CN117881420A - Temperature-controllable RNA immunotherapeutic agent for cancer - Google Patents

Abstract

The present disclosure relates to mRNA encoding a cancer antigen, self-replicating RNA, and temperature-sensitive self-replicating RNA. The RNA construct is suitable for cancer immunotherapy in a mammalian subject, such as a human subject.

Description

Temperature-controllable RNA immunotherapeutic agent for cancer

Cross Reference to Related Applications

The present application claims the benefit of U.S. provisional application Ser. No. 63/390,216, filed on 7.18, 2022, U.S. provisional application Ser. No. 63/341,318, filed on 5.12, 2022, and U.S. provisional application Ser. No. 63/240,280, filed on 9.2, 2021, each of which is hereby incorporated by reference in its entirety.

Submission of electronic sequence Listing

The contents of the electronic sequence listing (699442001540 seqlist. Xml; size: 25,374 bytes; and date of creation: 2022, 8, 30 days) are incorporated herein by reference in their entirety.

Technical Field

The present disclosure relates to mRNA encoding a cancer antigen, self-replicating RNA, and temperature-sensitive self-replicating RNA. The RNA construct is suitable for cancer immunotherapy in a mammalian subject, such as a human subject.

Background

Immunotherapy is effective in treating cancer and has been more widely used. One therapeutic strategy is to inject an immunogenic composition comprising an antigen expressed in tumor cells into a cancer patient. Tumor Associated Antigens (TAAs) are expressed in tumor cells but also in embryonic cells or at low levels in normal cells. Tumor Specific Antigens (TSAs) are also known as neoantigens, which are expressed only in tumor cells and are usually expressed by mutated genes in tumor cells. Cancer immunotherapy relies on the induction of Cytotoxic T Lymphocyte (CTL) responses against cancer cells.

There is a need in the art for cancer therapies that induce an effective TAA or TSA specific cellular immune response to destroy tumor cells expressing TAA or TSA.

Disclosure of Invention

The present disclosure relates to the use of cancer antigens (TAA and/or TSA) for inducing a cellular immune response against cancer cells. In some embodiments, a temperature-controllable self-replicating RNA vaccine platform is utilized. In exemplary embodiments, the WT1 protein is expressed in a host cell by temperature controlled self-replicating RNA (c-srRNA) to induce an effective cellular immune response against WT1 expressing tumor cells. c-srRNA is also referred to herein as temperature sensitive self-replicating RNA (srRNA). Importantly, c-srRNA-WT1 immunotherapeutic (EXG-5101) was found to inhibit tumor growth, even reduce the size of established tumors, in preclinical models. Thus, the c-srRNA platform described herein is a suitable vector for expressing a tumor-associated antigen (TAA) (e.g., WT1, NY-ESO-1, MAGEA3, BIRC5 (also known as survivin), PRAME) or a tumor-specific antigen (TSA) (also known as a neoantigen). In some embodiments, c-srRNA is used to express a fusion protein of two or more TAA, TSA, or a combination of TAA and TSA.

In other embodiments, the disclosure provides compositions 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.

Drawings

FIG. 1 shows a schematic representation of the mechanism by which the immune response of cells (CD4+ and CD8+ T cells) is induced following intradermal injection of temperature controlled self-replicating RNA (referred to herein as "c-srRNA" or "srRNAts").

FIG. 2 shows a schematic representation of cancer antigens expressed from temperature-controlled self-replicating RNA (c-srRNA). In an exemplary embodiment, the coding region of the human nephroblastoma (WT 1) protein is a gene of interest (GOI) inserted into the c-srRNA. The EXG-5101 antigen is a fusion protein comprising a signal peptide sequence (CD 5-SP) from human CD5 antigen as shown in SEQ ID NO. 1 and an amino acid sequence (isoform D, genBank No. NM-024326.6, NCBI No. NP-077744.4) of human WT1 protein as shown in SEQ ID NO. 1. The coding sequence for WT1 isoform D has a non-AUG (CUG) translation initiation codon.

Fig. 3 shows a schematic diagram of an exemplary method for stimulating an immune response against a cancer antigen in a human subject. c-srRNA is functional at allowable temperatures (e.g., 30-35℃.), while at non-allowable temperatures (e.g.,>and has no function at 37 ℃). The temperature at or slightly below the surface of the human body (surface body temperature) (about 31 ℃ -34 ℃) is below the core body temperature of the human body (37 ℃). The c-srRNA is delivered directly to cells of a subject at allowable surface body temperature by intradermal and subcutaneous administration.

FIG. 4 shows the testing of EXG-5101mRNA vaccine in a syngeneic mouse tumor model.

FIG. 5 shows a graph of tumor growth in BALB/c mice injected with Placebo (PBO), 5 μg or 25 μg of EXG-5101mRNA vaccine. Mean and standard deviation (error bars) of five mice per group (n=5) are shown. By day 7 post tumor inoculation, all three groups of mice had tumors. However, by day 25 post tumor inoculation (day 18 post injection), tumor growth was delayed in a dose-dependent manner in mice injected with EXG-5101mRNA vaccine. In contrast, tumors continued to grow in placebo-injected mice.

FIGS. 6A-6B show induction of tumor-associated antigen-reactive cellular immune responses by intradermal injection of EXG-5101mRNA (temperature-controllable self-replicating RNA encoding the human WT1 gene). Fig. 6A shows the experimental procedure. Fig. 6B shows the results of ELISpot assay of splenocytes obtained from five (n=5) mice, each of which has been immunized by intradermal injection of 25 μg EXG-5101 or placebo (buffer only). The left panel shows the frequency of interferon-gamma (IFN-gamma) Spot Forming Cells (SFCs) per 1x 10. Gamma.6 splenocytes which have been stimulated by a pool of 110 peptides (15 mer with 11 amino acid overlap: JPT Peptide Technologies, catalog number PM-WT 1) covered with human WT1 protein. The right panel shows the frequency of interleukin-4 (IL-4) SFC per 1x10≡6 splenocytes that had been stimulated by a pool of 110 peptides (15 mer with 11 amino acid overlap: JPT Peptide Technologies, catalog number PM-WT 1) covering the human WT1 protein. The mean and standard deviation (error bars) for each group are shown.

FIG. 7 shows a schematic representation of a fusion protein comprising multiple tumor-associated antigens expressed from temperature-controllable self-replicating RNA (c-srRNA). In an exemplary embodiment, the EXG-5105 antigen is a fusion protein comprising: a signal peptide sequence (CD 5-SP) from the human CD5 antigen as shown in SEQ NO. 1; the amino acid sequence of the human WT1 protein shown in SEQ ID NO. 2 [ isoform D, genBank No. NM-024326.6, NCBI No. NP-077744.4: the coding sequence of WT1 isoform D has a non-AUG (CUG) translation initiation codon ]; the amino acid sequence of the human BIRC5 (also called survivin) protein shown in SEQ ID NO. 3 (GenBank No. NM-001168); the amino acid sequence of the human NY-ESO-1 protein shown in SEQ NO. 4 (GenBank No. NM-001327); the amino acid sequence of the human MAGEA3 protein shown in SEQ NO. 5 (GenBank No. NM-005362); and the amino acid sequence of human PRAME protein shown in 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 plus TAA fusion protein is shown as SEQ ID NO. 8.

FIGS. 8A-8F show induction of tumor-associated antigen-reactive cellular immune responses by intradermal injection of EXG-5105mRNA (encoding human WT1 gene, human BIRC5 (survivin), human NY-ESO-1, human MAGEA3 and human PRAME temperature-controllable self-replicating RNA). Fig. 8A shows the experimental procedure. On day 0, a total of 10 BALB/c female mice were used for the experiment; five mice received an intradermal injection of 25 μg EXG-5105 each, and five mice received an intradermal injection of placebo (buffer only). On day 14, spleen cells were collected from each mouse and tested for immune responses against WT1 and NY-ESO-1 as exemplary antigens encoded on the EXG-5105mRNA vaccine by ELISPot assay. FIG. 8B shows the frequency of cytokine (left, interferon-gamma [ IFN-gamma ]; right, interleukin-4 [ IL-4 ]) Spot Forming Cells (SFC) per 1x10≡6 spleen cells that have been stimulated by a pool of 110 peptides (15-mer with 11 amino acid overlap: JPT Peptide Technologies, catalog number PM-WT 1) covered with human WT1 protein. FIG. 8C shows the frequency of cytokine (left, interferon-gamma [ IFN-gamma ]; right, interleukin-4 [ IL-4 ]) Spot Forming Cells (SFC) per 1x 10-6 spleen cells that have been stimulated by pools of peptides (15-mer with 11 amino acid overlaps: miltenyi Biotec, catalog No. 130-095-380) covered with human NY-ESO-1 protein. FIG. 8D shows the frequency of cytokine (left, interferon-gamma [ IFN-gamma ]; right, interleukin-4 [ IL-4 ]) Spot Forming Cells (SFC) per 1x10≡splenocytes that have been stimulated by pools of peptides (15 mer with 11 amino acid overlap: JPT PepMix MAGEA3, uniProt ID: P43357, catalog number PM-MAGEA 3) covered with human MAGEA3 protein. FIG. 8E shows the frequency of cytokine (left, interferon-gamma [ IFN-gamma ]; right, interleukin-4 [ IL-4 ]) Spot Forming Cells (SFC) per 1x 10-6 spleen cells that have been stimulated by pools of peptides (15-mer with 11 amino acid overlap: JPT PepMix survivin-1,UniProt ID:O15392, catalog number PM-survivin) covered with human BIRC5 (survivin) protein. FIG. 8F shows the frequency of cytokine (left, interferon-gamma [ IFN-gamma ]; right, interleukin-4 [ IL-4 ]) Spot Forming Cells (SFCs) per 1x10≡splenocyte that had been stimulated by pools of peptides (15 mer with 11 amino acid overlap: JPT PepMix PRAME (OIP 4), uniProt ID: P43357, catalog number PM-OIP 4) covered with human PRAME protein.

Figures 9A-9B show comparative srRNA constructs for T cell inducibility. Fig. 9A shows an experimental procedure. On day 0, mice were injected intradermally with 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 spleen cells were isolated for ELISpot assay against RBD proteins. FIG. 9B shows the number of IFN-. Gamma.spot forming cells (SFCs) in 1X 10. Sup.6 spleen cells from immunized mice re-stimulated by culture in spleen cells in the presence or absence of a pool of 53 peptides (15 mer with 11 amino acid overlap) covering SARS-CoV-2RBD (original strain). The mean and standard deviation (error bars) for each group are shown.

Detailed Description

Cancer immunotherapy is expected to be best achieved by relying primarily on immunogenic compositions that induce cellular immunity (i.e., T cell-induced 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). Accordingly, the present disclosure provides a cell-immune based platform for cancer immunotherapy. The wilms tumor protein 1 (WT 1) is a Tumor Associated Antigen (TAA) that is expressed in a broad range of tumors, but only in embryonic tissue and very limited cell types in adults. Thus, in some embodiments, c-srRNA encodes WT1. In some embodiments, c-srRNA encodes BIRC5 (also known as survivin). In some embodiments, 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.

mRNA immune treatment platform based on cell immunity

Vaccine platforms are described in part in the earlier patent application by Elixirgen [ PCT/US20/67506, now published as WO 2021/138447A1 ]. The vaccine platform is optimized to induce cellular immunity, which is made possible by combining prior knowledge of vaccine biology with temperature-controlled self-replicating mRNA (c-srRNA) based on alphaviruses such as Venezuelan Equine Encephalitis Virus (VEEV). The terms c-srRNA and srRNA are used interchangeably throughout this disclosure, with srRNA1ts2 (described in WO 2021/138447 A1) being an exemplary embodiment. By incorporating small amino acid changes in the alphavirus replicase that provide temperature sensitivity, c-srRNA is based on srRNA, which is also known as self-amplified mRNA (saRNA or SAM). The c-srRNA of Elixirgen is functional in the allowable temperature range of about 30℃to 35℃but not functional at non-allowable temperatures equal to or higher than about 37 ℃. It has all the benefits of the mRNA platform: no genome integration, rapid development and deployment, and simple GMP (good manufacturing process) processes, as well as the additional advantages of the srRNA platform (i.e. the precursor of our c-srRNA platform) compared to the mRNA platform, in particular longer expression [ Johanning et al, 1995] and higher immunogenicity at lower doses [ Brito et al, 2014]. However, this simple temperature-controllable feature makes it possible to bring together many of the desirable features of T cell-induced vaccines as briefly described below.

Briefly, srRNA1ts2 is a temperature sensitive, self-replicating VEEV-based RNA replicon developed for transient expression of heterologous proteins. Temperature sensitivity was conferred by inserting five amino acid residues into the nonstructural protein 2 (nsP 2) of VEEV. The nsP2 protein is a helicase/protease that, together with nsP1, nsP3 and nsP4, constitutes a VEEV replicase. srRNA1ts2 does not contain VEEV structural proteins (capsids, E1, E2 and E3). The disclosure of WO 2021/138447 A1 of Elixirgen Therapeutics, inc. In particular, example 3 of WO 2021/138447 A1, FIG. 12 and SEQ ID NOS 29-49 are hereby incorporated by reference.

General techniques and definitions

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.

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

The phrase "comprising" as used herein is open ended, meaning that such embodiments may include additional elements. In contrast, the phrase "consisting of … …" is closed, indicating that these embodiments do not include additional elements (except trace amounts of impurities). The phrase "consisting essentially of … …" is partially enclosed, meaning that the embodiments may further include elements that do not substantially alter the essential characteristics of the embodiments.

The term "about" as used herein with respect to a value encompasses from 90% to 110% of the value (e.g., a molecular weight of about 5,000 daltons when used with respect to chitosan oligosaccharides refers to 4,500 daltons to 5,500 daltons).

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

Unless otherwise specified, 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. The polypeptide may comprise natural amino acid residues or a combination of natural and unnatural amino acid residues. These terms also include post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, and the like. In some aspects, the polypeptide may contain modifications with respect to native or native sequences so long as the protein retains the desired activity (e.g., antigenicity).

The terms "isolated" and "purified" as used herein refer to materials that are removed from at least one component with which they are naturally associated (e.g., from their original environment). The term "isolated" when used in reference to a recombinant protein refers to a protein that has been removed from the medium of the host cell that produced the protein. In some embodiments, the isolated protein (e.g., WT1 protein) is at least 75%, 90%, 95%, 96%, 97%, 98% or 99% pure as determined by HPLC.

An "effective amount" or "sufficient amount" of a substance is an amount sufficient to achieve a beneficial or desired result, including a clinical result, and thus, the "effective amount" depends on the context of its application. In the context of administering a composition of the present disclosure comprising an mRNA encoding an antigen, an effective amount contains an mRNA sufficient to stimulate an immune response, preferably a cellular immune response to the antigen.

In the present disclosure, the terms "individual" and "subject" refer to mammals. "mammal" includes, but is not limited to, humans, non-human primates (e.g., monkeys), farm animals, sports animals, rodents (e.g., mice and rats), and pets (e.g., dogs and cats). In some embodiments, the subject is a human subject.

As used herein, the term "dose" with respect to a composition comprising mRNA encoding an antigen refers to the measured portion of the composition that is 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 mRNA encoding an antigen to stimulate an immune response to the antigen in the subject.

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

Conversely, "suppressing" a response or parameter includes reducing and/or suppressing the response or parameter when compared to a condition that is otherwise identical except for the parameter of interest or alternatively when compared to another condition. For example, "inhibition" of an immune response (e.g., a Th2 response) means a decrease in the response. Depending on the measured parameters, the decrease may be from 2-fold to 200-fold, from 5-fold to 500-fold or more, from 10-fold to 1000-fold or more, or from 2, 5, 10, 50 or 100-fold to 200, 500, 1,000, 2,000, 5,000 or 10,000-fold.

The relative terms "higher" and "lower" refer to a measurable increase or decrease in response or parameter, respectively, when compared to an otherwise identical condition except for the parameter of interest or alternatively compared to another condition. For example, "higher antibody titer" refers to an antigen-reactive antibody titer that is at least 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold higher than an antigen-reactive antibody titer as a result of administering a composition of the present disclosure comprising an mRNA encoding an antigen, as compared to an antigen-reactive antibody titer as a result of a control condition (e.g., administering a comparative composition comprising no mRNA or no control mRNA encoding an antigen). Similarly, "lower antibody titer" refers to an antigen-reactive antibody titer that is at least 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold lower than an antigen-reactive antibody titer as a result of administration of a composition of the present disclosure comprising an mRNA encoding an antigen as a result of control conditions (e.g., administration of a comparative composition comprising no mRNA or comprising a control mRNA that does not encode an antigen).

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

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

As used herein, "percent amino acid sequence identity (%)" and "percent identity" and "sequence identity" when used with respect to an amino acid sequence (reference polypeptide sequence) are defined as the percentage of amino acid residues in a candidate sequence (e.g., a subject antigen) that are identical to amino acid residues in the reference polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity and not considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining the percent amino acid sequence identity may be accomplished in a variety of ways well known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MegAlign (DNASTAR) software. One skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithms needed to achieve maximum alignment over the full length of the compared sequences.

Amino acid substitutions may include substitution of one amino acid in the polypeptide with another amino acid. Amino acid substitutions can be introduced into the antigen of interest and the product screened for desired activity (e.g., increased stability and/or immunogenicity).

Amino acids can generally be grouped according to the following common side chain characteristics:

(1) Hydrophobicity: norleucine, met, ala, val, leu, ile;

(2) Neutral hydrophilicity: cys, ser, thr, asn, gln;

(3) Acid: asp, glu;

(4) Alkaline: his, lys, arg;

(5) Residues that affect chain orientation: gly, pro; and

(6) Aromatic: trp, tyr, phe.

Conservative amino acid substitutions will involve the exchange of a member of one of these classes with another member of the same class. Non-conservative amino acid substitutions will involve exchanging members of one of these classes with members of the other class.

As used herein, the term "excipient" refers to a compound that is present in a composition comprising an active ingredient (e.g., mRNA encoding an antigen). Pharmaceutically acceptable excipients are inert pharmaceutical compounds and may include, for example, solvents, bulking agents, buffers, tonicity adjusters and preservatives (Pramantick et al, pharma Times,45:65-77,2013). In some embodiments, the compositions of the present disclosure include excipients that function as one or more of solvents, bulking agents, buffers, and tonicity adjusters (e.g., sodium chloride in saline may act as an aqueous carrier and tonicity adjuster). Optimizing intradermal delivery for cellular immunity

Intradermal vaccination results in long-term cellular immunity and increased immunogenicity [ hicking and Jones,2009]. Human skin (epidermis and dermis) is rich in Antigen Presenting Cells (APCs), including langerhans cells and dermal Dendritic Cells (DCs). Intradermal vaccination is known to be 5 to 10 times more effective than subcutaneous or intramuscular vaccination because it targets APCs [ hicking and Jones,2009], and this targeting also activates the T cell immune pathway to obtain long-term immunity. By intradermal injection, c-srRNA is taken up mainly by skin APCs, where it replicates, produces antigen, digests the antigen into peptides, and presents these peptides to T cells (FIG. 1). Peptides presented by this pathway stimulate MHC-I restricted cd8+ killer T cells. In an alternative pathway, APCs also ingest antigens produced by nearby skin cells. Peptides presented by this pathway stimulate MHC-II restricted cd4+ helper T cells.

Problems of intradermal injection and our solution

The following are the potential problems we find and the solutions we provide for our c-srRNA platform.

(1) One key unrecognized obstacle to the use of srRNA as an intradermal vaccine platform is that both mRNA and srRNA do not express antigen well at skin temperature [ PCT/US20/67506]. Not intuitively, the temperature of human skin (about 30 ℃ -35 ℃) is lower than human core body temperature (about 37 ℃); this means that the vectors and platforms developed at 37 ℃ are not optimal for intradermal injection. One innovation of our c-srRNA platform is that it strongly expresses antigen at skin temperature [ PCT/US20/67506]. In addition, this temperature control also minimizes safety risks caused by unintended systemic distribution of c-srRNA, because after the temperature of c-srRNA rises above its allowable threshold (as it moves closer to the body core), c-srRNA is deactivated. In other words, the c-srRNA platform expresses an antigen that is most suitable for intradermal injection compared to mRNA and srRNA, and it additionally has safety features: the ability of the vector to spread and develop in other areas of the subject's body is limited or inactivated.

(2) Another challenge of intradermal vaccination is the lack of suitable additives. Because adjuvants such as aluminum salts and oil-in-water are too locally reactive when delivered by the intradermal route, no adjuvants are incorporated into clinically approved intradermal vaccines, resulting in lower immunogenicity [ hicking and Jones,2009]. Lipid Nanoparticles (LNPs) for intramuscularly administered mRNA and srRNA vaccines are also oil-in-water, which may cause skin reactivities and increase the risk of allergic reactions to LNP components such as PEG. Our c-srRNA platform is a solution to this problem because it is injected as naked c-srRNA (without LNP, without adjuvant). First, self-replication of RNA within cells, particularly APCs, induces strong innate immunity that replaces the primary function of adjuvants. Second, the literature and our own data demonstrate that, particularly for intradermal injection, naked mRNA/srrrna is equally effective in producing antigen compared to electroporation of mRNA/srrrna [ Johansson et al 2012] and electroporation of a combination of mRNA/srrrna and LNP [ Golombek et al 2018 ].

(3) A third challenge is the limited number of precedents for intradermal vaccines. Only BCG vaccine is routinely administered intradermally. One way we have reduced the use of intradermal injection barriers is to use specialized devices such as MicronJet600 (NanoPass) and Immucise (Terumo), which can now be used to achieve easy, consistent intradermal injections. These devices are also good candidates for mass production and deployment. However, intradermal injection by Mantoux technology using standard needles and syringes is also an option due to the relatively high cost of these special devices.

Design of suitable antigens

Tumor Associated Antigens (TAAs) are expressed in tumor cells but also in embryonic cells or at low levels in normal cells. The national cancer institute (National Cancer Institute) selected 75 cancer antigens suitable for cancer therapy targets (Cheever et al, 2009). For example, the wilms tumor protein 1 (WT 1) is listed as the most promising among the 75 cancer antigens identified by the national cancer institute (Cheever et al, 2009). WT1 is expressed in a wide range of tumors, but only in embryonic tissue and in very limited cell types in adults. For example, WT1 is expressed in most leukemias (AML, ALL), pancreatic cancer, lung cancer, and glioblastomas. 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. The list also contains NY-ESO-1 (example 2) and MAGEA3 (example 3). Based on our c-srRNA platform, any TAA can be used as an antigen for cancer vaccines. Any combination of these TAAs may also be used as fusion proteins or as separately expressed proteins (example 4).

Recently, genomic sequencing of patient-derived tumor cells has become commonplace. Such efforts have generally identified protein products or peptides that are unique to the tumor due to mutations in the tumor genome. These Tumor Specific Antigens (TSAs) are also known as neoantigens, which are ideal targets for cancer vaccines. Based on our c-srRNA platform, a single TSA or a fusion of more than one TSA can be used as an antigen for a cancer vaccine (example 5).

Chitosan-enhances in vivo gene expression

Rnase inhibitors (proteins purified from human placenta) slightly enhance immunogenicity against antigens encoded on C-srRNA, most likely by enhancing antigen expression from C-srRNA in vivo when injected intradermally into mice (see, e.g., figure 25C of WO 2021/138447 A1). Rnase inhibitors can protect c-srRNA from rnase-mediated degradation in vivo. However, it is desirable to find alternative agents that can enhance the expression of a gene of interest (GOI) in vivo for therapeutic purposes, because it is difficult to use protein-based rnase inhibitors as excipients in injectable products.

Low molecular weight chitosan (molecular weight about 6 kDa) has been shown to inhibit RNase activity with an inhibition constant in the range of 30-220nM (Yakovlev et al Biochem Biophys Res Commun,357 (3): 584-8, 2007). Two different chitosan oligomers have recently been tested: chitosan oligomer (CAS number 9012-76-4; molecular weight.ltoreq.5 kDa,.gtoreq.75% deacetylation: heppe Medical Chitosan GmbH: product number 44009) and chitosan oligosaccharide lactate (CAS number 148411-57-8; molecular weight about 5kDa, >90% deacetylation: sigma-Aldrich: product number 523682). Surprisingly, it was found that even very low levels (as low as 0.001. Mu.g/mL) of chitosan oligomer (about 0.2nM: yakovlev et al, supra, 1/100 of the inhibition constant found in 2007) were able to enhance the expression of the luciferase encoded on c-srRNA by about 10-fold (data not shown). Similar enhancement of GOI expression was achieved by up to 0.5. Mu.g/mL of chitosan oligomer and 0.1. Mu.g/mL of chitosan oligosaccharide lactate.

Chitosan has been used as a nucleotide (DNA and RNA) delivery vehicle because it can form complexes or nanoparticles (reviewed in bussmann et al Adv Drug Deliv Rev,65 (9): 1234-70,2013; and Cao et al Drugs,17:381, 2019). However, it is notable that the enhanced GOI expression achieved by the chitosan oligomer is unlikely to be mediated by nanoparticle or complex formation of c-srRNA and chitosan oligomer. First, such low concentrations of chitosan oligomers do not allow for complex formation with RNA. Second, chitosan oligomer was added to c-srRNA immediately prior to intradermal injection, and thus, there was insufficient time for complex formation.

Since chitosan oligomers enhance GOI expression in vivo at much lower concentrations than are effective as rnase inhibitors in vitro (Yakovlev et al, supra, 2007), it is contemplated that such enhanced GOI expression by chitosan oligomers may not be mediated by its rnase inhibition mechanism. For example, chitosan oligomers may promote the incorporation of c-srRNA into cells, which may enhance GOI expression from c-srRNA. Nevertheless, this unexpected finding should provide an effective means for enhancing the in vivo therapeutic expression of GOI encoded on c-srRNA.

Detailed description of the illustrated embodiments

1. A composition for stimulating an immune response against a cancer antigen in a mammalian subject, the composition comprising an excipient and a temperature-sensitive self-replicating RNA comprising an Open Reading Frame (ORF) encoding a fusion protein and an alphavirus replicon lacking viral structural protein coding regions, wherein the ORF comprises from 5 'to 3':

(i) A nucleotide sequence encoding a mammalian signal peptide; and

(ii) A nucleotide sequence encoding a cancer antigen,

wherein the temperature sensitive self-replicating RNA is capable of expressing the fusion protein at a permissive temperature and not expressing the fusion protein at a non-permissive temperature.

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 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 at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID No. 7.

8. The composition of embodiment 2, wherein the TAA comprises 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 at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID No. 3.

10. The composition of embodiment 2, wherein the TAA comprises an 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 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 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 of any of embodiments 1-16, wherein the mammalian signal peptide is a signal peptide of a surface protein expressed in mammalian antigen presenting cells.

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 at least 90% or 95% identical to SEQ ID No. 1.

19. The composition of any one of embodiments 1-18, 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. The composition of any one of embodiments 1-20, wherein the alphavirus replicon comprises a non-structural protein coding region having 12-18 nucleotide insertions resulting in expression of non-structural protein 2 (nsP 2) comprising 4 to 6 additional amino acids between β sheet 5 and β sheet 6 of the nsP 2.

22. The composition of embodiment 21, wherein the additional amino acid comprises the sequence of SEQ ID No. 14 (TGAAA).

23. The composition according to embodiment 22, wherein the amino acid sequence of nsP2 comprises SEQ ID No. 12.

24. The composition according to embodiment 23, wherein the amino acid sequence of nsP2 comprises a sequence selected from the group consisting of SEQ ID No. 9, SEQ ID No. 10 and SEQ ID No. 11.

25. The composition according to embodiment 24, wherein the amino acid sequence of nsP2 comprises SEQ ID No. 11.

26. The composition of any of embodiments 1-25, wherein the allowed temperature is 30 ℃ to 36 ℃, or 31 ℃ to 35 ℃, or 32 ℃ to 34 ℃, or 33 ℃ ± 0.5 ℃, and the non-allowed temperature is 37 ℃ ± 0.5 ℃, optionally wherein the allowed temperature is 31 ℃ to 35 ℃, and the non-allowed temperature is at least 37 ℃ ± 0.5 ℃.

27. The composition of any one of embodiments 1-26, wherein the composition does not comprise lipid nanoparticles.

28. The composition of any one of embodiments 1-27, wherein the composition further comprises chitosan.

29. A method for stimulating an immune response against a cancer antigen in a mammalian subject, the method comprising administering to a mammalian subject the composition of any one of embodiments 1-28 to stimulate an immune response against 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 with the cancer antigen.

34. The method of any one of embodiments 29-33, wherein the mammalian subject is a human subject.

35. A kit, the kit comprising:

(i) The composition of any one of embodiments 1-28; and

(ii) A device for intradermal delivery of the composition to a mammalian subject.

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

Examples

Abbreviations: APC (antigen presenting cell); BIRC5 (containing baculovirus IAP repeat 5 or survivin); IL-4 (interleukin-4); IFN-gamma (interferon gamma); MAGEA3 (melanoma-associated antigen 3); ORF (open reading frame); PBO (placebo); NY-ESO-1 (new york esophageal squamous cell carcinoma protein 1 or CTAG 1B); PRAME (melanoma preferential expression antigen); SFC (spot forming cells); srrrnats (temperature sensitive self-replicating RNA or c-srRNA temperature controllable self-replicating RNA); TAA (tumor associated antigen); TSA (tumor specific antigen); and WT1 (nephroblastoma protein 1).

EXAMPLE 1 immunotherapy against WT1 expressing tumors

This example describes the following findings: when expressed from intradermally injected temperature-controllable self-replicating RNAs, the human wilms' tumor protein 1 (WT 1) protein induces an effective cellular immune response in BALB/c mice. Remarkably, the EXG-5101RNA construct induced the elimination of mouse mammary tumor cells expressing human WT1 in a syngeneic mouse cancer model.

Materials and methods

BALB/c inbred female mice.

EXG-5101mRNA was produced by in vitro transcription of a temperature controlled self-replicating RNA vector (srRNA 1ts2[ PCT/US2020/067506 ]), which encodes a fusion protein comprising a human CD5 signal peptide fused to a human WT1 protein (fig. 2). The WT1 protein of EXG-5101 is encoded by isoform D starting with a non-AUG (CUG) translation initiation codon.

4T1 mammary tumor cells (ATCC No. CRL-2539) were derived from BALB/c mice and are known to mimic human breast cancer (stage IV).

Fig. 4 shows the experimental procedure. 4T1 tumor cells were transfected with plasmid DNA encoding the human nephroblastoma protein 1 (WT 1) 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 mammary fat pads of BALB/c mice (day 0 post tumor inoculation). On day 7, placebo (PBO), 5 μg or 25 μg of EXG-5101mRNA (day 0 post-inoculation) was administered intradermally. Tumor sizes were measured on day 5, day 8 (day 0 post-vaccination), day 25 (day 18 post-vaccination), and day 32 (day 25 post-vaccination).

Results and conclusions

FIG. 5 shows tumor growth in BALB/c mice injected with Placebo (PBO), 5 μg or 25 μg of EXG-5101mRNA vaccine. Mean and standard deviation (error bars) of five mice per group (n=5) are shown. By day 7 post tumor inoculation, all three groups of mice had tumors. However, by day 25 (day 18 post-vaccination), tumor growth was inhibited in dose-dependent fashion in mice injected with EXG-5101mRNA, while tumor growth continued in placebo-injected mice.

FIGS. 6A-6B show induction of tumor-associated antigen-reactive cellular immune responses by intradermal injection of EXG-5101 mRNA. As shown in FIG. 6A, BALB/c mice were injected intradermally with 25 μg EXG-5101 or Placebo (PBO) on day 0. Splenocytes were collected from these mice on day 14 and used for ELISpot assays. FIG. 6B shows the results of the ELISPot assay as a frequency of IFN-. Gamma.or IL-4 Spot Forming Cells (SFC) per 1X10≡6 spleen cells that have been stimulated by pools of 110 peptides (15 mers with 11 amino acid overlaps) covered with human WT1 protein. IFN-gamma secreting cells represent CD8+ T cells and CD4+ Th1 cells, which are thought to be cell-mediated (cellular) immune responses, while IL-4 secreting cells represent CD4+ Th2 cells. Thus, the results indicate that EXG-5101 induces cellular immunity against human WT1 protein.

In summary, intradermally injected EXG-5101mRNA immunotherapeutic agents inhibited tumor growth or reduced tumor size of WT1 expressing tumors in a dose-dependent manner in a syngeneic mouse model of breast cancer. In addition, intradermal injected EXG-5101mRNA immunotherapeutic induced cellular immunity against human WT1 protein in the mouse model.

EXAMPLE 2 immunotherapy against NY-ESO-1 expressing tumors

This example describes an evaluation of whether intradermal injection of c-srRNA encoding human NY-ESO-1 was able to induce a cellular immune response against human NY-ESO-1 expressing mouse breast tumor cells in a syngeneic mouse cancer model.

Materials and methods

BALB/c inbred female mice.

The c-srRNA-NY-EOS 1mRNA is produced by in vitro transcription of a temperature controlled self-replicating RNA vector (srRNA 1ts2[ PCT/US20/67506 ]), which encodes a fusion protein comprising a human CD5 signal peptide fused to a human NY-ESO-1 protein. NY-ESO-1 is also known as cancer/testis antigen 1B (CTAG 1B) (NM-001327).

4T1 mammary tumor cells (ATCC No. CRL-2539) were derived from BALB/c mice and are known to mimic human breast cancer (stage IV).

4T1 tumor cells were transfected with plasmid DNA encoding human NY-ESO-1 (also known as cancer/testis antigen 1B (CTAG 1B) (NM-001327)) driven by the CMV promoter as a selectable marker for the neomycin resistance gene. Stable transformants of 4T1 cells expressing the human NY-ESO-1 gene were isolated by G418 selection. Cells were injected into mammary fat pads of BALB/c mice (day 0 post tumor inoculation). On day 7, placebo (PBO), 5 μg or 25 μg g c-srRNA-NY-ESO-1mRNA (day 0 post-inoculation) was administered intradermally. Tumor size was measured at several time points after vaccination.

Results and conclusions

Intradermal injection of c-srRNA-NY-ESO-1mRNA immunotherapeutic agents is expected 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.

EXAMPLE 3 immunotherapy against MAGEA 3-expressing tumors

This example describes an evaluation of whether intradermal injection of c-srRNA encoding human MAGE family member A3 (MAGEA 3) was able to induce a cellular immune response against human MAGEA3 expressing mouse breast tumor cells in a syngeneic mouse cancer model.

Materials and methods

BALB/c inbred female mice.

The c-srRNA mRNA-MAGEA3 is produced by in vitro transcription of a temperature controlled self-replicating RNA vector (srRNA 1ts2[ PCT/US20/67506 ]) which encodes a fusion protein comprising a human CD5 signal peptide fused to a human MAGE family member A3 (MAGEA 3) protein (NM-005362).

4T1 mammary tumor cells (ATCC No. CRL-2539) were derived from BALB/c mice and are known to mimic human breast cancer (stage IV).

4T1 tumor cells were transfected with plasmid DNA encoding human MAGEA3 (NM-005362) driven by the CMV promoter as a selectable marker for the neomycin resistance gene. Stable transformants of 4T1 cells expressing human MAGEA3 were isolated by G418 selection. Cells were injected into mammary fat pads of BALB/c mice (day 0 post tumor inoculation). On day 7, placebo (PBO), 5 μg or 25 μg g c-srRNA-MAGEA3 mRNA (day 0 post-inoculation) was administered intradermally. Tumor size was measured at several time points after vaccination.

Results and conclusions

Intradermal injection of c-srRNA-MAGEA3 mRNA immunotherapeutic agents is expected 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.

EXAMPLE 4 immunotherapy against tumors expressing two or more tumor-associated antigens (TAA)

This example describes the following findings: intradermal injection of c-srRNA encoding fusion proteins comprising WT1, NY-ESO-1, BIRC5, MAGEA3 and PRAME induced potent cellular immune responses against TAA of the fusion proteins in BALB/c mice.

Materials and methods

BALB/c inbred female mice.

FIG. 7 shows a schematic representation of EXG-5105 vaccine, which is a c-srRNA mRNA encoding fusion proteins of human WT1, NY-ESO-1, BIRC5, MAGEA3 and PRAME with signal peptide sequences derived from human CD5 gene (srRNA 1ts2[ PCT/US20/67506 ]).

4T1 mammary tumor cells (ATCC: CRL-2539) are derived from BALB/c, which are known to mimic human breast cancer (stage IV).

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

Results and conclusions

FIG. 8A shows an experimental procedure for checking the immunogenicity of EXG-5105mRNA vaccine. On day 0, BALB/c mice received intradermal injections of 25 μg of EXG-5105 or Placebo (PBO). Splenocytes were collected from these mice on day 14 and used for ELISpot assays. EXG-5105 encodes a fusion protein comprising human WT1, NY-ESO-1, BIRC5, MAGEA3 and PRAME. Thus, intradermal injection of EXG-5105 is expected to induce cellular immunity against all five TAAs simultaneously. In fact, the results shown in FIGS. 8B-8F indicate that this is true. FIG. 8B shows the results of the ELISPot assay as a frequency of IFN-. Gamma.or IL-4 Spot Forming Cells (SFC) per 1X10≡6 spleen cells that had been stimulated by pools of 110 peptides (15 mers with 11 amino acid overlaps) covered with human WT1 protein. IFN-gamma secreting cells represent CD8+ T cells and CD4+ Th1 cells, which indicate a cell-mediated (cellular) immune response, while IL-4 secreting cells represent CD4+ Th2 cells. Thus, the results indicate that EXG-5105 induces cellular immunity against human WT1 protein. Similarly, FIG. 8C shows that the result of the ELISPot assay is the frequency of IFN-. Gamma.or IL-4 Spot Forming Cells (SFC) per 1X 10. Sup.6 spleen cells that have been stimulated by pools of peptides (15 mers with 11 amino acid overlaps) that are covered with human NY-ESO-1 protein. The results indicate that EXG-5105 induces cellular immunity against the human NY-ESO-1 protein and the human WT1 protein. Similarly, FIGS. 8D, 8E and 8F show the frequency of cytokine (left, interferon-gamma [ IFN-gamma ]; right, interleukin-4 [ IL-4 ]) Spot Forming Cells (SFC) per 1x 10A 6 spleen cells, which have been stimulated by pools of peptides covered with human MAGEA3 protein, human BIRC5 (survivin) protein and human PRAME protein, respectively. Interestingly, a more potent cellular immune response was elicited against MAGEA3 and PRAME (both of which are expressed fairly exclusively in tumors and testes) compared to that against WT1, NY-ESO-1 and BIRC5 (all of which are expressed in tumors and some other tissues).

In summary, intradermally injected EXG-5105mRNA immunotherapeutic agents induced cellular immunity against the different components of the fusion protein in the syngeneic mouse cancer model. In addition, intradermal injection of EXG-5105mRNA vaccine was expected to inhibit growth of tumor cells expressing human WT1, NY-ESO-1, BIRC5, MAGEA3 and PRAME in vivo.

EXAMPLE 5 immunotherapeutic Agents against tumors expressing tumor-specific antigen (TSA)

This example describes the following findings: intradermal injection of srRNAts encoding neoantigens induces a cellular immune response against the neoantigen in BALB/c mice in a syngeneic mouse cancer model.

Materials and methods

BALB/c inbred female mice.

srRNA ts mRNA encoding a novel antigen having a signal peptide sequence derived from the human CD5 gene (srRNA 1ts2[ PCT/US20/67506 ]).

4T1 mammary tumor cells (ATCC: CRL-2539) are derived from BALB/c, which are known to mimic human breast cancer (stage IV).

The 4T1 tumor cell line was transfected with three plasmid DNA encoding human neoantigen driven by the CMV promoter and a selectable marker for G418 (neomycin). Stable transformants of 4T1 cells expressing human neoantigen were isolated after G418 selection. Cells were injected into mammary fat pads of BALB/c mice. Placebo (PBO), 5 μg or 25 μg of srRNAts-neoantigen mRNA vaccine was administered intradermally. Subsequently, tumor size was measured.

Results and conclusions

In the syngeneic mouse cancer model, intradermally injected srRNAts-neoantigen mRNA vaccines inhibit the growth of tumor cells expressing human neoantigens and eliminate tumors in a dose-dependent manner.

Example 6 comparison of self-replicating RNA for T cell inducibility

This example describes the following findings: intradermal injection of srrrnats constructs encoding antigens induces a cellular immune response against the antigen in mice.

Materials and methods

C57BL/6 mice.

Three different temperature-controllable self-replicating RNA vectors (c-srrrna) and control self-replicating RNA vectors (c-srrrna) were tested. The features of srRNA are summarized in Table 6-1. IFN- α/β sensitivity of parent VEEV strains has been previously reported (Spots et al, J Viol,72:10286-10291,1998). c-srRNA1 was based on the TRD strain of VEEV, but was modified to have an A16D substitution (TC 83 mutation) and a P778S substitution. c-srRNA3 was also based on the TRD strain of VEEV, but without A16D and P778S substitutions. srRNA4 was based on the V198 strain of VEEV isolated from humans. All three c-srRNA vectors contained the same 5 amino acid insertions within the nsP2 protein of VEEV for temperature controllability as previously described (see U.S. Pat. No. 11,421,248 to Ko, examples 3, 21 and 22, incorporated herein by reference). All four srrnas encode antigens that lack a signal peptide sequence (SARS-CoV-2 spike protein receptor binding domain).

TABLE 6-1.SrRNA characterization

RNA	ts-mutant	VEEV
			srRNA0	Whether or not	TRD
c-srRNA1	Is that	TRD/TC-83
			c-srRNA3	Is that	TRD
c-srRNA4	Is that	V198

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

Preparation of srRNA. All srRNA is produced by in vitro transcription. NEB 10-beta competent E.coli (C3019H/C3019I) was transformed with plasmid DNA and cultured in Luria Broth with 100. Mu.g/mL ampicillin. Purified plasmid DNA was linearized by MluI. In Vitro Transcription (IVT) of c-srRNA with Cap1 and poly A was performed using plasmid DNA in vitro transcription using T7 RNA polymerase and Cleancap AU (Trilink) according to the manufacturer's protocol.

srRNA was injected into the skin of mice. Mice were randomized and Mao Tiguang on hind limbs to expose skin one day prior to injection. 5 μg or 25 μg srRNA reconstituted in ringer's Lactate (LR) solution was injected intradermally onto shaved skin.

Results and conclusions

C57BL/6 mice received one of srRNA or placebo as naked RNA (without lipid nanoparticles or transfection reagent) by intradermal injection (fig. 9A). As expected, cellular immunity assessed by the presence of antigen-specific IFN- γ secreting T cells had been induced on day 14 post-vaccination (fig. 9B). The T cell response induced by c-srRNA1 was stronger than that induced by standard non-temperature controllable srRNA 0. Furthermore, the T cell response induced by both c-srRNA3 and c-srRNA4 was stronger than the response induced by srRNA0 and c-srRNA 1. Remarkably, the T cell response induced by both c-srRNA3 and c-srRNA4 was about 3 times greater than that induced by c-srRNA 1. This difference is expected to be due to the fact that the parent VEEV sequences of c-srRNA3 and c-srRNA4 are more resistant to inhibition by type I interferon than the parent VEEV sequences of c-srRNA 1.

Reference to the literature

References referred to in this disclosure include: PCT/US2020/067506of Elixirgen Therapeutics,Inc; 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 sample-inch counts.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(s)

SEQ ID NO:1

Human CD5 Signal peptide

MPMGSLQPLATLYLLGMLVASCLG

SEQ ID NO:2

Human nephroblastoma protein (NM_ 024426.6)

MDFLLLQDPASTCVPEPASQHTLRSGPGCLQQPEQQGVRDPGGIWAKLGAAEASAERLQGRRSRGASGSEPQQMGSDVRDLNALLPAVPSLGGGGGCALPVSGAAQWAPVLDFAPPGASAYGSLGGPAPPPAPPPPPPPPPHSFIKQEPSWGGAEPHEEQCLSAFTVHFSGQFTGTAGACRYGPFGPPPPSQASSGQARMFPNAPYLPSCLESQPAIRNQGYSTVTFDGTPSYGHTPSHHAAQFPNHSFKHEDPMGQQGSLGEQQYSVPPPVYGCHTPTDSCTGSQALLLRTPYSSDNLYQMTSQLECMTWNQMNLGATLKGVAAGSSSSVKWTEGQSNHSTGYESDNHTTPILCGAQYRIHTHGVFRGIQDVRRVPGVAPTLVRSASETSEKRPFMCAYPGCNKRYFKLSHLQMHSRKHTGEKPYQCDFKDCERRFSRSDQLKRHQRRHTGVKPFQCKTCQRKFSRSDHLKTHTRTHTGKTSEKPFSCRWPSCQKKFARSDELVRHHNMHQRNMTKLQLAL

SEQ ID NO:3

Human BIRC5 (another name survivin) protein (NM-001168)

MGAPTLPPAWQPFLKDHRISTFKNWPFLEGCACTPERMAEAGFIHCPTENEPDLAQCFFCFKELEGWEPDDDPIEEHKKHSSGCAFLSVKKQFEELTLGEFLKLDRERAKNKIAKETNNKKKEFEETAEKVRRAIEQLAAMD

SEQ ID NO:4

Human NY-ESO-1 protein (NM-001327)

MQAEGRGTGGSTGDADGPGGPGIPDGPGGNAGGPGEAGATGGRGPRGAGAARASGPGGGAPRGPHGGAASGLNGCCRCGARGPESRLLEFYLAMPFATPMEAELARRSLAQDAPPLPVPGVLLKEFTVSGNILTIRLTAADHRQLQLSISSCLQQLSLLMWITQCFLPVFLAQPPSGQRR

SEQ ID NO:5

Human MAGEA3 protein (NM-005362)

MPLEQRSQHCKPEEGLEARGEALGLVGAQAPATEEQEAASSSSTLVEVTLGEVPAAESPDPPQSPQGASSLPTTMNYPLWSQSYEDSSNQEEEGPSTFPDLESEFQAALSRKVAELVHFLLLKYRAREPVTKAEMLGSVVGNWQYFFPVIFSKASSSLQLVFGIELMEVDPIGHLYIFATCLGLSYDGLLGDNQIMPKAGLLIIVLAIIAREGDCAPEEKIWEELSVLEVFEGREDSILGDPKKLLTQHFVQENYLEYRQVPGSDPACYEFLWGPRALVETSYVKVLHHMVKISGGPHISYPPLHEWVLREGEE

SEQ ID NO:6

Human PRAME protein (NM-001291715)

MERRRLWGSIQSRYISMSVWTSPRRLVELAGQSLLKDEALAIAALELLPRELFPPLFMAAFDGRHSQTLKAMVQAWPFTCLPLGVLMKGQHLHLETFKAVLDGLDVLLAQEVRPRRWKLQVLDLRKNSHQDFWTVWSGNRASLYSFPEPEAAQPMTKKRKVDGLSTEAEQPFIPVEVLVDLFLKEGACDELFSYLIEKVKRKKNVLRLCCKKLKIFAMPMQDIKMILKMVQLDSIEDLEVTCTWKLPTLAKFSPYLGQMINLRRLLLSHIHASSYISPEKEEQYIAQFTSQFLSLQCLQALYVDSLFFLRGRLDQLLRHVMNPLETLSITNCRLSEGDVMHLSQSPSVSQLSVLSLSGVMLTDVSPEPLQALLERASATLQDLVFDECGITDDQLLALLPSLSHCSQLTTLSFYGNSISISALQSLLQHLIGLSNLTHVLYPVPLESYEDIHGTLHLERLAYLHARLRELLCELGRPSMVWLSANPCPHCGDRTFYDPEPILCPCFMPN

SEQ ID NO:7

Artificial protein: fusion LDFLLLQDPASTCVPEPASQHTLRSGPGCLQQPEQQGVRDPGGIWAKLGAAEASAERLQGRRSRGASGSEPQQMGSDVRDLNALLPAVPSLGGGGGCALPVSGAAQWAPVLDFAPPGASAYGSLGGPAPPPAPPPPPPPPPHSFIKQEPSWGGAEPHEEQCLSAFTVHFSGQFTGTAGACRYGPFGPPPPSQASSGQARMFPNAPYLPSCLESQPAIRNQGYSTVTFDGTPSYGHTPSHHAAQFPNHSFKHEDPMGQQGSLGEQQYSVPPPVYGCHTPTDSCTGSQALLLRTPYSSDNLYQMTSQLECMTWNQMNLGATLKGVAAGSSSSVKWTEGQSNHSTGYESDNHTTPILCGAQYRIHTHGVFRGIQDVRRVPGVAPTLVRSASETSEKRPFMCAYPGCNKRYFKLSHLQMHSRKHTGEKPYQCDFKDCERRFSRSDQLKRHQRRHTGVKPFQCKTCQRKFSRSDHLKTHTRTHTGKTSEKPFSCRWPSCQKKFARSDELVRHHNMHQRNMTKLQLALMGAPTLPPAWQPFLKDHRISTFKNWPFLEGCACTPERMAEAGFIHCPTENEPDLAQCFFCFKELEGWEPDDDPIEEHKKHSSGCAFLSVKKQFEELTLGEFLKLDRERAKNKIAKETNNKKKEFEETAEKVRRAIEQLAAMDMQAEGRGTGGSTGDADGPGGPGIPDGPGGNAGGPGEAGATGGRGPRGAGAARASGPGGGAPRGPHGGAASGLNGCCRCGARGPESRLLEFYLAMPFATPMEAELARRSLAQDAPPLPVPGVLLKEFTVSGNILTIRLTAADHRQLQLSISSCLQQLSLLMWITQCFLPVFLAQPPSGQRRMPLEQRSQHCKPEEGLEARGEALGLVGAQAPATEEQEAASSSSTLVEVTLGEVPAAESPDPPQSPQGASSLPTTMNYPLWSQSYEDSSNQEEEGPSTFPDLESEFQAALSRKVAELVHFLLLKYRAREPVTKAEMLGSVVGNWQYFFPVIFSKASSSLQLVFGIELMEVDPIGHLYIFATCLGLSYDGLLGDNQIMPKAGLLIIVLAIIAREGDCAPEEKIWEELSVLEVFEGREDSILGDPKKLLTQHFVQENYLEYRQVPGSDPACYEFLWGPRALVETSYVKVLHHMVKISGGPHISYPPLHEWVLREGEEMERRRLWGSIQSRYISMSVWTSPRRLVELAGQSLLKDEALAIAALELLPRELFPPLFMAAFDGRHSQTLKAMVQAWPFTCLPLGVLMKGQHLHLETFKAVLDGLDVLLAQEVRPRRWKLQVLDLRKNSHQDFWTVWSGNRASLYSFPEPEAAQPMTKKRKVDGLSTEAEQPFIPVEVLVDLFLKEGACDELFSYLIEKVKRKKNVLRLCCKKLKIFAMPMQDIKMILKMVQLDSIEDLEVTCTWKLPTLAKFSPYLGQMINLRRLLLSHIHASSYISPEKEEQYIAQFTSQFLSLQCLQALYVDSLFFLRGRLDQLLRHVMNPLETLSITNCRLSEGDVMHLSQSPSVSQLSVLSLSGVMLTDVSPEPLQALLERASATLQDLVFDECGITDDQLLALLPSLSHCSQLTTLSFYGNSISISALQSLLQHLIGLSNLTHVLYPVPLESYEDIHGTLHLERLAYLHARLRELLCELGRPSMVWLSANPCPHCGDRTFYDPEPILCPCFMPN of WT1, BIRC5, NY-ESO-1, MAGEA3 and PRAME proteins

SEQ ID NO:8

Artificial protein: fusion of human CD5 (signal peptide only), WT1, BIRC5, NY-ESO-1, MAGEA3 and PRAME proteins

MPMGSLQPLATLYLLGMLVASCLGLDFLLLQDPASTCVPEPASQHTLRSGPGCLQQPEQQGVRDPGGIWAKLGAAEASAERLQGRRSRGASGSEPQQMGSDVRDLNALLPAVPSLGGGGGCALPVSGAAQWAPVLDFAPPGASAYGSLGGPAPPPAPPPPPPPPPHSFIKQEPSWGGAEPHEEQCLSAFTVHFSGQFTGTAGACRYGPFGPPPPSQASSGQARMFPNAPYLPSCLESQPAIRNQGYSTVTFDGTPSYGHTPSHHAAQFPNHSFKHEDPMGQQGSLGEQQYSVPPPVYGCHTPTDSCTGSQALLLRTPYSSDNLYQMTSQLECMTWNQMNLGATLKGVAAGSSSSVKWTEGQSNHSTGYESDNHTTPILCGAQYRIHTHGVFRGIQDVRRVPGVAPTLVRSASETSEKRPFMCAYPGCNKRYFKLSHLQMHSRKHTGEKPYQCDFKDCERRFSRSDQLKRHQRRHTGVKPFQCKTCQRKFSRSDHLKTHTRTHTGKTSEKPFSCRWPSCQKKFARSDELVRHHNMHQRNMTKLQLALMGAPTLPPAWQPFLKDHRISTFKNWPFLEGCACTPERMAEAGFIHCPTENEPDLAQCFFCFKELEGWEPDDDPIEEHKKHSSGCAFLSVKKQFEELTLGEFLKLDRERAKNKIAKETNNKKKEFEETAEKVRRAIEQLAAMDMQAEGRGTGGSTGDADGPGGPGIPDGPGGNAGGPGEAGATGGRGPRGAGAARASGPGGGAPRGPHGGAASGLNGCCRCGARGPESRLLEFYLAMPFATPMEAELARRSLAQDAPPLPVPGVLLKEFTVSGNILTIRLTAADHRQLQLSISSCLQQLSLLMWITQCFLPVFLAQPPSGQRRMPLEQRSQHCKPEEGLEARGEALGLVGAQAPATEEQEAASSSSTLVEVTLGEVPAAESPDPPQSPQGASSLPTTMNYPLWSQSYEDSSNQEEEGPSTFPDLESEFQAALSRKVAELVHFLLLKYRAREPVTKAEMLGSVVGNWQYFFPVIFSKASSSLQLVFGIELMEVDPIGHLYIFATCLGLSYDGLLGDNQIMPKAGLLIIVLAIIAREGDCAPEEKIWEELSVLEVFEGREDSILGDPKKLLTQHFVQENYLEYRQVPGSDPACYEFLWGPRALVETSYVKVLHHMVKISGGPHISYPPLHEWVLREGEEMERRRLWGSIQSRYISMSVWTSPRRLVELAGQSLLKDEALAIAALELLPRELFPPLFMAAFDGRHSQTLKAMVQAWPFTCLPLGVLMKGQHLHLETFKAVLDGLDVLLAQEVRPRRWKLQVLDLRKNSHQDFWTVWSGNRASLYSFPEPEAAQPMTKKRKVDGLSTEAEQPFIPVEVLVDLFLKEGACDELFSYLIEKVKRKKNVLRLCCKKLKIFAMPMQDIKMILKMVQLDSIEDLEVTCTWKLPTLAKFSPYLGQMINLRRLLLSHIHASSYISPEKEEQYIAQFTSQFLSLQCLQALYVDSLFFLRGRLDQLLRHVMNPLETLSITNCRLSEGDVMHLSQSPSVSQLSVLSLSGVMLTDVSPEPLQALLERASATLQDLVFDECGITDDQLLALLPSLSHCSQLTTLSFYGNSISISALQSLLQHLIGLSNLTHVLYPVPLESYEDIHGTLHLERLAYLHARLRELLCELGRPSMVWLSANPCPHCGDRTFYDPEPILCPCFMPN

SEQ ID NO:9

Artificial protein: c-srRNA1 nsP2

GSVETPRGLIKVTSYDGEDKIGSYAVLSPQAVLKSEKLSCIHPLAEQVIVITHSGRKGRYAVEPYHGKVVVPEGHAIPVQDFQALSESATIVYNEREFVNRYLHHIATHGGALNTDEEYYKTVKPSEHDGEYLYDIDRKQCVKKELVTGLGLTGELVDPPFHEFAYESLRTRPAAPYQVPTIGVYGVPGSGKSGIIKSAVTKKDLVVSAKKENCAEIIRDVKKMKGLDVNARTVDSVLLNGCKHPVETLYIDEAFACHAGTLRALIAIIRPKKAVLCGDPKQCGFFNMMCLKVHFNHEICTQVFHKSISRRCTKSVTSVVSTLFYDKKMRTTNPKETKIVIDTTGSTKPKQDDLILTCFRGWVKQLQIDYKGNEIMTAAASQGLTRKGVYAVRYKVNENPLYAPTSEHVNVLLTRTEDRIVWKTLAGDPWIKTLTAKYPGNFTATIEEWQAEHDAIMRHILERPDPTDVFQNKANVCWAKALVPVLKTAGIDMTTEQWNTVDYFETDKAHSAEIVLNQLCVRFFGLDLDSGLFSAPTVPLSIRNNHWDNSPSPNMYGLNKEVVRQLSRRYPQLPRAVATGRVYDMNTGAAATGTLRNYDPRINLVPVNRRLPHALVLHHNEHPQSDFSSFVSKLKGRTVLVVGEKLSVPGKMVDWLSDRPEATFRARLDLGIPGDVPKYDIIFVNVRTPYKYHHYQQCEDHAIKLSMLTKKACLHLNPGGTCVSIGYGYADRASESIIGAIARQFKFSRVCKPKSSLEETEVLFVFIGYDRKARTHNSYKLSSTLTNIYTGSRLHEAGC

SEQ ID NO:10

Artificial protein: c-srRNA3 nsP2

GSVETPRGLIKVTSYAGEDKIGSYAVLSPQAVLKSEKLSCIHPLAEQVIVITHSGRKGRYAVEPYHGKVVVPEGHAIPVQDFQALSESATIVYNEREFVNRYLHHIATHGGALNTDEEYYKTVKPSEHDGEYLYDIDRKQCVKKELVTGLGLTGELVDPPFHEFAYESLRTRPAAPYQVPTIGVYGVPGSGKSGIIKSAVTKKDLVVSAKKENCAEIIRDVKKMKGLDVNARTVDSVLLNGCKHPVETLYIDEAFACHAGTLRALIAIIRPKKAVLCGDPKQCGFFNMMCLKVHFNHEICTQVFHKSISRRCTKSVTSVVSTLFYDKKMRTTNPKETKIVIDTTGSTKPKQDDLILTCFRGWVKQLQIDYKGNEIMTAAASQGLTRKGVYAVRYKVNENPLYAPTSEHVNVLLTRTEDRIVWKTLAGDPWIKTLTAKYPGNFTATIEEWQAEHDAIMRHILERPDPTDVFQNKANVCWAKALVPVLKTAGIDMTTEQWNTVDYFETDKAHSAEIVLNQLCVRFFGLDLDSGLFSAPTVPLSIRNNHWDNSPSPNMYGLNKEVVRQLSRRYPQLPRAVATGRVYDMNTGAAATGTLRNYDPRINLVPVNRRLPHALVLHHNEHPQSDFSSFVSKLKGRTVLVVGEKLSVPGKMVDWLSDRPEATFRARLDLGIPGDVPKYDIIFVNVRTPYKYHHYQQCEDHAIKLSMLTKKACLHLNPGGTCVSIGYGYADRASESIIGAIARQFKFSRVCKPKSSLEETEVLFVFIGYDRKARTHNPYKLSSTLTNIYTGSRLHEAGC

SEQ ID NO:11

Artificial protein: c-srRNA4 nsP2

GSVETPRGLIKVTSYAGEDKIGSYAVLSPQAVLKSEKLSCIHPLAEQVIVITHSGRKGRYAVEPYHGKVVVPEGHAIPVQDFQALSESATIVYNEREFVNRYLHHIATHGGALNTDEEYYKTVKPSEHDGEYLYDIDRKQCVKKELVTGLGLTGELVDPPFHEFAYESLRTRPAAPYQVPTIGVYGVPGSGKSGIIKSAVTKKDLVVSAKKENCAEIIRDVKKMKGLDVNARTVDSVLLNGCKHPVETLYIDEAFACHAGTLRALIAIIRPKKAVLCGDPKQCGFFNMMCLKVHFNHEICTQVFHKSISRRCTKSVTSVVSTLFYDKRMRTTNPKETKIEIDTTGSTKPKQDDLILTCFRGWVKQLQIDYKGNEIMTAAASQGLTRKGVYAVRYKVNENPLYAPTSEHVNVLLTRTEDRIVWKTLAGDPWIKTLTAKYPGNFTATIEEWQAEHDAIMRHILERPDPTDVFQNKANVCWAKALVPVLKTAGIDMTTEQWNTVDYFETDKAHSAEIVLNQLCVRFFGLDLDSGLFSAPTVPLSIRNNHWDNSPSPNMYGLNKEVVRQLSRRYPQLPRAVATGRVYDMNTGAAATGTLRNYDPRINLVPVNRRLPHALVLHHNEHPQSDFSSFVSKLKGRTVLVVGEKLSVPGKKVDWLSDQPEATFRARLDLGIPGDVPKYDIVFINVRTPYKYHHYQQCEDHAIKLSMLTKKACLHLNPGGTCVSIGYGYADRASESIIGAIARQFKFSRVCKPKSSHEETEVLFVFIGYDRKARTHNPYKLSSTLTNIYTGSRLHEAGC

SEQ ID NO:12

Artificial protein: c-srRNA nsP2 consensus sequence

GSVETPRGLIKVTSY[A/D]GEDKIGSYAVLSPQAVLKSEKLSCIHPLAEQVIVITHSGRKGRYAVEPYHGKVVVPEGHAIPVQDFQALSESATIVYNEREFVNRYLHHIATHGGALNTDEEYYKTVKPSEHDGEYLYDIDRKQCVKKELVTGLGLTGELVDPPFHEFAYESLRTRPAAPYQVPTIGVYGVPGSGKSGIIKSAVTKKDLVVSAKKENCAEIIRDVKKMKGLDVNARTVDSVLLNGCKHPVETLYIDEAFACHAGTLRALIAIIRPKKAVLCGDPKQCGFFNMMCLKVHFNHEICTQVFHKSISRRCTKSVTSVVSTLFYDK[K/R]MRTTNPKETKI[V/E]IDTTGSTKPKQDDLILTCFRGWVKQLQIDYKGNEIMTAAASQGLTRKGVYAVRYKVNENPLYAPTSEHVNVLLTRTEDRIVWKTLAGDPWIKTLTAKYPGNFTATIEEWQAEHDAIMRHILERPDPTDVFQNKANVCWAKALVPVLKTAGIDMTTEQWNTVDYFETDKAHSAEIVLNQLCVRFFGLDLDSGLFSAPTVPLSIRNNHWDNSPSPNMYGLNKEVVRQLSRRYPQLPRAVATGRVYDMNTGAAATGTLRNYDPRINLVPVNRRLPHALVLHHNEHPQSDFSSFVSKLKGRTVLVVGEKLSVPGK[M/K]VDWLSD[R/Q]PEATFRARLDLGIPGDVPKYDI[I/V]F[V/I]NVRTPYKYHHYQQCEDHAIKLSMLTKKACLHLNPGGTCVSIGYGYADRASESIIGAIARQFKFSRVCKPKSS[L/H]EETEVLFVFIGYDRKARTHN[P/S]YKLSSTLTNIYTGSRLHEAGC

SEQ ID NO:13

>VEEV：srRNA0

GSVETPRGLIKVTSYAGEDKIGSYAVLSPQAVLKSEKLSCIHPLAEQVIVITHSGRKGRYAVEPYHGKVVVPEGHAIPVQDFQALSESATIVYNEREFVNRYLHHIATHGGALNTDEEYYKTVKPSEHDGEYLYDIDRKQCVKKELVTGLGLTGELVDPPFHEFAYESLRTRPAAPYQVPTIGVYGVPGSGKSGIIKSAVTKKDLVVSAKKENCAEIIRDVKKMKGLDVNARTVDSVLLNGCKHPVETLYIDEAFACHAGTLRALIAIIRPKKAVLCGDPKQCGFFNMMCLKVHFNHEICTQVFHKSISRRCTKSVTSVVSTLFYDKKMRTTNPKETKIVIDTTGSTKPKQDDLILTCFRGWVKQLQIDYKGNEIMTAAASQGLTRKGVYAVRYKVNENPLYAPTSEHVNVLLTRTEDRIVWKTLAGDPWIKTLTAKYPGNFTATIEEWQAEHDAIMRHILERPDPTDVFQNKANVCWAKALVPVLKTAGIDMTTEQWNTVDYFETDKAHSAEIVLNQLCVRFFGLDLDSGLFSAPTVPLSIRNNHWDNSPSPNMYGLNKEVVRQLSRRYPQLPRAVATGRVYDMNTGTLRNYDPRINLVPVNRRLPHALVLHHNEHPQSDFSSFVSKLKGRTVLVVGEKLSVPGKMVDWLSDRPEATFRARLDLGIPGDVPKYDIIFVNVRTPYKYHHYQQCEDHAIKLSMLTKKACLHLNPGGTCVSIGYGYADRASESIIGAIARQFKFSRVCKPKSSLEETEVLFVFIGYDRKARTHNPYKLSSTLTNIYTGSRLHEAGC

SEQ ID NO:14

Artificial protein: TS insertion

TGAAA

Claims (36)

1. A composition for stimulating an immune response against a cancer antigen in a mammalian subject, the composition comprising an excipient and a temperature-sensitive self-replicating RNA comprising an Open Reading Frame (ORF) encoding a fusion protein and an alphavirus replicon lacking viral structural protein coding regions, wherein the ORF comprises from 5 'to 3':

(i) A nucleotide sequence encoding a mammalian signal peptide; and

(ii) A nucleotide sequence encoding a cancer antigen,

wherein the temperature sensitive self-replicating RNA is capable of expressing the fusion protein at a permissive temperature and not expressing the fusion protein at a non-permissive temperature.

2. The composition of claim 1, wherein the cancer antigen comprises a Tumor Associated Antigen (TAA).

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 at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID No. 2.

6. 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. 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 at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID No. 7.

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

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 at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID No. 3.

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

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 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. The composition of claim 14, wherein the amino acid sequence of the PRAME antigen comprises SEQ ID No. 6 or an amino acid sequence 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 of any one of claims 1-16, wherein the mammalian signal peptide is a signal peptide of a surface protein expressed in mammalian antigen presenting cells.

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 at least 90% or 95% identical to SEQ ID No. 1.

19. The composition of any one of claims 1-18, wherein the alphavirus is selected from venezuelan equine encephalitis virus, sindbis virus, and semliki forest virus.

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

21. The composition of any one of claims 1-20, wherein the alphavirus replicon comprises a non-structural protein coding region having 12-18 nucleotide insertions resulting in expression of non-structural protein 2 (nsP 2) comprising 4 to 6 additional amino acids between β sheet 5 and β sheet 6 of the nsP 2.

22. The composition of claim 21, wherein the additional amino acid comprises the sequence of SEQ ID No. 14 (TGAAA).

23. The composition of claim 22, wherein the amino acid sequence of nsP2 comprises SEQ ID No. 12.

24. The composition of claim 23, wherein the amino acid sequence of nsP2 comprises a sequence selected from the group consisting of SEQ ID No. 9, SEQ ID No. 10, and SEQ ID No. 11.

25. The composition of claim 24, wherein the amino acid sequence of nsP2 comprises SEQ ID No. 11.

26. The composition of any one of claims 1-25, wherein the allowed temperature is 30 ℃ to 36 ℃, or 31 ℃ to 35 ℃, or 32 ℃ to 34 ℃, or 33 ℃ ± 0.5 ℃, and the non-allowed temperature is 37 ℃ ± 0.5 ℃, optionally wherein the allowed temperature is 31 ℃ to 35 ℃, and the non-allowed temperature is at least 37 ℃ ± 0.5 ℃.

27. The composition of any one of claims 1-26, wherein the composition does not comprise lipid nanoparticles.

28. The composition of any one of embodiments 1-27, wherein the composition further comprises chitosan.

29. A method for stimulating an immune response against a cancer antigen in a mammalian subject, the method comprising administering the composition of any one of claims 1-28 to a mammalian subject to stimulate an immune response against the cancer antigen in the mammalian subject.

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

31. The method of claim 29 or claim 30, wherein the immune response comprises a cellular immune response reactive with mammalian cells expressing 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. The method of claim 32, wherein the immune response further comprises a humoral immune response reactive with the cancer antigen.

34. The method of any one of claims 29-33, wherein the mammalian subject is a human subject.

35. A kit, the kit comprising:

(i) The composition of any one of claims 1-28; and

(ii) A device for intradermal delivery of the composition to a mammalian subject.

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