Epitope selection methods

Abstract

Presented herein are methods and systems that allow for identification and selection of antigens, e.g., for cancer immunotherapy, such as for inclusion in personalized cancer vaccines (PCVs), use in T-cell therapies, T-cell receptor (TCR)-based therapies, etc.

Description

Attorney Docket No.: 2013237-1617EPITOPE SELECTION METHODSCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and benefit of U. S. Provisional Application No.63 / 777,979, filed on March 26, 2025, the content of which is hereby incorporated by reference herein in its entirety.BACKGROUND

[0002] Cancer is a primary cause of mortality, accounting for 1 in 4 of all deaths. Despite recent advances in the field of cancer immunotherapy there remains no single, broadly applicable treatment. Molecular heterogeneity of tumors renders many therapies ineffective for cancer patients.SUMMARY

[0003] Presented herein are methods and systems that allow for identification and selection of antigen epitope targets (e.g., neoantigen epitope targets and / or non-neoantigen epitope targets) for cancer immunotherapy, such as for inclusion in personalized cancer vaccines (PC Vs), use in T-cell therapies, T-cell receptor (TCR)-based therapies, etc.

[0004] In one aspect, the disclosure features a method of selecting shared antigen epitopes, e.g., for inclusion in a construct. In some embodiments, the construct is a vaccine construct. In some embodiments, the vaccine construct is for immunotherapy, e.g., for a cancer therapy. In some embodiments, the construct is for use in and / or administration to a subject. In some embodiments, the method comprises: (a) obtaining (e.g., receiving, accessing, and / or generating) (e.g., by a processor of a computing device) a candidate epitope list identifying a plurality of candidate epitopes (e.g., non-neoepitopes) and MHC presentation and immunogenicity data comprising, for each particular candidate epitope of the plurality, (i) and (ii) as follows: (i) a corresponding MHC presentation score representing a known and / or predicted likelihood and / or strength of binding between the particular candidate epitope and an MHC of the subject; and (ii) a corresponding immunogenicity score representing a known and / or predicted immunogenicity of the particular candidate epitope; (b) for each candidate epitope of at least a portion of the plurality of candidate epitopes, determining (e.g., by the processor) a corresponding rank (e.g.,Page 1 of 18313406582vlAttorney Docket No.: 2013237-1617relative to other candidate epitopes of the plurality) based at least in part on the MHC presentation and immunogenicity data; (c) selecting (e.g., by the processor) a subset of the plurality of candidate epitopes for inclusion in the polyepitopic construct based at least in part on their corresponding rankings as a set of shared antigen epitopes; and optionally (d) storing and / or providing (e.g., for display and / or further processing) (e.g., by the processor) the set of shared antigen epitopes.

[0005] In some embodiments, for each of at least a portion of the candidate epitopes, the corresponding MHC presentation score classifies the candidate epitope as a known T-cell epitope and / or a known ligand.

[0006] In some embodiments, for each of at least a portion of the candidate epitopes, the corresponding MHC presentation score represents a predicted MHC binding of the candidate epitope. In some embodiments, the MHC presentation score is a numerical value that quantifies a predicted binding (and / or presentation) strength and / or likelihood of binding. In some embodiments, the MHC presentation score is a percentile rank classifying the candidate epitope as a predicted strong, intermediate, or weak binder.

[0007] In some embodiments, for each of at least a portion (e.g., up to all) of the candidate epitopes, the corresponding immunogenicity score categorizes the candidate epitope according to a level of evidence of immunogenicity (e.g., and, optionally, safety) (e.g., based on pre-existing data).

[0008] In some embodiments, the method further comprises: identifying (e.g., by the processor) one or more of the candidate epitopes in a proteome of the subject (e.g., a human reference proteome and / or a curated / modified portion thereof, e.g., that excludes a gene-specific whitelist that includes (e.g., manually) curated homologous sequences including sequences from annotated pseudogenes); and filtering (e.g., by the processor) the plurality of candidate epitopes to exclude the one or more candidate epitopes identified the proteome of the subject (e.g., if at least a part of a candidate out of the plurality of candidate polynucleotide sequences matches to an 8-mer long sequence in the proteome data).

[0009] In some embodiments, the method comprises: identifying (e.g., by the processor) one or more of the candidate epitopes (e.g., alone or in combination, e.g., as in a combined cluster comprising two or more candidate epitopes) as matching a potency linker sequence; and filtering Page 2 of 18313406582vlAttorney Docket No.: 2013237-1617(e.g., by the processor) the plurality of candidate epitopes to exclude those candidate epitopes identified as matching a potency linker sequence.

[0010] In some embodiments, the method comprises: identifying (e.g., by the processor) one or more of the candidate epitopes as matching a non-immunogenic epitope of an epitope exclusion list, said epitope exclusion list comprising a listing of (e.g., a plurality of) epitopes previously (e.g., repeatedly) identified as non-immunogenic; and filtering (e.g., by the processor) the plurality of candidate epitopes to exclude the one or more candidate epitopes identified as matching a non-immunogenic epitope of the epitope exclusion list (e.g., setting their corresponding MHC prediction score to -1 to identify them as to be excluded).

[0011] In some embodiments, the method further comprise [e.g., at step (b) or (c)] subdividing (e.g., by the processor), the plurality of candidate epitopes into a plurality of batches according to their corresponding MHC presentation scores [e.g., each batch associated a particular classification of the candidate epitopes (e.g., a list of “known” epitopes, the list of “known” ligands) and / or a particular range of MHC binding data (e.g., predicted )]. In some embodiments provided methods comprise [e.g., at step (b) or (c)] subdividing (e.g., by the processor), the plurality of candidate epitopes into a plurality of batches, each batch associated a particular classification of the candidate epitopes including (i) a list of “known” epitopes, (ii) a list of “known” MHC ligands and (iii) predicted MHC ligands having a particular range of MHC binding scores. In some embodiments, each batch is associated with one or more (e.g., one or two) tiers, each tier associated with, and comprising candidate epitopes having, a particular immunogenicity categorization (e.g., as described herein, e.g., in paragraphs above).

[0012] In some embodiments, the method further comprise determining, for one or more candidate epitopes, a corresponding target cluster by: selecting (e.g., by the processor) a first candidate epitope as a target core; identifying (e.g., by the processor) one or more additional candidate epitopes (e.g., different from the first candidate epitope selected as the target core) belonging to a same transcript window as, and overlapping (e.g., by at least one amino acid) with, the first candidate epitope; and extending the target core to include the one or more ligands, thereby forming a target cluster [e.g., and checking if a final length of the core does not exceed 40 amino acids and / or if immunogenicity associated data of the core satisfies immunogenicity associated thresholds of associated batch].Page 3 of 18313406582vlAttorney Docket No.: 2013237-1617

[0013] In some embodiments, two cores of a plurality of candidate cores are combined into a combined core if the two cores overlap by at least one amino acid and correspond to a same transcript window (e.g., if the two cores belong to a same batch) (e.g., wherein a rank of the combined core is determined by a best rank of the two cores) (e.g., if a length of the combined core is longer than 40 amino acids, the combined core is split).

[0014] In some embodiments, the method comprises selecting candidate epitopes for inclusion in the construct in an iterative fashion, based at least in part on each candidate epitope’s corresponding rank (e.g., and on a pre-defined maximal number of target clusters and / or cores available for inclusion in the construct and / or a maximum construct length) (e.g., and, in certain embodiments, checking target linker junctions to confirm the do not comprise hits to high risk genes).

[0015] In some embodiments, the method comprises updating a rank of one or more candidate epitopes while selecting the subset for inclusion in the construct (e.g., to ensure inclusion of epitopes from a diversity of antigens).

[0016] In some embodiments, step (c) comprises: selecting (e.g., by the processor) two or more candidate epitopes [e.g., within a particular batch] from a same first antigen; determining (e.g., by the processor) a number of candidate epitopes selected from the first antigen to be greater than or equal to a threshold value; and reducing (e.g., by the processor) a rank of at least a portion of remaining candidate epitopes from the first antigen [e.g., all remaining candidate epitopes from the first antigen and within the particular batch], thereby downranking remaining candidate epitopes from the first antigen.

[0017] In another aspect, the disclosure features a method of selecting neoantigen epitopes, e.g., for inclusion in a construct. In some embodiments, the construct is a vaccine construct. In some embodiments, the vaccine construct is for immunotherapy, e.g., a cancer therapy. In some embodiments, the construct is for use in and / or administration to a subject. In some embodiments, the method comprises: (a) obtaining a candidate epitope list identifying a plurality of candidate epitopes, MHC presentation data, and expression data comprising, for each particular candidate epitope of the plurality, (i) and (ii) as follows: (i) a corresponding MHC presentation score representing a known and / or predicted likelihood and / or strength of binding between the particular candidate epitope and an MHC of the subject; and (ii) a corresponding Page 4 of 18313406582vlAttorney Docket No.: 2013237-1617expression score representing measured expression in the subject; (b) for each candidate epitope of at least a portion of the plurality of candidate epitopes, determining (e.g., by the processor) a corresponding rank (e.g., relative to other candidate epitopes of the plurality) based at least in part on the MHC presentation and expression data; (c) selecting (e.g., by the processor) a subset of the plurality of candidate epitopes for inclusion in the poly epitopic construct based at least in part on their corresponding rankings as a set of neoantigen epitopes; and optionally (d) storing and / or providing (e.g., for display and / or further processing) (e.g., by the processor) the set of neoantigen epitopes.

[0018] In some embodiments, the method further comprises obtaining germline DNA sequence from the subject and tumor DNA sequence from the subject.

[0019] In some embodiments, the method further comprises comparing germline DNA sequence from the subject and tumor DNA sequence from the subject to obtain the candidate epitope list.

[0020] In some embodiments, the method further comprises detecting one or more somatic mutations (e.g., within the tumor DNA sequence) (e.g., SNVs, InDeis, fusions).

[0021] In some embodiments, the method further comprises obtaining and / or determining the subject’s HLAtype, e.g., from the germline DNA sequence.

[0022] In some embodiments, the method comprises determining for each candidate epitope a corresponding variant allele frequency (e.g., a VAF of a e.g., somatic mutation encoding the candidate epitope) (e.g., and selecting the candidate epitopes based at least in part on their corresponding VAFs).

[0023] In some embodiments, for each of at least a portion of the candidate epitopes, the corresponding MHC presentation score classifies the candidate epitope as a known T-cell epitope and / or a known ligand.

[0024] In some embodiments, for each of at least a portion of the candidate epitopes, the corresponding MHC presentation score represents a predicted MHC binding of the candidate epitope. In some embodiments, the MHC presentation score is a numerical value that quantifies a predicted binding strength and / or likelihood of binding. In some embodiments, the MHC presentation score is a percentile rank classifying the candidate epitope as a predicted strong, intermediate, or weak binder.Page 5 of 18313406582vlAttorney Docket No.: 2013237-1617

[0025] In some embodiments, the method comprises: identifying (e.g., by the processor) one or more of the candidate epitopes (and / or one or more candidate epitope-linker combinations) in a proteome of the subject (e.g., a human reference proteome and / or a curated / modified portion thereof, e.g., that excludes a gene-specific whitelist that includes (e.g., manually) curated homologous sequences including sequences from annotated pseudogenes); and filtering (e.g., by the processor) the plurality of candidate epitopes to exclude the one or more candidate epitopes identified the proteome of the subject (e.g., if at least a part of a candidate out of the plurality of candidate polynucleotide sequences matches to an 8-mer long sequence in the proteome data).

[0026] In some embodiments, the method comprises: identifying (e.g., by the processor) one or more of the candidate epitopes (and / or one or more candidate epitope-linker combinations) as matching a sequence of a (e.g., predefined) list of tissue specific expression risk factors (e.g., for autoimmunity); and filtering (e.g., by the processor) the plurality of candidate epitopes to exclude the one or more candidate epitopes identified as matching a sequence of the tissue specific expression risk factors list.

[0027] In some embodiments, the method comprises determining, for each particular candidate epitope of the plurality of candidate epitopes, the corresponding MHC presentation score based at least in part on, for the particular candidate epitope, a corresponding expression score representing measured expression in the subject.

[0028] In another aspect, the disclosure features a method of selecting shared antigen epitopes, e.g., for inclusion in a construct. In some embodiments, the construct is a vaccine construct. In some embodiments, the vaccine construct is for immunotherapy, e.g., for a cancer therapy. In some embodiments, the construct is for use in and / or administration to a subject. In some embodiments, the method comprises: (a) obtaining a candidate epitope list identifying a plurality of candidate epitopes, MHC presentation data, and expression data comprising, for each particular candidate epitope of the plurality, (i) and (ii) as follows: (i) a corresponding MHC presentation score representing a known and / or predicted likelihood and / or strength of binding between the particular candidate epitope and an MHC of the subject; and (ii) a corresponding expression score representing measured expression (e.g., of a transcript and / or gene from which a shared antigen comprising the candidate epitope derives) in the subject, wherein the measured expression exceeds a predetermined threshold level; (b) for each candidate epitope of at least a Page 6 of 18313406582vlAttorney Docket No.: 2013237-1617portion of the plurality of candidate epitopes, determining (e.g., by the processor) a corresponding rank (e.g., relative to other candidate epitopes of the plurality) based at least in part on the MHC presentation and expression data; (c) selecting (e.g., by the processor) a subset of the plurality of candidate epitopes for inclusion in the poly epitopic construct based at least in part on their corresponding rankings as a set of shared antigen epitopes; and optionally (d) storing and / or providing (e.g., for display and / or further processing) (e.g., by the processor) the set of shared antigen epitopes.

[0029] In some embodiments, the method further comprises obtaining germline DNA sequence from the subject and tumor DNA sequence from the subject.

[0030] In some embodiments, the method further comprises comparing germline DNA sequence from the subject and tumor DNA sequence from the subject, e.g., to identify one or more germline mutations and one or more somatic mutations. In some embodiments, the plurality of candidate epitopes do not comprise a somatic mutation.

[0031] In some embodiments, the method further comprises obtaining and / or determining the subject’s HLAtype, e.g., from the germline DNA sequence.

[0032] In some embodiments, for each of at least a portion of the candidate epitopes, the corresponding MHC presentation score classifies the candidate epitope as a known T-cell epitope and / or a known ligand.

[0033] In some embodiments, for each of at least a portion of the candidate epitopes, the corresponding MHC presentation score represents a predicted MHC binding of the candidate epitope. In some embodiments, the MHC presentation score is a numerical value that quantifies a predicted binding strength and / or likelihood of binding. In some embodiments, the MHC presentation score is a percentile rank classifying the candidate epitope as a predicted strong, intermediate, or weak binder.

[0034] In some embodiments, the method comprises determining the corresponding MHC presentation score by selecting it from a lookup table of precomputed presentation scores (e.g., the lookup table comprising precomputed peptide-MHC binding scores for peptide-allele combinations derived from wild-type sequences from all genes within the list of non-neoantigens together with the 100 most frequent alleles occurring in the human population).Page 7 of 18313406582vlAttorney Docket No.: 2013237-1617

[0035] In some embodiments, the method comprises: identifying (e.g., by the processor) one or more of the candidate epitopes in a proteome of the subject (e.g., a human reference proteome and / or a curated / modified portion thereof, e.g., that excludes a gene-specific whitelist that includes (e.g., manually) curated homologous sequences including sequences from annotated pseudogenes); and filtering (e.g., by the processor) the plurality of candidate epitopes to exclude the one or more candidate epitopes identified the proteome of the subject (e.g., if at least a part of a candidate out of the plurality of candidate polynucleotide sequences matches to an 8-mer long sequence in the proteome data).

[0036] In some embodiments, the method comprises: identifying (e.g., by the processor) one or more of the candidate epitopes (e.g., alone or in combination, e.g., as in a target cluster comprising two or more candidate epitopes) as matching a potency linker sequence; and filtering (e.g., by the processor) the plurality of candidate epitopes to exclude those candidate epitopes identified as matching a potency linker sequence.

[0037] In some embodiments, the method comprises: identifying (e.g., by the processor) one or more of the candidate epitopes as matching a non-immunogenic epitope of an epitope exclusion list, said epitope exclusion list comprising a listing of (e.g., a plurality of) epitopes previously (e.g., repeatedly) identified as non-immunogenic; and filtering (e.g., by the processor) the plurality of candidate epitopes to exclude the one or more candidate epitopes identified as matching a non-immunogenic epitope of the epitope exclusion list (e.g., setting their corresponding MHC prediction score to -1 to identify them as to be excluded).

[0038] In some aspects, the present disclosure features a method of selecting neoantigen epitopes for inclusion in a construct [e.g., a vaccine construct (e.g., for immunotherapy, for a cancer therapy)] for a subject, said provided methods comprising: (a) obtaining a candidate epitope list identifying a plurality of candidate epitopes, MHC presentation data comprising, for each particular candidate epitope of the plurality, a corresponding MHC presentation score representing a known and / or predicted likelihood and / or strength of binding between the particular candidate epitope and an MHC of the subject; and (b) for each candidate epitope of at least a portion of the plurality of candidate epitopes, determining (e.g., by the processor) a corresponding rank (e.g., relative to other candidate epitopes of the plurality) based at least in part on the MHC presentation data; (c) selecting (e.g., by the processor) a subset of the plurality Page 8 of 18313406582vlAttorney Docket No.: 2013237-1617of candidate epitopes for inclusion in the polyepitopic construct based at least in part on their corresponding rankings as a set of neoantigen epitopes; and (d) storing and / or providing (e.g., for display and / or further processing) (e.g., by the processor) the set of neoantigen epitopes.

[0039] In some embodiments, the method comprise determining, for each particular candidate epitope of the plurality of candidate epitopes, the corresponding MHC presentation score based at least in part on, for the particular candidate epitope, a corresponding expression score representing measured expression in the subject.

[0040] In another aspect, the disclosure features a system comprising a processor of a computing device and a memory having instructions stored thereon, wherein the instructions, when executed by the processor, cause the processor to perform any of the methods disclosed herein (e.g., in paragraphs above).BRIEF DESCRIPTION OF THE DRAWING

[0041] FIG. 1 is a schematic depicting an exemplary bioinformatics workflow for detecting antigen based neotherapeutic target epitopes (“dANTe pipeline”), as described in Example 1. Abbreviations: BRCA = Breast invasive carcinoma; COAD = Colon adenocarcinoma; HLA = Human Leukocyte antigen; HNSC = Head and Neck squamous cell carcinoma; MHC = Major histocompatibility complex; TAA = Tumor associated antigen; WES = Whole exome sequencing.

[0042] FIG.2 is a bar chart depicting frequency of fusion events in 45 target genes across patients; bars are colored (cross-hatched in black and white figures) according to indications for patients having fusion events in a given target gene. Abbreviations: OV = Ovarian serous cystadenocarcinoma (associated with HOXB13 fusion events); PRAD = Prostate adenocarcinoma (associated with ACPP and / or KLK3 fusion events); SKCM = Skin Cutaneous Melanoma (associated with PMEL and / or TYR fusion events); TNBC MERIT = Triple negative breast carcinoma_Mutanome engineered RNA immuno-therapy (associated with ACTLS fusion events).

[0043] FIG.3 is a box plot depicting number of non-neoantigen transcripts from an exemplary non-neoantigen input list passing expression filters per indication. Individual patients are shown as dots. Abbreviations: BRCA = Breast invasive carcinoma; COAD = Colon adenocarcinoma; HNSC = Head and neck squamous cell carcinoma; KIRP = Kidney renal papillary cell carcinoma; LIHC = Liver hepatocellular carcinoma; LU AD = Lung adenocarcinoma; LUSC = Page 9 of 18313406582vlAttorney Docket No.: 2013237-1617Lung squamous cell carcinoma; OV = Ovarian serous cystadenocarcinoma; PAAD = Pancreatic adenocarcinoma; PRAD = Prostate adenocarcinoma; READ = Rectum adenocarcinoma; SKCM = Skin Cutaneous Melanoma; STAD = Stomach adenocarcinoma; TNBC MERIT = Triple negative breast carcinoma Mutanome engineered RNA immuno-therapy.

[0044] FIG.4 is a series of bar charts depicting fractions of patients with at least one transcript of a given gene from the non-neoantigen input list passing a gene-specific expression cutoff filter, according to indication. Abbreviations as in preceding figure.

[0045] FIG. 5 is a box plot depicting number of mutation events per patient within transcript regions defined by the exemplary input list of non-neoantigens, according to indication. Each dot represents a patient. Somatic mutations are shown in dotted hatching (lighter shade (yellow) in color figures), germline mutations are shown in striped hatching (darker shade (red) in color figures). Abbreviations as in preceding figure.

[0046] FIG.6 is a box plot depicting number of 9-mer MHC I potential non-neoepitope targets by indication. Dots represent individual patients. Abbreviations as in preceding figure.

[0047] FIG. 7 depicts a correlation between number of transcripts after expression filtering and number of generated non-neoepitope potential targets. Each dot represents an individual patient. R = 0.78, p < 2.2 x 10-16, Pearson correlation. Abbreviations as in preceding figure.

[0048] FIG. 8A is a series of bar charts depicting contribution of individual genes to a non-neoepitope target pool for a subset of indications. Data are pooled across different patients. X-axis represents individual genes, y-axis presents number of generated targets per gene. Only targets for 9-mers are shown. Abbreviations as in preceding figure.

[0049] FIG. 8B is a series of bar charts depicting contribution of individual genes to a non-neoepitope target pool for a subset of indications. Data are pooled across different patients. X-axis represents individual genes, y-axis presents number of generated targets per gene. Only targets for 9-mers are shown. Abbreviations as in preceding figure.

[0050] FIG.9 is a bar chart depicting number of targets per gene contributing to a non-neoepitope target pool. Only genes are displayed which have at least one target in one patient after expression filtering. Abbreviations as in preceding figure.Page 10 of 18313406582vlAttorney Docket No.: 2013237-1617

[0051] FIG. 10 depicts an exemplary view of independent mapping of generated non-neoepitope targets to a respective transcript sequence (transcript uc010aap.2) for a single patient (COAD patient TCGA-4N-A93T). X-axis represents the combined (spliced) exonic regions for a single transcript in the exemplary non-neoantigen input list according to a sliding window of potential target sequences (n=108 windows). Abbreviations: SNP = Single nucleotide polymorphism.

[0052] FIG. 11 is a box plot depicting fractions of non-neoepitope targets by indication for which precomputed MHC I binding scores were stored in a Lookup table to reduce computational cost. Abbreviations as in previous figures.

[0053] FIG. 12 is a box plot depicting number of expressed genes after filtering for each indication (N = 10-16 patients per indication). Abbreviations as in preceding figure.

[0054] FIG. 13 is a scatter plot depicting variant allele frequency (VAF) for neoepitope targets in tumor tissue or normal tissue. Each dot represents an individual neoepitope target mutation. Abbreviations as in preceding figure.

[0055] FIG. 14 is a scatter plot depicting tumor content (x-axis) versus total number of somatic mutations (y-axis). Tumor content does not correlate with number of detected somatic mutations. Each dot represents an individual patient.

[0056] FIG. 15 is a box plot depicting number of total somatic mutations by indication; Y-axis represents the average+ / -SD of mutation events (n = 10-16 patients per indication).Abbreviations as in previous figures.

[0057] FIG. 16 is a bar chart depicting average + / - SD of selected somatic mutation events (i.e., neoepitope targets) remaining after prioritization, by indication (n = 10-16 patients per indication). Abbreviations as in preceding figure.

[0058] FIG. 17 is a 2D plot depicting absolute frequency (y-axis) of occurrence of a neoepitope target for a given target gene (x-axis) across all patients (n = 159 patients).

[0059] FIG. 18 is a bar chart depicting number of unique target genes across different cancer indications. Between 100 and 500 unique target genes were found (n = 10-16 patients per indication). Abbreviations as in previous figures.Page 11 of 18313406582vlAttorney Docket No.: 2013237-1617

[0060] FIG. 19 is a bar chart depicting number of neoepitope target genes for 30 common oncogenic genes in the full neoepitope target gene space, with different colors (cross-hatching in black and white figures) for different indications.

[0061] FIG.20 is a scatter plot depicting number of SNVs detected across several cancer indications. Each dot represents a single patient. SNVs were detected using aligned DNA reads of tumor and WT samples. Abbreviations as in previous figures.

[0062] FIG.21 is a scatter plot depicting number of InDels detected across several cancer indications. Each dot represents a single patient. InDels were detected using Strelka.Abbreviations as in preceding figure.

[0063] FIG.22 is a block flow diagram showing an exemplary bioinformatics workflow for detecting antigen based neotherapeutic target epitopes (“dANTe pipeline”), as described in Example 1 (HLA- human leukocyte antigen; pMHC - peptide-major histocompatibility complex; TAA - tumor associated antigen; WES - whole exome sequence).

[0064] FIG.23 is a block flow diagram showing a sANTe algorithm to divide targets into different batches according to known epitopes, known ligands or ligands predicted to be presented, according to an illustrative embodiment (b - batch; MHC I / II - major histocompatibility complex I / II).

[0065] FIG.24 is a schematic showing targets, target clusters, and combined target clusters, according to an illustrative embodiment.

[0066] FIG.25 is a block flow diagram showing selection process of known epitopes from the immune epitope database (IEDB), according to an illustrative embodiment.

[0067] FIG.26A is a block flow diagram showing downranking of target clusters and respective thresholds, according to an illustrative embodiment.

[0068] FIG.26B is a block flow diagram showing downranking of target clusters, according to an illustrative embodiment.

[0069] FIG.26C is a block flow diagram showing downranking of target clusters, according to an illustrative embodiment.Page 12 of 18313406582vlAttorney Docket No.: 2013237-1617

[0070] FIG.27 is a box plot showing relative frequency of antigen candidate peptides present in a proteome, according to an illustrative embodiment.

[0071] FIG.28 is a box plot showing final target cluster lengths per patient across indications for different sANTe clustering approaches, according to an illustrative embodiment.

[0072] FIG.29 is a box plot showing a number of predicted strong MHC I ligands below a threshold of 0.03 in selected target clusters per patient for various indication, according to an illustrative embodiment.

[0073] FIG.30 is a box plot showing a number of final target clusters per patient for various indications, according to an illustrative embodiment.

[0074] FIG.31 is a bar chart showing percentage of final selected target clusters from a given antigen of a target list across various indications, according to an illustrative embodiment.

[0075] FIG.32 is a bar chart showing percentage of patients whose antigen expression passed a respective expression threshold across various indications, according to an illustrative embodiment.

[0076] FIG.33A is a bar chart showing fractions of final target clusters per indication derived from a specific antigen of a target list with a number on top of the bars representing the number of final target clusters, according to an illustrative embodiment.

[0077] FIG.33B is a bar chart showing fractions of final target clusters per indication derived from a specific antigen of a target list with a number on top of the bars representing the number of final target clusters, according to an illustrative embodiment.

[0078] FIG.34 is a box plot showing a number of final target clusters available from tier one to tier three antigens and selected using two different sANTe approaches: “No penalty” approach, “Clusters batch” approach, and “Available genes” - a median between two approaches, according to an illustrative embodiment.

[0079] FIG.35 is a 2D plot showing final target clusters and available antigens for each analyzed lung adenocarcinoma (LU AD) patient, according to an illustrative embodiment.Page 13 of 18313406582vlAttorney Docket No.: 2013237-1617

[0080] FIG.36 is a 2D plot showing final target clusters and available antigens for each analyzed lung squamous cell carcinoma (LUSC) patient, according to an illustrative embodiment.

[0081] FIG.37 is a 2D plot showing final target clusters and available antigens for each analyzed ovarian serous cystadenocarcinoma (OV) patient, according to an illustrative embodiment.

[0082] FIG.38 is a 2D plot showing final target clusters and available antigens for each analyzed prostate adenocarcinoma (PRAD) patient, according to an illustrative embodiment.

[0083] FIG.39 is a 2D plot showing final target clusters and available antigens for each analyzed skin cutaneous melanoma (SKCM) patient, according to an illustrative embodiment.

[0084] FIG.40 is a 2D plot showing final target clusters and available antigens for each analyzed triple negative breast carcinoma (TNBC) patient, according to an illustrative embodiment.

[0085] FIG.41 A is a block flow diagram showing a method for selecting neoantigen epitopes, according to an illustrative embodiment.

[0086] FIG.4 IB is a block flow diagram showing a method for selecting non-neoantigen epitopes, according to an illustrative embodiment.

[0087] FIG.42 is a block flow diagram showing a method for identifying polynucleotide sequences of antigens, according to an illustrative embodiment.

[0088] FIG.43 is a diagram showing a division of candidate polynucleotide sequences into batches, according to an illustrative embodiment.

[0089] FIG.44A is a diagram showing ordering of candidate polynucleotide sequences within a batch, according to an illustrative embodiment.

[0090] FIG.44B is a diagram showing ordering of candidate polynucleotide sequences within batches, according to an illustrative embodiment.

[0091] FIG.45 is a diagram showing determination of placement of candidate polynucleotide sequences in a poly epitopic construct using placement costs, according to an illustrative embodiment.Page 14 of 18313406582vlAttorney Docket No.: 2013237-1617

[0092] FIG.46 is a block diagram of an exemplary cloud computing environment, used in certain embodiments.

[0093] FIG.47 is a block diagram of an example computing device and an example mobile computing device used in certain embodiments.DEFINITIONS

[0094] About: The term “about”, when used herein in reference to a value, refers to a value that is similar, in context to the referenced value. In general, those skilled in the art, familiar with the context, will appreciate the relevant degree of variance encompassed by “about” in that context. For example, in some embodiments, the term “about” may encompass a range of values that within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the referred value.

[0095] Agent: As used herein, the term “agent,” may refer to a physical entity. In some embodiments, an agent may be characterized by a particular feature and / or effect. For example, as used herein, the term “therapeutic agent” refers to a physical entity has a therapeutic effect and / or elicits a desired biological and / or pharmacological effect. In some embodiments, an agent may be a compound, molecule, or entity of any chemical class including, for example, a small molecule, polypeptide, nucleic acid, saccharide, lipid, metal, or a combination or complex thereof. In some embodiments, part or all of an agent may be depicted herein as a chemical structure, or may be described using chemical nomenclature and / or with reference to general principles of organic chemistry, e.g., in accordance with the Periodic Table of Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed; “Organic Chemistry”, Thomas Sorrell, University Science Books, Sausalito: 1999, and / or “March’s Advanced Organic Chemistry”, 5th Ed., Ed.: Smith, M. B. and March, J., John Wiley & Sons, New York: 2001, the entire contents of which are hereby incorporated by reference. Unless otherwise stated or clear from context, chemical structures depicted herein may be considered to reference or include one or more, or all, stereoisomeric (e.g., enantiomeric or diastereomeric) forms of the structure, and / or one or more, or all, geometric or conformational isomeric forms of the structure. For example, unless otherwise indicated or clear, both R and S configurations of a stereocenter may be contemplated in embodiments of the disclosure. In some embodiments, a compound may be described and / or utilized as a particular single stereochemical isomer; alternatively or additionally, in some Page 15 of 18313406582vlAttorney Docket No.: 2013237-1617embodiments, such a compound may be described and / or utilized as a combination (e.g., a mixture) of one or more enantiomeric (e.g., diastereomeric) forms (e.g., as a racemic preparation). Analogously, in some embodiments, a single geometric isomer may be described and / or utilized; in some embodiments, a combination (e.g, a mixture) of geometric (or conformational) isomers may be described and / or utilized. Unless otherwise stated or clear from context, all tautomeric forms of provided compounds are within the scope of the disclosure. Still further, unless otherwise indicated or clear from context, in some embodiments, a particular chemical compound (e.g., as may be represented by a depicted chemical structure) may be described and / or utilized in an alternative isotopic form - i.e., in a form in which one or more atoms is isotopically altered (e.g., so that a hydrogen is replaced by deuterium or tritium, and / or a carbon is replaced by 13C- or 14C-. Thus, in some embodiments, a particular compound may be described and / or utilized as or in an isotopically enriched preparation.

[0096] Amino acid'. In its broadest sense, as used herein, the term “amino acid” refers to a compound and / or substance that can be, is, or has been incorporated into a polypeptide chain, e.g., through formation of one or more peptide bonds. In some embodiments, an amino acid has the general structure H2N–C(H)(R)–COOH. In some embodiments, an amino acid is a naturally-occurring amino acid. In some embodiments, an amino acid is a non-natural amino acid; in some embodiments, an amino acid is a D-amino acid; in some embodiments, an amino acid is an L-amino acid. “Standard amino acid” refers to any of the twenty standard L-amino acids commonly found in naturally occurring peptides. “Nonstandard amino acid” refers to any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or obtained from a natural source. In some embodiments, an amino acid, including a carboxy-and / or amino-terminal amino acid in a polypeptide, can contain a structural modification as compared with the general structure above. For example, in some embodiments, an amino acid may be modified by methylation, amidation, acetylation, pegylation, glycosylation, phosphorylation, and / or substitution (e.g., of the amino group, the carboxylic acid group, one or more protons, and / or the hydroxyl group) as compared with the general structure. In some embodiments, such modification may, for example, alter the circulating half-life of a polypeptide containing the modified amino acid as compared with one containing an otherwise identical unmodified amino acid. In some embodiments, such modification does not significantly alter a relevant activity of a polypeptide containing the modified amino acid, as compared with one Page 16 of 18313406582vlAttorney Docket No.: 2013237-1617containing an otherwise identical unmodified amino acid. As will be clear from context, in some embodiments, the term “amino acid” may be used to refer to a free amino acid; in some embodiments it may be used to refer to an amino acid residue of a polypeptide.

[0097] Antigen: term “antigen”, as used herein, refers to an agent that elicits an immune response; and / or (ii) an agent that binds to a T cell receptor (e.g., when presented by an MHC molecule) or to an antibody. In some embodiments, an antigen elicits a humoral response (e.g., including production of antigen-specific antibodies); in some embodiments, an elicits a cellular response (e.g., involving T-cells whose receptors specifically interact with the antigen). In some embodiments, and antigen binds to an antibody and may or may not induce a particular physiological response in an organism. In general, an antigen may be or include any chemical entity such as, for example, a small molecule, a nucleic acid, a polypeptide, a carbohydrate, a lipid, a polymer (in some embodiments other than a biologic polymer [e.g., other than a nucleic acid or amino acid polymer) etc. In some embodiments, an antigen is or comprises a polypeptide. In some embodiments, an antigen is or comprises a glycan. Those of ordinary skill in the art will appreciate that, in general, an antigen may be provided in isolated or pure form, or alternatively may be provided in crude form (e.g., together with other materials, for example in an extract such as a cellular extract or other relatively crude preparation of an antigen-containing source). In some embodiments, antigens utilized in accordance with the present invention are provided in a crude form. In some embodiments, an antigen is a recombinant antigen.

[0098] Associated'. Two events or entities are “associated” with one another, as that term is used herein, if the presence, level, degree, type and / or form of one is correlated with that of the other. For example, a particular entity (e.g., polypeptide, genetic signature, metabolite, microbe, etc.) is considered to be associated with a particular disease, disorder, or condition, if its presence, level and / or form correlates with incidence of, susceptibility to, severity of, stage of, etc. the disease, disorder, or condition (e.g., across a relevant population). In some embodiments, two or more entities are physically “associated” with one another if they interact, directly or indirectly, so that they are and / or remain in physical proximity with one another. In some embodiments, two or more entities that are physically associated with one another are covalently linked to one another; in some embodiments, two or more entities that are physically associated with one another are not covalently linked to one another but are non-covalently associated, for example by means ofPage 17 of 18313406582vlAttorney Docket No.: 2013237-1617hydrogen bonds, van der Waals interaction, hydrophobic interactions, magnetism, and combinations thereof.

[0099] Cancer. The term “cancer” is used herein to generally refer to a disease or condition in which cells of a tissue of interest exhibit relatively abnormal, uncontrolled, and / or autonomous growth, so that they exhibit an aberrant growth phenotype characterized by a significant loss of control of cell proliferation. In some embodiments, cancer may comprise cells that are precancerous (e.g., benign), malignant, pre-metastatic, metastatic, and / or non-metastatic. In some embodiments, cancer may be characterized by a solid tumor. In some embodiments, cancer may be characterized by a hematologic tumor. In general, examples of different types of cancers known in the art include, for example, triple negative breast cancer (TNBC), hematopoietic cancers including leukemias, lymphomas (Hodgkin’s and non-Hodgkin’s), myelomas and myeloproliferative disorders; sarcomas, melanomas, adenomas, carcinomas of solid tissue, squamous cell carcinomas of the mouth, throat, larynx, and lung, liver cancer, genitourinary cancers such as prostate, cervical, bladder, uterine, and endometrial cancer and renal cell carcinomas, bone cancer, pancreatic cancer, skin cancer, cutaneous or intraocular melanoma, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, head and neck cancers, ovarian cancer, breast cancer, glioblastomas, colorectal cancer, gastro-intestinal cancers and nervous system cancers, benign lesions such as papillomas, and the like.

[0100] Comparable: As used herein, the term “comparable” refers to two or more agents, entities, situations, sets of conditions, etc., that may not be identical to one another but that are sufficiently similar to permit comparison therebetween so that one skilled in the art will appreciate that conclusions may reasonably be drawn based on differences or similarities observed. In some embodiments, comparable sets of conditions, circumstances, individuals, or populations are characterized by a plurality of substantially identical features and one or a small number of varied features. Those of ordinary skill in the art will understand, in context, what degree of identity is required in any given circumstance for two or more such agents, entities, situations, sets of conditions, etc., to be considered comparable. For example, those of ordinary skill in the art will appreciate that sets of circumstances, individuals, or populations are comparable to one another when characterized by a sufficient number and type of substantiallyPage 18 of 18313406582vlAttorney Docket No.: 2013237-1617identical features to warrant a reasonable conclusion that differences in results obtained or phenomena observed under or with different sets of circumstances, individuals, or populations are caused by or indicative of the variation in those features that are varied.

[0101] Corresponding to: As used herein, the term “corresponding to” refers to a relationship between two or more entities. For example, the term “corresponding to” may be used to designate the position / identity of a structural element in a compound or composition relative to another compound or composition (e.g., to an appropriate reference compound or composition). For example, in some embodiments, a monomeric residue in a polymer (e.g., an amino acid residue in a polypeptide or a nucleic acid residue in a polynucleotide) may be identified as “corresponding to” a residue in an appropriate reference polymer. For example, those of ordinary skill will appreciate that, for purposes of simplicity, residues in a polypeptide are often designated using a canonical numbering system based on a reference related polypeptide, so that an amino acid “corresponding to” a residue at position 190, for example, need not actually be the 190thamino acid in a particular amino acid chain but rather corresponds to the residue found at 190 in the reference polypeptide; those of ordinary skill in the art readily appreciate how to identify “corresponding” amino acids. For example, those skilled in the art will be aware of various sequence alignment strategies, including software programs such as, for example, BLAST, CS-BLAST, CUSASW++, DIAMOND, FASTA, GGSEARCH / GLSEARCH, Genoogle, HMMER, HHpred / HHsearch, IDF, Infernal, KLAST, USEARCH, parasail, PSI-BLAST, PSI-Search, ScalaBLAST, Sequilab, SAM, SSEARCH, SWAPHI, SWAPHI-LS, SWIMM, or SWIPE that can be utilized, for example, to identify “corresponding” residues in polypeptides and / or nucleic acids in accordance with the present disclosure. Those of skill in the art will also appreciate that, in some instances, the term “corresponding to” may be used to describe an event or entity that shares a relevant similarity with another event or entity (e.g., an appropriate reference event or entity). To give but one example, a gene or protein in one organism may be described as “corresponding to” a gene or protein from another organism in order to indicate, in some embodiments, that it plays an analogous role or performs an analogous function and / or that it shows a particular degree of sequence identity or homology, or shares a particular characteristic sequence element.Page 19 of 18313406582vlAttorney Docket No.: 2013237-1617

[0102] Dosing regimen Those skilled in the art will appreciate that the term “dosing regimen” (or “therapeutic regimen”) may be used to refer to a set of unit doses (typically more than one) that are administered individually to a subject, typically separated by periods of time. In some embodiments, a given therapeutic agent has a recommended dosing regimen, which may involve one or more doses.

[0103] Encode". As used herein, the term “encode” or “encoding” refers to sequence information of a first molecule that guides production of a second molecule having a defined sequence of nucleotides (e.g., a polyribonucleotide) or a defined sequence of amino acids. For example, a DNA molecule can encode an RNA molecule (e.g., by a transcription process that includes a DNA-dependent RNA polymerase enzyme). An RNA molecule can encode a polypeptide (e.g., by a translation process). Thus, a gene, a cDNA, or an RNA molecule encodes a polypeptide if transcription and translation of RNA corresponding to that gene produces the polypeptide in a cell or other biological system. In some embodiments, a coding region of a polyribonucleotide encoding a target antigen refers to a coding strand, the nucleotide sequence of which is identical to the polyribonucleotide sequence of such a target antigen. In some embodiments, a coding region of a polyribonucleotide encoding a target antigen refers to a non-coding strand of such a target antigen, which may be used as a template for transcription of a gene or cDNA.

[0104] Epitope: As used herein, the term “epitope” refers to a moiety that is specifically recognized by an immune system (e.g., an immune system component) of a subject. For example, in some embodiments, an epitope may be a moiety that is specifically recognized by a T cell, a B cell, an immunoglobulin (e.g., antibody or receptor), immunoglobulin (e.g., antibody or receptor), binding component or an aptamer. In some embodiments, an epitope is comprised of a plurality of chemical atoms or groups on an antigen. In some embodiments, such chemical atoms or groups are surface-exposed when the antigen adopts a relevant three-dimensional conformation. In some embodiments, such chemical atoms or groups are physically near to each other in space when the antigen adopts such a conformation. In some embodiments, at least some such chemical atoms groups are physically separated from one another when the antigen adopts an alternative conformation (e.g., is linearized).

[0105] Expression". As used herein, the term “expression” of a nucleic acid sequence refers to the generation of a gene product from the nucleic acid sequence. In some embodiments, a gene Page 20 of 18313406582vlAttorney Docket No.: 2013237-1617product can be a transcript, e.g., a polyribonucleotide as provided herein. In some embodiments, a gene product can be a polypeptide. In some embodiments, expression of a nucleic acid sequence involves one or more of the following: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, etc.); (3) translation of an RNA into a polypeptide or protein; and / or (4) post-translational modification of a polypeptide or protein.

[0106] Homology. As used herein, the term “homology” or “homolog” refers to the overall relatedness between polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. In some embodiments, polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules are considered to be “homologous” to one another if their sequences are at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical. In some embodiments, polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules are considered to be “homologous” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% similar (e.g., containing residues with related chemical properties at corresponding positions). For example, as is well known by those of ordinary skill in the art, certain amino acids are typically classified as similar to one another as “hydrophobic” or “hydrophilic” amino acids, and / or as having “polar” or “non-polar” side chains. Substitution of one amino acid for another of the same type may often be considered a “homologous” substitution.

[0107] Identity. As used herein, the term “identity” refers to the overall relatedness between polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. In some embodiments, polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules are considered to be “substantially identical” to one another if their sequences are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical. Calculation of the percent identity of two nucleic acid or polypeptide sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second sequence for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In certain embodiments, the length of a sequence aligned for comparison purposes is at leastPage 21 of 18313406582vlAttorney Docket No.: 2013237-161730%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or substantially 100% of the length of a reference sequence. The nucleotides at corresponding positions are then compared. When a position in the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the 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 needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller, 1989, which has been incorporated into the ALIGN program (version 2.0). In some exemplary embodiments, nucleic acid sequence comparisons made with the ALIGN program use a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. The percent identity between two nucleotide sequences can, alternatively, be determined using the GAP program in the GCG software package using an NWSgapdna.CMP matrix.

[0108] Increased, Induced, or Reduced. As used herein, these terms or grammatically comparable comparative terms, indicate values that are relative to a comparable reference measurement. For example, in some embodiments, an assessed value achieved with a provided composition (e.g., a pharmaceutical composition) may be “increased” relative to that obtained with a comparable reference composition. Alternatively or additionally, in some embodiments, an assessed value achieved in a subject may be “increased” relative to that obtained in the same subject under different conditions (e.g., prior to or after an event; or presence or absence of an event such as administration of a composition (e.g., a pharmaceutical composition) as described herein, or in a different, comparable subject (e.g., in a comparable subject that differs from the subject of interest in prior exposure to a condition, e.g., absence of administration of a composition (e.g., a pharmaceutical composition) as described herein.). In some embodiments, comparative terms refer to statistically relevant differences (e.g., that are of a prevalence and / or magnitude sufficient to achieve statistical relevance). Those skilled in the art will be aware, or will readily be able to determine, in a given context, a degree and / or prevalence of difference that is required or sufficient to achieve such statistical significance. In some embodiments, the term Page 22 of 18313406582vlAttorney Docket No.: 2013237-1617“reduced” or equivalent terms refers to a reduction in the level of an assessed value by at least 5%, at least 10%, at least 20%, at least 50%, at least 75% or higher, as compared to a comparable reference. In some embodiments, the term “reduced” or equivalent terms refers to a complete or essentially complete inhibition, i.e., a reduction to zero or essentially to zero. In some embodiments, the term “increased” or “induced” refers to an increase in the level of an assessed value by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 80%, at least 100%, at least 200%, at least 500%, or higher, as compared to a comparable reference.

[0109] In order: As used herein with reference to a polynucleotide or polyribonucleotide, “in order” refers to the order of features from 5' to 3' along the polynucleotide or polyribonucleotide. As used herein with reference to a polypeptide, “in order” refers to the order of features moving from the N-terminal-most of the features to the C-terminal-most of the features along the polypeptide. “In order” does not mean that no additional features can be present among the listed features. For example, if Features A, B, and C of a polynucleotide are described herein as being “in order, Feature A, Feature B, and Feature C,” this description does not exclude, e.g., Feature D being located between Features A and B.

[0110] Individualized shared tumor antigen". As used herein, an “individualized shared tumor antigen” is a shared tumor antigen that is expressed in an individual subject.

[0111] Individualized shared tumor antigen epitope". As used herein, an “individualized shared tumor antigen epitope” is a shared tumor antigen epitope that is expressed in an individual subject.

[0112] Linker. As used herein, the term “linker” refers to a portion of a polypeptide that connects different regions, portions, or antigens to one another.

[0113] Lipid". As used herein, the terms “lipid” and “lipid-like material” are broadly defined as molecules which comprise one or more hydrophobic moieties or groups and optionally also one or more hydrophilic moieties or groups. Molecules comprising hydrophobic moieties and hydrophilic moieties are also typically denoted as amphiphiles.

[0114] Neoantigen: As used herein, the term “neoantigen” refers to an antigen that is not present in a reference, such as a normal non-cancerous or germline cell, but is present in a cancerPage 23 of 18313406582vlAttorney Docket No.: 2013237-1617cell. In some embodiments, a neoantigen includes one or more mutations relative to a corresponding antigen present in a normal non-cancerous or germline cell.

[0115] Neoantigen epitope: As used herein, the term “neoantigen epitope” refers to an epitope that is not present in a reference, such as a normal non-cancerous or germline cell, but is present in a cancer cell.

[0116] Non-neoantigen: As used herein, the term “non-neoantigen” refers to a tumor antigen that is not a neoantigen. In some embodiments, a non-neoantigen is a shared tumor antigen. In some embodiments, a non-neoantigen is an individualized shared tumor antigen.

[0117] Non-neoantigen epitope: As used herein, the term “non-neoantigen epitope” refers to a tumor epitope that is not a neoantigen epitope. In some embodiments, a non-neoantigen epitope is a shared tumor antigen epitope. In some embodiments, a non-neoantigen epitope is an individualized shared tumor antigen epitope.

[0118] Nucleic acid / Polynucleotide". As used herein, the term “nucleic acid” refers to a polymer of at least 10 nucleotides or more. In some embodiments, a nucleic acid is or comprises DNA. In some embodiments, a nucleic acid is or comprises RNA. In some embodiments, a nucleic acid is or comprises peptide nucleic acid (PNA). In some embodiments, a nucleic acid is or comprises a single stranded nucleic acid. In some embodiments, a nucleic acid is or comprises a double-stranded nucleic acid. In some embodiments, a nucleic acid comprises both single and double-stranded portions. In some embodiments, a nucleic acid comprises a backbone that comprises one or more phosphodiester linkages. In some embodiments, a nucleic acid comprises a backbone that comprises both phosphodiester and non-phosphodiester linkages. For example, in some embodiments, a nucleic acid may comprise a backbone that comprises one or more phosphorothioate or 5'-N-phosphoramidite linkages and / or one or more peptide bonds, e.g., as in a “peptide nucleic acid”. In some embodiments, a nucleic acid comprises one or more, or all, natural residues (e.g., adenine, cytosine, deoxyadenosine, deoxy cytidine, deoxy guanosine, deoxythymidine, guanine, thymine, uracil). In some embodiments, a nucleic acid comprises on or more, or all, non-natural residues. In some embodiments, a non-natural residue comprises a nucleoside analog (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3 -methyl adenosine, 5 -methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5 -fluorouridine, C5 -iodouridine, C5-propynyl-uridine, C5 - Page 24 of 18313406582vlAttorney Docket No.: 2013237-1617propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 6-0-methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof). In some embodiments, a non-natural residue comprises one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) as compared to those in natural residues. In some embodiments, a nucleic acid has a nucleotide sequence that encodes a functional gene product such as an RNA or polypeptide. In some embodiments, a nucleic acid has a nucleotide sequence that comprises one or more introns. In some embodiments, a nucleic acid may be prepared by isolation from a natural source, enzymatic synthesis (e.g., by polymerization based on a complementary template, e.g., in vivo or in vitro), reproduction in a recombinant cell or system, or chemical synthesis. In some embodiments, a nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, or 20,000 or more residues or nucleotides long.

[0119] Pharmaceutically effective amount". The term “pharmaceutically effective amount” or “therapeutically effective amount” refers to the amount which achieves a desired reaction or a desired effect alone or together with further doses. In the case of the treatment of a particular disease (e.g., cancer), a desired reaction in some embodiments relates to inhibition of the course of the disease (e.g., cancer). In some embodiments, such inhibition may comprise slowing down the progress of a disease (e.g., cancer) and / or interrupting or reversing the progress of the disease (e.g., cancer). In some embodiments, a desired reaction in a treatment of a disease (e.g., cancer) may be or comprise delay or prevention of the onset of a disease (e.g., cancer) or a condition (e.g., a cancer associated condition). An effective amount of a composition (e.g., a pharmaceutical composition) described herein will depend, for example, on disease (e.g., cancer) or a condition (e.g., a cancer associated condition) to be treated, the severity of such a disease (e.g., cancer) or a condition (e.g., a cancer associated condition), individual parameters of the patient, including, e.g., age, physiological condition, size and weight, the duration of treatment, the type of an accompanying therapy (if present), the specific route of administration and similar factors. Accordingly, doses of a composition (e.g., a pharmaceutical composition) described Page 25 of 18313406582vlAttorney Docket No.: 2013237-1617herein may depend on various of such parameters. In the case that a reaction in a patient is insufficient with an initial dose, higher doses (or effectively higher doses achieved by a different, more localized route of administration) may be used.

[0120] Polypeptide'. As used herein, the term “polypeptide” refers to a polymeric chain of amino acids. In some embodiments, a polypeptide has an amino acid sequence that occurs in nature. In some embodiments, a polypeptide has an amino acid sequence that does not occur in nature. In some embodiments, a polypeptide has an amino acid sequence that is engineered in that it is designed and / or produced through action of the hand of man. In some embodiments, a polypeptide may comprise or consist of natural amino acids, non-natural amino acids, or both. In some embodiments, a polypeptide may comprise or consist of only natural amino acids or only non-natural amino acids. In some embodiments, a polypeptide may comprise D-amino acids, L-amino acids, or both. In some embodiments, a polypeptide may comprise only D-amino acids. In some embodiments, a polypeptide may comprise only L-amino acids. In some embodiments, a polypeptide may include one or more pendant groups or other modifications, e.g., modifying or attached to one or more amino acid side chains, at the polypeptide’s N-terminus, at the polypeptide’s C-terminus, or any combination thereof. In some embodiments, such pendant groups or modifications comprise acetylation, amidation, lipidation, methylation, pegylation, etc., including combinations thereof. In some embodiments, a polypeptide may be cyclic, and / or may comprise a cyclic portion. In some embodiments, a polypeptide is not cyclic and / or does not comprise any cyclic portion. In some embodiments, a polypeptide is linear. In some embodiments, a polypeptide may be or comprise a stapled polypeptide. In some embodiments, the term “polypeptide” may be appended to a name of a reference polypeptide, activity, or structure; in such instances it is used herein to refer to polypeptides that share the relevant activity or structure and thus can be considered to be members of the same class or family of polypeptides. For each such class, the present specification provides and / or those skilled in the art will be aware of exemplary polypeptides within the class whose amino acid sequences and / or functions are known; in some embodiments, such exemplary polypeptides are reference polypeptides for the polypeptide class or family. In some embodiments, a member of a polypeptide class or family shows significant sequence homology or identity with, shares a common sequence motif (e.g., a characteristic sequence element) with, and / or shares a common activity (in some embodiments at a comparable level or within a designated range) with a Page 26 of 18313406582vlAttorney Docket No.: 2013237-1617reference polypeptide of the class; in some embodiments with all polypeptides within the class). For example, in some embodiments, a member polypeptide shows an overall degree of sequence homology or identity with a reference polypeptide that is at least about 30-40%, and is often greater than about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more and / or includes at least one region (e.g., a conserved region that may in some embodiments be or comprise a characteristic sequence element) that shows very high sequence identity, often greater than 90% or even 95%, 96%, 97%, 98%, or 99%. Such a conserved region usually encompasses at least 3-4 and often up to 35 or more amino acids; in some embodiments, a conserved region encompasses at least one stretch of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or more contiguous amino acids. In some embodiments, a relevant polypeptide may comprise or consist of a fragment of a parent polypeptide.

[0121] Prevent". As used herein, the term “prevent” or “prevention” when used in connection with the occurrence of a disease, disorder, and / or condition, refers to reducing the risk of developing the disease, disorder and / or condition and / or to delaying onset of one or more characteristics or symptoms of the disease, disorder or condition. Prevention may be considered complete when onset of a disease, disorder or condition has been delayed for a predefined period of time.

[0122] Reference". As used herein, the term “reference” describes a standard or control relative to which a comparison is performed. For example, in some embodiments, an agent, animal, individual, population, sample, sequence or value of interest is compared with a reference or control agent, animal, individual, population, sample, sequence or value. In some embodiments, a reference or control is tested and / or determined substantially simultaneously with the testing or determination of interest. In some embodiments, a reference or control is a historical reference or control, optionally embodied in a tangible medium. Typically, as would be understood by those skilled in the art, a reference or control is determined or characterized under comparable conditions or circumstances to those under assessment. Those skilled in the art will appreciate when sufficient similarities are present to justify reliance on and / or comparison to a particular possible reference or control.Page 27 of 18313406582vlAttorney Docket No.: 2013237-1617

[0123] Ribonucleic acid (RNA) or Polyribonucleotide'. As used herein, the term “ribonucleic acid,” “RNA,” or “polyribonucleotide” refers to a polymer of ribonucleotides. In some embodiments, an RNA is single stranded. In some embodiments, an RNA is double stranded. In some embodiments, an RNA comprises both single and double stranded portions. In some embodiments, an RNA can comprise a backbone structure as described in the definition of “Nucleic acid / Polynucleotide” above. An RNA can be a regulatory RNA (e.g., siRNA, microRNA, etc.), or a messenger RNA (mRNA). In some embodiments, an RNA is a mRNA. In some embodiments, where an RNA is a mRNA, a RNA typically comprises at its 3' end a poly(A) region. In some embodiments, where an RNA is a mRNA, an RNA typically comprises at its 5' end an art- recognized cap structure, e.g., for recognizing and attachment of a mRNA to a ribosome to initiate translation. In some embodiments, a RNA is a synthetic RNA. Synthetic RNAs include RNAs that are synthesized in vitro (e.g., by enzymatic synthesis methods and / or by chemical synthesis methods).

[0124] Ribonucleotide'. As used herein, the term “ribonucleotide” encompasses unmodified ribonucleotides and modified ribonucleotides. For example, unmodified ribonucleotides include the purine bases adenine (A) and guanine (G), and the pyrimidine bases cytosine (C) and uracil (U). Modified ribonucleotides may include one or more modifications including, but not limited to, for example, (a) end modifications, e.g., 5' end modifications (e.g., phosphorylation, dephosphorylation, conjugation, inverted linkages, etc.), 3' end modifications (e.g., conjugation, inverted linkages, etc.), (b) base modifications, e.g., replacement with modified bases, stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, or conjugated bases, (c) sugar modifications (e.g., at the 2' position or 4' position) or replacement of the sugar, and (d) internucleoside linkage modifications, including modification or replacement of the phosphodiester linkages. The term “ribonucleotide” also encompasses ribonucleotide triphosphates including modified and non-modified ribonucleotide triphosphates.

[0125] RNA lipid nanoparticle'. As used herein, the term “RNA lipid nanoparticle” refers to a nanoparticle comprising at least one lipid and RNA molecule(s), e.g., one or more polyribonucleotides as provided herein. In some embodiments, an RNA lipid nanoparticle comprises at least one cationic amino lipid. In some embodiments, an RNA lipid nanoparticle comprises at least one cationic amino lipid, at least one helper lipid, and at least one polymer-Page 28 of 18313406582vlAttorney Docket No.: 2013237-1617conjugated lipid (e.g., PEG-conjugated lipid). In various embodiments, RNA lipid nanoparticles as described herein can have an average size (e.g., Z-average) of about 100 nm to 1000 nm, or about 200 nm to 900 nm, or about 200 nm to 800 nm, or about 250 nm to about 700 nm. In some embodiments of the present disclosure, RNA lipid nanoparticles can have a particle size (e.g., Z-average) of about 30 nm to about 200 nm, or about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 to about 90 nm, about 80 nm to about 90 nm, or about 70 nm to about 80 nm. In some embodiments, an average size of lipid nanoparticles is determined by measuring the average particle diameter. In some embodiments, RNA lipid nanoparticles may be prepared by mixing lipids with RNA molecules described herein.

[0126] Secretory signal: As used herein, the term “secretory signal” refers to an amino acid sequence motif that targets associated polypeptides for translocation to a secretory pathway.

[0127] Shared tumor antigen. As used herein, the term “shared tumor antigen” refers to a tumor antigen expressed by a large fraction of cancers. In some embodiments, a “shared tumor antigen” is a tumor antigen expressed by a large fraction of cancers of the same type and / or a large fraction of cancers of different types. In some embodiments, a “shared tumor antigen” is a tumor antigen shared by a large fraction of different subjects having the same cancer type and / or different cancer types. With reference to “shared tumor antigen”, the term “large fraction” refers to at least 15%.

[0128] Shared tumor antigen epitope. As used herein, the term “shared tumor antigen epitope” refers to an epitope of and / or derived from a shared tumor antigen.

[0129] Subject'. As used herein, the term “subject” refers to an organism to be administered with a composition described herein, e.g., for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, domestic pets, etc.) and humans. In some embodiments, a subject is a human subject. In some embodiments, a subject is suffering from a disease, disorder, or condition (e.g., cancer and / or a cancer-associated condition). In some embodiments, a subject is susceptible to a disease, disorder, or condition (e.g., cancer and / or a cancer-associated condition). In some embodiments, a subject displays one or more symptoms or characteristics of a disease,Page 29 of 18313406582vlAttorney Docket No.: 2013237-1617disorder, or condition (e.g., cancer and / or a cancer-associated condition). In some embodiments, a subject displays one or more non-specific symptoms of a disease, disorder, or condition (e.g., cancer and / or a cancer-associated condition). In some embodiments, a subject does not display any symptom or characteristic of a disease, disorder, or condition (e.g., cancer and / or a cancer-associated condition). In some embodiments, a subject is someone with one or more features characteristic of susceptibility to or risk of a disease, disorder, or condition (e.g., cancer and / or a cancer-associated condition). In some embodiments, a subject is a patient. In some embodiments, a subject is an individual to whom diagnosis and / or therapy is and / or has been administered.

[0130] Suffering from'. An individual who is “suffering from” a disease, disorder, and / or condition (e.g., cancer and / or a cancer-associated condition) has been diagnosed with and / or displays one or more symptoms of a disease, disorder, and / or condition.

[0131] Susceptible to: An individual who is “susceptible to” a disease, disorder, and / or condition (e.g., cancer and / or a cancer-associated condition) is one who has a higher risk of developing the disease, disorder, and / or condition (e.g., cancer and / or a cancer-associated condition) than does a member of the general public. In some embodiments, an individual who is susceptible to a disease, disorder and / or condition (e.g., cancer and / or a cancer-associated condition) may not have been diagnosed with the disease, disorder, and / or condition (e.g., cancer and / or a cancer-associated condition). In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition (e.g., cancer and / or a cancer-associated condition) may exhibit symptoms of the disease, disorder, and / or condition (e.g., cancer and / or a cancer-associated condition). In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition (e.g., cancer and / or a cancer-associated condition) may not exhibit symptoms of the disease, disorder, and / or condition (e.g., cancer and / or a cancer-associated condition). In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition (e.g., cancer and / or a cancer-associated condition) will develop the disease, disorder, and / or condition (e.g., cancer and / or a cancer-associated condition). In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition (e.g., cancer and / or a cancer-associated condition) will not develop the disease, disorder, and / or condition (e.g., cancer and / or a cancer-associated condition).Page 30 of 18313406582vlAttorney Docket No.: 2013237-1617

[0132] Therapy. The term “therapy” refers to an administration or delivery of an agent or intervention that has a therapeutic effect and / or elicits a desired biological and / or pharmacological effect (e.g., has been demonstrated to be statistically likely to have such effect when administered to a relevant population). In some embodiments, a therapeutic agent or therapy is any substance that can be used to alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and / or reduce incidence of one or more symptoms or features of a disease, disorder, and / or condition (e.g., cancer and / or a cancer-associated condition). In some embodiments, a therapeutic agent or therapy is a medical intervention that can be performed to alleviate, relieve, inhibit, present, delay onset of, reduce severity of, and / or reduce incidence of one or more symptoms or features of a disease, disorder, and / or condition.

[0133] Treat: As used herein, the term “treat,” “treatment,” or “treating” refers to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and / or reduce incidence of one or more symptoms or features of a disease, disorder, and / or condition (e.g., cancer and / or a cancer-associated condition). Treatment may be administered to a subject who does not exhibit signs of a disease, disorder, and / or condition (e.g., cancer and / or a cancer-associated condition). In some embodiments, treatment may be administered to a subject who exhibits only early signs of the disease, disorder, and / or condition (e.g., cancer and / or a cancer-associated condition), for example for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and / or condition. In some embodiments, treatment may be administered to a subject at a later-stage of disease, disorder, and / or condition (e.g., cancer and / or a cancer-associated condition).DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0134] The present disclosure provides, in part, systems and methods for detection of candidate non-neoantigen epitope targets and candidate neoepitope targets (collectively, candidate neotherapeutic target epitopes). System and methods for detecting candidate neotherapeutic target epitopes disclosed herein may employ a variety of data sources, including, e.g., patient tumor DNA, tumor RNA, and germline DNA.Page 31 of 18313406582vlAttorney Docket No.: 2013237-1617

[0135] The present disclosure provides an insight that a modular and flexible system for detection of candidate neotherapeutic target epitopes can provide a highly heterogenous space of possible targets which is also tailored toward an individual patient.

[0136] Accordingly, the disclosure provides and / or utilizes methods of identifying and / or selecting both neoepitopes and non-neoepitopes (e.g., shared tumor antigen epitopes), e.g., for use in a cancer therapy or cancer vaccine.I. Selection of EpitopesA. Detecting Antigen-based Neotherapeutic Target Epitopes (“dANTe”)

[0137] In some embodiments, non-neoantigen epitopes and / or neoantigen epitopes are selected as depicted schematically in FIG. 1 (referred to herein as “dANTe” pipeline).

[0138] In certain embodiments, a method 4100 for selecting neoantigen epitopes may include steps as shown in FIG. 41A. For example, method 4100 can include one or more of: step 4102, comprising obtaining whole exome sequenced matched germline DNA and tumor DNA and tumor RNAseq data; step 4104, comprising performing HLA typing using DNA reads from germline sequences; step 4106, comprising aligning DNA reads to a reference genome; step 4108, comprising detecting mutations in sequences; step 4110, comprising annotating sequences to describe regions / sites of interest; step 4112, comprising computing predicted peptide-HLA I and II binding / presentation scores; step 4114, comprising ranking neoepitope targets based on scores and expression values; and / or step 4116, comprising storing and / or providing the ranked list of neoepitope targets. In some embodiments, method 4100 includes step 4102, comprising obtaining whole exome sequenced matched germline DNA and tumor DNA and tumor RNAseq data; step 4104, comprising performing HLA typing using DNA reads from germline sequences; step 4106; comprising aligning DNA reads to a reference genome; step 4108, comprising detecting mutations in sequences; step 4110, comprising annotating sequences to describe regions / sites of interest; step 4112, comprising computing predicted peptide-HLA I and II binding / presentation scores; step 4114, comprising ranking neoepitope targets based on scores and expression values, and step 4116, comprising storing and / or providing the ranked list of neoepitope targets.Page 32 of 18313406582vlAttorney Docket No.: 2013237-1617

[0139] In certain embodiments, a method 4150 for selecting non-neoantigen epitopes may include steps as shown in FIG.41B. For example, method 4150 can include submethods 4150a and 4150b. In some embodiments, submethod 4150a includes one or more of: step 4151a, comprising providing an input list of tumor-associated antigen targets and threshold expression levels; step 4152a, comprising pre-computing HLA binding for targets on an input list; and / or step 4153a, comprising filtering for transcripts exceeding threshold expression level. In some embodiments, submethod 4150a includes step 4151a, comprising providing an input list of tumor-associated antigen targets and threshold expression levels, followed by step 4152a, comprising pre-computing HLA binding for targets on an input list, followed by step 4153a, comprising filtering for transcripts exceeding threshold expression level. In some embodiments, submethod 4150a includes step 4151a, comprising providing an input list of tumor-associated antigen targets and threshold expression levels, followed by step 4153a, comprising filtering for transcripts exceeding threshold expression level, followed by step 4152a, comprising precomputing HLA binding for targets on an input list. In some embodiments, submethod 4150b includes one or more of: step 4151b, comprising obtaining whole exome sequenced matched germline DNA and tumor DNA and tumor RNAseq data; step 4152b, comprising performing HLA typing using DNA reads from germline sequences; step 4153b, comprising aligning DNA reads to a reference genome; step 4154b, comprising detecting mutations in exonic regions; step 4155b, comprising generating non-neoepitope target sequences; step 4156b, comprising annotating sequences to describe regions / sites of interest; step 4157b, comprising computing predicted peptide-HLA I and II binding / presentation scores; and / or step 4158b, comprising storing and / or providing a list of non-neoepitope targets with scores and expression values. In some embodiments, submethod 4150b includes: step 4151b, comprising obtaining whole exome sequenced matched germline DNA and tumor DNA and tumor RNAseq data; step 4152b, comprising performing HLA typing using DNA reads from germline sequences; step 4153b, comprising aligning DNA reads to a reference genome; step 4154b, detecting mutations in exonic regions; step 4155b, comprising generating non-neoepitope target sequences; step 4156b, comprising annotating sequences to describe regions / sites of interest; step 4157b, comprising computing predicted peptide-HLA I and II binding / presentation scores; and step 4158b, comprising storing and / or providing a list of non-neoepitope targets with scores and expression values, and includes step 4152a, comprising pre-computing HLA binding for targets on the input Page 33 of 18313406582vlAttorney Docket No.: 2013237-1617list, and step 4153a, comprising filtering for transcripts exceeding threshold expression level (e.g., step 4152a before step 4153a or step 4153a before step 4152a) after step 4151b, step 4152b, step 4153b, step 4154b, step 4156b, or step 4157b.

[0140] In some embodiments, non-neoantigen epitopes are selected using a list of tumor-associated antigen gene targets known to be associated with multiple indications (e.g., different cancers). In some embodiments, each of the gene targets on a list of tumor-associated antigen gene targets is associated with a predefined expression threshold (e.g., a level above which the tumor-associated antigen gene target is considered to be expressed in a given patient or sample).

[0141] In some embodiments, expression of each of the genes on a list of tumor-associated antigen gene targets is determined for a patient or sample using, e.g., a method described herein; expression of each of the gene targets is compared to a predefined threshold expression level; and the list of tumor-associated antigen gene targets used to select non-neoantigen epitopes is limited to those gene targets for which expression exceeds the respective predefined threshold.

[0142] In some embodiments, selection of non-neoantigen epitopes and / or neoantigen epitopes includes a step of whole exome sequencing and / or RNA sequencing of tumor cells and / or normal (i.e., non-cancerous) cells from a subject.

[0143] In some embodiments, selection of non-neoantigen epitopes and / or neoantigen epitopes includes a step of HLA typing or HLA calling (using, e.g., a method described herein).

[0144] In some embodiments, selection of non-neoantigen epitopes and / or neoantigen epitopes includes a step of detecting one or more somatic mutations (e.g., a single nucleotide variant (SNV) and / or an InDei) using, for example, a method described herein.

[0145] In some embodiments, selection of non-neoantigen epitopes and / or neoantigen epitopes includes a step of detecting one or more germline variants (using, e.g., a method described herein). In some embodiments, germline variants are detected by computation using results of a preceding step (e.g., in the vicinity of detected somatic mutations or within exonic regions of an exemplary input list of tumor-associated antigen gene targets).

[0146] In some embodiments, selection of non-neoantigen epitopes and / or neoantigen epitopes includes a step of detecting gene fusions using, e.g., a method described herein. In somePage 34 of 18313406582vlAttorney Docket No.: 2013237-1617embodiments, selection of non-neoantigen epitopes and / or neoantigen epitopes includes a step of deprioritizing target sequences in which gene fusions were detected.

[0147] In some embodiments, selection of non-neoantigen epitopes and / or neoantigen epitopes includes a step of annotating non-neoantigen epitopes and / or neoantigen epitope sequences with mutational information obtained using one or more steps described herein.

[0148] In some embodiments, step of determining expression of each of the genes on a list of tumor-associated antigen gene targets, as described herein, is performed before sequencing and / or mutational analysis. In some embodiments, step of determining expression of each of the genes on a list of tumor-associated antigen gene targets, as described herein, is performed after sequencing and / or mutational analysis.

[0149] In some embodiments, selection of non-neoantigen epitopes includes a step of deriving peptide non-neoantigen sequences by generating all possible peptide combinations based on detected mutation events and corresponding phasing of relevant alleles, using, e.g., a method described herein. In some embodiments, peptide non-neoantigen sequences containing a somatic mutation are discarded.

[0150] In some embodiments, selection of non-neoantigen epitopes and / or neoantigen epitopes includes a step of predicting HLA binding and presentation using a predictive model with expression and cleavage features. In some embodiments, HLA binding predictions may be precomputed for known peptide-allele combinations (e.g., those associated with tumor-associated antigen gene targets and / or known alleles), and precomputed binding predictions may be obtained by querying the precomputed binding predictions. In some embodiments, HLA binding predictions may be computed “on-line” by a predictive model (e.g., for unknown alleles and / or mutations).

[0151] In some embodiments, predicting HLA binding and presentation includes a step of creating a Lookup table of precomputed peptide-MHC binding scores for selecting non-neoantigen epitopes using a list of tumor-associated antigen gene targets. In some embodiments, querying a Lookup table to obtain a precomputed peptide-MHC binding score reduces computational time and / or expense associated with prediction of peptide-MHC binding scores used for selecting non-neoantigen epitopes. In some embodiments, a Lookup table used for selecting non-neoantigen epitopes contains precomputed peptide-MHC binding scores for Page 35 of 18313406582vlAttorney Docket No.: 2013237-1617peptide-allele combinations derived from a list of tumor-associated antigen gene targets and frequently occurring alleles in the human population.

[0152] In some embodiments, a selected non-neoantigen epitope is validated by comparison of its sequence to a reference sequence, such as a corresponding exonic reference transcript sequence. In some embodiments, successful validation by comparison of a non-neoantigen epitope sequence to a reference sequence includes up to two mismatches (e.g., to consider single nucleotide polymorphisms (SNPs) in the non-neoantigen epitope sequence).

[0153] In some embodiments, selection of neoantigen epitopes includes a step of ranking neoantigen epitope sequences based on MHC I and / or MHC II presentation and / or expression values. In some embodiments, selection of neoantigen epitopes includes a step of selecting a group of epitopes in which some epitopes have InDeis and some epitopes have SNVs. In some embodiments, selection of neoantigen epitopes includes a step of selecting a group of epitopes in which some epitopes have high MHC I presentation scores and some epitopes have high MHC II presentation scores.

[0154] In some embodiments, a selected neoantigen epitope is validated by comparison of its sequence to a reference proteome. In some embodiments, a selected neoantigen epitope is discarded (i.e., no longer considered selected) when a full match to its sequence is found in a reference proteome.B. Selecting Antigen-based Neotherapeutic Target Epitopes (“sANTe”)

[0155] In some embodiments, an additional framework may be applied to further select epitopes. For example, once antigens of a subject are selected (e.g., from genomic data from the subject) (e.g., using “dANTe” framework described herein), a framework referred to herein as Selecting Antigen-based Neotherapeutic Target Epitopes (“sANTe”) may be applied to further select epitopes, e.g., for inclusion in a vaccine.

[0156] In certain embodiments, a method 4200 for selecting polynucleotide sequences of antigens for inclusion in a vaccine (e.g., for a cancer therapy) may include the following steps as shown in FIG. 42. At step 4202, a candidate epitope list (e.g., candidate polynucleotide sequences) identifying a plurality of candidate epitopes and MHC presentation and immunogenicity data may be obtained. For each candidate epitope of the plurality of candidatePage 36 of 18313406582vlAttorney Docket No.: 2013237-1617epitopes, the MHC presentation and immunogenicity data may comprise: (i) a corresponding MHC presentation score representing a known and / or predicted likelihood and / or strength of binding between the particular candidate epitope and an MHC of the subject; and (ii) a corresponding immunogenicity score representing a known and / or predicted immunogenicity of the particular candidate epitope. At step 4204, for each candidate epitope, a corresponding rank based at least in part on the MHC presentation and immunogenicity data may be determined. At step 4206, a subset of the plurality of candidate epitopes may be selected based at least in part on the corresponding rank as a set of target epitopes. At step 4208, the set of target epitopes may be stored and / or provided (e.g., for display and / or further processing).1. Candidate Polynucleotide Sequences and Immune Response Data

[0157] Each candidate of a plurality of candidate polynucleotide sequences may comprise: DNA sequence, RNA sequence, polypeptide sequence, or any combination thereof. Each candidate of a plurality of candidate polynucleotide sequences may comprise: gene associated data (e.g., a gene identifier for a gene from to which the candidate belongs to), transcript associated data (e.g., a transcript identifier for a transcript from which the candidate originates from), proteome associated data (e.g., values associated with matching at least a part of the candidate to a proteome data of a subject, a Boolean value describing whether the candidate matches to a proteome data, a Boolean value describing whether a candidate within 3 positions upstream or downstream matches to a proteome data), or any combination thereof.

[0158] Each candidate of a plurality of candidate polynucleotide sequences may comprise expected (e.g., as derived from the literature and experiments) and / or predicted (e.g., as determined in silico) immunogenicity associated data (e.g., related to immune response processes, e.g., cleavage, HLA binding, expression, presentation). For example, immunogenicity associated data may comprise an immunogenicity tier (e.g., from highest to lowest evidence), gene expression associated data (e.g., expression value in transcripts per million of the associated gene, gene expression cut-off), associated allele data (e.g., allele name, major histocompatibility complex (MHC) class), HLA binding associated data (e.g., predicted percentile rank, cut-off value for the binding percentile rank), presentation at a cellular surface associated data (e.g., predicted presentation percentile rank), or any combination thereof. The HLA binding associatedPage 37 of 18313406582vlAttorney Docket No.: 2013237-1617data may be used to bin each candidate of a plurality of candidate polynucleotide sequences into various binding categories (e.g., strong, intermediate, and weak).

[0159] In certain embodiments, methods and systems of the present disclosure may evaluate and / or select candidate polynucleotide sequences, for example, for inclusion in a polyepitopic construct by analyzing, for each of a plurality of candidate polynucleotide sequences, corresponding polypeptide sequences and / or associated data such as one or more of MHC presentation data, immunogenicity data, and expression data. A candidate polynucleotide sequence may be, for example, a DNA sequence or an RNA sequence that encodes a particular candidate peptide epitope, such as a neoantigen epitope and / or a non-neoantigen epitope.

[0160] Accordingly, (e.g., computer-implemented) methods and systems of the present disclosure, may obtain a candidate epitope list that identifies a plurality of candidate epitopes, rank, and select particular candidate epitopes to include in a construct. As described herein, candidate epitopes selected for inclusion in a construct may, for example, be encoded by a polynucleotide construct. In certain embodiments, ranking and selection of candidate epitopes may be performed based on polypeptide sequences of candidate epitopes, polynucleotide sequences encoding particular candidate epitopes, as well as additional data, such as MHC presentation data, immunogenicity data, and / or expression data.

[0161] Accordingly, in certain embodiments, a candidate epitope list obtained and evaluated via methods and systems described herein may represent individual candidate epitopes, via one or more of: a polypeptide sequence (e.g., of a peptide epitope), a polynucleotide sequence (e.g., a DNA and / or RNA sequence encoding a peptide epitope), and a (e.g., unique) identifier, such as an alphanumeric code that identifies a transcript and / or a portion thereof (e.g., a UCSC transcript ID and, optionally, a e.g., number identifying a starting position of a peptide within a sequence window). A candidate epitope list may comprise, for each of a plurality of candidate epitopes, one or more of a polypeptide sequence, polynucleotide sequence, and an identifier. In certain embodiments, any of the aforementioned identifying information for candidate epitopes may be stored in one or more data tables, such that a candidate epitope list may comprise or point to (e.g., reference memory addresses of), for each candidate epitope, one or more corresponding data tables.Page 38 of 18313406582vlAttorney Docket No.: 2013237-1617

[0162] In certain embodiments, a candidate epitope list identifies or comprises MHC presentation data for a particular candidate epitope. In certain embodiments, for a particular candidate epitope, MHC presentation data comprises a corresponding MHC presentation score that represents a known and / or predicted likelihood, and / or strength of binding between the particular candidate epitope and an MHC molecule of the subject (e.g., under an assumption that peptides undergo an intracellular processing stage). In certain embodiments, a predicted strength or likelihood of binding between a candidate epitope and an MHC molecule includes or accounts for impact of intracellular processing steps (e.g., peptide cleavage), for example accounting for multiple steps in MHC presentation. In certain embodiments, for a particular candidate epitope, an MHC presentation score may classify the particular candidate epitope as belonging to one of a (e.g., predefined) number of categories. These categories may include, for example, (i) a known epitope class, such as a known T-cell epitope, (ii) a known ligand class, and (ii) one or more predicted binding level classes, such as strong, intermediate, and weak. In certain embodiments, additionally or alternatively, a MHC presentation score is a value, such as a numerical value (e.g., a floating point number) that quantifies a predicted and / or measured level of MHC binding (e.g., under an assumption that peptides undergo an intracellular processing stage). In certain embodiments, a MHC presentation score is a value, such as a numerical value, that quantifies a binding likelihood (e.g., under an assumption that peptides undergo an intracellular processing stage).

[0163] In certain embodiments, a candidate epitope list identifies immunogenicity data for a particular candidate epitope. In certain embodiments, for a particular candidate epitope, immunogenicity data comprises a corresponding immunogenicity score representing a known and / or predicted immunogenicity of the particular candidate epitope. In some embodiments, an immunogenicity score for a particular candidate epitope may be an associated immunogenicity categorization, such as an immunogenicity tier that classifies the particular candidate epitope according to a level of evidence for immunogenicity. In certain embodiments, immunogenicity tiers may be selected based on a manually curated categorization of genes into different groups of expected immunogenicity. A particular candidate epitope may, accordingly, be associated with (e.g., assigned) an immunogenicity tier (e.g., a manually curated categorization) of the gene that encodes it. Examples of certain immunogenicity tiers are described in further detail below.Page 39 of 18313406582vlAttorney Docket No.: 2013237-1617

[0164] In certain embodiments, a candidate epitope list identifies expression data for a particular candidate epitope. In certain embodiments, for a particular candidate epitope, expression data comprises a corresponding expression score representing a measured expression in the subject. An expression score for a particular candidate epitope may be a measured expression value (e.g., in transcripts per million, TPM) of a gene from which a transcript encoding the candidate epitope (e.g., encoding an antigen comprising the candidate epitope) derives. In certain embodiments, an expression score for a particular candidate epitope may be a relative value, for example, based at least in part on a gene-specific expression cutoff. For example, measured expression values for candidate epitopes originating (e.g., encoded by) a particular gene may be evaluated, for example normalized by, a gene-specific cutoff value for that particular gene. In this way, in certain embodiments, an expression fold change may be determined for a particular candidate epitope as the measured expression (e.g., in TPM) divided by the gene-specific cutoff value (e.g., of the gene encoding that particular candidate epitope). Gene-specific cutoff values may vary from gene to gene, and may be obtained from a list or table (e.g., internal data, curated from experiments, literature, etc.).2. Ranking and Filtering Candidate Polynucleotide SequencesFiltering Steps

[0165] In certain embodiments, a plurality of candidate epitopes, such as candidate polynucleotide sequences, can be filtered using proteome data from a subject. Proteome data may be, for example, a reference proteome, such as a human reference proteome. For example, epitope match in the proteome data may be related to a certain degree of abundance of the epitope in healthy tissue. For each candidate of at least a part of a plurality of candidate polynucleotide sequences, the candidate may be filtered (e.g., removed from subsequent analysis) if an associated polypeptide sequence (e.g., at least a part of it) matches to a sequence (e.g., 8-mer) from proteome data. The proteome data may be (e.g., manually, automatically) curated (e.g., processed) (e.g., to exclude a gene specific whitelist that contains, e.g., homologous sequences including sequences from annotated pseudogenes).

[0166] For example, as described herein, a candidate epitope list may identify or comprise, for each candidate epitope, a polynucleotide sequence along with an associated polypeptide sequence (e.g., encoded by the polynucleotide sequence). In certain embodiments, for a Page 40 of 18313406582vlAttorney Docket No.: 2013237-1617candidate epitope, an associated polypeptide sequence (e.g., of the epitope) may be compared with sequences (e.g., polypeptide sequences) from proteome data for the subject (e.g., a human reference proteome and / or a curated / modified portion thereof, e.g., that excludes a genespecific whitelist that includes (e.g., manually) curated homologous sequences including sequences from annotated pseudogenes). In certain embodiments, if particular candidate epitope’s associated polypeptide sequence is determined to match a sequence from proteome data, the particular candidate epitope may be filtered, e.g., so as to exclude it from further consideration. Matches between associated polypeptide sequences and proteome data may be determined, for example, by comparing the associated polypeptide sequence with subsequences, e.g., of polypeptides, from the proteome data.

[0167] In certain embodiments, one or more candidate epitopes are checked against potency linker sequences and, e.g., filtered / excluded if they match one or more potency linker sequences. A potency linker comprises a peptide sequence that can be detected in a potency assay (a potency linker sequence), for example as described in PCT Publication No.WO / 2024 / 180054 “Linker Sequence Potency Assays for Multiple Coding Nucleic Acids,” published September 6, 2024, the content of which is incorporated by reference herein in its entirety. Accordingly, in certain embodiments, filtering candidate epitopes to exclude those that comprise a potency linker sequence avoids the peptide sequence that is detected in the potency assay (e.g., the potency linker sequence) appearing in other, e.g., unintended parts of a construct (e.g., the candidate epitope sequence). For example, presence of potency linker sequences outside of a potency linker, e.g., in a candidate epitope itself, could hinder the ability to unambiguously determine potency (e.g., protein abundance in an assay), e.g., as described in PCT Publication No. WO / 2024 / 180054 “Linker Sequence Potency Assays for Multiple Coding Nucleic Acids,” published September 6, 2024. In certain embodiments, a combination of multiple candidate epitopes, such as in a combined target cluster as described in further detail herein, are compared against potency linker sequences. In certain embodiments, if at least a portion of a combined target cluster is identified as matching a potency linker sequence, the identified target cluster may be resolved (e.g., separated) into its single, constituent, clusters.

[0168] In certain embodiments, candidate epitopes are compared against an epitope exclusion list. An epitope exclusion list may comprise a listing of (e.g., a plurality of) epitopes previouslyPage 41 of 18313406582vlAttorney Docket No.: 2013237-1617(e.g., repeatedly) identified as non-immunogenic. Accordingly, certain candidate epitopes may be identified as matching a non-immunogenic epitope of the epitope exclusion list and excluded from further analysis. For example, in certain embodiments, candidate epitopes matching a non-immunogenic epitope of the epitope exclusion list may have their corresponding MHC prediction score set to -1, e.g., thereby identifying them as to be excluded.

[0169] Candidate epitopes may be matched against other sequences, e.g., in proteome data, potency linker sequences, epitope sequences of an epitope exclusion list, and the like, alone or in combination with a linker sequence.Ranking Candidates

[0170] In certain embodiments, a plurality of candidate polynucleotide sequences comprises a plurality of candidate cores. Each core of a plurality of candidate cores may be defined as an overlap of each of the plurality of candidate polynucleotide sequences with “known” epitopes, corresponding “known” HLA ligands, epitope-allele pairs corresponding to the strongest binding. In other words, candidate cores may correspond to parts of candidate sequences that are associated with the strongest binding.

[0171] In certain embodiments, a plurality of candidate polynucleotide sequences is ranked according to associated immunogenicity tier, HLA binding associated data, “known” epitopes data, “known” ligands data, gene expression level, or any combination thereof. The “known” epitopes data may refer to a list of epitopes (e.g., obtained from public, such as immune epitope database (IEDB), and private results) with evidence for immunogenicity (e.g., evidence that epitope specific T cells kill tumor cell lines or autologous tumor cells that endogenously express associated antigen, evidence that epitope specific T cells kill target cells transduced / transfected with associate antigen and are reported in more than one publication, experimental immunogenicity data with ex vivo enzyme-linked immunospot assay (ELISpot) and multimer data or T-cell receptor data). Certain epitopes may be excluded from the list of epitopes (e.g., negative experimental data, assay validity is questionable, only reported in one publication). The “known” ligands data may refer to a list of MHC class I and II ligands (e.g., obtained from public and private mass spectrometry data). The list of MHC ligands may be filtered to exclude ligand-allele pairs with low binding.Page 42 of 18313406582vlAttorney Docket No.: 2013237-1617

[0172] In certain embodiments, corresponding ranking comprises dividing a plurality of candidate polynucleotide sequences 4302 into batches 4304a, 4304b, and 4304c, as shown in FIG. 43. A batch may comprise candidate polynucleotide sequences with a same immunogenicity tier. A batch may comprise candidate polynucleotide sequences with two consecutive immunogenicity tiers. A batch may comprise candidate polynucleotide sequences with “known” epitopes. A batch may comprise candidate polynucleotide sequences with “known” ligands. A batch may comprise candidate polynucleotide sequences with a same binding category.

[0173] Batches (e.g., with associated candidate polynucleotide sequences) may be ranked by the associated tiers. Batches may be ranked by “known” epitopes, “known” ligands, and associated binding categories, for example, as shown in FIG.23.

[0174] For example, as described herein, for a particular candidate epitope, an MHC presentation score may classify the particular candidate epitope as (i) a known epitope, such as a known T-cell epitope, (ii) a known ligand, or may categorize and / or (iii) quantify a level of a peptide-MHC presentation (e.g., predicted MHC -I or MHC -II binding). A plurality of candidate epitopes may, accordingly, be subdivided into batches according to their corresponding MHC presentation scores. Each batch may comprise those candidate epitopes having a same MHC presentation score. For example, one or more batches may comprise candidate epitopes whose MHC presentation score classifies them as known epitopes, one or more batches may comprise candidate epitopes whose MHC presentation score classifies them as known ligands, and one or more batches may comprise candidate epitopes having a predicted peptide-MHC binding level within a particular range or range of relative values (e.g., a particular percentile).

[0175] In certain embodiments, subdividing candidate epitopes into batches may be used to rank and / or select candidate epitopes, for example for inclusion in a poly epitopic construct. For example, candidate epitopes classified as known epitopes may be ranked (e.g., so as to be preferentially selected) above candidate epitopes classified as known ligands. In certain embodiments, candidate epitopes classified as known epitopes and / or known ligands may be ranked (e.g., so as to be preferentially selected) above other candidate epitopes, e.g., not identified as known epitopes and / or known ligands.Page 43 of 18313406582vlAttorney Docket No.: 2013237-1617

[0176] In certain embodiments, candidate epitopes may be associated with (e.g., assigned to) a tier (e.g., an immunogenicity tier) that categorizes an immunogenicity of a candidate epitope, for example based on immunogenicity data. For example, a particular candidate epitope may be associated with a particular one of a finite, discrete, number of tiers based on a level of evidence of immunogenicity, safety, and / or tumor cell killing. For example, as described in further detail herein, e.g., in Example 2, candidate epitopes may be associated with one of five tiers based on evidence collected in internal and / or reported trials. In certain embodiments, other numbers of tiers may be used, for example two tiers, five tiers, ten tiers, etc.

[0177] In certain embodiments, various antigens, such as shared tumor antigens, may be associated with a particular tier and thereby used to associate candidate epitopes with corresponding tiers. For example, a particular antigen may be associated with a specific tier and, accordingly, candidate epitopes of that particular antigen may be thereby associated with that same specific tier.

[0178] In certain embodiments, antigens and / or candidate epitopes thereof are associated with one of five tiers. In certain embodiments, a first tier (e.g., tier 1) comprises those antigens and / or candidate epitopes thereof having (e.g., characterized by) a highest evidence for immunogenicity and / or safety in clinical trials, as well as, in certain embodiments, evidence for tumor cell killing. In certain embodiments, a second tier (e.g., tier 2) comprises cancer-testis and / or tumor specific antigens, and / or candidate epitopes thereof, with evidence for immunogenicity and / or differentiation antigens and / or candidate epitopes thereof with observed strong immunogenicity. In certain embodiments, a third tier (e.g., tier 3) comprises differentiation antigens and / or candidate epitopes thereof with evidence for immunogenicity. In certain embodiments, a fourth tier (e.g., tier 4) comprises cancer-testis antigens, tumor specific antigens, and / or differentiation antigens without evidence for immunogenicity. In certain embodiments, a fifth tier (e.g., tier 5) comprises tumor associated antigens with evidence for immunogenicity, but broader normal tissue expression pattern. In certain embodiments, candidate epitopes are ranked based at least in part on their associated tier. For example, candidate epitopes associated with the first tier (e.g., tier 1 candidate epitopes) may be ranked above candidate epitopes associated with the second tier (e.g., tier 2 candidate epitopes), which in turn may be ranked above candidate epitopes associated with the third tier (e.g., tier 3 candidate epitopes), and so on.Page 44 of 18313406582vlAttorney Docket No.: 2013237-1617

[0179] In certain embodiments, antigens (e.g., shared tumor antigens) and / or candidate epitopes thereof (e.g., shared tumor antigen epitopes) may be assigned to particular tiers based at least in part on a particular indication, such as a cancer type, determined for a particular subject. In certain embodiments, antigens and / or candidate epitopes thereof may be assigned to tiers based on the genes that encode them.

[0180] For example, in certain embodiments a subject is determined to have lung squamous cell carcinoma (LUSC). In certain embodiments, a subject is determined to have LUSC and a first tier of antigens comprises an antigen encoded by a MAGEA3. In certain embodiments, a subject is determined to have LUSC and a second tier of antigens comprises one or more antigens encoded by one or more of the following: ACTL8, CTAG2, CXorf61, MAGEA1, MAGEA2, MAGEA4, MAGEA9B, MAGEC1, MAGEC2, PLAC1. In certain embodiments, a subject is determined to have LUSC and a third tier of antigens comprises one or more antigens encoded by one or more of the following: CEACAM5, IGF2BP3, MLANA, and PRAME. In certain embodiments, a subject is determined to have LUSC and a fourth tier of antigens comprises antigens encoded by one or more of the following: BRDT, IGF2BP1, PAEP, and ZFP42. In certain embodiments, a subject is determined to have LUSC and a fifth tier of antigens comprises antigens encoded by one or more of the following: BIRC5, DEPDC1, FLT1, KDR, KIF20A, TP53, and TPBG

[0181] In certain embodiments, a subject is determined to have ovarian serous cystadenocarcinoma (OV). In certain embodiments, a subject is determined to have OV and a first tier of antigens comprises an antigen encoded by a CTAG1 A. In certain embodiments a subject is determined to have OV and a second tier of antigens comprises one or more antigens encoded by one or more of the following: ACTL8, CLDN6, CTAG2, MAGEA1, MAGEA4, MAGEA9B, MAGEC2, PLCA1, and TPTE. In certain embodiments a subject is determined to have OV and a third tier of antigens comprises one or more antigens encoded by one or more of the following: IGF2BP3 and PRAME. In certain embodiments a subject is determined to have OV and a fourth tier of antigens comprises antigens encoded by one or more of the following: PAEP and ZFP42. In certain embodiments, a subject is determined to have OV and a fifth tier of antigens comprises antigens encoded by one or more of the following: BIRC5, DEPDC1, FLT1, KDR, KIF20A, TP53, and TPBG.Page 45 of 18313406582vlAttorney Docket No.: 2013237-1617

[0182] In certain embodiments, a subject is determined to have prostate adenocarcinoma (PRAD). In certain embodiments, a subject is determined to have PRAD and no antigens and / or candidate epitopes thereof are associated with a first tier. In certain embodiments a subject is determined to have PRAD and a second tier of antigens comprises an antigen encoded by a H0XB13. In certain embodiments a subject is determined to have PRAD and a third tier of antigens comprises one or more antigens encoded by one or more of the following: ACPP, KLK2, KLK3, NKX3-1, and PRAME. In certain embodiments a subject is determined to have PRAD and a fourth tier of antigens comprises one or more antigens encoded by one or more of the following: PAGE4 and TDRD1. In certain embodiments a subject is determined to have PRAD and a fifth tier of antigens comprises antigens encoded by one or more of the following: BIRC5, DEPDC1, FLT1, KDR, KIF20A, TP53, and TPBG.

[0183] In certain embodiments, a subject is determined to have skin cutaneous melanoma (SKCM). In certain embodiments a subject is determined to have SKCM and a first tier of antigens comprises an antigen encoded by one or more of the following: CTAG1 A and MAGEA3. In certain embodiments a subject is determined to have SKCM and a second tier of antigens comprises one or more antigens encoded by one or more of the following: ACTL8, CTAG2, CXorf61, MAGEA1, MAGEA2, MAGEA4, MAGEA9B, MAGEC1, MAGEC2, and TPTE. In certain embodiments a subject is determined to have SKCM and a third tier of antigens comprises one or more antigens encoded by one or more of the following: IGF2BP3, MLANA, PMEL, PRAME, and TYR. In certain embodiments a subject is determined to have SKCM and a fourth tier of antigens comprises antigens encoded by one or more of the following: PAEP and TDRD1. In certain embodiments, a subject is determined to have SKCM and a fifth tier of antigens comprises antigens encoded by one or more of the following: BIRC5, DEPDC1, FLT1, KDR, KIF20A, TP53, and TPBG

[0184] In certain embodiments, a subject is determined to have triple negative breast carcinoma (TNBC). In certain embodiments a subject is determined to have TNBC and a first tier of antigens comprises an antigen encoded by one or more of the following: CTAG1 A and MAGEA3. In certain embodiments a subject is determined to have TNBC and a second tier of antigens comprises one or more antigens encoded by one or more of the following: ACTL8, CLDN6, CTAG2, CXorf61, MAGEA2, MAGEA4, MAGEA9B, MAGEC1, MAGEC2, andPage 46 of 18313406582vlAttorney Docket No.: 2013237-1617TPTE. In certain embodiments a subject is determined to have TNBC and a third tier of antigens comprises one or more antigens encoded by one or more of the following: ANKRD30A, GUCY2C, IGF2BP3, MLANA, and PRAME. In certain embodiments a subject is determined to have TNBC and a fourth tier of antigens comprises antigens encoded by one or more of the following: EDDM3B, TDRD1, and ZFP42. In certain embodiments, a subject is determined to have TNBC and a fifth tier of antigens comprises antigens encoded by one or more of the following: BIRC5, DEPDC1, FLT1, KDR, KIF20A, TP53, and TPBG

[0185] In certain embodiments, candidate epitopes may be subdivided into a plurality of batches based on their corresponding MHC presentation scores and associated tiers. In this way, in certain embodiments, each batch may be associated with one or more tiers, each tier associated with and comprising candidate epitopes having a particular immunogenicity categorization. In certain embodiments, each batch is associated with up to two (e.g., one or two) tiers.

[0186] In certain embodiments, candidate epitopes are ranked according to batches, tiers, and / or combinations thereof. For example, a first batch-tier combination may comprise candidate epitopes classified as known T-cell epitopes and assigned to a first tier, e.g., comprising those antigens and / or candidate epitopes thereof having (e.g., characterized by) a highest evidence for immunogenicity and / or safety in clinical trials, as well as, in certain embodiments, evidence for tumor cell killing. A second batch-tier combination may comprise candidate epitopes classified as known ligands and assigned to the first tier [e.g., tier 1, comprising those antigens and / or candidate epitopes thereof having (e.g., characterized by) a highest evidence for immunogenicity and / or safety in clinical trials, as well as, in certain embodiments, evidence for tumor cell killing], A third batch-tier combination may comprise candidate epitopes classified as strong predicted binders, e.g., according to a predicted MHC binding value (e.g., 0.5 or less percentile) and assigned to the first tier. A fourth batch-tier combination may comprise candidate epitopes that are classified as known T-cell epitopes and associated with a second and third tier (e.g., tiers 2 and 3). A fifth batch-tier combination may comprise candidate epitopes that are classified as known ligands and associated with a second and third tier (e.g., tiers 2 and 3). A sixth batch-tier combination may comprise candidate epitopes that classified strong predicted binders and associated with a second and third tier (e.g., tiers 2 and 3).Page 47 of 18313406582vlAttorney Docket No.: 2013237-1617

[0187] In this way, in certain embodiments, batch-tier combinations may be used to prioritize candidate epitopes based on (i) MHC presentation data - e.g., evidence and / or predictions of MHC presentation (e.g., binding) and (ii) immunogenicity data - e.g., evidence of immunogenicity. For example, for candidate epitopes associated with a same particular tier, and, accordingly, a same level of favorable immunogenicity evidence, those candidate epitopes having more favorable MHC presentation scores and / or classifications (e.g., known T-cell epitopes) may be ranked ahead of others.

[0188] In certain embodiments, additionally or alternatively, candidate epitopes are ranked based at least in part on their corresponding expression scores. For example, in certain embodiments, within each batch-tier combination, candidate epitopes may be ranked based at least in part on their expression scores such as, for each candidate epitope, a corresponding expression fold change. In certain embodiments, for example within a batch-tier combination, candidate epitopes may be split into two or more groups based on their expression scores, in certain embodiments, in comparison with a median expression fold change calculated across potential target genes. For example, within a batch-tier combination, candidate epitopes may be split into two groups based on their expression fold change. For example, a median expression fold change across all potential target genes may be determined, and candidate epitopes having an expression fold change greater than or equal to the median ranked first (e.g., in a first group). In certain embodiments, within each expression group, candidate epitopes may be ranked based on one or more of their peptide-MHC prediction values, expression fold changes, and transcript windows, and / or combinations thereof.Target Cores and Clusters

[0189] In certain embodiments, each core of a plurality of candidate cores is extended by an overlap of at least one amino acid between the core and a plurality of candidate polynucleotide sequences. As such, a core may be extended on each terminus as shown in FIG.24. Without wishing to be bound to any theory, longer candidate sequences may be preferrable, for example, to aid in overcoming immune tolerance, improving stability of a final construct, and / or being associated with a stronger immunogenicity. A core with extensions may need to satisfy peptide-MHC prediction threshold of a given batch (e.g., and in case of batches with “known” epitopes and “known” ligands, a threshold of strong binders may be assumed). A core may be checked to Page 48 of 18313406582vlAttorney Docket No.: 2013237-1617not exceed a length of 40 amino acids. Extensions may be checked to be derived from a same transcript window as a core.

[0190] In certain embodiments, each core of a plurality of candidate cores is extended by flanks (e.g., on each terminus, e.g., by up to three flanking amino acids).

[0191] In certain embodiments, two cores of a plurality of candidate cores are combined if the two cores overlap by at least one amino acid and correspond to a same transcript window. Only cores from a same batch may be considered for combining. Only cores from a same batch and with a same binding category may be considered for combining. A core with a best ranking out of two cores may determine ranking of a combined core. If a length of a combined core is longer than 40 amino acids, the combined core may be split (e.g., preserving original cores without extensions).

[0192] In certain embodiments, a plurality of candidate polynucleotide sequences is filtered to remove any duplicates (e.g., partial duplicates) (e.g., by collapsing two or more candidates into one candidate while preserving associated data) [e.g., while keeping candidates with a best parameter (e.g., rank, predicted presented ligands, peptide-MHC prediction value, expression value)]. For example, identical candidates from different transcripts of a same gene may be collapsed into one candidate while keeping data about associated transcripts. For example, identical candidates or candidates with identical cores may be discarded per antigen while keeping a candidate with a best parameter (e.g., rank, predicted presented ligands, peptide-MHC prediction value, expression value). For example, each candidate of the plurality of candidate polynucleotide sequences that is a substring of another candidate (e.g., of a same antigen) may be filtered out.

[0193] In certain embodiments, a plurality of candidate polynucleotide sequences is filtered using potency linker data.

[0194] For example, in certain embodiments, a first candidate epitope is selected as a target core. In certain embodiments, one or more flanking amino acids are added at one or both ends of a polypeptide sequence of a target core. In certain embodiments, a target core is extended by addition of ligands to create a target cluster. For example, a polypeptide sequence of a target core may be compared with polypeptide sequences of other candidate epitopes, for example within a particular batch and / or batch-tier combination. In certain embodiments, one or more Page 49 of 18313406582vlAttorney Docket No.: 2013237-1617ligands - e.g., other candidate epitopes - are identified as overlapping with (e.g., by at least one amino acid) the particular candidate epitope, and the (e.g., polypeptide sequence) of the particular target core can be extended to include the identified one or more ligands to form a target cluster. In certain embodiments, one or more ligands are also identified as belonging to (e.g., derived from) a same transcript as the candidate epitope selected as the target core. In certain embodiments, extension of a target core by addition of one or more ligands is checked to ensure that a resultant cluster’s polypeptide sequence is less than a threshold number of amino acids, for example 40 amino acids in length or less.

[0195] In certain embodiments, two or more target cores and / or target clusters may be combined if the two cores and / or clusters overlap by at least one amino acid and correspond to (e.g., are derived from) a same transcript window.Selecting for Antigen Diversity

[0196] In certain embodiments, a plurality of candidate polynucleotide sequences is ranked based on variety of associated antigens. Variety of antigens may be associated with a stronger immune response due to, for example, broadened immune response and a higher chance of overcoming immune tolerance. For example, if N (e.g., two) candidates 4402 in a batch 4404 correspond to a same antigen, remaining candidates corresponding to the same antigen may be downranked to an end of the batch as shown in FIG. 44A. For example, if K (e.g., two, three) candidates 4412 in a batch 4414a correspond to a same antigen and correspond to “known” epitopes or “known” ligands, remaining candidates corresponding to the same antigen may be downranked to an end of another batch (e.g., tier three, tier five) as shown in FIG. 44B. The values of N, K may be associated with a number of antigens in a specific set of batches.

[0197] For example, as described herein, methods and systems of the present disclosure may select candidate epitopes (e.g., individual candidate epitopes, e.g., target clusters formed via combination of multiple candidate epitopes) for inclusion in a construct, such as a polyepitopic construct as described herein, to provide for epitopes derived from a variety of antigens. For example, in certain embodiments, if more than a threshold number of candidate epitopes and / or clusters from a same antigen are selected, a rank of remaining candidate epitopes and / or target clusters from the same antigen may be updated, for example downranked, to prioritize selectionPage 50 of 18313406582vlAttorney Docket No.: 2013237-1617of candidate epitopes (e.g., target clusters comprising multiple candidate epitopes) from other, different antigens.3. Selecting Candidate Polynucleotide Sequences

[0198] In certain embodiments, for each candidate of a plurality of candidate polynucleotide sequences 4502, a placement cost is determined for candidate placement 4508 in a vaccine construct 4506 as shown in FIG.45. For each candidate, a placement cost represents an array of values 4504 associated with a potential candidate placement position in a vaccine construct. The placement cost may be associated with a rank of a candidate. For example, a placement cost may be determined as a rank of a candidate multiplied by 50. The placement cost may be associated with a length of a candidate and a placement position on a vaccine construct. For example, if a length of a candidate is below 17 amino acids, one is added to candidate placement cost for positions three and six. For example, if a candidate length exceeds 27 amino acids, one is added for every additional amino acid for a first and a last placement positions. The placement cost may be associated with linkers 4510 (e.g., in between candidates, before a first place, after a last place). For example, linkers at specific positions may be pre-defined and incompatible with certain candidates. A candidate at positions with an incompatible linker may receive a placement cost of 100,000.

[0199] In certain embodiments, for each candidate of a plurality of candidate polynucleotide sequences, compatibility with various linkers is determined. For example, a combination of a linker - candidate - linker may be checked to contain 8-mer of a homology to maximum expression risk genes. A candidate without any allowable linker may be discarded.

[0200] In certain embodiments, a length of a vaccine construct is 1,282 base pairs. In certain embodiments, a minimum number of candidates for a vaccine is eight. In certain embodiments, a maximum number of candidates for a vaccine is determined by linker availability. For example, a maximum number of candidates may be 18. If a total number of candidates in a selected subset is less than 8, a construction of a vaccine construct may not be attempted. If a total number of candidates in a selected subset is less than 8 and at least three candidates correspond to different cores, the at least three candidates may be duplicated in alternative scheme based on their ranking until eight candidates are reached (e.g., 1-2-3-1-2-3-1-2). APage 51 of 18313406582vlAttorney Docket No.: 2013237-1617placement cost of 1000 may be added to a first duplication of a candidate and 2000 to a second duplication of the candidate.

[0201] In certain embodiments, placement of candidates a plurality of candidate polynucleotide sequences in a construct is determined using an optimization algorithm (e.g., Munkers algorithm) based on placement costs (e.g., minimizing an overall placement cost, keeping an overall placement cost below 100,000). If optimization was unsuccessful, a candidate with a highest placement cost may be discarded and an optimization is performed again (e.g., if number of candidates allows).C. Additional Methods

[0202] In certain embodiments, one or more steps of dANTe and / or sANTe pipelines described herein may be used in conjunction with one or more approaches for assessing and / or predicting neoantigen epitope characteristics relevant for use cancer immunotherapy, such as confirming / checking whether mutations are non-synonymous [i.e., such that a resultant mutated codon is non-synonymous (encodes a different amino acid) with respect to the original, nonmutated codon] particular expression level, immunogenicity data and / or prediction data, such as predictions of efficacy along various steps in a major histocompatibility complex (MHC) binding pathway, such as cleavage, surface presentation, binding affinity, and the like. Various approaches for evaluating and selecting neoantigens based e.g., immunogenicity, expression and the like, which may be used in connection with technologies of the present disclosure are described, e.g., WO2011 / 143656, WO2012 / 159754, WO2014 / 082729, WO2014 / 180569, WO2015 / 014869, WO2016 / 128376, WO2017 / 194610, WO2018 / 015433, WO2018 / 148671, WO2018 / 224405, W02020 / 132586, and / or other publications such as: Laumont et al., Nature Communications 7.1 (2016); Pataskar, etal., Nature 603.7902 (2022); Bartok, et al., Nature 590.7845 (2021); Ouspenskaia, et al., Nature Biotechnology 40.2 (2022); Abelin et al., Immunity.2017 Feb 21;46(2):315-326; Abelin etal., Immunity. 2019 Oct 15; 51(4): 766-779. el 7 each of which is incorporated by reference herein in its entirety.

[0203] For example, in certain embodiments, MHC -I and / or MHC-II binding is experimentally assessed; in some embodiments it is predicted. In some embodiments, predicted MHC -I and / or MHC-II binding is determined using techniques neonmhc 1 and / or neonmhc2, which predict and / or characterize likelihood of MHC class I and MHC class II binding, respectively.Page 52 of 18313406582vlAttorney Docket No.: 2013237-1617Alternatively or additionally, in some embodiments, an MHC-peptide presentation prediction algorithm or MHC-peptide presentation predictor is or comprises NetMHCpan or NetMHCIIpan. In some embodiments, a hidden Markov model approach may be utilized for MHC-peptide presentation prediction and / or characterization. In some embodiments, the peptide prediction model MARIA may be utilized. In some embodiments, NetMHCpan is not utilized to predict or characterize likelihood of MHC binding. In some embodiments, the peptide prediction model MARIA is not utilized to predict or characterize likelihood of MHC binding. In some embodiments, neither NetMHCpan nor NetMHCIIpan is utilized to predict or characterize likelihood of MHC binding. In some embodiments, an MHC-peptide presentation prediction algorithm or MHC-peptide presentation predictor is or comprises RECON® (Real-time Epitope Computation for ONcology), which offers high quality MHC-peptide presentation prediction based on expression, processing and binding capabilities. See, for example, Abelin et al., Immunity 21:315, 2017; Abelin et al., Immunity 15:766, 2019, each of which is incorporated herein by reference in its entirety.

[0204] In some embodiments, expression level is experimentally determined (e.g., in a model system or in cancer patients). In some embodiments, expression level is a reported level (e.g., in a published or presented report). In some embodiments, expression level is assessed as RNA (e.g., via RNASeq or whole exome sequencing). In some embodiments, expression level is assessed as protein.

[0205] In certain embodiments, for example, candidate characteristics, such as MHC -I and / or MHC -II binding predictions, expression levels, and clonality may be evaluated and used for target selection in a stepwise fashion, for example by first identifying a highest priority collection of candidate mutations based on one or more methods described herein, and then ranking those candidates according to MHC binding predictions, followed by expression levels, or vice-a-versa. In certain embodiments, a process flow such as this may repeat, for example, moving onto, and ranking and selecting from a second-highest priority collection of candidates using MHC binding and / or expression level estimates or determined values, and so on, for example until a desired number of neoantigen epitopes and / or non-neoantigen epitopes, e.g., for inclusion in a vaccine, are obtained.Page 53 of 18313406582vlAttorney Docket No.: 2013237-1617

[0206] In some embodiments, a shared tumor antigen epitope is from ACTL8, ANKRD30A, CBX2, CLDN6, CST9, CST9L, EDDM3b, KK-LC-1, KLK2, KLK3 (PSA), LRRC26, MAGEA1, MAGEA3, MAGEA4, MAGEA5, MAGEA9b, MAGEB4, MAGEC1, NY-ESO-1, PLAC1, PRAME, SPANXE, SSX4, TPTE, TP53, orXAGE5.

[0207] In some embodiments, a shared tumor antigen is one that is known to be particularly highly expressed in tumor sample(s) as compared with non-tumor sample(s) (e.g., of the same tissue). In some such embodiments, the tumor antigen may have an amino acid sequence that is identical to that of a protein expressed in non-tumor cells, but the protein may be consistently overexpressed in tumor cells (e.g., of a particular type and / or stage, etc.). In some embodiments, a shared tumor antigen may include a mutation (relative to protein found in non-tumor samples) that is preferentially associated with (e.g., found in) tumor cells rather than non-tumor cells (e.g., of the same cell type).II. Vaccine Constructs

[0208] In certain embodiments, technologies of the present disclosure include and / or utilize constructs and / or methods for producing them based on subsets of neoantigen epitopes and / or non-neoantigen epitopes selected via a method described herein (e.g., dANTe and / or sANTe). As described in further detail herein, these constructs and or methods for producing them may include, without limitation, constructs (e.g., poly epitopic constructs) that may be used as personalized cancer vaccines as well as personalized T-cell therapies, TCR therapies, and the like.

[0209] In certain embodiments, technologies of the present disclosure may include enriched T-cell populations and / or tumor infiltrated lymphocyte (TIL) populations and / or methods for producing them. Enriched T-cell and / or TIL populations may be tailored using neoepitope prioritization technologies of the present disclosure to bind specifically to complexes comprising at least a portion of a neoepitope selected via a method described herein. In certain embodiments, said complexes also comprise a major histocompatibility complex (MHC) protein expressed by cancer cells (e.g., of a patient) and / or antigen presenting cells (APCs) of the patient. T cell and / or TIL populations may be obtained from blood, or other sample types.Certain example approaches for enriching populations to T-cells for binding neoepitopes are described in further in WO2019094642A1; WO2020227546A1; WO2022036137A1;Page 54 of 18313406582vlAttorney Docket No.: 2013237-1617W02023064930A1; WO2024124222A1, the content of each of which is incorporated herein in its entirety.

[0210] Additionally or alternatively, technologies of the present disclosure may be used for producing, and / or include, T-cell receptors capable of specifically binding a complex comprising at least a portion of a neoepitope selected according to approaches described herein. TCRs may be natural TCRs or engineered, e.g., chimeric TCRs (CAR). TCRs may be based on blood or based on TCRs from tumor infiltrating lymphocytes.

[0211] In some embodiments, a vaccine construct is a polyepitopic vaccine construct that includes up to about 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 50 neoantigen epitopes and / or up to about 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 50 non-neoantigen (e.g., shared tumor antigen) epitopes. In some embodiments, a polyepitopic vaccine construct includes at least one non-neoantigen (e.g., shared tumor antigen) epitope from each of about 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or 30 different non-neoantigens (e.g., shared tumor antigens) and / or includes at least one neoantigen epitope from each of about 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or 30 different neoantigens). In some embodiments, a poly epitopic vaccine construct includes shared tumor antigen epitopes that match a subject’s HLA.A. Secretory Signals

[0212] In some embodiments, a vaccine construct described herein includes a secretory signal, e.g., that is functional in mammalian cells. In some embodiments, a secretory signal comprises or consists of a human secretory signal. In some embodiments, a secretory signal comprises or consists of a non-human secretory signal. In some embodiments, a heterologous secretory signal comprises or consists of a viral secretory signal. In some embodiments, a viral secretory signal comprises or consists of an HSV secretory signal (e.g., an HSV-1 or HSV-2 secretory signal). In some embodiments, an HSV secretory signal comprises or consists of an HSV glycoprotein D (gD) secretory signal. In some embodiments, a secretory signal comprises or consists of an Ebola virus secretory signal. In some embodiments, an Ebola virus secretory signal comprises or consists of an Ebola virus spike glycoprotein (SGP) secretory signal.

[0213] In some embodiments, a secretory signal is characterized by a length of about 15 to 30 amino acids.Page 55 of 18313406582vlAttorney Docket No.: 2013237-1617

[0214] In many embodiments, a secretory signal is positioned at the N-terminus of a vaccine construct described herein. In some embodiments, a secretory signal preferably allows transport of a vaccine construct with which it is associated into a defined cellular compartment, preferably a cell surface, endoplasmic reticulum (ER) or endosomal-lysosomal compartment.

[0215] In some embodiments, a secretory signal is selected from an S1S2 secretory signal (aa 1-19), an immunoglobulin secretory signal (aa 1-22), a human SPARC secretory signal, a human insulin isoform 1 secretory signal, a human albumin secretory signal, etc. Those skilled in the art will be aware of other secretory signal such as, for example, as disclosed in W02017 / 081082 (e.g., SEQ ID NOs: 1-1115 and 1728, or fragments variants thereof, as disclosed in W02017 / 081082). In some embodiments, a secretory signal is a secretory signal as described in Kreiter, Sebastian, et al. " Increased antigen presentation efficiency by coupling antigens to MHC class I trafficking signals." The Journal of Immunology, 180.1 (2008): 309-318., the content of which is incorporated herein in its entirety, e.g., the mmsec secretory signal. In some embodiments, a vaccine construct described herein does not comprise a secretory signal.

[0216] In some embodiments, a secretory signal is one listed in Table 1, or a secretory signal having 1, 2, 3, 4, or 5 amino acid differences relative thereto. In some embodiments, a signal sequence is selected from those included in the Table 1 below and / or those encoded by the sequences in Table 2 below.Table 1: Exemplary secretory signalsSignal Sequence (Amino Acid)HSV-1 gD SP MGGAAARLGAVILFVVIVGLHGVRSKY(SEQ ID NO: 1)HSV-2 gD SP MGRLTSGVGTAALLWAVGLRWCA (SEQID NO: 2)HSV-2 MGRLTSGVGTAALLWAVGLRWCAKYA(SEQ ID NO: 3)Csp (isolate 3D7) MMRKLAILSVSSFLFVEA (SEQ ID NO: 4)HSV-1 gD SP MGGAAARLGAVILFVVIVGLHGVRGKY(SEQ ID NO: 5)Page 56 of 18313406582vlAttorney Docket No.: 2013237-1617Signal Sequence (Amino Acid)Ebola spike glycoprotein GP MGVTGILQLPRDRFKRTSFFLWVIILFQRTFS (SEQ ID NO: 6)SARS-CoV-2-S MFVFLVLLPLVSSQCVNLT (SEQ ID NO: 7) human Ig heavy chain signal MDWIWRILFLVGAATGAHSQM (SEQ ID NO: peptide (huSec) 8)HuIgGk signal peptide METPAQLLFLLLLWLPDTTG (SEQ ID NO: 9) IgE heavy chain epsilon- 1 signal MDWTWILFLVAAATRVHS (SEQ ID NO: 10) peptideJapanese encephalitis PRM signal MLGSNSGQRVVFTILLLLVAPAYS (SEQ ID sequence NO: 11)VSVg protein signal sequence MKCLLYLAFLFIGVNCA (SEQ ID NO: 12) TRIO MCRGLSAVLILLVSLSAQLHVVVG (SEQ ID NO: 13)human Ig heavy chain signal MELGLSWIFLLAILKGVQC (SEQ ID NO: 14) peptidehuman Ig heavy chain signal MELGLRWVFLVAILEGVQC (SEQ ID NO: 15) peptidehuman Ig heavy chain signal MKHLWFFLLLVAAPRWVLS (SEQ ID NO: 16) peptidehuman Ig heavy chain signal MDWTWRILFLVAAATGAHS (SEQ ID NO: 17) peptidehuman Ig heavy chain signal MDWTWRFLFWAAATGVQS (SEQ ID NO: peptide 18)human Ig heavy chain signal MEFGLSWLFLVAILKGVQC (SEQ ID NO: 19) peptidehuman Ig heavy chain signal MEFGLSWVFLVALFRGVQC (SEQ ID NO: 20) peptidehuman Ig heavy chain signal MDLLHKNMKHLWFFLLLVAAPRWVLS (SEQ peptide ID NO: 21)Page 57 of 18313406582vlAttorney Docket No.: 2013237-1617Signal Sequence (Amino Acid)human Ig kappa chain signal MDMRVPAQLLGLLLLWLSGARC (SEQ ID peptide NO: 22)human Ig kappa chain signal MKYLLPTAAAGLLLLAAQPAMA (SEQ ID peptide NO: 23)Table 2: Exemplary polynucleotide sequences encoding secretory signals Signal Sequence (Nucleotide)HSV-1 gD SP wild-type ATGGGGGGGGCTGCCGCCAGGTTGGGGGC CGTGATTTTGTTTGTCGTCATAGTGGGCCT CCATGGGGTCCGCAGCAAATAT (SEQ ID NO: 24)HSV-1 gD SP OptlO nt sequence ATGGGAGGAGCCGCCGCCAGACTGGGAG CCGTGATCCTGTTCGTGGTGATCGTGGGAC TGCATGGAGTGAGAAGCAAGTAC (SEQ ID NO: 25)SARS-CoV-2-S ATGTTTGTGTTTCTTGTGCTGCTGCCTCTT GTGTCTTCTCAGTGTGTGAATTTGACA(SEQ ID NO: 26)human Ig heavy chain signal ATGGATTGGATTTGGAGAATCCTGTTCCTC peptide (huSec) GTGGGAGCCGCTACAGGAGCCCACTCCCA GATG (SEQ ID NO: 27)human Ig heavy chain signal ATGGAGTTGGGACTGAGCTGGATTTTCCTT peptide TTGGCTATTTTAAAAGGTGTCCAGTGT (SEQ ID NO: 28)human Ig heavy chain signal ATGGAACTGGGGCTCCGCTGGGTTTTCCTT peptide GTTGCTATTTTAGAAGGTGTCCAGTGT (SEQ ID NO: 29)human Ig heavy chain signal ATGAAACACCTGTGGTTCTTCCTCCTGCTG peptide GTGGCAGCTCCCAGATGGGTCCTGTCC (SEQ ID NO: 30)Page 58 of 18313406582vlAttorney Docket No.: 2013237-1617Signal Sequence (Nucleotide)human Ig heavy chain signal ATGGACTGGACCTGGAGGATCCTCTTCTTG peptide GTGGCAGCAGCAACAGGTGCCCACTCG (SEQ ID NO: 31)human Ig heavy chain signal ATGGACTGGACCTGGAGGTTCCTCTTTGT peptide GGTGGCAGCAGCTACAGGTGTCCAGTCC(SEQ ID NO: 32)human Ig heavy chain signal ATGGAGTTTGGGCTGAGCTGGCTTTTTCTT peptide GTGGCGATTCTAAAAGGTGTCCAGTGT(SEQ ID NO: 33)human Ig heavy chain signal ATGGAGTTTGGGCTGAGCTGGGTTTTCCTC peptide GTTGCTCTTTTTAGAGGTGTCCAGTGT(SEQ ID NO: 34)human Ig heavy chain signal ATGGACCTCCTGCACAAGAACATGAAACA peptide CCTGTGGTTCTTCCTCCTCCTGGTGGCAGC TCCCAGATGGGTGCTGTCC (SEQ ID NO: 35) human Ig kappa chain signal ATGGACATGAGGGTCCCTGCTCAGCTCCT peptide GGGGCTCCTGCTGCTCTGGCTCTCAGGTG CCAGATGT (SEQ ID NO: 36)human Ig kappa chain signal ATGAAATACCTATTGCCTACGGCAGCCGCT peptide GGATTGTTATTACTCGCGGCCCAGCCGGCC ATGGCC (SEQ ID NO: 37)B. Trafficking Signals

[0217] In some embodiments, a vaccine construct described herein includes a trafficking signal. In some embodiments, an MHC trafficking domain is or comprises a transmembrane region and a cytoplasmic region of a chain of an MHC molecule (e.g., a MHC Class I molecule), for example, in some embodiments as described in the International Patent Publication Number WO 2005 / 038030, the contents of which are incorporated herein by reference in their entireties for the purposes described herein. In some embodiments, an MHC trafficking domain is or comprises an MHC Class I trafficking domain. In some embodiments, an MHC class I trafficking domain (MITD) comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence ofPage 59 of 18313406582vlAttorney Docket No.: 2013237-1617IVGIVAGLAVLAVVVIGAVVATVMCRRKSSGGKGGSYSQAASSDSAQGSDVSLTA (SEQ ID NO: 38). In some embodiments, an MHC class I trafficking domain (MITD) comprises an amino acid sequence that is identical to the amino acid sequence of IVGIVAGLAVLAVVVIGAVVATVMCRRKSSGGKGGSYSQAASSDSAQGSDVSLTA (SEQ ID NO: 38).C. Linkers

[0218] In some embodiments, a vaccine construct, e.g., a polyepitopic vaccine construct described herein, includes one or more linkers, e.g., between epitopes in the construct and / or before or after the last construct. In some embodiments, a linker is or comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids. In some embodiments, a linker is or comprises no more than about 30, 25, 20, 15, 10 or fewer amino acids. A linker can include any amino acid sequence and is not limited to any particular amino acids. In some embodiments, a linker comprises one or more glycine (G) amino acids. In some embodiments, a linker comprises one or more serine (S) amino acids. In some embodiments, a linker includes amino acids selected based on a cleavage predictor to generate highly-cleavable linkers.

[0219] In some embodiments, a linker is or comprises S-G4-S-G4-S (SEQ ID NO: 40). In some embodiments, a linker is or comprises GSPGSGSGS (SEQ ID NO: 41). In some embodiments, a linker is or comprises GGSGGGGSGG (SEQ ID NO: 42). In some embodiments, a linker is one presented in Table 3. In some embodiments, a linker is or comprises a sequence as set forth in W02017 / 081082, which is incorporated herein by reference in its entirety (see SEQ ID NOs: 1509-1565, or a fragment or variant thereof, of W02017 / 081082).

[0220] In some embodiments, one or more linker sequences may comprise cleavage sequences. In some embodiments, a linker may have a length of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid. In some embodiments, a linker of not more than about 30, 25, 20, 15, 10 or fewer amino acids is used. In general, any amino acid may be present as a linker sequence. In some embodiments, a linker or cleavage sequence contains a lysine (K). In some embodiments, a linker or cleavage sequence contains an arginine (R). In some embodiments, a linker or cleavage sequence contains a methionine (M). In some embodiments, a linker or cleavage sequence contains a tyrosine (Y). In some embodiments, a linker is designed to comprise amino acids based on a cleavage predictor to generate highly-cleavable sequences peptide sequences, and is a novel and effective Page 60 of 18313406582vlAttorney Docket No.: 2013237-1617way of delivering immunogenic epitopes in a vaccine setting. In some embodiments, the epitope distribution and their juxtaposition encoded in a polyepitopic vaccine construct are so designed to facilitate cleavage sequences contributed by the amino acid sequences of the epitopes and / or the flanking or linking residues and thereby using minimal linker sequences. Some exemplary cleavage sequences may be one or more of FRAC (SEQ ID NO: 43), KRCF (SEQ ID NO: 44), KKRY (SEQ ID NO: 45), ARMA (SEQ ID NO: 46), RRSG (SEQ ID NO: 47), MRAC (SEQ ID NO: 48), KMCG (SEQ ID NO: 49), ARCA (SEQ ID NO: 50), KKQG (SEQ ID NO: 51), YRSY (SEQ ID NO: 52), SFMN (SEQ ID NO: 53), FKAA (SEQ ID NO: 54), KRNG (SEQ ID NO: 55), YNSF (SEQ ID NO: 56), KKNG (SEQ ID NO: 57), RRRG (SEQ ID NO: 58), KRYS (SEQ ID NO: 59), and ARYA (SEQ ID NO: 60).

[0221] In some embodiments, a polyepitopic vaccine construct described herein comprises a linker between each shared tumor antigen epitope and / or between each neoantigen epitope. In some embodiments, linkers included in a particular polyepitopic vaccine construct described herein are the same. In some embodiments, two or more linkers in a particular poly epitopic vaccine construct described herein are different.

[0222] Exemplary linker sequences are provided in the following Table 3.Table 3: Exemplary linker sequences.Linker Sequence (Amino Acid)SGGGGSGGGGS (SEQ ID NO: 40)GSPGSGSGS (SEQ ID NO: 41)GGSGGGGSGG (SEQ ID NO: 42)GGS GGGS (SEQ ID NO: 62)GGGGSGGGGSGGGGS (SEQ ID NO: 63)AGNRVRRSVG (SEQ ID NO: 64)GSGSGS (SEQ ID NO: 65)GGSGGGGSGG (SEQ ID NO: 66)GGSLGGGGSG (SEQ ID NO: 67)FRAC (SEQ ID NO: 43)Page 61 of 18313406582vlAttorney Docket No.: 2013237-1617Linker Sequence (Amino Acid)KRCF (SEQ ID NO: 44)KKRY (SEQ ID NO: 45)ARMA (SEQ ID NO: 46)RRSG (SEQ ID NO: 47)MRAC (SEQ ID NO: 48)KMCG (SEQ ID NO: 49)ARCA(SEQ ID NO: 50)KKQG(SEQ ID NO: 51)YRSY (SEQ ID NO: 52)SFMN (SEQ ID NO: 53)FKAA (SEQ ID NO: 54)KRNG (SEQ ID NO: 55)YNSF (SEQ ID NO: 56)KKNG (SEQ ID NO: 57)RRRG (SEQ ID NO: 58)KRYS (SEQ ID NO: 59)ARYA (SEQ ID NO: 60)III. PolyribonucleotidesA. Exemplary Polyribonucleotides Features

[0223] In some embodiments, the disclosure provides polyribonucleotides that encode one or more vaccine constructs, e.g., one or more poly epitopic vaccine constructs, described herein. In some embodiments, polyribonucleotides described herein can comprise a nucleotide sequence that encodes a 5’UTR of interest and / or a 3’ UTR of interest. In some embodiments, polynucleotides described herein can comprise a nucleotide sequence that encodes a polyA tail. In some embodiments, polyribonucleotides described herein may comprise a 5’ cap, which may be incorporated during transcription, or joined to a polyribonucleotide post-transcription. In some embodiments, a first polyribonucleotide encoding a first polyepitopic and a second polyribonucleotide encoding a second poly epitopic vaccine comprise the same 5’ cap, cap proximal sequence(s), 5’ UTR, linker sequences, 3’ UTR, and poly(A) tail. In somePage 62 of 18313406582vlAttorney Docket No.: 2013237-1617embodiments, a first polyribonucleotide encoding a first polyepitopic and a second polyribonucleotide encoding a second poly epitopic vaccine comprise one or more different 5’ cap, cap proximal sequence(s), 5’ UTR, linker sequences, 3’ UTR, and / or poly(A) tail. In some embodiments, the 5’ cap, cap proximal sequence(s), 5’ UTR, linker sequences, 3’ UTR, and poly(A) tail cause innate immune stimulation and / or are intrinsic TLR7 / 8 agonists.1. 5' Cap

[0224] A structural feature of mRNAs is cap structure at five-prime end (5’). Natural eukaryotic mRNA comprises a 7-methylguanosine cap linked to the mRNA via a 5' to 5 '-triphosphate bridge resulting in cap0 structure (m7GpppN). In most eukaryotic mRNA and some viral mRNA, further modifications can occur at the 2' -hydroxy-group (2’-OH) (e.g, the 2'-hydroxyl group may be methylated to form 2'-0-Me) of the first and subsequent nucleotides producing “capl” and “cap2” five-prime ends, respectively). Diamond, et al., (2014) Cytokine & growth Factor Reviews, 25:543-550, which is incorporated herein by reference in its entirety, reported that capO-mRNA cannot be translated as efficiently as capl -mRNA in which the role of 2'-0-Me in the penultimate position at the mRNA 5’ end is determinant. Lack of the 2'-O-met has been shown to trigger innate immunity and activate IFN response. Daffis, et al. (2010) Nature, 468:452-456; andZtist etal. (2011) Nature Immunology, 12:137-143, each of which is incorporated herein by reference in its entirety.

[0225] RNA capping is well researched and is described, e.g., in Decroly E et al. (2012) Nature Reviews 10: 51-65; and in Ramanathan A. et al., (2016) Nucleic Acids Res; 44(16): 7511-7526, the entire contents of each of which is hereby incorporated by reference. For example, in some embodiments, a 5 ’-cap structure which may be suitable in the context of the present invention is a capO (methylation of the first nucleobase, e.g., m7GpppN), capl (additional methylation of the ribose of the adjacent nucleotide of m7GpppN), cap2 (additional methylation of the ribose of the 2nd nucleotide downstream of the m7GpppN), cap3 (additional methylation of the ribose of the 3rd nucleotide downstream of the m7GpppN), cap4 (additional methylation of the ribose of the 4th nucleotide downstream of the m7GpppN), ARCA (“anti-reverse cap analogue”), modified ARCA (e.g. phosphothioate modified ARCA), inosine, N1 -methyl-guanosine, 2’ -fluoroguanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.Page 63 of 18313406582vlAttorney Docket No.: 2013237-1617

[0226] The term “5'-cap” as used herein refers to a structure found on the 5'-end of an RNA, e.g., mRNA, and generally includes a guanosine nucleotide connected to an RNA, e.g., mRNA, via a 5'- to 5 '-triphosphate linkage (also referred to as Gppp or G(5')ppp(5')). In some embodiments, a guanosine nucleoside included in a 5’ cap may be modified, for example, by methylation at one or more positions (e.g., at the 7-position) on a base (guanine), and / or by methylation at one or more positions of a ribose. In some embodiments, a guanosine nucleoside included in a 5’ cap comprises a 3’0 methylation at a ribose (3’0MeG). In some embodiments, a guanosine nucleoside included in a 5’ cap comprises methylation at the 7-position of guanine (m7G). In some embodiments, a guanosine nucleoside included in a 5’ cap comprises methylation at the 7-position of guanine and a 3’ O methylation at a ribose (m7(3’OMeG)). It will be understood that the notation used in the above paragraph, e.g, “(m273’°)G” or “m7(3’OMeG)”, applies to other structures described herein.

[0227] In some embodiments, providing an RNA with a 5'-cap disclosed herein may be achieved by in vitro transcription, in which a 5'-cap is co-transcriptionally expressed into an RNA strand, or may be attached to an RNA post-transcriptionally using capping enzymes. In some embodiments, co-transcriptional capping with a cap disclosed improves the capping efficiency of an RNA compared to co-transcriptional capping with an appropriate reference comparator. In some embodiments, improving capping efficiency can increase a translation efficiency and / or translation rate of an RNA, and / or increase expression of an encoded polypeptide. In some embodiments, alterations to polynucleotides generates a non-hydrolyzable cap structure which can, for example, prevent decapping and increase RNA half-life.

[0228] In some embodiments, a utilized 5’ caps is a capO, a capl, or cap2 structure. See, e.g, FIG. 1 of Ramanathan Ketal., and FIG. 1 of Decroly E et al., each of which is incorporated herein by reference in its entirety. In some embodiments, an RNA described herein comprises a capl structure. In some embodiments, an RNA described herein comprises a cap2.

[0229] In some embodiments, an RNA described herein comprises a cap0 structure. In some embodiments, a cap0 structure comprises a guanosine nucleoside methylated at the 7-position of guanine ((m7)G). In some embodiments, such a cap0 structure is connected to an RNA via a 5'-to 5 '-triphosphate linkage and is also referred to herein as (m7)Gppp. In some embodiments, a cap0 structure comprises a guanosine nucleoside methylated at the 2’ -position of the ribose of Page 64 of 18313406582vlAttorney Docket No.: 2013237-1617guanosine. In some embodiments, a cap0 structure comprises a guanosine nucleoside methylated at the 3 ’-position of the ribose of guanosine. In some embodiments, a guanosine nucleoside included in a 5’ cap comprises methylation at the 7-position of guanine and at the 2’-position of the ribose ((m272’°)G). In some embodiments, a guanosine nucleoside included in a 5’ cap comprises methylation at the 7-position of guanine and at the 2’-position of the ribose ((m273’ °)G).

[0230] In some embodiments, a capl structure comprises a guanosine nucleoside methylated at the 7-position of guanine ((m7)G) and optionally methylated at the 2’ or 3’ position of the ribose, and a 2’0 methylated first nucleotide in an RNA ((m2’°)Ni). In some embodiments, a capl structure comprises a guanosine nucleoside methylated at the 7-position of guanine ((m7)G) and the 3’ position of the ribose, and a 2’0 methylated first nucleotide in an RNA ((m2'°)Ni). In some embodiments, a capl structure is connected to an RNA via a 5'- to 5 '-triphosphate linkage and is also referred to herein as, e.g., ((m7)Gppp(2'°)Ni) or (m273’^Gppp^'^Ni), wherein Ni is as defined and described herein. In some embodiments, a capl structure comprises a second nucleotide, N2, which is at position 2 and is chosen from A, G, C, or U, e.g., (m7)Gppp(2‘ °)NipN2 or (m273■°)Gppp(2'°)NipN2, wherein each of Ni and N2 is as defined and described herein.

[0231] In some embodiments, a cap2 structure comprises a guanosine nucleoside methylated at the 7-position of guanine ((m7)G) and optionally methylated at the 2’ or 3’ position of the ribose, and a 2’0 methylated first and second nucleotides in an RNA ((m2'°)Nip(m2’°)N2). In some embodiments, a cap2 structure comprises a guanosine nucleoside methylated at the 7-position of guanine ((m7)G) and the 3’ position of the ribose, and a 2’0 methylated first and second nucleotide in an RNA. In some embodiments, a cap2 structure is connected to an RNA via a 5'-to 5 '-triphosphate linkage and is also referred to herein as, e.g., ((m7)Gppp(2'°)Nip(2'°)N2) or (m273■°)Gppp(2,'0)Nip(2''0)N2), wherein each of Ni and N2 is as defined and described herein.

[0232] In some embodiments, the 5’ cap is a dinucleotide cap structure. In some embodiments, the 5’ cap is a dinucleotide cap structure comprising Ni, wherein Ni is as defined and described herein. In some embodiments, the 5’ cap is a dinucleotide cap G*Ni, wherein Ni is as defined above and herein, and G* comprises a structure of formula (I):Page 65 of 18313406582vlAttorney Docket No.: 2013237-1617(I)or a salt thereof,whereineach R2and R3is -OH or -OCH3; andX is O or S.

[0233] In some embodiments, R2is -OH. In some embodiments, R2is -OCH3. In some embodiments, R3is -OH. In some embodiments, R3is -OCH3. In some embodiments, R2is -OH and R3is -OH. In some embodiments, R2is -OH and R3is -CH3. In some embodiments, R2is -CH3 and R3is -OH. In some embodiments, R2is -CH3 and R3is -CH3.

[0234] In some embodiments, X is O. In some embodiments, X is S.

[0235] In some embodiments, the 5’ cap is a dinucleotide cap0 structure (e.g., (m7)GpppNi, (m27’2’-°)GpppNi, (m27’3’-°)GpppNi, (m7)GppSpNi, (m27’2’-°)GppSpNi, or (m27’3’-°)GppSpNi), wherein Ni is as defined and described herein. In some embodiments, the 5’ cap is a dinucleotide cap0 structure (e.g., (m7)GpppNi, (m272'°)GpppNi, (m273'°)GpppNi, (m7)GppSpNi, (m272'°)GppSpNi, or (m273'°)GppSpNi), wherein Ni is G. In some embodiments, the 5’ cap is a dinucleotide cap0 structure (e.g., (m7)GpppNi, (m272'°)GpppNi, (m273'°)GpppNi, (m7)GppSpNi, (m272'°)GppSpNi, or (m273'°)GppSpNi), wherein Ni is A, U, or C. In some embodiments, the 5’ cap is a dinucleotide capl structure (e.g., (m7)Gppp(m2’°)Ni, (m272'°)Gppp(m2’-°)Ni, (m27’3'°)Gppp(m2'°)Ni, (m7)GppSp(m2’-°)Ni, (m27’2’-°)GppSp(m2’- °)Ni, or (m273'°)GppSp(m2’°)Ni), wherein Ni is as defined and described herein. In some embodiments, the 5’ cap is selected from the group consisting of (m7)GpppG (“EcapO”), (m7)Gppp(m2’°)G (“Ecapl”), (m273'°)GpppG (“ARC A” or “DI”), and (m272'°)GppSpG (“beta-S-ARCA”). In some embodiments, the 5’ cap is (m7)GpppG (“EcapO”), having a structure:Page 66 of 18313406582vlAttorney Docket No.: 2013237-1617or a salt thereof.

[0236] In some embodiments, the 5’ cap is (m7)Gppp(m2’°)G (“Ecapl”), having a structure:or a salt thereof

[0237] In some embodiments, the 5’ cap is (m27’3'°)GpppG (“ARC A” or “DI”), having a structure:or a salt thereof

[0238] In some embodiments, the 5’ cap is (m27’2'°)GppSpG (“beta-S-ARCA”), having a structure:Page 67 of 18313406582vlAttorney Docket No.: 2013237-1617or a salt thereof.

[0239] In some embodiments, the 5’ cap is a trinucleotide cap structure. In some embodiments, the 5’ cap is a trinucleotide cap structure comprising NiplS, wherein Ni and N2 are as defined and described herein. In some embodiments, the 5’ cap is a dinucleotide cap G*NipN2, wherein Ni and N2 are as defined above and herein, and G* comprises a structure of formula (I):(I)or a salt thereof, wherein R2, R3, and X are as defined and described herein.

[0240] In some embodiments, the 5’ cap is a trinucleotide cap0 structure (e.g. (m7)GpppNipN2, (m27’2'°)GpppNipN2, or (m273'°)GpppNipN2), wherein Ni and N2 are as defined and described herein). In some embodiments, the 5’ cap is a trinucleotide capl structure (e.g., (m7)Gppp(m2’ °)NipN2, (m27’2'°)Gppp(m2’°)NipN2, (m27’3'°)Gppp(m2'°)NipN2), wherein Ni and N2 are as defined and described herein. In some embodiments, the 5’ cap is a trinucleotide cap2 structure (e.g., (m7)Gppp(m2’-0)Nip(m2’-°)N2, (m27’2’-0)Gppp(m2’-0)Nip(m2’-°)N2, (m27’3’-°)Gppp(m2’-°)Nip(m2’°)N2), wherein Ni and N2 are as defined and described herein. In some embodiments, the 5’ cap is selected from the group consisting of (m27’3'°)Gppp(m2'°)ApG (“CleanCap AG”, “CC413”), (m273'°)Gppp(m2'°)GpG (“CleanCap GG”), (m7)Gppp(m2'°)ApG, (m7)Gppp(m2’ °)GpG, (m273’-°)Gppp(m26’2’-°)ApG, and (m7)Gppp(m2’-°)ApU.Page 68 of 18313406582vlAttorney Docket No.: 2013237-1617

[0241] In some embodiments, the 5’ cap is (m273'°)Gppp(m2'°)ApG (“CleanCap AG”, “CC413”), having a structure:or a salt thereof.

[0242] In some embodiments, the 5’ cap is (m273'°)Gppp(m2'°)GpG (“CleanCap GG”), having a structure:or a salt thereof.

[0243] In some embodiments, the 5’ cap is (m7)Gppp(m2'°)ApG, having a structure:Page 69 of 18313406582vlAttorney Docket No.: 2013237-1617or a salt thereof.

[0244] In some embodiments, the 5’ cap is (m7)Gppp(m2'°)GpG, having a structure:OH OHor a salt thereof.

[0245] In some embodiments, the 5’ cap is (m27’3'°)Gppp(m26’2’°)ApG, having a structure:Page 70 of 18313406582vlAttorney Docket No.: 2013237-1617or a salt thereof.

[0246] In some embodiments, the 5’ cap is (m7)Gppp(m2'°)ApU, having a structure:OH OHor a salt thereof.

[0247] In some embodiments, the 5’ cap is a tetranucleotide cap structure. In some embodiments, the 5’ cap is a tetranucleotide cap structure comprising N₁pN₂pN₃, wherein Ni, N2, and N3 are as defined and described herein. In some embodiments, the 5’ cap is a tetranucleotide cap G*N₁pN₂pN₃, wherein Ni, N2, and N3 are as defined above and herein, and G* comprises a structure of formula (I):Page 71 of 18313406582vlAttorney Docket No.: 2013237-1617(I)or a salt thereof, wherein R2, R3, and X are as defined and described herein.

[0248] In some embodiments, the 5’ cap is a tetranucleotide cap0 structure (e.g.(m7)GpppN₁pN₂pN₃, (m27’2'°)GpppNipN2pN3, or (m27’3'°)GpppNiN2pN3), wherein Ni, N2, and N3 are as defined and described herein). In some embodiments, the 5’ cap is a tetranucleotide Capl structure (e.g., (m7)Gppp(m2'-O)N₁pN₂pN₃, (m27’2'°)Gppp(m2'°)NipN2pN3, (m27’3’ °)Gppp(m2'°)NipN2N3), wherein Ni, N2, and N3 are as defined and described herein. In some embodiments, the 5’ cap is a tetranucleotide Cap2 structure (e.g., (m7)Gppp(m2'°)Nip(m2’ °)N2pN3, (m27’2’-0)Gppp(m2’-0)Nip(m2’-°)N2pN3, (m27’3’-0)Gppp(m2’-0)Nip(m2’-°)N2pN3), wherein Ni, N2, and N3 are as defined and described herein. In some embodiments, the 5’ cap is selected from the group consisting of (m27’3'°)Gppp(m2'°)Ap(m2'°)GpG, (m273'°)Gppp(m2’ °)Gp(m2’-°)GpC, (m7)Gppp(m2'-O)Ap(m2'-O)UpA, and (m7)Gppp(m2’-0)Ap(m2’-°)GpG

[0249] In some embodiments, the 5’ cap is (m27,3'-O)Gppp(m2'-O)Ap(m2'-O)GpG, having a structure:Page 72 of 18313406582vlAttorney Docket No.: 2013237-1617or a salt thereof.

[0250] In some embodiments, the 5’ cap is (m27,3'-O)Gppp(m2'-O)Gp(m2'-O)GpC, having a structure:or a salt thereof.Page 73 of 18313406582vlAttorney Docket No.: 2013237-1617

[0251] In some embodiments, the 5’ cap is (m7)Gppp(m2'°)Ap(m2'°)UpA, having a structure:or a salt thereof.

[0252] In some embodiments, the 5’ cap is (m7)Gppp(m2'°)Ap(m2'°)GpG, having a structure:Page 74 of 18313406582vlAttorney Docket No.: 2013237-1617or a salt thereof.2. Cap Proximal Sequences

[0253] In some embodiments, a 5’ UTR utilized in accordance with the present disclosure comprises a cap proximal sequence, e.g., as disclosed herein. In some embodiments, a cap proximal sequence comprises a sequence adjacent to a 5’ cap. In some embodiments, a cap proximal sequence comprises nucleotides in positions +1, +2, +3, +4, and / or +5 of an RNA polynucleotide.

[0254] In some embodiments, a cap structure comprises one or more polynucleotides of a cap proximal sequence. In some embodiments, a cap structure comprises an m7Guanosine cap and nucleotide +1 (Ni) of an RNA polynucleotide. In some embodiments, a cap structure comprises an m7Guanosine cap and nucleotide +2 (N2) of an RNA polynucleotide. In some embodiments, a cap structure comprises an m7Guanosine cap and nucleotides +1 and +2 (Ni and N2) of an RNA polynucleotide. In some embodiments, a cap structure comprises an m7Guanosine cap and nucleotides +1, +2, and +3 (Ni, N2, and N3) of an RNA polynucleotide.

[0255] Those skilled in the art, reading the present disclosure, will appreciate that, in some embodiments, one or more residues of a cap proximal sequence (e.g., one or more of residues +1, +2, +3, +4, and / or +5) may be included in an RNA by virtue of having been included in a cap entity (e.g., a capl or cap2 structure, etc.); alternatively, in some embodiments, at least some of the residues in a cap proximal sequence may be enzymatically added (e.g., by a polymerase such as a T7 polymerase). For example, in certain exemplified embodiments where a m27’3’ °Gppp(mi2'°)ApG cap is utilized, +1 (i.e., Ni) and +2 (i.e. N2) are the (m12'-O)A and G residues of the cap, and +3, +4, and +5 are added by polymerase (e.g., T7 polymerase).

[0256] In some embodiments, the 5’ cap is a dinucleotide cap structure, wherein the cap proximal sequence comprises Ni of the 5’ cap, where Ni is any nucleotide, e.g, A, C, G or U. In some embodiments, the 5’ cap is a trinucleotide cap structure (e.g, the trinucleotide cap structures described above and herein), wherein the cap proximal sequence comprises Ni and N2 of the 5’ cap, wherein Ni and N2 are independently any nucleotide, e.g, A, C, G or U. In some embodiments, the 5’ cap is a tetranucleotide cap structure (e.g., the trinucleotide cap structures described above and herein), wherein the cap proximal sequence comprises Ni, N2, and N3 of the 5’ cap, wherein Ni, N2, and N3 are any nucleotide, e.g, A, C, G or U.Page 75 of 18313406582vlAttorney Docket No.: 2013237-1617

[0257] In some embodiments, e.g., where the 5’ cap is a dinucleotide cap structure, a cap proximal sequence comprises Ni of the 5’ cap, and N2, N3, N4 and N5, wherein Ni to N5 correspond to positions +1, +2, +3, +4, and / or +5 of an RNA polynucleotide. In some embodiments, e.g., where the 5’ cap is a trinucleotide cap structure, a cap proximal sequence comprises Ni and N2 of the 5’ cap, and N3, N4 and N5, wherein Ni to N5 correspond to positions +1, +2, +3, +4, and / or +5 of an RNA polynucleotide. In some embodiments, e.g., where the 5’ cap is a tetranucleotide cap structure, a cap proximal sequence comprises Ni, N2, and N3 of the 5’ cap, and N4 and N5, wherein Ni to N5 correspond to positions +1, +2, +3, +4, and / or +5 of an RNA polynucleotide.

[0258] In some embodiments, Ni is A. In some embodiments, Ni is C. In some embodiments, Ni is G. In some embodiments, Ni is U. In some embodiments, N2 is A. In some embodiments, N2 is C. In some embodiments, N2 is G. In some embodiments, N2 is U. In some embodiments, N3 is A. In some embodiments, N3 is C. In some embodiments, N3 is G. In some embodiments, N3 is U. In some embodiments, N4 is A. In some embodiments, N4 is C. In some embodiments, N4 is G. In some embodiments, N4 is U. In some embodiments, N5 is A. In some embodiments, N5 is C. In some embodiments, N5 is G. In some embodiments, N5 is U. It will be understood that, each of the embodiments described above and herein (e.g., for Ni through N5) may be taken singly or in combination and / or may be combined with other embodiments of variables described above and herein (e.g., 5’ caps).3. 5’ UTR

[0259] In some embodiments, a nucleic acid (e.g., DNA, RNA) utilized in accordance with the present disclosure comprises a 5'-UTR. In some embodiments, 5’-UTR may comprise a plurality of distinct sequence elements; in some embodiments, such plurality may be or comprise multiple copies of one or more particular sequence elements (e.g., as may be from a particular source or otherwise known as a functional or characteristic sequence element). In some embodiments a 5’ UTR comprises multiple different sequence elements.

[0260] The term “untranslated region” or “UTR” is commonly used in the art to refer to a region in a DNA molecule which is transcribed but is not translated into an amino acid sequence, or to the corresponding region in an RNA polynucleotide, such as an mRNA molecule. An untranslated region (UTR) can be present 5' (upstream) of an open reading frame (5'-UTR)Page 76 of 18313406582vlAttorney Docket No.: 2013237-1617and / or 3' (downstream) of an open reading frame (3'-UTR). As used herein, the terms “five prime untranslated region” or “5' UTR” refer to a sequence of a polyribonucleotide between the 5' end of the polyribonucleotide (e.g., a transcription start site) and a start codon of a coding region of the polyribonucleotide. In some embodiments, “5' UTR” refers to a sequence of a polyribonucleotide that begins at the 5' end of the polyribonucleotide (e.g., a transcription start site) and ends one nucleotide (nt) before a start codon (usually AUG) of a coding region of the polyribonucleotide, e.g., in its natural context. In some embodiments, a 5' UTR comprises a Kozak sequence. A 5'-UTR is downstream of the 5'-cap (if present), e.g., directly adjacent to the 5'-cap. In some embodiments, a 5’ UTR disclosed herein comprises a cap proximal sequence, e.g., as defined and described herein. In some embodiments, a cap proximal sequence comprises a sequence adjacent to a 5’ cap.

[0261] Exemplary 5’ UTRs include a human alpha globin (hAg) 5 ’UTR or a fragment thereof, a TEV 5’ UTR or a fragment thereof, a HSP705’ UTR or a fragment thereof, or a c-Jun 5’ UTR or a fragment thereof.

[0262] In some embodiments, an RNA disclosed herein comprises a hAg 5’ UTR or a fragment thereof.

[0263] In some embodiments, an RNA disclosed herein comprises a 5’ UTR having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a 5’ UTR with the sequence AGAATAAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACC (SEQ ID NO: 68). In some embodiments, an RNA disclosed herein comprises a 5’ UTR having the sequence AGAATAAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACC (SEQ ID NO: 68).4. PolyA Tail

[0264] In some embodiments, a polynucleotide (e.g., DNA, RNA) disclosed herein comprises a polyadenylate (polyA) sequence, e.g, as described herein. In some embodiments, a polyA sequence is situated downstream of a 3'-UTR, e.g, adjacent to a 3'-UTR.Page 77 of 18313406582vlAttorney Docket No.: 2013237-1617

[0265] As used herein, the term “poly(A) sequence” or “poly-A tail” refers to an uninterrupted or interrupted sequence of adenylate residues which is typically located at the 3 '-end of an RNA polynucleotide. Poly(A) sequences are known to those of skill in the art and may follow the 3’-UTR in the RNAs described herein. An uninterrupted poly(A) sequence is characterized by consecutive adenylate residues. In nature, an uninterrupted poly(A) sequence is typical. In some embodiments, polynucleotides disclosed herein comprise an uninterrupted Poly(A) sequence. In some embodiments, polynucleotides disclosed herein comprise interrupted Poly(A) sequence. In some embodiments, RNAs disclosed herein can have a poly(A) sequence attached to the free 3'-end of the RNA by a template-independent RNA polymerase after transcription or a poly(A) sequence encoded by DNA and transcribed by a template-dependent RNA polymerase.

[0266] It has been demonstrated that a poly(A) sequence of about 120 A nucleotides has a beneficial influence on the levels of RNA in transfected eukaryotic cells, as well as on the levels of protein that is translated from an open reading frame that is present upstream (5’) of the poly(A) sequence (Holtkamp et al., 2006, Blood, vol. 108, pp. 4009-4017, which is herein incorporated by reference).

[0267] In some embodiments, a poly(A) sequence in accordance with the present disclosure is not limited to a particular length; in some embodiments, a poly(A) sequence is any length. In some embodiments, a poly(A) sequence comprises, essentially consists of, or consists of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 A nucleotides, and, in particular, about 120 A nucleotides. In this context, "essentially consists of' means that most nucleotides in the poly(A) sequence, typically at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% by number of nucleotides in the poly(A) sequence are A nucleotides, but permits that remaining nucleotides are nucleotides other than A nucleotides, such as U nucleotides (uridylate), G nucleotides (guanylate), or C nucleotides (cytidylate). In this context, "consists of' means that all nucleotides in the poly(A) sequence, i.e., 100% by number of nucleotides in the poly(A) sequence, are A nucleotides. The term “A nucleotide” or “A” refers to adenylate.

[0268] In some embodiments, a poly(A) sequence is attached during RNA transcription, e.g., during preparation of in vitro transcribed RNA, based on a DNA template comprising repeated Page 78 of 18313406582vlAttorney Docket No.: 2013237-1617dT nucleotides (deoxythymidylate) in the strand complementary to the coding strand. The DNA sequence encoding a poly(A) sequence (coding strand) is referred to as poly(A) cassette.

[0269] In some embodiments, the poly(A) cassette present in the coding strand of DNA essentially consists of dA nucleotides, but is interrupted by a random sequence of the four nucleotides (dA, dC, dG, and dT). Such random sequence may be 5 to 50, 10 to 30, or 10 to 20 nucleotides in length. Such a cassette is disclosed in WO 2016 / 005324 Al, hereby incorporated by reference. Any poly(A) cassette disclosed in WO 2016 / 005324 Al may be used in accordance with the present disclosure. A poly(A) cassette that essentially consists of dA nucleotides, but is interrupted by a random sequence having an equal distribution of the four nucleotides (dA, dC, dG, dT) and having a length of e.g., 5 to 50 nucleotides shows, on DNA level, constant propagation of plasmid DNA in E. coli and is still associated, on RNA level, with the beneficial properties with respect to supporting RNA stability and translational efficiency is encompassed. In some embodiments, the poly(A) sequence contained in an RNA polynucleotide described herein essentially consists of A nucleotides, but is interrupted by a random sequence of the four nucleotides (A, C, G, U). Such random sequence may be 5 to 50, 10 to 30, or 10 to 20 nucleotides in length.

[0270] In some embodiments, no nucleotides other than A nucleotides flank a poly(A) sequence at its 3'-end, i.e., the poly(A) sequence is not masked or followed at its 3'-end by a nucleotide other than A.

[0271] In some embodiments, the poly(A) sequence may comprise at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly(A) sequence may essentially consist of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly(A) sequence may consist of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly(A) sequence comprises at least 100 nucleotides. In some embodiments, the poly(A) sequence comprises about 150 nucleotides. In some embodiments, the poly(A) sequence comprises about 120 nucleotides.

[0272] In some embodiments, a poly A tail comprises a specific number of Adenosines, such as about 50 or more, about 60 or more, about 70 or more, about 80 or more, about 90 or more, Page 79 of 18313406582vlAttorney Docket No.: 2013237-1617about 100 or more, about 120, or about 150 or about 200. In some embodiments a poly A tail of a string construct may comprise 200 A residues or less. In some embodiments, a poly A tail of a string construct may comprise about 200 A residues. In some embodiments, a poly A tail of a string construct may comprise 180 A residues or less. In some embodiments, a poly A tail of a string construct may comprise about 180 A residues. In some embodiments, a poly A tail may comprise 150 residues or less.

[0273] In some embodiments, RNA comprises a poly(A) sequence comprising the nucleotide sequence of AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCATATGACTAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 70), or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCATATGACTAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 70). In some embodiments, a poly(A) tail comprises a plurality of A residues interrupted by a linker. In some embodiments, a linker comprises the nucleotide sequence GCATATGAC.5. 3' UTR

[0274] In some embodiments, an RNA utilized in accordance with the present disclosure comprises a 3 '-UTR. As used herein, the terms “three prime untranslated region,” “3' untranslated region,” or “3' UTR” refer to a sequence of an mRNA molecule that begins following a stop codon of a coding region of an open reading frame sequence. In some embodiments, the 3' UTR begins immediately after a stop codon of a coding region of an open reading frame sequence, e.g., in its natural context. In other embodiments, the 3' UTR does not begin immediately after stop codon of the coding region of an open reading frame sequence, e.g., in its natural context. The term “3'-UTR” does preferably not include the poly(A) sequence. Thus, the 3'-UTR is upstream of the poly(A) sequence (if present), e.g. directly adjacent to the poly(A) sequence.

[0275] In some embodiments, an RNA disclosed herein comprises a 3’ UTR comprising an F element and / or an I element. In some embodiments, a 3’ UTR or a proximal sequence thereto Page 80 of 18313406582vlAttorney Docket No.: 2013237-1617comprises a restriction site. In some embodiments, a restriction site is a BamHI site. In some embodiments, a restriction site is a Xhol site.

[0276] In some embodiments, an RNA construct comprises an F element. In some embodiments, a F element sequence is a 3’-UTR of amino-terminal enhancer of split (AES).

[0277] In some embodiments, an RNA disclosed herein comprises a 3’ UTR having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a 3 ’ UTR with the sequence of CTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTC TCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACCACCTC TGCTAGTTCCAGACACCTCCCAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTA GCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAA CTAAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACC (SEQ ID NO: 69). In some embodiments, an RNA disclosed herein comprises a 3’ UTR with the sequence of CTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTC TCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACCACCTC TGCTAGTTCCAGACACCTCCCAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTA GCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAA CTAAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACC (SEQ ID NO: 69).

[0278] In some embodiments, a 3’UTR is an FI element as described in W02017 / 060314, which is herein incorporated by reference in its entirety.B. RNA Formats

[0279] At least three distinct formats useful for RNA compositions (e.g., pharmaceutical compositions) have been developed, namely non-modified uridine containing mRNA (uRNA), nucleoside-modified mRNA (modRNA), and self-amplifying mRNA (saRNA). Each of these platforms displays unique features. In general, in all three formats, RNA is capped, contains open reading frames (ORFs) flanked by untranslated regions (UTR), and have a polyA-tail at the 3' end. An ORF of an uRNA or modRNA encode a polyepitopic vaccine construct described herein. An saRNA has multiple ORFs.Page 81 of 18313406582vlAttorney Docket No.: 2013237-1617

[0280] In some embodiments, the RNA described herein may have modified nucleosides. In some embodiments, the RNA comprises a modified nucleoside in place of at least one (e.g., every) uridine.

[0281] The term “uracil,” as used herein, describes one of the nucleobases that can occur in the nucleic acid of RNA. The structure of uracil is:

[0282] The term “uridine,” as used herein, describes one of the nucleosides that can occur in RNA. The structure of uridine is:

[0283] UTP (uridine 5 ’-triphosphate) has the following structure:oo O 0o.... p 11..o... p li..o...p II...o..I. I:. I.o o o

[0284] Pseudo-UTP (pseudouridine 5 ’-triphosphate) has the following structure:Page 82 of 18313406582vlAttorney Docket No.: 2013237-1617OH OH

[0285] ‘ ‘Pseudouridine” is one example of a modified nucleoside that is an isomer of uridine, where the uracil is attached to the pentose ring via a carbon-carbon bond instead of a nitrogencarbon glycosidic bond.

[0286] Another exemplary modified nucleoside is N1 -methyl-pseudouridine (ml ), which has the structure:

[0287] Nl-methyl-pseudo-UTP has the following structure:OOH OH

[0288] Another exemplary modified nucleoside is 5-methyl-uridine (m5U), which has the structure:Page 83 of 18313406582vlAttorney Docket No.: 2013237-1617

[0289] In some embodiments, one or more uridine in the RNA described herein is replaced by a modified nucleoside. In some embodiments, the modified nucleoside is a modified uridine.

[0290] In some embodiments, RNA comprises a modified nucleoside in place of at least one uridine. In some embodiments, RNA comprises a modified nucleoside in place of each uridine.

[0291] In some embodiments, the modified nucleoside is independently selected from pseudouridine (y), Nl-methyl-pseudouridine (ml\| / ), and 5-methyl-uridine (m5U). In some embodiments, the modified nucleoside comprises pseudouridine (\p)- In some embodiments, the modified nucleoside comprises Nl-methyl-pseudouridine (ml\| / ). In some embodiments, the modified nucleoside comprises 5-methyl-uridine (m5U). In some embodiments, RNA may comprise more than one type of modified nucleoside, and the modified nucleosides are independently selected from pseudouridine (y), Nl-methyl-pseudouridine (ml\| / ), and 5-methyl-uridine (m5U). In some embodiments, the modified nucleosides comprise pseudouridine (y) and Nl-methyl-pseudouridine (ml\| / ). In some embodiments, the modified nucleosides comprise pseudouridine (y) and 5-methyl-uridine (m5U). In some embodiments, the modified nucleosides comprise Nl-methyl-pseudouridine (ml\| / ) and 5-methyl-uridine (m5U). In some embodiments, the modified nucleosides comprise pseudouridine (y), Nl-methyl-pseudouridine (ml\| / ), and 5-methyl-uridine (m5U).

[0292] In some embodiments, the modified nucleoside replacing one or more, e.g., all, uridine in the RNA may be any one or more of 3-methyl-uridine (m3U), 5-methoxy-uridine (mo5U), 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 4-thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho5U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), uridine 5-oxyacetic acid (cmo5U), uridine 5-oxyacetic acid methyl ester (mcmo5U), 5-carboxymethyl-uridine (cm5U), 1 -carboxymethyl-Page 84 of 18313406582vlAttorney Docket No.: 2013237-1617pseudouridine, 5-carboxyhydroxymethyl-uridine (chm5U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm5U), 5 -methoxy carbonylmethyl-uridine (mcm5U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm5s2U), 5-aminomethyl-2-thio-uridine (nm5s2U), 5-methylaminomethyl-uridine (mnm5U), 1-ethyl-pseudouridine, 5-methylaminomethyl-2-thio-uridine (mnm5s2U), 5-methylaminomethyl-2-seleno-uridine (mnm5se2U), 5-carbamoylmethyl-uridine (ncm5U), 5-carboxymethylaminomethyl-uridine (cmnm5U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm5s2U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (rm5U), 1 -taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine(im5s2U), 1 -taurinomethyl-4-thio-pseudouridine), 5-methyl-2-thio-uridine (m5s2U), 1 -methyl-4-thio-pseudouridine (mls4\| / ), 4-thio-l-methyl-pseudouridine, 3-methyl-pseudouridine (m3\| / ), 2-thio-l-methyl-pseudouridine, 1 -methyl- 1 -deaza-pseudouridine, 2-thio-l -methyl- 1 -deaza-pseudouri dine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m5D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1 -methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp3U), l-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp3 \| / ), 5-(isopentenylaminomethyl)uridine (inm5U), 5-(isopentenylaminomethyl)-2-thio-uridine (inm5s2U), a-thio-uridine, 2'-O-methyl-uridine (Um), 5,2'-O-dimethyl-uridine (m5Um), 2'-O-methyl-pseudouridine (\| / m), 2-thio-2'-O-methyl-uridine (s2Um), 5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcm5Um), 5-carbamoylmethyl-2'-O-methyl-uridine (ncm5Um), 5-carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm5Um), 3,2'-O-dimethyl-uridine (m3Um), 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm5Um), 1 -thio-uridine, deoxythymidine, 2'-F-ara-uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5-(2-carbomethoxyvinyl) uridine, 5-[3-(l-E-propenylamino)uridine, or any other modified uridine known in the art.

[0293] In some embodiments, the RNA comprises other modified nucleosides or comprises further modified nucleosides, e.g., modified cytidine. For example, in some embodiments, in the RNA 5 -methylcytidine is substituted partially or completely, preferably completely, for cytidine. In some embodiments, the RNA comprises 5-methylcytidine and one or more selected from pseudouridine (y), N1 -methyl-pseudouridine (ml\| / ), and 5-methyl-uridine (m5U). In some embodiments, the RNA comprises 5-methylcytidine and N1 -methyl-pseudouridine (ml\| / ). InPage 85 of 18313406582vlAttorney Docket No.: 2013237-1617some embodiments, the RNA comprises 5 -methylcytidine in place of each cytidine and Nl-methyl-pseudouridine (ml\| / ) in place of each uridine.

[0294] In some embodiments of the present disclosure, the RNA is “replicon RNA” or simply a “replicon,” in particular “self-replicating RNA” or “self-amplifying RNA.” In one particularly preferred embodiment, the replicon or self-replicating RNA is derived from or comprises elements derived from a single-stranded (ss) RNA virus, in particular a positive-stranded ssRNA virus, such as an alphavirus. Alphaviruses are typical representatives of positive-stranded RNA viruses. Alphaviruses replicate in the cytoplasm of infected cells (for review of the alphaviral life cycle see Jose et al., Future Microbiol., 2009, vol. 4, pp. 837-856, which is incorporated herein by reference in its entirety). The total genome length of many alphaviruses typically ranges between 11,000 and 12,000 nucleotides, and the genomic RNA typically has a 5’-cap, and a 3 ’ poly(A) tail. The genome of alphaviruses encodes non-structural proteins (involved in transcription, modification and replication of viral RNA and in protein modification) and structural proteins (forming the virus particle). There are typically two open reading frames (ORFs) in the genome. The four non-structural proteins (nsPl-nsP4) are typically encoded together by a first ORF beginning near the 5' terminus of the genome, while alphavirus structural proteins are encoded together by a second ORF which is found downstream of the first ORF and extends near the 3 ’ terminus of the genome. Typically, the first ORF is larger than the second ORF, the ratio being roughly 2:1. In cells infected by an alphavirus, only the nucleic acid sequence encoding non-structural proteins is translated from the genomic RNA, while the genetic information encoding structural proteins is translatable from a subgenomic transcript, which is an RNA molecule that resembles eukaryotic messenger RNA (mRNA; Gould et al., 2010, Antiviral Res., vol. 87 pp. 111-124). Following infection, i.e. at early stages of the viral life cycle, the (+) stranded genomic RNA directly acts like a messenger RNA for the translation of the open reading frame encoding the non-structural poly-protein (nsP1234).

[0295] Alphavirus-derived vectors have been proposed for delivery of foreign genetic information into target cells or target organisms. In simple approaches, a first ORF encodes an alphavirus-derived RNA-dependent RNA polymerase (replicase), which upon translation mediates self-amplification of the RNA. A second ORF encoding alphaviral structural proteins is replaced by an open reading frame encoding a polyepitopic vaccine construct described herein.Page 86 of 18313406582vlAttorney Docket No.: 2013237-1617Alphavirus-based trans-replication systems rely on alphavirus nucleotide sequence elements on two separate nucleic acid molecules: one nucleic acid molecule encodes a viral replicase, and the other nucleic acid molecule is capable of being replicated by said replicase in trans (hence the designation trans-replication system). Trans-replication requires the presence of both these nucleic acid molecules in a given host cell. The nucleic acid molecule capable of being replicated by the replicase in trans must comprise certain alphaviral sequence elements to allow recognition and RNA synthesis by the alphaviral replicase.

[0296] Features of a non-modified uridine platform may include, for example, one or more of intrinsic adjuvant effect, as well as good tolerability and safety. Features of modified uridine (e.g., pseudouridine) platform may include reduced adjuvant effect, blunted immune innate immune sensor activating capacity and thus good tolerability and safety. Features of selfamplifying platform may include, for example, long duration of protein expression, good tolerability and safety, higher likelihood for efficacy with very low vaccine dose.

[0297] The present disclosure provides particular RNA constructs optimized, for example, for improved manufacturability, encapsulation, expression level (and / or timing), etc. Certain components are discussed below, and certain preferred embodiments are exemplified herein. C. Codon Optimization and GC Enrichment

[0298] As used herein, the term “codon-optimized” refers to alteration of codons in a coding region of a nucleic acid molecule (e.g., a polyribonucleotide) to reflect the typical codon usage of a host organism (e.g., a subject receiving a nucleic acid molecule (e.g., a polyribonucleotide)) without preferably altering the amino acid sequence encoded by the nucleic acid molecule.Within the context of the present disclosure, in some embodiments, coding regions are codon-optimized for optimal expression in a subject to be treated using the RNA molecules described herein. In some embodiments, codon-optimization may be performed such that codons for which frequently occurring tRNAs are available are inserted in place of “rare codons.” In some embodiments, codon-optimization may include increasing guanosine / cytosine (G / C) content of a coding region of RNA described herein as compared to the G / C content of the corresponding coding sequence of a wild type RNA, wherein the amino acid sequence encoded by the RNA is preferably not modified compared to the amino acid sequence.Page 87 of 18313406582vlAttorney Docket No.: 2013237-1617

[0299] In some embodiments, a coding sequence (also referred to as a “coding region”) is codon optimized for expression in the subject to whom a composition (e.g., a pharmaceutical composition) is to be administered (e.g., a human). Thus, in some embodiments, sequences in such a polynucleotide (e.g., a polyribonucleotide) may differ from wild type sequences encoding the relevant antigen or fragment or epitope thereof, even when the amino acid sequence of the antigen or fragment or epitope thereof is wild type.

[0300] In some embodiments, strategies for codon optimization for expression in a relevant subject (e.g., a human), and even, in some cases, for expression in a particular cell or tissue.

[0301] Various species exhibit particular bias for certain codons of a particular amino acid. Without wishing to be bound by any one theory, codon bias (differences in codon usage between organisms) often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, among other things, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell may generally be a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes may be tailored for optimal gene expression in a given organism based on codon optimization. Codon usage tables are available, for example, at the " Codon Usage Database" available at kazusa.orjp / codon / and these tables may be adapted in a number of ways. Computer algorithms for codon optimizing a particular sequence for expression in a particular subject or its cells are also available, such as Gene Forge (Aptagen; Jacobus, PA), are also available.

[0302] In some embodiments, a polynucleotide (e.g., a polyribonucleotide) of the present disclosure is codon optimized, wherein the codons in the polynucleotide (e.g., the polyribonucleotide) are adapted to human codon usage (herein referred to as “human codon optimized polynucleotide”). Codons encoding the same amino acid occur at different frequencies in a subject, e.g., a human. Accordingly, in some embodiments, the coding sequence of a polynucleotide of the present disclosure is modified such that the frequency of the codons encoding the same amino acid corresponds to the naturally occurring frequency of that codon according to the human codon usage, e.g., as shown in Table 4. For example, in the case of the amino acid Ala, the wild type coding sequence is preferably adapted in a way that the codon “GCC” is used with a frequency of 0.40, the codon “GCT” is used with a frequency of 0.28, the Page 88 of 18313406582vlAttorney Docket No.: 2013237-1617codon “GCA” is used with a frequency of 0.22 and the codon “GCG” is used with 30 a frequency of 0.10 etc. (see Table 4). Accordingly, in some embodiments, such a procedure (as exemplified for Ala) is applied for each amino acid encoded by the coding sequence of a polynucleotide to obtain sequences adapted to human codon usage.Table 4: Human codon usage table with frequencies indicated for each amino acid.Amino acid Codon Frequency Amino acid Codon Frequency Ala GCG 0.10 Pro CCG 0.11Ala GCA 0.22 Pro CCA 0.27Ala GCT 0.28 Pro CCT 0.29Ala GCC* 0.40 Pro CCC* 0.33Cys TGT 0.42 Gin CAG* 0.73Cys TGC* 0.58 Gin CAA 0.27Asp GAT 0.44 Arg AGG 0.22Asp GAC* 0.56 Arg AGA* 0.21Glu GAG* 0.59 Arg CGG 0.19Glu GAA 0.41 Arg CGA 0.10Phe TTT 0.43 Arg CGT 0.09Phe TTC* 0.57 Arg CGC 0.19Gly GGG 0.23 Ser AGT 0.14Gly GGA 0.26 Ser AGC* 0.25Gly GGT 0.18 Ser TCG 0.06Gly GGC* 0.33 Ser TCA 0.15His CAT 0.41 Ser TCT 0.1BHis CAC* 0.59 Ser TCC 0.23lie ATA 0.14 Thr ACG 0.12lie ATT 0.35 Thr ACA 0.27lie ATC* 0.52 Thr ACT 0.23Lys AAG* 0.60 Tor ACC* 0.3BLys AAA 0.40 Vai GTG* 0.4BLeu TTG 0.12 Vai GTA 0.10Leu TTA 0.06 Vai GTT 0.17Leu CTG* 0.43 Vai GTC 0.25Leu CTA 0.07 Trp TGG* 1Leu CTT 0.12 Tyr TAT 042Lou CTC 0.20 Tyr TAC* 0.5BMet ATG* 1 Stop TGA* 0 61Asn AAT 0.44 Stop TAG 0.17Asn AAC* 0.56 Stop TAA 0.22Page 89 of 18313406582vlAttorney Docket No.: 2013237-1617

[0303] Certain strategies for codon optimization and / or G / C enrichment for human expression are described in W02002 / 098443, which is incorporated by reference herein in its entirety. In some embodiments, a coding sequence may be optimized using a multiparametric optimization strategy. In some embodiments, optimization parameters may include parameters that influence protein expression, which can be, for example, impacted on a transcription level, an mRNA level, and / or a translational level. In some embodiments, exemplary optimization parameters include, but are not limited to transcription-level parameters (including, e.g., GC content, consensus splice sites, cryptic splice sites, SD sequences, TATA boxes, termination signals, artificial recombination sites, and combinations thereof); mRNA-level parameters (including, e.g., RNA instability motifs, ribosomal entry sites, repetitive sequences, and combinations thereof); translation-level parameters (including, e.g., codon usage, premature poly(A) sites, ribosomal entry sites, secondary structures, and combinations thereof); or combinations thereof. In some embodiments, a coding sequence may be optimized by a GeneOptimizer algorithm as described in Fath et al. “Multiparameter RNA and Codon Optimization: A Standardized Tool to Assess and Enhance Autologous Mammalian Gene Expression” PLoS ONE 6(3): el7596; Rabb et al., “The GeneOptimizer Algorithm: using a sliding window approach to cope with the vast sequence space in multiparameter DNA sequence optimization” Systems and Synthetic Biology (2010) 4:215-225; and Graft et al. “Codon-optimized genes that enable increased heterologous expression in mammalian cells and elicit efficient immune responses in mice after vaccination of naked DNA” Methods Mol Med (2004) 94:197-210, the entire content of each of which is incorporated herein for the purposes described herein. In some embodiments, a coding sequence may be optimized by Eurofins’ adaption and optimization algorithm “GENEius” as described in Eurofins’ Application Notes: Eurofins’ adaption and optimization software “GENEius” in comparison to other optimization algorithms, the entire content of which is incorporated by reference for the purposes described herein.

[0304] In some embodiments, a coding sequence utilized in accordance with the present disclosure has G / C content that is increased compared to a wild type coding sequence for a polyepitopic vaccine construct described herein, or a portion thereof. In some embodiments, guanosine / cytidine (G / C) content of a coding region is modified relative to a wild type coding sequence for a poly epitopic vaccine construct described herein, but the amino acid sequence encoded by the polyribonucleotide not modified.Page 90 of 18313406582vlAttorney Docket No.: 2013237-1617

[0305] Without wishing to be bound by any particular theory, it is proposed that GC enrichment may improve translation of a payload sequence. Typically, sequences having an increased G (guanosine) / C (cytidine) content are more stable than sequences having an increased A (adenosine) / U (uridine) content. In respect to the fact that several codons code for one and the same amino acid (so-called degeneration of the genetic code), the most favorable codons for the stability can be determined (so-called alternative codon usage). Depending on the amino acid to be encoded by a polyribonucleotide, there are various possibilities for modification of the ribonucleic acid sequence, compared to its wild type sequence. In particular, codons which contain A and / or U nucleosides can be modified by substituting these codons by other codons, which code for the same amino acids but contain no A and / or U or contain a lower content of A and / or U nucleosides.

[0306] In some embodiments, G / C content of a coding region of a polyribonucleotide described herein is increased by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, or even more compared to the G / C content of the coding region prior to codon optimization, e.g., of the wild type RNA. In some embodiments, G / C content of a coding region of a polyribonucleotide described herein is decreased by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, or even more compared to the G / C content of the coding region prior to codon optimization, e.g., of the wild type RNA.

[0307] In some embodiments, stability and translation efficiency of a polyribonucleotide may incorporate one or more elements established to contribute to stability and / or translation efficiency of the polyribonucleotide; exemplary such elements are described, for example, in PCT / EP2006 / 009448 incorporated herein by reference. In some embodiments, to increase expression of a polyribonucleotide used according to the present disclosure, a polyribonucleotide may be modified within the coding region, i.e., the sequence encoding the expressed peptide or protein, without altering the sequence of the expressed peptide or protein, for example so as to increase the GC-content to increase mRNA stability and / or to perform a codon optimization and, thus, enhance translation in cells.IV. RNA Delivery Technologies

[0308] Provided polyribonucleotides may be delivered for therapeutic applications described herein using any appropriate methods known in the art, including, e.g., delivery as naked RNAs,Page 91 of 18313406582vlAttorney Docket No.: 2013237-1617or delivery mediated by viral and / or non-viral vectors, polymer-based vectors, lipid compositions, nanoparticles (e.g., lipid nanoparticles, polymeric nanoparticles, lipid-polymer hybrid nanoparticles, etc.), and / or peptide-based vectors. See, e.g., Wadhwa et al.“Opportunities and Challenges in the Delivery of mRNA-Based Vaccines” Pharmaceutics (2020) 102 (27 pages), the content of which is incorporated herein by reference, for information on various approaches that may be useful for delivery polyribonucleotides described herein.

[0309] In some embodiments, one or more polyribonucleotides can be formulated with lipid nanoparticles for delivery (e.g., administration).

[0310] In some embodiments, lipid nanoparticles can be designed to protect polyribonucleotides from extracellular RNases and / or engineered for systemic delivery of the RNAto target cells (e.g., liver cells). In some embodiments, such lipid nanoparticles may be particularly useful to deliver polyribonucleotides when polyribonucleotides are intravenously or intramuscularly administered to a subject.A. Lipid Compositions1. Lipids and Lipid-Like Materials

[0311] The terms "lipid" and "lipid-like material" are broadly defined herein as molecules which comprise one or more hydrophobic moieties or groups and optionally also one or more hydrophilic moieties or groups. Molecules comprising hydrophobic moieties and hydrophilic moieties are also frequently denoted as amphiphiles. Lipids are usually poorly soluble in water. In an aqueous environment, the amphiphilic nature allows the molecules to self-assemble into organized structures and different phases. One of those phases consists of lipid bilayers, as they are present in vesicles, multilamellar / unilamellar liposomes, or membranes in an aqueous environment. Hydrophobicity can be conferred by the inclusion of a polar groups that include, but are not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups and such groups substituted by one or more aromatic, cycloaliphatic, or heterocyclic group(s). The hydrophilic groups may comprise polar and / or charged groups and include carbohydrates, phosphate, carboxylic, sulfate, amino, sulfhydryl, nitro, hydroxyl, and other like groups.

[0312] Often, an amphiphilic compound has a polar head attached to a long hydrophobic tail. In some embodiments, the polar portion is soluble in water, while the non-polar portion is insolublePage 92 of 18313406582vlAttorney Docket No.: 2013237-1617in water. In addition, the polar portion may have either a formal positive charge, or a formal negative charge. Alternatively, the polar portion may have both a formal positive and a negative charge, and be a zwitterion or inner salt. For purposes of the disclosure, the amphiphilic compound can be, but is not limited to, one or a plurality of natural or non-natural lipids and lipid-like compounds.

[0313] A "lipid-like material" is a substance that is structurally and / or functionally related to a lipid but may not be considered a lipid in a strict sense. For example, the term includes compounds that are able to form amphiphilic layers as they are present in vesicles, multilamellar / unilamellar liposomes, or membranes in an aqueous environment and includes surfactants, or synthesized compounds with both hydrophilic and hydrophobic moieties.Generally speaking, the term refers to molecules, which comprise hydrophilic and hydrophobic moieties with different structural organization, which may or may not be similar to that of lipids.

[0314] Specific examples of amphiphilic compounds that may be included in an amphiphilic layer include, but are not limited to, phospholipids, aminolipids and sphingolipids.

[0315] Generally, lipids may be divided into eight categories: fatty acids, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, polyketides (derived from condensation of ketoacyl subunits), sterols and prenol lipids (derived from condensation of isoprene subunits). Although the term "lipid" is sometimes used as a synonym for fats, fats are a subgroup of lipids called triglycerides. Lipids also encompass molecules such as fatty acids and their derivatives (including tri-, di-, monoglycerides, and phospholipids), as well as sterol-containing metabolites such as cholesterol.

[0316] Fatty acids are a diverse group of molecules made of a hydrocarbon chain that terminates with a carboxylic acid group; this arrangement confers the molecule with a polar, hydrophilic end, and a nonpolar, hydrophobic end that is insoluble in water. The carbon chain, typically between four and 24 carbons long, may be saturated or unsaturated, and may be attached to functional groups containing oxygen, halogens, nitrogen, and sulfur. If a fatty acid contains a double bond, there is the possibility of either a cis or trans geometric isomerism, which significantly affects the molecule's configuration. Cis-double bonds cause the fatty acid chain to bend, an effect that is compounded with more double bonds in the chain. Other major lipid classes in the fatty acid category are the fatty esters and fatty amides.Page 93 of 18313406582vlAttorney Docket No.: 2013237-1617

[0317] Glycerolipids are composed of mono-, di-, and tri-substituted glycerols, the best-known being the fatty acid triesters of glycerol, called triglycerides. The word "triacylglycerol" is sometimes used synonymously with "triglyceride". In these compounds, the three hydroxyl groups of glycerol are each esterified, typically by different fatty acids. Additional subclasses of glycerolipids are represented by glycosylglycerols, which are characterized by the presence of one or more sugar residues attached to glycerol via a glycosidic linkage.

[0318] Glycerophospholipids are amphipathic molecules (containing both hydrophobic and hydrophilic regions) that contain a glycerol core linked to two fatty acid-derived "tails" by ester linkages and to one "head" group by a phosphate ester linkage. Examples of glycerophospholipids, usually referred to as phospholipids (though sphingomyelins are also classified as phospholipids) are phosphatidylcholine (also known as PC, GPCho or lecithin), phosphatidylethanolamine (PE or GPEtn) and phosphatidylserine (PS or GPSer).

[0319] Sphingolipids are members of a complex family of compounds that share a common structural feature, a sphingoid base backbone. The major sphingoid base in mammals is commonly referred to as sphingosine. Ceramides (N-acyl-sphingoid bases) are a major subclass of sphingoid base derivatives with an amide-linked fatty acid. The fatty acids are typically saturated or mono-unsaturated with chain lengths from 16 to 26 carbon atoms. The major phosphosphingolipids of mammals are sphingomyelins (ceramide phosphocholines), whereas insects contain mainly ceramide phosphoethanolamines and fungi have phytoceramide phosphoinositols and mannose-containing headgroups. The glycosphingolipids are a diverse family of molecules composed of one or more sugar residues linked via a glycosidic bond to the sphingoid base. Examples of these are the simple and complex glycosphingolipids such as cerebrosides and gangliosides.

[0320] Sterols, such as cholesterol and its derivatives, or tocopherol and its derivatives, are important components of membrane lipids, along with the glycerophospholipids and sphingomyelins.

[0321] Saccharolipids are compounds in which fatty acids are linked directly to a sugar backbone, forming structures that are compatible with membrane bilayers. In the saccharolipids, a monosaccharide substitutes for the glycerol backbone present in glycerolipids and glycerophospholipids. The most familiar saccharolipids are the acylated glucosamine precursors Page 94 of 18313406582vlAttorney Docket No.: 2013237-1617of the Lipid A component of the lipopolysaccharides in Gram-negative bacteria. Typical lipid A molecules are disaccharides of glucosamine, which are derivatized with as many as seven fatty-acyl chains. The minimal lipopolysaccharide required for growth in E. coli is Kdo2-Lipid A, a hexa-acylated disaccharide of glucosamine that is glycosylated with two 3-deoxy-D-manno-octulosonic acid (Kdo) residues.

[0322] Polyketides are synthesized by polymerization of acetyl and propionyl subunits by classic enzymes as well as iterative and multimodular enzymes that share mechanistic features with the fatty acid synthases. They comprise a large number of secondary metabolites and natural products from animal, plant, bacterial, fungal and marine sources, and have great structural diversity. Many polyketides are cyclic molecules whose backbones are often further modified by glycosylation, methylation, hydroxylation, oxidation, or other processes.

[0323] Lipids and lipid-like materials may be cationic, anionic or neutral. Neutral lipids or lipid-like materials exist in an uncharged or neutral zwitterionic form at a selected pH.

[0324] In some embodiments, suitable lipids or lipid-like materials for use in the present disclosure include those described in W02020 / 128031 and US20200163878, the entire contents of each of which are incorporated herein by reference for the purposes described herein.2. Cationic or cationically ionizable lipids or lipid-like materials

[0325] In some embodiments cationic or cationically ionizable lipids or lipid-like materials contemplated for use herein include any cationic or cationically ionizable lipids or lipid-like materials which are able to electrostatically bind nucleic acid. In one embodiment, cationic or cationically ionizable lipids or lipid-like materials contemplated for use herein can be associated with nucleic acid, e.g. by forming complexes with the nucleic acid or forming vesicles in which the nucleic acid is enclosed or encapsulated.

[0326] Cationic lipids or lipid-like materials are characterized in that they have a net positive charge (e.g., at a relevant pH). Cationic lipids or lipid-like materials bind negatively charged nucleic acid by electrostatic interaction. Generally, cationic lipids possess a lipophilic moiety, such as a sterol, an acyl chain, a diacyl or more acyl chains, and the head group of the lipid typically carries the positive charge.Page 95 of 18313406582vlAttorney Docket No.: 2013237-1617

[0327] In certain embodiments, a cationic lipid or lipid-like material has a net positive charge only at certain pH, in particular acidic pH, while it has preferably no net positive charge, preferably has no charge, i.e., it is neutral, at a different, preferably higher pH such as physiological pH. This ionizable behavior is thought to enhance efficacy through helping with endosomal escape and reducing toxicity as compared with particles that remain cationic at physiological pH.

[0328] In some embodiments, a cationic or cationically ionizable lipid or lipid-like material comprises a head group which includes at least one nitrogen atom (N) which is positive charged or capable of being protonated.

[0329] Examples of cationic lipids include, but are not limited to l,2-dioleoyl-3-trimethylammonium propane (DOTAP); N, N-dimethyl-2, 3 -dioleyloxypropylamine (DODMA), l,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), 3-(N — (N', N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), dimethyldioctadecylammonium (DDAB); l,2-dioleoyl-3-dimethylammonium-propane (DODAP); l,2-diacyloxy-3-dimethylammonium propanes; l,2-dialkyloxy-3 -dimethylammonium propanes; dioctadecyldimethyl ammonium chloride (DODAC), l,2-distearyloxy-N, N-dimethyl-3-aminopropane (DSDMA), 2,3-di(tetradecoxy)propyl-(2-hydroxyethyl)-dimethylazanium (DMRIE), l,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), l,2-dimyristoyl-3-trimethylammonium propane (DMTAP), l,2-dioleyloxypropyl-3-dimethyl-hydroxy ethyl ammonium bromide (DORIE), and 2,3 -dioleoyloxy- N-[2(spermine carboxamide)ethyl]-N, N-dimethyl-l-propanamium trifluoroacetate (DOSPA), l,2-dilinoleyloxy-N, N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N, N-dimethylaminopropane (DLenDMA), dioctadecylamidoglycyl spermine (DOGS), 3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-l-(cis,cis-9,12-oc-tadecadienoxy)propane (CLinDMA), 2-[5'-(cholest-5-en-3-beta-oxy)-3'-oxapentoxy)-3-dimethyl-l-(cis,cis-9',12'-octadecadienoxy)propane (CpLinDMA), N, N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), l,2-N, N'-dioleylcarbamyl-3 -dimethylaminopropane (DOcarbDAP), 2,3-Dilinoleoyloxy-N, N-dimethylpropylamine (DLinDAP), l,2-N, N'-Dilinoleylcarbamyl-3 -dimethylaminopropane (DLincarbDAP), 1,2-Dilinoleoylcarbamyl-3 -dimethylaminopropane (DLinCDAP), 2,2-dilinoleyl-4-dimethylaminomethyl-[l,3]-dioxolane (DLin-K-DMA), 2,2-dilinoleyl-4-dimethylaminoethyl-Page 96 of 18313406582vlAttorney Docket No.: 2013237-1617[l,3]-dioxolane (DLin-K-XTC2-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[l,3]-dioxolane (DLin-KC2-DMA), heptatriaconta-6,9,28,31 -tetraen- 19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA), N-(2-Hydroxyethyl)-N, N-dimethyl-2,3-bis(tetradecyloxy)-l-propanaminium bromide (DMRIE), (±)-N-(3-aminopropyl)-N, N-dimethyl-2,3-bis(cis-9-tetradecenyloxy)-l -propanaminium bromide (GAP-DMORIE), (±)-N-(3-aminopropyl)-N, N-dimethyl-2,3-bis(dodecyloxy)-l-propanaminium bromide (GAP-DLRIE), (±)-N-(3-aminopropyl)-N, N-dimethyl-2,3-bis(tetradecyloxy)-l -propanaminium bromide (GAP -DMRIE), N-(2-Aminoethyl)-N, N-dimethyl-2,3-bis(tetradecyloxy)-l -propanaminium bromide (βAE-DMRIE), N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium (DOBAQ), 2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA), l,2-dimyristoyl-3-dimethylammonium-propane (DMDAP), 1,2-dipalmitoy 1-3 -dimethylammonium-propane (DPD AP), Nl-[2-((lS)-l-[(3 -aminopropy l)amino] -4-[di(3-amino-propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide (MVE5), 1,2-dioleoyl-sn-glycero-3 -ethylphosphocholine (DOEPC), 2,3-bis(dodecyloxy)-N-(2-hydroxyethyl)-N, N-dimethylpropan-l-amonium bromide (DLRIE), N-(2-aminoethyl)-N, N-dimethyl-2,3-bis(tetradecyloxy)propan-l -aminium bromide (DMORIE), di((Z)-non-2-en-l-yl) 8,8'-((((2(dimethylamino)ethyl)thio)carbonyl)azanediyl)dioctanoate (ATX), N, N-dimethyl-2,3-bis(dodecyloxy)propan-l -amine (DLDMA), N, N-dimethyl-2,3-bis(tetradecyloxy)propan-l-amine (DMDMA), Di((Z)-non-2-en-l -yl)-9-((4-(dimethylaminobutanoyl)oxy)heptadecanedioate (L319), N-Dodecyl-3-((2-dodecylcarbamoyl-ethyl)-{2-[(2-dodecylcarbamoyl-ethyl)-2-{(2-dodecylcarbamoyl-ethyl)-[2-(2-dodecylcarbamoyl-ethylamino)-ethyl]-amino}-ethylamino)propionamide (lipidoid 98N12-5), 1-[2-[bis(2-hydroxydodecyl)amino]ethyl-[2-[4-[2-[bis(2-hydroxydodecyl)amino]ethyl]piperazin-1-yl]ethyl]amino]dodecan-2-ol (lipidoid C12-200), LIPOFECTIN® (commercially available cationic liposomes comprising DOTMA and 1,2-dioleoyl-sn-3-phosphoethanolamine (DOPE), from GIBCO / BRL, Grand Island, N.Y.);LIPOFECTAMINE® (commercially available cationic liposomes comprising N-(1-(2,3-dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA) and (DOPE), from GIBCO / BRL); and TRANSFECTAM® (commercially available cationic lipids comprising dioctadecylamidoglycyl carboxyspermine (DOGS) in ethanol from Promega Corp., Madison, Wis.) or any combination of any of the foregoing. Further suitable cationic lipids for use in the present disclosure include those Page 97 of 18313406582vlAttorney Docket No.: 2013237-1617described in W02020 / 128031 and US20200163878, the entire contents of each of which are incorporated herein by reference for the purposes described herein. Further suitable cationic lipids for use in the present disclosure include those described in WO2010 / 053572 (including C12-200 described at paragraph

[0225] ) and W02012 / 170930, both of which are incorporated herein by reference for the purposes described herein. Additional suitable cationic lipids for use in the present disclosure include HGT4003, HGT5000, HGT5001, HGT5001, HGT5002 (see US20150140070A1).

[0330] In some embodiments, formulations that are useful for pharmaceutical compositions (e.g., immunogenic compositions, e.g., vaccines) compositions as described herein can comprise at least one cationic lipid. Representative cationic lipids include, but are not limited to, 1,2-dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleyoxy-3morpholinopropane (DLin-MA), l,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleylthio-3 -dimethylaminopropane (DLin-S-DMA), 1 -linoleoyl-2-linoleyloxy- 3 dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyloxy-3 -trimethylaminopropane chloride salt (DLin-TMA. CI), 1,2-dilinoleoyl-3 -trimethylaminopropane chloride salt (DLin- TAP. CI), 1,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), 3-(N, Ndilinoleylamino)-l,2-propanediol (DLinAP), 3-(N, N-dioleylamino)-l,2-propanediol (DOAP), 1,2-dilinoleyloxo-3-(2-N, N-dimethylamino)ethoxypropane (DLin-EG-DMA), and 2,2-dilinoleyl-4-dimethylaminomethyl-[l,3]-dioxolane (DLin-K-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[l,3]-dioxolane (DLin-KC2-DMA); dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA); MC3 (US20100324120).

[0331] In some embodiments, amino or cationic lipids useful in accordance with the present disclosure have at least one protonatable or deprotonatable group, such that the lipid is positively charged at a pH at or below physiological pH (e.g. pH 7.4), and neutral at a second pH, preferably at or above physiological pH. It will, of course, be understood that the addition or removal of protons as a function of pH is an equilibrium process, and that the reference to a charged or a neutral lipid refers to the nature of the predominant species and does not require that all of lipids have to be present in the charged or neutral form. Lipids having more than one protonatable or deprotonatable group, or which are zwitterionic, are not excluded and may likewise suitable in the context of the present invention.Page 98 of 18313406582vlAttorney Docket No.: 2013237-1617

[0332] In some embodiments, a protonatable lipid has a pKa of the protonatable group in the range of about 4 to about 11, e.g., a pKa of about 5 to about 7.

[0333] In some embodiments, a cationic lipid may comprise from about 10 mol % to about 100 mol %, about 20 mol % to about 100 mol %, about 30 mol % to about 100 mol %, about 40 mol % to about 100 mol %, or about 50 mol % to about 100 mol % of total lipid present in a lipid composition utilized in accordance with the present disclosure.3. Additional lipids or lipid-like materials

[0334] In some embodiments, formulations utilized in accordance with the present disclosure may comprise lipids or lipid-like materials other than cationic or cationically ionizable lipids or lipid-like materials, i.e., non-cationic lipids or lipid-like materials (including non-cationically ionizable lipids or lipid-like materials). Collectively, anionic and neutral lipids or lipid-like materials are referred to herein as non-cationic lipids or lipid-like materials. In some embodiments, optimizing a formulation of nucleic acid particles by addition of other hydrophobic moieties, such as cholesterol and lipids, in addition to an ionizable / cationic lipid or lipid-like material may, for example, enhance particle stability and efficacy of nucleic acid delivery.

[0335] In some embodiments, a lipid or lipid-like material may be incorporated which may or may not affect the overall charge of particles. In certain embodiments, such lipid or lipid-like material is a non-cationic lipid or lipid-like material.

[0336] In some embodiments, a non-cationic lipid may comprise, e.g., one or more anionic lipids and / or neutral lipids. An "anionic lipid" is negatively charged (e.g., at a selected pH).

[0337] A "neutral lipid" exists either in an uncharged or neutral zwitterionic form (e.g., at a selected pH). In some embodiments, a formulation comprises one of the following neutral lipid components: (1) a phospholipid, (2) cholesterol or a derivative thereof; or (3) a mixture of a phospholipid and cholesterol or a derivative thereof. Examples of cholesterol derivatives include, but are not limited to, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'- hydroxybutyl ether, tocopherol and derivatives thereof, and mixtures thereof.Page 99 of 18313406582vlAttorney Docket No.: 2013237-1617

[0338] Specific exemplary phospholipids that can be used include, but are not limited to, phosphatidylcholines, phosphatidylethanolamines, phosphatidylglycerols, phosphatidic acids, phosphatidylserines or sphingomyelin. Such phospholipids include in particular diacylphosphatidylcholines, such as distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphatidylcholine (DLPC), palmitoyloleoyl-phosphatidylcholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), l-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1 -hexadecyl-sn-glycero-3-phosphocholine (Cl 6 Lyso PC) and phosphatidylethanolamines, in particular diacylphosphatidylethanolamines, such as dioleoylphosphatidylethanolamine (DOPE), distearoyl-phosphatidylethanolamine (DSPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), dilauroyl-phosphatidylethanolamine (DLPE), diphytanoyl-phosphatidylethanolamine (DPyPE), and further phosphatidylethanolamine lipids with different hydrophobic chains.

[0339] In certain embodiments, a formulation utilized in accordance with the present disclosure includes DSPC or DSPC and cholesterol.

[0340] In certain embodiments, formulations utilized in accordance with the present disclosure include both a cationic lipid and an additional (non-cationic) lipid.

[0341] In some embodiments, formulations herein include a polymer conjugated lipid such as a pegylated lipid. " Pegylated lipids" comprise both a lipid portion and a polyethylene glycol portion. Pegylated lipids are known in the art.

[0342] Without wishing to be bound by theory, the amount of (total) cationic lipid compared to the amount of other lipid(s) in formulation may affect important characteristics, such as charge, particle size, stability, tissue selectivity, and bioactivity of the nucleic acid. In some embodiments, the molar ratio of the at least one cationic lipid to the at least one additional lipid is from about 10:0 to about 1:9, about 4:1 to about 1:2, or about 3:1 to about 1:1.Page 100 of 18313406582vlAttorney Docket No.: 2013237-1617

[0343] In some embodiments, a non-cationic lipid, in particular a neutral lipid, (e.g., one or more phospholipids and / or cholesterol) may comprise from about 0 mol % to about 90 mol %, from about 0 mol % to about 80 mol %, from about 0 mol % to about 70 mol %, from about 0 mol % to about 60 mol %, or from about 0 mol % to about 50 mol %, of the total lipid present in a formulation.4. Lipoplex Particles

[0344] In certain embodiments of the present disclosure, the RNA described herein may be present in RNA lipoplex particles.

[0345] An " RNA lipoplex particle" contains lipid, in particular cationic lipid, and RNA.Electrostatic interactions between positively charged liposomes and negatively charged RNA results in complexation and spontaneous formation of RNA lipoplex particles. Positively charged liposomes may be generally synthesized using a cationic lipid, such as DOTMA, and additional lipids, such as DOPE. In one embodiment, a RNA lipoplex particle is a nanoparticle.

[0346] In certain embodiments, RNA lipoplex particles include both a cationic lipid and an additional lipid. In an exemplary embodiment, the cationic lipid is DOTMA and the additional lipid is DOPE.

[0347] In some embodiments, the molar ratio of the at least one cationic lipid to the at least one additional lipid is from about 10:0 to about 1:9, about 4: 1 to about 1:2, or about 3: 1 to about 1:1. In specific embodiments, the molar ratio may be about 3:1, about 2.75:1, about 2.5:1, about 2.25:1, about 2:1, about 1.75:1, about 1.5:1, about 1.25:1, or about 1:1. In an exemplary embodiment, the molar ratio of the at least one cationic lipid to the at least one additional lipid is about 2:1.

[0348] In some embodiments, RNA lipoplex particles have an average diameter that in one embodiment ranges from about 200 nm to about 1000 nm, from about 200 nm to about 800 nm, from about 250 to about 700 nm, from about 400 to about 600 nm, from about 300 nm to about 500 nm, or from about 350 nm to about 400 nm. In specific embodiments, the RNA lipoplex particles have an average diameter of about 200 nm, about 225 nm, about 250 nm, about 275 nm, about 300 nm, about 325 nm, about 350 nm, about 375 nm, about 400 nm, about 425 nm, about 450 nm, about 475 nm, about 500 nm, about 525 nm, about 550 nm, about 575 nm, about 600Page 101 of 18313406582vlAttorney Docket No.: 2013237-1617nm, about 625 nm, about 650 nm, about 700 nm, about 725 nm, about 750 nm, about 775 nm, about 800 nm, about 825 nm, about 850 nm, about 875 nm, about 900 nm, about 925 nm, about 950 nm, about 975 nm, or about 1000 nm. In an embodiment, the RNA lipoplex particles have an average diameter that ranges from about 250 nm to about 700 nm. In another embodiment, the RNA lipoplex particles have an average diameter that ranges from about 300 nm to about 500 nm. In an exemplary embodiment, the RNA lipoplex particles have an average diameter of about 400 nm.

[0349] RNA lipoplex particles and compositions comprising RNA lipoplex particles described herein are useful for delivery of RNA to a target tissue after parenteral administration, in particular after intravenous administration. The RNA lipoplex particles may be prepared using liposomes that may be obtained by injecting a solution of the lipids in ethanol into water or a suitable aqueous phase. In one embodiment, the aqueous phase has an acidic pH. In one embodiment, the aqueous phase comprises acetic acid, e.g., in an amount of about 5 mM.Liposomes may be used for preparing RNA lipoplex particles by mixing the liposomes with RNA. In one embodiment, the liposomes and RNA lipoplex particles comprise at least one cationic lipid and at least one additional lipid. In one embodiment, the at least one cationic lipid comprises l,2-di-O-octadecenyl-3 -trimethylammonium propane (DOTMA) and / or 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP). In one embodiment, the at least one additional lipid comprises l,2-di-(9Z-octadecenoyl)-sn-glycero-3 -phosphoethanolamine (DOPE), cholesterol (Choi) and / or l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). In one embodiment, the at least one cationic lipid comprises l,2-di-O-octadecenyl-3 -trimethylammonium propane (DOTMA) and the at least one additional lipid comprises l,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE). In one embodiment, the liposomes and RNA lipoplex particles comprise l,2-di-O-octadecenyl-3 -trimethylammonium propane (DOTMA) and l,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE).

[0350] Spleen targeting RNA lipoplex particles are described in WO 2013 / 143683, herein incorporated by reference. It has been found that RNA lipoplex particles having a net negative charge may be used to preferentially target spleen tissue or spleen cells such as antigen-presenting cells, in particular dendritic cells. Accordingly, following administration of the RNA lipoplex particles, RNA accumulation and / or RNA expression in the spleen occurs. Thus, RNAPage 102 of 18313406582vlAttorney Docket No.: 2013237-1617lipoplex particles of the disclosure may be used for expressing RNA in the spleen. In an embodiment, after administration of the RNA lipoplex particles, no or essentially no RNA accumulation and / or RNA expression in the lung and / or liver occurs. In one embodiment, after administration of the RNA lipoplex particles, RNA accumulation and / or RNA expression in antigen presenting cells, such as professional antigen presenting cells in the spleen occurs. Thus, RNA lipoplex particles of the disclosure may be used for expressing RNA in such antigen presenting cells. In one embodiment, the antigen presenting cells are dendritic cells and / or macrophages.5. Lipid Nanoparticles (LNPs)

[0351] In some embodiments, nucleic acid such as RNA described herein is administered in the form of lipid nanoparticles (LNPs). In some embodiments, LNPs may comprise any lipid capable of forming a particle to which the one or more nucleic acid molecules are attached, or in which the one or more nucleic acid molecules are encapsulated.

[0352] In some embodiments, an LNP comprises one or more cationic lipids, and one or more stabilizing lipids. Stabilizing lipids include neutral lipids and pegylated lipids.

[0353] In some embodiments, an LNP comprises a cationic lipid, a neutral lipid, a sterol, a polymer conjugated lipid; and an RNA, encapsulated within or associated with the lipid nanoparticle.

[0354] In some embodiments, a neutral lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE, DOPG, DPPG, POPE, DPPE, DMPE, DSPE, and SM. In some embodiments, the neutral lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM. In some embodiments, the neutral lipid is DSPC.

[0355] In some embodiments, a sterol is cholesterol.

[0356] In some embodiments, a polymer conjugated lipid is a pegylated lipid. In some embodiments, a pegylated lipid has the following structure:Page 103 of 18313406582vlAttorney Docket No.: 2013237-1617R13or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein:R12and R13are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds; and w has a mean value ranging from 30 to 60. In some embodiments, R12and R13are each independently straight, saturated alkyl chains containing from 12 to 16 carbon atoms. In some embodiments, w has a mean value ranging from 40 to 55. In some embodiments, the average w is about 45. In some embodiments, R12and R13are each independently a straight, saturated alkyl chain containing about 14 carbon atoms, and w has a mean value of about 45.

[0357] In some embodiments, a pegylated lipid is DMG-PEG 2000, e.g., having the following structure:Un

[0358] In some embodiments, a cationic lipid component of LNPs has the structure of Formula (III):R3^G3, L1. / N. ^L2R1^G1<32R2(III)or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein:Page 104 of 18313406582vlAttorney Docket No.: 2013237-1617one of L1or L2is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)X-, -S-S-, -C(=O)S-, SC(=O)-, -NRaC(=O)-, -C(=O)NRa-, NRaC(=O)NRa-, -OC(=O)NRa- or -NRaC(=O)O-, and the other of L1or L2is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)X-, -S-S-, -C(=O)S-, SC(=O)-, -NRaC(=O)-, -C(=O)NRa-, NRaC(=O)NRa-, -OC(=O)NRa- or -NRaC(=O)O- or a direct bond;G1and G2are each independently unsubstituted C1-C12 alkylene or C1-C12 alkenylene;G3is C1-C24 alkylene, C1-C24 alkenylene, C3-C8 cycloalkylene, C3-C8 cycloalkenylene;Rais H or C1-C12 alkyl;R1and R2are each independently C6-C24 alkyl or C6-C24 alkenyl;R3is H, OR5, CN, -C(=O)OR4, -OC(=O)R4or -NR5C(=O)R4;R4is C1-C12 alkyl;R5is H or C1-C6 alkyl; andx is 0, 1 or 2.

[0359] In some of the foregoing embodiments of Formula (III), the lipid has one of the following structures (IIIA) or (IIIB):(IIIA) (IIIB)wherein:A is a 3 to 8-membered cycloalkyl or cycloalkylene ring;R6is, at each occurrence, independently H, OH or C1-C24 alkyl;n is an integer ranging from 1 to 15.

[0360] In some of the foregoing embodiments of Formula (III), the lipid has structure (IIIA), and in other embodiments, the lipid has structure (IIIB).Page 105 of 18313406582vlAttorney Docket No.: 2013237-1617

[0361] In other embodiments of Formula (III), the lipid has one of the following structures (IIIC) or (IIID):wherein y and z are each independently integers ranging from 1 to 12.

[0362] In any of the foregoing embodiments of Formula (III), one of L1or L2is -O(C=O)-. For example, in some embodiments each of L1and L2are -O(C=O)-. In some different embodiments of any of the foregoing, L1and L2are each independently -(C=O)O- or -O(C=O)-. For example, in some embodiments each of L1and L2is -(C=O)O-.

[0363] In some different embodiments of Formula (III), the lipid has one of the following structures (IIIE) or (IIIF):(IIIE) (IIIF)

[0364] In some of the foregoing embodiments of Formula (III), the lipid has one of the following structures (IIIG), (IIIH), (IIII), or (IIIJ):(IIIG) (IIIH)Page 106 of 18313406582vlAttorney Docket No.: 2013237-1617(IIII) (IIIJ)

[0365] In some of the foregoing embodiments of Formula (III), n is an integer ranging from 2 to 12, for example from 2 to 8 or from 2 to 4. For example, in some embodiments, n is 3, 4, 5 or 6. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 6.

[0366] In some other of the foregoing embodiments of Formula (III), y and z are each independently an integer ranging from 2 to 10. For example, in some embodiments, y and z are each independently an integer ranging from 4 to 9 or from 4 to 6.

[0367] In some of the foregoing embodiments of Formula (III), R6is H. In other of the foregoing embodiments, R6is C1-C24 alkyl. In other embodiments, R6is OH.

[0368] In some embodiments of Formula (III), G3is unsubstituted. In other embodiments, G3 is substituted. In various different embodiments, G3is linear C1-C24 alkylene or linear C1-C24 alkenylene.

[0369] In some other foregoing embodiments of Formula (III), R1or R2, or both, is C6-C24 alkenyl. For example, in some embodiments, R1and R2each, independently have the following structure:R7aR7bwherein:R7aand R7bare, at each occurrence, independently H or C1-C12 alkyl; anda is an integer from 2 to 12,Page 107 of 18313406582vlAttorney Docket No.: 2013237-1617wherein R7a, R7band a are each selected such that R1and R2each independently comprise from 6 to 20 carbon atoms. For example, in some embodiments a is an integer ranging from 5 to 9 or from 8 to 12.

[0370] In some of the foregoing embodiments of Formula (III), at least one occurrence of R7ais H. For example, in some embodiments, R7ais H at each occurrence. In other different embodiments of the foregoing, at least one occurrence of R7bis C1-C8 alkyl. For example, in some embodiments, C1-C8 alkyl is methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tertbutyl, n-hexyl or n-octyl.

[0371] In different embodiments of Formula (III), R1or R2, or both, has one of the following structures:

[0372] In some of the foregoing embodiments of Formula (III), R3is OH,CN, -C(=O)OR4, -OC(=O)R4or -NHC(=O)R4. In some embodiments, R4is methyl or ethyl.

[0373] In various different embodiments, the cationic lipid of Formula (III) has one of the structures set forth in in Table 5 below.Table 5: Exemplary Compounds of Formula (HI).Page 108 of 18313406582vlAttorney Docket No.: 2013237-1617No. StructureI0III-20I0III-300III-4100HO\X / X^'N\III-5100HO^^N>III-610H ~YXX\XX-^^I0III-70Page 109 of 18313406582vlAttorney Docket No.: 2013237-1617No. StructureI0III-BTo 0Q _OH I 0III-9O=\ \\ o0( °\° — \ / III- 10OIII- 11III- 120III- 13 HO^N / XJ0Page 110 of 183 13406582vlAttorney Docket No.: 2013237-1617No. StructureIII- 14XooIII- 15 Z i—\ O ' - ° \ \ 7 \ ) \oo 2 °=) )° )= \° \= — ' ° ' —\ ° ' —) ) \ / ) \° °=o°=(o=\2 O — y / \ / ^ 7 °oo==III- 16 / ^o=Z z— '—> Z.—' 'Z.—'X X X X o o o oIII- 17III- 1BIII- 19HI-2000Page 111 of 183 13406582vlAttorney Docket No.: 2013237-1617No. StructureIII-21Io0III-2210( o= \o / (o=\I ° — \ / 0III-2300III-24100III-2510I0III-260Page 112 of 18313406582vlAttorney Docket No.: 2013237-1617No. StructureIII-27XoZ— yI0III-2BO \^ } O=0O\ / (O= / \I0III-290OH I 0III-300I0III-310H<\ JIII-32 l00Page 113 of 183 13406582vlAttorney Docket No.: 2013237-1617

[0374] In various different embodiments, a cationic lipid has one of the structures set forth in Table 6 below.Table 6: Exemplary Cationic Lipid Structures.No. StructureABPage 114 of 18313406582vlAttorney Docket No.: 2013237-1617

[0375] In some embodiments, an LNP comprises a cationic lipid that is an ionizable lipid-like material (lipidoid). In some embodiments, a cationic lipid has the following structure:

[0376] In some embodiments, lipid nanoparticles can have an average size (e.g., mean diameter) of about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 70 to about 90 nm, or about 70 nm to about 80 nm. In some embodiments, lipid Page 115 of 18313406582vlAttorney Docket No.: 2013237-1617nanoparticles in accordance with the present disclosure can have an average size (e.g., mean diameter) of about 50 nm to about 100 nm. In some embodiments, lipid nanoparticles may have an average size (e.g., mean diameter) of about 50 nm to about 150 nm. In some embodiments, lipid nanoparticles may have an average size (e.g., mean diameter) of about 60 nm to about 120 nm. In some embodiments, lipid nanoparticles in accordance with the present disclosure can have an average size (e.g., mean diameter) of about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm. The term “average diameter” or “mean diameter” refers to the mean hydrodynamic diameter of particles as measured by dynamic laser light scattering (DLS) with data analysis using the so-called cumulant algorithm, which provides as results the so-called Z-average with the dimension of a length, and the poly dispersity index (PI), which is dimensionless (Koppel, D., J. Chem. Phys. 57, 1972, pp 4814-4820, ISO 13321, which is herein incorporated by reference). Here “average diameter,” “mean diameter,” “diameter,” or “size” for particles is used synonymously with this value of the Z-average.

[0377] In some embodiments, lipid nanoparticles described herein may exhibit a polydispersity index less than about 0.5, less than about 0.4, less than about 0.3, or about 0.2 or less. By way of example, lipid nanoparticles can exhibit a poly dispersity index in a range of about 0.1 to about 0.3 or about 0.2 to about 0.3. The “poly dispersity index” is preferably calculated based on dynamic light scattering measurements by the so-called cumulant analysis as mentioned in the definition of the “average diameter.” Under certain prerequisites, it can be taken as a measure of the size distribution of an ensemble of ribonucleic acid nanoparticles (e.g., ribonucleic acid nanoparticles).

[0378] Lipid nanoparticles described herein can be characterized by an “N / P ratio,” which is the molar ratio of cationic (nitrogen) groups (the “N” in N / P) in the cationic polymer to the anionic (phosphate) groups (the “P” in N / P) in RNA. It is understood that a cationic group is one that is either in cationic form (e.g., N+), or one that is ionizable to become cationic. Use of a single number in an N / P ratio (e.g., an N / P ratio of about 5) is intended to refer to that number over 1, e.g., an N / P ratio of about 5 is intended to mean 5:1. In some embodiments, a lipid nanoparticle described herein has an N / P ratio greater than or equal to 5. In some embodiments, a lipid nanoparticle described herein has an N / P ratio that is about 5, 6, 7, 8, 9, or 10. In somePage 116 of 18313406582vlAttorney Docket No.: 2013237-1617embodiments, an N / P ratio for a lipid nanoparticle described herein is from about 10 to about 50. In some embodiments, an N / P ratio for a lipid nanoparticle described herein is from about 10 to about 70. In some embodiments, an N / P ratio for a lipid nanoparticle described herein is from about 10 to about 120.B. Exemplary Methods of Making Lipid Nanoparticles

[0379] Lipids and lipid nanoparticles comprising nucleic acids and their method of preparation are known in the art, including, e.g., as described in U. S. Patent Nos. 8,569,256, 5,965,542 and U. S. Patent Publication Nos. 2016 / 0199485, 2016 / 0009637, 2015 / 0273068, 2015 / 0265708, 2015 / 0203446, 2015 / 0005363, 2014 / 0308304, 2014 / 0200257, 2013 / 086373, 2013 / 0338210, 2013 / 0323269, 2013 / 0245107, 2013 / 0195920, 2013 / 0123338, 2013 / 0022649, 2013 / 0017223, 2012 / 0295832, 2012 / 0183581, 2012 / 0172411, 2012 / 0027803, 2012 / 0058188, 2011 / 0311583, 2011 / 0311582, 2011 / 0262527, 2011 / 0216622, 2011 / 0117125, 2011 / 0091525, 2011 / 0076335, 2011 / 0060032, 2010 / 0130588, 2007 / 0042031, 2006 / 0240093, 2006 / 0083780, 2006 / 0008910, 2005 / 0175682, 2005 / 017054, 2005 / 0118253, 2005 / 0064595, 2004 / 0142025, 2007 / 0042031, 1999 / 009076 and PCT Pub. Nos. WO 99 / 39741, WO 2018 / 081480, WO 2017 / 004143, WO 2017 / 075531, WO 2015 / 199952, WO 2014 / 008334, WO 2013 / 086373, WO 2013 / 086322, WO 2013 / 016058, WO 2013 / 086373, W02011 / 141705, and WO 2001 / 07548, the full disclosures of which are herein incorporated by reference in their entirety for the purposes described herein.

[0380] For example, in some embodiments, cationic lipids, neutral lipids (e.g., DSPC, and / or cholesterol) and polymer-conjugated lipids can be solubilized in ethanol at a pre-determined molar ratio (e.g., ones described herein). In some embodiments, lipid nanoparticles (lipid nanoparticle) are prepared at a total lipid to polyribonucleotides weight ratio of approximately 10: 1 to 30: 1. In some embodiments, such polyribonucleotides can be diluted to 0.2 mg / mL in acetate buffer. In some embodiments, preformed lipid nanoparticles (pre-LNPs) are prepared by mixing (i) lipids in an organic solvent with (ii) an acidified aqueous phase. In some embodiments, the resulting pre-LNPs are subjected to buffer exchange (e.g., to remove the organic solvent) prior to mixing with polyribonucleotide solutions, thereby forming pre-LNPs. In some embodiments, pre-LNPs are mixed with a polyribonucleotide solution to form polyribonucleotide lipid nanoparticles (RNA-LNPs).Page 117 of 18313406582vlAttorney Docket No.: 2013237-1617

[0381] In some embodiments, using an ethanol injection technique, a colloidal lipid dispersion comprising polyribonucleotides can be formed as follows: an ethanol solution comprising lipids, such as cationic lipids, neutral lipids, and polymer-conjugated lipids, is injected into an aqueous solution comprising polyribonucleotides (e.g., ones described herein).

[0382] In some embodiments, lipid and polyribonucleotide solutions can be mixed at room temperature by pumping each solution at controlled flow rates into a mixing unit, for example, using piston pumps. In some embodiments, the flow rates of a lipid solution and a RNA solution into a mixing unit are maintained at a ratio of 1:3. Upon mixing, nucleic acid-lipid particles are formed as the ethanolic lipid solution is diluted with aqueous polyribonucleotides. The lipid solubility is decreased, while cationic lipids bearing a positive charge interact with the negatively charged RNA.

[0383] In some embodiments, a solution comprising RNA-encapsulated lipid nanoparticles can be processed by one or more of concentration adjustment, buffer exchange, formulation, and / or filtration.

[0384] In some embodiments, RNA-encapsulated lipid nanoparticles can be processed through filtration.

[0385] In some embodiments, particle size and / or internal structure of lipid nanoparticles (with or without RNAs) may be monitored by appropriate techniques such as, e.g., small-angle X-ray scattering (SAXS) and / or transmission electron cryomicroscopy (CryoTEM).V. Pharmaceutical Compositions

[0386] The present disclosure provides compositions, e.g., pharmaceutical compositions comprising one or more polyribonucleotides described herein. Pharmaceutical formulations may additionally comprise a pharmaceutically acceptable excipient, which, as used herein, includes any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington's The Science and Practice of Pharmacy, 21st Edition, A. R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2006; incorporated herein by reference) discloses various excipients used in formulating pharmaceutical compositions and known techniques for the preparationPage 118 of 18313406582vlAttorney Docket No.: 2013237-1617thereof. Except insofar as any conventional excipient medium is incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated to be within the scope of this disclosure.

[0387] In some embodiments, an excipient is approved for use in humans and for veterinary use. In some embodiments, an excipient is approved by the United States Food and Drug Administration. In some embodiments, an excipient is pharmaceutical grade. In some embodiments, an excipient meets the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and / or the International Pharmacopoeia.

[0388] Pharmaceutically acceptable excipients used in the manufacture of pharmaceutical compositions include, but are not limited to, inert diluents, dispersing and / or granulating agents, surface active agents and / or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and / or oils. Such excipients may optionally be included in pharmaceutical formulations. Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and / or perfuming agents can be present in the composition, according to the judgment of the formulator.

[0389] General considerations in the formulation and / or manufacture of pharmaceutical agents may be found, for example, in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference).

[0390] In some embodiments, pharmaceutical compositions provided herein may be formulated with one or more pharmaceutically acceptable carriers or diluents as well as any other known adjuvants and excipients in accordance with conventional techniques such as those disclosed in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference).

[0391] Pharmaceutical compositions described herein can be administered by appropriate methods known in the art. As will be appreciated by a skilled artisan, the route and / or mode of administration may depend on a number of factors, including, e.g., but not limited to stability and / or pharmacokinetics and / or pharmacodynamics of pharmaceutical compositions described herein.Page 119 of 18313406582vlAttorney Docket No.: 2013237-1617

[0392] In some embodiments, pharmaceutical compositions described herein are formulated for parenteral administration, which includes modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intradermal, subcutaneous, subcuticular, or intraarticular injection and infusion. In preferred embodiments, pharmaceutical compositions described herein are formulated for intravenous, intramuscular, or subcutaneous administration.

[0393] In some embodiments, pharmaceutical compositions described herein are formulated for intravenous administration. In some embodiments, pharmaceutically acceptable excipients that may be useful for intravenous administration include sterile aqueous solutions or dispersions and sterile powders for preparation of sterile injectable solutions or dispersions.

[0394] Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, lipid nanoparticles, or other ordered structure suitable to high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. In some embodiments, prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

[0395] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization and / or microfiltration. In some embodiments, pharmaceutical compositions can be prepared as described herein and / or methods known in the art.

[0396] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the presence of microorganisms may be ensured both by sterilization procedures, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into Page 120 of 18313406582vlAttorney Docket No.: 2013237-1617pharmaceutical compositions described herein. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

[0397] Formulations of pharmaceutical 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 active ingredient(s) into association with a diluent or another excipient and / or one or more other accessory ingredients, and then, if necessary and / or desirable, shaping and / or packaging the product into a desired single- or multi-dose unit.

[0398] A pharmaceutical composition in accordance with the present disclosure may be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as a plurality of single unit doses. As used herein, a “unit dose” is discrete amount of the pharmaceutical composition comprising a predetermined amount of at least one RNA product produced using a system and / or method described herein.

[0399] Relative amounts of polyribonucleotides encapsulated in lipid nanoparticles, a pharmaceutically acceptable excipient, and / or any additional ingredients in a pharmaceutical composition can vary, depending upon the subject to be treated, target cells, diseases or disorders, and may also further depend upon the route by which the composition is to be administered.

[0400] In some embodiments, pharmaceutical compositions described herein are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art. Actual dosage levels of the active ingredients (e.g., polyribonucleotides encapsulated in lipid nanoparticles) in the pharmaceutical compositions described herein may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present disclosure employed, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and / or materials used in combination with the particular compositions employed, the age, sex, weight, condition, generalPage 121 of 18313406582vlAttorney Docket No.: 2013237-1617health and prior medical history of the patient being treated, and like factors well known in the medical arts.

[0401] A physician having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, a physician could start doses of active ingredients (e.g., polyribonucleotides encapsulated in lipid nanoparticles) employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[0402] In some embodiments, a pharmaceutical composition is formulated (e.g., but not limited to, for intravenous, intramuscular, or subcutaneous administration) to deliver a dose of about 5 mg RNA / kg.

[0403] In some embodiments, a pharmaceutical composition described herein may further comprise one or more additives, for example, in some embodiments that may enhance stability of such a composition under certain conditions. Examples of additives may include but are not limited to salts, buffer substances, preservatives, and carriers. For example, in some embodiments, a pharmaceutical composition may further comprise a cryoprotectant (e.g., sucrose) and / or an aqueous buffered solution, which may in some embodiments include one or more salts, including, e.g., alkali metal salts or alkaline earth metal salts such as, e.g., sodium salts, potassium salts, and / or calcium salts.

[0404] In some embodiments, a pharmaceutical composition provided herein is a preservative-free, sterile RNA-lipid nanoparticle dispersion in an aqueous buffer for intravenous or intramuscular administration.

[0405] Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions that are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and / or perform such modification with merely ordinary, if any, experimentation.Page 122 of 18313406582vlAttorney Docket No.: 2013237-1617VI. Treatment Methods

[0406] In some embodiments, one or more pharmaceutical compositions comprising one or more vaccine constructs described herein (e.g., one or more polyribonucleotides described herein) can be taken up by target cells (e.g., dendritic cells) for translation of polyepitopic-encoding RNA(s) thereby, inducing CD4+ and / or CD8+ T cell immunity against one or more of the epitopes. Accordingly, another aspect of the present disclosure relates to methods of using pharmaceutical compositions described herein. For example, one aspect provided herein is a method comprising administering a provided pharmaceutical composition to a subject suffering from cancer. In some embodiments, a provided pharmaceutical composition is administered by intravenous injection or infusion.

[0407] In some embodiments, technologies of the present disclosure may be administered to subjects according to a particular dosing regimen. In some embodiments, a dosing regimen may involve a single administration; in some embodiments, a dosing regimen may comprise one or more “booster” administrations after the initial administration. In some embodiments, initial and boost doses are the same amount; in some embodiments they differ. In some embodiments, two or more booster doses are administered. In some embodiments, a plurality of doses are administered at regular intervals. In some embodiments, periods of time between doses become longer.

[0408] In some embodiments, administered pharmaceutical compositions (e.g., immunogenic compositions, e.g., vaccines) comprising RNA constructs that encode one or more vaccine constructs are administered in RNA doses of from about 0.1 pg to about 300 pg, about 0.5 pg to about 200 pg, or about 1 pg to about 100 pg, such as about 1 pg, about 3 pg, about 10 pg, about 30 pg, about 50 pg, or about 100 pg. In some embodiments, an saRNA construct is administered at a lower dose (e.g., 2, 4, 5, 10 fold or more lower) than a modRNA or uRNA construct.

[0409] In some embodiments, a first booster dose is administered within about six months of the initial dose, and preferably within about 5, 4, 3, 2, or 1 months. In some embodiments, a first booster dose is administered in a time period that begins about 1, 2, 3, or 4 weeks after the first dose, and ends about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks of the first dose (e.g., between about 1 and about 12 weeks after the first dose, or between about 2 or 3 weeks and about 5 and 6 weeks after the first dose, or about 3 weeks or about 4 weeks after the first dose).Page 123 of 18313406582vlAttorney Docket No.: 2013237-1617

[0410] In some embodiments, a plurality of booster doses (e.g., 2, 3, or 4) doses are administered within 6 months of the first dose, or within 12 months of the first dose.

[0411] In some embodiments, an RNA dose is about 60 pg or lower, 50 pg or lower, 40 pg or lower, 30 pg or lower, 20 pg or lower, 10 pg or lower, 5 pg or lower, 2.5 pg or lower, or 1 pg or lower. In some embodiments, an RNA dose is about 0.25 pg, at least 0.5 pg, at least 1 pg, at least 2 pg, at least 3 pg, at least 4 pg, at least 5 pg, at least 10 pg, at least 20 pg, at least 30 pg, or at least 40 pg. In some embodiments, an RNA dose is about 0.25 pg to 60 pg, 0.5 pg to 55 pg, 1 pg to 50 pg, 5 pg to 40 pg, or 10 pg to 30 pg may be administered per dose. In some embodiments, an RNA dose is about 30 pg. In some embodiments, at least two such doses are administered. For example, a second dose may be administered about 21 days following administration of the first dose. In some embodiments, a first booster dose is administered about one month after an initial dose. In some such embodiments, at least one further booster is administered at one-month interval(s). In some embodiments, after 2 or 3 boosters, a longer interval is introduced and no further booster is administered for at least 6, 9, 12, 18, 24, or more months. In some embodiments, a single further booster is administered after about 18 months.

[0412] In some embodiments, provided treatment methods are administered to subjects in combination with one or more other therapies (e.g., surgery, radiation, chemotherapy, other biologic therapy, etc..VII. Methods of Manufacture

[0413] Individual polyribonucleotides can be produced by methods known in the art. For example, in some embodiments, polyribonucleotides can be produced by in vitro transcription, for example, using a DNA template. A plasmid DNA used as a template for in vitro transcription to generate a polyribonucleotide described herein is also within the scope of the present disclosure. In some embodiments, two or more different polyribonucleotides described herein are produced together by in vitro transcription. In some embodiments, two or more different polyribonucleotides described herein are produced separately by in vitro transcription and are subsequently formulated together.

[0414] A DNA template is used for in vitro RNA synthesis in the presence of an appropriate RNA polymerase (e.g., a recombinant RNA-polymerase such as a T7 RNA-polymerase) with ribonucleotide triphosphates (e.g., ATP, CTP, GTP, UTP). In some embodiments,Page 124 of 18313406582vlAttorney Docket No.: 2013237-1617polyribonucleotides (e.g., ones described herein) can be synthesized in the presence of modified ribonucleotide triphosphates. By way of example only, in some embodiments, pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), or 5-methyl-uridine (m5U) can be used to replace uridine triphosphate (UTP). In some embodiments, pseudouridine (ψ) can be used to replace uridine triphosphate (UTP). In some embodiments, N1-methyl-pseudouridine (m1ψ) can be used to replace uridine triphosphate (UTP). In some embodiments, 5-methyl-uridine (m5U) can be used to replace uridine triphosphate (UTP).

[0415] As will be clear to those skilled in the art, during in vitro transcription, an RNA polymerase (e.g., as described and / or utilized herein) typically traverses at least a portion of a single-stranded DNA template in the 3'— > 5' direction to produce a single-stranded complementary RNA in the 5'— > 3' direction.

[0416] In some embodiments where a polyribonucleotide comprises a polyA tail, one of those skill in the art will appreciate that such a polyA tail may be encoded in a DNA template, e.g., by using an appropriately tailed PCR primer, or it can be added to a polyribonucleotide after in vitro transcription, e.g., by enzymatic treatment (e.g., using a poly(A) polymerase such as an E. coli Poly(A) polymerase). Suitable poly(A) tails are described herein above. For example, in some embodiments, a poly(A) tail comprises a nucleotide sequence of AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCATATGACTAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 70). In some embodiments, a poly(A) tail comprises a plurality of A residues interrupted by a linker. In some embodiments, a linker comprises the nucleotide sequence GCATATGAC.

[0417] In some embodiments, those skilled in the art will appreciate that addition of a 5' cap to an RNA (e.g., mRNA) can facilitate recognition and attachment of the RNA to a ribosome to initiate translation and enhances translation efficiency. Those skilled in the art will also appreciate that a 5' cap can also protect an RNA product from 5' exonuclease mediated degradation and thus increases half-life. Methods for capping are known in the art; one of ordinary skill in the art will appreciate that in some embodiments, capping may be performed after in vitro transcription in the presence of a capping system (e.g., an enzyme-based capping system such as, e.g., capping enzymes of vaccinia virus). In some embodiments, a cap may be Page 125 of 18313406582vlAttorney Docket No.: 2013237-1617introduced during in vitro transcription, along with a plurality of ribonucleotide triphosphates such that a cap is incorporated into a polyribonucleotide during transcription (also known as co-transcriptional capping). In some embodiments, a GTP fed-batch procedure with multiple additions in the course of the reaction may be used to maintain a low concentration of GTP in order to effectively cap the RNA. In some embodiments, in vitro transcription is performed in a single reaction (i.e., “one pot”) non-fedbatch procedure. Suitable 5' cap are described herein above. For example, in some embodiments, a 5' cap comprises m7(3'OMeG)(5')ppp(5')(2'OMeA)pG.

[0418] Following RNA transcription, a DNA template is digested. In some embodiments, digestion can be achieved with the use of DNase I under appropriate conditions.

[0419] In some embodiments, in-vitro transcribed polyribonucleotides may be provided in a buffered solution, for example, in a buffer such as HEPES, a phosphate buffer solution, a citrate buffer solution, an acetate buffer solution; in some embodiments, such solution may be buffered to a pH within a range of, for example, about 6.5 to about 7.5; in some embodiments approximately 7.0. In some embodiments, production of polyribonucleotides may further include one or more of the following steps: purification, mixing, filtration, and / or filling.

[0420] In some embodiments, polyribonucleotides can be purified (e.g., in some embodiments after in vitro transcription reaction), for example, to remove components utilized or formed in the course of the production, like, e.g., proteins, DNA fragments, and / or or nucleotides. Various nucleic acid purifications that are known in the art can be used in accordance with the present disclosure. Certain purification steps may be or include, for example, one or more of precipitation, column chromatography (including, e.g., but not limited to anionic, cationic, hydrophobic interaction chromatography (HIC)), solid substrate-based purification (e.g., magnetic bead-based purification). In some embodiments, polyribonucleotides may be purified using magnetic bead-based purification, which in some embodiments may be or comprise magnetic bead-based chromatography. In some embodiments, polyribonucleotides may be purified using hydrophobic interaction chromatography (HIC) and / or diafiltration. In some embodiments, polyribonucleotides may be purified using HIC followed by diafiltration.

[0421] In some embodiments, dsRNA may be obtained as side product during in vitro transcription. In some such embodiments, a second purification step may be performed to Page 126 of 18313406582vlAttorney Docket No.: 2013237-1617remove dsRNA contamination. For example, in some embodiments, cellulose materials (e.g., microcrystalline cellulose) may be used to remove dsRNA contamination, for example in some embodiments in a chromatographic format. In some embodiments, cellulose materials (e.g., microcrystalline cellulose) can be pretreated to inactivate potential RNase contamination, for example in some embodiments by autoclaving followed by incubation with aqueous basic solution, e.g., NaOH. In some embodiments, cellulose materials may be used to purify polyribonucleotides according to methods described in WO 2017 / 182524, the entire content of which is incorporated herein by reference.

[0422] In some embodiments, a batch of polyribonucleotides may be further processed by one or more steps of filtration and / or concentration. For example, in some embodiments, polyribonucleotide(s), for example, after removal of dsRNA contamination, may be further subject to diafiltration (e.g., in some embodiments by tangential flow filtration), for example, to adjust the concentration of polyribonucleotides to a desirable RNA concentration and / or to exchange buffer to a drug substance buffer.

[0423] In some embodiments, polyribonucleotides may be processed through 0.2 pm filtration before they are filled into appropriate containers.

[0424] In some embodiments, polyribonucleotides and compositions thereof may be manufactured in accordance with a process as described herein, or as otherwise known in the art.

[0425] In some embodiments, polyribonucleotides and compositions thereof may be manufactured at a large scale. For example, in some embodiments, a batch of polyribonucleotides can be manufactured at a scale of greater than 1 g, greater than 2 g, greater than 3 g, greater than 4 g, greater than 5 g, greater than 6 g, greater than 7 g, greater than 8 g, greater than 9 g, greater than 10 g, greater than 15 g, greater than 20 g, or higher.

[0426] In some embodiments, RNA quality control may be performed and / or monitored at any time during production process of polyribonucleotides and / or compositions comprising the same. For example, in some embodiments, RNA quality control parameters, including one or more of RNA identity (e.g., sequence, length, and / or RNA natures), RNA integrity, RNA concentration, residual DNA template, and residual dsRNA, may be assessed and / or monitored after each or certain steps of a polyribonucleotide manufacturing process, e.g, after in vitro transcription, and / or each purification step.Page 127 of 18313406582vlAttorney Docket No.: 2013237-1617

[0427] In some embodiments, the stability of polyribonucleotides (e.g., produced by in vitro transcription) and / or compositions comprising polyribonucleotides can be assessed under various test storage conditions, for example, at room temperatures vs. fridge or sub-zero temperatures over a period of time (e.g., at least 3 months, at least 6 months, at least 9 months, at least 12 months, or longer). In some embodiments, polyribonucleotides (e.g., ones described herein) and / or compositions thereof may be stored stable at a fridge temperature (e.g., about 4°C to about 10°C) for at least 1 month or longer including, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, or at least 12 months or longer. In some embodiments, polyribonucleotides (e.g., ones described herein) and / or compositions thereof may be stored stable at a sub-zero temperature (e.g., -20°C or below) for at least 1 month or longer including, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, or at least 12 months or longer. In some embodiments, polyribonucleotides (e.g., ones described herein) and / or compositions thereof may be stored stable at room temperature (e.g., at about 25°C) for at least 1 month or longer.

[0428] In some embodiments, one or more assessments may be utilized during manufacture, or other preparation or use of polyribonucleotides (e.g., as a release test).

[0429] In some embodiments, one or more quality control parameters may be assessed to determine whether polyribonucleotides described herein meet or exceed acceptance criteria (e.g., for subsequent formulation and / or release for distribution). In some embodiments, such quality control parameters may include, but are not limited to RNA integrity, RNA concentration, residual DNA template and / or residual dsRNA. Certain methods for assessing RNA quality are known in the art; for example, one of skill in the art will recognize that in some embodiments, one or more analytical tests can be used for RNA quality assessment. Examples of such certain analytical tests may include but are not limited to gel electrophoresis, UV absorption, and / or PCR assay.

[0430] In some embodiments, a batch of polyribonucleotides may be assessed for one or more features as described herein to determine next action step(s). For example, a batch of polyribonucleotides can be designated for one or more further steps of manufacturing and / or Page 128 of 18313406582vlAttorney Docket No.: 2013237-1617formulation and / or distribution if RNA quality assessment indicates that such a batch of polyribonucleotides meet or exceed the relevant acceptance criteria. Otherwise, an alternative action can be taken (e.g., discarding the batch) if such a batch of polyribonucleotides does not meet or exceed the acceptance criteria.

[0431] In some embodiments, a batch of polyribonucleotides that satisfy assessment results can be utilized for one or more further steps of manufacturing and / or formulation and / or distribution.VIII. DNA Constructs

[0432] Among other things, the present disclosure provides DNA constructs, for example that may encode one or more vaccine constructs as described herein, or components thereof. In some embodiments, DNA constructs provided by and / or utilized in accordance with the present disclosure are comprised in a vector.

[0433] Non-limiting examples of a vector include plasmid vectors, cosmid vectors, phage vectors such as lambda phage, viral vectors such as retroviral, adenoviral or baculoviral vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), or Pl artificial chromosomes (PAC). In some embodiments, a vector is an expression vector. In some embodiments, a vector is a cloning vector. In general, a vector is a nucleic acid construct that can receive or otherwise become linked to a nucleic acid element of interest (e.g., a construct that is or encodes a payload, or that imparts a particular functionality, etc.).

[0434] Expression vectors, which may be plasmid or viral or other vectors, can include an expressible sequence of interest (e.g., a coding sequence) that is functionally linked with one or more control elements (e.g., promoters, enhancers, transcription terminators, etc.). Typically, such control elements are selected for expression in a system of interest. In some embodiments, a system is ex vivo (e.g., an in vitro transcription system); in some embodiments, a system is in vivo (e.g., a bacterial, yeast, plant, insect, fish, vertebrate, mammalian cell or tissue, etc.).

[0435] Cloning vectors are generally used to modify, engineer, and / or duplicate (e.g., by replication in vivo, for example in a simple system such as bacteria or yeast, or in vitro, such as by amplification such as polymerase chain reaction or other amplification process). In some embodiments, a cloning vector may lack expression signals.Page 129 of 18313406582vlAttorney Docket No.: 2013237-1617

[0436] In many embodiments, a vector may include replication elements such as primer binding site(s) and / or origin(s) of replication. In many embodiments, a vector may include insertion or modification sites such as restriction endonuclease recognition sites and / or guide RNA binding sites, etc.

[0437] In some embodiments, a vector is a viral vector (e.g., an AAV vector). In some embodiments, a vector is a non-viral vector. In some embodiments, a vector is a plasmid.

[0438] Those skilled in the art are aware of a variety of technologies useful for the production of recombinant polynucleotides (e.g., DNA or RNA) as described herein. For example, restriction digestion, reverse transcription, amplification (e.g., by polymerase chain reaction), Gibson assembly, etc., are well established and useful tools and technologies. Alternatively or additionally, certain nucleic acids may be prepared or assembled by chemical and / or enzymatic synthesis. In some embodiments, a combination of known methods is utilized to prepare a recombinant polynucleotide.

[0439] In some embodiments, polynucleotide(s) of the present disclosure are included in a DNA construct (e.g., a vector) amenable to transcription and / or translation.

[0440] In some embodiments, an expression vector comprises a polynucleotide that encodes a vaccine construct of the present disclosure operatively linked to a sequence or sequences that control expression (e.g., promoters, start signals, stop signals, polyadenylation signals, activators, repressors, etc.}. In some embodiments, a sequence or sequences that control expression are selected to achieve a desired level of expression. In some embodiments, more than one sequence that controls expression (e.g., promoters) are utilized. In some embodiments, more than one sequence that controls expression (e.g., promoters) are utilized to achieve a desired level of expression of a plurality of polynucleotides that encode a plurality proteins and / or polypeptides. In some embodiments, a plurality of vaccine constructs are expressed from the same vector (e.g., a bi-cistronic vector, a tri-cistronic vector, multi-cistronic). In some embodiments, a plurality of vaccine constructs are expressed, each of which is expressed from a separate vector.

[0441] In some embodiments, an expression vector comprising a polynucleotide of the present disclosure is used to produce a RNA and / or protein and / or polypeptide in a host cell. In some embodiments, a host cell may be in vitro (e.g, a cell line) - for example a cell or cell line (e.g, Human Embryonic Kidney (HEK cells), Chinese Hamster Ovary cells, etc. suitable for Page 130 of 18313406582vlAttorney Docket No.: 2013237-1617producing polynucleotides of the present disclosure and proteins and / or polypeptides encoded by said polynucleotides.

[0442] In some embodiments, an expression vector is an RNA expression vector. In some embodiments, an RNA expression vector comprises a polynucleotide template used to produce a RNA in cell-free enzymatic mix. In some embodiments, an RNA expression vector comprising a polynucleotide template is enzymatically linearized prior to in vitro transcription. In some embodiments, a polynucleotide template is generated through PCR as a linear polynucleotide template. In some embodiments, a linearized polynucleotide is mixed with enzymes suitable for RNA synthesis, RNA capping and / or purification. In some embodiments, the resulting RNA is suitable for producing polyepitopic vaccine constructs encoded by the RNA.

[0443] A variety of methods are known in the art to introduce an expression vector into host cells. In some embodiments, a vector may be introduced into host cells using transfection. In some embodiments, transfection is completed, for example, using calcium phosphate transfection, lipofection, or polyethylenimine-mediated transfection. In some embodiments, a vector may be introduced into a host cell using transduction.

[0444] In some embodiments, transformed host cells are cultured following introduction of a vector into a host cell to allow for expression of said recombinant polynucleotides. In some embodiments, a transformed host cells are cultured for at least 12 hours, 16 hours, 20 hours, 24 hours, 28 hours, 32 hours, 36 hours 40 hours, 44 hours, 48 hours, 52 hours, 56 hours, 60 hours, 64 hours, 68 hours, 72 hours or longer. Transformed host cells are cultured in growth conditions (e.g., temperature, carbon-dioxide levels, growth medium) in accordance with the requirements of a host cell selected. A skilled artisan would recognize culture conditions for host cells selected are well known in the art.IX. Computer System and Network Environment

[0445] As shown in FIG. 46, an implementation of a network environment 4600 for use in providing systems and methods as described herein is shown and described. In brief overview, referring now to FIG.46, a block diagram of an exemplary cloud computing environment 4600 is shown and described. The cloud computing environment 4600 may include one or more resource providers 4602a, 4602b, 4602c (collectively, 4602). Each resource provider 4602 may include computing resources. In some implementations, computing resources may include any Page 131 of 18313406582vlAttorney Docket No.: 2013237-1617hardware and / or software used to process data. For example, computing resources may include hardware and / or software capable of executing algorithms, computer programs, and / or computer applications. In some implementations, exemplary computing resources may include application servers and / or databases with storage and retrieval capabilities. Each resource provider 4602 may be connected to any other resource provider 4602 in the cloud computing environment 4600. In some implementations, the resource providers 4602 may be connected over a computer network 4608. Each resource provider 4602 may be connected to one or more computing device 4604a, 4604b, 4604c (collectively, 4604), over the computer network 4608.

[0446] The cloud computing environment 4600 may include a resource manager 4606. The resource manager 4606 may be connected to the resource providers 4602 and the computing devices 4604 over the computer network 4608. In some implementations, the resource manager 4606 may facilitate the provision of computing resources by one or more resource providers 4602 to one or more computing devices 4604. The resource manager 4606 may receive a request for a computing resource from a particular computing device 4604. The resource manager 4606 may identify one or more resource providers 4602 capable of providing the computing resource requested by the computing device 4604. The resource manager 4606 may select a resource provider 4602 to provide the computing resource. The resource manager 4606 may facilitate a connection between the resource provider 4602 and a particular computing device 4604. In some implementations, the resource manager 4606 may establish a connection between a particular resource provider 4602 and a particular computing device 4604. In some implementations, the resource manager 4606 may redirect a particular computing device 4604 to a particular resource provider 4602 with the requested computing resource.

[0447] FIG.47 shows an example of a computing device 4700 and a mobile computing device 4750 that can be used to implement the techniques described in this disclosure. The computing device 4700 is intended to represent various forms of digital computers, such as laptops, desktops, workstations, personal digital assistants, servers, blade servers, mainframes, and other appropriate computers. The mobile computing device 4750 is intended to represent various forms of mobile devices, such as personal digital assistants, cellular telephones, smart-phones, and other similar computing devices. The components shown here, their connections andPage 132 of 18313406582vlAttorney Docket No.: 2013237-1617relationships, and their functions, are meant to be examples only, and are not meant to be limiting.

[0448] The computing device 4700 includes a processor 4702, a memory 4704, a storage device 4706, a high-speed interface 4708 connecting to the memory 4704 and multiple high-speed expansion ports 4710, and a low-speed interface 4712 connecting to a low-speed expansion port 4714 and the storage device 4706. Each of the processor 4702, the memory 4704, the storage device 4706, the high-speed interface 4708, the high-speed expansion ports 4710, and the low-speed interface 4712, are interconnected using various busses, and may be mounted on a common motherboard or in other manners as appropriate. The processor 4702 can process instructions for execution within the computing device 4700, including instructions stored in the memory 4704 or on the storage device 4706 to display graphical information for a GUI on an external input / output device, such as a display 4716 coupled to the high-speed interface 4708. In other implementations, multiple processors and / or multiple buses may be used, as appropriate, along with multiple memories and types of memory. Also, multiple computing devices may be connected, with each device providing portions of the necessary operations (e.g., as a server bank, a group of blade servers, or a multi-processor system). Thus, as the term is used herein, where a plurality of functions are described as being performed by “a processor”, this encompasses embodiments wherein the plurality of functions are performed by any number of processors (one or more) of any number of computing devices (one or more). Furthermore, where a function is described as being performed by “a processor”, this encompasses embodiments wherein the function is performed by any number of processors (one or more) of any number of computing devices (one or more) (e.g., in a distributed computing system).

[0449] The memory 4704 stores information within the computing device 4700. In some implementations, the memory 4704 is a volatile memory unit or units. In some implementations, the memory 4704 is a non-volatile memory unit or units. The memory 4704 may also be another form of computer-readable medium, such as a magnetic or optical disk.

[0450] The storage device 4706 is capable of providing mass storage for the computing device 4700. In some implementations, the storage device 4706 may be or contain a computer-readable medium, such as a floppy disk device, a hard disk device, an optical disk device, or a tape device, a flash memory or other similar solid state memory device, or an array of devices,Page 133 of 18313406582vlAttorney Docket No.: 2013237-1617including devices in a storage area network or other configurations. Instructions can be stored in an information carrier. The instructions, when executed by one or more processing devices (for example, processor 4702), perform one or more methods, such as those described above. The instructions can also be stored by one or more storage devices such as computer- or machine-readable mediums (for example, the memory 4704, the storage device 4706, or memory on the processor 4702).

[0451] The high-speed interface 4708 manages bandwidth-intensive operations for the computing device 4700, while the low-speed interface 4712 manages lower bandwidth-intensive operations. Such allocation of functions is an example only. In some implementations, the highspeed interface 4708 is coupled to the memory 4704, the display 4716 (e.g., through a graphics processor or accelerator), and to the high-speed expansion ports 4710, which may accept various expansion cards (not shown). In the implementation, the low-speed interface 4712 is coupled to the storage device 4706 and the low-speed expansion port 4714. The low-speed expansion port 4714, which may include various communication ports (e.g., USB, Bluetooth®, Ethernet, wireless Ethernet) may be coupled to one or more input / output devices, such as a keyboard, a pointing device, a scanner, or a networking device such as a switch or router, e.g., through a network adapter.

[0452] The computing device 4700 may be implemented in a number of different forms, as shown in the figure. For example, it may be implemented as a standard server 4720, or multiple times in a group of such servers. In addition, it may be implemented in a personal computer such as a laptop computer 4722. It may also be implemented as part of a rack server system 4724. Alternatively, components from the computing device 4700 may be combined with other components in a mobile device (not shown), such as a mobile computing device 4750. Each of such devices may contain one or more of the computing device 4700 and the mobile computing device 4750, and an entire system may be made up of multiple computing devices communicating with each other.

[0453] The mobile computing device 4750 includes a processor 4752, a memory 4764, an input / output device such as a display 4754, a communication interface 4766, and a transceiver 4768, among other components. The mobile computing device 4750 may also be provided with a storage device, such as a micro-drive or other device, to provide additional storage. Each of Page 134 of 18313406582vlAttorney Docket No.: 2013237-1617the processor 4752, the memory 4764, the display 4754, the communication interface 4766, and the transceiver 4768, are interconnected using various buses, and several of the components may be mounted on a common motherboard or in other manners as appropriate.

[0454] The processor 4752 can execute instructions within the mobile computing device 4750, including instructions stored in the memory 4764. The processor 4752 may be implemented as a chipset of chips that include separate and multiple analog and digital processors. The processor 4752 may provide, for example, for coordination of the other components of the mobile computing device 4750, such as control of user interfaces, applications run by the mobile computing device 4750, and wireless communication by the mobile computing device 4750.

[0455] The processor 4752 may communicate with a user through a control interface 4758 and a display interface 4756 coupled to the display 4754. The display 4754 may be, for example, a TFT (Thin-Film-Transistor Liquid Crystal Display) display or an OLED (Organic Light Emitting Diode) display, or other appropriate display technology. The display interface 4756 may comprise appropriate circuitry for driving the display 4754 to present graphical and other information to a user. The control interface 4758 may receive commands from a user and convert them for submission to the processor 4752. In addition, an external interface 4762 may provide communication with the processor 4752, so as to enable near area communication of the mobile computing device 4750 with other devices. The external interface 4762 may provide, for example, for wired communication in some implementations, or for wireless communication in other implementations, and multiple interfaces may also be used.

[0456] The memory 4764 stores information within the mobile computing device 4750. The memory 4764 can be implemented as one or more of a computer-readable medium or media, a volatile memory unit or units, or a non-volatile memory unit or units. An expansion memory 4774 may also be provided and connected to the mobile computing device 4750 through an expansion interface 4772, which may include, for example, a SIMM (Single In Line Memory Module) card interface. The expansion memory 4774 may provide extra storage space for the mobile computing device 4750, or may also store applications or other information for the mobile computing device 4750. Specifically, the expansion memory 4774 may include instructions to carry out or supplement the processes described above, and may include secure information also. Thus, for example, the expansion memory 4774 may be provide as a security Page 135 of 18313406582vlAttorney Docket No.: 2013237-1617module for the mobile computing device 4750, and may be programmed with instructions that permit secure use of the mobile computing device 4750. In addition, secure applications may be provided via the SIMM cards, along with additional information, such as placing identifying information on the SIMM card in a non-hackable manner.

[0457] The memory may include, for example, flash memory and / or NVRAM memory (nonvolatile random access memory), as discussed below. In some implementations, instructions are stored in an information carrier. The instructions, when executed by one or more processing devices (for example, processor 4752), perform one or more methods, such as those described above. The instructions can also be stored by one or more storage devices, such as one or more computer- or machine-readable mediums (for example, the memory 4764, the expansion memory 4774, or memory on the processor 4752). In some implementations, the instructions can be received in a propagated signal, for example, over the transceiver 4768 or the external interface 4762

[0458] The mobile computing device 4750 may communicate wirelessly through the communication interface 4766, which may include digital signal processing circuitry where necessary. The communication interface 4766 may provide for communications under various modes or protocols, such as GSM voice calls (Global System for Mobile communications), SMS (Short Message Service), EMS (Enhanced Messaging Service), or MMS messaging (Multimedia Messaging Service), CDMA (code division multiple access), TDMA (time division multiple access), PDC (Personal Digital Cellular), WCDMA (Wideband Code Division Multiple Access), CDMA2000, or GPRS (General Packet Radio Service), among others. Such communication may occur, for example, through the transceiver 4768 using a radio-frequency. In addition, short-range communication may occur, such as using a Bluetooth®, Wi-Fi™, or other such transceiver (not shown). In addition, a GPS (Global Positioning System) receiver module 4770 may provide additional navigation- and location-related wireless data to the mobile computing device 4750, which may be used as appropriate by applications running on the mobile computing device 4750.

[0459] The mobile computing device 4750 may also communicate audibly using an audio codec 4760, which may receive spoken information from a user and convert it to usable digital information. The audio codec 4760 may likewise generate audible sound for a user, such as Page 136 of 18313406582vlAttorney Docket No.: 2013237-1617through a speaker, e.g., in a handset of the mobile computing device 4750. Such sound may include sound from voice telephone calls, may include recorded sound (e.g., voice messages, music files, etc.) and may also include sound generated by applications operating on the mobile computing device 4750.

[0460] The mobile computing device 4750 may be implemented in a number of different forms, as shown in the figure. For example, it may be implemented as a cellular telephone 4780. It may also be implemented as part of a smart-phone 4782, personal digital assistant, or other similar mobile device.

[0461] Various implementations of the systems and techniques described here can be realized in digital electronic circuitry, integrated circuitry, specially designed ASICs (application specific integrated circuits), computer hardware, firmware, software, and / or combinations thereof. These various implementations can include implementation in one or more computer programs that are executable and / or interpretable on a programmable system including at least one programmable processor, which may be special or general purpose, coupled to receive data and instructions from, and to transmit data and instructions to, a storage system, at least one input device, and at least one output device.

[0462] These computer programs (also known as programs, software, software applications or code) include machine instructions for a programmable processor, and can be implemented in a high-level procedural and / or object-oriented programming language, and / or in assembly / machine language. As used herein, the terms machine-readable medium and computer-readable medium refer to any computer program product, apparatus and / or device (e.g., magnetic discs, optical disks, memory, Programmable Logic Devices (PLDs)) used to provide machine instructions and / or data to a programmable processor, including a machine-readable medium that receives machine instructions as a machine-readable signal. The term machine-readable signal refers to any signal used to provide machine instructions and / or data to a programmable processor.

[0463] To provide for interaction with a user, the systems and techniques described here can be implemented on a computer having a display device (e.g., a CRT (cathode ray tube) or LCD (liquid crystal display) monitor) for displaying information to the user and a keyboard and a pointing device (e.g., a mouse o...

Claims

Attorney Docket No.: 2013237-1617CLAIMS1. A method of selecting shared antigen epitopes for inclusion in a construct for a subject, the method comprising:(a) obtaining a candidate epitope list identifying a plurality of candidate epitopes and MHC presentation and immunogenicity data comprising, for each particular candidate epitope of the plurality, (i) and (ii) as follows:(i) a corresponding MHC presentation score representing a known and / or predicted likelihood and / or strength of binding between the particular candidate epitope and an MHC of the subject; and(ii) a corresponding immunogenicity score representing a known and / or predicted immunogenicity of the particular candidate epitope;(b) for each candidate epitope of at least a portion of the plurality of candidate epitopes, determining a corresponding rank based at least in part on the MHC presentation and immunogenicity data;(c) selecting a subset of the plurality of candidate epitopes for inclusion in the polyepitopic construct based at least in part on their corresponding rankings as a set of shared antigen epitopes; and(d) storing and / or providing the set of shared antigen epitopes.

2. The method of claim 1, wherein, for each of at least a portion of the candidate epitopes, the corresponding MHC presentation score classifies the candidate epitope as a known T-cell epitope and / or a known ligand.

3. The method of claim 1 or 2, wherein, for each of at least a portion of the candidate epitopes, the corresponding MHC presentation score represents a predicted MHC binding of the candidate epitope.

4. The method of any one of claims 1-3, wherein for each of at least a portion of the candidate epitopes, the corresponding immunogenicity score categorizes the candidate epitope according to a level of evidence of immunogenicity.Page 175 of 18313406582vlAttorney Docket No.: 2013237-16175. The method of any one of the preceding claims, comprising:identifying one or more of the candidate epitopes in a proteome of the subject; and filtering the plurality of candidate epitopes to exclude the one or more candidate epitopes identified in the proteome of the subject.

6. The method of any one of the preceding claims, comprising:identifying one or more of the candidate epitopes as matching a potency linker sequence; andfiltering the plurality of candidate epitopes to exclude those candidate epitopes identified as matching a potency linker sequence.

7. The method of any one of the preceding claims, comprising:identifying one or more of the candidate epitopes as matching a non-immunogenic epitope of an epitope exclusion list, said epitope exclusion list comprising a listing of epitopes previously identified as non-immunogenic; andfiltering the plurality of candidate epitopes to exclude the one or more candidate epitopes identified as matching a non-immunogenic epitope of the epitope exclusion list.

8. The method of any one of the preceding claims, comprising subdividing, the plurality of candidate epitopes into a plurality of batches according to their corresponding MHC presentation scores.

9. The method of any one of the preceding claims, comprising subdividing, the plurality of candidate epitopes into a plurality of batches, each batch associated with a particular classification of the candidate epitopes including (i) a list of “known” epitopes, (ii) a list of “known” MHC ligands and (iii) predicted MHC ligands having a particular range of MHC binding scores.Page 176 of 18313406582vlAttorney Docket No.: 2013237-161710. The method of claim 8 or 9, wherein, each batch is associated with one or more tiers, each tier associated with, and comprising candidate epitopes having, a particular immunogenicity categorization.

11. The method of any one of the preceding claims, comprising determining, for one or more candidate epitopes, a corresponding target cluster by:selecting a first candidate epitope as a target core;identifying one or more additional candidate epitopes belonging to a same transcript window as, and overlapping with, the first candidate epitope; andextending the target core to include the one or more ligands, thereby forming a target cluster.

12. The method of any one of the preceding claims, wherein two cores of a plurality of candidate cores are combined into a combined core if the two cores overlap by at least one amino acid and correspond to a same transcript window.

13. The method of any one of the preceding claims, comprising selecting candidate epitopes for inclusion in the construct in an iterative fashion, based at least in part on each candidate epitope’s corresponding rank.

14. The method of any one of the preceding claims, comprising updating a rank of one or more candidate epitopes while selecting the subset for inclusion in the construct.

15. The method of any one of the preceding claims, wherein step (c) comprises: selecting two or more candidate epitopes from a same first antigen; determining a number of candidate epitopes selected from the first antigen to be greater than or equal to a threshold value; andreducing a rank of at least a portion of remaining candidate epitopes from the first antigen, thereby downranking remaining candidate epitopes from the first antigen.Page 177 of 18313406582vlAttorney Docket No.: 2013237-161716. A system comprising a processor of a computing device and a memory having instructions stored thereon, wherein the instructions, when executed by the processor, cause the processor to perform the method of any one of claims 1-15.

17. A method of selecting neoantigen epitopes for inclusion in a construct for a subject, the method comprising:(a) obtaining a candidate epitope list identifying a plurality of candidate epitopes, MHC presentation data and expression data comprising, for each particular candidate epitope of the plurality, (i) and (ii) as follows:(i) a corresponding MHC presentation score representing a known and / or predicted likelihood and / or strength of binding between the particular candidate epitope and an MHC of the subject; and(ii) a corresponding expression score representing measured expression in the subject;(b) for each candidate epitope of at least a portion of the plurality of candidate epitopes, determining a corresponding rank based at least in part on the MHC presentation and expression data;(c) selecting a subset of the plurality of candidate epitopes for inclusion in the polyepitopic construct based at least in part on their corresponding rankings as a set of neoantigen epitopes; and(d) storing and / or providing the set of neoantigen epitopes.

18. The method of claim 17, further comprising obtaining germline DNA sequence from the subject and tumor DNA sequence from the subject.

19. The method of claim 18, further comprising comparing germline DNA sequence from the subject and tumor DNA sequence from the subject to obtain the candidate epitope list.

20. The method of claim 19, comprising detecting one or more somatic mutations.Page 178 of 18313406582vlAttorney Docket No.: 2013237-161721. The method of any one of claims 18-20, further comprising determining the subject’s HLAtype from the germline DNA sequence.

22. The method of any one of the preceding claims, comprising determining for each candidate epitope a corresponding variant allele frequency.

23. The method of any one of claims 17-22, wherein, for each of at least a portion of the candidate epitopes, the corresponding MHC presentation score represents a predicted MHC binding of the candidate epitope.

24. The method of any one of claims 17-23 comprising:identifying one or more of the candidate epitopes in a proteome of the subject; and filtering the plurality of candidate epitopes to exclude the one or more candidate epitopes identified the proteome of the subject.

25. The method of any one of claims 17-24, comprising:identifying one or more of the candidate epitopes as matching a sequence of a list of tissue specific expression risk factors; andfiltering the plurality of candidate epitopes to exclude the one or more candidate epitopes identified as matching a sequence of the tissue specific expression risk factors list.

26. The method of any one of claims 17-25 comprising determining, for each particular candidate epitope of the plurality of candidate epitopes, the corresponding MHC presentation score based at least in part on, for the particular candidate epitope, a corresponding expression score representing measured expression in the subject.

27. A method of selecting shared antigen epitopes for inclusion in a construct for a subject, the method comprising:(a) obtaining a candidate epitope list identifying a plurality of candidate epitopes, MHC presentation data, and expression data comprising, for each particular candidate epitope of the plurality, (i) and (ii) as follows:Page 179 of 18313406582vlAttorney Docket No.: 2013237-1617(i) a corresponding MHC presentation score representing a known and / or predicted likelihood and / or strength of binding between the particular candidate epitope and an MHC of the subject; and(ii) a corresponding expression score representing measured expression in the subject, wherein the measured expression exceeds a predetermined threshold level; (b) for each candidate epitope of at least a portion of the plurality of candidate epitopes, determining a corresponding rank based at least in part on the MHC presentation and expression data;(c) selecting a subset of the plurality of candidate epitopes for inclusion in the polyepitopic construct based at least in part on their corresponding rankings as a set of shared antigen epitopes; and(d) storing and / or providing the set of shared antigen epitopes.

28. The method of claim 27, further comprising obtaining germline DNA sequence from the subject and tumor DNA sequence from the subject.

29. The method of 28, further comprising comparing germline DNA sequence from the subject and tumor DNA sequence from the subject to identify one or more germline mutations and one or more somatic mutations.

30. The method of claim 27, wherein the plurality of candidate epitopes do not comprise a somatic mutation.

31. The method of any one of claims 26-28, further comprising determining the subject’s HLAtype from the germline DNA sequence.

32. The method of any one of claims 27-31, wherein, for each of at least a portion of the candidate epitopes, the corresponding MHC presentation score classifies the candidate epitope as a known T-cell epitope and / or a known ligand.Page 180 of 18313406582vlAttorney Docket No.: 2013237-161733. The method of any one of claims 27-32, wherein, for each of at least a portion of the candidate epitopes, the corresponding MHC presentation score represents a predicted MHC binding of the candidate epitope.

34. The method of claim 33, comprising determining the corresponding MHC presentation score by selecting it from a lookup table of precomputed presentation scores.

35. The method of any one of claims 27-34, comprising:identifying one or more of the candidate epitopes in a proteome of the subject; and filtering the plurality of candidate epitopes to exclude the one or more candidate epitopes identified the proteome of the subject.

36. The method of any one of claims 27-35, comprising:identifying one or more of the candidate epitopes as matching a potency linker sequence; andfiltering the plurality of candidate epitopes to exclude those candidate epitopes identified as matching a potency linker sequence.

37. The method of any one of claims 27-36, comprising:identifying one or more of the candidate epitopes as matching a non-immunogenic epitope of an epitope exclusion list, said epitope exclusion list comprising a listing of epitopes previously identified as non-immunogenic; andfiltering the plurality of candidate epitopes to exclude the one or more candidate epitopes identified as matching a non-immunogenic epitope of the epitope exclusion list.

38. A method of selecting neoantigen epitopes for inclusion in a construct for a subject, the method comprising:(a) obtaining a candidate epitope list identifying a plurality of candidate epitopes, MHC presentation data comprising, for each particular candidate epitope of the plurality, a corresponding MHC presentation score representing a known and / or predicted likelihood and / or strength of binding between the particular candidate epitope and an MHC of the subject; and Page 181 of 18313406582vlAttorney Docket No.: 2013237-1617(b) for each candidate epitope of at least a portion of the plurality of candidate epitopes, determining a corresponding rank based at least in part on the MHC presentation data;(c) selecting a subset of the plurality of candidate epitopes for inclusion in the polyepitopic construct based at least in part on their corresponding rankings as a set of neoantigen epitopes; and(d) storing and / or providing the set of neoantigen epitopes.

39. The method of claim 38 comprising determining, for each particular candidate epitope of the plurality of candidate epitopes, the corresponding MHC presentation score based at least in part on, for the particular candidate epitope, a corresponding expression score representing measured expression in the subject.

40. A system comprising a processor of a computing device and a memory having instructions stored thereon, wherein the instructions, when executed by the processor, cause the processor to perform the method of any one of claims 17-39.Page 182 of 18313406582vl

Images