AU2024263122A1 - Compositions containing polynucleotide and polypeptide amphiphiles and methods of use thereof - Google Patents
Compositions containing polynucleotide and polypeptide amphiphiles and methods of use thereofInfo
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Abstract
Disclosed herein are compounds including an immunomodulatory (e.g., immuno-stimulatory or immuno-inhibitory) nucleic acid sequence, a polypeptide, and an albumin-binding domain, as well as pharmaceutically acceptable salts thereof. Furthermore, disclosed herein are methods for inducing an immune response in a subject, and methods of administering such compounds to induce an immune response in a subject.
Description
ATTORNEY DOCKET: 51026-051WO2 PATENT COMPOSITIONS CONTAINING POLYNUCLEOTIDE AND POLYPEPTIDE AMPHIPHILES AND METHODS OF USE THEREOF BACKGROUND OF THE INVENTION Vaccines are used to stimulate an immune response in an individual to provide protection against and/or treatment for a particular disease. Some vaccines include an antigen to induce an immune response. Immune responses as a result of vaccination have made an enormous contribution to both human and animal health. Since the invention of the first vaccine in 1796, vaccines have come to be considered the most successful method for preventing many infectious diseases by provoking an immune response in a subject. According to the World Health Organization, immunization currently prevents 2-3 million deaths every year across all age groups. The purpose of vaccination is to generate a strong and lasting immune response providing long-term protection against infection. However, many vaccines do not currently induce optimal immunity. There remains a need to develop new and improved compositions and methods for inducing immune responses in a subject thereof. SUMMARY OF THE INVENTION The disclosure provides compounds, pharmaceutically acceptable salts thereof, pharmaceutical compositions, and kits including a compound that includes an immunomodulatory y nucleic acid sequence, an albumin-binding domain, and a polypeptide. The disclosure further provides methods of inducing an immune response in a subject by administering the compounds and salts thereof described herein. In an aspect, the disclosure provides a compound comprising an immunostimulatory nucleic acid sequence, an albumin-binding domain, and a polypeptide, or a pharmaceutically acceptable salt thereof. In some embodiments, 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 by a linker 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 a further embodiment, the albumin-binding domain is bonded to the 5’ end of the immunostimulatory nucleic acid sequence, and wherein the polypeptide is linked by a linker to the 3’ end of the immunostimulatory nucleic acid sequence. In another embodiment, the albumin-binding domain is linked by a linker to the 5’ end of the immunostimulatory nucleic acid sequence and the polypeptide is linked by a linker to the 3’ end of the immunostimulatory nucleic acid sequence. In another aspect, the 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 the compound comprising an immunostimulatory nucleic acid sequence, an albumin-binding domain, and a polypeptide, or a pharmaceutically acceptable salt thereof to the subject.
ATTORNEY DOCKET: 51026-051WO2 PATENT In one embodiment, the polypeptide is bonded or linked to the 5’ end of the immunostimulatory nucleic acid sequence, and the albumin-binding domain is bonded or linked to the polypeptide. In another embodiment, the polypeptide is linked by a linker to the 5’ end of the immunostimulatory nucleic acid sequence, and the albumin-binding domain is bonded to the polypeptide. In a further embodiment, the polypeptide is bonded to the 5’ end of the immunostimulatory nucleic acid sequence, and the albumin- binding domain is linked by a linker to the polypeptide. In another embodiment, the polypeptide is linked by a linker to the 5’ end of the immunostimulatory nucleic acid sequence and the albumin-binding domain is linked by a linker to the polypeptide. In some embodiments, the polypeptide includes an N-terminal modification. In some embodiments, the N-terminal modification is the addition of an acetylcysteine. In some embodiments, the N-terminal modification is the addition of a des-aminocysteine homolog. In some embodiments, the des- aminocysteine homolog is 3-mercaptopropionic acid or mercaptoacetic acid. In some embodiments, the disclosure provides a compound or pharmaceutically acceptable salt thereof 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. In some embodiments, the immunostimulatory nucleic acid sequence is a poly-deoxyadenosine (poly-dA) nucleic acid sequence. In some embodiments, the immunostimulatory nucleic acid sequence is a poly-deoxythymidine (poly-dT) nucleic acid sequence. In some embodiments, the compound or pharmaceutically acceptable salt thereof comprises 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 poly-dT nucleic acid sequence include the same number of nucleotides. In some embodiments, the immunostimulatory nucleic acid sequence is a poly- deoxyguanosine(poly-dG) nucleic acid sequence. In some embodiments, the immunostimulatory nucleic acid sequence is a poly-deoxycytosine (poly-dC) nucleic acid sequence. In some embodiments, the compound or pharmaceutically acceptable salt thereof comprises a poly-dG nucleic acid sequence and a poly-dC nucleic acid sequence. In some embodiments, the poly-dG nucleic acid sequence and the poly- dC nucleic acid sequence hybridize to form a double-stranded DNA sequence. In some embodiments, the poly-dG nucleic acid sequence and poly-dC nucleic acid sequence include the same number of nucleotides. In some embodiments, the poly-dA nucleic acid sequence, poly-dT nucleic acid sequence, poly- dC nucleic acid sequence, and /or poly-dG nucleic acid sequence 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 90 and 100 nucleotides). In some embodiments, the poly-dA nucleic acid sequence, poly-dT nucleic acid sequence, poly-dC nucleic acid sequence, and /or poly-dG nucleic acid sequence 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 nucleic acid sequence, poly-dT nucleic acid sequence, poly-dC nucleic acid sequence, and /or poly-dG nucleic acid sequence comprise between 30
ATTORNEY DOCKET: 51026-051WO2 PATENT 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 nucleic acid sequence, poly-dT nucleic acid sequence, poly-dC nucleic acid sequence, and /or poly-dG nucleic acid sequence comprise 30, 40, 50, 75, or 100 nucleotides. In some embodiments, the poly-dA nucleic acid sequence and poly-dT nucleic acid sequence comprise the same number of nucleotides. In some embodiments, the poly-dG nucleic acid sequence and poly-dC nucleic acid sequence comprise the same number of nucleotides. 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 between 100% and 51% (e.g., between 100% and 60%, 100% and 70%, 100% and 80%, 100% and 90%, 100% and 95%, 95% and 51%, 90% and 51%, 80% and 51%, 70% and 51%, and 60% and 51%) dA nucleic acid residues and between 0% and 49% (e.g., 0% and 45%, 0% and 40%, 0% and 30%, 0% and 20%, 0 % and 10%, and 0% and 5%, 5% and 49%, 10% and 49%, 20% and 49%, 30% and 49%, and 40% and 49%) dT, dC, and/or dG nucleic acid residues. In some embodiments, the poly-dT nucleic acid sequence comprises between 100% and 51% (e.g., between 100% and 60%, 100% and 70%, 100% and 80%, 100% and 90%, 100% and 95%, 95% and 51%, 90% and 51%, 80% and 51%, 70% and 51%, and 60% and 51%) dT nucleic acid residues and between 0% and 49% (e.g., 0% and 45%, 0% and 40%, 0% and 30%, 0% and 20%, 0 % and 10%, and 0% and 5%, 5% and 49%, 10% and 49%, 20% and 49%, 30% and 49%, and 40% and 49%) dA, dC, and/or dG nucleic acid residues. In some embodiments, the poly-dG nucleic acid sequence comprises between 100% and 51% (e.g., between 100% and 60%, 100% and 70%, 100% and 80%, 100% and 90%, 100% and 95%, 95% and 51%, 90% and 51%, 80% and 51%, 70% and 51%, and 60% and 51%) dG nucleic acid residues and between 0% and 49% (e.g., 0% and 45%, 0% and 40%, 0% and 30%, 0% and 20%, 0 % and 10%, and 0% and 5%, 5% and 49%, 10% and 49%, 20% and 49%, 30% and 49%, and 40% and 49%) dC, dA, or dT nucleic acid residues. In some embodiments, the poly-dC nucleic acid sequence comprises between 100% and 51% (e.g., between 100% and 60%, 100% and 70%, 100% and 80%, 100% and 90%, 100% and 95%, 95% and 51%, 90% and 51%, 80% and 51%, 70% and 51%, and 60% and 51%) dC nucleic acid residues and between 0% and 49% (e.g., 0% and 45%, 0% and 40%, 0% and 30%, 0% and 20%, 0 % and 10%, and 0% and 5%, 5% and 49%, 10% and 49%, 20% and 49%, 30% and 49%, and 40% and 49%) dG, dA, or dT nucleic acid residues. 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 linkages in the sequence of SEQ ID NO: 25 are phosphorothioate, including the link between the diacyl lipid and the 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). In some embodiments, at least one internucleotide group 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. In some embodiments, all internucleotide groups connecting the nucleotides in the poly-dA nucleic acid sequence, poly-dT nucleic acid sequence, poly-dC nucleic acid
ATTORNEY DOCKET: 51026-051WO2 PATENT sequence, or poly-dG nucleic acid sequence are phosphorothioate. In some embodiments, the albumin- binding domain is bonded to the 5’ end of the poly-dA nucleic acid sequence, poly-dT nucleic acid sequence, poly-dG nucleic acid sequence, or poly-dC nucleic acid sequence. In some embodiments, the polypeptide is bonded to the 3’ end of the poly-dA nucleic acid sequence, poly-dT nucleic acid sequence, poly-dC nucleic acid sequence or poly-dG nucleic acid sequence. In some embodiments, the polypeptide is an antigen, or a fragment thereof. In some embodiments, the polypeptide is derived from tumors or viral or bacterial source. In some embodiments, the antigen, or fragment thereof, is a tumor-associated antigen. In some embodiments, the polypeptide is an influenza antigen, or fragment thereof. In some embodiments, the polypeptide is an Influenza nucleoprotein, or fragment thereof. In some embodiments, the polypeptide is a coronavirus antigen, or fragment thereof. In some embodiments, the polypeptide is a coronavirus spike protein, or fragment thereof. In some embodiments, the polypeptide is a coronavirus nucleocapsid protein, or fragment thereof. In some embodiments, the polypeptide includes or consists of the amino acid sequence of SEQ ID NO: 23. In some embodiments, the polypeptide is a neoantigen, or fragment thereof. In some embodiments, the albumin-binding domain is a lipid. In some embodiments, the lipid is a diacyl lipid. In some embodiments, the diacyl lipid comprises acyl chains 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. In some embodiments, the lipid is 1,2-distearoyl-sn- glycero-3-phosphatidylethanolamine (DSPE). In some embodiments, 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. In some embodiments, the linker comprises "N" polyethylene glycol units, wherein N is between 4-50. In some embodiments, the linker comprises PEG4-amido-PEG4. In some embodiments, the immunostimulatory nucleic acid sequence is bonded or linked by a linker to the following lipid, where the nucleotide linkage is as shown below:
or a salt thereof, wherein X is O (phosphodiester) or S (phosphorothioate).
ATTORNEY DOCKET: 51026-051WO2 PATENT In another aspect, the disclosure provides a compound comprising an immunostimulatory nucleic acid sequence, an albumin-binding domain, and a functional group, or a pharmaceutically acceptable salt thereof. In some embodiments, the functional group comprises a maleimide, a dithio(2-pyridyldithio), a cyclooctene, a cyclooctyne, an aldehyde, an azide, or an alkyne. In some embodiments, albumin-binding domain is bonded or linked to the 5’ end of the immunostimulatory nucleic acid sequence. In some embodiments, the albumin-binding domain is bonded or linked to the 3’ end of the immunostimulatory nucleic acid sequence. In another aspect, the disclosure provides a compound comprising an immunostimulatory nucleic acid sequence, an albumin-binding domain, and a functional group, 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 the compound comprising an immunostimulatory nucleic acid sequence, an albumin-binding domain, and a functional group, or a pharmaceutically acceptable salt thereof to the subject. In some embodiments, the polypeptide and albumin-binding domain are bonded or linked to the 5’ end of the immunostimulatory nucleic acid sequence. In some embodiments, the polypeptide and albumin-binding domain are bonded or linked to the 3’ end of the immunostimulatory nucleic acid sequence. In some embodiments, the immunostimulatory nucleic acid sequence is a poly-deoxyadenosine (poly-dA) nucleic acid sequence. In some embodiments, the immunostimulatory nucleic acid sequence is a poly-deoxythymidine (poly-dT) nucleic acid sequence. In some embodiments, the compound or pharmaceutically acceptable salt thereof comprises 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 poly-dT nucleic acid sequence comprise the same number of nucleotides. In some embodiments, the immunostimulatory nucleic acid sequence is a poly- deoxyguanosine(poly-dG) nucleic acid sequence. In some embodiments, the immunostimulatory nucleic acid sequence is a poly-deoxycytosine (poly-dC) nucleic acid sequence. In some embodiments, the compound or pharmaceutically acceptable salt thereof comprises a poly-dG nucleic acid sequence and a poly-dC nucleic acid sequence. In some embodiments, the poly-dG nucleic acid sequence and the poly- dC nucleic acid sequence hybridize to form a double-stranded DNA sequence. In some embodiments, the poly-dG nucleic acid sequence and poly-dC nucleic acid sequence comprise the same number of nucleotides. In some embodiments, the poly-dA nucleic acid sequence, poly-dT nucleic acid sequence, poly- dC nucleic acid sequence, and /or poly-dG nucleic acid sequence 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 90 and 100 nucleotides). In some embodiments, the poly-dA nucleic acid sequence, poly-dT nucleic acid sequence, poly-dC nucleic acid sequence, and /or poly-dG nucleic acid sequence 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 nucleic acid sequence, poly-dT nucleic acid sequence, poly-dC nucleic acid sequence, and /or poly-dG nucleic acid sequence comprise between 30
ATTORNEY DOCKET: 51026-051WO2 PATENT 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 nucleic acid sequence, poly-dT nucleic acid sequence, poly-dC nucleic acid sequence, and /or poly-dG nucleic acid sequence comprise 30, 40, 50, 75, or 100 nucleotides. 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 between 100% and 51% (e.g., between 100% and 60%, 100% and 70%, 100% and 80%, 100% and 90%, 100% and 95%, 95% and 51%, 90% and 51%, 80% and 51%, 70% and 51%, and 60% and 51%) dA nucleic acid residues and between 0% and 49% (e.g., 0% and 45%, 0% and 40%, 0% and 30%, 0% and 20%, 0 % and 10%, and 0% and 5%, 5% and 49%, 10% and 49%, 20% and 49%, 30% and 49%, and 40% and 49%) dT, dC, and/or dG) nucleic acid residues. In some embodiments, the poly-dT nucleic acid sequence comprises between 100% and 51% (e.g., between 100% and 60%, 100% and 70%, 100% and 80%, 100% and 90%, 100% and 95%, 95% and 51%, 90% and 51%, 80% and 51%, 70% and 51%, and 60% and 51%)dT nucleic acid residues and between 0% and 49% (e.g., 0% and 45%, 0% and 40%, 0% and 30%, 0% and 20%, 0 % and 10%, and 0% and 5%, 5% and 49%, 10% and 49%, 20% and 49%, 30% and 49%, and 40% and 49%) dA, dC, and/or dG nucleic acid residues. In some embodiments, the poly-dG nucleic acid sequence comprises between 100% and 51% (e.g., between 100% and 60%, 100% and 70%, 100% and 80%, 100% and 90%, 100% and 95%, 95% and 51%, 90% and 51%, 80% and 51%, 70% and 51%, and 60% and 51%) dG nucleic acid residues and between 0% and 49% (e.g., 0% and 45%, 0% and 40%, 0% and 30%, 0% and 20%, 0 % and 10%, and 0% and 5%, 5% and 49%, 10% and 49%, 20% and 49%, 30% and 49%, and 40% and 49%) dC, dA, or dT nucleic acid residues. In some embodiments, the poly-dC nucleic acid sequence comprises between 100% and 51% (e.g., between 100% and 60%, 100% and 70%, 100% and 80%, 100% and 90%, 100% and 95%, 95% and 51%, 90% and 51%, 80% and 51%, 70% and 51%, and 60% and 51%) dC nucleic acid residues and between 0% and 49% (e.g., 0% and 45%, 0% and 40%, 0% and 30%, 0% and 20%, 0 % and 10%, and 0% and 5%, 5% and 49%, 10% and 49%, 20% and 49%, 30% and 49%, and 40% and 49%) dG, dA, or dT nucleic acid residues. In some embodiments, the immunostimulatory 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 embodiment, the CpG sequence is 5'-TCCATGACGTTCCTGACGTT-3' (SEQ ID NO: 29). In some embodiments, at least one internucleotide group 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. In some embodiments, all internucleotide groups 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. In some embodiments, the albumin-binding domain is a lipid. In some embodiments, the lipid is a diacyl lipid. In some embodiments, the diacyl lipid comprises acyl chains comprising 12-30 hydrocarbon
ATTORNEY DOCKET: 51026-051WO2 PATENT 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-distearoyl-sn- glycero-3-phosphatidylethanolamine (DSPE). In some embodiments, the immunostimulatory nucleic acid sequence is bonded or linked by a linker to the following lipid, where the nucleotide linkage is as shown below:
or a salt thereof, wherein X is O or S. 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 at the by way of a PEG4 linker to the maleimide group. In another aspect, the disclosure provides, a compound comprising an immunostimulatory nucleic acid sequence, a linker, and a functional group or a pharmaceutically acceptable salt thereof. In some embodiments, the functional group comprises a maleimide, a dithio(2-pyridyldithio), a cyclooctene, a cyclooctyne, an aldehyde, an azide, or an alkyne. In another aspect, the disclosure provides a compound comprising an immunostimulatory nucleic acid sequence, a linker, and a functional group 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 the compound comprising an immunostimulatory nucleic acid sequence, a linker, and a functional group or a pharmaceutically acceptable salt thereof to the subject. In some embodiments, the immunostimulatory nucleic acid sequence is a poly-deoxyadenosine (poly-dA) nucleic acid sequence. In some embodiments, the immunostimulatory nucleic acid sequence is a poly-deoxythymidine (poly-dT) nucleic acid sequence. In some embodiments, the compound or pharmaceutically acceptable salt thereof comprises 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 poly-dT nucleic acid sequence comprise the same number of nucleotides. In some embodiments, the immunostimulatory nucleic acid sequence is a poly- deoxyguanosine(poly-dG) nucleic acid sequence. In some embodiments, the immunostimulatory nucleic acid sequence is a poly-deoxycytosine (poly-dC) nucleic acid sequence. In some embodiments, the compound or pharmaceutically acceptable salt thereof comprises a poly-dG nucleic acid sequence and a poly-dC nucleic acid sequence. In some embodiments, the poly-dG nucleic acid sequence and the poly-dC nucleic acid sequence hybridize to form a double-stranded DNA sequence. In some embodiments, the poly-dG nucleic acid
ATTORNEY DOCKET: 51026-051WO2 PATENT sequence and poly-dC nucleic acid sequence comprise the same number of nucleotides. In some embodiments, the poly-dA nucleic acid sequence, poly-dT nucleic acid sequence, poly-dC nucleic acid sequence, and /or poly-dG nucleic acid sequence comprise between 30 and 100 (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) nucleotides. In some embodiments, the poly-dA nucleic acid sequence, poly-dT nucleic acid sequence, poly-dC nucleic acid sequence, and /or poly-dG nucleic acid sequence 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 nucleic acid sequence, poly-dT nucleic acid sequence, poly-dC nucleic acid sequence, and /or poly-dG nucleic acid sequence 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 nucleic acid sequence, poly-dT nucleic acid sequence, poly-dC nucleic acid sequence, and /or poly-dG nucleic acid sequence comprise 30, 40, 50, 75, or 100 nucleotides. 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 between 100% and 51% (e.g., between 100% and 60%, 100% and 70%, 100% and 80%, 100% and 90%, 100% and 95%, 95% and 51%, 90% and 51%, 80% and 51%, 70% and 51%, and 60% and 51%) dA nucleic acid residues and between 0% and 49% (e.g., 0% and 45%, 0% and 40%, 0% and 30%, 0% and 20%, 0 % and 10%, and 0% and 5%, 5% and 49%, 10% and 49%, 20% and 49%, 30% and 49%, and 40% and 49%) dT, dC, and/or dG nucleic acid residues. In some embodiments, the poly-dT nucleic acid sequence comprises between 100% and 51% (e.g., between 100% and 60%, 100% and 70%, 100% and 80%, 100% and 90%, 100% and 95%, 95% and 51%, 90% and 51%, 80% and 51%, 70% and 51%, and 60% and 51%) dT nucleic acid residues and between 0% and 49% (e.g., 0% and 45%, 0% and 40%, 0% and 30%, 0% and 20%, 0 % and 10%, and 0% and 5%, 5% and 49%, 10% and 49%, 20% and 49%, 30% and 49%, and 40% and 49%) dA, dC, and/or dG nucleic acid residues. In some embodiments, the poly-dG nucleic acid sequence comprises between 100% and 51% (e.g., between 100% and 60%, 100% and 70%, 100% and 80%, 100% and 90%, 100% and 95%, 95% and 51%, 90% and 51%, 80% and 51%, 70% and 51%, and 60% and 51%) dG nucleic acid residues and between 0% and 49% (e.g., 0% and 45%, 0% and 40%, 0% and 30%, 0% and 20%, 0 % and 10%, and 0% and 5%, 5% and 49%, 10% and 49%, 20% and 49%, 30% and 49%, and 40% and 49%) dC, dA, or dT nucleic acid residues. In some embodiments, the poly-dC nucleic acid sequence comprises between 100% and 51% (e.g., between 100% and 60%, 100% and 70%, 100% and 80%, 100% and 90%, 100% and 95%, 95% and 51%, 90% and 51%, 80% and 51%, 70% and 51%, and 60% and 51%) dC nucleic acid residues and between 0% and 49% (e.g., 0% and 45%, 0% and 40%, 0% and 30%, 0% and 20%, 0 % and 10%, and 0% and 5%, 5% and 49%, 10% and 49%, 20% and 49%, 30% and 49%, and 40% and 49%) dG, dA, or dT nucleic acid residues. In some embodiments, the immunostimulatory 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
ATTORNEY DOCKET: 51026-051WO2 PATENT NO: 27). In some embodiments, the CpG sequence is 5'-TCCATGACGTTCCTGACGTT-3' (SEQ ID NO: 29). In some embodiments, at least one internucleotide group 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. In some embodiments, all internucleotide groups 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. In some embodiments, the linker is as shown below:
or a salt thereof, wherein X is O or S. 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 linked to the PEG4 which is bonded or linked to the maleimide group. In another aspect, the disclosure provides a compound comprising an immunoinhibitory nucleic acid sequence, an albumin-binding domain, and a polypeptide, or a pharmaceutically acceptable salt thereof. In some embodiments, wherein albumin-binding domain is bonded or linked to the 5’ end of the immunoinhibitory nucleic acid sequence, and wherein the polypeptide is bonded or linked to the 3’ end of the immunoinhibitory nucleic acid sequence. In some embodiments, the polypeptide is bonded or linked to the 5’ end of the immunoinhibitory nucleic acid sequence, and wherein albumin-binding domain is bonded or linked to the 3’ end of the immunoinhibitory nucleic acid sequence. In some embodiments, the polypeptide and albumin-binding domain both are bonded or linked to the 5’ end of the immunoinhibitory nucleic acid sequence. In some embodiments, the polypeptide and albumin-binding domain both are bonded or linked to the 3’ end of the immunoinhibitory nucleic acid sequence. In another aspect, the disclosure provides a compound comprising an immunoinhibitory 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 the compound comprising an immunoinhibitory nucleic acid sequence, an albumin-binding domain, and a polypeptide, or a pharmaceutically acceptable salt thereof to the subject. In some embodiments, the polypeptide comprises an N-terminal modification. In some embodiments, the N-terminal modification is the addition of an acetylcysteine. In some embodiments, the N-terminal modification is the addition of a des-aminocysteine homolog. In some embodiments, the des- aminocysteine homolog is 3-mercaptopropionic acid or mercaptoacetic acid. In some embodiments, the immunoinhibitory nucleic acid sequence is an A151 nucleic acid sequence. In some embodiments, the immunoinhibitory nucleic acid sequence has a nucleic acid sequence of TTAGG (SEQ ID NO: 28).
ATTORNEY DOCKET: 51026-051WO2 PATENT In some embodiments, the polypeptide is an antigen, or a fragment thereof. In some embodiments, the antigen is derived from a tumor or a viral or bacterial source. In some embodiments, the antigen, or fragment thereof, is a tumor-associated antigen. In some embodiments, the antigen is an influenza antigen, or fragment thereof. In some embodiments, the antigen is an influenza nucleoprotein, or fragment thereof. In some embodiments, the antigen is a coronavirus antigen, or fragment thereof. In some embodiments, the antigen is a coronavirus spike protein, or fragment thereof. In some embodiments, the antigen is a coronavirus nucleocapsid protein, or fragment thereof. In some embodiments, the albumin-binding domain is a lipid. In some embodiments, the lipid is a diacyl lipid. In some embodiments, the diacyl lipid comprises acyl chains 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. In some embodiments, the lipid is 1,2-distearoyl-sn- glycero-3-phosphatidylethanolamine (DSPE). In some embodiments, 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. In some embodiments, the linker comprises "N" polyethylene glycol units, wherein N is between 4-50. In some embodiments, the linker comprises PEG4-amido-PEG4. In some embodiments, the immunoinhibitory nucleic acid sequence is bonded or linked by a linker to the following lipid, where the nucleotide linkage is as shown below:
or a salt thereof, wherein X is O or S.
ATTORNEY DOCKET: 51026-051WO2 PATENT In another aspect, the disclosure provides a compound comprising an immunoinhibitory nucleic acid sequence, an albumin-binding domain, and a functional group, or a pharmaceutically acceptable salt thereof. In some embodiments, the functional group comprises a maleimide, a dithio(2-pyridyldithio), a cyclooctene, a cyclooctyne, an aldehyde, an azide, or an alkyne. In some embodiments, albumin-binding domain is bonded or linked to the 5’ end of the immunoinhibitory nucleic acid sequence. In some embodiments, albumin-binding domain is bonded or linked to the 3’ end of the immunoinhibitory nucleic acid sequence. In some embodiments, the immunoinhibitory nucleic acid sequence is an A151 nucleic acid sequence. In some embodiments, the immunoinhibitory nucleic acid sequence has a nucleic acid sequence of TTAGG (SEQ ID NO: 28). In another aspect, the disclosure a compound comprising an immunoinhibitory nucleic acid sequence, an albumin-binding domain, and a functional group, 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 the compound comprising an immunoinhibitory nucleic acid sequence, an albumin-binding domain, and a functional group, or a pharmaceutically acceptable salt thereof to the subject. In some embodiments, the albumin-binding domain is a lipid. In some embodiments, the lipid is a diacyl lipid. In some embodiments, the diacyl lipid comprises acyl chains 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. In some embodiments, the lipid is 1,2-distearoyl-sn- glycero-3-phosphatidylethanolamine (DSPE). In some embodiments, the immunoinhibitory nucleic acid sequence is bonded or linked by a linker to the following lipid, where the nucleotide linkage is as shown below:
or a salt thereof, wherein X is O or S. In another aspect, the disclosure provides a compound comprising an immunoinhibitory nucleic acid sequence, a linker, and a functional group or a pharmaceutically acceptable salt thereof. In some embodiments, the functional group comprises a maleimide, a dithio(2-pyridyldithio), a cyclooctene, a cyclooctyne, an aldehyde, an azide, or an alkyne. In some embodiments, the immunoinhibitory nucleic acid sequence is an A151 nucleic acid sequence. In some embodiments,the immunoinhibitory nucleic acid sequence has a nucleic acid sequence of TTAGG (SEQ ID NO: 28). In another aspect, the disclosure a compound comprising an immunoinhibitory nucleic acid sequence, a linker, and a functional group 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 the compound comprising an immunoinhibitory nucleic acid sequence, a linker, and a functional group or a pharmaceutically acceptable salt thereof to the subject. In some embodiments, 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. In
ATTORNEY DOCKET: 51026-051WO2 PATENT some embodiments, the linker comprises "N" polyethylene glycol units, wherein N is between 4-50. In some embodiments, the linker comprises PEG4-amido-PEG4. In some embodiments, the linker is as shown below:
or a salt thereof, wherein X is O or S. In another aspect, the disclosure provides a method of inducing an immune response against an antigen in a subject, the method comprising administering any one of the compounds or a pharmaceutically acceptable salt thereof described herein to the subject. In another aspect, the disclosure provides the compounds or a pharmaceutically acceptable salt thereof described herein for use in a method of inducing an immune response against an antigen in a subject, wherein the method comprises administering any one of the compounds described herein to the subject. In some embodiments, further comprising 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 fragment thereof. In some embodiments, the polypeptide is derived from tumors or viral or bacterial sources. In some embodiments, the antigen, or fragment thereof, is a tumor-associated antigen. In some embodiments, the antigen is an influenza antigen, or fragment thereof. In some embodiments, the antigen is an Influenza nucleoprotein, or fragment thereof. In some embodiments, the antigen is a coronavirus antigen, or fragment thereof. In some embodiments, the antigen is a coronavirus spike protein, or fragment thereof. In some embodiments, the antigen is a coronavirus nucleocapsid protein, or fragment thereof. In another aspect, the disclosure provides the use of one of the compounds or a pharmaceutically acceptable salt thereof described herein to induce an immune response against an antigen in a subject, wherein any one of the compounds described herein is to be administered to the subject. In some embodiments, an adjuvant is to 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 fragment thereof. In some embodiments, the polypeptide is derived from tumors or viral or bacterial sources. In some embodiments, the antigen, or fragment thereof, is a tumor-associated antigen. In some embodiments, the antigen is an influenza antigen, or fragment thereof. In some embodiments, the antigen is an Influenza nucleoprotein, or fragment thereof. In some embodiments, the antigen is a coronavirus antigen, or fragment thereof. In some embodiments, the antigen is a coronavirus spike protein, or fragment thereof. In some embodiments, the antigen is a coronavirus nucleocapsid protein, or fragment thereof. In some embodiments, the compound or a pharmaceutically acceptable salt thereof is administered or is to be administered subcutaneously. In some embodiments, the compound or a pharmaceutically acceptable salt thereof is administered transmucosally. In some embodiments, the compound or a pharmaceutically acceptable salt thereof is administered buccally. In some embodiments,
ATTORNEY DOCKET: 51026-051WO2 PATENT 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. In another aspect, the disclosure provides a pharmaceutical composition comprising any one of the compounds or a pharmaceutically acceptable salt thereof described herein and a pharmaceutically acceptable carrier. In another aspect, the disclosure provides a kit comprising any one of the compounds or a pharmaceutically acceptable salt thereof described herein. BRIEF DESCRIPTION OF THE DRAWINGS FIG.1A is a drawing of a single-stranded amphiphile (AMP) including the CpG-7909 nucleic acid sequence (SEQ ID NO: 25) bonded to a polypeptide sequence having an amino acid sequence of SEQ ID NO: 23 and conjugated to albumin-binding domain. FIG.1B is a drawing of a single-stranded amphiphile (AMP) and double-stranded AMP including the CpG-7909 nucleic acid sequence (SEQ ID NO: 25) conjugated to albumin-binding domain and a PEG linker modified with a maleimide functional group. FIG.2 is a graph showing the splenocyte IFNγ co-culture ELISpot responses of C57Bl6 mice that were administered a vaccine including a soluble or an amphiphilic antigen or adjuvant of a soluble or amphiphilic chimera as shown in FIG.1. FIG.3 is a graph showing the amount of CD8 cells that are specific to the gp100 antigen isolated from the peripheral blood collected from C57BL/6J mice (n= 5 per group) that were administered a vaccine including a soluble or an amphiphilic antigen or adjuvant of a soluble or amphiphilic chimera as shown in FIG.1. FIG.4 is a graph showing the percentage of cytokines, including (from top to bottom in each column) IFNγ and TNFα, only TNFα, and only IFNγ, found in peripheral blood cells collected C57BL/6J mice (n= 5 per group) that were administered a vaccine including a soluble or an amphiphilic antigen or adjuvant of a soluble or amphiphilic chimera as shown in FIG.1. FIG.5 is a graph showing the frequency of intracellular cytokine production, including, from top to bottom in each column, IFNγ and TNFα, only TNFα, and only IFNγ, in CD8+ T cells isolated from perfuse lung tissue in C57BL/6J mice (n = 5 per group) that were administered a vaccine including a soluble or an amphiphilic antigen or adjuvant of a soluble or amphiphilic chimera as shown in FIG.1. FIG.6 is a drawing of a single-stranded AMP-dT nucleic acid sequence bonded to a polypeptide sequence having an amino acid sequence of SEQ ID NO: 23 and conjugated to an albumin-binding domain. FIG.7 is a graph showing the splenocyte IFNγ co-culture ELISpot responses of C57Bl6 mice that were administered a vaccine including a soluble or an amphiphilic antigen or adjuvant of a soluble or amphiphilic chimera as shown in FIG.6. FIG.8 is a graph showing the amount of CD8 cells that are specific to the Influenza NP antigen isolated from the peripheral blood collected from C57BL/6J mice (n= 5 per group) that were administered
ATTORNEY DOCKET: 51026-051WO2 PATENT a vaccine including a soluble or an amphiphilic antigen or adjuvant of a soluble or amphiphilic chimera as shown in FIG.6. FIG.9 is a graph showing the frequency of cytokines in CD8+ T cells, including (from top to bottom in each column) IFNγ and TNFα, only TNFα, and only IFNγ, found in peripheral blood cells collected C57BL/6J mice (n= 5 per group) that were administered a including a soluble or an amphiphilic antigen or adjuvant of a soluble or amphiphilic chimera as shown in FIG.6. FIG.10 is a graph showing the frequency of intracellular cytokine production, including, from top to bottom in each column, IFNγ and TNFα, only TNFα, and only IFNγ, in CD8+ T cells isolated from perfuse lung tissue in C57BL/6J mice that were administered a vaccine including a soluble or an amphiphilic antigen or adjuvant of a soluble or amphiphilic chimera as shown in FIG.6. Definitions Terms used in the claims and specification are defined as set forth below unless otherwise specified. It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. As used herein, "about" will be understood by persons of ordinary skill and will vary to some extent depending on the context in which it is used. If there are uses of the term which are not clear to persons of ordinary skill given the context in which it is used, "about" will mean up to plus or minus 10% of the particular value. As used herein, the term "adjuvant" refers to a compound that, with a specific immunogen or antigen, will augment or otherwise alter or modify the resultant immune response. Modification of the immune response includes intensification or broadening the specificity of either or both antibody and cellular immune responses. Modification of the immune response can also mean decreasing or suppressing certain antigen-specific immune responses. In certain embodiments, the adjuvant is a cyclic dinucleotide. In some embodiments, the adjuvant is an immunomodulatory oligonucleotide as described herein. "Amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, y-carboxyglutamate, and phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified polypeptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that function in a manner similar to a naturally occurring amino acid. Amino acids can be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, can be referred to by their commonly accepted single-letter codes.
ATTORNEY DOCKET: 51026-051WO2 PATENT An "amino acid substitution" refers to the replacement of at least one existing amino acid residue in a predetermined amino acid sequence (an amino acid sequence of a starting polypeptide) with a second, different "replacement" amino acid residue. An "amino acid insertion" refers to the incorporation of at least one additional amino acid into a predetermined amino acid sequence. While the insertion will usually consist of the insertion of one or two amino acid residues, the present larger "peptide insertions," can be made, e.g., by insertion of about three to about five or even up to about ten, fifteen, or twenty amino acid residues. The inserted residue(s) may be naturally occurring or non-naturally occurring as disclosed above. An "amino acid deletion" refers to the removal of at least one amino acid residue from a predetermined amino acid sequence. As used herein, "amphiphile" or "amphiphilic" refers to a conjugate comprising a hydrophilic head group and a hydrophobic tail, thereby forming an amphiphilic conjugate. In some embodiments, an amphiphile conjugate comprises an immunostimulatory poly-dA, poly-dT, poly-dC, poly-dG, or CpG sequence, a polypeptide, and one or more hydrophobic lipid tails. The term "ameliorating" refers to any therapeutically beneficial result in the treatment of a disease state, e.g., influenza and SARS-CoV-2, including prophylaxis, lessening in the severity or progression, remission, or cure thereof. As used herein, "cancer antigen" refers to (i) tumor- specific antigens, (ii) tumor- associated antigens, (iii) cells that express tumor- specific antigens, (iv) cells that express tumor- associated antigens, (v) embryonic antigens on tumors, (vi) autologous tumor cells, (vii) tumor- specific membrane antigens, (viii) tumor- associated membrane antigens, (ix) growth factor receptors, (x) growth factor ligands, and (xi) any other type of antigen or antigen-presenting cell or material that is associated with a cancer. A polypeptide or amino acid sequence "derived from" a designated polypeptide or protein or a "polypeptide fragment" refers to the origin of the polypeptide. Preferably, the polypeptide or amino acid sequence which is derived or is a fragment of is from a particular sequence that has an amino acid sequence that is essentially identical to that 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 which is otherwise identifiable to one of ordinary skill in the art as having its origin in the sequence. Polypeptides derived from or that are fragments of another polypeptide may have one or more mutations relative to the starting polypeptide, e.g., one or more amino acid residues which have been substituted with another amino acid residue or which has one or more amino acid residue insertions or deletions. A polypeptide can comprise an amino acid sequence which is not naturally occurring. Such variants necessarily have less than 100% sequence identity or similarity with the starting molecule. In a preferred embodiment, the variant will have an amino acid sequence from about 75% to less than 100% amino acid sequence identity or similarity with the amino acid sequence of the starting polypeptide, more preferably from about 80% to less than 100%, more preferably from about 85% to less than 100%, more preferably from about 90% to less than 100% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) and most preferably from about 95% to less than 100%, e.g., over the length of the variant molecule. In one embodiment, there is one amino acid difference between a starting polypeptide sequence and the sequence derived therefrom. Identity or similarity with respect to this sequence is defined herein
ATTORNEY DOCKET: 51026-051WO2 PATENT as the percentage of amino acid residues in the candidate sequence that are identical (i.e., same residue) with the starting amino acid residues, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. As used herein, the term "cytotoxic T lymphocyte (CTL) response" refers to an immune response induced by cytotoxic T cells. CTL responses are mediated primarily by CD8+ T cells. As used herein, the term "effective dose" or "effective dosage" is defined as an amount sufficient to achieve or at least partially achieve the desired effect. The term "therapeutically effective dose" is defined as an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. Amounts effective for this use will depend upon the severity of the disorder being treated and the general state of the patient's own immune system. As used herein, "immune cell" is a cell of hematopoietic origin and that plays 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 particular embodiments, the immune cell is T cell. As used herein, "immune response" refers to a response made by the immune system of an organism to a substance, which includes but is not limited to foreign or self proteins. Three general types of "immune response" include mucosal, humoral, and cellular immune responses. For example, the immune response can include the activation, expansion, and/or increased proliferation of an immune cell. 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, developing immunity, developing hypersensitivity to an antigen, the response of antigen-specific lymphocytes to antigen, clearance of an infectious agent, and transplant or graft rejection. As used herein, an “immunoinhibitory oligonucleotide” is an oligonucleotide that can inhibit (e.g., reduce or prevent) an immune response. As used herein, an "immunomodulatory oligonucleotide" is an immunostimulatory oligonucleotide or an immunoinhibitory oligonucleotide. As used herein, an “immunostimulatory oligonucleotide” is an oligonucleotide that can stimulate (e.g., induce or enhance) an immune response. The terms "inducing an immune response" and "enhancing an immune response" are used interchangeably and refer to the stimulation of an immune response (i.e., either passive or adaptive) to a particular antigen. The term "induce" as used with respect to inducing complement dependent cytotoxicity (CDC) or antibody-dependent cellular cytotoxicity (ADCC) refer to the stimulation of particular direct cell killing mechanisms. As used herein, a subject "in need of prevention," "in need of treatment," or "in need thereof," refers to one, who by the judgment of an appropriate medical practitioner (e.g., a doctor, a nurse, or a nurse practitioner in the case of humans; a veterinarian in the case of non-human mammals), would reasonably benefit from a given treatment (such as treatment with a composition comprising an amphiphilic ligand conjugate).
ATTORNEY DOCKET: 51026-051WO2 PATENT The term "in vivo" refers to processes that occur in a living organism. The term "in vitro" refers to processes that occur outside a living organism, such as in a test tube, flask, or culture plate. As used herein, the terms "linked," "operably linked," "fused," or "fusion," are used interchangeably. These terms refer to the joining together of two more elements or components or domains, by an appropriate means including chemical conjugation or recombinant DNA technology. Methods of chemical conjugation (e.g., using heterobifunctional crosslinking agents) are known in the art as are methods of recombinant DNA technology. The term "lipid" refers to a biomolecule that is soluble in nonpolar solvents and insoluble in water. Lipids are often 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), prenol lipids, saccharolipids, and polyketides. In some embodiments, the lipid suitable for the amphiphilic ligand conjugates of the disclosure binds to human serum albumin under physiological conditions. In some embodiments, the lipid suitable for the amphiphilic ligand conjugates of the disclosure inserts into a cell membrane under physiological conditions. In some embodiments, the lipid binds albumin and inserts into a cell membrane under physiological conditions. In some embodiments, the lipid is a diacyl lipid. In some embodiments, the diacyl lipid includes at least 12 carbons. In some embodiments, the diacyl lipid includes 12-30 hydrocarbon units, 14-25 hydrocarbon units, or 16-20 hydrocarbon units. In some embodiments, the diacyl lipid includes 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 carbons. "Nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences and as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base 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, modifications at the second base can also be conservative. The term nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene. Polynucleotides of the present invention can be composed of any polyribonucleotide or polydeoxribonucleotide, which can be unmodified RNA or DNA or modified RNA or DNA. For example, polynucleotides can be composed of single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that can be single-stranded or, more typically, double-stranded or a mixture of single- and double stranded regions. In addition, the polynucleotide can be composed of triple-stranded regions comprising RNA or DNA or both RNA and DNA. A polynucleotide can also contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons. "Modified" bases include, for example, tritylated bases and unusual
ATTORNEY DOCKET: 51026-051WO2 PATENT bases such as inosine. A variety of modifications can be made to DNA and RNA; thus, "polynucleotide" embraces chemically, enzymatically, or metabolically modified forms. In some embodiments, the polypeptides of the invention are encoded by a nucleotide sequence. Nucleotide sequences of the invention can be useful for a number of applications, including: cloning, gene therapy, protein expression and purification, mutation introduction, DNA vaccination of a host in need thereof, antibody generation for, e.g., passive immunization, PCR, primer and probe generation, and the like. As used herein, "parenteral administration," "administered parenterally," and other grammatically equivalent phrases, refer to modes of administration other than enteral and topical administration, usually by injection, and include, without limitation, intravenous, intranasal, intraocular, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intracerebral, intracranial, intracarotid and intrasternal injection and infusion. As generally used herein, "pharmaceutically acceptable" refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues, organs, and/or bodily fluids of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit/risk ratio. The term “pharmaceutically acceptable salt,” as used herein, means any pharmaceutically acceptable salt of a conjugate, oligonucleotide, or polypeptide disclosed herein. Pharmaceutically acceptable salts of any of the compounds and nucleic acid sequences described herein may include those that are within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without undue toxicity, irritation, allergic response and are commensurate with a reasonable benefit/risk ratio. 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 in Pharmaceutical Salts: Properties, Selection, and Use (Eds. P.H. Stahl and C.G. Wermuth), Wiley-VCH, 2008. The salts can be prepared in situ during the final isolation and purification of the compounds described herein or separately by reacting a free base group with a suitable acid. Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2- hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate salts, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like, as well as nontoxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like. References to the compounds, nucleic acids, conjugates, oligonucleotides, or polypeptides in the claims and elsewhere herein optionally include pharmaceutically acceptable salts thereof unless otherwise indicated or not applicable.
ATTORNEY DOCKET: 51026-051WO2 PATENT As used herein, the term “physiological conditions” refers to the in vivo condition of a subject. In some embodiments, physiological condition refers to a neutral pH (e.g., pH between 6-8). As used herein, the term “peptide” refers to a polymer having 30 or fewer amino acid residues. "Polypeptide," "peptide", and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer. As used herein, the term "subject" or "mammal" or "patient" includes any human or non-human animal. For example, the methods and compositions of the present invention can be used to treat a subject with a disease or condition. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, mice, horses, pigs, cows, chickens, amphibians, reptiles, etc. The term "sufficient amount" or "amount sufficient to" means an amount sufficient to produce a desired effect, e.g., an amount sufficient to reduce the diameter of a tumor. The term "T cell" refers to a type of white blood cell that can be distinguished from other white blood cells by the presence of a T cell receptor on the cell surface. There are several subsets of T cells, including, but not limited to, T helper cells ( a.k.a. TH cells or CD4+ T cells) and subtypes, including TH, TH2, TH3, TH17, TH9, and TFH cells, cytotoxic T cells (i.e., Tc cells, CD8+ T cells, cytotoxic T lymphocytes, T-killer cells, killer T cells), memory T cells and subtypes, including central memory T cells (TCM cells), effector memory T cells (TEM and TEMRA cells), and resident memory T cells (TRM cells), regulatory T cells (a.k.a. Treg cells or suppressor T cells) and subtypes, including CD4+ FOXP3+ Treg cells, CD4+FOXP3- Treg cells, Tr1 cells, Th3 cells, and Treg17 cells, natural killer T cells (a.k.a. NKT cells), mucosal associated invariant T cells (MAITs), and gamma delta T cells (γδ T cells), including Vγ9/Vδ2 T cells. Any one or more of the aforementioned or unmentioned T cells may be the target cell type for a method of use of the invention. The terms "treat," "treating," and "treatment," as used herein, refer to therapeutic or preventative measures described herein. The methods of "treatment" employ administration to a subject, in need of such treatment, a poly-dA nucleic acid sequence and/or poly-dT nucleic acid sequence and an albumin- binding domain of the present disclosure. In some embodiments, a poly-dA nucleic acid sequence, poly- dT, poly-dG, and/or poly-dC nucleic acid sequence conjugated to an albumin-binding domain is administered to a subject in need of an enhanced immune response against a particular antigen or a subject who ultimately may acquire such a disorder, in order to prevent, cure, delay, reduce the severity of, or ameliorate one or more symptoms of the disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. The term "tumor- associated antigen" refers to an antigen that is produced in a tumor and can be detected by the immune system to trigger an immune response. Tumor-associated antigens have been identified in many human cancers including lung, skin, hematologic, brain, liver, breast, rectal, bladder, and stomach cancers. As used herein, "vaccine" refers to a formulation which contains an amphiphilic construct described herein, optionally combined with an adjuvant, which is in a form that is capable of being administered to a vertebrate and which induces a protective immune response sufficient to induce
ATTORNEY DOCKET: 51026-051WO2 PATENT immunity to prevent and/or ameliorate a disease or condition (e.g., influenza or SARS-CoV-2) and/or to reduce at least one symptom of a disease or condition (e.g., influenza or SARS-CoV-2). Typically, the vaccine comprises a conventional saline or buffered aqueous solution medium in which a composition as described herein is suspended or dissolved. In this form, a composition as described herein is used to prevent, ameliorate, or otherwise treat an infection or disease. Upon introduction into a host, the vaccine provokes an immune response including, but not limited to, the inducing a protective immune response to induce immunity to prevent and/or ameliorate a disease or condition (e.g., influenza or SARS-CoV-2) and/or to reduce at least one symptom of a disease or condition. DETAILED DESCRIPTION The disclosure provides compounds including an immunomodulatory (e.g., immuno-stimulatory or immuno-inhibitory) nucleic acid sequence conjugated, either directly or by a linker, with an albumin- binding domain and a polypeptide. The disclosure provides that the immunomodulatory (e.g., immuno- stimulatory or immuno-inhibitory) nucleic acid sequence may include a poly-adenosine (poly-dA) nucleic acid sequence, a poly-thymidine (poly-dT) nucleic acid sequence, a poly-deoxyguanosine (poly-dG) nucleic acid sequence, and/or a poly-deoxycytosine (poly-dC) nucleic acid sequence, as well as CpG nucleic acid sequences, conjugated with an albumin-binding domain. Furthermore, the disclosure provides pharmaceutical compositions and kits including the immunomodulatory (e.g., immuno- stimulatory or immuno-inhibitory) nucleic acid conjugated to a polypeptide and to an albumin-binding domain. The disclosure further provides methods of inducing an immune response in a subject by administering the compounds or pharmaceutically acceptable salts thereof described herein. Polypeptides Described herein are compounds including a polypeptide conjugated to an immunomodulatory (e.g., immuno-stimulatory or immuno-inhibitory) nucleic acid and conjugated to an albumin-binding domain. In some embodiments, the polypeptide may be antigenic protein or polypeptide, or a fragment thereof (e.g., an epitope). The polypeptide or peptide may 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) in length, including for example, 5 amino acids, 10 amino acids, 15 amino acids, 20 amino acids, 25 amino acids, 30 amino acids, 35 amino acids, 40 amino acids, 45 amino acids, or 50 amino acids. In some embodiments, a polypeptide can be greater than 50 amino acids in length. In some embodiments, the polypeptide can be > 100 amino acids in length. The protein or polypeptide can be any protein or polypeptide that can induce or increase the ability of the immune system to develop antibodies and T-cell responses to the protein or polypeptide. The polypeptide may be an antigen derived from a virus, bacterium, parasite, plant, protozoan, fungus, tissue or transformed cell such as a cancer or leukemic cell and can be a whole cell or immunogenic component thereof, e.g., cell wall components or molecular components thereof. Suitable antigens are known in the art and are available from commercial government and scientific sources. In one embodiment, the polypeptides are whole inactivated or attenuated organisms. These
ATTORNEY DOCKET: 51026-051WO2 PATENT organisms may be infectious organisms, such as viruses, parasites, and bacteria. These organisms may also be tumor cells. The polypeptides may be purified or partially purified polypeptides derived from tumors or viral or bacterial sources. The polypeptides can be recombinant polypeptides produced by expressing DNA encoding the polypeptide in a heterologous expression system. In some embodiments, the polypeptide may be modified at the N-terminus. In some embodiments, the polypeptide may be modified at the C-terminus. For example, the polypeptide may be modified with an acetylcysteine residue at the N-terminus. In some embodiments, the polypeptide may be modified with an aminocysteine homologue at the N-terminus. For example, the polypeptide may be modified with 3-mercaptopropionic acid or mercaptoacetic acid at the N-terminus. Antigens may be provided as single antigens or may be provided in combination. Antigens may also be provided as complex mixtures of polypeptides or nucleic acids. Exemplary antigens are provided below. Viral Antigens A viral antigen can be isolated from any virus including, but not limited to, a virus from any of the following viral families: Arenaviridae, Arterivirus, Astroviridae, Baculoviridae, Badnavirus, Barnaviridae, Birnaviridae, Bromoviridae, Bunyaviridae, Caliciviridae, Capillovirus, Carlavirus, Cauliniovirus, Circoviridae, Closterovirus, Comoviridae, Coronaviridae (e.g., Coronavirus, such as severe acute respiratory syndrome (SARS) virus; e.g., SARS-CoV-2), Corticoviridae, Cystoviridae, Deltavirus, Dianthovirus, Enamovirus, Filoviridae (e.g., Marburg virus and Ebola virus (e.g., Zaire, Reston, Ivory Coast, or Sudan strain)), Flaviviridae, (e.g., Hepatitis C virus, Dengue virus 1, Dengue virus 2, Dengue virus 3, and Dengue virus 4), Hepadnaviridae, Herpesviridae (e.g., Human herpesvirus 1, 3, 4, 5, and 6, and Cytomegalovirus), Hypoviridae, Iridoviridae, Leviviridae, Lipothrixviridae, Microviridae, Orthomyxoviridae (e.g., Influenzavirus A and B and C), Papovaviridae, Paramyxoviridae (e.g., measles, mumps, and human respiratory syncytial virus), Parvoviridae, Picornaviridae (e.g., poliovirus, rhinovirus, hepatovirus, and aphthovirus), Poxviridae (e.g., vaccinia and smallpox virus), Reoviridae (e.g., rotavirus), Retroviridae (e.g., lentivirus, such as human immunodeficiency virus (HIV) 1 and HIV 2), Rhabdoviridae (for example, rabies virus, measles virus, respiratory syncytial virus, etc.), Togaviridae (for example, 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. Viral antigens may be derived from a particular strain such as a papilloma virus, a herpes virus, e.g., herpes simplex 1 and 2; a hepatitis virus, for example, hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), the delta hepatitis D virus (HDV), hepatitis E virus (HEV) and hepatitis G virus (HGV), the tick-borne encephalitis viruses; parainfluenza, varicella-zoster, cytomeglavirus, Epstein- Barr, rotavirus, rhinovirus, adenovirus, coxsackieviruses, equine encephalitis, Japanese encephalitis, yellow fever, Rift Valley fever, and lymphocytic choriomeningitis. In some embodiments, compounds described herein include an immunomodulatory (e.g., immuno-stimulatory or immuno-inhibitory) nucleic acid conjugated to a polypeptide and conjugated to an albumin-binding domain. The polypeptide may be an antigen, or a fragment thereof. For example, the polypeptide may be derived from tumors (e.g., a tumor-associated antigen) or viral or bacterial sources. The polypeptide may be an antigen, and the antigen may be an influenza antigen, or fragment thereof.
ATTORNEY DOCKET: 51026-051WO2 PATENT For example, the antigen may be an influenza nucleoprotein, or fragment thereof. Specifically, 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 to: MASQGTKRSYEQMETDGERQNATEIRASVGKMIGGIGRFYIQMCTELKLSDYEGRLIQN SLTIERMVLSAFDERRNKYLEEHPSAGKDPKKTGGPIYRRVNGKWMRELILYDKEEIRRI WRQANNGDDATAGLTHMMIWHSNLNDATYQRTRALVRTGMDPRMCSLMQGSTLPRR SGAAGAAVKGVGTMVMELVRMIKRGINDRNFWRGENGRKTRIAYERMCNILKGKFQTA AQKAMMDQVRESRNPGNAEFEDLTFLARSALILRGSVAHKSCLPACVYGPAVASGYDF EREGYSLVGIDPFRLLQNSQVYSLIRPNENPAHKSQLVWMACHSAAFEDLRVLSFIKGT KVLPRGKLSTRGVQIASNENMETMESSTLELRSRYWAIRTRSGGNTNQQRASAGQISIQ PTFSVQRNLPFDRTTIMAAFNGNTEGRTSDMRTEIIRMMESARPEDVSFQGRGVFELSD EKAASPIVPSFDMSNEGSYFFGDNAEEYDN (SEQ ID NO: 22) 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 to SEQ ID NO: 22. In some embodiments, the influenza nucleoprotein has a polypeptide sequence of SEQ ID NO: 22, or fragment thereof. The antigen may be a coronavirus antigen, or fragment thereof. For example, the antigen may be a coronavirus spike protein, or fragment thereof. Bacterial Antigens 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, Hemophilus influenza type B (HIB), Hyphomicrobium, Legionella, Leptspirosis, Listeria, Meningococcus A, B and C, Methanobacterium, Micrococcus, Myobacterium, Mycoplasma, Myxococcus, Neisseria, Nitrobacter, Oscillatoria, Prochloron, Proteus, Pseudomonas, Phodospirillum, Rickettsia, Salmonella, Shigella, Spirillum, Spirochaeta, Staphylococcus, Streptococcus, Streptomyces, Sulfolobus, Thermoplasma, Thiobacillus, and Treponema, Vibrio, and Yersinia. Parasite Antigens Parasite antigens can be obtained from parasites such as, but not limited to, an antigen derived from Cryptococcus neoformans, Histoplasma capsulatum, Candida albicans, Candida tropicalis, Nocardia asteroides, Rickettsia ricketsii, Rickettsia typhi, Mycoplasma pneumoniae, Chlamydial psittaci, Chlamydial trachomatis, Plasmodium falciparum, Trypanosoma brucei, Entamoeba histolytica, Toxoplasma gondii, Trichomonas vaginalis and Schistosoma mansoni. These include Sporozoan antigens, Plasmodian antigens, such as all or part of a Circumsporozoite protein, a Sporozoite surface protein, a liver stage antigen, an apical membrane associated protein, or a Merozoite surface protein. Allergens and Environmental Antigens The antigen can be an allergen or environmental antigen, such as, but not limited to, an antigen derived from naturally occurring allergens such as pollen allergens (tree-, herb, weed-, and grass pollen
ATTORNEY DOCKET: 51026-051WO2 PATENT allergens), insect allergens (inhalant, saliva and venom allergens), animal hair and dandruff allergens, and food allergens. Important pollen allergens from trees, grasses and herbs originate from the taxonomic orders of Fagales, Oleales, Pinales and platanaceae including e.g., birch (Betula), alder (Alnus), hazel (Corylus), hornbeam (Carpinus) and olive (Olea), cedar (Cryptomeria and Juniperus), Plane tree (Platanus), the order of Poales including e.g., grasses of the genera Lolium, Phleum, Poa, Cynodon, Dactylis, Holcus, Phalaris, Secale, and Sorghum, the orders of Asterales and Urticales including i.a. herbs of the genera Ambrosia, Artemisia, and Parietaria. Other allergen antigens that may be used include allergens from house dust mites of the genus Dermatophagoides and Euroglyphus, storage mite e.g. Lepidoglyphys, Glycyphagus and Tyrophagus, those from cockroaches, midges and fleas e.g. Blatella, Periplaneta, Chironomus and Ctenocepphalides, those from mammals such as cat, dog and horse, birds, venom allergens including such originating from stinging or biting insects such as those from the taxonomic order of Hymenoptera including bees (superfamily Apidae), wasps (superfamily Vespidea), and ants (superfamily Formicoidae). Still other allergen antigens that may be used include inhalation allergens from fungi such as from the genera Alternaria and Cladosporium. Cancer Antigens A cancer antigen is an antigen that is typically expressed preferentially by cancer cells (i.e., it is expressed at higher levels in cancer cells than on non-cancer cells) and in some instances it is expressed solely by cancer cells. The cancer antigen may be expressed within a cancer cell or on the surface of the cancer cell. The cancer antigen may be a tumor- associated antigen. The cancer antigen 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-zeta 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), MAGE-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, α-fetoprotein, E-cadherin, α- catenin, β-catenin, γ-catenin, p120ctn, gp100Pmel117, PRAME, NY-ESO-1, cdc27, adenomatous polyposis coli protein (APC), fodrin, Connexin 37, Ig-idiotype, p15, gp75, GM2 ganglioside, GD2 ganglioside, human papilloma virus proteins, Smad family of tumor antigens, 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. 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 to AVGALEGPRNQDWLGVPRQL (SEQ ID NO: 23).
ATTORNEY DOCKET: 51026-051WO2 PATENT Immunomodulatory Nucleic Acid Sequences The compounds described herein include an immunomodulatory (e.g., immuno-stimulatory or immuno-inhibitory) nucleic acid sequence. The compound may include an immuno-inhibitory nucleic acid sequence. For example, the immune-inhibitory nucleic acid sequence may have a nucleic acid sequence of TTAGGG (SEQ ID NO: 28). The compound may include an immunostimulatory nucleic acid sequence. The immunostimulatory nucleic acid sequence may be a poly-adenosine nucleic acid sequence (poly-dA), a poly-thymidine nucleic acid sequence (poly-dT,) a polycytosine (poly-dC) nucleic acid sequence, a polyguanosine (poly-dG) nucleic acid sequence, or a CpG sequence. Polyadenosine and Polythymidine Nucleic Acid Sequences Poly-deoxyribonucleic acids include a strand of nucleic acids that may be single or double stranded. The poly-deoxyribonucleic acid may include only one type of nucleic acid in a single strand of nucleic acids (e.g., poly-dA and poly-dT). A poly-dA nucleic acid sequence may include only adenosine nucleobases. In some embodiments, the poly-dA nucleic acid sequence includes a mixture of adenosine and thymidine or other nucleic acid residues. For example, in some embodiments, the poly-dA nucleic acid sequence may be composed of between 100% and 51% of adenosine nucleic acid residues and between 0% and 49% thymidine nucleic acid residues. For example, in some embodiments, the poly-dA nucleic acid sequence may be composed of 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 10% and 5%) of adenosine 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 90% and 95%) thymidine, guanosine, and/or cytosine nucleic acid residues. In some embodiments, the nucleic acid sequence may include alternating dA and dT residues. A poly-dT sequence may include only thymidine nucleobases. In some embodiments, the poly-dT nucleic acid sequence includes a mixture of thymidine and adenosine or other nucleic acid residues. For example, in some embodiments, the poly-dT nucleic acid sequence may be composed of 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 10% and 5%) of thymidine 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 90% and 95%) a mixture of adenosine, guanosine, or cytosine nucleic acid residues. The poly-dA single single-stranded DNA sequence may include 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 95 and 100) nucleotides. In some embodiments, the poly-dA single-single stranded DNA sequence includes between 75 and 150 (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 140 and 150) nucleotides. In some embodiments, the poly- dA single-single stranded DNA sequence includes 75, 80, 85, 90, 95, or 100 nucleotides. In some embodiments, the poly-dA includes 50 nucleotides (dA50)
ATTORNEY DOCKET: 51026-051WO2 PATENT The poly-dT single single-stranded DNA sequence may include 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 95 and 100) nucleotides. In some embodiments, the poly-dT single-single stranded DNA sequence includes between 75 and 150 (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 140 and 150) nucleotides. In some embodiments, the poly- dT single-single stranded DNA sequence includes 75, 80, 85, 90, 95, or 100 nucleotides. In some embodiments, the poly-dT includes 50 nucleotides (dT50). The poly-dA and poly-dT double-stranded DNA sequence may include 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 95 and 100) nucleotides. In some embodiments, the poly-dA and poly-dT nucleic acids sequences include between 75 and 150 (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 140 and 150) nucleotides. In some embodiments, the poly- dA and poly-dT include the same number of nucleotides. In some embodiments, the poly-dA and poly-dA single-singles stranded DNA sequences of the double-stranded DNA sequence each includes 75, 80, 85, 90, 95, or 100 nucleotides. In some embodiments, the poly-dA and poly-dT include 50 nucleotides each (dA50:dT50). Reference to poly-deoxyribonucleic acids described herein, is to be understood as including pharmaceutically acceptable salts thereof. Polyguanosine and Polycytosine Nucleic Acid Sequences Poly-deoxyribonucleic acids include a strand of nucleic acids that may be single or double stranded. The poly-deoxyribonucleic acid may include only one type of nucleic acid in a single strand of nucleic acids (e.g., polyguanosine (poly-dG) and polycytosine (poly-dC)). A poly-dG nucleic acid sequence may include only guanosine nucleobases. In some embodiments, the poly-dG nucleic acid sequence includes a mixture of guanosine and cytosine nucleic acid residues. For example, in some embodiments, the poly-dG nucleic acid sequence may be composed of between 100% and 51% of guanosine nucleic acid residues and between 0% and 49% cytosine nucleic acid residues. A poly-dC sequence may include only cytosine nucleobases. In some embodiments, the poly-dC nucleic acid sequence includes a mixture of cytosine and guanosine or other nucleic acid residues. For example, in some embodiments, the poly-dC nucleic acid sequence may be composed of 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 10% and 5%) of cytosine 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 90% and 95%) of a mixture of adenosine, guanosine, or thymidine nucleic acid residues, or in some embodiments, the poly-dG nucleic acid may include 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 10% and 5%) of guanosine nucleic acid residues and
ATTORNEY DOCKET: 51026-051WO2 PATENT 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 90% and 95%) of a mixture of adenosine, cytosine, or thymidine nucleic acid residues. The poly-dG single-stranded DNA sequence may include 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 95 and 100) nucleotides. In some embodiments, the poly-dG single stranded DNA sequence includes between 75 and 150 (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 140 and 150) nucleotides. In some embodiments, the poly-dG single stranded DNA sequence includes 75, 80, 85, 90, 95, or 100 nucleotides. The poly-dC single-stranded DNA sequence may include 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 95 and 100) nucleotides. In some embodiments, the poly-dC single stranded DNA sequence includes between 75 and 150 (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 140 and 150) nucleotides. In some embodiments, the poly-dC single stranded DNA sequence includes 75, 80, 85, 90, 95, or 100 nucleotides. The poly-dG and poly-dC double-stranded DNA sequence may include 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 95 and 100) nucleotides. In some embodiments, the poly-dG and poly-dC nucleic acids sequences include between 75 and 150 (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 140 and 150) nucleotides. In some embodiments, the poly- dG and poly-dC include the same number of nucleotides. In some embodiments, the poly-dG and poly- dC single stranded DNA sequences of the double-stranded DNA sequence each includes 75, 80, 85, 90, 95, or 100 nucleotides. In some embodiments, the poly-dG and poly-dC strands of nucleic acids are complementary to one another. CpG Sequences CpG ODNs are short synthetic single-stranded DNA molecules containing unmethylated CpG dinucleotides in particular sequence contexts. CpG ODNs possess a partially or completely phosphorothioated (PS) backbone, as opposed to the natural phosphodiester (PO) backbone in DNA molecules. Three major classes of stimulatory CpG ODNs have been identified based on structural characteristics and activity on human peripheral blood mononuclear cells (PBMCs), in particular B cells and plasmacytoid dendritic cells (pDCs). These three classes are Class A (Type D), Class B (Type K), and Class C. CpG1826 and CpG7909 both are in CpG class B. Class B CpG ODNs contain a full PS backbone with one or more CpG dinucleotides. They strongly activate B cells and TLR9-dependent NF-κB signaling but weakly stimulate IFN-α secretion. CpG7909 has the following sequence: 5’-TCGTCGTTTTGTCGTTTTGTCGTT-3’ (SEQ ID NO: 25)
ATTORNEY DOCKET: 51026-051WO2 PATENT 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 linkages in the CpG sequence are phosphorothioate, including the link between the diacyl lipid and the oligodeoxynucleotide. Reference to poly-deoxyribonucleic acids described herein, is to be understood as including pharmaceutically acceptable salts thereof. Amphiphilic Poly-Deoxyribonucleic Acids Amphiphilic poly-deoxyribonucleic acids include a strand of nucleic acids 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 certain embodiments, the amphiphilic poly-deoxyribonucleic acid is a poly- deoxyadenosine (AMP-dA) strand of nucleic acids conjugated to an albumin-binding domain, e.g., a lipid. In certain embodiments, the amphiphilic poly-deoxyribonucleic acid is a poly-deoxythymidine (AMP-dT) strand of nucleic acids conjugated to an albumin-binding domain, e.g., a lipid. In certain embodiments, the amphiphilic poly-deoxyribonucleic acid is a poly-deoxyguanosine (AMP-dG) strand of nucleic acids conjugated to an albumin-binding domain, e.g., a lipid. In certain embodiments, the amphiphilic poly- deoxyribonucleic acid is a poly-deoxycytosine (AMP-dC) strand of nucleic acids conjugated to an albumin-binding domain, e.g., a lipid. In some embodiments, the compounds described herein include both a dA and a dT nucleic acid sequence, or a dG and a dC, and an albumin-binding domain (e.g., AMP-dA:dT,AMP-dT:dA, AMP-dG:dC, and AMP-dC:dG). The dA nucleic acid sequence and the dT nucleic acid sequence may hybridize to form a double-stranded DNA sequence (e.g., dA:dT and dT:dA). Likewise, the dG nucleic acid sequence and the dC nucleic acid sequence may hybridize to form a double-stranded DNA sequence (e.g., dG:dC and dC:dG). Examples of the poly-dA and/or poly-dT compounds of the disclosure are shown in FIG.1 and FIG.54. In some embodiments, the compounds described herein include a mixture of both dA and dT nucleic acid residues or a mixture of dG and dC nucleic acid residues. For example, the compound may include alternating dA and dT nucleic acid residues. The length of the poly-dA nucleic acid sequence may include between 30 and 150 nucleotides (e.g., between 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 140 and 150 nucleotides). For example, the length of the poly-dA nucleic acid sequence may 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 90 and 100 nucleotides). In some embodiments, the length of the poly-dA nucleic acid sequence may 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 90 and 100 nucleotides). In some embodiments, the length of the poly-dA nucleic acid sequence may 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 45 and 50 nucleotides). In some embodiments, the length of the poly-dA nucleic acid sequence may be 30, 35, 40, 45, 50, 55, 60,
ATTORNEY DOCKET: 51026-051WO2 PATENT 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 poly-dA nucleic acid sequence may be 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleotides. Furthermore, the disclosure provides a poly-dA single single- stranded DNA sequence including 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 95 and 100) nucleotides. The length of the poly-dT nucleic acid sequence may include between 30 and 150 nucleotides (e.g., between 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 140 and 150 nucleotides). For example, the length of the poly-dT nucleic acid sequence may 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 90 and 100 nucleotides). In some embodiments, the length of the poly-dT nucleic acid sequence may 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 90 and 100 nucleotides). In some embodiments, the length of the poly-dT nucleic acid sequence may 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 45 and 50 nucleotides). In some embodiments, the length of the poly-dT nucleic acid sequence may 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 poly-dT nucleic acid sequence may be 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleotides. Furthermore, the disclosure provides a poly-dT single single- stranded DNA sequence including 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 95 and 100) nucleotides. The length of both the poly-dA nucleic acid sequence and the poly-dT nucleic acid sequence may both include between 30 and 150 nucleotides (e.g., between 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 140 and 150 nucleotides). For example, the length of both the poly-dA nucleic acid sequence and the poly-dT nucleic acid sequence may 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 90 and 100 nucleotides). In some embodiments, the length of both the poly-dA nucleic acid sequence and the poly-dT nucleic acid sequence may 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 90 and 100 nucleotides). In some embodiments, the length of both the poly-dA nucleic acid sequence and the poly-dT nucleic acid sequence may 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 45 and 50 nucleotides). In some embodiments, the length of both the poly-dA nucleic acid sequence and the poly-dT nucleic acid sequence may be 30, 40, 50, 75, or 100 nucleotides. In some embodiments, the poly-dA nucleic acid sequence and poly-dT nucleic acid sequence include the same number of nucleotides. Furthermore, the disclosure provides a poly-dA and poly-dT double-stranded DNA sequence including between 30 and 100 (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
ATTORNEY DOCKET: 51026-051WO2 PATENT and 100, and 90 and 100) nucleotides. In some embodiments, the poly-dA and poly-dT include the same number of nucleotides. The length of the poly-dG nucleic acid sequence may include between 30 and 150 nucleotides (e.g., between 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 140 and 150 nucleotides). For example, the length of the poly-dG nucleic acid sequence may 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 90 and 100 nucleotides). In some embodiments, the length of the poly-dG nucleic acid sequence may 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 90 and 100 nucleotides). In some embodiments, the length of the poly-dG nucleic acid sequence may 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 45 and 50 nucleotides). In some embodiments, the length of the poly-dG nucleic acid sequence may 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 poly-dG nucleic acid sequence may be 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleotides. Furthermore, the disclosure provides a poly-dG single single-stranded DNA sequence including 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 95 and 100) nucleotides. The length of the poly-dC nucleic acid sequence may include between 30 and 150 nucleotides (e.g., between 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 140 and 150 nucleotides). For example, the length of the poly-dC nucleic acid sequence may 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 90 and 100 nucleotides). In some embodiments, the length of the poly-dC nucleic acid sequence may 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 90 and 100 nucleotides). In some embodiments, the length of the poly-dC nucleic acid sequence may 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 45 and 50 nucleotides). In some embodiments, the length of the poly-dC nucleic acid sequence may 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 poly-dC nucleic acid sequence may be 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleotides. Furthermore, the disclosure provides a poly-dC single single- stranded DNA sequence including 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 95 and 100) nucleotides. The length of both the poly-dG nucleic acid sequence and the poly-dC nucleic acid sequence may both include between 30 and 150 nucleotides (e.g., between 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 140 and 150 nucleotides). For example, the length of both the poly-dG nucleic acid
ATTORNEY DOCKET: 51026-051WO2 PATENT sequence and the poly-dC nucleic acid sequence may 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 90 and 100 nucleotides). In some embodiments, the length of both the poly-dG nucleic acid sequence and the poly-dC nucleic acid sequence may 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 90 and 100 nucleotides). In some embodiments, the length of both the poly-dG nucleic acid sequence and the poly-dC nucleic acid sequence may 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 45 and 50 nucleotides). In some embodiments, the length of both the poly-dG nucleic acid sequence and the poly-dC nucleic acid sequence may be 30, 40, 50, 75, or 100 nucleotides. In some embodiments, the poly-dG nucleic acid sequence and poly-dC nucleic acid sequence include the same number of nucleotides. Furthermore, the disclosure provides a poly-dG and poly-dC double-stranded DNA sequence including between 30 and 100 (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 90 and 100) nucleotides. In some embodiments, the poly-dG and poly-dC include the same number of nucleotides. The length of the alternating poly-dA and poly-dT nucleic acid sequence may include between 30 and 150 nucleotides (e.g., between 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 140 and 150 nucleotides). For example, the length of the poly-dA nucleic acid sequence may 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 90 and 100 nucleotides). In some embodiments, the length of the alternating poly-dA and poly-dT nucleic acid sequence may 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 90 and 100 nucleotides). In some embodiments, the length of the poly-dA nucleic acid sequence may 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 45 and 50 nucleotides). In some embodiments, the length of the poly-dA nucleic acid sequence may 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 sequence may be 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleotides. Furthermore, the disclosure provides an alternating poly-dA and poly-dT single single-stranded DNA sequence including 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 95 and 100) nucleotides. In some embodiments, between 50% and 100% (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%) of the internucleotide groups connecting the nucleotides in the poly-dA nucleic acid sequence and/or poly-dT nucleic acid sequence are phosphorothioate linkages. In some embodiments, between 1 and 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) of the internucleotide groups connecting the nucleotides in the poly-dA nucleic acid sequence and/or poly-dT nucleic acid sequence are phosphodiester linkages and the remaining internucleotide groups connecting the nucleotides of the poly-dA nucleic acid sequence and/or poly-dT nucleic acid sequence are phosphorothioate linkages. For example, the between 1 and 5 linkages (e.g.,
ATTORNEY DOCKET: 51026-051WO2 PATENT 1, 2, 3, 4, or 5) linkages at the 5’ and/or 3’ end of the poly-dA and/or poly-dT nucleic acid sequences are phosphodiester linkages, and all the remaining linkages are phosphorothioate linkages. In some embodiments, all internucleotide groups connecting the nucleotides in the poly-dA nucleic acid sequence and/or poly-dT nucleic acid sequence are phosphorothioate linkages. In some embodiments, between 50% and 100% (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%) of the internucleotide groups connecting the nucleotides in the poly-dG nucleic acid sequence and/or poly-dC nucleic acid sequence are phosphorothioate linkages. In some embodiments, between 1 and 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) of the internucleotide groups connecting the nucleotides in the poly-dG nucleic acid sequence and/or poly-dC nucleic acid sequence are phosphodiester linkages and the remaining internucleotide groups connecting the nucleotides of the poly-dG nucleic acid sequence and/or poly-dC nucleic acid sequence are phosphorothioate linkages. For example, the between 1 and 5 linkages (e.g., 1, 2, 3, 4, or 5) linkages at the 5’ and/or 3’ end of the poly-dG and/or poly-dC nucleic acid sequences are phosphodiester linkages, and all the remaining linkages are phosphorothioate linkages. In some embodiments, all internucleotide groups connecting the nucleotides in the poly-dG nucleic acid sequence and/or poly-dC nucleic acid sequence are phosphorothioate linkages. Reference to poly-deoxyribonucleic acids described herein, as well as amphiphiles including the poly-deoxyribonucleic acids, is to be understood as including pharmaceutically acceptable salts thereof. Lipids The compounds described herein include immunomodulatory (e.g., immuno-stimulatory or immuno-inhibitory) nucleic acid sequences that are conjugated to a polypeptide and a lipid. In some embodiments, the lipid is bonded to the 5’ end of a poly-dA nucleic acid sequence. In some embodiments, the lipid is bonded to the 5’ end of a poly-dT nucleic acid sequence. In some embodiments, the lipid is bonded to the 5’ end of a poly-dG nucleic acid sequence. In some embodiments, the lipid is bonded to the 5’ end of a poly-dC nucleic acid sequence. In some embodiments, the lipid is bonded to the 5’ end of a CpG nucleic acid sequence. In some embodiments, the lipid is bonded to the 3’ end of a nucleic acid sequence described herein. The lipid can be linear, branched, or cyclic. Examples of preferred lipids include, but are not limited to, fatty acids with aliphatic tails of 3-30 carbons including, but not limited to, linear unsaturated and saturated fatty acids, branched saturated and unsaturated fatty acids, and fatty acids 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. In certain embodiments, the lipid is a diacyl lipid or two-tailed lipid. In some embodiments, the tails in the diacyl lipid contain from about 12 to about 30 carbons (e.g., 1314, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29). In some embodiments the tails in the diacyl lipid contain about 14 to about 25 carbons (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24). In some embodiments, the tails of the diacyl lipid contain from about 16 to about 20 carbons (e.g., 17, 18, or 19). In some embodiments, the diacyl lipid comprises 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 carbons.
ATTORNEY DOCKET: 51026-051WO2 PATENT The carbon tails of the diacyl lipid can be saturated, unsaturated, or combinations thereof. The tails can be coupled to the head group via ester bond linkages, amide bond linkages, thioester bond linkages, or combinations thereof. In a particular embodiment, the diacyl lipids are phosphate lipids, glycolipids, sphingolipids, or combinations thereof. In some embodiments, the lipid is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE). In some embodiments, the immunomodulatory (e.g., immuno-stimulatory or immuno-inhibitory) nucleic acid sequence is bonded or linked by a linker to the following lipid, where the nucleotide linkage is as shown below:
, or a salt thereof, wherein X is O or S. The immunomodulatory (e.g., immuno-stimulatory or immuno-inhibitory) nucleic acid sequence may be directly bonded to the lipid. Alternatively, the immunomodulatory (e.g., immuno-stimulatory or immuno-inhibitory) nucleic sequence may be linked to the lipid through a linker. Reference to lipids herein, as well as amphiphiles including the lipid, is to be understood as including pharmaceutically acceptable salts thereof. Linkers In some embodiments, the compound includes an immunomodulatory (e.g., immuno-stimulatory or immuno-inhibitory) nucleic acid sequence conjugated to a polypeptide and linked to an albumin-binding domain, e.g., a lipid, by a linker. 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 ability of the albumin-binding domain to insert into the plasma membrane of cells, such as cells in the tissue adjacent to the injection site. The linker can also reduce or prevent the ability of the amphiphilic immunomodulatory (e.g., immuno-stimulatory or immuno-inhibitory) nucleic acid sequence from non-specifically associating with extracellular matrix proteins at the site of administration. For the amphiphilic immunomodulatory (e.g., immuno-stimulatory or immuno-inhibitory) nucleic acid sequence to be trafficked efficiently to the lymph node, it should remain soluble. A polar block linker may be included between the immunomodulatory (e.g., immuno-stimulatory or immuno-inhibitory) nucleic acid sequence and the albumin-binding domain to which it is conjugated to increase solubility of the amphiphilic immunomodulatory (e.g., immuno-stimulatory or immuno-inhibitory) nucleic acid sequence. The length and composition of the linker can be adjusted based on the albumin-binding domain and immunomodulatory (e.g., immuno-stimulatory or immuno-inhibitory) nucleic acid sequence selected. For example, in certain embodiments, the polynucleotide itself may be polar enough to ensure solubility; for example, polynucleotides that are 10, 15, 20, or more nucleotides in length. Therefore, in some embodiments, no additional linker is required. However, in certain cases, it can be desirable to include a linker that mimics the effect of a polar oligonucleotide. A linker can be used as part of any of albumin-
ATTORNEY DOCKET: 51026-051WO2 PATENT binding domain conjugates described herein, for example, lipid-oligonucleotide conjugates and lipid- peptide conjugates, which reduce cell membrane insertion/preferential portioning onto albumin. Suitable linkers include, but are not limited to, oligonucleotides such as those discussed above, including a string of nucleic acids, a hydrophilic polymer including but not limited to poly(ethylene glycol) (MW: 500 Da to 20,000 Da), polyacrylamide (MW: 500 Da to 20,000 Da), 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; polysaccharides, including but not limited to, dextran (MW: 1,000 Da to 2,000,000 Da), or combinations thereof. The hydrophobic albumin- binding domain and the linker/immunomodulatory (e.g., immuno-stimulatory or immuno-inhibitory) nucleic acid sequence are covalently linked. The covalent bond may be a non-cleavable linkage or a cleavable linkage. The non-cleavable linkage can include an amide bond or phosphate bond, and the cleavable linkage can include a disulfide bond, acid-cleavable linkage, ester bond, anhydride bond, biodegradable bond, or enzyme-cleavable linkage. 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, compound includes an immunomodulatory (e.g., immuno-stimulatory or immuno-inhibitory) nucleic acid sequence and a hydrophobic albumin-binding domain linked by a polyethylene glycol (PEG) molecule or a derivative or analog thereof. In some embodiments, compounds described herein contain a an immunomodulatory (e.g., immuno-stimulatory or immuno-inhibitory) nucleic acid sequence conjugated to a polypeptide and linked to PEG which is in turn linked to a hydrophobic albumin-binding domain, e.g., a lipid. The precise number of PEG units depends on the albumin-binding domain and the cargo, however, typically, a linker can 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 linker may be a PEG linker 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 units (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 45 and 50 units). In some embodiments, the linker has between about 45 and 55 PEG units. For example, in some embodiments, the linker has 48 PEG units. In some embodiments, the linker includes a PEG4-amido-PEG4 linker. In some embodiments, the linker may include one or more alkyl groups. In some embodiments, the linker may include a combination of alkyl groups and PEG groups. For example, the linker may be a hexylamine-PEG4 linker. As discussed above, in some embodiments, the linker is an oligonucleotide which includes a string of nucleic acids. In some embodiments, the compounds described herein include an immunomodulatory (e.g., immuno-stimulatory or immuno-inhibitory) nucleic sequence linked to a string of nucleic acids, which is in turn linked to a hydrophobic albumin-binding domain, e.g., a lipid. The linker can be any sequence, for example, the sequence of the oligonucleotide can be a random sequence, or a sequence specifically chosen for its molecular or biochemical properties (e.g., highly polar). In some
ATTORNEY DOCKET: 51026-051WO2 PATENT embodiments, the linker includes 20 one or more series of consecutive adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U), or analog thereof. In some embodiments, the linker consists of a series of consecutive adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U), or analog thereof. In some embodiments, the string of nucleic acids includes between 1 and 50 nucleic acid residues. In some embodiments, the string of nucleic acids includes between 5 and 30 nucleic acid residues. In some embodiments, the linker includes one or more guanines, for example between 1-10 guanines. In some embodiments, the linker is an oligonucleotide that includes a string of amino acids. In some embodiments, the amphiphilic immunomodulatory (e.g., immuno-stimulatory or immuno-inhibitory) nucleic acid sequence conjugated to a polypeptide is linked to string of amino acids, which is in turn linked to a hydrophobic albumin-binding domain, e.g., a lipid. The linker can have any amino acid sequence, for example, the sequence of the oligonucleotide can be a random sequence, or a sequence chosen for its molecular or biochemical properties (e.g., high flexibility). In some embodiments, the linker includes a series of glycine residue to form a polyglycine linker. In some embodiments, the linker includes an amino acid sequence of (Gly)n, wherein n may 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: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), GGAGGGAG (SEQ ID NO:16), GGSGGGSG (SEQ ID NO:17), GGAGGGAGGGAG (SEQ ID NO:18), GGSGGGSGGGSG (SEQ ID NO:19), GGGGAGGGGAGGGGA (SEQ ID NO:20), and GGGGSGGGGSGGGGS (SEQ ID NO:21). Methods of Conjugation Described herein are compounds including an immunomodulatory (e.g., immuno-stimulatory or immuno-inhibitory) nucleic acid sequence and an albumin-binding domain, wherein the immunomodulatory (e.g., immuno-stimulatory or immuno-inhibitory) domain includes a functional group (FIG.1B). Also described herein are compounds including an immunomodulatory (e.g., immuno- stimulatory or immuno-inhibitory) nucleic acid sequence and an albumin-binding domain, wherein the albumin-binding domain includes a functional group. The functional group may be a maleimide, a dithio(2-pyridyldithio), a cyclooctene, a cyclooctyne, an aldehyde, an azide, or an alkyne. In some embodiments, the functional group is capable of reacting with a polypeptide to conjugate the immunomodulatory (e.g., immuno-stimulatory or immuno-inhibitory) nucleic acid sequence to the polypeptide. In some embodiments, the functional group is capable of reacting with a polypeptide to conjugate the albumin-binding domain to the polypeptide. The polypeptide may be modified with N-terminal cysteine, acetyl-cysteine, sulfydryl, trans- cyclooctene, cyclooctyne, azide or alkyne for the conjugation with an immunomodulatory (e.g., immuno- stimulatory or immuno-inhibitory) nucleic acid sequence and an albumin-binding domain. In some embodiments, the polypeptide is modified with C-terminal cysteine, azide or alkyne for the conjugation with an immunomodulatory (e.g., immuno-stimulatory or immuno-inhibitory) nucleic acid sequence and an albumin-binding domain. In some embodiments, the internal cysteine or lysine of a polypeptide is used
ATTORNEY DOCKET: 51026-051WO2 PATENT for the conjugation with an immunomodulatory (e.g., immuno-stimulatory or immuno-inhibitory) nucleic acid sequence and an albumin-binding domain. The immunomodulatory (e.g., immuno-stimulatory or immuno-inhibitory) domain or the albumin- binding domain may be bonded or linked to a linker. In some embodiments, the linker includes a functional group. In some embodiments, the functional group is capable of conjugating to a polypeptide. For example, the immunomodulatory (e.g., immuno-stimulatory or immuno-inhibitory) nucleic acid sequence may be linked to a linker, wherein the linker is modified with a functional group. In some embodiments, the albumin-binding domain may be linked to a linker, wherein the linker is modified with a functional group. In some embodiments, the linker may be a PEG linker. In some embodiments, the immunomodulatory (e.g., immuno-stimulatory or immuno-inhibitory) oligonucleotide is conjugated to the polypeptide by way of a reaction between a dithio group and a free thiol group. Exemplary methods for preparing the immunomodulatory (e.g., immuno-stimulatory or immuno-inhibitory) nucleic acid sequences and albumin-binding domains with a functional group are provided below in Examples 3-5. Adjuvants In some embodiments, a pharmaceutical composition described herein may be administered with one or more adjuvants. An adjuvant refers to a substance that causes stimulation of the immune system. In this context, an adjuvant is used to enhance an immune response to one or more antigens. An adjuvant may be administered to a subject before, in combination with, or after administration of the antigens. In some embodiments, an additional adjuvant is administered to the subject in combination with the immunomodulatory (e.g., immuno-stimulatory or immuno-inhibitory) nucleic acid sequence conjugated to a polypeptide and conjugated to an albumin-binding domain described herein. In some embodiments, an adjuvant may be conjugated to an albumin-binding domain, e.g., a lipid. The adjuvant may be without limitation lipids (e.g., monophosphoryl lipid A (MPLA)), alum (e.g., aluminum hydroxide, aluminum phosphate); Freund’s adjuvant; saponins purified from the bark of the Q. saponaria tree such as QS21 (a glycolipid that elutes in the 21st peak with HPLC fractionation; Antigenics, Inc., Worcester, Mass.); poly[di(carboxylatophenoxy)phosphazene (PCPP polymer; Virus Research Institute, USA), Flt3 ligand, Leishmania elongation factor (a purified Leishmania protein; Corixa Corporation, Seattle, Wash.), ISCOMS (immunostimulating complexes which contain mixed saponins, lipids and form virus-sized particles with pores that can hold antigen; CSL, Melbourne, Australia), Pam3Cys, SB-AS4 (SmithKline Beecham adjuvant system #4 which contains alum and MPL; SBB, Belgium), non-ionic block copolymers that form micelles such as CRL 1005 (these contain a linear chain of hydrophobic polyoxypropylene flanked by chains of polyoxyethylene, Vaxcel, Inc., Norcross, Ga.), and Montanide IMS (e.g., IMS1312, water-based nanoparticles combined with a soluble immunostimulant, Seppic), and CDNs (cyclic di- nucleotides). Adjuvants may be toll-like receptor (TLR) ligands. Adjuvants that act through TLR3 include without limitation double-stranded RNA. Adjuvants that act through TLR4 include without limitation derivatives of lipopolysaccharides such as monophosphoryl lipid A (MPLA; Ribi ImmunoChem Research, Inc., Hamilton, Mont.), muramyl dipeptide (MDP; Ribi), and threonyl-muramyl dipeptide (t-MDP; Ribi); OM-174 (a glucosamine disaccharide related to lipid A; OM Pharma SA, Meyrin, Switzerland). Adjuvants
ATTORNEY DOCKET: 51026-051WO2 PATENT that act through TLR5 include without limitation flagellin. Adjuvants that act through TLR7 and/or TLR8 include single-stranded RNA, oligoribonucleotides (ORN), synthetic low molecular weight compounds such as imidazoquinolinamines (e.g., imiquimod (R-837), resiquimod (R-848)). Adjuvants 5 acting through TLR9 include DNA of viral or bacterial origin, or synthetic oligodeoxynucleotides (ODN), such as CpG ODN. For example, the CpG ODN may have a sequence of 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 linkages of the CpG may be all phosphorothioate linkages. Another adjuvant class is phosphorothioate containing molecules such as phosphorothioate nucleotide analogs and nucleic acids containing phosphorothioate backbone linkages. Pharmaceutical Compositions Described herein are pharmaceutical compositions of the disclosure including any one of the immunomodulatory (e.g., immuno-stimulatory or immuno-inhibitory) nucleic acid sequences conjugated to a polypeptide and conjugated to an albumin-binding domain. In addition to a therapeutic amount of the immunomodulatory (e.g., immuno-stimulatory or immuno-inhibitory) nucleic acid sequences conjugated to a polypeptide and conjugated to an albumin-binding domain described herein, the pharmaceutical compositions may contain a pharmaceutically acceptable carrier or excipient, which can be formulated by methods known to those skilled in the art. Pharmaceutically acceptable salts of the components are also included, as described herein. Acceptable carriers and excipients in the pharmaceutical compositions of the immunomodulatory (e.g., immuno-stimulatory or immuno-inhibitory) nucleic acid sequences conjugated a polypeptide and conjugated to an albumin-binding domain described herein are nontoxic to recipients at the dosages and concentrations employed. In certain embodiments, the formulation material(s) are for subcutaneous (s.c.) and/or intravenous (i.v.) administration. In some embodiments the formulation material(s) are for intramuscular administration. In some embodiments, administration is by inhalation or intranasal administration. In some embodiments, the formulation material(s) are for intraperitoneal, topical, or oral administration. In some embodiments, the pharmaceutical composition can contain formulation materials for modifying, maintaining, or preserving, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition. In some embodiments, suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, methionine, sodium sulfite or sodium hydrogen- sulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrates, HEPES, TAE, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta- cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, sucrose, mannose or dextran); proteins (such as human serum albumin, gelatin, dextran, and immunoglobulins); coloring, flavoring and diluting agents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as hexamethonium chloride, octadecyldimethylbenzyl ammonium chloride, resorcinol, and benzalkonium
ATTORNEY DOCKET: 51026-051WO2 PATENT chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancing agents (such as sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides, preferably sodium or potassium chloride, mannitol sorbitol); delivery vehicles; diluents; excipients and/or pharmaceutical adjuvants. (Remington's Pharmaceutical Sciences, 18th Edition, A. R. Gennaro, ed., Mack Publishing Company (1995). In some embodiments, the optimal pharmaceutical composition will be determined by one skilled in the art depending upon, for example, the intended route of administration, delivery format and desired dosage. See, for example, Remington's Pharmaceutical Sciences, supra. In some embodiments, such compositions may influence the physical state, stability, rate of in vivo release and rate of in vivo clearance of the amphiphilic conjugate. In some embodiments, the primary vehicle or carrier in a pharmaceutical composition, including the immunomodulatory (e.g., immuno-stimulatory or immuno-inhibitory) nucleic acid sequence conjugated a polypeptide and conjugated to an albumin-binding domain described herein can be either aqueous or non-aqueous in nature. For example, in some embodiments, a suitable vehicle or carrier can be water for injection, physiological saline solution, or artificial cerebrospinal fluid, possibly supplemented with other materials common in compositions for parenteral administration. In some embodiments, the saline includes isotonic phosphate-buffered saline. In certain embodiments, neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. In some embodiments, pharmaceutical compositions include Tris buffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, which can further include sorbitol or a suitable substitute therefore. In some embodiments, a composition including the immunomodulatory (e.g., immuno-stimulatory or immuno-inhibitory) nucleic acid sequence conjugated to a polypeptide and conjugated to an albumin-binding domain described herein can be prepared for storage by mixing the selected composition having the desired degree of purity with optional formulation agents (Remington's Pharmaceutical Sciences, supra) in the form of a lyophilized cake or an aqueous solution. Further, in some embodiments, the composition including the immunomodulatory (e.g., immuno-stimulatory or immuno-inhibitory) nucleic acid sequence conjugated to a polypeptide and conjugated to an albumin-binding domain described herein can be formulated as a lyophilizate using appropriate excipients such as sucrose. In some embodiments, the pharmaceutical composition may be selected for parenteral delivery. The preparation of such pharmaceutically acceptable compositions is within the ability of one skilled in the art. In some embodiments, the formulation components are present in concentrations that are acceptable to the site of administration. In some embodiments, buffers are used to maintain the composition at physiological pH or at a slightly lower pH, typically within a pH range of from about 5 to about 8. In some embodiments, when parenteral administration is contemplated, a therapeutic composition can be in the form of a pyrogen-free, parenterally acceptable aqueous solution including the immunomodulatory (e.g., immuno-stimulatory or immuno-inhibitory) nucleic acid sequences conjugated to
ATTORNEY DOCKET: 51026-051WO2 PATENT a polypeptide and conjugated to an albumin-binding domain described herein in a pharmaceutically acceptable vehicle. In some embodiments, a vehicle for parenteral injection is sterile distilled water in which an immunomodulatory (e.g., immuno-stimulatory or immuno-inhibitory) nucleic acid sequence conjugated to a polypeptide and conjugated to an albumin-binding domain described herein is formulated as a sterile, isotonic solution, properly preserved. In some embodiments, the preparation can involve the formulation of the desired molecule with an agent, such as injectable microspheres, bio-erodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads or liposomes, that can provide for the controlled or sustained release of the product which can then be delivered via a depot injection. In some embodiments, hyaluronic acid can also be used, and can have the effect of promoting sustained duration in the circulation. In some embodiments, implantable drug delivery devices can be used to introduce the desired molecule. The pharmaceutical composition may be administered in therapeutically effective amount such as to induce an immune response. The therapeutically effective amount of the immunomodulatory (e.g., immuno-stimulatory or immuno-inhibitory) nucleic acid sequence conjugated to a polypeptide and conjugated to an albumin-binding domain described herein included in the pharmaceutical preparations may be determined by one of skill in art, such that the dosage (e.g., a dose within the range of 0.01-100 mg/kg of body weight) induces an immune response in the subject. Vectors may be used as in vivo nucleic acid delivery vehicle include, but are not limited to, retroviral vectors, adenoviral vectors, poxviral vectors (e.g., vaccinia viral vectors, such as Modified Vaccinia Ankara (MVA)), adeno-associated viral vectors, and alphaviral vectors. In some embodiments, a vector can include internal ribosome entry site (IRES) that allows the expression of multiple coronavirus antigens (e.g., a coronavirus spike protein, a polypeptide thereof, or a nucleic acid sequence encoding the same) described herein. Other vehicles and methods for nucleic acid delivery are described in US Patent Nos.5,972,707, 5,697,901, and 6,261,554, each of which is incorporated by reference herein in its entirety. Other methods of producing pharmaceutical compositions are described in, e.g., US Patent Nos. 5,478,925, 8,603,778, 7,662,367, and 7,892,558, all of which are incorporated by reference herein in their entireties. In some embodiments, a pharmaceutical composition described herein may be administered with one or more adjuvants. Routes, Dosage, and Timing of Administration Pharmaceutical compositions of the disclosure that contain the immunomodulatory (e.g., immuno-stimulatory or immuno-inhibitory) nucleic acid sequence conjugated to a polypeptide and conjugated to an albumin-binding domain described herein as the therapeutic agent may be formulated for parenteral administration, subcutaneous administration, intravenous administration, intramuscular administration, intranasal administration, or inhalation. 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 therapeutic proteins are known in the art. See, for example, US Patent Nos.6,174,529, 6,613,332, 8,518,869, 7,402,155, and 6,591,129, and US Patent Application
ATTORNEY DOCKET: 51026-051WO2 PATENT Publication Nos. US20140051634, WO1993000077, and US20110184145, the disclosures of which are incorporated by reference in their entireties. One or more of these methods may be used to administer a pharmaceutical composition of the invention that contains an immunomodulatory (e.g., immuno-stimulatory or immuno-inhibitory) nucleic acid sequence conjugated to a polypeptide and conjugated to an albumin-binding domain. For injectable formulations, various effective pharmaceutical carriers are known in the art. See, e.g., Pharmaceutics and Pharmacy Practice, J. B. Lippincott Company, Philadelphia, Pa., Banker and Chalmers, eds., pages 238- 250 (1982), and ASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages 622-630 (1986). The dosage of the pharmaceutical compositions of the invention depends on factors including the route of administration and the physical characteristics, e.g., age, weight, general health, of the subject. Typically, the amount of an immunomodulatory (e.g., immuno-stimulatory or immuno-inhibitory) nucleic acid sequence conjugated to a polypeptide and conjugated to an albumin-binding domain described herein contained within a single dose may be an amount that effectively induces an immune response in the subject without inducing significant toxicity. A pharmaceutical composition of the invention may include a dosage of an immunomodulatory (e.g., immuno-stimulatory or immuno-inhibitory) nucleic acid sequence conjugated to a polypeptide and conjugated to an albumin-binding domain described herein 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 a more specific embodiment, about 0.1 to about 100 mg. The dosage may be adapted by the clinician in accordance with the different parameters of the subject. Pharmaceutical compositions of the invention that contain the immunomodulatory (e.g., immuno- stimulatory or immuno-inhibitory) nucleic acid sequence conjugated to a polypeptide and conjugated to an albumin-binding domain may be administered to a subject in need thereof, for example, one or more times (e.g., 1-10 times or more) daily, weekly, monthly, biannually, annually, or as medically necessary. Methods of Inducing an Immune Response The disclosure provides methods of inducing an immune response against an antigen in subject. The method includes administering any one of the compounds described herein and an antigen to the subject. In some embodiments, the disclosure provides a method of inducing an immune response against an antigen in subject by administering any one of the immunomodulatory (e.g., immuno- stimulatory or immuno-inhibitory) nucleic acid sequence conjugated to a polypeptide conjugated to an albumin-binding domain to the subject and further administering an adjuvant to the subject. In some embodiments, the immunomodulatory (e.g., immuno-stimulatory or immuno-inhibitory) nucleic acid sequence conjugated to a polypeptide and conjugated to an albumin-binding domain may be administered without one or more additional adjuvants. In some embodiments, the method includes administering to the subject a therapeutically effective amount of the immunomodulatory (e.g., immuno-stimulatory or immuno-inhibitory) nucleic sequence conjugated to a polypeptide and conjugated to an albumin-binding domain described herein. In some embodiments, the immunomodulatory (e.g., immuno-stimulatory or immuno-inhibitory) nucleic acid sequence conjugated to a polypeptide and conjugated to an albumin-binding domain is administered
ATTORNEY DOCKET: 51026-051WO2 PATENT substantially simultaneously. In some embodiments, the immunomodulatory (e.g., immuno-stimulatory or immuno-inhibitory) nucleic acid sequence conjugated to a polypeptide and conjugated to an albumin- binding domain is administered separately. The polypeptide may be an antigen, or a fragment thereof. For example, the polypeptide may be derived from tumors (e.g., a tumor-associated antigen) or viral or bacterial sources. In some embodiments, the antigen is an influenza antigen, or fragment thereof. In some embodiments, the antigen is an influenza nucleoprotein, or 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 to 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 to SEQ ID NO: 22. In some embodiments, the Influenza nucleoprotein comprises a polypeptide sequence of SEQ ID NO: 22. In some embodiments, the antigen is a coronavirus antigen, or fragment thereof. For example, the antigen is a coronavirus spike protein, or fragment thereof, or a coronavirus nucleocapsid protein, or fragment thereof. In some embodiments, one or more of the components administered is a pharmaceutically acceptable salt of the indicated component, as described herein. In some embodiments, the immune response is protective against an infection. For example, the immune response may be protective again an Influenza infection or a SARS-CoV-2 infection. In some embodiments, the immune response is protective against COVID-19 disease. In some embodiments, the disclosure provides a method of inducing an immune response against an antigen in subject by administering any one of the compounds or pharmaceutically acceptable salts described herein subcutaneously to the subject. In some embodiments, the disclosure provides a method of inducing an immune response against an antigen in subject by administering the antigen intramuscularly, subcutaneously, intravenously, intraperitoneally, topically, orally/buccally, sublingually, transmucosally, intranasally, or by inhalation to the subject. In some embodiments, the subject is a mammal. For example, the subject may be a human. Kits A kit can include the immunomodulatory (e.g., immuno-stimulatory or immuno-inhibitory) nucleic acid sequence conjugated to polypeptide and conjugated to an albumin-binding domains disclosed herein and instructions for use. The kits may include, in a suitable container, an immunomodulatory (e.g., immuno-stimulatory or immuno-inhibitory) nucleic acid sequence conjugated to a polypeptide and conjugated to an albumin-binding domain, one or more controls, and various buffers, reagents, enzymes and other standard ingredients well known in the art. In some embodiments, the kits further include an adjuvant. Accordingly, in some embodiments, the immunomodulatory (e.g., immuno-stimulatory or immuno-inhibitory) nucleic acid sequence conjugated to a polypeptide and conjugated to an albumin- binding domain is in a vial In some embodiments, the poly-dA and/or poly-dT nucleic acid sequences, the poly-dG and/or poly-dC nucleic acid sequences, the poly-dA and/or poly-dT nucleic acid sequences conjugated to an albumin-binding domain, the poly-dG and/or poly-dC nucleic acid sequences conjugated to an albumin-binding domain or ISD or immunostimulatory HSV sequence conjugated to an albumin-
ATTORNEY DOCKET: 51026-051WO2 PATENT binding domain, and the adjuvant 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. The container can include at least one vial, well, test tube, flask, bottle, syringe, or other container means, into which the immunomodulatory (e.g., immuno-stimulatory or immuno-inhibitory) nucleic acid sequence conjugated to a polypeptide and conjugated to an albumin-binding domain and in some instances, suitably aliquoted. When an additional component is provided, the kit can contain additional containers into which this compound may be placed. The kits can also include a means for containing the immunomodulatory (e.g., immuno-stimulatory or immuno-inhibitory) nucleic acid sequence conjugated to a polypeptide and conjugated to an albumin-binding domain and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained. Containers and/or kits can include labeling with instructions for use and/or warnings. In some embodiments, the disclosure provides a kit including a medicament including a composition including an immunomodulatory (e.g., immuno-stimulatory or immuno-inhibitory) nucleic acid sequence conjugated to a polypeptide and conjugated to an albumin-binding domain, an optional pharmaceutically acceptable carrier, and a package insert including instructions for administration of the medicament alone or in combination with a composition including an adjuvant and an optional pharmaceutically acceptable carrier, for treating, delaying progression of, or preventing a disease or condition (e.g., Influenza or SARS-CoV-2), wherein the immunomodulatory (e.g., immuno-stimulatory or immuno-inhibitory) nucleic acid sequence conjugated to a polypeptide and conjugated to an albumin- binding domain, and optionally a linker. The polypeptide may be an antigen, or a fragment thereof. For example, the polypeptide may be derived from tumors (e.g., a tumor-associated antigen) or viral or bacterial sources. In some embodiments, the polypeptide may be an influenza antigen, or fragment thereof. In some embodiments, the polypeptide may be an influenza nucleoprotein, or fragment thereof. In some embodiments, the polypeptide may be a coronavirus antigen, or fragment thereof. In some embodiments, the disclosure provides a kit including a container including a composition including an immunomodulatory (e.g., immuno-stimulatory or immuno-inhibitory) nucleic acid sequence conjugated to a polypeptide and conjugated to an albumin-binding domain, an optional pharmaceutically acceptable carrier, and a package insert including instructions for administration of composition vaccine in a subject, wherein the immunomodulatory (e.g., immuno-stimulatory or immuno-inhibitory) nucleic acid sequence conjugated to a polypeptide and conjugate to an albumin-binding domain and optionally includes a linker. In some embodiments, the kit further includes an adjuvant and instructions for administration of the adjuvant. In some embodiments of the kits, one of more of the components of the kits is a pharmaceutically acceptable salt of the component as described herein. EXAMPLES The following examples, which are intended to illustrate, rather than limit, the disclosure, are put forth to provide those of ordinary skill in the art with a description of how the compositions and methods
ATTORNEY DOCKET: 51026-051WO2 PATENT described herein may be used, made, and evaluated. The examples are intended to be purely exemplary of the disclosure and are not intended to limit the scope of what the inventors regard as their invention. Example 1. Effect of CpG Polypeptide Chimera on Immunogenicity This experiment aimed to determine if the conjugation of gp100 polypeptide-antigen with CpG adjuvant will affect the immunogenicity in comparison to the respective admixed vaccine, and if AMP- conjugation of the chimera (FIG.1) can increase its immunogenicity over the soluble form. 5 groups of 5 C57BL/6J mice each were administered a vaccine including the components of Table 1. Table 1. Summary of Vaccine Administration in Mice
Gp100 stock solutions were dissolved in limulus amebocyte lysate (LAL) H2O at a polypeptide concentration of 0.84 mg/mL. Final injections were diluted with 1x PBS. The mice were injected with a solution that contained 10 μg polypeptide/100 μL. Both the soluble and amphiphilic chimeras were conjugated at a 1:1 polypeptide:adjuvant ratio. The concentrations of chimeras were determined according to Table 2 as shown below. Each injection of chimera contained 10 μg of polypeptide. This corresponded to 4.31 nmol of adjuvant (as calculated in Table 3). The vaccine components are described in Table 4. Adjuvant stock solutions were generated by resuspending in limulus amebocyte lysate (LAL) H2O. Final injections were diluted with 1x Phosphate-buffered saline (PBS) such that the (AMP-)CpG had a concentration of 4.3 nmol/100 μL injection. 4.3 nmol is the equivalent to the mass present in the chimera at 10 µg of polypeptide. Table 2: UV-Vis Determination of Concentration
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ATTORNEY DOCKET: 51026-051WO2 PATENT Table 3: Vaccine Component Ratios
The immunizations were administered subcutaneously (SC) into the tail base of female B6 mice, bilaterally with 50 μL per side. A booster dose was given at roughly 2-week intervals. SC injections ensured that the vaccine was optimally delivered into lymph nodes via natural lymph drainage, and bi- weekly injections were determined to be optimal in immune response. Table 4: Vaccine Components
An ICS (Intracellular Stain) Assay for TNFα and IFNγ was performed on blood and lung samples 7 days post dose 2 (booster dose) (FIG.4 and FIG.5). The cells were surface stained for CD4 and CD8; intracellularly stained for CD3 (see Table 5 for antibody information). The ICS samples were activated for about 19-20 hours (in the presence of Brefeldin A and Monensin) with 2 μg/ml of gp100 (EGP) polypeptide (see Table 6). Table 5: Antibodies used for ICS
ATTORNEY DOCKET: 51026-051WO2 PATENT
Table 6: Re-Stimulation Polypeptides
Tetramer analysis was performed 7 days post booster dose using the Tetramer/PE - H-2Db gp100 (EGSRNQDWL) (SEQ ID NO: 24) (FIG.3). ELISpot analysis for IFNγ was performed on splenocytes 7 days after booster dose administration. Splenocytes (0.1x106 cells/well) were activated with 2 μg/ml of gp100 (EGP) polypeptide (see Table 6). IFNγ ELISpot plates were stimulated overnight (FIG.2). The AMP-conjugation of polypeptide and adjuvants increased their immunogenicity compared to their soluble forms as measured by ELISpot of splenocytes and ICS of PBMCs and lung-associated lymphocytes. Additionally, the conjugation of polypeptide to CpG7909 also increased the compound’s immunogenicity compared to the respective admixed components. This was likely due to the increased size of the molecule or other mechanisms, and thus better clearance to the lymph node, as well as that antigen presenting cells (APCs) were exposed to polypeptide and adjuvant at the same time. Moreover, when the chimeras in turn were AMP-conjugated, this increased the compound’s immunogenicity even further, due to the compounding effect of having covalently linked polypeptide-CpG pairs, and the benefit that AMP-conjugation provided to delivery to and retention in the lymph nodes as well as activation of APCs. Additionally, conjugation of a polypeptide to oligonucleotide increases the aqueous solubility of the polypeptide dramatically, especially the insoluble polypeptide, which allows the formulation of the polypeptide for dosing. Example 2. Effect of Poly-Deoxythymidine Polypeptide Chimera on Immunogenicity This experiment was designed to determine if conjugation of gp100 polypeptide-antigen with the poly-deoxythymidine adjuvant would affect immunogenicity in comparison to the respective admixed vaccine, and if conjugation with an amphiphile to the chimera (FIG.6) increased its immunogenicity over the soluble form. 5 groups of 5 C57BL/6J mice each were administered a vaccine including the components of Table 7. Table 7. Summary of Vaccine Administration in Mice
ATTORNEY DOCKET: 51026-051WO2 PATENT The gp100 stock solutions were made by dissolving in limulus amebocyte lysate (LAL) H2O at a polypeptide concentration of 0.84 mg/mL. Final injections were diluted with 1x PBS such that the injections contained 10 μg polypeptide/100 μL. Both soluble and amphiphilic chimeras were conjugated at 1:1 polypeptide:adjuvant ratio and the concentrations of chimeras were determined according to Table 8. Each injection of the chimera contained 10 μg of polypeptide. This corresponded to 4.31 nmol of adjuvant (as calculated in Table 9). The vaccine components are described in Table 10. The adjuvant stock solutions were made by resuspending in limulus amebocyte lysate (LAL) H2O. The final injections were diluted with 1x Phosphate-buffered saline (PBS) such that the concentration of (AMP-)dT was 4.3 nmol/100 μL injection (4.3 nmol is the equivalent to the mass present in the chimera at 10 µg of polypeptide).
Table 9: Vaccine Component Ratios
The immunizations were administered subcutaneously (SC) into the tail base of female B6 mice, bilaterally with 50 μL per side. A booster dose was given at roughly 2-week intervals. SC injections ensured that the vaccine was optimally delivered into lymph nodes via natural lymph drainage, and bi- weekly injections were determined to be optimal in immune response. Table 10: Vaccine Components
ATTORNEY DOCKET: 51026-051WO2 PATENT
An ICS (Intracellular Stain) Assay for TNFα and IFNγ was performed on blood and lung samples 7 days post dose 2 (booster dose) (FIG.9 and FIG.10). The cells were surface stained for CD4 and CD8; intracellularly stained for CD3 (see Table 11 for antibody information). The ICS samples were activated for about 19-20 hours (in the presence of Brefeldin A and Monensin) with 2 μg/ml of gp100 (EGP) polypeptide (see Table 12). Table 11: Antibodies used for ICS
Table 12: Re-Stimulation Polypeptides
Tetramer analysis was performed 7 days post booster dose using the Tetramer/PE - H-2Db gp100 (EGSRNQDWL) (SEQ ID NO: 24) (FIG.8). ELISpot analysis for IFNγ was performed on splenocytes 7 days after booster dose administration. Splenocytes (0.1x106 cells/well) were activated with 2 μg/ml of gp100 (EGP) polypeptide (see Table 12). IFNγ ELISpot plates were stimulated overnight (FIG.7). AMP-conjugation of the polypeptide-DNA-chimeras increased their immunogenicity compared to soluble chimeras as well as the AMP-conjugated polypeptide and DNA admixes. These data support that covalently linking the antigen to the DNA-adjuvant on the AMP-platform increases these constructs’ immunogenicity. The constructs described in Examples 1 and 2 were prepared as follows. 1. Preparation of CpG-PEG4-Mal (3)
ATTORNEY DOCKET: 51026-051WO2 PATENT 5.0 mg CpG-3’-amine (MW 7893.3) was dissolved in 500 ^L 20 mM pH 8.0 phosphate buffer, followed by addition of 5 mg NHS-PEG4-Mal (MW 513.5, 15-fold). The mixture was incubated at room temperature for 30 min. and purified by RP-HPLC [Phenylhexyl semi-prep column (10mmx 150mm), 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 isolated by lyophilization to provide CpG-PEG4-Mal 3. 2. Preparation of AMP-CpG-PEG4-Mal (4) 5.0 mg Amp-CpG-3’-amine (MW 8595.5) was dissolved in 500 ^L 20 mM pH 8.0 phosphate buffer, followed by addition of 5 mg 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-prep column (10mmx 150mm), 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 isolated by lyophilization. Product was confirmed by MALDI-TOF MS (molecular weight: calculated 8993.9, determined 8999.9). 3. Preparation of a CpG-Peptide (Maleimide Linkage), CpG-PEG4-gp100 (5a) CpG-PEG4-Mal 3 (1 equiv.) and gp100 polypeptide (1.5 equiv.) containing an N-terminal acetyl- cysteine residue was reacted at room temperature for 1 hour. The mixture was purified by RP-HPLC [Phenylhexyl semi-prep column (10mmx 150mm), 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 isolated by lyophilization and salt-exchanged to sodium salt to provide 5. Product was confirmed by MALDI-TOF MS (molecular weight: calculated 10612.9, determined 10613.5). 4. Preparation of an AMP-CpG-Peptide (Maleimide-AcCys Linkage), AMP-CpG-PEG4-gp100 (1a) AMP-CpG-PEG4-Mal 4 (1 equiv.) and gp100 polypeptide (1.5 equiv.) containing an N-terminal acetyl- cysteine residue was reacted at room temperature for 1 hour. The mixture was purified by RP-HPLC [Phenylhexyl semi-prep column (10mmx 150mm), 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 isolated by lyophilization and salt-exchanged to sodium salt to provide 1a. Product was confirmed by MALDI-TOF MS (molecular weight: calculated 11315.1, determined 11317.3). 5. Preparation a CpG-Peptide (Dithio (SS) Linkage), CpG-PEG4-gp100 (5b) 2.0 mg CpG-3’-amine (MW 7893.3) was dissolved in 200 ^L 20 mM pH 8.0 phosphate buffer, followed by addition of 1.5 mg PDP-PEG4-NHS ((2-pyridyldithio-)propionyl-PEG4-NHS, MW 559.6, 10- fold). The mixture was incubated at room temperature for 2 hours. Excess PDP-PEG4-NHS was removed by salt-exchange with a NAP-5 column to provide CpG-PEG4-PDP (6) that was reacted with gp100 polypeptide (1.5 equiv.) containing a N-terminal acetylcysteine residue at room temperature for 1 hour. The mixture was purified by RP-HPLC HPLC [Phenylhexyl semi-prep column (10mmx 150mm), 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 isolated by lyophilization. Product was confirmed by MALDI-TOF MS (molecular weight: calculated 10548.8, determined 10547.1). 6. Preparation Amp-CpG-Peptide (Maleimide-Cys Linkage), AMP-CpG-PEG4-gp100 (1b) Amp-CpG-PEG4-Mal (1 equiv.) and gp100 polypeptide (1.5 equiv.) containing a N-terminal cysteine residue was reacted at room temperature for 1 hour. The mixture was purified by RP-HPLC HPLC [Phenylhexyl semi-prep column (10mmx 150mm), mobile phase A (50 mM TEA pH 5.8), mobile phase B
ATTORNEY DOCKET: 51026-051WO2 PATENT (100% MeCN), flow rate (3 mL/min), gradient (10-90% over 30 min)] and isolated by lyophilization. Product was confirmed by MALDI-TOF MS (molecular weight: calculated 11273, determined 11270). 7. Preparation dT50-Peptide (Maleimide-AcCys Linkage), dT50-PEG4-gp100 (6) 10.0 mg dT50-3’-amine (MW 16129.8) was dissolved in 500 ^L 20 mM pH 8.0 phosphate buffer, followed by addition of 3.2 mg NHS-PEG4-Mal (MW 513.5, 10-fold). The mixture was desalted after 30 min with a NAP-5 column, before reacting with gp100 polypeptide (2.0 equiv.) containing a N-terminal acetyl-cysteine residue at room temperature for 1 hour. The mixture was purified by RP-HPLC [Biobasic- C4 column (4.6mmx 250mm), mobile phase A (50 mM TEA pH 5.8), mobile phase B (100% MeCN), gradient (10-30% over 30 min)] and fraction isolated by lyophilization to provide 6. Product was confirmed by MALDI-TOF MS (molecular weight: calculated 11849.7, determined 11850.0). 8. Preparation AMP-dT50-Peptide (Maleimide Linkage-AcCys), AMP-dT50-PEG4-gp100 (2) 6.5 mg AMP-dT50-3’-amine (MW 16832.2) was dissolved in 500 ^L 20 mM pH 8.0 phosphate buffer, followed by addition of 3 mg NHS-PEG4-Mal (MW 513.5, 10 equiv.). The mixture was incubated at room temperature for 30 min and desalted with a NAP-5 column, before reacting with gp100 polypeptide (1.5 equiv.) containing a N-terminal Acetyl-cysteine residue at room temperature for 1 hour. The mixture was purified by RP-HPLC [Phenylhexyl semi-prep column (10mmx 150mm), 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 fraction isolated by lyophilization to provide 2. Product was confirmed by MALDI-TOF MS (molecular weight: calculated 19551.8, determined 19540). Example 3. Strategy for Preparation of 5’ AMP Nucleotides The preparation of 5’ amphiphilic nucleotides is described in this example. This process is performed according to the reactions described in Scheme 1 where N-(2-hydroxy-3- octadecanoylaminopropyl)octadecanamide 7 is subjected to oxidative conditions such as, but not limited to, Moffit, Swern, Dess-Martin periodinane, TPAP, PDC, and PPC, provides N,N'-(2-oxopropane-1,3- diyl)distearamide 8. Treatment of 8 with a cyanide source such as, but not limited to, sodium cyanide, lithium cyanide, potassium cyanide, trimethylsilyl cyanide, and acetone cyanohydrin with catalytic acid 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(III) triflate provides cyanohydrin 9. Nitrile reduction employing reagents such as, but not limited to, hydrogen with metal catalyst such as, but not limited to, palladium, palladium hydroxide, nickel, Raney nickel, nickel dichloride, platinum (IV) oxide, or with metal hydrides such as, but not limited to, sodium tetraborohydrate or lithium tetraborohydrate, or with borane-tetrahydrofuran or tris(perflourophenyl)borane/trimethylsilane, provides the primary amine 10. Conversion of amine 10 to amide 12 may be achieved by treatment with known PEGylated, base- labile, protected amines 11 containing PEG-units of, but not limited to, n = 0-12, and containing an activated ester such as, but not limited to, N-hydroxysuccinimide ester or perfluorophenol ester or as a carboxylic acid that may be activated with reagents such as, but not limited to, p-toluenesulfonyl chloride, pivaloyl chloride, 2,4,6-trichlorobenzoyl chloride, N,N'-dicyclohexane carbodiimide, (1-Ethyl-3-(3- dimethylaminopropyl)carbodiimide, propanephosphonic acid anhydride, and (1-
ATTORNEY DOCKET: 51026-051WO2 PATENT [Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, hexafluorophosphate azabenzotriazole tetramethyl uronium and contains base-labile N protecting groups such as, but not limited to, 1,3-dithian-2-ylmethoxycarbonyl, dimethyl-1,3-dithian-2-ylmethoxycarbonyl, trifluoroacety and 9-fluorenylmethyloxycarbonyl. Formation of phosphoramidite 13 may be achieved by treating 12 with P(III) reagents such as, but not limited to, 3-((chloro(diisopropylamino)phosphaneyl)oxy)propanenitrile (X = Cl) or 3- ((bis(diisopropylamino)phosphaneyl)oxy)propanenitrile (X = isopropylamide). Phosphoramidite 13 may be coupled to an oligonucleotide using the solid phase synthesis (SPS) P(III) method as the last nucleotide 5’ coupling cycle. Catalyst for coupling include, but are not limited to,1H-tetrazole, 5-ethylthio-1H-tetrazole, 2-benzylthiotetrazole, and 4,5-dicyanoimidazole. Thiolation may be achieved by treatment with, but not limited to, 3-(dimethylaminomethylidene)amino-3H-1,2,4- dithiazole-3-thione, 3H-1,2-benzodithiol-3-one 1,1-dioxide, N,N,N'N'-tetraethylthiuram disulfide, and 3- amino-1,2,4-dithiazole-5-thione. Deprotection of the oligonucleotide and “NH-PG” group may be achieved by reacting with, but not limited to, amine bases such as t-butylamine, diethylamine and piperidine followed by oligonucleotide cleavage and deprotection, purification and isolation using techniques commonly employed in oligonucleotide SPS provides compound 12. Addition of the maleimide functionality required for antigen conjugation may be achieved by treatment of 12 with available maleimide fragments such as, but not limited to, 15 containing PEG-units of, 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 may be activated with reagents such as, but not limited, p-toluenesulfonyl chloride, pivaloyl chloride, 2,4,6-trichlorobenzoyl chloride, N,N'-dicyclohexane carbodiimide, (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide, propanephosphonic acid anhydride, and (1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5- b]pyridinium 3-oxide hexafluorophosphate, hexafluorophosphate azabenzotriazole tetramethyl uranium, to provide penultimate compound 16. Treatment of 16 with thiol containing antigens provides target compound 17 via sulfhydryl- maleimide conjugate addition.
ATTORNEY DOCKET: 51026-051WO2 PATENT
Example 4. Strategy for Preparation of 5’ AMP-Antigen Nucleotides The preparation of 5’ amphiphilic antigens is described in this example. This process is performed according to the reactions described in Scheme 2 where the preparation of 5’ amphiphilic antigens is described in this example. Compound 12 as described in Example 3 may be treated with known compounds 18 possessing PEG-units with, but not limited to, n = 0-12, and where LG is defined as a leaving group such as, but not limited to, mesylate, tosylate, chloride, bromide or iodide, and where P is an alcohol protecting group such as, but not limited to, acetate, trimethylsilyl, t-butyldimethylsilyl, benzyl, and p-methoxybenzyl, benzyl carbonate, allyl carbonate, triphenylmethyl, p- methoxyphenyldiphenylmethyl, tetrahydopyranyl and methoxymethyl and Y is, but not limited to, CH2 or CH2NH(CO)CH2. Deprotection of the alcohol P group by treatment with, but not limited to, mild acid (0.1- 0.5 N HCl), toluenesulfonic acid, or acetic acid or alternatively base hydrolysis such as, but not limited to potassium carbonate/methanol or sodium hydroxide, or alternatively hydrogenolysis such as, but not limited to hydrogen or hydrogen source with palladium, palladium hydroxide, nickel, Raney nickel, nickel dichloride, platinum (IV) oxide, or alternatively fluoride induced desilylation with, but not limited to tetrabutylammonium fluoride, hydrogen fluoride or hydrogen fluoride triethylamine, or alternatively by oxidative cleavage with, but not limited to, 2,3-dichloro-5,6-dicyanobenzoquinone provides compound 19. Formation of phosphoramidite 20 may be achieved by treating 19 with P(III) reagents such as, but not limited to, 3-((chloro(diisopropylamino)phosphaneyl)oxy)propanenitrile (X = Cl) or 3- ((bis(diisopropylamino)phosphaneyl)oxy)propanenitrile (X = isopropylamide).
ATTORNEY DOCKET: 51026-051WO2 PATENT Phosphoramidite 20 may be coupled to an oligonucleotide using the solid phase synthesis (SPS) P(III) method as the last nucleotide 5’ coupling cycle. Catalyst for coupling include, but are not limited to,1H-tetrazole, 5-ethylthio-1H-tetrazole, 2-benzylthiotetrazole, and 4,5-dicyanoimidazole. Thiolation may be achieved by treatment with, but not limited to, 3-(dimethylaminomethylidene)amino-3H-1,2,4- dithiazole-3-thione, 3H-1,2-benzodithiol-3-one 1,1-dioxide, N,N,N'N'-tetraethylthiuram disulfide, and 3- amino-1,2,4-dithiazole-5-thione. Deprotection of the oligonucleotide and “NH-PG” group may be achieved by reacting with, but not limited to, amine bases such t-butylamine, diethylamine and piperidine followed by oligonucleotide cleavage and deprotection, purification and isolation using techniques commonly employed in oligonucleotide SPS provides compound 21. Addition of the maleimide functionality required for antigen conjugation may be achieved by treatment of 21 with available maleimide fragment 15, functionalized with PEG-units between, but not limited to, m = 0-12, containing an activated ester such as N-hydrocysuccinimide ester and perfluorophenol ester or as a carboxylic acid that may be activated with, but not limited, reagents such as p-toluene sulfonyl chloride, pivaloyl chloride, 2,4,6-trichlorobenzoyl chloride, N,N'-dicyclohexane carbodiimide, (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide, propanephosphonic acid anhydride,and (1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, hexafluorophosphate azabenzotriazole tetramethyl uranium, to provide penultimate compound 22. Treatment of 22 with thiol containing antigens provides target compound 23 via sulfhydryl- maleimide conjugate addition.
ATTORNEY DOCKET: 51026-051WO2 PATENT Scheme 2.
Additionally, the process may be performed according to the reactions described in Scheme 3 where the preparation of 5’ amphiphilic antigens is described. Compound 24, where R1 together with oxo-amine result in a carbamate such as, but not limited to, t-butyl, O-benzyl, O-allyl, O-trimethylsilylethyl, may be subjected to oxidative conditions such as, but not limited to, Moffit, Swern, Dess-Martin periodinane, TPAP, PDC, and PPC, provides ketone-carbamate 25. Treatment of 25 with a cyanide source such as, but not limited to, sodium cyanide, lithium cyanide, potassium cyanide, trimethylsilyl cyanide, and acetone cyanohydrin with catalytic acid 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(III) triflate provides the cyanohydrin 26. Nitrile reduction employing reagents such as, but not limited to, hydrogen with metal catalyst such as, but not limited to, palladium, palladium hydroxide, nickel, Raney nickel, nickel dichloride, platinum (IV) oxide, or with metal hydrides such as, but not limited to, sodium tetraborohydrate or lithium tetraborohydrate, or with borane-tetrahydofuran or tris(perflourophenyl)borane/trimethylsilane, provides the primary amine 27.
ATTORNEY DOCKET: 51026-051WO2 PATENT Conversion of amine 27 to amide 28 may be achieved by treatment with known PEGylated, base- labile, protected amines 11 containing PEG-units of, but not limited to, n = 0-12, and containing an activated ester such as, but not limited to, N-hydrocysuccinimide ester or perfluorophenol ester or as a carboxylic acid that may be activated with reagents such as, but not limited to, p-toluenesulfonyl chloride, pivaloyl chloride, 2,4,6-trichlorobenzoyl chloride, N,N'-dicyclohexane carbodiimide, (1-Ethyl-3-(3- dimethylaminopropyl)carbodiimide, propanephosphonic acid anhydride, and (1- [Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, hexafluorophosphate azabenzotriazole tetramethyl uronium and contains base-labile N protecting groups such as, but not limited to, 1,3-dithian-2-ylmethoxycarbonyl, dimethyl-1,3-dithian-2-ylmethoxycarbonyl, trifluoroacety and 9-fluorenylmethyloxycarbonyl. Deprotection of carbamate 28, may 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 donor such as, but not limited to, ammonium formate or cyclohexadiene and metal catalyst such as, but not limited to, palladium, palladium hydroxide, nickel, Raney nickel, nickel dichloride, platinum (IV) oxide, or fluoride sources such as, but not limited to, tetrabutylammonium fluoride, hydrogen fluoride, or hydrogen fluoride triethylamine, to provide diamine 29. Amidation of 29 may be achieved by treatment with stearic acid activated esters such as, but not limited to, the N-hydrocysuccinimide ester and perfluorophenol ester or directly with stearoic acid with reagents such as, but not limited to, p-toluenesulfonyl chloride, pivaloyl chloride, 2,4,6-trichlorobenzoyl chloride, N,N'-dicyclohexane carbodiimide, (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide, propanephosphonic acid anhydride, and (1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5- b]pyridinium 3-oxide hexafluorophosphate, hexafluorophosphate azabenzotriazole tetramethyl uranium, or directly from available stearoyl chloride to provide compound 12.
ATTORNEY DOCKET: 51026-051WO2 PATENT Scheme 3.
Additionally, the process may be performed according to the reactions described in Scheme 4 where compound 28 (as described above) may be treated with known compounds 18 possessing PEG- units with, but not limited to, n = 0-12, and where LG is defined as a leaving group such as, but not limited to, mesylate, tosylate, chloride, bromide or iodide, and where P is an alcohol protecting group such as, but not limited to, acetate, trimethylsilyl, t-butyldimethylsilyl, benzyl, and p-methoxybenzyl, benzyl carbonate, allyl carbonate, and Y is, but not limited to, CH2 or CH2NH(CO)CH2 provides compound 29. Deprotection of carbamate 29, may 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 donor such as, but not limited to, ammonium formate or cyclohexadiene and metal catalyst such as, but not limited to, palladium, palladium hydroxide, nickel, Raney nickel, nickel dichloride, platinum (IV) oxide, or fluoride sources such as, but not limited to, tetrabutylammonium fluoride, hydrogen fluoride, or hydrogen fluoride triethylamine, provides diamine 30. Amidation of 30 may be achieved by treatment with stearic acid activated esters such as, but not limited to, the N-hydrocysuccinimide ester and perfluorophenol ester or directly with stearoic acid with reagents such as, but not limited to, p-toluene sulfonyl chloride, pivaloyl chloride, 2,4,6-trichlorobenzoyl chloride, N,N'-dicyclohexane carbodiimide, (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide, propanephosphonic acid anhydride, and (1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5- b]pyridinium 3-oxide hexafluorophosphate, hexafluorophosphate azabenzotriazole tetramethyl uranium, or directly from available stearoyl chloride provides compound 31. Deprotection of the hydroxyl P-group of 31 by treatment with, but not limited to, mild acid (0.1-0.5 N HCl), toluenesulfonic acid, or acetic acid or alternatively base hydrolysis such as, but not limited to potassium carbonate/methanol or sodium hydroxide, or alternatively hydrogenolysis such as, but not
ATTORNEY DOCKET: 51026-051WO2 PATENT limited to hydrogen or hydrogen source with palladium, palladium hydroxide, nickel, Raney nickel, nickel dichloride, platinum (IV) oxide, or alternatively fluoride induced desilylation with, but not limited to tetrabutylammonium fluoride, hydrogen fluoride or hydrogen fluoride triethylamine, or alternatively by oxidative cleavage with, but not limited to, 2,3-dichloro-5,6-dicyanobenzoquinone provides compound 32 Compound 32 may be treated with, but not limited to, P(III) reagents such as 3- ((chloro(diisopropylamino)phosphaneyl)oxy)propanenitrile (X = Cl) or 3- ((bis(diisopropylamino)phosphaneyl)oxy)propanenitrile (X = isopropylamide) to provide phosphoramidite 20. Scheme 4.
Additionally, the strategy may be achieved according to the reactions described in Scheme 5 where an orthogonally-functionalized, branched synthon such as, but not limited to, amino acid derivative 33 and amine 34 undergo polypeptide coupling using reagents such as, but not limited to, N,N’- diisopropylcarbodiimide, N,N’dicyclohexylcarbodiimide and 1-ethyl-3-(3- dimethylaminopropyl)carbodiimide, with activators such as, but not limited to, 1-Hydroxybenzotriazole and 3-hydroxytriazolo[4,5-b]pyridine to provide 35. Alternatively compounds 33 and 34 are treated with benzotriazole N-oxide reagents such as, but not limited to, 3-[bis(dimethylamino)methyliumyl]-3H-benzotriazol-1-oxide hexafluorophosphate, ((1H- benzo[d][1,2,3]triazol-1-yl)oxy)tris(dimethylamino)phosphonium hexafluorophosphate(V), or with and azabenzotriazole N-oxide reagents such as, but not limited to 1-[bis(dimethylamino)methylene]-1H-1,2,3- triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, (benzotriazol-1-yloxy)tripyrrolidinophosphonium
ATTORNEY DOCKET: 51026-051WO2 PATENT hexafluorophosphate, or (7-azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate, to provide compound 35. Acid catalyzed deprotection results in the diamino alcohol 36. Amidation of 36 may be achieved by treatment with stearic acid activated esters such as, but not limited to, the N-hydrocysuccinimide ester and perfluorophenol ester or directly with stearoic acid with reagents such as, but not limited to, p- toluenesulfonyl chloride, pivaloyl chloride, 2,4,6-trichlorobenzoyl chloride, N,N'-dicyclohexane carbodiimide, (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide, propanephosphonic acid anhydride, and (1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, hexafluorophosphate azabenzotriazole tetramethyl uranium, or directly from available stearoyl chloride to provide compound 37. Formation of phosphoramidite 38 may be achieved by treating 37 with P(III) reagents such as, but not limited to, 3-((chloro(diisopropylamino)phosphaneyl)oxy)propanenitrile (X = Cl) or 3- ((bis(diisopropylamino)phosphaneyl)oxy)propanenitrile (X = isopropylamide). An analogous series of reactions described in Scheme 1 (compounds 13 to 17) provides analogous 5’ AMP-Antigen Nucleotides. Scheme 5.
Example 5. Strategy for Preparation of 3’ AMP-Antigen Nucleotides The preparation of 3’ amphiphilic antigens is described in this example. This process is performed according to the reactions described in Scheme 6 where deprotection of base-labile protecting group (PG) of 12 (Example 3) with amine bases such as, but not limited to, diethylamine, t-butylamine, or piperidine, provides a primary amine. Treatment of the primary amine with available maleimides 15 (Example 3) functionalized with PEG-units between, but not limited to, m = 0-12, and containing an activated ester such as N-hydrocysuccinimide ester and perfluorophenol ester or as a carboxylic acid that may be activated with, but not limited, reagents such as p-toluenesulfonyl chloride, pivaloyl chloride, 2,4,6-trichlorobenzoyl chloride, N,N'-dicyclohexane carbodiimide, (1-Ethyl-3-(3- dimethylaminopropyl)carbodiimide, propanephosphonic acid anhydride, and (1- [Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, hexafluorophosphate azabenzotriazole tetramethyl uranium, provides compound 28.
ATTORNEY DOCKET: 51026-051WO2 PATENT Etherification of 39 may accomplished with known compounds 29 possessing PEG-units with, but not limited to, p = 0-12, and where LG is defined as a leaving group such as, but not limited to, mesylate, tosylate, chloride, bromide or iodide, and Y is, but not limited to, CH2 or CH2NH(CO)CH2 and where R2 is an acid labile group such as, but not limited to t-butyl, allyl, benzyl, p-methoxybenzyl, tetrahydrapyranyl or alternatively a silyl group such as, but not limited to t-butyldimethylsilyl ort-butyldiphenylsilyl provides 40. Hydrolysis of 40 under acidic conditions such as, but not limited to, acetic acid, trifluoroacetic acid, or hydrogen chloride or alternatively with fluoride source such as, but not limited to, tetrabutylammonium fluoride, hydrogen fluoride or hydrogen fluoride triethylamine provides compound 41. Activation of the carboxylic acid 41 with reagents such as, but not limited to, p-toluene sulfonyl chloride, pivaloyl chloride, 2,4,6-trichlorobenzoyl chloride, N,N'-dicyclohexane carbodiimide, (1-Ethyl-3-(3- dimethylaminopropyl)carbodiimide, propanephosphonic acid anhydride, and (1- [Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, hexafluorophosphate azabenzotriazole tetramethyl uranium, and treatment with 3’-amine functionalized oligonucleotide and oligonucleotide with pendent 3’-amine provide 42. Treatment of 42 with thiol containing antigens provide target compound 43 via sulfhydryl- maleimide conjugate addition.
ATTORNEY DOCKET: 51026-051WO2 PATENT Scheme 6.
Additionally, the strategy may be achieved according to the reactions described in Scheme 7. Treatment of orthogonally functionalized branched synthon such as, but not limited to, compound 44 with amine 34 (Scheme 5) with polypeptide coupling conditions such as, but not limited to N,N’- diisopropylcarbodiimide, N,N’dicyclohexylcarbodiimide and 1-ethyl-3-(3- dimethylaminopropyl)carbodiimide, with activators such as, but not limited to, 1-Hydroxybenzotriazole and 3-hydroxytriazolo[4,5-b]pyridine to provide compound 45. Alternatively compounds 44 and 35 are treated with benzotriazole N-oxide reagents such as, but not limited to, 3-[bis(dimethylamino)methyliumyl]-3H-benzotriazol-1-oxide hexafluorophosphate, ((1H- benzo[d][1,2,3]triazol-1-yl)oxy)tris(dimethylamino)phosphonium hexafluorophosphate(V), or with and azabenzotriazole N-oxide reagents such as, but not limited to 1-[bis(dimethylamino)methylene]-1H-1,2,3- triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, (benzotriazol-1-yloxy)tripyrrolidinophosphonium
ATTORNEY DOCKET: 51026-051WO2 PATENT hexafluorophosphate, or (7-azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate, to provide compound 45. Deprotection of 45 with bases such as, but not limited to, t-butylamine, diethylamine and piperidine followed by addition of available maleimide fragment 15, functionalized with PEG-units between, but not limited to, m = 0-12, and containing an activated ester such as N-hydrocysuccinimide ester and perfluorophenol ester or as a carboxylic acid that may be activated with, but not limited to, reagents such as p-toluene sulfonyl chloride, pivaloyl chloride, 2,4,6-trichlorobenzoyl chloride, N,N'- dicyclohexane carbodiimide, (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide, propanephosphonic acid anhydride,and (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, hexafluorophosphate azabenzotriazole tetramethyl uranium, provides compound 46. Deprotection under acidic conditions such as, but not limited to, trifluoracetic acid or hydrogen chloride followed by activation of the resulting carboxylic acid with reagents such as, but not limited to p- toluene sulfonic anhydride, pivaloyl chloride, 2,4,6-trichlorobenzoyl chloride, N,N'-dicyclohexane carbodiimide, (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide, propanephosphonic acid anhydride, and (1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, hexafluorophosphate azabenzotriazole tetramethyl uranium, and treatment with 3’-amine functionalized oligonucleotide such as, but not limited to, CpG-3’-amine provide compound 47 that serve as synthon for thiol-containing antigens. Scheme 7.
ATTORNEY DOCKET: 51026-051WO2 PATENT Numbered Embodiments 1. A compound comprising an immunostimulatory nucleic acid sequence, an albumin-binding domain, and a polypeptide, or a pharmaceutically acceptable salt thereof. 2. The compound or pharmaceutically acceptable salt thereof of embodiment 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 pharmaceutically acceptable salt thereof of embodiment 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 pharmaceutically acceptable salt thereof of embodiment 1, wherein the polypeptide and the albumin-binding domain both are bonded or linked to the 5’ end of the immunostimulatory nucleic acid sequence. 5. The compound of or pharmaceutically acceptable salt thereof embodiment 1, wherein the polypeptide and the albumin-binding domain both are bonded or linked to the 3’ end of the immunostimulatory nucleic acid sequence. 6. The compound or pharmaceutically acceptable salt thereof of any one of embodiments 1-5, wherein the polypeptide comprises an N-terminal modification. 7. The compound or pharmaceutically acceptable salt thereof of embodiment 6, wherein the N-terminal modification is the addition of an acetylcysteine. 8. The compound or pharmaceutically acceptable salt thereof of embodiment 7, wherein the N-terminal modification is the addition of a des-aminocysteine homolog. 9. The compound or pharmaceutically acceptable salt thereof of embodiment 8, wherein the des- aminocysteine homolog is 3-mercaptopropionic acid or mercaptoacetic acid. 10. The compound or pharmaceutically acceptable salt thereof of any one of embodiments 1-9, wherein the immunostimulatory nucleic acid sequence is a poly-deoxyadenosine (poly-dA) nucleic acid sequence. 11. The compound or pharmaceutically acceptable salt thereof of any one of embodiments 1-9, wherein the immunostimulatory nucleic acid sequence is a poly-deoxythymidine (poly-dT) nucleic acid sequence. 12. The compound or pharmaceutically acceptable salt thereof of embodiment 10 or 11, wherein the compound comprises a poly-dA nucleic acid sequence and a poly-dT nucleic acid sequence. 13. The compound or pharmaceutically acceptable salt thereof of embodiment 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 pharmaceutically acceptable salt thereof of embodiment 12 or 13, wherein the poly-dA nucleic acid sequence and poly-dT nucleic acid sequence comprise the same number of nucleotides. 15. The compound or pharmaceutically acceptable salt thereof of any one of embodiments 1-9, wherein the immunostimulatory nucleic acid sequence is a poly-deoxyguanosine(poly-dG) nucleic acid sequence.
ATTORNEY DOCKET: 51026-051WO2 PATENT 16. The compound or pharmaceutically acceptable salt thereof of any one of embodiments 1-9, wherein the immunostimulatory nucleic acid sequence is a poly-deoxycytosine (poly-dC) nucleic acid sequence. 17. The compound or pharmaceutically acceptable salt thereof of embodiment 15 or 16, wherein the compound comprises a poly-dG nucleic acid sequence and a poly-dC nucleic acid sequence. 18. The compound or pharmaceutically acceptable salt thereof of embodiment 17, 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 or pharmaceutically acceptable salt thereof of embodiment 17 or 18, wherein the poly-dG nucleic acid sequence and poly-dC nucleic acid sequence comprise the same number of nucleotides. 20. The compound or pharmaceutically acceptable salt thereof of any one of embodiments 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 comprise between 30 and 100 nucleotides. 21. The compound or pharmaceutically acceptable salt thereof of embodiment 20, wherein the poly-dA nucleic acid sequence, poly-dT nucleic acid sequence, poly-dC nucleic acid sequence, and /or poly-dG nucleic acid sequence comprise between 50 and 100 nucleotides. 22. The compound or pharmaceutically acceptable salt thereof of embodiment 20, wherein the poly-dA nucleic acid sequence, poly-dT nucleic acid sequence, poly-dC nucleic acid sequence, and /or poly-dG nucleic acid sequence comprise between 30 and 50 nucleotides. 23. The compound or pharmaceutically acceptable salt thereof of embodiment 20, wherein the poly-dA nucleic acid sequence, poly-dT nucleic acid sequence, poly-dC nucleic acid sequence, and /or poly-dG nucleic acid sequence comprise 30, 40, 50, 75, or 100 nucleotides. 24. The compound or pharmaceutically acceptable salt thereof of any one of embodiments 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. 25. The compound or pharmaceutically acceptable salt thereof of embodiment 24, 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 or pharmaceutically acceptable salt thereof of embodiment 24, 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 or pharmaceutically acceptable salt thereof of embodiment 24, 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 or pharmaceutically acceptable salt thereof of embodiment 24, 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 or pharmaceutically acceptable salt thereof of any one of embodiments 1-24, wherein the immunostimulatory nucleic acid sequence is a CpG sequence.
ATTORNEY DOCKET: 51026-051WO2 PATENT 30. The compound or pharmaceutically acceptable salt thereof of embodiment 29, wherein the CpG sequence is sequence 5’-TCGTCGTTTTGTCGTTTTGTCGTT-3’ (SEQ ID NO:25). 31. The compound or pharmaceutically acceptable salt thereof of embodiment 29, wherein the CpG sequence is 5’-TGACTGTGAACGTTCGAGATGA-3’ (SEQ ID NO: 26). 32. The compound or pharmaceutically acceptable salt thereof of embodiment 29, wherein the CpG sequence is 5’-TCGTCGTTTTCGGCGCGCGCCG-3’ (SEQ ID NO: 27). 33. The compound or pharmaceutically acceptable salt thereof of embodiment 29, wherein the CpG sequence is 5'-TCCATGACGTTCCTGACGTT-3' (SEQ ID NO: 29). 34. The compound or pharmaceutically acceptable salt thereof of any one of embodiments 1-28, wherein at least one internucleotide group 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. 35. The compound or pharmaceutically acceptable salt thereof of any one of embodiments 1-28, wherein all internucleotide groups 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. 36. The compound or pharmaceutically acceptable salt thereof of any one of embodiments 1-35, wherein the polypeptide is an antigen, or a fragment thereof. 37. The compound or pharmaceutically acceptable salt thereof of embodiment 36, wherein the antigen is derived from a tumor or a viral or bacterial source. 38. The compound or pharmaceutically acceptable salt thereof of embodiment 36, wherein the antigen, or fragment thereof, is a tumor-associated antigen. 39. The compound or pharmaceutically acceptable salt thereof of embodiment 36, wherein the antigen is an influenza antigen, or fragment thereof. 40. The compound or pharmaceutically acceptable salt thereof of embodiment 39, wherein the antigen is an influenza nucleoprotein, or fragment thereof. 41. The compound or pharmaceutically acceptable salt thereof of embodiment 37, wherein the antigen is a coronavirus antigen, or fragment thereof. 42. The compound or pharmaceutically acceptable salt thereof of embodiment 41, wherein the antigen is a coronavirus spike protein, or fragment thereof. 43. The compound or pharmaceutically acceptable salt thereof of embodiment 41, wherein the antigen is a coronavirus nucleocapsid protein, or fragment thereof. 44. The compound or pharmaceutically acceptable salt thereof of any one of embodiments 1-43, wherein the albumin-binding domain is a lipid. 45. The compound or pharmaceutically acceptable salt thereof of embodiment 44, wherein the lipid is a diacyl lipid. 46. The compound or pharmaceutically acceptable salt thereof of embodiment 45, wherein the diacyl lipid comprises acyl chains 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.
ATTORNEY DOCKET: 51026-051WO2 PATENT 47. The compound or pharmaceutically acceptable salt thereof of embodiment 45 or embodiment 46, wherein the lipid is 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine (DSPE). 48. The compound or pharmaceutically acceptable salt thereof of any one of embodiments 1-47, 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 or pharmaceutically acceptable salt thereof of embodiment 48, wherein the linker comprises "N" polyethylene glycol units, wherein N is between 4-50. 50. The compound or pharmaceutically acceptable salt thereof of embodiment 49, wherein the linker comprises PEG4-amido-PEG4. 51. The compound or pharmaceutically acceptable salt thereof of any one of embodiments 1-50, wherein the immunostimulatory nucleic acid sequence is bonded or linked by a linker to the following lipid, where the nucleotide linkage is as shown below:
or a salt thereof, wherein X is O or S. 52. A compound or 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 or pharmaceutically acceptable salt thereof of embodiment 52, 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 or pharmaceutically acceptable salt thereof of embodiment 52 or 53, wherein the albumin-binding domain is bonded or linked to the 5’ end of the immunostimulatory nucleic acid sequence. 55. The compound or pharmaceutically acceptable salt thereof of embodiment 52 or 53, wherein the albumin-binding domain is bonded or linked to the 3’ end of the immunostimulatory nucleic acid sequence. 56. The compound or pharmaceutically acceptable salt thereof of any one of embodiments 52-55, wherein the immunostimulatory nucleic acid sequence is a poly-deoxyadenosine (poly-dA) nucleic acid sequence. 57. The compound or pharmaceutically acceptable salt thereof of any one of embodiments 52-55, wherein the immunostimulatory nucleic acid sequence is a poly-deoxythymidine (poly-dT) nucleic acid sequence.
ATTORNEY DOCKET: 51026-051WO2 PATENT 58. The compound or pharmaceutically acceptable salt thereof of embodiment 56 or 57, wherein the compound comprises a poly-dA nucleic acid sequence and a poly-dT nucleic acid sequence. 59. The compound or pharmaceutically acceptable salt thereof of embodiment 58, 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 or pharmaceutically acceptable salt thereof of embodiment 58 or 59, wherein the poly-dA nucleic acid sequence and poly-dT nucleic acid sequence comprise the same number of nucleotides. 61. The compound or pharmaceutically acceptable salt thereof of any one of embodiments 52-55, wherein the immunostimulatory nucleic acid sequence is a poly-deoxyguanosine(poly-dG) nucleic acid sequence. 62. The compound or pharmaceutically acceptable salt thereof of any one of embodiments 52-55, wherein the immunostimulatory nucleic acid sequence is a poly-deoxycytosine (poly-dC) nucleic acid sequence. 63. The compound or pharmaceutically acceptable salt thereof of embodiment 61 or 62, wherein the compound comprises a poly-dG nucleic acid sequence and a poly-dC nucleic acid sequence. 64. The compound or pharmaceutically acceptable salt thereof of embodiment 63, 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 or pharmaceutically acceptable salt thereof of embodiment 63 or 64, wherein the poly-dG nucleic acid sequence and poly-dC nucleic acid sequence comprise the same number of nucleotides. 66. The compound or pharmaceutically acceptable salt thereof of any one of embodiments 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 comprise between 30 and 100 nucleotides. 67. The compound or pharmaceutically acceptable salt thereof of embodiment 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 comprise between 50 and 100 nucleotides. 68. The compound or pharmaceutically acceptable salt thereof of embodiment 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 comprise between 30 and 50 nucleotides. 69. The compound or pharmaceutically acceptable salt thereof of embodiment 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 comprise 30, 40, 50, 75, or 100 nucleotides. 70. The compound or pharmaceutically acceptable salt thereof of any one of embodiments 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. 71. The compound or pharmaceutically acceptable salt thereof of embodiment 70, 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.
ATTORNEY DOCKET: 51026-051WO2 PATENT 72. The compound or pharmaceutically acceptable salt thereof of embodiment 70, 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 or pharmaceutically acceptable salt thereof of embodiment 70, 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 or pharmaceutically acceptable salt thereof of embodiment 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. 75. The compound or pharmaceutically acceptable salt thereof of any one of embodiments 52-70, wherein the immunostimulatory nucleic acid sequence is a CpG sequence. 76. The compound or pharmaceutically acceptable salt thereof of embodiment 75, wherein the CpG sequence is sequence 5’-TCGTCGTTTTGTCGTTTTGTCGTT-3’ (SEQ ID NO:25). 77. The compound or pharmaceutically acceptable salt thereof of embodiment 75, wherein the CpG sequence is 5’-TGACTGTGAACGTTCGAGATGA-3’ (SEQ ID NO: 26). 78. The compound or pharmaceutically acceptable salt thereof of embodiment 75, wherein the CpG sequence is 5’-TCGTCGTTTTCGGCGCGCGCCG-3’ (SEQ ID NO: 27). 79. The compound or pharmaceutically acceptable salt thereof of embodiment 75, wherein the CpG sequence is 5'-TCCATGACGTTCCTGACGTT-3' (SEQ ID NO: 29). 80. The compound or pharmaceutically acceptable salt thereof of any one of embodiments 52-74, wherein at least one internucleotide group 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 or pharmaceutically acceptable salt thereof of any one of embodiments 52-74, wherein all internucleotide groups 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 or pharmaceutically acceptable salt thereof of any one of embodiments 52-81, wherein the albumin-binding domain is a lipid. 83. The compound or pharmaceutically acceptable salt thereof of embodiment 82, wherein the lipid is a diacyl lipid. 84. The compound or pharmaceutically acceptable salt thereof of embodiment 83, wherein the diacyl lipid comprises acyl chains 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 or pharmaceutically acceptable salt thereof of embodiment 83 or embodiment 84, wherein the lipid is 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine (DSPE). 86. The compound or pharmaceutically acceptable salt thereof of any one of embodiments 52-85, wherein the immunostimulatory nucleic acid sequence is bonded or linked by a linker to the following lipid, where the nucleotide linkage is as shown below:
ATTORNEY DOCKET: 51026-051WO2 PATENT
or a salt thereof, wherein X is O or S. 87. The compound or pharmaceutically acceptable salt thereof of embodiment 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 by way of a PEG4 linker to the maleimide group. 88. A compound comprising an immunostimulatory nucleic acid sequence, a linker, and a functional group or a pharmaceutically acceptable salt thereof. 89. The compound or pharmaceutically acceptable salt thereof of embodiment 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 pharmaceutically acceptable salt thereof of embodiment 88 or 89, wherein the immunostimulatory nucleic acid sequence is a poly-deoxyadenosine (poly-dA) nucleic acid sequence. 91. The compound or pharmaceutically acceptable salt thereof of any one of embodiments 88 or 89, wherein the immunostimulatory nucleic acid sequence is a poly-deoxythymidine (poly-dT) nucleic acid sequence. 92. The compound or pharmaceutically acceptable salt thereof of embodiment 90 or 91, wherein the compound comprises a poly-dA nucleic acid sequence and a poly-dT nucleic acid sequence. 93. The compound or pharmaceutically acceptable salt thereof of embodiment 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 pharmaceutically acceptable salt thereof of embodiment 92 or 93, wherein the poly-dA nucleic acid sequence and poly-dT nucleic acid sequence comprise the same number of nucleotides. 95. The compound or pharmaceutically acceptable salt thereof of embodiment 88 or 89, wherein the immunostimulatory nucleic acid sequence is a poly-deoxyguanosine(poly-dG) nucleic acid sequence. 96. The compound or pharmaceutically acceptable salt thereof of embodiment 88 or 89, wherein the immunostimulatory nucleic acid sequence is a poly-deoxycytosine (poly-dC) nucleic acid sequence 97. The compound or pharmaceutically acceptable salt thereof of embodiment 95 or 96, wherein the compound comprises a poly-dG nucleic acid sequence and a poly-dC nucleic acid sequence. 98. The compound or pharmaceutically acceptable salt thereof of embodiment 97, wherein the poly-dG nucleic acid sequence and the poly-dC nucleic acid sequence hybridize to form a double-stranded DNA sequence.
ATTORNEY DOCKET: 51026-051WO2 PATENT 99. The compound or pharmaceutically acceptable salt thereof of embodiment 95 or 96, wherein the poly-dG nucleic acid sequence and poly-dC nucleic acid sequence comprise the same number of nucleotides. 100. The compound or pharmaceutically acceptable salt thereof of any one of embodiments 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 comprise between 30 and 100 nucleotides. 101. The compound or pharmaceutically acceptable salt thereof of embodiment 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 comprise between 50 and 100 nucleotides. 102. The compound or pharmaceutically acceptable salt thereof of embodiment 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 comprise between 30 and 50 nucleotides. 103. The compound or pharmaceutically acceptable salt thereof of embodiment 101, wherein the poly-dA nucleic acid sequence, poly-dT nucleic acid sequence, poly-dC nucleic acid sequence, and /or poly-dG nucleic acid sequence comprise 30, 40, 50, 75, or 100 nucleotides. 104. The compound or pharmaceutically acceptable salt thereof of any one of embodiments 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. 105. The compound or pharmaceutically acceptable salt thereof of embodiment 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. 106. The compound or pharmaceutically acceptable salt thereof of embodiment 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. 107. The compound or pharmaceutically acceptable salt thereof of embodiment 104, 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 or pharmaceutically acceptable salt thereof of embodiment 104, wherein the poly-dC nucleic acid sequence comprises between 100% fand 51% dC nucleic acid residues and between 0% and 49% dG, dA, or dT nucleic acid residues. 109. The compound or pharmaceutically acceptable salt thereof of embodiment 88, wherein the immunostimulatory nucleic acid sequence is a CpG sequence. 110. The compound or pharmaceutically acceptable salt thereof of embodiment 109, wherein the CpG sequence is sequence 5’-TCGTCGTTTTGTCGTTTTGTCGTT-3’ (SEQ ID NO:25). 111. The compound or pharmaceutically acceptable salt thereof of embodiment 109, wherein the CpG sequence is 5’-TGACTGTGAACGTTCGAGATGA-3’ (SEQ ID NO: 26) 112. The compound or pharmaceutically acceptable salt thereof of embodiment 109, wherein the CpG sequence is 5’-TCGTCGTTTTCGGCGCGCGCCG-3’ (SEQ ID NO: 27). 113. The compound or pharmaceutically acceptable salt thereof of embodiment 109, wherein the CpG sequence is 5'-TCCATGACGTTCCTGACGTT-3' (SEQ ID NO: 29).
ATTORNEY DOCKET: 51026-051WO2 PATENT 114. The compound or pharmaceutically acceptable salt thereof of any one of embodiments 88-113, wherein at least one internucleotide group 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. 115. The compound or pharmaceutically acceptable salt thereof of any one of embodiments 88-114, wherein all internucleotide groups 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. 116. The compound or pharmaceutically acceptable salt thereof of any one of embodiments 88-115, wherein the linker is as shown below:
or a salt thereof, wherein X is O or S. 117. The compound or pharmaceutically acceptable salt thereof of embodiment 88, 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 or linked to the PEG4 which is bonded or linked to the maleimide group. 118. A compound comprising an immunoinhibitory nucleic acid sequence, an albumin-binding domain, and a polypeptide, or a pharmaceutically acceptable salt thereof. 119. The compound or pharmaceutically acceptable salt thereof of embodiment 118, wherein the albumin-binding domain is bonded or linked to the 5’ end of the immunoinhibitory nucleic acid sequence, and wherein the polypeptide is bonded or linked to the 3’ end of the immunoinhibitory nucleic acid sequence. 120. The compound or pharmaceutically acceptable salt thereof of embodiment 118, wherein the polypeptide is bonded or linked to the 5’ end of the immunoinhibitory nucleic acid sequence, and wherein the albumin-binding domain is bonded or linked to the 3’ end of the immunoinhibitory nucleic acid sequence. 121. The compound or pharmaceutically acceptable salt thereof of embodiment 118, wherein the polypeptide and the albumin-binding domain are bonded or linked to the 5’ end of the immunoinhibitory nucleic acid sequence. 122. The compound or pharmaceutically acceptable salt thereof of embodiment 118, wherein the polypeptide and the albumin-binding domain are bonded or linked to the 3’ end of the immunoinhibitory nucleic acid sequence. 123. The compound or pharmaceutically acceptable salt thereof of embodiment any one of embodiments 118-122, wherein the polypeptide comprises an N-terminal modification.
ATTORNEY DOCKET: 51026-051WO2 PATENT 124. The compound or pharmaceutically acceptable salt thereof of embodiment 123, wherein the N- terminal modification is the addition of an acetylcysteine. 125. The compound or pharmaceutically acceptable salt thereof of embodiment 124, wherein the N- terminal modification is the addition of a des-aminocysteine homolog. 126. The compound or pharmaceutically acceptable salt thereof of embodiment 125, wherein the des- aminocysteine homolog is 3-mercaptopropionic acid or mercaptoacetic acid. 127. The compound or pharmaceutically acceptable salt thereof of any one of embodiments 118-126, wherein the immunoinhibitory nucleic acid sequence is an A151 nucleic acid sequence. 128. The compound or pharmaceutically acceptable salt thereof of embodiment 127, wherein the immunoinhibitory nucleic acid sequence has a nucleic acid sequence of TTAGG (SEQ ID NO: 28). 129. The compound or pharmaceutically acceptable salt thereof of any one of embodiments 118-128, wherein the polypeptide is an antigen, or a fragment thereof. 130. The compound or pharmaceutically acceptable salt thereof of embodiment 129, wherein the antigen is derived from a tumor or a viral or bacterial source. 131. The compound or pharmaceutically acceptable salt thereof of embodiment 129, wherein the antigen, or fragment thereof, is a tumor-associated antigen. 132. The compound or pharmaceutically acceptable salt thereof of embodiment 129, wherein the antigen is an influenza antigen, or fragment thereof. 133. The compound or pharmaceutically acceptable salt thereof of embodiment 132, wherein the antigen is an influenza nucleoprotein, or fragment thereof. 134. The compound or pharmaceutically acceptable salt thereof of embodiment 130, wherein the antigen is a coronavirus antigen, or fragment thereof. 135. The compound or pharmaceutically acceptable salt thereof embodiment 134, wherein the antigen is a coronavirus spike protein, or fragment thereof. 136. The compound or pharmaceutically acceptable salt thereof of embodiment 134, wherein the antigen is a coronavirus nucleocapsid protein, or fragment thereof. 137. The compound or pharmaceutically acceptable salt thereof of any one of embodiments 118-136, wherein the albumin-binding domain is a lipid. 138. The compound or pharmaceutically acceptable salt thereof of embodiment 137, wherein the lipid is a diacyl lipid. 139. The compound or pharmaceutically acceptable salt thereof of embodiment 138, wherein the diacyl lipid comprises acyl chains 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. 140. The compound or pharmaceutically acceptable salt thereof of embodiment 138 or embodiment 139, wherein the lipid is 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine (DSPE). 141. The compound or pharmaceutically acceptable salt thereof of any one of embodiments 118-140, 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.
ATTORNEY DOCKET: 51026-051WO2 PATENT 142. The compound or pharmaceutically acceptable salt thereof of embodiment 141, wherein the linker comprises "N" polyethylene glycol units, wherein N is between 4-50. 143. The compound or pharmaceutically acceptable salt thereof of embodiment 142, wherein the linker comprises PEG4-amido-PEG4. 144. The compound or pharmaceutically acceptable salt thereof of any one of embodiments 118-143, wherein the immunoinhibitory nucleic acid sequence is bonded or linked by a linker to the following lipid, where the nucleotide linkage is as shown below:
or a salt thereof, wherein X is O or S. 145. A compound comprising an immunoinhibitory nucleic acid sequence, an albumin-binding domain, and a functional group, or a pharmaceutically acceptable salt thereof. 146. The compound or pharmaceutically acceptable salt thereof of embodiment 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 pharmaceutically acceptable salt thereof of embodiment 145 or 146, wherein the albumin-binding domain is bonded or linked to the 5’ end of the immunoinhibitory nucleic acid sequence. 148. The compound or pharmaceutically acceptable salt thereof of embodiment 145 or 146, wherein the albumin-binding domain is bonded or linked to the 3’ end of the immunoinhibitory nucleic acid sequence. 149. The compound or pharmaceutically acceptable salt thereof of any one of embodiments 145-148, wherein the immunoinhibitory nucleic acid sequence is an A151 nucleic acid sequence. 150. The compound or pharmaceutically acceptable salt thereof of embodiment 149, wherein the immunoinhibitory nucleic acid sequence has a nucleic acid sequence of TTAGG (SEQ ID NO: 28). 151. The compound or pharmaceutically acceptable salt thereof of any one of embodiments 145-150, wherein the albumin-binding domain is a lipid. 152. The compound or pharmaceutically acceptable salt thereof of embodiment 151, wherein the lipid is a diacyl lipid. 153. The compound or pharmaceutically acceptable salt thereof of embodiment 152, wherein the diacyl lipid comprises acyl chains 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. 154. The compound or pharmaceutically acceptable salt thereof of embodiment 152 or embodiment 153, wherein the lipid is 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine (DSPE).
ATTORNEY DOCKET: 51026-051WO2 PATENT 155. The compound or pharmaceutically acceptable salt thereof of any one of embodiments 145-154, wherein the immunoinhibitory nucleic acid sequence is bonded or linked by a linker to the following lipid, where the nucleotide linkage is as shown below:
or a salt thereof, wherein X is O or S. 156. A compound comprising an immunoinhibitory nucleic acid sequence, a linker, and a functional group or a pharmaceutically acceptable salt thereof. 157. The compound or pharmaceutically acceptable salt thereof of embodiment 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 embodiment 156 or 157, wherein the immunoinhibitory nucleic acid sequence is an A151 nucleic acid sequence. 159. The compound or pharmaceutically acceptable salt thereof of embodiment 158, wherein the immunoinhibitory nucleic acid sequence has a nucleic acid sequence of TTAGG (SEQ ID NO: 28). 160. The compound or pharmaceutically acceptable salt thereof of any one of embodiments 156-159, 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 embodiment 160, wherein the linker comprises "N" polyethylene glycol units, wherein N is between 4-50. 162. The compound or pharmaceutically acceptable salt thereof of embodiment 161, wherein the linker comprises PEG4-amido-PEG4. 163. The compound or pharmaceutically acceptable salt thereof of any one of embodiments 156-162, wherein the linker is as shown below:
or a salt thereof, wherein X is O or S.
ATTORNEY DOCKET: 51026-051WO2 PATENT 164. A method of inducing an immune response against an antigen in a subject, the method comprising administering a compound or pharmaceutically acceptable salt thereof of any one of embodiments 1-163 to the subject. 165. The method of embodiment 164, further comprising administering an adjuvant to the subject. 166. The method of embodiment 164 or 165, wherein the polypeptide is an antigen, or a fragment thereof. 167. The method of embodiment 166, wherein the antigen is derived from a tumor or a viral or bacterial source. 168. The method of embodiment 166, wherein the antigen, or fragment thereof, is a tumor-associated antigen. 169. The method of embodiment 166, wherein the antigen is an influenza antigen, or fragment thereof. 170. The method of embodiment 169, wherein the antigen is an influenza nucleoprotein, or fragment thereof. 171. The method of embodiment 167, wherein the antigen is a coronavirus antigen, or fragment thereof. 172. The method of embodiment 171, wherein the antigen is a coronavirus spike protein, or fragment thereof. 173. The method of embodiment 171, wherein the antigen is a coronavirus nucleocapsid protein, or fragment thereof. 174. The method of any of one of embodiments 164-173, wherein the compound or pharmaceutically acceptable salt thereof is administered subcutaneously. 175. The method of any of one of embodiments 164-173, wherein the compound or pharmaceutically acceptable salt thereof is administered transmucosally. 176. The method of embodiment 175, wherein the compound or pharmaceutically acceptable salt thereof is administered intranasally. 177. The method of any one of embodiments 164-176, wherein the subject is a mammal. 178. The method of embodiment 177, wherein the subject is a human. 179. A pharmaceutical composition comprising compound or pharmaceutically acceptable salt thereof of any one of embodiments 1-163 and a pharmaceutically acceptable carrier. 180. A kit comprising a compound or pharmaceutically acceptable salt thereof of any one of embodiments 1-163 and instructions for administration. Other Embodiments Various modifications and variations of the described compositions, methods, and uses of the invention 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 connection with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the art are intended to be within the scope of the invention.
ATTORNEY DOCKET: 51026-051WO2 PATENT All publications, patents, and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety. What is claimed is:
Claims
ATTORNEY DOCKET: 51026-051WO2 PATENT CLAIMS 1. A compound comprising an immunostimulatory nucleic acid sequence, an albumin-binding domain, and a polypeptide, or a pharmaceutically acceptable salt thereof. 2. The compound or 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 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 pharmaceutically acceptable salt thereof of claim 1, wherein the polypeptide and the albumin-binding domain both are bonded or linked to the 5’ end of the immunostimulatory nucleic acid sequence. 5. The compound of or pharmaceutically acceptable salt thereof claim 1, wherein the polypeptide and the albumin-binding domain both are bonded or linked to the 3’ end of the immunostimulatory nucleic acid sequence. 6. The compound or pharmaceutically acceptable salt thereof of claim 1, wherein the polypeptide comprises an N-terminal modification. 7. The compound or pharmaceutically acceptable salt thereof of claim 6, wherein the N-terminal modification is the addition of an acetylcysteine. 8. The compound or pharmaceutically acceptable salt thereof of claim 7, wherein the N-terminal modification is the addition of a des-aminocysteine homolog. 9. The compound or pharmaceutically acceptable salt thereof of claim 8, wherein the des-aminocysteine homolog is 3-mercaptopropionic acid or mercaptoacetic acid. 10. The compound or pharmaceutically acceptable salt thereof of claim 1, wherein the immunostimulatory nucleic acid sequence is a poly-deoxyadenosine (poly-dA) nucleic acid sequence. 11. The compound or pharmaceutically acceptable salt thereof of claim 1, wherein the immunostimulatory nucleic acid sequence is a poly-deoxythymidine (poly-dT) nucleic acid sequence.
ATTORNEY DOCKET: 51026-051WO2 PATENT 12. The compound or 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 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 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 pharmaceutically acceptable salt thereof of claim 1, wherein the immunostimulatory nucleic acid sequence is a poly-deoxyguanosine(poly-dG) nucleic acid sequence. 16. The compound or pharmaceutically acceptable salt thereof of claim 1, wherein the immunostimulatory nucleic acid sequence is a poly-deoxycytosine (poly-dC) nucleic acid sequence. 17. The compound or 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 or pharmaceutically acceptable salt thereof of claim 17, 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 or pharmaceutically acceptable salt thereof of claim 17, wherein the poly-dG nucleic acid sequence and poly-dC nucleic acid sequence comprise the same number of nucleotides. 20. The compound or pharmaceutically acceptable salt thereof of claim 1, wherein the poly-dA nucleic acid sequence, poly-dT nucleic acid sequence, poly-dC nucleic acid sequence, and /or poly-dG nucleic acid sequence comprise between 30 and 100 nucleotides. 21. The compound or pharmaceutically acceptable salt thereof of claim 20, wherein the poly-dA nucleic acid sequence, poly-dT nucleic acid sequence, poly-dC nucleic acid sequence, and /or poly-dG nucleic acid sequence comprise between 50 and 100 nucleotides. 22. The compound or pharmaceutically acceptable salt thereof of claim 20, wherein the poly-dA nucleic acid sequence, poly-dT nucleic acid sequence, poly-dC nucleic acid sequence, and /or poly-dG nucleic acid sequence comprise between 30 and 50 nucleotides. 23. The compound or pharmaceutically acceptable salt thereof of claim 20, wherein the poly-dA nucleic acid sequence, poly-dT nucleic acid sequence, poly-dC nucleic acid sequence, and /or poly-dG nucleic acid sequence comprise 30, 40, 50, 75, or 100 nucleotides.
ATTORNEY DOCKET: 51026-051WO2 PATENT 24. The compound or pharmaceutically acceptable salt thereof of claim 10, 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 or pharmaceutically acceptable salt thereof of claim 24, 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 or pharmaceutically acceptable salt thereof of claim 24, 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 or pharmaceutically acceptable salt thereof of claim 24, 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 or pharmaceutically acceptable salt thereof of claim 24, 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 or pharmaceutically acceptable salt thereof of claim 1, wherein the immunostimulatory nucleic acid sequence is a CpG sequence. 30. The compound or pharmaceutically acceptable salt thereof of claim 29, wherein the CpG sequence is sequence 5’-TCGTCGTTTTGTCGTTTTGTCGTT-3’ (SEQ ID NO:25). 31. The compound or pharmaceutically acceptable salt thereof of claim 29, wherein the CpG sequence is 5’-TGACTGTGAACGTTCGAGATGA-3’ (SEQ ID NO: 26). 32. The compound or pharmaceutically acceptable salt thereof of claim 29, wherein the CpG sequence is 5’-TCGTCGTTTTCGGCGCGCGCCG-3’ (SEQ ID NO: 27). 33. The compound or pharmaceutically acceptable salt thereof of claim 29, wherein the CpG sequence is 5'-TCCATGACGTTCCTGACGTT-3' (SEQ ID NO: 29). 34. The compound or pharmaceutically acceptable salt thereof of claim 1, wherein at least one internucleotide group 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.
ATTORNEY DOCKET: 51026-051WO2 PATENT 35. The compound or pharmaceutically acceptable salt thereof of claim 1, wherein all internucleotide groups 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. 36. The compound or pharmaceutically acceptable salt thereof of claim 1, wherein the polypeptide is an antigen, or a fragment thereof. 37. The compound or pharmaceutically acceptable salt thereof of claim 36, wherein the antigen is derived from a tumor or a viral or bacterial source. 38. The compound or pharmaceutically acceptable salt thereof of claim 36, wherein the antigen, or fragment thereof, is a tumor-associated antigen. 39. The compound or pharmaceutically acceptable salt thereof of claim 36, wherein the antigen is an influenza antigen, or fragment thereof. 40. The compound or pharmaceutically acceptable salt thereof of claim 39, wherein the antigen is an influenza nucleoprotein, or fragment thereof. 41. The compound or pharmaceutically acceptable salt thereof of claim 37, wherein the antigen is a coronavirus antigen, or fragment thereof. 42. The compound or pharmaceutically acceptable salt thereof of claim 41, wherein the antigen is a coronavirus spike protein, or fragment thereof. 43. The compound or pharmaceutically acceptable salt thereof of claim 41, wherein the antigen is a coronavirus nucleocapsid protein, or fragment thereof. 44. The compound or pharmaceutically acceptable salt thereof of claim 1, wherein the albumin-binding domain is a lipid. 45. The compound or pharmaceutically acceptable salt thereof of claim 44, wherein the lipid is a diacyl lipid. 46. The compound or pharmaceutically acceptable salt thereof of claim 45, wherein the diacyl lipid comprises acyl chains 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. 47. The compound or pharmaceutically acceptable salt thereof of claim 45 or claim 46, wherein the lipid is 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine (DSPE).
ATTORNEY DOCKET: 51026-051WO2 PATENT 48. The compound or pharmaceutically acceptable salt thereof of claim 1, 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 or pharmaceutically acceptable salt thereof of claim 48, wherein the linker comprises "N" polyethylene glycol units, wherein N is between 4-50. 50. The compound or pharmaceutically acceptable salt thereof of claim 49, wherein the linker comprises PEG4-amido-PEG4. 51. The compound or pharmaceutically acceptable salt thereof of claim 1, wherein the immunostimulatory nucleic acid sequence is bonded or linked by a linker to the following lipid, where the nucleotide linkage is as shown below:
or a salt thereof, wherein X is O or S. 52. A compound or 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 or pharmaceutically acceptable salt thereof of claim 52, 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 or pharmaceutically acceptable salt thereof of claim 52 or 53, wherein the albumin- binding domain is bonded or linked to the 5’ end of the immunostimulatory nucleic acid sequence. 55. The compound or pharmaceutically acceptable salt thereof of claim 52 or 53, wherein the albumin- binding domain is bonded or linked to the 3’ end of the immunostimulatory nucleic acid sequence. 56. The compound or pharmaceutically acceptable salt thereof of claim 52, wherein the immunostimulatory nucleic acid sequence is a poly-deoxyadenosine (poly-dA) nucleic acid sequence.
ATTORNEY DOCKET: 51026-051WO2 PATENT 57. The compound or pharmaceutically acceptable salt thereof of claim 52, wherein the immunostimulatory nucleic acid sequence is a poly-deoxythymidine (poly-dT) nucleic acid sequence. 58. The compound or pharmaceutically acceptable salt thereof of claim 56 or 57, wherein the compound comprises a poly-dA nucleic acid sequence and a poly-dT nucleic acid sequence. 59. The compound or pharmaceutically acceptable salt thereof of claim 58, 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 or pharmaceutically acceptable salt thereof of claim 58, wherein the poly-dA nucleic acid sequence and poly-dT nucleic acid sequence comprise the same number of nucleotides. 61. The compound or pharmaceutically acceptable salt thereof of claim 52, wherein the immunostimulatory nucleic acid sequence is a poly-deoxyguanosine(poly-dG) nucleic acid sequence. 62. The compound or pharmaceutically acceptable salt thereof of claim 52, wherein the immunostimulatory nucleic acid sequence is a poly-deoxycytosine (poly-dC) nucleic acid sequence. 63. The compound or pharmaceutically acceptable salt thereof of claim 61 or 62, wherein the compound comprises a poly-dG nucleic acid sequence and a poly-dC nucleic acid sequence. 64. The compound or pharmaceutically acceptable salt thereof of claim 63, 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 or pharmaceutically acceptable salt thereof of claim 63, wherein the poly-dG nucleic acid sequence and poly-dC nucleic acid sequence comprise the same number of nucleotides. 66. The compound or pharmaceutically acceptable salt thereof of claim 52, wherein the poly-dA nucleic acid sequence, poly-dT nucleic acid sequence, poly-dC nucleic acid sequence, and /or poly-dG nucleic acid sequence comprise between 30 and 100 nucleotides. 67. The compound or pharmaceutically acceptable salt thereof of claim 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 comprise between 50 and 100 nucleotides. 68. The compound or pharmaceutically acceptable salt thereof of claim 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 comprise between 30 and 50 nucleotides.
ATTORNEY DOCKET: 51026-051WO2 PATENT 69. The compound or pharmaceutically acceptable salt thereof of claim 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 comprise 30, 40, 50, 75, or 100 nucleotides. 70. The compound or pharmaceutically acceptable salt thereof of claim 56, 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 or pharmaceutically acceptable salt thereof of claim 70, 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 or pharmaceutically acceptable salt thereof of claim 70, 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 or pharmaceutically acceptable salt thereof of claim 70, 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 or pharmaceutically acceptable salt thereof of claim 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. 75. The compound or pharmaceutically acceptable salt thereof of claim 52, wherein the immunostimulatory nucleic acid sequence is a CpG sequence. 76. The compound or pharmaceutically acceptable salt thereof of claim 75, wherein the CpG sequence is sequence 5’-TCGTCGTTTTGTCGTTTTGTCGTT-3’ (SEQ ID NO:25). 77. The compound or pharmaceutically acceptable salt thereof of claim 75, wherein the CpG sequence is 5’-TGACTGTGAACGTTCGAGATGA-3’ (SEQ ID NO: 26). 78. The compound or pharmaceutically acceptable salt thereof of claim 75, wherein the CpG sequence is 5’-TCGTCGTTTTCGGCGCGCGCCG-3’ (SEQ ID NO: 27). 79. The compound or pharmaceutically acceptable salt thereof of claim 75, wherein the CpG sequence is 5'-TCCATGACGTTCCTGACGTT-3' (SEQ ID NO: 29).
ATTORNEY DOCKET: 51026-051WO2 PATENT 80. The compound or pharmaceutically acceptable salt thereof of claim 52, wherein at least one internucleotide group 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 or pharmaceutically acceptable salt thereof of claim 52, wherein all internucleotide groups 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 or pharmaceutically acceptable salt thereof of claim 52, wherein the albumin-binding domain is a lipid. 83. The compound or pharmaceutically acceptable salt thereof of claim 82, wherein the lipid is a diacyl lipid. 84. The compound or pharmaceutically acceptable salt thereof of claim 83, wherein the diacyl lipid comprises acyl chains 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 or pharmaceutically acceptable salt thereof of claim 83 or claim 84, wherein the lipid is 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine (DSPE). 86. The compound or pharmaceutically acceptable salt thereof of claim 52, wherein the immunostimulatory nucleic acid sequence is bonded or linked by a linker to the following lipid, where the nucleotide linkage is as shown below:
or a salt thereof, wherein X is O or S. 87. The compound or 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 by way of a PEG4 linker to the maleimide group.
ATTORNEY DOCKET: 51026-051WO2 PATENT 88. A compound comprising an immunostimulatory nucleic acid sequence, a linker, and a functional group or a pharmaceutically acceptable salt thereof. 89. The compound or 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 pharmaceutically acceptable salt thereof of claim 88 or 89, wherein the immunostimulatory nucleic acid sequence is a poly-deoxyadenosine (poly-dA) nucleic acid sequence. 91. The compound or pharmaceutically acceptable salt thereof of any one of claims 88 or 89, wherein the immunostimulatory nucleic acid sequence is a poly-deoxythymidine (poly-dT) nucleic acid sequence. 92. The compound or 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 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 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 pharmaceutically acceptable salt thereof of claim 88 or 89, wherein the immunostimulatory nucleic acid sequence is a poly-deoxyguanosine(poly-dG) nucleic acid sequence. 96. The compound or pharmaceutically acceptable salt thereof of claim 88 or 89, wherein the immunostimulatory nucleic acid sequence is a poly-deoxycytosine (poly-dC) nucleic acid sequence. 97. The compound or 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 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 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.
ATTORNEY DOCKET: 51026-051WO2 PATENT 100. The compound or 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 comprise between 30 and 100 nucleotides. 101. The compound or 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 comprise between 50 and 100 nucleotides. 102. The compound or 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 comprise between 30 and 50 nucleotides. 103. The compound or pharmaceutically acceptable salt thereof of claim 101, wherein the poly-dA nucleic acid sequence, poly-dT nucleic acid sequence, poly-dC nucleic acid sequence, and /or poly-dG nucleic acid sequence comprise 30, 40, 50, 75, or 100 nucleotides. 104. The compound or pharmaceutically acceptable salt thereof of claim 90, 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 or pharmaceutically acceptable salt thereof of claim 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. 106. The compound or pharmaceutically acceptable salt thereof of claim 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. 107. The compound or pharmaceutically acceptable salt thereof of claim 104, 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 or pharmaceutically acceptable salt thereof of claim 104, wherein the poly-dC nucleic acid sequence comprises between 100% fand 51% dC nucleic acid residues and between 0% and 49% dG, dA, or dT nucleic acid residues. 109. The compound or pharmaceutically acceptable salt thereof of claim 88, wherein the immunostimulatory nucleic acid sequence is a CpG sequence. 110. The compound or pharmaceutically acceptable salt thereof of claim 109, wherein the CpG sequence is sequence 5’-TCGTCGTTTTGTCGTTTTGTCGTT-3’ (SEQ ID NO:25).
ATTORNEY DOCKET: 51026-051WO2 PATENT 111. The compound or pharmaceutically acceptable salt thereof of claim 109, wherein the CpG sequence is 5’-TGACTGTGAACGTTCGAGATGA-3’ (SEQ ID NO: 26) 112. The compound or pharmaceutically acceptable salt thereof of claim 109, wherein the CpG sequence is 5’-TCGTCGTTTTCGGCGCGCGCCG-3’ (SEQ ID NO: 27). 113. The compound or pharmaceutically acceptable salt thereof of claim 109, wherein the CpG sequence is 5'-TCCATGACGTTCCTGACGTT-3' (SEQ ID NO: 29). 114. The compound or pharmaceutically acceptable salt thereof of claim 88, wherein at least one internucleotide group 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. 115. The compound or pharmaceutically acceptable salt thereof of claim 88, wherein all internucleotide groups 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. 116. The compound or pharmaceutically acceptable salt thereof of claim 88, wherein the linker is as shown below:
or a salt thereof, wherein X is O or S. 117. The compound or pharmaceutically acceptable salt thereof of claim 88, 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 or linked to the PEG4 which is bonded or linked to the maleimide group. 118. A compound comprising an immunoinhibitory nucleic acid sequence, an albumin-binding domain, and a polypeptide, or a pharmaceutically acceptable salt thereof. 119. The compound or pharmaceutically acceptable salt thereof of claim 118, wherein the albumin- binding domain is bonded or linked to the 5’ end of the immunoinhibitory nucleic acid sequence, and wherein the polypeptide is bonded or linked to the 3’ end of the immunoinhibitory nucleic acid sequence.
ATTORNEY DOCKET: 51026-051WO2 PATENT 120. The compound or pharmaceutically acceptable salt thereof of claim 118, wherein the polypeptide is bonded or linked to the 5’ end of the immunoinhibitory nucleic acid sequence, and wherein the albumin- binding domain is bonded or linked to the 3’ end of the immunoinhibitory nucleic acid sequence. 121. The compound or pharmaceutically acceptable salt thereof of claim 118, wherein the polypeptide and the albumin-binding domain are bonded or linked to the 5’ end of the immunoinhibitory nucleic acid sequence. 122. The compound or pharmaceutically acceptable salt thereof of claim 118, wherein the polypeptide and the albumin-binding domain are bonded or linked to the 3’ end of the immunoinhibitory nucleic acid sequence. 123. The compound or pharmaceutically acceptable salt thereof of claim 118, wherein the polypeptide comprises an N-terminal modification. 124. The compound or pharmaceutically acceptable salt thereof of claim 123, wherein the N-terminal modification is the addition of an acetylcysteine. 125. The compound or pharmaceutically acceptable salt thereof of claim 124, wherein the N-terminal modification is the addition of a des-aminocysteine homolog. 126. The compound or pharmaceutically acceptable salt thereof of claim 125, wherein the des- aminocysteine homolog is 3-mercaptopropionic acid or mercaptoacetic acid. 127. The compound or pharmaceutically acceptable salt thereof of claim 118, wherein the immunoinhibitory nucleic acid sequence is an A151 nucleic acid sequence. 128. The compound or pharmaceutically acceptable salt thereof of claim 127, wherein the immunoinhibitory nucleic acid sequence has a nucleic acid sequence of TTAGG (SEQ ID NO: 28). 129. The compound or pharmaceutically acceptable salt thereof of claim 118, wherein the polypeptide is an antigen, or a fragment thereof. 130. The compound or pharmaceutically acceptable salt thereof of claim 129, wherein the antigen is derived from a tumor or a viral or bacterial source. 131. The compound or pharmaceutically acceptable salt thereof of claim 129, wherein the antigen, or fragment thereof, is a tumor-associated antigen.
ATTORNEY DOCKET: 51026-051WO2 PATENT 132. The compound or pharmaceutically acceptable salt thereof of claim 129, wherein the antigen is an influenza antigen, or fragment thereof. 133. The compound or pharmaceutically acceptable salt thereof of claim 132, wherein the antigen is an influenza nucleoprotein, or fragment thereof. 134. The compound or pharmaceutically acceptable salt thereof of claim 130, wherein the antigen is a coronavirus antigen, or fragment thereof. 135. The compound or pharmaceutically acceptable salt thereof claim 134, wherein the antigen is a coronavirus spike protein, or fragment thereof. 136. The compound or pharmaceutically acceptable salt thereof of claim 134, wherein the antigen is a coronavirus nucleocapsid protein, or fragment thereof. 137. The compound or pharmaceutically acceptable salt thereof of claim 118, wherein the albumin- binding domain is a lipid. 138. The compound or pharmaceutically acceptable salt thereof of claim 137, wherein the lipid is a diacyl lipid. 139. The compound or pharmaceutically acceptable salt thereof of claim 138, wherein the diacyl lipid comprises acyl chains 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. 140. The compound or pharmaceutically acceptable salt thereof of claim 138 or claim 139, wherein the lipid is 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine (DSPE). 141. The compound or pharmaceutically acceptable salt thereof of claim 118, 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 or pharmaceutically acceptable salt thereof of claim 141, wherein the linker comprises "N" polyethylene glycol units, wherein N is between 4-50. 143. The compound or pharmaceutically acceptable salt thereof of claim 142, wherein the linker comprises PEG4-amido-PEG4.
ATTORNEY DOCKET: 51026-051WO2 PATENT 144. The compound or pharmaceutically acceptable salt thereof of claim 118, wherein the immunoinhibitory nucleic acid sequence is bonded or linked by a linker to the following lipid, where the nucleotide linkage is as shown below:
or a salt thereof, wherein X is O or S. 145. A compound comprising an immunoinhibitory nucleic acid sequence, an albumin-binding domain, and a functional group, or a pharmaceutically acceptable salt thereof. 146. The compound or 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 pharmaceutically acceptable salt thereof of claim 145 or 146, wherein the albumin-binding domain is bonded or linked to the 5’ end of the immunoinhibitory nucleic acid sequence. 148. The compound or pharmaceutically acceptable salt thereof of claim 145 or 146, wherein the albumin-binding domain is bonded or linked to the 3’ end of the immunoinhibitory nucleic acid sequence. 149. The compound or pharmaceutically acceptable salt thereof of claim 145, wherein the immunoinhibitory nucleic acid sequence is an A151 nucleic acid sequence. 150. The compound or pharmaceutically acceptable salt thereof of claim 149, wherein the immunoinhibitory nucleic acid sequence has a nucleic acid sequence of TTAGG (SEQ ID NO: 28). 151. The compound or pharmaceutically acceptable salt thereof of claim 145, wherein the albumin- binding domain is a lipid. 152. The compound or pharmaceutically acceptable salt thereof of claim 151, wherein the lipid is a diacyl lipid.
ATTORNEY DOCKET: 51026-051WO2 PATENT 153. The compound or pharmaceutically acceptable salt thereof of claim 152, wherein the diacyl lipid comprises acyl chains 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. 154. The compound or pharmaceutically acceptable salt thereof of claim 152 or claim 153, wherein the lipid is 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine (DSPE). 155. The compound or pharmaceutically acceptable salt thereof of claim 145, wherein the immunoinhibitory nucleic acid sequence is bonded or linked by a linker to the following lipid, where the nucleotide linkage is as shown below:
or a salt thereof, wherein X is O or S. 156. A compound comprising an immunoinhibitory nucleic acid sequence, a linker, and a functional group or a pharmaceutically acceptable salt thereof. 157. The compound or 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 immunoinhibitory nucleic acid sequence is an A151 nucleic acid sequence. 159. The compound or pharmaceutically acceptable salt thereof of claim 158, wherein the immunoinhibitory 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" polyethylene glycol units, wherein N is between 4-50.
ATTORNEY DOCKET: 51026-051WO2 PATENT 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 shown below:
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 a compound or pharmaceutically acceptable salt thereof of claim 1 to the subject. 165. The method of claim 164, further comprising administering an adjuvant to the subject. 166. The method of claim 164 or 165, wherein the polypeptide is an antigen, or a fragment thereof. 167. The method of claim 166, wherein the antigen is derived from a tumor or a 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.
ATTORNEY DOCKET: 51026-051WO2 PATENT 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 compound or pharmaceutically acceptable salt thereof of claim 1 and a pharmaceutically acceptable carrier. 180. A kit comprising a compound or pharmaceutically acceptable salt thereof of claim 1 and instructions for administration.
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| US202363461974P | 2023-04-26 | 2023-04-26 | |
| US63/461,974 | 2023-04-26 | ||
| PCT/US2024/026241 WO2024226775A2 (en) | 2023-04-26 | 2024-04-25 | Compositions containing polynucleotide and polypeptide amphiphiles and methods of use thereof |
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| WO2010002940A2 (en) * | 2008-06-30 | 2010-01-07 | Dynavax Technologies Corporation | Heterogeneous synthesis of multivalent chimeric immunomodulatory compounds using platform based molecules |
| EP3563872A1 (en) * | 2012-04-05 | 2019-11-06 | Massachusetts Institute Of Technology | Immunostimulatory compositions and methods of use thereof |
| EP3882257A1 (en) * | 2020-03-20 | 2021-09-22 | Ospedale San Raffaele S.r.l. | Ngr conjugates and uses thereof |
| US20230263882A1 (en) * | 2020-06-26 | 2023-08-24 | Elicio Therapeutics, Inc. | Compositions and methods for inducing an immune response against coronavirus |
| WO2023022973A2 (en) * | 2021-08-16 | 2023-02-23 | Elicio Therapeutics, Inc. | Compositions containing polynucleotide amphiphiles and methods of use thereof |
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