Temperature-controllable RNA immunotherapy for cancer

Abstract

The present disclosure relates to mRNA, self-replicating RNA, and temperature-sensitive self-replicating RNA constructs encoding cancer antigens, which RNA constructs are suitable for cancer immunotherapy in mammalian subjects, such as human subjects.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 390,216, filed July 18, 2022, U.S. Provisional Patent Application No. 63 / 341,318, filed May 12, 2022, and U.S. Provisional Patent Application No. 63 / 240,280, filed September 2, 2021, each of which is incorporated by reference in its entirety herein.

[0002] Submission of Electronic Sequence Listing The contents of the electronic sequence listing (699442001540SEQLIST.xml, size: 25374 bytes, created on August 30, 2022) are incorporated herein by reference in their entirety.

[0003] The present disclosure relates to mRNA, self-replicating RNA, and temperature-sensitive self-replicating RNA constructs encoding cancer antigens, which RNA constructs are suitable for cancer immunotherapy in mammalian subjects, such as human subjects. [Background technology]

[0004] Immunotherapy can be effective in the treatment of cancer and is becoming more widely used. One therapeutic strategy is to inject an immunogenic composition containing an antigen expressed in tumor cells into a cancer patient. Tumor-associated antigens (TAA) are expressed in tumor cells, but also in germ cells, or at low levels in normal cells. Tumor-specific antigens (TSA), also called neo-antigens, 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 a cytotoxic T lymphocyte (CTL) response 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 use of cancer antigens (TAA and / or TSA) to induce cellular immune responses against cancer cells. In some embodiments, a temperature-controllable self-replicating RNA vaccine platform is utilized. In an exemplary embodiment, WT1 protein is expressed in host cells from a temperature-controllable self-replicating RNA (c-srRNA) to induce a strong cellular immune response against WT1-expressing tumor cells. The c-srRNA is also referred to herein as a temperature-sensitive self-replicating RNA (srRNAts). Importantly, the c-srRNA-WT1 immunotherapeutic agent (EXG-5101) was found to inhibit tumor growth and even reduce the size of established tumors in preclinical models. Thus, the c-srRNA platform described herein is a suitable vector for the expression of tumor-associated antigens (TAA), such as WT1, NY-ESO-1, MAGEA3, BIRC5 (also known as SURVIVIN), PRAME, or tumor-specific antigens (TSA) (also known as neo-antigens). In some embodiments, the c-srRNA is used to express a fusion protein of two or more TAAs, TSAs, or a combination of TAAs and TSAs.

[0007] Among other embodiments, the present disclosure provides a composition comprising an excipient and a temperature-controllable self-replicating RNA (c-srRNA). In some embodiments, the composition comprises chitosan. In some embodiments, the chitosan is a low molecular weight (about 3-5 kDa) chitosan oligosaccharide, such as chitosan oligosaccharide lactate. In some embodiments, the composition does not comprise a liposome or lipid nanoparticle. [Brief description of the drawings]

[0008] [Figure 1] FIG. 1 shows a schematic diagram of the mechanism for induction of cellular (CD4+ and CD8+ T cell) immune responses following intradermal injection of temperature-controllable self-replicating RNA (referred to herein as "c-srRNA" or "srRNAts").

[0009] [Diagram 2] 1 shows a schematic diagram of a cancer antigen expressed from a temperature-controllable self-replicating RNA (c-srRNA). In an exemplary embodiment, the coding region of human Wilms' tumor (WT1) protein is the gene of interest (GOI) inserted into the c-srRNA. The EXG-5101 antigen is a fusion protein comprising a signal peptide sequence from human CD5 antigen (CD5-SP) shown as SEQ ID NO: 1 and the amino acid sequence of human WT1 protein shown as SEQ ID NO: 1 (isoform D, GenBank number NM_024426.6, NCBI number NP_077744.4). The coding sequence of WT1 isoform D has a non-AUG (CUG) translation initiation codon.

[0010] [Diagram 3] Schematic diagram of an exemplary method for stimulating an immune response to a cancer antigen in a human subject. c-srRNA is functional at permissive temperatures (e.g., 30-35° C.) but not functional at non-permissive temperatures (e.g., 37° C. or higher). The temperature at or just below the surface of the human body (surface body temperature) is about 31-34° C., which is lower than the core body temperature of the human body, which is about 37° C. c-srRNA is delivered by intradermal and subcutaneous administration directly to the cells of the subject at the permissive surface temperature.

[0011] [Figure 4] Illustrates testing of EXG-5101 mRNA vaccine in a syngeneic mouse tumor model.

[0012] [Diagram 5] Graph of tumor growth in BALB / c mice injected with placebo (PBO), 5 μg or 25 μg EXG-5101 mRNA vaccine. Mean and standard deviation (error bars) of 5 mice (n=5) are shown for each group. By day 7 after tumor inoculation, tumors had developed in mice from all three groups. However, by day 25 after tumor inoculation (day 18 after injection), tumor growth was dose-dependently delayed in mice injected with EXG-5101 mRNA vaccine. In contrast, tumors continued to grow in mice injected with placebo.

[0013] [Figure 6A] Figures 6A and 6B show the induction of tumor-associated antigen-reactive cellular immune responses by intradermal injection of EXG-5101 mRNA (temperature-controllable self-replicating RNA encoding the human WT1 gene). Figure 6A illustrates the experimental procedure. [Figure 6B] Figures 6A and 6B show the induction of tumor-associated antigen-reactive cellular immune responses by intradermal injection of EXG-5101 mRNA (temperature-controllable self-replicating RNA encoding the human WT1 gene). Figure 6B shows the results of an ELISpot assay of splenocytes obtained from five mice (n=5) each immunized by intradermal injection of 25 μg of EXG-5101 or placebo (buffer only). The left panel shows the frequency of interferon gamma (IFN-γ) spot-forming cells (SFC) per 1×106 splenocytes stimulated with a pool of 110 peptides covering the human WT1 protein (15-mers with 11 amino acid overlap: JPT Peptide Technologies, catalog number PM-WT1). The right panel shows the frequency of interleukin-4 (IL-4) SFC per 1×106 splenocytes stimulated with a pool of 110 peptides covering the human WT1 protein (15-mers with 11 amino acid overlap: JPT Peptide Technologies, Cat. No. PM-WT1). The mean and standard deviation (error bars) are shown for each group.

[0014] [Figure 7]FIG. 1 shows a schematic diagram of a fusion protein containing multiple tumor-associated antigens expressed from a temperature-controllable self-replicating RNA (c-srRNA). In an exemplary embodiment, the EXG-5105 antigen is a fusion protein comprising a signal peptide sequence from human CD5 antigen (CD5-SP) shown as SEQ ID NO:1, the amino acid sequence of human WT1 protein [isoform D, GenBank No. NM_024426.6, NCBI No. NP_077744.4: the coding sequence for WT1 isoform D has a non-AUG (CUG) translation initiation codon] shown as SEQ ID NO:2, the amino acid sequence of human BIRC5 (also known as SURVIVIN) protein shown as SEQ ID NO:3 (GenBank No. NM_001168), the amino acid sequence of human NY-ESO-1 protein shown as SEQ ID NO:4 (GenBank No. NM_001327), the amino acid sequence of human MAGEA3 protein shown as SEQ ID NO:5 (GenBank No. NM_005362), and the amino acid sequence of human PRAME protein shown as SEQ ID NO:6 (GenBank No. NM_001291715). The amino acid sequence of the TAA fusion protein is shown as SEQ ID NO:7, and the amino acid sequence of the CD5-SP+TAA fusion protein is shown as SEQ ID NO:8.

[0015] [Figure 8A] 8A-8F show the induction of tumor-associated antigen-reactive cellular immune responses by intradermal injection of EXG-5105 mRNA (temperature-controllable self-replicating RNA encoding a fusion protein of human WT1 gene, human BIRC5 (SURVIVIN), human NY-ESO-1, human MAGEA3, and human PRAME). FIG. 8A illustrates the experimental procedure. On day 0, a total of 10 BALB / c female mice were used in the experiment. Five mice were administered 25 μg of EXG-5105 each by intradermal injection, and five mice were administered placebo (buffer only) by intradermal injection. On day 14, splenocytes were collected from each mouse and tested for immune responses against WT1 and NY-ESO-1 as exemplary antigens encoded on the EXG-5105 mRNA vaccine by ELISpot assay. [Figure 8B]Figures 8A-8F show the induction of tumor-associated antigen-reactive cellular immune responses by intradermal injection of EXG-5105 mRNA (a temperature-controllable self-replicating RNA encoding a fusion protein of the human WT1 gene, human BIRC5 (SURVIVIN), human NY-ESO-1, human MAGEA3, and human PRAME). Figure 8B shows the frequency of cytokine (left, interferon gamma [IFN-γ]; right, interleukin-4 [IL-4]) spot-forming cells (SFCs) per 1 x 106 splenocytes stimulated with a pool of 110 peptides covering the human WT1 protein (15-mers with 11 amino acid overlap: JPT Peptide Technologies, catalog number PM-WT1). [Figure 8C] Figures 8A-8F show the induction of tumor-associated antigen-reactive cellular immune responses by intradermal injection of EXG-5105 mRNA (a temperature-controllable self-replicating RNA encoding a fusion protein of the human WT1 gene, human BIRC5 (SURVIVIN), human NY-ESO-1, human MAGEA3, and human PRAME). Figure 8C shows the frequency of cytokine (left, interferon gamma [IFN-γ]; right, interleukin-4 [IL-4]) spot-forming cells (SFCs) per 1 x 106 splenocytes stimulated with a pool of peptides covering the human NY-ESO-1 protein (15-mers with 11 amino acid overlap: Miltenyi Biotec, catalog no. 130-095-380). [Figure 8D]Figures 8A-8F show the induction of tumor-associated antigen-reactive cellular immune responses by intradermal injection of EXG-5105 mRNA (a temperature-controllable self-replicating RNA encoding a fusion protein of human WT1 gene, human BIRC5 (SURVIVIN), human NY-ESO-1, human MAGEA3, and human PRAME). Figure 8D shows the frequency of cytokine (left, interferon gamma [IFN-γ]; right, interleukin-4 [IL-4]) spot-forming cells (SFCs) per 1 x 106 splenocytes stimulated with a pool of peptides covering the human MAGEA3 protein (15-mers with 11 amino acid overlap: JPT PepMix MAGEA3, UniProt ID: P43357, catalog number PM-MAGEA3). [Figure 8E] Figures 8A-8F show the induction of tumor-associated antigen-reactive cellular immune responses by intradermal injection of EXG-5105 mRNA (a temperature-controllable self-replicating RNA encoding a fusion protein of the human WT1 gene, human BIRC5 (SURVIVIN), human NY-ESO-1, human MAGEA3, and human PRAME). Figure 8E shows the frequency of cytokine (left, interferon gamma [IFN-γ]; right, interleukin-4 [IL-4]) spot-forming cells (SFCs) per 1 x 106 splenocytes stimulated with a pool of peptides covering the human BIRC5 (SURVIVIN) protein (15-mers with 11 amino acid overlap: JPT PepMix Survivin-1, UniProt ID: O15392, catalog number PM-Survivin). [Figure 8F]Figures 8A-8F show the induction of tumor-associated antigen-reactive cellular immune responses by intradermal injection of EXG-5105 mRNA (a temperature-controllable self-replicating RNA encoding a fusion protein of human WT1 gene, human BIRC5 (SURVIVIN), human NY-ESO-1, human MAGEA3, and human PRAME). Figure 8F shows the frequency of cytokine (left, interferon gamma [IFN-γ]; right, interleukin-4 [IL-4]) spot-forming cells (SFCs) per 1 x 106 splenocytes stimulated with a pool of peptides covering the human PRAME protein (15-mers with 11 amino acid overlap: JPT PepMix PRAME (OIP4), UniProt ID: P43357, catalog number PM-OIP4).

[0016] [Figure 9A] Figure 9A and Figure 9B show a comparison of srRNA constructs for T cell inducibility. Figure 9A illustrates the experimental procedure. On day 0, mice were intradermally injected with either placebo (PBO, buffer only), srRNA0, c-srRNA1, c-srRNA3, or c-srRNA4. srRNA0, c-srRNA1, c-srRNA3, and c-srRNA4 encode the same RBD of SARS-CoV-2. On day 14, mice were sacrificed and splenocytes were isolated for ELISpot assay against RBD protein. [Figure 9B] Figures 9A and 9B show a comparison of srRNA constructs for T cell inducibility. Figure 9B shows the number of IFN-γ spot-forming cells (SFCs) in 1×106 splenocytes from immunized mice restimulated by culturing in splenocytes in the presence or absence of a pool of 53 peptides (15-mers with 11 amino acid overlap) covering the SARS-CoV-2 RBD (original strain). The mean and standard deviation (error bars) are shown for each group. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] Cancer immunotherapy is primarily based on immunogenic compositions that rely on the induction of cell-mediated immunity (i.e., CD8+ Killer T cells and CD4 + This is believed to be best achieved by a T cell-inducing vaccine involving 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 called neo-antigens). Thus, the present disclosure provides a cellular 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 a further embodiment, the c-srRNA encodes one, two, three, four, or all five cancer antigens of the group consisting of WT1, BIRC5, NY-ESO-1, MAGEA3, and PRAME.

[0018] 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, which is made possible by combining existing knowledge of vaccine biology with temperature-controllable self-replicating mRNA (c-srRNA) based on alphaviruses such as Venezuelan Equine Encephalitis Virus (VEEV). The terms c-srRNA and srRNAts are used interchangeably throughout this disclosure, with srRNA1ts2 (described in WO 2021 / 138447(A1)) being an exemplary embodiment. 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 about 30-35°C, but not at non-permissive temperatures of about 37°C and above. This platform has all the benefits of the mRNA platform, including no genomic integration, rapid development and deployment, and simple GMP (Good Manufacturing Process) processes, while at the same time having the additional advantages of the srRNA platform (i.e., the precursor to our c-srRNA platform) compared to the mRNA platform, in particular longer expression [Johanning et al., 1995] and higher immunogenicity at lower dosages [Brito et al., 2014]. However, this simple temperature controllable feature allows many desirable features of a T cell-inducing vaccine to be co-opted, as briefly described below.

[0019] Briefly, srRNA1ts2 is a temperature-sensitive self-replicating VEEV-based RNA replicon developed for the transient expression of heterologous proteins. Temperature sensitivity is conferred by the insertion of five amino acid residues within the nonstructural protein 2 (nsP2) of VEEV. 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.

[0020] 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.

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

[0022] 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 partially closed and indicates that such embodiments may include further elements that do not substantially alter the basic characteristics of such embodiments.

[0023] The term "about" as used herein with respect to a value includes 90% to 110% of that value (e.g., when used with respect to chitosan oligosaccharide, a molecular weight of about 5,000 daltons refers to 4,500 daltons to 5,500 daltons).

[0024] 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, moieties thereof, and combinations thereof. In the context of the present disclosure, the term "antigen" refers to a polypeptide or protein antigen, typically at least 8 amino acid residues in length, which may include one or more post-translational modifications.

[0025] The terms "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues and are not limited to a particular length unless otherwise specified. A polypeptide can include natural amino acid residues or a combination of natural and non-natural 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).

[0026] As used herein, the terms "isolated" and "purified" refer to a material that is removed from at least one component that is naturally associated with it (e.g., removed from its original environment). When used in reference to a recombinant protein, the term "isolated" refers to a protein that is 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.

[0027] An "effective amount" or "sufficient amount" of a substance is an amount sufficient to effect beneficial or desired results, including clinical results, and as such, "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.

[0028] In this disclosure, the terms "individual" and "subject" refer to a mammal. "Mammals" include, but are 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.

[0029] The term "dose" as used herein with respect to a composition comprising an mRNA encoding an antigen refers to a measured portion of the 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.

[0030] "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.

[0031] Conversely, "inhibition" of a response or parameter includes reducing and / or suppressing that response or parameter when compared to otherwise identical conditions, 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- to 200-fold, 5- to 500-fold or more, 10- 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.

[0032] 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 that includes an mRNA encoding an antigen that is at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 times higher than the antigen-reactive antibody titer resulting from a control condition (e.g., administration of a comparative composition that does not include an mRNA or that includes 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 that includes an mRNA encoding the antigen that is at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 times lower than the antigen-reactive antibody titer resulting from administration of a composition of the present disclosure that includes an mRNA encoding the antigen.

[0033] As used herein, the term "immunization" refers to the process of increasing a mammalian subject's response to an antigen, thus improving its ability to resist or overcome infection and / or resist disease.

[0034] As used herein, the term "vaccination" refers to the introduction of a vaccine into the body of a mammalian subject.

[0035] As used herein, "percent (%) amino acid sequence identity" and "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 the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps as necessary to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in a variety of ways that are 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 required to achieve maximum alignment over the entire length of the sequences being compared.

[0036] 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, such as increased stability and / or immunogenicity.

[0037] 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.

[0038] Conservative amino acid substitutions involve exchanging a member of one of these classes for another member of the same class. Non-conservative amino acid substitutions involve exchanging a member of one of these classes for a member of another class.

[0039] As used herein, the term "excipient" refers to a compound present in a composition that includes 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).

[0040] Optimizing intradermal delivery for cell-mediated immunity Intradermal vaccination results in long-lasting cellular immunity and increased immunogens [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, as it targets APCs [Hickling and Jones, 2009], and such 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 antigens into peptides, and presents these peptides to T cells (Figure 1). Peptides presented via this pathway are expressed by MHC-I-restricted CD8 + In another pathway, APCs also take up antigens produced by nearby skin cells. Peptides presented via this pathway are expressed by MHC-II-restricted CD4 + Stimulates helper T cells.

[0041] Problems with Intradermal Injection and Our Solution Here is a potential problem that we have identified and the solution that our c-srRNA platform offers.

[0042] (1) An unrecognized critical hurdle for the application of srRNA such as in intradermal vaccine platforms is that both mRNA and srRNA do not express antigens sufficiently at skin temperature [International Application No. PCT / US20 / 67506]. Confusingly, the temperature of human skin (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]. Moreover, 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 body core). In other words, the c-srRNA platform expresses the best antigens for intradermal injection compared to mRNA and srRNA, and it further has safety features such that the vector's ability to spread and become produced in other areas of the subject's body is limited or inactivated.

[0043] (2) Another challenge of intradermal vaccination is the lack of suitable additives. Adjuvants (e.g., aluminum salts and oil-in-water) are too locally reactogenic when delivered by the intradermal route, so no adjuvants have been incorporated into clinically approved intradermal vaccines, resulting in lower immunogenicity [Hickling and Jones, 2009]. Lipid nanoparticles (LNPs) used for intramuscularly administered mRNA and srRNA vaccines are also oil-in-water type, which may cause skin reactogenicity and increase the risk of allergic reactions to LNP components such as PEG. Our c-srRNA platform is a solution to this problem, since it is injected as naked c-srRNA (no LNP, no adjuvant). First, self-replication of RNA in cells, especially APCs, induces strong 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.

[0044] (3) The 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 to adopt intradermal injection is by using specialized devices, such as MicronJet600 (NanoPass) and Immucise (Terumo), that are currently available to allow easy and consistent intradermal injection. These devices are also good candidates for large-scale production and deployment. However, these specialized devices are relatively expensive, so intradermal injection with the Mantoux technique using a standard needle and syringe is also an option.

[0045] Designing suitable antigens Tumor-associated antigens (TAA) are expressed in tumor cells, but also in germ cells, or expressed at low levels in normal cells. The National Cancer Institute selected 75 cancer antigens suitable for targeting 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 in germ tissues, and only in very limited cell types in adults. For example, WT1 is expressed in most leukemias (AML, ALL), pancreatic cancer, lung cancer, and glioblastoma. WT1 peptides have been used as antigens for cancer vaccines in many preclinical and clinical trials. The use of WT1 is shown in Example 1. This list also includes NY-ESO-1 (Example 2) and MAGEA3 (Example 3). Any TAA may be used as an antigen for our c-srRNA platform-based cancer vaccine. Any combination of these TAAs may also be used as fusion proteins or separately expressed proteins (Example 4).

[0046] In recent years, it has become common to perform genomic sequencing of tumor cells from patients. Such efforts often identify tumor-specific protein products or peptides 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 our c-srRNA platform-based cancer vaccine (Example 5).

[0047] Chitosan enhancement of gene expression in vivo RNase inhibitors (proteins purified from human placenta) slightly enhance immunogenicity against antigens encoded on c-srRNA, presumably by enhancing expression of the antigen from c-srRNA in vivo when injected intradermally in mice (see, for example, Figure 25C of WO 2021 / 138447(A1)). RNase inhibitors may protect c-srRNA from RNase-mediated degradation in vivo. However, because it is difficult to use protein-based RNase inhibitors as excipients in injectable products, it is desirable to find alternative agents that can enhance expression of genes of interest (GOI) in vivo for therapeutic purposes.

[0048] Low molecular weight chitosan (molecular weight about 6 kDa) has been shown to inhibit the activity of RNase with an inhibition constant in the range of 30-220 nM (Yakovlev et al., Biochem Biophys Res Commun commun, 357(3):J.584-8, 2007). Two different chitosan oligomers have been recently tested: chitosan oligomer (CAS 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 about 5 kDa, >90% deacetylated: Sigma-Aldrich: product no. 523682). Surprisingly, it was found that even very low levels of chitosan oligomers, 0.001 μg / mL (about 1 / 100 of the inhibition constant found by Yakovlev et al., supra, 2007), were able to enhance the expression of luciferase encoded on c-srRNA by about 10-fold (data not shown). Similar enhancement of GOI expression was achieved by chitosan oligomers up to 0.5 μg / mL and by chitosan oligosaccharide lactate at 0.1 μg / mL.

[0049] 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 is noteworthy that the enhancement of GOI expression by chitosan oligomers is unlikely to be mediated by nanoparticles or complex formation between c-srRNA and chitosan oligomers. First, such low concentrations of chitosan oligomers do not allow complex formation with RNA. Second, there is not enough time for complex formation by adding chitosan oligomers to c-srRNA just before intradermal injection.

[0050] Since 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 their RNase inhibitory mechanism. For example, chitosan oligomers can promote the uptake of c-srRNA into cells, thereby enhancing the expression of GOI from c-srRNA. Nevertheless, this surprising finding provides an effective means to enhance the therapeutic expression of GOIs encoded on c-srRNA in vivo. Enumerated Embodiments 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, the ORF comprising, 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 a fusion protein at the permissive temperature but not at the non-permissive temperature. composition. 2. The composition of embodiment 1, wherein the cancer antigen comprises a tumor-associated antigen (TAA). 3. The composition of embodiment 2, wherein the TAA comprises a WT1 antigen, a NY-ESO-1 antigen, a MAGEA3 antigen, a BIRC5 (SURVIVIN) antigen, a PRAME antigen, or a combination thereof. 4. The composition of embodiment 2, wherein the TAA comprises a WT1 antigen. 5. The composition of embodiment 4, wherein the amino acid sequence of the WT1 antigen comprises SEQ ID NO:2, or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:2. 6. The composition of embodiment 2, wherein the TAA is a TAA fusion protein comprising a WT1 antigen, a NY-ESO-1 antigen, a MAGEA3 antigen, a BIRC5 antigen, and a PRAME antigen. 7. The composition of embodiment 6, wherein the amino acid sequence of the TAA fusion protein comprises SEQ ID NO:7, or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:7. 8. The composition of embodiment 2, wherein the TAA comprises a BIRC5 antigen. 9. The composition of embodiment 8, wherein the amino acid sequence of the BIRC5 antigen comprises SEQ ID NO:3, or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:3. 10. The composition of embodiment 2, wherein the TAA comprises a NY-ESO-1 antigen. 11. The composition of embodiment 10, wherein the amino acid sequence of the NY-ESO-1 antigen comprises SEQ ID NO:4 or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:4. 12. The composition of embodiment 2, wherein the TAA comprises a MAGEA3 antigen. 13. The composition of embodiment 12, wherein the amino acid sequence of the MAGEA3 antigen comprises SEQ ID NO:5, or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:5. 14. The composition of embodiment 2, wherein the TAA comprises a PRAME antigen. 15. The composition of embodiment 14, wherein the amino acid sequence of the PRAME antigen comprises SEQ ID NO:6 or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:6. 16. The composition of embodiment 1, wherein the cancer antigen comprises a neoantigen. 17. The composition according to any one of embodiments 1 to 16, wherein the mammalian signal peptide is a signal peptide of a surface protein expressed in a mammalian antigen-presenting cell. 18. The composition of embodiment 17, 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. 19. The composition of any one of embodiments 1 to 18, wherein the alphavirus is selected from the group consisting of Venezuelan equine encephalitis virus, Sindbis virus, and Semliki Forest virus. 20. The composition of embodiment 19, wherein the alphavirus is Venezuelan Equine Encephalitis virus. 21. A composition described in any one of embodiments 1 to 20, 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), comprising 4 to 6 additional amino acids between beta sheet 5 and beta sheet 6 of nsP2. 22. The composition of embodiment 21, wherein the additional amino acids comprise the sequence of SEQ ID NO: 14 (TGAAA). 23. The composition of embodiment 22, wherein the amino acid sequence of nsP2 comprises SEQ ID NO: 12. 24. The composition of embodiment 23, 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. 25. The composition of embodiment 24, wherein the amino acid sequence of nsP2 comprises SEQ ID NO:11. 26. The composition of any one of embodiments 1 to 25, wherein the acceptable 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-acceptable temperature is 37°C±0.5°C, optionally wherein the acceptable temperature is 31°C to 35°C and the non-acceptable temperature is at least 37°C±0.5°C. 27. A composition according to any one of the preceding embodiments, wherein the composition does not comprise lipid nanoparticles. 28. The composition of any one of the preceding embodiments, wherein the composition further comprises chitosan. 29. 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 1 to 28, thereby stimulating an immune response to the cancer antigen in the mammalian subject. 30. The method of embodiment 29, wherein the composition is administered intradermally. 31. The method of embodiment 29 or embodiment 30, wherein the immune response comprises a cellular immune response reactive with mammalian cells expressing the cancer antigen. 32. The method of embodiment 31, 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. 33. The method of embodiment 32, wherein the immune response further comprises a humoral immune response reactive to a cancer antigen. 34. The method of any one of embodiments 29-33, wherein the mammalian subject is a human subject. 35. (i) a composition according to any one of embodiments 1 to 28, (ii) a device for intradermal delivery of the composition to a mammalian subject; and Including the kit. 36. The kit of embodiment 35, wherein the device comprises a syringe and a needle. EXAMPLES

[0051] Abbreviations: APC (antigen-presenting cell); BIRC5 (baculovirus IAP repeat containing 5 or SURVIVIN); 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 (preferentially expressed antigen in melanoma); SFC (spot forming cell); srRNAts (temperature-sensitive self-replicating RNA or c-srRNA temperature-regulatable self-replicating RNA); TAA (tumor-associated antigen); TSA (tumor-specific antigen); and WT1 (Wilms' tumor 1).

[0052] Example 1. Immunotherapy for WT1-expressing tumors This example describes the finding that human Wilms' tumor 1 (WT1) protein induces a strong cellular immune response in BALB / c mice when expressed from temperature-controllable self-replicating RNA injected intradermally. Surprisingly, the EXG-5101 RNA construct induces elimination of mouse mammary tumor cells expressing human WT1 in a syngeneic mouse cancer model.

[0053] Materials and Methods BALB / c inbred female mouse.

[0054] EXG-5101 mRNA was produced by in vitro transcription of a temperature-controllable self-replicating RNA vector (srRNA1ts2 [International Application No. PCT / US2020 / 067506]) encoding a fusion protein containing the human CD5 signal peptide fused to the human WT1 protein (Figure 2). The WT1 protein in EXG-5101 is encoded by isoform D, which starts from a non-AUG (CUG) translation initiation codon.

[0055] 4T1 mammary tumor cells (ATCC number CRL-2539) were derived from BALB / c mice and are known to recapitulate human breast cancer (stage IV).

[0056] Figure 4 illustrates the experimental procedure. 4T1 tumor cells were transfected with plasmid DNA encoding human Wilms' tumor 1 (WT1) protein isoform D (NM_024426.6) driven by the CMV promoter and a neomycin resistance gene as a selectable marker. Stable transformants of 4T1 cells expressing human WT1 were isolated by G418 selection. Cells were injected into the mammary fat pad of BALB / c mice (day 0 after tumor inoculation). On day 7, either placebo (PBO), 5 μg or 25 μg of EXG-5101 mRNA was administered intradermally (day 0 after vaccination). Tumor sizes were measured on days 5, 8 (day 0 after vaccination), 25 (day 18 after vaccination), and 32 (day 25 after vaccination).

[0057] Results and Conclusions Figure 5 shows tumor growth in BALB / c mice injected with placebo (PBO), 5 μg or 25 μg of EXG-5101 mRNA vaccine. The mean and standard deviation (error bars) of 5 mice (n=5) are shown for each group. By day 7 after tumor inoculation, mice in all three groups developed tumors. However, by day 25 (day 18 after vaccination), tumor growth was dose-dependently suppressed in mice injected with EXG-5101 mRNA, while tumors continued to grow in mice injected with placebo.

[0058] Figures 6A and 6B show the induction of tumor-associated antigen-reactive cellular immune responses by intradermal injection of EXG-5101 mRNA. As shown in Figure 6A, BALB / c mice were intradermally injected with either 25 μg of EXG-5101 or placebo (PBO) on day 0. Splenocytes were collected from these mice on day 14 and used for ELISpot assay. Figure 6B shows the induction of tumor-associated antigen-reactive cellular immune responses by intradermal injection of EXG-5101 mRNA. Splenocytes were collected from 1 × 10 mice stimulated with a pool of 110 peptides (15-mers with 11 amino acid overlap) covering the human WT1 protein. 6 Results of the ELISpot assay are shown as the frequency of IFN-γ or IL-4 spot-forming cells (SFC) per splenocyte. IFN-γ-secreting cells are CD8 + T cells and CD4+ They correspond to Th1 cells and are thought to be involved in cell-mediated immune responses, whereas IL-4-secreting cells are CD4 + These cells correspond to Th2 cells. Therefore, the results indicate that EXG-5101 induced cellular immunity against human WT1 protein.

[0059] In conclusion, intradermally injected EXG-5101 mRNA immunotherapy dose-dependently inhibits tumor growth or reduces tumor size of WT1-expressing tumors in a syngeneic mouse model of breast cancer.Furthermore, intradermally injected EXG-5101 mRNA immunotherapy induces cellular immunity against human WT1 protein in the mouse model.

[0060] Example 2. Immunotherapy for tumors expressing NY-ESO-1 This example describes an evaluation of whether intradermally injected c-srRNA encoding human NY-ESO-1 can induce a cellular immune response against mouse mammary tumor cells expressing human NY-ESO-1 in a syngeneic mouse cancer model.

[0061] Materials and Methods BALB / c inbred female mouse.

[0062] c-srRNA-NY-EOS1 mRNA is produced by in vitro transcription of a temperature-controllable self-replicating RNA vector (srRNA1ts2 [International Application No. PCT / US20 / 67506]) encoding a fusion protein containing the human CD5 signal peptide fused to the human NY-ESO-1 protein. NY-ESO-1 is also known as cancer / testis antigen 1B (CTAG1B) (NM_001327).

[0063] 4T1 mammary tumor cells (ATCC number CRL-2539) were derived from BALB / c mice and are known to recapitulate human breast cancer (stage IV).

[0064] 4T1 tumor cells are transfected with plasmid DNA encoding human NY-ESO-1, also known as cancer / testis antigen 1B (CTAG1B) (NM_001327), driven by the CMV promoter, and a neomycin resistance gene as a selectable marker. Stable transformants of 4T1 cells expressing the human NY-ESO-1 gene are isolated by G418 selection. The cells are injected into the mammary fat pad of BALB / c mice (day 0 after tumor inoculation). On day 7, either placebo (PBO), 5 μg or 25 μg of c-srRNA-NY-ESO-1 mRNA are administered intradermally (day 0 after vaccination). Tumor size is measured at several time points after vaccination.

[0065] Results and Conclusions Intradermally injected c-srRNA-NY-ESO-1 mRNA immunotherapy appears to inhibit tumor growth or reduce tumor size of NY-ESO-1-expressing tumors in a dose-dependent manner in a syngeneic mouse model of breast cancer.

[0066] Example 3. Immunotherapy for tumors expressing MAGEA3 This example describes the evaluation of whether intradermally injected c-srRNA encoding human MAGE family member A3 (MAGEA3) can induce a cellular immune response against mouse mammary tumor cells expressing human MAGEA3 in a syngeneic mouse cancer model.

[0067] Materials and Methods BALB / c inbred female mouse.

[0068] c-srRNA mRNA-MAGEA3 is produced by in vitro transcription of a temperature-controllable self-replicating RNA vector (srRNA1ts2 [International Application No. PCT / US20 / 67506]) encoding a fusion protein containing the human CD5 signal peptide fused to the human MAGE family member A3 (MAGEA3) protein (NM_005362).

[0069] 4T1 mammary tumor cells (ATCC number CRL-2539) were derived from BALB / c mice and are known to recapitulate human breast cancer (stage IV).

[0070] 4T1 tumor cells are transfected with plasmid DNA encoding human MAGEA3 (NM_005362) driven by the CMV promoter and a neomycin resistance gene as a selectable marker. Stable transformants of 4T1 cells expressing human MAGEA3 are isolated by G418 selection. Cells are injected into the mammary fat pad of BALB / c mice (day 0 post-tumor inoculation). On day 7, either placebo (PBO), 5 μg or 25 μg of c-srRNA-MAGEA3 mRNA are administered intradermally (day 0 post-vaccination). Tumor size is measured at several time points post-vaccination.

[0071] Results and Conclusions Intradermally injected c-srRNA-MAGEA3 mRNA immunotherapy appears to inhibit tumor growth or reduce tumor size of MAGEA3-expressing tumors in a dose-dependent manner in a syngeneic mouse model of breast cancer.

[0072] Example 4. Immunotherapy for tumors expressing two or more tumor-associated antigens (TAA) This example describes the finding that intradermally injected c-srRNA encoding fusion proteins, including WT1, NY-ESO-1, BIRC5, MAGEA3, and PRAME, induced strong cellular immune responses against the TAAs of the fusion proteins in BALB / c mice.

[0073] Materials and Methods BALB / c inbred female mouse.

[0074] FIG. 7 shows a schematic diagram of the EXG-5105 vaccine, which is a c-srRNA mRNA (srRNA1ts2 [International Application No. PCT / US20 / 67506]) encoding a fusion protein of human WT1, NY-ESO-1, BIRC5, MAGEA3 and PRAME with a signal peptide sequence derived from the human CD5 gene.

[0075] 4T1 mammary tumor cells were derived from BALB / c (ATCC: CRL-2539) and are known to recapitulate human breast cancer (stage IV).

[0076] 4T1 tumor cell line was transfected with three plasmid DNAs encoding human WT1, BIRC5, NY-ESO-1, MAGEA3 and PRAME, respectively, driven by CMV promoter and selectable marker for G418 (neomycin). Stable transformants of 4T1 cells expressing human WT1, BIRC5, NY-ESO-1, MAGEA3 and PRAME were isolated after G418 selection. Cells were injected into the mammary fat pad of BALB / c mice. Either placebo (PBO), 5 μg or 25 μg of EXG-5105 mRNA vaccine was administered intradermally. Tumor size was then measured.

[0077] Results and Conclusions Figure 8A shows the experimental procedure for investigating the immunogenicity of EXG-5105 mRNA vaccine. BALB / c mice were intradermally injected with either 25 μg EXG-5105 or placebo (PBO) on day 0. Splenocytes were collected from these mice on day 14 and used for ELISpot assays. EXG-5105 encodes a fusion protein that includes human WT1, NY-ESO-1, BIRC5, MAGEA3, and PRAME. As such, intradermal injection of EXG-5105 is expected to induce cellular immunity against all five of these TAAs simultaneously. Indeed, the results shown in Figures 8B-8F show that this was the case. Figure 8B shows that 1 × 10 IgG antibodies stimulated with a pool of 110 peptides (15-mers with 11 amino acid overlap) covering the human WT1 protein were immunized with 1 × 10 IgG antibodies stimulated with a pool of 110 peptides (15-mers with 11 amino acid overlap) covering the human WT1 protein.6 Results of the ELISpot assay are shown as the frequency of IFN-γ or IL-4 spot-forming cells (SFC) per splenocyte. IFN-γ-secreting cells are CD8 + T cells and CD4 + They correspond to Th1 cells and are responsible for cell-mediated immune responses, whereas IL-4-secreting cells are responsible for CD4 + These correspond to Th2 cells. Thus, the results indicate that EXG-5105 induced cellular immunity against human WT1 protein. Similarly, Figure 8C shows that 1 × 10 cells stimulated with a pool of peptides (15-mers with 11 amino acid overlap) covering the human NY-ESO-1 protein were positive for WT1 protein. 6 The results of the ELISpot assay are shown as the frequency of IFN-γ or IL-4 spot-forming cells (SFC) per splenocyte. The results show that EXG-5105 induced cellular immunity against human NY-ESO-1 protein as well as human WT1 protein. Similarly, Figures 8D, 8E, and 8F show the results of 1 x 10 IL-4 spot-forming cells stimulated with pools of peptides covering human MAGEA3 protein, human BIRC5 (SURVIVIN) protein, and human PRAME protein, respectively. 6 The frequency of cytokine (left, interferon gamma [IFN-γ]; right, interleukin-4 [IL-4]) spot-forming cells (SFCs) per splenocyte is shown. Interestingly, stronger cellular immune responses were elicited against MAGEA3 and PRAME, all of which are expressed exclusively in tumors and testis, than against WT1, NY-ESO-1, and BIRC5, all of which are expressed in tumors as well as several other tissues.

[0078] In conclusion, intradermally injected EXG-5105 mRNA immunotherapy induces cellular immunity against individual components of the fusion protein in syngeneic mouse cancer models.Furthermore, intradermally injected EXG-5105 mRNA vaccine is predicted to suppress the growth of tumor cells expressing human WT1, NY-ESO-1, BIRC5, MAGEA3, and PRAME in vivo.

[0079] Example 5. Immunotherapy for tumors expressing tumor-specific antigens (TSAs) This example describes the finding that intradermally injected srRNAts encoding neoantigens induce cellular immune responses against neoantigens in BALB / c mice in a syngeneic mouse cancer model.

[0080] Materials and Methods BALB / c inbred female mouse.

[0081] The srRNAts mRNA (srRNA1ts2 [International Application No. PCT / US20 / 67506]) encodes a neoantigen with a signal peptide sequence derived from the human CD5 gene.

[0082] 4T1 mammary tumor cells were derived from BALB / c (ATCC: CRL-2539) and are known to recapitulate human breast cancer (stage IV).

[0083] 4T1 tumor cell line was transfected with three plasmid DNAs encoding human neoantigens driven by CMV promoter and a selectable marker for G418 (neomycin). Stable transformants of 4T1 cells expressing human neoantigens were isolated after G418 selection. Cells were injected into the mammary fat pad of BALB / c mice. Either placebo (PBO), 5 μg or 25 μg of srRNAts neoantigen mRNA vaccine was administered intradermally. Tumor size was then measured.

[0084] Results and Conclusions Intradermally injected srRNAts neoantigen mRNA vaccine suppresses the growth of tumor cells expressing human neoantigens and dose-dependently eliminates tumors in a syngeneic mouse cancer model.

[0085] Example 6. Comparison of self-replicating RNAs for T cell inducibility This example describes the finding that intradermally injected srRNAts constructs encoding an antigen induce a cellular immune response against that antigen in mice.

[0086] Materials and Methods C57BL / 6 mice.

[0087] Three different temperature-controllable self-replicating RNA vectors (c-srRNAs) and a control self-replicating RNA vector (c-srRNA) were tested. The characteristics of the srRNAs are summarized in Table 6-1. The IFN-α / β sensitivity of the parental VEEV strain was 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 have an A16D substitution (TC83 mutation) and a 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 in the nsP2 protein of VEEV for temperature regulation as previously described (see U.S. Patent No. 11,421,248 by Ko, Examples 3, 21 and 22, incorporated herein by reference). All four srRNAs encode an antigen (SARS-CoV-2 spike protein receptor binding domain) lacking a signal peptide sequence. [Table 1]

[0088] The nucleotide sequence of the VEEV genome is 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).

[0089] Preparation of srRNA. All srRNAs were produced by in vitro transcription. NEB 10-beta Competent E. coli (C3019H / C3019I) were transformed with plasmid DNA and grown in Luria broth containing 100 μg / mL ampicillin. Purified plasmid DNA was linearized with MluI. In vitro transcription (IVT) of c-srRNA with Cap1 and polyA was performed using in vitro transcription of plasmid DNA using T7 RNA polymerase with Cleancap AU (Trilink) according to the manufacturer's protocol.

[0090] Injection of srRNA into mouse skin. Mice were randomly divided into groups, and 1 day before injection, the hind limbs were shaved to expose the skin. 5 μg or 25 μg of srRNA reconstituted in lactated Ringer's (LR) solution was injected intradermally into the shaved skin.

[0091] Results and Conclusions C57BL / 6 mice were administered either srRNA as naked RNA (no lipid nanoparticles or transfection reagent) or a placebo by intradermal injection (Figure 9A). As expected, cellular immunity, assessed by the presence of antigen-specific IFN-γ-secreting T cells, was already induced by day 14 after vaccination (Figure 9B). T cell responses induced by c-srRNA1 were stronger than those induced by standard non-thermoregulatory srRNA0. Furthermore, T cell responses induced by both c-srRNA3 and c-srRNA4 were stronger than those induced by srRNA0 and c-srRNA1. Surprisingly, T cell responses induced by both c-srRNA3 and c-srRNA4 were approximately three-fold higher than those induced by c-srRNA1. This difference may be due to the parental VEEV sequences of c-srRNA3 and c-srRNA4 being more resistant to suppression by type I interferon than the parental VEEV sequence of c-srRNA1.

[0092] References References relevant to this disclosure include, but are not limited to, International Application No. PCT / US2020 / 067506 to Elixirgen Therapeutics; Brito et al., Mol Ther. 22(12):2118-2129, 2014; Cheever et al., Clin Cancer Res. 15:5323-5337, 2009; Golombek et al., Mol Ther Nucleic Acids. 11:382-392, 2018; Hickling et al., "Intradermal Delivery of Vaccines: A review of the literature and the potential for development for use in low-and middle-income countries." PATH / WHO August 27, 2009; Johanning et al., Nucleic Acids Res. 23(9):1495-501, 1995; and Johansson et al., PLoS One.7(1):e29732,2012. [Sequence Listing]

[0093] SEQ ID NO:1 >Human CD5 signal peptide [ka]

[0094] SEQ ID NO:2 >Human Wilms Tumor Protein (NM_024426.6) [ka]

[0095] SEQ ID NO:3 > Human BIRC5 (also known as survivin) protein (NM_001168) [ka]

[0096] SEQ ID NO:4 >Human NY-ESO-1 protein (NM_001327) [ka]

[0097] SEQ ID NO:5 >Human MAGEA3 protein (NM_005362) [ka]

[0098] SEQ ID NO:6 >Human PRAME protein (NM_001291715) [ka]

[0099] SEQ ID NO:7 >Artificial proteins: fusion of WT1, BIRC5, NY-ESO-1, MAGEA3, and PRAME proteins [ka]

[0100] SEQ ID NO:8 >Artificial proteins: fusion of human CD5 (signal peptide only), WT1, BIRC5, NY-ESO-1, MAGEA3, and PRAME proteins [ka]

[0101] SEQ ID NO:9 >Protein:c-srRNA1 nsP2 [ka]

[0102] SEQ ID NO:10 >Protein:c-srRNA3 nsP2 [ka]

[0103] SEQ ID NO:11 >Protein:c-srRNA4 nsP2 [ka]

[0104] SEQ ID NO:12 >Artificial Protein: c-srRNA nsP2 Consensus [ka]

[0105] SEQ ID NO:13 >VEEV:srRNA0 [ka]

[0106] SEQ ID NO:14 >Artificial protein: ts insertion T.G.A.A.

Claims

1. 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; composition.

2. The composition of claim 1 , wherein the cancer antigen comprises a tumor-associated antigen (TAA).

3. 3. The composition of claim 2, wherein the TAA comprises a WT1 antigen, a NY-ESO-1 antigen, a MAGEA3 antigen, a BIRC5 (survivin) antigen, a PRAME antigen, or a combination thereof.

4. The composition of claim 2 , wherein the TAA comprises a WT1 antigen.

5. The composition of claim 4, wherein the amino acid sequence of the WT1 antigen comprises SEQ ID NO:2 or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:

2.

6. 3. The composition of claim 2, wherein the TAA is a TAA fusion protein comprising a WT1 antigen, a NY-ESO-1 antigen, a MAGEA3 antigen, a BIRC5 antigen, and a PRAME antigen.

7. 7. The composition of claim 6, wherein the amino acid sequence of the TAA fusion protein comprises SEQ ID NO:7, or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:

7.

8. The composition of claim 2 , wherein the TAA comprises a BIRC5 antigen.

9. 9. The composition of claim 8, wherein the amino acid sequence of the BIRC5 antigen comprises SEQ ID NO:3, or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:

3.

10. The composition of claim 2, wherein the TAA comprises the NY-ESO-1 antigen.

11. 11. The composition of claim 10, wherein the amino acid sequence of the NY-ESO-1 antigen comprises SEQ ID NO:4, or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:

4.

12. The composition of claim 2 , wherein the TAA comprises a MAGEA3 antigen.

13. The composition of claim 12, wherein the amino acid sequence of the MAGEA3 antigen comprises SEQ ID NO:5 or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:

5.

14. The composition of claim 2 , wherein the TAA comprises a PRAME antigen.

15. 15. The composition of claim 14, wherein the amino acid sequence of the PRAME antigen comprises SEQ ID NO:6, or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:

6.

16. The composition of claim 1 , wherein the cancer antigen comprises a neoantigen.

17. The composition according to any one of claims 1 to 16, wherein the mammalian signal peptide is a signal peptide of a surface protein expressed in a mammalian antigen-presenting cell.

18. 18. The composition of claim 17, 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.

19. 17. The composition of any one of claims 1 to 16, wherein the alphavirus is selected from the group consisting of Venezuelan equine encephalitis virus, Sindbis virus, and Semliki Forest virus.

20. 20. The composition of claim 19, wherein the alphavirus is Venezuelan equine encephalitis virus.

21. 17. The composition of any one of claims 1 to 16, 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), comprising 4 to 6 additional amino acids between beta sheet 5 and beta sheet 6 of said nsP2.

22. 22. The composition of claim 21, wherein the additional amino acids comprise the sequence of SEQ ID NO: 14 (TGAAA).

23. 23. The composition of claim 22, wherein the amino acid sequence of nsP2 comprises SEQ ID NO:

12.

24. 24. The composition of claim 23, 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.

25. 25. The composition of claim 24, wherein the amino acid sequence of nsP2 comprises SEQ ID NO:

11.

26. 17. The composition of any one of claims 1 to 16, 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.

27. The composition of any one of claims 1 to 16, wherein the composition does not contain lipid nanoparticles.

28. The composition of any one of claims 1 to 16, wherein the composition further comprises chitosan.

29. 17. A method for stimulating an immune response to a cancer antigen in a mammalian subject, the method comprising administering to the mammalian subject the composition of any one of claims 1 to 16, thereby stimulating an immune response to said cancer antigen in said mammalian subject.

30. 30. The method of claim 29, wherein the composition is administered intradermally.

31. 30. The method of claim 29, wherein the immune response comprises a cellular immune response reactive with mammalian cells that express the cancer antigen.

32. The method of claim 31 , 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.

33. 33. The method of claim 32, wherein the immune response further comprises a humoral immune response reactive with the cancer antigen.

34. 30. The method of claim 29, wherein the mammalian subject is a human subject.

35. (i) a composition according to any one of claims 1 to 16; (ii) a device for intradermal delivery of said composition to a mammalian subject; and Includes a kit.

36. 36. The kit of claim 35, wherein the device comprises a syringe and a needle.

37. A composition for stimulating an immune response to a cancer antigen in a mammalian subject, comprising an excipient and an mRNA 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 WT1 antigen, a NY-ESO-1 antigen, a MAGEA3 antigen, a BIRC5 antigen, and a PRAME antigen; Including, composition.

38. The composition described in claim 37, wherein the amino acid sequence of the fusion protein comprises SEQ ID NO: 7 or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:

7.

39. An mRNA comprising an alphavirus replicon lacking a viral structural protein coding region, wherein the alphavirus replicon comprises a nonstructural protein coding region encoding nonstructural protein 2 (nsP2), and the amino acid sequence of the nsP2 comprises SEQ ID NO: 10 or SEQ ID NO:

11.

40. The mRNA described in claim 39, wherein the mRNA includes an open reading frame (ORF) encoding a cancer antigen.

41. The mRNA described in claim 40, wherein the cancer antigen comprises a neoantigen.

42. An mRNA described in any one of claims 39 to 41, wherein the amino acid sequence of nsP2 includes sequence number 11.

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