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WO2025260013A1 - Compositions et procédés d'adjuvants à base d'arnm pour vaccins à arnm - Google Patents

Compositions et procédés d'adjuvants à base d'arnm pour vaccins à arnm

Info

Publication number
WO2025260013A1
WO2025260013A1 PCT/US2025/033590 US2025033590W WO2025260013A1 WO 2025260013 A1 WO2025260013 A1 WO 2025260013A1 US 2025033590 W US2025033590 W US 2025033590W WO 2025260013 A1 WO2025260013 A1 WO 2025260013A1
Authority
WO
WIPO (PCT)
Prior art keywords
antigen
mrna
peptide
nanoparticle
forms
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/033590
Other languages
English (en)
Inventor
Jeffrey ISHIZUKA
Alex FREY
Kelly OLINO
Frankie SCALLO
David Braun
Therese CORDERO-DUMIT
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Yale University
Original Assignee
Yale University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Yale University filed Critical Yale University
Publication of WO2025260013A1 publication Critical patent/WO2025260013A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • 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
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55516—Proteins; Peptides
    • 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
    • A61K2039/55522—Cytokines; Lymphokines; Interferons
    • A61K2039/55527—Interleukins
    • 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
    • A61K2039/80—Vaccine for a specifically defined cancer
    • A61K2039/876—Skin, melanoma
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies

Definitions

  • the disclosed invention is generally in the field of vaccine design and specifically in the area of enhancing the efficacy and applicability of mRNA vaccines.
  • adjuvant typically refers to substances that are added for the purpose of increasing the local inflammatory response and thereby increase the immunogenicity of the vaccine and enhance long term protection. This concept originated from observations that the efficacy of early vaccines varied significantly from batch to batch, and that the presence of contaminants often paradoxically increased protection from infection.
  • adjuvants that range from metal salts (e.g.
  • mRNA vaccines have recently changed the landscape of treatment and prophylaxis in the fields of infectious disease and cancer.
  • adjuvant signals may include not just generalized triggers for inflammation, but coordinated combinations of discrete molecular programs designed to enable or suppress particular immune functions and drive specific subsets of immune cells. Therefore, it is an object of the invention to provide enhanced mRNA vaccine reagents that impart enhanced immunogenicity.
  • mRNA vaccines encoding one or more peptide antigens for expression in a host cell can be enhanced by the addition of one or more mRNA sequences encoding one or more peptide adjuvant(s) for expression of the adjuvant(s) in the same host cell.
  • Compositions of vaccines including mRNA sequences encoding peptide antigen(s) and peptide adjuvant(s) are described.
  • the peptide antigen(s) and peptide adjuvant(s) can be encoded on the same or different mRNA molecule.
  • the vaccines include a single mRNA molecule encoding one or more antigens and one or more adjuvants, for expression in the same host cell.
  • Nanoparticles encapsulating the mRNA sequences encoding peptide antigen(s) and peptide adjuvant(s) or immuno-signaling molecules are provided for administration to a subject in vivo. Methods of vaccinating a subject including administering to the subject a composition including mRNA sequences encoding peptide antigen(s) and peptide adjuvant(s) or immuno-signaling molecules are also described.
  • compositions of synthetic messenger ribonucleic acid (mRNA) molecules including one or more ribonucleic acid sequences encoding one or more peptide antigens, and one or more ribonucleic acid sequences encoding one or more peptide adjuvant(s) and/or other immuno- modulatory agent(s), are described.
  • the mRNA is configured to express the peptide antigen(s) and the peptide adjuvant(s) and/or other immuno-modulatory agent(s) as distinct polypeptides within a mammalian cell.
  • the mRNA(s) encoding each of the antigen(s) and the mRNA(s) encoding the adjuvant(s) are connected via one or more cleavable linker.
  • cleavable linkers include a P2A linker and a Furin cleavage site.
  • the Furin cleavage site excludes the linker amino acids.
  • the Furin cleavage site and the P2A linker are joined by a linker including the amino acid residues GSG.
  • the synthetic mRNA includes one or more of a single 5’ Cap 1 structure, a poly Adenylated 3’ tail structure, a single 5’ untranslated region (5’ UTR), and/or a single 3’ untranslated region (3’ UTR).
  • the one or more ribonucleic acid sequences encoding one or more peptide antigen(s) further include a ribonucleic acid sequence encoding a polypeptide targeting motif.
  • An exemplary polypeptide targeting motif enhances binding, uptake or processing of the antigen by an antigen presenting cell (APC).
  • one or more ribonucleic acid sequences 455738066.1 2 encoding a peptide antigen(s) is fused with a sequence encoding a polypeptide targeting motif.
  • an amino-terminus of a peptide antigen is coupled to a carboxyl terminus of a targeting motif.
  • the polypeptide targeting motif includes a Clec9a binding motif.
  • the synthetic mRNA includes, from 5’ to 3’: a 5’Cap 1 structure; a 5’ untranslated region (5’UTR); a sequence encoding a first polypeptide; a first Furin cleavage sequence; a first P2A linker sequence; a sequence encoding a second polypeptide; a 3’untranslated region (3’UTR); and a poly Adenylated 3’tail.
  • a first polypeptide encodes the peptide antigen and a second polypeptide encodes a peptide adjuvant or other immuno-modulatory agent.
  • a first polypeptide encodes the peptide adjuvant or other immuno-modulatory agent and a second polypeptide encodes a peptide antigen.
  • the synthetic mRNA includes, from 5’ to 3’: a 5’ Cap 1 structure; a 5’untranslated region (5’ UTR); a sequence encoding a first polypeptide; a first Furin cleavage sequence; a first P2A linker sequence; a sequence encoding a second polypeptide; a second Furin cleavage sequence; a second P2A linker sequence; a sequence encoding a third polypeptide; a 3’untranslated region (3’ UTR); and a poly Adenylated 3’ tail.
  • the first or the second polypeptide encodes a first peptide antigen
  • the first or the second polypeptide encodes a first peptide adjuvant or other immuno-modulatory agent
  • the third polypeptide encodes a second peptide adjuvant or other immuno-modulatory agent(s) or a second peptide antigen.
  • Compositions of nanoparticles including the described synthetic mRNAs are also provided.
  • the synthetic mRNA is encapsulated within the nanoparticle.
  • Compositions of cells including the described nanoparticles mRNAs are also provided.
  • synthetic nanoparticles for delivery of therapeutic and/or prophylactic agents to a subject include the described synthetic ribonucleic acid sequences encoding one or more peptide antigens and one or more ribonucleic acid sequences encoding one or more peptide adjuvants.
  • the ribonucleic acids are encapsulated within the nanoparticle, and/or the one or more ribonucleic acid sequences encoding the one or more antigens and the one or more adjuvants are included within the same ribonucleic acid.
  • the ribonucleic acid sequence(s) includes between approximately 100 and 15,000 bases, inclusive.
  • the ribonucleic acid sequence(s) encode two or more species of protein antigens. In some forms, the ribonucleic acid sequence(s) encode two or more species of protein adjuvants. In some forms, one or more peptide antigen(s) is derived from a source selected from the group including an infectious agent, a parasite, a cancer and a peptide allergen. For example, in some forms, one or more peptide antigen(s) is derived from an infectious agent selected from a virus, a fungi, a protozoan and a bacterium. In certain forms, one or more peptide antigen is derived from a virus.
  • one or more peptide antigen(s) is derived from a virus selected from a filovirus, an arenavirus, a coronavirus an orthopoxvirus and an orthomyxovirus.
  • viruses include Merkel Cell Polyomavirus (MCPyV), Influenza virus, Ebola virus, Zika Virus, and SARS-CoV-2.
  • MCPyV Merkel Cell Polyomavirus
  • one or more peptide antigen(s) is derived from a fungus, for example, Candida albicans, Aspergillus fumigatus, Candida auris, Cryptococcus neoformans, or Candida glabrata.
  • one or more peptide antigen(s) is, or is derived from a cancer antigen, for example, alpha- actinin-4, Alphafetoprotein (AFP), Bcr-Abl fusion protein, Carcinoembryonic antigen (CEA), CA-125, Casp-8, beta-catenin, cdc27, cdk4, cdkn2a, coa-1, dek-can fusion protein, epithelial tumor antigen, EF2, ETV6-AML1 fusion protein, LDLR-fucosyltransferaseAS fusion protein, HLA-A2, HLA-A11, hsp70-2, KIAAO205, Mart2, Mum-1, 2, and 3, neo-PAP, myosin class I, OS-9, pml-RARa fusion protein, PTPRK, K-ras, N-ras, Triosephosphate isomeras, Bage-1, Gage 3,4,5,6,7,
  • one or more peptide antigen(s) is derived from a bacterium, for example, Bacillus Spp, Yersinia Spp, Spirochetes Spp, Rickettsia Spp, Staphylococcus aureus, or Escherichia coli.
  • one or more peptide antigen(s) is a tolerogenic antigen, optionally derived from an allergen.
  • one or more peptide antigen(s) is derived from a protozoan.
  • Exemplary peptide antigen(s) that may be included in the described compositions include a cytokine, a TLR agonist, a pattern recognition receptor, a chemotactic factor, and a transcriptional regulator.
  • Exemplary cytokines include IL-2, IL-7 and IL-15.
  • Pharmaceutical composition including the described synthetic mRNAs, and/or nanoparticles encapsulating the described synthetic mRNAs, and/or cells including the described mRNAs, together with a pharmaceutically acceptable excipient, are also described.
  • the pharmaceutical compositions further include one or more additional active agents. 455738066.1 4 Methods of using the described compositions for inducing an immune response to a peptide antigen in a subject are also provided.
  • the methods include administering to the subject the described pharmaceutical composition(s) in an amount effective to induce or stimulate an immune response in the subject to the peptide antigen(s).
  • the immune response to the peptide antigen is effective to treat or prevent an infection and/or disease in the subject caused by the organism or agent from which the antigen is derived.
  • the peptide antigen is effective to treat or prevent a tumor, for example, whereby the antigen is an autologous tumor antigen.
  • the peptide antigen is effective to treat or prevent a bacterial infection, for example, whereby the antigen is derived from a pathogenic bacterium.
  • the peptide antigen is effective to treat or prevent a viral infection, for example, whereby the antigen is derived from a pathogenic virus.
  • the peptide antigen is effective to treat or prevent a fungal infection, for example, whereby the antigen is derived from a pathogenic fungus.
  • the peptide antigen is effective to treat or prevent a protozoan infection, for example, whereby the antigen is derived from a pathogenic protozoan.
  • composition is administered to the patient via intramuscular, intravenous, subcutaneous or intraperitoneal injection.
  • the subject has a disease or disorder, or has been identified as being at increased risk of getting a disease or disorder.
  • the subject has cancer, or has been identified as being at increased risk of getting a cancer.
  • the subject has an infectious disease, or has been identified as being at increased risk of getting an infectious disease.
  • Kits including the described synthetic mRNAs, or the described nanoparticles of any are also provided.
  • the kit further includes instructions for performing the described methods.
  • a nucleic acid including the nucleotide sequence set forth in SEQ ID NO:18 is also provided.
  • FIG. 1 is a diagram of a plasmid designed to encode a functional mRNA subunit of human IL-7.
  • the coding sequence for mRNA subunit containing IL-7 gene is present between MluI and XbaI restriction sites and is under the control of a T7 promoter.
  • the T7 promoter sequence is modified to end with an AGG sequence for compatibility with Trilink (San Diego, 455738066.1 5 CA) CleanCap 5’ mRNA capping technology.5’ untranslated region (UTR) contains a synthetic sequence optimized for high efficiency translation.
  • FIGS. 2A-2D are plots showing elevated levels of cytokines in HEK 293T cells after transfection of lipid nanoparticles (LNP) containing mRNA adjuvants.
  • LNP lipid nanoparticles
  • the cytokine output was determined using ELISA.
  • Fig.2A matched cytokine XCL1
  • Fig.2B IL-7
  • Fig.2C IL-2
  • IRF3 Fig.2D
  • Figure 3 is a plot showing evaluation of adjuvant immunomodulatory effects in vivo.
  • LTA Large T antigen
  • Figures 4A-4D are plots showing the effects of adjuvant IL-7 on immune memory formation. Spleen of mice treated with LTA alone or LTA + IL-7 were processed and analyzed for immune cells populations using flow cytometry.
  • LTA + IL-7 treatments have increased total T cells as well as an increased proportion of CD8+ T cells that are enriched in memory markers (Fig.4B and Fig.4D) compared to LTA alone (Fig.4A and Fig.4C).
  • Figure 5 is a plasmid diagram designed to encode single functional mRNA subunit containing target antigen (LTA) linked to adjuvant IL-2.
  • LTA target antigen
  • a self-cleaving P2A linker with a Furin cleavage site is included that produces full length antigen and functional full-length cytokine as separate end products.
  • the LTA-P2A-IL2 mRNA template Sequence is present between MluI and XbaI restriction sites and is under the control of a T7 promoter.
  • Figure 6 is a plasmid diagram designed to encode single functional mRNA subunit containing target antigen (LTA) linked to adjuvants, IL-7 and IL-2.
  • LTA target antigen
  • a self-cleaving P2A linker followed by a self-cleaving T2A linker with a Furin cleavage site is included that produces full length antigen and functional full-length cytokines (IL-7 and IL-2) as separate end products.
  • the LTA-P2A-IL7-T2A-IL2 mRNA template Sequence is present between MluI and XbaI restriction sites and is under the control of a T7 promoter.
  • Figures 7A-7B are a pair of plots showing production of cytokine with linked mRNA constructs.
  • LTA-P2A-IL7, LTA-P2A-IL2, and LTA-P2A-IL7-T2A-IL2 mRNA were transfected into HEK 293T cells. No transfection (NS), lipofectamine (LIPO), and LTA are used as control samples. The cytokine output was determined using ELISA. High levels of IL-2 (Fig. 7A) and IL-7 (Fig.7B) production are observed with single and double linked constructs compared to other samples.
  • Figures 8A-8B are graphs of the efficacy of the IL-7 + antigen co-encoded vaccine for developing CD8+ T cell memory, showing %Dextramer(+) CD8+ T Cells (Fig.8A) and %Dextramer(+) IL7+CD8+CD3+ (Fig.8B), respectively, for each of placebo, LTA-vaccine (LTA) and LTA+IL-7-containing vaccine (LTA+IL-7) treatment groups, respectively.
  • Figure 9 is a graph of the effect of the IL-2+IL -7+antigen-encoded vaccines on the frequency of antigen-specific T cells, showing CD8+ tetramer+ per mg for each of Ova, IL-7 only, IL-2 only, Ova+IL-7, Ova+IL-2 or Ova+IL-7+IL-2 mRNA encoded immunotherapies, respectively.
  • Figures 10A-10B are graphs of Blue Fluorescent Protein (BFP)+ cell frequency over time (hours) for each of 293T cells (Fig.10A) and MuTuDCs (Fig.10B), respectively, transfected.
  • BFP Blue Fluorescent Protein
  • vector refers to a nucleic acid molecule or polynucleotide, such as a replicating RNA, plasmid, phage, or cosmid, into which another nucleic acid sequence segment may be inserted so as to bring about the replication of the inserted segment.
  • the described vectors can be expression vectors.
  • expression vector refers to a vector that includes one or more expression control sequences.
  • gene refers to isolated or modified nucleic acid sequences, including both RNA and DNA, that encode genetic information for the synthesis of a whole RNA, a whole protein, or any portion of such whole RNA or whole protein. Genes that are not naturally part of a particular organism's genome are referred to as “foreign genes”, “heterologous genes” or “exogenous genes” and genes that are naturally a part of a particular organism's genome are referred to as “endogenous genes”.
  • endogenous genes genes that are naturally a part of a particular organism's genome.
  • RNA nucleic acid molecule at least complementary in part to a region of one of the two nucleic acid strands of the gene.
  • expression also refers to the translation from said RNA nucleic acid molecule to give a protein or polypeptide or a portion thereof.
  • antigen refers to any substance (e.g., peptide, protein, nuclei acid, lipid, small molecule, such as a moiety expressed by or otherwise associated with a pathogen or cancerous or pre-cancerous cell) that serves as a target for the receptors of an adaptive immune response.
  • the antigen may be a structural component of a pathogen, cancerous or pre-cancerous cell.
  • pathogen refers to an organism or other entity that causes a disease.
  • pathogens can be prions, viruses, prokaryotes such as bacteria, eukaryotes such as protozoa and fungi.
  • a pathogen can be the source of an antigen to which an adaptive immune response can be generated.
  • polypeptide includes proteins and fragments thereof. Polypeptides are described as amino acid residue sequences. Those sequences are written left to right in the direction from the amino (N) to the carboxyl (C) terminus.
  • amino acid residue sequences are denominated by either a three letter or a single letter code as indicated as follows: Alanine (Ala, A), Arginine (Arg, R), Asparagine (Asn, N), Aspartic Acid (Asp, D), Cysteine (Cys, C), Glutamine (Gln, Q), Glutamic Acid (Glu, E), Glycine (Gly, G), Histidine (His, H), Isoleucine (Ile, I), Leucine (Leu, L), Lysine (Lys, K), Methionine (Met, M), Phenylalanine (Phe, F), Proline (Pro, P), Serine (Ser, S), Threonine (Thr, T), Tryptophan (Trp, W), Tyrosine (Tyr, Y), and Valine (Val, V).
  • the term “antibody” refers to any immunoglobulin, whether natural or wholly or partially synthetically produced. All derivatives thereof which maintain specific binding ability are also included in the term. The term also covers any protein having a binding domain which is homologous or largely homologous to an immunoglobulin binding domain. Such proteins may be derived from natural sources, or partly or wholly synthetically produced. An antibody may be monoclonal or polyclonal. An antibody may be a member of any immunoglobulin class, including any of the human classes: IgG, IgM, IgA, IgD, and IgE.
  • antibody fragment or “characteristic portion of an antibody” are used interchangeably and refer to any derivative of an antibody which is less than full-length.
  • An antibody fragment can retain at least a significant portion of the full-length antibody's specific binding ability. Examples of such antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, scFv, Fv, dsFv diabody, and Fd fragments.
  • Antibody fragment also include Fc fragments.
  • An antibody fragment may be produced by any means. For example, an antibody fragment may be enzymatically or chemically produced by fragmentation of an intact antibody 455738066.1 8 and/or it may be recombinantly produced from a gene encoding the partial antibody sequence.
  • an antibody fragment may be wholly or partially synthetically produced.
  • An antibody fragment may optionally comprise a single chain antibody fragment.
  • an antibody fragment may include multiple chains which are linked together, for example, by disulfide linkages.
  • An antibody fragment may optionally include a multimolecular complex.
  • a functional antibody fragment will typically include at least about 50 amino acids and more typically will include at least about 200 amino acids.
  • the terms “individual,” “subject,” and “patient” are used interchangeably, and refer to a mammal, including, but not limited to, humans, rodents, such as mice and rats, and other laboratory animals.
  • the terms “approximately” or “about” in reference to a number are generally taken to include numbers that fall within a range of 5%, 10%, 15%, or 20% in either direction (greater than or less than) of the number unless otherwise stated or otherwise evident from the context (except where such number would be less than 0% or exceed 100% of a possible value).
  • the term “associated with” refers to the state of two or more entities which are linked by a direct or indirect covalent or non-covalent interaction. In some forms, an association is covalent. In some forms, a covalent association is mediated by a linker moiety.
  • an association is non-covalent (e.g., charge interactions, affinity interactions, metal coordination, physical adsorption, host-guest interactions, hydrophobic interactions, TT stacking interactions ( ⁇ - ⁇ stacking interactions), hydrogen bonding interactions, van der Waals interactions, magnetic interactions, electrostatic interactions, dipole-dipole interactions, etc.).
  • an entity e.g., immunomodulatory agent, targeting moiety, immunostimulatory agent, nanoparticle, etc.
  • an entity e.g., immunomodulatory agent, targeting moiety, immunostimulatory agent, nanoparticle, etc.
  • an entity e.g., immunomodulatory agent, targeting moiety, immunostimulatory agent, nanoparticle, etc.
  • the entity may be associated with the surface of, encapsulated within, surrounded by, and/or distributed throughout a lipid bilayer, lipid monolayer, polymeric matrix, etc. of an inventive vaccine nanocarrier.
  • the term “cell type” refers to a form of cell having a distinct set of morphological, biochemical, and/or functional characteristics that define the cell type.
  • a cell type can be defined with varying levels of specificity.
  • T cells and B cells are distinct cell types, which can be distinguished from one another but share certain features that are characteristic of the broader “lymphocyte” cell type of which both are members.
  • cells of different types may be distinguished from one 455738066.1 9 another based on their differential expression of a variety of genes which are referred to in the art as “markers” of a particular cell type or types (e.g., cell types of a particular lineage).
  • markers e.g., cell types of a particular lineage.
  • cells of different types may be distinguished from one another based on their differential functions.
  • a “cell type-specific marker” is a gene product or modified version thereof that is expressed at a significantly greater level by one or more cell types than by all or most other cell types and whose expression is characteristic of that cell type. Many cell-type specific markers are recognized as such in the art.
  • the term “in vitro” refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc., rather than within an organism (e.g., animal, plant, and/or microbe).
  • the term “in vivo” refers to events that occur within an organism (e.g., animal, plant, and/or microbe).
  • the terms “immunologic”, “immunological” or “immune” response refer to the development of a beneficial humoral (antibody mediated) and/or a cellular (mediated by antigen- specific T cells or their secretion products) response directed against an immunogen in a recipient patient.
  • Such a response can be an active response induced by administration of immunogen or a passive response induced by administration of antibody or primed T-cells.
  • the relative contributions of humoral and cellular responses to the protective or therapeutic effect of an immunogen can be distinguished by separately isolating antibodies and T-cells from an immunized syngeneic animal and measuring protective or therapeutic effect in a second subject.
  • the term “immunostimulatory agent” refers to an agent that modulates an immune response to an antigen but is not the antigen or derived from the antigen.
  • “Modulate”, as used herein, refers to inducing, enhancing, suppressing, directing, or redirecting an immune response.
  • Such agents include immunostimulatory agents that stimulate (or boost) an immune response to an antigen but, as defined above, is not the antigen or derived from the antigen.
  • Immunostimulatory agents therefore, include adjuvants.
  • the immunostimulatory agent is on the surface of the nanocarrier and/or is encapsulated within the nanocarrier.
  • the immunostimulatory agent on the surface of the nanocarrier is different from the immunostimulatory agent encapsulated within the nanocarrier.
  • the nanocarrier includes more than one type of immunostimulatory agent. In some forms, the more than one type of immunostimulatory agent act on different pathways. Examples of immunostimulatory agents include those provided elsewhere herein.
  • nucleic acid in its broadest sense, refers to any compound and/or substance that is or can be incorporated into an oligonucleotide chain.
  • a nucleic acid is a compound and/or substance that is or can be incorporated into an 455738066.1 10 oligonucleotide chain via a phosphodiester linkage.
  • nucleic acid refers to individual nucleic acid residues (e.g., nucleotides and/or nucleosides).
  • nucleic acid refers to an oligonucleotide chain including individual nucleic acid residues.
  • nucleic acid encompasses RNA as well as single and/or double-stranded DNA and/or cDNA, any natural or synthetic linear and sequential arrays of nucleotides and nucleosides, for example, replicating RNA (repRNA), messenger RNA (mRNA), small interfering RNA (siRNA), transfer RNA (tRNA), microRNA (miRNA), guide strand RNA (sgRNA), polynucleotides, oligo-nucleotides, oligo-nucleosides and derivatives thereof.
  • repRNA replicating RNA
  • mRNA messenger RNA
  • siRNA small interfering RNA
  • tRNA transfer RNA
  • miRNA transfer RNA
  • sgRNA guide strand RNA
  • nucleic acids may be collectively referred to as “constructs,” or “plasmids.”
  • nucleic acid DNA
  • RNA Ribonucleic acid
  • nucleic acid analogs i.e., analogs having other than a phosphodiester backbone.
  • peptide nucleic acids which are known in the art and have peptide bonds instead of phosphodiester bonds in the backbone, are considered within the scope of the present invention.
  • nucleotide sequence encoding an amino acid sequence includes all nucleotide sequences that are degenerate versions of each other and/or encode the same amino acid sequence. Nucleotide sequences that encode proteins and/or RNA may include introns.
  • Nucleic acids can be purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, etc. Where appropriate, e.g., in the case of chemically synthesized molecules, nucleic acids can include nucleoside analogs such as analogs having chemically modified bases or sugars, backbone modifications, etc. A nucleic acid sequence is presented in the 5' to 3' direction unless otherwise indicated.
  • the term “nucleic acid segment” is used herein to refer to a nucleic acid sequence that is a portion of a longer nucleic acid sequence. In many forms, a nucleic acid segment includes at least 3, 4, 5, 6, 7, 8, 9, l 0, or more residues.
  • a nucleic acid is or includes natural nucleosides (e.g., adenine (A); cytosine (C); guanine (G); and thymine (T) (uracil (U) for thymine (T) when the polynucleotide is RNA), deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine); nucleoside analogs (e.g., 2-aminoadenosine, 2- thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5- fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynylcyt
  • nucleotide sequences are provided using 455738066.1 11 character representations recommended by the International Union of Pure and Applied Chemistry (IUPAC) or a subset thereof.
  • the set of characters is (A, C, G, T, U) for adenosine, cytidine, guanosine, thymidine, and uridine respectively.
  • the set of characters is (A, C, G, T, U, I, X, ⁇ ) for adenosine, cytidine, guanosine, thymidine, uridine, inosine, uridine, xanthosine, pseudouridine respectively.
  • the set of characters is (A, C, G, T, U, I, X, ⁇ , R, Y, N) for adenosine, cytidine, guanosine, thymidine, uridine, inosine, uridine, xanthosine, pseudouridine, unspecified purine, unspecified pyrimidine, and unspecified nucleotide respectively.
  • a “particle” refers to any entity having a diameter of less than 10 microns ( ⁇ m). Typically, particles have a longest dimension (e.g., diameter) of 1000 nm or less. In some forms, particles have a diameter of 300 nm or less.
  • Particles include microparticles, nanoparticles, and pico-particles.
  • nanoparticles have a diameter of 200 nm or less.
  • nanoparticles have a diameter of 100 nm or less.
  • nanoparticles have a diameter of 50 nm or less.
  • nanoparticles have a diameter of 30 nm or less.
  • nanoparticles have a diameter of 20 nm or less.
  • nanoparticles have a diameter of 10 nm or less.
  • particles can be a matrix of polymers.
  • particles can be a non-polymeric particle (e.g., a metal particle, quantum dot, ceramic, inorganic material, bone, etc.). Particles may also be liposomes and/or micelles. As used herein, the term “nanoparticle” refers to any particle having a diameter of less than 1000 run.
  • the nanocarriers of the compositions provided herein, in some forms, have a mean geometric diameter that is less than 500 nm. In some forms, the nanocarriers have mean geometric diameter that is greater than 50 nm but less than 500 nm.
  • the mean geometric diameter of a population of nanocarriers is about 60 nm, 75 nm, 100 nm, 125 nm, 150 nm, 175 nm, 200 nm, 225 run, 250 nm, 275 nm, 300 nm, 325 nm, 350 run, 375 nm, 400 nm, 425 nm, 450 nm, or 475 nm.
  • the mean geometric diameter is between 100-400 nm, 100-300 nm, 100-250 nm, or 100-200 run.
  • the mean geometric diameter is between 60-400 nm, 60-350 run, 60-300 nm, 60-250 nm, or 60-200 nm. In some forms, the mean geometric diameter is between 75-250 nm. In some forms, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more of the nanocarriers of a population of nanocarriers have a diameter that is less than 500 nM. In some forms, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more of the nanocarriers of a population of nanocarriers have a diameter that is greater than 50 nm but less than 500 nm.
  • the nanocarriers of a population of nanocarriers have a diameter of about 60 nm, 75 nm, 100 nm, 125 nm, 150 nm, 175 nm, 200 nm, 225 nm, 250 nm, 275 nm, 300 nm, 325 nm, 350 nm, 375 nm, 400 nm, 425 nm, 450 nm, or 475 nm.
  • 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more of the nanocarriers of a population of nanocarriers have a diameter that is between 100-400 nm, 100-300 nm, 100-250 nm, or 100- 200 nm. In some forms, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more of the nanocarriers of a population of nanocarriers have a diameter that is between 60-400 nm, 60-350 nm, 60-300 nm, 60-250 nm, or 60-200 nm.
  • the term “poorly immunogenic antigen” refers to an antigen that does not trigger any or a sufficient level of a desired immune response. “Sufficient”, as used herein, refers to the ability to elicit a detectable or protective immune response when administered in a composition that does not employ a nanocarrier described herein, e.g., as free antigen mixed with adjuvant in the absence of a nanocarrier.
  • the desired immune response is to treat or prevent a disease or condition.
  • the desired immune response is to alleviate one or more symptoms of a disease or condition. Poorly immunogenic antigens include, but are not limited to, self antigens, small molecules, and carbohydrates.
  • self antigen refers to a normal substance in the body of an animal that when an immune response against the antigen within the animal is triggered, autoimmunity (e.g., an autoimmune disease) can result.
  • a self antigen can be a protein or peptide, lipoprotein, lipid, carbohydrate, or a nucleic acid.
  • the nucleic acid can be a DNA or RNA.
  • Self antigens include, but are not limited to enzymes, structural proteins, secreted proteins, cell surface receptors, and cytokines. In some forms, the self antigen is a cytokine, and the cytokine is TNF, IL-1, or IL-6.
  • the self antigen is cholesteryl ester transfer protein (CETP), a serum protein responsible for cholesterol transfer from high-density lipoprotein (HDL) to low-density lipoprotein cholesterol (LDL), the AP protein associated with Alzheimer's, a proteolytic enzyme that processes the pathological form of the AP protein, LDL associated with atherosclerosis, or a coreceptor for HIV-1.
  • the proteolytic enzyme that processes the pathological form of the AP protein is beta-secretase.
  • the LDL associated with atherosclerosis is oxidized or minimally modified.
  • the coreceptor for HIV-1 is CCR5.
  • a “small molecule” is understood in the art to be an organic molecule that is less than about 2000 g/mol in size. In some forms, the small molecule is less than about 1500 g/mol or less than about 1000 g/mol. In some forms, the small molecule is less than about 800 g/mol or less than about 500 g/mol. In some forms, small molecules are non-polymeric and/or non-oligomeric. In some forms, small molecules are not proteins, peptides, or amino acids. In 455738066.1 13 some forms, small molecules are not nucleic acids or nucleotides. In some forms, small molecules are not saccharides or polysaccharides.
  • T cell antigen refers to any antigen that is recognized by and triggers an immune response in a T cell (e.g., an antigen that is specifically recognized by a T cell receptor on a T cell via presentation of the antigen or portion thereof bound to a major histocompatibility complex molecule (MHC).
  • MHC major histocompatibility complex molecule
  • an antigen that is a T cell antigen is also a B cell antigen.
  • the T cell antigen is not also a B cell antigen.
  • T cells antigens generally are proteins or peptides.
  • T cell antigens may be an antigen that stimulates a CD8+ T cell response, a CD4+ T cell response, or both.
  • targets refers to any entity that is capable of specifically binding to a particular targeting moiety.
  • targets are specifically associated with one or more particular tissue types.
  • targets are specifically associated with one or more particular cell types. For example, a cell type specific marker is typically expressed at levels at least 2 fold greater in that cell type than in a reference population of cells.
  • the cell type specific marker is present at levels at least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, at least 50 fold, at least 100 fold, or at least 1000 fold greater than its average expression in a reference population. Detection or measurement of a cell type specific marker may make it possible to distinguish the cell type or types of interest from cells of many, most, or all other types.
  • a target can include a protein, a carbohydrate, a lipid, and/or a nucleic acid, as described herein. A substance is considered to be “targeted” for the purposes described herein if it specifically binds to a target.
  • a targeting moiety specifically binds to a target under stringent conditions.
  • An inventive nanocarrier, such as a vaccine nanocarrier, including a targeting moiety is considered to be “targeted” if the targeting moiety specifically binds to a target, thereby delivering the entire nanocarrier to a specific organ, tissue, cell, and/or subcellular locale.
  • targeting moiety refers to any moiety that binds to a component of a cell. In some forms, the targeting moiety specifically binds to a component of a cell.
  • the targeting moiety is encapsulated within the nanocarrier. In still other forms, the targeting moiety is associated with the nanocarrier. In some forms, the targeting moiety is covalently associated with the nanocarrier. In other forms, the targeting moiety is non-covalently associated with the nanocarrier. In yet other forms, the targeting moiety binds a receptor expressed on the surface of a cell. The targeting moiety, in some forms, binds a soluble receptor. In some forms, the soluble receptor is a complement protein or a pre-existing antibody. In further forms, the targeting moiety is for delivery of the nanocarrier to antigen presenting cells, T cells, or B cells. In some forms, the antigen presenting cells are macrophages.
  • the term “therapeutically effective amount” means an amount of a therapeutic, prophylactic, and/or diagnostic agent (e.g., inventive vaccine nanocarrier) that is sufficient, when administered to a subject suffering from or susceptible to a disease, disorder, and/or condition, to treat, alleviate, ameliorate, relieve, alleviate symptoms of, prevent, delay onset of, inhibit progression of, reduce severity of, and/or reduce incidence of the disease, disorder, and/or condition.
  • inventive vaccine nanocarrier e.g., inventive vaccine nanocarrier
  • the term “therapeutic agent” refers to any agent that, when administered to a subject, has a therapeutic, prophylactic, and/or diagnostic effect and/or elicits a desired biological and/or pharmacological effect.
  • the term “treating” refers to partially or completely alleviating, ameliorating, relieving, delaying onset of, inhibiting progression of, reducing severity of, and/or reducing incidence of one or more symptoms or features of a particular disease, disorder, and/or condition.
  • “treating” a microbial infection may refer to inhibiting survival, growth, and/or spread of the microbe.
  • Treatment may be administered to a subject who does not exhibit 455738066.1 15 signs of a disease, disorder, and/or condition and/or to a subject who exhibits only early signs of a disease, disorder, and/or condition for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and/or condition.
  • treatment includes delivery of an inventive vaccine nanocarrier to a subject.
  • the term “vaccine nanocarrier” refers to an entity including at least one immunomodulatory agent or immunostimulatory agent.
  • a vaccine nanocarrier includes at least two types of immunomodulatory agents.
  • the immunomodulatory agents are antigens
  • the vaccine nanocarrier includes 2, 3, 4, 5, 6, 7, 8, 9, 10, or more antigens.
  • T cell stimulation may be assayed by monitoring antigen- induced production of cytokines, antigen- induced proliferation of T cells, and/or antigen- induced changes in protein expression.
  • B cell stimulation may be assayed by monitoring antibody titers, antibody affinities, antibody performance in neutralization assays, class-switch recombination, affinity maturation of antigen-specific antibodies, development of memory B cells, development of long-lived plasma cells that can produce large amounts of high-affinity antibodies for extended periods of time, germinal center reactions, and/or antibody performance in neutralization assays.
  • biocompatible refers to one or more materials that are neither themselves toxic to the host (e.g., an animal or human), nor degrade (if the polymer degrades) at a rate that produces monomeric or oligomeric subunits or other byproducts at toxic concentrations in the host. 455738066.1 16
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the therapeutic compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
  • synthetic messenger ribonucleic acid refers to a non-naturally occurring messenger ribonucleic acid sequence, for example, including two or more open reading frames encoding two or more distinct polypeptides within a single mRNA molecule.
  • the described synthetic mRNAs may include natural nucleotides, synthetic nucleotides (such as nucleotide analogs), or mixtures thereof.
  • Exemplary synthetic mRNAs include a peptide antigen and a peptide adjuvant co-encoded on the same mRNA molecule, configured for co-expression of both the peptide antigen and the peptide adjuvant as distinct peptides within a host cell.
  • compositions Compositions of synthetic messenger ribonucleic acid (mRNA) including one or more ribonucleic acid sequences encoding one or more peptide antigens, and/or one or more ribonucleic acid sequences encoding one or more peptide adjuvants, and/or one or more other immune-modulatory agent peptide(s) for the expression of peptide antigen and peptide adjuvant, and optionally one or more other immuno-modulatory peptides in the same host cell are described.
  • mRNA messenger ribonucleic acid
  • the ribonucleic acid is configured to express the peptide antigen(s) and the peptide adjuvant(s) and optionally other immuno-modulatory peptides as distinct polypeptides within a mammalian cell.
  • the synthetic mRNA includes one or more ribonucleic acid sequences encoding one or more antigens and one or more ribonucleic acid sequences encoding one or more adjuvants and optionally one or more ribonucleic acid sequences encoding one or more other immuno-modulatory peptides within the same ribonucleic acid molecule.
  • compositions include mRNA adjuvants co- encapsulated with mRNA antigens, or mRNA adjuvants and optionally other immuno- modulatory peptides, co-encoded on the same mRNA strand as mRNA encoding a vaccine antigen.
  • mRNA adjuvants co- encapsulated with mRNA antigens
  • mRNA adjuvants and optionally other immuno- modulatory peptides co-encoded on the same mRNA strand as mRNA encoding a vaccine antigen.
  • compositions of synthetic mRNA sequences encoding peptide antigen(s) and peptide adjuvant(s) are described.
  • the peptide antigen(s) and peptide adjuvant(s) can be encoded on the same or different mRNA molecules.
  • a synthetic messenger ribonucleic acid includes (a) one or more ribonucleic acid sequences encoding one or more peptide antigens; and (b) one or more ribonucleic acid sequences encoding one or more peptide adjuvants, whereby the ribonucleic acid is configured to express the peptide antigen(s) and the peptide adjuvant(s) as distinct polypeptides within a mammalian cell.
  • the synthetic mRNA includes a single 5’ untranslated region (5’ UTR), and a single 3’untranslated region (3’ UTR).
  • the compositions of synthetic messenger ribonucleic acid (mRNA) include one or more ribonucleic acid sequences encoding one or more peptide adjuvants and/or Immuno-signaling agents.
  • the term “adjuvant” encompasses an immune-stimulatory molecule that is not an antigen per se.
  • the “adjuvant” may be any molecule that provides a co- stimulatory affect in a subject in addition to an antigen-specific immune response in the same subject.
  • immuno-signaling agent(s) encompasses one or more molecules that provide one or more biological signals to provide specific immune functions or effects.
  • a multiplicity of immuno-signaling agents are combined to program specific immune functions or effect.
  • a multiplicity of signals are combined together in a desired combination to provide one or more desired specific immune functions or effects.
  • the multiplicity includes a peptide adjuvant as one of the immuno-signaling agents.
  • the described peptide adjuvants can act via components of the innate and/or adaptive immune systems.
  • the adjuvants act directly or indirectly to direct, stimulate, increase, reduce, prevent minimize and/or otherwise impact one or more of the biological functions of an immune effector cell, such as a dendritic cell, a macrophage cell, a glial cell, a CD4+ helper T cell, a CD8+ cytotoxic T cell, or other immune cells or processes.
  • an immune effector cell such as a dendritic cell, a macrophage cell, a glial cell, a CD4+ helper T cell, a CD8+ cytotoxic T cell, or other immune cells or processes.
  • exemplary peptide “adjuvant” molecules include cytokines, pattern recognition receptors, chemotactic factors, transcriptional regulators, and Toll-Like Receptors (TLRs).
  • adjuvants can include mutant versions of genes or de novo genetic sequences, as long as these sequences directly or through coding for peptide sequences induce specific immune functions or effects.
  • Cytokines In some forms, the peptide adjuvant molecule is a cytokine molecule. Exemplary cytokine molecules include interleukins and tumor necrosis factors.
  • IL-2 Interleukin-2
  • the peptide adjuvant molecule(s) is or includes Interleukin-2 (IL-2).
  • IL-2 is a key cytokine involved in the activation and expansion of T cells, cells particularly cytotoxic T cells (CD8+ T cells) and natural killer (NK) cells (Berraondo et al., British Journal of cancer, 10:6-15, 2019).
  • the T cells and NK cells play a central role in recognizing and killing cancer cells.
  • IL-2 enhances the immune response against cancer.
  • IL-2 is an approximately 15.5 kDa glycoprotein, which includes 4 antiparallel amphipathic alpha helices.
  • IL-2 is predominantly produced by antigen-activated Th1 CD4 T cells and to a lesser extent by CD8 T cells, natural killer (NK) cells, and NK T cells (Conlon et al., Journal of interferon & cytokine research, 39:1, 2019).
  • An exemplary nucleic acid Coding Sequence for IL-2 is as follows: ATGTACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTGCACTTGTCACAAACAGTGCAC CTACTTCAAGTTCTACAAAGAAAACACAGCTACAACTGGAGCATTTACTGCTGGATTTACAGAT GATTTTGAATGGAATTAATAATTACAAGAATCCCAAACTCACCAGGATGCTCACATTTAAGTTT 455738066.1 20 TACATGCCCAAGAAGGCCACAGAACTGAAACATCTTCAGTGTCTGGAAGAAGAACTCAAACCTC TGGAGGAAGTGCTAAATTTAGCTCAAAGCAAAAACTTTCACTTAAGACCCAGGGACTTAATCAG CAATATCAACGTAATAGTTCTGGAACTAAAGGGATCTGAAACAACATTCATGTGTGAATATGCT GATGAGACAGCAACCATTGTAGAATTTCTGAACAGATGGATTACCTTTTGTCAAAGCATCATCT CAACACTGACTTGA (SEQ ID NO:1).
  • IL-2 An exemplary amino acid sequence for IL-2 is as follows: MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKF YMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYA DETATIVEFLNRWITFCQSIISTLT (SEQ ID NO:2) b.
  • Interleukin-7 IL-7
  • the peptide adjuvant molecule(s) is or includes Interleukin-7.
  • IL-7 is another common gamma chain cytokine, produced by nonhematopoietic stromal and epithelial cells: bone marrow stromal cells, MHC II+ thymic epithelial cells, liver and intestinal epithelial cells, keratinocytes, follicular DCs, smooth muscle cells, and sometimes by DCs and macrophages (Conlon, et al., Journal of interferon & cytokine research, 39:1, 2019).
  • An exemplary nucleic acid sequence for IL-7 ATGTTCCATGTTTCTTTTAGGTATATCTTTGGACTTCCTCCCCTGATCCTTGTTCTGTTGCCAG TAGCATCATCTGATTGTGATATTGAAGGTAAAGATGGCAAACAATATGAGAGTGTTCTAATGGT CAGCATCGATCAATTATTGGACAGCATGAAAGAAATTGGTAGCAATTGCCTGAATAATGAATTT AACTTTTTTAAAAGACATATCTGTGATGCTAATAAGGAAGGTATGTTTTTATTCCGTGCTGCTC GCAAGTTGAGGCAATTTCTTAAAATGAATAGCACTGGTGATTTTGATCTCCACTTATTAAAAGT TTCAGAAGGCACAACAATACTGTTGAACTGCACTGGCCAGGTTAAAGGAAGAAAACCAGCTGCC CTGGGTGAAGCCCAACCAACAAAGAGTTTGGAAGAAAATAAATCTTTAAAGGAACAGAAAAC TGAATGACTTGTTTCCTAAAGACTATTACAAGATAAAAACTTGTTGTTG
  • Interleukin-15 In some forms, the peptide adjuvant molecule(s) is or includes Interleukin-15.
  • IL-15 is a 14-to-15 kDa member of the 4-alpha-helix bundle family of cytokines. IL-15 is controlled at the level of transcription, but especially at the level of translation, so that although IL-15 message is 455738066.1 21 widely distributed, it is produced predominantly by DCs, epithelial cells, and monocytes (Conlon et al., Journal of interferon & cytokine research, 39:1, 2019).
  • An exemplary nucleic acid sequence for IL-15 ATGAGAATTTCGAAACCACATTTGAGAAGTATTTCCATCCAGTGCTACTTGTGTTTACTTCTAA ACAGTCATTTTCTAACTGAAGCTGGCATTCATGTCTTCATTTTGGGCTGTTTCAGTGCAGGGCT TCCTAAAACAGAAGCCAACTGGGTGAATGTAATAAGTGATTTGAAAAAAATTGAAGATCTTATT CAATCTATGCATATTGATGCTACTTTATATACGGAAAGTGATGTTCACCCCAGTTGCAAAGTAA CAGCAATGAAGTGCTTTCTCTTGGAGTTACAAGTTATTTCACTTGAGTCCGGAGATGCAAGTAT TCATGATACAGTAGAAAATCTGATCATCCTAGCAAACAACAGTTTGTCTTCTAATGGGAATGTA ACAGAATCTGGATGCAAAGAATGTGAGGAACTGGAGGAAAAAAAAAATATTAAAGAATTTTTGCAGA GTTTTGTACATATTGTCCAAATGTTCATCAACACTTCTTGA (SEQ ID NO
  • IL-15 An exemplary amino acid sequence for IL-15: MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLI QSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNV TESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO:6).
  • Interleukin-21 IL-21
  • the peptide adjuvant molecule(s) is or includes Interleukin-21.
  • IL-21 is composed of 4-alpha-helical bundles (Spolski and Leonard, 2008).
  • IL-21 has a major role in B cell differentiation into plasma cells, in the development of T follicular helper (Tfh) cells, promotes the development of Th17 cells, and enhances the survival, antiviral activity, and antitumor activity of CD8+ T cells.
  • Tfh T follicular helper
  • the peptide adjuvant molecule(s) is or includes Interferon-alpha.
  • This family of cytokines has been used for the treatment of several hematological malignancies and solid tumors at high doses to exploit their direct pro-apoptotic/antiproliferative activity on tumor cells.
  • the peptide adjuvant molecule(s) is or includes Interlekin-10.
  • IL-10 is released by innate and adaptive immune cells to fine-tune the activity of pro-inflammatory cytokines.
  • IL-10 is considered to be an immunosuppressive cytokine as it can decrease the antigen presenting activity of DCs and inhibit the cytotoxic and cytokine release functions performed by T and NK lymphocytes.
  • IL-10 activity In some forms, the peptide adjuvant molecule(s) is or includes TNF- ⁇ .
  • TNF- ⁇ is a pro- inflammatory cytokine produced mainly by myeloid-derived cells such as monocytes, macrophages and DCs, although many other cells such as T lymphocytes, endothelial cells, adipocytes and fibroblasts can also produce this cytokine under stressful conditions.
  • myeloid-derived cells such as monocytes, macrophages and DCs
  • TNF- ⁇ is mainly considered as a mediator of anti-tumor immune responses, and several immunotherapies have shown depleted anti-tumor efficacy when co-administered with TNF- ⁇ antagonists. ii.
  • the peptide adjuvant molecule(s) is or includes a Pattern recognition receptor (PRR)
  • PRRs are a class of receptors that play a crucial role in the innate immune system by recognizing specific patterns associated with pathogens or cellular damage. While PRRs are traditionally associated with host defense against pathogens, their role in cancer treatment has gained increasing attention due to their ability to recognize and respond to tumor-derived molecules or stressed/damaged cells. Activation of these receptors in cancer cells can lead to the production of type I interferons and pro-inflammatory cytokines, which can enhance anti-tumor immune responses.
  • the peptide adjuvant molecule molecule(s) is or includes a Chemotactic Factor.
  • a chemotactic factor also known as a chemotaxin or chemoattractant, is a substance that induces the movement of cells, typically immune cells, towards a higher concentration of the substance. These factors play a crucial role in directing the migration of immune cells to sites of infection, inflammation, or tissue damage. Chemotactic factors are often small molecules, peptides, or proteins that are released by cells in response to various stimuli, such as microbial products, pro-inflammatory cytokines, or tissue injury.
  • chemotactic factors can also contribute to the recruitment of immune cells to the tumor microenvironment. This recruitment can have both pro-tumor and anti-tumor effects, depending on the specific immune cell types involved and the local context within the tumor. a.
  • XCL1 In some forms, the peptide adjuvant molecule(s) is or includes XCL1.
  • XCL1 is a chemotactic factor secreted by activated NK cells, activated Th1-polarized CD4+ T cells, and activated CD8+ T cells, and co-secreted with IFN- ⁇ , MIP-1 ⁇ , MIP-1 ⁇ , and RANTES and is thus part of the Th1 immune defense (Kroczek, et al., Front Immunol.2018; 9: 2806).
  • the primary function of XCL1 is to attract and activate immune cells, directing their migration to specific sites within the body.
  • XCL1 binds to its receptor, XCR1 (X-C motif chemokine receptor 1), which is primarily expressed on dendritic cells and a subset of activated T cells.
  • XCL1 Upon binding, XCL1 induces chemotaxis of XCR1-expressing cells towards the source of XCL1.
  • XCL1 has been implicated in various immune responses, including the regulation of T cell and NK cell function, dendritic cell migration and activation, and the generation of adaptive immune responses. It plays a role in the communication between different immune cell types and contributes to the coordination of immune responses against pathogens, tumors, and other threats.
  • An exemplary nucleic acid sequence for XCL1 ATGAGACTTCTCATCCTGGCCCTCCTTGGCATCTGCTCTCTCACTGCATACATTGTGGAAGGTG TAGGGAGTGAAGTCTCAGATAAGAGGACCTGTGTGAGCCTCACTACCCAGCGACTGCCGGTTAG CAGAATCAAGACCTACACCATCACGGAAGGCTCCTTGAGAGCAGTAATTTTTATTACCAAACGT GGCCTAAAAGTCTGTGCTGATCCACAAGCCACATGGGTGAGAGACGTGGTCAGGAGCATGGACA GGAAATCCAACACCAGAAATAACATGATCCAGACCAAGCCAACAGGAACCCAGCAATCGACCAA TACAGCTGTGACTCTGACTGGCTAG (SEQ ID NO:7).
  • An exemplary amino acid sequence XCL1 MRLLILALLGICSLTAYIVEGVGSEVSDKRTCVSLTTQRLPVSRIKTYTITEGSLRAVIFITKR GLKVCADPQATWVRDVVRSMDRKSNTRNNMIQTKPTGTQQSTNTAVTLTG (SEQ ID NO:8).
  • the encoded XCL1 molecule is a truncated variant of XCL1, for example, that lacks one or more amino acid residues of SEQ ID NO:8.
  • the encoded XCL1 molecule is a truncated variant of XCL1 that lacks 1, 2, 3, 4, 5, 6, 7, 8, 9,10, or more than 10, such as 11, 12, 13, 14, 15, 16 up to 20 contiguous residues from the amino (NH-), or carboxyl (CO-) terminus, or both the (NH-) and carboxyl (CO-) terminus of the sequence set forth in SEQ ID NO:8.
  • the truncated form of XCL1 peptide has an increased function relative to the full-length sequence of SEQ ID NO:8.
  • the peptide adjuvant molecule is a transcriptional regulator peptide.
  • the peptide adjuvant molecule molecule(s) is or includes the transcriptional regulator peptide interferon regulator factor 3 (IRF3).
  • IRF3 is a well- characterized signaling mediator/transcription factor that is important for innate antiviral response. Upon detection of viral RNA or other viral components, IRF-3 is activated and translocates to the nucleus, where it induces the expression of type I interferons (IFNs) and other antiviral genes.
  • Type I IFNs in turn, stimulate the expression of numerous genes involved in antiviral defense mechanisms.
  • IRF-3 also exerts tumor-suppressive effects in certain contexts.
  • IRF-3-mediated activation of type I IFN signaling can promote antitumor immune responses by enhancing the recognition and elimination of cancer cells by immune cells.
  • IRF-3 can contribute to immune surveillance mechanisms that detect and eliminate cancerous cells.
  • Activation of IRF- 3 in response to cellular stress or oncogenic signaling pathways can lead to the production of pro-inflammatory cytokines and chemokines that recruit immune cells to the tumor microenvironment, where they can exert antitumor effects.
  • An exemplary nucleic acid sequence IRF3 ATGGGAACCCCAAAGCCACGGATCCTGCCCTGGCTGGTGTCGCAGCTGGACCTGGGGCAACTGG AGGGCGTGGCCTGGGTGAACAAGAGCCGCACGCGCTTCCGCATCCCTTGGAAGCACGGCCTACG GCAGGATGCACAGCAGGAGGATTTCGGAATCTTCCAGGCCTGGGCCGAGGCCACTGGTGCATAT GTTCCCGGGAGGGATAAGCCAGACCTGCCAACCTGGAAGAGGAATTTCCGCTCTGCCCTCAACC GCAAAGAAGGGTTGCGTTTAGCAGAGGACCGGAGCAAGGACCCTCACGACCCACATAAAATCTA 455738066.1 27 CGAGTTTGTGAACTCAGGAGTTGGGGACTTTTCCCAGCCAGACACCTCTCCGGACACCAATGGt ggaggcagtacttctgatacccagGAAGACATTCTGGATGAGTTACTGGGTAACATGGTGTTGG CCACTGGCCCAGATCCGGGACCCCCAAGCC
  • the peptide adjuvant molecule(s) is or includes a Toll-Like Receptor Agonist (TLR).
  • TLRs play a vital role in activating immune responses. TLRs recognize conserved pathogen-associated molecular patterns (PAMPs) expressed on a wide array of microbes, as well as endogenous DAMPs released from stressed or dying cells. The engagement of specific TLRs on cancer cells can impact tumor growth by various mechanisms, including inducing apoptosis and potentiating the effects of chemotherapy (Kaczanowska, et al., J Leukoc Biol.2013 Jun; 93(6): 847–863).
  • PAMPs pathogen-associated molecular patterns
  • compositions of synthetic messenger ribonucleic acid include one or more ribonucleic acid sequences encoding one or more other immunomodulatory peptides. 455738066.1 29 In some forms, the exogenous nucleic acid sequence encodes a peptide having an immunomodulatory function in vivo.
  • the compositions of mRNAs include two or more immunomodulatory or co-signaling agents, such as signaling pairs.
  • the described compositions include two or more mRNA sequences encoding two peptide molecules that act as ligand-binding pairs.
  • the ligand-binding pairs can be immuno-receptors and ligands thereof, or they can be receptors and co-receptors.
  • the mRNAs when the mRNAs encode cell-surface bound molecules, the mRNAs can encode an entire mature protein, for example, an intracellular component, trans-membrane component and an extracellular component of a given protein or receptor.
  • the mRNAs encode only a part of the mature protein, such as only the transmembrane and/or extracellular or intracellular component or only the extracellular component.
  • Multiple peptides function as immunomodulating agents, by either blocking the immune response or stimulating the immune response to generate tolerance.
  • Knowledge of B- or T-cell epitopes along with conformational constraints is important in the design of peptide-based immunomodulating agents.
  • immunomodulating agents are biological agents targeting TNF- ⁇ ; IL-1; and/or co-stimulation blockers.
  • modulation or blocking of adhesion and costimulatory molecules reduces the inflammatory cell accumulation and modifies the process of inflammation.
  • one immune-modulatory agent is a non-adjuvant immune-stimulatory agent.
  • one agent is all or part of a major histocompatibility complex (MHC) molecule, such as a class I MHC or a class II MHC.
  • MHC major histocompatibility complex
  • An exemplary pair or group of antigen and immuno-stimulatory agents can include, for example, an mRNA encoding all or part of an MHC peptide, and an mRNA encoding a cognate MHC antigenic peptide, for example, that binds to within the cognate binding groove of the MHC.
  • one immune-modulatory agent is all or part of a cognate antigen of an MHC molecule, such as a 7-12 amino acid polypeptide that can be recognized and bound to a class I major histocompatibility complex (MHCI) molecule, or a larger polypeptide that can be recognized by class II major histocompatibility complex (MHCII) molecule.
  • one immune-modulatory agent is 455738066.1 30 a peptide that inhibits or otherwise moderates the interaction between one immune receptor and another, such as a costimulatory molecule or cell adhesion molecule pair.
  • one immune-modulatory agent is a peptide that inhibits or blocks the CD80-CD28/CD152 interaction.
  • the immune- modulatory agent is a polyproline peptide that inhibits or blocks the CD80-CD28/CD152 interaction.
  • one immune-modulatory agent is a peptide that inhibits or blocks the ICAM-1-LFA-1 interaction.
  • one immune-modulatory agent is a peptide that inhibits or blocks the CD2-LFA3 (CD58) interaction.
  • one immune-modulatory agent is a peptide that inhibits or blocks the CD28/CD152-CD80/CD86 interaction.
  • one immune- modulatory agent is a peptide that inhibits or blocks the CD80-CD28/CD152 interaction.
  • the ligand-binding pairs include CD40 and CD40L for DC activation and antibody production. In some forms, the ligand-binding pairs include Flt3L-Flt3 for DC activation. In some forms, the ligand-binding pairs include OX40-OX40L.
  • one immune-modulatory agent is a peptide GITR. In some forms, one immune-modulatory agent is a peptide CD137. In some forms, one immune-modulatory agent is a peptide Fc- receptor version or nano body blocker of all of the inhibitory signals (PD1, TIGIT, TIM3, LAG3, etc.). In some forms, one immune-modulatory agent is a Nano body agonist. a.
  • an immune-modulatory agent peptide is or includes a peptide that targets and/or selectively binds to a desired target cell or receptor, for example, to initiate an immune response.
  • exemplary targeting agents include MHC molecules that target and bind to T cell receptors (TCRs) at the surface of T cells to initiate antigen-specific immune responses, and other molecules that enhance and/or initiate immune responses in vivo.
  • TCRs T cell receptors
  • CLec9 Targeting Molecules In some forms, the peptide adjuvant molecule(s) is or includes a peptide that targets and/or selectively binds to CLec9A.
  • CLec9A is a group V C-type lectin-like receptor located in the “Dectin-1 cluster” of related receptors, which are encoded within the natural killer (NK)-gene complex.
  • CLEC9A Is a Novel Activation C-type Lectin-like Receptor Expressed on BDCA3+ Dendritic Cells and a Subset of Monocytes.
  • a CLec9A binding agent is a peptide having at least one targeting moiety that specifically binds Clec9A. In various forms, these Clec9A binding agents bind to, but do not functionally modulate (e.g. partially or fully neutralize) Clec9A.
  • the present Clec9A binding agents have use in, for instance, directly or indirectly recruiting a Clec9A-expressing cell to a site of interest while still allowing the 455738066.1 31 Clec9A-expressing cell to signal via Clec9A (e.g., the binding of the Clec9A binding agent does not reduce or eliminate Clec9A signaling at the site of interest).
  • These ligands can be used to target therapeutic agents or detectable labels to Clec9A expressing cells, such as dendritic cells.
  • Exemplary Clec9A binding agents are described in WO/2017/134305, the contents of which are incorporated herein in their entirety. 2.
  • compositions of synthetic messenger ribonucleic acid include one or more ribonucleic acid sequences encoding one or more peptide antigens.
  • the exogenous nucleic acid sequence encodes a vaccine antigen.
  • An antigen can include any protein or peptide that is foreign to the subject organism.
  • Preferred antigens can be presented at the surface of antigen presenting cells (APC) of a subject for surveillance by immune effector cells, such as leucocytes expressing the CD4 receptor (CD4 T cells) and Natural Killer (NK) cells.
  • APC antigen presenting cells
  • CD4 T cells CD4 receptor
  • NK Natural Killer
  • the antigen is of viral, bacterial, protozoan, fungal, or animal origin.
  • the antigen is a cancer antigen.
  • Cancer antigens can be antigens expressed only on tumor cells and/or required for tumor cell survival. Certain antigens are recognized by those skilled in the art as immuno-stimulatory (i.e., stimulate effective immune recognition) and provide effective immunity to the organism or molecule from which they derive.
  • B cell antigens can be peptides, proteins, polysaccharides, saccharides, lipids, nucleic acids, small molecules (alone or with a hapten) or combinations thereof.
  • T cell antigens are proteins or peptides.
  • Antigens may be provided as single antigens or may be provided in combination. Antigens may also be provided as complex mixtures of polypeptides or nucleic acids.
  • Tumor Antigens In some forms, the antigen(s) is or includes a tumor antigen.
  • tumor antigens There are many classes of tumor antigens, including, but not limited to, oncogene expression products, alternatively spliced expression products, mutated gene products, over-expressed gene products, aberrantly expressed 455738066.1 32 gene products, antigens produced by an oncogenic viruses, oncofetal antigens, as well as proteins with altered cell surface glycolipids, and proteins having altered glycosylation profiles.
  • Exemplary tumor antigens included or encoded by Compositions including one or more mRNA antigens and one or more mRNA adjuvants include tumor-associated or tumor-specific antigens, such as, but not limited to, alpha-actinin-4, Alphafetoprotein (AFP), Bcr-Abl fusion protein, Carcinoembryonic antigen (CEA), CA-125, Casp-8, beta-catenin, cdc27, cdk4, cdkn2a, coa-1, dek-can fusion protein, epithelial tumor antigen, EF2, ETV6-AML1 fusion protein, LDLR-fucosyl transferase AS fusion protein, HLA-A2, HLA-A11, hsp70-2, KIAAO205, Mart2, Mum-1, 2, and 3, neo-PAP, myosin class I, OS-9, pml-RARa fusion protein, PTPRK, K-ras, N- ra
  • an exemplary tumor antigen is the model melanoma tumor antigen Trp1.
  • the tumor antigen is the gene product of a gene that is normally expressed during embryogenesis, and whose expression in normal adult tissues is limited, such as an “oncofetal” protein, or an alternatively-spliced variant of a normal protein.
  • Oncofetal antigens are proteins which are typically present only during fetal development but are found in adults with certain kinds of cancer. These proteins are often measurable in the blood of individuals with cancer and may be used to both diagnose and follow treatment of the tumors. Therefore, in some forms, the compositions including one or more mRNA antigens and one or more mRNA adjuvants include or encode one or more oncofetal proteins.
  • an exemplary oncofetal protein is the Hmga2 protein.
  • the tumor antigen is the gene product of a gene that is normally expressed or not expressed by cells, but whose sequence has been altered by mutation, genetic recombination, altered splicing or another process leading to a distinct protein coding sequence 455738066.1 33 or post-translational modification of a coding sequence and a consequently distinct peptide- MHC complex presented on the surface of a tumor compared with non-tumor cells of the same tissue of origin, here termed a ‘neoantigen’.
  • a neoantigen is specific to a subject. ii.
  • the antigen(s) is or includes a viral antigen.
  • a viral antigen can be isolated from any virus including, but not limited to, a virus from any of the following viral families: Arenaviridae, Arterivirus, Astroviridae, Baculoviridae, Badnavirus, Barnaviridae, Birnaviridae, Bromoviridae, Bunyaviridae, Caliciviridae, Capillovirus, Carlavirus, Caulimovirus, Circoviridae, Closterovirus, Comoviridae, Coronaviridae (e.g., Coronavirus, such as severe acute respiratory syndrome (SARS) virus such as SARS-CoV-1 and SARS-CoV- 2), Corticoviridae, Cystoviridae, Deltavirus, Dianthovirus, Enamovirus, Filoviridae (e.g., Marburg virus and Ebola virus (EBOV) (e.g., Zaire, Reston, Ivor
  • Suitable viral antigens also include all or part of Dengue protein M, Dengue protein E, Dengue D1NS1, Dengue D1NS2, and Dengue D1NS3.
  • Viral antigens may be derived from a particular strain such as a papilloma virus, a herpes virus, i.e.
  • herpes simplex 1 and 2 a hepatitis virus, for example, hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), the delta hepatitis D virus (HDV), hepatitis E virus (HEV) and hepatitis G virus (HGV), the tick-borne encephalitis viruses; parainfluenza, varicella-zoster, cytomeglavirus, Epstein-Barr, rotavirus, rhinovirus, adenovirus, coxsackieviruses, equine encephalitis, Japanese encephalitis, yellow fever, Rift Valley fever, and lymphocytic choriomeningitis.
  • HAV hepatitis A virus
  • HBV hepatitis B virus
  • HCV hepatitis C virus
  • HDV delta hepatitis D virus
  • HEV hepatitis E virus
  • HGV hepatitis G
  • the viral antigen is derived from one or more viruses from the Orthomyxovirus family, for example, the Influenza virus A, Influenza virus B, Influenza virus C, Isavirus, Thogotovirus and Quaranjavirus.
  • influenza A virus subtypes include H1N1, H1N2, H3N2, H3N1, H5N1, H2N2, and H7N7.
  • influenza virus antigens include one or more proteins or glycoproteins such as hemagglutinin, such as HA1 and HA2 subunits, neurominidase, viral RNA polymerase, such as one or more of PB1, PB2 PA and PB1-F2, 455738066.1 34 reverse transcriptase, capsid protein, non-structured proteins, such as NS1 and NEP, nucleoprotein, matrix proteins, such as M1 and M2 and pore proteins.
  • Influenza A virus antigens include one or more of the Hemagglutinin (HA) or Neuraminidase (NA) glycoproteins or fragments of the HA or NA, including the antigenic sites of the Hemagglutinin HA1 glycoprotein.
  • Compositions including one or more mRNA antigens and one or more mRNA adjuvants include RNA encoding the influenza A/WSN/33 HA protein.
  • the viral antigen in derived from one or more viruses from the genus Ebolavirus, for example, the Zaire ebolavirus (EBOV), Sudan ebolavirus (SUDV), Ta ⁇ Forest ebolavirus (TAFV), Reston ebolavirus (RESTV), and Bundibugyo ebolavirus (BDBV).
  • compositions including one or more mRNA antigens and one or more mRNA adjuvants include RNA, such as repRNA, encoding the Zaire ebolavirus glycoprotein (GP), or one or more fragments of the Zaire ebolavirus glycoprotein (GP).
  • RNA such as repRNA
  • the viral antigen in derived from one or more viruses from the genus Flavivirus, for example, the Zika virus (ZIKV).
  • the antigen is an MCPyV antigen.
  • antigens are vaccines formed therefrom are disclosed in PCT/US2023/084086, which is specifically incorporated by references herein in its entirety.
  • MCPyV is a non-enveloped, double-stranded DNA virus that is a part of the normal microbiome for a majority of individuals in endemic areas (Kervarrec T, et al., Front Oncol. 2019 Jun 10;9:451). Recent investigation has shown that a coding region for a truncated form of the viral Large T Antigen (LTA) and its isoform the Small T Antigen (STA) are integrated into the genome of MCPyV associated MCC and causes malignant transformation through mechanisms including an interaction between LTA and the Retinoblastoma Protein (RB1).
  • LTA Large T Antigen
  • STA Small T Antigen
  • the viral antigen is viral LTA derived from MCPyV.
  • the viral antigen derived from LTA of MCPyV includes the amino acid sequence of SEQ ID NO:21, MDLVLNRKEREALCKLLEIAPNCYGNIPLMKAAFKRSCLKHHPDKGGNPVIMMELNTLWSKFQQ NIHKLRSDFSMFDEVDEAPIYGTTKFKEWWRSGGFSFGKAYEYGPNPHGTNSRSRKPSSNASRG APSGSSPPHSQSSSSGYGSFSASQASDSQSRGPDIPPEHHEEPTSSSGSSSREETTNSGRESST PNGTSVPRNSSRTDGTWEDLFCDESLSSPEPPSSSEEPEEPPSSRSSPRQPPSSSAEEASSSQF TD (SEQ ID NO:21), or a fragment or variant thereof with e.g., at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% sequence identity thereto.
  • the viral antigen is STA derived from MCPyV, or an antigenic fragment thereof.
  • the viral antigen derived from STA of MCPyV includes the amino acid sequence of SEQ ID NO:22: MDLVLNRKEREALCKLLEIAPNCYGNIPLMKAAFKRSCLKHHPDKGGNPVIMMELNTLWSKFQQ NIHKLRSDFSMFDEVSTKFPWEEYGTLKDYMQSGYNARFCRGPGCMLKQLRDSKCACISCKLSR QHCSLKTLKQKNCLTWGECFCYQCFILWFGFPPTWESFDWWQKTLEETDYCLLHLHLF (SEQ ID NO:22), or a fragment or variant thereof with e.g., at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% sequence identity thereto.
  • the antigen(s) is or includes an endogenous retroelement antigen.
  • Endogenous retroelement-derived nucleic acids activate innate immune pathways and contribute to pathologies such as systemic lupus erythematosus and Aicardi–Goutines syndrome. It also enhances responses to poorly immunogenic antigens, such as T cell-independent type 2 antigens or tumors.
  • Endogenous retroelement antigens can originate from any endogenous retroelement that is native to the human genome including, but not limited to, Endogenous Retrovirsues (ERVs), Long Interspersed Nuclear Elements (LINEs), and Short Interspersed Nuclear Elements (SINEs).
  • ERV genes include ERV-W1 env (Syncytin-1), HERV-E (also known as ERVE-4), ERV-Fc1 env, ERV-K env and ERV-K pol.
  • ERV-W1 env Synchronization-1
  • HERV-E also known as ERVE-4
  • ERV-Fc1 env ERV-K env
  • ERV-K env ERV-K env
  • ERV-K pol Exemplary endogenous retroelement antigens are described in Braun, et al., Nat Med.2020 Jun; 26(6): pp909–918, the contents of which are incorporated herein in their entirety.
  • Bacterial Antigens can originate from any bacteria including, but not limited to, Actinomyces, Anabaena, Bacillus, Bacteroides, Bdellovibrio, Bordetella, Borrelia, Campylobacter, Caulobacter, Chlamydia, Chlorobium, Chromatium, Clostridium, Corynebacterium, Cytophaga, Deinococcus, Escherichia, Francisella, Halobacterium, Heliobacter, Haemophilus, Hemophilus influenza type B (HIB), Hyphomicrobium, Legionella, Leptspirosis, Listeria, Meningococcus A, B and C, Methanobacterium, Micrococcus, Myobacterium, Mycoplasma, Myxococcus, Neisseria, Nitrobacter, Oscillatoria, Prochloron, Proteus,
  • the antigen(s) is or includes a parasite antigen.
  • parasite allergens include but are not limited to, Cryptococcus neoformans, Histoplasma capsulatum, Candida albicans, Candida tropicalis, Nocardia asteroides, Rickettsia ricketsii, Rickettsia typhi, Mycoplasma pneumoniae, Chlamydial psittaci, Chlamydial trachomatis, Plasmodium falciparum, Trypanosoma brucei, Entamoeba histolytica, Toxoplasma gondii, Trichomonas vaginalis and Schistosoma mansoni.
  • the parasite antigen is one or more antigens from a protozoan, such as one or more protozoans from the genus Toxoplasma, for example T. gondii and species from a related genus, such as Neospora, Hammondia, Frenkelia, Isospora and Sarcocystis.
  • a protozoan such as one or more protozoans from the genus Toxoplasma
  • T. gondii and species from a related genus, such as Neospora, Hammondia, Frenkelia, Isospora and Sarcocystis.
  • gondii include the GRA6, ROP2A, ROP18, SAG1, SAG2A and AMA1 gene products.
  • the antigen(s) is or includes a tolerogenic antigen.
  • Exemplary tolerogenic antigens are known in the art. See, for example, U.S. Published Application No.2014/0356384.
  • the tolerogenic antigen is derived from a therapeutic agent protein to which tolerance is desired. Examples are protein drugs in their wild type, e.g., human factor VIII or factor IX, to which patients did not establish central tolerance because they were deficient in those proteins; or nonhuman protein drugs, used in a human.
  • protein drugs that are glycosylated in nonhuman forms due to production, or engineered protein drugs, e.g., having non-native sequences that can provoke an unwanted immune response.
  • examples of tolerogenic antigens that are engineered therapeutic proteins not naturally found in humans including human proteins with engineered mutations, e.g., mutations to improve pharmacological characteristics.
  • examples of tolerogenic antigens that have nonhuman glycosylation include proteins produced in yeast or insect cells.
  • Tolerogenic antigens can be from proteins that are administered to humans that are deficient in the protein. Deficient means that the patient receiving the protein does not naturally produce enough of the protein.
  • the proteins may be proteins for which a patient is genetically deficient.
  • Such proteins include, for example, antithrombin-III, protein C, factor VIII, factor IX, growth hormone, somatotropin, insulin, pramlintide acetate, mecasermin (IGF-1), ⁇ - gluco cerebrosidase, alglucosidase-alpha, laronidase ( ⁇ -L-iduronidase), idursuphase (iduronate- 2-sulphatase), galsulphase, agalsidase-beta ( ⁇ -galactosidase), ⁇ -1 proteinase inhibitor, and albumin. 455738066.1 37
  • the tolerogenic antigen can be from therapeutic antibodies and antibody-like molecules, including antibody fragments and fusion proteins with antibodies and antibody fragments.
  • the tolerogenic antigen can be from proteins that are nonhuman.
  • proteins include adenosine deaminase, pancreatic lipase, pancreatic amylase, lactase, botulinum toxin type A, botulinum toxin type B, collagenase, hyaluronidase, papain, L-Asparaginase, rasburicase, lepirudin, streptokinase, anistreplase (anisoylated plasminogen streptokinase activator complex), antithymocyte globulin, crotalidae polyvalent immune Fab, digoxin immune serum Fab, L-arginase, and L-methionase.
  • adenosine deaminase pancreatic lipase, pancreatic amylase, lactase, botulinum toxin type A, botulinum toxin type B, collagenase, hyaluronidase, papain, L-A
  • Tolerogenic antigens include those from human allograft transplantation antigens. Examples of these antigens are the subunits of the various MHC class I and MHC class II haplotype proteins, and single-amino-acid polymorphisms on minor blood group antigens including RhCE, Kell, Kidd, Duffy and Ss.
  • the tolerogenic antigen can be a self-antigen against which a patient has developed an autoimmune response or may develop an autoimmune response. Examples are proinsulin (diabetes), collagens (rheumatoid arthritis), myelin basic protein (multiple sclerosis).
  • Type 1 diabetes mellitus is an autoimmune disease whereby T cells that recognize islet proteins have broken free of immune regulation and signal the immune system to destroy pancreatic tissue.
  • Numerous protein antigens that are targets of such diabetogenic T cells have been discovered, including insulin, GAD65, chromogranin-A, among others.
  • T1D it would be useful to induce antigen-specific immune tolerance towards defined diabetogenic antigens to functionally inactivate or delete the diabetogenic T cell clones. Tolerance and/or delay of onset or progression of autoimmune diseases may be achieved for various of the many proteins that are human autoimmune proteins, a term referring to various autoimmune diseases wherein the protein or proteins causing the disease are known or can be established by routine testing.
  • a patient is tested to identify an autoimmune protein and an antigen is created for use in a molecular fusion to create immunotolerance to the protein.
  • an antigen or choosing an antigen from or derived from, one or more of the following proteins.
  • insulin In type 1 diabetes mellitus, several primary antigens have been identified: insulin, proinsulin, preproinsulin, glutamic acid decarboxylase-65 (GAD-65), GAD-67, insulinoma-associated protein 2 (IA-2), and insulinoma-associated protein 2 beta (IA- 455738066.1 38 213); other antigens include ICA69, ICA12 (SOX-13), carboxypeptidase H, Imogen 38, GLIMA 38, chromogranin-A, FISP-60, carboxypeptidase E, peripherin, glucose transporter 2, hepatocarcinoma-intestine-pancreas/pancreatic associated protein, S100 ⁇ , glial fibrillary acidic protein, regenerating gene II, pancreatic duodenal homeobox 1, dystrophia myotonica kinase, islet-specific glucose-6-phosphatase catalytic subunit-related protein, and SST G-protein coupled receptors 1-5.
  • primary antigens include thyroglobulin (TG), thyroid peroxidase (TPO) and thyrotropin receptor (TSHR); other antigens include sodium iodine symporter (NIS) and megalin.
  • TG thyroglobulin
  • TPO thyroid peroxidase
  • TSHR thyrotropin receptor
  • NIS sodium iodine symporter
  • an antigen is insulin-like growth factor 1 receptor.
  • a primary antigen is calcium sensitive receptor.
  • primary antigens include 21-hydroxylase, 17 ⁇ -hydroxylase, and P450 side chain cleavage enzyme (P450scc); other antigens include ACTH receptor, P450c21 and P450c17.
  • primary antigens include FSH receptor and alpha-enolase.
  • primary antigens include pituitary gland-specific protein factor (PGSF) 1a and 2; another antigen is type 2 iodothyronine deiodinase.
  • primary antigens include myelin basic protein, myelin oligodendrocyte glycoprotein and proteolipid protein.
  • a primary antigen In rheumatoid arthritis, a primary antigen is collagen II. In immunogastritis, a primary antigen is H+, K+-ATPase. In pernicious angemis, a primary antigen is intrinsic factor. In celiac disease, primary antigens are tissue transglutaminase and gliadin. In vitiligo, a primary antigen is tyrosinase, and tyrosinase related protein 1 and 2. In myasthenia gravis, a primary antigen is acetylcholine receptor.
  • primary antigens are desmoglein 3, 1 and 4; other antigens include pemphaxin, desmocollins, plakoglobin, perplakin, desmoplakins, and acetylcholine receptor.
  • primary antigens include BP180 and BP230; other antigens include plectin and laminin 5.
  • primary antigens include endomysium and tissue transglutaminase.
  • a primary antigen is collagen VII.
  • primary antigens include matrix metalloproteinase 1 and 3, the collagen- specific molecular chaperone heat-shock protein 47, fibrillin-1, and PDGF receptor; other antigens include Scl-70, U1 RNP, Th/To, Ku, Jo1, NAG-2, centromere proteins, topoisomerase I, nucleolar proteins, RNA polymerase I, II and III, PM-Slc, fibrillarin, and B23.
  • a primary antigen is U1snRNP.
  • the primary antigens are nuclear antigens SS-A and SS-B; other antigens include fodrin, poly(ADP-ribose) polymerase and topoisomerase.
  • primary antigens include nuclear proteins including SS-A, high mobility group box 1 (HMGB1), nucleosomes, histone 455738066.1 39 proteins and double-stranded DNA.
  • primary antigens include glomerular basement membrane proteins including collagen IV.
  • a primary antigen is cardiac myosin.
  • autoimmune polyglandular syndrome type 1 Other autoantigens revealed in autoimmune polyglandular syndrome type 1 include aromatic L-amino acid decarboxylase, histidine decarboxylase, cysteine sulfonic acid decarboxylase, tryptophan hydroxylase, tyrosine hydroxylase, phenylalanine hydroxylase, hepatic P450 cytochromes P4501A2 and 2A6, SOX-9, SOX-10, calcium-sensing receptor protein, and the type 1 interferons interferon alpha, beta and omega.
  • the tolerogenic antigen is a foreign antigen against which a patient has developed an unwanted immune response. Examples are food antigens.
  • Some embodiments include testing a patient to identify foreign antigen and creating a molecular fusion that comprises the antigen and treating the patient to develop immunotolerance to the antigen or food.
  • foods and/or antigens are provided. Examples are from peanut: conarachin (Ara h 1), allergen II (Ara h 2), arachis agglutinin, conglutin (Ara h 6); from apple: 31 kda major allergen/disease resistance protein homolog (Mal d 2), lipid transfer protein precursor (Mal d 3), major allergen Mal d 1.03D (Mal d 1); from milk: .alpha.-lactalbumin (ALA), lactotransferrin; from kiwi: actinidin (Act c 1, Act d 1), phytocystatin, thaumatin-like protein (Act d 2), kiwellin (Act d 5); from mustard: 2S albumin (Sin a 1), 11 S globulin (S
  • the antigen is an allergen or environmental antigen.
  • allergens and environmental antigens include but are not limited to, an antigen derived from naturally occurring allergens such as pollen allergens (tree-, herb, weed-, and grass pollen allergens), insect allergens (inhalant, saliva and venom allergens), animal hair and dandruff allergens, and food allergens.
  • pollen allergens tree-, herb, weed-, and grass pollen allergens
  • insect allergens inhalant, saliva and venom allergens
  • animal hair and dandruff allergens include i.a.
  • birch (Betula), alder (Alnus), hazel (Corylus), hornbeam (Carpinus) and olive (Olea), cedar (Cryptomeriaand Juniperus), Plane tree (Platanus), the order of Poales including, e.g., grasses of the genera Lolium, Phleum, Poa, Cynodon, Dactylis, Holcus, Phalaris, Secale, and Sorghum, the orders of Asterales and Urticales including e.g., herbs of the genera Ambrosia, Artemisia, and Parietaria.
  • allergen antigens include allergens from house dust mites of the genus Dermatophagoides and Euroglyphus, storage mite, e.g., Lepidoglyphys, Glycyphagus and Tyrophagus, those from cockroaches, midges and fleas, e.g., Blatella, Periplaneta, Chironomus and Ctenocepphalides, those from mammals such as cat, dog and horse, birds, venom allergens including such originating from stinging or biting insects such as those from the taxonomic order of Hymenoptera including bees (superfamily Apidae), wasps (superfamily Vespidea), and ants (superfamily Formicoidae).
  • allergen antigens that may be used include inhalation allergens from fungi such as from the genera Alternaria and Cladosporium.
  • exemplary food allergens include cow's milk (e.g., lactose), eggs, nuts, shellfish, fish, and legumes (peanuts and soybeans), fruits and vegetables such as tomatoes.
  • the compositions including one or more mRNA antigens and one or more mRNA adjuvants can include one or more immune-modulatory molecules, or one or more nucleic acids encoding immune-modulatory molecules to direct the immune response specifically toward a Th1 (cellular) or Th2 (humoral) polarization for the delivered allergen. 3.
  • compositions include or encode one or more molecules that function to direct, target or otherwise improve recognition and uptake of the encoded antigens by immune cells, such as antigen presenting cells (APCs).
  • compositions of synthetic messenger ribonucleic acid (mRNA) include one or more ribonucleic acid sequences encoding one or more amino acid sequences that are linked to sequence(s) encoding antigen(s), and which target the linked antigen(s) to APCs, such as dendritic cells (DCs), and/or improve the uptake and/or presentation of linked antigen(s) by an APC.
  • APCs antigen presenting cells
  • the targeting polypeptide can be 455738066.1 41 coupled directly or indirectly to an encoded antigen peptide.
  • the targeting polypeptide can be bound to either the “amino” (-NH) terminus, or the “carboxyl” (-COOH) terminus of the antigen polypeptide.
  • the targeting polypeptide is coupled to the antigen polypeptide via a linker, such as a flexible linker. i. Clec9a-targeting approaches
  • the compositions encode antigens linked to Clec9A-targeting sequences.
  • Clec9a also known as DNGR-1, is a C-type lectin receptor expressed on mouse CD8 ⁇ + DCs and plasmacytoid DCs (pDCs) and is also selectively expressed on human BDCA3+ DCs. Clec9a specifically recognizes actin filaments (F-actin) exposed on necrotic cells, leading to the subsequent routing of necrotic cells into the cross-priming pathway.
  • the described compositions including an mRNA encoding antigen also include an mRNA encoding a Clec9a-binding peptide. Typically, the Clec9a-binding peptide is linked to the antigen.
  • the compositions include a single mRNA encoding antigen and a Clec9a-binding peptide, such that the antigen peptide is expressed as a polypeptide that also encodes a Clec9a-binding domain.
  • the compositions include one or more mRNA(s) encoding one or more antigen(s) and a Clec9a-binding peptide, and one or more adjuvants and/or immuno-modulatory molecules.
  • the one or more antigen peptide is expressed as a polypeptide that also encodes a Clec9a-binding domain.
  • An exemplary polypeptide that selectively binds to Clec9a includes the amino acid sequence: WPRFHSSVRHTH (SEQ ID NO:23).
  • An exemplary nucleic acid sequence for a polypeptide that selectively binds to Clec9a includes the sequence:tggccgcgctttcatagcagcgtgcgccatacccat (SEQ ID NO:24).
  • compositions of synthetic messenger ribonucleic acid (mRNA) including one or more ribonucleic acid sequences encoding one or more peptide antigens and one or more ribonucleic acid sequences encoding one or more peptide adjuvants include therapeutic, prophylactic and diagnostic agents enclosed or encapsulated within non-toxic, non- immunogenic, biodegradable nanoparticles and have a size amenable for uptake by eukaryotic cells in vivo for the efficient delivery of the therapeutic, prophylactic and diagnostic agents to the intracellular space.
  • the delivery vehicles can protect therapeutic, prophylactic and diagnostic agents from immune surveillance and degradation in the body and enhance the serum half-life of the encapsulated active-agents.
  • the delivery vehicles can optionally include one or more targeting motifs to enhance specificity and uptake by target cells. 455738066.1 42 1.
  • Lipid Nanoparticles the compositions of synthetic messenger ribonucleic acid (mRNA) including one or more ribonucleic acid sequences encoding one or more peptide antigens and one or more ribonucleic acid sequences encoding one or more peptide adjuvants is encapsulated within lipid nanoparticles.
  • mRNA messenger ribonucleic acid
  • lipid nanoparticles have emerged as successful candidates for delivering mRNA, with lipid nanoparticle-based mRNA vaccines being actively used in clinical settings to combat COVID-19. (Hou et al., Nature Reviews Materials, volume 6, pages1078–1094, 2021).
  • the compositions of lipid nanoparticles typically involve various lipid components such as phospholipids, cholesterol, and other lipid modifiers. These components are often formulated in specific ratios to achieve desired properties such as stability, encapsulation efficiency, and target-specific delivery.
  • lipid nanoparticles may include surface modifications such as PEGylation to enhance circulation time and reduce immune response. Overall, the composition of lipid nanoparticles can be tailored based on the intended application and desired characteristics for efficient delivery of therapeutic agents such as mRNA.
  • the lipid particles may be formed from a combination of more than one lipid, for example, a charged lipid may be combined with a lipid that is non-ionic or uncharged at physiological pH.
  • Representative neutral and anionic lipids include, but are not limited to, sterols and lipids such as cholesterol, phospholipids, lysolipids, lysophospholipids, sphingolipids or pegylated lipids.
  • Neutral and anionic lipids include, but are not limited to, phosphatidylcholine (PC) (such as egg PC, soy PC), including 1 ,2-diacyl-glycero-3-phosphocholines; phosphatidylserine (PS), phosphatidylglycerol, phosphatidylinositol (PI); glycolipids; sphingophospholipids such as sphingomyelin and sphingoglycolipids (also known as 1-ceramidyl glucosides) such as ceramide galactopyranoside, gangliosides and cerebrosides; fatty acids, sterols, containing a carboxylic acid group for example, cholesterol.
  • PC phosphatidylcholine
  • PS phosphatidylserine
  • PS phosphatidylglycerol
  • PI phosphatidylinositol
  • glycolipids sphingophospholipids such as
  • Representative cationic lipids include, but are not limited to, N-[1-(2,3- dioleoyloxy)propyl]-N,N,N-trimethyl ammonium salts, referred to as TAP lipids, for example, methylsulfate salt.
  • Representative TAP lipids include, but are not limited to, DOTAP (dioleoyl- ), DMTAP (dimyristoyl-), DPTAP (dipalmitoyl-), and DSTAP (distearoyl-).
  • cationic lipids in the liposomes include, but are not limited to, dimethyldioctadecyl ammonium bromide (DDAB), 1 ,2-diacyloxy-3-trimethylammonium propanes, N-[1-(2,3- dioloyloxy)propyl]- ⁇ , ⁇ -dimethyl amine (DODAP), 1 ,2-diacyloxy-3-dimethylammonium 455738066.1 43 propanes, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 1 ,2- dialkyloxy-3-dimethylammonium propanes, dioctadecylamidoglycylspermine (DOGS), 3 -[N- (N',N'-dimethylamino-ethane)carbamoyl]cholesterol (DC-Chol); 2,3-dioleoyloxy-N-
  • the cationic lipids can be 1-[2-(acyloxy)ethyl]2-alkyl(alkenyl)-3-(2-hydroxyethyl)- imidazolinium chloride derivatives, for example, 1-[2-(9(Z)-octadecenoyloxy)ethyl]-2-(8(Z)- heptadecenyl-3-(2-hydroxyethyl)imidazolinium chloride (DOTIM), and 1-[2- (hexadecanoyloxy)ethyl]-2-pentadecyl-3-(2-hydroxyethyl)imidazolinium chloride (DPTIM).
  • DOTIM 1-[2-(hexadecanoyloxy)ethyl]-2-pentadecyl-3-(2-hydroxyethyl)imidazolinium chloride
  • the cationic lipids can be 2,3-dialkyloxypropyl quaternary ammonium compound derivatives containing a hydroxyalkyl moiety on the quaternary amine, for example, 1 ,2- dioleoyl-3-dimethyl-hydroxyethyl ammonium bromide (DORI), 1 ,2-dioleyloxypropyl-3- dimethyl-hydroxyethyl ammonium bromide (DORIE), 1 ,2-dioleyloxypropyl-3-dimetyl- hydroxypropyl ammonium bromide (DORIE-HP), 1 ,2-dioleyl-oxy-propyl-3-dimethyl- hydroxybutyl ammonium bromide (DORIE-HB), 1 ,2-dioleyloxypropyl-3-dimethyl- hydroxypentyl ammonium bromide (DORIE-Hpe), 1 ,2-dimyristyloxypropyl-3-dimethyl-
  • the particle or particle core is a lipid micelle.
  • Lipid micelles can be formed, for instance, as a water-in-oil emulsion with a lipid surfactant.
  • An emulsion is a blend of two immiscible phases wherein a surfactant is added to stabilize the dispersed droplets.
  • the lipid micelle is a microemulsion.
  • a microemulsion is a thermodynamically stable system composed of at least water, oil and a lipid surfactant producing a transparent and thermodynamically stable system whose droplet size is less than 1 micron, from about 10 nm to about 500 nm, or from about 10 nm to about 250 nm.
  • Lipid micelles are generally useful for encapsulating hydrophobic active agents, including hydrophobic therapeutic agents, hydrophobic prophylactic agents, or hydrophobic diagnostic agents. 455738066.1 44 b.
  • Liposomes In some forms, the particle or particle core is a liposome. Liposomes are small vesicles composed of an aqueous medium surrounded by lipids arranged in spherical bilayers. Liposomes can be classified as small uni-lamellar vesicles, large uni-lamellar vesicles, or multi- lamellar vesicles. Multi-lamellar liposomes contain multiple concentric lipid bilayers.
  • Liposomes can be used to encapsulate targeted agents, by trapping hydrophilic agents in the aqueous interior or between bilayers, or by trapping hydrophobic agents within the bilayer.
  • the lipid micelles and liposomes typically have an aqueous center.
  • the aqueous center can contain water or a mixture of water and alcohol.
  • Representative alcohols include, but are not limited to, methanol, ethanol, propanol, (such as isopropanol), butanol (such as n-butanol, isobutanol, sec-butanol, tert-butanol, pentanol (such as amyl alcohol, isobutyl carbinol), hexanol (such as 1-hexanol, 2-hexanol, 3-hexanol), heptanol (such as 1-heptanol, 2-heptanol, 3-heptanol and 4-heptanol) or octanol (such as 1-octanol) or a combination thereof.
  • methanol such as isopropanol
  • butanol such as n-butanol, isobutanol, sec-butanol, tert-butanol
  • pentanol such as amyl alcohol, isobut
  • liposomes are prepared from long chain fatty acids and phytosterol formulations.
  • Solid Lipid Particles In some forms, the particle is a solid lipid particle, or includes a solid lipid core. Solid lipid particles present an alternative to the colloidal micelles and liposomes. Solid lipid particles are typically submicron in size, i.e., from about 10 nm to about 1 micron, from 10 nm to about 500 nm, or from 10 nm to about 250 nm. Solid lipid particles are formed of lipids that are solids at room temperature. They are derived from oil-in-water emulsions, by replacing the liquid oil by a solid lipid.
  • Solid lipids include, but are not limited to, higher saturated alcohols, higher fatty acids, sphingolipids, synthetic esters, and mono-, di-, and triglycerides of higher saturated fatty acids.
  • Solid lipids can include aliphatic alcohols having 10-40, preferably 12-30 carbon atoms, such as cetostearyl alcohol.
  • Solid lipids can include higher fatty acids of 10-40, preferably 12-30 carbon atoms, such as stearic acid, palmitic acid, decanoic acid, and behenic acid.
  • Solid lipids can include glycerides, including monoglycerides, diglycerides, and triglycerides, of higher saturated fatty acids having 10-40, preferably 12-30 carbon atoms, such as glyceryl monostearate, glycerol behenate, glycerol palmitostearate, glycerol trilaurate, tricaprin, trilaurin, trimyristin, tripalmitin, tristearin, and hydrogenated castor oil.
  • Representative solid lipids can include cetyl palmitate or beeswax. Cyclodextrin can also be used.
  • compositions of lipid nanoparticles include SM-102, 1,2-DSPC, Cholesterol, and DMG-PEG in a lipid molar ratio of 50:10:38:1.5. 455738066.1 45 C.
  • Formulations and Carriers The composition of the invention can be formulated in pharmaceutical compositions. These compositions can include, in addition to the described nanoparticles including mRNA(s) encoding two or more peptides, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer, or other materials well known to those skilled in the art. Such materials should typically be non-toxic and should not typically interfere with the efficacy of the active ingredient.
  • compositions for oral administration can be in tablet, capsule, powder or liquid form.
  • a tablet can include a solid carrier such as gelatin or an adjuvant.
  • Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil, or synthetic oil. Physiological saline solution, dextrose, or other saccharide solution or glycols such as ethylene glycol, propylene glycol, or polyethylene glycol can be included.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the pharmaceutical composition (e.g., immunogenic or vaccine formulation) is administered.
  • Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, ethanol and the like. Examples of suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin.
  • the formulation should be selected according to the mode of administration.
  • the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity, and stability.
  • a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity, and stability.
  • isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, or Lactated Ringer's Injection.
  • Preservatives, stabilizers, buffers, antioxidants, and/or other additives can be included, as required.
  • Administration is preferably in a “therapeutically effective amount” or “prophylactically effective amount” (as the case can be, although prophylaxis can be considered therapy), this being sufficient to show benefit to the individual.
  • the actual amount administered, and rate and time-course of administration, will depend on the nature and severity of disease being treated. Prescription of treatment, e.g., decisions on dosage etc., is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and 455738066.1 46 other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in the latest edition of Remington's Pharmaceutical Science, Mack Publishing Company, Easton, Pa. (“Remington's”). 1.
  • compositions including mRNA(s) encoding one, two, or more peptides optionally encapsulated within a nanoparticle are formulated in a pharmaceutical composition.
  • Pharmaceutical compositions including the described compositions, together with one or more further adjuvants and/or additional active agents, and the combination thereof are provided.
  • D. Exemplary Immunogenic Compositions and Vaccines mRNA compositions, including nanoparticles encapsulating the mRNAs, can deliver exogenous proteins and/or nucleic acids to a subject to stimulate desired immune responses in the subject.
  • the delivery of antigen and adjuvants via the mRNA compositions, including nanoparticles encapsulating the mRNAs confers protective immunity to infectious agents such as viruses and bacteria.
  • Methods for vaccination using mRNA adjuvants and mRNA antigens within mRNA compositions, including nanoparticles encapsulating the mRNAs are provided which allow potent and persistent presentation of antigen to the immune system without stimulating IFN responses early upon injection. A strong IFN response would likely impede alphaviral replication and thus limit antigen dose over time. (Zhang, et al., J Virol., 81, 11246-11255, (2007); White, et al., J Virol., 75, 3706-3718 (2001)).
  • antigens can be substituted with the antigens described in the study conducted herein. These could include antigens from pathogenic microbes such as viruses, bacteria, fungi, protozoa, as well as cancer antigens. Therefore, the mRNA compositions, including nanoparticles encapsulating the mRNAs, can serve as a safe and effective platform for the delivery vaccine agents, such as RNA-encoded antigens and neo-antigens, targeting many different pathogenic microbes.
  • compositions of mRNA(s) optionally encapsulated within nanoparticles, such as lipid nanoparticles (LNP) results in the production of antibodies and other biomolecules capable of recognizing and neutralizing the antigen.
  • Antigen that has been arrayed on the surface of antigen-presenting cells (APC) can be presented to a “helper” T cell, such as an antigen-specific naive CD4+ T cell, or directly to a CD8+ T cell by cross presentation on MHC class I molecules.
  • APC antigen-presenting cells
  • T cell receptor TCR
  • the mRNA compositions, including nanoparticles encapsulating the mRNAs can be used to initiate, moderate or enhance a humoral and/or cellular immunity to an encoded antigen.
  • the mRNA compositions, including nanoparticles encapsulating the mRNAs deliver exogenous nucleic acids and/or proteins in an amount effective to induce, enhance or otherwise moderate the biological activities of immune cells, such as macrophages, B-cells, T-cells, dendritic cells and NK cells.
  • administration of the mRNA compositions, including nanoparticles encapsulating the mRNAs, including nucleic acid sequences encoding an antigen and an adjuvant to a subject confers immunity to the antigen to the subject.
  • Immunity can manifest in the production of a reservoir of memory T cells (i.e., memory CD8+ T cells) and/or antigen- specific B cells in the subject sufficient to provide rapid immune cellular and/or humoral immune responses to repeat exposure of the antigen.
  • administration of the mRNA compositions, including nanoparticles encapsulating the mRNAs, including nucleic acid sequences encoding an antigen and an adjuvant confers protection against infection or disease caused by the organism(s) from which the antigen is derived.
  • administration of the mRNA compositions, including nanoparticles encapsulating the mRNAs, including nucleic acid sequences encoding an antigen to a subject enhances the uptake and delivery of antigen to the antigen presenting cells of the subject relative to administration of equal amounts of the antigen or nucleic acid encoding the antigen alone. Therefore, administration of antigen to a subject via the mRNA compositions, including nanoparticles encapsulating the mRNAs, can enhance the immune response to the antigen in the subject relative to administration of equal amounts of the antigen or nucleic acid encoding the antigen alone.
  • mRNA compositions including nanoparticles encapsulating the mRNAs, can increase, prolong or otherwise enhance presentation of the encoded antigen at the surface of antigen presenting cells of the subject.
  • Vaccines can be administered prophylactically or therapeutically.
  • Vaccines can also be administered according to a vaccine schedule.
  • a vaccine schedule is a series of vaccinations, including the timing of all doses. Many vaccines require multiple doses for maximum effectiveness, either to produce sufficient initial immune response or to boost response that fades over time.
  • Vaccine schedules are known in the art, and are designed to achieve maximum effectiveness.
  • the adaptive immune response to one or more antigen delivered in the mRNA compositions, including nanoparticles encapsulating the mRNAs can be monitored using methods known in the art to measure the effectiveness of the vaccination protocol.
  • mRNA compositions, including nanoparticles encapsulating the mRNAs deliver exogenous proteins and/or nucleic acids to a subject and stimulate immune responses 455738066.1 48 specifically to antigen that is expressed and biologically-processed by the host cells. Therefore, methods for vaccination using nucleic acids encoding antigens deliver by the mRNA compositions, including nanoparticles encapsulating the mRNAs, can provide immunity to antigen including post-translational modifications specific to the host subject.
  • vaccination using nucleic acids encoding antigen delivered by the mRNA compositions, including nanoparticles encapsulating the mRNAs can provide immunity to proteins containing post-translational modifications native to the host, such as glycosylation, lipidation (including myristoylation, palmitoylation, isoprenylation), sulfation, oxidation, phosphorylation, adenylation, methylation and amidation.
  • the immune responses generated by a subject to these post-translationally-modified peptide antigens can be the same or different to immune responses raised in the same host against the non-natively expressed or post-translationally modified form of the same antigenic peptide.
  • mRNA compositions can include two or more different antigens and/or two or more different adjuvants.
  • vaccines include combinations of different types of mRNAs, such as those encode different antigens or adjuvants, each with different antigen/adjuvant production kinetics.
  • multiplexing RNAs with different kinetics nanoparticles encapsulating mRNA as vaccines can be engineered to express one or more antigens at different times following administration.
  • the multiplexed mRNA vaccines can include a mixture of distinct mRNA compositions, including nanoparticles encapsulating the mRNAs, each enclosing different RNA species, or alternatively each nanoparticle can be engineered to include more than one RNA species.
  • mRNA compositions, including nanoparticles encapsulating the mRNAs are engineered to include one species of RNA, such as a single species of mRNAs encoding a single antigen
  • vaccines can be designed by mixing a desired amount of each nanoparticle to create a combined nanoparticle vaccine, having the desired expression kinetics.
  • mRNA vaccines are self-limiting and only produce antigens/adjuvants for a finite amount of time until the host eliminates the mRNA and all vaccine products are cleared by the body. Antigen production occurs in host cell cytoplasm, and the genetic material in the nucleus of the cell is never manipulated.
  • mRNA compositions, including nanoparticles encapsulating the mRNAs, including two or more different mRNAs expressing the different antigens/adjuvants can be used to induce expression of the antigen in the cells of a recipient with different expression kinetics.
  • the delivery of two or more different 455738066.1 49 mRNAs expressing the different antigens/adjuvants can give rise to a different serum half-life of the expression product.
  • the encapsulated agent of mRNA compositions, including nanoparticles encapsulating the mRNAs is engineered to include more than one RNA species, to provide expression of antigens over prolonged or defined periods of time following administration.
  • mRNA compositions, including nanoparticles encapsulating the mRNAs can include mRNAs encoding the same or different antigens/adjuvants to provide distinct and different expression kinetics for the antigen(s) within a subject.
  • the mRNA compositions including nanoparticles encapsulating the mRNAs deliver cargo, such as RNA neo-antigens, to a subject to provide two or more distinct phases of expression of the antigen in the subject.
  • cargo such as RNA neo-antigens
  • mRNAs encoding an antigen can give rise to a first “expression phase” of an antigen associated with translation of one RNA species, leading to an increase in serum concentration of the antigen.
  • a second “expression phase” of the same or different antigen, associated with translation of a second RNA species leads to a subsequent increase in serum concentration of antigen.
  • mRNA compositions including nanoparticles encapsulating the mRNAs, including two or more different mRNAs expressing antigen are used for vaccination
  • primary i.e., “prime”
  • boost secondary or further subsequent
  • mRNA compositions, including nanoparticles encapsulating the mRNAs, enclosing more than one mRNA can provide “Self-boosting” vaccines.
  • the nanoparticle such as a lipid nanoparticle (LNP) vehicles can be designed to enter cells and deliver therapeutic, prophylactic and diagnostic agents at a certain rate, for example, by modification of the composition of the mRNA compositions, including nanoparticles encapsulating the mRNAs to alter the serum half-life of the nanoparticle in vivo.
  • mRNA compositions, including nanoparticles encapsulating the mRNAs are delivered to more than one bodily locations, for example by the same or different administration routes, leading to different serum half-life and cellular-uptake kinetics of the nanoparticle and subsequent differences in the location and rate of antigen expression.
  • mRNA compositions including nanoparticles each encapsulating two or more species of mRNAs are used to deliver mRNAs to express antigens/adjuvants in the cells of a subject
  • a smaller molar amount of each species of the mRNAs is required to produce 455738066.1 50 the same antigen-specific immune response in the subject, as compared to the molar amount of each species of mRNA delivered by mRNA compositions including nanoparticles each encapsulating only one of the two species of mRNAs each expressing one of the same antigens/adjuvant, to produce the same antigen-specific immune response.
  • a smaller amount of mRNA compositions including nanoparticles each encapsulating one or more RNAs molecule including two or more species of mRNA sequences, each encoding one or more antigen and one or more adjuvant, can be required to produce an antigen-specific immune response in a subject, as compared to the amount of mRNA compositions, including two or more nanoparticles each encapsulating one mRNA molecule encoding one the same antigen or adjuvant.
  • the described mRNA compositions including nanoparticles each encapsulating one or more RNA molecules, each encoding two or more species of mRNA sequences can be used to induce an antigen-specific immune response in a subject that reduces any undesirable effects associated with the introduction of the mRNA compositions, including nanoparticles encapsulating the mRNAs into a subject.
  • Methods for screening of different vaccine agents delivered by the mRNA compositions, including nanoparticles encapsulating the mRNAs are also provided.
  • the methods include vaccinating a subject with an mRNA composition, including nanoparticles encapsulating the mRNAs, including one or more distinct antigens/adjuvants or nucleic acids encoding antigens and adjuvants and assessing the immune response in the subject.
  • the adaptive immune response to one or more antigens delivered in the mRNA composition, including nanoparticles encapsulating the mRNAs can be monitored using methods known in the art to measure the effectiveness of the vaccination protocol. For example, the duration and extent of antigen- specific T-cell activation can be used as a marker for antigen expression kinetics. Antigen-serum concentration can also be used to determine antigen-expression kinetics. 1.
  • compositions including Multiplexed mRNAs
  • the compositions include multiple mRNA sequences, for example, as part of a single polycistronic mRNA, encoding an antigen and one or more non-antigen immune- modulatory agents.
  • the polycistronic mRNA is enclosed or encapsulated within a particle, such as a nanoparticle, for example, a lipid nanoparticle (LNP).
  • compositions include multiple mRNA sequences, for example, multiple polycistronic mRNAs encapsulated within the same particle, such as the same nanoparticle.
  • the compositions include 2 or more, such as 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 species of mRNAs, each encoding the same or different species of polypeptide.
  • a single polycistronic mRNA includes 2 or more, such as 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 species of mRNAs, each encoding the same or different species of polypeptide.
  • a single particle, such as a nanoparticle includes 1 or more polycistronic mRNA molecules, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 polycistronic mRNA molecules.
  • Each polycistronic mRNA molecule can encode 2 or more than 2 species of polypeptide for expression in a target cell. Therefore, compositions including a nanoparticle, such as an LNP, encapsulating 1 or more polycistronic mRNA molecules, such as 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 polycistronic mRNA molecules are also described. In some forms, the described compositions encode 2, 3, 4, 5, 6,7, 8, 9, 10 or more than 10, up to 20 different antigenic peptides and/or adjuvant peptides and/or immune-modulatory peptides, for delivery to and expression within the same cell(s).
  • compositions include a single nanoparticle including or encapsulating one or more mRNA molecule encoding a single species of polypeptide (i.e., a single adjuvant peptide, or a single antigen peptide, or a single immunomodulatory peptide), together with one or more polycistronic mRNA molecules encoding two or more peptides (i.e., one or more adjuvant peptide, and/or one or more antigen peptide, and/or one or more immunomodulatory peptide).
  • polypeptide i.e., a single adjuvant peptide, or a single antigen peptide, or a single immunomodulatory peptide
  • polycistronic mRNA molecules encoding two or more peptides (i.e., one or more adjuvant peptide, and/or one or more antigen peptide, and/or one or more immunomodulatory peptide).
  • the described compositions provide a platform for the multiplexing the delivery and expression of multiple species of immune peptides within the same cell in vivo, for example, to stimulate antigen-specific (i.e., cellular) immunity in a host subject and simultaneously boost or otherwise control non-specific (i.e., humoral) immune responses in the same subject.
  • the multiplexing of mRNA transcripts within the same particle and/or within the same RNA molecule according to the described compositions increases the biological immune processes that are generated, increased, reduced, or otherwise controlled within a host as compared to providing the same number of transcripts as a multiplicity of single mRNAs encoding the same peptides, but each delivered within separate particles to the same host.
  • providing a single particle including 2 species of peptides (i.e., one antigen and one adjuvant) to a host cell in vivo induces a stronger immune response in the host that providing the same mRNA encoding the same antigen in a first particle and the same mRNA encoding the same adjuvant as a second particle to the same host. It may be that the simultaneous processing, translation and/or post-translational modification of multiple species of mRNAs within a single cell enhances the biological activity of immune cells within the host as compared with processing the mRNAs delivered as two separate particles, for example, within two different cells of the same host.
  • the multiplexing of mRNAs encoding two peptides (e.g., one antigen peptide and one adjuvant peptide) within the same polycistronic mRNA molecule increases the biological immune processes that are generated, increased, reduced, or otherwise controlled within a host as compared to providing the same number of transcripts as single mRNAs, e.g., 455738066.1 52 encoding the same species of peptides, delivered within the same particle to the same host.
  • transcripts from the same polycistronic mRNA provides a more robust translation of equal amounts of each peptide within the cell, and/or provides a more effective immunomodulatory effect as compared with processing of a multiplicity of single mRNA molecules.
  • the combination of encoded antigen peptide(s), adjuvant peptide(s) and immune-modulatory agent peptide(s) that are encoded by encapsulated mRNA(s) according to the disclosure are effective to induce formation of a tertiary lymphoid structure when expressed within a host in vivo.
  • TLSs Tertiary lymphoid structures
  • ectopic lymphoid organs mainly include chemo-attracting B cells, T cells, and supporting dendritic cells (DCs).
  • DCs dendritic cells
  • Mature TLSs exhibit functional organization for the optimal development and collaboration of adaptive immune response, delivering an augmented effect on the tumor microenvironment (TME) Emerging evidence indicates that effector cells never fight alone during an immune response. Based on their presence in the TME, the localization, organization, and other cell types of the immune cell lineage can all greatly alter the tumor-associated immune response. It is worth pointing out that the compartmentalized organization defined by the physical separation of immune/non-immune clusters could independently determine the outcome of tumor sufferers.
  • TLSs tertiary lymphoid structures
  • DCs dendritic cells
  • SLOs secondary lymphoid organs
  • compositions include mRNA(s) encoding one or more of vascular cell adhesion molecule 1 (VCAM1), intercellular adhesion molecule 1 (ICAM1), mucosal vascular addressin cell adhesion molecule 1 (MAdCAM1), peripheral lymph node addressin (PNAd)) and/or one or more chemokines (e.g., chemokine (C-C motif) ligand 19 (CCL19), CCL21, and chemokine (C-X-C motif) ligand 13 (CXCL13)).
  • VCAM1 vascular cell adhesion molecule 1
  • ICAM1 intercellular adhesion molecule 1
  • MAdCAM1 mucosal vascular addressin cell adhesion molecule 1
  • PNAd peripheral lymph node addressin
  • the compositions include one or more mRNA(s) encoding one or more molecules that promote, stimulate, enhance or otherwise generate vascularization and/or immune cell recruitment at or 455738066.1 53 within a tertiary lymphoid structure.
  • the compositions include one or more mRNA(s) encoding one or more molecules that promote B cell recruitment, together with the downstream process for lymphoid neogenesis, including but not limited to vessel differentiation, chemokine induction, T/B segregation, and germinal center (GC) formation.
  • Specific signals thought to be involved in TLS formation include receptors such as lymphotoxin alpha, lymphotoxin beta, CXCL13 and TNF, as well as type I IFNs.
  • the combination of encoded antigen peptide(s), adjuvant peptide(s) and immune- modulatory agent peptide(s) include one or more of lymphotoxin alpha, lymphotoxin beta, CXCL13 and TNF, and/or type I IFNs.
  • the DNA vectors contains the following elements: (i) a prokaryotic origin of replication, so that the vector may be amplified in a prokaryotic host; (ii) a gene encoding a marker which allows selection of prokaryotic host cells that contain the vector (e.g., a gene encoding antibiotic resistance); and (iii) at least one gene encoding a desired mRNA or protein located adjacent to a transcriptional promoter capable of directing the expression of the gene.
  • nucleic acid for producing an mRNA includes a nucleotide sequence encoding a peptide antigen and/or adjuvant or immunomodulatory agent and optionally one or more additional elements linked thereto.
  • the elements include but are not limited to elements that enhance transcription or translation.
  • Non-limiting exemplary elements are discussed below and include, but are not limited to, signal peptide, promoter region such as T7 promoter, TRILINK CAP site, traditional KOZAK sequence, 5’ untranslated region (UTR), 3’ UTR, and poly(A) tail. It will be appreciated that some of these elements are present only for the purpose of transcription (e.g., promoter, CAP site, UTRs, etc.), and thus, may be absent from an mRNA construct and expressed protein.
  • nucleic acid stability and/or translation e.g., TRILINK CAP site, KOZAK sequence, poly(A) tail
  • protein trafficking and/or purification e.g., signal sequence and purification tags
  • these elements and others are disclosed in all combinations, and can be selectively present or absent from the constructs depending on the particular composition at a particular time, e.g., DNA or RNA vector construct, mRNA, or protein.
  • a signal peptide is incorporated to improve transcription of one or more mRNAs encoding one or more antigen and/or adjuvant peptides.
  • the nucleic acid sequence also includes one or more signal peptides. 455738066.1 54
  • the nucleic acid includes a promoter sequence. In one form, the nucleic acid includes a T7 promoter sequence. In another form, the nucleic acid includes a T3 promoter. In a further form, the nucleic acid includes a SP6 promoter. In some forms, the nucleic acid includes a 5’ untranslated region (UTR) sequence. In some forms, the nucleic acid includes a 3’ UTR sequence.
  • the isolated nucleic acid sequence includes nucleotide sequence encoding the antigen such as the LTA protein of MCPyV, and/or immunogenic domains and fragments thereof, and (i) signal peptide at the 5’ end of the nucleotide sequence encoding the antigen, (ii) one or more restriction sites flanking either or both side of the nucleotide sequence, (iii) promoter region such as T7 promoter, (iv) TRILINK CAP site, (v) a KOZAK sequence, (vi) 5’ UTR, (vii) 3’ UTR, and (viii) poly(A) tail.
  • the antigen such as the LTA protein of MCPyV
  • signal peptide at the 5’ end of the nucleotide sequence encoding the antigen
  • promoter region such as T7 promoter
  • TRILINK CAP site a KOZAK sequence
  • DNA constructs that encode a functional mRNA subunit for antigens and adjuvants are designed.
  • a T7 promoter sequence was added immediately following the first restriction site for compatibility with commercial in-vitro transcription kits. This T7 promoter sequence was modified to end with an AGG sequence for compatibility with Trilink (San Diego, CA) CleanCap 5’ mRNA capping technology.
  • UTR untranslated region
  • a synthetic sequence was chosen based on previously published screen (Cao, et al., Nat Commun 12, 41382021).
  • DNA constructs capable of encoding antigen and adjuvants are co- encapsulated for in vivo delivery. Linked constructs that contain coding regions for both the antigens of interest and adjuvants as a single functional subunit can be designed.
  • antigen constructs are linked to the adjuvant constructs using a self-cleaving linker, such as a 2A peptide linker, with addition of a Furin cleavage site that excludes the linker amino acids and produces full length antigen and functional full-length adjuvants as separate end products of a single mRNA.
  • Furin is a recombinant, ubiquitous subtilisin-like proprotein convertase with a minimal cleavage site of Arg-X-X-Arg, preferably the site Arg-X-Lys/Arg-Arg.
  • An exemplary furin cleavage nucleotide sequence is CGGGCCAAGCGG (SEQ ID NO:34).
  • two or more separate adjuvant transcripts can be linked using a self-cleaving P2A linker followed by a self-cleaving T2A linker with Furin cleavage sites that exclude linker amino acids. 455738066.1 55
  • these DNA sequences can be cloned into a vector such as pUC57-Simple or pUC57-Simple-BsaI-BsmBI-Free vector with an antibiotic resistance gene and be amplified into a prokaryotic host.
  • the template DNA can be extracted from the base plasmids by cutting at the appropriate restriction sites.
  • Template DNAs can be separated from plasmid DNAs based on fragment size by gel electrophoresis and be purified from agarose gel using Gel DNA recovery kit. Purified, double stranded, linearized template DNA can then be used for mRNA production via in vitro transcription methods. mRNA is 5’ capped for increased stability and translational efficiency. The mRNA products are then purified using RNA purifying kits and these purified functional mRNA can then be stored in sterile water at -80 degrees. The cloning platform demonstrated here can be used to substitute any suitable antigen transcript combined with any suitable adjuvant transcript(s) in a fully customizable model depending on the indication and clinical intent (i.e., prophylactic vs. therapeutic use). 1.
  • the described messenger ribonucleic acid (mRNA) molecules typically include a linker between the two or more sequences encoding different polypeptides.
  • linker sequences are inserted between multiple protein-coding sequences (ORFs) on a single mRNA transcript to ensure each protein is independently expressed.
  • ORFs protein-coding sequences
  • these linkers facilitate ribosomal skipping or peptide cleavage, allowing the ribosome to move to the next ORF without translating the linker itself.
  • the linkers are expressed and include a cleavage site, such as a protease cleavage site. Any linkers designed for expression of a polycistronic construct can be incorporated into the described synthetic mRNAs.
  • linkers can be fusion linkers, proteolytic cleavage sites, internal ribosomal entry sites (IRESs), or self-cleaving 2 A peptides.
  • the linkers can be “read through” linkers, such as tandem “Gly-Gly-Ser” or “Gly-Ser” repeats that act as “fusion peptides”, and/or self-cleaving 2A peptides, or they can be non-read through linkers, such as an IRES sequence.
  • a linker is a 2A peptide.
  • 2A peptides include polypeptides of about 18 to 22 contiguous amino acids, which can induce “ribosomal skipping” during translation, i.e., to cause the ribosome to “skip” over the peptide sequence during translation, effectively separating the two proteins.2A peptides are naturally present in various viruses, and include the sequence D-X-E-X-NPGP (SEQ ID NO:26).
  • the described synthetic mRNAs include a single ORF including a sequence encoding one or more 2A peptides to generate polyproteins by causing the ribosome to fail at making a peptide bond, and then resume translation.
  • Exemplary 2A peptide amino acid sequences include: EGRGSLLTCGDVEENPGP (“T2A”; SEQ ID NO:27); ATNFSLLKQAGDVEENPGP (“P2A”; SEQ ID NO:28); QCTNYALLKLAGDVESNPGP (“E2A”; SEQ ID NO:29); and VKQTLNFDLLKLAGDVESNPGP (“F2A”; SEQ ID NO:30).
  • a 2A peptide sequence is located between a first and second ORF encoding a polypeptide.
  • more than one 2A sequence is located between a first and second ORF encoding a polypeptide, for example, two or three 2A sequences in tandem may be included.
  • An exemplary nucleotide sequence for a T2A sequence includes: gagggcaggggaagtcttctaacatgcggggacgtggaggaaaatcccggccca (SEQ ID NO:31).
  • Any of the described 2A linkers can include a “GSG” (Gly-Ser-Gly) sequence on the N- terminal of a 2A peptide, for example, to assist with efficiency.
  • a linker is an internal ribosome entry site (IRES) or Ubx-IRES.
  • IRES sequence is an RNA element that allows for translation initiation in a cap-independent manner, as part of the greater process of protein synthesis.
  • IRES elements allow ribosomes to engage the mRNA and begin translation independently of the 5' cap. IRES sequences are known in the art (see, e.g., Zhao et al., Genomics Proteomics Bioinformatics.2020 Apr;18(2):129-139. doi: 10.1016/j.gpb.2020.03.001).
  • IRES nucleotide sequences include: aaaaacttccggcgggaaccggaaggtgcggtggcactcacggaatctcgggtcttctgacgtg ccgggcgggaagatgtcatcattgccaagaagagcgaaagtacaggtccaggatgtggtactga aagatgaattttctttcattctctgagttatcatctgcctctgagttatcatctgcctctgaaga (SEQ ID NO:32); and aaaatccaccccaacaatcgctgtgtgccgctttagtgcgctcgcgtcggctctacctgcgtg cttgctctcccaaccccggacacccggcttcgactggt tata
  • a synthetic mRNA prepared according to the described methods includes, from 5’ to 3’ (i) a 5’ Cap 1 structure; (ii) a 5’ untranslated region (5’ UTR); 455738066.1 57 (iii) a sequence encoding a first polypeptide; (iv) a first Furin cleavage sequence; (v) a first linker sequence, such as a P2A linker; (vi) a sequence encoding a second polypeptide; (vii) a 3’ untranslated region (3’ UTR); and (viii) a poly Adenylated 3’tail; whereby one of the first or the second polypeptide encodes the peptide antigen; and whereby one of the first or the second polypeptide encodes a peptide adjuvant.
  • the synthetic mRNA prepared according to the described methods includes, from 5’ to 3’ (i) a 5’ Cap 1 structure; (ii) a 5’ untranslated region (5’ UTR); (iii) a sequence encoding a first polypeptide; (iv) a first Furin cleavage sequence; (v) a first linker sequence, such as a P2A linker, optionally including an N-terminal Gly-Ser-Gly; (vi) a sequence encoding a second polypeptide; (vii) a second Furin cleavage sequence; (viii) a second linker sequence, such as a P2A linker, optionally including an N- terminal Gly-Ser-Gly; (ix) a sequence encoding a third polypeptide; (x) a 3’ untranslated region (3’ UTR); and (xi) a poly Adenylated 3’ tail; whereby the first or the second polypeptide encodes a first peptide
  • n1-methyl-pseudo UTP is also substituted for stability, and/or to limit immunogenicity of the resulting mRNA compositions in vivo.
  • base substitution is 100%. In other forms, base substitution is in an amount from about 10% to about 99%, inclusive.
  • An exemplary synthetic mRNA includes a cleavable linker configured between two coding mRNA sequences encoding a first and second, and/or second and third polypeptides.
  • An exemplary cleavable linker includes a furin recognition sequence, a Gly-Ser-Gly polypeptide, and a P2A linker peptide.
  • An exemplary cleavable linker polypeptide construct includes: RAKRGSGEGRGSLLTCGDVEENPGP (SEQ ID NO:34), with the furin cleavage sequence indicated in bold font, the Gly-Ser-Gly indicated in italic font, and the P2A peptide indicated in uppercase font, respectively.
  • a corresponding nucleic acid sequence encoding the cleavable linker polypeptide construct of SEQ ID NO:34 includes: cgggccaagcggggctccggcgagggcaggggggaagtcttctaacatgcggggacgtggaggaaa atcccggccca (SEQ ID NO:35).
  • modified nucleotides include, but are not limited to diaminopurine, S 2 T, 5- fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4- acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2- thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6- isopentenyladenine, 1 -methylguanine, 1 -methylinosine, 2,2-dimethylguanine, 2- methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7- methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D- mannosylqueos
  • Nucleotides within the describe synthetic mRNAs may also be modified at the base moiety (e.g., at one or more atoms that typically are available to form a hydrogen bond with a complementary nucleotide and/or at one or more atoms that are not typically capable of forming a hydrogen bond with a complementary nucleotide), sugar moiety or phosphate backbone.
  • the described synthetic mRNAs include Locked nucleic acids (LNA).
  • LNA is a family of conformationally locked nucleotide analogues which, amongst other benefits, imposes truly unprecedented affinity and very high nuclease resistance to DNA and RNA oligonucleotides (Wahlestedt C, et al., Proc. Natl Acad. Sci. USA, 975633–5638 (2000); Braasch, DA, et al., Chem. Biol.81–7 (2001); Kurreck J, et al., Nucleic Acids Res.301911–1918 (2002)). 455738066.1 59
  • the described synthetic mRNAs include Peptide nucleic acids (PNA).
  • PNA is a nucleic acid analog in which the sugar phosphate backbone of natural nucleic acid has been replaced by a synthetic peptide backbone usually formed from N-(2-amino-ethyl)-glycine units, resulting in an achiral and uncharged mimic (Nielsen, et al., Science 254, 1497-1500 (1991)). It is chemically stable and resistant to hydrolytic (enzymatic) cleavage.
  • the described synthetic mRNAs include a combination of natural or synthetic ribonucleotides, PNAs, LNAs, or any combination thereof.
  • the described synthetic mRNAs include one or more modified nucleotides that provide nuclease resistant nucleotide linkages, such as phosphorothioate (PS) bonds which substitute a sulfur atom for a non-bridging oxygen in the phosphate backbone of an oligonucleotide; 2'-O-Methyl (2'OMe), a naturally occurring post-transcriptional modification of RNA that prevents attack by single-stranded endonucleases; 2' fluoro bases, that include a fluorine-modified ribose which increases binding affinity and also confers some relative nuclease resistance relative to native RNA; inverted dT and/or ddT incorporated at the 3′ end of an oligonucleotide, leading to a 3'-3' linkage that will inhibit degradation by 3' exonucleases; and phosphorylation of the 3′ end of the nucleotides to inhibit degradation by some 3′-exonuclea
  • PS
  • nanoparticles such as lipid nanoparticles can be used to encapsulate the mRNAs for delivery of mRNAs into a subject.
  • lipid nanoparticles containing SM-102, 1,2-DSPC, Cholesterol, and DMG-PEG in a lipid molar ration of 50:10:38.5:1.5 are assembled and incorporated with mRNA using a rapid solvent injection mixing technique.
  • mRNA can be first combined with 50 mM sodium acetate solution at pH 5.0.
  • This solution can be then stirred in a sterile container at 700 rpm and the ethanolic mixture can be rapidly injected into the acidic solution and mixed for an additional 30 minutes to create homogenous nanoparticles containing the functional mRNA units.
  • Placebo containing all components except for mRNA can be prepared using the same process.
  • mRNA compositions, including nanoparticles encapsulating the mRNAs can then dialyzed against 500 volumes of sterile PBS.
  • the nanoparticle solution can be then drawn into sterile 1mL syringes and stored at 4 degrees until use. IV.
  • compositions for example including a synthetic messenger ribonucleic acid (mRNA) including (a) one or more ribonucleic acid sequences encoding one or more peptide antigens; and (b) one or more ribonucleic acid sequences encoding one or more peptide adjuvants optionally packaged in nanoparticles, are provided.
  • mRNA messenger ribonucleic acid
  • the methods induce an immune response to a peptide antigen in a subject by administering to the subject the pharmaceutical composition including a synthetic messenger ribonucleic acid (mRNA) including (a) one or more ribonucleic acid sequences encoding one or more peptide antigens; and (b) one or more ribonucleic acid sequences encoding one or more peptide adjuvants, whereby the ribonucleic acid is configured to express the peptide antigen(s) and the peptide adjuvant(s) as distinct polypeptides within a mammalian cell.
  • mRNA synthetic messenger ribonucleic acid
  • compositions to treat one or more diseases or disorders in a subject by administering the compositions of synthetic mRNA(s) to the subject are also provided. Any of the methods can include the selection of a subject and/or a suitable control. Suitable routes of administration, dosages and effective amounts are also described. A.
  • Methods of Treatment of treating a subject by administering to the subject an effective amount of the described compositions, for example, including a synthetic messenger ribonucleic acid (mRNA) including one or more ribonucleic acid sequences encoding one or more peptide antigens; and one or more ribonucleic acid sequences encoding one or more peptide adjuvants mRNAs and/or nanoparticles encapsulating the synthetic mRNAs for treating or preventing one or more diseases in the subject are provided.
  • mRNA messenger ribonucleic acid
  • the methods administer an amount of the described mRNA compositions to raise an immune response to the antigenic component in the subject, whereby the encoded adjuvant component simultaneously enhances, directs or otherwise influences the size, scope and/or downstream response to the antigen in the subject.
  • the mRNAs encode one or more antigen(s) associated with a disease or pathological state in the subject, and the methods administer the composition to treat or ameliorate the disease or pathological state in the subject.
  • the methods administer an mRNA encoding a cancer antigen to a subject to treat or ameliorate one or more symptoms of the cancer in the subject.
  • the methods administer an mRNA encoding an antigen derived from a disease state or a pathogen to a subject to prevent or delay the onset of one or more symptoms of the disease or an infection with the pathogen in the subject. Therefore, in some forms, the methods administer the compositions prophylactically to the subject. Methods for using the described compositions including one or more mRNA antigens and one or more mRNA adjuvants as a vaccine for inducing or stimulating an immune response to the encoded antigen(s) in a subject are provided.
  • the methods are effective to induce an immune response to a peptide antigen(s) in a subject, for example, whereby the immune response is effective to treat or prevent an infection and/or disease in the subject caused by the organism or agent from which the 455738066.1 61 antigen is derived.
  • exemplary diseases and/or infections include proliferative diseases such as cancer, and infectious diseases, such as those caused by a pathogenic bacterium, or a pathogenic virus, or a pathogenic fungus, or a pathogenic protozoan.
  • the methods are effective to treat a subject having a disease, disorder, or condition by administering to the subject an effective amount of a synthetic mRNA including (a) one or more ribonucleic acid sequences encoding one or more peptide antigens; and (b) one or more ribonucleic acid sequences encoding one or more peptide adjuvants to treat or reduce a symptom of a disease, disorder, or condition in the subject.
  • compositions including one or more mRNA antigens and one or more mRNA adjuvants can deliver protein and/or nucleic acid antigen to a subject in an amount effective to vaccinate the subject from one or more diseases and disorders.
  • the Compositions including one or more mRNA antigens and one or more mRNA adjuvants can serve as a vaccination platform for a wide variety of microbial pathogens, such as bacterial, viral, fungal and protozoan pathogens.
  • the target of the vaccine could be a type of cancer cell as a cancer treatment. Alternately, the target could be any of a large number of microbial pathogens.
  • Exemplary diseases that can be vaccinated against include disease for which vaccines are currently available.
  • Compositions including one or more mRNA antigens and one or more mRNA adjuvants can serve as a platform for inducing immunological tolerance to a subject to one or more allergens, such as food allergens and environmental allergens.
  • the recipient subject has a disease or disorder, or has been identified as being at increased risk of getting a disease or disorder.
  • the subject has cancer, or has been identified as being at increased risk of getting a cancer.
  • the subject has an infectious disease, or has been identified as being at increased risk of getting an infectious disease. a.
  • compositions including one or more mRNA antigens and one or more mRNA adjuvants can be used to immunize a subject against cancer.
  • the methods treat or prevent cancer or other proliferative diseases or disorders in a subject identified as having, or at risk of having cancer or other proliferative disease or disorder.
  • Cancer is a disease of genetic instability, allowing a cancer cell to acquire the hallmarks proposed by Hanahan and Weinberg, including (i) self-sufficiency in growth signals; (ii) insensitivity to anti-growth signals; (iii) evading apoptosis; (iv) sustained angiogenesis; (v) tissue 455738066.1 62 invasion and metastasis; (vi) limitless replicative potential; (vii) reprogramming of energy metabolism; and (viii) evading immune destruction (Cell.,144:646–674, (2011)).
  • Tumors which can be treated in accordance with the disclosed methods, are classified according to the embryonic origin of the tissue from which the tumor is derived.
  • Carcinomas are tumors arising from endodermal or ectodermal tissues such as skin or the epithelial lining of internal organs and glands. Sarcomas, which arise less frequently, are derived from mesodermal connective tissues such as bone, fat, and cartilage.
  • the leukemias and lymphomas are malignant tumors of hematopoietic cells of the bone marrow. Leukemias proliferate as single cells, whereas lymphomas tend to grow as tumor masses. Malignant tumors may show up at numerous organs or tissues of the body to establish a cancer.
  • compositions including one or more mRNA antigens and one or more mRNA adjuvants are administered to a subject diagnosed with cancer (i.e., as a therapeutic vaccine), or to a subject having a predisposition or risk of developing cancer (i.e., as a prophylactic vaccine).
  • compositions of compositions including one or more mRNA antigens and one or more mRNA adjuvants are administered to a cancer patient in addition to one or more additional therapeutic agents.
  • bioinformatics is used to sequence each patient’s unique tumor exome to identify neoantigens.
  • compositions and methods of treatment thereof are generally suited for treatment of carcinomas, sarcomas, lymphomas and leukemias.
  • the described compositions and methods are useful for treating, or alleviating subjects having benign or malignant tumors by delaying or inhibiting the growth/proliferation or viability of tumor cells in a subject, reducing the number, growth or size of tumors, inhibiting or reducing metastasis of the tumor, and/or inhibiting or reducing symptoms associated with tumor development or growth.
  • the types of cancer that can be treated with the provided compositions and methods include, but are not limited to, cancers such as vascular cancer such as multiple myeloma, adenocarcinomas and sarcomas, of bone, bladder, brain, breast, cervical, colorectal, esophageal, kidney, liver, lung, nasopharangeal, pancreatic, prostate, skin, stomach, and uterine.
  • cancers such as vascular cancer such as multiple myeloma, adenocarcinomas and sarcomas, of bone, bladder, brain, breast, cervical, colorectal, esophageal, kidney, liver, lung, nasopharangeal, pancreatic, prostate, skin, stomach, and uterine.
  • the compositions are used to treat multiple cancer types concurrently.
  • the compositions can also be used to treat metastases or tumors at multiple locations.
  • compositions including one or more mRNA antigens and one or more mRNA adjuvants include one or more tumor antigens or one or more nucleic acids expressing a 455738066.1 63 tumor antigen.
  • the compositions including one or more mRNA antigens and one or more mRNA adjuvants can be used to provide immunity and therapeutic activity against tumor cells and non-tumor cells located within a tumor or a tumor environment.
  • Compositions including one or more mRNA antigens and one or more mRNA adjuvants can be formulated to provide protective and/or therapeutic activity against solid tumors and cancers of the blood.
  • tumor cells include, but are not limited to, tumor cells of cancers, including leukemias including, but not limited to, acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemias such as myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemia leukemias and myelodysplastic syndrome, chronic leukemias such as but not limited to, chronic myelocytic (granulocytic) leukemia, chronic lymphocytic leukemia, hairy cell leukemia; polycythemia vera; lymphomas such as, but not limited to, Hodgkin’s disease, non-Hodgkin’s disease; multiple myelomas such as, but not limited to, smoldering multiple myeloma, non-secretory myeloma, osteosclerotic myeloma, plasma cell leukemia, solitary plasmacytoma and extramedul
  • Cancers that can be prevented, treated or otherwise diminished by the Compositions including one or more mRNA antigens and one or more mRNA adjuvants include myxosarcoma, osteogenic sarcoma, endotheliosarcoma, lymphangioendotheliosarcoma, mesothelioma, synovioma, hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchogenic carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, and gastric cancer (for a review of such disorders, see Fishman et al., 1985, Medicine, 2d Ed., J.B.
  • compositions including one or more mRNA 455738066.1 65 antigens and one or more mRNA adjuvants can be used to immunize a subject against one or more cancers for which no alternative vaccine is available.
  • compositions including one or more mRNA antigens and one or more mRNA adjuvants can deliver antigens to the APC of a subject in an amount effective to vaccinate the subject from one or more infectious diseases caused by a wide variety of microbial pathogens, such as bacterial, viral, fungal and protozoan pathogens.
  • microbial pathogens such as bacterial, viral, fungal and protozoan pathogens.
  • the target of the vaccine could be any of a large number of microbial pathogens.
  • Exemplary diseases that can be vaccinated against include disease for which vaccines are currently available, including Anthrax; Diseases (e.g., cervical cancer, cancer of the esophagus) caused by Human Papillomavirus (HPV); Diphtheria; Hepatitis A; Hepatitis B; Haemophilus influenzae type b (Hib); Influenza viruses (Flu); Japanese encephalitis (JE); Lyme disease; Measles; Meningococcal; Monkeypox; Mumps; Pertussis; Pneumococcal; Polio; Rabies; Rotavirus; Rubella; Shingles (Herpes Zoster); Smallpox; Tetanus; Toxoplasmosis; Typhoid; Tuberculosis (TB); Varicella (Chickenpox); Yellow Fever.
  • Anthrax e.g., cervical cancer, cancer of the esophagus
  • HPV Human Papillomavirus
  • compositions including one or more mRNA antigens and one or more mRNA adjuvants can be used to immunize a subject against an infectious disease or pathogen, for example, for vaccination against an infectious disease or pathogen to which no alternative vaccine is available/
  • infectious disease or pathogen for example, for vaccination against an infectious disease or pathogen to which no alternative vaccine is available/
  • Exemplary diseases include, but are not limited to, malaria, streptococcus, Ebola Zaire, HIV, Herpes virus, hepatitis C, Middle East Respiratory Syndrome (MERS), Sleeping sickness, Severe Acute Respiratory Syndrome (SARS), rhinovirus, chicken pox, Hendra, NIPA virus, Zika Virus, and others.
  • the disease is a pathogen that infects non-mammalian subjects, such as birds.
  • Exemplary avian subjects include domesticated birds (i.e., poultry), such as chickens, ducks, geese, pheasants and other commercial fowl, or pet birds such as parakeets and parrots.
  • domesticated birds i.e., poultry
  • poultry such as chickens, ducks, geese, pheasants and other commercial fowl
  • pet birds such as parakeets and parrots.
  • bacterial hybrid vectors can be useful to vaccinate birds against Infectious Bursal Disease (IBD).
  • IBD also known as Gumboro disease, a viral disease affecting the Bursa of Fabricius of young chickens.
  • compositions and methods disclosed herein may also be used to promote tolerance.
  • Tolerogenic therapy aims to induce immune tolerance where there is pathological or undesirable activation of the normal immune response.
  • Such embodiments may also include co- administration of an immunosuppressive agent such as rapamycin.
  • Tolerogenic vaccines deliver antigens with the purpose of suppressing immune responses (e.g., induce or increase a suppressive immune response) and promoting robust long-term antigen-specific immune tolerance.
  • compositions and methods disclosed herein are also useful for controlling the immune response to an antigen.
  • the compositions are used as part of a tolerizing vaccine.
  • the disclosed compositions are used to treat an inflammatory response or autoimmune disorder in a subject.
  • the disclosed methods can be used to prophylactically or therapeutically inhibit, reduce, alleviate, or permanently reverse one or more symptoms of an inflammatory response or autoimmune disorder.
  • An inflammatory response or autoimmune disorder can be inhibited or reduced in a subject by administering to the subject an effective amount of a composition in vivo, or cells modulated by the composition ex vivo.
  • Representative inflammatory responses and autoimmune diseases that can be inhibited or treated include, but are not limited to, rheumatoid arthritis, systemic lupus erythematosus, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison’s disease, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome (alps), autoimmune thrombocytopenic purpura (ATP), Bechet’s disease, bullous pemphigoid, cardiomyopathy, celiac sprue-dermatitis, chronic fatigue syndrome immune deficiency, syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, cicatricial pemphigoid, cold agglutinin disease, Crest syndrome, Crohn’s disease, Dego’s disease, dermatomyositis, dermatomyositis – juvenile, discoid l
  • compositions including one or more mRNA antigens and one or more mRNA adjuvants can be used to immunize a subject against an allergen.
  • the compositions and methods can therefore be tolerogenic vaccines.
  • the compositions including one or more mRNA antigens and one or more mRNA adjuvants can be administered to a subject diagnosed with an allergy or to a subject having a predisposition to an allergy.
  • the compositions of compositions including one or more mRNA antigens and one or more mRNA adjuvants are administered to a patient having an allergy in addition to one or more additional therapeutic agents. Allergies are abnormal reactions of the immune system that occur in response to otherwise harmless substances.
  • An allergy is a type of immune reaction in which the immune system responds to foreign microorganisms or particles by producing specific antibodies capable of binding to allergens such as pollen, dust, animal hairs, etc.
  • Allergic reactions that can be treated include delayed hypersensitivity reactions and immediate hypersensitivity reactions.
  • Allergies that can be treated include allergic responses in the skin, such as dermatitis, the upper airways and eyes, such as allergic rhinitis, hay fever, asthma, and conjunctivitis (pink eye) in the gastrointestinal tract, such as food allergies, and blood stream, such as urticaria and hives, angioedema, anaphylaxis, or atopic dermatitis.
  • allergic responses in the skin such as dermatitis, the upper airways and eyes, such as allergic rhinitis, hay fever, asthma, and conjunctivitis (pink eye) in the gastrointestinal tract, such as food allergies, and blood stream, such as urticaria and hives, angioedema
  • mRNA compositions, including nanoparticles encapsulating the mRNAs can be formulated into compositions including suitable excipient for administering the nanoparticles into the body of a subject.
  • mRNA compositions, including nanoparticles encapsulating the mRNAs are formulated in a carrier or excipient suitable for delivery into a subject by injection, for example, via intramuscular (i.m.) intravenous (i.v.), subcutaneous (s.c.), intraperitoneal (i.p.), or via skin scarification.
  • Typical carriers are saline, phosphate buffered saline, glucose solutions, and other injectable carriers.
  • formulations including mRNA compositions, with or without delivery vehicles such as nanoparticles encapsulating the mRNAs are described.
  • the mRNA compositions, including nanoparticles encapsulating the mRNAs can be formulated into pharmaceutical compositions including one or more pharmaceutically acceptable carriers.
  • Pharmaceutical compositions can be formulated for different mechanisms of administration, according to the desired purpose of the mRNA composition, including nanoparticles encapsulating the mRNAs, and the intended use.
  • compositions formulated for administration by parenteral intramuscular, intraperitoneal, intravenous (IV), intraocular or subcutaneous injection), topical or transdermal (either passively or using iontophoresis or electroporation) routes of administration or using bioerodible inserts are described.
  • parenteral intramuscular, intraperitoneal, intravenous (IV), intraocular or subcutaneous injection
  • topical or transdermal either passively or using iontophoresis or electroporation
  • routes of administration or using bioerodible inserts are described.
  • mRNA compositions, including nanoparticles encapsulating the mRNAs are formulated for administration in an aqueous solution, by parenteral injection.
  • the formulation may also be in the form of a suspension or emulsion.
  • compositions including effective amounts of an active agent, targeting moiety, and optional a delivery vehicle and optionally include pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, and/or carriers.
  • Such compositions include the diluents sterile water, buffered saline of various buffer content (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength and optionally additives such as detergents and solubilizing agents (e.g., TWEEN® 20, TWEEN® 80 also referred to as polysorbate 20 or 80), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), and preservatives (e.g., Thimersol, benzyl alcohol) and bulking substances (e.g., lactose, mannitol).
  • buffered saline of various buffer content e.g., Tris-HCl, acetate, phosphate
  • non-aqueous solvents or vehicles examples include propylene glycol, polyethylene glycol, vegetable oils, such as olive oil and corn oil, gelatin, and injectable organic esters such as ethyl oleate.
  • the formulations may be lyophilized and redissolved/resuspended immediately before use.
  • the formulation may be sterilized by, for example, filtration through a bacteria retaining filter, by incorporating sterilizing agents into the compositions, by irradiating the compositions, or by heating the compositions.
  • Compositions of mRNA compositions, including nanoparticles encapsulating the mRNAs can be formulated for application topically, by instillation or by inhalation.
  • mRNA compositions including nanoparticles encapsulating the mRNAs, are formulated for administration to the mucosa, such as the lungs, mouth, eyes, lungs, nasal, oral (sublingual, buccal), vaginal, or rectal mucosa.
  • Formulations for administration to the mucosa will typically be spray dried drug particles, which may be incorporated into a tablet, gel, capsule, suspension or emulsion. Standard pharmaceutical excipients are available from any formulator.
  • the mRNA compositions, including nanoparticles encapsulating the mRNAs are formulated for delivery to the skin, for example, by direct application to the surface 455738066.1 69 of diseased, or damaged or ruptured skin.
  • mRNA compositions including nanoparticles encapsulating the mRNAs are formulated for delivery to a wound or site of surgery.
  • Compositions formulated for topical delivery can include one or more penetration enhancers.
  • the mRNA compositions, including nanoparticles encapsulating the mRNAs are formulated for pulmonary delivery, such as intranasal administration or oral inhalation.
  • the respiratory tract is the structure involved in the exchange of gases between the atmosphere and the blood stream.
  • the upper and lower airways are called the conducting airways.
  • the terminal bronchi divide into respiratory bronchiole, which then lead to the ultimate respiratory zone, the alveoli, or deep lung.
  • the deep lung, or alveoli is the primary target of inhaled therapeutic aerosols for systemic drug delivery.
  • Therapeutic agents that are active in the lungs can be administered systemically and targeted via pulmonary absorption.
  • aerosol refers to any preparation of a fine mist of particles, which can be in solution or a suspension, whether or not it is produced using a propellant. Aerosols can be produced using standard techniques, such as ultra-sonication or high-pressure treatment. Carriers for pulmonary formulations can be divided into those for dry powder formulations and for administration as solutions. Aerosols for the delivery of therapeutic agents to the respiratory tract are known in the art.
  • the formulation can be formulated into a solution, e.g., water or isotonic saline, buffered or un- buffered, or as a suspension, for intranasal administration as drops or as a spray.
  • a solution e.g., water or isotonic saline, buffered or un- buffered, or as a suspension
  • such solutions or suspensions are isotonic relative to nasal secretions and of about the same pH, ranging e.g., from about pH 4.0 to about pH 7.4 or, from pH 6.0 to pH 7.0.
  • Buffers should be physiologically compatible and include, simply by way of example, phosphate buffers.
  • phosphate buffers One skilled in the art can readily determine a suitable saline content and pH for an innocuous aqueous solution for nasal and/or upper respiratory administration.
  • Compositions can be delivered to the lungs while inhaling and traverse across the lung epithelial lining to the blood stream when delivered either as an aerosol or spray dried particles having an aerodynamic diameter of less than about 5 microns.
  • Dry powder formulations (“DPFs”) with large particle size have improved flowability characteristics, such as less aggregation, easier aerosolization, and potentially less phagocytosis.
  • Dry powder aerosols for inhalation therapy are generally produced with mean diameters primarily in the range of less than 5 microns, although a preferred range is between one and ten microns in aerodynamic diameter. Large “carrier” particles (containing no drug) have been co- delivered with therapeutic aerosols to aid in achieving efficient aerosolization among other possible benefits.
  • compositions of Compositions including one or more mRNA antigens and one or more mRNA adjuvants are administered to a subject in a therapeutically effective amount.
  • an effective amount or “therapeutically effective amount” means a dosage sufficient to treat, inhibit, or alleviate one or more symptoms of the disorder being treated or to otherwise provide a desired pharmacologic and/or physiologic effect.
  • the precise dosage will vary according to a variety of factors such as subject-dependent variables (e.g., age, immune system health, etc.), the disease or disorder, and the treatment being effected.
  • subject-dependent variables e.g., age, immune system health, etc.
  • information will emerge regarding appropriate dosage levels for treatment of various conditions in various patients, and the ordinary skilled worker, considering the therapeutic context, age, and general health of the recipient, will be able to ascertain proper dosing.
  • the selected dosage depends upon the desired therapeutic effect, on the route of administration, and on the duration of the treatment desired.
  • compositions are formulated to achieve a modified prokaryotic cell serum level of between about 1 and about 1,000 ⁇ M. It has been established that an effective dose range (total mRNA content antigen + adjuvant) in mice is from about 10 ⁇ g to about 30 ⁇ g, inclusive per dose, with a preferred dose of about 15 ⁇ g. In some forms, a dosage effective in humans is from about 100 ⁇ g per dose to about 500 ⁇ g per dose, inclusive.
  • the effective dose of the described synthetic mRNA(s) is an amount equivalent to the amount of an approved mRNA therapeutic.
  • compositions including one or more mRNA antigens and one or more mRNA adjuvants can be in an amount effective to deliver antigen to a subject and induce the proliferation and clonal expansion of B cells, T cells or induce the migratory or chemotactic activity of macrophages. Therefore, in some forms, the compositions including one or more mRNA antigens and one or more mRNA adjuvants including encapsulated agent are in an amount effective to stimulate a primary immune response to an antigen in a subject.
  • the effective amount of compositions including one or more mRNA antigens and 455738066.1 71 one or more mRNA adjuvants does not induce significant cytotoxicity in the cells of a subject compared to an untreated control subject.
  • the amount of compositions including one or more mRNA antigens and one or more mRNA adjuvants is effective to prevent or reduce the infection or onset of a disease or disorder in a subject compared to an untreated control.
  • the compositions including one or more mRNA antigens and one or more mRNA adjuvants are in an amount effective to decrease the amount of expression of a target gene, or to prevent or decrease the serum concentration of a target gene product in a subject.
  • compositions including one or more mRNA antigens and one or more mRNA adjuvants are in an amount effective to induce presentation of an antigen by antigen-presenting cells.
  • compositions including one or more mRNA antigens and one or more mRNA adjuvants can be in an amount effective to induce T cell activation in response to an exogenous polypeptide encoded by a gene delivered to the cells of a subject by the compositions including one or more mRNA antigens and one or more mRNA adjuvants.
  • the one or more compositions including one or more mRNA antigens and one or more mRNA adjuvants are in an amount effective to decrease the amount of antigen required to stimulate a robust or protective immune response to the antigen in a subject.
  • the compositions including one or more mRNA antigens and one or more mRNA adjuvants can be effective to induce the production or antibodies to an antigen encoded by the compositions including one or more mRNA antigens and one or more mRNA adjuvants.
  • the compositions including one or more mRNA antigens and one or more mRNA adjuvants can be effective to enhance the amount of antigen-specific immune cells in a subject.
  • compositions including one or more mRNA antigens and one or more mRNA adjuvants can be effective to induce several signaling pathways controlling cellular immune activities, including cellular proliferation, chemotaxis and actin reorganization.
  • the effective amount of compositions including one or more mRNA antigens and one or more mRNA adjuvants does not cause cytotoxicity.
  • the effective amount of compositions including one or more mRNA antigens and one or more mRNA adjuvants to provide adaptive immunity to an encoded antigen or allergen should not generate a significant systemic increase in inflammatory cytokine production, including IFN.
  • compositions including one or more mRNA antigens and one or more mRNA adjuvants can be used to immunize a subject against a cancer, an infectious disease or an allergen using only a single dose. Therefore, in some forms, only a single administration is required with no boosting. In other forms, enhanced or prolonged immunity, such as protective 455738066.1 72 immunity, is achieved when one or more additional doses are used to boost the immune response to a first or previous administration. Typically, the absence of a cytokine response to the nanoparticle delivery vehicles in vivo precludes the development of anti-vector immunity.
  • compositions including one or more mRNA antigens and one or more mRNA adjuvants can be carried out repeatedly using the same or different doses of the compositions including one or more mRNA antigens and one or more mRNA adjuvants containing the same or different RNAs encoding antigens, for example, to provide immunity to a variety of different antigens or allergens in the same subject.
  • Combination Therapies Compositions including one or more mRNA antigens and one or more mRNA adjuvants can be administered alone, or in combination with one or more additional active agent(s), as part of a therapeutic or prophylactic treatment regime.
  • compositions including one or more mRNA antigens and one or more mRNA adjuvants can be administered on the same day, or a different day than the second active agent.
  • compositions including one or more mRNA antigens and one or more mRNA adjuvants can be administered on the first, second, third, or fourth day, or combinations thereof.
  • the term “combination” or “combined” is used to refer to either concomitant, simultaneous, or sequential administration of two or more agents. Therefore, the combinations can be administered either concomitantly (e.g., as an admixture), separately but simultaneously (e.g., via separate intravenous lines into the same subject), or sequentially (e.g., one of the compounds or agents is given first followed by the second).
  • the additional prophylactic or therapeutic agents can be vaccines for a specific antigen.
  • the antigen can be the same or different to that encoded by the compositions including one or more mRNA antigens and one or more mRNA adjuvants.
  • the compositions including one or more mRNA antigens and one or more mRNA adjuvants are useful as an agent to enhance the immune response to an antigen in a subject relative to the immune response raised to the same antigen in the absence of the nanoparticle delivery vehicles.
  • compositions including one or more mRNA antigens and one or more mRNA adjuvants are used to induce an immune response, for example, to one or more antigens or allergens encoded by RNA encapsulated within the compositions including one or more mRNA antigens and one or more mRNA adjuvants
  • the administration of an effective amount of the compositions including one or more mRNA antigens and one or more mRNA adjuvants does not require the co-administration of an adjuvant to elicit the desired immune response. Therefore, in 455738066.1 73 some forms, the compositions including one or more mRNA antigens and one or more mRNA adjuvants are administered in the absence of an adjuvant, or immuno-stimulatory molecule.
  • compositions including one or more mRNA antigens and one or more mRNA adjuvants can be compared to a control.
  • Suitable controls are known in the art and include, for example, untreated cells or an untreated subject.
  • the control is untreated tissue from the subject that is treated, or from an untreated subject.
  • the cells or tissue of the control are derived from the same tissue as the treated cells or tissue.
  • an untreated control subject suffers from, or is at risk from the same disease or condition as the treated subject.
  • an untreated control subject does not raise an immune response to an antigen.
  • compositions and methods can be compared to a control, such as a subject vaccinated with a different antigens/adjuvants or combination of antigens and adjuvants using the same or different delivery vehicles, same or different protocol, vaccination schedule or administration.
  • a suitable control is an unvaccinated subject.
  • a synthetic messenger ribonucleic acid (mRNA) molecule including (a) one or more ribonucleic acid sequences encoding one or more peptide antigen(s); and (b) one or more ribonucleic acid sequences encoding one or more peptide adjuvant(s) and/or other immuno-modulatory agent, wherein the ribonucleic acid is configured to express the peptide antigen(s) and the peptide adjuvant(s) and/or other immuno-modulatory agent(s) as distinct polypeptides within a mammalian cell.
  • the synthetic mRNA molecule of paragraph 1 wherein the one or more ribonucleic acid sequences encoding one or more peptide antigen(s) further include a ribonucleic acid sequence encoding a polypeptide targeting motif, wherein the polypeptide targeting motif enhances binding, uptake or processing of the antigen by an antigen presenting cell (APC).
  • APC antigen presenting cell
  • the synthetic mRNA of paragraph 6 wherein the one or more linker(s) include a P2A linker and a Furin cleavage site, and wherein the Furin cleavage site excludes the linker amino acids.
  • the synthetic mRNA of paragraph 7 wherein the Furin cleavage site and the P2A linker are joined by a linker including amino acid residues GSG.
  • the synthetic mRNA of any one of paragraphs 1-8 including a single 5’ Cap 1 structure.
  • the synthetic mRNA of any one of paragraphs 1-8 including a poly Adenylated 3’ tail structure.
  • the synthetic mRNA of any one of paragraphs 1-10 including a single 5’ untranslated region (5’ UTR). 12.
  • the synthetic mRNA of paragraph 1 or 2, including from 5’ to 3’ (i) a 5’Cap 1 structure; (ii) a 5’untranslated region (5’UTR); (iii) a sequence encoding a first polypeptide; (iv) a first Furin cleavage sequence; (v) a first P2A linker sequence; (vi) a sequence encoding a second polypeptide; (vii) a 3’untranslated region (3’UTR); and (viii) a poly Adenylated 3’tail; wherein one of the first or the second polypeptide encodes the peptide antigen and optionally a targeting motif; and wherein one of the first or the second polypeptide encodes a peptide adjuvant or other immuno-modulatory agent.
  • the synthetic mRNA of paragraph 1 or 2, including from 5’ to 3’ (i) a 5’ Cap 1 structure; (ii) a 5’untranslated region (5’ UTR); (iii) a sequence encoding a first polypeptide; (iv) a first Furin cleavage sequence; (v) a first P2A linker sequence; (vi) a sequence encoding a second polypeptide; (vii) a second Furin cleavage sequence; (viii) a second P2A linker sequence; (ix) a sequence encoding a third polypeptide; (x) a 3’untranslated region (3’ UTR); and (xi) a poly Adenylated 3’ tail; wherein the first or the second polypeptide encodes a first peptide antigen and optionally a targeting motif, wherein the first or the second polypeptide encodes a first peptide adjuvant and/or other immuno-modulatory agent, and wherein the third
  • MCPyV Merkel Cell Polyomavirus
  • Influenza virus Influenza virus
  • Ebola virus Zika Virus
  • SARS-Cov-2 SARS-Cov-2.
  • peptide antigen(s) is derived from a cancer selected from the group including alpha-actinin-4, Alphafetoprotein (AFP), Bcr-Abl fusion protein, Carcinoembryonic antigen (CEA), CA-125, Casp-8, beta-catenin, cdc27, cdk4, cdkn2a, coa-1, dek-can fusion protein, epithelial tumor antigen, EF2, ETV6-AML1 fusion protein, LDLR-fucosyltransferaseAS fusion protein, HLA-A2, HLA-A11, hsp70-2, KIAAO205, Mart2, Mum-1, 2, and 3, neo-PAP, myosin class I, OS-9, pml-RARa fusion protein, PTPRK, K- ras,
  • the method of paragraph 41 or 42, wherein the antigen is a tumor antigen. 44. The method of paragraph 43, wherein the tumor antigen is an autologous tumor antigen. 45. The method of paragraph 41 or 42, wherein the antigen is derived from a pathogenic bacterium. 46. The method of paragraph 41 or 42, wherein the antigen is derived from a pathogenic virus. 47. The method of paragraph 41 or 42, wherein the antigen is derived from a pathogenic fungus. 48. The method of paragraph 41 or 42, wherein the antigen is derived from a pathogenic protozoan. 49.
  • a method of treating a subject having a disease, disorder, or condition including administering to the subject an effective amount of the pharmaceutical composition of paragraph 40 to treat or reduce a symptom of the disease, disorder, or condition in the subject.
  • IL-2 was selected for its role as a pro-inflammatory cytokine that potentiates T cell differentiation to cytotoxic and memory populations when present with antigen stimulation.
  • the nucleic acid coding sequence used for IL-2 was as follows: ATGTACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTGCACTTGTCACAAACAGTGCAC CTACTTCAAGTTCTACAAAGAAAACACAGCTACAACTGGAGCATTTACTGCTGGATTTACAGAT GATTTTGAATGGAATTAATAATTACAAGAATCCCAAACTCACCAGGATGCTCACATTTAAGTTT TACATGCCCAAGAAGGCCACAGAACTGAAACATCTTCAGTGTCTGGAAGAAGAACTCAAACCTC TGGAGGAAGTGCTAAATTTAGCTCAAAGCAAAAACTTTCACTTAAGACCCAGGGACTTAATCAG CAATATCAACGTAATAGTTCTGGAACTAAAGGGATCTGAAACAACATTCATGTGTGAATATGCT GATGAGACAGCAACCATTGTAGAATTTCTGAACAGATGGATTACCTTTTGTCAAAGCATCATCT CAACACTGACTTGA (SEQ ID NO:1).
  • the nucleic acid coding sequence used for IL-7 was as follows: ATGTTCCATGTTTCTTTTAGGTATATCTTTGGACTTCCTCCCCTGATCCTTGTTCTGTTGCCAG TAGCATCATCTGATTGTGATATTGAAGGTAAAGATGGCAAACAATATGAGAGTGTTCTAATGGT CAGCATCGATCAATTATTGGACAGCATGAAAGAAATTGGTAGCAATTGCCTGAATAATGAATTT AACTTTTTTAAAAGACATATCTGTGATGCTAATAAGGAAGGTATGTTTTTATTCCGTGCTGCTC GCAAGTTGAGGCAATTTCTTAAAATGAATAGCACTGGTGATTTTGATCTCCACTTATTAAAAGT TTCAGAAGGCACAACAATACTGTTGAACTGCACTGGCCAGGTTAAAGGAAGAAAACCAGCTGCC CTGGGTGAAGCCCAACCAACAAAGAGTTTGGAAGAAAATAAATCTTTAAAGGAACAGAAAAAAC TGAATGACTTGTTTCCTAAAGACTATTACAAGATAAAAAAAA
  • IL-7 The amino acid sequence used for IL-7 was as follows: MFHVSFRYIFGLPPLILVLLPVASSDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEF NFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAA LGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH (SEQ ID NO:4).
  • IL-15 was selected for its role in activating and stimulating proliferation of T and NK cells. IL-15 also plays a key role in maintaining long term antigen-specific memory populations.
  • the nucleic acid coding sequence used for IL-15 was as follows: ATGAGAATTTCGAAACCACATTTGAGAAGTATTTCCATCCAGTGCTACTTGTGTTTACTTCTAA ACAGTCATTTTCTAACTGAAGCTGGCATTCATGTCTTCATTTTGGGCTGTTTCAGTGCAGGGCT TCCTAAAACAGAAGCCAACTGGGTGAATGTAATAAGTGATTTGAAAAAAATTGAAGATCTTATT CAATCTATGCATATTGATGCTACTTTATATACGGAAAGTGATGTTCACCCCAGTTGCAAAGTAA CAGCAATGAAGTGCTTTCTCTTGGAGTTACAAGTTATTTCACTTGAGTCCGGAGATGCAAGTAT TCATGATACAGTAGAAAATCTGATCATCCTAGCAAACAACAGTTTGTCTTCTAATGGGAATGTA ACAGAATCTGGATGCAAAGAATGTGAGGAACTGGAGGAAAAAAAAAATATTAAAGAATTTTTGCAGA GTTTTGTACATATTGTCCAAATGTTCATCAACACTTCTTGA
  • the amino acid sequence used for IL-15 was as follows: MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLI QSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNV TESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO:6).
  • XCL1 was selected for its role as a chemotactic factor for conventional dendritic cells and for stimulating antigen uptake and cross presentation.
  • the nucleic acid coding sequence used for XCL1 was as follows: ATGAGACTTCTCATCCTGGCCCTCCTTGGCATCTGCTCTCTCACTGCATACATTGTGGAAGGTG TAGGGAGTGAAGTCTCAGATAAGAGGACCTGTGTGAGCCTCACTACCCAGCGACTGCCGGTTAG CAGAATCAAGACCTACACCATCACGGAAGGCTCCTTGAGAGCAGTAATTTTTATTACCAAACGT GGCCTAAAAGTCTGTGCTGATCCACAAGCCACATGGGTGAGAGACGTGGTCAGGAGCATGGACA GGAAATCCAACACCAGAAATAACATGATCCAGACCAAGCCAACAGGAACCCAGCAATCGACCAA TACAGCTGTGACTCTGACTGGCTAG (SEQ ID NO:7).
  • the amino acid sequence used for XCL1 was as follows: MRLLILALLGICSLTAYIVEGVGSEVSDKRTCVSLTTQRLPVSRIKTYTITEGSLRAVIFITKR GLKVCADPQATWVRDVVRSMDRKSNTRNNMIQTKPTGTQQSTNTAVTLTG (SEQ ID NO:8).
  • IRF3 was selected for its role as a transcriptional regulator of cellular response to viral infection that activates innate immune pathways and leads to production of type 1 interferons.
  • the nucleic acid coding sequence used for IRF3 was as follows: ATGGGAACCCCAAAGCCACGGATCCTGCCCTGGCTGGTGTCGCAGCTGGACCTGGGGCAACTGG AGGGCGTGGCCTGGGTGAACAAGAGCCGCACGCGCTTCCGCATCCCTTGGAAGCACGGCCTACG GCAGGATGCACAGCAGGAGGATTTCGGAATCTTCCAGGCCTGGGCCGAGGCCACTGGTGCATAT GTTCCCGGGAGGGATAAGCCAGACCTGCCAACCTGGAAGAGGAATTTCCGCTCTGCCCTCAACC GCAAAGAAGGGTTGCGTTTAGCAGAGGACCGGAGCAAGGACCCTCACGACCCACATAAAATCTA CGAGTTTGTGAACTCAGGAGTTGGGGACTTTTCCCAGCCAGACACCTCTCCGGACACCAATGGt ggaggcagtacttctgatacccagGAAGACATTCTGGATGAGTTACTGGGTAACATGGTGTTGG CCACTGGCCCAGATCCGGGACCCCCAAGCC
  • IRF3-5D A constitutively active version of the IRF3 protein, known as IRF3-5D, with the following mutations: S396D/S398D/S402D/T404D/S405D has also been previously described, and was selected and shown below as a potential adjuvant (Lin, et al., Molecular and Cellular Biology, 19:4, 2465-2474 (1999)). This protein was also included in the panel.
  • the nucleic acid coding sequence used for IRF3-5D was as follows: ATGGGAACCCCAAAGCCACGGATCCTGCCCTGGCTGGTGTCGCAGCTGGACCTGGGGCAACTGG AGGGCGTGGCCTGGGTGAACAAGAGCCGCACGCGCTTCCGCATCCCTTGGAAGCACGGCCTACG GCAGGATGCACAGCAGGAGGATTTCGGAATCTTCCAGGCCTGGGCCGAGGCCACTGGTGCATAT GTTCCCGGGAGGGATAAGCCAGACCTGCCAACCTGGAAGAGGAATTTCCGCTCTGCCCTCAACC GCAAAGAAGGGTTGCGTTTAGCAGAGGACCGGAGCAAGGACCCTCACGACCCACATAAAATCTA CGAGTTTGTGAACTCAGGAGTTGGGGACTTTTCCCAGCCAGACACCTCTCCGGACACCAATGGt ggaggcagtacttctgatacccagGAAGACATTCTGGATGAGTTACTGGGTAACATGGTGTTGG CCACTGG CCACTCCCAGATCCGGGACC
  • MDA5 was selected for its role as a pattern recognition receptor that binds double stranded RNA and initiates activation of anti-viral innate immune programs through IRF3 and IRF7, leading to type 1 interferon production.
  • the nucleic acid coding sequence used for MDA5 was as follows: ATGTCGAATGGGTATTCCACAGACGAGAATTTCCGCTATCTCATCTCGTGCTTCAGGGCCAGGG TGAAAATGTACATCCAGGTGGAGCCTGTGCTGGACTACCTGACCTTTCTGCCTGCAGAGGTGAA GGAGCAGATTCAGAGGACAGTCGCCACCTCCGGGAACATGCAGGCAGTTGAACTGCTGCTGAGC ACCTTGGAGAAGGGAGTCTGGCACCTTGGTTGGACTCGGGAATTCGTGGAGGCCCTCCGGAGAA CCGGCAGCCCTCTGGCCGCCCGCTACATGAACCCTGAGCTCACGGACTTGCCCTCTCCATCGTT TGAGAACGCTCATGATGAATATCTCCAACTGCTGAACCTCCTTCAG
  • G495R mutated (G495R) form of MDA5 that is constitutively active and associated with Aicardi-Goutines syndrome has been previously described, and was also selected and shown below as a potential adjuvant (CA-MDA5) (Rice, et al., Nat Genet.46(5):503-509 (2014)).
  • the nucleic acid coding sequence used for CA-MDA5 was as follows: ATGTCGAATGGGTATTCCACAGACGAGAATTTCCGCTATCTCATCTCGTGCTTCAGGGCCAGGG TGAAAATGTACATCCAGGTGGAGCCTGTGCTGGACTACCTGACCTTTCTGCCTGCAGAGGTGAA GGAGCAGATTCAGAGGACAGTCGCCACCTCCGGGAACATGCAGGCAGTTGAACTGCTGCTGAGC ACCTTGGAGAAGGGAGTCTGGCACCTTGGTTGGACTCGGGAATTCGTGGAGGCCCTCCGGAGAA CCGGCAGCCCTCTGGCCGCCCGCTACATGAACCCTGAGCTCACGGACTTGCCCTCTCCATCGTT TGAGAACGCTCATGATGAATATCTCCAACTGCTGAACCTCCTTCAGCCCACTCTGGTGGACAAG CTTCTAGTTAGAGACGTCTTGGATAAGTGCATGGAGGAGGAACTGTTGACAATTGAAGACAGAA ACCGGATTGCTGCTGCAGAAAACAATGGAAATGAAT
  • This sequence was generated by artificial intelligence to be optimized for high efficiency translation and was among the highest performers in their 5’ UTR screen. Natural 3’ UTRs from each potential adjuvant were used. In cases where the natural 3’ UTR exceeded 100 bases, the length was shortened to only include the first 100 bases. A 100 base polyadenylation sequence was added following the 3’UTR.
  • the restriction site sequences are indicated in italic uppercase font; the T7 promoter and Trilink cap site “AG” is indicated within a box; the 5’UTR sequence is indicated in bold lowercase font, the traditional kozak sequience is indicated in bold italic uppercase font, the 3’UTR sequence is indicated in bold uppercase font and the polyA tail is indicated in lowercase font.
  • mRNA Production The template DNA was extracted from the base plasmids by cutting at restriction sites MluI and XbaI. Template DNA was separated from plasmid DNA based on fragment size by gel electrophoresis and was purified from agarose gel using a Zymo Research (Irvine, CA) Zymoclean Gel DNA recovery kit.
  • a test transfection was performed in HEK 293T cells.250,000 cells were plated in a 12 well tissue culture plate with 2 mL complete DMEM cell culture media.2 ug of mRNA was transfected using lipofectamine 2000 (Thermo, Waltham Ma.) per manufacturer protocol. No transfection and lipofectamine alone served as controls.18 hours following transfection, the supernatant was collected and tested for the cytokine output of interest using ELISA.
  • lipid nanoparticles containing SM-102, 1,2-DSPC, Cholesterol, and DMG-PEG in a lipid molar ration of 50:10:38.5:1.5 were assembled and incorporated with mRNA using a rapid solvent injection mixing technique.
  • mRNA was first combined with 50mM sodium acetate solution at pH 5.0. This solution was then stirred in a sterile container at 700rpm and the ethanolic mixture was rapidly injected into the acidic solution and mixed for an additional 30 minutes to create homogenous nanoparticles containing the functional mRNA units.
  • Placebo containing all components except for mRNA were prepared using the same process.
  • Compositions including one or more mRNA antigens and one or more mRNA adjuvants were then dialyzed against 500 volumes of sterile PBS.
  • the nanoparticle solution was then drawn into sterile 1mL syringes and stored at 4 degrees until use. Animal studies mRNA therapeutic vaccine response in a murine melanoma model was used to test the efficacy of adjuvant candidates.
  • LTA Large T antigen
  • MCPyV Merkel Cell Polyomavirus
  • mice from the above experiment that were treated with LTA alone or LTA + IL-7 were euthanized on day 30 and their and spleens were processed for analysis of immune populations by flow cytometry.
  • Spleens from the LTA + IL-7 treatment group were found to have an increase in total 455738066.1 90 T cells as well as an increased proportion of CD8+ T cells.
  • CD8+ T cells were enriched in memory markers IL7Ra and CD62L, indicating an expansion of both the overall memory compartment and the central memory compartment, suggesting the potential of the COMET approach to improve systemic immunity (FIGS.4A-4D).
  • the MCPyV LTA antigen transcript can be linked to the IL-2 transcript using a self-cleaving P2A linker with addition of a Furin cleavage site that excludes the linker amino acids and produces full length antigen and functional full-length cytokine as separate end products of a single mRNA (FIG.5).
  • two or more separate adjuvant transcripts can be linked using a self- cleaving P2A linker followed by a self-cleaving T2A linker with Furin cleavage sites that exclude linker amino acids.
  • the MCPyV LTA antigen can be linked to IL-7 and IL- 2 in the following sequence: LTA-P2A-IL7-T2A-IL2, which would result in full length antigen, functional full-length IL-7, and functional full length IL-2 as separate end products of a single mRNA (FIG.6).
  • LTA-P2A-IL7-T2A-IL2 which would result in full length antigen, functional full-length IL-7, and functional full length IL-2 as separate end products of a single mRNA (FIG.6).
  • These sequences can be cloned into a pUC57-Simple plasmid and used as a template for mRNA production by in-vitro transcription as described above. It is important to note that the cloning platform demonstrated here can theoretically be used to substitute any suitable antigen transcript combined with any suitable adjuvant transcript(s) in a fully customizable model depending on the indication and clinical intent (i.e.
  • the nucleic acid sequence of the DNA template encoding the LTA-P2A-IL2 mRNA is as follows: GAGCTCACGCGTTAATACGACTCACTATAAGacccaagctggctagcgtttaaacttaagcttg tgttacacaagggaagaaaagccgctgccgcactccgagtgtGCCACCATGTTCGTGTTCCTGG TGCTGCTGCCCCTGGTGAGCAGCCAGTGCGTGGATTTAGTCCTAAATAGGAAAGAAAGAGAGGC TCTCTGCAAGCTTTTAGAGATTGCTCCTAATTGTTATGGCAACATCCCTCTGATGAAAGCTGCT TTCAAAAGAAGCTGCTTAAAGCATCACCCTGATAAAGGGGGAAATCCTGTTATAATGATGGAAT TGAACACCCTTTGGAGCAAATTCCAGCAAAATATCCACAAGCTCAGAAGTGACTTCTCTATGTT TGAT
  • the nucleic acid sequence of the DNA template encoding the LTA-P2A-IL7-T2A-IL2 mRNA is as follows: GAGCTCACGCGTTAATACGACTCACTATAAGacccaagctggctagcgtttaaacttaagcttg 455738066.1 92 tgttacacaagggaagaaaagccgctgccgcactccgagtgtGCCACCATGTTCGTGTTCCTGG TGCTGCTGCCCCTGGTGAGCAGCCAGTGCGTGGATTTAGTCCTAAATAGGAAAGAAAGAGAGGC TCTCTCTGCAAGCTTTTAGAGATTGCTCCTAATTGTTATGGCAACATCCCTCTGATGAAAGCTGCT TTCAAAAGAAGCTGCTTAAAGCATCACCCTGATAAAGGGGGAAATCCTGTTATAATGATGGAAT TGAACACCCTTTGGAGCAAATTCCAGCAAAATATCCACAAGCTCAGAAGTGACTTC
  • the T7 promoter and Trilink cap site “AG” is indicated within a box; the 5’UTR sequence is indicated in bold lowercase font, the traditional Kozak sequence is indicated in bold italic uppercase font; Signal peptide is highlighted in dashed underlining and in uppercase font; Furin Cleavage site is highlighted in dashed underlining and in uppercase italics font; Linker sequence is highlighted in gray and in uppercase bold font; the 3’UTR sequence is indicated in bold uppercase font and the polyA tail is indicated in lowercase font.
  • LTA-P2A-IL7, LTA- P2A-IL2, and LTA-P2A-IL7-T2A-IL2 mRNA were transfected into HEK 293T cells.
  • 500k 293T cells were plated in 6 well plates with 2 mL complete DMEM media with 10% FBS, and each construct above was transfected at doses based on antigen weight, such that each well received the equivalent of 2 ⁇ g of antigen mRNA but different total doses depending on the portion of mRNA that encode adjuvanting cytokines.
  • a B16-OVA model will allow for identification of antigen specific T cells in the tumor, peripheral blood, and spleen of treated mice by flow cytometry using H-2K(b)- tetramers including the SIINFEKL (SEQ ID NO:20) peptide antigen.
  • OVA specific antigen presenting cells can also be identified by flow cytometry using an antibody directed against the Class 1 – SIINFEKL complex.
  • 455738066.1 94 OVA specific antibody levels can be tracked.
  • This approach could also be used in autoimmunity or autoinflammatory disease or allergy, in this case providing antigen in the context of explicitly tolerizing signals (e.g. in the case of MS providing myelin sheath protein along with regulatory T cell development signals such as TGFbeta or IL-10).
  • tolerizing mRNA vaccines is known in the art with a focus on the tolerogenic properties of nanoparticles and the mRNA itself, rather than programming the expansion of tolerogenic immune populations via adjuvant mRNA signals (Krienke, et al., Science 371, 145–153 (2021)).
  • Example 4 antigen+IL-7 vaccines increase antigen-specific memory CD8+ T cells compared with antigen-only vaccines
  • Linked constructs were designed as described in Example 3. Specifically, constructs including IL-7 linked to antigen were prepared. 15 ⁇ g of mRNA vaccine was used to treat B16-LTA tumor-bearing mice on d6 and day 13 after tumor inoculation (d13). On d15, mice were sacrificed and spleens were examined for antigen-specific and memory phenotype CD8+ T cells.
  • constructs including IL-7 linked to IL-2 and antigen were prepared.
  • B16-Ova tumor-bearing mice were treated on day 6 and day 13 after tumor inoculation with Ova, IL-7 only, IL-2 only, Ova+IL-7, Ova+IL-2 or Ova+IL-7+IL-2 mRNA encoded immunotherapies.
  • mice were sacrificed and spleens were dissected and evaluated via flow cytometry including evaluation via tetramers for the immunodominant epitope, SIINFEKL (SEQ ID NO:20).
  • SIINFEKL SEQ ID NO:20
  • compositions of synthetic messenger ribonucleic acid including one or more ribonucleic acid sequences encoding one or amino acid sequences that target linked antigens to antigen presenting cells (APCs) such as dendritic cells (DCs), or improve the uptake and presentation of those antigens by APCs were prepared and tested.
  • APCs antigen presenting cells
  • DCs dendritic cells
  • Clec9a is a protein expressed on APCs including dendritic cells.
  • An mRNA approach to co-encode a Clec9a-binding peptide linked to antigen was developed.
  • BFP Blue Fluorescent Protein
  • SP signal peptide
  • 293T cells were transfected with Blue Fluorescent Protein-containing (BFP), signal peptide- BFP-containing (SP-BFP), Clec9a-Binding-Peptide-BFP-containing (CBP-BFP) or SP-CBP- BFP mRNAs. Following transfection, 293T cells were mixed in a 1:1 ratio with MuTuDCs and incubated as shown prior to flow cytometry. 455738066.1 96
  • the CBP amino acid sequence that was used is:WPRFHSSVRHTH (SEQ ID NO:23).
  • each of the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D.
  • any subset or combination of these is also specifically contemplated and disclosed.
  • the sub-group of A-E, B-F, and C-E are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D.
  • use of “can” in this way is meant to positively state the option or capability while also leaving open that the option or capability could be absent in other forms or embodiments of the object or condition referred to.
  • use of the word “may” indicate an option or capability of the object or condition referred to.
  • use of “may” in this way is meant to positively state the option or capability while also leaving open that the option or capability could be absent in other forms or embodiments of the object or condition referred to.
  • use of “may” herein does not refer to an unknown or doubtful feature of an object or condition. Ranges can be expressed herein as from “about” one particular value, and/or to "about” another particular value.
  • ranges refer both to the recited range as a range and as a collection of individual numbers from and including the first endpoint to and including the second endpoint.
  • any of the individual numbers can be selected as one form of the quantity, value, or feature to which the range refers.
  • a range describes a set of numbers or values from and including the first endpoint to and including the second endpoint from which a single member of the set (i.e. a single number) can be selected as the quantity, value, or feature to which the range refers. The foregoing applies regardless of whether in particular cases some or all of these embodiments are explicitly disclosed.
  • any component, or subgroup of components can be either specifically included for or excluded from use or included in or excluded from a list of components.
  • Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the method and compositions described herein. Such equivalents are intended to be encompassed by the following claims. 455738066.1 100

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Abstract

Il a été établi que l'efficacité de vaccins à ARNm codant pour un ou plusieurs antigènes peptidiques pour l'expression dans une cellule hôte peut être améliorée par l'ajout d'une ou de plusieurs séquences d'ARNm codant pour un ou plusieurs adjuvants peptidiques pour l'expression de l'adjuvant dans ladite cellule hôte. L'invention concerne des compositions de vaccins comprenant des séquences d'ARNm codant pour un ou plusieurs antigènes peptidiques et un ou plusieurs adjuvants peptidiques. Le ou les antigènes peptidiques et le ou les adjuvants peptidiques peuvent être codés sur la même molécule d'ARNm ou une molécule d'ARNm différente. Dans certaines formes, les vaccins comprennent une seule molécule d'ARNm codant pour un antigène et un adjuvant, pour l'expression dans ladite cellule hôte. L'invention concerne également des nanoparticules encapsulant les séquences d'ARNm codant pour un ou plusieurs antigènes peptidiques et un ou plusieurs adjuvants peptidiques pour une administration à un sujet in vivo. L'invention concerne en outre des procédés de vaccination d'un sujet comprenant l'administration au sujet d'une composition comprenant des séquences d'ARNm codant pour un ou plusieurs antigènes peptidiques et un ou plusieurs adjuvants peptidiques.
PCT/US2025/033590 2024-06-13 2025-06-13 Compositions et procédés d'adjuvants à base d'arnm pour vaccins à arnm Pending WO2025260013A1 (fr)

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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140356384A1 (en) 2010-08-10 2014-12-04 Ecole Polytechnique Federale De Lausanne (Epfl) Erythrocyte-binding therapeutics
WO2017081082A2 (fr) * 2015-11-09 2017-05-18 Curevac Ag Molécules d'acide nucléique optimisées
WO2017134305A1 (fr) 2016-02-05 2017-08-10 Orionis Biosciences Nv Agents de signalisation bispécifiques et leurs utilisations
WO2021216647A1 (fr) * 2020-04-22 2021-10-28 Nutcracker Therapeutics, Inc. Nanoparticules de traitement d'arnm
US20220387573A1 (en) * 2019-06-27 2022-12-08 Etherna Immunotherapies Nv Combination therapy
CN116948967A (zh) * 2022-07-21 2023-10-27 四川康德赛医疗科技有限公司 一种基因修饰的树突状细胞及其应用
WO2023235749A2 (fr) * 2022-06-01 2023-12-07 Flag Bio, Inc. Adjuvants arn, procédés et utilisations de ceux-ci

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140356384A1 (en) 2010-08-10 2014-12-04 Ecole Polytechnique Federale De Lausanne (Epfl) Erythrocyte-binding therapeutics
WO2017081082A2 (fr) * 2015-11-09 2017-05-18 Curevac Ag Molécules d'acide nucléique optimisées
WO2017134305A1 (fr) 2016-02-05 2017-08-10 Orionis Biosciences Nv Agents de signalisation bispécifiques et leurs utilisations
US20220387573A1 (en) * 2019-06-27 2022-12-08 Etherna Immunotherapies Nv Combination therapy
WO2021216647A1 (fr) * 2020-04-22 2021-10-28 Nutcracker Therapeutics, Inc. Nanoparticules de traitement d'arnm
WO2023235749A2 (fr) * 2022-06-01 2023-12-07 Flag Bio, Inc. Adjuvants arn, procédés et utilisations de ceux-ci
CN116948967A (zh) * 2022-07-21 2023-10-27 四川康德赛医疗科技有限公司 一种基因修饰的树突状细胞及其应用

Non-Patent Citations (28)

* Cited by examiner, † Cited by third party
Title
BERRAONDO ET AL., BRITISH JOURNAL OF CANCER, vol. 10, 2019, pages 6 - 15
BRAASCH, DA ET AL., CHEM. BIOL., vol. 81-7, 2001
BRAUN ET AL., NAT MED, vol. 26, no. 6, June 2020 (2020-06-01), pages 909 - 918
BRAUN ET AL., NATURE MEDICINE, 2020
CAO ET AL.: "High-throughput 5' UTR engineering for enhanced protein production in non-viral gene therapies", NAT COMMUN, vol. 12, 2021, pages 4138
CELL, vol. 144, 2011, pages 646 - 674
CHERKASOVA ET AL., CANCER RESEARCH, 2017
CONLON ET AL., JOURNAL OF INTERFERON & CYTOKINE RESEARCH, vol. 39, no. 1, 2019, pages 1
DIAS JUNIOR ET AL., TRENDS IN MICROBIOLOGY, vol. 27, no. 1, 2019, pages 75 - 85
FISHMAN ET AL.: "Medicine", 1985, J.B. LIPPINCOTT CO.
GETTS ET AL., MABS, vol. 2, no. 6, 2010, pages 682 - 694
HESBACHER S ET AL., ONCOTARGET, vol. 7, no. 22, 31 May 2016 (2016-05-31), pages 32956 - 68
HOU ET AL., NATURE REVIEWS MATERIALS, vol. 6, 2021, pages 1078 - 1094
KACZANOWSKA ET AL., J LEUKOC BIOL, vol. 93, no. 6, June 2013 (2013-06-01), pages 847 - 863
KERVARREC T ET AL., FRONT ONCOL, vol. 9, 10 June 2019 (2019-06-10), pages 451
KRIENKE ET AL.: "A noninflammatory mRNA vaccine for treatment of experimental autoimmune encephalomyelitis", SCIENCE, vol. 371, 2021, pages 145 - 153, XP093150254, DOI: 10.1126/science.aay3638
KROCZEK ET AL., FRONT IMMUNOL, vol. 9, 2018, pages 2806
KURRECK J ET AL., NUCLEIC ACIDS RES, 2002, pages 301911 - 1918
NIELSEN ET AL., SCIENCE, vol. 254, 1991, pages 1497 - 1500
PHILADELPHIAMURPHY ET AL.: "Viking Penguin", 1997, PENGUIN BOOKS U.S.A., INC., article "Informed Decisions: The Complete Book of Cancer Diagnosis, Treatment, and Recovery"
RICE ET AL., NAT GENET, vol. 46, no. 5, 2014, pages 503 - 509
RICE ET AL.: "Gain-of-function mutations in IFIH1 cause a spectrum of human disease phenotypes associated with upregulated type I interferon signaling", NAT GENET, vol. 46, no. 5, May 2014 (2014-05-01), pages 503 - 509
RONGTUAN ET AL.: "Structural and Functional Analysis of Interferon Regulatory Factor 3: Localization of the Transactivation and Autoinhibitory Domains", MOLECULAR AND CELLULAR BIOLOGY, vol. 19, no. 4, 1999, pages 2465 - 2474, XP002110456
SHAIMARDANOVA ALISA A. ET AL: "Production and Application of Multicistronic Constructs for Various Human Disease Therapies", PHARMACEUTICS, vol. 11, no. 11, 6 November 2019 (2019-11-06), pages 580, XP055776188, DOI: 10.3390/pharmaceutics11110580 *
WAHLESTEDT C ET AL., PROC. NATL ACAD. SCI., 2000, pages 975633 - 5638
WHITE ET AL., J VIROL, vol. 75, 2001, pages 3706 - 3718
ZHANG ET AL., J VIROL, vol. 81, 2007, pages 11246 - 11255
ZHAO ET AL., GENOMICS PROTEOMICS BIOINFORMATICS, vol. 18, no. 2, April 2020 (2020-04-01), pages 129 - 139

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