RNA cancer vaccines

Abstract

To provide cancer ribonucleic acid (RNA) vaccines, and methods of using the vaccines and compositions comprising the vaccines, in particular, concatemeric mRNA cancer vaccines encoding several cancer epitopes on a single mRNA construct, i.e. poly-epitope mRNA constructs or poly-neo-epitope constructs.SOLUTION: The disclosure further relates to p53 and KRAS mutations, as well as incorporation of immune enhancers such as STING, e.g. mRNA constructs further encoding an immune stimulator or adjuvant. The disclosure further relates to inclusion of universal T cell epitopes, such as tetanus or diphtheria toxins to elicit an enhanced immune response.SELECTED DRAWING: None

Description

Technical field

[0001] Cross-reference of related applications This application is filed under 35 U.S.C. 119(e) under U.S. Provisional Application No. 62 / 453,444, entitled "RNA CANCER VACCINES," filed February 1, 2017; Provisional Application No. 62 / 453,465 entitled ``IMMUNOMODULATORY THERAPEUTIC MRNA COMPOSITIONS ENCODING ACTIVATING ONCOGENE MUTATION PEPTIDES'' and Provisional Application No. 62 / 558,238 entitled ``CONCATAMERIC RNA CANCER VACCINES'' filed September 13, 2017. The contents of each of these applications are incorporated herein by reference in their entirety. [Background technology]

[0002] Recent theories in 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, there is no single mechanism that predominates for the majority of clinical cases. However, the common mechanism suggests that cancer vaccines may provide a universal solution for cancer treatment.

[0003] Cancer vaccines include preventive or prophylactic vaccines (intended to prevent the development of cancer in healthy individuals), and therapeutic vaccines (intended to treat existing cancers by strengthening the body's natural defenses against cancer). ) is included. Cancer prevention vaccines can target infectious agents that induce or contribute to the development of cancer, for example, to prevent the induction of cancer by infectious diseases. Gardasil® and Cervarix® are two examples of commercially available prophylactic vaccines. Each vaccine protects against infection with HPV. Other preventive cancer vaccines may target host proteins or fragments that are predicted to increase the likelihood that an individual will develop 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 vaccines on the market or in development (eg, cancer vaccines) are based on whole microorganisms, protein antigens, peptides, polysaccharides, or deoxyribonucleic acid (DNA) vaccines, and combinations thereof. DNA vaccination is one technique used to stimulate humoral and cellular immune responses to antigens. Direct injection of genetically engineered DNA (eg, naked plasmid DNA) into a living host produces antigen directly from a small number of host cells and generates a protective immune response. However, this technology is beset by potential problems of DNA integration into the genome of the vaccine, including the possibility of insertional mutagenesis, which can result in activation of oncogenes or inhibition of tumor suppressor genes. [Means to solve the problem]

[0006] Described herein is an RNA (e.g., messenger RNA (mRNA)) ribonucleic acid (RNA) cancer vaccine that can safely induce the body's cellular machinery to produce nearly any cancer protein or fragment thereof of interest. provided by. In some embodiments, the RNA is modified RNA. Using the RNA vaccines of the present disclosure, for example, it is possible to induce a balanced immune response against cancer, including both cell-mediated and humoral immunity, without the risk that insertional mutagenesis may occur. can.

[0007] RNA vaccines may be utilized in a variety of settings depending on the prevalence of cancer or the extent or level of unmet medical need. RNA vaccines may be utilized to treat and / or prevent cancer at various stages or degrees of metastasis. Compared to alternative anti-cancer treatments, including cancer vaccines, RNA vaccines have superior properties in generating much higher antibody titers and generating responses faster. Without wishing to be bound by theory, it is believed that because RNA vaccines incorporate natural cellular machinery, RNA vaccines that are mRNA polynucleotides are better designed to generate the appropriate protein conformation upon translation. it is conceivable that. Unlike traditional treatments and vaccines that are manufactured ex vivo and can induce undesirable cellular responses, RNA vaccines are presented in cell lines in a more natural manner.

[0008] RNA vaccines are ribonucleic acid (RNA) polynucleotides 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). may include. Other embodiments include 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] The present disclosure provides, in some embodiments, an mRNA cancer vaccine of one or more mRNAs, each having an open reading frame encoding a cancer antigen peptide epitope, formulated into lipid nanoparticles and a pharmaceutically acceptable carrier. or an excipient, where the mRNA vaccine encodes between 5 and 100 peptide epitopes, and at least two of the peptide epitopes are personalized cancer antigens.

[0010] The present disclosure provides, in some embodiments, one or more peptide epitopes each having one or more open reading frames encoding 1-500 peptide epitopes that are personalized cancer antigens and a universal type II T cell epitope. An mRNA cancer vaccine is provided that includes lipid nanoparticles containing mRNA.

[0011] The present disclosure provides, in some embodiments, mRNA cancer vaccines comprising lipid nanoparticles comprising one or more of the following: (a) 1-500 peptide epitopes that are personalized cancer antigens; (b) one or more mRNAs each having one or more open reading frames encoding a universal type II T cell epitope; (b) one or more mRNAs each having one or more open reading frames encoding an activating oncogene mutant peptide; (c) one or more mRNAs each having an open reading frame encoding a cancer antigen peptide epitope; The mRNA vaccine encodes between 5 and 100 peptide epitopes, at least two of the peptide epitopes are personalized cancer antigens, and optionally the mRNA further encodes universal type II T cell epitopes. (d) one or more mRNAs each having an open reading frame encoding a cancer antigen peptide epitope, wherein the mRNA vaccine encodes between 5 and 100 peptide epitopes; at least three of the peptide epitopes are compound variants, at least two of the peptide epitopes 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 epitopes are ILMQYIKANSKFIGI (tetanus toxin; SEQ ID NO: 226), FNNFTVSFWLRVPKVSASHLE (tetanus toxin; SEQ ID NO: 227), QYIKANSKFIGITE (tetanus toxin; SEQ ID NO: 228), QSIALSSLMVAQAIP (diphtheria toxin; 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 universal type II T cell epitope throughout the mRNA. In other embodiments, the universal type II T cell epitope is repeated 1-20 times in the mRNA. In one embodiment, the universal type II T cell epitopes differ from each other throughout the mRNA. In some embodiments, the universal type II T cell epitope is located between all cancer antigen peptide epitopes. In another embodiment, the universal type II T cell epitopes are located between every other cancer antigen peptide epitope. In one embodiment, the universal type II T cell epitopes are located between every second 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, and optionally (iii) the KRAS mutation is a G13 mutation; and optionally, 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 variant peptides; ) at least two of the peptide epitopes are separated from each other by one glycine, and optionally all of the peptide epitopes are separated from each other by one glycine; (C) the concatemers are separated from each other by one glycine; (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 conventional cancer antigen; (iii) repeat polymorphisms include recurrent somatic cancer mutations in p53; (iv) recurrent somatic cancer mutations in p53 are (A) adjacent to codon position T125; Mutations in the canonical 5' splice site that cause 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 that induces a retained intron with the peptide sequence TAKSVTCTVSCPEGLASMRLQCLAVSPCISFVWNFGIPLHPLASCQCFFIVYPLNV (SEQ ID NO: 232) containing 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 induces a retained intron with the peptide sequence EYFTLQVLSLGTSYQVESFQSNTQNAVFFLTVLPAIGAFAAIRGQ (SEQ ID NO: 236) containing the epitope CTMFCQLAK (SEQ ID NO: 240); )(HLA-A * 11:01), epitope KSVTCTMF (SEQ ID NO: 241) (HLA-B * 58:01); and / or (D) a canonical 5' splice adjacent to codon position 224. Mutation in the epitope VPYEPPEVW (SEQ ID NO: 243) (HLA-B * 53:01, HLA-B * 51:01), epitope LTVPSTAW (SEQ ID NO: 244) (HLA-B * 58:01, HLA-B * A mutation inducing a potential alternative intronic 5' splice site generating a novel spanning peptide sequence VPYEPPEVWLALTVPPSTAWAA (SEQ ID NO: 242) containing (transcribed codon positions are standard (with reference to ENST00000269305 (SEQ ID NO: 245), which is a full-length p53 transcript); and / or (v) the mRNA cancer vaccine is free of stabilizers.

[0016] In some embodiments, the one or more mRNAs further include an open reading frame encoding an immunopotentiator. In other embodiments, the immunopotentiator is formulated into lipid nanoparticles. In one embodiment, the immunopotentiating agent is formulated into separate lipid nanoparticles. In some embodiments, the immunopotentiator 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 a constitutively active human STING polypeptide comprises the nucleotide sequence set forth in SEQ ID NO: 170. In some embodiments, the mRNA encoding a constitutively active human STING polypeptide comprises a 3'UTR with an 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 include a 5'UTR comprising the nucleotide sequence set forth in SEQ ID NO: 176. In one embodiment, each of the one or more mRNAs includes a polyA tail. In one embodiment, the polyA tail comprises about 100 nucleotides. In some embodiments, each of the one or more mRNAs includes a 5' cap 1 structure.

[0018] In some embodiments, one or more mRNAs include 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 45-55 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 epitope is in the form of a concatemeric cancer antigen composed of 2-100 peptide epitopes; optionally, the concatemeric cancer antigen is composed of 5-100 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) each peptide epitope c) the mRNAs encoding each peptide epitope are linked to each other with a single nucleotide linker; d) each peptide epitope is contain 25-35 amino acids and contain centrally located SNP variations; e) at least 30% of the peptide epitope has 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 have the highest affinity for class II MHC molecules derived from the subject; h) the mRNA encodes 45-55 peptide epitopes; i) the mRNA encodes 52 peptide epitopes; j) the peptide epitope 50% of the peptide epitope has binding affinity for class I MHC and 50% of the peptide epitope has binding affinity for class II MHC; k) the mRNA encoding the peptide epitope is the peptide epitopes are arranged in an order that minimizes pseudo-epitopes; l) at least 30% of the peptide epitopes are MHC class I binding peptides that are 15 amino acids long; and / or m) at least 30% of the peptide epitope is a 21 amino acid long MHC class II binding peptide.

[0021] In some embodiments, the present disclosure provides one or more lipid nanoparticles each having one or more open reading frames encoding 45-55 peptide epitopes that are personalized cancer antigens, formulated into lipid nanoparticles. The present invention provides an mRNA cancer vaccine containing mRNA.

[0022] In some embodiments, the present disclosure provides one or more lipid nanoparticles each having one or more open reading frames encoding 45-55 peptide epitopes that are personalized cancer antigens, formulated into lipid nanoparticles. optionally at least one of the peptide epitopes is an activated oncogene variant peptide or a conventional cancer antigen, and optionally at least three of the peptide epitopes are complex variants. , at least two of the peptide epitopes are point mutations, providing an mRNA cancer vaccine.

[0023] In some embodiments, the one or more mRNAs encode 48-54 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.

[0024] In another embodiment, the peptide epitope is in the form of a concatemeric cancer antigen composed of 2-100 peptide epitopes; optionally, the concatemeric cancer antigen is composed of 5-100 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) each peptide epitope c) the mRNAs encoding each peptide epitope are linked to each other with a single nucleotide linker; d) each peptide epitope is contain 25-35 amino acids and contain centrally located SNP variations; e) at least 30% of the peptide epitope has 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 have the highest affinity for class II MHC molecules derived from the subject; h) the mRNA encodes 45-55 peptide epitopes; i) the mRNA encodes 52 peptide epitopes; j) the peptide epitope 50% of the peptide epitope has binding affinity for class I MHC and 50% of the peptide epitope has binding affinity for class II MHC; k) the mRNA encoding the peptide epitope is the peptide epitopes are arranged in an order that minimizes pseudo-epitopes; l) at least 30% of the peptide epitopes are MHC class I binding peptides that are 15 amino acids long; and / or m) at least 30% of the peptide epitope is a 21 amino acid long MHC class II binding peptide.

[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 universal type II T cell epitopes. In another embodiment, the mRNA cancer vaccine encodes 1-20 universal type II T cell epitopes.

[0026] In some embodiments, the universal type II T cell epitopes are 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 universal type II T cell epitope throughout the mRNA. In some embodiments, the universal type II T cell epitope is repeated 1-20 times in the mRNA. In another embodiment, the universal type II T cell epitopes differ from each other across the entire mRNA. In one embodiment, the universal type II T cell epitope is located between all peptide epitopes. In some embodiments, the universal type II T cell epitopes are located between every other cancer antigen peptide epitope. In one embodiment, the universal type II T cell epitopes are located between every second peptide epitope.

[0028] In some embodiments, the one or more mRNAs further include an open reading frame encoding an immunopotentiator. In one embodiment, the immunopotentiating agent is formulated into lipid nanoparticles. In another embodiment, the immunopotentiator is formulated into separate lipid nanoparticles. In some embodiments, the immunopotentiator 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 a 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, and optionally (iii) the KRAS mutation is a G13 mutation; and optionally, 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 variant peptides; ) at least two of the peptide epitopes are separated from each other by one glycine, and optionally all of the peptide epitopes are separated from each other by one glycine; (C) the concatemers are separated from each other by one glycine; (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 conventional cancer antigen; (iii) repeat polymorphisms include recurrent somatic cancer mutations in p53; (iv) recurrent somatic cancer mutations in p53 are (A) adjacent to codon position T125; Mutations in the canonical 5' splice site that cause 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 that induces a retained intron with the peptide sequence TAKSVTCTVSCPEGLASMRLQCLAVSPCISFVWNFGIPLHPLASCQCFFIVYPLNV (SEQ ID NO: 232) containing 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 induces a retained intron with the peptide sequence EYFTLQVLSLGTSYQVESFQSNTQNAVFFLTVLPAIGAFAAIRGQ (SEQ ID NO: 236) containing the epitope CTMFCQLAK (SEQ ID NO: 240); )(HLA-A * 11:01), epitope KSVTCTMF (SEQ ID NO: 241) (HLA-B * 58:01); and / or (D) a canonical 5' splice adjacent to codon position 224. Mutation in the epitope VPYEPPEVW (SEQ ID NO: 243) (HLA-B * 53:01, HLA-B * 51:01), epitope LTVPSTAW (SEQ ID NO: 244) (HLA-B * 58:01, HLA-B * A mutation inducing a potential alternative intronic 5' splice site generating a novel spanning peptide sequence VPYEPPEVWLALTVPPSTAWAA (SEQ ID NO: 242) containing (transcribed codon positions are standard (with reference to ENST00000269305 (SEQ ID NO: 245), which is a full-length p53 transcript); and / or (v) the mRNA cancer vaccine is free of stabilizers.

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

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

[0034] In some embodiments, the immune response includes (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) a cellular or Includes humoral immune responses.

[0035] In one embodiment, an mRNA cancer vaccine comprises a single mRNA construct that encodes both a peptide epitope and a polypeptide that enhances the immune response to a personalized cancer antigen. In another embodiment, the peptide epitope is in the form of a concatemeric cancer antigen composed of 2-100 peptide epitopes; optionally, the concatemeric cancer antigen is composed of 5-100 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) each peptide epitope c) the mRNAs encoding each peptide epitope are linked to each other with a single nucleotide linker; d) each peptide epitope is contain 25-35 amino acids and contain centrally located SNP variations; e) at least 30% of the peptide epitope has 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 have the highest affinity for class II MHC molecules derived from the subject; h) the mRNA encodes 45-55 peptide epitopes; i) the mRNA encodes 52 peptide epitopes; j) the peptide epitope 50% of the peptide epitope has binding affinity for class I MHC and 50% of the peptide epitope has binding affinity for class II MHC; k) the mRNA encoding the peptide epitope is the peptide epitopes are arranged in an order that minimizes pseudo-epitopes; l) at least 30% of the peptide epitopes are MHC class I binding peptides that are 15 amino acids long; and / or m) at least 30% of the peptide epitope is a 21 amino acid long MHC class II binding peptide.

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

[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 has one or more mutations selected from the group consisting of V147L, N154S, V155M, R284M, R284K, R284T, E315Q, R375A, and combinations thereof. including. In another embodiment, the constitutively active human STING polypeptide comprises the 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 into the same or different lipid nanoparticles. In another embodiment, each mRNA encoding a cancer personalized cancer antigen is formulated into the same or different lipid nanoparticles. In some embodiments, each mRNA encoding a polypeptide that enhances the immune response to a personalized cancer antigen is formulated into 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. Formulated into particles. 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 an immune response to the personalized cancer antigen is are formulated into the same lipid nanoparticles as the respective mRNAs encoding them. 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 a different lipid nanoparticle. The same lipid nanoparticles are formulated with the respective mRNA encoding cancer antigens.

[0041] In some embodiments, the peptide epitope is a T cell epitope and / or a B cell epitope. In other embodiments, the peptide epitope comprises 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 epitope is optimized for binding strength to the subject's MHC. In other embodiments, the TCR face of each epitope has less 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 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; , the G12 KRAS mutation is selected from G12D, G12V, G12S, G12C, G12A, and G12R KRAS mutations; (iii) the KRAS mutation is a G13 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 variant peptides; (B) at least two of the peptide epitopes are separated from each other by one glycine, and optionally all of the peptide epitopes are separated from each other by one glycine; (C) the concatemers are 3 to 10 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 (iii) the repeat polymorphism includes a recurrent somatic cancer mutation in p53; (iv) the recurrent somatic cancer mutation in p53 is (A) adjacent to codon position T125; Mutations in the canonical 5' splice site, resulting in 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) Mutation in the canonical 5' splice site adjacent to codon position 331 that induces a retained intron with the peptide sequence TAKSVTCTVSCPEGLASMRLQCLAVSPCISFVWNFGIPLHPLASCQCFFIVYPLNV (SEQ ID NO: 232) containing the epitope LQVLSLGTSY (SEQ ID NO: 237); )(HLA-B * 15:01), epitope FQSNTQNAVF (SEQ ID NO: 238) (HLA-B * (C) Mutation in the canonical 3' splice site adjacent to codon position 126 that induces a retained intron with the peptide sequence EYFTLQVLSLGTSYQVESFQSNTQNAVFFLTVLPAIGAFAIRGQ (SEQ ID NO: 236) containing the epitope CTMFCQLAK (SEQ ID NO: 240); )(HLA-A * 11:01), epitope KSVTCTMF (SEQ ID NO: 241) (HLA-B * 58:01); and / or (D) a canonical 5' splice adjacent to codon position 224. Mutation in the epitope VPYEPPEVW (SEQ ID NO: 243) (HLA-B * 53:01, HLA-B * 51:01), epitope LTVPSTAW (SEQ ID NO: 244) (HLA-B * 58:01, HLA-B * A mutation inducing a potential alternative intronic 5' splice site generating a novel spanning peptide sequence VPYEPPEVWLALTVPPSTAWAA (SEQ ID NO: 242) containing (transcribed codon positions are standard (with reference to ENST00000269305 (SEQ ID NO: 245), which is a full-length p53 transcript); and / or (v) the mRNA cancer vaccine is free of stabilizers.

[0048] In some embodiments, the lipid nanoparticles have a mole of about 20-60% ionizable amino lipids: 5-25% neutral lipids: 25-55% sterols; 0.5-15% PEG-modified lipids. Optionally, the ionizable amino lipid is a cationic lipid. In one embodiment, the lipid nanoparticles include a molar ratio of about 50% Compound 25: about 10% DSPC: about 38.5% cholesterol; about 1.5% PEG-DMG. In another embodiment, the ionizable amino lipid is, for example, 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylamino Consists of butyrate (DLin-MC3-DMA) and di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319) selected from the group. In some embodiments, the lipid nanoparticles include 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 endogenous proteins.

[0050] In another embodiment, the mRNA further comprises an open reading frame encoding an immune checkpoint modulator. 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 modulator. In another embodiment, the immune checkpoint modulator is an inhibitory checkpoint polypeptide. In some embodiments, the inhibitory checkpoint polypeptide is PD1, PD-L1, CTLA4, TIM-3, VISTA, A2AR, B7-H3, B7-H4, BTLA, IDO, KIR, LAG3, or Prevents combinations.

[0051] In some embodiments, the checkpoint inhibitor polypeptide is an antibody. In one embodiment, the inhibitory checkpoint polypeptide is 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; The antibody is selected from specifically binding anti-PD-L1 antibodies or antigen-binding fragments thereof, 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 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- selected from the group consisting of methoxyuridine, and 2'-O-methyluridine.

[0053] The present disclosure, in another aspect, provides a method of vaccinating a subject with cancer, comprising administering to the subject an mRNA cancer vaccine as described above.

[0054] In some embodiments, the mRNA vaccine is administered to the subject at a dose level sufficient to deliver between 10 μg and 400 μg of the mRNA vaccine. In one embodiment, 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. In another embodiment, the mRNA vaccine is administered to the 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 by intradermal, intramuscular, and / or subcutaneous administration. In another embodiment, the mRNA vaccine is administered by intramuscular administration.

[0055] In some embodiments, the method further comprises administering an additional cancer therapeutic agent, optionally the additional cancer therapeutic agent being 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 is PD1, PD-L1, CTLA4, TIM-3, VISTA, A2AR, B7-H3, B7-H4, BTLA, IDO, KIR, LAG3, or a combination thereof. inhibit. In some embodiments, the checkpoint inhibitor polypeptide is an antibody. In other embodiments, the inhibitory checkpoint polypeptide is 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-PD1 antibody or antigen-binding fragment thereof that specifically binds to PD1, The antibody is selected from specifically binding anti-PD-L1 antibodies or antigen-binding fragments thereof, 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 dosage level sufficient to deliver 100-300 mg. In some embodiments, the immune checkpoint modulator is administered to the subject at a dosage 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 the mRNA vaccine administration.

[0057] In some embodiments, the cancer is non-small cell lung cancer (NSCLC), small cell lung cancer, melanoma, bladder urothelial carcinoma, HPV-negative head and neck squamous cell carcinoma (HNSCC), and microsatellite high frequency (MSI H) / solid malignant tumors that are mismatch repair (MMR) deficient. In one embodiment, the NSCLC is free of EGFR sensitivity mutations and / or ALK translocations. In another embodiment, the solid malignancy that is microsatellite frequent (MSI H) / mismatch repair (MMR) deficient is selected from the group consisting of colorectal cancer, gastric adenocarcinoma, esophageal adenocarcinoma, and endometrial cancer. Ru. In some embodiments, the cancer includes the pancreas, peritoneum, large intestine, small intestine, biliary tract, lung, endometrium, ovary, reproductive tract, gastrointestinal tract, cervix, stomach, urinary tract, colon, rectum, and hematopoietic system. and lymphoid tissue cancer.

[0058] The present disclosure provides, in some embodiments, an mRNA cancer vaccine of one or more mRNAs, each having an open reading frame encoding a cancer antigen peptide epitope, formulated into lipid nanoparticles and a pharmaceutically acceptable carrier. or an excipient, where 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 invention provides an mRNA cancer vaccine having one or more mRNAs, each having an open reading frame encoding a cancer antigen peptide epitope, and a pharmaceutically acceptable carrier or excipient. In mRNA cancer vaccines, mRNA vaccines encode between 5 and 100 peptide epitopes, at least 3 of the peptide epitopes are compound variants, and at least 2 of the peptide epitopes are point mutations. be.

[0060] In some embodiments, the lipid nanoparticles have a molar ratio of 20-60% cationic lipids: 5-25% non-cationic lipids: 25-55% sterols; 0.5-15% PEG-modified lipids. include. In some embodiments, the cationic lipid is, for example, 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyl (DLin-MC3-DMA), and the group consisting of di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319) selected from. In other embodiments, the lipid nanoparticles include 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] The vaccine of some embodiments is an mRNA with an open reading frame encoding a concatemeric cancer antigen composed of 5-100 peptide epitopes. In yet other embodiments, at least two of the peptide epitopes are separated from each other by one glycine. In yet other embodiments, the concatemeric cancer antigen comprises 20-40 peptide epitopes. In some embodiments, all of the peptide epitopes are separated from each other by one 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 embodiments includes 25-35 amino acids and includes a centrally located SNP variation.

[0064] In some embodiments, at least 30% of the peptide epitopes have the highest affinity for class I MHC molecules from the subject. In yet other embodiments, at least 30% of the peptide epitopes have the highest affinity for class II MHC molecules from the subject. In yet other embodiments, at least 50% of the peptide epitopes have a 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 other embodiments, the mRNA encoding the peptide epitopes is 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 variation, with 15 contiguous amino acids on either side of the SNP variation.

[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. A recall antigen can 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 include a repeat somatic cancer mutation in p53. The one or more recurrent somatic cancer mutations in p53 are, in some embodiments, (A) mutations in the canonical 5' splice site adjacent to codon position T125, comprising 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) Mutation in the canonical 5' splice site adjacent to codon position 331 that induces a retained intron with the peptide sequence TAKSVTCTVSCPEGLASMRLQCLAVSPCISFVWNFGIPLHPLASCQCFFIVYPLNV (SEQ ID NO: 232) containing the epitope LQVLSLGTSY (SEQ ID NO: 237); )(HLA-B * 15:01), epitope FQSNTQNAVF (SEQ ID NO: 238) (HLA-B * (C) Mutation in the canonical 3' splice site adjacent to codon position 126 that induces a retained intron with the peptide sequence EYFTLQVLSLGTSYQVESFQSNTQNAVFFLTVLPAIGAFAIRGQ (SEQ ID NO: 236) containing the epitope CTMFCQLAK (SEQ ID NO: 240); )(HLA-A * 11:01), epitope KSVTCTMF (SEQ ID NO: 241) (HLA-B * 58:01); and / or (D) a canonical 5' splice adjacent to codon position 224. Mutation in the epitope VPYEPPEVW (SEQ ID NO: 243) (HLA-B * 53:01, HLA-B * 51:01), epitope LTVPSTAW (SEQ ID NO: 244) (HLA-B * 58:01, HLA-B * A mutation inducing a potential alternative intronic 5' splice site generating a novel spanning peptide sequence VPYEPPEVWLALTVPPSTAWAA (SEQ ID NO: 242) containing (transcribed codon positions are standard ENST00000269305 (SEQ ID NO: 245), which is a full-length p53 transcript.

[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 from the group consisting of PD-1, TIM-3, VISTA, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, and LAG3. An antibody or fragment thereof that specifically binds to a selected molecule. 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 include stabilizing agents.

[0073] In some embodiments, the mRNA includes at least one chemical modification. Chemical modifications include 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 may be selected from the group consisting of O-methyluridine.

[0074] In other aspects, methods of vaccinating a subject are provided. The method involves administering an mRNA vaccine disclosed herein to a subject having cancer.

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

[0076] In some embodiments, mRNA vaccines are administered by intradermal, intramuscular, and / or subcutaneous administration. In some embodiments, mRNA vaccines are administered by intramuscular administration.

[0077] In some embodiments, the method further comprises administering an additional cancer therapeutic agent, optionally the additional cancer therapeutic agent being 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 from the group consisting of PD-1, TIM-3, VISTA, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, and LAG3. An antibody or fragment thereof that specifically binds to a selected molecule. 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 dosage level sufficient to deliver 100-300 mg. In some embodiments, the immune checkpoint modulator is administered to the subject at a dosage 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 the mRNA vaccine administration.

[0081] In some embodiments, the cancer is non-small cell lung cancer (NSCLC), small cell lung cancer, melanoma, bladder urothelial carcinoma, HPV-negative head and neck squamous cell carcinoma (HNSCC), and microsatellite high frequency (MSI H) / solid malignant tumors that are mismatch repair (MMR) deficient. In some embodiments, the NSCLC is free of EGFR sensitivity mutations and / or ALK translocations. In some embodiments, the solid malignancy that is microsatellite frequent (MSI H) / mismatch repair (MMR) deficient is selected from the group consisting of colorectal cancer, gastric adenocarcinoma, esophageal adenocarcinoma, and endometrial cancer. be done. In some embodiments, the cancer includes the pancreas, peritoneum, large intestine, small intestine, biliary tract, lung, endometrium, ovary, reproductive tract, gastrointestinal tract, cervix, stomach, urinary tract, colon, rectum, and hematopoietic system. and lymphoid tissue cancer.

[0082] In other embodiments, methods of preparing mRNA cancer vaccines are provided. The method includes isolating a sample from a subject, identifying multiple cancer antigens in the sample, determining immunogenic epitopes from the multiple cancer antigens, and identifying open reading frames encoding the cancer antigens. and preparing an mRNA cancer vaccine having an mRNA cancer vaccine. In another aspect of the invention, a method for producing mRNA encoding a concatemeric cancer antigen comprising 1000-3000 nucleotides is provided. The method is (a) A polynucleotide comprising 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, conjugated to a solid carrier. binding to nucleotides, (b) ligation of the 3' end of the second polynucleotide to the 5' end of the first polynucleotide under suitable conditions, the suitable conditions comprising a DNA ligase, whereby the first a ligation product produced by (c) ligation of the 5' end of a third polynucleotide comprising a 3'-UTR to the 3' end of the first ligation product under appropriate conditions, wherein the appropriate conditions ligation, thereby producing a second ligation product; (d) releasing the second ligation product from the solid support; , thereby producing mRNA encoding a concatemeric cancer antigen containing 1000 to 3000 nucleotides.

[0083] In other aspects, the invention is an mRNA cancer vaccine comprising concatemeric cancer antigens that can be prepared according to the methods described herein.

[0084] According to other embodiments of the invention, methods of treating a subject with a personalized mRNA cancer vaccine are provided. The method involves the identification of a set of neoepitopes by analyzing the patient's transcriptome and / or the patient's exome derived from a sample, and the binding strength with MHC, to generate a patient-specific mutanome. Mutanomic selection of a set of neoepitopes for vaccines based on MHC binding diversity, predicted immunogenicity, low autoreactivity, presence of activating oncogene mutations and / or T cell reactivity and preparing an mRNA vaccine encoding the set of neoepitopes and administering the mRNA vaccine to the subject within two months of isolation of the sample from the subject. In some embodiments, identifying comprises analyzing the patient transcriptome and / or patient exome obtained from the subject-derived sample. In some embodiments, the subject-derived sample is a biological sample, eg, a biopsy. In some embodiments, the method further includes isolating the sample from the subject. In some embodiments, identifying includes analyzing tissue-specific expression in available databases.

[0085] In another aspect of the invention, methods are 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 is a. Identification of patient-specific mutanome by analysis of patient transcriptome and patient exome; b. Evaluation of gene or transcript level expression in patient RNA sequencing; variant call confidence score; allele-specific expression based on RNA sequencing; conservative and non-conservative amino acid substitutions; Comparison of; Location of point mutations (Centering Score for increased TCR involvement); Location of point mutations (Anchoring Score for differences in binding to HLA); Selfness: Patient core epitope homology (<100%) with WES data; IC50 to HLA-A and HLA-B for 8-mer to 11-mer; IC50 to HLA-DRB1 for 15-mer to 20-mer; broad binding score (promiscuity IC50 for HLA-C for 8-mer to 11-mer; IC50 for HLA-DRB3-5 for 15-mer to 20-mer; IC50 for HLA-DRB3-5 for 15-mer to 20-mer IC50 for HLA-DQB1 / A1; IC50 for HLA-DPB1 / A1 for 15-mer to 20-mer; Comparison of ratio between class I and class II; HLA-A allotype, HLA-B allotype, and based on at least three of the following: HLA-DRB1 allotype diversity; ratio comparison of point mutations and complex epitopes (e.g., frameshifts); pseudoepitope HLA binding score; presence and / or abundance of RNA sequencing reads; selection of a subset of 15-500 neoepitopes from the mutanome using weighted values ​​for neoepitopes; c. Selection of a set of neoepitopes for use in a personalized mRNA cancer vaccine from the subset based on the highest weighted value, the set of neoepitopes comprising between 15 and 40 neoepitopes; include.

[0086] The present disclosure, in some embodiments, is an mRNA cancer vaccine of one or more mRNAs each having an open reading frame encoding a cancer antigen peptide epitope, wherein the mRNA further comprises a miRNA binding site. In some embodiments, the vaccine encodes 5-100 peptide epitopes.

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

[0088] Yet other embodiments 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. A vaccination method is provided in which the RNA polynucleotide does not contain stabilizing elements and no adjuvant is co-formulated or co-administered with the vaccine.

[0089] In other embodiments, the invention is directed to vaccination of 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. A composition or a method for vaccinating a subject, and 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 RNA polynucleotide 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 μg per dose. ~50μg, 40~60μg, 60~80μg, 60~100μg, 50~100μg, 80~120μg, 40~120μg, 40~150μg, 50~150μg, 50~200μg, 80~200μg, 100~200μg, 120~250μg , 150~250μg, 180~280μg, 200~300μg, 50~300μg, 80~300μg, 100~300μg, 40~300μg, 50~350μg, 100~350μg, 200~350μg, 300~350μg, 320~400μg, 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 a 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 RNA polynucleotide included in the nucleic acid vaccine administered to the subject is 25 micrograms. In some embodiments, the dose of RNA polynucleotide included in the nucleic acid vaccine administered to the subject is 100 micrograms. In some embodiments, the dose of RNA polynucleotide included in the nucleic acid vaccine administered to the subject is 50 micrograms. In some embodiments, the dose of RNA polynucleotide included in the nucleic acid vaccine administered to the subject is 75 micrograms. In some embodiments, the dose of RNA polynucleotide included in the nucleic acid vaccine administered to the subject is 150 micrograms. In some embodiments, the dose of RNA polynucleotide included in the nucleic acid vaccine administered to the subject is 400 micrograms. In some embodiments, the dose of RNA polynucleotide included in the nucleic acid vaccine administered to the subject is 200 micrograms. In some embodiments, the RNA polynucleotide accumulates at 100 times higher levels in regional lymph nodes compared to distal lymph nodes. In other embodiments, the nucleic acid vaccine is chemically modified, and in other embodiments, the nucleic acid vaccine is not chemically modified.

[0091] In some embodiments, an effective amount is a total dose of 1-100 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 once or twice in total. In some embodiments, the effective amount is a 100 μg dose administered to a subject a total of two times. In some embodiments, an effective amount can be administered to a subject once or twice or more, from 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 30μg, 5μg~40μg, 5μg~50μg, 10μg~15μg, 10μg~20μg, 10μg~25μg, 10μg~30μg, 10μg~40μg, 10μg~50μg, 10μg~60μg, 15μg~20μg, 15μg~25μg, 15μg~30μg g, 15μg~40μg, 15μg~50μg, 20μg~25μg, 20μg~30μg, 20μg~40μg20μg~50μg, 20μg~60μg, 20μg~70μg, 20μg~75μg, 30μg~35μg, 30μg~40μg, 30μg~45μg30μ g~50μg, 30μg~ The doses are 60μg, 30μg~70μg, and 30μg~75μg.

[0092] Aspects of the invention provide nucleic acid vaccines comprising one or more RNA polynucleotides having an open reading frame encoding a first antigenic polypeptide and a pharmaceutically acceptable carrier or excipient. , the RNA polynucleotide does not contain stabilizing elements and the vaccine does not contain an adjuvant. In some embodiments, the stabilizing element is a histone stem loop. In some embodiments, the stabilizing element is a nucleic acid sequence with increased GC content compared to the wild-type sequence.

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

[0094] Other embodiments include 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 binds the first antigen polypeptide. Nucleic acid vaccines are provided that encode and require at least 10 times fewer RNA polynucleotides in the vaccine than would be required for an unmodified mRNA vaccine to produce equivalent antibody titers.

[0095] In an aspect of the invention there is also provided a vaccine unit for use, said unit having an open reading frame comprising at least one chemical modification or optionally no chemical modification, wherein the open reading frame is linked to a first antigen. 10ug to 400ug of one or more RNA polynucleotides encoding a 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] Embodiments of the invention provide kits that include vials containing the mRNA cancer vaccines 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 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 lipids is 20 mg / mL.

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

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

[0100] In aspects of the invention, methods for vaccinating a subject are provided, the method comprising administering one or more RNA polynucleotides having an open reading frame encoding a first antigenic polypeptide in an amount effective for vaccinating a subject. comprising administering to a subject a single dose of 25ug / kg to 400ug / kg of a nucleic acid vaccine comprising:

[0101] In some embodiments, the present disclosure is an mRNA cancer vaccine that can include an activating cancer gene mutation as an antigen. In some embodiments, the activating oncogene 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, e.g., the G12 KRAS mutation is selected from G12D, G12V, and G12S KRAS mutations. In other embodiments, the KRAS mutation is a G13 mutation, eg, the G13 KRAS mutation is a G13D KRAS mutation. In some embodiments, the activating oncogene mutation is an H-RAS or N-RAS mutation.

[0102] In some embodiments, one of skill in the art will select the KRAS mutation, HLA subtype and tumor type and prepare a therapeutic KRAS vaccine based on the guidelines described herein. In some embodiments, the KRAS mutation is selected from G12C, G12V, G12D, G13D. In some embodiments, the HLA subtype is A * 02:01,C * 07:01,C * 04:01,C * Selected from 07:02. In some embodiments, the tumor type is selected from colorectal, pancreatic, pulmonary, 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 activated oncogene variant peptides. In some embodiments, at least two of the peptide epitopes are separated from each other by one glycine. In some embodiments, the concatemers include 3-10 activating oncogene variant peptides. In some such embodiments, all of the peptide epitopes are separated from each other by one 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 consists of PD-1, TIM-3, VISTA, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR and LAG3. An antibody or a fragment thereof that specifically binds to a molecule selected from the group. 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 include stabilizing agents.

[0108] In some embodiments, the mRNA comprises a molar ratio of about 20-60% cationic lipids: 5-25% non-cationic lipids: 25-55% sterols; 0.5-15% PEG-modified lipids. Formulated in a lipid nanoparticle carrier, such as a lipid nanoparticle carrier. Cationic lipids include, 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).

[0109] In some embodiments, the mRNA includes at least one chemical modification. Chemical modifications include 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 may be selected from the group consisting of O-methyluridine.

[0110] In other aspects, methods of treating a subject are provided. The method involves administering to a subject having cancer an mRNA cancer vaccine of any one of the embodiments described above. In some embodiments, an mRNA cancer vaccine is administered in combination with a cancer therapeutic. In some embodiments, an mRNA cancer vaccine is administered in combination with an inhibitory checkpoint polypeptide. For example, in some embodiments, the mRNA cancer vaccine is selected from the group consisting of PD-1, TIM-3, VISTA, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, and LAG3. An antibody or a fragment thereof that specifically binds to a molecule that is

[0111] The methods provided herein can be used to treat subjects with cancer. In some embodiments, the cancer includes the pancreas, peritoneum, large intestine, small intestine, biliary tract, lung, endometrium, ovary, reproductive tract, gastrointestinal tract, cervix, stomach, urinary tract, colon, rectum, and hematopoietic system. and lymphoid tissue cancer. In some embodiments, the cancer is colorectal cancer.

[0112] In some embodiments, the dose of the mRNA cancer vaccine administered to the subject is 1-5 μg, 5-10 μg, 10-15 μg, 15-20 μg, 10-25 μg, 20-25 μg, 20-50 μg, per dose. 30~50μg, 40~50μg, 40~60μg, 60~80μg, 60~100μg, 50~100μg, 80~120μg, 40~120μg, 40~150μg, 50~150μg, 50~200μg, 80~200μg, 100~ 200μg, 120~250μg, 150~250μg, 180~280μg, 200~300μg, 50~300μg, 80~300μg, 100~300μg, 40~300μg, 50~350μg, 100~350μg, 200~350μg, 300~350μg , 320-400μg, 40-380μg, 40-100μg, 100-400μg, 200-400μg, or 300-400μg. 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 the subject. In some embodiments, a 100 microgram dose of the mRNA cancer vaccine is administered to the subject. In some embodiments, a 50 microgram dose of the mRNA cancer vaccine is administered to the subject. In some embodiments, a 75 microgram dose of the mRNA cancer vaccine is administered to the subject. In some embodiments, a 150 microgram dose of the mRNA cancer vaccine is administered to the subject. In some embodiments, a 400 microgram dose of the mRNA cancer vaccine is administered to the subject. In some embodiments, a 200 microgram dose of the mRNA cancer vaccine is administered to the subject. In some embodiments, the mRNA cancer vaccine accumulates at 100 times higher levels in regional lymph nodes compared to distal 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, an effective amount is a total dose of 1-100 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 once or twice in total. In some embodiments, the effective amount is a 100 μg dose administered to a subject a total of two times. In some embodiments, an effective amount can be administered to a subject once or twice or more, from 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 30μg, 5μg~40μg, 5μg~50μg, 10μg~15μg, 10μg~20μg, 10μg~25μg, 10μg~30μg, 10μg~40μg, 10μg~50μg, 10μg~60μg, 15μg~20μg, 15μg~25μg, 15μg~30μg g, 15μg~40μg, 15μg~50μg, 20μg~25μg, 20μg~30μg, 20μg~40μg20μg~50μg, 20μg~60μg, 20μg~70μg, 20μg~75μg, 30μg~35μg, 30μg~40μg, 30μg~45μg30μ g~50μg, 30μg~ The doses are 60μg, 30μg~70μg, and 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, the method comprising: (a) an open reading frame encoding the cancer antigen according to any one of claims 1 to 103; (b) binding a first polynucleotide comprising a first polynucleotide comprising a 5'-UTR and a second polynucleotide comprising a 5'-UTR to a polynucleotide complexed to a solid support; (c) ligation to the 5' end of a polynucleotide under suitable conditions, the suitable conditions comprising a DNA ligase, whereby a first ligation product is produced; and (c) a 3'-UTR. ligation of the 5' end of a third polynucleotide comprising an RNA ligation product to the 3' end of a first ligation product under suitable conditions, the suitable conditions comprising an RNA ligase, whereby a second (d) releasing a second ligation product from a solid support, thereby producing an mRNA encoding a concatemeric cancer antigen comprising 1000 to 3000 nucleotides. provide.

[0116] An aspect of the invention is a method of treating a subject with a personalized mRNA cancer vaccine, comprising: identification of a set of neoepitopes to generate a patient-specific mutanome; Mutanome selection of neoepitope sets for vaccines based on diversity, predicted immunogenicity, low autoreactivity, and / or T cell reactivity and mRNA vaccines encoding neoepitope sets and administering the mRNA vaccine to the subject within two months of isolation of the sample from the subject.

[0117] An embodiment of the invention is 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, comprising: (a) Identification of patient-specific mutanome by analysis of transcriptome and patient exome; and (b) expression evaluation of gene or transcript levels in patient RNA sequencing; variant call confidence score; RNA Allele-specific expression based on sequencing; comparison of conservative and non-conservative amino acid substitutions; location of point mutations (Centering Score for increased TCR involvement); location of point mutations (Centering Score for increased TCR involvement); Anchoring Score); Selfness: core epitope homology (<100%) with patient WES data; HLA-A and HLA-B for 8-mer to 11-mer 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 ~20 mers; IC50 for HLA-DPB1 / A1 for 15 mers ~20 mers; Comparison of ratio between class I and class II; HLA-A allotypes covered in patients, HLA- B allotype and HLA-DRB1 allotype diversity; ratio comparison of point mutations to complex epitopes; pseudoepitope HLA binding score; neoepitope based on at least three of the following: presence and / or abundance of RNA sequencing reads (c) selection of a subset of 15-500 neoepitopes from the mutanome using weighted values ​​for selecting a set of neoepitopes, the set of neoepitopes comprising from 15 to 40 neoepitopes.

[0118] An embodiment of the invention is 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, comprising: (a) generating an RNA sequencing sample from the tumor to generate a set of RNA sequencing reads; (b) compiling a total number of nucleotide sequences from all RNA sequencing reads; (c) the tumor sample; Comparison of sequence information with a corresponding database of normal tissues of the same tissue type; and (d) a set of neoepitopes from the subset for use in personalized mRNA cancer vaccines based on the highest weighted value. wherein the set of neoepitopes includes 15 to 40 neoepitopes.

[0119] Details of various embodiments of the invention are set forth in the description below. 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, and such are illustrated in the accompanying drawings, in which like reference characters refer to different Refers to the same part throughout the viewpoint. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of various embodiments of the invention. [Brief explanation of drawings]

[0121]

Figure 1

Figure 2

[0122] Embodiments of the present disclosure provide RNA (eg, mRNA) vaccines 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 vaccination with DNA. be able to. In some embodiments, the vaccine comprises at least one RNA (eg, mRNA) polynucleotide having an open reading frame encoding a cancer antigen. In some embodiments, the vaccine comprises at least one RNA (e.g., , mRNA) polynucleotides. In another embodiment, the vaccine comprises at least one RNA (e.g., , mRNA) polynucleotides. In some embodiments, the vaccine comprises at least one RNA (eg, mRNA) polynucleotide having an open reading frame encoding a cancer antigen (eg, an activating oncogene variant peptide).

[0123] Although many attempts have been made to generate functional RNA vaccines, including mRNA cancer vaccines, the therapeutic efficacy of these RNA vaccines remains to be fully established. Quite surprisingly, the inventors have discovered a class of formulations for delivering mRNA vaccines that elicit significantly enhanced and in many ways synergistic immune responses, including enhanced T cell responses. . Vaccines of the invention include conventional cancer vaccines as well as personalized cancer vaccines. In some embodiments, 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 for the delivery of siRNA in various animal models as well as humans. Considering the findings obtained in connection with the delivery of siRNA by lipid nanoparticle formulations, the fact that lipid nanoparticles, as opposed to liposomes, are useful in cancer vaccines is quite surprising. Therapeutic delivery of siRNA formulated in lipid nanoparticles typically results in decreased antigen production and impaired immune responses, resulting in transient IgM responses and associated undesirable inflammatory responses. It has been observed that In contrast to the findings observed with siRNA, the lipid nanoparticle-mRNA cancer vaccine formulation described herein does not result in a transient IgM response, but rather an increase in IgG levels sufficient for prophylactic and therapeutic methods. has been shown to occur. The lipid nanoparticles of the present invention are not liposomes. Liposomes, as used herein, are lipid-based structures that have a lipid bilayer or monolayer shell and contain a nucleic acid payload within the core.

[0125] The generation of cancer antigens that elicit the desired immune response (eg, T cell response) against target polypeptide sequences in vaccine development remains a challenging task. The present invention includes techniques that overcome the hurdles associated with such vaccine development. Using the technology of the present invention, one can tailor the desired immune response by selecting appropriate T-cell cancer epitopes or B-cell cancer epitopes and formulating epitopes or antigens for effective delivery in vivo. It is possible to guide the Additionally or alternatively, the immune response is further stimulated 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 epitope or antigen. Can be strengthened.

[0126] Additionally or alternatively, the mRNA vaccine may include an activating oncogene mutant peptide (eg, a KRAS mutant peptide). Previous studies have shown that there are limits to the ability to produce T cells specific for oncogenic mutations. Most of this research was conducted in the context of the most common HLA allele (A2, which occurs 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 have been found in many cancers. The ability to target these mutations and generate enough T cells to kill tumors could have broad applications in cancer therapy. It is quite surprising that antigen delivery using mRNA has such significant advantages compared to peptide vaccine delivery. Accordingly, the present invention includes, in some embodiments, the surprising finding that activated oncogenic mutant antigens delivered in vivo in the form of mRNA significantly enhance the effectiveness of cancer treatment.

[0127] HLA class I molecules are highly polymorphic transmembrane glycoproteins composed of two polypeptide chains (heavy and light chains). Human major histocompatibility complex, which is a human leukocyte type antigen, is specific to each individual and has genetic characteristics. Class I heavy chains are encoded by three genes: HLA-A, HLA-B and HLA-C. HLA class I molecules are important in establishing the immune response 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. Start. Altered levels of HLA class I antigen production are a widespread phenomenon in malignant tumors and are accompanied by marked suppression of antitumor T cell function. This is one of the main mechanisms cancer cells use to evade immune surveillance. Downregulation of HLA class I antigen levels was detected in 90% of NSCLC tumors (n=65). HLA reduction or loss 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] Mounting evidence suggests that tumor cells use two general strategies to evade immune surveillance: immunoselection (less immunogenic tumor cell variants) and immunodestruction (destruction of the immune system). It suggests that. A correlation has been shown between changes in HLA class I antigens and the presence of 12 KRAS codon mutations, indicating that 12 KRAS codon mutations have an inductive effect on HLA class I antigen regulation in cancer progression. It suggests possibility. High frequency cancer mutations are predicted to bind to HLA class I alleles with high affinity (IC50≦50nM)7 and may be 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 the tumor. Checkpoint inhibitors enhance the action of mRNAs encoding activated oncogenic peptides by eliminating some of the obstacles to promoting immune responses, thereby allowing activated T cells to mount an efficient immune response against tumors. It will be possible to promote

[0130] It has been discovered that the mRNA vaccines described herein have several advantages compared to current vaccines. First, delivery by lipid nanoparticles (LNPs) is superior to other formulations including liposome- or protamine-based approaches described in the literature. The use of LNPs allows 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 invention are at least 10 times, at least 20 times, at least 40 times, at least 50 times, at least 100 times, at least 500 times, or at least 1,000 times more effective than conventional vaccines. Are better.

[0131] Although many attempts have been made to generate functional RNA vaccines, including mRNA vaccines and self-replicating RNA vaccines, the therapeutic efficacy of these RNA vaccines is still not fully established. Quite surprisingly, the delivery of mRNA vaccines in vivo elicits significantly enhanced and in many ways synergistic immune responses, including enhanced antigen production and production of functional antibodies with neutralizing capacity. In accordance with aspects of the present invention, the inventors have discovered a class of formulations for. These results can be achieved even when significantly lower doses of mRNA are administered compared to those used in other classes of lipid-based formulations. The formulations of the invention were unexpectedly shown to elicit a significant immune response in vivo, sufficient to establish the effectiveness of a functional mRNA vaccine as a prophylactic and therapeutic agent. Additionally, self-replicating RNA vaccines utilize the viral replication pathway to deliver enough RNA to cells to generate an immune response. The formulations of the invention produce sufficient protein to elicit a strong immune response without the need for viral replication. Therefore, the mRNA of the present invention is not self-replicating RNA and does not contain elements necessary for viral replication.

[0132] In some embodiments, the present invention includes the surprising finding that lipid nanoparticle (LNP) formulations significantly enhance the efficacy of mRNA vaccines, including chemically modified and unmodified mRNA vaccines. Furthermore, immunogenicity for epitopes was found to be similar regardless of the total number of epitopes contained within the construct. The epitopes contained in the 52-mer construct have similar immunogenicity compared to the 20-mer construct as measured by epitope-specific IFNγ responses. It was completely unexpected that increasing the length of the mRNA was shown to have no deleterious effect on the immunogenicity of the epitope. This also indicates a complete readthrough of the concatemers, as the last epitopes encoded by the 20-mer and 52-mer (SIINFEKL, SEQ ID NO: 231) were equivalent. It was also surprisingly found that antigen-specific responses to class I epitopes are increased when the vaccine is formulated with a constitutively active immunopotentiator.

[0133] The LNPs used in the studies described herein have been previously used for the delivery of siRNA in various animal models as well as in humans. In view of the findings obtained in connection with the delivery of siRNA by LNP formulations, the fact that LNPs are useful in vaccines is quite surprising. Therapeutic delivery of siRNA formulated in LNPs typically results in decreased antigen production and impaired immune responses as a result of transient IgM responses and associated undesirable inflammatory responses. It has been observed. In contrast to the findings observed with siRNA, it is demonstrated herein that the LNP-mRNA formulations of the present invention do not result in a transient IgM response, but rather an increase in IgG levels sufficient for prophylactic and therapeutic methods. is shown.

[0134] mRNA cancer vaccines offer a unique therapeutic alternative to peptide-based or DNA vaccines. When an mRNA cancer vaccine is delivered to cells, the mRNA is processed by intracellular machinery to produce polypeptides, which in turn trigger an immune response against the tumor. It can be an immunosensitive fragment that has the ability to stimulate.

[0135] In some embodiments, mRNA cancer vaccines may be administered with anti-cancer therapeutics, including, but not limited to, conventional cancer vaccines. mRNA cancer vaccines and anti-cancer therapeutics can be combined to further enhance immunotherapeutic responses. The mRNA cancer vaccine and other therapeutic agents may be administered simultaneously or sequentially. When other therapeutic agents are administered at the same time, they can be administered in the same formulation or in separate formulations, but the administration is carried out at the same time. When the administration of the other therapeutic agent and the mRNA cancer vaccine are temporally separate, the other therapeutic agent is administered sequentially with each other and sequentially with the mRNA cancer vaccine. The time interval between administrations of such compounds can be on the order of minutes, or can be longer, such as hours, days, weeks, months, and the like. Other therapeutic agents include, but are not limited to, anti-cancer therapeutics, adjuvants, cytokines, antibodies, antigens, and the like.

[0136] The cancer vaccines described herein have at least one 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). Contains one ribonucleic acid (RNA) polynucleotide. The antigenic peptide may be a personalized cancer antigen epitope, and / or a recurrent antigen. In some preferred embodiments, the vaccine is a combination of multiple epitopes of each of the above. Thus, the cancer vaccine may be a conventional cancer vaccine or a personalized cancer vaccine or a mixture thereof. Conventional cancer vaccines are vaccines that contain cancer antigens known to be commonly 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 on non-cancerous cells. Many tumor mutations are known in the art.

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

[0138] A personalized vaccine may, for example, contain RNA encoding one or more known cancer antigens specific for a tumor or cancer antigens specific for each subject, such antigens containing neoepitopes or subject-specific epitopes or Referred to as subject-specific antigens (referred to as individualized antigens). A "subject-specific cancer antigen" is an antigen that has been identified to be expressed on a particular patient's tumor. It is common that subject-specific cancer antigens typically may or may not be present in the tumor sample. It is not or rarely expressed in non-cancerous cells, or its expression in non-cancerous cells is markedly reduced compared to that in cancerous cells, and may interfere with the immune response induced upon vaccination. The inducing tumor-associated antigens are called neoepitopes. It is assumed that neoepitopes, such as tumor-associated antigens, are completely foreign to the body and therefore do not generate an immune response against healthy tissue or are masked by protective components of the immune system. In some embodiments, neoepitope-based personalized vaccines are desirable because such vaccine formulations will maximize specificity for patient-specific tumors. Neoepitopes resulting from mutations include point mutations, which are nonsynonymous mutations that result in a different amino acid in the protein, and modification or deletion of the stop codon, leading to the translation of a longer protein with a novel tumor-specific sequence at the C-terminus. Read-through mutations, splice site mutations that result in unique tumor-specific protein sequences due to the inclusion of introns in the mature mRNA, and chromosomal rearrangements (i.e., gene fusions that result in chimeric proteins with tumor-specific sequences at the junction of two proteins) ), frameshift mutations or deletions resulting in new open reading frames with novel tumor-specific protein sequences, and translocations. Accordingly, in some embodiments, the mRNA cancer vaccine comprises at least two cancer antigens that include mutations selected from the group consisting of frameshift mutations and recombination or any of the other mutations described herein. .

[0139] Methods for generating personalized cancer vaccines generally involve the identification of mutations, e.g. using deep sequencing techniques of nucleic acids or proteins, and e.g. validated peptide-MHC binding prediction algorithms, or which may bind to a patient's HLA alleles and determine whether the tumor Identification of neoepitopes by applying other analytical techniques to generate a set of candidate T cell epitopes based on mutations present in optional demonstration that the demonstration or candidate neoepitope binds to HLA proteins present on the tumor surface and development of a vaccine. The mRNA cancer vaccine of the present invention can contain multiple copies of a single neoepitope, multiple different neoepitopes based on a single type of mutation, i.e. point mutations, multiple different neoepitopes based on different variant types, tumor-associated Neoepitopes and other antigens such as antigens or recall antigens may be included.

[0140] Examples of mutation identification techniques include, but are not limited to, dynamic allele-specific hybridization (DASH), microplate array diagonal gel electrophoresis (MADGE), pyrosequencing, and oligonucleotides. Generating small signal molecules by specific ligation, the TaqMan system, and various DNA "chip" techniques, namely Affymetrix SNP chips, as well as invasive cleavage, followed by mass spectrometry, or immobilized padlock probes and rolling circle amplification. Includes methods based on implementation.

[0141] Nucleic acid or protein deep sequencing techniques are known in the art. Any type of array parsing method can be used. Nucleic acid sequencing may be performed on the whole tumor genome, tumor exome (protein-coding DNA), tumor transcriptome, or exosomes. Real-time, single-molecule-by-synthesis sequencing technology relies on the detection of fluorescent nucleotides each time they are incorporated into a nascent DNA strand that is complementary to the template being sequenced. Other rapid high-throughput sequencing methods exist. Protein sequencing may be performed on the tumor proteome. Additionally, protein mass spectrometry may 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 may be compared to a known control set or to sequencing analysis performed on normal tissue of the patient.

[0142] Accordingly, 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 that are useful in inducing tumor-specific immune responses in a subject. Optionally, some of these neoepitopes bind with greater affinity to class I HLA proteins compared to the wild-type peptide and / or have the ability to activate anti-tumor CD8 T cells. Others bind to class II and activate CD4+ T helper cells. Although the important role that class I antigens play in vaccines is recognized, it is important to note 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. It has been discovered in the present invention that this can be achieved.

[0143] MHC class I proteins are present on the surface of almost every cell in the body, including most tumor cells. Antigens, usually originating from endogenous proteins or intracellular pathogens, are loaded onto MHC class I proteins, and these antigens are then presented 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 that has the ability to specifically bind to MHC / peptide complexes.

[0144] Using computer algorithms it is possible to predict potential neoepitopes, i.e. after binding of class I or class II MHC molecules in the form of a peptide presenting complex, in this form A peptide sequence recognized by the T cell receptor of T lymphocytes. 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 were used to refine the list of neoepitopes selected based on algorithmic predictions using routine in vitro assays performed in the laboratory, such as Elispot assays. good.

[0146] The mRNA cancer vaccine of the present invention is a composition comprising a pharmaceutical composition. The present invention also includes methods for selecting, designing, preparing, manufacturing, formulating, and / or using mRNA cancer vaccines. Systems, processes, equipment, and kits for selecting, designing, and / or utilizing the mRNA cancer vaccines described herein are also provided.

[0147] The mRNA vaccines of the invention may include one or more cancer antigens. In some embodiments, the mRNA vaccine is 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 vaccine is 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 vaccine is comprised of no more than 1000, no more than 900, no more than 500, no more than 100, no more than 75, no more than 50, no more than 40, no more than 30, no more than 20, or no more than 100 cancer antigens. In yet other embodiments, the mRNA vaccine comprises 3-100, 5-100, 10-100, 15-100, 20-100, 25-100, 30-100, 35-100, 40-100, 45-100 , 50~100, 55~100, 60~100, 65~100, 70~100, 75~100, 80~100, 90~100, 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 have 100 to 1,000 cancer antigens.

[0148] In some embodiments, mRNA cancer vaccines and vaccination methods may be used to target epitopes or antigens based on specific mutations (neoepitopes) and those expressed by cancer-germline genes (antigens common to tumors found in multiple patients). )including.

[0149] As used herein, an epitope, also known as an antigenic determinant, is a portion of an antigen that, in appropriate circumstances, is recognized by the immune system, specifically by an antibody, B cell, or T cell. It is part of the antigen. Epitopes include B cell epitopes and T cell epitopes. B-cell epitopes are peptide sequences required for recognition by specific antibody-producing B cells. B cell epitope refers to the specific region of an antigen that is recognized by an antibody. The portion of an antibody that binds to an epitope is called a paratope. Epitopes can be conformational epitopes or linear epitopes based on structure and interaction with paratopes. A linear or continuous epitope is defined by the primary amino acid sequence of a particular region of a protein. Sequences that interact with antibodies are located in the protein consecutively next to each other, and epitopes can usually be mimicked by a single peptide. A conformational epitope is an epitope defined by the conformation of a native protein. Such epitopes can be continuous or discontinuous, ie, the components of the epitope can be located in different parts of the protein, and these different parts are in close proximity to each other in the folded, native protein structure.

[0150] T cell epitopes are peptide sequences associated with proteins present on APCs and required for recognition by specific T cells. T cell epitopes are processed intracellularly and then presented on the surface of APCs, where they bind to MHC molecules including MHC class II and MHC class I. Peptide epitopes can be of any length suitable for epitopes. In some embodiments, the peptide epitope is 9-30 amino acids. In other embodiments, the length is 9-22, 9-29, 9-28, 9-27, 9-26, 9-25, 9-24, 9-23, 9-21, 9-20, 9~19, 9~18, 10~22, 10~21, 10~20, 11~22, 22~21, 11~20, 12~22, 12~21, 12~20, 13~22, 13~ 21, 13-20, 14-19, 15-18, or 16-17 amino acids.

[0151] In some embodiments, the peptide epitopes include 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 about 10%: about 90%, about 20%: about 80%, about 30%: about 70%, about 40%: about 60%, about 50%: about 50%, about 60%: about 40%, about 70%: about 30%, about 80%: about 20%, about 90%: about 10% MHC class 1 epitope: MHC class The ratio selected from II epitopes. In one embodiment, the ratio of MHC class I epitopes: MHC class II epitopes is 3:1. In some embodiments, the ratio of MHC class II epitopes to MHC class I epitopes is about 10%: about 90%, about 20%: about 80%, about 30%: about 70%, about 40%: about 60%, about 50%: about 50%, about 60%: about 40%, about 70%: about 30%, about 80%: about 20%, about 90%: about 10% MHC class II epitope: MHC class The ratio selected from 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 cancer vaccine T cell epitope comprises 8-11 amino acids. In some embodiments, the cancer vaccine B cell epitope comprises 13-17 amino acids.

[0152] In other embodiments, cancer vaccines of the invention include 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 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 These include, but are not limited to, 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 vaccine comprises 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. In other embodiments, the one or more universal type II T cell epitope(s) are 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 They are scattered everywhere.

[0153] In some embodiments, the cancer vaccines of the invention comprise multiple peptide epitope antigens arranged with a single nucleotide spacer between the epitopes or directly with respect to each other without a spacer between the epitopes. Contains an mRNA vaccine encoding. Multiple epitope antigens include a mixture of MHC class I and MHC class II epitopes. For example, a multiple peptide epitope antigen has the following structure: (X-G-X) 1-10 (G-Y-G-Y) 1-10 (G-X-G-X) 0-10 (G-Y-G-Y) 0-10 ,(X-G) 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 with a length of 10 to 40 amino acids, Y is an MHC class II epitope with a length of 10 to 40 amino acids, and G is glycine).

[0154] In some embodiments, the cancer vaccines of the present invention provide mRNAs encoding multiple peptide epitope antigens in which a centrally located single nucleotide polymorphism (SNP) mutation is located and adjacent amino acids are present on either side of the SNP mutation. Including vaccines. In some embodiments, the number of flanking amino acids on either side of the centrally located SNP variation 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 a SNP flanked by two class I sequences, each sequence containing 7 amino acids. In another embodiment, the cancer vaccine epitope comprises a SNP flanked by two class II sequences, each sequence containing 10 amino acids. In some embodiments, an epitope can include 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 mRNA encoding a polypeptide that stimulates or enhances an immune response to one or more cancer antigens of interest. Such mRNAs that enhance the immune response against the cancer antigen(s) of interest are referred to herein as immunopotentiator mRNA constructs or immunopotentiator mRNAs, and include chemically modified mRNAs (mRNAs). include. The immunopotentiators of the present disclosure enhance immune responses to antigens of interest in a subject. The immune response that is enhanced may be a cellular response, a humoral response, or both. As used herein, "cell-mediated" immune responses are intended to encompass immune responses that involve or are mediated by T cells, whereas "humoral" immune responses are those that involve or are mediated by B cells. It is intended to encompass the immune response to Immune enhancers include, 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 dendritic cell development, activity or recruitment; and (vi) Immune response can be enhanced by any combination of (i) to (vi).

[0156] As used herein, "stimulating type I interferon pathway signaling" refers to activating one or more components of the type I interferon signaling pathway (e.g., activating the pathway by altering component phosphorylation, dimerization, etc.), stimulating transcription from interferon-sensitive response elements (ISREs) and / or stimulating transcription from type I interferons (e.g., IFN-α , IFN-β, IFN-ε, IFN-κ and / or IFN-ω). As used herein, "stimulating NFkB pathway signaling" refers to activating one or more components of the NFkB signaling pathway (e.g., phosphorylating such components). oxidation, dimerization, etc.), stimulating transcription from NFkB sites and / or stimulating the production of gene products whose expression is regulated by NFkB. It is intended that As used herein, "stimulating an inflammatory response" refers to stimulating the production of inflammatory cytokines (including, but not limited to, type I interferon, IL-6 and / or TNFα). is intended to encompass. As used herein, "stimulating the development, activity or recruitment of dendritic cells" refers to directly or indirectly stimulating the maturation, proliferation and / or functional activity of dendritic cells. is intended to be inclusive.

[0157] In some embodiments, the present disclosure provides methods for inducing adaptive immunity (e.g., by stimulating type I interferon production), stimulating an inflammatory response, stimulating NFkB signaling, and A polypeptide that stimulates or enhances an immune response in a subject in need thereof (e.g., enhances a subject's immune response) by stimulating the development, activity, or recruitment of dendritic cells (DCs). Provides mRNA encoding the peptide. In some embodiments, administration of an immune enhancer mRNA to a subject in need thereof increases 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 are enhanced. In some embodiments, administration of the immunopotentiator mRNA stimulates cytokine production (e.g., inflammatory cytokine production) and stimulates the cancer antigen-specific CD8 + stimulates effector cell responses and antigen-specific CD4 + Stimulates helper cell responses and effector memory CD62L lo Increase T cell populations, stimulate B cell activity, or stimulate antigen-specific antibody production (including combinations of the foregoing responses). In some embodiments, administration of the immunopotentiator mRNA stimulates cytokine production (e.g., inflammatory cytokine production) and stimulates antigen-specific CD8 + Stimulating effector cell responses. In some embodiments, administration of the immunopotentiator mRNA stimulates cytokine production (e.g., inflammatory cytokine production) and stimulates antigen-specific CD4 + Stimulating helper cell responses. In some embodiments, administration of the immunopotentiator mRNA stimulates cytokine production (e.g., inflammatory cytokine production) and inhibits effector memory CD62L. lo Increases T cell population. In some embodiments, administration of the immunopotentiator mRNA stimulates cytokine production (eg, inflammatory cytokine production) and stimulates B cell activity or stimulates antigen-specific antibody production.

[0158] In one embodiment, the immunopotentiator is a cancer antigen-specific CD8 + Increase effector cell responses (cell-mediated immunity). For example, but not limited to, immune enhancers include 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 increases CD8+ T cell cytokine production (e.g., IFN-γ, TNFα and / or IL-2 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% (CD8+ T cell cytokine production in the absence of immunopotentiators) ). For example, T cells obtained from a treated 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, secretory measurements of cytokine production (e.g., ELISA or (by any other suitable method for determining the amount of cytokines in the supernatant known in the art) and / or of the percentage of CD8+ T cells that are positive for intracellular staining (ICS) for cytokines. Includes decisions. 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., (see ). In one embodiment, the immunopotentiator increases 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. (Immune enhancer (compared to the percentage of CD8+ T cells positive for ICS for cytokine(s) in the absence of).

[0159] In yet another embodiment, the immune-enhancing agent 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 immuno-enhancing agent. +Increase the percentage of T cells. For example, the immunopotentiator increases the percentage of CD8+ T cells in the total T cell population by at least 5%, or at least 10%, or at least 15% compared to the percentage of CD8+ T cells in the absence of the immunopotentiator. % 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%. 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, fluorescent cell sorting. (FACS) or magnetically activated cell sorting (MACS).

[0160] In another embodiment, the immunopotentiating agent is tumor-specific, as determined by a decrease in tumor volume in vivo in the presence of the immunopotentiating agent compared to tumor volume in the absence of the immunopotentiating agent. Increases immune cell response. For example, the immunoenhancing agent increases the 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 It can be reduced by at least 35% or at least 40% or at least 45% or at least 50%. Measurements 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) by comparing, e.g., the amount of antigen-specific antibody production in the absence of the immunopotentiator. Increase by increasing. For example, an immunopotentiator increases antigen-specific antibody production by at least 5%, or at least 10%, or at least 15%, or at least 20%, or at least 25% compared to antigen-specific antibody production in the absence of the immunopotentiator. % or at least 30% or at least 35% or at least 40% or at least 45% or at least 50%. In one embodiment, antigen-specific IgG production is assessed. Antigen-specific antibody production is described in the art, including, but not limited to, ELISA, RIA, and the like to measure antigen-specific antibody (e.g., IgG) levels in a sample (e.g., a serum sample). can be evaluated using well-established methods.

[0162] In another embodiment, the immune enhancer is the effector memory CD62L lo Increases T cell population. For example, an immunopotentiator may increase CD62L in CD8+ T cells. lo Total % of T cells can be increased. Among other features, effects memory CD62L lo T cell populations have been shown to have important functions in lymphocyte trafficking (see, eg, Schenkel, J.M. and Masopust, D. (2014) Immunity 41:886-897). In various embodiments, the immunopotentiator enhances effector memory CD62L in CD8+ T cells in response to an antigen. lo The total percentage of T cells (CD62L in the CD8+ T cell population in the absence of immunopotentiators) lo (compared to the total percentage of T cells) 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% can be done. Effector memory CD62L in CD8+ T cells lo The 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 cell separation. Act (MACS) is included.

[0163] The ability of an immunopotentiator mRNA construct to enhance immune responses to cancer antigens can be evaluated in mouse model systems known in the art. In one embodiment, an immunocompetent mouse model system is used. In one embodiment, the mouse model system comprises C57 / Bl6 mice (eg, to assess 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 (eg, to assess B cell responses, such as antigen-specific antibody responses).

[0164] In one embodiment, the immunopotentiator 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 immunopotentiator is not a TLR, but rather a molecule within the TLR signaling pathway downstream from the receptor itself.

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

[0166] In one embodiment, the disclosed mRNA encoding an immunopotentiator is formulated within lipid nanoparticles. In one embodiment, the lipid nanoparticle further comprises mRNA encoding a cancer antigen. In one embodiment, lipid nanoparticles are administered to a subject to enhance an immune response to a cancer antigen in the subject. Suitable nanoparticles and methods of use are discussed further below.

[0167] Immune enhancer mRNA that stimulates type I interferon In some embodiments, the present disclosure stimulates an immune response against an antigen of interest by stimulating or enhancing type I interferon pathway signaling, thereby stimulating or enhancing type I interferon (IFN) production. Alternatively, an immunoenhancing agent mRNA encoding a polypeptide that enhances the immunity is provided. It is well established that type I IFN signaling is required for successful induction of antitumor or antimicrobial adaptive immunity (e.g., Fuertes, M.B. et al. (2013) Trends Immunol. 34:67-73 checking). Production of type I IFNs (including IFN-α, IFN-β, IFN-ε, IFN-κ and IFN-ω) is involved in the clearance of microbial infections such as viral infections. Additionally, host cell DNA (e.g., derived from cells that have undergone loss or dying) can induce type I interferon production, and the type I IFN signaling pathway is involved in the generation of anti-tumor adaptive immunity. It is also recognized that However, many pathogens and cancer cells have evolved mechanisms to reduce or suppress type I interferon responses. Therefore, activating (including stimulating and / or enhancing) the type I IFN signaling pathway in a subject in need thereof by providing the immune enhancer mRNA of the present disclosure to the subject may be useful for cancer. stimulate or enhance a subject's immune response in a variety of clinical situations, including the treatment of pathogenic infections and in the enhancement of vaccine responses that produce 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 effects. The standard outcome of in vivo production of type I IFN is the activation of antimicrobial cellular programs and the generation of innate and adaptive immune responses. Type I IFN induces a cell-intrinsic antimicrobial state in infected cells and neighboring cells, limiting the spread of pathogens, especially viral pathogens. Type I IFNs also modulate innate immune cell activation (eg, dendritic cell maturation) and promote antigen presentation and natural killer cell function. Type I IFNs also promote high affinity antigen-specific T 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 via autocrine signaling (Montoya et al., (2002) Blood 99:3263-3271). Type I IFN exposure increases the expression of chemokine receptors and adhesion molecules (e.g., promoting DC migration to draining lymph nodes), co-stimulatory molecules, and MHC class I and class II antigen presentation. Facilitates DC maturation. 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, largely depending 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 revealed that MHC-I expression, a requirement for optimal T cell stimulation, differentiation, expansion, and cytolytic activity, is upregulated in response to type I IFN in multiple cell types. (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 IFN can exert a strong co-stimulatory effect on CD8 T cells, increasing 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 effect 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). 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. has been done. 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, Includes TLR-9, RIG-1, DAI, and IFI16.

[0173] Thus, in one embodiment, the immunopotentiator 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 immunopotentiators. STING (interferon gene stimulator; also known as transmembrane protein 173 (TMEM173), IRF3 activation regulator (MITA), methionine-proline-tyrosine-serine (MPYS), and ER IFN-stimulating factor (ERIS)) A 379-amino acid endoplasmic reticulum (ER)-localized transmembrane protein that functions as a signaling molecule that modulates the transcription of immune response genes, including type I IFN and proinflammatory 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 adapter linking cytosolic detection of DNA to the TBK1 / IRF3 / type I IFN signaling axis. The signaling adapter 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). Initially characterized as a ubiquitous bacterial second messenger, CDN is now a pathogen-associated molecule that activates the TBK1 / IRF3 / type I IFN signaling axis through direct interaction with STING. It is known to constitute a class of pattern molecules (PAMPs). STING can sense aberrant DNA species and / or CDNs in the cytosol of cells, including CDNs derived from bacteria and / or CDNs derived from the host protein cyclic GMP-AMP synthase (cGAS). cGAS protein is a DNA sensor that produces cGAMP in response to 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 CDN, STING dimerizes, undergoes conformational changes, and promotes complex formation with TANK-binding kinase 1 (TBK1) (Ouyang et al., (2012) Immunity 36(6):1073 -1086). This complex translocates to the perinuclear Golgi 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 acts 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 CDN agonists of STING as vaccine adjuvants or immunomodulatory agents that can induce humoral and cellular immune responses (Dubensky et al., (2013) Ther Adv Vaccines 1(4):131-143 and references therein). In an earlier study, administration of the CDN c-di-GMP alleviated Staphylococcus aureus infection in vivo and reduced the number of bacterial cells recovered in a murine infection model, whereas in vitro, c-di-GMP GMP was shown to have no observable inhibitory or bactericidal effects on bacterial cells. This suggests that the decrease in bacterial cells is 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). Recent studies have shown that formulation of a synthetic CDN derivative molecule together with a granulocyte-macrophage colony-stimulating factor (GM-CSF)-producing cancer vaccine (termed STINGVAX) significantly improves cancer treatment compared to immunization with the GM-CSF vaccine alone. It has been shown to induce enhanced antitumor effects in vivo in animal models for human use (Fu et al., (2015) Sci Transl Med 7(283):283ra52). This suggests that CDN is a powerful vaccine adjuvant.

[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, the mutant STING allele was shown to potently stimulate the induction of type I IFN (Liu et al.,(2014)N Engl J Med 371:507-518;Jeremiah et al. 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 are modified mRNAs (mRNAs) encoding constitutively active forms of STING, including mutant human STING isoforms, for use as immunopotentiators as described herein. It is. MRNA encoding constitutively active forms of STING, including mutant human STING isoforms, are set forth in the sequence listing herein. The amino acid residue numbering of the mutant human STING polypeptides used herein is that used for the 379 amino acid residue wild-type human STING (isoform 1), available in the art as Genbank accession number NP_938023. correspond to what is happening.

[0180] Accordingly, in one aspect, the present 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 variant is encoded by the nucleotide sequence set forth in SEQ ID NO: 199. In one embodiment, the mmRNA comprises a 3'UTR sequence that includes the miR122 binding site shown in SEQ ID NO: 209.

[0181] In other aspects, the disclosure provides mmRNA encoding a mutant human STING protein 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 the R284T mutation and, for example, 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 the R284M mutation and, for example, 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 the R284K mutation and, for example, 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. Ru.

[0182] In other aspects, the disclosure provides mmRNA encoding a mutant human STING protein having a mutation, such as an amino acid substitution at amino acid residue 154, such as the N154S mutation. In certain embodiments, the mutant human STING protein has the N154S mutation and, for example, 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 other aspects, the present disclosure provides an mmRNA encoding a mutant human STING protein having a mutation, such as an amino acid substitution at amino acid residue 147, such as the V147L mutation. In certain embodiments, the mutant human STING protein having the 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 mmRNA encoding a mutant human STING protein having a mutation, such as an amino acid substitution at amino acid residue 315, such as the E315Q mutation. In certain embodiments, the mutant human STING protein with the 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 mmRNA encoding a mutant human STING protein having a mutation, such as an amino acid substitution at amino acid residue 375, such as the R375A mutation. In certain embodiments, the mutant human STING protein with the 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 disclosure provides mmRNA encoding a mutant human STING protein having one or more of the aforementioned mutations or a combination of two, three, four, or more. Accordingly, in one aspect, the present disclosure provides a mutant human STING having one or more mutations selected from the group consisting of V147L, N154S, V155M, R284T, R284M, R284K, E315Q and R375A, and combinations thereof. Provides mmRNA that encodes proteins. In other aspects, the present disclosure provides mutations having a combination of mutations selected from the group consisting of: V155M and R284T; V155M and R284M; V155M and R284K; V155M and V147L; provides mRNA encoding the type human STING protein.

[0187] In other aspects, the present disclosure provides 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. Provides mmRNA encoding. In other aspects, the disclosure provides mmRNA encoding a mutant human STING protein having V155M, V147L and N154S mutations. In other aspects, the disclosure provides an mmRNA encoding a mutant human STING protein having a V155M, V147L, N154S mutation and, optionally, a mutation at amino acid 284. In yet other aspects, the disclosure provides an mmRNA encoding a mutant human STING protein having a V155M, V147L, N154S mutation and a mutation at amino acid 284 selected from R284T, R284M and R284K. In other aspects, the disclosure provides mmRNA encoding mutant human STING proteins having V155M, V147L, N154S, and R284T mutations. In other aspects, the disclosure provides mmRNA encoding mutant human STING proteins having V155M, V147L, N154S, and R284M mutations. In other aspects, the disclosure provides mmRNA encoding mutant human STING proteins having V155M, V147L, N154S, and R284K mutations.

[0188] In other embodiments, the present disclosure provides combinations of mutations at amino acid residues 147, 154, 155 and optionally 284, particularly mutations having amino acid substitutions such as V147L, N154S, V155M, optionally R284M. provides mRNA encoding the type human STING protein. In certain embodiments, the mutant human STING protein has 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 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 a mutant human STING protein that is a constitutively active truncated form of a full-length 379 amino acid wild-type protein, such as a constitutively active human STING polypeptide consisting of amino acids 137-379. Provides mmRNA encoding.

[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 to pseudo-APCs. Antigen presentation is a critical step in the initiation, amplification, and duration of an immune response. In this process, antigen fragments are presented to T cells via the major histocompatibility complex (MHC) or human leukocyte antigens (HLA), thereby driving an antigen-specific immune response. For immunoprophylaxis and immunotherapy, enhancement of this response is important for improving efficacy. RNA vaccines of the invention may be designed or enhanced to promote efficient antigen presentation. One way to enhance processing and presentation by APCs is to improve the targeting of RNA vaccines to antigen presenting cells (APCs). Another approach involves activation of APC cells with immunostimulatory formulations and / or components.

[0191] Alternatively, methods for reprogramming non-APCs to become on the verge of becoming APCs may be used with the RNA vaccines of the invention. Importantly, most of the cells that take up mRNA preparations and are the targets of their therapeutic action are not APCs. Therefore, designing methods to convert these cells into APCs would be advantageous for efficacy. Provided herein are methods and techniques for delivering RNA vaccines, such as mRNA vaccines, to cells while also promoting a shift from non-APCs to APCs. In some embodiments, mRNA encoding an APC reprogramming molecule is included in or co-administered with an RNA vaccine.

[0192] APC reprogramming molecules, as used herein, are molecules that promote the transition of non-APC cells to an APC-like phenotype. The APC-like phenotype is a property that enables MHC class II processing. Therefore, APC cells with an APC-like phenotype have increased MHC class II processing capacity compared to identical cells without one or more exogenous molecules (APC reprogramming molecules). Cells that contain multiple exogenous molecules. In some embodiments, the APC reprogramming molecule is a chaperone protein such as CIITA (a central regulator of MHC class II expression), CLIP, HLA-DO, HLA-DM (an enhancer of antigen fragment loading into MHC class II). ), and / or costimulatory molecules (enhancers of T cell antigen recognition and T cell activation) such as CD40, CD80, CD86, etc.

[0193] CIITA proteins are transactivators that enhance transcriptional activation of MHC class II genes by interacting with a conserved set of DNA-binding proteins that associate with class II promoter regions (Steimle et al., 1993 ,Cell 75:135-146). The transcriptional activation function of CIITA is said to be located in the amino-terminal acidic domain (amino acids 26 to 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 CITTA 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 a full-length CIITA, CIP104, or other related molecule, or an active fragment thereof, where such active fragment has amino acids 26-137 of CIITA, or sequence identity thereto. amino acids 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 the subject in the form of mRNA encoding the APC reprogramming molecule. Accordingly, RNA vaccines of the invention may include 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 the one or more antigens is in a separate formulation 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 the subject the same number of times as the mRNA encoding the APC reprogramming molecule. In other embodiments, the mRNA encoding one or more antigens is administered to the subject a different number of times than the mRNA encoding the APC reprogramming molecule. For example, mRNA encoding an APC reprogramming molecule may be administered before mRNA encoding one or more antigens. The mRNA encoding the APC reprogramming molecule may be administered immediately before, at least 1 hour, at least 1 day, at least 1 week, or at least 1 month before the mRNA encoding the antigen.

[0195] Alternatively, mRNA encoding an APC reprogramming molecule may be administered after mRNA encoding one or more antigens. The mRNA encoding the APC reprogramming molecule may be administered immediately, at least 1 hour, at least 1 day, at least 1 week, or at least 1 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, an mRNA vaccine may include a recall antigen, sometimes referred to as a memory antigen. Recall antigens are antigens that an individual has encountered before and in which memory lymphocytes are already present. In some embodiments, the recall antigen can be an infectious disease antigen that the individual may have encountered, such as an influenza antigen. Recall antigens help promote a stronger immune response.

[0197] The antigen or neoepitope selected for inclusion in an mRNA vaccine will typically be a binding peptide with high affinity. In some embodiments, the antigen or neoepitope binds to the HLA protein with high affinity compared to the wild-type peptide. In some embodiments, the antigen or neoepitope has an IC50 of at least less than 5000 nM, at least less than 500 nM, at least less than 250 nM, at least less than 200 nM, at least less than 150 nM, at least less than 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 moderate to high affinity, and their affinity can be determined experimentally using biochemical assays of HLA binding. will be selected for testing. The cancer antigen can be a personalized cancer antigen. A personalized RNA cancer vaccine may, for example, contain RNA encoding one or more known cancer antigens that are specific to the respective subject's tumor or cancer antigens, such antigens being neoepitopic or subject-specific. called a target epitope or target-specific antigen. A "subject-specific cancer antigen" is an antigen that has been identified to be expressed on a particular patient's tumor. It is common that subject-specific cancer antigens typically may or may not be present in the tumor sample. It is not or rarely expressed in non-cancerous cells, or its expression in non-cancerous cells is markedly reduced compared to that in cancerous cells, and may interfere with the immune response induced upon vaccination. The inducing tumor-associated antigens are called neoepitopes. It is assumed that neoepitopes, such as tumor-associated antigens, are completely foreign to the body and therefore do not generate an immune response against healthy tissue or are masked by protective components of the immune system. In some embodiments, neoepitope-based personalized RNA cancer vaccines are desirable because such vaccine formulations will maximize specificity for patient-specific tumors. Neoepitopes resulting from mutations include point mutations, which are nonsynonymous mutations that result in a different amino acid in the protein, and modification or deletion of the stop codon, leading to the translation of a longer protein with a novel tumor-specific sequence at the C-terminus. Read-through mutations, splice site mutations that result in unique tumor-specific protein sequences due to the inclusion of introns in the mature mRNA, and chromosomal rearrangements (i.e., gene fusions that result in chimeric proteins with tumor-specific sequences at the junction of two proteins) ), frameshift mutations or deletions resulting in new open reading frames with novel tumor-specific protein sequences, and translocations. Accordingly, in some embodiments, the RNA cancer vaccine comprises at least one cancer antigen comprising a mutation selected from the group consisting of frameshift mutations and recombination or any of the other mutations described herein. .

[0198] Methods for producing personalized RNA cancer vaccines generally include identification of mutations using, for example, nucleic acid or protein deep sequencing techniques and, for example, validated peptide-MHC binding prediction algorithms, or which may bind to patient's HLA alleles; Identification of neoepitopes applying other analytical techniques to generate a set of candidate T cell epitopes based on mutations present in the tumor and optional targeting of selected neoepitopes by antigen-specific T cells. or the optional demonstration that a candidate neoepitope binds to an HLA protein present on the tumor surface, and the development of a vaccine. The RNA cancer vaccine of the present invention can contain multiple copies of a single neoepitope, multiple different neoepitopes based on a single type of mutation, i.e. point mutations, multiple different neoepitopes based on different variant types, tumor-associated Neoepitopes and other antigens such as antigens or recall antigens may be included.

[0199] Examples of mutation identification techniques include, but are not limited to, dynamic allele-specific hybridization (DASH), microplate array diagonal gel electrophoresis (MADGE), pyrosequencing, and oligonucleotides. Generating small signal molecules by specific ligation, the TaqMan system, and various DNA "chip" techniques, namely Affymetrix SNP chips, as well as invasive cleavage, followed by mass spectrometry, or immobilized padlock probes and rolling circle amplification. Includes methods based on implementation.

[0200] Nucleic acid or protein deep sequencing techniques are known in the art. Any type of array parsing method can be used. Nucleic acid sequencing may be performed on the whole tumor genome, tumor exome (protein-coding DNA), tumor transcriptome, or exosomes. Real-time, single-molecule-by-synthesis sequencing technology relies on the detection of fluorescent nucleotides each time they are incorporated into a nascent DNA strand that is complementary to the template being sequenced. Other rapid high-throughput sequencing methods exist. Protein sequencing may be performed on the tumor proteome. Additionally, protein mass spectrometry may 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 may be compared to a known control set or to sequencing analysis performed on normal tissue of the patient.

[0201] Accordingly, the present invention relates to methods for identifying and / or detecting neoepitopes of antigens, such as T cell epitopes. Specifically, the invention provides methods for identifying and / or detecting tumor-specific neoepitopes that are useful in inducing tumor-specific immune responses in a subject. Optionally, such neoepitopes have the ability to bind to class I HLA proteins with increased affinity compared to the wild-type peptide and / or to activate anti-tumor CD8 T cells. Identical mutations in any particular gene are rarely found throughout a tumor.

[0202] MHC class I proteins are present on the surface of almost every cell in the body, including most tumor cells. Antigens, usually originating from endogenous proteins or intracellular pathogens, are loaded onto MHC class I proteins, and these antigens are then presented 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 that has the ability to specifically bind to MHC / peptide complexes.

[0203] Using computer algorithms, it is possible to predict potential neoepitopes, such as T cell epitopes, i.e. those bound by class I or class II MHC molecules in the form of peptide-presenting complexes. In this form, the peptide sequence is recognized by the T cell receptor of T lymphocytes. 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 were used to refine the list of neoepitopes selected based on algorithmic predictions using routine in vitro assays performed in the laboratory, such as Elispot assays. good. Neoepitope vaccines, methods of their use and preparation are fully described in PCT / US2016 / 044918, which is incorporated herein by reference in its entirety.

[0205] Activating oncogene variant peptides selected for inclusion in RNA cancer vaccines are typically high affinity binding peptides. In some embodiments, the activating oncogene variant peptide binds to the HLA protein with high affinity compared to the wild type peptide. In some embodiments, the activating oncogene variant peptide has an IC50 of at least less than 5000 nM, at least less than 500 nM, at least less than 250 nM, at least less than 200 nM, at least less than 150 nM, at least less than 100 nM, at least less than 50 nM, or less. . Typically, peptides with a predicted IC50 of less than 50 nM are generally considered to be binding peptides with moderate to high affinity, and their affinity can be determined experimentally using biochemical assays of HLA binding. will be selected for testing.

[0206] In personalized cancer vaccines, subject-specific cancer antigens may be identified in a patient sample. For example, the sample can be a tissue sample or a tumor sample. For example, a sample of one or more tumor cells may be examined for the presence of a subject-specific cancer antigen. Tumor samples may be examined using whole genome, exome, or transcriptome analysis to identify subject-specific cancer antigens.

[0207] Alternatively, subject-specific cancer antigens may be identified in the subject's exosomes. Once an antigen for a vaccine is identified in a subject's exosomes, such antigen is said to be representative of the subject's exosomal antigens.

[0208] Exosomes are small microvesicles secreted by cells and typically have a diameter of approximately 30-100 nm. Exosomes are classically formed by inward invagination, and the last endosomal membrane is constricted and detached, resulting in the formation of multivesicular bodies (MVBs) containing many small vesicles with lipid bilayers. Each contains a sample of the cytoplasm of the parent cell. When MVBs fuse with the cell membrane, these exosomes are released from the cell and delivered into the blood, urine, cerebrospinal fluid, or other body fluids. Exosomes can be recovered from any of these biological fluids for further analysis.

[0209] Nucleic acids within exosomes have a role as biomarkers for tumor antigens. An advantage of exosome analysis to identify subject-specific cancer antigens is that this method eliminates the need to perform a biopsy. This can be particularly beneficial when a patient needs to undergo several rounds of treatment, including identification of cancer antigens 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. Examples of methods are described, for example, in US 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, the biological sample may suitably include bodily fluids derived from the subject. A body fluid can be a fluid isolated from any location in a subject's body, preferably a peripheral location, such as, but not limited to, blood, plasma, serum, urine, sputum, spinal fluid, brain fluid, etc. Spinal fluid, pleural effusion, nipple aspirate fluid, lymph fluid, fluid originating from the respiratory tract, intestinal tract, and genitourinary tract, lacrimal fluid, saliva, breast milk, fluid originating from the lymphatic system, semen, cerebrospinal fluid, fluid in internal organs, ascites, Includes tumor cyst fluid, amniotic fluid, and combinations thereof.

[0212] In some embodiments, cancer progression can be monitored to identify changes in expressed antigens. Accordingly, in some embodiments, the method further comprises identifying at least two cancer antigens from a sample of a subject to obtain a second set of cancer antigens; administration of an mRNA vaccine having an encoding open reading frame to the subject is performed at least one month after administration of the cancer mRNA vaccine. In some embodiments, the mRNA vaccine with open reading frames encoding the second set of antigens is administered 2 months, 3 months after the mRNA vaccine with open reading frames encoding the first set of cancer antigens. , administered to the subject after 4 months, 5 months, 6 months, 8 months, 10 months, or 1 year. In other embodiments, the mRNA vaccines having open reading frames encoding the second set of antigens are administered 1.5 years, 2 years later than the mRNA vaccines having open reading frames encoding the first set of cancer antigens. , administered to the subject after 2 1 / 2 years, 3 1 / 2 years, 3 1 / 2 years, 4 years, 4 1 / 2 years, or 5 years.

[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 "repeat" or "hotspot" mutations are often shown to have a "driver" role in tumors, causing some change in cancer cell functions that are important for tumor initiation, maintenance, or metastasis. Therefore, it is what tumors are selected for to evolve. In addition to their importance in tumor biology and therapy, recurrent mutations provide opportunities for precision medicine, where patient populations are stratified into groups that are more likely to respond to specific treatments. . These specific treatments include, but are not limited to, those that target the mutant protein itself.

[0214] Regarding recurrent mutations, much effort and research has focused on non-synonymous (or "missense") single nucleotide variants (SNVs), whereas synonymous (or "silent"), splice site, Population analyzes have revealed that a variety of highly complex (non-SNV) mutation categories, such as multiple nucleotide variants, insertions, and deletions, can also occur with high frequency.

[0215] The p53 gene (official symbol TP53) is mutated more frequently in human cancers than any other gene. For most mutations that occur in p53, the genomic location is unique to only one or a few patients, so these mutations cannot be used as repeating neoantigens for therapeutic vaccines designed for specific patient populations. was shown by a large-scale cohort study. Surprisingly, however, a small subset of the p53 locus does indeed show a pattern of "hot spots", where some positions of the gene are mutated with relatively high frequency. Notably, most of these recurrently mutated regions occur near exon-intron boundaries, disrupting standard nucleotide sequence motifs recognized by the mRNA splicing machinery. Mutations in splicing motifs can result in changes in the final mRNA sequence, even if no change in local amino acid sequence is predicted (ie, synonymous or intronic mutations). Therefore, even though these mutations can alter mRNA splicing in an unpredictable manner and have significant functional effects on the translated protein, they are classified as "non-" by common annotation tools. It is often annotated as "coding" and ignored without further analysis. If alternatively spliced ​​isoforms undergo in-frame sequence changes (i.e., no PTC occurs), these isoforms can escape elimination by NMD and are rapidly expressed and processed. and is presented on the cell surface by the HLA system. Furthermore, alternative splicing resulting from mutations is usually "cryptic", ie, not expressed in normal tissues, and therefore can be recognized by T cells as non-self neoantigens.

[0216] In some embodiments, the invention provides neoantigenic peptide sequences derived from certain recurrent somatic cancer mutations in p53 (including, but not limited to, missense SNVs) that often result in alternative splicing; These neoantigenic peptide sequences are intended for use as targets for therapeutic vaccination. In some embodiments, mutations that result in neoantigenic peptides and / or HLA-restricted epitopes in mRNA splicing events include mutations in the canonical 5' splice site adjacent to codon position T125, including epitope AVSPCISFVW (sequence Number 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) containing the peptide sequence TAKSVTCTVSCPEGLASMRLQCLAVSPCISFVWNFGIPLHPLASCQCFFIVYPLNV (SEQ ID NO: 232).

[0217] In some embodiments, mutations that result in neoantigenic peptides and / or HLA-restricted epitopes in mRNA splicing events include mutations in the canonical 5' splice site adjacent to codon position 331, including epitope LQVLSLGTSY (sequence Number 237)(HLA-B * 15:01), epitope FQSNTQNAVF (SEQ ID NO: 238) (HLA-B * 15:01) containing the peptide sequence EYFTLQVLSLGTSYQVESFQSNTQNAVFFLTVLPAIGAFAIRGQ (SEQ ID NO: 236).

[0218] In some embodiments, mutations that result in neoantigenic peptides and / or HLA-restricted epitopes in mRNA splicing events include mutations in the canonical 3' splice site adjacent to codon position 126, including mutations in the canonical 3' splice site adjacent to codon position 126, including epitope CTMFCQLAK (sequence Number 240)(HLA-A * 11:01), epitope KSVTCTMF (SEQ ID NO: 241) (HLA-B * A mutation is included that induces a potential alternative exon 3' splice site creating a novel spanning peptide sequence AKSVTCTMFCQLAK (SEQ ID NO: 239) containing 58:01).

[0219] In some embodiments, mutations that result in neoantigenic peptides and / or HLA-restricted epitopes in mRNA splicing events include mutations in the canonical 5' splice site adjacent to codon position 224, including epitope VPYEPPEVW (sequence Number 243)(HLA-B * 53:01, HLA-B * 51:01), epitope LTVPSTAW (SEQ ID NO: 244) (HLA-B * 58:01, HLA-B * A mutation is included that induces a potential alternative intronic 5' splice site creating a novel spanning peptide sequence VPYEPPEVWLALTVPPSTAWAA (SEQ ID NO: 242) containing 57:01).

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

[0221] Mutations to obtain neoepitopes of prior art peptide vaccines are typically obtained from patient DNA sequencing data. On the other hand, mRNA expression is a more direct measure of the global space of possible neoepitopes. For example, some tumor-specific neoepitopes may arise from splicing alterations, insertions / deletions resulting in frameshifts (InDels), alternative promoters, or epigenetic modifications, which are not easily achieved using only exome sequencing data. is not identified. For neoantigen vaccines, identifying these complex variant types is of potential value as it increases the number of epitopes that can bind to patient-specific HLA allotypes. Furthermore, complex variants are more immunogenic and may result in more effective immune responses against tumors due to their differences from self-proteins compared to variants resulting from single amino acid changes.

[0222] In some embodiments, the invention involves methods of identifying complex patient-specific mutations and formulating these mutations into effective personalized mRNA vaccines. The method involves the use of short read RNA sequencing. The main 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 through alternative splicing and other mechanisms. Sequencing reads are significantly shorter than full-length mRNA transcripts, making it difficult to map read sets to their correct corresponding isoforms within known gene annotation models. As a result, complex variants that deviate from known gene annotations (common in cancer) can be difficult to discover with standard approaches. However, the present disclosure involves the identification of short peptides rather than the exact exon composition of the full-length transcript. A method to identify short peptides representative of these complex variants 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 can analyze 50 codons, or 41 different peptide epitopes of length 9 (27 nucleotides) when capturing a coding region. . Therefore, we used a simple, computationally scalable operation to count all 27-mers from an RNA-sequencing sample, and the results were obtained for normal tissue derived from the same sample or from RNA-sequencing of normal tissue. Comparisons can be made against a 27-mer pre-computed database (eg GTEx).

[0224] An mRNA vaccine containing predicted neoepitopes from RNA-sequencing data is determined by 1) counting all possible 27-mers from all RNA-sequencing reads of a tumor sample and 2) open reading frames of each read. 3) by aligning the transcriptome with any portion of its entire read; and 3) predicting the 27-mer counts in a database of matched normal samples and / or normal tissues derived from the same tissue type. Add confidence to neoepitope predictions by comparing with corresponding 27-mer counts and 4) in the presence of somatic mutations found in the same gene using DNA sequencing data from the same tumor. It can be created by. Regarding point (4), mutations can often lead to transcriptional or splicing changes, resulting in changes in the mRNA sequence that cannot be predicted directly from the mutation itself. For example, splice site mutations can be expected to cause exon skipping, but it cannot be known with certainty which downstream exon will be selected by the local splicing machinery.

[0225] In one embodiment, the invention provides an mRNA vaccine comprising a concatemeric polyepitope construct or a set of individual epitope constructs comprising an open reading frame (ORF) encoding neoantigenic peptides 1-4.

[0226] In one embodiment, the invention provides selective administration of a vaccine comprising or encoding peptides 1-4 based on whether the patient's tumor contains any of the above mutations.

[0227] In one embodiment, the invention determines whether 1) the patient's tumor contains any of the above mutations, and 2) the corresponding HLA allele that the patient's normal HLA type is predicted to bind to the resulting neoantigen. Provide selective administration of vaccines based on the dual criteria of whether the vaccine contains the following:

[0228] It has been discovered that the mRNA vaccines described herein have several advantages compared to current vaccines. First, delivery by lipid nanoparticles (LNPs) is superior to other formulations, including liposome- or protamine-based approaches, described in the literature and does not require additional adjuvants. The use of LNPs allows 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 invention are at least 10 times, at least 20 times, at least 40 times, at least 50 times, at least 100 times, at least 500 times, or at least 1,000 times more effective than conventional vaccines. Are better.

[0229] Although many attempts have been made to generate functional RNA vaccines, including mRNA vaccines and self-replicating RNA vaccines, the therapeutic efficacy of these RNA vaccines is still not fully established. Quite surprisingly, the delivery of mRNA vaccines in vivo elicits significantly enhanced and in many ways synergistic immune responses, including enhanced antigen production and production of functional antibodies with neutralizing capacity. In accordance with aspects of the present invention, the inventors have discovered a class of formulations for. These results can be achieved even when significantly lower doses of mRNA are administered compared to those used in other classes of lipid-based formulations. The formulations of the invention were unexpectedly shown to elicit a significant immune response in vivo, sufficient to establish the effectiveness of a functional mRNA vaccine as a prophylactic and therapeutic agent. Additionally, self-replicating RNA vaccines utilize the viral replication pathway to deliver enough RNA to cells to generate an immune response. The formulations of the invention produce sufficient protein to elicit a strong immune response without the need for viral replication. Therefore, the mRNA of the present invention is not self-replicating RNA and does not contain elements necessary for viral replication.

[0230] In some embodiments, the present invention includes the surprising finding 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 inducing an enhanced immune response, the formulations of the invention generate an immune response more rapidly with 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 formulation of the present invention is superior to other vaccines even when the dose of mRNA is low compared to other vaccines.

[0231] The LNPs used in the studies described herein have been previously used for the delivery of siRNA in various animal models as well as in humans. In view of the findings obtained in connection with the delivery of siRNA by LNP formulations, the fact that LNPs are useful in vaccines is quite surprising. Therapeutic delivery of siRNA formulated in LNPs typically results in decreased antigen production and impaired immune responses as a result of transient IgM responses and associated undesirable inflammatory responses. It has been observed. In contrast to the findings observed with siRNA, it is demonstrated herein that the LNP-mRNA formulations of the present invention do not result in a transient IgM response, but rather an increase in IgG levels sufficient for prophylactic and therapeutic methods. is shown.

[0232] Nucleic acid / polynucleotide Cancer vaccines provided herein include at least one (or more than one) ribonucleic acid (RNA) polynucleotide 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 that includes a polymer of nucleotides. Such polymers are called polynucleotides.

[0233] Nucleic acids (also referred to as polynucleotides) include, for example, ribonucleic acids (RNA), deoxyribonucleic acids (DNA), threose nucleic acids (TNA), glycol nucleic acids (GNA), peptide nucleic acids (PNA), locked nucleic acids (β-D- LNA with ribo configuration, α-LNA with α-L-ribo configuration (diastereomers 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) encodes (at least one) polypeptide (an amino acid polymer of natural origin, an amino acid polymer of non-natural origin, or a modified amino acid polymer) and is translated into Refers to any polynucleotide from which a polypeptide can be produced in vitro, in vivo, in situ, or ex vivo.

[0235] The basic components of an mRNA molecule typically include at least one coding region, a 5' untranslated region (UTR), a 3' UTR, a 5' cap, and a poly A tail. Although the polynucleotides of the present disclosure can function as mRNA, they have functional and / or structural design features that help overcome the existing problems of efficiently expressing polypeptides using nucleic acid-based therapeutics. It can be distinguished from wild-type mRNA in that it has

[0236] In some embodiments, the cancer vaccine RNA polynucleotides are 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 cancer vaccine RNA polynucleotide encodes at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 antigen polypeptides. In some embodiments, the cancer vaccine RNA polynucleotide encodes at least 100 or at least 200 antigen polypeptides. In some embodiments, the cancer vaccine RNA polynucleotides are 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 antigens encodes a polypeptide.

[0237] In some embodiments, the cancer vaccine RNA polynucleotides are 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 activating oncogene mutant peptides. In some embodiments, the cancer vaccine RNA polynucleotide encodes at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 activating oncogene variant peptides. In some embodiments, the cancer vaccine RNA polynucleotide encodes at least 100 or at least 200 activating oncogene variant peptides. In some embodiments, the cancer vaccine RNA polynucleotides are 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 activity Encodes a mutated oncogene peptide.

[0238] In some embodiments, 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, matching the target codon frequency to that of the host organism to maintain proper folding, biasing GC content to increase mRNA stability or reduce secondary structure. minimization of tandem repeat codons and base sequences that could impair gene assembly or expression; customization of transcriptional and translational control regions; insertion or removal of protein transport sequences; addition, removal, or shuffling of protein domains; insertion or removal of restriction enzyme sites; modification of ribosome binding sites and mRNA degradation sites; to enable proper folding of the various domains of the protein. Codon optimization may be used to modulate the rate of translation of a polynucleotide, or to reduce or eliminate problematic secondary structure within a polynucleotide. Codon optimization tools, algorithms, and services are known in the art and include, but are not limited to, the services provided 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 is a naturally occurring or wild-type sequence (e.g., a naturally occurring or wild-type sequence that encodes a polypeptide or protein of interest (e.g., an antigenic protein or polypeptide)). share less than 95% sequence identity with the mRNA sequence). In some embodiments, the codon-optimized sequence is a naturally occurring or wild-type sequence (e.g., a naturally occurring or wild-type sequence that encodes a polypeptide or protein of interest (e.g., an antigenic protein or polypeptide)). share less than 90% sequence identity with the mRNA sequence). In some embodiments, the codon-optimized sequence is a naturally occurring or wild-type sequence (e.g., a naturally occurring or wild-type sequence that encodes a polypeptide or protein of interest (e.g., an antigenic protein or polypeptide)). share less than 85% sequence identity with the mRNA sequence). In some embodiments, the codon-optimized sequence is a naturally occurring or wild-type sequence (e.g., a naturally occurring or wild-type sequence that encodes a polypeptide or protein of interest (e.g., an antigenic protein or polypeptide)). share less than 80% sequence identity with the mRNA sequence). In some embodiments, the codon-optimized sequence is a naturally occurring or wild-type sequence (e.g., a naturally occurring or wild-type sequence that encodes a polypeptide or protein of interest (e.g., an antigenic protein or polypeptide)). share less than 75% sequence identity with the mRNA sequence).

[0240] In some embodiments, the codon-optimized sequence is a naturally occurring or wild-type sequence (e.g., a naturally occurring or wild-type sequence that encodes a polypeptide or protein of interest (e.g., an antigenic protein or polypeptide)). share 65% to 85% (eg, about 67% to about 85%, or about 67% to about 80%) sequence identity with the mRNA sequence). In some embodiments, the codon-optimized sequence is a naturally occurring or wild-type sequence (e.g., a naturally occurring or wild-type sequence that encodes a polypeptide or protein of interest (e.g., an antigenic protein or polypeptide)). It shares 65% to 75% or about 80% sequence identity with the mRNA sequence).

[0241] In some embodiments, the codon-optimized RNA may have, for example, enhanced levels 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 is functional compared to nucleic acids containing large amounts of adenine (A) and thymine (T) or uracil (U) nucleotides. may be more stable. WO02 / 098443 discloses a pharmaceutical composition containing mRNA stabilized by sequence modification of the translation region. Because the genetic code is degenerate, modifications work by replacing existing codons with codons that promote increased RNA stability without changing the resulting amino acids. This technique is not limited to RNA coding regions.

[0242] Antigen / antigen polypeptide In some embodiments, a cancer polypeptide (eg, an activated oncogene variant peptide) is longer than 5 amino acids and shorter than 50 amino acids. In some embodiments, the 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. A polypeptide can be a single molecule or a complex of multiple molecules such as a dimer, trimer, or tetramer. Polypeptides can also include single-chain polypeptides or multi-chain polypeptides, such as antibodies or insulin, and the polypeptides can be associated or linked. Most commonly, disulfide bonds are found in multi-chain polypeptides. The term polypeptide can also be applied to amino acid polymers in which at least one amino acid residue is an artificial chemical analog of the corresponding naturally occurring amino acid.

[0243] The term "polypeptide variant" refers to a molecule that differs in its amino acid sequence 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 as compared to a native or reference sequence. Typically, variants have at least 50% identity to the native or reference sequence. In some embodiments, the variant shares 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 mimetic" is one that contains at least one amino acid that is assumed to mimic the activation sequence. For example, glutamate can act as a phosphoro-threonine and / or phosphoro-serine mimetic. Alternatively, a mutant mimetic can be one that loses activity or is an inactivated product containing the mimetic, for example, phenylalanine can serve as an inactivating substitution for tyrosine, or alanine can act as an inactivating replacement for serine. Can act as an inactivating substitution.

[0245] "Ortholog" refers to a gene in different species that has evolved from a common ancestral gene through speciation. Orthologs usually retain the same function during evolution. Identification of orthologs is critical for performing reliable gene function predictions in newly sequenced genomes.

[0246] "Analogs" are intended to include polypeptide variants that differ by one or more amino acid changes, such amino acid changes e.g. Substitutions, additions, or deletions of amino acid residues that still remain.

[0247] The present disclosure provides several types of compositions based on polynucleotides or polypeptides, including variants and derivatives. These include, for example, variants and derivatives having substitutions, insertions, deletions, and covalent bonds. The term "derivative" is used interchangeably with the term "variant", but generally refers to a molecule that is modified and / or altered in any manner compared to a reference or starting molecule.

[0248] Accordingly, polynucleotides encoding peptides or polypeptides that contain substitutions, insertions, and / or additions, deletions, and covalent modifications with respect to the reference sequence, specifically the polypeptide sequences disclosed herein, Included within the scope of disclosure. For example, a sequence tag or an amino acid such as one or more lysines can be added to a peptide sequence (eg, at the N-terminus or C-terminus). Sequence tags can be used to detect, purify, or localize peptides. Lysine can be used to improve peptide solubility or to achieve biotin labeling. Alternatively, a truncated sequence may be created by optionally deleting amino acid residues located in the carboxy-terminal and amino-terminal regions of the amino acid sequence of the peptide or protein. Depending on the use of the sequence, e.g. expression of the sequence as part of a soluble longer sequence or expression of the sequence as part of a longer sequence linked to a solid support, certain amino acids (e.g. The C-terminal residue or the N-terminal residue) may alternatively be deleted.

[0249] When referring to a polypeptide, a "substitutional variant" is one in which at least one amino acid residue in the native or starting sequence has been removed and a different amino acid has been inserted at the same position where it was present. Substitutions can be single, where only one amino acid in the molecule is replaced, or substitutions can be multiple, where two or more amino acids are replaced in the same molecule.

[0250] As used herein, the term "conservative amino acid substitution" refers to the substitution of an amino acid normally occurring in a sequence with a different amino acid of similar size, charge, or polarity. Examples of conservative substitutions include substitution of a non-polar (hydrophobic) residue such as isoleucine, valine, and leucine for another non-polar residue. Similarly, examples of conservative substitutions include replacing one polar (hydrophilic) residue with another, such as replacing arginine with lysine, replacing glutamine with asparagine, and replacing glycine with serine. is included. Additionally, the substitution of one 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, is an additional conservative substitution. This is an example. Examples of non-conservative substitutions include non-polar (hydrophobic) amino acid residues such as isoleucine, valine, leucine, alanine, methionine, and polar (hydrophilic) residues such as cysteine, glutamine, glutamic acid, or lysine; and / or substitution of polar residues with non-polar residues.

[0251] When referring to polypeptides or polynucleotides, "features" are defined as molecular components based on different amino acid sequences or different nucleotides, respectively. Characteristics of polypeptides encoded by polynucleotides include surface appearances, local conformational shapes, folds, loops, half-loops, domains, half-domains, regions, termini, or any combination thereof.

[0252] When referring to a polypeptide, the term "domain" as used herein refers to one or more identifiable structural or functional characteristics or properties (e.g., as a site for protein-protein interaction). refers to a motif of a polypeptide that has the ability to function (binding ability).

[0253] When referring to a polypeptide, the term "site" as used herein with respect to amino acid-based embodiments is used interchangeably with "amino acid residue" and "amino acid side chain." When referring to a polynucleotide, the term "site" as used herein with respect to nucleotide-based embodiments is used interchangeably with "nucleotide." A site corresponds to a position within a peptide or within a polypeptide or within a polynucleotide that can be modified, manipulated, altered, derivatized, or altered within a polypeptide or polynucleotide-based molecule.

[0254] When referring to a polypeptide or polynucleotide, the term "termini" or "terminus" as used herein refers to the terminus of the polypeptide or polynucleotide, respectively. Such terminus is not limited to only the first or last position of the polypeptide or polynucleotide, but may additionally include amino acids or nucleotides of the terminal region. Molecules based on polypeptides have an N-terminus (free amino group (NH 2 ) 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 held together by disulfide bonds or non-covalent forces (multimers, oligomers). These proteins have multiple N-termini and C-termini. Alternatively, the terminus of the polypeptide may be modified, optionally starting from or terminating with a non-polypeptide-based moiety, such as an organic complex.

[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 of a reference protein that is greater than 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or 100 amino acids in length (amino acid residues compared to the reference polypeptide sequence) provided herein are polypeptide sequences that are identical except that they are shorter by at least one less. In another example, 10, 20, 30 have 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% identity to any of the sequences described herein. , 40, 50, or 100 amino acids can be utilized in accordance with the present disclosure. In some embodiments, the polypeptide has two, three, four, five, six, seven, eight, or more mutations set forth in any of the sequences provided or referenced herein. Contains 9, 10, or 11 or more. In another example, a stretch of 20, 30, 40, 50, or 100 amino acids that has greater than 80%, greater than 90%, greater than 95%, or 100% identity to any of the sequences described herein. has less than 80%, less than 75%, less than 70%, less than 65%, or less than 60% identity to any of the sequences described herein. Proteins having stretches of , 20, 25, or 30 amino acids can be utilized in accordance with the present disclosure.

[0256] A polypeptide or polynucleotide molecule of the present disclosure may be a reference molecule (e.g., a reference polypeptide or a reference polynucleotide), such as a molecule described in the art (e.g., an engineered or designed molecule or a wild-type molecule). ) may share some sequence similarity or identity with The term "identity" as known in the art refers to the relatedness between the sequences of two or more polypeptides or polynucleotides, such relatedness determined by sequence comparison. In the art, identity also refers to the degree of sequence relatedness between them, with such degree of relatedness determined by the number of matches between two or more amino acid residues or strings of nucleic acid residues. Identity is a measure of the percent identity between two sequences with fewer gaps (if any) in the alignment performed by a particular mathematical model or computer program (eg, an "algorithm"). The identity of related peptides can be easily calculated by known methods. "% Identity" applied to a polypeptide or polynucleotide sequence refers to the candidate amino acid sequence or candidate nucleic acid sequence after alignment of the sequences and introduction of gaps, if necessary, have been performed to achieve the maximum percent identity. It is defined as the percentage of residues (amino acid residues or nucleic acid residues) in a sequence that are identical to residues in the amino acid or nucleic acid sequence of a second sequence. Methods and computer programs for alignment purposes are well known in the art. It is understood that identity depends on the calculation of percent identity, but the value can vary due to gaps and penalties introduced in the calculation. Generally, a variant of a particular polynucleotide or polypeptide is at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65% relative to a particular reference polynucleotide or polypeptide. , 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%, at least 99%, but less than 100% sequence identity, 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) is included. 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 general 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.). The Fast Optimal Global Sequence Alignment Algorithm (FOGSAA) has been developed very recently, which is said to perform fast global alignment of nucleotide and protein sequences compared to other optimal global alignment methods, including the Needleman-Wunsch algorithm. Tools are described elsewhere herein, particularly in the definition of "identity" below.

[0257] The term "homology" as used herein refers to an overall relationship between polymeric molecules such as, for example, nucleic acid molecules (eg, DNA and / or RNA molecules) and / or polypeptide molecules. Polymer molecules (e.g., nucleic acid molecules (e.g., DNA and / or RNA molecules) and / or polypeptide molecules) that share acceptable limits of similarity or identity as determined by alignment to find residue matches are called homologous. Homology is a qualitative term that describes the relationship 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, the polymer molecules have 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% of their sequence. are considered "homologous" to each other if they are 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 or polypeptide sequences). at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 99% for at least one stretch in which the polypeptides encoded by the two polynucleotide sequences contain at least 20 amino acids Two polynucleotide sequences are considered homologous if even % are the same. In some embodiments, homologous polynucleotide sequences are characterized by the 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 the ability to encode a stretch containing at least 4-5 uniquely specified amino acids. Two protein sequences are homologous if the proteins are at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identical for at least one stretch containing at least 20 amino acids. it is conceivable that.

[0258] Homology implies that the sequences being compared 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 protein sequence) that is related to a second amino acid or nucleic acid sequence by descent from a common ancestral sequence. Point. The term "homolog" can also apply to relationships between genes and / or proteins separated by events of speciation, or relationships between genes and / or proteins separated by events of gene duplication. "Orthologs" are genes (or proteins) in different species that have evolved from a common ancestral gene (or protein) through speciation. Typically, orthologs retain the same function during evolution. "Paralogs" are genes (or proteins) that are related by duplication within the genome. Orthologs retain the same function over the course of evolution, while paralogs develop new functions even though a relationship to the original one exists.

[0259] The term "identity" refers to the overall relatedness between polymeric molecules such as, for example, polynucleotide molecules (eg, DNA 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 for the purpose of optimizing the comparison (e.g. Gaps can be introduced in one or both of the sequence and the second nucleic acid sequence, and non-identical sequences can be ignored for comparison purposes). In certain embodiments, the length of the sequences aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, of the length of the reference sequence. At least 90%, at least 95%, or 100%. Nucleotides at corresponding nucleotide positions are then compared. Molecules are identical at a position in a first sequence when that position is occupied by the same nucleotide as that at the corresponding position in the second sequence. The percent identity between two sequences is a function of the number of identical positions the sequences share, taking into account the number of gaps that need to be introduced and the length of each gap so that the alignment of the two sequences is optimal. becomes. Sequence comparison and determination of percent identity between two sequences can be accomplished using mathematical algorithms. For example, 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 Percent identity between two nucleic acid sequences can be determined using methods such as those described in Devereux, J., eds., M Stockton Press, New York, 1991; , 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 used in the ALIGN program (version 2.0). A PAM120 weighted residue table, a gap length penalty of 12, and a gap penalty of 4 are used. Alternatively, the percent identity between two nucleic acid sequences can be determined using the GAP program of the GCG software package using the NWSgapdna.CMP matrix. Methods commonly used to determine 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 documents are incorporated herein by reference. Identity determination techniques 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, an RNA (e.g., mRNA) vaccine of the present disclosure comprises at least one ribonucleic acid (RNA) polypeptide having an open reading frame encoding at least one respiratory polyhedrovirus (RSV) antigen polypeptide. The RNA contains at least one chemical modification.

[0261] The terms "chemical modification" and "chemically modified" refer to ribonucleosides or deoxyribonucleosides containing adenosine (A), guanosine (G), uridine (U), thymidine (T), or cytidine (C). , refers to a modification in at least one of its location, pattern, percentage, or population. Generally, such terms do not refer to ribonucleotide modifications in the cap portion that naturally occurs at the 5' end of the mRNA.

[0262] Modifications of polynucleotides include, but are not limited to, those described herein, and include modifications including, but not specifically limited to, chemical modifications. A polynucleotide (e.g., an RNA polynucleotide, such as an mRNA polynucleotide) may contain modifications of naturally occurring, non-naturally occurring origin, or a polynucleotide may contain a combination of naturally occurring and non-naturally occurring modifications. . A polynucleotide can include any useful modification of, for example, a sugar, nucleobase, or internucleoside linkage (eg, to a linked phosphate, phosphodiester linkage, or phosphodiester backbone).

[0263] The term "modification" with respect to polypeptides refers to modifications 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 combination of substitutions and insertions.

[0264] In some embodiments, a polynucleotide (eg, an RNA polynucleotide, such as an mRNA polynucleotide) comprises a variety of different modifications. In some embodiments, a particular region of a polynucleotide comprises one, two, or more (optionally different) nucleoside or nucleotide modifications. In some embodiments, a modified RNA polynucleotide (eg, a modified mRNA polynucleotide) that is introduced into a cell or organism exhibits reduced degradation in the cell or organism, respectively, as compared to an unmodified polynucleotide. In some embodiments, modified RNA polynucleotides (eg, modified mRNA polynucleotides) introduced into a cell or organism may exhibit reduced immunogenicity (eg, reduced innate response) in the cell or organism, respectively.

[0265] In some embodiments, polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) include non-naturally modified nucleotides that are introduced during or after synthesis of the polynucleotide to achieve a desired function or property. include. Modifications may be present in internucleotide linkages, purine or pyrimidine bases, or sugars. Modifications may be introduced at the end of the chain or anywhere else on the chain using chemical synthesis or polymerase enzymes. Any region of a polynucleotide may be chemically modified.

[0266] In some embodiments, a polynucleotide (eg, RNA, eg, mRNA) of the invention comprises a chemically modified nucleobase. The invention includes modified polynucleotides, including polynucleotides described herein (eg, polynucleotides comprising nucleotide sequences encoding one or more cancer epitope polypeptides). A modified polynucleotide may be chemically modified and / or structurally modified. When a polynucleotide of the invention is chemically and / or structurally modified, the polynucleotide may be referred to as a "modified polynucleotide."

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

[0268] Modified polynucleotides disclosed herein can contain a variety of different modifications. In some embodiments, a modified polynucleotide contains one, two, or more (optionally different) nucleoside or nucleotide modifications. In some embodiments, a modified polynucleotide that is introduced into a cell has one or more desired properties compared to an unmodified polynucleotide, such as improved protein expression, decreased immunogenicity, or can show reduced degradation in

[0269] In some embodiments, a polynucleotide of the invention (eg, a polynucleotide comprising a nucleotide sequence encoding one or more cancer epitope polypeptides) is structurally modified. As used herein, "structural" modifications include insertions, deletions, duplications, inversions, etc. of two or more linked nucleosides in a polynucleotide without significant chemical modification of the nucleotides themselves. or is a randomized modification. Structural modifications are chemical in nature and are therefore chemical modifications, since chemical bonds are necessarily broken and re-established to bring about the structural modification. However, structural modifications result in different nucleotide sequences. 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, a structural modification has occurred in the polynucleotide as a result of the insertion of the dinucleotide "CC".

[0270] In some embodiments, polynucleotides of the invention are chemically modified. As used herein with respect to polynucleotides, the term "chemically modified" or, as appropriate, "chemically modified" refers to adenosine (A), guanosine (G), uridine (U), or cytidine (C). Refers to a modification in one or more of the positions, patterns, percentages or populations of ribonucleosides or deoxyribonucleosides that include. Generally, as used herein, such terms are not intended to refer to ribonucleotide modifications in the naturally occurring cap portion of the 5' end of the mRNA.

[0271] In some embodiments, polynucleotides of the invention are produced by uniform chemical modification of all or any of the same nucleoside types, or by simple downward titration of the same starting modifications on all or any of the same nucleoside types. population of modifications, or all arbitrary chemical modifications of the same nucleoside type with random incorporation (such as when all uridines are replaced by uridine analogues, e.g. pseudouridine or 5-methoxyuridine). obtain. In another embodiment, a polynucleotide may 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, etc.). ).

[0272] Modified nucleotide base pairing refers not only to the 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. Also encompasses pairings, by arrangement of a hydrogen bond donor and a hydrogen bond acceptor, between a non-standard base and a standard base, or between two complementary non-standards, such as in a polynucleotide with at least one chemical modification. Hydrogen bonding between base structures becomes possible. One example of such pairing of non-standard bases is the base pairing between the modified nucleotides inosine and adenine, cytosine, or uracil. Any combination of bases / sugars or linkers may be incorporated into the polynucleotides of the present disclosure.

[0273] Those skilled in the art will appreciate that, unless otherwise specified, the polynucleotide sequences described in this application list a "T" in a representative DNA sequence; will recognize that it is replaced by "U".

[0274] Modifications of polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) useful in the compositions, methods, and synthetic processes of the present disclosure, including but not limited to chemical modifications, include, but are not limited to, the following: Includes: Uniform nucleotides, nucleosides, and nucleobases: 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-methyladenosine, 2-methylthio-N6-threonylcarbamoyl Adenosine, 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, 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'-a-aminoadenosine TP, 2'-deoxy-2'- a-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'-a-ethynyladenosine TP, 2-aminoadenine, 2-aminoadenosine TP, 2-amino-ATP, 2'-a-trifluoromethyladenosine TP, 2-azidoadenosine TP, 2'-b-ethynyladenosine TP, 2-bromoadenosine TP, 2'-b-trifluoromethyladenosine TP, 2-chloroadenosine TP, 2'-deoxy-2',2'-difluoroadenosine TP, 2'-deoxy-2'-a- Mercaptoadenosine TP, 2'-deoxy-2'-a-thiomethoxyadenosine TP, 2'-deoxy-2'-b-aminoadenosine TP, 2'-deoxy-2'-b-azidoadenosine TP, 2'- Deoxy-2'-b-bromoadenosine TP, 2'-deoxy-2'-b-chloroadenosine TP, 2'-deoxy-2'-b-fluoroadenosine TP, 2'-deoxy-2'-b-iodo Adenosine 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 TP, 3-deazaadenosine TP, 4'-azidoadenosine TP, 4'-carbocyclic adenosine TP, 4'-ethynyladenosine TP, 5'-homo-adenosine TP, 8- Aza-ATP, 8-bromo-adenosine TP, 8-trifluoromethyladenosine 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-dimethyl Cytidine, 5-formyl-2'-O-methylcytidine, lycidine, 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'-a-aminocytidine TP, 2'-deoxy-2'-a-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, 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'-a-mercaptocytidine TP, 2'-deoxy-2'-a-thiomethoxycytidine TP, 2'-deoxy-2'-b-aminocytidine TP, 2'-deoxy-2' -b-azidocytidine TP, 2'-deoxy-2'-b-bromocytidine TP, 2'-deoxy-2'-b-chlorocytidine TP, 2'-deoxy-2'-b-fluorocytidine TP, 2' -deoxy-2'-b-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-CTP, 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-methylguanosine, 2'-O-methylguanosine, 2'-O-ribosylguanosine (phosphate), 2'-O-methylguanosine, 2'-O-ribosylguanosine (phosphate), 7-aminomethyl-7-deazaguanosine, 7-cyano-7-deazaguanosine, alkaeosin, methylwaiosin , 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'-a-aminoguanosine TP, 2'-deoxy-2'-a-azide Guanosine TP, 6(methyl)guanine, 6-(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, 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-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'- a-mercaptoguanosine TP, 2'-deoxy-2'-a-thiomethoxyguanosine TP, 2'-deoxy-2'-b-aminoguanosine TP, 2'-deoxy-2'-b-azidoguanosine TP, 2 '-deoxy-2'-b-bromoguanosine TP, 2'-deoxy-2'-b-chloroguanosine TP, 2'-deoxy-2'-b-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'-homo-guanosine TP, 8-bromo-guanosine TP, 9-deazaguanosine TP, N2-isobutyl-guanosine TP, 1-methylinosine, inosine, 1,2'- O-dimethylinosine, 2'-O-methylinosine, 7-methylinosine, 2'-O-methylinosine, epoxycuosine, galactosyl-cuosine, mannosylcuosine, cuosine, allyamino-thymidine, azathymidine, Deazathymidine, deoxy-thymidine, 2'-O-methyluridine, 2-thiouridine, 3-methyluridine, 5-carboxymethyluridine, 5-hydroxyuridine, 5-methyluridine, 5-taurinomethyl-2-thiouridine, 5-tauridine Nomethyluridine, 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-carboxy Hydroxymethyluridine, 5-carboxyhydroxymethyluridine methyl ester, 5-carboxymethylaminomethyl-2'-O-methyluridine, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyl-2-thiouridine, 5-Carboxymethylaminomethyluridine, 5-carboxymethylaminomethyluridine, 5-carbamoylmethyluridine TP, 5-methoxycarbonylmethyl-2'-O-methyluridine, 5-methoxycarbonylmethyl-2-thiouridine, 5-methoxy Carbonylmethyluridine, 5-methyluridine, ), 5-methoxyuridine, 5-methyl-2-thiouridine, 5-methylaminomethyl-2-selenouridine, 5-methylaminomethyl-2-thiouridine, 5-methylaminomethyl Uridine, 5-methyldihydrouridine, 5-oxyacetic acid-uridine TP, 5-oxyacetic acid-methyl ester-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)-2,4-(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-(aminoalkyl) Amino-carbonylethylenyl)-2-(thio)-pseudouracil, 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine TP, 1-methyl-3-(3-amino-3-carboxy propyl)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'fluro-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'-a-aminouridine TP, 2'-deoxy-2'-a- Azidouridine TP, 2-methylpseudouridine, 3(3amino-3carboxypropyl)uracil, 4(thio)pseudouracil, 4-(thio)pseudouracil, 4-(thio)uracil, 4-thiouracil, 5(1, 3-Diazole-1-alkyl)uracil, 5(2-aminopropyl)uracil, 5(aminoalkyl)uracil, 5(dimethylaminoalkyl)uracil, 5(guanidiniumalkyl)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 -(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-pseudouridine, 2-thio-dihydro-pseudouridine, 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)pseudo Uridine TP, 1-(2,4,6-trimethyl-benzyl)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-tri Fluoromethoxybenzyl)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- inyl)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-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 -methoxy-benzyl)pseudo-UTP, 1-(4-methoxy-phenyl)pseudo-UTP, 1-(4-methylbenzyl)pseudouridine TP, 1-(4-methyl-benzyl)pseudo-UTP, 1-( 4-Nitrobenzyl)pseudouridine TP, 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-acetyl pseudouridine 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-allyl pseudouridine TP, 1-aminomethyl-pseudo-UTP, 1-ben, Zoylpseudouridine TP, 1-benzyloxymethylpseudouridine TP, 1-benzyl-pseudo-UTP, 1-biotinyl-PEG2-pseudouridine TP, 1-biotinyl pseudouridine TP, 1-butyl-pseudo-UTP, 1-cyano Methylpseudouridine 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-homoallyl pseudouridine TP, 1-hydroxymethyl pseudouridine TP, 1-iso- Propyl-pseudo-UTP, 1-Me-2-thio-pseudo-UTP, 1-Me-4-thio-pseudo-UTP, 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-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 UTP, 1-methyl-6-trifluoromethyl-pseudo-UTP, 1-morpholinomethyl-pseudo-uridine TP, 1-pentyl-pseudo-UTP, 1-phenyl-pseudo-UTP, 1-pivaloyl pseudo-uridine TP, 1 -Propargyl pseudouridine TP, 1-propyl-pseudo-UTP, 1-propynyl-pseudo-uridine, 1-p-tolyl-pseudo-UTP, 1-tert-butyl-pseudo-UTP, 1-thiomethoxymethyl pseudouridine 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-thio-uridine, 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-phenylethynyl uridine TP, 5-trideuteromethyl-6-deuterouridine TP, 5-trifluoromethyl-uridine TP, 5-vinylalauridine 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 -UTP, 6-ethoxy-pseudo-UTP, 6-ethylcarboxylate-pseudo-UTP, 6-ethyl-pseudo-UTP, 6-fluoro-pseudo-UTP, 6-formyl-pseudo-UTP, 6-hydroxyamino- pseudo-UTP, 6-hydroxy-pseudo-UTP, 6-iodo-pseudo-UTP, 6-iso-propyl-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 -Trifluoromethyl-pseudo-UTP, alpha-thio-pseudo-UTP, pseudouridine 1-(4-methylbenzenesulfonic acid) TP, pseudouridine 1-(4-methylbenzoic acid) TP, pseudouridine TP1-[3 -(2-ethoxy)]propionic acid, pseudouridine TP1-[3-{2-(2-[2-(2-ethoxy)-ethoxy]-ethoxy)-ethoxy}]propionic acid, pseudouridine TP1-[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, pseudouridine -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, wybutocin, hydroxywybutocin, isowyocin, peroxywybutocin, intermediate hydroxywybutocin, 4 -Demethylwyosin, 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) )-naphthalene, 2(amino)purine, 2,4,5-(trimethyl)phenyl, 2'methyl, 2'amino, 2'azido, 2'fluro-cytidine, 2'methyl, 2'amino, 2'azide , 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-aminopyridopyrimidine -3-yl, 2-oxo-pyridopyrimidin-3-yl, 2-pyridinone, 3 nitropyrrole, 3-(methyl)-7-(propynyl)isocarbostyryl, 3-(methyl)isocarbostyryl, 4 -(fluoro)-6-(methyl)benzimidazole, 4-(methyl)benzimidazole, 4-(methyl)indolyl, 4,6-(dimethyl)indolyl, 5-nitroindole, 5-substituted pyrimidine, 5-(methyl) Isocarbostyryl, 5-nitroindole, 6-(aza)pyrimidine, 6-(azo)thymine, 6-(methyl)-7-(aza)indolyl, 6-chloro-purine, 6-phenyl-pyrrolo-pyrimidine- 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)-phenoxazine -1-yl, 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaza) )-2-(oxo)-phenoxazin-1-yl, 7-(aza)indolyl, 7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)- phenoxazinyl-yl, 7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 7-(guanidinium Alkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-(guanidiniumalkylhydroxy)-1,3-(Diaza)-2-(oxo)-phenoxazin-1-yl, 7-(guanidiniumalkyl-hydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1 -yl, 7-(guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-(propynyl)isocarbostyryl, 7-(propynyl)isocarbo Styryl, 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, iso Carbostyryl, Isoguanisine, N2-substituted purines, N6-methyl-2-amino-purines, N6-substituted purines, N-alkylated derivatives, naphthalenyl, nitrobenzimidazolyl, nitroimidazolyl, nitroindazolyl, nitropyrazolyl, Nubularine, O6-substituted purine, O-alkylated derivative, ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidine-2-, on-3-yl, ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, oxoformycin TP, para-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-one -3-yl, para-substituted-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, pentacenyl, phenanthracenyl, phenyl, propynyl-7-(aza)indolyl, pyrenyl, pyrido Pyrimidin-3-yl, pyridopyrimidin-3-yl, 2-oxo-7-amino-pyridopyrimidin-3-yl, pyrrolo-pyrimidin-2-one-3-yl, pyrrolopyrimidinyl, pyrrolopyridinyl, stilbenzyl ( Stilbenzyl), substituted 1,2,4-triazole, tetracenyl, Tubercidine, xanthine, xanthosine-5'-TP, 2-thio-zebularine, 5-aza-2-thio-zebularine, 7-deaza-2- Amino-purine, pyridine-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 T.P.

[0275] In some embodiments, the polynucleotide (e.g., an RNA polynucleotide, such as an mRNA polynucleotide) comprises at least two (e.g., two, three, four, or more) of the aforementioned modified nucleobases. including combinations of

[0276] In some embodiments, the mRNA includes 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-dihydroshu-uridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-Methoxy-2-thio-psudouridine, 4-methoxy-psudouridine, 4-thio-1-methyl-psudouridine, 4-thio-psudouridine, 5-aza-uridine, dihydroshu-douridine, 5-Methyluridine, 5-methoxyuridine, 2'-O-methyluridine, 1-methyl-psudouridine (m1ψ), 1-ethyl-psudouridine (e1ψ), 5-methoxy-uridine (mo5U), 5 -Methyl-cytidine (m5C), α-thio-guanosine, α-thio-adenosine, 5-cyanouridine, 4'-thiouridine 7-deaza-adenine, 1-methyl-adenosine (m1A), 2-methyl-adenine ( m2A), N6-methyl-adenosine (m6A), and 2,6-diaminopurine, (I), 1-methyl-inosine (m1I), wyosine (imG), methylwyosine (mimG), 7-deaza-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-lycidine, 2-selenouridine, 3-(3-amino-3-carboxypropyl)-5,6 -dihydrouridine, 3-(3-amino-3-carboxypropyl)psudouridine, 3-methylsudouridine, 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-carboxymethyl Aminomethyl-2-geranylthiouridine, 5-carboxymethylaminomethyl-2-selenouridine, 5-cyanomethyluridine, 5-hydroxycytidine, 5-methylaminomethyl-2-geranylthiouridine, 7-aminocarboxypropyl- Demethylwyosin, 7-aminocarboxypropylwyosin, 7-aminocarboxypropylwyosin methyl ester, 8-methyladenosine, N4,N4-dimethylcytidine, N6-formyladenosine, N6-hydroxymethyladenosine, agmatidine, cyclic N6-threonylcarbamoyladenosine, glutamyl-cu-osine, methylated intermediate hydroxywaibutocin, N4,N4,2'-O-trimethylcytidine, geranylated 5-methylaminomethyl-2-thiouridine, geranylated 5- selected from the group consisting of 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 pseudouridine, 1-methyl-pseudouridine, 1-ethyl-pseudouridine, 5-methylcytosine, 5-methoxyuridine, and combinations thereof. selected from the group consisting of. In some embodiments, the polynucleotide (e.g., an RNA polynucleotide, such as an mRNA polynucleotide) comprises at least two (e.g., two, three, four, or more) of the aforementioned modified nucleobases. including combinations of

[0277] In some embodiments, the mRNA is a uracil-modified sequence that includes an ORF encoding one or more cancer epitope polypeptides, where the mRNA is a uracil-modified sequence that includes a chemically modified nucleobase, e.g. Contains methoxyuracil. In certain embodiments of the invention, when the 5-methoxyuracil base is connected to a ribose sugar, as in polynucleotides, 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% is 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 where at least 95% of the uracil in the polynucleotide is 5-methoxyuracil, the overall uracil content is such that the mRNA elicits little or no immune response while providing suitable protein expression levels. Can be adjusted. In some embodiments, the uracil content of the ORF is from about 105% to about 145%, from about 105% to about 140%, from about 110% to about the theoretical minimum uracil content (%Utm) in the corresponding wild-type ORF. Approximately 140%, approximately 110% to approximately 145%, approximately 115% to approximately 135%, approximately 105% to approximately 135%, approximately 110% to approximately 135%, approximately 115% to approximately 145%, or approximately 115% to approximately It is 140%. 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%Utm. %, about 145%, or about 150%. In this context, the term "uracil" may refer to 5-methoxyuracil and / or natural uracil.

[0279] In some embodiments, the uracil content in the ORF of the mRNA encoding one or more cancer epitope polypeptides of the invention is less than about 50%, less than about 40%, about less than 30%, less than about 20%, less than about 15%, or less than about 12%. 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 the 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" may refer to 5-methoxyuracil and / or natural uracil.

[0280] In a further embodiment, the ORF of the mRNA encoding one or more cancer epitope polypeptides of the invention comprises 5-methoxyuracil and the corresponding wild-type nucleotide sequence encoding one or more cancer epitope polypeptides. It has a controlled uracil content containing lower amounts of uracil pairs (UU) and / or uracil triplets (UUU) and / or uracil quadruplets (UUUU). In some embodiments, the ORF of the mRNA encoding one or more cancer epitope polypeptides of the invention does not contain any uracil pairs and / or uracil triplets and / or uracil quadruplets. In some embodiments, the number of uracil pairs and / or uracil triplets and / or uracil quadruplets occurs below a certain threshold, e.g., in the ORF of an mRNA encoding one or more cancer epitope polypeptides. 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 , reduced to 18 or less, 19 or less, or 20 or less. In specific embodiments, the ORF of the mRNA encoding one or more cancer epitope polypeptides of the invention is less than 20, less than 19, less than 18, less than 17, less than 16, less than 15, less than 14, less than 13 , less than 12, less than 11, less than 10, less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, less than 3, less than 2 or less 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 a further embodiment, the ORF of the mRNA encoding one or more cancer epitope polypeptides of the invention comprises 5-methoxyuracil and the corresponding wild-type nucleotide sequence encoding one or more cancer epitope polypeptides. It has a controlled uracil content containing less uracil-rich clusters than the uracil-rich clusters. In some embodiments, the ORF of an mRNA encoding one or more cancer epitope polypeptides of the invention is a uracil that corresponds to a corresponding uracil in the corresponding wild-type nucleotide sequence encoding one or more cancer epitope polypeptides. Contains uracil-rich clusters that are shorter than the length of rich clusters.

[0282] In further embodiments, lower 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% of the codons in the ORF of the mRNA that includes 5-methoxyuracil encoding one or more cancer epitope polypeptides; 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%, respectively, at an alternative codon that has a codon frequency lower than the codon frequency of the substituted codon in the synonymous codon set. Replaced. The ORF is also regulated for uracil content, as described above. In some embodiments, at least one codon in the ORF of the mRNA encoding one or more cancer epitope polypeptides is substituted with an alternative codon that has a lower codon frequency than the codon frequency of the substituted codon in the synonymous codon set. be done.

[0283] In some embodiments, modulating the uracil content of an ORF of an mRNA containing 5-methoxyuracil that encodes one or more cancer epitope polypeptides improves the uracil content of one or more cancer epitope polypeptides when administered to mammalian cells. The expression level of the epitope polypeptide is higher than the expression level of one or more cancer epitope polypeptides derived 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 contains at least 95% 5-methoxyuracil and the uracil content is about the theoretical minimum. 160%, about 170%, about 180%, about 190%, or about 200% compared to the corresponding mRNA. In still other embodiments, the expression level of the one or more cancer epitope polypeptides when administered to mammalian cells is increased compared to the corresponding mRNA, wherein at least about 50% of uracil; At least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% is 1-methylpseudouracil or pseudouracil. In some embodiments, the mammalian cell is a mouse cell, rat cell, or rabbit cell. In other embodiments, the mammalian cell is a monkey cell or a human cell. In some embodiments, the human cells are HeLa cells, BJ fibroblasts, 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%. do.

[0284] In some embodiments, a 5-methoxyuracil-containing mRNA ORF that encodes one or more cancer epitope polypeptides exhibits increased stability upon modulating uracil content. In some embodiments, the mRNA exhibits increased stability within the cell compared to the stability of the corresponding wild-type mRNA under the same conditions. In some embodiments, the mRNA exhibits increased stability, including increased nuclease resistance, thermostability, and / or secondary structure stabilization. In some embodiments, the increased stability exhibited by the mRNA is determined by determining the half-life of the mRNA (e.g., in plasma, cells, or tissue samples) and / or by determining the area under the curve of protein expression over time by the mRNA. (AUC) (eg, 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 invention induces a detectably lower immune response (e.g., innate or acquired immunity) compared to the immune response induced by the corresponding wild-type mRNA under the same conditions. do. In other embodiments, an mRNA of the present disclosure encodes one or more cancer epitope polypeptides, but compared to an immune response induced under the same conditions by an mRNA that does not contain 5-methoxyuracil, or A detectably lower immune response compared to the immune response induced under the same conditions by an mRNA that encodes one or more cancer epitope polypeptides and contains 5-methoxyuracil, but whose uracil content is not regulated. (e.g., innate or acquired immunity). Innate immune responses can be manifested by increased expression of pro-inflammatory cytokines, activation of intracellular PRRs (RIG-I, MDA5, etc.), cell death, and / or cessation or reduction of protein translation. In some embodiments, a reduction in the innate immune response is associated with a decrease in the innate immune response after one or more administrations of an mRNA of the invention to a cell. , by expression or activity levels of interferon-regulated genes such as toll-like receptors (e.g., TLR7 and TLR8), and / or by cell death. can be measured by the decrease in

[0286] In some embodiments, expression of type 1 interferon by a mammalian cell in response to an mRNA of the present disclosure is determined by the expression of a type 1 interferon by the corresponding wild-type mRNA, which encodes one or more cancer epitope polypeptides but includes 5-methoxyuracil. or at least 10%, 20%, 30%, 40% compared to an mRNA that encodes one or more cancer epitope polypeptides and contains 5-methoxyuracil, but whose uracil content is not regulated. , 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9%, or more than 99.9%. 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 lower than that of the corresponding wild-type mRNA, which encodes one or more cancer epitope polypeptides, but which does not contain 5-methoxyuracil. 10%, 25% higher than the frequency of cell death observed with mRNAs that do not contain 5-methoxyuracil or that encode one or more cancer epitope polypeptides and that contain 5-methoxyuracil but whose uracil content is not regulated. , 50%, 75%, 85%, 90%, 95%, or less than 95%. 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 a 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 the uracils in the mRNA are at least about 95% 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 In some embodiments, the ORF encoding one or more cancer epitope polypeptides has an ORF G / C content (absolute or relative amount) of at least about 40% compared to the corresponding wild-type ORF. Further modified to decrease. In yet other embodiments, the ORF encoding one or more cancer epitope polypeptides contains less than 20 non-phenylalanine uracil pairs and / or triplets. In some embodiments, at least one codon in the ORF of the mRNA encoding one or more cancer epitope polypeptides is further comprised of an alternative codon that has a codon frequency lower than that of the substituted codon in the synonymous codon set. Replaced. In some embodiments, at least about 95% of the uracil in the mRNA is 5-methoxyuracil, and the uracil content of the ORF is between about 115% and about 135% of the theoretical minimum uracil content in the corresponding wild-type ORF. The expression of one or more cancer epitope polypeptides encoded by an mRNA containing an ORF is at least about 10-fold when compared to the expression of one or more cancer epitope polypeptides from the corresponding wild-type mRNA. To increase. In some embodiments, the mRNA comprises an open ORF, wherein the uracil in the mRNA is at least about 95% 5-methoxyuracil, and the uracil content of the ORF is equal to that in the corresponding wild-type ORF. The mRNA has a minimum uracil content of about 115% to about 135%, and the mRNA does not substantially induce an innate immune response in the mammalian cells into which it is introduced.

[0288] In certain embodiments, the chemical modification is present on a nucleobase in a polynucleotide (eg, 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 1-methyl-pseudouridine (m1ψ), 1-ethyl-pseudouridine (e1ψ), 5- selected from the group consisting of 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 (eg, two, three, four, or more) of the aforementioned modified nucleobases.

[0289] In some embodiments, the polynucleotide (eg, an RNA polynucleotide, such as an mRNA polynucleotide) comprises pseudouridine (ψ) and 5-methyl-cytidine (m5C). In some embodiments, the polynucleotide (eg, an RNA polynucleotide, such as an mRNA polynucleotide) comprises 1-methyl-pseudouridine (m1ψ). In some embodiments, the polynucleotide (eg, an RNA polynucleotide, such as an mRNA polynucleotide) comprises 1-ethyl-pseudouridine (e1ψ). In some embodiments, the polynucleotide (eg, an RNA polynucleotide, such as an mRNA polynucleotide) comprises 1-methyl-pseudouridine (m1ψ) and 5-methyl-cytidine (m5C). In some embodiments, the polynucleotide (eg, an RNA polynucleotide, such as an mRNA polynucleotide) comprises 1-ethyl-pseudouridine (e1ψ) and 5-methyl-cytidine (m5C). In some embodiments, the polynucleotide (eg, an RNA polynucleotide, such as an mRNA polynucleotide) comprises 2-thiouridine (s2U). In some embodiments, the polynucleotide (eg, an RNA polynucleotide, such as an mRNA polynucleotide) comprises 2-thiouridine and 5-methyl-cytidine (m5C). In some embodiments, the polynucleotide (eg, an RNA polynucleotide, such as an mRNA polynucleotide) comprises methoxy-uridine (mo5U). In some embodiments, the polynucleotide (eg, an RNA polynucleotide, such as an mRNA polynucleotide) comprises 5-methoxy-uridine (mo5U) and 5-methyl-cytidine (m5C). In some embodiments, the polynucleotide (eg, an RNA polynucleotide, such as an mRNA polynucleotide) comprises 2'-O-methyluridine. In some embodiments, the polynucleotide (eg, an RNA polynucleotide, such as an mRNA polynucleotide) comprises 2'-O-methyluridine and 5-methyl-cytidine (m5C). In some embodiments, the polynucleotide (eg, an RNA polynucleotide, such as an mRNA polynucleotide) comprises N6-methyl-adenosine (m6A). In some embodiments, the polynucleotide (eg, an RNA polynucleotide, such as an mRNA polynucleotide) comprises N6-methyl-adenosine (m6A) and 5-methyl-cytidine (m5C).

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

[0291] In some embodiments, the chemically modified nucleoside in the open reading frame is 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 with 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 with modified uridines include 1-methyl-pseudouridine (m1ψ), 1-ethyl-pseudouridine (e1ψ), 5-methoxyuridine, 2-thiouridine, 5-cyanouridine, 2'- Includes O-methyluridine and 4'-thiouridine.

[0294] In some embodiments, the modified nucleobase is a modified adenine. Examples of nucleobases and nucleosides with modified adenine include 7-deaza-adenine, 1-methyl-adenosine (m1A), 2-methyl-adenosine (m2A), N6-methyl-adenosine (m6A) and 2,6- Contains diaminopurine.

[0295] In some embodiments, the modified nucleobase is a modified guanine. Examples of nucleobases and nucleosides with modified guanines 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, 7-methyl- Contains 8-oxo-guanosine.

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

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

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

[0299] In some embodiments, a polynucleotide (e.g., an RNA polynucleotide, such as an mRNA polynucleotide) is uniformly modified (e.g., completely modified, modified throughout the entire sequence) to obtain a particular modification. ). For example, a polynucleotide 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 exchange with modified residues 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 with 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 that are useful in the compositions of the present disclosure, including backbone modifications, include: 3'-alkylene phosphonates, 3'-aminophosphoroamidates, alkene-containing backbones, aminoalkylphosphoroamidates. , aminoalkylphosphotriester, boranophosphate, -CH 2 -O-N(CH 3 )-CH 2 -,-CH 2 -N(CH 3 )-N(CH 3 )-CH 2 -,-CH 2 -NH-CH 2 -, chiral phosphonate, chiral phosphorothioate, formacetyl and thioformacetyl skeleton, methylene (methylimino), methyleneformacetyl and thioformacetyl skeleton, methyleneimino and methylenehydrazino skeleton, morpholino bond, -N(CH 3 )-CH 2 -CH 2 -, heteroatom-containing oligonucleoside internucleoside linkages, phosphinates, phosphoramidates, phosphorodithioates, phosphorothioate internucleoside linkages, phosphorothioates, phosphotriesters, PNA, siloxane skeletons, sulfamate skeletons, sulfide sulfoxide and sulfone skeletons, sulfonates and These include, but are not limited to, sulfonamide backbones, thionoalkylphosphonates, thionoalkylphosphotriesters, and thionophosphoramidates.

[0305] Modified nucleosides and nucleotides (eg, building block molecules) that can be incorporated into polynucleotides (eg, RNA or mRNA as described herein) can be modified on sugars of ribonucleic acids. 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 1-6 Alkoxy, optionally substituted C 1-12 (heterocyclyl)oxy; sugar (e.g., ribose, pentose, or any described herein); polyethylene glycol (PEG), -O(CH 2 CH 2 O) n CH 2 CH 2 OR (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 , 1~8, 1~10, 1~16, 1~20, 2~4, 2~8, 2~10, 2~16, 2~20, 4~8, 4~10, 4~16, and 4~20);2'-hydroxyl is C 1-6 Alkylene bridge or C 1-6 A "locked" nucleic acid (LNA) that is connected to the 4'-carbon of the same ribose sugar by a heteroalkylene bridge (examples of bridges include methylene, propylene, ether, or amino bridges); as defined herein 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 an oxygen. Non-limiting examples of modified nucleotides include substitution of oxygen in the ribose (e.g., substitution with S, Se, or alkylene such as methylene or ethylene); addition of a double bond (e.g., substitution of the ribose with cyclopentenyl or cyclohexenyl); ); ring reduction of the ribose (e.g., to form a four-membered cyclobutane or oxetane); ring expansion of the ribose (e.g., anhydrohexitol, altritol, mannitol, cyclohexanyl, cyclohexenyl, and with an additional carbon or heteroatom such as morpholino and also forming a 6- or 7-membered ring with a phosphoroamidate backbone); polycyclic forms (e.g. tricyclo; and "unlocked" forms, e.g. Glycol nucleic acids (GNA) (e.g., R-GNA or S-GNA, where the ribose is replaced by a glycol unit attached to a phosphodiester bond), threose nucleic acids (where the ribose is replaced by a glycol unit attached to a phosphodiester bond), threose nucleic acids (where the ribose is ), and peptide nucleic acids (PNA, in which the ribose and phosphodiester backbones are replaced by 2-amino-ethyl-glycine bonds). may contain one or more carbons with a stereochemical configuration opposite to the chemical configuration. Thus, a polynucleotide molecule may include as a sugar a nucleotide containing, for example, arabinose. Such sugar modifications are As taught in Patent Publication Nos. WO2013052523 and WO2014093924, the contents of each of these documents are incorporated herein by reference in their entirety.

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

[0308] Polynucleotides of the present disclosure may be partially modified or fully modified over the entire length of the molecule. For example, one or more or all or a given type of nucleotide (e.g., a purine or pyrimidine, or any one or more or all of A, G, U, C) can be added to a polynucleotide of the invention or at its location. It may be uniformly modified in a given predetermined sequence region (eg, mRNA containing a poly A tail or mRNA excluding a poly A tail). In some embodiments, all nucleotides X in a polynucleotide of the present disclosure (or a given sequence region thereof) are modified nucleotides, wherein or A+G, A+U, A+C, G+U, G+C, U+C, A+G+U, A+G+C, G+U+C, or A+G+ It can be one of the combinations C.

[0309] Polynucleotides contain from about 1% to about 100% modified nucleotides (with respect to total nucleotide content or with respect to one or more types of nucleotides, i.e., any one or more of A, G, U, or C). or any intervening percentage (e.g. 1%~20%, 1%~25%, 1%~50%, 1%~60%, 1%~70%, 1%~80%, 1%~90% , 1%~95%, 10%~20%, 10%~25%, 10%~50%, 10%~60%, 10%~70%, 10%~80%, 10%~90%, 10 %~95%, 10%~100%, 20%~25%, 20%~50%, 20%~60%, 20%~70%, 20%~80%, 20%~90%, 20%~ 95%, 20%~100%, 50%~60%, 50%~70%, 50%~80%, 50%~90%, 50%~95%, 50%~100%, 70%~80% , 70%~90%, 70%~95%, 70%~100%, 80%~90%, 80%~95%, 80%~100%, 90%~95%, 90%~100%, and 95%~100%). It is understood that any remaining proportions correspond to the presence of unmodified A, G, U, or C.

[0310] The polynucleotide contains a minimum of 1% to a maximum of 100% modified nucleotides, or at least 5% modified nucleotides, at least 10% modified nucleotides, at least 25% modified nucleotides, at least 50% modified nucleotides, at least It may contain modified nucleotides in any intervening proportion, such as 80%, or at least 90% modified nucleotides. For example, a polynucleotide can include a modified pyrimidine, such as a modified uracil or a 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 the polynucleotide are modified uracils (e.g., 5-substituted uracils). ) is exchanged with The modified uracil can be exchanged by a compound with a single unique structure or by multiple compounds with different structures (e.g., two, three, four, or more unique structures). Can be exchanged. 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 modified cytosines (e.g., 5-substituted cytosines) is exchanged with A modified cytosine 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). Can be exchanged.

[0311] Thus, in some embodiments, the RNA vaccine comprises a 5'UTR element, an optionally codon-optimized open reading frame, and a 3'UTR element, a poly(A) sequence and / or a polyadenylation signal, and the RNA , not chemically modified.

[0312] In some embodiments, the modified nucleobase is a modified uracil. Examples of nucleobases and nucleosides with modified uracil include pseudouridine (ψ), pyridin-4-onribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2 -thio-uridine(s 2 U), 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-oxyacetic acid (cmo 5 U), uridine 5-oxyacetic 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-thio-uridine (mcm 5 s 2 U), 5-aminomethyl-2-thio-uridine (nm 5 s 2 U), 5-methylaminomethyl-uridine (mnm 5 U), 5-methylaminomethyl-2-thio-uridine (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-thio-uridine (cmnm 5 s 2 U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (τm 5 U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine (τm 5 s 2 U), 1-taurinomethyl-4-thio-pseudouridine, 5-methyl-uridine (m 5 U, i.e. with 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-pseudo Uridine, 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-thio-uridine (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 5 Um), 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 Contains -[3-(1-E-propenylamino)]uridine.

[0313] In some embodiments, the modified nucleobase is a modified cytosine. Examples of nucleobases and nucleosides with 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, lycidine (k 2 C), 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 (m4 Cm), 5-formyl-2’-O-methyl-cytidine (f 5 Cm), N4,N4,2’-O-trimethyl-cytidine (m 4 2 Cm), 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 with modified adenine 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 6 A), N6,N6-dimethyl-adenosine (m 6 2 A), N6-hydroxynorbarylcarbamoyl-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 2 Am), 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 with modified guanine include inosine (I), 1-methyl-inosine (m 1 I), wyoscin (imG), methyl-wyoscin (mimG), 4-demethyl-wyoscin (imG-14), isoyosin (imG2), wybutocin (yW), peroxywyoscin (o 2 yW), hydroxywybutocin (OhyW), intermediate hydroxywybutocin (OhyW) * ), 7-deaza-guanosine, queuosine (Q), epoxycuosine (oQ), galactosyl-cuosine (galQ), mannosyl-cuosine (manQ), 7-cyano-7-deaza-guanosine (preQ) 0 ), 7-aminomethyl-7-deaza-guanosine (preQ 1 ), arcaeosin (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 2 G), 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 2 Gm), 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. It will be done.

[0316] In vitro transcription of RNA (e.g. mRNA) Cancer vaccines of the present disclosure include at least one RNA polynucleotide, such as mRNA (eg, modified mRNA). mRNA, for example, is transcribed in vitro from template DNA; such 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 poly A 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 200-3,000 nucleotides. For example, polynucleotides are 200~500, 200~1000, 200~1500, 200~3000, 500~1000, 500~1500, 500~2000, 500~3000, 1000~1500, 1000~2000, 1000~3000, It may contain 1500-3000, or 2000-3000 nucleotides.

[0318] In another aspect, the invention relates to a method for preparing an mRNA cancer vaccine by IVT method. In vitro transcription (IVT) methods allow template-guided synthesis of RNA molecules of almost arbitrary sequence. The size range of RNA molecules that can be synthesized using IVT methods ranges from short oligonucleotides to long nucleic acid polymers of several thousand bases. The IVT method enables the synthesis of large amounts (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 D.C.: ASM Press, 2007.262-299). Generally, IVT utilizes a DNA template characterized by a promoter sequence located upstream of the sequence of interest. Promoter sequences are most commonly of bacteriophage origin (eg, T7, T3, or SP6 promoter sequences), but many other promoter sequences are acceptable, including those designed de novo. Typically, transcription of the DNA template is best accomplished by using an RNA polymerase that corresponds to the 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 is generally initiated on double-stranded DNA, but can also proceed on single-stranded DNA.

[0319] It is to be understood that the mRNA vaccines of the present disclosure, such as, for example, mRNA encoding a cancer antigen or, for example, an activated oncogene variant peptide, may be prepared using any suitable synthetic method. For example, in some embodiments, the mRNA vaccines of the present disclosure are prepared using IVT from single-stranded bottom-strand DNA as a template and a complementary oligonucleotide that serves as a promoter. 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, single-stranded bottom strand DNA is linearized from a circular template. The single-stranded bottom-strand DNA template generally includes a promoter sequence, eg, a bacteriophage promoter sequence, to facilitate IVT. Methods for preparing RNA using single-stranded bottom strand DNA and a top strand that is a promoter-complementary oligonucleotide are known in the art. Exemplary methods include, but are not limited to, a DNA bottom strand that is a template and a top strand that is a promoter-complementary oligonucleotide (e.g., a T7 promoter-complementary oligonucleotide, a T3 promoter-complementary oligonucleotide, or an SP6 promoter-complementary oligonucleotide). After annealing is performed with (nucleotides), 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 methods can also be performed using double-stranded DNA templates. For example, in some embodiments, double-stranded DNA templates are prepared by extending complementary oligonucleotides to generate complementary DNA strands using strand extension techniques available in the art. Ru. 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 that is a promoter-complementary oligonucleotide and subjected to PCR. The top strand is elongated and a double-stranded DNA template is generated. Alternatively, or in addition, top strand DNA comprising a sequence complementary to the bottom strand promoter sequence and complementary to a sequence encoding one or more epitopes of interest is annealed to the bottom strand promoter oligonucleotide; By subjecting it to a PCR-like process, the bottom strand is extended and a double-stranded DNA template is generated. In some embodiments, the number of PCR-like cycles ranges from 1 to 20 cycles, such as from 3 to 10 cycles. In some embodiments, the double-stranded DNA template is completely or partially synthesized by chemical synthesis methods. Double-stranded DNA templates can be subjected to in vitro transcription as described herein.

[0321] In another aspect, an mRNA vaccine of the present disclosure, e.g., an mRNA encoding a cancer antigen or epitope, is prepared using two DNA strands that are complementary throughout their overlapping sequences, such that the complementary portion is Single-stranded overhangs (ie, sticky ends) may be left behind when annealed. Such single-stranded overhangs can be made double-stranded by elongating using the other strand as a template, thereby producing double-stranded DNA. In some cases, this primer extension method can lengthen the ORF that will be incorporated into the template DNA sequence, for example compared to the size that will be incorporated into the template DNA sequence obtained by top-strand DNA synthesis methods. It is. In the primer extension method, part of the 3' end of the first strand (5" to 3' direction) is complementary to part of the 3' end of the second strand (3' to 5' direction). In some such embodiments, the single-stranded first strand DNA includes a promoter (e.g., T7, T3, or SP6) sequence, optionally a 5'-UTR, and a portion of an ORF or In some embodiments, the single-stranded second strand DNA includes a sequence that is complementary to part or all of the ORF (e.g., a portion of the 5' end of the ORF). (complementary to the 3' end), and optionally a 3'-UTR, a termination sequence, and / or a poly(A) tail. In RNA preparation methods using two synthetic DNA strands, overlapping complementary After annealing of the two strands with the moieties is performed, primer extension can be performed where the strands are extended using one or more PCR-like cycles to produce a double-stranded DNA template. In such embodiments, the number of PCR-like cycles ranges from 1 to 20 cycles, such as from 3 to 10 cycles. Such double-stranded DNA is amenable to in vitro transcription as described herein. can be provided.

[0322] In another aspect, the mRNA vaccines of the present disclosure, e.g., mRNA encoding a cancer antigen or epitope, are synthesized using synthetic double-stranded linear DNA molecules such as gBlocks® (Integrated DNA Technologies, Coralville, Iowa). may be prepared using as a double-stranded DNA template. The advantage of such synthetic double-stranded linear DNA molecules is that they provide longer templates from which mRNA can be generated. For example, the size range of gBlocks® can be 45-1000 (eg, 125-750 nucleotides). In some embodiments, the synthetic double-stranded linear DNA template includes a full-length 5'-UTR, a full-length 3'-UTR, or both. A full-length 5'-UTR can be up to 100 nucleotides in length, eg, about 40-60 nucleotides in length. A full-length 3'-UTR can be up to 300 nucleotides in length, eg, about 100-150 nucleotides in length.

[0323] To facilitate the generation of longer constructs, two or more double-stranded linear DNA molecules and / or gene fragments designed with overlapping sequences on their 3' strands can be used as known in the art. may be assembled together using any method described. For example, a mesophilic exonuclease can be used to cleave bases from the 5' ends of these double-stranded DNA fragments, followed by annealing of the newly formed complementary single-stranded 3' ends before polymerase-dependent Any single-strand gaps may be filled by extension and finally the DNA segments may be covalently joined by DNA ligase to perform the Gibson Assembly™ method (Synthetic Genomics, Inc., La Jolla, CA). .

[0324] In another aspect, mRNA vaccines of the present disclosure, such as, for example, mRNA encoding a cancer antigen or epitope, may be prepared using chemical synthesis of RNA. The method involves annealing a first polynucleotide comprising an open reading frame encoding a polypeptide and a second polynucleotide comprising a 5'-UTR to a complementary polynucleotide complexed to a solid support. Thereafter, the 3' end of the second polynucleotide is ligated to the 5' end of the first polynucleotide under appropriate conditions. Suitable conditions include the use of DNA ligase. The ligation reaction produces a first ligation product. Thereafter, the 5' end of a third polynucleotide containing a 3'-UTR is ligated to the 3' end of the first ligation product under appropriate conditions. Suitable conditions for the second ligation reaction include RNA ligase. A second ligation reaction produces a second ligation product. By releasing the second ligation product from the solid support, mRNA encoding the polypeptide of interest is generated. In some embodiments, the mRNA is 30-1000 nucleotides.

[0325] The mRNA encoding the polypeptide of interest is prepared by combining a first polynucleotide containing an open reading frame encoding the polypeptide and a second polynucleotide containing a 3'-UTR with a complementary polynucleotide complexed onto a solid carrier. They may also be prepared by binding to nucleotides. The 5' end of the second polynucleotide is ligated to the 3' end of the first polynucleotide under appropriate conditions. Suitable conditions include DNA ligase. The method produces a first ligation product. A second ligation product is generated by ligating a third polynucleotide containing a 5'-UTR to the first ligation product under appropriate conditions. Suitable conditions include RNA ligases such as T4RNA. By releasing the second ligation product from the solid support, mRNA encoding the polypeptide of interest is generated.

[0326] In some embodiments, the first polynucleotide is characterized by a 5'-triphosphate and a 3'-OH. In other embodiments, the second polynucleotide comprises 3'-OH. In yet other embodiments, the third polynucleotide comprises 5'-triphosphate and 3'-OH. The second polynucleotide may also include a 5'-cap structure. The method may further include the step of ligating a fourth polynucleotide comprising a poly-A region to the 3' end of the third polynucleotide. The fourth polynucleotide can include a 5'-triphosphate.

[0327] The method may or may not include reverse phase purification. The method may also include a washing step, in which the solid support is washed to remove unreacted polynucleotides. The solid carrier can be, for example, a capture resin. In some embodiments, the method includes purification with dT.

[0328] According to the present disclosure, the template DNA encoding the mRNA vaccines of the present disclosure includes an open reading frame (ORF) encoding one or more cancer epitopes. In some embodiments, the template DNA includes an ORF of up to 1000 nucleotides, such as about 10-350 nucleotides, about 30-300 nucleotides, or about 50-250 nucleotides. In some embodiments, the template DNA includes an ORF of about 150 nucleotides. In some embodiments, the template DNA includes an ORF of about 200 nucleotides.

[0329] In some embodiments, the IVT transcript is purified from the 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 to purify RNA, such as, for example, the MEGACLEAR™ kit (Ambion, Austin, TX). mRNA can be quantified using methods known in the art, including, but not limited to, using commercially available equipment such as the NanoDrop. Purified mRNA can be analyzed by agarose gel electrophoresis, for example, to confirm that the RNA size is correct and / or to confirm that the RNA has not been degraded.

[0330] Untranslated region (UTR) The untranslated region (UTR) is the region of the nucleic acid 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., ribonucleic acid (RNA), e.g., messenger RNA (mRNA)) comprising an open reading frame (ORF) encoding one or more cancer antigens or epitopes , UTR (eg, 5'UTR or a functional fragment thereof, 3'UTR or a functional fragment thereof, or a combination thereof).

[0331] A UTR may be homologous or heterologous to the coding region in the polynucleotide. In some embodiments, the UTR is homologous to an ORF encoding one or more cancer epitope polypeptides. In some embodiments, the UTR is heterologous to an ORF encoding one or more cancer epitope polypeptides. In some embodiments, the polynucleotide comprises two or more 5'UTRs or functional fragments thereof, each having the same or different nucleotide sequences. 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 functional fragment thereof, the 3'UTR or functional fragment thereof, or any combination thereof is sequence optimized.

[0333] In some embodiments, the 5'UTR or functional fragment thereof, the 3'UTR or functional fragment thereof, or any combination thereof, has at least one chemically modified nucleobase, e.g., 5-methoxy Contains uracil.

[0334] A UTR may have a regulatory role, eg, characteristics that confer stability, localization and / or increase or decrease translation efficiency. A polynucleotide comprising a UTR can be administered to a cell, tissue, or organ, and one or more regulatory characteristics can be measured using conventional methods. In some embodiments, a functional fragment of the 5'UTR or 3'UTR comprises one or more regulatory features of the full-length 5'UTR or 3'UTR, respectively.

[0335] The natural 5'UTR is characterized by its involvement in translation initiation. Natural 5'UTRs 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 is 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". ). The 5'UTR is also known to form secondary structures involved in elongation factor binding.

[0336] Polynucleotide stability and protein production can be enhanced by manipulating characteristics typically found in genes that are abundantly expressed in particular target organs. For example, introducing the 5'UTR of mRNAs expressed in the liver, such as albumin, serum amyloid A, apolipoprotein A / B / E, transferrin, alpha-fetoprotein, erythropoietin, or factor VIII, can be used in liver cell lines or in the liver. can enhance the expression of a polynucleotide. Similarly, muscle (e.g., MyoD, myosin, myoglobin, myogenin, herculin), endothelial cells (e.g., Tie-1, CD36), bone marrow 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 derived from other tissue-specific mRNAs can be used to improve expression in that tissue.

[0337] In some embodiments, the UTR is selected from a family of transcripts whose proteins have a common function, structure, characteristic, or property. For example, the encoded polypeptide may represent a family of proteins (i.e., at least one function, structure, characteristic, localization, origin, or expression pattern) that is expressed in a particular cell, tissue, or at a certain time during development. shared family). New polynucleotides can be created by replacing a UTR from either a gene or mRNA with any other UTR from the same or a different protein family.

[0338] In some embodiments, the 5'UTR and 3'UTR can be heterologous. In some embodiments, the 5'UTR may be from a different species than the 3'UTR. In some embodiments, the 3'UTR may be 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 document is incorporated herein by reference in its entirety) describes the present invention as a flanking region to an ORF. Provides a list of example UTRs that can be used in polynucleotides.

[0340] Examples of UTRs in this application include, but are not limited to, one or more 5'UTRs and / or 3'UTRs derived from the following nucleic acid sequences: such as α-globin or β-globin. Globin (e.g., Xenopus, mouse, rabbit, or human globin); strong Kozak translation initiation signal; CYBA (e.g., human cytochrome B-245α polypeptide); albumin (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., Pneumonia B virus), Sindbis virus, or PAV barley yellow dwarf virus); heat shock proteins (e.g., hsp70); translation initiation factors (e.g., elF4G); 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. 5'UTR of TOP genes lacking the 5'TOP motif (oligopyrimidine tract)); ribosomes protein large 32 (L32); ribosomal proteins (e.g., human or mouse ribosomal proteins, such as rps9); ATP synthases (e.g., ATP5A1 or mitochondrial H + -β subunit of ATP synthase); growth hormone (e.g. bovine (bGH) or human (hGH)); elongation factors (e.g. elongation factor 1 alpha 1 (EEF1A1)); manganese superoxide dismutase (MnSOD); muscle cell enhancer Factor 2A (MEF2A); β-F1-ATPase, creatine kinase, myoglobin, granulocyte colony-stimulating factor (G-CSF); collagen (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 nucleobindin (eg, Nucb1).

[0341] Other examples of 5'UTR and 3'UTR 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):4009-4017;Kormann et al.,Nat.Biotechnol.2011 29(2):154-157 ;Poleganov et al.,Hum.Gen.Ther.2015 26(11):751-766;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 et 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;WO20 07 / 036366;WO2011 / 015347;WO2012 / 072096;WO2013 / 143555; WO2014 / 071963;WO2013 / 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, but are not limited thereto. The contents of each of these documents are incorporated herein by reference in their entirety.

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

[0343] In some embodiments, the 3'UTR is a β-globin 3'UTR; a CYBA 3'UTR; an albumin 3'UTR; a growth hormone (GH) 3'UTR; a VEEV 3'UTR; 3'UTR of hepatitis virus (HBV); 3'UTR of α-globin; 3'UTR of DEN; 3'UTR of PAV barley yellow dwarf virus (BYDV-PAV); 3'UTR of elongation factor 1 α1 (EEF1A1) ;3'UTR of manganese superoxide dismutase (MnSOD);3'UTR of mitochondrial H(+)-ATP synthase β subunit (β-mRNA);3'UTR of GLUT1;3'UTR of MEF2A;β-F1 -3'UTR of 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 this document 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 list the start and end sites of 5'UTR and 3'UTR, and these The contents of each of the documents are incorporated herein by reference in their entirety. In Table 21, each 5'UTR (5'-UTR-005~5'-UTR68511) is identified at its start and end sites relative to its native or wild-type (homologous) transcript. (ENST; Identifier used in the ENSEMBL database).

[0345] Wild-type UTRs derived from any gene or mRNA can be incorporated into the polynucleotides of the invention. In some embodiments, a UTR can be modified relative to a wild-type or native UTR to generate a variant UTR, for example, by changing the orientation or position of the UTR relative to the ORF, or by adding additional It is carried out by nucleotide incorporation, nucleotide deletion, nucleotide substitution or transfer. In some embodiments, a variant of the 5' or 3' UTR includes, for example, a variant of the wild-type UTR, or one or more nucleotides added to or removed from the end of the UTR. Variants are available.

[0346] Additionally, one or more synthetic UTRs can also be used in combination with one or more non-synthetic UTRs. See, eg, Mandal and Rossi, Nat.Protoc.2013 8(3):568-82 and the sequences available from www.addgene.org / Derrick_Rossi / . The contents of each of these are incorporated herein by reference in their entirety. A UTR or portion thereof may be placed in the same orientation as the transcript from which it is selected, or the orientation or position may be altered. Accordingly, a 5'UTR and / or a 3'UTR may 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, eg, a 5'UTR or a 3'UTR in a double, triple or quadruple. For example, a double UTR includes two copies of the same UTR either in series or substantially in series. For example, the 3'UTR of double β-globin 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 are 5'UTR sequences comprising any of SEQ ID NOS: 247-271 and / or 3'UTR sequences comprising any of SEQ ID NOs: 272-302. 'at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96% for sequences selected from the group consisting of 'UTR sequences as well as any combination thereof; , at least about 97%, at least about 98%, at least about 99%, or about 100% identical.

[0350] Polynucleotides of the invention may contain combinations of features. For example, the ORF may be flanked by a 5'UTR containing a strong Kozak translation initiation signal and / or a 3'UTR containing an oligo(dT) sequence for template 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). (incorporated into the specification).

[0351] It is also within the scope of the invention to have patterned UTRs. As used herein, "patterned UTR" includes repeating or alternating patterns that repeat once, twice, or three or more times, such as ABABAB or AABBAABBAABB or ABCABCABC or variations thereof. 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 intronic sequences can be incorporated into polynucleotides of the invention. Incorporation of intronic sequences can increase protein production and polynucleotide expression levels. In some embodiments, a polynucleotide of the invention comprises an internal ribosome entry site (IRES) instead of or in addition to a UTR (e.g., Yakubov et al., Biochem. Biophys. Res. .2010 394(1):189-193, the contents of which are incorporated herein by reference in their entirety). In some embodiments, a polynucleotide of the invention comprises the 5' end of rubella virus (RV) genomic RNA and / or the 5' open reading frame of rubella virus (RV) RNA encoding a nonstructural protein. 5' and / or 3' sequences, respectively associated with the 3' end, or deletion derivatives thereof (see, e.g., Pogue et al., J. Virol. 67(12):7106-7117; The contents of the document are incorporated herein by reference in their entirety). Viral capsid sequences, such as the 5' portion of the capsid sequence, can also be used as translation enhancers (e.g., 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 these documents are incorporated herein by reference in their entirety). In some embodiments, the polynucleotide includes an IRES in place of the 5'UTR sequence. In some embodiments, the polynucleotide includes 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, the UTR also comprises at least one translational enhancer polynucleotide, translational enhancer element, or translational enhancer element (collectively referred to as a "TEE") that increases the amount of polypeptide or protein produced from the polynucleotide. nucleic acid sequences). As a non-limiting example, TEEs include those described in US2009 / 0226470, which is incorporated herein by reference in its entirety, and which are otherwise known in the art. As a non-limiting example, a TEE can be located between a transcriptional promoter and an initiation codon. In some embodiments, the 5'UTR includes a TEE.

[0354] In one aspect, a TEE is a conserved element in a UTR that can facilitate translational activity of a nucleic acid, such as, 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. This document is incorporated herein by reference in its entirety.

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

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

Claims

1. An mRNA comprising an open reading frame (ORF) encoding 5 to 500 peptide epitopes in a head-to-tail arrangement; Lipid nanoparticles comprising 20-60 mol % of an ionizable cationic lipid, 5-25 mol % of a non-cationic lipid, 25-55 mol % of a sterol, and 0.5-15 mol % of a PEG-modified lipid.

1. A personalized mRNA cancer vaccine for treating cancer in a human patient, comprising: The peptide epitope is (a) identifying neoepitopes that are expressed in tumor samples from said patient but not in normal tissues from said patient; (b) determining whether the identified neoepitopes include one or more neoepitopes comprising a mutation selected from a recurrent p53 mutation, a KRAS mutation, and an NRAS mutation; and (c) the following characteristics: (i) the peptide epitope is selected from identified neoepitopes; (ii) the peptide epitope is not self-reactive; (iii) each peptide epitope contains at least one of the following types of mutations: insertion, deletion, substitution, and frameshift; and (iv) at least one of the peptide epitopes is an MHC class I epitope and at least one of the peptide epitopes is an MHC class II epitope. selecting a peptide epitope encoded by an ORF having and selected by a method comprising: When the neoepitopes include one or more neoepitopes comprising any of recurrent p53 mutations, KRAS mutations, or NRAS mutations, the selecting step comprises selecting at least one of the one or more neoepitopes comprising a recurrent p53 mutation, KRAS mutation, or NRAS mutation; In the ORF, the peptide epitopes are linked to each other directly or via a linker; and A personalized mRNA cancer vaccine, wherein the mRNA comprises 1-methylpseudouridine.

2. The personalized mRNA cancer vaccine of claim 1, wherein one or more of the peptide epitopes are selected from neoepitopes comprising recurrent p53 mutations, KRAS mutations, or NRAS mutations.

3. The personalized mRNA cancer vaccine of claim 2, wherein one or more of the peptide epitopes comprise (i) a KRAS mutation independently selected from a G12 mutation and a G13 mutation, and / or (ii) an NRAS mutation independently selected from a Q61 mutation.

4. An individualized mRNA cancer vaccine as described in claim 1 or 2, wherein one or more of the peptide epitopes are abundant in tumor samples.

5. An individualized mRNA cancer vaccine as described in claim 1 or 2, wherein one or more of the peptide epitopes are present in large amounts in tumor samples.

6. The personalized mRNA cancer vaccine described in claim 1 or 2, wherein at least two of the peptide epitopes contain the same type of mutation.

7. The personalized mRNA cancer vaccine described in claim 1 or 2, wherein the peptide epitope is an indicator of one or more of the exome of a tumor sample and the transcriptome of a tumor sample.

8. The personalized mRNA cancer vaccine described in claim 1 or 2, wherein one or more of the peptide epitopes include conservative amino acid substitutions.

9. The personalized mRNA cancer vaccine described in claim 8, wherein each of the peptide epitopes comprises a non-conservative amino acid substitution.

10. The personalized mRNA cancer vaccine described in claim 1 or 2, wherein one or more of the peptide epitopes include non-conservative amino acid substitutions.

11. The personalized mRNA cancer vaccine described in claim 10, wherein each of the peptide epitopes comprises a non-conservative amino acid substitution.

12. The personalized mRNA cancer vaccine described in claim 1 or 2, wherein each peptide epitope is 9 to 29 amino acids in length.

13. The personalized mRNA cancer vaccine described in claim 1 or 2, wherein at least 30% of the peptide epitopes are MHC class I epitopes.

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

15. An individualized mRNA cancer vaccine as described in claim 1 or 2, wherein the ratio of MHC class I epitopes to MHC class II epitopes is 1:1, 2:1, 3:1, 4:1, or 5:

1.

16. The personalized mRNA cancer vaccine of claim 1 or 2, wherein one or more of the peptide epitopes comprises a centrally located SNP mutation.

17. The personalized mRNA cancer vaccine described in claim 1 or 2, wherein two or more of the peptide epitopes are linked to each other via a linker.

18. The personalized mRNA cancer vaccine described in claim 1 or 2, wherein two or more of the peptide epitopes are directly linked to each other without a linker.

19. The personalized mRNA cancer vaccine described in claim 1 or 2, wherein one or more of the peptide epitopes exhibit T cell reactivity.

20. The personalized mRNA cancer vaccine of claim 19, wherein the method for selecting the peptide epitopes further comprises identifying neoepitopes that exhibit T cell reactivity.

21. The personalized mRNA cancer vaccine of claim 1 or 2, wherein at least 50% of the peptide epitopes have predicted binding affinity for one or more selected from HLA-A, HLA-B, and DRB1.

22. The personalized mRNA cancer vaccine described in claim 1 or 2, wherein the ORF encodes up to 50 epitopes.

23. The personalized mRNA cancer vaccine of claim 22, wherein the ORF encodes 20 to 35 epitopes.

24. The personalized mRNA cancer vaccine of claim 1 or 2, wherein the peptide epitopes are positioned to minimize spurious epitopes.

25. The mRNA of claim 25, wherein: (a) a polyA tail, or a polyA tail comprising 100 nucleotides; (b) a 5'-end cap; (c) a 5′UTR, and (d) 3′UTR 3. The personalized mRNA cancer vaccine of claim 1 or 2, having one or more of the following characteristics selected from:

26. The personalized mRNA cancer vaccine of claim 1 or 2, wherein the mRNA comprises one or more chemical modifications 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-methoxyuridine, and 2'-O-methyluridine.

27. The personalized mRNA cancer vaccine of claim 26, wherein the mRNA is modified with N1-methylpseudouridine throughout its entire sequence.

28. The personalized mRNA cancer vaccine of claim 1 or 2, wherein the lipid nanoparticles comprise 50 mol% ionizable cationic lipids, 10 mol% non-cationic lipids, 38.5 mol% sterols, and 1.5 mol% PEG-modified lipids.

29. The lipid nanoparticles of claim 2, wherein the ionizable cationic lipid is a compound having the formula (I): 【Chemistry 1】 (In the formula, R 1 is selected from the group consisting of C 5-30 alkyl, C 5-20 alkenyl, and —R″M′R′; R 2 and R 3 are independently selected from the group consisting of C 1-14 alkyl and C 2-14 alkenyl; R 4 is (CH 2 ) n Q, where Q is —OR and n is selected from 1, 2, 3, 4, and 5; Each R 5 is H; Each R 6 is H; M and M′ are independently selected from C(O)O and OC(O); R 7 is H; R is H, R' is selected from the group consisting of C 1-18 alkyl and C 2-18 alkenyl; R" is selected from the group consisting of C3-14 alkyl and C3-14 alkenyl; m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13. or a salt thereof.

30. The compound of formula (I) comprising Compound 1: 【Chemistry 2】 30. The personalized mRNA cancer vaccine of claim 29, having the structure:

31. The personalized mRNA cancer vaccine of claim 30, wherein the lipid nanoparticles comprise a molar ratio of about 50% compound 1, about 10% DSPC, about 38.5% cholesterol, and about 1.5% PEG-DMG.

32. A personalized mRNA cancer vaccine as described in claim 1 or 2 for treating cancer in a patient, for use in combination therapy with an immune checkpoint modulator.

33. The personalized mRNA cancer vaccine described in claim 32, wherein the immune checkpoint modulator is an anti-PD-1 antibody.

34. The personalized mRNA cancer vaccine of claim 33, wherein the anti-PD-1 antibody is pembrolizumab.

35. A method for preparing a personalized mRNA cancer vaccine for treating cancer in a human patient, comprising: The personalized mRNA cancer vaccine comprises an mRNA comprising an open reading frame (ORF) encoding 5 to 100 peptide epitopes arranged in a head-to-tail configuration; The method comprises: (a) identifying neoepitopes that are expressed in tumor samples from said patient but not in normal tissues from said patient; (b) determining whether the identified neoepitopes include one or more neoepitopes comprising any of the mutations selected from recurrent p53 mutations, KRAS mutations, and NRAS mutations; and (c) the following characteristics: (i) the peptide epitope is selected from identified neoepitopes; (ii) the peptide epitope is not self-reactive; (iii) each peptide epitope contains at least one of the following types of mutations: insertion, deletion, substitution, and frameshift; and (iv) at least one of the peptide epitopes is an MHC class I epitope and at least one of the peptide epitopes is an MHC class II epitope. selecting a peptide epitope encoded by an ORF having selecting a peptide epitope by a method comprising: When the neoepitopes include one or more neoepitopes comprising a recurrent p53 mutation, a KRAS mutation, or an NRAS mutation, the selecting step comprises selecting at least one of the one or more neoepitopes comprising a recurrent p53 mutation, a KRAS mutation, or an NRAS mutation.

36. The method described in claim 35, further comprising a step of preparing mRNA, wherein the mRNA is modified with N1-methylpseudouridine throughout its entire sequence.

37. The method of claim 36, further comprising formulating the mRNA in lipid nanoparticles, wherein the lipid nanoparticles comprise 20-60 mol% ionizable cationic lipid, 5-25 mol% non-cationic lipid, 25-55 mol% sterol, and 0.5-15 mol% PEG-modified lipid.

38. The method of any one of claims 35 to 37, wherein one or more of the peptide epitopes are selected from neoepitopes comprising recurrent p53 mutations, KRAS mutations, or NRAS mutations.

39. The method of claim 38, wherein one or more of the peptide epitopes include one or more selected from (i) KRAS mutations independently selected from G12 mutations and G13 mutations, and / or (ii) NRAS mutations independently selected from Q61 mutations.

40. The peptide epitope having the following characteristics: each peptide epitope is 9 to 29 amino acids in length; At least 30% of the peptide epitopes are MHC class I epitopes. At least 50% of the peptide epitopes are MHC class I epitopes. the ratio of MHC class I epitopes to MHC class II epitopes is at least 1:1, 2:1, 3:1, 4:1, or 5:1; one or more of said peptide epitopes comprises conservative amino acid substitutions; one or more of said peptide epitopes comprises non-conservative amino acid substitutions; one or more of the peptide epitopes comprises a centrally located SNP variant; two or more of the peptide epitopes are linked to one another via a linker; and Two or more of the peptide epitopes are directly linked to each other without a linker.

39. The method of claim 35 or 38, comprising one or more of:

41. The method described in claim 35 or 38, wherein the method for selecting peptide epitopes further comprises identifying neoepitopes that exhibit T cell reactivity, and one or more of the peptide epitopes exhibit T cell reactivity.

42. The method described in claim 35 or 38, wherein the ORF encodes 20 to 35 epitopes.

43. The method of claim 35 or 38, wherein the peptide epitopes are positioned to minimize spurious epitopes.

44. The lipid nanoparticles, wherein the ionizable cationic lipid is a compound having the formula (I): 【Transformation 3】 (In the formula, R 1 is selected from the group consisting of C 5-30 alkyl, C 5-20 alkenyl, and —R″M′R′; R 2 and R 3 are independently selected from the group consisting of C 1-14 alkyl and C 2-14 alkenyl; R 4 is (CH 2 ) n Q, where Q is —OR and n is selected from 1, 2, 3, 4, and 5; Each R 5 is H; Each R 6 is H; M and M′ are independently selected from C(O)O and OC(O); R 7 is H; R is H, R' is selected from the group consisting of C 1-18 alkyl and C 2-18 alkenyl; R" is selected from the group consisting of C3-14 alkyl and C3-14 alkenyl; m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13. or a salt thereof.

45. The compound of formula (I) comprising Compound 1: 【Chemistry 4】 45. The method of claim 44, having the structure:

46. The method described in claim 45, wherein the lipid nanoparticles comprise a molar ratio of about 50% compound 1, about 10% DSPC, about 38.5% cholesterol, and about 1.5% PEG-DMG.

47. A method for selecting a set of neoepitopes for use in a personalized mRNA cancer vaccine comprising an mRNA comprising an open reading frame (ORF) encoding 5 to 100 peptide epitopes, comprising: The method comprises: (a) identifying neoepitopes that are expressed in tumor samples from a patient but not in normal tissues from said patient; (b) determining whether the identified neoepitopes include one or more neoepitopes comprising a mutation selected from any of recurrent p53 mutations, KRAS mutations, and NRAS mutations; and (c) the following characteristics: (i) the peptide epitope is selected from identified neoepitopes; (ii) the peptide epitope is not self-reactive; (iii) each peptide epitope contains at least one of the following types of mutations: insertion, deletion, substitution, and frameshift; and (iv) at least one of the peptide epitopes is an MHC class I epitope and at least one of the peptide epitopes is an MHC class II epitope. selecting a peptide epitope encoded by an ORF having Including, When the neoepitopes include one or more neoepitopes comprising a recurrent p53 mutation, a KRAS mutation, or an NRAS mutation, the selecting step comprises selecting at least one of the one or more neoepitopes comprising a recurrent p53 mutation, a KRAS mutation, or an NRAS mutation.

48. The peptide epitope having the following characteristics: each peptide epitope is 9 to 29 amino acids in length; At least 30% of the peptide epitopes are MHC class I epitopes. At least 50% of the peptide epitopes are MHC class I epitopes. the ratio of MHC class I epitopes to MHC class II epitopes is at least 1:1, 2:1, 3:1, 4:1, or 5:1; one or more of said peptide epitopes comprises conservative amino acid substitutions; one or more of said peptide epitopes comprises non-conservative amino acid substitutions; one or more of the peptide epitopes comprises a centrally located SNP variant; two or more of the peptide epitopes are linked to one another via a linker; and Two or more of the peptide epitopes are directly linked to each other without a linker.

48. The method of claim 47, comprising one or more of:

49. The method of claim 47 or 48, wherein one or more of the peptide epitopes are selected from neoepitopes comprising recurrent p53 mutations, KRAS mutations, or NRAS mutations.

50. The method of claim 49, wherein one or more of the peptide epitopes include one or more selected from (i) KRAS mutations independently selected from G12 mutations and G13 mutations, and / or (ii) NRAS mutations independently selected from Q61 mutations.

51. The method described in claim 47 or 48, wherein the peptide epitope is an indicator of one or more of the exome of a tumor sample and the transcriptome of a tumor sample.

52. The method described in claim 47 or 48, wherein the method for selecting peptide epitopes further comprises identifying neoepitopes that exhibit T cell reactivity, and one or more of the peptide epitopes exhibit T cell reactivity.

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