Synthetic RIG-I-like receptor agonists
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, including RLR agonists packaged in vims like particles (VLPs), in methods for treating, or ameliorating one or more symptoms of, a disorder (e.g., cancer).
1 . A composition comprising:
(a) a virus-like particle; and
(b) at least one synthetic RIG-I like receptor (RLR) agonist that specifically binds to a RLR,
wherein the RLR 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 polynucleotide comprises a 5′ diphosphate or triphosphate moiety, or derivative or analog thereof,
wherein the RLR agonist comprises a [AUCG]n repeat motif, wherein n=3, and wherein:
(a) the 5′ most AUCG repeat motif is preceded by CG;
(b) the 5′ most AUCG repeat motif is preceded by IG; or
(c) the 5′ most AUCG repeat is preceded by GG and wherein each G in the AUCG motif is substituted by inosine; and
wherein the at least one RLR agonist is packaged in the virus-like particle.
2 . The composition of claim 1 , wherein the linker is a nucleotide linker or a non-nucleotide linker.
3 . The composition of claim 2 , wherein the nucleotide linker comprises a tetraloop comprising the sequence UUCG.
4 . The composition of claim 2 , wherein the non-nucleotide linker is a hexaethylene glycol linker or a C9 alkyl linker.
5 . The composition of claim 1 , wherein the virus-like particle comprises recombinant proteins of an RNA-phage, wherein said RNA-phage is selected from the group consisting of: (a) bacteriophage Qβ; (b) bacteriophage R17; (c) bacteriophage fr; (d) bacteriophage GA; (e) bacteriophage SP; (f) bacteriophage MS2; (g) bacteriophage M11; (h) bacteriophage MX1; (i) bacteriophage NL95; (j) bacteriophage f2; (k) bacteriophage PP7; and (1) bacteriophage AP205.
6 . The composition of claim 5 , wherein the virus-like particle comprises recombinant proteins, or fragments thereof, of RNA-phage Qβ.
7 . The composition of claim 1 , wherein the virus-like particle comprises RNA-phage QB coat proteins each having an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 112.
8 . The composition of claim 1 wherein the RLR agonist is non-covalently bound to the virus-like particle.
9 . The composition of claim 1 , further comprising at least one antigen or antigenic determinant bound to the virus-like particle.
10 . A pharmaceutical composition, comprising the composition of claim 1 and a pharmaceutically acceptable carrier.
11 . A kit comprising the composition of claim 1 and instructions for use.
12 . A method of stimulating an immune response, treating or delaying progression of a cancer, or inhibiting tumor growth in a subject in need thereof, comprising administering to the subject the composition of claim 1 .
13 . The method of claim 12 , wherein the 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, 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, and a combination thereof.
14 . The method of claim 13 , wherein the opsonizing agent is an opsonizing antibody.
15 . The method of claim 13 , wherein the 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 composition.
16 . The method of claim 13 , wherein the one or more additional therapeutic agents is a PD-1/PD-L1 antagonist, a TIM-3 antagonist, a VISTA antagonist, an adenosine A2AR antagonist, a B7-H3 antagonist, a B7-H4 antagonist, a BTLA antagonist, a CTLA-4 antagonist, an IDO antagonist, a KIR antagonist, a LAG-3 antagonist, a Toll-like receptor 3 (TLR3) agonist, a Toll-like receptor 7 (TLR7) agonist, a Toll-like receptor 9 (TLR9) agonist, or an agonist comprising a polypeptide (e.g, antibody, or antigen binding portion thereof) that specifically binds to CD137 (4-1BB) or CD134 (OX40).
17 . A method of producing the composition of claim 1 , comprising:
(a) disassembling the virus-like particle, optionally removing nucleic acids of the disassembled virus-like particle;
(b) adding the RLR agonist; and
(c) reassembling the virus-like particle.
18 . The method of claim 17 , further comprising purifying the composition after reassembly.
19 . The method of claim 17 , further comprising (d) binding an antigen or antigenic determinant to the virus-like particle.
20 . The composition of claim 1 , wherein the first polynucleotide comprises SEQ ID NO: 50 and the second polynucleotide comprises SEQ ID NO: 81.
21 . The composition of claim 1 , wherein the first polynucleotide comprises SEQ ID NO: 61 and the second polynucleotide comprises SEQ ID NO: 91.
22 . The composition of claim 1 , wherein the first polynucleotide comprises SEQ ID NO: 59 and the second polynucleotide comprises SEQ ID NO: 89.