Antigens for cancer immunotherapy

Abstract

The present disclosure relates to the expression of a fusion protein for cancer immunotherapy in a mammalian subject, such as a human subject. In particular, the present disclosure relates to mRNA, self-replicating RNA and temperature-sensitive self-replicating RNA encoding a variety of tumor-associated and / or tumor-specific antigens.

Description

Cross - Reference to Related Applications

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 427,424, filed on November 22, 2022, which is hereby incorporated by reference in its entirety. Reference to Electronic Sequence Listing

[0002] The contents of the electronic sequence listing (699442001740SEQLIST.xml; size: 92,598 bytes; creation date: November 20, 2023) are hereby incorporated by reference in its entirety. Technical Field

[0003] The present disclosure relates to the expression of fusion proteins for cancer immunotherapy in mammalian subjects such as human subjects. In particular, the present disclosure relates to mRNAs, self - replicating RNAs, and temperature - sensitive self - replicating RNAs encoding multiple tumor - associated and / or tumor - specific antigens. Background Art

[0004] Immunotherapy can effectively treat cancer and has been more widely applied. One treatment strategy is to inject an immunogenic composition containing antigens expressed in tumor cells into cancer patients. Tumor - associated antigens (TAAs) are expressed in tumor cells but also in embryonic cells or at low levels in normal cells. Tumor - specific antigens (TSAs) (also known as neoantigens) are only expressed in tumor cells and are usually expressed by genes mutated in tumor cells. Cancer immunotherapy relies on inducing a cytotoxic T lymphocyte (CTL) response against cancer cells.

[0005] There is a need in the art for cancer immunotherapies that induce effective TAA - or TSA - specific cellular immune responses to destroy tumor cells expressing TAAs or TSAs. Summary of the Invention

[0006] The present disclosure relates to expressing cancer antigens (TAA and / or TSA) to induce a cellular immune response against cancer cells. In some embodiments, the cancer antigen is encoded by mRNA. In some embodiments, a temperature-controlled self-replicating RNA vaccine platform is utilized. In an exemplary embodiment, the cancer antigen is expressed in a host cell by temperature-controlled self-replicating RNA (c-srRNA) to induce an effective cellular immune response against tumor cells expressing the cancer antigen. The c-srRNA is also referred to herein as temperature-sensitive self-replicating RNA (srRNAts). The c-srRNA platform described herein is a suitable vector for expressing tumor-associated antigens (TAA) or tumor-specific antigens (TSA) (also referred to as neoantigens). In some embodiments, the TAA is selected from, but not limited to, NY-ESO-1, MAGEA3, TYR, TPTE (also referred to as PTEN2), or a combination thereof. In some embodiments, the TSA is a cancer protein, such as a mutant Ras GTPase. In some embodiments, the mutant Ras GTPase is KRAS having activating substitutions at one or more of G12, G13, and Q61. The c-srRNA is used to express a fusion protein of two or more TAAs, TSAs, or a combination of TAA and TSA.

[0007] In other embodiments, the present disclosure provides a composition comprising an excipient and temperature-controlled self-replicating RNA (c-srRNA). In some embodiments, the composition comprises chitosan. In some embodiments, the chitosan is low molecular weight (about 3-5 kDa) chitosan oligosaccharide, such as chitosan oligosaccharide lactate. In some embodiments, the composition does not comprise liposomes or lipid nanoparticles. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1A schematic diagram showing an exemplary method for designing a fusion protein of one or more mutant 9-mer peptides is shown. As an example, six common mutations (G12D, G12V, G12R, G12C, G12A, and G12S) of the human KRAS proto-oncogene (GenBank accession number NM_001369786) are used. Step 1: Take nine amino acids (9-mer) from the mutant residue (D in the case of the G12D example) in both the N-terminal and C-terminal directions to identify a 17-amino acid (17-mer) peptide sequence that contains the mutant residue (in this case D) in the center. If the mutant amino acid is close to the end of the N-terminal or C-terminal, one side of the peptide sequence can be shorter than 9-mer. Step 2: Repeat the same procedure for other mutations. In this example, six 17-mer peptides are generated from six common mutations (G12D, G12V, G12R, G12C, G12A, and G12S) of the human KRAS proto-oncogene. 17-mer peptides can be identified from mutations at other positions of the same protein (e.g., KRAS) or other oncoproteins (e.g., human TP53 tumor protein p53). Step 3: Generate a fusion protein of six 17-mer peptides. Generally, no additional amino acids are inserted between the 17-mer peptide sequences. Alternatively, a non-immunogenic glycine / serine linker can be inserted between one or more 17-mer peptides. The order of each 17-mer peptide can be changed relative to the order shown as SEQ ID NO:29 in this example.

[0009] Figure 2 A schematic diagram showing an exemplary method for designing a fusion protein of one or more mutant 15-mer peptides is shown. As an example, three common mutations (Q61H, Q61K, and Q61R) of the human KRAS proto-oncogene (GenBank accession number NM_001369786) are used. Step 1: Take 15 amino acids (15-mer) from the mutant amino acid (H in the case of the Q61H example) in both the N-terminal and C-terminal directions to identify a 29-amino acid (29-mer) peptide sequence that contains the mutant amino residue (in this case H) in the center. If the mutant amino acid is close to the end of the N-terminal or C-terminal, one side of the peptide sequence can be shorter than 15-mer. Step 2: Repeat the same procedure for other mutations. In this example, three 29-mer peptides are generated from three common mutations (Q61H, Q61K, and Q61R) of the human KRAS proto-oncogene. 29-mer peptides can be obtained from mutations at other positions of the same protein (e.g., KRAS) or other proteins (e.g., human TP53 tumor protein p53). Step 3: Generate a fusion protein of 29-mer peptides. Generally, no additional amino acids are inserted between the 29-mer peptide sequences. Alternatively, a non-immunogenic glycine / serine linker can be inserted between one or more 29-mer peptides. The order of each 29-mer peptide can be changed relative to the order shown as SEQ ID NO:35 in this example.

[0010] Figure 3A Schematic diagram of an exemplary fusion protein (TSA-5109) comprising a 17-mer peptide containing a mutation from the human KRAS proto-oncogene (GenBank accession number NM_001369786). Figure 3B Amino acid sequences of 13 different 17-mer peptides derived from 13 common mutations (G12D, G12V, G12R, G12C, G12A, G12S, G13D, G13C, G13P, G13S, Q61H, Q61K, and Q61R) of the human KRAS proto-oncogene, which peptides are included in TSA-5109. Figure 3C Amino acid sequence of the TSA-5109 fusion protein containing the human CD5 signal peptide (CD5sp) sequence at the N-terminus. The amino acid sequence of the TSA-5109 fusion protein without the CD5sp sequence is shown as SEQ ID NO:17, and the amino acid sequence of the TSA-5109 fusion protein with the CD5sp sequence is shown as SEQ ID NO:18.

[0011] Figure 4A Schematic diagram of an exemplary fusion protein (TSA-5111) comprising a 29-mer peptide containing a mutation from the human KRAS proto-oncogene (GenBank accession number NM_001369786). Figure 4B Amino acid sequences of 13 different 29-mer peptides derived from 13 common mutations (G12D, G12V, G12R, G12C, G12A, G12S, G13D, G13C, G13P, G13S, Q61H, Q61K, and Q61R) of the human KRAS proto-oncogene, which peptides are included in TSA-5111. Figure 4C Amino acid sequence of the TSA-5111 fusion protein containing the human CD5 signal peptide (CD5sp) sequence at the N-terminus. The amino acid sequence of the TSA-5111 fusion protein without the CD5sp sequence is shown as SEQ ID NO:19, and the amino acid sequence of the TSA-5111 fusion protein with the CD5sp sequence is shown as SEQ ID NO:20.

[0012] Figure 5A schematic diagram showing a fusion protein comprising multiple tumor-associated antigens is presented. In some embodiments, the tumor-associated antigens are expressed by a temperature-controllable self-replicating RNA (c-srRNA). In an exemplary embodiment, the TAA-5107 antigen is a fusion protein comprising: a signal peptide sequence from human CD5 antigen (CD5-SP) as shown in SEQ NO:1; the amino acid sequence of human NY-ESO-1 protein (GenBank accession number NM_001327) as shown in SEQ NO:4; the amino acid sequence of human MAGEA3 protein (GenBank accession number NM_005362) as shown in SEQ NO:5; the amino acid sequence of human TYR protein (GenBank accession number NM_000372); and the amino acid sequence of human transmembrane phosphatase with tensin homology (TPTE) protein (GenBank accession number NM_199261). The amino acid sequence of the TAA-5107 fusion protein (without CD5 SP) is shown in SEQ ID NO:15, and the amino acid sequence of the CD5-SP plus TAA fusion protein is shown in SEQ ID NO:16.

[0013] Figure 6 A schematic diagram showing an exemplary method for stimulating an immune response against cancer antigens in a human subject is presented. The c-srRNA is functional at a permissive temperature (e.g., 30°C - 35°C), but non-functional at a non-permissive temperature (e.g., ≥37°C). The temperature at or just below the human body surface (surface body temperature), which is around 31°C - 34°C, is lower than the core body temperature of the human body, which is around 37°C. The c-srRNA is directly delivered by intradermal and subcutaneous administration to the cells of a subject at the permissive surface body temperature.

[0014] Figures 7A - 7C A schematic diagram showing the inhibition of tumor growth by the EXG-5111 vaccine, which expresses the TSA-5111 antigen in vivo, is presented. BALB / c female mice received two intradermal doses of 100 μg of EXG-5111, two weeks apart. Two weeks later (day 0), the mice received 3 x 10^5 cells of CT26 murine colon carcinoma cells (ATCC CRL-2638), which are known to have a G12D mutation in the KRAS proto-oncogene. Figure 7A A schematic diagram showing the increase in tumor size of 15 mice that received intradermal placebo (PBO) injections is presented. Mice that met the euthanasia criteria due to tumor size or ulceration were sacrificed. In most mice, the tumors grew rapidly, and by day 28 after tumor injection, only one mouse survived. Figure 7BShows the increase in tumor size of 15 mice that received intradermal injection of EXG-5111 vaccine. Tumor growth was inhibited in mice that received EXG-5111 vaccine compared to the PBO group. By day 28 after tumor injection, 7 mice survived. Figure 7C Shows a comparison of tumor growth in the PBO and EXG-5111 groups, where the mean ± SEM of each group is shown. Inhibition of tumor growth by EXG-5111 was statistically significant at days 11, 14, 22, and 25, as indicated by the asterisks. Detailed Description

[0015] Cancer immunotherapy is expected to be best achieved through immunogenic compositions that mainly rely on induction of cellular immunity (i.e., T cell-inducing vaccines involving CD8+ cytotoxic T cells and CD4+ helper T cells). The present disclosure provides mRNAs, self-replicating RNAs (srRNAs), and temperature-controllable self-replicating RNAs (c-srRNAs) encoding one or more cancer antigens such as tumor-associated antigens (TAAs) and tumor-specific antigens (TSAs, also known as neoantigens). Accordingly, the present disclosure provides a cell-mediated immunity-based platform for cancer immunotherapy. Wilms tumor protein 1 (WT1) is a tumor-associated antigen (TAA) that is expressed in a wide range of tumors but only in embryonic tissues and very limited cell types in adults. Thus, in some embodiments, the c-srRNA encodes WT1. In some embodiments, the c-srRNA encodes BIRC5 (also known as survivin). In some embodiments, the c-srRNA encodes NY-ESO-1. In some embodiments, the c-srRNA encodes MAGEA3. In some embodiments, the c-srRNA encodes PRAME. In further embodiments, the c-srRNA encodes one, two, three, four, or all five cancer antigens from the group consisting of WT1, BIRC5, NY-ESO-1, MAGEA3, and PRAME. Cell-Mediated Immunity-Based mRNA Immunotherapy Platform

[0016] The vaccine platform is described in part in Elixirgen's earlier patent application [PCT / US20 / 67506, now published as WO 2021 / 138447 A1]. This vaccine platform was optimized to induce cellular immunity, which was made possible by combining existing knowledge of vaccine biology with temperature-controllable self-replicating mRNA (c-srRNA) based on alphaviruses such as Venezuelan equine encephalitis virus (VEEV). The terms c-srRNA and srRNAts are used interchangeably throughout this disclosure, and srRNA1ts2 (described in WO 2021 / 138447 A1) is an exemplary embodiment. The c-srRNA is based on srRNA, which is also known as self-amplifying mRNA (saRNA or SAM), by incorporating small amino acid changes that confer temperature sensitivity in the alphavirus replicase. Elixirgen's c-srRNA is functional within a permissive temperature range of approximately 30°C - 35°C, but is non-functional at non-permissive temperatures of approximately 37°C or higher. It has all the benefits of the mRNA platform: no genomic integration, rapid development and deployment, simple GMP (Good Manufacturing Practice) processes, and additional advantages over the srRNA platform (i.e., the predecessor of our c-srRNA platform) compared to the mRNA platform, particularly longer expression [Johanning et al., 1995] and higher immunogenicity at lower doses [Brito et al., 2014]. However, this simple temperature-controllable feature makes it possible to bring together many of the desired features of T cell-inducing vaccines, as briefly described below.

[0017] Briefly, srRNA1ts2 is a VEEV-based temperature-sensitive self-replicating RNA replicon developed for the transient expression of heterologous proteins. Temperature sensitivity is conferred by inserting five amino acid residues within the non-structural protein 2 (nsP2) of VEEV. The nsP2 protein is a helicase / protease that together with nsP1, nsP3, and nsP4 constitutes the VEEV replicase. The disclosure of WO 2021 / 138447 A1 of Elixirgen Therapeutics, Inc. is hereby incorporated by reference. Specifically, Examples 3, Figure 12, and SEQ ID NOs. 29 - 49 of WO 2021 / 138447 A1 are hereby incorporated by reference.

[0018] Exemplary vectors include three different temperature - controllable self - replicating RNA vectors (c - srRNA) and a control self - replicating RNA vector (c - srRNA). The characteristics of srRNA suitable for use in the compositions and methods of the present disclosure are summarized in Table I. The IFN - α / β sensitivity of the parental VEEV strain has been previously reported (Spotts et al., J Viol, 72:10286 - 10291, 1998). c - srRNA1 is based on the TRD strain of VEEV, but is modified to have an A16D substitution (TC83 mutation) and a P778S substitution. c - srRNA3 is also based on the TRD strain of VEEV, but does not have the A16D and P778S substitutions. srRNA4 is based on the V198 strain of VEEV isolated from humans. All three c - srRNA vectors contain the same 5 - amino - acid insertion within the nsP2 protein of VEEV to achieve temperature controllability, as previously described (see U.S. Patent No. 11,421,248 to Ko, Examples 3, 21, and 22, incorporated herein by reference). Table I. srRNA characteristics RNA ts mutant VEEV srRNA0 No TRD c-srRNA1 Yes TRD / TC-83 c-srRNA3 Yes TRD c-srRNA4 Yes V198

[0019] The nucleotide sequences of the VEEV genome are publicly available in GenBank: the TRD strain, GenBank accession number L01442.2; and the TC - 83 strain, GenBank accession number L01443.1. The amino acid sequences of the nsP2 proteins of srRNA are disclosed herein: srRNA0 (SEQ ID NO:13); c - srRNA1 (SEQ ID NO:9); c - srRNA3 (SEQ ID NO:10); c - srRNA4 (SEQ ID NO:11); and the c - srRNA consensus sequence (SEQ ID NO:12). General Techniques and Definitions

[0020] Unless otherwise indicated, the practice of the present disclosure will employ conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art.

[0021] As used herein and in the appended claims, unless otherwise indicated, the singular forms "a", "an", and "the" include plural referents. For example, "an" excipient includes one or more excipients.

[0022] As used herein, the phrase "comprising" is open-ended, indicating that such embodiments can include additional elements. In contrast, the phrase "consisting of" is closed-ended, indicating that such embodiments do not include additional elements (except for trace impurities). The phrase "consisting essentially of" is partially closed-ended, indicating that such embodiments can further include elements that do not materially alter the basic characteristics of such embodiments.

[0023] As used herein, the term "about" when used in connection with a value encompasses from 90% to 110% of that value (e.g., when used in connection with chitosan oligosaccharides, a molecular weight of about 5,000 daltons refers to from 4,500 daltons to 5,500 daltons).

[0024] The term "antigen" refers to a substance that is recognized and specifically bound by an antibody or a T cell antigen receptor. Antigens can include peptides, polypeptides, proteins, glycoproteins, polysaccharides, complex carbohydrates, sugars, gangliosides, lipids, and phospholipids; portions thereof and combinations thereof. In the context of the present disclosure, the term "antigen" generally refers to a polypeptide or protein antigen that is at least eight amino acid residues in length and can contain one or more post-translational modifications.

[0025] The terms "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues and are not limited to a certain length unless otherwise specified. A polypeptide can contain natural amino acid residues or a combination of natural and non-natural amino acid residues. The terms also include post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, etc. In some aspects, a polypeptide can contain modifications with respect to the native or natural sequence, so long as the protein retains the desired activity (e.g., antigenicity).

[0026] As used herein, the terms "isolated" and "purified" refer to a material that has been removed from at least one component with which it is naturally associated (e.g., removed from its original environment). When used in connection with a recombinant protein, the term "isolated" refers to a protein that has been removed from the culture medium of the host cell in which the protein was produced. In some embodiments, the isolated protein (e.g., the WT1 protein) is at least 75%, 90%, 95%, 96%, 97%, 98%, or 99% pure, as determined by HPLC.

[0027] An "effective amount" or "sufficient amount" of a substance is an amount sufficient to achieve a beneficial or desired result, including a clinical outcome, and thus, an "effective amount" depends on the context in which it is applied. In the case of administering a composition of the present disclosure comprising mRNA encoding an antigen, an effective amount contains mRNA sufficient to stimulate an immune response (preferably a cellular immune response against the antigen).

[0028] The term "treating" or "treatment" of a disease refers to implementing a protocol that can include administering one or more agents to an individual (human or other individual) in an effort to alleviate the signs or symptoms of the disease. Thus, "treating" or "treatment" does not require complete alleviation of signs or symptoms, does not require a cure, and specifically includes protocols that have only a palliative effect on the individual. As used herein and as is well known in the art, "treatment" is a method for obtaining a beneficial or desired result (including a clinical result). Beneficial or desired clinical results include, but are not limited to, alleviating or improving one or more symptoms, reducing the severity of the disease, stabilizing (i.e., not worsening) the disease state, preventing the spread of the disease, delaying or slowing the progression of the disease, improving or alleviating the disease state, and remission. "Treatment" can also mean prolonging the survival time of a cancer patient compared to the expected survival time of a control patient not receiving treatment. "Palliating" a disease or disorder means that the degree and / or the undesired clinical manifestations of the disease or disorder are alleviated, and / or the time course of the progression of the disease or disorder is slowed, compared to the expected untreated outcome.

[0029] In the present disclosure, the terms "individual" and "subject" refer to a mammal. "Mammal" includes, but is not limited to, humans, non-human primates (e.g., monkeys), livestock, sport animals, rodents (e.g., mice and rats), and pets (e.g., dogs and cats). In some preferred embodiments, the subject is a human subject.

[0030] As used herein with respect to a composition comprising an mRNA encoding an antigen, the term "dose" refers to the measured portion administered (administered or received) by a subject in any one administration. Administering a composition of the present disclosure to a subject in need thereof includes administering an effective amount of the composition comprising an mRNA encoding an antigen to stimulate an immune response against the antigen in the subject.

[0031] "Stimulation" of a response or parameter includes eliciting and / or enhancing the response or parameter when compared to conditions that are otherwise identical except for the parameter of interest or alternatively when compared to another condition (e.g., increased antigen-specific cytokine secretion after administration of a composition comprising or encoding an antigen compared to administration of a control composition that does not comprise or encode the antigen). For example, "stimulation" of an immune response (e.g., a Th1 response) means an increase in the response. Depending on the parameter being measured, the increase can be from 2-fold to 200-fold or more, from 5-fold to 500-fold or more, from 10-fold to 1000-fold or more, or from 2, 5, 10, 50, or 100-fold to 200, 500, 1,000, 5,000, or 10,000-fold.

[0032] In contrast, "suppression" of a response or parameter involves a decrease and / or repression of the response or parameter when compared to a condition that is otherwise identical except for the parameter of interest or alternatively when compared to another condition. For example, "suppression" of an immune response (e.g., a Th2 response) means a decrease in the response. Depending on the parameter being measured, the decrease can be from 2-fold to 200-fold, from 5-fold to 500-fold or more, from 10-fold to 1000-fold or more, or from 2, 5, 10, 50 or 100-fold to 200, 500, 1,000, 2,000, 5,000 or 10,000-fold.

[0033] The relative terms "higher" and "lower" refer, respectively, to a measurable increase or decrease in a response or parameter when compared to a condition that is otherwise identical except for the parameter of interest or alternatively when compared to another condition. For example, "higher antibody titer" means that the antigen-reactive antibody titer as a result of administering a composition of the present disclosure comprising mRNA encoding an antigen is at least 2, 3, 4, 5, 6, 7, 8, 9 or 10-fold higher than the antigen-reactive antibody titer as a result of a control condition (e.g., administering a comparative composition that does not contain mRNA or contains a control mRNA that does not encode an antigen). Similarly, "lower antibody titer" means that the antigen-reactive antibody titer as a result of a control condition (e.g., administering a comparative composition that does not contain mRNA or contains a control mRNA that does not encode an antigen) is at least 2, 3, 4, 5, 6, 7, 8, 9 or 10-fold lower than the antigen-reactive antibody titer as a result of administering a composition of the present disclosure comprising mRNA encoding an antigen.

[0034] As used herein in connection with an agent (e.g., an RNA molecule), the term "temperature-sensitive" refers to an agent that is active at a "permissive temperature" but has reduced activity at a higher and / or lower "non-permissive temperature".

[0035] As used herein, the term "permissive temperature" refers to any temperature at which the activity of a temperature-sensitive agent of the present disclosure is induced. Generally, the permissive temperature is not the normal body temperature of a subject. The normal body temperature of a human subject is about 37°C ± 0.5°C. Depending on the temperature-sensitive agent, the permissive temperature can be a temperature that is higher or lower than the normal body temperature of the subject. In some aspects, the permissive temperature range for a temperature-sensitive agent is from 30°C to 36°C. In some embodiments, the permissive temperature is from about 31°C to about 35°C or 32°C to 34°C (33°C ± 1.0°C). In some preferred embodiments, the permissive temperature is 33°C ± 0.5°C. Thus, in some embodiments, the non-permissive temperature of a temperature-sensitive self-replicating RNA of the present disclosure is higher than 36°C. In some preferred embodiments, the non-permissive temperature is 37°C ± 0.5°C.

[0036] As used herein, the term "non-permissive temperature" refers to any temperature under which the activity of a temperature-sensitive agent of the present disclosure is not induced. The activity of a temperature-sensitive agent is not induced when it is at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, or at least 50% lower than the activity level at the optimal permissive temperature. Generally, the non-permissive temperature is the normal body temperature of the subject. Depending on the temperature-sensitive agent, the non-permissive temperature can also be a temperature that is higher (e.g., 38 °C and above) or lower (e.g., below 30 °C) than the normal body temperature of the subject.

[0037] As used herein, the term "immunization" refers to a process that increases the response of a mammalian subject to an antigen and thus improves its ability to resist or overcome infection and / or to resist disease.

[0038] The term "vaccination" as used herein refers to the introduction of a vaccine into the body of a mammalian subject.

[0039] As used herein, when used in reference to an amino acid sequence (reference polypeptide sequence), "percent amino acid sequence identity (%)", "percent identity", and "sequence identity" are defined as the percentage of amino acid residues in a candidate sequence (e.g., a subject antigen) that are identical to the amino acid residues in the reference polypeptide sequence after aligning the sequences and introducing gaps (if necessary) to achieve the maximum percent sequence identity and not considering any conservative substitutions as part of the sequence identity. The alignment for the purpose of determining the percent amino acid sequence identity can be achieved in various ways within the skill in the art, such as using publicly available computer software, such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. One of ordinary skill in the art can determine the appropriate parameters for aligning the sequences, including any algorithms required to achieve the maximum alignment over the full length of the sequences being compared.

[0040] Throughout the present disclosure, exemplary amino acid sequences are shown as sequence identifiers. Some claimed embodiments are described by reference to the percent identity they have to the exemplary amino acid sequences. When compared and aligned over a comparison window or specified region to obtain maximum correspondence, two amino acid sequences are substantially the same if they have at least 90% identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over the specified region or over their entire sequences when not specified). For the purposes of the present disclosure and the claims, the BLASTP sequence comparison algorithm with default parameters is used to align amino acid sequences to determine sequence identity.

[0041] Algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, described in Altschul et al., J Mol Biol, 215:403-410, 1990; and Altschul et al., Nucleic Acids Res. 25:3389-3402, 1977, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI) website. The algorithms involve first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that match or satisfy some positive-valued threshold score T when aligned with words of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits serve as seeds for initiating a search to find longer HSPs that contain them. The word hits are then extended in both directions along each sequence as long as the cumulative alignment score can be increased. For nucleotide sequences, parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for a mismatched residue; always <0) are used to calculate the cumulative score. For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction is halted when the cumulative alignment score falls by the quantity X from its maximum achieved value; when the cumulative score goes to zero or below due to the accumulation of one or more negative-scoring residue alignments; or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses a word length (W) of 28, an expectation value (E) of 10, M = 1, N = -2, and comparison of both strands as default settings. For amino acid sequences, the BLASTP program uses a word length (W) of 3, an expectation value (E) of 10, and the BLOSUM62 scoring matrix as default settings (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1989).

[0042] An amino acid substitution can include replacing one amino acid in a polypeptide with another amino acid. An amino acid substitution can be introduced into a target antigen, and the resulting product can be screened for a desired activity (e.g., increased stability and / or immunogenicity).

[0043] Amino acids can generally be grouped according to the following common side chain characteristics: (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basic: His, Lys, Arg; (5) Residues affecting chain orientation: Gly, Pro; and (6) Aromatic: Trp, Tyr, Phe.

[0044] Conservative amino acid substitutions will involve exchanging a member of one of these categories with another member of the same category. Non-conservative amino acid substitutions will involve exchanging a member of one of these categories with a member of another category.

[0045] As used herein, the term "excipient" refers to a compound present in a composition comprising an active ingredient (e.g., mRNA encoding an antigen). A pharmaceutically acceptable excipient is an inert pharmaceutical compound and can include, for example, solvents, fillers, buffers, tonicity regulators, and preservatives (Pramanick et al., Pharma Times, 45:65 - 77, 2013). In some embodiments, the compositions of the present disclosure comprise excipients that serve as one or more of a solvent, filler, buffer, and tonicity regulator (e.g., sodium chloride in saline can serve both as an aqueous vehicle and as a tonicity regulator). Optimized for intradermal delivery for cellular immunity

[0046] Intradermal vaccination results in durable cellular immunity and increased immunogenicity [Hickling and Jones, 2009]. Human skin (epidermis and dermis) is rich in antigen-presenting cells (APCs), including Langerhans cells and dermal dendritic cells (DCs). Intradermal vaccination is known to be 5 to 10 times more effective than subcutaneous or intramuscular vaccination because it targets APCs [Hickling and Jones, 2009], and this targeting also activates the T cell immune pathway for durable immunity. By intradermal injection, c-srRNA is mainly taken up by skin APCs, where it replicates, produces antigen, digests the antigen into peptides, and presents these peptides to T cells ( Figure 1 ). Peptides presented by this pathway stimulate MHC-I-restricted CD8+ cytotoxic T cells. In an alternative pathway, APCs also take up antigen produced by nearby skin cells. Peptides presented by this pathway stimulate MHC-II-restricted CD4+ helper T cells. Problems and solutions with intradermal injection

[0047] Here are the potential problems we have found and the solutions provided by our c-srRNA platform.

[0048] (1) A key unrecognized obstacle to the application of srRNA as an intradermal vaccine platform is that neither mRNA nor srRNA expresses antigens well at skin temperature [PCT / US20 / 67506]. Counterintuitively, the temperature of human skin (about 30°C - 35°C) is lower than human core body temperature (about 37°C); this means that vectors and platforms developed at 37°C are not optimal for intradermal injection. An innovation of our c-srRNA platform is that it strongly expresses antigens at skin temperature [PCT / US20 / 67506]. Additionally, this temperature control also minimizes the safety risk caused by the unintended systemic distribution of c-srRNA, because once the temperature of c-srRNA rises above its permissive threshold (when it moves closer to the body core), it becomes inactivated. In other words, compared with mRNA and srRNA, the c-srRNA platform best expresses antigens for intradermal injection, and it additionally has a safety feature: the ability of the vector to spread and produce in other regions of the subject's body is restricted or inactivated.

[0049] (2) Another challenge for intradermal vaccination is the lack of suitable additives. Since adjuvants such as aluminum salts and oil-in-water are too locally reactogenic when delivered by the intradermal route, no adjuvant has been incorporated into clinically approved intradermal vaccines, resulting in lower immunogenicity [Hickling and Jones, 2009]. The lipid nanoparticles (LNPs) of mRNA and srRNA vaccines for intramuscular administration are also oil-in-water, which may cause skin reactogenicity and increase the risk of allergic reactions to LNP components such as PEG. Our c-srRNA platform is the solution to this problem because it is injected as naked c-srRNA (without LNP, without adjuvant). First, the self-replication of RNA within cells (especially APCs) induces strong innate immunity, which replaces the main function of adjuvants. Second, the literature and our own data demonstrate that, especially for intradermal injection, naked mRNA / srRNA is equally effective in generating antigens compared to electroporation of mRNA / srRNA [Johansson et al., 2012] and the combination of mRNA / srRNA and LNP [Golombek et al., 2018].

[0050] (3) The third challenge is the limited number of precedents for intradermal vaccines. Only Bacillus Calmette-Guérin has been routinely administered intradermally. One way we reduce the barriers to intradermal injection is to use specialized devices such as the MicronJet600 (NanoPass) and Immucise (Terumo), which now enable simple, consistent intradermal injection. These devices are also good candidates for large-scale production and deployment. However, due to the relatively high cost of these special devices, intradermal injection using the Mantoux technique with standard needles and syringes is also an option. Design of suitable antigens

[0051] Tumor-associated antigens (TAAs) are expressed in tumor cells, but also in embryonic cells or at low levels in normal cells. The National Cancer Institute has selected 75 cancer antigens suitable as targets for cancer therapy (Cheever et al., 2009). For example, Wilms' tumor protein 1 (WT1) was ranked as the most promising among the 75 cancer antigens identified by the National Cancer Institute (Cheever et al., 2009). WT1 is expressed in a wide range of tumors, but only in embryonic tissues and very limited cell types in adults. For example, WT1 is expressed in most leukemias (AML, ALL), pancreatic cancer, lung cancer, and glioblastoma. Other TAAs can be used as one or more antigens for cancer vaccines based on the c-srRNA platform described herein. Multiple TAAs expressed as fusion proteins (Example 3) or multiple TAAs expressed individually can also be used.

[0052] Recently, genomic sequencing of patient-derived tumor cells has become common. Such efforts typically identify protein products or peptides that are unique to them due to mutations in the tumor genome. These tumor-specific antigens (TSAs) (also called neoantigens) are ideal targets for cancer vaccines. A single TSA or a fusion of more than one TSA can be used as an antigen for cancer vaccines based on the c-srRNA platform described herein (Examples 1 and 2).

[0053] Most tumor-specific antigens (TSAs) contain one or more specific mutations, usually a single amino acid change compared to the normal protein. For example, the change from glycine (G) to aspartic acid (D) at position 12 of KRAS (G12D) is common in human cancers. The challenge here is how to design such an antigen that elicits strong T cell immunity against the specific mutation rather than the wild type (normal protein), especially CD8+ cytotoxic lymphocytes. To activate CD8+ cytotoxic lymphocytes, dendritic cells need to present MHC class I molecules loaded with short peptides (usually 9-mers, i.e., 9 amino acids). The design of a 17-mer peptide containing the mutated amino acid in the center is shown in Figure 1 Step 1. In this way, any 9-mer peptide processed from the 17-mer peptide contains the mutated amino acid, and thus, these 9-mer peptides are specific for the mutant protein (neoantigen). If the mutated amino acid is close to the end of the N-terminus or C-terminus, one side of the sequence can be shorter than 9-mers. Repeat this process for other mutations to identify multiple 17-mer sequences containing the mutation of interest, as shown in Figure 1as shown in Step 2. 17-mer peptide sequences can be identified from other mutations at the same position of the same oncoprotein (e.g., G12D, G12V, and G12R of the KRAS protein), mutations at other positions of the same oncoprotein (e.g., Q61H, Q61K of the KRAS protein), and / or mutations in other oncoproteins (e.g., R175H, R248Q, and R273H of the human TP53 tumor protein p53). Finally, the 17-mer peptides are ligated or expressed as a recombinant fusion protein, such as Figure 1 as shown in Step 3. Generally, no additional amino acids are inserted between the 17-mer peptide sequences. Alternatively, a non-immunogenic glycine / serine linker can be inserted between one or more 17-mer peptide sequences. The order of each 17-mer peptide sequence can be altered relative to the exemplary fusion protein described in Example 1. Thus, an exemplary embodiment includes an mRNA molecule encoding a KRAS polyprotein, wherein each neoantigen peptide of the polyprotein is 17 amino acids in length. However, in a further embodiment, the mRNA molecule can encode a KRAS polyprotein, wherein each neoantigen peptide of the polyprotein is from 16 to 24 amino acids in length (16-mer, 17-mer, 18-mer, 19-mer, 20-mer, 21-mer, 22-mer, 23-mer, or 24-mer).

[0054] Effective activation of CD8+ cytotoxic lymphocytes may generally require activation of CD4+ helper T cells. For this purpose, dendritic cells also need to present MHC class II molecules loaded with longer peptides (usually 15-mers, i.e., 15 amino acids). The design of 29-mer peptides containing the mutated amino acid in the center is shown in Figure 2 Step 1. In this way, any 15-mer peptide processed from the 29-mer peptide contains the mutated amino acid and, thus, these 15-mer peptides are specific for the mutant protein (neoantigen). Peptides processed from the 29-mer include 9-mer peptides that are loaded on MHC class I molecules. Many of these 9-mer peptides include the mutated amino acid, but some 9-mer peptides are wild-type. If the mutated amino acid is near the N-terminal or C-terminal end, one side of the sequence can be shorter than 15-mers. This process is repeated for other mutations to identify multiple 29-mer sequences containing the mutation of interest, such as Figure 2 as shown in Step 2. 29-mer peptide sequences can be identified from other mutations at the same position of the same oncoprotein (e.g., G12D, G12V, and G12R of the KRAS protein), mutations at other positions of the same oncoprotein (e.g., Q61H, Q61K of the KRAS protein), and / or mutations in other oncoproteins (e.g., R175H, R248Q, and R273H of the human TP53 tumor protein p53). Finally, the 29-mer peptides are ligated or expressed as a fusion protein, such as Figure 2As shown in Step 3. Generally, no additional amino acids are inserted between the 29-mer peptide sequences. Alternatively, a non-immunogenic glycine / serine linker can be inserted between one or more 29-mer peptide sequences. The order of each 29-mer peptide sequence can be altered relative to the exemplary fusion protein described in Example 2. Thus, exemplary embodiments include mRNA molecules encoding a KRAS multi-protein, wherein each neoantigen peptide of the multi-protein is 29 amino acids in length. However, in further embodiments, the mRNA molecule can encode a KRAS multi-protein, wherein each neoantigen peptide of the multi-protein is from 26 to 34 amino acids in length (26-, 27-, 28-, 29-, 30-, 31-, 32-, 33-, or 34-mers). Chitosan enhances gene expression in vivo

[0055] RNase inhibitor (a protein purified from human placenta) slightly enhances the immunogenicity against the antigen encoded on c-srRNA, most likely by enhancing the in vivo expression of the antigen from c-srRNA when intradermally injected into mice (see, for example, FIG. 25C of WO 2021 / 138447A1). The RNase inhibitor can protect c-srRNA from RNase-mediated degradation in vivo. However, it is desirable to find alternative agents that can enhance the expression of a gene of interest (GOI) in vivo for therapeutic purposes, as it is difficult to use a protein-based RNase inhibitor as an excipient in an injectable product.

[0056] It has been shown that low molecular weight chitosan (molecular weight approximately 6 kDa) can inhibit the activity of RNase, with an inhibition constant in the range of 30 - 220 nM (Yakovlev et al., Biochem Biophys Res Commun, 357(3):584 - 8, 2007). Two different chitosan oligomers were recently tested: chitosan oligomer (CAS number 9012 - 76 - 4; molecular weight ≤5 kDa, deacetylation ≥75%; Heppe Medical Chitosan GmbH: product number 44009) and chitosan oligosaccharide lactylation (CAS number 148411 - 57 - 8; molecular weight approximately 5 kDa, deacetylation >90%; Sigma - Aldrich: product number 523682). Surprisingly, it was found that even very low levels of chitosan oligomer, as low as 0.001 μg / mL (approximately 0.2 nM: approximately 1 / 100 of the inhibition constant found by Yakovlev et al., ibid, 2007) were able to enhance the expression of luciferase encoded on c-srRNA by approximately 10-fold (data not shown). Chitosan oligomer up to 0.5 μg / mL and chitosan oligosaccharide lactylation at 0.1 μg / mL achieved a similar enhancement of GOI expression.

[0057] Chitosan has been used as a delivery vehicle for nucleotides (DNA and RNA) because it can form complexes or nanoparticles (reviewed in Buschmann et al., Adv Drug Deliv Rev, 65(9):1234 - 70, 2013; and Cao et al., Drugs, 17:381, 2019). However, it is noteworthy that the enhancement of GOI expression by chitosan oligomers is unlikely to be mediated by nanoparticle or complex formation of c-srRNA and chitosan oligomers. First, such a low concentration of chitosan oligomers does not allow complex formation with RNA. Second, chitosan oligomers were added to c-srRNA immediately before intradermal injection and thus, there was not enough time to form complexes.

[0058] Since chitosan oligomers enhance GOI expression in vivo at much lower concentrations compared to the effective concentrations as RNase inhibitors in vitro (Yakovlev et al., ibid, 2007), it is conceivable that this enhanced GOI expression by chitosan oligomers may not be mediated by its RNase inhibitory mechanism. For example, chitosan oligomers can promote the incorporation of c-srRNA into cells, thereby enhancing the expression of GOI from c-srRNA. Nevertheless, this surprising finding should provide effective means to enhance the therapeutic expression of GOI encoded on c-srRNA in vivo. Enumerated embodiments 1. An RNA molecule comprising an open reading frame (ORF) encoding a fusion protein, wherein the ORF comprises, from 5' to 3': (i) a nucleotide sequence encoding a mammalian signal peptide; and (ii) a nucleotide sequence encoding a cancer antigen, wherein the cancer antigen comprises a KRAS polyprotein and the amino acid sequence of the KRAS polyprotein comprises: a) a first segment comprising: MTEYKLVVVGAX1GVGKSALTIQLIQNXaXbXcXdXeXf MTEYKLVVVGAX2GVGKSALTIQLIQNXaXbXcXdXeXf MTEYKLVVVGAX3GVGKSALTIQLIQNXaXbXcXdXeXf MTEYKLVVVGAX4GVGKSALTIQLIQNXaXbXcXdXeXf MTEYKLVVVGAX5GVGKSALTIQLIQNXaXbXcXdXeXf MTEYKLVVVGAX6GVGKSALTIQLIQNXaXbXcXdXeXf (SEQ ID NO:53); b) A second segment, said second segment comprising: MTEYKLVVVGAGX 10 VGKSALTIQLIQNHXaXbXcXdXeXf MTEYKLVVVGAGX 11 VGKSALTIQLIQNHXaXbXcXdXeXf MTEYKLVVVGAGX 12 VGKSALTIQLIQNHXaXbXcXdXeXf MTEYKLVVVGAGX 13 VGKSALTIQLIQNHXaXbXcXdXeXf (SEQ ID NO:54); and c) A third segment, said third segment comprising: DGETCLLDILDTAGX7EEYSAMRDQYMRTGXaXbXcXdXeXf DGETCLLDILDTAGX8EEYSAMRDQYMRTGXaXbXcXdXeXf DGETCLLDILDTAGX9EEYSAMRDQYMRTGXaXbXcXdXeXf (SEQ ID NO:55), wherein said first segment, said second segment and said third segment are arranged in any order, wherein X1, X2, X3, X4, X5 and X6 are independently selected from D, V, R, C, A and S, wherein X7, X8 and X9 are independently selected from H, K and R, wherein X 10 , X 11 , X 12 and X 13 are independently selected from D, C, P and S, and wherein Xa, Xb, Xc, Xd, Xe and Xf are independently selected from G, S and absent. 2. The RNA molecule according to embodiment 1, wherein the amino acid sequence of the KRAS multi-protein comprises SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 32, SEQ ID NO: 33, and SEQ ID NO: 34. 3. The RNA molecule according to embodiment 2, wherein the amino acid sequence of the KRAS multi-protein comprises residues 25 - 375 of SEQ ID NO: 20. 4. An RNA molecule, the RNA molecule comprising an open reading frame (ORF) encoding a fusion protein, wherein the ORF comprises, from 5' to 3': (i) a nucleotide sequence encoding a mammalian signal peptide; and (ii) a nucleotide sequence encoding a cancer antigen, wherein the cancer antigen comprises a KRAS multi-protein, and the amino acid sequence of the KRAS multi-protein comprises: a) a first segment, the first segment comprising: YKLVVVGAX1GVGKSALTXaXbXcXdXeXfYKLVVVGAX2GV GKSALTXaXbXcXdXeXf YKLVVVGAX3GVGKSALTXaXbXcXdXeXfYKLVVVGAX4GV GKSALTXaXbXcXdXeXf YKLVVVGAX5GVGKSALTXaXbXcXdXeXfYKLVVVGAX6GV GKSALT(SEQ ID NO: 56), b) a second segment, the second segment comprising: KLVVVGAGX 10 VGKSALTIXaXbXcXdXeXfKLVVVGAGX 11 VG KSALTIXaXbXcXdXeXf KLVVVGAGX 12 VGKSALTIXaXbXcXdXeXfKLVVVGAGX 13 VG KSALTI(SEQ ID NO: 57), and c) a third segment, the third segment comprising: LDILDTAGX7HEEYSAMRDXaXbXcXdXeXfLDILDTAGX8HEE YSAMRDXaXbXcXdXeXf LDILDTAGX9HEEYSAMRD(SEQ ID NO:58), wherein the first segment, the second segment, and the third segment are arranged in any order, wherein X1, X2, X3, X4, X5, and X6 are independently selected from D, V, R, C, A, and S, wherein X7, X8, and X9 are independently selected from H, K, and R, wherein X 10 , X 11 , X 12 and X 13 are independently selected from D, C, P, and S, and wherein Xa, Xb, Xc, Xd, Xe, and Xf are independently selected from G, S, and absent. 5. The RNA molecule according to embodiment 4, wherein the amino acid sequence of the KRAS multi-protein comprises SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, and SEQ ID NO:42. 6. The RNA molecule according to embodiment 5, wherein the amino acid sequence of the KRAS multi-protein comprises residues 25-245 of SEQ ID NO:18. 7. An RNA molecule comprising an open reading frame (ORF) encoding a fusion protein, wherein the ORF comprises, from 5' to 3': (i) a nucleotide sequence encoding a mammalian signal peptide; and (ii) a nucleotide sequence encoding a cancer antigen, wherein the cancer antigen comprises a KRAS multi-protein, and the amino acid sequence of the KRAS multi-protein comprises: YKLVVVGAX1GVGKSALTXaXbXcXdXeXfYKLVVVGAX2GV GKSALTXaXbXcXdXeXf YKLVVVGAX3GVGKSALTXaXbXcXdXeXfYKLVVVGAX4GV GKSALTXaXbXcXdXeXf YKLVVVGAX5GVGKSALTXaXbXcXdXeXfYKLVVVGAX6GVGKSALT (SEQ ID NO:56), wherein X1, X2, X3, X4, X5 and X6 are independently selected from D, V, R, C, A and S, and wherein Xa, Xb, Xc, Xd, Xe and Xf are independently selected from G, S and absent. 8. The RNA molecule according to embodiment 7, wherein the amino acid sequence of the KRAS multi-protein comprises SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27 and SEQ ID NO:28. 9. The RNA molecule according to embodiment 8, wherein the amino acid sequence of the KRAS multi-protein comprises SEQ ID NO:29. 10. An RNA molecule comprising an open reading frame (ORF) encoding a fusion protein, wherein the ORF comprises, from 5' to 3': (i) a nucleotide sequence encoding a mammalian signal peptide; and (ii) a nucleotide sequence encoding a cancer antigen, wherein the cancer antigen comprises a KRAS multi-protein, and the amino acid sequence of the KRAS multi-protein comprises: DGETCLLDILDTAGX7EEYSAMRDQYMRTGXaXbXcXdXeXf DGETCLLDILDTAGX8EEYSAMRDQYMRTGXaXbXcXdXeXf DGETCLLDILDTAGX9EEYSAMRDQYMRTGXaXbXcXdXeXf (SEQ ID NO:55), wherein X7, X8 and X9 are independently selected from H, K and R, and wherein Xa, Xb, Xc, Xd, Xe and Xf are independently selected from G, S and absent. 11. The RNA molecule according to embodiment 10, wherein the amino acid sequence of the KRAS multi-protein comprises SEQ ID NO:32, SEQ ID NO:33 and SEQ ID NO:34. 12. The RNA molecule according to embodiment 11, wherein the amino acid sequence of the KRAS multi-protein comprises SEQ ID NO:35. 13. An RNA molecule, the RNA molecule comprising an open reading frame (ORF) encoding a fusion protein, wherein the ORF comprises, from 5' to 3': (i) a nucleotide sequence encoding a mammalian signal peptide; and (ii) a nucleotide sequence encoding a cancer antigen, wherein the cancer antigen comprises a KRAS polyprotein, and the amino acid sequence of the KRAS polyprotein comprises: KLVVVGAGX 10 VGKSALTIXaXbXcXdXeXfKLVVVGAGX 11 VG KSALTIXaXbXcXdXeXf KLVVVGAGX 12 VGKSALTIXaXbXcXdXeXfKLVVVGAGX 13 VG KSALTI (SEQ ID NO:57), wherein X 10 、X 11 、X 12 and X 13 are independently selected from D, C, P, and S, and wherein Xa, Xb, Xc, Xd, Xe, and Xf are independently selected from G, S, and absent. 14. The RNA molecule according to embodiment 13, wherein the amino acid sequence of the KRAS polyprotein comprises SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:39. 15. An RNA molecule, the RNA molecule comprising an open reading frame (ORF) encoding a fusion protein, wherein the ORF comprises, from 5' to 3': (i) a nucleotide sequence encoding a mammalian signal peptide; and (ii) a nucleotide sequence encoding a cancer antigen, wherein the cancer antigen comprises a KRAS polyprotein, and the amino acid sequence of the KRAS polyprotein comprises: LDILDTAGX7HEEYSAMRDXaXbXcXdXeXfLDILDTAGX8HEEYSAMRDXaXbXcXdXeXf LDILDTAGX9HEEYSAMRD (SEQ ID NO:58), wherein X7, X8, and X9 are independently selected from H, K, and R, and wherein Xa, Xb, Xc, Xd, Xe, and Xf are independently selected from G, S, and absent. 16. The RNA molecule according to embodiment 15, wherein the amino acid sequence of the KRAS multi-protein comprises SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO: 42. 17. The RNA molecule according to embodiment 8, embodiment 14, or embodiment 16, wherein the amino acid sequence of the KRAS multi-protein comprises residues 25-245 of SEQ ID NO: 18. 18. An RNA molecule comprising an open reading frame (ORF) encoding a fusion protein, wherein the ORF comprises, from 5' to 3': (i) a nucleotide sequence encoding a mammalian signal peptide; and (ii) a nucleotide sequence encoding a cancer antigen, wherein the cancer antigen comprises a KRAS multi-protein, and the amino acid sequence of the KRAS multi-protein comprises: MTEYKLVVVGAX1GVGKSALTIQLIQNXaXbXcXdXeXf MTEYKLVVVGAX2GVGKSALTIQLIQNXaXbXcXdXeXf MTEYKLVVVGAX3GVGKSALTIQLIQNXaXbXcXdXeXf MTEYKLVVVGAX4GVGKSALTIQLIQNXaXbXcXdXeXf MTEYKLVVVGAX5GVGKSALTIQLIQNXaXbXcXdXeXf MTEYKLVVVGAX6GVGKSALTIQLIQNXaXbXcXdXeXf (SEQ ID NO: 53), wherein X1, X2, X3, X4, X5, and X6 are independently selected from D, V, R, C, A, and S, and wherein Xa, Xb, Xc, Xd, Xe, and Xf are independently selected from G, S, and absent. 19. The RNA molecule according to embodiment 18, wherein the amino acid sequence of the KRAS multi-protein comprises SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, and SEQ ID NO: 48. 20. An RNA molecule comprising an open reading frame (ORF) encoding a fusion protein, wherein the ORF comprises, from 5' to 3': (i) a nucleotide sequence encoding a mammalian signal peptide; and (ii) a nucleotide sequence encoding a cancer antigen, wherein the cancer antigen comprises a KRAS polyprotein, and the amino acid sequence of the KRAS polyprotein comprises: MTEYKLVVVGAGX 10 VGKSALTIQLIQNHXaXbXcXdXeXf MTEYKLVVVGAGX 11 VGKSALTIQLIQNHXaXbXcXdXeXf MTEYKLVVVGAGX 12 VGKSALTIQLIQNHXaXbXcXdXeXf MTEYKLVVVGAGX 13 VGKSALTIQLIQNHXaXbXcXdXeXf(SEQ ID NO:54), wherein X 10 , X 11 , X 12 and X 13 are independently selected from D, C, P, and S, and wherein Xa, Xb, Xc, Xd, Xe, and Xf are independently selected from G, S, and absent. 21. The RNA molecule according to embodiment 20, wherein the amino acid sequence of the KRAS polyprotein comprises SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, and SEQ ID NO:52. 22. The RNA molecule according to embodiment 11, embodiment 19, or embodiment 21, wherein the amino acid sequence of the KRAS polyprotein comprises residues 25 - 375 of SEQ ID NO:20. 23. The RNA molecule according to any one of embodiments 1 - 22, wherein the mammalian signal peptide is a signal peptide of a surface protein expressed in mammalian antigen - presenting cells. 24. The RNA molecule according to embodiment 23, wherein the mammalian signal peptide is a CD5 signal peptide, and the amino acid sequence of the CD5 signal peptide comprises SEQ ID NO:1 or an amino acid sequence that is at least 90% or 95% identical to SEQ ID NO:1. 25. The RNA molecule according to any one of embodiments 1 - 24, the RNA molecule comprises at least one modified nucleoside, optionally wherein the at least one modified nucleoside comprises pseudouridine. 26. A DNA template for an RNA molecule according to any one of embodiments 1-25, optionally wherein a first restriction enzyme cleavage site is present upstream of the nucleotide sequence encoding the mammalian signal peptide, and a second restriction enzyme cleavage site is present downstream of the nucleotide sequence encoding the cancer antigen. 27. An expression vector comprising the DNA template according to embodiment 26. 28. A host cell comprising the expression vector according to embodiment 27. 29. The RNA molecule according to any one of embodiments 1-25, wherein the RNA molecule is a self-replicating RNA. 30. A composition for stimulating an immune response against a cancer antigen in a mammalian subject, the composition comprising an excipient and the temperature-sensitive self-replicating RNA according to embodiment 29, wherein the self-replicating RNA is a temperature-sensitive RNA further comprising an alphavirus replicon lacking a viral structural protein coding region, and wherein the temperature-sensitive self-replicating RNA is capable of expressing the fusion protein at a permissive temperature but not at a non-permissive temperature. 31. A composition for stimulating an immune response against a cancer antigen in a mammalian subject, the composition comprising an excipient and a temperature-sensitive self-replicating RNA, the temperature-sensitive self-replicating RNA comprising an open reading frame (ORF) encoding a fusion protein and an alphavirus replicon lacking a viral structural protein coding region, wherein the ORF comprises, from 5' to 3': (i) a nucleotide sequence encoding a mammalian signal peptide; and (ii) a nucleotide sequence encoding a cancer antigen, wherein the temperature-sensitive self-replicating RNA is capable of expressing the fusion protein at a permissive temperature but not at a non-permissive temperature, and the cancer antigen comprises NY-ESO-1 antigen, MAGEA3 antigen, TYR antigen, and TPTE antigen. 32. The composition according to embodiment 31, wherein the mammalian signal peptide is a signal peptide of a surface protein expressed in mammalian antigen-presenting cells. 33. The composition according to embodiment 32, wherein the mammalian signal peptide is a CD5 signal peptide, and the amino acid sequence of the CD5 signal peptide comprises SEQ ID NO:1 or an amino acid sequence that is at least 90% or 95% identical to SEQ ID NO:1. 34. The composition according to embodiment 32, wherein the amino acid sequence of the fusion protein comprises SEQ ID NO:16 or an amino acid sequence that is at least 90% or 95% identical to SEQ ID NO:16. 35. A composition according to any one of embodiments 30 - 34, wherein the alphavirus is selected from Venezuelan equine encephalitis virus, Sindbis virus, and Semliki Forest virus. 36. The composition according to embodiment 35, wherein the alphavirus is Venezuelan equine encephalitis virus. 37. A composition according to any one of embodiments 30 - 36, wherein the alphavirus replicon comprises a non - structural protein - coding region having an insertion of 12 - 18 nucleotides, the insertion resulting in the expression of non - structural protein 2 (nsP2), and the non - structural protein 2 comprises 4 to 6 additional amino acids between β - sheet 5 and β - sheet 6 of the nsP2. 38. The composition according to embodiment 37, wherein the additional amino acids comprise the sequence of SEQ ID NO:14 (TGAAA). 39. The composition according to embodiment 38, wherein the amino acid sequence of the nsP2 comprises SEQ ID NO:12. 40. The composition according to embodiment 39, wherein the amino acid sequence of the nsP2 comprises a sequence selected from SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11. 41. The composition according to embodiment 40, wherein the amino acid sequence of the nsP2 comprises SEQ ID NO:11. 42. A composition according to any one of embodiments 30 - 41, wherein the permissive temperature is from 30°C to 36°C, or 31°C to 35°C, or 32°C to 34°C, or 33°C ± 0.5°C, and the non - permissive temperature is 37°C ± 0.5°C, optionally wherein the permissive temperature is from 31°C to 35°C, and the non - permissive temperature is at least 37°C ± 0.5°C. 43. A composition according to any one of embodiments 30 - 42, wherein the composition does not comprise lipid nanoparticles. 44. A composition according to any one of embodiments 30 - 43, wherein the composition further comprises chitosan. 45. A method for stimulating an immune response against a cancer antigen in a mammalian subject, the method comprising administering to the mammalian subject a composition according to any one of embodiments 30 - 44 to stimulate an immune response against the cancer antigen in the mammalian subject. 46. The method according to embodiment 45, wherein the composition is administered intradermally. 47. The method according to embodiment 45 or embodiment 46, wherein the immune response comprises a cellular immune response reactive with mammalian cells expressing the cancer antigen. 48. The method according to embodiment 47, wherein the cellular immune response comprises one or both of a cancer antigen-specific cytotoxic T lymphocyte response and a cancer antigen-specific helper T lymphocyte response. 49. The method according to embodiment 48, wherein the immune response further comprises a humoral immune response reactive with the cancer antigen. 50. The method according to any one of embodiments 45-49, wherein the mammalian subject is a human subject. 51. A kit comprising: (i) A composition according to any one of embodiments 30-44; and (ii) A device for intradermal delivery of the composition to a mammalian subject. 52. The kit according to embodiment 51, wherein the device comprises a syringe and a needle. 53. A method for expressing a fusion protein, the method comprising contacting mammalian cells with an RNA molecule according to any one of embodiments 1-25. 54. The method according to embodiment 53, wherein the contacting is in vitro. 55. The method according to embodiment 53, wherein the contacting is in vivo. 56. A method for treating cancer, the method comprising administering to a mammalian subject in need thereof an effective amount of a composition according to any one of embodiments 30-44 to treat the cancer. 57. The method according to embodiment 56, wherein the cells of the cancer express a KRAS oncogene comprising a substitution at one or more of positions 12, 13, and 61 of KRAS. 58. The method according to embodiment 56, wherein the cells of the cancer express one or more of the NY-ESO-1 antigen, the MAGEA3 antigen, the TYR antigen, and the TPTE antigen. 59. The method according to any one of embodiments 56-58, wherein the composition is administered intradermally. 60. A fusion protein encoded by an RNA molecule according to any one of embodiments 1-24, or a mature form of the fusion protein after cleavage of the signal peptide. Examples

[0059] Abbreviations: APC (antigen-presenting cell); BIRC5 (baculoviral IAP repeat-containing protein 5 or survivin); GOI (gene of interest); IL-4 (interleukin-4); IFN-γ (interferon γ); MAGEA3 (melanoma-associated antigen 3); ORF (open reading frame); PBO (placebo); NY-ESO-1 (New York esophageal squamous cell carcinoma protein 1 or CTAG1B); PRAME (preferentially expressed antigen of melanoma); SFC (spot-forming cell); srRNAts (temperature-sensitive self-replicating RNA = c-srRNA temperature-controllable self-replicating RNA); TAA (tumor-associated antigen); TPTE (transmembrane phosphatase with tensin homology); TSA (tumor-specific antigen); TYR (tyrosinase); and WT1 (Wilms tumor protein 1). Example 1. Immunotherapy against tumors expressing KRAS mutations

[0060] This example describes the generation of a fusion protein containing multiple KRAS substitutions based on the design principle shown in Figure 1 . The KRAS protein with substitutions at one or more of positions 12, 13, and 61 is an exemplary tumor-specific antigen (TSA). Materials and Methods

[0061] BALB / c inbred female mice.

[0062] EXG-5109 mRNA was produced by in vitro transcription of a plasmid containing a temperature-controllable self-replicating RNA expression cassette (c-srRNA3), which encodes a fusion protein (TSA-5109) that contains 13 different 17-mer peptides derived from 13 common mutations (G12D, G12V, G12R, G12C, G12A, G12S, G13D, G13C, G13P, G13S, Q61H, Q61K, and Q61R) of the human KRAS proto-oncogene. A schematic diagram of the fusion protein is shown in Figure 3A . The amino acid sequences of the 17-mer peptides are shown in Figure 3B , and the amino acid sequence of the fusion protein containing the human CD5 signal peptide is shown in Figure 3C and as shown in SEQ ID NO:18.

[0063] Wild-type and mutant peptides shown in Table 1-1 were used to restimulate T cells in splenocyte samples obtained from immunized mice. Table 1-1. Wild-type and mutant 25-mer KRAS peptides Peptide Sequence SEQ ID NO: G12G wild type MTEYKLVVVGAGGVGKSALTIQLIQ 59 G12D substitution MTEYKLVVVGADGVGKSALTIQLIQ 60 G12V substitution MTEYKLVVVGAVGVGKSALTIQLIQ 61 G12C substitution MTEYKLVVVGACGVGKSALTIQLIQ 62

[0064] The CT26 murine colon cancer cell line (ATCC CRL-2638) is derived from the BALB / c mouse strain and is known to have a G12D mutation in the KRAS proto-oncogene. The murine KRAS protein sequence is identical to the human KRAS protein in this region and thus the EXG-5109 vaccine developed for humans can be tested in mice. CT26 cells were injected into BALB / c mice to form syngeneic tumors. Placebo (PBO), 5 μg or 25 μg of the EXG-5109 mRNA vaccine was administered intradermally. Subsequently, tumor size was measured. Results and Conclusion

[0065] It is expected that the intradermally administered EXG-5109 mRNA immunotherapeutic agent elicits a strong cellular immune response against the mutant KRAS protein that contains substitutions at positions 12, 13, and / or 61 of human KRAS. In the ELISpot assay, splenocytes from immunized mice are expected not to respond to the G12G wild-type peptide but to respond to the G12D, G12V, and G12C mutant peptides. In addition, it is expected that the intradermally administered EXG-5109 mRNA immunotherapeutic agent inhibits tumor growth of CT26 murine colon cancer cells in a syngeneic cancer mouse model. Example 2. Immunotherapy against tumors expressing KRAS mutations

[0066] This example describes the generation of a fusion protein containing multiple KRAS substitutions based on the design principle shown in Figure 2 KRAS proteins with substitutions at one or more of positions 12, 13, and 61 are exemplary tumor-specific antigens (TSAs). Materials and Methods

[0067] BALB / c inbred female mice.

[0068] EXG-5111 mRNA was generated by in vitro transcription of a plasmid containing a temperature-controllable self-replicating RNA expression cassette (c-srRNA3) that encodes a fusion protein (TSA-5111) containing 13 different peptides (each 26 - 29 amino acids in length) derived from 13 common mutations (G12D, G12V, G12R, G12C, G12A, G12S, G13D, G13C, G13P, G13S, Q61H, Q61K, and Q61R) of the human KRAS proto-oncogene. A schematic of the fusion protein is shown in Figure 4A The amino acid sequences of the 26 - 29-mer peptides are shown in Figure 4B and the amino acid sequence of the fusion protein containing the human CD5 signal peptide is shown in Figure 4C and as shown in SEQ ID NO:20.

[0069] The wild-type and mutant peptides shown in Table 1-1 were used to restimulate T cells in splenocyte samples obtained from immunized mice.

[0070] The CT26 murine colon cancer cell line (ATCC CRL-2638) is derived from the BALB / c mouse strain and is known to have a G12D mutation in the KRAS proto-oncogene. The murine KRAS protein sequence is identical to the human KRAS protein in this region and thus the EXG-5111 vaccine developed for humans can be tested in mice. CT26 cells were injected into BALB / c mice to form syngeneic tumors. Placebo (PBO), 5 μg or 25 μg of the EXG-5111 mRNA vaccine was administered intradermally. Subsequently, tumor size was measured. Results and Conclusions

[0071] It was expected that the intradermally injected EXG-5111 immunotherapeutic agent would elicit a strong cellular immune response against mutant KRAS proteins that contain substitutions at positions 12, 13, and / or 61 of human KRAS. In the ELISpot assay, splenocytes from immunized mice were expected not to respond to the G12G wild-type peptide but to respond to the G12D, G12V, and G12C mutant peptides.

[0072] As expected, the intradermally injected EXG-5111 mRNA immunotherapeutic agent inhibited tumor growth of CT26 murine colon cancer cells in a syngeneic cancer mouse model. BALB / c female mice received two intradermal doses of 100 μg of EXG-5111, two weeks apart. Two weeks later (day 0), the mice received 3 x 10^5 cells of CT26 murine colon cancer cells (ATCC CRL-2638), which are known to have a G12D mutation in the KRAS proto-oncogene. Figure 7A The increase in tumor size (volume) of 15 mice that received intradermal placebo (PBO) injections is shown. Mice that met the euthanasia criteria due to tumor size or ulceration were sacrificed. Overall, in placebo-treated mice, tumors grew rapidly and by day 28 after tumor injection, only one mouse survived. Figure 7B The increase in tumor size (volume) of 15 mice that received intradermal EXG-5111 vaccine injections is shown. Compared to PBO, tumor growth was inhibited and slower in mice that received the EXG-5111 vaccine. By day 28 after tumor injection, 7 mice survived. Figure 7C A comparison of the PBO and EXG-5111 groups is shown, where the mean ± SEM of each group is shown in the figure and the number of surviving mice in each group is shown below. The inhibition of tumor growth by EXG-5111 was statistically significant at days 11, 14, 22, and 25. Example 3. Immunotherapy for tumors expressing multiple tumor-associated antigens (TAAs)

[0073] This example describes the evaluation of whether intradermal injection of c-srRNA can induce an effective cellular immune response against TAAs of a fusion protein in BALB / c mice, where the c-srRNA encodes a fusion protein (TAA-5107) containing the human CD5 signal peptide, NY-ESO-1, MAGEA3, TYR, and TPTE. Materials and Methods

[0074] BALB / c inbred female mice.

[0075] Figure 5 A schematic diagram of the EXG-5107 vaccine is shown. EXG-5107 mRNA was produced by in vitro transcription of a plasmid containing a temperature-controllable self-replicating RNA expression cassette (c-srRNA3), which encodes a fusion protein (TAA-5107) containing the human CD5 signal peptide, NY-ESO-1, MAGEA3, TYR, and TPTE. The amino acid sequence of the fusion protein containing the human CD5 signal peptide is shown in SEQ ID NO: 16.

[0076] Placebo (PBO) or 25 μg of the EXG-5107 vaccine was administered intradermally. Subsequently, the cellular immunity against TAAs of the TAA-5107 fusion protein was evaluated by ELISpot assay. Results and Conclusions

[0077] It was expected that the intradermal injection of the EXG-5107 mRNA immunotherapeutic agent would elicit a strong cellular immune response against the different components (NY-ESO-1, MAGEA3, TYR, and TPTE) of the fusion protein. References

[0078] References related to the present disclosure include: PCT / US2022 / 075789 and PCT / US2020 / 067506 of Elixirgen Therapeutics, Inc., the examples of which are incorporated herein by reference. Additional references related to the present disclosure include: Brito et al., Mol Ther. 22(12):2118-2129, 2014; Cheever et al., Clin Cancer Res. 15:5323-5337, 2009; Golombek et al., Mol Ther Nucleic Acids. 11:382-392, 2018; Hickling et al., Intradermal Delivery of Vaccines: A review of the literature and the potential for development for use in low- and middle-income countries. PATH / WHO August 27, 2009; Johanning et al., Nucleic Acids Res. 23(9):1495-501, 1995; and Johansson et al., PLoS One. 7(1):e29732, 2012. Sequence SEQ ID NO:1 >Human CD5 signal peptide MPMGSLQPLATLYLLGMLVASCLG SEQ ID NO:2 >Human nephroblastoma protein (NM_024426.6) MDFLLLQDPASTCVPEPASQHTLRSGPGCLQQPEQQGVRDPGGIWAKLGAAEASAERLQGRRSRGASGSEPQQMGSDVRDLNALLPAVPSLGGGGGCALPVSGAAQWAPVLDFAPPGASAYGSLGGPAPPPAPPPPPPPPPHSFIKQEPSWGGAEPHEEQCLSAFTVHFSGQFTGTAGACRYGPFGPPPPSQASSGQARMFPNAPYLPSCLESQPAIRNQGYSTVTFDGTPSYGHTPSHHAAQFPNHSFKHEDPMGQQGSLGEQQYSVPPPVYGCHTPTDSCTGSQALLLRTPYSSDNLYQMTSQLECMTWNQMNLGATLKGVAAGSSSSVKWTEGQSNHSTGYESDNHTTPILCGAQYRIHTHGVFRGIQDVRRVPGVAPTLVRSASETSEKRPFMCAYPGCNKRYFKLSHLQMHSRKHTGEKPYQCDFKDCERRFSRSDQLKRHQRRHTGVKPFQCKTCQRKFSRSDHLKTHTRTHTGKTSEKPFSCRWPSCQKKFARSDELVRHHNMHQRNMTKLQLAL SEQ ID NO:3 > Homo sapiens BIRC5 (also known as survivin) protein (NM_001168) MGAPTLPPAWQPFLKDHRISTFKNWPFLEGCACTPERMAEAGFIHCPTENEPDLAQCFFCFKELEGWEPDDDPIEEHKKHSSGCAFLSVKKQFEELTLGEFLKLDRERAKNKIAKETNNKKKEFEETAEKVRRAIEQLAAMD SEQ ID NO:4 > Homo sapiens NY-ESO-1 protein (NM_001327) MQAEGRGTGGSTGDADGPGGPGIPDGPGGNAGGPGEAGATGGRGPRGAGAARASGPGGGAPRGPHGGAASGLNGCCRCGARGPESRLLEFYLAMPFATPMEAELARRSLAQDAPPLPVPGVLLKEFTVSGNILTIRLTAADHRQLQLSISSCLQQLSLLMWITQCFLPVFLAQPPSGQRR SEQ ID NO:5 > Homo sapiens MAGEA3 protein (NM_005362) MPLEQRSQHCKPEEGLEARGEALGLVGAQAPATEEQEAASSSSTLVEVTLGEVPAAESPDPPQSPQGASSLPTTMNYPLWSQSYEDSSNQEEEGPSTFPDLESEFQAALSRKVAELVHFLLLKYRAREPVTKAEMLGSVVGNWQYFFPVIFSKASSSLQLVFGIELMEVDPIGHLYIFATCLGLSYDGLLGDNQIMPKAGLLIIVLAIIAREGDCAPEEKIWEELSVLEVFEGREDSILGDPKKLLTQHFVQENYLEYRQVPGSDPACYEFLWGPRALVETSYVKVLHHMVKISGGPHISYPPLHEWVLREGEE SEQ ID NO:6 > Homo sapiens PRAME protein (NM_001291715) MERRRLWGSIQSRYISMSVWTSPRRLVELAGQSLLKDEALAIAALELLPRELFPPLFMAAFDGRHSQTLKAMVQAWPFTCLPLGVLMKGQHLHLETFKAVLDGLDVLLAQEVRPRRWKLQVLDLRKNSHQDFWTVWSGNRASLYSFPEPEAAQPMTKKRKVDGLSTEAEQPFIPVEVLVDLFLKEGACDELFSYLIEKVKRKKNVLRLCCKKLKIFAMPMQDIKMILKMVQLDSIEDLEVTCTWKLPTLAKFSPYLGQMINLRRLLLSHIHASSYISPEKEEQYIAQFTSQFLSLQCLQALYVDSLFFLRGRLDQLLRHVMNPLETLSITNCRLSEGDVMHLSQSPSVSQLSVLSLSGVMLTDVSPEPLQALLERASATLQDLVFDECGITDDQLLALLPSLSHCSQLTTLSFYGNSISISALQSLLQHLIGLSNLTHVLYPVPLESYEDIHGTLHLERLAYLHARLRELLCELGRPSMVWLSANPCPHCGDRTFYDPEPILCPCFMPN SEQ ID NO:7 >Fusions of WT1, BIRC5, NY-ESO-1, MAGEA3 and PRAME SEQ ID NO:8 >Artificial: Fusion of human CD5 (signal peptide only), WT1, BIRC5, NY-ESO-1, MAGEA3 and PRAME SEQ ID NO:9 >Artificial: c-srRNA1 nsP2 GSVETPRGLIKVTSYDGEDKIGSYAVLSPQAVLKSEKLSCIHPLAEQVIVITHSGRKGRYAVEPYHGKVVVPEGHAIPVQDFQALSESATIVYNEREFVNRYLHHIATHGGALNTDEEYYKTVKPSEHDGEYLYDIDRKQCVKKELVTGLGLTGELVDPPFHEFAYESLRTRPAAPYQVPTIGVYGVPGSGKSGIIKSAVTKKDLVVSAKKENCAEIIRDVKKMKGLDVNARTVDSVLLNGCKHPVETLYIDEAFACHAGTLRALIAIIRPKKAVLCGDPKQCGFFNMMCLKVHFNHEICTQVFHKSISRRCTKSVTSVVSTLFYDKKMRTTNPKETKIVIDTTGSTKPKQDDLILTCFRGWVKQLQIDYKGNEIMTAAASQGLTRKGVYAVRYKVNENPLYAPTSEHVNVLLTRTEDRIVWKTLAGDPWIKTLTAKYPGNFTATIEEWQAEHDAIMRHILERPDPTDVFQNKANVCWAKALVPVLKTAGIDMTTEQWNTVDYFETDKAHSAEIVLNQLCVRFFGLDLDSGLFSAPTVPLSIRNNHWDNSPSPNMYGLNKEVVRQLSRRYPQLPRAVATGRVYDMNTGAAATGTLRNYDPRINLVPVNRRLPHALVLHHNEHPQSDFSSFVSKLKGRTVLVVGEKLSVPGKMVDWLSDRPEATFRARLDLGIPGDVPKYDIIFVNVRTPYKYHHYQQCEDHAIKLSMLTKKACLHLNPGGTCVSIGYGYADRASESIIGAIARQFKFSRVCKPKSSLEETEVLFVFIGYDRKARTHNSYKLSSTLTNIYTGSRLHEAGC SEQ ID NO:10 >Artificial: c-srRNA3 nsP2 GSVETPRGLIKVTSYAGEDKIGSYAVLSPQAVLKSEKLSCIHPLAEQVIVITHSGRKGRYAVEPYHGKVVVPEGHAIPVQDFQALSESATIVYNEREFVNRYLHHIATHGGALNTDEEYYKTVKPSEHDGEYLYDIDRKQCVKKELVTGLGLTGELVDPPFHEFAYESLRTRPAAPYQVPTIGVYGVPGSGKSGIIKSAVTKKDLVVSAKKENCAEIIRDVKKMKGLDVNARTVDSVLLNGCKHPVETLYIDEAFACHAGTLRALIAIIRPKKAVLCGDPKQCGFFNMMCLKVHFNHEICTQVFHKSISRRCTKSVTSVVSTLFYDKKMRTTNPKETKIVIDTTGSTKPKQDDLILTCFRGWVKQLQIDYKGNEIMTAAASQGLTRKGVYAVRYKVNENPLYAPTSEHVNVLLTRTEDRIVWKTLAGDPWIKTLTAKYPGNFTATIEEWQAEHDAIMRHILERPDPTDVFQNKANVCWAKALVPVLKTAGIDMTTEQWNTVDYFETDKAHSAEIVLNQLCVRFFGLDLDSGLFSAPTVPLSIRNNHWDNSPSPNMYGLNKEVVRQLSRRYPQLPRAVATGRVYDMNTGAAATGTLRNYDPRINLVPVNRRLPHALVLHHNEHPQSDFSSFVSKLKGRTVLVVGEKLSVPGKMVDWLSDRPEATFRARLDLGIPGDVPKYDIIFVNVRTPYKYHHYQQCEDHAIKLSMLTKKACLHLNPGGTCVSIGYGYADRASESIIGAIARQFKFSRVCKPKSSLEETEVLFVFIGYDRKARTHNPYKLSSTLTNIYTGSRLHEAGC SEQ ID NO:11 > Artificial: c-srRNA4 nsP2 GSVETPRGLIKVTSYAGEDKIGSYAVLSPQAVLKSEKLSCIHPLAEQVIVITHSGRKGRYAVEPYHGKVVVPEGHAIPVQDFQALSESATIVYNEREFVNRYLHHIATHGGALNTDEEYYKTVKPSEHDGEYLYDIDRKQCVKKELVTGLGLTGELVDPPFHEFAYESLRTRPAAPYQVPTIGVYGVPGSGKSGIIKSAVTKKDLVVSAKKENCAEIIRDVKKMKGLDVNARTVDSVLLNGCKHPVETLYIDEAFACHAGTLRALIAIIRPKKAVLCGDPKQCGFFNMMCLKVHFNHEICTQVFHKSISRRCTKSVTSVVSTLFYDKRMRTTNPKETKIEIDTTGSTKPKQDDLILTCFRGWVKQLQIDYKGNEIMTAAASQGLTRKGVYAVRYKVNENPLYAPTSEHVNVLLTRTEDRIVWKTLAGDPWIKTLTAKYPGNFTATIEEWQAEHDAIMRHILERPDPTDVFQNKANVCWAKALVPVLKTAGIDMTTEQWNTVDYFETDKAHSAEIVLNQLCVRFFGLDLDSGLFSAPTVPLSIRNNHWDNSPSPNMYGLNKEVVRQLSRRYPQLPRAVATGRVYDMNTGAAATGTLRNYDPRINLVPVNRRLPHALVLHHNEHPQSDFSSFVSKLKGRTVLVVGEKLSVPGKKVDWLSDQPEATFRARLDLGIPGDVPKYDIVFINVRTPYKYHHYQQCEDHAIKLSMLTKKACLHLNPGGTCVSIGYGYADRASESIIGAIARQFKFSRVCKPKSSHEETEVLFVFIGYDRKARTHNPYKLSSTLTNIYTGSRLHEAGC SEQ ID NO:12 > Artificial: Consensus sequence of c-srRNA nsP2 GSVETPRGLIKVTSY[A / D]GEDKIGSYAVLSPQAVLKSEKLSCIHPLAEQVIVITHSGRKGRYAVEPYHGKVVVPEGHAIPVQDFQALSESATIVYNEREFVNRYLHHIATHGGALNTDEEYYKTVKPSEHDGEYLYDIDRKQCVKKELVTGLGLTGELVDPPFHEFAYESLRTRPAAPYQVPTIGVYGVPGSGKSGIIKSAVTKKDLVVSAKKENCAEIIRDVKKMKGLDVNARTVDSVLLNGCKHPVETLYIDEAFACHAGTLRALIAIIRPKKAVLCGDPKQCGFFNMMCLKVHFNHEICTQVFHKSISRRCTKSVTSVVSTLFYDK[K / R]MRTTNPKETKI[V / E]IDTTGSTKPKQDDLILTCFRGWVKQLQIDYKGNEIMTAAASQGLTRKGVYAVRYKVNENPLYAPTSEHVNVLLTRTEDRIVWKTLAGDPWIKTLTAKYPGNFTATIEEWQAEHDAIMRHILERPDPTDVFQNKANVCWAKALVPVLKTAGIDMTTEQWNTVDYFETDKAHSAEIVLNQLCVRFFGLDLDSGLFSAPTVPLSIRNNHWDNSPSPNMYGLNKEVVRQLSRRYPQLPRAVATGRVYDMNTGAAATGTLRNYDPRINLVPVNRRLPHALVLHHNEHPQSDFSSFVSKLKGRTVLVVGEKLSVPGK[M / K]VDWLSD[R / Q]PEATFRARLDLGIPGDVPKYDI[I / V]F[V / I]NVRTPYKYHHYQQCEDHAIKLSMLTKKACLHLNPGGTCVSIGYGYADRASESIIGAIARQFKFSRVCKPKSS[L / H]EETEVLFVFIGYDRKARTHN[P / S]YKLSSTLTNIYTGSRLHEAGC SEQ ID NO:13 >VEEV: srRNA0 GSVETPRGLIKVTSYAGEDKIGSYAVLSPQAVLKSEKLSCIHPLAEQVIVITHSGRKGRYAVEPYHGKVVVPEGHAIPVQDFQALSESATIVYNEREFVNRYLHHIATHGGALNTDEEYYKTVKPSEHDGEYLYDIDRKQCVKKELVTGLGLTGELVDPPFHEFAYESLRTRPAAPYQVPTIGVYGVPGSGKSGIIKSAVTKKDLVVSAKKENCAEIIRDVKKMKGLDVNARTVDSVLLNGCKHPVETLYIDEAFACHAGTLRALIAIIRPKKAVLCGDPKQCGFFNMMCLKVHFNHEICTQVFHKSISRRCTKSVTSVVSTLFYDKKMRTTNPKETKIVIDTTGSTKPKQDDLILTCFRGWVKQLQIDYKGNEIMTAAASQGLTRKGVYAVRYKVNENPLYAPTSEHVNVLLTRTEDRIVWKTLAGDPWIKTLTAKYPGNFTATIEEWQAEHDAIMRHILERPDPTDVFQNKANVCWAKALVPVLKTAGIDMTTEQWNTVDYFETDKAHSAEIVLNQLCVRFFGLDLDSGLFSAPTVPLSIRNNHWDNSPSPNMYGLNKEVVRQLSRRYPQLPRAVATGRVYDMNTGTLRNYDPRINLVPVNRRLPHALVLHHNEHPQSDFSSFVSKLKGRTVLVVGEKLSVPGKMVDWLSDRPEATFRARLDLGIPGDVPKYDIIFVNVRTPYKYHHYQQCEDHAIKLSMLTKKACLHLNPGGTCVSIGYGYADRASESIIGAIARQFKFSRVCKPKSSLEETEVLFVFIGYDRKARTHNPYKLSSTLTNIYTGSRLHEAGC SEQ ID NO:14 >Artificial protein: TS insertion TGAAA SEQ ID NO:15 >Artificial (TAA-5107 without CD5 signal peptide) SEQ ID NO:16 >Artificial (TAA-5107 with CD5 signal peptide) SEQ ID NO:17 >Artificial (TSA-5109 without CD5 signal peptide) YKLVVVGADGVGKSALTYKLVVVGAVGVGKSALTYKLVVVGARGVGKSALTYKLVVVGACGVGKSALTYKLVVVGAAGVGKSALTYKLVVVGASGVGKSALTKLVVVGAGDVGKSALTIKLVVVGAGCVGKSALTIKLVVVGAGPVGKSALTIKLVVVGAGSVGKSALTILDILDTAGHEEYSAMRDLDILDTAGKEEYSAMRDLDILDTAGREEYSAMRD SEQ ID NO:18 >Artificial (TSA-5109 with CD5 signal peptide) 245AA MPMGSLQPLATLYLLGMLVASCLGYKLVVVGADGVGKSALTYKLVVVGAVGVGKSALTYKLVVVGARGVGKSALTYKLVVVGACGVGKSALTYKLVVVGAAGVGKSALTYKLVVVGASGVGKSALTKLVVVGAGDVGKSALTIKLVVVGAGCVGKSALTIKLVVVGAGPVGKSALTIKLVVVGAGSVGKSALTILDILDTAGHEEYSAMRDLDILDTAGKEEYSAMRDLDILDTAGREEYSAMRD SEQ ID NO:19 >Artificial (TSA-5111 without CD5 signal peptide) MTEYKLVVVGADGVGKSALTIQLIQNMTEYKLVVVGAVGVGKSALTIQLIQNMTEYKLVVVGARGVGKSALTIQLIQNMTEYKLVVVGACGVGKSALTIQLIQNMTEYKLVVVGAAGVGKSALTIQLIQNMTEYKLVVVGASGVGKSALTIQLIQNMTEYKLVVVGAGDVGKSALTIQLIQNHMTEYKLVVVGAGCVGKSALTIQLIQNHMTEYKLVVVGAGPVGKSALTIQLIQNHMTEYKLVVVGAGSVGKSALTIQLIQNHDGETCLLDILDTAGHEEYSAMRDQYMRTGDGETCLLDILDTAGKEEYSAMRDQYMRTGDGETCLLDILDTAGREEYSAMRDQYMRTG SEQ ID NO:20 >Artificial (TSA-5111 with CD5 signal peptide) 375AA MPMGSLQPLATLYLLGMLVASCLGMTEYKLVVVGADGVGKSALTIQLIQNMTEYKLVVVGAVGVGKSALTIQLIQNMTEYKLVVVGARGVGKSALTIQLIQNMTEYKLVVVGACGVGKSALTIQLIQNMTEYKLVVVGAAGVGKSALTIQLIQNMTEYKLVVVGASGVGKSALTIQLIQNMTEYKLVVVGAGDVGKSALTIQLIQNHMTEYKLVVVGAGCVGKSALTIQLIQNHMTEYKLVVVGAGPVGKSALTIQLIQNHMTEYKLVVVGAGSVGKSALTIQLIQNHDGETCLLDILDTAGHEEYSAMRDQYMRTGDGETCLLDILDTAGKEEYSAMRDQYMRTGDGETCLLDILDTAGREEYSAMRDQYMRTG SEQ ID NO:21 >WT-KRAS-32AA MTEYKLVVVGAGGVGKSALTIQLIQNHFVDEY SEQ ID NO:22 >G12D-32AA MTEYKLVVVGADGVGKSALTIQLIQNHFVDEY SEQ ID NO:23 >G12D-17AA YKLVVVGADGVGKSALT SEQ ID NO:24 >G12V-17AA YKLVVVGAVGVGKSALT SEQ ID NO:25 >G12R-17AA YKLVVVGARGVGKSALT SEQ ID NO:26 >G12C-17AA YKLVVVGACGVGKSALT SEQ ID NO:27 >G12A-17AA YKLVVVGAAGVGKSALT SEQ ID NO:28 >G12S-17AA YKLVVVGASGVGKSALT SEQ ID NO:29 >Artificial-G12X-Multi-protein-102AA YKLVVVGADGVGKSALTYKLVVVGAVGVGKSALTYKLVVVGARGVGKSALTYKLVVV GACGVGKSALTYKLVVVGAAGVGKSALTYKLVVVGASGVGKSALT SEQ ID NO:30 >WT-KRAS-39AA KQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLC SEQ ID NO:31 >Q61H-KRAS-39AA KQVVIDGETCLLDILDTAGHEEYSAMRDQYMRTGEGFLC SEQ ID NO:32 >Q61H-KRAS-29AA DGETCLLDILDTAGHEEYSAMRDQYMRTG SEQ ID NO:33 >Q61K-KRAS-29AA DGETCLLDILDTAGKEEYSAMRDQYMRTG SEQ ID NO:34 >Q61R-KRAS-29AA DGETCLLDILDTAGREEYSAMRDQYMRTG SEQ ID NO:35 >Artificial-Q61X-Multi-protein-87AA DGETCLLDILDTAGHEEYSAMRDQYMRTGDGETCLLDILDTAGKEEYSAMRDQYMRT GDGETCLLDILDTAGREEYSAMRDQYMRTG SEQ ID NO:36 >G13D-KRAS-17AA KLVVVGAGDVGKSALTI SEQ ID NO:37 >G13C-KRAS-17AA KLVVVGAGCVGKSALTI SEQ ID NO:38 >G13P-KRAS-17AA KLVVVGAGPVGKSALTI SEQ ID NO:39 >G13S-KRAS-17AA KLVVVGAGSVGKSALTI SEQ ID NO:40 >Q61H-KRAS-17AA LDILDTAGHEEYSAMRD SEQ ID NO:41 >Q61K-KRAS-17AA LDILDTAGKEEYSAMRD SEQ ID NO:42 >Q61R-KRAS-17AA LDILDTAGREEYSAMRD SEQ ID NO:43 >G12D-KRAS-26AA MTEYKLVVVGADGVGKSALTIQLIQN SEQ ID NO:44 >G12V-KRAS-26AA MTEYKLVVVGAVGVGKSALTIQLIQN SEQ ID NO:45 >G12R-KRAS-26AA MTEYKLVVVGARGVGKSALTIQLIQN SEQ ID NO:46 >G12C-KRAS-26AA MTEYKLVVVGACGVGKSALTIQLIQN SEQ ID NO:47 >G12A-KRAS-26AA MTEYKLVVVGAAGVGKSALTIQLIQN SEQ ID NO:48 >G12S-KRAS-26AA MTEYKLVVVGASGVGKSALTIQLIQN SEQ ID NO:49 >G13D-KRAS-27AA MTEYKLVVVGAGDVGKSALTIQLIQNH SEQ ID NO:50 >G13C-KRAS-27AA MTEYKLVVVGAGCVGKSALTIQLIQNH SEQ ID NO:51 >G13P-KRAS-27AA MTEYKLVVVGAGPVGKSALTIQLIQNH SEQ ID NO:52 >KRAS-G13S-27AA MTEYKLVVVGAGSVGKSALTIQLIQNH SEQ ID NO:53 >Artificial MTEYKLVVVGAX1GVGKSALTIQLIQNXaXbXcXdXeXf MTEYKLVVVGAX2GVGKSALTIQLIQNXaXbXcXdXeXf MTEYKLVVVGAX3GVGKSALTIQLIQNXaXbXcXdXeXf MTEYKLVVVGAX4GVGKSALTIQLIQNXaXbXcXdXeXf MTEYKLVVVGAX5GVGKSALTIQLIQNXaXbXcXdXeXf MTEYKLVVVGAX6GVGKSALTIQLIQNXaXbXcXdXeXf, wherein X1, X2, X3, X4, X5, and X6 are independently selected from D, V, R, C, A, and S, and wherein Xa, Xb, Xc, Xd, Xe, and Xf are independently selected from G, S, and absent. SEQ ID NO:54 >Artificial MTEYKLVVVGAGX 10 VGKSALTIQLIQNHXaXbXcXdXeXf MTEYKLVVVGAGX 11 VGKSALTIQLIQNHXaXbXcXdXeXf MTEYKLVVVGAGX 12 VGKSALTIQLIQNHXaXbXcXdXeXf MTEYKLVVVGAGX 13 VGKSALTIQLIQNHXaXbXcXdXeXf, wherein X 10 , X 11 , X 12 and X 13 are independently selected from D, C, P, and S, and wherein Xa, Xb, Xc, Xd, Xe, and Xf are independently selected from G, S, and absent. SEQ ID NO:55 >Artificial DGETCLLDILDTAGX7EEYSAMRDQYMRTGXaXbXcXdXeXf DGETCLLDILDTAGX8EEYSAMRDQYMRTGXaXbXcXdXeXf DGETCLLDILDTAGX9EEYSAMRDQYMRTGXaXbXcXdXeXf, wherein X7, X8, and X9 are independently selected from H, K, and R, and wherein Xa, Xb, Xc, Xd, Xe, and Xf are independently selected from G, S, and absent. SEQ ID NO:56 ARTIFICIAL YKLVVVGAX1GVGKSALTXaXbXcXdXeXfYKLVVVGAX2GVGKSALTXaXbXcXdXeXf YKLVVVGAX3GVGKSALTXaXbXcXdXeXfYKLVVVGAX4GVGKSALTXaXbXcXdXeXf YKLVVVGAX5GVGKSALTXaXbXcXdXeXfYKLVVVGAX6GVGKSALT, wherein X1, X2, X3, X4, X5 and X6 are independently selected from D, V, R, C, A and S, and wherein Xa, Xb, Xc, Xd, Xe and Xf are independently selected from G, S and absent. SEQ ID NO:57 >ARTIFICIAL KLVVVGAGX 10 VGKSALTIXaXbXcXdXeXfKLVVVGAGX 11 VGKSALTIXaXbXcXdXeXf KLVVVGAGX 12 VGKSALTIXaXbXcXdXeXfKLVVVGAGX 13 VGKSALTI, wherein X 10 , X 11 , X 12 and X 13 are independently selected from D, C, P and S, and wherein Xa, Xb, Xc, Xd, Xe and Xf are independently selected from G, S and absent. SEQ ID NO:58 >ARTIFICIAL LDILDTAGX7HEEYSAMRDXaXbXcXdXeXfLDILDTAGX8HEEYSAMRDXaXbXcXdXeXf LDILDTAGX9HEEYSAMRD, wherein X7, X8 and X9 are independently selected from H, K and R, and wherein Xa, Xb, Xc, Xd, Xe and Xf are independently selected from G, S and absent. SEQ ID NO:59 >G12G MTEYKLVVVGAGGVGKSALTIQLIQ SEQ ID NO:60 G12D MTEYKLVVVGADGVGKSALTIQLIQ SEQ ID NO:61 G12VMTEYKLVVVGAVGVGKSALTIQLIQ SEQ ID NO:62 G12C MTEYKLVVVGACGVGKSALTIQLIQ

Claims

1. An RNA molecule, the RNA molecule comprising an open reading frame (ORF) encoding a fusion protein, wherein the ORF comprises, from 5' to 3': (i) a nucleotide sequence encoding a mammalian signal peptide; and (ii) a nucleotide sequence encoding a cancer antigen, wherein the cancer antigen comprises a KRAS polyprotein, and the amino acid sequence of the KRAS polyprotein comprises: a) a first segment, the first segment comprising: MTEYKLVVVGAX1GVGKSALTIQLIQNXaXbXcXdXeXf MTEYKLVVVGAX2GVGKSALTIQLIQNXaXbXcXdXeXf MTEYKLVVVGAX3GVGKSALTIQLIQNXaXbXcXdXeXf MTEYKLVVVGAX4GVGKSALTIQLIQNXaXbXcXdXeXf MTEYKLVVVGAX5GVGKSALTIQLIQNXaXbXcXdXeXf MTEYKLVVVGAX6GVGKSALTIQLIQNXaXbXcXdXeXf (SEQ ID NO:53); b) a second segment, the second segment comprising: MTEYKLVVVGAGX 10 VGKSALTIQLIQNHXaXbXcXdXeXf MTEYKLVVVGAGX 11 VGKSALTIQLIQNHXaXbXcXdXeXf MTEYKLVVVGAGX 12 VGKSALTIQLIQNHXaXbXcXdXeXf MTEYKLVVVGAGX 13 VGKSALTIQLIQNHXaXbXcXdXeXf(SEQ ID NO:54); and c) a third segment, the third segment comprising: DGETCLLDILDTAGX7EEYSAMRDQYMRTGXaXbXcXdXeXf DGETCLLDILDTAGX8EEYSAMRDQYMRTGXaXbXcXdXeXf DGETCLLDILDTAGX9EEYSAMRDQYMRTGXaXbXcXdXeXf (SEQ ID NO:55), wherein the first segment, the second segment and the third segment are arranged in any order, wherein X1, X2, X3, X4, X5 and X6 are independently selected from D, V, R, C, A and S, wherein X7, X8 and X9 are independently selected from H, K and R, wherein X 10 , X 11 , X 12 and X 13 are independently selected from D, C, P, and S, and wherein Xa, Xb, Xc, Xd, Xe and Xf are independently selected from G, S and absent.

2. The RNA molecule according to claim 1, wherein the amino acid sequence of the KRAS polyprotein comprises SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:32, SEQ ID NO:33 and SEQ ID NO:

34.

3. The RNA molecule according to claim 2, wherein the amino acid sequence of the KRAS polyprotein comprises residues 25-375 of SEQ ID NO:

20.

4. An RNA molecule, the RNA molecule comprising an open reading frame (ORF) encoding a fusion protein, wherein the ORF comprises, from 5' to 3': (i) A nucleotide sequence encoding a mammalian signal peptide; and (ii) A nucleotide sequence encoding a cancer antigen, wherein the cancer antigen comprises a KRAS polyprotein, and the amino acid sequence of the KRAS polyprotein comprises: a) A first segment, the first segment comprising: YKLVVVGAX1GVGKSALTXaXbXcXdXeXfYKLVVVGAX2GV GKSALTXaXbXcXdXeXf YKLVVVGAX3GVGKSALTXaXbXcXdXeXfYKLVVVGAX4GV GKSALTXaXbXcXdXeXf YKLVVVGAX5GVGKSALTXaXbXcXdXeXfYKLVVVGAX6GV GKSALT(SEQ ID NO:56), b) A second segment, the second segment comprising: KLVVVGAGX 10 VGKSALTIXaXbXcXdXeXfKLVVVGAGX 11 VG KSALTIXaXbXcXdXeXf KLVVVGAGX 12 VGKSALTIXaXbXcXdXeXfKLVVVGAGX 13 VG KSALTI (SEQ ID NO:57), and c) A third segment, the third segment comprising: LDILDTAGX7HEEYSAMRDXaXbXcXdXeXfLDILDTAGX8HEE YSAMRDXaXbXcXdXeXf LDILDTAGX9HEEYSAMRD(SEQ ID NO:58), wherein the first segment, the second segment and the third segment are arranged in any order, wherein X1, X2, X3, X4, X5 and X6 are independently selected from D, V, R, C, A and S, wherein X7, X8 and X9 are independently selected from H, K and R, wherein X 10 , X 11 , X 12 and X 13 are independently selected from D, C, P and S, and wherein Xa, Xb, Xc, Xd, Xe and Xf are independently selected from G, S and absent.

5. The RNA molecule according to claim 4, wherein the amino acid sequence of the KRAS polyprotein comprises SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ IDNO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41 and SEQID NO:

42.

6. The RNA molecule according to claim 5, wherein the amino acid sequence of the KRAS polyprotein comprises residues 25-245 of SEQ ID NO:

18.

7. An RNA molecule, the RNA molecule comprising an open reading frame (ORF) encoding a fusion protein, wherein the ORF comprises from 5' to 3': (i) A nucleotide sequence encoding a mammalian signal peptide; and (ii) A nucleotide sequence encoding a cancer antigen, wherein the cancer antigen comprises a KRAS polyprotein, and the amino acid sequence of the KRAS polyprotein comprises: YKLVVVGAX1GVGKSALTXaXbXcXdXeXfYKLVVVGAX2GV GKSALTXaXbXcXdXeXf YKLVVVGAX3GVGKSALTXaXbXcXdXeXfYKLVVVGAX4GV GKSALTXaXbXcXdXeXf YKLVVVGAX5GVGKSALTXaXbXcXdXeXfYKLVVVGAX6GV GKSALT(SEQ ID NO:56), wherein X1, X2, X3, X4, X5 and X6 are independently selected from D, V, R, C, A, and S, and wherein Xa, Xb, Xc, Xd, Xe and Xf are independently selected from G, S, and absent.

8. The RNA molecule according to claim 7, wherein the amino acid sequence of the KRAS multi-protein comprises SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, and SEQ ID NO:

28.

9. The RNA molecule according to claim 8, wherein the amino acid sequence of the KRAS multi-protein comprises SEQ ID NO:

29.

10. An RNA molecule, the RNA molecule comprising an open reading frame (ORF) encoding a fusion protein, wherein the ORF comprises, from 5' to 3': (i) a nucleotide sequence encoding a mammalian signal peptide; and (ii) a nucleotide sequence encoding a cancer antigen, wherein the cancer antigen comprises a KRAS multi-protein, and the amino acid sequence of the KRAS multi-protein comprises: DGETCLLDILDTAGX7EEYSAMRDQYMRTGXaXbXcXdXeXf DGETCLLDILDTAGX8EEYSAMRDQYMRTGXaXbXcXdXeXf DGETCLLDILDTAGX9EEYSAMRDQYMRTGXaXbXcXdXeXf(SEQ ID NO:55), wherein X7, X8, and X9 are independently selected from H, K, and R, and wherein Xa, Xb, Xc, Xd, Xe and Xf are independently selected from G, S, and absent.

11. The RNA molecule according to claim 10, wherein the amino acid sequence of the KRAS multi-protein comprises SEQ ID NO:32, SEQ ID NO:33, and SEQ ID NO:

34.

12. The RNA molecule according to claim 11, wherein the amino acid sequence of the KRAS multi-protein comprises SEQ ID NO:

35.

13. An RNA molecule, the RNA molecule comprising an open reading frame (ORF) encoding a fusion protein, wherein the ORF comprises, from 5' to 3': (i) a nucleotide sequence encoding a mammalian signal peptide; and (ii) a nucleotide sequence encoding a cancer antigen, wherein the cancer antigen comprises a KRAS multi-protein, and the amino acid sequence of the KRAS multi-protein comprises: KLVVVGAGX 10 VGKSALTIXaXbXcXdXeXfKLVVVGAGX 11 VG KSALTIXaXbXcXdXeXf KLVVVGAGX 12 VGKSALTIXaXbXcXdXeXfKLVVVGAGX 13 VG KSALTI (SEQ ID NO:57), wherein X 10 、X 11 、X 12 and X 13 are independently selected from D, C, P, and S, and wherein Xa, Xb, Xc, Xd, Xe and Xf are independently selected from G, S, and absent.

14. The RNA molecule according to claim 13, wherein the amino acid sequence of the KRAS multi-protein comprises SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO:

39.

15. An RNA molecule comprising an open reading frame (ORF) encoding a fusion protein, wherein the ORF comprises, from 5' to 3': (i) a nucleotide sequence encoding a mammalian signal peptide; and (ii) a nucleotide sequence encoding a cancer antigen, wherein the cancer antigen comprises a KRAS multi-protein, and the amino acid sequence of the KRAS multi-protein comprises: LDILDTAGX7HEEYSAMRDXaXbXcXdXeXfLDILDTAGX8HEEYSAMRDXaXbXcXdXeXf LDILDTAGX9HEEYSAMRD (SEQ ID NO: 58), wherein X7, X8 and X9 are independently selected from H, K and R, and wherein Xa, Xb, Xc, Xd, Xe and Xf are independently selected from G, S and absent.

16. The RNA molecule according to claim 15, wherein the amino acid sequence of the KRAS multi-protein comprises SEQ ID NO: 40, SEQ ID NO: 41 and SEQ ID NO:

42.

17. The RNA molecule according to claim 8, claim 14 or claim 16, wherein the amino acid sequence of the KRAS multi-protein comprises residues 25-245 of SEQ ID NO:

18.

18. An RNA molecule comprising an open reading frame (ORF) encoding a fusion protein, wherein the ORF comprises, from 5' to 3': (i) a nucleotide sequence encoding a mammalian signal peptide; and (ii) a nucleotide sequence encoding a cancer antigen, wherein the cancer antigen comprises a KRAS multi-protein, and the amino acid sequence of the KRAS multi-protein comprises: MTEYKLVVVGAX1GVGKSALTIQLIQNXaXbXcXdXeXf MTEYKLVVVGAX2GVGKSALTIQLIQNXaXbXcXdXeXf MTEYKLVVVGAX3GVGKSALTIQLIQNXaXbXcXdXeXf MTEYKLVVVGAX4GVGKSALTIQLIQNXaXbXcXdXeXf MTEYKLVVVGAX5GVGKSALTIQLIQNXaXbXcXdXeXf MTEYKLVVVGAX6GVGKSALTIQLIQNXaXbXcXdXeXf (SEQ ID NO: 53), wherein X1, X2, X3, X4, X5 and X6 are independently selected from D, V, R, C, A and S, and wherein Xa, Xb, Xc, Xd, Xe and Xf are independently selected from G, S and absent.

19. The RNA molecule according to claim 18, wherein the amino acid sequence of the KRAS multi-protein comprises SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, and SEQ ID NO:

48.

20. An RNA molecule comprising an open reading frame (ORF) encoding a fusion protein, wherein the ORF comprises, from 5' to 3': (i) a nucleotide sequence encoding a mammalian signal peptide; and (ii) a nucleotide sequence encoding a cancer antigen, wherein the cancer antigen comprises a KRAS multi-protein, and the amino acid sequence of the KRAS multi-protein comprises: MTEYKLVVVGAGX 10 VGKSALTIQLIQNHXaXbXcXdXeXf MTEYKLVVVGAGX 11 VGKSALTIQLIQNHXaXbXcXdXeXf MTEYKLVVVGAGX 12 VGKSALTIQLIQNHXaXbXcXdXeXf MTEYKLVVVGAGX 13 VGKSALTIQLIQNHXaXbXcXdXeXf(SEQ ID NO:54), wherein X 10 、X 11 、X 12 and X 13 are independently selected from D, C, P and S, and wherein Xa, Xb, Xc, Xd, Xe, and Xf are independently selected from G, S, and absent.

21. The RNA molecule according to claim 20, wherein the amino acid sequence of the KRAS multi-protein comprises SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, and SEQ ID NO:

52.

22. The RNA molecule according to claim 11, claim 19, or claim 21, wherein the amino acid sequence of the KRAS multi-protein comprises residues 25 - 375 of SEQ ID NO:

20.

23. The RNA molecule according to any one of claims 1 - 22, wherein the mammalian signal peptide is a signal peptide of a surface protein expressed in mammalian antigen-presenting cells.

24. The RNA molecule according to claim 23, wherein the mammalian signal peptide is a CD5 signal peptide, and the amino acid sequence of the CD5 signal peptide comprises SEQ ID NO: 1 or an amino acid sequence that is at least 90% or 95% identical to SEQ ID NO:

1.

25. The RNA molecule according to any one of claims 1 - 24, the RNA molecule comprising at least one modified nucleoside, optionally wherein the at least one modified nucleoside comprises pseudouridine.

26. A DNA template for the RNA molecule according to any one of claims 1 - 25, optionally wherein a first restriction enzyme cleavage site is present upstream of the nucleotide sequence encoding the mammalian signal peptide, and a second restriction enzyme cleavage site is present downstream of the nucleotide sequence encoding the cancer antigen.

27. An expression vector comprising the DNA template according to claim 26.

28. A host cell comprising the expression vector according to claim 27.

29. The RNA molecule according to any one of claims 1 - 25, wherein the RNA molecule is a self-replicating RNA.

30. A composition for stimulating an immune response against a cancer antigen in a mammalian subject, the composition comprising an excipient and a temperature-sensitive self-replicating RNA according to claim 29, wherein the self-replicating RNA is a temperature-sensitive RNA further comprising an alphavirus replicon lacking a viral structural protein coding region, and wherein the temperature-sensitive self-replicating RNA is capable of expressing the fusion protein at a permissive temperature but not at a non-permissive temperature.

31. A composition for stimulating an immune response against a cancer antigen in a mammalian subject, the composition comprising an excipient and a temperature-sensitive self-replicating RNA, the temperature-sensitive self-replicating RNA comprising an open reading frame (ORF) encoding a fusion protein and an alphavirus replicon lacking a viral structural protein coding region, wherein the ORF comprises, from 5' to 3': (i) a nucleotide sequence encoding a mammalian signal peptide; and (ii) a nucleotide sequence encoding a cancer antigen, wherein the temperature-sensitive self-replicating RNA is capable of expressing the fusion protein at a permissive temperature but not at a non-permissive temperature, and the cancer antigen comprises NY-ESO-1 antigen, MAGEA3 antigen, TYR antigen, and TPTE antigen.

32. The composition according to claim 31, wherein the mammalian signal peptide is a signal peptide of a surface protein expressed in mammalian antigen-presenting cells.

33. The composition according to claim 32, wherein the mammalian signal peptide is a CD5 signal peptide, and the amino acid sequence of the CD5 signal peptide comprises SEQ ID NO:1 or an amino acid sequence that is at least 90% or 95% identical to SEQ ID NO:

1.

34. The composition according to claim 32, wherein the amino acid sequence of the fusion protein comprises SEQ ID NO:16 or an amino acid sequence that is at least 90% or 95% identical to SEQ ID NO:

16.

35. The composition according to any one of claims 30-34, wherein the alphavirus is selected from Venezuelan equine encephalitis virus, Sindbis virus, and Semliki Forest virus.

36. The composition according to claim 35, wherein the alphavirus is Venezuelan equine encephalitis virus.

37. The composition according to any one of claims 30-36, wherein the alphavirus replicon comprises a non-structural protein coding region with an insertion of 12-18 nucleotides, the insertion resulting in the expression of non-structural protein 2 (nsP2), and the non-structural protein 2 comprises 4 to 6 additional amino acids between beta-sheet 5 and beta-sheet 6 of the nsP2.

38. The composition according to claim 37, wherein the additional amino acids comprise the sequence of SEQ ID NO:14 (TGAAA).

39. The composition according to claim 38, wherein the amino acid sequence of the nsP2 comprises SEQ ID NO:

12.

40. The composition according to claim 39, wherein the amino acid sequence of nsP2 comprises a sequence selected from SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:

11.

41. The composition according to claim 40, wherein the amino acid sequence of nsP2 comprises SEQ ID NO:

11.

42. The composition according to any one of claims 30-41, wherein the permissive temperature is from 30°C to 36°C, or 31°C to 35°C, or 32°C to 34°C, or 33°C ± 0.5°C, and the non-permissive temperature is 37°C ± 0.5°C, optionally wherein the permissive temperature is from 31°C to 35°C, and the non-permissive temperature is at least 37°C ± 0.5°C.

43. The composition according to any one of claims 30-42, wherein the composition does not contain lipid nanoparticles.

44. The composition according to any one of claims 30-43, wherein the composition further comprises chitosan.

45. A method for stimulating an immune response against a cancer antigen in a mammalian subject, the method comprising administering to the mammalian subject a composition according to any one of claims 30-44 to stimulate an immune response against the cancer antigen in the mammalian subject.

46. The method according to claim 45, wherein the composition is administered intradermally.

47. The method according to claim 45 or claim 46, wherein the immune response comprises a cellular immune response reactive with mammalian cells expressing the cancer antigen.

48. The method according to claim 47, wherein the cellular immune response comprises one or both of a cancer antigen-specific cytotoxic T lymphocyte response and a cancer antigen-specific helper T lymphocyte response.

49. The method according to claim 48, wherein the immune response further comprises a humoral immune response reactive with the cancer antigen.

50. The method according to any one of claims 45-49, wherein the mammalian subject is a human subject.

51. A kit, the kit comprising: (i) a composition according to any one of claims 30-44; and (ii) a device for intradermal delivery of the composition to a mammalian subject.

52. The kit according to claim 51, wherein the device comprises a syringe and a needle.

53. A method for expressing a fusion protein, the method comprising contacting mammalian cells with an RNA molecule according to any one of claims 1-25.

54. The method according to claim 53, wherein the contacting is in vitro.

55. The method according to claim 53, wherein the contacting is in vivo.

56. A method for treating cancer, the method comprising administering to a mammalian subject in need thereof an effective amount of a composition according to any one of claims 30-44 to treat the cancer.

57. The method according to claim 56, wherein the cells of the cancer express a KRAS oncogene comprising a substitution at one or more of positions 12, 13, and 61 of KRAS.

58. The method according to claim 56, wherein the cells of the cancer express one or more of the NY-ESO-1 antigen, the MAGEA3 antigen, the TYR antigen, and the TPTE antigen.

59. The method according to any one of claims 56-58, wherein the composition is administered intradermally.

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