively;
(xii) SEQ ID NO: 48 and 79, respectively;
(xiii) SEQ ID NO: 49 and 80, respectively;
(xiv) SEQ ID NO: 50 and 81, respectively;
(xv) SEQ ID NO: 51 and 82, respectively;
(xvi) SEQ ID NO: 52 and 83, respectively;
(xvii) SEQ ID NO: 53 and 84, respectively;
(xviii) SEQ ID NO: 54 and 85, respectively;
(xix) SEQ ID NO: 55 and 86, respectively;
(xx) SEQ ID NO: 56 and 87, respectively;
(xxi) SEQ ID NO: 57 and 88, respectively;
(xxii) SEQ ID NO: 58 and 89, respectively;
(xxiii) SEQ ID NO: 59 and 89, respectively;
(xxiv) SEQ ID NO: 60 and 90, respectively;
(xxv) SEQ ID NO: 61 and 91, respectively;
(xxvi) SEQ ID NO: 62 and 92, respectively;
(xxvii) SEQ ID NO: 63 and 91, respectively;
(xxviii) SEQ ID NO: 64 and 93, respectively;
(xxix) SEQ ID NO: 65 and 94, respectively;
(xxx) SEQ ID NO: 66 and 95, respectively;
(xxxi) SEQ ID NO: 67 and 96, respectively; and
(xxxii) SEQ ID NO: 63 and 97, respectively.
In some aspects, the disclosure provides an RLR agonist that
specifically binds to RLRs, wherein the agonist comprises a
blunt-ended, hairpin RNA comprising at least one or more
nucleotides comprising inosine which base pairs with cytidine, and
wherein the agonist comprises the nucleotide sequence selected from
the group consisting of SEQ ID NOs: 22, 23 and 25.
In some aspects, the disclosure provides an RLR agonist that
specifically binds to RLRs, wherein the agonist comprises a
blunt-ended, hairpin RNA comprising at least one or more
nucleotides comprising inosine which base pairs with cytidine,
wherein the agonist comprises the formula
5'-(N.sub.1-N.sub.2-X.sub.1)-L-(X.sub.2-N.sub.3-N.sub.4)-3',
wherein (N.sub.1-N.sub.2-X.sub.1) comprises a first polynucleotide
and (X.sub.2-N.sub.3-N.sub.4) comprises a second polynucleotide,
and wherein the first polynucleotide and the second polynucleotide
comprise the nucleotide sequences selected from the group
consisting of:
(i) SEQ ID NO: 58 and 89, respectively;
(ii) SEQ ID NO: 59 and 89, respectively; and
(iii) SEQ ID NO: 61 and 91, respectively.
In some aspects, the disclosure provides an RLR agonist that
specifically binds to RLRs, wherein the agonist comprises a
blunt-ended, hairpin RNA comprising a non-nucleotide linker,
wherein the agonist comprises the formula
5'-(N.sub.1-N.sub.2-X.sub.1)-L-(X.sub.2-N.sub.3-N.sub.4)-3',
wherein (N.sub.1-N.sub.2-X.sub.1) comprises a first polynucleotide
and (X.sub.2-N.sub.3-N.sub.4) comprises a second polynucleotide,
and wherein the first polynucleotide and the second polynucleotide
comprise the nucleotide sequences selected from the group
consisting of:
(i) SEQ ID NO: 37 and 68, respectively;
(ii) SEQ ID NO: 38 and 69, respectively;
(iii) SEQ ID NO: 39 and 70, respectively;
(iv) SEQ ID NO: 40 and 71, respectively;
(v) SEQ ID NO: 41 and 72, respectively;
(vi) SEQ ID NO: 42 and 73, respectively;
(vii) SEQ ID NO: 43 and 74, respectively;
(viii) SEQ ID NO: 44 and 75, respectively;
(ix) SEQ ID NO: 45 and 76, respectively;
(x) SEQ ID NO: 46 and 77, respectively;
(xi) SEQ ID NO: 47 and 78, respectively;
(xii) SEQ ID NO: 48 and 79, respectively;
(xiii) SEQ ID NO: 49 and 80, respectively;
(xiv) SEQ ID NO: 50 and 81, respectively;
(xv) SEQ ID NO: 51 and 82, respectively;
(xvi) SEQ ID NO: 52 and 83, respectively;
(xvii) SEQ ID NO: 53 and 84, respectively;
(xviii) SEQ ID NO: 54 and 85, respectively;
(xix) SEQ ID NO: 55 and 86, respectively;
(xx) SEQ ID NO: 56 and 87, respectively;
(xxi) SEQ ID NO: 57 and 88, respectively;
(xxii) SEQ ID NO: 58 and 89, respectively;
(xxiii) SEQ ID NO: 59 and 89, respectively;
(xxiv) SEQ ID NO: 60 and 90, respectively;
(xxv) SEQ ID NO: 61 and 91, respectively;
(xxvi) SEQ ID NO: 62 and 92, respectively;
(xxvii) SEQ ID NO: 63 and 91, respectively;
(xxviii) SEQ ID NO: 64 and 93, respectively;
(xxix) SEQ ID NO: 65 and 94, respectively;
(xxx) SEQ ID NO: 66 and 95, respectively;
(xxxi) SEQ ID NO: 67 and 96, respectively; and
(xxxii) SEQ ID NO: 63 and 97, respectively.
In some embodiments of the RLR agonists provided by the disclosure,
the nucleotide sequence comprising the RLR agonist is not
complementary to a genomic DNA sequence or mRNA sequence, wherein
the RLR agonist does not participate in RNA interference, and
wherein the RLR agonist does not silence gene expression.
In some aspects, the disclosure provides a pharmaceutical
composition for stimulating an immune response, treating or
delaying progression of a cancer, or reducing or inhibiting tumor
growth in a subject in need thereof, comprising an RLR agonist
provided by the disclosure, and a pharmaceutically acceptable
carrier. In some embodiments, the RLR agonist is formulated in a
polyethylenimine (PEI) carrier. In some embodiments, the PEI
carrier is JetPEI.RTM..
In some aspects, the disclosure provides a method to increase
RLR-mediated production of one or more cytokines in a cell, the
method comprising contacting the cell with an RLR agonist provided
by the disclosure, wherein the RLR agonist increases RLR-mediated
cytokine production in a cell. In some embodiments, the RLR agonist
increases RLR-mediated type I interferon (e.g., IFN-.alpha.,
IFN-.beta.) production in a cell. In some embodiments, the RLR
agonist increases RLR-mediated IL-1.beta. production in a cell. In
some embodiments, the RLR agonist increases RLR-mediated IP-10
production in a cell. In some embodiments, the RLR agonist
increases RLR-mediated IL-6, IL-12p70, MCP-1 and/or MIP-1.beta.
production in a cell.
In some aspects, the disclosure provides a method to increase
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 disclosure, wherein the agonist increases
RLR-mediated expression of one or more interferon-stimulated genes
in a cell.
In some aspects, the disclosure provides method to increase
RLR-dependent intracellular signaling in a cell, the method
comprising contacting the cell with an RLR agonist provided by the
disclosure, wherein the agonist increases RLR-dependent
intracellular signaling.
In some aspects, the 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 disclosure.
In some aspects, the disclosure provides a method of treating or
delaying progression of a cancer in a subject, the method
comprising administering to the subject an effective amount of an
RLR agonist or a pharmaceutical composition provided by the
disclosure.
In some aspects, the 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 a pharmaceutical composition provided by the
disclosure.
In some aspects, the disclosure provides a method for stimulating
an immune response, treating or delaying progression of a cancer,
or inhibiting tumor growth in a subject in need thereof, the method
comprising administering to the subject an effective amount of an
RLR agonist or a pharmaceutical composition provided by the
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 or delaying progression
of the cancer, or inhibiting growth of the tumor.
In some embodiments of the methods provided by the disclosure, an
RLR agonist or pharmaceutical composition provided by the
disclosure is administered in combination with one or more
additional therapeutic agents, wherein the one or more additional
therapeutic agents is selected from the group consisting of: a
chemotherapy, a targeted anti-cancer therapy, an oncolytic drug, a
cell death-inducing agent, an opsonizing agent (e.g., an opsonizing
antibody) a cytotoxic agent, an immune-based therapy, a cytokine,
an activator or agonist of a costimulatory molecule, an inhibitor
of an inhibitory molecule, a vaccine, a cellular immunotherapy, or
a combination thereof.
In some embodiments, an RLR agonist or pharmaceutical composition
provided by the disclosure is administered preceding or subsequent
to administration of the one or more additional therapeutic agents
or wherein the one or more additional therapeutic agents is
administered concurrently with, preceding or subsequent to the
administration of the agonist or pharmaceutical composition.
In some embodiments, the one or more additional therapeutic agents
is a PD-1/PD-L1 antagonist, a TIM-3 antagonist, a VISTA antagonist,
an adenosine A2AR antagonist, a B7-H3 antagonist, a B7-H4
antagonist, a BTLA antagonist, a CTLA-4 antagonist, an IDO
antagonist, a KIR antagonist, a LAG-3 antagonist, a Toll-like
receptor 3 (TLR3) agonist, a Toll-like receptor 7 (TLR7) agonist, a
Toll-like receptor 9 (TLR9) agonist.
In some embodiments, the one or more additional therapeutic agents
is an agonist comprising an polypeptide (e.g., antibody, or antigen
binding portion thereof) that specifically binds to CD137
(4-1BB).
In some embodiments, the one or more additional therapeutic agents
is an agonist comprising an polypeptide (e.g., antibody, or antigen
binding portion thereof) that specifically binds to CD134
(OX40).
In some embodiments, the one or more additional therapeutic agents
is a PD-1/PD-L1 antagonist. In some embodiments, the PD-1/PD-L1
antagonist is selected from the group consisting of: PDR001,
KEYTRUDA.RTM. (pembrolizumab), OPDIVO.RTM. (nivolumab),
pidilizumab, MEDI0680, REGN2810, TSR-042, PF-06801591, and AMP-224.
In some embodiments, the PD-1/PD-L1 antagonist is selected from the
group consisting of: FAZ053, TENCENTRIQ.RTM. (atezolizumab),
BAVENCIO.RTM. (avelumab), IMFINZI.RTM. (durvalumab), and
BMS-936559.
In some embodiments, the one or more additional therapeutic agents
is a TIM-3 antagonist.
In some embodiments, the one or more additional therapeutic agents
is a VISTA antagonist.
In some embodiments, the one or more additional therapeutic agents
is an adenosine A2AR antagonist.
In some embodiments, the one or more additional therapeutic agents
is a B7-H3 antagonist.
In some embodiments, the one or more additional therapeutic agents
is a B7-H4 antagonist.
In some embodiments, the one or more additional therapeutic agents
is a BTLA antagonist.
In some embodiments, the one or more additional therapeutic agents
is a CTLA-4 antagonist.
In some embodiments, the one or more additional therapeutic agents
is a IDO antagonist.
In some embodiments, the one or more additional therapeutic agents
is a KIR antagonist.
In some embodiments, the one or more additional therapeutic agents
is a LAG-3 antagonist.
In some embodiments, the one or more additional therapeutic agents
is a Toll-like receptor 3 (TLR3) agonist. In some embodiments, the
TLR3 agonist is polyinosinic:polycytidylic acid (poly I:C). In some
embodiments, the TLR3 agonist is HILTONOL.RTM. (poly ICLC). In some
embodiments, the TLR3 agonist is polyadenylic-polyuridylic acid
(poly A:U). In some embodiments, the TLR3 agonist is RIBOXXIM.RTM.
(RGIC.RTM.100). In some embodiments, the TLR3 agonist is
RIBOXXON.RTM. (RGIC.RTM.50 bioconjugate). In some embodiments, the
TLR3 agonist is RIBOXXOL (RGIC.RTM.50).
In some embodiments, the one or more additional therapeutic agents
is a Toll-like receptor 7 (TLR7) agonist. 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).
In some embodiments, the one or more additional therapeutic agents
is a Toll-like receptor 9 (TLR9) agonist. 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).
In some aspects, the disclosure provides a use of an RLR agonist or
of a pharmaceutical composition provided by the disclosure, for
stimulating an immune response, treating or delaying progression of
a cancer, or inhibiting tumor growth in a subject in need thereof,
optionally for use in combination with one or more additional
therapeutic agents.
In some aspects, the disclosure provides a use of an RLR agonist or
a pharmaceutical composition provided by the disclosure, in the
manufacture of a medicament for stimulating an immune response,
treating or delaying progression of a cancer, or inhibiting tumor
growth in a subject in need thereof, optionally for use in
combination with one or more additional therapeutic agents.
In some aspects, the disclosure provides a kit comprising an RLR
agonist or a pharmaceutical composition provided by the disclosure
and instructions for use in stimulating an immune response in a
subject, or treating or delaying progression of a cancer, or
inhibiting tumor growth in a subject, optionally with instructions
for use in combination with one or more additional therapeutic
agents.
In some embodiments of a use or a kit provided by the disclosure,
an RLR agonist or a pharmaceutical composition provided by the
disclosure is administered in combination with one or more
additional therapeutic agents, wherein the one or more additional
therapeutic agents is selected from the group consisting of: a
chemotherapy, a targeted anti-cancer therapy, an oncolytic drug, a
cell death-inducing agent, an opsonizing agent (e.g., an opsonizing
antibody) a cytotoxic agent, an immune-based therapy, a cytokine,
an activator of a costimulatory molecule, an inhibitor of an
inhibitory molecule, a vaccine, a cellular immunotherapy, or a
combination thereof.
In some embodiments of a use or a kit provided by the disclosure,
an RLR agonist or a pharmaceutical composition provided by the
disclosure is administered preceding or subsequent to
administration of the one or more additional therapeutic agents or
wherein the one or more additional therapeutic agents is
administered concurrently with, preceding or subsequent to the
administration of the agonist or pharmaceutical composition.
In some embodiments of a use or a kit provided by the disclosure,
the one or more additional therapeutic agents is a PD-1/PD-L1
antagonist, a TIM-3 antagonist, a VISTA antagonist, an adenosine
A2AR antagonist, a B7-H3 antagonist, a B7-H4 antagonist, a BTLA
antagonist, a CTLA-4 antagonist, an IDO antagonist, a KIR
antagonist, a LAG-3 antagonist, a Toll-like receptor 3 (TLR3)
agonist, a Toll-like receptor 7 (TLR7) agonist, a Toll-like
receptor 9 (TLR9) agonist.
In some embodiments of a use or kit provided by the disclosure, the
one or more additional therapeutic agents is an agonist comprising
an polypeptide (e.g., antibody, or antigen binding portion thereof)
that specifically binds to CD137 (4-1BB).
In some embodiments of a use or kit provided by the disclosure, the
disclosure the one or more additional therapeutic agents is an
agonist comprising an polypeptide (e.g., antibody, or antigen
binding portion thereof) that specifically binds to CD134
(OX40).
BRIEF DESCRIPTION OF THE DRAWINGS
The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
FIG. 1 provides a bar graph depicting the quantification of IFN-a
secretion from human PBMC treated with 3 different concentrations
of RIG-I-like receptor agonists comprising various
modifications.
DETAILED DESCRIPTION
Overview
The RIG-I-like receptors (RLRs) are a family of cytosolic pattern
recognition receptors that are essential for detecting viral RNA
and initiating the innate immune response. The RLR family includes
three members: Retinoic acid-inducible gene I (RIG-I), Melanoma
differentiation-associated gene 5 (MDA5), and Laboratory of
genetics and physiology 2 (LGP2). These receptors are expressed in
both immune and non-immune cell types and regulate signaling
pathways that promote the IRF3-, IRF7-dependent expression of type
I and type III interferons (IFNs), and the NF-kappa B-dependent
expression of pro-inflammatory cytokines.
All three RLR family receptors have a DExD/H box RNA helicase
domain with ATPase activity. This domain along with the adjacent
C-terminal domain is required for RNA binding. In addition, the
C-terminal domains of RIG-I and LGP2 have been shown to act as
repressor domains, ensuring that the receptors remain in an
inactive conformation until they are bound by an activating
RNA.
The present disclosure provides RLR agonists comprising synthetic
RNA molecules that fold to form a duplexed, dsRNA and that comprise
one or more sequence motifs that provides one or more improved
biological activities.
Definitions
Terms used in the claims and specification are defined as set forth
below unless otherwise specified. In the case of direct conflict
with a term used in a parent provisional patent application, the
term used in the instant application shall control.
It must be noted that, as used in the specification and the
appended claims, the singular forms "a," "an" and "the" include
plural referents unless the context clearly dictates otherwise.
Further, unless otherwise required by context, singular terms shall
include pluralities and plural terms shall include the
singular.
About: As used herein, the term "about" (alternatively
"approximately") will be understood by persons of ordinary skill
and will vary to some extent depending on the context in which it
is used. If there are uses of the term which are not clear to
persons of ordinary skill given the context in which it is used,
"about" will mean up to plus or minus 10% of the particular
value.
Agonist: As used herein, the term "agonist" is used in its broadest
sense and encompasses any molecule or compound that partially or
fully promotes, induces, increases, and/or activates a biological
activity of a native polypeptide disclosed herein. Agonist
molecules according to the disclosure may include nucleic acids
(e.g., oligonucleotides, polynucleotides), antibodies or
antigen-binding fragments, fragments or amino acid sequence
variants of native polypeptides, peptides, oligonucleotides,
lipids, carbohydrates, and small organic molecules. In some
embodiments, activation in the presence of the 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% higher 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 disclosure. For example, these methods
include, but are not limited to, binding assays such as
enzyme-linked immuno-absorbent assay (ELISA), Forte Bio.COPYRGT.
systems, fluorescence polarization (FP) assay, and radioimmunoassay
(RIA). These assays determine the ability of an agonist to bind the
polypeptide of interest (e.g., a receptor or ligand) and therefore
indicate the ability of the agonist to promote, increase or
activate the activity of the polypeptide. Efficacy of an agonist
can also be determined using functional assays, such as the ability
of an agonist to activate or promote the function of the
polypeptide. For example, a functional assay may comprise
contacting a polypeptide with a candidate agonist molecule and
measuring a detectable change in one or more biological activities
normally associated with the polypeptide. The potency of an agonist
is usually defined by its EC.sub.50 value (concentration required
to activate 50% of the agonist response). The lower the EC.sub.50
value the greater the potency of the agonist and the lower the
concentration that is required to activate the maximum biological
response.
Ameliorating: As used herein, the term "ameliorating" refers to any
therapeutically beneficial result in the treatment of a disease
state, e.g., cancer, including prophylaxis, lessening in the
severity or progression, remission, or cure thereof.
Amino acid: 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 the 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,
.gamma.-carboxyglutamate, and O-phosphoserine. Amino acid analogs
refers to compounds that have the same basic chemical structure as
a naturally occurring amino acid, i.e., an a carbon that is bound
to a hydrogen, a carboxyl group, an amino group, and an R group,
e.g., homoserine, norleucine, methionine sulfoxide, methionine
methyl sulfonium. Such analogs have modified R groups (e.g.,
norleucine) or modified peptide backbones, but retain the same
basic chemical structure as a naturally occurring amino acid. Amino
acid mimetics refers to chemical compounds that have a structure
that is different from the general chemical structure of an amino
acid, but that function in a manner similar to a naturally
occurring amino acid.
Amino acids can 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. Nucleotides,
likewise, can be referred to by their commonly accepted
single-letter codes.
Amino acid substitution: As used herein, an "amino acid
substitution" refers to the replacement of at least one existing
amino acid residue in a predetermined amino acid sequence (an amino
acid sequence of a starting polypeptide) with a second, different
"replacement" amino acid residue. An "amino acid insertion" refers
to the incorporation of at least one additional amino acid into a
predetermined amino acid sequence. While the insertion will usually
consist of the insertion of one or two amino acid residues, larger
"peptide insertions," can also be made, e.g. insertion of about
three to about five or even up to about ten, fifteen, or twenty
amino acid residues. The inserted residue(s) may be naturally
occurring or non-naturally occurring as disclosed above. An "amino
acid deletion" refers to the removal of at least one amino acid
residue from a predetermined amino acid sequence.
Base Composition: As used herein, the term "base composition"
refers to the proportion of the total nucleotides of a nucleic acid
(e.g., an RNA) consisting of guanine (or hypoxanthine)+cytosine
and/or uracil (or thymine)+adenine nucleobases.
Base Pair: As used herein, the term "base pair" refers to two
nucleobases on opposite complementary polynucleotide strands, or
regions of the same strand, that interact via the formation of
specific hydrogen bonds. As used herein, the term "Watson-Crick
base pairing", used interchangeably with "complementary base
pairing", refers to a set of base pairing rules, wherein a purine
always binds with a pyrimidine such that the nucleobase adenine (A)
forms a complementary base pair with thymine (T) and guanine (G)
forms a complementary base pair with cytosine (C) in DNA molecules.
In RNA molecules, thymine is replaced by uracil (U), which, similar
to thymine (T), forms a complementary base pair with adenine (A).
The complementary base pairs are bound together by hydrogen bonds
and the number of hydrogen bonds differs between base pairs. As in
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.
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 "non-canonical base pairing". A "wobble base pair" is
a pairing between two nucleobases in RNA molecules that does not
follow Watson-Crick base pair rules. For example, inosine is a
nucleoside that is structurally similar to guanosine, but is
missing the 2-amino group. Inosine is able to form two hydrogen
bonds with each of the four natural nucleobases (Oda et al., (1991)
Nucleic Acids Res 19:5263-5267) and it is often used by researchers
as a "universal" base, meaning that it can base pair with all the
naturally-occurring or canonical bases. The four main wobble base
pairs are the guanine-uracil (G-U) base pair, the
hypoxanthine-uracil (I-U) base pair, the hypoxanthine-adenine (I-A)
base pair, and the hypoxanthine-cytosine (I-C) base pair. In order
to maintain consistency of nucleic acid nomenclature, "I" is used
for hypoxanthine because hypoxanthine is the nucleobase of inosine;
nomenclature otherwise follows the names of nucleobases and their
corresponding nucleosides (e.g., "G" for both guanine and
guanosine--as well as for deoxyguanosine). The thermodynamic
stability of a wobble base pair is comparable to that of a
Watson-Crick base pair. Wobble base pairs play a role in the
formation of secondary structure in RNA molecules.
In one aspect, the 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 (I-C base pair); that is, inosine can be
substituted for guanosine but cannot be substituted for the other
nucleosides.
Biologically active: As used herein, the phrase "biologically
active" refers to a characteristic of any substance that has
activity in a biological system and/or organism. For instance, a
substance that, when administered to an organism, has a biological
effect on that organism, is considered to be biologically active
and thus have "biological activity". In particular embodiments,
where a nucleic acid is biologically active, a portion of that
nucleic acid that shares at least one biological activity of the
whole nucleic acid is typically referred to as a "biologically
active" portion.
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 connected with one
another, by whatever means including chemical conjugation,
recombinant techniques or enzymatic activity, either directly or
via one or more additional moieties that serves as a linking agent,
to form a structure that is sufficiently stable so that the
moieties remain physically associated under the conditions in which
the structure is used, e.g., physiological conditions.
Complementary: As used herein, the term "complementary" or
"complementarity" refers to a relationship between the sequence of
nucleotides comprising two polynucleotide strands, or regions of
the same polynucleotide strand, and the formation of a duplex
comprising the strands or regions, wherein the extent of
consecutive base pairing between the two strands or regions is
sufficient for the generation of a duplex structure. It is known
that adenine (A) forms specific hydrogen bonds, or "base pairs",
with thymine (T) or uracil (U). Similarly, it is known that a
cytosine (C) base pairs with guanine (G). It is also known that
non-canonical nucleobases (e.g., inosine) can hydrogen bond with
natural bases. A sequence of nucleotides comprising a first strand
of a polynucleotide, or a region, portion or fragment thereof, is
said to be "sufficiently complementary" to a sequence of
nucleotides comprising a second strand of the same or a different
nucleic acid, or a region, portion, or fragment thereof, if, when
the first and second strands are arranged in an antiparallel
fashion, the extent of base pairing between the two strands
maintains the duplex structure under the conditions in which the
duplex structure is used (e.g., physiological conditions in a
cell). It should be understood that complementary strands or
regions of polynucleotides can include some base pairs that are
non-complementary. Complementarity may be "partial," in which only
some of the nucleobases comprising the polynucleotide are matched
according to base pairing rules. Or, there may be "complete" or
"total" complementarity between the nucleic acids. Although the
degree of complementarity between polynucleotide strands or regions
has significant effects on the efficiency and strength of
hybridization between the strands or regions, it is not required
for two complementary polynucleotides to base pair at every
nucleotide position. In some embodiments, a first polynucleotide is
100% or "fully" complementary to a second polynucleotide and thus
forms a base pair at every nucleotide position. In some
embodiments, a first polynucleotide is not 100% complementary
(e.g., is 90%, or 80% or 70% complementary) and contains mismatched
nucleotides at one or more nucleotide positions. While perfect
complementarity is often desired, some embodiments can include one
or more but preferably 6, 5, 4, 3, 2, or 1 mismatches.
Contacting: As used herein, the term "contacting" means
establishing a physical connection between two or more entities.
For example, contacting a cell with an agent (e.g. an RNA, a lipid
nanoparticle composition, or other pharmaceutical composition of
the disclosure) means that the cell and the agent are made to share
a physical connection. Methods of contacting cells with external
entities both in vivo, in vitro, and ex vivo are well known in the
biological arts. In exemplary embodiments of the disclosure, the
step of contacting a mammalian cell with a composition (e.g., an
isolated RNA, nanoparticle, or pharmaceutical composition of the
disclosure) is performed in vivo. For example, contacting a lipid
nanoparticle composition and a cell (for example, a mammalian cell)
which may be disposed within an organism (e.g., a mammal) may be
performed by any suitable administration route (e.g., parenteral
administration to the organism, including intravenous,
intramuscular, intradermal, and subcutaneous administration). For a
cell present in vitro, a composition (e.g., a lipid nanoparticle or
an isolated RNA) and a cell may be contacted, for example, by
adding the composition to the culture medium of the cell and may
involve or result in transfection. Moreover, more than one cell may
be contacted by an agent.
Denaturation: As used herein, the term "denaturation" refers to the
process by which the hydrogen bonding between base paired
nucleotides in a nucleic acid is disrupted, resulting in the loss
of secondary and/or tertiary nucleic acid structure (e.g. the
separation of previously annealed strands). Denaturation can occur
by the application of an external substance, energy, or biochemical
process to a nucleic acid.
Antigen presenting cell: The term "antigen presenting cell" or
"APC" is a cell that displays foreign antigen complexed with MHC on
its surface. T cells recognize this complex using T cell receptor
(TCR). Examples of APCs include, but are not limited to, dendritic
cells (DCs), peripheral blood mononuclear cells (PBMC), monocytes
(such as THP-1), B lymphoblastoid cells (such as C1R.A2, 1518
B-LCL) and monocyte-derived dendritic cells (DCs). Some APCs
internalize antigens either by phagocytosis or by receptor-mediated
endocytosis.
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 result in apoptosis can lead to observable
and characteristic morphological changes to a cell, including
membrane blebbing, cell volume shrinkage, chromosomal DNA
condensation and fragmentation, and mRNA decay. A common method to
identify cells, including T cells, undergoing apoptosis is to
expose 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 process of apoptosis.
Blunt-end: As used herein, the term "blunt-end" "blunt-ended"
refers to the structure of an end of a duplexed or double-stranded
nucleic acid, wherein both complementary strands comprising the
duplex terminate, at least at one end, in a base pair. Hence,
neither strand comprising the duplex extends further from the end
than the other.
Cancer antigen: As used herein, "cancer antigen" refers to (i)
tumor-specific antigens, such as neoantigens, (ii) tumor-associated
antigens, (iii) cells that express tumor-specific antigens, (iv)
cells that express 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 that is associated with a cancer.
Carcinoma: As used herein, the term "carcinoma" is art recognized
and refers to malignancies of epithelial or endocrine tissues
including respiratory system carcinomas, gastrointestinal system
carcinomas, genitourinary system carcinomas, testicular carcinomas,
breast carcinomas, prostatic carcinomas, endocrine system
carcinomas, and melanomas. The RIG-I-like receptor (RLR) agonists
described herein can be used to treat patients who have, who are
suspected of having, or who may be at high risk for developing any
type of cancer, including renal carcinoma or melanoma. Exemplary
carcinomas include those forming from tissue of the cervix, lung,
prostate, breast, head and neck, colon and ovary. The term also
includes carcinosarcomas, which include malignant tumors composed
of carcinomatous and sarcomatous tissues. An "adenocarcinoma"
refers to a carcinoma derived from glandular tissue or in which the
tumor cells form recognizable glandular structures.
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 mediated
primarily by CD8+ T cells.
Duplex: As used herein, the term "duplex" refers to a structure
formed by complementary strands of a double-stranded
polynucleotide, or complementary regions of a single-stranded
polynucleotide that folds back on itself. The duplex structure of a
nucleic acid arises as a consequence of complementary nucleotide
sequences being bound together, or hybridizing, by base pairing
interactions.
EC.sub.50: As used herein, the term "EC.sub.50" refers to the
concentration of an agonist which induces a response, either in an
in vitro or an in vivo assay, which is 50% of the maximal response,
i.e., halfway between the maximal response and the baseline.
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 the desired effect.
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 comprised of complementary
nucleotide strands that base pair to form a duplex that terminates
at one end in a nucleotide linker comprising a loop of unpaired
nucleotides (e.g., a tetraloop) comprising unpaired nucleotides or
in a non-nucleotide linker comprising a flexible chemical moiety
(e.g., ethylene glycol), either of which connects the complementary
nucleotide strands. RNA hairpins may differ 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 in the actual
nucleotide sequence. RNA hairpins may provide one or more
functions, including, but not limited to, guiding the overall
folding of an RNA molecule comprising the hairpin, determining
interactions in a ribozyme, protecting messenger RNA (e.g., mRNA)
from degradation, serving as a recognition motif or structure for
RNA binding proteins and acting as a substrate for enzymatic
reactions. Further description of RNA hairpin structures and
functions 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 regions of the hairpin RNAs comprising the
RLR agonists provided by the disclosure terminate in a blunt end
with a 5' triphosphate or diphosphate.
In need: As used herein, a subject "in need of prevention," "in
need of treatment," or "in need thereof," refers to one, who by the
judgment of an appropriate medical practitioner (e.g., a doctor, a
nurse, or a 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).
Linker: As used herein, the term "linker" (alternatively "tether"
or "spacer") refers to a moiety that covalently connects, attaches
or couples two polynucleotide strands or regions together. As used
herein, a linker comprising nucleotides is referred to as a
"nucleotide linker" (e.g. a tetraloop). As used herein, the term
"non-nucleotide linker" refers to a linker comprising a chemical
moiety and that does not comprise a nucleotide. Non-limiting
examples of non-nucleotide linkers include linkers comprising
ethylene glycol (e.g. hexaethylene glycol), alkyl chains (e.g. C9
alkyl linker), and stilbene diether. 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.
LGP2: As used herein, the term "LGP2" refers to the Laboratory of
Genetics and Physiology 2 polypeptide, a specific member of the
RIG-I-like receptor family 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 set forth in Table 4 (SEQ ID
NO: 100) and here:
TABLE-US-00001 (NCBP Accession Number: NP_077024.2)
MELRSYQWEVIMPALEGKNIIIWLPTGAGKTRAAAYVAKRHLETVDGAKV
VVLVNRVHLVTQHGEEFRRMLDGRWTVTTLSGDMGPRAGFGHLARCHDLL
ICTAELLQMALTSPEEEEHVELTVFSLIVVDECHHTHKDTVYNVIMSQYL
ELKLQRAQPLPQVLGLTASPGTGGASKLDGAINHVLQLCANLDTWCIMSP
QNCCPQLQEHSQQPCKQYNLCHRRSQDPFGDLLKKLMDQIHDHLEMPELS
RKFGTQMYEQQVVKLSEAAALAGLQEQRVYALHLRRYNDALLIHDTVRAV
DALAALQDFYHREHVTKTQILCAERRLLALFDDRKNELAHLATHGPENPK
LEMLEKILQRQFSSSNSPRGIIFTRTRQSAHSLLLWLQQQQGLQTVDIRA
QLLIGAGNSSQSTHMTQRDQQEVIQKFQDGTLNLLVATSVAEEGLDIPHC
NVVVRYGLLTNEISMVQARGRARADQSVYAFVATEGSRELKRELINEALE
TLMEQAVAAVQKMDQAEYQAKIRDLQQAALTKRAAQAAQRENQRQQFPVE
HVQLLCINCMVAVGHGSDLRKVEGTHHVNVNPNFSNYYNVSRDPVVINKV
FKDWKPGGVISCRNCGEVWGLQMIYKSVKLPVLKVRSMLLETPQGRIQAK
KWSRVPFSVPDFDFLQHCAENLSDLSLD
Local administration: As used herein, "local administration" or
"local delivery," refers to delivery that does not rely upon
transport of the composition or agent to its intended target tissue
or site via the vascular system. For example, the composition may
be delivered by injection or implantation of the composition or
agent or by injection or implantation of a device containing the
composition or agent. Following local administration in the
vicinity of a target tissue or site, the composition or agent, or
one or more components thereof, may diffuse to the intended target
tissue or site.
MDA5: As used herein, the term "MDA5" refers to the Melanoma
Differentiation-Associated Protein 5 polypeptide, a specific member
of the RIG-I-like receptor family and is encoded by the IFIH1 gene
in humans. Alternative names and acronyms for MDA5 in the art
include AGS7, Hlcd, IDDM19, MDA-5, RLR-2, SGMRT1, and interferon
induced with helicase C domain 1. An exemplary amino acid sequence
of full-length human MDA5 is set forth in Table 4 (SEQ ID NO: 99)
and here:
TABLE-US-00002 (NCBI Accession Number: NP_071451.2)
MSNGYSTDENFRYLISCFRARVKMYIQVEPVLDYLTFLPAEVKEQIQRTV
ATSGNMQAVELLLSTLEKGVWHLGWTREFVEALRRTGSPLAARYMNPELT
DLPSPSFENAHDEYLQLLNLLQPTLVDKLLVRDVLDKCMEEELLTIEDRN
RIAAAENNGNESGVRELLKRIVQKENWFSAFLNVLRQTGNNELVQELTGS
DCSESNAEIENLSQVDGPQVEEQLLSTTVQPNLEKEVWGMENNSSESSFA
DSSVVSESDTSLAEGSVSCLDESLGHNSNMGSDSGTMGSDSDEENVAARA
SPEPELQLRPYQMEVAQPALEGKNIIICLPTGSGKTRVAVYIAKDHLDKK
KKASEPGKVIVLVNKVLLVEQLFRKEFQPFLKKWYRVIGLSGDTQLKISF
PEVVKSCDIIISTAQILENSLLNLENGEDAGVQLSDFSLIIIDECHHTNK
EAVYNNIMRHYLMQKLKNNRLKKENKPVIPLPQILGLTASPGVGGATKQA
KAEEHILKLCANLDAFTIKTVKENLDQLKNQIQEPCKKFAIADATREDPF
KEKLLEIMTRIQTYCQMSPMSDFGTQPYEQWAIQMEKKAAKEGNRKERVC
AEHLRKYNEALQINDTIRMIDAYTHLETFYNEEKDKKFAVIEDDSDEGGD
DEYCDGDEDEDDLKKPLKLDETDRFLMTLFFENNKMLKRLAENPEYENEK
LTKLRNTIMEQYTRTEESARGIIFTKTRQSAYALSQWITENEKFAEVGVK
AHHLIGAGHSSEFKPMTQNEQKEVISKFRTGKINLLIATTVAEEGLDIKE
CNIVIRYGLVTNEIAMVQARGRARADESTYVLVAHSGSGVIEHETVNDFR
EKMMYKAIHCVQNMKPEEYAHKILELQMQSIMEKKMKTKRNIAKHYKNNP
SLITFLCKNCSVLACSGEDIHVIEKMHHVNMTPEFKELYIVRENKALQKK
CADYQINGEIICKCGQAWGTMMVHKGLDLPCLKIRNFVVVFKNNSTKKQY
KKWVELPITFPNLDYSECCLFSDED
Modified: As used herein "modified" or "modification" refers to a
changed state or change in structure resulting from a modification
of a polynucleotide, e.g., RNA. Polynucleotides may be modified in
various ways including chemically, structurally, and/or
functionally. For example, the RNA molecules of the present
disclosure may be modified by the incorporation of a non-natural
base or a sequence motif, comprising a functional sequence or
secondary structure, that provides a biological activity. In one
embodiment, the RNA is modified by the introduction of non-natural
or chemically-modified bases, nucleosides and/or nucleotides, e.g.,
as it relates to the natural ribonucleotides A, U, G, and C.
Naturally-occurring: As used herein, the term "naturally-occurring"
as applied to an object refers to the fact that an object can be
found in nature. For example, a polypeptide or polynucleotide
sequence, or components thereof 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.
Nucleic acid: As used herein, the term "nucleic acid" refers to
deoxyribonucleotides or ribonucleotides and polymers or oligomers
thereof in either single- or double-stranded form. Unless
specifically limited, the term encompasses nucleic acids containing
known analogues of natural nucleotides that have similar binding
properties as the reference nucleic acid and are metabolized in a
manner similar to naturally occurring nucleotides. Polymers of
nucleotides are referred to as "polynucleotides". Exemplary nucleic
acids or polynucleotides of the disclosure include, but are not
limited to, ribonucleic acids (RNAs), deoxyribonucleic acids
(DNAs), DNA-RNA hybrids, RNAi-inducing agents, RNAi agents, siRNAs,
shRNAs, miRNAs, antisense RNAs, ribozymes, catalytic DNA, RNAs that
induce triple helix formation, threose nucleic acids (TNAs), glycol
nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic
acids (LNAs, including LNA having a .beta.-D-ribo configuration,
.alpha.-LNA having an .alpha.-L-ribo configuration (a diastereomer
of LNA), 2'-amino-LNA having a 2'-amino functionalization, and
2'-amino-.alpha.-LNA having a 2'-amino functionalization) or
hybrids thereof.
Polynucleotides used herein can be composed of any
polyribonucleotide or polydeoxribonucleotide, which can be
unmodified RNA or DNA or modified RNA or DNA. For example,
polynucleotides can be composed of single- and double-stranded DNA,
DNA that is a mixture of single- and double-stranded regions,
single- and double-stranded RNA, and RNA that is mixture of single-
and double-stranded regions, hybrid molecules comprising DNA and
RNA that can be single-stranded or, more typically, double-stranded
or a mixture of single- and double-stranded regions. In addition,
the polynucleotide can be composed of triple-stranded regions
comprising RNA or DNA or both RNA and DNA. A 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, tritylated bases. "Modified nucleosides"
include, for example, as inosine and thymine, when the latter is
found in or comprises RNA. A variety of modifications can be made
to DNA and RNA; thus, "polynucleotide" embraces chemically,
enzymatically, or metabolically modified forms.
Nucleic Acid Structure: As used herein, the term "nucleic acid
structure" refers to the arrangement or organization of atoms,
chemical constituents, elements, motifs, and/or sequence of
nucleobases that comprise a nucleic acid (e.g. an RNA) and/or can
refer to the two-dimensional or three-dimensional state of a
nucleic acid. Accordingly, the term "RNA structure" refers to the
arrangement or organization of atoms, chemical constituents,
elements, motifs, and/or sequence of nucleobases comprising an RNA
molecule (e.g. an mRNA) and/or can refer to the two-dimensional
and/or three dimensional state of an RNA molecule. Nucleic acid
structure can be further demarcated into four organizational
categories referred to herein as "molecular structure", "primary
structure", "secondary structure", and "tertiary structure" based
on increasing organizational complexity.
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 the naturally occurring purines and
pyrimidines that confer improved properties (e.g. binding affinity,
nuclease resistance, chemical stability) to a nucleic acid or a
portion or segment thereof. Adenine, cytosine, guanine, thymine,
and uracil are the primary or canonical nucleobases predominately
found in natural nucleic acids. Other natural, non-natural,
non-canonical and/or synthetic nucleobases, can be incorporated
into nucleic acids, such as those disclosed herein.
Nucleoside/Nucleotide: As used herein, the term "nucleoside" refers
to a compound containing a sugar molecule (e.g., a ribose in RNA or
a deoxyribose in DNA), or derivative or analog thereof, covalently
linked to a nucleobase (e.g., a purine or pyrimidine), or a
derivative or analog thereof (also referred to herein as
"nucleobase"). As used herein, the term "nucleotide" refers to a
nucleoside covalently linked to a phosphate group. As used herein,
the term "ribonucleoside" refers to a nucleoside that comprise a
ribose and a nucleobase (e.g., adenosine (A), cytidine (C),
guanosine (G), 5-methyluridine (m.sup.5U), uridine (U), or inosine
(I)).
Operably linked: As used herein, a nucleic acid, or 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 fragment or portion
thereof.
Polynucleotide/oligonucleotide: As used herein, the terms
"polynucleotide" and "oligonucleotide" are used interchangeably and
refer to a single-stranded or double-stranded polymer or oligomer
of nucleotides or nucleoside monomers consisting of
naturally-occurring bases, sugars and intersugar (backbone)
linkages. The terms "polynucleotide" and "oligonucleotide" also
includes polymers and oligomers comprising non-naturally occurring
bases, sugars and intersugar (backbone) linkages, or portions
thereof, which function similarly. Polynucleotides are not limited
to any particular length of nucleotide sequence, as the term
"polynucleotides" encompasses polymeric forms of nucleotides of any
length. Short polynucleotides are typically referred to in the art
as "oligonucleotides". In the context of the present disclosure,
such modified or substituted polynucleotides and oligonucleotides
are often preferred over native forms because the modification
increases one or more desirable 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, the agonists of the
disclosure comprise polynucleotides and oligonucleotides that
contain at least one region of modified nucleotides that confers
one or more beneficial properties or increases biological activity
(e.g., increased nuclease resistance, increased uptake into cells,
increased duplex stability, increased binding affinity to a target
polypeptide).
Palindromic sequence: As used herein, the term "palindromic
sequence" (alternatively "palindrome") refers to a sequence of
nucleotides that is self-complementary; wherein the sequence of
nucleotides in the 5' to 3' direction is the same as the sequence
of nucleotides comprising the complementary strand, when read in
the 5' to 3'. For example, the sequence 5'-ACCTAGGT-3' is a
palindromic sequence because its complementary sequence,
3'-TGGATCCA-5', when read in the 5' to 3' direction, is the same as
the original sequence. In contrast, the sequence 5'-AGTGGCTG-3' is
not a palindromic sequence because its complementary sequence,
3'-TCACCGAC-5', when read in the 5' to 3' direction, is not the
same as the original sequence.
In one embodiment, the agonist is comprised of a first
oligonucleotide, wherein the sequence of the first oligonucleotide
is a palindromic sequence. In another embodiment, the agonist is
comprised of a first oligonucleotide, wherein the first
oligonucleotide comprises a palindromic sequence.
Palindromic sequences in preferred oligonucleotides of the
invention preferably include both the 5' end of the oligonucleotide
and the 3' end of the oligonucleotide, thus forming a blunt end. In
one embodiment of the invention the oligonucleotide comprises a
single palindromic sequence and in another more preferred
embodiment of the invention the oligonucleotide comprises two
complementary palindromes interrupted by an intervening sequence,
spacer, or linker that connects the 2 palindromes within 1 or 2
different oligonucleotides so as to form a hairpin duplex with a
blunt end.
Parenteral administration: As used herein, "parenteral
administration," "administered parenterally," and other
grammatically equivalent phrases, refer to modes of administration
other than enteral and topical administration, usually by
injection, and include, without limitation, intravenous,
intranasal, intraocular, intramuscular, intraarterial, intrathecal,
intracapsular, intraorbital, intracardiac, intradermal,
intraperitoneal, transtracheal, subcutaneous, subcuticular,
intraarticular, subcapsular, subarachnoid, intraspinal, epidural,
intracerebral, intracranial, intracarotid and intrasternal
injection and infusion.
Patient: As used herein, the term "patient" includes human and
other mammalian subjects that receive either prophylactic or
therapeutic treatment.
Percent identity: As used herein, the term "percent identity," in
the context of two or more nucleic acid or polypeptide sequences,
refers to two or more sequences or subsequences that have a
specified percentage of nucleotides or amino acid residues that are
the same, when compared and aligned for maximum correspondence, as
measured using one of the sequence comparison algorithms described
below (e.g., BLASTP and BLASTN or other algorithms available to
persons of skill) or by visual inspection. Depending on the
application, the "percent identity" can exist over a region of the
sequence being compared, e.g., over a functional domain, or,
alternatively, exist over the full length of the two sequences to
be compared. For sequence comparison, typically one sequence acts
as a reference sequence to which test sequences are compared. When
using a sequence comparison algorithm, test and reference sequences
are input into a computer, subsequence coordinates are designated,
if necessary, and sequence algorithm program parameters are
designated. The sequence comparison algorithm then calculates the
percent sequence identity for the test sequence(s) relative to the
reference sequence, based on the designated program parameters. The
percent identity between two sequences is a function of the number
of identical positions shared by the sequences (i.e., % homology=#
of identical positions/total # of positions.times.100), taking into
account the number of gaps, and the length of each gap, which need
to be introduced for optimal alignment of the two sequences. The
comparison of sequences and determination of percent identity
between two sequences can be accomplished using a mathematical
algorithm, as described in the non-limiting examples below.
Optimal alignment of sequences for comparison can be conducted,
e.g., by the local homology algorithm of Smith & Waterman, Adv.
Appl. Math. 2:482 (1981), by the homology alignment algorithm of
Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search
for similarity method of Pearson & Lipman, Proc. Nat'l. Acad.
Sci. USA 85:2444 (1988), by computerized implementations of these
algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin
Genetics Software Package, Genetics Computer Group, 575 Science
Dr., Madison, Wis.), or by visual inspection (see generally Ausub
el et al., infra).
One example of an algorithm that is suitable for determining
percent sequence identity and sequence similarity is the BLAST
algorithm, which is described in Altschul et al., J. Mol. Biol.
215:403-410 (1990). Software for performing BLAST analyses 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 a
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 (CABIOS,
4:11-17 (1989)) which has been 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. Mol. Biol. (48):444-453 (1970))
algorithm which has been incorporated into the GAP program in the
GCG software package (available at http://www.gcg.com), using
either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of
16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or
6.
The nucleic acid and protein sequences of the present disclosure
can further be used as a "query sequence" to perform a search
against public databases to, for example, identify related
sequences. Such searches can be performed using the NBLAST
and)(BLAST programs (version 2.0) of Altschul, et al. (1990) J.
Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed
with the NBLAST program, score=100, wordlength=12 to obtain
nucleotide sequences homologous to the nucleic acid molecules of
the invention. BLAST protein searches can be performed with the
XBLAST program, score=50, wordlength=3 to obtain amino acid
sequences homologous to the protein molecules of the invention. To
obtain gapped alignments for comparison purposes, Gapped BLAST can
be utilized as described in Altschul et al., (1997) Nucleic Acids
Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST
programs, the default parameters of the respective programs (e.g.,
XBLAST and NBLAST) can be used. See
http://www.ncbi.nlm.nih.gov.
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 bodily fluids of human beings and animals
without excessive toxicity, irritation, allergic response, or other
problems or complications commensurate with a reasonable
benefit/risk ratio.
Pharmaceutically acceptable carrier: As used herein, the term
"pharmaceutically acceptable carrier" refers to, and includes, any
and all solvents, dispersion media, coatings, antibacterial and
antifungal agents, isotonic and absorption delaying agents, and the
like that are physiologically compatible. The compositions can
include a pharmaceutically acceptable salt, e.g., an acid addition
salt or a base addition salt (see, e.g., Berge et al. (1977) J
Pharm Sci 66:1-19).
Phosphate: The term "phosphate" as used herein means a salt or
ester of phosphoric acid. Polyphosphates are salts or esters of
polymeric oxyanions formed from tetrahedral PO.sub.4 (phosphate)
structural units linked together by sharing 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 disclosure provides a
RIG-I-like receptor agonist comprising a diphosphate moiety, or a
derivative or analog thereof, linked to the 5' terminus. In some
embodiments, the disclosure provides a RIG-I-like receptor agonist
comprising a triphosphate moiety, or a derivative or analog
thereof, linked to the 5' terminus. In some embodiments, the
derivative or analog thereof is a phosphate bioisostere.
Phosphate bioisostere: As used herein, the term "phosphate
bioisostere" (alternatively "phosphate mimic") refers to chemical
substituents or groups with similar physical or chemical properties
to phosphate and which produce broadly similar biological
properties to phosphate, including biphosphate 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 making significant changes in
chemical structure. The use of bioisosteres is widespread in drug
development and is used, for example, to reduce toxicity, change
bioavailability, or modify the activity or metabolism of the
parental or lead compound (see e.g., Rye and Baell (2005) Curr Med
Chem 12(26):3127-3141; Elliot et al., (2012) MedChemCom
3(7):735-751, which are incorporated herein by reference in their
entirety).
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 residue is an artificial chemical
mimetic of a corresponding naturally occurring amino acid, as well
as to naturally occurring amino acid polymers and non-naturally
occurring amino acid polymer.
Preventing: As used herein, the term "preventing" when used in
relation to a condition, refers to administration of a composition
which reduces the frequency of, or delays the onset of, symptoms of
a medical condition in a subject relative to a subject which does
not receive the composition.
Purified: As used herein, the term "purified" or "isolated" as
applied to any of the proteins (antibodies or fragments) 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) which naturally accompany it,
e.g., other proteins, lipids, and nucleic acid in a prokaryote
expressing the proteins. 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.
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 distinct RIG-I-like
receptor ligands, wherein the relationship is the relative
agonistic effect of the reference ligand and the one or more
distinct RIG-I-like receptor ligands. As used herein, the term
connotes a RIG-I-like receptor ligand or agonist that is useful in
a test or assay, such as those described herein, (e.g., an IFN
induction assay), as a competitor, wherein the assay is useful for
the discovery, identification or development, of one or more
distinct agonists that bind to RIG-I-like receptors.
RIG-I: As used herein, the term "RIG-I" refers to the Retinoic
Acid-Inducible Gene I polypeptide, a specific member of the
RIG-I-like receptor family 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 set forth in Table 4 (SEQ ID NO: 98) and here:
TABLE-US-00003 (NCBI Accession Number: NP_055129.2)
MTTEQRRSLQAFQDYIRKTLDPTYILSYMAPWFREEEVQYIQAEKNNKGP
MEAATLFLKFLLELQEEGWFRGFLDALDHAGYSGLYEAIESWDFKKIEKL
EEYRLLLKRLQPEFKTRIIPTDIISDLSECLINQECEEILQICSTKGMMA
GAEKLVECLLRSDKENWPKTLKLALEKERNKFSELWIVEKGIKDVETEDL
EDKMETSDIQIFYQEDPECQNLSENSCPPSEVSDTNLYSPFKPRNYQLEL
ALPAMKGKNTIICAPTGCGKTFVSLLICEHHLKKFPQGQKGKVVFFANQI
PVYEQQKSVFSKYFERHGYRVTGISGATAENVPVEQIVENNDIIILTPQI
LVNNLKKGTIPSLSIFTLMIFDECHNTSKQHPYNMIMFNYLDQKLGGSSG
PLPQVIGLTASVGVGDAKNTDEALDYICKLCASLDASVIATVKHNLEELE
QVVYKPQKFFRKVESRISDKFKYIIAQLMRDTESLAKRICKDLENLSQIQ
NREFGTQKYEQWIVTVQKACMVFQMPDKDEESRICKALFLYTSHLRKYND
ALIISEHARMKDALDYLKDFFSNVRAAGFDEIEQDLTQRFEEKLQELESV
SRDPSNENPKLEDLCFILQEEYHLNPETITILFVKTRALVDALKNWIEGN
PKLSFLKPGILTGRGKTNQNTGMTLPAQKCILDAFKASGDHNILIATSVA
DEGIDIAQCNLVILYEYVGNVIKMIQTRGRGRARGSKCFLLTSNAGVIEK
EQINMYKEKMMNDSILRLQTWDEAVFREKILHIQTHEKFIRDSQEKPKPV
PDKENKKLLCRKCKALACYTADVRVIEECHYTVLGDAFKECFVSRPHPKP
KQFSSFEKRAKIFCARQNCSHDWGIHVKYKTFEIPVIKIESFVVEDIATG
VQTLYSKWKDFHFEKIPFDPAEMSK
RIG-I-like receptor: As used herein, the term "RIG-I-like receptor"
(abbreviate as "RLR") refers to any member of a family of DExD/H
box RNA helicases that function as cytoplasmic pattern recognition
sensors of pathogen-associated molecular patterns (PAMPs) typically
found in viral RNA. Upon ligand binding, RLRs signal downstream
transcription factor activation to drive type 1 interferon (IFN)
production and antiviral gene expression that elicits an
intracellular immune response to control virus infection. Three RLR
members have been identified: RIG-I (retinoic acid-inducible gene
I), MDA5 (melanoma differentiation associated factor 5), and LGP2
(laboratory of genetics and physiology 2 and a homolog of mouse
D11lgp2) (Loo and Gale (2011) Immunity 34(5):680-692).
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., an RNA) that binds to
RIG-I-like receptors (RLRs) and partially or fully promotes,
induces, increases, and/or activates a biological activity,
response, and/or downstream pathway(s) mediated by RLR signaling or
other RLR-mediated function. Examples of RIG-I-receptor agonists
are provided herein.
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 local segment or
portion thereof, that is persistently maintained under
physiological conditions and characterized by a low free energy
state. Typical examples of stable RNA secondary structures include
duplexes, hairpins, and stem-loops. Stable RNA secondary structures
are known in the art to exhibit various biological activities. The
term "stable" as used in reference to a polynucleotide duplex,
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.
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,
dog, cow, chickens, amphibians, reptiles, etc.
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 subsets
of T cells, including, but not limited to, T helper cells (a.k.a.
TH cells or CD4+ T cells) and subtypes, including TH1, TH2, TH3,
TH17, TH9, and TFH cells, cytotoxic T cells (a.k.a 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 (a.k.a. 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 (a.k.a. NKT cells), mucosal
associated invariant T cells (MAITs), and gamma delta T cells
(.gamma..delta. T cells), including V.gamma.9/V.delta.2 T cells.
Any one or more of the aforementioned or unmentioned T cells may be
the target cell type for a method of use of the invention.
T cell activation: As used herein, the term "T cell activation" or
"activation of T cells" refers to a cellular process in which
mature T cells, which express antigen-specific T cell receptors on
their surfaces, recognize their cognate antigens and respond by
entering the cell cycle, secreting cytokines or lytic enzymes, and
initiating or becoming competent to perform cell-based effector
functions. T cell activation requires at least two signals to
become fully activated. The first occurs after engagement of the T
cell antigen-specific receptor (TCR) by the antigen-major
histocompatibility complex (MEW), and the second by subsequent
engagement of co-stimulatory molecules (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.
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.
Tetraloop: As used herein, the term "tetraloop" refers to a type of
four-base loop motif found in hairpin or stem-loop RNA secondary
structures that cap duplexes at one end, linking the two strands
comprising the duplex, and provide stability to the hairpin
structure.
Therapeutic agent: As used herein, the term "therapeutic agent"
refers to any agent that, when administered to a subject, has a
therapeutic, diagnostic, and/or prophylactic effect and/or elicits
a desired biological and/or pharmacological effect.
Therapeutically effective amount: As used herein, the terms
"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 the desired biological or medical response, such as,
for example, curing or at least partially arresting the condition
or disease and its complications in a patient already suffering
from the disease (e.g., an improvement in one or more symptoms of a
cancer). Amounts effective for this use will depend on the severity
of the disorder being treated and the general state of the
patient's own immune system.
Treat: The terms "treat," "treating," and "treatment," as used
herein, refer to therapeutic or preventative measures described
herein. The methods of "treatment" employ administration to a
subject, in need of such treatment, a human antibody of the present
disclosure, for example, a subject in need of an enhanced immune
response against a particular antigen or a subject who ultimately
may acquire such a disorder, in order to prevent, cure, delay,
reduce the severity of, or ameliorate one or more symptoms of the
disorder or recurring disorder, or in order to prolong the survival
of a subject beyond that expected in the absence of such
treatment.
Tumor microenvironment: As used herein, the term "tumor
microenvironment" (alternatively "cancer microenvironment";
abbreviated TME) refers to the cellular environment or milieu in
which the tumor or neoplasm exists, including surrounding blood
vessels as well as non-cancerous cells including, but not limited
to, immune cells, fibroblasts, bone marrow-derived inflammatory
cells, and lymphocytes. Signaling molecules and the extracellular
matrix also comprise the TME. The tumor and the surrounding
microenvironment are closely related and interact constantly.
Tumors can influence the microenvironment by releasing
extracellular signals, promoting tumor angiogenesis and inducing
peripheral immune tolerance, while the immune cells in the
microenvironment can affect the growth and evolution of tumor
cells.
Unless otherwise defined, all technical and scientific terms used
herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this disclosure pertains.
Preferred methods and materials are described below, although
methods and materials similar or equivalent to those described
herein can also be used in the practice or testing of the presently
disclosed methods and compositions. All publications, patent
applications, patents, and other references mentioned herein are
incorporated by reference in their entirety.
Equivalents and Scope
Those skilled in the art will recognize, or be able to ascertain
using no more than routine experimentation, many equivalents to the
specific embodiments, described herein. The scope of the present
disclosure is not intended to be limited to the above Description,
but rather is as set forth in the appended claims.
In the claims articles such as "a," "an," and "the" may mean one or
more than one unless indicated to the contrary or otherwise evident
from the context. Claims or descriptions that include "or" between
one or more members of a group are considered satisfied if one,
more than one, or all of the group members are present in, employed
in, or otherwise relevant to a given product or process unless
indicated to the contrary or otherwise evident from the context.
The disclosure includes embodiments in which exactly one member of
the group is present in, employed in, or otherwise relevant to a
given product or process. The disclosure includes embodiments in
which more than one, or all of the group members are present in,
employed in, or otherwise relevant to a given product or process.
Furthermore, it is to be understood that the disclosure encompasses
all variations, combinations, and permutations in which one or more
limitations, elements, clauses, descriptive terms, etc., from one
or more of the listed claims is introduced into another claim. For
example, any claim that is dependent on another claim can be
modified to include one or more limitations found in any other
claim that is dependent on the same base claim. Furthermore, where
the claims recite a composition, it is to be understood that
methods of using the composition for any of the purposes disclosed
herein are included, and methods of making the composition
according to any of the methods of making disclosed herein or other
methods known in the art are included, unless otherwise indicated
or unless it would be evident to one of ordinary skill in the art
that a contradiction or inconsistency would arise.
Where elements are presented as lists, e.g., in Markush group
format, it is to be understood that each subgroup of the elements
is also disclosed, and any element(s) can be removed from the
group. It should it be understood that, in general, where the
invention, or aspects of the invention, is/are referred to as
comprising particular elements, features, etc., certain embodiments
of the invention or aspects of the invention consist, or consist
essentially of, such elements, features, etc. For purposes of
simplicity those embodiments have not been specifically set forth
in haec verba herein.
It is also noted that the term "comprising" is intended to be open
and permits but does not require the inclusion of additional
elements or steps. When the term "comprising" is used herein, the
term "consisting of" is thus also encompassed and disclosed
Where ranges are given, endpoints are included. Furthermore, it is
to be understood that unless otherwise indicated or otherwise
evident from the context and understanding of one of ordinary skill
in the art, values that are expressed as ranges can assume any
specific value or subrange within the stated ranges in different
embodiments of the invention, to the tenth of the unit of the lower
limit of the range, unless the context clearly dictates
otherwise.
In addition, it is to be understood that any particular embodiment
of the present invention that falls within the prior art may be
explicitly excluded from any one or more of the claims. Since such
embodiments are deemed to be known to one of ordinary skill in the
art, they may be excluded even if the exclusion is not set forth
explicitly herein. Any particular embodiment of the compositions of
the invention (e.g., any nucleic acid or protein encoded thereby;
any method of production; any method of use; etc.) can be excluded
from any one or more claims, for any reason, whether or not related
to the existence of prior art.
All cited sources, for example, references, publications,
databases, database entries, and art cited herein, are incorporated
into this application by reference, even if not expressly stated in
the citation. In case of conflicting statements of a cited source
and the instant application, the statement in the instant
application shall control.
RIG-I-Like Receptors and Their Ligands
The present disclosure provides synthetic RNA ligands that
specifically bind to RIG-I-like receptors (RLRs) and agonize RLRs
(RLR agonists). In some aspects, the disclosure provides RLR
agonists that are useful for the treatment of cancer. In some
aspects, the disclosure provides RLR agonists that are useful for
the treatment of infectious disease. In some embodiments, the RLR
agonists induce cytokine production. In some embodiments, the RLR
agonists increase the number of CD8+ T cells in the tumor
microenvironment. In some embodiments, the RLR agonists induce
protective anti-tumor immunity
RIG-I-like receptors (RLRs) comprise a family of DExD/H box RNA
helicases that function as cytosolic pattern recognition receptors
(PRRs) that sense the presence of pathogenic agents via the
recognition of pathogen-associated molecular patterns (PAMPs). In
particular, the intracellular presence of non-self (e.g., viral)
RNA is sensed by an infected cell via binding of the RNA to RLRs
and results in the initiation and modulation of antiviral immunity.
Like most viral RNAs, endogenous mRNA and RNA polymerase III
transcripts are also 5'-triphosphorylated, but eukaryotic mRNAs
possess a 5' cap structure linked to a guanosine methylated at N7
that prevents RIG-I activation. These structural differences
between viral and self RNAs, together with differences in
intracellular localization, are thought to enable the effective
function of RIG-I as a defense against viral infection by the
preferential detection of viral RNA. The molecular recognition and
binding of non-self RNA ligands to RLRs propagates specific
intracellular signal events culminating in the activation of
transcription factors that drive type 1 interferon (IFN) production
and antiviral gene expression. The RLR-mediated induction of IFN
and inflammatory cytokines production as well as antiviral gene
expression elicits an immune response to control virus infection
(Yoneyama et al., (2015) Curr Opin Immunol 32:48-53).
Three RLR family members have been identified: RIG-I (retinoic
acid-inducible gene I)--the founding member and best characterized
of RLR family, MDA5 (melanoma differentiation associated factor 5),
and LGP2 (laboratory of genetics and physiology 2 and a homolog of
mouse D11lgp2). RIG-I is an important component of the innate
immune system and plays a critical role in the defense against
infection by RNA viruses. In contrast to the Toll-like receptors
TLR3, TLR7, TLR8, and TLR9, that detect nucleic acids in the
endosomes of a subset of immune cells, RIG-I is a cytosolic innate
immune receptor that is 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 RNAs (Hornung
et al., (2006) Science 314(5801):994-997; Pichlmair et al., (2006)
Science 314(5801):997-1001).
High-resolution structures of RIG-I/ligand complexes have provided
the molecular detail of RIG-I binding to RNA ligands, specifically
to the activating ligand, double-stranded 5'-triphosphorylated RNA
(ppp-dsRNA) (Civril et al., (2011) EMBO Reports 12(11):1127-1134;
Jiang et al., (2011) Nature 479(7373):423-427; Kowalinski et al.,
(2011) Cell 147(2):423-435; Lu et al., (2010) Structure 18(8):
1032-1043; Luo et al., (2011) Cell 147(2) 409-422; Wang et al.,
(2010) Nature Structural & Molecular Biology 17(7):781-787;
Hornung et al., (2006) Science 314(5801):994-997; Pichlmair et al.,
(2006) Science 314(5801):997-1001; Schlee et al., (2009) Immunity
31(1):25-34).). The crystal structures of RIG-FRNA complexes show
protein binding to the backbone, not the bases, suggesting that the
RNA sequence may not affect RIG-I binding or that RNA sequence may
exhibit as of yet uncharacterized effects or activity. To date,
evidence for sequence-dependent differential interaction or
affinity with, and activation of, RIG-I-like receptors is not
described in the art (Schlee and Hartmann (2010) Molecular Therapy
18(7): 1254-1262).
Accordingly, the disclosure provides synthetic RIG-I-like receptor
(RLR) agonists comprising non-naturally occurring, synthetic, and
or engineered RLR RNA ligands. In some aspects, the disclosure
provides an RLR agonist that specifically binds to a RIG-I-like
receptor (RLR), wherein the agonist comprises a blunt-ended,
hairpin RNA comprising a first polynucleotide connected to a second
polynucleotide by a linker, wherein the first polynucleotide is
sufficiently complementary to the second polynucleotide to form a
duplex, wherein the duplex comprises less than 19 base pairs,
wherein the 5' most nucleotide of the first oligonucleotide
comprises a 5' diphosphate or triphosphate moiety, or derivative or
analog thereof, and 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.
In some embodiments, the RLR agonists of the disclosure comprise a
sequence motif, wherein the sequence motif is selected from the
group consisting of:
(i) a GT-repeat motif;
(ii) a GA-repeat motif;
(iii) a AUCG-repeat motif;
(iv) an AU-repeat motif;
(v) a dipyrimidine motif;
(vi) a dipurine motif;
(vii) a pyrimidine triplet motif;
(viii) a purine triplet motif;
(ix) a palindromic sequence motif; and
(x) a combination of any of (i)-(ix).
In some embodiments, the RLR agonists of the disclosure comprise at
least one improved biological activity, wherein the improved
biological activity is selected from:
(i) an increase in RLR-mediated cytokine production;
(ii) an increase in RLR-mediated expression of
interferon-stimulated genes;
(iii) an increase in RLR-mediated intracellular signaling;
(iv) an increase in binding affinity to RLRs; and
(v) a combination of any of (i)-(iv).
In some embodiments, the RLR agonists of the disclosure comprise a
sequence motif, wherein the sequence motif is a GT-repeat motif
comprises a sequence of <19, about 15-18, about 15, about 10-15,
about 10, about 5-10, about 5, about 4 about 18, 17, 16, 15, 14,
13, 12, 11, 10, 9, 8, 7, 6, 5, or 4 guanine and thymine
nucleotides, or derivatives or analogs thereof. In some
embodiments, the GT-repeat motif is [GT].sub.n, wherein n=2 to 9.
In some embodiments, the GT-repeat motif is [GT].sub.7. In some
embodiments, the GT-repeat motif is [GT].sub.3, and wherein the
GT-repeat motif is followed by a purine triplet and UCG,
respectively. In some embodiments, the purine triplet is GGA.
In some embodiments, the sequence motif is a GA-repeat motif
comprises a sequence of <19, about 15-18, about 15, about 10-15,
about 10, about 5-10, about 5, about 4 about 18, 17, 16, 15, 14,
13, 12, 11, 10, 9, 8, 7, 6, 5, or 4 guanine and adenine
nucleotides, or derivatives or analogs thereof. In some
embodiments, the GA-repeat motif is [GA].sub.n, where n=2 to 9. In
some embodiments, the GA-repeat motif is [GA].sub.7.
In some embodiments, the RLR agonists of the disclosure comprise a
sequence motif, wherein the sequence motif is a AUCG-repeat motif
comprising a sequence of <19, about 16, about 12-16, about 12,
about 8-12, about 6, about 16, 12, 8 adenine, uracil, cytosine, and
guanine nucleotides, or derivatives or analogs thereof.
In some embodiments, the AUCG-repeat motif is [AUCG].sub.n, where
n=2 to 4. In some embodiments, the AUCG-repeat motif is
[AUCG].sub.3.
In some embodiments, the AUCG-repeat 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 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.
In some embodiments, the RLR agonists of the disclosure comprise an
AUCG-repeat motif, wherein one or more uridine nucleosides (U) are
substituted with a modified nucleoside. In some embodiments,
wherein the modified nucleoside is ribothymidine (T). In some
embodiments, the AUGC-repeat motif is [AUCG].sub.3, wherein the one
or more uridine nucleosides (U) comprising the AUCG-repeat motif
are substituted with a modified nucleoside, wherein the modified
nucleoside is ribothymidine (T). In some embodiments, the
AUGC-repeat motif is [AUCG].sub.3, wherein the one or more uridine
nucleosides (U) comprising the AUCG-repeat motif are substituted
with a modified nucleoside, wherein the modified nucleoside is
ribothymidine (T), and wherein the AUGC-repeat motif is preceded by
GG.
In some embodiments, the RLR agonists of the disclosure comprise an
AUCG-repeat motif, wherein one or more guanosine nucleosides (G)
are substituted with a modified nucleoside. In some embodiments,
the modified nucleoside is inosine (I). In some embodiments, the
AUGC-repeat motif is [AUCG].sub.3, wherein the one or more
guanosine nucleosides (G) comprising the AUCG-repeat motif are
substituted with a modified nucleoside, wherein the modified
nucleoside is ribothymidine (T), and wherein the AUGC-repeat motif
is preceded by GG.
In some embodiments, the RLR agonists of the disclosure comprise a
AUCG-repeat motif, wherein the motif is preceded by a IG. In some
embodiments, the AUCG-repeat motif is [AUCG].sub.3 and is preceded
by a IG.
In some embodiments, the RLR agonists of the disclosure comprise an
AUCG-repeat, wherein one or more guanosine nucleosides (G) are
substituted with an inosine (I), wherein the AUCG-repeat is
preceded by an inosine (I). In some embodiments, the guanosine
nucleosides (G) comprising the AUCG-repeat are substituted with an
inosine (I), wherein the AUCG-repeat is preceded by an inosine (I),
wherein the 5' most nucleotide of the first polynucleotide
comprises inosine (I).
In some embodiments, the 5' most nucleotide of the first
oligonucleotide comprises inosine (I).
In some embodiments, the RLR agonists of the disclosure comprise a
AUCG-repeat sequence motif, wherein the AUCG-repeat motif is
[AUCG].sub.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.
In some embodiments, the RLR agonists of the disclosure comprise a
palindromic sequence, wherein the palindromic sequence comprises a
sequence of <19, about 15-18, about 15, about 10-15, about 10,
about 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or 4
nucleotides, or derivatives or analogs thereof, linked in any order
that results in a palindrome.
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].sub.n, where n=2 to 3. In some
embodiments, the AU-repeat motif is [AU].sub.2.
In some aspects, the disclosure provides an RLR agonist that
specifically binds to a RLRs, wherein the agonist comprises a
blunt-ended, hairpin RNA comprising at least one or more
nucleotides comprising inosine which base pairs with cytidine, and
wherein the agonist comprises the formula:
5'-(N.sub.1-N.sub.2-X.sub.1)-L-(X.sub.2-N.sub.3-N.sub.4)-3',
wherein
(i) (N.sub.1-N.sub.2-X.sub.1) comprises a first polynucleotide
comprising linked nucleotides N.sub.1, N.sub.2 and X.sub.1;
(ii) (X.sub.2-N.sub.3-N.sub.4) comprises a second polynucleotide
comprising linked nucleotides X.sub.2, N.sub.3 and N.sub.4;
(iii) N.sub.1, N.sub.2, N.sub.3 and N.sub.4 each comprise a single
nucleotide comprising a nucleoside selected from the group
consisting of: adenosine, guanosine, cytidine, 5-methyluridine,
uridine and inosine;
(iv) N.sub.1 base pairs with N.sub.4;
(v) N.sub.2 base pairs with N.sub.3;
(vi) N.sub.1 comprises a 5' diphosphate or triphosphate moiety, or
derivative or analog thereof;
(vii) X.sub.1 and X.sub.2 are each oligonucleotides comprising
nucleosides selected from the group consisting of: adenosine,
guanosine, cytidine, 5-methyluridine, uridine and inosine;
(viii) X.sub.1 is complementary to X2;
(ix) X.sub.1 and X.sub.2 are each 12 nucleotides to 16 nucleotides
in length and are the same length, and;
(x) L is a linker that covalently links the first polynucleotide
and the second polynucleotide.
In other aspects, the disclosure provides a synthetic RIG-I-like
receptor (RLR) agonist that specifically binds to RIG-I-like
receptors (RLRs), wherein the agonist comprises a blunt-ended,
hairpin RNA comprising a non-nucleotide linker, and wherein the
agonist comprises the formula:
5'-(N.sub.1-N.sub.2-X.sub.1)-L-(X.sub.2-N.sub.3-N.sub.4)-3',
wherein
(i) (N.sub.1-N.sub.2-X.sub.1) comprises a first polynucleotide
comprising linked nucleotides N.sub.1, N.sub.2 and X.sub.1;
(ii) (X.sub.2-N.sub.3-N.sub.4) comprises a second polynucleotide
comprising linked nucleotides X.sub.2, N.sub.3 and N.sub.4;
(iii) N.sub.1, N.sub.2, N.sub.3 and N.sub.4 each comprise a single
nucleotide comprising a nucleoside selected from the group
consisting of: adenosine, guanosine, cytidine, 5-methyluridine,
uridine and inosine;
(iv) N.sub.1 base pairs with N.sub.4;
(v) N.sub.2 base pairs with N.sub.3;
(vi) N.sub.1 comprises a 5' diphosphate or triphosphate moiety, or
derivative or analog thereof;
(vii) X.sub.1 and X.sub.2 are each oligonucleotides comprising
nucleosides selected from the group consisting of: adenosine,
guanosine, cytidine, 5-methyluridine, uridine and inosine;
(viii) X.sub.1 is complementary to X.sub.2;
(ix) X.sub.1 and X.sub.2 are each 12 nucleotides to 16 nucleotides
in length and are the same length, and;
(x) L is the non-nucleotide linker that covalently links the first
polynucleotide and the second polynucleotide.
In some embodiments, inosine, if present in the RLR agonist, base
pairs with cytidine.
In some embodiments, the linker (L) is a nucleotide linker or a
non-nucleotide linker.
In some aspects, the disclosure provides an RLR agonist that
specifically binds to RLRs, wherein the agonist comprises a
blunt-ended, hairpin RNA comprising a nucleotide or non-nucleotide
linker. RNA hairpins are among the most common RNA secondary
structural elements, wherein the hybridized portion or "stem" of
the hairpin are frequently capped by RNA tetraloops. RNA tetraloops
are composed of characteristic four-loop nucleotides that form a
compact and stable structure. While they can be formed by many
different nucleotide sequences, UNCG (N=A, C, G, or U), GNRA (R=A
or G), and CUUG tetraloops are found most often. Tetraloops usually
help initiate RNA-folding processes and provide sites for tertiary
contacts within or between RNAs and for protein binding, thereby
facilitating the assembly of ribonucleoprotein particles. Further
description of tetraloops can be found in Cheong, H., Kim, N. and
Cheong, C. (2015). RNA Structure: Tetraloops. In eLS, John Wiley
& Sons, Ltd (Ed.), which is incorporated herein by reference in
its entirety.
Accordingly, in some embodiments, the RLR agonists of the
disclosure comprise a nucleotide linker comprising a tetraloop. In
some embodiments, the nucleotide sequence of the tetraloop is
selected from the group consisting of: (a) UNCG, wherein N=A, C, G,
or U; (b) GNRA, wherein N=A, C, G, or U, and wherein R=A or G; (c)
ANYA, wherein N=A, C, G, or U, and wherein Y=C or T; (d) CUYG,
wherein Y=C or T; (e) UMAC, wherein M=A or C; and (f) CUUG.
In some embodiments, the nucleotide linker comprises the nucleotide
sequence UUUGAU or UGUUU. In some embodiments, the sequence of the
tetraloop is UUCG. In some embodiments, the sequence of the
tetraloop is GAUC. In some embodiments, the nucleotide linker
comprises the nucleotide sequence UUUGAU. In some embodiments, the
nucleotide linker comprises the nucleotide sequence UGUUU.
In other aspects, the RLR agonists of the disclosure comprise a
non-nucleotide linker. As described herein nucleic acid loops
(e.g., tetraloops) are a common element found in nucleic acid
secondary structure. Nucleotide loops arise in folded domains
occurring in intrastrand duplexes. Synthetic nucleic acids designed
to contain hairpin loops comprising non-nucleotide linking groups
(e.g., non-nucleotide linkers) can replace several nucleotides
bridging a folded duplex structure. Non-nucleotide groups have been
used as linkers in non-folded structures as well. Such linking
groups may be useful replacements of natural nucleotide linkers
(e.g., tetraloops). For example, they can shorten the synthesis of
nucleic acid with a desired secondary structure by several steps,
since one relatively long non-nucleotide linking group replaces
several individual nucleotides which may normally constitute a
loop. Such non-natural loops or linkers (e.g., non-nucleotide
linkers) can confer resistance to degradation by nucleases which
would ordinarily act on a natural loop structure in biological
contexts (e.g., in a cell or in the circulation of a subject upon
administration). A non-nucleotide linking group also has the
potential to provide a more stable folded structure than occurs
with the nucleotide loops and or linkers. Further description of
non-nucleotide linkers can be found in Rumney and Kool (1995) J Am
Chem Soc 117:5635-5646, which is incorporated herein by reference
in its entirety.
Accordingly, in some embodiments, the RLR agonists of the
disclosure comprise a non-nucleotide linker selected from the group
consisting of: (a) an ethylene glycol linker; and (b) an alkyl
linker.
In some embodiments, the non-nucleotide linker is a hexaethylene
glycol linker. In some embodiments, the non-nucleotide linker is a
C9 alkyl linker.
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 is selected from: a
phosphonate, a thiophosphonate, a phosphorothioate, a sulfate, a
sulfonate, a sulfamate, a thiazolidinone, a carboxylate, a
malonate, a boronic acid, a benzoxaborole, a boranophosphate, a
squaramide.
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
to the internucleotide linkages or to the polynucleotide
backbone.
In some aspects, the RLR agonist of the disclosure exhibits at
least one or more of the following properties: (a) specifically
binds to one or more RLRs (e.g. RIG-I, MDA5 and/or LGP2); (b)
increases RLR-mediated cytokine production; (c) increases
RLR-mediated expression of interferon-stimulated genes (ISGs); (d)
increases RLR-dependent intracellular signaling; (e) increases
stability of the duplex; (f) increases binding affinity to RLRs;
(g) decreases off-target binding; (h) increases biological
half-life; (i) increases biodistribution and bioavailability; (j)
increases and/or enhances uptake into cells and/or tissues; (k)
decreases immunogenicity; and (l) a combination of any of
(a)-(k).
In some aspects, the disclosure provides a synthetic RIG-I-like
receptor (RLR) agonist that specifically binds to a RIG-I-like
receptor (RLR), wherein the agonist comprises a blunt-ended,
hairpin RNA comprising the formula:
5'-(N.sub.1-N.sub.2-X.sub.1)-L-(X.sub.2-N.sub.3-N.sub.4)-3',
wherein
(i) (N.sub.1-N.sub.2-X.sub.1) comprises a first polynucleotide
comprising linked nucleotides N.sub.1, N.sub.2 and X.sub.1;
(ii) (X.sub.2-N.sub.3-N.sub.4) comprises a second polynucleotide
comprising linked nucleotides X.sub.2, N.sub.3 and N.sub.4;
(iii) N.sub.1, N.sub.2, N.sub.3 and N.sub.4 each comprise a single
nucleotide comprising a nucleoside selected from the group
consisting of: adenosine, guanosine, cytidine, 5-methyluridine,
uridine and inosine;
(iv) N.sub.1 base pairs with N.sub.4;
(v) N.sub.2 base pairs with N.sub.3;
(vi) N.sub.1 comprises a 5' diphosphate or triphosphate moiety, or
derivative or analog thereof;
(vii) X.sub.1 and X.sub.2 are each oligonucleotides comprising
nucleosides selected from the group consisting of: adenosine,
guanosine, cytidine, 5-methyluridine, uridine and inosine;
(viii) X.sub.1 is complementary to X.sub.2;
(ix) X.sub.1 and X.sub.2 are each 12 nucleotides to 16 nucleotides
in length and are the same length, and;
(x) L is a linker that operably links the first polynucleotide and
the second polynucleotide,
wherein at least one of N1, N2, N3, and N4 is inosine and/or at
least one of X1 and/or X2 comprises at least one inosine
nucleoside, and wherein the inosine nucleoside base pairs with
cytidine in the hairpin RNA.
In some embodiments, N1 comprises inosine and N4 comprises
cytidine. In some embodiments, N1 comprises cytidine and N4
comprises inosine. In some embodiments, N2 comprise inosine and N3
comprises cytidine. In some embodiments, N2 comprises cytidine and
N3 comprises inosine. In some embodiments, N1 comprises guanosine.
In some embodiments, N2 comprises guanosine. In some embodiments,
N1 comprises cytidine. In some embodiments, N2 comprises cytidine.
In some embodiments, N1 and N2 comprise guanosine and N.sub.3 and
N.sub.4 comprise cytidine. In some embodiments, N1 and N2 comprise
cytidine and N.sub.3 and N.sub.4 comprise guanosine. In some
embodiments, N1 and N2 comprise inosine and N.sub.3 and N.sub.4
comprise cytidine. In some embodiments, N1 and N2 comprise cytidine
and N.sub.3 and N.sub.4 comprise inosine. In some embodiments, N1
comprises inosine and N4 comprises cytidine, and X1 and/or X2 each
comprise at least one inosine. In some embodiments, N2 comprises
inosine and N3 comprises cytidine, and X1 and/or X2 each comprise
at least one inosine. In some embodiments, N1 and N2 comprise
guanosine N.sub.3 and N.sub.4 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 cytidine and N3 and N4 comprise guanosine, 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 inosine and no guanosine nucleosides. In some
embodiments, N1 and N2 comprise cytidine and N.sub.3 and N.sub.4
comprise guanosine, and X1 and X2 each comprise inosine and no
guanosine nucleosides. In some embodiments, N1 and N2 comprise
inosine and N3 and N4 comprise cytidine, and X1 and/or X2 each
comprise at least one inosine. In some embodiments, N1 and N2
comprise inosine and N3 and N4 comprise cytidine, and X1 and X2
each comprise at least one inosine. In some embodiments, N1 and N2
comprise cytidine and N3 and N4 comprise inosine, and X1 and/or X2
each comprise at least one inosine. In some embodiments, N1 and N2
comprise inosine and N3 and N4 comprise cytidine, and X1 and X2
comprise inosine and no guanosine nucleosides. In some embodiments,
N1 and N2 comprise cytidine and N.sub.3 and N.sub.4 comprise
inosine, and X1 and X2 comprise inosine and no guanosine
nucleosides. In some embodiments, X1 and X2 are each 12 nucleotides
and comprise 1, 2, 3 or 4 inosine nucleosides. In some embodiments,
X1 and X2 are each 13 nucleotides and comprise 1, 2, 3, 4 or 5
inosine nucleosides. In some embodiments, X1 and X2 are each 14
nucleotides and comprise 1, 2, 3, 4, 5 or 6 inosine nucleosides. In
some embodiments, X1 and X2 are each 15 nucleotides and comprise 1,
2, 3, 4, 5, 6, or 7 inosine nucleosides. In some embodiments, X1
and X2 are each 16 nucleotides and each comprise 1, 2, 3, 4, 5, 6,
7, or 8 inosine nucleosides. In some embodiments, X1 and X2 are
each 12 nucleotides and comprise at least 10%, 20%, 30% or 40%
inosine nucleosides.
In some aspects, the disclosure provides a synthetic RIG-I-like
receptor (RLR) agonist that specifically binds to a RIG-I-like
receptor (RLR), wherein the agonist comprises a blunt-ended,
hairpin RNA comprising the formula:
5'-(N.sub.1-N.sub.2-X.sub.1)-L-(X.sub.2-N.sub.3-N.sub.4)-3',
wherein
(i) (N.sub.1-N.sub.2-X.sub.1) comprises a first polynucleotide
comprising linked nucleotides N.sub.1, N.sub.2 and X.sub.1;
(ii) (X.sub.2-N.sub.3-N.sub.4) comprises a second polynucleotide
comprising linked nucleotides X.sub.2, N.sub.3 and N.sub.4;
(iii) N.sub.1, N.sub.2, N.sub.3 and N.sub.4 each comprise a single
nucleotide comprising a nucleoside selected from the group
consisting of: adenosine, guanosine, cytidine, 5-methyluridine,
uridine and inosine;
(iv) N.sub.1 base pairs with N.sub.4;
(v) N.sub.2 base pairs with N.sub.3;
(vi) N.sub.1 comprises a 5' diphosphate or triphosphate moiety, or
derivative or analog thereof;
(vii) X.sub.1 comprises a sequence motif [AUCN5].sub.x, wherein
N.sub.5 is comprises guanosine or inosine, wherein x is an integer
whose value indicates the number of sequence motifs, and wherein
x=3 or 4;
(viii) X.sub.2 comprises a sequence motif [CN.sub.6AU].sub.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;
(ix) L is a linker that operably links the first polynucleotide and
the second polynucleotide,
optionally, wherein at least one of N1, N2, N3, and N4 is inosine,
and wherein the inosine nucleoside base pairs with cytidine in the
hairpin RNA. In some embodiments, N5 comprises inosine and N6
comprises inosine. In some embodiments, N5 comprises guanosine and
N6 comprises inosine. In some embodiments, N5 comprises inosine and
N6 comprises guanosine. In some embodiments, N5 comprises guanosine
(G) and N6 comprises guanosine (G). In some embodiments, x=3 and
y=3. In some embodiments, x=4 and y=4. In some embodiments, N1
comprises inosine (I) and N4 comprises cytidine (C). In some
embodiments, N2 comprises inosine (I) and N3 comprises cytidine
(C). In some embodiments, N3 comprises inosine (I) and N2 comprises
cytidine (C). In some embodiments, N4 comprises inosine (I) and N1
comprises cytidine (C). In some embodiments, N1 comprises guanosine
(G). In some embodiments, N2 comprises guanosine (G). In some
embodiments, N1 comprises cytidine (C). In some embodiments, N2
comprises cytidine (C). In some embodiments, N1 and N2 comprise
guanosine (G) and N.sub.3 and N.sub.4 comprise cytidine (C). In
some embodiments, N1 and N2 comprise cytidine (C) and N.sub.3 and
N.sub.4 comprise guanosine (G). In some embodiments, N1 and N2
comprise inosine (I) and N.sub.3 and N.sub.4 comprise cytidine (C).
In some embodiments, N1 and N2 comprise cytidine (C) and N.sub.3
and N.sub.4 comprise inosine (I).
In some embodiments, the linker (L) is a nucleotide linker or a
non-nucleotide linker. In some embodiments, the linker (L) is a
nucleotide linker comprising a tetraloop, wherein the nucleotide
sequence of the tetraloop is selected from the group consisting of:
(a) UNCG, wherein N=A, C, G, or U; (b) GNRA, wherein N=A, C, G, or
U, and wherein R=A or G; (c) ANYA, wherein N=A, C, G, or U, and
wherein Y=C or T; (d) CUYG, wherein Y=C or T; (e) UMAC, wherein M=A
or C; and (f) CUUG.
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.
In some embodiments, the linker (L) is a nucleotide linker
comprising a tetraloop, wherein the sequence of the tetraloop is
UUCG. In some embodiments, the sequence of the tetraloop is
GAUC.
In some embodiments, the linker (L) is a non-nucleotide linker
selected from the group consisting of: (a) an ethylene glycol
linker; and (b) an alkyl linker.
In some embodiments, the non-nucleotide linker is a hexaethylene
glycol linker. In some embodiments, the the non-nucleotide linker
is a C9 alkyl linker.
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 is selected from: a
phosphonate, a thiophosphonate, a phosphorothioate, a sulfate, a
sulfonate, a sulfamate, a thiazolidinone, a carboxylate, a
malonate, a boronic acid, a benzoxaborole, a boranophosphate, a
squaramide.
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 to the internucleotide linkages or to the
polynucleotide backbone.
In some embodiments, the RLR agonist exhibits at least one or more
of the following properties: (a) specifically binds to one or more
RLRs (e.g. RIG-1, MDA5 and/or LGP2); (b) increases RLR-mediated
cytokine production; (c) increases RLR-mediated expression of
interferon-stimulated genes (ISGs); (d) increases RLR-dependent
intracellular signaling; (e) increases stability of the duplex; (f)
increases binding affinity to RLRs; (g) decreases off-target
binding; (h) increases biological half-life; (i) increases
biodistribution and bioavailability; (j) increases and/or enhances
uptake into cells and/or tissues; (k) decreases immunogenicity; and
(l) a combination of any of (a)-(k).
In some aspects, the disclosure provides a synthetic RIG-I-like
receptor (RLR) agonist that specifically binds to a RIG-I-like
receptor (RLR), wherein the agonist comprises a blunt-ended,
hairpin RNA comprising a 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 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, and wherein the agonist comprises the nucleotide sequence
selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 and 36.
In some aspects, the disclosure provides a synthetic RIG-I-like
receptor (RLR) agonist that specifically binds to a RIG-I-like
receptor (RLR), wherein the agonist comprises a blunt-ended,
hairpin RNA comprising a 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 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, and wherein the first polynucleotide and the second
polynucleotide comprise the nucleotide sequences selected from the
group consisting of:
(i) SEQ ID NO: 37 and 68, respectively;
(ii) SEQ ID NO: 38 and 69, respectively;
(iii) SEQ ID NO: 39 and 70, respectively;
(iv) SEQ ID NO: 40 and 71, respectively;
(v) SEQ ID NO: 41 and 72, respectively;
(vi) SEQ ID NO: 42 and 73, respectively;
(vii) SEQ ID NO: 43 and 74, respectively;
(viii) SEQ ID NO: 44 and 75, respectively;
(ix) SEQ ID NO: 45 and 76, respectively;
(x) SEQ ID NO: 46 and 77, respectively;
(xi) SEQ ID NO: 47 and 78, respectively;
(xii) SEQ ID NO: 48 and 79, respectively;
(xiii) SEQ ID NO: 49 and 80, respectively;
(xiv) SEQ ID NO: 50 and 81, respectively;
(xv) SEQ ID NO: 51 and 82, respectively;
(xvi) SEQ ID NO: 52 and 83, respectively;
(xvii) SEQ ID NO: 53 and 84, respectively;
(xviii) SEQ ID NO: 54 and 85, respectively;
(xix) SEQ ID NO: 55 and 86, respectively;
(xx) SEQ ID NO: 56 and 87, respectively;
(xxi) SEQ ID NO: 57 and 88, respectively;
(xxii) SEQ ID NO: 58 and 89, respectively;
(xxiii) SEQ ID NO: 59 and 89, respectively;
(xxiv) SEQ ID NO: 60 and 90, respectively;
(xxv) SEQ ID NO: 61 and 91, respectively;
(xxvi) SEQ ID NO: 62 and 92, respectively;
(xxvii) SEQ ID NO: 63 and 91, respectively;
(xxviii) SEQ ID NO: 64 and 93, respectively;
(xxix) SEQ ID NO: 65 and 94, respectively;
(xxx) SEQ ID NO: 66 and 95, respectively;
(xxxi) SEQ ID NO: 67 and 96, respectively; and
(xxxii) SEQ ID NO: 63 and 97, respectively.
In some aspects, the disclosure provides a synthetic RIG-I-like
receptor (RLR) agonist that specifically binds to a RIG-I-like
receptor (RLR), wherein the agonist comprises a blunt-ended,
hairpin RNA comprising at least one or more nucleotides comprising
inosine which base pairs with cytidine, and wherein the agonist
comprises the nucleotide sequence selected from the group
consisting of SEQ ID NOs: 22, 23 and 25.
In some aspects, the disclosure provides a synthetic RIG-I-like
receptor (RLR) agonist that specifically binds to a RIG-I-like
receptor (RLR), wherein the agonist comprises a blunt-ended,
hairpin RNA comprising at least one or more nucleotides comprising
inosine which base pairs with cytidine, wherein the agonist
comprises the formula
5'-(N.sub.1-N.sub.2-X.sub.1)-L-(X.sub.2-N.sub.3-N.sub.4)-3',
wherein (N.sub.1-N.sub.2-X.sub.1) comprises a first polynucleotide
and (X2-N.sub.3-N.sub.4) comprises a second polynucleotide, and
wherein the first polynucleotide and the second polynucleotide
comprise the nucleotide sequences selected from the group
consisting of: (i) SEQ ID NO: 58 and 89, respectively; (ii) SEQ ID
NO: 59 and 89, respectively; and (iii) SEQ ID NO: 61 and 91,
respectively.
In some aspects, the disclosure provides a synthetic RIG-I-like
receptor (RLR) agonist that specifically binds to RIG-I-like
receptors (RLRs), wherein the agonist comprises a blunt-ended,
hairpin RNA comprising a non-nucleotide linker, wherein the agonist
comprises the formula
5'-(N.sub.1-N.sub.2-X.sub.1)-L-(X.sub.2-N.sub.3-N.sub.4)-3',
wherein (N.sub.1-N.sub.2-X.sub.1) comprises a first polynucleotide
and (X.sub.2-N.sub.3-N.sub.4) comprises a second polynucleotide,
and wherein the first polynucleotide and the second polynucleotide
comprise the nucleotide sequences selected from the group
consisting of:
(i) SEQ ID NO: 37 and 68, respectively;
(ii) SEQ ID NO: 38 and 69, respectively;
(iii) SEQ ID NO: 39 and 70, respectively;
(iv) SEQ ID NO: 40 and 71, respectively;
(v) SEQ ID NO: 41 and 72, respectively;
(vi) SEQ ID NO: 42 and 73, respectively;
(vii) SEQ ID NO: 43 and 74, respectively;
(viii) SEQ ID NO: 44 and 75, respectively;
(ix) SEQ ID NO: 45 and 76, respectively;
(x) SEQ ID NO: 46 and 77, respectively;
(xi) SEQ ID NO: 47 and 78, respectively;
(xii) SEQ ID NO: 48 and 79, respectively;
(xiii) SEQ ID NO: 49 and 80, respectively;
(xiv) SEQ ID NO: 50 and 81, respectively;
(xv) SEQ ID NO: 51 and 82, respectively;
(xvi) SEQ ID NO: 52 and 83, respectively;
(xvii) SEQ ID NO: 53 and 84, respectively;
(xviii) SEQ ID NO: 54 and 85, respectively;
(xix) SEQ ID NO: 55 and 86, respectively;
(xx) SEQ ID NO: 56 and 87, respectively;
(xxi) SEQ ID NO: 57 and 88, respectively;
(xxii) SEQ ID NO: 58 and 89, respectively;
(xxiii) SEQ ID NO: 59 and 89, respectively;
(xxiv) SEQ ID NO: 60 and 90, respectively;
(xxv) SEQ ID NO: 61 and 91, respectively;
(xxvi) SEQ ID NO: 62 and 92, respectively;
(xxvii) SEQ ID NO: 63 and 91, respectively;
(xxviii) SEQ ID NO: 64 and 93, respectively;
(xxix) SEQ ID NO: 65 and 94, respectively;
(xxx) SEQ ID NO: 66 and 95, respectively;
(xxxi) SEQ ID NO: 67 and 96, respectively; and
(xxxii) SEQ ID NO: 63 and 97, respectively.
In some aspects, the disclosure provides RLR agonists wherein the
nucleotide sequence comprising the agonist is not complementary to
a genomic DNA sequence or mRNA sequence, wherein the RLR agonist
does not participate in RNA interference, and wherein the RLR
agonist does not silence gene expression.
RLR Agonists Comprising Modified Nucleobases, Nucleosides, or
Nucleotides
In some embodiments, an RLR agonist of the disclosure comprises one
or more modified nucleobases, nucleosides, or nucleotides. In some
embodiments, modified RLR agonists may have useful properties,
including enhanced stability, intracellular retention, enhanced
target binding, and/or an increase in induction of the innate
immune response of a cell into which the RLR agonist is introduced,
as compared to a reference unmodified RLR agonist. Therefore, use
of modified RLR agonists may enhance the efficiency of target
binding, intracellular retention of nucleic acids, as well as
possess reduced immunogenicity. In one embodiment, the agonist
provided by the disclosure is comprised of one or more
oligonucleotides that comprise at least one region modified to
increase target binding affinity. Affinity of an oligonucleotide
for its target polypeptide (e.g. an RLR receptor) can be determined
by, for example, measuring the degree of fluorescence polarization
(FP) upon binding of a fluorescently-labeled oligonucleotide to its
target (Moerke (2009) Curr Protoc Chem Biol 1(1):1-15).
In another embodiment, the RLR agonist provided by the disclosure
is comprised of at least one oligonucleotide comprising 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 routinely determined by measuring
the Tm of the duplex, which is the temperature at which the two
oligonucleotide strands comprising the duplex dissociate;
dissociation is detected spectrophotometrically. The higher the Tm,
the greater the stability of the duplex.
In one embodiment, the region of the oligonucleotide which is
modified to increase duplex stability comprises at least one
nucleotide modified at the 2' position of the sugar, most
preferably a 2'-O-alkyl, 2-O-alkyl-O-alkyl or 2'-fluoro-modified
nucleotide. In another embodiment, an oligonucleotide comprising an
RLR agonist is also modified to enhance nuclease resistance. Cells
contain a variety of exo- and endo-nucleases which can degrade
nucleic acids. A number of nucleotide and nucleoside modifications
have been shown to make the oligonucleotide into which they are
incorporated more resistant to nuclease digestion than an
unmodified oligonucleotide. Nuclease resistance is routinely
measured by incubating oligonucleotides with cellular extracts or
isolated nuclease solutions and measuring the extent of intact
oligonucleotide remaining over time, usually by gel
electrophoresis. Oligonucleotides which have been modified to
enhance their nuclease resistance survive intact for a longer time
than unmodified oligonucleotides. A variety of oligonucleotide
modifications have been demonstrated to enhance or confer nuclease
resistance. Oligonucleotides which contain at least one
phosphorothioate modification are presently more preferred. In some
cases, oligonucleotide modifications which enhance target binding
affinity are also, independently, able to enhance nuclease
resistance (De Mesmaeker et al., 1995, Acc. Chem. Res.
28:366-374).
Specific examples of some preferred oligonucleotides envisioned for
this invention include those containing modified backbones, for
example, phosphorothioates, phosphotriesters, methyl phosphonates,
short chain alkyl or cycloalkyl intersugar linkages or short chain
heteroatomic or heterocyclic intersugar linkages. Most preferred
are oligonucleotides with phosphorothioate backbones (including
those synthesized in a stereo-specific manner) and those with
heteroatom backbones, particularly CH2-NH--O--CH2,
CH2-N(CH3)-O--CH2 [known as a methylene(methylimino) or MMI
backbone], CH2-O--N(CH3)-CH2, CH2-N(CH3)-N(CH3)-CH2 and
O--N(CH3)-CH2-CH2 backbones, wherein the native phosphodiester
backbone is represented as O--P--O--CH2). The amide backbones
disclosed by De Mesmaeker et al. (1995, Acc. Chem. Res. 28:366-374)
are also preferred. Oligonucleotides may also contain one or more
substituted sugar moieties. Preferred oligonucleotides comprise one
of the following at the 2' position: OH, SH, SCH3, F, OCN,
OCH3OCH3, OCH3O(CH2)nCH3, O(CH2)nNH2 or O(CH2)nCH3 where n is from
1 to about 10; C1 to C10 lower alkyl, alkoxyalkoxy (also known in
the art as O-alkyl-O-alkyl), substituted lower alkyl, alkaryl or
aralkyl; Cl; Br; CN; CF3; OCF3; O, S--, or N-alkyl; O--O, S--, or
N-alkenyl; SOCH3; SO2CH3; ONO2; NO2; N3; NH2; heterocycloalkyl;
heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted
silyl; an RNA cleaving group; a reporter group; an intercalator; a
group for improving the pharmacokinetic properties of an
oligonucleotide; or a group for improving the pharmacodynamic
properties of an oligonucleotide and other substituents having
similar properties. A preferred modification includes
2'-methoxyethoxy [2'-O--CH2CH2OCH3, also known as
2'-O-(2-methoxyethyl) or 2'-MOE] (Martin et al., Helv. Chim. Acta,
1995, 78, 486). Other preferred modifications include 2'-methoxy
(2'-O--CH3), 2'-propoxy (2'-OCH2CH2CH3) and 2'-fluoro (2'-F).
Similar modifications may also be made at other positions on the
oligonucleotide, particularly the 3' position of the sugar on the
3' terminal nucleotide and the 5' position of 5' terminal
nucleotide. Oligonucleotides may also have sugar mimetics such as
cyclobutyls in place of the pentofuranosyl group.
Oligonucleotides may also include, additionally or alternatively,
nucleobase (often referred to in the art simply as "base")
modifications or substitutions. As used herein, "unmodified" or
"natural" nucleobases include adenine (A), guanine (G), thymine
(T), cytosine (C) and uracil (U). Modified nucleobases include
nucleobases found only infrequently or transiently in natural
nucleic acids, e.g., hypoxanthine, 6-methyladenine, 5-me
pyrimidines, particularly 5-methylcytosine (also referred to as
5-methyl-2'deoxycytosine and often referred to in the art as
5-me-C), 5-hydroxymethylcytosine (HMC), glycosyl HMC and
gentobiosyl HMC, as well as synthetic nucleobases, e.g.,
2-aminoadenine, 2-thiouracil, 2-thiothymine, 5-bromouracil,
5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine,
N6(6-aminohexyl)adenine and 2,6-diaminopurine. Kornberg, A., DNA
Replication, W. H. Freeman & Co., San Francisco, 1980, pp75-77;
Gebeyehu, G., et al., 1987, Nucl. Acids Res. 15:4513). A
"universal" base known in the art, e.g., inosine, may be included.
5-me-C substitutions have been shown to increase nucleic acid
duplex stability by 0.6-1.2.degree. C. (Sanghvi, Y. S., in Crooke,
S. T. and Lebleu, B., eds., Antisense Research and Applications,
CRC Press, Boca Raton, 1993, pp. 276-278) and are presently
preferred base substitutions.
Another modification of the oligonucleotides of the invention
involves chemically linking to the oligonucleotide one or more
moieties or conjugates which enhance the activity or cellular
uptake of the oligonucleotide. Such moieties include but are not
limited to lipid moieties such as a cholesterol moiety, a
cholesteryl moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA,
1989, 86, 6553), cholic acid (Manoharan et al., Bioorg. Med. Chem.
Let., 1994, 4, 1053), a thioether, e.g., hexyl-S-tritylthiol
(Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306; Manoharan
et al., Bioorg. Med. Chem. Let., 1993, 3, 2765), a thiocholesterol
(Oberhauser et al., Nucl. Acids Res., 1992, 20, 533), an aliphatic
chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et
al., EMBO J., 1991, 10, 111; Kabanov et al., FEBS Lett., 1990, 259,
327; Svinarchuk et al., Biochimie, 1993, 75, 49), a phospholipid, a
polyamine or a polyethylene glycol chain (Manoharan et al.,
Nucleosides & Nucleotides, 1995, 14, 969), or adamantane acetic
acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651).
Oligonucleotides comprising lipophilic 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.
The oligonucleotides of the invention may be provided as prodrugs,
which comprise one or more moieties which are cleaved off,
generally in the body, to yield an active oligonucleotide. One
example of a prodrug approach is described by Imbach et al. in WO
Publication 94/26764.
It is not necessary for all positions in a given oligonucleotide to
be uniformly modified, and in fact more than one of the
aforementioned modifications may be incorporated in a single
oligonucleotide or even at within a single nucleoside within an
oligonucleotide.
The oligonucleotides in accordance with this invention preferably
are from about 8 to about 50 nucleotides in length. In the context
of this invention it is understood that this encompasses
non-naturally occurring oligomers as hereinbefore described, having
8 to 50 monomers.
The oligonucleotides used in accordance with this invention may be
conveniently and routinely made through the well-known technique of
solid phase synthesis. Equipment for such synthesis is sold by
several vendors including Applied Biosystems. Any other means for
such synthesis may also be employed; the actual synthesis of the
oligonucleotides is well within the knowledge and ability of one of
ordinary skill in the art. It is also well known to use similar
techniques to prepare other oligonucleotides such as the
phosphorothioates and alkylated derivatives. It is also well known
to use similar techniques and commercially available modified
amidites and controlled-pore glass (CPG) products such as biotin,
fluorescein, acridine or psoralen-modified amidites and/or CPG
(available from Glen Research, Sterling Va.) to synthesize
fluorescently labeled, biotinylated or other modified
oligonucleotides such as cholesterol-modified oligonucleotides.
In some embodiments, an RLR agonist includes one or more (e.g., 1,
2, 3 or 4) different modified nucleobases, nucleosides, or
nucleotides. In some embodiments, an RLR agonist includes one or
more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70,
80, 90, 100, or more) different modified nucleobases, nucleosides,
or nucleotides. In some embodiments, the modified RLR agonist may
have reduced degradation in a cell into which the RLR agonist is
introduced, relative to a corresponding unmodified RLR agonist.
In some embodiments, the modified nucleobase is a modified uracil.
Exemplary nucleobases and nucleosides having a modified uracil
include pseudouridine (.psi.), pyridin-4-one ribonucleoside,
5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine
(s.sup.2U), 4-thio-uridine (s.sup.4U), 4-thio-pseudouridine,
2-thio-pseudouridine, 5-hydroxy-uridine (ho.sup.5U),
5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridineor
5-bromo-uridine), 3-methyl-uridine (m.sup.3U), 5-methoxy-uridine
(mo.sup.5U), uridine 5-oxyacetic acid (cmo.sup.5U), uridine
5-oxyacetic acid methyl ester (mcmo.sup.5U),
5-carboxymethyl-uridine (cm.sup.5U), 1-carboxymethyl-pseudouridine,
5-carboxyhydroxymethyl-uridine (chm.sup.5U),
5-carboxyhydroxymethyl-uridine methyl ester (mchm.sup.5U),
5-methoxycarbonylmethyl-uridine (mcm.sup.5U),
5-methoxycarbonylmethyl-2-thio-uridine (mcm.sup.5 s.sup.2U),
5-aminomethyl-2-thio-uridine (nm.sup.5 s.sup.2U), 5-methyl
aminomethyl-uridine (mnm.sup.5U), 5-methyl
aminomethyl-2-thio-uridine (mnm.sup.5 s.sup.2U), 5-methyl
aminomethyl-2-seleno-uridine (mnm.sup.5se.sup.2U),
5-carbamoylmethyl-uridine (ncm.sup.5U),
5-carboxymethylaminomethyl-uridine (cmnm.sup.5U), 5-carboxymethyl
aminomethyl-2-thio-uridine (cmnm.sup.5 s.sup.2U),
5-propynyl-uridine, 1-propynyl-pseudouridine,
5-taurinomethyl-uridine (.tau.m.sup.5U),
1-taurinomethyl-pseudouridine,
5-taurinomethyl-2-thio-uridine(.tau.m.sup.5 s.sup.2U),
1-taurinomethyl-4-thio-pseudouridine, 5-methyl-uridine (m.sup.5U,
i.e., having the nucleobase deoxythymine), 1-methyl-pseudouridine
(m.sup.1.psi.), 5-methyl-2-thio-uridine (m.sup.5 s.sup.2U),
1-methyl-4-thio-pseudouridine (m.sup.1s.sup.4.psi.),
4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine
(m.sup.3.psi.), 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.sup.5D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine,
2-methoxy-uridine, 2-methoxy-4-thio-uridine,
4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine,
N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine
(acp.sup.3U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine
(acp.sup.3 .psi.), 5-(isopentenylaminomethyl)uridine (inm.sup.5U),
5-(isopentenylaminomethyl)-2-thio-uridine (inm.sup.5 s.sup.2U),
.alpha.-thio-uridine, 2'-O-methyl-uridine (Um),
5,2'-O-dimethyl-uridine (m.sup.5Um), 2'-O-methyl-pseudouridine
(.psi.m), 2-thio-2'-O-methyl-uridine (s.sup.2Um),
5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcm.sup.5Um),
5-carbamoylmethyl-2'-O-methyl-uridine (ncm.sup.5Um),
5-carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm.sup.5Um),
3,2'-O-dimethyl-uridine (m.sup.3Um), and
5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm.sup.5Um),
1-thio-uridine, deoxythymidine, 2'-F-ara-uridine, 2'-F-uridine,
2'-OH-ara-uridine, 5-(2-carbomethoxyvinyl) uridine, and
5-[3-(1-E-propenylamino)]uridine.
In some embodiments, the modified nucleobase is a modified
cytosine. Exemplary nucleobases and nucleosides having a modified
cytosine include 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine,
3-methyl-cytidine (m.sup.3C), N4-acetyl-cytidine (ac.sup.4C),
5-formyl-cytidine (f.sup.5C), N4-methyl-cytidine (m.sup.4C),
5-methyl-cytidine (m.sup.5C), 5-halo-cytidine (e.g.,
5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm.sup.5C),
1-methyl-pseudoisocytidine, pyrrolo-cytidine,
pyrrolo-pseudoisocytidine, 2-thio-cytidine (s.sup.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, zebularine, 5-aza-zebularine,
5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine,
2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine,
4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine,
lysidine (k.sub.2C), .alpha.-thio-cytidine, 2'-O-methyl-cytidine
(Cm), 5,2'-O-dimethyl-cytidine (m.sup.5Cm),
N4-acetyl-2'-O-methyl-cytidine (ac.sup.4Cm),
N4,2-O-dimethyl-cytidine (m.sup.4Cm), 5-formyl-2'-O-methyl-cytidine
(f.sup.5Cm), N4,N4,2'-O-trimethyl-cytidine (m.sup.4.sub.2Cm),
1-thio-cytidine, 2'-F-ara-cytidine, 2'-F-cytidine, and
2'-OH-ara-cytidine.
In some embodiments, the modified nucleobase is a modified adenine.
Exemplary nucleobases and nucleosides having a modified adenine
include .alpha.-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.sup.1A), 2-methyl-adenine (m.sup.2A),
N6-methyl-adenosine (m.sup.6A), 2-methylthio-N6-methyl-adenosine
(ms.sup.2m.sup.6A), N6-isopentenyl-adenosine (i.sup.6A),
2-methylthio-N6-isopentenyl-adenosine (ms.sup.2i.sup.6A),
N6-(cis-hydroxyisopentenyl)adenosine (io.sup.6A),
2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine
(ms.sup.2io.sup.6A), N6-glycinylcarbamoyl-adenosine (g.sup.6A),
N6-threonylcarbamoyl-adenosine (t.sup.6A),
N6-methyl-N6-threonylcarbamoyl-adenosine (m.sup.6t.sup.6A),
2-methylthio-N6-threonylcarbamoyl-adenosine (ms.sup.2g.sup.6A),
N6,N6-dimethyl-adenosine (m.sup.6.sub.2A),
N6-hydroxynorvalylcarbamoyl-adenosine (hn.sup.6A),
2-methylthio-N6-hydroxynorvalylcarbamoyl-adenosine
(ms.sup.2hn.sup.6A), N6-acetyl-adenosine (ac.sup.6A),
7-methyl-adenine, 2-methylthio-adenine, 2-methoxy-adenine,
.alpha.-thio-adenosine, 2'-O-methyl-adenosine (Am),
N6,2'-O-dimethyl-adenosine (m.sup.6Am),
N6,N6,2'-O-trimethyl-adenosine (m.sup.6.sub.2Am),
1,2'-O-dimethyl-adenosine (m.sup.1Am), 2'-O-ribosyladenosine
(phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1-thio-adenosine,
8-azido-adenosine, 2'-F-ara-adenosine, 2'-F-adenosine,
2'-OH-ara-adenosine, and
N6-(19-amino-pentaoxanonadecyl)-adenosine.
In some embodiments, the modified nucleobase is a modified guanine.
Exemplary nucleobases and nucleosides having a modified guanine
include .alpha.-thio-guanosine, inosine (I), 1-methyl-inosine
(m.sup.1I), wyosine (imG), methylwyosine (mimG), 4-demethyl-wyosine
(imG-14), isowyosine (imG2), wybutosine (yW), peroxywybutosine
(o.sub.2yW), hydroxywybutosine (OhyW), undermodified
hydroxywybutosine (OhyW*), 7-deaza-guanosine, queuosine (Q),
epoxyqueuosine (oQ), galactosyl-queuosine (galQ),
mannosyl-queuosine (manQ), 7-cyano-7-deaza-guanosine (preQ.sub.0),
7-aminomethyl-7-deaza-guanosine (preQ.sub.1), archaeosine
(G.sup.+), 7-deaza-8-aza-guanosine, 6-thio-guanosine,
6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine,
7-methyl-guanosine (m.sup.7G), 6-thio-7-methyl-guanosine,
7-methyl-inosine, 6-methoxy-guanosine, 1-methyl-guanosine
(m.sup.1G), N2-methyl-guanosine (m.sup.2G),
N2,N2-dimethyl-guanosine (m.sup.22G), N2,7-dimethyl-guanosine
(m.sup.2,7G), N2, N2,7-dimethyl-guanosine (m.sup.2,2,7G),
8-oxo-guanosine, 7-methyl-8-oxo-guanosine,
1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine,
N2,N2-dimethyl-6-thio-guanosine, .alpha.-thio-guanosine,
2'-O-methyl-guanosine (Gm), N2-methyl-2'-O-methyl-guanosine
(m.sup.2Gm), N2,N2-dimethyl-2'-O-methyl-guanosine
(m.sup.2.sub.2Gm), 1-methyl-2'-O-methyl-guanosine (m.sup.1Gm),
N2,7-dimethyl-2'-O-methyl-guanosine (m.sup.2,7Gm),
2'-O-methyl-inosine (Im), 1,2'-O-dimethyl-inosine (m.sup.1Im),
2'-O-ribosylguanosine (phosphate) (Gr(p)), 1-thio-guanosine,
O6-methyl-guanosine, 2'-F-ara-guanosine, and 2'-F-guanosine.
In some embodiments, an RLR agonist of the disclosure includes a
combination of one or more of the aforementioned modified
nucleobases (e.g., a combination of 2, 3 or 4 of the aforementioned
modified nucleobases.)
In certain embodiments, an RLR agonist of the disclosure is
uniformly modified (i.e., fully modified, modified through-out the
entire sequence) for a particular modification. For example, an RLR
agonist can be uniformly modified with 5-methyl-cytidine
(m.sup.5C), meaning that all cytosine residues in the mRNA sequence
are replaced with 5-methyl-cytidine (m.sup.5C). Similarly, an RLR
agonist of the disclosure can be uniformly modified for any type of
nucleoside residue present in the sequence by replacement with a
modified residue such as those set forth above.
Examples of nucleoside modifications and combinations thereof that
may be present in an RLR agonist of the present disclosure include,
but are not limited to, those described in PCT Patent Application
Publications: WO2012045075, WO2014081507, WO2014093924,
WO2014164253, and WO2014159813.
The RLR agonists of the disclosure can include a combination of
modifications to the sugar, the nucleobase, and/or the
internucleoside linkage. These combinations can include any one or
more modifications described herein.
Examples of modified nucleosides and modified nucleoside
combinations are provided below in Table 1 and Table 2. These
combinations of modified nucleotides can be used to form the RLR
agonists of the disclosure. In certain embodiments, the modified
nucleosides may be partially or completely substituted for the
natural nucleotides of the RLR agonists of the disclosure. As a
non-limiting example, the natural nucleotide uridine may be
substituted with a modified nucleoside described herein. In another
non-limiting example, the natural nucleoside uridine may be
partially substituted (e.g., about 0.1%, 1%, 5%, 10%, 15%, 20%,
25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
90%, 95% or 99.9% of the natural uridines) with at least one of the
modified nucleoside disclosed herein.
TABLE-US-00004 TABLE 1 Combinations of Nucleoside Modifications
Modified Nucleotide Modified Nucleotide Combination
.alpha.-thio-cytidine .alpha.-thio-cytidine/5-iodo-uridine
.alpha.-thio-cytidine/N1-methyl-pseudouridine
.alpha.-thio-cytidine/.alpha.-thio-uridine
.alpha.-thio-cytidine/5-methyl-uridine
.alpha.-thio-cytidine/pseudo-uridine about 50% of the cytosines are
.alpha.-thio-cytidine pseudoisocytidine
pseudoisocytidine/5-iodo-uridine
pseudoisocytidine/N1-methyl-pseudouridine
pseudoisocytidine/.alpha.-thio-uridine
pseudoisocytidine/5-methyl-uridine pseudoisocytidine/pseudouridine
about 25% of cytosines are pseudoisocytidine
pseudoisocytidine/about 50% of uridines are N1-methyl-pseudouridine
and about 50% of uridines are pseudouridine pseudoisocytidine/about
25% of uridines are N1-methyl-pseudouridine and about 25% of
uridines are pseudouridine pyrrolo-cytidine
pyrrolo-cytidine/5-iodo-uridine
pyrrolo-cytidine/N1-methyl-pseudouridine
pyrrolo-cytidine/.alpha.-thio-uridine
pyrrolo-cytidine/5-methyl-uridine pyrrolo-cytidine/pseudouridine
about 50% of the cytosines are pyrrolo-cytidine 5-methyl-cytidine
5-methyl-cytidine/5-iodo-uridine
5-methyl-cytidine/N1-methyl-pseudouridine
5-methyl-cytidine/.alpha.-thio-uridine
5-methyl-cytidine/5-methyl-uridine 5-methyl-cytidine/pseudouridine
about 25% of cytosines are 5-methyl-cytidine about 50% of cytosines
are 5-methyl-cytidine 5-methyl-cytidine/5-methoxy-uridine
5-methyl-cytidine/5-bromo-uridine 5-methyl-cytidine/2-thio-uridine
5-methyl-cytidine/about 50% of uridines are 2- thio-uridine about
50% of uridines are 5-methyl-cytidine/ about 50% of uridines are
2-thio-uridine N4-acetyl-cytidine N4-acetyl-cytidine/5-iodo-uridine
N4-acetyl-cytidine/N1-methyl-pseudouridine
N4-acetyl-cytidine/.alpha.-thio-uridine
N4-acetyl-cytidine/5-methyl-uridine
N4-acetyl-cytidine/pseudouridine about 50% of cytosines are
N4-acetyl-cytidine about 25% of cytosines are N4-acetyl-cytidine
N4-acetyl-cytidine/5-methoxy-uridine
N4-acetyl-cytidine/5-bromo-uridine
N4-acetyl-cytidine/2-thio-uridine about 50% of cytosines are
N4-acetyl-cytidine/ about 50% of uridines are 2-thio-uridine
TABLE-US-00005 TABLE 2 Modified Nucleosides and Combinations
Thereof 1-(2,2,2-Trifluoroethyl)pseudo-UTP 1-Ethyl-pseudo-UTP
1-Methyl-pseudo-U-alpha-thio-TP 1-methyl-pseudouridine TP, ATP,
GTP, CTP 1-methyl-pseudo-UTP/5-methyl-CTP/ATP/GTP
1-methyl-pseudo-UTP/CTP/ATP/GTP 1-Propyl-pseudo-UTP 25%
5-Aminoallyl-CTP + 75% CTP/25% 5-Methoxy-UTP + 75% UTP 25%
5-Aminoallyl-CTP + 75% CTP/75% 5-Methoxy-UTP + 25% UTP 25%
5-Bromo-CTP + 75% CTP/25% 5-Methoxy-UTP + 75% UTP 25% 5-Bromo-CTP +
75% CTP/75% 5-Methoxy-UTP + 25% UTP 25% 5-Bromo-CTP + 75%
CTP/1-Methyl-pseudo-UTP 25% 5-Carboxy-CTP + 75% CTP/25%
5-Methoxy-UTP + 75% UTP 25% 5-Carboxy-CTP + 75% CTP/75%
5-Methoxy-UTP + 25% UTP 25% 5-Ethyl-CTP + 75% CTP/25% 5-Methoxy-UTP
+ 75% UTP 25% 5-Ethyl-CTP + 75% CTP/75% 5-Methoxy-UTP + 25% UTP 25%
5-Ethynyl-CTP + 75% CTP/25% 5-Methoxy-UTP + 75% UTP 25%
5-Ethynyl-CTP + 75% CTP/75% 5-Methoxy-UTP + 25% UTP 25%
5-Fluoro-CTP + 75% CTP/25% 5-Methoxy-UTP + 75% UTP 25% 5-Fluoro-CTP
+ 75% CTP/75% 5-Methoxy-UTP + 25% UTP 25% 5-Formyl-CTP + 75%
CTP/25% 5-Methoxy-UTP + 75% UTP 25% 5-Formyl-CTP + 75% CTP/75%
5-Methoxy-UTP + 25% UTP 25% 5-Hydroxymethyl-CTP + 75% CTP/25%
5-Methoxy-UTP + 75% UTP 25% 5-Hydroxymethyl-CTP + 75% CTP/75%
5-Methoxy-UTP + 25% UTP 25% 5-Iodo-CTP + 75% CTP/25% 5-Methoxy-UTP
+ 75% UTP 25% 5-Iodo-CTP + 75% CTP/75% 5-Methoxy-UTP + 25% UTP 25%
5-Methoxy-CTP + 75% CTP/25% 5-Methoxy-UTP + 75% UTP 25%
5-Methoxy-CTP + 75% CTP/75% 5-Methoxy-UTP + 25% UTP 25%
5-Methyl-CTP + 75% CTP/25% 5-Methoxy-UTP + 75% 1-Methyl- pseudo-UTP
25% 5-Methyl-CTP + 75% CTP/25% 5-Methoxy-UTP + 75% UTP 25%
5-Methyl-CTP + 75% CTP/50% 5-Methoxy-UTP + 50% 1-Methyl- pseudo-UTP
25% 5-Methyl-CTP + 75% CTP/50% 5-Methoxy-UTP + 50% UTP 25%
5-Methyl-CTP + 75% CTP/5-Methoxy-UTP 25% 5-Methyl-CTP + 75% CTP/75%
5-Methoxy-UTP + 25% 1-Methyl- pseudo-UTP 25% 5-Methyl-CTP + 75%
CTP/75% 5-Methoxy-UTP + 25% UTP 25% 5-Phenyl-CTP + 75% CTP/25%
5-Methoxy-UTP + 75% UTP 25% 5-Phenyl-CTP + 75% CTP/75%
5-Methoxy-UTP + 25% UTP 25% 5-Trifluoromethyl-CTP + 75% CTP/25%
5-Methoxy-UTP + 75% UTP 25% 5-Trifluoromethyl-CTP + 75% CTP/75%
5-Methoxy-UTP + 25% UTP 25% 5-Trifluoromethyl-CTP + 75%
CTP/1-Methyl-pseudo-UTP 25% N4-Ac-CTP + 75% CTP/25% 5-Methoxy-UTP +
75% UTP 25% N4-Ac-CTP + 75% CTP/75% 5-Methoxy-UTP + 25% UTP 25%
N4-Bz-CTP + 75% CTP/25% 5-Methoxy-UTP + 75% UTP 25% N4-Bz-CTP + 75%
CTP/75% 5-Methoxy-UTP + 25% UTP 25% N4-Methyl-CTP + 75% CTP/25%
5-Methoxy-UTP + 75% UTP 25% N4-Methyl-CTP + 75% CTP/75%
5-Methoxy-UTP + 25% UTP 25% Pseudo-iso-CTP + 75% CTP/25%
5-Methoxy-UTP + 75% UTP 25% Pseudo-iso-CTP + 75% CTP/75%
5-Methoxy-UTP + 25% UTP 25% 5-Bromo-CTP/75% CTP/Pseudo-UTP 25%
5-methoxy-UTP/25% 5-methyl-CTP/ATP/GTP 25%
5-methoxy-UTP/5-methyl-CTP/ATP/GTP 25% 5-methoxy-UTP/75%
5-methyl-CTP/ATP/GTP 25% 5-methoxy-UTP/CTP/ATP/GTP 25%
5-metoxy-UTP/50% 5-methyl-CTP/ATP/GTP 2-Amino-ATP 2-Thio-CTP
2-thio-pseudouridine TP, ATP, GTP, CTP 2-Thio-pseudo-UTP 2-Thio-UTP
3-Methyl-CTP 3-Methyl-pseudo-UTP 4-Thio-UTP 50% 5-Bromo-CTP + 50%
CTP/1-Methyl-pseudo-UTP 50% 5-Hydroxymethyl-CTP + 50%
CTP/1-Methyl-pseudo-UTP 50% 5-methoxy-UTP/5-methyl-CTP/ATP/GTP 50%
5-Methyl-CTP + 50% CTP/25% 5-Methoxy-UTP + 75% 1-Methyl- pseudo-UTP
50% 5-Methyl-CTP + 50% CTP/25% 5-Methoxy-UTP + 75% UTP 50%
5-Methyl-CTP + 50% CTP/50% 5-Methoxy-UTP + 50% 1-Methyl- pseudo-UTP
50% 5-Methyl-CTP + 50% CTP/50% 5-Methoxy-UTP + 50% UTP 50%
5-Methyl-CTP + 50% CTP/5-Methoxy-UTP 50% 5-Methyl-CTP + 50% CTP/75%
5-Methoxy-UTP + 25% 1-Methyl- pseudo-UTP 50% 5-Methyl-CTP + 50%
CTP/75% 5-Methoxy-UTP + 25% UTP 50% 5-Trifluoromethyl-CTP + 50%
CTP/1-Methyl-pseudo-UTP 50% 5-Bromo-CTP/50% CTP/Pseudo-UTP 50%
5-methoxy-UTP/25% 5-methyl-CTP/ATP/GTP 50% 5-methoxy-UTP/50%
5-methyl-CTP/ATP/GTP 50% 5-methoxy-UTP/75% 5-methyl-CTP/ATP/GTP 50%
5-methoxy-UTP/CTP/ATP/GTP 5-Aminoallyl-CTP
5-Aminoallyl-CTP/5-Methoxy-UTP 5-Aminoallyl-UTP 5-Bromo-CTP
5-Bromo-CTP/5-Methoxy-UTP 5-Bromo-CTP/1-Methyl-pseudo-UTP
5-Bromo-CTP/Pseudo-UTP 5-bromocytidine TP, ATP, GTP, UTP
5-Bromo-UTP 5-Carboxy-CTP/5-Methoxy-UTP 5-Ethyl-CTP/5-Methoxy-UTP
5-Ethynyl-CTP/5-Methoxy-UTP 5-Fluoro-CTP/5-Methoxy-UTP
5-Formyl-CTP/5-Methoxy-UTP 5-Hydroxy-methyl-CTP/5-Methoxy-UTP
5-Hydroxymethyl-CTP 5-Hydroxymethyl-CTP/1-Methyl-pseudo-UTP
5-Hydroxymethyl-CTP/5-Methoxy-UTP 5-hydroxymethyl-cytidine TP, ATP,
GTP, UTP 5-Iodo-CTP/5-Methoxy-UTP 5-Me-CTP/5-Methoxy-UTP 5-Methoxy
carbonyl methyl-UTP 5-Methoxy-CTP/5-Methoxy-UTP 5-methoxy-uridine
TP, ATP, GTP, UTP 5-methoxy-UTP 5-Methoxy-UTP
5-Methoxy-UTP/N6-Isopentenyl-ATP 5-methoxy-UTP/25%
5-methyl-CTP/ATP/GTP 5-methoxy-UTP/5-methyl-CTP/ATP/GTP
5-methoxy-UTP/75% 5-methyl-CTP/ATP/GTP 5-methoxy-UTP/CTP/ATP/GTP
5-Methyl-2-thio-UTP 5-Methylaminomethyl-UTP
5-Methyl-CTP/5-Methoxy-UTP 5-Methyl-CTP/5-Methoxy-UTP(cap 0)
5-Methyl-CTP/5-Methoxy-UTP(No cap) 5-Methyl-CTP/25% 5-Methoxy-UTP +
75% 1-Methyl-pseudo-UTP 5-Methyl-CTP/25% 5-Methoxy-UTP + 75% UTP
5-Methyl-CTP/50% 5-Methoxy-UTP + 50% 1-Methyl-pseudo-UTP
5-Methyl-CTP/50% 5-Methoxy-UTP + 50% UTP
5-Methyl-CTP/5-Methoxy-UTP/N6-Me-ATP 5-Methyl-CTP/75% 5-Methoxy-UTP
+ 25% 1-Methyl-pseudo-UTP 5-Methyl-CTP/75% 5-Methoxy-UTP + 25% UTP
5-Phenyl-CTP/5-Methoxy-UTP 5-Trifluoro-methyl-CTP/5-Methoxy-UTP
5-Trifluoromethyl-CTP 5-Trifluoromethyl-CTP/5-Methoxy-UTP
5-Trifluoromethyl-CTP/1-Methyl-pseudo-UTP
5-Trifluoromethyl-CTP/Pseudo-UTP 5-Trifluoromethyl-UTP
5-trifluromethylcytidine TP, ATP, GTP, UTP 75% 5-Aminoallyl-CTP +
25% CTP/25% 5-Methoxy-UTP + 75% UTP 75% 5-Aminoallyl-CTP + 25%
CTP/75% 5-Methoxy-UTP + 25% UTP 75% 5-Bromo-CTP + 25% CTP/25%
5-Methoxy-UTP + 75% UTP 75% 5-Bromo-CTP + 25% CTP/75% 5-Methoxy-UTP
+ 25% UTP 75% 5-Carboxy-CTP + 25% CTP/25% 5-Methoxy-UTP + 75% UTP
75% 5-Carboxy-CTP + 25% CTP/75% 5-Methoxy-UTP + 25% UTP 75%
5-Ethyl-CTP + 25% CTP/25% 5-Methoxy-UTP + 75% UTP 75% 5-Ethyl-CTP +
25% CTP/75% 5-Methoxy-UTP + 25% UTP 75% 5-Ethynyl-CTP + 25% CTP/25%
5-Methoxy-UTP + 75% UTP 75% 5-Ethynyl-CTP + 25% CTP/75%
5-Methoxy-UTP + 25% UTP 75% 5-Fluoro-CTP + 25% CTP/25%
5-Methoxy-UTP + 75% UTP 75% 5-Fluoro-CTP + 25% CTP/75%
5-Methoxy-UTP + 25% UTP 75% 5-Formyl-CTP + 25% CTP/25%
5-Methoxy-UTP + 75% UTP 75% 5-Formyl-CTP + 25% CTP/75%
5-Methoxy-UTP + 25% UTP 75% 5-Hydroxymethyl-CTP + 25% CTP/25%
5-Methoxy-UTP + 75% UTP 75% 5-Hydroxymethyl-CTP + 25% CTP/75%
5-Methoxy-UTP + 25% UTP 75% 5-Iodo-CTP + 25% CTP/25% 5-Methoxy-UTP
+ 75% UTP 75% 5-Iodo-CTP + 25% CTP/75% 5-Methoxy-UTP + 25% UTP 75%
5-Methoxy-CTP + 25% CTP/25% 5-Methoxy-UTP + 75% UTP 75%
5-Methoxy-CTP + 25% CTP/75% 5-Methoxy-UTP + 25% UTP 75%
5-methoxy-UTP/5-methyl-CTP/ATP/GTP 75% 5-Methyl-CTP + 25% CTP/25%
5-Methoxy-UTP + 75% 1-Methyl- pseudo-UTP 75% 5-Methyl-CTP + 25%
CTP/25% 5-Methoxy-UTP + 75% UTP 75% 5-Methyl-CTP + 25% CTP/50%
5-Methoxy-UTP + 50% 1-Methyl- pseudo-UTP 75% 5-Methyl-CTP + 25%
CTP/50% 5-Methoxy-UTP + 50% UTP 75% 5-Methyl-CTP + 25%
CTP/5-Methoxy-UTP 75% 5-Methyl-CTP + 25% CTP/75% 5-Methoxy-UTP +
25% 1-Methyl- pseudo-UTP 75% 5-Methyl-CTP + 25% CTP/75%
5-Methoxy-UTP + 25% UTP 75% 5-Phenyl-CTP + 25% CTP/25%
5-Methoxy-UTP + 75% UTP 75% 5-Phenyl-CTP + 25% CTP/75%
5-Methoxy-UTP + 25% UTP 75% 5-Trifluoromethyl-CTP + 25% CTP/25%
5-Methoxy-UTP + 75% UTP 75% 5-Trifluoromethyl-CTP + 25% CTP/75%
5-Methoxy-UTP + 25% UTP 75% 5-Trifluoromethyl-CTP + 25%
CTP/1-Methyl-pseudo-UTP 75% N4-Ac-CTP + 25% CTP/25% 5-Methoxy-UTP +
75% UTP 75% N4-Ac-CTP + 25% CTP/75% 5-Methoxy-UTP + 25% UTP 75%
N4-Bz-CTP + 25% CTP/25% 5-Methoxy-UTP + 75% UTP 75% N4-Bz-CTP + 25%
CTP/75% 5-Methoxy-UTP + 25% UTP 75% N4-Methyl-CTP + 25% CTP/25%
5-Methoxy-UTP + 75% UTP 75% N4-Methyl-CTP + 25% CTP/75%
5-Methoxy-UTP + 25% UTP 75% Pseudo-iso-CTP + 25% CTP/25%
5-Methoxy-UTP + 75% UTP 75% Pseudo-iso-CTP + 25% CTP/75%
5-Methoxy-UTP + 25% UTP 75% 5-Bromo-CTP/25% CTP/1-Methyl-pseudo-UTP
75% 5-Bromo-CTP/25% CTP/Pseudo-UTP 75% 5-methoxy-UTP/25%
5-methyl-CTP/ATP/GTP 75% 5-methoxy-UTP/50% 5-methyl-CTP/ATP/GTP 75%
5-methoxy-UTP/75% 5-methyl-CTP/ATP/GTP 75%
5-methoxy-UTP/CTP/ATP/GTP 8-Aza-ATP Alpha-thio-CTP CTP/25%
5-Methoxy-UTP + 75% 1-Methyl-pseudo-UTP CTP/25% 5-Methoxy-UTP + 75%
UTP CTP/50% 5-Methoxy-UTP + 50% 1-Methyl-pseudo-UTP CTP/50%
5-Methoxy-UTP + 50% UTP CTP/5-Methoxy-UTP CTP/5-Methoxy-UTP (cap 0)
CTP/5-Methoxy-UTP(No cap) CTP/75% 5-Methoxy-UTP + 25%
1-Methyl-pseudo-UTP CTP/75% 5-Methoxy-UTP + 25% UTP CTP/UTP(No cap)
N1-Me-GTP N4-Ac-CTP N4Ac-CTP/1-Methyl-pseudo-UTP
N4Ac-CTP/5-Methoxy-UTP N4-acetyl-cytidine TP, ATP, GTP, UTP
N4-Bz-CTP/5-Methoxy-UTP N4-methyl CTP N4-Methyl-CTP/5-Methoxy-UTP
Pseudo-iso-CTP/5-Methoxy-UTP PseudoU-alpha-thio-TP pseudouridine
TP, ATP, GTP, CTP pseudo-UTP/5-methyl-CTP/ATP/GTP UTP-5-oxyacetic
acid Me ester Xanthosine
According to the disclosure, polynucleotides of the disclosure may
be synthesized to comprise the combinations or single modifications
of Table 1 or Table 2.
Where a single modification is listed, the listed nucleoside or
nucleotide represents 100 percent of that A, U, G or C nucleotide
or nucleoside having been modified. Where percentages are listed,
these represent the percentage of that particular A, U, G or C
nucleobase triphosphate of the total amount of A, U, G, or C
triphosphate present. For example, the combination: 25%
5-Aminoallyl-CTP+75% CTP/25% 5-Methoxy-UTP+75% UTP refers to a
polynucleotide where 25% of the cytosine triphosphates are
5-Aminoallyl-CTP while 75% of the cytosines are CTP; whereas 25% of
the uracils are 5-methoxy UTP while 75% of the uracils are UTP.
Where no modified UTP is listed then the naturally occurring ATP,
UTP, GTP and/or CTP is used at 100% of the sites of those
nucleotides found in the polynucleotide. In this example all of the
GTP and ATP nucleotides are left unmodified.
Methods of Making RLR Agonists
RLR agonists of the present disclosure may be produced by means
available in the art, including but not limited to in vitro
transcription (IVT) and synthetic methods. Enzymatic (IVT),
solid-phase, liquid-phase, combined synthetic methods, small region
synthesis, and ligation methods may be utilized. In one embodiment,
RLR agonists are made using IVT enzymatic synthesis methods.
Methods of making 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. Accordingly, the present disclosure also includes
polynucleotides, e.g., DNA, constructs and vectors that may be used
to in vitro transcribe an RLR agonist described herein.
Non-natural modified nucleobases may be introduced into
polynucleotides, e.g., RNA, during synthesis or post-synthesis. In
certain embodiments, modifications may be on internucleoside
linkages, purine or pyrimidine bases, or sugar. In particular
embodiments, the modification may be introduced at the terminal of
a polynucleotide chain or anywhere else in the polynucleotide
chain; with chemical synthesis or with a polymerase enzyme.
Examples of modified nucleic acids and their synthesis are
disclosed in PCT application No. PCT/US2012/058519. Synthesis of
modified polynucleotides is also described in Verma and Eckstein,
Annual Review of Biochemistry, vol. 76, 99-134 (1998).
Either enzymatic or chemical ligation methods may be used to
conjugate polynucleotides or their regions with different
functional moieties, such as targeting or delivery agents,
fluorescent labels, liquids, nanoparticles, etc. Conjugates of
polynucleotides and modified polynucleotides are reviewed in
Goodchild, Bioconjugate Chemistry, vol. 1(3), 165-187 (1990). The
synthesis of oligonucleotides, polynucleotides, and conjugations
and ligations thereof, is 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.
Pharmaceutical Compositions and Formulations
In certain embodiments, the invention provides for a pharmaceutical
composition comprising an RLR agonist with a pharmaceutically
acceptable diluent, carrier, solubilizer, emulsifier, preservative
and/or adjuvant.
In certain embodiments, acceptable formulation materials preferably
are nontoxic to recipients at the dosages and concentrations
employed. In certain embodiments, the formulation material(s) are
for s.c. and/or I.V. administration. In certain embodiments, the
pharmaceutical composition can contain formulation materials for
modifying, maintaining or preserving, for example, the pH,
osmolality, viscosity, clarity, color, isotonicity, odor,
sterility, stability, rate of dissolution or release, adsorption or
penetration 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);
antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite
or sodium hydrogen-sulfite); buffers (such as borate, bicarbonate,
Tris-HCl, citrates, phosphates or other organic acids); bulking
agents (such as mannitol or glycine); chelating agents (such as
ethylenediamine tetraacetic acid (EDTA)); complexing agents (such
as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or
hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides;
disaccharides; and other carbohydrates (such as glucose, mannose or
dextrins); proteins (such as serum albumin, gelatin or
immunoglobulins); coloring, flavoring and diluting agents;
emulsifying agents; hydrophilic polymers (such as
polyvinylpyrrolidone); low molecular weight polypeptides;
salt-forming counterions (such as sodium); preservatives (such as
benzalkonium chloride, benzoic acid, salicylic acid, thimerosal,
phenethyl alcohol, methylparaben, propylparaben, chlorhexidine,
sorbic acid or hydrogen peroxide); solvents (such as glycerin,
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, tyloxapal); stability enhancing agents (such as
sucrose or sorbitol); tonicity enhancing agents (such as alkali
metal halides, preferably sodium or potassium chloride, mannitol
sorbitol); delivery vehicles; diluents; excipients and/or
pharmaceutical adjuvants. (Remington's Pharmaceutical Sciences,
18th Edition, A. R. Gennaro, ed., Mack Publishing Company (1995).
In certain embodiments, the formulation comprises PBS; 20 mM NaOAC,
pH 5.2, 50 mM NaCl; and/or 10 mM NAOAC, pH 5.2, 9% Sucrose. In
certain embodiments, the optimal pharmaceutical composition will be
determined by one skilled in the art depending upon, for example,
the intended route of administration, delivery format and desired
dosage. See, for example, Remington's Pharmaceutical Sciences,
supra. In certain embodiments, such compositions may influence the
physical state, stability, rate of in vivo release and/or rate of
in vivo clearance of the RLR agonist.
In certain embodiments, the primary vehicle or carrier in a
pharmaceutical composition can be either aqueous or non-aqueous in
nature. For example, in certain embodiments, a suitable vehicle or
carrier can be water for injection, physiological saline solution
or artificial cerebrospinal fluid, possibly supplemented with other
materials common in compositions for parenteral administration. In
certain embodiments, the saline comprises isotonic
phosphate-buffered saline. In certain embodiments, neutral buffered
saline or saline mixed with serum albumin are further exemplary
vehicles. In certain embodiments, pharmaceutical compositions
comprise Tris buffer of about pH 7.0-8.5, or acetate buffer of
about pH 4.0-5.5, which can further include sorbitol or a suitable
substitute therefore. In certain embodiments, a composition
comprising an RLR agonist can be prepared for storage by mixing the
selected composition having the desired degree of purity with
optional formulation agents (Remington's Pharmaceutical Sciences,
supra) in the form of a lyophilized cake or an aqueous solution.
Further, in certain embodiments, a composition comprising an RLR
agonist can be formulated as a lyophilizate using appropriate
excipients such as sucrose.
In certain embodiments, the pharmaceutical composition can be
selected for parenteral delivery. In certain embodiments, the
compositions can be selected for inhalation or for delivery through
the digestive tract, such as orally. The preparation of such
pharmaceutically acceptable compositions is within the ability of
one skilled in the art.
In certain embodiments, the formulation components are present in
concentrations that are acceptable to the site of administration.
In certain embodiments, buffers are used to maintain the
composition at physiological pH or at a slightly lower pH,
typically within a pH range of from about 5 to about 8.
In certain embodiments, when parenteral administration is
contemplated, a therapeutic composition can be in the form of a
pyrogen-free, parenterally acceptable aqueous solution comprising
an RLR agonist, in a pharmaceutically acceptable vehicle. In
certain embodiments, a vehicle for parenteral injection is sterile
distilled water in which an RLR agonist is formulated as a sterile,
isotonic solution, and properly preserved. In certain embodiments,
the preparation can involve the formulation of the desired molecule
with a delivery vehicle or agent, such as injectable microspheres,
bio-erodible particles, polymeric compounds (such as polylactic
acid, polyglycolic acid or polyethylenimine (e.g. JetPEI.RTM.)),
beads or liposomes, that can provide for the controlled or
sustained release of the product which can then be delivered via a
depot injection. In certain embodiments, hyaluronic acid can also
be used, and can have the effect of promoting sustained duration in
the circulation. In certain embodiments, implantable drug delivery
devices can be used to introduce the desired molecule.
In certain embodiments, a pharmaceutical composition can be
formulated for inhalation. In certain embodiments, an RLR agonist
can be formulated as a dry powder for inhalation. In certain
embodiments, an inhalation solution comprising an RLR agonist can
be formulated with a propellant for aerosol delivery. In certain
embodiments, solutions can be nebulized. Pulmonary administration
is further described in PCT application No. PCT/US94/001875, which
describes pulmonary delivery of chemically modified proteins.
In certain embodiments, it is contemplated that formulations can be
administered orally. In certain embodiments, an RLR agonist that is
administered in this fashion can be formulated with or without
those carriers customarily used in the compounding of solid dosage
forms such as tablets and capsules. In certain embodiments, a
capsule can be designed to release the active portion of the
formulation at the point in the gastrointestinal tract when
bioavailability is maximized and pre-systemic degradation is
minimized. In certain embodiments, at least one additional agent
can be included to facilitate absorption of an RLR agonist. In
certain embodiments, diluents, flavorings, low melting point waxes,
vegetable oils, lubricants, suspending agents, tablet
disintegrating agents, and binders can also be employed.
In certain embodiments, a pharmaceutical composition can involve an
effective quantity of an RLR agonist in a mixture with non-toxic
excipients which are suitable for the manufacture of tablets. In
certain embodiments, by dissolving the tablets in sterile water, or
another appropriate vehicle, solutions can be prepared in unit-dose
form. In certain embodiments, suitable excipients include, but are
not limited to, inert diluents, such as calcium carbonate, sodium
carbonate or bicarbonate, lactose, or calcium phosphate; or binding
agents, such as starch, gelatin, or acacia; or lubricating agents
such as magnesium stearate, stearic acid, or talc.
Additional pharmaceutical compositions will be evident to those
skilled in the art, including formulations involving an RLR agonist
in sustained- or controlled-delivery formulations. In certain
embodiments, techniques for formulating a variety of other
sustained- or controlled-delivery means, such as liposome carriers,
bio-erodible microparticles or porous beads and depot injections,
are also known to those skilled in the art. See for example, PCT
Application No. PCT/US93/00829 which describes the controlled
release of porous polymeric microparticles for the delivery of
pharmaceutical compositions. In certain embodiments,
sustained-release preparations can include semipermeable polymer
matrices in the form of shaped articles, e.g. films, or
microcapsules. Sustained release matrices can include polyesters,
hydrogels, polylactides (U.S. Pat. No. 3,773,919 and EP 058,481),
copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman
et al., Biopolymers, 22:547-556 (1983)), poly
(2-hydroxyethyl-methacrylate) (Langer et al., J. Biomed. Mater.
Res., 15: 167-277 (1981) and Langer, Chem. Tech., 12:98-105
(1982)), ethylene vinyl acetate (Langer et al., supra) or
poly-D(-)-3-hydroxybutyric acid (EP 133,988). In certain
embodiments, sustained release compositions can also include
liposomes, which can be prepared by any of several methods known in
the art. See, e.g., Eppstein et al, Proc. Natl. Acad. Sci. USA,
82:3688-3692 (1985); EP 036,676; EP 088,046 and EP 143,949.
The pharmaceutical composition to be used for in vivo
administration typically is sterile. In certain embodiments, this
can be accomplished by filtration through sterile filtration
membranes. In certain embodiments, where the composition is
lyophilized, sterilization using this method can be conducted
either prior to or following lyophilization and reconstitution. In
certain embodiments, the composition for parenteral administration
can be stored in lyophilized form or in a solution. In certain
embodiments, parenteral compositions generally are placed into a
container having a sterile access port, for example, an intravenous
solution bag or vial having a stopper pierceable by a hypodermic
injection needle.
In certain embodiments, once the pharmaceutical composition has
been formulated, it can be stored in sterile vials as a solution,
suspension, gel, emulsion, solid, or as a dehydrated or lyophilized
powder. In certain embodiments, such formulations can be stored
either in a ready-to-use form or in a form (e.g., lyophilized) that
is reconstituted prior to administration.
In certain embodiments, kits are provided for producing a
single-dose administration unit. In certain embodiments, the kit
can contain both a first container having a dried protein and a
second container having an aqueous formulation. In certain
embodiments, kits containing single and multi-chambered pre-filled
syringes (e.g., liquid syringes and lyosyringes) are included.
In certain embodiments, the effective amount of a pharmaceutical
composition comprising an RLR agonist to be employed
therapeutically will depend, for example, upon the therapeutic
context and objectives. One skilled in the art will appreciate that
the appropriate dosage levels for treatment, according to certain
embodiments, will thus vary depending, in part, upon the molecule
delivered, the indication for which an RLR agonist is being used,
the route of administration, and the size (body weight, body
surface or organ size) and/or condition (the age and general
health) of the patient. In certain embodiments, the clinician can
titer the dosage and modify the route of administration to obtain
the optimal therapeutic effect.
In certain embodiments, the frequency of dosing will take into
account the pharmacokinetic parameters of an RLR agonist in the
formulation used. In certain embodiments, a clinician will
administer the composition until a dosage is reached that achieves
the desired effect. In certain embodiments, the composition can
therefore be administered as a single dose or as two or more doses
(which may or may not contain the same amount of the desired
molecule) over time, or as a continuous infusion via an
implantation device or catheter. Further refinement of the
appropriate dosage is routinely made by those of ordinary skill in
the art and is within the ambit of tasks routinely performed by
them. In certain embodiments, appropriate dosages can be
ascertained through use of appropriate dose-response data.
In certain embodiments, the route of administration of the
pharmaceutical composition is in accord with known methods, e.g.
orally, through injection by intravenous, intraperitoneal,
intracerebral (intra-parenchymal), intracerebroventricular,
intramuscular, subcutaneously, intraocular, intraarterial,
intraportal, or intralesional routes; by sustained release systems
or by implantation devices. In certain embodiments, the
compositions can be administered by bolus injection or continuously
by infusion, or by implantation device. In certain embodiments,
individual elements of the combination therapy may be administered
by different routes.
In certain embodiments, the composition can be administered locally
via implantation of a membrane, sponge or another appropriate
material onto which the desired molecule has been absorbed or
encapsulated. In certain embodiments, where an implantation device
is used, the device can be implanted into any suitable tissue or
organ, and delivery of the desired molecule can be via diffusion,
timed-release bolus, or continuous administration. In certain
embodiments, it can be desirable to use a pharmaceutical
composition comprising an RLR agonist in an ex vivo manner. In such
instances, cells, tissues and/or organs that have been removed from
the patient are exposed to a pharmaceutical composition comprising
an RLR agonist after which the cells, tissues and/or organs are
subsequently implanted back into the patient.
In certain embodiments, an RLR agonist can be delivered by
implanting certain cells that have been genetically engineered,
using methods such as those described herein, to express and
secrete the agonist. In certain embodiments, such cells can be
animal or human cells, and can be autologous, heterologous, or
xenogeneic. In certain embodiments, the cells can be immortalized.
In certain embodiments, in order to decrease the chance of an
immunological response, the cells can be encapsulated to avoid
infiltration of surrounding tissues. In certain embodiments, the
encapsulation materials are typically biocompatible, semi-permeable
polymeric enclosures or membranes that allow the release of the
protein product(s) but prevent the destruction of the cells by the
patient's immune system or by other detrimental factors from the
surrounding tissues.
In some aspects, the disclosure provides a pharmaceutical
composition comprising an RLR agonist according to the disclosure
for stimulating an immune response, treating or delaying
progression of a cancer, or reducing or inhibiting tumor growth in
a subject in need thereof, and a pharmaceutically acceptable
carrier. In some embodiments, the RLR agonist is formulated in a
polyethylenimine (PEI) carrier. In some embodiments, the PEI
carrier is JetPEI.RTM..
Applications
The compositions described herein can be used in diagnostic and
therapeutic applications. For example, detectably-labeled RLR
agonists can be used in assays to detect the presence or amount of
the target protein in a sample (e.g., a biological sample). The
compositions can be used in in vitro assays for studying inhibition
of target function (e.g. RLR-mediated cellular signaling or
response). In some embodiments, e.g., in which the compositions
bind to and activate a target (e.g. a protein or polypeptide), the
compositions can be used as positive controls in assays designed to
identify additional novel compounds that also induce activity of
the target protein or polypeptide and/or are otherwise are useful
for treating a disorder associated with the target protein or
polypeptide. For example, a RLR-activating composition can be used
as a positive control in an assay to identify additional compounds
(e.g., small molecules, aptamers, or antibodies) that induce,
increase, or stimulate RLR function. The compositions can also be
used in therapeutic methods as elaborated on below.
Kits
A kit can include an RLR agonist as disclosed herein, and
instructions for use. The kits may comprise, in a suitable
container, an RLR agonist, one or more controls, and various
buffers, reagents, enzymes and other standard ingredients well
known in the art.
The container can include at least one vial, well, test tube,
flask, bottle, syringe, or other container means, into which an RLR
agonist may be placed, and in some instances, suitably aliquoted.
Where an additional component is provided, the kit can contain
additional containers into which this component may be placed. The
kits can also include a means for containing an RLR agonist and any
other reagent containers in close confinement for commercial sale.
Such containers may include injection or blow-molded plastic
containers into which the desired vials are retained. Containers
and/or kits can include labeling with instructions for use and/or
warnings.
In some aspects, the disclosure provides a kit comprising an RLR
agonist provided by the disclosure, or comprising a pharmaceutical
composition provided by the disclosure and instructions for use in
stimulating an immune response in a subject, or treating or
delaying progression of a cancer, or inhibiting tumor growth in a
subject, optionally with instructions for use in combination with
one or more additional therapeutic agents.
In some embodiments, the agonist or pharmaceutical composition is
administered in combination with one or more additional therapeutic
agents, wherein the one or more additional therapeutic agents is
selected from the group consisting of: a chemotherapy, a targeted
anti-cancer therapy, an oncolytic drug, a cell death-inducing
agent, an opsonizing agent (e.g., an opsonizing antibody) a
cytotoxic agent, an immune-based therapy, a cytokine, an activator
of a costimulatory molecule, an inhibitor of an inhibitory
molecule, a vaccine, a cellular immunotherapy, or a combination
thereof.
In some embodiments, the RLR agonist or pharmaceutical composition
is administered preceding or subsequent to administration of the
one or more additional therapeutic agents or wherein the one or
more additional therapeutic agents is administered concurrently
with, preceding or subsequent to the administration of the RLR
agonist or pharmaceutical composition.
In some embodiments, the one or more additional therapeutic agents
is a PD-1/PD-L1 antagonist, a TIM-3 antagonist, a VISTA antagonist,
an adenosine A2AR antagonist, a B7-H3 antagonist, a B7-H4
antagonist, a BTLA antagonist, a CTLA-4 antagonist, an IDO
antagonist, a KIR antagonist, a LAG-3 antagonist, a toll-like
receptor 3 (TLR3) agonist, a toll-like receptor 7 (TLR7) agonist, a
toll-like receptor 9 (TLR9) agonist.
In some embodiments, the one or more additional therapeutic agents
is an agonist comprising an polypeptide (e.g, antibody, or antigen
binding portion thereof) that specifically binds to CD137
(4-1BB).
In some embodiments, the one or more additional therapeutic agents
is an agonist comprising an polypeptide (e.g., antibody, or antigen
binding portion thereof) that specifically binds to CD134
(OX40).
Methods of Use
The compositions of the present invention have numerous in vitro
and in vivo utilities involving the detection and/or quantification
of RLRs and/or the agonism of RLR function.
The above-described compositions are useful in, inter alia, methods
for treating or preventing a variety of cancers or infectious
diseases in a subject. The compositions can be administered to a
subject, e.g., a human subject, using a variety of methods that
depend, in part, on the route of administration. The route can be,
e.g., intravenous injection or infusion (IV), subcutaneous
injection (SC), intradermal injection (ID), intraperitoneal (IP)
injection, intramuscular injection (IM), intratumoral injection
(IT) or intrathecal injection. The injection can be in a bolus or a
continuous infusion.
Administration can be achieved by, e.g., local infusion, injection,
or by means of an implant. The implant can be of a porous,
non-porous, or gelatinous material, including membranes, such as
sialastic membranes, or fibers. The implant can be configured for
sustained or periodic release of the composition to the subject.
See, e.g., U.S. Patent Application Publication No. 20080241223;
U.S. Pat. Nos. 5,501,856; 4,863,457; and 3,710,795; EP488401; and
EP 430539, the disclosures of each of which are incorporated herein
by reference in their entirety. The composition can be delivered to
the subject by way of an implantable device based on, e.g.,
diffusive, erodible, or convective systems, e.g., osmotic pumps,
biodegradable implants, electrodiffusion systems, electroosmosis
systems, vapor pressure pumps, electrolytic pumps, effervescent
pumps, piezoelectric pumps, erosion-based systems, or
electromechanical systems.
In some embodiments, an RLR agonist is therapeutically delivered to
a subject by way of local administration.
A suitable dose of an RLR agonist described herein, which dose is
capable of treating or preventing cancer in a subject, can depend
on a variety of factors including, e.g., the age, sex, and weight
of a subject to be treated and the particular inhibitor compound
used. Other factors affecting the dose administered to the subject
include, e.g., the type or severity of the cancer or infectious
disease. For example, a subject having metastatic melanoma may
require administration of a different dosage of an RLR agonist than
a subject with glioblastoma. Other factors can include, e.g., other
medical disorders concurrently or previously affecting the subject,
the general health of the subject, the genetic disposition of the
subject, diet, time of administration, rate of excretion, drug
combination, and any other additional therapeutics that are
administered to the subject. It should also be understood that a
specific dosage and treatment regimen for any particular subject
will also depend upon the judgment of the treating medical
practitioner (e.g., doctor or nurse). Suitable dosages are
described herein.
A pharmaceutical composition can include a therapeutically
effective amount of an RLR agonist thereof described herein. Such
effective amounts can be readily determined by one of ordinary
skill in the art based, in part, on the effect of the administered
RLR agonist, or the combinatorial effect of the RLR agonist and one
or more additional active agents, if more than one agent is used. A
therapeutically effective amount of an RLR agonist described herein
can also vary according to factors such as the disease state, age,
sex, and weight of the individual, and the ability of the agonist
(and one or more additional active agents) to elicit a desired
response in the individual, e.g., reduction in tumor growth. For
example, a therapeutically effective amount of an RLR agonist can
inhibit (lessen the severity of or eliminate the occurrence of)
and/or prevent a particular disorder, and/or any one of the
symptoms of the particular disorder known in the art or described
herein. A therapeutically effective amount is also one in which any
toxic or detrimental effects of the composition are outweighed by
the therapeutically beneficial effects.
Suitable human doses of any of the RLR agonists described herein
can further be evaluated in, e.g., Phase I dose escalation studies.
See, e.g., van Gurp et al. (2008) Am J Transplantation 8 (8):
1711-1718; Hanouska et al. (2007) Clin Cancer Res 13(2, part
1):523-531; and Hetherington et al. (2006) Antimicrobial Agents and
Chemotherapy 50(10): 3499-3500.
In some embodiments, the composition contains any of the RLR
agonists described herein and one or more (e.g., two, three, four,
five, six, seven, eight, nine, 10, or 11 or more) additional
therapeutic agents such that the composition as a whole is
therapeutically effective. For example, a composition can contain
an RLR agonist described herein and an alkylating agent, wherein
the agonist and agent are each at a concentration that when
combined are therapeutically effective for treating or preventing a
cancer (e.g., melanoma) in a subject.
Toxicity and therapeutic efficacy of such compositions can be
determined by known pharmaceutical procedures in cell cultures or
experimental animals (e.g., animal models of any of the cancers
described herein). These procedures can be used, e.g., for
determining the LD.sub.50 (the dose lethal to 50% of the
population) and the ED.sub.50 (the dose 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 the ratio LD.sub.50/ED.sub.50. An RLR agonist that
exhibits a high therapeutic index is preferred. While 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 to minimize potential damage to normal cells
and, thereby, reduce side effects.
The data obtained from the cell culture assays and animal studies
can be used in formulating a range of dosage for use in humans. For
an RLR agonist described herein, the therapeutically effective dose
can be estimated initially from cell culture assays. A dose can be
formulated in animal models to achieve a circulating plasma
concentration range that includes the EC.sub.50 (i.e., the
concentration of the agonist which achieves a half-maximal
inhibition of symptoms) as determined in cell culture. Such
information can be used to more accurately determine useful doses
in humans. Levels in plasma may be measured, for example, by high
performance liquid chromatography. In some embodiments, e.g., where
local administration (e.g., to the eye or a joint) is desired, cell
culture or animal modeling can be used to determine a dose required
to achieve a therapeutically effective concentration within the
local site.
In some embodiments, the methods can be performed in conjunction
with other therapies for cancer or infectious disease. For example,
the composition can be administered to a subject at the same time,
prior to, or after, radiation, surgery, targeted or cytotoxic
chemotherapy, chemoradiotherapy, hormone therapy, immunotherapy,
gene therapy, cell transplant therapy, precision medicine, genome
editing therapy, or other pharmacotherapy.
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 myelocytic leukemia, myeloblasts
promyelocyte myelomonocytic monocytic erythroleukemia, chronic
leukemia, chronic myelocytic (granulocytic) leukemia, chronic
lymphocytic leukemia, mantle cell lymphoma, primary central nervous
system lymphoma, Burkitt's lymphoma, marginal zone B cell lymphoma,
polycythemia vera, Hodgkin's disease, non-Hodgkin's disease,
multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain
disease, solid tumors, sarcomas, and carcinomas, fibrosarcoma,
myxosarcoma, liposarcoma, chrondrosarcoma, osteogenic sarcoma,
osteosarcoma, chordoma, angiosarcoma, endotheliosarcoma,
lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma,
mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma,
colon sarcoma, colorectal carcinoma, pancreatic cancer, breast
cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,
basal cell carcinoma, adenocarcinoma, sweat gland carcinoma,
sebaceous gland carcinoma, papillary carcinoma, papillary
adenocarcinomas, cystadenocarcinoma, medullary carcinoma,
bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct
carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's
tumor, cervical cancer, uterine cancer, testicular tumor, lung
carcinoma, small cell lung carcinoma, non-small cell lung
carcinoma, bladder carcinoma, epithelial carcinoma, glioma,
astrocytoma, medulloblastoma, craniopharyngioma, ependymoma,
pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma,
menangioma, melanoma, neuroblastoma, retinoblastoma, nasopharyngeal
carcinoma, esophageal carcinoma, basal cell carcinoma, biliary
tract cancer, bladder cancer, bone cancer, brain and central
nervous system (CNS) cancer, cervical cancer, choriocarcinoma,
colorectal cancers, connective tissue cancer, cancer of the
digestive system, endometrial cancer, esophageal cancer, eye
cancer, head and neck cancer, gastric cancer, intraepithelial
neoplasm, kidney cancer, larynx cancer, liver cancer, lung cancer
(small cell, large cell), melanoma, neuroblastoma; oral cavity
cancer (for example lip, tongue, mouth and pharynx), ovarian
cancer, pancreatic cancer, rectal cancer; cancer of the respiratory
system, sarcoma, skin cancer, stomach cancer, testicular cancer,
thyroid cancer, uterine cancer, and cancer of the urinary
system.
In some aspects, the disclosure provides a method to increase
RLR-mediated production of one or more cytokines in a cell, the
method comprising contacting the cell with an RLR agonist provided
by the disclosure, wherein the agonist increases RLR-mediated
cytokine production in a cell.
In some aspects, the disclosure provides a method to increase
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 disclosure, wherein the agonist increases
RLR-mediated expression of one or more interferon-stimulated genes
in a cell.
In some aspects, the disclosure provides a method to increase
RLR-dependent intracellular signaling in a cell, the method
comprising contacting the cell with an RLR agonist provided by the
disclosure, wherein the agonist increases RLR-dependent
intracellular signaling.
In some aspects, the 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 disclosure, or a pharmaceutical composition provided by the
disclosure.
In some aspects, the disclosure provides a method of treating or
delaying progression of a cancer in a subject, the method
comprising administering to the subject an effective amount of an
RLR agonist provided by the disclosure, or a pharmaceutical
composition provided by the disclosure.
In some aspects, the 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 disclosure, or a pharmaceutical
composition provided by the disclosure.
In some aspects, the disclosure provides a method for stimulating
an immune response, treating or delaying progression of a cancer,
or inhibiting tumor growth in a subject in need thereof, the method
comprising administering to the subject an effective amount of an
RLR agonist provided by the disclosure, or a pharmaceutical
composition provided by the 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 or delaying progression
of the cancer, or inhibiting growth of the tumor.
Combinations of RLR Agonists with Additional Therapeutic Agents
In some embodiments, an RLR agonist described herein can be
administered to a subject as a monotherapy. Alternatively, the RLR
agonist can be administered to a subject as a combination therapy
with another treatment, e.g., another treatment for a cancer. For
example, the combination therapy can include administering to the
subject (e.g., a human patient) one or more additional agents that
provide a therapeutic benefit to a subject who has, or is at risk
of developing, cancer.
In some embodiments of the methods provided by the disclosure, the
RLR agonist or pharmaceutical composition is administered in
combination with one or more additional therapeutic agents, wherein
the one or more additional therapeutic agents is selected from the
group consisting of: a chemotherapy, a targeted anti-cancer
therapy, an oncolytic drug, a cell death-inducing agent, an
opsonizing agent (e.g., an opsonizing antibody) a cytotoxic agent,
an immune-based therapy, a cytokine, an activator or agonist of a
costimulatory molecule, an inhibitor of an inhibitory molecule, a
vaccine, a cellular immunotherapy, or a combination thereof.
In some embodiments, the RLR agonist or pharmaceutical composition
is administered preceding or subsequent to administration of the
one or more additional therapeutic agents or wherein the one or
more additional therapeutic agents is administered concurrently
with, preceding or subsequent to the administration of the agonist
or pharmaceutical composition.
In some embodiments, the one or more additional therapeutic agents
is a PD-1/PD-L1 antagonist, a TIM-3 antagonist, a VISTA antagonist,
an adenosine A2AR antagonist, a B7-H3 antagonist, a B7-H4
antagonist, a BTLA antagonist, a CTLA-4 antagonist, an IDO
antagonist, a KIR antagonist, a LAG-3 antagonist, a toll-like
receptor 3 (TLR3) agonist, a toll-like receptor 7 (TLR7) agonist, a
toll-like receptor 9 (TLR9) agonist.
Combination with Chemotherapeutic Agents
Chemotherapeutic agents suitable for combination and/or
co-administration with compositions of the present invention
include, for example: taxol, cytochalasin B, gramicidin D, ethidium
bromide, emetine, mitomycin, etoposide, tenoposide, vincristine,
vinblastine, colchicin, doxorubicin, daunorubicin,
dihydroxyanthrancindione, mitoxantrone, mithramycin, actinomycin D,
1-dehydrotestosterone, glucocorticoids, procaine, tetracaine,
lidocaine, propranolol, and puromycin and analogs or homologs
thereof. Further agents include, for example, antimetabolites
(e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine,
5-fluorouracil decarbazine), alkylating agents (e.g.
mechlorethamine, thioTEPA, chlorambucil, melphalan, carmustine
(BSNU), lomustine (CCNU), cyclophosphamide, busulfan,
dibromomannitol, streptozotocin, mitomycin C, cis-dichlordiamine
platinum (II) (DDP), procarbazine, altretamine, cisplatin,
carboplatin, oxaliplatin, nedaplatin, satraplatin, or triplatin
tetranitrate), anthracycline (e.g. daunorubicin (formerly
daunomycin) and doxorubicin), antibiotics (e.g. dactinomcin
(formerly actinomycin), bleomycin, mithramycin, and anthramycin
(AMC)), and anti-mitotic agents (e.g. vincristine and vinblastine)
and temozolomide.
Combination with PD-1/PD-L1 Antagonists
In some embodiments, a RLR agonist, or pharmaceutical compositions
thereof, provided by the disclosure is combined (e.g., administered
in combination) with one or more PD-1/PD-L1 antagonist that
specifically binds to human PD-1 or PD-L1 and inhibits PD-1/PD-L1
biological activity and/or downstream pathway(s) and/or cellular
processed mediated by human PD-1/PD-L1 signaling or other human
PD-1/PD-L1-mediated functions.
Accordingly, provided herein are PD-1/PD-L1 antagonists that
directly or allosterically block, antagonize, suppress, inhibit or
reduce PD-1/PD-L1 biological activity, including downstream
pathways and/or cellular processes mediated by PD-1/PD-L1
signaling, such as receptor binding and/or elicitation of a
cellular response to PD-1/PD-L1. Also provided herein are
PD-1/PD-L1 antagonists that reduce the quantity or amount of human
PD-1 or PD-L1 produced by a cell or subject.
In some embodiments, the disclosure provides a PD-1/PD-L1
antagonist that binds human PD-1 and prevents, inhibits or reduces
PD-L1 binding to PD-1. In some aspects, the PD-1/PD-L1 antagonist
binds to the mRNA encoding PD-1 or PD-L1 and prevents translation.
In some embodiments, the PD-1/PD-L1 antagonist binds to the mRNA
encoding PD-1 or PD-L1 and causes degradation and/or turnover.
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 binding of PD-1 to PD-L1, PD-L2, or to both PD-L1
and PD-L2. In some embodiments, the PD-1/PD-L1 antagonist blocks
binding of PD-1 to PD-L1. In some embodiments, the PD-1/PD-L1
antagonist blocks 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 PD-1. In some embodiments, the PD-1/PD-L1 antagonist
specifically binds PD-L1. In some embodiments, the PD-1/PD-L1
antagonist specifically binds PD-L2.
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.
In some embodiments, the PD-1/PD-L1 antagonist is an isolated
monoclonal antibody (mAb), or antigen binding fragment thereof,
which specifically binds to PD-1 or PD-L1. In some embodiments, the
PD-1/PD-L1 antagonist is an antibody or antigen binding fragment
thereof that specifically binds to human PD-1. In some embodiments,
the PD-1/PD-L1 antagonist is an antibody or antigen binding
fragment thereof that specifically binds to human PD-L1. In some
embodiments, the PD-1/PD-L1 antagonist is an antibody or antigen
binding fragment 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 antigen binding fragment that binds to human PD-1
and inhibits the binding of PD-L1 to PD-1.
Several immune checkpoint antagonists that inhibit or disrupt the
interaction between PD-1 and either one or both of its ligands
PD-L1 and PD-L2 are in clinical development or are currently
available to clinicians for treating cancer.
Examples of anti-human PD-1 monoclonal antibodies, or antigen
binding fragments thereof, that may comprise the PD-1/PD-L1
antagonist in any of the compositions, methods, and uses provided
by the disclosure include, but are not limited to: KEYTRUDA.RTM.
(pembrolizumab, MK-3475, h409A11; see U.S. Pat. Nos. 8,952,136,
8,354,509, 8,900,587, and EP2170959, all of which are included
herein by reference in their entirety; Merck), OPDIVO.RTM.
(nivolumab, BMS-936558, MDX-1106, ONO-4538; see U.S. Pat. Nos.
7,595,048, 8,728,474, 9,073,994, 9,067,999, EP1537878, U.S. Pat.
Nos. 8,008,449, 8,779,105, and EP2161336, all of which are included
herein by reference in their entirety; Bristol Myers Squibb),
MEDI0680 (AMP-514), BGB-A317 and BGB-108 (BeiGene), 244C8 and 388D4
(see WO2016106159, which is incorporated herein by reference in its
entirety; Enumeral Biomedical), PDR001 (Novartis), and REGN2810
(Regeneron). Accordingly, in some embodiments the PD-1/PD-L1
antagonist is pembrolizumab. In some embodiments, the PD-1/PD-L1
antagonist is nivolumab.
Examples of anti-human PD-L1 monoclonal antibodies, or antigen
binding fragments thereof, that may comprise the PD-1/PD-L1
antagonist in any of the compositions, methods, and uses provided
by the disclosure include, but are not limited to: BAVENCIO.RTM.
(avelumab, MSB0010718C, see WO2013/79174, which is incorporated
herein by reference in its entirety; Merck/Pfizer), IMFINZI.RTM.
(durvalumab, MEDI4736), TECENTRIQ.RTM. (atezolizumab, MPDL3280A,
RG7446; see WO2010/077634, which is incorporated herein by
reference in its entirety; Roche), MDX-1105 (BMS-936559, 12A4; see
U.S. Pat. No. 7,943,743 and WO2013/173223, both of which are
incorporated herein by reference in their entirety; Medarex/BMS),
and FAZ053 (Novartis). Accordingly, 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.
In some embodiments, the PD-1/PD-L1 antagonist is an immunoadhesin
that specifically bind to human PD-1 or human PD-L1, e.g., a fusion
protein containing the extracellular or PD-1 binding portion of
PD-L1 or PD-L2 fused to a constant region such as an 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.
It will be understood by one of ordinary skill that any PD-1/PD-L1
antagonist which binds to PD-1 or PD-L1 and disrupts the PD-1/PD-L1
signaling pathway, is suitable for compositions, methods, and uses
disclosed herein.
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 Discov Today 21(6):1027-1036.
In some embodiments of the methods provided by the 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, KEYTRUDA.RTM. (pembrolizumab), OPDIVO.RTM. (nivolumab),
pidilizumab, MEDI0680, REGN2810, TSR-042, PF-06801591, and AMP-224.
In some embodiments, the PD-1/PD-L1 antagonist is selected from the
group consisting of: FAZ053, TENCENTRIQ.RTM. (atezolizumab),
BAVENCIO.RTM. (avelumab), IMFINZI.RTM. (durvalumab), and
BMS-936559.
Combinations with TIM-3 Antagonist
In some embodiments, an RLR agonist, or pharmaceutical compositions
thereof, provided by the disclosure is combined (e.g., administered
in combination) with a TIM-3 antagonist. The TIM-3 antagonist may
be an antibody, an antigen binding fragment thereof, an
immunoadhesin, a fusion protein, or an oligopeptide. In some
embodiments, the TIM-3 antagonist is chosen from MGB453 (Novartis),
TSR-022 (Tesaro), or LY3321367 (Eli Lilly).
Combinations with LAG-3 Antagonist
In some embodiments, an RLR agonist, or pharmaceutical compositions
thereof, provided by the disclosure is combined (e.g., administered
in combination) with a LAG-3 antagonist. The LAG-3 antagonist may
be an antibody, an antigen binding fragment thereof, an
immunoadhesin, a fusion protein, or oligopeptide. In some
embodiments, the LAG-3 inhibitor is chosen from LAG525 (Novartis),
BMS-986016 (Bristol-Myers Squibb), TSR-033 (Tesaro), MK-4280 (Merck
& Co), or REGN3767 (Regeneron).
Combinations with Toll-Like Receptor (TLR) Agonists
In some embodiments, an RLR agonist, or pharmaceutical composition
thereof, provided by the disclosure is combined (e.g. administered
in combination) with a TLR agonist.
Toll-like receptors (TLRs) are a family of germline-encoded
transmembrane proteins that facilitate pathogen recognition and
activation of the innate immune system. (Hoffmann et al., (1999)
Science 284:1313-1318; Rock et al., (1998) Proc Natl Acad Sci USA
95:588-593). TLRs are pattern recognition receptors (PRRs), and are
expressed by cells of the innate immune system. Examples of known
ligands for TLRs include gram positive bacteria (TLR-2), bacterial
endotoxin (TLR-4), flagellin protein (TLR-5), bacterial DNA
(TLR-9), double-stranded RNA and poly I:C (TLR-3), and yeast
(TLR-2). In vivo activation of TLRs initiates an innate immune
response involving specific cytokines, chemokines and growth
factors. While all TLRs can activate certain intracellular
signaling molecules such as nuclear factor kappa beta (NF-.kappa.B)
and mitogen activated protein kinases (MAP kinases), the specific
set of cytokines and chemokines released appears to be unique for
each TLR. TLR7, 8, and 9 comprise a subfamily of TLRs which are
located in endosomal or lysosomal compartments of immune cells such
as dendritic cells and monocytes. In contrast 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 recognition of microbial nucleic acids, such as single
stranded RNA.
Small, low-molecular weight (less than 400 Daltons) synthetic
imidazoquinoline compounds which resemble the purine nucleotides
adenosine and guanosine were the first TLR7 and TLR8 agonists to be
identified. A number of these compounds have demonstrated
anti-viral and anti-cancer properties. For example, the TLR7
agonist imiquimod (ALDARA.TM.) was approved by the U.S. Food and
Drug Administration as a topical agent for the treatment of skin
lesions caused by certain strains of the human papillomavirus.
Imiquimod may also be useful for the treatment of primary skin
cancers and cutaneous tumors such as basal cell carcinomas,
keratoacanthomas, actinic keratoses, and Bowen's disease. The
TLR7/8 agonist resiquimod (R-848) is being evaluated as a topical
agent for the treatment of human genital herpes.
TLR agonists according to the disclosure can be any TLR agonist.
For example, a TLR agonist can encompass a natural or synthetic TLR
ligand, a mutein or derivative of a TLR ligand, a peptide mimetic
of a TLR ligand, a small molecule that mimics the biological
function of a TLR ligand, or an antibody that stimulates a TLR
receptor. A TLR ligand is any molecule that binds to a TLR.
In some embodiments, an RLR agonist, or pharmaceutical composition
thereof, provided by the 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.
In some embodiments, an RLR agonist provided by the disclosure is
combined with a TLR3 agonist. A TLR3 agonist is an agonist that
causes a signaling response through TLR3. Exemplary TLR3 agonists
include, but are not limited to, polyinosinic:polycytidylic acid
(poly I:C), HILTONOL.RTM. (poly ICLC), polyadenylic-polyuridylic
acid (poly A:U), RIBOXXIM.RTM. (RGIC.RTM.100), RIBOXXON.RTM.
(RGIC.RTM.50 bioconjugate), and RIBOXXOL.RTM. (RGIC.RTM.50).
In some embodiments, an RLR agonist provided by the disclosure is
combined with polyinosinic:polycytidylic acid (poly I:C). In some
embodiments, the RLR agonist is combined with HILTONOL.RTM. (poly
ICLC). In some embodiments, the RLR agonist is combined with
polyadenylic-polyuridylic acid (poly A:U). In some embodiments, the
RLR agonist is combined with RIBOXXIM.RTM. (RGIC.RTM.100). In some
embodiments, the RLR agonist is combined with RIBOXXON.RTM.
(RGIC.RTM.50 bioconjugate). In some embodiments, the RLR agonist is
combined with RIBOXXOL.RTM. (RGIC.RTM.50).
In some embodiments, an RLR agonist provided by the disclosure is
combined with a TLR7 agonist. A TLR7 agonist is an agonist that
causes a signaling response through TLR7. Non-limiting examples of
TLR7 agonists include single stranded RNA (ssRNA), loxoribine (a
guanosine analogue derivatized at positions N7 and C8),
imidazoquinoline compounds (e.g., imiquimod and resiquimod), or
derivatives thereof. Further exemplary TLR7 agonists include, but
are not limited to, GS-9620 (Vesatolimod), imiquimod (ALDARA.TM.),
and resiquimod (R-848).
In some embodiments, an RLR agonist provided by the disclosure is
combined with GS-9620 (Vesatolimod). In some embodiments, the RLR
agonist is combined with imiquimod (ALDARA.TM.). In some
embodiments, the RLR agonist is combined with resiquimod
(R-848).
In some embodiments, an RLR agonists provided by the disclosure is
combined with a TLR9 agonist. A TLR9 agonist is an agonist that
causes a signaling response through TLR9. Exemplary TLR9 agonists
include, but are not limited to, CpG oligodeoxynucleotides (GpG
ODNs). In some embodiments, the CpG ODN is a Class A CpG ODN (CpG-A
ODN), a Class B CpG ODN (CpG-B ODN), or a Class C CpG ODN (CpG-B
ODN).
In some embodiments, an RLR agonist provided by the 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).
Other Combinations
In some embodiments, an RLR agonist, or pharmaceutical compositions
thereof, provided by the disclosure is combined (e.g., administered
in combination) with a VISTA antagonist, an adenosine A2AR
antagonist, a B7-H3 antagonist, a B7-H4 antagonist, a BTLA
antagonist, a CTLA-4 antagonist, an IDO antagonist, or a KIR
antagonist
In some embodiments, an RLR agonist, or pharmaceutical compositions
thereof, provided by the disclosure is combined (e.g., administered
in combination) with an agonist comprising an polypeptide (e.g,
antibody, or antigen binding portion thereof) that specifically
binds to CD137 (4-1BB).
In some embodiments, an RLR agonist, or pharmaceutical compositions
thereof, provided by the disclosure is combined (e.g., administered
in combination) with an agonist comprising an polypeptide (e.g.,
antibody, or antigen binding portion thereof) that specifically
binds to CD134 (OX40).
An RLR agonist described herein can replace or augment a previously
or currently administered therapy. For example, upon treating with
an RLR agonist, administration of the one or more additional active
agents can cease or diminish, e.g., be administered at lower levels
or dosages. In some embodiments, administration of the previous
therapy can be maintained. In some embodiments, a 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.
Monitoring a subject (e.g., a human patient) for an improvement in
a cancer, as defined herein, means evaluating the subject for a
change in a disease parameter, e.g., a reduction in tumor growth.
In some embodiments, the evaluation is performed at least one (1)
hour, e.g., at least 2, 4, 6, 8, 12, 24, or 48 hours, or at least 1
day, 2 days, 4 days, 10 days, 13 days, 20 days or more, or at least
1 week, 2 weeks, 4 weeks, 10 weeks, 13 weeks, 20 weeks or more,
after an administration. The subject can be evaluated in one or
more of the following periods: prior to beginning of treatment;
during the treatment; or after one or more elements of the
treatment have been administered. Evaluation can include evaluating
the need for further treatment, e.g., evaluating whether a dosage,
frequency of administration, or duration of treatment should be
altered. It can also include evaluating the need to add or drop a
selected therapeutic modality, e.g., adding or dropping any of the
treatments for a cancer described herein.
In some embodiments, an RLR agonist described herein is
administered to modulate a T-cell response in a patient, for
example, by increasing T-cell activation and/or proliferation.
Enhancement of T cell proliferation, IFN production and secretion,
and/or the cytolytic activity of T cells may be beneficial to
patients in need thereof to treat a disease or condition.
Accordingly, in some embodiments, an RLR agonist of the present
disclosure is administered to a patent in need thereof to induce or
increase T-cell activation, enhance T cell proliferation, induce
the production and/or secretion of IFN, and/or induce a cytolytic T
cell response.
In some embodiments, an RLR agonist described herein, can be
employed in methods of detection and/or quantification of human
RLRs in a biological sample. Accordingly, an RLR agonist, as
described herein, is used to diagnose, prognose, and/or determine
progression of disease (e.g., cancer) in a patient.
While the present disclosure has been described with reference to
the specific embodiments thereof, it should be understood by those
skilled in the art that various changes may be made and equivalents
may be substituted without departing from the true spirit and scope
of the disclosure. In addition, many modifications may be made to
adapt a particular situation, material, composition of matter,
process, process step or steps, to the objective, spirit and scope
of the present disclosure. All such modifications are intended to
be within the scope of the disclosure.
EXAMPLES
The disclosure will be more fully understood by reference to the
following examples. They should not, however, be construed as
limiting the scope of the disclosure. It is understood that the
examples and embodiments described herein are for illustrative
purposes only and that various modifications or changes in light
thereof will be suggested to persons skilled in the art and are to
be included within the spirit and purview of this application and
scope of the appended claims.
Example 1
Transfection of HuPBMCs with RLR Agonists Induces Cytokine
Production In Vitro
To determine the effect of RLR agonists comprising various
modification on cytokine induction, the ability of RLR agonists to
induce cytokine production was assessed in vitro. Human peripheral
blood mononuclear cells (huPBMCs) were prepared from two healthy
donors and seeded at a density of 2.times.10.sup.5 cells/well in a
standard 96-well tissue culture plate in 100 .mu.L of RPMI 1640
cell culture medium supplemented with fetal calf serum (FCS),
L-glutamine, and Pen/Strep. Independent transfections of huPBMCs
with RLR agonists, as indicated in FIG. 1, was carried out using
Lipofectamine2000 as the transfection reagent (except for G10 and
ODN2216, where direct incubation was applied, data not shown).
Cells were incubated for 24 h at 37C in a humidified incubator
followed by harvesting of cell culture supernatant. Supernatants
were immediately frozen and stored at -20C. Samples were thawed
once for analysis of cytokines IFN-.alpha.2a (FIG. 1), as well as
IL-1.beta., IP-10, IL-6, IL-12p70, MCP-1 and MIP-1.beta. (data not
shown) using a U-Plex MSD platform according to the manufacturer's
instructions. FIG. 1 shows the dose-dependent induction of
IFN-.alpha. secretion from human PBMC treated with novel candidate
RLR agonists comprising various modifications and/or sequence
motifs. RLR agonists we added at either 10 nM, 2 nM, or 0.4 nM. The
amount of IFN-.alpha.2a released by the cells in response to RLR
agonist transfection is given in pg/mL.
Tables 3 and 4 show the sequences of each RLR agonist. Table 3 also
shows the sequence and number corresponding to each compound tested
in FIG. 1. For examples, compound X25224 in FIG. 1 corresponds to
"RIG7" which comprises a first oligonucleotide comprising SEQ ID
NO: 42 linked via a linker "UUCG" to a second oligonucleotide
comprising SEQ ID NO: 73 and has a 5' diphosphate moiety. The
sequence of RIG7 is also set forth as SEQ ID NO: 6 in Table 4.
TABLE-US-00006 TABLE 3 OLIGONUCLEOTIDE COMBINATION TABLE First
Second Oligonu- Oli- 5' cleotide gonucleotide Phosphate RNA (FO)
(SO) Linker Moiety RIG 2a (X32671) 37 68 UUCG -- RIG 2b (X25217) 37
68 UUCG pp RIG 3a (X32666) 38 69 UUCG -- RIG 3b (X25218) 38 69 UUCG
pp RIG 4 (X25219) 39 70 UUCG pp RIG 5 (X25221) 40 71 UUCG pp RIG 6
(X25222) 41 72 UUCG pp RIG 7 (X25224) 42 73 UUCG pp RIG 8 (X25225)
43 74 UUCG pp RIG 9 (X25226) 44 75 UUCG pp RIG 10 (X25227) 45 76
UUCG pp RIG 11 (X25228) 46 77 UUCG pp RIG 12 (X25229) 47 78 UUCG pp
RIG 13a (X32667) 48 79 UUCG -- RIG 13b (X25230) 48 79 UUCG pp RIG
13c (X24921) 48 79 UUCG ppp RIG 14 (X25231) 49 80 UUCG pp RIG 15a
(X32665) 50 81 UUCG -- RIG 15b (X25232) 50 81 UUCG pp RIG 15c
(X24923) 50 81 UUCG ppp RIG 16 (X25233) 51 82 UUCG pp RIG 18
(X25234) 52 83 UUCG pp RIG 20a (X32750) 53 84 UUCG -- RIG 20b
(X25235) 53 84 UUCG pp RIG 21 (X25237) 54 85 UUCG pp RIG 22a
(X32672) 55 86 UUCG -- RIG 22b (X25239) 55 86 UUCG pp RIG 24a
(X25241) 56 87 UUCG pp RIG 24b (X25240) 56 87 UUCG ppp RIG 25
(X25242) 57 88 UUCG pp RIG 26 (X25243) 58 89 UUCG pp RIG 27a
(X32669) 59 89 UUCG -- RIG 27b (X25244) 59 89 UUCG pp RIG 27c
(X24935) 59 89 UUCG ppp RIG 28a (X25245) 60 90 UUCG pp RIG 28b
(X24936) 60 90 UUCG ppp RIG 35a (X32670) 61 91 UUCG -- RIG 35b
(X25247) 61 91 UUCG pp RIG 36 (X24945) 62 92 UUCG pp RIG 37a
(X25249) 63 91 UUUGAU pp RIG 37b (X25248) 63 91 UUUGAU ppp RIG 38a
(X32668) 63 91 UGUUU -- RIG 38b (X25251) 63 91 UGUUU pp RIG 39
(X25253) 63 91 GAUC pp RIG 40 (X25255) 64 93 GAUC pp RIG 41
(X25257) 65 94 GAUC pp RIG 42 (X25259) 64 93 UUCG pp RIG 43a
(X32673) 63 91 (C9) -- RIG 43b (X25261) 63 91 (C9) pp RIG 44
(X25263) 63 91 (HEG) pp RIG 47 (X25265) 66 95 UUCG pp RIG 48
(X25267) 67 96 UUCG pp RIG 49a (X25269) 63 97 UUCG pp RIG 49b
(X25268) 63 97 UUCG ppp RIG 50a (14L) 63 91 UUCG -- (X32664) RIG
50b (14L) 63 91 UUCG pp (X24943) RIG 50c (14L) 63 91 UUCG ppp
(X24907) (--) indicates no 5' phosphate; (pp) indicates 5'
diphosphate; (ppp) indicates 5' triphosphate
TABLE-US-00007 TABLE 4 SEQUENCE LISTING SEQ ID NO Description
Sequence 1 RIG 2 GGATCGATCGATCGUUCGCGATCGATCGATCC Nucleic acid
sequence 2 RIG 3 GGAUCGAUCGAUAUUUCGAUAUCGAUCGAUCC Nucleic acid
sequence 3 RIG 4 GCGCGCGCGCGCGCUUCGGCGCGCGCGCGCGC Nucleic acid
sequence 4 RIG 5 GGCGGCGCGCCGCCUUCGGGCGGCGCGCCGCC Nucleic acid
sequence 5 RIG 6 GGCGGCGGCGGCGGUUCGCCGCCGCCGCCGCC Nucleic acid
sequence 6 RIG 7 GGCGGCCGCCCGCGUUCGCGCGGGCGGCCGCC Nucleic acid
sequence 7 RIG 8 CGACGUCGACGUCGUUCGCGACGUCGACGUCG Nucleic acid
sequence 8 RIG 9 GCACGUCGACGUGCUUCGGCACGUCGACGUGC Nucleic acid
sequence 9 RIG 10 GGACGUCGACGUCCUUCGGGACGUCGACGUCC Nucleic acid
sequence 10 RIG 11 GGUCGCGACCAUAUUUCGAUAUGGUCGCGACC Nucleic acid
sequence 11 RIG 12 GGAUACGUCGACGUUUCGACGUCGACGUAUCC Nucleic acid
sequence 12 RIG 13 GAGAGAGAGAGAGAUUCGUCUCUCUCUCUCUC Nucleic acid
sequence 13 RIG 14 GAGUCUAGACUCCGUUCGCGGAGUCUAGACUC Nucleic acid
sequence 14 RIG 15 CGAUCGAUCGAUCGUUCGCGAUCGAUCGAUCG Nucleic acid
sequence (RIG 45) 15 RIG 16 CCAUCGAUCGAUCGUUCGCGAUCGAUCGAUGG
Nucleic acid sequence 16 RIG 18 GAAUCGAUCGAUCGUUCGCGAUCGAUCGAUUC
Nucleic acid sequence 17 RIG 20 GGGAUCGAUCGUUCGCGAUCGAUCCC Nucleic
acid sequence 18 RIG 21 CCCCCGAUCGAUCGUUCGCGAUCGAUCGGGGG Nucleic
acid sequence 19 RIG 22 GTGTGTGTGTGTGTUUCGACACACACACACAC Nucleic
acid sequence 20 RIG 24 GTGTGTGGAUCGAUUUCGAUCGAUCCACACAC Nucleic
acid sequence 21 RIG 25 GGAICGAICGAICGUUCGCGAICGAICGAICC Nucleic
acid sequence 22 RIG 26 IIAUCIAUCIAUCIUUCGCIAUCIAUCIAUCC Nucleic
acid sequence 23 RIG 27 GGAUCIAUCIAUCIUUCGCIAUCIAUCIAUCC Nucleic
acid sequence 24 RIG 28 GGIUCGIUCGIUCGUUCGCGIUCGIUCGIUCC Nucleic
acid sequence 25 RIG 35 IGAUCGAUCGAUCGUUCGCGAUCGAUCGAUCC Nucleic
acid sequence 26 RIG 36 AUCGAUCGAUCGUUCGCGAUCGAUCGAU Nucleic acid
sequence 27 RIG 37 GGAUCGAUCGAUCGUUUGAUCGAUCGAUCGAU Nucleic CC acid
sequence 28 RIG 38 GGAUCGAUCGAUCGUGUUUCGAUCGAUCGAUC Nucleic C acid
sequence 29 RIG 39 GGAUCGAUCGAUCGGAUCCGAUCGAUCGAUCC Nucleic acid
sequence 30 RIG 40 GGCAUGCGACCUCUGUUUGAUCAAACAGAGGU Nucleic
CGCAUGCC acid sequence 31 RIG 41 GGCAUGCGACCUCUGAUCAGAGGUCGCAUGCC
Nucleic acid sequence 32 RIG 42 GGCAUGCGACCUCUGUUUUUCGAAACAGAGGU
Nucleic CGCAUGCC acid sequence 33 RIG 47
TGCUCGAUCGAUCGUUCGCGAUCGAUCGAGCA Nucleic acid sequence 34 RIG 48
TCGUCGAUCGAUCGUUCGCGAUCGAUCGACGA Nucleic acid sequence 35 RIG 49
GGAUCGAUCGAUCGUUCGTGAUCGAUCGAUGG Nucleic acid sequence 36 RIG 50
GGAUCGAUCGAUCGUUCGCGAUCGAUCGAUCC Nucleic acid sequence (14L) 37 FO1
GGATCGATCGATCG Nucleic acid sequence 38 FO2 GGAUCGAUCGAUAU Nucleic
acid sequence 39 FO3 GCGCGCGCGCGCGC Nucleic acid sequence 40 FO4
GGCGGCGCGCCGCC Nucleic acid sequence 41 FO5 GGCGGCGGCGGCGG Nucleic
acid sequence 42 FO6 GGCGGCCGCCCGCG Nucleic acid sequence 43 FO7
CGACGUCGACGUCG Nucleic acid sequence 44 FO8 GCACGUCGACGUGC Nucleic
acid sequence 45 FO9 GGACGUCGACGUCC Nucleic acid sequence 46 FO10
GGUCGCGACCAUAU Nucleic acid sequence 47 FO11 GGAUACGUCGACGU Nucleic
acid sequence 48 FO12 GAGAGAGAGAGAGA Nucleic acid sequence 49 FO13
GAGUCUAGACUCCG
Nucleic acid sequence 50 FO14 CGAUCGAUCGAUCG Nucleic acid sequence
51 FO15 CCAUCGAUCGAUCG Nucleic acid sequence 52 FO16 GAAUCGAUCGAUCG
Nucleic acid sequence 53 FO17 GGGAUCGAUCG Nucleic acid sequence 54
FO18 CCCCCGAUCGAUCG Nucleic acid sequence 55 FO19 GTGTGTGTGTGTGT
Nucleic acid sequence 56 FO20 GTGTGTGGAUCGAU Nucleic acid sequence
57 FO21 GGAICGAICGAICG Nucleic acid sequence 58 FO22 IIAUCIAUCIAUCI
Nucleic acid sequence 59 FO23 GGAUCIAUCIAUCI Nucleic acid sequence
60 FO24 GGIUCGIUCGIUCG Nucleic acid sequence 61 FO25 IGAUCGAUCGAUCG
Nucleic acid sequence 62 FO26 AUCGAUCGAUCG Nucleic acid sequence 63
FO27 GGAUCGAUCGAUCG Nucleic acid sequence 64 FO28
GGCAUGCGACCUCUGUUU Nucleic acid sequence 65 FO29 GGCAUGCGACCUCU
Nucleic acid sequence 66 FO30 TGCUCGAUCGAUCG Nucleic acid sequence
67 FO31 TCGUCGAUCGAUCG Nucleic acid sequence 68 SO1 CGATCGATCGATCC
Nucleic acid sequence 69 SO2 AUAUCGAUCGAUCC Nucleic acid sequence
70 SO3 GCGCGCGCGCGCGC Nucleic acid sequence 71 SO4 GGCGGCGCGCCGCC
Nucleic acid sequence 72 SO5 CCGCCGCCGCCGCC Nucleic acid sequence
73 SO6 CGCGGGCGGCCGCC Nucleic acid sequence 74 SO7 CGACGUCGACGUCG
Nucleic acid sequence 75 SO8 GCACGUCGACGUGC Nucleic acid sequence
76 SO9 GGACGUCGACGUCC Nucleic acid sequence 77 SO10 AUAUGGUCGCGACC
Nucleic acid sequence 78 SO11 ACGUCGACGUAUCC Nucleic acid sequence
79 SO12 UCUCUCUCUCUCUC Nucleic acid sequence 80 SO13 CGGAGUCUAGACUC
Nucleic acid sequence 81 SO14 CGAUCGAUCGAUCG Nucleic acid sequence
82 SO15 CGAUCGAUCGAUGG Nucleic acid sequence 83 SO16 CGAUCGAUCGAUUC
Nucleic acid sequence 84 SO17 CGAUCGAUCCC Nucleic acid sequence 85
SO18 CGAUCGAUCGGGGG Nucleic acid sequence 86 SO19 ACACACACACACAC
Nucleic acid sequence 87 SO20 AUCGAUCCACACAC Nucleic acid sequence
88 SO21 CGAICGAICGAICC Nucleic acid sequence 89 SO22 CIAUCIAUCIAUCC
Nucleic acid sequence 90 SO23 CGIUCGIUCGIUCC Nucleic acid sequence
91 SO24 CGAUCGAUCGAUCC Nucleic acid sequence 92 SO25 CGAUCGAUCGAU
Nucleic acid sequence 93 SO26 AAACAGAGGUCGCAUGCC Nucleic acid
sequence 94 SO27 AGAGGUCGCAUGCC Nucleic acid sequence 95 SO28
CGAUCGAUCGAGCA Nucleic acid sequence 96 SO29 CGAUCGAUCGACGA Nucleic
acid sequence 97 SO30 TGAUCGAUCGAUGG Nucleic acid sequence 98 Human
MTTEQRRSLQAFQDYIRKTLDPTYILSYMAPW RIG-I
FREEEVQYIQAEKNNKGPMEAATLFLKFLLEL Amino
QEEGWFRGFLDALDHAGYSGLYEAIESWDFKK acid
IEKLEEYRLLLKRLQPEFKTRIIPTDIISDLS sequence
ECLINQECEEILQICSTKGMMAGAEKLVECLL RSDKENWPKTLKLALEKERNKFSELWIVEKGI
KDVETEDLEDKMETSDIQIFYQEDPECQNLSE
NSCPPSEVSDTNLYSPFKPRNYQLELALPAMK GKNTIICAPTGCGKTFVSLLICEHHLKKFPQG
QKGKVVFFANQIPVYEQQKSVFSKYFERHGYR VTGISGATAENVPVEQIVENNDIIILTPQILV
NNLKKGTIPSLSIFTLMIFDECHNTSKQHPYN MIMFNYLDQKLGGSSGPLPQVIGLTASVGVGD
AKNTDEALDYICKLCASLDASVIATVKHNLEE LEQVVYKPQKFFRKVESRISDKFKYIIAQLMR
DTESLAKRICKDLENLSQIQNREFGTQKYEQW IVTVQKACMVFQMPDKDEESRICKALFLYTSH
LRKYNDALIISEHARMKDALDYLKDFFSNVRA AGFDEIEQDLTQRFEEKLQELESVSRDPSNEN
PKLEDLCFILQEEYHLNPETITILFVKTRALV DALKNWIEGNPKLSFLKPGILTGRGKTNQNTG
MTLPAQKCILDAFKASGDHNILIATSVADEGI DIAQCNLVILYEYVGNVIKMIQTRGRGRARGS
KCFLLTSNAGVIEKEQINMYKEKKMNDSILRL QTWDEAVFREKILHIQTHEKFIRDSQEKPKPV
PDKENKKLLCRKCKALACYTADVRVIEECHYT VLGDAFKECFVSRPHPKPKQFSSFEKRAKIFC
ARQNCSHDWGIHVKYKTFEIPVIKIESFVVED IATGVQTLYSKWKDFHFEKIPFDPAEMSK 99
Human MSNGYSTDENFRYLISCFRARVKMYIQVEPVL MDA5
DYLTFLPAEVKEQIQRTVATSGNMQAVELLLS Amino
TLEKGVWHLGWTREFVEALRRTGSPLAARYMN acid
PELTDLPSPSFENAHDEYLQLLNLLQPTLVDK sequence
LLVRDVLDKCMEEELLTIEDRNRIAAAENNGN ESGVRELLKRIVQKENWFSAFLNVLRQTGNNE
LVQELTGSDCSESNAEIENLSQVDGPQVEEQL LSTTVQPNLEKEVWGMENNSSESSFADSSVVS
ESDTSLAEGSVSCLDESLGHNSNMGSDSGTMG SDSDEENVAARASPEPELQLRPYQMEVAQPAL
EGKNIIICLPTGSGKTRVAVYIAKDHLDKKKK ASEPGKVIVLVNKVLLVEQLFRKEFQPFLKKW
YRVIGLSGDTQLKISFPEVVKSCDIIISTAQI LENSLLNLENGEDAGVQLSDFSLIIIDECHHT
NKEAVYNNIMRHYLMQKLKNNRLKKENKPVIP LPQILGLTASPGVGGATKQAKAEEHILKLCAN
LDAFTIKTVKENLDQLKNQIQEPCKKFAIADA TREDPFKEKLLEIMTRIQTYCQMSPMSDFGTQ
PYEQWAIQMEKKAAKEGNRKERVCAEHLRKYN EALQINDTIRMIDAYTHLETFYNEEKDKKFAV
IEDDSDEGGDDEYCDGDEDEDDLKKPLKLDET DRFLMTLFFENNKMLKRLAENPEYENEKLTKL
RNTIMEQYTRTEESARGIIFTKTRQSAYALSQ WITENEKFAEVGVKAHHLIGAGHSSEFKPMTQ
NEQKEVISKFRTGKINLLIATTVAEEGLDIKE CNIVIRYGLVTNEIAMVQARGRARADESTYVL
VAHSGSGVIEHETVNDFREKMMYKAIHCVQNM KPEEYAHKILELQMQSIMEKKMKTKRNIAKHY
KNNPSLITFLCKNCSVLACSGEDIHVIEKMHH VNMTPEFKELYIVRENKALQKKCADYQINGEI
ICKCGQAWGTMMVHKGLDLPCLKIRNFVVVFK NNSTKKQYKKWVELPITFPNLDYSECCLFSDE D
100 Human MELRSYQWEVIMPALEGKNIIIWLPTGAGKTR LGP2
AAAYVAKRHLETVDGAKVVVLVNRVHLVTQHG Amino
EEFRRMLDGRWTVTTLSGDMGPRAGFGHLARC acid
HDLLICTAELLQMALTSPEEEEHVELTVFSLI sequence
VVDECHHTHKDTVYNVIMSQYLELKLQRAQPL PQVLGLTASPGTGGASKLDGAINHVLQLCANL
DTWCIMSPQNCCPQLQEHSQQPCKQYNLCHRR SQDPFGDLLKKLMDQIHDHLEMPELSRKFGTQ
MYEQQVVKLSEAAALAGLQEQRVYALHLRRYN DALLIHDTVRAVDALAALQDFYHREHVTKTQI
LCAERRLLALFDDRKNELAHLATHGPENPKLE MLEKILQRQFSSSNSPRGIIFTRTRQSAHSLL
LWLQQQQGLQTVDIRAQLLIGAGNSSQSTHMT QRDQQEVIQKFQDGTLNLLVATSVAEEGLDIP
HCNVVVRYGLLTNEISMVQARGRARADQSVYA FVATEGSRELKRELINEALETLMEQAVAAVQK
MDQAEYQAKIRDLQQAALTKRAAQAAQRENQR QQFPVEHVQLLCINCMVAVGHGSDLRKVEGTH
HVNVNPNFSNYYNVSRDPVVINKVFKDWKPGG VISCRNCGEVWGLQMIYKSVKLPVLKVRSMLL
ETPQGRIQAKKWSRVPFSVPDFDFLQHCAENL SDLSLD
SEQUENCE LISTINGS
1
100132DNAArtificial SequenceSynthetic RIG 2 Nucleic acid sequence
1ggatcgatcg atcguucgcg atcgatcgat cc 32232RNAArtificial
SequenceSynthetic RIG 3 Nucleic acid sequence 2ggaucgaucg
auauuucgau aucgaucgau cc 32332RNAArtificial SequenceSynthetic RIG 4
Nucleic acid sequence 3gcgcgcgcgc gcgcuucggc gcgcgcgcgc gc
32432RNAArtificial SequenceSynthetic RIG 5 Nucleic acid sequence
4ggcggcgcgc cgccuucggg cggcgcgccg cc 32532RNAArtificial
SequenceSynthetic RIG 6 Nucleic acid sequence 5ggcggcggcg
gcgguucgcc gccgccgccg cc 32632RNAArtificial SequenceSynthetic RIG 7
Nucleic acid sequence 6ggcggccgcc cgcguucgcg cgggcggccg cc
32732RNAArtificial SequenceSynthetic RIG 8 Nucleic acid sequence
7cgacgucgac gucguucgcg acgucgacgu cg 32832RNAArtificial
SequenceSynthetic RIG 9 Nucleic acid sequence 8gcacgucgac
gugcuucggc acgucgacgu gc 32932RNAArtificial SequenceSynthetic RIG
10 Nucleic acid sequence 9ggacgucgac guccuucggg acgucgacgu cc
321032RNAArtificial SequenceSynthetic RIG 11 Nucleic acid sequence
10ggucgcgacc auauuucgau auggucgcga cc 321132RNAArtificial
SequenceSynthetic RIG 12 Nucleic acid sequence 11ggauacgucg
acguuucgac gucgacguau cc 321232RNAArtificial SequenceSynthetic RIG
13 Nucleic acid sequence 12gagagagaga gagauucguc ucucucucuc uc
321332RNAArtificial SequenceSynthetic RIG 14 Nucleic acid sequence
13gagucuagac uccguucgcg gagucuagac uc 321432RNAArtificial
SequenceSynthetic RIG 15 Nucleic acid sequence (RIG 45)
14cgaucgaucg aucguucgcg aucgaucgau cg 321532RNAArtificial
SequenceSynthetic RIG 16 Nucleic acid sequence 15ccaucgaucg
aucguucgcg aucgaucgau gg 321632RNAArtificial SequenceSynthetic RIG
18 Nucleic acid sequence 16gaaucgaucg aucguucgcg aucgaucgau uc
321726RNAArtificial SequenceSynthetic RIG 20 Nucleic acid sequence
17gggaucgauc guucgcgauc gauccc 261832RNAArtificial
SequenceSynthetic RIG 21 Nucleic acid sequence 18cccccgaucg
aucguucgcg aucgaucggg gg 321932DNAArtificial SequenceSynthetic RIG
22 Nucleic acid sequence 19gtgtgtgtgt gtgtuucgac acacacacac ac
322032DNAArtificial SequenceSynthetic RIG 24 Nucleic acid sequence
20gtgtgtggau cgauuucgau cgauccacac ac 322132RNAArtificial
SequenceSynthetic RIG 25 Nucleic acid sequencemisc_feature(4)..(4)n
is inosinemisc_feature(8)..(8)n is inosinemisc_feature(12)..(12)n
is inosinemisc_feature(22)..(22)n is inosinemisc_feature(26)..(26)n
is inosinemisc_feature(30)..(30)n is inosine 21ggancgancg
ancguucgcg ancgancgan cc 322232RNAArtificial SequenceSynthetic RIG
26 Nucleic acid sequencemisc_feature(1)..(2)n is
inosinemisc_feature(6)..(6)n is inosinemisc_feature(10)..(10)n is
inosinemisc_feature(14)..(14)n is inosinemisc_feature(20)..(20)n is
inosinemisc_feature(24)..(24)n is inosinemisc_feature(28)..(28)n is
inosine 22nnaucnaucn aucnuucgcn aucnaucnau cc 322332RNAArtificial
SequenceSynthetic RIG 27 Nucleic acid sequencemisc_feature(6)..(6)n
is inosinemisc_feature(10)..(10)n is inosinemisc_feature(14)..(14)n
is inosinemisc_feature(20)..(20)n is inosinemisc_feature(24)..(24)n
is inosinemisc_feature(28)..(28)n is inosine 23ggaucnaucn
aucnuucgcn aucnaucnau cc 322432RNAArtificial SequenceSynthetic RIG
28 Nucleic acid sequencemisc_feature(3)..(3)n is
inosinemisc_feature(7)..(7)n is inosinemisc_feature(11)..(11)n is
inosinemisc_feature(21)..(21)n is inosinemisc_feature(25)..(25)n is
inosinemisc_feature(29)..(29)n is inosine 24ggnucgnucg nucguucgcg
nucgnucgnu cc 322532RNAArtificial SequenceSynthetic RIG 35 Nucleic
acid sequencemisc_feature(1)..(1)n is inosine 25ngaucgaucg
aucguucgcg aucgaucgau cc 322628RNAArtificial SequenceSynthetic RIG
36 Nucleic acid sequence 26aucgaucgau cguucgcgau cgaucgau
282734RNAArtificial SequenceSynthetic RIG 37 Nucleic acid sequence
27ggaucgaucg aucguuugau cgaucgaucg aucc 342833RNAArtificial
SequenceSynthetic RIG 38 Nucleic acid sequence 28ggaucgaucg
aucguguuuc gaucgaucga ucc 332932RNAArtificial SequenceSynthetic RIG
39 Nucleic acid sequence 29ggaucgaucg aucggauccg aucgaucgau cc
323040RNAArtificial SequenceSynthetic RIG 40 Nucleic acid sequence
30ggcaugcgac cucuguuuga ucaaacagag gucgcaugcc 403132RNAArtificial
SequenceSynthetic RIG 41 Nucleic acid sequence 31ggcaugcgac
cucugaucag aggucgcaug cc 323240RNAArtificial SequenceSynthetic RIG
42 Nucleic acid sequence 32ggcaugcgac cucuguuuuu cgaaacagag
gucgcaugcc 403332DNAArtificial SequenceSynthetic RIG 47 Nucleic
acid sequence 33tgcucgaucg aucguucgcg aucgaucgag ca
323432DNAArtificial SequenceSynthetic RIG 48 Nucleic acid sequence
34tcgucgaucg aucguucgcg aucgaucgac ga 323532DNAArtificial
SequenceSynthetic RIG 49 Nucleic acid sequence 35ggaucgaucg
aucguucgtg aucgaucgau gg 323632RNAArtificial SequenceSynthetic RIG
50 Nucleic acid sequence (14L) 36ggaucgaucg aucguucgcg aucgaucgau
cc 323714DNAArtificial SequenceSynthetic FO1 Nucleic acid sequence
37ggatcgatcg atcg 143814RNAArtificial SequenceSynthetic FO2 Nucleic
acid sequence 38ggaucgaucg auau 143914RNAArtificial
SequenceSynthetic FO3 Nucleic acid sequence 39gcgcgcgcgc gcgc
144014RNAArtificial SequenceSynthetic FO4 Nucleic acid sequence
40ggcggcgcgc cgcc 144114RNAArtificial SequenceSynthetic FO5 Nucleic
acid sequence 41ggcggcggcg gcgg 144214RNAArtificial
SequenceSynthetic FO6 Nucleic acid sequence 42ggcggccgcc cgcg
144314RNAArtificial SequenceSynthetic FO7 Nucleic acid sequence
43cgacgucgac gucg 144414RNAArtificial SequenceSynthetic FO8 Nucleic
acid sequence 44gcacgucgac gugc 144514RNAArtificial
SequenceSynthetic FO9 Nucleic acid sequence 45ggacgucgac gucc
144614RNAArtificial SequenceSynthetic FO10 Nucleic acid sequence
46ggucgcgacc auau 144714RNAArtificial SequenceSynthetic FO11
Nucleic acid sequence 47ggauacgucg acgu 144814RNAArtificial
SequenceSynthetic FO12 Nucleic acid sequence 48gagagagaga gaga
144914RNAArtificial SequenceSynthetic FO13 Nucleic acid sequence
49gagucuagac uccg 145014RNAArtificial SequenceSynthetic FO14
Nucleic acid sequence 50cgaucgaucg aucg 145114RNAArtificial
SequenceSynthetic FO15 Nucleic acid sequence 51ccaucgaucg aucg
145214RNAArtificial SequenceSynthetic FO16 Nucleic acid sequence
52gaaucgaucg aucg 145311RNAArtificial SequenceSynthetic FO17
Nucleic acid sequence 53gggaucgauc g 115414RNAArtificial
SequenceSynthetic FO18 Nucleic acid sequence 54cccccgaucg aucg
145514DNAArtificial SequenceSynthetic FO19 Nucleic acid sequence
55gtgtgtgtgt gtgt 145614DNAArtificial SequenceSynthetic FO20
Nucleic acid sequence 56gtgtgtggau cgau 145714RNAArtificial
SequenceSynthetic FO21 Nucleic acid sequencemisc_feature(4)..(4)n
is inosinemisc_feature(8)..(8)n is inosinemisc_feature(12)..(12)n
is inosine 57ggancgancg ancg 145814RNAArtificial SequenceSynthetic
FO22 Nucleic acid sequencemisc_feature(1)..(2)n is
inosinemisc_feature(6)..(6)n is inosinemisc_feature(10)..(10)n is
inosinemisc_feature(14)..(14)n is inosine 58nnaucnaucn aucn
145914RNAArtificial SequenceSynthetic FO23 Nucleic acid
sequencemisc_feature(6)..(6)n is inosinemisc_feature(10)..(10)n is
inosinemisc_feature(14)..(14)n is inosine 59ggaucnaucn aucn
146014RNAArtificial SequenceSynthetic FO24 Nucleic acid
sequencemisc_feature(3)..(3)n is inosinemisc_feature(7)..(7)n is
inosinemisc_feature(11)..(11)n is inosine 60ggnucgnucg nucg
146114RNAArtificial SequenceSynthetic FO25 Nucleic acid
sequencemisc_feature(1)..(1)n is inosine 61ngaucgaucg aucg
146212RNAArtificial SequenceSynthetic FO26 Nucleic acid sequence
62aucgaucgau cg 126314RNAArtificial SequenceSynthetic FO27 Nucleic
acid sequence 63ggaucgaucg aucg 146418RNAArtificial
SequenceSynthetic FO28 Nucleic acid sequence 64ggcaugcgac cucuguuu
186514RNAArtificial SequenceSynthetic FO29 Nucleic acid sequence
65ggcaugcgac cucu 146614DNAArtificial SequenceSynthetic FO30
Nucleic acid sequence 66tgcucgaucg aucg 146714DNAArtificial
SequenceSynthetic FO31 Nucleic acid sequence 67tcgucgaucg aucg
146814DNAArtificial SequenceSynthetic SO1 Nucleic acid sequence
68cgatcgatcg atcc 146914RNAArtificial SequenceSynthetic SO2 Nucleic
acid sequence 69auaucgaucg aucc 147014RNAArtificial
SequenceSynthetic SO3 Nucleic acid sequence 70gcgcgcgcgc gcgc
147114RNAArtificial SequenceSynthetic SO4 Nucleic acid sequence
71ggcggcgcgc cgcc 147214RNAArtificial SequenceSynthetic SO5 Nucleic
acid sequence 72ccgccgccgc cgcc 147314RNAArtificial
SequenceSynthetic SO6 Nucleic acid sequence 73cgcgggcggc cgcc
147414RNAArtificial SequenceSynthetic SO7 Nucleic acid sequence
74cgacgucgac gucg 147514RNAArtificial SequenceSynthetic SO8 Nucleic
acid sequence 75gcacgucgac gugc 147614RNAArtificial
SequenceSynthetic SO9 Nucleic acid sequence 76ggacgucgac gucc
147714RNAArtificial SequenceSynthetic SO10 Nucleic acid sequence
77auauggucgc gacc 147814RNAArtificial SequenceSynthetic SO11
Nucleic acid sequence 78acgucgacgu aucc 147914RNAArtificial
SequenceSynthetic SO12 Nucleic acid sequence 79ucucucucuc ucuc
148014RNAArtificial SequenceSynthetic SO13 Nucleic acid sequence
80cggagucuag acuc 148114RNAArtificial SequenceSynthetic SO14
Nucleic acid sequence 81cgaucgaucg aucg 148214RNAArtificial
SequenceSynthetic SO15 Nucleic acid sequence 82cgaucgaucg augg
148314RNAArtificial SequenceSynthetic SO16 Nucleic acid sequence
83cgaucgaucg auuc 148411RNAArtificial SequenceSynthetic SO17
Nucleic acid sequence 84cgaucgaucc c 118514RNAArtificial
SequenceSynthetic SO18 Nucleic acid sequence 85cgaucgaucg gggg
148614RNAArtificial SequenceSynthetic SO19 Nucleic acid sequence
86acacacacac acac 148714RNAArtificial SequenceSynthetic SO20
Nucleic acid sequence 87aucgauccac acac 148814RNAArtificial
SequenceSynthetic SO21 Nucleic acid sequencemisc_feature(4)..(4)n
is inosinemisc_feature(8)..(8)n is inosinemisc_feature(12)..(12)n
is inosine 88cgancgancg ancc 148914RNAArtificial SequenceSynthetic
SO22 Nucleic acid sequencemisc_feature(2)..(2)n is
inosinemisc_feature(6)..(6)n is inosinemisc_feature(10)..(10)n is
inosine 89cnaucnaucn aucc 149014RNAArtificial SequenceSynthetic
SO23 Nucleic acid sequencemisc_feature(3)..(3)n is
inosinemisc_feature(7)..(7)n is inosinemisc_feature(11)..(11)n is
inosine 90cgnucgnucg nucc 149114RNAArtificial SequenceSynthetic
SO24 Nucleic acid sequence 91cgaucgaucg aucc 149212RNAArtificial
SequenceSynthetic SO25 Nucleic acid sequence 92cgaucgaucg au
129318RNAArtificial SequenceSynthetic SO26 Nucleic acid sequence
93aaacagaggu cgcaugcc 189414RNAArtificial SequenceSynthetic SO27
Nucleic acid sequence 94agaggucgca ugcc 149514RNAArtificial
SequenceSynthetic SO28 Nucleic acid sequence 95cgaucgaucg agca
149614RNAArtificial SequenceSynthetic SO29 Nucleic acid sequence
96cgaucgaucg acga 149714DNAArtificial SequenceSynthetic SO30
Nucleic acid sequence 97tgaucgaucg augg 1498925PRTHomo
sapiensmisc_featureHuman RIG-I Amino acid sequence 98Met Thr Thr
Glu Gln Arg Arg Ser Leu Gln Ala Phe Gln Asp Tyr Ile1 5 10 15Arg Lys
Thr Leu Asp Pro Thr Tyr Ile Leu Ser Tyr Met Ala Pro Trp 20 25 30Phe
Arg Glu Glu Glu Val Gln Tyr Ile Gln Ala Glu Lys Asn Asn Lys 35 40
45Gly Pro Met Glu Ala Ala Thr Leu Phe Leu Lys Phe Leu Leu Glu Leu
50 55 60Gln Glu Glu Gly Trp Phe Arg Gly Phe Leu Asp Ala Leu Asp His
Ala65 70 75 80Gly Tyr Ser Gly Leu Tyr Glu Ala Ile Glu Ser Trp Asp
Phe Lys Lys 85 90 95Ile Glu Lys Leu Glu Glu Tyr Arg Leu Leu Leu Lys
Arg Leu Gln Pro 100 105 110Glu Phe Lys Thr Arg Ile Ile Pro Thr Asp
Ile Ile Ser Asp Leu Ser 115 120 125Glu Cys Leu Ile Asn Gln Glu Cys
Glu Glu Ile Leu Gln Ile Cys Ser 130 135 140Thr Lys Gly Met Met Ala
Gly Ala Glu Lys Leu Val Glu Cys Leu Leu145 150 155 160Arg Ser Asp
Lys Glu Asn Trp Pro Lys Thr Leu Lys Leu Ala Leu Glu 165 170 175Lys
Glu Arg Asn Lys Phe Ser Glu Leu Trp Ile Val Glu Lys Gly Ile 180 185
190Lys Asp Val Glu Thr Glu Asp Leu Glu Asp Lys Met Glu Thr Ser Asp
195 200 205Ile Gln Ile Phe Tyr Gln Glu Asp Pro Glu Cys Gln Asn Leu
Ser Glu 210 215 220Asn Ser Cys Pro Pro Ser Glu Val Ser Asp Thr Asn
Leu Tyr Ser Pro225 230 235 240Phe Lys Pro Arg Asn Tyr Gln Leu Glu
Leu Ala Leu Pro Ala Met Lys 245 250 255Gly Lys Asn Thr Ile Ile Cys
Ala Pro Thr Gly Cys Gly Lys Thr Phe 260 265 270Val Ser Leu Leu Ile
Cys Glu His His Leu Lys Lys Phe Pro Gln Gly 275 280 285Gln Lys Gly
Lys Val Val Phe Phe Ala Asn Gln Ile Pro Val Tyr Glu 290 295 300Gln
Gln Lys Ser Val Phe Ser Lys Tyr Phe Glu
Arg His Gly Tyr Arg305 310 315 320Val Thr Gly Ile Ser Gly Ala Thr
Ala Glu Asn Val Pro Val Glu Gln 325 330 335Ile Val Glu Asn Asn Asp
Ile Ile Ile Leu Thr Pro Gln Ile Leu Val 340 345 350Asn Asn Leu Lys
Lys Gly Thr Ile Pro Ser Leu Ser Ile Phe Thr Leu 355 360 365Met Ile
Phe Asp Glu Cys His Asn Thr Ser Lys Gln His Pro Tyr Asn 370 375
380Met Ile Met Phe Asn Tyr Leu Asp Gln Lys Leu Gly Gly Ser Ser
Gly385 390 395 400Pro Leu Pro Gln Val Ile Gly Leu Thr Ala Ser Val
Gly Val Gly Asp 405 410 415Ala Lys Asn Thr Asp Glu Ala Leu Asp Tyr
Ile Cys Lys Leu Cys Ala 420 425 430Ser Leu Asp Ala Ser Val Ile Ala
Thr Val Lys His Asn Leu Glu Glu 435 440 445Leu Glu Gln Val Val Tyr
Lys Pro Gln Lys Phe Phe Arg Lys Val Glu 450 455 460Ser Arg Ile Ser
Asp Lys Phe Lys Tyr Ile Ile Ala Gln Leu Met Arg465 470 475 480Asp
Thr Glu Ser Leu Ala Lys Arg Ile Cys Lys Asp Leu Glu Asn Leu 485 490
495Ser Gln Ile Gln Asn Arg Glu Phe Gly Thr Gln Lys Tyr Glu Gln Trp
500 505 510Ile Val Thr Val Gln Lys Ala Cys Met Val Phe Gln Met Pro
Asp Lys 515 520 525Asp Glu Glu Ser Arg Ile Cys Lys Ala Leu Phe Leu
Tyr Thr Ser His 530 535 540Leu Arg Lys Tyr Asn Asp Ala Leu Ile Ile
Ser Glu His Ala Arg Met545 550 555 560Lys Asp Ala Leu Asp Tyr Leu
Lys Asp Phe Phe Ser Asn Val Arg Ala 565 570 575Ala Gly Phe Asp Glu
Ile Glu Gln Asp Leu Thr Gln Arg Phe Glu Glu 580 585 590Lys Leu Gln
Glu Leu Glu Ser Val Ser Arg Asp Pro Ser Asn Glu Asn 595 600 605Pro
Lys Leu Glu Asp Leu Cys Phe Ile Leu Gln Glu Glu Tyr His Leu 610 615
620Asn Pro Glu Thr Ile Thr Ile Leu Phe Val Lys Thr Arg Ala Leu
Val625 630 635 640Asp Ala Leu Lys Asn Trp Ile Glu Gly Asn Pro Lys
Leu Ser Phe Leu 645 650 655Lys Pro Gly Ile Leu Thr Gly Arg Gly Lys
Thr Asn Gln Asn Thr Gly 660 665 670Met Thr Leu Pro Ala Gln Lys Cys
Ile Leu Asp Ala Phe Lys Ala Ser 675 680 685Gly Asp His Asn Ile Leu
Ile Ala Thr Ser Val Ala Asp Glu Gly Ile 690 695 700Asp Ile Ala Gln
Cys Asn Leu Val Ile Leu Tyr Glu Tyr Val Gly Asn705 710 715 720Val
Ile Lys Met Ile Gln Thr Arg Gly Arg Gly Arg Ala Arg Gly Ser 725 730
735Lys Cys Phe Leu Leu Thr Ser Asn Ala Gly Val Ile Glu Lys Glu Gln
740 745 750Ile Asn Met Tyr Lys Glu Lys Met Met Asn Asp Ser Ile Leu
Arg Leu 755 760 765Gln Thr Trp Asp Glu Ala Val Phe Arg Glu Lys Ile
Leu His Ile Gln 770 775 780Thr His Glu Lys Phe Ile Arg Asp Ser Gln
Glu Lys Pro Lys Pro Val785 790 795 800Pro Asp Lys Glu Asn Lys Lys
Leu Leu Cys Arg Lys Cys Lys Ala Leu 805 810 815Ala Cys Tyr Thr Ala
Asp Val Arg Val Ile Glu Glu Cys His Tyr Thr 820 825 830Val Leu Gly
Asp Ala Phe Lys Glu Cys Phe Val Ser Arg Pro His Pro 835 840 845Lys
Pro Lys Gln Phe Ser Ser Phe Glu Lys Arg Ala Lys Ile Phe Cys 850 855
860Ala Arg Gln Asn Cys Ser His Asp Trp Gly Ile His Val Lys Tyr
Lys865 870 875 880Thr Phe Glu Ile Pro Val Ile Lys Ile Glu Ser Phe
Val Val Glu Asp 885 890 895Ile Ala Thr Gly Val Gln Thr Leu Tyr Ser
Lys Trp Lys Asp Phe His 900 905 910Phe Glu Lys Ile Pro Phe Asp Pro
Ala Glu Met Ser Lys 915 920 925991025PRTHomo
sapiensmisc_featureHuman MDA5 Amino acid sequence 99Met Ser Asn Gly
Tyr Ser Thr Asp Glu Asn Phe Arg Tyr Leu Ile Ser1 5 10 15Cys Phe Arg
Ala Arg Val Lys Met Tyr Ile Gln Val Glu Pro Val Leu 20 25 30Asp Tyr
Leu Thr Phe Leu Pro Ala Glu Val Lys Glu Gln Ile Gln Arg 35 40 45Thr
Val Ala Thr Ser Gly Asn Met Gln Ala Val Glu Leu Leu Leu Ser 50 55
60Thr Leu Glu Lys Gly Val Trp His Leu Gly Trp Thr Arg Glu Phe Val65
70 75 80Glu Ala Leu Arg Arg Thr Gly Ser Pro Leu Ala Ala Arg Tyr Met
Asn 85 90 95Pro Glu Leu Thr Asp Leu Pro Ser Pro Ser Phe Glu Asn Ala
His Asp 100 105 110Glu Tyr Leu Gln Leu Leu Asn Leu Leu Gln Pro Thr
Leu Val Asp Lys 115 120 125Leu Leu Val Arg Asp Val Leu Asp Lys Cys
Met Glu Glu Glu Leu Leu 130 135 140Thr Ile Glu Asp Arg Asn Arg Ile
Ala Ala Ala Glu Asn Asn Gly Asn145 150 155 160Glu Ser Gly Val Arg
Glu Leu Leu Lys Arg Ile Val Gln Lys Glu Asn 165 170 175Trp Phe Ser
Ala Phe Leu Asn Val Leu Arg Gln Thr Gly Asn Asn Glu 180 185 190Leu
Val Gln Glu Leu Thr Gly Ser Asp Cys Ser Glu Ser Asn Ala Glu 195 200
205Ile Glu Asn Leu Ser Gln Val Asp Gly Pro Gln Val Glu Glu Gln Leu
210 215 220Leu Ser Thr Thr Val Gln Pro Asn Leu Glu Lys Glu Val Trp
Gly Met225 230 235 240Glu Asn Asn Ser Ser Glu Ser Ser Phe Ala Asp
Ser Ser Val Val Ser 245 250 255Glu Ser Asp Thr Ser Leu Ala Glu Gly
Ser Val Ser Cys Leu Asp Glu 260 265 270Ser Leu Gly His Asn Ser Asn
Met Gly Ser Asp Ser Gly Thr Met Gly 275 280 285Ser Asp Ser Asp Glu
Glu Asn Val Ala Ala Arg Ala Ser Pro Glu Pro 290 295 300Glu Leu Gln
Leu Arg Pro Tyr Gln Met Glu Val Ala Gln Pro Ala Leu305 310 315
320Glu Gly Lys Asn Ile Ile Ile Cys Leu Pro Thr Gly Ser Gly Lys Thr
325 330 335Arg Val Ala Val Tyr Ile Ala Lys Asp His Leu Asp Lys Lys
Lys Lys 340 345 350Ala Ser Glu Pro Gly Lys Val Ile Val Leu Val Asn
Lys Val Leu Leu 355 360 365Val Glu Gln Leu Phe Arg Lys Glu Phe Gln
Pro Phe Leu Lys Lys Trp 370 375 380Tyr Arg Val Ile Gly Leu Ser Gly
Asp Thr Gln Leu Lys Ile Ser Phe385 390 395 400Pro Glu Val Val Lys
Ser Cys Asp Ile Ile Ile Ser Thr Ala Gln Ile 405 410 415Leu Glu Asn
Ser Leu Leu Asn Leu Glu Asn Gly Glu Asp Ala Gly Val 420 425 430Gln
Leu Ser Asp Phe Ser Leu Ile Ile Ile Asp Glu Cys His His Thr 435 440
445Asn Lys Glu Ala Val Tyr Asn Asn Ile Met Arg His Tyr Leu Met Gln
450 455 460Lys Leu Lys Asn Asn Arg Leu Lys Lys Glu Asn Lys Pro Val
Ile Pro465 470 475 480Leu Pro Gln Ile Leu Gly Leu Thr Ala Ser Pro
Gly Val Gly Gly Ala 485 490 495Thr Lys Gln Ala Lys Ala Glu Glu His
Ile Leu Lys Leu Cys Ala Asn 500 505 510Leu Asp Ala Phe Thr Ile Lys
Thr Val Lys Glu Asn Leu Asp Gln Leu 515 520 525Lys Asn Gln Ile Gln
Glu Pro Cys Lys Lys Phe Ala Ile Ala Asp Ala 530 535 540Thr Arg Glu
Asp Pro Phe Lys Glu Lys Leu Leu Glu Ile Met Thr Arg545 550 555
560Ile Gln Thr Tyr Cys Gln Met Ser Pro Met Ser Asp Phe Gly Thr Gln
565 570 575Pro Tyr Glu Gln Trp Ala Ile Gln Met Glu Lys Lys Ala Ala
Lys Glu 580 585 590Gly Asn Arg Lys Glu Arg Val Cys Ala Glu His Leu
Arg Lys Tyr Asn 595 600 605Glu Ala Leu Gln Ile Asn Asp Thr Ile Arg
Met Ile Asp Ala Tyr Thr 610 615 620His Leu Glu Thr Phe Tyr Asn Glu
Glu Lys Asp Lys Lys Phe Ala Val625 630 635 640Ile Glu Asp Asp Ser
Asp Glu Gly Gly Asp Asp Glu Tyr Cys Asp Gly 645 650 655Asp Glu Asp
Glu Asp Asp Leu Lys Lys Pro Leu Lys Leu Asp Glu Thr 660 665 670Asp
Arg Phe Leu Met Thr Leu Phe Phe Glu Asn Asn Lys Met Leu Lys 675 680
685Arg Leu Ala Glu Asn Pro Glu Tyr Glu Asn Glu Lys Leu Thr Lys Leu
690 695 700Arg Asn Thr Ile Met Glu Gln Tyr Thr Arg Thr Glu Glu Ser
Ala Arg705 710 715 720Gly Ile Ile Phe Thr Lys Thr Arg Gln Ser Ala
Tyr Ala Leu Ser Gln 725 730 735Trp Ile Thr Glu Asn Glu Lys Phe Ala
Glu Val Gly Val Lys Ala His 740 745 750His Leu Ile Gly Ala Gly His
Ser Ser Glu Phe Lys Pro Met Thr Gln 755 760 765Asn Glu Gln Lys Glu
Val Ile Ser Lys Phe Arg Thr Gly Lys Ile Asn 770 775 780Leu Leu Ile
Ala Thr Thr Val Ala Glu Glu Gly Leu Asp Ile Lys Glu785 790 795
800Cys Asn Ile Val Ile Arg Tyr Gly Leu Val Thr Asn Glu Ile Ala Met
805 810 815Val Gln Ala Arg Gly Arg Ala Arg Ala Asp Glu Ser Thr Tyr
Val Leu 820 825 830Val Ala His Ser Gly Ser Gly Val Ile Glu His Glu
Thr Val Asn Asp 835 840 845Phe Arg Glu Lys Met Met Tyr Lys Ala Ile
His Cys Val Gln Asn Met 850 855 860Lys Pro Glu Glu Tyr Ala His Lys
Ile Leu Glu Leu Gln Met Gln Ser865 870 875 880Ile Met Glu Lys Lys
Met Lys Thr Lys Arg Asn Ile Ala Lys His Tyr 885 890 895Lys Asn Asn
Pro Ser Leu Ile Thr Phe Leu Cys Lys Asn Cys Ser Val 900 905 910Leu
Ala Cys Ser Gly Glu Asp Ile His Val Ile Glu Lys Met His His 915 920
925Val Asn Met Thr Pro Glu Phe Lys Glu Leu Tyr Ile Val Arg Glu Asn
930 935 940Lys Ala Leu Gln Lys Lys Cys Ala Asp Tyr Gln Ile Asn Gly
Glu Ile945 950 955 960Ile Cys Lys Cys Gly Gln Ala Trp Gly Thr Met
Met Val His Lys Gly 965 970 975Leu Asp Leu Pro Cys Leu Lys Ile Arg
Asn Phe Val Val Val Phe Lys 980 985 990Asn Asn Ser Thr Lys Lys Gln
Tyr Lys Lys Trp Val Glu Leu Pro Ile 995 1000 1005Thr Phe Pro Asn
Leu Asp Tyr Ser Glu Cys Cys Leu Phe Ser Asp 1010 1015 1020Glu Asp
1025100678PRTHomo sapiensmisc_featureHuman LGP2 Amino acid sequence
100Met Glu Leu Arg Ser Tyr Gln Trp Glu Val Ile Met Pro Ala Leu Glu1
5 10 15Gly Lys Asn Ile Ile Ile Trp Leu Pro Thr Gly Ala Gly Lys Thr
Arg 20 25 30Ala Ala Ala Tyr Val Ala Lys Arg His Leu Glu Thr Val Asp
Gly Ala 35 40 45Lys Val Val Val Leu Val Asn Arg Val His Leu Val Thr
Gln His Gly 50 55 60Glu Glu Phe Arg Arg Met Leu Asp Gly Arg Trp Thr
Val Thr Thr Leu65 70 75 80Ser Gly Asp Met Gly Pro Arg Ala Gly Phe
Gly His Leu Ala Arg Cys 85 90 95His Asp Leu Leu Ile Cys Thr Ala Glu
Leu Leu Gln Met Ala Leu Thr 100 105 110Ser Pro Glu Glu Glu Glu His
Val Glu Leu Thr Val Phe Ser Leu Ile 115 120 125Val Val Asp Glu Cys
His His Thr His Lys Asp Thr Val Tyr Asn Val 130 135 140Ile Met Ser
Gln Tyr Leu Glu Leu Lys Leu Gln Arg Ala Gln Pro Leu145 150 155
160Pro Gln Val Leu Gly Leu Thr Ala Ser Pro Gly Thr Gly Gly Ala Ser
165 170 175Lys Leu Asp Gly Ala Ile Asn His Val Leu Gln Leu Cys Ala
Asn Leu 180 185 190Asp Thr Trp Cys Ile Met Ser Pro Gln Asn Cys Cys
Pro Gln Leu Gln 195 200 205Glu His Ser Gln Gln Pro Cys Lys Gln Tyr
Asn Leu Cys His Arg Arg 210 215 220Ser Gln Asp Pro Phe Gly Asp Leu
Leu Lys Lys Leu Met Asp Gln Ile225 230 235 240His Asp His Leu Glu
Met Pro Glu Leu Ser Arg Lys Phe Gly Thr Gln 245 250 255Met Tyr Glu
Gln Gln Val Val Lys Leu Ser Glu Ala Ala Ala Leu Ala 260 265 270Gly
Leu Gln Glu Gln Arg Val Tyr Ala Leu His Leu Arg Arg Tyr Asn 275 280
285Asp Ala Leu Leu Ile His Asp Thr Val Arg Ala Val Asp Ala Leu Ala
290 295 300Ala Leu Gln Asp Phe Tyr His Arg Glu His Val Thr Lys Thr
Gln Ile305 310 315 320Leu Cys Ala Glu Arg Arg Leu Leu Ala Leu Phe
Asp Asp Arg Lys Asn 325 330 335Glu Leu Ala His Leu Ala Thr His Gly
Pro Glu Asn Pro Lys Leu Glu 340 345 350Met Leu Glu Lys Ile Leu Gln
Arg Gln Phe Ser Ser Ser Asn Ser Pro 355 360 365Arg Gly Ile Ile Phe
Thr Arg Thr Arg Gln Ser Ala His Ser Leu Leu 370 375 380Leu Trp Leu
Gln Gln Gln Gln Gly Leu Gln Thr Val Asp Ile Arg Ala385 390 395
400Gln Leu Leu Ile Gly Ala Gly Asn Ser Ser Gln Ser Thr His Met Thr
405 410 415Gln Arg Asp Gln Gln Glu Val Ile Gln Lys Phe Gln Asp Gly
Thr Leu 420 425 430Asn Leu Leu Val Ala Thr Ser Val Ala Glu Glu Gly
Leu Asp Ile Pro 435 440 445His Cys Asn Val Val Val Arg Tyr Gly Leu
Leu Thr Asn Glu Ile Ser 450 455 460Met Val Gln Ala Arg Gly Arg Ala
Arg Ala Asp Gln Ser Val Tyr Ala465 470 475 480Phe Val Ala Thr Glu
Gly Ser Arg Glu Leu Lys Arg Glu Leu Ile Asn 485 490 495Glu Ala Leu
Glu Thr Leu Met Glu Gln Ala Val Ala Ala Val Gln Lys 500 505 510Met
Asp Gln Ala Glu Tyr Gln Ala Lys Ile Arg Asp Leu Gln Gln Ala 515 520
525Ala Leu Thr Lys Arg Ala Ala Gln Ala Ala Gln Arg Glu Asn Gln Arg
530 535 540Gln Gln Phe Pro Val Glu His Val Gln Leu Leu Cys Ile Asn
Cys Met545 550 555 560Val Ala Val Gly His Gly Ser Asp Leu Arg Lys
Val Glu Gly Thr His 565 570 575His Val Asn Val Asn Pro Asn Phe Ser
Asn Tyr Tyr Asn Val Ser Arg 580 585 590Asp Pro Val Val Ile Asn Lys
Val Phe Lys Asp Trp Lys Pro Gly Gly 595 600 605Val Ile Ser Cys Arg
Asn Cys Gly Glu Val Trp Gly Leu Gln Met Ile 610 615 620Tyr Lys Ser
Val Lys Leu Pro Val Leu Lys Val Arg Ser Met Leu Leu625 630 635
640Glu Thr Pro Gln Gly Arg Ile Gln Ala Lys Lys Trp Ser Arg Val Pro
645 650 655Phe Ser Val Pro Asp Phe Asp Phe Leu Gln His Cys Ala Glu
Asn Leu 660 665 670Ser Asp Leu Ser Leu Asp 675
* * * * *
References