Cancer Vaccines

Abstract

The problem that this invention aims to solve is to provide a ribonucleic acid (RNA) cancer vaccine that is RNA (e.g., messenger RNA (mRNA)) that can safely induce the body's cellular machinery to produce almost any cancer protein or fragment thereof of interest. The present invention relates to a vaccine comprising an mRNA having an open reading frame encoding a cancer antigen and formulated in a lipid nanoparticle, and an mRNA having an open reading frame encoding an immune checkpoint regulator.

Description

[Technical Field]

[0001] Related Applications This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 62 / 245,129, filed October 22, 2015, U.S. Provisional Application No. 62 / 245,031, filed October 22, 2015, U.S. Provisional Application No. 62 / 247,317, filed October 28, 2015, U.S. Provisional Application No. 62 / 247,472, filed October 28, 2015, and U.S. Provisional Application No. 62 / 368,810, filed July 29, 2016, the contents of each of which are incorporated herein by reference in their entirety. [Background technology]

[0002] Cancer vaccines include preventative or prophylactic vaccines (intended to prevent the onset of cancer in healthy individuals) and therapeutic vaccines (intended to treat existing cancer by strengthening the body's natural defenses against cancer). Preventive cancer vaccines may target infectious agents that induce or contribute to the onset of cancer, for example, to prevent cancer induction by infectious disease. Gardasil® and Cervarix® are two examples of commercially available preventive vaccines. Each vaccine protects against infection by HPV. Other preventive cancer vaccines may target host proteins or fragments that are predicted to increase an individual's likelihood of developing cancer in the future.

[0003] Most vaccines available on the market or under development are based on whole microorganisms, protein antigens, peptides, polysaccharides, or deoxyribonucleic acid (DNA) vaccines, as well as combinations thereof. DNA vaccination is a technique used to stimulate humoral and cellular immune responses to antigens. Direct injection of engineered DNA (e.g., naked plasmid DNA) into a living host results in direct production of antigens by a small number of host cells, generating a protective immune response. However, this technique faces potential problems with DNA integration into the vaccine genome, including the possibility of insertional mutagenesis, which can result in the activation of oncogenes or the inhibition of tumor suppressor genes. Summary of the Invention

[0004] Provided herein are ribonucleic acid (RNA) cancer vaccines that are RNA (e.g., messenger RNA (mRNA)) that can safely induce the body's cellular machinery to produce almost any cancer protein or fragment thereof of interest. In some embodiments, the RNA is modified RNA. The RNA vaccines of the present disclosure can be used to induce a balanced immune response against cancer, including both cellular and humoral immunity, without the risk of, for example, insertional mutagenesis.

[0005] RNA vaccines may be used in a variety of settings depending on the prevalence of cancer or the extent or level of unmet medical need. RNA vaccines may be used to treat and / or prevent cancer at various stages or degrees of metastasis. Compared to alternative anticancer treatments, including cancer vaccines, RNA vaccines have superior properties in that they generate much higher antibody titers and generate responses more quickly. Without being bound by theory, because RNA vaccines incorporate natural cellular machinery, RNA vaccines, which are mRNA polynucleotides, are thought to be well-designed to generate the appropriate protein conformation upon translation. Unlike conventional vaccines, which are produced ex vivo and may induce undesirable cellular responses, RNA vaccines are presented to cell lines in a more natural manner.

[0006] Some embodiments of the present disclosure provide cancer vaccines comprising at least one ribonucleic acid (RNA) polynucleotide having an open reading frame encoding at least one cancer antigen polypeptide or immunogenic fragment thereof (e.g., an immunogenic fragment capable of inducing an immune response against cancer). Other embodiments comprise at least one ribonucleic acid (RNA) polynucleotide having an open reading frame encoding two or more antigens or epitopes capable of inducing an immune response against cancer.

[0007] In some aspects, the present invention provides a vaccine comprising an mRNA having an open reading frame encoding a cancer antigen and an mRNA having an open reading frame encoding an immune checkpoint modulator. In some embodiments, the immune checkpoint modulator is an inhibitory checkpoint polypeptide. In some embodiments, the inhibitory checkpoint polypeptide is an antibody or fragment thereof that specifically binds to a molecule selected from the group consisting of PD-1, TIM-3, VISTA, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, and LAG3. In some embodiments, the inhibitory checkpoint polypeptide is an anti-CTLA4 antibody or an anti-PD1 antibody. The vaccine optionally comprises lipid nanoparticles. In some embodiments, a subject is administered a vaccine of an mRNA having an open reading frame encoding a cancer antigen. In other embodiments, a checkpoint inhibitor is administered 3 to 10 weeks later. In some embodiments, a checkpoint inhibitor is administered 4 weeks later.

[0008] In another aspect, the present invention provides a personalized cancer vaccine comprising a lipid nanoparticle carrier and mRNA having an open reading frame encoding at least two cancer antigens, wherein the at least two cancer antigens are patient-specific cancer antigens. In some embodiments, the lipid nanoparticles have an average diameter of 50 to 200 nm.

[0009] In yet another aspect, the present invention is a personalized cancer vaccine of mRNA having an open reading frame encoding at least two cancer antigens, wherein the at least two cancer antigens are representative of antigens of a patient. In some embodiments, the patient's antigens are antigens identified from the patient's exosomes. In some embodiments, a single mRNA encodes the cancer antigen. In other embodiments, multiple mRNAs encode the cancer antigens.

[0010] In other embodiments, each mRNA may encode 5-10 cancer antigens or a single cancer antigen. In some embodiments, the mRNA encodes 2-100 cancer antigens. In other embodiments, the mRNA encodes 10-100, 20-100, 50-100, 100-200, 300-400, 500-600, 600-700, 700-800, 900-1,000, or 1,000-10,000 cancer antigens.

[0011] In some embodiments, a) The mRNAs encoding each cancer antigen are interspersed with cleavage-sensitive sites, b) the mRNAs encoding each cancer antigen are directly linked to each other without a linker; c) the mRNAs encoding each cancer antigen are linked to each other by a single nucleotide linker; d) each cancer antigen comprises 25-35 amino acids and a centrally located SNP mutation; e) at least 30% of the cancer antigens have the highest affinity for class I MHC molecules derived from the subject; f) at least 30% of the cancer antigens have highest affinity for class II MHC molecules derived from the subject; g) at least 50% of the cancer antigens have a predicted binding affinity for HLA-A, HLA-B, and / or DRB1 of IC>500 nM; h) the mRNA encodes 20 cancer antigens; i) 50% of the cancer antigens have binding affinity for class I MHC and 50% of the cancer antigens have binding affinity for class II MHC, and / or j) The mRNAs encoding the cancer antigens are arranged so that the order of the cancer antigens is such that the order minimizes pseudo-epitopes.

[0012] In some embodiments, each cancer antigen comprises 31 amino acids and includes a centrally located SNP mutation with 15 flanking amino acids on either side of the SNP mutation.

[0013] In some embodiments, the vaccine is a personalized cancer vaccine and the cancer antigen is a subject-specific cancer antigen. In some embodiments, the subject-specific cancer antigen can be a representative of the exome of a tumor sample from the subject, or a representative of the transcriptome of a tumor sample from the subject. In some embodiments, the subject-specific cancer antigen can be a representative of the exosome of the subject.

[0014] In some embodiments, the open reading frame further encodes one or more conventional cancer antigens. In some embodiments, the conventional cancer antigen is a non-mutated antigen. In some embodiments, the conventional cancer antigen is a mutated antigen.

[0015] In some embodiments, the mRNA vaccine further comprises an mRNA having an open reading frame encoding one or more conventional cancer antigens.

[0016] In some embodiments, a single mRNA encodes the cancer antigen. In other embodiments, multiple mRNAs encode the cancer antigens. In some embodiments, each cancer antigen is 10-50 amino acids in length. In other embodiments, each cancer antigen is 15-20 amino acids in length. In other embodiments, the cancer antigen is 20-50, 25-100, 100-200, 200-300, 300-400, 400-500, 500-1,000, or 1,000-10,000 amino acids in length.

[0017] In some embodiments, the vaccine further comprises an adjuvant.

[0018] Some embodiments of the present disclosure provide a cancer vaccine comprising at least one ribonucleic acid (RNA) polynucleotide having an open reading frame encoding at least one cancer polypeptide, at least one 5'-end cap, and at least one chemical modification, the cancer vaccine being formulated in a lipid nanoparticle. In some embodiments, the 5'-end cap is 7mG(5')ppp(5')NlmpNp.

[0019] In some embodiments, the at least one chemical modification is selected from pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methoxyuridine, and 2'-O-methyluridine. In some embodiments, the degree of incorporation of chemically modified nucleotides is optimized to improve the immune response to the vaccine formulation.

[0020] In some embodiments, the lipid nanoparticles comprise a cationic lipid, a PEG-modified lipid, a sterol, and a non-cationic lipid. In some embodiments, the cationic lipid is an ionizable cationic lipid, the non-cationic lipid is a neutral lipid, and the sterol is cholesterol. In some embodiments, the cationic lipid is selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319).

[0021] In some embodiments, the lipid nanoparticle formulation comprises an immunopotentiator (eg, a TLR agonist) to enhance the immunogenicity of the vaccine (formulation).

[0022] In some embodiments, 100% of the uracils in the open reading frame have a chemical modification. In some embodiments, the chemical modification is at the 5-position of the uracil. In some embodiments, the chemical modification is N1-methylpseudouridine.

[0023] In another embodiment, the vaccine includes or is co-administered with an mRNA encoding an APC reprogramming molecule, which can be CIITA, a chaperone protein (e.g., CLIP, HLA-DO, HLA-DM), a costimulatory molecule (e.g., CD40, CD80, CD86), or a CIITA fragment (e.g., amino acids 26-137 of CIITA, or a protein with 80% sequence identity to CIITA).

[0024] In another aspect, a method is provided for eliciting an immune response in a subject by identifying at least two cancer antigens from a sample from the subject and administering to the subject an mRNA vaccine having open reading frames encoding the at least two cancer antigens, wherein the at least two cancer antigens comprise a mutation selected from the group consisting of a frameshift mutation and a recombination.

[0025] In some embodiments, cancer antigens are identified from a subject's exosomes. In some embodiments, 2 to 100 antigens are identified from exosomes. In other embodiments, the mRNA vaccine has an open reading frame encoding 2 to 100 antigens. A single mRNA or multiple mRNAs may encode the antigens.

[0026] In some embodiments, the antigen is a cancer antigen. The cancer antigen may have a mutation selected from a point mutation, a frameshift mutation, and a recombination. The method may further include confirming that the cancer antigen is subject-specific by exome analysis. In some embodiments, the method may further include confirming that the cancer antigen is subject-specific by transcriptome analysis.

[0027] In some embodiments, the method also includes identifying at least two cancer antigens from the subject's sample to obtain a second set of cancer antigens, performed at least one month after administration of the mRNA vaccine, and administering to the subject an mRNA vaccine having an open reading frame encoding the second set of cancer antigens to the subject. In other embodiments, the subject's sample is a tumor sample.

[0028] In another aspect, the present invention includes a method for eliciting an immune response in a subject by identifying at least two cancer antigens from a sample from the subject to obtain a first set of cancer antigens, administering to the subject an mRNA vaccine having open reading frames encoding the first set of cancer antigens, and, at least one month after administration of the mRNA vaccine, identifying at least two cancer antigens from a sample from the subject to obtain a second set of cancer antigens, and administering to the subject an mRNA vaccine having open reading frames encoding the second set of cancer antigens.

[0029] In some embodiments, the mRNA vaccine having an open reading frame encoding the second set of antigens is administered to a subject 6 months to 1 year after the mRNA vaccine having an open reading frame encoding the first set of cancer antigens, hi other embodiments, the mRNA vaccine having an open reading frame encoding the second set of antigens is administered to a subject 1 to 2 years after the mRNA vaccine having an open reading frame encoding the first set of cancer antigens.

[0030] In some embodiments, a single mRNA has an open reading frame encoding the cancer antigen. In other embodiments, multiple mRNAs encode the antigens. In some embodiments, the second set of cancer antigens comprises 2 to 100 antigens. In other embodiments, the cancer antigens have a mutation selected from a point mutation, a frameshift mutation, and a recombination.

[0031] In another aspect, the present invention includes a method for eliciting an immune response in a subject by identifying at least two cancer antigens from a sample from the subject, administering to the subject mRNA having open reading frames encoding the at least two cancer antigens, and administering to the subject a cancer therapeutic agent. In some embodiments, the cancer therapeutic agent is a targeted therapy. The targeted therapy can be a BRAF inhibitor such as vemurafenib (PLX4032) or dabrafenib.

[0032] In other embodiments, the cancer therapeutic agent is a T cell therapeutic agent. The T cell therapeutic agent can be a checkpoint inhibitor such as an anti-PD-1 antibody or an anti-CTLA-4 antibody. In some embodiments, the anti-PD-1 antibody is BMS-936558 (nivolumab). In other embodiments, the anti-CTLA-4 antibody is ipilimumab. In other embodiments, the T cell therapeutic agent is OX40L. In still other embodiments, the cancer therapeutic agent is a vaccine comprising a population-based tumor-specific antigen.

[0033] In other embodiments, the cancer therapeutic is a vaccine comprising an mRNA having an open reading frame encoding one or more conventional cancer antigens.

[0034] In some embodiments, the mRNA having an open reading frame encoding at least two cancer antigens is administered to the subject simultaneously with the cancer therapeutic agent. In some embodiments, the mRNA having an open reading frame encoding at least two cancer antigens is administered to the subject before the administration of the cancer therapeutic agent. In some embodiments, the mRNA having an open reading frame encoding at least two cancer antigens is administered to the subject after the administration of the cancer therapeutic agent.

[0035] Another aspect of the present invention provides a method for producing an mRNA cancer vaccine, comprising combining an mRNA having an open reading frame encoding a cancer antigen with a lipid nanoparticle formulation and administering the mRNA cancer vaccine to a subject within 24 hours of combining. In some embodiments, the mRNA cancer vaccine is administered to a subject within 12 hours of combining. In other embodiments, the mRNA cancer vaccine is administered to a subject within 1 hour of combining. In some embodiments, the mRNA cancer vaccine encodes 2 to 100 cancer antigens or 10 to 100 cancer antigens.

[0036] In some embodiments, the vaccine is a personalized cancer vaccine and the cancer antigen is a subject-specific cancer antigen.

[0037] In some embodiments, a single mRNA encodes the cancer antigen. In other embodiments, multiple mRNAs encode the cancer antigens. In other embodiments, each mRNA encodes 5-10 cancer antigens or a single cancer antigen. In yet other embodiments, each cancer antigen is 10-50 amino acids in length, or 15-20 amino acids in length.

[0038] In another aspect of the present invention, a kit is provided. The kit includes a container containing a lipid nanoparticle formulation, a container containing a vaccine formulation, and instructions for adding a personalized mRNA cancer vaccine to the vaccine formulation to generate a personalized mRNA cancer vaccine formulation and mixing the personalized mRNA cancer vaccine formulation with the lipid nanoparticle formulation within 24 hours prior to administration to a subject. In some embodiments, the kit includes mRNA having an open reading frame encoding 2 to 100 cancer antigens.

[0039] Further provided herein is the use of a cancer vaccine in the manufacture of a medicament for use in a method for inducing an antigen-specific immune response in a subject, the method comprising administering to the subject the cancer vaccine in an amount effective to generate an antigen-specific immune response.

[0040] In another aspect, there is provided a method for treating cancer in a subject in need thereof by identifying at least two cancer antigens from exosomes isolated from a subject, generating an mRNA vaccine having open reading frames encoding the antigens based on the identified antigens, and administering the mRNA vaccine to the subject, wherein the mRNA vaccine induces a tumor-specific immune response in the subject, thereby treating the cancer in the subject.

[0041] In another aspect, the present invention is an RNA vaccine that can be prepared according to a method that includes identifying at least two cancer antigens from exosomes isolated from a subject, and generating, based on the identified antigens, an mRNA vaccine having open reading frames encoding the antigens.

[0042] In one embodiment of the present invention, a method for inducing an immune response in a subject against a cancer antigen is provided. The method comprises administering to the subject an RNA vaccine comprising at least one RNA polynucleotide having an open reading frame encoding at least one antigen polypeptide or an immunogenic fragment thereof, thereby inducing in the subject an immune response specific to the antigen polypeptide or an immunogenic fragment thereof, wherein the titer of anti-antigen polypeptide antibodies in the subject is increased after vaccination compared to the titer of anti-antigen polypeptide antibodies in subjects vaccinated with a dose of a conventional vaccine that is prophylactically effective against cancer. An "anti-antigen polypeptide antibody" is a serum antibody that specifically binds to an antigen polypeptide.

[0043] A prophylactically effective dose is a therapeutically effective dose that inhibits the progression of cancer at a clinically acceptable level. In some embodiments, a therapeutically effective dose is the dose described in the package insert for the vaccine. As used herein, a conventional vaccine refers to a vaccine other than the mRNA vaccine of the present invention. For example, conventional vaccines include, but are not limited to, live microbial vaccines, killed microbial vaccines, subunit vaccines, protein antigen vaccines, DNA vaccines, etc. In exemplary embodiments, a conventional vaccine is one that has received regulatory approval and / or is registered with a national drug regulatory agency, such as, for example, the U.S. Food and Drug Administration (FDA) or the European Medicines Agency (EMA).

[0044] In some embodiments, the titer of anti-antigen polypeptide antibodies in a subject increases by 1 log to 10 log after vaccination compared to the titer of anti-antigen polypeptide antibodies in a subject vaccinated with a conventional vaccine at a dose that is prophylactically effective against cancer.

[0045] In some embodiments, the titer of anti-antigen polypeptide antibodies in a subject increases by 1 log after vaccination compared to the titer of anti-antigen polypeptide antibodies in a subject vaccinated with a prophylactically effective dose of a conventional vaccine against cancer.

[0046] In some embodiments, the titer of anti-antigen polypeptide antibodies in a subject increases by 2 logs after vaccination compared to the titer of anti-antigen polypeptide antibodies in a subject vaccinated with a prophylactically effective dose of a conventional vaccine against cancer.

[0047] In some embodiments, the titer of anti-antigen polypeptide antibodies in a subject increases by 3 logs after vaccination compared to the titer of anti-antigen polypeptide antibodies in a subject vaccinated with a prophylactically effective dose of a conventional vaccine against cancer.

[0048] In some embodiments, the titer of anti-antigen polypeptide antibodies in a subject increases by 5 logs after vaccination compared to the titer of anti-antigen polypeptide antibodies in a subject vaccinated with a prophylactically effective dose of a conventional vaccine against cancer.

[0049] In some embodiments, the titer of anti-antigen polypeptide antibodies in a subject increases by 10 logs after vaccination compared to the titer of anti-antigen polypeptide antibodies in a subject vaccinated with a prophylactically effective dose of a conventional vaccine against cancer.

[0050] In another aspect of the present invention, there is provided a method for eliciting an immune response in a subject against a cancer antigen, the method comprising administering to the subject an RNA vaccine comprising at least one RNA polynucleotide having an open reading frame encoding at least one antigenic polypeptide or immunogenic fragment thereof, thereby inducing in the subject an immune response specific to the antigenic polypeptide or immunogenic fragment thereof, wherein the immune response in the subject is equivalent to the immune response in a subject vaccinated with a conventional vaccine against the cancer antigen at a dose level 2- to 100-fold higher than that of the RNA vaccine.

[0051] In some embodiments, the immune response in a subject is comparable to the immune response in a subject vaccinated with a conventional vaccine at twice the dose level compared to the RNA vaccine.

[0052] In some embodiments, the immune response in a subject is comparable to the immune response in a subject vaccinated with a conventional vaccine at three times the dose level compared to the RNA vaccine.

[0053] In some embodiments, the immune response in a subject is comparable to the immune response in a subject vaccinated with a conventional vaccine at four times the dose level compared to the RNA vaccine.

[0054] In some embodiments, the immune response in a subject is comparable to the immune response in a subject vaccinated with a conventional vaccine at a 5-fold dose level compared to the RNA vaccine.

[0055] In some embodiments, the immune response in a subject is comparable to the immune response in a subject vaccinated with a conventional vaccine at a 10-fold higher dose level compared to the RNA vaccine.

[0056] In some embodiments, the immune response in a subject is comparable to the immune response in a subject vaccinated with a conventional vaccine at a 50-fold higher dose level compared to the RNA vaccine.

[0057] In some embodiments, the immune response in a subject is comparable to the immune response in a subject vaccinated with a conventional vaccine at a 100-fold dose level compared to the RNA vaccine.

[0058] In some embodiments, the immune response in a subject is comparable to the immune response in a subject vaccinated with a conventional vaccine at a dose level 10-1000 times higher than that of the RNA vaccine.

[0059] In some embodiments, the immune response in a subject is comparable to the immune response in a subject vaccinated with a conventional vaccine at a dose level 100-1000 times higher than that of the RNA vaccine.

[0060] In other embodiments, the immune response is assessed by determining antibody titers in the subject.

[0061] In another aspect, the invention includes a method of eliciting an immune response in a subject against a cancer antigen by administering to the subject an RNA vaccine comprising at least one RNA polynucleotide having an open reading frame encoding at least one cancer antigen polypeptide or immunogenic fragment thereof, thereby inducing in the subject an immune response specific to the antigen polypeptide or immunogenic fragment thereof, wherein the immune response in the subject is induced 2 days to 10 weeks earlier than the immune response induced in a subject vaccinated with a prophylactically effective dose of a conventional vaccine against the cancer antigen. In some embodiments, the immune response in the subject is comparable to that induced in a subject vaccinated with a prophylactically effective dose of a conventional vaccine at a dose level 2-fold to 100-fold higher than that of the RNA vaccine.

[0062] In some embodiments, an immune response is induced in a subject two days earlier than the immune response induced in a subject vaccinated with a prophylactically effective dose of a conventional vaccine.

[0063] In some embodiments, an immune response is induced in a subject three days earlier than the immune response induced in a subject vaccinated with a prophylactically effective dose of a conventional vaccine.

[0064] In some embodiments, an immune response is induced in a subject one week earlier than the immune response induced in a subject vaccinated with a prophylactically effective dose of a conventional vaccine.

[0065] In some embodiments, an immune response is induced in a subject two weeks earlier than the immune response induced in a subject vaccinated with a prophylactically effective dose of a conventional vaccine.

[0066] In some embodiments, an immune response is induced in a subject three weeks earlier than the immune response induced in a subject vaccinated with a prophylactically effective dose of a conventional vaccine.

[0067] In some embodiments, an immune response is induced in a subject five weeks earlier than the immune response induced in a subject vaccinated with a prophylactically effective dose of a conventional vaccine.

[0068] In some embodiments, an immune response is induced in a subject 10 weeks earlier than the immune response induced in a subject vaccinated with a prophylactically effective dose of a conventional vaccine.

[0069] A method for inducing an immune response in a subject against cancer by administering to the subject a cancer RNA vaccine having an open reading frame encoding a first antigen polypeptide, wherein the RNA polynucleotide does not contain a stabilizing element and an adjuvant is not co-formulated or co-administered with the vaccine.

[0070] In yet another aspect, the invention includes a method for producing mRNA encoding a concatemeric cancer antigen comprising 1000 to 3000 nucleotides, the method comprising: (a) binding a first polynucleotide comprising an open reading frame encoding a concatemeric cancer antigen and a second polynucleotide comprising a 5'-UTR to a polynucleotide complexed to a solid support; (b) ligating the 3' end of the second polynucleotide to the 5' end of the first polynucleotide under suitable conditions, where the suitable conditions include a DNA ligase, thereby generating a first ligation product; (c) ligating the 5' end of a third polynucleotide comprising a 3'-UTR to the 3' end of the first ligation product under suitable conditions, where the suitable conditions include an RNA ligase, thereby generating a second ligation product; (d) liberation of the second ligation product from the solid support; and This is due to This results in the production of mRNAs encoding concatemeric cancer antigens containing 1000 to 3000 nucleotides.

[0071] In some embodiments of any one of the provided compositions or methods, the mRNA encodes one or more repeat polymorphisms. In some embodiments, the one or more repeat polymorphisms comprise recurrent somatic cancer mutations in p53. In some such embodiments, the one or more recurrent somatic cancer mutations in p53 are: (1) A mutation in the canonical 5' splice site adjacent to codon position T125, which encodes the epitope AVSPCISFVW (SEQ ID NO: 2) (HLA-B * 57:01, HLA-B * 58:01), epitope HPLASCQCFF (SEQ ID NO: 3) (HLA-B * 35:01, HLA-B * 53:01), epitope FVWNFGIPL (SEQ ID NO: 4) (HLA-A * 02:01, HLA-A * 02:06, HLA-B * 35:01), a mutation that induces a retained intron with the peptide sequence TAKSVTCTVSCPEGLASMRLQCLAVSPCISFVWNFGIPLHPLASCQCFFIVYPLNV (SEQ ID NO: 1), (2) a mutation in the canonical 5' splice site adjacent to codon position 331, which encodes the epitope LQVLSLGTSY (SEQ ID NO: 6) (HLA-B * 15:01), epitope FQSNTQNAVF (SEQ ID NO: 7) (HLA-B * 15:01), a mutation that introduces a retained intron with the peptide sequence EYFTLQVLSLGTSYQVESFQSNTQNAVFFLTVLPAIGAFAIRGQ (SEQ ID NO: 5), (3) a mutation in the canonical 3' splice site adjacent to codon position 126, which encodes the epitope CTMFCQLAK (SEQ ID NO: 9) (HLA-A * 11:01), epitope KSVTCTMF (SEQ ID NO: 10) (HLA-B * a mutation that introduces a cryptic alternative exon 3' splice site generating a novel spanning peptide sequence AKSVTCTMFCQLAK (SEQ ID NO: 8) containing the nucleotide sequence 58:01; and / or (4) a mutation in the canonical 5' splice site adjacent to codon position 224, which encodes the epitope VPYEPPEVW (SEQ ID NO: 12) (HLA-B * 53:01, HLA-B * 51:01), epitope LTVPPSTAW (SEQ ID NO: 13) (HLA-B * 58:01, HLA-B * a mutation introducing a cryptic alternative intron 5' splice site generating a novel spanning peptide sequence VPYEPPEVWLALTVPPSTAWAA (SEQ ID NO: 11) containing the nucleotide sequence 57:01; is selected from the group consisting of Transcription codon positions are referenced to ENST00000269305 (SEQ ID NO: 14), the standard full-length p53 transcript derived from Ensembl's v83 human genome annotation.

[0072] In one embodiment, the present invention provides a cancer treatment vaccine comprising mRNA encoding an open reading frame (ORF) encoding one or more of the neoantigen peptides (1)-(4). In one embodiment, the present invention provides for the selective administration of a vaccine comprising or encoding one or more of the peptides (1)-(4) based on whether a patient's tumor contains any of the above mutations. In one embodiment, the present invention provides for the selective administration of a vaccine based on the dual criteria of whether a subject's tumor contains any of the above mutations and whether the subject's normal HLA type contains the corresponding HLA allele predicted to bind to the resulting neoantigen.

[0073] In another aspect of the present invention, a kit for preparing an mRNA cancer vaccine is provided, comprising one or more containers containing one or more polynucleotides comprising a 5'-ORF, one or more polynucleotides comprising a 3'-ORF, one or more polynucleotides comprising a poly(A) tail, a ligase enzyme, and instructions for ligating one or more polynucleotides comprising an ORF encoding a patient-specific epitope to one or more polynucleotides comprising a 5'-ORF, a 3'-ORF, and a poly(A) tail.

[0074] Another aspect of the invention provides a method for treating a subject with a personalized mRNA cancer vaccine by isolating a sample from a subject, analyzing the patient's transcriptome and / or exome from the sample to identify a set of neoepitopes, selecting a set of neoepitopes for a vaccine from the mutanome based on MHC binding strength, MHC binding diversity, predicted immunogenicity, low autoreactivity, and / or T cell reactivity, preparing an mRNA vaccine encoding the set of neoepitopes, and administering the mRNA vaccine to the subject within two months of isolating the sample from the subject. In some embodiments, the mRNA vaccine is administered to the subject within one month of isolating the sample from the subject.

[0075] In another aspect, the invention includes a method for identifying a set of neoepitopes for use in a personalized mRNA cancer vaccine having one or more polynucleotides encoding the set of neoepitopes, the method comprising: a. Identification of patient-specific mutanomes through analysis of the patient transcriptome and the patient exome; b. Gene or transcript level expression assessment in patient RNA sequencing, variant call confidence score, allele-specific expression based on RNA sequencing, conservative vs. non-conservative amino acid substitutions, point mutation location (Centering Score for increased TCR engagement), point mutation location (Anchoring Score for differential HLA binding), selfness: core epitope homology (<100%) with patient WES data, IC50 for 8-mer to 11-mer against HLA-A and HLA-B, IC50 for 15-mer to 20-mer against HLA-DRB1, broad binding score and selecting a subset of 15 to 500 neoepitopes from the mutanome using weightings for the neoepitopes based on at least three of: the HLA-C Score (i.e., the number of patient HLAs predicted to bind), the IC50 for HLA-C for 8-11 mers, the IC50 for HLA-DRB3-5 for 15-20 mers, the IC50 for HLA-DQB1 / A1 for 15-20 mers, and the IC50 for HLA-DPB1 / A1 for 15-20 mers; a comparison of the class I to class II ratio; the diversity of HLA-A, HLA-B, and HLA-DRB1 allotypes covered in the patient; a comparison of the ratio of point mutations to compound epitopes (e.g., frameshifts); and / or the pseudoepitope HLA binding score. c. selecting a set of neoepitopes from the subset based on the highest weighted value for use in a personalized mRNA cancer vaccine, wherein the set of neoepitopes comprises 15-40 neoepitopes; This is due to the following.

[0076] In some embodiments, the nucleic acid vaccines described herein are chemically modified. In other embodiments, the nucleic acid vaccines are unmodified.

[0077] In yet another aspect, compositions and methods for vaccinating a subject are provided, comprising administering to the subject a nucleic acid vaccine comprising one or more RNA polynucleotides having an open reading frame encoding a first antigenic polypeptide or a concatemeric polypeptide, wherein the RNA polynucleotides do not comprise a stabilizing element, and no adjuvant is co-formulated or co-administered with the vaccine.

[0078] In another aspect, the present invention provides a composition or method for vaccinating a subject, comprising administering to the subject a nucleic acid vaccine comprising one or more RNA polynucleotides having an open reading frame encoding a first antigenic polypeptide, wherein the dose of the nucleic acid vaccine administered to the subject is between 10 μg / kg and 400 μg / kg. In some embodiments, the dose of the RNA polynucleotide is between 1 μg / kg and 5 μg / kg, 10 μg / kg and 15 μg / μg, 10 μg / kg and 20 μg / kg, 20 μg / kg and 50 μg / μg, 30 μg / kg and 50 μg, 40 μg / μg, 40 μg / μg, 60 μg / μg, 60 μg / μg, 50 μg / μg, 80 μg / μg, 40 μg / μg, 40 μg / μg, 50 μg / μg, 50 μg / μg, 50 μg / μg, 50 μg / μg, 50 μg / μg, 50 μg / μg, 50 μg / μg, 50 μg / μg, 50 μg / μg, 8 ... , 100-200 μg, 120-250 μg, 150-250 μg, 180-280 μg, 200-300 μg, 50-300 μg, 80-300 μg, 100-300 μg, 40-300 μg, 50-350 μg, 100-350 μg, 200-350 μg, 300-350 μg, 320-400 μg, 40-380 μg, 40-100 μg, 100-400 μg, 200-400 μg, or 300-400 μg. In some embodiments, the nucleic acid vaccine is administered to the subject by intradermal or intramuscular injection. In some embodiments, the nucleic acid vaccine is administered to the subject on day 0. In some embodiments, a second dose of the nucleic acid vaccine is administered to the subject on day 21.

[0079] In some embodiments, the dose of the RNA polynucleotide contained in the nucleic acid vaccine administered to a subject is 25 micrograms. In some embodiments, the dose of the RNA polynucleotide contained in the nucleic acid vaccine administered to a subject is 100 micrograms. In some embodiments, the dose of the RNA polynucleotide contained in the nucleic acid vaccine administered to a subject is 50 micrograms. In some embodiments, the dose of the RNA polynucleotide contained in the nucleic acid vaccine administered to a subject is 75 micrograms. In some embodiments, the dose of the RNA polynucleotide contained in the nucleic acid vaccine administered to a subject is 150 micrograms. In some embodiments, the dose of the RNA polynucleotide contained in the nucleic acid vaccine administered to a subject is 400 micrograms. In some embodiments, the dose of the RNA polynucleotide contained in the nucleic acid vaccine administered to a subject is 200 micrograms. In some embodiments, the RNA polynucleotide accumulates at 100-fold higher levels in local lymph nodes compared to distal lymph nodes. In other embodiments, the nucleic acid vaccine is chemically modified; in other embodiments, the nucleic acid vaccine is not chemically modified.

[0080] In some embodiments, the present invention provides a nucleic acid vaccine comprising one or more RNA polynucleotides having an open reading frame encoding a first antigen polypeptide or a concatemeric polypeptide, and a pharmaceutically acceptable carrier or excipient, wherein the RNA polynucleotides do not comprise a stabilizing element, and the vaccine does not comprise an adjuvant. In some embodiments, the stabilizing element is a histone stem loop. In some embodiments, the stabilizing element is a nucleic acid sequence with increased GC content compared to a wild-type sequence.

[0081] In some embodiments, the antibody titers generated by the mRNA vaccines of the present invention are neutralizing antibody titers. In some embodiments, the neutralizing antibody titers are higher than those generated by protein vaccines. In other embodiments, the neutralizing antibody titers generated by the mRNA vaccines of the present invention are higher than those generated by protein vaccines containing an adjuvant. In still other embodiments, the neutralizing antibody titer generated by the mRNA vaccine of the present invention is 1,000 to 10,000, 1,200 to 10,000, 1,400 to 10,000, 1,500 to 10,000, 1,000 to 5,000, 1,000 to 4,000, 1,800 to 10,000, 2,000 to 10,000, 2,000 to 5,000, 2,000 to 3,000, 2,000 to 4,000, 3,000 to 5,000, 3,000 to 4,000, or 2,000 to 2,500. The neutralizing titer is typically expressed as the highest serum dilution required to achieve a 50% reduction in plaque counts.

[0082] In preferred aspects, the vaccines of the present invention (e.g., mRNA vaccines encapsulated in LNPs) generate prophylactically and / or therapeutically effective levels, concentrations, and / or titers of antigen-specific antibodies in the blood or serum of vaccinated subjects. The term antibody titer, as defined herein, refers to the amount of antigen-specific antibodies generated in a subject, e.g., a human subject. In exemplary embodiments, antibody titers are expressed as the reciprocal of the highest dilution (in a serial dilution series) that still gives a positive result. In exemplary embodiments, antibody titers are determined or measured by enzyme-linked immunosorbent assay (ELISA). In exemplary embodiments, antibody titers are determined or measured by a neutralization assay, e.g., a microneutralization assay. In certain aspects, antibody titer measurements are expressed as ratios, such as 1:40, 1:100, etc.

[0083] In exemplary embodiments of the invention, an effective vaccine generates antibody titers of greater than 1:40, greater than 1:100, greater than 1:400, greater than 1:1000, greater than 1:2000, greater than 1:3000, greater than 1:4000, greater than 1:500, greater than 1:6000, greater than 1:7500, or greater than 1:10000. In exemplary embodiments, antibody titers are generated or achieved within 10 days after vaccination, within 20 days after vaccination, within 30 days after vaccination, within 40 days after vaccination, or within 50 days or more after vaccination. In exemplary embodiments, titers are generated or achieved after a single dose of vaccine is administered to a subject. In other embodiments, titers are generated or achieved after multiple doses, for example, a first dose and a second dose (e.g., a booster dose).

[0084] In exemplary aspects of the invention, antigen-specific antibodies are measured in μg / ml, or in IU / L (International Units per liter) or mIU / ml (milli-International Units per ml). In exemplary embodiments of the invention, effective vaccines are produced at >0.5 μg / ml, >0.1 μg / ml, >0.2 μg / ml, >0.35 μg / ml, >0.5 μg / ml, >1 μg / ml, >2 μg / ml, >5 μg / ml, or >10 μg / ml. In exemplary embodiments of the invention, effective vaccines are produced at >10 mIU / ml, >20 mIU / ml, >50 mIU / ml, >100 mIU / ml, >200 mIU / ml, >500 mIU / ml, or >1000 mIU / ml. In exemplary embodiments, the antibody level or concentration is generated or achieved within 10 days after vaccination, within 20 days after vaccination, within 30 days after vaccination, within 40 days after vaccination, or within 50 days or more after vaccination. In exemplary embodiments, the level or concentration is generated or achieved after a single dose of vaccine is administered to the subject. In other embodiments, the level or concentration is generated or achieved after multiple doses, for example, after a first dose and a second dose (e.g., a booster dose). In exemplary embodiments, the antibody level or concentration is determined or measured by enzyme-linked immunosorbent assay (ELISA). In exemplary embodiments, the antibody level or concentration is determined or measured by a neutralization assay, for example, a microneutralization assay. Also provided are nucleic acid vaccines comprising one or more RNA polynucleotides having an open reading frame encoding a first antigenic polypeptide or a concatemeric polypeptide, with a stabilizing element or formulated with an adjuvant, wherein the RNA polynucleotides are present in a formulation intended for in vivo administration to a host to induce higher and longer-lasting antibody titers compared to those elicited by mRNA vaccines encoding the first antigenic polypeptide. In some embodiments, the RNA polynucleotides are formulated to generate neutralizing antibodies within one week of a single administration. In some embodiments, the adjuvant is selected from cationic peptides and immunostimulatory nucleic acids.In some embodiments, the cationic peptide is protamine.

[0085] In some embodiments, nucleic acid vaccines are provided that include one or more RNA polynucleotides having an open reading frame that includes at least one chemical modification or optionally no nucleotide modifications, where the open reading frame encodes a first antigenic polypeptide or a concatemeric polypeptide, and the RNA polynucleotides are present in a formulation intended for in vivo administration to a host, such that the level of antigen expression in the host significantly exceeds the level of antigen expression produced by an mRNA vaccine that has a stabilizing element or is formulated with an adjuvant and encodes the first antigenic polypeptide.

[0086] In other aspects, nucleic acid vaccines are provided that include one or more RNA polynucleotides having an open reading frame that includes at least one chemical modification or optionally no nucleotide modifications, the open reading frame encoding a first antigenic polypeptide or a concatemeric polypeptide, wherein the vaccine requires at least 10-fold less RNA polynucleotide than would be required for generating comparable antibody titers from an unmodified mRNA vaccine. In some embodiments, the RNA polynucleotide is present in a dose of 25-100 micrograms.

[0087] Aspects of the invention also provide vaccine units of use, the units comprising 10 μg to 400 μg of one or more RNA polynucleotides having an open reading frame containing at least one chemical modification or optionally no nucleotide modifications, the open reading frame encoding a first antigenic polypeptide or a concatemeric polypeptide, and a pharmaceutically acceptable carrier or excipient formulated for delivery to a human subject. In some embodiments, the vaccine further comprises cationic lipid nanoparticles.

[0088] In some embodiments, the vaccine is administered to an individual via a route selected from the group consisting of intramuscular administration, intradermal administration, and subcutaneous administration. In some embodiments, the administering step comprises contacting the subject's muscle tissue with a device suitable for injecting the composition. In some embodiments, the administering step comprises contacting the subject's muscle tissue with a device suitable for injecting the composition in combination with electroporation.

[0089] In an embodiment of the present invention, a method of vaccinating a subject is provided, the method comprising administering to the subject a single dose of 25 μg / kg to 400 μg / kg of a nucleic acid vaccine comprising one or more RNA polynucleotides having an open reading frame encoding a first antigenic polypeptide or a concatemeric polypeptide in an amount effective for vaccinating the subject.

[0090] In another aspect, a nucleic acid vaccine is provided, comprising one or more RNA polynucleotides having an open reading frame comprising at least one chemical modification, the open reading frame encoding a first antigenic polypeptide or a concatemeric polypeptide, wherein the vaccine requires at least 10-fold less RNA polynucleotide than an unmodified mRNA vaccine would require to generate comparable antibody titers. In some embodiments, the RNA polynucleotide is present in a dose of 25-100 micrograms.

[0091] In another aspect, a nucleic acid vaccine is provided that comprises an LNP-formulated RNA polynucleotide having an open reading frame that does not contain any nucleotide modifications (unmodified), the open reading frame encoding a first antigen polypeptide or a concatemeric polypeptide, wherein the vaccine requires at least 10-fold less RNA polynucleotide than would be required for generating equivalent antibody titers with an unmodified mRNA vaccine that is not formulated in LNP. In some embodiments, the RNA polynucleotide is present in a dose of 25-100 micrograms.

[0092] The data shown in the examples show that the use of the formulation of the present invention significantly enhances immune response.Surprisingly, in contrast to the prior art reports that chemically unmodified mRNA is preferably formulated and used in a carrier for vaccine production, it is disclosed herein that chemically modified mRNA-LNP vaccines require much lower effective mRNA doses than unmodified mRNA, that is, compared to when unmodified mRNA is formulated in a carrier other than LNP, the effective mRNA dose required is 10 times lower.Both the chemically modified RNA vaccines and unmodified RNA vaccines of the present invention generate better immune response than mRNA vaccines formulated in different lipid carriers.

[0093] In other aspects, the invention encompasses methods of treating an elderly subject 60 years of age or older, the method comprising administering to the subject a nucleic acid vaccine comprising one or more RNA polynucleotides having an open reading frame encoding an antigenic polypeptide or a concatemeric polypeptide in an amount effective to vaccinate the subject.

[0094] In other aspects, the invention encompasses methods of treating a young subject 17 years of age or younger, the method comprising administering to the subject a nucleic acid vaccine comprising one or more RNA polynucleotides having an open reading frame encoding an antigenic polypeptide or a concatemeric polypeptide in an amount effective to vaccinate the subject.

[0095] In another aspect, the invention encompasses a method of treating an adult subject, the method comprising administering to the subject a nucleic acid vaccine comprising one or more RNA polynucleotides having an open reading frame encoding an antigenic polypeptide or a concatemeric polypeptide in an amount effective to vaccinate the subject.

[0096] In some aspects, the present invention includes methods of vaccinating a subject with a combination vaccine comprising at least two nucleic acid sequences encoding antigens, wherein the vaccine dose is a combined therapeutic dose and the dose of each individual nucleic acid encoding the antigen is a sub-therapeutic dose. In some embodiments, the combined dose of RNA polynucleotides in the nucleic acid vaccine administered to a subject is 25 micrograms. In some embodiments, the combined dose of RNA polynucleotides in the nucleic acid vaccine administered to a subject is 100 micrograms. In some embodiments, the combined dose of RNA polynucleotides in the nucleic acid vaccine administered to a subject is 50 micrograms. In some embodiments, the combined dose of RNA polynucleotides in the nucleic acid vaccine administered to a subject is 75 micrograms. In some embodiments, the combined dose of RNA polynucleotides in the nucleic acid vaccine administered to a subject is 150 micrograms. In some embodiments, the combined dose of RNA polynucleotides in the nucleic acid vaccine administered to a subject is 400 micrograms. In some embodiments, the fractional therapeutic dose of each individual nucleic acid encoding an antigen is 1 microgram, 2 micrograms, 3 micrograms, 4 micrograms, 5 micrograms, 6 micrograms, 7 micrograms, 8 micrograms, 9 micrograms, 10 micrograms, 11 micrograms, 12 micrograms, 13 micrograms, 14 micrograms, 15 micrograms, 16 micrograms, 17 micrograms, 18 micrograms, 19 micrograms, or 20 micrograms. In other embodiments, the nucleic acid vaccine is chemically modified; in other embodiments, the nucleic acid vaccine is not chemically modified.

[0097] Details of various embodiments of the invention are set forth in the description that follows. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

[0098] The foregoing and other objects, features, and advantages will become apparent from the following description of specific embodiments of the invention, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of various embodiments of the invention. [Brief explanation of the drawings]

[0099] [Figure 1] 1A-1D show the results of an assay to demonstrate CD8 responses specific to mRNA vaccine antigens. [Figure 2] 1 shows the results of an assay demonstrating that the mRNA vaccine induced antigen-specific effector / memory CD8 T cell responses. [Figure 3] FIG. 1 is a schematic diagram showing multi-factorial considerations for antigen design of mRNA-based neoepitopes. [Figure 4] 1 is a table showing multi-factorial considerations for antigen design of mRNA-based neoepitopes. [Figure 5] The epitopes encoded by the mRNAs were validated by a FACS-based assay in MCF7 (HLA*201) cells. Specific presentation of MHC1 / mutant-MART1 peptides in MCF7 cells was detected by anti-mutant-MART1 TCR markers. The sequences correspond, from top to bottom, to SEQ ID NOs: 15 to 19. [Figure 6]Figures 6A and 6B are schematic diagrams illustrating peptide epitopes. The polypeptide in Figure 6A contains two or more epitopes. The epitopes can be the same or different sequences, all T cell epitopes, all B cell epitopes, or a combination of both. The diagram in Figure 6B shows peptide epitopes with various terminal units to enhance MHC processing of the peptide. [Figure 7] 1 shows an exemplary T cell response elicited with mRNA encoding a concatemer of 20 epitopes. The mRNA concatemer induced both class I and class II T cell responses. [Figure 8A] Figure 1 shows exemplary T cell responses elicited by mRNAs encoding concatemers with epitopes at different positions. CA80 and CA81 encode 20 identical epitopes known to elicit T cell responses, including five class II epitopes, ten mouse class I epitopes, one mouse positive control (SIINFEKL (SEQ ID NO: 22) (derived from ovalbumin)), and four human (HLA-A2) epitopes (not shown). CA80 and CA81 differ only in the relative positions of these distinct epitopes. [Figure 8B] 1 shows an exemplary correlation between interferon-gamma spot-forming units (SFU) and CD8+IFN-γ+ responses. [Figure 9A] 1 shows an exemplary dose response elicited with mRNA encoding concatemeric CA-80. Fewer hits were observed as the vaccine dose was reduced. [Figure 9B] 1 shows an exemplary dose response elicited with mRNA encoding concatemeric CA-80. Fewer hits were observed as the vaccine dose was reduced. [Figure 10] An exemplary comparison of T cell responses to known epitopes when immunized with a single 20-mer and multiple 5-mers is shown. T cell responses to known epitopes were comparable when vaccinated as a single 20-mer or three 3-mers. A trend toward slightly higher T cell responses was observed when immunized with multiple 5-mers. [Figure 11] An exemplary comparison of T cell responses to class I epitopes alone or in the presence of class II support is shown. Five known class I epitopes were administered as 5-mers alone (no class II support) or together with five known class II epitopes (class II support), and the T cell responses to the five known class I epitopes were compared. The latter group also contained an additional 5-mer of the known class I epitope. The T cell responses to the known class I epitope were stronger in the presence of a 5-mer containing the known class II epitope. [Figure 12] Figure 1 shows exemplary T cell responses observed upon vaccination with a concatemer vaccine formulated with MC3 or Compound 25. CA-81 (containing 15 known murine epitopes) was formulated in MC3 and Compound 25. T cell responses to each epitope in the vaccine were measured and responses were compared between the two formulations. [Figure 13] FIG. 1 is a schematic diagram illustrating the mRNA components of mRNA-1. [Figure 14] 1 is a diagram illustrating the general molecular sequence of mRNA-1, where the patient-specific coding region is indicated by reference as (N). The sequence corresponds to SEQ ID NO: 20 and SEQ ID NO: 21. [Figure 15] FIG. 1 is a block diagram illustrating a computer system on which some embodiments may be implemented. [Figure 16] FIG. 1 is a schematic diagram showing the frequency of splice site mutations by tumor type (upper panel) and p53 mutations by position (lower panel), excluding silent mutations. [Figure 17] Schematic diagram showing hotspot splice sites and silent mutations that lead to the generation of retained introns and cryptic splicing sites. RNA sequencing confirmed that several mutation sites generated retained introns or cryptic splicing sites. Two representative peptides derived from mutations contained multiple HLA-A2-binding epitopes that did not match any other sites in the coding genome. DETAILED DESCRIPTION OF THE INVENTION

[0100] Detailed Description In embodiments of the present disclosure, RNA (e.g., mRNA) vaccines are provided that include polynucleotides encoding cancer antigens. The cancer RNA vaccines provided herein can be used to induce a balanced immune response, including cellular and / or humoral immunity, without many of the risks associated with DNA vaccination. In some embodiments, the vaccine includes at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding a cancer antigen.

[0101] Although many attempts have been made to generate functional RNA vaccines, including mRNA cancer vaccines, the therapeutic efficacy of these RNA vaccines has not yet been fully established.Surprisingly, the inventors have discovered a class of formulations for delivering mRNA vaccines that induce significantly enhanced and in many respects synergistic immune responses, including enhanced T cell responses.The vaccines of the present invention include conventional cancer vaccines and personalized cancer vaccines.In some aspects, the present invention includes the surprising finding that lipid nanoparticle formulations significantly enhance the efficacy of mRNA vaccines, including chemically modified and unmodified mRNA vaccines.

[0102] The lipid nanoparticles used in the test described herein have been used in various animal models and human for the delivery of siRNA.Considering the findings obtained in relation to the delivery of siRNA by lipid nanoparticle formulation, the fact that lipid nanoparticles are useful in vaccines is very surprising.It has been observed that the therapeutic delivery of siRNA formulated in lipid nanoparticles typically reduces antigen production and impairs immune response, resulting in undesirable inflammatory responses associated with transient IgM responses.In contrast to the findings observed with siRNA, lipid nanoparticle-mRNA vaccine formulations have been shown to not produce transient IgM responses, but rather promote IgG levels, which are sufficient for prophylactic and therapeutic methods.

[0103] In vaccine development, generating cancer antigens that induce the desired immune response (e.g., T cell response) against the target polypeptide sequence remains a challenging task. The present invention includes technology that overcomes the hurdles associated with vaccine development. Using the technology of the present invention, it is possible to tailor and direct the desired immune response by selecting an appropriate T cell or B cell cancer epitope and formulating the epitope or antigen for effective delivery in vivo.

[0104] Thus, the present invention relates to mRNA vaccines, which are described in International Patent Application No. PCT / US2015 / 027400, filed April 23, 2015, which is incorporated herein by reference in its entirety.

[0105] mRNA cancer vaccines offer a unique therapeutic option as an alternative to peptide-based or DNA vaccines. Once delivered to cells, the mRNA is processed by the intracellular machinery to produce polypeptides that can be processed into immunoreactive fragments capable of stimulating an immune response against tumors.

[0106] The cancer vaccines described herein comprise at least one ribonucleic acid (RNA) polynucleotide having an open reading frame encoding at least one cancer antigen polypeptide or an immunogenic fragment thereof (e.g., an immunogenic fragment capable of inducing an immune response against cancer). The cancer vaccine can be a conventional cancer vaccine or a personalized cancer vaccine. A conventional cancer vaccine is a vaccine that contains a cancer antigen known to be generally found in cancers or tumors, or a cancer antigen known to be found in a specific type of cancer or tumor. An antigen expressed in or by tumor cells is referred to as a "tumor-associated antigen." A specific tumor-associated antigen may or may not be expressed in non-cancerous cells. Many tumor mutations are known in the art.

[0107] A personalized vaccine can contain, for example, RNA encoding one or more known tumor or cancer antigens specific to each subject; such antigens are referred to as neoepitopes, subject-specific epitopes, or subject-specific antigens. A "subject-specific cancer antigen" is an antigen identified to be expressed in a specific patient's tumor. Subject-specific cancer antigens typically may or may not be present in tumor samples. A tumor-associated antigen that is not expressed, or is rarely expressed, in non-cancerous cells, or whose expression in non-cancerous cells is significantly reduced compared to that in cancerous cells and that induces an immune response upon vaccination is referred to as a neoepitope. Because neoepitopes, such as tumor-associated antigens, are completely foreign to the body, they are expected to not generate an immune response against healthy tissue or be masked by the immune system's defense components. In some embodiments, personalized vaccines based on neoepitopes are desirable because such vaccine formulations maximize specificity for patient-specific tumors. Mutational neoepitopes can arise from point mutations, which are nonsynonymous mutations that result in different amino acids in the protein; readthrough mutations, which result in the modification or deletion of a stop codon and lead to the translation of an elongated protein with a novel tumor-specific sequence at the C-terminus; splice site mutations, which result in the inclusion of an intron in the mature mRNA, resulting in a unique tumor-specific protein sequence; chromosomal rearrangements (i.e., gene fusions), which result in a chimeric protein with a tumor-specific sequence at the junction of two proteins; frameshift mutations or deletions, which result in a new open reading frame with a novel tumor-specific protein sequence; and translocations. Thus, in some embodiments, the mRNA cancer vaccine comprises at least two cancer antigens that contain mutations selected from the group consisting of frameshift mutations and recombinations or any of the other mutations described herein.

[0108] Methods for generating personalized cancer vaccines generally include identifying mutations, e.g., using nucleic acid or protein deep sequencing techniques, identifying neoepitopes, e.g., applying validated peptide-MHC binding prediction algorithms or other analytical techniques to generate a set of candidate T cell epitopes based on mutations present in the tumor that can bind to the patient's HLA alleles, optionally demonstrating that antigen-specific T cells target the selected neoepitopes or that the candidate neoepitopes bind to HLA proteins present on the tumor surface, and developing the vaccine. The mRNA cancer vaccines of the present invention can include multiple copies of a single neoepitope, multiple different neoepitopes based on a single type of mutation, i.e., point mutation, multiple different neoepitopes based on various mutation types, neoepitopes such as tumor-associated antigens or recall antigens, and other antigens.

[0109] Examples of mutation identification techniques include, but are not limited to, dynamic allele-specific hybridization (DASH), microplate array diagonal gel electrophoresis (MADGE), pyrosequencing, oligonucleotide-specific ligation, the TaqMan system, and various DNA "chip" technologies, i.e., Affymetrix SNP chips, as well as methods based on generating small signal molecules by invasive cleavage followed by mass spectrometry, or immobilized padlock probes and rolling circle amplification.

[0110] Deep sequencing techniques for nucleic acids or proteins are known in the art. Any type of sequence analysis method can be used. Nucleic acid sequencing can be performed on the entire tumor genome, tumor exome (protein-coding DNA), tumor transcriptome, or exosome. Real-time single-molecule sequencing by synthesis technology utilizes the detection of fluorescent nucleotides as they are incorporated into nascent DNA strands complementary to the template being sequenced. Other rapid high-throughput sequencing methods exist. Protein sequencing can be performed on tumor proteomes. Furthermore, protein mass spectrometry can be used to identify or verify the presence of mutant peptides bound to MHC proteins present in tumor cells. Peptides can be identified using mass spectrometry after acid elution from tumor cells or from HLA molecules immunoprecipitated from tumors. Sequencing results can be compared to a known control set or to sequencing analysis performed on the patient's normal tissue.

[0111] Thus, the present invention relates to methods for identifying and / or detecting neoepitopes of antigens, such as T cell epitopes. Specifically, the present invention provides methods for identifying and / or detecting tumor-specific neoepitopes that are useful in inducing tumor-specific immune responses in subjects. Optionally, such neoepitopes bind with stronger affinity to class I HLA proteins compared to wild-type peptides and / or have the ability to activate anti-tumor CD8+ T cells. Identical mutations in any particular gene are rarely found across tumors.

[0112] MHC class I proteins are present on the surface of almost all cells in the body, including most tumor cells. Antigens, usually originating from endogenous proteins or intracellular pathogens, are loaded onto MHC class I proteins, which then present these antigens to cytotoxic T lymphocytes (CTLs). T cell receptors have the ability to recognize and bind to peptides complexed with MHC class I molecules. Each cytotoxic T lymphocyte expresses a unique T cell receptor capable of specifically binding to MHC / peptide complexes.

[0113] Computer algorithms can be used to predict potential neoepitopes, such as T cell epitopes, i.e., peptide sequences that are recognized by the T cell receptor of T lymphocytes in the form of a peptide-presenting complex after binding of class I or class II MHC molecules. Examples of programs useful for identifying peptides that will bind to MHC include, for example, Lonza Epibase, SYFPEITHI (Rammensee et al., Immunogenetics, 50 (1999), 213-219), and HLA_BIND (Parker et al., J. Immunol., 152 (1994), 163-175).

[0114] Once a putative neoepitope is selected, it can be further tested using in vitro and / or in vitro assays. Isolates from each patient may be used to refine the list of neoepitopes selected based on algorithmic predictions using routine in vitro assays performed in laboratories, such as Elispot assays.

[0115] The mRNA cancer vaccines of the present invention are compositions, including pharmaceutical compositions. The present invention also encompasses methods for selecting, designing, preparing, manufacturing, formulating, and / or using mRNA cancer vaccines. Systems, processes, devices, and kits for selecting, designing, and / or utilizing the mRNA cancer vaccines described herein are also provided.

[0116] The mRNA vaccines of the present invention can include one or more cancer antigens. In some embodiments, the mRNA vaccines are composed of three or more, four or more, five or more, six or more, seven or more, eight or more, or nine or more antigens. In other embodiments, the mRNA vaccines are composed of 1,000 or fewer, 900 or fewer, 500 or fewer, 100 or fewer, 75 or fewer, 50 or fewer, 40 or fewer, 30 or fewer, 20 or fewer, or 100 or fewer cancer antigens. In still other embodiments, the mRNA vaccines are composed of 3 to 100, 5 to 100, 10 to 100, 15 to 100, 20 to 100, 25 to 100, 30 to 100, 35 to 100, 40 to 100, 45 to 100, 50 to 100, 55 to 100, 60 to 100, 65 to 100, 70 to 100, 75 to 100, 80 to 100, 90 or fewer cancer antigens. having ~100, 5-50, 10-50, 15-50, 20-50, 25-50, 30-50, 35-50, 40-50, 45-50, 100-150, 100-200, 100-300, 100-400, 100-500, 50-500, 50-800, 50-1,000, or 100-1,000 cancer antigens.

[0117] In some embodiments, mRNA cancer vaccines and vaccination methods include epitopes or antigens (neoepitopes) based on specific mutations and those expressed in cancer germline genes (antigens common to tumors found in multiple patients).

[0118] As used herein, an epitope, also known as an antigenic determinant, is a portion of an antigen that is recognized by the immune system, specifically by antibodies, B cells, or T cells, in the appropriate context. Epitopes include B cell epitopes and T cell epitopes. A B cell epitope is a peptide sequence required for recognition by specific antibody-producing B cells. A B cell epitope refers to the specific region of an antigen recognized by an antibody. The portion of an antibody that binds to an epitope is called a paratope. Epitopes can be conformational or linear epitopes based on their structure and interaction with the paratope. Linear or continuous epitopes are defined by the primary amino acid sequence of a specific region of a protein. The sequences that interact with antibodies are located contiguously adjacent to each other in the protein, and epitopes can usually be mimicked by a single peptide. A conformational epitope is an epitope defined by the native protein's three-dimensional structure. Such epitopes may be contiguous or discontinuous, that is, components of the epitope may be located in different parts of the protein, which are in close proximity to each other in the folded, native protein structure.

[0119] A T cell epitope is a peptide sequence that associates with a protein present on an APC and is required for recognition by a specific T cell. T cell epitopes are processed intracellularly and then presented on the surface of an APC, where they bind to MHC molecules, including MHC class II and MHC class I. Peptide epitopes can be of any suitable length for an epitope. In some embodiments, the peptide epitope is 9 to 30 amino acids long. In other embodiments, the length is 9 to 22, 9 to 29, 9 to 28, 9 to 27, 9 to 26, 9 to 25, 9 to 24, 9 to 23, 9 to 21, 9 to 20, 9 to 19, 9 to 18, 10 to 22, 10 to 21, 10 to 20, 11 to 22, 22 to 21, 11 to 20, 12 to 22, 12 to 21, 12 to 20, 13 to 22, 13 to 21, 13 to 20, 14 to 19, 15 to 18, or 16 to 17 amino acids.

[0120] In some embodiments, the peptide epitopes comprise at least one MHC class I epitope and at least one MHC class II epitope. In some embodiments, at least 10% of the epitopes are MHC class I epitopes. In some embodiments, at least 20% of the epitopes are MHC class I epitopes. In some embodiments, at least 30% of the epitopes are MHC class I epitopes. In some embodiments, at least 40% of the epitopes are MHC class I epitopes. In some embodiments, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% of the epitopes are MHC class I epitopes. In some embodiments, at least 10% of the epitopes are MHC class II epitopes. In some embodiments, at least 20% of the epitopes are MHC class II epitopes. In some embodiments, at least 30% of the epitopes are MHC class II epitopes. In some embodiments, at least 40% of the epitopes are MHC class II epitopes. In some embodiments, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% of the epitopes are MHC class II epitopes. In some embodiments, the ratio of MHC class I epitopes to MHC class II epitopes is selected from about 10%:about 90%, about 20%:about 80%, about 30%:about 70%, about 40%:about 60%, about 50%:about 50%, about 60%:about 40%, about 70%:about 30%, about 80%:about 20%, or about 90%:about 10% MHC class I epitopes:MHC class II epitopes. In some embodiments, the ratio of MHC class II epitopes to MHC class I epitopes is a ratio selected from about 10%:about 90%, about 20%:about 80%, about 30%:about 70%, about 40%:about 60%, about 50%:about 50%, about 60%:about 40%, about 70%:about 30%, about 80%:about 20%, about 90%:about 10% MHC class II epitopes:MHC class I epitopes.In some embodiments, at least one of the peptide epitopes of the cancer vaccine is a B cell epitope. In some embodiments, the T cell epitope of the cancer vaccine comprises 8-11 amino acids. In some embodiments, the B cell epitope of the cancer vaccine comprises 13-17 amino acids.

[0121] In some embodiments, the cancer vaccines of the present invention comprise mRNA vaccines encoding multiple peptide epitope antigens, either spaced apart by a single nucleotide spacer between the epitopes or directly spaced apart without any spacers between the epitopes. The multiple epitope antigens include a mixture of MHC class I epitopes and MHC class II epitopes. For example, the multiple peptide epitope antigens may be: (XGX) 1-10 (GYGY) 1-10 (GXGX) 0-10 (GYGY) 0-10 , (XG) 1-10 (GY) 1-10 (GX) 0-10 (GY) 0-10 , (XGXGX) 1-10 (GYGY) 1-10 (XGX) 0-10 (GYGY) 0-10 , (XGX) 1-10 (GYGYGY) 1-10 (XGX) 0-10 (GYGY) 0-10 , (XGXGXGX) 1-10 (GYGY) 1-10 (XGX) 0-10 (GYGY) 0-10 , (XGX) 1-10 (GYGYGYGY) 1-10 (XGX) 0-10 (GYGY) 0-10 , (X) 1-10 (Y) 1-10 (X) 0-10 (Y) 0-10 , (Y) 1-10 (X) 1-10 (Y)0-10 (X) 0-10 、(XX) 1-10 (Y) 1-10 (X) 0-10 (Y) 0-10 、(YY) 1-10 (XX) 1-10 (Y) 0-10 (X) 0-10 、(X) 1-10 (YY) 1-10 (X) 0-10 (Y) 0-10 、(XXX) 1-10 (YYY) 1-10 (XX) 0-10 (YY) 0-10 、(YYY) 1-10 (XXX) 1-10 (YY) 0-10 (XX) 0-10 、(XY) 1-10 (Y) 1-10 (X)1 -10 (Y)1 -10 、(YX) 1-10 (Y) 1-10 (X)1 -10 (Y)1 -10 、(YX) 1-10 (X) 1-10 (Y)1 -10 (Y)1 -10 、(Y-G-Y) 1-10 (G-X-G-X) 1-10 (G-Y-G-Y) 0-10 (G-X-G-X) 0-10 、(Y-G) 1-10 (G-X) 1-10 (G-Y) 0-10 (G-X) 0-10 、(Y-G-Y-G-Y) 1-10 (G-X-G-X) 1-10 (Y-G-Y) 0-10 (G-X-G-X) 0-10 、(Y-G-Y) 1-10 (G-X-G-X-G-X) 1-10 (Y-G-Y) 0-10 (G-X-G-X) 0-10 、(Y-G-Y-G-Y-G-Y) 1-10 (G-X-G-X) 1-10 (Y-G-Y) 0-10 (G-X-G-X) 0-10, (YGY) 1-10 (GXGXGXGX) 1-10 (YGY) 0-10 (GXGX) 0-10 , (XY) 1-10 (YX) 1-10 (XY) 0-10 (YX) 0-10 , (YX) 1-10 (XY) 1-10 (Y) 0-10 (X) 0-10 , (YY) 1-10 (X) 1-10 (Y) 0-10 (X) 0-10 , (XY) 1-10 (XY) 1-10 (X) 0-10 (X) 0-10 , (Y) 1-10 (YX) 1-10 (X) 0-10 (Y) 0-10 , (XYX) 1-10 (YXX) 1-10 (YX) 0-10 (YY) 0-10 , or (YYX) 1-10 (XXY) 1-10 (YX) 0-10 (XY) 0-10 and a polypeptide having the structure: X is an MHC class I epitope between 10 and 40 amino acids in length, Y is an MHC class II epitope between 10 and 40 amino acids in length, and G is glycine.

[0122] In some embodiments, RNA vaccines can be combined with agents to promote the production of antigen-presenting cells (APCs), for example, by converting non-APCs into pseudo-APCs. Antigen presentation is a critical step in the initiation, amplification, and duration of immune responses. In this process, antigen fragments are presented to T cells via major histocompatibility complexes (MHC) or human leukocyte antigens (HLA), thereby promoting antigen-specific immune responses. For immunoprophylaxis and immunotherapy, enhancing this response is important for improving efficacy. The RNA vaccines of the present invention may be designed or enhanced to promote efficient antigen presentation. One method for enhancing APC processing and presentation is to improve targeting of the RNA vaccine to antigen-presenting cells (APCs). Another approach involves activating APC cells with immunostimulatory agents and / or components.

[0123] Alternatively, methods for reprogramming non-APCs to become APCs may be used with the RNA vaccines of the present invention. Importantly, most of the cells that take up mRNA formulations and are the targets of their therapeutic effects are not APCs. Therefore, designing a method to convert these cells into APCs would be beneficial for efficacy. Provided herein are methods and approaches for delivering RNA vaccines, such as mRNA vaccines, to cells while also promoting the shift from non-APCs to APCs. In some embodiments, mRNA encoding APC reprogramming molecules is included in the RNA vaccine or co-administered with the RNA vaccine.

[0124] As used herein, an APC reprogramming molecule is a molecule that promotes the transition of a non-APC cell to an APC-like phenotype. An APC-like phenotype is a property that enables MHC class II processing. Thus, an APC cell with an APC-like phenotype is a cell that possesses one or more exogenous molecules (APC reprogramming molecules) and has enhanced MHC class II processing capability compared to the same cell without the one or more exogenous molecules. In some embodiments, the APC reprogramming molecule is a chaperone protein (enhancer of antigen fragment loading onto MHC class II), such as CIITA (a central regulator of MHC class II expression), CLIP, HLA-DO, or HLA-DM, and / or a costimulatory molecule (enhancer of T cell antigen recognition and T cell activation), such as CD40, CD80, or CD86.

[0125] The CIITA protein is a transactivator that enhances transcriptional activation of MHC class II genes by interacting with a conserved set of DNA-binding proteins associated with class II promoter regions (Steimle et al., 1993, Cell 75:135-146). The transcriptional activation function of CIITA has been localized to its amino-terminal acidic domain (amino acids 26-137). The protein that interacts with CIITA is called CIITA-interacting protein 104 (also referred to herein as CIP104) and is encoded by a nucleic acid molecule. Both CIITA and CIP104 have been shown to enhance transcription from MHC class II promoters and are therefore useful as APC reprogramming molecules of the present invention. In some embodiments, the APC reprogramming molecule is full-length CIITA, CIP104, or other related molecules, or an active fragment thereof, such as amino acids 26-137 of CIITA, or amino acids that have at least 80% sequence identity thereto and that maintain the ability to enhance transcriptional activation of MHC class II genes.

[0126] In a preferred embodiment, the APC reprogramming molecule is delivered to a subject in the form of an mRNA encoding the APC reprogramming molecule. Thus, the RNA vaccine of the present invention may include an mRNA encoding an APC reprogramming molecule. In some embodiments, the mRNA is monocistronic. In other embodiments, the mRNA is polycistronic. In some embodiments, the mRNA encoding one or more antigens is present in a formulation separate from the mRNA encoding the APC reprogramming molecule. In other embodiments, the mRNA encoding one or more antigens is present in the same formulation as the mRNA encoding the APC reprogramming molecule. In some embodiments, the mRNA encoding one or more antigens is administered to a subject at the same time as the mRNA encoding the APC reprogramming molecule. In other embodiments, the mRNA encoding one or more antigens is administered to a subject at a different time than the mRNA encoding the APC reprogramming molecule. For example, the mRNA encoding the APC reprogramming molecule may be administered before the mRNA encoding one or more antigens. The mRNA encoding the APC reprogramming molecule may be administered immediately before, at least one hour before, at least one day before, at least one week before, or at least one month before the mRNA encoding the antigen.

[0127] Alternatively, the mRNA encoding the APC reprogramming molecule may be administered after the mRNA encoding one or more antigens. The mRNA encoding the APC reprogramming molecule may be administered immediately after, at least one hour, at least one day, at least one week, or at least one month after the mRNA encoding the antigen. In some embodiments, the antigen is a cancer antigen, such as a patient-specific antigen. In other embodiments, the antigen is an antigen of an infectious disease.

[0128] In some embodiments, the mRNA vaccine may include a recall antigen, sometimes referred to as a memory antigen. A recall antigen is an antigen that an individual has previously encountered and for which memory lymphocytes already exist. In some embodiments, the recall antigen may be an infectious disease antigen that the individual may have encountered, such as an influenza antigen. Recall antigens help promote a more robust immune response.

[0129] The antigen or neoepitope selected for inclusion in the mRNA vaccine will typically be a binding peptide with high affinity. In some aspects, the antigen or neoepitope binds to HLA proteins with higher affinity than the wild-type peptide. In some embodiments, the antigen or neoepitope has an IC50 of at least 5000 nM, at least 500 nM, at least 250 nM, at least 200 nM, at least 150 nM, at least 100 nM, at least 50 nM, or less. Typically, peptides with a predicted IC50 of less than 50 nM are generally considered to be binding peptides with medium to high affinity, and will be selected for experimental testing of their affinity using biochemical assays of HLA binding.

[0130] In personalized cancer vaccine, target-specific cancer antigen can be identified in patient's sample.For example, sample can be tissue sample or tumor sample.For example, can check the presence of target-specific cancer antigen in one or more tumor cell samples.Can check tumor sample by using whole genome, exome or transcriptome analysis to identify target-specific cancer antigen.

[0131] Alternatively, subject-specific cancer antigens may be identified in the exosomes of a subject. Once antigens for a vaccine are identified in the exosomes of a subject, such antigens are said to be representative of the exosomal antigens of the subject.

[0132] Exosomes are small microvesicles secreted by cells, typically measuring approximately 30-100 nm in diameter. Exosomes classically form from invagination, resulting in the pinching and shedding of the terminal endosomal membrane, resulting in the formation of multivesicular bodies (MVBs) rich in small lipid bilayer vesicles, each of which contains a sample of the parent cell's cytoplasm. Upon fusion of the MVB with the plasma membrane, these exosomes are released from the cell and delivered to the blood, urine, cerebrospinal fluid, or other bodily fluids. Exosomes can be recovered from any of these biological fluids for further analysis.

[0133] Nucleic acids in exosomes serve as biomarkers for tumor antigens. An advantage of exosome analysis for identifying target-specific cancer antigens is that this method eliminates the need for biopsies. This can be particularly beneficial when patients need to undergo several rounds of treatment, including cancer antigen identification and vaccination.

[0134] Many methods for isolating exosomes from biological samples have been described in the art. For example, the following methods can be used: differential centrifugation, low-speed centrifugation, anion exchange and / or gel permeation chromatography, sucrose density gradient or organelle electrophoresis, magnetic activated cell sorting (MACS), nanomembrane ultrafiltration centrifugation, density gradient isolation using Percoll, and the use of microfluidic devices. Exemplary methods are described, for example, in U.S. Patent Publication No. 2014 / 0212871.

[0135] The term "biological sample" refers to a sample containing biological materials such as DNA, RNA, and proteins. In some embodiments, a biological sample may suitably comprise a bodily fluid derived from a subject. A bodily fluid may be a liquid isolated from any location, preferably a peripheral location, of a subject's body, including, but not limited to, blood, plasma, serum, urine, sputum, spinal fluid, cerebrospinal fluid, pleural fluid, nipple aspirate, lymphatic fluid, fluids derived from the respiratory tract, intestinal tract, and genitourinary tract, tears, saliva, breast milk, fluids derived from the lymphatic system, semen, cerebrospinal fluid, organ system fluids, ascites, tumor cyst fluid, amniotic fluid, and combinations thereof.

[0136] In some embodiments, cancer progression can be monitored to identify changes in expressed antigens. Thus, in some embodiments, the method further comprises: identifying at least two cancer antigens from the subject's sample to obtain a second set of cancer antigens; and administering to the subject an mRNA vaccine having an open reading frame encoding the second set of cancer antigens at least one month after the administration of the cancer mRNA vaccine. In some embodiments, the mRNA vaccine having an open reading frame encoding the second set of antigens is administered to the subject 2 months, 3 months, 4 months, 5 months, 6 months, 8 months, 10 months, or 1 year after the mRNA vaccine having an open reading frame encoding the first set of cancer antigens. In other embodiments, the mRNA vaccine having an open reading frame encoding the second set of antigens is administered to the subject 1.5 years, 2 years, 2.5 years, 3 years, 3.5 years, 4 years, 4.5 years, or 5 years after the mRNA vaccine having an open reading frame encoding the first set of cancer antigens.

[0137] Hotspot mutations as neoantigens In cancer population analyses, certain mutations occur in a higher proportion of patients than would be expected by chance. These "recurrent" or "hotspot" mutations are often shown to have a "driver" role in tumors, resulting in some alteration of cancer cell functions important for tumor initiation, maintenance, or metastasis, and therefore being selected for as tumors evolve. In addition to their importance in tumor biology and treatment, recurrent mutations offer opportunities for precision medicine, where patient populations are segmented into groups likely to respond to specific treatments. These specific treatments include, but are not limited to, those targeting the mutant protein itself.

[0138] Although much effort and research into recurrent mutations has focused on non-synonymous (or "missense") single nucleotide variants (SNVs), population analyses have revealed that a variety of more complex (non-SNV) mutation classes, including synonymous (or "silent"), splice site, multi-nucleotide variants, insertions, and deletions, can also occur frequently.

[0139] The p53 gene (officially designated TP53) mutates more frequently in human cancers than any other gene. Large-scale cohort studies have shown that the genomic locations of most p53 mutations are unique to only one or a few patients, preventing their use as recurrent neoantigens for therapeutic vaccines designed for specific patient populations. Surprisingly, however, a small subset of the p53 locus does exhibit a "hotspot" pattern, where several positions in the gene mutate with relatively high frequency. Remarkably, the majority of these recurrently mutated regions reside near exon-intron boundaries, disrupting standard nucleotide sequence motifs recognized by the mRNA splicing machinery. Mutations in splicing motifs can alter the final mRNA sequence even if the local amino acid sequence is not expected to change (i.e., synonymous or intronic mutations). Therefore, although these mutations can alter mRNA splicing in unpredictable ways and have significant functional consequences for the translated protein, they are often annotated as "non-coding" by common annotation tools and ignored without further analysis. If alternatively spliced ​​isoforms contain in-frame sequence changes (i.e., no PTCs), they can escape NMD elimination and are rapidly expressed, processed, and presented on the cell surface by the HLA system. Furthermore, alternative splicing resulting from mutations is usually "cryptic," i.e., not expressed in normal tissues, and therefore can be recognized by T cells as non-self neoantigens.

[0140] In some aspects, the present invention provides neo-antigenic peptide sequences derived from certain recurrent somatic cancer mutations in p53 (including but not limited to missense SNVs), which often result in alternative splicing, and which are intended for use as targets for therapeutic vaccination. In some embodiments, mutations that result in mRNA splicing events that generate neo-antigenic peptides and / or HLA-restricted epitopes include a mutation in the canonical 5' splice site adjacent to codon position T125, which results in the epitope AVSPCISFVW (SEQ ID NO: 2) (HLA-B * 57:01, HLA-B * 58:01), epitope HPLASCQCFF (SEQ ID NO: 3) (HLA-B * 35:01, HLA-B * 53:01), epitope FVWNFGIPL (SEQ ID NO: 4) (HLA-A * 02:01, HLA-A * 02:06, HLA-B * 35:01), a mutation is included that introduces a retained intron with the peptide sequence TAKSVTCTVSCPEGLASMRLQCLAVSPCISFVWNFGIPLHPLASCQCFFIVYPLNV (SEQ ID NO: 1).

[0141] In some embodiments, the mutations that result in a splicing event of mRNA that generates a neo-antigenic peptide and / or an HLA-restricted epitope include a mutation in the canonical 5' splice site adjacent to codon position 331 that results in the epitope LQVLSLGTSY (SEQ ID NO: 6) (HLA-B * 15:01), epitope FQSNTQNAVF (SEQ ID NO: 7) (HLA-B * 15:01) includes a mutation that introduces a retained intron with the peptide sequence EYFTLQVLSLGTSYQVESFQSNTQNAVFFLTVLPAIGAFAIRGQ (SEQ ID NO: 5).

[0142] In some embodiments, mutations that result in a splicing event of an mRNA that generates a neo-antigenic peptide and / or an HLA-restricted epitope include a mutation in the canonical 3' splice site adjacent to codon position 126 that encodes the epitope CTMFCQLAK (SEQ ID NO: 9) (HLA-A * 11:01), epitope KSVTCTMF (SEQ ID NO: 10) (HLA-B * The exon contains a mutation that introduces a cryptic alternative exon 3' splice site generating a novel spanning peptide sequence AKSVTCTMFCQLAK (SEQ ID NO: 8) containing the nucleotide sequence 58:01.

[0143] In some embodiments, mutations that result in a splicing event of an mRNA that generates a neo-antigenic peptide and / or an HLA-restricted epitope include a mutation in the canonical 5' splice site adjacent to codon position 224 that results in the epitope VPYEPPEVW (SEQ ID NO: 12) (HLA-B * 53:01, HLA-B * 51:01), epitope LTVPPSTAW (SEQ ID NO: 13) (HLA-B * 58:01, HLA-B * The present invention includes a mutation that introduces a potential alternative intron 5' splice site generating a novel spanning peptide sequence VPYEPPEVWLALTVPPSTAWAA (SEQ ID NO: 11) containing the nucleotide sequence 57:01.

[0144] In the preceding sequences, the transcription codon positions refer to ENST00000269305 (SEQ ID NO: 14), the standard full-length p53 transcript from Ensembl's v83 human genome annotation.

[0145] In one embodiment, the present invention provides an mRNA vaccine comprising a concatemeric polyepitope construct or a set of individual epitope constructs comprising open reading frames (ORFs) encoding neo-antigenic peptides 1-4.

[0146] In one embodiment, the invention provides for the selective administration of a vaccine comprising or encoding peptides 1-4 based on whether a patient's tumor contains any of the above mutations.

[0147] In one embodiment, the present invention provides for selective administration of vaccines based on two criteria: 1) whether the patient's tumor contains any of the above mutations, and 2) whether the patient's normal HLA type contains the corresponding HLA alleles predicted to bind to the resulting neoantigen.

[0148] The mRNA vaccines described herein have been discovered to offer several advantages over current vaccines. First, delivery via lipid nanoparticles (LNPs) is superior to other formulations, including liposomes or protamine-based approaches described in the literature, and does not require additional adjuvants. LNPs enable efficient delivery of chemically modified or unmodified mRNA vaccines. Both modified and unmodified LNP-formulated mRNA vaccines are significantly superior to conventional vaccines. In some embodiments, the mRNA vaccines of the present invention are at least 10-fold, at least 20-fold, at least 40-fold, at least 50-fold, at least 100-fold, at least 500-fold, or at least 1,000-fold superior to conventional vaccines.

[0149] Although numerous attempts have been made to generate functional RNA vaccines, including mRNA vaccines and self-replicating RNA vaccines, the therapeutic efficacy of such RNA vaccines has yet to be fully established. Surprisingly, in accordance with an embodiment of the present invention, the inventors have discovered a class of formulations for in vivo delivery of mRNA vaccines that elicit significantly enhanced and in many respects synergistic immune responses, including enhanced antigen production and the production of functional antibodies with neutralizing capacity. These results can be achieved even when significantly lower doses of mRNA are administered compared to the mRNA doses used in other classes of lipid-based formulations. The formulations of the present invention have been shown to unexpectedly elicit significant immune responses in vivo, sufficient to establish the efficacy of functional mRNA vaccines as prophylactic and therapeutic agents. Furthermore, self-replicating RNA vaccines generate immune responses by utilizing the viral replication pathway to deliver sufficient RNA to cells. The formulations of the present invention produce sufficient protein to induce a potent immune response without requiring viral replication. Therefore, the mRNAs of the present invention are not self-replicating RNA and do not contain elements necessary for viral replication.

[0150] In some aspects, the present invention encompasses the surprising discovery that lipid nanoparticle (LNP) formulations significantly enhance the efficacy of mRNA vaccines, including chemically modified and unmodified mRNA vaccines. The efficacy of mRNA vaccines formulated in LNPs was investigated in vivo using several different tumor antigens. In addition to eliciting enhanced immune responses, the formulations of the present invention generate immune responses more rapidly and at lower doses of antigen compared to other tested vaccines. Furthermore, compared to vaccines formulated in different carriers, the mRNA-LNP formulations of the present invention generate superior immune responses, both quantitatively and qualitatively. Furthermore, the mRNA-LNP formulations of the present invention outperform other vaccines, even when the mRNA dose is lower compared to the other vaccines.

[0151] The LNP used in the test described herein has been used for the delivery of siRNA in various animal models and humans before.Considering the findings obtained in relation to the delivery of siRNA by LNP formulation, the fact that LNP is useful in vaccines is very surprising.It has been observed that the therapeutic delivery of siRNA formulated in LNP typically reduces antigen production and impairs immune response, resulting in undesirable inflammatory responses associated with transient IgM responses.In contrast to the findings observed with siRNA, the LNP-mRNA formulation of the present invention is shown herein to not produce transient IgM responses, but to increase IgG levels, which is sufficient for prophylactic and therapeutic methods.

[0152] Nucleic Acids / Polynucleotides The cancer vaccines provided herein comprise at least one (or more) ribonucleic acid (RNA) polynucleotides having an open reading frame encoding at least one cancer antigen polypeptide. The term "nucleic acid," in its broadest sense, includes any compound and / or substance comprising a polymer of nucleotides. Such polymers are referred to as polynucleotides.

[0153] The nucleic acid (also called polynucleotide) may be or may comprise, for example, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA including LNA having a β-D-ribo configuration, α-LNA having an α-L-ribo configuration (a diastereomer of LNA), 2′-amino-functionalized 2′-amino-LNA, and 2′-amino-functionalized 2′-amino-α-LNA), ethylene nucleic acid (ENA), cyclohexenyl nucleic acid (CeNA), or chimeras or combinations thereof.

[0154] In some embodiments, polynucleotides of the present disclosure function as messenger RNA (mRNA). "Messenger RNA" (mRNA) refers to any polynucleotide that encodes (at least one) polypeptide (naturally occurring amino acid polymer, non-naturally occurring amino acid polymer, or modified amino acid polymer) and can be translated to produce the encoded polypeptide in vitro, in vivo, in situ, or ex vivo.

[0155] The basic components of an mRNA molecule typically include at least a coding region, a 5' untranslated region (UTR), a 3' UTR, a 5' cap, and a poly A tail. The polynucleotides of the present disclosure can function as mRNAs, but can be distinguished from wild-type mRNAs in that they possess functional and / or structural design features that help overcome existing problems in efficiently expressing polypeptides using nucleic acid-based therapeutics.

[0156] In some embodiments, the RNA polynucleotide of the cancer vaccine encodes 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 to 4, 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, 4 to 5, 5 to 10, 5 to 9, 5 to 8, 5 to 7, 5 to 6, 6 to 10, 6 to 9, 6 to 8, 6 to 7, 7 to 10, 7 to 9, 7 to 8, 8 to 10, 8 to 9, or 9 to 10 antigenic polypeptides. In some embodiments, the RNA polynucleotide of the cancer vaccine encodes at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 antigenic polypeptides. In some embodiments, the RNA polynucleotide of the cancer vaccine encodes at least 100 or at least 200 antigenic polypeptides. In some embodiments, the RNA polynucleotide of the cancer vaccine encodes 1-10, 5-15, 10-20, 15-25, 20-30, 25-35, 30-40, 35-45, 40-50, 1-50, 1-100, 2-50, or 2-100 antigenic polypeptides.

[0157] In some embodiments, the polynucleotides of the present disclosure are codon-optimized. Codon optimization methods are known in the art and may be used as provided herein. In some embodiments, codon optimization may be used to match the codon frequency of a target with that of a host organism to maintain proper folding, bias the GC content to improve mRNA stability or reduce secondary structure, minimize tandem repeat codons and base sequences that may impair gene assembly or expression, customize transcriptional and translational control regions, insert or remove protein transport sequences, remove / add post-translational modification sites (e.g., glycosylation sites) in the encoded protein, add, remove, or shuffle protein domains, insert or remove restriction enzyme sites, modify ribosome binding sites and mRNA degradation sites, adjust the translation rate to allow proper folding of various domains of a protein, or reduce or eliminate problematic secondary structures within a polynucleotide. Codon optimization tools, algorithms, and services are known in the art, including, but not limited to, services offered by GeneArt (Life Technologies), DNA2.0 (Menlo Park, CA), and / or proprietary methods. In some embodiments, open reading frame (ORF) sequences are optimized using an optimization algorithm.

[0158] In some embodiments, the codon-optimized sequence shares less than 95% sequence identity with a naturally occurring or wild-type sequence (e.g., a naturally occurring or wild-type mRNA sequence encoding a polypeptide or protein of interest (e.g., an antigenic protein or polypeptide)). In some embodiments, the codon-optimized sequence shares less than 90% sequence identity with a naturally occurring or wild-type sequence (e.g., a naturally occurring or wild-type mRNA sequence encoding a polypeptide or protein of interest (e.g., an antigenic protein or polypeptide)). In some embodiments, the codon-optimized sequence shares less than 85% sequence identity with a naturally occurring or wild-type sequence (e.g., a naturally occurring or wild-type mRNA sequence encoding a polypeptide or protein of interest (e.g., an antigenic protein or polypeptide)). In some embodiments, the codon-optimized sequence shares less than 80% sequence identity with a naturally occurring or wild-type sequence (e.g., a naturally occurring or wild-type mRNA sequence encoding a polypeptide or protein of interest (e.g., an antigenic protein or polypeptide)). In some embodiments, the codon-optimized sequence shares less than 75% sequence identity with a naturally occurring or wild-type sequence (e.g., a naturally occurring or wild-type mRNA sequence encoding a polypeptide or protein of interest (e.g., an antigenic protein or polypeptide)).

[0159] In some embodiments, the codon-optimized sequence shares 65% to 85% (e.g., about 67% to about 85% or about 67% to about 80%) sequence identity with a naturally occurring or wild-type sequence (e.g., a naturally occurring or wild-type mRNA sequence encoding a polypeptide or protein of interest (e.g., an antigenic protein or polypeptide)). In some embodiments, the codon-optimized sequence shares 65% to 75% or about 80% sequence identity with a naturally occurring or wild-type sequence (e.g., a naturally occurring or wild-type mRNA sequence encoding a polypeptide or protein of interest (e.g., an antigenic protein or polypeptide)).

[0160] In some embodiments, codon-optimized RNA can have, for example, an enhanced level of G / C. The G / C content of a nucleic acid molecule can affect RNA stability. RNA with increased amounts of guanine (G) and / or cytosine (C) residues can be functionally more stable compared to nucleic acids containing large amounts of adenine (A) and thymine (T) or uracil (U) nucleotides. WO 02 / 098443 discloses pharmaceutical compositions containing mRNA stabilized by sequence modification of the coding region. Because the genetic code is degenerate, modifications work by replacing existing codons with codons that promote improved RNA stability without changing the resulting amino acid. This approach is not limited to the coding region of RNA.

[0161] Antigens / antigenic polypeptides In some embodiments, the cancer antigen polypeptide is longer than 25 amino acids and shorter than 50 amino acids. Thus, polypeptides include gene products, naturally occurring polypeptides, synthetic polypeptides, and homologs, orthologs, paralogs, fragments, and other equivalent forms, variants, and analogs of the foregoing. Polypeptides can be single molecules or multimolecular complexes, such as dimers, trimers, or tetramers. Polypeptides can also include single-chain polypeptides or multi-chain polypeptides, such as antibodies or insulin, which can be associated or linked. Disulfide bonds are most commonly found in multi-chain polypeptides. The term polypeptide can also apply to amino acid polymers in which at least one amino acid residue is an artificial chemical analog of a corresponding naturally occurring amino acid.

[0162] The term "polypeptide variant" refers to a molecule whose amino acid sequence differs from a native or reference sequence. Amino acid sequence variants may have substitutions, deletions, and / or insertions at certain positions within the amino acid sequence compared to the native or reference sequence. Typically, variants share at least 50% identity to the native or reference sequence. In some embodiments, variants share at least 80% or at least 90% identity with the native or reference sequence.

[0163] In some embodiments, "mutant mimetics" are provided. As used herein, the term "mutant mimetics" includes at least one amino acid that is expected to mimic an activating sequence. For example, glutamate can act as a mimic of phosphoro-threonine and / or phosphoro-serine. Alternatively, mutant mimetics can lose activity or result in inactivated products containing the mimic; for example, phenylalanine can act as an inactivating substitution for tyrosine, or alanine can act as an inactivating substitution for serine.

[0164] "Orthologs" refer to genes in different species that evolved from a common ancestral gene through speciation. Orthologs usually retain the same function during evolution. Identification of orthologs is crucial for reliable gene function prediction in newly sequenced genomes.

[0165] "Analog" is intended to include polypeptide variants that differ by one or more amino acid changes, such as substitution, addition, or deletion of an amino acid residue, that still retain one or more of the properties of the parent or starting polypeptide.

[0166] The present disclosure provides several types of polynucleotide- or polypeptide-based compositions, including variants and derivatives. These include, for example, variants and derivatives with substitutions, insertions, deletions, and covalent bonds. The term "derivative" is used interchangeably with the term "variant," but generally refers to a molecule that has been modified and / or changed in any way compared to a reference or starting molecule.

[0167] Thus, polynucleotides encoding peptides or polypeptides containing substitutions, insertions, and / or additions, deletions, and covalent modifications with respect to a reference sequence, particularly a polypeptide sequence disclosed herein, are within the scope of this disclosure. For example, a sequence tag or one or more amino acids, such as lysine, can be added to a peptide sequence (e.g., at the N- or C-terminus). The sequence tag can be used for peptide detection, purification, or location. Lysine can be used to improve peptide solubility or enable biotin labeling. Alternatively, amino acid residues located in the carboxy- and amino-terminal regions of a peptide or protein amino acid sequence can be optionally deleted to produce truncated sequences. Depending on the intended use of the sequence, for example, expression of the sequence as part of a longer soluble sequence or as part of a longer sequence linked to a solid support, certain amino acids (e.g., C- or N-terminal residues) can alternatively be deleted.

[0168] When referring to a polypeptide, a "substitutional variant" is one in which at least one of the amino acid residues in a native or starting sequence has been removed and a different amino acid inserted in the same position where it was originally located. The substitutions can be single, where only one amino acid in the molecule has been substituted, or they can be multiple, where two or more amino acids have been substituted in the same molecule.

[0169] As used herein, the term "conservative amino acid substitution" refers to the substitution of an amino acid normally present in a sequence with a different amino acid of similar size, charge, or polarity. Examples of conservative substitutions include the substitution of a non-polar (hydrophobic) residue, such as isoleucine, valine, and leucine, for another non-polar residue. Similarly, examples of conservative substitutions include the substitution of one polar (hydrophilic) residue for another, such as the substitution of arginine for lysine, glutamine for asparagine, and glycine for serine. Furthermore, the substitution of a basic residue, such as lysine, arginine, or histidine, for another, or the substitution of one acidic residue, such as aspartic acid or glutamic acid, for another, are additional examples of conservative substitutions. Examples of non-conservative substitutions include substitutions of a non-polar (hydrophobic) amino acid residue such as isoleucine, valine, leucine, alanine, or methionine for a polar (hydrophilic) residue such as cysteine, glutamine, glutamic acid, or lysine, and / or a polar residue for a non-polar residue.

[0170] When referring to a polypeptide or polynucleotide, a "feature" is defined as a molecular entity based on a different amino acid sequence or a molecular entity based on a different nucleotide, respectively. Features of a polypeptide encoded by a polynucleotide include a surface appearance, a local conformational shape, a fold, a loop, a half-loop, a domain, a half-domain, a site, an end, or any combination thereof.

[0171] As used herein, the term "domain," when referring to a polypeptide, refers to a motif in a polypeptide having one or more identifiable structural or functional features or characteristics (e.g., binding ability to serve as a site for protein-protein interaction).

[0172] As used herein with reference to amino acid-based embodiments, the term "site" when referring to a polypeptide is used synonymously with "amino acid residue" and "amino acid side chain." As used herein with reference to nucleotide-based embodiments, the term "site" is used synonymously with "nucleotide." A site corresponds to a position within a peptide or polypeptide or polynucleotide that can be modified, manipulated, altered, derivatized, or changed within a polypeptide- or polynucleotide-based molecule.

[0173] As used herein, the terms "terminus" and "terminus" when referring to a polypeptide or polynucleotide refer to the end of the polypeptide or polynucleotide, respectively. Such termini are not limited to the first or last position of the polypeptide or polynucleotide, but may include additional amino acids or nucleotides in the terminal region. Polypeptide-based molecules can be characterized as having both an N-terminus (terminated by an amino acid with a free amino group (NH)) and a C-terminus (terminated by an amino acid with a free carboxyl group (COOH)). In some cases, proteins are composed of multiple polypeptide chains (multimers, oligomers) held together by disulfide bonds or noncovalent forces. Such proteins have multiple N- and C-termini. Alternatively, the ends of polypeptides may be modified, and as a result, in some cases, begin or end with non-polypeptide-based moieties, such as organic complexes.

[0174] Protein fragments, functional protein domains, and homologous proteins recognized by those skilled in the art are also considered to be within the scope of polypeptides of interest. For example, any protein fragment (meaning a polypeptide sequence that is identical except for being at least one less amino acid residue as compared to the reference polypeptide sequence) of a reference protein that is 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more than 100 amino acids in length is provided herein. In another example, any protein containing a stretch of 20, 30, 40, 50, or 100 amino acids that is 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% identical to any of the sequences described herein can be utilized in accordance with the present disclosure. In some embodiments, a polypeptide contains two, three, four, five, six, seven, eight, nine, ten, or eleven or more of the mutations set forth in any of the sequences provided or referenced herein. In another example, any protein comprising a stretch of 20, 30, 40, 50, or 100 amino acids that is more than 80%, more than 90%, more than 95%, or 100% identical to any of the sequences described herein, and having a stretch of 5, 10, 15, 20, 25, or 30 amino acids that is less than 80%, less than 75%, less than 70%, less than 65%, or less than 60% identical to any of the sequences described herein may be utilized in accordance with the present disclosure.

[0175] The polypeptide or polynucleotide molecules of the present disclosure may share a degree of sequence similarity or sequence identity with a reference molecule (e.g., a reference polypeptide or polynucleotide), such as a molecule described in the art (e.g., an engineered or designed molecule or a wild-type molecule). The term "identity," as known in the art, refers to the relatedness between two or more polypeptide or polynucleotide sequences, as determined by sequence comparison. In the art, identity also refers to the degree of sequence relatedness between them, as determined by the number of matches between strings of two or more amino acid or nucleic acid residues. Identity is a measure of the percentage of identical matches between two sequences, reducing gaps (if any) in the alignment performed by a specific mathematical model or computer program (e.g., an "algorithm"). The identity of related peptides can be easily calculated by known methods. "Percent identity," as applied to a polypeptide or polynucleotide sequence, is defined as the percentage of residues (amino acid or nucleic acid residues) in a candidate amino acid or nucleic acid sequence that are identical to the residues in the amino acid or nucleic acid sequence of a second sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity. Methods and computer programs for alignment are well known in the art. It is understood that identity is dependent on the calculation of percent identity, but may vary due to gaps and penalties introduced in the calculation.Typically, a particular polynucleotide or polypeptide variant will have at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity to a particular reference polynucleotide or polypeptide, as determined by sequence alignment programs and parameters described herein and known to those skilled in the art. Such alignment tools include those in the BLAST suite (Stephen F. Altschul, et al. (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs," Nucleic Acids Res. 25:3389-3402). Another common local alignment technique is based on the Smith-Waterman algorithm (Smith, T. F. & Waterman, M. S. (1981) "Identification of common molecular subsequences," J. Mol. Biol. 147:195-197). A common global alignment technique based on dynamic programming is the Needleman-Wunsch algorithm (Needleman, S. B. & Wunsch, C. D. (1970) "A general method applicable to the search for similarities in the amino acid sequences of two proteins," J. Mol. Biol. 48:443-453). A fast optimal global sequence alignment algorithm (FOGSAA) has been developed very recently, which is said to perform global alignment of nucleotide and protein sequences faster than other optimal global alignment methods, including the Needleman-Wunsch algorithm.Tools are described elsewhere herein, and in particular in the definition of "identity" below.

[0176] The term "homology," as used herein, refers to the overall relatedness between polymeric molecules, such as, for example, nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules. Polymeric molecules (e.g., nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules) that share an acceptable level of similarity or identity, as determined by alignment of matching residues, are said to be homologous. Homology is a qualitative term that describes the relatedness between molecules and can be based on quantitative similarity or identity. Similarity or identity is a quantitative term that defines the degree of sequence identity between two compared sequences. In some embodiments, polymeric molecules are considered to be "homologous" to one another if their sequences are at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical or similar. The term "homologous" necessarily refers to a comparison between at least two sequences (polynucleotide sequences or polypeptide sequences). Two polynucleotide sequences are considered to be homologous if the polypeptides they encode are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or even 99% identical over at least one stretch containing at least 20 amino acids. In some embodiments, homologous polynucleotide sequences are characterized by their ability to encode a stretch containing at least 4-5 uniquely specified amino acids. For polynucleotide sequences less than 60 nucleotides in length, homology is determined by their ability to encode a stretch containing at least 4-5 uniquely specified amino acids. Two protein sequences are considered to be homologous if the proteins are at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identical over at least one stretch containing at least 20 amino acids.

[0177] Homology implies that the compared sequences have diverged in evolution from a common origin. The term "homolog" refers to a first amino acid or nucleic acid sequence (e.g., a gene (DNA or RNA) sequence or a protein sequence) that is related to a second amino acid or nucleic acid sequence by descent from a common ancestral sequence. The term "homolog" can also apply to the relationship between genes and / or proteins that have separated by the event of speciation or the relationship between genes and / or proteins that have separated by the event of gene duplication. An "ortholog" is a gene (or protein) from different species that evolved from a common ancestral gene (or protein) by speciation. Typically, orthologs retain the same function during evolution. A "paralog" is a gene (or protein) that is related by duplication within a genome. Orthologs retain the same function during evolution, while paralogs develop new functions, even if related to their ancestors.

[0178] The term "identity" refers to the overall relatedness between polymer molecules, such as, for example, polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules. Calculation of the percent identity of two polynucleic acid sequences can be performed, for example, by aligning the two sequences to optimize comparison (e.g., gaps can be introduced into one or both of the first and second nucleic acid sequences to optimize alignment, and non-identical sequences can be ignored for comparison purposes). In certain embodiments, the length of the sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the length of the reference sequence. Nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that need to be introduced to optimize the alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.For example, the percent identity between two nucleic acid sequences can be determined using methods such as those described in Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991, each of which is incorporated herein by reference. For example, the percent identity between two nucleic acid sequences can be determined using the algorithm of Meyers and Miller (CABIOS, 1989, 4:11-17), which is incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the percent identity between two nucleic acid sequences can be determined using the GAP program in the GCG software package, using the NWSgapdna.CMP matrix. Commonly used methods for determining percent identity between sequences include, but are not limited to, those disclosed in Carillo, H., and Lipman, D., SIAM J Applied Math., 48:1073 (1988), which is incorporated herein by reference. Techniques for determining identity are incorporated into publicly available computer programs.Examples of computer software for determining homology between two sequences include, but are not limited to, the GCG program package, Devereux, J., et al., Nucleic Acids Research, 12(1), 387 (1984), BLASTP, BLASTN, and FASTA (Altschul, S. F. et al., J. Molec. Biol., 215, 403 (1990)).

[0179] chemical modification In some embodiments, the RNA (e.g., mRNA) vaccines of the present disclosure comprise at least one ribonucleic acid (RNA) polynucleotide having an open reading frame encoding at least one respiratory syncytial virus (RSV) antigenic polypeptide, wherein the RNA comprises at least one chemical modification.

[0180] The terms "chemical modification" and "chemically modified" refer to modifications in at least one of the position, pattern, percentage, or population of ribonucleosides or deoxyribonucleosides containing adenosine (A), guanosine (G), uridine (U), thymidine (T), or cytidine (C). Generally, these terms do not refer to ribonucleotide modifications in the cap moiety that naturally occurs at the 5' end of an mRNA.

[0181] Modifications of polynucleotides include, but are not limited to, those described herein, including, but not limited to, chemical modifications. Polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) can contain naturally occurring modifications, non-naturally occurring modifications, or polynucleotides can contain a combination of naturally occurring and non-naturally occurring modifications. Polynucleotides can include, for example, any useful modification of the sugar, nucleobase, or internucleoside linkage (e.g., to the linking phosphate, phosphodiester bond, or phosphodiester backbone).

[0182] The term "modified" with respect to a polypeptide refers to a modification to the standard set of 20 amino acids. A polypeptide provided herein is also considered "modified" if it contains an amino acid substitution, insertion, or a combination of substitution and insertion.

[0183] In some embodiments, a polynucleotide (e.g., an RNA polynucleotide such as an mRNA polynucleotide) comprises a variety of different modifications. In some embodiments, specific regions of a polynucleotide comprise one, two, or more (optionally different) nucleoside or nucleotide modifications. In some embodiments, modified RNA polynucleotides (e.g., modified mRNA polynucleotides) introduced into a cell or organism exhibit reduced degradation in the cell or organism, respectively, compared to unmodified polynucleotides. In some embodiments, modified RNA polynucleotides (e.g., modified mRNA polynucleotides) introduced into a cell or organism may exhibit reduced immunogenicity (e.g., reduced innate response) in the cell or organism, respectively.

[0184] In some embodiments, polynucleotides (e.g., RNA polynucleotides such as mRNA polynucleotides) contain non-naturally occurring modified nucleotides that are introduced during or after the synthesis of the polynucleotide to achieve a desired function or property. Modifications can be in the internucleotide linkage, the purine or pyrimidine base, or the sugar. Modifications can be introduced at the end of the chain or anywhere else in the chain using chemical synthesis or polymerase enzymes. Any region of a polynucleotide can be chemically modified.

[0185] The present disclosure provides polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) of modified nucleosides and nucleotides. A "nucleoside" refers to a compound comprising a sugar molecule (e.g., pentose or ribose) or a derivative thereof in combination with an organic base (e.g., a purine or pyrimidine) or a derivative thereof (also referred to herein as a "nucleobase"). A "nucleotide" refers to a nucleoside comprising a phosphate group. Modified nucleotides may be synthesized by any useful method, e.g., chemical, enzymatic, or recombinant, to include one or more modified or unnatural nucleosides. A polynucleotide may comprise one or more regions of linked nucleosides. Such regions may have a variety of backbone linkages. The linkages may be standard phosphodiester linkages, in which case the polynucleotide is considered to comprise a region of nucleotides.

[0186] Modified nucleotide base pairing encompasses not only standard adenosine-thymine, adenosine-uracil, or guanosine-cytosine base pairs, but also base pairs formed between nucleotides containing non-standard or modified bases and / or modified nucleotides, where the arrangement of hydrogen bond donors and hydrogen bond acceptors allows hydrogen bonding between the non-standard and standard bases, or between two complementary non-standard base structures, such as in polynucleotides having at least one chemical modification. One example of such non-standard base pairing is base pairing between the modified nucleotide inosine and adenine, cytosine, or uracil. Any combination of base / sugar or linker may be incorporated into the polynucleotides of the present disclosure.

[0187] Modifications of polynucleotides (e.g., RNA polynucleotides such as mRNA polynucleotides), including but not limited to chemical modifications, useful in the compositions, vaccines, methods, and synthetic processes of the present disclosure include, but are not limited to, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-methyladenosine, 2-methylthio-N6-threonylcarbamoyladenosine, N6-glycinylcarbamoyladenosine, N6-isopentenyladenosine, N6-methyladenosine, N6-threonylcarbamoyladenosine, 1,2'-O-dimethyladenosine, 1-methyladenosine, 2'-O-methyladenosine, 2'-O-ribosyladenosine (phosphate), 2 ... Adenosine, 2-methylthio-N6-isopentenyladenosine, 2-methylthio-N6-hydroxynorvalylcarbamoyladenosine, 2'-O-methyladenosine, 2'-O-ribosyladenosine (phosphate), isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, N6,2'-O-dimethyladenosine, N6,2'-O-dimethyladenosine adenosine, N6,N6,2'-O-trimethyladenosine, N6,N6-dimethyladenosine, N6-acetyladenosine, N6-hydroxynorvalylcarbamoyl adenosine, N6-methyl-N6-threonylcarbamoyl adenosine, 2-methyladenosine, 2-methylthio-N6-isopentenyladenosine, 7-deaza-adenosine, N1-methyl-adenosine, N6,N6(dimethyl)adenine, N6-cis-hydroxy-isopentenyl-adenosine, α-thio-adenosine, 2(amino)adenine, 2(aminopropyl)adenine, 2(methylthio)N6(isopentenyl)adenine, 2-(alkyl)adenine, 2-(aminoalkyl)adenine, 2-(aminopropyl)adenine, 2-(halo)adenine, 2-(halo)adenine, 2-(propyl)adenine, 2'-amino-2'-deoxy-ATP, 2' -azido-2'-deoxy-ATP, 2'-deoxy-2'-α-aminoadenosine TP, 2'-deoxy-2'-α-azidoadenosine TP, 6(alkyl)adenine, 6(methyl)adenine, 6-(alkyl)adenine, 6-(methyl)adenine, 7(deaza)adenine, 8(alkenyl)adenine, 8(alkynyl)adenine, 8(amino)adenine, 8(thioalkyl)adenine, 8-(alkenyl)adenine, 8-(alkyl)adenine, 8-(alkynyl)adenine, 8-(amino)adenine, 8-(halo)adenine, 8-(hydroxyl)adenine, 8-(thioalkyl)adenine, 8-(thiol)adenine, 8-azido-adenosine, azaadenine, deazaadenine, N6(methyl)adenine, N6-(isopentyl)adenine, 7-deaza-8-aza-adenosine, 7-methyladenine, 1-deazaadenosine TP, 2'fluoro-N6-Bz-deoxyadenosine TP, 2' -OMe-2-amino-ATP, 2'O-methyl-N6-Bz-deoxyadenosine TP, 2'-α-ethynyl adenosine TP, 2-aminoadenine, 2-amino adenosine TP, 2-amino-ATP, 2'-α-trifluoromethyl adenosine TP, 2-azidoadenosine TP, 2'-β-ethynyl adenosine TP, 2-bromoadenosine TP, 2'-β-trifluoromethyl adenosine TP, 2-chloroadenosine TP, 2'-deoxy-2',2'-Difluoroadenosine TP, 2'-deoxy-2'-α-mercaptoadenosine TP, 2'-deoxy-2'-α-thiomethoxyadenosine TP, 2'-deoxy-2'-β-aminoadenosine TP, 2'-deoxy-2'-β-azidoadenosine TP, 2'-deoxy-2'-β-bromoadenosine TP, 2'-deoxy-2'-β-chloroadenosine TP, 2'-deoxy-2'-β-fluoroadenosine TP, 2'-deoxy-2'-β-iodoadenosine TP, 2'-Deoxy-2'-b-mercaptoadenosine TP, 2'-deoxy-2'-b-thiomethoxyadenosine TP, 2-fluoroadenosine TP, 2-iodoadenosine TP, 2-mercaptoadenosine TP, 2-methoxy-adenine, 2-methylthio-adenine, 2-trifluoromethyladenosine TP, 3-deaza-3-bromoadenosine TP, 3-deaza-3-chloroadenosine TP, 3-deaza-3-fluoroadenosine TP, 3-deaza-3-iodoadenosine T P, 3-deazaadenosine TP, 4'-azidoadenosine TP, 4'-carbocyclic adenosine TP, 4'-ethynyl adenosine TP, 5'-homo-adenosine TP, 8-aza-ATP, 8-bromo-adenosine TP, 8-trifluoromethyl adenosine TP, 9-deazaadenosine TP, 2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 7-deaza-8-aza-2-aminopurine, 2,6-diaminopurine, 7-deaza 8-Aza-adenine, 7-deaza-2-aminopurine, 2-thiocytidine, 3-methylcytidine, 5-formylcytidine, 5-hydroxymethylcytidine, 5-methylcytidine, N4-acetylcytidine, 2'-O-methylcytidine, 2'-O-methylcytidine, 5,2'-O-dimethylcytidine, 5-formyl-2'-O-methylcytidine, lysidine, N4,2'-O-dimethylcytidine, N4-acetyl-2'-O-methylcytidine, N4-methylcytidine, N4,N4-dimethyl-2'-OMe-cytidine TP, 4-methylcytidine, 5-aza-cytidine, pseudo-iso-cytidine, pyrrolo-cytidine, α-thio-cytidine, 2-(thio)cytosine, 2'-amino-2'-deoxy-CTP, 2'-azido-2'-deoxy-CTP, 2'-deoxy-2'-α-aminocytidine TP, 2'-deoxy-2'-α-azidocytidine TP, 3(deaza)5(aza)cytosine, 3(methyl)cytosine, 3-(alkyl)cytosine, 3-(deaza)5(aza)cytosine, 3- (Methyl)cytidine, 4,2'-O-dimethylcytidine, 5-(halo)cytosine, 5-(methyl)cytosine, 5-(propynyl)cytosine, 5-(trifluoromethyl)cytosine, 5-(alkyl)cytosine, 5-(alkynyl)cytosine, 5-(halo)cytosine, 5-(propynyl)cytosine, 5-(trifluoromethyl)cytosine, 5-bromo-cytidine, 5-iodo-cytidine, 5-propynylcytosine, 6-(azo)cytosine, 6-aza-cytidine, azacytosine, deazacytosine, N4(acetyl)cytosine 1-methyl-1-deaza-pseudoisocytidine, 1-methyl-pseudoisocytidine, 2-methoxy-5-methyl-cytidine, 2-methoxy-cytidine, 2-thio-5-methyl-cytidine, 4-methoxy-1-methyl-pseudoisocytidine, 4-methoxy-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-pseudoisocytidine, 5-aza-zebularine, 5-methyl-zebularine, pyrrolo-pseudoisocytidine Socytidine, Zebularine, (E)-5-(2-bromo-vinyl)cytidine TP, 2,2'-anhydro-cytidine TP hydrochloride, 2'fluoro-N4-Bz-cytidine TP, 2'fluoro-N4-acetyl-cytidine TP, 2'-O-methyl-N4-acetyl-cytidine TP, 2'O-methyl-N4-Bz-cytidine TP, 2'-a-ethynylcytidine TP, 2'-a-trifluoromethylcytidine TP, 2'-b-ethynylcytidine TP, 2'-b-trifluoromethylcytidine TP, 2'-deoxy-2',2'-Difluorocytidine TP, 2'-deoxy-2'-α-mercaptocytidine TP, 2'-deoxy-2'-α-thiomethoxycytidine TP, 2'-deoxy-2'-β-aminocytidine TP, 2'-deoxy-2'-β-azidocytidine TP, 2'-deoxy-2'-β-bromocytidine TP, 2'-deoxy-2'-β-chlorocytidine TP, 2'-deoxy-2'-β-fluorocytidine TP, 2'-deoxy-2'-β-iodocytidine TP, 2'-deoxy 2'-b-mercaptocytidine TP, 2'-deoxy-2'-b-thiomethoxycytidine TP, 2'-O-methyl-5-(1-propynyl)cytidine TP, 3'-ethynylcytidine TP, 4'-azidocytidine TP, 4'-carbocyclic cytidine TP, 4'-ethynylcytidine TP, 5-(1-propynyl)ara-cytidine TP, 5-(2-chloro-phenyl)-2-thiocytidine TP, 5-(4-amino-phenyl)-2-thiocytidine TP, 5-aminoallyl-C TP, 5-cyanocytidine TP, 5-ethynylara-cytidine TP, 5-ethynylcytidine TP, 5'-homo-cytidine TP, 5-methoxycytidine TP, 5-trifluoromethyl-cytidine TP, N4-amino-cytidine TP, N4-benzoyl-cytidine TP, pseudoisocytidine, 7-methylguanosine, N2,2'-O-dimethylguanosine, N2-methylguanosine, wyosine, 1,2'-O-dimethylguanosine, 1-methyl Guanosine, 2'-O-methylguanosine, 2'-O-ribosylguanosine (phosphate), 2'-O-methylguanosine, 2'-O-ribosylguanosine (phosphate), 7-aminomethyl-7-deazaguanosine, 7-cyano-7-deazaguanosine, archaeosine, methylwiosine, N2,7-dimethylguanosine, N2,N2,2'-O-trimethylguanosine, N2,N2,7-trimethylguanosine, N2,N2-dimethylguanosine, N2,7,2'-O-trimethylguanosine, 6-thio-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, N1-methyl-guanosine, α-thio-guanosine, 2(propyl)guanine, 2-(alkyl)guanine, 2'-amino-2'-deoxy-GTP, 2'-azido-2'-deoxy-GTP, 2'-deoxy-2'-α-aminoguanosine TP, 2'-deoxy-2'-α-azidoguanosine TP, 6(methyl)guanosine, 6-(azido)guanosine (alkyl)guanine, 6-(methyl)guanine, 6-methyl-guanosine, 7-(alkyl)guanine, 7-(deaza)guanine, 7-(methyl)guanine, 7-(alkyl)guanine, 7-(deaza)guanine, 7-(methyl)guanine, 8-(alkyl)guanine, 8-(alkynyl)guanine, 8-(halo)guanine, 8-(thioalkyl)guanine, 8-(alkenyl)guanine, 8-(alkyl)guanine, 8-(alkynyl)guanine, 8-(amino)guanine guanine, 8-(halo)guanine, 8-(hydroxyl)guanine, 8-(thioalkyl)guanine, 8-(thiol)guanine, azaguanine, deazaguanine, N(methyl)guanine, N-(methyl)guanine, 1-methyl-6-thio-guanosine, 6-methoxy-guanosine, 6-thio-7-deaza-8-aza-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-methyl-guanosine, 7-deaza-8-aza-guanosine, 7-methyl-guanosine, 8-oxo-guanosine, N2,N2-dimethyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, 1-Me-GTP, 2'fluoro-N2-isobutyl-guanosine TP, 2'O-methyl-N2-isobutyl-guanosine TP, 2'-a-ethynylguanosine TP, 2'-a-trifluoromethylguanosine TP, 2'-b-ethynylguanosine TP, 2'-b-trifluoromethylguanosine TP, 2'-deoxy-2',2'-Difluoroguanosine TP, 2'-deoxy-2'-α-mercaptoguanosine TP, 2'-deoxy-2'-α-thiomethoxyguanosine TP, 2'-deoxy-2'-β-aminoguanosine TP, 2'-deoxy-2'-β-azidoguanosine TP, 2'-deoxy-2'-β-bromoguanosine TP, 2'-deoxy-2'-β-chloroguanosine TP, 2'-deoxy-2'-β-fluoroguanosine TP, 2'-deoxy-2'-β-iodoguanosine TP, 2'-deo, 2'-b-hydroxy-2'-b-mercaptoguanosine TP, 2'-deoxy-2'-b-thiomethoxyguanosine TP, 4'-azidoguanosine TP, 4'-carbocyclic guanosine TP, 4'-ethynylguanosine TP, 5'-homoguanosine TP, 8-bromoguanosine TP, 9-deazaguanosine TP, N2-isobutylguanosine TP, 1-methylinosine, inosine, 1,2'-O-dimethylinosine, 2'-O-methylinosine, 7-methylinosine, 2'-O-methylinosine, epoxyqueuosine, galactosylqueuosine, mannosylqueuosine, queuosine, allyamino-thymidine, azathymidine, deazathymidine, deoxythymidine, 2'-O-methyluridine, 2-thiouridine, 3-methyluridine, 5-carboxymethyluridine, 5-hydroxy- roxyuridine, 5-methyluridine, 5-taurinomethyl-2-thiouridine, 5-taurinomethyluridine, dihydrouridine, pseudouridine, (3-(3-amino-3-carboxypropyl)uridine, 1-methyl-3-(3-amino-5-carboxypropyl)pseudouridine, 1-methylpseudouridine, 1-ethyl-pseudouridine, 2'-O-methyluridine, 2'-O-methylpseudouridine, 2'-O-methyluridine, 2-thio-2'-O-methyluridine, 3-(3-amino-3-carboxypropyl)uridine, 3,2'-O-dimethyluridine, 3-methyl-pseudouridine TP, 4-thiouridine, 5-(carboxyhydroxymethyl)uridine, 5-(carboxyhydroxymethyl)uridine methyl ester, 5,2'-O-dimethyluridine, 5,6-Dihydro-uridine, 5-aminomethyl-2-thiouridine, 5-carbamoylmethyl-2'-O-methyluridine, 5-carbamoylmethyluridine, 5-carboxyhydroxymethyluridine, 5-carboxyhydroxymethyluridine methyl ester, 5-carboxymethylaminomethyl-2'-O-methyluridine, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluridine, 5-carboxymethylaminomethyluridine, 5-carbomethylaminomethyluridine, 5-carbomethylaminomethyluridine, Bamoylmethyluridine TP, 5-methoxycarbonylmethyl-2'-O-methyluridine, 5-methoxycarbonylmethyl-2-thiouridine, 5-methoxycarbonylmethyluridine, 5-methyluridine, ), 5-methoxyuridine, 5-methyl-2-thiouridine, 5-methylaminomethyl-2-selenouridine, 5-methylaminomethyl-2-thiouridine, 5-methylaminomethyluridine, 5-methyldihydrouridine, 5-oxyacetic acid-uridine TP, 5-oxyacetic acid-methylester-uridine TP, N1-methyl-pseudo- Uracil, N1-ethyl-pseudo-uracil, uridine 5-oxyacetic acid, uridine 5-oxyacetic acid methyl ester, 3-(3-amino-3-carboxypropyl)-uridine TP, 5-(iso-pentenylaminomethyl)-2-thiouridine TP, 5-(iso-pentenylaminomethyl)-2'-O-methyluridine TP, 5-(iso-pentenylaminomethyl)uridine TP, 5-propynyluracil, α-thio-uridine, 1(aminoalkylamino-carbonylethylenyl)-2(thio)-pseudouracil, 1(aminoalkylaminocarbonylethylenyl)-2(thio)-uridine ... 1(aminocarbonylethylenyl)-2,4-(dithio)pseudouracil, 1(aminoalkylaminocarbonylethylenyl)-4(thio)pseudouracil, 1(aminoalkylaminocarbonylethylenyl)-pseudouracil, 1(aminocarbonylethylenyl)-2(thio)-pseudouracil, 1(aminocarbonylethylenyl)-2,4-(dithio)pseudouracil, 1(aminocarbonylethylenyl)-4(thio)pseudouracil, 1(aminocarbonylethylenyl)-pseudouracil, 1-substituted 2(thio)-pseudouracil, 1-substituted 2,4-(dithio)pseudouracil, 1-substituted 4(thio)pseudouracil, 1-substituted pseudouracil, 1-(aminoalkylamino-carbonylethylenyl)-2-(thio)-pseudouracil, 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine TP, 1-methyl-3-(3-amino-3-carboxypropyl)pseudo-UTP, 1-methyl-pseudo-UTP, 1-ethyl-pseudo-UTP, 2(thio)pseudouracil, 2'-deoxyuridine , 2'fluorouridine, 2-(thio)uracil, 2,4-(dithio)pseudouracil, 2'methyl, 2'amino, 2'azido, 2'fluoro-guanosine, 2'-amino-2'-deoxy-UTP, 2'-azido-2'-deoxy-UTP, 2'-azido-deoxyuridine TP, 2'-O-methylpseudouridine, 2'deoxyuridine, 2'fluorouridine, 2'-deoxy-2'-α-aminouridine TP, 2'-deoxy-2'-α-azidouridine TP, 2-methyl Tyrpseudouridine, 3(3-amino-3-carboxypropyl)uracil, 4(thio)pseudouracil, 4-(thio)pseudouracil, 4-(thio)uracil, 4-thiouracil, 5(1,3-diazolyl-1-alkyl)uracil, 5(2-aminopropyl)uracil, 5(aminoalkyl)uracil, 5(dimethylaminoalkyl)uracil, 5(guanidinium alkyl)uracil, 5(methoxycarbonylmethyl)-2-(thio)uracil, 5(methoxycarbonyl -methyl)uracil, 5(methyl)2(thio)uracil, 5(methyl)2,4(dithio)uracil, 5(methyl)4(thio)uracil, 5(methylaminomethyl)-2(thio)uracil, 5(methylaminomethyl)-2,4(dithio)uracil, 5(methylaminomethyl)-4(thio)uracil, 5(propynyl)uracil, 5(trifluoromethyl)uracil, 5-(2-aminopropyl)uracil, 5-(alkyl)-2-(thio)pseudouracil, 5-(alkyl)-2,4(dithio)pseudouracil, 5-(alkyl)-4(thio)pseudouracil, 5-(alkyl)pseudouracil, 5-(alkyl)uracil, 5-(alkynyl)uracil, 5-(allylamino)uracil, 5-(cyanoalkyl)uracil, 5-(dialkylaminoalkyl)uracil, 5-(dimethylaminoalkyl)uracil, 5-(guanidiniumalkyl)uracil, 5-(halo)uracil, 5-(1,3-diazole-1-alkyl)uracil, 5-(methoxy)uracil, 5-( 5-(methoxycarbonylmethyl)-2-(thio)uracil, 5-(methoxycarbonyl-methyl)uracil, 5-(methyl)2(thio)uracil, 5-(methyl)2,4(dithio)uracil, 5-(methyl)4(thio)uracil, 5-(methyl)-2-(thio)pseudouracil, 5-(methyl)-2,4(dithio)pseudouracil, 5-(methyl)-4(thio)pseudouracil, 5-(methyl)pseudouracil, 5-(methylaminomethyl)-2(thio)uracil, 5-(methylaminomethyl)-2,4(dithio)uracil, 5-(methylaminomethyl)-4-(thio)uracil, 5-(propynyl)uracil, 5-(trifluoromethyl)uracil, 5-aminoallyl-uridine, 5-bromo-uridine, 5-iodo-uridine, 5-uracil, 6(azo)uracil, 6-(azo)uracil, 6-aza-uridine, allyamino-uracil, azauracil, deazauracil, N3(methyl)uracil, pseudo-UTP-1-2-ethanoic acid, pseudouracil, 4-thio-pseudo-U TP, 1-carboxymethyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 1-propynyl-uridine, 1-taurinomethyl-1-methyl-uridine, 1-taurinomethyl-4-thio-uridine, 1-taurinomethyl-pseudouridine, 2-methoxy-4-thio-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine , 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, (±)1-(2-hydroxypropyl)pseudouridine TP, (2R)-1-(2-hydroxypropyl)pseudouridine TP, (2S)-1-(2-hydroxypropyl)pseudouridine TP, (E)-5-(2-bromo-vinyl)ara-uridine TP, (E)- -5-(2-Bromo-vinyl)uridine TP, (Z)-5-(2-Bromo-vinyl)ara-uridine TP, (Z)-5-(2-Bromo-vinyl)uridine TP, 1-(2,2,2-trifluoroethyl)-pseudo-UTP, 1-(2,2,3,3,3-pentafluoropropyl)pseudouridine TP, 1-(2,2-diethoxyethyl)pseudouridine TP, 1-(2,4,6-trimethylbenzyl)pseudouridine TP, 1-(2,4,6-trimethylbenzyl)pseudo-UTP, 1-(2,4,6-trimethyl-phenyl)pseudo-UTP, 1-(2-amino-2-carboxyethyl)pseudo-UTP, 1-(2-amino-ethyl)pseudo-UTP, 1-(2-hydroxyethyl)pseudouridine TP, 1-(2-methoxyethyl)pseudouridine TP, 1-(3,4-bis-trifluoromethoxybenzyl)pseudouridine TP, 1-(3,4-dimethoxybenzyl)pseudouridine TP, 1-(3-amino-3-carboxypropyl)pseudo -UTP, 1-(3-amino-propyl)pseudo-UTP, 1-(3-cyclopropyl-prop-2-ynyl)pseudouridine TP, 1-(4-amino-4-carboxybutyl)pseudo-UTP, 1-(4-amino-benzyl)pseudo-UTP, 1-(4-amino-butyl)pseudo-UTP, 1-(4-amino-phenyl)pseudo-UTP, 1-(4-azidobenzyl)pseudouridine TP, 1-(4-bromobenzyl)pseudouridine TP, 1-(4 1-(4-chlorobenzyl)pseudouridine TP, 1-(4-fluorobenzyl)pseudouridine TP, 1-(4-iodobenzyl)pseudouridine TP, 1-(4-methanesulfonylbenzyl)pseudouridine TP, 1-(4-methoxybenzyl)pseudouridine TP, 1-(4-methoxybenzyl)pseudouridine TP, 1-(4-methoxybenzyl)pseudouridine TP, 1-(4-methoxyphenyl)pseudouridine TP, 1-(4-methylbenzyl)pseudouridine TP, 1-(4-methylbenzyl)pseudouridine TP pseudo-UTP, 1-(4-nitrobenzyl)pseudo-UTP, 1-(4-nitro-benzyl)pseudo-UTP, 1(4-nitro-phenyl)pseudo-UTP, 1-(4-thiomethoxybenzyl)pseudouridine TP, 1-(4-trifluoromethoxybenzyl)pseudouridine TP, 1-(4-trifluoromethylbenzyl)pseudouridine TP, 1-(5-amino-pentyl)pseudo-UTP, 1-(6-amino-hexyl)pseudo-UTP, 1,6-dimethyl-pseudo-UTP, 1-[3-(2-{2-[2-(2-aminoethoxy)-ethoxy]-ethoxy}-ethoxy)-propionyl]pseudouridine TP, 1-{3-[2-(2-aminoethoxy)-ethoxy]-propionyl}pseudouridine TP, 1-acetylpseudouridine TP, 1-alkyl-6-(1-propynyl)-pseudouridine TP, 1-alkyl-6-(2-propynyl)-pseudouridine TP, 1-alkyl-6-allyl-pseudouridine TP, 1-alkyl-6-ethynyl-pseudouridine TP, 1-alkyl-6-homoallyl-pseudouridine TP, 1-alkyl-6-vinyl-pseudouridine TP, 1-allylpseudouridine TP, 1-aminomethyl-pseudouridine TP, 1-benzoylpseudouridine TP, -benzyloxymethylpseudouridine TP, 1-benzyl-pseudo-UTP, 1-biotinyl-PEG2-pseudouridine TP, 1-biotinylpseudouridine TP, 1-butyl-pseudo-UTP, 1-cyanomethylpseudouridine TP, 1-cyclobutylmethyl-pseudo-UTP, 1-cyclobutyl-pseudo-UTP, 1-cycloheptylmethyl-pseudo-UTP, 1-cycloheptyl-pseudo-UTP, 1-cyclohexylmethyl-pseudo-UTP, 1-cyclohexyl-pseudo-UTP, 1-cyclooctylmethyl 1-cyclooctyl-pseudo-UTP, 1-cyclopentylmethyl-pseudo-UTP, 1-cyclopentyl-pseudo-UTP, 1-cyclopropylmethyl-pseudo-UTP, 1-cyclopropyl-pseudo-UTP, 1-ethyl-pseudo-UTP, 1-hexyl-pseudo-UTP, 1-homoallylpseudouridine TP, 1-hydroxymethylpseudouridine TP, 1-isopropyl-pseudo-UTP, 1-Me-2-thio-pseudo-UTP, 1-Me-4-thio-pseudo-UTP, 1-Me-allyl sulfa-thio-pseudo-UTP, 1-methanesulfonylmethylpseudouridine TP, 1-methoxymethylpseudouridine TP, 1-methyl-6-(2,2,2-trifluoroethyl)pseudo-UTP, 1-methyl-6-(4-morpholino)-pseudo-UTP, 1-methyl-6-(4-thiomorpholino)-pseudo-UTP, 1-methyl-6-(substituted phenyl)pseudo-UTP, 1-methyl-6-amino-pseudo-UTP, 1-methyl-6-azido-pseudo-UTP, 1-methyl-6-bromo-pseudo-UTP, 1-methyl-6 -butyl-pseudo-UTP, 1-methyl-6-chloro-pseudo-UTP, 1-methyl-6-cyano-pseudo-UTP, 1-methyl-6-dimethylamino-pseudo-UTP, 1-methyl-6-ethoxy-pseudo-UTP, 1-methyl-6-ethylcarboxylate-pseudo-UTP, 1-methyl-6-ethyl-pseudo-UTP, 1-methyl-6-fluoro-pseudo-UTP, 1-methyl-6-formyl-pseudo-UTP, 1-methyl-6-hydroxyamino-pseudo-UTP, 1-methyl-6-hydroxy-pseudo-UTP,1-methyl-6-iodo-pseudo-UTP, 1-methyl-6-iso-propyl-pseudo-UTP, 1-methyl-6-methoxy-pseudo-UTP, 1-methyl-6-methylamino-pseudo-UTP, 1-methyl-6-phenyl-pseudo-UTP, 1-methyl-6-propyl-pseudo-UTP, 1-methyl-6-tert-butyl-pseudo-UTP, 1-methyl-6-trifluoromethoxy-pseudo-UTP, 1-methyl-6-trifluoromethyl-pseudo-UTP, 1-morpholinomethylpseudouridine TP, 1-pentyl-pseudo-UTP -UTP, 1-phenyl-pseudo-UTP, 1-pivaloylpseudouridine TP, 1-propargylpseudouridine TP, 1-propyl-pseudo-UTP, 1-propynyl-pseudouridine, 1-p-tolyl-pseudo-UTP, 1-tert-butyl-pseudo-UTP, 1-thiomethoxymethylpseudouridine TP, 1-thiomorpholinomethylpseudouridine TP, 1-trifluoroacetylpseudouridine TP, 1-trifluoromethyl-pseudo-UTP, 1-vinylpseudouridine TP, 2,2'-anhydro-uridine TP , 2'-bromo-deoxyuridine TP, 2'-F-5-methyl-2'-deoxy-UTP, 2'-OMe-5-Me-UTP, 2'-OMe-pseudo-UTP, 2'-a-ethynyluridine TP, 2'-a-trifluoromethyluridine TP, 2'-b-ethynyluridine TP, 2'-b-trifluoromethyluridine TP, 2'-deoxy-2',2'-difluorouridine TP, 2'-deoxy-2'-a-mercaptouridine TP, 2'-deoxy-2'-a-thiomethoxyuridine TP, 2'-deoxy-2'-b-aminouridine TP, 2 '-Deoxy-2'-b-azidouridine TP, 2'-deoxy-2'-b-bromouridine TP, 2'-deoxy-2'-b-chlorouridine TP, 2'-deoxy-2'-b-fluorouridine TP, 2'-deoxy-2'-b-iodouridine TP, 2'-deoxy-2'-b-mercaptouridine TP, 2'-deoxy-2'-b-thiomethoxyuridine TP, 2-methoxy-4-thiouridine, 2-methoxyuridine, 2'-O-methyl-5-(1-propynyl)uridine TP, 3-alkyl-pseudo-UTP, 4'-azidouridine TP,4'-Carbocyclic Uridine TP, 4'-Ethynyl Uridine TP, 5-(1-Propynyl)ara-uridine TP, 5-(2-Furanyl)uridine TP, 5-Cyanouridine TP, 5-Dimethylaminouridine TP, 5'-Homo-uridine TP, 5-Iodo-2'-fluoro-deoxyuridine TP, 5-Phenylethynyluridine TP, 5-Trideuteromethyl-6-deuterouridine TP, 5-Trifluoromethyl-uridine TP, 5-Vinylarauridine TP, 6-(2,2,2-Trifluoroethyl)-pseudo-UTP, 6-(4-morpholino)-pseudo-UTP, 6-(4-thiomorpholino)-pseudo-UTP, 6-(substituted-phenyl)-pseudo-UTP, 6-amino-pseudo-UTP, 6-azido-pseudo-UTP, 6-bromo-pseudo-UTP, 6-butyl-pseudo-UTP, 6-chloro-pseudo-UTP, 6-cyano-pseudo-UTP, 6-dimethylamino-pseudo-UTP, 6-ethoxy-pseudo-UTP, 6-ethylcarboxylate-pseudo-UTP, 6-ethyl-pseudo-UTP, 6-fluoro-pseudo-UTP -UTP, 6-formyl-pseudo-UTP, 6-hydroxyamino-pseudo-UTP, 6-hydroxy-pseudo-UTP, 6-iodo-pseudo-UTP, 6-isopropyl-pseudo-UTP, 6-methoxy-pseudo-UTP, 6-methylamino-pseudo-UTP, 6-methyl-pseudo-UTP, 6-phenyl-pseudo-UTP, 6-phenyl-pseudo-UTP, 6-propyl-pseudo-UTP, 6-tert-butyl-pseudo-UTP, 6-trifluoromethoxy-pseudo-UTP, 6-trifluoro methyl-pseudo-UTP, alpha-thio-pseudo-UTP, pseudouridine 1-(4-methylbenzenesulfonic acid) TP, pseudouridine 1-(4-methylbenzoic acid) TP, pseudouridine TP 1-[3-(2-ethoxy)]propionic acid, pseudouridine TP 1-[3-{2-(2-[2-(2-ethoxy)-ethoxy]-ethoxy)-ethoxy}]propionic acid, pseudouridine TP 1-[3-{2-(2-[2-{2(2-ethoxy)-ethoxy}-ethoxy]-ethoxy)-ethoxy}]propionic acid,Pseudouridine TP1-[3-{2-(2-[2-ethoxy]-ethoxy)-ethoxy}]propionic acid, pseudouridine TP1-[3-{2-(2-ethoxy)-ethoxy}]propionic acid, pseudouridine TP1-methylphosphonic acid, pseudouridine TP1-methylphosphonic acid diethyl ester, pseudo-UTP-N1-3-propionic acid, pseudo-UTP-N1-4-butanoic acid, pseudo-UTP-N1-5-pentanoic acid, pseudo-UTP-N1-6-hexanoic acid, pseudo-UTP-N1-7-heptanoic acid, pseudo-UTP- N1-methyl-p-benzoic acid, pseudo-UTP-N1-p-benzoic acid, wybutosin, hydroxywybutosin, isowybutosin, peroxywybutosin, intermediate hydroxywybutosin, 4-demethylwybutosin, 2,6-(diamino)purine, 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl; 1,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 1,3,5-(triaza)-2,6-(dioxa-naphthiazin-1-yl Talen, 2(amino)purine, 2,4,5-(trimethyl)phenyl, 2'methyl, 2'amino, 2'azido, 2'fluro-cytidine, 2'methyl, 2'amino, 2'azido, 2'fluro-adenine, 2'methyl, 2'amino, 2'azido, 2'fluro-uridine, 2'-amino-2'-deoxyribose, 2-amino-6-chloro-purine, 2-aza-inosinyl, 2'-azido-2'-deoxyribose, 2'fluoro-2'-deoxyribose, 2'-fluoro-modified base, 2'-O-methyl-ribose, 2-oxo-7-aminopyridopyrimidin benzoin-3-yl, 2-oxo-pyridopyrimidin-3-yl, 2-pyridinone, 3-nitropyrrole, 3-(methyl)-7-(propynyl)isocarbostyril, 3-(methyl)isocarbostyril, 4-(fluoro)-6-(methyl)benzimidazole, 4-(methyl)benzimidazole, 4-(methyl)indolyl, 4,6-(dimethyl)indolyl, 5-nitroindole, 5-substituted pyrimidine, 5-(methyl)isocarbostyril, 5-nitroindole, 6-(aza)pyrimidine, 6-(azo)thymine, 6-(methyl)-7-(aza)indolyl,6-Chloro-purine, 6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl phenoxazin-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-(aza)indolyl, 7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazinyl, 7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3- (Aza)-phenoxazin-1-yl, 7-(guanidinium alkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-(guanidinium alkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-(guanidinium alkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-(guanidinium alkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-(propynyl)isocarbostyril, 7-(propynyl)isocarbostyril, propynyl-7-(aza)indolyl, 7- deaza-inosinyl, 7-substituted 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-substituted 1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 9-(methyl)-imidizopyridinyl, aminoindolyl, anthracenyl, bis-ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, bis-ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, difluorotolyl, hypoxanthine, imidizopyridinyl, inosinyl, isocarbostyril,Isoguanisine, N2-substituted purines, N6-methyl-2-amino-purine, N6-substituted purines, N-alkylated derivatives, naphthalenyl, nitrobenzimidazolyl, nitroimidazolyl, nitroindazolyl, nitropyrazolyl, nubularine, O6-substituted purines, O-alkylated derivatives, ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, ortho-substituted-, 6-Phenyl-pyrrolo-pyrimidin-2-one-3-yl, oxoformycin TP, para-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, para-substituted-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, pentacenyl, phenanthracenyl, phenyl, propynyl-7-(aza)indolyl, pyrenyl, pyridopyrimidin-3-yl, pyridopyrimidin-3-yl, 2-oxo-7-amino No-pyridopyrimidin-3-yl, pyrrolo-pyrimidin-2-one-3-yl, pyrrolopyrimidinyl, pyrrolopyridinyl, stilbenzyl, substituted 1,2,4-triazoles, tetracenyl, tubercidine, xanthine, xanthosine-5'-TP, 2-thio-zebularine, 5-aza-2-thio-zebularine, 7-deaza-2-amino-purine, pyridin-4-one ribonucleoside, 2-amino-riboside-TP, formycin A TP, formycin B TP, Pyrrolosine TP, 2'-OH-ara-adenosine TP, 2'-OH-ara-cytidine TP, 2'-OH-ara-uridine TP, 2'-OH-ara-guanosine TP, 5-(2-carbomethoxyvinyl)uridine TP, and N6-(19-amino-pentaoxanonadecyl)adenosine TP.

[0188] In some embodiments, a polynucleotide (e.g., an RNA polynucleotide such as an mRNA polynucleotide) comprises a combination of at least two (e.g., two, three, four, or more) of the foregoing modified nucleobases.

[0189] In some embodiments, modified nucleobases in a polynucleotide (e.g., an RNA polynucleotide such as an mRNA polynucleotide) are pseudouridine (ψ), 2-thiouridine (s2U), 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methoxyuridine, 2'-O-methyluridine, 1-methyl-pseudouridine (m1ψ), 1-ethyl-pseudouridine (e1ψ), ...methyluridine, 5-methyl- cytidine (mo5U), 5-methyl-cytidine (m5C), α-thio-guanosine, α-thio-adenosine, 5-cyanouridine, 4'-thiouridine, 7-deaza-adenine, 1-methyl-adenosine (m1A), 2-methyl-adenine (m2A), N6-methyl-adenosine (m6A), and 2,6-diaminopurine, (I), 1-methyl-inosine (m1I), wyosine (imG), methylwyosine (mimG), 7-deaza-adenine (m1A), 2-methyl-adenine (m2A), N6-methyl-adenosine (m6A), and 2,6-diaminopurine (I). Aza-guanosine, 7-cyano-7-deaza-guanosine (preQ0), 7-aminomethyl-7-deaza-guanosine (preQ1), 7-methyl-guanosine (m7G), 1-methyl-guanosine (m1G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 2,8-dimethyladenosine, 2-geranylthiouridine, 2-lysidine, 2-selenouridine, 3-(3-amino-3-carboxypropyl)-5,6-Dihydrouridine, 3-(3-amino-3-carboxypropyl)pseudouridine, 3-methylpseudouridine, 5-(carboxyhydroxymethyl)-2'-O-methyluridine methyl ester, 5-aminomethyl-2-geranylthiouridine, 5-aminomethyl-2-selenouridine, 5-aminomethyluridine, 5-carbamoylhydroxymethyluridine, 5-carbamoylmethyl-2-thiouridine, 5-carboxymethyl-2-thiouridine, 5-carboxymethylaminomethyl-2-geranylthiouridine, 5-carboxymethylaminomethyl-2-selenouridine, 5-cyanomethyluridine, 5-hydroxycytidine, 5-methylaminomethyl-2-geranylthiouridine, 7 In some embodiments, the at least one chemically modified nucleoside is selected from the group consisting of 5-aminocarboxypropyl-demethylwyosine, 7-aminocarboxypropylwyosine, 7-aminocarboxypropylwyosine methyl ester, 8-methyladenosine, N4,N4-dimethylcytidine, N6-formyladenosine, N6-hydroxymethyladenosine, agmatidine, cyclic N6-threonylcarbamoyladenosine, glutamyl-qeuosine, methylated intermediate hydroxywyosine, N4,N4,2'-O-trimethylcytidine, geranylated 5-methylaminomethyl-2-thiouridine, geranylated 5-carboxymethylaminomethyl-2-thiouridine, Q base, preQ0 base, preQ1 base, and combinations of two or more thereof. In some embodiments, the at least one chemically modified nucleoside is selected from the group consisting of pseudouridine, 1-methyl-pseudouridine, 1-ethyl-pseudouridine, 5-methylcytosine, 5-methoxyuridine, and combinations thereof. In some embodiments, a polyribonucleotide (e.g., an RNA polyribonucleotide, such as an mRNA polyribonucleotide) comprises a combination of at least two (e.g., two, three, four, or more) of the aforementioned modified nucleobases. In some embodiments, a polynucleotide (e.g., an RNA polynucleotide, such as an mRNA polynucleotide) comprises a combination of at least two (e.g., two, three, four, or more) of the aforementioned modified nucleobases.

[0190] In some embodiments, modified nucleobases in a polynucleotide (e.g., an RNA polynucleotide such as an mRNA polynucleotide) are selected from the group consisting of 1-methyl-pseudouridine (m1ψ), 1-ethyl-pseudouridine (e1ψ), 5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C), pseudouridine (ψ), α-thio-guanosine, and α-thio-adenosine. In some embodiments, a polyribonucleotide comprises a combination of at least two (e.g., two, three, four, or more) of the foregoing modified nucleobases, including, but not limited to, chemical modifications.

[0191] In some embodiments, a polynucleotide (e.g., an RNA polynucleotide such as an mRNA polynucleotide) comprises pseudouridine (ψ) and 5-methyl-cytidine (mC). In some embodiments, a polyribonucleotide (e.g., an RNA such as an mRNA) comprises 1-methyl-pseudouridine (mψ). In some embodiments, a polyribonucleotide (e.g., an RNA such as an mRNA) comprises 1-ethyl-pseudouridine (eψ). In some embodiments, a polyribonucleotide (e.g., an RNA such as an mRNA) comprises 1-methyl-pseudouridine (mψ) and 5-methyl-cytidine (mC). In some embodiments, a polyribonucleotide (e.g., an RNA such as an mRNA) comprises 1-ethyl-pseudouridine (eψ) and 5-methyl-cytidine (mC). In some embodiments, a polyribonucleotide (e.g., an RNA such as an mRNA) comprises 2-thiouridine (sU). In some embodiments, polyribonucleotides (e.g., RNA such as mRNA) comprise 2-thiouridine and 5-methyl-cytidine (m5C). In some embodiments, polyribonucleotides (e.g., RNA such as mRNA) comprise methoxy-uridine (mo5U). In some embodiments, polyribonucleotides (e.g., RNA such as mRNA) comprise 5-methoxy-uridine (mo5U) and 5-methyl-cytidine (m5C). In some embodiments, polyribonucleotides (e.g., RNA such as mRNA) comprise 2'-O-methyluridine. In some embodiments, polyribonucleotides (e.g., RNA such as mRNA) comprise 2'-O-methyluridine and 5-methyl-cytidine (m5C). In some embodiments, polyribonucleotides (e.g., RNA such as mRNA) comprise N6-methyl-adenosine (m6A). In some embodiments, the polyribonucleotide (eg, RNA such as mRNA) comprises N6-methyl-adenosine (m6A) and 5-methyl-cytidine (m5C).

[0192] In some embodiments, polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) are uniformly modified to incorporate a particular modification (e.g., complete modification, modification across the entire sequence). For example, polynucleotides can be uniformly modified with 1-methyl-pseudouridine, meaning that all of the uridine residues in the mRNA sequence are replaced with 1-methyl-pseudouridine. Similarly, polynucleotides can be uniformly modified so that any type of nucleoside residue is present in the sequence by replacement with modified residues such as those described above.

[0193] Examples of nucleobases and nucleosides having modified cytosines include N4-acetyl-cytidine (ac4C), 5-methyl-cytidine (m5C), 5-halo-cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm5C), 1-methyl-pseudoisocytidine, 2-thio-cytidine (s2C), and 2-thio-5-methyl-cytidine.

[0194] In some embodiments, the modified nucleobase is a modified uridine. Examples of nucleobases and nucleosides having modified uridines include 1-methyl-pseudouridine (m1ψ), 1-ethyl-pseudouridine (e1ψ), 5-methoxyuridine, 2-thiouridine, 5-cyanouridine, 2'-O-methyluridine, and 4'-thiouridine.

[0195] In some embodiments, the modified nucleobase is a modified adenine. Examples of nucleobases and nucleosides having modified adenines include 7-deaza-adenine, 1-methyl-adenosine (m1A), 2-methyl-adenine (m2A), and N6-methyl-adenosine (m6A).

[0196] In some embodiments, the modified nucleobase is a modified guanine. Examples of nucleobases and nucleosides having modified guanine include inosine (I), 1-methyl-inosine (m1I), wyosine (imG), methylwyosine (mimG), 7-deaza-guanosine, 7-cyano-7-deaza-guanosine (preQ0), 7-aminomethyl-7-deaza-guanosine (preQ1), 7-methyl-guanosine (m7G), 1-methyl-guanosine (m1G), 8-oxo-guanosine, and 7-methyl-8-oxo-guanosine.

[0197] The polynucleotides of the present disclosure may be partially modified or completely modified throughout the entire length of the molecule. For example, one or more or all or a given type of nucleotide (e.g., purines or pyrimidines, or one or more or all of A, G, U, C) may be uniformly modified in a polynucleotide of the present disclosure or in a given sequence region thereof (e.g., an mRNA including a polyA tail or an mRNA excluding a polyA tail). In some embodiments, all nucleotides X in a polynucleotide of the present disclosure (or in a given sequence region thereof) are modified nucleotides, and X can be any one of nucleotide A, nucleotide G, nucleotide U, nucleotide C, or any one of the following combinations: A+G, A+U, A+C, G+U, G+C, U+C, A+G+U, A+G+C, G+U+C, or A+G+C.

[0198] Polynucleotides may contain modified nucleotides (with respect to the total nucleotide content or with respect to any one or more types of nucleotides, i.e., A, G, U, or C) from about 1% to about 100% or any intervening percentage (e.g., 1% to 20%, 1% to 25%, 1% to 50%, 1% to 60%, 1% to 70%, 1% to 80%, 1% to 90%, 1% to 95%, 10% to 20%, 10% to 25%, 10% to 50%, 10% to 60%, 10% to 70%, 10% to 80%, 10 ...100%, 10% to 100%, 10% to 100%, 10% to 100%, 10% to 100%, 10% to 100%, 10% to 100%, 10% to 100%, It may include 0%, 10% to 95%, 10% to 100%, 20% to 25%, 20% to 50%, 20% to 60%, 20% to 70%, 20% to 80%, 20% to 90%, 20% to 95%, 20% to 100%, 50% to 60%, 50% to 70%, 50% to 80%, 50% to 90%, 50% to 95%, 50% to 100%, 70% to 80%, 70% to 90%, 70% to 95%, 70% to 100%, 80% to 90%, 80% to 95%, 80% to 100%, 90% to 95%, 90% to 100%, and 95% to 100%. It is understood that any remaining percentage corresponds to the presence of unmodified A, G, U, or C.

[0199] A polynucleotide can contain modified nucleotides from as little as 1% to as much as 100% or any intervening percentage, such as at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, or at least 90%. For example, a polynucleotide can contain modified pyrimidines, such as modified uracil or modified cytosine. In some embodiments, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90%, or 100% of the uracils in a polynucleotide are replaced with modified uracils (e.g., 5-substituted uracils). The modified uracils can be replaced by a compound with a single unique structure, or by multiple compounds with different structures (e.g., two, three, four, or more unique structures). In some embodiments, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90%, or 100% of the cytosines in the polynucleotide are replaced with modified cytosines (e.g., 5-substituted cytosines). The modified cytosines can be replaced by a compound having a single unique structure, or by multiple compounds having different structures (e.g., two, three, four, or more unique structures).

[0200] Thus, in some embodiments, the RNA vaccine comprises a 5'UTR element, optionally a codon-optimized open reading frame, and a 3'UTR element, a poly(A) sequence and / or a polyadenylation signal, and the RNA is not chemically modified.

[0201] In some embodiments, the modified nucleobase is a modified uracil. Examples of nucleobases and nucleosides having modified uracil include pseudouridine (ψ), pyridin-4-one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s 2U), 4-thio-uridine (s 4 U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho 5 U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), 3-methyl-uridine (m 3 U), 5-methoxy-uridine (mo 5 U), uridine 5-hydroxyacetic acid (cmo 5 U), uridine 5-hydroxyacetic acid methyl ester (mcmo 5 U), 5-carboxymethyl-uridine (cm 5 U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm 5 U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm 5 U), 5-methoxycarbonylmethyl-uridine (mcm 5 U), 5-methoxycarbonylmethyl-2-thiouridine (mcm 5 s 2 U), 5-aminomethyl-2-thio-uridine (nm 5 s 2 U), 5-methylaminomethyl-uridine (mnm 5 U), 5-methylaminomethyl-2-thiouridine (mnm 5 s 2 U), 5-methylaminomethyl-2-seleno-uridine (mnm 5 se 2 U), 5-carbamoylmethyl-uridine (ncm 5 U), 5-carboxymethylaminomethyl-uridine (cmnm 5 U), 5-carboxymethylaminomethyl-2-thiouridine (cmnm 5 s 2 U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (τm 5 U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thiouridine (τm 5 s 2U), 1-taurinomethyl-4-thio-pseudouridine, 5-methyl-uridine (m 5 U, i.e., having the nucleobase deoxythymine), 1-methyl-pseudouridine (m 1 ψ), 1-ethyl-pseudouridine (e1ψ), 5-methyl-2-thio-uridine (m 5 s 2 U), 1-methyl-4-thio-pseudouridine (m 1 s 4 ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m 3 ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m 5 D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp 3 U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp 3 ψ), 5-(isopentenylaminomethyl)uridine (inm 5 U), 5-(isopentenylaminomethyl)-2-thiouridine (inm 5 s 2 U), α-thio-uridine, 2'-O-methyl-uridine (Um), 5, 2'-O-dimethyl-uridine (m 5 Um), 2'-O-methyl-pseudouridine (ψm), 2-thio-2'-O-methyl-uridine (s 2 Um), 5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcm 5 Um), 5-carbamoylmethyl-2'-O-methyl-uridine (ncm 5 Um), 5-carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm 5Um), 3,2'-O-dimethyl-uridine (m 3 Um), and 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm 5 Um), 1-thio-uridine, deoxythymidine, 2'-F-ara-uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5-(2-carbomethoxyvinyl)uridine, and 5-[3-(1-E-propenylamino)]uridine.

[0202] In some embodiments, the modified nucleobase is a modified cytosine. Examples of nucleobases and nucleosides having modified cytosines include 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine (m 3 C), N4-acetyl-cytidine (ac 4 C), 5-formyl-cytidine (f 5 C), N4-methyl-cytidine (m 4 C), 5-methyl-cytidine (m 5 C), 5-halo-cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm 5 C), 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine (s 2 C), 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, lysidine (k2C), α-thio-cytidine, 2'-O-methyl-cytidine (Cm), 5,2'-O-dimethyl-cytidine (m 5 Cm), N4-acetyl-2'-O-methyl-cytidine (ac 4 Cm), N4, 2'-O-dimethyl-cytidine (m 4Cm), 5-formyl-2'-O-methyl-cytidine (f 5 Cm), N4,N4,2'-O-trimethyl-cytidine (m 4 2Cm), 1-thio-cytidine, 2'-F-ara-cytidine, 2'-F-cytidine, and 2'-OH-ara-cytidine.

[0203] In some embodiments, the modified nucleobase is a modified adenine. Examples of nucleobases and nucleosides having modified adenines include 2-amino-purine, 2,6-diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenosine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyl-adenosine (m 1 A), 2-methyl-adenine (m 2 A), N6-methyl-adenosine (m 6 A), 2-methylthio-N6-methyl-adenosine (ms 2 m 6 A), N6-isopentenyl-adenosine (i 6 A), 2-methylthio-N6-isopentenyl-adenosine (ms 2 i 6 A), N6-(cis-hydroxyisopentenyl)adenosine (io 6 A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine (ms 2 io 6 A), N6-glycinylcarbamoyl-adenosine (g 6 A), N6-threonylcarbamoyl-adenosine (t 6 A), N6-methyl-N6-threonylcarbamoyl-adenosine (m 6 t 6 A), 2-methylthio-N6-threonylcarbamoyl-adenosine (ms 2 g 6A), N6,N6-dimethyl-adenosine (m 6 2A), N6-hydroxynorvalylcarbamoyl-adenosine (hn 6 A), 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenosine (ms 2 hn 6 A), N6-acetyl-adenosine (ac 6 A), 7-methyl-adenine, 2-methylthio-adenine, 2-methoxy-adenine, α-thio-adenosine, 2'-O-methyl-adenosine (Am), N6,2'-O-dimethyl-adenosine (m 6 Am), N6,N6,2'-O-trimethyl-adenosine (m 6 2Am), 1,2'-O-dimethyl-adenosine (m 1 Am), 2'-O-ribosyladenosine (phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1-thio-adenosine, 8-azido-adenosine, 2'-F-ara-adenosine, 2'-F-adenosine, 2'-OH-ara-adenosine, and N6-(19-amino-pentaoxanonadecyl)-adenosine.

[0204] In some embodiments, the modified nucleobase is a modified guanine. Examples of nucleobases and nucleosides having modified guanine include inosine (I), 1-methyl-inosine (m), 1 I), wyosine (imG), methylwyosine (mimG), 4-demethyl-wyosine (imG-14), isowyosine (imG2), wyobutosine (yW), peroxywyobutosine (o2yW), hydroxywyobutosine (OhyW), intermediate hydroxywyobutosine (OhyW) * ), 7-deaza-guanosine, queuosine (Q), epoxyqueuosine (oQ), galactosyl-queuosine (galQ), mannosyl-queuosine (manQ), 7-cyano-7-deaza-guanosine (preQ0), 7-aminomethyl-7-deaza-guanosine (preQ1), archaeosine (G +), 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine (m 7 G), 6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine, 1-methyl-guanosine (m 1 G), N2-methyl-guanosine (m 2 G), N2,N2-dimethyl-guanosine (m 2 2G), N2,7-dimethyl-guanosine (m 2,7 G), N2,N2,7-dimethyl-guanosine (m 2,2,7 G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine, α-thio-guanosine, 2'-O-methyl-guanosine (Gm), N2-methyl-2'-O-methyl-guanosine (m 2 Gm), N2,N2-dimethyl-2'-O-methyl-guanosine (m 2 2Gm), 1-methyl-2'-O-methyl-guanosine (m 1 Gm), N2,7-dimethyl-2'-O-methyl-guanosine (m 2,7 Gm), 2'-O-methyl-inosine (Im), 1,2'-O-dimethyl-inosine (m 1 Im), 2'-O-ribosylguanosine(phosphate) (Gr(p)), 1-thio-guanosine, O6-methyl-guanosine, 2'-F-ara-guanosine, and 2'-F-guanosine.

[0205] In vitro transcription of RNA (e.g., mRNA) The cancer vaccines of the present disclosure include at least one RNA polynucleotide, such as an mRNA (e.g., a modified mRNA). The mRNA is transcribed in vitro, for example, from a template DNA; such a template DNA is referred to as an "in vitro transcription template." In some embodiments, the in vitro transcription template encodes a 5' untranslated (UTR) region, includes an open reading frame, and encodes a 3' UTR and a polyA tail. The specific nucleic acid sequence composition and length of the in vitro transcription template will depend on the mRNA encoded by the template.

[0206] "5' untranslated region" (UTR) refers to the region in an mRNA that is located immediately upstream (i.e., 5') of the start codon (i.e., the first codon in an mRNA transcript to be translated by ribosomes) and does not encode a polypeptide.

[0207] "3' untranslated region" (UTR) refers to the region in an mRNA that is located immediately downstream (i.e., 3') of the stop codon (i.e., the codon that conveys the information for terminating translation in an mRNA transcript) and does not encode a polypeptide.

[0208] An "open reading frame" is a contiguous stretch of DNA beginning with a start codon (e.g., methionine (ATG)) and ending with a stop codon (e.g., TAA, TAG, or TGA) that encodes a polypeptide.

[0209] A "poly A tail" is a region located downstream in an mRNA (e.g., immediately downstream (i.e., 3') of the 3' UTR) and containing multiple consecutive adenosine monophosphates. A poly A tail can contain 10 to 300 adenosine monophosphates. For example, a poly A tail can contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 adenosine monophosphates. In some embodiments, a poly A tail contains 50 to 250 adenosine monophosphates. In biologically relevant situations (e.g., in cells, in vivo), poly(A) tails function to protect mRNA from enzymatic degradation, e.g., in the cytoplasm, and aid in the termination of transcription, transport of mRNA from the nucleus, and translation.

[0210] In some embodiments, the polynucleotide comprises 200 to 3,000 nucleotides. For example, the polynucleotide may comprise 200 to 500, 200 to 1,000, 200 to 1,500, 200 to 3,000, 500 to 1,000, 500 to 1,500, 500 to 2,000, 500 to 3,000, 1,000 to 1,500, 1,000 to 2,000, 1,000 to 3,000, 1,500 to 3,000, or 2,000 to 3,000 nucleotides.

[0211] In another aspect, the present invention relates to a method for preparing an mRNA cancer vaccine by IVT. In vitro transcription (IVT) allows for the template-guided synthesis of RNA molecules of almost any sequence. The size range of RNA molecules that can be synthesized using IVT ranges from short oligonucleotides to long nucleic acid polymers of several thousand bases. IVT allows for the synthesis of large quantities (e.g., microgram to milligram amounts) of RNA transcripts (Beckert et al., Synthesis of RNA by in vitro transcription, Methods Mol Biol. 703:29-41 (2011); Rio et al., RNA: A Laboratory Manual. Cold Spring Harbor: Cold Spring Harbor Laboratory Press, 2011, 205-220; Cooper, Geoffery M., The Cell: A Molecular Approach. 4th ed. Washington DC: ASM Press, 2007, 262-299). Generally, IVT utilizes a DNA template characterized by a promoter sequence located upstream of the sequence of interest. The promoter sequence is most commonly of bacteriophage origin (e.g., T7, T3, or SP6 promoter sequence), but many other promoter sequences, including those designed de novo, are also acceptable. Typically, transcription of a DNA template is best achieved by using an RNA polymerase that corresponds to a specific bacteriophage promoter sequence. Examples of RNA polymerases include, but are not limited to, T7 RNA polymerase, T3 RNA polymerase, or SP6 RNA polymerase, among others. IVT generally initiates on double-stranded DNA, but can also proceed on a single strand.

[0212] It should be understood that mRNA vaccines of the present disclosure, such as mRNA encoding cancer antigens, may be prepared using any suitable synthesis method. For example, in some embodiments, mRNA vaccines of the present disclosure are prepared using IVT from a single-stranded bottom-strand DNA template and a complementary oligonucleotide that serves as a promoter. The single-stranded bottom-strand DNA can serve as a DNA template for in vitro transcription of RNA and may be obtained, for example, from a plasmid, a PCR product, or chemical synthesis. In some embodiments, the single-stranded bottom-strand DNA is linearized from a circular template. The single-stranded bottom-strand DNA template generally contains a promoter sequence, such as a bacteriophage promoter sequence, to facilitate IVT. Methods for preparing RNA using single-stranded bottom-strand DNA and a top-strand that is a promoter-complementary oligonucleotide are known in the art. In an exemplary method, but not limited to, a bottom strand of DNA template is annealed to a top strand of a promoter-complementary oligonucleotide (e.g., a T7 promoter-complementary oligonucleotide, a T3 promoter-complementary oligonucleotide, or an SP6 promoter-complementary oligonucleotide), and then IVT is performed using an RNA polymerase corresponding to the promoter sequence, such as, for example, T7 RNA polymerase, T3 RNA polymerase, or SP6 RNA polymerase.

[0213] IVT can also be performed using a double-stranded DNA template. For example, in some embodiments, a double-stranded DNA template is prepared by extending a complementary oligonucleotide to generate a complementary DNA strand using strand extension techniques available in the art. In some embodiments, a single-stranded bottom-strand DNA template containing a promoter sequence and a sequence encoding one or more epitopes of interest is annealed to a top-strand promoter-complementary oligonucleotide and subjected to a PCR-like process to extend the top strand and generate a double-stranded DNA template. Alternatively, or in addition, a top-strand DNA that is complementary to the bottom-strand promoter sequence and contains a sequence complementary to a sequence encoding one or more epitopes of interest is annealed to a bottom-strand promoter oligonucleotide and subjected to a PCR-like process to extend the bottom strand and generate a double-stranded DNA template. In some embodiments, the number of PCR-like cycles ranges from 1 to 20 cycles, e.g., 3 to 10 cycles. In some embodiments, the double-stranded DNA template is synthesized, in whole or in part, by chemical synthesis. The double-stranded DNA template can be subjected to in vitro transcription as described herein.

[0214] In another embodiment, the mRNA vaccines of the present disclosure, such as mRNA encoding a cancer antigen, may be prepared using two DNA strands whose overlapping sequences are complementary throughout, leaving single-stranded overhangs (i.e., sticky ends) when the complementary portions anneal. These single-stranded overhangs can be made double-stranded by extending them using the other strand as a template, thereby generating double-stranded DNA. In some cases, this primer extension method can lengthen the ORF to be incorporated into the template DNA sequence, for example, compared to the size incorporated into the template DNA sequence obtained by top-strand DNA synthesis. In the primer extension method, a portion of the 3' end of the first strand (5' to 3' direction) is complementary to a portion of the 3' end of the second strand (3' to 5' direction). In some such embodiments, the single-stranded first-strand DNA may include a promoter (e.g., T7, T3, or SP6) sequence, optionally a 5'-UTR, and part or all of the ORF (e.g., part of the 5' end of the ORF). In some embodiments, the single-stranded second-strand DNA may include a sequence complementary to part or all of the ORF (e.g., a portion complementary to the 3' end of the ORF), and optionally a 3'-UTR, a termination sequence, and / or a poly(A) tail. In methods for preparing RNA using two synthetic DNA strands, annealing of the two strands with overlapping complementary portions may be performed, followed by primer extension using one or more PCR-like cycles to extend the strands and generate a double-stranded DNA template. In some embodiments, the number of PCR-like cycles ranges from 1 to 20 cycles, e.g., 3 to 10 cycles. Such double-stranded DNA may be subjected to in vitro transcription as described herein.

[0215] In another aspect, mRNA vaccines of the present disclosure, e.g., mRNA encoding a cancer antigen, may be prepared using synthetic double-stranded linear DNA molecules, such as gBlocks® (Integrated DNA Technologies, Coralville, Iowa), as double-stranded DNA templates. The advantage of such synthetic double-stranded linear DNA molecules is that they provide longer templates from which mRNA can be generated. For example, gBlocks® may range in size from 45 to 1000 (e.g., 125 to 750 nucleotides). In some embodiments, the synthetic double-stranded linear DNA template comprises a full-length 5'-UTR, a full-length 3'-UTR, or both. The full-length 5'-UTR may be up to 100 nucleotides in length, e.g., about 40 to 60 nucleotides in length. The full-length 3'-UTR may be up to 300 nucleotides in length, e.g., about 100 to 150 nucleotides in length.

[0216] To facilitate the generation of longer constructs, two or more double-stranded linear DNA molecules and / or gene fragments designed to have overlapping sequences on the 3' strand may be assembled together using methods known in the art, such as using a mesophilic exonuclease to cleave bases from the 5' ends of such double-stranded DNA fragments, followed by annealing of the newly formed complementary single-stranded 3' ends, polymerase-dependent extension to fill any single-stranded gaps, and finally covalently joining the DNA segments with DNA ligase, in the Gibson Assembly™ method (Synthetic Genomics, Inc., La Jolla, CA).

[0217] In another embodiment, mRNA vaccines of the present disclosure, such as mRNA encoding a cancer antigen, may be prepared using chemical synthesis of RNA. The method involves annealing a first polynucleotide containing an open reading frame encoding a polypeptide and a second polynucleotide containing a 5'-UTR to complementary polynucleotides complexed to a solid support. The 3' end of the second polynucleotide is then ligated to the 5' end of the first polynucleotide under appropriate conditions. Suitable conditions include the use of a DNA ligase. The ligation reaction generates a first ligation product. The 3' end of a third polynucleotide containing a 3'-UTR is then ligated to the 3' end of the first ligation product under appropriate conditions. Suitable conditions for the second ligation reaction include the use of an RNA ligase. The second ligation reaction generates a second ligation product. The second ligation product is then released from the solid support to generate an mRNA encoding the polypeptide of interest. In some embodiments, the mRNA is between 30 and 1000 nucleotides.

[0218] An mRNA encoding a polypeptide of interest may be prepared by ligating a first polynucleotide containing an open reading frame encoding the polypeptide and a second polynucleotide containing a 3'-UTR to complementary polynucleotides complexed to a solid support. The 5'-end of the second polynucleotide is ligated to the 3'-end of the first polynucleotide under suitable conditions, including the use of a DNA ligase. A first ligation product is generated by the method. A third polynucleotide containing a 5'-UTR is ligated to the first ligation product under suitable conditions, including the use of an RNA ligase, such as T4 RNA. The second ligation product is released from the solid support to generate an mRNA encoding the polypeptide of interest.

[0219] In some embodiments, the first polynucleotide is characterized by a 5'-triphosphate and a 3'-OH. In yet other embodiments, the second polynucleotide comprises a 3'-OH. In yet other embodiments, the third polynucleotide comprises a 5'-triphosphate and a 3'-OH. The second polynucleotide may also comprise a 5'-cap structure. The method may further comprise a step of ligating a fourth polynucleotide comprising a poly-A region to the 3' end of the third polynucleotide. The fourth polynucleotide may comprise a 5'-triphosphate.

[0220] The method may or may not include reverse-phase purification. The method may also include a wash step, in which the solid support is washed to remove unreacted polynucleotides. The solid support may be, for example, a capture resin. In some embodiments, the method includes purification by dT.

[0221] According to the present disclosure, the template DNA encoding the mRNA vaccine of the present disclosure comprises an open reading frame (ORF) encoding one or more cancer epitopes. In some embodiments, the template DNA comprises an ORF of up to 1000 nucleotides, e.g., about 10-350 nucleotides, about 30-300 nucleotides, or about 50-250 nucleotides. In some embodiments, the template DNA comprises an ORF of about 150 nucleotides. In some embodiments, the template DNA comprises an ORF of about 200 nucleotides.

[0222] In some embodiments, IVT transcripts are purified from components of the IVT reaction mixture after the reaction has occurred. For example, the crude IVT mixture may be treated with RNase-free DNase to digest the original template. mRNA can be purified using methods known in the art, including, but not limited to, precipitation using organic solvents or column-based purification methods. Commercially available kits are available for purifying RNA, such as the MEGACLEAR™ kit (Ambion, Austin, TX). mRNA can be quantified using methods known in the art, including, but not limited to, using commercially available instruments such as the NanoDrop. Purified mRNA can be analyzed, for example, by agarose gel electrophoresis to confirm the RNA is the correct size and / or to confirm that the RNA has not been degraded.

[0223] In addition to other structural features such as a 5'-cap structure or a 3'-poly(A) tail, the template DNA may contain one or more stabilizing elements, including, but not limited to, a 5'-terminal untranslated region (UTR) (5'UTR) and / or a 3'-terminal untranslated region (UTR) (3'UTR). In some embodiments, the template DNA contains a 5'-UTR of about 1-30 nucleotides, e.g., about 5-25 nucleotides or about 10-20 nucleotides. In some embodiments, the template DNA contains a 5'-UTR of 13 nucleotides. In some embodiments, the template DNA does not contain a 5'-UTR. In some embodiments, the template DNA contains a 3'-UTR of about 1-60 nucleotides, e.g., about 10-50 nucleotides. In some embodiments, the template DNA contains a 3'-UTR of 40 nucleotides. In some embodiments, the template DNA does not contain a 3'-UTR. In some embodiments, the template DNA comprises a 3'-poly(A) tail of 1 to 150 nucleotides, for example, a 3'-poly(A) tail of 10 to 100 nucleotides (e.g., 30 nucleotides). Such stabilizing elements may be included in the DNA to be transcribed in the IVT reaction, or may be synthesized separately and added to the resulting RNA obtained from the IVT reaction.

[0224] A 3'-poly(A) tail may be added to the RNA of the present disclosure. Methods for adding poly(A) tails are well known in the art. Such methods include, but are not limited to, catalysis with poly(A) polymerase or periodate treatment. Alternatively, or in addition, the poly(A) tail can be synthesized separately and then added to the RNA using any suitable technique, such as click chemistry, ortho-click chemistry, solulink, or other bioconjugation chemistries known to those skilled in the art.

[0225] The RNA of the present disclosure may be capped with 7-methylguanosine (m7G). Methods for adding m7G caps are well known in the art. Examples include, but are not limited to, co-transcriptional incorporation of anti-reverse cap analog (ARCA) using RNA polymerase such as T7 polymerase. Commercially available kits, such as the HiScribe™ T7 ARCA mRNA kit (New England BioLabs), are available for generating mRNA containing ARCA using T7.

[0226] According to the present disclosure, two regions or portions of a polynucleotide may be linked or ligated using, for example, triphosphate chemistry. In some embodiments, a first region or portion of 100 or fewer nucleotides is chemically synthesized with a 5' monophosphate and the terminal 3'-OH is removed or blocked. If the region is longer than 80 nucleotides, it may be synthesized as two or more strands that are then chemically linked by ligation. If the first region or portion is synthesized as a non-positionally modified region or portion using IVT, it may then be converted to a 5' monophosphate, followed by capping of the 3' end. The monophosphate protecting group may be selected from any known in the art. The second region or portion of the polynucleotide may be synthesized using either chemical synthesis or IVT methods, for example, as described herein. IVT methods may include the use of an RNA polymerase that can utilize a primer with a modified cap. Alternatively, the cap may be chemically synthesized and coupled to the IVT region or portion.

[0227] For ligation methods, it should be noted that ligation with DNA T4 ligase followed by treatment with DNAse (to remove the DNA splint required for DNA T4 ligase activity) will readily prevent the formation of unwanted ligation products.

[0228] It is not necessary for the entire polynucleotide to be made with a phosphate-sugar backbone, and if one of the regions or portions encodes a polypeptide, it is preferred that such region or portion comprises a phosphate-sugar backbone.

[0229] Ligation may be performed using any suitable technique, such as enzymatic ligation, click chemistry, ortho-click chemistry, solulink, or other bioconjugation chemistries known to those of skill in the art. In some embodiments, ligation is guided by a complementary oligonucleotide splint. In some embodiments, ligation is performed without a complementary oligonucleotide splint.

[0230] In another aspect, the present invention relates to kits for preparing mRNA cancer vaccines via IVT. For personalized cancer vaccines, it is important to identify patient-specific mutations and vaccinate patients with one or more neoepitopes. In such vaccines, the antigen(s) encoded by the mRNA ORF are patient-specific. The 5' and 3' ends of the RNA encoding the antigen(s) may be more broadly applicable because they contain untranslated and stabilizing regions common to many RNAs. Among other things, the present disclosure provides kits containing one or more portions of a chimeric polynucleotide, such as one or more 5' and / or 3' regions of an RNA, which may be combined with an ORF encoding a patient-specific epitope. For example, the kit may include a polynucleotide containing one or more of a 5'-ORF, a 3'-ORF, and a poly(A) tail. In some embodiments, each polynucleotide component is present in an individual container. In other embodiments, multiple polynucleotide components are present together in a single container. In some embodiments, the kit includes a ligase enzyme. In some embodiments, the provided kits include instructions for use, ie, for ligating the epitope-encoding ORF to one or more other components included in the kit, such as, for example, the 5'-ORF, the 3'-ORF, and / or the poly(A) tail.

[0231] The present methods for generating personalized cancer vaccines involve identifying mutations in tissue samples using techniques such as the deep nucleic acid or protein sequencing methods described herein. In some embodiments, initial identification of mutations is performed in a patient's transcriptome. Data derived from the patient's transcriptome is compared with sequence information derived from the patient's exome to identify patient- and tumor-specific expression mutations. This comparison results in a dataset of putative neoepitopes, referred to as a mutanome. The mutanome may contain approximately 100 to 10,000 candidate mutations per patient. The mutanome is then subjected to data exploration analysis using a set of queries or algorithms to identify an optimal set of mutations for generating a neoantigen vaccine. In some embodiments, an mRNA neoantigen vaccine is designed and manufactured. The patient is then treated with the vaccine.

[0232] The neoantigen vaccine can be a polycistronic vaccine containing multiple neoepitopes, or one or more single RNA vaccines, or a combination thereof.

[0233] In some embodiments, the entire process from initiating the mutation identification process to initiating patient treatment is accomplished in less than two months. In other embodiments, the entire process is accomplished in seven weeks, six weeks, five weeks, four weeks, three weeks, two weeks, or less than one week. In some embodiments, the entire process is performed in less than 30 days.

[0234] The mutation identification process can include both transcriptome analysis and exome analysis, or only transcriptome analysis, or only exome analysis. In some embodiments, transcriptome analysis is performed first, and exome analysis is performed second. Analysis is performed on biological or tissue samples. In some embodiments, the biological or tissue sample is a blood sample or a serum sample. In other embodiments, the sample is a tissue bank sample or an EBV-transformed B cell.

[0235] It has been recognized and understood that optimal neoepitopes for inclusion in an mRNA vaccine may be evaluated and / or selected by analyzing certain characteristics of cancer-associated mutations. For example, at a given time, one or more of several characteristics may be evaluated or weighted to select a set of neoepitopes for inclusion in the vaccine. Characteristics of a neoepitope or set of neoepitopes may include, for example, gene or transcript level expression assessment in patient RNA sequencing or other nucleic acid analysis, tissue-specific expression in available databases, known oncogenes / tumor suppressors, variant call confidence score, allele-specific expression based on RNA sequencing, conservative vs. non-conservative amino acid substitutions, point mutation location (Centering Score for increased TCR engagement), point mutation location (Anchoring Score for differential HLA binding), selfness: core epitope homology (<100%) with patient WES data, IC50 for HLA-A and HLA-B for 8-mers to 11-mers, IC50 for HLA-DRB1 for 15-mers to 20-mers, broad binding score, and promiscuity score. These may include the IC50 for HLA-C for 8-mers to 11-mers, the IC50 for HLA-DRB3-5 for 15-mers to 20-mers, the IC50 for HLA-DQB1 / A1 for 15-mers to 20-mers, and the IC50 for HLA-DPB1 / A1 for 15-mers to 20-mers, a comparison of the proportion of class I to class II, the diversity of HLA-A allotypes, HLA-B allotypes, and HLA-DRB1 allotypes covered in the patient, a comparison of the proportion of point mutations to compound epitopes (e.g., frameshifts), and / or pseudoepitope HLA binding scores.

[0236] In some embodiments, the characteristics of cancer-associated mutant cancers used to identify optimal epitopes are characteristics related to the type of mutation, the abundance of the mutation in patient samples, immunogenicity, lack of autoreactivity, and the nature of the peptide composition.

[0237] The type of mutation should be determined and considered as a deciding factor in whether the putative epitope should be included in the vaccine. The type of mutation can vary. In some instances, it may be desirable to include multiple mutations of different types in a single vaccine. In other instances, a single type of mutation may be more desirable. The value for a particular mutation can be weighted and calculated. In some embodiments, the particular mutation is a single nucleotide polymorphism (SNP). In some embodiments, the particular mutation is a compound variant, e.g., a peptide sequence resulting from intron retention, complex splicing events, or insertion / deletion mutations that change the reading frame of the sequence.

[0238] The abundance of mutations in patient samples may also be scored to determine whether a putative epitope should be included in a vaccine: abundant mutations may promote a more robust immune response.

[0239] Immunogenicity considerations are an important factor in selecting optimal neoepitopes for inclusion in vaccines. Immunogenicity may be evaluated, for example, by analyzing the neoepitope's MHC-binding ability, HLA broadness, mutation location, predicted T cell reactivity, actual T cell reactivity, specific conformations and structures leading to solvent exposure, and specific amino acid representation. Known algorithms, such as the NetMHC prediction algorithm, can be used to predict the ability of peptides to bind to common HLA-A and HLA-B alleles. Structural evaluation of MHC-bound peptides may be performed using in silico 3D analysis and / or protein docking programs. Predicted epitope structures upon binding to MHC molecules, such as those obtained from the Rosetta algorithm, may be used to evaluate the degree of solvent exposure of amino acid residues in the epitope when bound to an MHC molecule. T cell reactivity may be evaluated experimentally using epitopes and T cells in vitro. Alternatively, T cell reactivity may be assessed using a T cell response / sequence dataset.

[0240] A key element for neoepitopes included in vaccines is the absence of self-reactivity. Putative neoepitopes may be screened to confirm that they arise, for example, as a result of genetic alterations in malignant cells and are exclusive to tumor tissue. Ideally, the epitope should not be present in the patient's normal tissue; therefore, self-similar epitopes are excluded from the dataset. To determine the absence of self-reactivity, a personalized coding genome may be used as a reference for comparison of neoantigen candidates. In some embodiments, the personalized coding genome is generated from personalized transcriptomes and / or exomes.

[0241] The nature of the peptide composition may be taken into account in designing epitopes, for example, each putative epitope may be given a score that compares the conserved and non-conserved amino acids found in the epitope.

[0242] In some embodiments, the analysis performed by the tools described herein may include comparisons of different sets of features obtained from patients at different times, i.e., before and after a therapeutic intervention, different sets of features obtained from different tissue samples, different sets of features obtained from different patients with similar tumors, etc. In some embodiments, the average peak value from one set of features may be compared to the average peak value from another set of features. For example, the average HLA binding values ​​may be compared between two different sets of distributions. The two sets of distributions may be determined over time periods separated by, for example, days, months, or years.

[0243] Furthermore, the inventors recognize and understand that the algorithms described herein can be used to collect and analyze such data for the characteristics of cancer mutations, which data is useful for identifying neoepitopes and sets of neoepitopes for developing personalized cancer vaccines.

[0244] In some embodiments, all annotated transcripts of tumor mutant peptides are included in vaccines according to the invention. In some embodiments, translations of RNAs identified in RNA sequencing are included in vaccines according to the invention.

[0245] It should be understood that concatemers of two or more peptides, such as two or more neoantigens, may create unintended new epitopes (false epitopes) at the peptide boundaries. To prevent or eliminate such false epitopes, class I allele hits may be screened across the peptide boundaries in the concatemer. In some embodiments, the order of peptides within the concatemer is shuffled to reduce or eliminate the formation of false epitopes. In some embodiments, linkers may be used between peptides to reduce or eliminate the formation of false epitopes, such as single amino acid linkers such as glycine. In some embodiments, anchor amino acids can be replaced with other amino acids, which will reduce or eliminate the formation of false epitopes. In some embodiments, peptides are truncated at the peptide boundaries within the concatemer to reduce or eliminate the formation of false epitopes.

[0246] In some embodiments, the multiple peptide epitope antigens are positioned and ordered to minimize pseudoepitopes. In other embodiments, the multiple peptide epitope antigens are polypeptides that do not contain pseudoepitopes. When the cancer antigen epitopes are arranged in a head-to-tail configuration in a concatemeric structure, a junction is formed between each cancer antigen epitope. This junction includes several amino acids, i.e., 1 to 10 amino acids, from the epitope present at the N-terminus of the peptide and several amino acids, i.e., 1 to 10 amino acids, from the C-terminus of the directly linked adjacent epitope. It is important that the junction is not an immunogenic peptide that could generate an immune response. In some embodiments, the junction forms a peptide sequence that binds to the target HLA protein for which a personalized cancer vaccine is designed with an IC50 of greater than about 50 nM. In other embodiments, the peptide sequence of the junction binds to the HLA protein of interest with an IC50 of greater than about 10 nM, about 150 nM, about 200 nM, about 250 nM, about 300 nM, about 350 nM, about 400 nM, about 450 nM, or about 500 nM.

[0247] A system for characterizing neoepitopes in accordance with the techniques described herein may take any suitable form, and embodiments are not limited in this regard. An example implementation of a computer system 900 that may be used in connection with some embodiments is shown in FIG. 15. One or more computer systems, such as computer system 900, may be used to perform any of the functionality described above. Computer system 900 may include one or more processors 910 and one or more computer-readable storage media (i.e., tangible and non-transitory computer-readable media), such as, for example, volatile storage device 920 and one or more non-volatile storage media 930, which may be formed from any suitable data storage medium. Processor 910 may control the writing and reading of data from volatile storage device 920 and non-volatile storage device 930 in any suitable manner, and embodiments are not limited in this regard. To perform any of the functionality described herein, the processor 910 may execute one or more instructions stored in one or more computer-readable storage media (e.g., volatile storage 920 and / or non-volatile storage 930), which may act as tangible and non-transitory computer-readable media for storing instructions for execution by the processor 910.

[0248] The above-described embodiments can be implemented in any of many ways. For example, the embodiments may be implemented using hardware, software, or a combination thereof. When implemented in software, the software code can be executed on any suitable processor or collection of processors, whether such processors are provided in a single computer or distributed among multiple computers. It should be understood that any element or collection of elements that perform the above-described functions can generally be considered to be one or more controllers that control the functions discussed above. The one or more controllers can be implemented in various ways, such as using dedicated hardware or general-purpose hardware (e.g., one or more processors) programmed with microcode or software to perform the above-described functions.

[0249] In this regard, one embodiment should be understood to include at least one computer-readable storage medium (i.e., at least one tangible and non-transitory computer-readable medium), such as computer memory (e.g., hard drive, flash memory, processor working memory, etc.), floppy disk, optical disk, magnetic tape, or other tangible and non-transitory computer-readable medium, encoded with a computer program (i.e., a plurality of instructions) that, when executed by one or more processors, performs the functions discussed above. The computer-readable storage medium is portable, such that the program stored thereon can be loaded onto any computer resource to implement the techniques discussed herein. Furthermore, it should be understood that reference to a computer program that, when executed, performs the functions discussed above is not limited to an application program running on a host computer. Rather, the term "computer program" is used herein in a generic sense to refer to any type of computer code (e.g., software or microcode) that can be used to program one or more processors to implement the techniques discussed above.

[0250] Treatment method Provided herein are compositions (e.g., pharmaceutical compositions), methods, kits, and reagents for the prevention and / or treatment of cancer in humans and other mammals. Cancer RNA vaccines can be used as therapeutic or prophylactic agents. Cancer RNA vaccines may be used in medical treatments to prevent and / or treat cancer. In exemplary embodiments, the cancer RNA vaccines of the present disclosure are used to provide prophylactic protection from cancer. Prophylactic protection from cancer can be achieved after administration of the cancer RNA vaccines of the present disclosure. The vaccines can be administered once, twice, three times, four times, or more times, although a single vaccine administration (optionally followed by a single booster administration) may be sufficient. It is more desirable to administer the vaccine to individuals with cancer to achieve a therapeutic response. Dosage may need to be adjusted accordingly.

[0251] Once the mRNA vaccine is synthesized, it is administered to patients. In some embodiments, the vaccine is administered at a schedule of up to 2 months, up to 3 months, up to 4 months, up to 5 months, up to 6 months, up to 7 months, up to 8 months, up to 9 months, up to 10 months, up to 11 months, up to 1 year, up to 1.5 years, up to 2 years, up to 3 years, or up to 4 years. The schedule can be the same or changed. In some embodiments, the schedule is once a week for the first 3 months, and then once a month.

[0252] The vaccine may be administered by any route, hi some embodiments, the vaccine is administered by intramuscular or intravenous routes.

[0253] The patient may be examined at any time during treatment to determine whether the mutations in the vaccine are still appropriate, and based on that analysis, the vaccine may be adjusted or reformulated to include one or more different mutations or to remove one or more mutations.

[0254] Therapeutic and Prophylactic Compositions For example, provided herein are compositions (e.g., pharmaceutical compositions), methods, kits, and reagents for the prevention, treatment, or diagnosis of cancer in humans or mammals. The cancer RNA vaccines can be used as therapeutic or prophylactic agents. The cancer RNA vaccines may be used in medical treatments to prevent and / or treat cancer. In some embodiments, the cancer vaccines of the present invention can be envisioned for use in priming immune effector cells, for example, to activate peripheral blood mononuclear cells (PBMCs) ex vivo, which are then infused (reinfused) into a subject.

[0255] In exemplary embodiments, a cancer vaccine comprising an RNA polynucleotide described herein can be administered to a subject (e.g., a mammalian subject, such as a human subject), and the RNA polynucleotide is translated in vivo to produce an antigenic polypeptide.

[0256] A cancer RNA vaccine can induce the translation of a polypeptide (e.g., an antigen or immunogen) in a cell, tissue, or organism. In exemplary embodiments, such translation occurs in vivo, although embodiments in which such translation occurs ex vivo, in culture, or in vitro can be envisioned. In exemplary embodiments, a cell, tissue, or organism is contacted with an effective amount of a composition comprising a cancer RNA vaccine, which comprises a polynucleotide having at least one translatable region encoding an antigen polypeptide.

[0257] An "effective amount" of a cancer RNA vaccine is provided based, at least in part, on the target tissue, target cell type, administration means, physical characteristics (e.g., nucleoside size and degree of modification) of the polynucleotide, as well as other components of the cancer RNA vaccine, and other determinants. Generally, an effective amount of a cancer RNA vaccine composition induces or enhances an immune response in response to antigen production in cells, and such antigen production is preferably more efficient compared to a composition comprising a corresponding unmodified polynucleotide encoding the same antigen or peptide antigen. Increased antigen production may be indicated by increased cell transfection (the rate at which the RNA vaccine is transfected into cells), increased protein translation from the polynucleotide, decreased nucleic acid degradation (e.g., indicated by an increased duration of protein translation from the modified polynucleotide), or a change in the antigen-specific immune response of host cells.

[0258] In some embodiments, RNA vaccines (including polynucleotides and the polypeptides they encode) according to the present disclosure may be used to treat cancer.

[0259] Cancer RNA vaccines may be administered prophylactically or therapeutically as part of an active immunization schedule to healthy individuals or individuals with early-stage cancer or active cancer after the onset of symptoms. In some embodiments, the amount of the RNA vaccine of the present disclosure provided to a cell, tissue, or subject may be an amount effective for immunoprophylaxis.

[0260] The cancer RNA vaccine may be administered together with other preventive or therapeutic compounds. As a non-limiting example, the preventive or therapeutic compound may be an adjuvant or a booster. When referring to a composition such as a vaccine, the term "booster" used herein refers to the additional administration of a preventive (vaccine) composition. A booster (or booster vaccine) may be given after the previous administration of a preventive composition. The time interval between the initial administration of the prophylactic composition and the booster administration may be, but is not limited to, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 36 hours, 2 days, 3 days, 4 days, It can be 5 days, 6 days, 1 week, 10 days, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 18 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, 25 years, 30 years, 35 years, 40 years, 45 years, 50 years, 55 years, 60 years, 65 years, 70 years, 75 years, 80 years, 85 years, 90 years, 95 years, or more than 99 years. In exemplary embodiments, the time interval between the initial administration of the prophylactic composition and the booster administration can be, but is not limited to, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 6 months, or 1 year.

[0261] In one embodiment, the polynucleotide may be administered intramuscularly or intradermally, similar to the administration of vaccines known in the art.

[0262] mRNA cancer vaccines may be used in various situations depending on the severity of the cancer or the degree or level of unmet medical need. As a non-limiting example, mRNA cancer vaccines may be used to treat cancer at any stage. Compared to commercially available anti-cancer vaccines, mRNA cancer vaccines have superior properties in terms of generating much higher antibody titers, generating T cell responses, and generating responses more quickly. Without being bound by theory, the inventors hypothesize that because mRNA cancer vaccines incorporate natural cellular machinery, mRNA cancer vaccines are well-designed to generate the appropriate protein conformation upon translation. Unlike conventional vaccines, which are produced ex vivo and may induce undesirable cellular responses, mRNA cancer vaccines are presented to cell lines in a more natural manner.

[0263] A non-limiting list of cancers that can be treated with mRNA cancer vaccines is provided below. Peptide epitopes or antigens can be derived from any antigen of such cancers or tumors. Such epitopes are referred to as cancer or tumor antigens. Cancer cells can express cell surface molecules differently during different phases of tumor progression. For example, cancer cells can express a cell surface antigen in a benign state, but downregulate that particular cell surface antigen during metastasis. Therefore, it is envisioned that tumor or cancer antigens can encompass antigens produced during any stage of cancer progression. The methods of the present invention can be tailored to accommodate such changes. For example, several different mRNA vaccines can be generated for a particular patient. For example, an initial vaccine can be used at the beginning of treatment. At some point thereafter, new mRNA vaccines can be generated and administered to the patient to cover different antigens being expressed.

[0264] In some embodiments, the tumor antigen is one of the following antigens: CD2, CD19, CD20, CD22, CD27, CD33, CD37, CD38, CD40, CD44, CD47, CD52, CD56, CD70, CD79, CD137, 4-IBB, 5T4, AGS-5, AGS-16, angiopoietin 2, B7.1, B7.2, B7DC, B7H1, B7H2, B7H3, BT-062, BTLA, CAIX, carcinoembryonic antigen, CTLA4, Cripto, ED-B, ErbB1, ErbB2, ErbB3, ErbB4, EGFL7, EpCAM, EphA2, EphA3, EphB2, FAP, fibronectin, folate receptor receptor, ganglioside GM3, GD2, glucocorticoid-inducible tumor necrosis factor receptor (GITR), gplOO, gpA33, GPNMB, ICOS, IGF1R, integrin av, integrin ανβ, LAG-3, Lewis Y, mesothelin, c-MET, MN, carbonic anhydrase IX, MUC1, MUC16, nectin-4, NKGD2, NOTCH, OX40, OX40L, PD-1, PDL1, PSCA, PSMA, RANKL, ROR1, ROR2, SLC44A4, syndecan-1, TACI, TAG-72, tenascin, TIM3, TRAILRl, TRAILR2, VEGFR-1, VEGFR-2, VEGFR-3, and their mutants.

[0265] Cancer or tumor includes, but is not limited to, neoplasia, malignancy, metastatic cancer, or any disease or disorder characterized by uncontrolled cell growth and therefore considered to be cancerous. Cancer can be primary or metastatic cancer. Specific cancers that can be treated according to the present invention include, but are not limited to, the following (for a review of such disorders, see Fishman et al., 1985, Medicine, 2d Ed., J.B. Lippincott Co., Philadelphia): Cancers include, but are not limited to, biliary tract cancer, bladder cancer, brain cancer including glioblastoma and medulloblastoma, breast cancer, cervical cancer, choriocarcinoma, colon cancer, endometrial cancer, esophageal cancer, gastric cancer, hematological neoplasms including acute lymphocytic leukemia and acute myeloid leukemia, multiple myeloma, AIDS-related leukemia and adult T-cell leukemia-lymphoma, intraepithelial neoplasia including Bowen's disease and Paget's disease, liver cancer, lung cancer, lymphomas including Hodgkin's disease and lymphocytic lymphoma, neuroblastoma, oral cancer including squamous cell carcinoma, epithelial cell carcinoma, ovarian cancer, including those arising from stromal, germ cell, and mesenchymal cells, pancreatic cancer, prostate cancer, rectal cancer, sarcomas, including leiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma, and osteosarcoma, skin cancer, including melanoma, Kaposi's sarcoma, basal cell carcinoma, and squamous cell carcinoma, testicular cancer, including embryonal tumors such as seminoma, nonseminoma, teratoma, and choriocarcinoma, stromal and germ cell tumors, thyroid cancer, including adenocarcinoma and medullary carcinoma of the thyroid, and renal cancer, including adenocarcinoma and Wilms' tumor. Commonly encountered cancers include breast cancer, prostate cancer, lung cancer, ovarian cancer, colorectal cancer, and brain cancer.

[0266] Provided herein are pharmaceutical compositions comprising cancer RNA vaccines and RNA vaccine compositions and / or conjugates, optionally in combination with one or more pharmaceutically acceptable excipients.

[0267] Cancer RNA vaccine can be formulated or administered alone, or can be formulated or administered in combination with one or more other components.For example, cancer RNA vaccine (vaccine composition) can contain other components, including but not limited to, adjuvant.In some embodiments, cancer RNA vaccine does not contain adjuvant (no adjuvant).

[0268] In other embodiments, the mRNA cancer vaccines described herein may be combined with any other treatment useful in treating a patient. For example, a patient may be treated with an mRNA cancer vaccine and an anti-cancer agent. Thus, in one embodiment, the methods of the present invention can be used in combination with one or more cancer treatments, such as anti-cancer agents, conventional cancer vaccines, chemotherapy, radiation therapy, etc. (e.g., simultaneously or as part of an overall treatment protocol). Parameters of cancer treatment that may vary include, but are not limited to, dosage, administration timing, or duration or treatment, and cancer treatments may vary in dosage, timing, or duration. Another treatment for cancer is surgery, which may be used alone or in combination with any of the aforementioned treatment methods. Any agent or treatment known to be useful or that has been or is currently being used to prevent or treat cancer (e.g., conventional cancer vaccines, chemotherapy, radiation therapy, surgery, hormonal therapy, and / or biological therapy / immunotherapy) can be used in combination with the compositions of the present invention in accordance with the invention described herein. Those skilled in the medical field can determine the appropriate treatment for a subject.

[0269] Examples of such agents (i.e., anti-cancer agents) include, but are not limited to, DNA-interactive agents (e.g., alkylating agents, such as nitrogen mustards (e.g., chlorambucil, cyclophosphamide, isofamide, mechlorethamine, melphalan, uracil mustard); aziridines such as thiotepa; methanesulfonate esters such as busulfan; nitrosoureas such as carmustine, lomustine, streptozocin; platinum complexes such as cisplatin, carboplatin; mitomycin, and procarbazine. bioreductive alkylating agents such as dacarbazine and altretamine; DNA strand breakers such as bleomycin; intercalating topoisomerase II inhibitors such as intercalators such as amsacrine, dactinomycin, daunorubicin, doxorubicin, idarubicin, mitoxantrone, and non-intercalators such as etoposide and teniposide; non-intercalating topoisomerase II inhibitors such as etoposide and teniposide; and DNA side chain inhibitors such as plicamydin. groove binders); antimetabolites (including, but not limited to, folate antagonists such as methotrexate and trimetrexate; pyrimidine antagonists such as fluorouracil, fluorodeoxyuridine, CB3717, azacitidine, and floxuridine; purine antagonists such as mercaptopurine, 6-thioguanine, pentostatin; sugar-modified analogs such as cytarabine and fludarabine; and ribonucleotide reductase inhibitors such as hydroxyurea); tubulin-interacting agents (including, but not limited to, colbitine (col cbicine, vincristine and vinblastine, both alkaloids, and paclitaxel and cytoxan); hormonal agents (including but not limited to estrogens, conjugated estrogens and ethinyl estradiol and diethylstilbesterol, chlortrianisen and idenestrol); progestins such as hydroxyprogesterone caproate, medroxyprogesterone, and megestrol;androgens such as testosterone, testosterone propionate; fluoxymesterone, methyltestosterone; corticosteroids such as prednisone, dexamethasone, methylprednisolone, and prednisolone; luteinizing hormone-releasing hormone agents or gonadotropin-releasing hormone antagonists such as leuprolide acetate and goserelin acetate; antihormonal antigens) (including, but not limited to, antiestrogens such as tamoxifen, antiandrogens such as flutamide; and antiadrenal agents such as mitotane and aminoglutethimide); cytokines (including, but not limited to, IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-18, TGF-beta, GM-CSF, M-CSF, G-CSF, TNF-alpha, TNF-beta, LAF, TCGF, ​​BCGF , TRF, BAF, BDG, MP, LIF, OSM, TMF, PDGF, IFN-α, IFN-β, IFN-γ, and uteroglobin (U.S. Pat. No. 5,696,092); anti-angiogenic agents (including, but not limited to, agents that inhibit VEGF (e.g., other neutralizing antibodies), soluble receptor constructs, tyrosine kinase inhibitors, antisense strategies, RNA aptamers and ribozymes against VEGF or VEGF receptors, immunotoxins and coaguligands, tumor vaccines, and antibodies);

[0270] Specific examples of anti-cancer agents that can be used in accordance with the methods of the present invention include, but are not limited to, acivicin, aclarubicin, acodazole hydrochloride, acronine, adozelesin, aldesleukin, altretamine, ambomycin, amethanthrone acetate, aminoglutethimide, amsacrine, anastrozole, anthramycin, asparaginase, asperlin, azacytidine, azetepa, azotomycin, batimastat, benzodepa, bicalutamide, bisantrene hydrochloride, bisnafide mesylate, biceresin, bleomycin sulfate, brequinar sodium, bropirimine, busulfan, cactinomycin, and calsterone. , caracemide, carbetimer, carboplatin, carmustine, carubicin hydrochloride, carzelesin, cedefingol, chlorambucil, ciloremycin, cisplatin, cladribine, crisnatol mesylate, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin hydrochloride, decitabine, dexorumaplatin, dezaguanine, dezaguanine mesylate, diaziquone, docetaxel, doxorubicin, doxorubicin hydrochloride, droloxifene, droloxifene citrate, dromostanolone propionate, duazomycin, edatrexate, eflomithine hydrochloride hydrochloride), elsamitrucin, enloplatin, enpromate, epipropizine, epirubicin hydrochloride, elbrozole, esorubicin hydrochloride, estramustine, estramustine phosphate sodium, etanidazole, etoposide, etoposide phosphate, etoprine, fadrozole hydrochloride, fazarabine, fenretinide, floxuridine, fludarabine phosphate, fluorouracil, flurocitabine, foskidone, fostriecinna thorium, gemcitabine, gemcitabine hydrochloride, hydroxyurea, idarubicin hydrochloride, ifosfamide, ilmofosine, interleukin II (including recombinant interleukin II or rIL2), interferon alpha-2a, interferon alpha-2b, interferon alpha-n1, interferon alpha-n3, interferon beta-Ia, interferon gamma-Ib, iproplatin, irinotecan hydrochloride,Lanreotide acetate, letrozole, leuprolide acetate, liarozole hydrochloride, lometrexol sodium, lomustine, losoxantrone hydrochloride, masoprocol, maytansine, mechlorethamine hydrochloride, megestrol acetate, melengestrol acetate, melphalan, menogaril, mercaptopurine, methotrexate, methotrexate sodium, metoprine, meturedepa, mitindomide, mitocalcin, mitochromine, mitogillin, mitomarcine, mitomycin, mitospel, mitotamine , mitoxantrone hydrochloride, mycophenolic acid, nocodazole, nogalamycin, ormaplatin, oxisuran, paclitaxel, pegaspargase, periomycin, pentamustine, peplomycin sulfate, perfosfamide, pipobroman, piposulfan, piroxantrone hydrochloride, plicamycin, promestane, porfimer sodium, porfiromycin, prednimustine, procarbazine hydrochloride, puromycin, puromycin hydrochloride, pyrazofurin, ribopurin, Rogletimide, safingol, safingol hydrochloride, semustine, simtrazene, sparfosate sodium, sparsomycin, spirogermanium hydrochloride, spiromustine, spiroplatin, streptonigrin, streptozocin, sulofenur, tallysomycin, tecogalan sodium, tegafur, teloxantrone hydrochloride, temoporfin, teniposide, teloxylon, testolactone, thiamiprine, thioguanine, thiotepa, tiazofurin, tirapazamine, toremif citrate These include phenanthrene, trestrone acetate, triciribine phosphate, trimetrexate, trimetrexate glucuronate, triptorelin, tubrozole hydrochloride, uracil mustard, uredepa, vapreotide, verteporfin, vinblastine sulfate, vincristine sulfate, vindesine, vindesine sulfate, vinepidine sulfate, vinglycinate sulfate, vinleurosine sulfate, vinorelbine tartrate, vinrocidine sulfate, vinzolidine sulfate, vorozole, zeniplatin, zinostatin, and zorubicin hydrochloride.

[0271] Other anti-cancer agents include, but are not limited to, 20-epi-1,25-dihydroxyvitamin D3, 5-ethynyluracil, angiogenesis inhibitors, anti-dorsalizing morphogenetic protein-1, ara-CDP-DL-PTBA, BCR / ABL antagonist, CaRestM3, CARN700, casein kinase inhibitor (ICOS), clotrimazole, collismycin A, collismycin B, combretastatin A4, crambecidin 816, cryptophycin 8, curacin A, dehydrodidemnin B, didemnin B, dihydro-5-azacytidine, dihydrotaxol, duocarmycin SA, kahalalide F, lamellarin-N triacetate, leucine ... Prolide + estrogen + progesterone, rissoclinamide 7, monophosphoryl lipid A + myobacterium cell wall sk, N-acetyldinaline, N-substituted benzamide, O6-benzylguanine, prasetin A, prasetin B, platinum complex, platinum compound, platinum-triamine complex, rhenium Re186 etidronate, RII retinamide, rubiginone B1, SarCNU, sarcophytol A, sargramostim, senescence inhibitor 1 These include benzodiazepine-derived inhibitor 1), spicamycin D, tallimustine, 5-fluorouracil, thrombopoietin, thymotrin, thyroid-stimulating hormone, variolin B, thalidomide, veraresol, veramine, verdin, verteporfin, vinorelbine, vinxaltine, vitaxin, zanoteron, zeniplatin, and zilascorub.

[0272] The present invention also encompasses the administration of compositions containing mRNA cancer vaccines in combination with radiation therapy, including the use of X-rays, gamma rays, and other radiation sources to destroy cancer cells. In a preferred embodiment, radiation therapy is administered as external beam radiation or teletherapy, in which radiation is directed from a distant source. In another preferred embodiment, radiation therapy is administered as internal therapy or brachytherapy, in which a radioactive source is placed inside the body in close proximity to the cancer cells or tumor mass.

[0273] In certain embodiments, appropriate antigen regimen is selected according to the type of cancer.For example, patients with ovarian cancer can be administered a preventively or therapeutically effective amount of the composition comprising mRNA cancer vaccine, and one or more other drugs useful for treating ovarian cancer can be combined in a preventively or therapeutically effective amount, such as but not limited to intraperitoneal radiotherapy such as P32 therapy, multi-regional and whole pelvic radiotherapy, cisplatin, paclitaxel (taxol) or docetaxel (taxotere) and cisplatin or carboplatin combination, cyclophosphamide and cisplatin combination, cyclophosphamide and carboplatin combination, 5-FU and leucovorin combination, etoposide, liposomal doxorubicin, gemcitabine or topotecan combination. Cancer treatments and their dosages, routes of administration, and recommended usage are known in the art and described in such publications as the Physician's Desk Reference (56th ed., 2002).

[0274] In some preferred embodiments of the present invention, the mRNA cancer vaccines are administered in conjunction with T cell activators, such as immune checkpoint modulators, including both stimulatory and inhibitory checkpoint molecules (i.e., anti-CTLA4 and anti-PD1 antibodies).

[0275] Stimulatory checkpoint inhibitors function by promoting checkpoint processes. Some stimulatory checkpoint molecules are members of the tumor necrosis factor (TNF) receptor superfamily (CD27, CD40, OX40, GITR, and CD137), while others belong to the B7-CD28 superfamily (CD28 and ICOS). OX40 (CD134) is involved in the proliferation of effector and memory T cells. Anti-OX40 monoclonal antibodies have been shown to be effective in the treatment of advanced cancers. MEDI0562 is a humanized OX40 agonist. GITR is a glucocorticoid-inducible TNFR family-related gene involved in T cell proliferation. Several antibodies against GITR have been shown to promote anticancer responses. ICOS is an inducible T cell costimulator and is important for T cell effector function. CD27 supports antigen-specific proliferation of naive T cells and is involved in the generation of T cell and B cell memory. Several agonistic anti-CD27 antibodies are in development. CD122 is the beta subunit of the interleukin-2 receptor. NKTR-214 is a CD122-biased binding immunostimulatory cytokine.

[0276] Inhibitory checkpoint molecules include, but are not limited to, PD-1, TIM-3, VISTA, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, and LAG3. CTLA-4, PD-1, and their ligands are members of the CD28-B7 family of costimulatory molecules that play important roles throughout all stages of T cell function and other cellular functions. CTLA-4 is cytotoxic T lymphocyte-associated protein 4 (CD152) and is involved in regulating T cell proliferation.

[0277] The PD-1 receptor is expressed on the surface of activated T cells (and B cells) and, under normal circumstances, binds to its ligands (PD-L1 and PD-L2) expressed on the surface of antigen-presenting cells such as dendritic cells or macrophages. This interaction signals T cells, causing them to inhibit. Cancer cells exploit this system by inducing high levels of PD-L1 expression on their surface. This allows cancer cells to dominate the PD-1 pathway, suppressing anti-cancer immune responses by switching off PD-1-expressing T cells that can invade the tumor microenvironment. Pembrolizumab (formerly known as MK-3475 and lambrolizumab, and marketed as Keytruda) is a human antibody used in cancer immunotherapy. This antibody targets the PD-1 receptor.

[0278] IDO (indoleamine-2,3-dioxygenase) is a tryptophan-degrading enzyme that suppresses T cells and NK cells, generates and activates Tregs and myeloid-derived suppressor cells, and promotes tumor angiogenesis. TIM-3 (T cell immunoglobulin domain and mucin domain 3) acts as a negative regulator of Th1 / Tc1 function by inducing cell death upon interaction with its ligand, galectin-9. VISTA is a V-domain Ig suppressor of T cell activation.

[0279] A checkpoint inhibitor is a molecule such as a monoclonal antibody, a humanized antibody, a fully human antibody, a fusion protein, or a combination thereof, or a small molecule. For example, a checkpoint inhibitor inhibits a checkpoint protein, and such a checkpoint protein can be CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, CHK2, A2aR, a B-7 family ligand, or a combination thereof. Ligands for checkpoint proteins include, but are not limited to, CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, CHK2, A2aR, and ligands of the B-7 family. In some embodiments, the anti-PD-1 antibody is BMS-936558 (nivolumab). In other embodiments, the anti-CTLA-4 antibody is ipilimumab (trade name Yervoy, formerly known as MDX-010 and MDX-101).

[0280] In some preferred embodiments, cancer therapeutics, including checkpoint regulators, are delivered in the form of mRNA encoding the cancer therapeutic, such as, for example, anti-PD1, cytokines, chemokines, or stimulatory receptors / ligands (e.g., OX40).

[0281] In some embodiments, the cancer therapeutic agent is a targeted therapy. The targeted therapy can be a BRAF inhibitor such as vemurafenib (PLX4032) or dabrafenib. The BRAF inhibitor can be PLX4032, PLX4720, PLX4734, GDC-0879, PLX4032, PLX-4720, PLX4734, or sorafenib tosylate. BRAF is a human gene that produces a protein called B-Raf, also known as the proto-oncogene B-Raf and v-Raf murine sarcoma viral oncogene homolog B1. The B-Raf protein is involved in intracellular signaling that is involved in inducing cell proliferation. Vemurafenib is a BRAF inhibitor and has been approved by the FDA for the treatment of late-stage melanoma.

[0282] In another embodiment, the T cell therapeutic agent is OX40L. OX40 is a member of the tumor necrosis factor / nerve growth factor receptor (TNFR / NGFR) family. OX40 may play a role in T cell activation and the regulation of differentiation, proliferation, or apoptosis of normal and malignant lymphoid cells.

[0283] In other embodiments, the cancer therapeutic agent is a cytokine. In yet other embodiments, the cancer therapeutic agent is a vaccine comprising a population-based tumor-specific antigen.

[0284] In other embodiments, the cancer therapeutic agent is a vaccine containing one or more conventional antigens expressed by cancer-germline genes (antigens common to tumors found in multiple patients, also referred to as "common cancer antigens"). In some embodiments, the conventional antigens are known to be found in cancers or tumors in general, or known to be found in specific types of cancers or tumors. In some embodiments, the conventional cancer antigens are non-mutated tumor antigens. In some embodiments, the conventional cancer antigens are mutated tumor antigens.

[0285] The p53 gene (officially designated TP53) mutates more frequently in human cancers than any other gene. Large-cohort studies have shown that the genomic locations of most p53 mutations are unique to only one or a few patients, preventing their use as recurrent neoantigens for therapeutic vaccines designed for specific patient populations. However, a small subset of the p53 locus does exhibit a "hotspot" pattern, where several positions in the gene are mutated with relatively high frequency. Strikingly, the majority of these recurrently mutated regions reside near exon-intron boundaries, disrupting standard nucleotide sequence motifs recognized by the mRNA splicing machinery (Figure 16). Mutations in splicing motifs can alter the final mRNA sequence even if the local amino acid sequence is not expected to change (i.e., synonymous or intronic mutations). Therefore, although these mutations can alter mRNA splicing in unpredictable ways and have significant functional consequences for the translated protein, they are often annotated as "non-coding" by common annotation tools and ignored without further analysis. If alternatively spliced ​​isoforms contain in-frame sequence changes (i.e., no pretermination codons (PTCs)), they can escape elimination by nonsense-mediated mRNA decay (NMD) and are rapidly expressed, processed, and presented on the cell surface by the HLA system. Furthermore, alternative splicing resulting from mutations is usually "cryptic," i.e., not expressed in normal tissues, and therefore can be recognized by T cells as non-self neoantigens.

[0286] In some instances, the cancer therapeutic is a vaccine that includes one or more neo-antigens that are repeat polymorphisms ("hot spot mutations"). For example, among other things, the present invention provides neo-antigen peptide sequences derived from certain recurrent somatic cancer mutations in p53. Examples of mutations and mRNA splicing events that result in neo-antigen peptides and HLA-restricted epitopes include, but are not limited to, those shown in Figure 17 and the following: (1) A mutation in the canonical 5' splice site adjacent to codon position T125, which encodes the epitope AVSPCISFVW (SEQ ID NO: 2) (HLA-B * 57:01, HLA-B * 58:01), epitope HPLASCQCFF (SEQ ID NO: 3) (HLA-B * 35:01, HLA-B * 53:01), epitope FVWNFGIPL (SEQ ID NO: 4) (HLA-A * 02:01, HLA-A * 02:06, HLA-B * 35:01), a mutation that induces a retained intron with the peptide sequence TAKSVTCTVSCPEGLASMRLQCLAVSPCISFVWNFGIPLHPLASCQCFFIVYPLNV (SEQ ID NO: 1), (2) a mutation in the canonical 5' splice site adjacent to codon position 331, which encodes the epitope LQVLSLGTSY (SEQ ID NO: 6) (HLA-B * 15:01), epitope FQSNTQNAVF (SEQ ID NO: 7) (HLA-B * 15:01), a mutation that introduces a retained intron with the peptide sequence EYFTLQVLSLGTSYQVESFQSNTQNAVFFLTVLPAIGAFAIRGQ (SEQ ID NO: 5), (3) a mutation in the canonical 3' splice site adjacent to codon position 126, which encodes the epitope CTMFCQLAK (SEQ ID NO: 9) (HLA-A * 11:01), epitope KSVTCTMF (SEQ ID NO: 10) (HLA-B *a mutation that introduces a cryptic alternative exon 3' splice site generating a novel spanning peptide sequence AKSVTCTMFCQLAK (SEQ ID NO: 8) containing the nucleotide sequence 58:01; and / or (4) a mutation in the canonical 5' splice site adjacent to codon position 224, which encodes the epitope VPYEPPEVW (SEQ ID NO: 12) (HLA-B * 53:01, HLA-B * 51:01), epitope LTVPPSTAW (SEQ ID NO: 13) (HLA-B * 58:01, HLA-B * A mutation introducing a cryptic alternative intron 5' splice site generating a novel spanning peptide sequence VPYEPPEVWLALTVPPSTAWAA (SEQ ID NO: 11) containing the nucleotide sequence 57:01. (The transcription codon positions are with reference to ENST00000269305 (SEQ ID NO: 14), the standard full-length p53 transcript from the Ensembl v83 human genome annotation.)

[0287] In one embodiment, the present invention provides a cancer treatment vaccine comprising mRNA encoding an open reading frame (ORF) encoding one or more of the neoantigen peptides (1)-(4). In one embodiment, the present invention provides for the selective administration of a vaccine comprising or encoding one or more of the peptides (1)-(4) based on whether a patient's tumor contains any of the above mutations. In one embodiment, the present invention provides for the selective administration of a vaccine based on the dual criteria of whether a subject's tumor contains any of the above mutations and whether the subject's normal HLA type contains the corresponding HLA allele predicted to bind to the resulting neoantigen.

[0288] In some embodiments, the cancer therapeutic vaccine comprises one or more mRNAs encoding one or more repeat polymorphisms. In some embodiments, the cancer therapeutic vaccine comprises one or more mRNAs encoding one or more patient-specific neoantigens. In some embodiments, the cancer therapeutic vaccine comprises one or more mRNAs encoding an immune checkpoint regulator. The one or more repeat polymorphisms, one or more patient-specific neoantigens, and / or one or more immune checkpoint regulators can be combined in any manner. For example, it may be desirable for one or more concatemeric constructs to encode one or more repeat polymorphisms, one or more patient-specific neoantigens, and / or one or more immune checkpoint regulators. In other instances, it may be desirable for one or more repeat polymorphisms, one or more patient-specific neoantigens, and / or one or more immune checkpoint regulators to be encoded by separate mRNA constructs. It should be understood that the one or more repeat polymorphisms, one or more patient-specific neoantigens, and / or one or more immune checkpoint regulators can be administered simultaneously or sequentially.

[0289] The mRNA cancer vaccine and the anti-cancer therapeutic agent can be combined to further enhance the immunotherapeutic response. The mRNA cancer vaccine and the other therapeutic agent may be administered simultaneously or sequentially. When the other therapeutic agent is administered simultaneously, it can be administered in the same formulation or in separate formulations, but the administration is simultaneous. When the other therapeutic agent and the mRNA cancer vaccine are administered temporally separately, the other therapeutic agent is administered consecutively with each other and with the mRNA cancer vaccine. The time interval between the administration of these compounds can be on the order of a few minutes, or it can be longer, such as several hours, days, weeks, or months. For example, in some embodiments, the time interval between the administration of these compounds is 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 8 hours, 12 hours, 24 hours, or longer. In some embodiments, the time interval between the administration of these compounds is 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days or more. In some embodiments, the time interval between the administration of these compounds is administered before the anti-cancer therapeutic agent. In some embodiments, the mRNA cancer vaccine is administered after the anti-cancer therapeutic agent.

[0290] Other therapeutic agents include, but are not limited to, anti-cancer therapeutic agents, adjuvants, cytokines, antibodies, antigens, and the like.

[0291] RNA vaccines may be formulated or administered in combination with one or more pharmaceutically acceptable excipients. In some embodiments, the vaccine composition comprises at least one additional active agent, such as, for example, a therapeutically active agent, a prophylactically active agent, or a combination of both. The vaccine composition may be sterile, pyrogen-free, or sterile and pyrogen-free. General considerations in the formulation and / or manufacture of pharmaceutical agents such as vaccine compositions may be found, for example, in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference in its entirety).

[0292] In some embodiments, the cancer RNA vaccine is administered to a human, human patient, or subject. For purposes of this disclosure, the phrase "active ingredient" generally refers to an RNA vaccine or a polynucleotide contained therein, such as, for example, an RNA polynucleotide (e.g., an mRNA polynucleotide) encoding an antigenic polypeptide.

[0293] Formulations of the vaccine compositions described herein may be prepared by any method now known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing into association the active ingredient (e.g., mRNA polynucleotide) with an excipient and / or one or more other accessory ingredients, followed, if necessary and / or desirable, by dividing the product into the desired single-dose or multi-dose units, shaping, and / or filling.

[0294] Cancer RNA vaccines can be formulated using one or more excipients to achieve the following: (1) improved stability, (2) increased transfection into cells, (3) sustained or delayed release (e.g., from a depot formulation), (4) altered biodistribution (e.g., targeting to specific tissues or cell types), (5) increased in vivo translation of the encoded protein, and / or (6) altered in vivo release profile of the encoded protein (antigen). In addition to conventional excipients such as any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersing or suspending aids, surfactants, isotonic agents, thickeners or emulsifiers, preservatives, etc., excipients can include, but are not limited to, lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, cells transfected with the cancer RNA vaccine (e.g., for transplantation into a subject), hyaluronidase, nanoparticle mimics, and combinations thereof.

[0295] stabilizing element Naturally occurring eukaryotic mRNA molecules have been shown to contain stabilizing elements, including but not limited to, their 5'-end untranslated region (UTR) (5'UTR) and / or their 3'-end untranslated region (UTR) (3'UTR), in addition to other structural features such as a 5'-cap structure or a 3'-poly(A) tail. Both the 5'UTR and the 3'UTR are typically transcribed from genomic DNA and become components of premature mRNAs. Characteristic structural features of mature mRNAs, such as the 5'-cap and the 3'-poly(A) tail, are usually added to transcribed (premature) mRNAs during mRNA processing. The 3'-poly(A) tail is typically a stretch of adenine nucleotides added to the 3' end of the transcribed mRNA. The 3'-poly(A) tail can contain up to approximately 400 adenine nucleotides. In some embodiments, the length of the 3'-poly(A) tail may be an essential factor for the stability of an individual mRNA.

[0296] In some embodiments, the RNA vaccine may contain one or more stabilizing elements. The stabilizing element may include, for example, a histone stem loop. Stem-loop binding protein (SLBP) was identified as a 32-kDa protein. SLBP associates with histone stem loops at the 3' end of histone messages in both the nucleus and the cytoplasm. SLBP expression levels are cell cycle-regulated, peaking during S phase, a period when histone mRNA levels also increase. This protein has been shown to be essential for efficient 3'-end processing of histone mRNA precursors by U7 snRNP. SLBP continues to associate with stem loops after processing and subsequently stimulates translation of mature histone mRNA into histone proteins in the cytoplasm. The RNA-binding domain of SLBP is conserved from metazoans to protozoans, and its binding to histone stem loops depends on the structure of the loop. The minimal binding site contains at least three nucleotides 5' and two nucleotides 3' of the stem loop.

[0297] In some embodiments, the RNA vaccine comprises a coding region, at least one histone stem loop, and optionally a poly(A) sequence or polyadenylation signal. The poly(A) sequence or polyadenylation signal generally enhances the expression level of the encoded protein. In some embodiments, the encoded protein is not a histone protein, a reporter protein (e.g., luciferase, GFP, EGFP, β-galactosidase, EGFP), or a marker or selection protein (e.g., alpha-globin, galactokinase, and xanthine:guanine phosphoribosyltransferase (GPT)).

[0298] In some embodiments, the combination of a poly(A) sequence or polyadenylation signal with at least one histone stem loop, which are naturally alternative mechanisms, acts synergistically to increase protein expression above levels observed with either of the individual elements. The synergistic effect of the combination of poly(A) and at least one histone stem loop has been shown to be independent of the order of the elements or the length of the poly(A) sequence.

[0299] In some embodiments, the RNA vaccine does not contain a histone downstream element (HDE). A "histone downstream element" (HDE) comprises a purine-rich polynucleotide stretch comprising approximately 15-20 nucleotides 3' of a naturally occurring stem-loop, which serves as a binding site for U7snRNA, which is involved in the processing of histone pre-mRNA into mature histone mRNA. Ideally, the nucleic acids of the present invention do not contain introns.

[0300] In some embodiments, RNA vaccines may or may not contain enhancer and / or promoter sequences, and these sequences may be modified or unmodified, activated or inactivated. In some embodiments, histone stem-loops are generally derived from histone genes. In histone stem-loops, two adjacent, partially or completely reverse-complementary short sequences separated by a spacer are base-paired intramolecularly to form a loop structure. The unpaired loop region typically lacks the ability to base-pair with any of the stem-loop elements. Stem-loops are an important component of many RNA secondary structures, so they occur more frequently in RNA, but can also exist in single-stranded DNA. The stability of a stem-loop structure generally depends on the length, the number of mismatches or bulges, and the base composition of the paired region. In some embodiments, wobble base-pairing (non-Watson-Crick base-pairing) can result. In some embodiments, at least one histone stem-loop sequence comprises a length of 15 to 45 nucleotides.

[0301] In other embodiments, the RNA vaccine may be one in which one or more sequences with high AU content are removed. These sequences are sometimes referred to as AURES, and are destabilizing sequences found in the 3'UTR. AURES may be removed from the RNA vaccine. Alternatively, AURES may be retained in the RNA vaccine.

[0302] Nanoparticle formulations In some embodiments, the cancer RNA vaccine is formulated in nanoparticles. In some embodiments, the cancer RNA vaccine is formulated in lipid nanoparticles. In some embodiments, the cancer RNA vaccine is formulated in a lipid-polycation complex, which is referred to as a cationic lipid nanoparticle. Formulation of lipid nanoparticles may be achieved by methods known in the art and / or the methods described in U.S. Publication No. 20120178702, which is incorporated herein by reference in its entirety. By way of non-limiting example, polycations may include, but are not limited to, polylysine, polyornithine, and / or polyarginine, as well as cationic peptides or polypeptides, such as the cationic peptides described in International Publication No. WO2012013326 or U.S. Patent Publication No. US20130142818, each of which is incorporated herein by reference in its entirety. In some embodiments, the cancer RNA vaccine is formulated in lipid nanoparticles comprising a non-cationic lipid, such as, but not limited to, cholesterol or dioleoylphosphatidylethanolamine (DOPE).

[0303] Lipid nanoparticle formulation can be influenced by biophysical parameters such as, but not limited to, the selection of cationic lipid components, the saturation degree of cationic lipid, the nature of PEGylation, the ratio of all components, and size.In one example by Semple et al. (Nature Biotech.2010 28:172-176 (incorporated herein in its entirety by reference)), lipid nanoparticle formulation is composed of 57.1% cationic lipid, 7.1% dipalmitoyl phosphatidylcholine, 34.3% cholesterol, and 1.4% PEG-c-DMA.In another example, changing the composition of cationic lipid can improve the delivery efficiency of siRNA to various antigen-presenting cells (Basha et al. Mol Ther.2011 19:2186-2200 (incorporated herein in its entirety by reference)).

[0304] In some embodiments, the lipid nanoparticle formulation may comprise 35-45% cationic lipid, 40-50% cationic lipid, 50-60% cationic lipid, and / or 55-65% cationic lipid. In some embodiments, the lipid to RNA (e.g., mRNA) ratio in the lipid nanoparticle may be 5:1 to 20:1, 10:1 to 25:1, 15:1 to 30:1, and / or at least 30:1.

[0305] In some embodiments, the proportion of PEG in the lipid nanoparticle formulation may be increased or decreased, and / or the carbon chain length of the PEG lipid may be changed from C14 to C18 to modify the pharmacokinetics and / or biodistribution of the lipid nanoparticle formulation. As a non-limiting example, the lipid nanoparticle formulation may include PEG-c-DOMG (R-3-[(ω-methoxy-poly(ethylene glycol)2000)carbamoyl)]-1,2-dimyristyloxypropyl-3-amine) (also referred to herein as PEG-DOMG) at a lipid molar ratio of 0.5% to 3.0%, 1.0% to 3.5%, 1.5% to 4.0%, 2.0% to 4.5%, 2.5% to 5.0%, and / or 3.0% to 6.0% relative to the cationic lipid, DSPC, and cholesterol. In some embodiments, PEG-c-DOMG may be replaced with a PEG lipid, such as, but not limited to, PEG-DSG (1,2-distearoyl-sn-glycerol, methoxypolyethylene glycol), PEG-DMG (1,2-dimyristoyl-sn-glycerol), and / or PEG-DPG (1,2-dipalmitoyl-sn-glycerol, methoxypolyethylene glycol). The cationic lipid may be selected from any lipid known in the art, such as, but not limited to, DLin-MC3-DMA, DLin-DMA, C12-200, and DLin-KC2-DMA.

[0306] In some embodiments, the cancer RNA vaccine formulation is a nanoparticle comprising at least one lipid. The lipid may be selected from, but is not limited to, DLin-DMA, DLin-K-DMA, 98N12-5, C12-200, DLin-MC3-DMA, DLin-KC2-DMA, DODMA, PLGA, PEG, PEG-DMG, PEGylated lipids, and aminoalcohol lipids. In some embodiments, the lipid may be a cationic lipid, such as, but not limited to, DLin-DMA, DLin-D-DMA, DLin-MC3-DMA, DLin-KC2-DMA, DODMA, and aminoalcohol lipids. The aminoalcohol cationic lipid may be a lipid described in U.S. Patent Publication No. US20130150625 and / or may be prepared by the methods described therein, which is incorporated herein by reference in its entirety. Non-limiting examples of cationic lipids include 2-amino-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-{[(9Z,2Z)-octadeca-9,12-dien-1-yloxy]methyl}propan-1-ol (compound 1 described in US20130150625), 2-amino-3-[(9Z)-octadec-9-en-1-yloxy]-2-{[(9Z)-octadec-9-en-1-yloxy]methyl}propan-1-ol (compound 2 ..., [(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-[(octyloxy)methyl]propan-1-ol (compound 3 described in US20130150625), and 2-(dimethylamino)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-{[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]methyl}propan-1-ol (compound 4 described in US20130150625), or any pharmaceutically acceptable salt or stereoisomer thereof.

[0307] Lipid nanoparticle formulations typically comprise lipids, particularly ionizable cationic lipids, as well as neutral lipids, sterols, and molecules capable of reducing particle aggregation, such as PEG or PEG-modified lipids, such as 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), or di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319).

[0308] In some embodiments, the lipid nanoparticle formulation consists essentially of: (i) at least one lipid selected from the group consisting of 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319); (ii) a neutral lipid selected from DSPC, DPPC, POPC, DOPE, and SM; (iii) a sterol, such as cholesterol; and (iv) a PEG-lipid, such as PEG-DMG or PEG-cDMA, in a molar ratio of 20-60% cationic lipid, 5-25% neutral lipid, 25-55% sterol, and 0.5-15% PEG-lipid.

[0309] In some embodiments, the lipid nanoparticle formulation comprises 25% to 75% on a molar basis, e.g., 35-65%, 45-65%, 60%, 57.5%, 50%, or 40% on a molar basis, of a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319).

[0310] In some embodiments, the lipid nanoparticle formulation comprises 0.5% to 15% neutral lipid on a molar basis, e.g., 3-12%, 5-10%, or 15%, 10%, or 7.5% neutral lipid on a molar basis. Examples of neutral lipids include, but are not limited to, DSPC, POPC, DPPC, DOPE, and SM. In some embodiments, the formulation comprises 5% to 50% sterol on a molar basis (e.g., 15-45%, 20-40%, 40%, 38.5%, 35%, or 31% on a molar basis). A non-limiting example of a sterol is cholesterol. In some embodiments, the lipid nanoparticle formulation comprises 0.5%-20% PEG or PEG-modified lipid on a molar basis (e.g., 0.5-10%, 0.5-5%, 1.5%, 0.5%, 1.5%, 3.5%, or 5% on a molar basis). In some embodiments, the PEG or PEG-modified lipid comprises PEG molecules with an average molecular weight of 2,000 Da. In some embodiments, the PEG or PEG-modified lipid comprises PEG molecules with an average molecular weight of less than 2,000 (e.g., approximately 1,500 Da, approximately 1,000 Da, or approximately 500 Da). Examples of PEG-modified lipids include, but are not limited to, PEG-distearoylglycerol (PEG-DMG) (also referred to herein as PEG-C14 or C14-PEG), PEG-cDMA (further discussed in Reyes et al. J. Controlled Release, 107, 276-287 (2005), the contents of which are incorporated herein by reference in their entirety).

[0311] In some embodiments, the lipid nanoparticle formulation comprises, on a molar basis, 25-75% cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 0.5-15% neutral lipid, 5-50% sterol, and 0.5-20% PEG or PEG-modified lipid.

[0312] In some embodiments, the lipid nanoparticle formulation comprises, on a molar basis, 35-65% cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 3-12% neutral lipid, 15-45% sterol, and 0.5-10% PEG or PEG-modified lipid.

[0313] In some embodiments, the lipid nanoparticle formulation comprises, on a molar basis, 45-65% cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 5-10% neutral lipid, 25-40% sterol, and 0.5-10% PEG or PEG-modified lipid.

[0314] In some embodiments, the lipid nanoparticle formulation comprises, on a molar basis, 60% cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 7.5% neutral lipid, 31% sterol, and 1.5% PEG or PEG-modified lipid.

[0315] In some embodiments, the lipid nanoparticle formulation comprises, on a molar basis, 50% cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 10% neutral lipid, 38.5% sterol, and 1.5% PEG or PEG-modified lipid.

[0316] In some embodiments, the lipid nanoparticle formulation comprises, on a molar basis, 50% cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 10% neutral lipid, 35% sterol, 4.5% or 5% PEG or PEG-modified lipid, and 0.5% targeting lipid.

[0317] In some embodiments, the lipid nanoparticle formulation comprises, on a molar basis, 40% cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 15% neutral lipid, 40% sterol, and 5% PEG or PEG-modified lipid.

[0318] In some embodiments, the lipid nanoparticle formulation comprises, on a molar basis, 57.2% cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 7.1% neutral lipid, 34.3% sterol, and 1.4% PEG or PEG-modified lipid.

[0319] In some embodiments, the lipid nanoparticle formulation comprises, on a molar basis, 57.5% PEG-cDMA as a cationic lipid selected from PEG lipids (PEG-cDMA is further discussed in Reyes et al. (J. Controlled Release, 107, 276-287 (2005)), the contents of which are incorporated herein by reference in their entirety), 7.5% neutral lipid, 31.5% sterol, and 3.5% PEG or PEG-modified lipid.

[0320] In some embodiments, the lipid nanoparticle formulation consists essentially of a lipid mixture in a molar ratio of 20-70% cationic lipid: 5-45% neutral lipid: 20-55% cholesterol: 0.5-15% PEG-modified lipid. In some embodiments, the lipid nanoparticle formulation consists essentially of a lipid mixture in a molar ratio of 20-60% cationic lipid: 5-25% neutral lipid: 25-55% cholesterol: 0.5-15% PEG-modified lipid.

[0321] In some embodiments, the molar ratio of lipids is 50 / 10 / 38.5 / 1.5 (mol % of cationic lipid / neutral lipid (e.g., DSPC) / cholesterol / PEG-modified lipid (e.g., PEG-DMG, PEG-DSG, or PEG-DPG)), 57.2 / 7.1 / 34.3 / 1.4 (mol % of cationic lipid / neutral lipid (e.g., DPPC) / cholesterol / PEG-modified lipid (e.g., PEG-cDMA)), 40 / 15 / 40 / 5 (mol % of cationic lipid / neutral lipid (e.g., DSPC) / cholesterol / PEG-modified lipid (e.g., PEG-DMG)), 50 / 10 / 35 / 4.5 / 0.5 (mol % of cationic lipid / neutral lipid (e.g., DSPC) / cholesterol / PEG-modified lipid (e.g., PEG- DSPC) / cholesterol / PEG-modified lipid (e.g., PEG-DMG)), 50 / 10 / 35 / 5 (cationic lipid / neutral lipid (e.g., DSPC) / cholesterol / PEG-modified lipid (e.g., PEG-DMG)), 40 / 10 / 40 / 10 (cationic lipid / neutral lipid (e.g., DSPC) / cholesterol / PEG-modified lipid (e.g., PEG-DMG or PEG-cDMA)), 35 / 15 / 40 / 10 (cationic lipid / neutral lipid (e.g., DSPC) / cholesterol / PEG-modified lipid (e.g., PEG-DMG or PEG-cDMA)), or 52 / 13 / 30 / 5 (cationic lipid / neutral lipid (e.g., DSPC) / cholesterol / PEG-modified lipid (e.g., PEG-DMG or PEG-cDMA)).

[0322] Examples of lipid nanoparticle compositions and methods for their preparation are described, without limitation, in, for example, Semple et al. (2010) Nat. Biotechnol. 28:172-176, Jayarama et al. (2012), Angew. Chem. Int. Ed., 51:8529-8533, and Maier et al. (2013) Molecular Therapy 21, 1570-1578 (the contents of each of which are incorporated herein by reference in their entirety).

[0323] In some embodiments, lipid nanoparticle formulations may comprise cationic lipids, PEG lipids, and structured lipids, and may optionally comprise non-cationic lipids. As a non-limiting example, lipid nanoparticles may comprise 40-60% cationic lipids, 5-15% non-cationic lipids, 1-2% PEG lipids, and 30-50% structured lipids. As another non-limiting example, lipid nanoparticles may comprise 50% cationic lipids, 10% non-cationic lipids, 1.5% PEG lipids, and 38.5% structured lipids. As yet another non-limiting example, lipid nanoparticles may comprise 55% cationic lipids, 10% non-cationic lipids, 2.5% PEG lipids, and 32.5% structured lipids. In some embodiments, the cationic lipid may be any of the cationic lipids described herein, including, but not limited to, DLin-KC2-DMA, DLin-MC3-DMA, and L319.

[0324] In some embodiments, the lipid nanoparticle formulations described herein may be lipid nanoparticles comprising four components. The lipid nanoparticles may comprise a cationic lipid, a non-cationic lipid, a PEG lipid, and a structured lipid. As a non-limiting example, the lipid nanoparticles may comprise 40-60% cationic lipid, 5-15% non-cationic lipid, 1-2% PEG lipid, and 30-50% structured lipid. As another non-limiting example, the lipid nanoparticles may comprise 50% cationic lipid, 10% non-cationic lipid, 1.5% PEG lipid, and 38.5% structured lipid. As yet another non-limiting example, the lipid nanoparticles may comprise 55% cationic lipid, 10% non-cationic lipid, 2.5% PEG lipid, and 32.5% structured lipid. In some embodiments, the cationic lipid may be any of the cationic lipids described herein, including, but not limited to, DLin-KC2-DMA, DLin-MC3-DMA, and L319.

[0325] In some embodiments, the lipid nanoparticle formulations described herein may comprise a cationic lipid, a non-cationic lipid, a PEG lipid, and a structured lipid. As a non-limiting example, the lipid nanoparticles may comprise 50% DLin-KC2-DMA as the cationic lipid, 10% DSPC as the non-cationic lipid, 1.5% PEG-DOMG as the PEG lipid, and 38.5% cholesterol as the structured lipid. As a non-limiting example, the lipid nanoparticles may comprise 50% DLin-MC3-DMA as the cationic lipid, 10% DSPC as the non-cationic lipid, 1.5% PEG-DOMG as the PEG lipid, and 38.5% cholesterol as the structured lipid. As a non-limiting example, the lipid nanoparticles may comprise 50% DLin-MC3-DMA as the cationic lipid, 10% DSPC as the non-cationic lipid, 1.5% PEG-DMG as the PEG lipid, and 38.5% cholesterol as the structured lipid. As yet another non-limiting example, the lipid nanoparticles comprise 55% L319 as the cationic lipid, 10% DSPC as the non-cationic lipid, 2.5% PEG-DMG as the PEG lipid, and 32.5% cholesterol as the structural lipid.

[0326] In some embodiments, the nanoparticles comprise a compound of formula (I): [ka] or a salt or isomer thereof, wherein: R1 is C 5-30 Alkyl, C 5-20 Alkenyl, -R * -YR", -YR", and -R"M'R'; R2 and R3 are H, C 1-14 Alkyl, C 2-14 Alkenyl, -R * YR”, -YR”, and -R * OR" or R2 and R3 together with the atoms to which they are attached form a heterocycle or carbocycle; R4 is C 3-6 Carbocyclic ring, -(CH2) n Q, -(CH2) n CHQR, -CHQR, -CQ(R)2, and unsubstituted C 1-6 alkyl, and Q is selected from the group consisting of carbocycle, heterocycle, -OR, -O(CH2) n N(R)2, -C(O)OR, -OC(O)R, -CX3, -CX2H, -CXH2, -CN, -N(R)2, -C(O)N(R)2, -N( R)C(O)R, -N(R)S(O)2R, -N(R)C(O)N(R)2, -N(R)C(S)N(R)2, -N(R)R8, -O(CH2) n -OR, -N(R)C(=NR9)N(R), -N(R)C(=CHR9)N(R), -OC(O)N(R), -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O)R, -N(OR)C(O)OR, -N(OR)C(O)N(R), -N(OR)C(S)N(R), -N(OR)C(=NR9)N(R), -N(OR)C(=CHR9)N(R), -C(=NR9)N(R), -C(=NR9)R, -C(O)N(R)OR, and -C(R)N(R)C(O)OR; R5 is C 1-3 Alkyl, C2-3 independently selected from the group consisting of alkenyl, and H; R6 is C 1-3 Alkyl, C 2-3 independently selected from the group consisting of alkenyl, and H; M and M' are independently selected from -C(O)O-, -OC(O)-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)-, -SS-, an aryl group, and a heteroaryl group; R7 is C 1-3 Alkyl, C 2-3 selected from the group consisting of alkenyl, and H; R8 is C 3-6 selected from the group consisting of carbocycles and heterocycles; R9 is H, CN, NO2, C 1-6 Alkyl, -OR, -S(O)2R, -S(O)2N(R)2, C 2-6 Alkenyl, C 3-6 selected from the group consisting of carbocycles, and heterocycles; R is C 1-3 Alkyl, C 2-3 independently selected from the group consisting of alkenyl, and H; R' is C 1-18 Alkyl, C 2-18 Alkenyl, -R * independently selected from the group consisting of Y-R″, -Y-R″, and H; R” are C 3-14 Alkyl and C 3-14 alkenyl; R * are respectively, C 1-12 Alkyl and C 2-12 alkenyl; Each Y is independently C 3-6 is a carbocyclic ring, Each X is independently selected from the group consisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13.

[0327] In some embodiments, a subset of compounds of Formula (I) are those in which R4 is -(CH2) n Q, -(CH2) n When Q is CHQR, -CHQR, or -CQ(R), it includes compounds in which (i) when n is 1, 2, 3, 4, or 5, Q is not -N(R), or (ii) when n is 1 or 2, Q is not a 5-, 6-, or 7-membered heterocycloalkyl.

[0328] In some embodiments, another subset of compounds of formula (I) is R1 is C 5-30 Alkyl, C 5-20 Alkenyl, -R * -YR", -YR", and -R"M'R'; R2 and R3 are H, C 1-14 Alkyl, C 2-14 Alkenyl, -R * YR”, -YR”, and -R * OR" or R2 and R3 together with the atoms to which they are attached form a heterocycle or a carbocycle; R4 is C 3-6 Carbocyclic ring, -(CH2) n Q, -(CH2) n CHQR, -CHQR, -CQ(R)2, and unsubstituted C 1-6 alkyl; and Q is selected from the group consisting of C 3-6 Carbocycle, 5-14 membered heteroaryl having one or more heteroatoms selected from N, O, and S, -OR, -O(CH2) n N(R)2, -C(O)OR, -OC(O)R, -CX3, -CX2H, -CXH2, -CN, -C(O)N(R)2, -N(R)C(O)R, -N( R)S(O)2R, -N(R)C(O)N(R)2, -N(R)C(S)N(R)2, -CRN(R)2C(O)OR, -N(R)R8, -O(CH2) nOR, -N(R)C(=NR9)N(R)2, -N(R)C(=CHR9)N(R)2, -OC(O)N(R)2, -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O)2R, -N(OR)C(O)OR, -N(OR)C(O)N(R)2, -N(OR)C(S)N(R)2, -N(OR)C(=NR9)N(R)2, -N(OR)C(=CHR9)N(R)2, -C(=NR9)N(R)2, -C(=NR9)R, -C(O)N(R)OR, as well as oxo(=O), OH, amino, mono- or di-alkylamino, and C 1-3 alkyl, 5-14 membered heterocycloalkyl having one or more heteroatoms selected from N, O, and S, each n being independently selected from 1, 2, 3, 4, and 5; R5 is C 1-3 Alkyl, C 2-3 independently selected from the group consisting of alkenyl, and H; R6 is C 1-3 Alkyl, C 2-3 independently selected from the group consisting of alkenyl, and H; M and M' are independently selected from -C(O)O-, -OC(O)-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)-, -SS-, an aryl group, and a heteroaryl group; R7 is C 1-3 Alkyl, C 2-3 selected from the group consisting of alkenyl, and H; R8, C 3-6 selected from the group consisting of carbocycles and heterocycles; R9 is H, CN, NO2, C 1-6 Alkyl, -OR, -S(O)2R, -S(O)2N(R)2, C 2-6 Alkenyl, C 3-6 selected from the group consisting of carbocycles, and heterocycles; R is C 1-3 Alkyl, C 2-3independently selected from the group consisting of alkenyl, and H; R' is C 1-18 Alkyl, C 2-18 Alkenyl, -R * independently selected from the group consisting of Y-R″, -Y-R″, and H; R” are C 3-14 Alkyl and C 3-14 alkenyl; R * are respectively, C 1-12 Alkyl and C 2-12 alkenyl; Y is independently C 3-6 is a carbocyclic ring, each X is independently selected from the group consisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13; The present invention includes the compound or a salt or isomer thereof.

[0329] In some embodiments, another subset of compounds of formula (I) is R1 is C 5-30 Alkyl, C 5-20 Alkenyl, -R * -YR", -YR", and -R"M'R'; R2 and R3 are H, C 1-14 Alkyl, C 2-14 Alkenyl, -R * YR”, -YR”, and -R * OR" or R2 and R3 together with the atoms to which they are attached form a heterocycle or a carbocycle; R4 is C 3-6 Carbocyclic ring, -(CH2) n Q, -(CH2) n CHQR, -CHQR, -CQ(R)2, and unsubstituted C 1-6 alkyl; and Q is selected from the group consisting of C 3-6Carbocycle, 5-14 membered heterocycle having one or more heteroatoms selected from N, O, and S, -OR, -O(CH2) n N(R)2, -C(O)OR, -OC(O)R, -CX3, -CX2H, -CXH2, -CN, -C(O)N(R)2, -N(R)C(O)R, -N( R)S(O)2R, -N(R)C(O)N(R)2, -N(R)C(S)N(R)2, -CRN(R)2C(O)OR, -N(R)R8, -O(CH2) n and (i) R4 is selected from the group consisting of -OR, -N(R)C(=NR9)N(R), -N(R)C(=CHR9)N(R), -OC(O)N(R), -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O)R, -N(OR)C(O)OR, -N(OR)C(O)N(R), -N(OR)C(S)N(R), -N(OR)C(=NR9)N(R), -N(OR)C(=CHR9)N(R), -C(=NR9)R, -C(O)N(R)OR, and -C(=NR9)N(R), each n is independently selected from 1, 2, 3, 4, and 5; Q is a 5-14 membered heterocycle; and (i) R4 is -(CH2) n Q (n is 1 or 2), or (ii) R4 is -(CH2) n or (iii) when R4 is -CHQR and -CQ(R)2, Q is either a 5- to 14-membered heteroaryl or an 8- to 14-membered heterocycloalkyl; R5 is C 1-3 Alkyl, C 2-3 independently selected from the group consisting of alkenyl, and H; R6 is C 1-3 Alkyl, C 2-3 independently selected from the group consisting of alkenyl, and H; M and M' are independently selected from -C(O)O-, -OC(O)-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)-, -SS-, an aryl group, and a heteroaryl group; R7 is C1-3 Alkyl, C 2-3 selected from the group consisting of alkenyl, and H; R8, C 3-6 selected from the group consisting of carbocycles and heterocycles; R9 is H, CN, NO2, C 1-6 Alkyl, -OR, -S(O)2R, -S(O)2N(R)2, C 2-6 Alkenyl, C 3-6 selected from the group consisting of carbocycles, and heterocycles; R is C 1-3 Alkyl, C 2-3 independently selected from the group consisting of alkenyl, and H; R' is C 1-18 Alkyl, C 2-18 Alkenyl, -R * independently selected from the group consisting of Y-R″, -Y-R″, and H; R” are C 3-14 Alkyl and C 3-14 alkenyl; R * are respectively, C 1-12 Alkyl and C 2-12 alkenyl; Y is independently C 3-6 is a carbocyclic ring, each X is independently selected from the group consisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13; The present invention includes the compound or a salt or isomer thereof.

[0330] In some embodiments, another subset of compounds of formula (I) is R1 is C 5-30 Alkyl, C 5-20 Alkenyl, -R * -YR", -YR", and -R"M'R'; R2 and R3 are H, C 1-14 Alkyl, C 2-14 Alkenyl, -R *YR”, -YR”, and -R * OR" or R2 and R3 together with the atoms to which they are attached form a heterocycle or a carbocycle; R4 is C 3-6 Carbocyclic ring, -(CH2) n Q, -(CH2) n CHQR, -CHQR, -CQ(R)2, and unsubstituted C 1-6 alkyl; and Q is selected from the group consisting of C 3-6 Carbocycle, 5-14 membered heteroaryl having one or more heteroatoms selected from N, O, and S, -OR, -O(CH2) n N(R)2, -C(O)OR, -OC(O)R, -CX3, -CX2H, -CXH2, -CN, -C(O)N(R)2, -N(R)C(O)R, -N( R)S(O)2R, -N(R)C(O)N(R)2, -N(R)C(S)N(R)2, -CRN(R)2C(O)OR, -N(R)R8, -O(CH2) n -OR, -N(R)C(=NR9)N(R), -N(R)C(=CHR9)N(R), -OC(O)N(R), -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O)R, -N(OR)C(O)OR, -N(OR)C(O)N(R), -N(OR)C(S)N(R), -N(OR)C(=NR9)N(R), -N(OR)C(=CHR9)N(R), -C(=NR9)R, -C(O)N(R)OR, and -C(=NR9)N(R), wherein each n is independently selected from 1, 2, 3, 4, and 5; R5 is C 1-3 Alkyl, C 2-3 independently selected from the group consisting of alkenyl, and H; R6 is C 1-3 Alkyl, C 2-3 independently selected from the group consisting of alkenyl, and H; M and M' are independently selected from -C(O)O-, -OC(O)-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)-, -SS-, an aryl group, and a heteroaryl group; R7 is C 1-3 Alkyl, C 2-3 selected from the group consisting of alkenyl, and H; R8, C 3-6 selected from the group consisting of carbocycles and heterocycles; R9 is H, CN, NO2, C 1-6 Alkyl, -OR, -S(O)2R, -S(O)2N(R)2, C 2-6 Alkenyl, C 3-6 selected from the group consisting of carbocycles, and heterocycles; R is C 1-3 Alkyl, C 2-3 independently selected from the group consisting of alkenyl, and H; R' is C 1-18 Alkyl, C 2-18 Alkenyl, -R * independently selected from the group consisting of Y-R″, -Y-R″, and H; R” are C 3-14 Alkyl and C 3-14 alkenyl; R * are respectively, C 1-12 Alkyl and C 2-12 alkenyl; Y is independently C 3-6 is a carbocyclic ring, each X is independently selected from the group consisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13; The present invention includes the compound or a salt or isomer thereof.

[0331] In some embodiments, another subset of compounds of formula (I) is R1 is C5-30 Alkyl, C 5-20 Alkenyl, -R * -YR", -YR", and -R"M'R'; R2 and R3 are H, C 2-14 Alkyl, C 2-14 Alkenyl, -R * YR”, -YR”, and -R * OR" or R2 and R3 together with the atoms to which they are attached form a heterocycle or a carbocycle; R4 is -(CH2) n Q or -(CH2) n CHQR, Q is -N(R)2, and n is selected from 3, 4, and 5; R5 is C 1-3 Alkyl, C 2-3 independently selected from the group consisting of alkenyl, and H; R6 is C 1-3 Alkyl, C 2-3 independently selected from the group consisting of alkenyl, and H; M and M' are independently selected from -C(O)O-, -OC(O)-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)-, -SS-, an aryl group, and a heteroaryl group; R7 is C 1-3 Alkyl, C 2-3 selected from the group consisting of alkenyl, and H; R is C 1-3 Alkyl, C 2-3 independently selected from the group consisting of alkenyl, and H; R' is C 1-18 Alkyl, C 2-18 Alkenyl, -R * independently selected from the group consisting of Y-R″, -Y-R″, and H; R” are C 3-14 Alkyl and C 3-14 alkenyl; R* are respectively, C 1-12 Alkyl and C 1-12 alkenyl; Y is independently C 3-6 is a carbocyclic ring, each X is independently selected from the group consisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13; The present invention includes the compound or a salt or isomer thereof.

[0332] In some embodiments, another subset of compounds of formula (I) is R1 is C 5-30 Alkyl, C 5-20 Alkenyl, -R * -YR", -YR", and -R"M'R'; R2 and R3 are C 1-14 Alkyl, C 2-14 Alkenyl, -R * YR”, -YR”, and -R * OR" or R2 and R3 together with the atoms to which they are attached form a heterocycle or a carbocycle; R4 is -(CH2) n Q, -(CH2) n -CHQR, -CHQR, and -CQ(R), where Q is -N(R), and n is selected from 1, 2, 3, 4, and 5; R5 is C 1-3 Alkyl, C 2-3 independently selected from the group consisting of alkenyl, and H; R6 is C 1-3 Alkyl, C 2-3 independently selected from the group consisting of alkenyl, and H; M and M' are independently selected from -C(O)O-, -OC(O)-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)-, -SS-, an aryl group, and a heteroaryl group; R7 is C 1-3 Alkyl, C 2-3 selected from the group consisting of alkenyl, and H; R is C 1-3 Alkyl, C 2-3 independently selected from the group consisting of alkenyl, and H; R' is C 1-18 Alkyl, C 2-18 Alkenyl, -R * independently selected from the group consisting of Y-R″, -Y-R″, and H; R” are C 3-14 Alkyl and C 3-14 alkenyl; R * are respectively, C 1-12 Alkyl and C 1-12 alkenyl; Y is independently C 3-6 is a carbocyclic ring, each X is independently selected from the group consisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13; The present invention includes the compound or a salt or isomer thereof.

[0333] In some embodiments, a subset of compounds of formula (I) is a compound of formula (IA): [ka] or a salt or isomer thereof, wherein l is selected from 1, 2, 3, 4, and 5; m is selected from 5, 6, 7, 8, and 9; M is a bond or M′; and R is an unsubstituted C 1-3 Alkyl, or -(CH2) nQ, where Q is OH, —NHC(S)N(R), —NHC(O)N(R), —N(R)C(O)R, —N(R)S(O)R, —N(R)R, —NHC(═NR)N(R), —NHC(═CHR)N(R), —OC(O)N(R), —N(R)C(O)OR, heteroaryl, or heterocycloalkyl; M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—, —P(O)(OR′)O—, —SS—, an aryl group, and a heteroaryl group; and R and R are independently selected from H, C 1-14 Alkyl, and C 2-14 alkenyl.

[0334] In some embodiments, a subset of compounds of formula (I) is a compound of formula (II): [ka] or a salt or isomer thereof, wherein l is selected from 1, 2, 3, 4, and 5; M is a bond or M′; and R is an unsubstituted C 1-3 Alkyl, or -(CH2) n Q, where n is 2, 3, or 4; Q is OH, —NHC(S)N(R)2, —NHC(O)N(R)2, —N(R)C(O)R, —N(R)S(O)2R, —N(R)R8, —NHC(═NR9)N(R)2, —NHC(═CHR9)N(R)2, —OC(O)N(R)2, —N(R)C(O)OR, heteroaryl, or heterocycloalkyl; M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—, —P(O)(OR′)O—, —SS—, an aryl group, and a heteroaryl group; and R2 and R3 are independently selected from H, C 1-14 Alkyl, and C 2-14 alkenyl.

[0335] In some embodiments, a subset of compounds of Formula (I) is a compound of Formula (IIa), Formula (IIb), Formula (IIc), or Formula (IIe): [ka] or a salt or isomer thereof, wherein R4 is as defined herein.

[0336] In some embodiments, a subset of compounds of formula (I) is a compound of formula (IId): [ka] or a salt or isomer thereof, wherein n is 2, 3, or 4; and m, R′, R″, and R2-R6 are as described herein. For example, R2 and R3 can each be C 5-14 Alkyl and C 5-14 alkenyl.

[0337] In some embodiments, a subset of compounds of Formula (I) is a compound of Formula (IIa), Formula (IIb), Formula (IIc), or Formula (IIe): [ka] or a salt or isomer thereof, wherein R4 is as defined herein.

[0338] In some embodiments, a subset of compounds of formula (I) is a compound of formula (IId): [ka] or a salt or isomer thereof, wherein n is 2, 3, or 4; and m, R′, R″, and R2-R6 are as described herein. For example, R2 and R3 can each be C 5-14 Alkyl and C 5-14 alkenyl.

[0339] In some embodiments, the compound of formula (I) is [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] is selected from the group consisting of:

[0340] In a further embodiment, the compound of formula (I) is [ka] is selected from the group consisting of:

[0341] In some embodiments, the compound of formula (I) is [ka] [ka] [ka] [ka] [ka] [ka] [ka]

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[0342] In some embodiments, the nanoparticles comprise the following compounds: [ka] or salts and isomers thereof.

[0343] In some embodiments, the disclosure features a nanoparticle composition that includes a lipid component that includes a compound described herein (e.g., a compound according to Formula (I), Formula (IA), Formula (II), Formula (IIa), Formula (IIb), Formula (IIc), Formula (IId), or Formula (IIe)).

[0344] In another aspect of the present invention, kits for achieving these methods are also provided. The kits include a container containing a lipid nanoparticle formulation, a container containing a vaccine formulation, and instructions for adding a personalized mRNA cancer vaccine to the vaccine formulation to generate a personalized mRNA cancer vaccine formulation and mixing the personalized mRNA cancer vaccine formulation with the lipid nanoparticle formulation within 24 hours prior to administration to a subject. In some embodiments, the kits include mRNAs having open reading frames encoding 2 to 100 cancer antigens.

[0345] The article comprises a pharmaceutical or diagnostic grade compound of the invention in one or more containers. The article may include instructions or labels that promote or describe the use of the compound of the invention.

[0346] "Promotion," as used herein, includes all methods of conducting business related to the compositions of the present invention for the treatment of cancer, including education, hospital and other clinical presentations, pharmaceutical industry activities including pharmaceutical sales, and any advertising or other promotional activities, including any form of written, oral, and electronic communication.

[0347] "Instructions" can be defined as promotional components and typically include written instructions on or associated with the packaging of the compositions of the present invention. Instructions can also include any oral or electronic instructions provided in any manner.

[0348] Thus, in some embodiments, pharmaceutical, diagnostic, or research kits may be assembled from the agents described herein to facilitate their use in therapeutic, diagnostic, or research applications. Kits may include one or more containers housing the components of the invention and instructions for use. Specifically, such kits may include one or more of the agents described herein, along with instructions describing the intended therapeutic use and proper administration of such agents. In certain embodiments, the agents in the kits may be in pharmaceutical formulations and dosages appropriate for a particular use and method of administration of the agent.

[0349] The kits may be designed to facilitate use of the methods described herein by a physician and may take many forms. Each of the components of the kit may be provided in liquid form (e.g., a solution) or solid form (e.g., a dry powder), if applicable. In certain cases, some of the compositions may be configurable or otherwise processable (e.g., to render them active), for example, by adding suitable solvents or other materials (e.g., water or cell culture medium), which may or may not be provided with the kit. "Instructions," as used herein, may be defined as instructional and / or promotional material and typically include written instructions on or associated with the packaging of the invention. Instructions may also include any oral or electronic instructions provided in any manner such that the user clearly recognizes that the instructions are associated with the kit, for example, through audiovisual (e.g., videotape, DVD, etc.), internet, and / or web-based communication, etc. The written description may be in a form prescribed by a government agency that regulates the manufacture, use, or sale of pharmaceutical or biological products, and the description may also reflect approval by the agency for manufacture, use, or sale for administration to humans.

[0350] The kit may include any one or more of the elements described herein in one or more containers. By way of example, in one embodiment, the kit may include instructions for mixing one or more components of the kit and / or isolating and mixing a sample and applying to a subject. The kit may include a container containing an agent described herein. The agent may be prepared aseptically, filled into a syringe, and delivered refrigerated. Alternatively, the agent may be contained in a vial or other storage container. Another agent prepared aseptically may be contained in a second container. Alternatively, the kit may include a premixed active agent delivered in a syringe, vial, tube, or other container.

[0351] The kits may have various forms, such as blister pouches, shrink-wrapped pouches, vacuum-sealable pouches, sealable thermoformed trays, or similar pouch or tray forms, and may include accessories loosely packaged in a pouch, one or more tubes, containers, boxes, or bags. The kits may be sterilized after the accessories are added, allowing the individual accessories within the container to remain unpackaged as they would normally be. The kits can be sterilized using any suitable sterilization technique, such as radiation sterilization, heat sterilization, or other sterilization methods known in the art. The kits may also include other components depending on the particular application, such as containers, cell culture media, salts, buffers, reagents, syringes, needles, cloths such as gauze for applying or removing disinfectants, disposable gloves, drug supports prior to administration, etc.

[0352] The compositions of the kit may be provided in any suitable form, such as, for example, a liquid solution or a dry powder. When the composition provided is a dry powder, the powder may be reconstituted by adding a suitable solvent, which may be provided together. In embodiments in which a liquid form of the composition is used, the liquid form may be concentrated or ready-to-use. The solvent will depend on the compound and mode of use or administration. Solvents suitable for drug compositions are well known, and information is available in the literature. The solvent will depend on the compound and mode of use or administration.

[0353] In one set of embodiments, the kit may include compartmentalized carrier means for sealingly receiving one or more container means, such as vials, tubes, and the like, each containing one of the separate elements to be used in the method. For example, one of the containers may contain a positive control for the assay. Additionally, the kit may include containers for other components, such as, for example, buffers useful in the assay.

[0354] The present invention also encompasses pharmaceutical products that are packaged and labeled. The article of manufacture includes a suitable unit dosage form in a suitable vessel or container, such as a glass vial or other container that is hermetically sealed. For dosage forms suitable for parenteral administration, the active ingredient is sterile and suitable for administration as a particulate-free solution. In other words, the present invention encompasses both parenteral solutions and lyophilized powders, each of which is sterile, the latter being suitable for reconstitution prior to injection. Alternatively, the unit dosage form may be a solid suitable for oral, transdermal, topical, or transmucosal delivery.

[0355] In preferred embodiments, the unit dosage form is suitable for intravenous, intramuscular, or subcutaneous delivery. The present invention therefore encompasses solutions, which are preferably sterile and suitable for each delivery route.

[0356] In another preferred embodiment, the compositions of the present invention are stored in a container together with a biocompatible surfactant or other protein, including but not limited to lecithin, taurocholate, and cholesterol, and other proteins, including but not limited to gamma globulin and serum albumin. More preferably, the compositions of the present invention are stored together with human serum albumin for human use and with bovine serum albumin for veterinary use.

[0357] As with any pharmaceutical product, the packaging materials and containers are designed to protect the stability of the product during storage and shipping. Additionally, the products of the present invention include instructions for use or other informational material that informs a physician, technician, or patient on how to appropriately prevent or treat the disease or disorder in question. In other words, the article of manufacture includes instruction means that indicate or suggest a dosing regimen, including, but not limited to, actual doses, monitoring procedures (such as how to monitor mean absolute lymphocyte count, tumor cell count, and tumor size), and other monitoring information.

[0358] More specifically, the present invention provides an article of manufacture comprising packaging material, such as a box, bottle, tube, vial, container, nebulizer, inhaler, intravenous infusion (IV) bag, envelope, and the like, and at least one unit dose form of a pharmaceutical agent contained within the packaging material. The present invention also provides an article of manufacture comprising packaging material, such as a box, bottle, tube, vial, container, nebulizer, inhaler, intravenous infusion (IV) bag, envelope, and the like, and at least one unit dose form of a respective pharmaceutical agent contained within the packaging material. The present invention further provides an article of manufacture comprising packaging material, such as a box, bottle, tube, vial, container, nebulizer, inhaler, intravenous infusion (IV) bag, envelope, and the like, and at least one unit dose form of a respective pharmaceutical agent contained within the packaging material. The present invention further provides an article of manufacture comprising a needle or syringe (preferably packaged in a sterile form) for injection of the formulation, and / or a packaged alcohol pad.

[0359] The relative amounts of active ingredient, pharmaceutically acceptable excipient, and / or any additional components in a vaccine composition can vary depending on the identity, size, and / or condition of the subject being treated, as well as the route by which the composition will be administered. For example, the composition can contain 0.1%-99% (w / w) active ingredient. By way of example, the composition can contain 0.1%-100% (w / w) active ingredient, e.g., 0.5-50% (w / w), 1-30% (w / w), 5-80% (w / w), or at least 80% (w / w).

[0360] In some embodiments, the desired therapeutic, diagnostic, prophylactic, or imaging effect can be achieved by administering 0.0001 mg / kg to 100 mg / kg, 0.001 mg / kg to 0.05 mg / kg, 0.005 mg / kg to 0.05 mg / kg, 0.001 mg / kg to 0.005 mg / kg, 0.05 mg / kg to 0.5 mg / kg, 0.01 mg / kg to 50 mg / kg, 0.1 mg / kg to 40 mg / kg, 0.5 mg / kg, or 10 mg / kg of the same or similar amount per subject's body weight per day. The RNA (e.g., mRNA) vaccine composition may be administered once or multiple times daily, weekly, monthly, etc. at a dosage level sufficient to deliver up to 30 mg / kg, 0.01 mg / kg to 10 mg / kg, 0.1 mg / kg to 10 mg / kg, or 1 mg / kg to 25 mg / kg (see, e.g., unit dose ranges set forth in International Publication No. WO2013078199, which is incorporated herein by reference in its entirety). The desired dose may be delivered three times daily, twice daily, once daily, once every two days, once every three days, once weekly, once every two weeks, once every three weeks, once every four weeks, once every two months, once every three months, once every six months, etc. In certain embodiments, the desired dose may be delivered using multiple administrations (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more administrations). When multiple administrations are used, a split-dosing regimen, such as those described herein, may be used. In some embodiments, the RNA vaccine composition may be administered at a dose level sufficient to deliver 0.0005 mg / kg to 0.01 mg / kg, e.g., about 0.0005 mg / kg to about 0.0075 mg / kg, e.g., about 0.0005 mg / kg, about 0.001 mg / kg, about 0.002 mg / kg, about 0.003 mg / kg, about 0.004 mg / kg, or about 0.005 mg / kg.In some embodiments, the RNA vaccine composition may be administered once or twice (or more) at a dose level sufficient to deliver 0.025 mg / kg to 0.250 mg / kg, 0.025 mg / kg to 0.500 mg / kg, 0.025 mg / kg to 0.750 mg / kg, or 0.025 mg / kg to 1.0 mg / kg.

[0361] In some embodiments, the RNA vaccine composition comprises 0.0100 mg, 0.025 mg, 0.050 mg, 0.075 mg, 0.100 mg, 0.125 mg, 0.150 mg, 0.175 mg, 0.200 mg, 0.225 mg, 0.250 mg, 0.275 mg, 0.300 mg, 0.325 mg, 0.350 mg, 0.375 mg, 0.400 mg, 0.425 mg, 0.450 mg, 0.475 mg, 0.500 mg, 0.525 mg, 0.550 mg, 0.575 mg, 0.600 mg, 0.625 mg, 0.650 mg, 0.675 mg, 0.700 mg, 0.725 mg, 0.750 mg, 0.77 The vaccine may be administered twice (e.g., on days 0 and 7, 0 and 14, 0 and 21, 0 and 28, 0 and 60, 0 and 90, 0 and 120, 0 and 150, 0 and 180, 0 and 3 months, 0 and 6 months, 0 and 9 months, 0 and 12 months, 0 and 18 months, 0 and 2 years, 0 and 5 years, or 0 and 10 years) at a total dose of 5 mg, 0.800 mg, 0.825 mg, 0.850 mg, 0.875 mg, 0.900 mg, 0.925 mg, 0.950 mg, 0.975 mg, or 1.0 mg, or at a dose level sufficient to deliver that total dose. The present disclosure encompasses higher and lower doses and higher and lower frequency administration. For example, the RNA vaccine composition may be administered three or four times.

[0362] In some embodiments, the RNA vaccine composition may be administered twice (e.g., on days 0 and 7, 0 and 14, 0 and 21, 0 and 28, 0 and 60, 0 and 90, 0 and 120, 0 and 150, 0 and 180, 0 and 3 months later, 0 and 6 months later, 0 and 9 months later, 0 and 12 months later, 0 and 18 months later, 0 and 2 years later, 0 and 5 years later, or 0 and 10 years later) at a total dose of 0.010 mg, 0.025 mg, 0.100 mg, or 0.400 mg, or at a dose level sufficient to deliver that total dose.

[0363] In some embodiments, the RNA vaccine intended for use in a method of vaccinating a subject is administered to the subject in an amount effective to vaccinate the subject, such as a single dose of 10 μg / kg to 400 μg / kg of the nucleic acid vaccine. In some embodiments, the RNA vaccine intended for use in a method of vaccinating a subject is administered to the subject in an amount effective to vaccinate the subject, such as a single dose of 10 μg to 400 μg of the nucleic acid vaccine.

[0364] In some embodiments, an RNA vaccine composition can include a polynucleotide described herein formulated in lipid nanoparticles comprising MC3, cholesterol, DSPC, and PEG2000-DMG, trisodium citrate buffer, sucrose, and water for injection. As a non-limiting example, a composition can include 2.0 mg / mL of a drug substance (e.g., a polynucleotide encoding a cancer antigen), 21.8 mg / mL of MC3, 10.1 mg / mL of cholesterol, 5.4 mg / mL of DSPC, 2.7 mg / mL of PEG2000-DMG, 5.16 mg / mL of trisodium citrate, 71 mg / mL of sucrose, and 1.0 mL of water for injection.

[0365] In some embodiments, the nanoparticles (e.g., lipid nanoparticles) have an average diameter of 10 to 500 nm, 20 to 400 nm, 30 to 300 nm, or 40 to 200 nm. In some embodiments, the nanoparticles (e.g., lipid nanoparticles) have an average diameter of 50 to 150 nm, 50 to 200 nm, 80 to 100 nm, or 80 to 200 nm.

[0366] Flagellin is a monomeric protein of approximately 500 amino acids whose polymerization forms flagella, which are involved in bacterial movement. Flagellin is expressed by various flagellated bacteria (e.g., Salmonella typhimurium) and non-flagellated bacteria (e.g., Escherichia coli). Flagellin detection by cells of the innate immune system (e.g., dendritic cells, macrophages) is mediated by Toll-like receptor 5 (TLR5) and the Nod-like receptors (NLRs), Ipaf and Naip5. TLRs and NLRs have been identified to play roles in activating both innate and adaptive immune responses. Therefore, flagellin exerts an adjuvant effect in vaccines.

[0367] Nucleotide and amino acid sequences encoding known flagellin polypeptides are publicly available in NCBI's GenBank database. Known flagellin sequences include those from S. Typhimurium, H. pylori, V. Cholera, S. marcesens, S. flexneri, T. pallidum, L. pneumophila, B. burgdorferei, C. difficile, R. meliloti, A. tumefaciens, R. lupini, B. clarridgeiae, P. mirabilis, B. subtilus, L. monocytogenes, P. aeruginosa, and E. coli, among others.

[0368] As used herein, a flagellin polypeptide refers to a full-length flagellin protein, immunogenic fragments thereof, and peptides having at least 50% sequence identity to a flagellin protein or an immunogenic fragment thereof. Examples of flagellin proteins include flagellins from Salmonella typhi (UniPro Accession Number: Q56086), Salmonella typhimurium (A0A0C9DG09), Salmonella enteritidis (A0A0C9BAB7), and Salmonella choleraesuis (Q6V2X8), as well as proteins having an amino acid sequence identified by any one of SEQ ID NOS: 420-422 (Table 66). In some embodiments, a flagellin polypeptide has at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity to a flagellin protein or an immunogenic fragment thereof.

[0369] In some embodiments, the flagellin polypeptide is an immunogenic fragment. An immunogenic fragment is a portion of a flagellin protein that elicits an immune response. In some embodiments, the immune response is a TLR5-mediated immune response. An example of an immunogenic fragment is a flagellin protein in which all or part of the hinge region has been deleted or replaced with other amino acids. For example, an antigenic polypeptide may be inserted into the hinge region. The hinge region is the hypervariable region of flagellin. The hinge region of flagellin is also referred to as the "D3 domain or region," "propeller domain or region," "hypervariable domain or region," and "variable domain or region." As used herein, "at least a portion of the hinge region" refers to any portion of the hinge region of flagellin, or the entire hinge region. In other embodiments, the immunogenic fragment of flagellin is a 20-, 25-, 30-, 35-, or 40-amino acid C-terminal fragment of flagellin.

[0370] The flagellin monomer is formed by domains D0 to D3. D0 and D1 form a stem, consisting of a long tandem alpha helix, and are highly conserved among different bacteria. The D1 domain contains several amino acid stretches useful for TLR5 activation. The entire D1 domain or one or more active regions within the domain are immunogenic fragments of flagellin. Examples of immunogenic regions within the D1 domain include residues 88-114 and 411-431 in FliC, a flagellin from Salmonella typhimurium. Among the 13 amino acids in the 88-100 region, at least six substitutions are tolerated between Salmonella flagellin and other flagellins, and TLR5 activation is still preserved even with these substitutions. Thus, immunogenic fragments of flagellin include a flagellin-like sequence that activates TLR5 and contains a 13 amino acid motif that is 53% or more identical to the 88-100 Salmonella sequence of FliC (LQRVRELAVQSAN (SEQ ID NO: 428)).

[0371] In some embodiments, RNA (e.g., mRNA) vaccines comprise RNA encoding a fusion protein of flagellin and one or more antigenic polypeptides. As used herein, "fusion protein" refers to the linkage of two components of a construct. In some embodiments, the carboxy terminus of an antigenic polypeptide is fused or linked to the amino terminus of a flagellin polypeptide. In other embodiments, the amino terminus of an antigenic polypeptide is fused or linked to the carboxy terminus of a flagellin polypeptide. Fusion proteins can include, for example, one, two, three, four, five, six, or more flagellin polypeptides linked to one, two, three, four, five, six, or more antigenic polypeptides. When two or more flagellin polypeptides and / or two or more antigenic polypeptides are linked, such constructs can be referred to as "multimers."

[0372] The components of the fusion protein may be directly linked to each other or may be linked via a linker. For example, the linker may be an amino acid linker. The amino acid linker encoded by the RNA (e.g., mRNA) vaccine to link the components of the fusion protein may include, for example, at least one member selected from the group consisting of lysine residues, glutamic acid residues, serine residues, and arginine residues. In some embodiments, the linker is 1 to 30, 1 to 25, 1 to 25, 5 to 10, 5, 15, or 5 to 20 amino acids in length.

[0373] In other embodiments, the RNA (e.g., mRNA) vaccine comprises at least two separate RNA polynucleotides, one encoding one or more antigenic polypeptides and the other encoding a flagellin polypeptide, and the at least two RNA polynucleotides may be formulated together in a carrier such as a lipid nanoparticle.

[0374] Liposomes, lipoplexes, and lipid nanoparticles The RNA vaccines of the present invention can be formulated using one or more liposomes, lipoplexes, or lipid nanoparticles. In one embodiment, the pharmaceutical composition of the RNA vaccine comprises liposomes. Liposomes are artificially prepared vesicles that may be composed primarily of lipid bilayers and may be used as delivery vehicles for administering nutrients and pharmaceutical formulations. Liposomes can vary in size, including, but not limited to, multilamellar vesicles (MLVs), which may be hundreds of nanometers in diameter and contain a series of concentric bilayers separated by narrow aqueous compartments; small unilamellar vesicles (SUVs), which may be less than 50 nm in diameter; and large unilamellar vesicles (LUVs), which may be 50-500 nm in diameter. Liposome design can include, but is not limited to, improving liposome attachment to unhealthy tissue or the use of opsonins or ligands to activate events such as, but not limited to, endocytosis. Liposomes can also contain low or high pH moieties to improve delivery of pharmaceutical formulations.

[0375] The formation of liposomes can depend on physicochemical characteristics, including, but not limited to, the pharmaceutical agent and liposome components to be encapsulated, the nature of the medium in which the lipid vesicles are dispersed, the effective concentration of the encapsulated substance and its potential toxicity, any additional processes involved during application and / or delivery of the vesicles, optimized size, polydispersity, and shelf life of the vesicles for the intended use, and batch-to-batch reproducibility and the possibility of large-scale production of safe and efficient liposome products.

[0376] Non-limiting examples include liposomes such as synthetic membrane vesicles that may be prepared by the methods, devices, and apparatus described in U.S. Patent Publication Nos. US20130177638, US20130177637, US20130177636, US20130177635, US20130177634, US20130177633, US20130183375, US20130183373, and US20130183372, the contents of each of which are incorporated herein by reference in their entirety.

[0377] In one embodiment, the pharmaceutical compositions described herein may include liposomes such as, but not limited to, those formed from 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA) liposomes, DiLa2 liposomes supplied by Marina Biotech (Bothell, WA), 1,2-dilinoleyloxy-3-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), and MC3...

Claims

1. an mRNA comprising an open reading frame (ORF) encoding at least one cancer antigen epitope and a compound of formula (I): 【Chemistry 1】 or a salt thereof; and a lipid nanoparticle comprising the mRNA cancer vaccine, During the ceremony, R １ But, C ５－３０ Alkyl, C ５－２０ alkenyl, and -R"M'R'; R ２ and R ３ But, C １－１４ Alkyl and C ２－１４ alkenyl, R ４ But -(CH ２ ) ｎ Q, Q is -OR and n is selected from 1, 2, 3, 4, and 5; R ５ are H, R ６ are H, M and M' are independently selected from -C(O)O- and -OC(O)-; R ７ is H, R is H, R' is C １－１８ Alkyl and C ２－１８ alkenyl, R” is C ３－１４ Alkyl and C ３－１４ alkenyl, and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13; mRNA cancer vaccines.

2. The mRNA cancer vaccine of claim 1 , wherein the mRNA comprises at least one chemical modification.

3. The mRNA cancer vaccine of claim 2, wherein the at least one chemical modification is N1-methylpseudouridine.

4. The mRNA cancer vaccine according to any one of claims 1 to 3, wherein the lipid nanoparticle is a lipid nanoparticle comprising 20-60% of an ionizable cationic lipid of formula (I), 5-25% of a neutral lipid, 25-55% of cholesterol, and 0.5-15% of a PEG-modified lipid.

5. The mRNA cancer vaccine of any one of claims 1 to 4, wherein the ORF encodes at least two cancer antigen epitopes.

6. The mRNA cancer vaccine of any one of claims 1 to 5, wherein the ORF encodes 5 to 100 cancer antigen epitopes.

7. The mRNA cancer vaccine of any one of claims 1 to 6, wherein the ORF encodes 15 to 50 cancer antigen epitopes.

8. The mRNA cancer vaccine of any one of claims 1 to 7, wherein the ORF encodes 20 to 50 cancer antigen epitopes.

9. The mRNA cancer vaccine of any one of claims 1 to 8, wherein the ORF encodes 25 to 50 cancer antigen epitopes.

10. The mRNA cancer vaccine of any one of claims 1 to 9, wherein the ORF encodes 30 to 50 cancer antigen epitopes.

11. An mRNA cancer vaccine described in any one of claims 1 to 10, wherein the ORF encodes 30 to 40 cancer antigen epitopes.

12. The mRNA cancer vaccine of any one of claims 5 to 11, wherein the at least two cancer antigen epitopes are arranged in a head-to-tail structure.

13. The mRNA cancer vaccine of any one of claims 6 to 12, wherein at least 50% of the cancer antigen epitopes specifically bind to one or more of HLA-A, HLA-B, and HLA-DR.

14. The mRNA cancer vaccine of claim 13, wherein at least 90% of the cancer antigen epitopes specifically bind to one or more of HLA-A, HLA-B, and HLA-DR.

15. The mRNA cancer vaccine of claim 14, wherein 100% of the cancer antigen epitopes specifically bind to one or more of HLA-A, HLA-B, and HLA-DR.

16. 16. The mRNA cancer vaccine of claim 15, wherein 100% of the cancer antigen epitopes specifically bind to one or more of HLA-A, HLA-B, HLA-DR, HLA-DRB1, HLA-C. HLA-A / A', HLA-B / B', HLA-C / C', HLA-DRB1 / B1', HLA-DRB345 / DRB3'4'5', HLA-DPA / B, HLA-DPA' / B', HLA-DPA' / B, HLA-DPA / B', HLA-DQA / B, HLA-DQA' / B', HLA-DQA' / B, and HLA-DQA / B'.

17. The mRNA cancer vaccine of any one of claims 5 to 16, wherein the cancer antigen epitope comprises at least one MHC class I epitope and at least one MHC class II epitope.

18. The mRNA cancer vaccine of claim 17, wherein at least 30% of the cancer antigen epitopes are MHC class I epitopes.

19. The mRNA cancer vaccine of claim 17 or 18, wherein at least 50% of the cancer antigen epitopes are MHC class I epitopes.

20. The mRNA cancer vaccine of any one of claims 17 to 19, wherein at least 60% of the cancer antigen epitopes are MHC class I epitopes.

21. The mRNA cancer vaccine of any one of claims 17 to 20, wherein at least 70% of the cancer antigen epitopes are MHC class I epitopes.

22. The mRNA cancer vaccine of any one of claims 17 to 21, wherein at least 80% of the cancer antigen epitopes are MHC class I epitopes.

23. the cancer antigen epitopes are all T cell epitopes, all B cell epitopes or a combination of both; 23. The mRNA cancer vaccine of any one of claims 5 to 22, optionally wherein the T cell epitope comprises 9-26 amino acids and / or the B cell epitope comprises 13-17 amino acids.

24. The mRNA cancer vaccine of claim 23, wherein the cancer antigen epitopes are all T-cell epitopes.

25. each cancer antigen epitope comprises 9-29 amino acids and contains a centrally located SNP mutation; Optionally, each cancer antigen epitope comprises 25 amino acids and includes a centrally located SNP mutation with 12 adjacent amino acids on either side of the SNP mutation. The mRNA cancer vaccine of any one of claims 1-24.

26. The compound of formula (I) 【Chemistry 2】 The mRNA cancer vaccine according to any one of claims 1 to 25,

27. The mRNA cancer vaccine of any one of claims 1 to 26, further comprising an immune checkpoint regulator.

28. 28. The mRNA cancer vaccine of claim 27, wherein the immune checkpoint modulator is an inhibitory checkpoint polypeptide.

29. 29. The mRNA cancer vaccine of claim 28, wherein the inhibitory checkpoint polypeptide is an antibody or fragment thereof that specifically binds to a molecule selected from the group consisting of PD-1, TIM-3, VISTA, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, and LAG3.

30. 29. The mRNA cancer vaccine of claim 28, wherein the inhibitory checkpoint polypeptide is an anti-CTLA4 antibody or an anti-PD1 antibody.

31. The mRNA cancer vaccine of claim 30, wherein the anti-PD1 antibody is pembrolizumab.

32. The method of claim 31, wherein the ORF comprises at least two cancer antigen epitopes, and the vaccine comprises: a) the cancer antigen epitopes encoded by the ORFs are linked via a linker; b) the cancer antigen epitopes encoded by the ORFs are directly linked to each other; and c) the cancer antigen epitopes encoded by the ORFs are linked to each other by a single amino acid linker; The mRNA cancer vaccine of any one of claims 1 to 31, further comprising one or more features selected from the group consisting of:

33. The mRNA cancer vaccine of any one of claims 1 to 32, wherein the ORF encodes 10 to 100 cancer antigen epitopes, and at least 10 of the cancer antigen epitopes encoded by the ORF are MHC class I epitopes.

34. The mRNA cancer vaccine of any one of claims 1 to 32, wherein the ORF encodes 20 to 100 cancer antigen epitopes, and at least 20 of the cancer antigen epitopes encoded by the ORF are MHC class I epitopes.

35. The mRNA cancer vaccine of any one of claims 1 to 34, wherein the mRNA further comprises a 5'-end cap that is 7mG(5')ppp(5')NlmpNp.

36. The mRNA cancer vaccine of any one of claims 1 to 35, wherein the mRNA does not contain a stabilizing element.

37. The mRNA cancer vaccine of any one of claims 1 to 36, comprising a recall antigen.

38. The mRNA cancer vaccine described in any one of claims 1 to 37, wherein the lipid nanoparticles comprise about 50 mol% of an ionizable cationic lipid of formula (I), about 10 mol% of a neutral lipid, about 38.5 mol% of cholesterol, and about 1.5 mol% of a PEG-modified lipid.

39. The compound of formula (I): 【Chemistry 3】 The mRNA cancer vaccine of claim 38, wherein the neutral lipid is DSPC and the PEG-modified lipid is PEG2000-DMG.

40. An mRNA cancer vaccine described in any one of claims 1 to 39, wherein at least one of the cancer antigen epitopes contains 9 to 29 amino acids.

41. 1. An mRNA vaccine for use in a method of eliciting an immune response in a subject, the method comprising identifying at least two cancer antigen epitopes from a biological sample isolated from the subject, and administering the mRNA vaccine to the subject; The mRNA vaccine comprises (i) an mRNA having an open reading frame (ORF) encoding at least two cancer antigen epitopes and (ii) a lipid particle, the lipid particle comprising a compound of formula (I): 【Chemistry 3】 or a salt thereof, During the ceremony, R １ But, C ５－３０ Alkyl, C ５－２０ alkenyl, and -R"M'R'; R ２ and R ３ But, C １－１４ Alkyl and C ２－１４ alkenyl, R ４ But -(CH ２ ) ｎ Q, Q is -OR and n is selected from 1, 2, 3, 4, and 5; R ５ are H, R ６ are H, M and M' are independently selected from -C(O)O- and -OC(O)-; R ７ is H, R is H, R' is C １－１８ Alkyl and C ２－１８ alkenyl, R” is C ３－１４ Alkyl and C ３－１４ alkenyl, and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13; mRNA vaccine.

42. The mRNA vaccine of claim 41 , wherein the mRNA comprises at least one chemical modification.

43. The mRNA vaccine of claim 42, wherein the at least one chemical modification is N1-methylpseudouridine.

44. The mRNA vaccine according to any one of claims 41 to 43, which is used in combination with a cancer therapeutic agent or an anticancer agent.

45. The mRNA vaccine of claim 44, wherein the cancer therapeutic or anti-cancer agent is selected from the group consisting of a targeted therapy and a T cell therapy, wherein the targeted therapy comprises a BRAF inhibitor and the T cell therapy comprises a checkpoint inhibitor.

46. The mRNA vaccine of claim 44, wherein the cancer therapeutic or anti-cancer agent comprises a checkpoint inhibitor.

47. The mRNA vaccine described in claim 46, wherein the checkpoint inhibitor is an anti-PD1 antibody.

48. An mRNA vaccine described in any one of claims 41 to 43, used in combination with a checkpoint inhibitor.

49. The mRNA vaccine described in claim 48, wherein the checkpoint inhibitor is an anti-PD1 antibody.

50. The mRNA vaccine of any one of claims 41 to 49, wherein the lipid particle is a lipid nanoparticle comprising 20-60% ionizable cationic lipid of formula (I), 5-25% neutral lipid, 25-55% cholesterol and 0.5-15% PEG-modified lipid.

51. The compound of formula (I) 【Chemistry 4】 The mRNA vaccine according to any one of claims 41 to 50,

52. The mRNA vaccine described in any one of claims 41 to 51, wherein the lipid particle comprises about 50 mol% ionizable cationic lipid of formula (I), about 10 mol% neutral lipid, about 38.5 mol% cholesterol and about 1.5 mol% PEG-modified lipid.

53. The compound of formula (I): 【Chemistry 5】 The mRNA vaccine of claim 52, wherein the neutral lipid is DSPC and the PEG-modified lipid is PEG2000-DMG.

54. The mRNA vaccine of any one of claims 41 to 53, wherein the ORF encodes 5 to 100 cancer antigen epitopes.

55. An mRNA vaccine described in any one of claims 41 to 53, wherein the ORF encodes 15 to 50 cancer antigen epitopes.

56. An mRNA vaccine described in any one of claims 41 to 53, wherein the ORF encodes 20 to 50 cancer antigen epitopes.

57. An mRNA vaccine described in any one of claims 41 to 53, wherein the ORF encodes 25 to 50 cancer antigen epitopes.

58. An mRNA vaccine described in any one of claims 41 to 53, wherein the ORF encodes 30 to 50 cancer antigen epitopes.

59. An mRNA vaccine described in any one of claims 41 to 53, wherein the ORF encodes 30 to 40 cancer antigen epitopes.

60. An mRNA vaccine described in any one of claims 41 to 59, wherein the at least two cancer antigen epitopes are arranged in a head-to-tail structure.

61. An mRNA vaccine described in any one of claims 41 to 60, wherein at least 50% of the cancer antigen epitopes specifically bind to one or more of HLA-A, HLA-B, and HLA-DR.

62. An mRNA vaccine described in any one of claims 41 to 60, wherein at least 90% of the cancer antigen epitopes specifically bind to one or more of HLA-A, HLA-B, and HLA-DR.

63. An mRNA vaccine described in any one of claims 41 to 60, wherein 100% of the cancer antigen epitopes specifically bind to one or more of HLA-A, HLA-B, and HLA-DR.

64. An mRNA vaccine described in any one of claims 41 to 63, wherein the cancer antigen epitope includes at least one MHC class I epitope and at least one MHC class II epitope.

65. An mRNA vaccine described in any one of claims 41 to 64, wherein at least 30% of the cancer antigen epitopes are MHC class I epitopes.

66. An mRNA vaccine described in any one of claims 41 to 64, wherein at least 50% of the cancer antigen epitopes are MHC class I epitopes.

67. An mRNA vaccine described in any one of claims 41 to 64, wherein at least 60% of the cancer antigen epitopes are MHC class I epitopes.

68. An mRNA vaccine described in any one of claims 41 to 64, wherein at least 70% of the cancer antigen epitopes are MHC class I epitopes.

69. An mRNA vaccine described in any one of claims 41 to 64, wherein at least 80% of the cancer antigen epitopes are MHC class I epitopes.

70. The cancer antigen epitopes are all T cell epitopes, all B cell epitopes, or a combination of both; 70. The mRNA vaccine of any one of claims 41-69, optionally wherein the T cell epitope comprises 9-26 amino acids and / or the B cell epitope comprises 13-17 amino acids.

71. An mRNA vaccine described in any one of claims 41 to 70, wherein the cancer antigen epitopes are all T-cell epitopes.

72. Each cancer antigen epitope comprises 9 to 29 amino acids and a centrally located SNP mutation; Optionally, each cancer antigen epitope comprises 25 amino acids and a centrally located SNP mutation with 12 adjacent amino acids on either side of the SNP mutation. The mRNA vaccine of any one of claims 41-71.

73. The vaccine comprising: a) the cancer antigen epitopes encoded by the ORFs are linked via a linker; b) the cancer antigen epitopes encoded by the ORFs are directly linked to each other; and c) the cancer antigen epitopes encoded by the ORFs are linked to each other by a single amino acid linker; 73. The mRNA vaccine of any one of claims 41 to 72, comprising one or more features selected from the group consisting of:

74. An mRNA vaccine described in any one of claims 41 to 73, wherein the ORF encodes 10 to 100 cancer antigen epitopes, and at least 10 of the cancer antigen epitopes encoded by the ORF are MHC class I epitopes.

75. The mRNA vaccine of any one of claims 41 to 74, wherein the ORF encodes 20 to 100 cancer antigen epitopes, and at least 20 of the cancer antigen epitopes encoded by the ORF are MHC class I epitopes.

76. The mRNA vaccine of any one of claims 41 to 75, wherein the mRNA further comprises a 5'-end cap that is 7mG(5')ppp(5')NlmpNp.

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