Personalized cancer vaccine epitope selection

Abstract

To provide optimized cancer vaccines, as well as methods of making the vaccines, methods of using the vaccines, and compositions comprising the vaccines, and further provide a computerized system for selecting nucleic acids to be incorporated into an optimized cancer vaccine.SOLUTION: The present invention provides a nucleic acid cancer vaccine, comprising one or more nucleic acids each having one or more open reading frames encoding 5 to 130 peptide epitopes, wherein each of the peptide epitopes are portions of personalized cancer antigens, wherein at least two of the peptide epitopes have different lengths.SELECTED DRAWING: None

Description

[Technical field]

[0001] Related Applications This application claims the benefit of priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 62 / 690,441, filed June 27, 2018, U.S. Provisional Application No. 62 / 757,045, filed November 7, 2018, U.S. Provisional Application No. 62 / 814,200, filed March 5, 2019, and U.S. Provisional Application No. 62 / 855,311, filed May 31, 2019, each of which is incorporated by reference in its entirety herein. [Background technology]

[0002] Recent theories in cancer evolution focus on three steps, including stress-induced genome instability, population diversity or heterogeneity, and genome-mediated macroevolution. This theory explains why most of the known molecular mechanisms can contribute to cancer, yet there is no single dominant mechanism in most clinical cases. However, a common mechanism suggests that cancer vaccines may provide a universal solution in the treatment of cancer.

[0003] Cancer vaccines include preventative or prophylactic vaccines, which are intended to prevent the development of cancer in healthy people, and therapeutic vaccines, which are intended to treat existing cancer by enhancing the body's natural defenses against cancer. Cancer preventative vaccines may target infectious agents that cause or contribute to the development of cancer, for example, to prevent infections from causing cancer. Gardasil® and Cervarix® are two examples of commercially available preventative vaccines that protect against HPV infection and resulting cancer. Other preventative cancer vaccines may target host proteins or fragments that are predicted to increase the likelihood that an individual will develop cancer in the future.

[0004] Many commercially available or developmental vaccines are based on whole microorganisms, protein antigens, peptides, or polysaccharides, and combinations thereof. Certain developmental vaccines are also based on nucleic acid vaccines (e.g., deoxyribonucleic acid (DNA) vaccines or ribonucleic acid (RNA) vaccines). Such nucleic acid vaccines are generally not optimized for maximum efficacy relative to their size or length. Summary of the Invention

[0005] Provided herein are nucleic acid (e.g., ribonucleic acid (RNA)) cancer vaccines that have maximized anti-cancer efficacy over a given length and include one or more nucleic acids that can induce the body's cellular machinery to produce a cancer protein or fragment thereof of interest. In some embodiments, the present disclosure also provides a method for making a nucleic acid cancer vaccine with maximized anti-cancer efficacy over a given length. In some embodiments, the present disclosure also provides a method for treating a patient with cancer with a cancer vaccine with maximized anti-cancer efficacy over a given length. Additionally, in certain embodiments, the present disclosure provides a computerized system for creating a nucleic acid cancer vaccine with maximized anti-cancer efficacy over a given length.

[0006] In one aspect, the disclosure provides a nucleic acid cancer vaccine comprising one or more nucleic acids each having one or more open reading frames encoding 5-130 peptide epitopes, each of the peptide epitopes being a portion of an individualized cancer antigen, and at least two peptide epitopes having different lengths. In another aspect, the disclosure provides a nucleic acid cancer vaccine comprising one or more nucleic acids each having one or more open reading frames encoding 5-130, 20-40, 30-35, or 34 peptide epitopes, each of the peptide epitopes being a portion of an individualized cancer antigen, and each of the peptide epitopes having different lengths. In another aspect, the disclosure provides a nucleic acid cancer vaccine comprising one or more nucleic acids each having one or more open reading frames encoding 5-130, 20-40, 30-35, or 34 peptide epitopes, each of the peptide epitopes being a portion of an individualized cancer antigen, and each of the peptide epitopes having equal lengths. In some embodiments, the cancer vaccine composition comprises one or more mRNAs having one or more open reading frames each encoding 34 peptide epitopes, 29 epitopes are MHC class I epitopes and 5 epitopes are MHC class II or MHC class I and II epitopes.

[0007] In some embodiments, the length of each peptide epitope is determined so that the anti-cancer efficacy of the nucleic acid cancer vaccine is maximized for one or more nucleic acids of a given length. In some embodiments, the minimum length of any peptide epitope is 8 amino acids. In some embodiments, the maximum length of any peptide epitope is 31 amino acids. In some embodiments, the minimum length of any or all of the peptide epitopes is 13 amino acids. In some embodiments, the maximum length of any or all of the peptide epitopes is 35 amino acids. In some embodiments, the length of any or all of the peptide epitopes is 25 amino acids.

[0008] In some embodiments, the cancer vaccine is a DNA cancer vaccine. In some embodiments, the cancer vaccine is an RNA cancer vaccine. In some embodiments, the cancer vaccine is an mRNA cancer vaccine and the one or more nucleic acids are mRNA. In some embodiments, the one or more mRNAs each comprise a 5' UTR and / or a 3' UTR. In some embodiments, the one or more mRNAs each comprise a polyA tail. In some embodiments, the polyA tail comprises about 100 nucleotides. In some embodiments, the one or more mRNAs each comprise a cap structure or a modified cap structure. In some embodiments, the cap structure or modified cap structure is a 5' cap structure, a 5' cap-0 structure, a 5' cap-1 structure, or a 5' cap-2 structure.

[0009] In some embodiments, one or more mRNAs comprise at least one chemical modification. In certain embodiments, the chemical modification is selected from the group consisting of pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methylpseudouridine, 5-methyluridine, 5-methoxyuridine, and 2'-0-methyluridine. In some embodiments, one or more mRNAs are fully modified.

[0010] In some embodiments, the one or more nucleic acids encode 3-10 peptide epitopes, 5-10 peptide epitopes, 10-20 peptide epitopes, 20-30 peptide epitopes, 30-40 peptide epitopes, 40-50 peptide epitopes, 50-60 peptide epitopes, 60-70 peptide epitopes, 70-80 peptide epitopes, 80-90 peptide epitopes, 90-100 peptide epitopes, 100-110 peptide epitopes, 110-120 peptide epitopes, or 120-130 peptide epitopes. In some embodiments, each of the peptide epitopes is encoded by a separate open reading frame. In some embodiments, the peptide epitopes are in the form of a concatemeric cancer antigen consisting of 3-130 peptide epitopes. In some embodiments, the cancer vaccine composition comprises one mRNA with one open reading frame encoding 15 peptide epitopes.

[0011] In some embodiments, one or more of the following conditions are met: a) the 3-130 peptide epitopes are interspersed with cleavage sensitive sites (e.g., a linker, such as a peptide linker that includes a cleavage sensitive site or a cleavage sensitive site as part of an adjacent epitope), and / or b) each peptide epitope is directly linked to each other without a linker, and / or c) each peptide epitope is linked to each other with a single amino acid linker, and / or d) each peptide epitope is linked to each other with a short linker, and / or e) each peptide epitope comprises 8-31 amino acids and comprises one or more SNP mutations, and / or f) each peptide epitope comprises 8-31 amino acids and comprises a mutation that results in a unique expressed peptide sequence, and / or g) at least 30% of the peptide epitopes have highest affinity for class I MHC molecules from the subject, and / or h) at least 30% of the peptide epitopes have highest affinity for class II MHC molecules from the subject. and / or i) none of the peptide epitopes have a highest affinity for a class II MHC molecule from the subject; and / or j) at least 50% of the peptide epitopes have an IC 50have a predicted binding affinity of <500 nM, and / or k) the nucleic acids encoding the peptide epitopes are arranged such that the peptide epitopes are ordered to minimize pseudo-epitopes, and / or l) the ratio of class I MHC molecule peptide epitopes to class II MHC molecule peptide epitopes is at least 1:1, 2:1, 3:1, 4:1, or 5:1, and / or m) no class II MHC molecule peptide epitopes are present. In other embodiments, at least 30% of the peptide epitopes have the highest affinity for class I MHC molecules and / or class II MHC class molecules from the subject. In other embodiments, at least 50% of the peptide epitopes have a probability percentile rank of greater than 0.5% for HLA-A, HLA-B, and / or DRB1. The probability percentile rank provides a threshold for determining strong binders and is a calculation of the percentage of scores in a frequency distribution that is equal to or lower than that.

[0012] In some embodiments, at least one of the peptide epitopes is a predicted T cell reactive epitope. In certain embodiments, at least one of the peptide epitopes is a predicted B cell reactive epitope. In some embodiments, the peptide epitopes include a combination of predicted T cell reactive epitopes and predicted B cell reactive epitopes. In some embodiments, the peptide epitopes are predicted T cell reactive epitopes and / or predicted B cell reactive epitopes. In some embodiments, at least one of the peptide epitopes is a predicted neo-epitope. In certain embodiments, at least one nucleic acid has an open reading frame encoding at least a fragment of one or more conventional cancer antigens or one or more cancer / testis antigens.

[0013] In some embodiments, each nucleic acid is formulated in a lipid nanoparticle. In some embodiments, each nucleic acid is formulated in a different lipid nanoparticle. In some embodiments, each nucleic acid is formulated in the same lipid nanoparticle.

[0014] In some embodiments, the total length of the one or more nucleic acids encodes a total protein length of 50-100 amino acids, 100-200 amino acids, 200-300 amino acids, 300-400 amino acids, 400-500 amino acids, 500-600 amino acids, 600-700 amino acids, 700-800 amino acids, 800-900 amino acids, 900-1000 amino acids, 1000-1100 amino acids, or 1100-1200 amino acids.

[0015] In some embodiments, the anti-cancer efficacy is calculated based at least in part on one or more factors selected from the group consisting of gene expression, RNA sequence, transcript abundance, DNA allele frequency, amino acid conservation, physiochemical similarity, cancer genes, predicted binding affinity to specific HLA alleles, clonality, binding efficiency, and presence of indels. In some embodiments, the one or more factors are input into a statistical model (e.g., a regression model (such as a linear regression model, a logistic regression model, a general linear model, etc.), a general linear model (such as a logistic regression model, a probit regression model, etc.), a random forest regression model, a neural network, a support vector machine, a Gaussian mixture model, a hierarchical Bayesian model, and / or any other suitable statistical model).

[0016] In another aspect, the disclosure provides a method of making a cancer vaccine, comprising: a) identifying 3-130 personalized cancer antigens for a patient; b) determining the anti-tumor efficacy of at least two peptide epitopes for each of the 3-130 personalized cancer antigens; and c) preparing a cancer vaccine, wherein the overall anti-cancer efficacy of the cancer vaccine is maximized for a given full-length cancer vaccine.

[0017] In another aspect, the present disclosure provides a method for treating a patient with cancer, comprising: a) analyzing a sample from the patient to identify one or more personalized cancer antigens; b) determining the anti-tumor efficacy of at least two peptide epitopes for each of the identified personalized cancer antigens; c) preparing a cancer vaccine, in which the overall anti-cancer efficacy of the cancer vaccine is maximized for a given full-length cancer vaccine; and d) administering the cancer vaccine to the patient. Optionally, any of the methods described herein may include the manufacture of the cancer vaccine.

[0018] In some embodiments, the cancer vaccine is a nucleic acid cancer vaccine that includes one or more nucleic acids, each having one or more open reading frames. In some embodiments, the cancer vaccine is a DNA cancer vaccine. In some embodiments, the cancer vaccine is an RNA cancer vaccine. In some embodiments, the cancer vaccine is an mRNA cancer vaccine. In some embodiments, the cancer vaccine is a peptide cancer vaccine.

[0019] In some embodiments, the cancer vaccine is administered at a dosage level sufficient to deliver 0.02-1.0 mg of the cancer vaccine to the subject. In some embodiments, the cancer vaccine is administered to the subject two, three, four or more times. In some embodiments, the cancer vaccine is administered by intradermal, intramuscular, intravascular, intratumoral, and / or subcutaneous administration. In some embodiments, the cancer vaccine is administered by intramuscular administration.

[0020] In certain embodiments, the methods and compositions described herein may be used with or for any type of cancer. In some embodiments, the cancer is selected from the group consisting of non-small cell lung cancer (NSCLC), small cell lung cancer, melanoma, bladder urothelial carcinoma, HPV-negative head and neck squamous cell carcinoma (HNSCC), solid malignancies that are microsatellite-high (MSI H) / mismatch repair (MMR) deficient, renal cancer, gastric cancer, and tumors with high tumor mutation burden. In some embodiments, the NSCLC lacks EGFR sensitizing mutations and / or ALK translocations. In some embodiments, the solid malignancies that are microsatellite-high (MSI H) / mismatch repair (MMR) deficient are selected from the group consisting of colorectal cancer, gastric adenocarcinoma, esophageal adenocarcinoma, and endometrial cancer. In some embodiments, the cancer is any one of melanoma, bladder cancer, HPV-negative HNSCC, NSCLC, SCLC, MSI-high tumors, or cancers with high tumor mutation burden (TMB).

[0021] In certain embodiments, the one or more mRNAs each comprise a 5'UTR and / or a 3'UTR. In some embodiments, the one or more mRNAs each comprise a polyA tail. In some embodiments, the polyA tail comprises about 100 nucleotides. In some embodiments, the one or more mRNAs each comprise a cap structure or a modified cap structure. In some embodiments, the cap structure or modified cap structure is a 5'cap structure, a 5'cap-0 structure, a 5'cap-1 structure, or a 5'cap-2 structure. In certain embodiments, the one or more mRNAs comprise at least one chemical modification. In some embodiments, the chemical modification is selected from the group consisting of pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methylpseudouridine, 5-methyluridine, 5-methoxyuridine, and 2'-0-methyluridine. In some embodiments, one or more mRNAs are fully modified.

[0022] In certain embodiments, the one or more nucleic acids encode 3-10 peptide epitopes, 5-10 peptide epitopes, 10-20 peptide epitopes, 20-30 peptide epitopes, 30-40 peptide epitopes, 40-50 peptide epitopes, 50-60 peptide epitopes, 60-70 peptide epitopes, 70-80 peptide epitopes, 80-90 peptide epitopes, 90-100 peptide epitopes, 100-110 peptide epitopes, 110-120 peptide epitopes, or 120-130 peptide epitopes. In some embodiments, each of the peptide epitopes is encoded by a separate open reading frame. In some embodiments, the peptide epitopes are in the form of a concatemeric cancer antigen consisting of 5-130 peptide epitopes.

[0023] In some embodiments, one or more of the following conditions are met: a) the 3-130 peptide epitopes are interspersed with cleavage-sensitive sites, and / or b) each peptide epitope is directly linked to each other without a linker, and / or c) each peptide epitope is linked to each other with a single amino acid linker, and / or d) each peptide epitope is linked to each other with a short linker, and / or e) each peptide epitope comprises 8-31 amino acids and includes one or more SNP mutations, and / or f) each peptide epitope comprises 8-31 amino acids and includes a mutation that results in a unique expressed peptide sequence, and / or g) at least 30% of the peptide epitopes have highest affinity for class I MHC molecules from the subject, and / or h) at least 30% of the peptide epitopes have highest affinity for class II MHC molecules from the subject, and / or i) none of the peptide epitopes have highest affinity for class II MHC molecules from the subject. and / or j) at least 50% of the peptide epitopes have an IC 50has a predicted binding affinity of <500 nM, and / or k) the nucleic acids encoding the peptide epitopes are arranged such that the peptide epitopes are ordered to minimize pseudo-epitopes, and / or l) the ratio of class I MHC molecule peptide epitopes to class II MHC molecule peptide epitopes is at least 1:1, 2:1, 3:1, 4:1, or 5:1, and / or m) there are no class II MHC molecule peptide epitopes.

[0024] In some embodiments, at least one of the peptide epitopes is a predicted T cell reactive epitope. In certain embodiments, at least one of the peptide epitopes is a predicted B cell reactive epitope. In some embodiments, the peptide epitopes include a combination of predicted T cell reactive epitopes and predicted B cell reactive epitopes. In certain embodiments, the peptide epitopes are predicted T cell reactive epitopes and / or predicted B cell reactive epitopes. In some embodiments, at least one of the peptide epitopes is a predicted neo-epitope. In some embodiments, at least one nucleic acid has an open reading frame encoding at least a fragment of one or more conventional cancer antigens or one or more cancer / testis antigens.

[0025] In some embodiments, each nucleic acid is formulated in a lipid nanoparticle. In some embodiments, each nucleic acid is formulated in a different lipid nanoparticle. In certain embodiments, each nucleic acid is formulated in the same lipid nanoparticle.

[0026] In some embodiments, the total length of the one or more nucleic acids encodes a total protein length of 50-100 amino acids, 100-200 amino acids, 200-300 amino acids, 300-400 amino acids, 400-500 amino acids, 500-600 amino acids, 600-700 amino acids, 700-800 amino acids, 800-900 amino acids, 900-1000 amino acids, 1000-1100 amino acids, or 1100-1200 amino acids. In some embodiments, the anti-cancer efficacy is calculated based, at least in part, on one or more factors selected from the group consisting of gene expression, RNA sequence, transcript abundance, DNA allele frequency, amino acid conservation, physiochemical similarity, cancer genes, predicted binding affinity to specific HLA alleles, clonality, binding efficiency, and presence of indels. In certain embodiments, the one or more factors are input into a statistical model (e.g., a regression model (such as a linear regression model, a logistic regression model, a general linear model, etc.), a general linear model (such as a logistic regression model, a probit regression model, etc.), a random forest regression model, a neural network, a support vector machine, a Gaussian mixture model, a hierarchical Bayesian model, and / or any other suitable statistical model).

[0027] In another aspect, the disclosure provides a computerized system for selecting nucleic acids for inclusion in a nucleic acid cancer vaccine having a maximum length, the system including: a communications interface configured to receive a plurality of sequences of nucleic acids encoding a plurality of peptide epitopes, each of the peptide epitopes being a portion of an individualized cancer antigen; at least one computer processor programmed to calculate, for each of the plurality of peptide epitopes, a score for each of a plurality of nucleic acids within the peptides, each of which includes at least one of the one or more peptide epitopes, and at least two of the nucleic acid sequences having different lengths; ranking the plurality of nucleic acid sequences within the plurality of peptides based on the calculated scores; and selecting nucleic acid sequences for inclusion in the vaccine based on the rankings and the maximum length vaccine.

[0028] In some embodiments, the minimum length of any peptide epitope is 8 amino acids. In some embodiments, the maximum length of any peptide epitope is 31 amino acids. In certain embodiments, the plurality of nucleic acids encodes 3-10 peptide epitopes, 5-10 peptide epitopes, 10-20 peptide epitopes, 20-30 peptide epitopes, 30-40 peptide epitopes, 40-50 peptide epitopes, 50-60 peptide epitopes, 60-70 peptide epitopes, 70-80 peptide epitopes, 80-90 peptide epitopes, 90-100 peptide epitopes, 100-110 peptide epitopes, 110-120 peptide epitopes, or 120-130 peptide epitopes.

[0029] In some embodiments, one or more of the following conditions are met: a) each peptide epitope comprises 8-31 amino acids and comprises one or more SNP mutations, and / or b) each peptide epitope comprises 8-31 amino acids and comprises a mutation resulting in a unique expressed peptide sequence, and / or c) at least 30% of the peptide epitopes have highest affinity for class I MHC molecules derived from the subject, and / or d) at least 30% of the peptide epitopes have highest affinity for class II MHC molecules derived from the subject, and / or e) none of the peptide epitopes have highest affinity for class II MHC molecules derived from the subject, and / or f) at least 50% of the peptide epitopes have IC for HLA-A, HLA-B, and / or DRB1. 50 and / or g) the ratio of class I MHC molecule peptide epitopes to class II MHC molecule peptide epitopes is at least 1:1, 2:1, 3:1, 4:1, or 5:1; and / or h) there are no class II MHC molecule peptide epitopes.

[0030] In some embodiments, at least one of the peptide epitopes is a predicted T cell reactive epitope. In some embodiments, at least one of the peptide epitopes is a predicted B cell reactive epitope. In some embodiments, the peptide epitopes include a combination of predicted T cell reactive epitopes and predicted B cell reactive epitopes. In certain embodiments, the peptide epitopes are predicted T cell reactive epitopes and / or predicted B cell reactive epitopes. In some embodiments, at least one of the peptide epitopes is a predicted neo-epitope. In some embodiments, at least one nucleic acid has an open reading frame encoding at least a fragment of one or more conventional cancer antigens or one or more cancer / testis antigens.

[0031] In some embodiments, the full length of the vaccine encodes a total protein length of 50-100 amino acids, 100-200 amino acids, 200-300 amino acids, 300-400 amino acids, 400-500 amino acids, 500-600 amino acids, 600-700 amino acids, 700-800 amino acids, 800-900 amino acids, 900-1000 amino acids, 1000-1100 amino acids, or 1100-1200 amino acids. In some embodiments, the score is calculated based, at least in part, on one or more factors selected from the group consisting of gene expression, RNA sequence, transcript abundance, DNA allele frequency, amino acid conservation, physiochemical similarity, cancer genes, predicted binding affinity to specific HLA alleles, clonality, binding efficiency, and presence of indels. In certain embodiments, the one or more factors are input into a statistical model (e.g., a regression model (such as a linear regression model, a logistic regression model, a general linear model, etc.), a general linear model (such as a logistic regression model, a probit regression model, etc.), a random forest regression model, a neural network, a support vector machine, a Gaussian mixture model, a hierarchical Bayesian model, and / or any other suitable statistical model).

[0032] In some embodiments, anti-tumor T cell responses are assessed for each neo-antigen, hi some embodiments, the assessment is based on the confidence of variant calls from WES and RNA-Seq data, mRNA transcript abundance from RNA-Seq data, variant allele frequency from WES and RNA-Seq data, and predicted HLA binding affinity from NetMHCpan and NetMHCIIpan.

[0033] In some embodiments, the patient's HLA allotype is identified and antigens predicted to bind to the patient's HLA are incorporated. In some embodiments, more weight may be assigned to predicted binders of HLA-A, -B, and DR (core targets) and less weight (but not zero) may be assigned to the patient's other HLA allotypes (complementary targets). Nearly all individuals have at least one HLA-A, -B, and DR functional allotype (i.e., core MHC allele), which are the restrictive elements for approximately 90% of all known human epitopes (Figure 5). HLA-C restricted or alloreactive T cells are rarely observed, and cell surface expression of HLA-C is 10% of that seen for HLA-A and B. The remaining complementary targets code for class II molecules and individuals may be null for the genes that encode them. Furthermore, four-digit precision typing of these complementary class II targets is often ambiguous, even using state-of-the-art NGS and other sequence-based typing methods. In some embodiments, if NGS-based allele typing for either the core or supplemental HLA targets is ambiguous, the allele(s) may not be considered when ranking the neo-antigens.

[0034] In some embodiments, an autologous check for each neo-antigen may be performed. In some embodiments, a patient-specific set of transcripts is created using protein-coding transcript amino acid sequences from a reference human genome annotation by aligning the sequences to the patient's own set of germline protein-coding variants. In some embodiments, this patient-specific exome (excluding the gene containing the neo-antigen) may be used to check each HLA class I-binding neo-antigen epitope (8-11-mer) for a 100% exact self-match. In some embodiments, using this tool, any neo-antigens identified elsewhere in the genome and / or transcriptome as a 100% self-match may be excluded from the mRNA construct.

[0035] All variants not excluded by the autologous check may be evaluated for inclusion in the design of patient-specific mRNA constructs. In some embodiments, predefined weights may be used rather than hard filters, based on the knowledge that MHC binding predictions are imperfect and RNA-Seq sensitivity may be limited by the tumor content of the biopsy and the depth of sequencing. [Brief description of the drawings]

[0036] [Figure 1] 1 is a table showing hotspot mutations by indication. [Diagram 2] A comparison of predicted % ranks for netMHCpan v3.0 vs. netMHCpan 4.0EL for HLA-A*02:01 is shown. Many peptides fall in and out of the 0.5% rank, which is generally considered to be the cutoff for "strong binders." [Diagram 3]Different methods of binding prediction are shown. A is a graph showing the homogeneity of predicted binders to major HLA alleles. Switching to percent rank (%Rank) leads to a more balanced distribution of predicted binders between different HLA alleles. B is a graph similarly showing the area under the curve (AUC) of different samples using different methods to predict MHC binding. The percent rank method was shown to improve prediction performance over other alternatives (e.g., IC50). [Figure 4A] Results of an in vivo immunogenicity study are shown. Comparable immune responses to class I epitopes were detected by the 20mer / 31 flank and 34mer / 25 flank vaccines, whereas the 40mer / 21 flank was undetectable at both the 3 and 10 μg doses. For several restimulations, only the 34mer construct showed a detectable response under the conditions tested. [Figure 4B] Results of an in vivo immunogenicity study are shown. Comparable immune responses to class I epitopes were detected by the 20mer / 31 flank and 34mer / 25 flank vaccines, whereas the 40mer / 21 flank was undetectable at both the 3 and 10 μg doses. For several restimulations, only the 34mer construct showed a detectable response under the conditions tested. [Figure 4C] Results of an in vivo immunogenicity study are shown. Comparable immune responses to class I epitopes were detected by the 20mer / 31 flank and 34mer / 25 flank vaccines, whereas the 40mer / 21 flank was undetectable at both the 3 and 10 μg doses. For several restimulations, only the 34mer construct showed a detectable response under the conditions tested. [Diagram 5]Core and complementary HLA targets of neoantigens are shown. A, Analysis of all known human T cell epitopes (positive in human T cell stimulation assays) using the Immune Epitope Database (IEDB, www.iedb.org / ) revealed a clear hierarchy of HLA-restricted elements with HLA-A, -B, and -DR accounting for approximately 90% of all described human epitopes in the database (n=8101). B, Restricting the IEDB search tool to only viral epitopes (n=4472) reinforced the apparent preference of T cells for these core class I and class II loci. This analysis suggests that neoantigen selection can be prioritized against mutations predicted to bind to the patient's HLA-A, -B, and -DRB1 allotypes. [Figure 6] Population analysis of somatic mutation burden. Distribution of nonsynonymous mutations in cancer histology cohorts from cBioPortal. Red, blue, and green lines represent 20, 34, and 100 mutations, respectively. [Figure 7A] We demonstrate the reproducibility of next generation sequencing (NGS) and bioinformatics system output using independent processing of four related tumor samples from a single patient. The primary tumor sample and three derived tumor cell lines were run through the NGS, variant calling, and bioinformatics systems. [Figure 7B] The reproducibility of next generation sequencing (NGS) and bioinformatics system output is shown using independent processing of four related tumor samples from a single patient. Minimal differences in the called variants between the four samples were observed. [Figure 7C] We show the reproducibility of next-generation sequencing (NGS) and bioinformatics system output using independent processing of four related tumor samples from a single patient. Correlation between the raw neoantigen scores of the 369 mutations identified (Spearman's rank correlation coefficient: tumor vs. line 1: ρ = 0.86, p = 1.92E-101, tumor vs. line 2: ρ = 0.84, p = 3.01E-89, and tumor vs. line 3: ρ = 0.84, p = 5.77E-91). [Figure 7D]Figure 1 shows the reproducibility of next generation sequencing (NGS) and bioinformatics system output using independent processing of four related tumor samples from a single patient. Venn diagram of common and unique neo-antigens selected for inclusion in representative mRNA sequences. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0037] An embodiment of the present disclosure provides a nucleic acid (e.g., DNA or RNA such as mRNA) vaccine comprising one or more nucleic acids having one or more open reading frames encoding a peptide epitope. As provided herein, a nucleic acid cancer vaccine encoding a peptide epitope of heterogeneous length may be used to induce a balanced immune response including cellular and / or humoral immunity. Methods of making a nucleic acid cancer vaccine with a given length of maximized anti-cancer efficacy are also provided herein, as well as a method of treating a patient with cancer with a cancer vaccine with a given length of maximized anti-cancer efficacy. In addition, a computerized system for making a nucleic acid cancer vaccine with a given length of maximized cancer efficacy is provided herein. The maximized anti-cancer efficacy can be determined by identifying a T cell activation or survival value, such as a maximum T cell activation or survival value, based on the length of the epitope or the nucleic acid encoding the epitope. The T cell activation or survival value can be determined using any method known in the art, for example, using a commercially available assay (Thermo Fisher Scientific, Promega Corporation, etc.). Typically, the T cell activation value is determined based on changes in cytokine expression levels, e.g., interferon gamma, associated with T cell activation or upregulation of cell surface activation markers such as 41BB and / or OX40. Survival values ​​can be assessed relative to survival in controls or population-based data on survival.

[0038] Although attempts have been made to produce nucleic acid cancer vaccines, such as RNA (e.g., mRNA) cancer vaccines, the efficacy of such vaccines remains variable.Very surprisingly, the inventors have discovered that the immune response to such cancer vaccines can be optimized through the evaluation and selection of peptide epitopes of various sizes (as opposed to the selection of peptide epitopes of uniform length / size) for inclusion in cancer vaccines.

[0039] The generation of cancer antigens that induce the desired immune response (e.g., T cell response) against the target polypeptide sequence in vaccine development remains a difficult task. The present invention involves a technology that overcomes the hurdles associated with vaccine development. In some embodiments, the nucleic acid vaccine of the present invention is superior to conventional vaccines (e.g., those that code for uniform length peptide epitopes) by at least 10-fold, 20-fold, 40-fold, 50-fold, 100-fold, 500-fold, or 1,000-fold.

[0040] As a non-limiting example, when an RNA (e.g., mRNA) nucleic acid cancer vaccine described herein is delivered to a cell, the RNA (e.g., mRNA) can be processed by intracellular machinery into polypeptides, which can then be processed into immune-sensitive fragments that can stimulate an immune response against a tumor or cancer cell population.

[0041] Peptide epitopes The nucleic acid cancer vaccine of the present disclosure can code for one or more peptide epitopes (parts of personalized cancer antigens). Parts of personalized cancer antigens are segments of personalized cancer antigens that are less than the full-length personalized cancer antigen. Personalized cancer antigens are tumor-specific antigens, also referred to as neo-antigens, that are present in the tumor of an individual and are not expressed or expressed at low levels in the normal non-cancerous tissue of the individual. The antigen may or may not be present in the tumor of other individuals.

[0042] In one embodiment, the nucleic acid cancer vaccine is composed of an open reading frame that may contain any number of peptide epitopes. In some embodiments, the nucleic acid cancer vaccine is composed of an open reading frame that may contain any number of peptide epitopes. In some embodiments, the nucleic acid cancer vaccine is composed of an open reading frame that may contain any number of peptide epitopes. In some embodiments, the nucleic acid cancer vaccine is composed of an open reading frame that may contain any number of peptide epitopes. In some embodiments, the nucleic acid cancer vaccine is composed of an open reading frame that may contain any number of peptide epitopes. In some embodiments, the nucleic acid cancer vaccine is composed of an open reading frame that may contain any number of peptide epitopes. In some embodiments, the nucleic acid cancer vaccine is composed of an open reading frame that may contain any number of peptide epitopes. or more, 50 or more, 55 or more, 60 or more, 65 or more, 70 or more, 75 or more, 80 or more, 85 or more, 90 or more, 95 or more, 100 or more, 105 or more, 110 or more, 115 or more, 120 or more, 125 or more, 130 or more, 135 or more, 140 or more, 145 or more, 150 or more, 155 or more, 160 or more, 165 or more, 170 or more, 175 or more, 180 or more, 185 or more, 190 or more, 195 or more, or 200 or more peptide epitopes. In other embodiments, the nucleic acid cancer vaccine is comprised of open reading frames encoding no more than 200, no more than 195, no more than 190, no more than 185, no more than 180, no more than 175, no more than 170, no more than 165, no more than 160, no more than 155, no more than 150, no more than 145, no more than 140, no more than 135, no more than 130, no more than 125, no more than 120, no more than 115, no more than 110, no more than 100, no more than 95, no more than 90, no more than 85, no more than 80, no more than 75, no more than 70, no more than 65, no more than 60, no more than 55, no more than 50, no more than 45, no more than 40, no more than 35, no more than 30, no more than 25, no more than 20, no more than 15, no more than 10, or no more than 5 peptide epitopes.In other embodiments, the nucleic acid cancer vaccines include up to 200, up to 195, up to 190, up to 185, up to 180, up to 175, up to 170, up to 165, up to 160, up to 155, up to 150, up to 145, up to 140, up to 135, up to 130, up to 125, up to 120, up to 115, up to 110, up to 100, up to 95, up to 96, up to 108, up to 109, up to 210, up to 211, up to 212, up to 213, up to 214, up to 215, up to 216, up to 217, up to 218, up to 219, up to 220, up to 225, up to 226, up to 228, up to 229, up to 230, up to 231, up to 232, up to 233, up to 234, up to 235, up to 236, up to 237, up to 238, up to 240, up to 245, up to 240, up to 245, up to 240, up to 245, up to 250, up to 255, up to 250, up to 255, up to 250, up to 260, up to 265, up to 265, up to 270, up to 275, up to 270, up to 275, up to 275, up to 280, up to 285, up to 285, up to 290, up to 30 up to 85, up to 80, up to 75, up to 70, up to 65, up to 60, up to 55, up to 50, up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10 peptide epitopes, up to 5 peptide epitopes, or up to 3 peptide epitopes.

[0043] In certain embodiments, the nucleic acid cancer vaccine comprises 3-10 peptide epitopes, 5-10 peptide epitopes, 10-20 peptide epitopes, 20-30 peptide epitopes, 30-40 peptide epitopes, 40-50 peptide epitopes, 50-60 peptide epitopes, 60-70 peptide epitopes, 70-80 peptide epitopes, 80-90 peptide epitopes, 90-100 peptide epitopes, 100-120 peptide epitopes, 100-140 peptide epitopes, 100-160 peptide epitopes, 100-200 peptide epitopes, 100-220 peptide epitopes, 100-240 peptide epitopes, 100-260 peptide epitopes, 100-30 peptide epitopes, 100-320 peptide epitopes, 100-40 peptide epitopes, 100-50 peptide epitopes, 100-60 peptide epitopes, 100-70 peptide epitopes, 100-80 peptide epitopes, 100-90 peptide epitopes, 100-140 peptide epitopes, 100-160 peptide epitopes, 100-240 peptide epitopes, 100-240 peptide epitopes, 100-320 peptide epitopes, 100-320 peptide epitopes, 100-40 peptide epitopes, 100-50 peptide epitopes, 100-60 peptide epitopes, 100-70 peptide epitopes, 100-80 peptide epitopes, 100-90 peptide epitopes, 100-140 peptide epitopes, 100-160 peptide epitopes, 100-240 peptide epitopes, 100 The nucleic acid sequence encoding the nucleic acid fragment may comprise one or more peptide epitopes, 100-110 peptide epitopes, 110-120 peptide epitopes, 120-130 peptide epitopes, 130-140 peptide epitopes, 140-150 peptide epitopes, 150-160 peptide epitopes, 160-170 peptide epitopes, 170-180 peptide epitopes, 180-190 peptide epitopes, or 190-200 peptide epitopes.

[0044] In certain embodiments, the nucleic acid cancer vaccine is 2-200, 5-200, 8-200, 10-200, 2-190, 5-190, 8-190, 10-190, 2-180, 5-180, 8-180, 10-180, 2-170, 5-170, 8-170, 10-170, 2-160, 5-160, 8-160, 10-160, 2-150, 5-150, 8-150, 10-150, 2-145, 5-145, 8-145, 10-145, 2-140, 5-140, 8-140, 10-140, 2-139, 5-139, 8-139, 10-139, 2~138, 5~138, 8~138, 10~138, 2~137, 5~137, 8~137, 10~137, 2~136, 5~136, 8~136, 10~136, 2~135, 5~135, 8~135, 10~135, 2~134, 5~134, 8~134, 10~134 , 2~133, 5~133, 8~133, 10~133, 2~132, 5~132, 8~132, 10~132, 2~131, 5~131, 8~131, 10~131, 2~130, 5~130, 8~130, 10~130, 2~129, 5~129, 8~129, 10~12 9, 2~128, 5~128, 8~128, 10~128, 2~127, 5~127, 8~127, 10~127, 2~126, 5~126, 8~126, 10~126, 2~125, 5~125, 8~125, 10~125, 2~124, 5~124, 8~124, 10~ 124, 2-123, 5-123, 8-123, 10-123, 2-122, 5-122, 8-122, 10-122, 2-121, 5-121, 8-121, 10-121, 2-120, 5-120, 8-120, 10-120, 2-119, 5-119, 8-119, 10 ~119, 2~118, 5~118, 8~118, 10~118, 2~117, 5~117, 8~117, 10~117, 2~116, 5~116, 8~116, 10~116, 2~115, 5~115, 8~115, 10~115, 2~114, 5~114, 8~114, 1 0~114, 2~113, 5~113, 8~113, 10~113, 2~112, 5~112, 8~112, 10~112, 2~111, 5~111, 8~111, 10~111, 2~110, 5~110, 8~110, 10~110, 2~100, 5~100, 8~100,Or it encodes 10 to 100 peptide epitopes.

[0045] In other embodiments, the nucleic acid cancer vaccine is 2-95, 5-95, 8-95, 10-95, 2-90, 5-90, 8-90, 10-85, 2-85, 5-85, 8-85, 10-85, 2-80, 5-80, 8-80, 10-80, 2-85, 5-85, 8-85, 10-85, 2-80, 5-80, 8-80, 10-80, 2-75, 5-75, 8-75, 10-75, 2-70, 5-70, 8-70, 10-70, 2-65, 5-65, 8-65, 10-65, 2-60, 5-60, 8-60, 10-60, 2-55, 5-55, 8~55, 10~55, 2~50, 5~50, 8~50, 10~50, 2~45, 5~45, 8~45, 10~45, 2~40, 5~40, 8~40, 10~40, 2~39, 5~39, 8~39, 10~39, 2~38, 5~38, 8~38, 10~38, 2~37, 5~37, 8~37, 10~37, 2~36, 5~36, 8~36, 10~36, 2~35, 5~35, 8~35, 10~35, 2~34, 5~34, 8~34, 10~34, 2~33, 5~33, 8~33, 10~33, 2~32, 5~32, 8~32, 10~ 32, 2-31, 5-31, 8-31, 10-31, 2-30, 5-30, 8-30, 10-30, 2-29, 5-29, 8-29, 10-29, 2-28, 5-28, 8-28, 10-28, 2-27, 5-27, 8-27, 10-27, 2-26, 5-26, 8-26, 10-26, 2-25, 5-25, 8-25, 10-25, 2-24, 5-24, 8-24, 10-24, 2-23, 5-23, 8-23, 10-23, 2-22, 5-22, 8-22, 10-22, 2-21, 5-21, 8-21, 10-21, 2-20, and encoding 5-20, 8-20, 10-20, 2-19, 5-19, 8-19, 10-19, 2-18, 5-18, 8-18, 10-18, 2-17, 5-17, 8-17, 10-17, 2-16, 5-16, 8-16, 10-16, 2-15, 5-15, 8-15, 10-15, 2-14, 5-14, 8-14, 10-14, 2-13, 5-13, 8-13, 10-13, 2-12, 5-12, 8-12, 10-12, 2-11, 5-11, 8-11, 10-11, 2-10, 5-10, or 8-10 peptide epitopes.

[0046] In still other embodiments, the nucleic acid cancer vaccine is 20 to 200, 30 to 200, 40 to 200, 50 to 200, 20 to 180, 30 to 180, 40 to 180, 50 to 180, 20 to 170, 30 to 170, 40 to 170, 50 to 170, 20 to 160, 30 to 160, 40 to 160, 20 to 150, 30 to 150, 40 to 150, 50 to 150, 20 The nucleic acid vaccine encodes up to 140, 30 to 140, 40 to 140, 50 to 140, 20 to 130, 20 to 130, 40 to 130, 50 to 130, 20 to 120, 30 to 120, 40 to 120, 50 to 120, 20 to 110, 30 to 110, 40 to 110, 50 to 110, 20 to 100, 30 to 100, 40 to 100, or 50 to 100 peptide epitopes. In one embodiment, the nucleic acid vaccine encodes 34 peptide epitopes.

[0047] In some embodiments, the nucleic acid cancer vaccines and vaccination methods described herein include an open reading frame that encodes an epitope or antigen based on a specific mutation (neoepitope) and / or a mutation expressed by a cancer germline gene (an antigen common to tumors found in multiple patients).

[0048] As used herein, an epitope, also known as an antigenic determinant, is a portion of an antigen that is recognized by the immune system, specifically by antibodies, B cells, or T cells, in the appropriate context. Epitopes can include B cell epitopes (e.g., predicted B cell reactive epitopes) and T cell epitopes (e.g., predicted T cell reactive epitopes). B cell epitopes (e.g., predicted B cell reactive epitopes) are peptide sequences required for recognition by specific antibody-producing B cells. B cell epitopes (e.g., predicted B cell reactive epitopes) refer to specific regions of an antigen that are recognized by antibodies. T cell epitopes (e.g., predicted T cell reactive epitopes) are peptide sequences that, in association with proteins on APCs, are required for recognition by specific T cells. T cell epitopes (e.g., predicted T cell reactive epitopes) are processed intracellularly and presented on the surface of APCs, where they bind to MHC molecules, including MHC class II and MHC class I molecules. The part of the antibody that binds to the epitope is called the paratope. The epitope can be a conformational epitope or a linear epitope, based on the structure and interaction with the paratope. A linear or continuous epitope is defined by the primary amino acid sequence of a particular region of the protein. The sequences that interact with the antibody are located sequentially adjacent to each other on the protein, and the epitope can usually be mimicked by a single peptide. A conformational epitope is an epitope that is defined by the conformation of the native protein. These epitopes can be continuous or discontinuous (i.e., perhaps the components of the epitope are located in different parts of the protein that are brought close to each other in the folded native protein structure).

[0049] Each peptide epitope can be any length that is reasonable for the epitope. In some embodiments, the length of each peptide epitope is not necessarily equal. In some embodiments, each peptide epitope in the nucleic acid cancer vaccine is different length. In certain embodiments, at least two (e.g., at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, and including up to and all) of the peptide epitopes in the nucleic acid cancer vaccine are different length.

[0050] In some embodiments, the length of at least one of the peptide epitopes is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, or at least 100 amino acids. In other embodiments, the length of at least one of the peptide epitopes is 100 or less, 95 or less, 90 or less, 85 or less, 80 or less, 75 or less, 70 or less, 65 or less, 60 or less, 55 or less, 50 or less, 45 or less, 40 or less, 35 or less, 30 or less, 25 or less, 20 or less, 15 or less, 14 or less, 13 or less, 12 or less, 11 or less, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less amino acids. In other embodiments, the length of at least one of the peptide epitopes is up to 100, up to 95, up to 90, up to 85, up to 80, up to 75, up to 70, up to 65, up to 60, up to 55, up to 50, up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, or up to 10 amino acids.

[0051] In some embodiments, each peptide epitope may be 5-100 amino acids in length (inclusive). In some embodiments, the length of at least one of the peptide epitopes is 5-100, 5-95, 5-90, 5-85, 5-80, 5-75, 5-70, 5-65, 5-60, 5-55, 5-50, 5-45, 5-40, 5-39, 5-38, 5-37, 5-36, 5-35, 5-34, 5-33, 5-32, 5-40, 5-50, 5-60, 5-7 ...85, 5-95, 5-90, 5-85, 5-90, 5-100, 5-120, 5-140, 5-160, 5-180, 5-190, 5-200, 5-210, 5-220, 5-230, 5-240, 5-250, 5-260, 5-270, 5-280, 5-290, 5-300, 5-310, 5-320, 5-330, 5-340, 5-350, 5 ~31, 5~30, 5~29, 5~28, 5~27, 5~26, 5~25, 5~24, 5~23, 5~22, 5~21, 5~20, 8~100, 8~95, 8~90, 8~85, 8~80, 8~75, 8~70, 8~65, 8~60, 8~55, 8~50, 8~45, 8~40, 8~39, 8~38, 8~37, 8~36, 8~35, 8~34, 8~33, 8~32, 8~31, 8~30, 8~29, 8~28, 8~27, 8~26, 8~25, 8~24, 8~23, 8~22, 8~21, 8~20, 10~100, 10~95, 10~90, 10~85, 10~80, 10~75, 10~70, 10~65, 10~60, 10~55, 10~ 50, 10-45, 10-40, 10-39, 10-38, 10-37, 10-36, 10-35, 10-34, 10-33, 10-32, 10-31, 10-30, 10-29, 10-28, 10-27, 10-26, 10-25, 10-24, 10-23, 10-22, 10-21, or 10-20 amino acids.

[0052] In some embodiments, each peptide epitope encoded by nucleic acid cancer vaccine can have different length.In certain embodiments, at least one of peptide epitopes has different length from another peptide epitope encoded by nucleic acid cancer vaccine.Each peptide epitope can be any length that is reasonable for the epitope.

[0053] In some embodiments, different percentages of the peptide epitope length are encoded by the nucleic acid. All percentages listed below can be approximate (i.e., within 5% of the amount listed). The use of the terms "approximately" and "about" are equivalent.

[0054] In some embodiments, the percentage of peptide epitope length encoded by a nucleic acid can be as follows: about 100% < 15 amino acids, about 0% > 15 amino acids, about 95% < 15 amino acids, about 5% > 15 amino acids, about 90% < 15 amino acids, about 10% > 15 amino acids, about 85% < 15 amino acids, about 15% > 15 amino acids, about 80% < 15 amino acids, about 20% > 15 amino acids, about 75% < 15 amino acids, about 25% > 15 amino acids, about 70% < 15 amino acids, about 30% > 15 amino acids, about 65% < 15 amino acids, about 35% > 15 amino acids, about 60% < 15 amino acids, about 40% > 15 amino acids, about 55% < 15 amino acids. 5%≧15 amino acids, about 45%≧15 amino acids, about 50%<15 amino acids, about 50%≧15 amino acids, about 45%<15 amino acids, about 55%≧15 amino acids, about 40%<15 amino acids, about 60%≧15 amino acids, about 35%<15 amino acids, about 65%≧15 amino acids, about 30%<15 amino acids, about 70%≧15 amino acids, about 25%<15 amino acids, about 75%≧15 amino acids, about 20%<15 amino acids, about 80%≧15 amino acids, about 15%<15 amino acids, about 85%≧15 amino acids, about 10%<15 amino acids, about 90%≧15 amino acids, about 5%<15 amino acids, about 95%≧15 amino acids, or about 0%<15 amino acids, about 100%≧15 amino acids.

[0055] In some embodiments, the percentage of peptide epitope length encoded by a nucleic acid can be as follows: about 100% < 17 amino acids, about 0% > 17 amino acids, about 95% < 17 amino acids, about 5% > 17 amino acids, about 90% < 17 amino acids, about 10% > 17 amino acids, about 85% < 17 amino acids, about 17% > 17 amino acids, about 80% < 17 amino acids, about 20% > 17 amino acids, about 75% < 17 amino acids, about 25% > 17 amino acids, about 70% < 17 amino acids, about 30% > 17 amino acids, about 65% < 17 amino acids, about 35% > 17 amino acids, about 60% < 17 amino acids, about 40% > 17 amino acids, about 55% < 17 amino acids. 17 amino acids, about 45%≧17 amino acids, about 50%<17 amino acids, about 50%≧17 amino acids, about 45%<17 amino acids, about 55%≧17 amino acids, about 40%<17 amino acids, about 60%≧17 amino acids, about 35%<17 amino acids, about 65%≧17 amino acids, about 30%<17 amino acids, about 70%≧17 amino acids, about 25%<17 amino acids, about 75%≧17 amino acids, about 20%<17 amino acids, about 80%≧17 amino acids, about 17%<17 amino acids, about 85%≧17 amino acids, about 10%<17 amino acids, about 90%≧17 amino acids, about 5%<17 amino acids, about 95%≧17 amino acids, or about 0%<17 amino acids, about 100%≧17 amino acids.

[0056] In some embodiments, the percentage of peptide epitope length encoded by a nucleic acid can be as follows: about 100% < 19 amino acids, about 0% > 19 amino acids, about 95% < 19 amino acids, about 5% > 19 amino acids, about 90% < 19 amino acids, about 10% > 19 amino acids, about 85% < 19 amino acids, about 19% > 19 amino acids, about 80% < 19 amino acids, about 20% > 19 amino acids, about 75% < 19 amino acids, about 25% > 19 amino acids, about 70% < 19 amino acids, about 30% > 19 amino acids, about 65% < 19 amino acids, about 35% > 19 amino acids, about 60% < 19 amino acids, about 40% > 19 amino acids, about 55% < 19 amino acids. 19 amino acids, about 45%≧19 amino acids, about 50%<19 amino acids, about 50%≧19 amino acids, about 45%<19 amino acids, about 55%≧19 amino acids, about 40%<19 amino acids, about 60%≧19 amino acids, about 35%<19 amino acids, about 65%≧19 amino acids, about 30%<19 amino acids, about 70%≧19 amino acids, about 25%<19 amino acids, about 75%≧19 amino acids, about 20%<19 amino acids, about 80%≧19 amino acids, about 19%<19 amino acids, about 85%≧19 amino acids, about 10%<19 amino acids, about 90%≧19 amino acids, about 5%<19 amino acids, about 95%≧19 amino acids, or about 0%<19 amino acids, about 100%≧19 amino acids.

[0057] In some embodiments, peptide epitopes lengths may fall into one of the following groups (totaling 100%): 8-12 amino acids, 13-17 amino acids, 18-21 amino acids, 22-26 amino acids, or 27-31 amino acids. About 0%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the peptide epitopes encoded by the open reading frames of the nucleic acid may be 8-12 amino acids in length. About 0%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the peptide epitopes encoded by the open reading frames of the nucleic acid can be 13-17 amino acids in length. About 0%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the peptide epitopes encoded by the open reading frames of the nucleic acid can be 18-21 amino acids in length. About 0%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the peptide epitopes encoded by the open reading frames of the nucleic acid can be 22-26 amino acids in length. About 0%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the peptide epitopes encoded by the open reading frames of the nucleic acid can be 27-31 amino acids in length. Some non-limiting examples of the percentage of peptide epitope length encoded by an open reading frame of a nucleic acid follow.

[0058] In some embodiments, the percentages of peptide epitope length encoded by the nucleic acid may be as follows: 50% 8-12 amino acids, 50% 13-17 amino acids, 0% 18-21 amino acids, 0% 22-26 amino acids, and 0% 27-31 amino acids; 0% 8-12 amino acids, 50% 13-17 amino acids, 50% 18-21 amino acids, 0% 22-26 amino acids, and 0% 27-31 amino acids; 0% 8-12 amino acids, 0% 13-17 amino acids, 50% 18-21 amino acids, 50% 22-26 amino acids, and 0% 27-31 amino acids; 0% 8-12 amino acids, 0% 13-17 amino acids, 50% 18-21 amino acids, 50% 22-26 amino acids, and 0% 27-31 amino acids; 0% 8-12 amino acids, 0% 13-17 amino acids, 0% 18-21 amino acids, 50% 22-26 amino acids, and 50% 27-31 amino acids; 13-17 amino acids, 50% 18-21 amino acids, 0% 22-26 amino acids, and 0% 27-31 amino acids; 50% 8-12 amino acids, 0% 13-17 amino acids, 0% 18-21 amino acids, 50% 22-26 amino acids, and 0% 27-31 amino acids; 50% 8-12 amino acids, 0% 13-17 amino acids, 0% 18-21 amino acids, 0% 22-26 amino acids, and 50% 27-31 amino acids; 0% 8-12 amino acids, 50% 13-17 amino acids, 50% 18-21 amino acids, 0% 22-26 amino acids, and 0% 27-31 amino acids; 0% 8-12 amino acids, 50% 13-17 amino acids, 50% 18-21 amino acids, 0% 22-26 amino acids, and 0% 27-31 amino acids; 0% 8-12 amino acids, 50% 13-17 amino acids, 0% 18-21 amino acids, 50% 22-26 amino acids, and 0% 27-31 amino acids; 0% 8-12 amino acids, 50% 13-17 amino acids, 0% 18-21 amino acids, 0% 22-26 amino acids, and 50% 27-31 amino acids; or 0% 8-12 amino acids, 0% 13-17 amino acids, 50% 18-21 amino acids, 0% 22-26 amino acids, and 50% 27-31 amino acids.

[0059] In some embodiments, the percentages of peptide epitope lengths encoded by the nucleic acid can be as follows: 10% 8-12 amino acids, 40% 13-17 amino acids, 40% 18-21 amino acids, 10% 22-26 amino acids, and 0% 27-31 amino acids; 10% 8-12 amino acids, 10% 13-17 amino acids, 40% 18-21 amino acids, 40% 22-26 amino acids, and 0% 27-31 amino acids; 40% 8-12 amino acids, 40% 13-17 amino acids, 10% 18-21 amino acids, 10% 22-26 amino acids, and 0% 27-31 amino acids; 10% 8-12 amino acids, 40% 13-17 amino acids, 10% 18-21 amino acids, 40% 22-26 amino acids, and 0% 27-31 amino acids; 8-12 amino acids, 10% 13-17 amino acids, 40% 18-21 amino acids, 10% 22-26 amino acids, and 0% 27-31 amino acids; 0% 8-12 amino acids, 10% 13-17 amino acids, 40% 18-21 amino acids, 40% 22-26 amino acids, and 10% 27-31 amino acids; 0% 8-12 amino acids, 10% 13-17 amino acids, 10% 18-21 amino acids, 40% 22-26 amino acids, and 40% 27-31 amino acids; 0% 8-12 amino acids, 40% 13-17 amino acids, 40% 18-21 amino acids, 10% 22-26 amino acids, and 10% 27-31 amino acids; 0% 8-12 amino acids, 10% 13-17 amino acids, 40% 18-21 amino acids, 10% 22-26 amino acids, and 40% 27-31 amino acids; 0% 8-12 amino acids, 40% 13-17 amino acids, 10% 18-21 amino acids, 40% 22-26 amino acids, and 10% 27-31 amino acids.

[0060] In some embodiments, the percentages of peptide epitope length encoded by the nucleic acid may be as follows: 25% 8-12 amino acids, 25% 13-17 amino acids, 25% 18-21 amino acids, 25% 22-26 amino acids, and 0% 27-31 amino acids; 25% 8-12 amino acids, 25% 13-17 amino acids, 25% 18-21 amino acids, 0% 22-26 amino acids, and 25% 27-31 amino acids; 25% 8-12 amino acids, 25% 13-17 amino acids, 0% 18-21 amino acids, 25% 22-26 amino acids, and 25% 27-31 amino acids; 25% 8-12 amino acids, 0% 13-17 amino acids, 25% 18-21 amino acids, 25% 22-26 amino acids, and 25% 27-31 amino acids; 0% 8-12 amino acids, 25% 13-17 amino acids, 25% 18-21 amino acids, 25% 22-26 amino acids, and 25% 27-31 amino acids.

[0061] In some embodiments, the percentages of peptide epitope lengths encoded by the nucleic acid can be as follows: 15% 8-12 amino acids, 15% 13-17 amino acids, 15% 18-21 amino acids, 15% 22-26 amino acids, and 40% 27-31 amino acids; 15% 8-12 amino acids, 15% 13-17 amino acids, 15% 18-21 amino acids, 15% 22-26 amino acids, and 40% 27-31 amino acids; 15% 8-12 amino acids, 15% 13-17 amino acids, 15% 18-21 amino acids, 15% 22-26 amino acids, and 40% 27-31 amino acids; 15% 8-12 amino acids, 15% 13-17 amino acids, 15% 18-21 amino acids, 15% 22-26 amino acids, and 40% 27-31 amino acids; 15% 8-12 amino acids, 15% 13-17 amino acids, 15% 18-21 amino acids, 15% 22-26 amino acids, and 40% 27-31 amino acids; 15% 8-12 amino acids, 15% 13-17 amino acids, 15% 18-21 amino acids, 15% 22-26 amino acids, and 40% 27-31 amino acids; 40% 8-12 amino acids, 15% 13-17 amino acids, 15% 18-21 amino acids, 15% 22-26 amino acids, and 15 ... 13-17 amino acids, 15% 18-21 amino acids, 15% 22-26 amino acids, and 15% 27-31 amino acids; 40% 8-12 amino acids, 15% 13-17 amino acids, 15% 18-21 amino acids, 15% 22-26 amino acids, and 15% 27-31 amino acids.

[0062] In some embodiments, the percentages of peptide epitope lengths encoded by the nucleic acid can be as follows: 10% 8-12 amino acids, 10% 13-17 amino acids, 10% 18-21 amino acids, 10% 22-26 amino acids, and 60% 27-31 amino acids; 10% 8-12 amino acids, 10% 13-17 amino acids, 10% 18-21 amino acids, 10% 22-26 amino acids, and 60% 27-31 amino acids; 10% 8-12 amino acids, 10% 13-17 amino acids, 10% 18-21 amino acids, 10% 22-26 amino acids, and 60% 27-31 amino acids; 10% 8-12 amino acids, 10% 13-17 amino acids, 10% 18-21 amino acids, 10% 22-26 amino acids, and 60% 27-31 amino acids; 10% 8-12 amino acids, 10% 13-17 amino acids, 10% 18-21 amino acids, 10% 22-26 amino acids, and 60% 27-31 amino acids; 10% 8-12 amino acids, 10% 13-17 amino acids, 10% 18-21 amino acids, 10% 22-26 amino acids, and 60% 27-31 amino acids; 60% 8-12 amino acids, 10% 13-17 amino acids, 10% 18-21 amino acids, 10% 22-26 amino acids, and 1 ... 13-17 amino acids, 10% 18-21 amino acids, 10% 22-26 amino acids, and 10% 27-31 amino acids; 60% 8-12 amino acids, 10% 13-17 amino acids, 10% 18-21 amino acids, 10% 22-26 amino acids, and 10% 27-31 amino acids.

[0063] In some embodiments, the percentages of peptide epitope lengths encoded by the nucleic acid can be as follows: 15% 8-12 amino acids, 20% 13-17 amino acids, 20% 18-21 amino acids, 15% 22-26 amino acids, and 30% 27-31 amino acids; 15% 8-12 amino acids, 15% 13-17 amino acids, 20% 18-21 amino acids, 20% 22-26 amino acids, and 30% 27-31 amino acids; 20% 8-12 amino acids, 20% 13-17 amino acids, 15% 18-21 amino acids, 15% 22-26 amino acids, and 30% 27-31 amino acids; 15% 8-12 amino acids, 20% 13-17 amino acids, 15% 18-21 amino acids, 20% 22-26 amino acids, and 30% 27-31 amino acids. 27-31 amino acids; 20% 8-12 amino acids, 15% 13-17 amino acids, 20% 18-21 amino acids, 15% 22-26 amino acids, and 30% 27-31 amino acids; 30% 8-12 amino acids, 15% 13-17 amino acids, 20% 18-21 amino acids, 20% 22-26 amino acids, and 15% 27-31 amino acids; 30% 8-12 amino acids, 15% 13-17 amino acids, 15% 18-21 amino acids, 20% 22-26 amino acids, and 20% 27-31 amino acids; 30% 8-12 amino acids, 20% 13-17 amino acids, 20% 18-21 amino acids, 15% 22-26 amino acids, and 15% 27-31 amino acids; 30% 8-12 amino acids, 20% 13-17 amino acids, 20% 18-21 amino acids, 15% 22-26 amino acids, and 15% 27-31 amino acids; 30% 8-12 amino acids, 15% 13-17 amino acids, 20% 18-21 amino acids, 15% 22-26 amino acids, and 20% 27-31 amino acids; 30% 8-12 amino acids, 20% 13-17 amino acids, 15% 18-21 amino acids, 20% 22-26 amino acids, and 15% 27-31 amino acids.

[0064] In some embodiments, the percentages of peptide epitope lengths encoded by the nucleic acid can be as follows: 35% 8-12 amino acids, 35% 13-17 amino acids, 10% 18-21 amino acids, 10% 22-26 amino acids, and 10% 27-31 amino acids; 10% 8-12 amino acids, 35% 13-17 amino acids, 35% 18-21 amino acids, 10% 22-26 amino acids, and 10% 27-31 amino acids; 10% 8-12 amino acids, 10% 13-17 amino acids, 35% 18-21 amino acids, 35% 22-26 amino acids, and 10% 27-31 amino acids; 10% 8-12 amino acids, 10% 13-17 amino acids, 35% 18-21 amino acids, 35% 22-26 amino acids, and 10% 27-31 amino acids; 10% 8-12 amino acids, 10% 13-17 amino acids, 10% 18-21 amino acids, 35% 22-26 amino acids, and 35% 27-31 amino acids; 35% 8-12 amino acids, 10% 13-17 amino acids, 35% 18-21 amino acids, 10% 22-26 amino acids, and 10% 27-31 amino acids; 35% 8-12 amino acids, 10% 13-17 amino acids, 10% 18-21 amino acids, 35% 22-26 amino acids, and 10% 27-31 amino acids; 35% 8-12 amino acids, 10% 13-17 amino acids, 10% 18-21 amino acids, 10% 22-26 amino acids, and 35% 27-31 amino acids; 10% 8-12 amino acids, 35% 13-17 amino acids, 10% 18-21 amino acids, 35% 22-26 amino acids, and 10% 27-31 amino acids; 10% 8-12 amino acids, 35% 13-17 amino acids, 10% 18-21 amino acids, 35% 22-26 amino acids, and 10% 27-31 amino acids; 10% 8-12 amino acids, 35% 13-17 amino acids, 10% 18-21 amino acids, 10% 22-26 amino acids, and 35% 27-31 amino acids.

[0065] In some embodiments, the percentage of peptide epitope length encoded by the nucleic acid may be as follows: 30% 8-12 amino acids, 30% 13-17 amino acids, 30% 18-21 amino acids, 5% 22-26 amino acids, and 5% 27-31 amino acids; 5% 8-12 amino acids, 30% 13-17 amino acids, 30% 18-21 amino acids, 30% 22-26 amino acids, and 5% 27-31 amino acids; 5% 8-12 amino acids, 5% 13-17 amino acids, 30% 18-21 amino acids, 30% 22-26 amino acids, and 30% 27-31 amino acids; 30% 8-12 amino acids, 5% 13-17 amino acids, 5% 18-21 amino acids, 30% 22-26 amino acids, and 30% 27-31 amino acids; 8-12 amino acids, 30% 13-17 amino acids, 5% 18-21 amino acids, 5% 22-26 amino acids, and 30% 27-31 amino acids; 5% 8-12 amino acids, 30% 13-17 amino acids, 5% 18-21 amino acids, 30% 22-26 amino acids, and 30% 27-31 amino acids; 5% 8-12 amino acids, 30% 13-17 amino acids, 30% 18-21 amino acids, 5% 22-26 amino acids, and 30% 27-31 amino acids; 30% 8-12 amino acids, 30% 13-17 amino acids, 5% 18-21 amino acids, 30% 22-26 amino acids, and 5% 27-31 amino acids; 30% 8-12 amino acids, 30% 13-17 amino acids, 5% 18-21 amino acids, 30% 22-26 amino acids, and 5% 27-31 amino acids; 30% 8-12 amino acids, 5% 13-17 amino acids, 30% 18-21 amino acids, 5% 22-26 amino acids, and 30% 27-31 amino acids.

[0066] In some embodiments, the percentage of peptide epitope length encoded by the nucleic acid can be as follows: 20% 8-12 amino acids, 20% 13-17 amino acids, 20% 18-21 amino acids, 20% 22-26 amino acids, and 20% 27-31 amino acids.

[0067] In some embodiments, the optimal length of the peptide epitope can be obtained via the following procedure: synthesize a V5 tag concatemer test protease site, introduce it into DC cells (e.g., using the RNA Squeeze procedure), lyse the cells, and then perform an anti-V5 Western blot to assess cleavage at the protease site.

[0068] The RNA Squeeze technique is an intracellular delivery method capable of delivering a variety of materials to a wide range of living cells. Cells are subjected to a microfluidic structure that induces rapid mechanical deformation. The deformation results in temporary membrane rupture and newly formed transient pores. The material is then passively diffused into the cell cytosol through the transient pores. The technique can be used with a variety of cell types, including primary fibroblasts, embryonic stem cells, and a host of immune cells, and has been shown to have relatively high viability in most applications and not damage sensitive materials such as quantum dots or proteins through its action. Sharei et al.,PNAS(2013);110(6):2082-7.

[0069] The peptide epitopes described herein can be encoded in any order in the nucleic acid. For example, each of the peptide epitopes can have a length that can be classified into one of the following groups (totaling 100%): 8-12 amino acids (represented by "A"), 13-17 amino acids (represented by "B"), 18-21 amino acids (represented by "C"), 22-26 amino acids (represented by "D"), or 27-31 amino acids (represented by "E"). One or more peptide epitopes of any group (e.g., 8-12 aa) can be encoded contiguously by the nucleic acid (e.g., the nucleic acid can encode two or more peptide epitopes of length "A" contiguously, which epitopes can be directly linked or indirectly linked as described elsewhere herein). In addition, peptide epitopes of different groups may be interspersed, and a nucleic acid may encode epitopes of different groups contiguous (e.g., a nucleic acid may encode a peptide epitope of length A next to a peptide epitope of length B, C, D, or E, and these epitopes may be directly linked or indirectly linked, as described elsewhere herein).

[0070] As non-limiting examples, peptide epitopes can be encoded in a nucleic acid as follows, or a nucleic acid can encode (at least in part) one of the following combinations of peptide epitopes: (A) 1~50 (B) 1~50 (C) 1~50 (D) 1~50 (E) 1~50 , (A) 1~50 (B) 1~50 (C) 1~50 (E) 1~50 (D) 1~50 , (A) 1~50 (B) 1~50 (D) 1~50 (C) 1~50 (E) 1~50 , (A) 1~50 (B) 1~50 (D) 1~50 (E) 1~50 (C) 1~50 , (A) 1~50 (B)1~50 (E) 1~50 (C) 1~50 (D) 1~50 、(A) 1~50 (B) 1~50 (E) 1~50 (D) 1~50 (C) 1~50 、(A) 1~50 (C) 1~50 (D) 1~50 (E) 1~50 (B) 1~50 、(A) 1~50 (C) 1~50 (D) 1~50 (B) 1~50 (E) 1~50 、(A) 1~50 (C) 1~50 (E) 1~50 (D) 1~50 (B) 1~50 、(A) 1~50 (C) 1~50 (E) 1~50 (B) 1~50 (D) 1~50 、(A) 1~50 (C) 1~50 (B) 1~50 (E) 1~50 (D) 1~50 、(A) 1~50 (C) 1~50 (B) 1~50 (D) 1~50 (E) 1~50 、(A) 1~50 (D) 1~50 (C) 1~50 (B) 1~50 (E) 1~50 、(A) 1~50 (D) 1~50 (C) 1~50 (E) 1~50 (B) 1~50 、(A) 1~50 (D) 1~50 (B) 1~50 (C) 1~50 (E) 1~50 、(A) 1~50 (D) 1~50 (B) 1~50 (E) 1~50 (C) 1~50 、(A) 1~50 (D) 1~50 (E) 1~50(B) 1~50 (C) 1~50 、(A) 1~50 (D) 1~50 (E) 1~50 (C) 1~50 (B) 1~50 、(A) 1~50 (E) 1~50 (C) 1~50 (B) 1~50 (D) 1~50 、(A) 1~50 (E) 1~50 (C) 1~50 (D) 1~50 (B) 1~50 、(A) 1~50 (E) 1~50 (B) 1~50 (C) 1~50 (D) 1~50 、(A) 1~50 (E) 1~50 (B) 1~50 (D) 1~50 (C) 1~50 、(A) 1~50 (E) 1~50 (D) 1~50 (B) 1~50 (C) 1~50 、(A) 1~50 (E) 1~50 (D) 1~50 (C) 1~50 (B) 1~50 、(B) 1~50 (A) 1~50 (C) 1~50 (D) 1~50 (E) 1~50 、(B) 1~50 (A) 1~50 (C) 1~50 (E) 1~50 (D) 1~50 、(B) 1~50 (A) 1~50 (D) 1~50 (C) 1~50 (E) 1~50 、(B) 1~50 (A) 1~50 (D) 1~50 (E) 1~50 (C) 1~50 、(B) 1~50 (A) 1~50 (E) 1~50 (C) 1~50 (D)1~50 、(B) 1~50 (A) 1~50 (E) 1~50 (D) 1~50 (C) 1~50 、(B) 1~50 (C) 1~50 (D) 1~50 (E) 1~50 (A) 1~50 、(B) 1~50 (C) 1~50 (D) 1~50 (A) 1~50 (E) 1~50 、(B) 1~50 (C) 1~50 (E) 1~50 (D) 1~50 (A) 1~50 、(B) 1~50 (C) 1~50 (E) 1~50 (A) 1~50 (D) 1~50 、(B) 1~50 (C) 1~50 (A) 1~50 (E) 1~50 (D) 1~50 、(B) 1~50 (C) 1~50 (A) 1~50 (D) 1~50 (E) 1~50 、(B) 1~50 (D) 1~50 (C) 1~50 (A) 1~50 (E) 1~50 、(B) 1~50 (D) 1~50 (C) 1~50 (E) 1~50 (A) 1~50 、(B) 1~50 (D) 1~50 (A) 1~50 (C) 1~50 (E) 1~50 、(B) 1~50 (D) 1~50 (A) 1~50 (E) 1~50 (C) 1~50 、(B) 1~50 (D) 1~50 (E) 1~50 (A) 1~50 (C) 1~50 、(B) 1~50(D) 1~50 (E) 1~50 (C) 1~50 (A) 1~50 、(B) 1~50 (E) 1~50 (C) 1~50 (A) 1~50 (D) 1~50 、(B) 1~50 (E) 1~50 (C) 1~50 (D) 1~50 (A) 1~50 、(B) 1~50 (E) 1~50 (A) 1~50 (C) 1~50 (D) 1~50 、(B) 1~50 (E) 1~50 (A) 1~50 (D) 1~50 (C) 1~50 、(B) 1~50 (E) 1~50 (D) 1~50 (A) 1~50 (C) 1~50 、(B) 1~50 (E) 1~50 (D) 1~50 (C) 1~50 (A) 1~50 、(C) 1~50 (B) 1~50 (A) 1~50 (D) 1~50 (E) 1~50 、(C) 1~50 (B) 1~50 (A) 1~50 (E) 1~50 (D) 1~50 、(C) 1~50 (B) 1~50 (D) 1~50 (A) 1~50 (E) 1~50 、(C) 1~50 (B) 1~50 (D) 1~50 (E) 1~50 (A) 1~50 、(C) 1~50 (B) 1~50 (E) 1~50 (A) 1~50 (D) 1~50 、(C) 1~50 (B) 1~50 (E)1~50 (D) 1~50 (A) 1~50 、(C) 1~50 (A) 1~50 (D) 1~50 (E) 1~50 (B) 1~50 、(C) 1~50 (A) 1~50 (D) 1~50 (B) 1~50 (E) 1~50 、(C) 1~50 (A) 1~50 (E) 1~50 (D) 1~50 (B) 1~50 、(C) 1~50 (A) 1~50 (E) 1~50 (B) 1~50 (D) 1~50 、(C) 1~50 (A) 1~50 (B) 1~50 (E) 1~50 (D) 1~50 、(C) 1~50 (A) 1~50 (B) 1~50 (D) 1~50 (E) 1~50 、(C) 1~50 (D) 1~50 (A) 1~50 (B) 1~50 (E) 1~50 、(C) 1~50 (D) 1~50 (A) 1~50 (E) 1~50 (B) 1~50 、(C) 1~50 (D) 1~50 (B) 1~50 (A) 1~50 (E) 1~50 、(C) 1~50 (D) 1~50 (B) 1~50 (E) 1~50 (A) 1~50 、(C) 1~50 (D) 1~50 (E) 1~50 (B) 1~50 (A) 1~50 、(C) 1~50 (D) 1~50 (E) 1~50 (A) 1~50(B) 1~50 、(C) 1~50 (E) 1~50 (A) 1~50 (B) 1~50 (D) 1~50 、(C) 1~50 (E) 1~50 (A) 1~50 (D) 1~50 (B) 1~50 、(C) 1~50 (E) 1~50 (B) 1~50 (A) 1~50 (D) 1~50 、(C) 1~50 (E) 1~50 (B) 1~50 (D) 1~50 (A) 1~50 、(C) 1~50 (E) 1~50 (D) 1~50 (B) 1~50 (A) 1~50 、(C) 1~50 (E) 1~50 (D) 1~50 (A) 1~50 (B) 1~50 、(D) 1~50 (B) 1~50 (C) 1~50 (A) 1~50 (E) 1~50 、(D) 1~50 (B) 1~50 (C) 1~50 (E) 1~50 (A) 1~50 、(D) 1~50 (B) 1~50 (A) 1~50 (C) 1~50 (E) 1~50 、(D) 1~50 (B) 1~50 (A) 1~50 (E) 1~50 (C) 1~50 、(D) 1~50 (B) 1~50 (E) 1~50 (C) 1~50 (A) 1~50 、(D) 1~50 (B) 1~50 (E) 1~50 (A) 1~50 (C) 1~50 、(D)1~50 (C) 1~50 (A) 1~50 (E) 1~50 (B) 1~50 、(D) 1~50 (C) 1~50 (A) 1~50 (B) 1~50 (E) 1~50 、(D) 1~50 (C) 1~50 (E) 1~50 (A ) 1~50 (B) 1~50 、(D) 1~50 (C) 1~50 (E) 1~50 (B) 1~50 (A) 1~50 、(D) 1~50 (C) 1~50 (B) 1~50 (E) 1~50 (A) 1~50 、(D) 1~50 (C) 1~50 (B) 1~50 (A) 1~50 (E) 1~50 、(D) 1~50 (A) 1~50 (C) 1~50 (B) 1~50 (E) 1~50 、(D) 1~50 (A) 1~50 (C) 1~50 (E) 1~50 (B) 1~50 、(D) 1~50 (A) 1~50 (B) 1~50 (C) 1~50 (E) 1~50 、(D) 1~50 (A) 1~50 (B) 1~50 (E) 1~50 (C) 1~50 、(D) 1~50 (A) 1~50 (E) 1~50 (B) 1~50 (C) 1~50 、(D) 1~50 (A) 1~50 (E) 1~50 (C) 1~50 (B) 1~50 、(D) 1~50 (E)1~50 (C) 1~50 (B) 1~50 (A) 1~50 、(D) 1~50 (E) 1~50 (C) 1~50 (A) 1~50 (B) 1~50 、(D) 1~50 (E) 1~50 (B) 1~50 (C) 1~50 (A) 1~50 、(D) 1~50 (E) 1~50 (B) 1~50 (A) 1~50 (C) 1~50 、(D) 1~50 (E) 1~50 (A) 1~50 (B) 1~50 (C) 1~50 、(D) 1~50 (E) 1~50 (A) 1~50 (C) 1~50 (B) 1~50 、(E) 1~50 (B) 1~50 (C) 1~50 (D) 1~50 (A) 1~50 、(E) 1~50 (B) 1~50 (C) 1~50 (A) 1~50 (D) 1~50 、(E) 1~50 (B) 1~50 (D) 1~50 (C) 1~50 (A) 1~50 、(E) 1~50 (B) 1~50 (D) 1~50 (A) 1~50 (C) 1~50 、(E) 1~50 (B) 1~50 (A) 1~50 (C) 1~50 (D) 1~50 、(E) 1~50 (B) 1~50 (A) 1~50 (D) 1~50 (C) 1~50 、(E) 1~50 (C) 1~50 (D) 1~50(A) 1~50 (B) 1~50 、(E) 1~50 (C) 1~50 (D) 1~50 (B) 1~50 (A) 1~50 、(E) 1~50 (C) 1~50 (A) 1~50 (D) 1~50 (B) 1~50 、(E) 1~50 (C) 1~50 (A) 1~50 (B) 1~50 (D) 1~50 、(E) 1~50 (C) 1~50 (B) 1~50 (A) 1~50 (D) 1~50 、(E) 1~50 (C) 1~50 (B) 1~50 (D) 1~50 (A) 1~50 、(E) 1~50 (D) 1~50 (C) 1~50 (B) 1~50 (A) 1~50 、(E) 1~50 (D) 1~50 (C) 1~50 (A) 1~50 (B) 1~50 、(E) 1~50 (D) 1~50 (B) 1~50 (C) 1~50 (A) 1~50 、(E) 1~50 (D) 1~50 (B) 1~50 (A) 1~50 (C) 1~50 、(E) 1~50 (D) 1~50 (A) 1~50 (B) 1~50 (C) 1~50 、(E) 1~50 (D) 1~50 (A) 1~50 (C) 1~50 (B) 1~50 、(E) 1~50 (A) 1~50 (C) 1~50 (B) 1~50 (D)1~50 , (E) 1~50 (A) 1~50 (C) 1~50 (D) 1~50 (B) 1~50 , (E) 1~50 (A) 1~50 (B) 1~50 (C) 1~50 (D) 1~50 , (E) 1~50 (A) 1~50 (B) 1~50 (D) 1~50 (C) 1~50 , (E) 1~50 (A) 1~50 (D) 1~50 (B) 1~50 (C) 1~50 or (E) 1~50 (A) 1~50 (D) 1~50 (C) 1~50 (B) 1~50 , Here, a peptide epitope of 8 to 12 amino acids is represented by "A", a peptide epitope of 13 to 17 amino acids is represented by "B", a peptide epitope of 18 to 21 amino acids is represented by "C", a peptide epitope of 22 to 26 amino acids is represented by "D", and a peptide epitope of 27 to 31 amino acids is represented by "E".

[0071] Any of the above combinations of peptide epitopes may be combined. For example, any of the nucleic acid cancer vaccines described herein may encode two or more of the listed peptide epitopes.

[0072] 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 peptide epitopes are MHC class I epitopes. In some embodiments, at least 20% of the peptide epitopes are MHC class I epitopes. In some embodiments, at least 30% of the peptide epitopes are MHC class I epitopes. In some embodiments, at least 40% of the peptide epitopes are MHC class I epitopes. In some embodiments, at least 0%, 60%, 70%, 80%, 90%, or 100% of the peptide epitopes are MHC class I epitopes. In some embodiments, none (0%) of the peptide epitopes are MHC class II epitopes. In some embodiments, at least 10% of the peptide epitopes are MHC class II epitopes. In some embodiments, at least 20% of the peptide epitopes are MHC class II epitopes. In some embodiments, at least 30% of the peptide epitopes are MHC class II epitopes. In some embodiments, at least 40% of the peptide epitopes are MHC class II epitopes. In some embodiments, at least 50%, 60%, 70%, 80%, 90%, or 100% of the peptide epitopes are MHC class II epitopes. In some embodiments, the ratio of MHC class I epitopes to MHC class II epitopes is selected from about 10%:about 90%, about 20%:about 80%, about 30%:about 70%, about 40%:about 60%, about 50%:about 50%, about 60%:about 40%, about 70%:about 30%, about 80%:about 20%, about 90%:about 10% MHC class I:MHC class II epitopes. In one embodiment, the ratio of MHC class I:MHC class II epitopes is 1:1. In one embodiment, the ratio of MHC class I:MHC class II epitopes is 2:1. In one embodiment, the ratio of MHC class I:MHC class II epitopes is 3:1. In one embodiment, the ratio of MHC class I:MHC class II epitopes is 4:1.In one embodiment, the ratio of MHC class I:MHC class II epitopes is 5:1. In some embodiments, the ratio of MHC class II epitopes to MHC class I epitopes is selected from about 10%:about 90%, about 20%:about 80%, about 30%:about 70%, about 40%:about 60%, about 50%:about 50%, about 60%:about 40%, about 70%:about 30%, about 80%:about 20%, about 90%:about 10% MHC class II:MHC class I epitopes. In one embodiment, the ratio of MHC class II:MHC class I epitopes is 1:1. In one embodiment, the ratio of MHC class II:MHC class I epitopes is 1:2. In one embodiment, the ratio of MHC class II:MHC class I epitopes is 1:3. In one embodiment, the ratio of MHC class II:MHC class I epitopes is 1:4. In one embodiment, the ratio of MHC class II:MHC class I epitopes is 1:5. In some embodiments, at least one of the peptide epitopes of the cancer vaccine is a B cell epitope. In some embodiments, one or more predicted T cell reactive epitopes of the cancer vaccine comprise 8-11 amino acids. In some embodiments, one or more predicted B cell reactive epitopes of the cancer vaccine comprise 13-17 amino acids.

[0073] The cancer vaccines of the present disclosure, in some embodiments, include mRNA vaccines encoding multiple peptide epitope antigens arranged directly together with a single amino acid spacer between the peptide epitopes, a short linker between the peptide epitopes, or no spacer between the peptide epitopes. The multiple epitope antigen may include a mixture of MHC class I epitopes and MHC class II epitopes. As a non-limiting example, the multiple peptide epitope antigen may be a polypeptide having the following structure: (XGX) 1~10 (GYGY) 1~10 (GXGX) 0~10 (GYGY) 0~10 , (XG) 1~10 (GY) 1~10 (GX) 0~10 (GY) 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 、(YGY) 1~10 (GXGX) 1~10 (GYGY) 0~10 (GXGX) 0~10 、(YG) 1~10 (GX) 1~10 (GY) 0~10 (GX) 0~10 、(YGYGY) 1~10 (GXGX) 1~10 (YGY) 0~10 (GXGX) 0~10 、(YGY) 1~10 (GXGXGX) 1~10 (YGY) 0~10 (GXGX) 0~10 、(YGYGYGY) 1~10 (GXGX) 1~10 (YGY) 0~10 (GXGX) 0~10 、(YGY) 1~10 (GXGXGXGX) 1~10 (YGY) 0~10 (GXGX) 0~10 、(XY) 1~10 (YX) 1~10 (XY) 0~10 (YX) 0~10 、(YX) 1~10 (XY) 1~10 (Y) 0~10 (X) 0~10 、(YY) 1~10 (X) 1~10 (Y) 0~10 (X) 0~10 、(XY) 1~10 (XY) 1~10 (X) 0~10 (X) 0~10 、(Y) 1~10 (YX) 1~10 (X) 0~10 (Y) 0~10 、(XYX) 1~10 (YXX) 1~10 (YX) 0~10 (YY) 0~10 、または(YYX) 1~10 (XXY) 1~10(YX) 0~10 (XY) 0~10 , wherein X is an MHC class I epitope 5-100 amino acids in length (e.g., any of the lengths described herein, including 8-31 amino acids), Y is an MHC class II epitope 5-100 amino acids in length (e.g., any of the lengths described herein, including 8-31 amino acids), and G is glycine.

[0074] The nucleic acid cancer vaccines of the present disclosure, in some aspects, include a nucleic acid encoding one or more peptide epitopes that contain a mutation that results in a unique expressed peptide sequence. In some embodiments, the mutation that results in a unique expressed peptide sequence can be, but is not limited to, an insertion, a deletion, a frameshift mutation, and / or a splicing variant. In some embodiments, the nucleic acid cancer vaccine encodes a plurality of peptide epitope antigens that contain one or more single nucleotide polymorphism (SNP) mutations with flanking amino acids on each side of the SNP mutation. In some embodiments, the number of flanking amino acids on each side of the SNP mutation can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, or 30. In some embodiments, the SNP mutation is centrally located and the number of flanking amino acids on each side of the SNP mutation is approximately the same. In other embodiments, the SNP mutation does not have an equal number of flanking amino acids on each side. In an embodiment, a cancer vaccine epitope comprises a SNP flanked by two class I sequences, each sequence comprising 7 amino acids. In another embodiment, a cancer vaccine epitope comprises a SNP flanked by two class II sequences, each sequence comprising 10 amino acids. In some embodiments, an epitope may comprise a centrally located SNP and flanks that are both class I sequences, both class II sequences, or one class I and one class II sequence.

[0075] In another embodiment, the peptide epitope is in the form of a concatemeric cancer antigen consisting of peptide epitopes. Any number of peptide epitopes may be used. In a particular embodiment, the peptide epitope is in the form of a concatemeric cancer antigen consisting of 5 to 200 peptide epitopes. In a particular embodiment, the peptide epitope is in the form of a concatemeric cancer antigen consisting of 3 to 130 peptide epitopes. In some embodiments, the concatemeric cancer antigen comprises one or more of the following: a) peptide epitopes (e.g., 3-200 or 3-130 peptide epitopes) interspersed with cleavage-sensitive sites, and / or b) each peptide epitope is directly linked to each other without a linker, and / or c) each peptide epitope is linked to each other with a single amino acid linker, and / or d) each peptide epitope is linked to each other with a short linker, and / or e) each peptide epitope comprises 8-31 amino acids and contains one or more SNP mutations (e.g., centrally located SNP mutations), and / or f) each peptide epitope comprises 8-31 amino acids and contains a mutation that results in a unique expressed peptide sequence, and / or g) at least 30% of the peptide epitopes have highest affinity for class I MHC molecules from the subject, and / or h) at least 30% of the peptide epitopes have highest affinity for class II MHC molecules from the subject. and / or i) none of the peptide epitopes have the highest affinity for a class II MHC molecule from the subject, and / or j) at least 50% of the peptide epitopes have a predicted binding affinity for HLA-A, HLA-B, and / or DRB1 of IC50<500nM, and / or k) the nucleic acids encoding the peptide epitopes are arranged such that the peptide epitopes are ordered to minimize pseudo epitopes, and / or l) the ratio of class I MHC molecule peptide epitopes to class II MHC molecule peptide epitopes is at least 1:1, 2:1, 3:1, 4:1, or 5:1, and / or m) there are no class II MHC molecule peptide epitopes.In some embodiments, a peptide epitope having the "highest affinity" for a class I MHC molecule specifically binds (i.e., binds with the highest affinity) to that class I MHC molecule. In some embodiments, a peptide epitope having the "highest affinity" for a class I MHC molecule has a higher binding affinity for that class I MHC molecule than for a class II MHC molecule. In some embodiments, a peptide epitope having the "highest affinity" for a class II MHC molecule specifically binds (i.e., binds with the highest affinity) to that class II MHC molecule. In some embodiments, a peptide epitope having the "highest affinity" for a class II MHC molecule has a higher binding affinity for that class II MHC molecule than for a class I MHC molecule.

[0076] It will be appreciated that concatemers of two or more peptides, for example two or more neo-antigens, may create unintended new epitopes (pseudo-epitopes) at peptide boundaries. To prevent or eliminate such pseudo-epitopes, class I alleles may be scanned for hits that cross peptide boundaries in the concatemer. In some embodiments, the peptide order within the concatemer is shuffled to reduce or eliminate pseudo-epitope formation. In some embodiments, linkers are used between the peptides, for example single amino acid linkers such as glycine, to reduce or eliminate pseudo-epitope formation. In some embodiments, anchor amino acids may be replaced with other amino acids that reduce or eliminate pseudo-epitope formation. In some embodiments, peptides are trimmed at peptide boundaries within the concatemer to reduce or eliminate pseudo-epitope formation.

[0077] In some embodiments, the multiple peptide epitope antigens are positioned and ordered to minimize pseudo epitopes. In other embodiments, the multiple peptide epitope antigens are polypeptides that do not contain pseudo epitopes. When the cancer antigen epitopes are arranged in a concatemeric structure in a head to tail formation, a junction is formed between each cancer antigen epitope. This includes some, i.e., 1-10 amino acids from the epitope on the N-terminus of the peptide, and some, i.e., 1-10 amino acids on the C-terminus of the adjacent directly linked epitope. It is important that the junction is not an immunogenic peptide that may generate an immune response. In some embodiments, the junction is such that the personalized cancer vaccine has an IC of greater than about 50 nM. 50 In other embodiments, the junction peptide sequence binds to the HLA protein of interest with an IC of greater than about 10 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM, 400 nM, 450 nM, or 500 nM. 50 Combine with.

[0078] Personalized cancer vaccines In some aspects, the present disclosure provides a nucleic acid cancer vaccine comprising one or more nucleic acids, each of which encodes at least one suitable cancer antigen, such as an individualized antigen specific to a cancer subject.

[0079] For example, a nucleic acid cancer vaccine may contain a nucleic acid encoding one or more cancer antigens specific to each subject, called neoepitopes. Antigens expressed in or by tumor cells are called "tumor-associated antigens". Certain tumor-associated antigens may or may not be expressed in non-cancerous cells. Many tumor mutations are known in the art. Tumor-associated antigens that are not expressed or are rarely expressed in non-cancerous cells, or whose expression in non-cancerous cells is sufficiently reduced compared to that in cancerous cells to induce an immune response induced upon vaccination, are called neoepitopes. Neoepitopes are completely foreign to the body and therefore do not generate an immune response against healthy tissues or are not masked by protective components of the immune system. In some embodiments, personalized vaccines based on neoepitopes are desirable because such vaccine formulations maximize the specificity for a patient's specific tumor. Mutation-derived neoepitopes can result from point mutations, nonsynonymous mutations leading to different amino acids in the protein, read-through mutations in which the stop codon is modified or deleted leading to the translation of a longer protein with a novel tumor-specific sequence at the C-terminus, splice site mutations leading to the inclusion of an intron in the mature mRNA and thus the unique tumor-specific protein sequence, chromosomal rearrangements (i.e., gene fusions) resulting in a chimeric protein with a tumor-specific sequence at the junction of two proteins, frameshift mutations or deletions resulting in a new open reading frame with a novel tumor-specific protein sequence, and / or translocations.

[0080] Methods for generating personalized cancer vaccines generally include, for example, identification of mutations using deep nucleic acid or protein sequencing techniques, generating a set of candidate T cell epitopes based on mutations present in the tumor that can bind to the patient's HLA alleles, for example, using the application of validated peptide-MHC binding prediction algorithms or other analytical techniques, any demonstration of antigen-specific T cells against the selected neoepitopes, or demonstration that the candidate neoepitopes bind to HLA proteins on the tumor surface, and developing a vaccine. Examples of techniques for identifying mutations include dynamic allele-specific hybridization (DASH), microplate array diagonal gel electrophoresis (MADGE), pyrosequencing, oligonucleotide-specific ligation, TaqMan systems, and various DNA "chip" technologies (e.g., Affymetrix SNP chips), and methods based on invasive cleavage to generate small signal molecules followed by mass spectrometry or immobilized padlock probes and rolling circle amplification.

[0081] Several deep nucleic acid and protein sequencing techniques are known in the art. Any type of sequence analysis method can be used. For example, nucleic acid sequencing can be performed on the entire tumor genome, tumor exome (protein-coding DNA), and / or tumor transcriptome. Real-time single molecule sequencing by synthesis technology relies on the detection of fluorescent nucleotides as they are incorporated into nascent strands of DNA complementary to the template being sequenced. Other rapid high-throughput sequencing methods also exist. Protein sequencing can be performed on the tumor proteome. In addition, protein mass spectrometry can be used to identify or verify the presence of mutant peptides bound to MHC proteins on tumor cells. Peptides can be acid eluted from tumor cells or HLA molecules immunoprecipitated from tumors and then identified using mass spectrometry. Sequencing results can be compared to known control sets or compared to sequencing analysis performed on the patient's normal tissue. In some embodiments, these neoepitopes can bind to class I HLA proteins with higher affinity than wild-type peptides and / or activate anti-tumor CD8 T cells. Identical mutations in any particular gene are found rarely across tumors.

[0082] MHC class I proteins are present on the surface of almost all cells in the body, including most tumor cells. MHC class I proteins are usually loaded with antigens derived from endogenous proteins or intracellular pathogens and then presented to cytotoxic T lymphocytes (CTLs). T cell receptors are capable of recognizing and binding peptides complexed with MHC class I molecules. Each cytotoxic T lymphocyte expresses a unique T cell receptor that can bind to a specific MHC / peptide complex.

[0083] Using computer algorithms, it is possible to predict potential neoepitopes, such as putative T cell reactive epitopes, i.e. peptide sequences, that are bound by class I or class II MHC molecules in the form of peptide-presenting complexes and then, in this form, recognized by the T cell receptors of T lymphocytes. Examples of programs useful for identifying peptides that 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).

[0084] Once putative neoepitopes are selected, they can be further tested using in vitro and / or in vivo assays. Conventional in vitro laboratory assays, such as Elispot assays, may be used with isolates from each patient to refine the list of neoepitopes selected based on the algorithm's predictions.

[0085] In some embodiments, the nucleic acid cancer vaccines and vaccination methods described herein may include peptide epitopes or antigens based on specific mutations (neoepitopes) and those expressed by cancer germline genes (referred to herein as "conventional cancer antigens" or "shared cancer antigens", antigens common to tumors found in multiple patients). In some embodiments, conventional antigens are those known to be found in cancers or tumors in general, or in particular types of cancers or tumors. In some embodiments, conventional cancer antigens are non-mutated tumor antigens. In some embodiments, conventional cancer antigens are mutated tumor antigens.

[0086] In some embodiments, the nucleic acid cancer vaccines and methods described herein may include peptide epitopes based on cancer / testis (CT) antigens. Cancer / testis antigen expression is restricted to male germ cells in healthy adults, but ectopic expression has been observed in tumor cells of several types of human cancers. Because male germ cells lack HLA class I molecules and cannot present antigens to T cells, cancer / testis antigens, when expressed in cancer cells, are generally considered neo-antigens and have the ability to elicit immune responses that are strictly cancer-specific. Cancer / testis antigens for use with the compositions and methods described herein include, but are not limited to, MAGEA1, MAGEA2, MAGEA3, MAGEA4, MAGEA5, MAGEA6, MAGEA8, MAGEA9, MAGEA10, MAGEA11, MAGEA12, BAGE, BAGE2, BAGE3, BAGE4, ​​BAGE5, MAGEB1, MAGEB2, MAGEB5, MAGEB6, MAGEB3, MAGEB4, MAGEB5, MAGEB6, MAGEB7, MAGEB8, MAGEB9, MAGEB10, MAGEB11, MAGEB12, MAGEB13, MAGEB14, MAGEB15, MAGEB16, MAGEB17, MAGEB18, MAGEB19, MAGEB20, MAGEB21, MAGEB22, MAGEB23, MAGEB24, MAGEB25, MAGEB26, MAGEB27, MAGEB28, MAGEB29 ... GEB4, GAGE1, GAGE2A, GAGE3, GAGE4, ​​GAGE5, GAGE6, GAGE7, GAGE8, SSX1, SSX2, SSX2b, SSX3, SSX4, CTAG1B, LAGE-1b, CTAG2, MAGEC1, MAGEC3, SYCP1, BRDT, MAGEC2, SPANXA1, SPANXB1, SPANXC, SPANXD, SPANXN1, SPANXN2, SPANXN3, SPAN XN4, SPANXN5, XAGE1D, XAGE1C, XAGE1B, XAGE1, XAGE2, XAGE3, XAGE-3b, XAGE-4 / RP11-167P23.2, XAGE5, DDX43, SAG E1, ADAM2, PAGE5, CT16.2, PAGE1, PAGE2, PAGE2B, PAGE3, PAGE4, ​​LIPI, VENTXP1, IL13RA2, TSP50, CTAGE1, CTAGE-2, C TAGE5, SPA17, ACRBP, CSAG1, CSAG2, DSCR8, MMA1b, DDX53, CTCFL, LUZP4, CASC5, TFDP3, JARID1B, LDHC, MORC1, DKKL 1, SPO11, CRISP2, FMR1NB, FTHL17, NXF2, TAF7L, TDRD1, TDRD6, TDRD4, TEX15, FATE1, TPTE, CT45A1, CT45A2, CT45A3,CT45A4、CT45A5、CT45A6、HORMAD1、HORMAD2、CT47A1、CT47A2、CT47A3、CT47A4、CT47A5、C T47A6、CT47A7、CT47A8、CT47A9、CT47A10、CT47A11、CT47B1、SLCO6A1、TAG、LEMD1、HSPB9、 CCDC110、ZNF165、SPACA3、CXorf48、THEG、ACTL8、NLRP4、COX6B2、LOC348120、CCDC33、LOC 196993、PASD1、LOC647107、TULP2、CT66 / AA884595、PRSS54、RBM46、CT69 / BC040308、CT70 / BI818097、SPINLW1、TSSK6、ADAM29、CCDC36、LOC440934、SYCE1、CPXCR1、TSPY3、TSGA10 LEFT、LEFT、SOUTH2、ARMC3、AKAP3、Cxorf61、PBK、C21orf99、OIP5、CEP290、CABYR、 SPAG9、MPHOSPH1、ROPN1、PLAC1、CALR3、PRM1、PRM2、CAGE1、TTK、LY6K、IMP-3、AKAP4、DPPA 2、KIAA0100、DCAF12、SEMG1、POTED、POTEB、POTE、POTEG、POTEB、POTEC、POTEB、GOLGAGL2 FA、CDCA1、PEPP2、OTOA、CCDC62、GPATCH2、CEP55、FAM46D、TEX14、CTNNA2、FAM133A、LOC130576、ANKRDIG45、ELOV MEFF1, TMEFF2, ARX, SPEF2, GPAT2, TMEM108, NOL4, PTPN20A, SPAG4, MAEL, RQCD1, PRAME, TEX101, SPATA19, ODF1, 、ODF4、ATAD2、ZNF645、MCAK、SPAG1、SPAG6、SPAG8、SPAG17、FB XO39、RGS22、サイクリンA1、C15orf60、CCDC83、TEKT5、NR6A1、TMPR SS12、TPPP2、PRSS55、DMRT1、EDAG、NDR、DNAJB8、CSAG3B、CTAG1A、GAGE12B、GAGE12C、GAGE12D、GAGE12E、GAGE12F、GAGE12F、GAGE12F.The antigen may be any such cancer / testis antigen known in the art, including GAGE12H, GAGE12I, GAGE12J, GAGE13, LOC728137, MAGEA2B, MAGEA9B / LOC728269, NXF2B, SPANXA2, SPANXB2, SPANXE, SSX4B, SSX5, SSX6, SSX7, SSX9, TSPY1D, TSPY1E, TSPY1F, TSPY1G, TSPY1H, TSPY1I, TSPY2, XAGE1E, XAGE2B / CTD-2267G17.3, and / or variants thereof.

[0087] In some embodiments, the nucleic acid cancer vaccine may further comprise one or more nucleic acids encoding one or more non-mutated tumor antigens. In some embodiments, the nucleic acid cancer vaccine may further comprise one or more nucleic acids encoding one or more mutated tumor antigens.

[0088] Many tumor antigens are known in the art. A cancer or tumor antigen (e.g., a conventional cancer antigen) for use with the compositions and methods described herein can be any such cancer or tumor antigen known in the art. In some embodiments, the cancer or tumor antigen (e.g., a conventional cancer antigen) is one of the following antigens: CD2, CD19, CD20, CD22, CD27, CD33, CD37, CD38, CD40, CD44, CD47, CD52, CD56, CD70, CD79, CD137, 4-IBB, 5T4, AGS-5, AGS-16, angiopoietin 2, B2M, B7.1, B7.2, B7DC, B7H1, B7H2, B7H3, BT-062, BT LA, CAIX, carcinoembryonic antigen, CTLA4, Cripto, ED-B, ErbBl, ErbB2, ErbB3, ErbB4, EGFL7, EpCAM, EphA2, EphA3, EphB2, FAP, fibronectin, folate receptor, ganglioside GM3, GD2, glucocorticoid-inducible tumor necrosis factor receptor (GITR), gpl00, gpA33, GPNMB, ICOS, IGF1R, integrin av, integrin ανβ, LAG-3, Lewis Y, mesothelin, c-MET, MN, carbonic anhydrase IX, MUC1, MUC16, Nectin-4, NKGD2, NOTCH, OX40, OX40L, PD-1, PDL1, PSCA, PSMA, RANKL, ROR1, ROR2, SLC44A4, Syndecan-1, TACI, TAG-72, Tenascin, TIM3, TRAILRl, TRAILR2, VEGFR-1, VEGFR-2, VEGFR-3, and / or variants thereof.

[0089] Epitopes can be identified using free or commercial databases (e.g., Lonza Epibase, Antitope). Such tools are useful for predicting the most immunogenic epitopes within a target antigen protein. Selected peptides may then be synthesized and screened with a human HLA panel, and the most immunogenic sequences are used to construct nucleic acids encoding the peptide epitope(s). One strategy for mapping epitopes of cytotoxic T cells is based on generating an equimolar mixture of four C-terminal peptides for each nominal 11-mer spanning the protein. This strategy will produce a library antigen containing all possible active CTL epitopes.

[0090] Neoepitopes can be designed to optimally bind to MHC to promote a robust immune response. In some embodiments, each peptide epitope comprises an antigenic region and an MHC stabilizing region. The MHC stabilizing region is a sequence that stabilizes the peptide in the MHC.

[0091] All MHC stabilizing regions within an epitope may be the same, or they may be different. The MHC stabilizing regions may be in the N-terminal portion of the peptide or the C-terminal portion of the peptide. Alternatively, the MHC stabilizing regions may be in the central region of the peptide.

[0092] The MHC stabilization region can be 5-10, 5-15, 8-10, 1-5, 3-7, or 3-8 amino acids long. In yet other embodiments, the antigenic region is 5-100 amino acids long. Peptides interact with MHC class I molecules by competitive affinity binding in the endoplasmic reticulum before they are presented on the cell surface. The affinity of an individual peptide is directly linked to its amino acid sequence and the presence of specific binding motifs at defined positions within the amino acid sequence. Peptides presented to the MHC are held by the floor of the peptide-binding groove in the central region of the α1 / α2 heterodimer (a molecule composed of two non-identical subunits). The sequence of residues in the floor of the peptide-binding groove determines the specific peptide residues to which it binds.

[0093] The optimal binding region can be identified by computer-assisted comparison of the affinity of the binding site (MHC pocket) for a particular amino acid at each amino acid within the binding site for each of the target epitopes to identify the ideal binder for all tested antigens. The MHC stabilizing region of the epitope can be identified using an amino acid prediction matrix of the data points of the binding site. The amino acid prediction matrix is ​​a table with a first and a second axis that define the data points. The prediction matrix can be generated as shown in Singh, H. and Raghava, GPS (2001), "ProPred: prediction of HLA-DR binding sites. "Bioinformatics, 17(12), 1236-37). In some embodiments, the prediction matrix is ​​based on evolutionary conservation, and in other embodiments, the prediction matrix uses biochemical similarity to examine how similar somatic amino acids are to germline amino acids (e.g., Kim et al., J Immunol. 2017:3360-3368). The similarity between the somatic and germline amino acids approximates how the mutation affects binding (e.g., T cell receptor recognition). In some embodiments, less similarity indicates improved binding (e.g., T cell receptor recognition).

[0094] In some embodiments, the MHC stabilization region is designed based on the particular MHC of the subject. In this way, the MHC stabilization region can be optimized for each patient.

[0095] Neoepitopes selected for inclusion in a cancer vaccine (e.g., a nucleic acid cancer vaccine) are typically high affinity binding peptides. In some aspects, the neoepitope binds to an HLA protein with greater affinity than the wild-type peptide. In some embodiments, the neoepitope has an IC of at least 5000 nM or less, at least 500 nM or less, at least 250 nM or less, at least 200 nM or less, at least 150 nM or less, at least 100 nM or less, at least 50 nM or less. 50 Typically, the predicted IC 50 Peptides <50 nM are generally considered to be medium- to high-affinity binding peptides and are selected to experimentally test their affinity using biochemical assays of HLA binding. Finally, it is determined whether the human immune system can mount an effective immune response against these mutated tumor antigens and thus effectively kill the tumor but not the normal cells.

[0096] In some embodiments, neoepitopes are 13 residues or less in length and may consist of about 8 to about 11 residues, particularly 9 or 10 residues. In other embodiments, neoepitopes may be designed to be longer. For example, neoepitopes may have extensions of 2 to 5 amino acids towards the N-terminus and C-terminus of each corresponding gene product. The use of longer peptides may allow for endogenous processing by patient cells, leading to more effective antigen presentation and induction of T cell responses.

[0097] Neoepitopes with desired activity may be modified as necessary to provide certain desired attributes, e.g., improved pharmacological properties, while increasing or at least substantially retaining all of the biological activity of the unmodified peptide to bind to the desired MHC molecule and activate the appropriate T or B cells. For example, neoepitopes may undergo various changes, such as either conservative or non-conservative substitutions, which may provide certain advantages in their use, e.g., improved MHC binding. Conservative substitutions refer to the replacement of an amino acid residue with another that is biologically and / or chemically similar, e.g., another hydrophobic residue, or another polar residue. Substitutions include combinations such as Gly, Ala; Val, Ile, Leu, Met; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; and Phe, Tyr. The effect of single amino acid substitutions may also be explored using D-amino acids. Such modifications can be made using well-known peptide synthesis procedures, as described, for example, in Merrifield, Science 232:341-347 (1986), Barany & Merrifield, The Peptides, Gross & Meienhofer, eds. (NY, Academic Press), pp. 1-284 (1979), and Stewart & Young, Solid Phase Peptide Synthesis, (Rockford, Ill., Pierce), 2d Ed. (1984).

[0098] Neoepitopes can also be modified by extending or decreasing the amino acid sequence of the compound, for example, by adding or deleting amino acids. Peptides, polypeptides, or analogs can also be modified by altering the order or composition of certain residues, and it is readily understood that certain amino acid residues essential for biological activity, e.g., amino acid residues at critical contact sites or conserved residues, generally will not be altered without adversely affecting biological activity.

[0099] Typically, a series of peptides with single amino acid substitutions are used to determine the effect of electrostatic charge, hydrophobicity, etc. on binding. For example, a series of positively (e.g., Lys or Arg) or negatively (e.g., Glu) charged amino acid substitutions are made along the length of the peptide to reveal different patterns of sensitivity to various MHC molecules and T-cell or B-cell receptors. In addition, multiple substitutions using small relatively neutral moieties such as Ala, Gly, Pro, or similar residues can be used. The substitutions can be homo- or hetero-oligomers. The number and type of residues substituted or added depend on the required spacing between essential contacts and certain functional attributes sought (e.g., hydrophobicity vs. hydrophilicity). Increased binding affinity to MHC molecules or T-cell receptors can also be achieved by such substitutions compared to the affinity of the parent peptide. In any case, such substitutions should be with amino acid residues or other molecular fragments selected to avoid, for example, steric and charge interferences that may disrupt binding.

[0100] Neoepitopes may also include isosteres of two or more residues within the neoepitope. An isostere, as defined herein, is a sequence of two or more residues that can be substituted with a second sequence because the conformation of the first sequence fits into the binding site specific for the second sequence. The term specifically includes peptide backbone modifications well known to those of skill in the art. Such modifications include modifications of the amide nitrogen, α-carbon, amide carbonyl, complete replacement of the amide bond, extensions, deletions, or backbone bridges. See generally Spatola, Chemistry and Biochemistry of Amino Acids, Peptides and Proteins, Vol. VII (Weinstein ed., 1983).

[0101] Immunogenicity considerations are an important component in the selection of optimal neoepitopes for inclusion in a vaccine. As a series of non-limiting examples, immunogenicity can be assessed by analyzing the neoepitopes' MHC binding capacity, HLA non-specificity, mutation location, predicted T cell reactivity, actual T cell reactivity, specific conformations and resulting solvent exposure, and representation of specific amino acids. Known algorithms such as the NetMHC prediction algorithm can be used to predict the ability of a peptide to bind to common HLA-A and -B alleles. In some embodiments, the NetMHC prediction algorithm uses the IC 50 In another embodiment, the NetMHC prediction algorithm uses percent rank and eluted ligand data to determine binding ability (Jurtz et al., J Immunol. 2017 Nov 1; 199(9): 3360-3368). As shown in Figures 2-3B, the percent rank method results in a more balanced distribution of predicted binders among different HLA alleles. Structural evaluation of MHC-binding peptides can also be performed in a computational three-dimensional analysis and / or protein docking program. Use of predicted epitope structures when bound to MHC molecules, such as those obtained from the Rosetta algorithm, can be used to evaluate the degree of solvent exposure of amino acid residues of the epitope when the epitope is bound to the MHC molecule. T cell reactivity can be experimentally evaluated with epitopes and T cells in vitro. Alternatively, T cell reactivity can be evaluated using T cell response / sequence datasets.

[0102] An important aspect of neoepitopes included in vaccines is the lack of self-reactivity. Putative neoepitopes may be screened to confirm that the epitope is restricted to tumor tissue, for example, resulting from genetic alterations in malignant cells. Ideally, the epitope should not be present in normal tissues of the patient, and thus self-similar epitopes are excluded from the data set. A personalized coding genome can be used as a reference for comparison of neoantigen candidates to determine the lack of self-reactivity. In some embodiments, a personalized coding genome is generated from a personalized transcriptome and / or exome.

[0103] The nature of peptide composition can also be considered in epitope design, for example, for each putative epitope a score can be provided regarding the value of conserved versus non-conserved amino acids found in the epitope.

[0104] In some embodiments, the analysis performed by the tools described herein may include comparing different sets of features obtained from a patient at different time points, i.e., before and after a therapeutic intervention, from different tissue samples, from different patients with similar tumors, etc. In some embodiments, the average of peak values ​​from one set of features may be compared to the average of peak values ​​from another set of features. For example, the average value of HLA binding may be compared between two different distribution sets. The two distribution sets may be determined for a time duration separated by, for example, days, months, or years.

[0105] A neoepitope characterization system according to the techniques described herein may take any suitable form, as embodiments are not limited in this respect. Any of the above-described functions may be implemented using one or more computer systems. The computer system may include one or more processors and one or more computer-readable storage media (i.e., tangible, non-transitory computer-readable media), which may be formed from any suitable data storage medium, such as a volatile storage medium and one or more non-volatile storage media. The processor may control writing data to and reading data from the volatile and non-volatile storage media in any suitable manner, as embodiments are not limited in this respect. To perform any of the functions described herein, the processor may execute one or more instructions stored in one or more computer-readable storage media (e.g., a volatile storage medium and / or a non-volatile storage medium), which may function as a tangible, non-transitory computer-readable medium that stores instructions for execution by the processor.

[0106] Preparation method In other aspects, the disclosure provides methods for preparing a cancer vaccine, the method comprising: a) identifying among personalized cancer antigens for a patient; b) determining the anti-tumor efficacy of at least two peptide epitopes for each of 3-130 personalized cancer antigens; and c) preparing a cancer vaccine in which the overall anti-cancer efficacy of the cancer vaccine is maximized over a given full length of the cancer vaccine (e.g., the predicted overall anti-cancer efficacy of the cancer vaccine is maximized).

[0107] Methods for generating cancer vaccines according to the present disclosure may include identification of mutations using techniques such as deep nucleic acid or protein sequencing methods as described herein of tissue samples. In some embodiments, an initial identification of mutations in a subject's (e.g., patient's) transcriptome is performed. Data from the subject's (e.g., patient's) transcriptome is compared to sequence information from the subject's (e.g., patient's) exome to identify expressed patient-specific and tumor-specific mutations. The comparison generates a data set of putative neoepitopes, referred to as a mutanome. The mutanome may include approximately 100-10,000 candidate mutations per patient. The mutanome is subjected to a series of queries or data-probing analyses using algorithms to identify an optimal set of mutations for the generation of a neoantigen vaccine. In some embodiments, an mRNA neoantigen vaccine is designed and manufactured. The patient is then treated with the vaccine. In certain embodiments, such neoantigen-containing vaccines may be polycistronic vaccines, including multiple neoepitopes or one or more single RNA vaccines or combinations thereof.

[0108] In some embodiments, the entire process from the start of the mutation identification process to the start of patient treatment is accomplished in less than 2 months. In other embodiments, the entire process is accomplished in 7 weeks or less, 6 weeks or less, 5 weeks or less, 4 weeks or less, 3 weeks or less, 2 weeks or less, or less than 1 week. In some embodiments, the entire method is performed in less than 30 days.

[0109] In personalized cancer vaccines, subject-specific cancer antigens may be identified in a patient sample. 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 comprise a bodily fluid from a subject. A bodily fluid may be a fluid isolated from any location in the subject's body, preferably a peripheral location, including, but not limited to, blood, plasma, serum, urine, sputum, spinal fluid, cerebrospinal fluid, pleural fluid, nipple aspirate, lymphatic fluid, respiratory, intestinal, and genitourinary tract fluids, tears, saliva, breast milk, fluids from the lymphatic system, semen, cerebrospinal fluid, intraorgan system fluids, ascites, tumor cyst fluid, amniotic fluid, and combinations thereof. In some embodiments, the sample may be a tissue sample or a tumor sample. For example, one or more samples of tumor cells may be tested for the presence of a subject-specific cancer antigen.

[0110] The process of identifying specific cancer antigens may involve both transcriptome and exome analysis, or only transcriptome or exome analysis. In some embodiments, transcriptome analysis is performed first and exome analysis is performed second. The analysis is performed on a biological or tissue sample. In some embodiments, the biological or tissue sample is a blood or serum sample. In other embodiments, the sample is a tissue bank sample or EBV transformation of B cells.

[0111] Alternatively, a subject-specific cancer antigen can be identified in the subject's exosomes. When an antigen of a vaccine is identified in a subject's exosomes, such an antigen is said to represent a subject's exosomal antigen.

[0112] Exosomes are small microvesicles shed by cells, typically with a diameter of approximately 30-100 nm. Exosomes classically form from invading and pinching the late endosomal membrane, resulting in the formation of multivesicular bodies (MVBs) filled with small lipid bilayer vesicles, each containing a sample of the parent cell's cytoplasm. Fusion of the MVB with the cell membrane releases these exosomes from the cell, delivering them into the blood, urine, cerebrospinal fluid, or other bodily fluids. Exosomes can be recovered from any of these biological fluids for further analysis.

[0113] Nucleic acids in exosomes serve as biomarkers for tumor antigens. The advantage of analyzing exosomes to identify target-specific cancer antigens is that the method avoids the need for biopsy. This may be particularly advantageous when patients need to have several rounds of therapy, including cancer antigen identification and vaccination.

[0114] Several methods of 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 organic electrophoresis, magnetic activated cell sorting (MACS), nanomembrane ultrafiltration concentration, Percoll gradient isolation, and microfluidic devices.Exemplary methods are described, for example, in US Patent Publication No. 2014 / 0212871.

[0115] Once the mRNA vaccine is synthesized, it is administered to the patient. In some embodiments, the vaccine is administered on a schedule of up to 2 months, up to 3 months, up to 4 months, up to 5 months, up to 6 months, up to 7 months, up to 8 months, up to 9 months, up to 10 months, up to 11 months, up to 1 year, up to 1.5 years, up to 2 years, up to 3 years, or up to 4 years. The schedule may be the same or different. In some embodiments, the schedule is once a week for the first 3 weeks, and once a month thereafter.

[0116] At any point in treatment, the patient can be tested to determine whether the mutations in the vaccine are still appropriate. Based on that analysis, the vaccine can be adjusted or reformulated to include one or more different mutations or to remove one or more mutations.

[0117] It is recognized and understood that optimal neoepitopes can be evaluated and / or selected for inclusion in a cancer vaccine by analyzing certain characteristics of cancer-associated mutations. Characteristics of a neoepitope or set of neoepitopes can include, for example, evaluation of gene or transcript level expression in patient RNA-seq or other nucleic acid analysis, tissue-specific expression in available databases, known cancer genes / tumor suppressors, confidence score of variant calling, RNA-seq allele-specific expression, conservative vs. non-conservative AA substitutions, location of point mutations (centering score for increased TCR binding), location of point mutations (anchoring score for differential HLA binding), autologous: less than 100% core epitope homology with patient WES data, 8mer-11mer HLA-A and -B IC 50 , 15mer to 20mer HLA-DRB1 IC 50 , non-specificity score (i.e., the number of patient HLAs predicted to bind), and HLA-C IC of 8mer to 11mer 50 , 15mer to 20mer HLA-DRB3-5 IC 50 , 15mer to 20mer HLA-DQB1 / A1 IC 50 , 15mer to 20mer HLA-DPB1 / A1 IC 50 , class I to class II ratio, diversity of patient HLA-A, -B, and DRB1 allotypes covered, percentage of point mutations versus complex epitopes (e.g., frameshifts), and / or pseudoepitope HLA binding scores.

[0118] In some embodiments, the characteristics of the cancer-associated mutations used to identify optimal neoepitopes are characteristics related to the type of mutation, the abundance of the mutation in patient samples, immunogenicity, lack of autoreactivity, and the nature of the peptide composition. The type of mutation should be determined and considered as a factor in determining whether the putative epitope should be included in the vaccine. The types of mutations can be varied. In some cases, it may be desirable to include multiple different types of mutations in a single vaccine. In other cases, a single type of mutation may be more desirable. The value of each specific mutation can be weighted and calculated. In some embodiments, the specific mutation is a single nucleotide polymorphism (SNP). In some embodiments, the specific mutation is a peptide sequence that results from a complex variant, such as intron retention, complex splicing events, or an insertion / deletion mutation that changes the reading frame of the sequence.

[0119] The abundance of mutations in patient samples can also be scored and factored into the determination of whether a putative epitope should be included in the vaccine: highly abundant mutations may promote a more robust immune response.

[0120] In some embodiments, the personalized mRNA cancer vaccine described herein can be used for the treatment of cancer.As one non-limiting example, the present disclosure provides a method for treating a patient with cancer, comprising: a) analyzing a sample from the patient to identify one or more personalized cancer antigens; b) determining the anti-tumor efficacy of at least two peptide epitopes for each of the identified personalized cancer antigens; c) preparing a cancer vaccine in which the total anti-cancer efficacy of the cancer vaccine is maximized for a given full-length cancer vaccine (e.g., the predicted total anti-cancer efficacy of the cancer vaccine is maximized); and d) administering the cancer vaccine to the patient.

[0121] Cancer vaccines (e.g., nucleic acid cancer vaccines) can be administered prophylactically or therapeutically to healthy individuals or to early stage cancer or late stage and / or metastatic cancer as part of an active immunization scheme. In one embodiment, an effective amount of a cancer vaccine (e.g., nucleic acid cancer vaccine) provided to a cell, tissue, or subject can be sufficient for immune activation, particularly antigen-specific immune activation.

[0122] In some embodiments, the cancer vaccine (e.g., nucleic acid cancer vaccine) may be administered together with an anti-cancer therapeutic agent. The cancer vaccine (e.g., nucleic acid cancer vaccine) and the anti-cancer therapeutic agent may be combined to further enhance the immunotherapeutic response. The cancer vaccine (e.g., nucleic acid cancer vaccine) and the other therapeutic agent may be administered simultaneously or sequentially. When the other therapeutic agents are administered simultaneously, they can be administered in the same or separate formulations, but are administered simultaneously. The other therapeutic agents are administered sequentially with each other and with the cancer vaccine (e.g., nucleic acid cancer vaccine) when the administration of the other therapeutic agent and the cancer vaccine (e.g., nucleic acid cancer vaccine) is temporally separated. The time separation between the administration of these compounds may be a few minutes, or it may be longer, for example, hours, days, weeks, months. The other therapeutic agents include, but are not limited to, anti-cancer therapeutic agents, adjuvants, cytokines, antibodies, antigens, etc.

[0123] In some embodiments, the progression of cancer can be monitored to identify changes in expressed antigens. Thus, in some embodiments, the method also includes identifying at least two cancer antigens from the subject's sample at least one month after administration of the cancer mRNA vaccine, producing a second set of cancer antigens, and administering to the subject an mRNA vaccine having an open reading frame encoding the second set of cancer antigens. In some embodiments, the mRNA vaccine having an open reading frame encoding the second set of antigens is administered to the subject 2 months, 3 months, 4 months, 5 months, 6 months, 8 months, 10 months, or 1 year after the mRNA vaccine having an open reading frame encoding the first set of cancer antigens. In other embodiments, the mRNA vaccine having an open reading frame encoding the second set of antigens is administered to the subject 1.5 years, 2 years, 2.5 years, 3 years, 3.5 years, 4 years, 4.5 years, or 5 years after the mRNA vaccine having an open reading frame encoding the first set of cancer antigens.

[0124] Hotspot mutations as neoantigens In cancer population analysis, certain mutations occur in a higher percentage of patients than would be expected by chance. These "recurrent" or "hotspot" mutations are often shown to have a "driver" role in tumors, leading to some changes in cancer cell functions that are important for tumor initiation, maintenance, or metastasis, and thus being selected in tumor evolution. In addition to their importance in tumor biology and therapy, they provide an opportunity for precision medicine, where recurrent mutations stratify patient populations into groups that are more likely to respond to certain therapies, including but not limited to targeting the mutant protein itself.

[0125] Thus, in some embodiments, the cancer vaccine further comprises one or more cancer hotspot neo-epitopes in addition to the personalized cancer epitopes. In some embodiments, the vaccine includes cancer hotspot mutations that occur above a threshold prevalence in the indication of interest. In some embodiments, the threshold prevalence is greater than 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%. The indications of interest include, but are not limited to, bladder urothelial carcinoma (BLCA), colon adenocarcinoma (COAD), esophageal carcinoma (ESCA), hepatocellular carcinoma (HCC), head and neck squamous cell carcinoma (HNSC), lung adenocarcinoma (LUAD), pancreatic adenocarcinoma (PAAD), prostate cancer (PRAD), rectal adenocarcinoma (READ), small cell lung cancer (SCLC), cutaneous melanoma (SKCM), serum ovarian carcinoma (SOC), gastric adenocarcinoma (STAD), and endometrial carcinoma (UEC). Exemplary mutations are provided in the table below, and an exemplary graph of hotspot mutations by indication is provided as FIG. [Table 1]

[0126] Although much of the effort and research on recurrent mutations has focused on nonsynonymous (or "missense") single nucleotide variants (SNVs), population analyses have revealed that a variety of more complex (non-SNV) variant classes, such as synonymous (or "silent"), splice site, multi-nucleotide variants, insertions, and deletions, can also occur with high frequency.

[0127] The p53 gene (official symbol TP53) is mutated more frequently than any other gene in human cancers. Large cohort studies have shown that for most p53 mutations, the genomic location is unique to one or only a few patients, and mutations cannot be used as recurrent neoantigens in therapeutic vaccines designed for specific patient populations. However, a small subset of p53 loci exhibits a "hotspot" pattern, where several positions within the gene are mutated at relatively high frequency. Strikingly, the majority of these recurrently mutated regions occur near exon-intron boundaries, disrupting canonical nucleotide sequence motifs recognized by the mRNA splicing machinery. Mutations in splicing motifs can alter the final mRNA sequence even if changes to the local amino acid sequence are not predicted (i.e., for synonymous or intronic mutations). Thus, these mutations are often annotated as "non-coding" by common annotation tools and ignored for further analysis, even though they can alter mRNA splicing in unpredictable ways and have profound functional consequences on the translated protein. If the alternatively spliced ​​isoform produces an in-frame sequence change (i.e., no PTC is produced), it avoids depletion by NMD and can be readily expressed, processed, and presented on the cell surface by the HLA system. Furthermore, the mutation-derived alternative splice is usually "cryptic," i.e., not expressed in normal tissues, and therefore can be recognized as a non-self neoantigen by T cells.

[0128] Mutations are typically derived from patient DNA sequencing data to derive neoepitopes for prior art peptide vaccines. However, mRNA expression is a more direct measure of the global space of possible neoepitopes. For example, some tumor-specific neoepitopes may arise from splicing changes, insertions / deletions (InDels) that result in frameshifts, alternative promoters, or epigenetic modifications that are not easily identified using exome sequencing data alone. In some embodiments, neoantigens from InDels are enriched for predicted high affinity binders to nsSNVs. Such neoantigens may be immunogenic. For example, frameshift InDels have been found to be significantly associated with checkpoint inhibitor response across three melanoma cohorts. All neoepitopes may be scored in the same manner as those neoepitopes arising from SNVs, although at most, one neoantigen candidate per InDel is included to avoid bias against InDels. There is untapped value in identifying these types of complex mutations for neoantigen vaccines. Because they increase the number of epitopes that can bind to a patient's unique HLA allotype, complex variants are more likely to be immunogenic and lead to a more effective immune response against tumors due to their differences with self-proteins compared to variants resulting from single amino acid changes.

[0129] In some aspects, the present invention involves a method for identifying patient-specific complex mutations and formulating these mutations into an effective personalized cancer vaccine (e.g., a nucleic acid cancer vaccine). The method involves the use of short-read RNA-Seq. A major challenge inherent in using short reads for RNA-seq is the fact that multiple mRNA transcript isoforms can be obtained from the same genomic locus due to alternative splicing and other mechanisms. Because the sequencing reads are much shorter than the full-length mRNA transcript, it becomes difficult to map a set of reads to the correct corresponding isoform within a known gene annotation model. As a result, complex variants that deviate from known gene annotations (which are common in cancer) can be difficult to discover by standard approaches. However, short peptides can be identified, rather than the exact exon composition of the full-length transcript. The method for identifying short peptides that may represent these complex mutations involves a short k-mer counting approach to neoepitope prediction of complex variants.

[0130] Nucleic Acids / Polynucleotides A cancer vaccine (e.g., a nucleic acid cancer vaccine), as provided herein, comprises at least one (one or more) nucleic acid having an open reading frame encoding at least one peptide epitope. The term "nucleic acid" in its broadest sense includes any compound and / or substance that comprises a polymer of nucleotides. These polymers are also referred to as polynucleotides.

[0131] The nucleic acid may be or include, for example, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA, LNA with a β-D-ribo configuration, α-LNA (a diastereomer of LNA) with an α-L-ribo configuration, 2′-amino-LNA with a 2′-amino functionalization, and 2′-amino-α-LNA with a 2′-amino functionalization), ethylene nucleic acid (ENA), cyclohexenyl nucleic acid (CeNA) or chimeras, or combinations thereof.

[0132] As a non-limiting example, when the DNA nucleic acid cancer vaccines described herein are delivered to a cell, the DNA is transcribed into RNA, the RNA is processed by intracellular machinery into a polypeptide, and the polypeptide can then be processed into an immunosensitive fragment that can stimulate an immune response against a tumor or cancer cell population. As a non-limiting example, when the RNA (e.g., mRNA) nucleic acid cancer vaccines described herein are delivered to a cell, the RNA (e.g., mRNA) is processed by intracellular machinery into a polypeptide, and the polypeptide can then be processed into an immunosensitive fragment that can stimulate an immune response against a tumor or cancer cell population.

[0133] In some embodiments, the nucleic acids of the present disclosure function as messenger RNA (mRNA). "Messenger RNA" (mRNA) refers to any nucleic acid that encodes (at least one) polypeptide (naturally occurring, non-naturally occurring, or modified polymer of amino acids) and can be translated in vitro, in vivo, in situ, or ex vivo to yield the encoded polypeptide.

[0134] The basic components of an mRNA molecule typically include at least a coding region, a 5' untranslated region (UTR), a 3'UTR, a 5' cap, and a poly A tail. The nucleic acids of the present disclosure may function as mRNAs, but may be distinguished from wild-type mRNAs in their functional and / or structural design features that serve to overcome existing problems of effective polypeptide expression using nucleic acid-based therapeutics.

[0135] In some embodiments, the polynucleotide of the present disclosure is codon-optimized. Codon optimization methods are known in the art and can be used as provided herein. In some embodiments, codon optimization can be used to match the codon frequency in target and host organisms to ensure proper folding; bias G / C content to increase mRNA stability or reduce secondary structure; minimize tandem repeat codon or base stretches that may impair gene assembly or expression; customize transcriptional and translational control regions; insert or remove protein transport sequences; remove / add post-translational modification sites (e.g., glycosylation sites) in encoded proteins; remove or shuffle protein domains; insert or delete restriction sites; modify ribosome binding sites and mRNA degradation sites; adjust the translation rate to allow various domains of the protein to fold properly; or reduce or eliminate problematic secondary structures in polynucleotides. Codon optimization tools, algorithms, and services are known in the art, and non-limiting examples include services and / or proprietary methods from GeneArt (Life Technologies), DNA2.0 (Menlo Park Calif.). In some embodiments, open reading frame (ORF) sequences are optimized using an optimization algorithm.

[0136] In some embodiments, the codon optimized sequence shares less than 95% sequence identity with a naturally occurring or wild-type sequence (e.g., a naturally occurring or wild-type mRNA sequence encoding a polypeptide or protein of interest (e.g., an antigenic protein or polypeptide)). In some embodiments, the codon optimized sequence shares less than 90% sequence identity with a naturally occurring or wild-type sequence (e.g., a naturally occurring or wild-type mRNA sequence encoding a polypeptide or protein of interest (e.g., an antigenic protein or polypeptide). In some embodiments, the codon optimized sequence shares less than 85% sequence identity with a naturally occurring or wild-type sequence (e.g., a naturally occurring or wild-type mRNA sequence encoding a polypeptide or protein of interest (e.g., an antigenic protein or polypeptide). In some embodiments, the codon optimized sequence shares less than 80% sequence identity with a naturally occurring or wild-type sequence (e.g., a naturally occurring or wild-type mRNA sequence encoding a polypeptide or protein of interest (e.g., an antigenic protein or polypeptide). In some embodiments, a codon-optimized sequence shares less than 75% sequence identity with a naturally occurring or wild-type sequence (e.g., a naturally occurring or wild-type mRNA sequence encoding a polypeptide or protein of interest (e.g., an antigenic protein or polypeptide)).

[0137] In some embodiments, the codon-optimized sequence shares 65% to 85% (e.g., about 67% to about 85%, or about 67% to about 80%) sequence identity with a naturally occurring or wild-type sequence (e.g., a naturally occurring or wild-type mRNA sequence encoding a polypeptide or protein of interest (e.g., an antigenic protein or polypeptide)). In some embodiments, the codon-optimized sequence shares 65% to 75%, or about 80% sequence identity with a naturally occurring or wild-type sequence (e.g., a naturally occurring or wild-type mRNA sequence encoding a polypeptide or protein of interest (e.g., an antigenic protein or polypeptide)).

[0138] In some embodiments, the codon-optimized RNA can be, for example, an RNA with an enhanced level of G / C. The G / C content of a nucleic acid molecule can affect the stability of the RNA. RNA with an increased amount of guanine (G) and / or cytosine (C) residues can be more functionally stable than nucleic acids containing large amounts of adenine (A) and thymine (T) or uracil (U) nucleotides. WO02 / 098443 discloses pharmaceutical compositions containing mRNA stabilized by sequence modifications in the translation region. Due to the degeneracy of the genetic code, the modifications work by replacing existing codons with ones that promote greater RNA stability without changing the resulting amino acid. The approach is limited to the coding region of the RNA.

[0139] Antigens / Antigen Polypeptides In some embodiments, each peptide epitope may be 5-100 amino acids in length (inclusive). In some embodiments, the length of at least one of the peptide epitopes is 5-100, 5-95, 5-90, 5-85, 5-80, 5-75, 5-70, 5-65, 5-60, 5-55, 5-50, 5-45, 5-40, 5-39, 5-38, 5-37, 5-36, 5-35, 5-34, 5-33, 5-32, 5-40, 5-50, 5-60, 5-7 ...85, 5-95, 5-90, 5-85, 5-80, 5-75, 5-70, 5-65, 5-60, 5-55, 5-50, 5-45, 5-40, 5-39, 5-38, 5-37, 5-36, 5-35, 5-34, 5-33, 5-32, 5-40, 5-50, 5-60, 5-75, 5-80, 5-95, 5-100, 5-120, 5-140, 5- ~31, 5~30, 5~29, 5~28, 5~27, 5~26, 5~25, 5~24, 5~23, 5~22, 5~21, 5~20, 8~100, 8~95, 8~90, 8~85, 8~80, 8~75, 8~70, 8~65, 8~60, 8~55, 8~50, 8~45, 8~40, 8~39, 8~38, 8~37, 8~36, 8~35, 8~34, 8~33, 8~32, 8~31, 8~30, 8~29, 8~28, 8~27, 8~26, 8~25, 8~24, 8~23, 8~22, 8~21, 8~20, 10~100, 10~95, 10~90, 10~85, 10~80, 10~75, 10~70, 10~65, 10~60, 10~55, 10~ 50, 10-45, 10-40, 10-39, 10-38, 10-37, 10-36, 10-35, 10-34, 10-33, 10-32, 10-31, 10-30, 10-29, 10-28, 10-27, 10-26, 10-25, 10-24, 10-23, 10-22, 10-21, or 10-20 amino acids.

[0140] In some embodiments, each peptide epitope encoded by nucleic acid cancer vaccine can have different length.In certain embodiments, at least one of peptide epitopes has different length from another peptide epitope encoded by nucleic acid cancer vaccine.Each peptide epitope can be any length that is reasonable for the epitope.

[0141] Polypeptides for use with the present disclosure include the aforementioned gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments, and other equivalents, variants, and analogs. Polypeptides can be single molecules or multi-molecular complexes such as dimers, trimers, or tetramers. Polypeptides can also include single-chain or multi-chain polypeptides such as antibodies or insulin, and can be associated or linked. Most commonly, disulfide bonds are found in multi-chain polypeptides. The term polypeptide can also apply to amino acid polymers, in which at least one amino acid residue is an artificial chemical analog of the corresponding naturally occurring amino acid.

[0142] The term "polypeptide variant" refers to a molecule whose amino acid sequence differs from a native or reference sequence. Amino acid sequence variants may have substitutions, deletions, and / or insertions at certain positions within the amino acid sequence compared to the native or reference sequence. Typically, variants have at least 50% identity to the native or reference sequence. In some embodiments, variants share at least 80% or at least 90% identity with the native or reference sequence.

[0143] In some embodiments, a "variant mimetic" is provided. As used herein, the term "variant mimetic" refers to one that contains at least one amino acid that mimics an activation sequence. For example, glutamate can function as a mimic of phosphoro-threonine and / or phosphoro-serine. Alternatively, the variant mimetic can result in inactivation or inactivation products that contain the mimic, for example, phenylalanine can function as an inactivating replacement for tyrosine, or alanine can function as an inactivating replacement for serine.

[0144] "Ortholog" refers to genes in different species that have evolved from a common ancestral gene through speciation. Orthologs usually retain the same function during evolution. Identification of orthologs is important for predicting the reliability of gene function in newly sequenced genomes.

[0145] "Analog" is meant to include polypeptide variants that differ by one or more amino acid alterations, including, for example, substitutions, additions, or deletions of amino acid residues that still retain one or more of the properties of the parent or starting polypeptide.

[0146] The present disclosure provides several types of polynucleotide or polypeptide-based compositions, including variants and derivatives. These include, for example, substitution, insertion, deletion, and covalent variants and derivatives. The term "derivative" is used interchangeably with the term "variant," but generally refers to a molecule that is modified and / or altered in any way relative to a reference or starting molecule.

[0147] Thus, polynucleotides encoding peptides or polypeptides containing substitutions, insertions and / or additions, deletions, and covalent modifications with respect to reference sequences, particularly the polypeptide sequences disclosed herein, are included within the scope of this disclosure. For example, sequence tags or amino acids, such as one or more lysines, can be added to the peptide sequence (e.g., at the N-terminus or C-terminus). Sequence tags can be used for peptide detection, purification, or localization. Lysines can be used to increase peptide solubility or enable biotinylation. Alternatively, amino acid residues located at the carboxy- and amino-terminal regions of the amino acid sequence of a peptide or protein can be optionally deleted to result in truncated sequences. Certain amino acids (e.g., C- or N-terminal residues) can alternatively be deleted depending on the use of the sequence, for example, expression of the sequence as part of a larger sequence that is soluble or linked to a solid support.

[0148] "Substitutional variants," when referring to polypeptides, are variants having at least one amino acid residue in a native or starting sequence, in which a different amino acid is inserted at the same position. The substitutions can be single (only one amino acid in the molecule has been substituted) or multiple (two or more amino acids have been substituted in the same molecule).

[0149] As used herein, the term "conservative amino acid substitution" refers to the replacement of an amino acid that is normally present in a sequence with a different amino acid that has a similar size, charge, or polarity. Examples of conservative substitutions include the replacement of a non-polar (hydrophobic) residue, such as isoleucine, valine, and leucine, with another non-polar residue. Similarly, examples of conservative substitutions include the replacement of one polar (hydrophobic) residue with another, such as between arginine and lysine, between glutamine and asparagine, and between glycine and serine. In addition, the replacement of a basic residue, such as lysine, arginine, or histidine, with another basic residue, or the replacement of one acidic residue, such as aspartic acid or glutamic acid, with another acidic residue, are additional examples of conservative substitutions. Examples of non-conservative substitutions include substitutions of a non-polar (hydrophobic) amino acid residue, such as isoleucine, valine, leucine, alanine, or methionine, for a polar (hydrophilic) residue, such as cysteine, glutamine, glutamic acid, or lysine, and / or substitutions of a polar residue for a non-polar residue.

[0150] A "feature," when referring to a polypeptide or polynucleotide, is defined as a distinct amino acid sequence-based or nucleotide-based component of a molecule, respectively. Features of a polypeptide encoded by a polynucleotide include surface expression, local conformational shape, folds, loops, half-loops, domains, half-domains, sites, termini, or any combination thereof.

[0151] The term "domain," as used herein when referring to a polypeptide, refers to a motif in a polypeptide having one or more identifiable structural or functional features or characteristics (e.g., binding ability, serving as a site for protein-protein interaction).

[0152] As used herein when referring to a polypeptide, the term "site" in relation to its amino acid-based embodiments is used synonymously with "amino acid residue" and "amino acid side chain." As used herein when referring to a polynucleotide, the term "site" in relation to its nucleotide-based embodiments is used synonymously with "nucleotide." A site represents a position within a peptide or polypeptide or polynucleotide that may be modified, manipulated, altered, derivatized, or changed within a polypeptide- or polynucleotide-based molecule.

[0153] As used herein, the term "terminus" or "terminus" refers to the end of a polypeptide or polynucleotide, respectively. Such ends are not limited to only the first or last site of a polypeptide or polynucleotide, but may include additional amino acids or nucleotides in the terminal region. Polypeptide-based molecules can be characterized as having both an N-terminus (terminated by an amino acid having a free amino group (NH2)) and a C-terminus (terminated by an amino acid having a free carboxyl group (COOH)). Proteins are sometimes formed of multiple polypeptide chains held together by disulfide bonds or non-covalent forces (multimers, oligomers). These proteins have multiple N-terminus and C-terminus. Alternatively, the ends of a polypeptide can be modified to optionally begin or end with a non-polypeptide-based moiety, such as an organic conjugate.

[0154] As will be appreciated by those skilled in the art, protein fragments, functional protein domains, and homologous proteins are also considered within the scope of the polypeptide of interest. For example, provided herein is any protein fragment that is 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more than 100 amino acids long (meaning a polypeptide sequence of at least one amino acid residue shorter than but otherwise identical to the reference polypeptide sequence). In another example, any protein that includes a 10, 20, 30, 40, 50, or 100 amino acid stretch that is 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% identical to any of the sequences described herein can be utilized in accordance with the present disclosure. In some embodiments, the polypeptide includes 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations as shown in any of the sequences provided or referenced herein. In another embodiment, any protein comprising a stretch of 20, 30, 40, 50, or 100 amino acids that is more than 80%, 90%, 95%, or 100% identical to any of the sequences described herein, which protein has a stretch of 5, 10, 15, 20, 25, or 30 amino acids that is less than 80%, 75%, 70%, 65%, or 60% identical to any of the sequences described herein.

[0155] A polypeptide or polynucleotide molecule of the present disclosure may share a degree of sequence similarity or "identity" with a reference molecule (e.g., a reference polypeptide or reference polynucleotide), for example, with a molecule described in the art (e.g., an engineered or designed molecule or a wild-type molecule). The term "identity" as known in the art refers to the relationship between the sequences of two or more polypeptides or polynucleotides (e.g., DNA molecules and / or RNA molecules) as determined by comparing the sequences. In the art, identity also means the degree of sequence correlation between them, as determined by the number of matches between two or more strings of amino acid or nucleic acid residues. Identity measures the percentage of identical matches between the smaller of two or more sequences with gap alignment (if any) addressed by a particular mathematical model or computer program (e.g., "algorithm"). The identity of related peptides can be easily calculated by known methods. "Percent identity" or "% identity" as applied to a polypeptide or polynucleotide sequence is defined as the percentage of residues (amino acid residues or nucleic acid residues) in a candidate amino acid or nucleic acid sequence that are identical to the residues in the amino acid or nucleic acid sequence of a second sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity. Methods and computer programs for alignment are well known in the art. It is understood that identity depends on the calculation of percent identity, but may have different values ​​due to gaps and penalties introduced in the calculation. Calculation of percent identity of two polynucleic acid sequences can be performed, for example, by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of the first and second nucleic acid sequences for optimal alignment, and non-identical sequences can be ignored for comparison purposes). In certain embodiments, the length of the sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the length of the reference sequence.The nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap, which must be introduced for optimal alignment of the two sequences. The comparison of sequences and the determination of the percent identity between two sequences can be achieved using a mathematical algorithm.

[0156] Generally, a variant of a particular polynucleotide or polypeptide has at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or less than 100% sequence identity to that particular reference polynucleotide or polypeptide as determined by sequence alignment programs and parameters described herein and known to those of skill in the art. For example, the percent identity between two nucleic acid sequences can be determined using methods such as those described in Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993, Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987, Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994, and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991 (each of which is incorporated herein by reference). For example, the percent identity between two nucleic acid sequences can be determined using the algorithm of Meyers and Miller (CABIOS, 1989, 4:11-17), which has been incorporated into the ALIGN program (version 2.0) using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. The percent identity between two nucleic acid sequences can alternatively be determined using the GAP program in the GCG software package, using the NWSgapdna.CMP matrix.Commonly used methods for determining percent identity between sequences include, but are not limited to, those disclosed in Carillo, H., and Lipman, D., SIAM J Applied Math., 48:1073 (1988), which is incorporated herein by reference. Techniques for determining identity are codified in publicly available computer programs. Exemplary 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 (Stephen F. Altschul, et al (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:3389-3402). Another popular local alignment technique is based on the Smith-Waterman algorithm (Smith, TF & Waterman, MS (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, SB & Wunsch, CD (1970) "A general method applicable to the search for similarities in the amino acid sequences of two proteins." J. Mol. Biol. 48:443-453). More recently, the Fast Optimal Global Sequence Alignment Algorithm (FOGSAA) has been developed, which is said to generate global alignments of nucleotide and protein sequences faster than other optimal global alignment methods, including the Needleman-Wunsch algorithm.

[0157] As used herein, the term "homology" refers to the overall correlation between polymer molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules. Polymer molecules (e.g., nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules) that share a threshold level of similarity or identity determined by alignment of matching residues are referred to as 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 match between two compared sequences. In some embodiments, polymer molecules are considered to be "homologous" to each other if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical or similar. The term "homologous" necessarily refers to a comparison between at least two sequences (polynucleotide or polypeptide sequences). Two polynucleotide sequences are considered to be homologous if the polypeptides they encode are at least 50%, 60%, 70%, 80%, 90%, 95%, or even 99% identical for at least one stretch of at least 20 amino acids. In some embodiments, homologous polynucleotide sequences are characterized by the ability to encode a stretch of 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 of at least 4-5 uniquely specified amino acids. Two protein sequences are considered to be homologous if the proteins are at least 50%, 60%, 70%, 80%, or 90% identical for at least one stretch of at least 20 amino acids.

[0158] Homology means that the compared sequences have diverged in evolution from a common origin. The term "homolog" refers to a first amino acid or nucleic acid sequence (e.g., a gene (DNA or RNA) or a protein sequence) that is related to a second amino acid or nucleic acid sequence in descending order from a common ancestral sequence. The term "homolog" can apply to the relationship of genes and / or proteins that have been separated by a speciation event or to the relationship of genes and / or proteins that have been separated by a gene duplication event. An "ortholog" is a gene (or protein) in different species that has evolved from a common ancestral gene (or protein) by speciation. Typically, an ortholog retains the same function during evolution. A "paralog" is a gene (or protein) that is related by duplication within a genome. An ortholog retains the same function during evolution, whereas a paralog evolves a new function, even if related to the original function.

[0159] chemical modification The modified nucleotide sequence encodes an epitope antigen polypeptide. In some embodiments, the nucleic acid cancer vaccine of the present invention comprises one or more chemically modified nucleic acid bases. The present invention includes modified polynucleotides, including the polynucleotides described herein (e.g., nucleic acids comprising nucleotide sequences encoding one or more cancer peptide epitopes). Modified nucleic acids can be chemically modified and / or structurally modified. When the nucleic acid of the present invention is chemically and / or structurally modified, the polynucleotide can be referred to as a "modified nucleic acid".

[0160] The present disclosure provides modified nucleosides and nucleotides of nucleic acids (e.g., RNA polynucleotides such as mRNA polynucleotides) that encode one or more cancer peptide epitopes. A "nucleoside" refers to a compound or derivative thereof that contains a sugar molecule (e.g., pentose or ribose) in combination with an organic base (e.g., a purine or pyrimidine) or a derivative thereof (also referred to herein as a "nucleobase"). A "nucleotide" refers to a nucleoside that includes a phosphate group. Modified nucleotides can be synthesized by any useful method, for example, chemically, enzymatically, or recombinantly, to include one or more modified or non-natural nucleosides. A nucleic acid can include a region or regions of linked nucleosides. Such regions can have variable backbone linkages. These linkages can be standard phosphodiester linkages, in which case the polynucleotide includes a region of nucleotides.

[0161] The modified nucleic acid disclosed herein may comprise a variety of distinct modifications.In some embodiments, modified polynucleotide comprises one, two or more (optionally different) nucleoside or nucleotide modifications.In some embodiments, modified polynucleotide introduced into cell may exhibit one or more desirable properties, such as, for example, improved protein expression in cell, reduced immunogenicity, or reduced degradation, compared with unmodified polynucleotide.

[0162] In some embodiments, the nucleic acids disclosed herein (e.g., nucleic acids encoding one or more peptide epitopes) are structurally modified. As used herein, a "structural" modification is when two or more linked nucleosides are inserted, deleted, duplicated, inverted, or randomized in a polynucleotide without significant chemical modification to the nucleotide itself. Since chemical bonds are necessarily broken and reformed to cause structural modifications, structural modifications are of a chemical nature and are therefore chemical modifications. However, structural modifications will result in a different sequence of nucleotides. For example, a polynucleotide "ATCG" can be chemically modified to "AT-5meC-G". The same polynucleotide can be structurally modified from "ATCG" to "ATCCCG", where a dinucleotide "CC" is inserted, resulting in a structural modification to the nucleic acid.

[0163] In some embodiments, the nucleic acid of the present disclosure is chemically modified. As used herein with respect to nucleic acid, the term "chemical modification" or, as appropriate, "chemically modified" refers to modification of adenosine (A), guanosine (G), uridine (U), or cytidine (C) ribo- or deoxyribonucleosides in one or more of their positions, patterns, percentages, or populations. Generally, as used herein, these terms are not intended to refer to ribonucleotide modifications in naturally occurring 5'-terminal mRNA cap moieties.

[0164] In some embodiments, the nucleic acids of the present disclosure may have uniform chemical modifications of all or any of the same nucleoside types, or a population of modifications resulting from simple downward titration of the same starting modifications of all or any of the same nucleoside types, or a measured percentage of all or any of the same nucleoside types but with random incorporation, e.g., all uridines are replaced by uridine analogs, e.g., pseudouridine or 5-methoxyuridine. In another embodiment, the polynucleotide may have uniform chemical modifications of two, three, or four of the same nucleoside types throughout the polynucleotide (e.g., all uridines and all cytosines are modified in the same way, etc.).

[0165] Modified nucleotide base pairing encompasses base pairs formed between nucleotides and / or modified nucleotides containing non-standard or modified bases, as well as standard adenosine-thymine, adenosine-uracil, or guanosine-cytosine base pairs, where the arrangement of hydrogen bond donors and hydrogen bond acceptors allows hydrogen bonding between a non-standard base and a standard base or between two complementary non-standard base structures. One example of such non-standard base pairing is base pairing between the modified nucleotide inosine and adenine, cytosine, or uracil. Any combination of bases / sugars or linkers may be incorporated into the polynucleotides of the present disclosure.

[0166] The skilled artisan will understand that, unless otherwise indicated, the nucleic acid sequences described in this application recite a "T" in a representative DNA sequence, but when the sequence represents RNA, the "T" is replaced with a "U."

[0167] Cancer vaccines of the present disclosure, in some embodiments, include at least one nucleic acid (e.g., RNA) having an open reading frame encoding at least one (e.g., 3-200 or 3-130) peptide epitope(s), where the nucleic acid includes nucleotides and / or nucleosides that can be standard (unmodified) or modified, as known in the art. In some embodiments, the nucleotides and nucleosides of the present disclosure include modified nucleotides or nucleosides. Such modified nucleotides and nucleosides can be naturally occurring modified nucleotides and nucleosides or non-naturally occurring modified nucleotides and nucleosides. Such modifications can include modifications at the sugar, backbone, or nucleobase moieties of the nucleotides and / or nucleosides, as recognized in the art.

[0168] In some embodiments, the naturally occurring modified nucleotides or nucleotides of the present disclosure are those that are commonly known or recognized in the art. Non-limiting examples of such naturally occurring modified nucleotides and nucleotides can be found, inter alia, in the widely recognized MODOMICS database.

[0169] In some embodiments, the non-naturally occurring modified nucleotides or nucleosides of the present disclosure are those that are commonly known or recognized in the art. Non-limiting examples of such non-naturally occurring modified nucleotides and nucleosides can be found, inter alia, in published U.S. application Nos. PCT / US2012 / 058519, PCT / US2013 / 075177, PCT / US2014 / 058897, PCT / US2014 / 058891, PCT / US2014 / 070413, PCT / US2015 / 36773, PCT / US2015 / 36759, PCT / US2015 / 36771, or PCT / IB2017 / 051367, all of which are incorporated herein by reference for this purpose.

[0170] Thus, the nucleic acids of the disclosure (e.g., DNA nucleic acids and RNA nucleic acids such as mRNA nucleic acids) can include standard nucleotides and nucleosides, naturally occurring nucleotides and nucleosides, non-naturally occurring nucleotides and nucleosides, or any combination thereof.

[0171] Nucleic acids of the disclosure (e.g., DNA nucleic acids and RNA nucleic acids such as mRNA nucleic acids) in some embodiments comprise a variety (more than one) different types of standard and / or modified nucleotides and nucleosides. In some embodiments, a particular region of a nucleic acid contains one, two, or more (optionally different) types of standard and / or modified nucleotides and nucleosides.

[0172] In some embodiments, modified RNA nucleic acids (e.g., modified mRNA nucleic acids) introduced into a cell or organism exhibit reduced degradation in the cell or organism, respectively, compared to unmodified nucleic acids comprising standard nucleotides and nucleosides.

[0173] In some embodiments, modified RNA nucleic acids (e.g., modified mRNA nucleic acids) introduced into a cell or organism may exhibit reduced immunogenicity (e.g., reduced innate response) in the cell or organism, respectively, compared to unmodified nucleic acids comprising standard nucleotides and nucleosides.

[0174] Nucleic acids (e.g., RNA nucleic acids, such as mRNA nucleic acids) in some embodiments include non-naturally modified nucleotides that are introduced during or after the synthesis of the nucleic acid to achieve a desired function or property. Modifications can be on the internucleotide linkage, the purine or pyrimidine base, or the sugar. Modifications can be introduced with chemical synthesis or with a polymerase enzyme at the end of the chain or anywhere within the chain. Any of the regions of the nucleic acid may be chemically modified.

[0175] The present disclosure provides modified nucleosides and nucleotides of nucleic acids (e.g., DNA nucleic acids or RNA nucleic acids such as mRNA nucleic acids). A "nucleoside" refers to a compound or derivative thereof that contains a sugar molecule (e.g., pentose or ribose) in combination with an organic base (e.g., a purine or pyrimidine) or a derivative thereof (also referred to herein as a "nucleobase"). A "nucleotide" refers to a nucleoside that includes a phosphate group. Modified nucleotides can be synthesized by any useful method, for example, chemically, enzymatically, or recombinantly, to include one or more modified or non-natural nucleosides. A nucleic acid can include a region or regions of linked nucleosides. Such regions can have variable backbone linkages. These linkages can be standard phosphodiester linkages, in which case the nucleic acid includes a region of nucleotides.

[0176] Modified nucleotide base pairing encompasses not only standard adenosine-thymine, adenosine-uracil, or guanosine-cytosine base pairs, but also base pairs formed between modified nucleotides containing nucleotides and / or non-standard or modified bases, where the arrangement of hydrogen bond donors and hydrogen bond acceptors allows hydrogen bonding between non-standard and standard bases or between two complementary non-standard base structures, for example, in those nucleic acids with at least one chemical modification. One example of such non-standard base pairing is base pairing between the modified nucleotide inosine and adenine, cytosine, or uracil. Any combination of base / sugar or linker can be incorporated into the nucleic acid of the present disclosure.

[0177] In some embodiments, modified nucleobases in a nucleic acid (e.g., an RNA nucleic acid such as an mRNA nucleic acid) include 1-methyl-pseudouridine (m1ψ), 1-ethyl-pseudouridine (e1ψ), 5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C), and / or pseudouridine (ψ). In some embodiments, modified nucleobases in a nucleic acid (e.g., an RNA nucleic acid such as an mRNA nucleic acid) include 5-methoxymethyluridine, 5-methylthiouridine, 1-methoxymethylpseudouridine, 5-methylcytidine, and / or 5-methoxycytidine. In some embodiments, a polynucleotide includes a combination of at least two (e.g., two, three, four, or more) of any of the foregoing modified nucleobases, including but not limited to chemical modifications.

[0178] In some embodiments, an RNA nucleic acid of the disclosure comprises 1-methyl-pseudouridine (m1ψ) substitutions at one or more or all uridine positions of the nucleic acid.

[0179] In some embodiments, an RNA nucleic acid of the disclosure comprises 1-methyl-pseudouridine (m1ψ) substitutions at one or more or all of the uridine positions of the nucleic acid and 5-methylcytidine substitutions at one or more or all of the cytidine positions of the nucleic acid.

[0180] In some embodiments, an RNA nucleic acid of the disclosure comprises pseudouridine (ψ) substitutions at one or more or all uridine positions of the nucleic acid.

[0181] In some embodiments, an RNA nucleic acid of the disclosure comprises pseudouridine (ψ) substitutions at one or more or all of the uridine positions of the nucleic acid and 5-methylcytidine substitutions at one or more or all of the cytidine positions of the nucleic acid.

[0182] In some embodiments, an RNA nucleic acid of the disclosure contains a uridine at one or more or all uridine positions of the nucleic acid.

[0183] In some embodiments, a nucleic acid (e.g., an RNA nucleic acid, such as an mRNA nucleic acid) is uniformly modified (e.g., completely modified, modified throughout the entire sequence) for a particular modification. For example, a nucleic acid can be uniformly modified with 1-methyl-pseudouridine, meaning that all uridine residues in the mRNA sequence are replaced with 1-methyl-pseudouridine. Similarly, a nucleic acid can be uniformly modified for any type of nucleoside residue present in the sequence by substitution with modified residues such as those described above.

[0184] The nucleic acid of the present disclosure may be partially or completely modified along the entire length of the molecule. For example, one or more or all or a given type of nucleotide (e.g., purine or pyrimidine, or any one or more or all of A, G, U, C) may be uniformly modified in the nucleic acid of the present disclosure, or in a given sequence region thereof (e.g., mRNA, including or excluding poly-A tail). In some embodiments, all nucleotides X in the nucleic acid of the present disclosure (or in the sequence region thereof) are modified nucleotides, where X can be any one of nucleotides A, G, U, C, or any one of combinations A+G, A+U, A+C, G+U, G+C, U+C, A+G+U, A+G+C, G+U+C, or A+G+C.

[0185] Nucleic acids may contain from about 1% to about 100% modified nucleotides (with respect to the overall nucleotide content or with respect to any one or more types of nucleotides, i.e., 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 will be understood that any remaining percentage is accounted for by the presence of unmodified A, G, U, or C.

[0186] The nucleic acid may contain as little as 1% and as much as 100% modified nucleotides, or any intervening percentage, for example, at least 5% modified nucleotides, at least 10% modified nucleotides, at least 25% modified nucleotides, at least 50% modified nucleotides, at least 80% modified nucleotides, or at least 90% modified nucleotides. For example, the nucleic acid may contain modified pyrimidines, such as modified uracil or 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 nucleic acid are replaced with modified uracils (e.g., 5-substituted uracils). The modified uracils may be replaced by a compound with a single unique structure, or may be replaced by multiple compounds with different structures (e.g., two, three, four, or more unique structures). In some embodiments, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90%, or 100% of the cytosines in a nucleic acid are replaced with modified cytosines (e.g., 5-substituted cytosines). The modified cytosines may be replaced by a compound having a single unique structure, or may be replaced by multiple compounds having different structures (e.g., two, three, four, or more unique structures).

[0187] In some embodiments, the nucleic acid may include any useful linker between nucleosides. Such linkers (including backbone modifications) useful in the compositions of the present disclosure include, but are not limited to, 3'-alkylene phosphonates, 3'-amino phosphoramidates, alkene-containing backbones, aminoalkyl phosphoramidates, aminoalkyl phosphotriesters, boranophosphates, -CH2-ON(CH3)-CH2-, -CH2-N(CH3)-N(CH3)-CH2-, -CH2-NH-CH2-, chiral phosphonates, chiral phosphorothioates, formacetyl and thioformacetyl backbones, methylene(methylimino), methyleneformacetyl and and thioformacetyl backbones, methyleneimino and methylenehydrazino backbones, morpholino linkages, -N(CH3)-CH2-CH2-, oligonucleosides with heteroatom internucleoside linkages, phosphinates, phosphoramidates, phosphorodithioates, phosphorothioate internucleoside linkages, phosphorothioates, phosphotriesters, PNAs, siloxane backbones, sulfamate backbones, sulfide sulfoxide and sulfone backbones, sulfonate and sulfonamide backbones, thionoalkylphosphonates, thionoalkylphosphotriesters, and thionophosphoramidates.

[0188] Modified nucleosides and nucleotides (e.g., building block molecules) that can be incorporated into nucleic acids (e.g., RNA or mRNA as described herein) can be modified on the sugar of the ribonucleic acid. For example, the 2' hydroxyl group (OH) can be modified or replaced with a number of different substituents. Exemplary substitutions at the 2' position include H, halo, optionally substituted C, aryl ... 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 C1~12 (heterocyclyl)oxy; sugar (e.g., ribose, pentose, or any of those described herein); polyethylene glycol (PEG), -O(CH2CH2O) n CH2CH2OR, where R is H or an optionally substituted alkyl and n is an integer from 0 to 20 (e.g., 0 to 4, 0 to 8, 0 to 10, 0 to 16, 1 to 4, 1 to 8, 1 to 10, 1 to 16, 1 to 20, 2 to 4, 2 to 8, 2 to 10, 2 to 16, 2 to 20, 4 to 8, 4 to 10, 4 to 16, and 4 to 20); 2'-hydroxyl is C 1~6 Alkylene or C 1~6 "Locked" Nucleic Acids (LNAs) are linked to the 4'-carbon of the same ribose sugar by a heteroalkylene bridge (wherein exemplary bridges include methylene, propylene, ether, or amino bridges); aminoalkyl; aminoalkoxy; amino; and amino acid.

[0189] Generally, RNA contains the sugar group ribose, which is a five-membered ring with oxygen. Exemplary non-limiting modified nucleotides include substitution of oxygen in ribose (e.g., with S, Se, or an alkylene such as methylene or ethylene); addition of a double bond (e.g., replacing ribose with cyclopentenyl or cyclohexenyl); ring contraction of ribose (e.g., forming a four-membered ring of cyclobutane or oxetane); ring expansion of ribose (e.g., forming a six- or seven-membered ring with additional carbon or heteroatoms, such as for anhydrohexitol, altritol, mannitol, cyclohexanyl, cyclohexenyl, and morpholino, which also have a phosphoramidate backbone); polycyclic forms (e.g., tricyclic, and "unlocked" forms such as glycol nucleic acid (GNA) (e.g., in which ribose is attached to a phosphodiester bond); Examples of sugar modifications include R-GNA or S-GNA, in which the ribose is replaced by an α-L-threofuranosyl-(3'→2')-linked glycol unit, TNA, in which the ribose is replaced by an α-L-threofuranosyl-(3'→2')-linked glycol unit, and peptide nucleic acid (PNA, in which a 2-amino-ethyl-glycine linkage replaces the ribose and phosphodiester backbone). The sugar group may also contain one or more carbons that have the opposite stereochemical configuration to that of the corresponding carbon in ribose. Thus, a polynucleotide molecule may include, for example, nucleotides that contain arabinose as the sugar. Such sugar modifications are described, for example, in International Patent Publication Nos. WO2013052523 and WO2014093924, the contents of each of which are incorporated herein by reference in their entirety for this purpose.

[0190] Nucleic acids of the disclosure (e.g., nucleic acids encoding one or more peptide epitopes, or functional fragments or variants thereof) can include combinations of modifications to the sugar, nucleobase, and / or internucleoside linkages. These combinations can include any one or more modifications described herein.

[0191] The nucleic acid cancer vaccine disclosed herein is a composition comprising pharmaceutical composition.The present disclosure also encompasses the method for selecting, designing, preparing, manufacturing, formulating and / or using the nucleic acid cancer vaccine provided herein.Also provided are systems (e.g., computerized systems), processes, devices and kits for selecting, designing and / or using the nucleic acid cancer vaccine described herein.

[0192] In vitro transcription of RNA (e.g., mRNA) Cancer vaccines of the present disclosure can include at least one nucleic acid (e.g., an RNA polynucleotide, such as an mRNA (message RNA) or an mmRNA (modified mRNA)). The mRNA is transcribed in vitro, for example, from a template DNA referred to as an "in vitro transcription template." In some embodiments, the in vitro transcription template encodes a 5' untranslated (UTR) region, contains an open reading frame, encodes a 3' UTR, and a polyA tail. The particular nucleic acid sequence composition and length of the in vitro transcription template depends on the mRNA encoded by the template.

[0193] In some embodiments, the nucleic acid comprises 15 to 3,000 nucleotides. For example, the polynucleotide may comprise 15 to 50, 15 to 100, 15 to 200, 15 to 300, 15 to 400, 15 to 500, 15 to 600, 15 to 700, 15 to 800, 15 to 900, 15 to 1000, 15 to 1200, 15 to 1400, 15 to 1500, 15 to 1800, 15 to 2000, 15 to 2500, 15 to 3000, 50 to 100, 50 ~200, 50~300, 50~400, 50~500, 50~600, 50~700, 50~800, 50~900, 50~1000, 50~1200, 50~1400, 50~1500, 50~1800, 50~2000, 50~2500, 50~3000, 100~200, 100~300, 100~400, 100~500, 100~60 0, 100~700, 100~800, 100~900, 100~1000, 100~1200, 100~1400, 100~1500, 100~1800, 100~2000, 100~2500, 100~3000, 200~300, 200~400, 200~500, 200~600, 200~700, 200,~800, 200~900, 2 The nucleic acid sequence may comprise 00-1000, 200-1500, 200-3000, 500-1000, 500-1500, 500-2000, 500-2500, 500-3000, 1000-1500, 1000-2000, 1000-2500, 1000-3000, 1500-3000, 2500-3000, or 2000-3000 nucleotides.

[0194] In another aspect, the present disclosure relates to a method for preparing nucleic acid cancer vaccines (e.g., mRNA cancer vaccines) by IVT method. In vitro transcription (IVT) method allows template-directed synthesis of RNA molecules of almost any sequence. The size of RNA molecules that can be synthesized using IVT method ranges from short oligonucleotides to long nucleic acid polymers of several thousand bases. IVT method allows synthesis of large amounts of RNA transcripts (e.g., microgram to milligram amounts). See Beckert et al., Synthesis of RNA by in vitro transcription, Methods Mol Biol. 703: 29-41 (2011); Rio et al. RNA: A Laboratory Manual. Cold Spring Harbor: Cold Spring Harbor Laboratory Press, 2011, 205-220.; Cooper, Geoffery M. The Cell: A Molecular Approach. 4th ed. Washington DC: ASM Press, 2007. 262-299 (each of which is incorporated herein by reference for this purpose). Generally, IVT utilizes a DNA template that features a promoter sequence upstream of the sequence of interest. The promoter sequence is most commonly of bacteriophage origin (e.g., T7, T3, or SP6 promoter sequence), but many other promoter sequences, including those designed de novo, are acceptable. Transcription of the DNA template is typically best accomplished by using an RNA polymerase that corresponds to a specific bacteriophage promoter sequence. Exemplary RNA polymerases include, but are not limited to, T7 RNA polymerase, T3 RNA polymerase, or SP6 RNA polymerase, among others. IVT generally initiates with dsDNA, but can proceed on a single strand.

[0195] It will be understood that the nucleic acid cancer vaccines (e.g., mRNA cancer vaccines) of the present disclosure, e.g., mRNA encoding a cancer antigen, can be made using any suitable synthesis method. For example, in some embodiments, the mRNA vaccines of the present disclosure are made using IVT from single-stranded bottom-stranded DNA as a template and a complementary oligonucleotide that serves as a promoter. The single-stranded bottom-stranded DNA can serve as a DNA template for in vitro transcription of RNA, and can be obtained, for example, from a plasmid, a PCR product, or chemical synthesis. In some embodiments, the single-stranded bottom-stranded DNA is linearized from a circular template. The single-stranded bottom-stranded DNA template generally includes a promoter sequence, e.g., a bacteriophage promoter sequence, to facilitate IVT. Methods for making RNA using single-stranded bottom-stranded DNA and top-stranded promoter complementary oligonucleotides are known in the art. Exemplary methods include, but are not limited to, annealing a DNA bottom strand template with a top strand promoter-complementary oligonucleotide (e.g., a T7 promoter-complementary oligonucleotide, a T3 promoter-complementary oligonucleotide, or a SP6 promoter-complementary oligonucleotide), followed by IVT using an RNA polymerase corresponding to the promoter sequence, e.g., aT7 RNA polymerase, T3 RNA polymerase, or SP6 RNA polymerase.

[0196] The IVT method can also be performed using a double-stranded DNA template. For example, in some embodiments, the double-stranded DNA template is made by extending a complementary oligonucleotide to generate a complementary DNA strand using strand extension techniques available in the art. In some embodiments, a single bottom strand DNA template containing a promoter sequence and a sequence encoding one or more peptide epitopes of interest is annealed to a top strand promoter complementary oligonucleotide and subjected to a PCR-like process to extend the top strand to generate a double-stranded DNA template. Alternatively or additionally, a top strand DNA that is complementary to the bottom strand promoter sequence and contains a sequence complementary to a sequence encoding one or more peptide epitopes of interest is annealed to a bottom strand promoter oligonucleotide and subjected to a PCR-like process to extend the bottom strand to generate a double-stranded DNA template. In some embodiments, the number of PCR-like cycles ranges from 1 to 20 cycles, e.g., 3 to 10 cycles. In some embodiments, the double-stranded DNA template is synthesized in whole or in part by chemical synthesis methods. The double-stranded DNA template can be subjected to in vitro transcription as described herein.

[0197] In another aspect, the nucleic acid cancer vaccines of the present disclosure, including, for example, mRNAs encoding peptide epitopes, can be made using two DNA strands that are complementary across the overlapping portions of their sequences, leaving single-stranded overhangs (i.e., sticky ends) when the complementary portions are annealed. These single-stranded overhangs can be made double-stranded by extension using the other strand as a template, thereby generating double-stranded DNA. In some cases, this primer extension method can allow for a larger ORF to be incorporated into the template DNA sequence, for example, compared to the size incorporated into the template DNA sequence obtained by the top strand DNA synthesis method. In the primer extension method, the 3' end portion of the first strand (5'-3' direction) is complementary to the 3' end portion of the second strand (3'-5' direction). In some such embodiments, the first single-stranded DNA can include a promoter (e.g., T7, T3, or SP6) sequence, optionally a 5'-UTR, and part or all of the ORF (e.g., the 5' end portion of the ORF). In some embodiments, the second single-stranded DNA may include a complementary sequence of part or all of the ORF (e.g., the portion complementary to the 3' end of the ORF), and optionally a 3'-UTR, a termination sequence, and / or a poly-A tail. A method of making RNA using two synthetic DNA strands may include annealing the two strands at the overlapping complementary portions, followed by extending the strands by primer extension using one or more PCR-like cycles to generate a double-stranded DNA template. In some embodiments, the number of PCR-like cycles ranges from 1 to 20 cycles, e.g., 3 to 10 cycles. Such double-stranded DNA may be subjected to in vitro transcription as described herein.

[0198] In another aspect, a nucleic acid vaccine of the present disclosure, including, for example, an mRNA encoding a peptide epitope, can be made using a synthetic double-stranded linear DNA molecule, such as gBlocks® (Integrated DNA Technologies, Coralville, Iowa), as a double-stranded DNA template. The advantage of such synthetic double-stranded linear DNA molecules is that they provide a longer template for generating mRNA. For example, the size of gBlocks® can range from 45 to 1000 (e.g., 125 to 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. The full-length 5′-UTR can be up to 100 nucleotides long, e.g., about 40 to 60 nucleotides. The full-length 3′-UTR can be up to 300 nucleotides long, e.g., about 100 to 150 nucleotides.

[0199] To facilitate the generation of longer constructs, two or more double-stranded linear DNA molecules and / or gene fragments designed with overlapping sequences on the 3' strand may be assembled together using methods known in the art. For example, the Gibson Assembly™ method (Synthetic Genomics, Inc., La Jolla, CA) can be performed using the use of neutrophil exonuclease to cleave bases from the 5' end of double-stranded DNA fragments, followed by annealing of the newly formed complementary single-stranded 3' ends, polymerase-dependent extension to fill any single-stranded gaps, and finally covalently joining the DNA segments with DNA ligase.

[0200] In another embodiment, the nucleic acid cancer vaccine of the present disclosure, including, for example, an mRNA encoding a peptide epitope, can be made using chemical synthesis of RNA. The method includes, for example, annealing a first polynucleotide including an open reading frame encoding a polypeptide and a second polynucleotide including a 5'-UTR to a complementary polynucleotide conjugated to a solid support. The 3' end of the second polynucleotide is then ligated to the 5' end of the first polynucleotide under suitable conditions. Suitable conditions include the use of a DNA ligase. The ligation reaction produces a first ligation product. The 5' end of a third polynucleotide including a 3'-UTR is then ligated to the 3' end of the first ligation product under suitable conditions. Suitable conditions for the second ligation reaction include an RNA ligase. The second ligation product is produced in a second ligation reaction. The second ligation product is released from the solid support to produce an mRNA encoding the polypeptide of interest. In some embodiments, the mRNA is between 30 and 1000 nucleotides.

[0201] An mRNA encoding one or more peptide epitopes can also be prepared by linking a first nucleic acid comprising an open reading frame encoding a nucleic acid to a second nucleic acid comprising a 3'-UTR to a complementary nucleic acid conjugated to a solid support. The 5' end of the second nucleic acid is ligated to the 3' end of the first nucleic acid under suitable conditions (e.g., with a DNA ligase). The method produces a first ligation product. A third nucleic acid comprising a 5'-UTR is ligated to the first ligation product under suitable conditions (e.g., with an RNA ligase, e.g., T4 RNA) to produce a second ligation product. The second ligation product is released from the solid support to produce an mRNA encoding one or more peptide epitopes.

[0202] In some embodiments, the first nucleic acid is characterized by a 5'-triphosphate and a 3'-OH. In other embodiments, the second nucleic acid comprises a 3'-OH. In yet other embodiments, the third nucleic acid comprises a 5'-triphosphate and a 3'-OH. The second nucleic acid may also comprise a 5'-cap structure. The method may also comprise the further step of ligating a fourth nucleic acid comprising a polyA region to the 3' end of the third nucleic acid. The fourth nucleic acid may comprise a 5'-triphosphate.

[0203] The method may or may not include reverse phase purification. The method may also include a wash step to wash the solid support to remove unreacted nucleic acid. The solid support may be, for example, a capture resin. In some embodiments, the method includes dT purification.

[0204] According to the present disclosure, the template DNA encoding the nucleic acid (e.g., mRNA) cancer vaccine of the present disclosure comprises an open reading frame (ORF) encoding one or more peptide epitopes. In some embodiments, the template DNA comprises an ORF of up to 1000 nucleotides, e.g., about 10-350, 30-300 nucleotides, or about 50-250 nucleotides. In some embodiments, the template DNA comprises an ORF of about 150 nucleotides. In some embodiments, the template DNA comprises an ORF of about 200 nucleotides.

[0205] In some embodiments, the IVT transcription products are purified from the components of the IVT reaction mixture after the reaction has occurred. For example, the crude IVT mixture can be treated with RNase-free DNase to digest the original template. Nucleic acids (e.g., mRNA) can be purified using methods known in the art, including, but not limited to, precipitation using organic solvents or column-based purification methods. Commercial kits are available for purifying RNA, e.g., MEGACLEAR™ kits (Ambion, Austin, TX). Nucleic acids (e.g., mRNA) can be quantified using methods known in the art, including, but not limited to, commercially available instruments, e.g., NanoDrop. Purified nucleic acids (e.g., mRNA) can be analyzed, for example, by agarose gel electrophoresis, to ensure that the nucleic acids are of the appropriate size and / or that no degradation of the nucleic acids has occurred.

[0206] Untranslated Regions (UTRs) An untranslated region (UTR) is a segment of nucleic acid before the start codon (5'UTR) and after the stop codon (3'UTR) that is not translated. In some embodiments, a nucleic acid (e.g., a ribonucleic acid (RNA), e.g., a messenger RNA (mRNA)) of the disclosure that includes an open reading frame (ORF) encoding one or more peptide epitopes further includes one or more UTRs (e.g., a 5'UTR or a functional fragment thereof, a 3'UTR or a functional fragment thereof, or a combination thereof).

[0207] The UTR may be homologous or heterologous to the coding region in the nucleic acid. In some embodiments, the UTR is homologous to the ORF encoding one or more peptide epitopes. In some embodiments, the UTR is heterologous to the ORF encoding one or more peptide epitopes. In some embodiments, the nucleic acid comprises two or more 5'UTRs or functional fragments thereof, each having the same or different nucleotide sequence. In some embodiments, the nucleic acid comprises two or more 3'UTRs or functional fragments thereof, each having the same or different nucleotide sequence.

[0208] In some embodiments, the 5'UTR or a functional fragment thereof, the 3'UTR or a functional fragment thereof, or any combination thereof, is sequence optimized.

[0209] In some embodiments, the 5'UTR or a functional fragment thereof, the 3'UTR or a functional fragment thereof, or any combination thereof comprises at least one chemically modified nucleobase, for example, 5-methoxyuracil.

[0210] UTRs can have features that provide a regulatory role, such as increased or decreased stability, localization, and / or translation efficiency. Nucleic acids containing UTRs can be administered to cells, tissues, or organisms, and one or more regulatory features can be measured using routine methods. In some embodiments, functional fragments of the 5'UTR or 3'UTR contain one or more regulatory features of the full-length 5' or 3'UTR, respectively.

[0211] Naturally occurring 5′ UTRs function in translation initiation. They contain signatures such as the Kozak sequence, which are commonly known to be involved in the process by which the ribosome initiates the translation of many genes. 5′ UTRs are also known to form secondary structures involved in elongation factor binding.

[0212] By engineering features typically found in abundantly expressed genes of specific target organs, nucleic acid stability and protein production can be enhanced. For example, introduction of the 5'UTR of liver-expressed mRNAs such as albumin, serum amyloid A, apolipoprotein A / B / E, transferrin, alpha-fetoprotein, erythropoietin, or factor VIII can enhance expression of the nucleic acid in hepatic cell lines or the liver. Similarly, use of 5'UTRs from other tissue-specific mRNAs to improve expression in that tissue is possible for 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).

[0213] In some embodiments, UTR is selected from a family of transcripts in which proteins share common functions, structures, characteristics, or properties.For example, the encoded polypeptide may belong to a family of proteins (i.e., share at least one function, structure, characteristic, localization, origin, or expression pattern) that are expressed in a particular cell, tissue, or at a certain time during development.The UTR from either gene or mRNA is exchanged with any other UTR from the same or different protein family to create new nucleic acid.

[0214] In some embodiments, the 5'UTR and the 3'UTR may 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.

[0215] International Patent Application No. PCT / US2014 / 021522 (Publication No. WO / 2014 / 164253) provides a list of exemplary UTRs that can be utilized as ORF flanking regions in the nucleic acids of the present disclosure, which publication is incorporated herein by reference for this purpose.

[0216] Additional exemplary UTRs that can be utilized in the nucleic acids of the present disclosure include globins, such as α- or β-globin (e.g., Xenopus, mouse, rabbit, or human globin); strong Kozak translation initiation signals; CYBA (e.g., human cytochrome b-245α polypeptide); albumins (e.g., human albumin 7); HSD17B4 (hydroxysteroid (17-β) dehydrogenase); viruses (e.g., tobacco etch virus (TEV), Venezuelan equine encephalitis virus (VEEV), dengue virus, cytomegalovirus (CMV, e.g., CMV immediate early 1 (IE1)), hepatitis viruses (e.g., hepatitis B virus), Sindbis virus, and the like. , 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 or β actin); GAPDH; tubulin; histones; citric acid cycle enzymes; topoisomerases (e.g., the 5´UTR of the TOP gene lacking a 5´TOP motif (oligopyrimidine tract)); ribosomal protein large 32 (L32); ribosomal proteins (e.g., human or mouse ribosomal proteins, such as rps9); ATP synthases (e.g., ATP5A1 or mitochondrial H +-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); myocyte enhancer factor 2A (MEF2A); β-F1-ATPase, creatine kinase, myoglobin, granulocyte colony-stimulating factor (G-CSF); collagen (e.g., collagen type I alpha 2 (Col1A2), collagen type I alpha 1 (Col1A1), collagen type VI alpha 2 (Col6A2), These include, but are not limited to, one or more 5'UTRs and / or 3'UTRs derived from the nucleic acid sequences of collagen type VI alpha 1 (Col6A1); ribophorin (e.g., ribophorin I (RPNI)); low density lipoprotein receptor-related protein (e.g., LRP1); cardiotrophin-like cytokine factor (e.g., Nnt1); calreticulin (Calr); procollagen-lysine, 2-oxoglutarate 5-dioxygenase 1 (Plod1); and nucleobindin (e.g., Nucb1).

[0217] In some embodiments, the 5'UTR is selected from the group consisting of β-globin 5'UTR; a 5'UTR containing a strong Kozak translation initiation signal; cytochrome b-245 alpha polypeptide (CYBA) 5'UTR; hydroxysteroid (17-beta) dehydrogenase (HSD17B4) 5'UTR; tobacco etch virus (TEV) 5'UTR; Venezuelan equine encephalitis virus (TEEV) 5'UTR; the 5' proximal open reading frame of rubella virus (RV) RNA encoding a nonstructural protein; dengue virus (DEN) 5'UTR; heat shock protein 70 (Hsp70) 5'UTR; eIF4G 5'UTR; GLUT1 5'UTR; functional fragments thereof, and any combination thereof.

[0218] In some embodiments, the 3'UTR is selected from the group consisting of β-globin 3'UTR; CYBA 3'UTR; albumin 3'UTR; growth hormone (GH) 3'UTR; VEEV 3'UTR; Hepatitis B virus (HBV) 3'UTR; α-globin 3'UTR; DEN 3'UTR; PAV barley yellow dwarf virus (BYDV-PAV) 3'UTR; elongation factor 1 alpha 1 (EEF1A1) 3'UTR; manganese superoxide dismutase (MnSOD) 3'UTR; β subunit of mitochondrial H(+)-ATP synthase (β-mRNA) 3'UTR; GLUT1 3'UTR; MEF2A 3'UTR; β-F1-ATPase 3'UTR; functional fragments thereof, and combinations thereof.

[0219] Wild-type UTR from any gene or mRNA can be incorporated into the nucleic acid of the present disclosure.In some embodiments, UTR can be modified relative to wild-type or natural UTR, for example, by changing the orientation or position of UTR relative to ORF, or by including additional nucleotides, deleting nucleotides, exchanging or rearranging nucleotides, to produce variant UTR.In some embodiments, variants of 5' or 3' UTR can be utilized, for example, mutants or variants of wild-type UTR, and one or more nucleotides are added or removed from the end of UTR.

[0220] Additionally, one or more synthetic UTRs may be used in combination with one or more non-synthetic UTRs. See, for example, Mandal and Rossi, Nat.Protoc.2013 8(3):568-82, and the sequences available at www.addgene.org / Derrick_Rossi / , the contents of each of which are incorporated herein by reference in their entirety. UTRs or portions thereof may be placed in the same orientation as their selected transcript, or the orientation or position may be altered. Thus, 5' and / or 3'UTRs may be inverted, shortened, extended, or combined with one or more other 5'UTRs or 3'UTRs.

[0221] In some embodiments, the nucleic acid may comprise multiple UTRs, such as double, triple, or quadruple 5'UTRs or 3'UTRs. For example, a double UTR comprises two copies of the same UTR, contiguous or substantially contiguous. For example, a double β-globin 3'UTR may be used (see, for example, US2010 / 0129877, the contents of which are incorporated herein by reference for this purpose).

[0222] The nucleic acids of the present disclosure may comprise a combination of features. For example, an ORF may be flanked by a 5'UTR that contains a strong Kozak translation initiation signal and / or a 3'UTR that contains an oligo(dT) sequence for templated addition of a polyA tail. The 5'UTR may comprise a first nucleic acid fragment and a second nucleic acid fragment from the same and / or a different UTR (see, e.g., US2010 / 0293625, which is incorporated herein by reference in its entirety for this purpose).

[0223] Other non-UTR sequences can be used as regions or subregions within the nucleic acids of the present disclosure. For example, introns or portions of intron sequences can be incorporated into the nucleic acids of the present disclosure. Incorporation of intron sequences can increase protein production as well as expression levels of the nucleic acids. In some embodiments, the nucleic acids of the present disclosure include an internal ribosome entry site (IRES) in place of or in addition to a UTR (see, e.g., Yakubov et al., Biochem. Biophys. Res. Commun. 2010 394(1):189-193, the contents of which are incorporated herein by reference in their entirety). In some embodiments, the nucleic acid includes an IRES in place of a 5'UTR sequence. In some embodiments, the nucleic acid includes an ORF and a viral capsid sequence. In some embodiments, the nucleic acid includes a synthetic 5'UTR in combination with a non-synthetic 3'UTR.

[0224] In some embodiments, the UTR also comprises at least one translation enhancer nucleic acid, translation enhancer element, or translation enhancer element(s) (collectively "TEE", which refers to a nucleic acid sequence that increases the amount of a polypeptide or protein produced from a polynucleotide. By way of non-limiting example, the TEE can include those described in US2009 / 0226470, which is incorporated herein by reference in its entirety for this purpose, and others known in the art. By way of non-limiting example, the TEE can be located between the transcription promoter and the start codon. In some embodiments, the 5' UTR comprises a TEE. In one aspect, a TEE is a conserved element in a UTR that can promote translational activity of a nucleic acid, such as, but not limited to, cap-dependent or cap-independent translation. In one non-limiting example, the TEE comprises a TEE sequence in the 5' leader of a Gtx homeodomain protein. See Chappell et al., PNAS 2004 101:9590-9594, which is incorporated herein by reference in its entirety for this purpose.

[0225] The term "translational enhancer polynucleotide" or "translational enhancer polynucleotide sequence" refers to a nucleic acid comprising one or more of the TEEs provided herein and / or known in the art (e.g., US6310197, US6849405, US7456273, US7183395, US2009 / 0226470, US2007 / 0048776, US2011 / 0124100, US2009 / 009 3049, US2013 / 0177581, WO2009 / 075886, WO2007 / 025008, WO2012 / 009644, WO2001 / 055371, WO1999 / 024595, EP2610341A1, and EP2610340A1, the contents of each of which are incorporated herein by reference in their entirety for this purpose), or variants, homologs, or functional derivatives thereof. In some embodiments, the nucleic acid of the present disclosure comprises one or more copies of a TEE. The TEE in the translation enhancer nucleic acid may be organized in one or more sequence segments. The sequence segment may contain one or more of the TEEs provided herein, each TEE being present in one or more copies. When multiple sequence segments are present in the translation enhancer nucleic acid, they may be homologous or heterologous. Thus, multiple sequence segments in a translation enhancer nucleic acid can contain the same or different types of TEEs provided herein, the same or different numbers of copies of each of the TEEs, and / or the same or different organization of the TEEs within each sequence segment. In one embodiment, a nucleic acid of the present disclosure comprises a translation enhancer nucleic acid sequence.

[0226] In some embodiments, the 5'UTR and / or 3'UTR comprising at least one TEE as described herein can be incorporated into a monocistronic sequence, such as, but not limited to, a vector system or a nucleic acid vector. In some embodiments, the 5'UTR and / or 3'UTR of the polynucleotide of the present disclosure comprises a TEE or a portion thereof as described herein. In some embodiments, the TEE in the 3'UTR can be the same and / or different from the TEE located in the 5'UTR.

[0227] In some embodiments, the 5'UTR and / or 3'UTR of a nucleic acid of the disclosure may comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, or more than 60 TEE sequences. In one embodiment, the 5'UTR of a nucleic acid of the disclosure may comprise 1-60, 1-55, 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 TEE sequence. The TEE sequences in the 5'UTR of the nucleic acid of the present disclosure can be the same or different TEE sequences. The combination of different TEE sequences in the 5'UTR of the nucleic acid of the present disclosure can include combinations in which two or more copies of any of the different TEE sequences are incorporated.

[0228] In some embodiments, the 5'UTR and / or 3'UTR comprises a spacer separating the two TEE sequences. As a non-limiting example, the spacer can be a 15 nucleotide spacer and / or other spacers known in the art (e.g., in multiples of 3 nucleotides). As another non-limiting example, the 5'UTR and / or 3'UTR comprises a TEE sequence spacer module that is repeated at least once, at least twice, at least three times, at least four times, at least five times, at least six times, at least seven times, at least eight times, at least nine times, at least ten times, or more than ten times in the 5'UTR and / or 3'UTR, respectively. In some embodiments, the 5'UTR and / or 3'UTR comprises a TEE sequence spacer module that is repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times.

[0229] 3′UTR and AU-rich elements In certain embodiments, a nucleic acid of the disclosure (eg, a nucleic acid encoding a peptide epitope of the disclosure) further comprises a 3'UTR.

[0230] The 3'-UTR is the section of an mRNA that immediately follows the translation stop codon and often contains regulatory regions that affect gene expression post-transcriptionally. Regulatory regions within the 3'-UTR can affect polyadenylation, translation efficiency, localization, and stability of the mRNA. In one embodiment, the 3'-UTR useful in the present disclosure contains binding sites for regulatory proteins or microRNAs. In some embodiments, the 3'-UTR has a silencer region that binds to a repressor protein and inhibits expression of the mRNA. In other embodiments, the 3'-UTR contains an AU-rich element (ARE). Proteins bind to the ARE to affect the stability or decay rate of the transcript in a localized manner or to affect translation initiation. In other embodiments, the 3'-UTR contains the sequence AAUAAA, which directs the addition of several hundred adenine residues, called a polyA tail, to the end of the mRNA transcript.

[0231] Native or wild-type 3'UTRs are known to have stretches of adenosines and uridines embedded in them. These AU-rich signatures are particularly common in genes with high turnover rates. Based on their sequence features and functional properties, AU-rich elements (AREs) can be separated into three classes (Chen et al, 1995): Class I AREs contain several dispersed copies of the AUUUA motif within the U-rich region. C-Myc and MyoD contain Class I AREs. Class II AREs have two or more overlapping UUAUUUA(U / A)(U / A) nonamers. Molecules containing this type of ARE include GM-CSF and TNF-a. Class III AREs do not contain the AUUUA motif. c-Jun and Myogenin are two well-studied examples of this class. While most proteins that bind to AREs are known to destabilize the messenger, members of the ELAV family, most notably HuR, have been documented to increase mRNA stability. HuR binds to all three classes of AREs. Engineering a HuR-specific binding site into the 3'UTR of a nucleic acid molecule results in HuR binding and thus stabilization of the message in vivo.

[0232] The introduction, removal, or modification of 3'UTR AU-rich elements (AREs) can be used to modulate the stability of the nucleic acids of the present disclosure. When engineering a specific nucleic acid, one or more copies of AREs can be introduced to reduce the stability of the nucleic acid of the present disclosure, thereby suppressing translation and reducing the production of the resulting protein. Similarly, AREs can be identified and removed or mutated to increase intracellular stability and thus increase the translation and production of the resulting protein. Transfection experiments can be performed in relevant cell lines using the nucleic acids of the present disclosure, and protein production can be assayed at various times after transfection. For example, cells can be transfected with different ARE engineered molecules and by using ELISA kits for the relevant proteins and assaying the protein produced at 6 hours, 12 hours, 24 hours, 48 ​​hours, and 7 days after transfection.

[0233] 5´ Capped Region The nucleic acid cancer vaccines described herein can be mRNA cancer vaccines that include one or more mRNAs having an open reading frame encoding a peptide epitope. Each of these mRNAs can have a 5' cap.

[0234] The 5' cap structure of native mRNA is involved in nuclear export, increases mRNA stability, and binds to mRNA cap-binding protein (CBP), which is involved in mRNA stability and translational competence in cells through the association of CBP with polyA-binding protein to form mature circular mRNA species. The cap further assists in the removal of the 5' proximal intron during mRNA splicing.

[0235] Endogenous mRNA molecules can be 5'-end capped, generating a 5'-ppp-5'-triphosphate bond between the terminal guanosine cap residue and the 5'-terminal transcribed sense nucleotide (cap) of the mRNA molecule. This 5'-guanylate cap can then be methylated to generate an N7-methyl-guanylate residue (cap-0). The ribose sugar of the terminal and / or pre-terminal transcribed nucleotide at the 5' end of the mRNA can also be optionally 2'-O-methylated (e.g., at the 2'-hydroxy group on the first ribose sugar (cap-1) or at the 2'-hydroxy groups on the first two ribose sugars (cap-2)). 5'-decapping and cleavage of the guanylate cap structure by hydrolysis can target nucleic acid molecules, such as mRNA molecules, for degradation.

[0236] In some embodiments, a nucleic acid of the disclosure (eg, a nucleic acid encoding a peptide epitope) incorporates a cap moiety.

[0237] In some embodiments, the nucleic acids of the present disclosure (e.g., nucleic acids encoding peptide epitopes) contain a non-hydrolyzable cap structure that prevents decapping and thus increases mRNA half-life. Because hydrolysis of the cap structure requires cleavage of the 5'-ppp-5' phosphorodiester bond, modified nucleotides can be used during the capping reaction. For example, vaccinia capping enzyme from New England Biolabs (Ipswich, MA) can be used with α-thio-guanosine nucleotides according to the manufacturer's instructions to create phosphorothioate bonds in the 5'-ppp-5' cap. Additional modified guanosine nucleotides, such as α-methyl-phosphonate and seleno-phosphate nucleotides, can be used.

[0238] Additional modifications include, but are not limited to, 2'-O-methylation of the ribose sugar of the 5'-terminus and / or 5'-preterminal nucleotide of a polynucleotide (as described above) on the 2'-hydroxyl group of the sugar ring. Multiple distinct 5'-cap structures may be used to generate the 5'-cap of a nucleic acid molecule, such as a polynucleotide that functions as an mRNA molecule. Cap analogs, also referred to herein as synthetic cap analogs, chemical caps, chemical cap analogs, or structural or functional cap analogs, differ in their chemical structure from the natural (i.e., endogenous, wild-type, or physical) 5'-cap while retaining cap function. Cap analogs may be chemically (i.e., non-enzymatically) or enzymatically synthesized and / or linked to the polynucleotides of the present disclosure.

[0239] For example, an anti-reverse cap analog (ARCA) cap contains two guanines linked by 5'-5'-triphosphate groups, with one guanine containing an N7 methyl group as well as a 3'-O-methyl group (i.e., N7,3'-O-dimethyl-guanosine-5'-triphosphate-5'-guanosine (m7G-3'mppp-G), which may equivalently be designated 3'O-Me-m7G(5')ppp(5')G). The 3'-O atom of the other unmodified guanine is linked to the 5' terminal nucleotide of the capped polynucleotide. The N7- and 3'-O-methylated guanine provide the terminal portion of the capped polynucleotide.

[0240] Another exemplary cap is mCAP, which is similar to ARCA but has a 2'-O-methyl group on the guanosine (i.e., N7,2'-O-dimethyl-guanosine-5'-triphosphate-5'-guanosine, m7Gm-ppp-G).

[0241] In some embodiments, the cap is a dinucleotide cap analog. As a non-limiting example, the dinucleotide cap analog can be modified at different phosphate positions with boranophosphate or phosphoroselenoate groups, such as the dinucleotide cap analogs described in U.S. Pat. No. 8,519,110, the contents of which are incorporated herein by reference in their entirety for this purpose.

[0242] In another embodiment, the cap is a cap analog and is an N7-(4-chlorophenoxyethyl) substituted dinucleotide form of a cap analog known in the art and / or described herein. Non-limiting examples of N7-(4-chlorophenoxyethyl) substituted dinucleotide forms of cap analogs include N7-(4-chlorophenoxyethyl)-G(5')ppp(5')G and N7-(4-chlorophenoxyethyl)-m3'-OG(5')ppp(5')G cap analogs (see, for example, the various cap analogs and methods of synthesizing cap analogs described in Kore et al. Bioorganic & Medicinal Chemistry 2013 21:4570-4574, the contents of which are incorporated herein by reference in their entirety for this purpose). In another embodiment, the cap analog of the present disclosure is a 4-chloro / bromophenoxyethyl analog.

[0243] Although cap analogs allow for concomitant capping of a polynucleotide or region thereof in an in vitro transcription reaction, up to 20% of the transcript may remain uncapped, which may lead to reduced translational competence and reduced cellular stability, as well as the structural difference of the cap analog from the endogenous 5'-cap structure of the nucleic acid produced by the endogenous cellular transcription machinery.

[0244] Nucleic acids of the present disclosure (e.g., nucleic acids encoding peptide antigens) can also be capped after production (by IVT or chemical synthesis) using enzymes to generate a more authentic 5'-cap structure. As used herein, the phrase "more authentic" refers to a feature that closely resembles or mimics an endogenous or wild-type feature, either structurally or functionally. That is, a "more authentic" feature is one that better represents an endogenous wild-type, natural, or physiological cellular function and / or structure, or is superior in one or more respects to the corresponding endogenous wild-type, natural, or physiological feature, compared to a synthetic feature or analog, etc. Non-limiting examples of more authentic 5' cap structures are those that have, among others, enhanced cap-binding protein binding, increased half-life, reduced susceptibility to 5' endonucleases, and / or reduced 5' decapping, compared to synthetic 5' cap structures (or wild-type, natural, or physiological 5' cap structures) known in the art. For example, recombinant vaccinia virus capping enzyme and recombinant 2'-O-methyltransferase enzyme can create a canonical 5'-5'-triphosphate bond between the 5'-terminal nucleotide of a polynucleotide and a guanine cap nucleotide, where the cap guanine contains an N7 methylation and the 5'-terminal nucleotide of the mRNA contains a 2'-O-methyl. Such a structure is called a Cap-1 structure. This cap results in higher translational competence and cellular stability, as well as reduced cellular pro-inflammatory cytokine activation, for example, compared to other 5' cap analog structures known in the art. Cap structures include, but are not limited to, 7mG(5')ppp(5')N, pN2p (Cap-0), 7mG(5')ppp(5')NlmpNp (Cap-1), and 7mG(5')-ppp(5')NlmpN2mp (Cap-2).

[0245] As a non-limiting example, capping a chimeric nucleic acid after production can be more efficient since nearly 100% of the chimeric nucleic acid can be capped, as opposed to about 80% when a cap analog is ligated to the chimeric nucleic acid during an in vitro transcription reaction.

[0246] In accordance with the present disclosure, the 5'-end cap can include an endogenous cap or a cap analog. In accordance with the present invention, the 5'-end cap can include a guanine analog. Useful guanine analogs include, but are not limited to, inosine, N1-methyl-guanosine, 2'fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

[0247] Poly A tail In some embodiments, the nucleic acids of the present disclosure (e.g., nucleic acids encoding peptide epitopes) further comprise a polyA tail. In further embodiments, terminal groups on the polyA tail may be incorporated for stabilization. In other embodiments, the polyA tail comprises a des-3' hydroxyl tail.

[0248] During RNA processing, long chains of adenine nucleotides (poly-A tails) can be added to nucleic acids such as mRNA molecules to increase stability. Immediately after transcription, the 3' end of the transcript can be cleaved to release a 3' hydroxyl. Poly-A polymerase then adds a chain of adenine nucleotides to the RNA. This process, called polyadenylation, adds a poly-A tail that can be, for example, between approximately 80 and approximately 250 residues long (including approximately 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 residues long). In some embodiments, the poly-A tail comprises about 100 nucleotides.

[0249] A polyA tail can also be added after the construct has been exported from the nucleus.

[0250] According to the present disclosure, terminal groups on the polyA tail can be incorporated for stabilization. The polynucleotides of the present disclosure can include des-3' hydroxyl tails. They can also include the structural moieties or 2'-O methyl modifications taught by Junjie Li et al. (Current Biology, Vol. 15, 1501-1507, August 23, 2005, the contents of which are incorporated herein by reference in their entirety for this purpose).

[0251] The nucleic acids of the present disclosure can be designed to encode transcripts with alternative poly-A tail structures, including histone mRNAs. According to Norbury, "terminal uridine has also been detected on human replication-dependent histone mRNAs. Turnover of these mRNAs is thought to be important to potentially prevent toxic histone accumulation following the completion or inhibition of chromosomal DNA replication. These mRNAs are distinguished by the absence of their 3' poly-A tails, a function assumed instead by a stable stem-loop structure and its cognate stem-loop binding protein (SLBP), the latter performing the same function as that of PABP on polyadenylated mRNAs" (Norbury, "Cytoplasmic RNA: a case of the tail wagging the dog," Nature Reviews Molecular Cell Biology; AOP, published online 29 August 2013; doi:10.1038 / nrm3645, the contents of which are incorporated herein by reference in their entirety for this purpose).

[0252] The length of the unique polyA tail provides certain advantages to the nucleic acids of the present disclosure. Generally, the length of the polyA tail, if present, is greater than 30 nucleotides in length. In another embodiment, the polyA tail is greater than 35 nucleotides in length (e.g., at least about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, or 3,000 nucleotides or more).

[0253] In some embodiments, the nucleic acid or region thereof comprises from about 15 to about 3,000 nucleotides (e.g., from 15 to 50, 15 to 100, 15 to 200, 15 to 300, 15 to 400, 15 to 500, 15 to 600, 15 to 700, 15 to 800, 15 to 900, 15 to 1000, 15 to 1200, 15 to 1400, 15 to 1500, 15 to 1800, 15 to 200 0, 15~2500, 15~3000, 50~100, 50~200, 50~300, 50~400, 50~500, 50~600, 50~700, 50~800, 50~900, 50~1000, 50~1200, 50~1400, 50~1500, 50~1800, 50~2000, 50~2500, 50~3000, 100~200, 100~300, 10 0~400, 100~500, 100~600, 100~700, 100~800, 100~900, 100~1000, 100~1200, 100~1400, 100~1500, 100~1800, 100~2000, 100~2500, 100~3000, 200~300, 200~400, 200~500, 200~600, 200~700, 200, 8 00, 200-900, 200-1000, 200-1500, 200-3000, 500-1000, 500-1500, 500-2000, 500-2500, 500-3000, 1000-1500, 1000-2000, 1000-2500, 1000-3000, 1500-3000, 2500-3000, or 2000-3000 nucleotides).

[0254] In some embodiments, the polyA tail is designed for the length of the entire nucleic acid or for the length of a particular region of the nucleic acid. This design can be based on the length of the coding region, the length of a particular feature or region, or can be based on the length of the final product expressed from the nucleic acid.

[0255] In this context, the polyA tail can be 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% longer than the nucleic acid or its features. The polyA tail can also be designated as a fraction of the nucleic acid to which it belongs. In this context, the polyA tail can be 10, 20, 30, 40, 50, 60, 70, 80, or 90% or more of the total length of the construct, the construct region or the total length of the construct minus the polyA tail. Additionally, conjugation of engineered binding sites of polyA binding proteins and nucleic acid sites can enhance expression.

[0256] Additionally, multiple separate nucleic acids can be linked together via PABP (polyA binding protein) through their 3' ends using modified nucleotides at the 3' end of the polyA tail. Transfection experiments can be performed in relevant cell lines and protein production can be assayed by ELISA at 12 hours, 24 hours, 48 ​​hours, 72 hours, and / or 7 days post-transfection.

[0257] In some embodiments, the nucleic acid of the present disclosure is designed to include a poly-AG quartet region. A G-quartet is a cyclic hydrogen-bonded array of four guanine nucleotides that can be formed by G-rich sequences in both DNA and RNA. In this embodiment, the G-quartet is incorporated at the end of a poly-A tail. The resulting nucleic acid is assayed at various time points for stability, protein production, and other parameters, including half-life. It has been discovered that the poly-AG quartet results in protein production from mRNA that is at least 75% equivalent to that seen using a 120-nucleotide poly-A tail alone.

[0258] Start codon region The invention also includes nucleic acids that contain both a start codon region and a nucleic acid described herein (e.g., a nucleic acid that contains a nucleotide sequence that encodes a peptide epitope). In some embodiments, the nucleic acids of the present disclosure can have a region that is similar to or functions similarly to a start codon region.

[0259] In some embodiments, translation of a nucleic acid may initiate on a codon other than the start codon AUG. Translation of a nucleic acid may initiate on alternative start codons, such as, but not limited to, ACG, AGG, AAG, CTG / CUG, GTG / GUG, ATA / AUA, ATT / AUU, TTG / UUG (see Touriol et al. Biology of the Cell 95 (2003) 169-178 and Matsuda and Mauro PLoS ONE, 2010 5:11, the contents of each of which are incorporated herein by reference in their entirety for this purpose).

[0260] As a non-limiting example, nucleic acid translation may begin on the alternative start codon ACG. As another non-limiting example, nucleic acid translation may begin on the alternative start codon CTG or CUG. As another non-limiting example, nucleic acid translation may begin on the alternative start codon GTG or GUG.

[0261] Nucleotides adjacent to a codon that initiates translation, such as, but not limited to, an initiation codon or an alternative initiation codon, are known to affect the translation efficiency, length, and / or structure of a nucleic acid. (See, e.g., Matsuda and Mauro PLoS ONE, 2010 5:11, the contents of which are incorporated herein by reference in their entirety for this purpose.) Masking any of the nucleotides adjacent to the codon that initiates translation can be used to alter the position of translation initiation, translation efficiency, length, and / or structure of a polynucleotide.

[0262] In some embodiments, a masking agent can be used near the start codon or alternative start codon to mask or hide the codon and reduce the possibility of translation initiation at the masked start codon or alternative start codon. Non-limiting examples of masking agents include antisense locked nucleic acid (LNA) nucleic acids and exon junction complexes (EJCs) (see, for example, Matsuda and Mauro (PLoS ONE, 2010 5:11), which describes masking agent LNA polynucleotides and EJCs, the contents of which are incorporated herein by reference in their entirety for this purpose).

[0263] In another embodiment, a masking agent can be used to mask the start codon of a nucleic acid to increase the likelihood that translation will initiate on an alternative start codon. In some embodiments, a masking agent can be used to mask the first start codon or an alternative start codon to increase the likelihood that translation will initiate on a start codon or an alternative start codon downstream of the masked start codon or alternative start codon.

[0264] In another embodiment, the start codon of the nucleic acid can be removed from the nucleic acid sequence to make the translation of the nucleic acid start at a codon that is not the start codon. The translation of the nucleic acid can start at the codon following the removed start codon, or at a downstream start codon or an alternative start codon. In a non-limiting example, the start codon ATG or AUG is removed as the first three nucleotides of the nucleic acid sequence to make the translation start at a downstream start codon or an alternative start codon. The nucleic acid sequence from which the start codon has been removed can further comprise at least one masking agent for the downstream start codon and / or the alternative start codon to control or attempt to control the start of translation, the length of the nucleic acid, and / or the structure of the nucleic acid.

[0265] Stop codon region The present disclosure also includes nucleic acids that include both a stop codon region and a nucleic acid described herein (e.g., a nucleic acid encoding a peptide epitope). In some embodiments, the nucleic acid of the present disclosure may include at least two stop codons before the 3' untranslated region (UTR). The stop codons may be selected from TGA, TAA, and TAG for DNA, or UGA, UAA, and UAG for RNA. In some embodiments, the nucleic acid of the present disclosure includes the stop codon TGA for DNA, or the stop codon UGA for RNA, and one additional stop codon. In further embodiments, the additional stop codon may be TAA or UAA. In another embodiment, the nucleic acid of the present disclosure includes three consecutive stop codons, four stop codons, or more.

[0266] Insertion and Substitution The present disclosure also includes nucleic acids of the present disclosure that further comprise insertions and / or substitutions.

[0267] In some embodiments, the 5'UTR of the nucleic acid may be replaced by the insertion of at least one region and / or string of nucleosides of the same base. The region and / or string of nucleotides may include, but is not limited to, at least 3, at least 4, at least 5, at least 6, at least 7, or at least 8 nucleotides, and the nucleotides may be natural and / or non-natural. As non-limiting examples, the group of nucleotides may include a string of 5-8 adenines, cytosines, thymines, any of the other nucleotides disclosed herein, and / or combinations thereof.

[0268] In some embodiments, the 5'UTR of a nucleic acid may be replaced by the insertion of at least two regions and / or strings of nucleotides of two different bases, such as, but not limited to, adenine, cytosine, thymine, any of the other nucleotides disclosed herein, and / or combinations thereof. For example, the 5'UTR may be replaced by the insertion of 5-8 adenine bases followed by the insertion of 5-8 cytosine bases. In another example, the 5'UTR may be replaced by the insertion of 5-8 cytosine bases followed by the insertion of 5-8 adenine bases.

[0269] In some embodiments, the nucleic acid may contain at least one substitution and / or insertion downstream of the transcription start site that can be recognized by an RNA polymerase. As a non-limiting example, at least one substitution and / or insertion may occur downstream of the transcription start site by substituting at least one nucleic acid in the region immediately downstream of the transcription start site (such as, but not limited to, +1 to +6). Alterations to the region of nucleotides immediately downstream of the transcription start site can affect the initiation rate, increase the apparent nucleotide triphosphate (NTP) reaction constant, and increase the dissociation of short transcripts from the transcription complex that corrects the initial transcription (Brieba et al, Biochemistry (2002) 41:5144-5149, which is incorporated herein by reference in its entirety for this purpose). Modification, substitution, and / or insertion of at least one nucleoside may cause silent mutations in the sequence or may cause mutations in the amino acid sequence.

[0270] In some embodiments, the nucleic acid may include a substitution of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, or at least 13 guanine bases downstream of the transcription start site.

[0271] In some embodiments, the nucleic acid may include at least one, at least two, at least three, at least four, at least five, or at least six guanine base substitutions in the region immediately downstream of the transcription start site. As a non-limiting example, if the nucleotides in the region are GGGAGA, the guanine bases may be substituted by at least one, at least two, at least three, or at least four adenine nucleotides. In another non-limiting example, if the nucleotides in the region are GGGAGA, the guanine bases may be substituted by at least one, at least two, at least three, or at least four cytosine bases. In another non-limiting example, if the nucleotides in the region are GGGAGA, the guanine bases may be substituted by at least one, at least two, at least three, or at least four thymines, and / or any of the nucleotides described herein.

[0272] In some embodiments, the nucleic acid may contain at least one substitution and / or insertion upstream of the start codon. For clarity, one skilled in the art will understand that the start codon is the first codon of a protein coding region, whereas the transcription start site is the site where transcription begins. The nucleic acid may contain, but is not limited to, at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or at least eight substitutions and / or insertions of nucleotide bases. Nucleotide bases may be inserted or substituted at at least one, at least two, at least three, at least four, or at least five positions upstream of the start codon. The inserted and / or substituted nucleotides may be the same base (e.g., all A, or all C, or all T, or all G), two different bases (e.g., A and C, A and T, or C and T), three different bases (e.g., A, C and T, or A, C and T), or at least four different bases.

[0273] As a non-limiting example, the guanine base upstream of the coding region in the nucleic acid can be replaced with adenine, cytosine, thymine, or any of the nucleotides described herein. In another non-limiting example, the replacement of the guanine base in the nucleic acid can be designed to leave one guanine base downstream of the transcription start site and before the start codon (see Esvelt et al. Nature (2011) 472 (7344): 499-503, the contents of which are incorporated herein by reference in their entirety for this purpose). As a non-limiting example, at least five nucleotides can be inserted downstream of the transcription start site but one position upstream of the start codon, and at least five nucleotides can be of the same base type.

[0274] According to the present disclosure, the two regions or portions of the chimeric nucleic acid can be joined or ligated, for example, using triphosphate chemistry. In some embodiments, a first region or portion of 100 nucleotides or less is chemically synthesized with a 5' monophosphate and a terminal 3' desOH or blocked OH. If the region is longer than 80 nucleotides, it can be synthesized as two or more strands that are then chemically linked by ligation. If the first region or portion is synthesized as a non-positionally modified region or portion using IVT, conversion to a 5' monophosphate with subsequent capping of the 3' end can follow. The monophosphate protecting group can be selected from any of those known in the art. The second region or portion of the chimeric nucleic acid can be synthesized, for example, using any of the chemical synthesis or IVT methods described herein. The IVT method can include the use of an RNA polymerase that can utilize a primer with a modified cap. Alternatively, a cap can be chemically synthesized and attached to the IVT region or portion.

[0275] For the ligation method, note that ligation with DNA T4 ligase followed by DNAse treatment (to eliminate the DNA splint required for DNA T4 ligase activity) should readily prevent unwanted formation of ligation products.

[0276] It is not necessary for the entire polynucleotide to be produced with a phosphate-sugar backbone. If one of the regions or portions encodes a polypeptide, it is preferred that such region or portion comprises a phosphate-sugar backbone.

[0277] Ligation can be performed using any suitable technique, such as enzymatic ligation, click chemistry, Orthoclick chemistry, SoluLINK, or other bioconjugate chemistries known to those of skill in the art. In some embodiments, ligation is directed by a complementary oligonucleotide splint. In some embodiments, ligation is performed without a complementary oligonucleotide splint.

[0278] Computerized Systems The above-described embodiments may be implemented in any of a number of ways. For example, the embodiments may be implemented using hardware, software, or a combination thereof. When implemented in software, the software code may be executed on any suitable processor or collection of processors, whether provided in a single computer or distributed among multiple computers. It should be understood that any component or collection of components performing the functions described above may be generally considered as one or more controllers that control the functions described above. The one or more controllers may be implemented in a number of ways, such as dedicated hardware or general-purpose hardware (e.g., one or more processors) that are programmed using microcode or software to perform the functions described above.

[0279] In this regard, it should be understood that one implementation aspect includes at least one computer-readable storage medium (i.e., at least one tangible, non-transitory computer-readable medium), such as a computer memory (e.g., a hard drive, a flash memory, a processor working memory, etc.), a floppy disk, an optical disk, a magnetic tape, or other tangible, non-transitory computer-readable medium, that is coded with a computer program (i.e., a plurality of instructions) and that, when executed on one or more processors, performs the functions described above. The computer-readable storage medium may be transportable such that the program stored thereon may be loaded onto any computer resource to implement the techniques discussed herein. In addition, it should be understood that reference to a computer program that, when executed, performs the functions described above is not limited to an application program running on a host computer. Rather, the term "computer program" is used in a general sense herein to refer to any type of computer code (e.g., software or microcode) that may be used to program one or more processors to implement the techniques discussed above.

[0280] As a non-limiting example, in one aspect, the present disclosure provides a computerized system for selecting nucleic acids for inclusion in a nucleic acid cancer vaccine having a maximum length, the system comprising: a communication interface configured to receive a plurality of sequences of nucleic acids encoding a plurality of peptide epitopes, each of the peptide epitopes being a portion of an individualized cancer antigen; and at least one computer processor programmed to calculate a score for each of the plurality of nucleic acids in the peptides, each of which comprises at least one of the one or more peptide epitopes, at least two of the nucleic acid sequences having different lengths, for each of the plurality of peptide epitopes, rank the plurality of nucleic acid sequences in the plurality of peptides based on the calculated scores, and select a nucleic acid sequence for inclusion in the vaccine based on the ranking and the maximum length of the vaccine. The score may be calculated by any means known in the art. As a set of non-limiting examples, the score may be calculated based at least in part on one or more factors selected from the group consisting of gene expression, RNA sequence, transcript abundance, DNA allele frequency, amino acid conservation, physiochemical similarity, cancer genes, predicted binding affinity to specific HLA alleles, clonality, binding efficiency, and presence in indels. In some embodiments, variant allele frequency (VAF) may be used. In one embodiment, the VAF cutoff is selected to be at a level where additive subclonal mutations are avoided, since contamination of tumor samples with adjacent normal tissues both reduces tumor purity and results in reduced (apparent) VAF. Thus, in cases of low tumor purity (e.g., average VAF less than 20%), the VAF cutoff is lowered (e.g., 10%-5%). In some embodiments, the VAF cutoff is less than 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less. In certain embodiments, one or more factors are input into a statistical model. In some embodiments, the statistical model may be a regression model (e.g., a linear regression model, a logistic regression model, a generalized linear model, etc.).In some embodiments, the statistical model may be a generalized linear model (e.g., a logistic regression model, a probit regression model, etc.). In some embodiments, the statistical model may be, for example, a random forest regression model, a neural network, a support vector machine, a Gaussian mixture model, a hierarchical Bayesian model, and / or any other public statistical model.

[0281] Treatment method Provided herein are compositions (e.g., pharmaceutical compositions), methods, kits, and reagents for the prevention and / or treatment of cancer in humans (e.g., subjects or patients) and other mammals. Nucleic acid cancer vaccines can be used as therapeutic or prophylactic agents in medicine to prevent and / or treat cancer. In exemplary aspects, the cancer vaccines of the present disclosure are used to provide prophylactic protection from cancer. Prophylactic protection from cancer can be achieved following administration of the cancer vaccines of the present disclosure. The vaccines can be administered once, twice, three times, four times, or more, although a single administration of the vaccine may be sufficient (optionally followed by a single booster). It may also be desirable to administer the vaccine to individuals with cancer to achieve a therapeutic response. Dosages may need to be adjusted accordingly.

[0282] Once the cancer vaccine (e.g., nucleic acid cancer vaccine) is synthesized, it is administered to the patient. In some embodiments, the vaccine is administered on a schedule of up to 2 months, up to 3 months, up to 4 months, up to 5 months, up to 6 months, up to 7 months, up to 8 months, up to 9 months, up to 10 months, up to 11 months, up to 1 year, up to 1.5 years, up to 2 years, up to 3 years, or up to 4 years. The schedule may be the same or may vary. In some embodiments, the schedule is once a week for the first 3 weeks, and once a month thereafter. The schedule may be determined or changed by a person skilled in the art (e.g., a physician) depending on the criteria of the individual patient or subject (e.g., weight, age, type of cancer, etc.).

[0283] The vaccine may be administered by any route, hi some embodiments, the vaccine is administered by intradermal, intramuscular, intravascular, intratumoral, and / or subcutaneous routes.

[0284] In some embodiments, the nucleic acid cancer vaccine may also be administered with an anti-cancer therapeutic agent. The nucleic acid cancer vaccine and the other therapeutic agent may be administered simultaneously or sequentially. When the other therapeutic agents are administered simultaneously, they can be administered in the same or separate formulations, but are administered simultaneously. The other therapeutic agents are administered sequentially with each other and with the nucleic acid cancer vaccine when the administration of the other therapeutic agent and the nucleic acid cancer vaccine is temporally separated. The time separation between the administration of these compounds may be a few minutes, or it may be longer, for example, a few hours, days, weeks, months. The other therapeutic agents include, but are not limited to, anti-cancer therapeutic agents, adjuvants, cytokines, antibodies, antigens, etc.

[0285] At any point in treatment, the patient can be tested to determine whether the mutations in the vaccine are still appropriate. Based on that analysis, the vaccine can be adjusted or reformulated to include one or more different mutations or to remove one or more mutations.

[0286] In an exemplary embodiment, a cancer vaccine containing an RNA polynucleotide described herein can be administered to a subject (e.g., a mammalian subject, such as a human subject), and the RNA polynucleotide is translated in vivo to produce an antigenic polypeptide.

[0287] Cancer vaccines can induce translation of polypeptides (e.g., antigens or immunogens) in cells, tissues, or organisms. In exemplary embodiments, such translation occurs in vivo, but embodiments in which such translation occurs ex vivo, in culture, or in vitro can be envisioned. In exemplary embodiments, cells, tissues, or organisms are contacted with an effective amount of a composition containing a cancer vaccine that contains a polynucleotide having at least one translatable region that encodes an antigen polypeptide.

[0288] An "effective amount" of a cancer RNA vaccine may be provided based at least in part on the target tissue, the target cell type, the means of administration, the physical characteristics of the polynucleotide (e.g., size and degree of modified nucleosides) and other components of the cancer vaccine, as well as other determinants. In general, an effective amount of a cancer vaccine composition provides an induced or enhanced immune response as a function of antigen production in cells, and is preferably more efficient than a composition containing a corresponding unmodified polynucleotide encoding the same antigen or peptide antigen. Increased antigen production may be demonstrated by increased cell transfection (percentage of cells transfected with the cancer vaccine), increased protein translation from the polynucleotide, decreased nucleic acid degradation (e.g., as demonstrated by an increased duration of protein translation from the modified polynucleotide), or a change in the antigen-specific immune response of the host cell.

[0289] Cancer vaccines may be administered prophylactically or therapeutically to healthy individuals as part of an active immunization scheme, or may be administered early in cancer or during advanced cancer after the onset of symptoms. In some embodiments, the amount of the RNA vaccine of the present disclosure provided to a cell, tissue, or subject may be an amount effective for immunoprophylaxis.

[0290] Cancer vaccines can be administered together with other preventive or therapeutic compounds. As a non-limiting example, the preventive or therapeutic compound can be an immune enhancer or booster. As used herein, when referring to a composition such as a vaccine, the term "booster" refers to an additional administration of a preventive (vaccine) composition. A booster (or booster vaccine) can be given after an earlier administration of a preventive composition. The time between the first administration of the prophylactic composition and the booster can be 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 36 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, The period may be, but is not limited to, 10 days, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 18 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, 25 years, 30 years, 35 years, 40 years, 45 years, 50 years, 55 years, 60 years, 65 years, 70 years, 75 years, 80 years, 85 years, 90 years, 95 years, or 99 years or more. In an exemplary embodiment, the administration time between the first administration of the prophylactic composition and the booster may be, but is not limited to, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 6 months, or 1 year.

[0291] Cancer vaccines can be used in a variety of settings, depending on the severity of the cancer or the degree or level of unmet medical need. As a non-limiting example, cancer vaccines can be used to treat any stage of cancer.

[0292] A non-limiting list of cancers that a cancer vaccine can treat is provided below. Peptide epitopes or antigens can be derived from any antigen of these cancers or tumors. Such epitopes can be referred to as cancer or tumor antigens. Cancer cells can differentially express cell surface molecules at different stages of tumor progression. For example, cancer cells can express a cell surface antigen in a benign state, but downregulate that particular cell surface antigen upon metastasis. Thus, it is envisioned that tumor or cancer antigens can encompass antigens produced at any stage of cancer progression. The methods of the present disclosure can be adjusted to accommodate these changes. For example, several different cancer vaccines can be generated for a particular patient. For example, the first vaccine can be used at the beginning of treatment. At a later time, new cancer vaccines can be generated and administered to the patient to account for the different antigens expressed.

[0293] In some embodiments, the tumor antigen is one of the following antigens: CD2, CD19, CD20, CD22, CD27, CD33, CD37, CD38, CD40, CD44, CD47, CD52, CD56, CD70, CD79, CD137, 4-IBB, 5T4, AGS-5, AGS-16, Angiopoietin 2, B7.1, B7.2, B7DC, B7H1, B7H2, B7H3, BT-062, BTLA, CAIX, Carcinoma Tumor folate receptor, ganglioside GM3, GD2, glucocorticoid-inducible tumor necrosis factor receptor (GITR), gpl00, gpA33, GPNMB, ICOS, IGF1R, integrin av, integrin ανβ, LAG-3, Lewis Y, mesothelin, c-MET, MN, carbonic anhydrase IX, MUC1, MUC16, Nectin-4, NKGD2, NOTCH, OX40, OX40L, PD-1, PDL1, PSCA, PSMA, RANKL, ROR1, ROR2, SLC44A4, Syndecan-1, TACI, TAG-72, Tenascin, TIM3, TRAILRl, TRAILR2, VEGFR-1, VEGFR-2, VEGFR-3, and their variants.

[0294] Cancer or tumor includes, but is not limited to, neoplasm, malignancy, metastasis, or any disease or disorder characterized by uncontrolled cell growth so as to be considered cancerous. Cancer can be primary or metastatic cancer. Specific cancers that can be treated according to the present disclosure include, but are not limited to, those listed below (for a review of such disorders, see Fishman et al., 1985, Medicine, 2d Ed., JBLippincott Co., Philadelphia). Cancers for use with the methods and compositions described herein include, but are not limited to, biliary tract cancer; bladder cancer; brain cancer, including glioblastoma and medulloblastoma; breast cancer; cervical cancer; choriocarcinoma; colon cancer; endometrial cancer; esophageal cancer; gastric cancer; hematological malignancies, including acute lymphocytic and myeloid leukemia; multiple myeloma; AIDS-related leukemia and adult T-cell leukemia lymphoma; intraepithelial neoplasia, including Bowen's disease and Paget's disease; liver cancer; lung cancer; lymphomas, including Hodgkin's disease and lymphocytic lymphoma; neuroblastoma; oral cancer, including squamous cell carcinoma; epithelial cell, stromal cell The cancers include, but are not limited to, ovarian cancer, including those derived from cysts, germ cells, and mesenchymal cells; pancreatic cancer; prostate cancer; rectal cancer; sarcomas, including leiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma, and osteosarcoma; skin cancer, including melanoma, Kaposi's sarcoma, basal cell carcinoma, and squamous cell carcinoma; reproductive tumors, such as testicular cancer, including seminoma, non-seminoma, and teratocarcinoma; tumors with high tumor mutation burden; choriomas; stromal tumors and germ cell tumors; thyroid cancer, including thyroid cancer and medullary carcinoma; and renal cancer, including adenocarcinoma and Wilms' tumor. In some embodiments, the cancer is any one of melanoma, bladder cancer, HPV-negative HNSCC, NSCLC, SCLC, MSI-high tumor, or TMB (tumor mutation burden) high cancer.

[0295] In some embodiments, the cancer is selected from the group consisting of non-small cell lung cancer (NSCLC), small cell lung cancer, melanoma, bladder urothelial carcinoma, HPV-negative head and neck squamous cell carcinoma (HNSCC), and solid malignancies that are microsatellite high (MSI H) / mismatch repair (MMR) deficient. In some embodiments, the NSCLC lacks an EGFR sensitizing mutation and / or an ALK translocation. In some embodiments, the solid malignancies that are microsatellite high (MSI H) / mismatch repair (MMR) deficient are selected from the group consisting of colorectal cancer, gastric adenocarcinoma, esophageal adenocarcinoma, and endometrial cancer.

[0296] Provided herein are cancer vaccines and pharmaceutical compositions comprising RNA vaccine compositions and / or complexes, optionally in combination with one or more pharma- ceutically acceptable excipients.Cancer vaccines can be formulated or administered alone or in combination with one or more other components described herein.

[0297] In other embodiments, the cancer vaccines described herein may be combined with any other therapy useful in treating a patient. For example, a patient may be treated with a cancer vaccine and an anti-cancer drug. Thus, in one embodiment, the methods of the present disclosure may be used in conjunction with one or more cancer therapeutics, such as in conjunction with (e.g., simultaneously or as part of an overall treatment procedure) an anti-cancer drug, a conventional cancer vaccine, chemotherapy, radiation therapy, etc. Parameters of the cancer treatment that may be varied include, but are not limited to, dosage, timing or duration of administration or therapy, and the cancer treatment may vary in dosage, timing, or duration. Another treatment for cancer is surgery, which may be utilized alone or in combination with any of the previous treatment methods. Any agent or therapy known to be useful or that has been or is currently being used for the prevention or treatment of cancer (e.g., conventional cancer vaccines, chemotherapy, radiation therapy, surgery, hormonal therapy, and / or biological therapy / immunotherapy) may be used in combination with the compositions of the present disclosure in accordance with the disclosure described herein. Those skilled in the art can determine the appropriate treatment for a subject.

[0298] Examples of such agents (i.e., anti-cancer agents) include, but are not limited to, alkylating agents (e.g., nitrogen mustards, e.g., chlorambucil, cyclophosphamide, isofamide, mechlorethamine, melphalan, uracil mustard; aziridines such as thiotepa; methanesulfonate esters such as busulfan; nitrosoureas such as carmustine, lomustine, streptozocin; platinum complexes such as cisplatin, carboplatin; mitomycin, and bioreductive alkylating agents such as procarbazine, dacarbazine, and altretamine); DNA strand breaking agents, such as bleomycin; intercalating topoisomerase II inhibitors, such as intercalators, e.g., amsacrine, dactinomycin, daunorubicin, doxorubicin, idarubicin, mitoxantrone, and non-intercalators, e.g., etoposide and teniposide; non-intercalating topoisomerase II inhibitors, e.g., etoposide and teniposide; and DNA minor groove binders, e.g., plicamydin; DNA interacting agents, including, but not limited to, methotrexate and trimetrexate. pyrimidine antagonists such as fluorouracil, fluorodeoxyuridine, CB3717, azacitidine, and floxuridine; purine antagonists such as mercaptopurine, 6-thioguanine, pentostatin; sugar-modified analogs such as cytarabine and fludarabine; and antimetabolites including ribonucleotide reductase inhibitors such as hydroxyurea; tubulin phase inhibitors including, but not limited to, colchicine, vincristine, and vinblastine, alkaloids, and both paclitaxel and cytoxan. Interacting agents; including but not limited to estrogens, conjugated estrogens, and ethinyl estradiol and diethylstilbesterol, chlortrianisene and idenestrol; progestins such as hydroxyprogesterone caproate, medroxyprogesterone, and megestrol; and androgens such as testosterone, testosterone propionate; fluoxymesterone, methyltestosterone; adrenal corticosteroids such as prednisone, dexamethasone, methylprednisolone, and prednisolone;Hormonal agents including leukocyte releasing hormone agents or gonadotropin releasing hormone antagonists, e.g., leuprolide acetate and goserelin acetate; antihormonal agents including, but not limited to, antiestrogens such as tamoxifen, antiandrogens such as flutamide, and antiadrenal agents such as mitotane and aminoglutethimide; anti-inflammatory agents including, but not limited to, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-18, TGF-β, GM-CSF, M-CSF, G-CSF, T cytokines, including NF-α, TNF-β, LAF, TCGF, ​​BCGF, TRF, BAF, BDG, MP, LIF, OSM, TMF, PDGF, IFN-α, IFN-β, IFN-γ, and uteroglobin (U.S. Patent No. 5,696,092); angiogenesis inhibitors, including, but not limited to, agents that inhibit VEGF (e.g., other neutralizing antibodies), soluble receptor constructs, tyrosine kinase inhibitors, antisense strategies, RNA aptamers and ribozymes against VEGF or VEGF receptors, immunotoxins and coagulants, tumor vaccines, and antibodies;

[0299] Specific examples of anti-cancer drugs that can be used in accordance with the methods of the present disclosure include acivicin, aclarubicin, acodazole hydrochloride, acronine, adozelesin, aldesleukin, altretamine, ambomycin, amethanthrone acetate, aminogluteimide, amsacrine, anastrozole, anthramycin, asparaginase, asperlin, azacytidine, azetepa, azotomycin, batimastat, benzodepa, bicalutamide, bisantrene hydrochloride, bisnafide dimesylate, bizelesin, bleomycin sulfate, brequinar sodium, and bropirimine. , busulfan, cactinomycin, calisterone, caracemide, carbetimer, carboplatin, carmustine, carubicin hydrochloride, carzelesin, cedefingol, chlorambucil, ciloremycin, cisplatin, cladribine, crisnatol mesylate, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin hydrochloride, decitabine, dexorumaplatin, dezaguanine, dezaguanine mesylate, diaziquone, docetaxel, doxorubicin, doxorubicin hydrochloride, droloxifene, droloxifene citrate, propionyl Dromostanolone hydrochloride, duazomycin, edatrexate, eflomitine hydrochloride, elsamitrucin, enloplatin, enpromidine, epipropizine, epirubicin hydrochloride, elbuzole, esorubicin hydrochloride, estramustine, estramustine sodium phosphate, etanisole, etoposide, etoposide phosphate, etopurine, fadrozole hydrochloride, fazarabine, fenretinide, floxinide, fludarabine phosphate, fluorouracil, flurocitabine, fosquidone, fostriecin sodium, gemcitabine, gemcitabine hydrochloride, hydroxyl cyclosporine, idarubicin hydrochloride, ifosfamide, irmofosine, interleukin II (including recombinant interleukin II, or rIL2), interferon alpha-2a, interferon alpha-2b, interferon alpha-n1, interferon alpha-n3, interferon beta-Ia, interferon gamma-Ib, iproplatin, irinotecan hydrochloride, lanreotide acetate, letrozole, leuprolide acetate, liorozole hydrochloride, lometrexol sodium, lomustine, losoxantrone hydrochloride, masoprocol, maytansine, mechlorethamine hydrochloride,Megestrol acetate, melengestrol acetate, melphalan, menogaril, mercaptopurine, methotrexate, methotrexate sodium, metoprine, meturedepa, mitindomide, mitocalcin, mitochromine, mitogillin, mitomarcine, mitomycin, mitospar, mitotane, mitoxantrone hydrochloride, mycophenolic acid, nocodazole, nogalamycin, ormaplatin, oxisuran, paclitaxel, pegaspargase, periodontitis mycin, pentamustine, peplomycin sulfate, perfosfamide, pipobroman, piposulfan, piroxantrone hydrochloride, plicamycin, promestane, porfimer sodium, porfiromycin, prednimustine, procarbazine hydrochloride, puromycin, puromycin hydrochloride, pyrazofurin, ribopurin, rogletimide, safingol, safingol hydrochloride, semustine, simtrazene, sparphosate sodium, spar Somycin, spirogermanium hydrochloride, spiromustine, spiroplatin, streptonigrin, streptozocin, sulofenur, tallysomycin, tecogalan sodium, tegafer, teroxantrone hydrochloride, temoporfin, teniposide, teroxylon, testolactone, thiamiprine, thioguanine, thiotepa, tiazofurin, tirapazamine, toremifene citrate, trestrone acetate, triciribine phosphate, trimetrexate, glucuronic acid These include, but are not limited to, trimetrexate ronate, triptorelin, tuburozole hydrochloride, uracil mustard, uredepa, vapreotide, verteporfin, vinblastine sulfate, vincristine sulfate, vindesine, vindesine sulfate, vinepidine sulfate, vinglisinate sulfate, vinleurosine sulfate, vinorelbine tartrate, vinrocidine sulfate, vinzolidine sulfate, vorozole, zeniplatin, zinostatin, and zorubicin hydrochloride.

[0300] Other anti-cancer drugs that may be used with the present compositions and methods include, but are not limited to: 20-epi-1,25 dihydroxyvitamin D3, 5-ethynyluracil, angiogenesis inhibitors, anti-dorsal morphogenetic protein-1, ara-CDP-DL-PTBA, BCR / ABL antagonists; CaRest M3, CARN 700, casein kinase inhibitor (ICO), clotrimazole, collismycin A, collismycin B, combretastatin A4, clambecidin 816, cryptophycin 8, curacin A, dehydrodidemnin B, didemnin B, dihydro-5-azacytidine, dihydrotaxol, duocarmycin SA, kahalalide F, lamellarin-N-triacetate, leuprolide + estrogen + progesterone, lysocrinamide 7, monophosphoryl lipid A + myobacterium cell wall sk, N-acetylglutamin, N-substituted benzamide, O6-benzyl Guanine, Prasetin A, Prasetin B, Platinum Complex, Platinum Compound, Platinum-Triamine Complex, Rhenium Re186 Etidronate, RII Retinamide, Rubiginone B1, SarCNU, Sarcophytol A, Sargramostim, Senescence derived inhibitor 1, Spicamycin D, Talimustine, 5-Fluorouracil, Thrombopoietin, Thymotrinan, Thyroid Stimulating Hormone, Variolin B, Satidomide, Veraresol, Veramine, Verdine, Verteporfin, Vinorelbine, Vinxartin, Vitaxin, Zanoterone, Zeniplatin, and Zilascorub.

[0301] The present disclosure also encompasses administering compositions comprising cancer vaccines in combination with radiation therapy, which includes the use of X-rays, gamma rays, and other radiation sources to destroy cancer cells. In certain embodiments, radiation therapy is administered as external beam radiation or teletherapy, where radiation is directed from a distant source. In other embodiments, radiation therapy is administered as internal therapy or brachytherapy, where a radiation source is placed inside the body close to the cancer cells or tumor mass.

[0302] In a specific embodiment, the appropriate anti-cancer regimen is selected (e.g., by a physician) according to the type of cancer. For example, a patient with ovarian cancer can be administered a prophylactically or therapeutically effective amount of a composition comprising a cancer vaccine in combination with a prophylactically or therapeutically effective amount of one or more other agents useful in ovarian cancer therapy, including, but not limited to, intraperitoneal radiation therapy, such as P32 therapy, whole abdominal and pelvic radiation therapy, combinations of cisplatin, paclitaxel (Taxol) or docetaxel (Taxotere) with cisplatin or carboplatin, combinations of cyclophosphamide with cisplatin, combinations of cyclophosphamide with carboplatin, combinations of 5-FU with leucovorin, etoposide, liposomal doxorubicin, gemcitabine or topotecan. Cancer therapies and their dosages, routes of administration, and recommended uses are known in the art and described in references such as the Physician's Desk Reference (56th ed., 2002).

[0303] In some embodiments of the present disclosure, the cancer vaccine is administered in conjunction with a T cell activator, such as an immune checkpoint modulator, including both stimulatory and inhibitory checkpoint molecules (e.g., anti-CTLA4 and / or anti-PD1 antibodies).

[0304] Stimulatory checkpoint inhibitors function by promoting checkpoint processes. Some stimulatory checkpoint molecules are members of the tumor necrosis factor (TNF) receptor superfamily (e.g., CD27, CD40, OX40, GITR, or CD137), while others belong to the B7-CD28 superfamily (e.g., CD28 or ICOS0). OX40 (CD134) is involved in the expansion of effector and memory T cells. Anti-OX40 monoclonal antibodies have been shown to be effective in the treatment of advanced cancers. MEDI0562 is a humanized OX40 agonist. GITR, glucocorticoids, and OX40 receptors are also involved in the treatment of advanced cancers. Id-inducible TNFR family-related genes are involved in T-cell proliferation. Several antibodies against GITR have been shown to promote antitumor responses. ICOS, inducible T-cell costimulator, is important in T-cell effector function. CD27 supports antigen-specific proliferation of naive T cells and participates in the generation of T-cell and B-cell memory. Several agonistic anti-CD27 antibodies are in development. CD122 is the interleukin-2 receptor beta subunit. NKTR-214 is a CD122-biased immunostimulatory cytokine.

[0305] Inhibitory checkpoint molecules include, but are not limited to, PD-1, TIM-3, VISTA, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, and LAG3. CTLA-4, PD-1, and its ligands are members of the CD28-B7 family of co-signaling molecules that play important roles throughout all stages of T cell and other cell functions. CTLA-4, cytotoxic T lymphocyte-associated protein 4 (CD152), is involved in the control of T cell proliferation.

[0306] The PD-1 receptor is expressed on the surface of activated T cells (and B cells) and, under normal circumstances, binds to its ligands (PD-L1 and PD-L2) expressed on the surface of antigen-presenting cells such as dendritic cells or macrophages. This interaction sends a signal to the T cell to inhibit it. Cancer cells exploit this system by driving high levels of PD-L1 expression on their surface. This allows them to gain control of the PD-1 pathway and turn off PD-1-expressing T cells that may enter the tumor microenvironment, thus suppressing the anti-cancer immune response. Pembrolizumab (formerly MK-3475 and lambrolizumab, trade name Keytruda) is a human antibody used in cancer immunotherapy that targets the PD-1 receptor.

[0307] The checkpoint inhibitor is a molecule such as a monoclonal antibody, a humanized antibody, a fully human antibody, a fusion protein, or a combination thereof, or a small molecule. For example, the checkpoint inhibitor inhibits a checkpoint protein, which may be CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, CHK2, A2aR, B-7 family ligand, or a combination thereof. The ligand of the checkpoint protein includes, but is not limited to, CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, CHK2, A2aR, and B-7 family ligand. In some embodiments, the anti-PD-1 antibody is BMS-936558 (nivolumab). In other embodiments, the anti-CTLA-4 antibody is ipilimumab (trade name Yervoy, formerly known as MDX-010 and MDX-101).

[0308] In some embodiments, the cancer therapeutic, including the checkpoint modulator, is delivered in the form of an mRNA that encodes the cancer therapeutic.

[0309] In some embodiments, the cancer therapeutic agent is a targeted therapy. The targeted therapy can be a BRAF inhibitor, such as vemurafenib (PLX4032) or dabrafenib. The BRAF inhibitor can be PLX 4032, PLX 4720, PLX 4734, GDC-0879, PLX 4032, PLX-4720, PLX 4734, and sorafenib tosylate. BRAF is a human gene that makes a protein called B-Raf, also called proto-oncogene B-Raf and v-Raf murine sarcoma viral oncogene homolog B1. The B-Raf protein is involved in sending signals within cells that are involved in inducing cell growth. Vemurafenib, a BRAF inhibitor, has been approved by the FDA for the treatment of late-stage melanoma.

[0310] In other embodiments, the cancer therapeutic agent is a cytokine. In yet other embodiments, the cancer therapeutic agent is a vaccine that includes a population-based tumor-specific antigen. In yet other embodiments, the cancer therapeutic agent is a vaccine that contains one or more conventional antigens expressed by cancer germline genes (common antigens in tumors found in multiple patients, also referred to as "shared cancer antigens"). In some embodiments, the conventional antigens are those known to be found in cancers or tumors in general, or in a particular type of cancer or tumor. In some embodiments, the conventional cancer antigen is a non-mutated tumor antigen. In some embodiments, the conventional cancer antigen is a mutated tumor antigen.

[0311] The p53 gene (official symbol TP53) is mutated more frequently than any other gene in human cancers. Large cohort studies have shown that for most p53 mutations, the genomic location is unique to one or a few patients, and mutations cannot be used as recurrent neoantigens in therapeutic vaccines designed for specific patient populations. However, a small subset of p53 loci exhibits a "hotspot" pattern (described elsewhere herein) in which several positions within the gene are mutated at relatively high frequency. Strikingly, the majority of these recurrently mutated regions occur near exon-intron boundaries, disrupting canonical nucleotide sequence motifs recognized by the mRNA splicing machinery.

[0312] Mutations in splicing motifs can alter the final mRNA sequence even if no changes to the local amino acid sequence are predicted (i.e., for synonymous or intronic mutations). Thus, these mutations are often annotated as "non-coding" by common annotation tools and ignored for further analysis, even though they can alter mRNA splicing in unpredictable ways and have profound functional consequences on the translated protein. If an alternatively spliced ​​isoform results in an in-frame sequence change (i.e., no pretermination codon (PTC) is produced), it can escape depletion by nonsense-mediated mRNA decay (NMD) and can be readily expressed, processed, and presented on the cell surface by the HLA system. Moreover, mutation-derived alternative splicing is usually "cryptic," i.e., not expressed in normal tissues, and therefore can be recognized by T cells as a non-self neoantigen.

[0313] In some cases, the cancer therapeutic is a vaccine that includes one or more neo-antigens that are recurrent polymorphisms ("hot spot mutations"). For example, among other things, the present disclosure provides neo-antigen peptide sequences that arise from certain recurrent somatic cancer mutations in p53.

[0314] formulation Cancer vaccines (e.g., nucleic acid cancer vaccines, such as mRNA cancer vaccines) can be formulated or administered in combination with one or more pharma- ceutically acceptable excipients. As a non-limiting set of examples, cancer vaccines can be formulated using one or more excipients to (1) increase stability, (2) increase cell transfection, (3) allow sustained or delayed release (e.g., from a depot formulation), (4) modify biodistribution (e.g., target specific tissues or cell types), (5) increase translation of the encoded protein in vivo, and / or (6) modify the release profile of the encoded protein (antigen) in vivo. In addition to any and all conventional excipients such as solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surfactants, isotonicity agents, thickening or emulsifying agents, preservatives, excipients may include, but are not limited to, lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, cells transfected with cancer vaccines (e.g., for implantation into a subject), hyaluronidase, nanoparticle mimetics, and combinations thereof.

[0315] In some embodiments, the vaccine composition comprises at least one additional active agent, e.g., a therapeutic active agent, a prophylactic active agent, or a combination of both. The vaccine composition can be sterile, pyrogen-free, or both sterile and pyrogen-free. General considerations in the formulation and / or manufacture of pharmaceuticals such as vaccine compositions can be found, for example, in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference in its entirety for this purpose).

[0316] In some embodiments, the cancer vaccine is administered to a human, human patient, or subject. For the purposes of this disclosure, the phrase "active ingredient" generally refers to the cancer vaccine or the nucleic acid contained therein, such as the RNA (e.g., mRNA) that encodes an antigenic polypeptide.

[0317] Formulations of the vaccine compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing into association the active ingredient (e.g., a nucleic acid such as mRNA) with an excipient and / or one or more other accessory ingredients, and then, as necessary and / or desired, dividing, shaping, and / or packaging the product into the desired single or multiple dose units.

[0318] Any formulation of the compositions disclosed herein may include one or more components in addition to those described above.For example, lipid compositions may include one or more permeation enhancing molecules, carbohydrates, polymers, surface modifiers (e.g., surfactants), or other components.For example, permeation enhancing molecules may be molecules described in US Patent Application Publication No. 2005 / 0222064.Carbohydrates may include monosaccharides (e.g., glucose) and polysaccharides (e.g., glycogen and its derivatives and analogs).

[0319] Polymers can be included and / or used to encapsulate or partially encapsulate the pharmaceutical compositions disclosed herein (e.g., pharmaceutical compositions in lipid nanoparticle form). The polymers can be biodegradable and / or biocompatible. The polymers can be selected from, but are not limited to, polyamines, polyethers, polyamides, polyesters, polycarbamates, polyureas, polycarbonates, polystyrenes, polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes, polyethyleneimines, polyisocyanates, polyacrylates, polyethacrylates, polyacrylonitriles, and polyarylates.

[0320] In some embodiments, the compositions disclosed herein can be formulated as lipid nanoparticles (LNPs).Accordingly, the present disclosure also provides a nanoparticle composition comprising (i) a lipid composition comprising a delivery agent, and (ii) a nucleic acid encoding one or more peptide epitopes.In such nanoparticle compositions, the lipid composition disclosed herein can encapsulate the nucleic acid encoding one or more peptide epitopes.

[0321] Nanoparticle compositions are typically sized to approximately a few micrometers or less and may contain lipid bilayers. Nanoparticle compositions include lipid nanoparticles (LNPs), liposomes (e.g., lipid vesicles), and lipoplexes. For example, nanoparticle compositions may be liposomes with lipid bilayers of 500 nm or less in diameter.

[0322] Nanoparticle compositions include, for example, lipid nanoparticles (LNPs), liposomes, and lipoplexes. In some embodiments, the nanoparticle compositions are vesicles that include one or more lipid bilayers. In certain embodiments, the nanoparticle compositions include two or more concentric bilayers separated by aqueous compartments. The lipid bilayers can be functionalized and / or cross-linked to each other. The lipid bilayers can include one or more ligands, proteins, or channels.

[0323] In one embodiment, the lipid nanoparticle comprises an ionic lipid, a structured lipid, a phospholipid, and an mRNA, hi some embodiments, the LNP comprises an ionic lipid, a PEG-modified lipid, a phospholipid, and a structured lipid.

[0324] The ratio between the lipid composition and the cancer vaccine can be about 10:1 to about 60:1 (weight / weight). In some embodiments, the ratio between the lipid composition and the nucleic acid is about 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1, 42:1, 43:1, 44:1, 45:1, 46:1, 47:1, 48:1, 49:1, 50:1, 51:1, 52:1, 53:1, 54:1, 55:1, 56:1, 57:1, 58:1, 59:1, 60:1, 61:1, 62:1, 63:1, 64:1, 65:1, 66:1, 67:1, 68:1, 69:1, 70:1, 71:1, 72:1, 73:1, 74:1, 75:1, 76:1, 77:1, 78:1, 79:1, 80:1, 81:1, 82:1, 83:1, 84:1, 85:1, 86:1, 87 The weight / weight ratio of the lipid composition to the cancer vaccine may be 4:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1, 42:1, 43:1, 44:1, 45:1, 46:1, 47:1, 48:1, 49:1, 50:1, 51:1, 52:1, 53:1, 54:1, 55:1, 56:1, 57:1, 58:1, 59:1, or 60:1 (weight / weight). In some embodiments, the weight / weight ratio of the lipid composition to the cancer vaccine is about 20:1 or about 15:1.

[0325] In one embodiment, a cancer vaccine (e.g., a nucleic acid cancer vaccine) has a lipid:polynucleotide weight ratio of 5:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, or 70:1, or a range or any of these ratios, for example, but not limited to, from 5:1 to about 10:1, from about 5:1 to about 15:1, from about 5:1 to about 20:1, from about 5:1 to about 25:1, from about 5:1 to about 30:1, from about 5:1 to about 35:1, from about 5:1 to about 40:1, from about 5:1 to about 45:1, from about 5:1 to about 50:1, from about 5:1 to about 55:1, from about 5:1 to about 60:1, from about 5:1 to about 70:1, or a range or any of these ratios. :1, about 10:1 to about 15:1, about 10:1 to about 20:1, about 10:1 to about 25:1, about 10:1 to about 30:1, about 10:1 to about 35:1, about 10:1 to about 40:1, about 10:1 to about 45:1, about 10:1 to about 50:1, about 10:1 to about 55:1, about 10:1 to about 60:1, about 10:1 to about 70:1, about 15 The lipid nanoparticles may be comprised in a ratio of about 15:1 to about 20:1, about 15:1 to about 25:1, about 15:1 to about 30:1, about 15:1 to about 35:1, about 15:1 to about 40:1, about 15:1 to about 45:1, about 15:1 to about 50:1, about 15:1 to about 55:1, about 15:1 to about 60:1, or about 15:1 to about 70:1.

[0326] In one embodiment, the cancer vaccine (e.g., a nucleic acid cancer vaccine) may be contained in the lipid nanoparticles at a concentration of approximately 0.1 mg / mL to 2 mg / mL, for example, but not limited to, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1.0 mg / mL, 1.1 mg / mL, 1.2 mg / mL, 1.3 mg / mL, 1.4 mg / mL, 1.5 mg / mL, 1.6 mg / mL, 1.7 mg / mL, 1.8 mg / mL, 1.9 mg / mL, 2.0 mg / mL), or greater than 2.0 mg / mL.

[0327] As generally defined herein, the term "lipid" refers to a small molecule with hydrophobic or amphiphilic properties. Lipids may be natural or synthetic. Examples of lipid classes include, but are not limited to, fats, waxes, sterol-containing metabolites, vitamins, fatty acids, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, and polyketides, and prenol lipids. In some cases, the amphiphilic properties of some lipids cause them to form liposomes, vesicles, or membranes in aqueous media.

[0328] In some embodiments, lipid nanoparticles (LNPs) may include ionic lipids. As used herein, the term "ionic lipid" has its customary meaning in the art and may refer to lipids that include one or more charged moieties. In some embodiments, ionic lipids may be positively or negatively charged. An ionic lipid may be positively charged, in which case it may be referred to as a "cationic lipid." In certain embodiments, an ionic lipid molecule may include an amine group and may be referred to as an ionic amino lipid. As used herein, a "charged moiety" is a chemical moiety that has a formal charge, e.g., monovalent (+1 or -1), divalent (+2 or -2), trivalent (+3 or -3), etc. A charged moiety may be anionic (i.e., negatively charged) or cationic (i.e., positively charged). Examples of positively charged moieties include amine groups (e.g., primary, secondary, and / or tertiary amines), ammonium groups, pyridinium groups, guanidine groups, and imidizolium groups. In certain embodiments, the charged moiety comprises an amine group. Examples of negatively charged groups or precursors thereof include carboxylate, sulfonate, sulfate, phosphonate, phosphate, hydroxyl, etc. The charge of the charged moiety may vary depending on environmental conditions in some cases, for example, a change in pH may change the charge of the moiety and / or make the moiety charged or uncharged. In general, the charge density of the molecule may be selected as desired. The ionic lipid may also be a compound disclosed in International Publication No. WO2017075531, WO2015199952, WO2013086354, or WO2013116126, or selected from formula CLI-CLXXXXII in U.S. Patent No. 7,404,969, each of which is incorporated herein by reference in its entirety for this purpose.

[0329] It should be understood that the term "charge" or "charged moiety" does not refer to a "partial negative charge" or a "partial positive charge" on a molecule. The terms "partial negative charge" and "partial positive charge" are given their customary meaning in the art. A "partial negative charge" can result when a functional group contains a bond that becomes polar such that electron density is attracted toward one atom of the bond, creating a partial negative charge on that atom. Those skilled in the art will generally recognize bonds that can become polar in this way.

[0330] In some embodiments, the ionic lipid is an ionic amino lipid, which is sometimes referred to in the art as an "ionic cationic lipid". In one embodiment, the ionic amino lipid can have a positively charged hydrophilic head and a hydrophobic tail connected via a linker structure. In addition, the ionic lipid can also be a lipid that contains a cyclic amine group.

[0331] The vaccines of the present disclosure are typically formulated into lipid nanoparticles. In some embodiments, the lipid nanoparticles comprise at least one ionic amino lipid, at least one non-cationic lipid, at least one sterol, and / or at least one polyethylene glycol (PEG)-modified lipid.

[0332] In some embodiments, the lipid nanoparticles comprise a molar ratio of ionizable amino lipids of 20-60%. For example, the lipid nanoparticles may comprise a molar ratio of ionizable amino lipids of 20-50%, 20-40%, 20-30%, 30-60%, 30-50%, 30-40%, 40-60%, 40-50%, or 50-60%. In some embodiments, the lipid nanoparticles comprise a molar ratio of ionizable amino lipids of 20%, 30%, 40%, 50, or 60%.

[0333] In some embodiments, the lipid nanoparticles comprise a molar ratio of 5-25% non-cationic lipid. For example, the lipid nanoparticles may comprise a molar ratio of 5-20%, 5-15%, 5-10%, 10-25%, 10-20%, 10-25%, 15-25%, 15-20%, or 20-25% non-cationic lipid. In some embodiments, the lipid nanoparticles comprise a molar ratio of 5%, 10%, 15%, 20%, or 25% non-cationic lipid.

[0334] In some embodiments, the lipid nanoparticles comprise a molar ratio of sterol of 25-55%. For example, the lipid nanoparticles may comprise a molar ratio of sterol of 25-50%, 25-45%, 25-40%, 25-35%, 25-30%, 30-55%, 30-50%, 30-45%, 30-40%, 30-35%, 35-55%, 35-50%, 35-45%, 35-40%, 40-55%, 40-50%, 40-45%, 45-55%, 45-50%, or 50-55%. In some embodiments, the lipid nanoparticles comprise a molar ratio of sterol of 25%, 30%, 35%, 40%, 45%, 50%, or 55%.

[0335] In some embodiments, the lipid nanoparticles comprise a molar ratio of 0.5-15% PEG-modified lipid. For example, the lipid nanoparticles may comprise a molar ratio of 0.5-10%, 0.5-5%, 1-15%, 1-10%, 1-5%, 2-15%, 2-10%, 2-5%, 5-15%, 5-10%, or 10-15%. In some embodiments, the lipid nanoparticles comprise a molar ratio of 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% PEG-modified lipid.

[0336] In some embodiments, the lipid nanoparticles comprise a molar ratio of 20-60% ionic lipid, 5-25% non-cationic lipid, 25-55% sterol, and 0.5-15% PEG-modified lipid.

[0337] In some embodiments, the ionic amino lipid of the present disclosure is a compound of formula (I): [ka] or a salt or isomer thereof, wherein R1 is C 5~30 Alkyl, C 5~20 alkenyl, -R*YR", -YR", and -R"M'R'; R2 and R3 are independently H, C 1~14 Alkyl, C 2~14 alkenyl, -R*YR", -YR", and -R*OR", or R2 and R3 together with the atom to which they are attached form a heterocycle or carbocycle; R4 is C 3~6 Carbocyclic ring, -(CH2) n Q, -(CH2) n CHQR, -CHQR, -CQ(R)2, and unsubstituted C 1~6 alkyl, where Q is a carbocycle, a heterocycle, -OR, -O(CH2) n N(R)2, -C(O)OR, -OC(O)R, -CX3, -CX2H, -CXH2, -CN, -N(R)2, -C(O)N(R)2, -N( R)C(O)R, -N(R)S(O)2R, -N(R)C(O)N(R)2, -N(R)C(S)N(R)2, -N(R)R8, -O(CH2) n n is independently selected from 1, 2, 3, 4, and 5; Each R5 is independently 1~3 Alkyl, C 2~3 alkenyl, and H; Each R6 is independently C 1~3 Alkyl, C 2~3alkenyl, and H; M and M' are independently selected from -C(O)O-, -OC(O)-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)2-, -SS-, an aryl group, and a heteroaryl group; R7 is C 1~3 Alkyl, C 2~3 alkenyl, and H; R8 is C 3~6 selected from the group consisting of carbocycles and heterocycles; R9 is H, CN, NO2, C 1~6 Alkyl, -OR, -S(O)2R, -S(O)2N(R)2, C 2~6 Alkenyl, C 3~6 selected from the group consisting of carbocycles and heterocycles; Each R is independently C 1~3 Alkyl, C 2~3 alkenyl, and H; Each R' is independently C 1~18 Alkyl, C 2~18 selected from the group consisting of alkenyl, -R*YR", -YR", and H; Each R″ is independently 3~14 Alkyl and C 3~14 alkenyl, Each R* is independently C 1~12 Alkyl and C 2~12 alkenyl, Each Y is independently C 3~6 is a carbocyclic ring, each X is independently selected from the group consisting of F, Cl, Br, and I; m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13.

[0338] In some embodiments, a subset of compounds of formula (I) includes compounds in which R4 is -(CH2) n Q, -(CH2) nWhen CHQR, -CHQR, or -CQ(R), compounds are included in which (i) Q is not -N(R) when n is 1, 2, 3, 4, or 5, or (ii) Q is not a 5-, 6-, or 7-membered heterocycloalkyl when n is 1 or 2.

[0339] In some embodiments, another subset of compounds of formula (I) includes R1 is C 5~30 Alkyl, C 5~20 alkenyl, -R*YR", -YR", and -R"M'R'; R2 and R3 are independently H, C 1~14 Alkyl, C 2~14 alkenyl, -R*YR", -YR", and -R*OR", or R2 and R3 together with the atom to which they are attached form a heterocycle or carbocycle; R4 is C 3~6 Carbocyclic ring, -(CH2) n Q, -(CH2) n CHQR, -CHQR, -CQ(R)2, and unsubstituted C 1~6 alkyl, where Q is selected from the group consisting of C 3~6 Carbocycles, N, O, and S, -OR, -O(CH2) n N(R)2, -C(O)OR, -OC(O)R, -CX3, -CX2H, -CXH2, -CN, -C(O)N(R)2, -N(R)C(O)R, -N( R)S(O)2R, -N(R)C(O)N(R)2, -N(R)C(S)N(R)2, -CRN(R)2C(O)OR, -N(R)R8, -O(CH2) n5-14 membered heteroaryl having one or more heteroatoms selected from OR, -N(R)C(=NR9)N(R)2, -N(R)C(=CHR9)N(R)2, -OC(O)N(R)2, -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O)2R, -N(OR)C(O)OR, -N(OR)C(O)N(R)2, -N(OR)C(S)N(R)2, -N(OR)C(=NR9)N(R)2, -N(OR)C(=CHR9)N(R)2, -C(=NR9)N(R)2, -C(=NR9)R, -C(O)N(R)OR, as well as oxo(=O), OH, amino, mono- or dialkylamino, and C 1~3 and each n is independently selected from 1, 2, 3, 4, and 5; Each R5 is independently 1~3 Alkyl, C 2~3 alkenyl, and H; Each R6 is independently selected from the group consisting of C 1~3 Alkyl, C 2~3 alkenyl, and H; M and M' are independently selected from -C(O)O-, -OC(O)-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)2-, -SS-, an aryl group, and a heteroaryl group; R7 is C 1~3 Alkyl, C 2~3 alkenyl, and H; R8 is C 3~6 selected from the group consisting of carbocycles and heterocycles; R9 is H, CN, NO2, C 1~6 Alkyl, -OR, -S(O)2R, -S(O)2N(R)2, C 2~6 Alkenyl, C 3~6 selected from the group consisting of carbocycles and heterocycles; Each R is independently C 1~3 Alkyl, C 2~3alkenyl, and H; Each R' is independently C 1~18 Alkyl, C 2~18 selected from the group consisting of alkenyl, -R*YR", -YR", and H; Each R″ is independently 3~14 Alkyl and C 3~14 alkenyl, Each R* is independently 1~12 Alkyl and C 2~12 alkenyl, Each Y is independently 3~6 is a carbocyclic ring, each X is independently selected from the group consisting of F, Cl, Br, and I; m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13; The compound, or a salt or isomer thereof is included.

[0340] In another embodiment, another subset of compounds of formula (I) includes R1 is C 5~30 Alkyl, C 5~20 alkenyl, -R*YR", -YR", and -R"M'R'; R2 and R3 are independently H, C 1~14 Alkyl, C 2~14 alkenyl, -R*YR", -YR", and -R*OR", or R2 and R3 together with the atom to which they are attached form a heterocycle or carbocycle; R4 is C 3~6 Carbocyclic ring, -(CH2) n Q, -(CH2) n CHQR, -CHQR, -CQ(R)2, and unsubstituted C 1~6 alkyl, wherein Q is selected from the group consisting of C 3~6 A 5-14 membered heterocyclic ring having one or more heteroatoms selected from a carbocyclic ring, N, O, and S, -OR, -O(CH2) nN(R)2, -C(O)OR, -OC(O)R, -CX3, -CX2H, -CXH2, -CN, -C(O)N(R)2, -N(R)C(O)R, -N( R)S(O)2R, -N(R)C(O)N(R)2, -N(R)C(S)N(R)2, -CRN(R)2C(O)OR, -N(R)R8, -O(CH2) n and (i) R4 is selected from -(CH2)N(R)OR, -N(R)C(=NR9)N(R)2, -N(R)C(=CHR9)N(R)2, -OC(O)N(R)2, -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O)2R, -N(OR)C(O)OR, -N(OR)C(O)N(R)2, -N(OR)C(S)N(R)2, -N(OR)C(=NR9)N(R)2, -N(OR)C(=CHR9)N(R)2, -C(=NR9)R, -C(O)N(R)OR, and -C(=NR9)N(R)2, each n is independently selected from 1, 2, 3, 4, and 5; Q is a 5-14 membered heterocycle; and (i) R4 is -(CH2) n Q (wherein n is 1 or 2), or (ii) R4 is -(CH2) n or (iii) when R4 is -CHQR and -CQ(R)2, Q is either a 5-14 membered heteroaryl or an 8-14 membered heterocycloalkyl; Each R5 is independently C 1~3 Alkyl, C 2~3 alkenyl, and H; Each R6 is independently selected from the group consisting of C 1~3 Alkyl, C 2~3 alkenyl, and H; M and M' are independently selected from -C(O)O-, -OC(O)-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)2-, -SS-, an aryl group, and a heteroaryl group; R7 is C 1~3 Alkyl, C 2~3 alkenyl, and H; R8 is C 3~6selected from the group consisting of carbocycles and heterocycles; R9 is H, CN, NO2, C 1~6 Alkyl, -OR, -S(O)2R, -S(O)2N(R)2, C 2~6 Alkenyl, C 3~6 selected from the group consisting of carbocycles and heterocycles; Each R is independently C 1~3 Alkyl, C 2~3 alkenyl, and H; Each R' is independently C 1~18 Alkyl, C 2~18 selected from the group consisting of alkenyl, -R*YR", -YR", and H; Each R″ is independently 3~14 Alkyl and C 3~14 alkenyl, Each R* is independently 1~12 Alkyl and C 2~12 alkenyl, Each Y is independently 3~6 is a carbocyclic ring, each X is independently selected from the group consisting of F, Cl, Br, and I; m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13; The compound, or a salt or isomer thereof is included.

[0341] In another embodiment, another subset of compounds of formula (I) includes R1 is C 5~30 Alkyl, C 5~20 alkenyl, -R*YR", -YR", and -R"M'R'; R2 and R3 are independently H, C 1~14 Alkyl, C 2~14 alkenyl, -R*YR", -YR", and -R*OR", or R2 and R3 together with the atom to which they are attached form a heterocycle or carbocycle; R4 is C 3~6 Carbocyclic ring, -(CH2) n Q, -(CH2)n CHQR, -CHQR, -CQ(R)2, and unsubstituted C 1~6 alkyl, where Q is selected from the group consisting of C 3~6 Carbocyclic ring, 5-14 membered heteroaryl having one or more heteroatoms selected from N, O, and S, -OR, -O(CH2) n N(R)2, -C(O)OR, -OC(O)R, -CX3, -CX2H, -CXH2, -CN, -C(O)N(R)2, -N(R)C(O)R, -N( R)S(O)2R, -N(R)C(O)N(R)2, -N(R)C(S)N(R)2, -CRN(R)2C(O)OR, -N(R)R8, -O(CH2) n wherein each n is independently selected from 1, 2, 3, 4, and 5; Each R5 is independently 1~3 Alkyl, C 2~3 alkenyl, and H; Each R6 is independently selected from the group consisting of C 1~3 Alkyl, C 2~3 alkenyl, and H; M and M' are independently selected from -C(O)O-, -OC(O)-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)2-, -SS-, an aryl group, and a heteroaryl group; R7 is C 1~3 Alkyl, C 2~3 alkenyl, and H; R8 is C 3~6 selected from the group consisting of carbocycles and heterocycles; R9 is H, CN, NO2, C 1~6 Alkyl, -OR, -S(O)2R, -S(O)2N(R)2, C 2~6 Alkenyl, C 3~6 selected from the group consisting of carbocycles and heterocycles; Each R is independently C 1~3 Alkyl, C 2~3 alkenyl, and H; Each R' is independently C 1~18 Alkyl, C 2~18 selected from the group consisting of alkenyl, -R*YR", -YR", and H; Each R″ is independently 3~14 Alkyl and C 3~14 alkenyl, Each R* is independently 1~12 Alkyl and C 2~12 alkenyl, Each Y is independently C 3~6 is a carbocyclic ring, each X is independently selected from the group consisting of F, Cl, Br, and I; m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13; The compound, or a salt or isomer thereof is included.

[0342] In another embodiment, another subset of compounds of formula (I) includes R1 is C 5~30 Alkyl, C 5~20 alkenyl, -R*YR", -YR", and -R"M'R'; R2 and R3 are independently H, C 2~14 Alkyl, C 2~14 alkenyl, -R*YR", -YR", and -R*OR", or R2 and R3 together with the atom to which they are attached form a heterocycle or carbocycle; R4 is -(CH2) n Q or -(CH2) n CHQR, where Q is -N(R)2 and n is selected from 3, 4, and 5; Each R5 is independently C 1~3 Alkyl, C 2~3 alkenyl, and H; Each R6 is independently selected from the group consisting of C 1~3 Alkyl, C 2~3 alkenyl, and H; M and M' are independently selected from -C(O)O-, -OC(O)-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)2-, -SS-, an aryl group, and a heteroaryl group; R7 is C 1~3 Alkyl, C 2~3 alkenyl, and H; Each R is independently C 1~3 Alkyl, C 2~3 alkenyl, and H; Each R' is independently C 1~18 Alkyl, C 2~18 selected from the group consisting of alkenyl, -R*YR", -YR", and H; Each R″ is independently 3~14 Alkyl and C 3~14 alkenyl, Each R* is independently 1~12 Alkyl and C 1~12 alkenyl, Each Y is independently 3~6 is a carbocyclic ring, each X is independently selected from the group consisting of F, Cl, Br, and I; m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13; The compound, or a salt or isomer thereof is included.

[0343] In another embodiment, another subset of compounds of formula (I) includes R1 is C 5~30 Alkyl, C 5~20alkenyl, -R*YR", -YR", and -R"M'R'; R2 and R3 are independently C 1~14 Alkyl, C 2~14 alkenyl, -R*YR", -YR", and -R*OR", or R2 and R3 together with the atom to which they are attached form a heterocycle or carbocycle; R4 is -(CH2) n Q, -(CH2) n -CHQR, -CHQR, and -CQ(R), where Q is -N(R) and n is selected from 1, 2, 3, 4, and 5; Each R5 is independently 1~3 Alkyl, C 2~3 alkenyl, and H; Each R6 is independently selected from the group consisting of C 1~3 Alkyl, C 2~3 alkenyl, and H; M and M' are independently selected from -C(O)O-, -OC(O)-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)2-, -SS-, an aryl group, and a heteroaryl group; R7 is C 1~3 Alkyl, C 2~3 alkenyl, and H; Each R is independently C 1~3 Alkyl, C 2~3 alkenyl, and H; Each R' is independently C 1~18 Alkyl, C 2~18 selected from the group consisting of alkenyl, -R*YR", -YR", and H; Each R″ is independently 3~14 Alkyl and C 3~14 alkenyl, Each R* is independently 1~12 Alkyl and C 1~12 alkenyl, Each Y is independently C 3~6 is a carbocyclic ring, each X is independently selected from the group consisting of F, Cl, Br, and I; m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13; The compound, or a salt or isomer thereof is included.

[0344] In some embodiments, a subset of compounds of formula (I) is a compound of formula (IA): [ka] or a salt or isomer thereof, wherein l is selected from 1, 2, 3, 4, and 5; m is selected from 5, 6, 7, 8, and 9; M is a bond or M'; and R is an unsubstituted C 1~3 Alkyl or -(CH2) n Q, where Q is OH, -NHC(S)N(R), -NHC(O)N(R), -N(R)C(O)R, -N(R)S(O)R, -N(R)R, -NHC(=NR)N(R), -NHC(=CHR)N(R), -OC(O)N(R), -N(R)C(O)OR, heteroaryl, or heterocycloalkyl; M and M' are independently selected from -C(O)O-, -OC(O)-, -C(O)N(R')-, -P(O)(OR')O-, -SS-, an aryl group, and a heteroaryl group; R and R are independently selected from H, C, 1~14 Alkyl, and C 2~14 alkenyl.

[0345] In some embodiments, a subset of compounds of formula (I) is a compound of formula (II): [ka] or a salt or isomer thereof, wherein l is selected from 1, 2, 3, 4, and 5; M1 is a bond or M'; and R4 is an unsubstituted C 1~3 Alkyl or -(CH2) nQ, where n is 2, 3, or 4; Q is OH, -NHC(S)N(R), -NHC(O)N(R), -N(R)C(O)R, -N(R)S(O)R, -N(R)R, -NHC(=NR)N(R), -NHC(=CHR)N(R), -OC(O)N(R), -N(R)C(O)OR, heteroaryl, or heterocycloalkyl; M and M' are independently selected from -C(O)O-, -OC(O)-, -C(O)N(R')-, -P(O)(OR')O-, -SS-, an aryl group, and a heteroaryl group; R and R are independently selected from H, C, or H; 1~14 Alkyl and C 2~14 alkenyl.

[0346] In some embodiments, a subset of compounds of formula (I) is of formula (IIa), (IIb), (IIc), or (IIe): [ka] or a salt or isomer thereof, wherein R4 is as described herein.

[0347] In some embodiments, a subset of compounds of formula (I) is a compound of formula (IId): [ka] or a salt or isomer thereof, wherein n is 2, 3, or 4; and m, R′, R″, and R2-R6 are as described herein. For example, each of R2 and R3 can be independently selected from the group consisting of C 5~14 Alkyl and C 5~14 alkenyl.

[0348] In some embodiments, the ionic cationic lipids of the present disclosure include compounds having the following structure: [ka]

[0349] In some embodiments, the non-cationic lipids of the present disclosure are 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3 ... glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2 cholesteryl hemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), sphingomyelin, and mixtures thereof.

[0350] In some embodiments, the PEG-modified lipids of the present disclosure include PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, and mixtures thereof. In some embodiments, the PEG-modified lipid is PEG-DMG, PEG-c-DOMG (also referred to as PEG-DOMG), PEG-DSG, and / or PEG-DPG.

[0351] In some embodiments, sterols of the present disclosure include cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, α-tocopherol, and mixtures thereof.

[0352] In some embodiments, the LNPs of the disclosure comprise an ionic amino lipid of Compound 1, the non-cationic lipid is DSPC, the structural lipid is cholesterol, and the PEG lipid is PEG-DMG.

[0353] In some embodiments, the LNPs of the disclosure comprise an N:P ratio of about 2:1 to about 30:1.

[0354] In some embodiments, the LNPs of the disclosure comprise an N:P ratio of about 6:1.

[0355] In some embodiments, the LNPs of the disclosure comprise an N:P ratio of about 3:1.

[0356] In some embodiments, the LNPs of the present disclosure comprise a weight / weight ratio of ionic cationic lipid component to RNA of about 10:1 to about 100:1.

[0357] In some embodiments, the LNPs of the present disclosure comprise a weight / weight ratio of ionic cationic lipid component to RNA of about 20:1.

[0358] In some embodiments, the LNPs of the present disclosure comprise a weight / weight ratio of ionic cationic lipid component to RNA of about 10:1.

[0359] In some embodiments, the LNPs of the present disclosure have an average diameter of about 50 nm to about 150 nm.

[0360] In some embodiments, the LNPs of the present disclosure have an average diameter of about 70 nm to about 120 nm.

[0361] In one embodiment, the lipid may be a cleavable lipid, such as those described in International Publication No. WO2012170889, the entire contents of which are incorporated herein by reference for this purpose. In one embodiment, the lipid may be synthesized by methods known in the art and / or described in International Publication No. WO2013086354, the contents of which are incorporated herein by reference in their entirety for this purpose.

[0362] Nanoparticle compositions can be characterized by a variety of methods. For example, microscopy (e.g., transmission electron microscopy or scanning electron microscopy) can be used to examine the morphology and size distribution of nanoparticle compositions. Dynamic light scattering or potentiometry (e.g., potentiometric titration) can be used to measure zeta potential. Dynamic light scattering can also be used to determine particle size. Instruments such as the Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK) can also be used to measure several properties of nanoparticle compositions, such as particle size, polydispersity index, and zeta potential.

[0363] Nanoparticle compositions can be characterized by a variety of methods. For example, microscopy (e.g., transmission electron microscopy or scanning electron microscopy) can be used to examine the morphology and size distribution of nanoparticle compositions. Dynamic light scattering or potentiometry (e.g., potentiometric titration) can be used to measure zeta potential. Dynamic light scattering can also be used to determine particle size. Instruments such as the Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK) can also be used to measure several properties of nanoparticle compositions, such as particle size, polydispersity index, and zeta potential.

[0364] The size of the nanoparticles can also help combat biological responses, such as, but not limited to, inflammation, or increase the biological effect of the polynucleotide. As used herein, "size" or "average size" in the context of a nanoparticle composition refers to the average diameter of the nanoparticle composition.

[0365] k...

Claims

1. A personalized messenger ribonucleic acid (mRNA) cancer vaccine, comprising: an mRNA having an open reading frame (ORF) encoding 5 to 50 peptide epitopes arranged in a head-to-tail formation; and Lipid nanoparticles comprising 20-60 mol% of an ionic 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; wherein said peptide epitopes are synthesized by the following steps: (a) identifying neoepitopes that are expressed in tumor samples from a patient but not in normal tissues from the patient; (b) determining whether the identified neoepitopes include one or more neoepitopes comprising a cancer hotspot mutation selected from a BRAF mutation, a PIK3CA mutation, an EGFR mutation, an FGFR3 mutation, an ERBB2 mutation, a PTEN mutation, and a BCOR mutation; and (c) selecting peptide epitopes encoded by ORFs having the following characteristics: (i) the peptide epitope is selected from the identified neoepitopes; (ii) the peptide epitope lacks autoreactivity; (iii) each peptide epitope has at least one of the following mutations: insertion, deletion, substitution, and frameshift mutation; (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; wherein, when the identified neoepitopes include one or more neoepitopes comprising cancer hotspot mutations selected from BRAF mutations, PIK3CA mutations, EGFR mutations, FGFR3 mutations, ERBB2 mutations, PTEN mutations, and BCOR mutations, selecting peptide epitopes comprises selecting one or more peptide epitopes from the neoepitopes comprising cancer hotspot mutations. and selected by a method comprising: In the ORF, the peptide epitopes are linked to each other directly or via a linker; An mRNA cancer vaccine, wherein the mRNA contains N1-methylpseudouridine.

2. The personalized mRNA cancer vaccine described in claim 1, wherein one or more of the peptide epitopes include one or more of the cancer hotspot mutations.

3. One or more of the peptide epitopes are BRAF mutations, including V600 mutations; PIK3CA mutations, including one or more selected from R88 mutations, E545 mutations, and H1047 mutations; EGFR mutations, including the L858 mutation; FGFR3 mutations, including S249 mutations; ERBB2 mutations, including S310 mutations; PTEN mutations, including R130 mutations, and BCOR mutations, including N1459 mutations; comprising one or more cancer hotspot mutations selected from The personalized mRNA cancer vaccine of claim 2.

4. A personalized messenger ribonucleic acid (mRNA) cancer vaccine, comprising: an mRNA having an open reading frame (ORF) encoding 5 to 50 peptide epitopes arranged in a head-to-tail formation; and Lipid nanoparticles comprising 20-60 mol% of an ionic 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; wherein said peptide epitopes are synthesized by the following steps: (a) identifying neoepitopes that are expressed in tumor samples from a patient but not in normal tissues from the patient; (b) determining whether the identified neoepitopes include one or more neoepitopes that include a p53 cancer hotspot mutation selected from the R175 mutation and the R282 mutation; and (c) selecting peptide epitopes encoded by ORFs having the following characteristics: (i) the peptide epitope is selected from the identified neoepitopes; (ii) the peptide epitope lacks autoreactivity; (iii) each peptide epitope has at least one of the following mutations: insertion, deletion, substitution, and frameshift mutation; (iv) at least one of said peptide epitopes is an MHC class I epitope and at least one of said peptide epitopes is an MHC class II epitope; wherein, when the identified neoepitopes include one or more neoepitopes comprising a p53 cancer hotspot mutation selected from an R175 mutation and an R282 mutation, selecting a peptide epitope comprises selecting one or more peptide epitopes from the neoepitopes comprising a p53 cancer hotspot mutation; and selected by a method comprising: In the ORF, the peptide epitopes are linked to each other directly or via a linker; An mRNA cancer vaccine, wherein the mRNA contains N1-methylpseudouridine.

5. The personalized mRNA cancer vaccine of claim 4, wherein one or more of the peptide epitopes comprise one or more p53 cancer hotspot mutations selected from R175 mutation and R282 mutation.

6. A personalized mRNA cancer vaccine described in any one of claims 1 to 5, wherein the ORF encodes 20 to 35 peptide epitopes.

7. An individualized mRNA cancer vaccine described in any one of claims 1 to 5, wherein at least 30% of the peptide epitopes are MHC class I peptide epitopes.

8. An individualized mRNA cancer vaccine described in any one of claims 1 to 5, wherein the step of identifying neoepitopes includes a step of identifying patient-specific mutanomes by analyzing the patient's transcriptome and the patient's exome.

9. A personalized mRNA cancer vaccine described in any one of claims 1 to 5, wherein each of the peptide epitopes has a length of 8 to 29 amino acids.

10. A personalized mRNA cancer vaccine described in any one of claims 1 to 5, wherein at least two of the peptide epitopes are linked to each other by a linker.

11. A personalized mRNA cancer vaccine described in any one of claims 1 to 5, wherein at least two of the peptide epitopes are directly linked to each other without a linker.

12. An individualized mRNA cancer vaccine described in any one of claims 1 to 5, wherein the mRNA is fully modified with N1-methylpseudouridine.

13. A personalized mRNA cancer vaccine described in any one of claims 1 to 5, wherein the ORF encodes 34 peptide epitopes.

14. A personalized mRNA cancer vaccine described in any one of claims 1 to 5, wherein the ratio of MHC class I peptide epitopes to MHC class II epitopes is at least 2:

1.

15. A personalized mRNA cancer vaccine described in any one of claims 1 to 5, wherein the ratio of MHC class I peptide epitopes to MHC class II epitopes is at least 3:

1.

16. A personalized mRNA cancer vaccine described in any one of claims 1 to 5, wherein the ratio of MHC class I peptide epitopes to MHC class II epitopes is at least 5:

1.

17. A personalized mRNA cancer vaccine described in any one of claims 1 to 5, wherein one or more of the peptide epitopes comprise 8 to 29 amino acids and include one or more single nucleotide polymorphism mutations.

18. A personalized mRNA cancer vaccine described in any one of claims 1 to 5, wherein the ORF encodes 5 to 40 peptide epitopes.

19. The mRNA (a) 5′UTR, (b) 3′UTR, (c) a poly A tail, and (d) a cap structure or modified cap structure selected from a 5' cap structure, a 5' cap-0 structure, a 5' cap-1 structure, and a 5' cap-2 structure. The personalized mRNA cancer vaccine of any one of claims 1 to 5, further comprising one or more features selected from:

20. The following conditions: a) at least 50% of said peptide epitopes have a probability percent rank of greater than 0.5% for HLA-A, HLA-B, and / or DRB1; and b) one or more of said peptide epitopes exhibit T cell reactivity; The personalized mRNA cancer vaccine of any one of claims 1 to 5, wherein one or more of the following are satisfied:

21. The personalized mRNA cancer vaccine described in any one of claims 1 to 5, wherein the ORF encoding the peptide epitopes is arranged such that the peptide epitopes are arranged to minimize pseudo-epitopes.

22. A personalized mRNA cancer vaccine described in any one of claims 1 to 5, wherein at least one of the peptide epitopes is a predicted T cell reactive epitope.

23. The personalized mRNA cancer vaccine described in any one of claims 1 to 5, wherein the peptide epitope comprises multiple different types of mutations selected from insertions, deletions, substitutions, and frameshift mutations.

24. The personalized mRNA cancer vaccine of any one of claims 1 to 5, wherein one or more of the peptide epitopes comprises a centrally located SNP mutation.

25. The method of claim 24, wherein the selection is (i) the lack of autoreactivity of the neoepitope; (ii) a mutation in the neoepitope; (iii) the abundance of mutations present in neoepitopes among the identified plurality of neoepitopes; (iv) the MHC-binding ability of the neoepitope; (v) the presence of conserved or non-conserved amino acids within the neoepitope; (vi) the presence of cancer hotspot mutations; based on weighting values ​​assigned to individual neoepitopes based on one or more individual neoepitope characteristics selected from: The personalized mRNA cancer vaccine according to any one of claims 1 to 5.

26. A method for producing a personalized mRNA cancer vaccine, comprising: (a) identifying neoepitopes that are expressed in tumor samples isolated from patients with cancer but are not expressed in normal tissues of the patients; (b) determining whether the identified neoepitopes comprise one or more neoepitopes comprising a cancer hotspot mutation selected from a BRAF mutation, a PIK3CA mutation, an EGFR mutation, an FGFR3 mutation, an ERBB2 mutation, a PTEN mutation, and a BCOR mutation; (c) selecting a peptide epitope from the plurality of neoepitopes, the peptide epitope having the following characteristics: (i) the peptide epitope is selected from the identified neoepitopes; (ii) the peptide epitope lacks autoreactivity; (iii) each peptide epitope has at least one of the following mutations: insertion, deletion, substitution, and frameshift mutation; (iv) at least one of said peptide epitopes is an MHC class I epitope and at least one of said peptide epitopes is an MHC class II epitope; wherein, when the identified neoepitopes include one or more neoepitopes comprising a cancer hotspot mutation selected from a BRAF mutation, a PIK3CA mutation, an EGFR mutation, an FGFR3 mutation, an ERBB2 mutation, a PTEN mutation, and a BCOR mutation, selecting a peptide epitope comprises selecting one or more peptide epitopes from the neoepitopes comprising the cancer hotspot mutation; (d) preparing an mRNA comprising an open reading frame (ORF) encoding the peptide epitope, wherein the mRNA comprises N1-methylpseudouridine; (e) formulating the mRNA with lipid nanoparticles, the lipid nanoparticles comprising 20-60 mol % ionic cationic lipids, 5-25 mol % non-cationic lipids, 25-55 mol % sterols, and 0.5-15 mol % PEG-modified lipids; A method comprising:

27. The peptide epitope or epitopes of claim 27, BRAF mutations, including V600 mutations; PIK3CA mutations, including one or more selected from R88 mutations, E545 mutations, and H1047 mutations; EGFR mutations, including the L858 mutation; FGFR3 mutations, including S249 mutations; ERBB2 mutations, including S310 mutations; PTEN mutations, including R130 mutations, and 27. The method of claim 26, comprising a cancer hotspot mutation selected from BCOR mutations, including the N1459 mutation.

28. A method for producing a personalized mRNA cancer vaccine, comprising: (a) identifying neoepitopes that are expressed in tumor samples isolated from patients with cancer but are not expressed in normal tissues of the patients; (b) determining whether the identified neoepitopes include one or more neoepitopes comprising a p53 cancer hotspot mutation selected from the R175 mutation and the R282 mutation; (c) selecting a peptide epitope from the plurality of neoepitopes, the peptide epitope having the following characteristics: (i) the peptide epitope is selected from the identified neoepitopes; (ii) the peptide epitope lacks autoreactivity; (iii) each peptide epitope has at least one of the following mutations: insertion, deletion, substitution, and frameshift mutation; (iv) at least one of said peptide epitopes is an MHC class I epitope and at least one of said peptide epitopes is an MHC class II epitope; wherein, when the identified neoepitopes include one or more neoepitopes comprising a p53 cancer hotspot mutation selected from an R175 mutation and an R282 mutation, selecting a peptide epitope comprises selecting one or more peptide epitopes from the neoepitopes comprising a p53 cancer hotspot mutation; (d) preparing an mRNA comprising an open reading frame (ORF) encoding the peptide epitope, wherein the mRNA comprises N1-methylpseudouridine; (e) formulating the mRNA with lipid nanoparticles, the lipid nanoparticles comprising 20-60 mol % ionic cationic lipids, 5-25 mol % non-cationic lipids, 25-55 mol % sterols, and 0.5-15 mol % PEG-modified lipids; A method comprising:

29. The method of claim 1, wherein the selection (i) lack of autoreactivity of the neoepitope; (ii) mutations in neoepitopes; (iii) the abundance of mutations present in the neoepitopes among the identified neoepitopes; (iv) the MHC binding ability of the neoepitope; and (v) the presence of conserved or non-conserved amino acids within the neoepitope; The method of any one of claims 26 to 28, wherein the neoepitope is based on one or more distinct neoepitope properties selected from:

30. A method described in any one of claims 26 to 28, wherein the mRNA is fully modified with N1-methylpseudouridine.

31. A method described in any one of claims 26 to 28, wherein the ORF encodes 20 to 35 peptide epitopes, or 34 peptide epitopes.

32. The mRNA (a) the 5′UTR and / or the 3′UTR, and / or (b) a polyA tail, and / or (c) a cap structure or modified cap structure selected from a 5' cap structure, a 5' cap-0 structure, a 5' cap-1 structure, and a 5' cap-2 structure; The method according to any one of claims 26 to 28, further comprising one or more of the following features:

33. A method according to any one of claims 26 to 28, wherein the ratio of MHC class I peptide epitopes to MHC peptide class II epitopes is at least 2:

1.

34. A method according to any one of claims 26 to 28, wherein the ratio of MHC class I peptide epitopes to MHC peptide class II epitopes is at least 3:

1.

35. A method according to any one of claims 26 to 28, wherein the ratio of MHC class I peptide epitopes to MHC peptide class II epitopes is at least 5:

1.

36. The following conditions: a) at least 50% of said peptide epitopes have a probability percent rank of greater than 0.5% for HLA-A, HLA-B, and / or DRB1; and b) one or more of said peptide epitopes exhibit T cell reactivity; The method according to any one of claims 26 to 28, wherein one or more of the following are satisfied:

37. The method of any one of claims 26 to 28, wherein at least one of the peptide epitopes is a predicted T cell reactive epitope.

38. A method described in any one of claims 26 to 28, wherein at least 30% of the peptide epitopes are MHC class I peptide epitopes.

39. A method described in any one of claims 26 to 28, wherein the ORF encoding the peptide epitopes is arranged so that the peptide epitopes are arranged to minimize pseudo-epitopes.

40. A method described in any one of claims 26 to 28, wherein the ORF encodes 5 to 40 peptide epitopes.

41. A method described in any one of claims 26 to 28, wherein one or more peptide epitopes comprise 8 to 29 amino acids and include one or more single nucleotide polymorphism (SNP) mutations.

42. A pharmaceutical composition for treating cancer in a patient, comprising the personalized mRNA cancer vaccine of any one of claims 1 to 5, wherein the personalized mRNA cancer vaccine is contained at a dosage level of 0.02 to 1.0 mg.

43. The pharmaceutical composition of claim 42, wherein the personalized mRNA cancer vaccine is contained at a dosage level of 1.0 mg.

44. The pharmaceutical composition of claim 43, wherein the personalized mRNA cancer vaccine is administered every three weeks.

45. The pharmaceutical composition described in claim 44, wherein the personalized mRNA cancer vaccine is administered in two, three, four or more divided doses.

46. ​​The pharmaceutical composition of any one of claims 42 to 45, wherein the personalized mRNA cancer vaccine is administered by intradermal, intramuscular, intravascular, intratumoral, and / or subcutaneous administration.

47. The pharmaceutical composition of any one of claims 42 to 45, wherein the cancer is selected from the group consisting of non-small cell lung cancer (NSCLC), small cell lung cancer, melanoma, bladder urothelial carcinoma, HPV-negative head and neck squamous cell carcinoma (HNSCC), microsatellite-high (MSI H) / mismatch repair (MMR)-deficient solid malignant tumors, renal cancer, gastric cancer, and tumors with a high tumor mutation burden.

48. The pharmaceutical composition described in any one of claims 42 to 45, wherein the personalized mRNA cancer vaccine is administered in combination with an immune checkpoint modulator.

49. The pharmaceutical composition described in claim 48, wherein the immune checkpoint modulator is an anti-PD-1 antibody.

50. The pharmaceutical composition described in claim 49, wherein the anti-PD-1 antibody is pembrolizumab.

51. The mRNA, the ORF encodes 5 to 40 peptide epitopes; each peptide epitope having a length of 8 to 29 amino acids; one or more of the peptide epitopes comprise a centrally located single nucleotide polymorphism (SNP) variation; at least 30% of the peptide epitopes are MHC class I peptide epitopes; and the mRNA is fully modified with N1-methylpseudouridine; 46. ​​The pharmaceutical composition of any one of claims 42 to 45, comprising one or more features selected from:

52. The mRNA (a) 5′UTR, (b) 3′UTR, (c) a polyA tail, and (d) a cap structure or modified cap structure selected from a 5' cap structure, a 5' cap-0 structure, a 5' cap-1 structure, and a 5' cap-2 structure; 52. The pharmaceutical composition of claim 51, further comprising one or more features selected from:

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