RNA cancer vaccines

Abstract

The present disclosure relates to cancer ribonucleic acid (RNA) vaccines and methods for using such vaccines and compositions comprising such vaccines. In particular, the present disclosure relates to concatemeric mRNA cancer vaccines that encode several cancer epitopes on a single mRNA construct, i.e., polyepitope mRNA constructs or polyneoepitope constructs. The present disclosure also relates to the incorporation of immune-enhancing agents such as p53 and KRAS mutations and STING, for example, the mRNA construct further encodes an immunostimulatory agent or adjuvant. The present disclosure also relates to the inclusion of universal T-cell epitopes, such as tetanus toxin or diphtheria toxin, which induce an enhanced immune response.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. §119(e) to the filing dates of U.S. Provisional Patent Application No. 62 / 453,444, entitled "RNA CANCER VACCINES," filed February 1, 2017; U.S. Provisional Patent Application No. 62 / 453,465, entitled "IMMUNOMODULATORY THERAPEUTIC MRNA COMPOSITIONS ENCODING ACTIVATING ONCOGENE MUTATION PEPTIDES," filed February 1, 2017; and U.S. Provisional Patent Application No. 62 / 558,238, entitled "CONCATAMERIC RNA CANCER VACCINES," filed September 13, 2017, the contents of each of which are incorporated herein by reference in their entirety. [Background technology]

[0002] Recent theories of cancer evolution focus on three stages, including stress-induced genomic instability, population diversity or heterogeneity, and genome-mediated macroevolution. This theory explains why, although most known molecular mechanisms may be involved in cancer, no single mechanism predominates for the majority of clinical cases. However, common mechanisms suggest that cancer vaccines may provide a universal solution for cancer treatment.

[0003] 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. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Prior et al.Cancer Res.2012 May 15;72(10):2457-2467 [Non-patent document 2] Diaz et al.The molecular evolution of acquired resistance to targeted EGFR blockade in colorectal cancers,Nature 486:537(2012) [Non-patent document 3] Misale et al.Emergence of KRAS muations and acquired resistance to anti-EGFR therapy in colorectal cancer,Nature 486:532(2012) Summary of the Invention [Problem to be solved by the invention]

[0005] Most commercially available or under development vaccines (e.g., cancer vaccines) 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 from 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. [Means for solving the problem]

[0006] 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.

[0007] RNA vaccines may be used in various settings depending on the prevalence of cancer or the degree 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 treatments and vaccines that are produced ex vivo and may induce undesirable cellular responses, RNA vaccines are presented to cell lines in a more natural manner.

[0008] An RNA vaccine may comprise a 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.

[0009] In some aspects, the present disclosure relates to an mRNA cancer vaccine of one or more mRNAs, each having an open reading frame encoding a cancer antigen peptide epitope, formulated in a lipid nanoparticle and a pharmaceutically acceptable carrier or excipient, wherein the mRNA vaccine encodes between 5 and 100 peptide epitopes, and at least two of the peptide epitopes are personalized cancer antigens.

[0010] In some embodiments, the present disclosure provides an mRNA cancer vaccine comprising lipid nanoparticles containing one or more mRNAs, each having one or more open reading frames encoding 1 to 500 peptide epitopes that are personalized cancer antigens and a universal type II T cell epitope.

[0011] In some embodiments, the present disclosure provides an mRNA cancer vaccine comprising lipid nanoparticles comprising one or more of the following: (a) one or more mRNAs, each having one or more open reading frames encoding 1 to 500 peptide epitopes that are personalized cancer antigens and a universal type II T cell epitope; (b) one or more mRNAs, each having an open reading frame encoding an activating oncogene mutant peptide, optionally wherein the mRNA further comprises a universal type II T cell epitope; (c) one or more mRNAs, each having an open reading frame encoding a cancer antigen peptide epitope. and / or (d) one or more mRNAs each having an open reading frame encoding a cancer antigen peptide epitope, wherein the mRNA encodes 5-100 peptide epitopes, at least three of which are compound variants and at least two of which are point mutations, and optionally the mRNA further comprises a universal type II T cell epitope. In some embodiments, the mRNA cancer vaccine encodes 1-20 universal type II T cell epitopes. In other embodiments, the universal type II T cell epitope is selected from the group consisting of ILMQYIKANSKFIGI (tetanus toxin; SEQ ID NO: 226), FNNFTVSFWLRVPKVSASHLE (tetanus toxin; SEQ ID NO: 227), QYIKANSKFIGITE (tetanus toxin; SEQ ID NO: 228) QSIALSSLMVAQAIP (diphtheria toxin; SEQ ID NO: 229), and AKFVAAWTLKAAA (pan-DR epitope; SEQ ID NO: 230).

[0012] In some embodiments, the universal type II T cell epitope is the same throughout the mRNA. In other embodiments, the universal type II T cell epitope is repeated 1 to 20 times in the mRNA. In one embodiment, the universal type II T cell epitopes are different from each other throughout the mRNA. In some embodiments, the universal type II T cell epitope is located between every cancer antigen peptide epitope. In another embodiment, the universal type II T cell epitope is located between every other cancer antigen peptide epitope. In one embodiment, the universal type II T cell epitope is located between every third cancer antigen peptide epitope.

[0013] In some embodiments, one or more of the following conditions are met: (i) the activating oncogene mutation is a KRAS mutation; (ii) the KRAS mutation is a G12 mutation, optionally wherein the G12 KRAS mutation is selected from a G12D, G12V, G12S, G12C, G12A, and G12R KRAS mutation; (iii) the KRAS mutation is a G13 mutation, optionally wherein the G13 KRAS mutation is a G13D KRAS mutation; and / or (iv) the activating oncogene mutation is an H-RAS or N-RAS mutation.

[0014] In some embodiments, one or more of the following conditions are met: (A) the mRNA has an open reading frame encoding a concatemer of two or more activating oncogene mutant peptides; (B) at least two of the peptide epitopes are separated from each other by a single glycine, and optionally, all of the peptide epitopes are separated from each other by a single glycine; (C) the concatemer comprises 3 to 10 activating oncogene mutant peptides; and / or (D) at least two of the peptide epitopes are directly linked to each other without a linker.

[0015] In certain embodiments, one or more of the following conditions are met: (i) at least one of the peptide epitopes is a conventional cancer antigen; (ii) at least one of the peptide epitopes is a repeat polymorphism; (iii) the repeat polymorphism comprises a recurrent somatic cancer mutation in p53; (iv) the recurrent somatic cancer mutation in p53 is (A) a mutation in the canonical 5' splice site adjacent to codon position T125, which is the epitope AVSPCISFVW (SEQ ID NO: 233) (HLA-B * 57:01, HLA-B * 58:01), epitope HPLASCQCFF (SEQ ID NO: 234) (HLA-B * 35:01, HLA-B * 53:01), epitope FVWNFGIPL (SEQ ID NO: 235) (HLA-A * 02:01, HLA-A * 02:06, HLA-B * (B) a mutation in the canonical 5' splice site adjacent to codon position 331, resulting in the epitope LQVLSLGTSY (SEQ ID NO: 237) (HLA-B * 15:01), epitope FQSNTQNAVF (SEQ ID NO: 238) (HLA-B * (C) a mutation in the canonical 3' splice site adjacent to codon position 126 that results in the epitope CTMFCQLAK (SEQ ID NO: 240) (HLA-A * 11:01), epitope KSVTCTMF (SEQ ID NO: 241) (HLA-B *and / or (D) a mutation in the canonical 5' splice site adjacent to codon position 224 that results in the epitope VPYEPPEVW (SEQ ID NO: 243) (HLA-B * 53:01, HLA-B * 51:01), epitope LTVPPSTAW (SEQ ID NO: 244) (HLA-B * 58:01, HLA-B * and / or (v) the mRNA cancer vaccine is selected from the group consisting of a mutation that induces a cryptic alternative intron 5' splice site generating a novel spanning peptide sequence VPYEPPEVWLALTVPPSTAWAA (SEQ ID NO: 242) containing the sequence: VPYEPPEVWLALTVPPSTAWAA (SEQ ID NO: 57:01) (transcription codon positions are with reference to ENST00000269305 (SEQ ID NO: 245), the standard full-length p53 transcript from Ensembl's v83 human genome annotation); and / or

[0016] In some embodiments, the one or more mRNAs further comprise an open reading frame encoding an immune-enhancing agent. In other embodiments, the immune-enhancing agent is formulated in a lipid nanoparticle. In one embodiment, the immune-enhancing agent is formulated in a separate lipid nanoparticle. In some embodiments, the immune-enhancing agent is a constitutively active human STING polypeptide. In one embodiment, the constitutively active human STING polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1. In another embodiment, the mRNA encoding the constitutively active human STING polypeptide comprises the nucleotide sequence set forth in SEQ ID NO: 170. In some embodiments, the mRNA encoding the constitutively active human STING polypeptide comprises a 3'UTR having a miR-122 microRNA-binding site. In one embodiment, the miR-122 microRNA-binding site comprises the nucleotide sequence set forth in SEQ ID NO: 175.

[0017] In some embodiments, the one or more mRNAs each comprise a 5' UTR comprising the nucleotide sequence set forth in SEQ ID NO: 176. In one embodiment, the one or more mRNAs each comprise a poly-A tail. In one embodiment, the poly-A tail comprises about 100 nucleotides. In some embodiments, the one or more mRNAs each comprise a 5' Cap 1 structure.

[0018] In some embodiments, one or more mRNAs comprise at least one chemical modification. In one embodiment, the chemical modification is N1-methylpseudouridine. In another embodiment, one or more mRNAs are fully modified with N1-methylpseudouridine.

[0019] In some embodiments, the one or more mRNAs encode 45 to 55 personalized cancer antigens. In one embodiment, the one or more mRNAs encode 52 personalized cancer antigens. In some embodiments, each personalized cancer antigen is encoded by a separate open reading frame. In another embodiment, the peptide epitopes are in the form of concatemeric cancer antigens composed of 2 to 100 peptide epitopes, optionally, the concatemeric cancer antigens are composed of 5 to 100 peptide epitopes.

[0020] In some embodiments, the concatemeric cancer antigen comprises: a) 2-100 peptide epitopes or 5-100 peptide epitopes interspersed with cleavage-sensitive sites; b) the mRNAs encoding each peptide epitope are directly linked to each other without a linker; c) the mRNAs encoding each peptide epitope are linked to each other by a single nucleotide linker; d) each peptide epitope comprises 25-35 amino acids and contains a centrally located SNP mutation; e) at least 30% of the peptide epitopes have the highest affinity for class I MHC molecules derived from the subject; f) at least 30% of the peptide epitopes have the highest affinity for class II MHC molecules derived from the subject; g) at least 50% of the peptide epitopes are HLA- h) the mRNA encodes 45-55 peptide epitopes; i) the mRNA encodes 52 peptide epitopes; j) 50% of the peptide epitopes have binding affinity for MHC class I and 50% of the peptide epitopes have binding affinity for MHC class II; k) the mRNA encoding the peptide epitopes is arranged such that the order of the peptide epitopes minimizes pseudo-epitopes; l) at least 30% of the peptide epitopes are 15 amino acids in length, MHC class I-binding peptides; and / or m) at least 30% of the peptide epitopes are 21 amino acids in length, MHC class II-binding peptides.

[0021] In some aspects, the present disclosure provides mRNA cancer vaccines comprising one or more mRNAs, each having one or more open reading frames encoding 45 to 55 peptide epitopes that are personalized cancer antigens, formulated in lipid nanoparticles.

[0022] In some aspects, the present disclosure provides mRNA cancer vaccines comprising one or more mRNAs each having one or more open reading frames encoding 45 to 55 peptide epitopes that are personalized cancer antigens, formulated in lipid nanoparticles, wherein optionally, at least one of the peptide epitopes is an activating oncogene mutant peptide or a conventional cancer antigen, and optionally, at least three of the peptide epitopes are complex variants and at least two of the peptide epitopes are point mutations.

[0023] In some embodiments, the one or more mRNAs encode 48 to 54 personalized cancer antigens. In one embodiment, the one or more mRNAs encode 52 personalized cancer antigens. In some embodiments, each of the personalized cancer antigens is encoded by a separate open reading frame.

[0024] In another embodiment, the peptide epitopes are in the form of concatemeric cancer antigens composed of 2 to 100 peptide epitopes, optionally the concatemeric cancer antigens are composed of 5 to 100 peptide epitopes. In some embodiments, the concatemeric cancer antigen comprises: a) 2-100 peptide epitopes or 5-100 peptide epitopes interspersed with cleavage-sensitive sites; b) the mRNAs encoding each peptide epitope are directly linked to each other without a linker; c) the mRNAs encoding each peptide epitope are linked to each other by a single nucleotide linker; d) each peptide epitope comprises 25-35 amino acids and contains a centrally located SNP mutation; e) at least 30% of the peptide epitopes have the highest affinity for class I MHC molecules derived from the subject; f) at least 30% of the peptide epitopes have the highest affinity for class II MHC molecules derived from the subject; g) at least 50% of the peptide epitopes are HLA- h) the mRNA encodes 45-55 peptide epitopes; i) the mRNA encodes 52 peptide epitopes; j) 50% of the peptide epitopes have binding affinity for MHC class I and 50% of the peptide epitopes have binding affinity for MHC class II; k) the mRNA encoding the peptide epitopes is arranged such that the order of the peptide epitopes minimizes pseudo-epitopes; l) at least 30% of the peptide epitopes are 15 amino acids in length, MHC class I-binding peptides; and / or m) at least 30% of the peptide epitopes are 21 amino acids in length, MHC class II-binding peptides.

[0025] In some embodiments, at least two of the peptide epitopes are separated from each other by a universal type II T cell epitope. In one embodiment, all of the peptide epitopes are separated from each other by a universal type II T cell epitope. In another embodiment, the mRNA cancer vaccine encodes 1 to 20 universal type II T cell epitopes.

[0026] In some embodiments, the universal type II T cell epitope is selected from the group consisting of ILMQYIKANSKFIGI (tetanus toxin; SEQ ID NO: 226), FNNFTVSFWLRVPKVSASHLE (tetanus toxin; SEQ ID NO: 227), QYIKANSKFIGITE (tetanus toxin; SEQ ID NO: 228) QSIALSSLMVAQAIP (diphtheria toxin; SEQ ID NO: 229), and AKFVAAWTLKAAA (pan-DR epitope; SEQ ID NO: 230).

[0027] In one embodiment, the universal type II T cell epitope is the same throughout the mRNA. In some embodiments, the universal type II T cell epitope is repeated 1 to 20 times in the mRNA. In another embodiment, the universal type II T cell epitopes are different from each other throughout the mRNA. In one embodiment, the universal type II T cell epitope is located between every peptide epitope. In some embodiments, the universal type II T cell epitope is located between every other cancer antigen peptide epitope. In one embodiment, the universal type II T cell epitope is located between every third peptide epitope.

[0028] In some embodiments, the one or more mRNAs further comprise an open reading frame encoding an immune enhancing agent. In one embodiment, the immune enhancing agent is formulated in a lipid nanoparticle. In another embodiment, the immune enhancing agent is formulated in a separate lipid nanoparticle. In some embodiments, the immune enhancing agent is a constitutively active human STING polypeptide. In one embodiment, the constitutively active human STING polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1. In another embodiment, the mRNA encoding the constitutively active human STING polypeptide comprises the nucleotide sequence set forth in SEQ ID NO: 170.

[0029] In some embodiments, one or more of the following conditions are met: (i) the activating oncogene mutation is a KRAS mutation; (ii) the KRAS mutation is a G12 mutation, optionally wherein the G12 KRAS mutation is selected from a G12D, G12V, G12S, G12C, G12A, and G12R KRAS mutation; (iii) the KRAS mutation is a G13 mutation, optionally wherein the G13 KRAS mutation is a G13D KRAS mutation; and / or (iv) the activating oncogene mutation is an H-RAS or N-RAS mutation.

[0030] In certain embodiments, one or more of the following conditions are met: (A) the mRNA has an open reading frame encoding a concatemer of two or more activating oncogene mutant peptides; (B) at least two of the peptide epitopes are separated from each other by a single glycine, and optionally, all of the peptide epitopes are separated from each other by a single glycine; (C) the concatemer contains between 3 and 10 activating oncogene mutant peptides; and / or (D) at least two of the peptide epitopes are directly linked to each other without a linker.

[0031] In specific embodiments, one or more of the following conditions are met: (i) at least one of the peptide epitopes is a conventional cancer antigen; (ii) at least one of the peptide epitopes is a repeat polymorphism; (iii) the repeat polymorphism comprises a recurrent somatic cancer mutation in p53; (iv) the recurrent somatic cancer mutation in p53 is (A) a mutation in the canonical 5' splice site adjacent to codon position T125, which is the epitope AVSPCISFVW (SEQ ID NO: 233) (HLA-B * 57:01, HLA-B * 58:01), epitope HPLASCQCFF (SEQ ID NO: 234) (HLA-B * 35:01, HLA-B * 53:01), epitope FVWNFGIPL (SEQ ID NO: 235) (HLA-A * 02:01, HLA-A * 02:06, HLA-B * (B) a mutation in the canonical 5' splice site adjacent to codon position 331, resulting in the epitope LQVLSLGTSY (SEQ ID NO: 237) (HLA-B * 15:01), epitope FQSNTQNAVF (SEQ ID NO: 238) (HLA-B * (C) a mutation in the canonical 3' splice site adjacent to codon position 126 that results in the epitope CTMFCQLAK (SEQ ID NO: 240) (HLA-A * 11:01), epitope KSVTCTMF (SEQ ID NO: 241) (HLA-B *and / or (D) a mutation in the canonical 5' splice site adjacent to codon position 224 that results in the epitope VPYEPPEVW (SEQ ID NO: 243) (HLA-B * 53:01, HLA-B * 51:01), epitope LTVPPSTAW (SEQ ID NO: 244) (HLA-B * 58:01, HLA-B * and / or (v) the mRNA cancer vaccine is selected from the group consisting of a mutation that induces a cryptic alternative intron 5' splice site generating a novel spanning peptide sequence VPYEPPEVWLALTVPPSTAWAA (SEQ ID NO: 242) containing the sequence: VPYEPPEVWLALTVPPSTAWAA (SEQ ID NO: 57:01) (transcription codon positions are with reference to ENST00000269305 (SEQ ID NO: 245), the standard full-length p53 transcript from Ensembl's v83 human genome annotation); and / or

[0032] Another aspect of the present disclosure is an mRNA cancer vaccine comprising lipid nanoparticles comprising: (i) one or more mRNAs each having one or more open reading frames encoding 1 to 500 peptide epitopes that are personalized cancer antigens; and (ii) an mRNA having an open reading frame encoding a polypeptide that enhances an immune response to the personalized cancer antigen, optionally wherein (i) and (ii) are present in a mass ratio of approximately 5:1.

[0033] Another aspect of the present disclosure is an mRNA cancer vaccine comprising lipid nanoparticles comprising: (i) one or more mRNAs each having one or more open reading frames encoding 1 to 500 peptide epitopes that are personalized cancer antigens; and (ii) an mRNA having an open reading frame encoding a polypeptide that enhances an immune response to the personalized cancer antigen, optionally wherein (i) and (ii) are present in a mass ratio of approximately 5:1; optionally, at least one of the peptide epitopes is an activating oncogene mutant peptide or a conventional cancer antigen; and optionally, at least three of the peptide epitopes are complex variants and at least two of the peptide epitopes are point mutations.

[0034] In some embodiments, the immune response comprises a cellular or humoral immune response characterized by (i) stimulating type I interferon pathway signaling, (ii) stimulating NFkB pathway signaling, (iii) stimulating an inflammatory response, (iv) stimulating cytokine production, or (v) stimulating dendritic cell development, activity, or recruitment, and (vi) any combination of (i)-(vi).

[0035] In one embodiment, the mRNA cancer vaccine comprises a single mRNA construct encoding both a peptide epitope and a polypeptide that enhances an immune response to a personalized cancer antigen. In another embodiment, the peptide epitope is in the form of a concatemeric cancer antigen composed of 2 to 100 peptide epitopes, and optionally, the concatemeric cancer antigen is composed of 5 to 100 peptide epitopes.

[0036] In some embodiments, the concatemeric cancer antigen comprises: a) 2-100 peptide epitopes or 5-100 peptide epitopes interspersed with cleavage-sensitive sites; b) the mRNAs encoding each peptide epitope are directly linked to each other without a linker; c) the mRNAs encoding each peptide epitope are linked to each other by a single nucleotide linker; d) each peptide epitope comprises 25-35 amino acids and contains a centrally located SNP mutation; e) at least 30% of the peptide epitopes have the highest affinity for class I MHC molecules derived from the subject; f) at least 30% of the peptide epitopes have the highest affinity for class II MHC molecules derived from the subject; g) at least 50% of the peptide epitopes are HLA- h) the mRNA encodes 45-55 peptide epitopes; i) the mRNA encodes 52 peptide epitopes; j) 50% of the peptide epitopes have binding affinity for MHC class I and 50% of the peptide epitopes have binding affinity for MHC class II; k) the mRNA encoding the peptide epitopes is arranged such that the order of the peptide epitopes minimizes pseudo-epitopes; l) at least 30% of the peptide epitopes are 15 amino acids in length, MHC class I-binding peptides; and / or m) at least 30% of the peptide epitopes are 21 amino acids in length, MHC class II-binding peptides.

[0037] In some embodiments, each peptide epitope comprises a centrally located SNP mutation with 15 flanking amino acids on either side of the SNP mutation.

[0038] In one embodiment, the polypeptide that enhances an immune response to at least one personalized cancer antigen in a subject is a constitutively active human STING polypeptide. In one embodiment, the constitutively active human STING polypeptide comprises one or more mutations selected from the group consisting of V147L, N154S, V155M, R284M, R284K, R284T, E315Q, R375A, and combinations thereof. In another embodiment, the constitutively active human STING polypeptide comprises a V155M mutation. In another embodiment, the constitutively active human STING polypeptide comprises the mutations R284M / V147L / N154S / V155M.

[0039] In some embodiments, each mRNA is formulated in the same or different lipid nanoparticles.In another embodiment, each mRNA that encodes cancer personalized cancer antigen is formulated in the same or different lipid nanoparticles.In some embodiments, each mRNA that encodes the polypeptide that enhances the immune response to personalized cancer antigen is formulated in the same or different lipid nanoparticles.

[0040] In some embodiments, each mRNA encoding a personalized cancer antigen is formulated into the same lipid nanoparticle, and each mRNA encoding a polypeptide that enhances the immune response to the personalized cancer antigen is formulated into a different lipid nanoparticle.In another embodiment, each mRNA encoding a personalized cancer antigen is formulated into the same lipid nanoparticle, and each mRNA encoding a polypeptide that enhances the immune response to the personalized cancer antigen is formulated into the same lipid nanoparticle as each mRNA encoding the personalized cancer antigen.In some embodiments, each mRNA encoding a personalized cancer antigen is formulated into a different lipid nanoparticle, and each mRNA encoding a polypeptide that enhances the immune response to the personalized cancer antigen is formulated into the same lipid nanoparticle as each mRNA encoding the personalized cancer antigen.

[0041] In some embodiments, the peptide epitopes are T cell epitopes and / or B cell epitopes. In other embodiments, the peptide epitopes comprise a combination of T cell epitopes and B cell epitopes. In one embodiment, at least one of the peptide epitopes is a T cell epitope. In another embodiment, at least one of the peptide epitopes is a B cell epitope.

[0042] In some embodiments, the peptide epitopes are optimized for binding strength to the MHC of interest, hi other embodiments, the TCR face of each epitope has low similarity to the endogenous protein.

[0043] In another embodiment, the mRNA cancer vaccine further comprises a recall antigen. In some embodiments, the recall antigen is an infectious disease antigen.

[0044] In one embodiment, the mRNA cancer vaccine further comprises an mRNA having an open reading frame encoding one or more conventional cancer antigens.

[0045] In one embodiment, one or more of the following conditions are met: (i) the activating oncogene mutation is a KRAS mutation; (ii) the KRAS mutation is a G12 mutation, optionally wherein the G12 KRAS mutation is selected from G12D, G12V, G12S, G12C, G12A, and G12R KRAS mutations; (iii) the KRAS mutation is a G13 mutation, optionally wherein the G13 KRAS mutation is a G13D KRAS mutation; and / or (iv) the activating oncogene mutation is an H-RAS or N-RAS mutation.

[0046] In one embodiment, one or more of the following conditions are met: (A) the mRNA has an open reading frame encoding a concatemer of two or more activating oncogene mutant peptides; (B) at least two of the peptide epitopes are separated from each other by a single glycine, and optionally, all of the peptide epitopes are separated from each other by a single glycine; (C) the concatemer contains 3 to 10 activating oncogene mutant peptides; and / or (D) at least two of the peptide epitopes are directly linked to each other without a linker.

[0047] In one embodiment, one or more of the following conditions are met: (i) at least one of the peptide epitopes is a conventional cancer antigen; (ii) at least one of the peptide epitopes is a repeat polymorphism; (iii) the repeat polymorphism comprises a recurrent somatic cancer mutation in p53; (iv) the recurrent somatic cancer mutation in p53 is (A) a mutation in the canonical 5' splice site adjacent to codon position T125, which is the epitope AVSPCISFVW (SEQ ID NO: 233) (HLA-B * 57:01, HLA-B * 58:01), epitope HPLASCQCFF (SEQ ID NO: 234) (HLA-B * 35:01, HLA-B * 53:01), epitope FVWNFGIPL (SEQ ID NO: 235) (HLA-A * 02:01, HLA-A * 02:06, HLA-B * (B) a mutation in the canonical 5' splice site adjacent to codon position 331, resulting in the epitope LQVLSLGTSY (SEQ ID NO: 237) (HLA-B * 15:01), epitope FQSNTQNAVF (SEQ ID NO: 238) (HLA-B *(C) a mutation in the canonical 3' splice site adjacent to codon position 126 that results in the epitope CTMFCQLAK (SEQ ID NO: 240) (HLA-A * 11:01), epitope KSVTCTMF (SEQ ID NO: 241) (HLA-B * and / or (D) a mutation in the canonical 5' splice site adjacent to codon position 224 that results in the epitope VPYEPPEVW (SEQ ID NO: 243) (HLA-B * 53:01, HLA-B * 51:01), epitope LTVPPSTAW (SEQ ID NO: 244) (HLA-B * 58:01, HLA-B * and / or (v) the mRNA cancer vaccine is selected from the group consisting of a mutation that induces a cryptic alternative intron 5' splice site generating a novel spanning peptide sequence VPYEPPEVWLALTVPPSTAWAA (SEQ ID NO: 242) containing the sequence: VPYEPPEVWLALTVPPSTAWAA (SEQ ID NO: 57:01) (transcription codon positions are with reference to ENST00000269305 (SEQ ID NO: 245), the standard full-length p53 transcript from Ensembl's v83 human genome annotation); and / or

[0048] In some embodiments, the lipid nanoparticles comprise a molar ratio of about 20-60% ionizable amino lipid: 5-25% neutral lipid: 25-55% sterol; and 0.5-15% PEG-modified lipid, where the ionizable amino lipid is optionally a cationic lipid. In one embodiment, the lipid nanoparticles comprise a molar ratio of about 50% Compound 25: about 10% DSPC: about 38.5% cholesterol; and about 1.5% PEG-DMG. In another embodiment, the ionizable amino lipid is selected from the group consisting of, for example, 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). In some embodiments, the lipid nanoparticles comprise a compound of Formula (I). In one embodiment, the compound of Formula (I) is Compound 25. In another embodiment, the lipid nanoparticles have a polydispersity value of less than 0.4. In some embodiments, the lipid nanoparticles have a net neutral charge at neutral pH values.

[0049] In one embodiment, the TCR face of each epitope has low similarity to the endogenous protein.

[0050] In another embodiment, the mRNA further comprises an open reading frame encoding an immune checkpoint regulator. In one embodiment, the mRNA cancer vaccine further comprises an additional cancer therapeutic agent, and optionally, the additional cancer therapeutic agent is an immune checkpoint regulator. In another embodiment, the immune checkpoint regulator is an inhibitory checkpoint polypeptide. In some embodiments, the inhibitory checkpoint polypeptide inhibits PD1, PD-L1, CTLA4, TIM-3, VISTA, A2AR, B7-H3, B7-H4, BTLA, IDO, KIR, LAG3, or a combination thereof.

[0051] In some embodiments, the checkpoint inhibitor polypeptide is an antibody. In one embodiment, the inhibitory checkpoint polypeptide is an antibody selected from an anti-CTLA4 antibody or antigen-binding fragment thereof that specifically binds to CTLA4, an anti-PD1 antibody or antigen-binding fragment thereof that specifically binds to PD1, an anti-PD-L1 antibody or antigen-binding fragment thereof that specifically binds to PD-L1, and combinations thereof. In one embodiment, the checkpoint inhibitor polypeptide is an anti-PD-L1 antibody selected from atezolizumab, avelumab, or durvalumab. In another embodiment, the checkpoint inhibitor polypeptide is an anti-CTLA-4 antibody selected from tremelimumab or ipilimumab. In some embodiments, the checkpoint inhibitor polypeptide is an anti-PD1 antibody selected from nivolumab or pembrolizumab.

[0052] In some embodiments, the chemical modification is selected from the group consisting of pseudouridine, N1-methylpseudouridine, 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-methyluridine, 5-methoxyuridine, and 2'-O-methyluridine.

[0053] In another aspect, the present disclosure provides a method of vaccinating a subject having cancer, comprising administering to the subject the mRNA cancer vaccine described above.

[0054] In some embodiments, the mRNA vaccine is administered to a subject at a dose level sufficient to deliver 10 μg to 400 μg of the mRNA vaccine. In one embodiment, the mRNA vaccine is administered to a subject at a dose level sufficient to deliver 0.033 mg, 0.1 mg, 0.2 mg, or 0.4 mg. In another embodiment, the mRNA vaccine is administered to a subject two, three, four, or more times. In some embodiments, the mRNA vaccine is administered once daily every three weeks. In one embodiment, the mRNA vaccine is administered intradermally, intramuscularly, and / or subcutaneously. In another embodiment, the mRNA vaccine is administered intramuscularly.

[0055] In some embodiments, the method further comprises administering an additional cancer therapeutic agent, optionally an immune checkpoint modulator to the subject. In one embodiment, the immune checkpoint modulator is an inhibitory checkpoint polypeptide. In another embodiment, the inhibitory checkpoint polypeptide inhibits PD1, PD-L1, CTLA4, TIM-3, VISTA, A2AR, B7-H3, B7-H4, BTLA, IDO, KIR, LAG3, or a combination thereof. In some embodiments, the checkpoint inhibitor polypeptide is an antibody. In other embodiments, the inhibitory checkpoint polypeptide is an antibody selected from an anti-CTLA4 antibody or antigen-binding fragment thereof that specifically binds to CTLA4, an anti-PD1 antibody or antigen-binding fragment thereof that specifically binds to PD1, an anti-PD-L1 antibody or antigen-binding fragment thereof that specifically binds to PD-L1, and combinations thereof. In some embodiments, the checkpoint inhibitor polypeptide is an anti-PD-L1 antibody selected from atezolizumab, avelumab, or durvalumab. In another embodiment, the checkpoint inhibitor polypeptide is an anti-CTLA-4 antibody selected from tremelimumab or ipilimumab. In other embodiments, the checkpoint inhibitor polypeptide is an anti-PD1 antibody selected from nivolumab or pembrolizumab.

[0056] In one embodiment, the immune checkpoint modulator is administered to the subject at a dose level sufficient to deliver 100-300 mg. In some embodiments, the immune checkpoint modulator is administered to the subject at a dose level sufficient to deliver 200 mg. In some embodiments, the immune checkpoint modulator is administered by intravenous infusion. In one embodiment, the immune checkpoint modulator is administered to the subject two, three, four, or more times. In some embodiments, the immune checkpoint modulator is administered to the subject on the same day as administration of the mRNA vaccine.

[0057] In some embodiments, the cancer is selected from the group consisting of non-small cell lung cancer (NSCLC), small cell lung cancer, melanoma, bladder urothelial carcinoma, HPV-negative head and neck squamous cell carcinoma (HNSCC), and solid malignancies that are microsatellite high (MSI H) / mismatch repair (MMR) deficient. In one embodiment, the NSCLC lacks an EGFR-sensitive mutation and / or an ALK translocation. In another embodiment, the solid malignancy that is microsatellite high (MSI H) / mismatch repair (MMR) deficient is selected from the group consisting of colorectal cancer, gastric adenocarcinoma, esophageal adenocarcinoma, and endometrial cancer. In some embodiments, the cancer is selected from cancers of the pancreas, peritoneum, large intestine, small intestine, biliary tract, lung, endometrium, ovary, reproductive tract, gastrointestinal tract, cervix, stomach, urinary tract, colon, rectum, and hematopoietic and lymphoid tissues.

[0058] In some aspects, the present disclosure relates to an mRNA cancer vaccine of one or more mRNAs, each having an open reading frame encoding a cancer antigen peptide epitope, formulated in a lipid nanoparticle and a pharmaceutically acceptable carrier or excipient, wherein the mRNA vaccine encodes between 5 and 100 peptide epitopes, and at least two of the peptide epitopes are personalized cancer antigens.

[0059] In another aspect, the present invention provides an mRNA cancer vaccine comprising one or more mRNAs each having an open reading frame encoding a cancer antigen peptide epitope, and a pharmaceutically acceptable carrier or excipient, wherein the mRNA vaccine encodes 5 to 100 peptide epitopes, at least three of the peptide epitopes being compound variants and at least two of the peptide epitopes being point mutations.

[0060] In some embodiments, the lipid nanoparticles comprise a molar ratio of 20-60% cationic lipid: 5-25% non-cationic lipid: 25-55% sterol: 0.5-15% PEG-modified lipid. In some embodiments, the cationic lipid is selected from the group consisting of, for example, 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). In other embodiments, the lipid nanoparticles comprise a compound of Formula (I). In some embodiments, the compound of Formula (I) is Compound 25.

[0061] In some embodiments, the lipid nanoparticles have a polydispersity value of less than 0.4. In some embodiments, the lipid nanoparticles have a net neutral charge at neutral pH values.

[0062] In some embodiments, the vaccine is an mRNA having an open reading frame encoding a concatemeric cancer antigen composed of 5 to 100 peptide epitopes. In still other embodiments, at least two of the peptide epitopes are separated from each other by a single glycine. In still other embodiments, the concatemeric cancer antigen comprises 20 to 40 peptide epitopes. In some embodiments, all of the peptide epitopes are separated from each other by a single glycine. In some embodiments, at least two of the peptide epitopes are directly linked to each other without a linker.

[0063] Each peptide epitope of the embodiment comprises 25-35 amino acids and includes a centrally located SNP mutation.

[0064] In some embodiments, at least 30% of the peptide epitopes have highest affinity for Class I MHC molecules from the subject. In yet other embodiments, at least 30% of the peptide epitopes have highest affinity for Class II MHC molecules from the subject. In yet other embodiments, at least 50% of the peptide epitopes have predicted binding affinity for HLA-A, HLA-B, and / or DRB1 of IC>500 nM.

[0065] In some embodiments, one or more mRNAs of the invention encode up to 20 peptide epitopes. In some embodiments, one or more mRNAs of the invention encode up to 50 epitopes. In some embodiments, one or more mRNAs of the invention encode up to 100 epitopes.

[0066] According to another embodiment, the mRNAs encoding the peptide epitopes are arranged such that the order of the peptide epitopes is in an order that minimizes pseudo-epitopes.

[0067] Each peptide epitope can contain 31 amino acids and includes a centrally located SNP mutation with 15 flanking amino acids on either side of the SNP mutation.

[0068] In some embodiments, the TCR face of each epitope has low similarity to the endogenous protein.

[0069] In yet other embodiments, the mRNA further comprises a recall antigen. The recall antigen may be an infectious disease antigen.

[0070] In other embodiments, at least one of the peptide epitopes is a conventional cancer antigen. The vaccine, in some embodiments, comprises an mRNA having an open reading frame encoding one or more repeat polymorphisms. The one or more repeat polymorphisms may comprise recurrent somatic cancer mutations in p53. The one or more recurrent somatic cancer mutations in p53, in some embodiments, are (A) a mutation in the canonical 5' splice site adjacent to codon position T125, encoding the epitope AVSPCISFVW (SEQ ID NO: 233) (HLA-B * 57:01, HLA-B * 58:01), epitope HPLASCQCFF (SEQ ID NO: 234) (HLA-B * 35:01, HLA-B * 53:01), epitope FVWNFGIPL (SEQ ID NO: 235) (HLA-A * 02:01, HLA-A * 02:06, HLA-B * (B) a mutation in the canonical 5' splice site adjacent to codon position 331, resulting in the epitope LQVLSLGTSY (SEQ ID NO: 237) (HLA-B * 15:01), epitope FQSNTQNAVF (SEQ ID NO: 238) (HLA-B *(C) a mutation in the canonical 3' splice site adjacent to codon position 126 that results in the epitope CTMFCQLAK (SEQ ID NO: 240) (HLA-A * 11:01), epitope KSVTCTMF (SEQ ID NO: 241) (HLA-B * and / or (D) a mutation in the canonical 5' splice site adjacent to codon position 224 that results in the epitope VPYEPPEVW (SEQ ID NO: 243) (HLA-B * 53:01, HLA-B * 51:01), epitope LTVPPSTAW (SEQ ID NO: 244) (HLA-B * 58:01, HLA-B * a mutation that introduces a cryptic alternative intron 5' splice site generating a novel spanning peptide sequence VPYEPPEVWLALTVPPSTAWAA (SEQ ID NO: 242) containing the sequence VPYEPPEVWLALTVPPSTAWAA (SEQ ID NO: 57:01) (transcription codon positions are with reference to ENST00000269305 (SEQ ID NO: 245), the standard full-length p53 transcript from Ensembl's v83 human genome annotation).

[0071] In some embodiments, the mRNA further comprises an open reading frame encoding an immune checkpoint modulator. In some embodiments, the mRNA cancer vaccine comprises 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 or anti-PD1 antibody. In some embodiments, the anti-PD-1 antibody is pembrolizumab.

[0072] In some embodiments, the mRNA cancer vaccine does not comprise a stabilizer.

[0073] In some embodiments, the mRNA comprises at least one chemical modification, which may be selected from the group consisting of pseudouridine, N1-methylpseudouridine, 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-methyluridine, 5-methoxyuridine, and 2'-O-methyluridine.

[0074] In another aspect, a method of vaccinating a subject is provided, the method involving administering to a subject having cancer an mRNA vaccine disclosed herein.

[0075] In some embodiments, the mRNA vaccine is administered to a subject at a dose level sufficient to deliver 10 μg to 400 μg of the mRNA vaccine. In some embodiments, the mRNA vaccine is administered to a subject at a dose level sufficient to deliver 0.033 mg, 0.1 mg, 0.2 mg, or 0.4 mg. In some embodiments, the mRNA vaccine is administered to a subject two, three, four, or more times. In some embodiments, the mRNA vaccine is administered once daily every three weeks.

[0076] In some embodiments, the mRNA vaccine is administered by intradermal, intramuscular, and / or subcutaneous administration. In some embodiments, the mRNA vaccine is administered by intramuscular administration.

[0077] In some embodiments, the method further comprises administering an additional cancer therapeutic agent, optionally wherein the additional cancer therapeutic agent is an immune checkpoint modulator to the subject. 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-PD1 antibody. In some embodiments, the anti-PD-1 antibody is pembrolizumab.

[0078] In some embodiments, the immune checkpoint modulator is administered to the subject at a dose level sufficient to deliver 100-300 mg, hi some embodiments, the immune checkpoint modulator is administered to the subject at a dose level sufficient to deliver 200 mg.

[0079] In some embodiments, the immune checkpoint modulator is administered by intravenous infusion.

[0080] In some embodiments, the immune checkpoint modulator is administered to the subject two, three, four or more times. In some embodiments, the immune checkpoint modulator is administered to the subject on the same day as administration of the mRNA vaccine.

[0081] In some embodiments, the cancer is selected from the group consisting of non-small cell lung cancer (NSCLC), small cell lung cancer, melanoma, bladder urothelial carcinoma, HPV-negative head and neck squamous cell carcinoma (HNSCC), and solid malignancies that are microsatellite high (MSI H) / mismatch repair (MMR) deficient. In some embodiments, the NSCLC lacks an EGFR-sensitive mutation and / or an ALK translocation. In some embodiments, the solid malignancies that are microsatellite high (MSI H) / mismatch repair (MMR) deficient are selected from the group consisting of colorectal cancer, gastric adenocarcinoma, esophageal adenocarcinoma, and endometrial cancer. In some embodiments, the cancer is selected from cancers of the pancreas, peritoneum, large intestine, small intestine, biliary tract, lung, endometrium, ovary, reproductive tract, gastrointestinal tract, cervix, stomach, urinary tract, colon, rectum, and hematopoietic and lymphoid tissues.

[0082] Another aspect of the present invention provides a method for preparing an mRNA cancer vaccine. The method involves isolating a sample from a subject, identifying multiple cancer antigens in the sample, determining immunogenic epitopes from the multiple cancer antigens, and preparing an mRNA cancer vaccine having an open reading frame encoding the cancer antigen. Another aspect of the present invention provides a method for generating mRNA encoding a concatemeric cancer antigen comprising 1,000 to 3,000 nucleotides. The method comprises: (a) binding of a first polynucleotide comprising an open reading frame encoding a cancer antigen according to any one of the preceding claims 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 thereby producing mRNA encoding a concatemeric cancer antigen containing 1000 to 3000 nucleotides.

[0083] In another aspect, the invention is an mRNA cancer vaccine comprising a concatemeric cancer antigen that can be prepared according to the methods described herein.

[0084] According to another aspect of the present invention, methods for treating a subject with a personalized mRNA cancer vaccine are provided. The methods include: identifying a set of neoepitopes by analyzing a patient's transcriptome and / or exome from a sample to generate a patient-specific mutanome; selecting a set of neoepitopes for the vaccine from the mutanome based on MHC binding strength, MHC binding diversity, predicted immunogenicity, low autoreactivity, presence of activating oncogene mutations, 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 identification includes analyzing the patient's transcriptome and / or exome from the subject-derived sample. In some embodiments, the subject-derived sample is a biological sample, e.g., a biopsy. In some embodiments, the method further includes isolating the sample from the subject. In some embodiments, the identification includes analyzing tissue-specific expression in available databases.

[0085] In another aspect of the invention, a method is provided 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-mers to HLA-A and HLA-B for 15-mers to HLA-DRB1 for 15-mers to 20-mers; broad binding score IC50 for HLA-C for 8-mers to 11-mers; IC50 for HLA-DRB3-5 for 15-mers to 20-mers; IC50 for HLA-DQB1 / A1 for 15-mers to 20-mers; IC50 for HLA-DPB1 / A1 for 15-mers to 20-mers; Class I to Class II ratio comparison; HLA-A allotype, HLA-B allotype, and HLA-DRB1 allotype diversity covered in patients; Point mutation to compound epitope (e.g., frameshift) ratio comparison; Pseudoepitope HLA Binding Score; Selection of a subset of 15 to 500 neoepitopes from the mutanome using a weighting value for neoepitopes based on at least three of the following: presence and / or abundance of RNA sequencing reads; 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 to 40 neoepitopes.

[0086] In some aspects, the present disclosure provides an mRNA cancer vaccine comprising one or more mRNAs, each having an open reading frame encoding a cancer antigen peptide epitope, wherein the mRNA further comprises an miRNA binding site. In some embodiments, the vaccine encodes between 5 and 100 peptide epitopes.

[0087] In some embodiments, the nucleic acid vaccines described herein are chemically modified. In other embodiments, the nucleic acid vaccines are unmodified.

[0088] Yet other aspects provide compositions and methods 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 cancer antigen epitope, wherein the RNA polynucleotides do not comprise a stabilizing element, and no adjuvant is co-formulated or co-administered with the vaccine.

[0089] 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 cancer antigen epitope, wherein the dose of the nucleic acid vaccine administered to the subject is 10 μg / kg to 400 μg / kg. In some embodiments, the dose of the RNA polynucleotide is 1 to 5 μg, 5 to 10 μg, 10 to 15 μg, 15 to 20 μg, 10 to 25 μg, 20 to 25 μg, 20 to 50 μg, 30 to 50 μg, 40 to 50 μg, 40 to 60 μg, 60 to 80 μg, 60 to 100 μg, 50 to 100 μg, 80 to 120 μg, 40 to 120 μg, 40 to 150 μg, 50 to 150 μg, 50 to 200 μg, or 80 to 200 μg per dose. , 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.

[0090] 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.

[0091] In some embodiments, the effective amount is a total dose of 1 to 100 μg. In some embodiments, the effective amount is a total dose of 100 μg. In some embodiments, the effective amount is a 25 μg dose administered to the subject a total of one or two times. In some embodiments, the effective amount is a 100 μg dose administered to the subject a total of two times. In some embodiments, the effective amount may be administered to the subject one or two or more times, such as 1 μg to 10 μg, 1 μg to 20 μg, 1 μg to 30 μg, 5 μg to 10 μg, 5 μg to 20 μg, 5 μg to 30 μg, 5 μg to 40 μg, 5 μg to 50 μg, 10 μg to 15 μg, 10 μg to 20 μg, 10 μg to 25 μg, 10 μg to 30 μg, 10 μg to 40 μg, 10 μg to 50 μg, 10 μg to 60 μg, 15 ... μg~20μg, 15μg~25μg, 15μg~30μg, 15μg~40μg, 15μg~50μg, 20μg~25μg, 20μg~30μg, 20μg~40μg20μg~50μg, 20μg~60μg , 20μg~70μg, 20μg~75μg, 30μg~35μg, 30μg~40μg, 30μg~45μg30μg~50μg, 30μg~60μg, 30μg~70μg, 30μg~75μg.

[0092] In some embodiments, the stabilizing element is a histone stem loop. In some embodiments, the stabilizing element is a nucleic acid sequence having an increased GC content compared to a wild-type sequence.

[0093] Embodiments provide nucleic acid vaccines comprising one or more RNA polynucleotides having an open reading frame that includes at least one chemical modification or optionally no chemical modification, wherein the open reading frame encodes a first antigenic polypeptide, wherein the RNA polynucleotides are present in a formulation for in vivo administration to a subject such that the level of antigen expression in the subject is significantly greater than the level of antigen expression provided by an mRNA vaccine encoding the first antigenic polypeptide that has a stabilizing element or is formulated with an adjuvant.

[0094] 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 chemical modification, where the open reading frame encodes a first antigenic polypeptide, wherein the vaccine requires at least 10-fold less RNA polynucleotide than an unmodified mRNA vaccine would require to generate equivalent antibody titers.

[0095] Aspects of the invention also provide a vaccine unit of use, the unit comprising 10 μg to 400 μg of one or more RNA polynucleotides having an open reading frame with at least one chemical modification or optionally no chemical modification, the open reading frame encoding a first antigenic 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.

[0096] Aspects of the present invention provide kits that include a vial containing an mRNA cancer vaccine disclosed herein. In some embodiments, the vial contains 0.1 mg to 1 mg of mRNA. In some embodiments, the vial contains 0.35 mg of mRNA. In some embodiments, the concentration of the mRNA is 1 mg / mL.

[0097] In some embodiments, the vial contains 5-15 mg of total lipid. In some embodiments, the vial contains 7 mg of total lipid. In some embodiments, the concentration of total lipid is 20 mg / mL.

[0098] In some embodiments, the mRNA cancer vaccine is liquid.

[0099] In some embodiments, the kit further comprises a syringe, hi some embodiments, the syringe is suitable for intramuscular administration.

[0100] 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 in an amount effective to vaccinate the subject.

[0101] In some aspects, the present disclosure is an mRNA cancer vaccine that can include an activating oncogenic mutation as an antigen. In some embodiments, the activating oncogenic mutation is a KRAS mutation. In some embodiments, the KRAS mutation is a G12 mutation. In some embodiments, the G12 KRAS mutation is selected from G12D, G12V, G12S, G12C, G12A, and G12R KRAS mutations, for example, the G12 KRAS mutation is selected from G12D, G12V, and G12S KRAS mutations. In other embodiments, the KRAS mutation is a G13 mutation, for example, the G13 KRAS mutation is a G13D KRAS mutation. In some embodiments, the activating oncogenic mutation is an H-RAS or N-RAS mutation.

[0102] In some embodiments, a skilled artisan will select a KRAS mutation, HLA subtype, and tumor type based on the guidelines provided herein to prepare a therapeutic KRAS vaccine. In some embodiments, the KRAS mutation is selected from G12C, G12V, G12D, and G13D. In some embodiments, the HLA subtype is A* 02:01, C * 07:01, C * 04:01, C * 07:02 In some embodiments, the tumor type is selected from colorectal, pancreatic, lung, and endometrioid.

[0103] In some embodiments, the HRAS mutation is a mutation in codon 12, codon 13, or codon 61. In some embodiments, the HRAS mutation is a 12V, 61L, or 61R mutation.

[0104] In some embodiments, the NRAS mutation is a mutation in codon 12, codon 13, or codon 61. In some embodiments, the NRAS mutation is a 12D, 13D, 61K, or 61R mutation.

[0105] Some embodiments of the present disclosure provide mRNA cancer vaccines comprising an mRNA having an open reading frame encoding a concatemer of two or more activating oncogene mutant peptides. In some embodiments, at least two of the peptide epitopes are separated from each other by a single glycine. In some embodiments, the concatemer comprises 3 to 10 activating oncogene mutant peptides. In some such embodiments, all of the peptide epitopes are separated from each other by a single glycine. In other embodiments, at least two of the peptide epitopes are directly linked to each other without a linker.

[0106] In some embodiments, the mRNA cancer vaccine further comprises a cancer therapeutic agent. In some embodiments, the mRNA cancer vaccine further comprises an inhibitory checkpoint polypeptide. For example, 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 other embodiments, the mRNA cancer vaccine further comprises a recall antigen. For example, in some embodiments, the recall antigen is an infectious disease antigen.

[0107] In some embodiments, the mRNA cancer vaccine does not comprise a stabilizer.

[0108] In some embodiments, mRNA is formulated in a lipid nanoparticle carrier, such as a lipid nanoparticle carrier comprising a molar ratio of approximately 20-60% cationic lipid, 5-25% non-cationic lipid, 25-55% sterol, and 0.5-15% PEG-modified lipid. The cationic lipid may be 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).

[0109] In some embodiments, the mRNA comprises at least one chemical modification, which may be selected from the group consisting of pseudouridine, N1-methylpseudouridine, 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-methyluridine, 5-methoxyuridine, and 2'-O-methyluridine.

[0110] In another aspect, a method of treating a subject is provided. The method involves administering to a subject having cancer the mRNA cancer vaccine of any one of the preceding embodiments. In some embodiments, the mRNA cancer vaccine is administered in combination with a cancer therapeutic agent. In some embodiments, the mRNA cancer vaccine is administered in combination with an inhibitory checkpoint polypeptide. For example, in some embodiments, the mRNA cancer vaccine 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.

[0111] The methods provided herein can be used to treat a subject with cancer. In some embodiments, the cancer is selected from cancers of the pancreas, peritoneum, large intestine, small intestine, biliary tract, lung, endometrium, ovary, reproductive tract, gastrointestinal tract, cervix, stomach, urinary tract, colon, rectum, and hematopoietic and lymphoid tissues. In some embodiments, the cancer is colorectal cancer.

[0112] In some embodiments, the dose of mRNA cancer vaccine administered to a subject is 1-5 μg, 5-10 μg, 10-15 μg, 15-20 μg, 10-25 μg, 20-25 μg, 20-50 μg, 30-50 μg, 40-50 μg, 40-60 μg, 60-80 μg, 60-100 μg, 50-100 μg, 80-120 μg, 40-120 μg, 40-150 μg, 50-150 μg, 50-200 μg, 80-20 μg, 90-100 μg, 100-150 μg, 100-20 ... The dose is 0 μg, 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 mRNA cancer vaccine is administered to the subject by intradermal or intramuscular injection. In some embodiments, the mRNA cancer vaccine is administered to the subject on day 0. In some embodiments, a second dose of the mRNA cancer vaccine is administered to the subject on day 21.

[0113] In some embodiments, a 25 microgram dose of the mRNA cancer vaccine is administered to a subject. In some embodiments, a 100 microgram dose of the mRNA cancer vaccine is administered to a subject. In some embodiments, a 50 microgram dose of the mRNA cancer vaccine is administered to a subject. In some embodiments, a 75 microgram dose of the mRNA cancer vaccine is administered to a subject. In some embodiments, a 150 microgram dose of the mRNA cancer vaccine is administered to a subject. In some embodiments, a 400 microgram dose of the mRNA cancer vaccine is administered to a subject. In some embodiments, a 200 microgram dose of the mRNA cancer vaccine is administered to a subject. In some embodiments, the mRNA cancer vaccine accumulates at 100-fold higher levels in local lymph nodes compared to distant lymph nodes. In other embodiments, the mRNA cancer vaccine is chemically modified, and in other embodiments, the mRNA cancer vaccine is not chemically modified.

[0114] In some embodiments, the effective amount is a total dose of 1 to 100 μg. In some embodiments, the effective amount is a total dose of 100 μg. In some embodiments, the effective amount is a 25 μg dose administered to the subject a total of one or two times. In some embodiments, the effective amount is a 100 μg dose administered to the subject a total of two times. In some embodiments, the effective amount may be administered to the subject one or two or more times, such as 1 μg to 10 μg, 1 μg to 20 μg, 1 μg to 30 μg, 5 μg to 10 μg, 5 μg to 20 μg, 5 μg to 30 μg, 5 μg to 40 μg, 5 μg to 50 μg, 10 μg to 15 μg, 10 μg to 20 μg, 10 μg to 25 μg, 10 μg to 30 μg, 10 μg to 40 μg, 10 μg to 50 μg, 10 μg to 60 μg, 15 ... μg~20μg, 15μg~25μg, 15μg~30μg, 15μg~40μg, 15μg~50μg, 20μg~25μg, 20μg~30μg, 20μg~40μg20μg~50μg, 20μg~60μg , 20μg~70μg, 20μg~75μg, 30μg~35μg, 30μg~40μg, 30μg~45μg30μg~50μg, 30μg~60μg, 30μg~70μg, 30μg~75μg.

[0115] An embodiment of the present invention is a method for producing mRNA encoding a concatemeric cancer antigen comprising 1000 to 3000 nucleotides, comprising: (a) binding a first polynucleotide comprising an open reading frame encoding the cancer antigen according to any one of claims 1 to 103 and a second polynucleotide comprising a 5'-UTR to a polynucleotide complexed to a solid support; and (b) ligating the 3' end of the second polynucleotide to the 5' end of the first polynucleotide under appropriate conditions, wherein the appropriate conditions include a DNA ligase, (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, the suitable conditions including an RNA ligase, thereby producing a second ligation product; and (d) releasing the second ligation product from the solid support, thereby producing an mRNA encoding a concatemeric cancer antigen comprising 1,000 to 3,000 nucleotides.

[0116] An embodiment of the present invention provides a method for treating a subject with a personalized mRNA cancer vaccine, comprising identifying a set of neoepitopes to generate a patient-specific mutanome; selecting a set of neoepitopes for the 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 a sample from the subject.

[0117] An embodiment of the present invention provides 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) identifying a patient-specific mutagenesis by analysis of the patient's transcriptome and the patient's exome; and (b) assessing gene or transcript level expression in the patient's RNA sequencing; variant call confidence scores; allele-specific expression based on RNA sequencing; comparison of conservative and non-conservative amino acid substitutions; location of point mutations (Centering Score for increased TCR engagement); location of point mutations (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; IC50 for HLA-C for 8-mers to 11-mers; IC50 for HLA-DRB3-5 for 15-mers to 20-mers; IC50 for HLA-DQB1 / A1 for 15-mers to 20-mers; IC50 for HLA-DPB1 / A1 for 15-mers to 20-mers; Class I vs. Class II ratio comparison; Coverage in patients The method includes (c) selecting a subset of 15 to 500 neoepitopes from the mutagenesis using weighting values ​​for the neoepitopes based on at least three of: diversity of HLA-A allotypes, HLA-B allotypes, and HLA-DRB1 allotypes; ratio comparison of point mutations and composite epitopes; pseudoepitope HLA binding score; and presence and / or abundance of RNA sequencing reads; and (d) selecting a set of neoepitopes from the subset based on the highest weighting value for use in a personalized mRNA cancer vaccine, wherein the set of neoepitopes includes 15 to 40 neoepitopes.

[0118] An embodiment of the present invention provides 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) generating an RNA sequencing sample from a patient's tumor to generate a set of RNA sequencing reads; (b) compiling a total number of nucleotide sequences from all the RNA sequencing reads; (c) comparing sequence information between the tumor sample and a corresponding database of normal tissues of the same tissue type; and (d) selecting a set of neoepitopes for use in the personalized mRNA cancer vaccine from the subset based on highest weighted value, wherein the set of neoepitopes comprises 15 to 40 neoepitopes.

[0119] 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.

[0120] 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]

[0121] [Figure 1] Confirmation of complete read-through of the concatemer is shown (SIINFEKL is SEQ ID NO: 231). [Figure 2] Antigen-specific responses against class I epitopes found in both constructs are shown. [Figure 3] Antigen-specific responses against class I epitopes exclusively found in the 52-mer construct are shown. [Figure 4]Antigen-specific responses to class II epitopes present in both constructs (left) and to class II epitopes present in the 52-mer construct (right) are shown. [Figure 5] FIG. 1 is a block diagram illustrating a computer system on which some embodiments may be implemented. [Figure 6] Figure 1 shows antigen-specific responses from mice immunized with mRNA encoding a concatemer of 52 mouse epitopes (additional epitope_4a_DX_RX_perm) in combination with STING immunopotentiator mRNA at various antigen and STING doses and antigen:STING ratios. Data shown are for in vitro restimulation with a peptide sequence corresponding to class II epitope RNA2 encoded within the concatemer. [Figure 7] Figure 1 shows antigen-specific responses from mice immunized with mRNA encoding a concatemer of 52 mouse epitopes (additional epitope_4a_DX_RX_perm) in combination with STING immunopotentiator mRNA at various antigen and STING doses and antigen:STING ratios. Data shown are for in vitro restimulation with a peptide sequence corresponding to class II epitope RNA3 encoded within the concatemer. [Figure 8] Antigen-specific responses from mice immunized with mRNA encoding a concatemer of 52 mouse epitopes (additional epitope_4a_DX_RX_perm) in combination with STING immunopotentiator mRNA at various antigen and STING doses and antigen:STING ratios are shown. Data shown are for in vitro restimulation with a peptide sequence corresponding to the class I epitope RNA7 encoded within the concatemer. [Figure 9]Antigen-specific responses from mice immunized with mRNA encoding a concatemer of 52 mouse epitopes (additional epitope_4a_DX_RX_perm) in combination with STING immunopotentiator mRNA at various antigen and STING doses and antigen:STING ratios are shown. Data shown are for in vitro restimulation with a peptide sequence corresponding to the class I epitope RNA13 encoded within the concatemer. [Figure 10] Antigen-specific responses from mice immunized with mRNA encoding a concatemer of 52 mouse epitopes (additional epitope_4a_DX_RX_perm) in combination with STING immunopotentiator mRNA at various antigen and STING doses and antigen:STING ratios are shown. Data shown are for in vitro restimulation with a peptide sequence corresponding to class I epitope RNA22 encoded within the concatemer. [Figure 11] Figure 1 shows antigen-specific responses from mice immunized with mRNA encoding a concatemer of 52 mouse epitopes (additional epitope_4a_DX_RX_perm) in combination with STING immunopotentiator mRNA at various antigen and STING doses and antigen:STING ratios. Data shown are for in vitro restimulation with a peptide sequence corresponding to the class II epitope RNA10 encoded within the concatemer. [Figure 12] 1 is a bar graph showing antigen-specific IFN-γ T responses from mice immunized with mRNA encoding a concatemer of 20 mouse epitopes (RNA31) in combination with a STING immunopotentiator mRNA, compared to standard adjuvant or unformulated (not encapsulated in LNPs). Data shown are for in vitro peptide restimulation with class II epitopes (RNA2 and RNA3) encoded within the concatemer. [Figure 13]1 is a bar graph showing antigen-specific IFN-γ T responses from mice immunized with mRNA encoding a concatemer of 20 mouse epitopes (RNA31) in combination with a STING immunopotentiator mRNA, compared to standard adjuvant or unformulated (not encapsulated in LNPs). Data shown are for in vitro peptide restimulation with class I epitopes encoded within the concatemer (RNA7, RNA10, and RNA13). [Figure 14] Figure 1 is a bar graph showing antigen-specific IFN-γ T responses from mice immunized with mRNA encoding a concatemer of 20 mouse epitopes (RNA31) in combination with a STING immunopotentiator mRNA, where the STING construct was administered simultaneously with the vaccine or 24 or 48 hours later. Data shown are for in vitro peptide restimulation with either class II epitopes (RNA2 and RNA3) or class I epitopes (RNA7, RNA10, RNA13) encoded within the concatemer. [Figure 15] 1 shows KRAS mutations in colorectal cancer identified in the COSMIC (2012) dataset. [Figure 16] Specificity of isoform-specific point mutations for HRAS is shown. Data representing the total number of tumors with each point mutation were collated from the COSMIC v52 release. Single base mutations resulting in each amino acid substitution are shown. The most frequent mutations for each isoform in each cancer type are highlighted in gray shading. H / L: Hematopoietic / Lymphoid. (Prior et al. Cancer Res. 2012 May 15;72(10):2457-2467). [Figure 17-1]Isoform-specific point mutation specificity for KRAS is shown. Data representing the total number of tumors with each point mutation were collated from the COSMIC v52 release. Single base mutations resulting in each amino acid substitution are shown. The most frequent mutations for each isoform in each cancer type are highlighted in gray shading. H / L: Hematopoietic / Lymphoid. (Prior et al. Cancer Res. 2012 May 15;72(10):2457-2467). [Figure 17-2] This is a continuation of Figure 17-1. [Figure 18] Specificity of isoform-specific point mutations for NRAS is shown. Data representing the total number of tumors with each point mutation were collated from the COSMIC v52 release. Single base mutations resulting in each amino acid substitution are shown. The most frequent mutations for each isoform in each cancer type are highlighted in gray shading. H / L: Hematopoietic / Lymphoid. (Prior et al. Cancer Res. 2012 May 15;72(10):2457-2467). [Figure 19-1] This indicates a secondary KRAS mutation after acquiring resistance to EGFR blockade (Diaz et al. The molecular evolution of acquired resistance to targeted EGFR blockade in colorectal cancers, Nature 486:537 (2012)). [Figure 19-2] This is a continuation of Figure 19-1. [Figure 20] This indicates secondary KRAS mutations after EGFR blockade (Misale et al. Emergence of KRAS mutations and acquired resistance to anti-EGFR therapy in colorectal cancer, Nature 486:532 (2012)). [Figure 21] NRAS and KRAS mutation frequencies in colorectal cancer identified using cBioPortal are shown. DETAILED DESCRIPTION OF THE INVENTION

[0122] 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 comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding a cancer antigen. In some embodiments, the vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having at least one open reading frame encoding a cancer antigen and at least one open reading frame encoding a universal type II T cell epitope. In another embodiment, the vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having at least one open reading frame encoding a cancer antigen and at least one open reading frame encoding an immune enhancer (e.g., adjuvant). In some embodiments, the vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding a cancer antigen (e.g., an activating oncogene mutant peptide).

[0123] 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.

[0124] The lipid nanoparticles used in the studies described herein have been previously used to deliver siRNA in various animal models and humans. Given the findings associated with the delivery of siRNA using lipid nanoparticle formulations, the fact that lipid nanoparticles, as opposed to liposomes, are useful in cancer vaccines is quite surprising. It has been observed that therapeutic delivery of siRNA formulated in lipid nanoparticles typically results in reduced antigen production and impaired immune responses, resulting in undesirable inflammatory responses associated with transient IgM responses. In contrast to the findings observed with siRNA, the lipid nanoparticle-mRNA cancer vaccine formulation described herein has been shown to not produce a transient IgM response, but rather to enhance IgG levels sufficient for prophylactic and therapeutic methods. The lipid nanoparticles of the present invention are not liposomes. As used herein, liposomes are lipid-based structures with a lipid bilayer or monolayer shell and a nucleic acid payload within the core.

[0125] Generating cancer antigens that elicit desired immune responses (e.g., T cell responses) against target polypeptide sequences remains a challenging task in vaccine development. The present invention includes technology that overcomes the hurdles associated with such vaccine development. Using the technology of the present invention, it is possible to tailor and direct a desired immune response by selecting appropriate T cell or B cell cancer epitopes and formulating the epitopes or antigens for effective delivery in vivo. Additionally or alternatively, the immune response can be further enhanced by selecting one or more universal type II T cell epitopes to be delivered in addition to the appropriate T cell and / or B cell cancer epitopes or antigens.

[0126] Additionally or alternatively, mRNA vaccines can include activating oncogenic mutant peptides (e.g., KRAS mutant peptides). Previous studies have shown that the ability to generate T cells specific for oncogenic mutations is limited. Most of this work was performed in the context of the most common HLA allele (A2, occurring in approximately 50% of Caucasians). More recent studies have explored the generation of point mutation-specific T cells in the context of less common HLA alleles (A11, C8). These findings have important implications for cancer treatment. Oncogenic mutations are found in many cancers. The ability to target these mutations and generate sufficient T cells to kill tumors could be widely applied to cancer therapy. It is quite surprising that antigen delivery using mRNA has such significant advantages over peptide vaccine delivery. Thus, in some embodiments, the present invention includes the surprising finding that activating oncogenic mutant antigens delivered in vivo in the form of mRNA significantly enhance the efficacy of cancer treatment.

[0127] HLA class I molecules are highly polymorphic transmembrane glycoproteins composed of two polypeptide chains (heavy and light chains). Human leukocyte antigens, the human major histocompatibility complex (HLC), are unique to each individual and are genetically characterized. The class I heavy chains are encoded by three genes: HLA-A, HLA-B, and HLA-C. HLA class I molecules are important for establishing immune responses by presenting endogenous antigens to T lymphocytes, thereby initiating a series of immune responses that lead to the elimination of tumor cells by cytotoxic T cells. Altered levels of HLA class I antigen production are widespread in malignant tumors and are associated with significant suppression of antitumor T cell function. This is one of the main mechanisms used by cancer cells to evade immune surveillance. Downregulated HLA class I antigen levels were detected in 90% of NSCLC tumors (n=65). Loss or loss of HLA was detected in 76% of pancreatic tumor samples (n=19). HLA class I antigen expression in colon cancer was dramatically reduced or undetectable in 96% of tumor samples (n=25).

[0128] A growing body of evidence suggests that tumor cells utilize two general strategies to evade immune surveillance: immune selection (low-immunogenic tumor cell variants) and immune subversion (subversion of the immune system). A correlation has been shown between changes in HLA class I antigens and the presence of 12 KRAS codon mutations, suggesting that these mutations may have an inducible effect on HLA class I antigen regulation during cancer progression. Frequent cancer mutations are predicted to bind to HLA class I alleles with high affinity (IC50 ≤ 50 nM), making them suitable for prophylactic cancer vaccines.

[0129] Therapeutic mRNA can be delivered alone or in combination with other cancer therapeutics, such as checkpoint inhibitors, which significantly enhance the immune response against tumors. Checkpoint inhibitors enhance the action of mRNAs encoding activating oncogenic peptides by removing some of the obstacles to promoting an immune response, thereby allowing activated T cells to efficiently promote an immune response against tumors.

[0130] The mRNA vaccines described herein have been found to be superior to current vaccines in several ways. First, delivery via lipid nanoparticles (LNPs) is superior to other formulations, including liposomes or protamine-based approaches described in the literature. LNPs allow for the 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.

[0131] 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.

[0132] In some aspects, the present invention includes the surprising discovery that lipid nanoparticle (LNP) formulations significantly enhance the efficacy of mRNA vaccines, including chemically modified and unmodified mRNA vaccines. Furthermore, immunogenicity against epitopes was found to be similar regardless of the total number of epitopes contained within the construct. Epitopes contained in 52-mer constructs have similar immunogenicity compared to 20-mer constructs, as measured by epitope-specific IFNγ responses. It was entirely unexpected that increasing mRNA length was shown to have no detrimental effect on epitope immunogenicity. The final epitopes encoded by the 20-mer and 52-mer (SIINFEKL, SEQ ID NO: 231) were comparable, indicating complete read-through of the concatemer. Surprisingly, antigen-specific responses against class I epitopes were also found to be enhanced when the vaccine was formulated with a structurally active immunopotentiator.

[0133] 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.

[0134] 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.

[0135] In some embodiments, mRNA cancer vaccines can be administered together with anti-cancer therapeutic agents, including, but not limited to, conventional cancer vaccines. mRNA cancer vaccines and anti-cancer therapeutic agents can be combined to further enhance immunotherapeutic responses. The mRNA cancer vaccine and other therapeutic agents can be administered simultaneously or sequentially. When other therapeutic agents are administered simultaneously, they can be administered in the same formulation or in separate formulations, but the administration is simultaneous. When the administration of other therapeutic agents and mRNA cancer vaccines is temporally separated, the other therapeutic agents are administered sequentially 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 can be longer, such as, for example, several hours, days, weeks, or months. Other therapeutic agents include, but are not limited to, anti-cancer therapeutic agents, adjuvants, cytokines, antibodies, antigens, etc.

[0136] 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 antigenic peptide can be a personalized cancer antigen epitope and / or a recurrent antigen. In some preferred embodiments, the vaccine is a combination of multiple epitopes of the above. Thus, the cancer vaccine can be a conventional cancer vaccine, a personalized cancer vaccine, or a mixture thereof. A conventional cancer vaccine is a vaccine that includes cancer antigens known to be generally found in cancers or tumors, or cancer antigens known to be found in specific types of cancers or tumors. Antigens expressed in or by tumor cells are referred to as "tumor-associated antigens." Certain tumor-associated antigens may or may not be expressed in non-cancerous cells. Many tumor mutations are known in the art.

[0137] Surprisingly, it was discovered that RNA-based multiepitope cancer vaccines, whether formulated as individual epitopes or concatemers, can provide optimal immune stimulation through a careful balance of MHC class I and MHC class II epitopes. RNA vaccines encoding both components are highly immunogenic.

[0138] A personalized vaccine may contain, for example, RNA encoding one or more known tumor-specific cancer antigens or cancer antigens specific to each subject; such antigens are referred to as neoepitopes, subject-specific epitopes, or subject-specific antigens (also referred to as personalized 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 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. Neoepitopes, such as tumor-associated antigens, are completely foreign to the body and therefore are expected not to generate an immune response against healthy tissue or to be shielded 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.

[0139] 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.

[0140] 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.

[0141] 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.

[0142] Thus, the present invention relates to methods for identifying and / or detecting neoepitopes of antigens. Specifically, the present invention provides methods for identifying and / or detecting tumor-specific neoepitopes useful in inducing tumor-specific immune responses in subjects. Optionally, some of these 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. Others bind to class II and activate CD4+ T helper cells. While the important role played by class I antigens in vaccines is recognized, the present invention has discovered that vaccines composed of a balance of class I and class II antigens actually produce stronger immune responses than vaccines based solely on class I or class II antigens.

[0143] 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.

[0144] Computer algorithms can be used to predict potential neoepitopes, i.e., peptide sequences that are recognized by the T cell receptors of T lymphocytes in the form of peptide-presenting complexes 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).

[0145] 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.

[0146] 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.

[0147] The mRNA vaccines of the present invention may comprise one or more cancer antigens. In some embodiments, the mRNA vaccines are comprised of 45 or more, 46 or more, 47 or more, 48 or more, 49 or more, 50 or more, 51 or more, 52 or more, 53 or more, 54 or more, or 55 or more antigens. In other embodiments, the mRNA vaccines are comprised of 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, or 9 or more antigens. In other embodiments, the mRNA vaccines are comprised of 1000 or less, 900 or less, 500 or less, 100 or less, 75 or less, 50 or less, 40 or less, 30 or less, 20 or less, or 100 or less cancer antigens. In still other embodiments, the mRNA vaccine comprises 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 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.

[0148] 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).

[0149] 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.

[0150] 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.

[0151] 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 one embodiment, the ratio of MHC class I epitopes to MHC class II epitopes is 3:1.In some embodiments, the ratio of MHC class II epitopes to MHC class I 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%, and about 90%:about 10% MHC class II epitopes:MHC class I epitopes. In one embodiment, the ratio of MHC class II epitopes:MHC class I epitopes is 1:3. 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.

[0152] In another aspect, the cancer vaccines of the present invention comprise mRNA vaccines encoding multiple peptide epitope antigens interspersed with one or more universal type II T cell epitopes. Universal type II T cell epitopes include, but are not limited to, ILMQYIKANSKFIGI (tetanus toxin; SEQ ID NO: 226), FNNFTVSFWLRVPKVSASHLE (tetanus toxin; SEQ ID NO: 227), QYIKANSKFIGITE (tetanus toxin; SEQ ID NO: 228), QSIALSSLMVAQAIP (diphtheria toxin; SEQ ID NO: 229), and AKFVAAWTLKAAA (pan-DR epitope (PADRE); SEQ ID NO: 230). In some embodiments, the mRNA vaccines contain the same universal type II T cell epitope. In other embodiments, the mRNA vaccines contain 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 different universal type II T cell epitopes. In some embodiments, one or more universal type II T cell epitope(s) are interspersed among all cancer antigens, hi other embodiments, one or more universal type II T cell epitope(s) are interspersed among every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 100 cancer antigens.

[0153] In some embodiments, the cancer vaccine of the present invention comprises an mRNA vaccine encoding multiple peptide epitope antigens, either separated by a single nucleotide spacer or directly separated by no spacer 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 have the following structure: (XGX) 1-10 (GYGY) 1-10 (GXGX) 0-10 (GYGY) 0-10 , (XG) 1-10(G-Y) 1-10 (G-X) 0-10 (G-Y) 0-10 、(X-G-X-G-X) 1-10 (G-Y-G-Y) 1-10 (X-G-X) 0-10 (G-Y-G-Y) 0-10 、(X-G-X) 1-10 (G-Y-G-Y-G-Y) 1-10 (X-G-X) 0-10 (G-Y-G-Y) 0-10 、(X-G-X-G-X-G-X) 1-10 (G-Y-G-Y) 1-10 (X-G-X) 0-10 (G-Y-G-Y) 0-10 、(X-G-X) 1-10 (G-Y-G-Y-G-Y-G-Y) 1-10 (X-G-X) 0-10 (G-Y-G-Y) 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 、(Y-G-Y) 1-10 (G-X-G-X-G-X-G-X) 1-10 (Y-G-Y) 0-10 (G-X-G-X) 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 (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).

[0154] In some aspects, the cancer vaccines of the present invention comprise mRNA vaccines encoding multiple peptide epitope antigens, each containing a centrally located single nucleotide polymorphism (SNP) mutation flanked by flanking amino acids. In some embodiments, the number of flanking amino acids on either side of the centrally located SNP mutation is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, or 30. In one embodiment, the cancer vaccine epitope comprises an SNP flanked by two Class I sequences, each containing 7 amino acids. In another embodiment, the cancer vaccine epitope comprises an SNP flanked by two Class II sequences, each containing 10 amino acids. In some embodiments, the epitope may comprise a centrally located SNP and flanking sequences that are both Class I sequences, both Class II sequences, or one Class I and one Class II sequence.

[0155] Immune enhancer mRNA One aspect of the present disclosure relates to mRNAs encoding polypeptides that stimulate or enhance an immune response to one or more cancer antigens of interest. Such mRNAs that enhance an immune response to cancer antigen(s) of interest are referred to herein as immune enhancer mRNA constructs or immune enhancer mRNAs, and include chemically modified mRNAs (mmRNAs). The immune enhancers of the present disclosure enhance the immune response to the antigen of interest in a subject. The enhanced immune response may be a cellular response, a humoral response, or both. As used herein, a "cellular" immune response is intended to encompass an immune response involving or mediated by T cells, while a "humoral" immune response is intended to encompass an immune response involving or mediated by B cells. An immune enhancer may, for example, (i) stimulating type I interferon pathway signaling; (ii) stimulating NFkB pathway signaling; (iii) stimulating an inflammatory response; (iv) stimulating cytokine production, or (v) stimulating the development, activity or recruitment of dendritic cells; and (vi) Any combination of (i) to (vi) can enhance the immune response.

[0156] As used herein, "stimulating type I interferon pathway signaling" is intended to encompass activating one or more components of the type I interferon signaling pathway (e.g., activating the pathway by altering the phosphorylation, dimerization, etc. of such components), stimulating transcription from an interferon-sensitive response element (ISRE), and / or stimulating the production or secretion of a type I interferon (e.g., IFN-α, IFN-β, IFN-ε, IFN-κ, and / or IFN-ω). As used herein, "stimulating NFkB pathway signaling" is intended to encompass activating one or more components of the NFkB signaling pathway (e.g., activating the pathway by altering the phosphorylation, dimerization, etc. of such components), stimulating transcription from an NFkB site, and / or stimulating the production of a gene product whose expression is controlled by NFkB. As used herein, "stimulating an inflammatory response" is intended to include stimulating the production of inflammatory cytokines (including, but not limited to, type I interferon, IL-6, and / or TNFα). As used herein, "stimulating the development, activity, or recruitment of dendritic cells" is intended to include directly or indirectly stimulating the maturation, proliferation, and / or functional activity of dendritic cells.

[0157] In some aspects, the present disclosure provides mRNAs encoding polypeptides that stimulate or enhance an immune response in a subject in need thereof (e.g., enhance a subject's immune response), e.g., by inducing adaptive immunity (e.g., by stimulating type I interferon production), stimulating an inflammatory response, stimulating NFkB signaling, and / or stimulating the development, activity, or recruitment of dendritic cells (DCs) in the subject. In some aspects, administration of the immune enhancer mRNA to a subject in need thereof enhances the subject's cellular immunity (e.g., T cell-mediated immunity), humoral immunity (e.g., B cell-mediated immunity), or both cellular and humoral immunity. In some aspects, administration of the immune enhancer mRNA stimulates cytokine production (e.g., inflammatory cytokine production), enhances cancer antigen-specific CD8 + Stimulates effector cell responses and stimulates antigen-specific CD4 + Stimulates helper cell responses and effector memory CD62L lo In some embodiments, administration of the immune enhancer mRNA stimulates cytokine production (e.g., inflammatory cytokine production) and increases the number of antigen-specific CD8 + In some embodiments, administration of the immune enhancer mRNA stimulates cytokine production (e.g., inflammatory cytokine production) and stimulates antigen-specific CD4 + Stimulates a helper cell response. In some embodiments, administration of the immune enhancer mRNA stimulates cytokine production (e.g., inflammatory cytokine production) and stimulates effector memory CD62L. lo Increases T cell populations. In some embodiments, administration of the immune enhancer mRNA stimulates cytokine production (e.g., inflammatory cytokine production) and stimulates B cell activity or stimulates antigen-specific antibody production.

[0158] In one embodiment, the immune enhancing agent is a cancer antigen-specific CD8 +Increase effector cell responses (cell-mediated immunity). For example, immune enhancers include, but are not limited to, CD8 + T cell proliferation and CD8 + Antigen-specific CD8, including T cell cytokine production +One or more indicators of effector cell activity can be increased. For example, in one embodiment, the immunopotentiator increases the production of IFN-γ, TNFα, and / or IL-2 by antigen-specific CD8+ T cells. In various embodiments, the immunopotentiator can increase CD8+ T cell cytokine production (e.g., IFN-γ, TNFα, and / or IL-2 production) in response to an antigen by at least 5%, or at least 10%, or at least 15%, or at least 20%, or at least 25%, or at least 30%, or at least 35%, or at least 40%, or at least 45%, or at least 50% (compared to CD8+ T cell cytokine production in the absence of the immunopotentiator). For example, T cells obtained from a subject can be stimulated in vitro with a cancer antigen, and CD8+ T cell cytokine production can be assessed in vitro. CD8+ T cell cytokine production can be determined by standard methods known in the art, including, but not limited to, measuring the amount of cytokine secreted (e.g., by ELISA or other suitable method for determining the amount of cytokine in the supernatant known in the art) and / or determining the percentage of CD8+ T cells that are positive for intracellular staining (ICS) for cytokines. For example, intracellular staining (ICS) of CD8+ T cells for expression of IFN-γ, TNFα, and / or IL-2 can be performed by methods known in the art (see, e.g., Examples). In one embodiment, the immune enhancer can increase the percentage of CD8+ T cells that are ICS positive for one or more cytokines (e.g., IFN-γ, TNFα and / or IL-2) in response to an antigen by at least 5%, or at least 10%, or at least 15%, or at least 20%, or at least 25%, or at least 30%, or at least 35%, or at least 40%, or at least 45%, or at least 50% (compared to the percentage of CD8+ T cells that are ICS positive for the cytokine(s) in the absence of the immune enhancer).

[0159] In yet another embodiment, the immunopotentiator increases the percentage of CD8+ T cells in the total T cell population (e.g., splenic T cells and / or PBMCs) compared to the percentage of CD8+ T cells in the absence of the immunopotentiator. For example, the immunopotentiator may increase the percentage of CD8+ T cells in the total T cell population by at least 5%, or at least 10%, or at least 15%, or at least 20%, or at least 25%, or at least 30%, or at least 35%, or at least 40%, or at least 45%, or at least 50%, compared to the percentage of CD8+ T cells in the absence of the immunopotentiator. The total percentage of CD8+ T cells in the total T cell population can be determined by standard methods known in the art, including, but not limited to, fluorescence-activated cell sorting (FACS) or magnetically activated cell sorting (MACS).

[0160] In another embodiment, the immunopotentiator enhances tumor-specific immune cell response, as determined by a decrease in tumor volume in vivo in the presence of the immunopotentiator, compared to the tumor volume in the absence of the immunopotentiator. For example, the immunopotentiator can reduce tumor volume by at least 5%, or at least 10%, or at least 15%, or at least 20%, or at least 25%, or at least 30%, or at least 35%, or at least 40%, or at least 45%, or at least 50%, compared to the tumor volume in the absence of the immunopotentiator. Measurement of tumor volume can be determined by methods well established in the art.

[0161] In another embodiment, the immunopotentiator increases B cell activity (humoral immune response), e.g., by increasing the amount of antigen-specific antibody production, compared to antigen-specific antibody production in the absence of the immunopotentiator. For example, the immunopotentiator can increase antigen-specific antibody production by at least 5%, or at least 10%, or at least 15%, or at least 20%, or at least 25%, or at least 30%, or at least 35%, or at least 40%, or at least 45%, or at least 50%, compared to antigen-specific antibody production in the absence of the immunopotentiator. In one embodiment, antigen-specific IgG production is assessed. Antigen-specific antibody production can be assessed by methods well established in the art, including, for example, but not limited to, ELISA, RIA, and the like, which measure antigen-specific antibody (e.g., IgG) levels in a sample (e.g., a serum sample).

[0162] In another embodiment, the immune enhancing agent is effector memory CD62L lo Increases T cell populations. For example, immune enhancers increase the expression of CD62L in CD8+ T cells. lo It can increase the total percentage of effector memory T cells, among other functions. lo T cell populations have been shown to have important functions in lymphocyte trafficking (see, e.g., Schenkel, J.M. and Masopust, D. (2014) Immunity 41:886-897). In various embodiments, the immune enhancer enhances effector memory CD62L expression in CD8+ T cells in response to antigen. lo The total percentage of T cells (CD62L in the CD8+ T cell population in the absence of immune enhancers) lo The effector memory CD62L expression in CD8+ T cells can be increased by at least 5%, or at least 10%, or at least 15%, or at least 20%, or at least 25%, or at least 30%, or at least 35%, or at least 40%, or at least 45%, or at least 50% (relative to the total percentage of T cells). loThe total percentage of T cells can be determined by standard methods known in the art, including, but not limited to, fluorescence activated cell sorting (FACS) or magnetic activated cell sorting (MACS).

[0163] The ability of an immune enhancer mRNA construct to enhance the immune response to a cancer antigen can be evaluated in a mouse model system known in the art. In one embodiment, an immunocompetent mouse model system is used. In one embodiment, the mouse model system comprises C57 / B16 mice (e.g., for evaluating antigen-specific CD8+ T cell responses to cancer antigens, such as those described in the Examples). In another embodiment, the mouse model system comprises BalbC mice or CD1 mice (e.g., for evaluating B cell responses, such as antigen-specific antibody responses).

[0164] In one embodiment, the immune potentiator polypeptides of the present disclosure function downstream of at least one Toll-like receptor (TLR), thereby enhancing the immune response. Thus, in one embodiment, the immune potentiator is not a TLR, but rather a molecule in the TLR signaling pathway downstream from the receptor itself.

[0165] In one embodiment, an mRNA of the present disclosure encoding an immune enhancing agent may include one or more modified nucleobases. Suitable modifications are discussed further below.

[0166] In one embodiment, the mRNA of the present disclosure that encodes an immune enhancing agent is formulated in lipid nanoparticles.In one embodiment, the lipid nanoparticles further comprise mRNA that encodes a cancer antigen.In one embodiment, the lipid nanoparticles are administered to a subject to enhance the immune response to the cancer antigen in the subject.Suitable nanoparticles and methods of use are further discussed below.

[0167] Immune-enhancing mRNA that stimulates type I interferon In some aspects, the present disclosure provides immune enhancer mRNAs encoding polypeptides that stimulate or enhance type I interferon pathway signaling, thereby stimulating or enhancing the production of type I interferons (IFNs), thereby stimulating or enhancing an immune response to an antigen of interest. It has been established that type I IFN signaling is required for successful induction of anti-tumor or anti-microbial adaptive immunity (see, e.g., Fuertes, MB et al. (2013) Trends Immunol. 34:67-73). The production of type I IFNs (including IFN-α, IFN-β, IFN-ε, IFN-κ, and IFN-ω) is involved in the elimination of microbial infections, such as viral infections. It has also been recognized that host cell DNA (e.g., derived from damaged or dying cells) can induce type I interferon production, and that the type I IFN signaling pathway is involved in the generation of anti-tumor adaptive immunity. However, many pathogens and cancer cells have evolved mechanisms to reduce or suppress type I interferon responses. Thus, activating (including stimulating and / or enhancing) the type I IFN signaling pathway in a subject in need thereof by providing the subject with an immune enhancer mRNA of the present disclosure stimulates or enhances the subject's immune response in a variety of clinical settings, including the treatment of cancer and pathogenic infections, and in enhancing vaccine responses to provide protective immunity.

[0168] Type I interferons (IFNs) are pro-inflammatory cytokines that are rapidly produced by multiple different cell types, typically during viral infection, and are known to have diverse actions. The standard consequence of in vivo production of type I IFNs is the activation of antimicrobial cellular programs and the development of innate and adaptive immune responses. Type I IFNs induce a cell-intrinsic antimicrobial state in infected and neighboring cells, limiting the spread of pathogens, particularly viral pathogens. Type I IFNs also regulate innate immune cell activation (e.g., dendritic cell maturation) and promote antigen presentation and natural killer cell function. Type I IFNs also promote high-affinity, antigen-specific T-cell and B-cell responses and the development of immunological memory (Ivashkiv and Donlin (2014) Nat Rev Immunol 14(1):36-49).

[0169] Type I IFN activates dendritic cells (DCs) and promotes their T cell stimulatory capacity through autocrine signaling (Montoya et al., (2002) Blood 99:3263-3271). Type I IFN exposure promotes DC maturation by increasing the expression of chemokine receptors and adhesion molecules (e.g., promoting DC migration to draining lymph nodes), costimulatory molecules, and MHC class I and class II antigen presentation. DCs that mature after type I IFN exposure can effectively stimulate protective T cell responses (Wijesundara et al., (2014) Front Immunol 29(412) and references therein).

[0170] Type I IFNs can promote or inhibit T cell activation, proliferation, differentiation, and survival, depending largely on the timing of type I IFN signaling relative to T cell receptor signaling (Crouse et al., (2015) Nat Rev Immunol 15:231-242). Early studies demonstrated that MHC-I expression, a requirement for optimal T cell stimulation, differentiation, expansion, and cytolytic activity, is upregulated in multiple cell types in response to type I IFN (Lindahl et al., (1976) J Infect Dis 133(Suppl):A66-A68; Lindahl et al., (1976) Proc Natl Acad Sci USA 17:1284-1287). Type I IFNs can exert a potent costimulatory effect on CD8 T cells, enhancing their proliferation and differentiation (Curtsinger et al., (2005) J Immunol 174:4465-4469; Kolumam et al., (2005) J Exp Med 202:637-650).

[0171] Similar to its effects on T cells, type I IFN signaling has both positive and negative effects on B cell responses, depending on the timing and context of exposure (Braun et al., (2002) Int Immunol 14(4):411-419; Lin et al., (1998) 187(1):79-87). The survival and maturation of immature B cells can be suppressed by type I IFN signaling. In contrast to immature B cells, type I IFN exposure has been shown to promote B cell activation, antibody production, and isotype switching after viral infection or experimental immunization (Le Bon et al., (2006) J Immunol 176:4:2074-2078; Swanson et al., (2010) J Exp Med 207:1485-1500).

[0172] A number of components have been established that are involved in type I IFN pathway signaling, including STING, interferon regulatory factors (such as IRF1, IRF3, IRF5, IRF7, IRF8, and IRF9), TBK1, IKKi, MyD88, and TRAM. Additional components involved in type I IFN pathway signaling include TRAF3, TRAF6, IRAK-1, IRAK-4, TRIF, IPS-1, TLR-3, TLR-4, TLR-7, TLR-8, TLR-9, RIG-1, DAI, and IFI16.

[0173] Thus, in one embodiment, the immune enhancer mRNA encodes any of the aforementioned components involved in type I IFN pathway signaling.

[0174] Immune enhancer mRNA encoding STING The present disclosure encompasses mRNAs (including mmRNAs) encoding STING, including constitutively active forms of STING, as immune-enhancing agents. STING (also known as stimulator of interferon genes; transmembrane protein 173 (TMEM173), regulator of IRF3 activation (MITA), methionine-proline-tyrosine-serine (MPYS), and ER IFN stimulator (ERIS)) is a 379-amino acid endoplasmic reticulum (ER)-localized transmembrane protein that functions as a signaling molecule that regulates the transcription of immune response genes, including type I IFN and pro-inflammatory cytokines (Ishikawa & Barber, (2008) Nature 455:647-678; Ishikawa et al., (2009) Nature 461:788-792; Barber (2010) Nat Rev Immunol 15(12):760-770).

[0175] STING functions as a signaling adaptor that links cytosolic detection of DNA to the TBK1 / IRF3 / type I IFN signaling axis. The signaling adaptor function of STING is activated through direct sensing of cyclic dinucleotides (CDNs). Examples of CDNs include cyclic di-GMP (guanosine 5'-monophosphate), cyclic di-AMP (adenosine 5'-monophosphate), and cyclic GMP-AMP (cGAMP). CDNs were initially characterized as ubiquitous bacterial second messengers but are now known to constitute a class of pathogen-associated molecular pattern molecules (PAMPs) that activate the TBK1 / IRF3 / type I IFN signaling axis through direct interaction with STING. STING can sense aberrant DNA species and / or CDNs in the cellular cytosol, including bacterial-derived CDNs and / or CDNs derived from the host protein cyclic GMP-AMP synthase (cGAS). The cGAS protein is a DNA sensor that produces cGAMP in response to the detection of DNA in the cytosol (Burdette et al., (2011) Nature 478:515-518; Sun et al., (2013) Science 339:786-791; Diner et al., (2013) Cell Rep 3:1355-1361; Ablasser et al., (2013) Nature 498:380-384).

[0176] Upon binding to CDNs, STING dimerizes and undergoes a conformational change, promoting the formation of a complex with TANK-binding kinase 1 (TBK1) (Ouyang et al., (2012) Immunity 36(6):1073-1086). This complex translocates to the perinuclear Golgi apparatus and delivers TBK1 to the endolysosomal compartment, where it phosphorylates IRF3 and NF-κB transcription factors (Zhong et al., (2008) Immunity 29:538-550). A recent study showed that STING functions as a scaffold by binding to both TBK1 and IRF3, specifically promoting the phosphorylation of IRF3 by TBK1 (Tanaka & Chen, (2012) Sci Signal 5(214):ra20). Activation of IRF3, IRF7 and NF-κB-dependent signaling pathways induces the production of cytokines such as type I IFNs and other immune response-related proteins, promoting anti-pathogen and / or anti-tumor activity.

[0177] Numerous studies have explored the use of STING CDN agonists as vaccine adjuvants or immunomodulators with the potential to elicit humoral and cellular immune responses (Dubensky et al., (2013) Ther Adv Vaccines 1(4):131-143 and references therein). Initial studies demonstrated that administration of the CDN c-di-GMP attenuated Staphylococcus aureus infection in vivo and reduced the number of bacterial cells recovered in a mouse infection model, whereas in vitro c-di-GMP had no observable inhibitory or bactericidal effect on bacterial cells, suggesting that the reduction in bacterial cells was due to an effect on the host immune system (Karaolis et al., (2005) Antimicrob Agents Chemother 49:1029-1038; Karaolis et al., (2007) Infect Immun 75:4942-4950). A recent study showed that formulating synthetic CDN derivative molecules with a granulocyte-macrophage colony-stimulating factor (GM-CSF)-producing cancer vaccine (termed STINGVAX) elicited enhanced antitumor effects in vivo in a cancer therapeutic animal model compared to immunization with the GM-CSF vaccine alone (Fu et al., (2015) Sci Transl Med 7(283):283ra52), suggesting that CDNs are potent vaccine adjuvants.

[0178] Mutant STING proteins resulting from polymorphisms mapped to the human TMEM173 gene have been described to exhibit gain-of-function or constitutively active phenotypes. When expressed in vitro, mutant STING alleles have been shown to potently stimulate the induction of type I IFNs (Liu et al., (2014) N Engl J Med 371:507-518; Jeremiah et al., (2014) J Clin Invest 124:5516-5520; Dobbs et al., (2015) Cell Host Microbe 18(2):157-168; Tang & Wang, (2015) PLoS ONE 10(3):e0120090; Melki et al., (2017) J Allergy Clin Immunol In Press; Konig et al., (2017) Ann Rheum Dis 76(2):468-472; Burdette et al. (2011) Nature 478:515-518).

[0179] Provided herein is a modified mRNA (mmRNA) encoding a constitutively active form of STING, including a mutant human STING isoform, for use as an immune enhancing agent as described herein. The mmRNA encoding a constitutively active form of STING, including a mutant human STING isoform, is set forth in the sequence listing herein. The amino acid residue numbering of the mutant human STING polypeptide used herein corresponds to that used for the 379 amino acid residue wild-type human STING (isoform 1), available in the art under Genbank accession number NP_938023.

[0180] Thus, in one aspect, the disclosure provides an mmRNA encoding a mutant human STING protein having an amino acid substitution, such as a mutation at amino acid residue 155, particularly the V155M mutation. In one embodiment, the mmRNA encodes the amino acid sequence set forth in SEQ ID NO: 1. In one embodiment, the STING V155M mutant is encoded by the nucleotide sequence set forth in SEQ ID NO: 199. In one embodiment, the mmRNA comprises a 3'UTR sequence containing the miR122 binding site set forth in SEQ ID NO: 209.

[0181] In other aspects, the disclosure provides mmRNAs encoding mutant human STING proteins having a mutation, such as an amino acid substitution at amino acid residue 284. Non-limiting examples of residue 284 substitutions include R284T, R284M, and R284K. In certain embodiments, the mutant human STING protein has an R284T mutation, e.g., has the amino acid sequence set forth in SEQ ID NO: 2 or is encoded by the nucleotide sequence set forth in SEQ ID NO: 200. In certain embodiments, the mutant human STING protein has an R284M mutation, e.g., has the amino acid sequence set forth in SEQ ID NO: 3 or is encoded by the nucleotide sequence set forth in SEQ ID NO: 201. In certain embodiments, the mutant human STING protein has an R284K mutation, e.g., has the amino acid sequence set forth in SEQ ID NO: 4 or 224 or is encoded by the nucleotide sequence set forth in SEQ ID NO: 202 or 225.

[0182] In other aspects, the disclosure provides mmRNAs encoding mutant human STING proteins having a mutation, such as an amino acid substitution at amino acid residue 154, such as an N154S mutation. In certain embodiments, the mutant human STING protein has the N154S mutation, e.g., has the amino acid sequence set forth in SEQ ID NO: 5 or is encoded by the nucleotide sequence set forth in SEQ ID NO: 203.

[0183] In yet another aspect, the disclosure provides mmRNA encoding a mutant human STING protein having a mutation, such as an amino acid substitution at amino acid residue 147, such as a V147L mutation. In certain embodiments, the mutant human STING protein having a V147L mutation has the amino acid sequence set forth in SEQ ID NO: 6 or is encoded by the nucleotide sequence set forth in SEQ ID NO: 204.

[0184] In other aspects, the disclosure provides mmRNAs encoding mutant human STING proteins having a mutation, such as an amino acid substitution at amino acid residue 315, such as an E315Q mutation. In certain embodiments, the mutant human STING protein having an E315Q mutation has the amino acid sequence set forth in SEQ ID NO: 7 or is encoded by the nucleotide sequence set forth in SEQ ID NO: 205.

[0185] In other aspects, the disclosure provides mmRNAs encoding mutant human STING proteins having a mutation, such as an amino acid substitution at amino acid residue 375, such as an R375A mutation. In certain embodiments, the mutant human STING protein having an R375A mutation has the amino acid sequence set forth in SEQ ID NO: 8 or is encoded by the nucleotide sequence set forth in SEQ ID NO: 206.

[0186] In other aspects, the present disclosure provides mmRNAs encoding mutant human STING proteins having one or more of the aforementioned mutations, or a combination of two, three, four, or more. Thus, in one aspect, the present disclosure provides mmRNAs encoding mutant human STING proteins having one or more mutations selected from the group consisting of V147L, N154S, V155M, R284T, R284M, R284K, E315Q, and R375A, and combinations thereof. In another aspect, the present disclosure provides mmRNAs encoding mutant human STING proteins having a combination of mutations selected from the group consisting of V155M and R284T; V155M and R284M; V155M and R284K; V155M and V147L; V155M and N154S; V155M and E315Q; and V155M and R375A.

[0187] In another aspect, the disclosure provides an mmRNA encoding a mutant human STING protein having V155M and one, two, three or more of the following mutations: R284T; R284M; R284K; V147L; N154S; E315Q; and R375A. In another aspect, the disclosure provides an mmRNA encoding a mutant human STING protein having V155M, V147L, and N154S mutations. In another aspect, the disclosure provides an mmRNA encoding a mutant human STING protein having V155M, V147L, N154S mutations and, optionally, a mutation at amino acid 284. In yet another aspect, the disclosure provides an mmRNA encoding a mutant human STING protein having V155M, V147L, N154S mutations and a mutation at amino acid 284 selected from R284T, R284M, and R284K. In another aspect, the present disclosure provides an mmRNA encoding a mutant human STING protein having the V155M, V147L, N154S, and R284T mutations. In another aspect, the present disclosure provides an mmRNA encoding a mutant human STING protein having the V155M, V147L, N154S, and R284M mutations. In another aspect, the present disclosure provides an mmRNA encoding a mutant human STING protein having the V155M, V147L, N154S, and R284K mutations.

[0188] In other embodiments, the disclosure provides mmRNAs encoding mutant human STING proteins having combinations of mutations at amino acid residues 147, 154, 155, and optionally 284, particularly amino acid substitutions such as V147L, N154S, V155M, and optionally R284M. In certain embodiments, the mutant human STING protein has the V147N, N154S, and V155M mutations, such as the amino acid sequence set forth in SEQ ID NO: 9, or is encoded by the nucleotide sequence set forth in SEQ ID NO: 207. In certain embodiments, the mutant human STING protein has the R284M, V147N, N154S, and V155M mutations, such as the amino acid sequence set forth in SEQ ID NO: 10, or is encoded by the nucleotide sequence set forth in SEQ ID NO: 208.

[0189] In another embodiment, the present disclosure provides an mmRNA encoding a mutant human STING protein that is a constitutively active truncated form of the full-length 379 amino acid wild-type protein, such as a constitutively active human STING polypeptide consisting of amino acids 137 to 379.

[0190] Antigen-presenting cell promoter 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 (pseudo-APCs). Antigen presentation is a critical step in the initiation, amplification, and duration of immune responses. In this process, fragments of antigens 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.

[0191] 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 techniques 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.

[0192] 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.

[0193] 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.

[0194] 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.

[0195] 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.

[0196] 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.

[0197] 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. The cancer antigen can be a personalized cancer antigen. A personalized RNA cancer vaccine may contain, for example, RNA encoding one or more known cancer antigens specific to each subject, such as tumor antigens or cancer antigens. 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 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 not to generate an immune response against healthy tissue or to be masked by the immune system's defense components. In some embodiments, personalized RNA cancer 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 a different amino acid 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 RNA cancer vaccine comprises at least one cancer antigen comprising a mutation selected from the group consisting of a frameshift mutation and a recombination or any of the other mutations described herein.

[0198] The method for generating personalized RNA cancer vaccines generally involves identifying mutations, e.g., using deep nucleic acid or protein 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 RNA 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.

[0199] 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.

[0200] 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.

[0201] 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 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 throughout tumors.

[0202] 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.

[0203] 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).

[0204] Once a putative neoepitope is selected, it can be further tested using in vitro and / or in vivo assays. Isolates from each patient can 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. Neoepitope vaccines, their methods of use, and methods for preparation are fully described in PCT / US2016 / 044918, which is incorporated herein by reference in its entirety.

[0205] The activated oncogene mutant peptide selected for inclusion in RNA cancer vaccine is typically a high affinity binding peptide.In some aspects, the activated oncogene mutant peptide binds to HLA protein with higher affinity than wild-type peptide.In some embodiments, the activated oncogene mutant peptide has an IC50 of at least less than 5000nM, at least less than 500nM, at least less than 250nM, at least less than 200nM, at least less than 150nM, at least less than 100nM, at least less than 50nM or less.Typically, peptides with predicted IC50 less than 50nM are generally considered to be binding peptides with medium to high affinity, and will be selected to experimentally test their affinity using biochemical assays of HLA binding.

[0206] 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.

[0207] 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.

[0208] 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.

[0209] 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.

[0210] 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.

[0211] 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.

[0212] 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.

[0213] 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.

[0214] 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.

[0215] 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.

[0216] 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 encodes the epitope AVSPCISFVW (SEQ ID NO: 233) (HLA-B * 57:01, HLA-B * 58:01), epitope HPLASCQCFF (SEQ ID NO: 234) (HLA-B * 35:01, HLA-B * 53:01), epitope FVWNFGIPL (SEQ ID NO: 235) (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: 232).

[0217] In some embodiments, the 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 331 that encodes the epitope LQVLSLGTSY (SEQ ID NO: 237) (HLA-B * 15:01), epitope FQSNTQNAVF (SEQ ID NO: 238) (HLA-B * 15:01), a mutation is included that introduces a retained intron having the peptide sequence EYFTLQVLSLGTSYQVESFQSNTQNAVFFLTVLPAIGAFAIRGQ (SEQ ID NO: 236).

[0218] In some embodiments, the 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: 240) (HLA-A * 11:01), epitope KSVTCTMF (SEQ ID NO: 241) (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: 239) containing the nucleotide sequence 58:01.

[0219] In some embodiments, the 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: 243) (HLA-B * 53:01, HLA-B * 51:01), epitope LTVPPSTAW (SEQ ID NO: 244) (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: 242) containing the nucleotide sequence 57:01.

[0220] In the preceding sequences, the transcription codon positions refer to ENST00000269305 (SEQ ID NO: 245), the standard full-length p53 transcript from Ensembl's v83 human genome annotation.

[0221] Neoepitopes for prior art peptide vaccines are typically derived from patient DNA sequencing data. mRNA expression, on the other hand, provides a more direct measure of the global space of possible neoepitopes. For example, some tumor-specific neoepitopes may arise from splicing alterations, frameshift insertions / deletions (InDELs), alternative promoters, or epigenetic modifications, and are not easily identified using exome sequencing data alone. For neoantigen vaccines, identifying these complex mutation types is potentially valuable because it increases the number of epitopes that can bind to patient-specific HLA allotypes. Furthermore, complex variants are more immunogenic and may elicit a more effective immune response against tumors due to their distinct characteristics from self-proteins compared to variants resulting from single amino acid changes.

[0222] In some aspects, the present invention involves methods for identifying complex patient-specific mutations and formulating these mutations into effective personalized mRNA vaccines. The methods involve the use of short-read RNA sequencing. A major challenge inherent in using short reads for RNA sequencing is the fact that multiple mRNA transcript isoforms can be obtained from the same genomic locus due to alternative splicing and other mechanisms. Because sequencing reads are significantly shorter than full-length mRNA transcripts, it becomes difficult to map read sets to the correct corresponding isoforms within known gene annotation models. As a result, complex variants (common in cancer) that deviate from known gene annotations can be difficult to discover using standard approaches. However, the present disclosure involves the identification of short peptides rather than the precise exon composition of full-length transcripts. The method for identifying short peptides representative of these complex mutations involves a short k-mer counting approach to neoepitope prediction of complex variants.

[0223] A typical next-generation sequencing read is 150 base pairs, which, when capturing coding regions, allows for the analysis of 50 codons, or 41 different peptide epitopes of length 9 (27 nucleotides). Therefore, a simple, computationally scalable operation can be used to count all 27-mers from an RNA-sequencing sample and compare the results against normal tissue derived from the same sample or against a pre-computed database of 27-mers obtained from RNA-sequencing of normal tissue (e.g., GTEx).

[0224] mRNA vaccines containing neoepitopes predicted from RNA sequencing data can be created by: 1) counting all possible 27-mers from all RNA sequencing reads of a tumor sample; 2) predicting the open reading frame of each read by aligning any portion of the entire read with the transcriptome; 3) comparing the 27-mer counts with those of corresponding 27-mers from matched normal samples and / or a database of normal tissues from the same tissue type; and 4) using DNA sequencing data from the same tumor to add confidence to the neoepitope prediction if somatic mutations in the same gene are present. Regarding point (4), mutations often cause transcriptional or splicing changes, resulting in changes in the mRNA sequence that cannot be directly predicted from the mutation itself. For example, a splice site mutation can be expected to cause exon skipping, but it is not possible to know with certainty which downstream exon will be selected by the local splicing mechanism.

[0225] 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.

[0226] 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.

[0227] 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.

[0228] 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.

[0229] 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.

[0230] 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 quantitatively and qualitatively superior immune responses. Furthermore, the mRNA-LNP formulations of the present invention outperform other vaccines, even when using lower mRNA doses compared to other vaccines.

[0231] 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.

[0232] 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.

[0233] 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.

[0234] 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.

[0235] 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.

[0236] In some embodiments, the RNA polynucleotide of the cancer vaccine encodes 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, 5-6, 6-10, 6-9, 6-8, 6-7, 7-10, 7-9, 7-8, 8-10, 8-9, or 9-10 antigenic polypeptides. In some embodiments, the RNA polynucleotide of the cancer vaccine encodes at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 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 to 10, 5 to 15, 10 to 20, 15 to 25, 20 to 30, 25 to 35, 30 to 40, 35 to 45, 40 to 50, 55 to 65, 60 to 70, 65 to 75, 70 to 80, 75 to 85, 80 to 90, 85 to 95, 90 to 100, 1 to 50, 1 to 100, 2 to 50, or 2 to 100 antigenic polypeptides.

[0237] 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 activating oncogene mutant peptides. In some embodiments, the RNA polynucleotide of the cancer vaccine encodes at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 activating oncogene mutant peptides. In some embodiments, the RNA polynucleotide of the cancer vaccine encodes at least 100 or at least 200 activating oncogene mutant peptides. 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, 55-65, 60-70, 65-75, 70-80, 75-85, 80-90, 85-95, 90-100, 1-50, 1-100, 2-50, or 2-100 activating oncogene mutant peptides.

[0238] 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.

[0239] 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)).

[0240] 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)).

[0241] 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.

[0242] Antigens / antigenic polypeptides In some embodiments, a cancer polypeptide (e.g., an activated oncogene mutant peptide) is longer than 5 amino acids and shorter than 50 amino acids. In some embodiments, a cancer polypeptide is longer than 25 amino acids and shorter than 50 amino acids. Thus, polypeptides include gene products, naturally occurring polypeptides, synthetic polypeptides, 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.

[0243] 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.

[0244] 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.

[0245] "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.

[0246] "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.

[0247] 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.

[0248] 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.

[0249] 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.

[0250] 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.

[0251] 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.

[0252] 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).

[0253] 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.

[0254] 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.

[0255] 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 than the reference polypeptide sequence) of a reference protein that is 5, 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 10, 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.

[0256] 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.

[0257] 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.

[0258] 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.

[0259] 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)).

[0260] chemical modification Modified nucleotide sequence encoding an epitope antigen polypeptide 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.

[0261] 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.

[0262] 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).

[0263] 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.

[0264] 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.

[0265] 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.

[0266] In some embodiments, polynucleotides of the invention (e.g., RNA, e.g., mRNA) comprise chemically modified nucleobases. The invention includes modified polynucleotides, including the polynucleotides described herein (e.g., polynucleotides comprising a nucleotide sequence encoding one or more cancer epitope polypeptides). Modified polynucleotides can be chemically modified and / or structurally modified. When a polynucleotide of the invention is chemically and / or structurally modified, the polynucleotide can be referred to as a "modified polynucleotide."

[0267] The present disclosure provides polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) of modified nucleosides and nucleotides that encode one or more cancer epitope polypeptides. A "nucleoside" refers to a compound containing 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 containing a phosphate group. Modified nucleotides can be synthesized by any useful method, e.g., chemical, enzymatic, or recombinant, to include one or more modified or unnatural nucleosides. A polynucleotide can include one or more regions of linked nucleosides. Such regions can have a variety of backbone linkages. The linkages can be standard phosphodiester linkages, in which case the polynucleotide is considered to include a region of nucleotides.

[0268] The modified polynucleotides disclosed herein can contain a variety of different modifications. In some embodiments, the modified polynucleotides contain one, two, or more (optionally different) nucleoside or nucleotide modifications. In some embodiments, the modified polynucleotides introduced into cells can exhibit one or more desirable properties, such as improved protein expression, reduced immunogenicity, or reduced degradation in cells, compared with unmodified polynucleotides.

[0269] In some embodiments, polynucleotides of the present invention (e.g., polynucleotides comprising a nucleotide sequence encoding one or more cancer epitope polypeptides) are structurally modified. As used herein, a "structural" modification is one in which two or more linked nucleosides are inserted, deleted, duplicated, inverted, or randomized in a polynucleotide without significant chemical modification of the nucleotides themselves. Because chemical bonds are necessarily broken and reconstructed to produce a structural modification, the structural modification is chemical in nature and is therefore a chemical modification. However, the structural modification results in a different nucleotide sequence. For example, the polynucleotide "ATCG" can be chemically modified to "AT-5meC-G." The same polynucleotide can be structurally modified from "ATCG" to "ATCCCG." In this case, the structural modification occurs in the polynucleotide as a result of the insertion of the dinucleotide "CC."

[0270] In some embodiments, the polynucleotides of the present invention are chemically modified. As used herein with respect to polynucleotides, the term "chemically modified" or, as appropriate, "chemically modified" refers to modifications in one or more of the position, pattern, percentage, or population of ribonucleosides or deoxyribonucleosides containing adenosine (A), guanosine (G), uridine (U), or cytidine (C). Generally, as used herein, such terms are not intended to refer to ribonucleotide modifications in the cap moiety that naturally occurs at the 5' end of mRNA.

[0271] In some embodiments, polynucleotides of the invention can have a uniform chemical modification of all or any of the same nucleoside types, or a population of modifications produced by simple downward titration of the same starting modification of all or any of the same nucleoside types, or a measured percentage of all optional chemical modifications of the same nucleoside types with random incorporation (such as when all uridines are replaced with uridine analogs, e.g., pseudouridine or 5-methoxyuridine). In another embodiment, a polynucleotide can have a uniform chemical modification of two, three, or four of the same nucleoside types throughout the polynucleotide (e.g., all uridines and all cytosines are modified in the same way, etc.).

[0272] 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.

[0273] Those of skill in the art will recognize that unless otherwise indicated, the polynucleotide sequences set forth in this application recite a "T" in a representative DNA sequence, but when the sequence represents RNA, the "T" is replaced with a "U."

[0274] Modifications of polynucleotides (e.g., RNA polynucleotides such as mRNA polynucleotides), including but not limited to chemical modifications, useful in the compositions, methods, and synthetic processes of the present disclosure include, but are not limited to, the following: uniform nucleotides, nucleosides, and nucleobases: 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-methyladenosine, 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 Chiladenosine, N6,N6,2'-O-trimethyladenosine, N6,N6-dimethyladenosine, N6-acetyladenosine, N6-hydroxynorvalylcarbamoyladenosine, N6-methyl-N6-threonylcarbamoyladenosine, 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', -b-iodoguanosine TP, 2'-deoxy-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 Lysine, 5-hydroxyuridine, 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-pseudo-uridine 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-UTP , 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)-pseudo-UTP, 1-alkyl-6-(2-propynyl)-pseudo-UTP, 1-alkyl-6-allyl-pseudo-UTP, 1-alkyl-6-ethynyl-pseudo-UTP, 1-alkyl-6-homoallyl-pseudo-UTP, 1-alkyl-6-vinyl-pseudo-UTP, 1-allylpseudouridine TP, 1-aminomethyl-pseudo-UTP, 1-ben, Zoylpseudouridine TP, 1-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-pseudo-UTP, 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 P, 1-Me-alpha-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-me 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-pyrimidine-2-, ion-3-yl, ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl, oxoformycin TP, para-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl, para-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl, pentacenyl, phenanthracenyl, phenyl, propynyl-7-(aza)indolyl, pyrenyl, pyridopyrimidin-3-yl, pyridopyrimidin-3-yl, 2-on Xo-7-amino-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.

[0275] 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.

[0276] In some embodiments, the mRNA comprises at least one chemically modified nucleoside, in some embodiments, the at least one chemically modified nucleoside is 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), 5-methoxy-uridine (mo5U), 5-methyl Chil-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-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-Q-wyosine, 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 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.

[0277] In some embodiments, the mRNA is a uracil-modified sequence containing an ORF encoding one or more cancer epitope polypeptides, wherein the mRNA comprises a chemically modified nucleobase, such as 5-methoxyuracil. In certain aspects of the present invention, as in polynucleotides, when a 5-methoxyuracil base is attached to a ribose sugar, the resulting modified nucleoside or nucleotide is referred to as 5-methoxyuridine. In some embodiments, the uracil in the polynucleotide is at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least 90%, at least 95%, at least 99%, or about 100% 5-methoxyuracil. In one embodiment, the uracil in the polynucleotide is at least 95% 5-methoxyuracil. In another embodiment, the uracil in the polynucleotide is 100% 5-methoxyuracil.

[0278] In embodiments in which at least 95% of the uracils in the polynucleotide are 5-methoxyuracil, the overall uracil content can be adjusted so that the mRNA provides suitable protein expression levels while eliciting little or no immune response. In some embodiments, the uracil content of the ORF is about 105% to about 145%, about 105% to about 140%, about 110% to about 140%, about 110% to about 145%, about 115% to about 135%, about 105% to about 135%, about 110% to about 135%, about 115% to about 145%, or about 115% to about 140% of the theoretical minimum uracil content (% UTM) in the corresponding wild-type ORF. In other embodiments, the uracil content of the ORF is about 117% to about 134% or 118% to 132% of % UTM. In some embodiments, the uracil content of the ORF encoding one or more cancer epitope polypeptides is about 115%, about 120%, about 125%, about 130%, about 135%, about 140%, about 145%, or about 150% of %Utm. In this context, the term "uracil" can refer to 5-methoxyuracil and / or natural uracil.

[0279] In some embodiments, the uracil content in the ORF of an mRNA encoding one or more cancer epitope polypeptides of the present invention is less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 15%, or less than about 12% of the total nucleobase content in the ORF. In some embodiments, the uracil content in the ORF is about 12% to about 25% of the total nucleobase content of the ORF. In other embodiments, the uracil content in the ORF is about 15% to about 17% of the total nucleobase content of the ORF. In one embodiment, the uracil content in the ORF of an mRNA encoding one or more cancer epitope polypeptides is less than about 20% of the total nucleobase content in the open reading frame. In this context, the term "uracil" can refer to 5-methoxyuracil and / or natural uracil.

[0280] In further embodiments, the ORF of an mRNA encoding one or more cancer epitope polypeptides of the present invention comprises 5-methoxyuracil and has a modulated uracil content that contains fewer uracil pairs (UU) and / or uracil triplets (UUU) and / or uracil quadruplets (UUUU) than the corresponding wild-type nucleotide sequence encoding one or more cancer epitope polypeptides. In some embodiments, the ORF of an mRNA encoding one or more cancer epitope polypeptides of the present invention does not contain any uracil pairs and / or uracil triplets and / or uracil quadruplets. In some embodiments, uracil pairs and / or uracil triplets and / or uracil quadruplets are reduced below a certain threshold, e.g., the number of occurrences in an mRNA ORF encoding one or more cancer epitope polypeptides is reduced to 1 or less, 2 or less, 3 or less, 4 or less, 5 or less, 6 or less, 7 or less, 8 or less, 9 or less, 10 or less, 11 or less, 12 or less, 13 or less, 14 or less, 15 or less, 16 or less, 17 or less, 18 or less, 19 or less, or 20 or less. In specific embodiments, an mRNA ORF encoding one or more cancer epitope polypeptides of the present invention contains fewer than 20, fewer than 19, fewer than 18, fewer than 17, fewer than 16, fewer than 15, fewer than 14, fewer than 13, fewer than 12, fewer than 11, fewer than 10, fewer than 9, fewer than 8, fewer than 7, fewer than 6, fewer than 5, fewer than 4, fewer than 3, fewer than 2, or fewer than 1 non-phenylalanine uracil pairs and / or triplets. In another embodiment, the ORF of the mRNA encoding one or more cancer epitope polypeptides does not contain any non-phenylalanine uracil pairs and / or triplets.

[0281] In further embodiments, the ORF of the mRNA encoding one or more cancer epitope polypeptides of the present invention contains 5-methoxyuracil and has a regulated uracil content that contains a smaller amount of uracil-rich cluster than the corresponding wild-type nucleotide sequence encoding one or more cancer epitope polypeptides. In some embodiments, the ORF of the mRNA encoding one or more cancer epitope polypeptides of the present invention contains a uracil-rich cluster that is shorter in length than the corresponding uracil-rich cluster in the corresponding wild-type nucleotide sequence encoding one or more cancer epitope polypeptides.

[0282] In another embodiment, low-frequency alternative codons are used.At least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or 100% of the codons in the ORF of mRNA that encode one or more cancer epitope polypeptides are replaced with alternative codons that have a lower codon frequency than the codon frequency of the replacement codon in the synonymous codon set, respectively.The uracil content of the ORF is also adjusted as described above. In some embodiments, at least one codon in the ORF of the mRNA encoding one or more cancer epitope polypeptides is replaced with an alternative codon that has a lower codon frequency than the codon frequency of the replacement codon in the synonymous codon set.

[0283] In some embodiments, modulating the uracil content of an mRNA ORF containing 5-methoxyuracil encoding one or more cancer epitope polypeptides results in higher expression levels of the one or more cancer epitope polypeptides when administered to mammalian cells than the expression levels of the one or more cancer epitope polypeptides from the corresponding wild-type mRNA. In other embodiments, the expression level of the one or more cancer epitope polypeptides when administered to mammalian cells is increased compared to a corresponding mRNA that contains at least 95% 5-methoxyuracil and has a uracil content of about 160%, about 170%, about 180%, about 190%, or about 200% of the theoretical minimum. In still other embodiments, the expression level of the one or more cancer epitope polypeptides when administered to mammalian cells is increased compared to a corresponding mRNA in which at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of the uracils are 1-methylpseudouracil or pseudouracil. In some embodiments, the mammalian cells are mouse cells, rat cells, or rabbit cells. In other embodiments, the mammalian cells are monkey cells or human cells. In some embodiments, the human cells are HeLa cells, BJ fibroblast cells, or peripheral blood mononuclear cells (PBMCs). In some embodiments, one or more cancer epitope polypeptides are expressed when the mRNA is administered to mammalian cells in vivo. In some embodiments, the mRNA is administered to a mouse, rabbit, rat, monkey, or human. In one embodiment, the mouse is a null mouse. In some embodiments, the mRNA is administered to the mouse in an amount of about 0.01 mg / kg, about 0.05 mg / kg, about 0.1 mg / kg, or about 0.15 mg / kg. In some embodiments, the mRNA is administered intravenously or intramuscularly. In other embodiments, one or more cancer epitope polypeptides are expressed when the mRNA is administered to mammalian cells in vitro.In some embodiments, expression is increased by at least about 2-fold, at least about 5-fold, at least about 10-fold, at least about 50-fold, at least about 500-fold, at least about 1500-fold, or at least about 3000-fold. In other embodiments, expression is increased by at least about 10%, about 20%, about 30%, about 40%, about 50%, 60%, about 70%, about 80%, about 90%, or about 100%.

[0284] In some embodiments, an mRNA ORF containing 5-methoxyuracil encoding one or more cancer epitope polypeptides exhibits increased stability when its uracil content is modulated. In some embodiments, the mRNA exhibits increased intracellular stability compared to the stability of the corresponding wild-type mRNA under the same conditions. In some embodiments, the mRNA exhibits increased stability, including improved nuclease resistance, thermal stability, and / or secondary structure stabilization. In some embodiments, the increased stability of an mRNA is measured by determining the half-life of the mRNA (e.g., in plasma, cells, or tissue samples) and / or determining the area under the curve (AUC) of protein expression by the mRNA over time (e.g., in vitro or in vivo). An mRNA is identified as having increased stability if its half-life and / or AUC exceeds the half-life and / or AUC of the corresponding wild-type mRNA under the same conditions.

[0285] In some embodiments, the mRNA of the present disclosure induces a detectably lower immune response (e.g., innate or adaptive immunity) compared to the immune response induced by the corresponding wild-type mRNA under the same conditions. In other embodiments, the mRNA of the present disclosure induces a detectably lower immune response (e.g., innate or adaptive immunity) compared to the immune response induced under the same conditions by an mRNA encoding one or more cancer epitope polypeptides but not containing 5-methoxyuracil, or compared to the immune response induced under the same conditions by an mRNA encoding one or more cancer epitope polypeptides and containing 5-methoxyuracil but with an unmodified uracil content. The innate immune response may be manifested by increased expression of pro-inflammatory cytokines, activation of intracellular PRRs (e.g., RIG-I, MDA5), cell death, and / or halted or reduced protein translation. In some embodiments, a reduction in the innate immune response can be measured by the expression or activity levels of type 1 interferons (e.g., IFN-α, IFN-β, IFN-κ, IFN-δ, IFN-ε, IFN-τ, IFN-ω, and IFN-ζ), or the expression of interferon-regulated genes such as toll-like receptors (e.g., TLR7 and TLR8), and / or by reduced cell death after one or more administrations of an mRNA of the invention to cells.

[0286] In some embodiments, expression of type 1 interferon by a mammalian cell in response to an mRNA of the present disclosure is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9%, or greater than 99.9% compared to the corresponding wild-type mRNA, an mRNA encoding one or more cancer epitope polypeptides but not containing 5-methoxyuracil, or an mRNA encoding one or more cancer epitope polypeptides and containing 5-methoxyuracil but with an unmodified uracil content. In some embodiments, the interferon is IFN-β. In some embodiments, the frequency of cell death resulting from administration of an mRNA of the present disclosure to a mammalian cell is 10%, 25%, 50%, 75%, 85%, 90%, 95%, or greater than 95% less than the frequency of cell death observed with the corresponding wild-type mRNA, an mRNA encoding one or more cancer epitope polypeptides but not containing 5-methoxyuracil, or an mRNA encoding one or more cancer epitope polypeptides and containing 5-methoxyuracil but with an unmodified uracil content. In some embodiments, the mammalian cell is a BJ fibroblast. In other embodiments, the mammalian cell is a splenocyte. In some embodiments, the mammalian cell is a mouse or rat cell. In other embodiments, the mammalian cell is a human cell. In one embodiment, the mRNA of the present disclosure does not substantially induce an innate immune response in the mammalian cell into which the mRNA is introduced.

[0287] In some embodiments, the polynucleotide is an mRNA comprising an ORF encoding one or more cancer epitope polypeptides, wherein at least about 95% of the uracil in the mRNA is 5-methoxyuracil, the uracil content of the ORF is about 115% to about 135% of the theoretical minimum uracil content in the corresponding wild-type ORF, and the uracil content in the ORF encoding one or more cancer epitope polypeptides is less than about 23% of the total nucleobase content in the ORF. In some embodiments, the ORF encoding one or more cancer epitope polypeptides is further modified to reduce the G / C content (absolute or relative) of the ORF by at least about 40% compared to the corresponding wild-type ORF. In still other embodiments, the ORF encoding one or more cancer epitope polypeptides contains fewer than 20 non-phenylalanine uracil pairs and / or triplets. In some embodiments, at least one codon in an ORF of an mRNA encoding one or more cancer epitope polypeptides is further substituted with an alternative codon that has a lower codon frequency than the replacement codon in the synonymous codon set. In some embodiments, expression of one or more cancer epitope polypeptides encoded by an mRNA comprising an ORF, wherein at least about 95% of the uracils in the mRNA are 5-methoxyuracil, and the uracil content of the ORF is about 115% to about 135% of the theoretical minimum uracil content in the corresponding wild-type ORF, is increased by at least about 10-fold when compared to expression of one or more cancer epitope polypeptides derived from the corresponding wild-type mRNA. In some embodiments, the mRNA comprises an open ORF, wherein at least about 95% of the uracils in the mRNA are 5-methoxyuracil, and the uracil content of the ORF is about 115% to about 135% of the theoretical minimum uracil content in the corresponding wild-type ORF, and the mRNA does not substantially induce an innate immune response in a mammalian cell into which the mRNA is introduced.

[0288] In certain embodiments, the chemical modification is present in a nucleobase in a polynucleotide (e.g., an RNA polynucleotide such as an mRNA polynucleotide). In some embodiments, the modified nucleobase in a polynucleotide (e.g., an RNA polynucleotide such as an mRNA polynucleotide) is 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, the polynucleotide comprises a combination of at least two (e.g., two, three, four, or more) of the foregoing modified nucleobases.

[0289] 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 polynucleotide (e.g., an RNA polynucleotide such as an mRNA polynucleotide) comprises 1-methyl-pseudouridine (mψ). In some embodiments, a polynucleotide (e.g., an RNA polynucleotide such as an mRNA polynucleotide) comprises 1-ethyl-pseudouridine (eψ). In some embodiments, a polynucleotide (e.g., an RNA polynucleotide such as an mRNA polynucleotide) comprises 1-methyl-pseudouridine (mψ) and 5-methyl-cytidine (mC). In some embodiments, a polynucleotide (e.g., an RNA polynucleotide such as an mRNA polynucleotide) comprises 1-ethyl-pseudouridine (eψ) and 5-methyl-cytidine (mC). In some embodiments, a polynucleotide (e.g., an RNA polynucleotide such as an mRNA polynucleotide) comprises 2-thiouridine (sU). In some embodiments, a polynucleotide (e.g., an RNA polynucleotide such as an mRNA polynucleotide) comprises 2-thiouridine and 5-methyl-cytidine (m5C). In some embodiments, a polynucleotide (e.g., an RNA polynucleotide such as an mRNA polynucleotide) comprises methoxy-uridine (mo5U). In some embodiments, a polynucleotide (e.g., an RNA polynucleotide such as an mRNA polynucleotide) comprises 5-methoxy-uridine (mo5U) and 5-methyl-cytidine (m5C). In some embodiments, a polynucleotide (e.g., an RNA polynucleotide such as an mRNA polynucleotide) comprises 2'-O-methyluridine. In some embodiments, a polynucleotide (e.g., an RNA polynucleotide such as an mRNA polynucleotide) comprises 2'-O-methyluridine and 5-methyl-cytidine (m5C). In some embodiments, a polynucleotide (e.g., an RNA polynucleotide such as an mRNA polynucleotide) comprises N6-methyl-adenosine (m6A).In some embodiments, a polynucleotide (eg, an RNA polynucleotide such as an mRNA polynucleotide) comprises N6-methyl-adenosine (m6A) and 5-methyl-cytidine (m5C).

[0290] In some embodiments, polynucleotides (e.g., RNA polynucleotides such as mRNA polynucleotides) are uniformly modified (e.g., completely modified, modified throughout the entire sequence) to obtain a particular modification. For example, polynucleotides can be uniformly modified with 5-methyl-cytidine (m5C), meaning that all cytosine residues in the mRNA sequence are replaced with 5-methyl-cytidine (m5C). As another example, polynucleotides can be uniformly modified with 1-methyl-pseudouridine, meaning that all 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 a modified residue such as any of those described above.

[0291] In some embodiments, the chemically modified nucleosides in the open reading frame are selected from the group consisting of uridine, adenine, cytosine, guanine, and any combination thereof.

[0292] In some embodiments, the modified nucleobase is a modified cytosine. 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.

[0293] 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.

[0294] 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), N6-methyl-adenosine (m6A), and 2,6-diaminopurine.

[0295] 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.

[0296] In some embodiments, the nucleobase-modified nucleotide in a polynucleotide (eg, an RNA polynucleotide such as an mRNA polynucleotide) is 5-methoxyuridine.

[0297] In some embodiments, a polynucleotide (eg, an RNA polynucleotide such as an mRNA polynucleotide) comprises a combination of at least two (eg, two, three, four, or more) modified nucleobases.

[0298] In some embodiments, a polynucleotide (eg, an RNA polynucleotide such as an mRNA polynucleotide) comprises 5-methoxyuridine (5mo5U) and 5-methyl-cytidine (m5C).

[0299] In some embodiments, polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) are uniformly modified (e.g., completely modified, modified throughout the entire sequence) to obtain a particular modification. For example, polynucleotides can be uniformly modified with 5-methoxyuridine, meaning that substantially all of the uridine residues in the mRNA sequence are replaced with 5-methoxyuridine. Similarly, polynucleotides can be uniformly modified so that any type of nucleoside residue is present in the sequence by replacement with a modified residue, such as any of those described above.

[0300] In some embodiments, the modified nucleobase is a modified cytosine.

[0301] In some embodiments, the modified nucleobase is a modified uracil. Examples of nucleobases and nucleosides having modified uracil include 5-methoxyuracil.

[0302] In some embodiments, the modified nucleobase is a modified adenine.

[0303] In some embodiments, the modified nucleobase is a modified guanine.

[0304] In some embodiments, a polynucleotide can include any useful linker between nucleosides. Such linkers, including backbone modifications, that are useful in the compositions of the present disclosure include: 3'-alkylene phosphonates, 3'-amino phosphoramidates, alkene-containing backbones, aminoalkyl phosphoramidates, aminoalkyl phosphotriesters, boranophosphates, -CH2-ON(CH3)-CH2-, -CH2-N(CH3)-N(CH3)-CH2-, -CH2-NH-CH2-, chiral phosphonates, chiral phosphorothioates, formacetyl and thioformacetyl backbones, methylene(methylimino), methyleneformacetyl and thioformacetyl backbones, methyl These include, but are not limited to, imino and methylenehydrazino backbones, morpholino linkages, -N(CH3)-CH2-CH2-, heteroatom-containing oligonucleoside internucleoside linkages, phosphinates, phosphoramidates, phosphorodithioates, phosphorothioate internucleoside linkages, phosphorothioates, phosphotriesters, PNAs, siloxane backbones, sulfamate backbones, sulfide sulfoxide and sulfone backbones, sulfonate and sulfonamide backbones, thionoalkylphosphonates, thionoalkylphosphotriesters, and thionophosphoramidates.

[0305] Modified nucleosides and nucleotides (e.g., building block molecules) that can be incorporated into polynucleotides (e.g., RNA or mRNA as described herein) can be modified on the sugar of the ribonucleic acid. For example, the 2' hydroxyl group (OH) can be modified or replaced with a number of different substituents. Examples of substitutions at the 2' position include H, halo, optionally substituted C, 1-6 Alkyl; optionally substituted C 1-6 Alkoxy; optionally substituted C 6-10 Aryloxy; optionally substituted C 3-8 Cycloalkyl; optionally substituted C 3-8 Cycloalkoxy; optionally substituted C 6-10 Aryloxy; optionally substituted C 6-10 Aryl-C 1-6Alkoxy, optionally substituted C 1-12 (heterocyclyl)oxy; sugars (e.g., ribose, pentose, or any described herein); polyethylene glycol (PEG), -O(CH2CH2O) n CH2CH2OR, where R is H or optionally substituted alkyl and n is an integer from 0 to 20 (e.g., 0 to 4, 0 to 8, 0 to 10, 0 to 16, 1 to 4, 1 to 8, 1 to 10, 1 to 16, 1 to 20, 2 to 4, 2 to 8, 2 to 10, 2 to 16, 2 to 20, 4 to 8, 4 to 10, 4 to 16, and 4 to 20); 1-6 Alkylene bridge or C 1-6 "Locked" nucleic acids (LNAs) connected to the 4'-carbon of the same ribose sugar by a heteroalkylene bridge (examples of bridges include methylene, propylene, ether, or amino bridges); aminoalkyl, as defined herein; aminoalkoxy, as defined herein; amino, as defined herein; and amino acid, as defined herein.

[0306] Generally, RNA contains the sugar group ribose, which is a five-membered ring with oxygen. Non-limiting examples of modified nucleotides include substitution of oxygen in the ribose (e.g., with S, Se, or an alkylene such as methylene or ethylene); addition of a double bond (e.g., substituting cyclopentenyl or cyclohexenyl for ribose); ring contraction of ribose (e.g., forming a four-membered cyclobutane or oxetane ring); ring expansion of ribose (e.g., forming a six- or seven-membered ring with additional carbon or heteroatoms and a phosphoramidate backbone, such as anhydrohexitol, altritol, mannitol, cyclohexanyl, cyclohexenyl, and morpholino); polycyclic forms (e.g., tricyclo; and "unlocked" forms, such as glycol nucleic acids (GNAs) (e.g., glycol nucleic acids in which the ribose is linked to a phosphodiester bond). Examples of such sugar modifications include R-GNA or S-GNA, in which the ribose is replaced by a threofuranosyl (3'→2') unit, threose nucleic acid (TNA, in which the ribose is replaced by α-L-threofuranosyl-(3'→2')), and peptide nucleic acid (PNA, in which the ribose and phosphodiester backbone are replaced by a 2-amino-ethyl-glycine linkage). The sugar group may also contain one or more carbons with the opposite stereochemical configuration to that of the corresponding carbon in ribose. Thus, a polynucleotide molecule may include, for example, arabinose-containing nucleotides as sugars. Such sugar modifications are taught in International Patent Publication Nos. WO2013052523 and WO2014093924, the contents of each of which are incorporated herein by reference in their entirety.

[0307] Polynucleotides of the invention (e.g., polynucleotides comprising a nucleotide sequence encoding one or more cancer epitope polypeptides or functional fragments or variants thereof) can include combinations of modifications to sugars, nucleobases, and / or internucleoside linkages, which combinations can include any one or more of the modifications described herein.

[0308] 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.

[0309] 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.

[0310] 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).

[0311] 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.

[0312] 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.

[0313] 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.

[0314] 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.

[0315] 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.

[0316] 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.

[0317] 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.

[0318] 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.

[0319] It should be understood that the mRNA vaccines of the present disclosure, e.g., mRNA encoding a cancer antigen or, e.g., an activated oncogene mutant peptide, may be prepared using any suitable synthesis method. For example, in some embodiments, the 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 includes a promoter sequence, e.g., a bacteriophage promoter sequence, to facilitate IVT. Methods for preparing RNA using a 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.

[0320] 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.

[0321] In another embodiment, the mRNA vaccine of the present disclosure, such as an mRNA encoding a cancer antigen or epitope, may be prepared using two DNA strands whose overlapping sequences are complementary throughout, leaving a single-stranded overhang (i.e., a sticky end) when the complementary portions are annealed. Such a single-stranded overhang can be made double-stranded by extending 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 primer extension, 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 an ORF (e.g., a portion of the 5' end of the ORF). In some embodiments, the single-stranded second-strand DNA may include a promoter (e.g., T7, T3, or SP6) sequence, optionally a 5'-UTR, and part or all of an ORF (e.g., a portion complementary to the 3' end of the ORF). The RNA may contain 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.

[0322] In another aspect, mRNA vaccines of the present disclosure, e.g., mRNA encoding a cancer antigen or epitope, 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.

[0323] 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).

[0324] In another embodiment, mRNA vaccines of the present disclosure, such as mRNA encoding a cancer antigen or epitope, 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.

[0325] 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.

[0326] In some embodiments, the first polynucleotide is characterized by a 5'-triphosphate and a 3'-OH. In 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.

[0327] 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.

[0328] 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, for example, 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.

[0329] 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.

[0330] Untranslated Regions (UTRs) An untranslated region (UTR) is a nucleic acid region of a polynucleotide that is not translated before the start codon (5'UTR) and after the stop codon (3'UTR). In some embodiments, a polynucleotide of the invention (e.g., a ribonucleic acid (RNA), e.g., a messenger RNA (mRNA)) comprising an open reading frame (ORF) encoding one or more cancer antigens or epitopes further comprises a UTR (e.g., a 5'UTR or a functional fragment thereof, a 3'UTR or a functional fragment thereof, or a combination thereof).

[0331] The UTR may be homologous or heterologous to the coding region in the polynucleotide. In some embodiments, the UTR is homologous to the ORF encoding one or more cancer epitope polypeptides. In some embodiments, the UTR is heterologous to the ORF encoding one or more cancer epitope polypeptides. In some embodiments, the polynucleotide comprises two or more 5' UTRs or functional fragments thereof, each of which has the same or different nucleotide sequence. In some embodiments, the polynucleotide comprises two or more 3' UTRs or functional fragments thereof, each of which has the same or different nucleotide sequence.

[0332] In some embodiments, the 5'UTR or a functional fragment thereof, the 3'UTR or a functional fragment thereof, or any combination thereof, is sequence optimized.

[0333] In some embodiments, the 5'UTR or a functional fragment thereof, the 3'UTR or a functional fragment thereof, or any combination thereof comprises at least one chemically modified nucleobase, for example, 5-methoxyuracil.

[0334] UTRs can have regulatory roles, e.g., characteristics that confer stability, localization, and / or increased or decreased translational efficiency. Polynucleotides containing UTRs can be administered to cells, tissues, or organs, and one or more regulatory characteristics can be measured using routine methods. In some embodiments, functional fragments of the 5' or 3' UTRs comprise one or more regulatory characteristics of the full-length 5' or 3' UTR, respectively.

[0335] Naturally occurring 5' UTRs are characterized by their involvement in translation initiation. They retain features such as the Kozak sequence, which is commonly known to be involved in the process by which translation of many genes is initiated by the ribosome. The Kozak sequence has the consensus CCR(A / G)CCAUGG (SEQ ID NO: 246), where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG) followed by another "G." 5' UTRs are also known to form secondary structures involved in the binding of elongation factors.

[0336] Polynucleotide stability and protein production can be enhanced by engineering features typically found in genes abundantly expressed in specific target organs. For example, introducing the 5'UTR of liver-expressed mRNAs, such as albumin, serum amyloid A, apolipoprotein A / B / E, transferrin, alpha-fetoprotein, erythropoietin, or factor VIII, can enhance polynucleotide expression in hepatic cell lines or the liver. Similarly, for muscle (e.g., MyoD, myosin, myoglobin, myogenin, herculin), endothelial cells (e.g., Tie-1, CD36), myeloid cells (e.g., C / EBP, AML1, G-CSF, GM-CSF, CD11b, MSR, Fr-1, i-NOS), leukocytes (e.g., CD45, CD18), adipose tissue (e.g., CD36, GLUT4, ACRP30, adiponectin), and lung epithelial cells (e.g., SP-A / B / C / D), 5' UTRs from other tissue-specific mRNAs can be used to improve expression in those tissues.

[0337] In some embodiments, UTRs are selected from a family of transcripts, whose proteins share a common function, structure, feature or characteristic. For example, the encoded polypeptide may belong to a family of proteins (i.e., a family that shares at least one function, structure, feature, localization, origin, or expression pattern) that are expressed in a particular cell, tissue, or at a certain time during development. UTRs from either a gene or mRNA can be exchanged with any other UTR from the same or different protein family to create new polynucleotides.

[0338] In some embodiments, the 5'UTR and 3'UTR may be heterologous. In some embodiments, the 5'UTR may be derived from a different species than the 3'UTR. In some embodiments, the 3'UTR may be derived from a different species than the 5'UTR.

[0339] Co-owned International Patent Application No. PCT / US2014 / 021522 (International Publication No. WO / 2014 / 164253, which is incorporated herein by reference in its entirety) provides a list of exemplary UTRs that can be utilized in the polynucleotides of the invention as flanking regions to the ORF.

[0340] Examples of UTRs of the present application include, but are not limited to, one or more 5'UTRs and / or 3'UTRs derived from the following nucleic acid sequences: globins, such as α-globin or β-globin (e.g., Xenopus, mouse, rabbit, or human globin); strong Kozak translation initiation signals; CYBA (e.g., human cytochrome b-245α polypeptide); albumins (e.g., human albumin 7); HSD17B4 (hydroxysteroid (17-β) dehydrogenase); viruses (e.g., tobacco etch virus (TEV), Venezuelan equine encephalitis virus (VEEV), dengue virus, cytomegalovirus (CMV) (e.g., CMV immediate early 1 (IE1)), hepatitis viruses (e.g., heat shock proteins (e.g., hsp70); translation initiation factors (e.g., eIF4G); glucose transporters (e.g., hGLUT1 (human glucose transporter 1)); actin (e.g., human α-actin or β-actin); GAPDH; tubulin; histones; citric acid cycle enzymes; topoisomerases (e.g., the 5'UTR of the TOP gene lacking the 5'TOP motif (oligopyrimidine tract)); ribosomal protein large 32 (L32); ribosomal proteins (e.g., human or mouse ribosomal proteins, such as rps9); ATP synthases (e.g., ATP5A1 or mitochondrial H +- the beta subunit of ATP synthase; growth hormone (e.g., bovine (bGH) or human (hGH)); elongation factor (e.g., elongation factor 1 α1 (EEF1A1)); manganese superoxide dismutase (MnSOD); myocyte enhancer factor 2A (MEF2A); β-F1-ATPase, creatine kinase, myoglobin, granulocyte colony-stimulating factor (G-CSF); collagens (e.g., type I collagen α2 (Col1A2), type I collagen α1 (Col1A1), type VI collagen α2 (Col6A2), type VI collagen α1 (Col6A1)); ribophorins (e.g., ribophorin I (RPNI)); low-density lipoprotein receptor-related protein (e.g., LRP1); cardiotrophin-like cytokine factors (e.g., Nnt1); calreticulin (Calr); procollagen-lysine, 2-oxoglutarate 5-dioxygenase 1 (Plod1); and nucleobioindins (e.g., Nucb1).

[0341] Other examples of 5’UTRs and 3’UTRs include Kariko et al., Mol. Ther. 2008 16(11):1833-1840; Kariko et al., Mol. Ther. 2012 20(5):948-953; Kariko et al., Nucleic Acids Res. 2011 39(21):e142; Strong et al., Gene Therapy 1997 4:624-627; Hansson et al., J. Biol. Chem. 2015 290(9):5661-5672; Yu et al., Vaccine 2007 25(10):1701-1711; Cafri et al., Mol. Ther. 2015 23(8):1391-1400; Andries et al., Mol. Pharm. 2012 9(8):2136-2145; Crowley et al., Gene Ther. 2015 Jun 30, doi:10.1038 / gt.2015.68; Ramunas et al., FASEB J. 2015 29(5):1930-1939; Wang et al., Curr. Gene Ther. 2015 15(4):428-435; Holtkamp et al., Blood 2006 108(13):4009-40l7; Kormann et al., Nat. Biotechnol. 2011 29(2):154-157; Poleganov et al., Hum. Gen. Ther. 2015 26(11):751-76; Warren et al., Cell Stem Cell 2010 7(5):618-630; Mandal and Rossi, Nat. Protoc. 2013 8(3):568-582; Holcik and Liebhaber, PNAS 1997 94(6):2410-2414; Ferizi et al., Lab Chip. 2015 15(17):3561-3571; Thess et al., Mol. Ther. 2015 23(9):1456-1464; Boros et al., PLoS One 2015 10(6):e0131141; Boros et al., J. Photochem. Photobiol. B.2013 129:93-99;Andries et al.,J.Control.Release 2015 217:337-344;Zinckgraf et al.,Vaccine 2003 21(15):1640-9;Garneau et al.,J.Virol.2008 82(2):880-892;Holden and Harris,Virology 2004 329(1):119-133;Chiu et al.,J.Virol.2005 79(13):8303-8315;Wang et al.,EMBO J.1997 16(13):4107-4116;Al-Zoghaibi et al.,Gene 2007 391(1-2):130-9;Vivinus et al.,Eur.J.Biochem.2001 268(7):1908-1917;Gan and Rhoads,J.Biol.Chem.1996 271(2):623-626;Boado et al.,J.Neurochem.1996 67(4):1335-1343;Knirsch and Clerch,Biochem.Biophys.Res.Commun.2000 272(1):164-168;Chung et al.,Biochemistry 1998 37(46):16298-16306;Izquierdo and Cuevza,Biochem.J.2000 346 Pt 3:849-855;Dwyer et al.,J.Neurochem.1996 66(2):449-458;Black and al.,Mol.Cell.Biol.1997 17(5):2756-2763;Izquierdo and Cuevza,Mol.Cell.Biol.1997 17(9):5255-5268;US8278036;US8748089;US8835108;US9012219;US2010 / 0129877;US2011 / 0065103;US2011 / 0086904;US2012 / 0195936;US2014 / 020675;US2013 / 0195967;US2014 / 029490;US2014 / 0206753;WO2007 / 036366;WO2011 / 015347;WO2012 / 072096;WO2013 / 143555;WO2014 / 071963;WO 2013 / 185067; WO2013 / 182623; WO2014 / 089486; WO2013 / 185069; WO2014 / 144196; WO2014 / 152659; 2014 / 152673; WO2014 / 152940; WO2014 / 152774; WO2014 / 153052; WO2014 / 152966, WO2014 / 152513; WO2015 / 101414; WO2015 / 101415; WO2015 / 062738; and WO2015 / 024667, the contents of each of which are incorporated herein by reference in their entirety. .

[0342] In some embodiments, the 5'UTR is selected from the group consisting of: the 5'UTR of beta-globin; a 5'UTR containing a strong Kozak translation initiation signal; the 5'UTR of cytochrome b-245 alpha polypeptide (CYBA); the 5'UTR of hydroxysteroid (17-beta) dehydrogenase (HSD17B4); the 5'UTR of tobacco etch virus (TEV); the 5'UTR of Venezuelan equine encephalitis virus (TEEV); the 5' open reading frame of rubella virus (RV) RNA encoding a nonstructural protein; the 5'UTR of dengue virus (DEN); the 5'UTR of heat shock protein 70 (Hsp70); the 5'UTR of eIF4G; the 5'UTR of GLUT1; a functional fragment thereof, and any combination thereof.

[0343] In some embodiments, the 3'UTR is selected from the group consisting of the 3'UTR of β-globin; the 3'UTR of CYBA; the 3'UTR of albumin; the 3'UTR of growth hormone (GH); the 3'UTR of VEEV; the 3'UTR of hepatitis B virus (HBV); the 3'UTR of α-globin; the 3'UTR of DEN; the 3'UTR of PAV barley yellow dwarf virus (BYDV-PAV); the 3'UTR of elongation factor 1 alpha 1 (EEF1A1); the 3'UTR of manganese superoxide dismutase (MnSOD); the 3'UTR of the β subunit of mitochondrial H(+)-ATP synthase (β-mRNA); the 3'UTR of GLUT1; the 3'UTR of MEF2A; the 3'UTR of β-F1-ATPase; functional fragments thereof, and any combination thereof.

[0344] Other examples of UTRs include, but are not limited to, one or more of the UTRs (including any combination of UTRs) disclosed in WO2014 / 164253, the contents of which are incorporated herein by reference in their entirety. Table 21 of U.S. Provisional Patent Application No. 61 / 775,509 and Table 22 of U.S. Provisional Patent Application No. 61 / 829,372 provide lists of start and end sites for 5'UTRs and 3'UTRs, the contents of each of which are incorporated herein by reference in their entirety. In Table 21, each 5'UTR (5'-UTR-005 to 5'-UTR68511) is identified by its start and end site relative to its native or wild-type (homologous) transcript (the identifier used in the ENST; ENSEMBL database).

[0345] The wild-type UTR from any gene or mRNA can be incorporated into the polynucleotide of the present invention.In some embodiments, UTR can be modified relative to wild-type or natural UTR to generate variant UTR, for example, by changing the orientation or position of UTR relative to ORF, or by incorporating additional nucleotides, deleting nucleotides, replacing nucleotides or transposition.In some embodiments, the variant of 5' or 3' UTR can be, for example, a mutant of wild-type UTR, or a variant in which one or more nucleotides are added or removed from the end of UTR.

[0346] Additionally, one or more synthetic UTRs can be used in combination with one or more non-synthetic UTRs. See, e.g., Mandal and Rossi, Nat. Protoc. 2013 8(3):568-82 and the sequences available at www.addgene.org / Derrick_Rossi / , the contents of each of which are incorporated herein by reference in their entirety. A UTR, or portion thereof, can be positioned in the same orientation as the transcript from which it was selected, or its orientation or position can be altered. Thus, the 5'UTR and / or 3'UTR can be inverted, shortened, extended, or combined with one or more other 5'UTRs or 3'UTRs.

[0347] In some embodiments, the polynucleotide comprises multiple UTRs, e.g., double, triple, or quadruple 5' or 3' UTRs. For example, a double UTR comprises two copies of the same UTR, either in tandem or substantially in tandem. For example, a double β-globin 3' UTR can be used (see US2010 / 0129877, the contents of which are incorporated herein by reference in their entirety).

[0348] In certain embodiments, a polynucleotide of the invention comprises a 5'UTR and / or a 3'UTR selected from any of the UTRs disclosed herein. In some embodiments, the 5'UTR and / or 3'UTR comprises: [Table 1-1] [Table 1-2]

[0349] In certain embodiments, the 5'UTR and / or 3'UTR sequences of the present invention comprise a nucleotide sequence that is at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to a sequence selected from the group consisting of a 5'UTR sequence comprising any of SEQ ID NOs: 247-271 and / or a 3'UTR sequence comprising any of SEQ ID NOs: 272-302, and any combination thereof.

[0350] Polynucleotides of the invention can comprise a combination of features. For example, an ORF can be flanked by a 5' UTR containing a strong Kozak translation initiation signal and / or a 3' UTR containing an oligo(dT) sequence for templated addition of a polyA tail. The 5' UTR comprises a first polynucleotide fragment and a second polynucleotide fragment derived from the same and / or different UTRs (see, e.g., US2010 / 0293625, which is incorporated herein by reference in its entirety).

[0351] It is also within the scope of the present invention to have patterned UTR. As used herein, "patterned UTR" includes repeating or alternating patterns, such as ABABAB or AABBABBAABB or ABCABCABC or variations thereof, that repeat once, twice, or three or more times. In these patterns, each letter A, B, or C represents a different UTR nucleic acid sequence.

[0352] Other non-UTR sequences can be used as regions or subregions within the polynucleotides of the invention. For example, introns or portions of intron sequences can be incorporated into the polynucleotides of the invention. Incorporation of intron sequences can increase protein production and polynucleotide expression levels. In some embodiments, the polynucleotides of the invention contain an internal ribosome entry site (IRES) instead of or in addition to a UTR (see, e.g., Yakubov et al., Biochem. Biophys. Res. Commun. 2010 394(1):189-193, the contents of which are incorporated herein by reference in their entirety). In some embodiments, polynucleotides of the invention comprise 5' and / or 3' sequences associated with the 5' and / or 3' ends, respectively, of rubella virus (RV) genomic RNA, including the 5' open reading frame of RV RNA encoding nonstructural proteins, or deletion derivatives thereof (see, e.g., Pogue et al., J. Virol. 67(12):7106-7117, the contents of which are incorporated herein by reference in their entirety). Viral capsid sequences, e.g., the 5' portions of capsid sequences, can also be used as translation enhancers (e.g., the Semliki Forest virus and Sindbis virus capsid RNAs described in Sjoberg et al., Biotechnology (NY) 1994 12(11):1127-1131 and Frolov and Schlesinger J. Virol. 1996 70(2):1182-1190, the contents of each of which are incorporated herein by reference in their entirety). In some embodiments, the polynucleotide comprises an IRES in place of the 5'UTR sequence. In some embodiments, the polynucleotide comprises an ORF and a viral capsid sequence. In some embodiments, the polynucleotide comprises a synthetic 5'UTR in combination with a non-synthetic 3'UTR.

[0353] In some embodiments, a UTR may also include at least one translation enhancer polynucleotide, translation enhancer element, or translation enhancer element (a nucleic acid sequence that increases the amount of polypeptide or protein produced from a polynucleotide, collectively referred to as a "TEE"). Non-limiting examples of TEEs include those described in US2009 / 0226470, which is incorporated herein by reference in its entirety, and others known in the art. ...

[0354] In one embodiment, a TEE is a conserved element in a UTR that can promote translational activity of a nucleic acid, including, but not limited to, cap-dependent or cap-independent translation. Conservation of these sequences has been found in 14 species, including humans. See, e.g., Panek et al., "An evolutionary conserved pattern of 18S rRNA sequence complementarity to mRNA 5'UTRs and its implications for eukaryotic gene translation regulation," Nucleic Acids Research 2013, doi:10.1093 / nar / gkt548, which is incorporated herein by reference in its entirety.

[0355] In one non-limiting example, the TEE comprises a TEE sequence in the 5'-leader of a Gtx homeodomain protein. See Chappell et al., PNAS 2004 101:9590-9594, which is incorporated herein by reference in its entirety.

[0356] In another non-limiting example, the TEE includes a TEE having one or more of the sequences of SEQ ID NOs: 1-35 of US2009 / 0226470, US2013 / 0177581, and WO2009 / 075886; SEQ ID NOs: 1-5 and 7-645 of WO2012 / 009644; and SEQ ID NO: 1 of WO1999 / 024595, US6310197, and US6849405, the contents of each of which are incorporated herein by reference in their entirety.

[0357] In some embodiments, the TEE is an internal ribosome entry site (IRES), HCV-IRES, or IRES element, such as, but not limited to, those described in US7468275, US2007 / 0048776, US2011 / 0124100, WO2007 / 025008, and WO2001 / 055369, the contents of each of which are incorporated herein by reference in their entirety. IRES elements may include, but are not limited to, Gtx sequences (e.g., Gtx9-nt, Gtx8-nt, Gtx7-nt) described in Chappell et al., PNAS 2004 101:9590-9594, Zhou et al., PNAS 2005 102:6273-6278, US2007 / 0048776, US2011 / 0124100, and WO2007 / 025008, the contents of each of which are incorporated herein by reference in their entirety.

[0358] "Translational enhancer polynucleotide" or "translational enhancer polynucleotide sequence" refers to a TEE provided herein and / or a TEE known in the art (e.g., US6310197, US6849405, US7456273, US7183395, US2009 / 0226470, US2007 / 0048776, US2011 / 0124100, US2009 / 0093049, US2013 / 01775 81, WO2009 / 075886, WO2007 / 025008, WO2012 / 009644, WO2001 / 055371, WO1999 / 024595, EP2610341A1, and EP2610340A1; the contents of each of these documents are incorporated herein by reference in their entirety. In some embodiments, the polynucleotides of the invention comprise one or more copies of a TEE. The TEEs in the translational enhancer polynucleotide can be organized into one or more sequence segments. A sequence segment can possess one or more of the TEEs provided herein, with each TEE being present in one or more copies. When multiple sequence segments are present in the translational enhancer polynucleotide, the sequence segments can be homogeneous or heterogeneous. Thus, multiple sequence segments in a translational enhancer polynucleotide can possess the same or different types of TEEs provided herein, the same or different numbers of respective copies of TEEs, and / or the same or different arrangements of TEEs within each sequence segment. In one embodiment, a polynucleotide of the invention comprises a translational enhancer polynucleotide sequence.

[0359] In some embodiments, the 5'UTR and / or 3'UTR of the polynucleotide of the invention may be any of the following: WO1999 / 024595, WO2012 / 009644, WO2009 / 075886, WO2007 / 025008, WO1999 / 024595, WO2001 / 055371, EP2610341A1, EP2610340A1, US6310197, US68494 05, US7456273, US7183395, US2009 / 0226470, US2011 / 0124100, US2007 / 0048776, US2009 / 0093049, or US2013 / 0177581, the contents of each of which are incorporated herein by reference in their entirety.

[0360] In some embodiments, the 5'UTR and / or 3'UTR of the polynucleotides of the invention are selected from the group consisting of US2009 / 0226470, US2007 / 0048776, US2013 / 0177581, US2011 / 0124100, WO1...

Claims

1. 1. An mRNA cancer vaccine, comprising: (a) mRNA having an open reading frame encoding 10 to 40 peptide epitopes that are personalized cancer antigens lipid nanoparticles comprising at least three of the peptide epitopes are complex variants resulting from intron retention, complex splicing events, or insertion / deletion mutations, and at least two of the peptide epitopes are point mutations; the mRNA comprises at least one chemical modification; The lipid nanoparticles comprise 20-60% ionizable amino lipid, 5-25% neutral lipid, 25-55% sterol, and 0.5-15% PEG-modified lipid.

2. The mRNA cancer vaccine of claim 1 , wherein the peptide epitopes include at least one MHC class I epitope and at least one MHC class II epitope.

3. The mRNA cancer vaccine of claim 1 or 2, wherein at least 50% of the peptide epitopes are MHC class I epitopes.

4. The mRNA cancer vaccine of claim 1 or 2, wherein at least 70% of the peptide epitopes are MHC class I epitopes.

5. The mRNA cancer vaccine of claim 1 or 2, wherein at least 80% of the peptide epitopes are MHC class I epitopes.

6. The mRNA cancer vaccine of claim 1 or 2, wherein at least 90% of the peptide epitopes are MHC class I epitopes.

7. The mRNA cancer vaccine of any one of claims 1 to 6, further comprising an mRNA encoding at least one activating oncogene mutant peptide.

8. an mRNA cancer vaccine, further comprising an mRNA having an open reading frame encoding a polypeptide that enhances an immune response to the personalized cancer antigen, Optionally, the mRNA having an open reading frame encoding 10 to 40 peptide epitopes that are personalized cancer antigens and the mRNA having an open reading frame encoding a polypeptide that enhances an immune response to said personalized cancer antigens are present in a mass ratio of approximately 5:1; 8. The mRNA cancer vaccine of claim 1, wherein optionally at least one of the peptide epitopes is an activating oncogene mutant peptide or a conventional cancer antigen.

9. (a) the mRNA comprises a 5'UTR comprising the nucleotide sequence set forth in SEQ ID NO: 176; (b) the mRNA comprises a polyA tail, optionally wherein the polyA tail comprises about 100 nucleotides; (c) the mRNA comprises a 5' Cap 1 structure, and / or (d) the at least one chemical modification is selected from the group consisting of pseudouridine, N1-methylpseudouridine, 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-methyluridine, 5-methoxyuridine, and 2'-O-methyluridine, and further optionally, the one or more mRNAs are fully modified with N1-methylpseudouridine; The mRNA cancer vaccine according to any one of claims 1 to 8.

10. (a) the mRNA encodes 15 or more personalized cancer antigens; and / or (b) the peptide epitope is in the form of a concatemeric cancer antigen; Optionally, the concatemeric cancer antigen comprises one or more of the following characteristics (i)-(vii): (i) the peptide epitopes are interspersed with cleavage-sensitive sites; (ii) the mRNAs encoding each peptide epitope are directly linked to each other without a linker; (iii) the mRNAs encoding each peptide epitope are linked to each other by a nucleotide linker encoding 1 to 30 amino acids; (iv) each peptide epitope comprises a centrally located SNP mutation with 6 to 15 adjacent amino acids on either side of the SNP mutation; (v) the mRNA encodes 20 to 40 cancer antigens; (vi) the mRNAs encoding the peptide epitopes are arranged such that the order of the peptide epitopes is in an order that minimizes spurious epitopes, and / or (vii) each peptide epitope comprises a centrally located SNP mutation, with 12 flanking amino acids on either side of the SNP mutation; The mRNA cancer vaccine according to any one of claims 1 to 9.

11. (a) the immune response is (i) stimulating type I interferon pathway signaling; (ii) stimulating NFkB pathway signaling; (iii) stimulating an inflammatory response; (iv) stimulating cytokine production; (v) stimulating the development, activity or recruitment of dendritic cells; or (vi) Any combination of (i) to (vi) or comprising a cellular or humoral immune response characterized by (b) the mRNA vaccine comprises a single mRNA construct encoding both the peptide epitope and the polypeptide that enhances an immune response to the personalized cancer antigen; The mRNA cancer vaccine of claim 8.

12. (i) the mRNA encoding a polypeptide that enhances an immune response to the personalized cancer antigen is formulated in a lipid nanoparticle that is different from the mRNA encoding the personalized cancer antigen; or (ii) mRNA encoding a polypeptide that enhances an immune response to a personalized cancer antigen is formulated in the same lipid nanoparticle as the mRNA encoding the personalized cancer antigen; The mRNA cancer vaccine according to any one of claims 8 to 11.

13. (a) the peptide epitope is a T cell epitope and / or a B cell epitope; (b) the peptide epitope comprises a combination of a T cell epitope and a B cell epitope; (c) at least one of the peptide epitopes is a T-cell epitope; (d) at least one of the peptide epitopes is a B-cell epitope; (e) the peptide epitope is optimized for binding strength to the subject's MHC; or (f) the mRNA vaccine further comprises an mRNA having an open reading frame encoding one or more conventional cancer antigens; The mRNA cancer vaccine according to any one of claims 1 to 12.

14. One or more of the following conditions: (a) at least one of the peptide epitopes is a conventional cancer antigen; (b) at least one of said peptide epitopes is a repeat polymorphism, and optionally said repeat polymorphism comprises a recurrent somatic cancer mutation in p53; (c) at least one of said peptide epitopes is an activating oncogene mutation peptide, said at least one activating oncogene mutation being a KRAS mutation, and optionally (i) the KRAS mutation is a G12 mutation, and optionally the G12 KRAS mutation is selected from a G12D, G12V, G12S, G12C, G12A, and G12R KRAS mutation; (ii) the KRAS mutation is a G13 mutation, and optionally the G13 KRAS mutation is a G13D KRAS mutation; and / or (iii) the activating oncogene mutation is an H-RAS or N-RAS mutation, and / or (d) the mRNA cancer vaccine does not contain a stabilizer. The mRNA cancer vaccine according to any one of claims 1 to 13, which satisfies the above.

15. (a) the lipid nanoparticles comprise a molar ratio of about 20-60% Compound 25:5-25% DSPC:25-55% cholesterol; 0.5-15% PEG-DMG; and optionally (i) the lipid nanoparticles comprise a molar ratio of about 50% Compound 25: about 10% DSPC: about 38.5% cholesterol; about 1.5% PEG-DMG; or (ii) the ionizable amino lipid is, for example, 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); (b) the lipid nanoparticles have a polydispersity value of less than 0.4; and / or (c) the lipid nanoparticles have a net neutral charge at a neutral pH value; The mRNA cancer vaccine according to any one of claims 1 to 14.

16. The lipid nanoparticles comprise a compound of formula (I), The compound of formula (I) 【Chemical 1】 The mRNA cancer vaccine according to any one of claims 1 to 15,

17. (a) the mRNA further comprises an open reading frame encoding an immune checkpoint regulator, and optionally (i) the immune checkpoint modulator is an inhibitory checkpoint polypeptide, or (ii) the immune checkpoint modulator is an inhibitory checkpoint polypeptide, and the inhibitory checkpoint polypeptide inhibits PD1, PD-L1, CTLA4, TIM-3, VISTA, A2AR, B7-H3, B7-H4, BTLA, IDO, KIR, LAG3, or a combination thereof; or (b) the mRNA cancer vaccine further comprises an additional cancer therapeutic agent, optionally, the additional cancer therapeutic agent is an immune checkpoint modulator, and further optionally, (i) the immune checkpoint modulator is an inhibitory checkpoint polypeptide, or (ii) the immune checkpoint modulator is an inhibitory checkpoint polypeptide, and the inhibitory checkpoint polypeptide inhibits PD1, PD-L1, CTLA4, TIM-3, VISTA, A2AR, B7-H3, B7-H4, BTLA, IDO, KIR, LAG3, or a combination thereof; The mRNA cancer vaccine according to any one of claims 1 to 16.

18. the inhibitory checkpoint polypeptide is an antibody, optionally the inhibitory checkpoint polypeptide is an antibody selected from an anti-CTLA4 antibody or antigen-binding fragment thereof that specifically binds to CTLA4, an anti-PD1 antibody or antigen-binding fragment thereof that specifically binds to PD1, an anti-PD-L1 antibody or antigen-binding fragment thereof that specifically binds to PD-L1, and combinations thereof, and further optionally (i) the checkpoint inhibitor polypeptide is an anti-PD-L1 antibody selected from atezolizumab, avelumab, or durvalumab; or (ii) the checkpoint inhibitor polypeptide is an anti-CTLA-4 antibody selected from tremelimumab or ipilimumab; The mRNA cancer vaccine of claim 17.

19. The mRNA cancer vaccine of claim 18, wherein the checkpoint inhibitor polypeptide is an anti-PD1 antibody selected from nivolumab or pembrolizumab.

20. The mRNA cancer vaccine of any one of claims 1 to 19, wherein at least two of the peptide epitopes encoded by the mRNA are directly linked to each other without a linker.

21. The mRNA cancer vaccine of any one of claims 1 to 20, wherein at least two of the peptide epitopes encoded by the mRNA are separated from each other by one glycine.

22. 22. The mRNA cancer vaccine of any one of claims 1 to 21, wherein the at least one chemical modification is selected from the group consisting of pseudouridine, N1-methylpseudouridine, 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-methyluridine, 5-methoxyuridine, and 2'-O-methyluridine.

23. The mRNA cancer vaccine of any one of claims 1 to 22, wherein the at least one chemical modification is N1-methylpseudouridine.

24. 24. An mRNA cancer vaccine for use in a method of vaccinating a subject having cancer, comprising administering to the subject the mRNA cancer vaccine of any one of claims 1 to 23, optionally wherein the mRNA vaccine is administered to the subject at a dose level sufficient to deliver 10 μg to 400 μg of the mRNA vaccine, and further optionally wherein the mRNA vaccine is administered to the subject at a dose level sufficient to deliver 0.033 mg, 0.1 mg, 0.2 mg, or 0.4 mg; and / or (i) the mRNA vaccine is administered to the subject two, three, four or more times; or (ii) An mRNA cancer vaccine, wherein the mRNA vaccine is administered once daily every three weeks.

25. The mRNA vaccine is administered by intradermal, intramuscular, and / or subcutaneous administration, optionally, the mRNA vaccine is administered by intramuscular administration. The mRNA cancer vaccine of claim 24.

26. The mRNA cancer vaccine of claim 24 or 25, wherein the method further comprises administering an additional cancer therapeutic agent, optionally wherein the additional cancer therapeutic agent is an immune checkpoint modulator.

27. The cancer is (i) the group consisting of non-small cell lung cancer (NSCLC), small cell lung cancer, melanoma, bladder urothelial carcinoma, HPV-negative head and neck squamous cell carcinoma (HNSCC), and solid malignancies that are microsatellite high (MSI H) / mismatch repair (MMR) deficient, optionally wherein the NSCLC lacks an EGFR-sensitive mutation and / or an ALK translocation, and / or the solid malignancy that is microsatellite high (MSI H) / mismatch repair (MMR) deficient is selected from the group consisting of colorectal cancer, gastric adenocarcinoma, esophageal adenocarcinoma, and endometrial carcinoma; and / or (ii) cancers of the pancreas, peritoneum, large intestine, small intestine, biliary tract, lung, endometrium, ovary, reproductive tract, gastrointestinal tract, cervix, stomach, urinary tract, colon, rectum, and hematopoietic and lymphoid tissues; The mRNA cancer vaccine of any one of claims 24 to 26, selected from the group consisting of:

28. The immune checkpoint modulator is an inhibitory checkpoint polypeptide, and optionally: (i) the inhibitory checkpoint polypeptide inhibits PD1, PD-L1, CTLA4, TIM-3, VISTA, A2AR, B7-H3, B7-H4, BTLA, IDO, KIR, LAG3, or a combination thereof; (ii) the inhibitory checkpoint polypeptide is an antibody, and further optionally, the inhibitory checkpoint polypeptide is an antibody selected from an anti-CTLA4 antibody or antigen-binding fragment thereof that specifically binds to CTLA4, an anti-PD1 antibody or antigen-binding fragment thereof that specifically binds to PD1, an anti-PD-L1 antibody or antigen-binding fragment thereof that specifically binds to PD-L1, and combinations thereof; (iii) the inhibitory checkpoint polypeptide is an anti-PD-L1 antibody selected from atezolizumab, avelumab, or durvalumab; or (iv) the inhibitory checkpoint polypeptide is an anti-CTLA-4 antibody selected from tremelimumab or ipilimumab; The mRNA cancer vaccine of claim 26.

29. The mRNA cancer vaccine of claim 26, wherein the immune checkpoint modulator is an inhibitory checkpoint polypeptide, and the inhibitory checkpoint polypeptide is an anti-PD1 antibody selected from nivolumab or pembrolizumab.

30. The mRNA cancer vaccine of any one of claims 24 to 29, wherein the immune checkpoint modulator is administered to the subject at a dose level sufficient to deliver 100-300 mg, and optionally, the immune checkpoint modulator is administered to the subject at a dose level sufficient to deliver 200 mg.

31. (a) the immune checkpoint modulator is administered by intravenous infusion. (b) the immune checkpoint modulator is administered to the subject two, three, four or more times; and / or (c) the immune checkpoint modulator is administered to the subject on the same day as administration of the mRNA vaccine.

32. The mRNA cancer vaccine of any one of claims 24 to 31, wherein the mRNA vaccine is administered by intramuscular administration.

33. 1. A method for producing mRNA encoding a concatemeric cancer antigen comprising 1000 to 3000 nucleotides, comprising: (a) binding a first polynucleotide comprising an open reading frame encoding a cancer antigen according to any one of claims 1 to 23 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, wherein 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, wherein the suitable conditions include an RNA ligase, thereby generating a second ligation product; (d) releasing the second ligation product from the solid support; and thereby producing mRNA encoding a concatemeric cancer antigen comprising said 1000 to 3000 nucleotides.

34. 1. An mRNA cancer vaccine comprising: lipid nanoparticles comprising 20-60% ionizable amino lipid, 5-25% neutral lipid, 25-55% sterol, and 0.5-15% PEG-modified lipid; and mRNA; (a) the mRNA has one or more open reading frames encoding 15 to 40 peptide epitopes that are personalized cancer antigens; (b) the open reading frame encodes one or more activating oncogene mutant peptides; (c) placing the peptide epitopes in an order that minimizes spurious epitopes; (d) at least 50% of the peptide epitopes encoded by the mRNA are MHC class I peptides; and (e) the mRNA comprises at least one chemical modification; mRNA cancer vaccines.

35. the activating oncogene mutant peptide comprises a KRAS mutation, an H-RAS mutation, an N-RAS mutation, or a combination thereof, and optionally (a) the KRAS mutation is a G12 and / or G13 mutation; (b) the KRAS mutation is a G12 mutation, and the G12 KRAS mutation is selected from G12D, G12V, G12S, G12C, G12A, and G12R KRAS mutations; and / or (c) the KRAS mutation comprises a G13D KRAS mutation; The mRNA cancer vaccine of claim 34.

36. 36. The mRNA cancer vaccine of claim 34 or 35, wherein the at least one chemical modification is selected from the group consisting of pseudouridine, N1-methylpseudouridine, 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-methyluridine, 5-methoxyuridine, and 2'-O-methyluridine.

37. The mRNA cancer vaccine of any one of claims 34 to 36, wherein the at least one chemical modification is N1-methylpseudouridine.

38. The mRNA cancer vaccine of any one of claims 34 to 36, wherein at least 70% of the peptide epitopes are MHC class I epitopes.

39. The mRNA cancer vaccine of any one of claims 34 to 36, wherein at least 80% of the peptide epitopes are MHC class I epitopes.

40. The mRNA cancer vaccine of any one of claims 34 to 36, wherein at least 90% of the peptide epitopes are MHC class I epitopes.

Images