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WO2023196936A1 - Polynucleotide cancer vaccine compositions and methods of using the same - Google Patents

Polynucleotide cancer vaccine compositions and methods of using the same Download PDF

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Publication number
WO2023196936A1
WO2023196936A1 PCT/US2023/065482 US2023065482W WO2023196936A1 WO 2023196936 A1 WO2023196936 A1 WO 2023196936A1 US 2023065482 W US2023065482 W US 2023065482W WO 2023196936 A1 WO2023196936 A1 WO 2023196936A1
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composition
vaccine
aspects
pharmaceutical composition
mage
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PCT/US2023/065482
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French (fr)
Inventor
Carlo IAVARONE
Majed Mohd MATAR
Subeena SOOD
John Henderson
Jessica KIM
Brian Jeffrey Sparks
Anthony Joseph RECUPERO
Michael Henry TARDUGNO
Khursheed Anwer
Jean D. Boyer
Kempaiah RAYAVARA
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Imunon Inc
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Imunon Inc
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Publication of WO2023196936A1 publication Critical patent/WO2023196936A1/en
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001154—Enzymes
    • A61K39/001156—Tyrosinase and tyrosinase related proteinases [TRP-1 or TRP-2]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5434—IL-12
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6093—Synthetic polymers, e.g. polyethyleneglycol [PEG], Polymers or copolymers of (D) glutamate and (D) lysine
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli

Definitions

  • the present disclosure relates generally to oncology, immunology, cancer vaccines, and gene therapy.
  • the disclosure relates to nucleic acidcontaining compositions and methods of using the same to generate an immune response to one or more tumor-associated antigens for treating, reducing the likelihood of, or preventing cancer in mammals.
  • Cancer vaccines are a type of cancer treatment based on immunotherapy. They work to boost the body's immune system to fight cancer. Vaccines that use tumor- associated antigens have been explored as treatment options for cancer. Cancer vaccines can use either shared tumor antigens expressed by many tumors or unique tumor antigens expressed by specific tumors. A challenge with tumor vaccines has been that tumors may contain many different cell types each with different surface antigens; and many tumors display few, if any, antigens that are foreign to the individual. Thus, recognition of tumor- associated and tumor-specific antigens by the immune system as well as the distinction between normal and tumor cells remain challenges for developing effective cancer vaccines.
  • Nucleic acid (DNA and RNA) vaccines are being developed for a variety of infectious diseases across the globe. Despite their attractiveness, issues such as suboptimal immunogenicity and effective delivery have been concerns with DNA vaccines.
  • compositions comprising: (a) a polynucleotide (e.g., an expression cassette) comprising an antigen nucleic acid which encodes (i) at least one tumor-associated antigen or an antigenic fragment thereof (e.g., a first tumor-associated antigen or an antigenic fragment thereof), wherein the antigen nucleic acid is operably linked to a promoter (e.g., a first promoter), and, optionally, (ii) a nucleic acid encoding an immune modifier (e.g., a cytokine).
  • the immune modifier is a cytokine.
  • the cytokine is IL-12.
  • the composition further comprises (b) a delivery component selected from the group consisting of a cationic polymer, a poly-inosinic-polycytidylic acid, a poloxamer, or derivative thereof.
  • the delivery component is a crown poloxamer.
  • the delivery component is a carrier for the polynucleotide.
  • the composition further comprises (c) an adjuvant.
  • the adjuvant comprises an aluminum or aluminum-salt based adjuvant, a stimulator of interferon genes (STING) agonist, or a combination thereof.
  • the composition stimulates an immune response to a tumor or target cancer cell.
  • the aluminum or aluminum-salt based adjuvant is selected from the group consisting of an aluminum phosphate, an aluminum hydroxide, an aluminum oxyhydroxide, a potassium aluminum sulfate [KA1(SO4)2], an aluminum bicarbonate, an aluminum hydroxyphosphate, an aluminum hydroxyphosphate sulfate, an aluminum chloride, an aluminum silicate, and any combination thereof.
  • the aluminum or aluminum-salt based adjuvant comprises an aluminum phosphate, an aluminum hydroxide, a potassium aluminum sulfate [KA1(SO4)2], an aluminum oxyhydroxide, or any combination thereof.
  • the aluminum or aluminum-salt based adjuvant comprises an aluminum phosphate or an aluminum hydroxide.
  • aluminum salt-based adjuvant is a mixture of aluminum hydroxide and magnesium hydroxide, a mixture of aluminum sulfate and sodium hydroxide, a mixture of aluminum sulfate and potassium hydroxide, a mixture of aluminum phosphate and magnesium hydroxide, aluminum phosphate and sodium hydroxide, aluminum phosphate and potassium hydroxide or a mixture of aluminum phosphate and aluminum hydroxide.
  • the adjuvant is MF59 (oil-in-water emulsion of squalene oil). In some aspects, the adjuvant is AS03 (a-tocopherol, squalenem and polysorbate 80 in an oil-in-water emulsion).
  • the STING agonist is selected from the group consisting of a cyclic di-nucleotides, a non-cyclic di-nucleotide small molecule, an amidobenzimidazole (ABZI), a flavonoid, a nanovaccine, an antibody drug conjugate, a bacterial vector, and an ENPP1 inhibitor.
  • the STING agonist is cyclic guanosine monophosphate (cGMP), cyclic adenosine monophosphate (cAMP), or cyclic guanosine monophosphate-adenosine monophosphate (cGAMP). In some aspects, the STING agonist is cGMP.
  • the antigen nucleic acid encodes at least one tumor-associated antigen or an antigenic fragment thereof, which can be an oncogene, a mutated tumor suppressor gene, an overexpressed or aberrantly expressed cellular protein, an antigen of an oncogenic virus, an oncofetal antigen, an altered cell surface glycolipid and/or glycoprotein, a cell type-specific differentiation antigen, or any combination thereof.
  • tumor-associated antigen or an antigenic fragment thereof which can be an oncogene, a mutated tumor suppressor gene, an overexpressed or aberrantly expressed cellular protein, an antigen of an oncogenic virus, an oncofetal antigen, an altered cell surface glycolipid and/or glycoprotein, a cell type-specific differentiation antigen, or any combination thereof.
  • the antigen nucleic acid encodes at least one tumor-associated antigen or an antigenic fragment thereof (e.g., a first tumor- associated antigen or an antigenic fragment thereof) selected from the group consisting of NYESO-1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A9, MAGE-A10, MAGE-CI, MAGE-C2, SSX, H0RMAD1, CXorf61, ACTL8, AKAP3,AKAP4, HOM- TES-85, OY-TES-1, HSP70-2, LAGE-1, PIWL1, PIWL2, PIWL3, PIWL4, PLU-1, PLAC1, SPAG9, SCP-1, TAG-1, TAG-2a, TAG-2b, TAG-2c, TRAG-3, KAP4, BAGE, BORIS, CT45, GAGE-1/2, PRAME, PAGE4, SP17, PASD1, p
  • the polynucleotide comprises an antigen nucleic acid sequence that is derived from a patient’s own nucleic acid sequence that encodes a NYESO-1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A9, MAGE-A10, MAGE-CI, MAGE-C2, SSX, HORMAD1, CXorf61, ACTL8, AKAP3,AKAP4, HOM-TES-85, OY- TES-1, HSP70-2, LAGE-1, PIWL1, PIWL2, PIWL3, PIWL4, PLU-1, PLAC1, SPAG9, SCP-1, TAG-1, TAG-2a, TAG-2b, TAG-2c, TRAG-3, KAP4, BAGE, BORIS, CT45, GAGE-1/2, PRAME, PAGE4, SP17, PASD1, p53, ras, B-RafHER-2/neu, mucin 1 (MUC1), epi
  • the polynucleotide (e.g., expression cassette) further comprises at least one additional antigen nucleic acid (e.g., a second antigen nucleic acid), which encodes at least one additional tumor-associated antigen or an antigenic fragment thereof (e.g., a second tumor-associated antigen or an antigenic fragment thereof).
  • additional antigen nucleic acid e.g., a second antigen nucleic acid
  • additional tumor-associated antigen or an antigenic fragment thereof e.g., a second tumor-associated antigen or an antigenic fragment thereof.
  • the at least one additional tumor-associated antigen or an antigenic fragment thereof is selected from the group consisting of a NYESO-1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A9, MAGE-A10, MAGE-CI, MAGE-C2, SSX, H0RMAD1, CXorf61, ACTL8, AKAP3,AKAP4, HOM-TES-85, OY-TES-1, HSP70-2, LAGE-1, PIWL1, PIWL2, PIWL3, PIWL4, PLU-1, PLAC1, SPAG9, SCP-1, TAG-1, TAG-2a, TAG-2b, TAG-2c, TRAG-3, KAP4, BAGE, BORIS, CT45, GAGE-1/2, PRAME, PAGE4, SP17, PASD1, p53, ras, B-RafHER-2/neu, mucin 1 (MUC1)
  • the at least one tumor-associated antigen or antigenic fragment thereof is/are selected from the group consisting of NYESO-1, CA-125, GP100, carcinoembryonic antigen (CEA), mucin 1, TRP-2, SP17, and any combinations thereof.
  • the at least one additional antigen nucleic acid (e.g., second antigen nucleic acid) of the polynucleotide is operably linked to the first promoter through an internal ribosome entry site (IRES) sequence.
  • IRES internal ribosome entry site
  • one antigenic nucleic acid of the polynucleotide is linked to a second antigenic nucleic acid through a 2A peptide.
  • the 2A peptide is a F2A, P2A, E2A or T2A peptide.
  • the at least one additional antigen nucleic acid (e.g., second antigen nucleic acid) of the polunucletoide is operably linked to one or more additional promoters (e.g., a second promoter).
  • the promoter e.g., first promoter
  • the one or more additional promoters e.g., second promoter
  • the polynucleotide further comprises one or more post- transcriptional regulatory elements.
  • the post-transcriptional regulatory element is a wood chuck hepatitis virus post-transcriptional regulatory element (WPRE).
  • the polynucleotide further comprises at least one 3' UTR poly(a) tail sequence operably linked to the first antigen nucleic acid, the second antigen nucleic acid, or any combination thereof.
  • the 3' UTR poly(a) tail sequence is a 3' UTR SV40 poly(a) tail sequence, a 3' UTR bovine growth hormone (bGH) poly(A) sequence, a 3' UTR actin poly(A) tail sequence, a 3' UTR hemoglobin poly(A) sequence, or combinations thereof.
  • bGH bovine growth hormone
  • the polynucleotide further comprises an enhancer sequence.
  • the enhancer sequence comprises a human actin enhancer sequence, a human myosin enhancer sequence, a human hemoglobin enhancer sequence, a human muscle creatine enhancer sequence, a viral enhancer sequence, a polynucleotide function enhancer sequence, or any combination thereof.
  • the enhancer sequence comprises a CMV intronic sequence, a P- actin intronic sequence, or the combination thereof. In some aspects, the enhancer sequence is a CMV intronic sequence. In some aspects, the enhancer sequence is a CMV intronic sequence, a SV40 enhancer sequence, a P-actin intronic sequence, or combinations thereof.
  • the polynucleotide is within a vector, wherein the vector is a DNA plasmid, a viral vector, a bacterial vector, a cosmid, or an artificial chromosome. In some aspects, the vector is a DNA plasmid.
  • the composition is a pharmaceutical composition comprising a pharmaceutically acceptable carrier.
  • the composition or pharmaceutical composition is a cancer vaccine.
  • the cationic polymer comprises a synthetic functionalized polymer, a P-amino ester, a lipid, a lipopolymer, or a chemical derivative thereof.
  • the synthetic functionalized polymer is a biodegradable crosslinked cationic multi-block copolymer.
  • the biodegradable cross-linked cationic multi-block copolymer is represented by the formula: (CP)xLyYz, wherein: (a) CP represents a cationic polymer containing at least one secondary amine group, wherein the cationic polymer has a number averaged molecular weight within the range of 1,000 to 25,000 Dalton, (b) Y represents a bifunctional biodegradable linker containing ester, amide, disulfide, or phosphate linages, (c) L represents a ligand, (d) x is an integer in the range from 1 to 20, (e) y is an integer in the range from 0 to 100, and (f) z is an integer in the range from 0 to 40.
  • CP represents a cationic polymer containing at least one secondary amine group, wherein the cationic polymer has a number averaged molecular weight within the range of 1,000 to 25,000 Dalton
  • Y represents a bifunctional biodegradable linker containing este
  • the cationic polymer comprises biodegradable cross-linked linear polyethyleneimine (LPEI).
  • the bifunctional biodegradable linker is hydrophilic and comprises a biodegradable linkage comprising a disulfide bond.
  • the bifunctional biodegradable linker is a dithiodipropionyl linker.
  • the biodegradable cross-linked cationic multi-block copolymer comprises LPEI and a dithiodipropionyl linker for cross-linking the multi-block copolymer, wherein the LPEI has an average molecular weight of 1,000 to 25,000 Dalton.
  • the biodegradable cross-linked cationic multi-block copolymer is covalently linked to at least one ligand.
  • the ligand is a targeting ligand selected from the group consisting of: a sugar moiety, a polypeptide, folate, and an antigen.
  • the sugar moiety is a monosaccharide or an oligosaccharide.
  • the monosaccharide is galactose.
  • the polypeptide is a glycoprotein, an antibody, an antibody fragment, a cell receptor, a cytokine receptor, or a growth factor receptor.
  • the growth factor receptor is an epidermal growth factor receptor.
  • the glycoprotein is transferrin or asialoorosomucoid (ASOR).
  • the antigen is an oncoviral antigen.
  • the biodegradable cross-linked cationic multi-block copolymer is covalently linked to polyethylene glycol (PEG) of molecular weight ranging from 500 to 20,000 Dalton.
  • PEG polyethylene glycol
  • the biodegradable cross-linked cationic multi-block copolymer is covalently linked to a fatty acyl chain selected from the group consisting of: oleic acid, palmitic acid, and stearic acid.
  • the biodegradable cross-linked cationic multi-block copolymer comprises at least one amine group that is electrostatically attracted to a polyanionic compound.
  • the polyanionic compound is a nucleic acid, wherein the biodegradable cross-linked cationic multi-block copolymer condenses the nucleic acid to form a compact structure.
  • the lipopolymer is a cationic lipopolymer comprising a PEI backbone covalently linked to a lipid or a PEG.
  • the PEI backbone is covalently linked to a lipid and a PEG.
  • the lipid and the PEG are directly attached to the PEI backbone by covalent bonds.
  • the lipid is attached to the PEI backbone through a PEG spacer.
  • the PEG has a molecular weight of between 50 to 20,000 Dalton.
  • the molar ratio of PEG to PEI is within a range of 0.1 : 1 to 500: 1.
  • the molar ratio of the lipid to the PEI is within a range of 0.1 : 1 to 500: 1.
  • the lipid is a cholesterol, a cholesterol derivative, a C12 to C18 fatty acid, or a fatty acid derivative.
  • the PEI is covalently linked to cholesterol and PEG, and wherein the average PEG:PEI:cholesterol molar ratio in the cationic lipopolymer is within the range of 1-5 PEG: 1 PEEO.4-1.5 cholesterol.
  • the PEI has a linear or branch configuration with a molecular weight of 100 to 500,000 Dalton.
  • the cationic lipopolymer further comprises a pendant functional moiety selected from the group consisting of: a receptor ligand, a membrane permeating agent, an endosomolytic agent, a nuclear localization sequence, and a pH sensitive endosomolytic peptide.
  • the cationic lipopolymer further comprises a targeting ligand, wherein the targeting ligand is directly attached to the PEI backbone or is attached through a PEG linker.
  • the targeting ligand is selected from the group consisting of: a sugar moiety, a polypeptide, folate, and an antigen.
  • the sugar moiety is a monosaccharide or an oligosaccharide.
  • the monosaccharide is galactose.
  • the polypeptide is a glycoprotein, an antibody, an antibody fragment, a cell receptor, a cytokine receptor, or a growth factor receptor.
  • the cationic polymer is present in an amount sufficient to produce a ratio of amine nitrogen in the cationic polymer to phosphate in the DNA plasmid vector from about 0.01 : 1 to about 50: 1. In some aspects, the ratio of amine nitrogen in the cationic polymer to phosphate in the DNA plasmid vector from about 1 : 10 to about 10: 1.
  • composition, pharmaceutical composition, or vaccine comprises about 0.1 mg/ml to about 10.0 mg/ml nucleic acid complexed with the cationic polymer.
  • the delivery component comprises a lipopoly amine with the following formula: (Staramine).
  • the delivery component comprises a mixture of the lipopolyamine and an alkylated derivative of the lipopolyamine.
  • the alkylated derivative of the lipopolyamine is a polyoxyalkylene, polyvinylpyrrolidone, polyacrylamide, polydimethylacrylamide, polyvinyl alcohol, dextran, poly (L-glutamic acid), styrene maleic anhydride, poly-N-(2- hydroxypropyl) methacrylamide, or polydivinylether maleic anhydride.
  • the alkylated derivative of the lipopolyamine has the following formula:
  • n represents an integer from 10 to 100 repeating units containing of 2-5 carbon atoms each.
  • the ratio of the lipopolyamine to the alkylated derivative of the lipopolyamine in the mixture is 1 : 1 to 10: 1.
  • the lipopolyamine is present in an amount sufficient to produce a ratio of amine nitrogen in the lipopolyamine to phosphate in the DNA plasmid vector from about 0.01 : 1 to about 50: 1.
  • the lipopolyamine is present in an amount sufficient to produce a ratio of amine nitrogen in the lipopolyamine to phosphate in the DNA plasmid vector from about 1 : 10 to about 10: 1.
  • the delivery component comprises a lipopolyamine with the following formula:
  • the delivery component comprises a mixture of the lipopolyamine and an alkylated derivative of the lipopolyamine.
  • the alkylated derivative of the lipopolyamine is a polyoxyalkylene, polyvinylpyrrolidone, polyacrylamide, polydimethylacrylamide, polyvinyl alcohol, dextran, poly (L-glutamic acid), styrene maleic anhydride, poly-N-(2- hydroxypropyl) methacrylamide, or polydivinylether maleic anhydride.
  • the ratio of the lipopolyamine to the alkylated derivative of the lipopolyamine in the mixture is 1 : 1 to 10: 1.
  • the lipolyamine is present in an amount sufficient to produce a ratio of amine nitrogen in the lipopolyamine to phosphate in the DNA plasmid vector from about 0.01 : 1 to about 50: 1.
  • the lipolyamine is present in an amount sufficient to produce a ratio of amine nitrogen in the lipopolyamine to phosphate in the DNA plasmid vector from about 1 : 10 to about 10: 1.
  • the delivery component comprises a poloxamer with the following formula: or a pharmaceutically acceptable salt thereof, wherein:
  • A represents an integer from 2 to 141;
  • B represents an integer from 16 to 67;
  • C represents an integer from 2 to 141;
  • RA and RC are the same or different, and are R'-L- or H;
  • L is a bond, — CO — , — CH2 — O — , or — O — CO — ;
  • R' is a metal chelator
  • At least one of RA and RC is R'-L-.
  • the R’ is covalently bound to the poloxamer (e.g., where a metal chelator (e.g., a crown) is covalently attached at different density to the poloxamer).
  • a metal chelator e.g., a crown
  • one metal chelator or two or more metal chelators is/are bound to the poloxamer.
  • 2-100 metal chelators are bound to the poloxamer.
  • the metal chelator is RNNH — , RN2N — , or (R" — (N(R") —
  • the metal chelator is a crown ether, a substituted-crown ether, a cryptand, or a substituted-cryptand.
  • the delivery component further comprises a PEG-PEI-cholesterol (PPC) lipopolymer, benzalkonium chloride (BAK), Omnifect, or a linear polyethyleneimine (LPEI).
  • PPC PEG-PEI-cholesterol
  • BAK benzalkonium chloride
  • Omnifect or a linear polyethyleneimine (LPEI).
  • LPEI linear polyethyleneimine
  • the LPEI is BD15-12.
  • the poloxamer e.g., crown poloxamer
  • the polynucleotide or DNA plasmid vector from about 0.01 % - about 5%.
  • the solution is co-formulated with a metal chelator (e.g., where the co-formulated metal chelator is a free metal chelator, which is formulated with the poloxamer (e.g., a non-crown poloxamer)).
  • a metal chelator e.g., where the co-formulated metal chelator is a free metal chelator, which is formulated with the poloxamer (e.g., a non-crown poloxamer)).
  • the co-formulated metal chelator is present in the solution at a concentration of about O.lmg/mL to about 20mg/mL.
  • the co-formulated metal chelator is crown ether, a substituted- crown ether, a cryptand, or a substituted-cryptand.
  • the metal chelator or co-formulated metal chelator is crown ether (Aza- 18 -crown-6) .
  • the delivery component comprises BD15-12.
  • the nucleotide to polymer (N:P) ratio is 0.1 : 1 to 5: 1.
  • the delivery component is PEG-PEI-cholesterol (PPC) lipopolymer.
  • PPC PEG-PEI-cholesterol
  • N:P nucleotide to polymer
  • the delivery component comprises Omnifect.
  • the nucleotide to polymer (N:P) ratio is 0.1 : 1 to 5: 1.
  • the delivery component comprises crown poloxamer connected by covalent bond directly or through a linker to an aluminum or aluminum-salt based adjuvant.
  • the delivery component comprises Staramine and mPEG modified Staramine.
  • the mPEG modified Staramine is Staramine- mPEG515 or Staramine-mPEGl 1.
  • the delivery component comprises Crossamine (N,N’ -di oleoyl tetrakis(aminomethyl)methane).
  • the delivery component is Starmine.
  • the nucleotide to polymer (N:P) ratio is 0.1 : 1 to 5: 1.
  • the delivery component is Crossamine.
  • the nucleotide to polymer (N:P) ratio is 0.1 : 1 to 5: 1.
  • the ratio of Staramine to mPEG modified Staramine is 10: 1.
  • the nucleotide to polymer (N:P) ratio is 0.01 : 1 to 5: 1.
  • the delivery component further comprises crown poloxamer.
  • the composition, pharmaceutical composition, or vaccine is stable at 0°C to 5°C for at least about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 monts, or about 12 months.
  • the composition, pharmaceutical composition, or vaccine is stable at 25°C for at least about 7 days, about 10 day, or about 14 days, or about 60 days.
  • the composition, pharmaceutical composition, or vaccine is stable at -20°C for at least about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 monts, or about 12 months.
  • the composition is lyophilized and is substantially free of aqueous components.
  • the composition is reconstituted with a diluent.
  • the diluent is water.
  • kits comprising the composition, pharmaceutical composition, or vaccine disclosed herein.
  • the kit further comprises a glass vial.
  • the kit further comprises instructions for using the composition or lyophilized composition in a method for inducing an immune response in a subject.
  • the kit further comprises instructions for using the composition or lyophilized composition in a method for preventing, reducing the incidence of, attenuating or treating a malignant disease in a subject.
  • the vaccine compositions provided herein are administered to a subject suffering from a malignant disease.
  • the vaccine compositions are adminsiered with an additional antineoplastic therapy.
  • the additional antineoplastic therapy includes any cancer therapy used to treat a cancer disclosed herein.
  • the vaccine compositions are administered with a radiation therapy.
  • the vaccine compositions are administered before a radiation therapy.
  • the vaccine compositions are administered after a radiation therapy.
  • the vaccine compositions are administered before a tumor surgery. In some aspects, the vaccine compositions are administered after a tumor surgery.
  • the antineoplastic therapy includes a chemotherapeutic agent, anti angiogenic agent, a PARP inhibitor, an antibody (e.g., checkpoint inhibitor), a PIPKinase inhibitor, a tyrosine kinase inhibitor, an adoptive cell therapy, or combinations thereof.
  • the antineoplastic therapy includes a radiation therapy, surgery, chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, or a combination of the foregoing.
  • the antineoplastic therapy may be in the form of adjuvant or neoadjuvant therapy.
  • the vaccine compositions are administered before tumor surgery. In some aspects, the vaccine compositions are administered after tumor surgery.
  • the antineoplastic cancer therapy includes the administration of a small molecule enzymatic inhibitor or anti-metastatic agent.
  • the antineoplastic cancer therapy is radiation therapy.
  • the antineoplastic therapy is a therapy targeting the PBK/AKT/mTOR pathway, a HSP90 inhibitor, a tubulin inhibitor, an apoptosis inhibitor, and/or a chemopreventative agent.
  • the antineoplastic therapy may be one or more of the chemotherapeutic agents known in the art.
  • the antineoplastic therapy is a therapy with a checkpoint inhibitor and comprises a therapeutically effective amount of a PD-1 or PD-L1 inhibitor, an inhibitor of CD274, an inhibitor of PDCD1, or an inhibitor of CD80, CD86, CD38, OD 01, TIGIT, C10orf54 (VISTA), ICOS, LAG3, CCL5, CXCL20 or any combination thereof and a pharmaceutically acceptable excipient.
  • the antineoplastic therapy is an anti angiogenic therapy and comprises bevacizumab, aflibercept, everolimus, lenalidomide, lenvatinib mesylate, pazopanib, ramucirumab, sunitinib, sorafenib, axitinib, regorafenib, cabozantinib, thalidomide, and/or vandetanib.
  • the antineoplastic therapy is a PI3Kinase inhibitor therapy and comprises idelalisib, copanlisib, duvelisib, alpelisib, umbralisib, buparlisib, copanlisib, dactolisib, duvelisib, parsaclisib, paxalisib, zandelisib, andor inbavolisib.
  • the antineoplastic therapy is a PARP inhibitor therapy and comprises talazoparib, veliparib, pamiparib, olaparib, rucaparib, CEP 9722, and/or E7016.
  • the antineoplastic therapy is a chemotherapy.
  • chemotherapy refers to the use of drugs to treat cancer.
  • a "chemotherapeutic agent” is used to connote a compound or composition that is administered in the treatment of cancer.
  • chemotherapeutic agents include, but are not limited to, alkylating agents, such as thiotepa and cyclosphosphamide; alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide, and trimethylolomelamine; acetogenins including bullatacin and bullatacinone; a camptothecin including the synthetic analogue topotecan; bryostatin; cally statin; CC-1065 including its adozelesin, carzelesin and bizelesin synthetic analogues; cryptophycins including cryptophycin 1 and cryptophycin 8; dolast
  • the antineoplastic therapy is a radiation therapy.
  • the cancer is selected from the group consisting of ovarian cancer, melanoma, colorectal cancer, liver cancer, pancreatic cancer, brain cancer, prostate cancer, bladder cancer, renal cancer, and hematological cancer.
  • Certain aspects of the disclosure are directed to methods of preventing, reducing the incidence of, attenuating or treating a tumor in a subject, the method comprising administering an effective amount of the composition, pharmaceutical composition, or cancer vaccine disclosed herein.
  • the composition, pharmaceutical composition, or cancer vaccine is administered to the subject by an intramuscular, intradermal, subcutaneous, intralymphatic, intraperitoneal, or intratumoral route of administration.
  • the composition, pharmaceutical composition, or cancer vaccine is administered once, twice, three times, four times, five times, six times, or between seven and twenty times.
  • the composition, pharmaceutical composition, or cancer vaccine is administered more than once in an interval of from 1 day to about 21 days; about 2 days to about 18 days; about 3 days to about 14 days; or about 4 days, about 7 days; about 10 days; about 14 days; about 21 days; about 28 days; about 35 days; about 42 days; about 49 days or about 56 days.
  • the composition, pharmaceutical composition, or vaccine is administered twice in an interval of about 4 days, about 7 days; about 10 days; about 14 days; about 21 days; about 28 days about 35 days or about 42 days.
  • the composition, pharmaceutical composition, or cancer vaccine is administered from about 2 times to about 20 times with intervals of about 7 days; about 10 days; about 14 days; about 21 days; about 28 days about 35 days or about 42 days between each administration.
  • the composition, pharmaceutical composition, or cancer vaccine is administered prophylactically more than once in an interval of from 1 day to about 21 days; about 2 days to about 18 days; about 3 days to about 14 days; or about 4 days, about 7 days; about 10 days; about 14 days; about 21 days; about 28 days; about 35 days; about 42 days; about 49 days or about 56 days.
  • the composition, pharmaceutical composition, or vaccine is administered prophylactically twice in an interval of about 4 days; about 7 days; about 10 days; about 14 days; about 21 days; about 28 days about 35 days or about 42 days.
  • composition, pharmaceutical composition, or cancer vaccine is administered prophylactically from about 2 times to about 20 times with intervals of about 4 days; about 7 days; about 10 days; about 14 days; about 21 days; about 28 days about 35 days or about 42 days between each administration.
  • the composition, pharmaceutical composition, or cancer vaccine is administered therapeutically more than once in an interval of from 1 day to about 21 days; about 2 days to about 18 days; about 3 days to about 14 days; or about 4 days, about 7 days; about 10 days; about 14 days; about 21 days; about 28 days; about 35 days; about 42 days; about 49 days or about 56 days.
  • the composition, pharmaceutical composition, or vaccine is administered therapeutically twice in an interval of about 4 days; about 7 days; about 10 days; about 14 days; about 21 days; about 28 days about 35 days or about 42 days.
  • the composition, pharmaceutical composition, or cancer vaccine is administered therapeutically from about 2 times to about 20 times with intervals of about 4 days; about 7 days; about 10 days; about 14 days; about 21 days; about 28 days about 35 days or about 42 days between each administration.
  • Certain aspects of the disclosure are directed to methods of making a vaccine, the method comprising the steps of: (a) combining the delivery component with the polynucleotide of the composition or pharmaceutical composition disclosed herein, (b) lyophilizing the combined delivery component and polynucleotide to a powder, and (c) reconstituting the powder with a diluent that comprises the adjuvant to form a cancer vaccine solution.
  • FIG. 1 shows a schematic of a vector construct comprising polynucleotide sequences of a gene encoding a tumor-associated antigen (TAA) under the control of promoter 1 (Prom-1), a gene encoding a second tumor-associated antigen (TAA-2) under the control of promoter 2 (Prom-2), and genes encoding interleukin 12 (IL- 12) p35 and IL- 12 p40 under the control of two cytomegalovirus (CMV) promoters in a plasmid backbone.
  • TAA tumor-associated antigen
  • TAA-2 tumor-associated antigen-2
  • IL- 12 interleukin 12
  • CMV cytomegalovirus
  • FIG. 2 shows a schematic of a vector construct comprising polynucleotide sequences of a gene encoding a tumor-associated antigen (TAA) under the control of promoter 1 (Prom-1), a gene encoding a second tumor-associated antigen (TAA-2) under the control of promoter 2 (Prom-2), genes encoding IL- 12 p35 and IL- 12 p40 under the control of two CMV promoters, and a gene encoding major histocompatibility complex class I (MHC I) under the control of promoter Z (Prom-Z) in a plasmid backbone.
  • TAA tumor-associated antigen
  • TAA-2 tumor-associated antigen-2
  • MHC I major histocompatibility complex class I
  • FIG. 3 shows a schematic of a vector construct comprising polynucleotide sequences of a gene encoding a tumor-associated antigen (TAA) under the control of promoter 1 (Prom-1), a gene encoding a second tumor-associated antigen (TAA-2) under the control of promoter 2 (Prom-2), genes encoding IL- 12 p35 and IL- 12 p40 under the control of two CMV promoters, and a gene encoding major histocompatibility complex class II (MHC II) under the control of promoter Z (Prom-Z) in a plasmid backbone.
  • TAA tumor-associated antigen
  • TAA-2 tumor-associated antigen-2
  • MHC II major histocompatibility complex class II
  • FIG. 4 shows a schematic of a vector construct comprising polynucleotide sequences of a gene encoding a tumor-associated antigen (TAA) under the control of promoter 1 (Prom-1), a gene encoding a second tumor-associated antigen (TAA-2) under the control of promoter 2 (Prom-2), and a gene encoding interleukin 2 (IL-2) under the control of a CMV promoter in a plasmid backbone.
  • TAA tumor-associated antigen
  • TAA-2 tumor-associated antigen
  • IL-2 interleukin 2
  • FIG. 5 shows a schematic of a vector construct comprising polynucleotide sequences of a gene encoding a tumor-associated antigen (TAA) under the control of promoter 1 (Prom-1), a gene encoding a second tumor-associated antigen (TAA-2) under the control of promoter 2 (Prom-2), a gene encoding IL-2 under the control of a CMV promoter, and a gene encoding MHC I under the control of promoter Z (Prom-Z) in a plasmid backbone.
  • TAA tumor-associated antigen
  • FIG. 6 shows a schematic of a vector construct comprising polynucleotide sequences of a gene encoding a tumor-associated antigen (TAA) under the control of promoter 1 (Prom-1), a gene encoding a second tumor-associated antigen (TAA-2) under the control of promoter 2 (Prom-2), a gene encoding IL-2 under the control of a CMV promoter, and a gene encoding MHC II under the control of promoter Z (Prom-Z) in a plasmid backbone.
  • TAA tumor-associated antigen
  • FIG. 7 shows a schematic of a vector construct comprising polynucleotide sequences of a gene encoding a tumor-associated antigen (TAA) under the control of promoter 1 (Prom-1), a gene encoding a second tumor-associated antigen (TAA-2) under the control of promoter 2 (Prom-2), a gene encoding IL-2 under the control of a CMV promoter, a gene encoding C-C motif chemokine ligand (CCL) 3 (CCL3) under the control of promoter X (Prom-X), and a gene encoding CCL4 under the control of promoter Y (Prom-Y) in a plasmid backbone.
  • TAA tumor-associated antigen
  • FIG. 8 shows a schematic of a vector construct comprising polynucleotide sequences of a gene encoding a tumor-associated antigen (TAA) under the control of promoter 1 (Prom-1), a gene encoding a second tumor-associated antigen (TAA-2) under the control of promoter 2 (Prom-2), and a gene encoding interleukin 15 (IL- 15) under the control of a CMV promoter in a plasmid backbone.
  • TAA tumor-associated antigen
  • TAA-2 tumor-associated antigen-2
  • IL- 15 interleukin 15
  • FIG. 9 shows a schematic of a vector construct comprising polynucleotide sequences of a gene encoding a tumor-associated antigen (TAA) under the control of promoter 1 (Prom-1), a gene encoding a second tumor-associated antigen (TAA-2) under the control of promoter 2 (Prom-2), a gene encoding IL- 15 under the control of a CMV promoter, and a gene encoding MHC I under the control of promoter Z (Prom-Z) in a plasmid backbone.
  • TAA tumor-associated antigen
  • FIG. 10 shows a schematic of a vector construct comprising polynucleotide sequences of a gene encoding a tumor-associated antigen (TAA) under the control of promoter 1 (Prom-1), a gene encoding a second tumor-associated antigen (TAA-2) under the control of promoter 2 (Prom-2), a gene encoding IL- 15 under the control of a CMV promoter, and a gene encoding MHC II under the control of promoter Z (Prom-Z) in a plasmid backbone.
  • TAA tumor-associated antigen
  • FIG. 11 shows a schematic of a vector construct comprising polynucleotide sequences of a gene encoding a tumor-associated antigen (TAA) under the control of promoter 1 (Prom-1), a gene encoding a second tumor-associated antigen (TAA-2) under the control of promoter 2 (Prom-2), a gene encoding IL- 15 under the control of a CMV promoter, a gene encoding CCL3 under the control of promoter X (Prom-X), and a gene encoding CCL4 under the control of promoter Y (Prom-Y) in a plasmid backbone.
  • TAA tumor-associated antigen
  • FIG. 12 shows a schematic of a vector construct comprising polynucleotide sequences of a gene encoding a tumor-associated antigen (TAA) under the control of promoter 1 (Prom-1), a gene encoding a second tumor-associated antigen (TAA-2) under the control of promoter 2 (Prom-2), a gene encoding CCL3 under the control of promoter X (Prom-X), and a gene encoding CCL4 under the control of promoter Y (Prom-Y) in a plasmid backbone.
  • TAA tumor-associated antigen
  • FIG. 13 shows a schematic of the constructs pVacl-pVac 5.
  • FIGs. 14A-14O show schematics of vectors, including pVac 6 to pVac 17, pVac64, and pVac65 vectors.
  • FIG. 14B shows a schematic of pVac6 comprising a polynucleotide sequence of a gene encoding a tumor-associated antigen (TAA) under the control of a hEFl-HTLV promoter with a SV40 enhancer and a SV40 poly A, a polynucleotide sequence of a gene encoding p40 under the control of a CMV promoter and a SV40 poly A, and a polynucleotide sequence of a gene encoding p35 under the control of a CMV promoter with a SV40 polyA.
  • TAA tumor-associated antigen
  • FIG. 14C shows a schematic of pVac7 comprising a polynucleotide sequence of a gene encoding a tumor-associated antigen (TAA) operably linked to a gene encoding a second tumor-associated antigen (TAA-2) by a IRES sequence and under the control of a hEFl-HTLV promoter with a SV40 polyA, a polynucleotide sequence of a gene encoding p40 under control of a CMV promoter and a SV40 polyA, and a polynucleotide sequence of a gene encoding p35 under control of a CMV promoter and a SV40 polyA.
  • TAA tumor-associated antigen
  • TAA-2 tumor-associated antigen-2
  • FIG. 14D shows a schematic of pVac8 comprising a polynucleotide sequence of a gene encoding a tumor-associated antigen (TAA) under the control of a EF-la promoter and a SV40 polyA.
  • TAA tumor-associated antigen
  • FIG. 14E shows a schematic of pVac9 comprising a polynucleotide sequence of a gene encoding a tumor-associated antigen (TAA) under the control of a EF-la promoter and a SV40 polyA, a polynucleotide sequence of a gene encoding p40 under the control of a CMV promoter and a SV40 polyA, and a polynucleotide sequence of a gene encoding a p35 under the control of a CMV promoter and a SV40 polyA.
  • TAA tumor-associated antigen
  • FIG. 14F shows a schematic of pVaclO comprising a polynucleotide sequence of a gene encoding a tumor-associated antigen (TAA) operably linked to a gene encoding a second tumor-associated antigen (TAA-2) by a IRES sequence and under the control of a EF-la promoter, a polynucleotide sequence of a gene encoding p40 under the control of a CMV promoter and a SV40 polyA, and a polynucleotide sequence of a gene encoding p35 under the control of a CMV promoter and a SV40 polyA.
  • TAA tumor-associated antigen
  • TAA-2 tumor-associated antigen-2
  • FIG. 14G shows a schematic of pVacl 1 comprising a polynucleotide sequence of a gene encoding a tumor-associated antigen (TAA) under the control of a EF- la and a SV40 polyA, a polynucleotide sequence of a gene encoding a second tumor- associated antigen (TAA-2) under the control of a CMV promoter and a SV40 polyA.
  • TAA tumor-associated antigen
  • FIG. 14H shows a schematic of pVacl2 comprising a polynucleotide sequence of a gene encoding a tumor-associated antigen (TAA) under the control of a CMV promoter and a SV40 polyA, and a polynucleotide sequence of a gene encoding a second tumor- associated antigen (TAA-2) under the control of a CMV promoter and a SV40 polyA.
  • FIG. 141 shows a schematic of pVacl3 comprising a polynucleotide sequence of a gene encoding a tumor-associated antigen (TAA) under the control of a EF-la promoter and a SV40 polyA.
  • TAA tumor-associated antigen
  • FIG. 14J shows a schematic of pVacl4 comprising a polynucleotide sequence of a gene encoding a tumor-associated antigen (TAA) under the control of a EF- la promoter and a SV40 polyA.
  • FIG. 14K shows a schematic of pVacl5 comprising a polynucleotide sequence of a gene encoding a tumor-associated antigen (TAA) under the control of a EF-la promoter and SV40 polyA and a polynucleotide sequence of a gene encoding a second tumor-associated antigen (TAA-2) under the control of a EF-la promoter and SV40 polyA.
  • TAA tumor-associated antigen
  • FIG. 14L shows a schematic of pVacl6 comprising a polynucleotide sequence of a gene encoding a tumor-associated antigen (TAA) under the control of a CMV promoter, SV40 enhancer, and SV40 polyA.
  • FIG. 14M shows a schematic of pVacl7 comprising a polynucleotide sequence of a gene encoding a tumor- associated antigen (TAA) under the control of a CMV promoter, SV40 enhancer, and SV40 polyA and a polynucleotide sequence of a gene encoding a second tumor- associated antigen (TAA-2) under the control of a CMV promoter, SV40 enhancer, and SV40 polyA.
  • TAA tumor-associated antigen
  • FIG. 14N shows a schematic of pVac64 comprising a polynucleotide sequence of a gene encoding Trp2 under the control of a CMV promoter with a SV40 enhancer and bGH polyA.
  • FIG. 140 shows a schematic of pVac65 comprising a polynucleotide sequence of a gene encoding human CTAG1B-NYES0-1 under the control of a CMV promoter with a SV40 enhancer and bGH polyA.
  • FIG. 15A is a DNA gel showing uncut pVac64 plasmid DNA or pVac64 plasmid DNA after restriction digestion with Xhol or Sacl.
  • FIG. 15B is a DNA gel showing uncut pVac65 plasmid DNA or pVac65 plasmid DNA after restriction digestion with Kpnl.
  • FIG. 16A is a graph showing the particle size of pVa64 plasmid DNA formulated with BD3.6-oleoyl, BD15-12, Crossamine, or poloxamer.
  • FIG. 16B is a graph showing the particle size of pVac65 plasmid DNA formulated with BD3.6-oleoyl, BD15-12, Crossamine, or poloxamer.
  • FIG. 17A is a graph showing the expression of Trp2 mRNA (FIG. 17A) after transfection of mammalian cells with pVac64 plasmid DNA formulated with BD3.6- oleoyl, BD-15, Crossamine, or poloxamer.
  • FIG. 17B is a graph showing the expression of NYESO-1 mRNA (FIG. 17B) after transfection of mammalian cells with pVac65 plasmid DNA formulated with BD3.6- oleoyl, BD-15, Crossamine, or poloxamer.
  • compositions including pharmaceutical compositions and vaccines, that comprise polynucleotides (e.g., DNA or mRNA), vectors, multi ci str onic mRNA vectors, DNA plasmid vectors of the disclosure to address, for example, the issue of suboptimal immunogenicity often associated with DNA-based cancer vaccine approaches on one or more levels.
  • polynucleotides e.g., DNA or mRNA
  • vectors e.g., multi ci str onic mRNA vectors
  • DNA plasmid vectors of the disclosure to address, for example, the issue of suboptimal immunogenicity often associated with DNA-based cancer vaccine approaches on one or more levels.
  • the composition can comprise polynucleotides that express one or more tumor-associated antigens and, optionally, express one or more immune modifiers (e.g., a cytokine).
  • the composition can comprise polynucleotides that express one or more tumor-associated antigens and interleukin- 12 (IL-12).
  • the composition comprising a polynucleotide encoding at least one tumor-associated antigen and IL- 12 inhibits an IgG response, but not a T cell response.
  • the composition comprising a polynucleotide that expresses one or more tumor-associated antigens further comprises a delivery agent.
  • the composition comprising a polynucleotide that expresses one or more tumor-associated antigens and a cytokine immune modifier (e.g., IL-12) further comprises a delivery agent.
  • the composition optionally comprises an adjuvant.
  • the compositions comprise an adjuvant or combination of adjuvants.
  • the adjuvant can comprise an aluminum or aluminum-salt based adjuvant.
  • the adjuvant can comprise a stimulator of interferon genes (STING) agonist.
  • the adjuvant can comprise an aluminum or aluminum-salt based adujvant and stimulator of interferon genes (STING) agonist.
  • the composition can comprise polynucleotides that co-express one or more immune modifier proteins such as cytokines and/or chemokines that augment the immune responses to the tumor-associated antigens.
  • the polynucleotides can include multiple tumor-associated antigens and/or multiple epitomes of a tumor-associated antigen instead of a single tumor-associated antigen, which can be co-expressed from the vectors disclosed herein to expand the spectrum of immunogenicity.
  • the vectors disclosed herein can be formulated with delivery systems (e.g., a cationic polymer, a poly-inosinic-polycylidylic acid, a poloxamer, or a derivative thereof) that protects the vector or polynucleotide from nuclease degradation and promote its translocation through cell compartments.
  • delivery systems e.g., a cationic polymer, a poly-inosinic-polycylidylic acid, a poloxamer, or a derivative thereof
  • the delivery systems disclosed herein can also be formulated to exhibit an additional adjuvant property to promote mobilization of antigen presenting cells to the site of vaccine delivery and antigen expression, thereby augmenting the uptake of the vaccine vector and the expressed tumor associated antigens into professional antigen presenting cells to elicit MHC Class I and MHC Class II presentation.
  • the compositions comprise an ALUM salt adjuvant.
  • the ALUM salt is selected from aluminum phosphate, aluminum hydroxide, potassium aluminum sulfate [KA1(SO4)2], aluminumcrystalline aluminum oxyhydroxide, aluminumaluminum hydroxyphosphate, amorphous aluminum hydroxyphosphate sulfate, aluminum chloride, aluminum silicate, and a mixture of aluminum hydroxide and magnesium hydroxide, a mixture of aluminum sulfate and sodium hydroxide ora mixture of aluminum sulfate and potassium hydroxide.
  • the adjuvant is MF59 (oil-in-water emulsion of squalene oil).
  • the adjuvant is AS03 (a-tocopherol, squalenem and polysorbate 80 in an oil-in-water emulsion).
  • the composition comprise a STING agonist.
  • the STING agonist is selected from a cyclic di-nucleotides, a non-cyclic di-nucleotide small molecule, an amidobenzimidazole, a nanovaccine, an antibody drug conjugate, a bacterial vector, and an ENPP1 inhibitor.
  • the STING agonist is a cyclic dinucleotide cGMP, cAMP, or cGMP-AMP. In some aspects, the STING agonist is cGMP.
  • the STING agonist is a cylic dinucleotide selected from ADU- S100, MK-1454, SB11285, BMS-986301, BI-STING (BI1387446), JNJ-67544412, 3’3’- cyclic AIMP, and GSK532.
  • the STING agonist is a non-cyclic dinucleotide small molecule selected from DMAXAA, ALG-031048, E7766, JNJ-‘6196, MK-21118, MSA-1, MSA-2, SNX281, SR-717, TAK676, TTI-10001, a Ryvu’s agonist, GF3-002, a Selvita agonist, CDR5500, CS-1010, CS-1018, CS-1020, and [2-ex] MSA-1.
  • the STNG agonist is a nanovaccine selected from PC7A nanoparticles, cGMP-nanoparticles, and ONM-500 nanoparticles.
  • the STING agonist is an antibody-drug conjugate.
  • the antibody drug conjugate is XMT-2056.
  • the STING agonist is an ENPP1 inhibitor selected from MV-626, SR-8314, SR-8291, and SR8541A.
  • the STING agonist is a bacterial vector.
  • the bacterial vector is a nonpathogenic E coli nissle expressing cyclic-di-AMP -producing enzymes.
  • the bacterial vector is SYNB1981.
  • the bacterial vector is an attenutated Salmonella Typhimurium strain enginerred to carry an inhibitory TREX-1 micro RNA.
  • the polynucleotide can comprise a nucleic acid sequence encoding one or more tumor-associated antigens (e.g., NYESO-1, MAGE-A1, MAGE- A2, MAGE-A3, MAGE-A4, MAGE-A9, MAGE-A10, MAGE-CI, MAGE-C2, SSX, HORMAD1, CXorf61, ACTL8, AKAP3,AKAP4, HOM-TES-85, OY-TES-1, HSP70-2, LAGE-1, PIWL1, PIWL2, PIWL3, PIWL4, PLU-l, PLAC1, SPAG9, SCP-1, TAG-1, TAG-2a, TAG-2b, TAG-2c, TRAG-3, KAP4, BAGE, BORIS, CT45, GAGE-1/2, PRAME, PAGE4, SP17, PASD1, p53, ras, B-RafHER-2/neu, mucin 1 (MUC1), epidermal growth
  • the one or more tumor associated antigens comprise tumor associated antigens from two or more tumors.
  • the vector further comprises a nucleic acid sequence encoding one or more immune modifier proteins (e.g., a cytokine).
  • the vector comprises a nucleic acid sequence encoding a tumor-associated antigen and, optionally, a second tumor-associated antigen.
  • the vector comprises a nucleic acid sequence encoding a tumor- associated antigen and a second tumor-associated antigen from a different tumor.
  • the present disclosure is directed to a composition
  • a polynucleotide e.g., a multicistronic DNA plasmid or multi ci str onic mRNA
  • an antigen nucleic acid which encodes a tumor-associated antigen (e.g., NYESO-1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A9, MAGE-A10, MAGE-CI, MAGE-C2, SSX, H0RMAD1, CXorf61, ACTL8, AKAP3,AKAP4, HOM-TES-85, OY- TES-1, HSP70-2, LAGE-1, PIWL1, PIWL2, PIWL3, PIWL4, PLU-l, PLAC1, SPAG9, SCP-1, TAG-1, TAG-2a, TAG-2b, TAG-2c, TRAG-3, KAP4, BAGE, BORIS, CT45, GAGE-1/2, PRAME, P
  • the present disclosure is directed to a composition
  • a composition comprising (a) a polynucleotide (e.g., a multicistronic DNA plasmid or multicistronic mRNA) comprising (i) an antigen nucleic acid which encodes a tumor-associated antigen (e.g., NYESO-1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A9, MAGE-A10, MAGE-CI, MAGE-C2, SSX, H0RMAD1, CXorf61, ACTL8, AKAP3,AKAP4, HOM-TES-85, OY-TES-1, HSP70-2, LAGE-1, PIWL1, PIWL2, PIWL3, PIWL4, PLU-l, PLAC1, SPAG9, SCP-1, TAG-1, TAG-2a, TAG-2b, TAG-2c, TRAG-3, KAP4, BAGE, BORIS, CT45, GAGE-1/2, PRAME, P
  • the present disclosure is directed to a composition
  • a composition comprising (a) a polynucleotide (e.g., a multicistronic DNA plasmid or multi ci str onic mRNA) comprising an antigen nucleic acid which encodes a tumor-associated antigen (e.g.
  • NYESO-1 MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A9, MAGE-A10, MAGE-CI, MAGE-C2, SSX, H0RMAD1, CXorf61, ACTL8, AKAP3,AKAP4, HOM-TES-85, OY- TES-1, HSP70-2, LAGE-1, PIWL1, PIWL2, PIWL3, PIWL4, PLU-l, PLAC1, SPAG9, SCP-1, TAG-1, TAG-2a, TAG-2b, TAG-2c, TRAG-3, KAP4, BAGE, BORIS, CT45, GAGE-1/2, PRAME, PAGE4, SP17, PASD1, p53, ras, B-RafHER-2/neu, mucin 1 (MUC1), epidermal growth factor receptor (EGFR), GP100, prostatic acid phosphatase, prostate-specific membrane antigen (PSMA), mammoglobin-A, carcinoembry
  • omposition comprises (a) a polynucleotide (e.g., a multicistronic DNA plasmid or multicistronic mRNA) comprising an antigen nucleic acid which encodes a tumor-associated antigen (e.g., NYESO-1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A9, MAGE-A10, MAGE-CI, MAGE-C2, SSX, H0RMAD1, CXorf61, ACTL8, AKAP3,AKAP4, HOM-TES-85, OY-TES-1, HSP70-2, LAGE-1, PIWL1, PIWL2, PIWL3, PIWL4, PLU-l, PLAC1, SPAG9, SCP-1, TAG-1, TAG-2a, TAG-2b, TAG-2c, TRAG-3, KAP4, BAGE, BORIS, CT45, GAGE-1/2, PRAME, PAGE4, SP17, PASD1, p53
  • the composition comprises (a) a polynucleotide (e.g., a multi ci stronic DNA plasmid or multi ci str onic mRNA) comprising an antigen nucleic acid which encodes a tumor- associated antigen (e.g, NYESO-1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A9, MAGE-A10, MAGE-CI, MAGE-C2, SSX, H0RMAD1, CXorf61, ACTL8, AKAP3,AKAP4, HOM-TES-85, OY-TES-1, HSP70-2, LAGE-1, PIWL1, PIWL2, PIWL3, PIWL4, PLU-1, PLAC1, SPAG9, SCP-1, TAG-1, TAG-2a, TAG-2b, TAG-2c, TRAG-3, KAP4, BAGE, BORIS, CT45, GAGE-1/2, PRAME, PAGE4, SP17, PASD1, p
  • the composition comprises (a) a polynucleotide (e.g., a multi ci stronic DNA plasmid or multi ci stronic mRNA) comprising an antigen nucleic acid which encodes a tumor-associated antigen (e.g, NYESO-1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A9, MAGE-A10, MAGE-CI, MAGE-C2, SSX, H0RMAD1, CXorf61, ACTL8, AKAP3,AKAP4, HOM-TES-85, OY-TES-1, HSP70-2, LAGE-1, PIWL1, PIWL2, PIWL3, PIWL4, PLU-1, PLAC1, SPAG9, SCP-1, TAG-1, TAG-2a, TAG-2b, TAG-2c, TRAG-3, KAP4, BAGE, BORIS, CT45, GAGE-1/2, PRAME, PAGE4, SP17, PASD1, p53
  • the multi ci stronic DNA or mRNA comprises one tumor- associated antigen and an immune modifier protein.
  • the multicistronic DNA or mRNA comprises at least two tumor- associated antigens.
  • the multicistronic DNA or mRNA comprises at least two tumor-associated antigens and an immune modifier protein.
  • the multicistronic DNA comprises at least two tumor-associated antigens and at least two immune modifier proteins.
  • the antigen nucleic acid (e.g., antigen nucleic acid) of the polynucleotide is operably linked to a promoter.
  • the polynucleotide further comprises one or more nucleic acids encoding an immune modifier protein.
  • the polynucleotide further comprises at least one additional antigen nucleic acid (e.g., second antigen nucleic acid) which encodes at least one additional tumor-associated antigen (e.g., a second tumor-associated antigen or an antigenic fragment thereof) and/or one additional cytokine immune modifier.
  • additional antigen nucleic acid e.g., second antigen nucleic acid
  • tumor-associated antigen e.g., a second tumor-associated antigen or an antigenic fragment thereof
  • the nucleic acid molecules of the present disclosure comprise one or more features that distinguish the present nucleic acid molecules form those that exist in nature e.g., comprising at least one gene encoding a tumor-associated antigen and a heterologous promoter).
  • the expression of multiple tumor-associated antigens expands the spectrum of immunogenicity, while the presence of an adjuvant (e.g., a STING agonist in the composition) and optionally the expression of at least one immune modifier protein augment the immune responses to the multiple tumor-associated antigens.
  • the tumor-associated antigens are selected from the group consisting of NYESO- 1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A9, MAGE-A10, MAGECI, MAGE-C2, SSX, H0RMAD1, CXorf61, ACTL8, AKAP3,AKAP4, HOM-TES-85, OY-TES-1, HSP70-2, LAGE-1, PIWL1, PIWL2, PIWL3, PIWL4, PLU-1, PLAC1, SPAG9, SCP-1, TAG-1, TAG-2a, TAG-2b, TAG-2c, TRAG-3, KAP4, BAGE, BORIS, CT45, GAGE-1/2, PRAME, PAGE4, SP17, PASD1, p53, ras, B-RafHER-2/neu, mucin 1 (MUC1), epidermal growth factor receptor (EGFR), GP100, prostatic acid phosphatase, prostate-specific membrane antigen (MUC1)
  • the composition comprises (i) a polynucleotide comprising: (a) a first nucleic acid encoding a first tumor-associated antigen (e.g., NYESO-1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A9, MAGE-A10, MAGE-CI, MAGE-C2, SSX, H0RMAD1, CXorf61, ACTL8, AKAP3,AKAP4, HOM-TES-85, OY-TES-1, HSP70-2, LAGE-1, PIWL1, PIWL2, PIWL3, PIWL4, PLU-l, PLAC1, SPAG9, SCP-1, TAG-1, TAG-2a, TAG-2b, TAG-2c, TRAG-3, KAP4, BAGE, BORIS, CT45, GAGE-1/2, PRAME, PAGE4, SP17, PASD1, p53, ras, B-RafHER-2/neu, mucin
  • the polynucleotide can include the elements as disclosed in any of FIGs. 1-12.
  • the vector constructs illustrated in any of FIGs 1-12 can modified to replace the “TAA Gene” (a first nucleotide sequence encoding a tumor- associated antigen) and the “TAA-2” (a second nucleotide sequence encoding a tumor- associated antigen) with nucleotide sequences encoding any combinations of tumor- associated antigens or antigenic fragment thereof disclosed herein.
  • the polynucleotide can include the elements as disclosed in any of FIGs. 14A-14O.
  • the vector constructs illustrated in any of FIGs. 14A- 14M can modified to replace the “TAA” (a first nucleotide sequence encoding a tumor- associated antigen) and the “TAA-2” (a second nucleotide sequence encoding a tumor- associated antigen) with nucleotide sequences encoding any combinations of tumor- associated antigens or antigenic fragment thereof disclosed herein.
  • the polynucleotide further comprises a fourth nucleic acid encoding a second immune modifier protein, wherein the fourth nucleic acid is operably linked to a fourth promoter.
  • the polynucleotide can comprise a first nucleic acid encoding a first tumor-associated antigen (e.g., NYESO-1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A9, MAGE-A10, MAGE-CI, MAGE-C2, SSX, H0RMAD1, CXorf61, ACTL8, AKAP3,AKAP4, HOM-TES-85, OY-TES-1, HSP70-2, LAGE-1, PIWL1, PIWL2, PIWL3, PIWL4, PLU-1, PLAC1, SPAG9, SCP-1, TAG-1, TAG-2a, TAG-2b, TAG-2c, TRAG-3, KAP4, BAGE, BORIS, CT45, GAGE-1/2, PRAME, PAGE4, SP17, PASD1, p53, ras, B-RafHER-2/neu, mucin 1 (MUC1), epidermal growth factor receptor
  • the polynucleotide further comprises a second nucleic acid encoding a second tumor-associated antigen (e.g. NYESO-1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A9, MAGE-A10, MAGE-CI, MAGE-C2, SSX, H0RMAD1, CXorf61, ACTL8, AKAP3,AKAP4, HOM- TES-85, OY-TES-1, HSP70-2, LAGE-1, PIWL1, PIWL2, PIWL3, PIWL4, PLU-1, PLAC1, SPAG9, SCP-1, TAG-1, TAG-2a, TAG-2b, TAG-2c, TRAG-3, KAP4, BAGE, BORIS, CT45, GAGE-1/2, PRAME, PAGE4, SP17, PASD1, p53, ras, B-RafHER-2/neu, mucin 1 (MUC1), epidermal growth factor receptor (EGFR),
  • the second nucleic acid is operably linked to the first promoter through an IRES sequence.
  • the first tumor-associated antigen and the second tumor-associated antigen are different portions of the same tumor-associated antigen.
  • the first tumor-associated antigen and the second tumor-associated antigen are different tumor-associated antigens, wherein the different tumor-associated antigens are derived from different tumors.
  • the first tumor-associated antigen and the second tumor-associated antigen are different tumor-associated antigens, wherein the different tumor-associated antigens are derived from the same tumors.
  • the polynucleotide further comprises a second promoter, and the second nucleic acid is operably linked to the second promoter.
  • compositions, vaccines, or pharmaceutical compositions further comprise a delivery component (e.g., a cationic polymer such as a biodegradable cross-linked cationic multi-block copolymer, a PEG-PEI-cholesterol (PPC) lipopolymer, a lipopolyamine, or a lipopolyamine derivative).
  • a delivery component e.g., a cationic polymer such as a biodegradable cross-linked cationic multi-block copolymer, a PEG-PEI-cholesterol (PPC) lipopolymer, a lipopolyamine, or a lipopolyamine derivative.
  • PPC PEG-PEI- cholesterol
  • the PEG-PEI- cholesterol (PPC) lipopolymer has an average PEG:PEI:cholesterol ratio of 2.5: 1 :0.6.
  • the delivery component exhibits an adjuvant property.
  • the adjuvant property of the delivery component promotes mobilization of antigen presenting cells to the site of vaccine delivery and antigen expression, thereby augmenting the uptake of the polynucleotide and the expressed antigens into professional antigen presenting cells to elicit MHC Class I and MHC Class II presentation.
  • the present disclosure is directed to vaccines or compositions comprising (i) a vector (e.g., a multi ci stronic DNA plasmid vector or a multi ci str onic messenger RNA (mRNA) vector) comprising a nucleic acid sequence encoding one or more tumor associated antigens (e.g., NYESO-1, MAGE-A1, MAGE- A2, MAGE-A3, MAGE-A4, MAGE-A9, MAGE-A10, MAGE-CI, MAGE-C2, SSX, H0RMAD1, CXorf61, ACTL8, AKAP3,AKAP4, HOM-TES-85, OY-TES-1, HSP70-2, LAGE-1, PIWL1, PIWL2, PIWL3, PIWL4, PLU-1, PLAC1, SPAG9, SCP-1, TAG-1, TAG-2a, TAG-2b, TAG-2c, TRAG-3, KAP4, BAGE, BORIS, CT45, GAGE-1/2,
  • the vector further comprises a nucleic acid sequence encoding one or more immune modifier proteins. In some aspects, the vector comprises a nucleic acid sequence encoding a tumor-associated antigen and, optionally, a second tumor-associated antigen. In some aspects, the vector comprises a nucleic acid sequence encoding a tumor associated antigen and a second tumor-associated antigen from a different tumor.
  • Some aspects relate to methods of eliciting humoral and/or cellular immune response against a tumor (e.g., an ovarian cancer, melanoma, colorectal cancer, liver cancer, pancreatic cancer, brain cancer, prostate cancer, bladder cancer, renal cancer, or hematological cancer) following in vivo administration of a vector or composition of the disclosure.
  • a tumor e.g., an ovarian cancer, melanoma, colorectal cancer, liver cancer, pancreatic cancer, brain cancer, prostate cancer, bladder cancer, renal cancer, or hematological cancer
  • the present disclosure is also directed to methods of inducing an immune response in a subject comprising administering an effective amount of any composition, pharmaceutical composition, vaccine, polynucleotide, or vector disclosed herein to the subject.
  • the present disclosure is also directed to methods of preventing, reducing the incidence of, attenuating or treating a tumor in a subject comprising administering an effective amount of any composition, polynucleotide, vector, pharmaceutical composition, or vaccine disclosed herein to the subject.
  • the tumor is an ovarian cancer, melanoma, colorectal cancer, liver cancer, pancreatic cancer, brain cancer, prostate cancer, bladder cancer, renal cancer, or hematological cancer.
  • the present disclosure is also directed to methods of making any composition, pharmaceutical composition, or vaccine disclosed herein.
  • the term “approximately” or “about” is applied herein to a particular value, the value without the term “approximately” or “about is also disclosed herein.
  • any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.
  • nucleic acids can be used interchangeably and refer to the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine; "RNA molecules”, including mRNA) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; "DNA molecules”), or any phosphoester analogs thereof, such as phosphorothioates and thioesters, in either single stranded form, or a double-stranded helix.
  • ribonucleosides adenosine, guanosine, uridine or cytidine
  • RNA molecules including mRNA
  • deoxyribonucleosides deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine
  • DNA molecules or any phosphoester analogs thereof, such as phospho
  • Single stranded nucleic acid sequences refer to single-stranded DNA (ssDNA) or single-stranded RNA (ssRNA). Double stranded DNA- DNA, DNA-RNA and RNA-RNA helices are possible.
  • nucleic acid molecule and in particular DNA or RNA molecule, refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear or circular DNA molecules (e.g., restriction fragments), plasmids, supercoiled DNA and chromosomes.
  • a "recombinant DNA molecule” is a DNA molecule that has undergone a molecular biological manipulation.
  • DNA includes, but is not limited to, cDNA, genomic DNA, DNA plasmid, synthetic DNA, and semi-synthetic DNA.
  • a "nucleic acid composition" of the disclosure comprises one or more nucleic acids as described herein.
  • RNA can be obtained by transcription of a DNA-sequence, e.g., inside a cell. In eukaryotic cells, transcription is typically performed inside the nucleus or the mitochondria. In vivo, transcription of DNA usually results in premature RNA, which has to be processed into messenger RNA (mRNA). Processing of the premature RNA, e.g., in eukaryotic organisms, comprises a variety of different posttranscriptional-modifications such as splicing, 5'-capping, polyadenylation, export from the nucleus or the mitochondria and the like. The sum of these processes is also called maturation of RNA.
  • mRNA messenger RNA
  • the mature mRNA usually provides the nucleotide sequence that can be translated into an amino acid sequence of a particular peptide, protein, or protein antigen.
  • a mature mRNA comprises a 5' cap, optionally a 5'-UTR, an open reading frame, optionally a 3'-UTR, and a poly(A) sequence.
  • multicistronic mRNA or “multicistronic mRNA vector,” as used herein, refers to an mRNA having two or more open reading frames.
  • An open reading frame in this context is a sequence of codons that is translatable into a polypeptide or protein.
  • the term “5'-cap,” as used herein, refers to an entity, typically a modified nucleotide entity, which generally “caps” the 5'-end of a mature mRNA.
  • a 5'-cap can typically be formed by a modified nucleotide, particularly by a derivative of a guanine nucleotide.
  • the 5'-cap is linked to the 5'-terminus via a 5'-5'-triphosphate linkage.
  • a 5'-cap can be methylated, e.g., m7GpppN, wherein N is the terminal 5' nucleotide of the nucleic acid carrying the 5'-cap, typically the 5'-end of an RNA.
  • poly(A) sequence also called “poly(A) tail” or “3'-poly(A) tail,” is typically understood to be a sequence of adenine nucleotides, e.g., of up to about 400 adenine nucleotides.
  • a poly(A) sequence can be located at the 3' end of an mRNA.
  • a poly(A) sequence can also be located within an mRNA or any other nucleic acid molecule, such as, e.g., in a vector, for example, in a vector serving as template for the generation of an RNA, preferably an mRNA, e.g., by transcription of the vector.
  • a poly (A) sequence is present in the 3'-UTR of the mRNA as defined herein.
  • a 3'-UTR sequence is part of an mRNA, which is located between the protein coding region (i.e. the open reading frame) and the 3' terminus of the mRNA molecule. If a 3 '-terminal poly(A) sequence ('poly(A) tail') was added to the RNA (e.g. by polyadenylation), then the term 3'-UTR can refer to that part of the molecule, which is located between the protein coding region and the 3'-terminal poly(A) sequence.
  • a 3'-UTR can also comprise a poly(A) sequence (e.g., a poly(A) sequence which is not located at the very 3' terminus of the RNA molecule).
  • a 3'-UTR of the mRNA is not translated into an amino acid sequence.
  • the 3'-UTR sequence is generally encoded by the gene, which is transcribed into the respective mRNA during the gene expression process.
  • the genomic sequence is first transcribed into pre-mature mRNA, which comprises optional introns.
  • the pre-mature mRNA is then further processed into mature mRNA in a maturation process.
  • This maturation process comprises the steps of 5' capping, splicing the pre-mature mRNA to excise optional introns and modifications of the 3 '-end, such as polyadenylation of the 3 '-end of the pre-mature mRNA and optional endo-/ or exonuclease cleavages etc.
  • a 3'-UTR corresponds to the sequence of a mature mRNA, which is located 3' to the stop codon of the protein coding region (e.g., immediately 3' to the stop codon of the protein coding region), and which extends to the 3' terminus of the RNA molecule or to the 5'-side of a 3' terminal poly(A) sequence (e.g., to the nucleotide immediately 5' to the 3' terminus or immediately 5' to the 3' terminal poly(A) sequence).
  • the term "corresponds to" means that the 3'-UTR sequence can be an RNA sequence, such as in the mRNA sequence used for defining the 3'-UTR sequence, or a DNA sequence, which corresponds to such RNA sequence.
  • a 3'-UTR of a gene such as “3'-UTR of alpha or beta globin” is the sequence, which corresponds to the 3'-UTR of the mature mRNA derived from this gene, i.e. the mRNA obtained by transcription of the gene and maturation of the pre-mature mRNA.
  • the term "3'-UTR of a gene” encompasses the DNA sequence and the RNA sequence of the 3'-UTR.
  • the 3'-UTR is derived from a gene that relates to an mRNA with an enhanced half-like (i.e., that provides a stable mRNA), for example a 3'-UTR of a gene selected from the group consisting of: albumin gene, an a-globin gene, a P-globin gene, a tyrosine hydroxylase gene, a lipoxygenase gene, and a collagen alpha gene, such as a collagen alpha 1(1) gene.
  • a 5'-UTR is typically understood to be a particular section of messenger RNA (mRNA). It is located 5' of the open reading frame of the mRNA. In some aspect, the 5'- UTR starts with the transcriptional start site and ends one nucleotide before the start codon of the open reading frame.
  • the 5'-UTR can comprise elements for controlling gene expression, also called regulatory elements. Such regulatory elements can be, for example, ribosomal binding sites or a 5'-Terminal Oligopyrimidine Tract.
  • the 5'-UTR can be posttranscriptionally modified, for example by addition of a 5'-cap.
  • a 5'-UTR corresponds to the sequence of a mature mRNA which is located between the 5' cap and the start codon.
  • the 5'-UTR corresponds to the sequence which extends from a nucleotide located 3' to the 5'-cap (e.g., from the nucleotide located immediately 3' to the 5 'cap) to a nucleotide located 5' to the start codon of the protein coding region (e.g., to the nucleotide located immediately 5' to the start codon of the protein coding region).
  • the nucleotide located immediately 3' to the 5' cap of a mature mRNA typically corresponds to the transcriptional start site.
  • the term “corresponds to” means that the 5'-UTR sequence can be an RNA sequence, such as in the mRNA sequence used for defining the 5'-UTR sequence, or a DNA sequence which corresponds to such RNA sequence.
  • the term "a 5'-UTR of a gene” is the sequence, which corresponds to the 5'-UTR of the mature mRNA derived from this gene.
  • transfecting refers to the transport of nucleic acids from the environment external to a cell to the internal cellular environment, with particular reference to the cytoplasm and/or cell nucleus.
  • nucleic acids can be delivered to cells either after being encapsulated within or adhering to one or more cationic polymer/nucleic acid complexes or being entrained therewith.
  • Particular transfecting instances deliver a nucleic acid to a cell nucleus.
  • Nucleic acids include DNA and RNA as well as synthetic congeners thereof.
  • nucleic acids include missense, antisense, nonsense, as well as protein producing nucleotides, on and off and rate regulatory nucleotides that control protein, peptide, and nucleic acid production.
  • they can be genomic DNA, cDNA, mRNA, tRNA, rRNA, hybrid sequences or synthetic or semi-synthetic sequences, and of natural or artificial origin.
  • the nucleic acid can be variable in size, ranging from oligonucleotides to chromosomes.
  • These nucleic acids can be of human, animal, vegetable, bacterial, viral, or synthetic origin. They can be obtained by any technique known to a person skilled in the art.
  • biodegradable or “biodegradation” is defined as the conversion of materials into less complex intermediates or end products by solubilization hydrolysis, or by the action of biologically formed entities which can be enzymes and other products of the organism.
  • peptide means peptides of any length and includes proteins.
  • polypeptide and oligopeptide are used herein without any particular intended size limitation, unless a particular size is otherwise stated.
  • a "derivative" of a carbohydrate includes, for example, an acid form of a sugar, e.g. glucuronic acid; an amine of a sugar, e.g. galactosamine; a phosphate of a sugar, e.g. mannose-6-phosphate; and the like.
  • inverted terminal repeat refers to a single stranded sequence of nucleotides followed downstream by its reverse complement.
  • the intervening sequence of nucleotides between the initial sequence and the reverse complement can be any length including zero.
  • administering refers to the physical introduction of a therapeutic agent e.g., nucleic acid molecules, vectors, compositions, and pharmaceutical compositions described herein) to a subject, using any of the various methods and delivery systems known to those skilled in the art.
  • a therapeutic agent e.g., nucleic acid molecules, vectors, compositions, and pharmaceutical compositions described herein
  • Exemplary routes of administration include intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intrasterna, oral, rectal, topical, epidermal, mucosal, intranasal, vaginal, rectal, sublingual administration, and combinations thereof.
  • Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods.
  • Treatment refers to any type of intervention or process performed on, or the administration of an active agent to, a subject with the objective of reversing, alleviating, ameliorating, inhibiting, slowing down, or preventing the onset, progression, development, severity, or recurrence of a symptom, complication, condition, or biochemical indicia associated with a disease (e.g., cancer).
  • a disease e.g., cancer
  • a "therapeutically effective amount,” “effective amount,” “therapeutic dose,” “effective dose,” or “effective dosage,” as used herein, means an amount or a dose that achieves a therapeutic goal, as described herein.
  • a therapeutically effective amount etc. can be administered in a single dose, or can be achieved by administration of multiple doses (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more doses).
  • the ability of a therapeutic agent to promote disease regression or inhibit the development or recurrence of the disease can be evaluated using a variety of methods known to the skilled practitioner, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by assaying the activity of the agent in in vitro assays.
  • the term “antineoplastic therapy”, refers to any therapeutic agent that is used to treat a neoplastic disease (e.g., cancer or noncancerous tumor).
  • the antineoplastic therapy includes, but is not limited to, a chemotherapeutic agent (list several chemo), an anti angiogenic agent (e.g., bevacizumab), a PARP inhibitor, an antibody (e.g., checkpoint inhibitor), a PIPKinase in hibitor, a tyrosine kinase inhibitor, or an adoptive cell therapy.
  • the terms “prevent,” “preventing,” “prevention,” “prophylactic treatment,” and the like refer to reducing the probability of developing a disease or condition in a subject, who does not have, but is at risk of or susceptible to developing a disease or condition.
  • an adjuvant refers to any component which improves the body's response to a vaccine.
  • an adjuvant can be ALUM, MF59 (oil-in- water emulsion of squalene oil), AS03 (a-tocopherol, squalenem and polysorbate 80 in an oil-in-water emulsion) or a STING agonist.
  • the term “ALUM” or “Alum” as use herein refers to an aluminum or aluminum-salt.
  • the ALUM is an aluminum or aluminum-salt based adjuvant, e.g., aluminum phosphate, aluminum hydroxide, potassium aluminum sulfate [KA1(SO4)2], aluminumcrystalline aluminum oxyhydroxide, aluminum hydroxyphosphate, amorphous aluminum hydroxyphosphate sulfate, aluminum chloride, aluminum silicate, and a mixture of aluminum hydroxide and magnesium hydroxide, a mixture of aluminum sulfate and sodium hydroxide ora mixture of aluminum sulfate and potassium hydroxide.
  • An ALUM can be an aluminum hydroxide gel or aluminum phosphate gel.
  • STING refers to the stimulator of interferon genes protein that is a pattern recognition receptor (PRR) that senses cyclic dinucleotides and induces the expression of type I interferon canonically via dendritic cells.
  • PRR pattern recognition receptor
  • STING agonist refers to a molecule that induces a STING pathway.
  • vaccines refer to an immunogenically active composition for the prophylaxis and/or treatment of diseases. Accordingly, in some aspects, vaccines are medicaments which comprise or deliver antigens and are intended to be used in humans or animals for generating specific defense and protective substance by vaccination
  • cancer vaccine refers to a vaccine that either treats existing cancer or prevents the development of cancer.
  • a cancer vaccine can use a patient’s pre-cancerous or cancer cells, separate proteins of the precancerous cells or cancer cells and immunize the patient against those proteins as antigens using a vaccine composition described herein.
  • tumor or “neoplasm” or “neoplastic disease” refers to an abnormal mass of tissue that forms when cells grow and divide more than they should or do not die when they should. Tumors may be benign (non-cancerous) or malignant (cancer). Benign tumors do not spread into, or invade nearby tissues or other parts of the body.
  • cancer refers to any disease characterized by abnormal cells dividing uncontrollably and spreading into, or invading, nearby tissues and/or other parts of a body through either the blood system or the lymph system.
  • the term “inducing immunity” or “immunogenically active” refers to the ability to stimulate an immune response, /. ⁇ ., to stimulate the production of antibodies, particularly humoral antibodies, or to stimulate a cell-mediated response.
  • the effective immunizing amount of the immunogenically active component(s) of this disclosure can vary and can be any amount sufficient to evoke an immune response and provide a protective immune response against a tumor.
  • a dosage unit comprising a polynucleotide (e.g., plasmid DNA) of the disclosure is contemplated. At least one dosage unit per patient is contemplated herein as a vaccination regimen. In some embodiments, two or more dosage units can be useful. The skilled artisan will quickly recognize that a particular quantity of vaccine composition per dosage unit, as well as the total number of dosage units per vaccination regimen, can be optimized, so long as an effective immunizing amount of the virus or a component thereof is ultimately delivered to the subject.
  • an “immunological response” to a substance such as a composition or vaccine is the development in the subject of a cellular and/or antibody-mediated immune response to a composition or vaccine of interest.
  • an “immunological response” includes but is not limited to one or more of the following effects: the production of antibodies, B cells, helper T cells, and/or cytotoxic T cells, directed specifically to an antigen or antigens included in the composition or vaccine of interest.
  • the subject can display either a therapeutic or protective immunological response so the tumor growth will be reduced and/or the clinical severity of the disease reduced. In some aspects, such protection can be demonstrated by either a reduction or lack of symptoms normally displayed by a tumor bearing subject, a quicker recovery time and/or a lowered tumor burden in the infected subject.
  • a “tumor-associated antigen” or “TAA” of the disclosure can be a protein or any other molecule that is found only on tumor cells and not, or to a lesser extent, on normal cells.
  • the tumor-associated antigens provided herein are proteins or polypeptides, or antigenic fragments thereof.
  • the tumor-associated antigens are only expressed on cancer cells and not present on normal cells.
  • the tumor-associated antigens are express on tumor cells and normal cells, but the expression of the tumor-associated antigens is higher on tumor cells.
  • a tumor-associated antigen can be, for example, a NYESO-1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE- A4, MAGE-A9, MAGE-A10, MAGE-CI, MAGE-C2, SSX, H0RMAD1, CXorf61, ACTL8, AKAP3,AKAP4, HOM-TES-85, OY-TES-1, HSP70-2, LAGE-1, PIWL1, PIWL2, PIWL3, PIWL4, PLU-1, PLAC1, SPAG9, SCP-1, TAG-1, TAG-2a, TAG-2b, TAG-2c, TRAG-3, KAP4, BAGE, BORIS, CT45, GAGE-1/2, PRAME, PAGE4, SP17, PASD1, p53, ras, B-RafHER-2/neu, mucin 1 (MUC1), epidermal growth factor receptor (EGFR), GP100, prostatic acid phosphatase, prostate-specific membrane antigen (PSMA),
  • the antigenic polypeptides of the disclosure can be full-length polypeptides or active fragments or variants thereof.
  • the term “active fragments” or “active variants” or “antigenic fragments” refers to fragments or variants that retain all or some of the antigenic nature of the polypeptide.
  • the present disclosure encompasses any tumor associated polypeptide, antigen, epitope or immunogen that elicits an immunogenic response in a subject.
  • the tumor associated polypeptide, antigen, epitope or immunogen can be any tumor associated polypeptide, antigen, epitope or immunogen, such as, but not limited to, a protein, peptide or fragment or variant thereof, that elicits, induces or stimulates a response in a subject.
  • the tumor associated polypeptide, antigen, epitope or immunogen can be derived from any tumor including, but not limited to, an ovarian cancer, melanoma, colorectal cancer, liver cancer, pancreatic cancer, brain cancer, prostate cancer, bladder cancer, renal cancer, or hematological cancer.
  • epitope refers to the site on an antigen or hapten to which specific B cells and/or T cells respond.
  • the term is also used interchangeably with “antigenic determinant” or “antigenic determinant site.”
  • Antibodies that recognize the same epitope can be identified in a simple immunoassay showing the ability of one antibody to block the binding of another antibody to a target antigen.
  • the term "pharmaceutical agent”, “pharmaceutical composition” or “drug” or any other similar term means any chemical or biological material or compound suitable for administration by the methods previously known in the art and/or by the methods taught in the present disclosure, which induce a desired biological or pharmacological effect, which can include but are not limited to (1) having a prophylactic effect on the organism and preventing an undesired biological effect such as preventing tumor growth or spread of cancer to other tissues, (2) alleviating a condition caused by a disease, for example, alleviating pain or inflammation caused as a result of disease, and/or (3) either alleviating, reducing, or completely eliminating a disease (e.g., cancer) from the organism.
  • the effect can be local or it can be systemic.
  • a “pharmaceutically acceptable carrier” refers to a carrier that can be administered to a subject, together with an agent, and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the agent.
  • the pharmaceutically acceptable carrier is an aqueous solvent, i.e., a solvent comprising water, optionally with additional co-solvents.
  • exemplary pharmaceutically acceptable carriers include water, buffer solutions in water (such as phosphate-buffered saline (PBS), and 5% dextrose in water (D5W).
  • the aqueous solvent further comprises dimethyl sulfoxide (DMSO), e.g., in an amount of about 1-4%, or 1-3%.
  • the pharmaceutically acceptable carrier is isotonic (i.e., has substantially the same osmotic pressure as a body fluid such as plasma).
  • a “subject” includes any human or non-human animal.
  • nonhuman animal includes, but is not limited to, vertebrates such as nonhuman primates, sheep, dogs, and rodents such as mice, rats, and guinea pigs.
  • the subject is a human.
  • the terms "subject” and “patient” are used interchangeably herein.
  • the subject suffers from a tumor or cancer.
  • telomere a gene product
  • mRNA messenger RNA
  • expression produces a "gene product.”
  • a gene product can be either a nucleic acid, e.g., a messenger RNA produced by transcription of a gene, or a polypeptide which is translated from a transcript.
  • Gene products described herein can further include nucleic acids with post transcriptional modifications, e.g., polyadenylation or splicing, or polypeptides with post translational modifications, e.g., methylation, glycosylation, the addition of lipids, association with other protein subunits, or proteolytic cleavage.
  • post transcriptional modifications e.g., polyadenylation or splicing
  • polypeptides with post translational modifications e.g., methylation, glycosylation, the addition of lipids, association with other protein subunits, or proteolytic cleavage.
  • the term “5'” or “5 prime” refers to the 5' end of a nucleic acid or nucleic acid sequence
  • the term “3'” or “3 prime” refer to the 3' end of nucleic acid or nucleic acid sequence.
  • nucleic acids refer to two or more sequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned (introducing gaps, if necessary) for maximum correspondence, not considering any conservative amino acid substitutions as part of the sequence identity.
  • percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software are known in the art that can be used to obtain alignments of amino acid or nucleotide sequences.
  • promoter refers to DNA sequence capable of controlling the expression of a coding sequence or functional RNA.
  • a coding sequence is located 3' to a promoter sequence. Promoters can be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters can direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental or physiological conditions.
  • Promoters that cause a gene to be expressed in most cell types at most times are commonly referred to as “constitutive promoters.” Promoters that cause a gene to be expressed in a specific cell type are commonly referred to as “cell-specific promoters” or “tissue-specific promoters.” Promoters that cause a gene to be expressed at a specific stage of development or cell differentiation are commonly referred to as “developmentally-specific promoters” or “cell differentiation-specific promoters.” Promoters that are induced and cause a gene to be expressed following exposure or treatment of the cell with an agent, biological molecule, chemical, ligand, light, or the like that induces the promoter are commonly referred to as “inducible promoters” or “regulatable promoters.” It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths can have identical promoter activity.
  • operably linked refers to genetic elements that are joined together in a manner that enables them to carry out their normal functions.
  • a gene is operably linked to a promoter when its transcription is under the control of the promoter and this transcription results in the production of the product encoded by the gene.
  • immune modifier protein refers to a protein that augments the immune response to a one or more antigens.
  • immune modifier proteins include, but are not limited to, a cytokine, a chemokine, major histocompatibility complex (MHC) class I (MHC I), MHC class II (MHC II), human leukocyte antigen (HLA)-DR isotype (HLA-DR), CD80, CD86, and any combination thereof.
  • the cytokine immune modifier proteins include, but are not limited to, interleukin (IL) 2 (IL-2), IL-12 p35, IL-12 p40, IL-12 p70, IL-15, IL-18, tumor necrosis factor alpha (TNFa), granulocyte-macrophage colony-stimulating factor (GM- CSF), interferon (IFN) a (IFN-a), and IFN-p.
  • IL interleukin
  • IL-2 interleukin 2
  • IL-12 p35 IL-12 p40
  • IL-12 p70 IL-15
  • IL-18 tumor necrosis factor alpha
  • GM- CSF granulocyte-macrophage colony-stimulating factor
  • IFN interferon
  • the chemokine immune modifier proteins include, but are not limited to, C-C motif chemokine ligand (CCL) 3 (CCL3), CCL4, CCL5, CCL21, CCL28, C-X-C motif chemokine ligand (CXCL) 10 (CXCL10), and any combination thereof.
  • the immune modifier proteins include one or more unmethylated cytosine-guanine dinucleotide-containing oligodeoxynucleotide (CpG).
  • the immune modifier proteins include one or more concatamers of non-coding 5'-C-phosphate-G-3' (CpG) dinucleotides.
  • the one or more concatamers of non-coding CpG dinucleotides activate the Tolllike receptor 9 (TLR9) signaling pathway.
  • the one or more concatamers of non-coding CpG dinucleotides comprise one or more concatamers of non-coding CpG dinucleotides previously reported in Bauer, A. et al., Nucleic Acids Research 38(12):3891-908 (2010); Cornelie, S. et al., Journal of Biological Chemistry
  • an immune modifier protein as disclosed herein can include a combination of immune modifier proteins, e.g., a cytokine or chemokine protein or their coding sequence either co-expressed from the same plasmid as the antigen or from a different plasmid.
  • immune modifier proteins e.g., a cytokine or chemokine protein or their coding sequence either co-expressed from the same plasmid as the antigen or from a different plasmid.
  • targeting ligand is intended to refer to ligands conjugated to a polymer either directly or via one or more spacer molecules. In some aspects, only a small portion of the available amino groups of the polymer is coupled to the ligand. In some aspects, the targeting ligands conjugated to the polymers direct the polymers-nucleic acid complex to bind to specific target cells and penetrate into such cells (e.g., epithelial cells, endothelial cells, hematopoietic cells, and the like).
  • the target ligands can also be an intracellular targeting element, enabling the transfer of the nucleic acid/drug to be guided towards certain favored cellular compartments (mitochondria, nucleus, and the like).
  • the ligand is a polypeptide, folate, and an antigen.
  • the polypeptide ligand is a glycoprotein (e.g., transferrin or asialoorosomucoid (ASOR)), an antibody, an antibody fragment, a cell receptor, a cytokine receptor, or a growth factor receptor (e.g., epidermal growth factor receptor).
  • the antigen ligand is a tumor-associated antigen, a viral antigen, a bacterial antigen, or a parasite antigen.
  • the ligand is a fusogenic agent (e.g., polymixin B and hemaglutinin HA2), a lysosomotrophic agent, or a nucleus localization signal (NLS) (e.g., T-antigen, and the like).
  • the ligand is a sugar moiety coupled to an amino group.
  • the sugar moiety is a mono- or oligo-saccharide, such as galactose, glucose, fucose, fructose, lactose, sucrose, mannose, cellobiose, nytrose, triose, dextrose, trehalose, maltose, galactosamine, glucosamine, galacturonic acid, glucuronic acid, and gluconic acid.
  • a mono- or oligo-saccharide such as galactose, glucose, fucose, fructose, lactose, sucrose, mannose, cellobiose, nytrose, triose, dextrose, trehalose, maltose, galactosamine, glucosamine, galacturonic acid, glucuronic acid, and gluconic acid.
  • antibody includes molecules or active fragments (i.e., antigen binding fragments) of molecules that bind to antigens. These active fragments can be derived from an antibody of the present disclosure by a number of techniques. For further description of general techniques for the isolation of active fragments of antibodies, see for example, Khaw, B. A. et al. J. Nucl. Med. 23: 1011-1019 (1982).
  • antibody also includes bispecific and chimeric antibodies and antibodies in nonmammalian species.
  • biodegradable linker or “biofunctional biodegradable linker,” as used herein, refers to a biodegradable linker containing ester, amide, disulfide, and/or phosphate linkages that is used to cross-link cationic multi-block copolymers.
  • the biodegradable linker is hydrophilic and comprises a biodegradable linkage comprising a disulfide bond.
  • the biodegradable linker is a dithiodipropionyl linker.
  • vector is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked; or an entity comprising such a nucleic acid molecule capable of transporting another nucleic acid.
  • the vector is a "plasmid,” which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.
  • the vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome.
  • such vectors include, but are not limited to: an adenoviral vector, an adeno- associated virus (AAV) vector, retroviral vector, a lentiviral vector, poxvirus vector, a baculovirus vector, a herpes viral vector, simian virus 40 (SV40), cytomegalovirus (CMV), mouse mammary tumor virus (MMTV), and Moloney murine leukemia virus.
  • AAV adeno- associated virus
  • retroviral vector retroviral vector
  • a lentiviral vector poxvirus vector
  • a baculovirus vector a herpes viral vector
  • SV40 simian virus 40
  • CMV cytomegalovirus
  • MMTV mouse mammary tumor virus
  • Moloney murine leukemia virus Moloney murine leukemia virus.
  • Certain vectors, or polynucleotides that are part of vectors are capable of autonomous replication in a host cell into which they are introduced (e.g.,
  • vectors e.g., non-episomal mammalian vectors
  • vectors can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • certain vectors are capable of directing the expression of genes to which they are operatively linked.
  • Such vectors are referred to herein as "recombinant expression vectors” (or simply, “expression vectors”).
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and vector can sometimes be used interchangeably, depending on the context, as the plasmid is the most commonly used form of vector.
  • viral vectors e.g., replication defective retroviruses, poxviruses, herpesviruses, baculoviruses, adenoviruses and adeno-associated viruses
  • viral vectors e.g., replication defective retroviruses, poxviruses, herpesviruses, baculoviruses, adenoviruses and adeno-associated viruses
  • polyxamer or “poloxamer backbone” refers to molecules having the general formula HO — (C H-tO CsHeO C H-tOk — H in which a and c are approximately equal. See, Handbook of Biodegradable Polymers, Chapter 12' "The Poloxamers: Their Chemistry andMedical Applications” authored by Lorraine E. Reeve. See also U.S. Publ. No. 2010/0004313, which is herein incorporated by reference in its entirety. Because the poloxamers are the products of a sequential series of reactions, the chain lengths of individual poloxamer blocks are statistical distributions about the average chain length.
  • the number of ethyleneoxy groups (a and c) and the number of propylenoxy groups (Z>) are meant to be averages.
  • Poloxamers are generally based on an amphiphilic triblock copolymer of ethylene oxide and propylene oxide, having a central hydrophobic chain of polypropylene oxide flanked by two hydrophilic chains of polyethylene oxide. Because the lengths of the polymer blocks of a poloxamer backbone can vary between various polymeric constructs, many different poloxamers are considered to be within the scope of the present disclosure. In one aspect, for example, the average molecular weight of the pol oxamer backbone can range from about 100 to about 100,000 Dalton.
  • the average molecular weight of the pol oxamer backbone can range from about 500 to about 50,000 Dalton. In yet another aspect, the average molecular weight of the pol oxamer backbone can range from about 1000 to about 20,000 Dalton.
  • the poloxamer backbone can also be described in terms of a ratio of ethylene oxide to propylene oxide. In another aspect, the ratio of ethylene oxide to propylene eoxide is from about 20: 1 to about 1 :20. For example, in one aspect the ratio of ethylene oxide to propylene oxide is from about 5: 1 to about 1 :5.
  • BD15-12 rerfers to a PEI-based co-block polymer that utilized biodegradable linkages.
  • compositions e.g., pharmaceutical compositions or vaccines.
  • the compositions comprise a polynucleotide as disclosed herein and an adjuvant.
  • the compositions comprise a polynucleotide as disclosed herein and a STING agonist.
  • the compositions comprise a polynucleotide as disclosed herein, an adjuvant, and a STING agonist.
  • the compositions further comprise a delivery component.
  • compositions e.g., pharmaceutical compositions or vaccines
  • the compositions comprise a polynucleotide as described herein, an adjuvant, and a delivery component.
  • compositions e.g., pharmaceutical compositions or vaccines
  • the compositions comprise a polynucleotide as described herein, a STING agonist, and a delivery component.
  • compositions e.g., pharmaceutical compositions or vaccines
  • the compositions comprise a polynucleotide as described herein, an adjuvant, a STING agonist, and a delivery component.
  • the adjuvant is selected from potassium aluminum sulfate [KA1(SO4)2], aluminum hydroxide, crystalline aluminum oxyhydroxide, aluminum phosphate, aluminum hydroxyphosphate, amorphous aluminum hydroxyphosphate sulfate, aluminum chloride, aluminum silicate, and a composition comprising aluminum hydroxide and magnesium hydroxide.
  • the aluminum adjuvant is an aluminum hydroxide gel or aluminum phosphate gel.
  • the aluminum adjuvant is a mixture of aluminum sulfate and sodium or potassium hydroxide.
  • the immune stimulator is a STING agonist.
  • STING agonist is cyclic AMP (cAMP).
  • STING agonist is cyclic- GMP-AMP (cGAMP).
  • the STING agonist is an amidobenzimidazole (ABZI).
  • the STING agonist is a cylic dinucleotide selected from ADU-
  • the STING agonist is a non-cyclic dinucleotide small molecule selected from DMAXAA, ALG-031048, E7766, JNJ-‘6196, MK-21118, MSA-1, MSA-2, SNX281, SR-717, TAK676, TTI-10001, a Ryvu’s agonist, GF3-002, a Selvita agonist, CDR5500, CS-1010, CS-1018, CS-1020, and [2-ex] MSA-1.
  • the STING agonist is a nanovaccine selected from PC7A nanoparticles, cGMP-nanoparticles, and ONM-500 nanoparticles.
  • the STING agonist is an antibody-drug conjugate.
  • the antibody drug conjugate is XMT-2056.
  • the STING agonist is an ENPP1 inhibitor selected from MV-626, SR-8314, SR-8291, and SR8541A.
  • the STING agonist is a bacterial vector.
  • the bacterial vector is a nonpathogenic E coli nissle expressing cyclic-di-AMP -producing enzymes.
  • the bacterial vector is SYNB1981.
  • the bacterial vector is an attenutated Salmonella Typhimurium strain enginerred to carry an inhibitory TREX-1 micro RNA.
  • compositions e.g., pharmaceutical compositions and vaccines
  • the compositions comprising any polynucleotide or vector described or exemplified herein.
  • the compositions e.g., pharmaceutical composition or vaccine
  • compositions e.g., pharmaceutical composition or vaccine
  • the second polynucleotide encodes a second antigen wherein the second antigen is operably linked to a promoter.
  • the second polynucleotide encodes at least one immune modifier protein.
  • the immune modifier protein is selected from the group consisting of IL-2, IL-12 p35, IL-12 p40, IL-12 p70, IL-15, IL-18, TNFa, GM-CSF, IFN-a, IFN-p, a chemokine, MHC I, MHC II, HLA-DR, CD80, and CD86, wherein the nucleic acid encoding the at least one immune modifier protein is operably linked to a promoter.
  • the compositions e.g., pharmaceutical composition or vaccine
  • a delivery component e.g., a cationic polymer, a poly-inosinic-polycytidylic acid, or a poloxamer.
  • the delivery component further comprises benzalkonium chloride.
  • compositions e.g., pharmaceutical composition or vaccine
  • the third polynucleotide encodes a third antigen wherein the third antigen is operably linked to a promoter.
  • the third polynucleotide encodes at least one immune modifier protein selected from the group consisting of IL-2, IL-12 p35, IL-12 p40, IL-12 p70, IL-15, IL- 18, TNFa, GM-CSF, IFN-a, IFN-p, a chemokine, MHC I, MHC II, HLA-DR, CD80, and CD86, wherein the nucleic acid encoding the at least one immune modifier protein is operably linked to a promoter.
  • the nucleic acid encoding the at least one immune modifier protein is operably linked to a promoter.
  • compositions e.g., pharmaceutical composition or vaccine
  • a delivery component e.g., a cationic polymer, a poly-inosinic-polycytidylic acid, or a poloxamer.
  • the delivery component further comprises benzalkonium chloride.
  • compositions e.g., pharmaceutical composition or vaccine
  • the fourth polynucleotide encodes a fourth antigen wherein the fourth antigen is operably linked to a promoter.
  • the fourth polynucleotide encodes at least one immune modifier protein selected from the group consisting of IL-2, IL-12 p35, IL-12 p40, IL-12 p70, IL-15, IL- 18, TNFa, GM-CSF, IFN-a, IFN-p, a chemokine, MHC I, MHC II, HLA-DR, CD80, and CD86, wherein the nucleic acid encoding the at least one immune modifier protein is operably linked to a promoter.
  • the nucleic acid encoding the at least one immune modifier protein is operably linked to a promoter.
  • compositions further comprises a delivery component (e.g., a cationic polymer, a poly-inosinic-polycytidylic acid, or a poloxamer).
  • delivery component further comprises benzalkonium chloride.
  • the compositions e.g., pharmaceutical compositions and vaccines
  • the compositions e.g., pharmaceutical composition or vaccine
  • further comprises a pharmaceutically acceptable carrier e.g., a pharmaceutically acceptable carrier.
  • the at least one immune modifier protein comprises one or more concatamers of non-coding 5'-C-phosphate-G-3' (CpG) dinucleotides.
  • the one or more concatamers of non-coding CpG dinucleotides activate the Toll-like receptor 9 (TLR9) signaling pathway.
  • the one or more concatamers of non-coding CpG dinucleotides comprise one or more concatamers of non-coding CpG dinucleotides previously reported in Bauer, A. et al., Nucleic Acids Research 38(12):3891-908 (2010); Cornelie, S.
  • compositions comprising any polynucleotide, multi ci str onic mRNA vector, or DNA plasmid vector described or exemplified herein.
  • compositions comprising (i) a vector (e.g., a multi ci stronic DNA plasmid vector or a multi ci stronic messenger RNA (mRNA) vector) comprising a nucleic acid sequence encoding one or more tumor-associated antigens; and (ii) a delivery component (e.g., a cationic polymer, a poly-inosinic- polycytidylic acid, or a poloxamer).
  • a delivery component e.g., a cationic polymer, a poly-inosinic- polycytidylic acid, or a poloxamer.
  • the compositsion further comprise (iii) an adjuvant.
  • the adjuvant comprises an aluminum or aluminum-salt based adjuvant, a stimulator of interferon genes (STING) agonist, or a combination thereof.
  • the adjuvant comprises an aluminum or aluminum-salt based adjuvant and a STING agonist.
  • the aluminum or aluminum-salt based adjuvant comprises an aluminum oxyhydroxide, an aluminum hydroxide, an aluminum phosphate, or any combination thereof.
  • the STING agonist is cyclic guanosine monophosphate (cGMP), cyclic adenosine monophosphate (cAMP), or cyclic guanosine monophosphate-adenosine monophosphate (cGAMP).
  • the vector further comprises a nucleic acid sequence encoding one or more immune modifier proteins.
  • the vector comprises a nucleic acid sequence encoding a tumor-associated antigen and, optionally, a second tumor- associated antigen.
  • Some aspects relate to methods of eliciting an immune response (e.g., T-cell response) against a tumor (e.g., ovarian cancer, melanoma, colorectal cancer, liver cancer, pancreatic cancer, brain cancer, prostate cancer, bladder cancer, renal cancer, or hematological cancer) following in vivo administration of a vector or composition of the disclosure.
  • Some aspects relate to methods of eliciting T cell (e.g., CD8+ T cell) immune response against a tumor (e.g., ovarian cancer, melanoma, colorectal cancer, liver cancer, pancreatic cancer, brain cancer, prostate cancer, bladder cancer, renal cancer, or hematological cancer) following in vivo administration of a multi ci str onic DNA plasmid, a multi ci stronic mRNA vector, or a composition comprising the same of the disclosure.
  • a tumor e.g., ovarian cancer, melanoma, colorectal cancer, liver cancer, pancreatic cancer, brain cancer, prostate cancer, bladder cancer, renal cancer, or hematological cancer
  • compositions including (i) a DNA plasmid vector comprising a DNA sequence of one or more antigens; (ii) a delivery component, such as a synthetic non-viral DNA carrier (e.g., a cationic polymer, a poly- inosinic-polycytidylic acid, or a poloxamer); and (iii) an adjuvant comprising an aluminum or aluminum-salt based adjuvant, a stimulator of interferon genes (STING) agonist, or a combination thereof.
  • a delivery component such as a synthetic non-viral DNA carrier (e.g., a cationic polymer, a poly- inosinic-polycytidylic acid, or a poloxamer)
  • an adjuvant comprising an aluminum or aluminum-salt based adjuvant, a stimulator of interferon genes (STING) agonist, or a combination thereof.
  • STING stimulator of interferon genes
  • the delivery component of the compositions disclosed herein may comprise any combination of delivery components disclosed herein.
  • compositions e.g., pharmaceutical compositions or vaccines
  • a delivery component and a polynucleotide comprising: a first nucleotide sequence, wherein the first nucleotide sequence encodes IL-12 p35 and is operably linked to a CMV promoter; a second nucleotide sequence, wherein the second nucleotide sequence encodes IL-12 p40 and is operably linked to a CMV promoter; a third nucleotide sequence, wherein the third nucleotide sequence encodes a first tumor- associated antigen and is operably linked to promoter 1; and a fourth nucleotide sequence, wherein the fourth nucleotide sequence encodes a second tumor-associated antigen and is operably linked to promoter 2.
  • the polynucleotide comprises: a 5' first nucleotide sequence, wherein the first nucleotide sequence encodes IL-12 p35 and is operably linked to a CMV promoter; a second nucleotide sequence positioned 3' to the first nucleotide sequence, wherein the second nucleotide sequence encodes IL- 12 p40 and is operably linked to a CMV promoter; a third nucleotide sequence positioned 3' to the second nucleotide sequence, wherein the third nucleotide sequence encodes a first tumor- associated antigen and is operably linked to promoter 1; and a fourth nucleotide sequence positioned 3' to the third nucleotide sequence, wherein the fourth nucleotide sequence encodes a second tumor-associated antigen and is operably linked to promoter 2.
  • the first, second, third, and fourth nucleotide sequences of the polynucleotide are configured as shown in FIG. 1.
  • compositions e.g., pharmaceutical compositions or vaccines
  • a delivery component and a polynucleotide comprising: a first nucleotide sequence, wherein the first nucleotide sequence encodes IL-12 p35 and is operably linked to a CMV promoter; a second nucleotide sequence, wherein the second nucleotide sequence encodes IL-12 p40 and is operably linked to a CMV promoter; a third nucleotide sequence, wherein the third nucleotide sequence encodes MHC I and is operably linked to promoter Z; a fourth nucleotide sequence, wherein the fourth nucleotide sequence encodes a first tumor-associated antigen and is operably linked to promoter 1, and a fifth nucleotide sequence, wherein the fifth nucleotide sequence encodes a second tumor-associated antigen and is operably linked to promoter 2.
  • the polynucleotide comprises: a 5' first nucleotide sequence, wherein the first nucleotide sequence encodes IL-12 p35 and is operably linked to a CMV promoter; a second nucleotide sequence positioned 3' to the first nucleotide sequence, wherein the second nucleotide sequence encodes IL- 12 p40 and is operably linked to a CMV promoter; a third nucleotide sequence positioned 3' to the second nucleotide sequence, wherein the third nucleotide sequence encodes MHC I and is operably linked to promoter Z; a fourth nucleotide sequence positioned 3' to the third nucleotide sequence, wherein the fourth nucleotide sequence encodes a first tumor-associated antigen and is operably linked to promoter 1, and a fifth nucleotide sequence positioned 3' to the fourth nucleotide sequence, wherein the fifth nucleotide sequence encodes a second tumor-associated antigen
  • compositions e.g., pharmaceutical compositions or vaccines
  • a delivery component and a polynucleotide comprising: a first nucleotide sequence, wherein the first nucleotide sequence encodes IL-12 p35 and is operably linked to a CMV promoter; a second nucleotide sequence, wherein the second nucleotide sequence encodes IL-12 p40 and is operably linked to a CMV promoter; a third nucleotide sequence, wherein the third nucleotide sequence encodes MHC II and is operably linked to promoter Z; a fourth nucleotide sequence, wherein the fourth nucleotide sequence encodes a first tumor-associated antigen and is operably linked to promoter 1, and a fifth nucleotide sequence, wherein the fifth nucleotide sequence encodes a second tumor-associated antigen and is operably linked to promoter 2.
  • the polynucleotide comprises: a 5' first nucleotide sequence, wherein the first nucleotide sequence encodes IL-12 p35 and is operably linked to a CMV promoter; a second nucleotide sequence positioned 3' to the first nucleotide sequence, wherein the second nucleotide sequence encodes IL- 12 p40 and is operably linked to a CMV promoter; a third nucleotide sequence positioned 3' to the second nucleotide sequence, wherein the third nucleotide sequence encodes MHC II and is operably linked to promoter Z; a fourth nucleotide sequence positioned 3' to the third nucleotide sequence, wherein the fourth nucleotide sequence encodes a first tumor-associated antigen and is operably linked to promoter 1, and a fifth nucleotide sequence positioned 3' to the fourth nucleotide sequence, wherein the fifth nucleotide sequence encodes a second tumor- associated anti
  • compositions e.g., pharmaceutical compositions or vaccines
  • a delivery component and a polynucleotide comprising: a first nucleotide sequence, wherein the first nucleotide sequence encodes IL-2 and is operably linked to a CMV promoter; a second nucleotide sequence, wherein the second nucleotide sequence encodes a first tumor-associated antigen and is operably linked to promoter 1; and a third nucleotide sequence, wherein the third nucleotide sequence encodes a second tumor-associated antigen and is operably linked to promoter 2.
  • the polynucleotide comprises: a 5' first nucleotide sequence, wherein the first nucleotide sequence encodes IL-2 and is operably linked to a CMV promoter; a second nucleotide sequence positioned 3' to the first nucleotide sequence, wherein the second nucleotide sequence encodes a first tumor-associated antigen and is operably linked to promoter 1; and a third nucleotide sequence positioned 3' to the second nucleotide sequence, wherein the third nucleotide sequence encodes a second tumor-associated antigen and is operably linked to promoter 2.
  • the first, second, and third nucleotide sequences of the polynucleotide are configured as shown in FIG. 4.
  • the compositions (e.g., pharmaceutical compositions or vaccines) of the disclosure comprise a delivery component and a polynucleotide comprising: a first nucleotide sequence, wherein the first nucleotide sequence encodes IL-2 and is operably linked to a CMV promoter; a second nucleotide sequence, wherein the second nucleotide sequence encodes MHC I and is operably linked to promoter Z; a third nucleotide sequence, wherein the third nucleotide sequence encodes a first tumor-associated antigen and is operably linked to promoter 1; and a fourth nucleotide sequence, wherein the fourth nucleotide sequence encodes a second tumor-associated antigen and is operably linked to promoter 2.
  • the polynucleotide comprises: a 5' first nucleotide sequence, wherein the first nucleotide sequence encodes IL-2 and is operably linked to a CMV promoter; a second nucleotide sequence positioned 3' to the first nucleotide sequence, wherein the second nucleotide sequence encodes MHC I and is operably linked to promoter Z; a third nucleotide sequence positioned 3' to the second nucleotide sequence, wherein the third nucleotide sequence encodes a first tumor-associated antigen and is operably linked to promoter 1; and a fourth nucleotide sequence positioned 3' to the third nucleotide sequence, wherein the fourth nucleotide sequence encodes a second tumor-associated antigen and is operably linked to promoter 2.
  • the first, second, third, and fourth nucleotide sequences of the polynucleotide are configured as shown in FIG. 5.
  • compositions e.g., pharmaceutical compositions or vaccines
  • a delivery component and a polynucleotide comprising: a first nucleotide sequence, wherein the first nucleotide sequence encodes IL-2 and is operably linked to a CMV promoter; a second nucleotide sequence, wherein the second nucleotide sequence encodes MHC II and is operably linked to promoter Z; a third nucleotide sequence, wherein the third nucleotide sequence encodes a first tumor-associated antigen and is operably linked to promoter 1; and a fourth nucleotide sequence, wherein the fourth nucleotide sequence encodes a second tumor-associated antigen and is operably linked to promoter 2.
  • the polynucleotide comprises: a 5' first nucleotide sequence, wherein the first nucleotide sequence encodes IL-2 and is operably linked to a CMV promoter; a second nucleotide sequence positioned 3' to the first nucleotide sequence, wherein the second nucleotide sequence encodes MHC II and is operably linked to promoter Z; a third nucleotide sequence positioned 3' to the second nucleotide sequence, wherein the third nucleotide sequence encodes a first tumor-associated antigen and is operably linked to promoter 1; and a fourth nucleotide sequence positioned 3' to the third nucleotide sequence, wherein the fourth nucleotide sequence encodes a second tumor-associated antigen and is operably linked to promoter 2.
  • the first, second, third, and fourth nucleotide sequences of the polynucleotide are configured as shown in FIG. 6.
  • compositions e.g., pharmaceutical compositions or vaccines
  • a delivery component and a polynucleotide comprising: a first nucleotide sequence, wherein the first nucleotide sequence encodes IL-2 and is operably linked to a CMV promoter; a second nucleotide sequence, wherein the second nucleotide sequence encodes CCL3 and is operably linked to promoter X; a third nucleotide sequence, wherein the third nucleotide sequence encodes CCL4 and is operably linked to promoter Y; a fourth nucleotide sequence, wherein the fourth nucleotide sequence encodes a first tumor-associated antigen and is operably linked to promoter 1, and a fifth nucleotide sequence, wherein the fifth nucleotide sequence encodes a second tumor- associated antigen and is operably linked to promoter 2.
  • the polynucleotide comprises: a 5' first nucleotide sequence, wherein the first nucleotide sequence encodes IL-2 and is operably linked to a CMV promoter; a second nucleotide sequence positioned 3' to the first nucleotide sequence, wherein the second nucleotide sequence encodes CCL3 and is operably linked to promoter X; a third nucleotide sequence positioned 3' to the second nucleotide sequence, wherein the third nucleotide sequence encodes CCL4 and is operably linked to promoter Y; a fourth nucleotide sequence positioned 3' to the third nucleotide sequence, wherein the fourth nucleotide sequence encodes a first tumor-associated antigen and is operably linked to promoter 1, and a fifth nucleotide sequence positioned 3' to the fourth nucleotide sequence, wherein the fifth nucleotide sequence encodes a second tumor-associated antigen and is operably linked to promoter
  • compositions e.g., pharmaceutical compositions or vaccines
  • a delivery component and a polynucleotide comprising: a first nucleotide sequence, wherein the first nucleotide sequence encodes IL-15 and is operably linked to a CMV promoter; a second nucleotide sequence, wherein the second nucleotide sequence encodes a first tumor-associated antigen and is operably linked to promoter 1; and a third nucleotide sequence, wherein the third nucleotide sequence encodes a second tumor-associated antigen and is operably linked to promoter 2.
  • the polynucleotide comprises: a 5' first nucleotide sequence, wherein the first nucleotide sequence encodes IL- 15 and is operably linked to a CMV promoter; a second nucleotide sequence positioned 3' to the first nucleotide sequence, wherein the second nucleotide sequence encodes a first tumor-associated antigen and is operably linked to promoter 1; and a third nucleotide sequence positioned 3' to the second nucleotide sequence, wherein the third nucleotide sequence encodes a second tumor-associated antigen and is operably linked to promoter 2.
  • the first, second, and third nucleotide sequences of the polynucleotide are configured as shown in FIG. 8.
  • compositions e.g., pharmaceutical compositions or vaccines
  • a delivery component and a polynucleotide comprising: a first nucleotide sequence, wherein the first nucleotide sequence encodes IL-15 and is operably linked to a CMV promoter; a second nucleotide sequence, wherein the second nucleotide sequence encodes MHC I and is operably linked to promoter Z; a third nucleotide sequence, wherein the third nucleotide sequence encodes a first tumor-associated antigen and is operably linked to promoter 1; and a fourth nucleotide sequence, wherein the fourth nucleotide sequence encodes a second tumor-associated antigen and is operably linked to promoter 2.
  • the polynucleotide comprises: a 5' first nucleotide sequence, wherein the first nucleotide sequence encodes IL-15 and is operably linked to a CMV promoter; a second nucleotide sequence positioned 3' to the first nucleotide sequence, wherein the second nucleotide sequence encodes MHC I and is operably linked to promoter Z; a third nucleotide sequence positioned 3' to the second nucleotide sequence, wherein the third nucleotide sequence encodes a first tumor-associated antigen and is operably linked to promoter 1; and a fourth nucleotide sequence positioned 3' to the third nucleotide sequence, wherein the fourth nucleotide sequence encodes a second tumor-associated antigen and is operably linked to promoter 2.
  • the first, second, third, and fourth nucleotide sequences of the polynucleotide are configured as shown in FIG. 9.
  • compositions e.g., pharmaceutical compositions or vaccines
  • a delivery component and a polynucleotide comprising: a first nucleotide sequence, wherein the first nucleotide sequence encodes IL-15 and is operably linked to a CMV promoter; a second nucleotide sequence, wherein the second nucleotide sequence encodes MHC II and is operably linked to promoter Z; a third nucleotide sequence, wherein the third nucleotide sequence encodes a first tumor-associated antigen and is operably linked to promoter 1; and a fourth nucleotide sequence, wherein the fourth nucleotide sequence encodes a second tumor-associated antigen and is operably linked to promoter 2.
  • the polynucleotide comprises: a 5' first nucleotide sequence, wherein the first nucleotide sequence encodes IL-15 and is operably linked to a CMV promoter; a second nucleotide sequence positioned 3' to the first nucleotide sequence, wherein the second nucleotide sequence encodes MHC II and is operably linked to promoter Z; a third nucleotide sequence positioned 3' to the second nucleotide sequence, wherein the third nucleotide sequence encodes a first tumor-associated antigen and is operably linked to promoter 1; and a fourth nucleotide sequence positioned 3' to the third nucleotide sequence, wherein the fourth nucleotide sequence encodes a second tumor-associated antigen and is operably linked to promoter 2.
  • the first, second, third, and fourth nucleotide sequences of the polynucleotide are configured as shown in FIG. 10.
  • compositions e.g., pharmaceutical compositions or vaccines
  • a delivery component and a polynucleotide comprising: a first nucleotide sequence, wherein the first nucleotide sequence encodes IL-15 and is operably linked to a CMV promoter; a second nucleotide sequence, wherein the second nucleotide sequence encodes CCL3 and is operably linked to promoter X; a third nucleotide sequence, wherein the third nucleotide sequence encodes CCL4 and is operably linked to promoter Y; a fourth nucleotide sequence, wherein the fourth nucleotide sequence encodes a first tumor-associated antigen and is operably linked to promoter 1, and a fifth nucleotide sequence, wherein the fifth nucleotide sequence encodes a second tumor- associated antigen and is operably linked to promoter 2.
  • the polynucleotide comprises: a 5' first nucleotide sequence, wherein the first nucleotide sequence encodes IL- 15 and is operably linked to a CMV promoter; a second nucleotide sequence positioned 3' to the first nucleotide sequence, wherein the second nucleotide sequence encodes CCL3 and is operably linked to promoter X; a third nucleotide sequence positioned 3' to the second nucleotide sequence, wherein the third nucleotide sequence encodes CCL4 and is operably linked to promoter Y; a fourth nucleotide sequence positioned 3' to the third nucleotide sequence, wherein the fourth nucleotide sequence encodes a first tumor-associated antigen and is operably linked to promoter 1, and a fifth nucleotide sequence positioned 3' to the fourth nucleotide sequence, wherein the fifth nucleotide sequence encodes a second tumor-associated antigen and is operably linked to a C
  • compositions e.g., pharmaceutical compositions or vaccines
  • a delivery component and a polynucleotide comprising: a first nucleotide sequence, wherein the first nucleotide sequence encodes CCL3 and is operably linked to promoter X; a second nucleotide sequence, wherein the second nucleotide sequence encodes CCL4 and is operably linked to promoter Y; a third nucleotide sequence, wherein the third nucleotide sequence encodes a first tumor-associated antigen and is operably linked to promoter 1; and a fourth nucleotide sequence, wherein the fourth nucleotide sequence encodes a second tumor-associated antigen and is operably linked to promoter 2.
  • the polynucleotide comprises: a 5' first nucleotide sequence, wherein the first nucleotide sequence encodes CCL3 and is operably linked to promoter X; a second nucleotide sequence positioned 3' to the first nucleotide sequence, wherein the second nucleotide sequence encodes CCL4 and is operably linked to promoter Y; a third nucleotide sequence positioned 3' to the second nucleotide sequence, wherein the third nucleotide sequence encodes a first tumor-associated antigen and is operably linked to promoter 1; and a fourth nucleotide sequence positioned 3' to the third nucleotide sequence, wherein the fourth nucleotide sequence encodes a second tumor- associated antigen and is operably linked to promoter 2.
  • the first, second, third, and fourth nucleotide sequences of the polynucleotide are configured as shown in FIG. 12.
  • one or more nucleotide sequences of the polynucleotide are configured as shown in FIGs. 14A-14O. In some aspects, two or more nucleotide sequences of the polynucleotide are configured as shown in FIGs. 14A-14O. In some aspects, three or more nucleotide sequences of the polynucleotide are configured as shown in FIGs. 14A-14O.
  • the first, second, and third nucleotide sequences of the polynucleotide are configured as shown in FIG. 14B. In some aspects, the first, second, third, and fourth nucleotide sequences of the polynucleotide are configured as shown in FIG. 14C. In some aspects, the first nucleotide sequence of the polynucleotide is configured as shown in FIG. 14D. In some aspects, the first, second, and third nucleotide sequences of the polynucleotide are configured as shown in FIG. 14E. In some aspects, the first, second, third, and fourth nucleotide sequences of the polynucleotide are configured as shown in FIG. 14F.
  • the first and second nucleotide sequences of the polynucleotide are configured as shown in FIG. 14G. In some aspects, the first and second nucleotide sequences of the polynucleotide are configured as shown in FIG. 14H. In some aspects, the first nucleotide sequence of the polynucleotide is configured as shown in FIG. 141. In some aspects, the first nucleotide sequence of the polynucleotide is configured as shown in FIG. 14J. In some aspects, the first and second nucleotide sequence of the polynucleotide is configured as shown in FIG. 14K. In some aspects, the first nucleotide sequence of the polynucleotide is configured as shown in FIG.
  • first and second nucleotide sequences of the polynucleotide are configured as shown in FIG. 14M. In some aspects, the first nucleotide sequence of the polynucleotide is configured as shown in FIG. 14N. In some aspects, the first nucleotide sequence of the polynucleotide is configured as shown in FIG. 140.
  • compositions e.g., pharmaceutical compositons or vaccines
  • the nucleotide sequences encode antigens to a virus, a bacteria or a parasite.
  • the nucleotide sequences encode one or more antigens comprise one or more tumor-associated antigens.
  • compositions can comprise a delivery component and a polynucleotides configured as shown in the vector constructs illustrated in any of FIGs. 14N-14O.
  • compositions e.g., pharmaceutical compositons or vaccines
  • nucleotide sequences encode antigens to a virus, a bacteria or a parasite.
  • nucleotide sequences encode one or more antigens comprise one or more tumor-associated antigens.
  • compositions e.g., pharmaceutical compositions or vaccines
  • a delivery component and a polynucleotide comprising: a first nucleotide sequence, wherein the first nucleotide sequence encodes a first tumor associated protein and is operably linked to a first promoter (e.g., a hEFl-HTLV promoter); a second nucleotide sequence, wherein the second nucleotide sequence encodes IL-12 p35 and is operably linked to a second promoter (e.g., a CMV promoter); and a third nucleotide sequence, wherein the third nucleotide sequence encodes IL- 12 p40 and is operably linked to a second promoter (e.g., a CMV promoter).
  • a first promoter e.g., a hEFl-HTLV promoter
  • a second nucleotide sequence wherein the second nucleotide sequence encodes IL-12 p35 and is operably linked
  • compositions e.g., pharmaceutical compositions or vaccines
  • a delivery component and a polynucleotide comprising: a first nucleotide sequence, wherein the first nucleotide sequence encodes a first tumor- associated antigen and is operably linked to a first promoter (e.g., a hEFl-HTLV promoter); a second nucleotide sequence, wherein the second nucleotide sequence encodes a second tumor-associated antigen and is operably linked to the first promoter through an IRES sequence; a third nucleotide sequence, wherein the third nucleotide sequence encodes IL-12 p35 and is operably linked to a second promoter (e.g., a CMV promoter); and a fourth nucleotide sequence, wherein the fourth nucleotide sequence encodes IL-12 p340 and is operably linked to a second promoter (e.g., a CMV promoter).
  • a first promoter e.g
  • compositions e.g., pharmaceutical compositions or vaccines
  • a delivery component and a polynucleotide comprising: a first nucleotide sequence, wherein the first nucleotide sequence encodes a first tumor- associated antigen and is operably linked to a first promoter (e.g., an EF-la promoter); a second nucleotide sequence, wherein the second nucleotide sequence encodes IL- 12 p35 and is operably linked to a second promoter (e.g., a CMV promoter); and a third nucleotide sequence, wherein the third nucleotide sequence encodes IL-12 p40 and is operably linked to a second promoter (e.g., a CMV promoter).
  • a first promoter e.g., an EF-la promoter
  • a second nucleotide sequence wherein the second nucleotide sequence encodes IL- 12 p35 and is operably linked to a second promote
  • compositions e.g., pharmaceutical compositions or vaccines
  • a delivery component and a polynucleotide comprising: a first nucleotide sequence, wherein the first nucleotide sequence encodes a first tumor- associated antigen and is operably linked to a first promoter (e.g., an EF-la promoter); a second nucleotide sequence, wherein the second nucleotide sequence encodes a second tumor-associated antigen and is operably linked to the first promoter through an IRES sequence; a third nucleotide sequence, wherein the third nucleotide sequence encodes IL- 12 p35 and is operably linked to a second promoter (e.g., a CMV promoter); and a fourth nucleotide sequence, wherein the fourth nucleotide sequence encodes IL-12 p340 and is operably linked to a second promoter (e.g., a CMV promoter).
  • a first promoter e.g., an
  • compositions e.g., pharmaceutical compositions or vaccines
  • the compositions comprise a delivery component and a polynucleotide comprising a first nucleotide sequence, wherein the first nucleotide sequence encodes a first tumor- associated antigen and is operably linked to a first promoter (e.g., an EF-la promoter
  • compositions e.g., pharmaceutical compositions or vaccines
  • the compositions comprise a delivery component and a polynucleotide comprising a first nucleotide sequence, wherein the first nucleotide sequence encodes a first tumor- associated antigen and is operably linked to a first promoter (e.g., an EF-la promoter); and a second nucleotide sequence, wherein the second nucleotide sequence encodes a second tumor-associated antigen and is operably linked to a second promoter (e.g., a CMV promoter).
  • a first promoter e.g., an EF-la promoter
  • a second nucleotide sequence e.g., a second tumor-associated antigen and is operably linked to a second promoter (e.g., a CMV promoter).
  • the composition e.g., pharmaceutical compositions or vaccines
  • the composition can comprise a delivery component, wherein the polynucleotide configured as shown in the vector constructs illustrated in any of FIGs. 1-14 can be modified to replace the the tumor-associated antigen- 1 or the tumor-associated antigen-2 and/or any additional tumor-associated antigen with nucleotide sequences encoding any combinations of tumor- associated antigens or antigenic fragment thereof disclosed herein.
  • the nucleotide sequences encode two or more tumor-associated antigens.
  • compositions can comprise an adjuvant and a polynucleotide configured as shown in the vector constructs illustrated in any of FIGs. 1-14 and/or vector constructs that have been modified to replace the tumor-associated antigen- 1 protein or the tumor-associated antigen-2 protein and/or an additional tumor-associated antigen with nucleotide sequences encoding any combinations of tumor-associated antigen or antigenic fragment thereof disclosed herein.
  • compositions can comprise a polynucleotide encoding any combinations of tumor-associated antigen or antigenic fragment thereof disclosed herein and an adjuvant selected from potassium aluminum sulfate [KA1(SO4)2], aluminum hydroxide, crystalline aluminum oxyhydroxide, aluminum phosphate, aluminum hydroxyphosphate, amorphous aluminum hydroxyphosphate sulfate, aluminum chloride, aluminum silicate, and a composition comprising aluminum hydroxide and magnesium hydroxide.
  • potassium aluminum sulfate K aluminum sulfate
  • Al hydroxide aluminum hydroxide
  • crystalline aluminum oxyhydroxide aluminum phosphate
  • aluminum hydroxyphosphate aluminum hydroxyphosphate
  • amorphous aluminum hydroxyphosphate sulfate aluminum chloride
  • aluminum silicate aluminum silicate
  • compositions can comprise a polynucleotide encoding any combinations of tumor-associated antigen or antigenic fragment thereof disclosed herein and a STING agonist.
  • compositions can comprise a polynucleotide encoding any combinations of tumor-associated antigen or antigenic fragment thereof disclosed herein and the STING agonist cyclic GMP (cGMP).
  • cGMP STING agonist cyclic GMP
  • compositions can comprise a polynucleotide encoding any combinations of tumor-associated antigen or antigenic fragment thereof disclosed herein and a STING agonist selected from ADU- S100, MK-1454, SB11285, BMS-986301, BI-STING (BI1387446), JNJ-67544412, 3’3’- cyclic AIMP, and GSK532.
  • compositions can comprise a polynucleotide encoding any combinations of tumor-associated antigen or antigenic fragment thereof disclosed herein and a STING agonist selected from DMAXAA, ALG-031048, E7766, JNJ-‘6196, MK-21118, MSA-1, MSA-2, SNX281, SR-717, TAK676, TTI-10001, a Ryvu’s agonist, GF3-002, a Selvita agonist, CDR5500, CS-1010, CS-1018, CS-1020, and [2-ex] MSA-1.
  • STING agonist selected from DMAXAA, ALG-031048, E7766, JNJ-‘6196, MK-21118, MSA-1, MSA-2, SNX281, SR-717, TAK676, TTI-10001, a Ryvu’s agonist, GF3-002, a Selvita agonist, CDR5500, CS-1010, CS-1018, CS-1020, and [
  • compositions can comprise a polynucleotide encoding any combinations of tumor-associated antigen or antigenic fragment thereof disclosed herein and a STNG agonist selected from a PC7A nanoparticle, a cGMP-nanoparticle, and a ONM-500 nanoparticle.
  • compositions can comprise a polynucleotide encoding any combinations of tumor-associated antigen or antigenic fragment thereof disclosed herein and a STING agonist that is an antibody-drug conjugate.
  • the antibody drug conjugate is XMT-2056.
  • compositions can comprise a polynucleotide encoding any combinations of tumor-associated antigen or antigenic fragment thereof disclosed herein and a STING agonist that is an ENPP1 inhibitor selected from MV-626, SR-8314, SR-8291, and SR8541A.
  • compositions can comprise a polynucleotide encoding any combinations of tumor-associated antigen or antigenic fragment thereof disclosed herein and a STING agonist that is a bacterial vector.
  • the bacterial vector is a nonpathogenic E coli nissle expressing cyclic-di- AMP-producing enzymes. In some aspects, the bacterial vector is SYNB1981. In some aspects, the bacterial vector is an attenutated Salmonella Typhimurium strain enginerred to carry an inhibitory TREX-1 micro RNA.
  • compositions can comprise a polynucleotide encoding any combinations of tumor-associated antigen or antigenic fragment thereof disclosed herein, an adjuvant and a STING agonist.
  • compositions can comprise a polynucleotide encoding any combinations of tumor-associated antigen or antigenic fragment thereof disclosed herein, an adjuvant selected from potassium aluminum sulfate [KA1(SO4)2], aluminum hydroxide, crystalline aluminum oxyhydroxide, aluminum phosphate, aluminum hydroxyphosphate, amorphous aluminum hydroxyphosphate sulfate, aluminum chloride, aluminum silicate, a composition comprising aluminum hydroxide and magnesium hydroxide and combinations thereof, and a STING agonist selected from cyclic GMP (cGMP), cAMP, and cGAMP.
  • an adjuvant selected from potassium aluminum sulfate [KA1(SO4)2]
  • aluminum hydroxide crystalline aluminum oxyhydroxide
  • aluminum phosphate aluminum hydroxyphosphate
  • amorphous aluminum hydroxyphosphate sulfate aluminum chloride
  • aluminum silicate aluminum silicate
  • a composition comprising aluminum hydroxide and magnesium hydroxide and combinations thereof
  • a STING agonist selected from
  • compositions can comprise a polynucleotide encoding any combinations of tumor-associated antigen or antigenic fragment thereof disclosed herein, an adjuvant selected from potassium aluminum sulfate [KA1(SO4)2], aluminum hydroxide, crystalline aluminum oxyhydroxide, aluminum phosphate, aluminum hydroxyphosphate, amorphous aluminum hydroxyphosphate sulfate, aluminum chloride, aluminum silicate, a composition comprising aluminum hydroxide and magnesium hydroxide and combinaions thereof, and a STING agonist selected from cyclic GMP (cGMP), ADU-S100, MK-1454, SB11285, BMS-986301, BI-STING (BI1387446), JNJ-67544412, 3’3’-cyclic AIMP, GSK532, DMAXAA, ALG-031048, E7766, JNJ-‘6196, MK-21118, MSA-1, MSA-2, SNX
  • compositions e.g., pharmaceutical composition or vaccine
  • a delivery component e.g., a cationic polymer, a poly-inosinic- polycytidylic acid, or a poloxamer.
  • the delivery component further comprises benzalkonium chloride.
  • compositions of the present disclosure e.g., pharmaceutical compositions, vaccines, vectors, and DNA plasmid vectors
  • compositions of the present disclosure can be formulated according to known methods for preparing pharmaceutically useful compositions.
  • Formulations are described in a number of sources which are well known and readily available to those skilled in the art.
  • Remington's Pharmaceutical Science (Martin EW [1995] Easton Pennsylvania, Mack Publishing Company, 19th Ed.) describes formulations, which can be used in connection with the subject disclosure.
  • Formulations suitable for parenteral administration include, for example, aqueous sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and nonaqueous sterile suspensions which can include suspending agents and thickening agents.
  • the formulations can be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and can be stored in a freeze dried (lyophilized) condition requiring only the condition of the sterile liquid carrier, for example, water for injections, prior to use.
  • sterile liquid carrier for example, water for injections
  • Extemporaneous injection solutions and suspensions can be prepared from sterile powder, granules, tablets, etc. It should be understood that in addition to the ingredients particularly mentioned above, the formulations of the subject disclosure can include other agents conventional in the art having regard to the type of formulation in question.
  • compositions or vaccines that can be safely stored for periods of time and reconstituted prior to use.
  • the composition, pharmaceutical composition, or vaccine of the disclosure is a lyophilized product, e.g., substantially free of aqueous components.
  • the lyophilized composition or vaccine is reconstituted in a diluent, e.g., prior to administration.
  • the lyophilized composition or vaccine is reconstituted in water.
  • compositions or vaccine comprising: (1) any polynucleotide or DNA plasmid vector disclosed herein; (2) a STING agonist; (3) an aluminum or aluminum salt based adjuvant; and (4) any delivery component disclosed herein, wherein the composition is substantially free of aqueous components.
  • a composition or vaccine of the disclosure is lyophilized.
  • the composition or vaccine is stable at 0°C to 5°C for at least about 1 month (or 30 days), 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 monts, or 12 months. In some aspects, the composition or vaccine is stable at 0°C to 5°C for at least 1 year, at least 2 years, at least
  • the composition or vaccine is stable at -20°C for at least 1 year, at least 2 years, at least 3 years, at least 4 years, at least 5 years, or at least 10 years. In some aspects, the composition or vaccine is stable at 25°C for at least about 7 days, about 10 day, or about 14 days. In some aspects, the composition or vaccine is lyophilized and is stable at 0°C to 5°C for at least about 1 month (or 30 days), 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 monts, or 12 months. In some aspects, the lyophilized composition or vaccine is stable at 0°C to 5°C for at least 1 year, at least 2 years, at least 3 years, at least
  • the lyophilized composition or vaccine is stable at -20°C for at least 1 year, at least 2 years, at least 3 years, at least 4 years, at least
  • the lyophilized composition or vaccine is stable at 25°C for at least about 7 days, about 10 day, or about 14 days.
  • the lyophilized composition or vaccine is reconstituted into a reconstituted composition or vaccine formulation for administration.
  • the reconstituted composition or vaccine is stable at 0°C to 5°C for at least about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, or about 6 months after reconstitution of the lyophilized composition or vaccine with a diluent (e.g., water).
  • the reconstiuted composition or vaccine is stable at -20°C for at least 1 year, at least 2 years, at least 3 years, at least 4 years, at least 5 years, or at least 10 years.
  • the reconstituted composition or vaccine is stable at 25°C for at least about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days after reconstitution of the lyophilized composition with a diluent.
  • the diluent is water.
  • the compositions of the subject disclosure can further comprise other components such as a pharmaceutically acceptable carrier and/or an immune modifier protein.
  • the immune modifier protein can be expressed in alternative polynucleotides, plasmids, or vectors or are delivered as proteins in combination with the compositions of the subject disclosure.
  • the immune modifier protein can be selected from the group consisting of: a- interferon (IFN-a), P-interferon (IFN-P), y-interferon, platelet derived growth factor (PDGF), TNFa, TNFP, GM-CSF, epidermal growth factor (EGF), cutaneous T cellattracting chemokine (CTACK), epithelial thymus-expressed chemokine (TECK), mucosae-associated epithelial chemokine (MEC), IL-12, IL-15, MHC, CD80, CD86 including IL- 15 having the signal sequence deleted and optionally including the signal peptide from IgE.
  • IFN-a interferon
  • IFN-P P-interferon
  • PDGF platelet derived growth factor
  • TNFa TNFa
  • TNFP TNFP
  • GM-CSF epidermal growth factor
  • EGF epidermal growth factor
  • CTL epidermal growth factor
  • CTACK epidermal growth factor
  • the immune modifier protein can be IL-12, IL-15, IL-28, CTACK, TECK, platelet derived growth factor (PDGF), TNFa, TNFP, GM-CSF, epidermal growth factor (EGF), IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-18, or a combination thereof.
  • genes that can be useful immune modifier proteins include those encoding: MCP-1, MIP-la, MIP-lp, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, pl50.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, mutant forms of IL-18, CD40, CD40L, vascular growth factor, fibroblast growth factor, IL-7, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Fit, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, DR6, Caspase ICE, Fos, c- jun, Sp-1, Ap-1, Ap-2, p
  • compositions of the disclosure can be formulated according to the mode of administration to be used.
  • an injectable vaccine pharmaceutical composition can be sterile, pyrogen free and particulate free.
  • An isotonic formulation or solution can be used.
  • Additives for isotonicity can include sodium chloride, dextrose, mannitol, sorbitol, and lactose.
  • the vaccine can comprise a vasoconstriction agent.
  • the isotonic solutions can include phosphate buffered saline.
  • Vaccine can further comprise stabilizers including gelatin and albumin. The stabilizers can allow the formulation to be stable at room or ambient temperature for extended periods of time, including LGS or polycations or polyanions.
  • the polynucleotides of the disclosure can include DNA or mRNA sequences (e.g., multicistronic DNA or multi ci str onic mRNA) for use in the compositions (e.g., pharmaceutical compositons and vaccines) disclosed herein.
  • DNA or mRNA sequences e.g., multicistronic DNA or multi ci str onic mRNA
  • compositions e.g., pharmaceutical compositons and vaccines
  • the present disclosure is directed to a polynucleotide can comprise a nucleic acid sequence encoding one or more tumor-associated antigens (e.g., NYESO-1, MAGE-A1, MAGE- A2, MAGE-A3, MAGE-A4, MAGE-A9, MAGE-A10, MAGE-CI, MAGE-C2, SSX, H0RMAD1, CXorf61, ACTL8, AKAP3,AKAP4, HOM-TES-85, OY-TES-1, HSP70-2, LAGE-1, PIWL1, PIWL2, PIWL3, PIWL4, PLU-1, PLAC1, SPAG9, SCP-1, TAG-1, TAG-2a, TAG-2b, TAG-2c, TRAG-3, KAP4, BAGE, BORIS, CT45, GAGE-1/2, PRAME, PAGE4, SP17, PASD1, p53, ras, B-RafHER-2/neu, mucin 1 (MUC1), epidermatitis,
  • the antigenic fragment of a tumor-associated antigen can be an extracellular region of the antigen.
  • the antigenic of CA-125 can be the extracellular region of CA-125 or a portion of the extracellular region of CA-125.
  • the vector further comprises a nucleic acid sequence encoding one or more immune modifier proteins.
  • the vector comprises a nucleic acid sequence encoding a tumor- associated antigen and, optionally, a second tumor-associated antigen.
  • the vector comprises a nucleic acid sequence encoding a tumor-associated antigen and a second tumor-associated antigen.
  • the nucleic acid sequence encodes a tumor-associated antigen and a second tumor-associated antigen from a different tumor.
  • the nucleic acid sequence encodes different portions of the same tumor- associated antigen, wherein the different portions of the same tumor-associated antigen are derived from the same tumor.
  • the present disclosure is directed to a polynucleotide comprising: (a) a antigen nucleic acid (e.g., first antigen nucleic acid) which encodes a tumor associated protein antigen (e.g., NYESO-1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE- A4, MAGE-A9, MAGE-A10, MAGE-CI, MAGE-C2, SSX, HORMAD1, CXorf61, ACTL8, AKAP3,AKAP4, HOM-TES-85, OY-TES-1, HSP70-2, LAGE-1, PIWL1, PIWL2, PIWL3, PIWL4, PLU-l, PLAC1, SPAG9, SCP-1, TAG-1, TAG-2a, TAG-2b, TAG-2c, TRAG-3, KAP4, BAGE, BORIS, CT45, GAGE-1/2, PRAME, PAGE4, SP17, PASD1, p53, ras
  • the antigen nucleic acid (e.g., first antigen nucleic acid) is operably linked to a first promoter.
  • the polynucleotide comprises two or more nucleic acids encoding an immune modifier protein.
  • each of the nucleic acids encoding an immune modifier protein encodes a different immune modifier protein.
  • the polynucleotides disclosed herein can comprise: (b) or (c) at least one additional antigen nucleic acid (e.g., second antigen nucleic acid) which encodes a second tumor associated protein antigen (e.g., NYESO-1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A9, MAGE-A10, MAGE-CI, MAGE-C2, SSX, H0RMAD1, CXorf61, ACTL8, AKAP3,AKAP4, HOM-TES-85, OY-TES-1, HSP70-2, LAGE-1, PIWL1, PIWL2, PIWL3, PIWL4, PLU-l, PLAC1, SPAG9, SCP-1, TAG-1, TAG-2a, TAG-2b, TAG-2c, TRAG-3, KAP4, BAGE, BORIS, CT45, GAGE-1/2, PRAME, PAGE4, SP17, PASD1, p53, ras,
  • the second tumor-associated antigen or antigenic fragment thereof comprises one or more portions of the tumor associated antigen.
  • the first tumor-associated antigen and the second tumor-associated antigen are tumor-associated antigen antigens from different tumors. In some aspects, the first tumor-associated antigen and the second tumor-associated antigens are different portions of the same tumor-associated antigen. In some aspects, the first tumor-associated antigen and the second tumor-associated antigen are different tumor-associated antigens, wherein the different tumor-associated antigens are derived from different tumors. In some aspects, the first tumor-associated antigen and the second tumor-associated antigen are different tumor-associated antigens, wherein the different tumor-associated antigens are derived from the same tumor.
  • the at least one additional antigen nucleic acid (e.g., second antigen nucleic acid) is operably linked to the first promoter through an internal ribosome entry site (IRES) sequence.
  • IRES sequence comprises a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 19.
  • the present disclosure is directed to a polynucleotide (e.g., multicistronic DNA or multi ci str onic mRNA) comprising: (a) an antigen nucleic acid (e.g., first antigen nucleic acid) which encodes a tumor-associated antigen or an antigenic fragment thereof; and (b) a nucleic acid encoding an immune modifier protein.
  • an antigen nucleic acid e.g., first antigen nucleic acid
  • the polynucleotide comprises at least two tumor-associated antigens.
  • the polynucleotide comprises at least two tumor-associated antigens and an immune modifier protein.
  • the polynucleotide comprises at least two tumor-associated antigens and at least two immune modifier proteinsln some aspects, the present disclosure is directed to a composition comprising (a) a polynucleotide (e.g., a multicistronic DNA plasmid or multicistronic mRNA) comprising an antigen nucleic acid which encodes a tumor-associated antigen (e.g., NYESO-1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A9, MAGE-A10, MAGE-CI, MAGE-C2, SSX, H0RMAD1, CXorf61, ACTL8, AKAP3,AKAP4, HOM-TES-85, OY-TES-1, HSP70-2, LAGE-1, PIWL1, PIWL2, PIWL3, PIWL4, PLU-1, PLAC1, SPAG9, SCP-1, TAG-1, TAG-2a, TAG-2b, TAG-2c, TRA
  • the composition comprises (a) a polynucleotide (e.g., a multicistronic DNA plasmid or multicistronic mRNA) comprising an antigen nucleic acid which encodes a tumor- associated antigen (e.g, NYESO-1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A9, MAGE-A10, MAGE-CI, MAGE-C2, SSX, H0RMAD1, CXorf61, ACTL8, AKAP3,AKAP4, HOM-TES-85, OY-TES-1, HSP70-2, LAGE-1, PIWL1, PIWL2, PIWL3, PIWL4, PLU-1, PLAC1, SPAG9, SCP-1, TAG-1, TAG-2a, TAG-2b, TAG-2c, TRAG-3, KAP4, BAGE, BORIS, CT45, GAGE-1/2, PRAME, PAGE4, SP17, PASD1, p53, ra
  • the composition comprises (a) a polynucleotide (e.g., a multi ci str onic DNA plasmid or multi ci str onic mRNA) comprising an antigen nucleic acid which encodes a tumor-associated antigen (e.g., NYESO-1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A9, MAGE-A10, MAGE-CI, MAGE-C2, SSX, H0RMAD1, CXorf61, ACTL8, AKAP3,AKAP4, HOM-TES-85, OY-TES-1, HSP70-2, LAGE-1, PIWL1, PIWL2, PIWL3, PIWL4, PLU-1, PLAC1, SPAG9, SCP-1, TAG-1, TAG-2a, TAG-2b, TAG-2c, TRAG-3, KAP4, BAGE, BORIS, CT45, GAGE-1/2, PRAME, PAGE4, SP17, PASD1,
  • the antigen nucleic acid (e.g., first antigen nucleic acid) is operably linked to a first promoter.
  • the polynucleotide comprises two or more nucleic acids encoding an immune modifier protein.
  • each of the nucleic acids encoding an immune modifier protein encodes a different immune modifier protein.
  • the polynucleotides disclosed herein can further comprise: (c) at least one additional antigen nucleic acid (e.g., second antigen nucleic acid) which encodes a tumor- associated antigen or an antigenic fragment thereof.
  • additional antigen nucleic acid e.g., second antigen nucleic acid
  • umor associated antigen protein or antigenic fragment thereof is selected from the group consisting of: NYESO-1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A9, MAGE-A10, MAGE-CI, MAGE-C2, SSX, H0RMAD1, CXorf61, ACTL8, AKAP3,AKAP4, HOM-TES-85, OY-TES-1, HSP70-2, LAGE-1, PIWL1, PIWL2, PIWL3, PIWL4, PLU-1, PLAC1, SPAG9, SCP-1, TAG-1, TAG-2a, TAG-2b, TAG-2c, TRAG-3, KAP4, BAGE, BORIS, CT45, GAGE-1/2, PRAME, PAGE4, SP17, PASD1, p53, ras, B-RafHER-2/neu, mucin 1 (MUC1), epidermal growth factor receptor (EGFR), GP100, prostatic acid phosphatase, prostate-specific membrane
  • the antigen nucleic acid encodes a tumor-associated antigen or an antigenic fragment thereof and the at least one additional antigen nucleic acid (e.g., second antigen nucleic acid) encodes a tumor- associated antigen or an antigenic fragment thereof from a different tumor.
  • the antigen nucleic acid e.g., first antigen nucleic acid
  • the at least one additional antigen nucleic acid e.g., second antigen nucleic acid
  • the at least one additional antigen nucleic acid (e.g., second antigen nucleic acid) is operably linked to the first promoter through an internal ribosome entry site (IRES) sequence.
  • IRES sequence comprises a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 19.
  • the polynucleotides disclosed herein can further comprise one or more second promoters.
  • the at least one additional antigen nucleic acid e.g., second antigen nucleic acid
  • one or more nucleic acids encoding an immune modifier protein is operably linked to the one or more second promoters.
  • one or more of the nucleic acids encoding an immune modifier protein is operably linked to the first promoter or the one or more second promoters through an internal ribosome entry site (IRES) sequence.
  • IRS internal ribosome entry site
  • the IRES sequence comprises a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 19.
  • the first promoter or the one or more second promoters is selected from the group consisting of: a cytomegalovirus (CMV) promoter (SEQ ID NO: 14), a Rouse sarcoma virus (RSV) promoter, a Moloney murine leukemia virus (Mo- MuLV) long terminal repeat (LTR) promoter, a mammalian elongation factor 1 (EFl) promoter, a cytokeratin 18 (CK18) promoter, a cytokeratin 19 (CK19) promoter, a simian virus 40 (SV40) promoter (SEQ ID NO: 15 or SEQ ID NO: 30), a murine U6 promoter, a skeletal a-actin promoter, a P-actin promoter, a murine phosphoglycerate kinase 1 (PGK1) promoter, a human PGK1 promoter, a CMV enhancer/chicken P-actin (CAG)
  • CMV cytome
  • the one or more second promoters is the CMV promoter. In some aspects, the one or more second promoters is a mammalian EFl promoter. In some aspects, the mammalian EFl promoter is a hEFl-HTLV promoter (SEQ ID NO: 18).
  • each of the nucleic acids which encodes an immune modifier protein is under the control of a promoter selected from the group consisting of a CMV promoter, an RSV promoter, a Mo-MuLV LTR promoter, a mammalian EFl promoter, a CK18 promoter, a CK19 promoter, an SV40 promoter, a murine U6 promoter, a skeletal a-actin promoter, a P-actin promoter, a murine PGK1 promoter, a human PGK1 promoter, a CAG promoter, and any combination thereof.
  • the mammalian EFl promoter is a hEFl-HTLV promoter.
  • each of the at least one additional antigen nucleic acid (e.g., second antigen nucleic acid)s is under the control of a promoter selected from the group consisting of a CMV promoter, an RSV promoter, a Mo-MuLV LTR promoter, a mammalian EFl promoter, a CK18 promoter, a CK19 promoter, an SV40 promoter, a murine U6 promoter, a skeletal a-actin promoter, a P-actin promoter, a murine PGK1 promoter, a human PGK1 promoter, a CAG promoter, and any combination thereof.
  • the mammalian EFl promoter is a hEFl-HTLV promoter.
  • the immune modifier protein is selected from the group consisting of: interleukin (IL) 2 (IL-2), IL-12 p35, IL-12 p40, IL-12 p70, IL-15, IL-18, tumor necrosis factor alpha (TNFa), granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon (IFN) a (IFN-a), IFN-P, a chemokine, major histocompatibility complex (MHC) class I (MHC I), MHC class II (MHC II), human leukocyte antigen (HLA)-DR isotype (HLA-DR), CD80, CD86, and any combination thereof.
  • IL interleukin
  • IL-2 interleukin 2
  • IL-12 p35 IL-12 p40
  • IL-12 p70 IL-15
  • IL-18 tumor necrosis factor alpha
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • the chemokine is selected from the group consisting of: C-C motif chemokine ligand (CCL) 3 (CCL3), CCL4, CCL5, CCL21, CCL28, C-X-C motif chemokine ligand (CXCL) 10 (CXCL10), and any combination thereof.
  • the immune modifier protein is an interleukin, e.g., IL-12.
  • IL-12 is composed of four alpha helices. It is a heterodimeric cytokine encoded by two separate genes, IL-12A (p35) and IL-12B (p40).
  • the active heterodimer (referred to as p70), and a homodimer of p40 are formed following protein synthesis.
  • the immune modifier protein is an IL-12 heterodimer (IL-12 p70) or an IL-12 homodimer (e.g., IL-12 p35 or IL-12 p40).
  • the IL-12 p35 immune modifier protein comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 21 (mouse IL-12 p35) or SEQ ID NO: 25 (human IL-12 p35).
  • the IL-12 p40 immune modifier protein comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 23 (mouse IL-12 p40) or SEQ ID NO: 27 (human IL-12 p40).
  • the nucleic acid encoding IL-12 p35 has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 20 (nucleic acid sequence encoding mouse IL-12 p35) or SEQ ID NO: 24 (nucleic acid sequence encoding human IL-12 p35).
  • the nucleic acid encoding IL-12 p40 has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 22 (nucleic acid sequence encoding mouse IL- 12 p40) or SEQ ID NO: 26 (nucleic acid sequence encoding human IL- 12 p40).
  • the immune modifier protein comprises one or more concatamers of non-coding 5'-C-phosphate-G-3' (CpG) dinucleotides.
  • the one or more concatamers of non-coding CpG dinucleotides activate the Toll-like receptor 9 (TLR9) signaling pathway.
  • the one or more concatamers of non-coding CpG dinucleotides comprise one or more concatamers of non-coding CpG dinucleotides previously reported in Bauer, A. et al., Nucleic Acids Research 38(12):3891-908 (2010); Cornelie, S.
  • polynucleotides encoding an immune modifier protein comprises a combination (i) a nucleic acid encoding an interleukin, and (ii) a nucleic acid encoding a major histocompatibility complex and/or a chemokine.
  • the polynucleotidesencoding an immune modifier protein comprises a nucleic acid encoding IL-12 p35, a nucleic acid encoding IL-12 p40, or the combination thereof.
  • the nucleic acid encoding IL- 12 p35 has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 20 (nucleic acid sequence encoding mouse IL- 12 p35) or SEQ ID NO: 24 (nucleic acid sequence encoding human IL-12 p35).
  • the nucleic acid encoding IL-12 p40 has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 22 (nucleic acid sequence encoding mouse IL- 12 p40) or SEQ ID NO: 26 (nucleic acid sequence encoding human IL- 12 p40).
  • the nucleic acid encoding an immune modifier protein further comprises a nucleic acid encoding MHC I, a nucleic acid encoding MHC II, or the combination thereof.
  • the polynucleotidesencoding an immune modifier protein comprises a nucleic acid encoding IL-12, a nucleic acid encoding IL-15, or the combination thereof.
  • the polynucleotidesencoding an immune modifier protein comprises a combination of a nucleic acid encoding IL-12 and a nucleic acid encoding IL-15.
  • the polynucleotides encoding an immune modifier protein comprises a nucleic acid encoding IL-2, a nucleic acid encoding IL-15, or the combination thereof.
  • the nucleic acid encoding an immune modifier protein further comprises a nucleic acid encoding MHC I, a nucleic acid encoding MHC II, a nucleic acid encoding CCL3, a nucleic acid encoding CCL4, any the combination thereof.
  • the polynucleotidesencoding an immune modifer comprises a nucleic acid encoding CCL3, a nucleic acid encoding CCL4, or the combination thereof.
  • the antigen nucleic acid (e.g., first antigen nucleic acid) of the polynucleotides disclosed herein can encode a full length tumor-associated antigen polypeptide or antigenic fragment thereof.
  • the antigen nucleic acid (e.g., first antigen nucleic acid) of the polynucleotide encodes at least 8, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 750, at least 1,000, or at least 1,250 contiguous amino acids of any one of SEQ ID NOs: 1, 3, 5, 7, 8, or 10.
  • the antigen nucleic acid (e.g., first antigen nucleic acid or second antigen nucleic acid) of the polynucleotide encodes a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 1, 3, 5, 7, 8, or 10.
  • the antigen nucleic acid (e.g., first antigen nucleic acid or second antigen nucleic acid) of the polynucleotide comprises a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 2, 4, 6, 9, 11, 28, or 29.
  • the antigen nucleic acid (e.g., first antigen nucleic acid) and the at least one additional antigen nucleic acid (e.g., second antigen nucleic acid) of the polynucleotides encode a tumor-associated antigen or antigenic fragments thereof from different tumors.
  • the antigen nucleic acid (e.g., first antigen nucleic acid) and the at least one additional antigen nucleic acid (e.g., second antigen nucleic acid) of the polynucleotides encode tumor-associated antigens or antigenic fragments thereof, wherein the tumor-associated antigens or antigenic fragments thereof are derived from different tumors.
  • the antigen nucleic acid (e.g., first antigen nucleic acid) and the at least one additional antigen nucleic acid (e.g., second antigen nucleic acid) of the polynucleotides encode tumor-associated antigens or antigenic fragments thereof, wherein the tumor-associated antigens or antigenic fragments thereof are derived from the same tumors
  • the antigen nucleic acid (e.g., first antigen nucleic acid) and the at least one additional antigen nucleic acid (e.g., second antigen nucleic acid) of the polynucleotides encode tumor-associated antigens or antigenic fragments thereof, wherein the tumor-associated antigens or antigenic fragments thereof are portions derived from the same tumor assoicated antigen.
  • the present disclosure is directed to a polynucleotide (e.g., multicistronic DNA or multi ci str onic mRNA) comprising an antigen nucleic acid (e.g., first antigen nucleic acid) which encodes a first tumor-associated antigen or an antigenic fragment thereof, wherein the antigen nucleic acid (e.g., first antigen nucleic acid) is operably linked to a first promoter.
  • an antigen nucleic acid e.g., first antigen nucleic acid
  • first antigen nucleic acid which encodes a first tumor-associated antigen or an antigenic fragment thereof
  • the antigen nucleic acid e.g., first antigen nucleic acid
  • the polynucleotide further comprises at least one additional antigen nucleic acid (e.g., second antigen nucleic acid) which encodes a second tumor- associated antigen or an antigenic fragment thereof.
  • the at least one additional antigen nucleic acid (e.g., second antigen nucleic acid) which encodes a second tumor-associated antigen is selected from the group consisting of NYESO-1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A9, MAGE-A10, MAGE-CI, MAGE-C2, SSX, H0RMAD1, CXorf61, ACTL8, AKAP3,AKAP4, HOM-TES-85, OY-TES-1, HSP70-2, LAGE-1, PIWL1, PIWL2, PIWL3, PIWL4, PLU-1, PLAC1, SPAG9, SCP-1, TAG-1, TAG-2a, TAG-2b, TAG-2c, TRA
  • the at least one additional antigen nucleic acid (e.g., second antigen nucleic acid) is operably linked to the first promoter through an internal ribosome entry site (IRES) sequence.
  • IRES sequence comprises a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 19.
  • the polynucleotide comprises one or more second promoters.
  • the at least one additional antigen nucleic acid e.g., second antigen nucleic acid
  • the first promoter or the one or more second promoters is selected from the group consisting of: a cytomegalovirus (CMV) promoter, a Rouse sarcoma virus (RSV) promoter, a Moloney murine leukemia virus (Mo-MuLV) long terminal repeat (LTR) promoter, a mammalian elongation factor 1 (EFl) promoter, a cytokeratin 18 (CK18) promoter, a cytokeratin 19 (CK19) promoter, a simian virus 40 (SV40) promoter, a murine U6 promoter, a skeletal a-actin promoter, a P-actin promoter, a murine phosphoglycerate kinase 1 (PGK1) promoter, a human PGK1 promoter, a CBA promoter, a CAG promoter, and any combination thereof.
  • the mammalian EFl promoter is a cytomegalovirus (CMV)
  • the at least one additional antigen nucleic acid is under the control of a promoter selected from the group consisting of a CMV promoter, an RSV promoter, a Mo-MuLV LTR promoter, a mammalian EFl promoter, a CK18 promoter, a CK19 promoter, an SV40 promoter, a murine U6 promoter, a skeletal a-actin promoter, a P-actin promoter, a murine PGK1 promoter, a human PGK1 promoter, a CBA promoter, a CAG promoter, and any combination thereof.
  • the mammalian EFl promoter is a hEFl-HTLV promoter.
  • the antigen nucleic acid encodes a full-length tumor-associated antigen or an antigenic fragment thereof.
  • the antigen nucleic acid e.g., first antigen nucleic acid
  • the antigen nucleic acid is operably linked to a promoter.
  • the antigen nucleic acid e.g., first antigen nucleic acid
  • the mammalian EFl promoter is a hEFl-HTLV promoter.
  • the polynucleotides disclosed herein can further comprise one or more post- transcriptional regulatory elements.
  • the post-translational regulatory element is positioned 3' to a coding region of the polynucleotide.
  • Non-limiting examples of post-transcriptional regulatory elements that are useful for the present disclosure include a mutated woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), microRNA binding site, DNA nuclear targeting sequence, or combinations thereof.
  • WPRE woodchuck hepatitis virus post-transcriptional regulatory element
  • microRNA binding site DNA nuclear targeting sequence, or combinations thereof.
  • the post-transcriptional regulatory element is a WPRE.
  • the polynucleotide can also comprise one or more polyadenylation (poly(a)) signals, which can be downstream of any protein coding sequence.
  • polyadenylation signals include but are not limited to a SV40 poly(a) tail (SEQ ID NO: 12), LTR poly(a) tail, bovine growth hormone (bGH) poly(a) tail (SEQ ID NO: 13), human growth hormone (hGH) poly(a) tail, or human P-globin poly(a) tail.
  • the SV40 polyadenylation signal can be a polyadenylation signal from a pCEP4 vector (Invitrogen, San Diego, CA).
  • the polynucleotides disclosed herein further comprise at least one 3' UTR poly(a) tail sequence operably linked to the antigen nucleic acid (e.g., first antigen nucleic acid), the at least one additional antigen nucleic acid (e.g., second antigen nucleic acid), the nucleic acid encoding an immune modifier protein, or any combination thereof.
  • the 3' UTR poly(a) tail sequence is a 3' UTR SV40 poly(a) tail sequence, a 3' UTR bovine growth hormone (bGH) poly(A) sequence, a 3' UTR actin poly(A) tail sequence, a 3' UTR hemoglobin poly(A) sequence, or combinations thereof.
  • the polynucleotides disclosed herein can further comprise at least one enhancer sequence upstream of any protein coding sequence.
  • the enhancer can be necessary for DNA expression.
  • the enhancer is a human actin enhancer, human myosin enhancer, human hemoglobin enhancer, human muscle creatine enhancer, or a viral enhancer such as one from CMV, HA, RSV or EB V.
  • the enhancer is a polynucleotide function enhancer as described in U.S. Patent Nos. 5,593,972, 5,962,428, and WO 94/016737.
  • the enhancer sequence is a CMV intronic sequence or a P-actin intronic sequence.
  • the enhancer sequence is a SV40 enhancer sequence (SEQ ID NO: 17).
  • the polynucleotides disclosed herein can further comprise one or more inverted terminal repeats (ITRs).
  • ITRs inverted terminal repeats
  • the polynucleotide comprises a first ITR and a second ITR.
  • the polynucleotide comprises a first ITR, e.g., a 5' ITR, and a second ITR, e.g., a 3' ITR.
  • ITRs are involved in parvovirus (e.g., adeno- associated virus (AAV)) DNA replication and rescue, or excision, from prokaryotic plasmids (Samulski et al., 1983, 1987; Senapathy et al., 1984; Gottlieb and Muzyczka, 1988).
  • ITRs appear to be the minimum sequences required for AAV proviral integration and for packaging of AAV DNA into virions (McLaughlin et al., 1988; Samulski et al., 1989). These elements are essential for efficient multiplication of a parvovirus genome.
  • the ITRs fold into a hairpin T-shaped structure.
  • the ITRs fold into non-T-shaped hairpin structures, e.g., into a U-shaped hairpin structure.
  • the ITRs that are useful for the present disclosure comprise an ITR from an AAV genome.
  • the ITR is an ITR of an AAV genome selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and any combination thereof.
  • the ITR is an ITR of the AAV2 genome.
  • the ITR is a synthetic sequence genetically engineered to include at its 5' and 3' ends ITRs derived from one or more of AAV genomes.
  • the ITR is not derived from an AAV genome.
  • the ITR is an ITR of a non- AAV.
  • the ITR is an ITR of a non- AAV genome from the viral family Parvoviridae selected from, but not limited to, the group consisting of Bocavirus, Dependovirus, Erythrovirus, Amdovirus, Parvovirus, Densovirus, Iteravirus, Contravirus, Aveparvovirus, Copiparvovirus, Protoparvovirus, Tetraparvovirus, Ambidensovirus, Brevidensovirus, Hepandensovirus, Penstyldensovirus and any combination thereof.
  • the ITR is derived from erythrovirus parvovirus B19 (human virus). In some aspects, the ITR is derived from a Muscovy duck parvovirus (MDPV) strain. In certain aspects, the MDPV strain is attenuated, e.g., MDPV strain FZ91-30. In some aspects, the MDPV strain is pathogenic, e.g., MDPV strain YY. In some aspects, the ITR is derived from a porcine parvovirus, e.g., porcine parvovirus U44978. In some aspects, the ITR is derived from a mice minute virus, e.g., mice minute virus U34256.
  • MDPV Muscovy duck parvovirus
  • the ITR is derived from a canine parvovirus, e.g., canine parvovirus M19296. In some aspects, the ITR is derived from a mink enteritis virus, e.g., mink enteritis virus D00765. In some aspects, the ITR is derived from a Dependoparvovirus. In certain aspects, the Dependoparvovirus is a Dependovirus Goose parvovirus (GPV) strain. In some aspects, the GPV strain is attenuated, e.g., GPV strain 82-0321V. In some aspects, the GPV strain is pathogenic, e.g., GPV strain.
  • GPV Dependovirus Goose parvovirus
  • the polynucleotides disclosed herein can also comprise a mammalian origin of replication (e.g., an Epstein Barr virus origin of replication) in order to maintain the vector extrachromosomally and produce multiple copies of the vector in a cell.
  • a mammalian origin of replication e.g., an Epstein Barr virus origin of replication
  • the polynucleotide is a multi ci str onic mRNA.
  • the multi ci stronic mRNA comprises a 5' cap and a 3' UTR poly(A) tail sequence.
  • the 3' UTR poly(a) tail sequence is a 3' UTR SV40 poly(a) tail sequence (SEQ ID NO: 13), a 3' UTR bovine growth hormone (bGH) poly(A) sequence (SEQ ID NO: 13), a 3' UTR actin poly(A) tail sequence, a 3' UTR hemoglobin poly(A) sequence, or any combinations thereof.
  • the multi ci stronic mRNA comprises a 5' UTR and/or a 3' UTR.
  • the vector constructs can comprise the elements as illustrated in any of FIGs 1-12.
  • the vector constructs illustrated in any of FIGs 1-12 can modified to replace the “TAA Gene” (a first nucleotide sequence encoding a tumor- associated antigen) and the “TAA-2 Gene” (a second nucleotide sequence encoding a tumor-associated antigen) with nucleotide sequences encoding any combinations of tumor-associated antigen or antigenic fragment thereof disclosed herein.
  • the vector constructs disclosed herein can be used to express DNA or mRNA.
  • the vector constructs can comprise the elements as illustrated in any of FIGs. 14A-14O.
  • the vector constructs illustrated in any of FIGs. 14A-14M can modified to replace the “TAA” (a first nucleotide sequence encoding a tumor-associated antigen) and the “TAA-2” (a second nucleotide sequence encoding a tumor-associated antigen) with nucleotide sequences encoding any combinations of tumor-associated antigen or antigenic fragment thereof disclosed herein.
  • the vector constructs disclosed herein can be used to express DNA or mRNA.
  • the polynucleotides disclosed herein can comprise: a nucleotide sequence, wherein the nucleotide sequence encodes a tumor-associated antigen (e.g., NYESO-1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A9, MAGE-A10, MAGE-CI, MAGE-C2, SSX, H0RMAD1, CXorf61, ACTL8, AKAP3,AKAP4, HOM- TES-85, OY-TES-1, HSP70-2, LAGE-1, PIWL1, PIWL2, PIWL3, PIWL4, PLU-1, PLAC1, SPAG9, SCP-1, TAG-1, TAG-2a, TAG-2b, TAG-2c, TRAG-3, KAP4, BAGE, BORIS, CT45, GAGE-1/2, PRAME, PAGE4, SP17, PASD1, p53, ras, B-RafHER-2/neu, mu
  • the polynucleotide further comprises a nucleic acid sequence encoding one or more immune modifier proteins. In some aspects, the polynucleotide comprises a nucleic acid sequence encoding a tumor-associated antigen and, optionally, a second tumor-associated antigen.
  • the promoter is selected from the group consisting of: a CMV promoter, an RSV promoter, a Mo-MuLV LTR promoter, a mammalian EFl promoter, a CK18 promoter, a CK19 promoter, an SV40 promoter, a murine U6 promoter, a skeletal a-actin promoter, a P-actin promoter, a murine PGK1 promoter, a human PGK1 promoter, a CAG promoter, and any combination thereof.
  • the mammalian EFl promoter is a hEFl-HTLV promoter.
  • promoter 1 is selected from the group consisting of: a CMV promoter, an RSV promoter, a Mo-MuLV LTR promoter, a mammalian EFl promoter, a CK18 promoter, a CK19 promoter, and any combination thereof.
  • the IRES sequence comprises a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 19.
  • vectors or constructs comprising any polynucleotide described or exemplified herein, wherein the vector is a DNA plasmid vector, a multi ci str onic mRNA vector, a viral vector, a bacterial vector, a cosmid, or an artificial chromosome.
  • the vector is selected from an AAV vector, adenoviral vector, retroviral vector, poxvirus vector, baculovirus vector, herpes viral vector, or combinations thereof.
  • the vectors are DNA plasmid vectors and multi ci stronic mRNA vectors comprising any polynucleotide described or exemplified herein.
  • Host cells comprising any polynucleotide, vector, DNA plasmid vector, or vaccine described herein.
  • Host cells include prokaryotic cells, lower eukaryotic cells such as yeast, other eukaryotic cells such as insect cells, and higher eukaryotic cells such as mammalian cells.
  • Mammalian host cells include, but are not limited to, CHO, VERO, BHK, Hela, MDCK, HEK 293, NIH 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NSO (a murine myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7O3O, COS (e.g., COS1 or COS), PER.C6, VERO, HsS78Bst, HEK-293T, HepG2, SP210, Rl.l, B-W, L-M, BSC1, BSC40, YB/20, BMT10, HBK, NSO, HT1080 and HsS78Bst cells.
  • COS e.g., COS1 or COS
  • PER.C6 VERO
  • HsS78Bst HEK-293T
  • HepG2 SP210
  • Rl.l B-
  • the recombinant host cells arc prepared by introducing the vectors, polynucleotides, or vaccines described herein into the cells by techniques readily available to the person of ordinary skill in the art. These include, but are not limited to, calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, lipofection, and other techniques such as those found in Sambrook. et al. (Molecular Cloning : A Laboratory Manual. 2nd. ed.. Cold Spring Harbor Laboratory. Cold Spring Harbor Laboratory Press. Cold Spring Harbor. NY (1989).
  • the disclosed compositions, pharmaceutical compositions, vaccines, vectors, multi ci stronic mRNA vectors, or DNA plasmid vectors further comprise a delivery component.
  • the delivery component is a non-viral delivery component or system based on “naked DNA” or formulated plasmid DNA.
  • the delivery component or system can be used to deliver mRNA.
  • the non-viral delivery component is a cationic polymer.
  • the cationic polymer is a synthetic functionalized polymer, a lipid, a lipopolymer, or a chemical derivative thereof.
  • Non-viral gene delivery components or systems based on "naked DNA” or formulated plasmid DNA, have potential benefits over viral vectors due to simplicity of use and lack of inciting a specific immune response.
  • a number of synthetic gene delivery systems have been described to overcome the limitations of naked DNA, including cationic lipids, peptides, and polymers.
  • non-viral delivery systems can be used for delivery of mRNA.
  • PEI polyethyleneimine
  • PEI endosomolytic activity which enables PEI to yield high transfection efficiency.
  • Commercial branched PEI is composed of 25% primary amines, 50% secondary amines and 25% tertiary amines.
  • the overall protonation level of PEI doubles from pH 7 to pH 5, which means in the endosome PEI becomes heavily protonated.
  • Protonation of PEI triggers chloride influx across the endosomal membrane, and water follows to counter the high ion concentration inside the endosome, which eventually leads to endosomal disruption from osmotic swelling and release of the entrapped DNA.
  • Because of its intrinsic endosomolytic activity, PEI generally does not require the addition of an endosomolytic agent for transfection. Due to these advantages PEI has been increasingly utilized in polymer functionalization strategies to create safer and more efficient delivery systems.
  • the delivery component is a cationic polymer.
  • the cationic polymer is a synthetic functionalized polymer, a lipid, a lipopolymer, or a chemical derivative thereof.
  • the cationic polymer is present in an amount sufficient to produce a ratio of amine nitrogen in the cationic polymer to phosphate in the DNA plasmid vector or multi ci str onic mRNA vector from about 0.1 : 1 to about 100: 1.
  • the ratio of amine nitrogen in the cationic polymer to phosphate in the DNA plasmid vector or multi ci str onic mRNA vector is from about 0.1 : 1 to about 10: 1.
  • the composition, pharmaceutical composition, or vaccine comprises about 0.5 mg/ml to about 5.0 mg/ml nucleic acid complexed with the cationic polymer.
  • the delivery component is a poloxamer or a derivative thereof. In some aspects, the poloxamer or derivative thereof is present in a solution with the polynucleotide, multi ci str onic mRNA vector, or DNA plasmid vector from about 0.1% to about 5% or about 0.5% - about 5%. In some aspects, the delivery component is a P-amino ester.
  • the polymer is present in a solution with the polynucleotide, multicistronic mRNA vector, or DNA plasmid vector from about 0.1% to about 5% or about 0.5% - about 5%.
  • the delivery component is a poly-inosinic-polycytidylic acid.
  • the poly-inosinic-polycytidylic acid is present in a solution with the polynucleotide, multicistronic mRNA vector, or DNA plasmid vector from about 0.1% to about 5% or about 0.5% - about 5%.
  • the delivery component is a biodegradable cross-linked cationic multi-block copolymer, for example any biodegradable cross-linked cationic multi-block copolymer disclosed in U.S. Patent No. 8,445,017.
  • the biodegradable cross-linked cationic multi-block copolymer is a biodegradable cross-linked cationic multi -block copolymer of linear poly(alkylenimine) (LPAI) and a hydrophilic linker, wherein said LPAI blocks are crossed linked together by said hydrophilic linker with biodegradable ester, amide, disulfide, or phosphate linkages bonds.
  • LPAI linear poly(alkylenimine)
  • the linear poly(alkylenimine) (LPAI) is a member selected from the group consisting of polyethyleneimine, polypropylenimine, aminoglycoside-polyamine, dideoxy-diamino- .beta.-cyclodextrin, spermine and spermidine.
  • the linear poly(alkylenimine) (LPAI) is linear poly(ethylenimine) (LPEI).
  • poly(alkylenimine) (LPAI) is 3.6kD of LPEI (BD3.6-oleoyl).
  • the cross-linked cationic multi-block copolymer is linked by the biodegradable linkers to other moieties such as, for example, fluorescent markers, lipids anchors or their derivatives, i.e., cholesterol, fatty acids or their derivatives.
  • the molecular weight of the linear PEI used in this dislosure is within the range of 1,000 to 25,000 Dalton.
  • the linear PEI blocks are preferably linked to one another via a diamide linkage utilizing a biodegradable disulfidediacid-derived linker, i.e., dithiodipropionate derivatives.
  • the molar ratio of the linker to the PEI is within a range of 1/1 to 5/1; the molar ratio of the lipid anchors to PEI is from 0/1 to 3/1.
  • the polymer is formulated as a polyammonium salt, preferably with a chloride counterion. Since the toxicity of PEI increases with an increase in its molecular weight, the use of lower molecular weight PEIs as blocks in the polymer provides an improved gene carrier for use as a general reagent for transfection of mammalian cells, and for the in vivo application of gene therapy.
  • the biodegradable, cross-linked cationic multi-block copolymer comprises low molecular weight linear PEI blocks and a dithioacid moiety, i.e., dithiodipropionic acid, as biodegradable linkers.
  • the biodegradable, cross-linked cationic multi-block copolymers are synthesized by cross-linking low molecular weight linear PEI units via a biodegradable disulfide linkage.
  • These biodegradable cross-linked cationic multi-block copolymers are water soluble and transfectionally superior (68-70 fold higher activity) to single block polymers. See U.S. Patent No. 8,445,017.
  • the linker to the polymer molecular weight ratio is ⁇ 0.2 which minimizes the dilution of polyamine polymer backbone.
  • the chemical bond between the linker and the polymer blocks is a disulfide bond which can be biodegraded more easily as compared to amide bonds.
  • Other biodegradable bonds can also be used in the present disclosure includes: phosphoesters, hydrazone, cis-asotinyl, urethane and poly(ethyl). Since any linker reacts in stepwise fashion, it can link either different blocks or the different areas of the same block (loop formation). The latter will favor the formation of a lightly cross-linked material with poor solubility due to multiple looping.
  • the cationic block copolymers are represented by the following formula: (CP)vL,Yz wherein CP represents a cationic polymer containing at least one secondary amine group, said CP polymer has a number averaged molecular weight within the range of 1,000 to 25,000 Dalton; Y represents a bifunctional biodegradable linker containing ester, amide, disulfide, or phosphate linkages; L represents a ligand; x is an integer in the range from 1 to 20; y is an integer from 0 to 100; and z is an integer in the range from 0 to 40.
  • the cationic polymer comprises linear polyethyleneimine (LPEI).
  • the LPEI is BD15-12, which has the following formula: wherein the PEIs are approximately 15,000 Da, and wherein there is an average of 12 crosslinkers per PEI.
  • the LPEI is Omnifect, which has the following formula:
  • the bifunctional biodegradable linker is hydrophilic and comprises a biodegradable linkage comprising a disulfide bond. In some aspects, the bifunctional biodegradable linker is a dithiodipropionyl linker.
  • the biodegradable cross-linked cationic multi-block copolymer comprises LPEI and a dithiodipropionyl linker for cross-linking the multi-block copolymer, wherein the LPEI has an average molecular weight of 1,000 to 25,000 Dalton.
  • the biodegradable cross-linked cationic multi-block copolymer is covalently linked to at least one ligand.
  • the biodegradable, cross-linked, cationic, multi-block copolymers of LPEI and lipopolymers of the present disclosure have amine groups that are electrostatically attracted to polyanionic compounds such as nucleic acids.
  • the cationic copolymer condenses DNA and forms compact structures.
  • low toxicity of monomeric degradation products after delivery of bioactive materials provides for gene carriers with reduced cytotoxicity and increased transfection efficiency.
  • the biodegradable cross-linked cationic multi-block copolymers are conjugated with tracers (for example, fluorescent markers) or ligands either directly or via spacer molecules. In some aspects, only a small portion of the available amino groups is coupled to the ligand.
  • the ligands conjugated to the polymers are targeting ligands that direct the polymers-nucleic acid complex to bind to specific target cells and penetrate into such cells.
  • the targeting ligands can also be an intracellular targeting element, enabling the transfer of the nucleic acid/drug to be guided towards certain favored cellular compartments (mitochondria, nucleus, and the like).
  • the targeting ligands conjugated to the polymers direct the polymers-nucleic acid complex to bind to specific target cells and penetrate into such cells (e.g., epithelial cells, endothelial cells, hematopoietic cells, and the like).
  • the target ligands can also be an intracellular targeting element, enabling the transfer of the nucleic acid/drug to be guided towards certain favored cellular compartments (mitochondria, nucleus, and the like).
  • the targeting ligand is a polypeptide, folate, and an antigen.
  • the polypeptide is a glycoprotein (e.g., transferrin or asialoorosomucoid (ASOR)), an antibody, an antibody fragment, a cell receptor, a cytokine receptor, or a growth factor receptor (e.g., epidermal growth factor receptor).
  • the antigen is a tumor-associated antigen, a viral antigen, a bacterial antigen, or a parasite antigen.
  • the ligand is a fusogenic agent (e.g., polymixin B and hemaglutinin HA2), a lysosomotrophic agent, or a nucleus localization signal (NLS) (e.g., T-antigen, and the like).
  • the ligand is a sugar moiety coupled to an amino group.
  • the sugar moiety is a mono- or oligo-saccharide, such as galactose, glucose, fucose, fructose, lactose, sucrose, mannose, cellobiose, nytrose, triose, dextrose, trehalose, maltose, galactosamine, glucosamine, galacturonic acid, glucuronic acid, and gluconic acid.
  • a mono- or oligo-saccharide such as galactose, glucose, fucose, fructose, lactose, sucrose, mannose, cellobiose, nytrose, triose, dextrose, trehalose, maltose, galactosamine, glucosamine, galacturonic acid, glucuronic acid, and gluconic acid.
  • the biodegradable cross-linked cationic multi-block copolymer is covalently linked to polyethylene glycol (PEG) of molecular weight ranging from 500 to 20,000 Dalton.
  • PEG polyethylene glycol
  • the biodegradable cross-linked cationic multi-block copolymer is covalently linked to a fatty acyl chain selected from the group consisting of: oleic acid, palmitic acid, and stearic acid.
  • the biodegradable cross-linked cationic multi-block copolymer comprises at least one amine group that is electrostatically attracted to a polyanionic compound.
  • the polyanionic compound is a nucleic acid, wherein the biodegradable cross-linked cationic multi -block copolymer condenses the nucleic acid to form a compact structure.
  • the biodegradable cross-linked cationic multi-block copolymer has the following formula: wherein A and B are such that the molecular weight of the individual linear polyethylenimine chains are from 5,000 to 25,000 Dalton; the intermolecular crosslinks connect approximately 5-10% of amines; the biodegradable crosslinks are dithiodipropionyl (each half composed of 3 carbon atoms) and can be from 1-10 carbon atoms.
  • the biodegradable cross-linked cationic polymer comprises 10,000 to 15,000 Dalton linear PEI covalently connected with a dithiopropionyl linkage (see, e.g., U.S. Patent No. 8,445,017).
  • the biodegradable cross-linked cationic polymer comprises 10,000 to 15,000 Dalton linear PEI covalently connected with a dithiopropionyl linkage (see, e.g., U.S. Patent No. 8,445,017) and is further conjugated to polyethyleneglycol (PEG) of molecular weight ranging from 500 to 20,000 Dalton.
  • the biodegradable cross-linked cationic polymer comprises 10,000 to 15,000 Dalton linear PEI covalently connected with a dithiopropionyl linkage (see, e.g., U.S. Patent No. 8,445,017) and is further conjugated to polyethyleneglycol (PEG) of molecular weight ranging from 500 to 20,000 Dalton.
  • the biodegradable cross-linked cationic polymer comprises 15,000 to 20,000 Dalton linear PEI covalently connected with a dithiopropionyl linkage (see, e.g., U.S. Patent No. 8,445,017). In some aspects, the biodegradable cross-linked cationic polymer comprises 15,000 to 20,000 Dalton linear PEI covalently connected with a dithiopropionyl linkage (see, e.g., U.S. Patent No. 8,445,017) and is further conjugated to polyethyleneglycol (PEG) of molecular weight ranging from 500 to 20,000 Dalton.
  • PEG polyethyleneglycol
  • the biodegradable cross-linked cationic multi-block copolymer is present in an amount sufficient to produce a ratio of amine nitrogen in the biodegradable cross-linked cationic multi-block copolymer to phosphate in the polynucleotide, multi ci str onic mRNA vector, or DNA plasmid vector from about 0.01 : 1 to about 50: 1 (e.g., about 0.01 : 1 to about 40: 1; about 0.01 : 1 to about 30: 1; about 0.01 : 1 to about 20: 1; about 0.01 : 1 to about 10: 1, or about 0.01 : 1 to about 5: 1).
  • the ratio of amine nitrogen in the biodegradable cross-linked cationic multi-block copolymer to phosphate in the polynucleotide, multi ci str onic mRNA vector, or DNA plasmid vector is from about 0.1 : 1 to about 50: 1 (e.g., about 0.1 : 1 to about 40: 1; about 0.1 : 1 to about 30: 1; about 0.1 : 1 to about 20:1; about 0.1 : 1 to about 10: 1, or about 0.1 : 1 to about 5:1).
  • the ratio of amine nitrogen in the biodegradable cross-linked cationic multi-block copolymer to phosphate in the polynucleotide, multi ci str onic mRNA vector, or DNA plasmid vector is from about 1 : 10 to about 10: 1.
  • the composition, pharmaceutical composition, or vaccine comprises about 0.1 mg/ml to about 10 mg/ml (e.g., about 0.1 mg/ml to about 5 mg/ml; about 0.5 mg/ml to about 10 mg/ml; or about 0.5 mg/ml to about 5 mg/ml) nucleic acid complexed with the biodegradable cross-linked cationic multi-block copolymer.
  • the composition, pharmaceutical composition, or vaccine comprises about 1 mg/ml to about 10 mg/ml (e.g., about 1 mg/ml to about 6 mg/ml; about 2 mg/ml to about 6 mg/ml; about 5 mg/ml to about 10 mg/ml; or about 6 mg/ml to about 10 mg/ml) nucleic acid complexed with the biodegradable cross-linked cationic multi-block copolymer.
  • VI.2 Cationic Lipopolymers Comprising a PEI Backbone
  • the delivery component is a cationic lipopolymer comprising a PEI backbone covalently linked to a lipid or a polyethylene glycol (PEG) as disclosed in U.S. Patent No. 7,964,571.
  • the PEI backbone is covalently linked to a lipid and a PEG.
  • the lipid and the PEG are directly attached to the PEI backbone by covalent bonds.
  • the lipid is attached to the PEI backbone through a PEG spacer.
  • the PEG has a molecular weight of between 50 to 20,000 Dalton.
  • the molar ratio of PEG to PEI is within a range of 0.1 : 1 to 500: 1.
  • the molar ratio of the lipid to the PEI is within a range of 0.1 : 1 to 500: 1.
  • the lipid is a cholesterol, a cholesterol derivative, a C12 to Cis fatty acid, or a fatty acid derivative.
  • the addition of PEG enhances the stability of the nucleic acid/polymer complexes in the biological milieu and allows for the incorporation of ligands (e.g., a targeting ligand) on to the PPC chain to improve the tissue selectivity of delivery.
  • ligands e.g., a targeting ligand
  • the cationic lipopolymer is a PEG:PEI: cholesterol (PPC) lipopolymer, which comprises a PEI backbone covalently linked to cholesterol and PEG.
  • the PEI is covalently linked to cholesterol and PEG, and wherein the average PEG:PEI:cholesterol molar ratio in the cationic lipopolymer is within the range of 1-5 PEG: 1 PEI:0.4-1.5 cholesterol.
  • the PEG-PEI-cholesterol (PPC) lipopolymer has an average PEG:PEI:cholesterol ratio of 2.5: 1 :0.6.
  • the PEI has a linear or branch configuration with a molecular weight of 100 to 500,000 Dalton.
  • the cationic lipopolymer further comprises a pendant functional moiety selected from the group consisting of: a receptor ligand, a membrane permeating agent, an endosomolytic agent, a nuclear localization sequence, and a pH sensitive endosomolytic peptide.
  • the cationic lipopolymer further comprises a targeting ligand, wherein the targeting ligand is directly attached to the PEI backbone or is attached through a PEG linker.
  • the targeting ligand is selected from the group consisting of: a sugar moiety, a polypeptide, folate, and an antigen.
  • the sugar moiety is a monosaccharide.
  • the monosaccharide is galactose.
  • the sugar moiety is an oligosaccharide.
  • the polypeptide is a glycoprotein, an antibody, an antibody fragment, a cell receptor, a cytokine receptor, or a growth factor receptor.
  • the growth factor receptor is an epidermal growth factor receptor.
  • the glycoprotein is transferrin or asialoorosomucoid (ASOR).
  • the antigen is a tumor-associated antigen, a viral antigen, a bacterial antigen, or a parasite antigen.
  • the cationic lipopolymer is present in an amount sufficient to produce a ratio of amine nitrogen in the cationic polymer to phosphate in the polynucleotide, multi ci str onic mRNA vector, or DNA plasmid vector from about 0.01 : 1 to about 50: 1 (e.g., about 0.01 : 1 to about 40:1; about 0.01 : 1 to about 30: 1; about 0.01 :1 to about 20:1; about 0.01 : 1 to about 10: 1, or about 0.01 :1 to about 5: 1).
  • the ratio of amine nitrogen in the cationic lipopolymer to phosphate in the polynucleotide, multi ci str onic mRNA vector, or DNA plasmid vector is from about 0.1 : 1 to about 50: 1 (e.g., about 0.1 : 1 to about 40: 1; about 0.1 : 1 to about 30: 1; about 0.1 : 1 to about 20: 1; about 0.1 : 1 to about 10: 1, or about 0.1 : 1 to about 5: 1).
  • the ratio of amine nitrogen in the cationic lipopolymer to phosphate in the polynucleotide, multi ci stronic mRNA vector, or DNA plasmid vector is from about 1 : 10 to about 10: 1.
  • the composition, pharmaceutical composition, or vaccine comprises about 0.1 mg/ml to about 10.0 mg/ml (e.g., about 0.1 mg/ml to about 5 mg/ml; about 0.5 mg/ml to about 10 mg/ml; or about 0.5 mg/ml to about 5 mg/ml) nucleic acid complexed with the cationic polymer.
  • the composition, pharmaceutical composition, or vaccine comprises about 1 mg/ml to about 10 mg/ml (e.g., about 1 mg/ml to about 6 mg/ml; about 2 mg/ml to about 6 mg/ml; about 5 mg/ml to about 10 mg/ml; or about 6 mg/ml to about 10 mg/ml) nucleic acid complexed with the cationic polymer.
  • the delivery component of the composition, pharmaceutical composition, or vaccine comprises a lipopolyamine with the following formula: (Staramine). [0346] In some aspects, the delivery component comprises a mixture of the lipopolyamine and an alkylated derivative of the lipopolyamine. In some aspects, the alkylated derivative of the lipopolyamine is a polyoxyalkylene, polyvinylpyrrolidone, polyacrylamide, polydimethylacrylamide, polyvinyl alcohol, dextran, poly (L-glutamic acid), styrene maleic anhydride, poly-N-(2-hydroxypropyl) methacrylamide, or polydivinylether maleic anhydride. In some aspects, the alkylated derivative of the lipopolyamine has the following formula:
  • n is an integer from 10 to 100 repeating units containing 2-5 carbon atoms each.
  • the ratio of the lipopolyamine to the alkylated derivative of the lipopolyamine in the mixture is 1 : 1 to 10: 1.
  • the lipopolyamine is present in an amount sufficient to produce a ratio of amine nitrogen in the lipopolyamine to phosphate in the polynucleotide, multi ci str onic mRNA vector, or DNA plasmid vector from about 0.01:1 to about 50:1 (e.g., about 0.01:1 to about 40:1; about 0.01:1 to about 30:1; about 0.01:1 to about 20:1; about 0.01:1 to about 10:1, or about 0.01:1 to about 5:1).
  • the ratio of amine nitrogen in the lipopolyamine to phosphate in the polynucleotide, multi ci stronic mRNA vector, or DNA plasmid vector is from about 0.1:1 to about 50:1 (e.g., about 0.1:1 to about 40:1; about 0.1:1 to about 30:1; about 0.1:1 to about 20:1; about 0.1:1 to about 10:1, or about 0.1:1 to about 5:1). In some aspects, the ratio of amine nitrogen in the lipopolyamine to phosphate in the polynucleotide, multi ci stronic mRNA vector, or DNA plasmid vector is from about 1 : 10 to about 10:1.
  • the delivery component of the composition, pharmaceutical composition, or vaccine comprises a lipopolyamine with the following formula: (Crossamine).
  • the delivery component comprises a mixture of the lipopolyamine and an alkylated derivative of the lipopolyamine.
  • the alkylated derivative of the lipopolyamine is a polyoxyalkylene, polyvinylpyrrolidone, polyacrylamide, polydimethylacrylamide, polyvinyl alcohol, dextran, poly (L-glutamic acid), styrene maleic anhydride, poly-N-(2-hydroxypropyl) methacrylamide, or polydivinylether maleic anhydride.
  • the ratio of the lipopolyamine to the alkylated derivative of the lipopolyamine in the mixture is 1:1 to 10:1.
  • the lipopolyamine is present in an amount sufficient to produce a ratio of amine nitrogen in the lipopolyamine to phosphate in the polynucleotide, multi ci stronic mRNA vector, or DNA plasmid vector from about 0.01 : 1 to about 50: 1 (e.g., about 0.01 : 1 to about 40: 1; about 0.01:1 to about 30:1; about 0.01:1 to about 20:1; about 0.01:1 to about 10:1, or about 0.01:1 to about 5:1).
  • a ratio of amine nitrogen in the lipopolyamine to phosphate in the polynucleotide, multi ci stronic mRNA vector, or DNA plasmid vector from about 0.01 : 1 to about 50: 1 (e.g., about 0.01 : 1 to about 40: 1; about 0.01:1 to about 30:1; about 0.01:1 to about 20:1; about 0.01:1 to about 10:1, or about 0.01:1 to about 5:1).
  • the ratio of amine nitrogen in the lipopolyamine to phosphate in the polynucleotide, multi ci stronic mRNA vector, or DNA plasmid vector is from about 0.1 : 1 to about 50: 1 (e.g., about 0.1 : 1 to about 40: 1; about 0.1:1 to about 30:1; about 0.1:1 to about 20:1; about 0.1:1 to about 10:1, or about 0.1:1 to about 5:1). In some aspects, the ratio of amine nitrogen in the lipopolyamine to phosphate in the polynucleotide, multi ci stronic mRNA vector, or DNA plasmid vector is from about 1:10 to about 10:1.
  • the composition, pharmaceutical composition, or vaccine comprises about 0.1 mg/ml to about 10.0 mg/ml (e.g., about 0.1 mg/ml to about 5 mg/ml; about 0.5 mg/ml to about 10 mg/ml; or about 0.5 mg/ml to about 5 mg/ml) nucleic acid complexed with the lipopolyamine or derivative thereof.
  • the composition, pharmaceutical composition, or vaccine comprises about 1 mg/ml to about 10 mg/ml (e.g., about 1 mg/ml to about 6 mg/ml; about 2 mg/ml to about 6 mg/ml; about 5 mg/ml to about 10 mg/ml; or about 6 mg/ml to about 10 mg/ml) nucleic acid complexed with the lipopolyamine or derivative thereof.
  • the polynucleotide, vector, multicistronic mRNA vector, or DNA plasmid vector of the disclosure is complexed with or encapsulated by a delivery component, e.g., a lipopolymer.
  • a delivery component e.g., a lipopolymer.
  • At least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the polynucleotide, vector, multicistronic mRNA vector, or DNA plasmid vector is encapsulated by the delivery component, e.g., a lipopolymer.
  • about 20% to about 100%, about 20% to about 80%, about 20% to about 60%, or about 40% to about 80% of the polynucleotide, vector, multicistronic mRNA vector, or DNA plasmid vector of the disclosure is encapsulated by the delivery component, e.g., a lipopolymer.
  • the delivery component forms a micellular structure.
  • the delivery component comprises a poloxamer back-bone having a metal chelator covalently coupled to at least one terminal end of the poloxamer backbone (e.g. Crown Poloxamer).
  • the metal chelator is coupled to at least two terminal ends of the poloxamer backbone.
  • At least one metal chelator is coupled to a poloxamer. In some aspects, at least two metal chelators are coupled to a poloxamer. In some aspects, 1-5, 5- 10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 50-55, 55-60, 60-65, 65-70, 70-75, 75-80, 80-85, 85-90, 90-95, or 95-100 metal chelators are coupled to a poloxamer.
  • the poloxamer backbone is a poloxamer backbone disclosed in U.S. Publ. No. 2010/0004313, which is herein incorporated by reference in its entirety.
  • the metal chelator is a metal chelator disclosed in U.S. Publ. No. 2010/0004313.
  • the delivery component of the composition, pharmaceutical composition, or vaccine comprises a polymer having the following formula: or pharmaceutically acceptable salts thereof, wherein:
  • A represents an integer from 2 to 141;
  • B represents an integer from 16 to 67;
  • C represents an integer from 2 to 141;
  • R 4 and R c are the same or different, and are R'-L- or H, wherein at least one of R 4 and R c is R'-L-;
  • L is a bond, — CO — , — CH2 — O — , or — O — CO — ;
  • R' is a metal chelator
  • the metal chelator is R? NH — , R.SN — , or (R" — (N(R") — CILCIL h — N — CH2CO — , wherein each x is independently 0-2, and wherein R" is HO2C— CH2— .
  • the delivery component comprises a poloxamer with the following formula: or a pharmaceutically acceptable salt thereof, wherein:
  • A represents an integer from 2 to 141;
  • B represents an integer from 16 to 67;
  • C represents an integer from 2 to 141;
  • RA and RC are the same or different, and are R'-L- or H;
  • L is a bond, — CO — , — CH2 — O — , or — O — CO — ;
  • R' is a metal chelator
  • At least one of RA and RC is R'-L-.
  • the R’ is covalently bound to the poloxamer (e.g., where a metal chelator (e.g., a crown) is covalently attached at different density to the poloxamer).
  • a metal chelator e.g., a crown
  • one metal chelator or two or more metal chelators is/are bound to the poloxamer.
  • 2-100 metal chelators are bound to the poloxamer.
  • the metal chelator is a crown ether selected from the group consisting of 12-crown-4, 15-crown-5, 18-crown-6, 20-crown-6, 21-crown-7, and 24- crown-8.
  • the crown ether is a substituted-crown ether, wherein the substituted-crown ether has:
  • the metal chelator is a cryptand, wherein the cryptand is selected from the group consisting of (1,2,2) cryptand, (2,2,2) cryptand, (2,2,3) cryptand, and (2,3,3) cryptand.
  • the cryptand is a substituted-cryptand, wherein the substituted cryptand has:
  • the delivery component is Crown Poloxamer (aza-crown-linked poloxamer), wherein the Crown Poloxamer comprises a polymer having the following formula:
  • a represents an integer of about 10 units; and b represents an integer of about 21 units; and wherein the total molecular weight of the polymer is about 2,000 Da to about 2,200 Da.
  • the crown poloxamer can be derivatized with a cationic molecule, a ligand, or other chemical entities.
  • the polymer or poloxamer (e.g., crown poloxamer)is present in a solution with the polynucleotide, multi ci str onic mRNA vector, or DNA plasmid vector from about 0.01% - about 5% or about 0.5% - about 5%.
  • the solution is co-formulated with a metal chelator (e.g., where the co-formulated metal chelator is a free metal chelator, which is formulated with the poloxamer (e.g., a non-crown poloxamer)).
  • a metal chelator e.g., where the co-formulated metal chelator is a free metal chelator, which is formulated with the poloxamer (e.g., a non-crown poloxamer)).
  • the co-formulated metal chelator is present in the solution at a concentration of about O.lmg/mL to about 20mg/mL.
  • the co-formulated metal chelator is crown ether, a substituted- crown ether, a cryptand, or a substituted-cryptand.
  • the metal chelator or co-formulated metal chelator is crown ether (Aza- 18 -crown-6) .
  • the delivery component further comprises benzalkonium chloride (BAK).
  • BAK benzalkonium chloride
  • the delivery component comprises BD15-12.
  • the ratio of nucleotide to BD15-12 polymer is 0.1 : 1 to 5: 1.
  • the delivery component comprises Omnifect. In some aspects, the ratio of nucleotide to Omnifect polymer (N:P) is 0.1 : 1 to5: l. [0375] In some aspects, the delivery component comprises Crown Poloxamer (azacrown-linked poloxamer). In some aspects, the ratio of nucleotide to Crown Poloxamer (N:P) is 0.1 : 1 to 5: 1. In some aspects, the delivery component comprises Crown Poloxamer and/or a PEG-PEI-cholesterol (PPC) lipopolymer. In some aspects, the delivery component comprises Crown Poloxamer and/or benzalkonium chloride. In some aspects, the delivery component comprises Crown Poloxamer and/or Omnifect. In some aspects, the delivery component comprises Crown Poloxamer and/or a linear polyethyleneimine (LPEI). In some aspects, the delivery component comprises Crown Poloxamer and/or BD15-12.
  • the delivery component comprises Crown Poloxamer and/or BD15-12.
  • the delivery component comprises Staramine and/or mPEG modified Staramine.
  • the mPEG modified Staramine is Staramine- mPEG515.
  • the mPEG modified Staramine is Staramine-mPEGl 1.
  • the ratio of Staramine to mPEG modified Staramine is 10: 1.
  • the nucleotide to polymer (N:P) ratio is 0.1 : 1 to 5: 1.
  • the delivery component comprises Staramine, mPEG modified Staramine, and/or Crown Poloxamer.
  • the delivery component comprises Staramine, Staramine-mPEG515, and/or Crown Poloxamer.
  • the delivery component comprises a Staramine, a Staramine-mPEGl 1, and/or a Crown Poloxamer.
  • the metal chelator is added directly to a solution comprising (i) the polynucleotide, multi ci stronic mRNA vector, or DNA plasmid vector as described herein, and (ii) a poloxamer.
  • the metal chelator is present in the solution at concentration of about O. lmg/mL to about 20mg/mL. In some aspects, metal chelator is present in the solution at concentration of about O.lmg/mL to about 20mg/mL, about O.
  • the metal chelator is crown ether, a substituted-crown ether, a cryptand, or a substituted-cryptand. In some aspects, the metal chelator is crown ether (Aza-18-crown- 6).
  • the metal chelator is added directly to a solution comprising (i) the polynucleotide, multi ci stronic mRNA vector, or DNA plasmid vector as described herein, (ii) a poloxamer, and (iii) an an adjuvant comprising an aluminum or aluminum- salt based adjuvant, a stimulator of interferon genes (STING) agonist, or a combination thereof.
  • the metal chelator is present in the solution at concentration of about O.lmg/mL to about 20mg/mL. In some aspects, metal chelator is present in the solution at concentration of about O.
  • the metal chelator is crown ether, a substituted-crown ether, a cryptand, or a substituted-cryptand. In some aspects, the metal chelator is crown ether (Aza-18-crown- 6). In some aspects, the adjuvant is AIPO4.
  • the metal chelator is added directly to a solution comprising (i) the polynucleotide, multi ci stronic mRNA vector, or DNA plasmid vector as described herein, (ii) a poloxamer, and (iii) an an adjuvant comprising an aluminum or aluminum- salt based adjuvant, or a combination thereof.
  • the metal chelator is present in the solution at concentration of about O.lmg/mL to about 20mg/mL. In some aspects, metal chelator is present in the solution at concentration of about 0. Img/mL to about 20mg/mL, about O.
  • the metal chelator is crown ether, a substituted-crown ether, a cryptand, or a substituted-cryptand.
  • the metal chelator is crown ether (Aza-18-crown-6).
  • the adjuvant is AIPO4.
  • the present disclosure also features containers comprising any polynucleotide, vector, vaccine, composition, or pharmaceutical composition described and exemplified herein.
  • the container is a glass vial.
  • kits comprising any polynucleotide, vector, vaccine, composition, or pharmaceutical composition described and exemplified herein.
  • the kits can be used to supply polynucleotides, vectors, vaccines, composition, pharmaceutical compositions, and other agents for use in diagnostic, basic research, or therapeutic methods, among others.
  • the kits comprise any one or more of the polynucleotides, vectors, vaccines, composition, or pharmaceutical compositions described or exemplified herein and instructions for using the one or more polynucleotides, vectors, vaccines, composition, or pharmaceutical compositions in a method for inducing an immune response in a subject.
  • kits comprise any one or more of the polynucleotides, vectors, vaccines, composition, or pharmaceutical compositions described or exemplified herein and instructions for using the one or more polynucleotides, vectors, vaccines, composition, or pharmaceutical compositions in a method for preventing, reducing the growth of, attenuating or treating a tumor in a subject.
  • the kit comprises a composition, pharmaceutical composition, or vaccine that is liquid and is stable at 0°C to 5°C for at least about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 monts, or about 12 months.
  • the liquid composition, pharmaceutical composition, or vaccine is stable at 25°C for at least about 7 days, about 10 day, or about 14 days, or about 60 days.
  • the kit comprises a composition, pharmaceutical composition, or vaccine that is liquid and is stable at -20°C for at least about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 monts, or about 12 months.
  • the kit comprises a composition, pharmaceutical composition, or vaccine that is lyophilized and is stable at 0°C to 5°C for at least about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 monts, or about 12 months.
  • the lyophilized and reconstituted composition, pharmaceutical composition, or vaccine is stable at 25°C for at least about 7 days, about 10 day, or about 14 days, or about 60 days.
  • the kit comprises a composition, pharmaceutical composition, or vaccine that is lyophilized and is stable at -20°C for at least about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 monts, or about 12 months.
  • the lyophilized composition is substantially free of aqueous components.
  • the kit further comprises a dilent and the composition is reconstituted with the diluent.
  • the diluent is water.
  • the present disclosure also features methods of inducing an immune response in a subject, the method comprising administering an effective amount of any polynucleotide, vector, multi ci str onic mRNA vector, DNA plasmid vector, composition, or pharmaceutical composition described or exemplified herein to the subject.
  • the immune response is to one or more tumor-associated antigens disclosed herein, e.g., NYESO-1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A9, MAGE-A10, MAGE-CI, MAGE-C2, SSX, H0RMAD1, CXorf61, ACTL8, AKAP3,AKAP4, HOM-TES-85, OY-TES-1, HSP70-2, LAGE-1, PIWL1, PIWL2, PIWL3, PIWL4, PLU-1, PLAC1, SPAG9, SCP-1, TAG-1, TAG-2a, TAG-2b, TAG-2c, TRAG-3, KAP4, BAGE, BORIS, CT45, GAGE-1/2, PRAME, PAGE4, SP17, PASD1, p53, ras, B-RafHER-2/neu, mucin 1 (MUC1), epidermal growth factor receptor (EGFR), GP100, prostatic acid phosphata
  • the present disclosure also features methods of preventing, reducing the incidence of, attenuating or treating a tumor (e.g., an ovarian cancer, melanoma, colorectal cancer, liver cancer, pancreatic cancer, brain cancer, prostate cancer, bladder cancer, renal cancer, or hematological cancer).
  • a tumor e.g., an ovarian cancer, melanoma, colorectal cancer, liver cancer, pancreatic cancer, brain cancer, prostate cancer, bladder cancer, renal cancer, or hematological cancer.
  • tumor in a subject, the method comprising administering an effective amount of any polynucleotide, vector, multi ci str onic mRNA vector, DNA plasmid vector, composition, or pharmaceutical composition described or exemplified herein to the subject.
  • the methods of the disclosure are directed to administration of a composition, pharmaceutical composition, or vaccine disclosed herein.
  • the process of administration can be varied, depending on the agent, or agents, and the desired effect.
  • the process of administration involves administering a therapeutic agent (e.g., any composition or pharmaceutical composition, polynucleotide, vector, vaccine, disclosed herein) to a patient in need of such treatment.
  • a therapeutic agent e.g., any composition or pharmaceutical composition, polynucleotide, vector, vaccine, disclosed herein
  • Methods of delivering compositions comprising DNA vaccines are described in U.S. Patent Nos. 4,945,050 and 5,036,006.
  • Administration can be accomplished by any means appropriate for the therapeutic agent, for example, by parenteral means of delivery.
  • Parenteral delivery can include for example, subcutaneous, intravenous, intramuscular, intra-arterial, intraperitoneal, intralymphatic, and injection into the tissue of an organ (e.g., intratumoral).
  • Administration can generally also include delivery with a pharmaceutically acceptable carrier, such as, for example, a buffer, a polypeptide, a peptide, a polysaccharide conjugate, a liposome, and/or a lipid, according to methods known in the art.
  • a pharmaceutically acceptable carrier such as, for example, a buffer, a polypeptide, a peptide, a polysaccharide conjugate, a liposome, and/or a lipid, according to methods known in the art.
  • Pulmonary delivery can include inhalation of the agent.
  • Catheter-based delivery can include delivery by iontr opheretic catheter-based delivery.
  • Oral delivery can include delivery of a coated pill, or administration of a liquid by mouth.
  • Administration can generally also include delivery with a pharmaceutically acceptable carrier, such as, for example, a buffer, a polypeptide, a peptide, a polysaccharide conjugate, a liposome, and/or a lipid, according to methods known in the art.
  • the viral vectors of the present disclosure are administered in an amount approximately corresponding to 10 2 to 10 14 PFU, 10 5 to 10 12 PFU, or 10 6 to IO 10 PFU per subject, calculated as the PFU of the viral vector.
  • the viral vectors of the present disclosure are administered by directly injecting a viral vector suspension prepared by suspending the viral vector in PBS (phosphate buffered saline) or saline into a local site (e.g., into the lung tissue, liver, muscle or brain), by nasal or respiratory inhalation, or by intravascular (e.g., intra-arterial, intravenous, and portal venous), intralymphatic, subcutaneous, intracutaneous, intradermal, or intraperitoneal administration.
  • PBS phosphate buffered saline
  • saline into a local site (e.g., into the lung tissue, liver, muscle or brain), by nasal or respiratory inhalation, or by intravascular (e.g., intra-arterial, intravenous, and portal ve
  • compositions comprising (i) a vector (e.g., a multi ci stronic DNA plasmid vector or a multi ci str onic messenger RNA (mRNA) vector) comprising a nucleic acid sequence encoding one or more tumor-associated antigens (e.g., NYESO-1, MAGE-A1, MAGE- A2, MAGE-A3, MAGE-A4, MAGE-A9, MAGE-A10, MAGE-CI, MAGE-C2, SSX, H0RMAD1, CXorf61, ACTL8, AKAP3,AKAP4, HOM-TES-85, OY-TES-1, HSP70-2, LAGE-1, PIWL1, PIWL2, PIWL3, PIWL4, PLU-1, PLAC1, SPAG9, SCP-1, TAG-1, TAG-2a, TAG-2b, TAG-2c, TRAG-3, KAP4, BAGE, BORIS,
  • a vector e.g., a multi ci stronic
  • the method further comprises obtaining a nucleic acid sequence of a subject’s tumor-associated antigens (e.g., NYESO-1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A9, MAGE-A10, MAGE-CI, MAGE-C2, SSX, H0RMAD1, CXorf61, ACTL8, AKAP3,AKAP4, HOM-TES-85, OY-TES-1, HSP70-2, LAGE-1, PIWL1, PIWL2, PIWL3, PIWL4, PLU-1, PLAC1, SPAG9, SCP-1, TAG-1, TAG-2a, TAG-2b, TAG-2c, TRAG-3, KAP4, BAGE, BORIS, CT45, GAGE-1/2, PRAME, PAGE4, SP17, PASD1, p53, ras, B-RafHER-2/neu, mucin 1 (MUC1), epidermal growth factor receptor (EGFR)
  • the adjuvant comprises an aluminum or aluminum-salt based adjuvant and a STING agonist.
  • the aluminum or aluminum-salt based adjuvant comprises an aluminum phosphate, an aluminum hydroxide, an aluminum oxyhydroxide, or any combination thereof.
  • the STING agonist is cyclic guanosine monophosphate (cGMP), cyclic adenosine monophosphate (cAMP), or cyclic guanosine monophosphate-ad enosine monophosphate (cGAMP).
  • the adjuvant is M59 (oil-in-water emulsion of squalene oil).
  • the adjuvant is AS03 (a-tocopherol, squalenem and polysorbate 80 in an oil-in-water emulsion).
  • the vector further comprises a nucleic acid sequence encoding one or more immune modifier proteins.
  • the vector comprises a nucleic acid sequence encoding a tumor-associated antigen and, optionally, a second tumor- associated antigen.
  • Some aspects relate to methods of eliciting humoral and/or cellular immune response against a tumor following in vivo administration of a multicistronic DNA plasmid, a multicistronic mRNA vector, or a composition comprising the same of the disclosure.
  • compositions including (i) a DNA plasmid vector comprising a DNA sequence of one or more antigens; (ii) a delivery component, such as a synthetic non-viral DNA carrier (e.g., a cationic polymer, a poly-inosinic-polycytidylic acid, or a poloxamer); and (iii) an adjuvant comprising an aluminum or aluminum-salt based adjuvant, a stimulator of interferon genes (STING) agonist, or a combination thereof.
  • a delivery component such as a synthetic non-viral DNA carrier (e.g., a cationic polymer, a poly-inosinic-polycytidylic acid, or a poloxamer)
  • an adjuvant comprising an aluminum or aluminum-salt based adjuvant, a stimulator of interferon genes (STING) agonist, or a combination thereof.
  • STING stimulator of interferon genes
  • the delivery component of the compositions disclosed herein may comprise any combination of delivery components disclosed herein.
  • the tumor is an ovarian cancer, melanoma, colorectal cancer, liver cancer, pancreatic cancer, brain cancer, prostate cancer, bladder cancer, renal cancer, or hematological cancer.
  • the one or more antigens comprise one or more tumor-associated antigens. In some aspects the one or more tumor-associated antigens comprise two or more portions of the same antigen.
  • compositions including (i) a multicistronic DNA plasmid vector comprising a DNA sequence of one or more tumor associated antigens and optionally a DNA sequence of one or more immune modifier proteins; (ii) a delivery component, such as a synthetic non-viral DNA carrier (e.g., a cationic polymer, a poly-inosinic-polycytidylic acid, or a poloxamer); and (iii) an adjuvant comprising an aluminum or aluminum-salt based adjuvant, a stimulator of interferon genes (STING) agonist, or a combination thereof.
  • a delivery component such as a synthetic non-viral DNA carrier (e.g., a cationic polymer, a poly-inosinic-polycytidylic acid, or a poloxamer)
  • an adjuvant comprising an aluminum or aluminum-salt based adjuvant, a stimulator of interferon genes (STING) agonist, or
  • compositions further comprise one or more immunopotentiators that are capable of activating the innate immunity system.
  • the immunopotentiators are selected from a non-coding DNA (e.g., concatamers of non-coding 5'-C-phosphate-G-3' (CpG) dinucleotides), a non-coding RNA, a small molecule, or any combination thereof.
  • compositions including (i) a multi ci stronic RNA vector comprising an RNA sequence of one or more tumor associated antigens and opionally an RNA sequence of one or more immune modifier proteins; (ii) a delivery component, such as a synthetic non-viral RNA carrier/adjuvant (e.g., a cationic polymer, a poly-inosinic-poly cytidylic acid, or a pol oxamer); and (iii) an adjuvant comprising an aluminum or aluminum-salt based adjuvant, a stimulator of interferon genes (STING) agonist, or a combination thereof.
  • a delivery component such as a synthetic non-viral RNA carrier/adjuvant (e.g., a cationic polymer, a poly-inosinic-poly cytidylic acid, or a pol oxamer)
  • an adjuvant comprising an aluminum or aluminum-salt based
  • Some aspects relate to methods of eliciting humoral and/or cellular immune response against a tumor-associated antigen following in vivo administration of a multi ci stronic DNA plasmid vector, a multi ci stronic mRNA vector, or a composition comprising the same of the disclosure.
  • compositions comprising a polynucleotide comprising: (a) an antigen nucleic acid (e.g., first antigen nucleic acid) which encodes a first tumor-associated antigen or an antigenic fragment thereof; and (b) a nucleic acid encoding an immune modifier protein.
  • the polynucleotide is multi ci stronic (e.g., a multi ci stronic DNA plasmid or a multi ci stronic messenger RNA (mRNA)).
  • mRNA messenger RNA
  • the polynucleotide is combined with a delivery component, such as a synthetic non-viral carrier.
  • the polynucleotide is combined with an adjuvant an adjuvant comprising an aluminum or aluminum-salt based adjuvant, a stimulator of interferon genes (STING) agonist, or a combination thereof.
  • a polynucleotide comprising: (a) an antigen nucleic acid (e.g., first antigen nucleic acid) which encodes a tumor-associated antigen or an antigenic fragment thereof, wherein the antigen nucleic acid (e.g., first antigen nucleic acid) is operably linked to a first promoter; and (b) a nucleic acid encoding an immune modifier protein.
  • the polynucleotide is a multi ci stronic DNA vector.
  • the first tumor-associated antigen is selected from the group consisting of: NYESO-1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A9, MAGE-A10, MAGE-CI, MAGE-C2, SSX, H0RMAD1, CXorf61, ACTL8, AKAP3,AKAP4, HOM-TES-85, OY-TES-1, HSP70-2, LAGE-1, PIWL1, PIWL2, PIWL3, PIWL4, PLU-1, PLAC1, SPAG9, SCP-1, TAG-1, TAG-2a, TAG-2b, TAG-2c, TRAG-3, KAP4, BAGE, BORIS, CT45, GAGE-1/2, PRAME, PAGE4, SP17, PASD1, p53, ras, B-RafHER-2/neu, mucin 1 (MUC1), epidermal growth factor receptor (EGFR), GP100, prostatic acid phosphatase, prostate
  • the polynucleotide comprises two or more nucleic acids encoding an immune modifier protein.
  • each of the nucleic acids encoding an immune modifier protein encodes a different immune modifier protein.
  • the two or more of the nucleic acids encoding an immune modifier protein encode IL- 12 p35 and IL- 12 p40.
  • the polynucleotide further comprises: (c) at least one additional antigen nucleic acid (e.g., second antigen nucleic acid) which encodes a second tumor- associated antigen or an antigenic fragment thereof.
  • additional antigen nucleic acid e.g., second antigen nucleic acid
  • the second tumor- associated antigen is selected from the group consisting of: NYESO-1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A9, MAGE-A10, MAGE-CI, MAGE-C2, SSX, H0RMAD1, CXorf61, ACTL8, AKAP3,AKAP4, HOM-TES-85, OY-TES-1, HSP70-2, LAGE-1, PIWL1, PIWL2, PIWL3, PIWL4, PLU-1, PLAC1, SPAG9, SCP-1, TAG-1, TAG-2a, TAG-2b, TAG-2c, TRAG-3, KAP4, BAGE, BORIS, CT45, GAGE-1/2, PRAME, PAGE4, SP17, PASD1, p53, ras, B-RafHER-2/neu, mucin 1 (MUC1), epidermal growth factor receptor (EGFR), GP100, prostatic acid phosphatase, prostatespecific membrane antigen
  • the polynucleotide further comprises: (b) or (c) at least one additional antigen nucleic acid (e.g., second antigen nucleic acid) which encodes a tumor- associated antigen or an antigenic fragment thereof.
  • additional antigen nucleic acid e.g., second antigen nucleic acid
  • the tumor-associated antigen is selected from the group consisting of: NYESO-1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A9, MAGE-A10, MAGE-CI, MAGE-C2, SSX, H0RMAD1, CXorf61, ACTL8, AKAP3,AKAP4, HOM-TES-85, OY-TES-1, HSP70-2, LAGE-1, PIWL1, PIWL2, PIWL3, PIWL4, PLU-1, PLAC1, SPAG9, SCP-1, TAG-1, TAG-2a, TAG-2b, TAG-2c, TRAG-3, KAP4, BAGE, BORIS, CT45, GAGE-1/2, PRAME, PAGE4, SP17, PASD1, p53, ras, B-RafHER-2/neu, mucin 1 (MUC1), epidermal growth factor receptor (EGFR), GP100, prostatic acid phosphatase, prostatespecific membrane antigen (MUC1)
  • the at least one additional antigen nucleic acid (e.g., second antigen nucleic acid) is operably linked to the first promoter through an internal ribosome entry site (IRES) sequence.
  • IRES sequence comprises a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 19.
  • the polynucleotide comprises one or more second promoters.
  • the at least one additional antigen nucleic acid e.g., second antigen nucleic acid
  • one or more nucleic acids encoding an immune modifier protein is operably linked to the one or more second promoters.
  • one or more of the nucleic acids encoding an immune modifier protein is operably linked to the first promoter or the one or more second promoters through an internal ribosome entry site (IRES) sequence.
  • IRS internal ribosome entry site
  • the IRES sequence comprises a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 19.
  • the first promoter or the one or more second promoters is selected from the group consisting of: a cytomegalovirus (CMV) promoter (SEQ ID NO: 14), a Rouse sarcoma virus (RSV) promoter, a Moloney murine leukemia virus (Mo- MuLV) long terminal repeat (LTR) promoter, a mammalian elongation factor 1 (EFl) promoter, a cytokeratin 18 (CK18) promoter, a cytokeratin 19 (CK19) promoter, a simian virus 40 (SV40) promoter (SEQ ID NO: 15 or SEQ ID NO: 30), a murine U6 promoter, a skeletal a-actin promoter, a P-actin promoter, a murine phosphoglycerate kinase 1 (PGK1) promoter, a human PGK1 promoter, a CBA promoter, a CAG promoter (SEQ ID NO: 14), a Ro
  • the mammalian EFl promoter is a hEFl-HTLV promoter (SEQ ID NO: 18).
  • the one or more second promoters is the CMV promoter.
  • each of the nucleic acids which encodes an immune modifier protein is under the control of a promoter selected from the group consisting of a CMV promoter, an RSV promoter, a Mo-MuLV LTR promoter, a mammalian EFl promoter, a CK18 promoter, a CK19 promoter, an SV40 promoter, a murine U6 promoter, a skeletal a-actin promoter, a P-actin promoter, a murine PGK1 promoter, a human PGK1 promoter, a CB A promoter, a CAG promoter, and any combination thereof.
  • the mammalian EFl promoter is a hEFl-HTLV promoter.
  • each of the at least one additional antigen nucleic acid (e.g., second antigen nucleic acid)s is under the control of a promoter selected from the group consisting of a CMV promoter, an RSV promoter, a Mo-MuLV LTR promoter, a mammalian EFl promoter, a CK18 promoter, a CK19 promoter, an SV40 promoter, a murine U6 promoter, a skeletal a-actin promoter, a P-actin promoter, a murine PGK1 promoter, a human PGK1 promoter, a CBA promoter, a CAG promoter, and any combination thereof.
  • the mammalian EFl promoter is a hEFl-HTLV promoter.
  • the immune modifier protein is selected from the group consisting of: interleukin (IL) 2 (IL-2), IL-12 p35, IL-12 p40, IL-12 p70, IL-15, IL-18, tumor necrosis factor alpha (TNFa), granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon (IFN) a (IFN-a), IFN-P, a chemokine, major histocompatibility complex (MHC) class I (MHC I), MHC class II (MHC II), human leukocyte antigen (HLA)-DR isotype (HLA-DR), CD80, CD86, and any combination thereof.
  • IL interleukin
  • IL-2 interleukin 2
  • IL-12 p35 IL-12 p40
  • IL-12 p70 IL-15
  • IL-18 tumor necrosis factor alpha
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • the chemokine is selected from the group consisting of: C-C motif chemokine ligand (CCL) 3 (CCL3), CCL4, CCL5, CCL21, CCL28, C-X-C motif chemokine ligand (CXCL) 10 (CXCL10), and any combination thereof.
  • the immune modifier protein comprises one or more concatamers of non-coding 5'-C-phosphate-G-3' (CpG) dinucleotides.
  • the one or more concatamers of non-coding CpG dinucleotides activate the Toll-like receptor 9 (TLR9) signaling pathway.
  • the one or more concatamers of non-coding CpG dinucleotides comprise one or more concatamers of non-coding CpG dinucleotides previously reported in Bauer, A. et al., Nucleic Acids Research 38( 12): 3891 -908 (2010); Cornelie, S.
  • the nucleic acid encoding an immune modifier protein comprises a combination (i) a nucleic acid encoding an interleukin, and (ii) a nucleic acid encoding a major histocompatibility complex and/or a chemokine.
  • the nucleic acid encoding an immune modifier protein comprises a nucleic acid encoding IL-12, a nucleic acid encoding IL-15, or the combination thereof.
  • the nucleic acid encoding an immune modifier protein comprises a combination of a nucleic acid encoding IL- 12 and a nucleic acid encoding IL-15.
  • the nucleic acid encoding an immune modifier protein comprises a nucleic acid encoding IL-2, a nucleic acid encoding IL- 15, a nucleic acid encoding MHC I, a nucleic acid encoding MHC II, a nucleic acid encoding CCL3, a nucleic acid encoding CCL4, or any combination thereof.
  • the nucleic acid encoding an immune modifier protein comprises a nucleic acid encoding MHC I, a nucleic acid encoding MHC II, a nucleic acid encoding CCL3, a nucleic acid encoding CCL4, or any combination thereof.
  • the nucleic acid encoding an immune modifier protein comprises a nucleic acid encoding CCL3, a nucleic acid encoding CCL4, or the combination thereof.
  • the polynucleotides or nucleic acids can comprise DNA or mRNA sequences.
  • the polynucleotide further comprises one or more post- transcriptional regulatory elements.
  • the post-transcriptional regulatory element is a wood chuck hepatitis virus post-transcriptional regulatory element (WPRE).
  • the polynucleotide further comprises at least one 3' UTR poly(a) tail sequence operably linked to the antigen nucleic acid (e.g., first antigen nucleic acid), the at least one additional antigen nucleic acid (e.g., second antigen nucleic acid), the nucleic acid encoding an immune modifier protein, or any combination thereof.
  • the 3' UTR poly(a) tail sequence is a 3' UTR SV40 poly(a) tail sequence (SEQ ID NO: 13), a 3' UTR bovine growth hormone (bGH) poly(A) sequence (SEQ ID NO: 13), a 3' UTR actin poly(A) tail sequence, a 3' UTR hemoglobin poly(A) sequence, or any combinations thereof.
  • the polynucleotide further comprises at least one enhancer sequence.
  • the enhancer sequence is a human actin enhancer sequence, a human myosin enhancer sequence, a human hemoglobin enhancer sequence, a human muscle creatine enhancer sequence, a viral enhancer sequence, or a polynucleotide function enhancer sequence.
  • the enhancer sequence is a CMV intronic sequence or a P-actin intronic sequence.
  • the enhancer sequence is a SV40 enhancer sequence (SEQ ID NO: 18).
  • the polynucleotide is a multi ci str onic mRNA comprising a 5' cap and a 3' UTR poly(A) tail sequence.
  • the 3' UTR poly(a) tail sequence is a 3' UTR SV40 poly(a) tail sequence (SEQ ID NO: 13), a 3' UTR bovine growth hormone (bGH) poly(A) sequence (SEQ ID NO: 13), a 3' UTR actin poly(A) tail sequence, a 3' UTR hemoglobin poly(A) sequence, or any combinations thereof.
  • the multicistronic mRNA comprises a 5' UTR and/or a 3' UTR.
  • a vector comprising any polynucleotide described or exemplified herein, wherein the vector is a DNA plasmid, a multicistronic mRNA, a viral vector, a bacterial vector, a cosmid, or an artificial chromosome.
  • the composition, pharmaceutical composition, or vaccine further comprises a second polynucleotide encoding at least one immune modifier protein selected from the group consisting of: IL-2, IL-12 p35, IL-12 p40, IL-12 p70, IL-15, IL- 18, TNFa, GM-CSF, IFN-a, IFN-p, a chemokine, MHC I, MHC II, HLA-DR, CD80, and CD86, wherein the polynucleotide encoding the at least one immune modifier protein is operably linked to a promoter.
  • the composition, pharmaceutical composition, or vaccine further comprises a delivery component.
  • a vector e.g., a plasmid vector
  • any polynucleotide e.g., DNA or mRNA
  • the plasmid vector is a DNA plasmid vector.
  • compositions, pharmaceutical composition, or vaccine comprising any polynucleotide, vector, multicistronic mRNA vector, or DNA plasmid vector described or exemplified herein.
  • compositions, pharmaceutical composition, or vaccine comprising: (a) a polynucleotide or a vector comprising the polynucleotide, and (b) a delivery component, wherein the polynucleotide comprises a antigen nucleic acid (e.g., first antigen nucleic acid) which encodes a first tumor- associated antigen or an antigenic fragment thereof, wherein the antigen nucleic acid (e.g., first antigen nucleic acid) is operably linked to a first promoter, optionally wherein the delivery component is a cationic polymer, a poly-inosinic-polycytidylic acid, or a poloxamer or derivative thereof.
  • a delivery component wherein the polynucleotide comprises a antigen nucleic acid (e.g., first antigen nucleic acid) which encodes a first tumor- associated antigen or an antigenic fragment thereof, wherein the antigen nucleic acid (e.g., first antigen
  • the delivery component is a cationic polymer, a poly-inosinic-polycytidylic acid, or a poloxamer or derivative thereof.
  • the antigen nucleic acid e.g., first antigen nucleic acid
  • the antigen nucleic acid which encodes a first tumor-associated antigen is selected from the group consisting of NYESO-1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A9, MAGE-A10, MAGE-CI, MAGE-C2, SSX, H0RMAD1, CXorf61, ACTL8, AKAP3,AKAP4, HOM-TES-85, OY-TES-1, HSP70-2, LAGE-1, PIWL1, PIWL2, PIWL3, PIWL4, PLU-1, PLAC1, SPAG9, SCP-1, TAG-1, TAG-2a, TAG-2b, TAG-2c, TRAG-3, KAP4, BAGE, BORIS, CT45
  • the delivery component of the compositions, pharmaceutical compositions, or vaccines may comprise any combination of delivery components disclosed herein.
  • the polynucleotide further comprises at least one additional antigen nucleic acid (e.g., second antigen nucleic acid) which encodes a second tumor- associated antigen or an antigenic fragment thereof.
  • the at least one additional antigen nucleic acid (e.g., second antigen nucleic acid) which encodes a second tumor-associated antigen is selected from the group consisting of a tumor-associated antigen and any antigenic fragment thereof.
  • the delivery component of the composition, pharmaceutical composition, or vaccine is a cationic polymer.
  • the cationic polymer is a synthetic functionalized polymer, a lipid, a lipopolymer, or a chemical derivative thereof.
  • the synthetic functionalized polymer is a biodegradable cross-linked cationic multi-block copolymer.
  • the biodegradable cross-linked cationic multi-block copolymer is represented by the formula: (CP)xLyYz, wherein: (a) CP represents a cationic polymer containing at least one secondary amine group, wherein the cationic polymer has a number averaged molecular weight within the range of 1,000 to 25,000 Dalton; (b) Y represents a bifunctional biodegradable linker containing ester, amide, disulfide, or - I l l - phosphate linages; (c) L represents a ligand; (d) x is an integer in the range from 1 to 20; (e) y is an integer in the range from 0 to 100; and (f) z is an integer in the range from 0 to 40.
  • the cationic polymer comprises linear polyethyleneimine (LPEI).
  • the cationic polymer comprising linear polyethyleneimine (LPEI) is BD15K-12, wherein the PEIs are approximately 15,000 Da, and wherein there is an average of 12 crosslinkers per PEI.
  • the bifunctional biodegradable linker is hydrophilic and comprises a biodegradable linkage comprising a disulfide bond. In some aspects, the bifunctional biodegradable linker is a dithiodipropionyl linker.
  • the biodegradable cross-linked cationic multi-block copolymer comprises LPEI and a dithiodipropionyl linker for cross-linking the multi-block copolymer, wherein the LPEI has an average molecular weight of 1,000 to 25,000 Dalton.
  • the biodegradable cross-linked cationic multi-block copolymer is covalently linked to at least one ligand.
  • the ligand is a targeting ligand selected from the group consisting of: a sugar moiety, a polypeptide, folate, and an antigen.
  • the sugar moiety is a monosaccharide.
  • the monosaccharide is galactose.
  • the sugar moiety is an oligosaccharide.
  • the polypeptide is a glycoprotein, an antibody, an antibody fragment, a cell receptor, a cytokine receptor, or a growth factor receptor.
  • the growth factor receptor is an epidermal growth factor receptor.
  • the glycoprotein is transferrin or asialoorosomucoid (ASOR).
  • the biodegradable cross-linked cationic multi-block copolymer is covalently linked to polyethylene glycol (PEG) of molecular weight ranging from 500 to 20,000 Dalton.
  • PEG polyethylene glycol
  • the biodegradable cross-linked cationic multi-block copolymer is covalently linked to a fatty acyl chain selected from the group consisting of: oleic acid, palmitic acid, and stearic acid.
  • the biodegradable cross-linked cationic multi-block copolymer comprises at least one amine group that is electrostatically attracted to a polyanionic compound.
  • the polyanionic compound is a nucleic acid, wherein the biodegradable cross-linked cationic multi -block copolymer condenses the nucleic acid to form a compact structure.
  • the delivery component of the composition, pharmaceutical composition, or vaccine is a cationic lipopolymer comprising a PEI backbone covalently linked to a lipid or a PEG.
  • the PEI backbone is covalently linked to a lipid and a PEG.
  • the lipid and the PEG are directly attached to the PEI backbone by covalent bonds.
  • the lipid is attached to the PEI backbone through a PEG spacer.
  • the PEG has a molecular weight of between 50 to 20,000 Dalton.
  • the molar ratio of PEG to PEI is within a range of 0.1 : 1 to 500: 1.
  • the molar ratio of the lipid to the PEI is within a range of 0.1 : 1 to 500: 1.
  • the lipid is a cholesterol, a cholesterol derivative, a C12 to C18 fatty acid, or a fatty acid derivative.
  • the PEI is covalently linked to cholesterol and PEG, and wherein the average PEG:PEI: cholesterol molar ratio in the cationic lipopolymer is within the range of 1-5 PEG: 1 PEI:0.4-1.5 cholesterol.
  • the PEI has a linear or branch configuration with a molecular weight of 100 to 500,000 Dalton.
  • the cationic lipopolymer further comprises a pendant functional moiety selected from the group consisting of: a receptor ligand, a membrane permeating agent, an endosomolytic agent, a nuclear localization sequence, and a pH sensitive endosomolytic peptide.
  • the cationic lipopolymer further comprises a targeting ligand, wherein the targeting ligand is directly attached to the PEI backbone or is attached through a PEG linker.
  • the targeting ligand is selected from the group consisting of: a sugar moiety, a polypeptide, folate, and an antigen.
  • the sugar moiety is a monosaccharide.
  • the monosaccharide is galactose.
  • the sugar moiety is an oligosaccharide.
  • the polypeptide is a glycoprotein, an antibody, an antibody fragment, a cell receptor, a cytokine receptor, or a growth factor receptor.
  • the growth factor receptor is an epidermal growth factor receptor.
  • the glycoprotein is transferrin or asialoorosomucoid (ASOR).
  • the cationic polymer of the composition, pharmaceutical composition, or vaccine is present in an amount sufficient to produce a ratio of amine nitrogen in the cationic polymer to phosphate in the polynucleotide, multicistronic mRNA vector, or DNA plasmid vector from about 0.01 : 1 to about 50: 1 (e.g., about 0.01 : 1 to about 40:1; about 0.01 : 1 to about 30:1; about 0.01 : 1 to about 20: 1; about 0.01 : 1 to about 10: 1, or about 0.01 : 1 to about 5: 1).
  • the ratio of amine nitrogen in the cationic polymer to phosphate in the polynucletide, multicistronic mRNA vector, or DNA plasmid vector is from about 0.1 : 1 to about 50: 1 (e.g., about 0.1 : 1 to about 40: 1; about 0.1 :1 to about 30: 1; about 0.1 : 1 to about 20: 1; about 0.1 :1 to about 10: 1, or about 0.1 : 1 to about 5:1). In some aspects, the ratio of amine nitrogen in the cationic polymer to phosphate in the polynucleotide, multi ci str onic mRNA vector, or DNA plasmid vector is from about 1 : 10 to about 10: 1.
  • the composition, pharmaceutical composition, or vaccine comprises about 0.1 mg/ml to about 10 mg/ml (e.g., about 0.1 mg/ml to about 5 mg/ml; about 0.5 mg/ml to about 10 mg/ml; or about 0.5 mg/ml to about 5 mg/ml) nucleic acid (e.g., DNA plasmid vector or multicistronic mRNA vector) complexed with the cationic polymer.
  • nucleic acid e.g., DNA plasmid vector or multicistronic mRNA vector
  • the composition, pharmaceutical composition, or vaccine comprises about 1 mg/ml to about 10 mg/ml (e.g., about 1 mg/ml to about 6 mg/ml; about 2 mg/ml to about 6 mg/ml; about 5 mg/ml to about 10 mg/ml; or about 6 mg/ml to about 10 mg/ml) nucleic acid (e.g., DNA plasmid vector or multicistronic mRNA vector) complexed with the cationic polymer.
  • nucleic acid e.g., DNA plasmid vector or multicistronic mRNA vector
  • the delivery component of the composition, pharmaceutical composition, or vaccine comprises a lipopolyamine with the following formula: (Staramine).
  • the delivery component comprises a mixture of the lipopolyamine and an alkylated derivative of the lipopolyamine.
  • the alkylated derivative of the lipopolyamine is a polyoxyalkylene, polyvinylpyrrolidone, polyacrylamide, polydimethylacrylamide, polyvinyl alcohol, dextran, poly (L-glutamic acid), styrene maleic anhydride, poly-N-(2-hydroxypropyl) methacrylamide, or polydivinylether maleic anhydride.
  • the alkylated derivative of the lipopolyamine has the following formula:
  • n an integer from 10 to 100 repeating units containing 2-5 carbon atoms each.
  • the ratio of the lipopolyamine to the alkylated derivative of the lipopolyamine in the mixture is 0.01:1 to 10:1.
  • the lipopolyamine is present in an amount sufficient to produce a ratio of amine nitrogen in the lipopolyamine to phosphate in the polynucleotide, multi ci stronic mRNA vector, or DNA plasmid vector from about 0.01:1 to about 50:1 (e.g., about 0.01:1 to about 40:1; about 0.01:1 to about 30:1; about 0.01:1 to about 20:1; about 0.01:1 to about 10:1, or about 0.01:1 to about 5:1).
  • the ratio of amine nitrogen in the lipopolyamine to phosphate in the polynucleotide, multi ci stronic mRNA vector, or DNA plasmid vector is from about 0.1:1 to about 50:1 (e.g., about 0.1:1 to about 40:1; about 0.1:1 to about 30:1; about 0.1:1 to about 20:1; about 0.1:1 to about 10:1, or about 0.1:1 to about 5:1). In some aspects, the ratio of amine nitrogen in the lipopolyamine to phosphate in the polynucleotide, multi ci stronic mRNA vector, or DNA plasmid vector is from about 0.01 : 10 to about 10:1.
  • the delivery component of the composition, pharmaceutical composition, or vaccine comprises a lipopolyamine with the following formula: (Crossamine).
  • the delivery component comprises a mixture of the lipopolyamine and an alkylated derivative of the lipopolyamine.
  • the alkylated derivative of the lipopolyamine is a polyoxyalkylene, polyvinylpyrrolidone, polyacrylamide, polydimethylacrylamide, polyvinyl alcohol, dextran, poly (L-glutamic acid), styrene maleic anhydride, poly-N-(2-hydroxypropyl) methacrylamide, or polydivinylether maleic anhydride.
  • the ratio of the lipopolyamine to the alkylated derivative of the lipopolyamine in the mixture is 0.01 : 1 to 10: 1.
  • the lipopolyamine is present in an amount sufficient to produce a ratio of amine nitrogen in the lipopolyamine to phosphate in the polynucleotide, multi ci str onic mRNA vector, or DNA plasmid vector from about 0.01 :1 to about 50: 1 (e.g., about 0.01 : 1 to about 40:1; about 0.01 : 1 to about 30:1; about 0.01 : 1 to about 20: 1; about 0.01 : 1 to about 10: 1, or about 0.01 : 1 to about 5: 1).
  • the ratio of amine nitrogen in the lipopolyamine to phosphate in the polynucleotide, multi ci str onic mRNA vector, or DNA plasmid vector is from about 0.1 : 1 to about 50: 1 (e.g., about 0.1 : 1 to about 40: 1; about 0.1 :1 to about 30: 1; about 0.1 : 1 to about 20: 1; about 0.1 :1 to about 10: 1, or about 0.1 : 1 to about 5:1). In some aspects, the ratio of amine nitrogen in the lipopolyamine to phosphate in the polynucleotide, multi ci str onic mRNA vector, or DNA plasmid vector is from about 1 : 10 to about 10: 1.
  • the delivery component of the composition, pharmaceutical composition, or vaccine comprises a poloxamer back-bone having a metal chelator covalently coupled to at least one terminal end of the poloxamer backbone.
  • the metal chelator is coupled to at least two terminal ends of the poloxamer backbone.
  • at least one metal chelator is coupled to a poloxamer.
  • at least two metal chelators are coupled to a poloxamer.
  • 1-5, 5- 10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 50-55, 55-60, 60-65, 65-70, 70-75, 75-80, 80-85, 85-90, 90-95, or 95-100 metal chelators are coupled to a poloxamer.
  • the poloxamer backbone is a poloxamer backbone disclosed in U.S. Publ. No. 2010/0004313, which is herein incorporated by reference in its entirety.
  • the metal chelator is a metal chelator disclosed in U.S. Publ. No. 2010/0004313.
  • the delivery component of the composition, pharmaceutical composition, or vaccine comprises a polymer having the following formula: and pharmaceutically acceptable salts thereof, wherein:
  • A represents an integer from 2 to 141;
  • B represents an integer from 16 to 67;
  • C represents an integer from 2 to 141;
  • RA and RC are the same or different, and are R'-L- or H, wherein at least one of RA and RC is R'-L-;
  • L is a bond, — CO — , — CH2 — O — , or — O — CO — ; and R' is a metal chelator.
  • the delivery component comprises a poloxamer with the following formula: or a pharmaceutically acceptable salt thereof, wherein:
  • A represents an integer from 2 to 141;
  • B represents an integer from 16 to 67;
  • C represents an integer from 2 to 141;
  • RA and RC are the same or different, and are R'-L- or H;
  • L is a bond, — CO — , — CH2 — O — , or — O — CO — ;
  • R' is a metal chelator
  • At least one of RA and RC is R'-L-.
  • the R’ is covalently bound to the poloxamer (e.g., where a metal chelator (e.g., a crown) is covalently attached at different density to the poloxamer).
  • a metal chelator e.g., a crown
  • one metal chelator or two or more metal chelators is/are bound to the poloxamer.
  • 2-100 metal chelators are bound to the poloxamer.
  • the metal chelator is RNNH — , RN2N — , or (R" — (N(R") — CH2CH2)x)2 — N — CH2CO — , wherein each x is independently 0-2, and wherein R" is HO2C — CH2 — .
  • the metal chelator is a crown ether selected from the group consisting of 12-crown-4, 15-crown-5, 18-crown-6, 20-crown-6, 21-crown-7, and 24-crown-8.
  • the crown ether is a substituted-crown ether, wherein the substituted crown ether has:
  • the metal chelator is a cryptand, wherein the cryptand is selected from the group consisting of (1,2,2) cryptand, (2,2,2) cryptand, (2,2,3) cryptand, and (2,3,3) cryptand.
  • the cryptand is a substituted-cryptand, wherein the substituted cryptand has:
  • the delivery component is Crown Poloxamer (aza-crown-linked poloxamer), wherein the Crown Poloxamer comprises a polymer having the following formula: or pharmaceutically acceptable salts thereof, wherein: a represents an integer of about 10 units; and b represents an integer of about 21 units; and wherein the total molecular weight of the polymer is about 2,000 Da to about 2,200 Da.
  • Crown Poloxamer aza-crown-linked poloxamer
  • the crown poloxamer can be derivatized with a cationic molecule, a ligand, or other chemical entities.
  • the polymer or poloxamer (e.g., crown poloxamer) is present in a solution with the polynucleotide, multi ci str onic mRNA vector, or DNA plasmid vector from about 0.01% - about 5% or about 0.5% - about 5%.
  • the solution is co-formulated with a metal chelator (e.g., where the co-formulated metal chelator is a free metal chelator, which is formulated with the poloxamer (e.g., a non-crown poloxamer)).
  • a metal chelator e.g., where the co-formulated metal chelator is a free metal chelator, which is formulated with the poloxamer (e.g., a non-crown poloxamer)).
  • the co-formulated metal chelator is present in the solution at a concentration of about O.lmg/mL to about 20mg/mL.
  • the co-formulated metal chelator is crown ether, a substituted- crown ether, a cryptand, or a substituted-cryptand.
  • the metal chelator or co-formulated metal chelator is crown ether (Aza- 18 -crown-6) .
  • the delivery component of the composition, pharmaceutical composition, or vaccine is a P-amino ester.
  • the polymer is present in a solution with the polynucleotide, multi ci str onic mRNA vector, or DNA plasmid vector from about 0.01% - about 5% or about 0.5% - about 5%.
  • the delivery component of the composition, pharmaceutical composition, or vaccine is a poly-inosinic-polycytidylic acid.
  • the poly- inosinic-polycytidylic acid is present in a solution with the polynucleotide, multi ci stronic mRNA vector, or DNA plasmid vector from about 0.01% - about 5% or about 0.5% - about 5%.
  • the delivery component further comprises benzalkonium chloride.
  • the delivery component comprises BD15-12.
  • the ratio of nucleotide to BD15-12 polymer is 0.1 : 1 to 5: 1.
  • the delivery component comprises Omnifect.
  • the ratio of nucleotide to Omnifect polymer (N:P) is 0.1 : 1 to 5: 1.
  • the delivery component comprises Crown Poloxamer (azacrown-linked poloxamer). In some aspects, the ratio of nucleotide to Crown Poloxamer (N:P) is 0.1 : 1 to 5: 1. In some aspects, the delivery component comprises Crown Poloxamer and a PEG-PEI-cholesterol (PPC) lipopolymer. In some aspects, the delivery component comprises Crown Poloxamer and benzalkonium chloride. In some aspects, the delivery component comprises Crown Poloxamer and Omnifect. In some aspects, the delivery component comprises Crown Poloxamer and a linear polyethyleneimine (LPEI). In some aspects, the delivery component comprises Crown Poloxamer and BD15-12.
  • PPC PEG-PEI-cholesterol
  • the delivery component comprises Staramine and mPEG modified Staramine.
  • the mPEG modified Staramine is Staramine- mPEG515.
  • the mPEG modified Staramine is Staramine-mPEGl 1.
  • the ratio of Staramine to mPEG modified Staramine is 0.1 : 1 to 10: 1.
  • the nucleotide to polymer (N:P) ratio is 0.1 : 1 to 5: 1.
  • the delivery component comprises Staramine, mPEG modified Staramine, and Crown Poloxamer.
  • the delivery component comprises Staramine, Staramine- mPEG515, and Crown Poloxamer.
  • the delivery component comprises Staramine, Staramine-mPEGl 1, and Crown Poloxamer.
  • the metal chelator is added directly to a solution comprising (i) the polynucleotide, multi ci stronic mRNA vector, or DNA plasmid vector as described herein, and (ii) a poloxamer.
  • the metal chelator is present in the solution at concentration of about O. lmg/mL to about 20mg/mL. In some aspects, metal chelator is present in the solution at concentration of about O.lmg/mL to about 20mg/mL, about O.
  • the metal chelator is crown ether, a substituted-crown ether, a cryptand, or a substituted-cryptand. In some aspects, the metal chelator is crown ether (Aza-18-crown- 6).
  • the metal chelator is added directly to a solution comprising (i) the polynucleotide, multi ci stronic mRNA vector, or DNA plasmid vector as described herein, (ii) a poloxamer, and (iii) an an adjuvant comprising an aluminum or aluminum- salt based adjuvant, a stimulator of interferon genes (STING) agonist, or a combination thereof.
  • the metal chelator is present in the solution at concentration of about O.lmg/mL to about 20mg/mL. In some aspects, metal chelator is present in the solution at concentration of about O.
  • the metal chelator is crown ether, a substituted-crown ether, a cryptand, or a substituted-cryptand. In some aspects, the metal chelator is crown ether (Aza-18-crown- 6). In some aspects, the adjuvant is AIPO4.
  • the metal chelator is added directly to a solution comprising (i) the polynucleotide, multi ci stronic mRNA vector, or DNA plasmid vector as described herein, (ii) a poloxamer, and (iii) an an adjuvant comprising an aluminum or aluminum- salt based adjuvant, or a combination thereof.
  • the metal chelator is present in the solution at concentration of about O.lmg/mL to about 20mg/mL. In some aspects, metal chelator is present in the solution at concentration of about 0. Img/mL to about 20mg/mL, about O.
  • the metal chelator is crown ether, a substituted-crown ether, a cryptand, or a substituted-cryptand.
  • the metal chelator is crown ether (Aza-18-crown-6).
  • the adjuvant is AIPO4.
  • composition, pharmaceutical composition, or vaccine is lyophilized.
  • a host cell comprising any polynucleotide, vector, multi ci stronic mRNA vector, DNA plasmid vector, composition, pharmaceutical composition, or vaccine described or exemplified herein.
  • the host cell is a eukaryotic host cell.
  • the host cell is a human host cell.
  • kits comprising any polynucleotide, vector, multi ci stronic mRNA vector, DNA plasmid vector, composition, pharmaceutical composition, vaccine, or lyophilized composition described or exemplified herein.
  • the kit further comprises a glass vial.
  • the kit further comprises instructions for using the polynucleotide, vector, multi ci stronic mRNA vector, DNA plasmid vector, composition, or pharmaceutical composition in a method for inducing an immune response in a subject.
  • the kit further comprise instruction for reconstituting the composition, pharmaceutical composition, vaccine or lyophilized vaccine.
  • the kit further comprises instructions for using the polynucleotide, vector, multi ci stronic mRNA vector, DNA plasmid vector, composition, pharmaceutical composition, vaccine, or lyophilized composition in a method for preventing, reducing the incidence of, attenuating or treating a tumor in a subject.
  • a method of inducing an immune response in a subject comprising administering an effective amount of any polynucleotide, vector, multi ci str onic mRNA vector, DNA plasmid vector, composition, pharmaceutical composition, or vaccine described or exemplified herein to the subject.
  • the immune response is to one or more antigens disclosed herein.
  • the immune response is a protective immune response.
  • the polynucleotide, vector, multi ci stronic mRNA vector, DNA plasmid vector, composition, pharmaceutical composition, or vaccine is administered to the subject by an intramuscular, subcutaneous, intralymphatic, intranasal, or intraperitoneal route of administration.
  • the immune response is to one or more antigens comprising one or more tumor-associated antigens.
  • the present disclosure also features methods of making any composition, pharmaceutical composition, or vaccine described or exemplified herein.
  • the methods comprise the steps of: (a) combining a delivery component disclosed herein with a polynucleotide disclosed herein, (b) lyophilizing the combined delivery component and polynucleotide to a powder, and (c) reconstituting the powder with a diluent to form a solution of nucleic acid complexed with the delivery component
  • the methods comprise the steps of: (a) combining a delivery component disclosed herein with a polynucleotide disclosed herein, (b) lyophilizing the combined delivery component and polynucleotide to a powder, and (c) reconstituting the powder with a diluent to form a solution of nucleic acid complexed with the delivery component, wherein the diluent comprises an adjuvant and/or a STING agonist.
  • the methods comprise combining an adjuvant with the diluent. In some aspects, the methods comprise combining a STING agonist with the diluent.
  • the methods comprise combining an adjuvant and/or a STING agonist with the reconstituted polynucleotide/delivery component solution. In some aspects, the methods comprise combining a STING agonist with the diluent and an adjuvant with reconstituted polynucleotide/delivery component solution. In some aspects, the methods comprise combining an adjuvant with the diluent and a STING agonist with the reconstituted polynucleotide/delivery component solution.
  • DNA plasmids vectors are constructed with the elements as shown in FIGs. 1-14.
  • the starting plasmid backbone used in vector construction contains multiple restriction sites that are utilized for subcloning the desired expression cassettes in order to generate the DNA plasmid constructs.
  • Each expression cassette that is cloned into the vector contains all of the necessary components required for expressing the functional protein or antigen (e.g promoter sequence, gene sequence, poly A tail sequence).
  • the general methodology consists of digesting the starting DNA plasmid backbone with the appropriate restriction digest enzyme.
  • the insert sequence is obtained from isolating and gel purifying a genetic sequence from a second DNA plasmid using appropriate restriction digest enzymes.
  • sequences to be inserted into the plasmid vector are generated synthetically or a combination of the previous two approaches can be used.
  • the starting backbone and insert sequences are mixed together and ligated using DNA ligase.
  • the resulting DNA plasmids are then transformed into e.coli using standard protocols and streaked onto LB agar plates containing the appropriate antibiotics. Bacterial colonies are subsequently isolated and grown in medium so that the plasmid can be purified and screened by restriction digest and gel electrophoresis in order to identify the colonies that have the correct plasmids.
  • Plasmid DNA vectors for expression of Trp2 under the control of a CMV promoter with a SV40 enhancer (pVac64; FIG. 14N). or NYESO-1 under the control of a CMV promoter with a SV40 enhancer were generated (pVac65; FIG. 140). Restriction digestion of pVac64 with Xhol (2 bands; 4380bp and 842bp) or Sacl (3 bands; 3990bp, 1268bp, and 414bp) produced bands of the correct size (FIG. 15A). Restriction digestion of pVac65 with Kpnl (2 bands; 2368bp and 2293bp) produced bands of the correct size (FIG. 15B).
  • the present disclosure can include a nucleic acid (e.g., a DNA plasmid vector or multi ci str onic mRNA vector) complexed with a biodegradable cross-linked cationic multi-block copolymer of the following formula: wherein: A and B are such that the molecular weight of the individual linear polyethylenimine chains are from 5,000 to 20,000 Dalton; the intermolecular crosslinks connect approximately 5-10% of amines; the biodegradable crosslinks are dithiodipropionyl (each half composed of 3 carbon atoms) and can be from 1-10 carbon atoms.
  • a nucleic acid e.g., a DNA plasmid vector or multi ci str onic mRNA vector
  • a and B are such that the molecular weight of the individual linear polyethylenimine chains are from 5,000 to 20,000 Dalton; the intermolecular crosslinks connect approximately 5-10% of amines; the biodegradable crosslinks
  • the biodegradable cross-linked cationic polymer comprises 10,000 to 15,000 Dalton linear PEI covalently connected with a dithiopropionyl linkage (see, e.g., U.S. Patent No. 8,445,017).
  • the polymer is dissolved in sterile water to give a final concentration of 3 mg/ml.
  • the DNA is dissolved in sterile water to give a final concentration of 1 mg/ml.
  • the two components are diluted separately with 5% glucose to a volume of 150 uL each, and then the plasmid DNA solution is added to the polymer solution.
  • the biodegradable cross-linked cationic polymer comprises 10,000 to 15,000 Dalton linear PEI covalently connected with a dithiopropionyl linkage (see, e.g., U.S. Patent No. 8,445,017) and is further conjugated to polyethyleneglycol (PEG) of molecular weight ranging from 500 to 20,000 Dalton.
  • PEG polyethyleneglycol
  • the resultant polymer is dissolved in sterile water to give a final concentration of 3 mg/ml.
  • the DNA is dissolved in sterile water to give a final concentration of 1 mg/ml.
  • the two components are diluted separately with 5% glucose to a volume of 150 uL each, and then the plasmid DNA solution is added to the polymer solution.
  • the biodegradable cross-linked cationic polymer comprises 10,000 to 15,000 Dalton linear PEI covalently connected with a dithiopropionyl linkage (see, e.g., U.S. Patent No. 8,445,017) and is further conjugated to polyethyleneglycol (PEG) of molecular weight ranging from 500 to 20,000 Dalton.
  • PEG polyethyleneglycol
  • the resultant polymer is dissolved in sterile water to give a final concentration of 3 mg/ml.
  • the DNA is dissolved in sterile water to give a final concentration of 1 mg/ml.
  • the two components are diluted separately with 5% glucose to a volume of 150 uL each, and then the plasmid DNA solution is added to the polymer solution.
  • the biodegradable cross-linked cationic polymer comprises 15,000 to 20,000 Dalton linear PEI covalently connected with a dithiopropionyl linkage (see, e.g., U.S. Patent No. 8,445,017).
  • the polymer is dissolved in sterile water to give a final concentration of 3 mg/ml.
  • the DNA is dissolved in sterile water to give a final concentration of 1 mg/ml.
  • the two components are diluted separately with 5% glucose to a volume of 150 uL each, and then the plasmid DNA solution is added to the polymer solution.
  • the biodegradable cross-linked cationic polymer comprises 15,000 to 20,000 Dalton linear PEI covalently connected with a dithiopropionyl linkage (see, e.g., U.S. Patent No. 8,445,017) and is further conjugated to polyethyleneglycol (PEG) of molecular weight ranging from 500 to 20,000 Dalton.
  • PEG polyethyleneglycol
  • the resultant polymer is dissolved in sterile water to give a final concentration of 3 mg/ml.
  • the DNA is dissolved in sterile water to give a final concentration of 1 mg/ml.
  • the two components are diluted separately with 5% glucose to a volume of 150 uL each, and then the plasmid DNA solution is added to the polymer solution.
  • DNA complexes with biodegradable cross-linked cationic polymer can be prepared at different ratios 1/15/1, 10/1, and 20/1 nitrogen/phosphate (N/P).
  • N/P nitrogen/phosphate
  • the complexes are diluted in a cuvette for measurement of particle size and the potential of the complex.
  • the electrophoretic mobility of the samples can be measured at 25°C, and at a wavelength of 657 nm and at a constant angle of 90° with a Particle sizer.
  • Example 3 Vector Formulation with the Delivery Carrier PPC
  • the DNA and PPC solutions are separately prepared at 5 mg/ml in water for injection and subsequently diluted to 0.3 mg/ml (DNA) and 1.108 mg/ml (PPC) in 3% lactose.
  • the DNA in lactose solution is added to the PPC in lactose solution using a micropipette to a nitrogen to phosphate ratio (N:P ratio) of 0.01 :1 to 11 : 1, and the formulation is incubated for 15 minutes at room temperature to allow the complexes to form.
  • the PPC/DNA complexes in 3% lactose are lyophilized using a FREEZONE freeze dry System from LABCONCO Corp. Kansas City, Mo. 500 ul of prepared formulation is added to 2 ml borosilicate glass vials which were then lyophilized using a freeze drying program consisting of the following segments:
  • the resultant lyophilized powder is reconstituted with 150 pl water for injection to make 0.5 mg/ml DNA.
  • Poloxamers are gently mixed with 1 mg/ml of nucleic acids in water or saline solution (0.15M) at variable concentrations.
  • Formulated poloxamer (5%)/plasmid solutions are analyzed by gel electrophoresis in order to verify interaction between formulated plasmid and poloxamer. Comparison between unformulated plasmid DNA and DNA formulated with poloxamer have the similar movement through the gel and therefore indicate no binding between plasmid DNA and poloxamer.
  • the formulated plasmid with poloxamer is used for gene transfer in mammalian cell or tissue.
  • X.5 Synthesis of Aza-crown-linked Poloxamer (Crown Pol oxamer)
  • An aza-crown-linked poloxamer (crown poloxamer) is constructed as follows. Poloxamer 124 (Pluronic L-44; 500 mg, 220 pmol) is dissolved in toluene (3ml), and the resulting solution is treated with 2 ml (4 mmol) of 2M phosgene solution in toluene. After 3 hrs at room temperature, the mixture is concentrated in vacuum, the residue is redissolved in 3 ml toluene and concentrated again. The residue is dissolved in dry chloroform (5 ml).
  • Vaccine plasmid DNA is prepared initially at 0.1 mg/mL with PEG-PEI- cholesterol (PPC; MW 4.2 kD), by mixing plasmid DNA with the polymer at (11 : 1) and (0.5: 1) (N:P) ratios in 5% dextrose solution, and the mixture is incubated at room temperature for 10 minutes to allow the formation of nanocomplexes. Then, pDNA/PPC nanocomplexes are concentrated to 1-5 mg/mL using Amicon Ultra Centrifugal Filters (Ultracel-3K MWCO). [0506] The electrophoretic mobility of the PPC/DNA complexes is determined by agarose gel electrophoresis at 70 V for one hour.
  • DNA integrity in the complexes is determined by incubation of the complexes with 50 pg of dextran sulfate or triton-X for 10 minutes at room temperature followed by gel electrophoresis at 100 Volts for one hour.
  • the particle size the nanocomplexes in Milli-Q water is determined at 657 nm at a constant angle of 90° by dynamic light scattering using a Malvern particle size analyzer. Osmolality of the formulation is determined using Fiske210 micro-sample osmometer, DNA quantification was performed using spectrophotometry, formulation pH was measured using Accumet research ARI 5 pH meter.
  • Vaccine plasmid DNA is prepared initially at 0.1 mg/mL with PEI-base copolymer, BD15-12 (15kD linear PEI; MW 26.5 kD), by mixing plasmid DNA with the polymer at 10:1 and 0.5: 1 N:P ratios in 5% dextrose solution, and the mixture is incubated at room temperature for 10 minutes to allow the formation of nanocomplexes. Then, BD15-12/pDNA nanocomplexes are concentrated to 2.5-5 mg/mL using Amicon Ultra Centrifugal Filters (Ultracel-3K MWCO)
  • the electrophoretic mobility of the BD15-12/DNA complexes is determined by agarose gel electrophoresis at 70 V for one hour.
  • the particle size of the nanocomplexes in Milli-Q water is determined at 657 nm at a constant angle of 90° by dynamic light scattering using a Malvern particle size analyzer.
  • Osmolality of the formulation is determined using Fiske 210 micro-sample osmometer, DNA quantification is performed using spectrophotometry, and formulation pH is measured using Accumet research ARI 5 pH meter.
  • Staramine Star-mPEG/ p Vaccine F ormul ati on :
  • Staramine Poly(ethylene glycol) methyl ether (mPEG)/pVaccine formulation is composed of Staramine (0.635 kD) liposomes and plasmid DNA.
  • Staramine alone or 10 1 mixtures of Staramine (Star) and Star-PEG515 (1.2 kD) are rotary-evaporated to a film. The flask of liposome film is held under high vacuum overnight.
  • the gel retardation assay is performed by loading the Staramine/plasmid DNA on 1% agarose gel and electrophoresed at 100 V for 1 hour. To release the plasmid DNA from the Staramine nanoparticles, 10% TritonX-100 is added to the complex and the solution is loaded on the agarose gel. Osmolality of the formulation is determined using Fiske210 micro-sample osmometer, DNA quantification is performed using spectrophotometry, and formulation pH was measured using Accumet research ARI 5 pH meters.
  • Vaccine plasmid DNA is prepared initially at 0.1 mg/mL with Omnifect (MW 7.3 kD), by mixing plasmid DNA with the polymer at 10: 1 and 0.5: 1 N:P ratios in 5% dextrose solution, and the mixture is incubated at room temperature for 10 minutes to allow the formation of nanocomplexes. Then, Omnifect/pDNA nanocomplexes are concentrated to 2.5-5 mg/mL using Amicon Ultra Centrifugal Filters (Ultracel-3K MWCO)
  • the electrophoretic mobility of the Omnifect/DNA complexes is determined by agarose gel electrophoresis at 70 V for one hour.
  • the particle size the nanocomplexes in Milli-Q water is determined at 657 nm at a constant angle of 90° by dynamic light scattering using a Malvern particle size analyzer.
  • DNA integrity in the complexes is determined by incubation of the complexes with 50 pg of dextran sulfate or triton-X for 10 minutes at room temperature followed by gel electrophoresis at 100 Volts for one hour.
  • Osmolality of the formulation is determined using Fiske210 micro-sample osmometer, DNA quantification is performed using spectrophotometry, and formulation pH is measured using Accumet research AR 15 pH meter.
  • the formulation is made in 20 mM Tris-8% sucrose instead of PBS or NaCl. Osmolality of the formulation was determined using Fiske210 micro-sample osmometer, DNA quantification is performed using spectrophotometry, and formulation pH was measured using Accumet research ARI 5 pH meter.
  • the required concentration of plasmid DNA to produce a final concentration of 0.01-0.5 mg/ml is made in 5% dextrose or PBS, the mixture was mixed by low speed vortexing and the required amount of cationic adjuvant carrier (Staramine, BD15-12, PPC, Omnifect, Benzalkonium chloride (BAK)) was add to plasmid DNA.
  • pDNA/PPC nanocomplexes are concentrated to 1-5 mg/mL using Amicon Ultra Centrifugal Filters (Ultracel-3K MWCO). Then crown poloxamer (/. ⁇ ., aza-crown-linked poloxamer) is add to the DNA solution to produce a final concentration of 0.1-5%.
  • Particle size of the CP- cationic carrier-pDNA complexes is measured with Malvern particle size analyzer sizer. Mixing crown poloxamer with plasmid DNA did not form any detectable nanoparticles by particle size measurements or gel electrophoresis. However, when cationic delivery systems are added to crown poloxamer/DNA formulation measurable nanoparticles are observed in the formulation.
  • the complexation efficiency is determined by gel retardation assay (Life Technologies, Carlsbad, CA). The gel retardation assay is performed by loading the formulated plasmid DNA on 1% agarose gel and electrophoresed at 100 V for 1 hour.
  • Osmolality of the formulation is determined using Fiske210 micro-sample osmometer, DNA quantification was performed using spectrophotometry, and formulation pH is measured using Accumet research ARI 5 pH meter.
  • Example 6 Formulations for Expression of Trp2 and NYESO-1 in Mammalian Cells [0517] To express Trp2 or NYESO-1 in mammalian cells, pVac64 or pVac65 plasmid DNA was formulated with BD3.6-oleoyl, BD15-12, Crossamine, or poloxamer.
  • Formulations comprising BD3.6-oleoyl, BD15-12, or Crossamine were able to produce particles (FIGs. 16A-16B).
  • Formulations comprising poloxamer did not produce measureable particles since poloxamer is non-condensing.
  • Mammalian cells were transfected with pVac64 plasmid DNA formulated with BD3.6-oleoyl, BD15-12, Crossamine, or poloxamer to express Trp2. Each formuation was able to generate Trp2 (FIG. 17A). Cells transfected with plasmid DNA formulated with BD3.6-oleoyl, BD 15-12, or Crossamine expressed higher levels of Trp2 compared to cells transfected with plasmid DNA formulated with poloxamer.
  • Mammalian cells were transfected with pVac65 plasmid DNA formulated with BD3.6-oleoyl, BD15-12, Crossamine, or poloxamer to express NYESO-1. Each formulation was able to generate NYESO-1 (FIG. 17B). Cells transfected with plasmid DNA formulated with BD3.6-oleoyl, BD15-12, or Crossamine expressed higher levels of NYESO-1 compared to cells transfected with plasmid DNA formulated with poloxamer.
  • a subcutaneous B16 melanoma model will be used for the evaluation of the therapies provided herein. Mice will be injected with Bl 6F 10 melanoma cells by subcutaneous injection and B16F10 cells will form a palpable tumor in about 5 to 10 days. The tumors will be grown to about 1 x 1 x 1-cm tumor in about 14 to 21 days. When allowed to grow larger, the tumors often become necrotic in the center, therefore mice will be sacrificed before this point. The typical dose used will be about 1 x io 5 cells/mouse, which is 1.5 to 2 times the minimal tumorigenic dose in normal mice.
  • mice will be administered the vaccine compositions provided herein before the tumor cells are injected to determine whether the vaccine compositions have a prophylactic effect.
  • mice will be administered the vaccine compositions provided herein together with the tumor cells to determine whether the vaccine compositions have a therapeutic effect.
  • mice will be administered the vaccine compositions provided herein at day 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14,15,16, 17, 18, 19, or 20 days after injection of the tumor cells.
  • mice will be administered the vaccine compositions provided herein after a tumor has been established to determine whether the vaccine compositions have a therapeutic effect.
  • C57BL6 mice will be injected with 5* 10 6 ID8 ovarian cancer cells or SKOV3 ovarian cancer cells into the peritoneal cavity via a left lower abdominal wall injection.
  • the tumors will be grown to about a 1 x 1 x 1-cm tumor.
  • mice will be administered the vaccine compositions provided herein before the tumor cells are injected to determine whether the vaccine compositions have a prophylactic effect.
  • mice will be administered the vaccine compositions provided herein together with the tumor cells to determine whether the vaccine compositions have a therapeutic effect.
  • mice will be administered the vaccine compositions provided herein at day 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14,15,16, 17, 18, 19, or 20 days after injection of the tumor cells.
  • mice will be administered the vaccine compositions provided herein after a tumor has been established to determine whether the vaccine compositions have a therapeutic effect.

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Abstract

Disclosed herein are immune stimulatory compositions, pharmaceutical compositions, and cancer vaccines comprising (a) a polynucleotide comprising (i) at least one antigen nucleic acid which encodes at least one tumor-associated antigen or an antigenic fragment thereof, wherein the antigen nucleic acid is operably linked to a promoter and, optionally, (ii) at least one nucleic acid which encodes an immune modifier (e.g., a cytokine); and (b) a delivery component selected from the group consisting of a cationic polymer, a poly-inosinic-polycytidylic acid, a poloxamer, or derivative thereof. The composition can futher comprises an adjuvant comprising an aluminum or aluminum-salt based adjuvant, and/or a stimulator of interferon genes (STING) agonist, or a combination thereof. Methods of production and therapeutic use of the same are also disclosed herein.

Description

POLYNUCLEOTIDE CANCER VACCINE COMPOSITIONS AND METHODS OF USING THE SAME
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional Application No. 63/328,232, filed April 6, 2022, which is hereby incorporated by reference in its entirety.
REFERENCE TO SEQUENCE LISTING SUMBITTED ELECTRONICALLY
[0002] The content of the electronically submitted sequence listing in ASCII text file (Name: 2437_078PC01_Seqlisting_ST26.txt; Size: 51,744 bytes; and Date of Creation: April 4, 2023, filed with the application is incorporated herein by reference in its entirety.
FIELD OF DISCLOSURE
[0003] The present disclosure relates generally to oncology, immunology, cancer vaccines, and gene therapy. In certain aspects, the disclosure relates to nucleic acidcontaining compositions and methods of using the same to generate an immune response to one or more tumor-associated antigens for treating, reducing the likelihood of, or preventing cancer in mammals.
BACKGROUND OF THE DISCLOSURE
[0004] Cancer vaccines are a type of cancer treatment based on immunotherapy. They work to boost the body's immune system to fight cancer. Vaccines that use tumor- associated antigens have been explored as treatment options for cancer. Cancer vaccines can use either shared tumor antigens expressed by many tumors or unique tumor antigens expressed by specific tumors. A challenge with tumor vaccines has been that tumors may contain many different cell types each with different surface antigens; and many tumors display few, if any, antigens that are foreign to the individual. Thus, recognition of tumor- associated and tumor-specific antigens by the immune system as well as the distinction between normal and tumor cells remain challenges for developing effective cancer vaccines. [0005] Nucleic acid (DNA and RNA) vaccines are being developed for a variety of infectious diseases across the globe. Despite their attractiveness, issues such as suboptimal immunogenicity and effective delivery have been concerns with DNA vaccines.
BRIEF SUMMARY OF THE DISCLOSURE
[0006] Certain aspects of the disclosure are directed to a composition comprising: (a) a polynucleotide (e.g., an expression cassette) comprising an antigen nucleic acid which encodes (i) at least one tumor-associated antigen or an antigenic fragment thereof (e.g., a first tumor-associated antigen or an antigenic fragment thereof), wherein the antigen nucleic acid is operably linked to a promoter (e.g., a first promoter), and, optionally, (ii) a nucleic acid encoding an immune modifier (e.g., a cytokine). In some aspects, the immune modifier is a cytokine. In some aspects, the cytokine is IL-12. In some aspects, the composition further comprises (b) a delivery component selected from the group consisting of a cationic polymer, a poly-inosinic-polycytidylic acid, a poloxamer, or derivative thereof. In some aspects, the delivery component is a crown poloxamer. In some aspects, the delivery component is a carrier for the polynucleotide. In some aspects, the composition further comprises (c) an adjuvant. In some aspects, the adjuvant comprises an aluminum or aluminum-salt based adjuvant, a stimulator of interferon genes (STING) agonist, or a combination thereof. In some aspects, the composition stimulates an immune response to a tumor or target cancer cell.
[0007] In some aspects, the aluminum or aluminum-salt based adjuvant is selected from the group consisting of an aluminum phosphate, an aluminum hydroxide, an aluminum oxyhydroxide, a potassium aluminum sulfate [KA1(SO4)2], an aluminum bicarbonate, an aluminum hydroxyphosphate, an aluminum hydroxyphosphate sulfate, an aluminum chloride, an aluminum silicate, and any combination thereof.
[0008] In some aspects, the aluminum or aluminum-salt based adjuvant comprises an aluminum phosphate, an aluminum hydroxide, a potassium aluminum sulfate [KA1(SO4)2], an aluminum oxyhydroxide, or any combination thereof.
[0009] In some aspects, the aluminum or aluminum-salt based adjuvant comprises an aluminum phosphate or an aluminum hydroxide. [0010] In some aspects, aluminum salt-based adjuvant is a mixture of aluminum hydroxide and magnesium hydroxide, a mixture of aluminum sulfate and sodium hydroxide, a mixture of aluminum sulfate and potassium hydroxide, a mixture of aluminum phosphate and magnesium hydroxide, aluminum phosphate and sodium hydroxide, aluminum phosphate and potassium hydroxide or a mixture of aluminum phosphate and aluminum hydroxide.
[0011] In some aspects, the adjuvant is MF59 (oil-in-water emulsion of squalene oil). In some aspects, the adjuvant is AS03 (a-tocopherol, squalenem and polysorbate 80 in an oil-in-water emulsion).
[0012] In some aspects, the STING agonist is selected from the group consisting of a cyclic di-nucleotides, a non-cyclic di-nucleotide small molecule, an amidobenzimidazole (ABZI), a flavonoid, a nanovaccine, an antibody drug conjugate, a bacterial vector, and an ENPP1 inhibitor.
[0013] In some aspects, the STING agonist is cyclic guanosine monophosphate (cGMP), cyclic adenosine monophosphate (cAMP), or cyclic guanosine monophosphate-adenosine monophosphate (cGAMP). In some aspects, the STING agonist is cGMP.
[0014] In some aspects, the antigen nucleic acid encodes at least one tumor-associated antigen or an antigenic fragment thereof, which can be an oncogene, a mutated tumor suppressor gene, an overexpressed or aberrantly expressed cellular protein, an antigen of an oncogenic virus, an oncofetal antigen, an altered cell surface glycolipid and/or glycoprotein, a cell type-specific differentiation antigen, or any combination thereof.
[0015] In some aspects, the antigen nucleic acid (e.g., first antigen nucleic acid) encodes at least one tumor-associated antigen or an antigenic fragment thereof (e.g., a first tumor- associated antigen or an antigenic fragment thereof) selected from the group consisting of NYESO-1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A9, MAGE-A10, MAGE-CI, MAGE-C2, SSX, H0RMAD1, CXorf61, ACTL8, AKAP3,AKAP4, HOM- TES-85, OY-TES-1, HSP70-2, LAGE-1, PIWL1, PIWL2, PIWL3, PIWL4, PLU-1, PLAC1, SPAG9, SCP-1, TAG-1, TAG-2a, TAG-2b, TAG-2c, TRAG-3, KAP4, BAGE, BORIS, CT45, GAGE-1/2, PRAME, PAGE4, SP17, PASD1, p53, ras, B-RafHER-2/neu, mucin 1 (MUC1), epidermal growth factor receptor (EGFR), GP100, prostatic acid phosphatase, prostate-specific membrane antigen (PSMA), mammoglobin-A, carcinoembryonic antigen (CEA), papillomavirus antigens E6/E7, heat-shock proteins, alpha fetoprotein, CDK, P-catenin, mesothelin, cancer antigen-125 (CA125), and any antigenic fragments thereof.
[0016] In some aspects, the polynucleotide comprises an antigen nucleic acid sequence that is derived from a patient’s own nucleic acid sequence that encodes a NYESO-1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A9, MAGE-A10, MAGE-CI, MAGE-C2, SSX, HORMAD1, CXorf61, ACTL8, AKAP3,AKAP4, HOM-TES-85, OY- TES-1, HSP70-2, LAGE-1, PIWL1, PIWL2, PIWL3, PIWL4, PLU-1, PLAC1, SPAG9, SCP-1, TAG-1, TAG-2a, TAG-2b, TAG-2c, TRAG-3, KAP4, BAGE, BORIS, CT45, GAGE-1/2, PRAME, PAGE4, SP17, PASD1, p53, ras, B-RafHER-2/neu, mucin 1 (MUC1), epidermal growth factor receptor (EGFR), GP100, prostatic acid phosphatase, prostate-specific membrane antigen (PSMA), mammoglobin-A, carcinoembryonic antigen (CEA), papillomavirus antigens E6ZE7, heat-shock proteins, alpha fetoprotein, CDK, P-catenin, mesothelin, or cancer antigen-125 (CA125), or any antigenic fragments thereof. In some aspects, by using a patient’s own nucleic acid sequence of tumor- associated antigens, a personalized vaccine composition is generated that specifically targets the patient’s specific tumor-associated antigens.
[0017] In some aspects, the polynucleotide (e.g., expression cassette) further comprises at least one additional antigen nucleic acid (e.g., a second antigen nucleic acid), which encodes at least one additional tumor-associated antigen or an antigenic fragment thereof (e.g., a second tumor-associated antigen or an antigenic fragment thereof).
[0018] In some aspects, the at least one additional tumor-associated antigen or an antigenic fragment thereof (e.g., a second tumor-associated antigen) is selected from the group consisting of a NYESO-1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A9, MAGE-A10, MAGE-CI, MAGE-C2, SSX, H0RMAD1, CXorf61, ACTL8, AKAP3,AKAP4, HOM-TES-85, OY-TES-1, HSP70-2, LAGE-1, PIWL1, PIWL2, PIWL3, PIWL4, PLU-1, PLAC1, SPAG9, SCP-1, TAG-1, TAG-2a, TAG-2b, TAG-2c, TRAG-3, KAP4, BAGE, BORIS, CT45, GAGE-1/2, PRAME, PAGE4, SP17, PASD1, p53, ras, B-RafHER-2/neu, mucin 1 (MUC1), epidermal growth factor receptor (EGFR), GP100, prostatic acid phosphatase, prostate-specific membrane antigen (PSMA), mammoglobin-A, carcinoembryonic antigen (CEA), papillomavirus antigens E6ZE7, heatshock proteins, alpha fetoprotein, CDK, P-catenin, mesothelin, cancer antigen- 125 (CA125), and any antigenic fragments thereof. [0019] In some aspects, the at least one tumor-associated antigen or antigenic fragment thereof (e.g., first tumor-associated antigen and/or the second tumor-associated antigen) is/are selected from the group consisting of NYESO-1, CA-125, GP100, carcinoembryonic antigen (CEA), mucin 1, TRP-2, SP17, and any combinations thereof.
[0020] In some aspects, the at least one additional antigen nucleic acid (e.g., second antigen nucleic acid) of the polynucleotide is operably linked to the first promoter through an internal ribosome entry site (IRES) sequence.
[0021] In some aspects, one antigenic nucleic acid of the polynucleotide is linked to a second antigenic nucleic acid through a 2A peptide. In some aspects, the 2A peptide is a F2A, P2A, E2A or T2A peptide.
[0022] In some aspects, the at least one additional antigen nucleic acid (e.g., second antigen nucleic acid) of the polunucletoide is operably linked to one or more additional promoters (e.g., a second promoter).
[0023] In some aspects, the promoter (e.g., first promoter) or the one or more additional promoters (e.g., second promoter) is/are selected from the group consisting of: a cytomegalovirus (CMV) promoter, a Rouse sarcoma virus (RSV) promoter, a Moloney murine leukemia virus (Mo-MuLV) long terminal repeat (LTR) promoter, a mammalian elongation factor 1 (EFl) promoter, a cytokeratin 18 (CK18) promoter, a cytokeratin 19 (CK19) promoter, a simian virus 40 (SV40) promoter, a murine U6 promoter, a skeletal a-actin promoter, a P-actin promoter, a murine phosphoglycerate kinase 1 (PGK1) promoter, a human PGK1 promoter, a CBA promoter, a CAG promoter, and any combination thereof.
[0024] In some aspects, the polynucleotide further comprises one or more post- transcriptional regulatory elements. In some aspects, the post-transcriptional regulatory element is a wood chuck hepatitis virus post-transcriptional regulatory element (WPRE).
[0025] In some aspects, the polynucleotide further comprises at least one 3' UTR poly(a) tail sequence operably linked to the first antigen nucleic acid, the second antigen nucleic acid, or any combination thereof.
[0026] In some aspects, the 3' UTR poly(a) tail sequence is a 3' UTR SV40 poly(a) tail sequence, a 3' UTR bovine growth hormone (bGH) poly(A) sequence, a 3' UTR actin poly(A) tail sequence, a 3' UTR hemoglobin poly(A) sequence, or combinations thereof.
[0027] In some aspects, the polynucleotide further comprises an enhancer sequence. In some aspects, the enhancer sequence comprises a human actin enhancer sequence, a human myosin enhancer sequence, a human hemoglobin enhancer sequence, a human muscle creatine enhancer sequence, a viral enhancer sequence, a polynucleotide function enhancer sequence, or any combination thereof.
[0028] In some aspects, the enhancer sequence comprises a CMV intronic sequence, a P- actin intronic sequence, or the combination thereof. In some aspects, the enhancer sequence is a CMV intronic sequence. In some aspects, the enhancer sequence is a CMV intronic sequence, a SV40 enhancer sequence, a P-actin intronic sequence, or combinations thereof.
[0029] In some aspects, the polynucleotide is within a vector, wherein the vector is a DNA plasmid, a viral vector, a bacterial vector, a cosmid, or an artificial chromosome. In some aspects, the vector is a DNA plasmid.
[0030] In some aspects, the composition is a pharmaceutical composition comprising a pharmaceutically acceptable carrier. In some aspects, the composition or pharmaceutical composition is a cancer vaccine.
[0031] In some aspects, the cationic polymer comprises a synthetic functionalized polymer, a P-amino ester, a lipid, a lipopolymer, or a chemical derivative thereof.
[0032] In some aspects, the synthetic functionalized polymer is a biodegradable crosslinked cationic multi-block copolymer.
[0033] In some aspects, the biodegradable cross-linked cationic multi-block copolymer is represented by the formula: (CP)xLyYz, wherein: (a) CP represents a cationic polymer containing at least one secondary amine group, wherein the cationic polymer has a number averaged molecular weight within the range of 1,000 to 25,000 Dalton, (b) Y represents a bifunctional biodegradable linker containing ester, amide, disulfide, or phosphate linages, (c) L represents a ligand, (d) x is an integer in the range from 1 to 20, (e) y is an integer in the range from 0 to 100, and (f) z is an integer in the range from 0 to 40.
[0034] In some aspects, the cationic polymer comprises biodegradable cross-linked linear polyethyleneimine (LPEI).
[0035] In some aspects, the bifunctional biodegradable linker is hydrophilic and comprises a biodegradable linkage comprising a disulfide bond.
[0036] In some aspects, the bifunctional biodegradable linker is a dithiodipropionyl linker. [0037] In some aspects, the biodegradable cross-linked cationic multi-block copolymer comprises LPEI and a dithiodipropionyl linker for cross-linking the multi-block copolymer, wherein the LPEI has an average molecular weight of 1,000 to 25,000 Dalton.
[0038] In some aspects, the biodegradable cross-linked cationic multi-block copolymer is covalently linked to at least one ligand.
[0039] In some aspects, the ligand is a targeting ligand selected from the group consisting of: a sugar moiety, a polypeptide, folate, and an antigen.
[0040] In some aspects, the sugar moiety is a monosaccharide or an oligosaccharide. In some aspects, the monosaccharide is galactose.
[0041] In some aspects, the polypeptide is a glycoprotein, an antibody, an antibody fragment, a cell receptor, a cytokine receptor, or a growth factor receptor. In some aspects, the growth factor receptor is an epidermal growth factor receptor. In some aspects, the glycoprotein is transferrin or asialoorosomucoid (ASOR).
[0042] In some aspects, the antigen is an oncoviral antigen.
[0043] In some aspects, the biodegradable cross-linked cationic multi-block copolymer is covalently linked to polyethylene glycol (PEG) of molecular weight ranging from 500 to 20,000 Dalton.
[0044] In some aspects, the biodegradable cross-linked cationic multi-block copolymer is covalently linked to a fatty acyl chain selected from the group consisting of: oleic acid, palmitic acid, and stearic acid.
[0045] In some aspects, the biodegradable cross-linked cationic multi-block copolymer comprises at least one amine group that is electrostatically attracted to a polyanionic compound.
[0046] In some aspects, the polyanionic compound is a nucleic acid, wherein the biodegradable cross-linked cationic multi-block copolymer condenses the nucleic acid to form a compact structure.
[0047] In some aspects, the lipopolymer is a cationic lipopolymer comprising a PEI backbone covalently linked to a lipid or a PEG. In some aspects, the PEI backbone is covalently linked to a lipid and a PEG. In some aspects, the lipid and the PEG are directly attached to the PEI backbone by covalent bonds. In some aspects, the lipid is attached to the PEI backbone through a PEG spacer. In some aspects, the PEG has a molecular weight of between 50 to 20,000 Dalton. In some aspects, the molar ratio of PEG to PEI is within a range of 0.1 : 1 to 500: 1. In some aspects, the molar ratio of the lipid to the PEI is within a range of 0.1 : 1 to 500: 1.
[0048] In some aspects, the lipid is a cholesterol, a cholesterol derivative, a C12 to C18 fatty acid, or a fatty acid derivative. In some aspects, the PEI is covalently linked to cholesterol and PEG, and wherein the average PEG:PEI:cholesterol molar ratio in the cationic lipopolymer is within the range of 1-5 PEG: 1 PEEO.4-1.5 cholesterol. In some aspects, the PEI has a linear or branch configuration with a molecular weight of 100 to 500,000 Dalton.
[0049] In some aspects, the cationic lipopolymer further comprises a pendant functional moiety selected from the group consisting of: a receptor ligand, a membrane permeating agent, an endosomolytic agent, a nuclear localization sequence, and a pH sensitive endosomolytic peptide.
[0050] In some aspects, the cationic lipopolymer further comprises a targeting ligand, wherein the targeting ligand is directly attached to the PEI backbone or is attached through a PEG linker. In some aspects, the targeting ligand is selected from the group consisting of: a sugar moiety, a polypeptide, folate, and an antigen. In some aspects, the sugar moiety is a monosaccharide or an oligosaccharide. In some aspects, the monosaccharide is galactose. In some aspects, the polypeptide is a glycoprotein, an antibody, an antibody fragment, a cell receptor, a cytokine receptor, or a growth factor receptor.
[0051] In some aspects, the cationic polymer is present in an amount sufficient to produce a ratio of amine nitrogen in the cationic polymer to phosphate in the DNA plasmid vector from about 0.01 : 1 to about 50: 1. In some aspects, the ratio of amine nitrogen in the cationic polymer to phosphate in the DNA plasmid vector from about 1 : 10 to about 10: 1.
[0052] In some aspects, composition, pharmaceutical composition, or vaccine comprises about 0.1 mg/ml to about 10.0 mg/ml nucleic acid complexed with the cationic polymer.
[0053] In some aspects, the delivery component comprises a lipopoly amine with the following formula:
Figure imgf000009_0001
(Staramine).
[0054] In some aspects, the delivery component comprises a mixture of the lipopolyamine and an alkylated derivative of the lipopolyamine.
[0055] In some aspects, the alkylated derivative of the lipopolyamine is a polyoxyalkylene, polyvinylpyrrolidone, polyacrylamide, polydimethylacrylamide, polyvinyl alcohol, dextran, poly (L-glutamic acid), styrene maleic anhydride, poly-N-(2- hydroxypropyl) methacrylamide, or polydivinylether maleic anhydride.
[0056] In some aspects, the alkylated derivative of the lipopolyamine has the following formula:
Figure imgf000010_0001
(methoxypolyethylene glycol (mPEG) modified Staramine), wherein n represents an integer from 10 to 100 repeating units containing of 2-5 carbon atoms each.
[0057] In some aspects, the ratio of the lipopolyamine to the alkylated derivative of the lipopolyamine in the mixture is 1 : 1 to 10: 1.
[0058] In some aspects, the lipopolyamine is present in an amount sufficient to produce a ratio of amine nitrogen in the lipopolyamine to phosphate in the DNA plasmid vector from about 0.01 : 1 to about 50: 1.
[0059] In some aspects, the lipopolyamine is present in an amount sufficient to produce a ratio of amine nitrogen in the lipopolyamine to phosphate in the DNA plasmid vector from about 1 : 10 to about 10: 1.
[0060] In some aspects, the delivery component comprises a lipopolyamine with the following formula:
Figure imgf000010_0002
[0061] In some aspects, the delivery component comprises a mixture of the lipopolyamine and an alkylated derivative of the lipopolyamine. [0062] In some aspects, the alkylated derivative of the lipopolyamine is a polyoxyalkylene, polyvinylpyrrolidone, polyacrylamide, polydimethylacrylamide, polyvinyl alcohol, dextran, poly (L-glutamic acid), styrene maleic anhydride, poly-N-(2- hydroxypropyl) methacrylamide, or polydivinylether maleic anhydride.
[0063] In some aspects, the ratio of the lipopolyamine to the alkylated derivative of the lipopolyamine in the mixture is 1 : 1 to 10: 1.
[0064] In some aspects, the lipolyamine is present in an amount sufficient to produce a ratio of amine nitrogen in the lipopolyamine to phosphate in the DNA plasmid vector from about 0.01 : 1 to about 50: 1.
[0065] In some aspects, the lipolyamine is present in an amount sufficient to produce a ratio of amine nitrogen in the lipopolyamine to phosphate in the DNA plasmid vector from about 1 : 10 to about 10: 1.
[0066] In some aspects, the delivery component comprises a poloxamer with the following formula:
Figure imgf000011_0001
or a pharmaceutically acceptable salt thereof, wherein:
A represents an integer from 2 to 141;
B represents an integer from 16 to 67;
C represents an integer from 2 to 141;
RA and RC are the same or different, and are R'-L- or H;
L is a bond, — CO — , — CH2 — O — , or — O — CO — ; and
R' is a metal chelator.
[0067] In some aspects, at least one of RA and RC is R'-L-.
[0068] In some aspects, the R’ is covalently bound to the poloxamer (e.g., where a metal chelator (e.g., a crown) is covalently attached at different density to the poloxamer).
[0069] In some aspects, one metal chelator or two or more metal chelators is/are bound to the poloxamer.
[0070] In some aspects, 2-100 metal chelators are bound to the poloxamer.
[0071] In some aspects, the metal chelator is RNNH — , RN2N — , or (R" — (N(R") —
CH2CH2)x)2 — N — CH2CO — , wherein each x is independently 0-2, and wherein R" is HO2C— CH2— . [0072] In some aspects, the metal chelator is a crown ether, a substituted-crown ether, a cryptand, or a substituted-cryptand.
[0073] In some aspects, the delivery component further comprises a PEG-PEI-cholesterol (PPC) lipopolymer, benzalkonium chloride (BAK), Omnifect, or a linear polyethyleneimine (LPEI). In some aspects, the LPEI is BD15-12.
[0074] In some aspects, the poloxamer (e.g., crown poloxamer) is present in a solution with the polynucleotide or DNA plasmid vector from about 0.01 % - about 5%.
[0075] In some aspects, the solution is co-formulated with a metal chelator (e.g., where the co-formulated metal chelator is a free metal chelator, which is formulated with the poloxamer (e.g., a non-crown poloxamer)).
[0076] In some aspects, the co-formulated metal chelator is present in the solution at a concentration of about O.lmg/mL to about 20mg/mL.
[0077] In some aspects, the co-formulated metal chelator is crown ether, a substituted- crown ether, a cryptand, or a substituted-cryptand.
[0078] In some aspects, the metal chelator or co-formulated metal chelator is crown ether (Aza- 18 -crown-6) .
[0079] In some aspects, the delivery component comprises BD15-12. In some aspects, the nucleotide to polymer (N:P) ratio is 0.1 : 1 to 5: 1.
[0080] In some aspects, the delivery component is PEG-PEI-cholesterol (PPC) lipopolymer. In some aspects, the nucleotide to polymer (N:P) ratio is 0.1 : 1 to 5: 1.
[0081] In some aspects, the delivery component comprises Omnifect. In some aspects, the nucleotide to polymer (N:P) ratio is 0.1 : 1 to 5: 1.
[0082] In some aspects, the delivery component comprises crown poloxamer connected by covalent bond directly or through a linker to an aluminum or aluminum-salt based adjuvant.
[0083] In some aspects, the delivery component comprises Staramine and mPEG modified Staramine. In some aspects, the mPEG modified Staramine is Staramine- mPEG515 or Staramine-mPEGl 1. In some aspects, the delivery component comprises Crossamine (N,N’ -di oleoyl tetrakis(aminomethyl)methane).
[0084] In some aspects, the delivery component is Starmine. In some aspects, the nucleotide to polymer (N:P) ratio is 0.1 : 1 to 5: 1.
[0085] In some aspects, the delivery component is Crossamine. In some aspects, the nucleotide to polymer (N:P) ratio is 0.1 : 1 to 5: 1. [0086] In some aspects, the ratio of Staramine to mPEG modified Staramine is 10: 1. In some aspects, the nucleotide to polymer (N:P) ratio is 0.01 : 1 to 5: 1.
[0087] In some aspects, the delivery component further comprises crown poloxamer.
[0088] In some aspects, the composition, pharmaceutical composition, or vaccine is stable at 0°C to 5°C for at least about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 monts, or about 12 months. In some aspects, the composition, pharmaceutical composition, or vaccine is stable at 25°C for at least about 7 days, about 10 day, or about 14 days, or about 60 days.
[0089] In some aspects, the composition, pharmaceutical composition, or vaccine is stable at -20°C for at least about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 monts, or about 12 months.
[0090] In some aspects, the composition is lyophilized and is substantially free of aqueous components. In some aspects, the composition is reconstituted with a diluent.In some aspects, the diluent is water.
[0091] Certain aspects of the disclosure are directed to a kit comprising the composition, pharmaceutical composition, or vaccine disclosed herein. In some aspects, the kit further comprises a glass vial. In some aspects, the kit further comprises instructions for using the composition or lyophilized composition in a method for inducing an immune response in a subject. In some aspects, the kit further comprises instructions for using the composition or lyophilized composition in a method for preventing, reducing the incidence of, attenuating or treating a malignant disease in a subject.
[0092] In some aspects, the vaccine compositions provided herein are administered to a subject suffering from a malignant disease. In some aspects, the vaccine compositions are adminsiered with an additional antineoplastic therapy. In some aspects, the additional antineoplastic therapy includes any cancer therapy used to treat a cancer disclosed herein. In some aspects, the vaccine compositions are administered with a radiation therapy. In some aspects, the vaccine compositions are administered before a radiation therapy. In some aspects, the vaccine compositions are administered after a radiation therapy.
[0093] In some aspects, the vaccine compositions are administered before a tumor surgery. In some aspects, the vaccine compositions are administered after a tumor surgery. [0094] In some aspects, the antineoplastic therapy includes a chemotherapeutic agent, anti angiogenic agent, a PARP inhibitor, an antibody (e.g., checkpoint inhibitor), a PIPKinase inhibitor, a tyrosine kinase inhibitor, an adoptive cell therapy, or combinations thereof.
[0095] In some aspects, the antineoplastic therapy includes a radiation therapy, surgery, chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, or a combination of the foregoing. The antineoplastic therapy may be in the form of adjuvant or neoadjuvant therapy. In some aspects, the vaccine compositions are administered before tumor surgery. In some aspects, the vaccine compositions are administered after tumor surgery.
[0096] In some aspects, the antineoplastic cancer therapy includes the administration of a small molecule enzymatic inhibitor or anti-metastatic agent. In some aspects, the antineoplastic cancer therapy is radiation therapy. In some aspects, the antineoplastic therapy is a therapy targeting the PBK/AKT/mTOR pathway, a HSP90 inhibitor, a tubulin inhibitor, an apoptosis inhibitor, and/or a chemopreventative agent. The antineoplastic therapy may be one or more of the chemotherapeutic agents known in the art.
[0097] In some aspects, the antineoplastic therapy is a therapy with a checkpoint inhibitor and comprises a therapeutically effective amount of a PD-1 or PD-L1 inhibitor, an inhibitor of CD274, an inhibitor of PDCD1, or an inhibitor of CD80, CD86, CD38, OD 01, TIGIT, C10orf54 (VISTA), ICOS, LAG3, CCL5, CXCL20 or any combination thereof and a pharmaceutically acceptable excipient.
[0098] In some aspects, the antineoplastic therapy is an anti angiogenic therapy and comprises bevacizumab, aflibercept, everolimus, lenalidomide, lenvatinib mesylate, pazopanib, ramucirumab, sunitinib, sorafenib, axitinib, regorafenib, cabozantinib, thalidomide, and/or vandetanib.
[0099] In some aspects, the antineoplastic therapy is a PI3Kinase inhibitor therapy and comprises idelalisib, copanlisib, duvelisib, alpelisib, umbralisib, buparlisib, copanlisib, dactolisib, duvelisib, parsaclisib, paxalisib, zandelisib, andor inbavolisib.
[0100] In some aspects, the antineoplastic therapy is a PARP inhibitor therapy and comprises talazoparib, veliparib, pamiparib, olaparib, rucaparib, CEP 9722, and/or E7016. [0101] In some aspects, the antineoplastic therapy is a chemotherapy. The term "chemotherapy" refers to the use of drugs to treat cancer. A "chemotherapeutic agent" is used to connote a compound or composition that is administered in the treatment of cancer. Examples of chemotherapeutic agents include, but are not limited to, alkylating agents, such as thiotepa and cyclosphosphamide; alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide, and trimethylolomelamine; acetogenins including bullatacin and bullatacinone; a camptothecin including the synthetic analogue topotecan; bryostatin; cally statin; CC-1065 including its adozelesin, carzelesin and bizelesin synthetic analogues; cryptophycins including cryptophycin 1 and cryptophycin 8; dolastatin; duocarmycin including the synthetic analogues, KW-2189 and CB 1-TM1; eleutherobin; pancrati statin; a sarcodictyin; spongistatin; nitrogen mustards, such as chlorambucil, chlomaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, and uracil mustard; nitrosureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and raninmustine; antibiotics, such as the enediyne antibiotics, e.g., calicheamicin, including calicheamicin gammall and calicheamicin omegall; dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores, aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin including morpholinodoxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolinodoxorubicin and deoxydoxorubicin; epirubicin; esorubicin; idarubicin; marcellomycin; mitomycins, such as mitomycin C, mycophenolic acid, nogalarnycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; anti-metabolites, such as methotrexate and 5 -fluorouracil (5-FU); folic acid analogues, such as denopterin, pteropterin, and trimetrexate; purine analogs, such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens, such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, and testolactone; anti-adrenals, such as mitotane and trilostane; folic acid replenisher, such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids, such as maytansine and ansamitocins; mitoguazone; itoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSKpolysaccharide complex; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2' ,2"- trichlorotriethylamine; trichothecenes including T-2 toxin, verracurin A, roridin A and anguidine; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; taxoids, e.g., paclitaxel and docetaxel gemcitabine; 6-thioguanine; mercaptopurine; platinum coordination complexes, such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan, e.g., CPT-11; topoisomerase inhibitor RFS 2000; difluorometlhylomithine (DMFO); retinoids, such as retinoic acid; capecitabine; carboplatin, procarbazine, plicomycin, gemcitabien, navelbine, farnesylprotein tansferase inhibitors, transplatinum, and pharmaceutically acceptable salts, acids, or derivatives of any of the above.
[0102] In some aspects, the antineoplastic therapy is a radiation therapy.
[0103] In some aspects, the cancer is selected from the group consisting of ovarian cancer, melanoma, colorectal cancer, liver cancer, pancreatic cancer, brain cancer, prostate cancer, bladder cancer, renal cancer, and hematological cancer.
[0104] Certain aspects of the disclosure are directed to methods of preventing, reducing the incidence of, attenuating or treating a tumor in a subject, the method comprising administering an effective amount of the composition, pharmaceutical composition, or cancer vaccine disclosed herein.
[0105] In some aspects, the composition, pharmaceutical composition, or cancer vaccine is administered to the subject by an intramuscular, intradermal, subcutaneous, intralymphatic, intraperitoneal, or intratumoral route of administration. In some aspects, the composition, pharmaceutical composition, or cancer vaccine is administered once, twice, three times, four times, five times, six times, or between seven and twenty times. In some aspects, the composition, pharmaceutical composition, or cancer vaccine is administered more than once in an interval of from 1 day to about 21 days; about 2 days to about 18 days; about 3 days to about 14 days; or about 4 days, about 7 days; about 10 days; about 14 days; about 21 days; about 28 days; about 35 days; about 42 days; about 49 days or about 56 days. In some aspects, the composition, pharmaceutical composition, or vaccine is administered twice in an interval of about 4 days, about 7 days; about 10 days; about 14 days; about 21 days; about 28 days about 35 days or about 42 days. In some aspects, the composition, pharmaceutical composition, or cancer vaccine is administered from about 2 times to about 20 times with intervals of about 7 days; about 10 days; about 14 days; about 21 days; about 28 days about 35 days or about 42 days between each administration.
[0106] In some aspects, the composition, pharmaceutical composition, or cancer vaccine is administered prophylactically more than once in an interval of from 1 day to about 21 days; about 2 days to about 18 days; about 3 days to about 14 days; or about 4 days, about 7 days; about 10 days; about 14 days; about 21 days; about 28 days; about 35 days; about 42 days; about 49 days or about 56 days. In some aspects, the composition, pharmaceutical composition, or vaccine is administered prophylactically twice in an interval of about 4 days; about 7 days; about 10 days; about 14 days; about 21 days; about 28 days about 35 days or about 42 days. In some aspects, the composition, pharmaceutical composition, or cancer vaccine is administered prophylactically from about 2 times to about 20 times with intervals of about 4 days; about 7 days; about 10 days; about 14 days; about 21 days; about 28 days about 35 days or about 42 days between each administration.
[0107] In some aspects, the composition, pharmaceutical composition, or cancer vaccine is administered therapeutically more than once in an interval of from 1 day to about 21 days; about 2 days to about 18 days; about 3 days to about 14 days; or about 4 days, about 7 days; about 10 days; about 14 days; about 21 days; about 28 days; about 35 days; about 42 days; about 49 days or about 56 days. In some aspects, the composition, pharmaceutical composition, or vaccine is administered therapeutically twice in an interval of about 4 days; about 7 days; about 10 days; about 14 days; about 21 days; about 28 days about 35 days or about 42 days. In some aspects, the composition, pharmaceutical composition, or cancer vaccine is administered therapeutically from about 2 times to about 20 times with intervals of about 4 days; about 7 days; about 10 days; about 14 days; about 21 days; about 28 days about 35 days or about 42 days between each administration.
[0108] Certain aspects of the disclosure are directed to methods of making a vaccine, the method comprising the steps of: (a) combining the delivery component with the polynucleotide of the composition or pharmaceutical composition disclosed herein, (b) lyophilizing the combined delivery component and polynucleotide to a powder, and (c) reconstituting the powder with a diluent that comprises the adjuvant to form a cancer vaccine solution.
BRIEF DESCRIPTION OF THE DRAWINGS
[0109] FIG. 1 shows a schematic of a vector construct comprising polynucleotide sequences of a gene encoding a tumor-associated antigen (TAA) under the control of promoter 1 (Prom-1), a gene encoding a second tumor-associated antigen (TAA-2) under the control of promoter 2 (Prom-2), and genes encoding interleukin 12 (IL- 12) p35 and IL- 12 p40 under the control of two cytomegalovirus (CMV) promoters in a plasmid backbone.
[0110] FIG. 2 shows a schematic of a vector construct comprising polynucleotide sequences of a gene encoding a tumor-associated antigen (TAA) under the control of promoter 1 (Prom-1), a gene encoding a second tumor-associated antigen (TAA-2) under the control of promoter 2 (Prom-2), genes encoding IL- 12 p35 and IL- 12 p40 under the control of two CMV promoters, and a gene encoding major histocompatibility complex class I (MHC I) under the control of promoter Z (Prom-Z) in a plasmid backbone.
[OHl] FIG. 3 shows a schematic of a vector construct comprising polynucleotide sequences of a gene encoding a tumor-associated antigen (TAA) under the control of promoter 1 (Prom-1), a gene encoding a second tumor-associated antigen (TAA-2) under the control of promoter 2 (Prom-2), genes encoding IL- 12 p35 and IL- 12 p40 under the control of two CMV promoters, and a gene encoding major histocompatibility complex class II (MHC II) under the control of promoter Z (Prom-Z) in a plasmid backbone.
[0112] FIG. 4 shows a schematic of a vector construct comprising polynucleotide sequences of a gene encoding a tumor-associated antigen (TAA) under the control of promoter 1 (Prom-1), a gene encoding a second tumor-associated antigen (TAA-2) under the control of promoter 2 (Prom-2), and a gene encoding interleukin 2 (IL-2) under the control of a CMV promoter in a plasmid backbone.
[0113] FIG. 5 shows a schematic of a vector construct comprising polynucleotide sequences of a gene encoding a tumor-associated antigen (TAA) under the control of promoter 1 (Prom-1), a gene encoding a second tumor-associated antigen (TAA-2) under the control of promoter 2 (Prom-2), a gene encoding IL-2 under the control of a CMV promoter, and a gene encoding MHC I under the control of promoter Z (Prom-Z) in a plasmid backbone.
[0114] FIG. 6 shows a schematic of a vector construct comprising polynucleotide sequences of a gene encoding a tumor-associated antigen (TAA) under the control of promoter 1 (Prom-1), a gene encoding a second tumor-associated antigen (TAA-2) under the control of promoter 2 (Prom-2), a gene encoding IL-2 under the control of a CMV promoter, and a gene encoding MHC II under the control of promoter Z (Prom-Z) in a plasmid backbone.
[0115] FIG. 7 shows a schematic of a vector construct comprising polynucleotide sequences of a gene encoding a tumor-associated antigen (TAA) under the control of promoter 1 (Prom-1), a gene encoding a second tumor-associated antigen (TAA-2) under the control of promoter 2 (Prom-2), a gene encoding IL-2 under the control of a CMV promoter, a gene encoding C-C motif chemokine ligand (CCL) 3 (CCL3) under the control of promoter X (Prom-X), and a gene encoding CCL4 under the control of promoter Y (Prom-Y) in a plasmid backbone.
[0116] FIG. 8 shows a schematic of a vector construct comprising polynucleotide sequences of a gene encoding a tumor-associated antigen (TAA) under the control of promoter 1 (Prom-1), a gene encoding a second tumor-associated antigen (TAA-2) under the control of promoter 2 (Prom-2), and a gene encoding interleukin 15 (IL- 15) under the control of a CMV promoter in a plasmid backbone.
[0117] FIG. 9 shows a schematic of a vector construct comprising polynucleotide sequences of a gene encoding a tumor-associated antigen (TAA) under the control of promoter 1 (Prom-1), a gene encoding a second tumor-associated antigen (TAA-2) under the control of promoter 2 (Prom-2), a gene encoding IL- 15 under the control of a CMV promoter, and a gene encoding MHC I under the control of promoter Z (Prom-Z) in a plasmid backbone. [0118] FIG. 10 shows a schematic of a vector construct comprising polynucleotide sequences of a gene encoding a tumor-associated antigen (TAA) under the control of promoter 1 (Prom-1), a gene encoding a second tumor-associated antigen (TAA-2) under the control of promoter 2 (Prom-2), a gene encoding IL- 15 under the control of a CMV promoter, and a gene encoding MHC II under the control of promoter Z (Prom-Z) in a plasmid backbone.
[0119] FIG. 11 shows a schematic of a vector construct comprising polynucleotide sequences of a gene encoding a tumor-associated antigen (TAA) under the control of promoter 1 (Prom-1), a gene encoding a second tumor-associated antigen (TAA-2) under the control of promoter 2 (Prom-2), a gene encoding IL- 15 under the control of a CMV promoter, a gene encoding CCL3 under the control of promoter X (Prom-X), and a gene encoding CCL4 under the control of promoter Y (Prom-Y) in a plasmid backbone.
[0120] FIG. 12 shows a schematic of a vector construct comprising polynucleotide sequences of a gene encoding a tumor-associated antigen (TAA) under the control of promoter 1 (Prom-1), a gene encoding a second tumor-associated antigen (TAA-2) under the control of promoter 2 (Prom-2), a gene encoding CCL3 under the control of promoter X (Prom-X), and a gene encoding CCL4 under the control of promoter Y (Prom-Y) in a plasmid backbone.
[0121] FIG. 13 shows a schematic of the constructs pVacl-pVac 5.
[0122] FIGs. 14A-14O show schematics of vectors, including pVac 6 to pVac 17, pVac64, and pVac65 vectors. FIG. 14B shows a schematic of pVac6 comprising a polynucleotide sequence of a gene encoding a tumor-associated antigen (TAA) under the control of a hEFl-HTLV promoter with a SV40 enhancer and a SV40 poly A, a polynucleotide sequence of a gene encoding p40 under the control of a CMV promoter and a SV40 poly A, and a polynucleotide sequence of a gene encoding p35 under the control of a CMV promoter with a SV40 polyA. FIG. 14C shows a schematic of pVac7 comprising a polynucleotide sequence of a gene encoding a tumor-associated antigen (TAA) operably linked to a gene encoding a second tumor-associated antigen (TAA-2) by a IRES sequence and under the control of a hEFl-HTLV promoter with a SV40 polyA, a polynucleotide sequence of a gene encoding p40 under control of a CMV promoter and a SV40 polyA, and a polynucleotide sequence of a gene encoding p35 under control of a CMV promoter and a SV40 polyA. FIG. 14D shows a schematic of pVac8 comprising a polynucleotide sequence of a gene encoding a tumor-associated antigen (TAA) under the control of a EF-la promoter and a SV40 polyA. FIG. 14E shows a schematic of pVac9 comprising a polynucleotide sequence of a gene encoding a tumor-associated antigen (TAA) under the control of a EF-la promoter and a SV40 polyA, a polynucleotide sequence of a gene encoding p40 under the control of a CMV promoter and a SV40 polyA, and a polynucleotide sequence of a gene encoding a p35 under the control of a CMV promoter and a SV40 polyA. FIG. 14F shows a schematic of pVaclO comprising a polynucleotide sequence of a gene encoding a tumor-associated antigen (TAA) operably linked to a gene encoding a second tumor-associated antigen (TAA-2) by a IRES sequence and under the control of a EF-la promoter, a polynucleotide sequence of a gene encoding p40 under the control of a CMV promoter and a SV40 polyA, and a polynucleotide sequence of a gene encoding p35 under the control of a CMV promoter and a SV40 polyA. FIG. 14G shows a schematic of pVacl 1 comprising a polynucleotide sequence of a gene encoding a tumor-associated antigen (TAA) under the control of a EF- la and a SV40 polyA, a polynucleotide sequence of a gene encoding a second tumor- associated antigen (TAA-2) under the control of a CMV promoter and a SV40 polyA. FIG. 14H shows a schematic of pVacl2 comprising a polynucleotide sequence of a gene encoding a tumor-associated antigen (TAA) under the control of a CMV promoter and a SV40 polyA, and a polynucleotide sequence of a gene encoding a second tumor- associated antigen (TAA-2) under the control of a CMV promoter and a SV40 polyA. FIG. 141 shows a schematic of pVacl3 comprising a polynucleotide sequence of a gene encoding a tumor-associated antigen (TAA) under the control of a EF-la promoter and a SV40 polyA. FIG. 14J shows a schematic of pVacl4 comprising a polynucleotide sequence of a gene encoding a tumor-associated antigen (TAA) under the control of a EF- la promoter and a SV40 polyA. FIG. 14K shows a schematic of pVacl5 comprising a polynucleotide sequence of a gene encoding a tumor-associated antigen (TAA) under the control of a EF-la promoter and SV40 polyA and a polynucleotide sequence of a gene encoding a second tumor-associated antigen (TAA-2) under the control of a EF-la promoter and SV40 polyA. FIG. 14L shows a schematic of pVacl6 comprising a polynucleotide sequence of a gene encoding a tumor-associated antigen (TAA) under the control of a CMV promoter, SV40 enhancer, and SV40 polyA. FIG. 14M shows a schematic of pVacl7 comprising a polynucleotide sequence of a gene encoding a tumor- associated antigen (TAA) under the control of a CMV promoter, SV40 enhancer, and SV40 polyA and a polynucleotide sequence of a gene encoding a second tumor- associated antigen (TAA-2) under the control of a CMV promoter, SV40 enhancer, and SV40 polyA. FIG. 14N shows a schematic of pVac64 comprising a polynucleotide sequence of a gene encoding Trp2 under the control of a CMV promoter with a SV40 enhancer and bGH polyA. FIG. 140 shows a schematic of pVac65 comprising a polynucleotide sequence of a gene encoding human CTAG1B-NYES0-1 under the control of a CMV promoter with a SV40 enhancer and bGH polyA.
[0123] FIG. 15A is a DNA gel showing uncut pVac64 plasmid DNA or pVac64 plasmid DNA after restriction digestion with Xhol or Sacl.
[0124] FIG. 15B is a DNA gel showing uncut pVac65 plasmid DNA or pVac65 plasmid DNA after restriction digestion with Kpnl.
[0125] FIG. 16A is a graph showing the particle size of pVa64 plasmid DNA formulated with BD3.6-oleoyl, BD15-12, Crossamine, or poloxamer.
[0126] FIG. 16B is a graph showing the particle size of pVac65 plasmid DNA formulated with BD3.6-oleoyl, BD15-12, Crossamine, or poloxamer.
[0127] FIG. 17A is a graph showing the expression of Trp2 mRNA (FIG. 17A) after transfection of mammalian cells with pVac64 plasmid DNA formulated with BD3.6- oleoyl, BD-15, Crossamine, or poloxamer.
[0128] FIG. 17B is a graph showing the expression of NYESO-1 mRNA (FIG. 17B) after transfection of mammalian cells with pVac65 plasmid DNA formulated with BD3.6- oleoyl, BD-15, Crossamine, or poloxamer.
DETAILED DESCRIPTION OF THE DISCLOSURE
I. Overview
[0129] Provided herein are compositions, including pharmaceutical compositions and vaccines, that comprise polynucleotides (e.g., DNA or mRNA), vectors, multi ci str onic mRNA vectors, DNA plasmid vectors of the disclosure to address, for example, the issue of suboptimal immunogenicity often associated with DNA-based cancer vaccine approaches on one or more levels.
[0130] In some aspects, the composition can comprise polynucleotides that express one or more tumor-associated antigens and, optionally, express one or more immune modifiers (e.g., a cytokine). In some aspects, the composition can comprise polynucleotides that express one or more tumor-associated antigens and interleukin- 12 (IL-12). In some aspects, the composition comprising a polynucleotide encoding at least one tumor-associated antigen and IL- 12 inhibits an IgG response, but not a T cell response.
[0131] In some aspects, the composition comprising a polynucleotide that expresses one or more tumor-associated antigens further comprises a delivery agent. In some aspects, the composition comprising a polynucleotide that expresses one or more tumor-associated antigens and a cytokine immune modifier (e.g., IL-12) further comprises a delivery agent. In some aspects, the composition optionally comprises an adjuvant. In some aspects, the compositions comprise an adjuvant or combination of adjuvants. In some aspects, the adjuvant can comprise an aluminum or aluminum-salt based adjuvant. In some aspects, the adjuvant can comprise a stimulator of interferon genes (STING) agonist. In some aspects, the adjuvant can comprise an aluminum or aluminum-salt based adujvant and stimulator of interferon genes (STING) agonist.
[0132] In some aspects, the composition can comprise polynucleotides that co-express one or more immune modifier proteins such as cytokines and/or chemokines that augment the immune responses to the tumor-associated antigens. In some aspects, the polynucleotides can include multiple tumor-associated antigens and/or multiple epitomes of a tumor-associated antigen instead of a single tumor-associated antigen, which can be co-expressed from the vectors disclosed herein to expand the spectrum of immunogenicity. In some aspects, to further improve vaccine efficiency, the vectors disclosed herein can be formulated with delivery systems (e.g., a cationic polymer, a poly-inosinic-polycylidylic acid, a poloxamer, or a derivative thereof) that protects the vector or polynucleotide from nuclease degradation and promote its translocation through cell compartments. In some aspects, the delivery systems disclosed herein can also be formulated to exhibit an additional adjuvant property to promote mobilization of antigen presenting cells to the site of vaccine delivery and antigen expression, thereby augmenting the uptake of the vaccine vector and the expressed tumor associated antigens into professional antigen presenting cells to elicit MHC Class I and MHC Class II presentation. See Greenland, J.R., et al., Molecular Therapy, 12(1): 164-70 (2005) (hereafter “Greenland 2005”) and Suschak, J. J., et al., Human Vaccines & Immunotherapeutics, 13(12):2837-48 (2017) (hereafter “Suschak 2017”).
[0133] In some aspects, the compositions comprise an ALUM salt adjuvant. In some aspects, the ALUM salt is selected from aluminum phosphate, aluminum hydroxide, potassium aluminum sulfate [KA1(SO4)2], aluminumcrystalline aluminum oxyhydroxide, aluminumaluminum hydroxyphosphate, amorphous aluminum hydroxyphosphate sulfate, aluminum chloride, aluminum silicate, and a mixture of aluminum hydroxide and magnesium hydroxide, a mixture of aluminum sulfate and sodium hydroxide ora mixture of aluminum sulfate and potassium hydroxide. In some aspects, the adjuvant is MF59 (oil-in-water emulsion of squalene oil). In some aspects, the adjuvant is AS03 (a-tocopherol, squalenem and polysorbate 80 in an oil-in-water emulsion).
[0134] In some aspects, the composition comprise a STING agonist. In some aspects, the STING agonist is selected from a cyclic di-nucleotides, a non-cyclic di-nucleotide small molecule, an amidobenzimidazole, a nanovaccine, an antibody drug conjugate, a bacterial vector, and an ENPP1 inhibitor.
[0135] In some aspects, the STING agonist is a cyclic dinucleotide cGMP, cAMP, or cGMP-AMP. In some aspects, the STING agonist is cGMP.
[0136] In some aspects, the STING agonist is a cylic dinucleotide selected from ADU- S100, MK-1454, SB11285, BMS-986301, BI-STING (BI1387446), JNJ-67544412, 3’3’- cyclic AIMP, and GSK532.
[0137] In some aspects, the STING agonist is a non-cyclic dinucleotide small molecule selected from DMAXAA, ALG-031048, E7766, JNJ-‘6196, MK-21118, MSA-1, MSA-2, SNX281, SR-717, TAK676, TTI-10001, a Ryvu’s agonist, GF3-002, a Selvita agonist, CDR5500, CS-1010, CS-1018, CS-1020, and [2-ex] MSA-1.
[0138] In some aspects, the STNG agonist is a nanovaccine selected from PC7A nanoparticles, cGMP-nanoparticles, and ONM-500 nanoparticles.
[0139] In some aspects, the STING agonist is an antibody-drug conjugate. In some aspects, the antibody drug conjugate is XMT-2056. In some aspects, the STING agonist is an ENPP1 inhibitor selected from MV-626, SR-8314, SR-8291, and SR8541A.
[0140] In some aspects, the STING agonist is a bacterial vector. In some aspects, the bacterial vector is a nonpathogenic E coli nissle expressing cyclic-di-AMP -producing enzymes. In some aspects, the bacterial vector is SYNB1981. In some aspect, the bacterial vector is an attenutated Salmonella Typhimurium strain enginerred to carry an inhibitory TREX-1 micro RNA.
[0141] In some aspects, the polynucleotide can comprise a nucleic acid sequence encoding one or more tumor-associated antigens (e.g., NYESO-1, MAGE-A1, MAGE- A2, MAGE-A3, MAGE-A4, MAGE-A9, MAGE-A10, MAGE-CI, MAGE-C2, SSX, HORMAD1, CXorf61, ACTL8, AKAP3,AKAP4, HOM-TES-85, OY-TES-1, HSP70-2, LAGE-1, PIWL1, PIWL2, PIWL3, PIWL4, PLU-l, PLAC1, SPAG9, SCP-1, TAG-1, TAG-2a, TAG-2b, TAG-2c, TRAG-3, KAP4, BAGE, BORIS, CT45, GAGE-1/2, PRAME, PAGE4, SP17, PASD1, p53, ras, B-RafHER-2/neu, mucin 1 (MUC1), epidermal growth factor receptor (EGFR), GP100, prostatic acid phosphatase, prostatespecific membrane antigen (PSMA), mammoglobin-A, carcinoembryonic antigen (CEA), papillomavirus antigens E6ZE7, heat-shock proteins, alpha fetoprotein, CDK, P-catenin, mesothelin, or cancer antigen-125 (CA125), an antigenic fragments thereof, or any combinations thereof). In some aspects, the one or more tumor associated antigens comprise tumor associated antigens from two or more tumors. In some aspects, the vector further comprises a nucleic acid sequence encoding one or more immune modifier proteins (e.g., a cytokine). In some aspects, the vector comprises a nucleic acid sequence encoding a tumor-associated antigen and, optionally, a second tumor-associated antigen. In some aspects, the vector comprises a nucleic acid sequence encoding a tumor- associated antigen and a second tumor-associated antigen from a different tumor.
[0142] In some aspects, the present disclosure is directed to a composition comprising a polynucleotide (e.g., a multicistronic DNA plasmid or multi ci str onic mRNA) comprising an antigen nucleic acid which encodes a tumor-associated antigen (e.g., NYESO-1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A9, MAGE-A10, MAGE-CI, MAGE-C2, SSX, H0RMAD1, CXorf61, ACTL8, AKAP3,AKAP4, HOM-TES-85, OY- TES-1, HSP70-2, LAGE-1, PIWL1, PIWL2, PIWL3, PIWL4, PLU-l, PLAC1, SPAG9, SCP-1, TAG-1, TAG-2a, TAG-2b, TAG-2c, TRAG-3, KAP4, BAGE, BORIS, CT45, GAGE-1/2, PRAME, PAGE4, SP17, PASD1, p53, ras, B-RafHER-2/neu, mucin 1 (MUC1), epidermal growth factor receptor (EGFR), GP100, prostatic acid phosphatase, prostate-specific membrane antigen (PSMA), mammoglobin-A, carcinoembryonic antigen (CEA), papillomavirus antigens E6ZE7, heat-shock proteins, alpha fetoprotein, CDK, P-catenin, mesothelin, or cancer antigen-125 (CA125), an antigenic fragments thereof, or any combinations thereof) and a cytokine immune modifier. In some aspects, the present disclosure is directed to a composition comprising (a) a polynucleotide (e.g., a multicistronic DNA plasmid or multicistronic mRNA) comprising (i) an antigen nucleic acid which encodes a tumor-associated antigen (e.g., NYESO-1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A9, MAGE-A10, MAGE-CI, MAGE-C2, SSX, H0RMAD1, CXorf61, ACTL8, AKAP3,AKAP4, HOM-TES-85, OY-TES-1, HSP70-2, LAGE-1, PIWL1, PIWL2, PIWL3, PIWL4, PLU-l, PLAC1, SPAG9, SCP-1, TAG-1, TAG-2a, TAG-2b, TAG-2c, TRAG-3, KAP4, BAGE, BORIS, CT45, GAGE-1/2, PRAME, PAGE4, SP17, PASD1, p53, ras, B-RafHER-2/neu, mucin 1 (MUC1), epidermal growth factor receptor (EGFR), GP100, prostatic acid phosphatase, prostatespecific membrane antigen (PSMA), mammoglobin-A, carcinoembryonic antigen (CEA), papillomavirus antigens E6ZE7, heat-shock proteins, alpha fetoprotein, CDK, P-catenin, mesothelin, cancer antigen-125 (CA125), an antigenic fragments thereof, or any combination thereof) and (ii) a cytokine immune modifier; and (b) a delivery agent. In some aspects, the present disclosure is directed to a composition comprising (a) a polynucleotide (e.g., a multicistronic DNA plasmid or multi ci str onic mRNA) comprising an antigen nucleic acid which encodes a tumor-associated antigen (e.g. NYESO-1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A9, MAGE-A10, MAGE-CI, MAGE-C2, SSX, H0RMAD1, CXorf61, ACTL8, AKAP3,AKAP4, HOM-TES-85, OY- TES-1, HSP70-2, LAGE-1, PIWL1, PIWL2, PIWL3, PIWL4, PLU-l, PLAC1, SPAG9, SCP-1, TAG-1, TAG-2a, TAG-2b, TAG-2c, TRAG-3, KAP4, BAGE, BORIS, CT45, GAGE-1/2, PRAME, PAGE4, SP17, PASD1, p53, ras, B-RafHER-2/neu, mucin 1 (MUC1), epidermal growth factor receptor (EGFR), GP100, prostatic acid phosphatase, prostate-specific membrane antigen (PSMA), mammoglobin-A, carcinoembryonic antigen (CEA), papillomavirus antigens E6ZE7, heat-shock proteins, alpha fetoprotein, CDK, P-catenin, mesothelin, cancer antigen-125 (CA125) or an antigenic fragments thereof, or any combination thereof) and a cytokine immune modifier; (b) a delivery agent; and (c) an adjuvant comprising an aluminum or aluminum-salt based adjuvant and/or a STING agonist. In some aspects, omposition comprises (a) a polynucleotide (e.g., a multicistronic DNA plasmid or multicistronic mRNA) comprising an antigen nucleic acid which encodes a tumor-associated antigen (e.g., NYESO-1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A9, MAGE-A10, MAGE-CI, MAGE-C2, SSX, H0RMAD1, CXorf61, ACTL8, AKAP3,AKAP4, HOM-TES-85, OY-TES-1, HSP70-2, LAGE-1, PIWL1, PIWL2, PIWL3, PIWL4, PLU-l, PLAC1, SPAG9, SCP-1, TAG-1, TAG-2a, TAG-2b, TAG-2c, TRAG-3, KAP4, BAGE, BORIS, CT45, GAGE-1/2, PRAME, PAGE4, SP17, PASD1, p53, ras, B-RafHER-2/neu, mucin 1 (MUC1), epidermal growth factor receptor (EGFR), GP100, prostatic acid phosphatase, prostatespecific membrane antigen (PSMA), mammoglobin-A, carcinoembryonic antigen (CEA), papillomavirus antigens E6ZE7, heat-shock proteins, alpha fetoprotein, CDK, P-catenin, mesothelin, or cancer antigen-125 (CA125)) or an antigenic fragments thereof, or any combination thereof) and a cytokine immune modifier; and (b) a delivery agent; and (c) an adjuvant comprising an aluminum or aluminum-salt based adjuvant. In some aspects, the composition comprises (a) a polynucleotide (e.g., a multi ci stronic DNA plasmid or multi ci str onic mRNA) comprising an antigen nucleic acid which encodes a tumor- associated antigen (e.g, NYESO-1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A9, MAGE-A10, MAGE-CI, MAGE-C2, SSX, H0RMAD1, CXorf61, ACTL8, AKAP3,AKAP4, HOM-TES-85, OY-TES-1, HSP70-2, LAGE-1, PIWL1, PIWL2, PIWL3, PIWL4, PLU-1, PLAC1, SPAG9, SCP-1, TAG-1, TAG-2a, TAG-2b, TAG-2c, TRAG-3, KAP4, BAGE, BORIS, CT45, GAGE-1/2, PRAME, PAGE4, SP17, PASD1, p53, ras, B-RafHER-2/neu, mucin 1 (MUC1), epidermal growth factor receptor (EGFR), GP100, prostatic acid phosphatase, prostate-specific membrane antigen (PSMA), mammoglobin-A, carcinoembryonic antigen (CEA), papillomavirus antigens E6ZE7, heatshock proteins, alpha fetoprotein, CDK, P-catenin, mesothelin, or cancer antigen- 125 (CA125)) or an antigenic fragments thereof, or any combination thereof) and a cytokine immune modifier; and (b) a delivery agent; and (c) an adjuvant comprising a STING agonist. In some aspects, the composition comprises (a) a polynucleotide (e.g., a multi ci stronic DNA plasmid or multi ci stronic mRNA) comprising an antigen nucleic acid which encodes a tumor-associated antigen (e.g, NYESO-1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A9, MAGE-A10, MAGE-CI, MAGE-C2, SSX, H0RMAD1, CXorf61, ACTL8, AKAP3,AKAP4, HOM-TES-85, OY-TES-1, HSP70-2, LAGE-1, PIWL1, PIWL2, PIWL3, PIWL4, PLU-1, PLAC1, SPAG9, SCP-1, TAG-1, TAG-2a, TAG-2b, TAG-2c, TRAG-3, KAP4, BAGE, BORIS, CT45, GAGE-1/2, PRAME, PAGE4, SP17, PASD1, p53, ras, B-RafHER-2/neu, mucin 1 (MUC1), epidermal growth factor receptor (EGFR), GP100, prostatic acid phosphatase, prostatespecific membrane antigen (PSMA), mammoglobin-A, carcinoembryonic antigen (CEA), papillomavirus antigens E6ZE7, heat-shock proteins, alpha fetoprotein, CDK, P- catenin, mesothelin, or cancer antigen- 125 (CAI 25)) or an antigenic fragments thereof, or any combination thereof) and a cytokine immune modifier; and (b) a delivery agent; and (c) an adjuvant comprising an aluminum or aluminum-salt based adjuvant and a STING agonist. In some aspects, the multi ci stronic DNA or mRNA comprises one tumor- associated antigen and an immune modifier protein. [0143] In some aspects, the multicistronic DNA or mRNA comprises at least two tumor- associated antigens. In some aspects, the multicistronic DNA or mRNA comprises at least two tumor-associated antigens and an immune modifier protein. In some aspects, the multicistronic DNA comprises at least two tumor-associated antigens and at least two immune modifier proteins. In some aspects, the antigen nucleic acid (e.g., antigen nucleic acid) of the polynucleotide is operably linked to a promoter. In some aspects, the polynucleotide further comprises one or more nucleic acids encoding an immune modifier protein. In some aspects, the polynucleotide further comprises at least one additional antigen nucleic acid (e.g., second antigen nucleic acid) which encodes at least one additional tumor-associated antigen (e.g., a second tumor-associated antigen or an antigenic fragment thereof) and/or one additional cytokine immune modifier.
[0144] As described herein, the nucleic acid molecules of the present disclosure comprise one or more features that distinguish the present nucleic acid molecules form those that exist in nature e.g., comprising at least one gene encoding a tumor-associated antigen and a heterologous promoter). Not to be bound by any theory, in some aspects, the expression of multiple tumor-associated antigens, expands the spectrum of immunogenicity, while the presence of an adjuvant (e.g., a STING agonist in the composition) and optionally the expression of at least one immune modifier protein augment the immune responses to the multiple tumor-associated antigens. In some aspects, the tumor-associated antigens are selected from the group consisting of NYESO- 1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A9, MAGE-A10, MAGECI, MAGE-C2, SSX, H0RMAD1, CXorf61, ACTL8, AKAP3,AKAP4, HOM-TES-85, OY-TES-1, HSP70-2, LAGE-1, PIWL1, PIWL2, PIWL3, PIWL4, PLU-1, PLAC1, SPAG9, SCP-1, TAG-1, TAG-2a, TAG-2b, TAG-2c, TRAG-3, KAP4, BAGE, BORIS, CT45, GAGE-1/2, PRAME, PAGE4, SP17, PASD1, p53, ras, B-RafHER-2/neu, mucin 1 (MUC1), epidermal growth factor receptor (EGFR), GP100, prostatic acid phosphatase, prostate-specific membrane antigen (PSMA), mammoglobin-A, carcinoembryonic antigen (CEA), papillomavirus antigens E6ZE7, heat-shock proteins, alpha fetoprotein, CDK, P-catenin, mesothelin, or cancer antigen-125 (CA125), or any combination thereof).
[0145] In some aspects, the composition comprises (i) a polynucleotide comprising: (a) a first nucleic acid encoding a first tumor-associated antigen (e.g., NYESO-1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A9, MAGE-A10, MAGE-CI, MAGE-C2, SSX, H0RMAD1, CXorf61, ACTL8, AKAP3,AKAP4, HOM-TES-85, OY-TES-1, HSP70-2, LAGE-1, PIWL1, PIWL2, PIWL3, PIWL4, PLU-l, PLAC1, SPAG9, SCP-1, TAG-1, TAG-2a, TAG-2b, TAG-2c, TRAG-3, KAP4, BAGE, BORIS, CT45, GAGE-1/2, PRAME, PAGE4, SP17, PASD1, p53, ras, B-RafHER-2/neu, mucin 1 (MUC1), epidermal growth factor receptor (EGFR), GP100, prostatic acid phosphatase, prostatespecific membrane antigen (PSMA), mammoglobin-A, carcinoembryonic antigen (CEA), papillomavirus antigens E6ZE7, heat-shock proteins, alpha fetoprotein, CDK, P-catenin, mesothelin, or cancer antigen-125 (CA125)) or an antigenic fragments thereof), wherein the first nucleic acid is operably linked to a first promoter; (b) a second nucleic acid encoding a second tumor-associated antigen (e.g., NYESO-1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A9, MAGE-A10, MAGE-CI, MAGE-C2, SSX, H0RMAD1, CXorf61, ACTL8, AKAP3,AKAP4, HOM-TES-85, OY-TES-1, HSP70-2, LAGE-1, PIWL1, PIWL2, PIWL3, PIWL4, PLU-l, PLAC1, SPAG9, SCP-1, TAG-1, TAG-2a, TAG-2b, TAG-2c, TRAG-3, KAP4, BAGE, BORIS, CT45, GAGE-1/2, PRAME, PAGE4, SP17, PASD1, p53, ras, B-RafHER-2/neu, mucin 1 (MUC1), epidermal growth factor receptor (EGFR), GP100, prostatic acid phosphatase, prostatespecific membrane antigen (PSMA), mammoglobin-A, carcinoembryonic antigen (CEA), papillomavirus antigens E6ZE7, heat-shock proteins, alpha fetoprotein, CDK, P-catenin, mesothelin, or cancer antigen-125 (CA125)) or an antigenic fragments thereof), wherein the second nucleic acid is operably linked to a second promoter; and optionally (c) a third nucleic acid encoding an immune modifier protein, wherein the third nucleic acid is operably linked to a third promoter; and (ii) and adjuvant comprising an aluminum or aluminum-salt based adjuvant and/or a STING agonist.
[0146] In some aspects, the polynucleotide can include the elements as disclosed in any of FIGs. 1-12. In some aspects, the vector constructs illustrated in any of FIGs 1-12 can modified to replace the “TAA Gene” (a first nucleotide sequence encoding a tumor- associated antigen) and the “TAA-2” (a second nucleotide sequence encoding a tumor- associated antigen) with nucleotide sequences encoding any combinations of tumor- associated antigens or antigenic fragment thereof disclosed herein.
[0147] In some aspects, the polynucleotide can include the elements as disclosed in any of FIGs. 14A-14O. In some aspects, the vector constructs illustrated in any of FIGs. 14A- 14M can modified to replace the “TAA” (a first nucleotide sequence encoding a tumor- associated antigen) and the “TAA-2” (a second nucleotide sequence encoding a tumor- associated antigen) with nucleotide sequences encoding any combinations of tumor- associated antigens or antigenic fragment thereof disclosed herein.
[0148] In some aspects, the polynucleotide further comprises a fourth nucleic acid encoding a second immune modifier protein, wherein the fourth nucleic acid is operably linked to a fourth promoter.
[0149] In some aspects, the polynucleotide can comprise a first nucleic acid encoding a first tumor-associated antigen (e.g., NYESO-1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A9, MAGE-A10, MAGE-CI, MAGE-C2, SSX, H0RMAD1, CXorf61, ACTL8, AKAP3,AKAP4, HOM-TES-85, OY-TES-1, HSP70-2, LAGE-1, PIWL1, PIWL2, PIWL3, PIWL4, PLU-1, PLAC1, SPAG9, SCP-1, TAG-1, TAG-2a, TAG-2b, TAG-2c, TRAG-3, KAP4, BAGE, BORIS, CT45, GAGE-1/2, PRAME, PAGE4, SP17, PASD1, p53, ras, B-RafHER-2/neu, mucin 1 (MUC1), epidermal growth factor receptor (EGFR), GP100, prostatic acid phosphatase, prostate-specific membrane antigen (PSMA), mammoglobin-A, carcinoembryonic antigen (CEA), papillomavirus antigens E6ZE7, heat-shock proteins, alpha fetoprotein, CDK, P-catenin, mesothelin, or cancer antigen-125 (CA125)) or an antigenic fragments thereof), wherein the first nucleic acid is operably linked to a first promoter. In some aspects, the polynucleotide further comprises a second nucleic acid encoding a second tumor-associated antigen (e.g. NYESO-1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A9, MAGE-A10, MAGE-CI, MAGE-C2, SSX, H0RMAD1, CXorf61, ACTL8, AKAP3,AKAP4, HOM- TES-85, OY-TES-1, HSP70-2, LAGE-1, PIWL1, PIWL2, PIWL3, PIWL4, PLU-1, PLAC1, SPAG9, SCP-1, TAG-1, TAG-2a, TAG-2b, TAG-2c, TRAG-3, KAP4, BAGE, BORIS, CT45, GAGE-1/2, PRAME, PAGE4, SP17, PASD1, p53, ras, B-RafHER-2/neu, mucin 1 (MUC1), epidermal growth factor receptor (EGFR), GP100, prostatic acid phosphatase, prostate-specific membrane antigen (PSMA), mammoglobin-A, carcinoembryonic antigen (CEA), papillomavirus antigens E6/E7, heat-shock proteins, alpha fetoprotein, CDK, P-catenin, mesothelin, or cancer antigen-125 (CA125)) or an antigenic fragments thereof). In some aspects, the second nucleic acid is operably linked to the first promoter through an IRES sequence. In some aspects, the first tumor- associated antigen and the second tumor-associated antigen are different portions of the same tumor-associated antigen. In some aspects, the first tumor-associated antigen and the second tumor-associated antigen are different tumor-associated antigens, wherein the different tumor-associated antigens are derived from different tumors. In some aspects, the first tumor-associated antigen and the second tumor-associated antigen are different tumor-associated antigens, wherein the different tumor-associated antigens are derived from the same tumors. In some aspects, the polynucleotide further comprises a second promoter, and the second nucleic acid is operably linked to the second promoter.
[0150] In some aspects, the compositions, vaccines, or pharmaceutical compositions further comprise a delivery component (e.g., a cationic polymer such as a biodegradable cross-linked cationic multi-block copolymer, a PEG-PEI-cholesterol (PPC) lipopolymer, a lipopolyamine, or a lipopolyamine derivative). In some aspects, the PEG-PEI- cholesterol (PPC) lipopolymer has an average PEG:PEI:cholesterol ratio of 2.5: 1 :0.6.
[0151] In some aspects, the delivery component exhibits an adjuvant property. Not to be bound by any theory, in some aspects, the adjuvant property of the delivery component promotes mobilization of antigen presenting cells to the site of vaccine delivery and antigen expression, thereby augmenting the uptake of the polynucleotide and the expressed antigens into professional antigen presenting cells to elicit MHC Class I and MHC Class II presentation. In some aspects, the present disclosure is directed to vaccines or compositions comprising (i) a vector (e.g., a multi ci stronic DNA plasmid vector or a multi ci str onic messenger RNA (mRNA) vector) comprising a nucleic acid sequence encoding one or more tumor associated antigens (e.g., NYESO-1, MAGE-A1, MAGE- A2, MAGE-A3, MAGE-A4, MAGE-A9, MAGE-A10, MAGE-CI, MAGE-C2, SSX, H0RMAD1, CXorf61, ACTL8, AKAP3,AKAP4, HOM-TES-85, OY-TES-1, HSP70-2, LAGE-1, PIWL1, PIWL2, PIWL3, PIWL4, PLU-1, PLAC1, SPAG9, SCP-1, TAG-1, TAG-2a, TAG-2b, TAG-2c, TRAG-3, KAP4, BAGE, BORIS, CT45, GAGE-1/2, PRAME, PAGE4, SP17, PASD1, p53, ras, B-RafHER-2/neu, mucin 1 (MUC1), epidermal growth factor receptor (EGFR), GP100, prostatic acid phosphatase, prostatespecific membrane antigen (PSMA), mammoglobin-A, carcinoembryonic antigen (CEA), papillomavirus antigens E6ZE7, heat-shock proteins, alpha fetoprotein, CDK, P- catenin,mesothelin, or cancer antigen-125 (CA125)), (ii) a delivery component (e.g., a cationic polymer, a poly-inosinic-polycytidylic acid, or a poloxamer), (iii) a STING agonist, and (iv) an ALUM adjuvant. In some aspects, the vector further comprises a nucleic acid sequence encoding one or more immune modifier proteins. In some aspects, the vector comprises a nucleic acid sequence encoding a tumor-associated antigen and, optionally, a second tumor-associated antigen. In some aspects, the vector comprises a nucleic acid sequence encoding a tumor associated antigen and a second tumor-associated antigen from a different tumor.
[0152] Some aspects relate to methods of eliciting humoral and/or cellular immune response against a tumor (e.g., an ovarian cancer, melanoma, colorectal cancer, liver cancer, pancreatic cancer, brain cancer, prostate cancer, bladder cancer, renal cancer, or hematological cancer) following in vivo administration of a vector or composition of the disclosure.
[0153] The present disclosure is also directed to methods of inducing an immune response in a subject comprising administering an effective amount of any composition, pharmaceutical composition, vaccine, polynucleotide, or vector disclosed herein to the subject. The present disclosure is also directed to methods of preventing, reducing the incidence of, attenuating or treating a tumor in a subject comprising administering an effective amount of any composition, polynucleotide, vector, pharmaceutical composition, or vaccine disclosed herein to the subject. In some aspects, the tumor is an ovarian cancer, melanoma, colorectal cancer, liver cancer, pancreatic cancer, brain cancer, prostate cancer, bladder cancer, renal cancer, or hematological cancer. The present disclosure is also directed to methods of making any composition, pharmaceutical composition, or vaccine disclosed herein.
II. Definitions
[0154] In order that the present disclosure can be more readily understood, certain terms are first defined. As used in this application, except as otherwise expressly provided herein, each of the following terms shall have the meaning set forth below. Additional definitions are set forth throughout the application.
[0155] The term "and/or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and/or" as used in a phrase such as "A and/or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the term "and/or" as used in a phrase such as "A, B, and/or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone). [0156] It is understood that wherever aspects are described herein with the language "comprising," otherwise analogous aspects described in terms of "consisting of and/or "consisting essentially of are also provided.
[0157] As used herein, the term "approximately" or "about," as applied to one or more values of interest, refers to a value that is similar to a stated reference value and within a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value). When the term "approximately" or "about" is applied herein to a particular value, the value without the term "approximately" or "about is also disclosed herein.
[0158] As described herein, any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.
[0159] As used herein, the terms "ug" and "uM" are used interchangeably with "pg" and "pM," respectively.
[0160] Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. The headings provided herein are not limitations of the various aspects of the disclosure, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.
[0161] The terms "nucleic acids," "nucleic acid molecules, "nucleotides," "nucleotide(s) sequence," and "polynucleotide" can be used interchangeably and refer to the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine; "RNA molecules", including mRNA) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; "DNA molecules"), or any phosphoester analogs thereof, such as phosphorothioates and thioesters, in either single stranded form, or a double-stranded helix. Single stranded nucleic acid sequences refer to single-stranded DNA (ssDNA) or single-stranded RNA (ssRNA). Double stranded DNA- DNA, DNA-RNA and RNA-RNA helices are possible. The term nucleic acid molecule, and in particular DNA or RNA molecule, refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear or circular DNA molecules (e.g., restriction fragments), plasmids, supercoiled DNA and chromosomes. In discussing the structure of particular double-stranded DNA molecules, sequences can be described herein according to the normal convention of giving only the sequence in the 5’ to 3’ direction along the non-transcribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA). A "recombinant DNA molecule" is a DNA molecule that has undergone a molecular biological manipulation. DNA includes, but is not limited to, cDNA, genomic DNA, DNA plasmid, synthetic DNA, and semi-synthetic DNA. A "nucleic acid composition" of the disclosure comprises one or more nucleic acids as described herein.
[0162] RNA can be obtained by transcription of a DNA-sequence, e.g., inside a cell. In eukaryotic cells, transcription is typically performed inside the nucleus or the mitochondria. In vivo, transcription of DNA usually results in premature RNA, which has to be processed into messenger RNA (mRNA). Processing of the premature RNA, e.g., in eukaryotic organisms, comprises a variety of different posttranscriptional-modifications such as splicing, 5'-capping, polyadenylation, export from the nucleus or the mitochondria and the like. The sum of these processes is also called maturation of RNA. The mature mRNA usually provides the nucleotide sequence that can be translated into an amino acid sequence of a particular peptide, protein, or protein antigen. Typically a mature mRNA comprises a 5' cap, optionally a 5'-UTR, an open reading frame, optionally a 3'-UTR, and a poly(A) sequence.
[0163] The term “multicistronic mRNA” or “multicistronic mRNA vector,” as used herein, refers to an mRNA having two or more open reading frames. An open reading frame in this context is a sequence of codons that is translatable into a polypeptide or protein.
[0164] The term “5'-cap,” as used herein, refers to an entity, typically a modified nucleotide entity, which generally “caps” the 5'-end of a mature mRNA. A 5'-cap can typically be formed by a modified nucleotide, particularly by a derivative of a guanine nucleotide. In some aspects, the 5'-cap is linked to the 5'-terminus via a 5'-5'-triphosphate linkage. A 5'-cap can be methylated, e.g., m7GpppN, wherein N is the terminal 5' nucleotide of the nucleic acid carrying the 5'-cap, typically the 5'-end of an RNA. The naturally occurring 5'-cap is m7GpppN. [0165] As used herein, a “poly(A) sequence,” also called “poly(A) tail” or “3'-poly(A) tail,” is typically understood to be a sequence of adenine nucleotides, e.g., of up to about 400 adenine nucleotides. A poly(A) sequence can be located at the 3' end of an mRNA. In some aspects, a poly(A) sequence can also be located within an mRNA or any other nucleic acid molecule, such as, e.g., in a vector, for example, in a vector serving as template for the generation of an RNA, preferably an mRNA, e.g., by transcription of the vector. In some aspects, a poly (A) sequence is present in the 3'-UTR of the mRNA as defined herein.
[0166] In some aspects, a 3'-UTR sequence is part of an mRNA, which is located between the protein coding region (i.e. the open reading frame) and the 3' terminus of the mRNA molecule. If a 3 '-terminal poly(A) sequence ('poly(A) tail') was added to the RNA (e.g. by polyadenylation), then the term 3'-UTR can refer to that part of the molecule, which is located between the protein coding region and the 3'-terminal poly(A) sequence. In some aspects, a 3'-UTR can also comprise a poly(A) sequence (e.g., a poly(A) sequence which is not located at the very 3' terminus of the RNA molecule). A 3'-UTR of the mRNA is not translated into an amino acid sequence. The 3'-UTR sequence is generally encoded by the gene, which is transcribed into the respective mRNA during the gene expression process. The genomic sequence is first transcribed into pre-mature mRNA, which comprises optional introns. The pre-mature mRNA is then further processed into mature mRNA in a maturation process. This maturation process comprises the steps of 5' capping, splicing the pre-mature mRNA to excise optional introns and modifications of the 3 '-end, such as polyadenylation of the 3 '-end of the pre-mature mRNA and optional endo-/ or exonuclease cleavages etc. In some aspects, a 3'-UTR corresponds to the sequence of a mature mRNA, which is located 3' to the stop codon of the protein coding region (e.g., immediately 3' to the stop codon of the protein coding region), and which extends to the 3' terminus of the RNA molecule or to the 5'-side of a 3' terminal poly(A) sequence (e.g., to the nucleotide immediately 5' to the 3' terminus or immediately 5' to the 3' terminal poly(A) sequence). The term "corresponds to" means that the 3'-UTR sequence can be an RNA sequence, such as in the mRNA sequence used for defining the 3'-UTR sequence, or a DNA sequence, which corresponds to such RNA sequence. In some aspects, the term "a 3'-UTR of a gene", such as "3'-UTR of alpha or beta globin", is the sequence, which corresponds to the 3'-UTR of the mature mRNA derived from this gene, i.e. the mRNA obtained by transcription of the gene and maturation of the pre-mature mRNA. The term "3'-UTR of a gene" encompasses the DNA sequence and the RNA sequence of the 3'-UTR. In some aspects, the 3'-UTR is derived from a gene that relates to an mRNA with an enhanced half-like (i.e., that provides a stable mRNA), for example a 3'-UTR of a gene selected from the group consisting of: albumin gene, an a-globin gene, a P-globin gene, a tyrosine hydroxylase gene, a lipoxygenase gene, and a collagen alpha gene, such as a collagen alpha 1(1) gene.
[0167] A 5'-UTR is typically understood to be a particular section of messenger RNA (mRNA). It is located 5' of the open reading frame of the mRNA. In some aspect, the 5'- UTR starts with the transcriptional start site and ends one nucleotide before the start codon of the open reading frame. The 5'-UTR can comprise elements for controlling gene expression, also called regulatory elements. Such regulatory elements can be, for example, ribosomal binding sites or a 5'-Terminal Oligopyrimidine Tract. The 5'-UTR can be posttranscriptionally modified, for example by addition of a 5'-cap. In some aspects, a 5'-UTR corresponds to the sequence of a mature mRNA which is located between the 5' cap and the start codon. In some aspects, the 5'-UTR corresponds to the sequence which extends from a nucleotide located 3' to the 5'-cap (e.g., from the nucleotide located immediately 3' to the 5 'cap) to a nucleotide located 5' to the start codon of the protein coding region (e.g., to the nucleotide located immediately 5' to the start codon of the protein coding region). The nucleotide located immediately 3' to the 5' cap of a mature mRNA typically corresponds to the transcriptional start site. The term "corresponds to" means that the 5'-UTR sequence can be an RNA sequence, such as in the mRNA sequence used for defining the 5'-UTR sequence, or a DNA sequence which corresponds to such RNA sequence. In some aspects, the term "a 5'-UTR of a gene", is the sequence, which corresponds to the 5'-UTR of the mature mRNA derived from this gene.
[0168] As used herein, the term "transfecting" or "transfection" refers to the transport of nucleic acids from the environment external to a cell to the internal cellular environment, with particular reference to the cytoplasm and/or cell nucleus. Without being bound by any particular theory, it is to be understood that nucleic acids can be delivered to cells either after being encapsulated within or adhering to one or more cationic polymer/nucleic acid complexes or being entrained therewith. Particular transfecting instances deliver a nucleic acid to a cell nucleus. Nucleic acids include DNA and RNA as well as synthetic congeners thereof. Such nucleic acids include missense, antisense, nonsense, as well as protein producing nucleotides, on and off and rate regulatory nucleotides that control protein, peptide, and nucleic acid production. In particular, but not limited to, they can be genomic DNA, cDNA, mRNA, tRNA, rRNA, hybrid sequences or synthetic or semi-synthetic sequences, and of natural or artificial origin. In addition, the nucleic acid can be variable in size, ranging from oligonucleotides to chromosomes. These nucleic acids can be of human, animal, vegetable, bacterial, viral, or synthetic origin. They can be obtained by any technique known to a person skilled in the art.
[0169] As used herein, the term "biodegradable" or "biodegradation" is defined as the conversion of materials into less complex intermediates or end products by solubilization hydrolysis, or by the action of biologically formed entities which can be enzymes and other products of the organism.
[0170] As used herein, "peptide" means peptides of any length and includes proteins. The terms "polypeptide" and "oligopeptide" are used herein without any particular intended size limitation, unless a particular size is otherwise stated.
[0171] As used herein, a "derivative" of a carbohydrate includes, for example, an acid form of a sugar, e.g. glucuronic acid; an amine of a sugar, e.g. galactosamine; a phosphate of a sugar, e.g. mannose-6-phosphate; and the like.
[0172] As used herein, the term "inverted terminal repeat" (or "ITR") refers to a single stranded sequence of nucleotides followed downstream by its reverse complement. The intervening sequence of nucleotides between the initial sequence and the reverse complement can be any length including zero.
[0173] "Administering" and similar terms refer to the physical introduction of a therapeutic agent e.g., nucleic acid molecules, vectors, compositions, and pharmaceutical compositions described herein) to a subject, using any of the various methods and delivery systems known to those skilled in the art. Exemplary routes of administration include intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intrasterna, oral, rectal, topical, epidermal, mucosal, intranasal, vaginal, rectal, sublingual administration, and combinations thereof. Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods. [0174] Treatment" or "therapy" of a subject refers to any type of intervention or process performed on, or the administration of an active agent to, a subject with the objective of reversing, alleviating, ameliorating, inhibiting, slowing down, or preventing the onset, progression, development, severity, or recurrence of a symptom, complication, condition, or biochemical indicia associated with a disease (e.g., cancer).
[0175] A "therapeutically effective amount," “effective amount,” "therapeutic dose," "effective dose," or "effective dosage," as used herein, means an amount or a dose that achieves a therapeutic goal, as described herein. One of ordinary skill in the art will further understand that a therapeutically effective amount etc. can be administered in a single dose, or can be achieved by administration of multiple doses (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more doses). The ability of a therapeutic agent to promote disease regression or inhibit the development or recurrence of the disease can be evaluated using a variety of methods known to the skilled practitioner, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by assaying the activity of the agent in in vitro assays.
[0176] As used herein, the term “antineoplastic therapy”, refers to any therapeutic agent that is used to treat a neoplastic disease (e.g., cancer or noncancerous tumor). In some aspects, the antineoplastic therapy includes, but is not limited to, a chemotherapeutic agent (list several chemo), an anti angiogenic agent (e.g., bevacizumab), a PARP inhibitor, an antibody (e.g., checkpoint inhibitor), a PIPKinase in hibitor, a tyrosine kinase inhibitor, or an adoptive cell therapy.
[0177] As used herein, the terms “prevent,” “preventing,” “prevention,” “prophylactic treatment,” and the like, refer to reducing the probability of developing a disease or condition in a subject, who does not have, but is at risk of or susceptible to developing a disease or condition.
[0178] As used herein, the term “adjuvant” refers to any component which improves the body's response to a vaccine. In some aspects, an adjuvant can be ALUM, MF59 (oil-in- water emulsion of squalene oil), AS03 (a-tocopherol, squalenem and polysorbate 80 in an oil-in-water emulsion) or a STING agonist.
[0179] As used herein, the term “ALUM” or “Alum” as use herein refers to an aluminum or aluminum-salt. In some aspects, the ALUM is an aluminum or aluminum-salt based adjuvant, e.g., aluminum phosphate, aluminum hydroxide, potassium aluminum sulfate [KA1(SO4)2], aluminumcrystalline aluminum oxyhydroxide, aluminum hydroxyphosphate, amorphous aluminum hydroxyphosphate sulfate, aluminum chloride, aluminum silicate, and a mixture of aluminum hydroxide and magnesium hydroxide, a mixture of aluminum sulfate and sodium hydroxide ora mixture of aluminum sulfate and potassium hydroxide. An ALUM can be an aluminum hydroxide gel or aluminum phosphate gel.
[0180] As used herein, the term “STING” refers to the stimulator of interferon genes protein that is a pattern recognition receptor (PRR) that senses cyclic dinucleotides and induces the expression of type I interferon canonically via dendritic cells.
[0181] As used herein, the term “STING agonist” refers to a molecule that induces a STING pathway.
[0182] As used herein, the terms “vaccine” or “vaccine composition” refer to an immunogenically active composition for the prophylaxis and/or treatment of diseases. Accordingly, in some aspects, vaccines are medicaments which comprise or deliver antigens and are intended to be used in humans or animals for generating specific defense and protective substance by vaccination
[0183] As used herein, the term “cancer vaccine” refers to a vaccine that either treats existing cancer or prevents the development of cancer. A cancer vaccine can use a patient’s pre-cancerous or cancer cells, separate proteins of the precancerous cells or cancer cells and immunize the patient against those proteins as antigens using a vaccine composition described herein.
[0184] As used herein, the term “tumor” or “neoplasm” or “neoplastic disease” refers to an abnormal mass of tissue that forms when cells grow and divide more than they should or do not die when they should. Tumors may be benign (non-cancerous) or malignant (cancer). Benign tumors do not spread into, or invade nearby tissues or other parts of the body.
[0185] As used herein, the term “cancer” refers to any disease characterized by abnormal cells dividing uncontrollably and spreading into, or invading, nearby tissues and/or other parts of a body through either the blood system or the lymph system.
[0186] A used herein, the term “inducing immunity” or “immunogenically active” refers to the ability to stimulate an immune response, /.< ., to stimulate the production of antibodies, particularly humoral antibodies, or to stimulate a cell-mediated response. For example, the ability to stimulate the production of circulating or secretory antibodies or the production of a cell-mediated response in local mucosal regions, peripheral blood, cerebral spinal fluid or the like. In some aspects, the effective immunizing amount of the immunogenically active component(s) of this disclosure can vary and can be any amount sufficient to evoke an immune response and provide a protective immune response against a tumor. A dosage unit comprising a polynucleotide (e.g., plasmid DNA) of the disclosure is contemplated. At least one dosage unit per patient is contemplated herein as a vaccination regimen. In some embodiments, two or more dosage units can be useful. The skilled artisan will quickly recognize that a particular quantity of vaccine composition per dosage unit, as well as the total number of dosage units per vaccination regimen, can be optimized, so long as an effective immunizing amount of the virus or a component thereof is ultimately delivered to the subject.
[0187] An “immunological response” to a substance such as a composition or vaccine is the development in the subject of a cellular and/or antibody-mediated immune response to a composition or vaccine of interest. Usually, an “immunological response” includes but is not limited to one or more of the following effects: the production of antibodies, B cells, helper T cells, and/or cytotoxic T cells, directed specifically to an antigen or antigens included in the composition or vaccine of interest. In some aspects, the subject can display either a therapeutic or protective immunological response so the tumor growth will be reduced and/or the clinical severity of the disease reduced. In some aspects, such protection can be demonstrated by either a reduction or lack of symptoms normally displayed by a tumor bearing subject, a quicker recovery time and/or a lowered tumor burden in the infected subject.
[0188] A “tumor-associated antigen” or “TAA” of the disclosure can be a protein or any other molecule that is found only on tumor cells and not, or to a lesser extent, on normal cells. In some aspects, the tumor-associated antigens provided herein are proteins or polypeptides, or antigenic fragments thereof. In some aspects, the tumor-associated antigens are only expressed on cancer cells and not present on normal cells. In some aspects, the tumor-associated antigens are express on tumor cells and normal cells, but the expression of the tumor-associated antigens is higher on tumor cells. A tumor-associated antigen can be, for example, a NYESO-1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE- A4, MAGE-A9, MAGE-A10, MAGE-CI, MAGE-C2, SSX, H0RMAD1, CXorf61, ACTL8, AKAP3,AKAP4, HOM-TES-85, OY-TES-1, HSP70-2, LAGE-1, PIWL1, PIWL2, PIWL3, PIWL4, PLU-1, PLAC1, SPAG9, SCP-1, TAG-1, TAG-2a, TAG-2b, TAG-2c, TRAG-3, KAP4, BAGE, BORIS, CT45, GAGE-1/2, PRAME, PAGE4, SP17, PASD1, p53, ras, B-RafHER-2/neu, mucin 1 (MUC1), epidermal growth factor receptor (EGFR), GP100, prostatic acid phosphatase, prostate-specific membrane antigen (PSMA), mammoglobin-A, carcinoembryonic antigen (CEA), papillomavirus antigens E6/E7, heat-shock proteins, alpha fetoprotein, CDK, P-catenin, mesothelin, or cancer antigen-125 (CA125), or any antigenic fragment thereof.
[0189] It is recognized that the antigenic polypeptides of the disclosure can be full-length polypeptides or active fragments or variants thereof. In some aspects, the term “active fragments” or “active variants” or “antigenic fragments” refers to fragments or variants that retain all or some of the antigenic nature of the polypeptide. Thus, in some aspects, the present disclosure encompasses any tumor associated polypeptide, antigen, epitope or immunogen that elicits an immunogenic response in a subject. The tumor associated polypeptide, antigen, epitope or immunogen can be any tumor associated polypeptide, antigen, epitope or immunogen, such as, but not limited to, a protein, peptide or fragment or variant thereof, that elicits, induces or stimulates a response in a subject. The tumor associated polypeptide, antigen, epitope or immunogen can be derived from any tumor including, but not limited to, an ovarian cancer, melanoma, colorectal cancer, liver cancer, pancreatic cancer, brain cancer, prostate cancer, bladder cancer, renal cancer, or hematological cancer.
[0190] The term “epitope” refers to the site on an antigen or hapten to which specific B cells and/or T cells respond. The term is also used interchangeably with “antigenic determinant” or “antigenic determinant site.” Antibodies that recognize the same epitope can be identified in a simple immunoassay showing the ability of one antibody to block the binding of another antibody to a target antigen.
[0191] As used herein, the term "pharmaceutical agent", "pharmaceutical composition" or "drug" or any other similar term means any chemical or biological material or compound suitable for administration by the methods previously known in the art and/or by the methods taught in the present disclosure, which induce a desired biological or pharmacological effect, which can include but are not limited to (1) having a prophylactic effect on the organism and preventing an undesired biological effect such as preventing tumor growth or spread of cancer to other tissues, (2) alleviating a condition caused by a disease, for example, alleviating pain or inflammation caused as a result of disease, and/or (3) either alleviating, reducing, or completely eliminating a disease (e.g., cancer) from the organism. The effect can be local or it can be systemic. [0192] A “pharmaceutically acceptable carrier” refers to a carrier that can be administered to a subject, together with an agent, and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the agent. In certain aspects, the pharmaceutically acceptable carrier is an aqueous solvent, i.e., a solvent comprising water, optionally with additional co-solvents. Exemplary pharmaceutically acceptable carriers include water, buffer solutions in water (such as phosphate-buffered saline (PBS), and 5% dextrose in water (D5W). In certain embodiments, the aqueous solvent further comprises dimethyl sulfoxide (DMSO), e.g., in an amount of about 1-4%, or 1-3%. In certain aspects, the pharmaceutically acceptable carrier is isotonic (i.e., has substantially the same osmotic pressure as a body fluid such as plasma).
[0193] A "subject" includes any human or non-human animal. The term "nonhuman animal" includes, but is not limited to, vertebrates such as nonhuman primates, sheep, dogs, and rodents such as mice, rats, and guinea pigs. In some aspects, the subject is a human. The terms "subject" and "patient" are used interchangeably herein. In some aspects, the subject suffers from a tumor or cancer.
[0194] The term "expression" as used herein refers to a process by which a polynucleotide produces a gene product, for example, a tumor-associated antigen or antigenic fragment thereof. In some aspects, it includes, without limitation, transcription of the polynucleotide into messenger RNA (mRNA) and the translation of an mRNA into a polypeptide. Expression produces a "gene product." As used herein, a gene product can be either a nucleic acid, e.g., a messenger RNA produced by transcription of a gene, or a polypeptide which is translated from a transcript. Gene products described herein can further include nucleic acids with post transcriptional modifications, e.g., polyadenylation or splicing, or polypeptides with post translational modifications, e.g., methylation, glycosylation, the addition of lipids, association with other protein subunits, or proteolytic cleavage.
[0195] As used herein, the term “5'” or “5 prime” refers to the 5' end of a nucleic acid or nucleic acid sequence, and the term “3'” or “3 prime” refer to the 3' end of nucleic acid or nucleic acid sequence.
[0196] The terms "identical" or percent "identity" in the context of two or more nucleic acids refer to two or more sequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned (introducing gaps, if necessary) for maximum correspondence, not considering any conservative amino acid substitutions as part of the sequence identity. The percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software are known in the art that can be used to obtain alignments of amino acid or nucleotide sequences.
[0197] As used herein, the term "promoter" refers to DNA sequence capable of controlling the expression of a coding sequence or functional RNA. In some aspects, a coding sequence is located 3' to a promoter sequence. Promoters can be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters can direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental or physiological conditions. Promoters that cause a gene to be expressed in most cell types at most times are commonly referred to as "constitutive promoters." Promoters that cause a gene to be expressed in a specific cell type are commonly referred to as "cell-specific promoters" or "tissue-specific promoters." Promoters that cause a gene to be expressed at a specific stage of development or cell differentiation are commonly referred to as "developmentally-specific promoters" or "cell differentiation-specific promoters." Promoters that are induced and cause a gene to be expressed following exposure or treatment of the cell with an agent, biological molecule, chemical, ligand, light, or the like that induces the promoter are commonly referred to as "inducible promoters" or "regulatable promoters." It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths can have identical promoter activity.
[0198] The term “operably linked” refers to genetic elements that are joined together in a manner that enables them to carry out their normal functions. For example, a gene is operably linked to a promoter when its transcription is under the control of the promoter and this transcription results in the production of the product encoded by the gene.
[0199] As used herein, the term “immune modifier protein” refers to a protein that augments the immune response to a one or more antigens. In some aspects, immune modifier proteins include, but are not limited to, a cytokine, a chemokine, major histocompatibility complex (MHC) class I (MHC I), MHC class II (MHC II), human leukocyte antigen (HLA)-DR isotype (HLA-DR), CD80, CD86, and any combination thereof. In some aspects, the cytokine immune modifier proteins include, but are not limited to, interleukin (IL) 2 (IL-2), IL-12 p35, IL-12 p40, IL-12 p70, IL-15, IL-18, tumor necrosis factor alpha (TNFa), granulocyte-macrophage colony-stimulating factor (GM- CSF), interferon (IFN) a (IFN-a), and IFN-p. In some aspects, the chemokine immune modifier proteins include, but are not limited to, C-C motif chemokine ligand (CCL) 3 (CCL3), CCL4, CCL5, CCL21, CCL28, C-X-C motif chemokine ligand (CXCL) 10 (CXCL10), and any combination thereof. In some aspects, the immune modifier proteins include one or more unmethylated cytosine-guanine dinucleotide-containing oligodeoxynucleotide (CpG).In some aspects, the immune modifier proteins include one or more concatamers of non-coding 5'-C-phosphate-G-3' (CpG) dinucleotides. In some aspects, the one or more concatamers of non-coding CpG dinucleotides activate the Tolllike receptor 9 (TLR9) signaling pathway. In some aspects, the one or more concatamers of non-coding CpG dinucleotides comprise one or more concatamers of non-coding CpG dinucleotides previously reported in Bauer, A. et al., Nucleic Acids Research 38(12):3891-908 (2010); Cornelie, S. et al., Journal of Biological Chemistry
279(15): 15124-9 (2004); Klinman, D. etal., J Immunol. 158(8):3635-9 (1997); Klinman, D. et al., Immunological Reviews 199(l):201-16 (2004); Luo, Z. et al., Mol Med Rep. 6(6): 1309-14 (2012); Bode, C. et al., Expert Rev Vaccines 10(4):499-511 (2011); and Kuo, T. et al., Scientific Reports 10:20085 (2020), each of which is incorporated by reference herein in its entirety. In some aspects, an immune modifier protein as disclosed herein can include a combination of immune modifier proteins, e.g., a cytokine or chemokine protein or their coding sequence either co-expressed from the same plasmid as the antigen or from a different plasmid. See, e.g., Hirao, L.A., et al, Vaccine, 26:3112-20 (2008); Kanagavelu, S.K., et al., Vaccine, 30:691-702 (2012); Ahlers, D.J., et al., Curr MolMed., 3:285-301 (2003); Jafarfzade, B.S., et al., Bratisl Lek Listy, 118:564-9 (2017); Ahler, J.D. and Belyakov, I.M., Eur J Immunol, 39:2657-69 (2009); Moore, A.C., et al., J Virol. 76:243-50 (2002); Barouch, D.H., et al., PNAS, 97:4192-7 (2000); Kalams, S.A., et al., J Infectious Disease, 208:818-29 (2013); Buchbinder, S., et al., PLOS One, 12(7):e0179597 (2017); Henke, A, et al., Intervirology, 49:249-52 (2006); and Yang, S.H., et al., Gen Ther., 13: 1110-7 (2006).
[0200] The term “targeting ligand,” as used herein, is intended to refer to ligands conjugated to a polymer either directly or via one or more spacer molecules. In some aspects, only a small portion of the available amino groups of the polymer is coupled to the ligand. In some aspects, the targeting ligands conjugated to the polymers direct the polymers-nucleic acid complex to bind to specific target cells and penetrate into such cells (e.g., epithelial cells, endothelial cells, hematopoietic cells, and the like). In some aspects, the target ligands can also be an intracellular targeting element, enabling the transfer of the nucleic acid/drug to be guided towards certain favored cellular compartments (mitochondria, nucleus, and the like). In some aspects, the ligand is a polypeptide, folate, and an antigen. In some aspects, the polypeptide ligand is a glycoprotein (e.g., transferrin or asialoorosomucoid (ASOR)), an antibody, an antibody fragment, a cell receptor, a cytokine receptor, or a growth factor receptor (e.g., epidermal growth factor receptor). In some aspects, the antigen ligand is a tumor-associated antigen, a viral antigen, a bacterial antigen, or a parasite antigen. In some aspects, the ligand is a fusogenic agent (e.g., polymixin B and hemaglutinin HA2), a lysosomotrophic agent, or a nucleus localization signal (NLS) (e.g., T-antigen, and the like). In some aspects, the ligand is a sugar moiety coupled to an amino group. In some aspects, the sugar moiety is a mono- or oligo-saccharide, such as galactose, glucose, fucose, fructose, lactose, sucrose, mannose, cellobiose, nytrose, triose, dextrose, trehalose, maltose, galactosamine, glucosamine, galacturonic acid, glucuronic acid, and gluconic acid.
[0201] The term “antibody” includes molecules or active fragments (i.e., antigen binding fragments) of molecules that bind to antigens. These active fragments can be derived from an antibody of the present disclosure by a number of techniques. For further description of general techniques for the isolation of active fragments of antibodies, see for example, Khaw, B. A. et al. J. Nucl. Med. 23: 1011-1019 (1982). The term “antibody” also includes bispecific and chimeric antibodies and antibodies in nonmammalian species.
[0202] The term “biodegradable linker” or “biofunctional biodegradable linker,” as used herein, refers to a biodegradable linker containing ester, amide, disulfide, and/or phosphate linkages that is used to cross-link cationic multi-block copolymers. In some aspects, the biodegradable linker is hydrophilic and comprises a biodegradable linkage comprising a disulfide bond. In some aspects, the biodegradable linker is a dithiodipropionyl linker.
[0203] The term "vector," as used herein, is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked; or an entity comprising such a nucleic acid molecule capable of transporting another nucleic acid. In some aspects, the vector is a "plasmid," which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. In some aspects, the vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. In some aspects, such vectors include, but are not limited to: an adenoviral vector, an adeno- associated virus (AAV) vector, retroviral vector, a lentiviral vector, poxvirus vector, a baculovirus vector, a herpes viral vector, simian virus 40 (SV40), cytomegalovirus (CMV), mouse mammary tumor virus (MMTV), and Moloney murine leukemia virus. Certain vectors, or polynucleotides that are part of vectors, are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication, and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" can sometimes be used interchangeably, depending on the context, as the plasmid is the most commonly used form of vector. However, also disclosed herein are other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, poxviruses, herpesviruses, baculoviruses, adenoviruses and adeno-associated viruses), which can serve equivalent functions.
[0204] As used herein, the term “poloxamer” or “poloxamer backbone” refers to molecules having the general formula HO — (C H-tO CsHeO C H-tOk — H in which a and c are approximately equal. See, Handbook of Biodegradable Polymers, Chapter 12' "The Poloxamers: Their Chemistry andMedical Applications" authored by Lorraine E. Reeve. See also U.S. Publ. No. 2010/0004313, which is herein incorporated by reference in its entirety. Because the poloxamers are the products of a sequential series of reactions, the chain lengths of individual poloxamer blocks are statistical distributions about the average chain length. Thus, the number of ethyleneoxy groups (a and c) and the number of propylenoxy groups (Z>) are meant to be averages. Poloxamers are generally based on an amphiphilic triblock copolymer of ethylene oxide and propylene oxide, having a central hydrophobic chain of polypropylene oxide flanked by two hydrophilic chains of polyethylene oxide. Because the lengths of the polymer blocks of a poloxamer backbone can vary between various polymeric constructs, many different poloxamers are considered to be within the scope of the present disclosure. In one aspect, for example, the average molecular weight of the pol oxamer backbone can range from about 100 to about 100,000 Dalton. In another aspect, the average molecular weight of the pol oxamer backbone can range from about 500 to about 50,000 Dalton. In yet another aspect, the average molecular weight of the pol oxamer backbone can range from about 1000 to about 20,000 Dalton. The poloxamer backbone can also be described in terms of a ratio of ethylene oxide to propylene oxide. In another aspect, the ratio of ethylene oxide to propylene eoxide is from about 20: 1 to about 1 :20. For example, in one aspect the ratio of ethylene oxide to propylene oxide is from about 5: 1 to about 1 :5.
[0205] As used herein, “BD15-12” rerfers to a PEI-based co-block polymer that utilized biodegradable linkages.
[0206] Various additional aspects of the disclosure are described, disclosed or illustrated in further detail in the following subsections.
III. Compositions
[0207] In some aspects, provided are compositions (e.g., pharmaceutical compositions or vaccines). In some aspects, the compositions comprise a polynucleotide as disclosed herein and an adjuvant. In some aspects, the compositions comprise a polynucleotide as disclosed herein and a STING agonist. In some aspects, the compositions comprise a polynucleotide as disclosed herein, an adjuvant, and a STING agonist. In some aspects, the compositions further comprise a delivery component.
[0208] In some aspects, the compositions (e.g., pharmaceutical compositions or vaccines) of the disclosure comprise a polynucleotide as described herein, an adjuvant, and a delivery component.
[0209] In some aspects, the compositions (e.g., pharmaceutical compositions or vaccines) of the disclosure comprise a polynucleotide as described herein, a STING agonist, and a delivery component.
[0210] In some aspects, the compositions (e.g., pharmaceutical compositions or vaccines) of the disclosure comprise a polynucleotide as described herein, an adjuvant, a STING agonist, and a delivery component.
[0211] In some aspects, the adjuvant is selected from potassium aluminum sulfate [KA1(SO4)2], aluminum hydroxide, crystalline aluminum oxyhydroxide, aluminum phosphate, aluminum hydroxyphosphate, amorphous aluminum hydroxyphosphate sulfate, aluminum chloride, aluminum silicate, and a composition comprising aluminum hydroxide and magnesium hydroxide.
[0212] In some aspects, the aluminum adjuvant is an aluminum hydroxide gel or aluminum phosphate gel.
[0213] In some aspects, the aluminum adjuvant is a mixture of aluminum sulfate and sodium or potassium hydroxide.
[0214] In some aspects, the immune stimulator is a STING agonist.
[0215] In some aspects, the STING agonist is cyclic GMP (cGMP). In some aspects, the
STING agonist is cyclic AMP (cAMP). In some aspects, the STING agonist is cyclic- GMP-AMP (cGAMP).
[0216] In some aspects, the STING agonist is an amidobenzimidazole (ABZI).
[0217] In some aspects, the STING agonist is a cylic dinucleotide selected from ADU-
S100, MK-1454, SB11285, BMS-986301, BI-STING (BI1387446), JNJ-67544412, 3’3’- cyclic AIMP, and GSK532.
[0218] In some aspects, the STING agonist is a non-cyclic dinucleotide small molecule selected from DMAXAA, ALG-031048, E7766, JNJ-‘6196, MK-21118, MSA-1, MSA-2, SNX281, SR-717, TAK676, TTI-10001, a Ryvu’s agonist, GF3-002, a Selvita agonist, CDR5500, CS-1010, CS-1018, CS-1020, and [2-ex] MSA-1.
[0219] In some aspects, the STING agonist is a nanovaccine selected from PC7A nanoparticles, cGMP-nanoparticles, and ONM-500 nanoparticles.
[0220] In some aspects, the STING agonist is an antibody-drug conjugate. In some aspects, the antibody drug conjugate is XMT-2056.
[0221] In some aspecst, the STING agonist is an ENPP1 inhibitor selected from MV-626, SR-8314, SR-8291, and SR8541A.
[0222] In some aspects, the STING agonist is a bacterial vector. In some aspects, the bacterial vector is a nonpathogenic E coli nissle expressing cyclic-di-AMP -producing enzymes. In some aspects, the bacterial vector is SYNB1981. In some aspect, the bacterial vector is an attenutated Salmonella Typhimurium strain enginerred to carry an inhibitory TREX-1 micro RNA. Also provided herein are compositions (e.g., pharmaceutical compositions and vaccines) comprising any polynucleotide or vector described or exemplified herein. In some aspects, the compositions (e.g., pharmaceutical composition or vaccine) further comprises a pharmaceutically acceptable carrier. In some aspects, the compositions (e.g., pharmaceutical composition or vaccine) further comprises a second polynucleotide. In some aspects, the second polynucleotide encodes a second antigen wherein the second antigen is operably linked to a promoter. In some aspects, the second polynucleotide encodes at least one immune modifier protein. In some aspects, the immune modifier protein is selected from the group consisting of IL-2, IL-12 p35, IL-12 p40, IL-12 p70, IL-15, IL-18, TNFa, GM-CSF, IFN-a, IFN-p, a chemokine, MHC I, MHC II, HLA-DR, CD80, and CD86, wherein the nucleic acid encoding the at least one immune modifier protein is operably linked to a promoter. In some aspects, the compositions (e.g., pharmaceutical composition or vaccine) further comprises a delivery component (e.g., a cationic polymer, a poly-inosinic-polycytidylic acid, or a poloxamer). In some aspects, the delivery component further comprises benzalkonium chloride.
[0223] In some aspects, the compositions (e.g., pharmaceutical composition or vaccine) further comprises a third polynucleotide. In some aspects, the third polynucleotide encodes a third antigen wherein the third antigen is operably linked to a promoter. In some aspects, the third polynucleotide encodes at least one immune modifier protein selected from the group consisting of IL-2, IL-12 p35, IL-12 p40, IL-12 p70, IL-15, IL- 18, TNFa, GM-CSF, IFN-a, IFN-p, a chemokine, MHC I, MHC II, HLA-DR, CD80, and CD86, wherein the nucleic acid encoding the at least one immune modifier protein is operably linked to a promoter. In some aspects, the compositions (e.g., pharmaceutical composition or vaccine) further comprises a delivery component (e.g., a cationic polymer, a poly-inosinic-polycytidylic acid, or a poloxamer). In some aspects, the delivery component further comprises benzalkonium chloride.
[0224] In some aspects, the compositions (e.g., pharmaceutical composition or vaccine) further comprises a fourth polynucleotide. In some aspects, the fourth polynucleotide encodes a fourth antigen wherein the fourth antigen is operably linked to a promoter. In some aspects, the fourth polynucleotide encodes at least one immune modifier protein selected from the group consisting of IL-2, IL-12 p35, IL-12 p40, IL-12 p70, IL-15, IL- 18, TNFa, GM-CSF, IFN-a, IFN-p, a chemokine, MHC I, MHC II, HLA-DR, CD80, and CD86, wherein the nucleic acid encoding the at least one immune modifier protein is operably linked to a promoter. In some aspects, the compositions (e.g., pharmaceutical composition or vaccine) further comprises a delivery component (e.g., a cationic polymer, a poly-inosinic-polycytidylic acid, or a poloxamer). In some aspects, the delivery component further comprises benzalkonium chloride. [0225] The compositions (e.g., pharmaceutical compositions and vaccines) can comprise any polynucleotide or vector described or exemplified herein. In some aspects, the compositions (e.g., pharmaceutical composition or vaccine) further comprises a pharmaceutically acceptable carrier.
[0226] In some aspects, the at least one immune modifier protein comprises one or more concatamers of non-coding 5'-C-phosphate-G-3' (CpG) dinucleotides. In some aspects, the one or more concatamers of non-coding CpG dinucleotides activate the Toll-like receptor 9 (TLR9) signaling pathway. In some aspects, the one or more concatamers of non-coding CpG dinucleotides comprise one or more concatamers of non-coding CpG dinucleotides previously reported in Bauer, A. et al., Nucleic Acids Research 38(12):3891-908 (2010); Cornelie, S. et al., Journal of Biological Chemistry 279(15): 15124-9 (2004); Klinman, D. etal., J Immunol. 158(8):3635-9 (1997); Klinman, D. et al., Immunological Reviews 199(l):201-16 (2004); Luo, Z. et al., Mol Med Rep. 6(6): 1309-14 (2012); Bode, C. et al., Expert Rev Vaccines 10(4):499-511 (2011); and Kuo, T. et al., Scientific Reports 10:20085 (2020), each of which is incorporated by reference herein in its entirety. Also provided herein are compositions (e.g., pharmaceutical compositions or vaccines) comprising any polynucleotide, multi ci str onic mRNA vector, or DNA plasmid vector described or exemplified herein.
[0227] Certain aspects of the disclosure relate to compositions comprising (i) a vector (e.g., a multi ci stronic DNA plasmid vector or a multi ci stronic messenger RNA (mRNA) vector) comprising a nucleic acid sequence encoding one or more tumor-associated antigens; and (ii) a delivery component (e.g., a cationic polymer, a poly-inosinic- polycytidylic acid, or a poloxamer). In some aspects, the compositsion further comprise (iii) an adjuvant. In some aspects, the adjuvant comprises an aluminum or aluminum-salt based adjuvant, a stimulator of interferon genes (STING) agonist, or a combination thereof. In some aspects, the adjuvant comprises an aluminum or aluminum-salt based adjuvant and a STING agonist. In some aspects, the aluminum or aluminum-salt based adjuvant comprises an aluminum oxyhydroxide, an aluminum hydroxide, an aluminum phosphate, or any combination thereof. In some aspects, the STING agonist is cyclic guanosine monophosphate (cGMP), cyclic adenosine monophosphate (cAMP), or cyclic guanosine monophosphate-adenosine monophosphate (cGAMP).
[0228] In some aspects, the vector further comprises a nucleic acid sequence encoding one or more immune modifier proteins. In some aspects, the vector comprises a nucleic acid sequence encoding a tumor-associated antigen and, optionally, a second tumor- associated antigen. Some aspects relate to methods of eliciting an immune response (e.g., T-cell response) against a tumor (e.g., ovarian cancer, melanoma, colorectal cancer, liver cancer, pancreatic cancer, brain cancer, prostate cancer, bladder cancer, renal cancer, or hematological cancer) following in vivo administration of a vector or composition of the disclosure.
[0229] Some aspects relate to methods of eliciting T cell (e.g., CD8+ T cell) immune response against a tumor (e.g., ovarian cancer, melanoma, colorectal cancer, liver cancer, pancreatic cancer, brain cancer, prostate cancer, bladder cancer, renal cancer, or hematological cancer) following in vivo administration of a multi ci str onic DNA plasmid, a multi ci stronic mRNA vector, or a composition comprising the same of the disclosure.
[0230] Certain aspects of the disclosure relate to compositions including (i) a DNA plasmid vector comprising a DNA sequence of one or more antigens; (ii) a delivery component, such as a synthetic non-viral DNA carrier (e.g., a cationic polymer, a poly- inosinic-polycytidylic acid, or a poloxamer); and (iii) an adjuvant comprising an aluminum or aluminum-salt based adjuvant, a stimulator of interferon genes (STING) agonist, or a combination thereof.
[0231] The delivery component of the compositions disclosed herein may comprise any combination of delivery components disclosed herein.
[0232] In some aspects, the compositions (e.g., pharmaceutical compositions or vaccines) of the disclosure comprise a delivery component and a polynucleotide comprising: a first nucleotide sequence, wherein the first nucleotide sequence encodes IL-12 p35 and is operably linked to a CMV promoter; a second nucleotide sequence, wherein the second nucleotide sequence encodes IL-12 p40 and is operably linked to a CMV promoter; a third nucleotide sequence, wherein the third nucleotide sequence encodes a first tumor- associated antigen and is operably linked to promoter 1; and a fourth nucleotide sequence, wherein the fourth nucleotide sequence encodes a second tumor-associated antigen and is operably linked to promoter 2. In some aspects, the polynucleotide comprises: a 5' first nucleotide sequence, wherein the first nucleotide sequence encodes IL-12 p35 and is operably linked to a CMV promoter; a second nucleotide sequence positioned 3' to the first nucleotide sequence, wherein the second nucleotide sequence encodes IL- 12 p40 and is operably linked to a CMV promoter; a third nucleotide sequence positioned 3' to the second nucleotide sequence, wherein the third nucleotide sequence encodes a first tumor- associated antigen and is operably linked to promoter 1; and a fourth nucleotide sequence positioned 3' to the third nucleotide sequence, wherein the fourth nucleotide sequence encodes a second tumor-associated antigen and is operably linked to promoter 2. In some aspects, the first, second, third, and fourth nucleotide sequences of the polynucleotide are configured as shown in FIG. 1.
[0233] In some aspects, the compositions (e.g., pharmaceutical compositions or vaccines) of the disclosure comprise a delivery component and a polynucleotide comprising: a first nucleotide sequence, wherein the first nucleotide sequence encodes IL-12 p35 and is operably linked to a CMV promoter; a second nucleotide sequence, wherein the second nucleotide sequence encodes IL-12 p40 and is operably linked to a CMV promoter; a third nucleotide sequence, wherein the third nucleotide sequence encodes MHC I and is operably linked to promoter Z; a fourth nucleotide sequence, wherein the fourth nucleotide sequence encodes a first tumor-associated antigen and is operably linked to promoter 1, and a fifth nucleotide sequence, wherein the fifth nucleotide sequence encodes a second tumor-associated antigen and is operably linked to promoter 2. In some aspects, the polynucleotide comprises: a 5' first nucleotide sequence, wherein the first nucleotide sequence encodes IL-12 p35 and is operably linked to a CMV promoter; a second nucleotide sequence positioned 3' to the first nucleotide sequence, wherein the second nucleotide sequence encodes IL- 12 p40 and is operably linked to a CMV promoter; a third nucleotide sequence positioned 3' to the second nucleotide sequence, wherein the third nucleotide sequence encodes MHC I and is operably linked to promoter Z; a fourth nucleotide sequence positioned 3' to the third nucleotide sequence, wherein the fourth nucleotide sequence encodes a first tumor-associated antigen and is operably linked to promoter 1, and a fifth nucleotide sequence positioned 3' to the fourth nucleotide sequence, wherein the fifth nucleotide sequence encodes a second tumor-associated antigen and is operably linked to promoter 2. In some aspects, the first, second, third, fourth, and fifth nucleotide sequences of the polynucleotide are configured as shown in FIG. 2.
[0234] In some aspects, the compositions (e.g., pharmaceutical compositions or vaccines) of the disclosure comprise a delivery component and a polynucleotide comprising: a first nucleotide sequence, wherein the first nucleotide sequence encodes IL-12 p35 and is operably linked to a CMV promoter; a second nucleotide sequence, wherein the second nucleotide sequence encodes IL-12 p40 and is operably linked to a CMV promoter; a third nucleotide sequence, wherein the third nucleotide sequence encodes MHC II and is operably linked to promoter Z; a fourth nucleotide sequence, wherein the fourth nucleotide sequence encodes a first tumor-associated antigen and is operably linked to promoter 1, and a fifth nucleotide sequence, wherein the fifth nucleotide sequence encodes a second tumor-associated antigen and is operably linked to promoter 2. In some aspects, the polynucleotide comprises: a 5' first nucleotide sequence, wherein the first nucleotide sequence encodes IL-12 p35 and is operably linked to a CMV promoter; a second nucleotide sequence positioned 3' to the first nucleotide sequence, wherein the second nucleotide sequence encodes IL- 12 p40 and is operably linked to a CMV promoter; a third nucleotide sequence positioned 3' to the second nucleotide sequence, wherein the third nucleotide sequence encodes MHC II and is operably linked to promoter Z; a fourth nucleotide sequence positioned 3' to the third nucleotide sequence, wherein the fourth nucleotide sequence encodes a first tumor-associated antigen and is operably linked to promoter 1, and a fifth nucleotide sequence positioned 3' to the fourth nucleotide sequence, wherein the fifth nucleotide sequence encodes a second tumor- associated antigen and is operably linked to promoter 2. In some aspects, the first, second, third, fourth, and fifth nucleotide sequences of the polynucleotide are configured as shown in FIG. 3.
[0235] In some aspects, the compositions (e.g., pharmaceutical compositions or vaccines) of the disclosure comprise a delivery component and a polynucleotide comprising: a first nucleotide sequence, wherein the first nucleotide sequence encodes IL-2 and is operably linked to a CMV promoter; a second nucleotide sequence, wherein the second nucleotide sequence encodes a first tumor-associated antigen and is operably linked to promoter 1; and a third nucleotide sequence, wherein the third nucleotide sequence encodes a second tumor-associated antigen and is operably linked to promoter 2. In some aspects, the polynucleotide comprises: a 5' first nucleotide sequence, wherein the first nucleotide sequence encodes IL-2 and is operably linked to a CMV promoter; a second nucleotide sequence positioned 3' to the first nucleotide sequence, wherein the second nucleotide sequence encodes a first tumor-associated antigen and is operably linked to promoter 1; and a third nucleotide sequence positioned 3' to the second nucleotide sequence, wherein the third nucleotide sequence encodes a second tumor-associated antigen and is operably linked to promoter 2. In some aspects, the first, second, and third nucleotide sequences of the polynucleotide are configured as shown in FIG. 4. [0236] In some aspects, the compositions (e.g., pharmaceutical compositions or vaccines) of the disclosure comprise a delivery component and a polynucleotide comprising: a first nucleotide sequence, wherein the first nucleotide sequence encodes IL-2 and is operably linked to a CMV promoter; a second nucleotide sequence, wherein the second nucleotide sequence encodes MHC I and is operably linked to promoter Z; a third nucleotide sequence, wherein the third nucleotide sequence encodes a first tumor-associated antigen and is operably linked to promoter 1; and a fourth nucleotide sequence, wherein the fourth nucleotide sequence encodes a second tumor-associated antigen and is operably linked to promoter 2. In some aspects, the polynucleotide comprises: a 5' first nucleotide sequence, wherein the first nucleotide sequence encodes IL-2 and is operably linked to a CMV promoter; a second nucleotide sequence positioned 3' to the first nucleotide sequence, wherein the second nucleotide sequence encodes MHC I and is operably linked to promoter Z; a third nucleotide sequence positioned 3' to the second nucleotide sequence, wherein the third nucleotide sequence encodes a first tumor-associated antigen and is operably linked to promoter 1; and a fourth nucleotide sequence positioned 3' to the third nucleotide sequence, wherein the fourth nucleotide sequence encodes a second tumor-associated antigen and is operably linked to promoter 2. In some aspects, the first, second, third, and fourth nucleotide sequences of the polynucleotide are configured as shown in FIG. 5.
[0237] In some aspects, the compositions (e.g., pharmaceutical compositions or vaccines) of the disclosure comprise a delivery component and a polynucleotide comprising: a first nucleotide sequence, wherein the first nucleotide sequence encodes IL-2 and is operably linked to a CMV promoter; a second nucleotide sequence, wherein the second nucleotide sequence encodes MHC II and is operably linked to promoter Z; a third nucleotide sequence, wherein the third nucleotide sequence encodes a first tumor-associated antigen and is operably linked to promoter 1; and a fourth nucleotide sequence, wherein the fourth nucleotide sequence encodes a second tumor-associated antigen and is operably linked to promoter 2. In some aspects, the polynucleotide comprises: a 5' first nucleotide sequence, wherein the first nucleotide sequence encodes IL-2 and is operably linked to a CMV promoter; a second nucleotide sequence positioned 3' to the first nucleotide sequence, wherein the second nucleotide sequence encodes MHC II and is operably linked to promoter Z; a third nucleotide sequence positioned 3' to the second nucleotide sequence, wherein the third nucleotide sequence encodes a first tumor-associated antigen and is operably linked to promoter 1; and a fourth nucleotide sequence positioned 3' to the third nucleotide sequence, wherein the fourth nucleotide sequence encodes a second tumor-associated antigen and is operably linked to promoter 2. In some aspects, the first, second, third, and fourth nucleotide sequences of the polynucleotide are configured as shown in FIG. 6.
[0238] In some aspects, the compositions (e.g., pharmaceutical compositions or vaccines) of the disclosure comprise a delivery component and a polynucleotide comprising: a first nucleotide sequence, wherein the first nucleotide sequence encodes IL-2 and is operably linked to a CMV promoter; a second nucleotide sequence, wherein the second nucleotide sequence encodes CCL3 and is operably linked to promoter X; a third nucleotide sequence, wherein the third nucleotide sequence encodes CCL4 and is operably linked to promoter Y; a fourth nucleotide sequence, wherein the fourth nucleotide sequence encodes a first tumor-associated antigen and is operably linked to promoter 1, and a fifth nucleotide sequence, wherein the fifth nucleotide sequence encodes a second tumor- associated antigen and is operably linked to promoter 2. In some aspects, the polynucleotide comprises: a 5' first nucleotide sequence, wherein the first nucleotide sequence encodes IL-2 and is operably linked to a CMV promoter; a second nucleotide sequence positioned 3' to the first nucleotide sequence, wherein the second nucleotide sequence encodes CCL3 and is operably linked to promoter X; a third nucleotide sequence positioned 3' to the second nucleotide sequence, wherein the third nucleotide sequence encodes CCL4 and is operably linked to promoter Y; a fourth nucleotide sequence positioned 3' to the third nucleotide sequence, wherein the fourth nucleotide sequence encodes a first tumor-associated antigen and is operably linked to promoter 1, and a fifth nucleotide sequence positioned 3' to the fourth nucleotide sequence, wherein the fifth nucleotide sequence encodes a second tumor-associated antigen and is operably linked to promoter 2. In some aspects, the first, second, third, fourth, and fifth nucleotide sequences of the polynucleotide are configured as shown in FIG. 7.
[0239] In some aspects, the compositions (e.g., pharmaceutical compositions or vaccines) of the disclosure comprise a delivery component and a polynucleotide comprising: a first nucleotide sequence, wherein the first nucleotide sequence encodes IL-15 and is operably linked to a CMV promoter; a second nucleotide sequence, wherein the second nucleotide sequence encodes a first tumor-associated antigen and is operably linked to promoter 1; and a third nucleotide sequence, wherein the third nucleotide sequence encodes a second tumor-associated antigen and is operably linked to promoter 2. In some aspects, the polynucleotide comprises: a 5' first nucleotide sequence, wherein the first nucleotide sequence encodes IL- 15 and is operably linked to a CMV promoter; a second nucleotide sequence positioned 3' to the first nucleotide sequence, wherein the second nucleotide sequence encodes a first tumor-associated antigen and is operably linked to promoter 1; and a third nucleotide sequence positioned 3' to the second nucleotide sequence, wherein the third nucleotide sequence encodes a second tumor-associated antigen and is operably linked to promoter 2. In some aspects, the first, second, and third nucleotide sequences of the polynucleotide are configured as shown in FIG. 8.
[0240] In some aspects, the compositions (e.g., pharmaceutical compositions or vaccines) of the disclosure comprise a delivery component and a polynucleotide comprising: a first nucleotide sequence, wherein the first nucleotide sequence encodes IL-15 and is operably linked to a CMV promoter; a second nucleotide sequence, wherein the second nucleotide sequence encodes MHC I and is operably linked to promoter Z; a third nucleotide sequence, wherein the third nucleotide sequence encodes a first tumor-associated antigen and is operably linked to promoter 1; and a fourth nucleotide sequence, wherein the fourth nucleotide sequence encodes a second tumor-associated antigen and is operably linked to promoter 2. In some aspects, the polynucleotide comprises: a 5' first nucleotide sequence, wherein the first nucleotide sequence encodes IL-15 and is operably linked to a CMV promoter; a second nucleotide sequence positioned 3' to the first nucleotide sequence, wherein the second nucleotide sequence encodes MHC I and is operably linked to promoter Z; a third nucleotide sequence positioned 3' to the second nucleotide sequence, wherein the third nucleotide sequence encodes a first tumor-associated antigen and is operably linked to promoter 1; and a fourth nucleotide sequence positioned 3' to the third nucleotide sequence, wherein the fourth nucleotide sequence encodes a second tumor-associated antigen and is operably linked to promoter 2. In some aspects, the first, second, third, and fourth nucleotide sequences of the polynucleotide are configured as shown in FIG. 9.
[0241] In some aspects, the compositions (e.g., pharmaceutical compositions or vaccines) of the disclosure comprise a delivery component and a polynucleotide comprising: a first nucleotide sequence, wherein the first nucleotide sequence encodes IL-15 and is operably linked to a CMV promoter; a second nucleotide sequence, wherein the second nucleotide sequence encodes MHC II and is operably linked to promoter Z; a third nucleotide sequence, wherein the third nucleotide sequence encodes a first tumor-associated antigen and is operably linked to promoter 1; and a fourth nucleotide sequence, wherein the fourth nucleotide sequence encodes a second tumor-associated antigen and is operably linked to promoter 2. In some aspects, the polynucleotide comprises: a 5' first nucleotide sequence, wherein the first nucleotide sequence encodes IL-15 and is operably linked to a CMV promoter; a second nucleotide sequence positioned 3' to the first nucleotide sequence, wherein the second nucleotide sequence encodes MHC II and is operably linked to promoter Z; a third nucleotide sequence positioned 3' to the second nucleotide sequence, wherein the third nucleotide sequence encodes a first tumor-associated antigen and is operably linked to promoter 1; and a fourth nucleotide sequence positioned 3' to the third nucleotide sequence, wherein the fourth nucleotide sequence encodes a second tumor-associated antigen and is operably linked to promoter 2. In some aspects, the first, second, third, and fourth nucleotide sequences of the polynucleotide are configured as shown in FIG. 10.
[0242] In some aspects, the compositions (e.g., pharmaceutical compositions or vaccines) of the disclosure comprise a delivery component and a polynucleotide comprising: a first nucleotide sequence, wherein the first nucleotide sequence encodes IL-15 and is operably linked to a CMV promoter; a second nucleotide sequence, wherein the second nucleotide sequence encodes CCL3 and is operably linked to promoter X; a third nucleotide sequence, wherein the third nucleotide sequence encodes CCL4 and is operably linked to promoter Y; a fourth nucleotide sequence, wherein the fourth nucleotide sequence encodes a first tumor-associated antigen and is operably linked to promoter 1, and a fifth nucleotide sequence, wherein the fifth nucleotide sequence encodes a second tumor- associated antigen and is operably linked to promoter 2. In some aspects, the polynucleotide comprises: a 5' first nucleotide sequence, wherein the first nucleotide sequence encodes IL- 15 and is operably linked to a CMV promoter; a second nucleotide sequence positioned 3' to the first nucleotide sequence, wherein the second nucleotide sequence encodes CCL3 and is operably linked to promoter X; a third nucleotide sequence positioned 3' to the second nucleotide sequence, wherein the third nucleotide sequence encodes CCL4 and is operably linked to promoter Y; a fourth nucleotide sequence positioned 3' to the third nucleotide sequence, wherein the fourth nucleotide sequence encodes a first tumor-associated antigen and is operably linked to promoter 1, and a fifth nucleotide sequence positioned 3' to the fourth nucleotide sequence, wherein the fifth nucleotide sequence encodes a second tumor-associated antigen and is operably linked to promoter 2. In some aspects, the first, second, third, fourth, and fifth nucleotide sequences of the polynucleotide are configured as shown in FIG. 11.
[0243] In some aspects, the compositions (e.g., pharmaceutical compositions or vaccines) of the disclosure comprise a delivery component and a polynucleotide comprising: a first nucleotide sequence, wherein the first nucleotide sequence encodes CCL3 and is operably linked to promoter X; a second nucleotide sequence, wherein the second nucleotide sequence encodes CCL4 and is operably linked to promoter Y; a third nucleotide sequence, wherein the third nucleotide sequence encodes a first tumor-associated antigen and is operably linked to promoter 1; and a fourth nucleotide sequence, wherein the fourth nucleotide sequence encodes a second tumor-associated antigen and is operably linked to promoter 2. In some aspects, the polynucleotide comprises: a 5' first nucleotide sequence, wherein the first nucleotide sequence encodes CCL3 and is operably linked to promoter X; a second nucleotide sequence positioned 3' to the first nucleotide sequence, wherein the second nucleotide sequence encodes CCL4 and is operably linked to promoter Y; a third nucleotide sequence positioned 3' to the second nucleotide sequence, wherein the third nucleotide sequence encodes a first tumor-associated antigen and is operably linked to promoter 1; and a fourth nucleotide sequence positioned 3' to the third nucleotide sequence, wherein the fourth nucleotide sequence encodes a second tumor- associated antigen and is operably linked to promoter 2. In some aspects, the first, second, third, and fourth nucleotide sequences of the polynucleotide are configured as shown in FIG. 12.
[0244] In some aspects, one or more nucleotide sequences of the polynucleotide are configured as shown in FIGs. 14A-14O. In some aspects, two or more nucleotide sequences of the polynucleotide are configured as shown in FIGs. 14A-14O. In some aspects, three or more nucleotide sequences of the polynucleotide are configured as shown in FIGs. 14A-14O.
[0245] In some aspects, the first, second, and third nucleotide sequences of the polynucleotide are configured as shown in FIG. 14B. In some aspects, the first, second, third, and fourth nucleotide sequences of the polynucleotide are configured as shown in FIG. 14C. In some aspects, the first nucleotide sequence of the polynucleotide is configured as shown in FIG. 14D. In some aspects, the first, second, and third nucleotide sequences of the polynucleotide are configured as shown in FIG. 14E. In some aspects, the first, second, third, and fourth nucleotide sequences of the polynucleotide are configured as shown in FIG. 14F. In some aspects, the first and second nucleotide sequences of the polynucleotide are configured as shown in FIG. 14G. In some aspects, the first and second nucleotide sequences of the polynucleotide are configured as shown in FIG. 14H. In some aspects, the first nucleotide sequence of the polynucleotide is configured as shown in FIG. 141. In some aspects, the first nucleotide sequence of the polynucleotide is configured as shown in FIG. 14J. In some aspects, the first and second nucleotide sequence of the polynucleotide is configured as shown in FIG. 14K. In some aspects, the first nucleotide sequence of the polynucleotide is configured as shown in FIG. 14L. In some aspects, the first and second nucleotide sequences of the polynucleotide are configured as shown in FIG. 14M. In some aspects, the first nucleotide sequence of the polynucleotide is configured as shown in FIG. 14N. In some aspects, the first nucleotide sequence of the polynucleotide is configured as shown in FIG. 140.
[0246] In some aspects, the compositions (e.g., pharmaceutical compositons or vaccines) can comprise a delivery component and a polynucleotides configured as shown in the vector constructs illustrated in any of FIGs 1-12, which can modified to replace the “TAA-1 Gene” (a first nucleotide sequence encoding a tumor-associated antigen) and the “TAA-2 Gene” (a second nucleotide sequence encoding a tumor-associated antigen) with nucleotide sequences encoding any combinations of tumor-associated antigen or antigenic fragment thereof disclosed herein. In some aspect, the nucleotide sequences encode antigens to a virus, a bacteria or a parasite. In some aspects, the nucleotide sequences encode one or more antigens comprise one or more tumor-associated antigens.
[0247] In some aspects, the compositions (e.g., pharmaceutical compositons or vaccines) can comprise a delivery component and a polynucleotides configured as shown in the vector constructs illustrated in any of FIGs. 14N-14O. In some aspects, the compositions (e.g., pharmaceutical compositons or vaccines) can comprise a delivery component and a polynucleotides configured as shown in the vector constructs illustrated in any of FIGs. 14A-14M, which can modified to replace the “TAA” (a first nucleotide sequence encoding a tumor-associated antigen) and the “TAA-2” (a second nucleotide sequence encoding a tumor-associated antigen) with nucleotide sequences encoding any combinations of tumor-associated antigen or antigenic fragment thereof disclosed herein. In some aspect, the nucleotide sequences encode antigens to a virus, a bacteria or a parasite. In some aspects, the nucleotide sequences encode one or more antigens comprise one or more tumor-associated antigens.
[0248] In some aspects, the compositions (e.g., pharmaceutical compositions or vaccines) of the disclosure comprise a delivery component and a polynucleotide comprising: a first nucleotide sequence, wherein the first nucleotide sequence encodes a first tumor associated protein and is operably linked to a first promoter (e.g., a hEFl-HTLV promoter); a second nucleotide sequence, wherein the second nucleotide sequence encodes IL-12 p35 and is operably linked to a second promoter (e.g., a CMV promoter); and a third nucleotide sequence, wherein the third nucleotide sequence encodes IL- 12 p40 and is operably linked to a second promoter (e.g., a CMV promoter).
[0249] In some aspects, the compositions (e.g., pharmaceutical compositions or vaccines) of the disclosure comprise a delivery component and a polynucleotide comprising: a first nucleotide sequence, wherein the first nucleotide sequence encodes a first tumor- associated antigen and is operably linked to a first promoter (e.g., a hEFl-HTLV promoter); a second nucleotide sequence, wherein the second nucleotide sequence encodes a second tumor-associated antigen and is operably linked to the first promoter through an IRES sequence; a third nucleotide sequence, wherein the third nucleotide sequence encodes IL-12 p35 and is operably linked to a second promoter (e.g., a CMV promoter); and a fourth nucleotide sequence, wherein the fourth nucleotide sequence encodes IL-12 p340 and is operably linked to a second promoter (e.g., a CMV promoter).
[0250] In some aspects, the compositions (e.g., pharmaceutical compositions or vaccines) of the disclosure comprise a delivery component and a polynucleotide comprising: a first nucleotide sequence, wherein the first nucleotide sequence encodes a first tumor- associated antigen and is operably linked to a first promoter (e.g., an EF-la promoter); a second nucleotide sequence, wherein the second nucleotide sequence encodes IL- 12 p35 and is operably linked to a second promoter (e.g., a CMV promoter); and a third nucleotide sequence, wherein the third nucleotide sequence encodes IL-12 p40 and is operably linked to a second promoter (e.g., a CMV promoter).
[0251] In some aspects, the compositions (e.g., pharmaceutical compositions or vaccines) of the disclosure comprise a delivery component and a polynucleotide comprising: a first nucleotide sequence, wherein the first nucleotide sequence encodes a first tumor- associated antigen and is operably linked to a first promoter (e.g., an EF-la promoter); a second nucleotide sequence, wherein the second nucleotide sequence encodes a second tumor-associated antigen and is operably linked to the first promoter through an IRES sequence; a third nucleotide sequence, wherein the third nucleotide sequence encodes IL- 12 p35 and is operably linked to a second promoter (e.g., a CMV promoter); and a fourth nucleotide sequence, wherein the fourth nucleotide sequence encodes IL-12 p340 and is operably linked to a second promoter (e.g., a CMV promoter).
[0252] In some aspects, the compositions (e.g., pharmaceutical compositions or vaccines) of the disclosure comprise a delivery component and a polynucleotide comprising a first nucleotide sequence, wherein the first nucleotide sequence encodes a first tumor- associated antigen and is operably linked to a first promoter (e.g., an EF-la promoter
[0253] In some aspects, the compositions (e.g., pharmaceutical compositions or vaccines) of the disclosure comprise a delivery component and a polynucleotide comprising a first nucleotide sequence, wherein the first nucleotide sequence encodes a first tumor- associated antigen and is operably linked to a first promoter (e.g., an EF-la promoter); and a second nucleotide sequence, wherein the second nucleotide sequence encodes a second tumor-associated antigen and is operably linked to a second promoter (e.g., a CMV promoter).
[0254] In some aspects, the composition (e.g., pharmaceutical compositions or vaccines) can comprise a delivery component, wherein the polynucleotide configured as shown in the vector constructs illustrated in any of FIGs. 1-14 can be modified to replace the the tumor-associated antigen- 1 or the tumor-associated antigen-2 and/or any additional tumor-associated antigen with nucleotide sequences encoding any combinations of tumor- associated antigens or antigenic fragment thereof disclosed herein. In some aspects, the nucleotide sequences encode two or more tumor-associated antigens.
[0255] In some aspects, the compositions (pharmaceutical compositions or vaccines) can comprise an adjuvant and a polynucleotide configured as shown in the vector constructs illustrated in any of FIGs. 1-14 and/or vector constructs that have been modified to replace the tumor-associated antigen- 1 protein or the tumor-associated antigen-2 protein and/or an additional tumor-associated antigen with nucleotide sequences encoding any combinations of tumor-associated antigen or antigenic fragment thereof disclosed herein.
[0256] In some aspects, the compositions (pharmaceutical compositions or vaccines) can comprise a polynucleotide encoding any combinations of tumor-associated antigen or antigenic fragment thereof disclosed herein and an adjuvant selected from potassium aluminum sulfate [KA1(SO4)2], aluminum hydroxide, crystalline aluminum oxyhydroxide, aluminum phosphate, aluminum hydroxyphosphate, amorphous aluminum hydroxyphosphate sulfate, aluminum chloride, aluminum silicate, and a composition comprising aluminum hydroxide and magnesium hydroxide.
[0257] In some aspects, the compositions (pharmaceutical compositions or vaccines) can comprise a polynucleotide encoding any combinations of tumor-associated antigen or antigenic fragment thereof disclosed herein and a STING agonist.
[0258] In some aspects, the compositions (pharmaceutical compositions or vaccines) can comprise a polynucleotide encoding any combinations of tumor-associated antigen or antigenic fragment thereof disclosed herein and the STING agonist cyclic GMP (cGMP).
[0259] In some aspects, the compositions (pharmaceutical compositions or vaccines) can comprise a polynucleotide encoding any combinations of tumor-associated antigen or antigenic fragment thereof disclosed herein and a STING agonist selected from ADU- S100, MK-1454, SB11285, BMS-986301, BI-STING (BI1387446), JNJ-67544412, 3’3’- cyclic AIMP, and GSK532.
[0260] In some aspects, the compositions (pharmaceutical compositions or vaccines) can comprise a polynucleotide encoding any combinations of tumor-associated antigen or antigenic fragment thereof disclosed herein and a STING agonist selected from DMAXAA, ALG-031048, E7766, JNJ-‘6196, MK-21118, MSA-1, MSA-2, SNX281, SR-717, TAK676, TTI-10001, a Ryvu’s agonist, GF3-002, a Selvita agonist, CDR5500, CS-1010, CS-1018, CS-1020, and [2-ex] MSA-1.
[0261] In some aspects, the compositions (pharmaceutical compositions or vaccines) can comprise a polynucleotide encoding any combinations of tumor-associated antigen or antigenic fragment thereof disclosed herein and a STNG agonist selected from a PC7A nanoparticle, a cGMP-nanoparticle, and a ONM-500 nanoparticle.
[0262] In some aspects, the compositions (pharmaceutical compositions or vaccines) can comprise a polynucleotide encoding any combinations of tumor-associated antigen or antigenic fragment thereof disclosed herein and a STING agonist that is an antibody-drug conjugate. In some aspects, the antibody drug conjugate is XMT-2056.
[0263] In some aspects, the compositions (pharmaceutical compositions or vaccines) can comprise a polynucleotide encoding any combinations of tumor-associated antigen or antigenic fragment thereof disclosed herein and a STING agonist that is an ENPP1 inhibitor selected from MV-626, SR-8314, SR-8291, and SR8541A. [0264] In some aspects, the compositions (pharmaceutical compositions or vaccines) can comprise a polynucleotide encoding any combinations of tumor-associated antigen or antigenic fragment thereof disclosed herein and a STING agonist that is a bacterial vector. In some aspects, the bacterial vector is a nonpathogenic E coli nissle expressing cyclic-di- AMP-producing enzymes. In some aspects, the bacterial vector is SYNB1981. In some aspects, the bacterial vector is an attenutated Salmonella Typhimurium strain enginerred to carry an inhibitory TREX-1 micro RNA.
[0265] In some aspects, the compositions (pharmaceutical compositions or vaccines) can comprise a polynucleotide encoding any combinations of tumor-associated antigen or antigenic fragment thereof disclosed herein, an adjuvant and a STING agonist.
[0266] In some aspects, the compositions (pharmaceutical compositions or vaccines) can comprise a polynucleotide encoding any combinations of tumor-associated antigen or antigenic fragment thereof disclosed herein, an adjuvant selected from potassium aluminum sulfate [KA1(SO4)2], aluminum hydroxide, crystalline aluminum oxyhydroxide, aluminum phosphate, aluminum hydroxyphosphate, amorphous aluminum hydroxyphosphate sulfate, aluminum chloride, aluminum silicate, a composition comprising aluminum hydroxide and magnesium hydroxide and combinations thereof, and a STING agonist selected from cyclic GMP (cGMP), cAMP, and cGAMP.
[0267] In some aspects, the compositions (pharmaceutical compositions or vaccines) can comprise a polynucleotide encoding any combinations of tumor-associated antigen or antigenic fragment thereof disclosed herein, an adjuvant selected from potassium aluminum sulfate [KA1(SO4)2], aluminum hydroxide, crystalline aluminum oxyhydroxide, aluminum phosphate, aluminum hydroxyphosphate, amorphous aluminum hydroxyphosphate sulfate, aluminum chloride, aluminum silicate, a composition comprising aluminum hydroxide and magnesium hydroxide and combinaions thereof, and a STING agonist selected from cyclic GMP (cGMP), ADU-S100, MK-1454, SB11285, BMS-986301, BI-STING (BI1387446), JNJ-67544412, 3’3’-cyclic AIMP, GSK532, DMAXAA, ALG-031048, E7766, JNJ-‘6196, MK-21118, MSA-1, MSA-2, SNX281, SR-717, TAK676, TTI-10001, a Ryvu’s agonist, GF3-002, a Selvita agonist, CDR5500, CS-1010, CS-1018, CS-1020, [2-ex] MSA-1, a PC7A nanoparticle, a cGMP-nanoparticle, and a ONM-500 nanoparticle, the XMT-2056 antibody-drug conjugate, an ENPP1 inhibitor selected from MV-626, SR-8314, SR-8291, and SR8541A, a bacterial vector selected from SYNB1981 and an attenutated Salmonella Typhimurium strain enginerred to carry an inhibitory TREX-1 micro RNA.
[0268] In some aspects, the compositions (e.g., pharmaceutical composition or vaccine) further comprise a delivery component (e.g., a cationic polymer, a poly-inosinic- polycytidylic acid, or a poloxamer). In some aspects, the delivery component further comprises benzalkonium chloride.
[0269] The polynucleotides and compositions of the present disclosure (e.g., pharmaceutical compositions, vaccines, vectors, and DNA plasmid vectors) can be formulated according to known methods for preparing pharmaceutically useful compositions.
[0270] Formulations are described in a number of sources which are well known and readily available to those skilled in the art. For example, Remington's Pharmaceutical Science (Martin EW [1995] Easton Pennsylvania, Mack Publishing Company, 19th Ed.) describes formulations, which can be used in connection with the subject disclosure. Formulations suitable for parenteral administration include, for example, aqueous sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and nonaqueous sterile suspensions which can include suspending agents and thickening agents. The formulations can be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and can be stored in a freeze dried (lyophilized) condition requiring only the condition of the sterile liquid carrier, for example, water for injections, prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powder, granules, tablets, etc. It should be understood that in addition to the ingredients particularly mentioned above, the formulations of the subject disclosure can include other agents conventional in the art having regard to the type of formulation in question.
[0271] The disclosure also provides lyophilized (or freeze-dried) compositions or vaccines that can be safely stored for periods of time and reconstituted prior to use. In some aspects, the composition, pharmaceutical composition, or vaccine of the disclosure is a lyophilized product, e.g., substantially free of aqueous components. In some aspects, the lyophilized composition or vaccine is reconstituted in a diluent, e.g., prior to administration. In some aspects, the lyophilized composition or vaccine is reconstituted in water. [0272] Some aspects are directed to a lyophilized composition or vaccine comprising: (1) any polynucleotide or DNA plasmid vector disclosed herein; (2) a STING agonist; (3) an aluminum or aluminum salt based adjuvant; and (4) any delivery component disclosed herein, wherein the composition is substantially free of aqueous components. In some aspects, a composition or vaccine of the disclosure is lyophilized.
[0273] In some aspects, the composition or vaccine is stable at 0°C to 5°C for at least about 1 month (or 30 days), 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 monts, or 12 months. In some aspects, the composition or vaccine is stable at 0°C to 5°C for at least 1 year, at least 2 years, at least
3 years, at least 4 years or at least 5 years. In some aspects, the composition or vaccine is stable at -20°C for at least 1 year, at least 2 years, at least 3 years, at least 4 years, at least 5 years, or at least 10 years. In some aspects, the composition or vaccine is stable at 25°C for at least about 7 days, about 10 day, or about 14 days. In some aspects, the composition or vaccine is lyophilized and is stable at 0°C to 5°C for at least about 1 month (or 30 days), 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 monts, or 12 months. In some aspects, the lyophilized composition or vaccine is stable at 0°C to 5°C for at least 1 year, at least 2 years, at least 3 years, at least
4 years or at least 5 years. In some aspects, the lyophilized composition or vaccine is stable at -20°C for at least 1 year, at least 2 years, at least 3 years, at least 4 years, at least
5 years, or at least 10 years. In some aspects, the lyophilized composition or vaccine is stable at 25°C for at least about 7 days, about 10 day, or about 14 days.
[0274] In some aspects, the lyophilized composition or vaccine is reconstituted into a reconstituted composition or vaccine formulation for administration. In some aspects, the reconstituted composition or vaccine is stable at 0°C to 5°C for at least about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, or about 6 months after reconstitution of the lyophilized composition or vaccine with a diluent (e.g., water). In some aspects, the reconstiuted composition or vaccine is stable at -20°C for at least 1 year, at least 2 years, at least 3 years, at least 4 years, at least 5 years, or at least 10 years. In some aspects, the reconstituted composition or vaccine is stable at 25°C for at least about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days after reconstitution of the lyophilized composition with a diluent. In some aspects, the diluent is water. [0275] The compositions of the subject disclosure can further comprise other components such as a pharmaceutically acceptable carrier and/or an immune modifier protein. The immune modifier protein can be expressed in alternative polynucleotides, plasmids, or vectors or are delivered as proteins in combination with the compositions of the subject disclosure. The immune modifier protein can be selected from the group consisting of: a- interferon (IFN-a), P-interferon (IFN-P), y-interferon, platelet derived growth factor (PDGF), TNFa, TNFP, GM-CSF, epidermal growth factor (EGF), cutaneous T cellattracting chemokine (CTACK), epithelial thymus-expressed chemokine (TECK), mucosae-associated epithelial chemokine (MEC), IL-12, IL-15, MHC, CD80, CD86 including IL- 15 having the signal sequence deleted and optionally including the signal peptide from IgE. The immune modifier protein can be IL-12, IL-15, IL-28, CTACK, TECK, platelet derived growth factor (PDGF), TNFa, TNFP, GM-CSF, epidermal growth factor (EGF), IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-18, or a combination thereof. Other genes that can be useful immune modifier proteins include those encoding: MCP-1, MIP-la, MIP-lp, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, pl50.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, mutant forms of IL-18, CD40, CD40L, vascular growth factor, fibroblast growth factor, IL-7, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Fit, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, DR6, Caspase ICE, Fos, c- jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, Inactive NIK, SAP K, SAP-1, JNK, interferon response genes, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, 0x40, 0x40 LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAPI, TAP2, functional fragments thereof, and combinations thereof.
[0276] In some aspects, compositions of the disclosure can be formulated according to the mode of administration to be used. For example, an injectable vaccine pharmaceutical composition can be sterile, pyrogen free and particulate free. An isotonic formulation or solution can be used. Additives for isotonicity can include sodium chloride, dextrose, mannitol, sorbitol, and lactose. The vaccine can comprise a vasoconstriction agent. The isotonic solutions can include phosphate buffered saline. Vaccine can further comprise stabilizers including gelatin and albumin. The stabilizers can allow the formulation to be stable at room or ambient temperature for extended periods of time, including LGS or polycations or polyanions.
IV. Polynucleotides
[0277] In some aspects, the polynucleotides of the disclosure can include DNA or mRNA sequences (e.g., multicistronic DNA or multi ci str onic mRNA) for use in the compositions (e.g., pharmaceutical compositons and vaccines) disclosed herein. In some aspects, the present disclosure is directed to a polynucleotide can comprise a nucleic acid sequence encoding one or more tumor-associated antigens (e.g., NYESO-1, MAGE-A1, MAGE- A2, MAGE-A3, MAGE-A4, MAGE-A9, MAGE-A10, MAGE-CI, MAGE-C2, SSX, H0RMAD1, CXorf61, ACTL8, AKAP3,AKAP4, HOM-TES-85, OY-TES-1, HSP70-2, LAGE-1, PIWL1, PIWL2, PIWL3, PIWL4, PLU-1, PLAC1, SPAG9, SCP-1, TAG-1, TAG-2a, TAG-2b, TAG-2c, TRAG-3, KAP4, BAGE, BORIS, CT45, GAGE-1/2, PRAME, PAGE4, SP17, PASD1, p53, ras, B-RafHER-2/neu, mucin 1 (MUC1), epidermal growth factor receptor (EGFR), GP100, prostatic acid phosphatase, prostatespecific membrane antigen (PSMA), mammoglobin-A, carcinoembryonic antigen (CEA), papillomavirus antigens E6ZE7, heat-shock proteins, alpha fetoprotein, CDK, P-catenin, mesothelin, or cancer antigen-125 (CA125)) or antigenic fragments thereof). In some aspects, the antigenic fragment of a tumor-associated antigen can be an extracellular region of the antigen. For example, the antigenic of CA-125 can be the extracellular region of CA-125 or a portion of the extracellular region of CA-125. In some aspects, the vector further comprises a nucleic acid sequence encoding one or more immune modifier proteins. In some aspects, the vector comprises a nucleic acid sequence encoding a tumor- associated antigen and, optionally, a second tumor-associated antigen. In some aspects, the vector comprises a nucleic acid sequence encoding a tumor-associated antigen and a second tumor-associated antigen. In some aspects, the nucleic acid sequence encodes a tumor-associated antigen and a second tumor-associated antigen from a different tumor. In some aspects, the nucleic acid sequence encodes different portions of the same tumor- associated antigen, wherein the different portions of the same tumor-associated antigen are derived from the same tumor.
[0278] In some aspects, the present disclosure is directed to a polynucleotide comprising: (a) a antigen nucleic acid (e.g., first antigen nucleic acid) which encodes a tumor associated protein antigen (e.g., NYESO-1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE- A4, MAGE-A9, MAGE-A10, MAGE-CI, MAGE-C2, SSX, HORMAD1, CXorf61, ACTL8, AKAP3,AKAP4, HOM-TES-85, OY-TES-1, HSP70-2, LAGE-1, PIWL1, PIWL2, PIWL3, PIWL4, PLU-l, PLAC1, SPAG9, SCP-1, TAG-1, TAG-2a, TAG-2b, TAG-2c, TRAG-3, KAP4, BAGE, BORIS, CT45, GAGE-1/2, PRAME, PAGE4, SP17, PASD1, p53, ras, B-RafHER-2/neu, mucin 1 (MUC1), epidermal growth factor receptor (EGFR), GP100, prostatic acid phosphatase, prostate-specific membrane antigen (PSMA), mammoglobin-A, carcinoembryonic antigen (CEA), papillomavirus antigens E6/E7, heat-shock proteins, alpha fetoprotein, CDK, P-catenin, mesothelin, or cancer antigen-125 (CA125)) or an antigenic fragment thereof; and (b) a nucleic acid encoding an immune modifier protein. In some aspects, the antigen nucleic acid (e.g., first antigen nucleic acid) is operably linked to a first promoter. In some aspects, the polynucleotide comprises two or more nucleic acids encoding an immune modifier protein. In some aspects, each of the nucleic acids encoding an immune modifier protein encodes a different immune modifier protein.
[0279] The polynucleotides disclosed herein can comprise: (b) or (c) at least one additional antigen nucleic acid (e.g., second antigen nucleic acid) which encodes a second tumor associated protein antigen (e.g., NYESO-1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A9, MAGE-A10, MAGE-CI, MAGE-C2, SSX, H0RMAD1, CXorf61, ACTL8, AKAP3,AKAP4, HOM-TES-85, OY-TES-1, HSP70-2, LAGE-1, PIWL1, PIWL2, PIWL3, PIWL4, PLU-l, PLAC1, SPAG9, SCP-1, TAG-1, TAG-2a, TAG-2b, TAG-2c, TRAG-3, KAP4, BAGE, BORIS, CT45, GAGE-1/2, PRAME, PAGE4, SP17, PASD1, p53, ras, B-RafHER-2/neu, mucin 1 (MUC1), epidermal growth factor receptor (EGFR), GP100, prostatic acid phosphatase, prostate-specific membrane antigen (PSMA), mammoglobin-A, carcinoembryonic antigen (CEA), papillomavirus antigens E6ZE7, heat-shock proteins, alpha fetoprotein, CDK, P-catenin, mesothelin, or cancer antigen-125 (CA125)) or an antigenic fragment thereof. In some aspects, the second tumor-associated antigen or antigenic fragment thereof comprises one or more portions of the tumor associated antigen. In some aspects, the first tumor-associated antigen and the second tumor-associated antigen are tumor-associated antigen antigens from different tumors. In some aspects, the first tumor-associated antigen and the second tumor-associated antigens are different portions of the same tumor-associated antigen. In some aspects, the first tumor-associated antigen and the second tumor-associated antigen are different tumor-associated antigens, wherein the different tumor-associated antigens are derived from different tumors. In some aspects, the first tumor-associated antigen and the second tumor-associated antigen are different tumor-associated antigens, wherein the different tumor-associated antigens are derived from the same tumor. In some aspects, the at least one additional antigen nucleic acid (e.g., second antigen nucleic acid) is operably linked to the first promoter through an internal ribosome entry site (IRES) sequence. In some aspects, the IRES sequence comprises a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 19.
[0280] In some aspects, the present disclosure is directed to a polynucleotide (e.g., multicistronic DNA or multi ci str onic mRNA) comprising: (a) an antigen nucleic acid (e.g., first antigen nucleic acid) which encodes a tumor-associated antigen or an antigenic fragment thereof; and (b) a nucleic acid encoding an immune modifier protein. In some aspects, the polynucleotide comprises at least two tumor-associated antigens. In some aspects, the polynucleotide comprises at least two tumor-associated antigens and an immune modifier protein. In some aspects, the polynucleotide comprises at least two tumor-associated antigens and at least two immune modifier proteinsln some aspects, the present disclosure is directed to a composition comprising (a) a polynucleotide (e.g., a multicistronic DNA plasmid or multicistronic mRNA) comprising an antigen nucleic acid which encodes a tumor-associated antigen (e.g., NYESO-1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A9, MAGE-A10, MAGE-CI, MAGE-C2, SSX, H0RMAD1, CXorf61, ACTL8, AKAP3,AKAP4, HOM-TES-85, OY-TES-1, HSP70-2, LAGE-1, PIWL1, PIWL2, PIWL3, PIWL4, PLU-1, PLAC1, SPAG9, SCP-1, TAG-1, TAG-2a, TAG-2b, TAG-2c, TRAG-3, KAP4, BAGE, BORIS, CT45, GAGE-1/2, PRAME, PAGE4, SP17, PASD1, p53, ras, B-RafHER-2/neu, mucin 1 (MUC1), epidermal growth factor receptor (EGFR), GP100, prostatic acid phosphatase, prostatespecific membrane antigen (PSMA), mammoglobin-A, carcinoembryonic antigen (CEA), papillomavirus antigens E6ZE7, heat-shock proteins, alpha fetoprotein, CDK, P-catenin, mesothelin, or cancer antigen-125 (CA125)) or an antigenic fragments thereof); and (b) an adjuvant comprising an aluminum or aluminum-salt based adjuvant. In some aspects, the composition comprises (a) a polynucleotide (e.g., a multicistronic DNA plasmid or multicistronic mRNA) comprising an antigen nucleic acid which encodes a tumor- associated antigen (e.g, NYESO-1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A9, MAGE-A10, MAGE-CI, MAGE-C2, SSX, H0RMAD1, CXorf61, ACTL8, AKAP3,AKAP4, HOM-TES-85, OY-TES-1, HSP70-2, LAGE-1, PIWL1, PIWL2, PIWL3, PIWL4, PLU-1, PLAC1, SPAG9, SCP-1, TAG-1, TAG-2a, TAG-2b, TAG-2c, TRAG-3, KAP4, BAGE, BORIS, CT45, GAGE-1/2, PRAME, PAGE4, SP17, PASD1, p53, ras, B-RafHER-2/neu, mucin 1 (MUC1), epidermal growth factor receptor (EGFR), GP100, prostatic acid phosphatase, prostate-specific membrane antigen (PSMA), mammoglobin-A, carcinoembryonic antigen (CEA), papillomavirus antigens E6ZE7, heatshock proteins, alpha fetoprotein, CDK, P-catenin, mesothelin, or cancer antigen- 125 (CA125)) or an antigenic fragments thereof); and (b) an adjuvant comprising a STING agonist. In some aspects, the composition comprises (a) a polynucleotide (e.g., a multi ci str onic DNA plasmid or multi ci str onic mRNA) comprising an antigen nucleic acid which encodes a tumor-associated antigen (e.g., NYESO-1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A9, MAGE-A10, MAGE-CI, MAGE-C2, SSX, H0RMAD1, CXorf61, ACTL8, AKAP3,AKAP4, HOM-TES-85, OY-TES-1, HSP70-2, LAGE-1, PIWL1, PIWL2, PIWL3, PIWL4, PLU-1, PLAC1, SPAG9, SCP-1, TAG-1, TAG-2a, TAG-2b, TAG-2c, TRAG-3, KAP4, BAGE, BORIS, CT45, GAGE-1/2, PRAME, PAGE4, SP17, PASD1, p53, ras, B-RafHER-2/neu, mucin 1 (MUC1), epidermal growth factor receptor (EGFR), GP100, prostatic acid phosphatase, prostatespecific membrane antigen (PSMA), mammoglobin-A, carcinoembryonic antigen (CEA), papillomavirus antigens E6ZE7, heat-shock proteins, alpha fetoprotein, CDK, P-catenin, mesothelin, or cancer antigen-125 (CA125)) or an antigenic fragments thereof); and (b) an adjuvant comprising an aluminum or aluminum-salt based adjuvant and a STING agonist. In some aspects, the antigen nucleic acid (e.g., first antigen nucleic acid) is operably linked to a first promoter. In some aspects, the polynucleotide comprises two or more nucleic acids encoding an immune modifier protein. In some aspects, each of the nucleic acids encoding an immune modifier protein encodes a different immune modifier protein.
[0281] The polynucleotides disclosed herein can further comprise: (c) at least one additional antigen nucleic acid (e.g., second antigen nucleic acid) which encodes a tumor- associated antigen or an antigenic fragment thereof. In some aspects, the t. umor associated antigen protein or antigenic fragment thereof is selected from the group consisting of: NYESO-1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A9, MAGE-A10, MAGE-CI, MAGE-C2, SSX, H0RMAD1, CXorf61, ACTL8, AKAP3,AKAP4, HOM-TES-85, OY-TES-1, HSP70-2, LAGE-1, PIWL1, PIWL2, PIWL3, PIWL4, PLU-1, PLAC1, SPAG9, SCP-1, TAG-1, TAG-2a, TAG-2b, TAG-2c, TRAG-3, KAP4, BAGE, BORIS, CT45, GAGE-1/2, PRAME, PAGE4, SP17, PASD1, p53, ras, B-RafHER-2/neu, mucin 1 (MUC1), epidermal growth factor receptor (EGFR), GP100, prostatic acid phosphatase, prostate-specific membrane antigen (PSMA), mammoglobin-A, carcinoembryonic antigen (CEA), papillomavirus antigens E6ZE7, heatshock proteins, alpha fetoprotein, CDK, P-catenin, mesothelin, or cancer antigen- 125 (CA125)). In some aspects, the antigen nucleic acid (e.g., first antigen nucleic acid) encodes a tumor-associated antigen or an antigenic fragment thereof and the at least one additional antigen nucleic acid (e.g., second antigen nucleic acid) encodes a tumor- associated antigen or an antigenic fragment thereof from a different tumor. In some aspects, the antigen nucleic acid (e.g., first antigen nucleic acid) encodes a tumor- associated antigen or an antigenic fragment thereof and the at least one additional antigen nucleic acid (e.g., second antigen nucleic acid) encodes a different tumor-associated antigen or an antigenic fragment thereof from the same tumor. In some aspects, the at least one additional antigen nucleic acid (e.g., second antigen nucleic acid) is operably linked to the first promoter through an internal ribosome entry site (IRES) sequence. In some aspects, the IRES sequence comprises a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 19.
[0282] The polynucleotides disclosed herein can further comprise one or more second promoters. In some aspects, the at least one additional antigen nucleic acid (e.g., second antigen nucleic acid) is operably linked to the one or more second promoters. In some aspects, one or more nucleic acids encoding an immune modifier protein is operably linked to the one or more second promoters. In some aspects, one or more of the nucleic acids encoding an immune modifier protein is operably linked to the first promoter or the one or more second promoters through an internal ribosome entry site (IRES) sequence. In some aspects, the IRES sequence comprises a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 19.
[0283] In some aspects, the first promoter or the one or more second promoters is selected from the group consisting of: a cytomegalovirus (CMV) promoter (SEQ ID NO: 14), a Rouse sarcoma virus (RSV) promoter, a Moloney murine leukemia virus (Mo- MuLV) long terminal repeat (LTR) promoter, a mammalian elongation factor 1 (EFl) promoter, a cytokeratin 18 (CK18) promoter, a cytokeratin 19 (CK19) promoter, a simian virus 40 (SV40) promoter (SEQ ID NO: 15 or SEQ ID NO: 30), a murine U6 promoter, a skeletal a-actin promoter, a P-actin promoter, a murine phosphoglycerate kinase 1 (PGK1) promoter, a human PGK1 promoter, a CMV enhancer/chicken P-actin (CAG) promoter (SEQ ID NO: 16), and any combination thereof. In some aspects, the one or more second promoters is the CMV promoter. In some aspects, the one or more second promoters is a mammalian EFl promoter. In some aspects, the mammalian EFl promoter is a hEFl-HTLV promoter (SEQ ID NO: 18).
[0284] In some aspects, each of the nucleic acids which encodes an immune modifier protein is under the control of a promoter selected from the group consisting of a CMV promoter, an RSV promoter, a Mo-MuLV LTR promoter, a mammalian EFl promoter, a CK18 promoter, a CK19 promoter, an SV40 promoter, a murine U6 promoter, a skeletal a-actin promoter, a P-actin promoter, a murine PGK1 promoter, a human PGK1 promoter, a CAG promoter, and any combination thereof. In some aspects, the mammalian EFl promoter is a hEFl-HTLV promoter.
[0285] In some aspects, each of the at least one additional antigen nucleic acid (e.g., second antigen nucleic acid)s is under the control of a promoter selected from the group consisting of a CMV promoter, an RSV promoter, a Mo-MuLV LTR promoter, a mammalian EFl promoter, a CK18 promoter, a CK19 promoter, an SV40 promoter, a murine U6 promoter, a skeletal a-actin promoter, a P-actin promoter, a murine PGK1 promoter, a human PGK1 promoter, a CAG promoter, and any combination thereof. In some aspects, the mammalian EFl promoter is a hEFl-HTLV promoter.
[0286] In some aspects, the immune modifier protein is selected from the group consisting of: interleukin (IL) 2 (IL-2), IL-12 p35, IL-12 p40, IL-12 p70, IL-15, IL-18, tumor necrosis factor alpha (TNFa), granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon (IFN) a (IFN-a), IFN-P, a chemokine, major histocompatibility complex (MHC) class I (MHC I), MHC class II (MHC II), human leukocyte antigen (HLA)-DR isotype (HLA-DR), CD80, CD86, and any combination thereof. In some aspects, the chemokine is selected from the group consisting of: C-C motif chemokine ligand (CCL) 3 (CCL3), CCL4, CCL5, CCL21, CCL28, C-X-C motif chemokine ligand (CXCL) 10 (CXCL10), and any combination thereof.
[0287] In some aspects, the immune modifier protein is an interleukin, e.g., IL-12. IL-12 is composed of four alpha helices. It is a heterodimeric cytokine encoded by two separate genes, IL-12A (p35) and IL-12B (p40). The active heterodimer (referred to as p70), and a homodimer of p40 are formed following protein synthesis. In some aspects, the immune modifier protein is an IL-12 heterodimer (IL-12 p70) or an IL-12 homodimer (e.g., IL-12 p35 or IL-12 p40).
[0288] In some aspects, the IL-12 p35 immune modifier protein comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 21 (mouse IL-12 p35) or SEQ ID NO: 25 (human IL-12 p35). In some aspects, the IL-12 p40 immune modifier protein comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 23 (mouse IL-12 p40) or SEQ ID NO: 27 (human IL-12 p40).
[0289] In some aspects, the nucleic acid encoding IL-12 p35 has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 20 (nucleic acid sequence encoding mouse IL-12 p35) or SEQ ID NO: 24 (nucleic acid sequence encoding human IL-12 p35). In some aspects, the nucleic acid encoding IL-12 p40 has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 22 (nucleic acid sequence encoding mouse IL- 12 p40) or SEQ ID NO: 26 (nucleic acid sequence encoding human IL- 12 p40).
[0290] In some aspects, the immune modifier protein comprises one or more concatamers of non-coding 5'-C-phosphate-G-3' (CpG) dinucleotides. In some aspects, the one or more concatamers of non-coding CpG dinucleotides activate the Toll-like receptor 9 (TLR9) signaling pathway. In some aspects, the one or more concatamers of non-coding CpG dinucleotides comprise one or more concatamers of non-coding CpG dinucleotides previously reported in Bauer, A. et al., Nucleic Acids Research 38(12):3891-908 (2010); Cornelie, S. el al., Journal of Biological Chemistry 279(15): 15124-9 (2004); Klinman, D. et al., J Immunol. 158(8):3635-9 (1997); Klinman, D. et al., Immunological Reviews 199(l):201-16 (2004); Luo, Z. et al., Mol Med Rep. 6(6): 1309-14 (2012); Bode, C. et al., Expert Rev Vaccines 10(4):499-511 (2011); and Kuo, T. et al., Scientific Reports 10:20085 (2020), each of which is incorporated by reference herein in its entirety.
[0291] In some aspects, polynucleotides encoding an immune modifier protein comprises a combination (i) a nucleic acid encoding an interleukin, and (ii) a nucleic acid encoding a major histocompatibility complex and/or a chemokine. [0292] In some aspects, the polynucleotidesencoding an immune modifier protein comprises a nucleic acid encoding IL-12 p35, a nucleic acid encoding IL-12 p40, or the combination thereof. In some aspects, the nucleic acid encoding IL- 12 p35 has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 20 (nucleic acid sequence encoding mouse IL- 12 p35) or SEQ ID NO: 24 (nucleic acid sequence encoding human IL-12 p35). In some aspects, the nucleic acid encoding IL-12 p40 has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 22 (nucleic acid sequence encoding mouse IL- 12 p40) or SEQ ID NO: 26 (nucleic acid sequence encoding human IL- 12 p40). In some aspects, the nucleic acid encoding an immune modifier protein further comprises a nucleic acid encoding MHC I, a nucleic acid encoding MHC II, or the combination thereof.
[0293] In some aspects, the polynucleotidesencoding an immune modifier protein comprises a nucleic acid encoding IL-12, a nucleic acid encoding IL-15, or the combination thereof.
[0294] In some aspects, the polynucleotidesencoding an immune modifier protein comprises a combination of a nucleic acid encoding IL-12 and a nucleic acid encoding IL-15.
[0295] In some aspects, the polynucleotides encoding an immune modifier protein comprises a nucleic acid encoding IL-2, a nucleic acid encoding IL-15, or the combination thereof. In some aspects, the nucleic acid encoding an immune modifier protein further comprises a nucleic acid encoding MHC I, a nucleic acid encoding MHC II, a nucleic acid encoding CCL3, a nucleic acid encoding CCL4, any the combination thereof.
[0296] In some aspects, the polynucleotidesencoding an immune modifer comprises a nucleic acid encoding CCL3, a nucleic acid encoding CCL4, or the combination thereof.
[0297] The antigen nucleic acid (e.g., first antigen nucleic acid) of the polynucleotides disclosed herein can encode a full length tumor-associated antigen polypeptide or antigenic fragment thereof. For example, in some aspects, the antigen nucleic acid (e.g., first antigen nucleic acid) of the polynucleotide encodes at least 8, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 750, at least 1,000, or at least 1,250 contiguous amino acids of any one of SEQ ID NOs: 1, 3, 5, 7, 8, or 10. In some aspects, the antigen nucleic acid (e.g., first antigen nucleic acid or second antigen nucleic acid) of the polynucleotide encodes a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 1, 3, 5, 7, 8, or 10. In some aspects, the antigen nucleic acid (e.g., first antigen nucleic acid or second antigen nucleic acid) of the polynucleotide comprises a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 2, 4, 6, 9, 11, 28, or 29.
[0298] In some aspects, the antigen nucleic acid (e.g., first antigen nucleic acid) and the at least one additional antigen nucleic acid (e.g., second antigen nucleic acid) of the polynucleotides encode a tumor-associated antigen or antigenic fragments thereof from different tumors. In some aspects, the antigen nucleic acid (e.g., first antigen nucleic acid) and the at least one additional antigen nucleic acid (e.g., second antigen nucleic acid) of the polynucleotides encode tumor-associated antigens or antigenic fragments thereof, wherein the tumor-associated antigens or antigenic fragments thereof are derived from different tumors. In some aspects, the antigen nucleic acid (e.g., first antigen nucleic acid) and the at least one additional antigen nucleic acid (e.g., second antigen nucleic acid) of the polynucleotides encode tumor-associated antigens or antigenic fragments thereof, wherein the tumor-associated antigens or antigenic fragments thereof are derived from the same tumors, In some aspects, the antigen nucleic acid (e.g., first antigen nucleic acid) and the at least one additional antigen nucleic acid (e.g., second antigen nucleic acid) of the polynucleotides encode tumor-associated antigens or antigenic fragments thereof, wherein the tumor-associated antigens or antigenic fragments thereof are portions derived from the same tumor assoicated antigen.
[0299] In some aspects, the present disclosure is directed to a polynucleotide (e.g., multicistronic DNA or multi ci str onic mRNA) comprising an antigen nucleic acid (e.g., first antigen nucleic acid) which encodes a first tumor-associated antigen or an antigenic fragment thereof, wherein the antigen nucleic acid (e.g., first antigen nucleic acid) is operably linked to a first promoter.
[0300] In some aspects, the polynucleotide further comprises at least one additional antigen nucleic acid (e.g., second antigen nucleic acid) which encodes a second tumor- associated antigen or an antigenic fragment thereof. In some aspects, the at least one additional antigen nucleic acid (e.g., second antigen nucleic acid) which encodes a second tumor-associated antigen is selected from the group consisting of NYESO-1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A9, MAGE-A10, MAGE-CI, MAGE-C2, SSX, H0RMAD1, CXorf61, ACTL8, AKAP3,AKAP4, HOM-TES-85, OY-TES-1, HSP70-2, LAGE-1, PIWL1, PIWL2, PIWL3, PIWL4, PLU-1, PLAC1, SPAG9, SCP-1, TAG-1, TAG-2a, TAG-2b, TAG-2c, TRAG-3, KAP4, BAGE, BORIS, CT45, GAGE-1/2, PRAME, PAGE4, SP17, PASD1, p53, ras, B-RafHER-2/neu, mucin 1 (MUC1), epidermal growth factor receptor (EGFR), GP100, prostatic acid phosphatase, prostatespecific membrane antigen (PSMA), mammoglobin-A, carcinoembryonic antigen (CEA), papillomavirus antigens E6ZE7, heat-shock proteins, alpha fetoprotein, CDK, P-catenin, mesothelin, and cancer antigen-125 (CA125),.
[0301] In some aspects, the at least one additional antigen nucleic acid (e.g., second antigen nucleic acid) is operably linked to the first promoter through an internal ribosome entry site (IRES) sequence. In some aspects, the IRES sequence comprises a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 19.
[0302] In some aspects, the polynucleotide comprises one or more second promoters. In some aspects, the at least one additional antigen nucleic acid (e.g., second antigen nucleic acid) is operably linked to the one or more second promoters.
[0303] In some aspects, the first promoter or the one or more second promoters is selected from the group consisting of: a cytomegalovirus (CMV) promoter, a Rouse sarcoma virus (RSV) promoter, a Moloney murine leukemia virus (Mo-MuLV) long terminal repeat (LTR) promoter, a mammalian elongation factor 1 (EFl) promoter, a cytokeratin 18 (CK18) promoter, a cytokeratin 19 (CK19) promoter, a simian virus 40 (SV40) promoter, a murine U6 promoter, a skeletal a-actin promoter, a P-actin promoter, a murine phosphoglycerate kinase 1 (PGK1) promoter, a human PGK1 promoter, a CBA promoter, a CAG promoter, and any combination thereof. In some aspects, the mammalian EFl promoter is a hEFl-HTLV promoter. In some aspects, the one or more second promoters is the CMV promoter.
[0304] In some aspects, the at least one additional antigen nucleic acid (e.g., second antigen nucleic acid) is under the control of a promoter selected from the group consisting of a CMV promoter, an RSV promoter, a Mo-MuLV LTR promoter, a mammalian EFl promoter, a CK18 promoter, a CK19 promoter, an SV40 promoter, a murine U6 promoter, a skeletal a-actin promoter, a P-actin promoter, a murine PGK1 promoter, a human PGK1 promoter, a CBA promoter, a CAG promoter, and any combination thereof. In some aspects, the mammalian EFl promoter is a hEFl-HTLV promoter.
[0305] In some aspects, the antigen nucleic acid (e.g., first antigen nucleic acid) encodes a full-length tumor-associated antigen or an antigenic fragment thereof. In some aspects, the antigen nucleic acid (e.g., first antigen nucleic acid) is operably linked to a promoter. In some aspects, the antigen nucleic acid (e.g., first antigen nucleic acid) is operably linked to a mammalian EFl promoter. In some aspects, the mammalian EFl promoter is a hEFl-HTLV promoter.
[0306] The polynucleotides disclosed herein can further comprise one or more post- transcriptional regulatory elements. In some aspects, the post-translational regulatory element is positioned 3' to a coding region of the polynucleotide. Non-limiting examples of post-transcriptional regulatory elements that are useful for the present disclosure include a mutated woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), microRNA binding site, DNA nuclear targeting sequence, or combinations thereof. In some aspects, the post-transcriptional regulatory element is a WPRE.
[0307] The polynucleotide can also comprise one or more polyadenylation (poly(a)) signals, which can be downstream of any protein coding sequence. Examples of polyadenylation signals include but are not limited to a SV40 poly(a) tail (SEQ ID NO: 12), LTR poly(a) tail, bovine growth hormone (bGH) poly(a) tail (SEQ ID NO: 13), human growth hormone (hGH) poly(a) tail, or human P-globin poly(a) tail. The SV40 polyadenylation signal can be a polyadenylation signal from a pCEP4 vector (Invitrogen, San Diego, CA). In some aspects, the polynucleotides disclosed herein further comprise at least one 3' UTR poly(a) tail sequence operably linked to the antigen nucleic acid (e.g., first antigen nucleic acid), the at least one additional antigen nucleic acid (e.g., second antigen nucleic acid), the nucleic acid encoding an immune modifier protein, or any combination thereof. In some aspects, the 3' UTR poly(a) tail sequence is a 3' UTR SV40 poly(a) tail sequence, a 3' UTR bovine growth hormone (bGH) poly(A) sequence, a 3' UTR actin poly(A) tail sequence, a 3' UTR hemoglobin poly(A) sequence, or combinations thereof.
[0308] The polynucleotides disclosed herein can further comprise at least one enhancer sequence upstream of any protein coding sequence. The enhancer can be necessary for DNA expression. In some aspects, the enhancer is a human actin enhancer, human myosin enhancer, human hemoglobin enhancer, human muscle creatine enhancer, or a viral enhancer such as one from CMV, HA, RSV or EB V. In some aspects, the enhancer is a polynucleotide function enhancer as described in U.S. Patent Nos. 5,593,972, 5,962,428, and WO 94/016737. In some aspects, the enhancer sequence is a CMV intronic sequence or a P-actin intronic sequence. In aspects, the enhancer sequence is a SV40 enhancer sequence (SEQ ID NO: 17).
[0309] The polynucleotides disclosed herein can further comprise one or more inverted terminal repeats (ITRs). In some aspects, the polynucleotide comprises a first ITR and a second ITR. In some aspects, the polynucleotide comprises a first ITR, e.g., a 5' ITR, and a second ITR, e.g., a 3' ITR. Typically, ITRs are involved in parvovirus (e.g., adeno- associated virus (AAV)) DNA replication and rescue, or excision, from prokaryotic plasmids (Samulski et al., 1983, 1987; Senapathy et al., 1984; Gottlieb and Muzyczka, 1988). In addition, ITRs appear to be the minimum sequences required for AAV proviral integration and for packaging of AAV DNA into virions (McLaughlin et al., 1988; Samulski et al., 1989). These elements are essential for efficient multiplication of a parvovirus genome. In some aspects, the ITRs fold into a hairpin T-shaped structure. In some aspects, the ITRs fold into non-T-shaped hairpin structures, e.g., into a U-shaped hairpin structure.
[0310] In some aspects, the ITRs that are useful for the present disclosure comprise an ITR from an AAV genome. In certain aspects, the ITR is an ITR of an AAV genome selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and any combination thereof. In some aspects, the ITR is an ITR of the AAV2 genome. In some aspects, the ITR is a synthetic sequence genetically engineered to include at its 5' and 3' ends ITRs derived from one or more of AAV genomes.
[0311] In some aspects, the ITR is not derived from an AAV genome. In some aspects, the ITR is an ITR of a non- AAV. In some aspects, the ITR is an ITR of a non- AAV genome from the viral family Parvoviridae selected from, but not limited to, the group consisting of Bocavirus, Dependovirus, Erythrovirus, Amdovirus, Parvovirus, Densovirus, Iteravirus, Contravirus, Aveparvovirus, Copiparvovirus, Protoparvovirus, Tetraparvovirus, Ambidensovirus, Brevidensovirus, Hepandensovirus, Penstyldensovirus and any combination thereof. In certain aspects, the ITR is derived from erythrovirus parvovirus B19 (human virus). In some aspects, the ITR is derived from a Muscovy duck parvovirus (MDPV) strain. In certain aspects, the MDPV strain is attenuated, e.g., MDPV strain FZ91-30. In some aspects, the MDPV strain is pathogenic, e.g., MDPV strain YY. In some aspects, the ITR is derived from a porcine parvovirus, e.g., porcine parvovirus U44978. In some aspects, the ITR is derived from a mice minute virus, e.g., mice minute virus U34256. In some aspects, the ITR is derived from a canine parvovirus, e.g., canine parvovirus M19296. In some aspects, the ITR is derived from a mink enteritis virus, e.g., mink enteritis virus D00765. In some aspects, the ITR is derived from a Dependoparvovirus. In certain aspects, the Dependoparvovirus is a Dependovirus Goose parvovirus (GPV) strain. In some aspects, the GPV strain is attenuated, e.g., GPV strain 82-0321V. In some aspects, the GPV strain is pathogenic, e.g., GPV strain.
[0312] The polynucleotides disclosed herein can also comprise a mammalian origin of replication (e.g., an Epstein Barr virus origin of replication) in order to maintain the vector extrachromosomally and produce multiple copies of the vector in a cell.
[0313] In some aspects, the polynucleotide is a multi ci str onic mRNA. In some aspects, the multi ci stronic mRNA comprises a 5' cap and a 3' UTR poly(A) tail sequence. In some aspects, the 3' UTR poly(a) tail sequence is a 3' UTR SV40 poly(a) tail sequence (SEQ ID NO: 13), a 3' UTR bovine growth hormone (bGH) poly(A) sequence (SEQ ID NO: 13), a 3' UTR actin poly(A) tail sequence, a 3' UTR hemoglobin poly(A) sequence, or any combinations thereof. In some aspects, the multi ci stronic mRNA comprises a 5' UTR and/or a 3' UTR.
[0314] In some aspects, the vector constructs can comprise the elements as illustrated in any of FIGs 1-12. In some aspects, the vector constructs illustrated in any of FIGs 1-12 can modified to replace the “TAA Gene” (a first nucleotide sequence encoding a tumor- associated antigen) and the “TAA-2 Gene” (a second nucleotide sequence encoding a tumor-associated antigen) with nucleotide sequences encoding any combinations of tumor-associated antigen or antigenic fragment thereof disclosed herein. In some aspects, the vector constructs disclosed herein can be used to express DNA or mRNA.
[0315] In some aspects, the vector constructs can comprise the elements as illustrated in any of FIGs. 14A-14O. In some aspects, the vector constructs illustrated in any of FIGs. 14A-14M can modified to replace the “TAA” (a first nucleotide sequence encoding a tumor-associated antigen) and the “TAA-2” (a second nucleotide sequence encoding a tumor-associated antigen) with nucleotide sequences encoding any combinations of tumor-associated antigen or antigenic fragment thereof disclosed herein. In some aspects, the vector constructs disclosed herein can be used to express DNA or mRNA. [0316] In some aspects, the polynucleotides disclosed herein can comprise: a nucleotide sequence, wherein the nucleotide sequence encodes a tumor-associated antigen (e.g., NYESO-1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A9, MAGE-A10, MAGE-CI, MAGE-C2, SSX, H0RMAD1, CXorf61, ACTL8, AKAP3,AKAP4, HOM- TES-85, OY-TES-1, HSP70-2, LAGE-1, PIWL1, PIWL2, PIWL3, PIWL4, PLU-1, PLAC1, SPAG9, SCP-1, TAG-1, TAG-2a, TAG-2b, TAG-2c, TRAG-3, KAP4, BAGE, BORIS, CT45, GAGE-1/2, PRAME, PAGE4, SP17, PASD1, p53, ras, B-RafHER-2/neu, mucin 1 (MUC1), epidermal growth factor receptor (EGFR), GP100, prostatic acid phosphatase, prostate-specific membrane antigen (PSMA), mammoglobin-A, carcinoembryonic antigen (CEA), papillomavirus antigens E6/E7, heat-shock proteins, alpha fetoprotein, CDK, P-catenin, mesothelin, or cancer antigen-125 (CA125)) and the nucleotide sequence is operably linked to a promoter. In some aspects, the polynucleotide further comprises a nucleic acid sequence encoding one or more immune modifier proteins. In some aspects, the polynucleotide comprises a nucleic acid sequence encoding a tumor-associated antigen and, optionally, a second tumor-associated antigen.
[0317] In some aspects, the promoter is selected from the group consisting of: a CMV promoter, an RSV promoter, a Mo-MuLV LTR promoter, a mammalian EFl promoter, a CK18 promoter, a CK19 promoter, an SV40 promoter, a murine U6 promoter, a skeletal a-actin promoter, a P-actin promoter, a murine PGK1 promoter, a human PGK1 promoter, a CAG promoter, and any combination thereof. In some aspects, the mammalian EFl promoter is a hEFl-HTLV promoter.In some aspects, promoter 1 is selected from the group consisting of: a CMV promoter, an RSV promoter, a Mo-MuLV LTR promoter, a mammalian EFl promoter, a CK18 promoter, a CK19 promoter, and any combination thereof.
[0318] In some aspects, the IRES sequence comprises a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 19.
V. Vectors and Host Cells,
[0319] Also provided herein are vectors or constructs comprising any polynucleotide described or exemplified herein, wherein the vector is a DNA plasmid vector, a multi ci str onic mRNA vector, a viral vector, a bacterial vector, a cosmid, or an artificial chromosome. In some aspects, the vector is selected from an AAV vector, adenoviral vector, retroviral vector, poxvirus vector, baculovirus vector, herpes viral vector, or combinations thereof.
[0320] In some aspects, the vectors are DNA plasmid vectors and multi ci stronic mRNA vectors comprising any polynucleotide described or exemplified herein.
[0321] Also provided herein are recombinant host cells comprising any polynucleotide, vector, DNA plasmid vector, or vaccine described herein. Host cells include prokaryotic cells, lower eukaryotic cells such as yeast, other eukaryotic cells such as insect cells, and higher eukaryotic cells such as mammalian cells. Mammalian host cells include, but are not limited to, CHO, VERO, BHK, Hela, MDCK, HEK 293, NIH 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NSO (a murine myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7O3O, COS (e.g., COS1 or COS), PER.C6, VERO, HsS78Bst, HEK-293T, HepG2, SP210, Rl.l, B-W, L-M, BSC1, BSC40, YB/20, BMT10, HBK, NSO, HT1080 and HsS78Bst cells. In some aspects, the recombinant host cells arc prepared by introducing the vectors, polynucleotides, or vaccines described herein into the cells by techniques readily available to the person of ordinary skill in the art. These include, but are not limited to, calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, lipofection, and other techniques such as those found in Sambrook. et al. (Molecular Cloning : A Laboratory Manual. 2nd. ed.. Cold Spring Harbor Laboratory. Cold Spring Harbor Laboratory Press. Cold Spring Harbor. NY (1989).
VI. Delivery Components
[0322] In some aspects, the disclosed compositions, pharmaceutical compositions, vaccines, vectors, multi ci stronic mRNA vectors, or DNA plasmid vectors further comprise a delivery component. In some aspects, the delivery component is a non-viral delivery component or system based on “naked DNA” or formulated plasmid DNA. In some aspects, the delivery component or system can be used to deliver mRNA. In some aspects, the non-viral delivery component is a cationic polymer. In some aspects, the cationic polymer is a synthetic functionalized polymer, a lipid, a lipopolymer, or a chemical derivative thereof. [0323] Non-viral gene delivery components or systems, based on "naked DNA" or formulated plasmid DNA, have potential benefits over viral vectors due to simplicity of use and lack of inciting a specific immune response. A number of synthetic gene delivery systems have been described to overcome the limitations of naked DNA, including cationic lipids, peptides, and polymers. Similarly, non-viral delivery systems can be used for delivery of mRNA.
[0324] Polymers have emerged as a viable alternative to current systems because their excellent molecular flexibility allows for complex modifications and incorporation of novel chemistries. Cationic polymers, such as poly(L-lysine) (PLL) and poly(L-arginine) (PLA), polyethyleneimine (PEI) have been widely studied as gene delivery candidates due to their ability to condense DNA, and promote DNA stability and transmembrane delivery. PEI efficiently condenses DNA into small narrowly distributed positively charged spherical complexes and can transfect cells in vitro and in vivo. PEI is similar to other cationic polymers in that the transfection activity of PEI increases with increasing polymer/DNA ratios. A distinct advantage of PEI over PLL is its endosomolytic activity which enables PEI to yield high transfection efficiency. Commercial branched PEI is composed of 25% primary amines, 50% secondary amines and 25% tertiary amines. The overall protonation level of PEI doubles from pH 7 to pH 5, which means in the endosome PEI becomes heavily protonated. Protonation of PEI triggers chloride influx across the endosomal membrane, and water follows to counter the high ion concentration inside the endosome, which eventually leads to endosomal disruption from osmotic swelling and release of the entrapped DNA. Because of its intrinsic endosomolytic activity, PEI generally does not require the addition of an endosomolytic agent for transfection. Due to these advantages PEI has been increasingly utilized in polymer functionalization strategies to create safer and more efficient delivery systems.
[0325] In some aspects, the delivery component is a cationic polymer. In some aspects, the cationic polymer is a synthetic functionalized polymer, a lipid, a lipopolymer, or a chemical derivative thereof. In some aspects, the cationic polymer is present in an amount sufficient to produce a ratio of amine nitrogen in the cationic polymer to phosphate in the DNA plasmid vector or multi ci str onic mRNA vector from about 0.1 : 1 to about 100: 1. In some aspects, the ratio of amine nitrogen in the cationic polymer to phosphate in the DNA plasmid vector or multi ci str onic mRNA vector is from about 0.1 : 1 to about 10: 1. In some aspects, the composition, pharmaceutical composition, or vaccine comprises about 0.5 mg/ml to about 5.0 mg/ml nucleic acid complexed with the cationic polymer. In some aspects, the delivery component is a poloxamer or a derivative thereof. In some aspects, the poloxamer or derivative thereof is present in a solution with the polynucleotide, multi ci str onic mRNA vector, or DNA plasmid vector from about 0.1% to about 5% or about 0.5% - about 5%. In some aspects, the delivery component is a P-amino ester. In some aspects, the polymer is present in a solution with the polynucleotide, multicistronic mRNA vector, or DNA plasmid vector from about 0.1% to about 5% or about 0.5% - about 5%. In some aspects, the delivery component is a poly-inosinic-polycytidylic acid. In some aspects, the poly-inosinic-polycytidylic acid is present in a solution with the polynucleotide, multicistronic mRNA vector, or DNA plasmid vector from about 0.1% to about 5% or about 0.5% - about 5%.
VI.1 Biodegradable Cross-linked Cationic Multi-block Copolymers
[0326] In some aspects, the delivery component is a biodegradable cross-linked cationic multi-block copolymer, for example any biodegradable cross-linked cationic multi-block copolymer disclosed in U.S. Patent No. 8,445,017. In some aspects, the biodegradable cross-linked cationic multi-block copolymer is a biodegradable cross-linked cationic multi -block copolymer of linear poly(alkylenimine) (LPAI) and a hydrophilic linker, wherein said LPAI blocks are crossed linked together by said hydrophilic linker with biodegradable ester, amide, disulfide, or phosphate linkages bonds. In some aspects, the linear poly(alkylenimine) (LPAI) is a member selected from the group consisting of polyethyleneimine, polypropylenimine, aminoglycoside-polyamine, dideoxy-diamino- .beta.-cyclodextrin, spermine and spermidine. In some aspects, the linear poly(alkylenimine) (LPAI) is linear poly(ethylenimine) (LPEI). In some aspects, poly(alkylenimine) (LPAI) is 3.6kD of LPEI (BD3.6-oleoyl).
[0327] In some aspects, the cross-linked cationic multi-block copolymer is linked by the biodegradable linkers to other moieties such as, for example, fluorescent markers, lipids anchors or their derivatives, i.e., cholesterol, fatty acids or their derivatives. In some aspects, the molecular weight of the linear PEI used in this dislosure is within the range of 1,000 to 25,000 Dalton. In some aspects, the linear PEI blocks are preferably linked to one another via a diamide linkage utilizing a biodegradable disulfidediacid-derived linker, i.e., dithiodipropionate derivatives. In some aspects, the molar ratio of the linker to the PEI is within a range of 1/1 to 5/1; the molar ratio of the lipid anchors to PEI is from 0/1 to 3/1. In some aspects, the polymer is formulated as a polyammonium salt, preferably with a chloride counterion. Since the toxicity of PEI increases with an increase in its molecular weight, the use of lower molecular weight PEIs as blocks in the polymer provides an improved gene carrier for use as a general reagent for transfection of mammalian cells, and for the in vivo application of gene therapy.
[0328] In some aspects, the biodegradable, cross-linked cationic multi-block copolymer comprises low molecular weight linear PEI blocks and a dithioacid moiety, i.e., dithiodipropionic acid, as biodegradable linkers. The biodegradable, cross-linked cationic multi-block copolymers are synthesized by cross-linking low molecular weight linear PEI units via a biodegradable disulfide linkage. These biodegradable cross-linked cationic multi-block copolymers are water soluble and transfectionally superior (68-70 fold higher activity) to single block polymers. See U.S. Patent No. 8,445,017.
[0329] In some aspects, the linker to the polymer molecular weight ratio is <0.2 which minimizes the dilution of polyamine polymer backbone. In some aspects, the chemical bond between the linker and the polymer blocks is a disulfide bond which can be biodegraded more easily as compared to amide bonds. Other biodegradable bonds can also be used in the present disclosure includes: phosphoesters, hydrazone, cis-asotinyl, urethane and poly(ethyl). Since any linker reacts in stepwise fashion, it can link either different blocks or the different areas of the same block (loop formation). The latter will favor the formation of a lightly cross-linked material with poor solubility due to multiple looping. The process disclosed in U.S. Patent No. 8,445,017 solves this problem by incorporating partial and reversible blocking/protection of nitrogen atoms in the LPEI blocks. Such LPEI functionalization also increases polymer solubility, facilitating the linking of LPEI blocks. This process also allows for convenient incorporation of pendant auxiliary ligands (for example, lipids, or fluorescent markers) onto a cationic polymer.
[0330] In some aspects, the cationic block copolymers are represented by the following formula: (CP)vL,Yz wherein CP represents a cationic polymer containing at least one secondary amine group, said CP polymer has a number averaged molecular weight within the range of 1,000 to 25,000 Dalton; Y represents a bifunctional biodegradable linker containing ester, amide, disulfide, or phosphate linkages; L represents a ligand; x is an integer in the range from 1 to 20; y is an integer from 0 to 100; and z is an integer in the range from 0 to 40. In some aspects, the cationic polymer comprises linear polyethyleneimine (LPEI). In some aspects, the LPEI is BD15-12, which has the following formula:
Figure imgf000085_0001
wherein the PEIs are approximately 15,000 Da, and wherein there is an average of 12 crosslinkers per PEI. In some aspects, the LPEI is Omnifect, which has the following formula:
Figure imgf000086_0001
wherein the PEIs are approximately 3,600 Da, wherein there is an average of 3 crosslinkers per PEI, and wherein there is an average of 1 PEG-lipid per PEI. In some aspects, the bifunctional biodegradable linker is hydrophilic and comprises a biodegradable linkage comprising a disulfide bond. In some aspects, the bifunctional biodegradable linker is a dithiodipropionyl linker.
[0331] In some aspects, the biodegradable cross-linked cationic multi-block copolymer comprises LPEI and a dithiodipropionyl linker for cross-linking the multi-block copolymer, wherein the LPEI has an average molecular weight of 1,000 to 25,000 Dalton. In some aspects, the biodegradable cross-linked cationic multi-block copolymer is covalently linked to at least one ligand.
[0332] In some aspects, the biodegradable, cross-linked, cationic, multi-block copolymers of LPEI and lipopolymers of the present disclosure have amine groups that are electrostatically attracted to polyanionic compounds such as nucleic acids. In some aspects, the cationic copolymer condenses DNA and forms compact structures. In addition, low toxicity of monomeric degradation products after delivery of bioactive materials provides for gene carriers with reduced cytotoxicity and increased transfection efficiency.
[0333] In some aspects, the biodegradable cross-linked cationic multi-block copolymers are conjugated with tracers (for example, fluorescent markers) or ligands either directly or via spacer molecules. In some aspects, only a small portion of the available amino groups is coupled to the ligand. The ligands conjugated to the polymers are targeting ligands that direct the polymers-nucleic acid complex to bind to specific target cells and penetrate into such cells. The targeting ligands can also be an intracellular targeting element, enabling the transfer of the nucleic acid/drug to be guided towards certain favored cellular compartments (mitochondria, nucleus, and the like). The targeting ligands conjugated to the polymers direct the polymers-nucleic acid complex to bind to specific target cells and penetrate into such cells (e.g., epithelial cells, endothelial cells, hematopoietic cells, and the like). The target ligands can also be an intracellular targeting element, enabling the transfer of the nucleic acid/drug to be guided towards certain favored cellular compartments (mitochondria, nucleus, and the like).
[0334] In some aspects, the targeting ligand is a polypeptide, folate, and an antigen. In some aspects, the polypeptide is a glycoprotein (e.g., transferrin or asialoorosomucoid (ASOR)), an antibody, an antibody fragment, a cell receptor, a cytokine receptor, or a growth factor receptor (e.g., epidermal growth factor receptor). In some aspects, the antigen is a tumor-associated antigen, a viral antigen, a bacterial antigen, or a parasite antigen. In some aspects, the ligand is a fusogenic agent (e.g., polymixin B and hemaglutinin HA2), a lysosomotrophic agent, or a nucleus localization signal (NLS) (e.g., T-antigen, and the like). In some aspects, the ligand is a sugar moiety coupled to an amino group. In some aspects, the sugar moiety is a mono- or oligo-saccharide, such as galactose, glucose, fucose, fructose, lactose, sucrose, mannose, cellobiose, nytrose, triose, dextrose, trehalose, maltose, galactosamine, glucosamine, galacturonic acid, glucuronic acid, and gluconic acid.
[0335] In some aspects, the biodegradable cross-linked cationic multi-block copolymer is covalently linked to polyethylene glycol (PEG) of molecular weight ranging from 500 to 20,000 Dalton. In some aspects, the biodegradable cross-linked cationic multi-block copolymer is covalently linked to a fatty acyl chain selected from the group consisting of: oleic acid, palmitic acid, and stearic acid. In some aspects, the biodegradable cross-linked cationic multi-block copolymer comprises at least one amine group that is electrostatically attracted to a polyanionic compound. In some aspects, the polyanionic compound is a nucleic acid, wherein the biodegradable cross-linked cationic multi -block copolymer condenses the nucleic acid to form a compact structure.
[0336] In some aspects, the biodegradable cross-linked cationic multi-block copolymer has the following formula:
Figure imgf000088_0001
wherein A and B are such that the molecular weight of the individual linear polyethylenimine chains are from 5,000 to 25,000 Dalton; the intermolecular crosslinks connect approximately 5-10% of amines; the biodegradable crosslinks are dithiodipropionyl (each half composed of 3 carbon atoms) and can be from 1-10 carbon atoms. In some aspects, the biodegradable cross-linked cationic polymer comprises 10,000 to 15,000 Dalton linear PEI covalently connected with a dithiopropionyl linkage (see, e.g., U.S. Patent No. 8,445,017). In some aspects, the biodegradable cross-linked cationic polymer comprises 10,000 to 15,000 Dalton linear PEI covalently connected with a dithiopropionyl linkage (see, e.g., U.S. Patent No. 8,445,017) and is further conjugated to polyethyleneglycol (PEG) of molecular weight ranging from 500 to 20,000 Dalton. In some aspects, the biodegradable cross-linked cationic polymer comprises 10,000 to 15,000 Dalton linear PEI covalently connected with a dithiopropionyl linkage (see, e.g., U.S. Patent No. 8,445,017) and is further conjugated to polyethyleneglycol (PEG) of molecular weight ranging from 500 to 20,000 Dalton. In some aspects, the biodegradable cross-linked cationic polymer comprises 15,000 to 20,000 Dalton linear PEI covalently connected with a dithiopropionyl linkage (see, e.g., U.S. Patent No. 8,445,017). In some aspects, the biodegradable cross-linked cationic polymer comprises 15,000 to 20,000 Dalton linear PEI covalently connected with a dithiopropionyl linkage (see, e.g., U.S. Patent No. 8,445,017) and is further conjugated to polyethyleneglycol (PEG) of molecular weight ranging from 500 to 20,000 Dalton.
[0337] In some aspects, the biodegradable cross-linked cationic multi-block copolymer is present in an amount sufficient to produce a ratio of amine nitrogen in the biodegradable cross-linked cationic multi-block copolymer to phosphate in the polynucleotide, multi ci str onic mRNA vector, or DNA plasmid vector from about 0.01 : 1 to about 50: 1 (e.g., about 0.01 : 1 to about 40: 1; about 0.01 : 1 to about 30: 1; about 0.01 : 1 to about 20: 1; about 0.01 : 1 to about 10: 1, or about 0.01 : 1 to about 5: 1). In some aspects, the ratio of amine nitrogen in the biodegradable cross-linked cationic multi-block copolymer to phosphate in the polynucleotide, multi ci str onic mRNA vector, or DNA plasmid vector is from about 0.1 : 1 to about 50: 1 (e.g., about 0.1 : 1 to about 40: 1; about 0.1 : 1 to about 30: 1; about 0.1 : 1 to about 20:1; about 0.1 : 1 to about 10: 1, or about 0.1 : 1 to about 5:1). In some aspects, the ratio of amine nitrogen in the biodegradable cross-linked cationic multi-block copolymer to phosphate in the polynucleotide, multi ci str onic mRNA vector, or DNA plasmid vector is from about 1 : 10 to about 10: 1.
[0338] In some aspects, the composition, pharmaceutical composition, or vaccine comprises about 0.1 mg/ml to about 10 mg/ml (e.g., about 0.1 mg/ml to about 5 mg/ml; about 0.5 mg/ml to about 10 mg/ml; or about 0.5 mg/ml to about 5 mg/ml) nucleic acid complexed with the biodegradable cross-linked cationic multi-block copolymer. In some aspects, the composition, pharmaceutical composition, or vaccine comprises about 1 mg/ml to about 10 mg/ml (e.g., about 1 mg/ml to about 6 mg/ml; about 2 mg/ml to about 6 mg/ml; about 5 mg/ml to about 10 mg/ml; or about 6 mg/ml to about 10 mg/ml) nucleic acid complexed with the biodegradable cross-linked cationic multi-block copolymer. VI.2 Cationic Lipopolymers Comprising a PEI Backbone
[0339] In some aspects, the delivery component is a cationic lipopolymer comprising a PEI backbone covalently linked to a lipid or a polyethylene glycol (PEG) as disclosed in U.S. Patent No. 7,964,571. In some aspects, the PEI backbone is covalently linked to a lipid and a PEG. In some aspects, the lipid and the PEG are directly attached to the PEI backbone by covalent bonds. In some aspects, the lipid is attached to the PEI backbone through a PEG spacer. In some aspects, the PEG has a molecular weight of between 50 to 20,000 Dalton. In some aspects, the molar ratio of PEG to PEI is within a range of 0.1 : 1 to 500: 1. In some aspects, the molar ratio of the lipid to the PEI is within a range of 0.1 : 1 to 500: 1. In some aspects, the lipid is a cholesterol, a cholesterol derivative, a C12 to Cis fatty acid, or a fatty acid derivative. The addition of PEG enhances the stability of the nucleic acid/polymer complexes in the biological milieu and allows for the incorporation of ligands (e.g., a targeting ligand) on to the PPC chain to improve the tissue selectivity of delivery. Ari U.S. Patent No. 7,964,571.
[0340] In some aspects, the cationic lipopolymer is a PEG:PEI: cholesterol (PPC) lipopolymer, which comprises a PEI backbone covalently linked to cholesterol and PEG. In some aspects, the PEI is covalently linked to cholesterol and PEG, and wherein the average PEG:PEI:cholesterol molar ratio in the cationic lipopolymer is within the range of 1-5 PEG: 1 PEI:0.4-1.5 cholesterol. In some aspects, the PEG-PEI-cholesterol (PPC) lipopolymer has an average PEG:PEI:cholesterol ratio of 2.5: 1 :0.6. In some aspects, the PEI has a linear or branch configuration with a molecular weight of 100 to 500,000 Dalton.
[0341] In some aspects, the cationic lipopolymer further comprises a pendant functional moiety selected from the group consisting of: a receptor ligand, a membrane permeating agent, an endosomolytic agent, a nuclear localization sequence, and a pH sensitive endosomolytic peptide.
[0342] In some aspects, the cationic lipopolymer further comprises a targeting ligand, wherein the targeting ligand is directly attached to the PEI backbone or is attached through a PEG linker. In some aspects, the targeting ligand is selected from the group consisting of: a sugar moiety, a polypeptide, folate, and an antigen. In some aspects, the sugar moiety is a monosaccharide. In some aspects, the monosaccharide is galactose. In some aspects, the sugar moiety is an oligosaccharide. In some aspects, the polypeptide is a glycoprotein, an antibody, an antibody fragment, a cell receptor, a cytokine receptor, or a growth factor receptor. In some aspects, the growth factor receptor is an epidermal growth factor receptor. In some aspects, the glycoprotein is transferrin or asialoorosomucoid (ASOR). In some aspects, the antigen is a tumor-associated antigen, a viral antigen, a bacterial antigen, or a parasite antigen.
[0343] In some aspects, the cationic lipopolymer is present in an amount sufficient to produce a ratio of amine nitrogen in the cationic polymer to phosphate in the polynucleotide, multi ci str onic mRNA vector, or DNA plasmid vector from about 0.01 : 1 to about 50: 1 (e.g., about 0.01 : 1 to about 40:1; about 0.01 : 1 to about 30: 1; about 0.01 :1 to about 20:1; about 0.01 : 1 to about 10: 1, or about 0.01 :1 to about 5: 1). In some aspects, the ratio of amine nitrogen in the cationic lipopolymer to phosphate in the polynucleotide, multi ci str onic mRNA vector, or DNA plasmid vector is from about 0.1 : 1 to about 50: 1 (e.g., about 0.1 : 1 to about 40: 1; about 0.1 : 1 to about 30: 1; about 0.1 : 1 to about 20: 1; about 0.1 : 1 to about 10: 1, or about 0.1 : 1 to about 5: 1). In some aspects, the ratio of amine nitrogen in the cationic lipopolymer to phosphate in the polynucleotide, multi ci stronic mRNA vector, or DNA plasmid vector is from about 1 : 10 to about 10: 1.
[0344] In some aspects, the composition, pharmaceutical composition, or vaccine comprises about 0.1 mg/ml to about 10.0 mg/ml (e.g., about 0.1 mg/ml to about 5 mg/ml; about 0.5 mg/ml to about 10 mg/ml; or about 0.5 mg/ml to about 5 mg/ml) nucleic acid complexed with the cationic polymer. In some aspects, the composition, pharmaceutical composition, or vaccine comprises about 1 mg/ml to about 10 mg/ml (e.g., about 1 mg/ml to about 6 mg/ml; about 2 mg/ml to about 6 mg/ml; about 5 mg/ml to about 10 mg/ml; or about 6 mg/ml to about 10 mg/ml) nucleic acid complexed with the cationic polymer.
VI.3 Lipopolyamines and Derivatives Thereof
[0345] In some aspects, the delivery component of the composition, pharmaceutical composition, or vaccine comprises a lipopolyamine with the following formula:
Figure imgf000091_0001
(Staramine). [0346] In some aspects, the delivery component comprises a mixture of the lipopolyamine and an alkylated derivative of the lipopolyamine. In some aspects, the alkylated derivative of the lipopolyamine is a polyoxyalkylene, polyvinylpyrrolidone, polyacrylamide, polydimethylacrylamide, polyvinyl alcohol, dextran, poly (L-glutamic acid), styrene maleic anhydride, poly-N-(2-hydroxypropyl) methacrylamide, or polydivinylether maleic anhydride. In some aspects, the alkylated derivative of the lipopolyamine has the following formula:
Figure imgf000092_0001
(methoxypolyethylene glycol (mPEG) modified Staramine), wherein n is an integer from 10 to 100 repeating units containing 2-5 carbon atoms each.
[0347] In some aspects, the alkylated derivative of the lipopolyamine has the following formula:
Figure imgf000092_0002
wherein n = 11 (Staramine-mPEG515). In some aspects, the alkylated derivative of the lipopolyamine has the following formula:
Figure imgf000092_0003
(Staramine-mPEGl 1).
[0348] In some aspects, the ratio of the lipopolyamine to the alkylated derivative of the lipopolyamine in the mixture is 1 : 1 to 10: 1. In some aspects, the lipopolyamine is present in an amount sufficient to produce a ratio of amine nitrogen in the lipopolyamine to phosphate in the polynucleotide, multi ci str onic mRNA vector, or DNA plasmid vector from about 0.01:1 to about 50:1 (e.g., about 0.01:1 to about 40:1; about 0.01:1 to about 30:1; about 0.01:1 to about 20:1; about 0.01:1 to about 10:1, or about 0.01:1 to about 5:1).
[0349] In some aspects, the ratio of amine nitrogen in the lipopolyamine to phosphate in the polynucleotide, multi ci stronic mRNA vector, or DNA plasmid vector is from about 0.1:1 to about 50:1 (e.g., about 0.1:1 to about 40:1; about 0.1:1 to about 30:1; about 0.1:1 to about 20:1; about 0.1:1 to about 10:1, or about 0.1:1 to about 5:1). In some aspects, the ratio of amine nitrogen in the lipopolyamine to phosphate in the polynucleotide, multi ci stronic mRNA vector, or DNA plasmid vector is from about 1 : 10 to about 10:1.
[0350] In some aspects, the delivery component of the composition, pharmaceutical composition, or vaccine comprises a lipopolyamine with the following formula:
Figure imgf000093_0001
(Crossamine).
[0351] In some aspects, the delivery component comprises a mixture of the lipopolyamine and an alkylated derivative of the lipopolyamine. In some aspects, the alkylated derivative of the lipopolyamine is a polyoxyalkylene, polyvinylpyrrolidone, polyacrylamide, polydimethylacrylamide, polyvinyl alcohol, dextran, poly (L-glutamic acid), styrene maleic anhydride, poly-N-(2-hydroxypropyl) methacrylamide, or polydivinylether maleic anhydride. In some aspects, the ratio of the lipopolyamine to the alkylated derivative of the lipopolyamine in the mixture is 1:1 to 10:1. In some aspects, the lipopolyamine is present in an amount sufficient to produce a ratio of amine nitrogen in the lipopolyamine to phosphate in the polynucleotide, multi ci stronic mRNA vector, or DNA plasmid vector from about 0.01 : 1 to about 50: 1 (e.g., about 0.01 : 1 to about 40: 1; about 0.01:1 to about 30:1; about 0.01:1 to about 20:1; about 0.01:1 to about 10:1, or about 0.01:1 to about 5:1). In some aspects, the ratio of amine nitrogen in the lipopolyamine to phosphate in the polynucleotide, multi ci stronic mRNA vector, or DNA plasmid vector is from about 0.1 : 1 to about 50: 1 (e.g., about 0.1 : 1 to about 40: 1; about 0.1:1 to about 30:1; about 0.1:1 to about 20:1; about 0.1:1 to about 10:1, or about 0.1:1 to about 5:1). In some aspects, the ratio of amine nitrogen in the lipopolyamine to phosphate in the polynucleotide, multi ci stronic mRNA vector, or DNA plasmid vector is from about 1:10 to about 10:1. [0352] In some aspects, the composition, pharmaceutical composition, or vaccine comprises about 0.1 mg/ml to about 10.0 mg/ml (e.g., about 0.1 mg/ml to about 5 mg/ml; about 0.5 mg/ml to about 10 mg/ml; or about 0.5 mg/ml to about 5 mg/ml) nucleic acid complexed with the lipopolyamine or derivative thereof. In some aspects, the composition, pharmaceutical composition, or vaccine comprises about 1 mg/ml to about 10 mg/ml (e.g., about 1 mg/ml to about 6 mg/ml; about 2 mg/ml to about 6 mg/ml; about 5 mg/ml to about 10 mg/ml; or about 6 mg/ml to about 10 mg/ml) nucleic acid complexed with the lipopolyamine or derivative thereof.
[0353] In some aspects, the polynucleotide, vector, multicistronic mRNA vector, or DNA plasmid vector of the disclosure is complexed with or encapsulated by a delivery component, e.g., a lipopolymer. In some aspects, the polynucleotide, vector, multicistronic mRNA vector, or DNA plasmid vector is encapsulated by a delivery component, e.g., a lipopolymer. In some aspects, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the polynucleotide, vector, multicistronic mRNA vector, or DNA plasmid vector is encapsulated by the delivery component, e.g., a lipopolymer. In some aspects, about 20% to about 100%, about 20% to about 80%, about 20% to about 60%, or about 40% to about 80% of the polynucleotide, vector, multicistronic mRNA vector, or DNA plasmid vector of the disclosure is encapsulated by the delivery component, e.g., a lipopolymer. In some aspects, the delivery component forms a micellular structure.
VI.4 Poloxamers and Derivatives Thereof
[0354] In some aspects, the delivery component comprises a poloxamer back-bone having a metal chelator covalently coupled to at least one terminal end of the poloxamer backbone (e.g. Crown Poloxamer). In some aspects, the metal chelator is coupled to at least two terminal ends of the poloxamer backbone.
[0355] In some aspects, at least one metal chelator is coupled to a poloxamer. In some aspects, at least two metal chelators are coupled to a poloxamer. In some aspects, 1-5, 5- 10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 50-55, 55-60, 60-65, 65-70, 70-75, 75-80, 80-85, 85-90, 90-95, or 95-100 metal chelators are coupled to a poloxamer.
[0356] In some aspects, the poloxamer backbone is a poloxamer backbone disclosed in U.S. Publ. No. 2010/0004313, which is herein incorporated by reference in its entirety. In some aspects, the metal chelator is a metal chelator disclosed in U.S. Publ. No. 2010/0004313. In some aspects, the delivery component of the composition, pharmaceutical composition, or vaccine comprises a polymer having the following formula:
Figure imgf000095_0001
or pharmaceutically acceptable salts thereof, wherein:
A represents an integer from 2 to 141;
B represents an integer from 16 to 67;
C represents an integer from 2 to 141;
R4 and Rc are the same or different, and are R'-L- or H, wherein at least one of R4 and Rc is R'-L-;
L is a bond, — CO — , — CH2 — O — , or — O — CO — ; and
R' is a metal chelator.
[0357] In some aspects, the metal chelator is R? NH — , R.SN — , or (R" — (N(R") — CILCIL h — N — CH2CO — , wherein each x is independently 0-2, and wherein R" is HO2C— CH2— .
[0358] In some aspects, the delivery component comprises a poloxamer with the following formula:
Figure imgf000095_0002
or a pharmaceutically acceptable salt thereof, wherein:
A represents an integer from 2 to 141;
B represents an integer from 16 to 67;
C represents an integer from 2 to 141;
RA and RC are the same or different, and are R'-L- or H;
L is a bond, — CO — , — CH2 — O — , or — O — CO — ; and
R' is a metal chelator.
[0359] In some aspects, at least one of RA and RC is R'-L-.
[0360] In some aspects, the R’ is covalently bound to the poloxamer (e.g., where a metal chelator (e.g., a crown) is covalently attached at different density to the poloxamer). [0361] In some aspects, one metal chelator or two or more metal chelators is/are bound to the poloxamer.
[0362] In some aspects, 2-100 metal chelators are bound to the poloxamer.
[0363] In some aspects, the metal chelator is a crown ether selected from the group consisting of 12-crown-4, 15-crown-5, 18-crown-6, 20-crown-6, 21-crown-7, and 24- crown-8. In some aspects, the crown ether is a substituted-crown ether, wherein the substituted-crown ether has:
(1) one or more of the crown ether oxygens independently replaced by NH or S,
(2) one or more of the crown ether — CH2 — CH2 — moieties replaced by — C6H4— , — CioHe— , or — CeHio— ,
(3) one or more of the crown ether — CH2 — O — CH2 — moieties replaced by — C4H2O— or — C5H3N— , or
(4) any combination thereof.
[0364] In some aspects, the metal chelator is a cryptand, wherein the cryptand is selected from the group consisting of (1,2,2) cryptand, (2,2,2) cryptand, (2,2,3) cryptand, and (2,3,3) cryptand. In some aspects, the cryptand is a substituted-cryptand, wherein the substituted cryptand has:
(1) one or more of the crypthand ether oxygens independently replaced by NH or S,
(2) one or more of the crown ether — CH2 — CH2 — moieties replaced by — C6H4— , — CioHe— , or — CeHio— ,
(3) one or more of the crown ether — CH2 — O — CH2 — moieties replaced by — C4H2O— or — C5H3N— , or
(4) any combination thereof.
[0365] In some aspects, the delivery component is Crown Poloxamer (aza-crown-linked poloxamer), wherein the Crown Poloxamer comprises a polymer having the following formula:
Figure imgf000097_0001
or pharmaceutically acceptable salts thereof, wherein: a represents an integer of about 10 units; and b represents an integer of about 21 units; and wherein the total molecular weight of the polymer is about 2,000 Da to about 2,200 Da.
[0366] In some aspects, the crown poloxamer can be derivatized with a cationic molecule, a ligand, or other chemical entities.
[0367] In some aspects, the polymer or poloxamer (e.g., crown poloxamer)is present in a solution with the polynucleotide, multi ci str onic mRNA vector, or DNA plasmid vector from about 0.01% - about 5% or about 0.5% - about 5%.
[0368] In some aspects, the solution is co-formulated with a metal chelator (e.g., where the co-formulated metal chelator is a free metal chelator, which is formulated with the poloxamer (e.g., a non-crown poloxamer)).
[0369] In some aspects, the co-formulated metal chelator is present in the solution at a concentration of about O.lmg/mL to about 20mg/mL.
[0370] In some aspects, the co-formulated metal chelator is crown ether, a substituted- crown ether, a cryptand, or a substituted-cryptand.
[0371] In some aspects, the metal chelator or co-formulated metal chelator is crown ether (Aza- 18 -crown-6) .
[0372] In some aspects, the delivery component further comprises benzalkonium chloride (BAK).
[0373] In some aspects, the delivery component comprises BD15-12. In some aspects, the ratio of nucleotide to BD15-12 polymer (N:P) is 0.1 : 1 to 5: 1.
[0374] In some aspects, the delivery component comprises Omnifect. In some aspects, the ratio of nucleotide to Omnifect polymer (N:P) is 0.1 : 1 to5: l. [0375] In some aspects, the delivery component comprises Crown Poloxamer (azacrown-linked poloxamer). In some aspects, the ratio of nucleotide to Crown Poloxamer (N:P) is 0.1 : 1 to 5: 1. In some aspects, the delivery component comprises Crown Poloxamer and/or a PEG-PEI-cholesterol (PPC) lipopolymer. In some aspects, the delivery component comprises Crown Poloxamer and/or benzalkonium chloride. In some aspects, the delivery component comprises Crown Poloxamer and/or Omnifect. In some aspects, the delivery component comprises Crown Poloxamer and/or a linear polyethyleneimine (LPEI). In some aspects, the delivery component comprises Crown Poloxamer and/or BD15-12.
[0376] In some aspects, the delivery component comprises Staramine and/or mPEG modified Staramine. In some aspects, the mPEG modified Staramine is Staramine- mPEG515. In some aspects, the mPEG modified Staramine is Staramine-mPEGl 1. In some aspects, the ratio of Staramine to mPEG modified Staramine is 10: 1. In some aspects, the nucleotide to polymer (N:P) ratio is 0.1 : 1 to 5: 1. In some aspects, the delivery component comprises Staramine, mPEG modified Staramine, and/or Crown Poloxamer. In some aspects, the delivery component comprises Staramine, Staramine-mPEG515, and/or Crown Poloxamer. In some aspects, the delivery component comprises a Staramine, a Staramine-mPEGl 1, and/or a Crown Poloxamer.
[0377] In some aspects, the metal chelator is added directly to a solution comprising (i) the polynucleotide, multi ci stronic mRNA vector, or DNA plasmid vector as described herein, and (ii) a poloxamer. In some aspects, the metal chelator is present in the solution at concentration of about O. lmg/mL to about 20mg/mL. In some aspects, metal chelator is present in the solution at concentration of about O.lmg/mL to about 20mg/mL, about O. lmg/mL to about Img/mL, about Img/mL to about 5mg/mL, about 5mg/mL to about lOmg/mL, about lOmg/mL to about 15mg/mL, or about 15mg/mL to about 20mg/mL. In some aspects, the metal chelator is crown ether, a substituted-crown ether, a cryptand, or a substituted-cryptand. In some aspects, the metal chelator is crown ether (Aza-18-crown- 6).
[0378] In some aspects, the metal chelator is added directly to a solution comprising (i) the polynucleotide, multi ci stronic mRNA vector, or DNA plasmid vector as described herein, (ii) a poloxamer, and (iii) an an adjuvant comprising an aluminum or aluminum- salt based adjuvant, a stimulator of interferon genes (STING) agonist, or a combination thereof. In some aspects, the metal chelator is present in the solution at concentration of about O.lmg/mL to about 20mg/mL. In some aspects, metal chelator is present in the solution at concentration of about O. lmg/mL to about 20mg/mL, about O.lmg/mL to about Img/mL, about Img/mL to about 5mg/mL, about 5mg/mL to about lOmg/mL, about lOmg/mL to about 15mg/mL, or about 15mg/mL to about 20mg/mL. In some aspects, the metal chelator is crown ether, a substituted-crown ether, a cryptand, or a substituted-cryptand. In some aspects, the metal chelator is crown ether (Aza-18-crown- 6). In some aspects, the adjuvant is AIPO4.
[0379] In some aspects, the metal chelator is added directly to a solution comprising (i) the polynucleotide, multi ci stronic mRNA vector, or DNA plasmid vector as described herein, (ii) a poloxamer, and (iii) an an adjuvant comprising an aluminum or aluminum- salt based adjuvant, or a combination thereof. In some aspects, the metal chelator is present in the solution at concentration of about O.lmg/mL to about 20mg/mL. In some aspects, metal chelator is present in the solution at concentration of about 0. Img/mL to about 20mg/mL, about O. lmg/mL to about Img/mL, about Img/mL to about 5mg/mL, about 5mg/mL to about lOmg/mL, about lOmg/mL to about 15mg/mL, or about 15mg/mL to about 20mg/mL. In some aspects, the metal chelator is crown ether, a substituted-crown ether, a cryptand, or a substituted-cryptand. In some aspects, the metal chelator is crown ether (Aza-18-crown-6). n some aspects, the adjuvant is AIPO4.
VII. Kits and Containers
[0380] The present disclosure also features containers comprising any polynucleotide, vector, vaccine, composition, or pharmaceutical composition described and exemplified herein. In some aspects, the container is a glass vial.
[0381] The present disclosure also features kits comprising any polynucleotide, vector, vaccine, composition, or pharmaceutical composition described and exemplified herein. The kits can be used to supply polynucleotides, vectors, vaccines, composition, pharmaceutical compositions, and other agents for use in diagnostic, basic research, or therapeutic methods, among others. In some aspects, the kits comprise any one or more of the polynucleotides, vectors, vaccines, composition, or pharmaceutical compositions described or exemplified herein and instructions for using the one or more polynucleotides, vectors, vaccines, composition, or pharmaceutical compositions in a method for inducing an immune response in a subject. In some aspects, the kits comprise any one or more of the polynucleotides, vectors, vaccines, composition, or pharmaceutical compositions described or exemplified herein and instructions for using the one or more polynucleotides, vectors, vaccines, composition, or pharmaceutical compositions in a method for preventing, reducing the growth of, attenuating or treating a tumor in a subject.
[0382] In some aspects, the kit comprises a composition, pharmaceutical composition, or vaccine that is liquid and is stable at 0°C to 5°C for at least about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 monts, or about 12 months. In some aspects, the liquid composition, pharmaceutical composition, or vaccine is stable at 25°C for at least about 7 days, about 10 day, or about 14 days, or about 60 days.
[0383] In some aspects, the kit comprises a composition, pharmaceutical composition, or vaccine that is liquid and is stable at -20°C for at least about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 monts, or about 12 months.
[0384] In some aspects, the kit comprises a composition, pharmaceutical composition, or vaccine that is lyophilized and is stable at 0°C to 5°C for at least about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 monts, or about 12 months. In some aspects, the lyophilized and reconstituted composition, pharmaceutical composition, or vaccine is stable at 25°C for at least about 7 days, about 10 day, or about 14 days, or about 60 days.
[0385] In some aspects, the kit comprises a composition, pharmaceutical composition, or vaccine that is lyophilized and is stable at -20°C for at least about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 monts, or about 12 months.
[0386] In some aspects, the lyophilized composition is substantially free of aqueous components. In some aspects, the kit further comprises a dilent and the composition is reconstituted with the diluent.In some aspects, the diluent is water. VIII. Methods of Using the Compositions, Vaccines, and Vectors
[0387] The present disclosure also features methods of inducing an immune response in a subject, the method comprising administering an effective amount of any polynucleotide, vector, multi ci str onic mRNA vector, DNA plasmid vector, composition, or pharmaceutical composition described or exemplified herein to the subject. In some aspects, the immune response is to one or more tumor-associated antigens disclosed herein, e.g., NYESO-1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A9, MAGE-A10, MAGE-CI, MAGE-C2, SSX, H0RMAD1, CXorf61, ACTL8, AKAP3,AKAP4, HOM-TES-85, OY-TES-1, HSP70-2, LAGE-1, PIWL1, PIWL2, PIWL3, PIWL4, PLU-1, PLAC1, SPAG9, SCP-1, TAG-1, TAG-2a, TAG-2b, TAG-2c, TRAG-3, KAP4, BAGE, BORIS, CT45, GAGE-1/2, PRAME, PAGE4, SP17, PASD1, p53, ras, B-RafHER-2/neu, mucin 1 (MUC1), epidermal growth factor receptor (EGFR), GP100, prostatic acid phosphatase, prostate-specific membrane antigen (PSMA), mammoglobin-A, carcinoembryonic antigen (CEA), papillomavirus antigens E6ZE7, heatshock proteins, alpha fetoprotein, CDK, P-catenin, mesothelin, or cancer antigen- 125 (CA125)).
[0388] The present disclosure also features methods of preventing, reducing the incidence of, attenuating or treating a tumor (e.g., an ovarian cancer, melanoma, colorectal cancer, liver cancer, pancreatic cancer, brain cancer, prostate cancer, bladder cancer, renal cancer, or hematological cancer).
[0389] tumor) in a subject, the method comprising administering an effective amount of any polynucleotide, vector, multi ci str onic mRNA vector, DNA plasmid vector, composition, or pharmaceutical composition described or exemplified herein to the subject.
[0390] In some aspects, the methods of the disclosure are directed to administration of a composition, pharmaceutical composition, or vaccine disclosed herein.
[0391] The process of administration can be varied, depending on the agent, or agents, and the desired effect. Thus, the process of administration involves administering a therapeutic agent (e.g., any composition or pharmaceutical composition, polynucleotide, vector, vaccine, disclosed herein) to a patient in need of such treatment. Methods of delivering compositions comprising DNA vaccines are described in U.S. Patent Nos. 4,945,050 and 5,036,006. [0392] Administration can be accomplished by any means appropriate for the therapeutic agent, for example, by parenteral means of delivery. Parenteral delivery can include for example, subcutaneous, intravenous, intramuscular, intra-arterial, intraperitoneal, intralymphatic, and injection into the tissue of an organ (e.g., intratumoral). Administration can generally also include delivery with a pharmaceutically acceptable carrier, such as, for example, a buffer, a polypeptide, a peptide, a polysaccharide conjugate, a liposome, and/or a lipid, according to methods known in the art.
[0393] Pulmonary delivery can include inhalation of the agent. Catheter-based delivery can include delivery by iontr opheretic catheter-based delivery. Oral delivery can include delivery of a coated pill, or administration of a liquid by mouth. Administration can generally also include delivery with a pharmaceutically acceptable carrier, such as, for example, a buffer, a polypeptide, a peptide, a polysaccharide conjugate, a liposome, and/or a lipid, according to methods known in the art.
[0394] In some aspects, the viral vectors of the present disclosure are administered in an amount approximately corresponding to 102 to 1014 PFU, 105 to 1012 PFU, or 106 to IO10 PFU per subject, calculated as the PFU of the viral vector. In some aspects, the viral vectors of the present disclosure are administered by directly injecting a viral vector suspension prepared by suspending the viral vector in PBS (phosphate buffered saline) or saline into a local site (e.g., into the lung tissue, liver, muscle or brain), by nasal or respiratory inhalation, or by intravascular (e.g., intra-arterial, intravenous, and portal venous), intralymphatic, subcutaneous, intracutaneous, intradermal, or intraperitoneal administration.
[0395] Certain aspects of the disclosure relate to method of administering to a subject a composition comprising (i) a vector (e.g., a multi ci stronic DNA plasmid vector or a multi ci str onic messenger RNA (mRNA) vector) comprising a nucleic acid sequence encoding one or more tumor-associated antigens (e.g., NYESO-1, MAGE-A1, MAGE- A2, MAGE-A3, MAGE-A4, MAGE-A9, MAGE-A10, MAGE-CI, MAGE-C2, SSX, H0RMAD1, CXorf61, ACTL8, AKAP3,AKAP4, HOM-TES-85, OY-TES-1, HSP70-2, LAGE-1, PIWL1, PIWL2, PIWL3, PIWL4, PLU-1, PLAC1, SPAG9, SCP-1, TAG-1, TAG-2a, TAG-2b, TAG-2c, TRAG-3, KAP4, BAGE, BORIS, CT45, GAGE-1/2, PRAME, PAGE4, SP17, PASD1, p53, ras, B-RafHER-2/neu, mucin 1 (MUC1), epidermal growth factor receptor (EGFR), GP100, prostatic acid phosphatase, prostatespecific membrane antigen (PSMA), mammoglobin-A, carcinoembryonic antigen (CEA), papillomavirus antigens E6ZE7, heat-shock proteins, alpha fetoprotein, CDK, P-catenin, mesothelin, or cancer antigen-125 (CA125)); (ii) a delivery component (e.g., a cationic polymer, a poly-inosinic-polycytidylic acid, or a poloxamer); and (iii) an adjuvant comprising an aluminum or aluminum-salt based adjuvant, a stimulator of interferon genes (STING) agonist, or a combination thereof.
[0396] In some aspects, the method further comprises obtaining a nucleic acid sequence of a subject’s tumor-associated antigens (e.g., NYESO-1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A9, MAGE-A10, MAGE-CI, MAGE-C2, SSX, H0RMAD1, CXorf61, ACTL8, AKAP3,AKAP4, HOM-TES-85, OY-TES-1, HSP70-2, LAGE-1, PIWL1, PIWL2, PIWL3, PIWL4, PLU-1, PLAC1, SPAG9, SCP-1, TAG-1, TAG-2a, TAG-2b, TAG-2c, TRAG-3, KAP4, BAGE, BORIS, CT45, GAGE-1/2, PRAME, PAGE4, SP17, PASD1, p53, ras, B-RafHER-2/neu, mucin 1 (MUC1), epidermal growth factor receptor (EGFR), GP100, prostatic acid phosphatase, prostatespecific membrane antigen (PSMA), mammoglobin-A, carcinoembryonic antigen (CEA), papillomavirus antigens E6ZE7, heat-shock proteins, alpha fetoprotein, CDK, P-catenin, mesothelin, or cancer antigen-125 (CA125)) and including the patient’s own nucleic acid sequences that encode for a tumor-associated antigen (e.g., NYESO-1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A9, MAGE-A10, MAGE-CI, MAGE-C2, SSX, H0RMAD1, CXorf61, ACTL8, AKAP3,AKAP4, HOM-TES-85, OY-TES-1, HSP70-2, LAGE-1, PIWL1, PIWL2, PIWL3, PIWL4, PLU-1, PLAC1, SPAG9, SCP-1, TAG-1, TAG-2a, TAG-2b, TAG-2c, TRAG-3, KAP4, BAGE, BORIS, CT45, GAGE-1/2, PRAME, PAGE4, SP17, PASD1, p53, ras, B-RafHER-2/neu, mucin 1 (MUC1), epidermal growth factor receptor (EGFR), GP100, prostatic acid phosphatase, prostatespecific membrane antigen (PSMA), mammoglobin-A, carcinoembryonic antigen (CEA), papillomavirus antigens E6ZE7, heat-shock proteins, alpha fetoprotein, CDK, P-catenin, mesothelin, or cancer antigen-125 (CA125) into the vector of the composition.
[0397] In some aspects, the adjuvant comprises an aluminum or aluminum-salt based adjuvant and a STING agonist. In some aspects, the aluminum or aluminum-salt based adjuvant comprises an aluminum phosphate, an aluminum hydroxide, an aluminum oxyhydroxide, or any combination thereof. In some aspects, the STING agonist is cyclic guanosine monophosphate (cGMP), cyclic adenosine monophosphate (cAMP), or cyclic guanosine monophosphate-ad enosine monophosphate (cGAMP). In some aspects, the adjuvant is M59 (oil-in-water emulsion of squalene oil). In some aspects, the adjuvant is AS03 (a-tocopherol, squalenem and polysorbate 80 in an oil-in-water emulsion).
[0398] In some aspects, the vector further comprises a nucleic acid sequence encoding one or more immune modifier proteins. In some aspects, the vector comprises a nucleic acid sequence encoding a tumor-associated antigen and, optionally, a second tumor- associated antigen. Some aspects relate to methods of eliciting humoral and/or cellular immune response against a tumor following in vivo administration of a vector or composition of the disclosure.
[0399] Some aspects relate to methods of eliciting humoral and/or cellular immune response against a tumor following in vivo administration of a multicistronic DNA plasmid, a multicistronic mRNA vector, or a composition comprising the same of the disclosure.
[0400] Certain aspects of the disclosure relate to administering compositions including (i) a DNA plasmid vector comprising a DNA sequence of one or more antigens; (ii) a delivery component, such as a synthetic non-viral DNA carrier (e.g., a cationic polymer, a poly-inosinic-polycytidylic acid, or a poloxamer); and (iii) an adjuvant comprising an aluminum or aluminum-salt based adjuvant, a stimulator of interferon genes (STING) agonist, or a combination thereof.
[0401] The delivery component of the compositions disclosed herein may comprise any combination of delivery components disclosed herein.
[0402] In some aspects, the tumor is an ovarian cancer, melanoma, colorectal cancer, liver cancer, pancreatic cancer, brain cancer, prostate cancer, bladder cancer, renal cancer, or hematological cancer.
[0403] In some aspects, the one or more antigens comprise one or more tumor-associated antigens. In some aspects the one or more tumor-associated antigens comprise two or more portions of the same antigen.
[0404] Certain aspects of the disclosure relate to administering compositions including (i) a multicistronic DNA plasmid vector comprising a DNA sequence of one or more tumor associated antigens and optionally a DNA sequence of one or more immune modifier proteins; (ii) a delivery component, such as a synthetic non-viral DNA carrier (e.g., a cationic polymer, a poly-inosinic-polycytidylic acid, or a poloxamer); and (iii) an adjuvant comprising an aluminum or aluminum-salt based adjuvant, a stimulator of interferon genes (STING) agonist, or a combination thereof. In some aspects, the compositions further comprise one or more immunopotentiators that are capable of activating the innate immunity system. In some aspects, the immunopotentiators are selected from a non-coding DNA (e.g., concatamers of non-coding 5'-C-phosphate-G-3' (CpG) dinucleotides), a non-coding RNA, a small molecule, or any combination thereof.
[0405] Certain aspects of the disclosure relate to administering compositions including (i) a multi ci stronic RNA vector comprising an RNA sequence of one or more tumor associated antigens and opionally an RNA sequence of one or more immune modifier proteins; (ii) a delivery component, such as a synthetic non-viral RNA carrier/adjuvant (e.g., a cationic polymer, a poly-inosinic-poly cytidylic acid, or a pol oxamer); and (iii) an adjuvant comprising an aluminum or aluminum-salt based adjuvant, a stimulator of interferon genes (STING) agonist, or a combination thereof.
[0406] Some aspects relate to methods of eliciting humoral and/or cellular immune response against a tumor-associated antigen following in vivo administration of a multi ci stronic DNA plasmid vector, a multi ci stronic mRNA vector, or a composition comprising the same of the disclosure.
[0407] Certain aspects of the disclosure relate to administering a composition comprising a polynucleotide comprising: (a) an antigen nucleic acid (e.g., first antigen nucleic acid) which encodes a first tumor-associated antigen or an antigenic fragment thereof; and (b) a nucleic acid encoding an immune modifier protein. In some aspects, the polynucleotide is multi ci stronic (e.g., a multi ci stronic DNA plasmid or a multi ci stronic messenger RNA (mRNA)). In some aspects, the polynucleotide is combined with a delivery component, such as a synthetic non-viral carrier. In some aspects, the polynucleotide is combined with an adjuvant an adjuvant comprising an aluminum or aluminum-salt based adjuvant, a stimulator of interferon genes (STING) agonist, or a combination thereof.
[0408] In some aspects, provided herein is a polynucleotide comprising: (a) an antigen nucleic acid (e.g., first antigen nucleic acid) which encodes a tumor-associated antigen or an antigenic fragment thereof, wherein the antigen nucleic acid (e.g., first antigen nucleic acid) is operably linked to a first promoter; and (b) a nucleic acid encoding an immune modifier protein. In some aspects, the polynucleotide is a multi ci stronic DNA vector.
[0409] In some aspects, the first tumor-associated antigen is selected from the group consisting of: NYESO-1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A9, MAGE-A10, MAGE-CI, MAGE-C2, SSX, H0RMAD1, CXorf61, ACTL8, AKAP3,AKAP4, HOM-TES-85, OY-TES-1, HSP70-2, LAGE-1, PIWL1, PIWL2, PIWL3, PIWL4, PLU-1, PLAC1, SPAG9, SCP-1, TAG-1, TAG-2a, TAG-2b, TAG-2c, TRAG-3, KAP4, BAGE, BORIS, CT45, GAGE-1/2, PRAME, PAGE4, SP17, PASD1, p53, ras, B-RafHER-2/neu, mucin 1 (MUC1), epidermal growth factor receptor (EGFR), GP100, prostatic acid phosphatase, prostate-specific membrane antigen (PSMA), mammoglobin-A, carcinoembryonic antigen (CEA), papillomavirus antigens E6ZE7, heatshock proteins, alpha fetoprotein, CDK, P-catenin, mesothelin, or cancer antigen- 125 (CA125)) or antigenic fragments thereof.
[0410] In some aspects, the polynucleotide comprises two or more nucleic acids encoding an immune modifier protein. In some aspects, each of the nucleic acids encoding an immune modifier protein encodes a different immune modifier protein. In some aspects, the two or more of the nucleic acids encoding an immune modifier protein encode IL- 12 p35 and IL- 12 p40.
[0411] In some aspects, the polynucleotide further comprises: (c) at least one additional antigen nucleic acid (e.g., second antigen nucleic acid) which encodes a second tumor- associated antigen or an antigenic fragment thereof. In some aspects, the second tumor- associated antigen is selected from the group consisting of: NYESO-1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A9, MAGE-A10, MAGE-CI, MAGE-C2, SSX, H0RMAD1, CXorf61, ACTL8, AKAP3,AKAP4, HOM-TES-85, OY-TES-1, HSP70-2, LAGE-1, PIWL1, PIWL2, PIWL3, PIWL4, PLU-1, PLAC1, SPAG9, SCP-1, TAG-1, TAG-2a, TAG-2b, TAG-2c, TRAG-3, KAP4, BAGE, BORIS, CT45, GAGE-1/2, PRAME, PAGE4, SP17, PASD1, p53, ras, B-RafHER-2/neu, mucin 1 (MUC1), epidermal growth factor receptor (EGFR), GP100, prostatic acid phosphatase, prostatespecific membrane antigen (PSMA), mammoglobin-A, carcinoembryonic antigen (CEA), papillomavirus antigens E6ZE7, heat-shock proteins, alpha fetoprotein, CDK, P-catenin, mesothelin, or cancer antigen-125 (CA125)) or antigenic fragment thereof.
[0412] In some aspects, the polynucleotide further comprises: (b) or (c) at least one additional antigen nucleic acid (e.g., second antigen nucleic acid) which encodes a tumor- associated antigen or an antigenic fragment thereof. In some aspects, the tumor-associated antigen is selected from the group consisting of: NYESO-1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A9, MAGE-A10, MAGE-CI, MAGE-C2, SSX, H0RMAD1, CXorf61, ACTL8, AKAP3,AKAP4, HOM-TES-85, OY-TES-1, HSP70-2, LAGE-1, PIWL1, PIWL2, PIWL3, PIWL4, PLU-1, PLAC1, SPAG9, SCP-1, TAG-1, TAG-2a, TAG-2b, TAG-2c, TRAG-3, KAP4, BAGE, BORIS, CT45, GAGE-1/2, PRAME, PAGE4, SP17, PASD1, p53, ras, B-RafHER-2/neu, mucin 1 (MUC1), epidermal growth factor receptor (EGFR), GP100, prostatic acid phosphatase, prostatespecific membrane antigen (PSMA), mammoglobin-A, carcinoembryonic antigen (CEA), papillomavirus antigens E6ZE7, heat-shock proteins, alpha fetoprotein, CDK, P-catenin, mesothelin, cancer antigen-125 (CA125) or antigenic fragment thereof.
[0413] In some aspects, the at least one additional antigen nucleic acid (e.g., second antigen nucleic acid) is operably linked to the first promoter through an internal ribosome entry site (IRES) sequence. In some aspects, the IRES sequence comprises a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 19.
[0414] In some aspects, the polynucleotide comprises one or more second promoters. In some aspects, the at least one additional antigen nucleic acid (e.g., second antigen nucleic acid) is operably linked to the one or more second promoters. In some aspects, one or more nucleic acids encoding an immune modifier protein is operably linked to the one or more second promoters. In some aspects, one or more of the nucleic acids encoding an immune modifier protein is operably linked to the first promoter or the one or more second promoters through an internal ribosome entry site (IRES) sequence. In some aspects, the IRES sequence comprises a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 19.
[0415] In some aspects, the first promoter or the one or more second promoters is selected from the group consisting of: a cytomegalovirus (CMV) promoter (SEQ ID NO: 14), a Rouse sarcoma virus (RSV) promoter, a Moloney murine leukemia virus (Mo- MuLV) long terminal repeat (LTR) promoter, a mammalian elongation factor 1 (EFl) promoter, a cytokeratin 18 (CK18) promoter, a cytokeratin 19 (CK19) promoter, a simian virus 40 (SV40) promoter (SEQ ID NO: 15 or SEQ ID NO: 30), a murine U6 promoter, a skeletal a-actin promoter, a P-actin promoter, a murine phosphoglycerate kinase 1 (PGK1) promoter, a human PGK1 promoter, a CBA promoter, a CAG promoter (SEQ ID NO: 16), and any combination thereof. In some aspects, the mammalian EFl promoter is a hEFl-HTLV promoter (SEQ ID NO: 18). In some aspects, the one or more second promoters is the CMV promoter. [0416] In some aspects, each of the nucleic acids which encodes an immune modifier protein is under the control of a promoter selected from the group consisting of a CMV promoter, an RSV promoter, a Mo-MuLV LTR promoter, a mammalian EFl promoter, a CK18 promoter, a CK19 promoter, an SV40 promoter, a murine U6 promoter, a skeletal a-actin promoter, a P-actin promoter, a murine PGK1 promoter, a human PGK1 promoter, a CB A promoter, a CAG promoter, and any combination thereof. In some aspects, the mammalian EFl promoter is a hEFl-HTLV promoter.
[0417] In some aspects, each of the at least one additional antigen nucleic acid (e.g., second antigen nucleic acid)s is under the control of a promoter selected from the group consisting of a CMV promoter, an RSV promoter, a Mo-MuLV LTR promoter, a mammalian EFl promoter, a CK18 promoter, a CK19 promoter, an SV40 promoter, a murine U6 promoter, a skeletal a-actin promoter, a P-actin promoter, a murine PGK1 promoter, a human PGK1 promoter, a CBA promoter, a CAG promoter, and any combination thereof. In some aspects, the mammalian EFl promoter is a hEFl-HTLV promoter.
[0418] In some aspects, the immune modifier protein is selected from the group consisting of: interleukin (IL) 2 (IL-2), IL-12 p35, IL-12 p40, IL-12 p70, IL-15, IL-18, tumor necrosis factor alpha (TNFa), granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon (IFN) a (IFN-a), IFN-P, a chemokine, major histocompatibility complex (MHC) class I (MHC I), MHC class II (MHC II), human leukocyte antigen (HLA)-DR isotype (HLA-DR), CD80, CD86, and any combination thereof. In some aspects, the chemokine is selected from the group consisting of: C-C motif chemokine ligand (CCL) 3 (CCL3), CCL4, CCL5, CCL21, CCL28, C-X-C motif chemokine ligand (CXCL) 10 (CXCL10), and any combination thereof.
[0419] In some aspects, the immune modifier protein comprises one or more concatamers of non-coding 5'-C-phosphate-G-3' (CpG) dinucleotides. In some aspects, the one or more concatamers of non-coding CpG dinucleotides activate the Toll-like receptor 9 (TLR9) signaling pathway. In some aspects, the one or more concatamers of non-coding CpG dinucleotides comprise one or more concatamers of non-coding CpG dinucleotides previously reported in Bauer, A. et al., Nucleic Acids Research 38( 12): 3891 -908 (2010); Cornelie, S. et al., Journal of Biological Chemistry 279(15): 15124-9 (2004); Klinman, D. et al., J Immunol. 158(8):3635-9 (1997); Klinman, D. et al., Immunological Reviews 199(l):201-16 (2004); Luo, Z. et al., Mol Med Rep. 6(6): 1309-14 (2012); Bode, C. et al., Expert Rev Vaccines 10(4):499-511 (2011); and Kuo, T. et al., Scientific Reports 10:20085 (2020), each of which is incorporated by reference herein in its entirety.
[0420] In some aspects, the nucleic acid encoding an immune modifier protein comprises a combination (i) a nucleic acid encoding an interleukin, and (ii) a nucleic acid encoding a major histocompatibility complex and/or a chemokine.
[0421] In some aspects, the nucleic acid encoding an immune modifier protein comprises a nucleic acid encoding IL-12, a nucleic acid encoding IL-15, or the combination thereof.
[0422] In some aspects, the nucleic acid encoding an immune modifier protein comprises a combination of a nucleic acid encoding IL- 12 and a nucleic acid encoding IL-15.
[0423] In some aspects, the nucleic acid encoding an immune modifier protein comprises a nucleic acid encoding IL-2, a nucleic acid encoding IL- 15, a nucleic acid encoding MHC I, a nucleic acid encoding MHC II, a nucleic acid encoding CCL3, a nucleic acid encoding CCL4, or any combination thereof.
[0424] In some aspects, the nucleic acid encoding an immune modifier protein comprises a nucleic acid encoding MHC I, a nucleic acid encoding MHC II, a nucleic acid encoding CCL3, a nucleic acid encoding CCL4, or any combination thereof.
[0425] In some aspects, the nucleic acid encoding an immune modifier protein comprises a nucleic acid encoding CCL3, a nucleic acid encoding CCL4, or the combination thereof.
[0426] In some aspects, the polynucleotides or nucleic acids can comprise DNA or mRNA sequences.
[0427] In some aspects, the polynucleotide further comprises one or more post- transcriptional regulatory elements. In some aspects, the post-transcriptional regulatory element is a wood chuck hepatitis virus post-transcriptional regulatory element (WPRE).
[0428] In some aspects, the polynucleotide further comprises at least one 3' UTR poly(a) tail sequence operably linked to the antigen nucleic acid (e.g., first antigen nucleic acid), the at least one additional antigen nucleic acid (e.g., second antigen nucleic acid), the nucleic acid encoding an immune modifier protein, or any combination thereof. In some aspects, the 3' UTR poly(a) tail sequence is a 3' UTR SV40 poly(a) tail sequence (SEQ ID NO: 13), a 3' UTR bovine growth hormone (bGH) poly(A) sequence (SEQ ID NO: 13), a 3' UTR actin poly(A) tail sequence, a 3' UTR hemoglobin poly(A) sequence, or any combinations thereof.
[0429] In some aspects, the polynucleotide further comprises at least one enhancer sequence. In some aspects, the enhancer sequence is a human actin enhancer sequence, a human myosin enhancer sequence, a human hemoglobin enhancer sequence, a human muscle creatine enhancer sequence, a viral enhancer sequence, or a polynucleotide function enhancer sequence. In some aspects, the enhancer sequence is a CMV intronic sequence or a P-actin intronic sequence. In aspects, the enhancer sequence is a SV40 enhancer sequence (SEQ ID NO: 18).
[0430] In some aspects, the polynucleotide is a multi ci str onic mRNA comprising a 5' cap and a 3' UTR poly(A) tail sequence. In some aspects, the 3' UTR poly(a) tail sequence is a 3' UTR SV40 poly(a) tail sequence (SEQ ID NO: 13), a 3' UTR bovine growth hormone (bGH) poly(A) sequence (SEQ ID NO: 13), a 3' UTR actin poly(A) tail sequence, a 3' UTR hemoglobin poly(A) sequence, or any combinations thereof. In some aspects, the multicistronic mRNA comprises a 5' UTR and/or a 3' UTR.
[0431] Also provided herein is a vector comprising any polynucleotide described or exemplified herein, wherein the vector is a DNA plasmid, a multicistronic mRNA, a viral vector, a bacterial vector, a cosmid, or an artificial chromosome.
[0432] In some aspects, the composition, pharmaceutical composition, or vaccine further comprises a second polynucleotide encoding at least one immune modifier protein selected from the group consisting of: IL-2, IL-12 p35, IL-12 p40, IL-12 p70, IL-15, IL- 18, TNFa, GM-CSF, IFN-a, IFN-p, a chemokine, MHC I, MHC II, HLA-DR, CD80, and CD86, wherein the polynucleotide encoding the at least one immune modifier protein is operably linked to a promoter.In some aspects, the composition, pharmaceutical composition, or vaccine further comprises a delivery component.
[0433] Also provided herein is a vector (e.g., a plasmid vector) comprising any polynucleotide (e.g., DNA or mRNA) described or exemplified herein. In some aspects, the plasmid vector is a DNA plasmid vector.
[0434] Also provided herein is a composition, pharmaceutical composition, or vaccine comprising any polynucleotide, vector, multicistronic mRNA vector, or DNA plasmid vector described or exemplified herein.
[0435] Also provided herein are methods of administering a composition, pharmaceutical composition, or vaccine comprising: (a) a polynucleotide or a vector comprising the polynucleotide, and (b) a delivery component, wherein the polynucleotide comprises a antigen nucleic acid (e.g., first antigen nucleic acid) which encodes a first tumor- associated antigen or an antigenic fragment thereof, wherein the antigen nucleic acid (e.g., first antigen nucleic acid) is operably linked to a first promoter, optionally wherein the delivery component is a cationic polymer, a poly-inosinic-polycytidylic acid, or a poloxamer or derivative thereof. In some aspects, the delivery component is a cationic polymer, a poly-inosinic-polycytidylic acid, or a poloxamer or derivative thereof. In some aspects, the antigen nucleic acid (e.g., first antigen nucleic acid) which encodes a first tumor-associated antigen is selected from the group consisting of NYESO-1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A9, MAGE-A10, MAGE-CI, MAGE-C2, SSX, H0RMAD1, CXorf61, ACTL8, AKAP3,AKAP4, HOM-TES-85, OY-TES-1, HSP70-2, LAGE-1, PIWL1, PIWL2, PIWL3, PIWL4, PLU-1, PLAC1, SPAG9, SCP-1, TAG-1, TAG-2a, TAG-2b, TAG-2c, TRAG-3, KAP4, BAGE, BORIS, CT45, GAGE-1/2, PRAME, PAGE4, SP17, PASD1, p53, ras, B-RafHER-2/neu, mucin 1 (MUC1), epidermal growth factor receptor (EGFR), GP100, prostatic acid phosphatase, prostatespecific membrane antigen (PSMA), mammoglobin-A, carcinoembryonic antigen (CEA), papillomavirus antigens E6ZE7, heat-shock proteins, alpha fetoprotein, CDK, P-catenin, mesothelin, or cancer antigen-125 (CA125)), and any antigenic fragment thereof. In some aspects, the delivery component further comprises benzalkonium chloride.
[0436] The delivery component of the compositions, pharmaceutical compositions, or vaccines may comprise any combination of delivery components disclosed herein.
[0437] In some aspects, the polynucleotide further comprises at least one additional antigen nucleic acid (e.g., second antigen nucleic acid) which encodes a second tumor- associated antigen or an antigenic fragment thereof. In some aspects, the at least one additional antigen nucleic acid (e.g., second antigen nucleic acid) which encodes a second tumor-associated antigen is selected from the group consisting of a tumor-associated antigen and any antigenic fragment thereof.
[0438] In some aspects, the delivery component of the composition, pharmaceutical composition, or vaccine is a cationic polymer. In some aspects, the cationic polymer is a synthetic functionalized polymer, a lipid, a lipopolymer, or a chemical derivative thereof. In some aspects, the synthetic functionalized polymer is a biodegradable cross-linked cationic multi-block copolymer.
[0439] In some aspects, the biodegradable cross-linked cationic multi-block copolymer is represented by the formula: (CP)xLyYz, wherein: (a) CP represents a cationic polymer containing at least one secondary amine group, wherein the cationic polymer has a number averaged molecular weight within the range of 1,000 to 25,000 Dalton; (b) Y represents a bifunctional biodegradable linker containing ester, amide, disulfide, or - I l l - phosphate linages; (c) L represents a ligand; (d) x is an integer in the range from 1 to 20; (e) y is an integer in the range from 0 to 100; and (f) z is an integer in the range from 0 to 40. In some aspects, the cationic polymer comprises linear polyethyleneimine (LPEI). In some aspects, the cationic polymer comprising linear polyethyleneimine (LPEI) is BD15K-12, wherein the PEIs are approximately 15,000 Da, and wherein there is an average of 12 crosslinkers per PEI. In some aspects, the bifunctional biodegradable linker is hydrophilic and comprises a biodegradable linkage comprising a disulfide bond. In some aspects, the bifunctional biodegradable linker is a dithiodipropionyl linker.
[0440] In some aspects, the biodegradable cross-linked cationic multi-block copolymer comprises LPEI and a dithiodipropionyl linker for cross-linking the multi-block copolymer, wherein the LPEI has an average molecular weight of 1,000 to 25,000 Dalton. In some aspects, the biodegradable cross-linked cationic multi-block copolymer is covalently linked to at least one ligand.
[0441] In some aspects, the ligand is a targeting ligand selected from the group consisting of: a sugar moiety, a polypeptide, folate, and an antigen. In some aspects, the sugar moiety is a monosaccharide. In some aspects, the monosaccharide is galactose. In some aspects, the sugar moiety is an oligosaccharide. In some aspects, the polypeptide is a glycoprotein, an antibody, an antibody fragment, a cell receptor, a cytokine receptor, or a growth factor receptor. In some aspects, the growth factor receptor is an epidermal growth factor receptor. In some aspects, the glycoprotein is transferrin or asialoorosomucoid (ASOR).
[0442] In some aspects, the biodegradable cross-linked cationic multi-block copolymer is covalently linked to polyethylene glycol (PEG) of molecular weight ranging from 500 to 20,000 Dalton. In some aspects, the biodegradable cross-linked cationic multi-block copolymer is covalently linked to a fatty acyl chain selected from the group consisting of: oleic acid, palmitic acid, and stearic acid. In some aspects, the biodegradable cross-linked cationic multi-block copolymer comprises at least one amine group that is electrostatically attracted to a polyanionic compound. In some aspects, the polyanionic compound is a nucleic acid, wherein the biodegradable cross-linked cationic multi -block copolymer condenses the nucleic acid to form a compact structure.
[0443] In some aspects, the delivery component of the composition, pharmaceutical composition, or vaccine is a cationic lipopolymer comprising a PEI backbone covalently linked to a lipid or a PEG. In some aspects, the PEI backbone is covalently linked to a lipid and a PEG. In some aspects, the lipid and the PEG are directly attached to the PEI backbone by covalent bonds. In some aspects, the lipid is attached to the PEI backbone through a PEG spacer. In some aspects, the PEG has a molecular weight of between 50 to 20,000 Dalton. In some aspects, the molar ratio of PEG to PEI is within a range of 0.1 : 1 to 500: 1. In some aspects, the molar ratio of the lipid to the PEI is within a range of 0.1 : 1 to 500: 1. In some aspects, the lipid is a cholesterol, a cholesterol derivative, a C12 to C18 fatty acid, or a fatty acid derivative. In some aspects, the PEI is covalently linked to cholesterol and PEG, and wherein the average PEG:PEI: cholesterol molar ratio in the cationic lipopolymer is within the range of 1-5 PEG: 1 PEI:0.4-1.5 cholesterol. In some aspects, the PEI has a linear or branch configuration with a molecular weight of 100 to 500,000 Dalton.
[0444] In some aspects, the cationic lipopolymer further comprises a pendant functional moiety selected from the group consisting of: a receptor ligand, a membrane permeating agent, an endosomolytic agent, a nuclear localization sequence, and a pH sensitive endosomolytic peptide.
[0445] In some aspects, the cationic lipopolymer further comprises a targeting ligand, wherein the targeting ligand is directly attached to the PEI backbone or is attached through a PEG linker. In some aspects, the targeting ligand is selected from the group consisting of: a sugar moiety, a polypeptide, folate, and an antigen. In some aspects, the sugar moiety is a monosaccharide. In some aspects, the monosaccharide is galactose. In some aspects, the sugar moiety is an oligosaccharide. In some aspects, the polypeptide is a glycoprotein, an antibody, an antibody fragment, a cell receptor, a cytokine receptor, or a growth factor receptor. In some aspects, the growth factor receptor is an epidermal growth factor receptor. In some aspects, the glycoprotein is transferrin or asialoorosomucoid (ASOR).
[0446] In some aspects, the cationic polymer of the composition, pharmaceutical composition, or vaccine is present in an amount sufficient to produce a ratio of amine nitrogen in the cationic polymer to phosphate in the polynucleotide, multicistronic mRNA vector, or DNA plasmid vector from about 0.01 : 1 to about 50: 1 (e.g., about 0.01 : 1 to about 40:1; about 0.01 : 1 to about 30:1; about 0.01 : 1 to about 20: 1; about 0.01 : 1 to about 10: 1, or about 0.01 : 1 to about 5: 1). In some aspects, the ratio of amine nitrogen in the cationic polymer to phosphate in the polynucletide, multicistronic mRNA vector, or DNA plasmid vector is from about 0.1 : 1 to about 50: 1 (e.g., about 0.1 : 1 to about 40: 1; about 0.1 :1 to about 30: 1; about 0.1 : 1 to about 20: 1; about 0.1 :1 to about 10: 1, or about 0.1 : 1 to about 5:1). In some aspects, the ratio of amine nitrogen in the cationic polymer to phosphate in the polynucleotide, multi ci str onic mRNA vector, or DNA plasmid vector is from about 1 : 10 to about 10: 1.
[0447] In some aspects, the composition, pharmaceutical composition, or vaccine comprises about 0.1 mg/ml to about 10 mg/ml (e.g., about 0.1 mg/ml to about 5 mg/ml; about 0.5 mg/ml to about 10 mg/ml; or about 0.5 mg/ml to about 5 mg/ml) nucleic acid (e.g., DNA plasmid vector or multicistronic mRNA vector) complexed with the cationic polymer. In some aspects, the composition, pharmaceutical composition, or vaccine comprises about 1 mg/ml to about 10 mg/ml (e.g., about 1 mg/ml to about 6 mg/ml; about 2 mg/ml to about 6 mg/ml; about 5 mg/ml to about 10 mg/ml; or about 6 mg/ml to about 10 mg/ml) nucleic acid (e.g., DNA plasmid vector or multicistronic mRNA vector) complexed with the cationic polymer.
[0448] In some aspects, the delivery component of the composition, pharmaceutical composition, or vaccine comprises a lipopolyamine with the following formula:
Figure imgf000114_0001
(Staramine).
[0449] In some aspects, the delivery component comprises a mixture of the lipopolyamine and an alkylated derivative of the lipopolyamine. In some aspects, the alkylated derivative of the lipopolyamine is a polyoxyalkylene, polyvinylpyrrolidone, polyacrylamide, polydimethylacrylamide, polyvinyl alcohol, dextran, poly (L-glutamic acid), styrene maleic anhydride, poly-N-(2-hydroxypropyl) methacrylamide, or polydivinylether maleic anhydride. In some aspects, the alkylated derivative of the lipopolyamine has the following formula:
Figure imgf000114_0002
(methoxypolyethylene glycol (mPEG) modified Staramine, wherein n represents an integer from 10 to 100 repeating units containing 2-5 carbon atoms each. In some aspects, , the alkylated derivative of the lipopolyamine has the following formula:
Figure imgf000115_0001
wherein n = 11 (Staramine-mPEG515). In some aspects, the alkylated derivative of the lipopolyamine has the following formula:
Figure imgf000115_0002
(Staramine-mPEGl 1).
[0450] In some aspects, the ratio of the lipopolyamine to the alkylated derivative of the lipopolyamine in the mixture is 0.01:1 to 10:1. In some aspects, the lipopolyamine is present in an amount sufficient to produce a ratio of amine nitrogen in the lipopolyamine to phosphate in the polynucleotide, multi ci stronic mRNA vector, or DNA plasmid vector from about 0.01:1 to about 50:1 (e.g., about 0.01:1 to about 40:1; about 0.01:1 to about 30:1; about 0.01:1 to about 20:1; about 0.01:1 to about 10:1, or about 0.01:1 to about 5:1). In some aspects, the ratio of amine nitrogen in the lipopolyamine to phosphate in the polynucleotide, multi ci stronic mRNA vector, or DNA plasmid vector is from about 0.1:1 to about 50:1 (e.g., about 0.1:1 to about 40:1; about 0.1:1 to about 30:1; about 0.1:1 to about 20:1; about 0.1:1 to about 10:1, or about 0.1:1 to about 5:1). In some aspects, the ratio of amine nitrogen in the lipopolyamine to phosphate in the polynucleotide, multi ci stronic mRNA vector, or DNA plasmid vector is from about 0.01 : 10 to about 10:1.
[0451] In some aspects, the delivery component of the composition, pharmaceutical composition, or vaccine comprises a lipopolyamine with the following formula:
Figure imgf000115_0003
(Crossamine). [0452] In some aspects, the delivery component comprises a mixture of the lipopolyamine and an alkylated derivative of the lipopolyamine. In some aspects, the alkylated derivative of the lipopolyamine is a polyoxyalkylene, polyvinylpyrrolidone, polyacrylamide, polydimethylacrylamide, polyvinyl alcohol, dextran, poly (L-glutamic acid), styrene maleic anhydride, poly-N-(2-hydroxypropyl) methacrylamide, or polydivinylether maleic anhydride. In some aspects, the ratio of the lipopolyamine to the alkylated derivative of the lipopolyamine in the mixture is 0.01 : 1 to 10: 1. In some aspects, the lipopolyamine is present in an amount sufficient to produce a ratio of amine nitrogen in the lipopolyamine to phosphate in the polynucleotide, multi ci str onic mRNA vector, or DNA plasmid vector from about 0.01 :1 to about 50: 1 (e.g., about 0.01 : 1 to about 40:1; about 0.01 : 1 to about 30:1; about 0.01 : 1 to about 20: 1; about 0.01 : 1 to about 10: 1, or about 0.01 : 1 to about 5: 1). In some aspects, the ratio of amine nitrogen in the lipopolyamine to phosphate in the polynucleotide, multi ci str onic mRNA vector, or DNA plasmid vector is from about 0.1 : 1 to about 50: 1 (e.g., about 0.1 : 1 to about 40: 1; about 0.1 :1 to about 30: 1; about 0.1 : 1 to about 20: 1; about 0.1 :1 to about 10: 1, or about 0.1 : 1 to about 5:1). In some aspects, the ratio of amine nitrogen in the lipopolyamine to phosphate in the polynucleotide, multi ci str onic mRNA vector, or DNA plasmid vector is from about 1 : 10 to about 10: 1.
[0453] In some aspects, the delivery component of the composition, pharmaceutical composition, or vaccine comprises a poloxamer back-bone having a metal chelator covalently coupled to at least one terminal end of the poloxamer backbone. In some aspects, the metal chelator is coupled to at least two terminal ends of the poloxamer backbone. In some aspects, at least one metal chelator is coupled to a poloxamer. In some aspects, at least two metal chelators are coupled to a poloxamer. In some aspects, 1-5, 5- 10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 50-55, 55-60, 60-65, 65-70, 70-75, 75-80, 80-85, 85-90, 90-95, or 95-100 metal chelators are coupled to a poloxamer.
[0454] In some aspects, the poloxamer backbone is a poloxamer backbone disclosed in U.S. Publ. No. 2010/0004313, which is herein incorporated by reference in its entirety. In some aspects, the metal chelator is a metal chelator disclosed in U.S. Publ. No. 2010/0004313. In some aspects, the delivery component of the composition, pharmaceutical composition, or vaccine comprises a polymer having the following formula:
Figure imgf000117_0001
and pharmaceutically acceptable salts thereof, wherein:
A represents an integer from 2 to 141;
B represents an integer from 16 to 67;
C represents an integer from 2 to 141;
RA and RC are the same or different, and are R'-L- or H, wherein at least one of RA and RC is R'-L-;
L is a bond, — CO — , — CH2 — O — , or — O — CO — ; and R' is a metal chelator.
[0455] In some aspects, the delivery component comprises a poloxamer with the following formula:
Figure imgf000117_0002
or a pharmaceutically acceptable salt thereof, wherein:
A represents an integer from 2 to 141;
B represents an integer from 16 to 67;
C represents an integer from 2 to 141;
RA and RC are the same or different, and are R'-L- or H;
L is a bond, — CO — , — CH2 — O — , or — O — CO — ; and
R' is a metal chelator.
[0456] In some aspects, at least one of RA and RC is R'-L-.
[0457] In some aspects, the R’ is covalently bound to the poloxamer (e.g., where a metal chelator (e.g., a crown) is covalently attached at different density to the poloxamer).
[0458] In some aspects, one metal chelator or two or more metal chelators is/are bound to the poloxamer.
[0459] In some aspects, 2-100 metal chelators are bound to the poloxamer.
[0460] In some aspects, the metal chelator is RNNH — , RN2N — , or (R" — (N(R") — CH2CH2)x)2 — N — CH2CO — , wherein each x is independently 0-2, and wherein R" is HO2C — CH2 — . In some aspects, the metal chelator is a crown ether selected from the group consisting of 12-crown-4, 15-crown-5, 18-crown-6, 20-crown-6, 21-crown-7, and 24-crown-8. In some aspects, the crown ether is a substituted-crown ether, wherein the substituted crown ether has:
(1) one or more of the crown ether oxygens independently replaced by NH or S,
(2) one or more of the crown ether — CH2 — CH2 — moieties replaced by — C6H4— , — C10H6— , or — C6H10— ,
(3) one or more of the crown ether — CH2 — O — CH2 — moieties replaced by — C4H2O— or — C5H3N— , or
(4) any combination thereof.
[0461] In some aspects, the metal chelator is a cryptand, wherein the cryptand is selected from the group consisting of (1,2,2) cryptand, (2,2,2) cryptand, (2,2,3) cryptand, and (2,3,3) cryptand. In some aspects, the cryptand is a substituted-cryptand, wherein the substituted cryptand has:
(1) one or more of the crypthand ether oxygens independently replaced by NH or S,
(2) one or more of the crown ether — CH2 — CH2 — moieties replaced by — C6H4— , — C10H6— , or — C6H10— ,
(3) one or more of the crown ether — CH2 — O — CH2 — moieties replaced by — C4H2O— or — C5H3N— , or
(4) any combination thereof.
[0462] In some aspects, the delivery component is Crown Poloxamer (aza-crown-linked poloxamer), wherein the Crown Poloxamer comprises a polymer having the following formula:
Figure imgf000118_0001
or pharmaceutically acceptable salts thereof, wherein: a represents an integer of about 10 units; and b represents an integer of about 21 units; and wherein the total molecular weight of the polymer is about 2,000 Da to about 2,200 Da.
[0463] In some aspects, the crown poloxamer can be derivatized with a cationic molecule, a ligand, or other chemical entities.
[0464] In some aspects, the polymer or poloxamer (e.g., crown poloxamer) is present in a solution with the polynucleotide, multi ci str onic mRNA vector, or DNA plasmid vector from about 0.01% - about 5% or about 0.5% - about 5%.
[0465] In some aspects, the solution is co-formulated with a metal chelator (e.g., where the co-formulated metal chelator is a free metal chelator, which is formulated with the poloxamer (e.g., a non-crown poloxamer)).
[0466] In some aspects, the co-formulated metal chelator is present in the solution at a concentration of about O.lmg/mL to about 20mg/mL.
[0467] In some aspects, the co-formulated metal chelator is crown ether, a substituted- crown ether, a cryptand, or a substituted-cryptand.
[0468] In some aspects, the metal chelator or co-formulated metal chelator is crown ether (Aza- 18 -crown-6) .
[0469] In some aspects, the delivery component of the composition, pharmaceutical composition, or vaccine is a P-amino ester. In some aspects, the polymer is present in a solution with the polynucleotide, multi ci str onic mRNA vector, or DNA plasmid vector from about 0.01% - about 5% or about 0.5% - about 5%.
[0470] In some aspects, the delivery component of the composition, pharmaceutical composition, or vaccine is a poly-inosinic-polycytidylic acid. In some aspects, the poly- inosinic-polycytidylic acid is present in a solution with the polynucleotide, multi ci stronic mRNA vector, or DNA plasmid vector from about 0.01% - about 5% or about 0.5% - about 5%.
[0471] In some aspects, the delivery component further comprises benzalkonium chloride.
[0472] In some aspects, the delivery component comprises BD15-12. In some aspects, the ratio of nucleotide to BD15-12 polymer (N:P) is 0.1 : 1 to 5: 1.
[0473] In some aspects, the delivery component comprises Omnifect. In some aspects, the ratio of nucleotide to Omnifect polymer (N:P) is 0.1 : 1 to 5: 1.
[0474] In some aspects, the delivery component comprises Crown Poloxamer (azacrown-linked poloxamer). In some aspects, the ratio of nucleotide to Crown Poloxamer (N:P) is 0.1 : 1 to 5: 1. In some aspects, the delivery component comprises Crown Poloxamer and a PEG-PEI-cholesterol (PPC) lipopolymer. In some aspects, the delivery component comprises Crown Poloxamer and benzalkonium chloride. In some aspects, the delivery component comprises Crown Poloxamer and Omnifect. In some aspects, the delivery component comprises Crown Poloxamer and a linear polyethyleneimine (LPEI). In some aspects, the delivery component comprises Crown Poloxamer and BD15-12.
[0475] In some aspects, the delivery component comprises Staramine and mPEG modified Staramine. In some aspects, the mPEG modified Staramine is Staramine- mPEG515. In some aspects, the mPEG modified Staramine is Staramine-mPEGl 1. In some aspects, the ratio of Staramine to mPEG modified Staramine is 0.1 : 1 to 10: 1. In some aspects, the nucleotide to polymer (N:P) ratio is 0.1 : 1 to 5: 1. In some aspects, the delivery component comprises Staramine, mPEG modified Staramine, and Crown Poloxamer. In some aspects, the delivery component comprises Staramine, Staramine- mPEG515, and Crown Poloxamer. In some aspects, the delivery component comprises Staramine, Staramine-mPEGl 1, and Crown Poloxamer.
[0476] In some aspects, the metal chelator is added directly to a solution comprising (i) the polynucleotide, multi ci stronic mRNA vector, or DNA plasmid vector as described herein, and (ii) a poloxamer. In some aspects, the metal chelator is present in the solution at concentration of about O. lmg/mL to about 20mg/mL. In some aspects, metal chelator is present in the solution at concentration of about O.lmg/mL to about 20mg/mL, about O. lmg/mL to about Img/mL, about Img/mL to about 5mg/mL, about 5mg/mL to about lOmg/mL, about lOmg/mL to about 15mg/mL, or about 15mg/mL to about 20mg/mL. In some aspects, the metal chelator is crown ether, a substituted-crown ether, a cryptand, or a substituted-cryptand. In some aspects, the metal chelator is crown ether (Aza-18-crown- 6).
[0477] In some aspects, the metal chelator is added directly to a solution comprising (i) the polynucleotide, multi ci stronic mRNA vector, or DNA plasmid vector as described herein, (ii) a poloxamer, and (iii) an an adjuvant comprising an aluminum or aluminum- salt based adjuvant, a stimulator of interferon genes (STING) agonist, or a combination thereof. In some aspects, the metal chelator is present in the solution at concentration of about O.lmg/mL to about 20mg/mL. In some aspects, metal chelator is present in the solution at concentration of about O. lmg/mL to about 20mg/mL, about O.lmg/mL to about Img/mL, about Img/mL to about 5mg/mL, about 5mg/mL to about lOmg/mL, about lOmg/mL to about 15mg/mL, or about 15mg/mL to about 20mg/mL. In some aspects, the metal chelator is crown ether, a substituted-crown ether, a cryptand, or a substituted-cryptand. In some aspects, the metal chelator is crown ether (Aza-18-crown- 6). In some aspects, the adjuvant is AIPO4.
[0478] In some aspects, the metal chelator is added directly to a solution comprising (i) the polynucleotide, multi ci stronic mRNA vector, or DNA plasmid vector as described herein, (ii) a poloxamer, and (iii) an an adjuvant comprising an aluminum or aluminum- salt based adjuvant, or a combination thereof. In some aspects, the metal chelator is present in the solution at concentration of about O.lmg/mL to about 20mg/mL. In some aspects, metal chelator is present in the solution at concentration of about 0. Img/mL to about 20mg/mL, about O. lmg/mL to about Img/mL, about Img/mL to about 5mg/mL, about 5mg/mL to about lOmg/mL, about lOmg/mL to about 15mg/mL, or about 15mg/mL to about 20mg/mL. In some aspects, the metal chelator is crown ether, a substituted-crown ether, a cryptand, or a substituted-cryptand. In some aspects, the metal chelator is crown ether (Aza-18-crown-6). n some aspects, the adjuvant is AIPO4.
[0479] In some aspects, the composition, pharmaceutical composition, or vaccine is lyophilized.
[0480] Also provided herein is a host cell comprising any polynucleotide, vector, multi ci stronic mRNA vector, DNA plasmid vector, composition, pharmaceutical composition, or vaccine described or exemplified herein. In some aspects, the host cell is a eukaryotic host cell. In some aspects, the host cell is a human host cell.
[0481] Also provided herein is a kit comprising any polynucleotide, vector, multi ci stronic mRNA vector, DNA plasmid vector, composition, pharmaceutical composition, vaccine, or lyophilized composition described or exemplified herein. In some aspects, the kit further comprises a glass vial. In some aspects, the kit further comprises instructions for using the polynucleotide, vector, multi ci stronic mRNA vector, DNA plasmid vector, composition, or pharmaceutical composition in a method for inducing an immune response in a subject. In some aspects, the kit further comprise instruction for reconstituting the composition, pharmaceutical composition, vaccine or lyophilized vaccine. In some aspects, the kit further comprises instructions for using the polynucleotide, vector, multi ci stronic mRNA vector, DNA plasmid vector, composition, pharmaceutical composition, vaccine, or lyophilized composition in a method for preventing, reducing the incidence of, attenuating or treating a tumor in a subject. [0482] Also provided herein is a method of inducing an immune response in a subject, the method comprising administering an effective amount of any polynucleotide, vector, multi ci str onic mRNA vector, DNA plasmid vector, composition, pharmaceutical composition, or vaccine described or exemplified herein to the subject. In some aspects, the immune response is to one or more antigens disclosed herein. In some aspects, the immune response is a protective immune response. In some aspects, the polynucleotide, vector, multi ci stronic mRNA vector, DNA plasmid vector, composition, pharmaceutical composition, or vaccine is administered to the subject by an intramuscular, subcutaneous, intralymphatic, intranasal, or intraperitoneal route of administration.
[0483] In some aspects, the immune response is to one or more antigens comprising one or more tumor-associated antigens.
IX. Methods of Making the Compositions or Vaccines
[0484] The present disclosure also features methods of making any composition, pharmaceutical composition, or vaccine described or exemplified herein.
[0485] In some aspects, the methods comprise the steps of: (a) combining a delivery component disclosed herein with a polynucleotide disclosed herein, (b) lyophilizing the combined delivery component and polynucleotide to a powder, and (c) reconstituting the powder with a diluent to form a solution of nucleic acid complexed with the delivery component
[0486] In some aspects, the methods comprise the steps of: (a) combining a delivery component disclosed herein with a polynucleotide disclosed herein, (b) lyophilizing the combined delivery component and polynucleotide to a powder, and (c) reconstituting the powder with a diluent to form a solution of nucleic acid complexed with the delivery component, wherein the diluent comprises an adjuvant and/or a STING agonist.
[0487] In some aspects, the methods comprise combining an adjuvant with the diluent. In some aspects, the methods comprise combining a STING agonist with the diluent.
[0488] In some aspects, the methods comprise combining an adjuvant and/or a STING agonist with the reconstituted polynucleotide/delivery component solution. In some aspects, the methods comprise combining a STING agonist with the diluent and an adjuvant with reconstituted polynucleotide/delivery component solution. In some aspects, the methods comprise combining an adjuvant with the diluent and a STING agonist with the reconstituted polynucleotide/delivery component solution. [0489] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Sambrook et al., ed. (1989) Molecular Cloning A Laboratory Manual (2nd ed.; Cold Spring Harbor Laboratory Press); Sambrook et al., ed. (1992) Molecular Cloning: A Laboratory Manual, (Cold Springs Harbor Laboratory, NY); D. N. Glover ed., (1985) DNA Cloning, Volumes I and II; Gait, ed. (1984) Oligonucleotide Synthesis; Mullis et al. U.S. Pat. No. 4,683,195; Hames and Higgins, eds. (1984) Nucleic Acid Hybridization; Hames and Higgins, eds. (1984) Transcription And Translation; Freshney (1987) Culture Of Animal Cells (Alan R. Liss, Inc.); Immobilized Cells And Enzymes (IRL Press) (1986); Perbal (1984) A Practical Guide To Molecular Cloning; the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Miller and Calos eds. (1987) Gene Transfer Vectors For Mammalian Cells, (Cold Spring Harbor Laboratory); Wu et al., eds., Methods In Enzymology, Vols. 154 and 155; Mayer and Walker, eds. (1987) Immunochemical Methods In Cell And Molecular Biology (Academic Press, London); Weir and Blackwell, eds., (1986) Handbook Of Experimental Immunology, Volumes I-IV; Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1986); ); Crooks, Antisense drug Technology: Principles, strategies and applications, 2nd Ed. CRC Press (2007) and in Ausubel et al. (1989) Current Protocols in Molecular Biology (John Wiley and Sons, Baltimore, Md.).
[0490] All of the references cited above, as well as all references cited herein and the amino acid or nucleotide sequences (e.g., GenBank numbers and/or Uniprot numbers), are incorporated herein by reference in their entireties.
[0491] The following examples are offered by way of illustration and not by way of limitation.
EXAMPLES
Example 1 : Vector Construction
[0492] DNA plasmids vectors are constructed with the elements as shown in FIGs. 1-14. The starting plasmid backbone used in vector construction contains multiple restriction sites that are utilized for subcloning the desired expression cassettes in order to generate the DNA plasmid constructs. Each expression cassette that is cloned into the vector contains all of the necessary components required for expressing the functional protein or antigen (e.g promoter sequence, gene sequence, poly A tail sequence). The general methodology consists of digesting the starting DNA plasmid backbone with the appropriate restriction digest enzyme. The insert sequence is obtained from isolating and gel purifying a genetic sequence from a second DNA plasmid using appropriate restriction digest enzymes. Alternatively, the sequences to be inserted into the plasmid vector are generated synthetically or a combination of the previous two approaches can be used. The starting backbone and insert sequences are mixed together and ligated using DNA ligase. The resulting DNA plasmids are then transformed into e.coli using standard protocols and streaked onto LB agar plates containing the appropriate antibiotics. Bacterial colonies are subsequently isolated and grown in medium so that the plasmid can be purified and screened by restriction digest and gel electrophoresis in order to identify the colonies that have the correct plasmids.
[0493] Plasmid DNA vectors for expression of Trp2 under the control of a CMV promoter with a SV40 enhancer (pVac64; FIG. 14N). or NYESO-1 under the control of a CMV promoter with a SV40 enhancer were generated (pVac65; FIG. 140). Restriction digestion of pVac64 with Xhol (2 bands; 4380bp and 842bp) or Sacl (3 bands; 3990bp, 1268bp, and 414bp) produced bands of the correct size (FIG. 15A). Restriction digestion of pVac65 with Kpnl (2 bands; 2368bp and 2293bp) produced bands of the correct size (FIG. 15B).
Example 2: Vector Formulation with the Delivery Component
[0494] The present disclosure can include a nucleic acid (e.g., a DNA plasmid vector or multi ci str onic mRNA vector) complexed with a biodegradable cross-linked cationic multi-block copolymer of the following formula:
Figure imgf000125_0001
wherein: A and B are such that the molecular weight of the individual linear polyethylenimine chains are from 5,000 to 20,000 Dalton; the intermolecular crosslinks connect approximately 5-10% of amines; the biodegradable crosslinks are dithiodipropionyl (each half composed of 3 carbon atoms) and can be from 1-10 carbon atoms.
[0495] In some aspects, the biodegradable cross-linked cationic polymer comprises 10,000 to 15,000 Dalton linear PEI covalently connected with a dithiopropionyl linkage (see, e.g., U.S. Patent No. 8,445,017). The polymer is dissolved in sterile water to give a final concentration of 3 mg/ml. The DNA is dissolved in sterile water to give a final concentration of 1 mg/ml. To make the polymer/DNA complex, the two components are diluted separately with 5% glucose to a volume of 150 uL each, and then the plasmid DNA solution is added to the polymer solution.
[0496] In some aspects, the biodegradable cross-linked cationic polymer comprises 10,000 to 15,000 Dalton linear PEI covalently connected with a dithiopropionyl linkage (see, e.g., U.S. Patent No. 8,445,017) and is further conjugated to polyethyleneglycol (PEG) of molecular weight ranging from 500 to 20,000 Dalton. The resultant polymer is dissolved in sterile water to give a final concentration of 3 mg/ml. The DNA is dissolved in sterile water to give a final concentration of 1 mg/ml. To make the polymer/DNA complex, the two components are diluted separately with 5% glucose to a volume of 150 uL each, and then the plasmid DNA solution is added to the polymer solution.
[0497] In some aspects, the biodegradable cross-linked cationic polymer comprises 10,000 to 15,000 Dalton linear PEI covalently connected with a dithiopropionyl linkage (see, e.g., U.S. Patent No. 8,445,017) and is further conjugated to polyethyleneglycol (PEG) of molecular weight ranging from 500 to 20,000 Dalton. The resultant polymer is dissolved in sterile water to give a final concentration of 3 mg/ml. The DNA is dissolved in sterile water to give a final concentration of 1 mg/ml. To make the polymer/DNA complex, the two components are diluted separately with 5% glucose to a volume of 150 uL each, and then the plasmid DNA solution is added to the polymer solution.
[0498] In some aspects, the biodegradable cross-linked cationic polymer comprises 15,000 to 20,000 Dalton linear PEI covalently connected with a dithiopropionyl linkage (see, e.g., U.S. Patent No. 8,445,017). The polymer is dissolved in sterile water to give a final concentration of 3 mg/ml. The DNA is dissolved in sterile water to give a final concentration of 1 mg/ml. To make the polymer/DNA complex, the two components are diluted separately with 5% glucose to a volume of 150 uL each, and then the plasmid DNA solution is added to the polymer solution.
[0499] In some aspects, the biodegradable cross-linked cationic polymer comprises 15,000 to 20,000 Dalton linear PEI covalently connected with a dithiopropionyl linkage (see, e.g., U.S. Patent No. 8,445,017) and is further conjugated to polyethyleneglycol (PEG) of molecular weight ranging from 500 to 20,000 Dalton. The resultant polymer is dissolved in sterile water to give a final concentration of 3 mg/ml. The DNA is dissolved in sterile water to give a final concentration of 1 mg/ml. To make the polymer/DNA complex, the two components are diluted separately with 5% glucose to a volume of 150 uL each, and then the plasmid DNA solution is added to the polymer solution.
[0500] Complex formation is allowed to proceed for 15 minutes at room temperature. To study the effect of the charge ratio on gene transfer, DNA complexes with biodegradable cross-linked cationic polymer can be prepared at different ratios 1/15/1, 10/1, and 20/1 nitrogen/phosphate (N/P). Following complex formation, the complexes are diluted in a cuvette for measurement of particle size and the potential of the complex. The electrophoretic mobility of the samples can be measured at 25°C, and at a wavelength of 657 nm and at a constant angle of 90° with a Particle sizer.
Example 3 : Vector Formulation with the Delivery Carrier PPC
[0501] Bench-scale production of highly concentrated liquid formulations of fully condensed nucleic acid with a cationic lipopolymer is prepared. This involves preparation of nucleic acid complexes with a cationic polymer followed by lyophilization and reconstitution to isotonic solutions. The nucleic acid used is a plasmid DNA, and the polymer comprises a PEI backbone covalently linked to PEG and cholesterol (PEG-PEI- cholesterol (“PPC”)). The molar ratio between PEG and PEI and between cholesterol and PEI is 0.5-10 and 0.1-10, respectively. First, the DNA and PPC solutions are separately prepared at 5 mg/ml in water for injection and subsequently diluted to 0.3 mg/ml (DNA) and 1.108 mg/ml (PPC) in 3% lactose. The DNA in lactose solution is added to the PPC in lactose solution using a micropipette to a nitrogen to phosphate ratio (N:P ratio) of 0.01 :1 to 11 : 1, and the formulation is incubated for 15 minutes at room temperature to allow the complexes to form. The PPC/DNA complexes in 3% lactose are lyophilized using a FREEZONE freeze dry System from LABCONCO Corp. Kansas City, Mo. 500 ul of prepared formulation is added to 2 ml borosilicate glass vials which were then lyophilized using a freeze drying program consisting of the following segments:
1) freezing segment (ramp 0.25°C/min, hold at 34°C. for 4 hours),
2) primary drying segment (hold at 34°C. for 24 hrs),
3) secondary drying segment (ramp to 20°C and hold for 24 hrs), and
4) ramp to 4°C. at 0.25°C/min.
[0502] The resultant lyophilized powder is reconstituted with 150 pl water for injection to make 0.5 mg/ml DNA.
Example 4: Vector Formulation with the Delivery Carrier Poloxamer
[0503] Poloxamers are gently mixed with 1 mg/ml of nucleic acids in water or saline solution (0.15M) at variable concentrations. Formulated poloxamer (5%)/plasmid solutions are analyzed by gel electrophoresis in order to verify interaction between formulated plasmid and poloxamer. Comparison between unformulated plasmid DNA and DNA formulated with poloxamer have the similar movement through the gel and therefore indicate no binding between plasmid DNA and poloxamer. The formulated plasmid with poloxamer is used for gene transfer in mammalian cell or tissue. X.5 Synthesis of Aza-crown-linked Poloxamer (Crown Pol oxamer)
Figure imgf000128_0001
[0504] An aza-crown-linked poloxamer (crown poloxamer) is constructed as follows. Poloxamer 124 (Pluronic L-44; 500 mg, 220 pmol) is dissolved in toluene (3ml), and the resulting solution is treated with 2 ml (4 mmol) of 2M phosgene solution in toluene. After 3 hrs at room temperature, the mixture is concentrated in vacuum, the residue is redissolved in 3 ml toluene and concentrated again. The residue is dissolved in dry chloroform (5 ml). To this solution was added aza-18-crown-6 [l-aza-4, 7, 10, 13, 16- pentaoxacyclooctadecane (125 mg, 500 pmol) and Hunig' s base (100 pl, 574 pmol). After 70 hrs the reaction mixture is concentrated in vacuum, the residue is re-dissolved in distilled water and dialyzed [membrane cutoff 1000 Da] against distilled water. Concentration of the dialyzate afforded 410 mg of the title compound. Proton NMR (D2O): 4.20 ppm (t, CH2OC=O); 3.7-3.5 ppm [(-CH2-CH2-O-), both crown and poloxamer)]; 3.4 ppm (m, crown CH2N); 1.1 ppm (m, poloxamer-(CH3)CH-CH2-).
Example 5: Cancer Vaccine Formulations
PPC/pVaccine Formulation
[0505] Vaccine plasmid DNA is prepared initially at 0.1 mg/mL with PEG-PEI- cholesterol (PPC; MW 4.2 kD), by mixing plasmid DNA with the polymer at (11 : 1) and (0.5: 1) (N:P) ratios in 5% dextrose solution, and the mixture is incubated at room temperature for 10 minutes to allow the formation of nanocomplexes. Then, pDNA/PPC nanocomplexes are concentrated to 1-5 mg/mL using Amicon Ultra Centrifugal Filters (Ultracel-3K MWCO). [0506] The electrophoretic mobility of the PPC/DNA complexes is determined by agarose gel electrophoresis at 70 V for one hour. DNA integrity in the complexes is determined by incubation of the complexes with 50 pg of dextran sulfate or triton-X for 10 minutes at room temperature followed by gel electrophoresis at 100 Volts for one hour. The particle size the nanocomplexes in Milli-Q water is determined at 657 nm at a constant angle of 90° by dynamic light scattering using a Malvern particle size analyzer. Osmolality of the formulation is determined using Fiske210 micro-sample osmometer, DNA quantification was performed using spectrophotometry, formulation pH was measured using Accumet research ARI 5 pH meter.
BD15-12/pVaccine Formulation
[0507] Vaccine plasmid DNA is prepared initially at 0.1 mg/mL with PEI-base copolymer, BD15-12 (15kD linear PEI; MW 26.5 kD), by mixing plasmid DNA with the polymer at 10:1 and 0.5: 1 N:P ratios in 5% dextrose solution, and the mixture is incubated at room temperature for 10 minutes to allow the formation of nanocomplexes. Then, BD15-12/pDNA nanocomplexes are concentrated to 2.5-5 mg/mL using Amicon Ultra Centrifugal Filters (Ultracel-3K MWCO)
[0508] The electrophoretic mobility of the BD15-12/DNA complexes is determined by agarose gel electrophoresis at 70 V for one hour. The particle size of the nanocomplexes in Milli-Q water is determined at 657 nm at a constant angle of 90° by dynamic light scattering using a Malvern particle size analyzer. Osmolality of the formulation is determined using Fiske 210 micro-sample osmometer, DNA quantification is performed using spectrophotometry, and formulation pH is measured using Accumet research ARI 5 pH meter.
Staramine : Star-mPEG/ p Vaccine F ormul ati on :
[0509] Staramine: Poly(ethylene glycol) methyl ether (mPEG)/pVaccine formulation is composed of Staramine (0.635 kD) liposomes and plasmid DNA. Staramine alone or 10: 1 mixtures of Staramine (Star) and Star-PEG515 (1.2 kD) are rotary-evaporated to a film. The flask of liposome film is held under high vacuum overnight. Water for injection is added to the film to give the desired Staramine concentration and bath sonicated for ~30 minutes using Branson Water Bath Sonicator model 2510 followed by a probe sonication for 5 minutes (using a continuous pulse sonication with an output wattage of 5-10 watts (rms) (Model 100; Fisher Scientific Sonic Desmembrator, Pittsburg, PA). The liposome solution is filtered through a 0.2-pm filter, diluted with 5% dextrose, and mixed with the desired amount of plasmid. Particle size of the complexes is measured with Malvern particle size analyzer sizer. The complexation efficiency is determined by gel retardation assay (Life Technologies, Carlsbad, CA). The gel retardation assay is performed by loading the Staramine/plasmid DNA on 1% agarose gel and electrophoresed at 100 V for 1 hour. To release the plasmid DNA from the Staramine nanoparticles, 10% TritonX-100 is added to the complex and the solution is loaded on the agarose gel. Osmolality of the formulation is determined using Fiske210 micro-sample osmometer, DNA quantification is performed using spectrophotometry, and formulation pH was measured using Accumet research ARI 5 pH meters.
Omnifect/pVaccine Formulation
[0510] Vaccine plasmid DNA is prepared initially at 0.1 mg/mL with Omnifect (MW 7.3 kD), by mixing plasmid DNA with the polymer at 10: 1 and 0.5: 1 N:P ratios in 5% dextrose solution, and the mixture is incubated at room temperature for 10 minutes to allow the formation of nanocomplexes. Then, Omnifect/pDNA nanocomplexes are concentrated to 2.5-5 mg/mL using Amicon Ultra Centrifugal Filters (Ultracel-3K MWCO)
[0511] The electrophoretic mobility of the Omnifect/DNA complexes is determined by agarose gel electrophoresis at 70 V for one hour. The particle size the nanocomplexes in Milli-Q water is determined at 657 nm at a constant angle of 90° by dynamic light scattering using a Malvern particle size analyzer. DNA integrity in the complexes is determined by incubation of the complexes with 50 pg of dextran sulfate or triton-X for 10 minutes at room temperature followed by gel electrophoresis at 100 Volts for one hour. Osmolality of the formulation is determined using Fiske210 micro-sample osmometer, DNA quantification is performed using spectrophotometry, and formulation pH is measured using Accumet research AR 15 pH meter.
Crown poloxamer/p Vaccine Formulation
[0512] The required concentration of plasmid DNA to produce a final concentration of 1- 5 mg/ml is made in PBS or NaCl, the mixture was mixed by low speed vortexing and the required amount of crown poloxamer (MW 2.2 Kd) is added to the DNA solution to produce a final concentration of 0.01-5%.
[0513] To store the formulation at -20° C or for lyophilization purposes, the formulation is made in 20 mM Tris-8% sucrose instead of PBS or NaCl. Osmolality of the formulation was determined using Fiske210 micro-sample osmometer, DNA quantification is performed using spectrophotometry, and formulation pH was measured using Accumet research ARI 5 pH meter.
Crown poloxamer/Cationic Carrier Adjuvant/pVaccine Formulation
[0514] The required concentration of plasmid DNA to produce a final concentration of 0.01-0.5 mg/ml is made in 5% dextrose or PBS, the mixture was mixed by low speed vortexing and the required amount of cationic adjuvant carrier (Staramine, BD15-12, PPC, Omnifect, Benzalkonium chloride (BAK)) was add to plasmid DNA. pDNA/PPC nanocomplexes are concentrated to 1-5 mg/mL using Amicon Ultra Centrifugal Filters (Ultracel-3K MWCO). Then crown poloxamer (/.< ., aza-crown-linked poloxamer) is add to the DNA solution to produce a final concentration of 0.1-5%. Particle size of the CP- cationic carrier-pDNA complexes is measured with Malvern particle size analyzer sizer. Mixing crown poloxamer with plasmid DNA did not form any detectable nanoparticles by particle size measurements or gel electrophoresis. However, when cationic delivery systems are added to crown poloxamer/DNA formulation measurable nanoparticles are observed in the formulation. The complexation efficiency is determined by gel retardation assay (Life Technologies, Carlsbad, CA). The gel retardation assay is performed by loading the formulated plasmid DNA on 1% agarose gel and electrophoresed at 100 V for 1 hour.
[0515] Osmolality of the formulation is determined using Fiske210 micro-sample osmometer, DNA quantification was performed using spectrophotometry, and formulation pH is measured using Accumet research ARI 5 pH meter.
Lyophilization Cycle
[0516] 1 ml of each formulation is aliquoted into 2 ml glass vials and placed into freeze- dryer (FREEZONE Triad freeze dry System from LABCONCO Corp. Kansas City, MO.). Vials are cooled to -45 °C for 16 hours, and then temperature is raised to -15°C before the start of the primary drying. After 24 hrs, the shelf temperature is raised to 0° C and kept under vacuum for another 24 hours. Finally, shelf temperature is raised to 25° C for 72 hours and vials are capped under vacuum at the end of secondary drying segment.
Example 6: Formulations for Expression of Trp2 and NYESO-1 in Mammalian Cells [0517] To express Trp2 or NYESO-1 in mammalian cells, pVac64 or pVac65 plasmid DNA was formulated with BD3.6-oleoyl, BD15-12, Crossamine, or poloxamer.
Formulations comprising BD3.6-oleoyl, BD15-12, or Crossamine were able to produce particles (FIGs. 16A-16B). Formulations comprising poloxamer did not produce measureable particles since poloxamer is non-condensing.
[0518] Mammalian cells were transfected with pVac64 plasmid DNA formulated with BD3.6-oleoyl, BD15-12, Crossamine, or poloxamer to express Trp2. Each formuation was able to generate Trp2 (FIG. 17A). Cells transfected with plasmid DNA formulated with BD3.6-oleoyl, BD 15-12, or Crossamine expressed higher levels of Trp2 compared to cells transfected with plasmid DNA formulated with poloxamer.
[0519] Mammalian cells were transfected with pVac65 plasmid DNA formulated with BD3.6-oleoyl, BD15-12, Crossamine, or poloxamer to express NYESO-1. Each formulation was able to generate NYESO-1 (FIG. 17B). Cells transfected with plasmid DNA formulated with BD3.6-oleoyl, BD15-12, or Crossamine expressed higher levels of NYESO-1 compared to cells transfected with plasmid DNA formulated with poloxamer.
Example 7: Melanoma Mouse Model
[0520] A subcutaneous B16 melanoma model will be used for the evaluation of the therapies provided herein. Mice will be injected with Bl 6F 10 melanoma cells by subcutaneous injection and B16F10 cells will form a palpable tumor in about 5 to 10 days. The tumors will be grown to about 1 x 1 x 1-cm tumor in about 14 to 21 days. When allowed to grow larger, the tumors often become necrotic in the center, therefore mice will be sacrificed before this point. The typical dose used will be about 1 x io5 cells/mouse, which is 1.5 to 2 times the minimal tumorigenic dose in normal mice.
[0521] In some experiments, mice will be administered the vaccine compositions provided herein before the tumor cells are injected to determine whether the vaccine compositions have a prophylactic effect. [0522] In some experiments, mice will be administered the vaccine compositions provided herein together with the tumor cells to determine whether the vaccine compositions have a therapeutic effect.
[0523] In some experiments, mice will be administered the vaccine compositions provided herein at day 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14,15,16, 17, 18, 19, or 20 days after injection of the tumor cells.
[0524] In some experiments, mice will be administered the vaccine compositions provided herein after a tumor has been established to determine whether the vaccine compositions have a therapeutic effect.
Example 8: Ovarian Cancer Mouse Model
[0525] C57BL6 mice will be injected with 5* 106 ID8 ovarian cancer cells or SKOV3 ovarian cancer cells into the peritoneal cavity via a left lower abdominal wall injection. The tumors will be grown to about a 1 x 1 x 1-cm tumor.
[0526] In some experiments, mice will be administered the vaccine compositions provided herein before the tumor cells are injected to determine whether the vaccine compositions have a prophylactic effect.
[0527] In some experiments, mice will be administered the vaccine compositions provided herein together with the tumor cells to determine whether the vaccine compositions have a therapeutic effect.
[0528] In some experiments, mice will be administered the vaccine compositions provided herein at day 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14,15,16, 17, 18, 19, or 20 days after injection of the tumor cells.
[0529] In some experiments, mice will be administered the vaccine compositions provided herein after a tumor has been established to determine whether the vaccine compositions have a therapeutic effect.
Table 1 : SEQUENCES
Figure imgf000133_0001
Figure imgf000134_0001
Figure imgf000135_0001
Figure imgf000136_0001
Figure imgf000137_0001
Figure imgf000138_0001
Figure imgf000139_0001
Figure imgf000140_0001
Figure imgf000141_0001
Figure imgf000142_0001
Figure imgf000143_0001

Claims

WHAT IS CLAIMED IS:
1. An composition comprising:
(a) a polynucleotide comprising (i) an antigen nucleic acid which encodes a tumor-associated antigen or an antigenic fragment thereof, wherein the antigen nucleic acid is operably linked to a promoter, and optionally (ii) a nucleic acid which encodes an cytokine immune modifier;
(b) a delivery component selected from the group consisting of a cationic polymer, a poly-inosinic-polycytidylic acid, a poloxamer, or derivative thereof; and
(c) optionally, an adjuvant.
2. The compositions of claim 1, wherein cytokine immune modifier is IL-12.
3. The composition of claim 1 or 2, wherein the delivery component is a crown poloxamer.
4. The composition of any one of claims 1-3, wherein the adjuvant comprises an aluminum or aluminum-salt based adjuvant, a stimulator of interferon genes (STING) agonist, a CpG, an oil-in-water emulsion of squalene oil, an oil-in-water emulsion comprising a-tocopherol, squalenem and polysorbate 80, or a combination thereof.
5. The compositon of claim 4, wherein the aluminum or aluminum-salt based adjuvant is selected from the group consisting of an aluminum phosphate, an aluminum hydroxide, aluminum oxyhydroxide, a potassium aluminum sulfate [KA1(SO4)2], an aluminum hydroxyphosphate, an aluminum hydroxyphosphate sulfate, an aluminum chloride, an aluminum silicate, and any combination thereof.
6. The composition of claim 4 or 5, wherein the STING agonist is selected from the group consisting of a cyclic di-nucleotides, a non-cyclic di-nucleotide small molecule, an amidobenzimidazole (ABZI), a flavonoid, a nanovaccine, an antibody drug conjugate, a bacterial vector, and an ENPP1 inhibitor.
7. The composition of any one of claims 4-6, wherein the STING agonist is cyclic guanosine monophosphate (cGMP), cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), an amidobenzimidazole, or flavonoid.
8. The composition of any one of claims 4-7, wherein the STING agonist is cGMP.
9. The composition of any one of claims 1-8, wherein the antigen nucleic acid of the polynucleotide which encodes a tumor-associated antigen is selected from the group consisting of a NYESO-1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A9, MAGE-A10, MAGE-CI, MAGE-C2, SSX, H0RMAD1, CXorf61, ACTL8, AKAP3,AKAP4, HOM-TES-85, OY-TES-1, HSP70-2, LAGE-1, PIWL1, PIWL2, PIWL3, PIWL4, PLU-1, PLAC1, SPAG9, SCP-1, TAG-1, TAG-2a, TAG-2b, TAG-2c, TRAG-3, KAP4, BAGE, BORIS, CT45, GAGE- 1/2, PRAME, PAGE4, SP17, PASD1, p53, ras, B-RafHER-2/neu, mucin 1 (MUC1), epidermal growth factor receptor (EGFR), GP100, prostatic acid phosphatase, prostate-specific membrane antigen (PSMA), mammoglobin-A, carcinoembryonic antigen (CEA), papillomavirus antigens E6/E7, heat-shock proteins, alpha fetoprotein, CDK, P-catenin, mesothelin, and cancer antigen- 125 (CA125), and any antigenic fragments thereof.
10. The composition of any one of claims 1-9, wherein the polynucleotide further comprises at least one additional antigen nucleic acid, which encodes a second tumor-associated antigen or an antigenic fragment thereof.
11. The composition of claim 10, wherein the second antigen nucleic acid of the polynucleotide which encodes a second tumor-associated antigen selected from the group consisting of a NYESO-1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A9, MAGE- A10, MAGE-CI, MAGE-C2, SSX, H0RMAD1, CXorf61, ACTL8, AKAP3,AKAP4, HOM- TES-85, OY-TES-1, HSP70-2, LAGE-1, PIWL1, PIWL2, PIWL3, PIWL4, PLU-1, PLAC1, SPAG9, SCP-1, TAG-1, TAG-2a, TAG-2b, TAG-2c, TRAG-3, KAP4, BAGE, BORIS, CT45, GAGE-1/2, PRAME, PAGE4, SP17, PASD1, p53, ras, B-RafHER-2/neu, mucin 1 (MUC1), epidermal growth factor receptor (EGFR), GP100, prostatic acid phosphatase, prostate-specific membrane antigen (PSMA), mammoglobin-A, carcinoembryonic antigen (CEA), papillomavirus antigens E6ZE7, heat-shock proteins, alpha fetoprotein, CDK, P-catenin, mesothelin, and cancer antigen- 125 (CAI 25), and any antigenic fragments thereof.
12. The composition of any one of claims 1-11, wherein the tumor-associated antigen and/or the second tumor-associated antigen is/are selected from the group consisting of NYESO- 1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A9, MAGE-A10, MAGE-CI, MAGE-C2, SSX, H0RMAD1, CXorf61, ACTL8, AKAP3,AKAP4, HOM-TES-85, OY-TES-1, HSP70-2, LAGE-1, PIWL1, PIWL2, PIWL3, PIWL4, PLU-1, PLAC1, SPAG9, SCP-1, TAG-1, TAG-2a, TAG-2b, TAG-2c, TRAG-3, KAP4, BAGE, BORIS, CT45, GAGE-1/2, PRAME, PAGE4, SP17, PASD1, p53, ras, B-RafHER-2/neu, mucin 1 (MUC1), epidermal growth factor receptor (EGFR), GP100, prostatic acid phosphatase, prostate-specific membrane antigen (PSMA), mammoglobin-A, carcinoembryonic antigen (CEA), papillomavirus antigens E6ZE7, heat-shock proteins, alpha fetoprotein, CDK, P-catenin, mesothelin, cancer antigen-125 (CA125), and antigenic fragments thereof, and any combination thereof.
13. The composition of claim 12, wherein the tumor-associated antigen and/or the second tumor-associated antigen is/are selected from the group consisting of: SP17, TRP-2 , NYESO-1, CEA, CA125, mucin 1, and GP100, antigenic fragments thereof, and any combination thereof.
14. The composition of any one of claims 10-13, wherein the tumor-associated antigen is selected from NYESO-1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE- A9, MAGE-A10, MAGE-CI, MAGE-C2, SSX, H0RMAD1, CXorf61, ACTL8, AKAP3,AKAP4, HOM-TES-85, OY-TES-1, HSP70-2, LAGE-1, PIWL1, PIWL2, PIWL3, PIWL4, PLU-1, PLAC1, SPAG9, SCP-1, TAG-1, TAG-2a, TAG-2b, TAG-2c, TRAG-3, KAP4, BAGE, BORIS, CT45, GAGE-1/2, PRAME, PAGE4, SP17, PASD1, p53, ras, B-RafHER-2/neu, mucin 1 (MUC1), epidermal growth factor receptor (EGFR), GP100, prostatic acid phosphatase, prostate-specific membrane antigen (PSMA), mammoglobin-A, carcinoembryonic antigen (CEA), papillomavirus antigens E6ZE7, heat-shock proteins, alpha fetoprotein, CDK, P-catenin, mesothelin, and cancer antigen-125 (CA125), or an antigenic fragment thereof, and wherein the second tumor-associated antigen is seleted from a NYESO-1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A9, MAGE-A10, MAGE-CI, MAGE-C2, SSX, H0RMAD1, CXorf61, ACTL8, AKAP3,AKAP4, HOM-TES-85, OY-TES-1, HSP70-2, LAGE- 1, PIWL1, PIWL2, PIWL3, PIWL4, PLU-1, PLAC1, SPAG9, SCP-1, TAG-1, TAG-2a, TAG-2b, TAG-2c, TRAG-3, KAP4, BAGE, BORIS, CT45, GAGE-1/2, PRAME, PAGE4, SP17, PASD1, p53, ras, B-RafHER-2/neu, mucin 1 (MUC1), epidermal growth factor receptor (EGFR), GP100, prostatic acid phosphatase, prostate-specific membrane antigen (PSMA), mammoglobin-A, carcinoembryonic antigen (CEA), papillomavirus antigens E6/E7, heat-shock proteins, alpha fetoprotein, CDK, P-catenin, mesothelin, and cancer antigen-125 (CA125), or an antigenic fragment thereof.
15. The composition of any one of claims 10-14, wherein the tumor-associated antigen is selected from SP17, TRP-2 , NYESO-1, CEA, CA125, mucin 1, and GP100 or an antigenic fragment thereof, and wherein the second tumor-assocaited antigen is sei eted from a NYESO-1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A9, MAGE-A10, MAGE-C1, MAGE-C2, SSX, H0RMAD1, CXorfbl, ACTL8, AKAP3,AKAP4, HOM-TES-85, OY-TES-1, HSP70-2, LAGE- 1, PIWL1, PIWL2, PIWL3, PIWL4, PLU-1, PLAC1, SPAG9, SCP-1, TAG-1, TAG-2a, TAG-2b, TAG-2c, TRAG-3, KAP4, BAGE, BORIS, CT45, GAGE-1/2, PRAME, PAGE4, SP17, PASD1, p53, ras, B-RafHER-2/neu, mucin 1 (MUC1), epidermal growth factor receptor (EGFR), GP100, prostatic acid phosphatase, prostate-specific membrane antigen (PSMA), mammoglobin-A, carcinoembryonic antigen (CEA), papillomavirus antigens E6/E7, heat-shock proteins, alpha fetoprotein, CDK, P-catenin, mesothelin, and cancer antigen-125 (CA125), or an antigenic fragment thereof.
16. The composition of any one of claims 10-15, wherein the second antigen nucleic acid of the polynucleotide is operably linked to the promoter through an internal ribosome entry site (IRES) sequence.
17. The composition of any one of claims 10-16, wherein the second antigen nucleic acid of the polunucleotide is operably linked to a second promoter.
18. The composition of any one of claims 1-17, wherein the promoter or the one or more second promoters is selected from the group consisting of: a cytomegalovirus (CMV) promoter, a Rouse sarcoma virus (RSV) promoter, a Moloney murine leukemia virus (Mo- MuLV) long terminal repeat (LTR) promoter, a mammalian elongation factor 1 (EFl) promoter, a cytokeratin 18 (CK18) promoter, a cytokeratin 19 (CK19) promoter, a simian virus 40 (SV40) promoter, a murine U6 promoter, a skeletal a-actin promoter, a P-actin promoter, a murine phosphoglycerate kinase 1 (PGK1) promoter, a human PGK1 promoter, a CBA promoter, a CAG promoter, and any combination thereof.
19. The composition of any one of claims 1-18, wherein the antigen nucleic acid of the polynucleotide encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 1, 3, 5, 7, 8, or 10.
20. The composition of any one of claims 1-19, wherein the antigen nucleic acid of the polynucleotide comprises a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 2, 4, 6, 9, 11, 28, or 29.
21. The composition of any one of claims 1-20, wherein the polynucleotide further comprises one or more post-transcriptional regulatory elements.
22. The composition of claim 21, wherein the post-transcriptional regulatory element is a wood chuck hepatitis virus post-transcriptional regulatory element (WPRE).
23. The polynucleotide of any one of claims 1-22, wherein the polynucleotide further comprises at least one 3' UTR poly(a) tail sequence operably linked to the antigen nucleic acid, the second antigen nucleic acid, or any combination thereof.
24. The composition of claim 23, wherein the 3' UTR poly(a) tail sequence is a 3' UTR SV40 poly(a) tail sequence, a 3' UTR bovine growth hormone (bGH) poly(A) sequence, a 3' UTR actin poly(A) tail sequence, a 3' UTR hemoglobin poly (A) sequence, or combinations thereof.
25. The composition of any one of claims 1-24, wherein the polynucleotide further comprises an enhancer sequence.
26. The composition of claim 25, wherein the enhancer sequence comprises a human actin enhancer sequence, a human myosin enhancer sequence, a human hemoglobin enhancer sequence, a human muscle creatine enhancer sequence, a viral enhancer sequence, a polynucleotide function enhancer sequence, or any combination thereof.
27. The composition of claim 25 or 26, wherein the enhancer sequence comprises a CMV intronic sequence, a P-actin intronic sequence, or the combination thereof.
28. The composition of claim 27, wherein the enhancer sequence is a CMV intronic sequence.
29. The composition of claim 25 or 26, wherein the enhancer sequence is a CMV intronic sequence, a SV40 enhancer sequence, a P-actin intronic sequence, or combinations thereof.
30. The composition of any one of claims 1-29, wherein the polynucleotide is within a vector, wherein the vector is a DNA plasmid, a viral vector, a bacterial vector, a cosmid, or an artificial chromosome.
31. The composition of claim 30, wherein the vector is a DNA plasmid.
32. The composition of claim 31, wherein the DNA plasmid vector is selected from the group consisting of: pVac 2, pVac 3, pVac 5, pVac 6, pVac 7, pVac 8, pVac 9, pVac 10, pVac 11, pVac 12, pVac 13, pVac 14, pVac 15, pVac 16, and pVac 17.
33. The composition of any one of claims 1-32 wherein the composition is a pharmaceutical composition comprising a pharmaceutically acceptable carrier.
34. The composition of any one of claims 1-33, wherein the composition is a vaccine.
35. The composition, pharmaceutical composition, or vaccine of any one of claims 1- 34, wherein the cationic polymer comprises a synthetic functionalized polymer, a P-amino ester, a lipid, a lipopolymer, or a chemical derivative thereof.
36. The composition, pharmaceutical composition, or vaccine of claim 35, wherein the synthetic functionalized polymer is a biodegradable cross-linked cationic multi-block copolymer.
37. The composition, pharmaceutical composition, or vaccine of claim 36, wherein the biodegradable cross-linked cationic multi-block copolymer is represented by the formula: (CP)xLyYz, wherein:
(a) CP represents a cationic polymer containing at least one secondary amine group, wherein the cationic polymer has a number averaged molecular weight within the range of 1,000 to 25,000 Dalton,
(b) Y represents a bifunctional biodegradable linker containing ester, amide, disulfide, or phosphate linkages,
(c) L represents a ligand,
(d) x is an integer in the range from 1 to 20,
(e) y is an integer in the range from 0 to 100, and
(f) z is an integer in the range from 0 to 40.
38. The composition, pharmaceutical composition, or vaccine of claim 37, wherein the cationic polymer comprises biodegradable cross-linked linear polyethyleneimine (LPEI).
39. The composition, pharmaceutical composition, or vaccine of claim 37 or 38, wherein the bifunctional biodegradable linker is hydrophilic and comprises a biodegradable linkage comprising a disulfide bond.
40. The composition, pharmaceutical composition, or vaccine of claim 37 or 38, wherein the bifunctional biodegradable linker is a dithiodipropionyl linker.
41. The composition, pharmaceutical composition, or vaccine of claim 36, wherein the biodegradable cross-linked cationic multi-block copolymer comprises LPEI and a dithiodipropionyl linker for cross-linking the multi-block copolymer, wherein the LPEI has an average molecular weight of 1,000 to 25,000 Dalton.
42. The composition, pharmaceutical composition, or vaccine of claim 41, wherein the biodegradable cross-linked cationic multi-block copolymer is covalently linked to at least one ligand.
43. The composition, pharmaceutical composition, or vaccine of claims 37-42, wherein the ligand is a targeting ligand selected from the group consisting of: a sugar moiety, a polypeptide, folate, and an antigen.
44. The composition, pharmaceutical composition, or vaccine of claim 43, wherein the sugar moiety is a monosaccharide or an oligosaccharide.
45. The composition, pharmaceutical composition, or vaccine of claim 44, wherein the monosaccharide is galactose.
46. The composition, pharmaceutical composition, or vaccine of claim 43, wherein the polypeptide is a glycoprotein, an antibody, an antibody fragment, a cell receptor, a cytokine receptor, or a growth factor receptor.
47. The composition, pharmaceutical composition, or vaccine of claim 46, wherein the growth factor receptor is an epidermal growth factor receptor.
48. The composition, pharmaceutical composition, or vaccine of claim 46, wherein the glycoprotein is transferrin or asialoorosomucoid (ASOR).
49. The composition, pharmaceutical composition, or vaccine of claim 43, wherein the antigen is a tumor-associated antigen, a bacterial antigen, or a parasite antigen.
50. The composition, pharmaceutical composition, or vaccine of any one of claims 36-49, wherein the biodegradable cross-linked cationic multi-block copolymer is covalently linked to polyethylene glycol (PEG) of molecular weight ranging from 500 to 20,000 Dalton.
51. The composition, pharmaceutical composition, or vaccine of any one of claims 36-50, wherein the biodegradable cross-linked cationic multi-block copolymer is covalently linked to a fatty acyl chain selected from the group consisting of: oleic acid, palmitic acid, and stearic acid.
52. The composition, pharmaceutical composition, or vaccine of any one of claims 36-51, wherein the biodegradable cross-linked cationic multi-block copolymer comprises at least one amine group that is electrostatically attracted to a polyanionic compound.
53. The composition, pharmaceutical composition, or vaccine of claim 52, wherein the polyanionic compound is a nucleic acid, wherein the biodegradable cross-linked cationic multi-block copolymer condenses the nucleic acid to form a compact structure.
54. The composition, pharmaceutical composition, or vaccine of claim 35, wherein the lipopolymer is a cationic lipopolymer comprising a PEI backbone covalently linked to a lipid or a PEG.
55. The composition, pharmaceutical composition, or vaccine of claim 54, wherein the PEI backbone is covalently linked to a lipid and a PEG.
56. The composition, pharmaceutical composition, or vaccine of claim 55, wherein the lipid and the PEG are directly attached to the PEI backbone by covalent bonds.
57. The composition, pharmaceutical composition, or vaccine of claim 55, wherein the lipid is attached to the PEI backbone through a PEG spacer.
58. The composition, pharmaceutical composition, or vaccine of any one of claims 54-57, wherein the PEG has a molecular weight of between 50 to 20,000 Dalton.
59. The composition, pharmaceutical composition, or vaccine of any one of claims 54-58, wherein the molar ratio of PEG to PEI is within a range of 0.1 : 1 to 500: 1.
60. The composition, pharmaceutical composition, or vaccine of any one of claims 54-58, wherein the molar ratio of the lipid to the PEI is within a range of 0.1 : 1 to 500: 1.
61. The composition, pharmaceutical composition, or vaccine of any one of claims 54-60, wherein the lipid is a cholesterol, a cholesterol derivative, a C12 to C18 fatty acid, or a fatty acid derivative.
62. The composition, pharmaceutical composition, or vaccine of claim 61, wherein the PEI is covalently linked to cholesterol and PEG, and wherein the average
PEG:PEI: cholesterol molar ratio in the cationic lipopolymer is within the range of 1-5 PEG: 1 PEEO.4-1.5 cholesterol.
63. The composition, pharmaceutical composition, or vaccine of any one of claims 54-62, wherein the PEI has a linear or branch configuration with a molecular weight of 100 to 500,000 Dalton.
64. The composition, pharmaceutical composition, or vaccine of any one of claims 54-63, wherein the cationic lipopolymer further comprises a pendant functional moiety selected from the group consisting of: a receptor ligand, a membrane permeating agent, an endosomolytic agent, a nuclear localization sequence, and a pH sensitive endosomolytic peptide.
65. The composition, pharmaceutical composition, or vaccine of any one of claims 54-64, wherein the cationic lipopolymer further comprises a targeting ligand, wherein the targeting ligand is directly attached to the PEI backbone or is attached through a PEG linker.
66. The composition, pharmaceutical composition, or vaccine of claim 65, wherein the targeting ligand is selected from the group consisting of: a sugar moiety, a polypeptide, folate, and an antigen.
67. The composition, pharmaceutical composition, or vaccine of claim 66, wherein the sugar moiety is a monosaccharide or an oligosaccharide.
68. The composition, pharmaceutical composition, or vaccine of claim 67, wherein the monosaccharide is galactose.
69. The composition, pharmaceutical composition, or vaccine of claim 66, wherein the polypeptide is a glycoprotein, an antibody, an antibody fragment, a cell receptor, a cytokine receptor, or a growth factor receptor.
70. The composition, pharmaceutical composition, or vaccine of any one of claims 1- 69, wherein the cationic polymer is present in an amount sufficient to produce a ratio of amine nitrogen in the cationic polymer to phosphate in the DNA plasmid vector from about 0.01:1 to about 50:1.
71. The composition, pharmaceutical composition, or vaccine of claim 70, wherein the ratio of amine nitrogen in the cationic polymer to phosphate in the DNA plasmid vector from about 1 : 10 to about 10:1.
72. The composition, pharmaceutical composition, or vaccine of any one of claims 1- 71, wherein the composition, comprises about 0.1 mg/ml to about 10.0 mg/ml nucleic acid complexed with the cationic polymer.
73. The composition, pharmaceutical composition, or vaccine of any one of claims 1- 34, wherein the delivery component comprises a lipopolyamine with the following formula:
Figure imgf000154_0001
(Staramine).
74. The composition, pharmaceutical composition, or vaccine of claim 73, wherein the delivery component comprises a mixture of the lipopolyamine and an alkylated derivative of the lipopolyamine.
75. The composition, pharmaceutical composition, or vaccine of claim 74, wherein the alkylated derivative of the lipopolyamine is a polyoxyalkylene, polyvinylpyrrolidone, polyacrylamide, polydimethylacrylamide, polyvinyl alcohol, dextran, poly (L-glutamic acid), styrene maleic anhydride, poly -N-(2 -hydroxypropyl) methacrylamide, or polydivinylether maleic anhydride.
76. The composition, pharmaceutical composition, or vaccine of claim 75, wherein the alkylated derivative of the lipopolyamine has the following formula:
Figure imgf000155_0001
(methoxypolyethylene glycol (mPEG) modified Staramine), wherein n represents an integer from 10 to 100 repeating units containing of 2-5 carbon atoms each.
77. The composition, pharmaceutical composition, or vaccine of claim 74 or 75, wherein the ratio of the lipopolyamine to the alkylated derivative of the lipopolyamine in the mixture is 1 : 1 to 10:1.
78. The composition, pharmaceutical composition, or vaccine of any one of claims 75-77, wherein the lipopolyamine is present in an amount sufficient to produce a ratio of amine nitrogen in the lipopolyamine to phosphate in the DNA plasmid vector from about 0.01 : 1 to about 50: 1.
79. The composition, pharmaceutical composition, or vaccine of claim 78, wherein the lipopolyamine is present in an amount sufficient to produce a ratio of amine nitrogen in the lipopolyamine to phosphate in the DNA plasmid vector from about 1 : 10 to about 10: 1.
80. The composition, pharmaceutical composition, or vaccine of any one of claims 1- 34, wherein the delivery component comprises a lipopolyamine with the following formula:
Figure imgf000155_0002
81. The composition, pharmaceutical composition, or vaccine of claim 80, wherein the delivery component comprises a mixture of the lipopolyamine and an alkylated derivative of the lipopolyamine.
82. The composition, pharmaceutical composition, or vaccine of claim 81, wherein the alkylated derivative of the lipopolyamine is a polyoxyalkylene, polyvinylpyrrolidone, polyacrylamide, polydimethylacrylamide, polyvinyl alcohol, dextran, poly (L-glutamic acid), styrene maleic anhydride, poly -N-(2 -hydroxypropyl) methacrylamide, or polydivinylether maleic anhydride.
83. The composition, pharmaceutical composition, or vaccine of claim 81 or 82, wherein the ratio of the lipopolyamine to the alkylated derivative of the lipopolyamine in the mixture is 1 : 1 to 10:1.
84. The composition, pharmaceutical composition, or vaccine of any one of claims 80-83, wherein the lipolyamine is present in an amount sufficient to produce a ratio of amine nitrogen in the lipopolyamine to phosphate in the DNA plasmid vector from about 0.01 : 1 to about 50: 1.
85. The composition, pharmaceutical composition, or vaccine of claim 84, wherein the lipolyamine is present in an amount sufficient to produce a ratio of amine nitrogen in the lipopolyamine to phosphate in the DNA plasmid vector from about 0.1 : 10 to about 10:0.1.
86. The composition, pharmaceutical composition, or vaccine of any one of claims 1- 34, wherein the delivery component comprises a poloxamer with the following formula:
Figure imgf000156_0001
or a pharmaceutically acceptable salt thereof, wherein:
A represents an integer from 2 to 141;
B represents an integer from 16 to 67;
C represents an integer from 2 to 141; RA and RC are the same or different, and are R'-L- or H;
L is a bond, — CO — , — CH2 — O — , or — O — CO — ; and
R' is a metal chelator.
87. The composition, pharmaceutical composition, or vaccine of claim 86, wherein at least one of RA and RC is R'-L-.
88. The composition, pharmaceutical composition, or vaccine of claim 86, wherein the R’ is covalently bound to the poloxamer.
89. The composition, pharmaceutical composition, or vaccine of claim 86-88, wherein one metal chelator or two or more metal chelators is/are bound to the poloxamer.
90. The composition, pharmaceutical composition, or vaccine of claim 86-89, wherein 2-100 metal chelators are bound to the poloxamer.
91. The composition of claim 86, wherein the metal chelator is RNNH — , RN2N — , or (R" — (N(R") — CH2CH2)x)2 — N — CH2CO — , wherein each x is independently 0-2, and wherein R" is HO2C — CH2 — .
92. The composition, pharmaceutical composition, or vaccine of any one of claims 86-91, wherein the metal chelator is a crown ether, a substituted-crown ether, a cryptand, or a sub stituted-cry ptand .
93. The composition, pharmaceutical composition, or vaccine of any one of claims 86-92, wherein the poloxamer is present in a solution with the polynucleotide or DNA plasmid vector from about 0.01% - about 5%.
94. The composition, pharmaceutical composition, or vaccine of claim 93, wherein the solution is co-formulated with a metal chelator.
95. The composition, pharmaceutical composition, or vaccine of claim 94, wherein the co-formulated metal chelator is present in the solution at a concentration of about 0. Img/mL to about 20mg/mL.
96. The composition, pharmaceutical composition, or vaccine of any one of claims 94-95, wherein the co-formulated metal chelator is crown ether, a substituted-crown ether, a cryptand, or a substituted-cryptand.
97. The composition, pharmaceutical composition, or vaccine of any one of claim 86- 96, wherein the metal chelator and/or co-formulated metal chelator is crown ether (Aza- 18- cr own-6).
98. The composition, pharmaceutical composition, or vaccine of any one of claims 37-97, wherein the delivery component comprises BD 15-12.
99. The composition, pharmaceutical composition, or vaccine of claim 98, wherein the nucleotide to polymer (N:P) ratio is 0.1 : 1 to 5: 1.
100. The composition, pharmaceutical composition, or vaccine of any one of claims 37-97, wherein the delivery component comprises Omnifect.
101. The composition, pharmaceutical composition, or vaccine of claim 100, wherein the nucleotide to polymer (N:P) ratio is 0.01 : 1 to 5: 1.
102. The composition, pharmaceutical composition, or vaccine of any one of claims 37-97, wherein the delivery component comprises Crown Poloxamer.
103. The composition, pharmaceutical composition, or vaccine of claim 102, wherein the nucleotide to polymer (N:P) ratio is 0.01 : 1 to 5: 1.
104. The composition, pharmaceutical composition, or vaccine of claim 102 or 103, wherein the delivery component further comprises a PEG-PEI-chole sterol (PPC) lipopolymer, benzalkonium chloride (BAK), Omnifect, or a linear polyethyleneimine (LPEI).
105. The composition, pharmaceutical composition, or vaccine of 104, wheren the LPEI is BD15-12.
106. The composition, pharmaceutical composition, or vaccine of any one of claims 37-97, wherein the delivery component comprises Staramine and mPEG modified Staramine.
107. The composition, pharmaceutical composition, or vaccine of claim 106, wherein the mPEG modified Staramine is Staramine-mPEG515.
108. The composition, pharmaceutical composition, or vaccine of claim 106, wherein the mPEG modified Staramine is Staramine-mPEGl 1.
109. The composition, pharmaceutical composition, or vaccine of any one of claims 106-108, wherein the ratio of Staramine to mPEG modified Staramine is 10: 1.
110. The composition, pharmaceutical composition, or vaccine of any one of claims 106-109, wherein the nucleotide to polymer (N:P) ratio is 0.01 : 1 to 5: 1.
111. The composition, pharmaceutical composition, or vaccine of any one of claims 106-110, wherein the delivery component further comprises crown pol oxamer.
112. The composition, pharmaceutical composition, or vaccine of any of claims 102-
111, wherein the Crown Pol oxamer is derivatized with a cationic molecule, a ligand, or other chemical entity.
113. The composition, pharmaceutical composition, or vaccine of any one of claims 1-
112, wherein the composition is stable at 0°C to 5°C for at least about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 12 months.
114. The composition, pharmaceutical composition, or vaccine of any one of claims 1- 113, wherein the composition is stable at 25°C for at least about 7 days, about 10 day, about 14 days, or about 60 days.
115. The composition, pharmaceutical composition, or vaccine of any one of claims 1- 112, wherein the composition is stable at -20°C for at least about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 24 months or about 36 months.
116. The composition, pharmaceutical composition, or vaccine of any one of claims 1- 115, wherein the composition is lyophilized and is substantially free of aqueous components.
117. The composition, pharmaceutical composition, or vaccine of claim 116, wherein the composition is reconstituted with a diluent.
118. The composition, pharmaceutical composition, or vaccine of claim 117, wherein the diluent is water.
119. A kit comprising the composition, pharmaceutical composition, or vaccine of any one of claims 1-118.
120. The kit of claim 119, further comprising a glass vial.
121. The kit of claim 118 or 119, further comprising instructions for using the composition or lyophilized composition in a method for inducing an immune response in a subject.
122. The kit of claim 118 or 119, further comprising instructions for using the composition or lyophilized composition in a method for preventing, reducing the incidence of, attenuating or treating a tumor in a subject.
123. The kit of claim 122, wherein the tumor is an ovarian cancer, melanoma, colorectal cancer, liver cancer, pancreatic cancer, brain cancer, prostate cancer, bladder cancer, renal cancer, or hematological cancer.
124. A method of inducing an immune response in a subject, the method comprising administering an effective amount of the composition, pharmaceutical composition, or vaccine of any one of claims 1-118 to the subject.
125. The method of claim 124, wherein the immune response is to one or more tumor- associated antigens.
126. The method of claim 118 or 125, wherein the immune response is a protective immune response.
127. The method of any one of claim 124-126, wherein the method further comprises administering a therapeutically effective amount of an additional antineoplastic therapy.
128. The method of claim 127, wherein the additional antineoplastic comprises a chemotherapeutic agent, an anti angiogenic agent, a PARP inhibitor, an antibody, a PI3 kinase inhibitor, a tyrosine kinase inhibitor, an adoptive cell therapy, or combinations thereof.
129. The method of claim 127 or 128, wherein the additional antineoplastic therapy is surgery or radiation therapy.
130. The method of claim 129, wherein the surgery is performed or the radiation therapy is administered before the administration of an effective amount of the composition, pharmaceutical composition, or vaccine.
131. The method of claim 129, wherein the surgery is performed or the radiation therapy is administered before the administration of an effective amount of the composition, pharmaceutical composition, or vaccine.
132. A method of preventing, reducing the incidence of, attenuating or treating a tumor in a subject, the method comprising administering an effective amount of the composition, pharmaceutical composition, or vaccine of any one of claims 1-118 to the subject.
133. The method of claim 132, wherein the tumor is an ovarian cancer, melanoma, colorectal cancer, liver cancer, pancreatic cancer, brain cancer, prostate cancer, bladder cancer, renal cancer, or hematological cancer.
134. The method of claim 132 or 133, wherein the tumor is an ovarian cancer or a melanoma.
135. The method of any one of claim 132-134, wherein the method further comprises administering a therapeutically effective amount of an additional antineoplastic therapy.
136. The method of claim 135, wherein the additional antineoplastic comprises a chemotherapeutic agent, an anti angiogenic agent, a PARP inhibitor, an antibody, a PI3 kinase inhibitor, a tyrosine kinase inhibitor, an adoptive cell therapy, or combinations thereof.
137. The method of claim 135 or 136, wherein the additional antineoplastic therapy is surgery or radiation therapy.
138. The method of claim 137, wherein the surgery is performed or the radiation therapy is administered before the administration of an effective amount of the composition, pharmaceutical composition, or vaccine.
139. The method of claim 137, wherein the surgery is performed or the radiation therapy is administered after the administration of an effective amount of the composition, pharmaceutical composition, or vaccine.
140. The method of any one of claims 124-139, wherein the composition is administered to the subject by an intramuscular, subcutaneous, intralymphatic, or intraperitoneal route of administration.
141. The method of any one of claims 124-140, wherein the additional antineoplastic therapy is administered to the subject by an intramuscular, subcutaneous, intralymphatic, or intraperitoneal route of administration.
142. A method of making a vaccine, the method comprising the steps of: (a) combining the delivery component with the polynucleotide of the composition or pharmaceutical composition of any one of claims 1-118, (b) lyophilizing the combined delivery component and polynucleotide to a powder, and (c) reconstituting the powder with a diluent that comprises the adjuvant to form a vaccine solution.
143. The method of claim 142 further comprising prior to (a) the steps of: (i) sequencing a subject’s nucleic acid sequence of a tumor-associated antigen, and (ii) generating an antigenic nucleic acid of the polynucleotide of the composition from the subject’s nucleic acid sequence of a tumor-associated antigen.
PCT/US2023/065482 2022-04-06 2023-04-06 Polynucleotide cancer vaccine compositions and methods of using the same Ceased WO2023196936A1 (en)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100004313A1 (en) * 2008-02-29 2010-01-07 Tbd Modified Poloxamers for Gene Expression and Associated Methods
US20190343942A1 (en) * 2016-04-22 2019-11-14 Curevac Ag Rna encoding a tumor antigen
US20200282032A1 (en) * 2016-05-27 2020-09-10 Etubics Corporation Neoepitope vaccine compositions and methods of use thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100004313A1 (en) * 2008-02-29 2010-01-07 Tbd Modified Poloxamers for Gene Expression and Associated Methods
US20190343942A1 (en) * 2016-04-22 2019-11-14 Curevac Ag Rna encoding a tumor antigen
US20200282032A1 (en) * 2016-05-27 2020-09-10 Etubics Corporation Neoepitope vaccine compositions and methods of use thereof

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