Antigens for Cancer Immunotherapy

Abstract

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 that encode multiple tumor-associated and / or tumor-specific antigens.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims priority to U.S. Provisional Patent Application No. 63 / 427,424, filed November 22, 2022, which is incorporated herein by reference in its entirety.

[0002] Electronic Sequence Listing Reference The contents of the electronic sequence listing (699442001740SEQLIST.xml, size: 92,598 bytes, created on: November 20, 2023) are incorporated herein by reference in their entirety.

[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 that encode multiple tumor-associated and / or tumor-specific antigens. [Background technology]

[0004] Immunotherapy can be effective in treating cancer and is becoming more widely used. One therapeutic strategy is to inject cancer patients with an immunogenic composition containing an antigen expressed in tumor cells. Tumor-associated antigens (TAAs) are expressed in tumor cells but are also expressed in germ cells or at low levels in normal cells. Tumor-specific antigens (TSAs), also called neoantigens, are expressed only in tumor cells and are often expressed from genes that are mutated in tumor cells. Cancer immunotherapy relies on the induction of cytotoxic T lymphocyte (CTL) responses against cancer cells.

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

[0006] The present disclosure relates to the expression of cancer antigens (TAA and / or TSA) that induce a cellular immune response against cancer cells. In some embodiments, the cancer antigen is encoded by mRNA. In some embodiments, a temperature-controllable, self-replicating RNA vaccine platform is utilized. In an exemplary embodiment, the cancer antigen is expressed in host cells from temperature-controllable, self-replicating RNA (c-srRNA) to induce a potent cellular immune response against cancer antigen-expressing tumor cells. 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 the expression of tumor-associated antigens (TAA) or tumor-specific antigens (TSA) (also known as neoantigens). In some embodiments, the TAA is selected from the group consisting of, but not limited to, NY-ESO-1, MAGEA3, TYR, TPTE (also known as PTEN2), or a combination thereof. In some embodiments, the TSA is an oncoprotein such as a mutant Ras GTPase. In some embodiments, the mutant Ras GTPase is KRAS with activating substitutions at one or more of G12, G13, and Q61. c-srRNA is used to express fusion proteins of two or more TAAs, TSAs, or combinations of TAAs and TSAs.

[0007] Among other embodiments, the present disclosure provides compositions comprising an excipient and temperature-controllable self-replicating RNA (c-srRNA). In some embodiments, the compositions comprise chitosan. In some embodiments, the chitosan is a low molecular weight (approximately 3-5 kDa) chitosan oligosaccharide, such as chitosan oligosaccharide lactate. In some embodiments, the compositions do not comprise liposomes or lipid nanoparticles. [Brief explanation of the drawings]

[0008] [Figure 1] A schematic diagram of an exemplary method for designing a fusion protein of mutant 9-mer peptide(s) is shown. Six common mutations (G12D, G12V, G12R, G12C, G12A, and G12S) of the human KRAS proto-oncogene (GenBank number NM_001369786) are used as an example. Step 1: 9 amino acids (9-mers) are taken from the mutated residue (D in the example of G12D) toward both the N- and C-terminus to identify a 17-amino acid (17-mer) peptide sequence containing the mutated residue (D in this case) in the middle. If the mutated amino acid is located near the N- or C-terminus, the peptide sequence on either side can be shorter than the 9-mer. Step 2: The same procedure is repeated 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 in the same protein (e.g., KRAS) or from mutations in other oncoproteins (e.g., the human TP53 oncoprotein p53). Step 3: Generate a fusion protein of six 17-mer peptides. Typically, 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 of the 17-mer peptides. The order of each 17-mer peptide may be altered from that shown in this example as SEQ ID NO:29. [Figure 2]A schematic diagram of an exemplary method for designing a fusion protein of mutant 15-mer peptide(s) is shown. As an example, three common mutations (Q61H, Q61K, and Q61R) in the human KRAS proto-oncogene (GenBank number NM_001369786) are used. Step 1: 15 amino acids (15-mer) are taken from the mutant amino acid (H in the example of Q61H) toward both the N-terminus and C-terminus to identify a 29-amino acid (29-mer) peptide sequence containing the mutant amino acid residue (H in this case) in the center. If the mutant amino acid is located near the N-terminus or C-terminus, the peptide sequence on either side can be shorter than the 15-mer. Step 2: The same procedure is repeated for other mutations. In this example, three 29-mer peptides are generated from three common mutations (Q61H, Q61K, and Q61R) in the human KRAS proto-oncogene. The 29-mer peptides can be derived from mutations at other positions in the same protein (e.g., KRAS) or from mutations in other proteins (e.g., the human TP53 tumor protein p53). Step 3: Generate a fusion protein of the 29-mer peptides. Typically, 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 may be altered from that shown in this example as SEQ ID NO:35. [Figure 3A] FIG. 1 shows a schematic diagram of an exemplary fusion protein (TSA-5109) containing a mutated 17mer peptide derived from the human KRAS proto-oncogene (GenBank number NM_001369786). [Figure 3B] The amino acid sequences of 13 different 17-mer peptides derived from 13 common mutations of the human KRAS proto-oncogene contained in TSA-5109 (G12D; G12V, G12R, G12C, G12A, G12S, G13D, G13C, G13P, G13S, Q61H, Q61K, and Q61R) are shown. [Figure 3C]

[0033] Figure 1 shows the amino acid sequences 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 set forth as SEQ ID NO:17, and the amino acid sequence of the TSA-5109 fusion protein with the CD5sp sequence is set forth as SEQ ID NO:18. [Figure 4A] FIG. 1 shows a schematic diagram of an exemplary fusion protein (TSA-5111) containing a mutated 29-mer peptide derived from the human KRAS proto-oncogene (GenBank number NM_001369786). [Figure 4B] The amino acid sequences of 13 different 29-mer peptides derived from 13 common mutations of the human KRAS proto-oncogene contained in TSA-5111 (G12D; G12V, G12R, G12C, G12A, G12S, G13D, G13C, G13P, G13S, Q61H, Q61K, and Q61R) are shown. [Figure 4C]

[0033] Figure 1 shows the amino acid sequences of the TSA-5111 fusion protein containing the human CD5 signal peptide (CD5sp) sequence at its N-terminus. The amino acid sequence of the TSA-5111 fusion protein without the CD5sp sequence is set forth as SEQ ID NO:19, and the amino acid sequence of the TSA-5111 fusion protein with the CD5sp sequence is set forth as SEQ ID NO:20. [Figure 5]1 shows a schematic diagram of a fusion protein comprising multiple tumor-associated antigens. In some embodiments, the tumor-associated antigens are expressed from 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) set forth as SEQ ID NO:1, the amino acid sequence of human NY-ESO-1 protein (GenBank No. NM_001327) set forth as SEQ ID NO:4, the amino acid sequence of human MAGEA3 protein (GenBank No. NM_005362) set forth as SEQ ID NO:5, the amino acid sequence of human TYR protein (GenBank No. NM_000372), and the amino acid sequence of human transmembrane phosphatase with tensin homology (TPTE) protein (GenBank No. NM_199261). The amino acid sequence of the TAA-5107 fusion protein (without CD5 SP) is set forth as SEQ ID NO:15, and the amino acid sequence of the CD5-SP+TAA fusion protein is set forth as SEQ ID NO:16.

[0009] [Figure 6] A schematic diagram of an exemplary method for stimulating an immune response to a cancer antigen in a human subject is shown. c-srRNA is functional at permissive temperatures (e.g., 30-35°C) but not at non-permissive temperatures (e.g., above 37°C). The temperature at or just below the surface of the human body (surface body temperature) is approximately 31-34°C, which is lower than the core body temperature, which is approximately 37°C. c-srRNA is delivered directly to the subject's cells at the permissive surface temperature by intradermal and subcutaneous administration. [Figure 7A]Figure 7 shows tumor growth suppression by the EXG-5111 vaccine, which expresses the TSA-5111 antigen in vivo. BALB / c female mice were intradermally administered 100 μg of EXG-5111 twice, two weeks apart. Two weeks later (day 0), the mice were administered 3×10^5 cells of CT26 mouse colon carcinoma cells (ATCC CRL-2638), known to harbor a G12D mutation in the KRAS proto-oncogene. Figure 7A shows the increase in tumor size in 15 mice that received intradermal placebo (PBO) injections. Mice that met the euthanasia criteria due to tumor size or ulceration were sacrificed. In most mice, tumors grew rapidly, and by day 28 after tumor injection, only one mouse survived. Figure 7B shows the increase in tumor size in 15 mice that received intradermal EXG-5111 vaccine injections. In contrast to the PBO group, tumor growth was suppressed in mice administered the EXG-5111 vaccine. By day 28 after tumor injection, seven mice survived. Figure 7C shows a comparison of tumor growth in the PBO and EXG-5111 groups, with the mean ± SEM for each group shown. The suppression of tumor growth by EXG-5111 was statistically significant on days 11, 14, 22, and 25, as indicated by asterisks. [Figure 7B]Figure 7 shows tumor growth suppression by the EXG-5111 vaccine, which expresses the TSA-5111 antigen in vivo. BALB / c female mice were intradermally administered 100 μg of EXG-5111 twice, two weeks apart. Two weeks later (day 0), the mice were administered 3×10^5 cells of CT26 mouse colon carcinoma cells (ATCC CRL-2638), known to harbor a G12D mutation in the KRAS proto-oncogene. Figure 7A shows the increase in tumor size in 15 mice that received intradermal placebo (PBO) injections. Mice that met the euthanasia criteria due to tumor size or ulceration were sacrificed. In most mice, tumors grew rapidly, and by day 28 after tumor injection, only one mouse survived. Figure 7B shows the increase in tumor size in 15 mice that received intradermal EXG-5111 vaccine injections. In contrast to the PBO group, tumor growth was suppressed in mice administered the EXG-5111 vaccine. By day 28 after tumor injection, seven mice survived. Figure 7C shows a comparison of tumor growth in the PBO and EXG-5111 groups, with the mean ± SEM for each group shown. The suppression of tumor growth by EXG-5111 was statistically significant on days 11, 14, 22, and 25, as indicated by asterisks. [Figure 7C]Figure 7 shows tumor growth suppression by the EXG-5111 vaccine, which expresses the TSA-5111 antigen in vivo. BALB / c female mice were intradermally administered 100 μg of EXG-5111 twice, two weeks apart. Two weeks later (day 0), the mice were administered 3×10^5 cells of CT26 mouse colon carcinoma cells (ATCC CRL-2638), known to harbor a G12D mutation in the KRAS proto-oncogene. Figure 7A shows the increase in tumor size in 15 mice that received intradermal placebo (PBO) injections. Mice that met the euthanasia criteria due to tumor size or ulceration were sacrificed. In most mice, tumors grew rapidly, and by day 28 after tumor injection, only one mouse survived. Figure 7B shows the increase in tumor size in 15 mice that received intradermal EXG-5111 vaccine injections. In contrast to the PBO group, tumor growth was suppressed in mice administered the EXG-5111 vaccine. By day 28 after tumor injection, seven mice survived. Figure 7C shows a comparison of tumor growth in the PBO and EXG-5111 groups, with the mean ± SEM for each group shown. The suppression of tumor growth by EXG-5111 was statistically significant on days 11, 14, 22, and 25, as indicated by asterisks. [Figure 7D] table DETAILED DESCRIPTION OF THE INVENTION

[0010] Cancer immunotherapy is believed to be best achieved by immunogenic compositions that primarily rely on the induction of cell-mediated immunity (i.e., T cell-inducing vaccines involving CD8+ killer T cells and CD4+ helper T cells). The present disclosure provides mRNA, self-replicating RNA (srRNA), and temperature-controllable self-replicating RNA (c-srRNA) encoding one or more cancer antigens, such as tumor-associated antigens (TAA) and tumor-specific antigens (TSA, also known as neoantigens). Thus, the present disclosure provides a cell-mediated immunity-based platform for cancer immunotherapy. Wilms' tumor 1 (WT1) is a tumor-associated antigen (TAA) that is expressed in a wide range of tumors, but only in very limited cell types in embryonic tissues and 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.

[0011] Cell-mediated immunity-based mRNA immunotherapy platform The vaccine platform is described in part in Elixirgen's prior patent application (International Application No. PCT / US20 / 67506, now published as WO 2021 / 138447(A1)). This vaccine platform is optimized to induce cell-mediated immunity 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, with srRNA1ts2 (described in WO 2021 / 138447(A1)) being an exemplary embodiment. The c-srRNA is based on srRNA, also known as self-amplifying mRNA (saRNA or SAM), by incorporating small amino acid changes in the alphavirus replicase that provide temperature sensitivity. Elixirgen's c-srRNA is functional in the permissive temperature range of approximately 30-35°C, but not at non-permissive temperatures above approximately 37°C. This platform offers all the benefits of the mRNA platform, including no genomic integration, rapid development and deployment, and simple GMP (Good Manufacturing Practice) processes, while simultaneously possessing the additional advantages of the srRNA platform (i.e., the precursor to our c-srRNA platform) compared to the mRNA platform, specifically longer expression [Johanning et al., 1995] and higher immunogenicity at lower dosages [Brito et al., 2014]. However, this simple temperature-controllable feature allows for the coexistence of many desirable features of a T cell-inducing vaccine, as briefly described below.

[0012] Briefly, srRNA1ts2 is a temperature-sensitive, self-replicating VEEV-based RNA replicon developed for transient expression of heterologous proteins. Temperature sensitivity is conferred by the insertion of five amino acid residues within VEEV nonstructural protein 2 (nsP2). The nsP2 protein is a helicase / proteinase that, together with nsP1, nsP3, and nsP4, constitutes the VEEV replicase. srRNA1ts2 does not contain the VEEV structural proteins (capsid, E1, E2, and E3). The disclosure of WO 2021 / 138447(A1) to Elixirgen Therapeutics Inc. is incorporated herein by reference. In particular, Example 3, FIG. 12, and SEQ ID NOs:29-49 of WO 2021 / 138447(A1) are incorporated herein by reference.

[0013] Exemplary vectors include three different temperature-controllable self-replicating RNA vectors (c-srRNAs) and a control self-replicating RNA vector (c-srRNA). The properties of srRNAs 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 Virol, 72:10286-10291, 1998). c-srRNA1 was based on the TRD strain of VEEV but was modified to contain the A16D substitution (TC83 mutation) and the P778S substitution. c-srRNA3 was also based on the TRD strain of VEEV but did not contain the A16D and P778S substitutions. srRNA4 was based on the V198 strain of VEEV isolated from a human. All three c-srRNA vectors contain the same five amino acid insertion within the VEEV nsP2 protein for temperature regulation, as previously described (see U.S. Pat. No. 11,421,248 to Ko, Examples 3, 21, and 22, incorporated herein by reference). [Table 1]

[0014] The nucleotide sequences of the VEEV genomes are disclosed in GenBank under GenBank No. L01442.2 for the TRD strain and GenBank No. L01443.1 for the TC-83 strain. 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); c-srRNA consensus (SEQ ID NO:12).

[0015] General Techniques and Definitions The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art.

[0016] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, "an" excipient includes one or more excipients.

[0017] As used herein, the phrase "comprising" is open-ended and indicates that such embodiments may include additional elements. In contrast, the phrase "consisting of" is closed and indicates that such embodiments do not include additional elements (except for minor impurities). The phrase "consisting essentially of" is part-closed and indicates that such embodiments may include additional elements that do not materially alter the basic characteristics of such embodiments.

[0018] The term "about" as used herein in reference to a value includes 90% to 110% of that value (e.g., when used in reference to a chitosan oligosaccharide, a molecular weight of about 5,000 daltons refers to 4,500 daltons to 5,500 daltons).

[0019] The term "antigen" refers to a substance that is specifically recognized and 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" typically refers to a polypeptide or protein antigen of at least eight amino acid residues in length, which may contain one or more post-translational modifications.

[0020] The terms "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues and are not limited to a particular length unless otherwise specified. A polypeptide can include natural amino acid residues or a combination of natural and unnatural amino acid residues. The term also includes post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, and the like. In some aspects, a polypeptide can include modifications relative to the native or naturally occurring sequence, so long as the protein maintains a desired activity (e.g., antigenicity).

[0021] As used herein, the terms "isolated" and "purified" refer to 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 with respect to a recombinant protein, the term "isolated" refers to a protein that has been removed from the culture medium of a host cell that produced the protein. In some embodiments, an isolated protein (e.g., a WT1 protein) is at least 75%, 90%, 95%, 96%, 97%, 98%, or 99% pure as determined by HPLC.

[0022] An "effective amount" or "sufficient amount" of a substance is an amount sufficient to bring about beneficial or desired results, including clinical results, and thus, an "effective amount" will vary depending on the context in which it is applied. In the context of administering a composition of the present disclosure that includes mRNA encoding an antigen, an effective amount contains sufficient mRNA to stimulate an immune response, preferably a cellular immune response to the antigen.

[0023] The term "treating" a disease or "treatment" of a disease refers to carrying out a protocol that may include administering one or more drugs to an individual (human or otherwise) to alleviate signs or symptoms of the disease. Thus, "treating" or "treatment" does not require complete relief of signs or symptoms, does not require a cure, and specifically includes protocols that have only a palliative effect on an individual. As used herein, and as well understood in the art, "treatment" is an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results include, but are not limited to, relief or amelioration of one or more symptoms, reduction in the extent of disease, stabilized (i.e., not worsening) disease, prevention of disease spread, delay or slowing of disease progression, improvement or palliation of the disease state, and remission. "Treatment" can also mean extending the survival of a cancer patient compared to the expected survival of a control patient not receiving treatment. To "ameliorate" a disease or disorder means to lessen the severity and / or undesirable clinical symptoms of the disease or disorder and / or slow the time course of progression of the disease or disorder compared to the expected outcome of untreated treatment.

[0024] In this disclosure, the terms "individual" and "subject" refer to a mammal. "Mammal" includes, but is not limited to, humans, non-human primates (e.g., monkeys), farm animals, 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.

[0025] The term "dose," as used herein with respect to a composition comprising an mRNA encoding an antigen, refers to a measured amount taken by (administered to or received by) a subject at any one time. Administering a composition of the present disclosure to a subject in need thereof includes administering an effective amount of a composition comprising an mRNA encoding an antigen to stimulate an immune response to the antigen in the subject.

[0026] "Stimulation" of a response or parameter includes eliciting and / or enhancing that response or parameter when compared to conditions that are otherwise the same except for the parameter of interest, or when compared to another condition (e.g., an increase in antigen-specific cytokine secretion following administration of a composition that contains or encodes an antigen compared to administration of a control composition that does not contain or encode the antigen). For example, "stimulation" of an immune response (e.g., a Th1 response) refers to an increase in the response. Depending on the parameter being measured, the increase can be 2-fold to 200-fold or more, 5-fold to 500-fold or more, 10-fold to 1000-fold or more, or 2-, 5-, 10-, 50-, or 100-fold to 200-, 500-, 1,000-, 5,000-, or 10,000-fold.

[0027] Conversely, "inhibition" of a response or parameter includes reducing and / or suppressing that response or parameter when compared to conditions that are otherwise the same except for the parameter of interest, or when compared to another condition. For example, "inhibition" of an immune response (e.g., a Th2 response) refers to a decrease in the response. Depending on the parameter being measured, the decrease can be 2-fold to 200-fold, 5-fold to 500-fold or more, 10-fold to 1000-fold or more, or 2-, 5-, 10-, 50-, or 100-fold to 200-, 500-, 1,000-, 2,000-, 5,000-, or 10,000-fold.

[0028] The relative terms "higher" and "lower" refer to a measurable increase or decrease, respectively, in a response or parameter when compared to conditions that are otherwise the same except for the parameter of interest, or when compared to another condition. For example, a "higher antibody titer" refers to an antigen-reactive antibody titer resulting from administration of a composition of the present disclosure comprising an mRNA encoding an antigen that is at least 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold higher than the antigen-reactive antibody titer resulting from control conditions (e.g., administration of a comparative composition that does not comprise mRNA or that comprises a control mRNA that does not encode the antigen). Similarly, a "lower antibody titer" refers to an antigen-reactive antibody titer resulting from administration of a composition of the present disclosure comprising an mRNA encoding the antigen that is at least 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold lower than the antigen-reactive antibody titer resulting from control conditions (e.g., administration of a comparative composition that does not comprise mRNA or that comprises a control mRNA that does not encode the antigen).

[0029] As used herein in reference to an agent (e.g., an RNA molecule), the term "temperature sensitive" refers to an agent that has activity at a "permissive temperature" but has reduced activity at higher and / or lower "non-permissive temperatures."

[0030] As used herein, the term "permissive temperature" refers to any temperature at which activity of a temperature-sensitive agent of the present disclosure is induced. Typically, the permissive temperature is not the subject's normal body temperature. Normal body temperature for a human subject is approximately 37°C ± 0.5°C. Depending on the temperature-sensitive agent, the permissive temperature may be higher or lower than the subject's normal body temperature. In some aspects, the permissive temperature for a temperature-sensitive agent ranges from 30°C to 36°C. In some embodiments, the permissive temperature is 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 for a temperature-sensitive self-replicating RNA of the present disclosure is greater than 36°C. In some preferred embodiments, the non-permissive temperature is 37°C ± 0.5°C.

[0031] As used herein, the term "non-permissive temperature" refers to any temperature at which the activity of a temperature-sensitive agent of the present disclosure is not induced. A temperature-sensitive agent is not induced if its activity is at least 95% lower, at least 90% lower, at least 85% lower, at least 80% lower, at least 75% lower, or at least 50% lower than the activity level at the optimal permissive temperature. Typically, the non-permissive temperature is the subject's normal body temperature. Depending on the temperature-sensitive agent, the non-permissive temperature can also be higher (e.g., 38°C or higher) or lower (e.g., less than 30°C) than the subject's normal body temperature.

[0032] As used herein, the term "immunization" refers to the process of mounting a mammalian subject's response to an antigen, thereby improving its ability to resist or overcome infection and / or resist disease.

[0033] As used herein, the term "vaccination" refers to the introduction of a vaccine into the body of a mammalian subject.

[0034] As used herein, "percent (%) amino acid sequence identity," "percent identity," and "sequence identity," when used in reference to an amino acid sequence (reference polypeptide sequence), are defined as the percentage of amino acid residues in a candidate sequence (e.g., a target antigen) that are identical to those in the reference polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity; any conservative substitutions are not considered as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximum alignment over the entire length of the sequences being compared.

[0035] Exemplary amino acid sequences are set forth in sequence identifiers throughout this disclosure. Some claimed embodiments are described by reference to the percent identity shared with exemplary amino acid sequences. Two amino acid sequences are substantially identical if, when compared and aligned for maximum correspondence over a comparison window or designated region, their amino acid sequences share at least 90% identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over a specified region, or, if not specified, over the entire sequence). For purposes of this disclosure and claims, amino acid sequences are aligned and sequence identity determined using the BLASTP sequence comparison algorithm with default parameters.

[0036] Suitable algorithms for determining percent sequence identity and percent sequence similarity are the BLAST algorithm and the BLAST 2.0 algorithm, 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 analysis is publicly available through the website of the National Center for Biotechnology Information (NCBI). This algorithm involves first identifying short words of length W in a query sequence and identifying high-scoring sequence pairs (HSPs) by either matching when aligned with words of the same length in database sequences or satisfying some positive threshold score T. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits serve as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using the parameters M (reward score for a pair of matching residues; always greater than 0) and N (penalty score for mismatching residues; always less than 0) for nucleotide sequences. For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of the word hits in each direction is stopped when: the cumulative alignment score falls by an amount X from its maximum achieved value; the cumulative score falls below zero due to the accumulation of one or more negative-scoring residue alignments; or 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 as defaults a word size (W) of 28, an expectation (E) of 10, M=1, N=-2, and a comparison of both strands.For amino acid sequences, the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1989).

[0037] Amino acid substitutions can include replacing one amino acid in a polypeptide with another amino acid. Amino acid substitutions can be introduced into an antigen of interest and the products can be screened for a desired activity (e.g., increased stability and / or immunogenicity).

[0038] Amino acids can be broadly classified according to the following common side chain properties: (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; (6) Aromatic: Trp, Tyr, Phe.

[0039] Conservative amino acid substitutions involve exchanging a member of one of these classes for another member of the same class, while non-conservative amino acid substitutions involve exchanging a member of one of these classes for a member of another class.

[0040] As used herein, the term "excipient" refers to a compound present in a composition containing an active ingredient (e.g., an mRNA encoding an antigen). Pharmaceutically acceptable excipients are inert pharmaceutical compounds and may include, for example, solvents, bulking agents, buffers, tonicity agents, and preservatives (Pramanick et al., Pharma Times, 45:65-77, 2013). In some embodiments, the compositions of the present disclosure include an excipient that functions as one or more of a solvent, bulking agent, buffer, and tonicity agent (e.g., sodium chloride in saline can serve as both an aqueous vehicle and a tonicity agent).

[0041] Optimizing intradermal delivery for cell-mediated immunity Intradermal vaccination results in long-lasting cellular immunity and enhanced 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 5–10 times more effective than subcutaneous or intramuscular vaccination because it targets APCs [Hickling and Jones, 2009]. This targeting also activates T cell immune pathways for long-lasting immunity. Upon intradermal injection, c-srRNA is primarily taken up by skin APCs, where it replicates, produces antigens, digests the antigens into peptides, and presents these peptides to T cells (Figure 1). Peptides presented via this pathway stimulate MHC-I-restricted CD8+ killer T cells. In another pathway, APCs also take up antigens produced by nearby skin cells. Peptides presented via this pathway stimulate MHC-II-restricted CD4+ helper T cells.

[0042] Intradermal injection problems and solutions Here is a potential problem we have identified and a solution our c-srRNA platform offers.

[0043] (1) A significant unrecognized hurdle for the application of srRNA, such as in intradermal vaccine platforms, is that both mRNA and srRNA do not adequately express antigens at skin temperature [International Application No. PCT / US20 / 67506]. Confoundingly, human skin temperature (approximately 30–35°C) is lower than human core body temperature (approximately 37°C). This means that vectors and platforms developed at 37°C are not optimal for intradermal injection. One innovation of our c-srRNA platform is that it strongly expresses antigens at skin temperature [International Application No. PCT / US20 / 67506]. Furthermore, this temperature control also minimizes safety risks caused by unintended systemic distribution of c-srRNA, because c-srRNA is inactivated when its temperature rises above its tolerance threshold (when c-srRNA approaches the deep body). In other words, the c-srRNA platform expresses the best antigen for intradermal injection compared to mRNA and srRNA, and it also has safety features that limit or inactivate the ability of the vector to spread and become produced in other areas of the subject's body.

[0044] (2) Another challenge with intradermal vaccination is the lack of suitable additives. Clinically approved intradermal vaccines do not incorporate any adjuvants because adjuvants such as aluminum salts and oil-in-water adjuvants are too reactogenic locally when delivered intradermally, resulting in reduced immunogenicity [Hickling and Jones, 2009]. Lipid nanoparticles (LNPs) used in intramuscularly administered mRNA and srRNA vaccines are also oil-in-water, which can cause skin reactogenicity and increase the risk of allergic reactions to LNP components such as PEG. Our c-srRNA platform, injected as naked c-srRNA (no LNPs or adjuvants), offers a solution to this problem. First, self-replication of RNA within cells, particularly APCs, induces potent innate immunity, which replaces the primary function of adjuvants. Second, the literature and our own data demonstrate that naked mRNA / srRNA produces antigens equally efficiently compared to electroporation of mRNA / srRNA [Johansson et al., 2012] and mRNA / srRNA combined with LNPs [Golombek et al., 2018], especially for intradermal injection.

[0045] (3) A third challenge is the limited number of precedents for intradermal vaccines. Only the BCG vaccine has been routinely administered intradermally. One way to lower the hurdle for adopting intradermal injection is through the use of specialized devices, such as the MicronJet 600 (NanoPass) and Immucise (Terumo), which are currently available to enable easy and consistent intradermal injection. These devices are also good candidates for large-scale production and deployment. However, because these specialized devices are relatively expensive, intradermal injection via the Mantoux technique using a standard needle and syringe is also an option.

[0046] Designing suitable antigens Tumor-associated antigens (TAA) are expressed in tumor cells but also in embryonic cells or at low levels in normal cells. The National Cancer Institute selected 75 cancer antigens suitable as targets for cancer therapy (Cheever et al., 2009). For example, Wilms' tumor 1 (WT1) was ranked as the most promising of 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 may be used as antigen(s) for the c-srRNA platform-based cancer vaccine described herein. It is also possible to use multiple TAAs expressed as a fusion protein (Example 3) or multiple TAAs expressed separately.

[0047] In recent years, it has become common to perform genomic sequencing of tumor cells from patients. Such efforts often identify protein products or peptides that are unique to tumors due to mutations in their genomes. These tumor-specific antigens (TSAs), also called neoantigens, are ideal targets for cancer vaccines. A single TSA or a fusion of multiple TSAs may be used as an antigen for the c-srRNA platform-based cancer vaccine described herein (Examples 1 and 2).

[0048] Most tumor-specific antigens (TSAs) contain specific mutations, often single amino acid changes, compared to the normal protein. For example, a glycine (G) to aspartic acid (D) change at position 12 of KRAS (G12D) is commonly found in human cancers. The challenge is how to design an antigen that elicits strong T cell immunity, particularly CD8+ cytotoxic lymphocytes, against this specific mutation but not against the wild-type (normal) protein. Activation of CD8+ cytotoxic lymphocytes requires dendritic cells to present MHC class I molecules loaded with short peptides (typically 9-mers, i.e., 9 amino acids). The design of a 17-mer peptide containing the mutated amino acid in its center is shown in Step 1 of Figure 1. In this way, any 9-mer peptides processed from the 17-mer peptide will contain the mutated amino acid, thus making these 9-mer peptides specific for the mutant protein (neo-antigen). If the mutated amino acid is located near the N- or C-terminus, the sequence on one side can be shorter than the 9-mer. This process is repeated for other mutations to identify multiple 17mer sequences containing the mutation of interest, as shown in step 2 of Figure 1. 17mer peptide sequences can be identified from other mutations at the same position in the same oncoprotein (e.g., G12D, G12V, and G12R in the KRAS protein), mutations at other positions in the same oncoprotein (e.g., Q61H and Q61K in the KRAS protein), and / or mutations in other oncoproteins (e.g., R175H, R248Q, and R273H in the human TP53 oncoprotein p53). Finally, these 17mer peptides are concatenated or expressed as a recombinant fusion protein, as shown in step 3 of Figure 1. Typically, no additional amino acids are inserted between the 17mer peptide sequences. Alternatively, a non-immunogenic glycine / serine linker can be inserted between one or more 17mer peptide sequences. The order of each 17mer peptide sequence may be altered from the exemplary fusion protein described in Example 1.Thus, exemplary embodiments include mRNA molecules that encode KRAS polyproteins in which each neo-antigenic peptide of the polyprotein is 17 amino acids in length. However, in further embodiments, the mRNA molecules may encode KRAS polyproteins in which each neo-antigenic peptide of the polyprotein is 16-24 amino acids in length (16-, 17-, 18-, 19-, 20-, 21-, 22-, 23-, or 24-mer).

[0049] Efficient activation of CD8+ cytotoxic lymphocytes often requires activation of CD4+ helper T cells. For this purpose, dendritic cells also need to present MHC class II molecules loaded with longer peptides (typically 15-mers, i.e., 15 amino acids). The design of a 29-mer peptide containing a mutant amino acid in the middle is shown in Step 1 of Figure 2. In this way, any 15-mer peptides processed from the 29-mer peptide will contain the mutant amino acid, thus making these 15-mer peptides specific for the mutant protein (neoantigen). The processed peptides from the 29-mer contain 9-mer peptides loaded onto MHC class I molecules. Many of these 9-mer peptides contain the mutant amino acid, but some are wild-type. If the mutant amino acid is located near the N- or C-terminus, the sequence on either side can be shorter than the 15-mer. This process is repeated for other mutations to identify multiple 29-mer sequences containing the desired mutation, as shown in Step 2 of Figure 2. 29-mer peptide sequences can be identified from other mutations at the same position in the same oncoprotein (e.g., G12D, G12V, and G12R in the KRAS protein), mutations at other positions in the same oncoprotein (e.g., Q61H and Q61K in the KRAS protein), and / or mutations in other oncoproteins (e.g., R175H, R248Q, and R273H in the human TP53 oncoprotein p53). Finally, these 29-mer peptides are concatenated or expressed as a fusion protein, as shown in step 3 of Figure 2. Typically, 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 may be altered from the exemplary fusion protein described in Example 2. Thus, exemplary embodiments include an mRNA molecule encoding a KRAS polyprotein, in which each neo-antigenic peptide of the polyprotein is 29 amino acids long.However, in further embodiments, the mRNA molecule may encode a KRAS polyprotein in which each neo-antigenic peptide of the polyprotein is 26 to 34 amino acids in length (26, 27, 28, 29, 30, 31, 32, 33, or 34-mer).

[0050] Enhancement of gene expression in vivo by chitosan RNase inhibitors (proteins purified from human placenta) slightly enhance the immunogenicity of antigens encoded on c-srRNA, presumably by enhancing the expression of antigens from c-srRNA in vivo when injected intradermally into mice (see, e.g., Figure 25C of WO 2021 / 138447(A1)). RNase inhibitors may protect c-srRNA from RNase-mediated degradation in vivo. However, due to the challenges of using protein-based RNase inhibitors as excipients in injectable products, it is desirable to find alternative agents that can enhance the expression of genes of interest (GOIs) in vivo for therapeutic purposes.

[0051] Low-molecular-weight chitosan (approximately 6 kDa) has been shown to inhibit RNase activity with inhibition constants ranging from 30 to 220 nM (Yakovlev et al., Biochem Biophys Res Commun, 357(3):584-8, 2007). Recently, two different chitosan oligomers have been tested: chitosan oligomer (catalog no. 9012-76-4; molecular weight ≤5 kDa, >75% deacetylated; Heppe Medical Chitosan GmbH; product no. 44009) and chitosan oligosaccharide lactate (CAS no. 148411-57-8; molecular weight approx. 5 kDa, >90% deacetylated; Sigma-Aldrich; product no. 523682). Surprisingly, even very low levels of chitosan oligomers, 0.001 μg / mL (approximately 0.2 nM, approximately 1 / 100 of the inhibition constant found by Yakovlev et al., supra, 2007), were found to be able to enhance the expression of luciferase encoded on c-srRNA by approximately 10-fold (data not shown). Similar enhancement of GOI expression was achieved with chitosan oligomers up to 0.5 μg / mL and chitosan oligosaccharide lactate at 0.1 μg / mL.

[0052] Chitosan has been used as a nucleotide (DNA and RNA) delivery vector due to its ability to 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 should be noted that the enhancement of GOI expression by chitosan oligomers is unlikely to be mediated by nanoparticle or complex formation between c-srRNA and chitosan oligomers. First, such low concentrations of chitosan oligomers do not allow complex formation with RNA. Second, chitosan oligomers are added to c-srRNA immediately before intradermal injection, which does not allow sufficient time for complex formation.

[0053] Because chitosan oligomers enhance GOI expression in vivo at concentrations much lower than their effective concentration as RNase inhibitors in vitro (Yakovlev et al., supra, 2007), it is possible that this enhanced GOI expression by chitosan oligomers is not mediated by its RNase inhibitory mechanism. For example, chitosan oligomers may promote the uptake of c-srRNA into cells, thereby enhancing GOI expression from c-srRNA. Nevertheless, this surprising finding provides an effective means for enhancing the therapeutic expression of GOIs encoded on c-srRNA in vivo. Enumerated Embodiments 1. An RNA molecule comprising an open reading frame (ORF) encoding a fusion protein, the ORF comprising, from 5' to 3': (i) a nucleotide sequence encoding a mammalian signal peptide; (ii) a nucleotide sequence encoding a cancer antigen; Including, The cancer antigen comprises a KRAS polyprotein, and the amino acid sequence of the KRAS polyprotein is a) a first segment, MTEYKLVVVGAX1GVGKSALTIQLIQNXaXbXcXdXeXf MTEYKLVVVGAX2GVGKSALTIQLIQNXaXbXcXdXeXf MTEYKLVVVGAX3GVGKSALTIQLIQNXaXbXcXdXeXf MTEYKLVVVGAX4GVGKSALTIQLIQNXaXbXcXdXeXf MTEYKLVVVGAX5GVGKSALTIQLIQNXaXbXcXdXeXf MTEYKLVVVGAX6GVGKSALTIQLIQNXaXbXcXdXeXf(SEQ ID NO:53); a first segment including: b) a second segment, MTEYKLVVVGAGX 10VGKSALTIQLIQNHXaXbXcXdXeXf MTEYKLVVVGAGX 11 VGKSALTIQLIQNHXaXbXcXdXeXf MTEYKLVVVGAGX 12 VGKSALTIQLIQNHXaXbXcXdXeXf MTEYKLVVVGAGX 13 VGKSALTIQLIQNHXaXbXcXdXeXf(SEQ ID NO:54) a second segment including: c) a third segment, DGETCLLDILDTAGX7EEYSAMRDQYMRTGXaXbXcXdXeXf DGETCLLDILDTAGX8EEYSAMRDQYMRTGXaXbXcXdXeXf DGETCLLDILDTAGX9EEYSAMRDQYMRTGXaXbXcXdXeXf(SEQ ID NO:55) a third segment including wherein the first segment, the second segment, and the third segment are arranged in any order; X1, X2, X3, X4, X5, and X6 are independently selected from D, V, R, C, A, and S; X7, X8, and X9 are independently selected from H, K, and R; X 10 , X 11 , X 12 , and X 13 is independently selected from D, C, P, and S; Xa, Xb, Xc, Xd, Xe, and Xf are independently selected from G, S, and absent; RNA molecule. 2. The RNA molecule of embodiment 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 of embodiment 2, wherein the amino acid sequence of the KRAS polyprotein comprises residues 25 to 375 of SEQ ID NO:20. 4. An RNA molecule comprising an open reading frame (ORF) encoding a fusion protein, the ORF comprising, from 5' to 3': (i) a nucleotide sequence encoding a mammalian signal peptide; (ii) a nucleotide sequence encoding a cancer antigen; Including, The cancer antigen comprises a KRAS polyprotein, and the amino acid sequence of the KRAS polyprotein is a) a first segment, YKLVVVGAX1GVGKSALTXaXbXcXdXeXfYKLVVVGAX2GVGKSALTXaXbXcXdXeXf YKLVVVGAX3GVGKSALTXaXbXcXdXeXfYKLVVVGAX4GVGKSALTXaXbXcXdXeXf YKLVVVGAX5GVGKSALTXaXbXcXdXeXfYKLVVVGAX6GVGKSALT(SEQ ID NO:56) a first segment including: b) a second segment, KLVVVGAGX 10 VGKSALTIXaXbXcXdXeXfKLVVVGAGX 11 VGKSALTIXaXbXcXdXeXf KLVVVGAGX 12 VGKSALTIXaXbXcXdXeXfKLVVVGAGX 13VGKSALTI (SEQ ID NO: 57) a second segment including: c) a third segment, LDILDTAGX7HEEYSAMRDXaXbXcXdXeXfLDILDTAGX8HEEYSAMRDXaXbXcXdXeXf LDILDTAGX9HEEYSAMRD(SEQ ID NO:58) a third segment including wherein the first segment, the second segment, and the third segment are arranged in any order; X1, X2, X3, X4, X5, and X6 are independently selected from D, V, R, C, A, and S; X7, X8, and X9 are independently selected from H, K, and R; X 10 , X 11 , X 12 , and X 13 is independently selected from D, C, P, and S; Xa, Xb, Xc, Xd, Xe, and Xf are independently selected from G, S, and absent; RNA molecule. 5. The RNA molecule of embodiment 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 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 of embodiment 5, wherein the amino acid sequence of the KRAS polyprotein comprises residues 25 to 245 of SEQ ID NO:18. 7. An RNA molecule comprising an open reading frame (ORF) encoding a fusion protein, the ORF comprising, from 5' to 3': (i) a nucleotide sequence encoding a mammalian signal peptide; (ii) a nucleotide sequence encoding a cancer antigen; Including, The cancer antigen comprises a KRAS polyprotein, and the amino acid sequence of the KRAS polyprotein is YKLVVVGAX1GVGKSALTXaXbXcXdXeXfYKLVVVGAX2GVGKSALTXaXbXcXdXeXf YKLVVVGAX3GVGKSALTXaXbXcXdXeXfYKLVVVGAX4GVGKSALTXaXbXcXdXeXf YKLVVVGAX5GVGKSALTXaXbXcXdXeXfYKLVVVGAX6GVGKSALT(SEQ ID NO:56) Including, wherein X1, X2, X3, X4, X5, and X6 are independently selected from D, V, R, C, A, and S; Xa, Xb, Xc, Xd, Xe, and Xf are independently selected from G, S, and absent; RNA molecule. 8. The RNA molecule of embodiment 7, 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, and SEQ ID NO:28. 9. The RNA molecule of embodiment 8, wherein the amino acid sequence of the KRAS polyprotein comprises SEQ ID NO:29. 10. An RNA molecule comprising an open reading frame (ORF) encoding a fusion protein, the ORF comprising, from 5' to 3': (i) a nucleotide sequence encoding a mammalian signal peptide; (ii) a nucleotide sequence encoding a cancer antigen; Including, The cancer antigen comprises a KRAS polyprotein, and the amino acid sequence of the KRAS polyprotein is DGETCLLDILDTAGX7EEYSAMRDQYMRTGXaXbXcXdXeXf DGETCLLDILDTAGX8EEYSAMRDQYMRTGXaXbXcXdXeXf DGETCLLDILDTAGX9EEYSAMRDQYMRTGXaXbXcXdXeXf(SEQ ID NO:55) Including, wherein X7, X8, and X9 are independently selected from H, K, and R; Xa, Xb, Xc, Xd, Xe, and Xf are independently selected from G, S, and absent; RNA molecule. 11. The RNA molecule of embodiment 10, wherein the amino acid sequence of the KRAS polyprotein comprises SEQ ID NO: 32, SEQ ID NO: 33, and SEQ ID NO: 34. 12. The RNA molecule of embodiment 11, wherein the amino acid sequence of the KRAS polyprotein comprises SEQ ID NO: 35. 13. An RNA molecule comprising an open reading frame (ORF) encoding a fusion protein, the ORF comprising, from 5' to 3': (i) a nucleotide sequence encoding a mammalian signal peptide; (ii) a nucleotide sequence encoding a cancer antigen; Including, The cancer antigen comprises a KRAS polyprotein, and the amino acid sequence of the KRAS polyprotein is KLVVVGAGX 10 VGKSALTIXaXbXcXdXeXfKLVVVGAGX 11 VGKSALTIXaXbXcXdXeXf KLVVVGAGX 12 VGKSALTIXaXbXcXdXeXfKLVVVGAGX 13 VGKSALTI (SEQ ID NO: 57) Including, X 10 , X 11 , X 12 , and X 13 is independently selected from D, C, P, and S; Xa, Xb, Xc, Xd, Xe, and Xf are independently selected from G, S, and absent; RNA molecule. 14. The RNA molecule of 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 comprising an open reading frame (ORF) encoding a fusion protein, the ORF comprising, from 5' to 3': (i) a nucleotide sequence encoding a mammalian signal peptide; (ii) a nucleotide sequence encoding a cancer antigen; Including, The cancer antigen comprises a KRAS polyprotein, and the amino acid sequence of the KRAS polyprotein is LDILDTAGX7HEEYSAMRDXaXbXcXdXeXfLDILDTAGX8HEEYSAMRDXaXbXcXdXeXf LDILDTAGX9HEEYSAMRD(SEQ ID NO:58) Including, wherein X7, X8, and X9 are independently selected from H, K, and R; Xa, Xb, Xc, Xd, Xe, and Xf are independently selected from G, S, and absent; RNA molecule. 16. The RNA molecule of embodiment 15, wherein the amino acid sequence of the KRAS polyprotein comprises SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO: 42. 17. The RNA molecule of embodiment 8, embodiment 14, or embodiment 16, wherein the amino acid sequence of the KRAS polyprotein comprises residues 25 to 245 of SEQ ID NO: 18. 18. An RNA molecule comprising an open reading frame (ORF) encoding a fusion protein, the ORF comprising, from 5' to 3': (i) a nucleotide sequence encoding a mammalian signal peptide; (ii) a nucleotide sequence encoding a cancer antigen; Including, The cancer antigen comprises a KRAS polyprotein, and the amino acid sequence of the KRAS polyprotein is 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; Xa, Xb, Xc, Xd, Xe, and Xf are independently selected from G, S, and absent; RNA molecule. 19. The RNA molecule of embodiment 18, 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, and SEQ ID NO:48. 20. An RNA molecule comprising an open reading frame (ORF) encoding a fusion protein, the ORF comprising, from 5' to 3': (i) a nucleotide sequence encoding a mammalian signal peptide; (ii) a nucleotide sequence encoding a cancer antigen; Including, The cancer antigen comprises a KRAS polyprotein, and the amino acid sequence of the KRAS polyprotein is MTEYKLVVVGAGX 10 VGKSALTIQLIQNHXaXbXcXdXeXf MTEYKLVVVGAGX 11VGKSALTIQLIQNHXaXbXcXdXeXf MTEYKLVVVGAGX 12 VGKSALTIQLIQNHXaXbXcXdXeXf MTEYKLVVVGAGX 13 VGKSALTIQLIQNHXaXbXcXdXeXf(SEQ ID NO:54) Including, where X 10 , X 11 , X 12 , and X 13 is independently selected from D, C, P, and S; Xa, Xb, Xc, Xd, Xe, and Xf are independently selected from G, S, and absent; RNA molecule. 21. The RNA molecule of 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 of embodiment 11, embodiment 19, or embodiment 21, wherein the amino acid sequence of the KRAS polyprotein comprises residues 25 to 375 of SEQ ID NO:20. 23. The RNA molecule according to any one of embodiments 1 to 22, wherein the mammalian signal peptide is a signal peptide of a surface protein expressed in a mammalian antigen-presenting cell. 24. The RNA molecule of 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 of any one of embodiments 1 to 24, comprising at least one modified nucleoside, optionally wherein said at least one modified nucleoside comprises pseudouridine. 26. A DNA template for an RNA molecule according to any one of embodiments 1 to 25, optionally comprising a first restriction enzyme site upstream of the nucleotide sequence encoding a mammalian signal peptide and a second restriction site downstream of the nucleotide sequence encoding a cancer antigen. 27. An expression vector comprising the DNA template of embodiment 26. 28. A host cell comprising an expression vector according to embodiment 27. 29. The RNA molecule according to any one of embodiments 1 to 25, wherein the RNA molecule is a self-replicating RNA. 30. A composition for stimulating an immune response to a cancer antigen in a mammalian subject, comprising an excipient and the temperature-sensitive self-replicating RNA of 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 the temperature-sensitive self-replicating RNA is capable of expressing a fusion protein at a permissive temperature but not at a non-permissive temperature. 31. A composition for stimulating an immune response to a cancer antigen in a mammalian subject, comprising an excipient and a 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; (ii) a nucleotide sequence encoding a cancer antigen; Including, the temperature-sensitive self-replicating RNA is capable of expressing a fusion protein at a permissive temperature but not at a non-permissive temperature; The composition, wherein the cancer antigens comprise NY-ESO-1 antigen, MAGEA3 antigen, TYR antigen, and TPTE antigen. 32. The composition of embodiment 31, wherein the mammalian signal peptide is a signal peptide of a surface protein expressed in a mammalian antigen-presenting cell. 33. The composition of 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 of 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. The composition of any one of embodiments 30-34, wherein the alphavirus is selected from the group consisting of Venezuelan equine encephalitis virus, Sindbis virus, and Semliki Forest virus. 36. The composition of embodiment 35, wherein the alphavirus is Venezuelan equine encephalitis virus. 37. The composition of any one of embodiments 30 to 36, wherein the alphavirus replicon comprises a nonstructural protein coding region having an insertion of 12 to 18 nucleotides that results in expression of nonstructural protein 2 (nsP2), and the nsP2 comprises 4 to 6 additional amino acids between beta sheet 5 and beta sheet 6 of the nsP2. 38. The composition of embodiment 37, wherein the additional amino acids comprise the sequence of SEQ ID NO: 14 (TGAAA). 39. The composition of embodiment 38, wherein the amino acid sequence of nsP2 comprises SEQ ID NO: 12. 40. The composition of embodiment 39, wherein the amino acid sequence of nsP2 comprises one sequence selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11. 41. The composition of embodiment 40, wherein the amino acid sequence of nsP2 comprises SEQ ID NO:11. 42. The composition of any one of embodiments 30-41, wherein the permissive temperature is 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, the permissive temperature is 31°C to 35°C and the non-permissive temperature is at least 37°C ± 0.5°C. 43. The composition of any one of embodiments 30-42, wherein the composition does not comprise lipid nanoparticles. 44. The composition of any one of embodiments 30-43, wherein the composition further comprises chitosan. 45. A method for stimulating an immune response to a cancer antigen in a mammalian subject, comprising administering to the mammalian subject a composition described in any one of embodiments 30 to 44, thereby stimulating an immune response to the cancer antigen in the mammalian subject. 46. ​​The method of embodiment 45, wherein the composition is administered intradermally. 47. The method of 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 of 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 of embodiment 48, wherein the immune response further comprises a humoral immune response reactive with a cancer antigen. 50. The method of any one of embodiments 45-49, wherein the mammalian subject is a human subject. 51. (i) a composition according to any one of embodiments 30 to 44; and (ii) a device for intradermal delivery of the composition to a mammalian subject; and Includes a kit. 52. The kit of embodiment 51, wherein the device comprises a syringe and a needle. 53. A method for expressing a fusion protein, the method comprising contacting a mammalian cell with an RNA molecule according to any one of embodiments 1 to 25. 54. The method of embodiment 53, wherein the contacting is in vitro. 55. The method of embodiment 53, wherein the contacting is in vivo. 56. A method for treating cancer, comprising administering to a mammalian subject in need thereof an effective amount of the composition of any one of embodiments 30-44 to treat cancer. 57. The method of embodiment 56, wherein 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 of embodiment 56, wherein cells of the cancer express one or more of the following antigens: NY-ESO-1 antigen, MAGEA3 antigen, TYR antigen, and TPTE antigen. 59. The method of 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 to 24, or the mature form of the fusion protein after cleavage of the signal peptide. [Example]

[0054] Abbreviations: APC (antigen-presenting cell); BIRC5 (baculovirus IAP repeat-containing 5 or SURVIVIN); GOI (gene of interest); IL-4 (interleukin-4); IFN-γ (interferon gamma); MAGEA3 (melanoma-associated antigen 3); ORF (open reading frame); PBO (placebo); NY-ESO-1 (New York esophageal squamous cell carcinoma 1 or CTAG1B); PRAME (antigen preferentially expressed in melanoma); SFC (spot-forming cell); srRNAts (temperature-sensitive self-replicating RNA = c-srRNA temperature-regulatable self-replicating RNA); TAA (tumor-associated antigen); TPTE (transmembrane phosphatase with tensin homology); TSA (tumor-specific antigen); TYR (tyrosinase); and WT1 (Wilms' tumor 1).

[0055] Example 1. Immunotherapy for tumors expressing KRAS mutations This example describes the production of fusion proteins containing multiple KRAS substitutions based on the design principles shown in Figure 1. KRAS proteins with substitutions at one or more of positions 12, 13, and 61 are exemplary tumor-specific antigens (TSAs).

[0056] Materials and Methods BALB / c inbred female mouse.

[0057] EXG-5109 mRNA was produced by in vitro transcription of a plasmid containing a temperature-controllable, self-replicating RNA expression cassette (c-srRNA3) encoding a fusion protein (TSA-5109) containing 13 different 17-mer peptides derived from 13 common mutations of the human KRAS proto-oncogene (G12D; G12V, G12R, G12C, G12A, G12S, G13D, G13C, G13P, G13S, Q61H, Q61K, and Q61R). 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 is set forth in SEQ ID NO: 18.

[0058] The wild-type and mutant peptides shown in Table 1-1 are used to restimulate T cells in splenocyte samples taken from immunized mice. [Table 2]

[0059] The CT26 mouse colon cancer cell line (ATCC CRL-2638) is derived from a BALB / c mouse strain and is known to have a G12D mutation in the KRAS proto-oncogene. The mouse KRAS protein sequence is identical to the human KRAS protein in this region, so the EXG-5109 vaccine developed for human use can be tested in mice. CT26 cells are injected into BALB / c mice to form syngeneic tumors. Either placebo (PBO), 5 μg, or 25 μg of EXG-5109 mRNA vaccine is administered intradermally. Tumor size is then measured.

[0060] Results and Conclusions Intradermally injected EXG-5109 mRNA immunotherapy is expected to induce a strong cellular immune response against mutant KRAS proteins containing substitutions at positions 12, 13, and / or 61 of human KRAS. In ELISpot assays, splenocytes from immunized mice are expected to respond to the G12G wild-type peptide but not to the G12D, G12V, and G12C mutant peptides. Furthermore, intradermally injected EXG-5109 mRNA immunotherapy is expected to suppress tumor growth of CT26 mouse colon cancer cells in a syngeneic cancer mouse model.

[0061] Example 2. Immunotherapy for tumors expressing KRAS mutations This example describes the production of fusion proteins containing multiple KRAS substitutions based on the design principles shown in Figure 2. KRAS proteins with substitutions at one or more of positions 12, 13, and 61 are exemplary tumor-specific antigens (TSAs).

[0062] Materials and Methods BALB / c inbred female mouse.

[0063] EXG-5111 mRNA was produced by in vitro transcription of a plasmid containing a temperature-controllable, self-replicating RNA expression cassette (c-srRNA3) encoding a fusion protein (TSA-5111) containing 13 different peptides (each 26-29 amino acids long) derived from 13 common mutations in the human KRAS proto-oncogene (G12D; G12V, G12R, G12C, G12A, G12S, G13D, G13C, G13P, G13S, Q61H, Q61K, and Q61R). A schematic diagram of the fusion protein is shown in Figure 4A. The amino acid sequences of the 26- and 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 is set forth in SEQ ID NO: 20.

[0064] The wild-type and mutant peptides shown in Table 1-1 were used to restimulate T cells in splenocyte samples taken from immunized mice.

[0065] The CT26 murine colon carcinoma cell line (ATCC CRL-2638) is derived from a BALB / c mouse strain and is known to harbor a G12D mutation in the KRAS proto-oncogene. The mouse KRAS protein sequence is identical to the human KRAS protein in this region, and therefore, the EXG-5111 vaccine developed for human use can be tested in mice. Syngeneic tumors were formed by injecting CT26 cells into BALB / c mice. Either placebo (PBO), 5 μg, or 25 μg of EXG-5111 mRNA vaccine was administered intradermally. Tumor size was then measured.

[0066] Results and Conclusions Intradermally injected EXG-5111 immunotherapeutic agent is expected to induce a strong cellular immune response against mutant KRAS proteins containing substitutions at positions 12, 13, and / or 61 of human KRAS. In an ELISpot assay, splenocytes from immunized mice are expected to respond to the G12G wild-type peptide but not to the G12D, G12V, and G12C mutant peptides.

[0067] As expected, intradermally injected EXG-5111 mRNA immunotherapy suppressed tumor growth of CT26 mouse colon cancer cells in a syngeneic mouse cancer model. BALB / c female mice were intradermally administered 100 μg of EXG-5111 twice, two weeks apart. Two weeks later (day 0), the mice were injected with 3×10^5 cells of CT26 mouse colon cancer cells (ATCC CRL-2638), known to harbor a G12D mutation in the KRAS proto-oncogene. Figure 7A shows the increase in tumor size (volume) in 15 mice that received intradermal placebo (PBO) injections. Mice that met euthanasia criteria due to tumor size or ulceration were sacrificed. Generally, tumors grew rapidly in placebo-treated mice, and by day 28 after tumor injection, only one mouse survived. Figure 7B shows the increase in tumor size (volume) in 15 mice that received intradermal EXG-5111 vaccine injections. In contrast to PBO, tumor growth was inhibited and slowed in mice that received the EXG-5111 vaccine. By day 28 after tumor injection, seven mice survived. Figure 7C shows a comparison between the PBO and EXG-5111 groups; the mean ± SEM for each group is shown graphically, and the number of surviving mice in each group is indicated at the bottom. Tumor growth inhibition by EXG-5111 was statistically significant on days 11, 14, 22, and 25.

[0068] Example 3. Immunotherapy for tumors expressing multiple tumor-associated antigens (TAA) This example describes the evaluation of whether intradermally injected c-srRNA encoding a fusion protein (TAA-5107) containing the human CD5 signal peptide, NY-ESO-1, MAGEA3, TYR, and TPTE can induce a strong cellular immune response against the TAA of the fusion protein in BALB / c mice.

[0069] Materials and Methods BALB / c inbred female mouse.

[0070] Figure 5 shows a schematic diagram of the EXG-5107 vaccine. EXG-5107 mRNA was produced by in vitro transcription of a plasmid containing a temperature-controllable self-replicating RNA expression cassette (c-srRNA3) encoding 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 set forth as SEQ ID NO: 16.

[0071] Either placebo (PBO) or 25 μg of EXG-5107 vaccine will be administered intradermally, followed by evaluation of cellular immunity against the TAA of the TAA-5107 fusion protein by ELISpot assay.

[0072] Results and Conclusions Intradermally injected EXG-5107 mRNA immunotherapy appears to induce strong cellular immune responses against the different components of the fusion protein (NY-ESO-1, MAGEA3, TYR, and TPTE).

[0073] References References relevant to this disclosure include International Application No. PCT / US2022 / 075789 and International Application No. PCT / US2020 / 067506 to Elixirgen Therapeutics, Inc., the examples of which are incorporated herein by reference. Further references relevant to this 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.

[0074] array SEQ ID NO:1 >Human CD5 signal peptide MPMGSLQPLATLYLLGMLVASCLG SEQ ID NO:2 >Human Wilms tumor protein (NM_024426.6) MDFLLLQDPASTCVPEPASQHTLRSGPGCLQQPEQQGVRDPGGIWAKLGAAEASAERLQGRRSRGASGSEPQQMGSDVRDLNALLPAVPSLGGGGGCALPVSGAAQWAPVLDFAPPGASAYGSLGGPAPP PAPPPPPPPPPHSFIKQEPSWGGAEPHEEQCLSAFTVHFSGQFTGTAGACRYGPFGPPPPSQASSGQARMFPNAPYLPSCLESQPAIRNQGYSTVTFDGTPSYGHTPSHHAAQFPNHSFKHEDPMGQQGSL GEQQYSVPPPVYGCHTPTDSCTGSQALLLRTPYSDNLYQMTSQLECMTWNQMNLGATLKGVAAGSSSSSVKWTEGQSNHSTGYESDNHTTPILCGAQYRIHTHGVFRGIQDVRVRVPGVAPTLVRSASETS EKRPFMCAYPGCNKRYFKLSHLQMHSRKHTGEKPYQCDFKDCERRFSRSDQLKRHQRRHTGVKPFQCKTCQRKFSRSDHLKTHTRTHTGKTSEKFSCRWPSCQKKFARSDELVRHHNMHQRNMTKLQLAL SEQ ID NO:3 >Human BIRC5 (also known as survivin) protein (NM_001168) MGAPTLPPAWQPFLKDHRISTFKNWPFLEGCACTPERMAEAGFIHCPTENEPDLAQCFFCFKELEGWEPDDDPIEEHKKHSSGCAFLSVKKQFEELTLGEFLKLDRERAKNKIAKETNNKKKEFEETAEKVRRAIEQLAAMD SEQ ID NO:4 >Human NY-ESO-1 protein (NM_001327) MQAEGRGTGGSTGDADGPGGPGIPDGPGGNAGGGPGEAGATGGRGPRGAGAARASGPGGGAPRGPHGGAASGLNGCCRCGARGPESRLLEFYLAMPFATPMEAELARRSLAQDAPPLPVPGVLLKEFTVSGNILTIRLTAADHRQLQLSISSCLQQLSLLMWITQCFLPVFLAQPPSGQRR SEQ ID NO:5 >Human MAGEA3 protein (NM_005362) MPLEQRSQHCKPEEGLEARGEALGLVGAQAPATEEQEAASSSSTLVEVTLGEVPAAESPDPPQSPQGASSLPTTMNYPLWSQSYEDSSNQEEEGPSTFPDLESEFQAALSRKVAELVHFLLLKYRAREPVTKAEMLGSVVGNWQYFFPVIFSKASSS LQLVFGIELMEVDPIGHLYIFATCLGLSYDGLLGDNQIMPKAGLLIIVLAIIAREGDCAPEEKIWEELSVLEVFEGREDSILGDPKKLLTQHFVQENYLEYRQVPGSDPACYEFLWGPRALVETSYVKVLHHMVKISGGPHISYPPLHEWVLREGEE SEQ ID NO:6 >Human PRAME protein (NM_001291715) MERRRLWGSIQSRYISMSVWTSPRRLVELAGQSLLKDEALAIAALELLPRELFPPLFMAAFDGRHSQTLKAMVQAWPFTCLPLGVLMKGQHLHLETFKAVLDGLDVLLAQEVRPRRWKLQVLDLRKNSHQDFWTVWSGNRASLYSFPEPEAAQPMTKKRKVDGLSTEAEQPFIPVEVLVDLFLKEGACDELFSYLIEKVKRKKNVLRLCCKKLKIFAMPMQDIKMILKMVQLDSIEDLEVTCTWKLPTLAKFSPYLGQMINLRRLLLSHIHASSYISPEKEEQYIAQFTSQFLSLQCLQALYVDSLFFLRGRLDQLLRHVMNPLETLSITNCRLSEGDVMHLSQSPSVSQLSVLSLSGVMLTDVSPEPLQALLERASATLQDLVFDECGITDDQLLALLPSLSHCSQLTTLSFYGNSISISALQSLLQHLIGLSNLTHVLYPVPLESYEDIHGTLHLERLAYLHARLRELLCELGRPSMVWLSANPCPHCGDRTFYDPEPILCPCFMPN SEQ ID NO:7 >Artificial: Fusion 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 [Table 3] SEQ ID NO:10 >Artificial: c-srRNA3 nsP2 [Table 4] SEQ ID NO:11 >Artificial: c-srRNA4 nsP2 [Table 5] SEQ ID NO:12 >Artificial:c-srRNA nsP2 consensus [Table 6] SEQ ID NO:13 >VEEV:srRNA0 GSVETPRGLIKVTSYAGEDKIGSYAVLSPQAVLKSEKLSCIHPLAEQVIVITHSGRKGRYAVEPYHGKVVVPEGHAIPVQDFQALSESATIVYNEREFVNRYLHHIATHGGALNTDEEYYKTVKPSEHDGEYLYDIDRKQCVKKELVTGLGLTGELVDPPFHEFAYESLRTRPAAPYQVPTIGVYGVPGSGKSGIIKS AVTKKDLVVSAKKENCAEIRDVKKMKGLDVNARTVDSVLLNGCKHPVETLYIDEAFACHAGTLRALIAIIRPKKAVLCGDPKQCGFFNMMCLKVHFNHEICTQVFHKSISRRCTKSVTSVVSTLFYDKKMRTTNPKETKIVIDTTGSTKPKQDDLILTCFRGWVKQLQIDYKGNEIMTAAASQGLTRKGVYAVRYKVN ENPLYAPTSEHVNVLLTRTEDRIVWKTLAGDPWIKTLTAKYPGNFTATIEEWQAEHDAIMRHILERPDPPTDVFQNKANVCWAKALVPVLKTAGIDMTTEQWNTVDYFETDKAHSAEIVLNQLCVRFFGLDLDSGLFSAPTVPLSIRNNHWDNSPSPNMYGLNKEVVRQLSRRYPQLPRAVATGRVYDMNTGTLRNYDP RINLVPVNRRLLPHALVLHHNEHPQSDFSSFVSKLKGRTVLVVGEKLSVPGKMVDWLSDRPEATFRARLDLGIPGDVPKYDIIFVNVRTPYKYHHYQQCEDHAIKLSMLTKKACLHLNPGGTCVSIGYGYADRASESIIGAIARQFKFSRVCKPKSSLEETEVLFVFIGYDRKARTHNPYKLSSTLTNIYTGSRLHEAGC SEQ ID NO:14 >Artificial protein: TS insertion T.G.A.A. 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) YKLVVVGADGVGKSALTYKLVVVGAVGVGKSALTYKLVVVGARGVGKSALTYKLVVVGACGVGKSALTYKLVVVGAAGVGKSALTYKLVVVGASGVGKSALTKLVVVGAG DVGKSALTIKLVVVGAGCVGKSALTIKLVVVGAGPVGKSALTIKLVVVGAGSVGKSALTILDILDTAGHEEYSAMRDLDILDTAGKEEYSAMRDLDILDTAGREEYSAMRD SEQ ID NO:18 > Artificial (TSA-5109 with CD5 signal peptide) 245 amino acids MPMGSLQPLATLYLLGMLVASCLGYKLVVVGADGVGKSALTYKLVVVGAVGVGKSALTYKLVVVGARGVGKSALTYKLVVVGACGVGKSALTYKLVVVGAAGVGKSALTYKLVVVGASGVGK SALTKLVVVGAGDVGKSALTIKLVVVGAGCVGKSALTIKLVVVGAGPVGKSALTIKLVVVGAGSVGKSALTILDILDTAGHEEYSAMRDLDILDTAGKEEYSAMRDLDILDTAGREEYSAMRD SEQ ID NO:19 > Artificial (TSA-5111 without CD5 signal peptide) MTEYKLVVVGADGVGKSALTIQLIQNMTEYKLVVVGAVGVGKSALTIQLIQNMTEYKLVVVGARGVGKSALTIQLIQNMTEYKLVVVGACGVGKSALTIQLIQNMTEYKLVVVGAAGVGKSALTIQLIQNMTEYKLVVVGASGVGKSALTIQLIQNMTEYKLVVVGAGDVGKSALTIQLIQNHMTEYKLVVVGAGCVGKSALTIQLIQNHMTEYKLVVVGAGPVGKSALTIQLIQNHMTEYKLVVVGAGSVGKSALTIQLIQNHDGETCLLDILDTAGHEEYSAMRDQYMRTGDGETCLLDILDTAGKEEYSAMRDQYMRTGDGETCLLDILDTAGREEYSAMRDQYMRTG SEQ ID NO:20 >Artificial (TSA - 5111 with CD5 signal peptide) 375 amino acids MPMGSLQPLATLYLLGMLVASCLGMTEYKLVVVGADGVGKSALTIQLIQNMTEYKLVVVGAVGVGKSALTIQLIQNMTEYKLVVVGARGVGKSALTIQLIQNMTEYKLVVVGACGVGKSALTIQLIQNMTEYKLVVVGAAGVGKSALTIQLIQNMTEYKLVVVGASGVGKSALTIQLIQNMTEYKLVVVGAGDVGKSALTIQLIQNHMTEYKLVVVGAGCVGKSALTIQLIQNHMTEYKLVVVGAGPVGKSALTIQLIQNHMTEYKLVVVGAGSVGKSALTIQLIQNHDGETCLLDILDTAGHEEYSAMRDQYMRTGDGETCLLDILDTAGKEEYSAMRDQYMRTGDGETCLLDILDTAGREEYSAMRDQYMRTG SEQ ID NO:21 >WT - KRAS - 32AA MTEYKLVVVGAGGVGKSALTIQLIQNHFVDEY SEQ ID NO:22 >G12D - 32AA MTEYKLVVVGADGVGKSALTIQLIQNHFVDEY SEQ ID NO:23 >G12D-17AA [Table 7] SEQ ID NO:24 >G12V-17AA [Table 8] SEQ ID NO:25 >G12R-17AA [Table 9] SEQ ID NO:26 >G12C-17AA [Table 10] SEQ ID NO:27 >G12A-17AA [Table 11] SEQ ID NO:28 >G12S-17AA [Table 12] SEQ ID NO:29 >Artificial-G12X-Polyprotein-102 Amino Acids YKLVVVGADGVGKSALTYKLVVVGAVGVGKSALTYKLVVVGARGVGKSALTYKLVVVGACGVGKSALTYKLVVVGAAGVGKSALTYKLVVVGASGVGKSALT SEQ ID NO:30 >WT-KRAS-39AA KQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLC SEQ ID NO:31 >Q61H-KRAS-39AA [Table 13] SEQ ID NO:32 >Q61H-KRAS-29AA [Table 14] SEQ ID NO:33 >Q61K-KRAS-29AA [Table 15] SEQ ID NO:34 >Q61R-KRAS-29AA [Table 16] SEQ ID NO:35 >Artificial-Q61X-Polyprotein-87 Amino Acids DGETCLLDILDTAGGHEEYSAMRDQYMRTGDGETCLLDILDTAGKEEYSAMRDQYMRTGDGETCLLDILDTAGREEYSAMRDQYMRTG SEQ ID NO:36 >G13D-KRAS-17AA [Table 17] SEQ ID NO:37 >G13C-KRAS-17AA [Table 18] SEQ ID NO:38 >G13P-KRAS-17AA [Table 19] SEQ ID NO:39 >G13S-KRAS-17AA [Table 20] SEQ ID NO:40 >Q61H-KRAS-17AA Table 21 SEQ ID NO:41 Q61K-KRAS-17AA Table 22 SEQ ID NO:42 Q61R-KRAS-17AA Table 23 SEQ ID NO:43 G12D-KRAS-26AA Table 24 SEQ ID NO:44 >G12V-KRAS-26AA Table 25 SEQ ID NO:45 G12R-KRAS-26AA Table 26 SEQ ID NO:46 G12C-KRAS-26AA Table 27 SEQ ID NO:47 G12A-KRAS-26AA Table 28 SEQ ID NO:48 G12S-KRAS-26AA Table 29 SEQ ID NO:49 >G13D-KRAS-27AA [Table 30] SEQ ID NO:50 >G13C-KRAS-27AA [Table 31] SEQ ID NO:51 >G13P-KRAS-27AA [Table 32] SEQ ID NO:52 >KRAS-G13S-27AA [Table 33] 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 Xa, Xb, Xc, Xd, Xe, and Xf are independently selected from G, S, and absent). SEQ ID NO:54 >Artificial MTEYKLVVVGAGX 10 VGKSALTIQLIQNHXaXbXcXdXeXf MTEYKLVVVGAGX 11VGKSALTIQLIQNHXaXbXcXdXeXf MTEYKLVVVGAGX 12 VGKSALTIQLIQNHXaXbXcXdXeXf MTEYKLVVVGAGX 13 VGKSALTIQLIQNHXaXbXcXdXeXf (where X 10 , X 11 , X 12 , and X 13 are independently selected from D, C, P, and S; and 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 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 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 (where X 10 , X 11 , X 12 , and X 13 are independently selected from D, C, P, and S; and 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 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. 1. An RNA molecule comprising an open reading frame (ORF) encoding a fusion protein, said ORF comprising, from 5' to 3': (i) a nucleotide sequence encoding a mammalian signal peptide; (ii) a nucleotide sequence encoding a cancer antigen; and Including, The cancer antigen comprises a KRAS polyprotein, and the amino acid sequence of the KRAS polyprotein is a) a first segment, MTEYKLVVVG^ １ - MTEYKLVVVG^ ２ - MTEYKLVVVG^ ３ - MTEYKLVVVG^ ４ - MTEYKLVVVG^ ５ - MTEYKLVVVGAX ６ GVGKSALTIQLIQNXaXbXcXdXeXf(SEQ ID NO:53); a first segment including: b) a second segment, MTEYKLVVVGAGX １０ VGKSALTIQLIQNHXaXbXcXdXeXf MTEYKLVVVGAGX １１ VGKSALTIQLIQNHXaXbXcXdXeXf MTEYKLVVVGAGX １２ VGKSALTIQLIQNHXaXbXcXdXeXf MTEYKLVVVGAGX １３ VGKSALTIQLIQNHXaXbXcXdXeXf(SEQ ID NO:54) a second segment including c) a third segment, DGETCLLDILDTAGX ７ EEYSAMRDQYMRTGXaXbXcXdXeXf DGETCLLDILDTAGX ８ EEYSAMRDQYMRTGXaXbXcXdXeXf DGETCLLDILDTAGX ９ EEYSAMRDQYMRTGXaXbXcXdXeXf(SEQ ID NO:55) a third segment including wherein the first segment, the second segment, and the third segment are arranged in any order; X １ , X ２ , X ３ , X ４ , X ５ , and X ６ is independently selected from D, V, R, C, A, and S; X ７ , X ８ , and X ９ is independently selected from H, K, and R; X １０ , X １１ , X １２ , and X １３ is independently selected from D, C, P, and S; Xa, Xb, Xc, Xd, Xe, and Xf are independently selected from G, S, and absent; RNA molecule.

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

20.

4. 1. An RNA molecule comprising an open reading frame (ORF) encoding a fusion protein, said ORF comprising, from 5' to 3': (i) a nucleotide sequence encoding a mammalian signal peptide; (ii) a nucleotide sequence encoding a cancer antigen; and Including, The cancer antigen comprises a KRAS polyprotein, and the amino acid sequence of the KRAS polyprotein is a) a first segment, YKLVVVGAX １ GVGKSALTXaXbXcXdXeXfYKLVVVGAX ２ GVGKSALTXaXbXcXdXeXf YKLVVVGAX ３ GVGKSALTXaXbXcXdXeXfYKLVVVGAX ４ GVGKSALTXaXbXcXdXeXf YKLVVVGAX ５ GVGKSALTXaXbXcXdXeXfYKLVVVGAX ６ GVGKSALT(SEQ ID NO:56) a first segment including: b) a second segment, KLVVVGAGX １０ VGKSALTIXaXbXcXdXeXfKLVVVGAGX １１ VGKSALTIXaXbXcXdXeXf KLVVVGAGX １２ VGKSALTIXaXbXcXdXeXfKLVVVGAGX １３ VGKSALTI(SEQ ID NO:57) a second segment including c) a third segment, LDILDTAGX ７ HEEYSAMRDAaAｬAcAtAeAfLDILDTAGA ８ HEEYSAMRDAaAAcATAeAf LDILDTAGX ９ HEEYSAMRD (SEQ ID NO:58) a third segment including wherein the first segment, the second segment, and the third segment are arranged in any order; X １ , X ２ , X ３ , X ４ , X ５ , and X ６ is independently selected from D, V, R, C, A, and S; X ７ , X ８ , and X ９ is independently selected from H, K, and R; X １０ , X １１ , X １２ , and X １３ is independently selected from D, C, P, and S; Xa, Xb, Xc, Xd, Xe, and Xf are independently selected from G, S, and absent; RNA molecule.

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

18.

7. 1. An RNA molecule comprising an open reading frame (ORF) encoding a fusion protein, said ORF comprising, from 5' to 3': (i) a nucleotide sequence encoding a mammalian signal peptide; (ii) a nucleotide sequence encoding a cancer antigen; and Including, The cancer antigen comprises a KRAS polyprotein, and the amino acid sequence of the KRAS polyprotein is YKLVVVGAX １ GVGKSALTXaXbXcXdXeXfYKLVVVGAX ２ GVGKSALTXaXbXcXdXeXf YKLVVVGAX ３ GVGKSALTXaXbXcXdXeXfYKLVVVGAX ４ GVGKSALTXaXbXcXdXeXf YKLVVVGAX ５ GVGKSALTXaXbXcXdXeXfYKLVVVGAX ６ GVGKSALT(SEQ ID NO:56) Including, Here, X １ , X ２ , X ３ , X ４ , X ５ , and X ６ is independently selected from D, V, R, C, A, and S; Xa, Xb, Xc, Xd, Xe, and Xf are independently selected from G, S, and absent; RNA molecule.

8. The RNA molecule of claim 7, 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, and SEQ ID NO:

28.

9. The RNA molecule of claim 8, wherein the amino acid sequence of the KRAS polyprotein comprises SEQ ID NO:

29.

10. 1. An RNA molecule comprising an open reading frame (ORF) encoding a fusion protein, said ORF comprising, from 5' to 3': (i) a nucleotide sequence encoding a mammalian signal peptide; (ii) a nucleotide sequence encoding a cancer antigen; and Including, The cancer antigen comprises a KRAS polyprotein, and the amino acid sequence of the KRAS polyprotein is DGETCLLDILDTAGX ７ EEYSAMRDQYMRTGXaXbXcXdXeXf DGETCLLDILDTAGX ８ EEYSAMRDQYMRTGXaXbXcXdXeXf DGETCLLDILDTAGX ９ EEYSAMRDQYMRTGXaXbXcXdXeXf(SEQ ID NO:55) Including, Here, X ７ , X ８ , and X ９ is independently selected from H, K, and R; Xa, Xb, Xc, Xd, Xe, and Xf are independently selected from G, S, and absent; RNA molecule.

11. The RNA molecule of claim 10, wherein the amino acid sequence of the KRAS polyprotein comprises SEQ ID NO: 32, SEQ ID NO: 33, and SEQ ID NO:

34.

12. The RNA molecule of claim 11 , wherein the amino acid sequence of the KRAS polyprotein comprises SEQ ID NO:

35.

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

14. 14. The RNA molecule of claim 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. 1. An RNA molecule comprising an open reading frame (ORF) encoding a fusion protein, said ORF comprising, from 5' to 3': (i) a nucleotide sequence encoding a mammalian signal peptide; (ii) a nucleotide sequence encoding a cancer antigen; and Including, The cancer antigen comprises a KRAS polyprotein, and the amino acid sequence of the KRAS polyprotein is LDILDTAGX ７ HEEYSAMRDAaAｬAcAtAeAfLDILDTAGA ８ HEEYSAMRDAaAAcATAeAf LDILDTAGX ９ HEEYSAMRD (SEQ ID NO:58) Including, Here, X ７ , X ８ , and X ９ is independently selected from H, K, and R; Xa, Xb, Xc, Xd, Xe, and Xf are independently selected from G, S, and absent; RNA molecule.

16. 16. The RNA molecule of claim 15, wherein the amino acid sequence of the KRAS polyprotein comprises SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO:

42.

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

18.

18. 1. An RNA molecule comprising an open reading frame (ORF) encoding a fusion protein, said ORF comprising, from 5' to 3': (i) a nucleotide sequence encoding a mammalian signal peptide; (ii) a nucleotide sequence encoding a cancer antigen; and Including, The cancer antigen comprises a KRAS polyprotein, and the amino acid sequence of the KRAS polyprotein is MTEYKLVVVG^ １ - MTEYKLVVVG^ ２ - MTEYKLVVVG^ ３ - MTEYKLVVVG^ ４ - MTEYKLVVVG^ ５ - MTEYKLVVVGAX ６ GVGKSALTIQLIQNXaXbXcXdXeXf (SEQ ID NO: 53), Here, X １ , X ２ , X ３ , X ４ , X ５ , and X ６ is independently selected from D, V, R, C, A, and S; Xa, Xb, Xc, Xd, Xe, and Xf are independently selected from G, S, and absent; RNA molecule.

19. 19. The RNA molecule of claim 18, 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, and SEQ ID NO:

48.

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

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

20.

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

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

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

27. 27. An expression vector comprising the DNA template of claim 26.

28. A host cell comprising the expression vector of claim 27.

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

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

31. 1. A composition for stimulating an immune response to a cancer antigen in a mammalian subject, comprising an excipient and a 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; (ii) a nucleotide sequence encoding a cancer antigen; and Including, the temperature-sensitive self-replicating RNA is capable of expressing the fusion protein at a permissive temperature but not at a non-permissive temperature; The composition, wherein the cancer antigens comprise NY-ESO-1 antigen, MAGEA3 antigen, TYR antigen, and TPTE antigen.

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

33. 33. The composition of 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. 33. The composition of 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. 35. The composition of any one of claims 30 to 34, wherein the alphavirus is selected from the group consisting of Venezuelan equine encephalitis virus, Sindbis virus, and Semliki Forest virus.

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

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

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

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

12.

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

11.

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

11.

42. 42. The composition of any one of claims 30 to 41, wherein the permissive temperature is 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 31°C to 35°C and the non-permissive temperature is at least 37°C±0.5°C.

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

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

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

46. 46. ​​The method of claim 45, wherein the composition is administered intradermally.

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

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

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

51. (i) a composition according to any one of claims 30 to 44; (ii) a device for intradermal delivery of said composition to a mammalian subject; and Includes a kit.

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

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

54. 54. The method of claim 53, wherein said contacting is in vitro.

55. 54. The method of claim 53, wherein the contacting is in vivo.

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

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

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

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

Images