Compositions containing polynucleotides and polypeptide amphiphiles and methods of use thereof

Abstract

Disclosed herein are compounds comprising an immunomodulatory (e.g., immunostimulatory or immunosuppressive) nucleic acid sequence, a polypeptide, and an albumin binding domain, and pharmaceutically acceptable salts thereof. In addition, disclosed herein are methods for inducing an immune response in a subject, as well as methods of administering such compounds to induce an immune response in a subject.

Description

Background Technology

[0001] Vaccines are used to stimulate an individual's immune response to provide protection and / or treatment against a specific disease. Some vaccines contain antigens to induce an immune response. The immune response generated by vaccination makes a significant contribution to human and animal health. Since the invention of the first vaccine in 1796, vaccines have been considered the most successful method of preventing a wide range of infectious diseases by eliciting an immune response in subjects. According to the World Health Organization, immunization currently prevents 2-3 million deaths annually across all age groups. The goal of vaccination is to generate a strong and long-lasting immune response, thereby providing long-term protection against infection. However, many vaccines currently fail to induce optimal immunity.

[0002] There is still a need to develop new and improved compositions and methods for inducing immune responses in their subjects. Summary of the Invention

[0003] This disclosure provides compounds, pharmaceutically acceptable salts thereof, pharmaceutical compositions, and kits comprising the compounds, said compounds comprising an immunomodulatory nucleic acid sequence, an albumin-binding domain, and a polypeptide. This disclosure further provides a method for inducing an immune response in a subject by administering the compounds described herein and their salts.

[0004] In one aspect, this disclosure provides a compound or a pharmaceutically acceptable salt thereof, the compound comprising an immunostimulatory nucleic acid sequence, an albumin-binding domain, and a polypeptide. In some embodiments, the albumin-binding domain is bonded or linked to the 5' end of the immunostimulatory nucleic acid sequence, and wherein the polypeptide is bonded or linked to the 3' end of the immunostimulatory nucleic acid sequence. In one embodiment, the albumin-binding domain is bonded to the 5' end of the immunostimulatory nucleic acid sequence, and the polypeptide is bonded to the 3' end of the immunostimulatory nucleic acid sequence. In another embodiment, the albumin-binding domain is linked to the 5' end of the immunostimulatory nucleic acid sequence via a adapter, and the polypeptide is bonded to the 3' end of the immunostimulatory nucleic acid sequence. In yet another embodiment, the albumin-binding domain is bonded to the 5' end of the immunostimulatory nucleic acid sequence, and wherein the polypeptide is linked to the 3' end of the immunostimulatory nucleic acid sequence via a adapter. In yet another embodiment, the albumin-binding domain is linked to the 5' end of the immunostimulatory nucleic acid sequence via a adapter, and the polypeptide is linked to the 3' end of the immunostimulatory nucleic acid sequence via a adapter. In another aspect, this disclosure provides a compound comprising an immunostimulatory nucleic acid sequence, an albumin-binding domain, and a polypeptide, or a pharmaceutically acceptable salt thereof, for use in a method of inducing an immune response against an antigen in a subject, wherein the method comprises administering to the subject the compound comprising the immunostimulatory nucleic acid sequence, the albumin-binding domain, and the polypeptide, or a pharmaceutically acceptable salt thereof.

[0005] In one embodiment, the polypeptide is bonded or linked to the 5' end of an immunostimulatory nucleic acid sequence, and an albumin-binding domain is bonded or linked to the polypeptide. In another embodiment, the polypeptide is linked to the 5' end of an immunostimulatory nucleic acid sequence via a adapter, and an albumin-binding domain is bonded to the polypeptide. In yet another embodiment, the polypeptide is bonded to the 5' end of an immunostimulatory nucleic acid sequence, and an albumin-binding domain is linked to the polypeptide via a adapter. In yet another embodiment, the polypeptide is linked to the 5' end of an immunostimulatory nucleic acid sequence via a adapter, and an albumin-binding domain is linked to the polypeptide via a adapter.

[0006] In some embodiments, the polypeptide comprises an N-terminal modification. In some embodiments, the N-terminal modification is the addition of acetylcysteine. In some embodiments, the N-terminal modification is the addition of a deaminocysteine ​​homolog. In some embodiments, the deaminocysteine ​​homolog is 3-mercaptopropionic acid or mercaptoacetic acid.

[0007] In some embodiments, this disclosure provides a compound or a pharmaceutically acceptable salt thereof, said compound comprising an immunostimulatory nucleic acid sequence, an albumin-binding domain, a linker, and a reactive functional group. In some embodiments, the reactive functional group is a maleimide functional group.

[0008] In some embodiments, the immunostimulatory nucleic acid sequence is a polydeoxyadenosine (poly-dA) nucleic acid sequence. In some embodiments, the immunostimulatory nucleic acid sequence is a polydeoxythymidine (poly-dT) nucleic acid sequence. In some embodiments, the compound or a pharmaceutically acceptable salt thereof comprises both a poly-dA nucleic acid sequence and a poly-dT nucleic acid sequence. In some embodiments, the poly-dA nucleic acid sequence and the poly-dT nucleic acid sequence hybridize to form a double-stranded DNA sequence. In some embodiments, the poly-dA nucleic acid sequence and the poly-dT nucleic acid sequence comprise the same number of nucleotides.

[0009] In some embodiments, the immunostimulatory nucleic acid sequence is a polydeoxyguanosine (polydG) nucleic acid sequence. In some embodiments, the immunostimulatory nucleic acid sequence is a polydeoxycytosine (polydC) nucleic acid sequence. In some embodiments, the compound or a pharmaceutically acceptable salt thereof comprises both the polydG and polydC nucleic acid sequences. In some embodiments, the polydG and polydC nucleic acid sequences hybridize to form a double-stranded DNA sequence. In some embodiments, the polydG and polydC nucleic acid sequences comprise the same number of nucleotides.

[0010] In some embodiments, the poly-dA, poly-dT, poly-dC, and / or poly-dG nucleic acid sequences comprise between 30 and 100 nucleotides (e.g., between 30 and 90, 30 and 80, 30 and 70, 30 and 60, 30 and 50, 30 and 40, 40 and 100, 50 and 100, 60 and 100, 70 and 100, 80 and 100, or between 90 and 100 nucleotides). In some embodiments, the poly-dA, poly-dT, poly-dC, and / or poly-dG nucleic acid sequences comprise between 50 and 100 nucleotides (e.g., between 50 and 90, 50 and 80, 50 and 70, 50 and 60, 60 and 100, 70 and 100, 80 and 100, or between 90 and 100 nucleotides). In some embodiments, the poly(dA), poly(dT), poly(dC), and / or poly(dG) nucleic acid sequences comprise between 30 and 50 nucleotides (e.g., between 30 and 45, 30 and 40, 30 and 35, 35 and 50, 40 and 50, or 45 and 50 nucleotides). In some embodiments, the poly(dA), poly(dT), poly(dC), and / or poly(dG) nucleic acid sequences comprise 30, 40, 50, 75, or 100 nucleotides. In some embodiments, the poly(dA) and poly(dT) nucleic acid sequences comprise the same number of nucleotides. In some embodiments, the poly(dG) and poly(dC) nucleic acid sequences comprise the same number of nucleotides.

[0011] In some embodiments, the poly-dA nucleic acid sequence, poly-dT nucleic acid sequence, poly-dG nucleic acid sequence and / or poly-dC nucleic acid sequence comprises a mixture of dA, dT, dC and / or dG nucleic acid residues. In some embodiments, the poly-dA nucleic acid sequence comprises 100% to 51% (e.g., 100% to 60%, 100% to 70%, 100% to 80%, 100% to 90%, 100% to 95%, 95% to 51%, 90% to 51%, 80% to 51%, 70% to 51%, and 60% to 51%) of dA nucleic acid residues, and 0% to 49% (e.g., 0% to 45%, 0% to 40%, 0% to 30%, 0% to 20%, 0% to 10%, and 0% to 5%, 5% to 49%, 10% to 49%, 20% to 49%, 30% to 49%, and 40% to 49%) of dT, dC, and / or dG nucleic acid residues. In some embodiments, the poly-dT nucleic acid sequence comprises 100% to 51% (e.g., 100% to 60%, 100% to 70%, 100% to 80%, 100% to 90%, 100% to 95%, 95% to 51%, 90% to 51%, 80% to 51%, 70% to 51%, and 60% to 51%) of dT nucleic acid residues, and 0% to 49% (e.g., 0% to 45%, 0% to 40%, 0% to 30%, 0% to 20%, 0% to 10%, and 0% to 5%, 5% to 49%, 10% to 49%, 20% to 49%, 30% to 49%, and 40% to 49%) of dA, dC, and / or dG nucleic acid residues. In some embodiments, the polydG nucleic acid sequence comprises 100% to 51% (e.g., 100% to 60%, 100% to 70%, 100% to 80%, 100% to 90%, 100% to 95%, 95% to 51%, 90% to 51%, 80% to 51%, 70% to 51%, and 60% to 51%) of dG nucleic acid residues and 0% to 49% (e.g., 0% to 45%, 0% to 40%, 0% to 30%, 0% to 20%, 0% to 10%, and 0% to 5%, 5% to 49%, 10% to 49%, 20% to 49%, 30% to 49%, and 40% to 49%) of dC, dA, or dT nucleic acid residues.In some embodiments, the poly-dC nucleic acid sequence comprises 100% to 51% (e.g., 100% to 60%, 100% to 70%, 100% to 80%, 100% to 90%, 100% to 95%, 95% to 51%, 90% to 51%, 80% to 51%, 70% to 51%, and 60% to 51%) of dC nucleic acid residues and 0% to 49% (e.g., 0% to 45%, 0% to 40%, 0% to 30%, 0% to 20%, 0% to 10%, and 0% to 5%, 5% to 49%, 10% to 49%, 20% to 49%, 30% to 49%, and 40% to 49%) of dG, dA, or dT nucleic acid residues.

[0012] In some embodiments, the immunostimulatory nucleic acid sequence is a CpG sequence. In some embodiments, the CpG sequence is 5'-TCGTCGTTTTGTCGTTTTGTCGTT-3' (SEQ ID NO: 25). In some embodiments, all bonds in the sequence of SEQ ID NO: 25 are phosphate thioesters, including the linkage between a diacyl lipid and an oligodeoxynucleotide. In some embodiments, the CpG sequence is 5'-TGACTGTGAACGTTCGAGATGA-3' (SEQ ID NO: 26). In some embodiments, the CpG sequence is 5'-TCGTCGTTTTCGGCGCGCGCCG-3' (SEQ ID NO: 27). In some embodiments, the CpG sequence is 5'-TCCATGACGTTCCTGACGTT-3' (SEQ ID NO: 29).

[0013] In some embodiments, at least one internucleotide group connecting the nucleotides in the poly(dA), poly(dT), poly(dC), or poly(dG) nucleic acid sequences is a phosphodiester. In some embodiments, all internucleotide groups connecting the nucleotides in the poly(dA), poly(dT), poly(dC), or poly(dG) nucleic acid sequences are phosphate thioesters. In some embodiments, the albumin-binding domain is bonded to the 5' end of the poly(dA), poly(dT), poly(dG), or poly(dC) nucleic acid sequence. In some embodiments, the polypeptide is bonded to the 3' end of the poly(dA), poly(dT), poly(dC), or poly(dG) nucleic acid sequence.

[0014] In some embodiments, the polypeptide is an antigen or a fragment thereof. In some embodiments, the polypeptide is derived from a tumor, virus, or bacteria. In some embodiments, the antigen or a fragment thereof is a tumor-associated antigen. In some embodiments, the polypeptide is an influenza antigen or a fragment thereof. In some embodiments, the polypeptide is an influenza nucleoprotein or a fragment thereof. In some embodiments, the polypeptide is a coronavirus antigen or a fragment thereof. In some embodiments, the polypeptide is a coronavirus spike protein or a fragment thereof. In some embodiments, the polypeptide is a coronavirus nucleocapsid protein or a fragment thereof. In some embodiments, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 23. In some embodiments, the polypeptide is a neoantigen or a fragment thereof.

[0015] In some embodiments, the albumin-binding domain is a lipid. In some embodiments, the lipid is a diacyllipide. In some embodiments, the diacyllipide comprises an acyl chain containing 12-30 hydrocarbon units, 14-25 hydrocarbon units, 16-20 hydrocarbon units, or 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 hydrocarbon units. In some embodiments, the lipid is 1,2-distearate-sn-glycerol-3-phosphatidylethanolamine (DSPE). In some embodiments, the albumin-binding domain and the polypeptide are bonded or linked by a linker selected from the group consisting of hydrophilic polymers, a string of hydrophilic amino acids, polysaccharides, and oligonucleotides, or combinations thereof. In some embodiments, the linker comprises “N” polyethylene glycol units, where N is between 4 and 50. In some embodiments, the linker comprises PEG4-acylamino-PEG4.

[0016] In some embodiments, the immune-stimulated nucleic acid sequence is bonded or linked to the following lipids via a linker, wherein the nucleotide bonds are as follows:

[0017]

[0018] Or its salts, wherein X is O (phosphodiester) or S (thiophosphate).

[0019] In another aspect, this disclosure provides a compound or a pharmaceutically acceptable salt thereof comprising an immunostimulatory nucleic acid sequence, an albumin-binding domain, and a functional group. In some embodiments, the functional group comprises maleimide, dithio(2-pyridyldithio), cyclooctene, cyclooctyne, an aldehyde, an azide, or an alkyne. In some embodiments, the albumin-binding domain is bonded to or linked to the 5' end of the immunostimulatory nucleic acid sequence. In some embodiments, the albumin-binding domain is bonded to or linked to the 3' end of the immunostimulatory nucleic acid sequence. In another aspect, this disclosure provides a compound or a pharmaceutically acceptable salt thereof comprising an immunostimulatory nucleic acid sequence, an albumin-binding domain, and a functional group for use in a method of inducing an immune response against an antigen in a subject, wherein the method comprises administering to the subject the compound or a pharmaceutically acceptable salt thereof comprising the immunostimulatory nucleic acid sequence, an albumin-binding domain, and a functional group.

[0020] In some embodiments, the peptide and albumin-binding domains are bonded to or linked to the 5' end of the immunostimulated nucleic acid sequence. In some embodiments, the peptide and albumin-binding domains are bonded to or linked to the 3' end of the immunostimulated nucleic acid sequence.

[0021] In some embodiments, the immunostimulatory nucleic acid sequence is a polydeoxyadenosine (poly-dA) nucleic acid sequence. In some embodiments, the immunostimulatory nucleic acid sequence is a polydeoxythymidine (poly-dT) nucleic acid sequence. In some embodiments, the compound or a pharmaceutically acceptable salt thereof comprises both a poly-dA nucleic acid sequence and a poly-dT nucleic acid sequence. In some embodiments, the poly-dA nucleic acid sequence and the poly-dT nucleic acid sequence hybridize to form a double-stranded DNA sequence. In some embodiments, the poly-dA nucleic acid sequence and the poly-dT nucleic acid sequence comprise the same number of nucleotides.

[0022] In some embodiments, the immunostimulatory nucleic acid sequence is a polydeoxyguanosine (polydG) nucleic acid sequence. In some embodiments, the immunostimulatory nucleic acid sequence is a polydeoxycytosine (polydC) nucleic acid sequence. In some embodiments, the compound or a pharmaceutically acceptable salt thereof comprises both the polydG and polydC nucleic acid sequences. In some embodiments, the polydG and polydC nucleic acid sequences hybridize to form a double-stranded DNA sequence. In some embodiments, the polydG and polydC nucleic acid sequences comprise the same number of nucleotides.

[0023] In some embodiments, the poly-dA, poly-dT, poly-dC, and / or poly-dG nucleic acid sequences comprise between 30 and 100 nucleotides (e.g., between 30 and 90, 30 and 80, 30 and 70, 30 and 60, 30 and 50, 30 and 40, 40 and 100, 50 and 100, 60 and 100, 70 and 100, 80 and 100, or between 90 and 100 nucleotides). In some embodiments, the poly-dA, poly-dT, poly-dC, and / or poly-dG nucleic acid sequences comprise between 50 and 100 nucleotides (e.g., between 50 and 90, 50 and 80, 50 and 70, 50 and 60, 60 and 100, 70 and 100, 80 and 100, or between 90 and 100 nucleotides). In some embodiments, the poly-dA, poly-dT, poly-dC, and / or poly-dG nucleic acid sequences comprise between 30 and 50 nucleotides (e.g., between 30 and 45, 30 and 40, 30 and 35, 35 and 50, 40 and 50, or 45 and 50 nucleotides). In some embodiments, the poly-dA, poly-dT, poly-dC, and / or poly-dG nucleic acid sequences comprise 30, 40, 50, 75, or 100 nucleotides.

[0024] In some embodiments, the poly-dA nucleic acid sequence, poly-dT nucleic acid sequence, poly-dG nucleic acid sequence and / or poly-dC nucleic acid sequence comprises a mixture of dA, dT, dC and / or dG nucleic acid residues. In some embodiments, the poly-dA nucleic acid sequence comprises 100% to 51% (e.g., 100% to 60%, 100% to 70%, 100% to 80%, 100% to 90%, 100% to 95%, 95% to 51%, 90% to 51%, 80% to 51%, 70% to 51%, and 60% to 51%) of dA nucleic acid residues, and 0% to 49% (e.g., 0% to 45%, 0% to 40%, 0% to 30%, 0% to 20%, 0% to 10%, and 0% to 5%, 5% to 49%, 10% to 49%, 20% to 49%, 30% to 49%, and 40% to 49%) of dT, dC, and / or dG nucleic acid residues. In some embodiments, the poly-dT nucleic acid sequence comprises 100% to 51% (e.g., 100% to 60%, 100% to 70%, 100% to 80%, 100% to 90%, 100% to 95%, 95% to 51%, 90% to 51%, 80% to 51%, 70% to 51%, and 60% to 51%) of dT nucleic acid residues, and 0% to 49% (e.g., 0% to 45%, 0% to 40%, 0% to 30%, 0% to 20%, 0% to 10%, and 0% to 5%, 5% to 49%, 10% to 49%, 20% to 49%, 30% to 49%, and 40% to 49%) of dA, dC, and / or dG nucleic acid residues. In some embodiments, the polydG nucleic acid sequence comprises 100% to 51% (e.g., 100% to 60%, 100% to 70%, 100% to 80%, 100% to 90%, 100% to 95%, 95% to 51%, 90% to 51%, 80% to 51%, 70% to 51%, and 60% to 51%) of dG nucleic acid residues and 0% to 49% (e.g., 0% to 45%, 0% to 40%, 0% to 30%, 0% to 20%, 0% to 10%, and 0% to 5%, 5% to 49%, 10% to 49%, 20% to 49%, 30% to 49%, and 40% to 49%) of dC, dA, or dT nucleic acid residues.In some embodiments, the poly-dC nucleic acid sequence comprises 100% to 51% (e.g., 100% to 60%, 100% to 70%, 100% to 80%, 100% to 90%, 100% to 95%, 95% to 51%, 90% to 51%, 80% to 51%, 70% to 51%, and 60% to 51%) of dC nucleic acid residues and 0% to 49% (e.g., 0% to 45%, 0% to 40%, 0% to 30%, 0% to 20%, 0% to 10%, and 0% to 5%, 5% to 49%, 10% to 49%, 20% to 49%, 30% to 49%, and 40% to 49%) of dG, dA, or dT nucleic acid residues.

[0025] In some embodiments, the immune-stimulating nucleic acid sequence is a CpG sequence. In some embodiments, the CpG sequence is sequence 5'-TCGTCGTTTTGTCGTTTTGTCGTT-3' (SEQ ID NO: 25). In some embodiments, the CpG sequence is 5'-TGACTGTGAACGTTCGAGATGA-3' (SEQ ID NO: 26). In some embodiments, the CpG sequence is 5'-TCGTCGTTTTCGGCGCGCGCCG-3' (SEQ ID NO: 27). In some embodiments, the CpG sequence is 5'-TCCATGACGTTCCTGACGTT-3' (SEQ ID NO: 29).

[0026] In some embodiments, at least one internucleotide group connecting the nucleotides in the poly(dA), poly(dT), poly(dC), or poly(dG) nucleic acid sequences is a phosphodiester. In some embodiments, all internucleotide groups connecting the nucleotides in the poly(dA), poly(dT), poly(dC), or poly(dG) nucleic acid sequences are phosphate thioesters.

[0027] In some embodiments, the albumin-binding domain is a lipid. In some embodiments, the lipid is a diacyllipid. In some embodiments, the diacyllipid comprises an acyl chain containing 12-30 hydrocarbon units, 14-25 hydrocarbon units, 16-20 hydrocarbon units, or 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 hydrocarbon units. In some embodiments, the lipid is 1,2-distearate-sn-glycerol-3-phosphatidylethanolamine (DSPE).

[0028] In some embodiments, the immune-stimulated nucleic acid sequence is bonded or linked to the following lipids via a linker, wherein the nucleotide bonds are as follows:

[0029]

[0030] Or its salt, wherein X is O or S.

[0031] In some embodiments, the immunostimulatory nucleic acid sequence is a CpG sequence, wherein the functional group is a maleimide group, and wherein the 5' end of the CpG sequence is bonded or linked to the 3' end of the albumin-binding domain, and the 3' end of the CpG sequence is linked to the maleimide group via a PEG4 linker.

[0032] In another aspect, this disclosure provides a compound or a pharmaceutically acceptable salt thereof comprising an immunostimulatory nucleic acid sequence, a linker, and a functional group. In some embodiments, the functional group comprises maleimide, dithio(2-pyridyldithio), cyclooctene, cyclooctyne, an aldehyde, an azide, or an alkyne. In another aspect, this disclosure provides a compound or a pharmaceutically acceptable salt thereof comprising an immunostimulatory nucleic acid sequence, a linker, and a functional group for use in a method of inducing an immune response against an antigen in a subject, wherein the method comprises administering to the subject the compound or a pharmaceutically acceptable salt thereof comprising the immunostimulatory nucleic acid sequence, a linker, and a functional group.

[0033] In some embodiments, the immunostimulatory nucleic acid sequence is a polydeoxyadenosine (poly-dA) nucleic acid sequence. In some embodiments, the immunostimulatory nucleic acid sequence is a polydeoxythymidine (poly-dT) nucleic acid sequence. In some embodiments, the compound or a pharmaceutically acceptable salt thereof comprises both a poly-dA nucleic acid sequence and a poly-dT nucleic acid sequence. In some embodiments, the poly-dA nucleic acid sequence and the poly-dT nucleic acid sequence hybridize to form a double-stranded DNA sequence. In some embodiments, the poly-dA nucleic acid sequence and the poly-dT nucleic acid sequence comprise the same number of nucleotides.

[0034] In some embodiments, the immunostimulatory nucleic acid sequence is a polydeoxyguanosine (polydG) nucleic acid sequence. In some embodiments, the immunostimulatory nucleic acid sequence is a polydeoxycytosine (polydC) nucleic acid sequence. In some embodiments, the compound or a pharmaceutically acceptable salt thereof comprises both the polydG and polydC nucleic acid sequences.

[0035] In some embodiments, the polydG and polydC nucleic acid sequences hybridize to form a double-stranded DNA sequence. In some embodiments, the polydG and polydC nucleic acid sequences contain the same number of nucleotides. In some embodiments, the polydA, polydT, polydC, and / or polydG nucleic acid sequences contain between 30 and 100 nucleotides (e.g., between 30 and 90, 30 and 80, 30 and 70, 30 and 60, 30 and 50, 30 and 40, 40 and 100, 50 and 100, 60 and 100, 70 and 100, 80 and 100, or 90 and 100 nucleotides). In some embodiments, the poly(dA), poly(dT), poly(dC), and / or poly(dG) nucleic acid sequences comprise between 50 and 100 nucleotides (e.g., between 50 and 90, 50 and 80, 50 and 70, 50 and 60, 60 and 100, 70 and 100, 80 and 100, or 90 and 100 nucleotides). In some embodiments, the poly(dA), poly(dT), poly(dC), and / or poly(dG) nucleic acid sequences comprise between 30 and 50 nucleotides (e.g., between 30 and 45, 30 and 40, 30 and 35, 35 and 50, 40 and 50, or 45 and 50 nucleotides). In some embodiments, the poly(dA), poly(dT), poly(dC), and / or poly(dG) nucleic acid sequences comprise 30, 40, 50, 75, or 100 nucleotides.

[0036] In some embodiments, the poly-dA nucleic acid sequence, poly-dT nucleic acid sequence, poly-dG nucleic acid sequence and / or poly-dC nucleic acid sequence comprises a mixture of dA, dT, dC and / or dG nucleic acid residues. In some embodiments, the poly-dA nucleic acid sequence comprises 100% to 51% (e.g., 100% to 60%, 100% to 70%, 100% to 80%, 100% to 90%, 100% to 95%, 95% to 51%, 90% to 51%, 80% to 51%, 70% to 51%, and 60% to 51%) of dA nucleic acid residues, and 0% to 49% (e.g., 0% to 45%, 0% to 40%, 0% to 30%, 0% to 20%, 0% to 10%, and 0% to 5%, 5% to 49%, 10% to 49%, 20% to 49%, 30% to 49%, and 40% to 49%) of dT, dC, and / or dG nucleic acid residues. In some embodiments, the poly-dT nucleic acid sequence comprises 100% to 51% (e.g., 100% to 60%, 100% to 70%, 100% to 80%, 100% to 90%, 100% to 95%, 95% to 51%, 90% to 51%, 80% to 51%, 70% to 51%, and 60% to 51%) of dT nucleic acid residues, and 0% to 49% (e.g., 0% to 45%, 0% to 40%, 0% to 30%, 0% to 20%, 0% to 10%, and 0% to 5%, 5% to 49%, 10% to 49%, 20% to 49%, 30% to 49%, and 40% to 49%) of dA, dC, and / or dG nucleic acid residues. In some embodiments, the polydG nucleic acid sequence comprises 100% to 51% (e.g., 100% to 60%, 100% to 70%, 100% to 80%, 100% to 90%, 100% to 95%, 95% to 51%, 90% to 51%, 80% to 51%, 70% to 51%, and 60% to 51%) of dG nucleic acid residues and 0% to 49% (e.g., 0% to 45%, 0% to 40%, 0% to 30%, 0% to 20%, 0% to 10%, and 0% to 5%, 5% to 49%, 10% to 49%, 20% to 49%, 30% to 49%, and 40% to 49%) of dC, dA, or dT nucleic acid residues.In some embodiments, the poly-dC nucleic acid sequence comprises 100% to 51% (e.g., 100% to 60%, 100% to 70%, 100% to 80%, 100% to 90%, 100% to 95%, 95% to 51%, 90% to 51%, 80% to 51%, 70% to 51%, and 60% to 51%) of dC nucleic acid residues and 0% to 49% (e.g., 0% to 45%, 0% to 40%, 0% to 30%, 0% to 20%, 0% to 10%, and 0% to 5%, 5% to 49%, 10% to 49%, 20% to 49%, 30% to 49%, and 40% to 49%) of dG, dA, or dT nucleic acid residues.

[0037] In some embodiments, the immune-stimulating nucleic acid sequence is a CpG sequence. In some embodiments, the CpG sequence is sequence 5'-TCGTCGTTTTGTCGTTTTGTCGTT-3' (SEQ ID NO: 25). In some embodiments, the CpG sequence is 5'-TGACTGTGAACGTTCGAGATGA-3' (SEQ ID NO: 26). In some embodiments, the CpG sequence is 5'-TCGTCGTTTTCGGCGCGCGCCG-3' (SEQ ID NO: 27). In some embodiments, the CpG sequence is 5'-TCCATGACGTTCCTGACGTT-3' (SEQ ID NO: 29).

[0038] In some embodiments, at least one internucleotide group connecting the nucleotides in the poly(dA), poly(dT), poly(dC), or poly(dG) nucleic acid sequences is a phosphodiester. In some embodiments, all internucleotide groups connecting the nucleotides in the poly(dA), poly(dT), poly(dC), or poly(dG) nucleic acid sequences are phosphate thioesters. In some embodiments, the connector is as follows:

[0039]

[0040] Or its salt, wherein X is O or S.

[0041] In some embodiments, the immunostimulatory nucleic acid sequence is a CpG sequence, wherein the linker is PEG4, wherein the functional group is maleimide, and wherein the 3' end of the CpG sequence is bonded or connected to the PEG4, wherein the PEG4 is bonded or connected to the maleimide group.

[0042] In another aspect, this disclosure provides a compound or a pharmaceutically acceptable salt thereof comprising an immunosuppressive nucleic acid sequence, an albumin-binding domain, and a polypeptide. In some embodiments, the albumin-binding domain is bonded or linked to the 5' end of the immunosuppressive nucleic acid sequence, and the polypeptide is bonded or linked to the 3' end of the immunosuppressive nucleic acid sequence. In some embodiments, the polypeptide is bonded or linked to the 5' end of the immunosuppressive nucleic acid sequence, and the albumin-binding domain is bonded or linked to the 3' end of the immunosuppressive nucleic acid sequence. In some embodiments, both the polypeptide and the albumin-binding domain are bonded or linked to the 5' end of the immunosuppressive nucleic acid sequence. In some embodiments, both the polypeptide and the albumin-binding domain are bonded or linked to the 3' end of the immunosuppressive nucleic acid sequence. In another aspect, this disclosure provides a compound or a pharmaceutically acceptable salt thereof comprising an immunosuppressive nucleic acid sequence, an albumin-binding domain, and a polypeptide for use in a method of inducing an immune response against an antigen in a subject, wherein the method comprises administering to the subject the compound or a pharmaceutically acceptable salt thereof comprising the immunosuppressive nucleic acid sequence, an albumin-binding domain, and a polypeptide.

[0043] In some embodiments, the polypeptide comprises an N-terminal modification. In some embodiments, the N-terminal modification is the addition of acetylcysteine. In some embodiments, the N-terminal modification is the addition of a deaminocysteine ​​homolog. In some embodiments, the deaminocysteine ​​homolog is 3-mercaptopropionic acid or mercaptoacetic acid. In some embodiments, the immunosuppressive nucleic acid sequence is an A151 nucleic acid sequence. In some embodiments, the immunosuppressive nucleic acid sequence has the nucleic acid sequence TTAGG (SEQ ID NO: 28).

[0044] In some embodiments, the polypeptide is an antigen or a fragment thereof. In some embodiments, the antigen is derived from a tumor, virus, or bacteria. In some embodiments, the antigen or a fragment thereof is a tumor-associated antigen. In some embodiments, the antigen is an influenza antigen or a fragment thereof. In some embodiments, the antigen is an influenza nucleoprotein or a fragment thereof. In some embodiments, the antigen is a coronavirus antigen or a fragment thereof. In some embodiments, the antigen is a coronavirus spike protein or a fragment thereof. In some embodiments, the antigen is a coronavirus nucleocapsid protein or a fragment thereof.

[0045] In some embodiments, the albumin-binding domain is a lipid. In some embodiments, the lipid is a diacyllipid. In some embodiments, the diacyllipid comprises an acyl chain containing 12-30 hydrocarbon units, 14-25 hydrocarbon units, 16-20 hydrocarbon units, or 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 hydrocarbon units. In some embodiments, the lipid is 1,2-distearate-sn-glycerol-3-phosphatidylethanolamine (DSPE).

[0046] In some embodiments, the albumin-binding domain and the polypeptide are bonded or linked by a linker selected from the group consisting of hydrophilic polymers, a string of hydrophilic amino acids, polysaccharides, and oligonucleotides, or combinations thereof. In some embodiments, the linker comprises “N” polyethylene glycol units, where N is between 4 and 50. In some embodiments, the linker comprises PEG4-amide-PEG4.

[0047] In some embodiments, the immunosuppressive nucleic acid sequence is bonded or linked to the following lipids via a linker, wherein the nucleotide bonds are as follows:

[0048]

[0049] Or its salt, wherein X is O or S.

[0050] In another aspect, this disclosure provides a compound or a pharmaceutically acceptable salt thereof comprising an immunosuppressive nucleic acid sequence, an albumin-binding domain, and a functional group. In some embodiments, the functional group comprises maleimide, dithio(2-pyridyldithio), cyclooctene, cyclooctyne, an aldehyde, an azide, or an alkyne. In some embodiments, the albumin-binding domain is bonded to or linked to the 5' end of the immunosuppressive nucleic acid sequence. In some embodiments, the albumin-binding domain is bonded to or linked to the 3' end of the immunosuppressive nucleic acid sequence. In some embodiments, the immunosuppressive nucleic acid sequence is an A151 nucleic acid sequence. In some embodiments, the immunosuppressive nucleic acid sequence has a nucleic acid sequence of TTAGG (SEQ ID NO: 28). In another aspect, this disclosure provides a compound or a pharmaceutically acceptable salt thereof comprising an immunosuppressive nucleic acid sequence, an albumin-binding domain, and a functional group for use in a method of inducing an immune response against an antigen in a subject, wherein the method comprises administering to the subject the compound or a pharmaceutically acceptable salt thereof comprising the immunosuppressive nucleic acid sequence, an albumin-binding domain, and a functional group.

[0051] In some embodiments, the albumin-binding domain is a lipid. In some embodiments, the lipid is a diacyllipid. In some embodiments, the diacyllipid comprises an acyl chain containing 12-30 hydrocarbon units, 14-25 hydrocarbon units, 16-20 hydrocarbon units, or 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 hydrocarbon units. In some embodiments, the lipid is 1,2-distearate-sn-glycerol-3-phosphatidylethanolamine (DSPE).

[0052] In some embodiments, the immunosuppressive nucleic acid sequence is bonded or linked to the following lipids via a linker, wherein the nucleotide bonds are as follows:

[0053]

[0054] Or its salt, wherein X is O or S.

[0055] In another aspect, this disclosure provides a compound or a pharmaceutically acceptable salt thereof comprising an immunosuppressive nucleic acid sequence, a linker, and a functional group. In some embodiments, the functional group comprises maleimide, dithio(2-pyridyldithio), cyclooctene, cyclooctyne, an aldehyde, an azide, or an alkyne. In some embodiments, the immunosuppressive nucleic acid sequence is an A151 nucleic acid sequence. In some embodiments, the immunosuppressive nucleic acid sequence has a nucleic acid sequence of TTAGG (SEQ ID NO: 28). In another aspect, this disclosure provides a compound or a pharmaceutically acceptable salt thereof comprising an immunosuppressive nucleic acid sequence, a linker, and a functional group for use in a method of inducing an immune response against an antigen in a subject, wherein the method comprises administering to the subject the compound or a pharmaceutically acceptable salt thereof comprising the immunosuppressive nucleic acid sequence, a linker, and a functional group.

[0056] In some embodiments, the connector is selected from the group consisting of hydrophilic polymers, a string of hydrophilic amino acids, polysaccharides, and oligonucleotides, or combinations thereof. In some embodiments, the connector comprises "N" polyethylene glycol units, wherein N is between 4 and 50. In some embodiments, the connector comprises PEG4-amide-PEG4.

[0057] In some embodiments, the connector is as follows:

[0058]

[0059] Or its salt, wherein X is O or S.

[0060] In another aspect, this disclosure provides a method for inducing an immune response against an antigen in a subject, the method comprising administering to the subject any of the compounds described herein or a pharmaceutically acceptable salt thereof. In another aspect, this disclosure provides compounds described herein or pharmaceutically acceptable salts thereof for use in a method for inducing an immune response against an antigen in a subject, wherein the method comprises administering to the subject any of the compounds described herein. In some embodiments, the method further comprises administering an adjuvant to the subject. In some embodiments, the polypeptide is an antigen or a fragment thereof. In some embodiments, the polypeptide is a neoantigen or a fragment thereof. In some embodiments, the polypeptide is derived from a tumor or viral or bacterial source. In some embodiments, the antigen or a fragment thereof is a tumor-associated antigen. In some embodiments, the antigen is an influenza antigen or a fragment thereof. In some embodiments, the antigen is an influenza nucleoprotein or a fragment thereof. In some embodiments, the antigen is a coronavirus antigen or a fragment thereof. In some embodiments, the antigen is a coronavirus spike protein or a fragment thereof. In some embodiments, the antigen is a coronavirus nucleocapsid protein or a fragment thereof.

[0061] On the other hand, this disclosure provides the use of any of the compounds described herein or a pharmaceutically acceptable salt thereof to induce an immune response against an antigen in a subject, wherein any of the compounds described herein will be administered to the subject. In some embodiments, an adjuvant will be administered to the subject. In some embodiments, the polypeptide is an antigen or a fragment thereof. In some embodiments, the polypeptide is a neoantigen or a fragment thereof. In some embodiments, the polypeptide is derived from a tumor or viral or bacterial source. In some embodiments, the antigen or a fragment thereof is a tumor-associated antigen. In some embodiments, the antigen is an influenza antigen or a fragment thereof. In some embodiments, the antigen is an influenza nucleoprotein or a fragment thereof. In some embodiments, the antigen is a coronavirus antigen or a fragment thereof. In some embodiments, the antigen is a coronavirus spike protein or a fragment thereof. In some embodiments, the antigen is a coronavirus nucleocapsid protein or a fragment thereof.

[0062] In some embodiments, the compound or a pharmaceutically acceptable salt thereof is administered subcutaneously or will be administered subcutaneously. In some embodiments, the compound or a pharmaceutically acceptable salt thereof is administered via a mucous membrane. In some embodiments, the compound or a pharmaceutically acceptable salt thereof is administered via the buccal membrane. In some embodiments, the compound or a pharmaceutically acceptable salt thereof is administered sublingually. In some embodiments, the compound or a pharmaceutically acceptable salt thereof is administered intramuscularly. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0063] In another respect, this disclosure provides a pharmaceutical composition comprising any of the compounds described herein or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0064] On the other hand, this disclosure provides a kit comprising any of the compounds described herein or a pharmaceutically acceptable salt thereof. Attached Figure Description

[0065] Figure 1A The diagram shows a single-stranded amphiphilic compound (AMP) comprising a CpG-7909 nucleic acid sequence (SEQ ID NO:25) that is bonded to a polypeptide sequence having the amino acid sequence of SEQ ID NO: 23 and conjugated to an albumin-binding domain.

[0066] Figure 1B The diagram shows a single-stranded amphiphilic compound (AMP) and a double-stranded AMP, which contain a CpG-7909 nucleic acid sequence (SEQ ID NO: 25) conjugated to an albumin-binding domain and a PEG linker modified with a maleimide functional group.

[0067] Figure 2 This is a graph showing the ELISpot response of spleen cells co-cultured with IFNγ in C57Bl6 mice, which were administered a vaccine containing an adjuvant comprising a soluble or amphiphilic antigen or a soluble or amphiphilic chimera as shown in Figure 1.

[0068] Figure 3 This is a graph showing the amount of CD8 cells specific to the gp100 antigen isolated from peripheral blood collected from C57BL / 6J mice (n = 5 per group) that were administered a vaccine containing an adjuvant of a soluble or amphiphilic antigen or a soluble or amphiphilic chimera as shown in Figure 1.

[0069] Figure 4 This is a graph showing the percentage of cytokines found in peripheral blood cells collected from C57BL / 6J mice (n = 5 per group), including (from top to bottom in each column) IFNγ and TNFα, TNFα only, and IFNγ only, the C57BL / 6J mice being administered a vaccine containing an adjuvant comprising a soluble or amphiphilic antigen or a soluble or amphiphilic chimera as shown in Figure 1.

[0070] Figure 5 This shows CD8 isolated from perfused lung tissue of C57BL / 6J mice (n = 5 per group). + A graph showing the frequency of intracellular cytokines produced in T cells, with each column of intracellular cytokines including IFNγ and TNFα, TNFα only, and IFNγ only from top to bottom. The C57BL / 6J mice were administered a vaccine containing an adjuvant comprising a soluble or amphiphilic antigen or a soluble or amphiphilic chimera as shown in Figure 1.

[0071] Figure 6 The diagram shows a single-stranded AMP-dT nucleic acid sequence, which is coupled to a polypeptide sequence having the amino acid sequence of SEQ ID NO: 23 and conjugated to an albumin-binding domain.

[0072] Figure 7 This is a graph showing the ELISpot response of spleen cells co-cultured with IFNγ from C57Bl6 mice, which were treated with IFNγ containing... Figure 6 Vaccines containing soluble or amphiphilic antigens or soluble or amphiphilic chimeric adjuvants.

[0073] Figure 8 This is a graph showing the amount of CD8 cells specific for influenza NP antigen isolated from peripheral blood collected from C57BL / 6J mice (n = 5 per group), said C57BL / 6J mice were administered with a drug containing... Figure 6 Vaccines containing soluble or amphiphilic antigens or soluble or amphiphilic chimeric adjuvants.

[0074] Figure 9 This shows the CD8+ found in peripheral blood cells collected from C57BL / 6J mice (n = 5 per group). + A graph showing the frequencies of cytokines in T cells, including (each column from top to bottom) IFNγ and TNFα, TNFα only, and IFNγ only, in C57BL / 6J mice administered with the following cytokines: Figure 6 Vaccines containing soluble or amphiphilic antigens or soluble or amphiphilic chimeric adjuvants.

[0075] Figure 10 This shows CD8 isolated from perfused lung tissue of C57BL / 6J mice. + A graph showing the frequency of intracellular cytokines produced in T cells. Each column for intracellular cytokines, from top to bottom, includes IFNγ and TNFα, TNFα only, and IFNγ only. The C57BL / 6J mice were administered cytokines containing... Figure 6 Vaccines containing soluble or amphiphilic antigens or soluble or amphiphilic chimeric adjuvants.

[0076] definition

[0077] Unless otherwise stated, the terms used in the claims and description are as defined below.

[0078] It must be noted that, unless the context clearly indicates otherwise, as used in this specification and the appended claims, the singular forms “a / an” and “the” include plural indicators.

[0079] As used herein, “about” will be understood by one of ordinary skill in the art and will vary to some extent depending on the context in which it is used. If the use of the term is unclear to one of ordinary skill in the art in the given context in which it is used, “about” will mean up to a specific value of plus or minus 10%.

[0080] As used herein, the term "adjuvant" refers to a compound that, when used with a specific immunogen or antigen, enhances or otherwise alters or modifies the resulting immune response. Modification of the immune response includes strengthening or amplifying the specificity of either or both antibody- and cell-mediated immune responses. Modification of the immune response can also mean reducing or inhibiting certain antigen-specific immune responses. In some embodiments, the adjuvant is a cyclic dinucleotide. In some embodiments, the adjuvant is an immunomodulatory oligonucleotide as described herein.

[0081] "Amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimics that function in a manner similar to that of naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that are subsequently modified, such as hydroxyproline, γ-carboxyglutamic acid, and phosphoserine. Amino acid analogs are compounds that have the same basic chemical structure as naturally occurring amino acids (i.e., carbon atoms bound to hydrogen, carboxyl groups, amino groups, and R groups), such as homoserine, ortholeucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (e.g., ortholeucine) or modified polypeptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimics are chemical compounds that have a structure different from the general chemical structure of amino acids but function in a manner similar to that of naturally occurring amino acids. Amino acids are represented herein by their commonly known three-letter symbols or single-letter symbols recommended by the IUPAC-IUB Biochemistry Nomenclature Committee. Similarly, nucleotides are represented by their commonly accepted single-letter codes.

[0082] "Amino acid substitution" refers to replacing at least one existing amino acid residue in a predetermined amino acid sequence (the amino acid sequence of the starting polypeptide) with a second, different "substitute" amino acid residue. "Amino acid insertion" refers to incorporating at least one additional amino acid into a predetermined amino acid sequence. While insertions typically consist of the insertion of one or two amino acid residues, larger "peptide insertions" of the present invention can be made, for example, by inserting about three to about five, or even up to about ten, fifteen, or twenty amino acid residues. One or more inserted residues can be naturally occurring or non-natural, as disclosed above. "Amino acid deletion" refers to removing at least one amino acid residue from a predetermined amino acid sequence.

[0083] As used herein, "amphiphilic" or "amphiphilic" refers to a conjugate comprising a hydrophilic head group and a hydrophobic tail, thereby forming an amphiphilic conjugate. In some embodiments, the amphiphilic conjugate comprises an immunostimulatory poly(dA), poly(dT), poly(dC), poly(dG), or CpG sequence, a polypeptide, and one or more hydrophobic lipid tails.

[0084] The term “improvement” refers to a beneficial outcome in the treatment of any disease state, such as influenza and SARS-CoV-2, including its prevention, reduction of severity or progression, remission or cure.

[0085] As used herein, “cancer antigen” means (i) tumor-specific antigen, (ii) tumor-associated antigen, (iii) a cell expressing a tumor-specific antigen, (iv) a cell expressing a tumor-associated antigen, (v) an embryonic antigen on a tumor, (vi) an autologous tumor cell, (vii) a tumor-specific membrane antigen, (viii) a tumor-associated membrane antigen, (ix) a growth factor receptor, (x) a growth factor ligand, and (xi) any other type of antigen or antigen-presenting cell or material associated with cancer.

[0086] The term "derived from" a specified polypeptide or protein polypeptide or amino acid sequence, or "peptide fragment," refers to the source of the polypeptide. Preferably, the derived or fragmented polypeptide or amino acid sequence is derived from a specific sequence having an amino acid sequence substantially identical to this sequence or a portion thereof. In some embodiments, the portion consists of at least 10-20 amino acids, preferably at least 20-30 amino acids, more preferably at least 30-50 amino acids, or otherwise identified by those skilled in the art as having its source in said sequence. A polypeptide derived from another polypeptide or a fragment of another polypeptide may have one or more mutations relative to the starting polypeptide, such as one or more amino acid residues being substituted by another amino acid residue or having one or more amino acid residues inserted or deleted.

[0087] The polypeptide may contain amino acid sequences that are not naturally occurring. Such variants must have less than 100% sequence identity or similarity to the starting molecule. In preferred embodiments, the variant will have an amino acid sequence that, for example, has about 75% to less than 100%, more preferably about 80% to less than 100%, more preferably about 85% to less than 100%, more preferably about 90% to less than 100% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) and most preferably about 95% to less than 100% amino acid sequence identity or similarity to the amino acid sequence of the starting polypeptide in length.

[0088] In one embodiment, there is an amino acid difference between the starting polypeptide sequence and the sequence derived therefrom. This sequence identity or similarity is defined herein as the percentage of amino acid residues in the candidate sequence that are identical (i.e., same residues) to the starting amino acid residue, after sequence alignment and, where necessary, introducing gaps to obtain the maximum percentage of sequence identity.

[0089] As used in this article, the term "cytotoxic T lymphocyte (CTL) response" refers to an immune response induced by cytotoxic T cells. CTL responses are primarily mediated by CD8+ T cells.

[0090] As used herein, the term "effective dose" or "effective dosage" is defined as the amount sufficient to achieve or at least partially achieve the desired effect.

[0091] The term "therapeutic effective dose" is defined as an amount sufficient to cure or at least partially suppress the disease and its complications in a patient with the existing condition. The effective dose used for this purpose will depend on the severity of the disorder being treated and the overall state of the patient's own immune system.

[0092] As used herein, "immune cells" are hematopoietic cells that play a role in the immune response. Immune cells include lymphocytes (e.g., B cells and T cells), natural killer cells, and myeloid cells (e.g., monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes). In a particular embodiment, the immune cell is a T cell.

[0093] As used herein, an “immune response” refers to the response of an organism’s immune system to substances, including but not limited to exogenous or autologous proteins. Three general types of “immune responses” include mucosal immune responses, humoral immune responses, and cellular immune responses. For example, an immune response may include the activation, expansion, and / or increased proliferation of immune cells. An immune response may also include at least one of the following: cytokine production, T cell activation and / or proliferation, granzyme or perforin production, activation of antigen-presenting cells or dendritic cells, antibody production, inflammation, immunization, hypersensitivity to antigens, antigen-specific lymphocyte responses to antigens, clearance of infectious agents, and transplant or graft rejection.

[0094] As used in this article, “immunosuppressive oligonucleotides” are oligonucleotides that can suppress (e.g., reduce or prevent) immune responses.

[0095] As used in this article, “immunomodulatory oligonucleotides” are either immunostimulatory oligonucleotides or immunosuppressive oligonucleotides.

[0096] As used in this article, “immunostimulatory oligonucleotides” are oligonucleotides that can stimulate (e.g., induce or enhance) an immune response.

[0097] The terms “inducing an immune response” and “enhancing an immune response” are used interchangeably and refer to stimulating an immune response against a specific antigen (i.e., passive or adaptive).

[0098] The term “inducing” in the context of induced complement-dependent cytotoxicity (CDC) or antibody-dependent cytotoxicity (ADCC) refers to the stimulation of a specific direct cell-killing mechanism.

[0099] As used herein, a subject who “requires prevention,” “requires treatment,” or “has a need for it” means a subject who, in the judgment of an appropriate medical practitioner (e.g., a doctor, nurse, or nurse practitioner in the case of humans; a veterinarian in the case of non-human mammals), would reasonably benefit from a given treatment (such as treatment with a composition containing an amphiphilic ligand conjugate).

[0100] The term "in vivo" refers to processes that occur within a living organism.

[0101] The term "in vitro" refers to processes that occur outside of a living organism, such as in a test tube, flask, or culture plate.

[0102] As used herein, the terms “connected,” “operably connected,” “fused,” or “fused” are used interchangeably. These terms refer to the joining of two or more elements, components, or domains together by appropriate means, including chemical conjugation or recombinant DNA techniques. Methods of chemical conjugation (e.g., using heterobifunctional crosslinking agents) are known in the art, as are methods of recombinant DNA techniques.

[0103] The term "lipid" refers to a biomolecule that is soluble in nonpolar solvents but insoluble in water. Lipids are generally described as hydrophobic or amphiphilic molecules, which allows them to form structures such as vesicles or membranes in aqueous environments. Lipids include fatty acids, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids (including cholesterol), isopentenol lipids, glycolipids, and polyketide compounds. In some embodiments, lipids suitable for the amphiphilic ligand conjugates disclosed herein bind to human serum albumin under physiological conditions. In some embodiments, lipids suitable for the amphiphilic ligand conjugates disclosed herein intercalate into cell membranes under physiological conditions. In some embodiments, lipids bind to albumin and intercalate into cell membranes under physiological conditions. In some embodiments, lipids are diacyl lipids. In some embodiments, diacyl lipids contain at least 12 carbons. In some embodiments, diacyl lipids contain 12-30 hydrocarbon units, 14-25 hydrocarbon units, or 16-20 hydrocarbon units. In some embodiments, the diacyllipid contains 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 carbons.

[0104] “Nucleic acid” refers to deoxyribonucleotides or ribonucleotides in single-stranded or double-stranded form and their polymers. Unless otherwise specified, the term covers nucleic acids containing known natural nucleotide analogs, which have similar binding properties to a reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a specific nucleic acid sequence also implicitly covers its conserved variants (e.g., degenerate codon substitutions) and complementary sequences, as well as explicitly indicated sequences. Specifically, degenerate codon substitution can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with a mixture of bases and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081, 1991; Ohtsuka et al., J. Biol. Chem. 260:2605-2608, 1985; and Cassol et al., 1992; Rossolini et al., Mal. Cell. Probes 8:91-98, 1994). For arginine and leucine, the modification at the second base can also be conserved. The term nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene.

[0105] The polynucleotides of the present invention can be composed of any polynucleotide or polydeoxynucleotide (which may be unmodified RNA or DNA or modified RNA or DNA). For example, polynucleotides can be composed of single-stranded and double-stranded DNA, DNA as a mixture of single-stranded and double-stranded regions, single-stranded and double-stranded RNA, and RNA as a mixture of single-stranded and double-stranded regions, or hybrid molecules comprising DNA and RNA that may be single-stranded or more typically double-stranded or a mixture of single-stranded and double-stranded regions. Additionally, polynucleotides can be composed of triple-stranded regions comprising RNA or DNA, or both RNA and DNA. Polynucleotides may also contain one or more modified bases or DNA or RNA backbones modified for stability or other reasons. “Modified” bases include, for example, triphenylmethylated bases and rare bases such as inosine. DNA and RNA can be modified in various ways; therefore, “polynucleotide” includes forms modified chemically, enzymatically, or metabolically. In some embodiments, the polypeptides of the present invention are encoded by a nucleotide sequence. The nucleotide sequences of this invention can be used in a variety of applications, including: cloning, gene therapy, protein expression and purification, mutation introduction, DNA vaccination of desired hosts, antibody generation for, for example, passive immunization, PCR, primer and probe generation, etc.

[0106] As used herein, “external administration,” “external administration,” and other grammatically equivalent phrases refer to administration methods other than intestinal and local administration, usually by injection, and including but not limited to intravenous, intranasal, intraocular, intramuscular, intraarterial, intrasheath, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, intracerebral, intracranial, intracarotid, and intrasternal injections and infusions.

[0107] As is commonly used in this article, “pharmaceutically acceptable” means, to the extent of reasonable medical judgment, those compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with human and animal tissues, organs, and / or body fluids without causing excessive toxicity, irritation, allergic reactions, or other problems or complications (in proportion to a reasonable benefit / risk ratio).

[0108] As used herein, the term "pharmaceuticalally acceptable salt" means any pharmaceutically acceptable salt of the conjugates, oligonucleotides, or peptides disclosed herein. Pharmaceutically acceptable salts of any compound and nucleic acid sequence described herein may include those suitable for use in tissue contact with humans and animals without excessive toxicity, irritation, anaphylactic reactions, and in proportion to a reasonable benefit / risk ratio, within the bounds of reasonable medical judgment. Pharmaceutically acceptable salts are well known in the art. For example, pharmaceutically acceptable salts are described in: Berge et al., J. Pharmaceutical Sciences 66:1-19, 1977 and Pharmaceutical Salts: Properties, Selection, and Use (edited by PHStahl and C.G. Wermuth), Wiley-VCH, 2008. Salts may be prepared in situ during the final isolation and purification of the compounds described herein or by reacting a free base group with a suitable acid. Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, hydrogen sulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, diglucuronate, dodecyl sulfate, ethanesulfonate, fumarate, glucohepanoate, glyceryl phosphate, hemisulfate, heptaate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, dihydroxynaphthalate, pectate, persulfate, 3-phenylpropionate, phosphate, picrate, neopentanoate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate, etc. Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, etc., as well as non-toxic ammonium, quaternary ammonium, and amine cations, including but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, etc. Unless otherwise indicated or applicable, references to compounds, nucleic acids, conjugates, oligonucleotides, or polypeptides in the claims and elsewhere herein optionally include their pharmaceutically acceptable salts.

[0109] As used herein, the term "physiological condition" refers to the physical condition of a subject. In some embodiments, physiological condition refers to neutral pH (e.g., pH between 6 and 8).

[0110] As used herein, the term “peptide” refers to a polymer having 30 or fewer amino acid residues.

[0111] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acid residues. The terminology applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acids, and applies to both naturally occurring and non-naturally occurring amino acid polymers.

[0112] As used herein, the terms “subject” or “mammal” or “patient” include any human or non-human animal. For example, the methods and compositions of the present invention can be used to treat a subject suffering from a disease or condition. The term “non-human animal” includes all vertebrates, such as mammals and non-mammals, including non-human primates, sheep, dogs, cats, mice, horses, pigs, cattle, chickens, amphibians, reptiles, etc.

[0113] The term “sufficient amount” or “enough amount to…” means an amount sufficient to produce the desired effect, such as an amount sufficient to reduce the diameter of a tumor.

[0114] The term "T cell" refers to a type of white blood cell that can be distinguished from other white blood cells by the presence of T cell receptors on its cell surface. Several subsets of T cells exist, including but not limited to T helper cells (also known as T cells). H Cells or CD4 + T cells) and subtypes (including T cells) H T H 2. T H 3. T H 17. T H 9 and T FH Cellular cells), cytotoxic T cells (i.e., Tc cells, CD8 cells), + T cells, cytotoxic T lymphocytes, T killer cells, cytotoxic T cells), memory T cells and subtypes (including central memory T cells (T cells)). CM T cells), effector memory T cells (T cells) EM and T EMRA (cells) and resident memory T cells (T cells) RM T cells), regulatory T cells (also known as Treg cells or suppressor T cells), and subtypes (including CD4+ cells) + FOXP3 + T reg Cells, CD4 + FOXP3 - T reg Cells, Tr1 cells, Th3 cells and T cells reg17 cells), natural killer T cells (also known as NKT cells), mucosa-associated inertial T cells (MAIT) and gamma delta T cells (including Vγ9 / Vδ2 T cells). Any one or more of the aforementioned or unmentioned T cells may be the target cell type of the method of use of the present invention.

[0115] As used herein, the terms “treat,” “treating,” and “treatment” refer to the treatment or preventative measures described herein. A method of “treatment” involves administering the disclosed poly(dA) and / or poly(dT) nucleic acid sequences and albumin-binding domains to a subject in need of such treatment. In some embodiments, poly(dA), poly(dT), poly(dG), and / or poly(dC) nucleic acid sequences conjugated to the albumin-binding domain are administered to a subject in need of an enhanced immune response against a specific antigen or to a subject who may eventually acquire such a disorder, to prevent, cure, delay, or reduce the severity of the disorder or relapse, or to improve one or more symptoms of the disorder or relapse, or to prolong the subject’s survival beyond what would be expected in the absence of such treatment.

[0116] The term "tumor-associated antigen" refers to antigens produced in tumors and that can be detected by the immune system to trigger an immune response. Tumor-associated antigens have been identified in many human cancers, including lung cancer, skin cancer, blood cancer, brain cancer, liver cancer, breast cancer, rectal cancer, bladder cancer, and stomach cancer.

[0117] As used herein, a “vaccine” means a formulation containing the amphiphilic construct described herein, optionally in combination with an adjuvant, said formulation being in a form capable of being administered to vertebrates and inducing a protective immune response sufficient to induce immunity to prevent and / or improve a disease or condition (e.g., influenza or SARS-CoV-2) and / or alleviate at least one symptom of a disease or condition (e.g., influenza or SARS-CoV-2). Typically, a vaccine comprises a conventional saline or buffered aqueous solution medium in which the composition described herein is suspended or dissolved. In this form, the composition described herein is used to prevent, improve, or otherwise treat an infection or disease. Upon introduction into a host, the vaccine elicits an immune response, including but not limited to inducing a protective immune response to induce immunity to prevent and / or improve a disease or condition (e.g., influenza or SARS-CoV-2) and / or alleviate at least one symptom of a disease or condition. Detailed Implementation

[0118] This disclosure provides compounds comprising immunomodulatory (e.g., immunostimulatory or immunosuppressive) nucleic acid sequences conjugated directly or via a linker to an albumin-binding domain and a peptide. The immunomodulatory (e.g., immunostimulatory or immunosuppressive) nucleic acid sequences may include polyadenosine (poly-dA), polythymidine (poly-dT), polydeoxyguanosine (poly-dG), and / or polydeoxycytosine (poly-dC), as well as CpG nucleic acid sequences conjugated to an albumin-binding domain. Furthermore, this disclosure provides pharmaceutical compositions and kits comprising immunomodulatory (e.g., immunostimulatory or immunosuppressive) nucleic acids conjugated to peptides and albumin-binding domains. This disclosure further provides a method for inducing an immune response in a subject, said method being performed by administering the compounds described herein or pharmaceutically acceptable salts thereof.

[0119] polypeptide

[0120] This article describes compounds comprising polypeptides conjugated to immunomodulatory (e.g., immunostimulatory or immunosuppressive) nucleic acids and albumin-binding domains. In some embodiments, the polypeptide may be an antigen protein or a polypeptide or a fragment thereof (e.g., an epitope).

[0121] The length of a polypeptide or peptide can be 2-100 amino acids (e.g., between 2 and 90, 2 and 80, 2 and 70, 2 and 60, 2 and 50, 2 and 40, 2 and 30, 2 and 20, 2 and 10, 10 and 100, 20 and 100, 30 and 100, 40 and 100, 50 and 100, 60 and 100, 70 and 100, 80 and 100, or 90 and 100 amino acids), including, for example, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids. In some embodiments, the length of the polypeptide can be greater than 50 amino acids. In some embodiments, the length of the polypeptide can be >100 amino acids.

[0122] Proteins or peptides can be any protein or peptide that can induce or increase the immune system’s ability to produce antibodies and T-cell responses against the protein or peptide.

[0123] The polypeptide can be an antigen derived from viruses, bacteria, parasites, plants, protozoa, fungi, tissues, or transformed cells (such as cancer cells or leukemia cells), and can be a whole cell or its immunogenic component, such as a cell wall component or its molecular component. Suitable antigens are known in the art and are available from commercial, governmental, and scientific sources. In one embodiment, the polypeptide is a whole inactivated or attenuated organism. These organisms can be infectious organisms, such as viruses, parasites, and bacteria. These organisms can also be tumor cells. The polypeptide can be a purified or partially purified polypeptide derived from a tumor or viral or bacterial source. The polypeptide can be a recombinant polypeptide produced by expressing DNA encoding the polypeptide in a heterologous expression system.

[0124] In some embodiments, the peptide may be modified at the N-terminus. In some embodiments, the peptide may be modified at the C-terminus. For example, the peptide may be modified at the N-terminus with an acetylcysteine ​​residue. In some embodiments, the peptide may be modified at the N-terminus with an aminocysteine ​​homologue. For example, the peptide may be modified at the N-terminus with 3-mercaptopropionic acid or mercaptoacetic acid.

[0125] Antigens may be provided as a single antigen or in combination. Antigens may also be provided as a complex mixture of peptides or nucleic acids. Exemplary antigens are provided below.

[0126] Viral antigens

[0127] Viral antigens can be isolated from any virus, including but not limited to viruses from any of the following virus families: Arenaviridae, Arterivirus, Astroviridae, Baculoviridae, Badnavirus, Barnaviridae, Birnaviridae, Bromoviridae, Bunyaviridae, Caliciviridae, Capillovirus, Carlavirus, Cauliniovirus, Circoviridae, Closterovirus, Comoviridae, and Coronaviridae (e.g., coronaviruses, such as Severe Acute Respiratory Syndrome (SARS) virus).For example, SARS-CoV-2), Corticoviridae, Cystoviridae, Deltavirus, Dianthovirus, Enamovirus, Filoviridae (e.g., Marburg virus and Ebola virus) (e.g., Zaire, Reston, Ivory Coast). (Coastal or Sudan strains), Flaviviridae (e.g., hepatitis C virus, dengue virus 1, dengue virus 2, dengue virus 3, and dengue virus 4), Hepadnaviridae, Herpesviridae (e.g., human herpesviruses 1, 3, 4, 5, and 6, and cytomegalovirus), Hypoviridae, Iridoviridae, Leviviridae, Lipothrixviridae, Microviridae, Orthomyxovirid The family classifications of viruses include: Papovaviridae (e.g., influenza A, B, and C), Paramyxoviridae (e.g., measles virus, mumps virus, and human respiratory syncytial virus), Parvoviridae, Picornaviridae (e.g., poliovirus, rhinovirus, hepatitis virus, and foot-and-mouth disease virus), Poxviridae (e.g., cowpox and smallpox viruses), Reoviridae (e.g., rotavirus), Retroviridae (e.g., lentiviruses such as human immunodeficiency virus (HIV) 1 and HIV 2), Rhabdoviridae (e.g., rabies virus, measles virus, respiratory syncytial virus, etc.), Togaviridae (e.g., rubella virus, dengue virus, etc.), and Totiviridae. Suitable viral antigens also include all or part of dengue protein M, dengue protein E, dengue D1NS1, dengue D1NS2, and dengue D1NS3.

[0128] Viral antigens can be derived from specific strains, such as papillomaviruses, herpesviruses, such as herpes simplex virus 1 and 2; hepatitis viruses, such as hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), hepatitis E virus (HEV), and hepatitis G virus (HGV), tick-borne encephalitis virus; parainfluenza virus, varicella-zoster virus, cytomegalovirus, Epstein-Barr virus, rotavirus, rhinovirus, adenovirus, coxsackieviruses, equine encephalitis, Japanese encephalitis, yellow fever, Rift Valley fever, and lymphocytic choroid plexus meningitis.

[0129] In some embodiments, the compounds described herein comprise immunomodulatory (e.g., immunostimulatory or immunosuppressive) nucleic acids conjugated to a polypeptide and to an albumin-binding domain. The polypeptide may be an antigen or a fragment thereof. For example, the polypeptide may be derived from a tumor (e.g., a tumor-associated antigen) or a viral or bacterial source. The polypeptide may be an antigen, and the antigen may be an influenza antigen or a fragment thereof. For example, the antigen may be an influenza nucleoprotein or a fragment thereof. In particular, the influenza nucleoprotein may comprise a polypeptide sequence having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with the following:

[0130] MASQGTKRSYEQMETDGERQNATEIRASVGKMIGGIGRFYIQMCTELKLSDYEGRLIQNSLTIERMVLSAFDERRNKYLEEHPSAGKDPKKTGGPIYRRVNGKWMRELILYDKEEIRRIWRQANN GDDATAGLTHMMIWHSNLNDATYQRTRALVRTGMDPRMCSLMQGSTLPRRSGAAGAAVKGVGTMVMELVRMIKRGINDRNFWRGENGRKTRIAYERMCNILKGKFQTAAQKAMMDQVRESRNPGNA EFEDLTFLARSALILRGSVAHKSCLPACVYGPAVASGYDFEREGYSLVGIDPFRLLQNSQVYSLIRPNENPAHKSQLVWMACHSAAFEDLRVLSFIKGTKVLPRGKLSTRGVQIASNENMETMES STLELRSRYWAIRTRSGGNTNQQRASAGQISIQPTFSVQRNLPFDRTTIMAAFNGNTEGRTSDMRTEIIRMMESARPEDVSFQGRGVFELSDEKAASPIVPSFDMSNEGSYFFGDNAEEYDN (SEQ ID NO: 22)

[0131] The influenza nucleoprotein may comprise a polypeptide sequence having at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with SEQ ID NO: 22. In some embodiments, the influenza nucleoprotein has the polypeptide sequence of SEQ ID NO: 22 or a fragment thereof.

[0132] An antigen can be a coronavirus antigen or a fragment thereof. For example, an antigen can be the coronavirus spike protein or a fragment thereof.

[0133] bacterial antigens

[0134] Bacterial antigens can originate from any bacteria, including but not limited to *Actinomyces*, *Anabaena*, *Bacillus*, *Bacteroides*, *Bdellovibrio*, *Bordetella*, *Borrelia*, *Campylobacter*, *Caulobacter*, *Chlamydia*, *Chlorobium*, *Chromatium*, *Clostridium*, *Corynebacterium*, *Cytophaga*, *Deinococcus*, *Escherichia*, *Francisella*, *Halobacterium*, *Heliobacter*, *Haemophilus*, and *Hemophilus influenzae type b*. B)(HIB), Hyphomicrobium, Legionella, Leptspirosis, Listeria, Meningococcus (groups A, B, and C), Methanobacterium, Micrococcus, Myobacterium, Mycoplasma, Myxococcus, Neisseria, Nitrobacter, Oscillatoria, Prochlorophyll The genera *Proteus*, *Pseudomonas*, *Rhodospirillum*, *Rickettsia*, *Salmonella*, *Shigella*, *Spirillum*, *Spirochaeta*, *Staphylococcus*, *Streptococcus*, *Streptomyces*, *Sulfolobus*, *Thermoplasma*, *Thiobacillus*, and *Treponema*.The genera *Vibrio* and *Yersinia*.

[0135] parasite antigen

[0136] Parasitic antigens can be obtained from parasites, such as, but not limited to, antigens derived from the following: Cryptococcus neoformans, Histoplasma capsulatum, Candida albicans, Candida tropicalis, Nocardia asteroides, Rickettsia ricketsii, Rickettsia typhi, Mycoplasma pneumoniae, Chlamydia psittaci, Chlamydia trachomatis, Plasmodium falciparum, Trypanosoma brucei, Entamoeba histolytica, Toxoplasma gondii, and Trichomonas vaginalis. (Vaginalis) and Schistosoma mansoni. These include sporozoan antigens, Plasmodium antigens, such as all or part of cyclosporin, sporozoan surface proteins, hepatic antigens, apical membrane-associated proteins, or merozoan surface proteins.

[0137] Allergens and environmental antigens

[0138] Antigens can be allergens or environmental antigens, such as, but not limited to, antigens derived from: naturally occurring allergens, such as pollen allergens (pollen allergens from trees, herbs, weeds, and grasses), insect allergens (inhalants, saliva, and venom allergens), animal dander and scalp allergens, and food allergens. Important pollen allergens from trees, grasses, and herbs originate from the following taxa: Fagales, Oleales, Pinales, and Platanaceae, including, for example, birch (Betula), alder (Alnus), hazel (Corylus), hornbeam (Carpinus), olive (Olea), cedar (Cryptomeria and Juniper), and London plane (Platanus). The order Poales includes, for example, grasses of the genera *Lolium*, *Phleum*, *Poa*, *Cynodon*, *Dactylis*, *Holcus*, *Phalaris*, *Secale*, and *Sorghum*; the orders Asterales and Urticales, especially including herbaceous plants of the genera *Ambrosia*, *Artemisia*, and *Parietaria*. Other allergen antigens that can be used include allergens from house dust mites (Dermatophagoides) and Euroglyphus, storage mites (e.g., Lepidoglyphys, Glycyphagus, and Tyrophagus); allergens from cockroaches, mosquitoes, and fleas, such as those from the genera Blatella, Periplaneta, Chironomus, and Ctenocepphalides; allergens from mammals (such as cats, dogs, and horses) and birds; and venom allergens, including those from insects that bite or sting, such as those from the following taxa: Hymenoptera, including bees (superfamily Apidae), wasps (superfamily Vespidea), and ants (superfamily Formicoidae). Other allergen antigens can also be used, including inhaled allergens such as those from fungi such as Alternaria and Cladosporium.

[0139] Cancer antigens

[0140] Cancer antigens are antigens that are typically preferentially expressed through cancer cells (i.e., their expression levels are higher in cancer cells than in non-cancer cells) and, in some cases, expressed only through cancer cells. Cancer antigens can be expressed within cancer cells or on the surface of cancer cells. Cancer antigens can be tumor-associated antigens. Cancer antigens can be MART-1 / Melan-A, gp100, adenosine deaminase-binding protein (ADAbp), FAP, cyclophilin b, colorectal-associated antigen (CRC)-0017-1A / GA733, carcinoembryonic antigen (CEA), CAP-1, CAP-2, etv6, AML1, prostate-specific antigen (PSA), PSA-1, PSA-2, PSA-3, prostate-specific membrane antigen (PSMA), T-cell receptor / CD3-ζ chain, and CD20. The cancer antigen may be selected from the group consisting of: MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), M AGE-Xp4 (MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-05, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9, BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1 CDK4, tyrosinase, p53, MUC family, HER2 / neu, p21ras, RCAS1, alpha-fetoprotein, E-cadherin, α-catenin, β-catenin, γ-catenin, p120ctn, gp100Pmel117, PRAME, NY-ESO-1, cdc27, adenomatous polyposis protein (APC), cytosine, gap junction protein 37, Ig-idiotype, p15, gp75, GM2 ganglioside, GD2 ganglioside, human papillomavirus protein, Smad tumor antigen family, Imp-1, P1A, EBV-encoded nuclear antigen (EBNA)-1, brain glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1 and CT-7, CD20 or c-erbB-2.

[0141] In some embodiments, the antigen may be a melanocyte-associated antigen. For example, the antigen may be a melanocyte-associated antigen having at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with AVGALEGPRNQDWLGVPRQL (SEQ ID NO: 23).

[0142] Immune regulatory nucleic acid sequence

[0143] The compounds described herein contain immunomodulatory (e.g., immunostimulatory or immunosuppressive) nucleic acid sequences. Compounds may contain immunosuppressive nucleic acid sequences. For example, an immunosuppressive nucleic acid sequence may have the nucleic acid sequence TTAGGG (SEQ ID NO: 28).

[0144] The compound may contain an immunostimulatory nucleic acid sequence. The immunostimulatory nucleic acid sequence may be a polyadenosine nucleic acid sequence (polydA), a polythymidine nucleic acid sequence (polydT), a polycytosine (polydC) nucleic acid sequence, a polyguanosine (polydG) nucleic acid sequence, or a CpG sequence.

[0145] Polyadenosine and polythymidine nucleic acid sequences

[0146] Polydeoxyribonucleic acid (PORNA) comprises nucleic acid chains that can be single-stranded or double-stranded. A PORNA may contain only one type of nucleic acid (e.g., poly-dA and poly-dT) within a single-stranded nucleic acid. A poly-dA nucleic acid sequence may contain only adenosine nucleobases. In some embodiments, the poly-dA nucleic acid sequence comprises a mixture of adenosine and thymidine or other nucleic acid residues. For example, in some embodiments, the poly-dA nucleic acid sequence may consist of between 100% and 51% adenosine nucleic acid residues and between 0% and 49% thymidine nucleic acid residues. For example, in some embodiments, the polydA nucleic acid sequence may consist of 100% to 5% (e.g., 100% to 10%, 100% to 30%, 100% to 50%, 100% to 60%, 100% to 70%, 100% to 80%, 100% to 90%, 100% to 95%, 95% to 5%, 90% to 5%, 80% to 5%, 70% to 5%, 60% to 5%, 50% to 5%, 30% to 5% or 10% to 5%) of adenosine nucleic acid residues and 0% to 95% (e.g., 0% to 75%, 0% to 50%, 0% to 25%, 0% to 10%, 10% to 95%, 25% to 95%, 50% to 95%, 75% to 95% or 90% to 95%) of thymidine, guanosine, and / or cytosine nucleic acid residues. In some embodiments, the nucleic acid sequence may contain alternating dA and dT residues. The poly-dT sequence may contain only thymidine nucleobases. In some embodiments, the poly-dT nucleic acid sequence contains a mixture of thymidine and adenosine or other nucleic acid residues. For example, in some embodiments, the polydT nucleic acid sequence may consist of a mixture of 100% to 5% (e.g., 100% to 10%, 100% to 30%, 100% to 50%, 100% to 60%, 100% to 70%, 100% to 80%, 100% to 90%, 100% to 95%, 95% to 5%, 90% to 5%, 80% to 5%, 70% to 5%, 60% to 5%, 50% to 5%, 30% to 5% or 10% to 5%) of thymidine nucleic acid residues and 0% to 95% (e.g., 0% to 75%, 0% to 50%, 0% to 25%, 0% to 10%, 10% to 95%, 25% to 95%, 50% to 95%, 75% to 95% or 90% to 95%) of adenosine, guanosine or cytosine nucleic acid residues.

[0147] A single-stranded DNA sequence of polydA may contain between 75 and 100 nucleotides (e.g., between 75 and 95, 75 and 90, 75 and 85, 75 and 80, 80 and 100, 85 and 100, 90 and 100, and between 95 and 100). In some embodiments, a single-stranded DNA sequence of polydA may contain between 75 and 150 nucleotides (e.g., between 75 and 140, 75 and 130, 75 and 120, 75 and 110, 75 and 100, 75 and 95, 75 and 90, 75 and 85, 75 and 80, 80 and 150, 85 and 150, 90 and 150, 95 and 150, 100 and 150, 110 and 150, 120 and 150, 130 and 150, and between 140 and 150). In some embodiments, the single-stranded DNA sequence of poly-dA comprises 75, 80, 85, 90, 95, or 100 nucleotides. In some embodiments, poly-dA comprises 50 nucleotides (dA50).

[0148] A single-stranded DNA sequence of polydT may contain between 75 and 100 nucleotides (e.g., between 75 and 95, 75 and 90, 75 and 85, 75 and 80, 80 and 100, 85 and 100, 90 and 100, and between 95 and 100). In some embodiments, a single-stranded DNA sequence of polydT may contain between 75 and 150 nucleotides (e.g., between 75 and 140, 75 and 130, 75 and 120, 75 and 110, 75 and 100, 75 and 95, 75 and 90, 75 and 85, 75 and 80, 80 and 150, 85 and 150, 90 and 150, 95 and 150, 100 and 150, 110 and 150, 120 and 150, 130 and 150, and between 140 and 150). In some embodiments, a single-stranded DNA sequence of polydT comprises 75, 80, 85, 90, 95, or 100 nucleotides. In some embodiments, polydT comprises 50 nucleotides (dT50).

[0149] The poly-dA and poly-dT double-stranded DNA sequences may contain between 75 and 100 nucleotides (e.g., between 75 and 95, 75 and 90, 75 and 85, 75 and 80, 80 and 100, 85 and 100, 90 and 100, and between 95 and 100). In some embodiments, the poly-dA and poly-dT nucleic acid sequences contain between 75 and 150 nucleotides (e.g., between 75 and 140, 75 and 130, 75 and 120, 75 and 110, 75 and 100, 75 and 95, 75 and 90, 75 and 85, 75 and 80, 80 and 150, 85 and 150, 90 and 150, 95 and 150, 100 and 150, 110 and 150, 120 and 150, 130 and 150, and between 140 and 150). In some embodiments, poly(dA) and poly(dT) comprise the same number of nucleotides. In some embodiments, the poly(dA) and poly(dT) single-stranded DNA sequences in the double-stranded DNA sequence each comprise 75, 80, 85, 90, 95, or 100 nucleotides. In some embodiments, poly(dA) and poly(dT) each comprise 50 nucleotides (dA50:dT50).

[0150] The polydeoxyribonucleic acid mentioned in this article should be understood to include its pharmaceutically acceptable salts.

[0151] Polyguanosine and polycytosine nucleic acid sequences

[0152] Polydeoxyribonucleic acid (PORNA) comprises nucleic acid chains that can be single-stranded or double-stranded. A PORNA may contain only one type of nucleic acid (e.g., polyguanosine (polydG) and polycytosine (polydC)) within its single-stranded nucleic acid. A polydG nucleic acid sequence may contain only guanosine nucleobases. In some embodiments, a polydG nucleic acid sequence comprises a mixture of guanosine and cytosine nucleic acid residues. For example, in some embodiments, a polydG nucleic acid sequence may consist of between 100% and 51% guanosine nucleic acid residues and between 0% and 49% cytosine nucleic acid residues. A polydC sequence may contain only cytosine nucleobases. In some embodiments, a polydC nucleic acid sequence comprises a mixture of cytosine and guanosine or other nucleic acid residues. For example, in some embodiments, the polydC nucleic acid sequence may be a mixture of 100% to 5% (e.g., 100% to 10%, 100% to 30%, 100% to 50%, 100% to 60%, 100% to 70%, 100% to 80%, 100% to 90%, 100% to 95%, 95% to 5%, 90% to 5%, 80% to 5%, 70% to 5%, 60% to 5%, 50% to 5%, 30% to 5%, or 10% to 5%) of cytosine nucleic acid residues and 0% to 95% (e.g., 0% to 75%, 0% to 50%, 0% to 25%, 0% to 10%, 10% to 95%, 25% to 95%, 50% to 95%, 75% to 95%, or 90% to 95%) of adenosine, guanosine, or thymidine nucleic acid residues. In some embodiments, the polydG nucleic acid may comprise between 100% and 5% (e.g., between 100% and 10%, 100% and 30%, 100% and 50%, 100% and 60%, 100% and 70%, 100% and 80%, 100% and 90%, 100% and 95%, 95% and 5%, 90% and 5%, 80% and 5%, 70% and 5%, 60% and 5%, 50% and 5%, 30% and 5%, or between 10% and 5%) of guanosine nucleic acid residues and between 0% and 95% (e.g., between 0% and 75%, 0% and 50%, 0% and 25%, 0% and 10%, 10% and 95%, 25% and 95%, 50% and 95%, 75% and 95%, or between 90% and 95%) of adenosine, cytosine, or thymidine nucleic acid residues.

[0153] The polydG single-stranded DNA sequence may contain between 75 and 100 nucleotides (e.g., between 75 and 95, 75 and 90, 75 and 85, 75 and 80, 80 and 100, 85 and 100, 90 and 100, and between 95 and 100). In some embodiments, the polydG single-stranded DNA sequence contains between 75 and 150 nucleotides (e.g., between 75 and 140, 75 and 130, 75 and 120, 75 and 110, 75 and 100, 75 and 95, 75 and 90, 75 and 85, 75 and 80, 80 and 150, 85 and 150, 90 and 150, 95 and 150, 100 and 150, 110 and 150, 120 and 150, 130 and 150, and between 140 and 150). In some embodiments, the polydG single-stranded DNA sequence comprises 75, 80, 85, 90, 95, or 100 nucleotides.

[0154] The polydC single-stranded DNA sequence may contain between 75 and 100 nucleotides (e.g., between 75 and 95, 75 and 90, 75 and 85, 75 and 80, 80 and 100, 85 and 100, 90 and 100, and between 95 and 100). In some embodiments, the polydC single-stranded DNA sequence contains between 75 and 150 nucleotides (e.g., between 75 and 140, 75 and 130, 75 and 120, 75 and 110, 75 and 100, 75 and 95, 75 and 90, 75 and 85, 75 and 80, 80 and 150, 85 and 150, 90 and 150, 95 and 150, 100 and 150, 110 and 150, 120 and 150, 130 and 150, and between 140 and 150). In some embodiments, the polydC single-stranded DNA sequence comprises 75, 80, 85, 90, 95, or 100 nucleotides.

[0155] The polydG and polydC double-stranded DNA sequences may contain between 75 and 100 nucleotides (e.g., between 75 and 95, 75 and 90, 75 and 85, 75 and 80, 80 and 100, 85 and 100, 90 and 100, and between 95 and 100). In some embodiments, the polydG and polydC nucleic acid sequences contain between 75 and 150 nucleotides (e.g., between 75 and 140, 75 and 130, 75 and 120, 75 and 110, 75 and 100, 75 and 95, 75 and 90, 75 and 85, 75 and 80, 80 and 150, 85 and 150, 90 and 150, 95 and 150, 100 and 150, 110 and 150, 120 and 150, 130 and 150, and between 140 and 150). In some embodiments, the polydG and polydC contain the same number of nucleotides. In some embodiments, the polydG and polydC single-stranded DNA sequences in the double-stranded DNA sequence each contain 75, 80, 85, 90, 95, or 100 nucleotides. In some embodiments, the polydG and polydC strands of the nucleic acid are complementary to each other.

[0156] CpG sequence

[0157] CpG ODNs are short, synthetic, single-stranded DNA molecules containing unmethylated CpG dinucleotides in a specific sequence background. Unlike the native phosphodiester (PO) backbone of DNA, CpG ODNs possess a partially or fully phosphorylated (PS) backbone. Based on structural characteristics and activity against human peripheral blood mononuclear cells (PBMCs), particularly B cells and plasmacytoid dendritic cells (pDCs), three main classes of stimulating CpG ODNs have been identified. These three main classes are class A (type D), class B (type K), and class C.

[0158] CpG1826 and CpG7909 both belong to class B CpGs. Class B CpG ODNs contain a complete PS backbone with one or more CpG dinucleotides. They strongly activate B cells and TLR9-dependent NF-κB signaling, but have a weaker stimulatory effect on IFN-α secretion. CpG7909 has the following sequence:

[0159] 5'-TCGTCGTTTTGTCGTTTTGTCGTT-3' (SEQ ID NO: 25)

[0160] In some embodiments, the CpG sequence is 5'-TGACTGTGAACGTTCGAGATGA-3' (SEQ ID NO: 26) (CpG 1018). In some embodiments, the CpG sequence is 5'-TCGTCGTTTTCGGCGCGCGCCG-3' (SEQ ID NO: 27) (CpG 2395). In some embodiments, the CpG sequence is 5'-TCCATGACGTTCCTGACGTT-3' (SEQ ID NO: 29) (CPG1826). In some embodiments, all bonds in the CpG sequence are phosphate thioesters, including linkages between diacyl lipids and oligodeoxynucleotides.

[0161] The polydeoxyribonucleic acid mentioned in this article should be understood to include its pharmaceutically acceptable salts.

[0162] Amphiphilic polydeoxyribonucleic acid

[0163] Amphiphilic polydeoxyribonucleic acid (PDRNA) comprises a nucleic acid chain conjugated directly or via a linker to a polypeptide and further conjugated directly or via a linker to an albumin-binding domain (e.g., a lipid). In some embodiments, the amphiphilic PDRNA is a polydeoxyadenosine (AMP-dA) chain of a nucleic acid conjugated to an albumin-binding domain (e.g., a lipid). In some embodiments, the amphiphilic PDRNA is a polydeoxythymidine (AMP-dT) chain of a nucleic acid conjugated to an albumin-binding domain (e.g., a lipid). In some embodiments, the amphiphilic PDRNA is a polydeoxyguanosine (AMP-dG) chain of a nucleic acid conjugated to an albumin-binding domain (e.g., a lipid). In some embodiments, the amphiphilic PDRNA is a polydeoxycytosine (AMP-dC) chain of a nucleic acid conjugated to an albumin-binding domain (e.g., a lipid).

[0164] In some embodiments, the compounds described herein comprise both dA and dT nucleic acid sequences, or dG and dC, and an albumin-binding domain (e.g., AMP-dA:dT, AMP-dT:dA, AMP-dG:dC, and AMP-dC:dG). The dA and dT nucleic acid sequences can hybridize to form a double-stranded DNA sequence (e.g., dA:dT and dT:dA). Similarly, the dG and dC nucleic acid sequences can hybridize to form a double-stranded DNA sequence (e.g., dG:dC and dC:dG). Examples of poly-dA and / or poly-dT compounds disclosed herein are illustrated in Figures 1 and 54. In some embodiments, the compounds described herein comprise a mixture of both dA and dT nucleic acid residues or a mixture of dG and dC nucleic acid residues. For example, the compounds may comprise alternating dA and dT nucleic acid residues.

[0165] The length of the poly(dA) nucleic acid sequence can include between 30 and 150 nucleotides (e.g., 30 and 140, 30 and 130, 30 and 120, 30 and 110, 30 and 100, 30 and 90, 30 and 80, 30 and 70, 30 and 60, 30 and 50, 30 and 40, 40 and 150, 50 and 150, 60 and 150, 70 and 150, 80 and 150, 90 and 150, 100 and 150, 110 and 150, 120 and 150, 130 and 150, and between 140 and 150 nucleotides). For example, the length of the poly(dA) nucleic acid sequence can be between 30 and 100 nucleotides (e.g., between 30 and 90, 30 and 80, 30 and 70, 30 and 60, 30 and 50, 30 and 40, 40 and 100, 50 and 100, 60 and 100, 70 and 100, 80 and 100, and between 90 and 100 nucleotides). In some embodiments, the length of the poly(dA) nucleic acid sequence can be between 50 and 100 nucleotides (e.g., between 50 and 90, 50 and 80, 50 and 70, 50 and 60, 60 and 100, 70 and 100, 80 and 100, and between 90 and 100 nucleotides). In some embodiments, the length of the poly(dA) nucleic acid sequence can be between 30 and 50 nucleotides (e.g., between 30 and 45, 30 and 40, 30 and 35, 35 and 50, 40 and 50, and between 45 and 50 nucleotides). In some embodiments, the length of the polydA nucleic acid sequence can be 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 nucleotides. In some embodiments, the length of the polydA nucleic acid sequence can be 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleotides. Furthermore, this disclosure provides a single-stranded polydA DNA sequence containing between 75 and 100 nucleotides (e.g., between 75 and 95, 75 and 90, 75 and 85, 75 and 80, 80 and 100, 85 and 100, 90 and 100, and 95 and 100).

[0166] The length of a polydT nucleic acid sequence can include between 30 and 150 nucleotides (e.g., 30 and 140, 30 and 130, 30 and 120, 30 and 110, 30 and 100, 30 and 90, 30 and 80, 30 and 70, 30 and 60, 30 and 50, 30 and 40, 40 and 150, 50 and 150, 60 and 150, 70 and 150, 80 and 150, 90 and 150, 100 and 150, 110 and 150, 120 and 150, 130 and 150, and between 140 and 150 nucleotides). For example, the length of the polydT nucleic acid sequence can be between 30 and 100 nucleotides (e.g., between 30 and 90, 30 and 80, 30 and 70, 30 and 60, 30 and 50, 30 and 40, 40 and 100, 50 and 100, 60 and 100, 70 and 100, 80 and 100, and between 90 and 100 nucleotides). In some embodiments, the length of the polydT nucleic acid sequence can be between 50 and 100 nucleotides (e.g., between 50 and 90, 50 and 80, 50 and 70, 50 and 60, 60 and 100, 70 and 100, 80 and 100, and between 90 and 100 nucleotides). In some embodiments, the length of the polydT nucleic acid sequence can be between 30 and 50 nucleotides (e.g., between 30 and 45, 30 and 40, 30 and 35, 35 and 50, 40 and 50, and between 45 and 50 nucleotides). In some embodiments, the length of the polydT nucleic acid sequence can be 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 nucleotides. In some embodiments, the length of the polydT nucleic acid sequence can be 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleotides. Furthermore, this disclosure provides a single-stranded polydT DNA sequence containing between 75 and 100 nucleotides (e.g., between 75 and 95, 75 and 90, 75 and 85, 75 and 80, 80 and 100, 85 and 100, 90 and 100, and 95 and 100).

[0167] Both poly-dA and poly-dT nucleic acid sequences can include 30 to 150 nucleotides in length (e.g., 30 to 140, 30 to 130, 30 to 120, 30 to 110, 30 to 100, 30 to 90, 30 to 80, 30 to 70, 30 to 60, 30 to 50, 30 to 40, 40 to 150, 50 to 150, 60 to 150, 70 to 150, 80 to 150, 90 to 150, 100 to 150, 110 to 150, 120 to 150, 130 to 150, and 140 to 150 nucleotides). For example, the length of both the poly-dA and poly-dT nucleic acid sequences can be between 30 and 100 nucleotides (e.g., between 30 and 90, 30 and 80, 30 and 70, 30 and 60, 30 and 50, 30 and 40, 40 and 100, 50 and 100, 60 and 100, 70 and 100, 80 and 100, and between 90 and 100 nucleotides). In some embodiments, the length of both the poly-dA and poly-dT nucleic acid sequences can be between 50 and 100 nucleotides (e.g., between 50 and 90, 50 and 80, 50 and 70, 50 and 60, 60 and 100, 70 and 100, 80 and 100, and between 90 and 100 nucleotides). In some embodiments, the length of both the poly-dA and poly-dT nucleic acid sequences can be between 30 and 50 nucleotides (e.g., between 30 and 45, 30 and 40, 30 and 35, 35 and 50, 40 and 50, and between 45 and 50 nucleotides). In some embodiments, the length of both the poly-dA and poly-dT nucleic acid sequences can be 30, 40, 50, 75, or 100 nucleotides. In some embodiments, the poly-dA and poly-dT nucleic acid sequences contain the same number of nucleotides. Furthermore, this disclosure provides poly-dA and poly-dT double-stranded DNA sequences containing between 30 and 100 nucleotides (e.g., between 30 and 90, 30 and 80, 30 and 70, 30 and 60, 30 and 50, 30 and 40, 40 and 100, 50 and 100, 60 and 100, 70 and 100, 80 and 100, and between 90 and 100). In some embodiments, poly(dA) and poly(dT) contain the same number of nucleotides.

[0168] The length of the polydG nucleic acid sequence can include between 30 and 150 nucleotides (e.g., 30 and 140, 30 and 130, 30 and 120, 30 and 110, 30 and 100, 30 and 90, 30 and 80, 30 and 70, 30 and 60, 30 and 50, 30 and 40, 40 and 150, 50 and 150, 60 and 150, 70 and 150, 80 and 150, 90 and 150, 100 and 150, 110 and 150, 120 and 150, 130 and 150, and between 140 and 150 nucleotides). For example, the length of the polydG nucleic acid sequence can be between 30 and 100 nucleotides (e.g., between 30 and 90, 30 and 80, 30 and 70, 30 and 60, 30 and 50, 30 and 40, 40 and 100, 50 and 100, 60 and 100, 70 and 100, 80 and 100, and between 90 and 100 nucleotides). In some embodiments, the length of the polydG nucleic acid sequence can be between 50 and 100 nucleotides (e.g., between 50 and 90, 50 and 80, 50 and 70, 50 and 60, 60 and 100, 70 and 100, 80 and 100, and between 90 and 100 nucleotides). In some embodiments, the length of the polydG nucleic acid sequence can be between 30 and 50 nucleotides (e.g., between 30 and 45, 30 and 40, 30 and 35, 35 and 50, 40 and 50, and between 45 and 50 nucleotides). In some embodiments, the length of the polydG nucleic acid sequence can be 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 nucleotides. In some embodiments, the length of the polydG nucleic acid sequence can be 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleotides. Furthermore, this disclosure provides a single-stranded polydG DNA sequence containing between 75 and 100 nucleotides (e.g., between 75 and 95, 75 and 90, 75 and 85, 75 and 80, 80 and 100, 85 and 100, 90 and 100, and 95 and 100).

[0169] The length of a polydC nucleic acid sequence can include between 30 and 150 nucleotides (e.g., 30 and 140, 30 and 130, 30 and 120, 30 and 110, 30 and 100, 30 and 90, 30 and 80, 30 and 70, 30 and 60, 30 and 50, 30 and 40, 40 and 150, 50 and 150, 60 and 150, 70 and 150, 80 and 150, 90 and 150, 100 and 150, 110 and 150, 120 and 150, 130 and 150, and between 140 and 150 nucleotides). For example, the length of the polydC nucleic acid sequence can be between 30 and 100 nucleotides (e.g., between 30 and 90, 30 and 80, 30 and 70, 30 and 60, 30 and 50, 30 and 40, 40 and 100, 50 and 100, 60 and 100, 70 and 100, 80 and 100, and between 90 and 100 nucleotides). In some embodiments, the length of the polydC nucleic acid sequence can be between 50 and 100 nucleotides (e.g., between 50 and 90, 50 and 80, 50 and 70, 50 and 60, 60 and 100, 70 and 100, 80 and 100, and between 90 and 100 nucleotides). In some embodiments, the length of the polydC nucleic acid sequence can be between 30 and 50 nucleotides (e.g., between 30 and 45, 30 and 40, 30 and 35, 35 and 50, 40 and 50, and between 45 and 50 nucleotides). In some embodiments, the length of the polydC nucleic acid sequence can be 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 nucleotides. In some embodiments, the length of the polydC nucleic acid sequence can be 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleotides. Furthermore, this disclosure provides polydC single-stranded DNA sequences comprising between 75 and 100 nucleotides (e.g., between 75 and 95, 75 and 90, 75 and 85, 75 and 80, 80 and 100, 85 and 100, 90 and 100, and 95 and 100).

[0170] Both polydG and polydC nucleic acid sequences can include 30 to 150 nucleotides in length (e.g., 30 to 140, 30 to 130, 30 to 120, 30 to 110, 30 to 100, 30 to 90, 30 to 80, 30 to 70, 30 to 60, 30 to 50, 30 to 40, 40 to 150, 50 to 150, 60 to 150, 70 to 150, 80 to 150, 90 to 150, 100 to 150, 110 to 150, 120 to 150, 130 to 150, and 140 to 150 nucleotides). For example, the length of both the polydG and polydC nucleic acid sequences can be between 30 and 100 nucleotides (e.g., between 30 and 90, 30 and 80, 30 and 70, 30 and 60, 30 and 50, 30 and 40, 40 and 100, 50 and 100, 60 and 100, 70 and 100, 80 and 100, and between 90 and 100 nucleotides). In some embodiments, the length of both the polydG and polydC nucleic acid sequences can be between 50 and 100 nucleotides (e.g., between 50 and 90, 50 and 80, 50 and 70, 50 and 60, 60 and 100, 70 and 100, 80 and 100, and between 90 and 100 nucleotides). In some embodiments, the length of both the polydG and polydC nucleic acid sequences can be between 30 and 50 nucleotides (e.g., between 30 and 45, 30 and 40, 30 and 35, 35 and 50, 40 and 50, and between 45 and 50 nucleotides). In some embodiments, the length of both the polydG and polydC nucleic acid sequences can be 30, 40, 50, 75, or 100 nucleotides. In some embodiments, the polydG and polydC nucleic acid sequences contain the same number of nucleotides. Furthermore, this disclosure provides polydG and polydC double-stranded DNA sequences containing between 30 and 100 nucleotides (e.g., between 30 and 90, 30 and 80, 30 and 70, 30 and 60, 30 and 50, 30 and 40, 40 and 100, 50 and 100, 60 and 100, 70 and 100, 80 and 100, and between 90 and 100). In some embodiments, polydG and polydC contain the same number of nucleotides.

[0171] The length of alternating poly-dA and poly-dT nucleic acid sequences can include between 30 and 150 nucleotides (e.g., 30 and 140, 30 and 130, 30 and 120, 30 and 110, 30 and 100, 30 and 90, 30 and 80, 30 and 70, 30 and 60, 30 and 50, 30 and 40, 40 and 150, 50 and 150, 60 and 150, 70 and 150, 80 and 150, 90 and 150, 100 and 150, 110 and 150, 120 and 150, 130 and 150, and between 140 and 150 nucleotides). For example, the length of the poly-dA nucleic acid sequence can be between 30 and 100 nucleotides (e.g., 30 and 90, 30 and 80, 30 and 70, 30 and 60, 30 and 50, 30 and 40, 40 and 100, 50 and 100, 60 and 100, 70 and 100, 80 and 100, and 90 and 100 nucleotides). In some embodiments, the length of alternating poly-dA and poly-dT nucleic acid sequences can be between 50 and 100 nucleotides (e.g., 50 and 90, 50 and 80, 50 and 70, 50 and 60, 60 and 100, 70 and 100, 80 and 100, and 90 and 100 nucleotides). In some embodiments, the length of the poly-dA nucleic acid sequence can be between 30 and 50 nucleotides (e.g., 30 and 45, 30 and 40, 30 and 35, 35 and 50, 40 and 50, and 45 and 50 nucleotides). In some embodiments, the length of the poly-dA nucleic acid sequence can be 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 nucleotides. In some embodiments, the length of the alternating poly-dA and poly-dT nucleic acid sequences can be 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleotides. In addition, this disclosure provides single-stranded DNA sequences of poly(dA) and poly(dT) containing alternating nucleotides between 75 and 100 (e.g., between 75 and 95, 75 and 90, 75 and 85, 75 and 80, 80 and 100, 85 and 100, 90 and 100, and between 95 and 100).

[0172] In some embodiments, the internucleotide groups connecting 50% to 100% of the nucleotides in the poly(dA) and / or poly(dT) nucleic acid sequences (e.g., 50% to 90%, 50% to 80%, 50% to 70%, 50% to 60%, 60% to 100%, 70% to 100%, 80% to 100%, or 90% to 100%) are phosphate thioester bonds. In some embodiments, the internucleotide groups connecting 1 to 10 nucleotides in the poly(dA) and / or poly(dT) nucleic acid sequences (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) are phosphodiester bonds, and the remaining internucleotide groups connecting the nucleotides in the poly(dA) and / or poly(dT) nucleic acid sequences are phosphate thioester bonds. For example, the bonds between 1 and 5 (e.g., 1, 2, 3, 4, or 5) at the 5' and / or 3' ends of the poly-dA and / or poly-dT nucleic acid sequences are phosphodiester bonds, and all remaining bonds are thiophosphate bonds. In some embodiments, all internucleotide groups linking the nucleotides in the poly-dA and / or poly-dT nucleic acid sequences are thiophosphate bonds.

[0173] In some embodiments, the internucleotide groups connecting 50% to 100% of the nucleotides in the polydG and / or polydC nucleic acid sequences (e.g., 50% to 90%, 50% to 80%, 50% to 70%, 50% to 60%, 60% to 100%, 70% to 100%, 80% to 100%, or 90% to 100%) are phosphate thioester bonds. In some embodiments, the internucleotide groups connecting 1 to 10 nucleotides in the polydG and / or polydC nucleic acid sequences (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) are phosphodiester bonds, and the remaining internucleotide groups connecting the nucleotides in the polydG and / or polydC nucleic acid sequences are phosphate thioester bonds. For example, the bonds between 1 and 5 (e.g., 1, 2, 3, 4, or 5) at the 5' and / or 3' ends of the polydG and / or polydC nucleic acid sequences are phosphodiester bonds, and all remaining bonds are thiophosphate bonds. In some embodiments, all internucleotide groups linking the nucleotides in the polydG and / or polydC nucleic acid sequences are thiophosphate bonds.

[0174] The references to polydeoxyribonucleic acid (PDA) and amphiphiles containing PDA should be understood to include their pharmaceutically acceptable salts.

[0175] lipids

[0176] The compounds described herein comprise immunomodulatory (e.g., immunostimulatory or immunosuppressive) nucleic acid sequences conjugated to peptides and lipids. In some embodiments, the lipid is bound to the 5' end of a poly(dA) nucleic acid sequence. In some embodiments, the lipid is bound to the 5' end of a poly(dT) nucleic acid sequence. In some embodiments, the lipid is bound to the 5' end of a poly(dG) nucleic acid sequence. In some embodiments, the lipid is bound to the 5' end of a poly(dC) nucleic acid sequence. In some embodiments, the lipid is bound to the 5' end of a CpG nucleic acid sequence. In some embodiments, the lipid is bound to the 3' end of a nucleic acid sequence described herein. The lipid may be linear, branched, or cyclic.

[0177] Examples of preferred lipids include, but are not limited to, fatty acids with an aliphatic tail having 3 to 30 carbons, including, but not limited to, straight-chain unsaturated and saturated fatty acids, branched-chain saturated and unsaturated fatty acids, and fatty acid derivatives such as fatty acid esters, fatty acid amides and fatty acid thioesters, diacyl lipids, cholesterol, cholesterol derivatives and steroid acids (such as bile acids), lipid A or combinations thereof.

[0178] In some embodiments, the lipid is a diacyllipid or a bi-tailed lipid. In some embodiments, the tail of the diacyllipid contains about 12 to about 30 (e.g., 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29) carbons. In some embodiments, the tail of the diacyllipid contains about 14 to about 25 (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24) carbons. In some embodiments, the tail of the diacyllipid contains about 16 to about 20 (e.g., 17, 18, or 19) carbons. In some embodiments, the diacyllipid comprises 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 carbons.

[0179] The carbon tail of a diacyl lipid can be saturated, unsaturated, or a combination thereof. The tail can be coupled to the head group via an ester bond, amide bond, thioester bond, or a combination thereof. In particular embodiments, the diacyl lipid is a phosphate lipid, glycolipid, sphingolipid, or a combination thereof.

[0180] In some embodiments, the lipid is 1,2-distearate-sn-glycerol-3-phosphate ethanolamine (DSPE).

[0181] In some embodiments, immunomodulatory (e.g., immunostimulatory or immunosuppressive) nucleic acid sequences are bonded or linked to the following lipids via adapters, wherein the nucleotide bonds are as follows:

[0182] ,

[0183] Or its salt, wherein X is O or S.

[0184] Immunomodulatory (e.g., immunostimulatory or immunosuppressive) nucleic acid sequences can bind directly to lipids. Alternatively, immunomodulatory (e.g., immunostimulatory or immunosuppressive) nucleic acid sequences can be linked to lipids via adapters.

[0185] Lipids and amphiphiles including lipids in this article should be understood to include their pharmaceutically acceptable salts.

[0186] connector

[0187] In some embodiments, the compound comprises an immunomodulatory (e.g., immunostimulatory or immunosuppressive) nucleic acid sequence conjugated to a polypeptide via a linker and linked to an albumin-binding domain (e.g., a lipid). The linker may be a hydrophilic polymer, a string of hydrophilic amino acids, a polysaccharide, and an oligonucleotide, or a combination thereof. The linker may reduce or prevent the albumin-binding domain from inserting into the plasma membrane of cells, such as cells in tissue adjacent to the injection site. The linker may also reduce or prevent the amphiphilic immunomodulatory (e.g., immunostimulatory or immunosuppressive) nucleic acid sequence from nonspecifically associating with extracellular matrix proteins at the site of administration. For efficient delivery of the amphiphilic immunomodulatory (e.g., immunostimulatory or immunosuppressive) nucleic acid sequence to lymph nodes, it should remain soluble. A polar blocking linker may be included between an immunomodulatory (e.g., immunostimulatory or immunosuppressive) nucleic acid sequence and its conjugated albumin-binding domain to increase the solubility of the amphiphilic immunomodulatory (e.g., immunostimulatory or immunosuppressive) nucleic acid sequence.

[0188] The length and composition of the linker can be tailored based on the selected albumin-binding domain and immunomodulatory (e.g., immunostimulatory or immunosuppressive) nucleic acid sequence. For example, in some embodiments, the polynucleotide itself may have sufficient polarity to ensure solubility; for example, a polynucleotide of 10, 15, 20, or more nucleotides in length. Therefore, in some embodiments, an additional linker is not required. However, in some cases, it may be desirable to include a linker that mimics the function of a polar oligonucleotide. The linker can be used as part of any albumin-binding domain conjugate described herein, such as lipid-oligonucleotide conjugates and lipid-peptide conjugates, thereby reducing cell membrane insertion / preferential dispensing to albumin.

[0189] Suitable linkers include, but are not limited to, oligonucleotides, such as those discussed above, including a string of nucleic acids; hydrophilic polymers, including but not limited to polyethylene glycol (MW: 500 Da to 20,000 Da), polyacrylamide (MW: 500 Da to 20,000 Da), and polyacrylic acid; a string of hydrophilic amino acids, such as serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, or combinations thereof; and polysaccharides, including but not limited to dextran (MW: 1,000 Da to 2,000,000 Da) or combinations thereof. A hydrophobic albumin-binding domain and a linker / immunomodulatory (e.g., immunostimulatory or immunosuppressive) nucleic acid sequence are covalently linked. The covalent bond can be an incleivable or cleavable bond. An incleivable bond can include an amide bond or a phosphate bond, and a cleavable bond can include a disulfide bond, an acid-cleavable bond, an ester bond, an anhydride bond, a biodegradable bond, or an enzyme-cleavable bond.

[0190] In some embodiments, the linker is one or more ethylene glycol (EG) units, more preferably two or more EG units (i.e., polyethylene glycol (PEG)). For example, in some embodiments, the compound includes an immunomodulatory (e.g., immunostimulatory or immunosuppressive) nucleic acid sequence and a hydrophobic albumin-binding domain linked by a polyethylene glycol (PEG) molecule or a derivative or analog thereof.

[0191] In some embodiments, the compounds described herein contain an immunomodulatory (e.g., immunostimulatory or immunosuppressive) nucleic acid sequence conjugated to a polypeptide and linked to a PEG, wherein the PEG is in turn linked to a hydrophobic albumin-binding domain (e.g., a lipid). The exact number of PEG units depends on the albumin-binding domain and the loading; however, typically, the linker may have between about 1 and about 100 PEG units (e.g., 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, 36, 37, 38, 39, 40, 41, 42, 43, ...). 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 PEG units). In some embodiments, the connector may be a PEG connector having between about 20 and about 80, between about 30 and about 70, or between about 40 and about 60 PEG units. In some embodiments, the number of PEG units is between 24 and 50 (e.g., between 24 and 45, 24 and 40, 24 and 35, 24 and 30, 30 and 50, 35 and 50, 40 and 50, and between 45 and 50). In some embodiments, the connector has between about 45 and 55 PEG units. For example, in some embodiments, the connector has 48 PEG units. In some embodiments, the connector comprises a PEG4-amide-PEG4 connector.

[0192] In some embodiments, the connector may contain one or more alkyl groups. In some embodiments, the connector may contain a combination of alkyl groups and PEG groups. For example, the connector may be a hexylamine-PEG4 connector.

[0193] As discussed above, in some embodiments, the adapter is an oligonucleotide comprising a string of nucleic acids. In some embodiments, the compounds described herein comprise an immunomodulatory (e.g., immunostimulatory or immunosuppressive) nucleic acid sequence linked to a string of nucleic acids, which in turn is linked to a hydrophobic albumin-binding domain (e.g., lipid). The adapter can be any sequence; for example, the oligonucleotide sequence can be a random sequence or a sequence specifically selected for its molecular or biochemical characteristics (e.g., high polarity). In some embodiments, the adapter comprises one or more sequential sequences of adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U), or analogues thereof. In some embodiments, the adapter consists of a sequential sequence of adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U), or analogues thereof.

[0194] In some embodiments, a nucleic acid string contains between 1 and 50 nucleic acid residues. In some embodiments, a nucleic acid string contains between 5 and 30 nucleic acid residues. In some embodiments, the linker contains one or more guanine residues, such as between 1 and 10 guanine residues.

[0195] In some embodiments, the linker is an oligonucleotide comprising a string of amino acids. In some embodiments, an amphiphilic immunomodulatory (e.g., immunostimulatory or immunosuppressive) nucleic acid sequence conjugated to a polypeptide is linked to a string of amino acids, which in turn are linked to a hydrophobic albumin-binding domain (e.g., lipid). The linker can have any amino acid sequence; for example, the sequence of the oligonucleotide can be a random sequence or a sequence selected for its molecular or biochemical properties (e.g., high flexibility). In some embodiments, the linker comprises a series of glycine residues to form a polyglycine linker. In some embodiments, the linker comprises (Gly) nThe amino acid sequence, where n can be between 2 and 20 residues. Examples of polyglycine linkers include, but are not limited to, GGG, GGGA (SEQ ID NO:1), GGGG (SEQ ID NO:2), GGGAG (SEQ ID NO:3), GGGAGG (SEQ ID NO:4), GGGAGGG (SEQ ID NO:5), GGAG (SEQ ID NO:6), GGSG (SEQ ID NO:7), AGGG (SEQ ID NO:8), SGGG (SEQ ID NO:8) NO:9), GGAGGA (SEQ ID NO:10), GGSGGS (SEQ ID NO:11), GGAGGAGGA (SEQ ID NO:12), GGSGGSGGS (SEQ ID NO:13), GGAGGAGGAGGA (SEQ ID NO:14), GGSGGSGGSGGS (SEQ ID NO:15), GGAGGAG (SEQ ID NO:16), GGSGGGSG (SEQ ID NO:15) NO:17), GGAGGAGGGAG (SEQ ID NO:18), GGGSGGGSGGGSG (SEQ ID NO:19), GGGGAGGGGAGGGGA (SEQ ID NO:20) and GGGGSGGGGSGGGGS (SEQ ID NO:21).

[0196] Joining methods

[0197] This article describes compounds comprising an immunomodulatory (e.g., immunostimulatory or immunosuppressive) nucleic acid sequence and an albumin-binding domain, wherein the immunomodulatory (e.g., immunostimulatory or immunosuppressive) domain comprises a functional group ( Figure 1B This document also describes compounds comprising an immunomodulatory (e.g., immunostimulatory or immunosuppressive) nucleic acid sequence and an albumin-binding domain, wherein the albumin-binding domain comprises a functional group. The functional group may be maleimide, dithio(2-pyridyldithio), cyclooctene, cyclooctyne, aldehyde, azide, or alkyne. In some embodiments, the functional group is capable of reacting with a peptide to conjugate the immunomodulatory (e.g., immunostimulatory or immunosuppressive) nucleic acid sequence to the peptide. In some embodiments, the functional group is capable of reacting with a peptide to conjugate the albumin-binding domain to the peptide.

[0198] The peptide may be modified with an N-terminal cysteine, acetylcysteine, thiol, trans-cyclooctene, cyclooctyne, azide, or alkyne to conjugate an immunomodulatory (e.g., immunostimulatory or immunosuppressive) nucleic acid sequence and an albumin-binding domain. In some embodiments, the peptide may be modified with a C-terminal cysteine, azide, or alkyne to conjugate an immunomodulatory (e.g., immunostimulatory or immunosuppressive) nucleic acid sequence and an albumin-binding domain. In some embodiments, an internal cysteine ​​or lysine residue of the peptide may be used to conjugate an immunomodulatory (e.g., immunostimulatory or immunosuppressive) nucleic acid sequence and an albumin-binding domain.

[0199] An immunomodulatory (e.g., immunostimulatory or immunosuppressive) domain or an albumin-binding domain may be bonded to or linked to a adapter. In some embodiments, the adapter includes functional groups. In some embodiments, the functional groups are capable of conjugating peptides. For example, an immunomodulatory (e.g., immunostimulatory or immunosuppressive) nucleic acid sequence may be linked to the adapter, wherein the adapter is modified with functional groups. In some embodiments, the adapter may be a PEG adapter.

[0200] In some embodiments, immunomodulatory (e.g., immunostimulatory or immunosuppressive) oligonucleotides are conjugated to peptides via a reaction between a dithio group and a free thiol group. Exemplary methods for preparing immunomodulatory (e.g., immunostimulatory or immunosuppressive) nucleic acid sequences, albumin-binding domains, and functional groups are provided in Examples 3-5 below.

[0201] adjuvant

[0202] In some embodiments, the pharmaceutical compositions described herein may be administered in combination with one or more adjuvants. An adjuvant is a substance that stimulates the immune system. In this context, an adjuvant is used to enhance an immune response to one or more antigens. The adjuvant may be administered to the subject before, in combination with, or after the administration of the antigen. In some embodiments, an additional adjuvant may be administered to the subject in combination with an immunomodulatory (e.g., immunostimulatory or immunosuppressive) nucleic acid sequence described herein that is conjugated to a peptide and an albumin-binding domain. In some embodiments, the adjuvant may be conjugated to an albumin-binding domain (e.g., a lipid). Adjuvants may be, but are not limited to, lipids (e.g., monophosphoryl lipid A (MPLA)), alum (e.g., aluminum hydroxide, aluminum phosphate); Freund's adjuvant; saponins purified from the bark of the soapberry tree (Q. saponaria), such as QS21 (glycolipid eluted in the 21st peak by HPLC fractionation; Antigenics, Inc., Worcester, Massachusetts); poly(di(carboxyphenoxy)phosphazene) (PCPP polymer; Virus Research Institute, USA), Flt3 ligand, Leishmania extension factor (purified Leishmania protein; Corixa Corporation, Seattle, Washington); ISCMS (an immunostimulatory complex containing a mixture of saponins and lipids and forming virus-sized particles with pores capable of containing antigens; CSL Corporation, Melbourne, Australia). Melbourne, Australia), Pam3Cys, SB-AS4 (SmithKline Beecham adjuvant system #4, containing alum and MPL; SBB, Belgium), micelle-forming nonionic block copolymers (such as CRL 1005) (these contain hydrophobic polyoxypropylene straight chains with polyoxyethylene chains attached to the sides, Vaxcel, Inc., Norcross, Georgia), and Montanide IMS (e.g., IMS1312, water-based nanoparticles in combination with soluble immunostimulants, Seppic), and CDN (cyclic dinucleotides).

[0203] Adjuvants can be toll-like receptor (TLR) ligands. Adjuvants acting via TLR3 include, but are not limited to, double-stranded RNA. Adjuvants acting via TLR4 include, but are not limited to, lipopolysaccharide derivatives such as monophospholipid A (MPLA: Ribi ImmunoChem Research, Inc., Hamilton, Montana), muramyl dipeptide (MDP; Ribi), and threonyl-muramyl dipeptide (t-MDP; Ribi); OM-174 (a lipid A-associated glucosamine disaccharide; OM Pharma SA, Meyrin, Switzerland). Adjuvants acting via TLR5 include, but are not limited to, flagellin. Adjuvants acting via TLR7 and / or TLR8 include single-stranded RNA, oligonucleotides (ORNs), and synthetic low molecular weight compounds such as imidazoquinolineamines (e.g., imiquimod (R-837), retsimote (R-848)). Adjuvants that function via TLR9 include viral or bacterial DNA, or synthetic oligodeoxynucleotides (ODNs), such as CpG ODN. For example, CpG ODN can have sequences such as TCGTCGTTTTGTCGTTTTGTCGTT-3' (SEQ ID NO: 25), 5'-TGACTGTGAACGTTCGAGATGA-3' (SEQ ID NO: 26), 5'-TCGTCGTTTTCGGCGCGCGCCG-3' (SEQ ID NO: 27), or 5'-TCCATGACGTTCCTGACGTT-3' (SEQ ID NO: 29). The bonds in CpG can be entirely phosphate thioester bonds.

[0204] Another class of adjuvants are molecules containing thiophosphates (such as thiophosphate nucleotide analogs) and nucleic acids containing thiophosphate backbone bonds.

[0205] Pharmaceutical Composition

[0206] This document describes pharmaceutical compositions comprising any of an immunomodulatory (e.g., immunostimulatory or immunosuppressive) nucleic acid sequence conjugated to a peptide and an albumin-binding domain. In addition to therapeutic amounts of the immunomodulatory (e.g., immunostimulatory or immunosuppressive) nucleic acid sequence conjugated to a peptide and an albumin-binding domain as described herein, the pharmaceutical compositions may contain pharmaceutically acceptable carriers or excipients, which may be formulated by methods known to those skilled in the art. Pharmaceutically acceptable salts of the components, as described herein, may also be included.

[0207] Acceptable carriers and excipients in pharmaceutical compositions of immunomodulatory (e.g., immunostimulatory or immunosuppressive) nucleic acid sequences conjugated to peptides and albumin-binding domains as described herein are non-toxic to the recipient at the employed doses and concentrations. In some embodiments, one or more formulation materials are used for subcutaneous (sc) and / or intravenous (iv) administration. In some embodiments, one or more formulation materials are used for intramuscular administration. In some embodiments, administration is by inhalation or intranasal administration. In some embodiments, one or more formulation materials are used for intraperitoneal, topical, or oral administration. In some embodiments, the pharmaceutical composition may contain formulation materials for altering, maintaining, or preserving, for example, the composition's pH, osmotic pressure, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption, or permeation. In some embodiments, suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine, or lysine); antimicrobial agents; antioxidants (such as ascorbic acid, methionine, sodium sulfite, or sodium bisulfite); buffers (such as borates, bicarbonates, Tris-HCl, citrates, HEPES, TAE, phosphates, or other organic acids); swelling agents (such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, β-cyclodextrin, or hydroxypropyl-β-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, sucrose, mannose, or dextran); proteins (such as human serum albumin, gelatin, dextran, and immunoglobulins); colorants, flavorings, and diluents; emulsifiers; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight peptides; and salts. Balanced ions (such as sodium); preservatives (such as hexamethyl diammonium chloride, octadecyl dimethyl benzyl ammonium chloride, resorcinol, and benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide); solvents (such as glycerol, propylene glycol, or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronic acid derivatives). ic), PEG, dehydrated sorbitol esters, polysorbate esters (such as polysorbate 20, polysorbate 80), triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancers (such as sucrose or sorbitol); tension enhancers (such as alkali metal halides, preferably sodium chloride or potassium chloride, mannitol, sorbitol); delivery media; diluents; excipients and / or pharmaceutical adjuvants.(Remington's Pharmaceutical Sciences, 18th Edition, AR Gennaro, editor, Mack Publishing Company (1995)). In some embodiments, the optimal pharmaceutical composition will be determined by those skilled in the art based on, for example, the intended route of administration, delivery form, and required dosage. See, for example, Remington's Pharmaceutical Sciences, ibid. In some embodiments, such compositions may affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the amphiphilic conjugate.

[0208] In some embodiments, the primary medium or carrier in a pharmaceutical composition comprising an immunomodulatory (e.g., immunostimulatory or immunosuppressive) nucleic acid sequence conjugated herein with a peptide and an albumin-binding domain may be aqueous or non-aqueous in nature. For example, in some embodiments, a suitable medium or carrier may be water for injection, physiological saline, or artificial cerebrospinal fluid, possibly supplemented with other materials common in compositions for parenteral administration. In some embodiments, the saline comprises isotonic phosphate-buffered saline. In some embodiments, neutral buffered saline or saline mixed with serum albumin are additional exemplary mediums. In some embodiments, the pharmaceutical composition comprises a Tris buffer at about pH 7.0–8.5, or an acetate buffer at about pH 4.0–5.5, which may further comprise sorbitol or a suitable alternative thereof. In some embodiments, compositions comprising the immunomodulatory (e.g., immunostimulatory or immunosuppressive) nucleic acid sequences conjugated herein with peptides and albumin-binding domains can be prepared for storage by mixing the selected composition, having the desired purity, with an optional formulation (Remington's Pharmaceutical Sciences, ibid.) in the form of a lyophilized cake or aqueous solution. Further, in some embodiments, compositions comprising the immunomodulatory (e.g., immunostimulatory or immunosuppressive) nucleic acid sequences conjugated herein with peptides and albumin-binding domains can be formulated as lyophilized products using suitable excipients (such as sucrose).

[0209] In some embodiments, the pharmaceutical composition may be selected for parenteral delivery. The preparation of such pharmaceutically acceptable compositions is within the capabilities of those skilled in the art.

[0210] In some embodiments, the formulation components are present at a concentration acceptable at the application site. In some embodiments, a buffer solution is used to maintain the composition at a physiological pH or slightly lower, typically in the pH range of about 5 to about 8.

[0211] In some embodiments, when considering parenteral administration, the therapeutic composition may be in the form of a pyrogen-free, parenterically acceptable aqueous solution containing, in a pharmaceutically acceptable medium, an immunomodulatory (e.g., immunostimulatory or immunosuppressive) nucleic acid sequence conjugated herein with a peptide and an albumin-binding domain. In some embodiments, the medium for parenteral injection is sterile distilled water in which the immunomodulatory (e.g., immunostimulatory or immunosuppressive) nucleic acid sequence conjugated herein with a peptide and an albumin-binding domain is formulated into a suitably preserved sterile isotonic solution. In some embodiments, the formulation may include a formulation of the desired molecule with reagents such as injectable microspheres, bio-erosive particles, polymeric compounds such as polylactic acid or polyglycolic acid, beads, or liposomes, which may provide controlled or sustained release of the product, which may then be delivered via reservoir injection. In some embodiments, hyaluronic acid may also be used and may have an effect that promotes duration of circulation. In some embodiments, an implantable drug delivery device may be used to introduce the desired molecule.

[0212] The pharmaceutical composition may be administered in a therapeutically effective amount to induce an immune response. The therapeutically effective amount of the immunomodulatory (e.g., immunostimulatory or immunosuppressive) nucleic acid sequence described herein, conjugated with a peptide and an albumin-binding domain, contained in the pharmaceutical formulation, can be determined by those skilled in the art such that said dose (e.g., a dose in the range of 0.01-100 mg / kg body weight) induces an immune response in a subject.

[0213] Vectors that can be used as in vivo nucleic acid delivery mediators include, but are not limited to, retroviral vectors, adenoviral vectors, poxvirus vectors (e.g., vaccinia virus vectors, such as modified Ankara vaccinia virus (MVA)), adeno-associated virus vectors, and alpha virus vectors. In some embodiments, the vector may include an internal ribosome entry site (IRES) that allows expression of one of the various coronavirus antigens described herein (e.g., coronavirus spike protein, its polypeptide, or nucleic acid sequence encoding it). Other mediators and methods for nucleic acid delivery are described in, for example, U.S. Patent Nos. 5,972,707, 5,697,901, and 6,261,554 (each of which is incorporated herein by reference in its entirety). Other methods for producing pharmaceutical compositions are described in, for example, U.S. Patent Nos. 5,478,925, 8,603,778, 7,662,367, and 7,892,558 (all of which are incorporated herein by reference in their entirety).

[0214] In some embodiments, the pharmaceutical compositions described herein may be administered together with one or more adjuvants.

[0215] Route of administration, dosage and time

[0216] Pharmaceutical compositions disclosed herein containing, as therapeutic agents, immunomodulatory (e.g., immunostimulatory or immunosuppressive) nucleic acid sequences conjugated herein with peptides and albumin-binding domains can be formulated for parenteral, subcutaneous, intravenous, intramuscular, intranasal, or inhalation administration. In some embodiments, the therapeutic agent is formulated for transmucosal administration. In some embodiments, the therapeutic agent is formulated for buccal administration. In some embodiments, the therapeutic agent is formulated for sublingual administration. Methods of administering the therapeutic protein are known in the art. See, for example, U.S. Patent Nos. 6,174,529, 6,613,332, 8,518,869, 7,402,155, and 6,591,129, and U.S. Patent Application Publication Nos. US20140051634, WO 1993000077, and US 20110184145, the disclosures of which are incorporated herein by reference in their entirety.

[0217] One or more of these methods can be used to administer the pharmaceutical compositions of the present invention, said pharmaceutical compositions containing an immunomodulatory (e.g., immunostimulatory or immunosuppressive) nucleic acid sequence conjugated to a polypeptide and an albumin-binding domain. Various effective drug carriers are known in the art for injectable formulations. See, for example, *Pharmaceutics and Pharmacy Practice*, JB Lippincott Company, Philadelphia, PA, eds., Banker and Chalmers, pp. 238-250 (1982), and *ASHP Handbook on Injectable Drugs*, Toissel, 4th edition, pp. 622-630 (1986). The dosage of the pharmaceutical compositions of the present invention depends on factors including the route of administration and the physical characteristics of the subject (e.g., age, weight, general health status). Typically, the amount of the polypeptide-conjugated and albumin-binding domain-conjugated immunomodulatory (e.g., immunostimulatory or immunosuppressive) nucleic acid sequence described herein contained in a single dose can be an amount that effectively induces an immune response in a subject without inducing significant toxicity. The pharmaceutical compositions of the present invention may comprise, in doses ranging from 0.001 to 500 mg (e.g., 0.01, 0.05, 0.1, 0.2, 0.3, 0.5, 0.7, 0.8, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 10 mg, 15 mg, 20 mg, 30 mg, 50 mg, 100 mg, 250 mg, or 500 mg) and, in more specific embodiments, from about 0.1 to about 100 mg of the polypeptide-conjugated and albumin-binding domain-conjugated immunomodulatory (e.g., immunostimulatory or immunosuppressive) nucleic acid sequence described herein. Clinicians may adjust the dosage according to different parameters of the subject.

[0218] The pharmaceutical composition of the present invention, containing an immunomodulatory (e.g., immunostimulatory or immunosuppressive) nucleic acid sequence conjugated to a polypeptide and an albumin-binding domain, may be administered to a subject in need once or more (e.g., 1-10 times or more) once, for example, daily, weekly, monthly, semi-annually, annually, or as medically necessary.

[0219] Methods of inducing immune responses

[0220] This disclosure provides a method for inducing an immune response against an antigen in a subject. The method includes administering any of the compounds described herein and an antigen to the subject.

[0221] In some embodiments, this disclosure provides a method for inducing an immune response against an antigen in a subject, the method being performed by administering to the subject any of an immunomodulatory (e.g., immunostimulatory or immunosuppressive) nucleic acid sequence conjugated with a peptide and an albumin-binding domain, and further administering an adjuvant. In some embodiments, the immunomodulatory (e.g., immunostimulatory or immunosuppressive) nucleic acid sequence conjugated with both a peptide and an albumin-binding domain may be administered without one or more additional adjuvants.

[0222] In some embodiments, the method includes administering to a subject a therapeutically effective amount of an immunomodulatory (e.g., immunostimulatory or immunosuppressive) nucleic acid sequence conjugated herein with a peptide and an albumin-binding domain. In some embodiments, the immunomodulatory (e.g., immunostimulatory or immunosuppressive) nucleic acid sequence conjugated with a peptide and an albumin-binding domain is administered substantially simultaneously. In some embodiments, the immunomodulatory (e.g., immunostimulatory or immunosuppressive) nucleic acid sequence conjugated with a peptide and an albumin-binding domain is administered alone.

[0223] The polypeptide can be an antigen or a fragment thereof. For example, the polypeptide can be derived from a tumor (e.g., a tumor-associated antigen) or from a viral or bacterial source. In some embodiments, the antigen is an influenza antigen or a fragment thereof. In some embodiments, the antigen is an influenza nucleoprotein or a fragment thereof. In some embodiments, the influenza nucleoprotein comprises a polypeptide sequence having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with SEQ ID NO: 22. In some embodiments, the influenza nucleoprotein comprises a polypeptide sequence having at least 95% (e.g., at least 96%, 97%, 98%, 99%, or 100%) sequence identity with SEQ ID NO: 22. In some embodiments, the influenza nucleoprotein comprises the polypeptide sequence of SEQ ID NO: 22. In some embodiments, the antigen is a coronavirus antigen or a fragment thereof. For example, the antigen is a coronavirus spike protein or a fragment thereof, or a coronavirus nucleocapsid protein or a fragment thereof.

[0224] In some embodiments, one or more components are pharmaceutically acceptable salts of the indicated components, as described herein.

[0225] In some embodiments, the immune response provides protection against infection. For example, the immune response can provide protection against influenza infection or SARS-CoV-2 infection.

[0226] In some embodiments, the immune response has a protective effect against COVID-19 disease.

[0227] In some embodiments, this disclosure provides a method for inducing an immune response against an antigen in a subject, the method being performed by subcutaneously administering any of the compounds described herein or a pharmaceutically acceptable salt thereof to the subject. In some embodiments, this disclosure provides a method for inducing an immune response against an antigen in a subject, the method being performed by administering the antigen intramuscularly, subcutaneously, intravenously, intraperitoneally, topically, orally / buccally, sublingually, transmucosally, intranasally, or by inhalation to the subject.

[0228] In some embodiments, the subject is a mammal. For example, the subject can be a human.

[0229] Reagent test kit

[0230] The kit may include the immunomodulatory (e.g., immunostimulatory or immunosuppressive) nucleic acid sequences conjugated with peptides and albumin-binding domains as disclosed herein, along with instructions for use. The kit may include, in a suitable container, the immunomodulatory (e.g., immunostimulatory or immunosuppressive) nucleic acid sequences conjugated with peptides and albumin-binding domains, one or more controls, and various buffers, reagents, enzymes, and other standard components well known in the art. In some embodiments, the kit further includes an adjuvant. Thus, in some embodiments, the immunomodulatory (e.g., immunostimulatory or immunosuppressive) nucleic acid sequences conjugated with peptides and albumin-binding domains are in vials. In some embodiments, poly(dA) and / or poly(dT) nucleic acid sequences, poly(dG) and / or poly(dC) nucleic acid sequences, poly(dA) and / or poly(dT) nucleic acid sequences conjugated with albumin-binding domains, poly(dG) and / or poly(dC) nucleic acid sequences conjugated with albumin-binding domains, or ISD or immunostimulatory HSV sequences conjugated with albumin-binding domains and adjuvants are in separate vials. In some embodiments, the antigen or nucleic acid encoding the antigen and adjuvant are in the same vial. In some embodiments, the antigen or nucleic acid encoding the antigen and the adjuvant are in separate vials.

[0231] The container may include at least one vial, well, test tube, flask, bottle, syringe, or other container device to which an immunomodulatory (e.g., immunostimulatory or immunosuppressive) nucleic acid sequence conjugated to a peptide and to an albumin-binding domain is contained, and in some cases appropriately aliquoted. When additional components are provided, the kit may contain additional containers to which such compounds can be placed. The kit may also include a device for containing an immunomodulatory (e.g., immunostimulatory or immunosuppressive) nucleic acid sequence conjugated to a peptide and to an albumin-binding domain, and any other tightly sealed reagent container for commercial sale. Such containers may include injection or blow-molded plastic containers in which the desired vials are held. The container and / or kit may include labels with instructions for use and / or warnings.

[0232] In some embodiments, this disclosure provides a kit comprising a drug and a package insert, the drug comprising a composition comprising an immunomodulatory (e.g., immunostimulatory or immunosuppressive) nucleic acid sequence conjugated to a peptide and conjugated to an albumin-binding domain, and optionally a pharmaceutically acceptable carrier, the package insert comprising instructions for administration of the drug alone or in combination with a composition comprising an adjuvant and optionally a pharmaceutically acceptable carrier, for the treatment of a disease or condition (e.g., influenza or SARS-CoV-2), for delaying the progression of a disease or condition (e.g., influenza or SARS-CoV-2), or for the prevention of a disease or condition (e.g., influenza or SARS-CoV-2), wherein the immunomodulatory (e.g., immunostimulatory or immunosuppressive) nucleic acid sequence is conjugated to a peptide and conjugated to an albumin-binding domain, and optionally comprises a linker. The peptide may be an antigen or a fragment thereof. For example, the peptide may be derived from a tumor (e.g., a tumor-associated antigen) or a viral or bacterial source. In some embodiments, the peptide may be an influenza antigen or a fragment thereof. In some embodiments, the peptide may be an influenza nucleoprotein or a fragment thereof. In some embodiments, the peptide may be a coronavirus antigen or a fragment thereof.

[0233] In some embodiments, this disclosure provides a kit comprising a container and a package insert, the container comprising a composition containing an immunomodulatory (e.g., immunostimulatory or immunosuppressive) nucleic acid sequence conjugated to a peptide and to an albumin-binding domain, and optionally a pharmaceutically acceptable carrier, the package insert comprising instructions for administering the composition vaccine in a subject, wherein the immunomodulatory (e.g., immunostimulatory or immunosuppressive) nucleic acid sequence is conjugated to a peptide and to an albumin-binding domain, and optionally includes a linker. In some embodiments, the kit further comprises an adjuvant and instructions for administering the adjuvant.

[0234] In some embodiments of the kit, one or more components of the kit are pharmaceutically acceptable salts of the components as described herein.

[0235] Example

[0236] The following examples are provided to illustrate, and not limit, this disclosure, to provide those skilled in the art with a description of how the compositions and methods described herein can be used, implemented, and evaluated. These examples are intended to be merely illustrative of this disclosure and are not intended to limit the scope of what inventors would consider their invention.

[0237] Example 1. Effect of CpG polypeptide chimeras on immunogenicity

[0238] This experiment aimed to determine whether the conjugation of gp100 peptide-antigen with CpG adjuvant affects immunogenicity compared to the corresponding mixed vaccine, and whether the AMP conjugation of the chimera (Figure 1) can increase its immunogenicity to that of the soluble form.

[0239] Five groups of C57BL / 6J mice (n=5 per group) were administered a vaccine containing the components listed in Table 1.

[0240] Table 1. Summary of mouse vaccine administration

[0241]

[0242] Gp100 stock solution was dissolved in horseshoe crab amoeboids (LAL) H2O at a peptide concentration of 0.84 mg / mL. The final injection solution was diluted with 1x PBS. Mice were injected with a solution containing 10 μg peptide / 100 μL.

[0243] Both soluble and amphiphilic chimeras were conjugated at a 1:1 peptide:adjuvant ratio. Chip concentrations were determined according to Table 2 below. Each injection contained 10 μg of peptide in the chimera. This corresponds to 4.31 nmol of adjuvant (calculated as in Table 3). Vaccine components are described in Table 4.

[0244] The adjuvant stock solution was prepared by resuspending the horseshoe crab amoeboids lysate (LAL) H₂O. The final injection solution was diluted with 1x phosphate-buffered saline (PBS) to achieve a (AMP-)CpG concentration of 4.3 nmol / 100 μL injection solution. 4.3 nmol corresponds to the mass present in the chimera at a concentration of 10 µg of peptide.

[0245] Table 2: UV-Vis Concentration Measurement

[0246]

[0247] -

[0248] Table 3: Vaccine Component Ratios

[0249]

[0250] Subcutaneous (SC) immunization was administered to the base of the tail of female B6 mice, 50 μL bilaterally. Booster doses were given approximately every two weeks. SC injections ensure optimal delivery of the vaccine to the lymph nodes via natural lymphatic drainage, and every two weeks was determined to be optimal for an immune response.

[0251] Table 4: Vaccine Components

[0252]

[0253] Seven days after dose 2 (boost dose), ICS (intracytoplasmic staining) assays targeting TNFα and IFNγ were performed on blood and lung samples. Figure 4 and Figure 5 Cell surface staining was performed for CD4 and CD8; intracellular staining was performed for CD3 (see Table 5 for antibody information). ICS samples were activated with 2 μg / ml gp100 (EGP) peptide for approximately 19–20 hours (in the presence of brevidin A and monensin) (see Table 6).

[0254] Table 5: Antibodies used in ICS

[0255]

[0256] Table 6: Restimulation peptides

[0257]

[0258] Seven days after the booster dose, tetramer / PE-H-2Db gp100 (EGSRNQDWL) (SEQ ID NO: 24) was used. Figure 3 Tetramer analysis was performed.

[0259] Seven days after booster dose administration, ELISpot analysis of spleen cells targeting IFNγ was performed. Spleen cells (0.1 x 10⁻⁶) were analyzed. 6 Cells / well) were activated with 2 μg / ml gp100 (EGP) peptide (see Table 6). IFNγ ELISpot plates were stimulated overnight. Figure 2 ).

[0260] AMP conjugation of peptides and adjuvants increases their immunogenicity compared to their soluble forms, as measured by ELISpot on spleen cells and ICS on PBMCs and lung-associated lymphoid cells. Additionally, conjugation of peptides to CpG7909 also increases the immunogenicity of the compounds compared to the corresponding mixed components. This may be due to increased molecular size or other mechanisms leading to better lymph node clearance, and simultaneous exposure of antigen-presenting cells (APCs) to both the peptide and adjuvant. Furthermore, when the chimera is re-AMP conjugated, this further increases the immunogenicity of the compounds due to the complex effect of the covalently linked peptide-CpG pair, the benefits of delivery to and retention in lymph nodes provided by AMP conjugation, and the activation of APCs. Furthermore, conjugation of peptides to oligonucleotides significantly improves the water solubility of peptides, especially insoluble peptides, thereby allowing for the formulation of peptides for administration.

[0261] Example 2. Effect of polydeoxythymidine polypeptide chimeras on immunogenicity

[0262] This experiment was designed to determine whether the conjugation of gp100 peptide-antigen to polydeoxythymidine adjuvant affects immunogenicity compared to corresponding mixed vaccines, and the conjugation of amphiphiles to chimeras. Figure 6 Does it increase its immunogenicity, making it higher than the soluble form?

[0263] Five groups of C57BL / 6J mice (n=5 per group) were administered a vaccine containing the components listed in Table 7.

[0264] Table 7. Summary of mouse vaccine administration

[0265]

[0266] The gp100 stock solution was prepared by dissolving the peptide in horseshoe crab amoeboid cell lysate (LAL) H2O at a concentration of 0.84 mg / mL. The final injection solution was diluted with 1x PBS to provide 10 μg peptide / 100 μL.

[0267] Both soluble and amphiphilic chimeras were conjugated at a 1:1 peptide:adjuvant ratio, and the concentration of the chimeras was determined according to Table 8. Each injection contained 10 μg of peptide in the chimera. This corresponds to 4.31 nmol of adjuvant (calculated as in Table 9). The vaccine components are described in Table 10.

[0268] The adjuvant stock solution was prepared by resuspending in horseshoe crab amoeboid cell lysate (LAL) H2O. The final injection solution was diluted with 1x phosphate-buffered saline (PBS) to achieve a (AMP-)dT concentration of 4.3 nmol / 100 μL injection solution (4.3 nmol is equivalent to the mass present in the chimera at 10 µg of peptide).

[0269] Table 8: UV-Vis Concentration Measurement

[0270]

[0271] -

[0272] Table 9: Vaccine Component Ratios

[0273]

[0274] Subcutaneous (SC) immunization was administered to the base of the tail of female B6 mice, 50 μL bilaterally. Booster doses were given approximately every two weeks. SC injections ensure optimal delivery of the vaccine to the lymph nodes via natural lymphatic drainage, and every two weeks was determined to be optimal for an immune response.

[0275] Table 10: Vaccine Components

[0276]

[0277] Seven days after dose 2 (boost dose), ICS (intracytoplasmic staining) assays targeting TNFα and IFNγ were performed on blood and lung samples. Figure 9 and Figure 10 Cell surface staining was performed for CD4 and CD8; intracellular staining was performed for CD3 (see Table 11 for antibody information). ICS samples were activated with 2 μg / ml gp100 (EGP) peptide for approximately 19–20 hours (in the presence of brevidin A and monensin) (see Table 12).

[0278] Table 11: Antibodies used in ICS

[0279]

[0280] Table 12: Restimulated peptides

[0281]

[0282] Seven days after the booster dose, tetramer / PE-H-2Db gp100 (EGSRNQDWL) (SEQ ID NO: 24) was used. Figure 8 Tetramer analysis was performed.

[0283] Seven days after booster dose administration, ELISpot analysis of spleen cells targeting IFNγ was performed. Spleen cells (0.1 x 10⁻⁶) were analyzed. 6 Cells / well) were activated with 2 μg / ml gp100 (EGP) peptide (see Table 12). IFNγ ELISpot plates were stimulated overnight. Figure 7 ).

[0284] Compared to soluble chimeras and AMP-conjugated peptide-DNA mixtures, AMP conjugation of peptide-DNA chimeras increased their immunogenicity. These data suggest that covalently linking antigens to DNA adjuvants on an AMP platform increases the immunogenicity of these constructs.

[0285] The constructs described in Examples 1 and 2 are prepared below.

[0286] 1. Preparation of CpG-PEG4-Mal(3)

[0287] 5.0 mg of CpG-3'-amine (MW 7893.3) was dissolved in 500 μL of 20 mM pH 8.0 phosphate buffer, and then 5 mg of NHS-PEG4-Mal (MW 513.5, 15-fold) was added. The mixture was incubated at room temperature for 30 min and purified by RP-HPLC [phenylhexyl semi-preparative column (10 mm x 150 mm), mobile phase A (25 mM TEA pH 8.0), mobile phase B (100% MeCN), flow rate (3 mL / min), gradient (10%-30% over 30 min)] and separated by lyophilization to obtain CpG-PEG4-Mal 3.

[0288] 2. Preparation of AMP-CpG-PEG4-Mal(4)

[0289] 5.0 mg of Amp-CpG-3'-amine (MW 8595.5) was dissolved in 500 μL of 20 mM pH 8.0 phosphate buffer, followed by the addition of 5 mg of NHS-PEG4-Mal (MW 513.5, 16-fold). The mixture was incubated at room temperature for 30 min. AMP-CpG-PEG4-Mal 4 was purified by RP-HPLC [phenylhexyl semi-preparative column (10 mm x 150 mm), mobile phase A (50 mMTEA pH 5.8), mobile phase B (100% MeCN), flow rate (3 mL / min), gradient (10%–90% over 30 min)] and separated by lyophilization. The product was confirmed by MALDI-TOF MS (molecular weight: calculated 8993.9, determined 8999.9).

[0290] 3. Preparation of CpG-peptide (maleimide bond) CpG-PEG4-gp100 (5a)

[0291] CpG-PEG4-Mal 3 (1 equivalent) and gp100 peptide containing N-terminal acetylcysteine ​​residues (1.5 equivalent) were reacted at room temperature for 1 hour. The mixture was purified by RP-HPLC [phenylhexyl semi-preparative column (10 mm x 150 mm), mobile phase A (50 mM TEA pH 5.8), mobile phase B (100% MeCN), flow rate (3 mL / min), gradient (10%–30% over 30 min)], and separated by lyophilization and salt exchange to sodium salt to give 5. The product was confirmed by MALDI-TOF MS (molecular weight: calculated 10612.9, determined 10613.5).

[0292] 4. Preparation of AMP-CpG-peptide (maleimide-AcCys bond) AMP-CpG-PEG4-gp100 (1a)

[0293] AMP-CpG-PEG4-Mal 4 (1 equivalent) and gp100 peptide containing N-terminal acetylcysteine ​​residues (1.5 equivalent) were reacted at room temperature for 1 hour. The mixture was purified by RP-HPLC [phenylhexyl semi-preparative column (10 mm x 150 mm), mobile phase A (50 mM TEA pH 5.8), mobile phase B (100% MeCN), flow rate (3 mL / min), gradient (10%-90% over 30 min)], and separated by lyophilization and salt exchange to sodium salt to give 1a. The product was confirmed by MALDI-TOF MS (molecular weight: calculated 11315.1, determined 11317.3).

[0294] 5. Preparation of CpG-peptide (disulfide (SS) bond) CpG-PEG4-gp100 (5b)

[0295] 2.0 mg of CpG-3'-amine (MW 7893.3) was dissolved in 200 μL of 20 mM pH 8.0 phosphate buffer, and then 1.5 mg of PDP-PEG4-NHS ((2-pyridyldithio-)propionyl-PEG4-NHS, MW 559.6, 10-fold) was added. The mixture was incubated at room temperature for 2 hours. Excess PDP-PEG4-NHS was removed by salt exchange using a NAP-5 column to obtain CpG-PEG4-PDP (6), which was reacted with a gp100 peptide (1.5 equivalents) containing an N-terminal acetylcysteine ​​residue at room temperature for 1 hour. The mixture was purified by RP-HPLC [phenylhexyl semi-preparative column (10 mm x 150 mm), mobile phase A (50 mM TEA pH 5.8), mobile phase B (100% MeCN), flow rate (3 mL / min), gradient (10%-30% over 30 min)] and separated by lyophilization. The product was confirmed by MALDI-TOF MS (molecular weight: calculated 10548.8, determined 10547.1).

[0296] 6. Preparation of Amp-CpG-peptide (maleimide-Cys bond) AMP-CpG-PEG4-gp100 (1b)

[0297] Amp-CpG-PEG4-Mal (1 equivalent) and gp100 peptide containing N-terminal cysteine ​​residues (1.5 equivalent) were reacted at room temperature for 1 hour. The mixture was purified by RP-HPLC [phenylhexyl semi-preparative column (10 mm x 150 mm), mobile phase A (50 mM TEA pH 5.8), mobile phase B (100% MeCN), flow rate (3 mL / min), gradient (10%-90% over 30 min)] and separated by lyophilization. The product was confirmed by MALDI-TOF MS (molecular weight: calculated 11273, determined 11270).

[0298] 7.dT 50 -peptide (maleimide-AcCys bond) dT 50 Preparation of PEG4-gp100(6)

[0299] 10.0 mg dT 50 3'-amine (MW 16129.8) was dissolved in 500 μL of 20 mM pH 8.0 phosphate buffer, followed by the addition of 3.2 mg NHS-PEG4-Mal (MW 513.5, 10-fold). After 30 min, the mixture was desalted using a NAP-5 column and then reacted with gp100 peptide (2.0 equivalents) containing N-terminal acetylcysteine ​​residues at room temperature for 1 h. The mixture was purified by RP-HPLC [Biobasic-C4 column (4.6 mm x 250 mm), mobile phase A (50 mM TEA pH 5.8), mobile phase B (100% MeCN), gradient (10%–30% over 30 min)], and the fractions were lyophilized to obtain 6. The product was confirmed by MALDI-TOF MS (molecular weight: calculated 11849.7, determined 11850.0).

[0300] 8.AMP-dT 50 -Peptide (maleimide bond -AcCys)AMP-dT 50 Preparation of PEG4-gp100(2)

[0301] 6.5 mg AMP-dT 503'-amine (MW 16832.2) was dissolved in 500 μL of 20 mM pH 8.0 phosphate buffer, followed by the addition of 3 mg NHS-PEG4-Mal (MW 513.5, 10 equivalents). The mixture was incubated at room temperature for 30 min and desalted using a NAP-5 column, then reacted with a gp100 peptide (1.5 equivalents) containing an N-terminal acetylcysteine ​​residue at room temperature for 1 h. The mixture was purified by RP-HPLC [phenylhexyl semi-preparative column (10 mm x 150 mm), mobile phase A (50 mM TEA pH 5.8), mobile phase B (100% MeCN), flow rate (3 mL / min), gradient (10%–90% over 30 min)], and the fractions were lyophilized to obtain product 2. The product was confirmed by MALDI-TOF MS (molecular weight: calculated 19551.8, determined 19540).

[0302] Example 3. Strategies for preparing 5' AMP nucleotides

[0303] The preparation of 5' amphiphilic nucleotides is described in this example. The process is carried out according to the reaction described in Scheme 1, wherein N-(2-hydroxy-3-octadecanoylaminopropyl)octadecanoamide 7 is subjected to oxidative conditions (such as, but not limited to, Moffit, Swern, Des Martin periodoyl alkyl, TPAP, PDC, and PPC) to give N,N'-(2-oxopropane-1,3-diyl)distearate amide 8.

[0304] Treatment of cyanide sources (such as, but not limited to, sodium cyanide, lithium cyanide, potassium cyanide, trimethylcyanosilane and acetone cyanohydrin) in the presence of catalytic acids (such as, but not limited to, hydrogen chloride, sodium bisulfite, acetic acid and Lewis acids (such as, but not limited to, zinc iodide and ytterbium trifluoromethanesulfonate (III)))8 yields cyanohydrin9.

[0305] The nitrile was reduced to primary amine 10 by means of a reagent (such as, but not limited to, hydrogen in the presence of a metal catalyst (such as, but not limited to, palladium, palladium hydroxide, nickel, Raney nickel, nickel dichloride, platinum dioxide (IV)) or a metal hydride (such as, but not limited to, sodium tetraborane hydrate or lithium tetraborane hydrate) or borane-tetrahydrofuran or tris(pentafluorophenyl)borane / trimethylsilane).

[0306] The conversion of amine 10 to amide 12 can be achieved by treatment with a known polyethylene glycol-modified, base-labile, protected amine 11 containing PEG units (but not limited to n = ...). 0-12) and contains an activated ester (such as, but not limited to, N-hydroxysuccinimide ester or perfluorophenol ester) or as a carboxylic acid that can be activated by a reagent such as, but not limited to, p-toluenesulfonyl chloride, pentanoyl chloride, 2,4,6-trichlorobenzoyl chloride, N,N'-dicyclohexanecarbodiimide, (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, propanephosphonic anhydride and (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, hexafluorophosphate azirzotriazole tetramethylurea, and the carboxylic acid contains a base-unstable N protecting group (such as, but not limited to, 1,3-dithiazide-2-ylmethoxycarbonyl, dimethyl-1,3-dithiazide-2-ylmethoxycarbonyl, trifluoroacetyl and 9-fluorenylmethyloxycarbonyl).

[0307] The formation of phosphoramide 13 can be achieved by treatment 12 with the following P(III) reagent, such as, but not limited to, 3-((chloro(diisopropylamino)phosphino)oxy)propionitrile (X = Cl) or 3-((bis(diisopropylamino)phosphino)oxy)propionitrile (X = isopropylamide).

[0308] Phosphoramide 13 can be coupled to an oligonucleotide using solid-phase synthesis (SPS) P(III) as the final nucleotide 5' coupling cycle. Catalysts used for coupling include, but are not limited to, 1H-tetrazole, 5-ethylthio-1H-tetrazole, 2-benzylthiotetrazole, and 4,5-dicyanimidazolium. Thiolization can be achieved by treatment with (but not limited to): 3-(dimethylaminomethylene)amino-3H-1,2,4-dithiazol-3-thione, 3H-1,2-benzodithiol-3-one 1,1-dioxide, N,N,N'N'-tetraethylthiuram disulfide, and 3-amino-1,2,4-dithiazol-5-thione. Deprotection of the oligonucleotide and the "NH-PG" group can be achieved by reaction with an amine base (but not limited to) (such as tert-butylamine, diethylamine, and piperidine), followed by oligonucleotide cleavage and deprotection, purification and separation using techniques commonly used in oligonucleotide SPS, yielding compound 12.

[0309] The addition of the maleimide functional group required for antigen conjugation can be achieved by the following treatment 12: a usable maleimide fragment, such as, but not limited to, 15, said 15 containing a PEG unit (but not limited to, m = 0-12) and containing an activated ester (such as, but not limited to, N-hydroxysuccinimide ester and perfluorophenol ester) or as a carboxylic acid that can be activated by a reagent such as, but not limited to, p-toluenesulfonyl chloride, pentanoyl chloride, 2,4,6-trichlorobenzoyl chloride, N,N'-dicyclohexanecarbodiimide, (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, propanephosphonic anhydride and (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, hexafluorophosphate azizobenzotriazole tetramethyluranium, to obtain the penultimate compound 16.

[0310] 16 was treated with a thiol-containing antigen, followed by conjugated addition of thiol-maleimide to obtain target compound 17.

[0311] Option 1.

[0312]

[0313] Example 4. Strategies for preparing 5' AMP-antigen nucleotides

[0314] The preparation of a 5' amphiphilic antigen is described in this example. This process is carried out according to the reaction described in Scheme 2, where the preparation of a 5' amphiphilic antigen is described in this example. Compound 12 as described in Example 3 can be treated with a known compound 18 having a PEG unit (but not limited to, n = 0-12), and wherein LG is defined as a leaving group, such as, but not limited to, methanesulfonate, toluenesulfonate, chloride, bromide, or iodide, and wherein P is an alcohol protecting group, such as, but not limited to, acetate, trimethylsilyl, tert-butyldimethylsilyl, benzyl, and p-methoxybenzyl, benzyl carbonate, allyl carbonate, triphenylmethyl, p-methoxyphenyldiphenylmethyl, tetrahydropyranyl, and methoxymethyl, and Y is, but not limited to, CH2 or CH2NH(CO)CH2. Deprotection of the P group of the alcohol is carried out by treatment with (but not limited to) a weak acid (0.1-0.5 N HCl), toluenesulfonic acid or acetic acid, or alternatively by alkaline hydrolysis (such as, but not limited to, potassium carbonate / methanol or sodium hydroxide), or alternatively by hydrogenolysis in the presence of palladium, palladium hydroxide, nickel, Raney nickel, nickel dichloride, platinum dioxide (IV) (such as, but not limited to, hydrogen or hydrogen source), or alternatively by fluoride-induced desilylation with (but not limited to) tetrabutylammonium fluoride, hydrogen fluoride or triethylamine hydrogen fluoride, or alternatively by oxidative cleavage with (but not limited to) 2,3-dichloro-5,6-dicyanobenzoquinone, to give compound 19.

[0315] The formation of phosphoramide 20 can be achieved by the following treatment 19: P(III) reagent, such as, but not limited to, 3-((chloro(diisopropylamino)phosphino)oxy)propionitrile (X = Cl) or 3-((bis(diisopropylamino)phosphino)oxy)propionitrile (X = isopropylamide).

[0316] Phosphoramide 20 can be coupled to an oligonucleotide using solid-phase synthesis (SPS) P(III) as the final nucleotide 5' coupling cycle. Catalysts used for coupling include, but are not limited to, 1H-tetrazole, 5-ethylthio-1H-tetrazole, 2-benzylthiotetrazole, and 4,5-dicyanimidazolium. Thiolization can be achieved by treatment with (but not limited to): 3-(dimethylaminomethylene)amino-3H-1,2,4-dithiazol-3-thione, 3H-1,2-benzodithiol-3-one 1,1-dioxide, N,N,N'N'-tetraethylthiuram disulfide, and 3-amino-1,2,4-dithiazol-5-thione. Deprotection of the oligonucleotide and the "NH-PG" group can be achieved by reaction with an amine base (but not limited to) (such as tert-butylamine, diethylamine, and piperidine), followed by oligonucleotide cleavage and deprotection, purification and separation using techniques commonly used in oligonucleotide SPS, yielding compound 21.

[0317] The addition of the maleimide functional group required for antigen conjugation can be achieved by the following treatment 21: a usable maleimide fragment 15, said maleimide fragment 15 being functionalized with a PEG unit (but not limited to, m = between 0 and 12), containing an activated ester (such as N-hydroxysuccinimide ester and perfluorophenol ester) or as a carboxylic acid that can be activated by a reagent (but not limited to), said reagent such as p-toluenesulfonyl chloride, pentanoyl chloride, 2,4,6-trichlorobenzoyl chloride, N,N'-dicyclohexanecarbodiimide, (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, propanephosphonic anhydride and (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, hexafluorophosphate azizobenzotriazole tetramethyluranium, to obtain the penultimate compound 22.

[0318] 22 was treated with a thiol-containing antigen, followed by conjugated addition of thiol-maleimide to give target compound 23.

[0319] Option 2.

[0320]

[0321] Alternatively, this process can be carried out according to the reaction described in Scheme 3, which describes the preparation of the 5' amphiphilic antigen.

[0322] Compound 24 (where R1, together with an oxoamine, produces a carbamate, such as, but not limited to, tert-butyl, O-benzyl, O-allyl, O-trimethylsilylethyl) can be subjected to oxidative conditions, such as, but not limited to, Moffit, Swern, Des Martin periodoyl alkyl, TPAP, PDC, and PPC, to give keto-carbamate 25.

[0323] Treatment of 25 with cyanide sources (such as, but not limited to, sodium cyanide, lithium cyanide, potassium cyanide, trimethylcyanosilane and acetone cyanohydrin) in the presence of catalytic acids (such as, but not limited to, hydrogen chloride, sodium bisulfite, acetic acid and Lewis acids (such as, but not limited to, zinc iodide and ytterbium trifluoromethanesulfonate (III))) yields cyanohydrin 26.

[0324] The nitrile was reduced to primary amine 27 by means of a reagent (such as, but not limited to, hydrogen in the presence of a metal catalyst (such as, but not limited to, palladium, palladium hydroxide, nickel, Raney nickel, nickel dichloride, platinum dioxide (IV)) or a metal hydride (such as, but not limited to, sodium tetraborane hydrate or lithium tetraborane hydrate) or borane-tetrahydrofuran or tri(pentafluorophenyl)borane / trimethylsilane).

[0325] The conversion of amine 27 to amide 28 can be achieved by treatment with a known polyethylene glycol-modified, base-labile, protected amine 11 containing PEG units (but not limited to n = ...). 0-12) and contains an activated ester (such as, but not limited to, N-hydroxysuccinimide ester or perfluorophenol ester) or as a carboxylic acid that can be activated by a reagent such as, but not limited to, p-toluenesulfonyl chloride, pentanoyl chloride, 2,4,6-trichlorobenzoyl chloride, N,N'-dicyclohexanecarbodiimide, (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, propanephosphonic anhydride and (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, hexafluorophosphate azirzotriazole tetramethylurea, and the carboxylic acid contains a base-unstable N protecting group (such as, but not limited to, 1,3-dithiazide-2-ylmethoxycarbonyl, dimethyl-1,3-dithiazide-2-ylmethoxycarbonyl, trifluoroacetyl and 9-fluorenylmethyloxycarbonyl).

[0326] Deprotection of carbamate 28 can be achieved by treatment with (but not limited to) acidic hydrolysis conditions (such as, but not limited to, TFA and HCl) or hydrogenolysis conditions (such as with hydrogen or hydrogen donors (such as, but not limited to, ammonium formate or cyclohexadiene) and metal catalysts (such as, but not limited to, palladium, palladium hydroxide, nickel, Raney nickel, nickel dichloride, platinum dioxide (IV))) or fluoride sources (such as, but not limited to, tetrabutylammonium fluoride, hydrogen fluoride or triethylamine hydrogen fluoride) to obtain diamine 29.

[0327] The amidation of 29 can be achieved by using an activated stearic acid ester (such as, but not limited to, N-hydroxysuccinimide ester and perfluorophenol ester) or directly with stearic acid and a reagent (such as, but not limited to, p-toluenesulfonyl chloride, pentanoyl chloride, 2,4,6-trichlorobenzoyl chloride, N,N'-dicyclohexanecarbodiimide, (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, propanephosphonic anhydride and (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, hexafluorophosphate azizobenzotriazole tetramethyluranium) or directly with a suitable stearyl chloride to give compound 12.

[0328] Option 3.

[0329]

[0330] Alternatively, the process can be carried out according to the reaction described in Scheme 4, wherein compound 28 (as described above) can be treated with a known compound 18 having a PEG unit (but not limited to, n = 0-12), and wherein LG is defined as a leaving group, such as, but not limited to, methanesulfonate, toluenesulfonate, chloride, bromide or iodide, and wherein P is an alcohol protecting group, such as, but not limited to, acetate, trimethylsilyl, tert-butyldimethylsilyl, benzyl, and p-methoxybenzyl, benzyl carbonate, allyl carbonate, and Y is, but not limited to, CH2 or CH2NH(CO)CH2, to obtain compound 29.

[0331] Deprotection of carbamate 29 can be achieved by treatment with (but not limited to) acidic hydrolysis conditions (such as, but not limited to, TFA and HCl) or hydrogenolysis conditions (such as with hydrogen or a hydrogen donor (such as, but not limited to, ammonium formate or cyclohexadiene) and a metal catalyst (such as, but not limited to, palladium, palladium hydroxide, nickel, Raney nickel, nickel dichloride, platinum dioxide (IV))) or a fluoride source (such as, but not limited to, tetrabutylammonium fluoride, hydrogen fluoride or triethylamine hydrogen fluoride) to obtain diamine 30.

[0332] The amidation of 30 can be achieved by using an activated stearic acid ester (such as, but not limited to, N-hydroxysuccinimide ester and perfluorophenol ester) or directly with stearic acid and a reagent (such as, but not limited to, p-toluenesulfonyl chloride, pivaloyl chloride, 2,4,6-trichlorobenzoyl chloride, N,N'-dicyclohexanecarbodiimide, (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, propanephosphonic anhydride and (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, hexafluorophosphate azizobenzotriazole tetramethyluranium) or directly with a suitable stearyl chloride to give compound 31.

[0333] The deprotection of the hydroxyl P group of compound 31 is carried out by treatment with (but not limited to) a weak acid (0.1-0.5 N HCl), toluenesulfonic acid, or acetic acid, or alternatively by alkaline hydrolysis (such as, but not limited to, potassium carbonate / methanol or sodium hydroxide), or alternatively by hydrogenolysis in the presence of palladium, palladium hydroxide, nickel, Raney nickel, nickel dichloride, or platinum dioxide (IV) (such as, but not limited to, hydrogen or a hydrogen source), or alternatively by fluoride-induced desilylation with (but not limited to) tetrabutylammonium fluoride, hydrogen fluoride, or triethylamine hydrogen fluoride, or alternatively by oxidative cleavage with (but not limited to) 2,3-dichloro-5,6-dicyanobenzoquinone, to give compound 32.

[0334] Compound 32 can be treated with (but not limited to) P(III) reagents, such as 3-((chloro(diisopropylamino)phosphinyl)oxy)propionitrile (X = Cl) or 3-((bis(diisopropylamino)phosphinyl)oxy)propionitrile (X = isopropylamide), to give phosphoramide 20.

[0335] Option 4.

[0336]

[0337] Alternatively, the strategy can be implemented according to the reaction described in Scheme 5, wherein orthogonally functionalized branched synthons (such as, but not limited to, amino acid derivatives 33 and amine 34) are coupled to peptides using reagents (such as, but not limited to, N,N'-diisopropylcarbodiimide, N,N'-dicyclohexylcarbodiimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) and activators (such as, but not limited to, 1-hydroxybenzotriazole and 3-hydroxytriazolo[4,5-b]pyridine) to obtain 35.

[0338] Alternatively, compounds 33 and 34 are treated with a benzotriazole N-oxide reagent (such as, but not limited to, 3-[bis(dimethylamino)methylonyl]-3H-benzotriazole-1-oxide hexafluorophosphate, ((1H-benzo[d][1,2,3]triazol-1-yl)oxy)tri(dimethylamino)phosphonium hexafluorophosphate (V)), or an azabenzotriazole N-oxide reagent (such as, but not limited to, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazol[4,5-b]pyridinium 3-oxide hexafluorophosphate, (benzotriazole-1-yloxy)tripyrrolylphosphonium hexafluorophosphate or (7-azabenzotriazole-1-yloxy)tripyrrolylphosphonium hexafluorophosphate) to obtain compound 35.

[0339] Acid-catalyzed deprotection yields diamino alcohol 36. Amidation of 36 can be achieved by using a stearic acid-activated ester (such as, but not limited to, N-hydroxysuccinimide ester and perfluorophenol ester) or directly with stearic acid and a reagent (such as, but not limited to, p-toluenesulfonyl chloride, pivaloyl chloride, 2,4,6-trichlorobenzoyl chloride, N,N'-dicyclohexanecarbodiimide, (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, propanephosphonic anhydride, and (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, hexafluorophosphate azizobenzotriazole tetramethyluranium) or directly with a suitable stearyl chloride to give compound 37.

[0340] The formation of phosphoramide 38 can be achieved by treatment 37 with a P(III) reagent, such as, but not limited to, 3-((chloro(diisopropylamino)phosphino)oxy)propionitrile (X = Cl) or 3-((bis(diisopropylamino)phosphino)oxy)propionitrile (X = isopropylamide). A series of similar reactions described in Scheme 1 (compounds 13 to 17) provide similar 5' AMP antigen nucleotides.

[0341] Option 5.

[0342]

[0343] Example 5. Strategies for preparing 3' AMP-antigen nucleotides

[0344] The preparation of the 3' amphiphilic antigen is described in this example. The process is carried out according to the reaction described in Scheme 6, wherein the base-unstable protecting group (PG) of 12 (Example 3) is deprotected with an amine base (such as, but not limited to, diethylamine, tert-butylamine, or piperidine) to obtain a primary amine. Compound 28 is provided by treating a primary amine with a available maleimide 15 (Example 3), said maleimide 15 being functionalized with a PEG unit (but not limited to, m = between 0 and 12) and containing an activated ester (such as N-hydroxysuccinimide ester and perfluorophenol ester) or as a carboxylic acid that can be activated by reagents (but not limited to), such as p-toluenesulfonyl chloride, pentanoyl chloride, 2,4,6-trichlorobenzoyl chloride, N,N'-dicyclohexanecarbodiimide, (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, propanephosphonic anhydride, and (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, hexafluorophosphate azizobenzotriazole tetramethyluranium.

[0345] The etherification of 39 can be accomplished using a known compound 29 having a PEG unit (but not limited to, p = 0-12), and wherein LG is defined as a leaving group, such as, but not limited to, methanesulfonate, toluenesulfonate, chloride, bromide or iodide, and Y is, but not limited to, CH2 or CH2NH(CO)CH2, and wherein R2 is an acid-labile group, such as, but not limited to, tert-butyl, allyl, benzyl, p-methoxybenzyl, tetrahydropyranyl, or alternatively silyl groups, such as, but not limited to, tert-butyldimethylsilyl or tert-butyldiphenylsilyl, to obtain 40.

[0346] Hydrolysis of 40 under acidic conditions (such as, but not limited to, acetic acid, trifluoroacetic acid, or hydrogen chloride) or alternatively with a source of hydrogen fluoride (such as, but not limited to, tetrabutylammonium fluoride, hydrogen fluoride, or triethylamine hydrogen fluoride) yields compound 41.

[0347] Carboxylic acid 41 was activated with reagents (such as, but not limited to, p-toluenesulfonyl chloride, pentanoyl chloride, 2,4,6-trichlorobenzoyl chloride, N,N'-dicyclohexanecarbodiimide, (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, propanephosphonic anhydride, and (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, hexafluorophosphate azizobenzotriazole tetramethyluranium) and treated with 3'-amine-functionalized oligonucleotides and oligonucleotides with 3'-amine side chains to obtain 42.

[0348] The target compound 43 was obtained by treating 42 with a thiol-containing antigen and then undergoing conjugated addition with a thiol-maleimide.

[0349] Option 6.

[0350]

[0351] Alternatively, the strategy can be implemented according to the reaction described in Scheme 7. Under polypeptide coupling conditions (such as, but not limited to, N,N'-diisopropylcarbodiimide, N,N'-dicyclohexylcarbodiimide, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide), in the presence of an activator (such as, but not limited to, 1-hydroxybenzotriazole and 3-hydroxytriazolo[4,5-b]pyridine), orthogonally functionalized branched synthons (such as, but not limited to, compound 44) ​​are treated with amine 34 (Scheme 5) to obtain compound 45.

[0352] Alternatively, compounds 44 and 35 are treated with a benzotriazole N-oxide reagent (such as, but not limited to, 3-[bis(dimethylamino)methylonyl]-3H-benzotriazole-1-oxide hexafluorophosphate, ((1H-benzo[d][1,2,3]triazol-1-yl)oxy)tri(dimethylamino)phosphonium hexafluorophosphate (V)), or an azabenzotriazole N-oxide reagent (such as, but not limited to, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazol[4,5-b]pyridinium 3-oxide hexafluorophosphate, (benzotriazole-1-yloxy)tripyrrolidinylphosphonium hexafluorophosphate, or (7-azabenzotriazole-1-yloxy)tripyrrolidinylphosphonium hexafluorophosphate) to obtain compound 45.

[0353] 45 is deprotected with a base (such as, but not limited to, tert-butylamine, diethylamine, and piperidine), and then a suitable maleimide fragment 15 is added, which is functionalized with a PEG unit (but not limited to, m = between 0 and 12) and contains an activated ester (such as N-hydroxysuccinimide ester and perfluorophenol ester) or as a carboxylic acid that can be activated by a reagent (but not limited to), such as p-toluenesulfonyl chloride, pentanoyl chloride, 2,4,6-trichlorobenzoyl chloride, N,N'-dicyclohexanecarbodiimide, (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, propanephosphonic anhydride, and (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, hexafluorophosphate azizobenzotriazole tetramethyluranium, to obtain compound 46.

[0354] The carboxylic acid is deprotected under acidic conditions (such as, but not limited to, trifluoroacetic acid or hydrogen chloride), and then activated with reagents (such as, but not limited to, p-toluenesulfonic anhydride, pentanoyl chloride, 2,4,6-trichlorobenzoyl chloride, N,N'-dicyclohexanecarbodiimide, (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, propanephosphonic anhydride and (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, hexafluorophosphate azizobenzotriazole tetramethyluranium), and treated with 3'-amine-functionalized oligonucleotides (such as, but not limited to, CpG-3'-amine) to give compound 47, which is used as a synthon containing a thiol antigen.

[0355] Option 7.

[0356]

[0357] Numbered Examples

[0358] 1. A compound or a pharmaceutically acceptable salt thereof, said compound comprising an immunostimulatory nucleic acid sequence, an albumin-binding domain, and a polypeptide.

[0359] 2. The compound as described in Example 1 or a pharmaceutically acceptable salt thereof, wherein the albumin-binding domain is bonded to or linked to the 5' end of the immunostimulatory nucleic acid sequence, and wherein the polypeptide is bonded to or linked to the 3' end of the immunostimulatory nucleic acid sequence.

[0360] 3. The compound as described in Example 1 or a pharmaceutically acceptable salt thereof, wherein the polypeptide is bonded or linked to the 5' end of the immunostimulatory nucleic acid sequence, and wherein the albumin-binding domain is bonded or linked to the 3' end of the immunostimulatory nucleic acid sequence.

[0361] 4. The compound as described in Example 1 or a pharmaceutically acceptable salt thereof, wherein both the polypeptide and the albumin-binding domain are bonded to or linked to the 5' end of the immunostimulated nucleic acid sequence.

[0362] 5. The compound as described in Example 1 or a pharmaceutically acceptable salt thereof, wherein both the polypeptide and the albumin-binding domain are bonded to or linked to the 3' end of the immunostimulated nucleic acid sequence.

[0363] 6. The compound of any one of Examples 1-5 or a pharmaceutically acceptable salt thereof, wherein the polypeptide comprises an N-terminal modification.

[0364] 7. The compound as described in Example 6 or a pharmaceutically acceptable salt thereof, wherein the N-terminal modification is the addition of acetylcysteine.

[0365] 8. The compound as described in Example 7 or a pharmaceutically acceptable salt thereof, wherein the N-terminal modification is the addition of a deaminocysteine ​​homolog.

[0366] 9. The compound as described in Example 8 or a pharmaceutically acceptable salt thereof, wherein the deaminocysteine ​​homolog is 3-mercaptopropionic acid or mercaptoacetic acid.

[0367] 10. The compound or a pharmaceutically acceptable salt thereof as described in any one of Examples 1-9, wherein the immunostimulatory nucleic acid sequence is a polydeoxyadenosine (poly-dA) nucleic acid sequence.

[0368] 11. The compound or a pharmaceutically acceptable salt thereof as described in any one of Examples 1-9, wherein the immunostimulatory nucleic acid sequence is a polydeoxythymidine (polydT) nucleic acid sequence.

[0369] 12. The compound or a pharmaceutically acceptable salt thereof as described in Example 10 or 11, wherein the compound comprises a poly(dA) nucleic acid sequence and a poly(dT) nucleic acid sequence.

[0370] 13. The compound as described in Example 12 or a pharmaceutically acceptable salt thereof, wherein the poly(dA) nucleic acid sequence and the poly(dT) nucleic acid sequence hybridize to form a double-stranded DNA sequence.

[0371] 14. The compound or a pharmaceutically acceptable salt thereof as described in Example 12 or 13, wherein the poly-dA nucleic acid sequence and the poly-dT nucleic acid sequence comprise the same number of nucleotides.

[0372] 15. The compound or a pharmaceutically acceptable salt thereof as described in any one of Examples 1-9, wherein the immunostimulatory nucleic acid sequence is a polydeoxyguanosine (polydG) nucleic acid sequence.

[0373] 16. The compound or a pharmaceutically acceptable salt thereof as described in any one of Examples 1-9, wherein the immunostimulatory nucleic acid sequence is a polydeoxycytosine (polydC) nucleic acid sequence.

[0374] 17. The compound or a pharmaceutically acceptable salt thereof as described in Example 15 or 16, wherein the compound comprises a polydG nucleic acid sequence and a polydC nucleic acid sequence.

[0375] 18. The compound as described in Example 17 or a pharmaceutically acceptable salt thereof, wherein the polydG nucleic acid sequence and the polydC nucleic acid sequence hybridize to form a double-stranded DNA sequence.

[0376] 19. The compound or a pharmaceutically acceptable salt thereof as described in Example 17 or 18, wherein the polydG nucleic acid sequence and the polydC nucleic acid sequence comprise the same number of nucleotides.

[0377] 20. The compound or a pharmaceutically acceptable salt thereof as described in any one of Examples 1-19, wherein the poly-dA nucleic acid sequence, poly-dT nucleic acid sequence, poly-dC nucleic acid sequence and / or poly-dG nucleic acid sequence comprises between 30 and 100 nucleotides.

[0378] 21. The compound as described in Example 20 or a pharmaceutically acceptable salt thereof, wherein the poly-dA nucleic acid sequence, poly-dT nucleic acid sequence, poly-dC nucleic acid sequence and / or poly-dG nucleic acid sequence comprises between 50 and 100 nucleotides.

[0379] 22. The compound as described in Example 20 or a pharmaceutically acceptable salt thereof, wherein the poly-dA nucleic acid sequence, poly-dT nucleic acid sequence, poly-dC nucleic acid sequence and / or poly-dG nucleic acid sequence comprises between 30 and 50 nucleotides.

[0380] 23. The compound as described in Example 20 or a pharmaceutically acceptable salt thereof, wherein the poly-dA nucleic acid sequence, poly-dT nucleic acid sequence, poly-dC nucleic acid sequence and / or poly-dG nucleic acid sequence comprises 30, 40, 50, 75 or 100 nucleotides.

[0381] 24. The compound or a pharmaceutically acceptable salt thereof as described in any one of Examples 10-23, wherein the poly-dA nucleic acid sequence, poly-dT nucleic acid sequence, poly-dG nucleic acid sequence and / or poly-dC nucleic acid sequence comprises a mixture of dA, dT, dC and / or dG nucleic acid residues.

[0382] 25. The compound as described in Example 24 or a pharmaceutically acceptable salt thereof, wherein the poly-dA nucleic acid sequence comprises between 100% and 51% dA nucleic acid residues and between 0% and 49% dT, dC, and / or dG nucleic acid residues.

[0383] 26. The compound as described in Example 24 or a pharmaceutically acceptable salt thereof, wherein the poly-dT nucleic acid sequence comprises between 100% and 51% dT nucleic acid residues and between 0% and 49% dA, dC, and / or dG nucleic acid residues.

[0384] 27. The compound as described in Example 24 or a pharmaceutically acceptable salt thereof, wherein the polydG nucleic acid sequence comprises between 100% and 51% dG nucleic acid residues and between 0% and 49% dC, dA, or dT nucleic acid residues.

[0385] 28. The compound as described in Example 24 or a pharmaceutically acceptable salt thereof, wherein the poly-dC nucleic acid sequence comprises between 100% and 51% dC nucleic acid residues and between 0% and 49% dG, dA, or dT nucleic acid residues.

[0386] 29. The compound or a pharmaceutically acceptable salt thereof as described in any one of Examples 1-24, wherein the immunostimulatory nucleic acid sequence is a CpG sequence.

[0387] 30. The compound as described in Example 29 or a pharmaceutically acceptable salt thereof, wherein the CpG sequence is sequence 5'-TCGTCGTTTTGTCGTTTTGTCGTT-3' (SEQ ID NO: 25).

[0388] 31. The compound as described in Example 29 or a pharmaceutically acceptable salt thereof, wherein the CpG sequence is 5'-TGACTGTGAACGTTCGAGATGA-3' (SEQ ID NO: 26).

[0389] 32. The compound as described in Example 29 or a pharmaceutically acceptable salt thereof, wherein the CpG sequence is 5'-TCGTCGTTTTCGGCGCGCGCCG-3' (SEQ ID NO: 27).

[0390] 33. The compound as described in Example 29 or a pharmaceutically acceptable salt thereof, wherein the CpG sequence is 5'-TCCATGACGTTCCTGACGTT-3' (SEQ ID NO: 29).

[0391] 34. The compound or a pharmaceutically acceptable salt thereof as described in any one of Examples 1-28, wherein at least one internucleotide group connecting the nucleotides in the poly-dA, poly-dT, poly-dC, or poly-dG nucleic acid sequences is a phosphodiester.

[0392] 35. The compound or a pharmaceutically acceptable salt thereof as described in any one of Examples 1-28, wherein all internucleotide groups connecting the nucleotides in the poly(dA), poly(dT), poly(dC), or poly(dG) nucleic acid sequences are phosphate thioesters.

[0393] 36. The compound or a pharmaceutically acceptable salt thereof as described in any one of Examples 1-35, wherein the polypeptide is an antigen or a fragment thereof.

[0394] 37. The compound or a pharmaceutically acceptable salt thereof as described in Example 36, wherein the antigen is derived from a tumor, virus, or bacterial source.

[0395] 38. The compound or a pharmaceutically acceptable salt thereof as described in Example 36, wherein the antigen or a fragment thereof is a tumor-associated antigen.

[0396] 39. The compound or a pharmaceutically acceptable salt thereof as described in Example 36, wherein the antigen is an influenza antigen or a fragment thereof.

[0397] 40. The compound as described in Example 39 or a pharmaceutically acceptable salt thereof, wherein the antigen is an influenza nucleoprotein or a fragment thereof.

[0398] 41. The compound as described in Example 37 or a pharmaceutically acceptable salt thereof, wherein the antigen is a coronavirus antigen or a fragment thereof.

[0399] 42. The compound as described in Example 41 or a pharmaceutically acceptable salt thereof, wherein the antigen is a coronavirus spike protein or a fragment thereof.

[0400] 43. The compound as described in Example 41 or a pharmaceutically acceptable salt thereof, wherein the antigen is a coronavirus nucleocapsid protein or a fragment thereof.

[0401] 44. The compound or a pharmaceutically acceptable salt thereof as described in any one of Examples 1-43, wherein the albumin-binding domain is a lipid.

[0402] 45. The compound as described in Example 44 or a pharmaceutically acceptable salt thereof, wherein the lipid is a diacyl lipid.

[0403] 46. ​​The compound of Example 45 or a pharmaceutically acceptable salt thereof, wherein the diacyl lipid comprises an acyl chain comprising 12-30 hydrocarbon units, 14-25 hydrocarbon units, 16-20 hydrocarbon units, or 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 hydrocarbon units.

[0404] 47. The compound or a pharmaceutically acceptable salt thereof as described in Example 45 or Example 46, wherein the lipid is 1,2-distearate-sn-glycerol-3-phosphatidylethanolamine (DSPE).

[0405] 48. The compound or a pharmaceutically acceptable salt thereof as described in any one of Examples 1-47, wherein the albumin-binding domain and the polypeptide are bonded or linked by a linker selected from the group consisting of hydrophilic polymers, a string of hydrophilic amino acids, polysaccharides and oligonucleotides or combinations thereof.

[0406] 49. The compound as described in Example 48 or a pharmaceutically acceptable salt thereof, wherein the connector comprises “N” polyethylene glycol units, wherein N is between 4 and 50.

[0407] 50. The compound as described in Example 49 or a pharmaceutically acceptable salt thereof, wherein the connector comprises PEG4-amide-PEG4.

[0408] 51. The compound or a pharmaceutically acceptable salt thereof as described in any one of Examples 1-50, wherein the immunostimulatory nucleic acid sequence is bonded or linked to the following lipid via a linker, wherein the nucleotide bond is as follows:

[0409]

[0410] or its salt,

[0411] Where X is O or S.

[0412] 52. A compound or a pharmaceutically acceptable salt thereof, said compound or a pharmaceutically acceptable salt thereof comprising an immunostimulatory nucleic acid sequence, an albumin-binding domain, and a functional group or a pharmaceutically acceptable salt thereof.

[0413] 53. The compound as described in Example 52 or a pharmaceutically acceptable salt thereof, wherein the functional group comprises maleimide, dithio(2-pyridyldithio), cyclooctene, cyclooctyne, aldehyde, azide or alkyne.

[0414] 54. The compound or a pharmaceutically acceptable salt thereof as described in Examples 52 or 53, wherein the albumin-binding domain is bonded to or linked to the 5' end of the immunostimulated nucleic acid sequence.

[0415] 55. The compound or a pharmaceutically acceptable salt thereof as described in Examples 52 or 53, wherein the albumin-binding domain is bonded to or linked to the 3' end of the immunostimulated nucleic acid sequence.

[0416] 56. The compound or a pharmaceutically acceptable salt thereof as described in any one of Examples 52-55, wherein the immunostimulatory nucleic acid sequence is a polydeoxyadenosine (poly-dA) nucleic acid sequence.

[0417] 57. The compound or a pharmaceutically acceptable salt thereof as described in any one of Examples 52-55, wherein the immunostimulatory nucleic acid sequence is a polydeoxythymidine (polydT) nucleic acid sequence.

[0418] 58. The compound or a pharmaceutically acceptable salt thereof as described in Examples 56 or 57, wherein the compound comprises a poly(dA) nucleic acid sequence and a poly(dT) nucleic acid sequence.

[0419] 59. The compound as described in Example 58 or a pharmaceutically acceptable salt thereof, wherein the poly(dA) nucleic acid sequence and the poly(dT) nucleic acid sequence hybridize to form a double-stranded DNA sequence.

[0420] 60. The compound or a pharmaceutically acceptable salt thereof as described in Examples 58 or 59, wherein the poly-dA nucleic acid sequence and the poly-dT nucleic acid sequence comprise the same number of nucleotides.

[0421] 61. The compound or a pharmaceutically acceptable salt thereof as described in any one of Examples 52-55, wherein the immunostimulatory nucleic acid sequence is a polydeoxyguanosine (polydG) nucleic acid sequence.

[0422] 62. The compound or a pharmaceutically acceptable salt thereof as described in any one of Examples 52-55, wherein the immunostimulatory nucleic acid sequence is a polydeoxycytosine (polydC) nucleic acid sequence.

[0423] 63. The compound or a pharmaceutically acceptable salt thereof as described in Examples 61 or 62, wherein the compound comprises a polydG nucleic acid sequence and a polydC nucleic acid sequence.

[0424] 64. The compound as described in Example 63 or a pharmaceutically acceptable salt thereof, wherein the polydG nucleic acid sequence and the polydC nucleic acid sequence hybridize to form a double-stranded DNA sequence.

[0425] 65. The compound or a pharmaceutically acceptable salt thereof as described in Examples 63 or 64, wherein the polydG nucleic acid sequence and the polydC nucleic acid sequence comprise the same number of nucleotides.

[0426] 66. The compound or a pharmaceutically acceptable salt thereof as described in any one of Examples 52-65, wherein the poly-dA nucleic acid sequence, poly-dT nucleic acid sequence, poly-dC nucleic acid sequence and / or poly-dG nucleic acid sequence comprises between 30 and 100 nucleotides.

[0427] 67. The compound or a pharmaceutically acceptable salt thereof as described in Example 66, wherein the poly-dA nucleic acid sequence, poly-dT nucleic acid sequence, poly-dC nucleic acid sequence and / or poly-dG nucleic acid sequence comprises between 50 and 100 nucleotides.

[0428] 68. The compound or a pharmaceutically acceptable salt thereof as described in Example 66, wherein the poly-dA nucleic acid sequence, poly-dT nucleic acid sequence, poly-dC nucleic acid sequence and / or poly-dG nucleic acid sequence comprises between 30 and 50 nucleotides.

[0429] 69. The compound as described in Example 66 or a pharmaceutically acceptable salt thereof, wherein the poly-dA nucleic acid sequence, poly-dT nucleic acid sequence, poly-dC nucleic acid sequence and / or poly-dG nucleic acid sequence comprises 30, 40, 50, 75 or 100 nucleotides.

[0430] 70. The compound or a pharmaceutically acceptable salt thereof as described in any one of Examples 56-69, wherein the poly-dA nucleic acid sequence, poly-dT nucleic acid sequence, poly-dG nucleic acid sequence and / or poly-dC nucleic acid sequence comprises a mixture of dA, dT, dC and / or dG nucleic acid residues.

[0431] 71. The compound as described in Example 70 or a pharmaceutically acceptable salt thereof, wherein the poly-dA nucleic acid sequence comprises between 100% and 51% dA nucleic acid residues and between 0% and 49% dT, dC, and / or dG nucleic acid residues.

[0432] 72. The compound as described in Example 70 or a pharmaceutically acceptable salt thereof, wherein the poly-dT nucleic acid sequence comprises between 100% and 51% dT nucleic acid residues and between 0% and 49% dA, dC, and / or dG nucleic acid residues.

[0433] 73. The compound or a pharmaceutically acceptable salt thereof as described in Example 70, wherein the polydG nucleic acid sequence comprises between 100% and 51% dG nucleic acid residues and between 0% and 49% dC, dA, or dT nucleic acid residues.

[0434] 74. The compound or a pharmaceutically acceptable salt thereof as described in Example 70, wherein the poly-dC nucleic acid sequence comprises between 100% and 51% dC nucleic acid residues and between 0% and 49% dG, dA, or dT nucleic acid residues.

[0435] 75. The compound or a pharmaceutically acceptable salt thereof as described in any one of Examples 52-70, wherein the immunostimulatory nucleic acid sequence is a CpG sequence.

[0436] 76. The compound as described in Example 75 or a pharmaceutically acceptable salt thereof, wherein the CpG sequence is sequence 5'-TCGTCGTTTTGTCGTTTTGTCGTT-3' (SEQ ID NO: 25).

[0437] 77. The compound as described in Example 75 or a pharmaceutically acceptable salt thereof, wherein the CpG sequence is 5'-TGACTGTGAACGTTCGAGATGA-3' (SEQ ID NO: 26).

[0438] 78. The compound as described in Example 75 or a pharmaceutically acceptable salt thereof, wherein the CpG sequence is 5'-TCGTCGTTTTCGGCGCGCGCCG-3' (SEQ ID NO: 27).

[0439] 79. The compound as described in Example 75 or a pharmaceutically acceptable salt thereof, wherein the CpG sequence is 5'-TCCATGACGTTCCTGACGTT-3' (SEQ ID NO: 29).

[0440] 80. The compound or a pharmaceutically acceptable salt thereof as described in any one of Examples 52-74, wherein at least one internucleotide group connecting the nucleotides in the poly(dA), poly(dT), poly(dC), or poly(dG) nucleic acid sequences is a phosphodiester.

[0441] 81. The compound or a pharmaceutically acceptable salt thereof as described in any one of Examples 52-74, wherein all internucleotide groups connecting the nucleotides in the poly(dA), poly(dT), poly(dC), or poly(dG) nucleic acid sequences are phosphate thioesters.

[0442] 82. The compound or a pharmaceutically acceptable salt thereof as described in any one of Examples 52-81, wherein the albumin-binding domain is a lipid.

[0443] 83. The compound as described in Example 82 or a pharmaceutically acceptable salt thereof, wherein the lipid is a diacyl lipid.

[0444] 84. The compound of Example 83 or a pharmaceutically acceptable salt thereof, wherein the diacyl lipid comprises an acyl chain comprising 12-30 hydrocarbon units, 14-25 hydrocarbon units, 16-20 hydrocarbon units, or 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 hydrocarbon units.

[0445] 85. The compound or a pharmaceutically acceptable salt thereof as described in Example 83 or Example 84, wherein the lipid is 1,2-distearate-sn-glycerol-3-phosphatidylethanolamine (DSPE).

[0446] 86. The compound or a pharmaceutically acceptable salt thereof as described in any one of Examples 52-85, wherein the immunostimulatory nucleic acid sequence is bonded or linked to the following lipid via a adapter, wherein the nucleotide bond is as follows:

[0447]

[0448] or its salt,

[0449] Where X is O or S.

[0450] 87. The compound as described in Example 52 or a pharmaceutically acceptable salt thereof, wherein the immunostimulatory nucleic acid sequence is a CpG sequence, wherein the functional group is a maleimide group, and wherein the 5' end of the CpG sequence is bonded to or connected to the albumin-binding domain, and the 3' end of the CpG sequence is connected to the maleimide group via a PEG4 linker.

[0451] 88. A compound or a pharmaceutically acceptable salt thereof, said compound comprising an immunostimulatory nucleic acid sequence, a linker, and a functional group.

[0452] 89. The compound or a pharmaceutically acceptable salt thereof as described in Example 88, wherein the functional group comprises maleimide, dithio(2-pyridyldithio), cyclooctene, cyclooctyne, aldehyde, azide or alkyne.

[0453] 90. The compound or a pharmaceutically acceptable salt thereof as described in Examples 88 or 89, wherein the immunostimulatory nucleic acid sequence is a polydeoxyadenosine (poly-dA) nucleic acid sequence.

[0454] 91. The compound or a pharmaceutically acceptable salt thereof as described in any one of Examples 88 or 89, wherein the immunostimulatory nucleic acid sequence is a polydeoxythymidine (polydT) nucleic acid sequence.

[0455] 92. The compound or a pharmaceutically acceptable salt thereof as described in Examples 90 or 91, wherein the compound comprises a poly(dA) nucleic acid sequence and a poly(dT) nucleic acid sequence.

[0456] 93. The compound as described in Example 92 or a pharmaceutically acceptable salt thereof, wherein the poly(dA) nucleic acid sequence and the poly(dT) nucleic acid sequence hybridize to form a double-stranded DNA sequence.

[0457] 94. The compound or a pharmaceutically acceptable salt thereof as described in Examples 92 or 93, wherein the poly-dA nucleic acid sequence and the poly-dT nucleic acid sequence comprise the same number of nucleotides.

[0458] 95. The compound or a pharmaceutically acceptable salt thereof as described in Examples 88 or 89, wherein the immunostimulatory nucleic acid sequence is a polydeoxyguanosine (polydG) nucleic acid sequence.

[0459] 96. The compound or a pharmaceutically acceptable salt thereof as described in Examples 88 or 89, wherein the immunostimulatory nucleic acid sequence is a polydeoxycytosine (polydC) nucleic acid sequence.

[0460] 97. The compound or a pharmaceutically acceptable salt thereof as described in Example 95 or 96, wherein the compound comprises a polydG nucleic acid sequence and a polydC nucleic acid sequence.

[0461] 98. The compound as described in Example 97 or a pharmaceutically acceptable salt thereof, wherein the polydG nucleic acid sequence and the polydC nucleic acid sequence hybridize to form a double-stranded DNA sequence.

[0462] 99. The compound or a pharmaceutically acceptable salt thereof as described in Examples 95 or 96, wherein the polydG nucleic acid sequence and the polydC nucleic acid sequence comprise the same number of nucleotides.

[0463] 100. The compound or a pharmaceutically acceptable salt thereof as described in any one of Examples 88-99, wherein the poly-dA nucleic acid sequence, poly-dT nucleic acid sequence, poly-dC nucleic acid sequence and / or poly-dG nucleic acid sequence comprises between 30 and 100 nucleotides.

[0464] 101. The compound or a pharmaceutically acceptable salt thereof as described in Example 100, wherein the poly-dA nucleic acid sequence, poly-dT nucleic acid sequence, poly-dC nucleic acid sequence and / or poly-dG nucleic acid sequence comprises between 50 and 100 nucleotides.

[0465] 102. The compound or a pharmaceutically acceptable salt thereof as described in Example 100, wherein the poly-dA nucleic acid sequence, poly-dT nucleic acid sequence, poly-dC nucleic acid sequence and / or poly-dG nucleic acid sequence comprises between 30 and 50 nucleotides.

[0466] 103. The compound as described in Example 101 or a pharmaceutically acceptable salt thereof, wherein the poly-dA nucleic acid sequence, poly-dT nucleic acid sequence, poly-dC nucleic acid sequence and / or poly-dG nucleic acid sequence comprises 30, 40, 50, 75 or 100 nucleotides.

[0467] 104. The compound or a pharmaceutically acceptable salt thereof as described in any one of Examples 90-103, wherein the poly-dA nucleic acid sequence, poly-dT nucleic acid sequence, poly-dG nucleic acid sequence and / or poly-dC nucleic acid sequence comprises a mixture of dA, dT, dC and / or dG nucleic acid residues.

[0468] 105. The compound or a pharmaceutically acceptable salt thereof as described in Example 104, wherein the poly-dA nucleic acid sequence comprises between 100% and 51% dA nucleic acid residues and between 0% and 49% dT, dC, and / or dG nucleic acid residues.

[0469] 106. The compound or a pharmaceutically acceptable salt thereof as described in Example 104, wherein the poly-dT nucleic acid sequence comprises between 100% and 51% dT nucleic acid residues and between 0% and 49% dA, dC, and / or dG nucleic acid residues.

[0470] 107. The compound or a pharmaceutically acceptable salt thereof as described in Example 104, wherein the polydG nucleic acid sequence comprises between 100% and 51% dG nucleic acid residues and between 0% and 49% dC, dA, or dT nucleic acid residues.

[0471] 108. The compound or a pharmaceutically acceptable salt thereof as described in Example 104, wherein the poly-dC nucleic acid sequence comprises between 100% and 51% dC nucleic acid residues and between 0% and 49% dG, dA, or dT nucleic acid residues.

[0472] 109. The compound as described in Example 88 or a pharmaceutically acceptable salt thereof, wherein the immunostimulatory nucleic acid sequence is a CpG sequence.

[0473] 110. The compound as described in Example 109 or a pharmaceutically acceptable salt thereof, wherein the CpG sequence is sequence 5'-TCGTCGTTTTGTCGTTTTGTCGTT-3' (SEQ ID NO: 25).

[0474] 111. The compound as described in Example 109 or a pharmaceutically acceptable salt thereof, wherein the CpG sequence is 5'-TGACTGTGAACGTTCGAGATGA-3' (SEQ ID NO: 26).

[0475] 112. The compound as described in Example 109 or a pharmaceutically acceptable salt thereof, wherein the CpG sequence is 5'-TCGTCGTTTTCGGCGCGCGCCG-3' (SEQ ID NO: 27).

[0476] 113. The compound as described in Example 109 or a pharmaceutically acceptable salt thereof, wherein the CpG sequence is 5'-TCCATGACGTTCCTGACGTT-3' (SEQ ID NO: 29).

[0477] 114. The compound or a pharmaceutically acceptable salt thereof as described in any one of Examples 88-113, wherein at least one internucleotide group connecting the nucleotides in the poly(dA), poly(dT), poly(dC), or poly(dG) nucleic acid sequences is a phosphodiester.

[0478] 115. The compound or a pharmaceutically acceptable salt thereof as described in any one of Examples 88-114, wherein all internucleotide groups connecting the nucleotides in the poly(dA), poly(dT), poly(dC), or poly(dG) nucleic acid sequences are phosphate thioesters.

[0479] 116. The compound or a pharmaceutically acceptable salt thereof as described in any one of Examples 88-115, wherein the connector is as follows:

[0480]

[0481] or its salt,

[0482] Where X is O or S.

[0483] 117. The compound of Example 88 or a pharmaceutically acceptable salt thereof, wherein the immunostimulatory nucleic acid sequence is a CpG sequence, wherein the linker is PEG4, wherein the functional group is maleimide, and wherein the 3' end of the CpG sequence is bonded to or connected to the PEG4, and the PEG4 is bonded to or connected to the maleimide group.

[0484] 118. A compound or a pharmaceutically acceptable salt thereof, said compound comprising an immunosuppressive nucleic acid sequence, an albumin-binding domain, and a polypeptide.

[0485] 119. The compound or a pharmaceutically acceptable salt thereof as described in Example 118, wherein the albumin-binding domain is bonded or linked to the 5' end of the immunosuppressive nucleic acid sequence, and wherein the polypeptide is bonded or linked to the 3' end of the immunosuppressive nucleic acid sequence.

[0486] 120. The compound or a pharmaceutically acceptable salt thereof as described in Example 118, wherein the polypeptide is bonded or linked to the 5' end of the immunosuppressive nucleic acid sequence, and wherein the albumin-binding domain is bonded or linked to the 3' end of the immunosuppressive nucleic acid sequence.

[0487] 121. The compound as described in Example 118 or a pharmaceutically acceptable salt thereof, wherein the polypeptide and the albumin-binding domain are bonded to or linked to the 5' end of the immunosuppressive nucleic acid sequence.

[0488] 122. The compound as described in Example 118 or a pharmaceutically acceptable salt thereof, wherein the polypeptide and the albumin-binding domain are bonded to or linked to the 3' end of the immunosuppressive nucleic acid sequence.

[0489] 123. The compound or a pharmaceutically acceptable salt thereof as described in any one of Examples 118-122, wherein the polypeptide comprises an N-terminal modification.

[0490] 124. The compound as described in Example 123 or a pharmaceutically acceptable salt thereof, wherein the N-terminal modification is the addition of acetylcysteine.

[0491] 125. The compound as described in Example 124 or a pharmaceutically acceptable salt thereof, wherein the N-terminal modification is the addition of a deaminocysteine ​​homolog.

[0492] 126. The compound as described in Example 125 or a pharmaceutically acceptable salt thereof, wherein the deaminocysteine ​​homolog is 3-mercaptopropionic acid or mercaptoacetic acid.

[0493] 127. The compound or a pharmaceutically acceptable salt thereof as described in any one of Examples 118-126, wherein the immunosuppressive nucleic acid sequence is an A151 nucleic acid sequence.

[0494] 128. The compound as described in Example 127 or a pharmaceutically acceptable salt thereof, wherein the immunosuppressive nucleic acid sequence has the nucleic acid sequence TTAGG (SEQ ID NO: 28).

[0495] 129. The compound or a pharmaceutically acceptable salt thereof as described in any one of Examples 118-128, wherein the polypeptide is an antigen or a fragment thereof.

[0496] 130. The compound or a pharmaceutically acceptable salt thereof as described in Example 129, wherein the antigen is derived from a tumor, virus, or bacterial source.

[0497] 131. The compound or a pharmaceutically acceptable salt thereof as described in Example 129, wherein the antigen or a fragment thereof is a tumor-associated antigen.

[0498] 132. The compound or a pharmaceutically acceptable salt thereof as described in Example 129, wherein the antigen is an influenza antigen or a fragment thereof.

[0499] 133. The compound or a pharmaceutically acceptable salt thereof as described in Example 132, wherein the antigen is an influenza nucleoprotein or a fragment thereof.

[0500] 134. The compound or a pharmaceutically acceptable salt thereof as described in Example 130, wherein the antigen is a coronavirus antigen or a fragment thereof.

[0501] 135. The compound as described in Example 134 or a pharmaceutically acceptable salt thereof, wherein the antigen is a coronavirus spike protein or a fragment thereof.

[0502] 136. The compound as described in Example 134 or a pharmaceutically acceptable salt thereof, wherein the antigen is a coronavirus nucleocapsid protein or a fragment thereof.

[0503] 137. The compound or a pharmaceutically acceptable salt thereof as described in any one of Examples 118-136, wherein the albumin-binding domain is a lipid.

[0504] 138. The compound as described in Example 137 or a pharmaceutically acceptable salt thereof, wherein the lipid is a diacyl lipid.

[0505] 139. The compound of Example 138 or a pharmaceutically acceptable salt thereof, wherein the diacyl lipid comprises an acyl chain comprising 12-30 hydrocarbon units, 14-25 hydrocarbon units, 16-20 hydrocarbon units, or 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 hydrocarbon units.

[0506] 140. The compound or a pharmaceutically acceptable salt thereof as described in Example 138 or Example 139, wherein the lipid is 1,2-distearate-sn-glycerol-3-phosphatidylethanolamine (DSPE).

[0507] 141. The compound or a pharmaceutically acceptable salt thereof as described in any one of Examples 118-140, wherein the albumin-binding domain and the polypeptide are bonded or linked by a linker selected from the group consisting of hydrophilic polymers, a string of hydrophilic amino acids, polysaccharides and oligonucleotides or combinations thereof.

[0508] 142. The compound or a pharmaceutically acceptable salt thereof as described in Example 141, wherein the connector comprises “N” polyethylene glycol units, wherein N is between 4 and 50.

[0509] 143. The compound as described in Example 142 or a pharmaceutically acceptable salt thereof, wherein the linker comprises PEG4-amide-PEG4.

[0510] 144. The compound or a pharmaceutically acceptable salt thereof as described in any one of Examples 118-143, wherein the immunosuppressive nucleic acid sequence is bonded or linked to the following lipid via a linker, wherein the nucleotide bond is as follows:

[0511]

[0512] or its salt,

[0513] Where X is O or S.

[0514] 145. A compound or a pharmaceutically acceptable salt thereof, said compound comprising an immunosuppressive nucleic acid sequence, an albumin-binding domain, and a functional group.

[0515] 146. The compound or a pharmaceutically acceptable salt thereof as described in Example 145, wherein the functional group comprises maleimide, dithio(2-pyridyldithio), cyclooctene, cyclooctyne, aldehyde, azide or alkyne.

[0516] 147. The compound or a pharmaceutically acceptable salt thereof as described in Examples 145 or 146, wherein the albumin-binding domain is bonded to or linked to the 5' end of the immunosuppressive nucleic acid sequence.

[0517] 148. The compound or a pharmaceutically acceptable salt thereof as described in Examples 145 or 146, wherein the albumin-binding domain is bonded to or linked to the 3' end of the immunosuppressive nucleic acid sequence.

[0518] 149. The compound or a pharmaceutically acceptable salt thereof as described in any one of Examples 145-148, wherein the immunosuppressive nucleic acid sequence is an A151 nucleic acid sequence.

[0519] 150. The compound as described in Example 149 or a pharmaceutically acceptable salt thereof, wherein the immunosuppressive nucleic acid sequence has the nucleic acid sequence TTAGG (SEQ ID NO: 28).

[0520] 151. The compound or a pharmaceutically acceptable salt thereof as described in any one of Examples 145-150, wherein the albumin-binding domain is a lipid.

[0521] 152. The compound as described in Example 151 or a pharmaceutically acceptable salt thereof, wherein the lipid is a diacyl lipid.

[0522] 153. The compound of Example 152 or a pharmaceutically acceptable salt thereof, wherein the diacyl lipid comprises an acyl chain comprising 12-30 hydrocarbon units, 14-25 hydrocarbon units, 16-20 hydrocarbon units, or 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 hydrocarbon units.

[0523] 154. The compound or a pharmaceutically acceptable salt thereof as described in Example 152 or Example 153, wherein the lipid is 1,2-distearate-sn-glycerol-3-phosphatidylethanolamine (DSPE).

[0524] 155. The compound or a pharmaceutically acceptable salt thereof as described in any one of Examples 145-154, wherein the immunosuppressive nucleic acid sequence is bonded or linked to the following lipid via a linker, wherein the nucleotide bond is as follows:

[0525]

[0526] or its salt,

[0527] Where X is O or S.

[0528] 156. A compound or a pharmaceutically acceptable salt thereof, said compound comprising an immunosuppressive nucleic acid sequence, a linker, and a functional group.

[0529] 157. The compound or a pharmaceutically acceptable salt thereof as described in Example 156, wherein the functional group comprises maleimide, dithio(2-pyridyldithio), cyclooctene, cyclooctyne, aldehyde, azide or alkyne.

[0530] 158. The compound or a pharmaceutically acceptable salt thereof as described in Examples 156 or 157, wherein the immunosuppressive nucleic acid sequence is the A151 nucleic acid sequence.

[0531] 159. The compound as described in Example 158 or a pharmaceutically acceptable salt thereof, wherein the immunosuppressive nucleic acid sequence has the nucleic acid sequence TTAGG (SEQ ID NO: 28).

[0532] 160. The compound or a pharmaceutically acceptable salt thereof as described in any one of Examples 156-159, wherein the linker is selected from the group consisting of: hydrophilic polymers, a string of hydrophilic amino acids, polysaccharides and oligonucleotides or combinations thereof.

[0533] 161. The compound or a pharmaceutically acceptable salt thereof as described in Example 160, wherein the connector comprises “N” polyethylene glycol units, wherein N is between 4 and 50.

[0534] 162. The compound as described in Example 161 or a pharmaceutically acceptable salt thereof, wherein the linker comprises PEG4-acylamino-PEG4.

[0535] 163. The compound or a pharmaceutically acceptable salt thereof as described in any one of Examples 156-162, wherein the connector is as follows:

[0536]

[0537] or its salt,

[0538] Where X is O or S.

[0539] 164. A method for inducing an immune response against an antigen in a subject, the method comprising administering to the subject a compound as described in any one of Examples 1-163 or a pharmaceutically acceptable salt thereof.

[0540] 165. The method as described in Example 164, the method further comprising administering an adjuvant to the subject.

[0541] 166. The method as described in Examples 164 or 165, wherein the polypeptide is an antigen or a fragment thereof.

[0542] 167. The method as described in Example 166, wherein the antigen is derived from a tumor, virus, or bacteria.

[0543] 168. The method as described in Example 166, wherein the antigen or a fragment thereof is a tumor-associated antigen.

[0544] 169. The method as described in Example 166, wherein the antigen is an influenza antigen or a fragment thereof.

[0545] 170. The method as described in Example 169, wherein the antigen is an influenza nucleoprotein or a fragment thereof.

[0546] 171. The method as described in Example 167, wherein the antigen is a coronavirus antigen or a fragment thereof.

[0547] 172. The method as described in Example 171, wherein the antigen is a coronavirus spike protein or a fragment thereof.

[0548] 173. The method as described in Example 171, wherein the antigen is a coronavirus nucleocapsid protein or a fragment thereof.

[0549] 174. The method as described in any one of Examples 164-173, wherein the compound or a pharmaceutically acceptable salt thereof is administered subcutaneously.

[0550] 175. The method as described in any one of Examples 164-173, wherein the compound or a pharmaceutically acceptable salt thereof is administered via a mucosa.

[0551] 176. The method as described in Example 175, wherein the compound or a pharmaceutically acceptable saline thereof is administered intranasally.

[0552] 177. The method as described in any one of Examples 164-176, wherein the subject is a mammal.

[0553] 178. The method as described in Example 177, wherein the subject is a human.

[0554] 179. A pharmaceutical composition comprising a compound as described in any one of Examples 1-163 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0555] 180. A kit comprising a compound as described in any one of Examples 1-163 or a pharmaceutically acceptable salt thereof, and instructions for use.

[0556] Other embodiments

[0557] Various modifications and variations to the compositions, methods, and uses described herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in conjunction with specific embodiments, it should be understood that the claimed invention should not be unduly limited to such specific embodiments. In fact, various modifications to the described modes of carrying out the invention that will be readily apparent to those skilled in the art are contemplated within the scope of the invention.

[0558] All publications, patents and patent applications are incorporated herein by reference in their entirety, to the same extent that each individual publication, patent or patent application is specifically and individually incorporated herein by reference in its entirety.

[0559] Claims.

Claims

1. A compound, or a pharmaceutically acceptable salt thereof, comprising an immunostimulatory nucleic acid sequence, an albumin binding domain, and a polypeptide.

2. The compound, or a pharmaceutically acceptable salt thereof, of claim 1, wherein the albumin binding domain is bonded or linked to the 5' end of the immunostimulatory nucleic acid sequence, and wherein the polypeptide is bonded or linked to the 3' end of the immunostimulatory nucleic acid sequence.

3. The compound, or a pharmaceutically acceptable salt thereof, of claim 1, wherein the polypeptide is bonded or linked to the 5' end of the immunostimulatory nucleic acid sequence, and wherein the albumin binding domain is bonded or linked to the 3' end of the immunostimulatory nucleic acid sequence.

4. The compound, or a pharmaceutically acceptable salt thereof, of claim 1, wherein both the polypeptide and the albumin binding domain are bonded or linked to the 5' end of the immunostimulatory nucleic acid sequence.

5. The compound, or a pharmaceutically acceptable salt thereof, of claim 1, wherein both the polypeptide and the albumin binding domain are bonded or linked to the 3' end of the immunostimulatory nucleic acid sequence.

6. The compound, or a pharmaceutically acceptable salt thereof, of claim 1, wherein the polypeptide comprises an N-terminal modification.

7. The compound, or a pharmaceutically acceptable salt thereof, of claim 6, wherein the N-terminal modification is the addition of acetylcysteine.

8. The compound, or a pharmaceutically acceptable salt thereof, of claim 7, wherein the N-terminal modification is the addition of a desamino cysteine analog.

9. The compound, or a pharmaceutically acceptable salt thereof, of claim 8, wherein the desamino cysteine analog is 3-mercaptopropionic acid or mercaptoacetic acid.

10. The compound, or a pharmaceutically acceptable salt thereof, of claim 1, wherein the immunostimulatory nucleic acid sequence is a polydeoxyadenosine (poly-dA) nucleic acid sequence.

11. The compound, or a pharmaceutically acceptable salt thereof, of claim 1, wherein the immunostimulatory nucleic acid sequence is a polydeoxythymidine (poly-dT) nucleic acid sequence.

12. The compound, or a pharmaceutically acceptable salt thereof, of claim 10 or 11, wherein the compound comprises a poly-dA nucleic acid sequence and a poly-dT nucleic acid sequence.

13. The compound, or a pharmaceutically acceptable salt thereof, of claim 12, wherein the poly-dA nucleic acid sequence and the poly-dT nucleic acid sequence hybridize to form a double-stranded DNA sequence.

14. The compound, or a pharmaceutically acceptable salt thereof, of claim 12, wherein the poly-dA nucleic acid sequence and poly-dT nucleic acid sequence comprise the same number of nucleotides.

15. The compound, or a pharmaceutically acceptable salt thereof, of claim 1, wherein the immunostimulatory nucleic acid sequence is a polydeoxyguanosine (poly-dG) nucleic acid sequence.

16. The compound, or a pharmaceutically acceptable salt thereof, of claim 1, wherein the immunostimulatory nucleic acid sequence is a polydeoxycytosine (poly-dC) nucleic acid sequence.

17. The compound, or a pharmaceutically acceptable salt thereof, of claim 15 or 16, wherein the compound comprises a poly-dG nucleic acid sequence and a poly-dC nucleic acid sequence.

18. The compound of claim 17, or a pharmaceutically acceptable salt thereof, wherein the poly-dG nucleic acid sequence and the poly-dC nucleic acid sequence hybridize to form a double stranded DNA sequence.

19. The compound of claim 17, or a pharmaceutically acceptable salt thereof, wherein the poly-dG nucleic acid sequence and poly-dC nucleic acid sequence comprise the same number of nucleotides.

20. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein the poly-dA nucleic acid sequence, poly-dT nucleic acid sequence, poly-dC nucleic acid sequence, and / or poly-dG nucleic acid sequence comprises between 30 and 100 nucleotides.

21. The compound of claim 20, or a pharmaceutically acceptable salt thereof, wherein the poly-dA nucleic acid sequence, poly-dT nucleic acid sequence, poly-dC nucleic acid sequence, and / or poly-dG nucleic acid sequence comprises between 50 and 100 nucleotides.

22. The compound of claim 20, or a pharmaceutically acceptable salt thereof, wherein the poly-dA nucleic acid sequence, poly-dT nucleic acid sequence, poly-dC nucleic acid sequence, and / or poly-dG nucleic acid sequence comprises between 30 and 50 nucleotides.

23. The compound of claim 20, or a pharmaceutically acceptable salt thereof, wherein the poly-dA nucleic acid sequence, poly-dT nucleic acid sequence, poly-dC nucleic acid sequence, and / or poly-dG nucleic acid sequence comprises 30, 40, 50, 75, or 100 nucleotides.

24. The compound of claim 10, or a pharmaceutically acceptable salt thereof, wherein the poly-dA nucleic acid sequence, poly-dT nucleic acid sequence, poly-dG nucleic acid sequence, and / or poly-dC nucleic acid sequence comprises a mixture of dA, dT, dC, and / or dG nucleic acid residues.

25. The compound of claim 24, or a pharmaceutically acceptable salt thereof, wherein the poly-dA nucleic acid sequence comprises between 100% and 51% dA nucleic acid residues and between 0% and 49% dT, dC, and / or dG nucleic acid residues.

26. The compound of claim 24, or a pharmaceutically acceptable salt thereof, wherein the poly-dT nucleic acid sequence comprises between 100% and 51% dT nucleic acid residues and between 0% and 49% dA, dC, and / or dG nucleic acid residues.

27. The compound of claim 24, or a pharmaceutically acceptable salt thereof, wherein the poly-dG nucleic acid sequence comprises between 100% and 51% dG nucleic acid residues and between 0% and 49% dC, dA, or dT nucleic acid residues.

28. The compound of claim 24, or a pharmaceutically acceptable salt thereof, wherein the poly-dC nucleic acid sequence comprises between 100% and 51% dC nucleic acid residues and between 0% and 49% dG, dA, or dT nucleic acid residues.

29. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein the immunostimulatory nucleic acid sequence is a CpG sequence.

30. The compound of claim 29, or a pharmaceutically acceptable salt thereof, wherein the CpG sequence is the sequence 5'-TCGTCGTTTTGTCGTTTTGTCGTT-3' (SEQ ID NO: 25).

31. The compound of claim 29, or a pharmaceutically acceptable salt thereof, wherein the CpG sequence is 5'-TGACTGTGAACGTTCGAGATGA-3' (SEQ ID NO: 26).

32. The compound of claim 29, or a pharmaceutically acceptable salt thereof, wherein the CpG sequence is 5'-TCGTCGTTTTCGGCGCGCGCCG-3' (SEQ ID NO: 27).

33. The compound of claim 29, or a pharmaceutically acceptable salt thereof, wherein the CpG sequence is 5'-TCCATGACGTTCCTGACGTT-3' (SEQ ID NO: 29).

34. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein at least one internucleotide group linking the nucleotides in the poly-dA nucleic acid sequence, poly-dT nucleic acid sequence, poly-dC nucleic acid sequence, or poly-dG nucleic acid sequence is a phosphodiester.

35. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein all internucleotide groups linking the nucleotides in the poly-dA nucleic acid sequence, poly-dT nucleic acid sequence, poly-dC nucleic acid sequence, or poly-dG nucleic acid sequence are phosphorothioate.

36. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein the polypeptide is an antigen or fragment thereof.

37. The compound of claim 36, or a pharmaceutically acceptable salt thereof, wherein the antigen is derived from a tumor or viral or bacterial source.

38. The compound of claim 36, or a pharmaceutically acceptable salt thereof, wherein the antigen or fragment thereof is a tumor-associated antigen.

39. The compound of claim 36, or a pharmaceutically acceptable salt thereof, wherein the antigen is an influenza antigen or fragment thereof.

40. The compound of claim 39, or a pharmaceutically acceptable salt thereof, wherein the antigen is an influenza nucleoprotein or fragment thereof.

41. The compound of claim 37, or a pharmaceutically acceptable salt thereof, wherein the antigen is a coronavirus antigen or fragment thereof.

42. The compound of claim 41, or a pharmaceutically acceptable salt thereof, wherein the antigen is a coronavirus spike protein or fragment thereof.

43. The compound of claim 41, or a pharmaceutically acceptable salt thereof, wherein the antigen is a coronavirus nucleocapsid protein or fragment thereof.

44. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein the albumin binding domain is a lipid.

45. The compound of claim 44, or a pharmaceutically acceptable salt thereof, wherein the lipid is a diacyl lipid.

46. The compound of claim 45, or a pharmaceutically acceptable salt thereof, wherein the diacyl lipid comprises an acyl chain comprising 12-30, 14-25, 16-20, or 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 hydrocarbon units.

47. The compound of claim 45 or claim 46, or a pharmaceutically acceptable salt thereof, wherein the lipid is l,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE).

48. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein the albumin binding domain and the polypeptide are bonded or linked by a linker, wherein the linker is selected from the group consisting of a hydrophilic polymer, a string of hydrophilic amino acids, a polysaccharide, and an oligonucleotide, or a combination thereof.

49. The compound of claim 48, or a pharmaceutically acceptable salt thereof, wherein the linker comprises "N" PEG units, wherein N is between 4-50.

50. The compound of claim 49, or a pharmaceutically acceptable salt thereof, wherein the linker comprises PEG4-amido-PEG4.

51. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein the immunostimulatory nucleic acid sequence is bonded or linked to the following lipid by a linker, wherein the nucleotide bond is shown below: or a salt thereof, wherein X is O or S.

52. A compound, or a pharmaceutically acceptable salt thereof, comprising an immunostimulatory nucleic acid sequence, an albumin binding domain, and a functional group, or a pharmaceutically acceptable salt thereof.

53. The compound of claim 52, or a pharmaceutically acceptable salt thereof, wherein the functional group comprises a maleimide, a dithio (2-pyridyldithio), a cyclooctene, a cyclooctyne, an aldehyde, an azide, or an alkyne.

54. The compound of claim 52 or 53, or a pharmaceutically acceptable salt thereof, wherein the albumin binding domain is bonded or linked to the 5' end of the immunostimulatory nucleic acid sequence.

55. The compound of claim 52 or 53, or a pharmaceutically acceptable salt thereof, wherein the albumin binding domain is bonded or linked to the 3' end of the immunostimulatory nucleic acid sequence.

56. The compound of claim 52, or a pharmaceutically acceptable salt thereof, wherein the immunostimulatory nucleic acid sequence is a polydeoxyadenosine (poly-dA) nucleic acid sequence.

57. The compound of claim 52, or a pharmaceutically acceptable salt thereof, wherein the immunostimulatory nucleic acid sequence is a polydeoxythymidine (poly-dT) nucleic acid sequence.

58. The compound of claim 56 or 57, or a pharmaceutically acceptable salt thereof, wherein the compound comprises a poly-dA nucleic acid sequence and a poly-dT nucleic acid sequence.

59. The compound of claim 58, or a pharmaceutically acceptable salt thereof, wherein the poly-dA nucleic acid sequence and the poly-dT nucleic acid sequence hybridize to form a double stranded DNA sequence.

60. The compound of claim 58, or a pharmaceutically acceptable salt thereof, wherein the poly-dA nucleic acid sequence and poly-dT nucleic acid sequence comprise the same number of nucleotides.

61. The compound of claim 52, or a pharmaceutically acceptable salt thereof, wherein the immunostimulatory nucleic acid sequence is a polydeoxyguanosine (poly-dG) nucleic acid sequence.

62. The compound of claim 52, or a pharmaceutically acceptable salt thereof, wherein the immune stimulatory nucleic acid sequence is a polydeoxy cytosine (poly-dC) nucleic acid sequence.

63. The compound of claim 61 or 62, or a pharmaceutically acceptable salt thereof, wherein the compound comprises a poly-dG nucleic acid sequence and a poly-dC nucleic acid sequence.

64. The compound of claim 63, or a pharmaceutically acceptable salt thereof, wherein the poly-dG nucleic acid sequence and the poly-dC nucleic acid sequence hybridize to form a double stranded DNA sequence.

65. The compound of claim 63, or a pharmaceutically acceptable salt thereof, wherein the poly-dG nucleic acid sequence and poly-dC nucleic acid sequence comprise the same number of nucleotides.

66. The compound of claim 52, or a pharmaceutically acceptable salt thereof, wherein the poly-dA nucleic acid sequence, poly-dT nucleic acid sequence, poly-dC nucleic acid sequence, and / or poly-dG nucleic acid sequence comprises between 30 and 100 nucleotides.

67. The compound of claim 66, or a pharmaceutically acceptable salt thereof, wherein the poly-dA nucleic acid sequence, poly-dT nucleic acid sequence, poly-dC nucleic acid sequence, and / or poly-dG nucleic acid sequence comprises between 50 and 100 nucleotides.

68. The compound of claim 66, or a pharmaceutically acceptable salt thereof, wherein the poly-dA nucleic acid sequence, poly-dT nucleic acid sequence, poly-dC nucleic acid sequence, and / or poly-dG nucleic acid sequence comprises between 30 and 50 nucleotides.

69. The compound of claim 66, or a pharmaceutically acceptable salt thereof, wherein the poly-dA nucleic acid sequence, poly-dT nucleic acid sequence, poly-dC nucleic acid sequence, and / or poly-dG nucleic acid sequence comprises 30, 40, 50, 75, or 100 nucleotides.

70. The compound of claim 56, or a pharmaceutically acceptable salt thereof, wherein the poly-dA nucleic acid sequence, poly-dT nucleic acid sequence, poly-dG nucleic acid sequence, and / or poly-dC nucleic acid sequence comprises a mixture of dA, dT, dC, and / or dG nucleic acid residues.

71. The compound of claim 70, or a pharmaceutically acceptable salt thereof, wherein the poly-dA nucleic acid sequence comprises between 100% and 51% dA nucleic acid residues and between 0% and 49% dT, dC, and / or dG nucleic acid residues.

72. The compound of claim 70, or a pharmaceutically acceptable salt thereof, wherein the poly-dT nucleic acid sequence comprises between 100% and 51% dT nucleic acid residues and between 0% and 49% dA, dC, and / or dG nucleic acid residues.

73. The compound of claim 70, or a pharmaceutically acceptable salt thereof, wherein the poly-dG nucleic acid sequence comprises between 100% and 51% dG nucleic acid residues and between 0% and 49% dC, dA, or dT nucleic acid residues.

74. The compound of claim 70, or a pharmaceutically acceptable salt thereof, wherein the poly-dC nucleic acid sequence comprises between 100% and 51% dC nucleic acid residues and between 0% and 49% dG, dA, or dT nucleic acid residues.

75. The compound of claim 52, or a pharmaceutically acceptable salt thereof, wherein the immunostimulatory nucleic acid sequence is a CpG sequence.

76. The compound of claim 75, or a pharmaceutically acceptable salt thereof, wherein the CpG sequence is the sequence 5'-TCGTCGTTTTGTCGTTTTGTCGTT-3' (SEQ ID NO: 25).

77. The compound of claim 75, or a pharmaceutically acceptable salt thereof, wherein the CpG sequence is 5'-TGACTGTGAACGTTCGAGATGA-3' (SEQ ID NO: 26).

78. The compound of claim 75, or a pharmaceutically acceptable salt thereof, wherein the CpG sequence is 5'-TCGTCGTTTTCGGCGCGCGCCG-3' (SEQ ID NO: 27).

79. The compound of claim 75, or a pharmaceutically acceptable salt thereof, wherein the CpG sequence is 5'-TCCATGACGTTCCTGACGTT-3' (SEQ ID NO: 29).

80. The compound of claim 52, or a pharmaceutically acceptable salt thereof, wherein at least one internucleotide linkage connecting the nucleotides in the poly-dA nucleic acid sequence, poly-dT nucleic acid sequence, poly-dC nucleic acid sequence, or poly-dG nucleic acid sequence is a phosphodiester.

81. The compound of claim 52, or a pharmaceutically acceptable salt thereof, wherein all internucleotide linkages connecting the nucleotides in the poly-dA nucleic acid sequence, poly-dT nucleic acid sequence, poly-dC nucleic acid sequence, or poly-dG nucleic acid sequence are phosphorothioate.

82. The compound of claim 52, or a pharmaceutically acceptable salt thereof, wherein the albumin binding domain is a lipid.

83. The compound of claim 82, or a pharmaceutically acceptable salt thereof, wherein the lipid is a diacyl lipid.

84. The compound of claim 83, or a pharmaceutically acceptable salt thereof, wherein the diacyl lipid comprises an acyl chain comprising 12-30 hydrocarbon units, 14-25 hydrocarbon units, 16-20 hydrocarbon units, or 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 hydrocarbon units.

85. The compound of claim 83 or claim 84, or a pharmaceutically acceptable salt thereof, wherein the lipid is l,2-distearoyl-sn-glycero-3-phosphatidylethanolamine (DSPE).

86. The compound of claim 52, or a pharmaceutically acceptable salt thereof, wherein the immunostimulatory nucleic acid sequence is bonded or linked to the following lipid through a linker, wherein the nucleotide bond is shown below: or a salt thereof, wherein X is O or S. ​ 87. The compound, or a pharmaceutically acceptable salt thereof, of claim 52, wherein the immunostimulatory nucleic acid sequence is a CpG sequence, wherein the functional group is a maleimide group, and wherein the 5’ end of the CpG sequence is bonded or linked to the albumin binding domain and the 3’ end of the CpG sequence is linked to the maleimide group by a PEG4 linker.

88. A compound, or a pharmaceutically acceptable salt thereof, comprising an immunostimulatory nucleic acid sequence, a linker, and a functional group.

89. The compound, or a pharmaceutically acceptable salt thereof, of claim 88, wherein the functional group comprises a maleimide, a dithio (2-pyridyldithio), a cyclooctene, a cyclooctyne, an aldehyde, an azide, or an alkyne.

90. The compound, or a pharmaceutically acceptable salt thereof, of claim 88 or 89, wherein the immunostimulatory nucleic acid sequence is a polydeoxyadenosine (poly-dA) nucleic acid sequence.

91. The compound, or a pharmaceutically acceptable salt thereof, of any one of claims 88 or 89, wherein the immunostimulatory nucleic acid sequence is a polydeoxythymidine (poly-dT) nucleic acid sequence.

92. The compound, or a pharmaceutically acceptable salt thereof, of claim 90, wherein the compound comprises a poly-dA nucleic acid sequence and a poly-dT nucleic acid sequence.

93. The compound, or a pharmaceutically acceptable salt thereof, of claim 92, wherein the poly-dA nucleic acid sequence and the poly-dT nucleic acid sequence hybridize to form a double stranded DNA sequence.

94. The compound, or a pharmaceutically acceptable salt thereof, of claim 92, wherein the poly-dA nucleic acid sequence and poly-dT nucleic acid sequence comprise the same number of nucleotides.

95. The compound, or a pharmaceutically acceptable salt thereof, of claim 88 or 89, wherein the immunostimulatory nucleic acid sequence is a polydeoxyguanosine (poly-dG) nucleic acid sequence.

96. The compound, or a pharmaceutically acceptable salt thereof, of claim 88 or 89, wherein the immunostimulatory nucleic acid sequence is a polydeoxycytosine (poly-dC) nucleic acid sequence.

97. The compound, or a pharmaceutically acceptable salt thereof, of claim 95, wherein the compound comprises a poly-dG nucleic acid sequence and a poly-dC nucleic acid sequence.

98. The compound, or a pharmaceutically acceptable salt thereof, of claim 97, wherein the poly-dG nucleic acid sequence and the poly-dC nucleic acid sequence hybridize to form a double stranded DNA sequence.

99. The compound, or a pharmaceutically acceptable salt thereof, of claim 95, wherein the poly-dG nucleic acid sequence and poly-dC nucleic acid sequence comprise the same number of nucleotides.

100. The compound, or a pharmaceutically acceptable salt thereof, of claim 88, wherein the poly-dA nucleic acid sequence, poly-dT nucleic acid sequence, poly-dC nucleic acid sequence, and / or poly-dG nucleic acid sequence comprises between 30 and 100 nucleotides.

101. The compound, or a pharmaceutically acceptable salt thereof, of claim 100, wherein the poly-dA nucleic acid sequence, poly-dT nucleic acid sequence, poly-dC nucleic acid sequence, and / or poly-dG nucleic acid sequence comprises between 50 and 100 nucleotides.

102. The compound of claim 100, or a pharmaceutically acceptable salt thereof, wherein the poly-dA nucleic acid sequence, poly-dT nucleic acid sequence, poly-dC nucleic acid sequence, and / or poly-dG nucleic acid sequence comprises between 30 and 50 nucleotides.

103. The compound of claim 101, or a pharmaceutically acceptable salt thereof, wherein the poly-dA nucleic acid sequence, poly-dT nucleic acid sequence, poly-dC nucleic acid sequence, and / or poly-dG nucleic acid sequence comprises 30, 40, 50, 75, or 100 nucleotides.

104. The compound of claim 90, or a pharmaceutically acceptable salt thereof, wherein the poly-dA nucleic acid sequence, poly-dT nucleic acid sequence, poly-dG nucleic acid sequence, and / or poly-dC nucleic acid sequence comprises a mixture of dA, dT, dC, and / or dG nucleic acid residues.

105. The compound of claim 104, or a pharmaceutically acceptable salt thereof, wherein the poly-dA nucleic acid sequence comprises between 100% and 51% dA nucleic acid residues and between 0% and 49% dT, dC, and / or dG nucleic acid residues.

106. The compound of claim 104, or a pharmaceutically acceptable salt thereof, wherein the poly-dT nucleic acid sequence comprises between 100% and 51% dT nucleic acid residues and between 0% and 49% dA, dC, and / or dG nucleic acid residues.

107. The compound of claim 104, or a pharmaceutically acceptable salt thereof, wherein the poly-dG nucleic acid sequence comprises between 100% and 51% dG nucleic acid residues and between 0% and 49% dC, dA, or dT nucleic acid residues.

108. The compound of claim 104, or a pharmaceutically acceptable salt thereof, wherein the poly-dC nucleic acid sequence comprises between 100% and 51% dC nucleic acid residues and between 0% and 49% dG, dA, or dT nucleic acid residues.

109. The compound of claim 88, or a pharmaceutically acceptable salt thereof, wherein the immunostimulatory nucleic acid sequence is a CpG sequence.

110. The compound of claim 109, or a pharmaceutically acceptable salt thereof, wherein the CpG sequence is the sequence 5'-TCGTCGTTTTGTCGTTTTGTCGTT-3' (SEQ ID NO: 25).

111. The compound of claim 109, or a pharmaceutically acceptable salt thereof, wherein the CpG sequence is 5'-TGACTGTGAACGTTCGAGATGA-3' (SEQ ID NO: 26).

112. The compound of claim 109, or a pharmaceutically acceptable salt thereof, wherein the CpG sequence is 5'-TCGTCGTTTTCGGCGCGCGCCG-3' (SEQ ID NO: 27).

113. The compound of claim 109, or a pharmaceutically acceptable salt thereof, wherein the CpG sequence is 5'-TCCATGACGTTCCTGACGTT-3' (SEQ ID NO: 29).

114. The compound of claim 88, or a pharmaceutically acceptable salt thereof, wherein at least one internucleotide group linking the nucleotides in the poly-dA nucleic acid sequence, poly-dT nucleic acid sequence, poly-dC nucleic acid sequence, or poly-dG nucleic acid sequence is phosphodiester.

115. The compound of claim 88, or a pharmaceutically acceptable salt thereof, wherein all internucleotide groups linking the nucleotides in the poly-dA nucleic acid sequence, poly-dT nucleic acid sequence, poly-dC nucleic acid sequence, or poly-dG nucleic acid sequence are phosphorothioate.

116. The compound of claim 88, or a pharmaceutically acceptable salt thereof, wherein the linker is shown below: or a salt thereof, wherein X is O or S.

117. The compound of claim 88, or a pharmaceutically acceptable salt thereof, wherein the immunostimulatory nucleic acid sequence is a CpG sequence, wherein the linker is PEG4, wherein the functional group is a maleimide, and wherein the 3’ end of the CpG sequence is bonded or linked to the PEG4, which is bonded or linked to the maleimide group.

118. A compound, or a pharmaceutically acceptable salt thereof, comprising an immunosuppressive nucleic acid sequence, an albumin binding domain, and a polypeptide.

119. The compound of claim 118, or a pharmaceutically acceptable salt thereof, wherein the albumin binding domain is bonded or linked to the 5’ end of the immunosuppressive nucleic acid sequence, and wherein the polypeptide is bonded or linked to the 3’ end of the immunosuppressive nucleic acid sequence.

120. The compound of claim 118, or a pharmaceutically acceptable salt thereof, wherein the polypeptide is bonded or linked to the 5’ end of the immunosuppressive nucleic acid sequence, and wherein the albumin binding domain is bonded or linked to the 3’ end of the immunosuppressive nucleic acid sequence.

121. The compound of claim 118, or a pharmaceutically acceptable salt thereof, wherein the polypeptide and the albumin binding domain are bonded or linked to the 5’ end of the immunosuppressive nucleic acid sequence.

122. The compound of claim 118, or a pharmaceutically acceptable salt thereof, wherein the polypeptide and the albumin binding domain are bonded or linked to the 3’ end of the immunosuppressive nucleic acid sequence.

123. The compound of claim 118, or a pharmaceutically acceptable salt thereof, wherein the polypeptide comprises an N-terminal modification.

124. The compound of claim 123, or a pharmaceutically acceptable salt thereof, wherein the N-terminal modification is the addition of acetylcysteine.

125. The compound of claim 124, or a pharmaceutically acceptable salt thereof, wherein the N-terminal modification is the addition of a desamino cysteine analog.

126. The compound of claim 125, or a pharmaceutically acceptable salt thereof, wherein the desamino cysteine analog is 3-mercaptopropionic acid or mercaptoacetic acid.

127. The compound of claim 118, or a pharmaceutically acceptable salt thereof, wherein the immunosuppressive nucleic acid sequence is an A151 nucleic acid sequence.

128. The compound of claim 127, or a pharmaceutically acceptable salt thereof, wherein the immunosuppressive nucleic acid sequence has the nucleic acid sequence of TTAGG (SEQ ID NO: 28).

129. The compound of claim 118, or a pharmaceutically acceptable salt thereof, wherein the polypeptide is an antigen or fragment thereof.

130. The compound of claim 129, or a pharmaceutically acceptable salt thereof, wherein the antigen is derived from a tumor or viral or bacterial source.

131. The compound of claim 129, or a pharmaceutically acceptable salt thereof, wherein the antigen or fragment thereof is a tumor-associated antigen.

132. The compound of claim 129, or a pharmaceutically acceptable salt thereof, wherein the antigen is an influenza antigen or fragment thereof.

133. The compound of claim 132, or a pharmaceutically acceptable salt thereof, wherein the antigen is an influenza nucleoprotein or fragment thereof.

134. The compound of claim 130, or a pharmaceutically acceptable salt thereof, wherein the antigen is a coronavirus antigen or fragment thereof.

135. The compound of claim 134, or a pharmaceutically acceptable salt thereof, wherein the antigen is a coronavirus spike protein or fragment thereof.

136. The compound of claim 134, or a pharmaceutically acceptable salt thereof, wherein the antigen is a coronavirus nucleocapsid protein or fragment thereof.

137. The compound of claim 118, or a pharmaceutically acceptable salt thereof, wherein the albumin binding domain is a lipid.

138. The compound of claim 137, or a pharmaceutically acceptable salt thereof, wherein the lipid is a diacyl lipid.

139. The compound of claim 138, or a pharmaceutically acceptable salt thereof, wherein the diacyl lipid comprises an acyl chain comprising 12-30, 14-25, 16-20, or 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 hydrocarbon units.

140. The compound of claim 138 or claim 139, or a pharmaceutically acceptable salt thereof, wherein the lipid is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE).

141. The compound of claim 118, or a pharmaceutically acceptable salt thereof, wherein the albumin binding domain and the polypeptide are bonded or linked by a linker, wherein the linker is selected from the group consisting of a hydrophilic polymer, a string of hydrophilic amino acids, a polysaccharide, and an oligonucleotide, or a combination thereof.

142. The compound of claim 141, or a pharmaceutically acceptable salt thereof, wherein the linker comprises "N" PEG units, wherein N is between 4-50.

143. The compound of claim 142, or a pharmaceutically acceptable salt thereof, wherein the linker comprises PEG4-amido-PEG4.

144. The compound, or a pharmaceutically acceptable salt thereof, of claim 118, wherein the immunosuppressive nucleic acid sequence is bonded or linked to the following lipid through a linker, wherein the nucleotide bond is shown below: or a salt thereof, wherein X is O or S.

145. A compound, or a pharmaceutically acceptable salt thereof, comprising an immunosuppressive nucleic acid sequence, an albumin binding domain, and a functional group.

146. The compound, or a pharmaceutically acceptable salt thereof, of claim 145, wherein the functional group comprises a maleimide, a dithio (2-pyridyldithio), a cyclooctene, a cyclooctyne, an aldehyde, an azide, or an alkyne.

147. The compound, or a pharmaceutically acceptable salt thereof, of claim 145 or 146, wherein the albumin binding domain is bonded or linked to the 5' end of the immunosuppressive nucleic acid sequence.

148. The compound, or a pharmaceutically acceptable salt thereof, of claim 145 or 146, wherein the albumin binding domain is bonded or linked to the 3' end of the immunosuppressive nucleic acid sequence.

149. The compound, or a pharmaceutically acceptable salt thereof, of claim 145, wherein the immunosuppressive nucleic acid sequence is an A151 nucleic acid sequence.

150. The compound, or a pharmaceutically acceptable salt thereof, of claim 149, wherein the immunosuppressive nucleic acid sequence has the nucleic acid sequence of TTAGG (SEQ ID NO: 28).

151. The compound, or a pharmaceutically acceptable salt thereof, of claim 145, wherein the albumin binding domain is a lipid.

152. The compound, or a pharmaceutically acceptable salt thereof, of claim 151, wherein the lipid is a diacyl lipid.

153. The compound, or a pharmaceutically acceptable salt thereof, of claim 152, wherein the diacyl lipid comprises an acyl chain comprising 12-30, 14-25, 16-20, or 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 hydrocarbon units.

154. The compound, or a pharmaceutically acceptable salt thereof, of claim 152 or claim 153, wherein the lipid is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE).

155. The compound, or a pharmaceutically acceptable salt thereof, of claim 145, wherein the immunosuppressive nucleic acid sequence is bonded or linked to the following lipid through a linker, wherein the nucleotide bond is shown below: or a salt thereof, wherein X is O or S.

156. A compound, or a pharmaceutically acceptable salt thereof, comprising an immunosuppressive nucleic acid sequence, a linker, and a functional group.

157. The compound, or a pharmaceutically acceptable salt thereof, of claim 156, wherein the functional group comprises a maleimide, a dithio (2-pyridyldithio), a cyclooctene, a cyclooctyne, an aldehyde, an azide, or an alkyne.

158. The compound or pharmaceutically acceptable salt thereof of claim 156 or 157, wherein the immunosuppressive nucleic acid sequence is an A151 nucleic acid sequence.

159. The compound or pharmaceutically acceptable salt thereof of claim 158, wherein the immunosuppressive nucleic acid sequence has a nucleic acid sequence of TTAGG (SEQ ID NO: 28).

160. The compound or pharmaceutically acceptable salt thereof of claim 156, wherein the linker is selected from the group consisting of a hydrophilic polymer, a string of hydrophilic amino acids, a polysaccharide, and an oligonucleotide, or a combination thereof.

161. The compound or pharmaceutically acceptable salt thereof of claim 160, wherein the linker comprises "N" PEG units, wherein N is between 4-50.

162. The compound or pharmaceutically acceptable salt thereof of claim 161, wherein the linker comprises PEG4-amido-PEG4.

163. The compound or pharmaceutically acceptable salt thereof of claim 156, wherein the linker is as follows: or a salt thereof, wherein X is O or S.

164. A method of inducing an immune response against an antigen in a subject, the method comprising administering to the subject a compound of claim 1 or a pharmaceutically acceptable salt thereof.

165. The method of claim 164, further comprising administering to the subject an adjuvant.

166. The method of claim 164 or 165, wherein the polypeptide is an antigen or fragment thereof.

167. The method of claim 166, wherein the antigen is derived from a tumor or viral or bacterial source.

168. The method of claim 166, wherein the antigen or fragment thereof is a tumor associated antigen.

169. The method of claim 166, wherein the antigen is an influenza antigen or fragment thereof.

170. The method of claim 169, wherein the antigen is an influenza nucleoprotein or fragment thereof.

171. The method of claim 167, wherein the antigen is a coronavirus antigen or fragment thereof.

172. The method of claim 171, wherein the antigen is a coronavirus spike protein or fragment thereof.

173. The method of claim 171, wherein the antigen is a coronavirus nucleocapsid protein or fragment thereof.

174. The method of claim 164, wherein the compound or pharmaceutically acceptable salt thereof is administered subcutaneously.

175. The method of claim 164, wherein the compound or pharmaceutically acceptable salt thereof is administered transmucosally.

176. The method of claim 175, wherein the compound or pharmaceutically acceptable salt thereof is administered intranasally.

177. The method of claim 164, wherein the subject is a mammal.

178. The method of claim 177, wherein the subject is a human.

179. A pharmaceutical composition comprising a compound of claim 1 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

180. A kit comprising a compound of claim 1, or a pharmaceutically acceptable salt thereof, and instructions for administration.

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