Compositions and methods of use thereof

Abstract

Disclosed herein are pharmaceutical compositions comprising RNA-based expression systems suitable for storage, as well as methods for making and using the same.

Description

[Background technology]

[0001] background Alphaviruses are a group of small, single-stranded, positive-sense RNA viruses implicated in numerous diseases in humans and other animals. See, for example, "The Alphaviruses: Gene Expression, Replication, and Evolution," Microbiological Reviews, September 1994, pp. 491-562; Jose et al. (Non-Patent Document 1) "A structural and functional perspective of alphavirus replication and assembly," Future Microbol., 2009, v. 4:837-856. Due to their high replication efficiency and specificity, alphaviruses have proven useful for engineering self-replicating RNA vectors for the expression of heterologous proteins in mammalian cells. See, for example, Frolov et al., "Alphavirus-based expression vectors: strategies and applications," PNAS, 1996, v. 93, pp. 11371-11377 (Non-Patent Document 3); Young Kim, et al., "Enhancement of protein expression by alphavirus replicons by designing self-replicating subgenomic RNAs," PNAS, 2014, v. 11:29, pp. 10708-10713 (Non-Patent Document 4). The stability of these RNA-based expression systems can decrease over time. Furthermore, ease of storage is hindered by the need to constantly keep these expression systems at impractical low temperatures. Therefore, there remains a need to develop pharmaceutical compositions containing these vector systems that are stable when stored. [Prior art documents] [Non-patent literature]

[0002] [Non-Patent Document 1] “The Alphaviruses: Gene Expression, Replication and Evolution,” Microbiological Reviews, September 1994, p. 491-562, Jose et al. [Non-patent document 2] “A structural and functional perspective of alphavirus replication and assembly,”Future Micriobol.,2009,v.4:837-856 [Non-patent document 3] Frolov et al., “Alphavirus-based expression vectors: strategies and applications”, PNAS, 1996, v.93, pp. 11371-11377 [Non-patent document 4] Young Kim, et al., “Enhancement of protein expression by alphavirus replicons by designing self-replicating subgenomic RNAs”, PNAS, 2014, v.11:29, pp.10708-10713 Summary of the Invention

[0003] overview Disclosed herein is a pharmaceutical composition comprising a lipid nanoparticle (LNP)-encapsulated self-amplifying alphavirus-based expression system or comprising a viral-based expression system, further comprising a buffer system and two or more stabilizing excipients. Further, the disclosure includes a method of eliciting an immune response in a subject by administering the pharmaceutical composition to the subject. [Brief explanation of the drawings]

[0004] [Figure 1A]1 shows six line graphs showing the concentration profiles of DP in various formulations as described in Example 2. [Figure 1B] 1 shows six line graphs showing the % encapsulation profile of DP in various formulations as described in Example 2. [Figure 2A] 1 shows six line graphs showing the size profile of DP in various formulations as described in Example 2. [Figure 2B] 1 shows six line graphs illustrating the polydispersity of DP in various formulations as described in Example 2. [Figure 3] 1 shows six line graphs illustrating the efficacy profile of DP in various formulations as described in Example 2. [Figure 4] 1 shows a bar graph depicting the full length profile of DP in various formulations as described in Example 2. [Figure 5] 5 shows five line graphs depicting the size profiles of samRNA-LNPs in various formulations as described in Example 3. [Figure 6] 5 shows five graphs illustrating the polydispersity profiles of samRNA-LNPs in various formulations as described in Example 3. [Figure 7] 5 shows five line graphs depicting the concentration profiles of samRNA-LNP in various formulations as described in Example 3. [Figure 8] 5 shows five line graphs depicting the full-length profile of samRNA-LNP in various formulations as described in Example 3. [Figure 9] 5 shows five graphs showing the full length profile of the formulation as described in Example 3. [Figure 10] 1 shows six graphs illustrating the efficacy profile of samRNA-LNP in various formulations as described in Example 3. DETAILED DESCRIPTION OF THE INVENTION

[0005] Detailed Description Provided herein are formulations of RNA-based expression systems that increase the stability of the RNA-based expression systems when stored for extended periods of time at specific temperatures as described herein, compared to other formulated or unformulated RNA-based expression systems.

[0006] The RNA-based expression system may include one or more RNA constructs encapsulated in lipid nanoparticles (LNPs). The present disclosure includes a variety of RNA-based expression systems. The RNA-based expression system may be a messenger RNA (mRNA)-based expression system, a circular (circRNA)-based expression system, a single-guide RNA (sgRNA)-based expression system, or a self-amplifying RNA (samRNA) expression system. In some embodiments, the RNA-based expression system is a messenger RNA (mRNA)-based expression system. In some embodiments, the RNA-based expression system is a circular (circRNA)-based expression system. In some embodiments, the RNA-based expression system is a single-guide RNA (sgRNA)-based expression system. In some embodiments, the RNA-based expression system is a self-amplifying RNA (samRNA) expression system.

[0007] The present disclosure includes an RNA-based expression system further comprising a buffer, an amino acid, and a cryoprotectant. In some embodiments, the formulations provided herein comprise an RNA-based expression system and a buffer. In some embodiments, the formulations provided herein comprise an RNA-based expression system and a buffer. In some embodiments, the formulations provided herein comprise an RNA-based expression system and an amino acid. In some embodiments, the formulations provided herein comprise an RNA-based expression system and a cryoprotectant.

[0008] The stability of the RNA-based expression systems and / or pharmaceutical compositions contemplated herein can be determined by assessing changes in one or more properties of the RNA-based expression system and / or pharmaceutical composition over time. For example, the stability of the RNA-based expression system and / or pharmaceutical composition can be assessed by one or more assays including particle size, PDI, samRNA concentration, encapsulation rate, samRNA full length profile (FLP), and potency. In some embodiments, the stability of the RNA-based expression system and / or pharmaceutical composition is assessed by assessing particle size. In some embodiments, the stability of the RNA-based expression system and / or pharmaceutical composition is assessed by assessing PDI. In some embodiments, the stability of the RNA-based expression system and / or pharmaceutical composition is assessed by assessing samRNA concentration. In some embodiments, the stability of the RNA-based expression system and / or pharmaceutical composition is assessed by assessing FLP. In some embodiments, the stability of the RNA-based expression system and / or pharmaceutical composition is assessed by assessing encapsulation rate. In some embodiments, the stability of the RNA-based expression system and / or pharmaceutical composition is assessed by assessing potency.

[0009] Formulations provided herein that allow for the stability of RNA-based expression and / or pharmaceutical compositions are determined after storage for periods as described herein. For example, the stability of an RNA-based expression and / or pharmaceutical composition is assessed after at least 1 day of storage. In some embodiments, the stability of an RNA-based expression and / or pharmaceutical composition is assessed after at least 2 days of storage. In some embodiments, the stability of an RNA-based expression and / or pharmaceutical composition is assessed after at least 3 days of storage. In some embodiments, the stability of an RNA-based expression and / or pharmaceutical composition is assessed after at least 4 days of storage. In some embodiments, the stability of an RNA-based expression and / or pharmaceutical composition is assessed after at least 5 days of storage. In some embodiments, the stability of an RNA-based expression and / or pharmaceutical composition is assessed after at least 6 days of storage. In some embodiments, the stability of an RNA-based expression and / or pharmaceutical composition is assessed after at least 1 week of storage. In some embodiments, the stability of an RNA-based expression and / or pharmaceutical composition is assessed after at least 2 weeks of storage. In some embodiments, the stability of an RNA-based expression and / or pharmaceutical composition is assessed after at least 3 weeks of storage. In some embodiments, the stability of the RNA-based expression and / or pharmaceutical composition is assessed after at least 1 month of storage. In some embodiments, the stability of the RNA-based expression and / or pharmaceutical composition is assessed after at least 2 months of storage. In some embodiments, the stability of the RNA-based expression and / or pharmaceutical composition is assessed after at least 3 months of storage. In some embodiments, the stability of the RNA-based expression and / or pharmaceutical composition is assessed after at least 4 months of storage. In some embodiments, the stability of the RNA-based expression and / or pharmaceutical composition is assessed after at least 5 months of storage. In some embodiments, the stability of the RNA-based expression and / or pharmaceutical composition is assessed after at least 6 months of storage.

[0010] The formulations provided herein allow for stability of RNA-based expression at temperatures described throughout this disclosure. For example, formulations comprising RNA-based expression as described herein can be stored at temperatures between -78°C and -25°C. In some embodiments, pharmaceutical compositions comprising RNA-based expression are stored at temperatures between -30°C and -10°C. In some embodiments, pharmaceutical compositions comprising RNA-based expression are stored at temperatures between -5°C and 15°C. In some embodiments, pharmaceutical compositions comprising RNA-based expression are stored at temperatures between 0°C and 10°C. In some embodiments, pharmaceutical compositions comprising RNA-based expression are stored at temperatures between 15°C and 35°C. In some embodiments, pharmaceutical compositions comprising RNA-based expression are stored at temperatures between 25°C and 35°C. In some embodiments, pharmaceutical compositions comprising RNA-based expression are stored at approximately -20°C. In some embodiments, pharmaceutical compositions comprising RNA-based expression are stored at approximately 5°C. In some embodiments, pharmaceutical compositions comprising RNA-based expression are stored at approximately 25°C.

[0011] definition In general, the terms used in the claims and this specification are intended to be interpreted to have their plain meaning as understood by one of ordinary skill in the art. Certain terms are defined below to provide additional clarity. In the event of a conflict between the plain meaning and a definition provided, the definition provided shall prevail.

[0012] As used herein, the term "pharmaceutical composition" is used interchangeably with the term "formulation."

[0013] The term "about" is used herein to mean approximately, within a range, roughly, or around. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the stated numerical values. In general, the term "about" is used herein to modify a numerical value above and below the stated value by a variance of 10%.

[0014] As used herein, the term "antigen" refers to a substance that stimulates an immune response. An antigen can be a neoantigen. An antigen can be a "shared antigen," which is an antigen found among a particular population, for example, a particular population of cancer patients.

[0015] As used herein, the term "neoantigen" refers to an antigen that has at least one alteration that distinguishes it from the corresponding wild-type antigen, for example, through a mutation in a tumor cell or a tumor cell-specific post-translational modification. A neoantigen can include a polypeptide sequence or a nucleotide sequence. Mutations can include frameshift or non-frameshift indels, missense or nonsense substitutions, splice site alterations, genomic rearrangements or gene fusions, or any genomic or expression alteration that results in a neoORF. Mutations can also include splice variants. Tumor cell-specific post-translational modifications can include aberrant phosphorylation. Tumor cell-specific post-translational modifications can also include spliced ​​antigens generated by the proteasome. See Liepe et al., A large fraction of HLA class I ligands are proteasome-generated spliced ​​peptides; Science. 2016 Oct 21;354(6310):354-358. Subjects can be identified for administration using various diagnostic methods, such as the patient selection methods described further below.

[0016] As used herein, the term "tumor antigen" is an antigen derived from a polypeptide that is known or found to be present in tumor cells or tissues of a subject, but not present in the subject's corresponding normal cells or tissues, or that has altered expression in tumor cells or cancerous tissues compared to normal cells or tissues.

[0017] As used herein, the term "antigen-based vaccine" refers to a vaccine composition that is based on one or more antigens, e.g., multiple antigens. Vaccines can be nucleotide-based (e.g., viral-based, RNA-based, or DNA-based), protein-based (e.g., peptide-based), or a combination thereof.

[0018] As used herein, the term "candidate antigen" is a mutation or other abnormality that gives rise to a sequence that may represent an antigen.

[0019] As used herein, the term "coding region" is the portion or portions of a gene that encodes a protein.

[0020] As used herein, the term "coding mutation" is a mutation that occurs in a coding region.

[0021] As used herein, the term "ORF" means open reading frame.

[0022] As used herein, the term "NEO-ORF" is a tumor-specific ORF that results from mutation or other abnormalities such as splicing.

[0023] As used herein, the term "missense mutation" is a mutation that results in the substitution of one amino acid for another.

[0024] As used herein, the term "nonsense mutation" is a mutation that results in the substitution of an amino acid with a stop codon or the removal of the canonical start codon.

[0025] As used herein, the term "frameshift mutation" is a mutation that causes a change in the frame of a protein.

[0026] As used herein, the term "indel" is an insertion or deletion of one or more nucleic acids.

[0027] As used herein, the term "percent identity" in the context of two or more nucleic acid or polypeptide sequences refers to two or more sequences or subsequences that have a specified percentage of nucleotides or amino acid residues that are the same when compared and aligned to closest correspondence using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to those of skill in the art) or as determined by visual inspection. Depending on the application, the percent "identity" can exist over a region of the sequences being compared, e.g., over a functional domain, or alternatively, over the entire length of the two sequences being compared.

[0028] For sequence comparison, typically, one sequence serves as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, and coordinates are subsequently designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence based on the designated program parameters. Alternatively, sequence similarity or difference can be established by the presence or absence of specific nucleotides or amino acids (e.g., sequence motifs) at selected sequence positions for the translated sequence.

[0029] Optimal alignment of sequences for comparison can be performed by the local homology algorithm of Smith and Waterman Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. MoL. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), and by visual inspection (see, e.g., generally, Ausubel et al., infra).

[0030] One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.

[0031] As used herein, the term "non-stop or read-through" refers to a mutation that results in the removal of the native stop codon.

[0032] As used herein, the term "epitope" is the specific portion of an antigen that is typically bound by an antibody or T-cell receptor.

[0033] As used herein, the term "immunogenic" is the ability to stimulate an immune response, for example, via T cells, B cells, or both.

[0034] As used herein, the terms "HLA binding affinity" and "MHC binding affinity" refer to the binding affinity between a particular antigen and a particular MHC allele.

[0035] As used herein, the term "bait" is a nucleic acid probe used to enrich for a specific sequence of DNA or RNA from a sample.

[0036] As used herein, the term "variant" refers to a difference between a nucleic acid of interest and a reference human genome used as a control.

[0037] As used herein, the term "variant calling" is an algorithmic determination of the presence of a variant, typically from sequencing.

[0038] As used herein, the term "polymorphism" refers to a germline variant, i.e., a variant found in all DNA-bearing cells of an individual.

[0039] As used herein, the term "somatic variant" is a variant that occurs in the non-germline cells of an individual.

[0040] As used herein, the term "allele" is a version of a gene or a version of a gene sequence or a version of a protein.

[0041] As used herein, the term "HLA type" refers to the complement of HLA gene alleles.

[0042] As used herein, the term "nonsense-mediated decay" or "NMD" is the degradation of mRNA by the cell due to a premature stop codon.

[0043] As used herein, the term "truncal mutation" is a mutation that originates early in the development of a tumor and is present in a significant proportion of the cells of the tumor.

[0044] As used herein, the term "subclonal mutation" refers to a mutation that originates late in the development of a tumor and is present in only a subset of tumor cells.

[0045] As used herein, the term "exome" refers to the subset of the genome that encodes proteins. The exome can be the collection of exons of the genome.

[0046] As used herein, the term "logistic regression" is a regression model for binary data from statistics in which the logit of the probability that the dependent variable is equal to 1 is modeled as a linear function of the dependent variable.

[0047] As used herein, the term "neural network" is a machine learning model for classification or regression that typically consists of multiple layers of linear transformations followed by element-wise nonlinearities trained via stochastic gradient descent and backpropagation.

[0048] As used herein, the term "proteome" is the set of all proteins expressed and / or translated by a cell, a group of cells, or an individual.

[0049] As used herein, the term "peptidome" refers to the set of all peptides presented by MHC-I or MHC-II on the cell surface. Peptidome can refer to the properties of a cell or a collection of cells (e.g., tumor peptidome, which refers to the combination of the peptidomes of all cells that comprise a tumor, or infectious disease peptidome, which refers to the combination of the peptidomes of all cells infected with an infectious disease).

[0050] As used herein, the term "ELISPOT" refers to enzyme-linked immunosorbent spot assay, a common method for monitoring immune responses in humans and animals.

[0051] As used herein, the term "dextramer" refers to a dextran-based peptide-MHC multimer used for antigen-specific T cell staining in flow cytometry.

[0052] As used herein, the term "tolerance or immune tolerance" is a state of immune non-responsiveness to one or more antigens, eg, self-antigens.

[0053] As used herein, the term "central tolerance" refers to tolerance that is affected in the thymus by deleting autoreactive T cell clones or by promoting their differentiation into immunosuppressive regulatory T cells (Tregs).

[0054] As used herein, the term "peripheral tolerance" is tolerance that is affected in the periphery by downregulating or activating autoreactive T cells that survive central tolerance, or by promoting the differentiation of these T cells into Tregs.

[0055] The term "sample" can include a single cell or multiple cells or cell fragments or an aliquot of bodily fluid obtained from a subject by means including venipuncture, excretion, ejaculation, massage, biopsy, needle aspiration, lavage sample, scraping, surgical incision, or intervention, or other means known in the art.

[0056] The term "subject" includes human or non-human cells, tissues, or organisms, whether in vivo, ex vivo, or in vitro, male or female. The term subject includes mammals, including humans.

[0057] The term "mammal" encompasses both humans and non-humans, and includes, but is not limited to, humans, non-human primates, canines, felines, murines, bovines, equines, and porcines.

[0058] The term "clinical factor" refers to a measure of a subject's condition, e.g., disease activity or severity. "Clinical factor" encompasses all markers of a subject's health status, including non-sample markers and / or other characteristics of the subject, such as, but not limited to, age and sex. A clinical factor can be a score, value, or set of values ​​that can be obtained from evaluation of a sample (or a population of samples) from a subject or subjects experiencing a determined condition. Clinical factors can also be predicted by markers and / or other parameters, such as gene expression surrogates. Clinical factors can include tumor type, tumor subtype, infection type, infection subtype, and smoking history.

[0059] The term "antigen-encoding nucleic acid sequence derived from a tumor" refers to nucleic acid sequences obtained from the tumor, e.g., via RT-PCR, or sequence data obtained by sequencing the tumor and then synthesizing nucleic acid sequences using the sequencing data, e.g., via various synthetic or PCR-based methods known in the art. Derived sequences can include nucleic acid sequence variants, such as sequence-optimized nucleic acid sequence variants (e.g., codon-optimized and / or otherwise optimized for expression), that encode the same polypeptide sequence as the corresponding native nucleic acid sequence obtained from the tumor.

[0060] The term "antigen-encoding nucleic acid sequence derived from an infection" refers to nucleic acid sequences obtained from infected cells or infectious disease organisms, e.g., via RT-PCR, or sequence data obtained by sequencing infected cells or infectious disease organisms and then synthesizing nucleic acid sequences using the sequencing data, e.g., via various synthetic or PCR-based methods known in the art. Derived sequences can include nucleic acid sequence variants, such as sequence-optimized nucleic acid sequence variants (e.g., codon-optimized and / or otherwise optimized for expression), that encode the same polypeptide sequence as the corresponding native infectious disease organism nucleic acid sequence. Derived sequences can include nucleic acid sequence variants that encode modified infectious disease organism polypeptide sequences having one or more (e.g., 1, 2, 3, 4, or 5) mutations relative to the native infectious disease organism polypeptide sequence. For example, a modified polypeptide sequence can have one or more missense mutations compared to the native polypeptide sequence of the infectious disease organism protein.

[0061] The term "alphavirus" refers to members of the Togaviridae family and are single-stranded, positive-sense RNA viruses. Alphaviruses are typically classified as Old World viruses, such as Sindbis virus, Ross River virus, Mayaro virus, Chikungunya virus, and Semliki Forest virus, or New World viruses, such as Eastern equine encephalitis, Aura virus, Fort Morgan virus, Venezuelan equine encephalitis, and its derivative TC-83. Alphaviruses are typically self-replicating RNA viruses.

[0062] The term "alphavirus backbone" refers to the minimal sequence(s) of an alphavirus that allows for autonomous replication of the viral genome. The minimal sequence can include conserved sequences for nonstructural protein-mediated amplification, the nonstructural protein 1 (nsP1) gene, the nsP2 gene, the nsP3 gene, the nsP4 gene, and polyA sequences, as well as sequences for expression of subgenomic viral RNA, including the subgenomic (e.g., 26S) promoter element.

[0063] The term "sequence for nonstructural protein-mediated amplification" includes alphavirus conserved sequence elements (CSEs) well known to those of skill in the art, including, but not limited to, alphavirus 5'UTR, 51-nt CSE, 24-nt CSE, subgenomic promoter sequences (e.g., 26S subgenomic promoter sequences), 19-nt CSE, and alphavirus 3'UTR.

[0064] The term "RNA polymerase" includes polymerases that catalyze the production of an RNA polynucleotide from a DNA template, including, but not limited to, bacteriophage-derived polymerases, including T3, T7, and SP6.

[0065] The term "lipid" includes hydrophobic and / or amphipathic molecules. Lipids can be cationic, anionic, or neutral. Lipids can be synthetic or naturally derived, and in some cases, biodegradable. Lipids can include cholesterol, phospholipids, lipid conjugates, including but not limited to polyethylene glycol (PEG) conjugates (PEGylated lipids), waxes, oils, glycerides, fats, and fat-soluble vitamins. Lipids can also include dilinoleylmethyl-4-dimethylaminobutyrate (MC3) and MC3-like molecules.

[0066] The term "lipid nanoparticle" or "LNP" includes vesicle-like structures, also known as liposomes, formed using a lipid-containing membrane surrounding an aqueous interior. Lipid nanoparticles include lipid-based compositions with a solid lipid core stabilized by surfactants. The core lipid can be fatty acids, acylglycerols, waxes, and mixtures of these surfactants. Biological membrane lipids, such as phospholipids, sphingomyelin, bile salts (sodium taurocholate), and sterols (cholesterol), can be utilized as stabilizers. Lipid nanoparticles can be formed using defined ratios of different lipid molecules, including, but not limited to, defined ratios of one or more cationic, anionic, or neutral lipids. Lipid nanoparticles can encapsulate molecules within their outer membrane shell, which can then be contacted with target cells to deliver the encapsulated molecules to the host cell cytosol. Lipid nanoparticles can be modified or functionalized with non-lipid molecules, including on their surface. Lipid nanoparticles can be unilamellar or multilamellar. Lipid nanoparticles can be complexed with nucleic acids. Unilamellar lipid nanoparticles can be complexed with nucleic acids, with the nucleic acid being in the aqueous interior. Multilamellar lipid nanoparticles can be complexed with nucleic acids, with the nucleic acid being in, formed between, or sandwiched between the aqueous interior.

[0067] Abbreviations: MHC: major histocompatibility complex, HLA: human leukocyte antigen or human MHC locus, NGS: next generation sequencing, PPV: positive predictive value, TSNA: tumor-specific neoantigen, FFPE: formalin-fixed paraffin-embedded, NMD: nonsense-mediated decay, NSCLC: non-small cell lung cancer, DC: dendritic cell.

[0068] It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0069] Unless specifically stated otherwise or clear from the context, the term "about" as used herein is understood to mean within normal tolerances in the art, e.g., within two standard deviations of the mean. About can be understood to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values ​​provided herein are modified by the term about.

[0070] Any terms not directly defined herein should be understood to have the meaning commonly associated with them as understood within the technical field of the present invention. Certain terms are discussed herein to provide additional guidance to the practitioner in describing the compositions, devices, methods, etc. of embodiments of the present invention and how to make or use them. It will be understood that the same thing may be referred to in more than one way. Consequently, alternative language and synonyms may be used for any one or more of the terms discussed herein. Whether a term is detailed or discussed herein is immaterial. Some synonyms or interchangeable methods, materials, etc. are provided. The recitation of one or several synonyms or equivalents does not exclude the use of other synonyms or equivalents unless explicitly stated. The use of examples, including examples of terms, is for illustrative purposes only and does not limit the scope and meaning of the embodiments of the present invention herein.

[0071] All references, issued patents, and patent applications cited within the body of this specification are hereby incorporated by reference in their entirety for all purposes.

[0072] antigen An antigen can comprise a nucleotide or a polypeptide. For example, an antigen can be an RNA sequence that encodes a polypeptide sequence. Thus, antigens useful in vaccines can comprise a nucleotide sequence or a polypeptide sequence.

[0073] Disclosed herein are isolated peptides comprising tumor-specific mutations identified by the methods disclosed herein, peptides comprising known tumor-specific mutations, and mutant polypeptides or fragments thereof identified by the methods disclosed herein. Neoantigen peptides can be described in the context of their coding sequences, where neoantigens comprise nucleotide sequences (e.g., DNA or RNA) that encode the associated polypeptide sequence.

[0074] Also disclosed herein are peptides derived from any polypeptide known or found to have altered expression in tumor cells or cancerous tissues compared to normal cells or tissues, for example, any polypeptide known or found to be abnormally expressed in tumor cells or cancerous tissues compared to normal cells or tissues. Suitable polypeptides from which antigenic peptides can be derived can be found, for example, in the COSMIC database. COSMIC screens comprehensive information on somatic mutations in human cancers. Peptides include tumor-specific mutations.

[0075] Also disclosed herein are peptides derived from any polypeptide associated with an infectious disease organism, an infection in a subject, or infected cells in a subject. Antigens can be derived from the nucleotide sequence or polypeptide sequence of an infectious disease organism. Polypeptide sequences of infectious disease organisms include, but are not limited to, pathogen-derived peptides, virus-derived peptides, bacterial-derived peptides, fungal-derived peptides, and / or parasite-derived peptides. Infectious disease organisms include, but are not limited to, severe acute respiratory syndrome-associated coronavirus (SARS), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Ebola, HIV, hepatitis B virus (HBV), influenza, hepatitis C virus (HCV), human papillomavirus (HPV), cytomegalovirus (CMV), chikungunya virus, respiratory syncytial virus (RSV), dengue virus, orthomyxoviridae, and tuberculosis.

[0076] Antigens can be selected that are predicted to be presented on the cell surface of cells such as tumor cells, infected cells, or immune cells, including professional antigen-presenting cells such as dendritic cells. Antigens can be selected that are predicted to be immunogenic.

[0077] One or more polypeptides encoded by the antigen nucleotide sequence have an IC of less than 1000 nM for an MHC class I peptide of 8-15, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in length. 50 The peptides may include at least one of: binding affinity to MHC having a specific binding affinity value, the presence of a sequence motif within or near the peptide that promotes proteasomal cleavage, and the presence or presence of a sequence motif that promotes TAP transport. For MHC class II peptides, the peptides may be 6-30, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids in length, and the presence of a sequence motif within or near the peptide promotes cleavage by extracellular or lysosomal proteases (e.g., cathepsins) or HLA binding catalyzed by HLA-DM.

[0078] One or more antigens may be presented on the surface of a tumor. One or more antigens may be presented on the surface of an infected cell.

[0079] The one or more antigens can be immunogenic in a subject having a tumor, e.g., capable of eliciting a T cell or B cell response in the subject. The one or more antigens can be immunogenic in a subject having or suspected of having an infection, e.g., capable of eliciting a T cell or B cell response in the subject. The one or more antigens can be immunogenic in a subject at risk of infection, e.g., capable of eliciting a T cell or B cell response in the subject that provides immunological protection (i.e., immunity) against the infection, e.g., capable of stimulating the production of memory T cells, memory B cells, or antibodies specific to the infection.

[0080] One or more antigens can elicit a B cell response, such as the production of antibodies that recognize the one or more antigens. Antibodies can recognize linear polypeptide sequences or can recognize secondary and tertiary structures. Thus, B cell antigens can include linear polypeptide sequences or polypeptides with secondary and tertiary structure, including, but not limited to, full-length proteins, protein subunits, protein domains, or any polypeptide sequence known or predicted to have secondary and tertiary structure.

[0081] One or more antigens that elicit an autoimmune response in a subject can be excluded from consideration in the context of generating a vaccine for the subject.

[0082] The size of the at least one antigenic peptide molecule (e.g., epitope sequence) can be about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, about 100, about 101, about 102 The antigenic peptide molecule can include, but is not limited to, about 4, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120 or more amino acid residues, and any range derivable therein. In certain embodiments, the antigenic peptide molecule is 50 amino acids or less.

[0083] Antigenic peptides and polypeptides can be 15 residues or less in length, typically about 8 to about 11 residues, particularly 9 or 10 residues, for MHC class I, and can contain 6 to 30 residues (inclusive) for MHC class II.

[0084] If desired, longer peptides can be designed in several ways. In one case, when the likelihood of peptide presentation on HLA alleles is predicted or known, longer peptides can consist of either (1) individual presented peptides with extensions of 2–5 amino acids toward the N- and C-termini of each corresponding gene product, or (2) concatenation of part or all of the presented peptide with the respective extension sequences. In another case, when sequencing reveals long (more than 10 residues) neo-epitope sequences present in tumors (e.g., due to frameshifts, readthrough, or intron inclusion resulting in novel peptide sequences), longer peptides can consist of the full range of novel tumor- or infectious disease-specific amino acids, thus avoiding the need for computational or in vitro test-based selection of shorter peptides presented by the strongest HLA alleles. In both cases, the use of longer peptides may allow endogenous processing by patient cells, resulting in more effective antigen presentation and elicitation of T cell responses.

[0085] Antigenic peptides and polypeptides can be presented on HLA proteins. In some embodiments, antigenic peptides and polypeptides are presented on HLA proteins with higher affinity than wild-type peptides. In some embodiments, antigenic peptides or polypeptides are presented on HLA proteins with higher affinity than wild-type peptides. 50 may be at least less than 5000 nM, at least less than 1000 nM, at least less than 500 nM, at least less than 250 nM, at least less than 200 nM, at least less than 150 nM, at least less than 100 nM, at least less than 50 nM or less.

[0086] In some embodiments, the antigenic peptides and polypeptides do not induce an autoimmune response and / or do not induce immune tolerance when administered to a subject.

[0087] Compositions comprising at least two or more antigenic peptides are also provided. In some embodiments, the compositions contain at least two distinct peptides. The at least two distinct peptides can be derived from the same polypeptide. Distinct polypeptides mean that the peptides vary in length, amino acid sequence, or both. The peptides can be derived from any polypeptide known or found to contain a tumor-specific mutation, or any polypeptide known or found to have altered expression in tumor cells or cancerous tissue compared to normal cells or tissues, e.g., any polypeptide known or found to be aberrantly expressed in tumor cells or cancerous tissue compared to normal cells or tissues. The peptides can be derived from any polypeptide known or suspected to be associated with an infectious disease organism, or any polypeptide known or found to have altered expression in infected cells compared to normal cells or tissues (e.g., an infectious disease polynucleotide or polypeptide, including an infectious disease polynucleotide or polypeptide with host cell-restricted expression). Suitable polypeptides from which antigenic peptides can be derived can be found, for example, in the COSMIC database or the AACR Genomics Evidence Neoplasia Information Exchange (GENIE) database. COSMIC compiles comprehensive information on somatic mutations in human cancers. AACR GENIE aggregates clinical-grade cancer genomic data and links them to clinical outcomes from tens of thousands of cancer patients. In some embodiments, the tumor-specific mutation is a driver mutation for a particular cancer type.

[0088] Antigenic peptides and polypeptides with desired activities or properties can be modified to provide certain desired attributes, e.g., improved pharmacological characteristics, while increasing or at least retaining substantially all of the biological activity of the unmodified peptide for binding to desired MHC molecules and activating appropriate T cells. For example, antigenic peptides and polypeptides can be subjected to various changes, such as conservative or non-conservative substitutions, which can confer certain advantages in their use, such as improved MHC binding, stability, or presentation. Conservative substitutions refer to the replacement of an amino acid residue with another that is biologically and / or chemically similar, e.g., one hydrophobic residue for another, or one polar residue for another. Substitutions include combinations such as Gly, Ala, Val, Ile, Leu, Met, Asp, Glu, Asn, Gln; Ser, Thr; Lys, Arg; and Phe, Tyr. The effects of single amino acid substitutions can also be examined using D-amino acids. Such modifications can be carried out using well-known peptide synthesis procedures, as described, for example, in Merrifield, Science 232:341-347 (1986), Barany & Merrifield, The Peptides, Gross & Meienhofer, eds. (NY, Academic Press), pp. 1-284 (1979), and Stewart & Young, Solid Phase Peptide Synthesis, (Rockford, III., Pierce), 2nd Ed. (1984).

[0089] Modification of peptides and polypeptides with various amino acid mimetics or unnatural amino acids can be particularly useful for increasing the stability of peptides and polypeptides in vivo. Stability can be assayed in several ways. For example, stability is tested using peptidases and various biological media, such as human plasma and serum. See, for example, Verhoef, et al., Eur. J. Drug Metab Pharmacokin. 11:291-302 (1986). Peptide half-life can be conveniently determined using a 25% human serum (v / v) assay. The protocol generally follows: Pooled human serum (type AB, non-heat-inactivated) is defatted by centrifugation before use. The serum is then diluted to 25% with RPMI tissue culture medium and used to test peptide stability. At predetermined time intervals, a small amount of the reaction solution is removed and added to either 6% aqueous trichloroacetic acid or ethanol. The cloudy reaction sample is cooled for 15 minutes (4°C) and then spun to pellet precipitated serum proteins. The presence of the peptide is then determined by reverse-phase HPLC using stability-specific chromatography conditions.

[0090] Peptides and polypeptides can be modified to provide desirable attributes other than improved serum half-life. For example, the ability of a peptide to induce CTL activity can be enhanced by linking it to a sequence containing at least one epitope capable of inducing a T helper cell response. The immunogenic peptide / T helper conjugate can be linked by a spacer molecule. The spacer is typically composed of relatively small, neutral molecules, such as amino acids or amino acid mimetics, that are substantially uncharged under physiological conditions. The spacer is typically selected from, for example, Ala, Gly, or other neutral spacers of nonpolar or neutral polar amino acids. It will be understood that, if present, the spacer need not be composed of the same residues and can thus be a hetero- or homo-oligomer. If present, the spacer is typically at least one or two residues, more usually three to six residues. Alternatively, the peptide can be linked to the T helper peptide without a spacer.

[0091] The antigenic peptide can be linked to the T helper peptide either directly or via a spacer at either the amino or carboxy terminus of the peptide. The amino terminus of either the antigenic peptide or the T helper peptide can be acylated. Exemplary T helper peptides include tetanus toxoid 830-843, influenza 307-319, malaria sporozoite 382-398, and 378-389.

[0092] Proteins or peptides can be produced by any technique known to those of skill in the art, including expressing the protein, polypeptide, or peptide via standard molecular biology techniques, isolating the protein or peptide from a natural source, or chemically synthesizing the protein or peptide. Nucleotide and protein, polypeptide, and peptide sequences corresponding to various genes have been previously disclosed and can be found in computerized databases known to those of skill in the art. One such database is the Genbank and GenPept databases of the National Center for Biotechnology Information, located on the National Institutes of Health website. The coding regions of known genes can be amplified and / or expressed using the techniques disclosed herein or as known to those of skill in the art. Alternatively, various commercially available preparations of proteins, polypeptides, and peptides are known to those of skill in the art.

[0093] In a further aspect, the antigen comprises a nucleic acid (e.g., a polynucleotide) encoding an antigenic peptide or a portion thereof. The polynucleotide can be, for example, DNA, cDNA, PNA, CNA, RNA (e.g., mRNA), either single-stranded and / or double-stranded, or a polynucleotide in a natural or stabilized form, e.g., a polynucleotide with a phosphonothioate backbone, or a combination thereof, with or without introns. A still further aspect provides an expression vector capable of expressing the polypeptide or a portion thereof. Expression vectors for different cell types are well known in the art and can be selected without undue experimentation. Generally, the DNA is inserted into an expression vector, such as a plasmid, in the proper orientation and correct reading frame for expression. If necessary, the DNA can be linked to appropriate transcriptional and translational regulatory control nucleotide sequences recognized by the desired host; such controls are generally available in expression vectors. The vector is then introduced into the host by standard techniques. Guidance can be found, for example, in Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

[0094] lipid nanoparticles In some embodiments, any of the above compositions further comprises a nanoparticle delivery vehicle. The nanoparticle delivery vehicle may be a lipid nanoparticle (LNP). In some embodiments, the LNP comprises an ionizable amino lipid. In some embodiments, the ionizable amino lipid comprises an MC3-like (dilinoleylmethyl-4-dimethylaminobutyrate) molecule. In some embodiments, the nanoparticle delivery vehicle encapsulates a neoantigen expression system.

[0095] In some embodiments, any of the above compositions further comprise a plurality of LNPs, the LNPs comprising a neoantigen expression system, a cationic lipid, a non-cationic lipid, and a conjugated lipid that inhibits aggregation of the LNPs, and at least about 95% of the LNPs in the plurality of LNPs either have a non-lamellar morphology or are electron dense.

[0096] In some embodiments, the non-cationic lipid is a mixture of (1) a phospholipid and (2) cholesterol or a cholesterol derivative.

[0097] In some embodiments, the conjugated lipid that inhibits aggregation of LNPs is a polyethylene glycol (PEG)-lipid conjugate. In some embodiments, the PEG-lipid conjugate is selected from the group consisting of PEG-diacylglycerol (PEG-DAG) conjugates, PEG-dialkyloxypropyl (PEG-DAA) conjugates, PEG-phospholipid conjugates, PEG-ceramide (PEG-Cer) conjugates, and mixtures thereof. In some embodiments, the PEG-DAA conjugate is PEG-didecyloxypropyl (C 10 ) conjugate, PEG-dilauryloxypropyl (C 12 ) conjugate, PEG-dimyristyloxypropyl (C 14 ) conjugate, PEG-dipalmityloxypropyl (C 16 ) conjugate, PEG-distearyloxypropyl (C 18 ) conjugates, and mixtures thereof.

[0098] In some embodiments, the neoantigen expression system is fully encapsulated in the LNP.

[0099] In some embodiments, the non-lamellar morphology of the LNPs is an inverted hexagonal (H II ) or cubic phase structure.

[0100] In some embodiments, the cationic lipids comprise about 10 mol% to about 50 mol% of the total lipids present in the LNP. In some embodiments, the cationic lipids comprise about 20 mol% to about 50 mol% of the total lipids present in the LNP. In some embodiments, the cationic lipids comprise about 20 mol% to about 40 mol% of the total lipids present in the LNP.

[0101] In some embodiments, the non-cationic lipids comprise between about 10 mol% and about 60 mol% of the total lipids present in the LNP. In some embodiments, the non-cationic lipids comprise between about 20 mol% and about 55 mol% of the total lipids present in the LNP. In some embodiments, the non-cationic lipids comprise between about 25 mol% and about 50 mol% of the total lipids present in the LNP.

[0102] In some embodiments, complex lipids comprise from about 0.5 mol% to about 20 mol% of the total lipids present in the LNP. In some embodiments, complex lipids comprise from about 2 mol% to about 20 mol% of the total lipids present in the LNP. In some embodiments, complex lipids comprise from about 1.5 mol% to about 18 mol% of the total lipids present in the LNP.

[0103] In some embodiments, greater than 95% of the LNPs have a non-lamellar morphology. In some embodiments, greater than 95% of the LNPs are electron-dense.

[0104] In some embodiments, any of the above compositions further comprise a plurality of LNPs, the LNPs comprising a cationic lipid comprising 50 mol% to 65 mol% of the total lipids present in the LNP, a complex lipid that inhibits aggregation of the LNP comprising 0.5 mol% to 2 mol% of the total lipids present in the LNP, and a non-cationic lipid comprising any of the following: a mixture of phospholipids comprising 4 mol% to 10 mol% of the total lipids present in the LNP and cholesterol or a derivative thereof comprising 30 mol% to 40 mol% of the total lipids present in the LNP, a mixture of phospholipids comprising 3 mol% to 15 mol% of the total lipids present in the LNP and cholesterol or a derivative thereof comprising 30 mol% to 40 mol% of the total lipids present in the LNP, or a mixture of up to 49.5 mol% of the total lipids present in the LNP and cholesterol or a derivative thereof comprising 30 mol% to 40 mol% of the total lipids present in the LNP.

[0105] In some embodiments, any of the above compositions further comprise a plurality of LNPs, the LNPs comprising cationic lipids comprising 50 mol% to 85 mol% of the total lipids present in the LNPs, complex lipids that inhibit aggregation of the LNPs comprising 0.5 mol% to 2 mol% of the total lipids present in the LNPs, and non-cationic lipids comprising 13 mol% to 49.5 mol% of the total lipids present in the LNPs.

[0106] In some embodiments, the phospholipid comprises dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), or a mixture thereof.

[0107] In some embodiments, the conjugated lipid comprises a polyethylene glycol (PEG)-lipid conjugate. In some embodiments, the PEG-lipid conjugate comprises a PEG-diacylglycerol (PEG-DAG) conjugate, a PEG-dialkyloxypropyl (PEG-DAA) conjugate, or a mixture thereof. In some embodiments, the PEG-DAA conjugate comprises a PEG-dimyristyloxypropyl (PEG-DMA) conjugate, a PEG-distearyloxypropyl (PEG-DSA) conjugate, or a mixture thereof. In some embodiments, the PEG portion of the conjugate has an average molecular weight of about 2,000 daltons.

[0108] In some embodiments, complex lipids comprise between 1 mol % and 2 mol % of the total lipids present in the LNP.

[0109] In some embodiments, the LNP is a compound having the structure of Formula I [ka] or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein L 1 and L 2 are each independently -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(0) x -, -SS-, -C(=O)S-, -SC(=O)-, -R a C(=O)-, -C(=O)R a -, -R a C(=O)R a -, -OC(=O)R a -, -R a C(=O)O- or a direct bond, and G 1 is C1-C2 alkylene, -(C=O)-, -O(C=O)-, -SC(=O)-, -R a C(=O)- or a direct bond: -C(=O)-, -(C=O)O-, -C(=O)S-, -C(=O)R a - or a direct bond, G is a C1-C6 alkylene, and R a is H or C1~C 12alkyl, and R 1a and R 1b is, at each occurrence, independently: (a) H or C1-C 12 alkyl, or (b) R 1a is H or C1~C 12 alkyl, and R 1b together with the carbon atom to which it is attached, the adjacent R 1b and together with the carbon atom to which it is attached form a carbon-carbon double bond; R 2a and R 2b is, at each occurrence, independently: (a) H or C1-C 12 alkyl, or (b) R 2a is H or C1~C 12 alkyl, and R 2b together with the carbon atom to which it is attached, the adjacent R 2b and together with the carbon atom to which it is attached form a carbon-carbon double bond; R 3a and R 3b is, at each occurrence, independently: (a) H or C1-C 12 alkyl, or (b) R 3a is H or C1~C 12 alkyl, and R 3b together with the carbon atom to which it is attached, form a carbon-carbon double bond with the adjacent R and the carbon atom to which it is attached, and R 4a and R 4b is, at each occurrence, independently: (a) H or C1-C 12 alkyl, or (b) R 4a is H or C1~C 12 alkyl, and R 4b together with the carbon atom to which it is attached, the adjacent R 4b and together with the carbon atom to which it is attached form a carbon-carbon double bond; R 5 and R 6 are each independently H or methyl, and R 7 is C4~C 20alkyl, and R 8 and R 9 are independently C1 to C 12 alkyl, or R 8 and R 9 together with the nitrogen atom to which they are attached form a 5-, 6-, or 7-membered heterocycle; a, b, c, and d are each independently an integer of 1 to 24; and x is 0, 1, or 2.

[0110] In some embodiments, the LNP is a compound having the structure of Formula II [ka] or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein L 1 and L 2 are each independently —O(C═O)—, —(C═O)O—, or a carbon-carbon double bond; R 1a and R 1b is, at each occurrence, independently: (a) H or C1-C 12 alkyl, or (b) R 1a is H or C1~C 12 alkyl, and R 1b together with the carbon atom to which it is attached, the adjacent R 1b and together with the carbon atom to which it is attached form a carbon-carbon double bond; R 2a and R 2b is, at each occurrence, independently: (a) H or C1-C 12 alkyl, or (b) R 2a is H or C1~C 12 alkyl, and R 2b together with the carbon atom to which it is attached, the adjacent R 2b and together with the carbon atom to which it is attached form a carbon-carbon double bond; R 3a and R 3b is, at each occurrence, independently: (a) H or C1-C 12 alkyl, or (b) R 3ais H or C1~C 12 alkyl, and R 3b together with the carbon atom to which it is attached, the adjacent R 3b and together with the carbon atom to which it is attached form a carbon-carbon double bond; R 4a and R 4b is, at each occurrence, independently: (a) H or C1-C 12 alkyl, or (b) R 4a is H or C1~C 12 alkyl, and R 4b together with the carbon atom to which it is attached, the adjacent R 4b and together with the carbon atom to which it is attached form a carbon-carbon double bond; R 5 and R 6 are each independently methyl or cycloalkyl, and R 7 is, at each occurrence, independently H or C1-C 12 alkyl, and R 8 and R 9 are each independently unsubstituted C1 to C 12 alkyl or R 8 and R 9 together with the nitrogen atom to which they are attached, form a 5-, 6-, or 7-membered heterocycle containing one nitrogen atom; a and d are each independently an integer of 0 to 24; b and c are each independently an integer of 1 to 24; and e is 1 or 2, with the proviso that R 1a , R 2a , R 3a or R 4a At least one of C1 to C 12 alkyl or L 1 Or L 2 at least one of is —O(C═O)— or —(C═O)O—, and R 1a and R 1b is not isopropyl when a is 6 and is n-butyl when a is 8.

[0111] In some embodiments, any of the above compositions further comprise one or more excipients, including a neutral lipid, a steroid, and a polymeric conjugate lipid. In some embodiments, the neutral lipid comprises at least one of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). In some embodiments, the neutral lipid is DSPC.

[0112] In some embodiments, the molar ratio of compound to neutral lipid ranges from about 2:1 to about 8:1.

[0113] In some embodiments, the steroid is cholesterol, hi some embodiments, the molar ratio of the compound to cholesterol ranges from about 2:1 to 1:1.

[0114] In some embodiments, the polymer-conjugated lipid is a PEGylated lipid. In some embodiments, the molar ratio of the compound to the PEGylated lipid ranges from about 100:1 to about 25:1. In some embodiments, the PEGylated lipid is PEG-DAG, PEG polyethylene (PEG-PE), PEG-succinyl-diacylglycerol (PEG-S-DAG), PEG-cer, or PEG dialkyloxypropylcarbamate. In some embodiments, the PEGylated lipid is represented by the following structure III: [ka] or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein R 10 and R 11are each independently a linear or branched, saturated or unsaturated alkyl chain containing 10 to 30 carbon atoms, the alkyl chain optionally being interrupted by one or more ester linkages, and z having an average value ranging from 30 to 60. In some embodiments, R 10 and R 11 are each independently a straight, saturated alkyl chain having 12 to 16 carbon atoms. In some embodiments, the average z is about 45.

[0115] In some embodiments, the LNPs self-assemble into non-bilayer structures when mixed with polyanionic nucleic acids. In some embodiments, the non-bilayer structures are 60 nm to 120 nm in diameter. In some embodiments, the non-bilayer structures are about 70 nm, about 80 nm, about 90 nm, or about 100 nm in diameter. In some embodiments, the nanoparticle delivery vehicle is about 100 nm in diameter.

[0116] Payload and Antigen The payload nucleic acid sequence can be any nucleic acid sequence desired to be delivered to a cell of interest. Generally, the payload is a nucleic acid sequence linked to a promoter that drives expression of the nucleic acid sequence. The payload nucleic acid sequence can encode a polypeptide (i.e., a nucleic acid sequence that can be transcribed and translated into a protein). Generally, a payload nucleic acid sequence that encodes a peptide can encode any protein desired to be expressed in a cell. Proteins include, but are not limited to, antigens (e.g., MHC class I epitopes, MHC class II epitopes, or epitopes that can stimulate a B-cell response), antibodies, cytokines, chimeric antigen receptors (CARs), T-cell receptors, or genome editing system components (e.g., nucleases used in genome editing systems). Genome editing systems include, but are not limited to, CRISPR systems, zinc finger systems, meganuclease systems, or TALEN systems. The payload nucleic acid sequence can be non-coding (i.e., a nucleic acid sequence that can be transcribed but not translated into a protein). Generally, a non-coding payload nucleic acid sequence can be any non-coding polynucleotide desired to be expressed in a cell. Non-coding polynucleotides include, but are not limited to, RNA interference (RNAi) polynucleotides (e.g., antisense oligonucleotides, shRNA, siRNA, miRNA, etc.) or genome editing polynucleotides (e.g., guide RNA [gRNA], single guide RNA [sgRNA], trans-activating CRISPR [tracrRNA], and / or CRISPR RNA [crRNA]). A payload nucleic acid sequence can encode two or more (e.g., 2, 3, 4, 5, or more) distinct polypeptides (e.g., two or more distinct epitope sequences linked to each other) or contain two or more distinct non-coding nucleic acid sequences (e.g., two or more different RNAi polynucleotides). A payload nucleic acid sequence can have a combination of polypeptide-encoding and non-coding nucleic acid sequences.

[0117] The vector may comprise 1 to 30 payload-encoding nucleic acid sequences, 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, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65 , 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more different payload-encoding nucleic acid sequences, 6, 7, 8, 9, 10, 11, 12, 13, or 14 different payload-encoding nucleic acid sequences, or 12, 13, or 14 different payload-encoding nucleic acid sequences. A payload-encoding nucleic acid sequence may refer to the payload-encoding portion of a "cassette." Cassette characteristics are described in more detail herein. A cassette may contain two or more payload-encoding nucleic acid sequences linked together within the cassette (e.g., as an illustrative, non-limiting example, linked antigen-encoding nucleic acid sequences encoding linked T cell epitopes).

[0118] The vector may contain 1 to 30 distinct payload-encoding nucleic acid sequences, 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, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 1 The cassette may comprise 5, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more distinct payload-encoding nucleic acid sequences, 6, 7, 8, 9, 10, 11, 12, 13, or 14 distinct payload-encoding nucleic acid sequences, or 12, 13, or 14 distinct payload-encoding nucleic acid sequences. A payload-encoding nucleic acid sequence can refer to the sequence of an individual payload sequence, e.g., as an illustrative, non-limiting example, each of the linked T-cell epitopes of two or more payload-encoding nucleic acid sequences linked together in a cassette.

[0119] The antigen can comprise a nucleotide or a polypeptide. For example, the antigen can be an RNA sequence that encodes a polypeptide sequence. Thus, the antigen useful for the vaccine can comprise a nucleotide sequence or a polypeptide sequence. Antigens that can be used for cancer vaccines are described in International Patent Application Publication No. WO / 2019 / 226941, which is incorporated herein by reference in its entirety for all purposes.

[0120] Disclosed herein are isolated peptides comprising tumor-specific mutations identified by the methods disclosed herein, peptides comprising known tumor-specific mutations, and mutant polypeptides or fragments thereof identified by the methods disclosed herein. Neoantigen peptides can be described in the context of their coding sequences, where neoantigens comprise nucleotide sequences (e.g., DNA or RNA) that encode the associated polypeptide sequence.

[0121] Also included herein are peptides derived from any polypeptide known or found to have altered expression in tumor cells or cancerous tissues compared to normal cells or tissues, e.g., any polypeptide known or found to be aberrantly expressed in tumor cells or cancerous tissues compared to normal cells or tissues. Suitable polypeptides from which antigenic peptides can be derived can be found, for example, in the COSMIC database. COSMIC screens comprehensive information on somatic mutations in human cancers. Peptides include tumor-specific mutations. Tumor antigens (e.g., shared tumor antigens and tumor neoantigens) can include, but are not limited to, those described in U.S. Patent No. 17 / 058,128, which is incorporated herein for all purposes. Antigenic peptides can be described in the context of their coding sequences, where the antigenic peptide includes a nucleotide sequence (e.g., DNA or RNA) that encodes the relevant polypeptide sequence.

[0122] One or more polypeptides encoded by the antigen nucleotide sequence have an IC of less than 1000 nM for an MHC class I peptide of 8-15, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in length. 50The peptides may include at least one of: binding affinity to MHC having a specific binding affinity value, the presence of a sequence motif within or near the peptide that promotes proteasomal cleavage, and the presence of a sequence motif that promotes TAP transport. For MHC class II peptides, the peptides may be 6-30, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids in length, and the presence of a sequence motif within or near the peptide promotes cleavage by extracellular or lysosomal proteases (e.g., cathepsins) or HLA-DM-catalyzed HLA binding.

[0123] One or more antigens can be presented on the surface of the tumor.

[0124] The one or more antigens can be immunogenic in a tumor-bearing subject, for example, capable of eliciting a T cell or B cell response in the subject.

[0125] One or more antigens that induce an autoimmune response in a subject can be excluded from consideration in the context of generating a vaccine for a tumor-bearing subject.

[0126] The size of the at least one antigenic peptide molecule can include, but is not limited to, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120 or more amino acid residues, and any range derivable therein. In certain embodiments, the antigenic peptide molecule is 50 amino acids or less.

[0127] Antigenic peptides and polypeptides can be 15 residues or less in length, typically about 8 to about 11 residues, particularly 9 or 10 residues, for MHC class I, and can contain 6 to 30 residues (inclusive) for MHC class II.

[0128] Antigenic peptides and polypeptides can be presented on HLA proteins. In some embodiments, antigenic peptides and polypeptides are presented on HLA proteins with higher affinity than wild-type peptides. In some embodiments, antigenic peptides or polypeptides are presented on HLA proteins with higher affinity than wild-type peptides. 50 may be at least less than 5000 nM, at least less than 1000 nM, at least less than 500 nM, at least less than 250 nM, at least less than 200 nM, at least less than 150 nM, at least less than 100 nM, at least less than 50 nM or less.

[0129] In some embodiments, the antigenic peptides and polypeptides do not induce an autoimmune response and / or do not induce immune tolerance when administered to a subject.

[0130] Compositions comprising at least two or more antigenic peptides are also provided. In some embodiments, the composition contains at least two distinct peptides. The at least two distinct peptides can be derived from the same polypeptide. Distinct polypeptides mean that the peptides vary in length, amino acid sequence, or both. The peptides can be derived from any polypeptide known or found to contain tumor-specific mutations, or any polypeptide known or found to have altered expression in tumor cells or cancerous tissues compared to normal cells or tissues, e.g., any polypeptide known or found to be aberrantly expressed in tumor cells or cancerous tissues compared to normal cells or tissues. Suitable polypeptides from which antigenic peptides can be derived can be found, for example, in the COSMIC database or the AACR Genomics Evidence Neoplasia Information Exchange (GENIE) database. COSMIC compiles comprehensive information on somatic mutations in human cancers. AACR GENIE compiles clinical-grade cancer genomic data and links it to clinical outcomes from tens of thousands of cancer patients. The peptides contain tumor-specific mutations. In some aspects, the tumor-specific mutation is a driver mutation for a particular cancer type.

[0131] Also disclosed herein are peptides derived from any polypeptide associated with an infectious disease organism, an infection in a subject, or infected cells in a subject. Antigens can be derived from the nucleotide sequence or polypeptide sequence of an infectious disease organism. Polypeptide sequences of infectious disease organisms include, but are not limited to, pathogen-derived peptides, virus-derived peptides, bacterial-derived peptides, fungal-derived peptides, and / or parasite-derived peptides. Infectious disease organisms include, but are not limited to, severe acute respiratory syndrome-associated coronavirus (SARS), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Ebola, HIV, hepatitis B virus (HBV), influenza, hepatitis C virus (HCV), human papillomavirus (HPV), cytomegalovirus (CMV), chikungunya virus, respiratory syncytial virus (RSV), dengue virus, orthomyxoviridae, and tuberculosis.

[0132] Disclosed herein are isolated peptides comprising infectious disease organism-specific antigens or epitopes identified by the methods disclosed herein, peptides comprising known infectious disease organism-specific antigens or epitopes, and mutant polypeptides or fragments thereof identified by the methods disclosed herein. Antigenic peptides can be described in the context of their coding sequences, where antigens include nucleotide sequences (e.g., DNA or RNA) that encode the relevant polypeptide sequence.

[0133] The vectors and related compositions described herein can be used to deliver antigens from any organism, including their toxins or other by-products, to prevent and / or treat infections or other adverse reactions associated with the organism or its by-products.

[0134] Antigens (e.g., encoded by cassettes) that can be incorporated into vaccines include immunogens useful for immunizing humans or non-human animals against viruses, such as pathogenic viruses that infect humans and non-human vertebrates. Antigens can be selected from various viral families. Examples of desirable viral families against which an immune response is desired include the picornavirus family, which includes the rhinovirus genus, which is responsible for approximately 50% of cold cases; the enterovirus genus, which includes human enteroviruses such as polypoliovirus, coxsackievirus, echovirus, and hepatitis A virus; and the aptovirus genus, which is primarily responsible for foot-and-mouth disease in non-human animals. Within the picornavirus family of viruses, target antigens include VP1, VP2, VP3, VP4, and VPG. Another viral family is the calcivirus family, which includes the Norwalk virus group, a virus that is an important causative agent of epidemic gastroenteritis. Yet another desirable viral family for use in targeting antigens to stimulate immune responses in humans and non-human animals is the Togavirus family, which includes the Alphavirus genus, which includes Sindbis virus, Ross River virus, and Venezuelan, Eastern, and Western equine encephalitis viruses, as well as Rubiviruses, including rubella virus. The Flaviviridae family includes the viruses of dengue fever, yellow fever, Japanese encephalitis, St. Louis encephalitis, and tick-borne encephalitis. Other target antigens can be generated from the hepatitis C virus or coronavirus family, which includes several non-human viruses, such as infectious bronchitis virus (poultry), porcine transmissible gastroenteritis virus (pigs), porcine hemagglutinating encephalomyelitis virus (pigs), feline infectious peritonitis virus (cats), feline enteric coronavirus (cats), canine coronavirus (dogs), and human respiratory coronaviruses, which can cause the common cold and / or non-A, B, or C hepatitis. Within the coronavirus family, target antigens include E1 (also called M or matrix protein), E2 (also called S or spike protein), E3 (also called HE or hemagglutinin-eluterose) glycoproteins (not present in all coronaviruses), or N (nucleocapsid).Still other antigens may target the rhabdovirus family, which includes the vesiculovirus genus (e.g., vesicular stomatitis virus) and common lyssaviruses (e.g., rabies virus). Within the rhabdovirus family, suitable antigens may be derived from the G protein or N protein. The filoviridae family includes hemorrhagic fever viruses such as Marburg virus and Ebola virus and may be suitable sources of antigens. The paramyxovirus family includes parainfluenza virus type 1, parainfluenza virus type 3, bovine parainfluenza virus type 3, rubulavirus (mumps virus), parainfluenza virus type 2, parainfluenza virus type 4, Newcastle disease virus (chicken), morbilliviruses including rinderpest, measles, and canine distemper, and pneumonia viruses including respiratory syncytial viruses (e.g., glyco- (G) protein and fusion (F) protein, the sequences of which are available from GenBank). Influenza virus is classified within the orthomyxovirus family and may be a suitable source of antigens (e.g., HA protein, N1 protein). The Bunyavirus family includes the Bunyavirus genus (California encephalitis, La Crosse), Phlebovirus genus (Rift Valley fever), Hantavirus genus (Puremara and Hemahagin fever viruses), Nairovirus genus (Nairobi sheep disease), and various unclassified Bungaviruses. The Arenavirus family provides the antigenic source for LCM and Lassa fever viruses. The Reovirus family includes the Reovirus genus, Rotavirus genus (causing acute gastroenteritis in children), Orbiviruses, and Carciviruses (Colorado tick fever, Lebombo (human), equine encephalopathy, bluetongue). The Retrovirus family includes the Oncovirinae subfamily, which encompasses human and veterinary diseases such as feline leukemia virus, HTLV-1 and HTLV-2, lentiviruses (including human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), equine infectious anemia virus, and spumaviruses). Among lentiviruses, many suitable antigens have been described and can be readily selected.Suitable HIV and SIV antigens include, but are not limited to, gag, pol, Vif, Vpx, VPR, Env, Tat, Nef, and Rev proteins, as well as various fragments thereof. For example, suitable fragments of the Env protein may include any of its subunits, such as gp120, gp160, or gp41, or smaller fragments thereof, e.g., at least about eight amino acids in length. Similarly, fragments of the tat protein may be selected. [See U.S. Pat. Nos. 5,891,994 and 6,193,981.] See also the HIV and SIV proteins described in D.H. Barouch et al., J. Virol., 75(5):2462-2467 (March 2001) and R.R. Amara, et al., Science, 292:69-74 (April 6, 2001). In another example, HIV and / or SIV immunogenic proteins or peptides may be used to form fusion proteins or other immunogenic molecules. See, for example, the HIV-1 Tat and / or Nef fusion proteins and immunization regimens described in WO 01 / 54719, published August 2, 2001, and WO 99 / 16884, published April 8, 1999. The present invention is not limited to the HIV and / or SIV immunogenic proteins or peptides described herein. Furthermore, various modifications to these proteins have been described or can be readily obtained by those skilled in the art. See, for example, the modified gag proteins described in U.S. Pat. No. 5,972,596. Furthermore, any desired HIV and / or SIV immunogens may be delivered alone or in combination. Such combinations may involve expression from a single vector or multiple vectors. The papovavirus family includes the Polyomavirinae subfamily (BKU and JCU viruses) and the Papillomavirinae subfamily (associated with the malignant progression of cancer or papilloma). The adenovirus family includes viruses that cause respiratory disease and / or enteritis (e.g., EX, AD7, ARD, OB). The parvovirus family includes feline parvovirus (feline enteritis), feline panleucopeniavirus, canine parvovirus, and porcine parvovirus.The herpesvirus family includes the Alphaherpesvirinae subfamily, which includes the genera Simplexvirus (HSVI, HSVII) and Varicellovirus (pseudorabies, varicella-zoster), and the Betaherpesvirinae subfamily, which includes the genera Cytomegalovirus (human CMV) and Muromegalovirus, as well as the Gammaherpesvirinae subfamily, which includes the genera Lymphocryptovirus, EBV (Burkitt's lymphoma), infectious rhinotracheitis, Marek's disease virus, and Rhadinovirus. The poxvirus family includes the Orthopoxvirus genera (Valiola (smallpox) and Vaccinia (cowpox)), the Chordopoxvirinae subfamily, which includes Parapoxvirus, Avipoxvirus, Capripoxvirus, Leporipoxvirus, and Suipoxvirus, and the Entomopoxvirinae subfamily. The hepadnavirus family includes Hepatitis B virus. One unclassified virus that may be a suitable source of antigen is Hepatitis Delta virus. Still other viral sources include avian infectious bursal disease virus and porcine reproductive and respiratory syndrome virus. The alphavirus family includes equine arteritis virus and various encephalitis viruses.

[0135] Antigens that can be incorporated into vaccines (e.g., encoded by cassettes) also include immunogens useful for immunizing humans or non-human animals against pathogens, including bacteria, fungi, parasitic microorganisms, or multicellular parasites that infect humans and non-human vertebrates. Examples of bacterial pathogens include pathogenic gram-positive cocci, including pneumococci, staphylococci, and streptococci. Pathogenic gram-negative cocci include meningococci and gonococci. Pathogenic enteric gram-negative bacilli include Enterobacteriaceae, Pseudomonas, Acinetobacter, and Enella, Melasidosis, Salmonella, Shigella, Haemophilus (Haemophilus influenzae, Haemophilus somnus), Moraxella, H. ducreyi (which causes chancroid), Brucella, Francella tularensis (which causes tularemia), Yersinia (Pasteurella), Streptobacillus monoriformis, and Spirirum. Gram-positive bacilli include Listeria monocytogenes, Erysipelothrix hosiopathy, Corynebacterium diphtheriae (diphtheria), cholera, Bacillus anthracis (anthrax), Bacillus donovanosis (granulomatous individuation), and Bacillus batonnelliosis. Diseases caused by pathogenic anaerobic bacteria include tetanus, botulism, other clostridia, tuberculosis, leprosy, and other mycobacteria.Specific bacterial species include, for example, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus faecalis, Moraxella catarrhalis, Helicobacter pylori, Neisseria meningitidis, Neisseria gonorrhoeae, Chlamydia trachomatis, Chlamydia pneumoniae, Chlamydia psitaxii, Bordetella pertussis, Salmonella typhimurium, Salmonella typhimurium, Vibrio cholerae, Escherichia coli, Shigella, Vibrio cholerae, and Corynebacterium diphtheriae. Pathogenic spirochete diseases include, but are not limited to, Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium intracellulare complex, Proteus mirabilis, Proteus vulgaris, Staphylococcus aureus, Clostridium tetani, Leptospira interrogans, Borrelia burgdorferi, Pasteurella haemolytica, Pasteurella multocida, Actinobacillus leuropneumoniae, and Mycoplasma garsepticum. Pathogenic spirochete diseases include syphilis, treponematosis (strawberry disease), pinta and endemic syphilis, and leptospirosis. Other infectious diseases caused by highly pathogenic bacteria and pathogenic fungi include actinomycosis, cardiomyopathy, cryptococcosis, blastomycosis, histoplasmosis, coccidioidomycosis, candidiasis, aspergillosis, mucormycosis, sporotrichosis, paracoccidioidomycosis, Petri's ellipsoidosis, torulosophytic mycosis, mycosis and chromomycosis, and dermatophytosis. Rickettsial infections include typhoid fever, Rocky Mountain spotted fever, Q fever, and rickettsialpox. Examples of chlamydial mycoplasma and chlamydial infections include mycoplasmal pneumonia, lymphogranuloma venereum, psittacosis, and perinatal chlamydial infections.Pathogenic eukaryotes include pathogenic protozoa and helminths and the infectious diseases they cause, including amebiasis, malaria, leishmaniasis (e.g., caused by Leishmania major), trypanosomiasis, toxoplasmosis (e.g., caused by Toxoplasma gondii), Pneumocystis carinii; Toxoplasma gondii, babesiosis, giardiasis (e.g., caused by Giardia), trichanthosis (e.g., caused by Trichomonas), filariasis, schistosomiasis (e.g., caused by Schistosoma blood flukes), nematode, trematode, flagellate, and tapeworm infections. Other parasitic infections may be caused by Ascaris, Trichuris, Cryptosporidium, and Pneumocystis carinii, among others.

[0136] Also disclosed herein are peptides derived from any polypeptide associated with an infectious disease organism, an infection in a subject, or infected cells in a subject. Antigens can be derived from the nucleic acid sequence or polypeptide sequence of an infectious disease organism. Polypeptide sequences of infectious disease organisms include, but are not limited to, pathogen-derived peptides, virus-derived peptides, bacterial-derived peptides, fungal-derived peptides, and / or parasite-derived peptides. Infectious disease organisms include, but are not limited to, severe acute respiratory syndrome-associated coronavirus (SARS), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Ebola, HIV, hepatitis B virus (HBV), influenza, hepatitis C virus (HCV), human papillomavirus (HPV), cytomegalovirus (CMV), chikungunya virus, respiratory syncytial virus (RSV), dengue virus, orthomyxoviridae, and tuberculosis.

[0137] Antigens can be selected that are predicted to be presented on the cell surface of cells such as tumor cells, infected cells, or immune cells, including professional antigen-presenting cells such as dendritic cells. Antigens can be selected that are predicted to be immunogenic.

[0138] One or more polypeptides encoded by the antigen nucleotide sequence have an IC of less than 1000 nM for an MHC class I peptide of 8-15, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in length. 50 The peptides may include at least one of: binding affinity to MHC having a specific binding affinity value, the presence of a sequence motif within or near the peptide that promotes proteasomal cleavage, and the presence or presence of a sequence motif that promotes TAP transport. For MHC class II peptides, the peptides may be 6-30, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids in length, and the presence of a sequence motif within or near the peptide promotes cleavage by extracellular or lysosomal proteases (e.g., cathepsins) or HLA-DM catalyzed HLA binding.

[0139] One or more antigens may be presented on the surface of a tumor. One or more antigens may be presented on the surface of an infected cell.

[0140] The one or more antigens can be immunogenic in a subject having a tumor, e.g., can stimulate a T cell response and / or a B cell response in the subject. The one or more antigens can be immunogenic in a subject having or suspected of having an infection, e.g., can stimulate a T cell response and / or a B cell response in the subject. The one or more antigens can be immunogenic in a subject at risk of infection, e.g., can stimulate a T cell response and / or a B cell response that provides immunological protection (i.e., immunity) against the infection, e.g., can stimulate the production of memory T cells, memory B cells, or antibodies specific for the infection.

[0141] One or more antigens can stimulate a B cell response, such as the production of antibodies that recognize one or more antigens (e.g., antibodies that recognize tumor or infectious disease antigens). Antibodies can recognize linear polypeptide sequences or secondary and tertiary structures. Thus, B cell antigens can include linear polypeptide sequences or polypeptides with secondary and tertiary structure, including, but not limited to, full-length proteins, protein subunits, protein domains, or any polypeptide sequence known or predicted to have secondary and tertiary structure. Antigens capable of stimulating a B cell response against tumor or infectious disease antigens can be antigens found on the surface of tumor cells or infectious disease organisms, respectively. Antigens capable of eliciting a B cell response against tumor or infectious disease antigens can be intracellular neoantigens expressed in tumors or infectious disease organisms, respectively.

[0142] The one or more antigens can include a combination of antigens capable of stimulating a T cell response (e.g., peptides containing predicted T cell epitope sequences) and separate antigens capable of stimulating a B cell response (e.g., full-length proteins, protein subunits, protein domains).

[0143] One or more antigens that stimulate an autoimmune response in a subject can be excluded from consideration in the context of generating a vaccine for the subject.

[0144] The size of the at least one antigenic peptide molecule (e.g., epitope sequence) can be about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, about 100, about 101, about 102 The antigenic peptide molecule can include, but is not limited to, about 4, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120 or more amino acid residues, and any range derivable therein. In certain embodiments, the antigenic peptide molecule is 50 amino acids or less.

[0145] Antigenic peptides and polypeptides can be 15 residues or less in length, typically containing about 8 to about 11 residues, particularly 9 or 10 residues, for MHC class I, and 6 to 30 residues (inclusive) for MHC class II.

[0146] If desired, longer peptides can be designed in several ways. In one case, when the likelihood of peptide presentation on HLA alleles is predicted or known, longer peptides can comprise either (1) individual presented peptides with extensions of 2–5 amino acids toward the N- and C-termini of each corresponding gene product, or (2) concatenation of part or all of the presented peptide with the respective extension sequences. In another case, when sequencing reveals long (more than 10 residues) neo-epitope sequences present in tumors (e.g., due to frameshifts, readthrough, or intron inclusion resulting in novel peptide sequences), longer peptides can (3) encompass the full range of novel tumor- or infectious disease-specific amino acids, thus avoiding the need for computational or in vitro test-based selection of shorter peptides presented by the strongest HLA. In both cases, the use of longer peptides may allow endogenous processing by patient cells, resulting in more effective antigen presentation and stimulation of T cell responses. Longer peptides can also include full-length proteins, protein subunits, protein domains, and combinations thereof of peptides, such as those expressed in tumors or infectious disease organisms, respectively. Longer peptides (e.g., full-length proteins, protein subunits, or protein domains) and combinations thereof can be included to stimulate a B cell response.

[0147] Antigenic peptides and polypeptides can be presented on HLA proteins. In some embodiments, antigenic peptides and polypeptides are presented on HLA proteins with higher affinity than wild-type peptides. In some embodiments, antigenic peptides or polypeptides are presented on HLA proteins with higher affinity than wild-type peptides. 50 may be at least less than 5000 nM, at least less than 1000 nM, at least less than 500 nM, at least less than 250 nM, at least less than 200 nM, at least less than 150 nM, at least less than 100 nM, at least less than 50 nM or less.

[0148] In some embodiments, the antigenic peptides and polypeptides do not induce an autoimmune response and / or do not induce immune tolerance when administered to a subject.

[0149] Compositions comprising at least two or more antigenic peptides are also provided. In some embodiments, the compositions contain at least two distinct peptides. The at least two distinct peptides can be derived from the same polypeptide. Distinct polypeptides mean that the peptides vary in length, amino acid sequence, or both. The peptides can include tumor-specific mutations. The tumor-specific peptides can be derived from any polypeptide known or found to contain a tumor-specific mutation, or any polypeptide known or found to have altered expression in tumor cells or cancerous tissue compared to normal cells or tissues, e.g., any polypeptide known or found to be aberrantly expressed in tumor cells or cancerous tissue compared to normal cells or tissues. The peptides can be derived from any polypeptide known or suspected to be associated with an infectious disease organism, or any polypeptide known or found to have altered expression in infected cells compared to normal cells or tissues (e.g., an infectious disease polynucleotide or polypeptide, including an infectious disease polynucleotide or polypeptide with host cell-restricted expression). Suitable polypeptides from which antigenic peptides can be derived can be found, for example, in the COSMIC database or the AACR Genomics Evidence Neoplasia Information Exchange (GENIE) database. COSMIC compiles comprehensive information on somatic mutations in human cancers. AACR GENIE aggregates clinical-grade cancer genomic data and links them to clinical outcomes from tens of thousands of cancer patients. In some embodiments, the tumor-specific mutation is a driver mutation for a particular cancer type.

[0150] Antigenic peptides and polypeptides with desired activities or properties can be modified to provide certain desired attributes, e.g., improved pharmacological characteristics, while increasing or at least retaining substantially all of the biological activity of the unmodified peptide for binding to desired MHC molecules and activating appropriate T cells. For example, antigenic peptides and polypeptides can be subjected to various changes, such as conservative or non-conservative substitutions, which can confer certain advantages in their use, such as improved MHC binding, stability, or presentation. Conservative substitutions refer to the replacement of an amino acid residue with another that is biologically and / or chemically similar, e.g., one hydrophobic residue for another, or one polar residue for another. Substitutions include combinations such as Gly, Ala, Val, Ile, Leu, Met, Asp, Glu, Asn, Gln, Ser, Thr, Lys, Arg, and Phe, Tyr, etc. The effects of single amino acid substitutions can also be examined using D-amino acids. Such modifications can be carried out using well-known peptide synthesis procedures, as described, for example, in Merrifield, Science 232:341-347 (1986), Barany & Merrifield, The Peptides, Gross & Meienhofer, eds. (NY, Academic Press), 1-284 (1979), and Stewart & Young, Solid Phase Peptide Synthesis, (Rockford, III., Pierce), 2nd Ed. (1984).

[0151] Modification of peptides and polypeptides with various amino acid mimetics or unnatural amino acids can be particularly useful for increasing the stability of peptides and polypeptides in vivo. Stability can be assayed in several ways. For example, stability is tested using peptidases and various biological media, such as human plasma and serum. See, for example, Verhoef, et al., Eur. J. Drug Metab Pharmacokin. 11:291-302 (1986). Peptide half-life can be conveniently determined using a 25% human serum (v / v) assay. The protocol generally follows: Pooled human serum (type AB, non-heat-inactivated) is defatted by centrifugation before use. The serum is then diluted to 25% with RPMI tissue culture medium and used to test peptide stability. At predetermined time intervals, a small amount of the reaction solution is removed and added to either 6% aqueous trichloroacetic acid or ethanol. The cloudy reaction sample is cooled for 15 minutes (4°C) and then spun to pellet precipitated serum proteins. The presence of the peptide is then determined by reverse-phase HPLC using stability-specific chromatography conditions.

[0152] Peptides and polypeptides can be modified to provide desirable attributes other than improved serum half-life. For example, the ability of a peptide to stimulate CTL activity can be enhanced by linking it to a sequence containing at least one epitope capable of stimulating a T helper cell response. The immunogenic peptide / T helper conjugate can be linked by a spacer molecule. The spacer is typically composed of relatively small, neutral molecules, such as amino acids or amino acid mimetics, that are substantially uncharged under physiological conditions. The spacer is typically selected from, for example, Ala, Gly, or other neutral spacers of nonpolar or neutral polar amino acids. It will be understood that, optionally, a spacer present need not be composed of the same residues and can thus be a hetero- or homo-oligomer. If present, the spacer is typically at least one or two residues, more usually three to six residues. Alternatively, the peptide can be linked to the T helper peptide without a spacer.

[0153] The antigenic peptide can be linked to the T helper peptide either directly or via a spacer at either the amino or carboxy terminus of the peptide. The amino terminus of either the antigenic peptide or the T helper peptide can be acylated. Exemplary T helper peptides include tetanus toxoid 830-843, influenza 307-319, malaria circumsporozoite 382-398, and 378-389.

[0154] Proteins or peptides can be produced by any technique known to those of skill in the art, including expressing the protein, polypeptide, or peptide via standard molecular biology techniques, isolating the protein or peptide from a natural source, or chemically synthesizing the protein or peptide. Nucleotide and protein, polypeptide, and peptide sequences corresponding to various genes have been previously disclosed and can be found in computerized databases known to those of skill in the art. One such database is the Genbank and GenPept databases of the National Center for Biotechnology Information, located on the National Institutes of Health website. The coding regions of known genes can be amplified and / or expressed using the techniques disclosed herein or as known to those of skill in the art. Alternatively, various commercially available preparations of proteins, polypeptides, and peptides are known to those of skill in the art.

[0155] In a further aspect, the antigen comprises a nucleic acid (e.g., a polynucleotide) encoding an antigenic peptide or a portion thereof. The polynucleotide can be, for example, DNA, cDNA, PNA, CNA, RNA (e.g., mRNA), either single-stranded and / or double-stranded, or a polynucleotide in a natural or stabilized form, e.g., a polynucleotide with a phosphonothioate backbone, or a combination thereof, with or without introns. The polynucleotide sequence encoding the antigen can be sequence-optimized to improve expression, such as by improving transcription, translation, post-transcriptional processing, and / or RNA stability. For example, the polynucleotide sequence encoding the antigen can be codon-optimized. As used herein, "codon optimization" refers to replacing infrequently used codons with frequently used synonymous codons with respect to the codon bias of a given organism. Polynucleotide sequences can be optimized to improve post-transcriptional processing and to reduce unintended splicing, for example, by removing splicing motifs (e.g., canonical and / or cryptic / non-canonical splice donor, branch, and / or acceptor sequences) and / or incorporating exogenous splicing motifs (e.g., splice donor, branch, and / or acceptor sequences) to bias favorable splicing events. Exogenous intron sequences include, but are not limited to, those derived from SV40 (e.g., SV40 mini-introns) and immunoglobulins (e.g., human β-globin genes). Exogenous intron sequences can be incorporated between the promoter / enhancer sequence and the antigen(s) sequence. Exogenous intron sequences for use in expression vectors are described in detail in Callendret et al. (Virology. 2007, July 5, 363(2):288-302), which is incorporated herein by reference for all purposes. Polynucleotide sequences can be optimized to improve transcript stability, for example, by removal of RNA instability motifs (eg, AU-rich elements and 3'UTR motifs) and / or repetitive nucleotide sequences.Polynucleotide sequences can be optimized to improve transcription accuracy, for example, by removing cryptic transcription initiators and / or terminators. Polynucleotide sequences can be optimized to improve translation and translation accuracy, for example, by removing cryptic AUG start codons, premature polyA sequences, and / or secondary structure motifs. Polynucleotide sequences can be optimized to improve nuclear export of transcripts, for example, by adding constitutive transport elements (CTEs), RNA transport elements (RTEs), or Woodchuck posttranscriptional regulatory elements (WPREs). Nuclear export signals for use in expression vectors are described in detail in Callendret et al. (Virology. July 5, 2007; 363(2):288-302), which is incorporated herein by reference for all purposes. Polynucleotide sequences can be optimized for GC content, for example, to reflect the average GC content of a given organism. Sequence optimization can balance one or more sequence properties, such as transcription, translation, posttranscriptional processing, and / or RNA stability. Sequence optimization can generate optimal sequences that balance transcription, translation, post-transcriptional processing, and RNA stability. Sequence optimization algorithms such as GeneArt (Thermo Fisher), Codon Optimization Tool (IDT), CoolTool (University of Singapore), and SGI-DNA (La Jolla California) are known to those skilled in the art. One or more regions of the antigen-encoding protein can be sequence-optimized separately.

[0156] A further embodiment provides an expression vector capable of expressing a polypeptide or a portion thereof. Expression vectors for different cell types are well known in the art and can be selected without undue experimentation. Generally, DNA is inserted into an expression vector, such as a plasmid, in the appropriate orientation and correct reading frame for expression. If necessary, the DNA can be linked to appropriate transcriptional and translational regulatory control nucleotide sequences recognized by the desired host, although such controls are generally available in expression vectors. The vector is then introduced into the host by standard techniques. Guidance can be found, for example, in Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

[0157] cassette The methods used for the selection of one or more payloads, cloning and construction of "cassettes," and their insertion into viral vectors are within the purview of those skilled in the art given the teachings provided herein. A "payload cassette" or "cassette" or "antigen cassette" refers to a combination of a selected payload or payloads (e.g., a payload-encoding nucleic acid sequence, e.g., an antigen-encoding nucleic acid sequence) with other regulatory elements necessary to transcribe the payload(s) and express the transcribed products. The selected payload or payloads can refer to separate payload sequences; for example, a payload-encoding nucleic acid sequence within a cassette can encode a payload-encoding nucleic acid sequence (or multiple payload-encoding nucleic acid sequences) such that the payloads are transcribed and expressed. The payload or payloads can be operably linked to regulatory elements in a manner that allows transcription. Such components include conventional regulatory elements capable of driving expression of the payload(s) in cells transfected with the viral vector. Thus, the payload cassette can also contain a selected promoter linked to the payload(s) and located along with any other regulatory elements within the selected viral sequence of the recombinant vector. The cassette can include one or more payloads, such as one or more sequences encoding any of the payloads described herein. The cassette can have one or more payload-encoding nucleic acid sequences, e.g., a cassette containing multiple payload-encoding nucleic acid sequences, each independently operably linked to a separate promoter and / or linked together using other multicistronic systems, e.g., 2A ribosome skipping sequence elements (e.g., E2A, P2A, F2A, or T2A sequences) or internal ribosome entry site (IRES) sequence elements. The linker can also have a cleavage site, such as a TEV or furin cleavage site. Linkers with cleavage sites can be used in combination with other elements, such as those in multicistronic systems.In a non-limiting illustrative example, a furin protease cleavage site can be used in combination with a 2A ribosomal skipping sequence element such that the furin protease cleavage site is configured to facilitate post-translational removal of the 2A sequence.

[0158] In cassettes containing two or more payload-encoding nucleic acid sequences, each payload-encoding nucleic acid sequence can be linked (e.g., in an illustrative, non-limiting example, linked payload-encoding nucleic acid sequences encoding linked T cell epitopes). In illustrative examples of multicistronic formats, the payload-encoding cassettes are configured as follows: (1) endogenous 26S promoter-payload1-T2A-payload2 protein, or (2) endogenous 26S promoter-payload1-26S promoter-payload2. In further illustrative examples of multicistronic formats, the SARS-CoV-2 payload-encoding cassettes are configured as follows: (1) endogenous 26S promoter-spike protein-T2A-membrane protein, or (2) endogenous 26S promoter-spike protein-26S promoter-linked T cell epitope.

[0159] In addition to the promoters derived from subgenomic alphaviruses described herein, additional promoters or promoter elements can be used. Useful promoters can be constitutive or regulated (inducible) promoters, allowing for control of the amount of expressed payload(s). For example, a desirable promoter is the cytomegalovirus immediate-early promoter / enhancer promoter [see, e.g., Boshart et al., Cell, 41:521-530 (1985)]. Another desirable promoter includes the Rous sarcoma virus long terminal repeat (LTR) promoter / enhancer. Yet another promoter / enhancer sequence is the chicken cytoplasmic beta-actin promoter [see, e.g., Takost et al., Nucl. Acids Res., 11(23):8287 (1983)]. Other suitable or desirable promoters can be selected by those skilled in the art.

[0160] The cassette can also contain nucleic acid sequences heterologous to the viral vector sequence, including sequences providing signals for efficient polyadenylation of the transcript (poly(A), poly-A, or pA) and introns with functional splice donor and acceptor sites. A common polyA sequence used in exemplary vectors of the present invention is derived from the papovavirus SV-40. A polyA sequence (e.g., a non-natural polyA) can generally be inserted into the cassette after the payload-based sequence and before the viral vector sequence. A common intron sequence can also be derived from SV-40 and is referred to as the SV-40T intron sequence. The cassette can also contain such an intron located between the promoter / enhancer sequence and the payload(s). The selection of these and other common vector elements is conventional (see, e.g., Sambrook et al., "Molecular Cloning. A Laboratory Manual," 2d ed., Cold Spring Harbor Laboratory, New York (1989) and references cited therein), and many such sequences are available from commercial and industrial sources as well as Genbank.

[0161] A cassette can have one or more payloads (e.g., one or more payload-encoding nucleic acid sequences). For example, a given cassette can include 1-10, 1-20, 1-30, 10-20, 15-25, 15-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more payloads. The payloads can be directly linked to each other. The payloads can also be linked to each other with a linker. The payloads can be in any orientation relative to each other, including NC or CN.

[0162] As described elsewhere herein, the cassette can be located at the site of any selected deletion in the viral vector, for example, the deleted structural proteins of the VEE backbone or the E1 or E3 gene region deletion of a ChAd-based vector, among others that can be selected.

[0163] A multicistronic samRNA vector can be described using the following formula to describe the ordered sequence of each element from the 5' to the 3' end: P1-(L5 b -N c -L3 d ) X -P2-(L5 b -N c -L3 d ) X -P a -(L5 b -N c -L3 d ) X -(G5 e -U f )Y-G3 g where P1 contains the SGP1 subgenomic promoter, P2 contains the SGP2 subgenomic promoter, and P a When a=0 or 1, N comprises a payload-encoding nucleic acid sequence, c=1, L5 comprises a 5' linker sequence, b=0 or 1, L3 comprises a 3' linker sequence, d=0 or 1, G5 comprises at least one nucleic acid sequence encoding a GPGPG amino acid linker (SEQ ID NO: 1), e=0 or 1, G3 comprises at least one nucleic acid sequence encoding a GPGPG amino acid linker (SEQ ID NO: 1), g=0 or 1, U comprises at least one MHC class II epitope-encoding nucleic acid sequence, f=1, X=1 to 400, and for each X, a corresponding N c is the corresponding payload-encoding nucleic acid sequence, where Y=0, 1, or 2, and for each Y, the corresponding U fare universal MHC class II epitope-encoding nucleic acid sequences, and optionally, the at least one universal sequence comprises at least one of tetanus toxoid and PADRE.

[0164] A payload-encoding sequence (e.g., a cassette, or one or more of the payload-encoding nucleic acid sequences within a cassette) can be described using the following formula to describe the ordered sequence of each element from the 5' end to the 3' end: P a -(L5 b -N c -L3 d ) X -(G5 e -U f ) Y -G3 g wherein P comprises a second promoter nucleotide sequence, a=0 or 1, c=1, N comprises one of the payload-derived nucleic acid sequences described herein (e.g., any of the antigen-encoding nucleic acid sequences described herein), L5 comprises a 5' linker sequence, b=0 or 1, L3 comprises a 3' linker sequence, d=0 or 1, G5 comprises one of at least one nucleic acid sequences encoding a GPGPG amino acid linker (SEQ ID NO: 1), e=0 or 1, G3 comprises one of at least one nucleic acid sequences encoding a GPGPG amino acid linker (SEQ ID NO: 1), g=0 or 1, U comprises one of at least one MHC class II epitope-encoding nucleic acid sequences, f=1, X=1 to 400, and for each X, a corresponding N c is the corresponding payload-encoding nucleic acid sequence, where Y=0, 1, or 2, and for each Y, the corresponding U f is (1) a universal MHC class II epitope-encoding nucleic acid sequence, optionally at least one of which comprises at least one of tetanus toxoid and PADRE, or (2) an MHC class II epitope-encoding nucleic acid sequence. In some embodiments, for each X, a corresponding N c are distinct payload-encoding nucleic acid sequences. In some embodiments, for each Y, a corresponding Uf are individual universal MHC class II epitope-encoding nucleic acid sequences or individual MHC class II antigen-encoding nucleic acid sequences. The above payload-encoding sequence formulas optionally describe only a portion of a cassette encoding linked payload sequences, such as linked T cell epitopes. For example, as an illustrative, non-limiting example, in a cassette encoding linked T cell epitopes and one or more full-length SARS-CoV-2 proteins, the above payload-encoding sequence formulas describe the linked T cell epitopes, and separately, the cassettes encode one or more full-length SARS-CoV-2 proteins, optionally linked using multicistronic systems such as 2A ribosomal skipping sequence elements (e.g., E2A, P2A, F2A, or T2A sequences) and / or internal ribosome entry site (IRES) sequence elements.

[0165] In one example, the elements present are b=1, d=1, e=1, g=1, h=1, X=18, Y=2, the vector backbone comprises a ChAdV68 vector, a=1, P is a CMV promoter, at least one second poly(A) sequence is present, the second poly(A) sequence is exogenous to the vector backbone, optionally the exogenous poly(A) sequence comprises an SV40 poly(A) signal sequence or a BGH poly(A) signal sequence, and each N is an MHC class I epitope, an MHC class II epitope, or an MHC class II epitope that is 7 to 15 amino acids in length. , an epitope capable of stimulating a B cell response, or a combination thereof, wherein L5 is a native 5' linker sequence encoding the native N-terminal amino acid sequence of the epitope, wherein the 5' linker sequence encodes a peptide that is at least 3 amino acids in length, L3 is a native 3' linker sequence encoding the native C-terminal amino acid sequence of the epitope, wherein the 3' linker sequence encodes a peptide that is at least 3 amino acids in length, and U is each of a PADRE class II sequence and a tetanus toxoid MHC class II sequence. The above payload-encoding sequence formulas optionally describe only a portion of the payload cassette that encodes linked epitope sequences, such as linked T cell epitopes.

[0166] In one example, the elements present are b=1, d=1, e=1, g=1, h=1, X=18, Y=2, the vector backbone comprises a Venezuelan equine encephalitis virus vector, a=0, the payload cassette is operably linked to an endogenous 26S promoter, and the at least one polyadenylation poly(A) sequence is a poly(A) sequence of at least 80 consecutive A nucleotides provided by the backbone, where each N is an MHC class I epitope, an MHC class II epitope, or an epitope that is 7-15 amino acids in length and stimulates a B cell response. L5 is a natural 5' linker sequence encoding the natural N-terminal amino acid sequence of the epitope, wherein the 5' linker sequence encodes a peptide that is at least 3 amino acids in length; L3 is a natural 3' linker sequence encoding the natural C-terminal amino acid sequence of the epitope, wherein the 3' linker sequence encodes a peptide that is at least 3 amino acids in length; and U is each of the PADRE class II sequence and the tetanus toxoid MHC class II sequence.

[0167] The payload cassette can be described using the following formula to describe the ordered sequence of each element from the 5' to the 3' end: (P a -(L5 b -N c -L3 d ) X ) Z -(P2 h -(G5 e -U f ) Y ) W -G3 g wherein P and P2 comprise promoter nucleotide sequences, N comprises an MHC class I epitope-encoding nucleic acid sequence, L5 comprises a 5' linker sequence, L3 comprises a 3' linker sequence, G5 comprises a nucleic acid sequence encoding an amino acid linker, G3 comprises one of at least one nucleic acid sequence encoding an amino acid linker, U comprises an MHC class II antigen-encoding nucleic acid sequence, and for each X, the corresponding Nc is an epitope-encoding nucleic acid sequence, and for each Y, the corresponding Uf is an MHC class II epitope-encoding nucleic acid sequence (e.g., a universal MHC class II epitope-encoding nucleic acid sequence). The universal sequence can comprise at least one of tetanus toxoid and PADRE. The universal sequence can comprise a tetanus toxoid peptide. The universal sequence can comprise a PADRE peptide. The universal sequence can comprise a tetanus toxoid and a PADRE peptide. The compositions and ordered sequences can be further defined by selecting the number of elements present, for example, a=0 or 1, b=0 or 1, c=1, d=0 or 1, e=0 or 1, f=1, g=0 or 1, h=0 or 1, X=1-400, Y=0, 1, 2, 3, 4 or 5, Z=1-400, and W=0, 1, 2, 3, 4 or 5.

[0168] In one example, the elements present include a=0, b=1, d=1, e=1, g=1, h=0, X=10, Y=2, Z=1, and W=1, indicating that no additional promoters are present (e.g., only promoter nucleotide sequences provided by the vector backbone, such as an RNA alphavirus or ChAdV backbone, are present), 10 MHC class I epitopes are present, a 5' linker for each N is present, a 3' linker for each N is present, two MHC class II epitopes are present, a linker connecting the two MHC class II epitopes is present, a linker connecting the 5' ends of the two MHC class II epitopes to the 3' linker of the final MHC class I epitope is present, and a linker connecting the 3' ends of the two MHC class II epitopes to the vector backbone (e.g., a ChAdV or RNA alphavirus backbone).

[0169] Examples of linking the 3' end of the cassette to a vector backbone (e.g., an RNA alphavirus backbone) include directly linking to a 3' UTR element provided by the vector backbone, such as a 3' 19-nt CSE. Examples of linking the 5' end of the cassette to a vector backbone (e.g., an RNA alphavirus backbone) include directly linking to a promoter or 5' UTR element of the vector backbone, such as a subgenomic promoter sequence (e.g., a 26S subgenomic promoter sequence), an alphavirus 5' UTR, a 51-nt CSE, or a 24-nt CSE.

[0170] Other examples include a=1, indicating the presence of a promoter other than the promoter nucleotide sequence provided by the vector backbone (e.g., ChAdV or RNA alphavirus backbone); a=1 and Z is greater than 1, indicating the presence of multiple promoters other than the promoter nucleotide sequence provided by the vector backbone, each driving expression of one or more distinct MHC Class I epitope-encoding nucleic acid sequences; h=1, describing the presence of a separate promoter driving expression of the MHC Class II epitope-encoding nucleic acid sequence; and g=0, indicating that the MHC Class II epitope-encoding nucleic acid sequence, if present, is directly linked to the vector backbone (e.g., ChAdV or RNA alphavirus backbone). For example, a ChAdV vector backbone can have the cassette placed under the control of a CMV promoter / enhancer.

[0171] Other examples include each MHC class I epitope present can have a 5' linker, a 3' linker, neither, or both. In examples where two or more MHC class I epitopes are present on the same antigen cassette, some MHC class I epitopes can have both a 5' linker and a 3' linker, while other MHC class I epitopes can have either a 5' linker and a 3' linker, or neither. In other examples where two or more MHC class I epitopes are present on the same antigen cassette, some MHC class I epitopes can have either a 5' linker or a 3' linker, while other MHC class I epitopes can have either a 5' linker and a 3' linker, or neither.

[0172] In instances where two or more MHC class II epitopes are present on the same antigen cassette, some MHC class II epitopes may have both a 5' linker and a 3' linker, while other MHC class II epitopes may have either a 5' linker or a 3' linker, or neither. In other instances where two or more MHC class II epitopes are present on the same antigen cassette, some MHC class II epitopes may have either a 5' linker or a 3' linker, while other MHC class II epitopes may have either a 5' linker or a 3' linker, or neither.

[0173] Other examples include each payload present can have neither a 5' linker nor a 3' linker, or both. In examples where two or more payloads are present on the same payload cassette, some payloads can have both a 5' linker and a 3' linker, while other payloads can have either a 5' linker and a 3' linker, or neither. In other examples where two or more payloads are present on the same payload cassette, some payloads can have either a 5' linker or a 3' linker, while other payloads can have either a 5' linker and a 3' linker, or neither.

[0174] The promoter nucleotide sequences P and / or P2 can be the same as the promoter nucleotide sequences provided by the vector backbone, such as the RNA alphavirus backbone. For example, the promoter sequences Pn and P2 provided by the RNA alphavirus backbone can each comprise a subgenomic promoter sequence (e.g., a 26S subgenomic promoter sequence) or a CMV promoter. The promoter nucleotide sequences P and / or P2 can be different from the promoter nucleotide sequences provided by the vector backbone (e.g., a ChAdV or RNA alphavirus backbone) and can be different from each other.

[0175] The 5' linker L5 can be a natural or non-natural sequence. Non-natural sequences include, but are not limited to, AAY, RR, and DPP. The 3' linker L3 can also be a natural or non-natural sequence. Additionally, L5 and L3 can both be natural sequences, both non-natural sequences, or one natural and the other non-natural. For each X, the amino acid linker can be 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, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 10 , 92, 93, 94, 95, 96, 97, 98, 99, 100 or more amino acids. For each X, the amino acid linker can also be at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 amino acids in length.For each X, the amino acid linker may also be selected from the group consisting of: 2-10, 2-15, 2-20, 2-25, 2-30, 2-40, 2-50, 3-10, 3-15, 3-20, 3-25, 3-30, 3-40, 3-50, 4-10, 4-15, 4-20, 4-25, 4-30, 4-40, 4-50, 5-10, 5-15, 5-20, The number of amino acids may be 5 to 25, 5 to 30, 5 to 40, 5 to 50, 6 to 10, 6 to 15, 6 to 20, 6 to 25, 6 to 30, 6 to 40, 6 to 50, 7 to 10, 7 to 15, 7 to 20, 7 to 25, 7 to 30, 7 to 40, 7 to 50, 8 to 10, 8 to 15, 8 to 20, 8 to 25, 8 to 30, 8 to 40, or 8 to 50.

[0176] For each Y, the amino acid linker G5 is 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, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, , 92, 93, 94, 95, 96, 97, 98, 99, 100 or more amino acids. For each Y, the amino acid linker can also be at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 amino acids in length. G5 also comes in lengths of 2-10, 2-15, 2-20, 2-25, 2-30, 2-40, 2-50, 3-10, 3-15, 3-20, 3-25, 3-30, 3-40, 3-50, 4-10, 4-15, 4-20, 4-25, 4-30, 4-40, 4-50, 5-10, 5-15, 5-20, 5-25, 5 The amino acid sequence may be from 1 to 30, 5 to 40, 5 to 50, 6 to 10, 6 to 15, 6 to 20, 6 to 25, 6 to 30, 6 to 40, 6 to 50, 7 to 10, 7 to 15, 7 to 20, 7 to 25, 7 to 30, 7 to 40, 7 to 50, 8 to 10, 8 to 15, 8 to 20, 8 to 25, 8 to 30, 8 to 40, or 8 to 50 amino acids.

[0177] The amino acid linker G3 is 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, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122 The amino acid sequence can be 3, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more amino acids. G3 can also be at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 amino acids in length. G3 also has lengths of 2-10, 2-15, 2-20, 2-25, 2-30, 2-40, 2-50, 3-10, 3-15, 3-20, 3-25, 3-30, 3-40, 3-50, 4-10, 4-15, 4-20, 4-25, 4-30, 4-40, 4-50, 5-10, 5-15, 5-20, 5-25, 5 The amino acid sequence may be from 1 to 30, 5 to 40, 5 to 50, 6 to 10, 6 to 15, 6 to 20, 6 to 25, 6 to 30, 6 to 40, 6 to 50, 7 to 10, 7 to 15, 7 to 20, 7 to 25, 7 to 30, 7 to 40, 7 to 50, 8 to 10, 8 to 15, 8 to 20, 8 to 25, 8 to 30, 8 to 40, or 8 to 50 amino acids.

[0178] For each X, each N can encode an MHC class I epitope, an MHC class II epitope, an epitope / antigen capable of stimulating a B cell response, or a combination thereof. For each X, each N can encode a combination of an MHC class I epitope, an MHC class II epitope, and an epitope / antigen capable of stimulating a B cell response. For each X, each N can encode a combination of an MHC class I epitope and an MHC class II epitope. For each X, each N can encode a combination of an MHC class I epitope and an epitope / antigen capable of stimulating a B cell response. For each X, each N can encode a combination of an MHC class II epitope and an epitope / antigen capable of stimulating a B cell response. For each X, each N can encode an MHC class II epitope. For each X, each N can encode an epitope / antigen capable of stimulating a B cell response. For each X, each N can encode an MHC class I epitope that is 7 to 15 amino acids in length. For each X, each N can also encode an MHC class I epitope that is 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, or 30 amino acids in length. For each X, each N can also encode an MHC class I epitope that is at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 amino acids in length. For each X, each N can encode an MHC class II epitope. For each X, each N can encode an epitope capable of stimulating a B cell response.

[0179] The cassettes, each comprising a respective cassette within a multicistronic system, can be at least 100, 200, 300, 400, 500, 600, 700, 800, or 900 nucleotides in length. The cassettes can be at least 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10,000 nucleotides in length. The cassettes can be at least 1000 nucleotides in length. The cassettes can be at least 2000 nucleotides in length. The cassettes can be at least 3000 nucleotides in length. The cassettes can be at least 4000 nucleotides in length. The cassettes can be at least 5000 nucleotides in length. The cassettes can be at least 6000 nucleotides in length. The cassettes can be at least 7000 nucleotides in length. The cassette can be at least 8,000 nucleotides in length. The cassette can be at least 9,000 nucleotides in length. The cassette can be 100-1,000, 100-2,000, 100-3,000, 100-4,000, 100-5,000, 100-6,000, 100-7,000, 100-8,000, 100-9,000, or 100-10,000 nucleotides in length. The cassette can be 500-1,000, 500-2,000, 500-3,000, 500-4,000, 500-5,000, 500-6,000, 500-7,000, 500-8,000, 500-9,000, or 500-10,000 nucleotides in length. The cassette can be 1000-2000, 1000-3000, 1000-4000, 1000-5000, 1000-6000, 1000-7000, 1000-8000, 1000-9000, or 1000-10000 nucleotides in length. The cassette can be about the length deleted from the alphavirus (e.g., the length of the deleted structural protein in the VEE backbone). The cassette can be less than the length deleted from the alphavirus. The cassette can be greater than the length deleted from the alphavirus.

[0180] For vectors containing multiple cassettes, the total length of all cassettes combined can be at least 100, 200, 300, 400, 500, 600, 700, 800, or 900 nucleotides in length. For vectors containing multiple cassettes, the total length of all cassettes combined can be at least 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000 nucleotides in length. For vectors containing multiple cassettes, the total length of all the combined cassettes can be 100 to 1,000, 100 to 2,000, 100 to 3,000, 100 to 4,000, 100 to 5,000, 100 to 6,000, 100 to 7,000, 100 to 8,000, 100 to 9,000, or 100 to 10,000 nucleotides in length. For vectors containing multiple cassettes, the total length of all the combined cassettes can be 500 to 1,000, 500 to 2,000, 500 to 3,000, 500 to 4,000, 500 to 5,000, 500 to 6,000, 500 to 7,000, 500 to 8,000, 500 to 9,000, or 500 to 10,000 nucleotides in length. For vectors containing multiple cassettes, the total length of all the combined cassettes can be 1000-2000, 1000-3000, 1000-4000, 1000-5000, 1000-6000, 1000-7000, 1000-8000, 1000-9000, or 1000-10000 nucleotides in length.

[0181] The cassette can be 700 nucleotides or less. The cassette can be 700 nucleotides or less and encode two distinct epitope-encoding nucleic acid sequences (e.g., encoding two distinct infectious disease- or tumor-derived nucleic acid sequences that encode immunogenic polypeptides). The cassette can be 700 nucleotides or less and encode at least two distinct epitope-encoding nucleic acid sequences. The cassette can be 700 nucleotides or less and encode three distinct epitope-encoding nucleic acid sequences. The cassette can be 700 nucleotides or less and encode at least three distinct epitope-encoding nucleic acid sequences. The cassette can be 700 nucleotides or less and comprise 1-10, 1-5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more payloads.

[0182] The cassette can be 375-700 nucleotides in length. The cassette can be 375-700 nucleotides in length and encode two distinct epitope-encoding nucleic acid sequences (e.g., encoding two distinct infectious disease- or tumor-derived nucleic acid sequences encoding immunogenic polypeptides). The cassette can be 375-700 nucleotides in length and encode at least two distinct epitope-encoding nucleic acid sequences. The cassette can be 375-700 nucleotides in length and encode three distinct epitope-encoding nucleic acid sequences. The cassette can be 375-700 nucleotides in length and encode at least three distinct epitope-encoding nucleic acid sequences. The cassette can be 375-700 nucleotides in length and comprise 1-10, 1-5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more payloads.

[0183] The cassettes can be no more than 600, 500, 400, 300, 200, or 100 nucleotides in length. The cassettes can be no more than 600, 500, 400, 300, 200, or 100 nucleotides in length and encode two distinct epitope-encoding nucleic acid sequences. The cassettes can be no more than 600, 500, 400, 300, 200, or 100 nucleotides in length and encode at least two distinct epitope-encoding nucleic acid sequences. The cassettes can be no more than 600, 500, 400, 300, 200, or 100 nucleotides in length and encode three distinct epitope-encoding nucleic acid sequences. The cassettes can be no more than 600, 500, 400, 300, 200, or 100 nucleotides in length and encode at least three distinct epitope-encoding nucleic acid sequences. Cassettes can be up to 600, 500, 400, 300, 200, or 100 nucleotides in length and can contain 1-10, 1-5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more payloads.

[0184] The cassette can be 375-600, 375-500, or 375-400 nucleotides in length. The cassette can be 375-600, 375-500, or 375-400 nucleotides in length and encode two distinct epitope-encoding nucleic acid sequences. The cassette can be 375-600, 375-500, or 375-400 nucleotides in length and encode at least two distinct epitope-encoding nucleic acid sequences. The cassette can be 375-600, 375-500, or 375-400 nucleotides in length and encode three distinct epitope-encoding nucleic acid sequences. The cassette can be 375-600, 375-500, or 375-400 nucleotides in length and encode at least three distinct epitope-encoding nucleic acid sequences. The cassettes can be 375-600, 375-500, or 375-400 nucleotides in length and can contain 1-10, 1-5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more payloads.

[0185] Vaccine Composition The vaccine composition may further comprise an adjuvant and / or a carrier. Examples of useful adjuvants and carriers are provided herein below. The composition may be associated with a carrier, such as a protein, or an antigen-presenting cell, such as a dendritic cell (DC), capable of presenting the peptide to T cells.

[0186] An adjuvant is any substance that, when incorporated into a vaccine composition, increases or otherwise modifies the immune response to a neoantigen. The carrier can be a scaffold, such as a polypeptide or polysaccharide, to which the neoantigen can associate. Optionally, the adjuvant is covalently or non-covalently conjugated.

[0187] The ability of an adjuvant to increase the immune response to an antigen is typically manifested by a significant or substantial increase in immune-mediated reactions or a reduction in disease symptoms. For example, an increase in humoral immunity is typically manifested by a significant increase in the titer of antibodies produced against the antigen, and an increase in T cell activity is typically manifested as an increase in cell proliferation, cytotoxicity, or cytokine secretion. Adjuvants can also alter the immune response, for example, by shifting a predominantly humoral or Th response to a predominantly cellular or Th response.

[0188] Suitable adjuvants include 1018 ISS, alum, aluminum salts, Amplivax, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX, JuvImmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTel vector system, PLG microparticles, resiquimod, SRL172, virosomes and other virus-like particles, YF-17D, VEGF Adjuvants include, but are not limited to, Aquila's QS21 stimulon (Aquila Biotech, Worcester, Mass., USA), which is derived from saponin, R848, beta-glucan, Pam3Cys, mycobacterial extracts, and synthetic bacterial cell wall mimics, as well as other proprietary adjuvants such as Ribi's Detox.Quil or Superfos. Adjuvants such as incomplete Freund's or GM-CSF are useful. Several immunological adjuvants specific for dendritic cells (e.g., MF59) and their preparations have been reported (Dupuis M, et al., Cell Immunol. 1998;186(1):18-27; Allison AC; Dev Biol Stand. 1998;92:3-11). Cytokines can also be used. Several cytokines influence dendritic cell migration to lymphoid tissues (e.g., TNF-alpha), accelerate dendritic cell maturation into efficient antigen-presenting cells for T lymphocytes (e.g., GM-CSF, IL-1, and IL-4) (U.S. Patent No. 5,849,589, which is incorporated herein by reference in its entirety), and act as immune adjuvants (e.g., IL-12) (Gabrilovich DI, et al., J Immunother Emphasis Tumor Immunol. 1996(6):414-418).

[0189] CpG immunostimulatory oligonucleotides have also been reported to enhance the effect of adjuvants in vaccine settings. Other TLR-binding molecules, such as RNA-binding TLR7, TLR8, and / or TLR9, can also be used.

[0190] Other examples of useful adjuvants include, but are not limited to, chemically modified CpG (e.g., CpR, Idera), poly(I:C) (e.g., polyi:CI2U), non-CpG bacterial DNA or RNA, and immunologically active small molecules and antibodies, such as cyclophosphamide, sunitinib, bevacizumab, Celebrex, NCX-4016, sildenafil, tadalafil, vardenafil, sorafinib, XL-999, CP-547632, pazopanib, ZD2171, AZD2171, ipilimumab, tremelimumab, and SC58175, which may act therapeutically and / or as adjuvants. The amounts and concentrations of adjuvants and additives can be readily determined by one of ordinary skill in the art without undue experimentation. Additional adjuvants include colony-stimulating factors such as granulocyte-macrophage colony-stimulating factor (GM-CSF, sargramostim).

[0191] A vaccine composition can include two or more different adjuvants. Additionally, a therapeutic composition can include any adjuvant material, including any of the above or a combination thereof. It is also contemplated that the vaccine and adjuvant can be administered together or separately in any suitable sequence.

[0192] The carrier (or excipient) can exist independently of the adjuvant. The function of the carrier can be, for example, to increase the activity or immunogenicity, particularly by increasing the molecular weight of the variant, to confer stability, to increase biological activity, or to increase serum half-life. Furthermore, the carrier can assist in the presentation of the peptide to T cells. The carrier can be any suitable carrier known to those skilled in the art, such as a protein or an antigen-presenting cell. Carrier proteins can be, but are not limited to, keyhole limpet hemocyanin, serum proteins such as transferrin, bovine serum albumin, human serum albumin, thyroglobulin, or ovalbumin, immunoglobulins, or hormones such as insulin or palmitic acid. For human immunization, the carrier is generally a physiologically acceptable carrier that is acceptable and safe for humans. However, tetanus toxoid and / or diphtheria toxoid are suitable carriers. Alternatively, the carrier can be a dextran, such as Sepharose.

[0193] buffer solution Exemplary buffering agents include, but are not limited to, citrate buffer, acetate buffer, phosphate buffer, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, d-gluconic acid, calcium glycerophosphate, calcium lactate, calcium lactobionate, propanoic acid, calcium levulinate, pentanoic acid, dicalcium phosphate, phosphoric acid, tricalcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dipotassium phosphate, monopotassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, disodium phosphate, monosodium phosphate, sodium phosphate mixtures, tromethamine, aminosulfonic acid buffers (e.g., HEPES), magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, and / or combinations thereof. The lubricant may be selected from the non-limiting group consisting of magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behanate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, and combinations thereof.

[0194] In some embodiments, the buffer is selected from the group consisting of citrate, succinate, malate, phosphate, histidine, glycine, MOPS, HEPES, Tris, and Bis-Tris. In some embodiments, the buffer is a citrate buffer. In some embodiments, the buffer is a succinate buffer. In some embodiments, the buffer is a malate buffer. In some embodiments, the buffer is a phosphate buffer. In some embodiments, the buffer is a histidine buffer. In some embodiments, the buffer is MOPS. In some embodiments, the buffer is HEPES. In some embodiments, the buffer is Tris. In some embodiments, the buffer is Bis-Tris.

[0195] In some embodiments, the buffer has a salt concentration of 1 to 15 nM. In some embodiments, the buffer has a salt concentration of 3 to 13 nM. In some embodiments, the buffer has a salt concentration of 5 to 11 nM. In some embodiments, the buffer has a salt concentration of 6 to 10 nM. In some embodiments, the buffer has a salt concentration of 7 to 9 nM. In some embodiments, the buffer has a salt concentration of 7.5 to 8.5 nM. In some embodiments, the buffer has a salt concentration of 7.8 to 8.2 nM.

[0196] pH In some embodiments, the buffer has a salt concentration of about 5.5 nM. In some embodiments, the buffer has a salt concentration of about 6.0 nM. In some embodiments, the buffer has a salt concentration of about 6.5 nM. In some embodiments, the buffer has a salt concentration of about 7.0 nM. In some embodiments, the buffer has a salt concentration of about 7.5 nM. In some embodiments, the buffer has a salt concentration of about 7.8 nM. In some embodiments, the buffer has a salt concentration of about 7.9 nM. In some embodiments, the buffer has a salt concentration of about 8.0 nM. In some embodiments, the buffer has a salt concentration of about 8.1 nM. In some embodiments, the buffer has a salt concentration of about 8.2 nM. In some embodiments, the buffer has a salt concentration of about 8.5 nM. In some embodiments, the buffer has a salt concentration of about 9.0 nM. In some embodiments, the buffer has a salt concentration of about 9.5 nM. In some embodiments, the buffer has a salt concentration of about 10.0 nM. In some embodiments, the buffer has a salt concentration of about 10.5 nM.

[0197] In some embodiments, the pharmaceutical composition has a pH of 6.0 to 9.2. In some embodiments, the pharmaceutical composition has a pH of 6.8 to 8.8. In some embodiments, the pharmaceutical composition has a pH of 7.0 to 8.6. In some embodiments, the pharmaceutical composition has a pH of 7.3 to 8.3. In some embodiments, the pharmaceutical composition has a pH of 7.4 to 8.2. In some embodiments, the pharmaceutical composition has a pH of 7.5 to 8.1. In some embodiments, the pharmaceutical composition has a pH of 7.6 to 8.0. In some embodiments, the pharmaceutical composition has a pH of 7.7 to 7.9.

[0198] In some embodiments, the pharmaceutical composition has a pH of about 5. In some embodiments, the pharmaceutical composition has a pH of about 5.5. In some embodiments, the pharmaceutical composition has a pH of about 6.0. In some embodiments, the pharmaceutical composition has a pH of about 7.0. In some embodiments, the pharmaceutical composition has a pH of about 7.5. In some embodiments, the pharmaceutical composition has a pH of about 7.6. In some embodiments, the pharmaceutical composition has a pH of about 7.7. In some embodiments, the pharmaceutical composition has a pH of about 7.8. In some embodiments, the pharmaceutical composition has a pH of about 7.9. In some embodiments, the pharmaceutical composition has a pH of about 8.0. In some embodiments, the pharmaceutical composition has a pH of about 8.1. In some embodiments, the pharmaceutical composition has a pH of about 8.2. In some embodiments, the pharmaceutical composition has a pH of about 8.3. In some embodiments, the pharmaceutical composition has a pH of about 8.5. In some embodiments, the pharmaceutical composition has a pH of about 9.0. In some embodiments, the pharmaceutical composition has a pH of about 9.5.

[0199] cryoprotectants In some embodiments, the cryoprotectant can be a compound used to protect the formulation from damage caused by cold, e.g., freezing. In some embodiments, the cryoprotectant can include a polyol, e.g., a carbohydrate, such as sucrose, trehalose, glucose, or 2-hydroxypropyl-α-cyclodextrin. A sugar alcohol, such as sorbitol, can also be included in the cryoprotectant. In some embodiments, the cryoprotectant can include a protein, peptide, or amino acid. For example, the cryoprotectant can include proline or hydroxyl proline. In some embodiments, the cryoprotectant can include an organic compound, such as glycerol, ethylene glycol, or propylene glycol. In some cases, the cryoprotectant can include a polymer, e.g., polyvinylpyrrolidone, polyethylene glycol, or gelatin or hydroxyethyl cellulose.

[0200] In some embodiments, the cryoprotectant is selected from the group consisting of ethanol, sucrose, maltose, lactose, glucose, galactose, trehalose, raffinose, other polyols, and polyhydric alcohols. In some embodiments, the cryoprotectant is a carbohydrate. In some embodiments, the cryoprotectant is selected from the group consisting of sucrose, maltose, lactose, glucose, galactose, trehalose, and raffinose. In some embodiments, the cryoprotectant is sucrose. In some embodiments, the cryoprotectant is glucose. In some embodiments, the cryoprotectant is galactose. In some embodiments, the cryoprotectant is trehalose. In some embodiments, the cryoprotectant is raffinose.

[0201] In some embodiments, the pharmaceutical composition comprises 6-19 wt% cryoprotectant. In some embodiments, the pharmaceutical composition comprises 7-18 wt% cryoprotectant. In some embodiments, the pharmaceutical composition comprises 8-17 wt% cryoprotectant. In some embodiments, the pharmaceutical composition comprises 9-16 wt% cryoprotectant. In some embodiments, the pharmaceutical composition comprises 10-15 wt% cryoprotectant. In some embodiments, the pharmaceutical composition comprises 11-14 wt% cryoprotectant.

[0202] In some embodiments, the pharmaceutical composition comprises about 6 wt% cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 7 wt% cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 8 wt% cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 9 wt% cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 10 wt% cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 11 wt% cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 12 wt% cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 13 wt% cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 14 wt% cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 15 wt% cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 16 wt% cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 17 wt% cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 18 wt% cryoprotectant.

[0203] amino acid In some aspects, the amino acid is selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, the amino acid is selected from the group consisting of arginine, histidine, and lysine. In some embodiments, the amino acid is arginine. In some embodiments, the amino acid is histidine. In some embodiments, the amino acid is lysine.

[0204] In some embodiments, the amino acids have a concentration of 50-100 mM. In some embodiments, the amino acids have a concentration of 60-90 mM. In some embodiments, the amino acids have a concentration of 65-85 nM. In some embodiments, the amino acids have a concentration of 70-80 mM. In some embodiments, the amino acids have a concentration of 71-79 mM. In some embodiments, the amino acids have a concentration of 72-78 nM. In some embodiments, the amino acids have a concentration of 73-77 mM. In some embodiments, the amino acids have a concentration of 74-76 mM.

[0205] In some embodiments, the amino acids are at a concentration of about 50 mM. In some embodiments, the amino acids are at a concentration of about 55 mM. In some embodiments, the amino acids are at a concentration of about 60 mM. In some embodiments, the amino acids are at a concentration of about 65 mM. In some embodiments, the amino acids are at a concentration of about 70 mM. In some embodiments, the amino acids are at a concentration of about 71 mM. In some embodiments, the amino acids are at a concentration of about 72 mM. In some embodiments, the amino acids are at a concentration of about 73 mM. In some embodiments, the amino acids are at a concentration of about 74 mM. In some embodiments, the amino acids are at a concentration of about 75 mM. In some embodiments, the amino acids are at a concentration of about 76 mM. In some embodiments, the amino acids are at a concentration of about 77 mM. In some embodiments, the amino acids are at a concentration of about 78 mM. In some embodiments, the amino acids are at a concentration of about 79 mM. In some embodiments, the amino acids are at a concentration of about 80 mM. In some embodiments, the amino acids are at a concentration of about 85 mM. In some embodiments, the amino acid is at a concentration of about 90 mM. In some embodiments, the amino acid is at a concentration of about 95 mM. In some embodiments, the amino acid is at a concentration of about 100 mM.

[0206] The vaccine can contain 1 to 30 peptides, 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, or 30 different peptides, 6, 7, 8, 9, 10, 11, 12, 13, or 14 different peptides, or 12, 13, or 14 different peptides. The peptides can include post-translational modifications. The vaccines contain 1 to 100 or more nucleotide sequences: 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, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, It may contain 4, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more different nucleotide sequences, 6, 7, 8, 9, 10, 11, 12, 13 or 14 different nucleotide sequences, or 12, 13 or 14 different nucleotide sequences. The vaccine contains 1–30 antigen sequences: 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, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, It may contain 3, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more different antigen sequences, 6, 7, 8, 9, 10, 11, 12, 13 or 14 different antigen sequences, or 12, 13 or 14 different antigen sequences.

[0207] The vaccines contain 1 to 30 antigen-encoding nucleic acid sequences: 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, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121 The cassette may contain 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more different antigen-encoding nucleic acid sequences, 6, 7, 8, 9, 10, 11, 12, 13, or 14 different antigen-encoding nucleic acid sequences, or 12, 13, or 14 different antigen-encoding nucleic acid sequences. An antigen-encoding nucleic acid sequence may refer to the antigen-encoding portion of an antigen "cassette." Features of antigen cassettes are described in more detail herein. The cassette may contain two or more antigen-encoding nucleic acid sequences linked together within the cassette (e.g., linked antigen-encoding nucleic acid sequences encoding linked T cell epitopes).

[0208] The vaccines contain 1 to 30 individual epitope-encoding nucleic acid sequences, 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, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, The cassette may contain 5, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more distinct epitope-encoding nucleic acid sequences, 6, 7, 8, 9, 10, 11, 12, 13, or 14 distinct epitope-encoding nucleic acid sequences, or 12, 13, or 14 distinct epitope-encoding nucleic acid sequences. An epitope-encoding nucleic acid sequence can refer to the sequence of individual epitope sequences, e.g., each of the linked T cell epitopes of two or more antigen-encoding nucleic acid sequences linked together within a cassette.

[0209] The vaccine can contain at least two repeats of an epitope-encoding nucleic acid sequence. As used herein, "repeat" (or interchangeably, "repeat") refers to two or more repeats of the same nucleic acid epitope-encoding nucleic acid sequence (including any 5' linker sequence and / or any 3' linker sequence described herein) within the antigen-encoding nucleic acid sequence. In one example, the antigen-encoding nucleic acid sequence portion of the cassette encodes at least two repeats of an epitope-encoding nucleic acid sequence. In a further non-limiting example, the antigen-encoding nucleic acid sequence portion of the cassette encodes two or more distinct epitopes, at least one of the distinct epitopes being encoded by at least two repeats of a nucleic acid sequence encoding a separate epitope (i.e., at least two distinct epitope-encoding nucleic acid sequences). In an illustrative non-limiting example, the antigen-encoding nucleic acid sequence comprises epitope-encoding sequence A(E A ), epitope coding sequence B (E B ) and epitope coding sequence C(EC ), and having at least one repeat of the distinct epitopes, are exemplified, but not limited to, by the following formula: 1. Repeat of one distinct epitope (repeat of epitope A): 1. E A -E B -E C -E A , or 2. E A -E A -E B -E C 2. Repeats of multiple distinct epitopes (repeats of epitopes A, B and C): 1. E A -E B -E C -E A -E B -E C , or 2. E A -E A -E B -E B -E C -E C 3. Multiple repeats of multiple distinct epitopes (repeats of epitopes A, B and C): 1. E A -E B -E C -E A -E B -E C -E A -E B -E C , or 2. E A -E A -E A -E B -E B -E B -E C -EC -E C

[0210] The above examples are not limiting, and an antigen-encoding nucleic acid sequence having at least one repeat of a distinct epitope can encode each of the distinct epitopes in any order or frequency. For example, the order and frequency can be, for example, in an example having epitopes A, B, and C, the sequence can be encoded by the formula E A -E B -E C -E C -E A -E B -E A -E C -E A -E C -E C -E B The epitopes may be randomly arranged according to the

[0211] Also provided herein is an antigen-encoding cassette, the antigen-encoding cassette having at least one antigen-encoding nucleic acid sequence described from the 5' end to the 3' end by the following formula: 1. (E x -(E N n ) y ) z wherein E represents a nucleotide sequence comprising a respective epitope-encoding nucleic acid sequence; n represents the number of separate, distinct epitope-encoding nucleic acid sequences and is any integer, including 0; E N represents a nucleotide sequence comprising a separate, distinct epitope-encoding nucleic acid sequence for each corresponding n, For each repetition of z, for each n, x=0 or 1, y=0 or 1, and at least one of x or y=1; and z=2 or more, and the antigen-encoding nucleic acid sequence is E, given E N or a combination thereof, including at least two repeats.

[0212] Each E or E Ncan independently comprise any epitope-encoding nucleic acid sequence described herein (e.g., peptides encoding infectious disease T cell epitopes and / or neoantigen epitopes). For example, each E or E N are independently expressed as L5 b -N c -L3 d ) from the 5' end to the 3' end, wherein N is each E or E N and c=1, L5 comprises a 5' linker sequence and b=0 or 1, and L3 comprises a 3' linker sequence and d=0 or 1. Epitopes and linkers that can be used are further described herein, see, e.g., VAAntigen Cassette.

[0213] Repeats of epitope-encoding nucleic acid sequences (including any 5' linker sequence and / or any 3' linker sequence) can be directly linearly linked to each other (e.g., E A -E A -...). Repeats of epitope-encoding nucleic acid sequences can be separated by one or more additional nucleotide sequences. Generally, repeats of epitope-encoding nucleic acid sequences can be separated by nucleotide sequences of any size applicable to the compositions described herein. In one example, repeats of epitope-encoding nucleic acid sequences can be separated by separate epitope-encoding nucleic acid sequences (e.g., E as described above). A -E B -E C -E A In examples where the repeats are separated by a single, separate and distinct epitope-encoding nucleic acid sequence, and each epitope-encoding nucleic acid sequence (including any 5 linker sequences and / or any 3 linker sequences) encodes a peptide 25 amino acids in length, the repeats may be separated by E A -E B -E A ...can be separated by 75 nucleotides, such as in an antigen-encoding nucleic acid represented by EA are separated by 75 nucleotides. In an illustrative example, in an antigen-encoding nucleic acid having the sequence VTNTEMFVTAPDNLGYMYEVQWPGQ (SEQ ID NO: 3)) and Trp2 (TQPQIANCSVYDFFVWLHYYSVRDT (SEQ ID NO: 4)) repeats of the 25-mer antigens Trp1 (VTNTEMFVTAPDNLGYMYEVQWPGQTQPQIANCSVYDFFVWLHYYSVRDTVTNTEMFVTAPDNLGYMYEVQWPGQTQPQIANCSVYDFFVWLHYYSVRDT (SEQ ID NO: 2)), the Trp1 repeats are separated by the 25-mer Trp2, and therefore the Trp1 epitope-encoding nucleic acid sequence repeats are separated by 75 nucleotides of the Trp2 epitope-encoding nucleic acid sequence. In examples where the repeats are separated by 2, 3, 4, 5, 6, 7, 8 or 9 separate and distinct epitope-encoding nucleic acid sequences, each epitope-encoding nucleic acid sequence (including any 5' linker sequence and / or any 3' linker sequence) encoding a peptide 25 amino acids in length, the repeats can be separated by 150, 225, 300, 375, 450, 525, 600 or 675 nucleotides, respectively.

[0214] In one embodiment, the different peptides and / or polypeptides or the nucleotide sequences encoding them are selected such that the peptides and / or polypeptides are capable of associating with different MHC molecules, such as different MHC class I molecules and / or different MHC class II molecules. In some aspects, a single vaccine composition comprises coding sequences for peptides and / or polypeptides capable of associating with the most frequently occurring MHC class I molecules and / or different MHC class II molecules. Thus, the vaccine composition can comprise different fragments capable of associating with at least two preferred, at least three preferred, or at least four preferred MHC class I molecules and / or different MHC class II molecules.

[0215] The vaccine composition can stimulate a specific cytotoxic T cell response and / or a specific helper T cell response.The vaccine composition can stimulate a specific cytotoxic T cell response and a specific helper T cell response.

[0216] The vaccine composition can stimulate a specific B cell response (eg, an antibody response).

[0217] The vaccine composition can stimulate a specific cytotoxic T cell response, a specific helper T cell response, and / or a specific B cell response. The vaccine composition can stimulate a specific cytotoxic T cell response and a specific B cell response. The vaccine composition can stimulate a specific helper T cell response and a specific B cell response. The vaccine composition can stimulate a specific cytotoxic T cell response, a specific helper T cell response, and a specific B cell response.

[0218] The vaccine composition may further comprise an adjuvant and / or a carrier. Examples of useful adjuvants and carriers are provided herein below. The composition may be associated with a carrier, e.g., a protein, or an antigen-presenting cell, e.g., a dendritic cell (DC), capable of presenting the peptide to T cells.

[0219] An adjuvant is any substance that, when incorporated into a vaccine composition, increases or otherwise modifies the immune response to an antigen. The carrier can be a scaffolding structure, such as a polypeptide or polysaccharide, to which the antigen can associate. Optionally, the adjuvant is covalently or non-covalently conjugated.

[0220] The ability of an adjuvant to increase the immune response to an antigen is typically manifested by a significant or substantial increase in immune-mediated reactions or a reduction in disease symptoms. For example, an increase in humoral immunity is typically manifested by a significant increase in the titer of antibodies produced against the antigen, and an increase in T cell activity is typically manifested as an increase in cell proliferation, cytotoxicity, or cytokine secretion. Adjuvants can also alter the immune response, for example, by shifting a predominantly humoral or Th response to a predominantly cellular or Th response.

[0221] Suitable adjuvants include 1018 ISS, alum, aluminum salts, Amplivax, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX, JuvImmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTel vector system, PLG microparticles, resiquimod, SRL172, virosomes and other virus-like particles, YF-17D, VEGF Adjuvants include, but are not limited to, Aquila's QS21 stimulon (Aquila Biotech, Worcester, Mass., USA), which is derived from saponin, R848, beta-glucan, Pam3Cys, mycobacterial extracts, and synthetic bacterial cell wall mimics, as well as other proprietary adjuvants, such as Ribi's Detox.Quil or Superfos. Adjuvants such as incomplete Freund's or GM-CSF are useful. Several immunological adjuvants specific for dendritic cells (e.g., MF59) and their preparations have been reported (Dupuis M, et al., Cell Immunol. 1998;186(1):18-27; Allison AC; Dev Biol Stand. 1998;92:3-11). Cytokines can also be used. Several cytokines influence dendritic cell migration to lymphoid tissues (e.g., TNF-alpha), accelerate dendritic cell maturation into efficient antigen-presenting cells for T lymphocytes (e.g., GM-CSF, IL-1, and IL-4) (U.S. Patent No. 5,849,589, which is incorporated herein by reference in its entirety), and act as immune adjuvants (e.g., IL-12) (Gabrilovich DI, et al., J Immunother Emphasis Tumor Immunol. 1996(6):414-418).

[0222] CpG immunostimulatory oligonucleotides have also been reported to enhance the effect of adjuvants in vaccine settings. Other TLR-binding molecules, such as RNA-binding TLR7, TLR8, and / or TLR9, can also be used.

[0223] Other examples of useful adjuvants include, but are not limited to, chemically modified CpG (e.g., CpR, Idera), poly(I:C) (e.g., polyi:CI2U), non-CpG bacterial DNA or RNA, and immunologically active small molecules and antibodies, such as cyclophosphamide, sunitinib, bevacizumab, Celebrex, NCX-4016, sildenafil, tadalafil, vardenafil, sorafinib, XL-999, CP-547632, pazopanib, ZD2171, AZD2171, ipilimumab, tremelimumab, and SC58175, which may act therapeutically and / or as adjuvants. The amounts and concentrations of adjuvants and additives can be readily determined by one of ordinary skill in the art without undue experimentation. Additional adjuvants include colony-stimulating factors such as granulocyte-macrophage colony-stimulating factor (GM-CSF, sargramostim).

[0224] A vaccine composition can include two or more different adjuvants. Additionally, a therapeutic composition can include any adjuvant material, including any of the above or a combination thereof. It is also contemplated that the vaccine and adjuvant can be administered together or separately in any suitable sequence.

[0225] The carrier (or excipient) can exist independently of the adjuvant. The function of the carrier can be, for example, to increase the molecular weight of a particular variant to increase activity or immunogenicity, to confer stability, to increase biological activity, or to increase serum half-life. Furthermore, the carrier can assist in presenting the peptide to T cells. The carrier can be any suitable carrier known to those skilled in the art, such as a protein or an antigen-presenting cell. Carrier proteins can be, but are not limited to, keyhole limpet hemocyanin, serum proteins such as transferrin, bovine serum albumin, human serum albumin, thyroglobulin, or ovalbumin, immunoglobulins, or hormones such as insulin or palmitic acid. For human immunization, the carrier is generally a physiologically acceptable carrier that is acceptable and safe for humans. However, tetanus toxoid and / or diphtheria toxoid are suitable carriers. Alternatively, the carrier can be dextran, e.g., Sepharose.

[0226] Cytotoxic T cells (CTLs) recognize antigens in the form of peptides bound to MHC molecules, rather than intact foreign antigens themselves. MHC molecules themselves are located on the cell surface of antigen-presenting cells. Therefore, CTL activation is possible when a trimeric complex of peptide antigen, MHC molecule, and APC is present. Correspondingly, immune responses can be enhanced when not only peptides are used to activate CTLs but also APCs bearing the respective MHC molecules are additionally added. Therefore, in some embodiments, the vaccine composition additionally contains at least one antigen-presenting cell.

[0227] Antigens can also be included in viral vector-based vaccine platforms such as vaccinia, fowlpox, self-replicating alphavirus, Maraba virus, adenovirus (see, e.g., Tatsis et al., Adenoviruses, Molecular Therapy (2004) 10, 616-629), or lentiviruses, including but not limited to any generation of second, third, or hybrid second / third generation lentiviruses and recombinant lentiviruses designed to target specific cell types or receptors (see, e.g., Hu et al., Immunization Delivered by Lentiviral Vectors for Cancer and Infectious Diseases, Immunol Rev. (2011) 239(1):45-61; Sakuma et al., Lentiviral vectors: basic to translational, Biochem J. (2012) 443(3):603-18; Cooper et al., Rescue of splicing-mediated intron loss maximizes expression in lentiviral vectors containing the human (See, e.g., ubiquitin C promoter, Nucl. Acids Res. (2015) 43(1):682-690; Zufferey et al., Self-Inactivating Lentivirus Vector for Safe and Efficient In Vivo Gene Delivery, J. Virol. (1998) 72(12):9873-9880.) Depending on the packaging capacity of the viral vector-based vaccine platform described above, this approach can deliver one or more nucleotide sequences encoding one or more antigenic peptides.The sequences may be flanked by non-mutated sequences, separated by linkers, or preceded by one or more sequences that target intracellular compartments (see, e.g., Gros et al., Prospective identification of neoantigen-specific lymphocytes in the peripheral blood of melanoma patients, Nat Med. (2016) 22(4):433-8; Stronen et al., Targeting of cancer neoantigens with donor-derived T cell receptor repertoires, Science. (2016) 352(6291):1337-41; Lu et al., Efficient identification of mutated cancer antigens recognized by T cells associated with durable tumor regressions, Clin Cancer Res. (2014) 20(13):3401-10). Upon introduction into a host, infected cells express the antigen, thereby stimulating a host immune (e.g., CTL) response against the peptide(s). Vaccine vectors and methods useful in immunization protocols are described, for example, in U.S. Patent No. 4,722,848. Another vector is BCG (Bacille Calmette Guerin). BCG vectors are described by Stover et al. (Nature 351:456-460 (1991)). A wide variety of other vaccine vectors useful for therapeutic administration of antigens or immunization, such as Salmonella typhi vectors, will be apparent to those skilled in the art from the disclosure herein.

[0228] Additional considerations for vaccine design and manufacturing Determination of a set of peptides covering all tumor subclones Truncal peptides, meaning those presented by all or most tumor subclones, can be prioritized for inclusion in the vaccine. Optionally, if there are no truncal peptides predicted to be presented and likely to be immunogenic, or if the number of truncal peptides predicted to be presented and likely to be immunogenic is small enough that additional non-truncal peptides can be included in the vaccine, additional peptides can be prioritized by estimating the number and identity of tumor subclones and selecting peptides to maximize the number of tumor subclones covered by the vaccine.

[0229] Antigen prioritization After all of the above antigen filters have been applied, more candidate antigens may still be available for vaccine inclusion than vaccine technology can support. Additionally, uncertainties may remain about various aspects of antigen analysis, and trade-offs may exist between different properties of candidate vaccine antigens. Therefore, instead of predefined filters at each step of the selection process, an integrated multidimensional model can be considered, placing candidate antigens in a space with at least the following axes and optimizing selection using an integrated approach:

[0230] Risk of autoimmunity or tolerance (germline risk) (lower risk of autoimmunity is typically preferred) Probability of sequencing artifacts (lower probability of artifacts is typically preferred) Probability of immunogenicity (higher probability of immunogenicity is typically preferred) Probability of presentation (higher probability of presentation is typically preferred) Gene expression (higher expression is typically preferred) HLA gene coverage (a greater number of HLA molecules involved in presenting a set of antigens may decrease the probability that tumors, infectious diseases, and / or infected cells will escape immune attack through downregulation or mutation of HLA molecules) HLA class coverage (covering both HLA-I and HLA-II may increase the chance of a therapeutic response and decrease the chance of avoiding tumors or infections)

[0231] Furthermore, optionally, an antigen can be deprioritized (e.g., excluded) from vaccination if it is predicted to be presented by a lost or inactivated HLA allele in either all or part of the patient's tumor or infected cells. Loss of an HLA allele can occur through somatic mutation, loss of heterozygosity, or homozygous deletion of the locus. Methods for detecting somatic mutation of HLA alleles are well known in the art, for example (Shukla et al., 2015). Methods for detecting somatic LOH and homozygous deletion (including HLA loci) have also been well described (Carter et al., 2012; McGranahan et al., 2017; Van Loo et al., 2010). An antigen can also be deprioritized if mass spectrometry data indicates that the predicted antigen is not presented by the predicted HLA allele.

[0232] Self-amplifying RNA vectors Generally, all self-amplifying RNA (SAM) vectors contain a self-amplifying backbone derived from a self-replicating virus. The term "self-amplifying backbone" refers to the minimal sequence(s) of a self-replicating virus that allows the viral genome to replicate autonomously. For example, the minimal sequence that allows alphavirus replication can include conserved sequences for nonstructural protein-mediated amplification (e.g., the nonstructural protein 1 (nsP1) gene, the nsP2 gene, the nsP3 gene, the nsP4 gene, and / or a polyA sequence). The self-amplifying backbone can also include sequences for expression of subgenomic viral RNA (e.g., the 26S promoter element for alphaviruses). samRNA vectors can be positive-sense or negative-sense RNA polynucleotides, such as vectors with backbones derived from positive-sense or negative-sense self-replicating viruses. Self-replicating viruses include, but are not limited to, alphaviruses, flaviviruses (e.g., Kunjin virus), measles virus, and rhabdoviruses (e.g., rabies virus and vesicular stomatitis virus). Examples of samRNA vector systems derived from self-replicating viruses are described in detail by Lundstrom (Molecules. 2018 Dec 13;23(12).pii:E3310.doi:10.3390 / molecules23123310), which is incorporated herein by reference for all purposes.

[0233] Alphavirus Biology Alphaviruses refer to members of the Togaviridae family and are single-stranded, positive-sense RNA viruses. Members are typically classified as Old World viruses, such as Sindbis virus, Ross River virus, Mayaro virus, chikungunya virus, and Semliki Forest virus, or New World viruses, such as Eastern equine encephalitis virus, Aura virus, Fort Morgan virus, and Venezuelan equine encephalitis virus and its derivative TC-83 (Strauss Microbial Review 1994). Natural alphavirus genomes are typically approximately 12 kb in length, with the first two-thirds containing genes encoding nonstructural proteins (nsPs) that form the RNA replication complex for autonomous replication of the viral genome, and the last third containing subgenomic expression cassettes encoding structural proteins for virion production (Frolov RNA 2001).

[0234] The model life cycle of alphaviruses involves several distinct steps (Strauss Microbiol Review 1994, Jose Future Microbiol 2009). After viral attachment to the host cell, the virion fuses with the membrane within an endocytic compartment, resulting in the eventual release of genomic RNA into the cytosol. The genomic RNA, which is in a positive-strand orientation and contains a 5' methylguanylate cap and a 3' poly(A) tail, is translated to produce nonstructural proteins nsP1-4, which form a replication complex. Early in infection, the positive strand is then replicated onto a negative-strand template by the complex. In the current model, the replication complex undergoes further processing as infection progresses, and the resulting processed complex switches to transcribing the negative strand into both full-length positive-strand genomic RNA and the 26S subgenomic positive-strand RNA containing the structural genes. Several conserved sequence elements (CSEs) in alphaviruses are identified that potentially play roles in various RNA replication steps, including the complement of the 5′ UTR in replication of plus-strand RNA from a minus-strand template, a 51-nt CSE in replication of minus-strand synthesis from a genomic template, a 24-nt CSE in the junction region between nsP and 26S RNA in transcription of subgenomic RNA from the minus strand, and a 3′ 19-nt CSE in minus-strand synthesis from a plus-strand template.

[0235] Following replication of the various RNA species, viral particles are then typically assembled in the natural life cycle of a virus. The 26S RNA is translated, and the resulting proteins are further processed to produce structural proteins, including capsid proteins, glycoproteins E1 and E2, and two small polypeptides, E3 and 6K (Strauss 1994). Encapsidation of the viral RNA occurs, packaging capsid proteins that are usually specific only to the genomic RNA, followed by virion assembly and membrane surface budding.

[0236] Alphaviruses as delivery vectors Alphaviruses (including alphavirus sequences, characteristics, and other elements) can be used to generate alphavirus-based delivery vectors (also called alphavirus vectors, alphavirus viral vectors, alphavirus vaccine vectors, self-replicating RNA (srRNA) vectors, or self-amplifying mRNA (SAM) vectors). Alphaviruses have previously been engineered for use as expression vector systems (Pushko 1997, Rheme 2004). Alphaviruses offer several advantages, particularly in vaccine settings where heterologous antigen expression may be desirable. Due to their ability to replicate autonomously in the host cytoplasm, alphavirus vectors generally produce high copy numbers of expression cassettes within cells, resulting in high levels of heterologous antigen production. Additionally, vectors are generally transient, resulting in improved biosafety and reduced induction of immune tolerance to the vector. The public also generally lacks pre-existing immunity to alphavirus vectors compared to other standard viral vectors, such as human adenoviruses. Alphavirus-based vectors also generally result in a cytotoxic response to infected cells. Cytotoxicity, to some extent, can be important in a vaccine environment to adequately stimulate an immune response to the expressed heterologous antigen. However, the desired degree of cytotoxicity can be a balancing act, and thus several attenuated alphaviruses have been developed, including the TC-83 strain of VEE. Thus, the exemplary antigen expression vectors described herein utilize an alphavirus backbone that allows for high-level antigen expression, stimulates a strong immune response to the antigen, does not stimulate an immune response to the vector itself, and can be used in a safe manner. Furthermore, antigen expression cassettes can be designed to stimulate different levels of immune response through optimization of the alphavirus sequences used by the vector, including, but not limited to, sequences derived from VEE or its attenuated derivative, TC-83.

[0237] Several expression vector design strategies have been engineered using alphavirus sequences (Pushko 1997). In one strategy, alphavirus vector design involves inserting a second copy of the 26S promoter sequence element downstream of the structural protein gene, followed by insertion of the heterologous gene (Frolov 1993). Thus, in addition to the native nonstructural and structural proteins, an additional subgenomic RNA expressing the heterologous protein is generated. In this system, all elements for the production of infectious virions are present, and therefore, repeated rounds of infection of the expression vector in uninfected cells can occur.

[0238] Another expression vector design utilizes a helper virus system (Pushko 1997). In this strategy, structural proteins are replaced by heterologous genes. Thus, after autonomous replication of viral RNA mediated by the still-intact nonstructural genes, the 26S subgenomic RNA provides expression of the heterologous protein. Traditionally, an additional vector expressing the structural proteins is then supplied in trans, such as by cotransfection of a cell line, to produce infectious virus. This system is described in detail in US Pat. No. 8,093,021, which is incorporated herein by reference in its entirety for all purposes. Helper vector systems offer the advantage of limiting the likelihood of infectious particle formation, thus improving biosafety. Furthermore, helper vector systems reduce total vector length, potentially improving replication and expression efficiency. Thus, the exemplary antigen expression vectors described herein can utilize an alphavirus backbone, in which structural proteins are replaced by an antigen cassette. The resulting vectors promote efficient expression by reducing overall expression vector size, while reducing biosafety concerns.

[0239] In vitro production of self-amplifying viruses A convenient technique well known in the art for producing RNA is in vitro transcription (IVT), in which a DNA template of the desired vector is first generated by techniques well known to those of skill in the art, including standard molecular biology techniques such as cloning, restriction digestion, ligation, gene synthesis (e.g., chemical and / or enzymatic synthesis), and polymerase chain reaction (PCR).

[0240] The DNA template contains an RNA polymerase promoter at the 5' end of the sequence desired to be transcribed into RNA (e.g., SAM). Promoters include, but are not limited to, bacteriophage polymerase promoters such as T3, T7, K11, or SP6. Depending on the particular RNA polymerase promoter sequence selected, additional 5' nucleotides may be transcribed in addition to the desired sequence. For example, a standard T7 promoter can be referenced by the sequence TAATACGACTCACTATAGG (SEQ ID NO: 5), and an IVT reaction using the DNA template TAATACGACTCACTATAGGN (SEQ ID NO: 6) to produce the desired sequence N would generate the mRNA sequence GG-N. Generally, without being bound by theory, T7 polymerase more efficiently transcribes RNA transcripts that begin with a guanosine. If additional 5' nucleotides are not desired (e.g., no additional GG), the RNA polymerase promoter contained in the DNA template can be a sequence that results in a transcript containing only the 5' nucleotide of the desired sequence, e.g., a samRNA with the native 5' sequence of the self-replicating virus from which the samRNA vector is derived. For example, a minimal T7 promoter can be referenced by the sequence TAATACGACTCACTATA (SEQ ID NO: 7), and an IVT reaction using the DNA template TAATACGACTCACTATAN (SEQ ID NO: 8) for production of the desired sequence N will generate mRNA sequence N. Similarly, a minimal SP6 promoter referenced by the sequence ATTTAGGTGACACTATA (SEQ ID NO: 9) can be used to generate transcripts without additional 5' nucleotides. In a typical IVT reaction, a DNA template is incubated with the appropriate RNA polymerase enzyme, buffer, and nucleotides (NTPs).

[0241] The resulting RNA polynucleotides can optionally be further modified, including, but not limited to, the addition of a 5' cap structure, such as 7-methylguanosine or related structures, and optionally modifying the 3' end to include a polyadenylic acid (polyA) tail. In a modified IVT reaction, the RNA is co-transcriptionally capped with a 5' cap structure by adding a cap analog during IVT. The cap analog is a dinucleotide (m 7 G-ppp-N) cap analog or trinucleotide (m 7 The cap analogs may include a trinucleotide cap analog (m-ppp-NN), where N represents a nucleotide or modified nucleotide (e.g., a ribonucleoside, including, but not limited to, adenosine, guanosine, cytidine, and uridine). Exemplary cap analogs and their use in IVT reactions are also described in detail in U.S. Pat. No. 10,519,189, which is incorporated herein for all purposes. As discussed, T7 polymerase more efficiently transcribes RNA transcripts that begin with guanosine. To improve transcription efficiency on templates that do not begin with guanosine, trinucleotide cap analogs (m-ppp-NN) may be used. 7 G-ppp-NN) can be used. Trinucleotide cap analogs can be used in place of dinucleotide cap analogs (m 7 It is possible to increase the transfer efficiency by 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20-fold or more relative to IVT reactions using G-ppp-N.

[0242] A 5' cap structure can also be added post-transcriptionally, such as by using the vaccinia capping system (eg, NEB catalog no. M2080) which contains mRNA 2'-O-methyltransferase and S-adenosylmethionine.

[0243] The resulting RNA polynucleotides can optionally be further modified alternatively or in addition to the capping techniques described, including, but not limited to, modifying the 3' end to include a polyadenylic acid (polyA) tail.

[0244] The RNA can then be purified using techniques well known in the art, such as phenol-chloroform extraction or column purification (eg, chromatography-based purification).

[0245] Lipid nanoparticle delivery An important aspect to consider in vaccine vector design is immunity to the vector itself (Riley 2017). This can be in the form of pre-existing immunity to the vector itself, such as with certain human adenovirus systems, or the development of immunity to the vector after administration of the vaccine. The latter is an important consideration when multiple administrations of the same vaccine are given, e.g., separate priming and boosting doses, or when the same vaccine vector system is used to deliver different antigen cassettes.

[0246] For alphavirus vectors, the standard delivery method is the aforementioned helper virus system, which provides the capsid, E1, and E2 proteins in trans to produce infectious viral particles. However, it is important to note that the E1 and E2 proteins are always the primary targets of neutralizing antibodies (Strauss 1994). Therefore, the effectiveness of using alphavirus vectors to deliver an antigen of interest to target cells can be reduced if the infectious particles are targeted by neutralizing antibodies.

[0247] An alternative to viral particle-mediated gene delivery is the use of nanomaterials to deliver expression vectors (Riley 2017). Importantly, nanomaterial vehicles can be fabricated from non-immunogenic materials, generally avoiding priming immunity to the delivery vector itself. These materials can include, but are not limited to, lipids, inorganic nanomaterials, and other polymeric materials. Lipids can be cationic, anionic, or neutral. Materials can be synthetic or naturally derived, and in some cases, biodegradable. Lipids can include fats, cholesterol, phospholipids, lipid conjugates, including, but not limited to, polyethylene glycol (PEG) conjugates (PEGylated lipids), waxes, oils, glycerides, and fat-soluble vitamins.

[0248] Lipid nanoparticles (LNPs) are attractive delivery systems due to the amphiphilic nature of lipids, which allows for the formation of membranes and vesicle-like structures (Riley 2017). These vesicles typically deliver expression vectors by absorbing into the membrane of target cells and releasing the nucleic acid into the cytosol. Furthermore, LNPs can be further modified or functionalized to facilitate targeting of specific cell types. Another consideration in LNP design is the balance between targeting efficiency and cytotoxicity. Lipid compositions generally contain specific mixtures of cationic, neutral, anionic, and amphiphilic lipids. In some cases, specific lipids are included to prevent LNP aggregation, prevent lipid oxidation, or provide functional chemical groups that facilitate the attachment of additional moieties. The lipid composition can affect overall LNP size and stability. In one example, the lipid composition contains dilinoleylmethyl-4-dimethylaminobutyrate (MC3) or an MC3-like molecule. MC3 and MC3-like lipid compositions can be formulated to contain one or more other lipids, such as PEG or PEG-conjugated lipids, sterols, or neutral lipids.

[0249] Nucleic acid vectors, e.g., expression vectors, directly exposed to serum can have several undesirable consequences, including degradation of the nucleic acid by serum nucleases or off-target stimulation of the immune system by released nucleic acids. Therefore, encapsulation of alphavirus vectors can be used to avoid degradation while also avoiding potential off-target effects. In certain examples, the alphavirus vector is completely encapsulated within the delivery vehicle, e.g., within the aqueous interior of the LNP. Encapsulation of the alphavirus vector within the LNP can be achieved by techniques well known to those skilled in the art, such as microfluidic mixing and droplet generation performed on a microfluidic droplet generator. Such devices include, but are not limited to, standard T-junction or flow-focusing devices. In one example, a desired lipid formulation, such as an MC3- or MC3-like-containing composition, is provided to the droplet generator in parallel with the alphavirus delivery vector and other desired agents, such that the delivery vector and desired agent are completely encapsulated within the MC3- or MC3-like-based LNP. In one example, the droplet generator can control the size range and size distribution of the generated LNPs. For example, LNPs can have a size ranging from 1 to 1000 nanometers in diameter, e.g., 1, 10, 50, 100, 500, or 1000 nanometers. Following droplet generation, the delivery vehicle encapsulating the expression vector can be processed or modified to prepare them for administration.

[0250] Conventional and pharmaceutically acceptable routes of administration include, but are not limited to, intranasal, intramuscular, intratracheal, subcutaneous, intradermal, rectal, oral, and other parenteral routes of administration. Routes of administration may be combined, if desired, or tailored depending on the immunogen or disease. For example, in the prevention of rabies, subcutaneous, intratracheal, and intranasal routes are preferred. The route of administration will depend primarily on the nature of the disease being treated.

[0251] The level of immunity to the antigen(s) can be monitored to determine the need for boosters, if any. After assessing antibody titers in the serum, for example, any booster immunizations may be desired. [Example]

[0252] Example 1: Short-term stability of samRNA-LNP DP in new formulations versus current TSM formulations Previously, self-amplifying RNA encapsulated in lipid nanoparticle (samRNA-LNP)-based drug products (DPs) was formulated and stored in TSM (5 mM Tris, 10 wt% sucrose, and 10 wt% maltose) buffer and shown to be stable for long-term storage at ≤60°C. Due to the limited clinical sites with storage capabilities below -60°C, it is desirable to test the stability of samRNA-LNP DPs in other potential formulations and at alternative temperature conditions that may enable distribution to clinical sites without storage capabilities below -60°C.

[0253] The samRNA-LNP DP stored in TSM is unstable when stored at temperatures above -60°C; the particles tend to increase in hydrodynamic size and exhibit time- and temperature-dependent degradation in the full-length profile of the encapsulated samRNA, with a concomitant loss of cellular potency. These observed undesirable changes in critical quality attributes in the DP when stored above -60°C in the current TSM formulation make storage of the DP above -60°C unsuitable for clinical administration. To find a formulation that is stable at storage temperatures above -60°C, two studies were evaluated: a short-term pH and excipient scouting study and a short-term stability robustness study.

[0254] The studies involved buffer exchanging the samRNA-LNP DP into the investigational buffer system via tangential flow filtration (99.9% buffer exchange) and evaluating the stability of the DP at various time points and temperatures in the investigational buffer system compared to the current TSM buffer system.

[0255] Short-term pH and excipient scouting study: samRNA-LNP construct stability from pH 5.5 to pH 8.0 using DP in TSM buffer as a control. The buffer formulations investigated were as follows: mod-GB6: 25 mM succinic acid, 12 wt% sucrose, 100 mM NaCl, 50 mM ArgHCl (pH 5.80) mod-GB7: 10 mM citric acid, 15 wt% sucrose, 50 mM ArgHCl (pH 5.5) mod-GB8: 20 mM histidine, 15 wt% sucrose, 100 mM NaCl, 50 mM ArgHCl (pH 6.1) mod-TSM1: 5 mM Tris, 15 wt% sucrose, 100 mM NaCl (pH 7.8) TSA-2: 5 mM Tris, 15 wt% sucrose, 50 mM ArgHCl (pH 7.8)

[0256] Short-term stability and robustness studies: The stability of the samRNA-LNP structure was assessed in the buffer solution of the lead formulation candidate from the previous study, as well as the stability of the samRNA DP in a modified formulation of the lead formulation candidate from the previous study (excipient components were varied over a narrow range to establish robustness and further refine the selection of the optimal formulation).

[0257] Example 2: Short-term pH and excipient scouting studies with samRNA DP Initially, the formulations (pH 5.5-8.0) were evaluated for short-term (3-month) stability to determine the optimal pH of the formulation matrix. The efficacy of the formulations was evaluated at various time points and temperatures, including accelerated storage conditions at 5°C, 25°C, and stressed storage conditions at 40°C. The short-term stability results of the study, as assessed by full-length profiles by capillary electrophoresis, concentration and encapsulation determination by absorbance spectroscopy, and size and polydispersity determination by dynamic light scattering, are shown in the graphs below.

[0258] The formulation composition, pH and osmolality of DP in the various investigated matrices are summarized in Table 1 below.

[0259] [Table 1]

[0260] Comparison of samRNA-LNP stability in samples incubated under various temperature conditions, including stressed and accelerated conditions, for up to one month (1M) showed the most promising data in TSA-2 buffer. The TSA-2 formulation was further evaluated for up to three months and demonstrated excellent stability at -80°C and -20°C for up to three months, excellent stability over multiple F / T events from either -80°C or -20°C storage conditions, and stability for up to two weeks under refrigerated conditions at 5°C, as shown by the data (Figures 1-4).

[0261] As seen from % encapsulation, DLS, and cell-based potency data from storage conditions at -80°C, -20°C, and 5°C, as well as F / T data for the TSA-2 buffer matrix, there was no increase in size, loss in potency, or decrease in % encapsulation efficiency over up to three months of storage. Furthermore, at refrigerated storage conditions at 5°C, acceptable critical quality attributes are maintained for up to two (2) weeks. The data support significant improvements in DP stability in the investigational formulation matrix of TSA-2 over the existing TSM formulation matrix. With the current TSM formulation matrix, a significant decrease in % encapsulation and an increase in size at elevated storage conditions of -20°C are observed within one month of storage.

[0262] The TSA-2 buffer was further analyzed in a second samRNA-LNP construct encoding a different cassette approximately 6 kb in size for assessment of the robustness of the formulation matrix.

[0263] Example 3: 3-month stability of samRNA-LNP DP in optimized TSA formulation Based on the results of TSA-2 buffer in the short-term stability studies summarized above, further optimization of excipient studies was initiated.

[0264] In this short-term stability study, slight changes to the amount of buffer Tris, stabilizing excipients sucrose and arginine, and formulation pH were evaluated. The resulting composition of the investigational formulated matrix was as follows:

[0265] TIFF2025540137000006.tif57159

[0266] The aforementioned formulations were used in short-term stability studies of buffer-exchanged samRNA-LNP DPs at incubation temperatures of -20°C, 5°C, 25°C, and 40°C. The formulations were evaluated for stability trends by particle size and polydispersity determination, concentration and encapsulation determination, encapsulated RNA full-length profile determination, and cellular efficacy. The resulting data are shown in Figures 5-10.

[0267] The efficacy of the Tris-sucrose-arginine-based formulation for DP was evaluated by various key product attributes (Figures 5-10). No appreciable changes in CQAs were observed as a function of multiple freeze-thaw (F / T) events from the intended storage temperature of -20°C. Furthermore, minor changes in buffer, cryoprotectant, and stabilizer amounts did not show any appreciable difference in the stability profile over 3 months of storage at -20°C. In fact, cell-based potency values ​​were maintained at greater than 50% for up to 3 months at -20°C and up to 1 month at 5°C.

[0268] Therefore, based on the data generated, the TSA-4 formulation (8 mM Tris, 13 wt% sucrose, 75 mM Arg) demonstrated the most consistent stability profile, as assessed by size, aggregation, concentration, encapsulation, full-length samRNA-LNP RNA profile, and cell-based efficacy, during stability evaluations at -80 °C, -20 °C for up to 3 months, and 5 °C for up to 1 month, when compared to TO (initial measurement). Therefore, these data indicate that the optimized Tris-sucrose-arginine formulation, TSA-4 (8 mM Tris, 13 wt% sucrose, 75 mM Arg), provides robust short-term samRNA-LNP stabilization at the intended storage condition of -20 °C. Therefore, based on the stability trend for up to 3 months at -20 °C, it is expected that the TSA formulation will enable long-term stability of samRNA-LNP DP at -20 °C.

[0269] Therefore, the TSA-4 formulation (8 mM Tris, 13 wt% sucrose, 75 mM Arg) was also evaluated as a formulation matrix to enable the production of lyophilized samRNA-LNP DPs that could be stored for long periods at 5°C.

[0270] The TSA-4 buffer and the samRNA-LNP DP formulated in TSA-4 buffer were analyzed to determine the critical temperature, Tg' (glass transition temperature), and T c The thermal properties (Tg' and Tg) are useful for designing a rational freezing cycle around the freeze-dried cake collapse temperature. C ) were characterized.

[0271] Lyo cycle development began with an initial assessment of the Lyo suitability of TSA-4 buffer by developing a basic lyophilization cycle using only TSA-4 buffer, followed by running a lyo cycle on samRNA-LNP product formulated in TSA-4 buffer.

[0272] Multiple iterations of lyophilization cycle optimization were performed (summarized in the table below) to narrow down a robust lyophilization cycle that allows short-term stability of DP in TSA formulations at 5° C. storage conditions.

[0273] TIFF2025540137000007.tif82159

[0274] The final lyophilized DP stability study of DP formulated in TSA-4 buffer (2 mL of 0.06 mg / mL DP filled in a 6 mL vial) was staged for short-term stability studies at various temperatures (-80°C, 5°C, 25°C, and 40°C) and storage conditions up to 3 months.

[0275] Data from the stability evaluation of the lyophilized DP are shown in the table below.

[0276] TIFF2025540137000008.tif167170

[0277] The freeze-dried DP exhibited a refined physical cake structure without structural defects such as cracks, meltback and micro-collapse.

[0278] The lyophilized samRNA-LNP DP also demonstrated excellent DP quality for up to 3 months at 5°C, with no change in any of the measured attributes, including size, encapsulation, FLP, and cellular potency.

[0279] Additionally, the refined cake allowed acceptable reconstitution (reconstitution time within 2 minutes) without any foaming that would result in the production of a corresponding liquid DP.

[0280] The lyophilized DP was also evaluated for the impact of transport of the DP at 5° C. Data generated from the transport study conducted at 5° C. mimicked the stability profile observed without transport (when stored at 5° C. in the development laboratory setting).

[0281] At accelerated and stress conditions of 25°C and 40°C, the data showed a trend towards product degradation (loss of full-length RNA profile) evidenced by a concomitant decrease in cellular efficacy with increasing duration.

[0282] However, current data still support acceptable DP attributes for up to 1 month at 25°C to allow for accidental exposure or product deviation in clinical practice from the recommended 5°C.

[0283] In summary, stability data generated from DP in TSA-4 buffer demonstrate robust stability for up to 3 months at 5°C and potential long-term stability for up to 2 years at 5°C storage conditions.

Claims

1. A pharmaceutical composition comprising an RNA-based expression system and further comprising a buffer, an amino acid, and a cryoprotectant.

2. 2. The pharmaceutical composition of claim 1, wherein the RNA-based expression system is selected from the group consisting of a messenger RNA (mRNA)-based expression system, a circular (circRNA)-based expression system, a single guide RNA (sgRNA)-based expression system, and a self-amplifying RNA (saMRNA) expression system.

3. 2. The pharmaceutical composition of claim 1, wherein the RNA-based expression system is a self-amplifying RNA (sa-mRNA) expression system.

4. 3. The pharmaceutical composition of claim 1, wherein the amino acid is selected from histidine, lysine, arginine, glutamine, and arginine, or a pharmaceutically acceptable salt thereof.

5. 7. The pharmaceutical composition of claim 6, wherein the amino acid is arginine.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the amino acid has a concentration of 10 to 140 mM.

7. The pharmaceutical composition according to any one of claims 1 to 5, wherein the amino acid has a concentration of 25 to 125 mM.

8. The pharmaceutical composition according to any one of claims 1 to 5, wherein the amino acid has a concentration of 50 to 100 mM.

9. The pharmaceutical composition according to any one of claims 1 to 5, wherein the amino acid has a concentration of 70 to 80 mM.

10. The pharmaceutical composition of any one of claims 1 to 5, wherein the amino acid has a concentration of about 75 mM.

11. The pharmaceutical composition according to any one of claims 1 to 10, wherein the composition has a pH of 6.5 to 9.

1.

12. The pharmaceutical composition of claim 11, wherein the pH is 7.3 to 8.

3.

13. 12. The pharmaceutical composition of claim 11, wherein the pH is about 7.

8.

14. 14. The pharmaceutical composition of any one of claims 1 to 13, wherein the buffer is selected from the group consisting of citrate, succinate, malate, phosphate, histidine, glycine, MOPS, HEPES, Tris, and Bis-Tris.

15. 15. The pharmaceutical composition of claim 14, wherein the buffer is Tris.

16. 15. The pharmaceutical composition of claim 14, wherein the buffer is not a phosphate buffer.

17. The pharmaceutical composition according to any one of claims 1 to 15, wherein the buffer has a concentration of 1 to 15 mM.

18. The pharmaceutical composition according to any one of claims 1 to 15, wherein the buffer has a concentration of 3 to 12 mM.

19. The pharmaceutical composition according to any one of claims 1 to 15, wherein the buffer has a concentration of 6 to 10 mM.

20. The pharmaceutical composition of any one of claims 1 to 15, wherein the buffer has a concentration of about 8.0 mM.

21. The pharmaceutical composition of any one of claims 1 to 15, wherein the buffer has a concentration greater than 5.0 mM.

22. The pharmaceutical composition according to any one of claims 1 to 20, comprising a cryoprotectant.

23. 23. The pharmaceutical composition of claim 22, wherein the cryoprotectant is selected from the group consisting of ethanol, sucrose, maltose, lactose, glucose, galactose, trehalose, raffinose, other polyols and polyhydric alcohols.

24. The pharmaceutical composition of any one of claims 22 to 23, wherein the cryoprotectant is 6 to 18 wt% of the pharmaceutical composition.

25. The pharmaceutical composition of any one of claims 22 to 23, wherein the cryoprotectant is 11 to 15 wt% of the pharmaceutical composition.

26. 24. The pharmaceutical composition of claim 22 or 23, wherein the cryoprotectant is about 13 wt% of the pharmaceutical composition.

27. The pharmaceutical composition of any one of claims 1 to 26, wherein the cryoprotectant is sucrose.

28. sAmRNA-based expression system, and 1. A pharmaceutical composition further comprising 70-80 mM arginine, 11-15 wt % sucrose, and 6-10 mM Tris, A pharmaceutical composition having a pH of 7.6 to 8.

0.

29. sAmRNA-based expression system, and 1. A pharmaceutical composition further comprising about 75 mM arginine, about 13 wt % sucrose, and about 8 mM Tris, A pharmaceutical composition having a pH of about 7.

8.

30. 30. The pharmaceutical composition of any one of claims 1 to 29, wherein the stability of the pharmaceutical composition is maintained at a temperature of at least -20°C, at least 5°C, or at least 25°C.

31. 31. The pharmaceutical composition of claim 30, wherein the stability is assessed by one or more assays including particle size, PDI, samRNA concentration, encapsulation rate, full length profile (FLP) of samRNA, and potency.

32. 32. The pharmaceutical composition of claim 31, wherein the potency of the RNA-based expression system of the pharmaceutical composition is greater than about 40% after storage compared to the potency before storage.

33. 33. The pharmaceutical composition of claim 32, stored at about -20°C.

34. 33. The pharmaceutical composition of claim 32, stored at about 5°C.

35. 33. The pharmaceutical composition of claim 32, stored at about 25°C.

36. The pharmaceutical composition according to any one of claims 32 to 35, which is stored for at least 1 day.

37. The pharmaceutical composition according to any one of claims 32 to 35, which is stored for at least 3 days.

38. The pharmaceutical composition of any one of claims 32 to 35, which is stored for at least 5 days.

39. The pharmaceutical composition of any one of claims 32 to 35, which is stored for at least one week.

40. The pharmaceutical composition of any one of claims 32 to 35, which is stored for at least 2 weeks.

41. The pharmaceutical composition according to any one of claims 32 to 35, which is stored for at least one month.

42. The pharmaceutical composition according to any one of claims 32 to 35, which is stored for at least 3 months.

43. The pharmaceutical composition according to any one of claims 32 to 35, which is stored for at least 6 months.

44. 44. The pharmaceutical composition of any one of claims 1 to 43, wherein the buffer is not a phosphate buffer.

45. The pharmaceutical composition according to any one of claims 1 to 43, which does not contain a phosphate buffer.

46. 46. ​​The pharmaceutical composition of any one of claims 1 to 45, in a liquid formulation.

47. The pharmaceutical composition of any one of claims 1 to 45, in a lyophilized formulation.

48. 48. The pharmaceutical composition of claim 47, wherein the water content of the pharmaceutical composition is less than 5 wt%.

49. SiO ２ 49. The pharmaceutical composition of any one of claims 1 to 48, stored in a container comprising:

50. 1. A method for eliciting an immune response in a subject, comprising:

49. A method comprising administering to said subject a composition according to any one of claims 1 to 48.

51. 51. The method of claim 50, wherein the composition is administered intramuscularly (IM), intradermally (ID), subcutaneously (SC), or intravenously (IV).

52. 52. The method of claim 51, wherein the composition is administered intramuscularly.

53. 53. The method of any one of claims 50-52, wherein the method further comprises administration of one or more immunomodulatory agents, optionally administered before, simultaneously with, or after administration of the composition or pharmaceutical composition.

54. 54. The method of claim 53, wherein the one or more immunomodulatory agents are selected from the group consisting of an anti-CTLA4 antibody or antigen-binding fragment thereof, an anti-PD-1 antibody or antigen-binding fragment thereof, an anti-PD-L1 antibody or antigen-binding fragment thereof, an anti-4-1BB antibody or antigen-binding fragment thereof, or an anti-OX-40 antibody or antigen-binding fragment thereof.

55. 55. The method of claim 53 or 54, wherein the immunomodulatory agent is administered intravenously (IV), intramuscularly (IM), intradermally (ID), or subcutaneously (SC).

56. 56. The method of claim 55, wherein the subcutaneous administration is near the site of administration of the composition or pharmaceutical composition or in proximity to one or more vector or composition-draining lymph nodes.

57. 57. The method of any one of claims 49 to 56, further comprising administering to the subject a second vaccine composition.

58. 58. The method of claim 57, wherein the second vaccine composition is administered before the administration of the composition of any one of claims 1 to 48.

59. 58. The method of claim 57, wherein the second vaccine composition is administered after administration of the composition of any one of claims 1 to 48.

60. The method of any one of claims 57 to 59, wherein the second vaccine composition is the same as the composition of any one of claims 1 to 48.

61. The method of any one of claims 57 to 59, wherein the second vaccine composition is different from the composition of any one of claims 1 to 48.

62. A method for preparing a pharmaceutical composition according to any one of claims 1 to 48, comprising a freeze-drying step.

63. 63. The method of claim 62, wherein the freeze-drying step comprises a secondary drying step.

64. 64. The method of claim 63, wherein the conditions of the secondary drying step comprise a shelf temperature of about 10°C.

65. 64. The method of claim 63, wherein the conditions of the secondary drying step comprise a shelf temperature of 5 to 15°C.

66. 66. The method of any one of claims 62 to 65, wherein the conditions of the secondary drying step comprise a vacuum of about 50 mTorr.

67. 66. The method of any one of claims 62 to 65, wherein the conditions of the secondary drying step comprise a vacuum of 25 to 75 mTorr.

68. The conditions of the secondary drying step are: a) a shelf temperature of about 10°C; b) a vacuum of about 50 mTorr; c) a ramp rate of about 0.1°C / min; and d) a duration of approximately 1040 minutes; 64. The method of claim 63, comprising:

69. 69. The method of any one of claims 63 to 68, wherein the freeze-drying step comprises a primary drying step.

70. 70. The method of claim 69, wherein the conditions of the primary drying step comprise a shelf temperature of about -37°C.

71. 70. The method of claim 69, wherein the conditions of the primary drying step comprise a shelf temperature of -62 to -12°C.

72. 72. The method of any one of claims 69 to 71, wherein the conditions of the primary drying step comprise a vacuum of about 50 mTorr.

73. 72. The method of any one of claims 69 to 71, wherein the conditions of the primary drying step comprise a vacuum of 25 to 75 mTorr.

74. 74. The method of any one of claims 63 to 73, wherein the freeze-drying step comprises a freezing step.

75. 75. The method of claim 74, wherein the conditions of the freezing step comprise a shelf temperature of about -55°C.

76. 75. The method of claim 74, wherein the conditions of the freezing step comprise a shelf temperature of -60 to -35°C.

77. The conditions of the primary drying step are: a) a shelf temperature of about -37°C; b) a vacuum of about 50 mTorr; c) a ramp rate of about 0.5°C / min; d) a duration of approximately 4800 minutes; 70. The method of claim 69, comprising:

78. A composition prepared by the method of any one of claims 63 to 77.

Images